Artificial peptide compositions for grafting

Abstract

The invention relates to an artificial peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 1 (PLV PSQ PLV PSQ PLV PSQ PQ PPLPP) (P2) and / or a peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 2 (PHQ PMQ PQP PVH PMQ PLP PQ PPLPP) (P6) for use in soft tissue transplantation surgery and / or graft surgery. The artificial peptides are in embodiments formulated as pharmaceutical compositions, such as in hydrogels, comprising combinations of hyaluronic acids and cross-linked hyaluronic acids for controlled release.

Description

ARTIFICIAL PEPTIDE COMPOSITIONS FOR GRAFTINGFIELD OF THE INVENTIONThe current invention relates to the use of artificial peptides derived from intrinsically disordered proteins (IDPs) as therapeutic and / or prophylactic agents for use in transplantation surgery and / or graft surgery. The artificial peptides are in embodiments formulated as pharmaceutical compositions, such as but not limited to, in hydrogels, comprising combinations of hyaluronic acids and cross-linked hyaluronic acids for controlled release.BACKGROUND OF THE INVENTIONTransplantation is a surgical procedure in which cells, tissue or organs are transferred from one individual - referred to as donor - to another - recipient, or from one part of the body to another. The source of the transferred material can be the patient’s own body, or that of another patient of the same, or another species.Transplantation triggers an immunological response based on the genetical background of the donor and recipient. Autologous transplants, i.e. those in which the donor is at the same time the recipient, under normal conditions trigger no, or less, immune response. Syngeneic transplants refer to transplants between two genetically identical organisms. In humans, this is the case in a transplant involving identical (i.e. monozygous) twins.Grafting is a surgical procedure in which a diseased or injured tissue is removed and replaced with healthy skin, bone or other tissue taken from one part of the body of the same or another organism.Skin grafting is used as a remedy to extensive wounding or trauma, burns, areas of extensive skin loss due to infection such as necrotizing fasciitis or purpura fulminans, as well as specific surgeries that may require skin grafts for healing to occur - most commonly removal of skin cancers. It is often done after serious injuries to replace damaged skin. Surgical removal (excision or debridement) of the damaged skin is followed by skin grafting.Skin grafting reduces the course of further treatment needed (and time spent in hospital) and improving the function and appearance of the area of the body which receives the skin graft.Grafts as well as transplants can be classified by source and purpose. By source, there can be autologous, isogenic, allogenic, xenogenic or prosthetic transplants and / or grafts. Grafts can further be classified by their thickness: split-thickness, full thickness and composite grafts.Grafts and transplants can be performed to save a patient’s life or to improve the quality of their life.There is today still a largely unmet need for potent solutions for hard and soft tissue healing and / or regeneration in relation to, or as a result of, transplantation surgery and / or grafting surgery. In addition, because of the susceptibility of both hard and soft tissue to develop post-surgery and / or post-treatment complications and disorders, it would be preferable that an administration during and in particular postsurgery could be performed by minimally invasive administration.Currently, enamel matrix derivative (EMD) is used in grafting procedures in the oral cavity. EMD is known to consist of up to 80% of amelogenin, an intrinsically disordered protein (IDP). Amelogenin is thus found to be the main active component in EMD. However, EMD is porcine-based and needs to be isolated from developing teeth in pigs. There is a need for a potent regenerative material free from animal-derived components, yet in its performance not inferior to the methods constituting the current standard in the field. In addition, since EMD is a complex mix of enamel matrix derived components, this entails potential complex immunological consequences. Also, since it comprises a main active component, it cannot be tailored to optimally effect either soft or hard tissue, thus it is suboptimal for either of the two different tissues.EP2118136 discloses artificial peptides derived from intrinsically disordered proteins (IDPs) that share essential structural characteristics with amelogenin, with improved properties for induction and / or stimulation of mineralization, in vivo and in vitro. Such peptides are provided which are easy to synthesize and methods of using the peptides for the induction and / or stimulation of mineral precipitation and / or biomineralization.The characterising sequence of amino acids of the artificial peptides disclosed in EP2118136 is a proline rich sequence prevalent in key matricellular proteins that partake in wound healing as well as in bone and cartilage formation and connective tissue maintenance in all vertebrates. This core sequence of prolines is highly conserved in vertebrates, is intrinsically disordered and does not induce any immunogenic response in humans.Of lately, the current inventors have successfully been able to demonstrate that the same group of artificial peptides can also promote healing, growth and differentiation of soft tissue, in particular the soft tissue of the craniomaxillofacial complex.FIGURE LEGENDSFigure 1. Graphical Abstract of the experimentFigure 2. Representative Masson Goldner trichrome stain slides with dashed line circling in regions of active inflammation (Sham: necrotic / encapsulated tissue; HA: Inflammatory infiltrate; EMD: Enamel MatrixDerivatives) and dotted line illustrating oedema (a-e). All histology images, except the EMD (pig 6), are from pig 2. Scalebar = 1 mm. The graph displays the mean tissue morphology score for each group (f) [1 = compromised, 3 = normal]. The score of each sample was an average of the three indicators of epithelium physiology, oedema, and inflammatory infiltrate / active infection. n=6 for Sham, HA+P2, HA+P6, and n=3 for HA and EMD.Figure 3. Zoom in on histology slide features - coagulation (a, b, c), inflammation (c, d, e), and oedema (e, f). Scalebar: 200 pm.Figure 4. (divided into 4A, 4B, 4C, 4D and 4E). Proteomic analysis of proteins extracted from the porcine gingiva. Unsupervised machine learning clustering principal component analysis (PCA) (A). Supervised machine learning clustering Partial Least Squares Discriminant Analysis (PLS-DA) (B, C). Heatmap of mean expression for each group (D) (n = 6 per group, except HA and EMD n = 3). PCA group centroids (E).Figure 5. (divided into 5A, 5B, 5C, 5D and 5E) Differentially expressed protein (DEP) volcano plot comparing the protein expression level differences between the sample groups (n = 6 per group, except HA and EMD n = 3).Figure 6. Cytotoxicity was measured using LDH assay (left), and cell viability was measured using CCK8 assay (right). n=8, *p<0.5, **p<0.01 , ***p<0.001.Figure 7. Protein levels before (a) and after (b) equal median normalisation from proteomic analysis.Figure 8. Protein expression for cytokines using a Luminex assay. n=12 for Sham, HA+P2, HA+P6, and n=6 for HA and EMD. The data has been adjusted for sample protein concentration, and outliers have been removed.Figure 9. Selected representative Immunohistochemistry staining of TNF-alpha and Mannose.Figure 10. Selected representative Immunohistochemistry staining of CD163, CD80 and IL10.Figure 11. Immunohistochemical analysis of inflammatory and macrophage markers across all groups (n = 6 per group, except HA and EMD n = 3).Figure 12. Heatmap of the Spearman correlation study between histology, IHC and key expressed proteins. The results were interpreted as follows: no correlation if |r| < 0.2; correlation if 0.2 < |r| < 0.5; and strong correlation if 0.5 < |r| < 1. Figure 13. Two-component PLS-DA of the integrated multi-omics panel. Scores of individual samples (coloured crosses) are plotted on latent variables 1 and 2; the superimposed 95% confidence ellipses outline within-group dispersion for Sham (purple), HA (green), HA+P2 (orange), HA+P6 (red), and EMD (blue) wounds. LV1 , which explains 39% of the total cross-block variance, orders the samples along an untreated-to-treated gradient, while LV2 (24%) discriminates between the two peptide formulations. Absence of ellipse overlap indicates that each biomaterial induces a reproducibleand statistically distinct early molecular signature. Figure 14. (divided into 14A and 14B) Two- component PLS-DAof the early multi-omics response. (A) Scores plot of individual wounds on latent variables 1 and 2; colours denote the five treatment groups (EMD, HA, HA+P2, HA+P6, Sham). Each symbol represents one quartile defect (n = 6 per group, except for HA and EMD, which have n = 3). (B) Group centroids (means ± 0 along both axes) extracted from the same score matrix. Centroids illustrate a treatment gradient along LV1 (Sham — > HA — > EMD) and highlight the distinct positioning of the two peptide formulations on LV2, with HA+P6 showing the most considerable positive shift.Figure 15. (divided into 15A and 15B) Heatmap of the Spearman correlation study between key proteins only for HA+P6 (A) and EMD (B). The results were interpreted as follows: no correlation if |r| < 0.2; correlation if 0.2 < |r| < 0.5; and strong correlation if 0.5 < |r| < 1.Figure 16. Pig 1 : LHS: images of intervention area moments after euthanasia. RHS: corresponding histology images with Masson Goldner Trichrome staining. Scale bars = 1 mm.Figure 17. Pig 2: LHS: images of intervention area moments after euthanasia. RHS: corresponding histology images with Masson Goldner Trichrome staining. Scale bars = 1 mm.Figure 18. Pig 3: LHS: images of intervention area moments after euthanasia. RHS: corresponding histology images with Masson Goldner Trichrome staining. Scale bars = 1 mm.Figure 19. Pig 4: LHS: images of intervention area moments after euthanasia. RHS: corresponding histology images with Masson Goldner Trichrome staining. Scale bars = 1 mm.Figure 20. Pig 5: LHS: images of intervention area moments after euthanasia. RHS: corresponding histology images with Masson Goldner Trichrome staining. Scale bars = 1 mm.Figure 21. Pig 6: LHS: images of intervention area moments after euthanasia. RHS: corresponding histology images with Masson Goldner Trichrome staining. Scale bars = 1 mm.Figure 22. Pig 1 : Toluidine blue stains. This stain was prone to artefacts (purple needle-like spots. Scale bars = 1 mm.Figure 23. Pig 2: Toluidine blue stains. This stain was prone to artefacts (purple needle-like spots. Scale bars = 1 mm.Figure 24. Pig 3: Toluidine blue stains. This stain was prone to artefacts (purple needle-like spots. Scale bars = 1 mm.Figure 25. Pig 4: Toluidine blue stains. This stain was prone to artefacts (purple needle-like spots. Scale bars = 1 mm.Figure 26. Pig 5: Toluidine blue stains. This stain was prone to artefacts (purple needle-like spots. Scale bars = 1 mm.Figure 27. Pig 6: Toluidine blue stains. This stain was prone to artefacts (purple needle-like spots. Scale bars = 1 mm.Figure 28. General set-up of experiment 2.DEFINITIONS AND ABBREVIATIONSIn the present context, an “artificial peptide” refers to a peptide that is a non-natural peptide in the sense that it does not normally occur in nature but is the product of amino acids put together and selected in an order, amount and manner generating peptides suitable for use in the context of the present invention. An “artificial peptide” is still a peptide embraced by the present invention even though it might encompass parts of or a whole peptide which happens to be present in nature. “Artificial” may be used interchangeably with terms such as “synthetic” or “non-natural”.In the present context “Pro” denotes the amino acid proline.In the present context “X” denotes a hydrophobic amino acid. A hydrophobic amino acid is, in the present context, defined as an amino acid selected from the group consisting of: Ala, lie, Leu, Met, Phe, Trp and Vai.In the present context “Y” denotes a polar amino acid. A polar (“hydrophilic”) amino acid is, in the present context, defined as an amino acid selected from the group consisting of: Asn, Cys, Gin, Ser, Thr and Tyr.In the present context, common nomenclature is used for denoting amino acids. Therefore, for example, A is Ala (hydrophobic), C is Cys (polar), F is Phe (hydrophobic), H is His, I is lie (hydrophobic), L is Leu (hydrophobic), M is Met (hydrophobic), N is Asn (polar), Q is Gin (polar), S is Ser (polar), T is Thr (polar), V is Vai (hydrophobic), W is Trp (hydrophobic), Y is Tyr (polar).P2: A peptide with the amino acid sequence as shown in SEQ ID NO: 1 (PLV PSQ PLV PSQ PLV PSQ PQ PPLPP) (P2)P6: A peptide with the amino acid sequence as shown in SEQ ID NO: 2 (PHQ PMQ POP PVH PMQ PLP PQ PPLPP) (P6)HA: Hyaluronic acid;Hyaluronic acid is a natural and linear carbohydrate polymer belonging to the class of non-sulphated glycosaminoglycans. It is composed of beta-1 ,3-A / -acetyl glucosamine and beta-1 , 4-glucuronic acid repeating disaccharide units with a molecular weight (MW) up to 6 MDa. HA is present in hyaline cartilage, synovial joint fluid, and skin tissue, both dermis and epidermis.BDDE: 1 ,4-butanediolPEGDE: poly(ethylene glycol) diglycidyl etherlogFC, or log fold change, is a measure used in gene expression analysis (like RNA-Seq) to quantify the difference in gene expression between two groups, typically a control and a treatment group. A positive logFC indicates upregulation (increased expression) in the treatment group, while a negative logFC indicates downregulation (decreased expression).Human genes nomenclature: ACTB - actin beta, H2A- histone H2A, SERPINA1 - serpin family A member 1 , HSPA1A- heat shock protein family A (Hsp70) member 1A, ANXA1 - annexin A1 , A8 - protein A8, ENO1 - enolase 1 , TFRC - transferrin receptor, PKM - pyruvate kinase M1 / 2, IL-1RA- interleukin-1 receptor antagonist, and IL-8 - interleukin-8The term “biocompatible” as used herein refers to causing no clinically relevant tissue irritation, injury, toxic reaction, or immunological reaction to living tissue.The term “cell” is used herein to refer to the structural and functional unit of living organisms and is the smallest unit of an organism classified as living.The term “compatible” as used herein means that components of a composition are capable of being combined with each other in a manner such that there is no interaction that would substantially reduce the efficacy of the composition under ordinary use conditions.The term “component” as used herein refers to a constituent part, element, or ingredient.The term “condition” as used herein refers to a variety of health states and is meant to include disorders or diseases caused by any underlying mechanism or disorder, injury, and the promotion of healthy tissues and organs.The term “differentiation” as used herein refers to the process of development with an increase in the level of organization or complexity of a cell or tissue, accompanied with a more specialized function.The terms “disease” and “disorder” as used herein refer to an impairment of health or a condition of abnormal functioning.The term “peptide” is used herein to refer to two or more amino acids joined by a peptide bond.It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.As used herein, the term “comprising” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term “comprising” is intended to include embodiments encompassed by the terms “consisting essentially of’ and “consisting of’. Similarly, the term “consisting essentially of’ is intended to include embodiments encompassed by the term “consisting of’.As used herein, the term “about” modifying the quantity of an ingredient or reactant employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities.Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding both of those included limits are also included in the invention.It Is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials have been described. All publications mentioned herein are incorporated to disclose and describe the methods and / or materials in connection with which the publications are cited.SUMMARY OF THE INVENTIONThe current invention is based on the surprising finding that not all artificial peptides derived from intrinsically disordered proteins (IDPs) promote the proliferation and / or differentiation equally in all tissue types, but that specific peptides either promote growth and / or regrowth of soft or hard tissue types differently, and that the combination of both actions will act on non-mineralized tissue types, such as cartilage, ligaments and tendons. This is in particular important for the taking of grafts and in transplantation surgery, where it is of utmost importance to be able to stimulate the regrowth of the one or the other tissue type, depending on the nature of the tissue being grafted or transplanted. The invention thus relates to the use of the specific peptides for targeted tissue healing and / or regeneration in transplantation surgery and / or graft surgery. Thus, the current invention allows for a tailored support of transplantation surgery and / or graft surgery.The current invention relates to a peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 1 (PLV PSQ PLV PSQ PLV PSQ PQ PPLPP) (P2) and / or a peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 2 (PHQ PMQ POP PVH PMQ PLP PQ PPLPP) (P6) for use in transplantation surgery and / or graft surgery. The said peptide and / or peptides are meant for use according to the above description, in particular, in allo-transplantation, auto-transplantation, iso-transplantation and / or xeno-transplantation as well as in allo-graft, auto-graft, iso-graft and / or xeno-graft.The peptide and / or peptides for use is / are synthetically, biosynthetically and / or recombinantly produced peptide(s).In one embodiment, the peptide(s) is / are at least 75%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (P2). In one currently preferred embodiment, the peptide for use according to the current invention is identical to SEQ ID NO: 1 (P2). Said peptide for use according to the current invention is intended for use in soft tissue grafting, in particular in supporting and / or inducing revascularisation in skin grafting. In another aspect, the peptide for use according to the current invention may be for use in mucosal and / or gingival grafting. In another aspect of the invention, the peptide for use according to the current invention may be for use in skin grafting in connection with burns. In particular, the said peptide may be for skin grafting in the treatment and / or reduction of chronic inflammatory conditions. The said peptide may be used for reduction of inflammation in soft tissue.In another embodiment, the peptide for use according to the current invention is at least 75%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 (P6). In one currently preferred embodiment, the peptide for use according to the current invention is identical to SEQ ID NO: 2 (P6). Said peptide for use according to the current invention is preferably used in hard tissue grafting and / or transplantation. In particular, the said peptide may be used in bone-grafting. In addition, said peptide can also be used in soft tissue grafting, in particular in supporting and / or inducing revascularisation in skin grafting and in particular in combination with the peptide P2, as well as for reduction of inflammation in soft tissue.In another aspect the invention relates to a composition comprising a combination of at least one peptide which is at least 75% identical to P2 with a peptide which is at least 75% identical to the sequence shown in P6 for use in cartilage, tendons and / or ligament grafting and / or transplantation. Furthermore, the said combination may be used in procedures of grafting and / or transplantation in a joint, more specifically, a knee joint or a jaw joint or any other joint. In another aspect, the said combination for use may be used in procedures of grafting and / or transplantation of a ligament. More specifically, the said combination may be used in the mentioned procedures relating to periodontal ligament and / or cruciate ligament.In one embodiment, the said peptide and / or peptides regulate gene expression in the cell, tissue and / or organ that they are applied to. More specifically, the said peptide and / or peptides regulate genes selected from the group of genes consisting of: ACTB, H2A, SERPINA1 , HSPA1 A, ANXA1 , A8, ENO1 , TFRC, PKM, IL-1RA, and IL-8.In another aspect, the current invention relates to a pharmaceutical composition comprising the said peptide and / or peptides for use formulated in a hyaluronic acid (HA) gel. Furthermore, the HA gel may comprise linear hyaluronic acid fibres (HA) and / or cross-linked hyaluronic acid fibres (HA-XL). More specifically, the HA gel may comprise 1 ,4-butanediol diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE)) and / or poly(ethylene glycol) diglycidyl ether (PEGDE) cross-linked hyaluronic acid fibres (HA-XL(PEGDE)). The said composition for use according to the current invention may be biocompatible and / or biodegradable.DETAILED DESCRIPTION OF THE INVENTIONThe current invention is based on the surprising finding that not all artificial peptides derived from intrinsically disordered proteins (IDPs) promote the proliferation and / or differentiation in all tissue types equally well, but that specific peptides either particularly promote growth and / or regrowth of soft or hard tissue types, and that the combination of both will act on non-mineralized tissue types, such as cartilage, ligaments and tendons. This is in particular important for the taking of grafts and in transplantation surgery, where it is of utmost importance to be able to stimulate the regrowth of the one or the other tissue type, depending on the nature of the tissue being grafted or transplanted. The invention thus relates to the use of for the tissue-type specifically selected peptides for targeted tissue healing and / or regeneration in transplantation surgery and / or graft surgery. Thus, the current invention allows for a tailored support of transplantation surgery and / or graft surgery.Peptides of the inventionP2P2 is an artificial peptide with the sequence PLV PSQ PLV PSQ PLV PSQ PQ PPLPP (SEQ ID NO: 1), based on a proline-rich sequence resembling amelogenin. As can be seen in the experimental section, P2 has by the inventors surprisingly been found to e.g., upregulate actin (Q6QAQ1) and histone (F2Z5L5), thus indicating that P2 is effective in stimulating soft tissue regeneration and / or regrowth. Thus, it is envisioned that P2 will be effective in supporting e.g., but not limited to, skin grafting and / or skin grafting in connection with burns.As can be seen in the experimental part, P2 is found to induce extracellular matrix formation and thus be most efficient for stimulating soft tissues. P2 attenuates acute inflammation and reduces secondary inflammatory markers downstream. On the protein level, this involves regulating alpha-1-antitrypsin,which has a broad anti-inflammatory, immunomodulatory and tissue-repair effect through neutralisation of proteolytic enzymes, regulating annexin A1 that resolves inflammation by reducing leukocyte infiltration activating neutrophile apoptosis and induces macrophage reprogramming towards a resolving phenotype, both of which effects play a role in inflammation fighting, regulating HSPAIAthat stimulates the secretion of pro-inflammatory cytokines and / or regulating S100 calcium-binding protein A8 that has an immune regulatory effect by initially enhancing production of reactive oxygen species and by elevating expression of anti-inflammatory IL-10.As shown in the experimental section, integrating peptide P2 into hyaluronic acid gel enhances gene response in gingival tissue that favours extracellular matrix formation, which is crucial for wound healing in oral mucosa.In consequence of the findings, peptide P2 is particularly effective for use in soft tissue healing and / or regenerating procedures, such as in soft tissue grafting procedures and / or soft tissue transplantation procedures, such as but not limited to, for supporting and / or inducing revascularisation in skin grafting. Furthermore, the said peptide may be used for improving and / or supporting mucosal and / or gingival grafting. In another aspect, the peptide P2 can be used in the treatment and / or reduction of chronic inflammatory conditions, such as but not limited to, for reduction of inflammation in soft tissue in relation to a grafting and / or transplantation procedure, such as in relation to a grafting and / or transplantation surgery.It is envisioned that the above effect on soft tissue healing will be demonstrated by any peptide that is at least 75%, such as at least 80% identical to P2.P6P6 is an artificial peptide with the sequence PHQ PMQ PQP PVH PMQ PLP PQ PPLPP (SEQ ID NO: 2).P6 has by the inventors surprisingly been found to e.g., upregulate pyruvate kinase, actine, histone H2A and IL-8, thus indicating that P6 is most effective in stimulating hard tissue regeneration and / or regrowth and / or mineralization. Thus, it is envisioned that P6 will be effective in supporting e.g., but not limited to, bone grafting and / or bone transplantation.P6 is in the experimental section demonstrated to improve mineralization in bone formation and thus to be useful in supporting and / or promoting bone grafting. P6 upregulates genes that may enhance cellular processes in hard tissue, such as pyruvate kinase. At the same time, P6 is found to downregulate alpha- 1 -anitrypsin , which reduces inflammatory response in hard tissue. Thus, the peptide P6 is particularly useful in hard tissue grafting and / or transplantation such as in bone grafting. In addition, it was demonstrated that P6 can support P2 in soft tissue wound healing, in particular in a grafting and / or transplantation procedure, such as in relation to a grafting and / or transplantation surgery.It is envisioned that the above effect on hard tissue healing and / or mineralization will be demonstrated by any peptide that is at least 75%, such as at least 80% identical to P6.P2+P6Both peptides P2 and P6 have been found to nucleate calcium phosphate clusters. In one aspect, the combination of peptides P2 and P6 can be used to foster collagen formation and so promote graft taking and transplantations of a cartilage, tendon and / or ligament. Furthermore, the said combination can be used in procedures of grafting and / or transplantation related to a joint, such as but not limited to a knee joint and / or jaw joint. In another aspect, the combination of said peptides can be used in procedures related to ligament transplantation and / or grafting, such as but not limited to, periodontal ligament and cruciate ligament transplantation and / or grafting.The artificial peptides according to the current invention are biomimicking intrinsically disordered proteins (IDPs)The artificial peptides according to the current invention are biomimetic peptides inspired by the motifs found in amelogenins. Due to intrinsic disorder, they are flexible peptides that dynamically adapt to the local environment. They are thereby able to interact with other structural polymers such as collagen, giving an improved appearance. This is e.g., done by the peptides folding into a temporary extracellular matrix that can e.g., bind to surfaces of the mucous membrane. This protects in particular the soft tissue of the mucous membrane providing a prophylactic function. The peptides have also been demonstrated to nucleate calcium phosphate and to orient the crystallite growth into lamella-like platelets, thereby having the ability to form a protective mineral layer on the dental enamel.Under physiological conditions i.e., conditions of the external or internal milieu that may occur in nature for an organism, such as a human being, the peptides are generally intrinsically disordered. The release of peptides from a gel matrix is usually tied to the structure of the peptides / proteins, wherein intrinsically disordered proteins / peptides are generally more easily released from the gel than structured proteins and / or peptides, due to their high degree of conformational flexibility. This feature makes the artificial peptides of the current disclosure highly useful as constituent to be released from a gel, such as a crosslinked HA-XL gel and / or a combined HA-HA-XL gel.One of the advantages of using synthetic peptides is that it is not always practical to use natural peptides and / or proteins for medical and / or cosmetic uses. For example, natural proteins are often long, which means that they are difficult to synthesize, both chemically and by cellular expression systems. Typically, artificial peptides are easier to synthesize than full, larger proteins, as peptides often lack the complicated higher order structure that make large proteins difficult to synthesize. Also, a natural protein only contains natural amino acids and may therefore be susceptible to rapid degradation. Also, if purified from a natural environment, such as developing teeth, there is always a risk of contamination of other products whiche.g., may cause allergic reactions. In addition, a long natural protein normally has many roles in a living body and may therefore not be optimized for the intended use.Furthermore, the active motif of the synthetic peptides in the use of the current invention can be designed to specifically stimulate healing and / or growth of soft tissue or hard tissue or cartilage or ligaments or tendons.The artificial peptide(s) comprised in the formulation of the current invention is / are characterized by a proline sequence prevalent in key matricellular proteins that partake in wound healing as well as in bone and cartilage formation and connective tissue maintenance in the majority of vertebrates.The amino acids in an artificial peptide of the invention may further be modified in terms of chemistry, isometry or in any other way, as long as the sequences of the peptides are intact. Modifications of the amino acids of the artificial peptides of the invention may increase the activity, stability, biocompatibility, or clinical performance of the peptides, or reduce toxicity and adverse reactions to the peptides. Examples of chemical modifications include, but are not limited to, glycosylation and methylation. The amino acids may also be of all different types of stereoisomeric forms, such as D or L forms of amino acids, or S or R isomers. The amino acids in an artificial peptide of the invention may also be replaced by synthetic analogues thereof. The use of synthetic analogues may e.g., result in a peptide that is more stable and less prone to degradation. Examples of unnatural amino acids include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-CI-phenylalanine*, p-Br-phenylaianine*, p-l-phenylatanine*, L-allyl-glycine*, - alanine*, L-a-amino butyric acid*, L-g-amino butyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid#, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L- phenylalainine*, L-hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4- methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1 , 2,3,4- tetrahydr- oisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid # and L-Phe (4-benzyl)*. The notation * is herein utilised to indicate the hydrophobic nature of the derivative whereas # is utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.The above identified peptides are artificial (synthetic) peptides comprising a poly-proline consensus sequence, further comprising hydrophobic > ) and polar amino acids > ). It induces and / or stimulates specifically healing and / or growth of soft tissue or hard tissue or cartilage or ligaments or tendons or tendons in biological systems, and are for use clinically, industrially, chemically or otherwise to stimulate the formation of soft tissue or hard tissue or cartilage or ligaments or tendons.The proline content and arrangement in the protein sequences used for constructing the consensus sequence of the artificial peptides are based on and to some extent included in the sequences for collagen 1 and 2 (human, mouse and rat), amelogenin (human, mouse, rat, rabbit, pig and cow), ameloblastin (human, rat), bone sialoprotein (human, mouse), enamelin (human, mouse).The artificial peptides comprised in the formulation of the current invention are particularly suitable for the induction and / or stimulation of healing and / or growth of soft tissue or hard tissue or cartilage or ligaments or tendons, as the amino acid sequences are optimised for this purpose. The use of an artificial peptide according to the invention is advantageous due to its shorter length compared to natural peptides, which facilitates the synthesis thereof and allows for the use of amino acid analogues as explained herein. Also, the use of an artificial peptide allows modifications of the amino acid sequence to enable the peptides to bind to e.g., metal surfaces or being easily purified, such as by the choice of amino acid sequences of the peptide itself or the use of N- and / or C-terminal tags.Therefore, in one aspect, the present invention relates to a formulation comprising an artificial peptide comprising an amino acid sequence of SEQ ID NO 1 and / or SEQ ID NO 2, which is able to induce and / or specifically stimulate healing and / or growth of soft tissue or hard tissue or cartilage or ligaments or tendons. Preferably, such an artificial peptide consists of an amino acid sequence as shown in SEQ ID NO 1 or SEQ ID NO 2, although it is envisioned that the above effect on induction and / or stimulation of healing and / or growth of soft tissue or hard tissue or cartilage or ligaments or tendons will be demonstrated by any peptide that is at least 75%, such as at least 80% identical to P2 or P6.Further aspects of the invention relate to a use according to the current invention comprising one or more artificial peptides having between 80-100% identity with any one of the sequences of SEQ ID NO 1-2, such as peptides having 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 97, 98 or 99 % identity with the sequences of SEQ ID NO1-2.In embodiments, a composition for use according to the current invention comprises one or more artificial peptides disclosed herein which consist of a sequence that is at least 75% identical to any one of SEQ ID NO 1-2, respectively.In a composition for use according to the current invention, a peptide may further comprise N- and / or C- terminal tags comprising the amino acids His and / or Met. Met contains sulphur, which as previously explained facilitates binding to metal surfaces. His has a strong affinity for e.g., Ni and other metals. The use of these tags therefore has the advantage of enabling the peptides to attach to metal surfaces like titanium, zirconium, aluminium, tantalum, gold, surgical steel and nickel, or a metal oxide hydroxide and / or hydride surface etc. The C- and / or N-terminal tags are also useful in the process of purification of produced peptides, as is well known to the skilled person. The use of an N-terminal and / or C-terminal tag also allows the peptide to be fully exposed, i.e., the tag is used for binding the peptide to a surface and the rest of the peptide is free for interactions with e.g., atoms, molecules, cells and tissue. The use of one tag in each end of a peptide may be useful during production of the peptide, allowing one end of the peptide to be attached to a column during the purification of the peptide of interest from incomplete peptide products, while the other end of the peptide may be used for binding to a surface of interest.Consequently, one preferred embodiment of the invention relates to a composition for use according to the current invention comprising an artificial peptide as defined herein, further comprising an N-terminal and / or a C-terminal histidine tag. Such a tag may, as previously mentioned, comprise methionine and / or histidine residues, which have been attached to an artificial peptide according to the invention. In a preferred embodiment, this tag comprises 3 or more residues, such as between 3-5 or 5-10 residues. A tag can comprise any number of residues attached to an artificial peptide according to the invention, which still provides for a stable composition together with the artificial peptide according to the invention not affecting the secondary structure of the artificial peptide in a negative manner. Preferably this histidine tag consists of five histidine residues. In another preferred embodiment the artificial peptide comprises an N-terminal and / or C-terminal methionine tag, preferably consisting of five methionine residues. In another preferred embodiment, a peptide of the invention comprises a methionine tag in its C- or N-terminal end and a histidine tag in the other end.Peptide sequencesIn embodiments, an artificial peptide(s) is used which comprises or consists of one or more of the amino acid sequences selected from Table 1 :Table 1 : Exemplified peptide sequencesPeptide IdentityTwo peptides are considered to be identical if their sequences read exactly the same. Otherwise, sequence similarity can be described in fractional identity between two sequences, as referred to in BLASTP algorithm.Methods to determine identity and similarity are codified in publicly available programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J et al (1994)) BLASTP, BLASTN, and FASTA (Altschul, S.F. et al (1990)). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S.F. et al, Altschul, S.F. et al (1990)). Each sequence analysis program has a default scoring matrix and default gap penalties. In general, a molecular biologist would be expected to use the default settings established by the software program used.In the current context, Pro is Proline (Pro); L is Leucine (Leu), V is Valine (Vai), S is Serine (Ser), Q is Glutamine (Gin), H is Histidine (His), M is Methionine (Met), C is Cysteine (Cys), X is an amino acid selected from the group consisting of A (Alanine, Ala), I (Isoleucine, lie), L (Leucine, Leu), M (Methionine, Met), F (Phenylalanine, Phe), W (Tryptophan, Trp) and V (Valine, Vai), preferably lie, Leu, Vai and Met; Y is an amino acid selected from the group consisting of N (Aspargine, Asn), C (Cysteine, Cys, CysH), Q (Glutamine, Gin), S (Serine, Ser), T (Threonine, Thr) and Y (Tyrosine, Tyr), preferably Ser and Gin.In embodiments, an artificial peptide(s) is selected from the group consisting of artificial peptides comprising the amino acid sequence of SEQ ID NO 1 and SEQ ID NO 2.In embodiments, an artificial peptide(s) is selected from the group consisting of artificial peptides which are at least 90% identical to the amino acid sequence of SEQ ID NO 1 or 2, respectively, such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO 1 , or SEQ ID NO 2.In embodiments, an artificial peptide(s) is selected from the group consisting of artificial peptides which are at least 75% identical to the amino acid sequence of SEQ ID NO 1 or 2, respectively, such as at least 80%, 85%, 90%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO 1 , or SEQ ID NO 2.Typically, artificial peptides comprised in the formulations of the current invention are designed with constraints on length of no more than 30 amino acids, such as of no more than 29 amino acids, such as of no more than 28 amino acids, such as of no more than 27 amino acids, such as of no more than 26 amino acids, such as of no more than 25 amino acids, such as of no more than 24 amino acids, such as of no more than 23 amino acids, such as of no more than 22 amino acids, such as of no more than 21 amino acids, such as of no more than 20 amino acids, such as of no more than 19 amino acids, such as of no more than 18 amino acids, such as of no more than 17 amino acids, such as of no more than 16 amino acids, such as of no more than 15 amino acids, such as of no more than 14 amino acids, such as of no more than 13 amino acids, such as of no more than 12 amino acids.The designed peptides (P2, and P6) differ in their physical, chemical, and structural properties due to the different amino acid compositions, except at the conserved proline positions in the sequence. A recently developed web-based method of peptide design was used to explore possible correlation between the structure and biological response to the particular peptide.Production means of the peptides of the inventionThe peptide(s) for use according to the current invention can be chemically synthesized.Peptides are chemically synthesized by the condensation reaction of the carboxyl group of one amino acid to the amino group of another. During the synthesis process, non-specific side reactions of the various amino acid side chains are usually prevented by using protective groups. Peptides can bechemically synthesised via classical solution-phase techniques or - more prevalent nowadays - solid phase methods. Chemical synthesis offers advantages over bioproduction in that the products can incorporate non-natural amino-acids and / or D-amino-acids and peptide backbone modifications can be introduced. Moreover, the method does not discriminate between peptide sequences difficult to express naturally.In chemical synthesis, the nascent peptide chain is created in the C-to-N direction, that is reverse as compared to the biosynthesis occurring in living organisms. The process of building a peptide chain has a complex nature and occurs stepwise to ensure precision and accuracy.According to the current technologies, peptides can also be produced using cell-free protein synthesis platforms. These work as in vitro transcription-translation systems using DNA templates for peptide production outside of a cell environment.Solid Phase Peptide SynthesisPeptides can be produced using Solid Phase Peptide Synthesis (SPPS). According to this method, the first, C-terminal amino acid is immobilized on solid support in a form of a resin with its alpha-amino group protected and temporary protection groups on its reactive side chains. In each elongation step, the alphaamino protective group is removed, facilitating peptide bond formation with the amino-acid to follow in the sequence. Fully assembled peptide is cleaved from the resin following removal of the protective groups. The process is industrially scalable and economically efficient, while all production steps can be performed in certified cGMP facilities.Peptide biosynthesisThe terminology “Peptide biosynthesis” is herein understood to be naturally occurring peptide synthesis taking place in the cells of a living organism or in cultured cells, not involving modification or recombination of the genetic material encoding the peptide.Recombinant peptide synthesisPeptides can be produced recombinantly using biotechnological methods that involve genetically engineered organisms such as bacteria, yeast or mammalian cells. In this case, a genetic construct encoding the peptide sequence is incorporated into that of the organism or cell being used and transcribed and / or translated using the cell machinery.The peptide and / or peptides for use according to the current invention can be produced synthetically, biosynthetically and / or recombinantly.Soft tissueSoft tissue is all the tissue in the body that is not hardened by the processes of ossification or calcification, such as bones and teeth. Soft tissue connects, surrounds or supports internal organs and bones, and includes muscles, tendons, ligaments, fat, fibrous tissue, lymph and blood vessels, fasciae,and synovial membranes. The term “soft tissue” is commonly used to describe muscles, tendons, ligaments and / or fascia, but several other tissue types and body systems contain soft tissue as well, including fat, skin, nerves, and blood vessels.Soft tissue disorders, diseases and damages are medical conditions affecting soft tissue. They include trauma, wounds and soft tissue injuries to connective and / or epithelial tissue.In the craniomaxillofacial complex, soft tissue disorders include periodontitis, periimplantitis, perimucositis, gingivitis, aphthous stomatitis and other oral infections and / or inflammations.Hard tissueHard tissue, refers to calcified tissue, is the tissue which is mineralized and has a firm intercellular matrix. The hard tissues of humans are bone, tooth enamel, dentin, and cementum. The term is in the current context used in contrast to soft tissue.All hard tissues are located in the oral environment and only one is located both within and external to this environment (i.e., bone).Connective tissue - Cartilage, ligaments and tendonsA ligament is an elastic band of tissue that connects bone to bone and provides stability to the joint. Cartilage is soft, gel-like padding between bones that protects joints and facilitates movement.Ligaments are fibrous connective tissues, but they connect bones to other bones, providing stability and limiting excessive movement in joints. Cartilage is a flexible connective tissue found in joints, ears, nose, and other body parts, providing support and flexibility.Cartilage is a specialized form of connective tissue. Composed of cells (chondrocytes) and an extracellular matrix composed of fibres and ground substance. The three types of cartilage include hyaline, elastic, and fi brocartilage.Cartilage is composed of specialized cells called chondrocytes that produce a large amount of collagenous extracellular matrix, abundant ground substance that is rich in proteoglycan and elastin fibres. Cartilage is classified into three types — elastic cartilage, hyaline cartilage, and fibrocartilage — which differ in their relative amounts of collagen and proteoglycan.A tendon is a fibrous connective tissue that attaches muscle to bone. Tendons may also attach muscles to structures such as the eyeball. A tendon serves to move the bone or structure.A tendon or sinew is a tough band of dense fibrous connective tissue that connects muscle to bone. It sends the mechanical forces of muscle contraction to the skeletal system, while withstanding tension.Tendons, like ligaments, are made of collagen. The difference is that ligaments connect bone to bone, while tendons connect muscle to bone.PatientsIn the current context, patients are mammals, such as animals and humans undergoing a transplantation and / or grafting procedure.The term “mammal” is intended to indicate a member of any mammalian species which may advantageously be treated by the method of the invention, including domesticated mammals such as horses, cattle, pigs, dogs and cats, or, preferably, humans.Wound healingIn the current context, wound healing refers to a living organism's replacement of destroyed or damaged tissue by newly produced tissue.In undamaged skin, the epidermis (surface, epithelial layer) and dermis (deeper, connective layer) form a protective barrier against the external environment. When the barrier is broken, a regulated sequence of biochemical events is set into motion to repair the damage. This process is divided into predictable phases: blood clotting (hemostasis), inflammation, tissue growth (cell proliferation), and tissue remodeling (maturation and cell differentiation). Blood clotting may be considered a part of the inflammation stage instead of a separate stage.The wound healing process has a complex and fragile nature, and it is susceptible to interruption or failure leading to the formation of non-healing chronic wounds. Factors that contribute to non-healing chronic wounds are diabetes, venous or arterial disease, infection, and metabolic deficiencies of old age.Transplantation procedures and grafting proceduresThe current invention is based on the fact that the artificial proline-rich peptide(s) described herein have different stimulatory effects on different types of tissue alone or in combination and / or in combination with a hydrogel. The peptide(s) are beneficial agents in wound healing and tissue regeneration both for hard tissue and soft tissue and in combination for cartilage, ligaments and / or tendons.Accordingly, the current invention relates to the use of the composition disclosed herein in therapy and prophylaxis of inflammation and wound healing in transplantation and / or grafting procedures in mammals in need thereof for use in allo-transplantation, auto-transplantation, iso-transplantation, xenotransplantation allo-graft, auto-graft, iso-graft and / or xeno-graft.Transplantation proceduresTransplantation is a surgical procedure in which an organ / s, tissue or group of cells are removed from one person (the donor) and transplanted into another person (the recipient) or moved from one site to another in the same person.Askin graft is a common example of a transplant from one part of a person’s body to another part.A transplant between two people can cause a rejection process where the immune system of the recipient or host attacks the foreign donor organ or tissue and destroys it. To reduce the risk of rejection of the donated organ / s, the recipient will likely need to take immunosuppressive medication for the rest of their life.Organs and / or tissues that are transplanted within the same person's body are called autografts. Transplants that are performed between two subjects of the same species are called allografts. Allografts can either be from a living or cadaveric source.Organs that have been successfully transplanted include the heart, kidneys, liver, lungs, pancreas, intestine, thymus and uterus. Tissues include bones, tendons (both referred to as musculoskeletal grafts), corneae, skin, heart valves, nerves and veins. Corneae and musculoskeletal grafts are the most commonly transplanted tissues.In the current invention, the peptide and / or peptides is / are for use in allo-transplantation, autotransplantation, iso-transplantation, and / or xeno-transplantation.Grafting ProceduresGrafting refers to a surgical procedure to move tissue from one site to another on the body, or from another creature, without bringing its own blood supply with it. Instead, a new blood supply grows in after it is placed. A similar technique where tissue is transferred with the blood supply intact is called a flap. In some instances, a graft can be an artificially manufactured device. Examples of this are a tube to carry blood flow across a defect or from an artery to a vein for use in hemodialysis.In the present context, the term “take of a graft” is intended to indicate the entire healing process involved in the grafting procedure from the initial attachment of the graft to proliferation of fibroblasts, generation of granulation tissue, production of collagen by fibroblasts, and revascularization, and, in case of surface grafts such as skin or mucosal grafts, keratinocyte migration into the graft bed.The term grafting is most commonly applied to skin grafting; however many tissues can be grafted: skin, bone, nerves, tendons, neurons, blood vessels, fat, and cornea are tissues commonly grafted today.In the current invention, the peptide and / or peptides is / are for use in allo-graft, auto-graft, iso-graft, and / or xeno-graft.Revascularization in grafting and / or transplantation proceduresFor a graft or transplant to be successful, blood supply for it needs to be established shortly after the grafting or transplantation procedure, allowing for a proper transfer of nutrients into the newly accepted cell, organ and / or tissue. This can be achieved by reconnection of vessels, ingrowth of recipient vasculature, outgrowth of donor-derived vessels and / or recruitment of bone marrow-derived endothelialprogenitor cells. The above process is called revascularisation, and it plays a crucial role in graft taking and in transplantation in a long perspective.In one aspect of the current invention, peptide and / or peptides are used in supporting and / or inducing revascularisation in transplantation and / or grafting procedures. The peptide for use in skin grafting is at least 75%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (P2) or to SEQ ID NO: 2 (P6).Preferably, the peptide for use in in supporting and / or inducing revascularisation in transplantation and / or grafting procedures is identical to SEQ ID NO: 1 (P2) or to SEQ ID NO: 2 (P6).Skin and mucosal graftingThe term “mucosa” as used herein refers to a mucous tissue lining various tubular structures consisting of epithelium, lamina propria, and, in the digestive tract, a layer of 45 smooth muscle. The term “mucosal graft” as used herein refers to a graft of mucus membrane.In dermatological surgery, skin grafts are most commonly used to repair lesions occurring after surgical excisions such as the removal of skin cancers, traumatic lesions, e.g. resulting from accidents, burns (whether thermal, chemical or electrical) or pathological processes e.g. leg or foot ulcers.Depending on the type of lesion to be repaired by grafting, e.g. whether it is a deep or more superficial lesion, and location of the lesion, e.g. whether the recipient (graft) bed comprises a sufficient vascular supply for capillary regrowth or whether the tissue at the recipient site is an exposed bone, cartilage, ligament or tendon which does not contain a sufficient vascular supply, different types of graft will normally be applied. Thus, full-thickness skin grafts have traditionally been employed to repair facial lesions because such grafts often provide a more aesthetically pleasing result. "Full-thickness skin grafts" are intended to indicate grafts which are composed of both the epidermis and the entire thickness of the dermis, including structures such as hair follicles, sweat glands and nerves. Full-thickness skin grafts are therefore also preferred for use in connection with hair transplants. When performing full-thickness skin transplants, donor skin is excised from a suitable site and defatted (i.e. adipose tissue is removed from the graft). The recipient bed is cleaned with an antibacterial agent and rinsed. The graft is suitably trimmed to the size of the recipient site and placed dermis down on the recipient bed. The graft is then secured by suturing and may be further immobilised by means of a suitable dressing or bandage. While full- thickness skin grafts tend to give the best results from an aesthetic point of view, graft take is often more difficult to obtain because revascularisation of the graft is required.Another type of graft is the split-thickness skin graft which is composed of the entire thickness of the epidermis and a partial-thickness dermis. They have the advantage of containing less tissue for revascularisation and are more likely to be successful on various types of recipient bed than full-thickness grafts. Split-thickness skin grafts are often used to cover more extensive lesions but are often less aesthetically attractive than the full- thickness grafts. To cover large lesions such as extensive burns, splitthickness grafts may be used as seed or mesh grafts which means that the graft is divided into smaller portions (such as strips) and placed on the lesion. New epithelial growth then takes place from each of the portions of skin grafted onto the lesion.In one aspect of the current invention, peptide and / or peptides are used in skin grafting. The peptide for use in skin grafting is at least 75%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (P2).Preferably, the peptide for use in skin grafting procedures is identical to SEQ ID NO: 1 (P2).Infections and inflammationsInfections and Inflammations remain among the most frequently encountered infections in surgery, and their severity ranges from mild cellulitis to severe, necrotizing infections with high incidences of morbidity and mortality. Most commonly, these disorders result from skin lesions in a susceptible host, but sometimes develop following hematogenous spread from a previously unknown focus.The artificial peptide(s) according to the current invention modulate acute and chronic inflammation in hard and in soft tissue, respectively. They rejuvenate senescent cells (e.g., from irradiation damage) and reduce pain and swelling when applied locally.In one aspect, the current invention thus relates to a peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 1 (PLV PSQ PLV PSQ PLV PSQ PQ PPLPP) (P2) and / or a peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 2 (PHQ PMQ POP PVH PMQ PLP PQ PPLPP) (P6) for use in skin grafting and / or in the treatment and / or reduction of chronic inflammatory conditions.In a preferred aspect, the current invention thus relates to a peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 1 (PLV PSQ PLV PSQ PLV PSQ PQ PPLPP) (P2) for reduction of inflammation in soft tissue.Compositions for use in the current inventionIn the current context, the wording composition is used interchangeably with formulation.Typically, a composition according to the current invention comprises a. 0.1-250 pg / mL, such as 0.1 , 1.0, 5.0, 10, 50, 100, 200 or 250 pg / mL of an artificial peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 1 (PLV PSQ PLV PSQ PLV PSQ PQ PPLPP) (P2) and / or a peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 2 (PHQ PMQ POP PVH PMQ PLP PQ PPLPP) (P6).In one aspect, the current invention relates to a pharmaceutical and / or a cosmetical formulation in the form of a gel, such as a hydro-gel, comprising a) an artificial peptide, or a combination of artificial peptides, characterized by a proline sequence prevalent in key matricellular proteins that partake inwound healing as well as in bone and cartilage formation and connective tissue maintenance in the majority of vertebrates and b) BDDE cross-linked hyaluronic fibres (HA-BDDE) and / or PEGDE crosslinked hyaluronic fibres (HA-PEGDE) for use in transplantation surgery and / or graft surgery.Typically, a composition according for use in transplantation surgery and / or graft surgery according to the current invention comprises a) 0.1-250 pg / mL, such as 0.1 , 1.0, 5.0, 10, 50, 100, 200 or 250 pg / mL of an artificial peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 1 (PLV PSQ PLV PSQ PLV PSQ PQ PPLPP) (P2) and / or a peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 2 (PHQ PMQ POP PVH PMQ PLP PQ PPLPP) (P6) and b) 1-40 mg / mL, such as 1.0, 2.5, 4, 10, 20, 25 or 40 mg / mL cross-linked Hyaluronic fibres (HA-XL).In embodiments, a composition for use in transplantation surgery and / or graft surgery according to the current invention comprises one or more artificial peptide(s) selected from the group consisting of artificial peptides comprising the amino acid sequence of SEQ ID NO 1 (peptide 2) and / or SEQ ID NO 2 (peptide 6).In embodiments, a composition for use in transplantation surgery and / or graft surgery according to the current invention comprises an artificial peptide comprising the amino acid sequence of SEQ ID NO 1 (P2).In embodiments, a composition for use in transplantation surgery and / or graft surgery according to the current invention comprises an artificial peptide comprising the amino acid sequence of SEQ ID NO 2 (P6).In embodiments, a composition for use in transplantation surgery and / or graft surgery according to the current invention comprises two or more artificial peptide(s) comprising the amino acid sequence of SEQ ID NO 1 (P2) or SEQ ID NO 2 (P6), respectively.A composition for use according to the current invention can comprise cross-linked Hyaluronic fibres (HA- XL) which comprise or consist of 1 ,4-butanediol diglycidyl ether cross-linked Hyaluronic fibres (HA- BDDE).In a currently preferred embodiment, a composition for use according to the current invention comprises a) 50 pg / mL of an artificial peptide with the amino acid sequence of SEQ ID NO 1 (P 2) and / or SEQ ID NO 2 (P 6), b) 20 mg / mL 1 ,4-butanediol diglycidyl ether cross-linked Hyaluronic fibres (HA-BDDE), and c. 2.5 mg / mL non-crosslinked Hyaluronic fibres (HA).In a composition for use according to the current invention, the Hyaluronic acid fibers are typically between 0.7-4.0 MDa, such as between 1.0-2.0, between 1.5-1.8, or between 3.0-3.3 MDa. In one embodiment the Hyaluronic acid fibers are between 3.0-3.3 MDa.Atypical composition for use according to the current invention is a hydrogel.A composition for use according to the current invention can further comprise one or more of a component selected from the group consisting of: a fluoride salt, such as a salt selected from the group consisting of NaF, CaF2 and ZnF2, Sorbitol, Xylitol, NaOH, HCL, Phosphate Buffer Solution (PBS) and Acetic Acid.A composition for use according to the current invention is stable / in gel form at RT for at least 1-2 years and / or in the body for at least 30 days.In one aspect, a composition for use according to the current invention releases the comprised artificial peptide at a controlled rate, such as of 1|_ig per hour.In embodiments, a composition for use according to the current invention can further comprise another active ingredient, such as, but not limited to, an active ingredient selected from the group consisting of growth factors, plasma rich fibrin / plasma and enamel matrix derivatives.One aspect relates to a composition comprising an artificial peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 1 (PLV PSQ PLV PSQ PLV PSQ PQ PPLPP) (P2) for use according to the current invention is intended for use in soft tissue healing, such as, but not limited to, for use in inducing neovascularization, for inducing reepithelization, for stimulating collagen production, and / or for promoting oriented collagen formation, in graft procedures and / or transplantation procedures.In one embodiment, a composition comprising an artificial peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 1 (PLV PSQ PLV PSQ PLV PSQ PQ PPLPP) (P2) induces protein regulation such as up- and downregulation in soft tissue. Examples of proteins that may be upregulated include: actin and histone H2A. Examples of proteins that may be downregulated include: alpha-1-antitrypsin, Hsp72, annexin A1 , S100 calcium binding protein A8, alpha-enolase, transferrin receptor protein 1.One aspect relates to a composition of an artificial peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 2 (PHQ PMQ POP PVH PMQ PLP PQ PPLPP) (P6) for use according to the current invention for use in hard tissue healing, such as, but not limited to, for use in graft procedures and / or transplantation procedures of bone.In one embodiment, a composition comprising an artificial peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 2 (PHQ PMQ POP PVH PMQ PLP PQ PPLPP) (P6) induces protein regulation such as up- and downregulation in hard tissue. Examples of proteins that may be upregulated include pyruvate kinase, actine, histone H2A and IL-8. Examples of proteins that may be downregulated include alpha-1-anitrypsin, which reduces inflammatory response in hard tissue.HydrogelsHydrogels are an extensively investigated class of biomaterials, and an increasing number of products have reached the clinic.Hydrogels represent a group of biomaterials consisting of water- swollen polymer or colloidal networks. Hydrogels are viscoelastic materials that have attracted attention in regenerative medicine due to their ability to structurally mimic the extracellular matrix (ECM), thereby creating a conducive environment for cell proliferation and tissue regeneration. The viscoelastic properties of hydrogels allow them to function as stem cell carriers or scaffolds for controlled drug release.EXAMPLESThe following examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. Considering the present invention and the general level of skill in the art, those of skill can appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.Example 11. Materials and methods:1.1 Test groups:Emdogain® (EMD) was acquired from Institute Straumann AG (Basel, Switzerland). Emdogain® consists of 30 mg / ml of enamel matrix derivative in an acidic propylene glycol alginate (PGA) gel and is intended for periodontal regeneration. It was used as supplied. Crosslinked hyaluronic acid (HA) alone, with 50 pg / ml P2 (HA+P2), and with 50 pg / ml P6 (HA+P6) was manufactured in accordance with ISO 13485:2016. The peptide concentration was based on the highest bioactivity displayed in former studies [ Rubert, M., et al., 2012; Ramis, J.M., et al., 2012; Villa, O., et al., 2015; Rubert, M., et al., 2011 ; Rubert, M., et al., 2013; Zhu, H., et al., 2020; Zhu, H., et al., 2020]. The peptide sequences of P2 and P6 are shown in Table 1. The hyaluronic acid was crosslinked with 1 ,4-Butanediol diglycidyl ether (BDDE) and mixed with 10% sodium hyaluronate, in a formulation comparable to that of HyaDENT BG®, although at an increased hyaluronic acid concentration of 22.5 mg / ml compared to 18 mg / ml.1.2 Animal handling:A total of 7 conventional pigs (Breeding farm, Spain) (age: 2 months, weight: 18.750-15.135 kg, quarantine period: 8 days) were used for this study. All the experiments were carried out according to the national legislation following the community guidelines after the authorisation of the competent, autonomous authority in the facilities available to the Rof Codina Foundation (CeBioVet facility, Lugo, Spain) for this purpose (Approval number 02 / 20 / LU001 from the Galician Government). The animals werekept as a group identified by subcutaneous microchips. They were housed in an area with natural light, air renewal and regulated temperature. The animals were fed a conventional granulated diet for their species and had access to water supply. During the study, they were visited daily by people trained in laboratory animal science.All procedures were performed using general anaesthesia. The animals were premedicated with an intramuscular combination of medetomidine 20 pg / kg (Sededorm; Vetpharma Animal Health S.L., Spain), ketamine 10 mg / kg (Ketamidor, Karizoo Laboratories S.A., Spain), midazolam 0.3 mg / kg (Midazolam Normon, Normon Laboratories S.A., Spain) and morphine 0.3 mg / kg (Morfina Serra, Serra Pamies Laboratories S.A., Spain) for sedation and pain control under veterinary care. Furthermore, an intravenous injection of meloxicam 0.2 mg / kg (Metacam, Boehringer Ingelheim S.A, Spain) was administrated to provide adequate analgesia. General anaesthesia was induced with intravenous administration of propofol 2-4 mg / kg (Propofol Lipuro, B. Braun VetCare S.A., Spain) and maintained with isoflurane (Vetflurane, VIRBAC Laboratories S.A., Spain) an inhalational anaesthetic agent. During anaesthesia, the animals were monitored via electrocardiography, capnography, pulse oximetry, and non- invasive blood pressure. Antibiotic prophylaxis was administered before the surgery, using cefazoline at 22 mg / kg (Cefazolina Normon, Normon Laboratories S.A., Spain). After surgery, one dose of amoxicillin (trihydrate) at 15 mg / kg (Amoxoil retard, SYVA laboratories S.A., Spain) was given to the pigs for 48 hours of antibiotic coverage. The animals were monitored daily and during the interventions by a veterinarian accredited and trained in the science of laboratory animals (categories B or C, functions a, b and c).1.3 Surgery and sample extraction:The gingival tissue of 3 premolars at each side, both upper and lower jaw, was detached from the teeth using a scapula, and any residual ligament was scraped off the teeth with a scalpel. Any residual periodontal ligament was gently removed with a spatula. Each gingival flap measured approximately 4 x 3 mm, with a depth that reached the periosteum and a split-thickness extension to the mucogingival junction. The inner epithelial lining of the gingival flaps was excised using a scalpel to ensure a consistent wound bed. Haemostasis was achieved prior to the application of the experimental gels. There was no postoperative oral hygiene protocols applied during the six days experiment.Each quadrant of the oral cavity was randomly assigned to one of the treatment groups. Sham, HA+P2, and HA+P6 treatments were applied in all six animals. HA and Emdogain® (commercially available EMD) were applied to three animals each. In the seventh animal, only EMD and HA treatments were used, with two application sites per treatment group assigned to the lower quadrants and one per group to the upper quadrants. Unfortunately, this animal was lost during the peri-anaesthetic period due to a bradyarrhythmic cardiac event.After six days, the remaining animals were euthanised following sedation with medetomidine (20 pg / kg, Sededorm; Vetpharma Animal Health S.L., Spain), ketamine (10 mg / kg, Ketamidor; Karizoo Laboratories S.A., Spain), and midazolam (0.3 mg / kg, Normon; Normon Laboratories S.A., Spain). Anesthesia was induced with propofol (2-4 mg / kg, Propofol Lipuro; B. Braun VetCare S.A., Spain) and followed by euthanasia via intravenous overdose of pentobarbital (200 mg / kg, Dolethal; Vetoquinol E.V.S.A., Spain). The gingival tissue samples were explanted, dissected, and fixed in 70% ethanol, then transferred to 4% paraformaldehyde (PFA) for 24 hours.The bleeding was allowed to stop before the gels were applied and randomly allocated to the 4 quartiles. Sham, HA+P2, and HA+P6 were used in all six animals, while HA and EMD were used in 3 animals each. After 6 days, the animals were euthanised after being sedated with medetomidine 20 pg / kg of body mass (Sededorm; Vetpharma Animal Health S.L., Spain), ketamine 10 mg / kg of body mass (Ketamidor, Karizoo Laboratories S.A., Spain) and midazolam 0.3 mg / kg of body mass (Normon, Normon Laboratories S.A., Spain), induced with propofol 2-4 mg / kg of body mass (Propofol Lipuro, B. Braun VetCare S.A., Spain), and then sacrificed by an intravenous overdose of pentobarbital 200 mg / kg of body mass (Dolethal, Vetoquinol E.V.S.A., Spain). The explanted gingival samples were dissected and fixed in 70% ethanol, followed by 24 hours in 4% paraformaldehyde (PFA).1.4 Histology and immunohistochemical preparation and histomorphological analysisAfter euthanasia, gingival tissues were harvested and fixed in phosphate-buffered 4% paraformaldehyde (PFA) (Cat. no. 158127, Sigma-Aldrich, St. Louis, MO, USA) at 4 °C for 24 hours. Samples were subsequently embedded in Technovit® 9100 NEW (Heraeus Kulzer, Hanau, Germany) following the manufacturer's protocol. Sectioning was performed using a rotary microtome (HM 355S, Thermo Fisher Scientific, Karlsruhe, Germany) at a thickness of 5-6 pm. Kawamoto's film method (SECTION-LAB Co. Ltd., Hiroshima, Japan) was applied during sectioning to maintain tissue integrity and minimise the detachment of biomaterial from the sections.Histological evaluation was conducted on toluidine blue and Masson-Goldner trichrome-stained sections to assess overall tissue morphology and inflammatory status. Inflammation grading was based on epithelial integrity, oedema, and inflammatory infiltration using a three-tier scale: 3 (normal, keratinised, non-inflamed), 2 (moderately altered), and 1 (compromised or infected). Two independent, blinded investigators scored the slides. For immunohistochemical analysis, sections were first treated with 3% hydrogen peroxide solution (Merck KGaA, Darmstadt, Germany) for 5 minutes to quench endogenous peroxidase activity. After rinsing in phosphate-buffered saline (PBS), non-specific binding was blocked using a commercial protein blocking solution (DAKO, X0909, Agilent Technologies, Santa Clara, CA, USA). Sections were then incubated overnight at 4 °C with primary antibodies: anti-CD80 (ab64116, Abeam, Cambridge, UK), anti-CD163 (ab182422, Abeam), anti-CD206 (ab64693, Abeam), anti-IL-10 (ab34843, Abeam), and anti-TNF-a (ab6671 , Abeam), all diluted in DAKO antibody diluent (S0809, Agilent Technologies).Following primary antibody incubation, the slides were treated with biotinylated goat anti-rabbit IgG secondary antibody (BA-1000, Vector Laboratories, Burlingame, CA, USA) and then processed using the VECTASTAIN Elite ABC Kit (PK-6100, Vector Laboratories) for signal amplification. Immunoreactivity was visualised using the NovaRED™ Peroxidase Substrate Kit (SK-4800, Vector Laboratories), and counterstained with haematoxylin (Cat. no. 6765009, Thermo Fisher Scientific, Waltham, MA, USA). Slides were mounted using Vitro-Clud® mounting medium (R. Langenbrinck GmbH, Emmendingen, Germany) and imaged using a Leica DM5500 microscope (Leica Microsystems GmbH, Wetzlar, Germany), then analysed in Imaged as earlier described [Malhan, D., et al., 2018].Before embedding, the samples underwent a dehydration process involving a series of alcohol and xylene baths. Thin sections of 5 pm thickness were prepared from the embedded samples using a motorised rotary microtome. The sections were stained with toluidine blue for general visualisation and Masson Goldner Trichrome, which highlights connective tissue and inflammatory components, for further evaluation. Inflammation was assessed based on the Masson Goldner Trichrome-stained slides by two experts, one of whom was blinded to the sample identity. Each sample was graded individually based on three criteria: epithelium, oedema, and inflammatory infiltration. The average of these individual scores provided the final inflammation score for each sample. A grading scale of 1 to 3 was used, with 3 indicating normal appearance, 2 suggesting partially compromised physiology, and 1 suggesting compromised physiology.1.5. Protein Extraction:After RNA extraction (samples degraded, see Limitations section), the remaining interphase and organic phases from TRIzol™ Reagent (ThermoFisher Scientific, Madrid, Spain) were submitted to protein extraction, following the manufacturer's guidance. Briefly, 0.3 mL of ethanol 100% (Labkem, Barcelona, Spain) was added to each sample for every 1 mL of TRIzol™. Tubes were centrifuged, and DNA pellets were stored at -20°C. Supernatants containing proteins were submitted to dialysis using Slide-A-Lyzer® dialysis cassettes (2000 MWCO, ThermoFisher Scientific, Madrid, Spain). Dialysis was performed against 3 changes of 0.1% SDS at 4°C, making the first change after 16 hours, the second after further 4 hours, and the last after 2 additional hours. The protein solution was centrifuged for 10 minutes at 10,000 x g at 4°C before transferring the supernatant into a new container. To enhance protein recovery, the remaining protein pellets were solubilised in 100 pL of a buffer containing Tris-HCI 0.05M (PanReac AppliChem, Monza, Italy), urea 4M (PanReac, Barcelona, Spain), and SDS 0.05% (Sigma-Aldrich, Darmstadt, Germany) at a pH 8, as described by Hummon et al. [Hummon, A.B., et al., 2007]. If necessary, heat (50°C) was applied to those samples remaining insoluble after buffer addition. The samples were then stored at -20°C for further processing.1.6 Protein quantification & LuminexAfter solubilisation, protein concentration was measured using the Pierce™ BCA Protein Assay Kit (ThermoScientific, Rockford, USA), following the manufacturer's guidance. In short, samples were diluted25-fold and loaded into a 96-well ELISA plate. Two different calibration curves were prepared for each protein fraction, supernatant and solubilised pellet, using as diluent 0.1% SDS or buffer (Tris-HCI 0.05M, urea 4M, and SDS 0.05%), respectively. Absorbance was read at 562 nm using a PowerWave HT plate spectrophotometer (Biotek). Luminex analysis was conducted using the commercial pre-configured immunoassay multiplex assay MILLIPLEX® Porcine Cytokine / Chemokine Magnetic Bead Panel (Merck, Darmstadt, Germany). The supernatants obtained as described above were processed according to the manufacturer’s protocols (Porcine Cytokine / Chemokine Magnetic Bead Panel Catalogue). Following 13 cytokines were analysed: GM-CSF, IFNy, IL-1a, IL-1 , IL-1Ra, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12, IL-18, TNF-a. Multianalyte profiling was performed using the Luminex200TM system (Luminex Corporation, Austin, TX, USA) after overnight incubation, and acquired fluorescence data were analysed by the XPONENT3.1 software (Luminex).1.7 Mass spectrometry & ProteomicsThe digested samples were analysed in a nanoElute system coupled to a timsTOF pro (Bruker). The peptides were separated by liquid chromatography in an Aurora Elite column (C18, 1 .5 pm beads, 75 pm inner diameter, 15 cm length; lonOptics) using a flow rate of 200 nL / min with 0.1% formic acid (solvent A) and 0.1% formic acid in acetonitrile (solvent B). A 60-minute gradient was used, starting from 2% solvent B to 35% at a column temperature of 50°C. The mass spectrometer was operated in data-dependent mode to isolate automatically and fragment multiple charged precursors (top 10). The data obtained from the MS were processed and quantified using label-free quantification methods. To ensure comparability across samples, the raw protein quantification data were normalised (Figure 7). The normalisation was performed using the “equalMedianNormalizations” function from the “DEqMS” package in the statistical software R, which adjusts the data to account for systematic differences across samples. The Iog2 transformation was applied to stabilise variance and make the data distribution more normal-like. This data identified the 40 most expressed proteins by evaluating median absolute deviation (MAD). Further, Principal Component Analysis (PCA- “factoextra” package) and Partial Least Squares Discriminant Analysis (PLS-DA- “mixOmics” package, freely available on Bioconductor (https: / / bioconductor.org) were applied. Lastly, differential expression analysis (DEA) was carried out using the “limma” package [Ritchie, M.E., et al., 2015]. This method identified differentially expressed proteins (DEPs) by fitting a linear model to the expression data and conducting empirical Bayes moderation of the standard errors. DEPs were determined based on a log2(|FoldChange (FC)|) threshold of 0.585 and a p-value threshold of 0.05. RStudio, Version: 2024.04.2+764, was used for plot visualisation.1.8 Multi-block confirmatory modelTo confirm whether the same covariation structure persisted after dimension reduction, the four data blocks — Histology, IHC, Luminex, and Proteome — were integrated using multi-block partial least-squares discriminant analysis (block-PLS-DA) with the DIABLO algorithm implemented in mixOmics (v6.24). An empirical design matrix with an off-diagonal weight of 0.1 linked the blocks while preventing overfitting.Two latent components were extracted — the minimum number that yielded stable classification accuracy in preliminary tuning. Model performance was evaluated using a leave-one-pig-out cross-validation procedure repeated ten times; the average overall accuracy, balanced error rate, and area under the ROC curve were recorded. Sample projections (scores) were plotted on the LV1-LV2 plane, and 95 % confidence ellipses were generated for each treatment group. Only variables retained by the DIABLO feature-selection procedure (keepX = 25 per block) were interpreted in the text. Group centroids and 95 % confidence ellipses were plotted from the resulting score matrix (x2distribution, 2 d.f., p = 0.05; matplotlib 3.8). To test the robustness of class separation, we computed the ratio of between- to within- cluster sums of squares (SSp / SSw) for the observed labels and 1 ,000 random re-labelling’s; the empirical p-value equals the proportion of permuted ratios at least as large as the observed one. Finally, the early profiles of HA+P6 and EMD were compared variable-by-variable with Welch’s t-test, and Benjamini adjusted the resulting p-values with the Hochberg procedure, controlling the false-discovery rate at q < 0.05 All analyses were carried out in R (version 4 3 ) based on scripts by Zhang and Datta [Zhang and Datta, 2023],1.9 In vitro biocompatibility testing:The biocompatibility of the gels was measured using in vitro cytotoxicity testing with an LDH-assay and cell viability using a CCK8Assay, conforming to the method suggested in ISO 10993-5

[2009] . The method has been described previously [Ovrebo, 0., et al., 2024]. In brief, a 24-well plate was seeded with 40.000 mouse pre-osteoblastic cells (MC3T3-E1) in each well and 1 ml cell medium. The gel was introduced using cell inserts with 0.4 pm PET membrane. For the LDH test, the positive control cells were killed by introducing Triton X-100 1 hour before the data was collected. EMD was excluded from this analysis due to availability and since its biocompatibility has been primarily established clinically.1.10 Statistics:Data distribution was first assessed for normality using the Kolmogorov-Smirnov test, followed by a Holm-Sidak method for normality verification, presenting data as means with standard deviation if normal distributed and median with interquartile range if not. Both one-way and two-way ANOVA and Tukey's post-hoc tests were used for comparing parametric datasets. Mann-Whitney test was used for the nonparametric dataset.These statistical analyses were conducted in a self-written Python script with dependencies on the SciPy library. Based on data from similar studies, a power analysis was performed in the software G*Power 3.1 [Faul, F., et al., 2009] to evaluate the number of animals to use. It was determined that the minimum sample should be 6 animals per group to obtain a study power of 85% and type I error of 0.05. Datasets were considered significantly different for p<0.05. All data obtained were analysed using GraphPad Prism version 10.1 (GraphPad Software Inc., CA, USA). Spearmen’s bivariate correlation was used to analyse the correlation plots. The results were interpreted as follows: no correlation if |r| < 0.2; correlation if 0.2 < |r| < 0.5; and strong correlation if 0.5 < |r| < 1. A negative r indicated a negative correlation, whereas a positive r indicated a positive correlation. All graphicalrepresentations were performed on GraphPad Prism and Biorender. A priori sample size estimation was conducted using G*Power version 3.1 [Faul, F., et al., 2009], informed by effect sizes reported in previous comparable studies. To achieve a statistical power (1-p) of 0.85 and a Type I error rate (a) of 0.05, a minimum of six samples per experimental group was calculated to be necessary. Statistical significance was defined as a p-value of less than 0.05 for all analyses. The study design and reporting adhere to the ARRIVE 2.0 guidelines for preclinical animal research as recommended by the EQUATOR Network.ResultsAll six animals appeared healthy during the six-day follow-up period, and there were no signs of systematic reactions or toxicity from the procedure or treatment. However, one animal was lost due to causes unrelated to the biomaterials used (peri-anaesthetic period due to a bradyarrhythmia cardiac event), resulting in an uneven number per group.1.11 Histology and histomorphometry:Masson Goldner Trichrome staining was employed to obtain a detailed description of the gingival tissue's morphology. While the stain is primarily intended for connective tissue colouration, we discovered that it is also helpful in detecting alterations in the epithelium, identifying oedema, and assessing inflammatory infiltrates and acute local infections. We assigned each feature a score ranging from 1 to 3, corresponding to compromised to normal physiology, before averaging the scores to determine an overall inflammatory score for each sample. The scoring table is presented below (Table 2).Table 2: Grading scale for only the histology samples. The sample score was the average of the three scorings: epithelium, oedema, and inflammatory infiltrate.Table 3: Grading of histology samples. Grading system: 1 (compromised), 2 (somewhat compromised), 3 (normal physiology). LL: Lower Left, LR: Lower Right, UL: Upper left: UR: Upper Right.Figure 2 presents representative histology slides for each group. Table 2 presents the scoring applied. Histology images along with digital images of the animals for the six pigs are provided as Figures 16-21 .The sham and EMD groups exhibited epithelial thinning, whereas the epithelial layer had completely vanished in the HA group. The application of HA+P6 demonstrated a healthy epithelium in its lower portion, while it was not present in its upper portion, which is attributed to the slicing direction employed during specimen preparation. HA+P2 exhibits a healthy epithelium with keratinisation throughout. It alsopresents epithelium on three sides, possibly due to slicing direction or the possibility that the gingiva failed to re-attach and new epithelialisation has occurred.Inflammation was evident in the sham group in the form of encapsulated necrotic cells (dashed oval). The HA group showed a significant inflammatory infiltrate in the upper left portion (red region in dashed ovals) and similar all the samples analysed from the same group. EMD exhibited signs of active infection with pus exiting the epithelium (opening in the epithelium - dashed oval). Oedema was observed in all groups, including Sham, HA+P6, HA, and EMD (dotted ovals).The HA+P2 group received a perfect 3 score, indicating normal histology (Figure 2f). The inclusion of P2 and P6 prevented an adverse effect on the tissue after gingival detachment, with indications of improved response compared to the clinical benchmark, EMD. Hyaluronic acid alone was the poorest performing group; however, due to a limited sample size, it was impossible to observe any significant differences between the groups.Overall, introducing peptides HA+P2 and HA+P6 demonstrated the most favourable results, with controlled inflammation and robust tissue healing, while HA consistently produced the poorest outcomes, marked by severe inflammation and tissue damage. Regarding the epithelium, HA+P2 and HA+P6 displayed strong, keratinised tissue with minimal irregularities. Sham and EMD showed variable results, with some samples exhibiting thinning or breaches. In contrast, HA consistently displayed weak, thin, and frequently breached epithelium. HA+P2 and HA+P6 maintained moderate levels of oedema that did not impede healing. Sham and EMD exhibited variable oedema, often associated with inflammation, while HA consistently presented severe oedema, frequently linked to poor healing and active infections. Regarding inflammatory infiltration, HA+P2 and HA+P6 effectively managed inflammation, with moderate infiltrate supporting tissue remodelling. Sham and EMD showed mild to moderate infiltrate, with signs of ongoing inflammation in some cases. HA displayed intense infiltration, often accompanied by active infections, necrotic tissue, and poor healing outcomes. This is reflected in the mean tissue morphology score displayed in Figure 2f.More detailed examples of the histological findings are displayed in Figure 3. The coagulum was observed in Figures 3a), 3b) and 3c). A significant difference was that inflammatory cells circled the coagulum for the HA +P2 sample, suggesting active remodelling. This was not observed for the HA or sham group. In 3c) to 3e), regions with inflammatory infiltration were observed, particularly for the HA groups - while extensive oedema was observed in 3e) and 3f). Interestingly, although the EMD displayed oedema, there was a limited indication of inflammatory infiltration (Fig. 3f). Toluidine blue stains are displayed in Figures 22-27.7.72 Immunohistochemistry and histomorphometry:Representative sections stained for the pro-inflammatory cytokine TNF-a (Figure 9A) and for mannose receptor (Figure 9B) reveal treatment-dependent modulation of the early inflammatory micro-environment.Sham defects exhibited a diffuse, low-to-moderate TNF-a signal and weak mannose-positive labelling, representing the baseline innate response to an ungrafted cavity. HA implantation markedly intensified TNF-a immunoreactivity throughout the defect, whereas mannose staining remained faint, denoting a sustained M1-skewed inflammatory profile elicited by the ceramic alone. HA+P2 reduced the overall TNF- a burden compared with unmodified HA while producing a marginal increase in mannose-positive cells, indicating partial attenuation of the pro-inflammatory milieu. HA+P6 further suppressed TNF-a expression to near-background levels. It elicited the most homogeneous man nose-receptor staining among the experimental groups, consistent with a pronounced shift toward an anti-inflammatory, M2-like phenotype. EMD mirrored the HA+P6 pattern, displaying minimal TNF-a and prominent mannose labelling, corroborating its known capacity to foster a pro-healing macrophage response. Collectively, peptide P6 and EMD most effectively dampened TNF-a-driven inflammation while promoting mannose-expressing macrophages, whereas unmodified HA sustained a robust pro-inflammatory state (Figure 9A and 9B).Representative immunohistochemical micrographs (scale bar = 1 mm) illustrate distinct immune profiles among the five experimental conditions. Sham defects displayed only scattered CD163-positive macrophages and limited CD80 immunoreactivity, with negligible IL-10 staining, reflecting the baseline inflammatory milieu of an un-grafted defect. HA grafts elicited a pronounced accumulation of CD163- positive cells throughout the defect, accompanied by markedly stronger CD80 labelling and a diffuse increase in IL-10 expression, indicative of a mixed M1 / M2 response to the ceramic alone (Figure 10A, 10B, and 10C). HA+P2 reduced the CD80 signal relative to HA while maintaining moderate CD163 staining; IL-10-positive cells were more abundant than in the sham but less uniformly distributed than in HA+P6, suggesting a partial shift toward a pro-resolution phenotype. HA+P6 sections exhibited the most homogeneous CD163 labelling, minimal CD80 immunoreactivity, and robust, widespread IL-10 staining, consistent with a predominantly M2-biased, anti-inflammatory environment. EMD exhibited a staining pattern comparable to HA+P6, with intense signals for CD163 and IL-10 and only faint positivity for CD80, supporting its established capacity to promote a pro-healing macrophage phenotype. Collectively, the peptide-modified HA (especially HA+P6) and EMD treatments favoured an M2-skewed response, whereas HA alone triggered a stronger mixed inflammatory reaction (Figure 10A ,10B and 10C).Immunohistochemical analysis revealed distinct effects of treatment groups on inflammatory and macrophage-related markers. HA treatment significantly increased TNF-a expression compared to the Sham group (p = 0.0026), suggesting an elevated pro-inflammatory response (Fig. 11 A). Notably, HA+P6 significantly reduced TNF-a levels relative to HA alone (p = 0.0164), indicating a potential antiinflammatory effect. No other treatments, including EMD, significantly altered TNF-a expression (Fig. 11A). For CD80 and CD163, markers of pro- and anti-inflammatory macrophage phenotypes, respectively, no significant differences were observed across any groups (p > 0.05), indicating limited modulation of macrophage polarisation under the tested conditions (Fig. 11C and 11 D). Mannose receptor expression was significantly increased in the HA+P6 group compared to HA (p = 0.0405), suggestingenhanced M2-like macrophage activity (Fig. 11B). All other comparisons were non-significant. Similarly, IL-10 expression remained unchanged across all groups, with no statistically significant differences detected (p > 0.05), indicating that anti-inflammatory cytokine modulation was not a dominant effect in this model (Fig. 11E).Proteomics analysis revealed that EMD treatment significantly upregulated several proteins involved in wound healing, matrix organisation, and cellular repair when compared to Sham and HA-based formulations. Protein levels were normalised before proteomic analysis-Partial-least-squares (PLS) score plots were generated to complement the unsupervised principal component analysis (PCA) and to visualise class separation among the five treatments (Sham, HA, HA+P2, HA+P6, and EMD). The first latent variable accounted for 60% of the total variance, while the second accounted for an additional 7%, resulting in a cumulative explanatory power of 67%. Because the sum exceeded the conventional 60 % threshold, the two components were sufficient to illustrate the multivariate structure (Figure 5A and 5B). The 95% confidence ellipses clarified how each formulation clustered in latent variable space. Although the confidence regions of adjacent groups overlapped at their margins, the centroids were displaced along orthogonal directions, indicating genuine compositional differences despite partial overlap of the ellipses. In particular, HA+P6 and EMD shifted positively along LV1 , whereas HA and Sham clustered on the negative side; HA+P2 occupied an intermediate position, mirroring its mixed inflammatory profile. The relative size of each ellipse reflected within-group dispersion rather than inter-group difference, with Sham displaying the narrowest spread and HA the widest, consistent with the histological variance reported above (Figure 4A and 4B). Overall, the supervised PLS results corroborated the PCA findings: peptide- enhanced gels, especially HA+P6, drive the early tissue proteome towards the EMD-like quadrant, whereas unmodified HA remains closer to the Sham control. The directional separation of biplot vectors therefore provides an additional layer of evidence that the proline-rich peptide P6 confers a distinct immunomodulatory signature. The heat map is plotted based on the top 40 proteins, as determined by the median absolute difference (MAD). The square of the distance from the data to the mean is used for the standard deviation (Figure 4D).To understand the difference between the groups as shown in the heatmap (Figure 4D), one must examine the individual protein levels. Differentially expressed protein (DEP) analysis was employed to identify the differences between the groups. Figure 5 presents a series of volcano plots illustrating the differential protein expression profiles across multiple treatment comparisons, including HA, HA+P2, HA+P6, and EMD versus Sham, as well as direct comparisons between treatments. Each plot displays the Iog2 fold change on the x-axis against the - log 10 adjusted p-value on the y-axis, allowing for the visualisation of both the magnitude and statistical significance of protein expression changes. Proteins positioned to the right represent those significantly upregulated, while those on the left are downregulated in each comparison. Coloured data points indicate statistically significant proteins, with the intensity of colour reflecting the adjusted p-value and dot size representing the magnitude of the effect. Notably,several proteins, such as thymosin p-10 (F2Z5L5), a histone H2B isoform (A0A287AEQ0), the keratin- associated protein I3L7Z6, and cytoplasmic actin (Q6QAQ1 ) are prominently labelled due to their substantial differential expression. The plots demonstrate distinct transcriptional responses across treatments. EMD and HA+P2 comparisons reveal a strong upregulation of regenerative and remodelling- associated proteins, whereas HA alone exhibits a more limited and predominantly downregulated profile. These patterns underscore the enhanced biological activity of peptide- and protein-enriched hydrogels in modulating protein expression relevant to regeneration, particularly in the early stages of wound healing (Figure 5).1.13 Inflammatory markersProteomics was applied to determine the upregulation / downregulation of proteins to understand the cascades activated (Figure 4 & Table 3). Prior to analysis, the protein levels were normalised (Figure 7). Unsupervised PCA analysis suggests that the primary and secondary principal component explains more than 70% of the variance (Figure 4A), and supervised PLS-DA analysis suggests they explain 67% of the variance (Figure 4B). From the heatmap in Figure 4D, a clear trend of downregulation of protein expression can be observed for EMD. At the same time, the other groups had some upregulation and some downregulations relative to each other.Differential expressed protein (DEP) analysis was applied to understand the differences between the groups focusing on the top 5 up / downregulated proteins (Figure 5) - a detailed description is provided in Table 3, and a summary is presented here. The HA+P2 group exhibited significant downregulation of proteins crucial for wound healing, including those involved in inflammation control (Alpha-1-antitrypsin, HSPA1A, Annexin A1 , S100 calcium-binding protein A8), cell migration (Alpha-enolase), proliferation (Transferrin receptor protein 1 ), and metabolism (Alpha-enolase), compared to the Sham group. In the HA versus Sham comparison, several genes essential for cellular repair and immune response were downregulated, indicating suppressed wound healing pathways, while one gene was upregulated. The HA+P6 versus Sham analysis identified up-regulated genes potentially enhancing cellular processes (Pyruvate kinase) and down-regulated genes suggesting suppression of critical functions (Alpha-1- antitrypsin). Comparisons between HA+P2 and HA revealed up-regulated genes in the HA+P2 group (Actin and Histone H2A), suggesting an improved wound healing response.Additionally, EMD showed a marked up-regulation of a specific gene over HA (Brain abundant membrane- attached signal protein), implying enhanced healing processes. At the same time, it downregulated the two genes Trypsin (P00761 ) and trypsinogen (A0A287B5W2) compared to HA+P2, suggesting a shift towards a more stable wound environment with less remodelling (Table 4).When compared to Sham, EMD significantly upregulated 19 proteins (adj. p < 0.16), including C3S7K5, F1SGI7, thymosin p-10 (F2Z5L5), and multiple histone-related proteins (A0A8W4FCQ5, A0A287AEQ0, F2Z5L2), indicating activation of pathways associated with proliferation, ECM remodelling, and cellularstress response. Compared to HA, one protein (A0A287ALA0) was moderately upregulated (logFC = 8.45), although this difference was not statistically significant after correction (adj. p = 0.85). Notably, compared to HA+P2, EMD downregulated trypsin-related proteins (P00761 , A0A287B5W2), suggesting a reduction in tissue remodelling activity and a shift toward a more stabilised wound environment. In the EMD vs. HA+P6 comparison, several proteins, including the extracellular-matrix protein collagen type I a- 2 chain (C3S7K5), a keratin-type II cytoskeletal isoform (I3LDS3), and a 14-3-3— like adaptor protein (F1SGI7), were significantly upregulated (logFC > 6.6; adj. p < 0.56), supporting enhanced ECM activity and cellular regeneration (Table 4).Table 4: Proteomics analysis of all up- and downregulated proteins only when compared to EMD. Proteins that are not significantly upregulated or downregulated (P-value > 0.05) in the comparison are excluded from this overview.Proteomics profiling of the HA+P2 treatment revealed significant transcriptional changes compared to both HA alone and the Sham control, indicating distinct biological activity associated with the proline-richpeptide. Compared to HA, HA+P2 upregulated 21 proteins (adj. p < 0.25), including those linked to cytoskeletal regulation (Q6QAQ1), chromatin organisation (F2Z5L5, F2Z5L2), and stress response (I3LVD5, A0A286ZWK2). Several of these proteins demonstrated high fold changes (logFC > 7), suggesting robust transcriptional activation. These data point toward an enhanced cellular remodelling and repair response following HA+P2 treatment. In contrast, when compared to Sham, HA+P2 significantly downregulated 11 proteins (adj. p < 0.46), including collagen I a-1 (F1SFA7), keratin-related proteins — (I3L7Z6), and iron-uptake receptor transferrin receptor 1 (P21753), many of which are involved in structural maintenance and inflammatory regulation. Notably, the downregulation of signalling adaptors included the 14-3-3 E isoform (K7GM40 / YWHAE) and an uncharacterised keratin-like protein Q95274 suggests a shift in epithelial and immune response pathways (Table 5).Table 5: Proteomics analysis of all up- and downregulated proteins only when compared to HA+P2.Proteins that are not significantly upregulated or downregulated (P-value > 0.05) in the comparison are excluded from this overview.Comparative Proteomics analysis revealed that HA+P6 treatment induces a distinct protein expression profile, particularly when compared to HA+P2 and Sham. In the HA+P6 vs. HA+P2 comparison, 13 proteins were significantly upregulated (adj. p < 0.47). The most highly upregulated protein was I3L7Z6 (logFC = 8.34), followed by others involved in epithelial regulation (K7GM40, F1SFA7), immune function (Q9XSD9, P21753), and structural repair (A0A287A2R9, F1 RMV7). These data suggest that HA+P6 promotes epithelial regeneration and tissue remodelling, potentially more effectively than HA+P2Relative to Sham, HA+P6 markedly increased an uncharacterised membrane protein (A0A8W4FHP1 ) and keratinocyte proline-rich protein (Q6DUB7), while it suppressed six others, notably the structural matrix component collagen type I a-1 chain (F1 SFA7), the signalling adaptor 14-3-3 <( / b (Q5XLD3), and another uncharacterised protein (A0A5G2QML3). The downregulation of F1SFA7, in particular (logFC = -6.19), may indicate the attenuation of early inflammation or modulation of cytoskeletal activity (Table 6).Table 6: Proteomics analysis of all up- and downregulated proteins only when compared to HA+P6.Proteins that are not significantly upregulated or downregulated (P-value > 0.05) in the comparison are excluded from this overview.Proteomics profiling of the HA group relative to Sham revealed a limited but biologically relevant set of differentially expressed proteins. Only one protein, P80310, was significantly upregulated (logFC = 5.78; adj. p = 0.28), indicating a modest activation of protein expression related to protein transport or metabolism. In contrast, nine proteins were significantly downregulated, including an uncharacterised cytoplasmic protein (P20303), thymosin p-10 (F2Z5L5) and its closely related thymosin homologue (F2Z5L2), with fold changes exceeding -10 and adjusted p-values < 0.07. These proteins are associated with chromatin structure, mitochondrial function, and metabolic regulation, suggesting that HA maysuppress early regenerative and metabolic pathways relative to Sham. Additional downregulated proteins such as mitochondrial oxidoreductase (A0A480KJB8) and dihydrolipoyl dehydrogenase, the E3 subunit of the pyruvate-dehydrogenase complex (P00346) also support a trend toward reduced metabolic activity (Table 7).Table 7: Proteomics analysis of all up-and downregulated proteins only when compared to HA. Proteins that are not significantly upregulated / downregulated (P-value: >0.05) in the comparison are excluded from this overview.Differentially expressed protein (DEP) analysis was employed to identify the differences between the groups, with a focus on the top 5 upregulated and downregulated proteins (Figure 5). The HA+P2 group exhibited significant downregulation of proteins crucial for wound healing, including those involved in inflammation control (Alpha-1-antitrypsin, HSPA1A, Annexin A1 , S100 calcium-binding protein A8), cell migration (Alpha-enolase), proliferation (Transferrin receptor protein 1), and metabolism (Alpha-enolase), compared to the Sham group. In the HA versus Sham comparison, several proteins essential for cellular repair and immune response were downregulated, indicating suppressed wound healing pathways, while one protein was upregulated. The HA+P6 versus Sham analysis identified up-regulated proteins potentially enhancing cellular processes (Pyruvate kinase) and down-regulated proteins suggesting suppression of critical functions (Alpha-1-antitrypsin). EMD treatment resulted in significant up-regulation of proteins associated with cell proliferation and migration (Pyruvate kinase) and extracellular matrix formation (Histone H2B and A0A8W4F6V7), indicating enhanced wound healing compared to the Sham group. Comparisons between HA+P2 and HA revealed upregulated proteins in the HA+P2 group, including Actin and Histone H2A, suggesting an improved wound-healing response. Additionally, EMD. showed a marked upregulation of a specific protein, Brain-abundant membrane-attached signal protein, compared to HA, implying enhanced healing processes. At the same time, EMD downregulated the twoproteins trypsin (P00761 ) and trypsinogen (A0A287B5W2) compared to HA+P2, suggesting a shift toward a more stable wound environment with less remodelling (Table 5).In addition to the proteomics, Luminex was applied to determine the expression of pre-selected inflammatory cytokines. Most of the expression was below the detection limit of the kit, and observable levels were achieved only for IL-1 ra, IL-6, IL-8, and IL-18 (Figure 5). IL-1 ra, an anti-inflammatory cytokine, was upregulated in P6 compared to P2. Additionally, IL-8 was upregulated in HA+P6 and HA alone compared to the sham group, which is associated with rapid wound healing (Figure 8).Spearman analysis revealed two opposing response programmes that dominated the early wound milieu. A tightly interconnected inflammatory cluster — CD80, TNF-a and the danger-associated proteases trypsin-1 and trypsinogen — showed strong positive inter-correlations (p == 0.47-0.52) and rose in parallel with oedema and inflammatory-cell infiltrate, while displaying inverse relationships with epithelial tongue length. Conversely, a reparative cluster containing apolipoprotein A-l, thymosin p-10, 14-3-3 E, keratin-2, and decorin was positively associated with epithelial continuity and with the M2-associated markers CD163 and mannose receptor (p = 0.72) but negatively correlated with the inflammatory proteases (p = - 0.63). Oedema and the composite “Inflammation” score fell between the two blocks, reflecting their mixed role in tissue damage and subsequent remodelling. Defects treated with HA+P6 or EMD aligned with the reparative axis — higher APOA1 / thymosin p-10, longer epithelium, lower CD80 and TNF-a — whereas plain HA clustered with sham wounds inside the protease-rich, epithelial-poor quadrant. Thus, the correlation structure confirms that proline-rich peptide enrichment, particularly P6, redirects the early gingival response from a TNF-a / TNF-a / trypsin-centred inflammatory state toward an APOA1 / keratin-dominated reparative phenotype (Figure 12). HA+P6 correlations are segregated into two opposing modules. A prohealing hub centred on the CD163 / mannose receptor rose in parallel with epithelial extension (p = 0.93), oedema resolution (p = 0.78), and normalisation of the global inflammation score (p = 0.89). This cluster included COL1A2, decorin and the cytoskeletal triad keratin-2, thymosin p-10 and 14-3-3 £ (0.67-0.90). The counter-module comprised TNF-a and trypsin-type DAMPs, mutually correlated (p = 1.00) and anticorrelated with CD163 (-0.66) and epithelial advance (-0.65); their only positive link was with CD80 (0.52). Thus, peptide enrichment insulates an M2 / collagen-remodelling programme from a residual M1 / TNF-a-trypsin axis. For EMD correlation, the matrix split even more cleanly into two mirror-image signatures. CD80, TNF-a, and trypsin DAMPs formed a single block (p = +1 ) that was perfectly anticorrelated (p = -1 ) with an M2-repair cluster headed by CD163, the mannose receptor, keratin-2, and COL1A2; decorin, thymosin p-10, and 14-3-3 £ tracked the latter. Oedema bridged the divide (± 0.87). This near-binary pattern indicates that EMD rapidly polarises the wound toward a CD163-rich, collagenizing, epithelializing phenotype while confining M1 / TNF-a activity to a discrete niche during the first postoperative week (Figure 14A and 14B).The Spearman correlation analysis revealed a pro-inflammatory axis for HA+P6 and EMD (Figure 15A and 15B). The reciprocal pro-resolution axis was driven by apolipoprotein A-l (APOA1 ), thymosin p-10(TMSB10), 14-3-3 £ (YWHAE) and Keratin-2 (KRT2); these proteins correlated positively with epithelial integrity and CD163 / mannose-receptor staining (p = 0.46-0.71 , q < 0.05) and negatively with the inflammatory block. To verify that a single variable set did not drive these correlations, the four data blocks were integrated using a two-component multi-block PLS-DA (DIABLO, with leave-one-pig-out cross-validation, achieving an overall accuracy of 87%), see Figure 14A and 14B. Two-component PLS- DA of the integrated histology, cytokine and proteomics matrix separated all five groups without centroid overlap (Fig. 13). LV1 captured the main variance, ordering the samples along an untreated-to-treated gradient (Sham » HA = HA+P2 » HA+P6 » EMD), whereas LV2 resolved the two peptide formulations, positioning HA+P6 above and HA+P2 just below the origin. A permutation test (1 ,000 random relabelling) confirmed that the between- and within-cluster sum-of-squares ratio (4.17) was unlikely to occur by chance (pperm = 0.047). 95% confidence ellipses were non-overlapping, indicating tight within-group reproducibility. Convergence of HA+P6 and EMD. Despite occupying distinct coordinates, the HA+P6 and EMD centroids lie closest in the latent space, suggesting partial convergence of their early molecular signatures. Univariate comparison, corrected for multiple testing (Benjamini-Hochberg, q < 0.05), identified only two significant protein differences: keratin-associated protein I3L7Z6 (higher in EMD) (Table 4) and an uncharacterised keratin-like protein Q95274 (higher in HA+P6) (Table 6). The scarcity of single-marker disparities, combined with the multivariate proximity of the centroids, indicates that peptide enrichment shifts HA+P6 toward an EMD-like immunomodulatory profile while retaining a discernible molecular identity. Collectively, these findings indicate that EMD induces a robust transcriptional program promoting wound stabilization, ECM deposition, and regulated inflammation, distinguishing it from HA- based treatments alone. HA+P6 promotes a transcriptional environment favouring epithelial remodelling and inflammation control, with a distinct regulatory pattern from both HA+P2 and Sham.Table 8: Univariate HA+P6 vs EMD comparison, After Benjamini-Hochberg correction (q < 0.05) only two proteins differed significantly between HA+P6 and EMDProtein Adj_pCD80 0.03CD163 0.03Mannose 0.03TNF-alpha 0.031.14 Determining expression levels of cytokinesExpression levels of pre-selected inflammatory cytokines were discerned using the Luminex platform. In most cases of the tested cytokines, the expression levels were below the detection limit of the kit. IL-1 ra, IL-6, IL-8, and IL-18 yielded measurable levels. IL-1 ra, an anti-inflammatory cytokine, was upregulated forP6 compared to P2, and IL-8 was upregulated for HA+P6 and HA alone compared to Sham, which is associated with rapid wound healing (Figure 8).1.15 In vitro biocompatibilityA combination of cytotoxicity testing and cell viability testing was used to characterise the biocompatibility of the gels (Figure 6). The HA+P6 group had significantly greater cell viability than the other groups, with a mean of 115% compared to the control, while HA had a mean of 98% and HA+P2 had a mean of 104%. All groups had cell viability according to ISO 10993.The two methods for measuring biocompatibility displayed opposite results. All the HA gel groups exhibit significantly higher cytotoxicity, with the means being 26% for HA, 43% for HA+P2, and 48% for HA+P6, where the ISO 10993-5 standards recommend cytotoxicity below 30%, i.e. the peptide groups had higher cytotoxicity than the recommended limit. For the viability testing, the HA+P6 group had significantly greater cell viability than the other groups, with a mean of 115% compared to the control, while HA had a mean of 98% and HA+P2 had a mean of 104%. For cell viability, the ISO 10993-5 standard recommends a viability of at least 70%, which all groups were well above.The histological score suggests that the inclusion of the proline-rich peptide improves the immunological response of crosslinked hyaluronic acid, comparable to EMD. There was also evidence of improved tissue remodelling were also observed with the peptides. Proteomics showed that P2 peptides enhanced the wound healing response compared to HA alone by upregulating actin (Q6QAQ1) and Histone H2A (F2Z5L5), while EMD promoted a more stable wound environment with less remodelling by downregulating trypsin (P00761) and Trypsinogen (A0A287B5W2), compared to HA+P2. Cytokine expression in the HA+Peptide groups was similar to EMD, with downregulation of IL-6 suggesting rapid wound healing. In vitro viability tests showed improved viability for both peptide groups compared to the control, although cytotoxicity was moderately high for the peptide groups. This cytotoxicity was not observed in vivo.DiscussionThis experiment aimed at determining if the early wound-healing response in gingival tissue could be enhanced by incorporating proline-rich peptides in a crosslinked hyaluronic acid gel. It was compared against a crosslinked hyaluronic acid gel and EMD (Emdogain®). The focus was on interpreting signs of inflammation from various biochemical assays.Gingival tissue has a sophisticated healing cascade. During the first 48-72 h after the trauma, such as a flap creation, healing is governed by the haemostatic-inflammatory response: a fibrin clot stabilises the wound while neutrophils and then macrophages clear debris and release cytokines that set the stage for repair, changes clinicians judge by bright-red colour, oedema and bleeding on gentle probing. By about day 4 the proliferative phase takes over; capillary-rich granulation tissue fills the defect and keratinocytes migrate centripetally, so epithelial coverage accelerates through the first week. A randomised clinical trialon four gingivectomy techniques found that surface epithelialisation was consistently complete between days 5 and 14, irrespective of scalpel, electrocautery or laser use. A more recent prospective study that photographed standardised 3 mm buccal wounds showed 0 % epithelialisation on day 3, 61-69 % on day 7 and 92-100 % by day 14, with >95 % wound closure at the two-week visit. The model used in this experiment describes if the early clot integrity and inflammation has been resolved, demonstrates granulation tissue and flap stability, and improved epithelial cover indicates accelerated conformation.There are currently a few products being used clinically to improve wound healing, enamel matrix derivative (EMD), with its major component amelogenin - a proline-rich, intrinsically disordered protein being the most prominent solution. When injected into the defect site, amelogenin self-aggregates into nanospheres, forming a temporary extracellular matrix (ECM) that facilitates tissue regeneration. Subsequently, the protein spheres are gradually digested by proteolytic enzymes, releasing peptides that interact with cellular receptors. These peptides, among other functions, regulate the expression of dentin sialoprotein, collagen I, BMP, and TGF-p. Clinically, EMD has demonstrated efficacy in regenerating the periodontal ligament, alveolar bone, and cementum, essential components for successful periodontal regeneration. Recently, new products utilising hyaluronic acid have entered clinical use, such as 8 mg / ml HA combined with xylitol and water as carriers, which in a gingival recession trial by Pilloni and colleagues [Pilloni, A., et al., 2023], upregulated LOX mRNA, MMP-1 protein, and TIMP1 but did not improve angiogenesis. After instrumentation, Pilloni and colleagues [Pilloni, A., et al., J. Periodontal., 2023; 94:354-363] also tested a combination of hyaluronic acid and polynucleotide for treating periodontal pockets in a split-mouth study. Throughout the 48-week follow-up period, both groups demonstrated clinical improvement, with no statistically significant difference between the control group (instrumentation alone) and the test group (instrumentation along with hyaluronic acid + polynucleotide). Although the mean PPD reduction was slightly greater in the test group (-2.08 ± 1.24 mm vs. -1.94 ± 1.19 mm), without statistical significance, claiming superiority is misleading.In the current experiment, proteomics performed three days after gingival detachment captured more than 650 porcine proteins and revealed that biomaterial chemistry, far more than inter-animal variability, dictated the early host response. EMD produced the most pro-healing signature, characterised by a broad rise in Keratin-2), ribosomal proteins, and extracellular-matrix glycoproteins, features that align well with its clinical reputation for accelerating epithelial resurfacing and forming a provisional matrix. HA evoked the most overt inflammatory profile at the opposite end of the spectrum. In contrast, the HA-P6 shifted the molecular milieu to the pro-resolution pattern seen with EMD. EMD has been shown earlier to inhibit epithelial cell growth.A closer look was taken at five well-established anti-inflammatory “gatekeepers” — apolipoprotein A-l (APOA1), Decorin, thymosin p-4, histidine-rich glycoprotein (HRG), and 14-3-3 EIn EMD-treated wounds, APOA1 and thymosin p-4 rose sharply while cationic trypsin fell by roughly seven log-fold, indicating an early curb on PAR-1 / 2 signalling and fibrin I ECM degradation. It has been demonstrated that trypsin inhibition attenuates TNF-a and IL-6, which is consistent with the literature showing that EMD can also attenuate pro-inflammatory cytokines such as IL-1 p, TNF-a and IL-6.By contrast, HA-P2 provoked a compensatory increase in decorin, HRG and 14-3-3 E when benchmarked to plain HA, yet still sat below sham levels for AP0A1 , thymosin p-4 and 14-3-3 £ — evidence that HA fails to resolve inflammation fully.HA-P6 reversed this deficiency, as AP0A1 and 14-3-3 £ rebounded, suggesting that the peptide can redirect the macrophage phenotype and restrain inflammasome activity.The selective spike of decorin in HA-P2, but not in EMD or HA-P6, flags a potential fibrosis risk because decorin simultaneously sequesters TGF-p and provides truncated TLR2 / 4 signals that appear when tissue damage is excessive.Pro-inflammatory drivers echoed the same hierarchy. S100A8 / 9 and ribosomal histones, both canonical damage-associated molecules, were higher for HA but were decreased by EMD and HA-P6. Conversely, the level of keratins K6 / K16 / K17 and actin-binding thymosins rose most in groups that exhibit faster epithelial restitution, again EMD and HA-P6, reinforcing the concept that inflammation resolution and epithelial migration are molecularly inseparable: AP0A1 , thymosin p-4 and 14-3-3 £ marking blunt neutrophil influx and inflammasome priming, thereby allowing keratinocytes to proliferate and migrate without protease stress. Immunohistochemistry aligned with, but also nuanced, the proteomic findings.On day 3, none of the biomaterials had yet shifted to an IL-10 profile. This cytokine typically peaks four to five days after wounding, consistent with the notion that the field is still undergoing an inflammatory-to- proliferative transition. The absence of an IL-10 rise, therefore, does not contradict the proteomic data; instead, the early modulation of AP0A1 , thymosin p-4 and 14-3-3 £ by EMD and HA-P6 is expected to pave the way for a later IL-10 surge once NF-KB and NLRP3 priming have been dampened.TNF-a staining, in contrast, had already diverged: plain HA showed a significant excess relative to sham, EMD and HA-P6, while HA-P6 significantly lowered TNF-a compared with HA. These histological observations align with the proteomics findings: the only material that failed to upregulate AP0A1 , thymosin p-4, or 14-3-3 £ — and that maintained high levels of S100A8 / 9 and trypsins — was the same material that sustained a TNF-a surplus.Together, the data points to three design principles. First, protease shielding is pivotal; the dramatic downregulation of trypsins by EMD suggests that enamel-matrix peptides may physically sequester or inhibit serine proteases, a property worth engineering into future coatings. Second, HA+P6 reinstates antiinflammatory gatekeepers and curtails TNF-a. Third, Decorin may serve as an early sentinel for fibrosis risk, rising only when an implant provokes excessive tissue stress.The correlation structure supports a model in which two partially opposing programmes dictate early gingival healing. A pro-inflammatory axis — marked by mannose receptor-positive macrophages, oedema, high TNF-a and trypsin-1 — appears in lesions where epithelial restitution is limited. Conversely, a proresolution axis — centred on thymosin p-10, AP0A1 and 14-3-3 E — emerges when epithelium has advanced, and TNF-a has already subsided. Decorin’s dual affiliation suggests that it is recruited early as a damage signal but then participates in matrix reorganisation once the protease burden is alleviated. Notably, the negative correlation between TNF-a and the pro-resolution proteins echoes the biomaterialspecific findings: EMD and HA-P6, which boosted thymosin p-10 and AP0A1 in the proteomic dataset, were also the only materials that prevented a TNF-a surge histologically. In contrast, HA, which failed to raise these gatekeepers, showed the highest TNF-a levels. Taken together, the data imply that successful biomaterials steer wounds away from a mannose+ / TNF-a-dominated milieu and toward an APOA1 / thymosin p-10 / 14-3-3 £-dominant environment, thereby enabling rapid epithelial closure and controlled collagen deposition.Limitations:The model chosen was an acute gingival detachment model, where the defect was created by cutting into the gingiva with a scalpel to assess early wound healing and gingival reattachment. The mode used is not suitable for assessing periodontal regeneration. Periodontal defects are created by bacterial-induced inflammation, resulting in a trauma response to the created defect, as opposed to a typical chronic inflammation. Since a scalpel was used to cut through the mucous membrane, the defect is prone to bacterial infections. It was cleaned with an antiseptic agent to reduce the likelihood of infection. Another aspect of the model is that it focuses on the short-term immunological response but not the complete remodelling phase; longitudinal sampling is required to link early signatures to final attachment strength. Pig keratinocyte kinetics are faster than those of humans, possibly magnifying early implant effects; parallel ex vivo human-gingiva explants would strengthen translational claims. A key limitation is that the gingival detachment model captures early soft tissue immunodynamics but does not replicate true periodontal regeneration with ligament and bone involvement. Due to the unforeseen loss of animals, one group had only three samples, fewer than the power analysis recommended. However, a significant difference between the groups was still observed. EMD was not included in the CCK-8 assay.Conclusion:The presented data supports the working hypothesis that a fully synthetic, peptide-enhanced hyaluronic acid (HA) gel can partially reproduce the early immunomodulatory signature traditionally attributed to an enamel-matrix derivative (EMD). In the acute porcine gingival-detachment model, the HA+P6 formulation attenuated TNF-a and trypsin-related danger signals, promoted CD163 / mannose-receptor expression, and up-regulated pro-resolution proteins such as apolipoprotein A-l, thymosin p-10 and 14-3-3 £ — changes that closely paralleled those observed with EMD. Plain cross-linked HA, by contrast, maintained a TNF-a / trypsin-dominated profile and showed little engagement of reparative pathways.These findings indicate that short proline-rich peptides can endow an HA carrier with an EMD-like, M2- skewed early response while retaining the material’s synthetic and animal-free character. The study is limited to soft tissue healing over six days and does not address ligament or bone regeneration; therefore, conclusions are confined to very early immunodynamics. Longer follow-up in true periodontal or osseous models will be required before the clinical potential of synthetic peptide-modified HA can be fully established.This early immunological in vivo model suggests that introducing proline-rich peptides (P2 or P6) to HA significantly enhances the inflammatory response, promoting rapid wound healing. The formulation's performance was on par with the market-leading product Emdogain® but with indications of a cascade that favours epithelial regeneration. The tissue's physiological appearance was better maintained, with no adverse inflammatory response observed in cytokine analysis. Both the peptide groups and Emdogain® attenuate acute inflammation, guiding tissue response towards favourable formation and remodelling. This experiment aimed at understanding if the early immunological response to a crosslinked hyaluronic acid-gel could be improved by incorporating proline-rich peptides mimicking the active sequence of EMD, and was controlled against a crosslinked hyaluronic acid-gel and EMD in Emdogain® withthe focus on interpreting signs of inflammation from histology, Luminex measurements, and proteomics.H istological analysis yielded an inflammatory score obtained by averaging grading from inflammatory infiltrate / acute infections, oedema, and epithelium appearance. No statistical significance between the groups was observed, primarily due to a limited data set. Contrary to what was reported in the literature, no anti-inflammatory response to HA was observed. The HA had a worse mean inflammatory response than the Sham control. EMD had a mean similar to Sham but with a lower variance. At the same time, the HA with the peptides showed an improved mean inflammation score, suggesting improved local response. The P6 mean was slightly higher than that of P2. Interestingly, including a small quantity of the proline-rich peptides (50 pg / ml) seems to improve the overall response of hyaluronic acid gel, yielding an improved inflammatory score compared to EMD. This is consistent with the in vivo results (rat oral mucosa using 37 pM EMD and P2) of Villa and colleagues [Villa, O., et al., 2015], where they observed from histology that EMD had a significantly better inflammation score than the placebo on day 1 , but P2 had a significantly better inflammation score than the placebo on days 3 and 7. Similar clinical results have also been observed for EMD. In a split-mouth clinical trial (n = 12) led by Villa, the investigators examined the difference in cytokine levels 7 and 14 days after open flap debridement surgery, where the test group was subsequently treated with EMD. A significant difference in IL-8 and PDGF-BB was demonstrated after 14 days, where EMD downregulated both. In this experiment, EMD group showed a lower IL-8 levels than the other groups, while IL-6 levels were higher. Still, it is higher than the other groups, while IL-6 is not significantly different. It appears that IL-6 is downregulated for HA+P6 and partly downregulated for HA+P2, suggesting a later stage in the wound healing cascade. Additionally, there was a comparable response among the different groups and EMD. The other tested cytokines were below thesensitivity limit of the kit, demonstrating one of the limitations of the Luminex method. Another major limitation of this method is that it only provides measures of pre-selected proteins, meaning that other up / downregulated cytokines are not detected resulting in an incomplete understanding of the cascades in place.The immunohistochemical analysis provided key insights into the inflammatory and immune-modulatory effects of the tested treatments. HA alone significantly increased TNF-a expression, suggesting a heightened pro-inflammatory response, whereas HA+P6 markedly reduced TNF-a levels, aligning more closely with the immunological profile observed for EMD. Although no significant changes were observed in CD80 or CD163, the upregulation of the Mannose receptor in the HA+P6 group indicates a potential shift toward an M2-like (anti-inflammatory) macrophage phenotype. Notably, IL-10 levels remained unchanged, suggesting that anti-inflammatory cytokine modulation may occur through macrophage surface markers rather than soluble mediators. These findings support the immunomodulatory potential of HA+P6 and highlight its ability to attenuate inflammation, a feature critical for promoting regenerative healing in periodontal applications.Notably, proteins related to inflammatory response and wound healing were downregulated in the HA+P2 group compared to the Sham group, indicating that the P2 peptide attenuates acute inflammation and reduces secondary inflammatory markers downstream. This includes Alpha-1-antitrypsin, which has a broad anti-inflammatory, immunomodulatory and tissue-repair effect through neutralisation of proteolytic enzymes, Annexin A1 that revolves inflammation by reducing leukocyte infiltration activating neutrophil apoptosis as well as inducing macrophage reprogramming towards a resolving phenotype , and S100 calcium-binding protein A8 that has an immune regulatory effect by initially enhance production of reactive oxygen species then by also elevate expression of anti-inflammatory IL-10. EMD also downregulates genetic signals, suggesting a milder inflammatory process. This effect may be due to EMD’s ability to inhibit epithelial cell growth, explaining reduced protein expression in gingival epithelium. The downregulation of genes was more significant for EMD than for the peptide groups, where higher activity could indicate faster healing, more inflammation, or more regeneration. It was explicitly shown that EMD downregulates trypsin and trypsinogen compared to HA+P2. It has been demonstrated that trypsin inhibition attenuates TNF-a and IL-6 , consistent with literature showing EMD can attenuate pro- inflammatory cytokines like IL-1 p, TNF-a, and IL-6, 2022]. Histological results suggest that peptide- enhanced hyaluronic acid improves healing, while the Sham group shows more inflammation. Integrating P2 into hyaluronic acid gel enhances gene response, favouring extracellular matrix formation, which is crucial for wound healing. Both peptides and EMD reduce chronic inflammation and inflammatory gene expression while upregulating genes related to extracellular matrix formation and remodelling. The more substantial attenuation of gene expression by EMD compared to peptide groups could be due to differences in potency, molar concentration, or EMDpreference for regenerating non-epithelial tissues likeperiodontal ligament, whereas peptide-enhanced hyaluronic acid gel favours epithelial tissue regeneration.Biocompatibility of the hyaluronic acid gels in vitro was also evaluated, using a cytotoxicity test and a cell viability test. All groups displayed positive results regarding cell viability; the HA+P6 group was significantly better than the other groups. The cytotoxicity results, however, were suboptimal, with the peptide groups having cytotoxicity above the recommended limit of 30% from ISO 10993-5. One possible explanation is that the peptides were produced with trifluoroacetate (TFA) as the counter-ion, which is mildly toxic in the short-term. No toxic response in vivo was observed. Also, in previous studies with the peptides, high cytotoxicity has been observed using an LDH assay, which was not observable when the same material was tested in vivo, suggesting that this method should not be used as the main indicator of biomaterial’s biocompatibility.In the trials, all groups were prone to bacterial infections, and in most of the low-scoring samples, there were indications of infections, which can have come from the bacterial flora in the mouths of the pigs. The European Federation of Periodontology (EFP) clinical guidelines recommend instrumentation and possibly cleaning with an antiseptic agent before applying a regenerative material. This could potentially have improved the outcome of this study. This was not done in this study due to lack of availability in the clinic and to reduce the number of variables.The literature on Emdogain® and HyaDENT BG® indicates that these solutions can provide clinical improvement, but a cost-benefit analysis should be conducted before recommending them to patients. The herein presented results suggests that crosslinked HA with proline-rich peptides provides a comparable or even improved short-term immunological response to EMD, which is promising for its clinical use. It has also been demonstrated that the peptides induce biomineralisation through the nucleation of apatite, which conforms with the prerequisite for intrinsic osteoinductivity (bone growth in the centre of the biomaterial). Furthermore, the crosslinking of the hyaluronic acid provides resistance against enzymatic degradation , which facilitates osteoconductivity (bone growth from defect edge into biomaterial) Hence, not only does the biomaterial (HA+P2 / P6) display an improved immunological tissue response, but it should also display osteoconductivity and osteoinductivity, which are ideal biomaterial properties for periodontal regeneration of intrabony defects. However, the ability of the peptide-enhanced gels to regenerate periodontal tissue needs to be confirmed appropriately in in vivo models and clinical trials.Indications are provided that proline-rich peptides can improve the short-term immunological response to crosslinked hyaluronic acid gels, making its performance comparable to the market-leading product, Emdogain®. The immunological response was first measured by grading histological images. While Emdogain® had a more stable performance in this trial, the mean score was improved for the peptide groups. The cytokine and biomarker response suggests that both the peptides and enamel matrix derivatives have the ability to attenuate acute inflammation, leading towards a cascade that favours ECMformation and remodelling. Specifically, the P2 peptides enhance the wound healing response compared to HA alone by upregulating actin (Q6QAQ1) and histone (F2Z5L5). At the same time, Emdogain® promotes a more stable wound environment with less remodelling by downregulating trypsin (P00761 ) and trypsinogen (A0A287B5W2) compared to HA+P2. HA+P6 yielded a comparable gene response to HA. There are further indications that the proline-rich peptide-enhanced hydrogels favour the regeneration of epithelial tissues, while Emdogain® can favour non-epithelial tissues. The results clearly show that the proline-rich peptides can be favourable biomolecules to induce periodontal tissue regeneration.Example 2Comprehensive proteomic evaluation of biomaterial-treated porcine skin woundsUsing data-independent acquisition (DIA) mass-spectrometry and the MS-DAP analysis workflow, the tissue proteome of 14 mm full-thickness porcine wounds treated with nine biomaterial or device formulations and an untreated sham control were profiled. After stringent peptide filtering (>3 quantified replicates per group) and normalisation (vsn + mode-between), 68 - 72 k peptides mapping to ~6.0 - 6.9 k proteins were retained per contrast. MS-EmpiRe was the most sensitive of the three statistical engines applied; at a 1 % FDR, it identified between 18 and 649 differentially abundant proteins (DAPs) per treatment. Across all treatments, leakage / serum proteins (ALB paralogues, APOB, APOH, A1BG), acute- phase / innate immunity factors (C3 / C5 / C7, SERPINH1 , PGlyRPI) and extracellular matrix (ECM) components (collagens VI / XII / XIV, EMILIN-1 , fibronectin, tenascin-XB) formed a conserved woundhealing signature. Treatment-specific patterns centred on lipid handling, keratinocyte differentiation, and myofibrillar proteins, suggesting divergent impacts on inflammation, re-epithelialization, and contractile remodelling.The general set-up of experiment 2 is shown in Figure 28.MethodologyPeptides P2 and P6 are as described herein.Test Groups:The hyaluronic acid (HA) was crosslinked with 1 ,4-Butanediol diglycidyl ether (BDDE) and mixed with 10% sodium hyaluronate in a formulation comparable to that of HyaDENT BG®).Emdogain® was procured from Institute Straumann AG, Basel, Switzerland. HA with 50 pg / ml P2 (HA+P2) and with 50 pg / ml P6 (HA+P6) was manufactured in accordance with ISO 13485:2016. The peptide concentration was based on the highest bioactivity displayed in former studies.Table 9Animal handling:A total of 6 conventional pigs (Breeding farm, Spain) (age: 2 months, weight: 18,750-15.135 kg, quarantine period: 8 days) were used for this study. All experiments were conducted in accordance with national legislation and community guidelines, following the authorisation of the competent, autonomous authority at the facilities available to the Rof Codina Foundation (CeBioVet facility, Lugo, Spain) for this purpose (Approval number 02 / 20 / LU001 from the Galician Government). The animals were kept as a group identified by subcutaneous microchips. They were housed in an area with natural light, fresh air, and a regulated temperature. The animals were fed a conventional granulated diet for their species and had access to water supply. During the study, they were visited daily by people trained in laboratory animal science.All procedures were performed using general anaesthesia. The animals were premedicated with an intramuscular combination of medetomidine 20 pg / kg (Sededorm; Vetpharma Animal Health S.L., Spain), ketamine 10 mg / kg (Ketamidor, Karizoo Laboratories S.A., Spain), midazolam 0.3 mg / kg (Midazolam Normon, Normon Laboratories S.A., Spain) and morphine 0.3 mg / kg (Morfina Serra, Serra Pamies Laboratories S.A., Spain) for sedation and pain control under veterinary care. Furthermore, an intravenous injection of meloxicam 0.2 mg / kg (Metacam, Boehringer Ingelheim S.A, Spain) was administrated to provide adequate analgesia. General anaesthesia was induced with the intravenous administration of propofol (2-4 mg / kg; Propofol Lipuro, B. Braun VetCare S.A., Spain) and maintained withisoflurane (Vetflurane, VIRBAC Laboratories S.A., Spain), an inhalational anaesthetic agent. During anaesthesia, the animals were monitored via electrocardiography, capnography, pulse oximetry, and non- invasive blood pressure. Antibiotic prophylaxis was administered before surgery using cefazoline at a dose of 22 mg / kg (Cefazolina Normon, Normon Laboratories S.A., Spain). After surgery, one dose of amoxicillin trihydrate (Amoxil retard, SYVA Laboratories S.A., Spain) at 15 mg / kg was administered to the pigs for 48 hours of antibiotic coverage. The animals were monitored daily and during the interventions by a veterinarian accredited and trained in the science of laboratory animals (categories B or C, functions a, b and c).Wound creation, treatment allocation and dressingThirty-six circular, full-thickness punch wounds (14 mm diameter) were created along the dorsal skin of each anaesthetised pig. Wounds were spaced > 20 mm apart to prevent mutual (field) influences and were randomly assigned to one of nine treatment arms. Immediately after haemostasis, the designated test formulation was applied to each wound and the site was occluded with a semi-permeable polyurethane film dressing (Tegaderm™).Two additional “indicator” wounds were generated on every animal to track overall healing kinetics; the inlife phase of the study ended once these indicator wounds had contracted to a residual diameter of 7 mm.Tissue collection, protein extraction and proteomic workflowProcedures identical to the previously described protocol (example 1are not repeated here: snapfreezing, urea lysis, Lys-C / trypsin digestion, DIA LC-MS / MS on a Q-Exactive HF-X, library-free analysis with DIA-NN, MS-EmpiRe statistics, g:Profiler GO enrichment, etc.)ResultsData Quality OverviewPeptide-identification confidence was uniform (median C-score == -3.6; 64 % identification rate) across 184 DIA runs. Per-sample peptide totals ranged from -100 k to -125 k, with no apparent batch or platelocation bias (QC section 1.2). Cumulative completeness revealed 37,200 peptides detected in 90% or more of the samples, indicating robust core coverage (QC 1.3.1 ). Principal-component analysis separated the samples primarily by treatment along the PC1 (17.4%) and PC2 (9.3%) axes (QC 1.7), justifying pairwise differential testing.Table 10: Differential abundance per treatmentNumbers shown are q < 0.01 unless otherwise indicated.Differential-detection (presence / absence) analysis further highlighted a recurring set of myofibrillar proteins (MYH1 / 2 / 4 / 7, MYBPC2, NEB) across HA-containing and HA + EMD treatments (30 - 46 extreme z-scores) consistent with deeper tissue capture or early contractile granulation.Table 11 : Cross-treatment similarities and differencesCommon molecular denominatorDespite formulation-specific nuances, every treatment shared a “serum leak + innate immunity + ECM repair” triad:1. Plasma-derived proteins (APOB, APOH, A1 BG) indicate vascular permeability and fibrin exudate.2. Early innate responders (complement factors, SERPINH1 , PGIyRP1 / FABP family) reflecting neutrophil recruitment and oxidative burst.3. Matrix scaffold proteins (collagens VI / XII / XIV, EMILIN-1 , fibronectin) marking fibroblast activation and provisional matrix deposition.This core programme underpins canonical second-intention healing and was merely modulated in amplitude by each biomaterial.GO-enrichment-guided pathway analysisThe Gene-Ontology (GO) over-representation screen distilled the 6 - 7 k quantified wound proteins into nine biological “pathway clusters”. Below, each treatment is revisited in the light of these clusters (FDR-corrected q< 0.05 unless indicated).Table 12Treatment-specific mechanistic portraits• VetricynVF - Dominated by the contractile apparatus cluster (25* enrichment), confirming that this hypochlorous acid formulation primarily accelerates myofibroblast recruitment and matrix tensioning.• HA only - Couples sarcomere (22*) and xenobiotic metabolism (24*) pathways, indicating that pure HA balances contractility with redox / homeostatic buffering during re-epithelialisation.• HA + P2 / HA + P6 - Share contractile and DNA-repair clusters; the P2 variant emphasises myosin assembly, while P6 accentuates SSB repair, suggesting complementary control of force generation and genomic integrity.• HA + P2 / P6 mix - Adds ECM organisation and cytokine activity, depicting a multifaceted progranulation environment (collagen cross-linking, controlled cytokine flux).• EMD only - Mirrors HA + EMD but with weaker amplitude; nonetheless, enriches striated-muscle contraction (35*) and enamel components, implying modest but focused contractile remodelling.Unified Pathway LandscapeAll biomaterials converge on a core wound-healing triad — serum leak, innate defence, and ECM repair — previously defined at the protein level. GO enrichment reveals that how each material bends this triad differs:1. Contractile bias (HA-containing gels, VetricynVF, EMD) — predicts faster closure through wound-edge tension.2. Genomic-stability bias (P2 / P6 gels) — predicts higher proliferative capacity with lower mutagenic risk.These clusters map seamlessly onto the differential-protein hierarchies described earlier, reinforcing the view that each biomaterial tunes, rather than overrides, the intrinsic porcine wound programme.Table 13: Comparative SummaryCandidate-level dissection of the differential proteomeStatistical landscapeApplying the experiment-wide thresholds that were also used in the MS-DAP quality report ( \log2FC| s 0.16 and q < 0.01 ) yielded 1 355-2 431 significant proteins per treatment. HA-based combinations that contained short-chain polyphosphates (HA+P2, HA+P6, and HA+P2 / P6) produced the broadest signatures (> 2,000 hits each), whereas single-component HA or EMD produced the smallest yet still sizable sets (~1 ,250-1 ,500).A 19-protein pan-treatment coreNineteen proteins passed significance in every material (Table 14). They assemble into four functional modules:Table 14The presence of this core, regardless of biomaterial, argues that wound contraction driven by a myofibroblast-like programme is a universal denominator of porcine skin repair in this model.Treatment-specific signaturesPolyphosphate-rich HA (HA+P2, HA+P2 / P6). These groups were dominated by fast-twitch sarcomeric isoforms (TNNT3 log2FC = +3.25; CKM log2FC +2.33 in HA+P2), together with a surge in glycolytic enzymes (PGAM2, PDHA1). The pattern suggests an anabolic, muscle-like contractile burst that may account for the fast closure previously noted macroscopically.Enamel-matrix derivative (EMD). A unique >30-fold rise in AMBN and co-enrichment of AMELX (detected only here) support a mineralisation-centred mechanism reminiscent of EMD behaviour in periodontal therapy.Concerted down-regulation of peroxisomal importThe peroxisome biogenesis factor PEX1 was the single most down-regulated protein across all materials (up to -3 log2fold change in HA+P2) . Suppressing PEX1 limits the oxidation of very long-chain fatty acids, thereby directing lipids toward membrane synthesis — consistent with the intense membrane trafficking demand during re-epithelialization.Pathway-level integration with GO enrichmentLinking the candidate list to the nine GO clusters reported earlier refines the mechanistic picture:1. Contractile fibre assembly and actin-myosin sliding. The sarcomeric core feeds directly into Cluster A (G0:0055003). Still, polyphosphate groups uniquely extend this axis with MYH4, MYH7, and MYBPC2 (detected by differential-detection z-scores), suggesting that chain length modulates the strength of the contractile phenotype.2. Calcium homeostasis and excitation-contraction coupling. CASQ1 / 2 integrates with Cluster B (G0:0006874), and their stoichiometric increase in HA+EMD hints at HA-mediated Ca2+buffering that enhances the EMD mineralisation cascade.3. Extracellular-matrix organisation. Collagens VI & XII, PRELP and EMILIN1 form a perpendicular axis in HA+P6 and PERIPREP REM, mapping onto Cluster C (G0:0030198) and explaining the histological impression of denser granulation tissue in these groups.4. Peroxisomal lipid handling. Uniform repression of PEX1 situates within Cluster E (G0:0007031 ), suggesting a cross-material strategy that spares reducing equivalents for collagen cross-linking rather than p-oxidation.Table 15: Synthesis: similarities and contrasts between biomaterialsImplications for material design1. Triggering a fast-twitch contractile programme (polyphosphates) appears most effective when rapid wound closure is paramount, but it carries a larger metabolite-demand footprint.2. EMD’s mineralisation axis is potentiated — not hindered — by HA, provided sufficient Ca2+buffering is available; this advocates for co-formulation strategies that stabilise local Ca2+.3. Universal PEX1 suppression cautions that systemic peroxisomal disorders could impair wound repair with any of these materials.ConclusionsGO-term clustering sharpens the protein-centric picture: muscle-contraction pathways dominate most HA-based and oxidising treatments; immune and antigen-processing pathways distinguish PERIPREP formulations; DNA-repair and ECM-assembly pathways mark mixed-weight HA gels.LIST OF REFERENCES1. EP21181362. Rubert, M., et al. Effect of alginate hydrogel containing polyproline-rich peptides on osteoblast differentiation. Biomedical Materials, 2012; 7.3. Ramis, J.M., et al. Effect of Enamel Matrix Derivative and of Proline-Rich Synthetic Peptides on the Differentiation of Human Mesenchymal Stem Cells Toward the Osteogenic Lineage. Tissue Engineering Part A, 2012; 18:1253-1263.4. Villa, O., et al. Proline-Rich Peptide Mimics Effects of Enamel Matrix Derivative on Rat Oral Mucosa Incisional Wound Healing. J. Periodontal., 2015; 86:1386-1395.5. Rubert, M., et al. Synthetic Peptides Analogue to Enamel Proteins Promote Osteogenic Differentiation of MC3T3-E1 and Mesenchymal Stem Cells. Journal of Biomaterials and Tissue Engineering, 2011 ; 1 :198-209.6. Rubert, M., et al. Effect of TiO2 scaffolds coated with alginate hydrogel containing a proline-rich peptide on osteoblast growth and differentiation in vitro. Journal of Biomedical Materials Research Part A, 2013; 101 :1768-1777.7. Zhu, H., et al. Xeno-Hybrid Bone Graft Releasing Biomimetic Proteins Promotes Osteogenic Differentiation of hMSCs. Front Cell Dev Biol, 2020; 8:619111.8. Zhu, H., et al. Xenohybrid Bone Graft Containing Intrinsically Disordered Proteins Shows Enhanced In Vitro Bone Formation. ACS Applied Bio Materials, 2020; 3:2263-2274.9. Hummon, A.B., et al. Isolation and solubilization of proteins after TRIzol® extraction of RNA and DNAfrom patient material following prolonged storage. Biotechniques, 2007; 42:467-472.10. Ritchie, M.E., et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic acids research, 2015; 43:e47-e47.11. International Organization for Standardization, ISO 10993-5:2009 Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity. 200912. Ovrebo, 0., et al. Characterisation and biocompatibility of crosslinked hyaluronic acid with BDDE and PEGDE for clinical applications. Reactive and Functional Polymers, 2024; 105920.13. Faul, F., et al. Statistical power analyses using G* Power 3.1 : Tests for correlation and regression analyses. Behav. Res. Methods, 2009; 41 :1149-1160.14. 0vreb0, 0., et al. Towards bone regeneration: Understanding the nucleating ability of proline-rich peptides in biomineralisation. Biomaterials Advances, 2024; 213801.15. Pilloni, A., et al. Clinical, histological, immunohistochemical, and biomolecular analysis of hyaluronic acid in early wound healing of human gingival tissues: A randomized, split-mouth trial. J. Periodontal., 2023.Pilloni, A., et al. Clinical effects of the adjunctive use of polynucleotide and hyaluronic acid-based gel in the subgingival re-instrumentation of residual periodontal pockets: A randomized, splitmouth clinical trial. J. Periodontal., 2023; 94:354-363. Malhan, D., et al. An Optimized Approach to Perform Bone Histomorphometry. Front Endocrinol (Lausanne), 2018; 9:666.

Claims

CLAIMS1. A peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO:1 (PLV PSQ PLV PSQ PLV PSQ PQ PPLPP) (P2) and / or a peptide with a sequence which is at least 75% identical to the sequence shown in SEQ ID NO: 2 (PHQ PMQ POP PVH PMQ PLP PQ PPLPP) (P6) for use in soft tissue transplantation surgery and / or soft tissue graft surgery.

2. The peptide and / or peptides for use according to claim 1 , wherein the peptide and / or peptides is / are for use in allo-transplantation, auto-transplantation, iso-transplantation, xenotransplantation, allo-graft, auto-graft, iso-graft and / or xeno-graft surgery of soft tissue.

3. The peptide and / or peptides for use according to claim 1 , wherein the peptide and / or peptides is / are synthetically, biosynthetically and / or recombinantly produced peptide(s).

4. The peptide for use according to any of the preceding claims, wherein the peptide is at least 80%, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (P2).

5. The peptide for use according to any of the preceding claims, wherein the peptide is identical to SEQ ID NO: 1 (P2).

6. The peptide for use according to any one of claims 1-3 wherein the peptide is at least 80%, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 (P6).

7. The peptide for use according to claim 6, wherein the peptide is identical to SEQ ID NO: 2 (P6).

8. The peptide for use according to any one of claims 4-7 for use in soft tissue grafting, for use in supporting and / or inducing revascularisation in skin grafting, for mucosal and / or gingival grafting, for skin grafting in connection with burns, for skin grafting in the treatment and / or reduction of chronic inflammatory conditions and / or for reduction of inflammation in soft tissue.

9. A combination for use of at least one peptide according to any one of claims 4-5 (P2), with at least one peptide according to any one of claims 6-7 (P6) for use in cartilage, ligament and / or tendon grafting and / or transplantation.

10. The combination for use according to claim 9, for use in procedures of grafting and / or transplantation in a joint, such as selected from the group consisting of knee joint and jaw joint, such as for use in procedures of grafting and / or transplantation of a ligament such as selected from the group consisting of periodontal ligament and cruciate ligament.11 . The peptide and / or peptides for use according to any one of the preceding claims, wherein the peptide(s) regulate(s) gene expression in the cell, tissue and / or organ that they are applied to, such as, wherein the regulated genes are selected from the group of genes consisting of: ACTB, H2A, SERPINA1, HSPA1A, ANXA1 , A8, ENO1 , TFRC, PKM, IL-1RA, and IL-8.

12. A pharmaceutical composition comprising the peptide and / or peptides for use according to any one of the preceding claims.

13. The composition for use according to claim 12, wherein the peptide and / or peptides are formulated in a hyaluronic acid (HA) gel, such as wherein the HA gel comprises linear hyaluronic acid fibres (HA) and cross-linked hyaluronic acid fibres (HA-XL), such as wherein the HA gel comprises 1 ,4-butanediol diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE)) and / or poly(ethylene glycol) diglycidyl ether (PEGDE) cross-linked hyaluronic acid fibres (HA- XL(PEGDE)).

14. The composition for use according to any one of claims 12-13, wherein the pharmaceutical composition is biocompatible and / or biodegradable.65

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