The FGF-2 polypeptides with improved thermal and oxidative stability, the process for preparing the FGF-2 polypeptides and use thereof

Abstract

The invention relates to a Fibroblast Growth Factor 2 (FGF-2) polypeptides with improved stability while maintaining biological activity compared to the wild-type of FGF-2. The invention provides the process for the preparation, stabilisation and the use thereof in biotechnological research and industrial applications, the pharmaceutical industry, cosmetics, clean meat industry, generation of organoids, 3D cell culture models, in combination with physical modalities and other related applications.

Description

[0001] THE FGF-2 POEYPEPTIDES WITH IMPROVED THERMAL AND OXIDATIVE STABILITY, THE PROCESS FOR PREPARING THE FGF-2 POLYPEPTIDES AND USE THEREOF

[0002] Field of the invention

[0003] [1] The invention relates to a Fibroblast Growth Factor 2 (FGF-2) polypeptide with improved stability, especially thermal and oxidative stability, compared to the wild-type FGF-2 and the use thereof in research and industrial applications, such as biotechnological research, medicine, pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models and other related applications.

[0004] Background of the invention

[0005]

[0002] Fibroblast growth factors (FGFs) are a family of cell signaling proteins. They are involved in a variety of processes, especially as key elements for normal development of animal cells. These growth factors bind to heparin and heparan sulphate and typically activate cell surface receptors.

[0006] [3] Fibroblast growth factors that signal through FGF receptors (FGFRs) regulate fundamental developmental pathways, including the regulation of angiogenesis and wound repair. FGFRs are expressed on many different cell types and regulate key cell processes, such as proliferation, differentiation and survival, which make FGF signaling susceptible to subversion by cancer cells.

[0007] [4] FGFs are secreted glycoproteins that are generally readily sequestered to the extracellular matrix, as well as the cell surface, by heparan sulphate proteoglycans (HPSGs). For cellular signaling, FGFs are released from the extracellular matrix by heparinases, proteases or specific FGF-binding proteins, and the liberated FGFs subsequently bind to cell surface HPSGs. Cell surface HPSGs also stabilize the FGF ligand-receptor interaction, forming a ternary complex with FGFR.

[0008] [5] FGF receptors signal as dimers, and ligand-dependent dimerization leads to a conformational shift in receptor structure that activates the intracellular kinase domain, resulting in intermolecular transphosphorylation of the tyrosine kinase domains and intracellular tail. Phosphorylated tyrosine residues on the receptor function as docking sites for adaptor proteins, which themselves may also be directly phosphorylated by FGFR, leading to the activation of multiple signal transduction pathways.

[0009]

[0006] The human FGF-2 gene encodes not one protein, but a complex set of isoforms. The secreted isoform is a single-chain, non-glycosylated polypeptide with 154 amino acids. The amino acid sequence of human FGF-2 is 99% homologous to that of bovine FGF-2 and has high homology with bovine and rodent FGF- 2, suggesting strong sequence conservation for structure and function.

[0010] [7] The stability of FGF-2 is widely accepted to be a major concern in the development of useful medicinal products or serum replacement in biotechnological research. The manufacturers usually state that solutions of reconstituted FGF-2 are stable for up to 12 months only when stored at -20 °C or lower. The reconstituted FGFs solutions are stable for only about a week at 4 °C and are recommended to be used within 24 h when at ambient temperatures (around 25 °C). Nevertheless, a 50% loss of functionality of FGF-2 solutions at a concentration of 72 pg / mL was observed after just 4 min at 25 °C. The functional half-life is decreased to 37, 33 and 10 min, as the storage temperature is increased to 37 °C, 42 °C and 50 °C, respectively.

[0011] [8] One of the challenges is to preserve the biological activity of FGF-2. Most efforts are directed at sustaining FGF-2 activity for cell culture research, or to develop sustained-release formulations of FGF- 2 for tissue engineering applications. For example, approaches to maintain the stability and biological activity of FGF-2 include modulating ionic interactions in solution and chemical modifications of FGF- 2.

[0012] [9] The addition of excipients to aqueous solutions of FGF-2 is among the simplest methods for FGF-2 stabilization by way of modulating ionic interactions in solution. Common strategies include the complexation of FGF-2 with its endogenous stabilizer, heparin or heparin-like polymers, or polycations. Ionic interactions between FGF-2 and the additives reduce the structural energy at the heparin-binding site, stabilize the FGF-2 native conformation and prolong its bioactivity in aqueous media. This method, unfortunately, has some disadvantages. For example, there are safety concerns because the pharmaceutical-grade heparins are isolated from porcine intestines and bovine lung tissues and the heparins are susceptible to batch-to-batch variability. This variability can induce immune responses or be contaminated or adulterated by natural and synthetic heparinoids that may lead to anaphylactoid type responses and death. Additionally, natural heparin is susceptible to degradation and desulphation by heparinases, which may adversely affect its effectiveness at stabilizing FGF-2.

[0013]

[0010] Methods of prolonging the bioactivity of FGF-2 in aqueous media include point mutations of the protein and covalent grafting of the protein onto scaffold materials.

[0014]

[0011] Mutant variants of the FGF-2 protein have been developed by aligning the wild-type FGF-2 protein sequence with stabilized FGF-1 mutant sequences, or by combining several individual stabilizing mutations identified by other investigators in the field.

[0015]

[0012] The identification of useful point mutations has been aided by computer modeling. Modeling has identified sequences that are important for protein functionality and contribute the greatest amount to structural free energy. Following an analysis of sequence conservation, mutations in regions that might compromise protein function, were avoided. Conversely, mutations that were likely to result in a decrease in protein free energy were promoted. The obtained FGF-2 mutants showed the lowest energy and had increased functional half-life.

[0016]

[0013] Several modified FGF-2 polypeptides with improved thermal stability in comparison with natural types have been recently published, but despite some advances within the state of the art, there is still a need to further improve the thermal and oxidative stability of the FGF-2 product while maintaining its biological activity.

[0014] The object of the invention is to prepare an FGF-2 variant with improved thermal and oxidative stability and to find an efficient, scalable and economically advantageous process for the production of FGF-2 with high yields.

[0017]

[0015] Furthermore, the demand for stabilized FGF-2 variants extends beyond traditional cell culture or tissue engineering applications. In particular, there is a growing need for stable and biologically active FGF-2 formulations suitable for dermatological and cosmetic uses, where the tissue is subjected to external stressors or therapeutic procedures. Environmental stressors such as ultraviolet radiation, pollution, and temperature fluctuations, as well as intentional procedures including laser treatments, radiofrequency therapies, chemical peels, and microneedling, can compromise the tissue’s structural integrity and barrier function, leading to irritation, inflammation, and delayed recovery.

[0018]

[0016] Conventional FGF-2 formulations are not suitable for such applications due to their instability at physiological and ambient temperatures, and thus there is critical need for improved variants. Therefore, there remains a significant need for FGF-2 variants with enhanced thermal and oxidative stability that retain biological functionality and can be effectively utilized to promote and / or accelerate skin regeneration following exposure to environmental stressors or dermatological interventions.

[0019] Brief summary of the invention

[0020]

[0017] The disadvantages of the solutions, according to the state of the art, are solved by the present invention that provides a thermostable oxidation resistant polypeptide that possesses FGF-2 activity and has increased stability while maintaining biological activity compared to the wild-type FGF-2.

[0021]

[0018] In one aspect of the invention, the product is a truncated thermostabilized oxidation resistant FGF-2 polypeptide with amino acid substitutions characterized by sequence SEQ ID NO: 4. According to the present invention, the FGF-2 polypeptide with SEQ ID NO: 4 has 78.1 % sequence identity to Homo sapiens FGF-2 (SEQ ID NO: 1) and 78.7 % sequence identity to Bos taurus FGF-2 (SEQ ID NO: 2). The product has increased thermal and oxidative stability compared to the wild-type FGF-2 and artificial state of the art FGF-2 polypeptides.

[0022]

[0019] In another aspect of the invention, the products are thermostabilized FGF-2 polypeptides with amino acid substitutions characterized by sequences SEQ ID NO: 5 to 10.

[0023]

[0020] The FGF-2 polypeptides according to the invention may comprise a fusion partner and / or an affinity tag in order to improve their certain properties, such as solubility or proteolytic stability, or to increase their expression yields, or to facilitate their subsequent processing. This approach may allow preparation and use of the FGF-2 polypeptides according to the invention on an industrial scale.

[0021] The invention also provides the process for preparation of the FGF-2 polypeptides and the use thereof in the pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models, and other related applications.

[0024]

[0022] Experiments have shown increased thermal and oxidative stability while maintaining biological activity compared to the Homo sapiens FGF-2. The biological activity of the FGF-2 products according to the invention has been tested by cell growth experiments, and the biological effects of commercially available FGF-2 and FGF-2 products according to the invention have been compared. According to the invention, the thermal stability of the FGF-2 polypeptides using the nano differential scanning fluorimetry (nanoDSF) method, and the effect of long-term storage on product stability have been investigated.

[0025]

[0023] In one aspect of the invention, an FGF-2 variant suitable for promoting and / or accelerating skin regeneration following exposure to external stressors and / or procedures that result in skin and / or tissue irritation and / or damage, is provided.

[0026]

[0024] The invention also provides the process for combination of the FGF-2 variant with at least one physical modality.

[0027]

[0025] In one aspect, the present invention provides a variant of FGF-2 protein with enhanced stability when stored in an aqueous solution comprising sulfate anion (SOF ). The sulfate anion may be supplied by one or more sulfate salts, including, without limitation, ammonium sulfate, potassium sulfate, sodium sulfate, magnesium sulfate, calcium sulfate, manganese(II) sulfate, ferrous sulfate, ferric sulfate, copper(II) sulfate, zinc sulfate, aluminum sulfate, and alums such as potassium alum (KA1(SO4)2), sodium alum (NaAl(SC>4)2), or ammonium alum (NFLA SCh^), including their hydrates and mixtures thereof. The use of sulfate anion as a stabilizing agent contributes to increased thermal stability, improved resistance to aggregation, and prolonged shelf-life of the variant of FGF-2 protein. This stabilization allows for the preparation of highly concentrated FGF-2 formulations without significant loss of activity or structural integrity over extended periods of storage.

[0028] Brief description of the several views of the drawings

[0029]

[0026] Fig. 1 - Sequences of wild type FGF-2 homologs (Homo sapiens, Bos taurus) and FGF-2 product according to the invention characterized by SEQ ID NO: 4.

[0030]

[0027] Fig. 2 - SDS-PAGE analysis of the expression of truncated thermostabilized FGF-2 variants with improved oxidation resistance in E. coli BL21 (DE3) strain. LI is the molecular weight standard. L2 is the FGF-2 polypeptide characterized by SEQ ID NO: 5. L3 is the FGF-2 polypeptide characterized by SEQ ID NO: 9. Expression was induced by addition of 1 mM IPTG and was performed at 20 °C for 24 h.

[0031]

[0028] Fig. 3 - SDS-PAGE analysis of the truncated thermostabilized FGF-2 with improved oxidation resistance characterized by SEQ ID NO: 5 after purification. (El) molecular weight standard, (L3) pellet of the lysate, (L4) clarified lysate, (L6) flow through after loading the supernatant onto HisTrap column, (L7) wash of HisTrap column, (L8) elution from HisTrap column, (E10) flow through after loading the product HiTrap SP HP column, (Li l) wash of HiTrap SP HP column, (L12,L13,L14) elution from HiTrap SP HP column. L2, L5, and L9 are without samples.

[0032]

[0029] Fig. 4 - Thermostability of truncated thermostabilized FGF-2 with improved oxidation resistance characterized by SEQ ID NO: 4 analyzed by nanoDSF. Changes in tryptophan emission at 330 and 350 nm were monitored, and the ratio 330 / 350 nm was plotted against the temperature. Representative thermal unfolding curve (top) and its first derivative analysis (bottom).

[0033]

[0030] Fig. 5 - Stability of truncated thermostabilized FGF-2 with improved oxidative stability characterized by SEQ ID NO: 4 after storage at -20 °C for 90 days evaluated by biological activity assay.

[0034]

[0031] Fig. 6 and 7 - Stability of truncated thermostabilized FGF-2 with improved oxidative stability characterized by SEQ ID NO: 4 after freeze thaw cycle by biological activity assay (cell growth) and evaluation of cell growth density by microscopy photo evaluation. Bright dots - cell density after seeding. Dark dots - cell densities before passage. Fig. 6 - evaluation after 1st freeze thaw cycle, Fig. 7 - evaluation after 2nd freeze thaw cycle.

[0035]

[0032] Fig. 8 - The results from the cultivation of cells, when state of the art thermostabilized FGF-2 characterized by SEQ ID NO: 3 and thermostabilized FGF-2 with improved oxidative stability according to the invention characterized by SEQ ID NO: 4 were compared. First column - medium with thermostabilized FGF-2 characterized by SEQ ID NO: 3; second column - negative control without FGF2; third column thermostabilized FGF-2 characterized by SEQ ID NO: 5, 100 ng / mL; fourth column- SEQ ID NO: 5, 10 ng / mL; fifth column - SEQ ID NO: 4, 100 ng / mL; sixth column - SEQ ID NO: 4, 10 ng / mL.

[0036]

[0033] Fig. 9 - SDS-PAGE analysis of the expression of truncated thermostabilized FGF-2 variants with improved oxidative stability in E. coli BL21 (DE3) strain: (LI) molecular weight standard; (L2) pellet of the lysate and (L3) clarified lysate of the FGF-2 polypeptide characterized by SEQ ID NO: 5; (L5) pellet of the lysate and (L6) clarified lysate of the FGF-2 polypeptide characterized by SEQ ID NO: 10; (L8) pellet of the lysate and (L9) clarified lysate of the FGF-2 polypeptide characterized by SEQ ID NO: 9; (Li l) pellet of the lysate and (L12) clarified lysate of the FGF-2 polypeptide characterized by SEQ ID NO: 8; (L14) pellet of the lysate and (L15) clarified lysate SEQ ID NO: 7. L4, L7, L10 and L13 are without samples. The expression was induced by addition of 1 mM IPTG and was performed at 20°C for 24 h.

[0037]

[0034] Fig. 10 - illustrates the cell cultivation system according to the invention.

[0038]

[0035] Fig. 11 - illustrates exemplary invasive energy delivery elements.

[0039]

[0036] Fig. 12 - illustrates exemplary local and global alignments of two amino acid sequences

[0040]

[0037] Fig. 13 - illustrates schematic example of treatment device which may provide treatment by one or more physical modalities prior, at the same time or after application of FGF-2 polypeptide (in any form).

[0041] Cross-reference to related applications

[0042]

[0038] The PCT application PCT / IB2023 / 061877 filed November 24, 2023, the PCT application PCT / IB2024 / 059990 filed , October 11, 2024, U.S. Provisional application 63 / 715,083 filed November 1, 2024, U.S. Provisional 63 / 810,945 filed May 23, 2025, and U.S. Provisional 63 / 875,084 filed September 3, 2025, are all incorporated herein by reference in their entirety.

[0043] Detailed description of the invention

[0044]

[0039] The disadvantages of the solutions according to state of the art are solved by the present invention, which provides thermostable oxidation resistant polypeptides possessing FGF-2 activity and having increased stability compared to the wild-type FGF-2 polypeptides and also to artificial FGF-2 variants according to the state of the art, while maintaining the biological activity. The invention also provides the process for preparation of the FGF-2 polypeptides and use thereof.

[0045]

[0040] In one aspect of the invention, the FGF-2 polypeptide may be interchangeable with the FGF-2 variant.

[0046]

[0041] In one aspect of the invention, the product is a thermostable FGF-2 polypeptide derived from Bos taurus FGF-2 (SEQ ID NO: 2) comprising at least one of the amino acid substitutions R31L, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, S109E and / or S121P.

[0047]

[0042] In one aspect of the invention, the product is a thermostable FGF-2 polypeptide comprising at least one amino acid substitutions from the group of R31L, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, S109E or S121P. In other aspects of the invention, the thermostable FGF-2 polypeptide may comprise at least two or more, or at least three or more amino acid substitutions from said group.

[0048]

[0043] In another aspect of the invention, the product is a thermostable FGF-2 polypeptide comprising at least one amino acid substitutions from the group of R31L, C34S, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, C101A, C101V, S109E and / or S121P.

[0044] In one aspect of the invention, the product is a thermostable FGF-2 polypeptide derived from SEQ ID NO: 2 comprising at least one amino acid substitutions from the group of R31L, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, S109E and / or S121P.

[0049]

[0045] In one aspect of the invention, the product is a thermostable FGF-2 polypeptide derived from SEQ ID NO: 2 comprising at least one amino acid substitutions from the group of R31L, C34S, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, C101A, C101V, S109E and / or S121P.

[0050]

[0046] The substitution of reactive surface cysteine for serine may provide higher resistance to oxidation and also higher long term stability of the FGF-2 polypeptide.

[0051]

[0047] In one aspect of the invention, the product is a truncated thermostabilized FGF-2 polypeptide characterized by sequence SEQ ID NO: 4 (Fig. 1). The sequence SEQ ID NO: 4 is derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2), with the following modifications: deletion of amino acids 1-20 at the N-terminus, and ten amino acid substitutions, specifically R31L, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, S109E and / or S121P.

[0052]

[0048] The FGF-2 polypeptides according to the invention may comprise a fusion partner and / or an affinity tag in order to improve their certain properties, such as solubility or proteolytic stability, or to increase their expression yields, or to facilitate their subsequent processing. This approach may allow preparation and use of the FGF-2 polypeptides according to the invention on an industrial scale.

[0053]

[0049] The affinity tag used may be, for example, a histidine tag. The histidine tag may comprise a cleavage site for 3C or TEV protease. The affinity tag may be added to the N-terminus of the protein. The sequence of the histidine tag may be, for example, MHHHHHHLEVLFQGP or MHHHHHHENLYFQG.

[0054]

[0050] The affinity tag or fusion partner used may be at least one sequence selected from sequences SEQ ID NO: 11 to 18.

[0055]

[0051] The FGF-2 products according to the invention may comprise FGF-2 polypeptide N-terminally or C- terminally fused with a fusion partner. An expression vector of FGF-2 polypeptide N-terminally or C- terminally fused with a fusion partner may be prepared. The fusion partner may be, for example, a protein, a polypeptide or a peptide, some of which are well known to those skilled in the art. The fusion partner used may be, for example, a maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin A (TrxA) or small ubiquitin-like modifier (SUMO).

[0056]

[0052] In another aspect of the invention, the product is a truncated thermostabilized oxidation resistant FGF- 2 polypeptide characterized by sequence SEQ ID NO: 5. The FGF-2 polypeptide comprises the following modifications: deletion of amino acids 1-20 at the N-terminus, and ten amino acids substitution R31L, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, S109E and S121P, and a histidine tag with a cleavage site for 3C protease added to the N-end of the protein.

[0057]

[0053] In another aspect of the invention, the FGF-2 polypeptides may be derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2), with deletion of amino acids 1 to 15 to 1 to 22 at the N-terminus. These polypeptides may comprise at least one of amino acid substitutions: R31E, V52T, E54D, H59F, C78S, E92Y, S94I, C96N, S109E, and / or S121P relative to SEQ ID NO: 2.

[0058]

[0054] In another aspect of the invention, the FGF-2 polypeptides may be derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2), with deletion of up to 22 amino acids at the N-terminus. These polypeptides may comprise at least one of amino acid substitutions: R31E, V52T, E54D, H59F, C78S, E92Y, S94I, C96N, S109E, and / or S121P relative to SEQ ID NO: 2.

[0059]

[0055] In another aspect of the invention, the FGF-2 polypeptides may comprise deletion of amino acids 1 to 15 to 1 to 22 at the N-terminus. These polypeptides may additionally comprise at least one amino acid substitution from the group of R31E, V52T, E54D, H59F, C78S, E92Y, S94I, C96N, S109E, and / or S121P relative to SEQ ID NO: 2.

[0060]

[0056] In another aspect of the invention, the FGF-2 polypeptides may comprise deletion of up to 22 amino acids at the N-terminus. These polypeptides may additionally comprise at least one amino acid substitution from the group of R31E, V52T, E54D, H59F, C78S, E92Y, S94I, C96N, S109E, and / or S121P relative to SEQ ID NO: 2.

[0061]

[0057] In another aspect of the invention, the FGF-2 polypeptides may comprise deletion of amino acids 1 to 15 to 1 to 22 at the N-terminus. These polypeptides may additionally comprise at least one amino acid substitution from the group of R31E, C34S, V52T, E54D, H59F, C78S, E92Y, S94I, C96N, C101A, C101V, S109E, and / or S121P relative to SEQ ID NO: 2.

[0062]

[0058] In another aspect of the invention, the FGF-2 polypeptides may comprise deletion of up to 22 amino acids at the N-terminus. These polypeptides may additionally comprise at least one amino acid substitution from the group of R31E, C34S, V52T, E54D, H59F, C78S, E92Y, S94I, C96N, C101A, C101V, S109E, and / or S121P relative to SEQ ID NO: 2.

[0063]

[0059] In another aspect of the invention, the FGF-2 polypeptides may be derived from SEQ ID NO: 2, with deletion of amino acids 1 to 15 to 1 to 22 at the N-terminus. These polypeptides may additionally comprise at least one amino acid substitution from the group of R31E, V52T, E54D, H59F, C78S, E92Y, S94I, C96N, S109E, and / or S121P relative to SEQ ID NO: 2.

[0064]

[0060] In another aspect of the invention, the FGF-2 polypeptides may be derived from SEQ ID NO: 2, with deletion of up to 22 amino acids the N-terminus. These polypeptides may additionally comprise at least one amino acid substitution from the group of R31L, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, S109E, and / or S121P relative to SEQ ID NO: 2.

[0065]

[0061] In another aspect of the invention, the FGF-2 polypeptides may be derived from SEQ ID NO: 2, with deletion of amino acids 1 to 15 to 1 to 22 at the N -terminus. These polypeptides may additionally comprise at least one amino acid substitution from the group of R31L, C34S, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, C101A, C101V, S109E, and / or S121P relative to SEQ ID NO: 2.

[0066]

[0062] In another aspect of the invention, the FGF-2 polypeptides may be derived from SEQ ID NO: 2, with deletion of up to 22 amino acids at the N-terminus. These polypeptides may additionally comprise at least one amino acid substitution from the group of R31L, C34S, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, C101A, C101V, S109E, and / or S121P relative to SEQ ID NO: 2.

[0067]

[0063] In another aspect of the invention, the FGF-2 polypeptides may be derived from SEQ ID NO: 2, with deletion of up to 22 amino acids at the N-terminus. These polypeptides may additionally comprise at least one amino acid substitution from the group of R31L, C34S, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, C101A, C101V, S109E, and / or S121P relative to SEQ ID NO: 2, and may have at least 90% sequence identity to SEQ ID NO: 2.

[0068]

[0064] In another aspect, the invention provides FGF-2 polypeptides derived from SEQ ID NO: 2, or a polypeptide having at least 90% sequence identity thereto, with deletion of up to 22 amino acids at the N-terminus, and optionally comprising at least one amino acid substitution selected from the group consisting of R31L, C34S, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, C101A, C101V, S109E, and / or S121P relative to SEQ ID NO: 2.

[0069]

[0065] The truncated thermostabilized oxidation resistant FGF-2 polypeptide (SEQ ID NO: 4), according to the present invention, has 78.1 % sequence identity to Homo sapiens FGF-2 (SEQ ID NO: 1) and 78.7 % sequence identity to Bos taurus FGF-2 (SEQ ID NO: 2).

[0070]

[0066] The present invention also comprises other FGF-2 polypeptides that are derived from the amino acid sequence of SEQ ID NO: 4, for example FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95%, or at least 98%, or at least 99% sequence identity to the sequence of the truncated thermostabilized FGF-2 polypeptide characterized by sequence SEQ ID NO: 4.

[0071]

[0067] The present invention also comprises FGF-2 polypeptides characterized by SEQ ID NO: 5 - 10 or other FGF-2 polypeptides according to the description.

[0072]

[0068] The present invention also comprises FGF-2 polypeptides characterized by SEQ ID NO: 19 - 21, wherein these FGF-2 polypeptides may be characterized by increased thermal resistance.

[0069] In another aspect of the invention, products are FGF-2 polypeptides, that are derived from the amino acid sequences of SEQ ID NO: 5 to 10, for example FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95%, or at least 98%, or at least 99% sequence identity to the sequences SEQ ID NO: 5 to 10.

[0073]

[0070] The present invention also comprises FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 4 comprising at least one of amino acid substitutions: R11L, V32T, E34D, H39F, L72Y, S74I, C76N, C58S, S89E and / or S101P.

[0074]

[0071] The thermostabilized oxidation resistant FGF-2 polypeptide, according to the invention, may comprise amino acid substitution R31L (arginine at position 31 substituted by leucine) and H59F (histidine at position 59 substituted by phenylalanine) in the amino acid sequence of the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2). In yet another aspect of the invention, the thermostabilized oxidation resistant FGF-2 polypeptide may comprise other amino acid substitutions than R31L and H59F substitution in SEQ ID NO: 2. In one aspect of the invention, the thermostabilized oxidation resistant FGF-2 polypeptide may comprise an R31 substitution with any appropriate amino acid, for example with isoleucine or valine, and an H59 substitution with any appropriate amino acid, for example with tryptophan or isoleucine.

[0075]

[0072] The present invention also comprises thermostabilized oxidation resistant FGF-2 polypeptides with the melting temperature (Tm) higher than or equal to 69 °C, or higher than or equal to 70 °C, or higher than or equal to 75 °C, or higher than or equal to 76 °C.

[0076]

[0073] The present invention also comprises thermostabilized oxidation resistant FGF-2 polypeptides with the melting temperature (Tm) higher than or equal to 69 °C, or higher than or equal to 70 °C, or higher than or equal to 73 °C, or higher than or equal to 74 °C, or higher than or equal to 75 °C, or higher than or equal to 76 °C.

[0077]

[0074] The melting temperature (Tm) of thermostabilized oxidation resistant FGF-2 polypeptides according to the present invention may be in the range of 68 to 82°C, or in the range of 70 to 80 °C, or in the range of 72 to 78 °C.

[0078]

[0075] As used herein, when referring to a parameter expressed as, for example a range from 0 to 1000, it is to be understood that both the lower limit 0 and the upper limit 1000 are explicitly included within the scope of the disclosure. The stated range encompasses all intermediate values between the specified limits, including any subranges or specific values that may be explicitly or implicitly derived from the broader range. Unless explicitly stated otherwise, all numerical values are to be interpreted as including the endpoints.

[0076] The process according to the invention may consist of the steps:

[0079] Transformation of competent E. coli cells with plasmid DNA carrying the relevant FGF-2 sequence

[0080] Selection and screening of the transformed cells Cultivation of the transformed cells Induction of FGF-2 production FGF-2 detection

[0081] FGF-2 isolation and purification

[0082] FGF-2 characterization.

[0083]

[0077] According to the process of the invention, the bacterial expression host system, for example E. coli bacterial expression host system, may be used for the production of FGF-2 polypeptides due to its low cost, well-known biochemistry and genetics, rapid growth, and good productivity. A bacterial expression system is ideal for FGF-2 production as FGF-2 is a relatively small and single-domain protein with a compact fold. Furthermore, crystal structures of FGF-FGFR-heparin complexes and molecular mechanisms of the action of FGF proteins suggest that no post -translational modifications or cofactors of FGF proteins are required for their proper function. Previously, FGF proteins purified from E. coli showed biological activity. Therefore, the bacterial expression host system appears to be ideal for the large-scale industrial production of FGF-2. The presence of rare codons may be addressed by using codon optimization.

[0084]

[0078] In one aspect of the invention, the strains used for the production of FGF-2 polypeptides according to the invention may be E. coli strains, for example, BL21 (DE3), BL21-Gold (DE3) and / or BL21- CodonPlus (DE3)-RIPL.

[0085]

[0079] The competent cells for the production of FGF-2 polypeptides may be transformed with plasmid DNA carrying the relevant FGF-2 sequence, encoding for example the FGF-2 polypeptide as defined by sequence SEQ ID NO: 4, or any other sequences according to the invention, e.g. SEQ ID NO: 5 to 10.

[0086]

[0080] The transformed cells may be plated onto solid selection media, for example suspension of cells may be plated on LB (Luria-Bertani) agar plates supplemented with antibiotics. The colonies are later, for example on next day, picked and transferred into liquid selection media, for example LB broth supplemented with antibiotics. The cells may be grown for several hours, for example overnight. The culture may be then placed into fresh liquid selection medium, for example LB broth supplemented with antibiotics, and incubated at temperature in the range of 15 to 45 °C, or in the range of 20 to 40 °C, or in the range of 30 to 37 °C. The antibiotic used may be, for example, kanamycin, or any other appropriate antibiotic.

[0081] When the optical density at 550 nm (OD550) in the range of 0.4 to 1.0, or in the range of 0.5 to 0.9, or in the range of 0.6 to 0.8 is reached, an inducing agent may be added, for example isopropyl [3-D- thiogalactoside (IPTG), or any other appropriate inducing agent may be added to cell cultures for induction of FGF-2 production. The concentration of IPTG may be in the range of 0.001 to 10 mM, or in the range of 0.01 to 1 mM, or in the range of 0.05 to 0.5 mM. The cell cultures may be incubated at a temperature in the range of 4 to 40 °C, or in the range of 15 to 30 °C, or in the range of 18 to 25°C. After the appropriate time of the incubation period, for example in the range 1 hour to 72 hours, or in the range of 2 hours to 48 hours, or in the range of 12 hours to 24 hours, the culture may be harvested, for example by centrifuging at 13,000 g, for example at room temperature.

[0087]

[0082] The cell pellets may be resuspended in 150 pl of lx lithium dodecyl sulphate (LDS) gel sample buffer and heated to the temperature, which may be in the range of 60 to 100 °C, or in the range of 80 to 98 °C, or in the range of 90 to 95 °C for the time period, which may be in the range of 1 to 60 minutes, or in the range of 2 to 30 minutes, or in the range of 3 to 10 minutes.

[0088]

[0083] The samples may be spun down at 13,000 g, for example at room temperature, and analyzed with, for example, gradient SDS-PAGE. The volume of the sample analyzed may be in the range of 1 pl to 15 pl, or in the range of 2 to 12 pl, or in the range of 5 to 10 pl.

[0089]

[0084] The purification of the FGF-2 polypeptide according to the invention may be carried out, for example, by the following procedure: Post-induction bacterial cells from one liter cultivation are harvested, for example by centrifugation at 5000 g for 20 minutes. The cell pellet is lysed, for example by addition of lysozyme and by sonication. The lysate is clarified by centrifugation, for example at 75, 000 g at 4 °C for 30 minutes, and filtered through a 0.22 pm membrane. The clarified lysate is loaded onto a HisTrap column. The FGF-2 polypeptide is eluted by imidazole and purified with cation exchange chromatography, for example using HiTrap SP Sepharose.

[0090]

[0085] The FGF-2 polypeptide may be analyzed by SDS-PAGE. The thermostability of the FGF-2 polypeptide may be analyzed, for example, by nanoDSF or DSF.

[0091]

[0086] The long-term stability of the FGF-2 polypeptides according to the invention may be tested. For example the FGF-2 polypeptide may be stored in a fridge, e.g. at 4 °C for 30 days, or at -20 °C temperature for 90 days, and the protein may be then analyzed by size-exclusion chromatography, SDS- PAGE or by biological activity test, as described in example 6.

[0092]

[0087] The stability analysis after one or more freeze-thaw cycles of the FGF-2 polypeptides according to the invention may be tested, for example by the process described in example 5 or by the biological activity test as described in example 6.

[0088] The biological activity of the FGF-2 polypeptides according to the invention may be tested and the achieved cell growth may be determined. Different concentrations of the FGF-2s may be tested, for example the concentration of FGF-2 may be 100, 10 or 1 ng / ml.

[0093]

[0089] The FGF-2 polypeptide according to the invention characterized by SEQ ID NO: 4 has similar biological activity as the state of the art FGF-2 polypeptide characterized by SEQ ID NO: 3, e.g. at concentrations of 100 ng / ml and 10 ng / ml, but possesses a significant improvement of the thermal stability and stability under oxidative conditions, which results in longer-term preservation of the biological activity.

[0094]

[0090] In one aspect, the invention may comprise proteins artificially designed through computational methods, including machine learning approaches, that can perform substantially the same function as the protein described herein, with at least partially similar sequence identity.

[0095]

[0091] As used herein, the term “sequence identity” may refer to the degree of similarity between two polynucleotide or polypeptide sequences, expressed as a percentage. The percentage may be calculated by using local or global alignment algorithms, optimally aligning the two polynucleotide or two polypeptide sequences.

[0096]

[0092] The local sequence alignment may be calculated as follows:

[0097] Sequence identity (%) = (Number of identical residues / Defined length in given alignment) x 100

[0098] In this context, residues refer to amino acids or nucleotides, depending on the type of compared sequences. Identical residues refer to positions where the residues (amino acids or nucleotides) are the same in both sequences at that alignment position. The defined length in the given alignment includes gaps and mismatches but may exclude overhangs. The local alignment may be calculated by using BLAST, FASTA, EMBOSS Water using Smith-Waterman algorithm, or any other appropriate tool for the local alignment.

[0099]

[0093] The local alignment may be calculated alternatively as follows:

[0100] Sequence identity (%) = (Number of identical residues aligned by local alignment / Total number of residues in the longer sequence) x 100

[0101] In this context, residues refer to amino acids or nucleotides, depending on the type of compared sequences. Identical residues are those where the same amino acid or nucleotide is present at corresponding positions within the aligned region identified through local alignment.

[0102] This calculation method differs from traditional local alignment methods that calculate identity based on the length of the given alignment itself. Instead, it normalizes the sequence identity by considering the total number of residues in the longer sequence involved in the comparison, including any gaps introduced during the alignment. This approach retains the characteristics of local alignment while allowing the resulting identity value to reflect the proportion of similarity in the context of the entire longer sequence. Suitable algorithms for this method may include customized local alignment tools or modified versions of established software like BLAST, FASTA, or EMBOSS Water.

[0103]

[0094] The global sequence alignment may be calculated as follows:

[0104] Sequence identity (%) = (Number of identical residues / Total number of residues in the sequence) x 100

[0105] In this context, residues refer to amino acids or nucleotides, depending on the nature of the compared sequences. Identical residues refer to positions where the residues (amino acids or nucleotides) are the same in both sequences at that alignment position. The total number of residues in the sequence represents the final length of the alignment after both sequences have been aligned. If the sequences differ in length, gaps are introduced to make both sequences equal in length. Therefore, the total number of residues corresponds to the length of the aligned sequences, which is always the same for both sequences in the global alignment. The global alignment may be calculated by using EMBOSS Needle, EMBOSS Stretcher, MUSCLE, BLAST using Needleman-Wunsch algorithm or any other appropriate tool for the global alignment.

[0106]

[0095] The global sequence alignment may be calculated alternatively as follows:

[0107] Sequence identity (%) = (Number of identical residues / Total number of residues in the shorter sequence) x 100

[0108] This calculation method differs from traditional global alignment methods that calculate identity based on the final length of the alignment after both sequences have been aligned. Instead, it normalizes the sequence identity by considering the total number of residues in the shorter sequence involved in the comparison. This approach retains the characteristics of local alignment while allowing the resulting identity value to reflect the proportion of similarity in the context of the entire longer sequence. Suitable algorithms for this method may include customized local alignment tools or modified versions of established software like EMBOSS Needle, EMBOSS Stretcher, MUSCLE, BLAST using Needleman- Wunsch algorithm or any other appropriate tool for global alignment.

[0109]

[0096] In another aspect of the invention, the sequence identity may be calculated alternatively, as follows:

[0110] Sequence identity (%) = Number of identical residues / Total number of residues in the claimed sequence) x 100

[0111] The claimed sequence may be the full sequence of amino acids or nucleotides claimed or a part of this sequence such as a domain or a motif relevant to the function of the claimed sequence. Global or local alignment algorithms may be used to calculate the alignment itself.

[0097] In one aspect of the invention, the alignment algorithms used for the calculation of sequence identity may apply alignment parameters comprising scoring matrices and gap penalties (gap-open, gapextension).

[0112]

[0098] In addition to the choice of alignment algorithm, scoring matrices may be utilized to evaluate the similarity between residues. The scoring matrix scheme assigns numerical values to matches and mismatches. At its most basic level, both nucleotide and protein alignments are scored by assigning a positive value for a match (e.g., +1) and a negative value for a mismatch (e.g., -1). Gaps are not scored as neutral events but are penalized separately by the combination of gap open and gap extension parameters. The final alignment score may be calculated as follows:

[0113] Alignment score = Sum of substitution scores - sum of gap penalties of all gaps, wherein the gap penalty follows an affine model and may be calculated as:

[0114] Gap penalty = (gap open) + (gap extension) x (k-1), where k represents the length of the gap.

[0115] For nucleotide sequence alignments (DNA or RNA), substitution matrices such as EDNAFULL, DNAfull, DNAmat, or NUC.4.4 may be employed, with the choice of nomenclature depending on the software package (e.g., EMBOSS, BLAST, or FASTA). These matrices provide a more biologically realistic evaluation of nucleotide similarity, particularly in analyses where mutational bias or evolutionary distance is relevant.

[0116] For protein sequence alignments, substitution matrices such as PAM (Point Accepted Mutation) and BLOSUM (Blocks Substitution Matrix) may be employed. BLOSUM62 or variance of the matrix (such as EBLOSUM62) is a commonly accepted standard that assigns empirically derived log-odds scores to amino acid substitutions observed in conserved protein blocks. Other matrices such as BLOSUM50, BLOSUM80, or PAM250 may be selected depending on the expected evolutionary distance between the compared proteins.

[0117]

[0099] In sequence alignment, insertions and deletions are modeled as gaps that are penalized to prevent overfitting of alignments. Two parameters are typically distinguished: a “gap open” penalty, which is applied when a new gap is introduced into the alignment, and a “gap extension” penalty, which is applied for each additional residue that extends an already existing gap. The selection of gap open and gap extension values may depend on the alignment algorithm used and the nature of the sequences compared. For example, typical gap open penalties may range from about 5 to 15 for protein sequences and from about 10 to 20 for nucleotide sequences, while gap extension penalties may typically range from about 0.1 to 2 for proteins and from about 0.5 to 5 for nucleotides. In most alignment algorithms, the total gap penalty follows an affine model, in which the penalty for a gap of length k residues is calculated as previously mentioned:

[0118] Gap penalty = (gap open) + (gap extension) x (k-1).

[0119] Higher gap open penalties tend to discourage introduction of new gaps, favoring alignments with fewer insertions or deletions, whereas lower penalties permit more flexible alignment accommodating potential evolutionary changes.

[0120]

[0100] By way of example, the sequence identity of protein sequences may be calculated by using scoring matrix EBLOSSUM62, with gap open 10 and gap extend 0.5 by implementation of local sequence alignment or global sequence alignment:

[0121] Sequences of beta-defensin 6 of Gallus gallus:

[0122] Sequence A: ILYLLLSVLFVVLQGVA (17 aa, SEQ ID NO: 23)

[0123] Sequence B (truncated and modified at the end): ILYLLLSVLGVADCED (16 aa, SEQ ID NO: 24)

[0124] The sequence identity is calculated as the number of identical residues divided by the defined alignment length. In this case, there are 12 identical residues out of 17 positions in the local alignment, corresponding to 70.6% identity, and 12 identical residues out of 21 positions in the global alignment, corresponding to 57.1% identity. The alignment of the sequences for local and global alignment are visualized in Fig. 12.

[0125]

[0101] Experimental conditions of testing biological activity may be as described in examples, or any other appropriate conditions set according to the needs of a person skilled in the art.

[0126]

[0102] The process for preparing the truncated thermostabilized FGF-2 and other FGF-2 derivatives, according to the invention, is appropriate for large-scale production. The process has been verified for example in 1 liter scale and provided about 10 - 40 mg of FGF-2 with very high purity, for example, 94%. The melting temperature (Tm) was determined, and the effect of the mutations (deletion at the N- terminus and nine substitutions in the FGF-2 chain) to increase FGF-2 stability has been evaluated.

[0127]

[0103] According to the invention, the FGF-2 polypeptides have been prepared in high yields. The products showed excellent thermal stability and very good long-term stability at 4 °C. The products may be frozen and successfully recovered.

[0128]

[0104] The FGF-2 polypeptides according to the invention may be successfully used in biotechnological research and industrial applications, medicine, the pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models and other related applications. These products according to the invention may be used, for example, for preparing cosmetic products, such as creams, gels, or lotions for improvement of the visual appearance of the skin and for skin rejuvenation.

[0129]

[0105] The FGF-2 polypeptides according to the invention may be used in many biotechnological processes, for example for cell cultivation or for biotechnological production of many different types of requested compounds, such as, for example, proteins, active pharmaceutical ingredients or antibodies.

[0130]

[0106] The FGF-2 polypeptides according to the invention may be used in cell cultivation processes for the purpose of preparing cultured meat products for human consumption or as a pet food. The FGF- 2 polypeptides according to the invention may be used as a component of culture media. These processes of cell cultivation may be carried out in a cell cultivation system 1, as depicted on Fig. 10. The cell cultivation system 1 may comprise at least one of: a cultivation device 2, formed for example by a production bioreactor, a cell harvesting device 3, a central control unit 4, or a monitoring device 5.

[0131]

[0107] The cultivation device 2, e.g. the production bioreactor, the cell harvesting device 3, the central control unit 4, and / or the monitoring device 5 may be in direct or indirect connection and / or communication with each other.

[0132]

[0108] The cell harvesting device 3 may comprise a filtration device, a centrifugation device, or any other appropriate device for harvesting of cells.

[0133]

[0109] Optionally the system may further comprise, for example, a seeding tank or a device for preparing a cultured meat composition (not depicted on Fig. 10).

[0134]

[0110] The thermostable oxidation resistant FGF-2 polypeptides according to the invention may be used as a component of the culture medium in many biotechnological processes, for example in cultivation of mammalian cells, for the purpose of preparing cultured meat products in clean meat industry. The thermostable oxidation resistant FGF-2 polypeptides according to the invention may be used as signaling compounds in the culture medium. The culture medium according to the invention may further comprise amino acids or their sources, in combination with at least one type of compounds that may be selected from a group comprising: sugars, fatty acids, vitamins and organic micronutrients, mineral compounds, supplements, such as for example iron supplementation compounds, organic amines, shear protectants, additional compounds, or any other appropriate compounds.

[0135]

[0111] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, wherein the composition may be a cosmetic composition, medical composition, pharmaceutical composition, dermatological composition, wound-healing composition, or any other appropriate composition.

[0112] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, optionally in combination with at least one additional ingredient from the group of solvents, stabilizers, emollients, humectants, occlusives, preservatives, penetration enhancers, antioxidants, surfactants, rheological additives, colorants, or any other ingredient commonly used in cosmetic, medical, dermatological, or pharmaceutical formulations.

[0136]

[0113] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, optionally in combination with at least two additional ingredients from the group of solvents, stabilizers, emollients, humectants, occlusives, preservatives, penetration enhancers, antioxidants, surfactants, rheological additives, colorants, or any other ingredient commonly used in cosmetic, medical, dermatological, or pharmaceutical formulations.

[0137]

[0114] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, optionally in combination with at least three additional ingredients from the group of solvents, stabilizers, emollients, humectants, occlusives, preservatives, penetration enhancers, antioxidants, surfactants, rheological additives, colorants, or any other ingredient commonly used in cosmetic, medical, dermatological, or pharmaceutical formulations.

[0138]

[0115] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, optionally in combination with at least four additional ingredients from the group of solvents, stabilizers, emollients, humectants, occlusives, preservatives, penetration enhancers, antioxidants, surfactants, rheological additives, colorants, or any other ingredient commonly used in cosmetic, medical, dermatological, or pharmaceutical formulations.

[0139]

[0116] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, optionally in combination with at least five additional ingredients from the group of solvents, stabilizers, emollients, humectants, occlusives, preservatives, penetration enhancers, antioxidants, surfactants, rheological additives, colorants, or any other ingredient commonly used in cosmetic, medical, dermatological, or pharmaceutical formulations.

[0140]

[0117] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, optionally in combination with at least six additional ingredients from the group of solvents, stabilizers, emollients, humectants, occlusives, preservatives, penetration enhancers, antioxidants, surfactants, rheological additives, colorants, or any other ingredient commonly used in cosmetic, medical, dermatological, or pharmaceutical formulations.

[0141]

[0118] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, optionally in combination with at least seven additional ingredients from the group of solvents, stabilizers, emollients, humectants, occlusives, preservatives, penetration enhancers, antioxidants, surfactants, rheological additives, colorants, or any other ingredient commonly used in cosmetic, medical, dermatological, or pharmaceutical formulations.

[0142]

[0119] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, optionally in combination with at least eight additional ingredients from the group of solvents, stabilizers, emollients, humectants, occlusives, preservatives, penetration enhancers, antioxidants, surfactants, rheological additives, colorants, or any other ingredient commonly used in cosmetic, medical, dermatological, or pharmaceutical formulations.

[0143]

[0120] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, optionally in combination with at least nine additional ingredients from the group of solvents, stabilizers, emollients, humectants, occlusives, preservatives, penetration enhancers, antioxidants, surfactants, rheological additives, colorants, or any other ingredient commonly used in cosmetic, medical, dermatological, or pharmaceutical formulations.

[0144]

[0121] In one aspect, the present invention may relate to compositions comprising an FGF-2 variant, optionally in combination with at least ten additional ingredients from the group of solvents, stabilizers, emollients, humectants, occlusives, preservatives, penetration enhancers, antioxidants, surfactants, rheological additives, colorants, or any other ingredient commonly used in cosmetic, medical, dermatological, or pharmaceutical formulations.

[0145]

[0122] Additional ingredients that may optionally be included in the composition of the invention with FGF-2 may comprise, but are not limited to, following: a.) at least one solvent or formulation base, such as water (aqua), ethanol, methanol, propylene glycol, polyethylene glycol (PEG), glycerin, isopropanol, any other solvent, or a combination thereof, including their derivatives, metabolites, or chemically modified forms, to dissolve, disperse, or stabilize the FGF-2; b.) at least one stabilizer, such as ethylenediaminetetraacetic acid (EDTA), glycerin, butylated hydroxytoluene (BHT), sodium gluconate, triethanol amine, tetrasodium glutamate diacetate (TSGD), phosphate buffers, any other stabilizer, or a combination thereof, including their derivatives, metabolites, or chemically modified forms, to maintain the biological activity of FGF-2; c.) at least one emollient (moisturizer), such as ammonium lactate, dimethicone, cyclopentasiloxane, cyclohexasiloxane, glycerin, extract of the aloe vera, lanolin, caprylic / capric triglycerides, squalane, coconut, jojoba, sesame, olive, almond or any plant-derived oils, paraffin, triethylhexanoin, beeswax, petrolatum, mineral oil, cocoa butter, shea butter, cetyl alcohol, stearic acid, isopropyl palmitate, hyaluronic acid, urea, candelilla wax, carnauba wax, ceramides, dimethicone, any other emollient, or a combination thereof, including their derivatives, metabolites, or chemically modified forms, to soften and smooth the skin; d.) at least one humectant, such as hyaluronic acid, sodium hyaluronate, glycerol, sorbitol, xylitol, aloe vera extract, propylene glycol, alpha hydroxy acids (e.g. lactic acid, glycolic acid), glyceryl triacetate, honey, lithium chloride, molasses, quillaia, castor oil, urea, ethylhexylglycerin, any other humectant, or a combination thereof, including their derivatives, metabolites, or chemically modified forms, to enhance moisture retention in the stratum corneum; e.) at least one occlusive, such as petrolatum, castor oil, shea wax, carnauba wax, silicone, any other occlusive, or a combination thereof, including their derivatives, metabolites, or chemically modified forms, to form a barrier on top of the skin to prevent moisture evaporation; f.) at least one preservative, such as formaldehyde, phenoxyethanol, ethylhexylglycerin, salicylic acid, sodium benzoate, benzoic acid, benzylate, benzyl ester, methylthiazolinone, methylchloroisothiazolinone, sodium dehydroacetate, potassium sorbate, parabens (e.g. ethylparaben, butylparaben, methylparaben, propylparaben, isobutylparaben, isopropylparaben or any other paraben molecule), formaldehyde -releasing preservatives (e.g. DMDM hydantoin, diazolydinal urea, imidiazoldinal urea, quaternium-15, bronopol), citric acid, dehydroacetic acid, essential oils, antimicrobial peptides, sorbic acid, triclocarban, triclosan, zinc pyrithione, plant extracts (e.g. ginger), any other preservative, or a combination thereof, including their derivatives, metabolites, or chemically modified forms, to inhibit the growth of microorganisms; g.) at least one antioxidant, such as tocopherol (vitamin E), ascorbic acid (vitamin C), retinol (vitamin A), vitamin D, vitamin K, thiamine (vitamin Bl), riboflavin (vitamin B2), niacin (vitamin B3), cyanocobalamin (vitamin B12), pantothetic acid (vitamin B5), biotin (vitamin B7), folic acid (vitamin B9), minerals (e.g. zinc, selenium, copper, manganese), arginine, compounds derived from plants (e.g. rosemary, green tea, marigold, damask rose, P. aureum, G. biloba or any other plant), estradiol, melatonin, glutathione, lycopene, lutein, resveratrol, lipoic acid, delphinidin, coenzyme Q10, caffeic acid, resveratrol, sodium carbonate, dibutyl hydroxy toluene, tert-butyl hydroquinone, propyl gallate, lecithin, any other antioxidant, or combinations thereof, including their derivatives, metabolites, or chemically modified forms, to protect the skin cells from oxidative damage caused by free radicals; h.) at least one penetration enhancer, including substances or delivery systems that facilitate increased transdermal or dermal penetration and / or provide sustained release of FGF-2. The penetration enhancers may include, for example, chemical enhancers such as dimethyl sulfoxide (DMSO), azones (e.g., laurocapram), pyrrolidones (e.g., 2 -pyrrolidone), alcohols (e.g., ethanol, decanol), glycols (e.g., propylene glycol), urea, hydrocarbons, and terpenes, including their derivatives, metabolites, or chemically modified forms. In addition, suitable delivery systems may comprise lipid-based carriers and vesicular systems such as liposomes, niosomes, ethosomes, transfersomes, oleosomes, alcohol- based vesicles, micelles, and lipid vesicles. Further examples include particulate carriers, such as microspheres, nanospheres, millimeter spheres, microcapsules, nanocapsules, microemulsions, nanoemulsions, microparticles, nanoparticles, and millimeter particles. Additional delivery platforms may include inclusion complexes, sponge -like systems, sustained-release formulations, or any other suitable penetration enhancer or delivery vehicle, or combinations thereof, capable of improving the delivery of FGF-2 into the skin layers; i.) at least one surfactant, such as: nonionic (e.g. octyl glucoside, decyl glucoside, coco glucoside, cetyl alcohol, steryl alcohol, glyceryl laurate, span, polyethylene glycol ester, lauryl glycol ether, polypropylene glycol ester), anionic (e.g. sodium lauryl sulfate, ammonium lauryl sulfate, sulfosuccinates, alkyl benzene sulfanate, acyl methyl taurates, acyl sarcocinates, propyl peptide condensates, monoglyceride sulfates, fatty glycerol, ether sulfanates), cationic (quaternary ammonium salts, cocamidopropyl betaine, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride), or amphoteric (e.g. cocamidopropyl betaine, lauryl betaine, cocoamphopropionate, cocamidopropyl hydroxy sultaine, soy lecithin, sodium lauraminopropionate) surfactants, including their derivatives, metabolites, or chemically modified forms, which may function to reduce surface tension between the cosmetic composition and the skin surface, enhance the spreadability of the composition, or facilitate the delivery of FGF-2 to the epidermal or dermal layers; and j.) at least one rheological additive from the group of, for example, thickening agents (e.g. hydrocolloids like xantham gum, guar gum, alginates, cellulose derivatives like carboxymethylcellulose, hydroxyethylcellulose, polymers like polyacrylamide, polyvinyl alcohol), gelling agents (e.g. agar, pectin, alginate), suspending agents (e.g. clay minerals, such as bentonite, kaolin, silica gel, polymers), rheology modifiers (e.g. bentonine, polyurethanes), anti -sedimentation agents (e.g. starch, gums), flow modifiers (e.g. silicone oils, alkylated polysaccharides), including their derivatives, metabolites, or chemically modified forms, to modify the flow and the viscosity properties of the cosmetic composition. k.) at least one colorant, which may include pigments, dyes, lakes, mineral colorants, or plant- derived colorants, such as iron oxides, titanium dioxide, zinc oxide, ultramarines, chromium oxide greens, manganese violet, ferric ferrocyanide, carmine, annatto extract, beta-carotene, or other FDA- or pharmacopeia-approved colorants for cosmetic, dermatological, or pharmaceutical use. Colorants may be included to impart a desirable color to the composition, to enhance product aesthetics, to indicate product usage, or for branding purposes. The colorants may be used individually or in combination, and may include derivatives, metabolites, or chemically modified forms thereof.

[0123] It should be understood that the categories and examples of ingredients listed above are intended to be illustrative and not limiting. The compositions may include any other pharmaceutically, dermatologically, or cosmetically acceptable ingredient having a function analogous to those described herein.

[0146]

[0124] In one aspect of the invention, the water (aqua) from the group of solvents may be ultrapure water, such as deionized, distilled, or reverse osmosis-treated water, typically used in cosmetic or pharmaceutical formulations. Such water may be substantially free from ionic, organic, and microbial contaminants, ensuring enhanced stability and compatibility of the composition with sensitive active ingredients.

[0147]

[0125] In one aspect of the invention, “solvent” refers to any pharmaceutically, dermatologically, or cosmetically acceptable liquid or semi-liquid medium capable of dissolving, suspending, diluting, or dispersing FGF-2 and / or one or more additional ingredients in the composition, thereby facilitating homogeneous distribution and effective delivery of the FGF-2 variant and other formulation components.

[0148]

[0126] In one aspect of the invention, “stabilizer” refers to any compound capable of maintaining or enhancing the biological activity of FGF-2 under storage or use conditions.

[0149]

[0127] In one aspect of the invention, "emollient" refers to any compound that softens, smooths, or moisturizes the skin by filling spaces between desquamating skin cells and forming an occlusive barrier to reduce water loss.

[0150]

[0128] In one aspect of the invention, "humectant" refers to any compound that attracts and retains moisture in the skin by promoting water absorption from the surrounding environment or underlying layers of the skin.

[0151]

[0129] In one aspect of the invention, "occlusive" refers to any compound that forms a hydrophobic barrier on the surface of the skin to prevent transepidermal water loss and maintain skin hydration.

[0152]

[0130] In one aspect of the invention, "preservative" refers to any compound that inhibits or retards the growth of microorganisms, thereby extending the shelf life and maintaining the safety of the composition.

[0153]

[0131] In one aspect of the invention, "antioxidant" refers to any compound that neutralizes reactive oxygen species or free radicals, thereby protecting FGF-2 and skin cells from oxidative damage.

[0154]

[0132] In one aspect of the invention, "penetration enhancer" refers to any compound, system, or mechanism that facilitates or improves the delivery of FGF-2 across the stratum corneum or into deeper layers of the skin.

[0133] In one aspect of the invention, "surfactant" refers to any compound that lowers surface tension between two substances, such as between the composition and the skin, and may enhance the spreadability or delivery of active ingredients.

[0155]

[0134] In one aspect of the invention, "rheological additive" refers to any compound that modifies the flow, viscosity, or texture of the composition to achieve desired application characteristics or stability.

[0156]

[0135] In one aspect of the invention, "colorant" refers to any pharmaceutically, dermatologically, or cosmetically acceptable pigment, dye, lake, or other coloring agent that imparts color to the composition for aesthetic enhancement, product identification, usage indication, or branding purposes, without adversely affecting the biological activity of FGF-2 or the overall safety and stability of the composition.

[0157]

[0136] In one aspect, the present invention relates to a composition comprising a FGF-2 variant in combination with at least one peptide or protein commonly used in cosmetic, dermatological, or pharmaceutical formulations, wherein the peptide or protein is selected from the group consisting of elastins, collagens, dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, oligopeptides, functional peptides promoting skin regeneration, hydration, elasticity, or extracellular matrix stimulation, or a combination thereof, including their derivatives, analogues, or chemically modified forms.

[0158]

[0137] In another aspect of the invention, the at least one peptide and / or protein may be chemically modified to enhance stability, bioavailability, or bioactivity. Such modifications may include, but are not limited to, acetylation, amidation, phosphorylation, PEGylation, glycosylation, methylation, lipidation, cyclization, isomerization, or combinations thereof.

[0159]

[0138] In a further aspect, the composition may additionally comprise one or more functional agents from the group of anti-inflammatory agents, skin- whitening or brightening agents, film-forming agents, and wound-healing agents. The anti-inflammatory agents may comprise, for example, bisabolol, allantoin, beta-glucan, or plant-derived extracts (e.g., chamomile, Centella asiatica). Skin-whitening or brightening agents may comprise niacinamide, arbutin, kojic acid, licorice extract, or ascorbyl derivatives. Film-forming agents may comprise polyvinylpyrrolidone (PVP), acrylates copolymers, or polysaccharide-based polymers. Such ingredients may be included individually or in combination to further enhance the cosmetic, dermatological, or pharmaceutical performance of the composition.

[0160]

[0139] In one aspect of the invention, the composition comprising FGF-2 may comprise at least one additional ingredient in a range of 10,000 ppm to 990,000 ppm, or in a range of 15,000 ppm to 980,000 ppm, or in a range of 20,000 ppm to 970,000 ppm, or in a range of 21,000 ppm to 960,000 ppm, or in a range of 25,000 ppm to 950,000 ppm, or in a range of 30,000 ppm to 900,000 ppm, or in a range of 40,000 ppm to 850,000 ppm, or in a range of 50,000 ppm to 800,000 ppm, or in a range of 60,000 ppm to 750,000 ppm, or in a range of 70,000 ppm to 700,000 ppm, all calculated relative to the total weight of the composition.

[0161]

[0140] In another aspect of the invention, the composition comprising FGF-2 may comprise for example the first additional ingredient in a range of 10000 ppm to 990000 ppm, the second additional ingredient in a range of 10000 ppm to 990000 ppm, the third additional ingredient in a range of 10000 ppm to 990000 ppm, the fourth additional ingredient in a range of 10000 ppm to 990000 ppm, the fifth additional ingredient in a range of 10000 ppm to 990000 ppm, all calculated relative to the total weight of the composition.

[0162]

[0141] In one aspect of the invention, the composition may comprise FGF-2 variant in a range of 500 ppm to 300000 ppm, or in a range of 500 ppm to 250000 ppm, or in a range of 500 ppm to 200000 ppm, or in a range of 500 ppm to 150000 ppm, or in a range of 500 ppm to 100000 ppm, or in a range of 500 ppm to 50000 ppm, all calculated relative to the total weight of the composition.

[0163]

[0142] In one aspect of the invention, the composition comprising the FGF-2 variant may comprise water in an amount of 600000 to 950000 ppm, one or more components comprising at least one fatty acid from the additional ingredients in an amount of 50000 to 350000 ppm, and the FGF-2 variant in an amount of 0.1 to 500 ppm, all calculated relative to the total weight of the composition.

[0164]

[0143] In one aspect of the invention, the composition comprising an FGF-2 variant may comprise:

[0165] (a) the FGF-2 variant in an amount ranging from 500 ppm to 300,000 ppm, or from 500 ppm to 250000 ppm, or from 500 ppm to 200000 ppm, or from 500 ppm to 150000 ppm, or from 500 ppm to 100000 ppm, or from 500 ppm to 50000 ppm; and

[0166] (b) at least one additional ingredient in an amount ranging from 10000 ppm to 990000 ppm, or from 15000 ppm to 980000 ppm, or from 20000 ppm to 970000 ppm, or from 21000 ppm to 960000 ppm, wherein all ppm may be calculated relative to the total weight of the composition; wherein the ratio of the concentration of the FGF-2 variant to the concentration of the additional ingredient ranges from approximately 1:2 to 1:2,000, depending on the desired composition characteristics, including but not limited to viscosity, stability, penetration enhancement, and therapeutic or cosmetic effect.

[0167]

[0144] In one aspect, the composition comprising an FGF-2 variant may comprise 30% to 99% by weight of one or more solvents.

[0168]

[0145] In one aspect, the composition comprising an FGF-2 variant may comprise: a.) 0.01% to 5% by weight of one or more stabilizers; b.) 1% to 30% by weight of one or more humectants; c.) 0.1% to 6% by weight of one or more preservatives; d.) 30% to 99% by weight of one or more solvents; and e.) 0.1% to 30% by weight of any other additional ingredient.

[0169] The specific percentages of the components may vary depending on the desired composition characteristics, including but not limited to viscosity, stability, penetration enhancement, or therapeutic or cosmetic effect.

[0170]

[0146] In one aspect of the invention, the composition comprising an FGF-2 variant may comprise: a) 100 ppm to 50000 ppm of one or more stabilizers; b) 10000 ppm to 300000 ppm of one or more humectants; c) 1000 ppm to 60000 ppm of one or more preservatives; d) 300000 ppm to 990000 ppm of one or more solvents; and e) 1000 ppm to 300000 ppm of one or more additional ingredients; wherein the specific concentration ranges may vary depending on the desired composition characteristics, including but not limited to viscosity, stability, penetration enhancement, or therapeutic or cosmetic effect. All ppm may be calculated relative to the total weight of the composition.

[0171]

[0147] In one aspect, the composition comprising an FGF-2 variant may comprise: a.) 0.01% to 5% by weight of one or more stabilizers; b.) 5% to 25% by weight of one or more occlusives; c.) 0.1% to 2% by weight of one or more preservatives; d.) 30% to 99% by weight of one or more solvents; and e.) 0.1% to 30% by weight of any other additional ingredient. The specific percentages of the components may vary depending on the desired composition characteristics, including but not limited to viscosity, stability, penetration enhancement, or therapeutic or cosmetic effect.

[0172]

[0148] In one aspect of the invention, the composition comprising an FGF-2 variant may comprise: a) 100 ppm to 50000 ppm of one or more stabilizers; b) 50000 ppm to 250000 ppm of one or more occlusives; c) 1000 ppm to 20000 ppm of one or more preservatives; d) 300000 ppm to 990000 ppm of one or more solvents; and e) 1000 ppm to 300000 ppm of one or more additional ingredients; wherein the specific concentration ranges may vary depending on the desired composition characteristics, including but not limited to viscosity, stability, penetration enhancement, or therapeutic or cosmetic effect. All ppm may be calculated relative to the total weight of the composition.

[0173]

[0149] In one aspect, the composition comprising an FGF-2 variant may comprise: a.) 0.01% to 5% by weight of one or more stabilizers; b.) 5% to 25% by weight of one or more occlusives; c.) 0.1% to 2% by weight of one or more preservatives; d.) 30% to 99% by weight of one or more solvents; and e.) 0.1% to 30% by weight of any other additional ingredient.

[0174] The specific percentages of the components may vary depending on the desired composition characteristics, including but not limited to viscosity, stability, penetration enhancement, or therapeutic or cosmetic effect.

[0175]

[0150] In one aspect of the invention, the composition comprising an FGF-2 variant may comprise: a) 100 ppm to 50000 ppm of one or more stabilizers; b) 50000 ppm to 250000 ppm of one or more occlusives; c) 1000 ppm to 20000 ppm of one or more preservatives; d) 300000 ppm to 990000 ppm of one or more solvents; and e) 1000 ppm to 300000 ppm of one or more additional ingredients; wherein the specific concentration ranges may vary depending on the desired composition characteristics, including but not limited to viscosity, stability, penetration enhancement, or therapeutic or cosmetic effect. All ppm may be calculated relative to the total weight of the composition.

[0176]

[0151] In one aspect, the composition comprising an FGF-2 variant may comprise: a.) 1% to 13% by weight of one or more penetration enhancers; b.) 5% to 35% by weight of one or more emollients; c.) 0.1% to 6% by weight of one or more preservatives; d.) 30% to 99% by weight of one or more solvents; and e.) 0.1% to 30% by weight of any other additional ingredient.

[0177] The specific percentages of the components may vary depending on the desired composition characteristics, including but not limited to viscosity, stability, penetration enhancement, or therapeutic or cosmetic effect.

[0178]

[0152] In one aspect of the invention, the composition comprising an FGF-2 variant may comprise: a) 10000 ppm to 130 000 ppm of one or more penetration enhancers; b) 50000 ppm to 350000 ppm of one or more emollients; c) 1000 ppm to 60000 ppm of one or more preservatives; d) 300000 ppm to 990000 ppm of one or more solvents; and e) 1000 ppm to 300000 ppm of one or more additional ingredients; wherein the specific concentration ranges may vary depending on the desired composition characteristics, including but not limited to viscosity, stability, penetration enhancement, or therapeutic or cosmetic effect. All ppm may be calculated relative to the total weight of the composition.

[0179]

[0153] In one aspect of the invention, the composition comprising FGF-2 may comprise: a.) at least one of: solvents, stabilizers, emollients, humectants, preservatives, or colorants; and b.) at least one of: penetration enhancers, antioxidants, surfactants, rheological additives, or occlusives.

[0180]

[0154] In one aspect of the invention, the composition comprising FGF-2 may comprise: a.) at least two of: solvents, stabilizers, emollients, humectants, preservatives, or colorants; and b.) at least two of: penetration enhancers, antioxidants, surfactants, rheological additives, or occlusives.

[0181]

[0155] In one aspect of the invention, the composition comprising FGF-2 may comprise: a.) at least three of: solvents, stabilizers, emollients, humectants, preservatives, or colorants; and b.) at least three of: penetration enhancers, antioxidants, surfactants, rheological additives, or occlusives.

[0182]

[0156] In one aspect of the invention, the composition comprising FGF-2 may comprise: a.) at least one of: solvents, stabilizers, emollients, humectants, preservatives, colorants, or peptides or proteins; and b.) at least one of: penetration enhancers, antioxidants, surfactants, rheological additives, occlusives, anti-inflammatory agents, skin- whitening or brightening agents, film-forming agents, or wound-healing agents.

[0183]

[0157] In one aspect of the invention, the composition comprising FGF-2 may comprise: a.) at least two of: solvents, stabilizers, emollients, humectants, preservatives, or colorants, or peptides or proteins; and b.) at least two of: penetration enhancers, antioxidants, surfactants, rheological additives, occlusives, anti-inflammatory agents, skin- whitening or brightening agents, film-forming agents, or wound-healing agents.

[0184]

[0158] In one aspect of the invention, the composition comprising FGF-2 may comprise: a.) at least three of: solvents, stabilizers, emollients, humectants, preservatives, or colorants, peptides or proteins; and b.) at least three of: penetration enhancers, antioxidants, surfactants, rheological additives, occlusives, anti-inflammatory agents, skin- whitening or brightening agents, film-forming agents, or wound-healing agents.

[0185]

[0159] In one aspect of the invention, the composition comprising FGF-2 may comprise: a.) at least four of: solvents, stabilizers, emollients, humectants, preservatives, or colorants, peptides or proteins; and b.) at least four of: penetration enhancers, antioxidants, surfactants, rheological additives, occlusives, anti-inflammatory agents, skin- whitening or brightening agents, film-forming agents, or wound-healing agents.

[0186]

[0160] In one aspect of the invention, the composition may be in the form of creams, lotions, gels, emulsions, serums, soap substituents, ointments, powders, paste, sprays, or any other suitable form for topical application to the skin, mucosal surfaces, ocular tissues, or other epithelial or connective tissues.

[0187]

[0161] The administration of FGF-2, as described herein, may be effective in accelerating and / or promoting tissue regeneration, soothing irritation, improving the appearance and texture of epithelial tissues, promoting tissue restoration, promoting tissue renewal, or any combination thereof, in tissues affected by external stressors and / or procedures resulting in damage and / or irritation. The administration of FGF-2 may, for example, stimulate keratinocyte proliferation, support extracellular matrix repair, and accelerate wound healing in epithelial tissues, including but not limited to the skin.

[0188]

[0162] In one aspect of the invention, the external stressors and / or procedures resulting in damaged and / or irritated epithelial tissue may comprise, but are not limited to: a.) mechanical irritation and / or damage, wherein mechanical irritation refers to physical stress or trauma caused by friction, pressure, stretching, abrasion, or any other mechanical force resulting in disruption of the epithelial barrier, leading to inflammation, micro-injuries, or impaired healing. Mechanical irritation and / or damage may be caused, for example, by shaving, dermabrasion, microneedling, exfoliation, or other forms of mechanical irritation and / or damage; b.) thermal stress, wherein thermal stress refers to exposure to abnormally high or low temperatures resulting in cellular stress, barrier disruption, inflammation, oxidative stress, heat shock responses, and / or tissue damage. Thermal stress may be caused, for example, by laser treatment, intense pulsed light (IPL) treatment, sun exposure (including ultraviolet (UV) radiation), micro wave treatment, or other thermal stress; c.) chemical exposure, wherein chemical exposure refers to contact with exogenous chemical agents that disrupt the epithelial barrier, induce cellular stress, inflammation, oxidative damage, or impair tissue regeneration. Chemical exposure may be caused, for example, by acids, alkalis, solvents, detergents, surfactants, alcohols, oxidizers, or environmental pollutants, including household cleaning agents, industrial chemicals, personal care products, occupational irritants, or other chemical agents; d.) environmental stress, wherein environmental stress refers to exposure to physical or chemical conditions that disrupt the epithelial barrier, induce oxidative stress, trigger inflammatory responses, or impair natural tissue regenerative processes. Environmental stress may be caused, for example, by ultraviolet (UV) radiation, air pollution, extreme temperatures, low humidity, wind exposure, blue light (high-energy visible light), tobacco smoke, dry climate, or other environmental factors; and e.) biological stress, wherein biological stress refers to tissue irritation, inflammation, and / or damage induced by biological agents, including but not limited to bacteria, viruses, fungi, protozoa, or parasites. Biological stress may result in disruption of epithelial or connective tissue integrity, induction of oxidative stress, activation of immune or inflammatory responses, or impaired healing processes. Biological stress may be caused, for example, by bacterial infections, viral infections (e.g., herpes simplex virus, human papillomavirus), fungal infections (e.g., Candida spp.), parasitic infestations, exposure to alterations of the microbiome balance (dysbiosis), or any other biological agent.

[0189]

[0163] The damage and / or irritation may affect one or more layers of epithelial or connective tissues, including, but not limited to, the epidermis, dermis, and hypodermis. The administration of FGF-2 may be beneficial in promoting the structural and functional recovery of these tissue layers, depending on the route of administration and the extent of tissue penetration.

[0190]

[0164] In another aspect, the present invention may provide for the use of an FGF-2 variant, administered prior to, during, and / or following dermatological, aesthetic, and / or therapeutic procedures that induce controlled injury or stress to epithelial and / or connective tissues for treatment benefit, e.g. therapeutic or cosmetic benefit. Such procedures may comprise, but are not limited to, microneedling, fractional laser therapy, radiofrequency treatment, microdermabrasion, chemical peeling, or photodynamic therapy.

[0191]

[0165] In another aspect, the present invention may provide for the use of an FGF-2 variant administered prior to, during, and / or following non-invasive or minimally invasive aesthetic procedures that temporarily affect the outer layers of the skin in order to improve its visual appearance, hydration, and texture. Such procedures may comprise, but are not limited to, microneedling, fractional laser therapy, radiofrequency treatment, microdermabrasion, superficial chemical peeling, or light-based cosmetic stimulation. The composition comprising FGF-2 is intended to support skin comfort and visual skin quality following such cosmetic treatments and does not serve a therapeutic or medical purpose.

[0192]

[0166] In one aspect of the invention, the microneedling into the tissue may be performed by needlelike energy delivery elements placed on the applicator, wherein the elements may deliver at least one of electromagnetic energy comprising radiofrequency (RF) energy, electric field.

[0193]

[0167] In one aspect of the invention, the RF energy may be delivered by needle-like energy delivery elements in a monopolar, a bipolar and / or a multipolar mode to create thermal damage in the treated tissue.

[0194]

[0168] Frequencies of the RF field delivered to the tissue may be in the range of 80 kHz to 5 GHz, or in the range of 0.1 MHz to 500 MHz or in the range of 0.2 MHz to 50 MHz, or in the range of 0.3 MHz to 30 MHz.

[0195]

[0169] The delivery of the RF field may be provided by at least one RF pulse having a specified time (tl), referred as a RF pulse duration, which may be in the range of 0.1 ms to 2500 ms, or in the range of 0.5 ms to 2000 ms, or in the range of 1 ms to 1500 ms.

[0196]

[0170] The current density of the radiofrequency field delivered by one energy delivery element may be in the range of 1 A / cm2to 300 A / cm2, or in the range of 5 A / cm2to 200 A / cm2, or in the range of 10 A / cm2to 150 A / cm2.

[0197]

[0171] An RF pulse voltage (amplitude) may be from IV to 1000V. A maximum energy may be dependent on the type of the energy delivery element and may for example be between 10 mJ and 500 mJ per needle -type energy delivery element or pin-type energy delivery element.

[0198]

[0172] The PEF may be delivered (applied) by needle-like energy delivery elements in a monopolar, a bipolar or a multipolar mode. It may be delivered in the form of electric pulses including monophasic (single polarity) pulses, symmetrical and / or asymmetrical biphasic pulses. The pulses may repeat from lx to lOOOOOx during a treatment or from lx to lOOOx or from lx to lOOx or from lx to 50x during one applicator application. The frequency of the high frequency pulses may vary from 1 Hz to 500 Hz or from 5 Hz to 250 Hz or from 10 Hz to 100 Hz. An amplitude (Um) of the monophasic pulses may vary from 10 V up to 15 kV, and the peak to peak amplitude of biphasic pulses may vary from 20 V to 30 kV.

[0199]

[0173] In a multipolar mode there is at least one first energy delivery element operating in a mode with first polarity and at least one second energy delivery element operating in a mode with a different polarity (which may be an opposite polarity) than the operating mode of the at least one first energy delivery element. A surface area or a sum of the surface areas of the at least one first energy delivery element may be significantly smaller than a surface area or a sum of the surface areas of the at least one second energy delivery element. The ratio between the surfaces may be from 2:3 to 1:100, or 3:5 to 1:70, or 1:2 to 1:40. The energy delivery elements with different polarities in the multipolar mode may be all surface energy delivery elements, all invasive energy delivery elements, or at least one energy delivery element having a first polarity may be a surface energy delivery element and at least one other energy delivery element having a different polarity than the first polarity may be an invasive energy delivery element.

[0200]

[0174] The PEF pulses may be combined with extra pre -pulses for example for tissue conditioning. Pulses may be delivered as single pulses or they may be repeated in at least one train, where parameters of the pulses may vary or may remain constant. Trains of pulses may be repeated as well and may create one or more bursts. A maximal amplitude of the pulses may depend on the target tissue, electrode size and / or electrode distance in order to create an electric field with a maximum electric field magnitude for example between 0.1 kV to 30 kV or between 0.2 kV to 5 kV or between 0.3 kV to 2 kV per cm in a target tissue.

[0201]

[0175] A duration of the pulse may vary from a nanosecond range to milliseconds range, for example from 2 ns to 10 ms, or from 10 ns to 1 ms or from 100 ns to 100 ps. The shape of the pulse may be for example a square, a curve similar to exponential discharge, a rectangle, a saw, a triangle, trapezoidal or a sinusoidal.

[0202]

[0176] Maximal power output may be 0.01 W to 500 W. The energy of the pulse may be from 0.01 J to 1 J or from 0.05 J to 0.7 J or from 0.1 J to 0.45 J. Energy delivered per area may vary from 0.01 J / mm2to 1 J / mm2or from 0.05 J / mm2to 0.7 J / mm2or from 0.1 J / mm2to 0.45 J / mm2. Energy delivered per volume may be from 0.01 J / mm3to 1 J / mm3, or from 0.05 J / mm3to 0.6 J / mm3, or from 0.1 J / mm3to 0.3 J / mm3.

[0203]

[0177] The adjustable parameters of the PEF pulse and / or the pulse protocol may include for example amplitude, pulse width, number of pulses, length of inter pulse pauses, length of inter train pauses and so on. The parameters may be set for example based on a distance between particular energy delivery elements, for example energy delivery elements creating a bipolar pair. In particular examples of applicator or distal tip of the applicator including invasive energy delivery elements, for example in the form of needles, the maximal amplitude may be needed to be restricted. For example maximal absolute maximal amplitude of the PEF pulse according to distance between needles in bi-polar pair may be restricted in following way - for needle distance of 3 mm to 6 mm, maximal amplitude may be set to 3000 V to 6000 V, for distance of 2 mm to 3 mm, maximal amplitude may be set to 2000 V to 3000 V, for distance of 1.5 mm to 2 mm, maximal amplitude may be set to 1300 V to 2200 V, for distance for 1 mm to 1.5 mm, maximal amplitude may be set to 1000 V to 1700 V.

[0204]

[0178] Fig. 13 shows schematic example of treatment device which may provide treatment by one or more physical modalities prior, at the same time or after application of FGF-2 polypeptide (in any form). The system for skin treatment 20 may apply a one or more physical modalities into the tissue. The system may include a power supply 21 connected to the an energy source 33 (e.g. electricity socket). The system for skin treatment 20 includes at least one applicator 24 which may be placed inside a main unit (its casing) or may be separated from the system for skin treatment 20 and connected by a cable to the main unit. The microprocessor control unit 22 with user interface 23 provides communication between the First physical modality treatment unit 25 and / or Second physical modality treatment unit unit 27. User interface 23 allows setting up the treatment parameters and also may provide the operator various treatment information. First physical modality treatment unit 25 (e.g. electrode, coil, diode, vacuum cup, roller, etc.) and / or Second physical modality treatment unit unit 27 (e.g. electrode, coil, diode, vacuum cup, roller, etc.) may be placed in at least one applicator 24. However the treatment units may also have separate applicators. The applicator 24 may preferably contain a sensor unit 26

[0205]

[0179] The sensor unit 26 may contain one or more sensors for sensing temperature, resistance, contact with skin or force applied to skin.

[0206]

[0180] The temperature sensor measures and monitors the temperature of the treated tissue. Temperature can be analysed by a microprocessor control unit 22. The temperature sensor may be a contact sensor, contactless sensor (e.g. infrared temperature sensor) or invasive sensor (e.g. a thermocouple) for precise temperature measuring of deep layers of soft tissue. The microprocessor control unit 22 may also use algorithms to calculate the deep or upper-most. A temperatures feedback system may control the temperature and based on set / pre-set limits, alert the operator in human perceptible form e.g. on the user interface 23. In a limit temperature condition, the device may be configured to adjust output power, activate cooling or stop the therapy.

[0207]

[0181] The temperature sensor measures and monitors the temperature of the treated tissue. Temperature can be analyzed by a microprocessor control unit 22. The temperature sensor may be a contact sensor, contactless sensor (e.g. infrared temperature sensor) or invasive sensor (e.g. a thermocouple) for precise temperature measuring of deep layers of soft tissue. The microprocessor control unit 22 may also use algorithms to calculate the deep or upper-most. A temperatures feedback system may control the temperature and based on set / pre-set limits, alert the operator in human perceptible form e.g. on the user interface 23. In a limit temperature condition, the device may be configured to adjust output power, activate cooling or stop the therapy.

[0208]

[0182] A resistance sensor may measure the skin resistance, since it may vary for different patients, as well as the humidity, wetness and sweat may influence the resistance and therefore the behaviour of the skin to the physical modality. Based on the measured skin resistance, the skin impedance may also be calculated.

[0183] The contact and / or force applied by the applicator on the skin surface may be measured piezoresistively, mechanically, optically, electrically, electromagnetically or magnetically. The measured information from the contact and / or force sensor may influence the start of the therapy or generation of First or Second physical modality treatment unit unit.

[0209]

[0184] The system for skin treatment 20 generates at least one physical modality enabling treatment (both medical or non-medical) of the tissue e.g. in the skin area. The connective tissue in the skin area contains layer epidermis 28 and dermis 29; white adipose tissue in hypodermis 30 and peritoneal cavity 32. The other soft tissue below the skin area e.g. muscular tissue 31 remains untreated and unharmed.

[0210]

[0185] The simultaneous or sequential application of one or more physical modalities in combination with the FGF-2 variant may transiently increase tissue permeability, stimulate microcirculation, modulate cellular signaling pathways, and enhance receptor activation, thereby promoting deeper penetration, improved bioavailability, and synergistically enhanced treatment effects, e.g. biological and / or cosmetic effects of the FGF-2 variant.

[0211]

[0186] In one aspect, the present invention may provide combined application of an FGF-2 variant with one or more physical modalities configured to induce controlled stimulation of tissue, e.g. epithelial and / or connective tissues. The physical modalities may comprise electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation) pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuumcompression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma-based energy, oxygen-based therapy, or any combination thereof.

[0212]

[0187] In one aspect of the invention, a method for providing combined application of an FGF-2 variant and at least one physical modality to enhance aesthetic properties of the skin and underlying tissues by promoting at least one of smoothness, firmness, elasticity, hydration and / or uniformity of tone and texture, wherein the physical modality may be from the group of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation, pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuumcompression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma-based energy, oxygen-based therapy, or any combination thereof.

[0188] In further aspects, any of the above-mentioned physical modalities may be applied individually or in combination with at least one other modality to produce synergistic effects with an FGF-2 variant.

[0213]

[0189] In one aspect, the present invention may provide combined application of an FGF-2 variant with one or more physical modalities to produce synergistic effect that would not be achievable by either component alone.

[0214]

[0190] In one aspect, the present invention provides a combined application of an FGF-2 variant with one or more physical modalities, resulting in at least one of the following effects: transiently increased tissue permeability, stimulation of microcirculation, modulation of cellular signaling pathways, and enhancement of receptor activation, thereby promoting deeper penetration, improved bioavailability, and synergistically enhanced treatment effects, e.g. biological and / or cosmetic effects of the FGF-2 variant, wherein the synergistic effect is not achievable by either component alone.

[0215] The synergic effect may comprise at least one of enhanced permeability and diffusion of FGF-2 variant into tissues, upregulation of FGF receptor (FGFR) expression, stimulation of fibroblast proliferation, enhanced collagen and elastin synthesis, induction of heat-shock protein production, reduction of inflammation and oxidative stress, accelerated epithelialization and wound healing, shortened recovery time and reduced post-procedural downtime, visible improvement in skin tone, texture and elasticity, visible reduction of wrinkles, scars, and uneven pigmentation.

[0216] The physical modality may be from the group of laser energy, radiofrequency energy, pulsed electric field energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling, electromagnetic field stimulation, magnetotherapy, diathermy, vacuum-compression therapy, photobiomodulation, cryogenic therapy or thermal energy, plasma-based energy, oxygen-based therapy, or any combination thereof.

[0217]

[0191] In another aspect of the invention, the delivery and / or dosing of the FGF-2 variant may be performed through or in coordination with the physical modalities described herein. The device may serve not only as a means of tissue stimulation but also as a vector for localized or controlled administration of the FGF-2 variant.

[0218]

[0192] The FGF-2 variant may be applied to the surface of the applicator, incorporated into a coupling or composition, embedded within a microneedle, coating, and / or reservoir of the device.

[0219]

[0193] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative) and at least one of radiofrequency (RF) energy, microwave energy, photobiomodulation, pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma -based energy, oxy gen -based therapy, thereby producing a synergistic effect not achievable by either component alone.

[0220]

[0194] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with radiofrequency (RF) energy and at least one of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), microwave energy, photobiomodulation, pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma -based energy, oxy gen -based therapy, thereby producing a synergistic effect not achievable by either component alone.

[0221]

[0195] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with pulsed electric field energy and at least one of laser energy, radiofrequency energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling, electromagnetic field stimulation, magnetotherapy, diathermy, vacuum-compression therapy, photobiomodulation, cryogenic therapy or thermal energy, plasma-based energy, or oxygen-based therapy, thereby producing a synergistic effect not achievable by either component alone.

[0222]

[0196] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with ultrasound energy and at least one of laser energy, radiofrequency energy, pulsed electric field energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling, electromagnetic field stimulation, magnetotherapy, diathermy, vacuum-compression therapy, photobiomodulation, cryogenic therapy or thermal energy, plasma-based energy, or oxygen-based therapy, thereby producing a synergistic effect not achievable by either component alone.

[0223]

[0197] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with acoustic shockwave energy and at least one of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation, pulsed electric field (PEF) energy, ultrasound energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, thermal energy ( e.g. cryogenic therapy or heat therapy), plasma-based energy, oxygen-based therapy , thereby producing a synergistic effect not achievable by either component alone.

[0198] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with mechanical microneedling or microperforation and at least one of electromagnetic field (e.g. light energy, laser energy (ablative or non -ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation, pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma -based energy, oxy gen -based therapy, thereby producing a synergistic effect not achievable by either component alone.

[0224]

[0199] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative) and at least one of radiofrequency (RF) energy, microwave energy, photobiomodulation, pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma -based energy, oxygen-based therapy, or any combination thereof., thereby producing a synergistic effect not achievable by either component alone.

[0225]

[0200] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with magnetotherapy and at least one of laser energy, radiofrequency energy, pulsed electric field energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling, electromagnetic field stimulation, diathermy, vacuum-compression therapy, photobiomodulation, cryogenic therapy or thermal energy, plasma-based energy, or oxygen-based therapy, thereby producing a synergistic effect not achievable by either component alone.

[0226]

[0201] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with diathermy and at least one of laser energy, radiofrequency energy, pulsed electric field energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling, electromagnetic field stimulation, magnetotherapy, vacuum-compression therapy, photobiomodulation, cryogenic therapy or thermal energy, plasma-based energy, or oxygen-based therapy, thereby producing a synergistic effect not achievable by either component alone.

[0227]

[0202] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with vacuum-compression therapy and at least one of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation, pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, thermal energy ( e.g. cryogenic therapy or heat therapy), plasma-based energy, oxygen-based therapy, thereby producing a synergistic effect not achievable by either component alone.

[0228]

[0203] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with photobiomodulation and at least one of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma -based energy, oxy gen -based therapy, thereby producing a synergistic effect not achievable by either component alone.

[0229]

[0204] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with thermal energy ( e.g. cryogenic therapy or heat therapy) and at least one of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation, pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, plasma-based energy, oxygen-based therapy, , thereby producing a synergistic effect not achievable by either component alone.

[0230]

[0205] In one aspect of the invention, the method may comprise applying an FGF-2 variant in combination with plasma-based energy and at least one of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation, pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), oxygen-based therapy, thereby producing a synergistic effect not achievable by either component alone.

[0231]

[0206] In one aspect of the invention, the method comprises applying an FGF-2 variant in combination with oxygen-based therapy and at least one of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation, pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma-based energy, thereby producing a synergistic effect not achievable by either component alone.

[0207] In one aspect, the present invention relates to the delivery of radiofrequency (RF) energy and / or pulsed electric field (PEF) energy to epithelial and / or connective tissues to induce controlled irritation and / or damage, thereby enhancing the uptake and biological effectiveness of FGF-2.

[0232]

[0208] In one aspect, the present invention relates to the delivery of radiofrequency (RF) energy and / or pulsed electric field (PEF) energy to epithelial and / or connective tissues to induce controlled thermal and / or electrical stimulation, resulting in transient cellular stress and increased tissue permeability, thereby enhancing the uptake and biological effectiveness of an FGF-2 variant.

[0233]

[0209] In one aspect of the invention, the RF and / or PEF procedure may be utilized according to a system, wherein the system may comprise one or more generators for producing RF signals (RF generator) and / or PEF signals (PEF generator), a control unit, an applicator, a power supply unit, a data storage unit, a switching unit, and a user interface. Optionally, the system may also include a cooling unit and / or a fluid delivery unit.

[0234]

[0210] The applicator may include at least one energy delivery element adapted for contact with or penetration into epithelial and / or connective tissue. The energy delivery element may be configured to generate and deliver an RF field and / or a PEF to the tissue. Energy delivery elements may be superficial (e.g., surface electrodes or pins) or invasive (e.g., needles, blades, or sharp projections), depending on the intended depth of energy delivery. Superficial elements may deliver energy without penetrating the tissue. Invasive elements may be adapted to penetrate tissues such as skin, hypodermis, dermis, adipose tissue, or mucosal tissues before delivering energy.

[0235]

[0211] The energy delivery elements may be made of conductive materials such as copper, gold, steel, titanium, platinum, platinum-iridium, silver, or other suitable materials.

[0236]

[0212] The at least one energy delivery element (203) may be insulated (601), uninsulated (602), or partially insulated (603) as shown in Fig. 11. In aspects where at least one energy delivery element (203) is an invasive energy delivery element (403) and has a form of a needle, partial insulation may involve insulating the shaft of a needle while leaving the tip exposed, with the exposed portion constituting, for example, from 0.1% to 20%, or from 0.5% to 15%, or from 1% to 10% of the needle length. Insulation materials may comprise polyimide, polyurethane, polyester, polyvinyl chloride (PVC), rubber, nylon, polyethylene, polypropylene, silicone, fiberglass, ceramic, ethylene propylene diene monomer (EPDM), different fluoropolymers like polytetrafluoroethylene (PTFE), or combinations thereof.

[0237]

[0213] In one aspect, the present invention may provide for the administration of an FGF-2 variant to the surface of epithelial and / or connective tissues, to the surface of the applicator, or to both, wherein the FGF-2 variant may be administered prior to, during, and / or following the delivery of radiofrequency energy and / or pulsed electric field energy.

[0214] In one aspect of the invention, RF energy may be delivered in a monopolar, a bipolar and / or a multipolar mode to treat (e.g. create thermal damage in the treated tissue) by an electrode (First or Second physical modality treatment unit unit).

[0238]

[0215] Frequencies of the RF field delivered to the tissue may be in the range of 80 kHz to 5 GHz, or in the range of 0.1 MHz to 500 MHz or in the range of 0.2 MHz to 50 MHz, or in the range of 0.3 MHz to 30 MHz.

[0239]

[0216] The delivery of the RF field may be provided by at least one RF pulse having a specified time (tl), referred as a RF pulse duration, which may be in the range of 0.1 ms to 2500 ms, or in the range of 0.5 ms to 2000 ms, or in the range of 1 ms to 1500 ms.

[0240]

[0217] The current density of the radiofrequency field delivered by one energy delivery element may be in the range of 1 A / cm2 to 300 A / cm2, or in the range of 5 A / cm2 to 200 A / cm2, or in the range of 10 A / cm2 to 150 A / cm2.

[0241]

[0218] An RF pulse voltage (amplitude) may be from 1 V to 1000 V, or from 2 V to 900 V, or from 50 V to 845 V, or from 132 V to 650 V.

[0242]

[0219] A maximum energy may be dependent on the type of the energy delivery element and may for example be between 10 mJ and 500 mJ per needle -type energy delivery element or pin-type energy delivery element.

[0243]

[0220] In monopolar mode, RF energy is delivered between active electrodes and an indifferent electrode with a larger surface area. In bipolar mode, RF energy is delivered between adjacent electrodes with opposite polarity. In multipolar mode, multiple electrodes of different polarities are used with surface area ratios ranging typically from 2:3 to 1:100.

[0244]

[0221] In monopolar mode, the RF energy may be delivered among one or more energy delivery elements placed on the applicator and / or one or more indifferent electrodes placed in the distance, for example on the skin of the patient and / or for example on the applicator. The one or more indifferent electrodes may have in some aspects a significantly larger surface area than the sum of the surface areas of the active energy delivery elements. In a bipolar mode the RF energy is applied between two or more, for example adjacent energy delivery elements with different polarities. In this example, the sum of the surfaces of the active energy delivery elements with the first polarity is similar to the sum of the surfaces of the active electrodes with the second polarity. In a multipolar mode, there is at least one first energy delivery element operating in a mode with a first polarity and a at least one second energy delivery element operating in a mode with a different polarity (which may be an opposite polarity) than the operating mode of the first at least one energy delivery element. A surface or a sum of the surfaces of the first at least one energy delivery element is significantly smaller than a surface or a sum of the surfaces of the second at least one energy delivery element.

[0245]

[0222] The parameters described herein are intended to be illustrative and not limiting, and may be adjusted according to the particular medical and / or cosmetic application, tissue type, and desired therapeutic outcome.

[0246]

[0223] In one aspect, the present invention relates to the delivery of PEF energy to epithelial and / or connective tissues using monopolar, bipolar, or multipolar mode.

[0247]

[0224] PEF energy may be delivered in the form of electric pulses including monophasic (single polarity) pulses, and symmetrical and / or asymmetrical biphasic pulses. The pulses may repeat from lx to lOOOOOx during a treatment, or from lx to lOOOx, or from lx to lOOx, or from lx to 50x during one applicator application.

[0248]

[0225] The frequency of the high frequency pulses may vary from 1 Hz to 500 Hz, or from 5 Hz to 250 Hz, or from 10 Hz to 100 Hz. An amplitude (Um) of the monophasic pulses may vary from 10 V up to 15 kV, and the peak to peak amplitude of biphasic pulses may vary from 20 V to 30 kV.

[0249]

[0226] In monopolar mode, the PEF field may be generated between one or more active energy delivery elements included in the applicator and one or more indifferent electrodes placed elsewhere (e.g., on the skin or the applicator itself). The indifferent electrodes may have a significantly larger surface area compared to the active electrodes.

[0250]

[0227] In bipolar mode, the PEF field may be generated between two or more adjacent energy delivery elements with opposite polarity. The surface area of the electrodes with the first polarity is preferably similar to the surface area of those with the second polarity.

[0251]

[0228] In multipolar mode there is at least one first energy delivery element operating in a mode with first polarity and at least one second energy delivery element operating in a mode with a different polarity (which may be an opposite polarity) than the operating mode of the at least one first energy delivery element. The surface area or the sum of the surface areas of the at least one first energy delivery element may be significantly smaller than the surface area or the sum of the surface areas of the at least one second energy delivery element. The ratio between the surfaces may be from 2:3 to 1 : 100, or 3:5 to 1 :70, or 1:2 to 1:40. The energy delivery elements with different polarities in the multipolar mode may be all surface energy delivery elements, all invasive energy delivery elements, or at least one energy delivery element having a first polarity may be a surface energy delivery element and at least one other energy delivery element having a different polarity than the first polarity may be an invasive energy delivery element.

[0229] The PEF pulses may be combined with extra pre -pulses, for example, for tissue conditioning. Pulses may be delivered as single pulses or they may be repeated in at least one train, where parameters of the pulses may vary or may remain constant. Trains of pulses may be repeated as well and may create one or more bursts.

[0252]

[0230] The maximal amplitude of the pulses may depend on the target tissue, electrode size and / or electrode distance in order to create an electric field with a maximum electric field magnitude for example between 0.1 kV to 30 kV or between 0.2 kV to 5 kV or between 0.3 kV to 2 kV per cm in a target tissue.

[0253]

[0231] The pulse duration may vary from a nanosecond range to milliseconds range, for example from 2 ns to 10 ms, or from 10 ns to 1 ms or from 100 ns to 100 ps.

[0254]

[0232] The pulse shape may be, for example, a square, a curve similar to exponential discharge, a rectangle, a saw, a triangle, trapezoidal and / or sinusoidal.

[0255]

[0233] The maximal power output may be in a range of 0.01 W to 500 W, or in a range of 0.5 W to 479 W, or in a range of 10 W to 400 W, or in a range of 200 W to 342W.

[0256]

[0234] The pulse energy may be in a range of 0.01 J to 1 J, or in a range of 0.05 J to 0.7 J, or in a range of 0.1 J to 0,45 J.

[0257]

[0235] The energy delivered per area may vary from 0.01 J / mm2 to 1 J / mm2 or from 0.05 J / mm2 to 0.7 J / mm2 or from 0.1 J / mm2 to 0.45 J / mm2. The energy delivered per volume may be from 0.01 J / mm3 to 1 J / mm3, or from 0.05 J / mm3 to 0.6 J / mm3, or from 0.1 J / mm3 to 0.3 J / mm3.

[0258]

[0236] The parameters of PEF pulses may be adjustable before or during treatment, depending on treatment requirements.

[0259]

[0237] The system may be adapted for use with multiple applicators or interchangeable distal applicator parts, differing in: the number of energy delivery elements, type (superficial vs. invasive), insulation (insulated, uninsulated, or partially insulated elements), dimensions of the elements, or spacing between the elements.

[0260]

[0238] The application of the at least one applicator and / or a treatment may include a noninvasive application of the RF field and / or the PEF. The RF field and / or the PEF may be in this example delivered via at least one superficial energy delivery element placed adjacent to the tissue. The energy delivery element in this example does not penetrate the tissue before, during or after the application of the at least one applicator or a treatment.

[0239] In examples with a plurality of energy delivery elements the distance between particular energy delivery elements may be from 0.5 mm to 10 mm, and it may be uniform or it may differ within an applicator. The invasive energy delivery elements in the form of needles may have a diameter for example from 0.05 mm to 2 mm, or from 0.1 mm to 1 mm or from 0.15 mm to 0.5 mm and again may be uniform or may differ within an applicator. The surface of the distal part of the applicator including the energy delivery elements may be flat or may be curved for example to conform to a specific part of a human body anatomy.

[0261]

[0240] In another aspect of the invention, the invasive energy delivery elements in the form of needles may penetrate the skin to a depth in a range of 1.5 mm to 4 mm, or in a range of 2 mm to 3.5 mm, or in a range of 2.5 mm to 3 mm.

[0262]

[0241] In another aspect of the invention, the delivery of radiofrequency energy and / or PEF energy may be combined with one or more additional physical modalities from the group of laser energy, radiofrequency energy, pulsed electric field energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling, electromagnetic field stimulation, magnetotherapy, diathermy, vacuum-compression therapy, photobiomodulation, cryogenic therapy or thermal energy, plasma-based energy, oxygen-based therapy, or any combination thereof. The combination of modalities may further enhance tissue permeability, microcirculation, and metabolic activity, thereby synergistically increasing the penetration and biological response to the FGF-2 variant.

[0263]

[0242] In one aspect of the invention, a method for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) inducing irritation and / or damage of the tissue by at least one external stressor or procedure; wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or and promote shortened post -procedure recovery time.

[0264]

[0243] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) inducing irritation and / or damage of the tissue by at least one external stressor or procedure; wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or and promote shortened post -procedure recovery time.

[0265]

[0244] In one aspect of the invention, a method for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) inducing irritation and / or damage of the skin by at least one external stressor from the group of mechanical irritation and / or damage, chemical exposure, or thermal stress; wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or promote shortened post -procedure recovery time.

[0266]

[0245] In one aspect of the invention, a method of use of an FGF-2, e.g. for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) inducing irritation and / or damage of the skin by at least one external stressor from the group of mechanical irritation and / or damage, chemical exposure, or thermal stress; wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or promote shortened post -procedure recovery time.

[0267]

[0246] In one aspect of the invention, a method for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) inducing irritation and / or damage of the skin by at least two of external stressors from the group of mechanical irritation and / or damage, chemical exposure, or thermal stress; wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or promote shortened post -procedure recovery time.

[0247] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) inducing irritation and / or damage of the skin by at least two of external stressors from the group of mechanical irritation and / or damage, chemical exposure, or thermal stress; wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or promote shortened post -procedure recovery time.

[0268]

[0248] In one aspect of the invention, a method for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering radiofrequency energy and / or pulsed electric field energy to the tissue using invasive or nonin vasive delivery elements, wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or promote shortened post -procedure recovery time.

[0269]

[0249] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering radiofrequency energy and / or pulsed electric field energy to the tissue using invasive or nonin vasive delivery elements, wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or promote shortened post -procedure recovery time.

[0270]

[0250] In one aspect of the invention, a method for a cosmetic procedure involving epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) applying at least one external cosmetic procedure or physical stimulus (e.g. laser, radiofrequency) intended to improve the aesthetic appearance of the skin; wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the application of FGF-2 may support treatment outcomes, e.g. cosmetic outcomes by improving hydration, visual skin smoothness, and / or subjective post-procedural comfort.

[0271]

[0251] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for cosmetic procedure involving epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) applying at least one external cosmetic procedure or physical stimulus (e.g. laser, radiofrequency) intended to improve the aesthetic appearance of the skin; wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the application of FGF-2 may support treatment outcomes, e.g. cosmetic outcomes by improving hydration, visual skin smoothness, and / or subjective post-procedural comfort.

[0272]

[0252] In one aspect, the present invention provides for the delivery of ablative or non-ablative laser energy, produced by a laser source, to epithelial and / or connective tissues to induce controlled irritation and / or damage, thereby promoting the uptake and biological effectiveness of an FGF-2 variant into deeper tissue layers.

[0273]

[0253] In one aspect, the present invention provides for the delivery of ablative or non-ablative laser energy to epithelial and / or connective tissues to produce controlled optical and thermal stimulation, resulting in mild, reversible biological stress that increases microcirculation, tissue permeability, and receptor responsiveness, thereby facilitating the enhanced penetration and effectiveness of an FGF-2 variant.

[0274]

[0254] In one aspect, the combined application of an ablative laser and an FGF-2 variant may produce a synergistic biological effect based on controlled micro-ablation of the epidermis and partial vaporization of superficial dermal structures. The laser energy, typically absorbed by water within the tissue, generates localized microthermal zones (MTZs) that result in precise microchannels extending through the stratum corneum and into the dermal layer. These microchannels transiently disrupt the skin barrier and promote direct transdermal diffusion of the FGF-2 variant into viable epidermal and dermal compartments. The surrounding areas of non-ablated tissue remain intact and may act as biological reservoirs of regenerative cells and cytokines, which, in combination with exogenous FGF-2, may accelerate epithelialization and / or stimulate fibroblast proliferation.

[0275]

[0255] The controlled thermal response associated with ablative laser exposure may further induce the expression of heat-shock proteins (HSP70, HSP90) and upregulation of fibroblast growth factor receptors (FGFR) in keratinocytes and fibroblasts. The synergy of micro-injury induced by the laser and the biological activity of FGF-2 may lead to accelerated dermal remodeling, increased collagen and elastin synthesis, improved microvascular perfusion, and visible improvement in skin texture, firmness, and / or tone.

[0276]

[0256] In one aspect, the combination of a non-ablative laser and an FGF-2 variant may produce a synergistic effect through controlled photothermal stimulation of the dermal tissue without removal of the epidermal surface. The absorbed laser energy may raise the temperature of the dermis up to approximately 49 °C, inducing mild, reversible cellular stress and enhancing microcirculation and tissue permeability. This photothermal effect may promote vasodilation and transient extracellular matrix relaxation, thereby facilitating the deeper penetration and more uniform diffusion of the FGF-2 variant through the epidermal barrier and into the dermis.

[0277]

[0257] The controlled thermal response associated with non-ablative laser exposure may further induce the expression of heat-shock proteins (HSP70, HSP90) and upregulation of fibroblast growth factor receptors (FGFR) in keratinocytes and fibroblasts. The synergy may result in enhanced fibroblast proliferation, collagen and elastin neosynthesis, angiogenesis, and overall dermal remodeling without surface ablation. The combined treatment improves skin elasticity, density, and hydration while minimizing downtime and post-procedure inflammation.

[0278]

[0258] In another aspect of the invention, the synergistic effect of an FGF-2 variant in combination with both ablative and non-ablative laser or light modalities may provide complementary treatment effect e.g. regenerative and cosmetic effects by First or Second physical modality treatment unit unit which may be e.g. diode lasers, Nd:YAG lasers, Er:YAG lasers, CO2 lasers, Ho:YAG lasers, Er,Cr:YSGG lasers, alexandrite lasers, ruby lasers, fiber lasers, titanium: sapphire lasers, excimer lasers, argon lasers, helium-neon lasers, solid-state lasers, pulsed dye lasers, quantum cascade lasers, LED arrays, intense pulsed light (IPL) sources, xenon flashlamps, superluminescent diodes (SLDs), low-level laser therapy (LLLT) sources, etc...

[0279]

[0259] In another aspect, the system for laser energy delivery may comprise one or more laser generators adapted to produce ablative and / or non-ablative laser energy, a control unit, at least one applicator, a power supply unit, a data storage unit, a cooling unit, a user interface, and optionally a fluid delivery unit.

[0260] The applicator may comprise at least one energy delivery element adapted for delivering laser energy to the surface of or into epithelial and / or connective tissues. The energy delivery element may be configured to emit laser energy at controlled parameters including wavelength, pulse energy, pulse duration, spot size, and fluence.

[0280]

[0261] The system may further be configured to deliver laser energy in single or multiple pulses, either sequentially, simultaneously, or in overlapping manner, using ablative and / or non-ablative laser sources.

[0281]

[0262] In another aspect, the energy of a single laser energy pulse produced by a laser source may be in a range of 0.1 J to 6 J, or in a range of 0.2 J to 5 J, or in a range of 0.3 J to 4 J. Additionally, the maximal energy may be in a range of 2 J to 4 J.

[0282]

[0263] Non-ablative laser energy may be delivered at a wavelength between 1000 nm and 3000 nm, preferably between 1200 nm and 1650 nm, or more preferably between 1520 nm and 1560 nm. Alternatively, the wavelength for non-ablative laser energy may fall within ranges from 9000 nm to 11000 nm or from 1700 nm to 4000 nm.

[0283]

[0264] In another aspect, the maximal pulse energy (i.e. energy released in a single pulse) of the non- ablative laser energy may be in a range of 1 J to 5 J, or in a range of 2 J to 4 J, or in a range of 2.5 J to 3.5 J, or in a range of 2.6 J to 3.4 J, or in a range of 2.7 J to 3.3 J.

[0284]

[0265] Ablative laser energy may be delivered at a wavelength between 2500 nm and 3500 nm, preferably between 2700 nm and 3200 nm, or more preferably between 2900 nm and 2980 nm. Alternatively, the ablative wavelength may be in the range from 9000 nm to 11000 nm or from 1700 nm to 4000 nm.

[0285]

[0266] In another aspect, the maximal pulse energy (i.e. energy released in a single pulse) of the ablative laser energy may be in a range of 1 J to 5 J, or in a range of 2 J to 4.5 J, or in a range of 3 J to 4 J, or in a range of 3.1 J to 3.9 J, or in a range of 3.2 J to 3.8 J.

[0286]

[0267] In one example, when using an ablative laser source and a non-ablative laser source in a sequential, overlapping, or simultaneous manner: the fluence per repetition (each laser firing once) may range from 0.1 J / cm2to 12 J / cm2, preferably from 0.2 J / cm2to 10 J / cm2, or from 0.4 J / cm2to 7 J / cm2; the combined energy per repetition may range from 0.2 J to 12 J, preferably from 0.4 J to 10 J, or from 0.6 J to 7.5 J; and the pulse duration for any laser source may range from 0.01 ms to 30 ms, preferably from 0.05 ms to 20 ms, or from 0.1 ms to 10 ms.

[0287]

[0268] The laser energy pulses may be delivered as single or multiple pulses, and may be applied in a sequential, simultaneous, or overlapping manner using both ablative and non-ablative laser sources.

[0288]

[0269] In one aspect, the laser energy pulse stack may comprise a plurality of consecutive laser energy pulses, wherein the number of pulses per stack may range from 1 to 100, preferably from 1 to 50, more preferably from 1 to 10, or from 2 to 100, 2 to 50, or 2 to 10.

[0289]

[0270] In one aspect, the firing frequency of the laser energy pulse stacks may range from 0.1 Hz to 50 Hz, preferably from 1 Hz to 40 Hz, or more preferably from 1.5 Hz to 30 Hz, or alternatively from 0.1 Hz to 10 Hz, from 0.5 Hz to 7 Hz, or from 1 Hz to 5 Hz.

[0290]

[0271] In one aspect, the overlapping time between consecutive laser energy pulses may be in the range of 0.1% to 99%, preferably from 0.5% to 95%, or from 1% to 90% of the shortest pulse length among the plurality of pulses.

[0291]

[0272] In one aspect, the output power of the apparatus operating in continuous mode may range from 10 W to 20,000 W, preferably from 50 W to 19,000 W, or from 100 W to 17,500 W.Alternatively, the continuous mode output power may range from 10 W to 2,500 W.

[0292]

[0273] In one aspect of the invention, the laser energy may be delivered in a fractional or patterned distribution, optionally randomized or sequentially scanned to control spatial overlap and minimize cumulative thermal damage.

[0293]

[0274] In another aspect of the invention, two or more laser sources or energy modalities may be synchronized to deliver combined energy pulses with controlled temporal overlap or delay.

[0294]

[0275] In another aspect of the invention, the laser source may be from the group of YAG, CO2, Nd:YAG, Er:Glass, Thulium, Diode, fractional, fiber, alexandrite, ruby laser, or any other laser source.

[0295]

[0276] In one aspect of the invention, a method for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering ablative and / or non-ablative laser energy into the tissue; wherein the steps may be performed in any order and / or the application of FGF-2 in step a.) may be performed concurrently with the external stressors or procedures b.), and wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or promote shortened post -procedure recovery time.

[0296]

[0277] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering ablative and / or non-ablative laser energy into the tissue; wherein the steps may be performed in any order and / or the application of FGF-2 in step a.) may be performed concurrently with the external stressors or procedures b.), and wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or promote shortened post -procedure recovery time.

[0297]

[0278] In one aspect, the present invention relates to the use of an FGF-2 variant to promote and / or accelerate the wound healing processes in epithelial and / or connective tissues.

[0298]

[0279] FGF-2 may stimulate key cellular activities involved in wound healing, including keratinocyte proliferation, fibroblast activation, angiogenesis, extracellular matrix remodeling, and re- epithelialization, thereby supporting tissue restoration and functional recovery.

[0299]

[0280] In another aspect, the FGF-2 variant may be used to enhance the healing of various types of wounds, including but not limited to: a.) acute wounds (e.g., surgical incisions, abrasions, lacerations, burns); b.) chronic wounds (e.g., diabetic ulcers, pressure ulcers, venous leg ulcers); c.) traumatic injuries (e.g., mechanical injuries, thermal injuries, chemical injuries); and d.) procedurally-induced wounds (e.g., microneedling, fractional laser treatment, radiofrequency ablation, pulsed electric field treatment).

[0281] In another aspect, the administration of an FGF-2 variant prior to, during, and / or following external procedures that induce controlled tissue irritation or damage (such as laser treatment, radiofrequency, microneedling, or PEF) may synergistically enhance wound healing by promoting faster cell proliferation, neovascularization, and ECM restoration while reducing inflammation and scar formation. Such a combination may result in improved healing outcomes, accelerated recovery times, and enhanced cosmetic and functional results in comparison to procedures performed without FGF-2 administration.

[0300]

[0282] In one aspect, the present invention may provide a method for promoting and / or accelerating hair growth comprising the administration of an FGF-2 variant to a subject in need thereof.

[0301]

[0283] In one aspect, the present invention provides for the use of an FGF-2 variant for transdermal delivery to epithelial and / or connective tissues.

[0302]

[0284] The FGF-2 variant may be administered using systems, compositions, or devices designed to enhance transdermal penetration, thereby facilitating delivery of the biologically active molecule across the stratum corneum and into deeper tissue layers to promote and / or accelerate tissue regeneration, wound healing, and restoration of tissue function.

[0303]

[0285] In another aspect, transdermal delivery of the FGF-2 variant may be facilitated by: a.) mechanical methods, including microneedling, dermabrasion, shaving, or other mechanical irritation and / or damage techniques as previously described; b.) physical methods, such as electroporation, ultrasound, iontophoresis, radiofrequency, pulsed electric fields, or laser-induced microchannels; and / or c.) chemical methods employing penetration enhancers as previously described.

[0304]

[0286] In one exemplary method, the administration of an FGF-2 variant is combined with the delivery of RF energy for promoting accelerated tissue regeneration, wherein: an FGF-2 variant may be reconstituted into a sterile aqueous composition comprising, for example, at least one stabilizer, at least one antioxidant and at least one humactant; the FGF-2 composition may be applied topically to the surface of epithelial and / or connective tissues, at a concentration in a range of 1 pg / mL to 1000 pg / mL, or in a range of 10 pg / mL to 800 pg / mL, or in a range of 24 pg / mL to 321 pg / mL;

[0305] RF energy may be delivered to the treatment site using an applicator comprising at least one invasive energy delivery element; the RF energy parameters may be set to comprise, for example, frequency: 0.3 MHz to 30 MHz, current density: 5 A / cm2to 200 A / cm2, RF pulse duration: 0.5 ms to 2000 ms, RF voltage amplitude: 50 V to 845 V ; and

[0306] The FGF-2 composition may be applied, for example, following the delivery of RF energy to enhance treatment outcomes, e.g. regenerative outcomes, wherein the treated tissue may optionally be covered with an occlusive dressing or a hydrogel patch comprising an additional FGF-2 composition to maintain hydration and promote sustained delivery.

[0307]

[0287] In one exemplary method, the administration of an FGF-2 variant may be combined with the application of non-ablative laser energy to enhance transdermal penetration and promote and / or accelerate tissue regeneration, wherein: an FGF-2 variant is formulated into, for example, a cream comprising at least one stabilizer and at least one humactant; a thin layer of the FGF-2 composition may be optionally applied to the treatment site 5 to 30 minutes prior to laser energy application to allow pre-conditioning of the tissue; laser energy may be applied to the treatment site using a laser source with the following parameters: wavelength: 1520 nm to 1560 nm, pulse energy: 2 J to 4 J, pulse duration: 0.05 ms; immediately following laser energy delivery, an additional amount of the FGF-2 composition may be applied to the treated area; and the treated area may optionally be massaged gently to enhance penetration of the FGF-2.

[0308]

[0288] In one aspect of the invention, the combined application of an FGF-2 variant with radiofrequency energy, pulsed electric field energy, and / or non-ablative laser energy may be used in a cosmetic procedure aimed at improving the aesthetic appearance and physiological quality of epithelial and / or connective tissues. The combined treatment may attenuate local inflammatory responses induced by energy-based stimulation, thereby minimizing erythema, edema, and discomfort typically associated with such procedures. Furthermore, the treatment may promote accelerated epithelial restoration and reorganization of the extracellular matrix, leading to improved skin smoothness, elasticity, and overall texture. As a result, the procedure may provide enhanced treatment outcomes, e.g. cosmetic outcomes, including a rejuvenated appearance and reduced post -procedure recovery time, compared to treatments performed without the FGF-2 variant.

[0309]

[0289] In one aspect of the invention, the application of radiofrequency energy, pulsed electric field energy, and / or non-ablative laser energy may transiently increase the permeability of cellular membranes and / or induce electroporation-like effects within the epithelial cells of the treated tissue. Such transient biophysical alterations facilitate enhanced transmembrane transport and intracellular uptake of the administered FGF-2 variant, thereby improving its local bioavailability and biological activity. Furthermore, the applied energy may induce upregulation of fibroblast growth factor receptors (FGFRs) and associated downstream signaling pathways in dermal and epidermal cells, leading to a potentiated cellular response to the exogenously supplied FGF-2 variant. As a result, the combined treatment produces a synergistic enhancement of treatment effects, e.g. regenerative and cosmetic effects relative to administration of the FGF-2 variant or energy-based therapy alone.

[0310]

[0290] In one aspect of the invention, the present disclosure relates to the delivery of ultrasound energy to epithelial and / or connective tissues to induce controlled mechanical and / or thermal effects, thereby enhancing the penetration and biological effectiveness of an FGF-2 variant. The ultrasound energy may be delivered in a continuous or pulsed mode to create localized mechanical vibration, acoustic cavitation, and / or microstreaming, which transiently increase membrane permeability and tissue perfusion through a process known as sonoporation, thereby facilitating diffusion of the administered FGF-2 variant into deeper tissue layers.

[0311]

[0291] In one aspect, the combined application of ultrasound energy and an FGF-2 variant may produce a synergistic effect through controlled mechanical and thermal stimulation of epithelial and / or connective tissues. The propagation of ultrasonic waves through the skin may induce acoustic cavitation, microstreaming, and / or localized micro vibrations within epidermal and dermal layers. These may transiently disrupt the phospholipid bilayer within epidermal cell membranes, and loosen tight junctions between adjacent cells, leading to ultrasound-induced sonoporation and increased transcellular and paracellular permeability. As a result, the diffusion and bioavailability of the FGF-2 variant within deeper tissue compartments are significantly enhanced, promoting a synergistic biological response.

[0312]

[0292] In one aspect of the invention, the system for ultrasound energy delivery may comprise one or more ultrasound generators configured to produce acoustic energy, a control unit, an applicator, a power supply unit, a cooling unit, a data storage unit, acoustic window, and a user interface. The ultrasound generator may operate in continuous wave mode or pulsed wave mode and may be configured to control the amplitude, frequency, pulse duration, and duty cycle of the delivered ultrasound energy.

[0313]

[0293] In another aspect of the invention, the system applying ultrasound energy may further optionally comprise a fluid delivery unit for administration of the FGF-2 variant prior to, during, or after the ultrasound application. The FGF-2 variant may be also applied locally directly to the target tissue region, to the surface of the applicator, or both, thereby enabling direct contact between the compound and the treated area.

[0294] The frequency of the ultrasound energy may range from 20 kHz to 25 GHz, or from 20 kHz to 1 GHz, or from 50 kHz to 250 MHz, or from 100 kHz to 100 MHz. In low-frequency applications (below

[0314] 1 MHz), the ultrasound may produce mechanical cavitation and microstreaming effects, while at higher frequencies (above 1 MHz) it may primarily produce controlled thermal stimulation.

[0315]

[0295] In another aspect of the invention, the frequency of the ultrasound energy may range from 20 kHz to 80 MHz, from 50 kHz to 50 MHz, or from 150 kHz to 20 MHz.

[0316]

[0296] The output power of the ultrasound energy on the surface of the applicator may be less than or equal to 20 W, or 15 W, or 10 W, or 5 W.

[0317]

[0297] The acoustic intensity delivered to the tissue may range from 0.05 W / cm2to 5 W / cm2, or from 0.1 W / cm2to 3 W / cm2, or from 0.2 W / cm2to 2 W / cm2.

[0318]

[0298] Ultrasound energy may provide energy flux on the surface of the applicator and / or on the surface of the treated tissue (e.g. skin) in the range of 0.001 W / cm2to 250 W / cm2, more preferably in the range of 0.005 W / cm2to 50 W / cm2, even more preferably in the range of 0.01 W / cm2to 25 W / cm2, or most preferably in the range of 0.05 W / cm2to 20 W / cm2.

[0319]

[0299] Ultrasound energy may be focused or defocused. Treatment depth of ultrasound energy may be in the range of 0.1 mm to 100 mm, 0.2 mm to 50 mm, 0.25 mm to 25 mm, or 0.3 mm to 15 mm. At a depth of 5 mm the ultrasound energy may provide an energy flux in the range of 0.01 W / cm2to 20 W / cm2or 0.05 W / cm2to 15 W / cm2.

[0320]

[0300] The ratio between the output power of ultrasound energy on the surface of the applicator and the output power of the ultrasound energy on the surface of the acoustic window (W / cm2) may be in the range of 0.001 to 1500, more preferably in the range of 0.002 to 500, even more preferably in the range of 0.005 to 250, even more preferably in the range of 0.01 to 100, or most preferably in the range of 0.2 to 50.

[0321]

[0301] An ultrasound beam may have a beam non -uniformity ratio (RBN) in the range of 0.1 to 20, or

[0322] 2 to 15, or 4 to 10. In addition, an ultrasound beam may have a beam non-uniformity ratio below 15 or 10. An ultrasound beam may be divergent, convergent and / or collimated. The temperature of tissue, skin and / or skin surface may be increased by application of ultrasound energy to a value in the range of 32° C to 90° C, of 35° C to 65° C, or of 37° C to 55° C.

[0323]

[0302] The ultrasound energy may be a targeted ultrasound energy which is able to provide effects similar to focused ultrasound energy into a bigger area. Targeted ultrasound energy is not limited to a small focus point, as focused ultrasound energy is. Targeted ultrasound energy may have a different effective radiating area, defined as beam cross-sectional area determined at a distance of 3 mm from the surface of the applicator and multiplied by a dimensionless factor equal to 1.354. The effective radiating area of targeted ultrasound energy may be in the range of 0.1 cm2to 1.5 cm2, more preferably in the range of 0.25 cm2to 1 cm2.

[0324]

[0303] The ultrasound beam of targeted ultrasound energy may have a beam non-uniformity ratio below 8. The treatment depth of targeted ultrasound energy may be in the range of 0.1 mm to 80 mm, or 0.2 mm to 75 mm, or 0.25 mm to 50 mm, or 0.25 mm to 25 mm.

[0325]

[0304] Targeted ultrasound energy may provide energy flux on the surface of the applicator and / or on the surface of the treated tissue (e.g. skin) in the range of 0.001 W / cm2to 200 W / cm2, more preferably in the range of 0.005 W / cm2to 50 W / cm2, even more preferably in the range of 0.01 W / cm2to 25 W / cm2, or most preferably in the range of 0.1 W / cm2to 20 W / cm2.

[0326]

[0305] The ultrasound energy may be transferred to the tissue through an acoustic window. In one embodiment, the RF electrode may act as the acoustic window. The acoustic window may have a surface area in the range of 0.01 cm2to 250 cm2, or 0.1 cm2to 150 cm2, or 0.2 cm2to 100 cm2, or 0.2 cm2to 80 cm2. The output power of targeted ultrasound energy on the surface of the applicator may be up to 2.5 W, or 4 W, or 10 W.

[0327]

[0306] The frequency of targeted ultrasound energy may be in a range of 100 kHz to 20 MHz, 200 kHz to 10 MHz, or 250 kHz to 8 MHz. Energy flux of the targeted ultrasound energy measured in water at 5 mm deep below the contact surface of the applicator may be in the range of 0.1 W / cm2to 15 W / cm2, 0.25 W / cm2to 10 W / cm2, or 0.5 W / cm2to 6 W / cm2.

[0328]

[0307] The temperature of the tissue 3 mm below the surface of the applicator providing targeted ultrasound energy may be increased to a value in the range of 39° C to 55° C, or 40° C to 50° C. Targeted ultrasound energy may be applied and provide treatment action to the epidermis, epidermal basal layer, dermis (including papillary dermis, reticular dermis) and / or hypodermis. The temperature of tissue, skin and / or skin surface may be increased by application of targeted ultrasound energy to a value in the range of 32° C to 90° C, 35° C to 65° C, or 37° C to 55° C.

[0329]

[0308] In one aspect, the system may be configured to deliver ultrasound energy in a pulsed mode with a duty cycle between 5% and 90%, or between 10% and 70%, or between 20% and 50%. The pulse repetition frequency may be in the range of 0.1 Hz to 1000 Hz, or from 1 Hz to 500 Hz, or from 5 Hz to 200 Hz. The duration of a single ultrasound pulse may be between 0.1 ms and 500 ms, or between 0.5 ms and 200 ms, or between 1 ms and 100 ms. The total exposure time per treatment area may range from 1 s to 600 s, depending on the desired depth and extent of tissue stimulation.

[0309] In one aspect, the ultrasound system may deliver energy in a focused or non-focused pattern. Focused ultrasound (for example, HIFU or MFU) may concentrate acoustic energy within a defined focal zone at specific tissue depths, creating microthermal zones (MTZs) that stimulate fibroblast proliferation, collagen remodeling, and neovascularization. Non-focused ultrasound may be applied over a broader surface area for enhancement of diffusion, hydration, and metabolic activity of the tissue, while supporting FGF-2 uptake.

[0330]

[0310] In one aspect, the ultrasound transducer may be combined with other physical modalities, such as laser irradiation, radiofrequency energy, or pulsed electric fields, within a multifunctional applicator. The control unit may coordinate delivery timing and power output among modalities to achieve synergistic effects. For example, sequential or simultaneous ultrasound and FGF-2 administration may transiently increase permeability and receptor responsiveness, while combined ultrasound and RF heating may further enhance tissue perfusion and metabolic activation.

[0331]

[0311] In one aspect, the combined treatment of ultrasound energy and FGF-2 variant may result in synergistic biological effects including, but not limited to, enhanced fibroblast proliferation, collagen and elastin neosynthesis, angiogenesis, accelerated wound closure, and reduced inflammatory response. The procedure may further improve the cosmetic appearance of the skin by enhancing smoothness, elasticity, and uniformity of texture, and by reducing visible signs of aging or scarring.

[0332]

[0312] In one aspect, the ultrasound system may comprise one or more sensors configured to monitor parameters of the treated tissue and / or of the coupling medium, including acoustic impedance, reflected wave intensity, temperature, or cavitation activity. The control unit may receive real-time feedback from the sensors and automatically adjust output power, frequency, or pulse duration of the ultrasound energy to maintain safe and effective treatment conditions and to achieve uniform energy deposition within the target tissue.

[0333]

[0313] In one aspect, the ultrasound system may be configured to operate in a multi -frequency or dualmode regime, wherein two or more frequencies are emitted either sequentially or simultaneously. The combination of low -frequency (for cavitation) and high-frequency (for controlled thermal stimulation) components may enable selective treatment of different tissue depths and provide optimized mechanical and thermal synergy with the administered FGF-2 variant.

[0334]

[0314] In one aspect, the ultrasound energy delivery may be synchronized with the administration or release of the FGF-2 variant. The synchronization may involve temporal alignment of ultrasound pulses with micro-dosing events, fluid jet pulses, or micro-reservoir openings in the applicator. This configuration may promote acoustic streaming and transient permeabilization of cell membranes exactly at the moment of compound delivery, thereby maximizing local uptake and receptor activation of the FGF-2 variant.

[0335]

[0315] In one aspect of the invention, a method for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering ultrasound energy into the tissue; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration of FGF-2 into deeper tissue layers, promote and / or accelerate tissue regeneration, and reduce post-procedure recovery time.

[0336]

[0316] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering ultrasound energy into the tissue; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration of FGF-2 into deeper tissue layers, promote and / or accelerate tissue regeneration, and reduce post -procedure recovery time.

[0337]

[0317] In one aspect, the invention relates to the delivery of acoustic shockwave energy to epithelial and / or connective tissues to induce controlled mechanical stimulation, thereby enhancing the penetration and biological effectiveness of an FGF-2 variant. The acoustic shockwaves may create transient mechanical stress, microcavitation, and shear forces within the tissue, resulting in temporary alteration of cell membrane permeability and activation of repair mechanisms that facilitate enhanced uptake of the FGF-2 variant and stimulation of regenerative processes.

[0338]

[0318] In one aspect, the present disclosure relates to the delivery of acoustic shockwave energy to epithelial and / or connective tissues to induce controlled mechanical stimulation, thereby enhancing tissue regeneration and improving the uptake of an FGF-2 variant. The acoustic shockwaves may be produced as single or repetitive high-pressure pulses that propagate through the skin and underlying dermal layers, creating transient mechanical deformation, cavitation, and shear forces within the extracellular matrix (ECM). These physical effects temporarily loosen and realign collagen and elastin fibers, resulting in partial remodeling of the ECM architecture and increased interstitial space. This transient mechanical relaxation facilitates the diffusion and distribution of bioactive compounds, including an administered FGF-2 variant, throughout deeper dermal compartments.

[0339]

[0319] In one aspect of the invention, the system for shockwave energy delivery may comprise one or more acoustic wave generators, a control unit, an applicator, a piezo-element, a power supply unit, and a user interface, or any other component. Optionally, the system may further comprise a cooling unit, an integrated coupling medium delivery unit, or a data storage unit for treatment tracking. The shockwave generator and / or piezo-element may produce either focused or radial acoustic waves, and the control unit may regulate the energy, frequency, pulse duration, and repetition rate of the delivered shockwaves.

[0340]

[0320] A power supply unit of the present invention may power the various electrical components of the apparatus, including but not limited to a condenser which discharges current into a piezo-element of an applicator of the present invention. The condenser may provide electrical voltage in a range from 1 kV to 90 kV, from 2 kV to 50 kV, from 5 kV to 25 kV, or from 7 kV to 15 kV.

[0341]

[0321] The applicator may include at least one transducer or projectile element configured to generate and deliver acoustic shockwaves to the surface of or into epithelial and / or connective tissues. The transducer may be electromagnetic, electrohydraulic, or piezoelectric in type. The applicator may further comprise a coupling membrane or fluid interface to ensure efficient acoustic transmission. The geometry of the applicator may be planar, concave, or convex, depending on the desired focal depth and target tissue volume.

[0342]

[0322] In some aspects of the invention, the piezo-element may have a volume of at least 1.5 cm3, or may have a volume in a range from 1.5 cm3to 600 cm3, from 1.5 cm3to 300 cm3, from 1.5 cm3to 160 cm3, from 1.5 cm3to 950 cm3, from 1.5 cm3to 60 cm3, from 3.5 cm3to 35 cm3, or from 3.5 cm3to 20 cm<

[0343]

[0323] The diameter of the piezo-element may be in a range from 1 cm to 20 cm, from 2 to 15 cm, or from 6 cm to 10 cm, wherein the diameter may be measured between the two most distant points of the active piezoelectric surface, or between the two outermost opposite points when measured perpendicularly to the central axis of the element.

[0344]

[0324] The frequency of the provided exciting electric current to the piezo-element may be in a range from 1 Hz to 40 Hz, from 2 Hz to 30 Hz, from 2 Hz to 20 Hz, from 2 Hz to 15 Hz, or from 4 Hz to 14 Hz.

[0345]

[0325] The duration of one shock wave pulse may be in a range from 200 ns to 30 ps, from 400 ns to 15 ps, or from 400 ns to 2.5 ps or from 800 ns to 1.5 ps. The FWHM of the shock wave positive phase may be from 50 ns to 20 ps, or from 50 ns to 10 ps, or from 50 ns to 2.5 ps, from 100 ns to 1.7 ps, or from 100 ns to 1.2 ps.

[0346]

[0326] The pulse width of a shock wave pulse positive phase may be in a range from 0.1 ps to 30 ps, from 0.5 ps to 10 ps, from 0.5 ps to 3 ps, from 0.7 ps to 2 ps, or from 0.8 ps to 1.7 ps. The rise time of a shock wave pulse may be in a range from 50 ns to 2000 ns, or from 50 ns to 1000 ns, or from 100 ns to 700 ns, or from 100 ns to 500 ns, or from 100 ns to 400 ns.

[0347]

[0327] In one aspect of the invention, an applicator of the present invention may provide a pressure to the patient’s body in a range from 5 MPa to 250 MPa, from 5 MPa to 200 MPa, from 5 MPa to 150 MPa, from 5 MPa to 120 MPa, from 8 MPa to 50 MPa, or from 10 MPa to 35 MPa at the focal volume.

[0348]

[0328] In one aspect of the invention, the acoustic shockwave energy may be delivered in the form of focused waves, radial (non-focused) waves, or a combination thereof, depending on the desired depth of penetration, energy distribution, and treatment outcome.

[0349]

[0329] The width of the focus volume at the horizontal maximum focal volume cross section (x- or y- axis) may be in a range from 0.1 mm to 100 mm, from 0.5 mm to 70 mm, from 1.5 mm to 50 mm, from 1.5 mm to 40 mm, from 1.5 mm to 15 mm, or from 2 mm to 10 mm. The depth of the focus volume at the vertical maximum focal volume cross section (z axis) may be in a range from 0.1 mm to 100 mm, from 1.5 mm to 100 mm, from 5 mm to 100 mm, from 10 mm to 60 mm, or from 15 mm to 40 mm.

[0350]

[0330] The energy of the shock wave pulse in at least part of the focal volume measured in water by needle hydrophone may be in a range from 0.01 mJ- mm2to 20 mJ- mm2, from 0.01 mJ- mm2to 15 mJ- mm2, from 0.01 mJ- mm2to 10 mJ- mm2, from 0.01 mJ- mm2to 7 mJ- mm2, from 0.03 mJ- mm2to 4 mJ- mm2, or from 0.03 mJ- mm2to 0.5 mJ- mm2in the focal volume.

[0351]

[0331] The size, shape or depth of the focal volume may be influenced by acoustic lens. The acoustic lens radius of curvature may be in a range from 30 mm to 350 mm, from 30 mm to 150 mm, from 40mm to 100 mm, or from 50 mm to 80 mm.

[0352]

[0332] The frequency of acoustic shockwaves may range from 1 Hz to 25 Hz, or from 2 Hz to 20 Hz, or from 3 Hz to 15 Hz. The peak pressure amplitude of each shockwave may range from 0.05 MPa to 10 MPa, or from 0.1 MPa to 5 MPa, or from 0.2 MPa to 2 MPa. The number of pulses per treatment may range from 100 to 20,000, preferably from 500 to 10,000, or from 1000 to 5000 pulses, depending on the size of the treatment area and desired intensity of stimulation. The pulse duration may be between 100 ns and 10 ps, preferably between 200 ns and 5 ps.

[0333] In one aspect of the invention, the system may allow for the adjustment of treatment parameters including energy flux density, typically ranging from 0.01 mJ / mm2to 2.0 mJ / mm2, or from 0.05 mJ / mm2to 1.0 mJ / mm2, or from 0.1 mJ / mm2to 0.6 mJ / mm2. The repetition frequency and energy per pulse may be modulated by the control unit during treatment to optimize mechanical stimulation and to prevent discomfort or excessive tissue loading. The system may also comprise feedback sensors for monitoring reflected acoustic signals or mechanical resistance to dynamically adjust energy output.

[0353]

[0334] In one aspect of the invention, the coupling medium used between the applicator and the tissue may comprise a conductive gel, hydrogel, or fluid film that may additionally serve as a carrier for an FGF-2 variant. During treatment, the propagation of shockwaves through this coupling layer may enhance local mixing and diffusion of the compound into the epidermal and dermal structures, thereby improving local concentration and bioavailability of the FGF-2 variant.

[0354]

[0335] In one aspect of the invention, the combined application of acoustic shockwave energy and an FGF-2 variant may induce a synergistic biological response characterized by stimulation of fibroblast proliferation, increased collagen and elastin synthesis, and promotion of angiogenesis and tissue oxygenation. The transient mechanical deformation of cells and extracellular matrix may further upregulate fibroblast growth factor receptors (FGFRs) and associated signaling pathways, amplifying the treatment effect, e.g. regenerative effect of the administered FGF-2 variant.

[0355]

[0336] In one aspect of the invention, the shockwave system may be configured to integrate additional energy sources such as radiofrequency, pulsed electric field, or laser energy within a single applicator. The control unit may coordinate the sequence and timing of energy delivery to achieve synergistic enhancement of FGF-2 transport and tissue stimulation. For example, application of ultrasound or RF heating immediately before or after shockwave delivery may further enhance microcirculation and compound penetration.

[0356]

[0337] In one aspect of the invention, the shockwave system may comprise one or more sensors configured to monitor operational and tissue parameters during treatment, including reflected acoustic wave intensity, tissue stiffness, impedance, or surface temperature. The control unit may analyze sensor data in real time and automatically adjust the energy flux density, pulse frequency, or focal depth of the shockwaves to ensure consistent and safe energy deposition while preventing excessive mechanical loading or heating of the tissue.

[0357]

[0338] In another aspect of the invention, to achieve the best results in the soft tissue by at least one radial shock wave, the energy flux density of the mechanical stimulation is preferably in the range between 0.001 mW- mm2and 160 mW- mm2, more preferably in the range between 0.001 mW- mm2and 100 mW- mm2, or most preferably in the range between 0.001 mW- mm2and 50 mW- mm2.

[0339] The electromagnetic field used for heating the soft tissue may be a radiofrequency field or microwave field, typically in the range of 0.1 MHz to 25 GHz, more preferably in the range from 0.1 MHz to 435 MHz, or most preferably in the range from 0.1 MHz to 28 MHz. All of the above mentioned waves may cause movement of charged particles e.g. ions, rotation of dipolar molecules or polarization of normally non polar particles and therefore increase the tissue temperature.

[0358]

[0340] The energy flux density of the electromagnetic field is preferably in the range between 0.01 mW- mm2and 10 000 mW- mm2, more preferably in the range between 0.1 mW- mm2and 5 000 mW- mm2, or most preferably in the range between 0.5 mW- mm2and 1 000 mW- mm2.

[0359]

[0341] The sum of energy flux density of the mechanical stimulation and electromagnetic field applied to the patient simultaneously, successively, or in overlap should be preferably above 0.1 mW- mm2, 1 mW- mm2, or 5 mW- mm2, generally up to a maximum of 100, 500, 1000, 5,000, 10,000 or 15,000 mW- mm2.

[0360]

[0342] In one aspect, the shockwave generator may be coupled with an automatic calibration and selftest module that adjusts the pressure output, verifies pulse count, and compensates for applicator wear or changes in acoustic coupling efficiency. Such calibration ensures reproducible energy delivery across multiple treatments and compliance with device safety standards.

[0361]

[0343] In one aspect of the invention, a method for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering acoustic shockwave energy into the tissue; wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or reduce post -procedure recovery time.

[0362]

[0344] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering acoustic shockwave energy into the tissue; wherein steps a.) and b.) may be performed in any order and / or may be performed concurrently, and wherein the treatment may enhance the penetration of FGF-2 into deeper tissue layers and promote and / or accelerate tissue regeneration and / or reduce post-procedure recovery time.

[0345] In one aspect, the present invention of vacuum pressure therapy relates to the delivery of positive and / or negative pressure modalities to epithelial and / or connective tissues to induce controlled mechanical stimulation and modulation of tissue perfusion, thereby enhancing the penetration and biological effectiveness of an FGF-2 variant. The alternating application of negative and positive pressure may create transient deformation of tissues, stimulate lymphatic flow and microcirculation, and facilitate transport of the FGF-2 variant into deeper tissue layers.

[0363]

[0346] In one aspect of the invention, the term vacuum compression therapy, pressure therapy, therapy and vacuum pressure therapy may be used interchangeably.

[0364]

[0347] According to one aspect of the invention, one or more therapy devices may be used to deliver pressure changes to the subject’s body part. The device may be used to treat various body parts, including limbs, arms, hands, legs, feet, trunk or thighs, abdomen, torso, lower body part, genital tissue (e.g. vaginal tissue, penile tissue), head, neck or others.

[0365]

[0348] The disclosed devices and related methods of therapy may include application of a pressure gradient by one or more pressure generators. The disclosed devices and related methods of therapy may include application of a pressure gradient by one or more pressure generators together with the application of at least one additional type of energy.

[0366]

[0349] The pressure gradient may be induced by a hyperbaric and / or hypobaric environment inside a therapy device or inside a part of the therapy device (e.g. therapy chamber). The pressure gradient may include at least one hypobaric period when the pressure inside the device or inside a part of the therapy device (e.g. therapy chamber) may be below atmospheric pressure and / or at least one hyperbaric period when the pressure inside the device or inside a part of the therapy device (e.g. therapy chamber) may be above the atmospheric pressure. Atmospheric pressure may be applied during an atmospheric pressure period. Atmospheric pressure may refer to the pressure outside of the device and / or a standard pressure defined as 1 bar (equal to 100 kPa), or 1 atm (101,325 Pa), or 101.3 kPa, or 101.33 kPa, or 101.6 kPa, or 101 kPa, or 99.99 kPa.

[0367]

[0350] The vacuum compression therapy (pressure therapy) device according to the invention may comprise light signaling. The light signaling may indicate the status of the therapy, may provide visual cues, feedback, or error signaling, or any other appropriate or requested status.

[0368]

[0351] The therapy may have a duration in the range from 1 minute to 180 minutes, or from 1 minute to 60 minutes, or from 5 minutes to 60 minutes, or from 10 minutes to 45 minutes or, or from 15 minutes to 30 minutes, or from 1 minute to 10 minutes, or from 10 minutes to 20 minutes, or from 15 minutes to 45 minutes, or from 20 minutes to 40 minutes.

[0352] The light signaling may comprise changes in color, for example red for error or stop, green for operational, or any other appropriate light signaling color.

[0369]

[0353] The running therapy may be signaled by a blue color.

[0370]

[0354] The light signaling may comprise an LED (Light Emitting Diode), e.g. LED strip, a display, OLED (Organic Light Emitting Diode) display, LCD displays (Liquid Crystal Display), signal light, fiber optic light guides, laser diodes, electroluminescent panels (EL Panels), light bars or strips, or any other appropriate light signaling component.

[0371]

[0355] The pressure difference between the pressure in the therapy chamber and the atmospheric pressure may be in a range from 0.001 kPa to 1000 kPa, from 0.01 kPa to 1000 kPa, from 0.01 kPa to 100 Pa, or from 0.1 kPa to 100 kPa, or from 1 kPa to 100 kPa, or from 1 kPa to 20 kPa, or from 10 kPa to 50 kPa, or from 5 kPa to 20 kPa, or from 10 kPa to 15 kPa, or from 20 kPa to 30 kPa.

[0372]

[0356] The vacuum compression therapy (pressure therapy) in combination with thermal treatment may be used for various aesthetic benefits, such as, for example, tightening and firming of the skin, improving skin texture and tone, body sculpting, cellulite reduction, post-liposuction care, buttock enhancement, strengthening of the connective tissue, body contouring, improvement of the body circulation, enhanced lymphatic drainage, muscle toning, muscle relaxation, increased blood flow, reducing pain, enhancing tissue healing, reducing inflammation, and others.

[0373]

[0357] The subject’s body part may be heated to a temperature in a range from 38 °C to 100 °C, or from 50 °C to 80 °C, or from 55 °C to 75 °C, or from 57 °C to 72 °C, or from 60 °C to 70 °C, or from 37.5 °C to 50 °C, or from 38 °C to 48 °C, or from 40 °C to 45 °C, or from 41 °C to 43 °C.

[0374]

[0358] The temperature at the output of the thermal treatment element may range from 38 °C to 200 °C, or from 38 °C to 150 °C, or from 38 °C to 100 °C, or from 38 °C to 80 °C, or from 38 °C to 60 °C, or from 38 °C to 50 °C, or from 38 °C to 45 °C, or from 45 °C to 100 °C, or from 50 °C to 100 °C.

[0375]

[0359] The thermal treatment may be provided by a flow of heated fluid (e.g. heated pressure fluid or heated different fluid than the pressure fluid) to the therapy unit 105. The heated fluid may have a flow in a range from 0.001 L / minute to 10,000 L / minute, or from 1 L / minute to 10,000 L / minute, or from 1 L / minute to 5000 L / minute, or from 50 L / minute to 1000 L / minute or from 100 L / minute to 1500 L / minute, or from 500 L / minute to 1500 L / minute, or from 800 L / minute to 1200 L / minute, or from 1000 L / minute to 1500 L / minute, or from 1100 L / minute to 1300 L / minute.

[0376]

[0360] In one aspect of the invention, the system for vacuum pressure therapy may comprise a pressure generation unit configured to produce adjustable negative and positive pressures, a control unit, an applicator with at least one suction chamber or cavity, a power supply unit, a pressure sensor, a treatment element, and a user interface. Optionally, the system may further include a data storage unit, a cooling unit, and / or a fluid delivery unit for the administration of the FGF-2 variant.

[0377]

[0361] The treatment element may comprise a therapeutic fluid. The therapeutic fluid may be a therapeutic gas, such as oxygen or carbon dioxide. The therapeutic fluid (e.g. therapeutic gas) may flow to the therapy unit by at least one pump or a nozzle. The therapeutic fluid (e.g. therapeutic gas) may be used to hasten the healing of lesions, wounds, or ulcers on the subject’s body part. The therapeutic fluid (e.g. therapeutic gas) may also be used to treat various medical, physiological, or aesthetic conditions.

[0378]

[0362] The treatment element may be used to treat the body part by an electromagnetic field. The electromagnetic field may provide a treatment energy to the body part.

[0379]

[0363] The treatment element may comprise a radiofrequency radiating element (e.g. a transducer, a coil, or an electrode). An electromagnetic field used for treating the tissue may be a radiofrequency field, typically in a range from 10 kHz to 300 GHz, or 100 kHz to 3 GHz, or 500 kHz to 3 GHz, or 400 kHz to 900 MHz, or 500 kHz to 900 MHz, or 500 kHz to 100 MHz, or 500 kHz to 50 MHz, or 650 kHz to 30 MHz, or 720 kHz to 8 MHz, or around 13.56 MHz, 40.68 MHz, 27.12 MHz, or 2.45 GHz. The wavelength I of the radiofrequency field may be in a range from 1 mm to 1000 km, or from 1 mm to 100 km, or from 1 mm to 30 km, or from 100 mm to 3 km, or from 100 mm to 600 m, or from to 600m, or from 333 mm to 750 m, or from 333 mm to 600 m, or from 3 m to 600 m, or from 6 m to 600 m, or from 10 m to 461 m, or from 37.5 m to 416 m, or from 1 mm to 10 km, or from 10 mm to 1 km, or from 100 mm to 100 m, or from 100 m to 1 km, or from 1 mm to 100 mm.

[0380]

[0364] The output power of the radiofrequency field may be in a range of 0.1 W to 1000 W, or 1 W to 400 W or 2 W to 370 W, or 5 W to 300 W, or 5 W to 250 W, or 8 W to 180 W, or 10 W to 150 W.

[0381]

[0365] The treatment element may comprise a radiofrequency radiating element (e.g. a transducer, a coil or an electrode). An electromagnetic field used for treating the tissue may be a radiofrequency field, typically in a range from 10 kHz to 300 GHz, or 100 kHz to 3 GHz, or 500 kHz to 3 GHz, or 400 kHz to 900 MHz, or 500 kHz to 900 MHz, or 500 kHz to 100 MHz, or 500 kHz to 50 MHz, or 650 kHz to 30 MHz, or 720 kHz to 8 MHz, or around 13.56 MHz, 40.68 MHz, 27.12 MHz, or 2.45 GHz. The wavelength I of the radiofrequency field may be in a range from 1 mm to 1000 km, or from 1 mm to 100 km, or from 1 mm to 30 km, or from 100 mm to 3 km, or from 100 mm to 600 m, or from to 600m, or from 333 mm to 750 m, or from 333 mm to 600 m, or from 3 m to 600 m, or from 6 m to 600 m, or from 10 m to 461 m, or from 37.5 m to 416 m, or from 1 mm to 10 km, or from 10 mm to 1 km, or from 100 mm to 100 m, or from 100 m to 1 km, or from 1 mm to 100 mm.

[0382]

[0366] The output power of the radiofrequency field may be in a range of 0.1 W to 1000 W, or 1 W to 400 W, or 2 W to 370 W, or 5 W to 300 W, or 5 W to 250 W, or 8 W to 180 W, or 10 W to 150 W.

[0367] The treatment element may comprise at least one microwave radiating element. An electromagnetic field used for treating the tissue may be a microwave field, typically in a range from 300 MHz to 300 GHz, or 400 MHz to 150 GHz, or 500 MHz to 100 GHz, or 700 MHz to 70 GHz, or 1 GHz to 30 GHz, or 1 GHz to 10 GHz.

[0383]

[0368] The wavelength X of the microwave field may range from 1 mm to 1 m, or 2 mm to 75 cm, or 3 mm to 60 cm, or 4.3 mm to 43 cm, or 1 cm to 30 cm, or 3 cm to 30 cm.

[0384]

[0369] The electromagnetic radiation may have a frequency within various ISM bands, such as, for example, 6.78 MHz, 13.56 MHz, 26.957 MHz, 40.66 MHz, 433.05 MHz, 902 MHz, 2.4 GHz, 5.725 GHz, 24 GHz, 61 GHz, 122 GHz, or 244 GHz.

[0385]

[0370] The treatment element may comprise a light radiating element (e.g. light emitting diode, laser, flashlamp, or a light bulb). The electromagnetic field used for treating the tissue may comprise light energy .

[0386]

[0371] The light may have a frequency in a range from 1 THz to 1500 THz, or from 10 THz to 1000 THz, or from 100 THz to 1000 THz, or from 400 THz to 790 THz.

[0387]

[0372] The wavelength X of the light may range from 100 nm to 15,000 nm, or from 200 nm to 12,000 nm, or from 300 nm to 11,000 nm, or from 400 nm to 10,600 nm, or from 380 nm to 750 nm, or from 200 nm to 1140 nm, or from 290 nm to 980 nm, or from 320 nm to 850 nm, or from 400 nm to 700 nm, or from 1000 nm to 11,000 nm, or from 1064 nm to 10,650 nm, or from 1900 nm to 5800 nm, or from 2080 nm to 4100 nm.

[0388]

[0373] The light treatment may involve irradiating a target area to be treated with radiation at one or more light wavelengths preferentially absorbed by the chromophores in a target area.

[0389]

[0374] The light treatment may provide heating, myorelaxation effects and / or biostimulation. Light in the range of 620 to 750 nm may be beneficial for local circulation enhancement or restoration of connective tissue. Light in the range of 400 to 500 nm may provide a bactericidal effect. Light in the range of 560 to 600 nm may stimulate tissue rejuvenation. Each of these types of light may be applied.

[0390]

[0375] Light may be monochromatic or polychromatic. Light may be applied in pulses with a duration in the range of 0. 1 ps to 10000 ms, or in the range of 1 ps to 5000 ms, or in the range of 2 ps to 2500 ms, or in the range of 5 ps to 1000 ms.

[0391]

[0376] The energy flux provided by the light may be in the range of 0.005 W-cm2to 75 W- cm2, or in the range of 0.01 W-cm2to 60 W-cm2, or in the range of 0.01 W-cm2to 50 W-cm2. Methods and devices may also include a spot size defined as an area of the surface of tissue treated by the light. The spot size may be in the range of 0.01 cm2to 1000 cm2, or in the range of 0.05 cm2to 800 cm2, or in the range of 0.1 cm2to 750 cm2.

[0392]

[0377] In another aspect, the device may comprise different pressure generators, e.g. one for positive pressure and second one for negative pressure generation.

[0393]

[0378] The pressure generator may be connected to the therapy unit by at least one fluid conduit (e.g. pipe, tube, or hose) or a system of fluid conduits.

[0394]

[0379] The pressure generator may be able to provide periods of various pressures in the therapy unit. The therapy unit may comprise periods of various pressures.

[0395]

[0380] The pressure generator may provide an alternating pressure, such that a first period of non- atmospheric pressure is followed by a second period of non -atmospheric pressure. The first period of non-atmospheric pressure may be a period with a pressure higher than atmospheric pressure. The second period of the non-atmospheric pressure may be a period with pressure lower than atmospheric pressure.

[0396]

[0381] The first period of non-atmospheric pressure may have a duration in a range from 0.001 seconds to 10,000 seconds, or from 0.1 seconds to 5000 seconds, or from 1 second to 1000 seconds, or from 1 second to 500 seconds, or from 1 second to 100 seconds, or from 1 second to 50 seconds, or from 1 second to 20 seconds, or from 5 seconds to 15 seconds, or from 1 seconds to 10 seconds, or from 1 second to 5 seconds, or from 2 seconds to 4 seconds, or from 5 to 10 seconds, or from 5 to 25 seconds, or from 3 to 7 seconds, or from 8 to 14 seconds, or from 10 to 60 seconds, or from 10 to 40 seconds, or from 10 to 30 seconds, or from 10 to 25 seconds, or from 10 to 20 seconds, or from 10 to 16 seconds.

[0397]

[0382] The second period of non-atmospheric pressure may have a duration in a range from 0.001 seconds to 10,000 seconds, or from 0.1 seconds to 5000 seconds, or from 1 second to 1000 seconds, or from 1 second to 500 seconds, or from 1 second to 100 seconds, or from 1 second to 50 seconds, or from 1 second to 20 seconds, or from 5 seconds to 15 seconds, or from 1 seconds to 10 seconds, or from 1 second to 5 seconds, or from 2 seconds to 4 seconds, or from 5 to 10 seconds, or from 5 to 25 seconds, or from 3 to 7 seconds, or from 8 to 14 seconds, or from 10 to 60 seconds, or from 10 to 40 seconds, or from 10 to 30 seconds, or from 10 to 25 seconds, or from 10 to 20 seconds, or from 10 to 16 seconds.

[0398]

[0383] The pressure difference between the pressure in the therapy chamber of the therapy unit 105 and the atmospheric pressure in the first period of non-atmospheric pressure may be in a range from 0.001 kPa to 1000 kPa, from 0.01 kPa to 1000 kPa, or from 0.01 kPa to 100 Pa, or from 0.1 kPa to 100 kPa, or from 1 kPa to 100 kPa, or from 1 kPa to 20 kPa, or from 10 kPa to 50 kPa, or from 5 kPa to 20 kPa, or from 10 kPa to 15 kPa, or from 20 kPa to 30 kPa.

[0384] The pressure difference between the pressure in the therapy chamber of the therapy unit 105 and the atmospheric pressure in the second period of non-atmospheric pressure may be in a range from 0.001 kPa to 1000 kPa, from 0.01 kPa to 1000 kPa, or from 0.01 kPa to 100 Pa, or from 0.1 kPa to 100 kPa, or from 1 kPa to 100 kPa, or from 1 kPa to 20 kPa, or from 10 kPa to 50 kPa, or from 5 kPa to 20 kPa, or from 10 kPa to 15 kPa, or from 20 kPa to 30 kPa.

[0399]

[0385] The pressure generator may provide an alternating pressure, such that a first period of non- atmospheric pressure is followed by a second period of atmospheric pressure. In one aspect, the first period of the non-atmospheric pressure may be a period with a pressure higher than the atmospheric pressure (hyperbaric period). In another aspect, the first period of the non-atmospheric pressure may be a period with pressure lower than the atmospheric pressure (hypobaric period).

[0400]

[0386] The first period of non-atmospheric pressure may have a duration in a range from 0.001 seconds to 10,000 seconds, or from 0.1 seconds to 5000 seconds, or from 1 second to 1000 seconds, or from 1 second to 500 seconds, or from 1 second to 100 seconds, or from 1 second to 50 seconds, or from 1 second to 20 seconds, or from 5 seconds to 15 seconds, or from 1 second to 10 seconds, or from 1 second to 5 seconds, or from 2 seconds to 4 seconds, or from 5 to 10 seconds, or from 5 to 25 seconds, or from 3 to 7 seconds, or from 8 to 14 seconds, or from 10 to 60 seconds, or from 10 to 40 seconds, or from 10 to 30 seconds, or from 10 to 25 seconds, or from 10 to 20 seconds, or from 10 to 16 seconds.

[0401]

[0387] The second period of atmospheric pressure may have a duration in a range from 0.001 seconds to 10,000 seconds, or from 0.1 seconds to 5000 seconds, or from 1 second to 1000 seconds, or from 1 second to 500 seconds, or from 1 second to 100 seconds, or from 1 second to 50 seconds, or from 1 second to 20 seconds, or from 5 seconds to 15 seconds, or from 1 second to 10 seconds, or from 1 second to 5 seconds, or from 2 seconds to 4 seconds, or from 5 to 10 seconds, or from 5 to 25 seconds, or from 3 to 7 seconds, or from 8 to 14 seconds, or from 10 to 60 seconds, or from 10 to 40 seconds, or from 10 to 30 seconds, or from 10 to 25 seconds, or from 10 to 20 seconds, or from 10 to 16 seconds.

[0402]

[0388] The pressure difference between the pressure in the therapy chamber of the therapy unit 105 and the atmospheric pressure in the first period of non-atmospheric pressure may be in a range from 0.001 kPa to 1000 kPa, from 0.01 kPa to 1000 kPa, or from 0.01 kPa to 100 Pa, or from 0.1 kPa to 100 kPa, or from 1 kPa to 100 kPa, or from 1 kPa to 20 kPa, or from 10 kPa to 50 kPa, or from 5 kPa to 20 kPa, or from 10 kPa to 15 kPa, or from 20 kPa to 30 kPa.

[0403]

[0389] The pressure generator may provide an alternating pressure, such that a first period of non- atmospheric pressure is followed by a second period of non-atmospheric pressure. The first period of non-atmospheric pressure may be a period with a pressure higher than atmospheric pressure and the second period of non-atmospheric pressure may be a period with a pressure higher than atmospheric pressure, wherein the pressure of the second period may be different from the first period of non- atmospheric pressure. In another aspect of the invention, the first period of non-atmospheric pressure may be a period with a pressure lower than atmospheric pressure and the second period of non- atmospheric pressure may be a period with pressure lower than atmospheric pressure, wherein the pressure of the second period may be different from the first period of non-atmospheric pressure.

[0404]

[0390] In one aspect of the invention, the pressure generator may provide an alternating pressure cycle comprising three periods, such that a first period of non-atmospheric pressure is followed by a second period of atmospheric pressure and a third period of non-atmospheric pressure. The first period may have a duration Tl, the second period may have duration T2, and the third period may have duration T3. The duration of the first period Tl may the be same or different than the duration of the second period T2, the duration of the second period T2 may be the same or different than the duration of the third period T3, and the duration of the first period Tl may be the same or different than the duration of the third period T3.

[0405]

[0391] The duration of at least one of Tl, T2 or T3 may be in a range from 0.001 seconds to 10,000 seconds, or from 0.1 seconds to 5000 seconds, or from 1 second to 1000 seconds, or from 1 second to 500 seconds, or from 1 second to 100 seconds, or from 1 second to 50 seconds, or from 1 second to 20 seconds, or from 5 seconds to 15 seconds, or from 1 second to 10 seconds, or from 1 second to 5 seconds, or from 2 seconds to 4 seconds, or from 5 to 10 seconds, or from 5 to 25 seconds, or from 3 to 7 seconds, or from 8 to 14 seconds, or from 10 to 60 seconds, or from 10 to 40 seconds, or from 10 to 30 seconds, or from 10 to 25 seconds, or from 10 to 20 seconds, or from 10 to 16 seconds.

[0406]

[0392] The applicator may comprise one or more contact surfaces or chambers configured to form a sealed interface with the treated tissue region. The contact surface may be planar, concave, convex, or anatomically contoured to correspond to the body region being treated. The applicator may further include flexible sealing elements, valves, and / or pressure ports adapted for dynamic alternation between suction and compression phases. In one aspect of the invention, the applicator may include transparent or optical windows to enable visual or optical monitoring of tissue deformation during therapy.

[0407]

[0393] The magnitude of negative pressure applied to the tissue may range from -100 kPa to -5 kPa, or preferably from -80 kPa to -20 kPa. The magnitude of positive compression pressure may range from +5 kPa to +50 kPa, or preferably from +10 kPa to +30 kPa. The frequency of vacuum-compression cycles may range from 0.05 Hz to 10 Hz, or preferably from 0.1 Hz to 5 Hz. The duration of individual suction or compression phases may vary from 0.1 s to 10 s, or from 0.2 s to 5 s, or from 0.5 s to 2 s.

[0408]

[0394] In one aspect, the system may be configured to deliver vacuum and compression phases in sequential, alternating, or overlapping cycles. The control unit may regulate the cycle amplitude, frequency, waveform (for example sinusoidal, trapezoidal, or pulsed), and total duration according to tissue type and desired treatment outcome. The system may further include feedback sensors for monitoring applied pressure, tissue deformation, and temperature, with real-time adjustment of operational parameters to maintain safe and consistent therapy conditions.

[0409]

[0395] In one aspect, the alternating negative and positive pressure may mechanically modulate local perfusion, capillary dilation, and interstitial fluid dynamics, leading to transient increases in tissue permeability and oxygenation. These physiological effects may improve uptake and receptor-mediated activity of the administered FGF-2 variant, enhance fibroblast and endothelial cell activity, and accelerate tissue regeneration.

[0410]

[0396] In one aspect, the vacuum-compression system may optionally integrate other physical modalities, including radiofrequency, pulsed electric field, ultrasound, or photobiomodulation energy sources, within a single multifunctional applicator. The control unit may synchronize pressure cycles with the energy delivery timing to further enhance tissue permeability, perfusion, and absorption of the FGF-2 variant.

[0411]

[0397] In one aspect, the combined application of vacuum-compression therapy and the FGF-2 variant may result in synergistic biological outcomes, including stimulation of angiogenesis, activation of fibroblast proliferation, accelerated epithelial recovery, and reduction of inflammation and edema. The therapy may improve skin texture, elasticity, and hydration, and may reduce post-procedural downtime in treatment applications, e.g. cosmetic and regenerative applications.

[0412]

[0398] In one aspect, the vacuum-compression system may comprise one or more sensors configured to monitor pressure, tissue displacement, impedance, or contact quality between the applicator and the tissue surface. The control unit may process sensor feedback in real time to automatically adjust negative and positive pressure amplitudes, cycle frequency, or duration to ensure consistent energy delivery and maintain patient comfort. The system may also include automatic leak detection or coupling verification to guarantee stable pressure conditions throughout the procedure.

[0413]

[0399] In one aspect of the invention, a method for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) applying cyclic vacuum and compression forces to the tissue; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration of the FGF-2 variant into deeper tissue layers, promote and / or accelerate tissue regeneration, and reduce post-procedure recovery time.

[0414]

[0400] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. in cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) applying cyclic vacuum and compression forces to the tissue;

[0415]

[0401] wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration of the FGF-2 variant into deeper tissue layers, promote and / or accelerate tissue regeneration, and reduce post -procedure recovery time.

[0416]

[0402] In one aspect, the invention relates to the delivery of photobiomodulation (PBM) energy, provided by a light-emitting source such as light-emitting diodes (LEDs) or other light -based devices, to epithelial and / or connective tissues. The PBM energy may be applied to induce controlled photochemical and photothermal effects, resulting in increased cellular metabolism, microcirculation, and tissue oxygenation, thereby enhancing the biological activity and effectiveness of an FGF-2 variant.

[0417]

[0403] In one aspect, the invention relates to the application of photobiomodulation (PBM) energy to epithelial tissues to stimulate cellular activity and enhance the biological response to an FGF-2 variant. The application of light in the visible to near-infrared spectrum (400-1200 nm) may be absorbed by mitochondria within epidermal cells, leading to an increase in mitochondrial activity and ATP production. This may result in enhanced cellular metabolism and an increase in the proliferative capacity of keratinocytes in the basal layer of the epidermis.

[0418]

[0404] The increased ATP production and improved cellular activity may also upregulate the expression of FGFRs, making epidermal cells more responsive to external growth factors such as FGF- 2. This synergistic effect facilitates greater receptor-mediated uptake of the FGF-2 variant, enhancing its biological activity. The combination of enhanced metabolic activity, increased receptor expression, and efficient FGF-2 uptake may result in accelerated regeneration of the epidermis, improved skin texture, and faster healing times for damaged and / or aging skin.

[0419]

[0405] In one aspect of the invention, the system for photobiomodulation energy delivery may comprise one or more light sources configured to emit light in continuous or pulsed mode, a control unit, a power supply unit, an applicator or light -emitting panel, and a user interface. Optionally, the system may include a data storage unit, a cooling or ventilation module, and a fluid delivery unit for administering the FGF-2 variant before, during, or after PBM treatment. The control unit may regulate the wavelength, pulse frequency, intensity, and duration of the emitted light according to predefined treatment parameters.

[0420]

[0406] The light source may be selected from the group consisting of red, infrared, near-infrared, blue, green, or mixed-spectrum LEDs, laser diodes, or other coherent and non-coherent light sources. The wavelength of the emitted light may be in a range from 400 nm to 1200 nm, in a range from 450 nm to 1100 nm, or in a range from 600 nm to 1000 nm. In one example, red light may be delivered in the range of 620 nm to 750 nm, near-infrared light in the range of 800 nm and 950 nm, and blue light in the range of 450 nm and 495 nm. The emission may be monochromatic, polychromatic, or broadband, depending on the desired biological response.

[0421]

[0407] The optical power density (irradiance) of the emitted PBM energy may be in a range from 1 mW / cm2to 500 mW / cm2, in a range from 5 mW / cm2to 200 mW / cm2, or in a range from 10 mW / cm2to 100 mW / cm2. The energy density (fluence) applied to the tissue may range from 0.1 J / cm2to 100 J / cm2, preferably from 0.5 J / cm2to 50 J / cm2, or more preferably from 1 J / cm2to 20 J / cm2. The total treatment duration may range from 10 seconds to 30 minutes, depending on target depth, wavelength, and desired biological effect.

[0422]

[0408] In one aspect, the system may be configured to deliver photobiomodulation energy in continuous wave or pulsed mode. In the pulsed mode, the duty cycle may range from 5% to 90%, preferably from 10% to 70%. The pulse repetition frequency may be in a range from 0.1 Hz to 10 kHz, in a range from 0.5 Hz to 5 kHz, or in a range from 1 Hz to 1 kHz. The control unit may modulate the pulse width, frequency, and intensity to optimize photonic stimulation while avoiding excessive heating or phototoxicity.

[0423]

[0409] In one aspect, the applicator or light -emitting panel may include arrays of LEDs arranged in planar, curved, or flexible geometries to conform to the surface of the body. The applicator may further comprise an optical diffuser, collimator, or lens system to ensure uniform illumination. The contact surface may include a transparent or semi-transparent membrane impregnated with the FGF-2 variant or with a biocompatible carrier formulation that may release the FGF-2 variant under light exposure or mild heating.

[0424]

[0410] In one aspect, the PBM energy may be applied sequentially or simultaneously with the administration of the FGF-2 variant. Light exposure may enhance mitochondrial cytochrome c oxidase activity, increase ATP production, and upregulate intracellular calcium signaling, thereby priming cells for receptor-mediated response to FGF-2. The combined treatment may result in enhanced fibroblast proliferation, collagen synthesis, and overall tissue rejuvenation compared to PBM or FGF-2 monotherapy.

[0425]

[0411] In one aspect, the photobiomodulation therapy may employ distinct wavelength combinations or sequential irradiation cycles to target multiple biological layers. For example, shorter wavelengths (blue and green) may act superficially to reduce inflammation and microbial load, whereas longer wavelengths (red and near-infrared) may penetrate deeper layers to stimulate fibroblast and endothelial activity, thereby supporting the synergistic effects of FGF-2 on dermal regeneration.

[0426]

[0412] In one aspect of the invention, a method for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering photobiomodulation energy using at least one light source emitting in the visible or nearinfrared range; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration of the FGF-2 variant into deeper tissue layers and promote and / or accelerate tissue regeneration, rejuvenation, and / or reduction of inflammation and oxidative stress.

[0427]

[0413] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering photobiomodulation energy using at least one light source emitting in the visible or nearinfrared range; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration of the FGF-2 variant into deeper tissue layers and promote and / or accelerate tissue regeneration, rejuvenation, and / or reduction of inflammation and oxidative stress.

[0428]

[0414] In one aspect, the invention relates to the application of electromagnetic fields (EMF), pulsed electromagnetic fields (PEMF), or magnetic fields to epithelial and / or connective tissues to induce controlled electrical and magnetic stimulation, thereby enhancing cellular activity and potentiating the biological effect of an FGF-2 variant. The electromagnetic stimulation may modulate ion channel activity, transmembrane potential, and intracellular signaling cascades that contribute to tissue regeneration and repair.

[0415] The magnetic field and / or RF field may be characterized by intensity. In the case of a magnetic field, the intensity may include magnetic flux density or amplitude of magnetic flux density. In the case of an RF field, the intensity may include energy flux density of the RF field or RF waves.

[0429]

[0416] In one aspect of the invention, the system for electromagnetic field stimulation may comprise one or more electromagnetic field or magnetic field generators, a control unit, a power supply unit, an applicator comprising one or more inductive coils, and a user interface. Optionally, the system may include a data storage module, temperature sensors, or impedance sensors for real-time feedback control. The control unit may regulate field intensity, pulse shape, repetition frequency, and exposure duration according to treatment parameters.

[0430]

[0417] One or more applicators and / or the additional treatment devices may include a magnetic field generating device (e.g. a magnetic coil), generating a magnetic field for a magnetic treatment. The magnetic field generating device may generate an RF field for the RF treatment. The electromagnetic field may produce frequencies with far different values. The magnetic field generating device may produce a dominant magnetic field vector for the magnetic treatment during lower frequencies of the produced electromagnetic field. Alternatively, the magnetic field generating device may produce a dominant electromagnetic field vector for the magnetic treatment during higher frequencies of electromagnetic field which may be used for the RF treatment. The magnetic field generating device in the high frequency electromagnetic field domain may provide an RF field similar to the RF field provided by the RF electrode. When one magnetic field generating device is used for providing both the RF treatment and the magnetic treatment, the difference between frequencies for the RF treatment and the magnetic treatment production may be in a range from 500 kHz to 5 GHz, or from 500 kHz to 2.5 GHz, or from 400 kHz to 800 kHz, or from 2 GHz to 2.5 GHz. Also, when one magnetic field generating device is used for providing both the RF treatment and the magnetic treatment, the frequencies for the RF treatment may correspond with frequencies in the range of 100 kHz to 3 GHz, 400 kHz to 900 MHz, or 500 kHz to 3 GHz.

[0431]

[0418] One or more applicators and / or additional treatment devices may include one or more RF electrodes and one or more magnetic field generating devices, wherein the RF electrodes have different characteristics, structure and / or design than the magnetic field generating device. The RF electrode may operate as a unipolar electrode, monopolar electrode, bipolar electrode, and / or as a multipolar electrode. One or more RF electrodes may be used for capacitive and / or inductive heating of a biological structure or body area.

[0432]

[0419] The applicator may include two bipolar RF electrodes. The bipolar electrodes may transfer the RF field between the two bipolar RF electrodes located in at least one applicator. Bipolar electrodes may increase safety and targeting of provided RF treatment, as compared to electrodes of monopolar type. Bipolar electrodes may provide electromagnetic field passing through a patient’s tissue located around and between RF electrodes, wherein due to impedance matching, it is possible to prevent the creation of standing electromagnetic waves in the patient’s tissue and prevent unwanted thermal injury of nontargeted tissue. Also, the distance between bipolar electrodes influences the depth of RF wave penetration allowing for enhanced targeting of the RF treatment.

[0433]

[0420] The applicator may include a monopolar RF electrode or additional monopolar electrodes. Monopolar electrodes may transfer radiofrequency energy between an active electrode and a passive electrode, wherein the active electrode may be part of the applicator and the passive electrode having larger surface area may be located at least 5 cm, 10 cm, or 20 cm from the applicator. A grounded electrode may be used as the passive electrode. The grounded electrode may be on the opposite side of the patient’s body to where the applicator is attached.

[0434]

[0421] The magnetic treatment may be provided by the magnetic field generating device. The magnetic field generating device may be made from a conductive material, such as a metal, for example copper. The magnetic field generating device may be formed as a coil of a variety of sizes and shapes. The magnetic field generating device may be a coil of multiple windings wherein one loop of the coil may include one or more wires. An individual loop of one or more wires may be insulated from the other turns or loops of one or more wires. Each loop of wiring in a magnetic coil may be called a turn. Further, individual wires in one turn or loop may be insulated from each other. The shape of the magnetic field generating device may be optimized with regard to the applicator size and design. The coil may be wound in order to match at least part of the applicator’s shape according to the applicator’s floor projection. The coil winding may be at least partially circular, oval and / or may have any other shape that matches the shape of the applicator or a portion thereof. The loops of winding may be stacked on top of each other, may be arranged side by side, or stacking of the winding may be combined side by side and on top of other windings. The coil may be flat.

[0435]

[0422] The total number of protrusions in one RF electrode regardless of the parallel cuts may be in the range of 5 to 1000, or of 10 to 600, or of 20 to 400, or of 50 to 400, or of 100 to 400 or of 15 to 200, or of 30 to 100, or of 40 to 150, or of 25 to 140.

[0436]

[0423] The total number of apertures or cutouts in one RF electrode regardless of the parallel cuts may be in the range of 5 to 1000, or of 10 to 600, or of 20 to 400, or of 50 to 400, or of 100 to 400 or of 15 to 200, or of 30 to 100, or of 40 to 150, or of 25 to 140.

[0437]

[0424] The number of apertures, cutouts and / or protrusions in one RF electrode located below the coil including its core may be in a range of 5 to 1000, or of 10 to 600, or of 20 to 400, or of 50 to 400, or of 100 to 400 or of 15 to 200, or of 30 to 100, or of 40 to 150, or of 25 to 140.

[0438]

[0425] The number of individual protrusions included in one RF electrode may be in a range of 1 to 8000, or of 2 to 8000, or of 5 to 8000, or of 3 to 5000, or of 5 to 1000, or of 5 to 500, or of 10 to 500, or of 5 to 220, or of 10 to 100, in an area of size 2 cm multiplied by 1 cm.

[0426] The electrode area of one or more RF electrodes in one applicator or one additional treatment device may be in a range from 1 cm2to 2500 cm2, or 25 cm2to 800 cm2, or 30 cm2to 600 cm2, or 30 cm2to 400 cm2, or from 50 cm2to 300 cm2, or from 40 cm2to 200 cm2according to the applicator’s floor projection.

[0439]

[0427] The magnetic induction produced by the system may range from 0.1 mT to 200 mT, preferably from 0.5 mT to 100 mT, or from 1 mT to 50 mT. The frequency of the applied field may range from 1 Hz to 100 kHz, preferably from 5 Hz to 50 kHz, or more preferably from 10 Hz to 20 kHz. The waveform of the electromagnetic pulses may be sinusoidal, square, trapezoidal, or exponentially decaying, with pulse durations ranging from 1 ps to 100 ms.

[0440]

[0428] The RF electrodes may be made of specific conductive materials reducing induction of unwanted physical effects in the RF electrode. Such materials may have relative permeability in a range of 4 to 1,000,000, or of 20 to 300,000, or of 200 to 250,000, or of 300 to 100,000, or of 300 to 18,000, or of 1,000 to 8,000. The material of the RF electrode may include carbon, aluminum, copper, nickel, cobalt, manganese, zinc, iron, titanium, silver, brass, platinum, palladium and / or others from which \alloys may be created, such as Mu-metal, permalloy, electrical steel, ferritic steel, ferrite, stainless steel, or the like. In addition, the RF electrode may be made from mixed metal oxides, fixed powder from metal oxides, or metal from m-metal elements to minimize induction of eddy currents and heating of the RF electrode, and also in order to minimize energy loss of time-varying magnetic field.

[0441]

[0429] In one aspect of the invention, the electromagnetic field may be applied as a static, alternating, or pulsed field. Static magnetic fields may produce constant low-level magnetomechanical stimulation, whereas pulsed or alternating fields may induce microcurrents and electromechanical effects in tissues. The electromagnetic field may be delivered via surface coils, Helmholtz-type coils, or flexible applicators configured to conform to the anatomical contours of the treated area.

[0442]

[0430] In one aspect of the invention, the system may be configured to deliver electromagnetic stimulation in combination with the administration of an FGF-2 variant. The magnetic field may be applied before, during, or after the compound administration to enhance cellular uptake and receptor activation. Exposure to electromagnetic fields may upregulate fibroblast growth factor receptors (FGFR1-3), vascular endothelial growth factor (VEGF), and nitric oxide (NO) signaling, resulting in enhanced microcirculation and metabolic activity.

[0443]

[0431] In one aspect of the invention, the electromagnetic field system may be configured for localized or whole -body treatment. Localized applicators may target specific anatomical regions such as the face, scalp, or joints, while whole-body systems may use multiple coils arranged within a mat, chamber, or wearable structure. The exposure duration may range from 1 minute to 60 minutes, or from 5 minutes to 30 minutes, depending on the treatment area and desired biological effect.

[0432] In one aspect of the invention, the system may allow modulation of field parameters during treatment, including pulse intensity, frequency, and polarity, based on real-time sensor feedback. The control unit may implement adaptive algorithms to maintain optimal magnetic flux density and minimize tissue overheating. The system may also include electromagnetic shielding and grounding mechanisms to ensure patient safety and electromagnetic compatibility.

[0444]

[0433] In one aspect of the invention, the combination of electromagnetic field stimulation and administration of an FGF-2 variant may produce synergistic biological effects including enhanced fibroblast proliferation, improved collagen and elastin synthesis, angiogenesis, and accelerated wound healing. The exposure to magnetic fields may modulate reactive oxygen species (ROS) balance and calcium ion homeostasis, sensitizing cells to the action of FGF-2 and amplifying its treatment benefits, e.g. regenerative and cosmetic benefits.

[0445]

[0434] In one aspect of the invention, electromagnetic field stimulation may be used in combination with other physical modalities, including radiofrequency, ultrasound, photobiomodulation, or vacuumcompression therapy, to achieve multimodal synergistic stimulation. The control unit may synchronize electromagnetic pulses with other energy sources and with the administration of the FGF-2 variant to maximize therapeutic efficacy and uniform tissue activation.

[0446]

[0435] In one aspect of the invention, a method for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) applying electromagnetic or pulsed electromagnetic field stimulation to the tissue; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration, bioavailability, and biological activity of the FGF-2 variant, leading to accelerated tissue regeneration, improved elasticity, and enhanced cosmetic outcomes.

[0447]

[0436] In one aspect of the invention, a method of use an FGF-2 variant, e.g. for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) applying electromagnetic or pulsed electromagnetic field stimulation to the tissue; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration, bioavailability, and biological activity of the FGF-2 variant, leading to accelerated tissue regeneration, improved elasticity, and enhanced cosmetic outcomes.

[0437] In one aspect of the invention, controlled delivery of cryogenic and / or thermal energy to epithelial and / or connective tissues to induce reversible temperature variations is provided to modulate cellular metabolism, perfusion, and / or permeability to enhance the biological effect of an FGF-2 variant. Controlled thermal cycling between cooling and heating phases may improve microcirculation, stimulate fibroblast activity, and facilitate diffusion and receptor activation of the administered FGF-2 variant.

[0448]

[0438] In one aspect of the invention, the application of cryogenic therapy may synergistically enhance the biological effectiveness of an FGF-2 variant by transiently reducing inflammation, decreasing edema, and stimulating vasodilation following the cooling phase. The cooling phase may reduce local tissue temperature to between, for example, -10 °C and +5 °C, causing vasoconstriction followed by reperfusion upon re -warming. This microcirculatory response may promote enhanced blood flow, oxygenation, and the delivery of an FGF-2 variant into the treated tissue. Additionally, the cooling phase may transiently increase cellular permeability in the epidermis, facilitating improved transdermal penetration of the FGF-2 variant. The synergistic effect of cryogenic therapy and delivery of an FGF-2 variant may ultimately improve skin elasticity, firmness, texture, and overall appearance.

[0449]

[0439] In one aspect of the invention, thermal modulation therapy may enhance the biological response to an FGF-2 variant by increasing dermal temperature and stimulating fibroblast activity. When tissue is exposed to temperatures ranging from 38 °C to 45 °C, controlled thermal heating induces collagen denaturation and remodeling, which provides immediate tissue tightening effects. Thermal modulation also increases the permeability of the extracellular matrix (ECM), creating a favorable environment for the delivery of FGF-2 deeper into the dermal layers. This heat-induced change in tissue structure facilitates enhanced penetration and bioavailability of the FGF-2 variant. The synergistic effect of thermal modulation and delivery of an FGF-2 variant may ultimately improve skin elasticity, firmness, texture, and overall appearance.

[0450]

[0440] In one aspect, the present invention relates to a device that includes one or more thermoelectric materials, or at least one thermal adjustment apparatus, placed near a surface, such as the skin of a user. The device may be configured to generate a series of thermal pulses in succession at the surface. This thermal pulsing, when suitably applied, may result in an enhanced thermal sensation for a user which, in some cases, may provide the user with a more pleasurable thermal experience than would otherwise be the case without the thermal pulsing.

[0451]

[0441] As further described herein, a thermal pulse may include a transient, reversible temperature change at a surface, where the temperature changes from an initial temperature to a second temperature, quickly followed by a return temperature change at the surface from the second temperature back to the initial temperature, or a temperature close to the initial temperature, all over a relatively short period of time (e.g., less than 120 seconds, or shorter).

[0442] For example, a thermal pulse may include a first temperature adjustment at a surface from a first temperature to a second temperature (e.g., at an average rate of 0.1 °C-10.0 °C. / second), and a second temperature adjustment at the surface from the second temperature to a third temperature (e.g., also at an average rate of 0.1 °C-10.0 °C. / second). In such a thermal pulse, the difference in magnitude between the first temperature and the third temperature may be less than 25% of the difference in magnitude between the first temperature and the second temperature. Further, in some cases, the magnitude of the first average rate may be greater than the magnitude of the second average rate.

[0452]

[0443] In some aspects, the invention is based on the unexpected discovery that varying the temperature at the surface of human skin in a certain manner, for example, by generating thermal pulses according to particular temperature profiles, may give rise to an enhanced heating or cooling effect for the individual. This enhanced thermal effect may be perceived by the individual in a way that is more pronounced when the temperature is pulsed back and forth in a reversible manner at the surface under short time durations (e.g., less than 120 seconds, less than 30 seconds), in comparison to if the temperature is gradually changed from one temperature to another at the surface over longer periods of time (e.g., over several minutes or hours). That is, when subject to thermal pulses in accordance with embodiments of the present disclosure, the perceived strength of this heating / cooling effect is comparable to actual changes in temperature that are much larger in magnitude and which may be applied, for example, at steady state.

[0453]

[0444] In some aspects, the invention is based on the unexpected discovery that, in particular, by generating thermal pulses at the surface of human skin having particular combinations of parameters, such as rates of temperature change, magnitudes of temperature change, pulse duration, etc., as described in more detail herein, the effects of adaptive desensitization are mitigated or otherwise reduced, and the perceived effects of cooling and / or heating are enhanced. As compared to the desensitization that may occur in a cooled or heated room, the devices described herein may continuously provide a user with an enhanced thermal experience, e.g., a pleasant feeling of cooling and / or heating, according to the preferences of the user. As noted above, due to the manner in which the thermal pulse is generated, when the device is in operation, a user may experience, or feel, a temperature sensation that is perceived to be greater in magnitude as compared to the actual magnitude in temperature change of the device at the surface of the skin.

[0454]

[0445] Each of the first and second temperature adjustments may be characterized by an average rate of between about 0.1 °C / sec and about 10.0 °C / sec. In some cases, the magnitude of the average rate of the first temperature adjustment is greater than the magnitude of the average rate of the second temperature adjustment. That is, the time period under which the surface adjacent the thermal adjustment apparatus to thermally relax or otherwise adjust from the second (pulsed) temperature to the third (return) temperature may be longer than the time period for the surface to initially step from the first (initial) temperature to the second (pulsed) temperature.

[0455]

[0446] In some aspects, each thermal pulse generated by the device may last for a time period of less than 120 seconds (e.g., 1-30 seconds) and may include a first initial temperature adjustment at a region of the thermoelectric material(s) (or suitable thermal adjustment apparatus) adjacent the surface from a first (initial) temperature to a second (pulsed) temperature, and a second return temperature adjustment at the region adjacent the surface, from the second (pulsed) temperature to a third (return) temperature.

[0456]

[0447] In one aspect, the system for cryogenic therapy and thermal energy may comprise a thermal energy generation unit, a control unit, an applicator with a temperature-controlled contact surface, a power supply unit, and a user interface. Optionally, the system may further include a cooling medium reservoir, a heating element, temperature sensors, and a data storage unit. The thermal modulation may be based on thermoelectric (Peltier) elements, circulating fluid systems, resistive heating elements, or cryogenic gas expansion.

[0457]

[0448] The applicator may include one or more contact surfaces or plates configured for thermal exchange with the tissue. The contact surfaces may be metallic, ceramic, or polymeric and may include integrated temperature sensors to monitor tissue-surface interface temperature. The geometry of the applicator may be planar, concave, convex, or anatomically contoured for optimal contact with the skin or mucosal tissue. The system may further comprise a flexible membrane or hydrogel layer acting as both a thermal interface and a carrier for an FGF-2 variant.

[0458]

[0449] In one aspect, the cooling mode may be configured to decrease tissue temperature to between -20 °C and +15 °C, preferably between -10 °C and +10 °C, or between 0 °C and +5 °C. The heating mode may increase tissue temperature to between +35 °C and +50 °C, preferably between +37 °C and +45 °C. The rate of temperature change may be between 0.1 °C / s and 10 °C / s. The duration of individual cooling or heating cycles may range from 1 s to 120 s, depending on the targeted tissue depth and physiological response.

[0459]

[0450] In one aspect, the system may be configured to perform cyclic thermal modulation, including alternating cooling and heating phases with controlled dwell times. The cycle frequency may range from 0.01 Hz to 5 Hz, preferably from 0.05 Hz to 1 Hz. The waveform of the thermal modulation may be sinusoidal, square, trapezoidal, or customized to achieve desired temperature gradients within the treated tissue volume.

[0460]

[0451] In one aspect, the control unit may include feedback algorithms to dynamically regulate the temperature of the applicator and tissue surface based on sensor data. Parameters such as surface temperature, tissue impedance, and perfusion rate may be monitored in real time, allowing adaptive control of power output, cycle duration, or phase ratio between cooling and heating to maintain safety and reproducibility of treatment conditions.

[0461]

[0452] In one aspect, cryogenic stimulation may additionally reduce local inflammation, edema, and nociception, creating favorable conditions for post-procedural recovery. Heating, on the other hand, may enhance collagen remodeling and fibroblast proliferation. The alternating or combined use of cooling and heating may therefore produce synergistic regenerative and cosmetic effects in combination with FGF-2 administration.

[0462]

[0453] In one aspect, the combined application of cryogenic therapy and / or thermal energy and an FGF-2 variant may result in accelerated tissue regeneration, enhanced fibroblast proliferation, improved collagen organization, and reduced inflammation. The procedure may further improve the aesthetic appearance of the skin by increasing elasticity, smoothness, and hydration, and by shortening posttreatment recovery time compared to conventional monotherapies.

[0463]

[0454] In one aspect, the cryogenic therapy and thermal energy system may be configured to deliver spatially selective temperature control, wherein deeper tissue layers are subjected to controlled heating while the epidermal surface is simultaneously cooled. This configuration allows targeted stimulation of dermal fibroblasts and collagen fibers without causing thermal injury to the epidermis, thus ensuring both safety and enhanced therapeutic outcomes.

[0464]

[0455] In one aspect, the applicator may include independent heating and cooling elements operating in coordinated or opposing phases. The heating element may be configured to generate thermal energy in the range of +37 °C to +50 °C, targeting tissue depths of 1 mm to 5 mm, while the cooling element may maintain the epidermal surface temperature between +5 °C and +25 °C. The control unit may dynamically regulate both temperature zones in real time based on sensor feedback to achieve a stable temperature gradient between the superficial and deeper tissue layers.

[0465]

[0456] In one aspect, the selective heating of deeper dermal layers may induce collagen denaturation and controlled remodeling, stimulate fibroblast proliferation, and promote angiogenesis. Simultaneous cooling of the epidermis may prevent discomfort, erythema, or epidermal dehydration, maintaining the integrity of the skin barrier during treatment. The combination of these effects creates an optimal physiological environment for enhanced absorption and receptor -mediated activity of the administered FGF-2 variant.

[0466]

[0457] In one aspect of the invention, a method for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) applying controlled cryogenic therapy and / or thermal energy to the tissue; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration of the FGF-2 variant, promote or accelerate tissue regeneration, and improve post-procedural healing and cosmetic outcomes.

[0467]

[0458] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) applying controlled cryogenic therapy and / or thermal energy to the tissue; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration of the FGF-2 variant, promote or accelerate tissue regeneration, and improve post-procedural healing and cosmetic outcomes.

[0468]

[0459] In one aspect, the invention relates to the delivery of plasma-based energy, preferably cold atmospheric plasma (CAP), to epithelial and / or connective tissues to induce controlled chemical and electrical stimulation, thereby enhancing tissue regeneration and improving the biological effectiveness of an FGF-2 variant. The plasma may contain reactive oxygen and nitrogen species (ROS / RNS), electrons, ions, and photons that transiently modify the cellular microenvironment, increasing membrane permeability and activating intracellular signaling pathways associated with repair and regeneration.

[0469]

[0460] In another aspect of the invention, the reactive oxygen and / or nitrogen species generated by plasma-based energy temporarily disrupt the phospholipid bilayer of epidermal cell membranes, thereby increasing transmembrane permeability and facilitating the diffusion of an FGF-2 variant into the tissue. This may result in enhanced intracellular accumulation and biological activity of the FGF-2 variant within the cells.

[0470]

[0461] In one aspect, the device for plasma energy delivery may comprise a plasma generation unit, a power supply, a control unit, a gas source, an applicator, and a user interface. Optionally, the system may further comprise a cooling module, a data storage unit, or a fluid delivery unit for administration of an FGF-2 variant. The control unit may regulate applied voltage, current, pulse duration, frequency, and gas flow rate to achieve stable plasma discharge and reproducible energy delivery.

[0471]

[0462] The voltage applied for plasma generation may range from 0.3 kV to 35 kV, preferably from 0.8 kV to 20 kV. The operating frequency of radiofrequency may range from 0.3 kHz to 50 kHz, more preferably from 0.8 kHz to 20 kHz. The gas flow rate may range from 0.1 dm3 / min to 25 dm3 / min, more preferably in the range of 0.5 dm3 / min to 18 dm3 / min, most preferably in the range of 0.8 dm3 / min to 15 dm3 / min. The plasma temperature may be maintained below 45 °C, preferably between 25 °C and 40 °C, to prevent thermal damage and ensure comfortable contact with the tissue surface.

[0472]

[0463] The light wavelength may be in the range of 400 nm to 2200 nm, more preferably in the range of 600 nm to 2050 nm, most preferably in the range of 800 nm to 1980 nm. In some aspects of the invention, the light wavelength may be in the range of 1025 nm to 1100 nm. In some aspects of the invention, the light wavelength may be in the range of 1400 nm to 1420 nm. In some aspects of the invention, the light wavelength may be in the range of 1835 to 1940 nm. In some aspects of the invention, the light wavelength may be in the range of 1835 to 1880 nm. In some aspects of the invention, the light wavelength may be in the range of 1880 to 1940 nm. The wavelength of the applied light may be close to 254 nm, 405 nm, 450 nm, 530 nm, 560 nm, 575 nm, 640 nm, 685 nm, 830 nm and / or 1064 nm. Term "close to" refers to deviation of not more than 20 %, more preferably 15 %, most preferably 10 % from the nominal wavelength.

[0473]

[0464] Pulse energy of the light may be in the range of 0.1 mJ to 100 mJ, more preferably in the range of 0.5 mJ to 75 mJ, most preferably in the range of 1 mJ to 50 mJ. Fluence of the light beam may be in the range of 0.1 J.cm2to 3000 J.cm2, more preferably in the range of 1 J.cm2to 1500 J.cm2, most preferably in the range of 5 J.cm2to 1000 J.cm2. Pulse width may be in the range of 0.01 ms to 1500 ms, more preferably in the range of 0.1 ms to 1000 ms, most preferably in the range of 1 ms to 750 ms. When more than one laser beam is used, the individual beams may be separated by a distance (center to center) of at least 0.1 mm, 0.3 mm, 0.5 mm or 1 mm. Light spot size may be in the range of 0.001 mm2to 600 mm2, more preferably in the range of 0.012 mm2to 500 mm2, most preferably in the range of 0.01 mm2to 400 mm2.

[0474]

[0465] Pulse duration of the electric energy may be in the range 0.1 ps to 100 ps. more preferably in the range of 0.5 ps to 80 ps, most preferably in the range of 1 ps to 60 ps.

[0475]

[0466] Transferring the energy into the tissue may include application of energy by the light guide where the energy may cause thermal damage. Thermal damage may include ablation and / or coagulation of the tissue.

[0476]

[0467] The next step may be delivery of a source gas and / or secondary gas from a gas supply adjacent to the energy delivery element and generation of the plasma. A source gas may be delivered to, and plasma may be generated by, a designated plasma generator (i.e. a plasma delivery element) which is distinct and / or separated from the light guide. Alternatively, the plasma may be generated by the light (e.g. laser).

[0477]

[0468] In one aspect of the invention, the applicator may comprise a nozzle, probe, or electrode assembly configured to generate plasma and direct it toward the treatment area. The applicator may include insulating layers or dielectric barriers to maintain non-thermal plasma conditions. The distance between the plasma outlet and the tissue surface may range from 1 mm to 20 mm, or from 2 mm to 10 mm, to achieve optimal plasma exposure and reactive species delivery.

[0478]

[0469] In one aspect of the invention, the control unit may modulate plasma parameters such as voltage amplitude, pulse duration, and gas composition to adjust the ratio of reactive oxygen and nitrogen species, UV emission intensity, and ion density. Such modulation allows selective targeting of biological effects, including antimicrobial sterilization, stimulation of fibroblast proliferation, and transient permeabilization of cellular membranes facilitating penetration of the FGF-2 variant.

[0479]

[0470] In one aspect of the invention, the plasma treatment may transiently increase local concentrations of nitric oxide and reactive oxygen species, resulting in activation of angiogenic and wound-healing pathways. When administered concurrently with the FGF-2 variant, these reactive species may enhance receptor sensitivity, stimulate fibroblast and keratinocyte proliferation, and amplify the treatment effects, e.g. regenerative and cosmetic effects of the treatment.

[0480]

[0471] In one aspect of the invention, the device for delivering plasma-based energy may be configured to integrate plasma-based energy with other physical modalities, such as radiofrequency, pulsed electric field, or photobiomodulation, within a single multifunctional device. The control unit may synchronize the plasma pulses with other energy outputs or with administration of the FGF-2 variant, thereby maximizing penetration, bioavailability, and biological response.

[0481]

[0472] In one aspect of the invention, the plasma treatment may also provide antimicrobial and antiseptic benefits by inactivating bacteria, fungi, and viruses on the skin surface, thereby reducing the risk of infection after cosmetic or regenerative procedures. This effect, combined with the regenerative activity of the FGF-2 variant, may contribute to improved wound healing and reduced post -treatment inflammation.

[0482]

[0473] In one aspect of the invention, the plasma energy may be applied in a scanning or pulsed mode, with exposure times per area ranging from 0.1 s to 60 s, depending on the desired treatment intensity. The control unit may regulate repetition frequency, pulse count, and energy per pulse to achieve uniform stimulation and prevent overtreatment. In some embodiments, the plasma exposure may be synchronized with micro-dosing or spraying of an FGF-2 variant-containing formulation.

[0483]

[0474] In one aspect of the invention, a method for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering cold atmospheric plasma energy to the tissue; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration of the FGF-2 variant into deeper tissue layers and promote and / or accelerate tissue regeneration, sterilization of the surface, and overall improvement of cosmetic outcomes.

[0484]

[0475] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering cold atmospheric plasma energy to the tissue; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration of the FGF-2 variant into deeper tissue layers and promote and / or accelerate tissue regeneration, sterilization of the surface, and overall improvement of cosmetic outcomes.

[0485]

[0476] In one aspect, the invention relates to the application of oxygen-based therapy to epithelial and / or connective tissues to enhance tissue oxygenation, metabolic activity, and regenerative capacity, thereby improving the biological efficacy of an FGF-2 variant. Oxygen-based stimulation may be provided through topical oxygen diffusion, transdermal oxygen jets, oxygen -enriched liquids, or systemic hyperbaric exposure.

[0486]

[0477] In one aspect of the invention, the system for oxygen-based therapy may comprise an oxygen generation unit or storage unit, a pressure control unit, a gas delivery system, an applicator configured for oxygen dispersion or injection, a power supply unit, and a user interface. Optionally, the system may further include a cooling module, a data storage unit, and / or a fluid delivery component for concurrent administration of an FGF-2 variant. The control unit may regulate oxygen pressure, flow rate, humidity, and temperature to achieve safe and consistent oxygen delivery to the tissue.

[0487]

[0478] In one aspect of the invention, the oxygen delivery may be topical, transdermal, or systemic. In topical or transdermal configurations, oxygen may be applied as a continuous or pulsed jet, a mist, or an aerosol directed onto the skin surface. The flow rate of oxygen or oxygen -enriched air may range from 0.1 L / min to 20 L / min, preferably from 0.5 L / min to 10 L / min. The gas temperature may be maintained between 20 °C and 40 °C to ensure comfort and optimize diffusion across the stratum corneum.

[0488]

[0479] In one aspect of the invention, the oxygen therapy may be provided under normobaric or hyperbaric conditions. In hyperbaric mode, oxygen pressure within a sealed chamber or localized applicator may range from 1.2 atm to 3.0 atm, preferably from 1.3 atm to 2.5 atm. The treatment duration may vary from 5 minutes to 120 minutes, depending on the targeted tissue depth and physiological response. The increased partial pressure of oxygen enhances diffusion into interstitial spaces, promoting the bioavailability and activity of an FGF-2 variant.

[0489]

[0480] In one aspect of the invention, the oxygen delivery applicator may include one or more nozzles, diffusers, microperforated membranes, or microporous films configured to disperse oxygen evenly over the treatment area. The applicator may be planar, concave, or flexible, allowing adaptation to different body surfaces. The system may further include integrated temperature and oxygen concentration sensors to provide feedback control and maintain the desired oxygen exposure level.

[0490]

[0481] In one aspect of the invention, an FGF-2 variant may be co-administered with oxygen flow or incorporated into a carrier formulation such as an oxygenated gel, hydrogel, serum, or mist. The oxygen stream may act as a physical vector to assist the transdermal penetration of the compound through convection and micropressure effects. In another aspect of the invention, oxygen enrichment may stabilize the FGF-2 variant in its active conformation and prevent oxidative degradation of formulation components.

[0491]

[0482] In one aspect of the invention, the oxygen delivery system may be configured to integrate additional physical modalities such as radiofrequency, ultrasound, or vacuum-compression therapy within a single applicator. The control unit may synchronize oxygen flow with these energy modalities and with the timing of FGF-2 variant administration to enhance tissue perfusion, penetration, and metabolic activity.

[0492]

[0483] In one aspect of the invention, oxygen exposure may increase mitochondrial oxidative phosphorylation and ATP production, leading to enhanced cell proliferation, differentiation, and protein synthesis. These effects synergize with FGF-2 signaling by stimulating fibroblast metabolism, collagen and elastin synthesis, and angiogenesis. Improved oxygenation further accelerates wound healing, tissue regeneration, and recovery from thermal or mechanical stress.

[0493]

[0484] In one aspect of the invention, oxygen-based therapy may also have antimicrobial and detoxifying effects due to the generation of reactive oxygen intermediates and improved local immune response. This contributes to a cleaner and more oxygen-rich tissue microenvironment, supporting FGF- 2-mediated regeneration and reducing the likelihood of post-procedural inflammation or infection.

[0494]

[0485] In one aspect of the invention, the combined application of oxygen-based therapy and an FGF- 2 variant may produce synergistic biological effects, including enhanced fibroblast proliferation, collagen and elastin synthesis, angiogenesis, and accelerated wound closure. The treatment may improve skin brightness, tone, and texture, reduce visible signs of aging, and shorten recovery time following aesthetic or regenerative procedures.

[0486] In one aspect of the invention, a method for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) applying oxygen-based therapy to the tissue in the form of oxygen flow, diffusion, jet, mist, or hyperbaric exposure; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration, oxygenation, and biological activity of the FGF-2 variant, thereby promoting tissue regeneration, rejuvenation, and recovery.

[0495]

[0487] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) applying oxygen-based therapy to the tissue in the form of oxygen flow, diffusion, jet, mist, or hyperbaric exposure; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance penetration, oxygenation, and biological activity of the FGF-2 variant, thereby promoting tissue regeneration, rejuvenation, and recovery.

[0496]

[0488] In one aspect of the invention, the present disclosure relates to the delivery of microwave energy to epithelial and / or connective tissues to induce controlled thermal and dielectric effects, thereby enhancing tissue regeneration and improving the biological effectiveness of an FGF-2 variant. Microwave energy may provide uniform and deep heating of biological tissues through oscillation of polar molecules, increasing local temperature, perfusion, and metabolic activity in the treated region.

[0497]

[0489] In one aspect of the invention, the system for microwave energy delivery may comprise a microwave generator, a power amplifier, a control unit, a waveguide or antenna assembly, a power supply unit, and a user interface. Optionally, the system may further include a data storage module, a cooling unit, and / or a fluid delivery component for administration of an FGF-2 variant. The control unit may regulate frequency, power output, pulse duration, and exposure time to achieve the desired heating depth and temperature distribution.

[0498]

[0490] An electromagnetic field used for heating the tissue may be a microwave field, typically in a range from 300 MHz to 300 GHz, 400 MHz to 150 GHz, 500 MHz to 100 GHz, 700 MHz to 70 GHz, 1 GHz to 30 GHz, or 1 GHz to 10 GHz. The wavelength of the electromagnetic field may range from 1 mm to 1 m, or 2 mm to 75 cm, or 3 mm to 60 cm, or 4.3 mm to 43 cm, or 1 cm to 30 cm, or 3 cm to 30 cm.

[0499]

[0491] An electromagnetic field used for heating the tissue may be a microwave field, typically in a range from 0.5 GHz to 7 GHz, from 1.3 GHz to 4.6 GHz, or from 2 GHz to 5.8 GHz. The wavelength of the electromagnetic field may be in a range from 4 cm to 0.6 m, or 6.5 cm to 23 cm, or 5.1 cm to 15 cm.

[0500]

[0492] The frequencies may be within various ISM bands, such as, for example, 26.957 MHz, 40.66 MHz, 433.05 MHz, 902 MHz, 2.4 GHz, 5.725 GHz, 24 GHz, 61 GHz, 122 GHz or 244 GHz.

[0501]

[0493] The microwave generator may operate at a frequency in a range of 400 MHz to 8 GHz, or in a range of 915 MHz to 2.5 GHz, or in a range of 1 GHz to 2 GHz or any other microwave frequencies. The output power may be in a range from 5 W to 500 W, or in a range from 10 W to 200 W, or in a range from 30 W to 150 W. The energy may be delivered continuously or in pulsed mode, with pulse durations between 10 ps and 1 s and duty cycles ranging from 5% to 90%. The exposure duration per treated area may range from 1 s to 300 s.

[0502]

[0494] In one aspect of the invention, the applicator may include one or more antennas, electrode, waveguide antenna or radiating elements configured to emit microwave energy in focused, collimated, or diffuse patterns. The antennas may be waveguide-based, patch-type, dipole, or horn configurations, optionally combined with dielectric lenses or reflectors for shaping of the electromagnetic field. The applicator may be designed for contact or non-contact use, depending on the anatomical region and desired treatment depth.

[0503]

[0495] As used herein, the term “treatment element” refers to any component or assembly configured to deliver energy or a physical stimulus to biological tissue during a treatment procedure. The treatment element may comprise, but is not limited to, one or more antennas, electrodes, transducers, emitters, waveguides, radiating elements, or other energy-delivery structures designed to emit electromagnetic, acoustic, thermal, mechanical, or optical energy in focused, collimated, or diffuse patterns.

[0504]

[0496] A treatment element may comprise various energy sources, such as, for example, an antenna, electrode, light source, ultrasound source, or other. The antenna may be rigid, at least partially flexible, or flexible. The antenna may be a dipole antenna, patch antenna, biconical antenna, turnstile antenna, multi-dipole antenna, bow-tie antenna, ring antenna, choke dipole antenna, modified dipole antenna, spiral antenna, helical antenna, monopole antenna, loop antenna, horn antenna, pyramidal horn antenna, waveguide antenna and / or others.

[0505]

[0497] In other aspects of the invention, two or more treatment elements may be arranged or operated together as a treatment array, allowing spatially distributed, sequential, or simultaneous energy delivery to the target tissue. The configuration of each treatment element (for example, waveguide-based, patchtype, dipole, horn, or piezoelectric) may be selected or interchanged depending on the energy modality, anatomical region, and desired treatment depth.

[0506]

[0498] The waveguide may be made out of a material with good electrical conductivity, such as copper, aluminum, brass, silver, gold, and others. The electrical conductivity of the waveguide material may be in range from 4- 106S / m to 7- 107S / m, or from 5- 106S / m to 7- 107S / m, or from 7- 106S / m to 7- 107S / m, or from 9- 106S / m to 7- 107S / m, or from 1 - 107S / m to 7- 107S / m, or from 1.5- 107S / m to 6.5- 107S / m, or from 1.60- 107S / m to 2- 107S / m, or from 3.3- 107S / m to 4- 107S / m, or from 3.7- 107S / m to 4- 107S / m, or from 4- 107S / m to 4.5- 107S / m, or from 5- 107S / m to 6- 107S / m, or from 5.9- 107S / m to 6- 107S / m, or from 6- 107S / m to 6.4- 107S / m.

[0507]

[0499] The waveguide may be hollow, partially loaded, or completely loaded with a waveguide loading material. The waveguide loading material may be, for example, air, pressurized air, or any dielectric material (e.g., ceramics).

[0508]

[0500] In one aspect of the invention, the applicator may provide a microwave energy to heat of the soft tissue within the body region to a temperature in a range of 37.5 °C to 100 °C, or 37.5 °C to 95 °C or 38 °C to 90 °C, or 38 °C to 80 °C, or 38 °C to 70 °C, or 38 °C to 65 °C, or 38 °C to 60 °C, or 40 °C to 70 °C, or 40 °C to 65 °C, or 42 °C to 50 °C.

[0509]

[0501] In another aspect of the invention, the applicator may provide a microwave energy to heat the body region combined with the cooling of the surface of the soft tissue (e.g. the surface of the skin) of the body region, thus creating a thermal reverse gradient in the body region. In that case, the surface of the soft tissue (e.g. the surface of the skin, such as the epidermis) may be cooled to room temperature, or in a range of 5 °C to 70 °C, or 10 °C to 60 °C, or 15 °C to 55 °C, or 20 °C to 50 °C, or 20 °C to 45 °C, or 25 °C to 40 °C, or 30 °C to 35 °C.

[0510]

[0502] In another case, the surface of the soft tissue (e.g. the surface of the skin, as the epidermis)may be cooled to room temperature, or in a range of 0 °C to 40 °C, or 2 °C to 30 °C, or 5 °C to 25 °C, or 8 °C to 21 °C, or 12 °C to 17 °C, or 13 °C to 15 °C.

[0511]

[0503] The first layer below the surface of the soft tissue, for example, a dermal layer (including at least one of collagen or elastin) may be heated to a temperature in a range of 37.5 °C to 90 °C, or 38 °C to 90 °C, or 42 °C to 90 °C, or 50 °C to 80 °C, or 55 °C to 75 °C, or 57 °C to 72 °C, or 60 °C to 70 °C, or 38 °C to 80 °C, or 38 °C to 70 °C, or 38 °C to 65 °C, or 38 °C to 60 °C, or 45 °C to 70 °C, or 45 °C to 65 °C.

[0504] The second layer, which may be even deeper than the first layer from the surface of the soft tissue, for example, SMAS, may be heated to a temperature in the range of 37.5 °C to 90 °C, or 38 °C to 90 °C, or 42 °C to 90 °C, or 50 °C to 80 °C, or 55 °C to 75 °C, or 57 °C to 72 °C, or 60 °C to 70 °C, or 38 °C to 80 °C, or 38 °C to 70 °C, or 38 °C to 65 °C, or 38 °C to 60 °C, or 45 °C to 70 °C, or 45 °C to 65 °C.

[0512]

[0505] In another aspect of the invention, the applicator may provide a treatment energy to heat adipose tissue to a temperature in a range of 37.5 °C to 90 °C, or 38°C to 90 °C, or 42 °C to 90 °C, or 50 °C to 80 °C, or 55 °C to 75 °C, or 57 °C to 72 °C, or 60 °C to 70°C, or 38 °C to 80 °C, or 38 °C to 70 °C, or 38 °C to 65 °C, or 38 °C to 60 °C, or 45 °C to 70 °C, or 45 °C to 65 °C.

[0513]

[0506] In another aspect of the invention, at least one treatment array may be configured to simultaneously activate treatment elements within the treatment array.

[0514]

[0507] In another aspect of the invention, at least one treatment array may be configured for the sequential activation of treatment elements within the treatment array. For example, one treatment element may be activated first, followed by the activation of a second treatment element, and subsequently by the activation of additional treatment elements in sequence.

[0515]

[0508] The length of the treatment element may be in a range from 0.1 mm to 1000 mm, or from 1 mm to 500 mm, or from 50 mm to 500 mm, or from 50 mm to 250 mm, or from 1 mm to 100 mm, or from 1 mm to 75 mm, or from 1 mm to 50 mm, or from 1 mm to 25 mm, or from 1 mm to 20 mm, or from 1 mm to 15 mm, or from 1 mm to 10 mm, or from 10 mm, to 50 mm, or from 10 mm to 30 mm, or from 20 mm to 80 mm, or from 20 mm to 50 mm, or from 5 mm to 10 mm, or from 0.1 mm to 10 mm.

[0516]

[0509] The width of the treatment element 301 may be in a range from 0.1 mm to 1000 mm, or from 1 mm to 500 mm, or from 50 mm to 500 mm, or from 50 mm to 250 mm, or from 1 mm to 100 mm, or from 1 mm to 75 mm, or from 1 mm to 50 mm, or from 1 mm to 25 mm, or from 1 mm to 20 mm, or from 1 mm to 15 mm, or from 1 mm to 10 mm, or from 10 mm, to 50 mm, or from 10 mm to 30 mm, or from 20 mm to 80 mm, or from 20 mm to 50 mm, or from 5 mm to 10 mm, or from 0.1 mm to 10 mm.

[0517]

[0510] The thickness of the treatment element may be in a range from 0.001 mm to 100 mm, or from 0.01 mm to 10 mm, or from 0.05 mm to 1 mm, or from 0.1 mm to 1 mm, or from 0.2 mm to 1 mm, or from 0.2 mm to 0.5 mm, or from 0.5 mm to 1 mm, or from 1 mm to 10 mm, or from 1 mm to 5 mm, or from 1.5 mm to 4 mm, or from 2 mm to 4 mm, or from 2 mm to 15 mm, or from 10 mm to 15 mm.

[0518]

[0511] The treatment element may comprise a surface area in a range from 0.01 mm2to l«106mm2, or from 0.01 mm2to 110,000 mm2, or from 1 mm2to 1* 10,000 mm2, or from 1 mm2to 2500 mm2, or from 4 mm2to 500 mm2, or from 4 mm2to 225 mm2, or from 25 mm2to 2500 mm2, or from 25 mm2to 1000 mm2, or from 25 mm2to 900 mm2, 0.25 mm2to 25 mm2, or from 1 mm2to 25 mm2, or from 1000 mm2to 5000 mm2, or from 1000 mm2to 4000 mm2, or from 1500 mm2to 3000 mm2, or from 2000 mm2to 2500 mm2, or from 2200 mm2to 2400 mm2.

[0519]

[0512] The treatment element may comprise a volume in a range from 1- 105 mm3to 1- 108mm3, or from 1 - 104mm3to 1- 105mm3, or from 0.05 mm3to 1- 10,000 mm3, or from 0.1 mm3to 10,000 mm3, or from 1 mm3to 7500 mm3, or from 1 mm3to 5000 mm3, or from 1 mm3to 2500 mm3, or from 1 mm to 1000 mm3, or from 1000 to 5000, or from 100 mm3to 10,000 mm3, or from 1000 mm3to 10,000 mm3, or from 5000 mm3to 10,000 mm3, or from 2500 mm3to 8000 mm3, or 5000 mm3to 50,000 mm3, or from 10,000 mm3to 40,000 mm3, or from 25,000 mm3to 35,000 mm3, or from 3000 mm3to 10,000 mm3, or from 4000 mm3to 9000 mm3, or from 5000 mm3to 9000 mm3, or from 7000 mm3to 8000 mm3, or from 7500 mm3to 8000 mm3.

[0520]

[0513] In one aspect, the microwave energy may be delivered to achieve selective heating of deeper dermal and subdermal structures while maintaining epidermal cooling. The tissue temperature may be elevated to between +37 °C and +50 °C, preferably between +40 °C and +45 °C, for durations of 1 s to 120 s. Integrated cooling elements such as liquid-cooled surfaces, air jets, or Peltier-based contact plates may protect the skin surface from overheating during treatment.

[0521]

[0514] In one aspect, the microwave energy delivery system may comprise temperature and impedance sensors for real-time feedback control. The control unit may dynamically adjust output power based on measured tissue impedance or reflected power to ensure consistent energy deposition and prevent excessive thermal load. The system may further include electromagnetic shielding and safety interlocks for patient protection.

[0522]

[0515] In one aspect, microwave energy may be used to precondition tissues before, during, or after administration of an FGF-2 variant. The controlled heating may increase local perfusion and vascular permeability, facilitating diffusion and receptor-mediated uptake of the compound. Enhanced temperature may also accelerate enzymatic activity, collagen remodeling, and fibroblast proliferation, thereby synergistically amplifying the biological effects of the FGF-2 variant.

[0523]

[0516] In one aspect, the combination of microwave energy and an FGF-2 variant may result in synergistic activation of cellular pathways including heat-shock protein expression, fibroblast growth factor receptor upregulation, and angiogenesis. This synergy may promote improved tissue elasticity, accelerated wound healing, and visible skin rejuvenation, while minimizing downtime and side effects.

[0524]

[0517] In one aspect, the system may integrate microwave energy delivery with additional modalities, such as radiofrequency, vacuum-compression, or photobiomodulation, within a single multifunctional applicator. The control unit may coordinate timing and power levels between modalities and synchronize the delivery of an FGF-2 variant with microwave exposure to maximize penetration and biological activation.

[0525]

[0518] In one aspect of the invention, a method for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering microwave energy to the tissue to induce controlled dielectric heating; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance the penetration, diffusion, and biological activity of the FGF-2 variant and promote and / or accelerate tissue regeneration, remodeling, and rejuvenation.

[0526]

[0519] In one aspect of the invention, a method of use of an FGF-2 variant, e.g. for a cosmetic and / or therapeutic procedure of epithelial and / or connective tissues may comprise: a.) administering an FGF-2 variant to the tissue surface, to the surface of an applicator, or to both; and b.) delivering microwave energy to the tissue to induce controlled dielectric heating; wherein steps a.) and b.) may be performed in any order and / or concurrently, and wherein the treatment may enhance the penetration, diffusion, and biological activity of the FGF-2 variant and promote and / or accelerate tissue regeneration, remodeling, and rejuvenation.

[0527]

[0520] In one aspect of the invention, the variant of FGF-2 protein may be stored in the formulation comprising sulfate anion, wherein the formulation may enhance the stability, pro-long shelf-life of FGF- 2 variant or a combination thereof.

[0528]

[0521] The sulfate anion in the formulation may be present at a concentration in the range of 15 mM to 30 mM, in the range of 20 mM to 27 mM, or in the range of 22 mM to 25 mM.

[0529]

[0522] In one aspect of the invention, the variant of FGF-2 polypeptide may be stored in the formulation comprising at least one sulfate stabilizer, wherein the formulation may enhance the stability, pro-long shelf-life of FGF-2 variant or a combination thereof.

[0530]

[0523] The sulfate stabilizer in the formulation may be present at a concentration in the range of 15 mM to 30 mM, in the range of 20 mM to 27 mM, or in the range of 22 mM to 25 mM.

[0531]

[0524] In one aspect of the invention, the sulfate anion may comprise one or more sulfate salts, including, without limitation, ammonium sulfate, potassium sulfate, sodium sulfate, magnesium sulfate, calcium sulfate, manganese(II) sulfate, ferrous sulfate, ferric sulfate, copper(II) sulfate, zinc sulfate, aluminum sulfate, and alums such as potassium alum (KA1(SO4)2), sodium alum (NaAl(S04)2), ammonium alum (NH4A1(SO4)2), their hydrates or mixtures thereof.

[0532]

[0525] In another aspect of the invention, the formulation may further comprise at least one buffering agent from the group comprising phosphate buffers (e.g., KH2PO4, K2HPO4), TRIS (tris(hydroxymethyl)aminomethane), HEPES (4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid), or a combination thereof.

[0533]

[0526] In another aspect of the invention, the formulation may further comprise one or more agents for adjusting the ionic strength, wherein the agent may comprise at least one inorganic salt from the group of sodium chloride, potassium chloride, or ammonium sulfate.

[0534]

[0527] In one aspect of the invention, the sulfate stabilizer may comprise sulfate anions and / or sulfate salts.

[0535]

[0528] In one aspect of the invention, the formulation comprising sulfate stabilizer may be part of the composition.

[0536]

[0529] The inorganic salt in the formulation may be present at a concentration in a range of 500 mM to 1400 mM, in the range of 700 mM to 1200 mM, or in the range of 900 mM to 1100 mM.

[0537]

[0530] The pH of the formulation may be in the range of 5.8 to 8, or in the range of 6 to 7.5, or in the range of 6.4 to 7.

[0538]

[0531] In one aspect of the invention, the FGF-2 variant in the formulation may be present at a concentration in the range from 0.1 mg / mL to 25 mg / mL, in the range from 1 mg / mL to 22 mg / mL, or in the range of 5 to 20 mg / mL, preferably 10 mg / mL to 18 mg / mL.

[0539]

[0532] In one aspect of the invention, the FGF-2 variant in the formulation may be stable at refrigerated temperatures (2 - 8°C), at room temperature (20 - 25°C), or at elevated temperatures up to 60°C for extended periods.

[0540]

[0533] In one aspect of the invention, the formulation maintains at least 85 - 115% of the initial biological activity of the FGF-2 variant after storage for 11 days at 37°C or 60°C. After 35 days at room temperature and 28 days at 4°C, the biological activity was maintained at the same value as its initial value.

[0541]

[0534] In another aspect of the invention, the formulation comprising sulfate anion, at least one buffering agent, at least one inorganic salt or a combination thereof may be used for storage of other proteins or peptides, including but not limited to growth factors, antimicrobial peptides, enzymes, cytokines, hormones, or any other suitable proteins or peptides.

[0542]

[0535] The growth factors may comprise one or more of the following: albumin, insulin, insulin-like growth factor (IGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), Transforming growth factor beta 1 (TGF beta 1), TGF beta 2, TGF beta 3, Fibroblast growth factor variants (FGF) besides FGF-2, or any other suitable growth factor, including but not limited to those known in the art.

[0543]

[0536] The antimicrobial peptides may comprise one or more of the following: defensins, alicin, cathelicidins, melittin or any other suitable antimicrobial peptide, including but not limited to those known in the art.

[0544] EXAMPLES

[0545] Example 1: preparation of the truncated thermostabilized oxidation resistant FGF-2 polypeptide characterized by sequence SEQ ID NO: 5

[0546]

[0537] The competent cells of three selected E. coli strains (BL21 (DE3), BL21-Gold (DE3) or BL21- CodonPlus (DE3)-RIPL) were transformed with the plasmid DNA of the truncated thermostabilized oxidation resistant construct of FGF-2 corresponding to SEQ ID NO: 5, and each cell suspension was plated on agar plates supplemented with kanamycin. The next day, two colonies from each plate were picked into 5 mL LB broth supplemented with kanamycin and grown overnight. The following day, 50 pL of each overnight culture was added into two fresh 5 mL of LB broth supplemented with kanamycin, and incubated at 220 rpm at 37 °C. After 70 minutes, the cell cultures were let to cool down to 25 °C, which took 20 minutes. IPTG was added to a final concentration of 1 mM to induce the production of truncated thermostabilized oxidation resistant FGF-2. Non-induced cell cultures were used as control samples. The cell cultures were incubated at 220 rpm at 25 °C for 24 hours. After the incubation period, 1 mL of each culture was pipetted into clean microcentrifuge tubes, and the cells were spun down at 13000 g at room temperature. The cell pellets were resuspended in 150 pL of lx lithium dodecyl sulphate (LDS) gel sample buffer and heated to 95 °C for 5 minutes. The samples were spun down at 13000 g at room temperature, and 10 pl of each sample was loaded onto gradient SDS-PAGE.

[0547]

[0538] Fig. 2 depicts the SDS-PAGE analysis of the expression of truncated thermostabilized FGF-2 variants with improved oxidation resistance in E. coli BL21 (DE3) strain. LI is the molecular weight standard. L2 - FGF-2 polypeptide characterized by SEQ ID NO: 5. L3 - FGF-2 polypeptide characterized by SEQ ID NO: 9. Expression was induced by addition of 1 mM IPTG and was performed at 20°C for 24 h.

[0539] The truncated thermostabilized FGF-2 characterized by SEQ ID NO: 4 was successfully expressed in all three E. coli strains, BL21 (DE3), BL21-Gold (DE3) or BL21-CodonPlus (DE3)-RIPL. The FGF-2 protein was clearly visible between 15 and 20 kDa in cell cultures induced with IPTG. No band corresponding to truncated thermostabilized FGF-2 was observed in non-induced cells. The best yield was observed in BL21 (DE3) cells.

[0548] Example 2: preparation of the truncated thermostabilized FGF-2 polypeptide characterized by sequence SEQ ID NO: 5 and the purification thereof on HisTrap FF column and Superdex200 Increase column

[0549]

[0540] BL21 (DE3) competent cells were transformed with the plasmid DNA of truncated thermostabilized oxidation resistant construct of FGF-2 and plated on agar plates supplemented with kanamycin. The next day, a colony was picked and grew overnight in 5 mL LB broth supplemented with kanamycin. The following day, 1 mL of the overnight culture was added into fresh 1 L LB broth supplemented with kanamycin. Cultures (2x 0.5 L) in 2 L Erlenmeyer flasks were incubated at 220 rpm at 37 °C. When the cell density reached OD550 of 0.6, the cultures were cooled down on ice to 25 °C, and this process took about 5 minutes. The final concentration of 1 mM IPTG was used to induce expression. After induction, the cells were grown at 220 rpm at 25°C. At 24 hours post-induction, the cells were harvested by centrifugation at 5 000 g for 20 minutes. The cell pellet was resuspended in 100 mL of cold buffer containing 30 mM N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) - HEPES (pH 7.5), 500 mM NaCl, 10 mM imidazole, 10 mM MgC12, 1% NP-40, 1 tablet of protease inhibitor SigmaFast, DNAse I (5 mg in total), and lysozyme (100 mg in total). The suspension was incubated on ice for 30 minutes. Then, the cells were lysed by sonication on ice for 8 min (10s On / 20s Off, amplitude 40%), and the lysate was clarified by centrifugation at 75 000 g at 4 °C for 30 minutes. The lysate was filtered through a 0.22 pm membrane and loaded at a 5 mL / min flow rate onto the HisTrap FF (5 mL) column that was equilibrated in 30 mM HEPES (pH 7.5), 500 mM NaCl and 10 mM imidazole. The FGF-2 was washed with a buffer containing 30 mM HEPES (pH 7.5), 500 mM NaCl and 40 mM imidazole and finally eluted with 30 mM HEPES (pH 7.5), 500 mM NaCl and 150 mM imidazole. The protein was concentrated to 1 mL using VivaSpin Turbo centrifugal concentrators with 10 kDa cut-off and diluted 20 x with buffer containing 30 mM HEPES (pH 7.5). The diluted protein was loaded onto a HiTrap SP Sepharose HP (5 mL) at a flow rate of 5 mL / min and eluted with a continuous salt gradient of buffer containing 30 mM HEPES (pH 7.5) and 1 M NaCl. The total gradient elution time was 40 minutes, and the gradient was set from 0 to 60 % of the 30 mM HEPES (pH 7.5) and 1 M NaCl. The flow rate was 3 mL / min.

[0550]

[0541] The results of purification of the FGF-2 characterized by SEQ ID NO: 5 were controlled using SDS-PAGE analysis (Fig. 3). After sonication, the lysate was centrifuged, and 2 pl of pellet (L3) and clarified lysate (L4) were loaded on the gel. The supernatant was then loaded onto a HisTrap column and flow through (L6), fraction after washing with a buffer containing 10 mM imidazole (L7), and finally with 150 mM imidazole (L8) were loaded on the gel. Eluted protein was then loaded on HiTrap SP Sepharose HP column and flow through (LIO), wash with 30 mM HEPES (Li l), and finally elution fractions (L12, L13, L14) were loaded on the gel and analyzed using SDS-PAGE.

[0551] Example 3: the thermostability assay

[0552]

[0542] The thermostability assay of FGF-2 was performed using nanoDSF in Prometheus NT.48 (NanoTemper). Standard NanoTemper capillaries were loaded with FGF-2 (1 mg / ml) in a buffer containing 15 mM HEPES (pH 7.5) and 150 mM NaCl. The measurement was performed in triplicates at temperatures from 20 °C to 90 °C with a temperature ramp of 1.5 °C / min. Changes in tryptophan emission at 330 and 350 nm were monitored, and the ratio 330 / 350 nm was plotted against the temperature.

[0553]

[0543] In one aspect of the invention, the TM of thermostabilized truncated FGF-2 characterized by sequence SEQ ID NO: 4 determined by nanoDSF was 76.7 °C (Fig. 4). Representative thermal unfolding curve (top) and its first derivative analysis, indicating the melting temperature of the protein (bottom) are shown.

[0554]

[0544] The experimental TM was about 23.2 °C higher than the Homo sapiens FGF-2 (SEQ ID NO: 1). This value indicates excellent thermal stability, which is in agreement with the contributions of individual substitutions.

[0555] Example 4: long term stability

[0556]

[0545] The thermo-stabilized truncated FGF-2 characterized by sequence SEQ ID NO: 5 was stored in a freezer at -20 °C for 90 days post production, and the protein was analyzed by testing of biological activity on mammalian cells. No loss of biological activity was detected after 90 days.

[0557]

[0546] Fig. 5 depicts the results of a biological activity test, after 90 days of storage in the freezer at - 20 °C, which shows that FGF-2 characterized by sequence SEQ ID NO: 5 shows higher biological activity over 4 passages in comparison to FGF-2 characterized by sequence SEQ ID NO: 3.

[0558] Example 5: stability analysis after freeze-thaw cycle

[0559]

[0547] Stability analysis after two freeze-thaw cycles of the FGF-2 characterized by SEQ ID NO: 5 was performed. Stability was determined by biological activity assay on mammalian cells with concentration of FGF-2 100 ng / mL. No loss of biological activity was observed after two freeze thaw cycles.

[0560]

[0548] Stability was determined by biological activity assay on mammalian cells with concentration of FGF-2 100 ng / mL. No loss of biological activity was observed after two freeze thaw cycles.

[0561]

[0549] Fig. 6 and Fig. 7 depict analysis of biological activity showing that after two freeze thaw cycles mammalian cells grew into comparable densities showing no significant loss of biological activity.

[0562] Example 6: testing the biological activity

[0563]

[0550] The biological effects of the FGF-2 polypeptides according to the invention (FGF-2, SEQ ID NO: 4 and SEQ ID NO: 5) were tested and compared with state of the art FGF-2 characterized by SEQ ID NO: 3 (Fig. 8). Two different concentrations of the FGF-2 polypeptides were tested: 100 and 10 ng / ml.

[0564]

[0551] In the case of the FGF-2 characterized by SEQ ID NO: 3, the achieved cell growth generally was comparable with the FGF-2 characterized by SEQ ID NO: 5 and FGF-2 characterized by SEQ ID NO: 4.

[0565]

[0552] Experimental details of testing biological activity:

[0566] • Cells: C2C12 (seeding density 4.000 cells per well)

[0567] • Cultivation vessel: 24-well plate

[0568] • Basal medium: Essential 8™ Basal Medium

[0569] • FGF-2 variants tested: o No FGF-2 o Commercial FGF-2 - concentrations 1, 10 and 100 ng / ml thermostabilized truncated FGF-2 (SEQ ID NO: 3) - concentrations 1, 10 and 100 ng / ml

[0570] • Cultivation time points: o Day 0 - seeding o Day 3 - cell counting o Day 7 - cell counting, end of experiment

[0571] Example 7: preparation of other thermostabilized FGF-2 polypeptides according to the invention (SEQ ID NO: 4 and 6-10)

[0572]

[0553] The thermostabilized FGF-2 polypeptides according to the invention corresponding to SEQ ID NO: 4 and 6 -10 were prepared according to the procedure described in example 1.

[0554] Fig. 9 - SDS-PAGE analysis of the expression of truncated thermostabilized FGF-2 variants with improved oxidation resistance in E. coli BL21 (DE3) strain: (LI) molecular weight standard; (L2) pellet of the lysate and (L3) clarified lysate of the FGF-2 polypeptide characterized by SEQ ID NO: 5; (L5) pellet of the lysate and (L6) clarified lysate of the FGF-2 polypeptide characterized by SEQ ID NO: 10; (L8) pellet of the lysate and (L9) clarified lysate of the FGF-2 polypeptide characterized by SEQ ID NO:9; (Li l) pellet of the lysate and (L12) clarified lysate of the FGF-2 polypeptide characterized by SEQ ID NO:8; (L14) pellet of the lysate and (L15) clarified lysate SEQ ID NO: 7. L 4, L7, LIO and L13 are without samples. Expression was induced by addition of 1 mM IPTG and the assay was performed at 20°C for 24 h.

[0573] Industrial Applicability

[0574] The FGF-2 polypeptides with improved thermal and oxidative stability according to the invention may be used in scientific research as well as in many industrial applications, for example, in biotechnological research, medicine, the pharmaceutical industry, cosmetics, the clean meat industry, the generation of organoids and 3D cell culture models, and in other related applications.

[0575] SEQUENCE LISTING:

[0576] SEQ ID NO: 1

[0577] Homo sapiens FGF-2

[0578] MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQ

[0579] LQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWY

[0580] VALKRTGQYKLGSKTGPGQKAILFLPMSAKS

[0581] SEQ ID NO: 2

[0582] Bos taurus FGF-2

[0583] MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQ

[0584] LQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYSSWY

[0585] VALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0586] SEQ ID NO: 3

[0587] FGF-2 polypeptide variant 1

[0588] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYL

[0589] AMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQK AILFLPMSAKS

[0590] SEQ ID NO: 4

[0591] FGF-2 polypeptide variant 2

[0592] GGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVSANRYLA

[0593] MKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKA ILFLPMSAKS

[0594] SEQ ID NO: 5

[0595] FGF-2 polypeptide variant 3

[0596] MHHHHHHLEVLFQGPFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAE

[0597] ERGVVSIKGVSANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALK

[0598] RTGQYKLGPKTGPGQKAILFLPMSAKS

[0599] SEQ ID NO: 6 FGF-2 polypeptide variant 4

[0600] MHHHHHHENLYFQGGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERG

[0601] VVSIKGVSANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTG

[0602] QYKLGPKTGPGQKAILFLPMSAKS

[0603] SEQ ID NO: 7

[0604] FGF-2 polypeptide variant 5

[0605] MHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLE

[0606] FPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKD

[0607] FETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFP

[0608] KLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGGGGSGGGGSGGGGSE

[0609] NLYFQGGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVSA

[0610] NRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTG

[0611] PGQKAILFLPMSAKS

[0612] SEQ ID NO: 8

[0613] FGF-2 polypeptide variant 6

[0614] MHHHHHHSSGMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQV

[0615] AATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEA LSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENG KYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSN IDTS KVNYGVTVLPTFKGQPS KPFVGVLS AGINA ASPNKELAKEFLENYLLTDEGLEAVNKD KPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTV DEALKDAQTGGGGSGGGGSGGGGSENLYFQGGHFKDPKLLYCKNGGFFLRIHPDGRVDGTR

[0616] DKSDPFIKLQLQAEERGVVSIKGVSANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTY RSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0617] SEQ ID NO: 9

[0618] FGF-2 polypeptide variant 7

[0619] MHHHHHHSSGMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFA

[0620] KRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGGHFKDPKLLYCKNGGF

[0621] FLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVSANRYLAMKEDGRLYAIKNVTDEC

[0622] FFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0623] SEQ ID NO: 10

[0624] Fusion protein of FGF-2 and TrxA

[0625] MHHHHHHSSGMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQG

[0626] KLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGGGGS GGGGSGGGGSENLYFQGGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEE

[0627] RGVVSIKGVSANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKR

[0628] TGQYKLGPKTGPGQKAILFLPMSAKS

[0629] SEQ ID NO: 11

[0630] Affinity tag

[0631] MHHHHHHENLYFQG

[0632] SEQ ID NO: 12

[0633] Affinity tag

[0634] MHHHHHHLEVLFQGP

[0635] SEQ ID NO: 13

[0636] Fusion partner

[0637] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGD

[0638] VKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSK

[0639] LPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAI

[0640] PQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD

[0641] SEQ ID NO: 14

[0642] Fusion partner

[0643] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIF

[0644] WAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLP

[0645] NPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVD

[0646] NAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV

[0647] TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKS

[0648] YEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT

[0649] SEQ ID NO: 15

[0650] Fusion partner

[0651] MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSL

[0652] RFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG

[0653] SEQ ID NO: 16 Fusion partner

[0654] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNID QNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA

[0655] SEQ ID NO: 17

[0656] Affinity tag

[0657] GGGGSGGGGSGGGGSENLYFQG

[0658] SEQ ID NO: 18

[0659] Affinity tag

[0660] MHHHHHHSSG

[0661] SEQ ID NO: 19

[0662] FGF-2 polypeptide variant 8

[0663] GGHFKDPKLLYSKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVSANRYLA

[0664] MKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKA ILFLPMSAKS

[0665] SEQ ID NO: 20

[0666] FGF-2 polypeptide variant 9

[0667] GGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVSANRYLA

[0668] MKEDGRLYAIKNVTDEAFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKA ILFLPMSAKS

[0669] SEQ ID NO: 21

[0670] FGF-2 polypeptide variant 10

[0671] GGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVSANRYLA

[0672] MKEDGRLYAIKNVTDESFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKA ILFLPMSAKS

[0673] SEQ ID NO: 22

[0674] TrxA polypeptide variant 1 MHHHHHHSSGMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQG

[0675] KLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGGGGS GGGGSGGGGSENLYFQ

[0676] SEQ ID NO: 23

[0677] Beta-defensin 6 of Gallus gallus

[0678] ILYLLLSVLFVVLQGVA

[0679] SEQ ID NO: 24

[0680] Modified and truncated Beta-defensin 6 of Gallus gallus

[0681] ILYLLLSVLGVADCED

Claims

Independent claims:

1. An FGF-2 polypeptide having at least 90 % sequence identity to SEQ ID NO: 4.

2. An FGF-2 polypeptide having at least 90 % sequence identity to at least one of SEQ ID NO: 5 to 22.

3. An FGF-2 polypeptide derived from SEQ ID NO: 4, wherein the polypeptide comprises at least one amino acid substitution from the group of R31L, C34S, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, C101A, C101V, S109E, and S121P.

4. An FGF-2 polypeptide derived from at least one of SEQ ID NO: 5 to 22, wherein the polypeptide comprises at least one amino acid substitution from the group of R31L, C34S, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, C101A, C101V, S109E, and S121P.

5. An FGF-2 polypeptide derived from SEQ ID NO: 3, or 5, or 6, wherein the polypeptide comprises a deletion of up to 22 amino acids at the N-terminus.

6. An FGF-2 polypeptide derived from SEQ ID NO: 4,7. wherein the polypeptide comprises a deletion of up to 22 amino acids at the N-terminus.

8. A composition comprising an FGF-2 polypeptide, wherein the composition comprises:(a) at least one of: solvents, stabilizers, emollients, humectants, preservatives, or colorants; and(b) at least one of: penetration enhancers, antioxidants, surfactants, rheological additives, or occlusives.

9. A method of administering an FGF-2 polypeptide, comprising:(a) applying an FGF-2 polypeptide to the tissue surface, to the surface of an applicator, or to both; and(b) inducing tissue irritation, tissue damage, or a both by at least one external stressor or procedure; wherein steps (a) and (b) are performed in any order.

10. A system for administering an FGF-2 polypeptide, comprising:(a) an FGF-2 polypeptide; and(b) a device that induce irritation of tissue, damage of tissue, or their combination by at least one external stressor or procedure, wherein the system is configured such that the application of the FGF-2 polypeptide and the induction of tissue irritation, tissue damage, or a combination thereof are performed in any order.

11. A method of administering an FGF-2 polypeptide, comprising: application of an FGF-2 polypeptide with one or more physical modalities consisting from the group of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation) pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma-based energy, oxygen-based therapy, or any combination thereof12. A system for administering an FGF-2 polypeptide, comprising:(a) an FGF-2 polypeptide; and(b) a device configured to deliver at least one physical modality from the group of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation) pulsed electric field (PEF) energy,ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma-based energy, oxygen-based therapy, or any combination thereof13. A formulation comprising: an FGF-2 polypeptide and a sulfate stabilizer.

14. A method of storing of the FGF-2 polypeptide in a formulation comprising a sulfate stabilizer.

15. A composition comprising an FGF-2 polypeptide and at least one additional ingredient, wherein:(a) the FGF-2 polypeptide is present in an amount ranging from 500 ppm to 300000 ppm, and(b) the at least one additional ingredient is present in an amount ranging from 10000 ppm to 990,000 ppm relative to a total weight of the composition, and wherein the ratio of the concentration of the FGF-2 variant to the concentration of the additional ingredient is between 1:2 and 1:2,000.

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