Biomimetic peptides and their use in bone regeneration

Abstract

The present invention relates to biomimetic peptides with a common pentapeptide sequence that enable bone regeneration by activating the hydroxyapatite (Hap) deposition process. Their use as medicines, especially in the field of bone, tooth (dentin and enamel) and periodontal regeneration, and in the functionalization of prostheses, screws, fixtures, inserts or supports made of metal, ceramic, or natural or synthetic polymeric materials, is also described. Pharmaceutical and cosmetic compositions comprising such peptides are further described, as are their use as medicines in bone, tooth and periodontal regeneration, their cosmetic use, and their use in the functionalization of prostheses, screws, fixtures, inserts or supports made of metal, ceramic, or natural or synthetic polymeric materials.

Description

[Technical field]

[0001] The present invention relates to biomimetic peptides with a common pentapeptide sequence that enable bone regeneration by activating the hydroxyapatite (Hap) deposition process. Their use as medicines, especially in the field of bone, tooth (dentin and enamel) and periodontal regeneration, and in the functionalization of prostheses, screws, fixtures, inserts or supports made of metal, ceramic, or natural or synthetic polymeric materials, is also described. Pharmaceutical and cosmetic compositions comprising such peptides are further described, as are their use as medicines in bone, tooth and periodontal regeneration, their cosmetic use, and their use in the functionalization of prostheses, screws, fixtures, inserts or supports made of metal, ceramic, or natural or synthetic polymeric materials. [Background technology]

[0002] The assembly of hydroxyapatite (HAp) and proteins is a key process underlying the formation of bone and teeth in vertebrates. The human body can fabricate these hierarchically structured organic-inorganic hybrid composites using organic molecules as regulators of HAp deposition.

[0003] HAp[Ca 10 (PO 4 ) 6 (OH) 2 HAp is the main component of bone, dentin and enamel. In these hard tissues, HAp combines with various types of proteins to form highly organized hierarchical structures with unique morphological, structural and mechanical properties.

[0004] Bone is made up of 65-70% mineral components, mainly HAp, and 30-35% organic molecules, including mainly type I collagen, various other non-collagenous proteins and glycosaminoglycans.

[0005] The organization of type I collagen is important for the deposition of HAp in bone tissue. Two α-1 and one α-2 chains of type I collagen constitute the basic unit of collagen fibrils, i.e., tropocollagen. Tropocollagen units form highly ordered structures, and the gap regions between fibrils, called "hole zones", which have high charge density, are the key sites where nucleation of mineral components occurs.

[0006] Non-collagenous proteins such as bone sialoprotein, osteonectin, osteopontin, osteocalcin, and dentin matrix proteins bind to tropocollagen and regulate HAp mineralization. These proteins contain a high density of acidic amino acid residues in their sequences, which are involved in the Ca 2+ It has a high affinity for ions.

[0007] Tooth enamel is composed of more than 95% by weight of organic and less than 1% of mineral components. Instead of type I collagen present in bone, the most important role of HAp mineralization in enamel is played by amelogenin with contributions of other proteins (ameloblastin, amelin, ceterin, enamelin, and matrix metalloproteinase 20). Self-assembled nanospheres of amelogenin and mineral prenucleation groups form composite nanoparticles, which then assemble into linear and parallel matrices, forming the elongated crystal bundles of enamel.

[0008] Repair of bone and tooth defects and / or gaps can be performed with autografts (use of bone from the patient himself, harvested from another body site) or allografts (bone material from a cadaver). In both cases, often insurmountable problems can arise due to lack of harvest sites (autografts) or immunogenicity problems (allografts).

[0009] For these reasons, the research and development of new materials for bone and tooth repair is a hot topic issue. With reference to the principles of tissue engineering, the regeneration of tissues and organs can be achieved by the implantation of engineered constructs. The success of the engineered constructs depends on the use of an appropriate combination of scaffolds, stimulating factors and, in some cases, cells.

[0010] Understanding the mechanisms underlying the mineralization of HAp with organic molecules has been a source of inspiration for the design of new functional materials. Many researchers have studied the formation of organic hybrids of HAp with organic molecules, mainly proteins, under biological conditions that mimic the biomineralization process.

[0011] As far as proteins are concerned, those related to the biomineralization of HAp in vivo have always been considered ideal candidates for the production of protein-HAp hybrids. In fact, the mineralization of HAp, including collagen and amelogenin, the organic components that regulate HAp deposition in bone and teeth, has been very thoroughly studied. Non-collagenous proteins and proteins other than amelogenin that help control HAp deposition in bone and enamel have also been used in the preparation of protein-HAp hybrids. All these organic-inorganic hybrids used as scaffolds for bone regeneration in the presence or absence of cells have provided positive results by promoting osteogenic differentiation of cells and improving the formation and integration of new bone.

[0012] The proteins used to generate HAp organic hybrids are not limited to those associated with bone and tooth mineralization. Silk proteins have been used as model proteins for HAp nucleation and deposition due to their high biocompatibility as well as mechanical and structural properties.

[0013] In addition to proteins, other natural polymers such as polysaccharides (e.g., chitin, chitosan, chondroitin sulfate) have attracted some interest as substrates capable of inducing and modulating HAp precipitation. Similarly, bacterial cellulose nanofibers have been used as a model in which collagen-based structures can be replicated to induce HAp deposition.

[0014] A very interesting alternative that has recently emerged is the use of biomimetic peptides as substrates that can activate HAp nucleation when incorporated into scaffolds for bone regeneration. Peptides can be easily produced both by direct synthesis of the sequence of interest and by genetic engineering methods. The sequence of interest can be designed in silico or derived from the active domains of extracellular matrix proteins and the large family of proteins directly involved in the process of in vivo bone mineralization (e.g. type I collagen, non-collagenous proteins, amelogenins, etc.) and then used directly to functionalize scaffolds for bone regeneration and / or to prepare peptide-HAp hybrids.

[0015] These biomimetic peptides may perform different functions within the matrix for bone regeneration: they may have osteoconductive activity and favor HAp nucleation and deposition, they may have osteoinductive activity and therefore favor the adhesion of cells, their proliferation and differentiation in osteoblastic cell lines, and they may also be involved in the process of angiogenesis, which represents a key step for in vivo bone regeneration. Depending on the function to be performed, the biomimetic peptides can be incorporated into the scaffold in different positions (on the surface, in the bulk) and in free or bound form, depending on whether their diffusion into the environment surrounding the implant is required or not.

[0016] Particular interest in the development of new materials for bone, periodontal and dental repair, together with the difficulties encountered in the case of autografts or allografts, underlies the need to identify new approaches for the repair of defects in the field of bone regeneration. Summary of the Invention [Problem to be solved by the invention]

[0017] The object of the present invention is therefore to provide novel biomimetic peptides that allow bone regeneration by activating and accelerating the HAp nucleation phenomenon, which do not show the drawbacks found in different peptides or proteins known for the same application. In particular, proteins belonging to the family of BMPs (bone morphogenetic proteins) have some drawbacks related to extreme in vivo reactivity and high production costs. Two different members of this family are commercially available: BMP-2 and BMP-7. Products containing BMP-7 received regulatory approval only for use in very limited areas, and were then withdrawn from the market after serious doubts about their safety arose. In fact, cases of various side effects have been reported, ranging from simple irritation to severe infections requiring restorative surgical intervention. Products based on BMP-2 (especially its recombinant version rH-BMP-2) are now instead on the market, representing the standard of care for a certain number of applications, often competing with the use of autologous bone. However, over time, a fairly large number of studies have highlighted many side effects (even some quite serious) that could be directly correlated with the use of this peptide and were not identified in the first clinical trials. Among the main ones, we can mention osteolysis, urinary problems, the appearance of pain, and, in the case of higher doses, an increased risk of developing tumors [EJCarragee et al.The Spine Journal 11(2011)471-491]. Also, products containing rH-BMP-2 are very costly. As far as peptides are concerned, instead, a sequence derived from collagen type 1 is currently used in the clinic; this sequence, known as P-15, has a very high capacity to stimulate cell adhesion [H.Nguyen et al.Biochemical and Biophysical Research Communications 311(2003)179-186]; however, to be effective in promoting bone tissue formation, it must be bound to a preformed support, usually the bone matrix, that provides the attracted cells with a favorable environment for their development and differentiation. [Means for solving the problem]

[0018] Thus, the present invention relates to a biomimetic peptide consisting of the general formula (I): Xaa1-Ser-Gly-Tyr-Glu-Tyr-Xaa2 (SEQ ID NO: 5) During the ceremony, - when Xaa1 is Val-Asn-Gly-Gly-Tyr (SEQ ID NO: 6), Xaa2 is Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe (SEQ ID NO: 7) or or Xaa2 is Ala-Trp; If -Xaa1 is not present, Xaa2 is Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe (SEQ ID NO: 7), and -Xaa1 is Gly-Pro-Tyr-Val-Ala-His-Gly-Gly-Tyr (SEQ ID NO: 8); Xaa2 is absent.

[0019] The biomimetic peptides described are of synthetic nature.

[0020] In another aspect, the present invention describes the use of the peptide as a medicament.

[0021] In another aspect, the present invention relates to the use of the peptides of the invention in bone, tooth (dentin and enamel) and periodontal regeneration.

[0022] In yet another aspect, the present invention describes a composition comprising one or more peptides, identical or different from each other, their salts, and an excipient or additive acceptable in the biomedical, cosmetic or pharmaceutical fields.

[0023] The use of a composition according to the invention as a medicament is also described.

[0024] In yet another aspect, the present invention describes the use of the compositions in bone, tooth (dentin and enamel) and periodontal regeneration.

[0025] In yet another aspect, the present invention describes the use of a pharmaceutical or cosmetic composition comprising the peptides described herein or for the surface functionalization of prostheses, screws, fixtures, inserts or supports made of metal, ceramic or natural or synthetic polymeric materials.

[0026] In a further embodiment, the general formula (I): The cosmetic use of a composition comprising a peptide having the sequence Xaa1-Ser-Gly-Tyr-Glu-Tyr-Xaa2 (SEQ ID NO: 5) as described by the present invention is described.

[0027] The dependent claims describe further embodiments of the invention.

[0028] The present invention will now be described in detail with reference to the accompanying drawings in which: [Brief description of the drawings]

[0029] [Figure 1] SEM images of fibroin foams. A: Peptide-free fibroin foam incubated for 14 days in SBF. B: Fibroin foam functionalized with a biomimetic peptide (SEQ ID NO: 3) incubated for 14 days in SBF. C: Higher magnification detail of image B showing the morphology of mineral deposits. D: Fibroin foam functionalized with a biomimetic peptide (SEQ ID NO: 4) incubated for 14 days in SBF. E: Fibroin foam functionalized with a biomimetic peptide (SEQ ID NO: 2) incubated for 14 days in SBF. F: Higher magnification detail of image E showing the accumulation of mineral deposits. [Diagram 2]IR spectra of fibroin foams. (a) Peptide-free fibroin foam not incubated in SBF. (b) Peptide-free fibroin foam incubated in SBF for 14 days. (c) Fibroin foam functionalized with a biomimetic peptide (SEQ ID NO: 3) incubated in SBF for 14 days. (d) Fibroin foam functionalized with a biomimetic peptide (SEQ ID NO: 4) incubated in SBF for 14 days. (e) Fibroin foam functionalized with a biomimetic peptide (SEQ ID NO: 2) incubated in SBF for 14 days. The area enclosed by the dashed rectangle identifies the spectral region 1100-900 cm-1 characteristic of the stretching vibrations of phosphate and carbonate groups. [Diagram 3] SEM / EDX spectrum of fibroin foam functionalized with a biomimetic peptide (SEQ ID NO: 4) incubated in SBF for 14 days. The top SEM image shows an area of ​​the sample where two crystal forms are present, with large sized, regularly shaped crystals dominating on the right (Object 10438) and crystalline aggregates consisting of associations of smaller sized crystals evident on the left (Object 10437). The chemical composition of the crystal "Object 10438" is dominated by the presence of the elements sodium (Na) and chlorine (Cl), with the complete absence of calcium (Ca) and phosphorus (P). In contrast, the aggregate "Object 10437" has strong signals for calcium (Ca) and phosphorus (P). Signals for the elements carbon (C), oxygen (O), nitrogen (N), gold (Au) and palladium (Pd), common to both spectra, reflect the organic nature of the fibroin foam (C, O, N) and the sample preparation method (Au / Pd sputtering). [Figure 4]Non-functionalized (SF) and biomimetic peptide (SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4) functionalized fibroin foams incubated in the absence (no MSC) or presence (+MSC) of human mesenchymal stem cells were maintained for 7 days in complete medium. Afterwards, the foams were decellularized and stained with von Kossa stain to highlight calcium deposits (A). The percentage of the area covered by von Kossa staining was calculated using ImageJ software and reported in graphs B and C. The amount of deposited calcium is always higher in fibroin foams functionalized with biomimetic peptide than in non-functionalized foams, both in the absence (B) and presence (C) of MSC cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention relates to the identification of novel biomimetic peptides of synthetic nature, which have the characteristic of enabling bone regeneration by activating and accelerating the process of HAp nucleation and deposition.

[0031] The biomimetic peptides are of the general formula (I): Xaa1-Ser-Gly-Tyr-Glu-Tyr-Xaa2 (SEQ ID NO: 5) During the ceremony, - when Xaa1 is Val-Asn-Gly-Gly-Tyr (SEQ ID NO: 6), Xaa2 is Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe (SEQ ID NO: 7) or or Xaa2 is Ala-Trp; If -Xaa1 is not present, Xaa2 is Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe (SEQ ID NO: 7), and -Xaa1 is Gly-Pro-Tyr-Val-Ala-His-Gly-Gly-Tyr (SEQ ID NO: 8); Xaa2 is absent.

[0032] All peptides described herein have in common the pentapeptide sequence of SEQ ID NO:5.

[0033] Advantageously, in a preferred embodiment, the peptide comprises: SEQ ID NO: 1 Val-Asn-Gly-Gly-Tyr-Ser-Gly-Tyr-Glu-Tyr-Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe, SEQ ID NO: 2 Ser-Gly-Tyr-Glu-Tyr-Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe, SEQ ID NO: 3 Gly-Pro-Tyr-Val-Ala-His-Gly-Gly-Tyr-Ser-Gly-Tyr-Glu-Tyr, and SEQ ID NO: 4 is selected from the group consisting of Val-Asn-Gly-Gly-Tyr-Ser-Gly-Tyr-Glu-Tyr-Ala-Trp.

[0034] All peptides of SEQ ID NO: 1-4 have in common the amino acid core of SEQ ID NO: 5. Without wishing to be bound by any theory, it is believed that it is precisely this five amino acid core (pentapeptide) that enables the peptides to have the capacity for bone regeneration by activating and accelerating the nucleation phenomenon of the HAp deposition process.

[0035] These peptides were identified by the inventors through studies in which peptides described in US Pat. No. 7,193,038, known to promote cell attachment and proliferation, were added to a silk fibroin matrix.

[0036] Matrices (especially electrospun surfaces, gels, pastes and foams) loaded with the peptides according to the invention promoted rapid initiation of the mineralization process with favorable cell proliferation and deposition of hydroxyapatite precursors, even on functionalized matrices incubated in an environment favorable for bone growth both in the presence and in the complete absence of cells.

[0037] To verify that this result was due to the presence of the peptide used for functionalization, experiments were performed in the same environment but with a non-functionalized matrix, and, as expected, there was no (significant) nucleation of mineral components.

[0038] A second aspect of the invention relates to the use as a medicament of a peptide having the general formula (I): Xaa1-Ser-Gly-Tyr-Glu-Tyr-Xaa2 (SEQ ID NO: 5) During the ceremony, - when Xaa1 is Val-Asn-Gly-Gly-Tyr (SEQ ID NO: 6), Xaa2 is Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe (SEQ ID NO: 7) or or Xaa2 is Ala-Trp; If -Xaa1 is not present, Xaa2 is Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe (SEQ ID NO: 7), and -Xaa1 is Gly-Pro-Tyr-Val-Ala-His-Gly-Gly-Tyr (SEQ ID NO: 8); Xaa2 is absent.

[0039] In yet another aspect, the use of peptides having general formula (I) according to the invention in bone, tooth (dentin and enamel) and periodontal regeneration as substrates for HAp nucleation is described.

[0040] In a preferred embodiment, said bone, dental and periodontal regeneration is in the treatment of bone, dental or periodontal pathologies or is required by specific surgical techniques.

[0041] In an even more preferred embodiment, said bone, tooth or periodontal pathology is selected from the group comprising fracture, bone gap, sarcoma, dentin hypersensitivity, pyorrhea and the surgical technique may comprise posterolateral spinal fusion, anterior cervical discectomy with fusion, posterior lumbar intervertebral fusion, transintervertebral lumbar intervertebral fusion, orthopedic endoprosthesis implantation, maxillary sinus lift, revision of orthopedic endoprosthesis, vertebroplasty, dental implantation.

[0042] In a fourth aspect, the present invention describes a composition comprising one or more peptides, identical or different from each other, their salts, and an excipient or additive acceptable in the biomedical, cosmetic or pharmaceutical field.

[0043] The compositions of the present invention may further comprise hyaluronic acid and / or salts thereof, chitosan, alginic acid, silk fibroin, propylene glycol, propylene glycol alginate, poloxamer, chondroitin sulfate, collagen, gelatin, elastin, polylactic acid (PLA), poly(lactic-co-glycolic) acid (PLGA), polyglycolic acid (PGA), polyvinyl alcohol (PVA), polycaprolactone (PCL), bioglass, hydroxyapatite and related compounds, calcium salts, decellularized bone matrix, pectin, sericin, cellulose and derivatives thereof, fibrin, and combinations thereof.

[0044] In a preferred embodiment, the pharmaceutical composition comprising one or more peptides, their salts, the same or different from each other, and pharmacologically acceptable excipients, is in the form of a gel, paste, membrane, fabric, foam, putty, powder, granule, compressed solid, film, or is contained / adsorbed within micellar or particulate elements, fibers, nanofibers, hollow fibers, hollow nanofibers, membranes, ceramics, nanotubes, porous and / or trabecular metal structures, textile products, nanobubbles, sol-gels, combinations of these systems, and composites containing these systems and combinations thereof. This composition in the form of a gel or paste can be a toothpaste.

[0045] In a further preferred embodiment, the cosmetic composition comprising one or more peptides, their salts, the same or different from each other, and excipients acceptable for cosmetic formulations, is in the form of a gel, paste, membrane, fabric, foam, putty, powder, granule, compressed solid, film, or is contained / adsorbed in micellar or particulate elements, fibers, nanofibers, hollow fibers, hollow nanofibers, membranes, ceramics, nanotubes, porous and / or trabecular metal structures, textile products, nanobubbles, sol-gels, combinations of these systems, and composites containing these systems and combinations thereof. This composition in the form of a gel or paste can be a toothpaste.

[0046] In yet another aspect, the present invention describes the use of the composition in bone, tooth and periodontal regeneration.

[0047] Peptides of general formula (I): Xaa1-Ser-Gly-Tyr-Glu-Tyr-Xaa2 (SEQ ID NO: 5) During the ceremony, - when Xaa1 is Val-Asn-Gly-Gly-Tyr (SEQ ID NO: 6), Xaa2 is Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe (SEQ ID NO: 7) or or Xaa2 is Ala-Trp; If -Xaa1 is not present, Xaa2 is Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe (SEQ ID NO: 7), and -Xaa1 is Gly-Pro-Tyr-Val-Ala-His-Gly-Gly-Tyr (SEQ ID NO: 8); Xaa2 is absent, They are described by the sequences SEQ ID NOs: 1 to 4 and are used for the functionalization of the silk fibroin matrix described above.

[0048] The use of a composition according to the invention as a medicament is also described.

[0049] In yet another aspect, the present invention describes the use of a pharmaceutical or cosmetic composition comprising a peptide as described herein for the surface functionalization of prostheses, screws, fixtures, inserts or supports made of metal, ceramic or natural or synthetic polymeric materials.

[0050] The present invention further describes the use of the peptides of the invention for the surface functionalization of prostheses, screws, fasteners, inserts or supports made of metal, ceramic or natural or synthetic polymer materials. In a further embodiment, the peptides of the general formula (I): The cosmetic use of a composition comprising a peptide having the sequence Xaa1-Ser-Gly-Tyr-Glu-Tyr-Xaa2 (SEQ ID NO: 5) as described by the present invention is described.

[0051] The following examples of embodiments of the present invention are reported below by way of illustration.

[0052] example Example 1: Synthesis of biomimetic peptides Several peptides were synthesized, including the pentapeptide of SEQ ID NO: 5. The most promising sequence that showed the ability to induce advanced mineralization by deposition of hydroxyapatite precursors is as follows: SEQ ID NO: 1 Val-Asn-Gly-Gly-Tyr-Ser-Gly-Tyr-Glu-Tyr-Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe, SEQ ID NO: 2 Ser-Gly-Tyr-Glu-Tyr-Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe, SEQ ID NO: 3 Gly-Pro-Tyr-Val-Ala-His-Gly-Gly-Tyr-Ser-Gly-Tyr-Glu-Tyr, and SEQ ID NO: 4 Val-Asn-Gly-Gly-Tyr-Ser-Gly-Tyr-Glu-Tyr-Ala-Trp.

[0053] The peptides were synthesized according to the solid phase peptide synthesis protocol using microwave. Wang resin was used as support. Each coupling was performed by working 5 times more than the initial loading of the resin, and N,N'-diisopropylcarbodiimide (DIC) (0.5 M in dimethylformamide (DMF)) and coupling agent Oxyma Pure® (1 M in DMF) were used as coupling agents. Cleavage from the resin was achieved using phenol, triisopropylsilane (TIS), thioanisole (TAN), H 2 A mixture of O and trifluoroacetic acid (TFA) (4 / 4 / 4 / 2 / 86) was used. The sequence was purified by semi-preparative RP-HPLC.

[0054] Example 2: Preparation of fibroin foam functionalized with biomimetic peptides The following procedure was adopted to prepare a matrix of fibroin functionalized with biomimetic peptides in the form of a lyophilized foam: a) To prepare the fibroin aqueous solution, 50 ml of a 9.3 M aqueous solution of lithium bromide was added to 5 g of degummed silk. It was incubated for 3 hours in a thermostatic bath at 60 °C. At the end of the incubation period, the solution was filtered, diluted with 100 ml of distilled water, transferred to a dialysis bag and dialyzed against distilled water for 2 days until the salt was completely removed. The aqueous solution of fibroin thus obtained was recovered from the dialysis bag and, after filtration, the biomimetic peptide was added; b) To prepare an aqueous solution of the biomimetic peptide, 50 mg of peptide was solubilized in 2.5 ml of distilled water. c) To prepare a fibroin solution containing biomimetic peptides, 2.5 ml of the aqueous solution of the biomimetic peptides was added to 10 ml of the aqueous solution of the fibroin.To prepare a fibroin solution without the biomimetic peptides (blank), 2.5 ml of water was added to 10 ml of the aqueous solution of the fibroin. d) To prepare the freeze-dried foam, 0.1 ml of solution (c) was taken with a micropipette, transferred into a container of corresponding volume and frozen at -20°C for 24 hours. The container was then placed in a freeze-dryer and freeze-dried for 48 hours until the ice was completely removed by sublimation. After freeze-drying, the foam was solidified by immersion in methanol and dried at room temperature.

[0055] The fibroin foam samples thus obtained were stored in a desiccator at room temperature until further use.

[0056] Example 3: Incubation of fibroin foam functionalized with biomimetic peptides in a favorable environment for bone growth (in the absence of cells) The solution in which the biomimetic peptide-functionalized foam was incubated was prepared according to the method of Kokubo and Takadama (Kokubo T., Takadama H. ​​How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 2006 27(15):2907-2915). This solution will be identified hereafter by the acronym SBF (Simulated Body Fluid).

[0057] Incubation of foams in SBF was performed under the following experimental conditions: -Foam weight: 4mg -Volume of SBF solution: 40ml -Temperature: 37℃ -Maximum incubation period: 14 days -Change of SBF solution: every 3 days

[0058] At the end of the incubation period, the foams were removed, gently rinsed with distilled water and allowed to dry at room temperature before subsequent analysis.

[0059] Example 4: Morphological characterization of fibroin foams incubated in SBF by scanning electron microscopy (SEM) Fibroin foam samples were fixed to SEM specimen holders (stubs) with a diameter of 1 cm using double-sided adhesive tape. The stubs were then sputter-coated by exposure in a gold / palladium plasma (Sputter Coater Desk IV, Denton Vacuum, LLC) and analyzed under a scanning electron microscope (Zeiss EVO MA10). The results obtained are reported in Figure 1.

[0060] All samples tested, non-functionalized fibroin foam (blank) and fibroin foam functionalized with biomimetic peptides, have surface deposits of material with polyhedral crystal-like structure, which appear much brighter than the background polymeric material that constitutes the foam. These properties (ordered structure and high response intensity upon exposure to the electron beam) are typical of inorganic materials.

[0061] The density of inorganic deposits is significantly higher in the functionalized fibroin foam samples than in the blank (non-functionalized fibroin foam).

[0062] Example 5: Characterization of fibroin foam incubated in SBF by infrared spectroscopy (ATR-FTIR) Fibroin foam samples were analyzed using an ALPHA FTIR spectrometer (Bruker) equipped with an ATR Platinum Diamond accessory in the wavelength range 4000–400 cm -1 In, 4 cm -1 The resulting spectrum is reported in FIG.

[0063] By comparing the spectra of the samples incubated in SBF (blank and foams functionalized with biomimetic peptides) with a reference sample (fibroin foam not incubated in SBF), it was found that the incubated samples exhibited a spectral range of 1100–900 cm that was not present in the reference. -1It can be observed that the sponge samples exhibit IR bands of 1000 nm to 1500 nm in diameter. These bands are due to the stretching vibrations of groups such as phosphate and carbonate. The presence of these IR bands is therefore due to the incubation method in SBF. All sponge samples incubated in SBF, both blank and those functionalized with biomimetic peptides, exhibit these IR bands. However, in accordance with the morphological observations reported in Example 4, it is interesting to observe that the intensity of the bands is clearly higher in the samples functionalized with biomimetic peptides, while in the blank it remains at the baseline level.

[0064] Example 6: Chemical characterization of fibroin foam incubated in SBF using scanning electron microscopy with X-ray detector (SEM / EDX) In order to characterize morphologically and compositionally the mineral structure constituted by calcium and phosphate salts, fibroin foam samples were analyzed by SEM / EDX, and the results obtained are reported in Figure 3.

[0065] As already highlighted in Figure 1 (images C and F), there are two types of mineral deposits, one consisting of single crystals and one consisting of crystalline aggregates of variable dimensions. The single crystals are mainly composed of sodium (Na) and chlorine (Cl), while the aggregates are mainly characterized by calcium (Ca) and phosphorus (P) signals, as well as magnesium (Mg) of lower intensity. It is noted that the signals of carbon (C), oxygen (O), nitrogen (N), gold (Au) and palladium (Pd) are common to all spectra, since they reflect the organic nature of the fibroin form (C, O, N) and the sample preparation mode (Au / Pd sputtering).

[0066] Example 7: Mineralization studies of fibroin foam incubated in the absence and presence of human mesenchymal stem cells Human mesenchymal stem cells (MSCs) were purchased from Lonza and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% MSC Stimulatory Supplement, 100 U / mL penicillin-streptomycin, and non-essential amino acids at 37°C and 5% CO 2The fibroin foam was placed in a 96-well plate, soaked in 70% ethanol for 30 min, washed three times with phosphate-buffered saline (PBS), soaked overnight in 100 U / mL penicillin-streptomycin, and washed three times with PBS. MSCs (10 4 Cells were seeded by pipetting 100 μL / well of fibroin foam at different points to improve cell distribution within the foam and allowed to adhere for 30 min. Afterwards, 150 μL of complete medium was added to each well. As a negative control, fibroin foams were kept in complete cell-free medium. The medium was changed every 2 days.

[0067] Calcium deposition on decellularized fibroin foams was revealed by von Kossa staining. Prior to cell lysis and protein extraction, foams were washed twice with PBS (without magnesium / calcium ions). Cell lysis was performed with 100 mM NaF, 1 mM NaVO 4 , HEPES buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% (v / v) glycerol, 1% (v / v) Triton X-100, 1.5 mM MgCl ), containing 1 μg / mL leupeptin and 1 μg / mL aprotinin. 2 , 1 mM EGTA). After 30 min on ice, the extract was centrifuged at 13,000 g for 10 min as cell lysate and stored at -20°C. The remaining deposited matrix was washed twice with PBS (without magnesium / calcium ions). To fix the matrix onto the fibroin foam, 25% formaldehyde was added in ultrapure water for 10 min at room temperature and then washed twice with ultrapure water. A 2.5% silver nitrate solution was added to the wells containing the fibroin foam, and then they were placed under UV light for 30 min. Finally, excess unreacted silver was removed using 5% sodium thiosulfate.

[0068] Human mesenchymal stem cells (MSCs) were cultured for 7 days in fibroin foam functionalized with biomimetic peptides, using non-functionalized fibroin foam as a reference. Acellular foams were incubated for a similar time. The degree of calcium deposition was studied by von Kossa staining and quantified by image analysis using ImageJ software. Briefly, images were converted to 16-bit grayscale images. The threshold was manually adjusted and the percentage of the area covered by signals above the threshold was measured.

[0069] As shown in Figure 4, fibroin foam functionalized with biomimetic peptides shows higher calcium deposition than non-functionalized foams. The amount of calcium deposited by the action of biomimetic peptides is higher in samples incubated in the presence of MSCs than in samples incubated without MSCs.

[0070] From the detailed description and the above examples, the advantages achieved by the peptides of the invention are clear: in particular, these peptides are surprisingly and advantageously suitable for use in bone, tooth and periodontal regeneration, and have been shown to favor rapid and effective mineralization.

Claims

1. A peptide comprising the general formula (I): Xaa1-Ser-Gly-Tyr-Glu-Tyr-Xaa2 (SEQ ID NO: 5) however, - when Xaa1 is Val-Asn-Gly-Gly-Tyr (SEQ ID NO: 6), Xaa2 is Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe (SEQ ID NO: 7), or Xaa2 is Ala-Trp; - if Xaa1 is absent, Xaa2 is Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe (SEQ ID NO: 7), and - when Xaa1 is Gly-Pro-Tyr-Val-Ala-His-Gly-Gly-Tyr (SEQ ID NO: 8), Xaa2 is absent and the peptide is a peptide selected from the group consisting of: SEQ ID NO: 1 Val-Asn-Gly-Gly-Tyr-Ser-Gly-Tyr-Glu-Tyr-Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe, SEQ ID NO: 2 Ser-Gly-Tyr-Glu-Tyr-Ala-Trp-Ser-Ser-Glu-Ser-Asp-Phe, SEQ ID NO: 3 Gly-Pro-Tyr-Val-Ala-His-Gly-Gly-Tyr-Ser-Gly-Tyr-Glu-Tyr, and SEQ ID NO: 4 Val-Asn-Gly-Gly-Tyr-Ser-Gly-Tyr-Glu-Tyr-Ala-Trp.

2. Use of the peptide described in claim 1 for the preparation of a medicine.

3. The use described in claim 2, wherein the medicine is for regenerating bones, teeth and periodontium.

4. 4. The use according to claim 3, wherein bone, tooth and periodontal regeneration is in the treatment of pathologies of said bone, tooth or periodontal tissues.

5. 5. The use according to claim 4, wherein the bone, tooth or periodontal pathology is selected from the group comprising fractures, bone clefts, sarcomas, dentin hypersensitivity, pyorrhea.

6. A composition comprising one or more peptides according to claim 1, their salts, and an excipient or additive acceptable in the biomedical, cosmetic or pharmaceutical fields.

7. 7. The composition of claim 6, further comprising hyaluronic acid and / or a salt thereof, chitosan, alginic acid, silk fibroin, propylene glycol, propylene glycol alginate, poloxamer, chondroitin sulfate, collagen, gelatin, elastin, polylactic acid (PLA), poly(lactic-co-glycolic) acid (PLGA), polyglycolic acid (PGA), polyvinyl alcohol (PVA), polycaprolactone (PCL), bioglass, hydroxyapatite, calcium salts, decellularized bone matrix, pectin, sericin, cellulose, fibrin, and combinations thereof.

8. 8. The composition of claim 7, wherein the composition is in the form of a gel, paste, foam, putty, powder, membrane, fabric, compressed solid granules or film, or is contained / adsorbed within micellar elements or particles, hollow fibers or nanofibers, membranes, ceramics or nanotubes, porous and / or trabecular metal structures, textile products, nanobubbles, sol-gels, or combinations thereof.

9. 10. Use of a composition comprising one or more peptides according to claim 1 for the preparation of a medicament.

10. Use of the composition according to claim 6 for bone, tooth and periodontal regeneration.

11. 7. Use of a composition according to claim 6 for the functionalisation of prostheses, screws, fixtures, inserts or supports in metal, ceramic or natural or synthetic polymer materials.

12. 7. The cosmetic use of the composition according to claim 6.

13. 10. Use of a peptide according to claim 1 for the functionalisation of prostheses, screws, fixtures, inserts or supports made of metal, ceramic or natural or synthetic polymer materials.

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