Scaffold for cartilage regeneration

Abstract

The invention relates to the fabrication of biocompatible scaffolds, in particular materials capable of inducing hypoxia in order to enhance cartilage regeneration by creating and maintaining an optimal microenvironment. Compositions and methods for their preparation and use thereof in the treatment of chondral and osteochondral diseases or lesions are described.

Description

[0001] SCAFFOLD FOR CARTILAGE REGENERATION

[0002] DESCRIPTION

[0003] FIELD OF THE INVENTION

[0004] The invention relates to the fabrication of biocompatible scaffolds, in particular materials capable of inducing hypoxia in order to enhance cartilage regeneration by creating and maintaining an optimal microenvironment. Compositions and methods for their preparation and use thereof in the treatment of chondral and osteochondral diseases or lesions are described.

[0005] STATE OF THE ART

[0006] Disorders affecting the osteoarticular regions encompass a wide variety of disorders, with a very high prevalence in the joints, in the strict sense. Here, mechanical stress (e.g., tendon or meniscal lesions) or pathological insults (e.g., rheumatoid arthritis - RA) induce the onset of chondral defects that rapidly degenerate, irreparably damaging the joint. Osteoarthritis (OA) is among the most common joint diseases and poses a severe socioeconomic burden to both patients and healthcare systems. It is estimated that over 250 million people worldwide are affected by symptomatic OA, which drastically limits patients’ ability to perform daily activities.

[0007] Recent epidemiological studies have highlighted that the prevalence of OA has increased annually, with a strong correlation with aging and obesity. Despite numerous efforts to identify a treatment for OA, no disease-modifying drug therapy has yet been developed. In this complex scenario, the only curative treatment remains total joint replacement (TJR), which is the most widely adopted final approach based on the surgical removal of damaged tissue and its replacement with artificial prostheses.

[0008] Unfortunately, the failure rate associated with TJR remains relatively high, mainly due to post-operative complications, such as periprosthetic infections or prosthesis misalignment. Furthermore, prostheses have a limited lifespan of approximately I Q- 25 years and often require revision surgeries, making TJR a challenge for young patients with post-traumatic OA. This is highly relevant, as up to 36% of athletes suffer from focal full-thickness chondral defects, and up to 89% of NBA players and 20% of American football players have articular cartilage abnormalities.

[0009] Recently, several surgical alternatives to TJR have been developed, often adopted as a first-line approach for early-stage OA. Among these alternatives, reparative cartilage techniques (RCTs), as microfracture, autologous chondrocyte implantation, and matrix-assisted autologous chondrocyte implantation (MACI), are the most representative.

[0010] The primary rationale for reparative cartilage techniques is to induce a pro- regenerative response that stimulates the formation of new, functional cartilage. Unfortunately, although clinical results suggest RCTs as promising alternatives to TJR, their long-term success rate is relatively low, with only 57% of patients showing complete healing after treatment. This is primarily due to a different biochemical composition of the newly formed cartilage, which is composed of fibrocartilaginous fibers rich in collagen I. This type of cartilage is less resistant to mechanical stress than native hyaline cartilage, which is primarily composed of a dense extracellular matrix (ECM) rich in glycosaminoglycans (GAGs), proteoglycans, and type II collagen.

[0011] The composition of hyaline cartilage is complemented by the presence of chondrocytes embedded in the ECM (articular chondrocytes - ACs), which are characterized by a low metabolism and, consequently, an overall limited self-healing capacity. For this reason, the molecular pathways involved in chondrocyte biology are of great interest to shed light on the poor regenerative capacity of ACs, which drastically limits hyaline cartilage self-repair.

[0012] Several molecular mechanisms have been considered, including autophagydependent and hypoxia-dependent pathways. Interestingly, the latter pathway has been reported to be predominant in the regulation of ACs phenotype and fate.

[0013] It is well known that articular cartilage is avascular and, therefore, constitutively hypoxic, and ACs metabolism has evolved to function optimally in an environment of 1-10% O2. This hypoxic environment was reproduced in several in vitro studies to improve our understanding of ACs biology. All these studies have demonstrated that a low oxygen tension contributes to the maintenance of the native ACs phenotype, promoting the expression of pro-anabolic chondral genes, such as type II collagen, which is the hallmark of hyaline cartilage.

[0014] Consequently, tissue engineering strategies were proposed that exploit oxygen concentration control to promote cartilage regeneration.

[0015] Recently, this approach has been applied to treat osteochondral and chondral lesions by fabricating scaffolds capable of modulating oxygen concentration at the implant site.

[0016] The common paradigm was the addition of oxygen-retaining agents, such as hemoglobin, myoglobin, dioxides, or peroxides, to the scaffolds.

[0017] However, despite the advanced technology of these approaches, the extent to which controlled oxygen release can influence the production of type II collagen has not been fully elucidated. Furthermore, oxidative stress induced by dioxides and peroxides can drastically reduce cell viability and proliferation. Consequently, several studies have reported a different approach based on the fabrication of 3D supports enriched with molecules, such as oxygen scavengers, which can lower oxygen concentrations. However, several disadvantages were observed with the use of these classes of compounds, such as low bioavailability and toxicity of metabolites thereof.

[0018] Within this complex landscape, the purpose of the present invention is to propose an approach that overcomes the limitations presented so far and exploits the pro- chondrogenic effects of hypoxia, by developing a scaffold-based approach applicable within a translational context for repairing joint chondral lesions.

[0019] Therefore, it is necessary to develop innovative scaffolds that can create and maintain an optimal hypoxic microenvironment, enhancing extracellular matrix deposition and cell viability, while avoiding the use of oxygen-sequestering drugs and molecules.

[0020] SUMMARY OF THE INVENTION

[0021] Despite its simple cytological composition, hyaline cartilage (HC) is a dynamic tissue that responds and adapts to biochemical and mechanical stimuli to perform its physiological function, thus ensuring proper joint function.

[0022] Despite their slow metabolism, chondrocytes primarily contribute to maintaining HC homeostasis and integrity. As main adverse effect, their non-proliferative phenotype hinders joint self-healing, making it difficult for damaged HC to regenerate without external intervention.

[0023] For this reason, several efforts were made to maximize the results of regenerative cartilage treatments (RCTs), which are effective but still cause undesirable post- surgical changes in chondrocyte phenotype. This phenomenon typically occurs after matrix implantation, triggering chondrocyte dedifferentiation, and ultimately leading to a decrease in the type ll / type I and X collagen ratio. This histological arrangement is characteristic of the fibrous / hypertrophic chondrocytes often observed in damaged HC affected by chondral defects.

[0024] Several molecular mechanisms have been implicated in the etiology of these undesirable changes in chondrocytes, with the cellular response to hypoxia being one of the most relevant. Indeed, hypoxia is one of the main promoters of type II collagen production in chondrocytes in vivo, an effect mediated by the s1 a subunit of hypoxia-inducible factor-1 a (HIF-1 a). In a normoxic environment, the oxygendependent enzyme prolyl hydroxylase (PHD) promotes the continued proteasomal degradation of HIF-1 a. However, under hypoxic conditions, the low oxygen concentrations inhibit PHD enzymes, ultimately leading to the stabilization of HIF- 1 a. The stabilized HIF-1 a protein then translocates to the nucleus, where it forms a heterodimer with HIF-1 (3, activating critical pathways for chondrocytes to adapt to the physiologically low-oxygen environment of articular cartilage, thus supporting cell survival and maintaining overall cartilage homeostasis.

[0025] Based on this knowledge, attempts were made in the past to produce scaffolds that mimic the hypoxic environment to provide proliferative stimuli to embedded cells. Deferoxamine (DFO) or inorganic ions were used to reduce the oxygen level inside these scaffolds. However, several disadvantages have limited the application of these approaches, including systemic toxicity and induction of DNA damage through oxidative stress.

[0026] The present invention was therefore conceived to overcome the limitations of these technologies, aiming to bridge the gap between beneficial and harmful effects. In particular, a composition was developed that delivers pro-regenerative hypoxic signals to human articular chondrocytes that are incorporated. This design aims to preserve the native chondrocyte phenotype, ultimately promoting in situ chondral injury repair.

[0027] The invention therefore concerns a composition comprising a polymeric scaffold and an unmodified gelatin functionalized with a fluorinated derivative.

[0028] The following exemplary scheme illustrates how the polymeric scaffold and an unmodified gelatin functionalized with a fluorinated derivative can be assembled.

[0029] Het = heterocycle (e.g., 1 ,2,4-oxadiazole, 1 ,3,4-oxadiazole, 1 ,2,4-triazole, thiazole, etc.)

[0030] FHC = fluorinated hydrophobic chain

[0031] Nu = nucleophile (e.g., -OH, -NH2, -SH groups belonging to macromolecules such as cellulose derivatives, collagen, gelatin, chitosan, etc.)

[0032] In the present invention, when reference is made to the term “composition,” it is intended to include a novel bioactive “scaffold,” “biomaterial,” or “implant.”

[0033] The composition is based on a matrix composed of gelatin methacrylate (GelMA), a gelatin derivative suitable for UV-mediated cross-linking, which increases the native structural stability of gelatin. GelMA has the following structure: https: / / www.sigmaaldrich.com / IT / it / substance / gelatinmethacryloyl12345987657srsl tid=AfmBOopEiBiK7062GAVmNQq2RgkMxrfnp21 QGGIqmvweHIBtjpppxl8o)

[0034] The GelMA matrix is enriched with hypoxia-inducing nuclei (GelOXA) composed of unmodified gelatin (uG) functionalized with a fluorinated oxadiazole (OXA), a class of compounds characterized by high affinity for oxygen.

[0035] In a second aspect, a method for the preparation of a GelMA / GelOXA composition comprising I described which comprises the following steps: a. Reacting pentadecafluoroheptyl amidoxime (IIIPAC: 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluoro-N’-hydroxyoctanimidamide)

[0036] H2N. ,C7F15N HO with pentafluorobenzyl chloride in the presence of pyridine, to obtain 0- pentafluorobenzoyl-pentadecafluoroheptyl amidoxime

[0037] (IUPAC: 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluoro-N’-

[0038] (pentafluorobenzoyloxy)octanim idam ide) b. Heating the O-pentafluorobenzoyl-pentadecafluoroheptyl amidoxime obtained from step a. at 160°C, allowing it to cool down, and dissolving the residue with hexane to obtain 3-pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4-oxadiazole (OXA). c. Mixing the 3-pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4-oxadiazole (OXA) with type A gelatin in triethylamine (TEA), and treating with ethyl acetate (centrifuge / lyophilize) to obtain GelOXA; and d. mixing the GelOXA from step c. with methacrylic gelatin (GelMA) to obtain GelMA / GelOXA.

[0039] In a third aspect, the invention concerns a product (scaffold, biomaterial, or implant) obtainable from the process according to the present invention.

[0040] In a fourth aspect, the use of the GelMA / GelOXA composition as a medicament is described herein.

[0041] In a fifth aspect, the present invention describes the use of the GelMA / GelOXA composition in the treatment of chondral and osteochondral diseases or lesions.

[0042] The invention has enabled the development of a hypoxic scaffold as an active matrix for treatments with cellularized, regenerative cartilage with human articular chondrocytes (hAC).

[0043] Surprisingly, GelMA / GelOXA has properties that allow to sequester oxygen.

[0044] GelMA / GelOXA is able to reduce oxygen concentration in a controlled manner, creating an optimal hypoxic microenvironment for hACs. Without being tied to any theory, this hypoxic microenvironment appears to better preserve the native chondrocyte phenotype, strikingly promoting the deposition of a new ECM rich in type II collagen, ultimately enabling functional cartilage regeneration.

[0045] The dependent claims describe specific embodiments of the invention.

[0046] DESCRIPTION OF THE FIGURES

[0047] The invention will now be described in detail, with reference to the attached Figures. Figure 1 : Synthesis of fluorinated 1 ,2,4-oxadiazole (OXA) and fluorinated gelatin- oxadiazole derivatives (GelOXA) (A). Solid-state NMR spectrum with magic angle spinning (MAS) of GelOXA (B).

[0048] Figure 2: hACs grown in a 24-well plate, imaged daily under bright field from day 0 to day 7 using a 4X objective (scale bar = 200 pm) and a 10X objective (scale bar = 100 pm) (A). Proliferation assay for hACs during 2D culture for 7 days, quantified using the Alamar blue assay (B). Friedman test followed by Dunn’s post-hoc test, ns = not significant.

[0049] Figure 3: Alcian blue staining of hACs cultured in 2D on day 0 (A, B) and day 7 (B, C) for GAG quantification. Scale bar = 100 pm (A, C) and 50 pm (B, D).

[0050] Figure 4: Type II collagen production by hACs cultured in 2D on day 0 (A, C) and day 7 (B, D), imaged after immunohistochemistry. Yellow staining = type II collagen. Blue staining = nuclei. White arrows = peri-Zextracellular diffusion of type II collagen. Scale bar = 100 pm (A, C) and 50 pm (B, D). Quantification of type II collagen on day 0 and day 7 (E). Unpaired t-test with Welch’s correction, **** p < 0.0001. For quantification, 9 images were randomly acquired on three independent replicates.

[0051] Figure 5: Live / Dead assay on hACs embedded in a 3D GelMA scaffold at day 0 (A) and day 9 after culture under hypoxic (B) or normoxic (C) conditions. Live / Dead assay on hACs embedded in a 3D GelMA / GelOXA scaffold after 9 days of culture under normoxic conditions. Scale bar = 100 pm.

[0052] Figure 6: Confocal microscopy images of hACs after 9 days of culture in the GelMA scaffold under hypoxia (A, D) and normoxic conditions (B, E).

[0053] Immunohistochemistry on hACs after 9 days of culture in the GelMA-GelOXA scaffold (C, F). Scale bar = 25 pm (A, B, and C). The arrows indicate type II collagen. Nuclei are also visible. Scale bar = 10 pm (D, E, and F). One-way ANOVA followed by Tukey’s post hoc test. For quantification, 5 images were randomly acquired on three independent replicates. *p < 0.05.

[0054] Figure 7: Images of hACs captured by confocal microscopy after 9 days of culture in a GelMA scaffold under hypoxic conditions (A, D) and normoxic conditions (B, E). Immunohistochemistry of hACs after 9 days of culture in the GelMA-GelOXA scaffold (C, F). Scale bar = 25 pm (A, B, and C). The arrows type X collagen. Nuclei are also visible. Scale bar = 10 m (D, E, and F). One-way ANOVA followed by Tukey’s post-hoc test. For quantification, 5 images were randomly acquired on three independent replicates. *p < 0.05.

[0055] Figure 8: Images of hACs captured by confocal microscopy after 9 days of culture in a GelMA scaffold under hypoxic conditions (A, D) and normoxic conditions (B, E). Immunohistochemistry of hACs after 9 days of culture in the GelMA-GelOXA scaffold (C, F). Scale bar = 25 pm (A, B, and C). The arrows indicate type I collagen. Nuclei are also visible. Scale bar = 10 pm (D, E, and F). One-way ANOVA followed by Tukey’s post-hoc test. For quantification, 5 images were acquired randomly on three independent replicates. *p < 0.05.

[0056] Figure 9: Images of hACs captured by confocal microscopy after 9 days of culture in a GelMA scaffold under hypoxic (A, D) and normoxic conditions (B, E). Immunohistochemistry of hACs after 9 days of culture in the GelMA-GelOXA scaffold (C, F). Scale bar = 25 pm (A, B, and C). The arrows type I collagen. Nuclei are also visible. Scale bar = 10 pm (D, E, and F). Friedman test followed by Dunn’s post-hoc test. For quantification, 5 images were randomly acquired on three independent replicates. *p < 0.05.

[0057] Figure 10: Experimental design timeline. Isolation and expansion were performed using serum-enriched culture medium (cFBS-M). The cFBS-M was replaced with serum-free, chondrospecific culture medium (cCM) after biofabrication on day -1 . The experiment began after 24 hours of recovery time.

[0058] Art. 170-bis, paragraphs 2, 3 and 4 of the Italian Industrial Property Code (IPC) With regard to the provisions of Art. 170-bis, paragraphs 2, 3 and 4 IPC, please note the following: the origin of the biological material used in the present patent application is specified in the application itself, and the handling of said biological material has been carried out in compliance with the obligations arising from national and Ell regulations).

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060] Osteoarthritis (OA) is a debilitating joint condition affecting millions of people worldwide, causing painful chondral defects (CDs) that compromise patients’ quality of life.

[0061] Currently, several cartilage reconstructive techniques (RCTs) (e.g., Matrix-Assisted Autologous Chondrocyte Implantation - MACI) have been developed to overcome the limitations of total joint replacement (TJR) in the treatment of chondral defects.

[0062] However, there is no consensus on the long-term efficacy of regenerative cartilage treatments (RCTs), as they do not provide adequate pro-regenerative stimuli to ensure complete CDs healing.

[0063] The present invention originates from the identification of a composition or scaffold capable of promoting CDs healing by providing pro-regenerative hypoxic signals at the cellular / tissue level, for use during RCTs.

[0064] In a first aspect, therefore, the invention relates to a composition or biomaterial comprising a polymeric scaffold and an unmodified gelatin functionalized with a fluorinated derivative, wherein the polymeric scaffold is selected from the group consisting of a hydrogel, an electrospun fiber, and a chitosan.

[0065] The polymeric scaffold of the invention may be a hydrogel scaffold or a non-hydrogel scaffold (e.g., an electrospun fiber) and, in a preferred form, such hydrogel scaffold is selected from the group consisting of a methacrylated gelatin (GelMA), a gelatin, and a chitosan, more preferably it is composed of a gelatin methacrylate matrix (GelMA) doped with hypoxic GelMA seeds functionalized with a fluorinated oxadiazole (GelOXA), which ensures the delivery of hypoxic signals to human articular chondrocytes (hACs) embedded in the scaffold, while the fluorinated derivative of the unmodified gelatin is preferably a fluorinated oxadiazole.

[0066] Gelatin is a biocompatible polymer derived from collagen that exhibits several reactive groups useful for chemical functionalization with OXA, whose affinity for oxygen is due to the presence of fluorine atoms directly or indirectly bonded to a nitrogen-based heterocyclic nucleus. Overall, this chemical-structural architecture perfectly aligns with the highly demanding experimental requirements of locally modulating oxygen concentration.

[0067] The fluorinating properties of OXA, whose long perfluoroalkyl chain (C7F15) linked to the 1 ,2,4-oxadiazole nucleus allows for facile fluorination via nucleophilic substitutions, were exploited.

[0068] In a preferred embodiment, in the composition of the invention:

[0069] - said fluorinated oxadiazole is 3-pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4- oxadiazole (OXA);

[0070] - said methacrylic gelatin is GelMA

[0071] - said unmodified gelatin is type A gelatin from porcine skin of formula

[0072] Gelatin

[0073] In a more preferred embodiment, the composition of the invention is a GelMA / GelOXA mixture.

[0074] The GelMA / GelOXA scaffold surprisingly preserves hACs viability, maintaining their native phenotype and significantly enhancing the production of type II collagen. Furthermore, a reduction in type I and type X collagen, characteristic of unhealthy cartilage, was observed. These results pave the way for regeneration of healthy, hybrid-like cartilage by providing hypoxic signals even under normoxic conditions.

[0075] The field of cartilage regeneration faces significant challenges linked to the limitations of current therapeutic approaches. In particular, the inability to maintain a favorable environment for chondrocytes during the repair process represents a crucial obstacle. Furthermore, existing technologies fail to effectively exploit the pro- chondrogenic effects of hypoxia, thus limiting functional cartilage regeneration.

[0076] Surprisingly, the GelMA / GelOXA scaffold allows to create and maintain an optimal hypoxic microenvironment, thus improving extracellular matrix deposition and cell viability.

[0077] In a second aspect, a method for the preparation of a GelMA / GelOXA composition is herein described, which comprises the steps of: a. Reacting pentadecafluoroheptyl amidoxime with pentafluorobenzyl chloride in the presence of pyridine to obtain O-pentafluorobenzoyl-pentadecafluoroheptyl amidoxime; b. Heating the O-pentafluorobenzoyl-pentadecafluoroheptyl amidoxime obtained from step a. to 160°C, allowing it to cool down, and dissolving the residue with hexane to obtain 3-pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4-oxadiazole (OXA); c. Mixing 3-pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4-oxadiazole (OXA) with a type A gelatin in triethylamine (TEA), and treating with ethyl acetate (centrifugation / freeze-drying) to obtain GelOXA; and d. Mixing the GelOXA from step c. with a methacrylic gelatin (GelMA) to obtain GelMA / GelOXA.

[0078] In a preferred embodiment of the method of the invention:

[0079] - the pentadecafluoroheptyl amidoxime from step a. is suspended in toluene;

[0080] - the step b. of heating to 160°C is performed for one hour;

[0081] 3-pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4-oxadiazole (OXA) is subsequently obtained by vacuum filtration;

[0082] - 3-pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4-oxadiazole (OXA) and gelatin type A from step c. are dissolved in DMSO; and

[0083] - GelOXA from step c. is obtained after centrifugation and lyophilization.

[0084] In an even more preferred embodiment, the method of the invention allows for the preparation of the GelMA / GelOXA composition populated with cells. To this end, the method includes an additional step: step e. of culturing cells in the GelMA / GelOXA support obtained from step d.

[0085] The method therefore allows for the preparation of a scaffold in which cells grow, while maintaining an optimal hypoxic microenvironment, thus enhancing extracellular matrix deposition and cell viability.

[0086] The scaffold design is based on GelOXA seeds as the active hypoxia-inducing component, dispersed in a GelMA matrix, which provides the main structural support.

[0087] As will be seen in the experimental part, the scaffold was cellularized with human articular chondrocytes (hACs) collected and cultured using a customized protocol. The scaffold of the invention can therefore be used and implanted in the patient populated with cells or without cells. In either case, it provides support for the cells present in the area to be treated.

[0088] In a third aspect, the invention concerns a product (scaffold, biomaterial, or implant) obtainable from the process according to the present invention.

[0089] The method described herein has led to the creation of a hypoxic scaffold that promotes tissue regeneration through an active matrix. In particular, the biomaterial exploits the control of oxygen concentration to promote cartilage regeneration.

[0090] In a fourth aspect, the use of the GelMA / GelOXA composition as a medicament is herein described.

[0091] In a fifth aspect, the present invention describes the use of the GelMA / GelOXA composition in the treatment of chondral and osteochondral diseases or lesions.

[0092] The invention has enabled the development of a hypoxic scaffold as an active matrix for treatments with cellularized regenerative cartilage with human articular chondrocytes (hACs).

[0093] Advantageously, in a preferred embodiment of the use of the GelMA / GelOXA composition, the chondral and osteochondral diseases are selected from the group consisting of osteoarthritis, osteoarthrosis, arthrosis, arthritis, and the chondral and osteochondral lesions are selected from the group consisting of traumatic and post- traumatic lesions.

[0094] Furthermore, the GelMA / GelOXA scaffold ability to deliver healing signals directly to the injury site has great potential for the treatment of OA and related CDs, potentially revolutionizing the fields of cartilage repair and regenerative medicine, and tissue engineering for cartilage regeneration using an innovative approach based on a scaffold capable of refining hypoxia in situ. This scaffold has demonstrated the advantage of creating a controlled and localized environment that mimics the native oxygen levels of healthy articular cartilage. Furthermore, the GelMA / GelOXA scaffold according to the invention has overcome the limitations described for other hypoxia-inducing materials, which induce only transient hypoxia and short-lived stabilization of HIF-1 a. Conversely, the GelMA / GelOXA scaffold has been shown to be effective in inducing and maintaining HIF-1 a for up to 9 days. This strong result is supported by evidence of increased type II collagen production coupled with reduced expression of undesirable hyaline cartilage markers (type l / X collagen). Remarkably, the scaffold promoted the deposition of a healthy, hyaline-like extracellular matrix even under normoxic conditions, suggesting its potential to deliver pro-regenerative signals directly to the injury site.

[0095] The GelMA / GelOXA scaffold surprisingly emerges as a promising strategy for cartilage regeneration in the treatment of chondral defects, maximizing the benefits of hypoxia in a controlled and targeted manner, thus promising significant results for future cartilage regeneration.

[0096] The following Examples of embodiments of the present invention are provided for illustrative purposes.

[0097] EXAMPLES

[0098] Example 1: Synthesis and Chemical-Physical Characterization of OXA and GelOXA Derivatives

[0099] Synthesis of 3-Pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4-oxadiazole (OXA) Pentadecafluoroheptyl amidoxime (Compound 1 , Figure 1A) (428 mg, 1 mmol) was suspended in toluene (10 mL), then pyridine (0.09 g, 1 mmol) and pentafluorobenzyl chloride (254 mg, 1.1 mmol) were added, and the mixture was stirred at room temperature overnight. The solvent was removed by evaporation, and the solid residue was filtered off by washing with water (3x20 mL), affording 0- pentafluorobenzoyl-pentadecafluoroheptyl amidoxime (intermediate, Compound 2 in Figure 1A) (603 mg, 97%, mp 154-6 °C). The solid was melted at 160 °C over 1 hour and, after cooling, treated with hexane. The filtrate was reduced under vacuum to obtain 3-pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4-oxadiazole (OXA, Figure 1A) (453 mg, 77%): mp, 36-8 °C.

[0100] Under these conditions, OXA can react with the nucleophilic groups of gelatin, primarily the -OH groups of hydroxyproline. Fluorinated, functionalized gelatin (GelOXA) was obtained in excellent yield after precipitation in ethyl acetate. The solid GelOXA was simply recovered after centrifugation, washed, and centrifuged twice.

[0101] The solvents and reagents were purchased from Sigma-Aldrich (reagent grade) and were not further purified. Type A gelatin (porcine skin, Sigma-Aldrich, 250 Bloom) was used.

[0102] Melting points were determined using a REICHART-THERMOVAR hot-stage apparatus.

[0103] Synthesis of Gelatin-Oxadiazole (GelOXA)

[0104] Gelatin (100 mg) was dissolved in DMSO (5 mL), and triethylamine (TEA) (55 pL) was added. The oxadiazole derivative obtained in the previous step (OXA) (100 mg) was dissolved in DMSO (1 mL) and added to half of the solution. After a few minutes, a yellowish gel formed which was left at room temperature overnight. Ethyl acetate (40 mL) was added to the mixture, and the resulting mixture was centrifuged (10 min, 4000 rpm) to remove the solvent and unreacted materials. The treatment was repeated twice. The residue was freeze-dried overnight, yielding 189 mg of GelOXA. Characterization of GelOXA by19F MAS-NMR,

[0105] Several techniques were used to verify the gelatin functionalization and evaluate the properties of GelOXA. Solid-state magic-angle spin (MAS)19F NMR spectroscopy is shown in Figure 1 B, clearly showing the presence of the oxadiazole compound fluorinated groups. The -CF3 group (approximately -77 ppm), the -CF2- groups (in the region from -110 to -125 ppm), and the fluorine atoms bonded to the aromatic ring (in the region from -130 to -165 ppm) are clearly visible.

[0106] The minimal changes in cell density observed by bright-field microscopy suggest that hACs do not exhibit significant proliferative activity throughout the entire experimental period. Cell density appeared slightly altered until day 2, followed by normalization over time until day 7 (Figure 2A). These results are consistent with data obtained from the Alamar Blue proliferation assay, indicating a non-significant modification of the native, non-proliferative phenotype of hACs (Figure 2B). hACs showed a good overall capacity to produce GAGs, which are among the most representative components of the extracellular matrix of hyaline cartilage. GAGs were identified after staining with Alcian Blue, a group of water-soluble, polyvalent basic dyes that can bind to sulfated and carboxylated mucopolysaccharides and sialomucins, resulting in a pigmentation ranging from light to intense blue. Remarkably, the slight but detectable blue coloration observed in hACs immediately after harvest on day 0 (Figure 3A, B) increased after 7 days of 2D culture (day 7) (Figure 3C, D).

[0107] The maintenance of the characteristic phenotype of hACs was confirmed by the presence of type II collagen, as demonstrated by immunocytochemical staining (Figure 4A, B).

[0108] Surprisingly, the amount of type II collagen increased significantly after 7 days of culture (Figure 4E), with visible diffusion into the pericellular and extracellular spaces (Figure 4C,D). These results strongly indicate a positive effect of the culture conditions on the preservation of the native phenotype of hACs, which produces an extracellular matrix rich in type II collagen.

[0109] Example 2: Effects of GelMA / GelOXA

[0110] Support of Cell Viability in Normoxic Conditions

[0111] The viability of hACs in 3D culture was assessed using the same Live / Dead kit used for 2D culture. hACs showed robust viability immediately after the support biofabrication, at day 0, as clearly indicated by the abundance of visible spots (green) in Figure 5A.

[0112] Cells in the GelMA support showed reduced viability after 9 days of 3D culture, as evidenced by the increased number of spots (red), indicated by the arrows, present after both hypoxic and normoxic culture (Figure 5B, C).

[0113] Conversely, culture in the GelMA / GelOXA support of the invention resulted in a significantly greater number of visible spots (green) compared to those (red) indicated by the arrows (Figure 5D), suggesting that the GelMA / GelOXA support provides a more suitable environment for viability of hACs in 3D.

[0114] Promoting hyaline cartilage-like ECM secretion through activation of hypoxiadependent pathways

[0115] Cells cultured in GelMA under hypoxic conditions showed increased production of type II collagen compared to cells cultured in GelMA under normoxic conditions. Remarkably, hACs cultured in the GelMA / GelOXA support under normoxic conditions exhibited a remarkable capacity to produce type II collagen, with a significantly increased amount compared to GelMA under normoxic conditions (negative control), as shown in Figures 6C and 6F. Importantly, there was no significant difference in type II collagen deposition between the culture in GelMA under hypoxic conditions (positive control) and in the GelMA / GelOXA support.

[0116] This suggests that GelMA / GelOXA supports can induce the production of a hyaline cartilage-like ECM rich in type II collagen even under normoxic conditions.

[0117] Conversely, collagen X immunofluorescence results showed that hACs incorporated into GelMA cultured under normoxic conditions produced higher amounts of type X collagen than hACs in GelMA cultured under hypoxic conditions or cells in GelMA / GelOXA cultured under normoxic conditions.

[0118] Deposition of collagen X, a selective marker for unhealthy hypertrophic chondrocytes, was observed in its characteristic perinuclear location in GelMA supports cultured under normoxic conditions. Interestingly, the amount of type X collagen in GelMA / GelOXA conditions was not significantly different from that in the positive control (GelMA in hypoxia), suggesting that the GelMA / GelOXA support may prevent hypertrophic differentiation of hACs under normoxic conditions.

[0119] Furthermore, the GelMA / GelOXA support showed a regulatory effect on type I collagen deposition, typical of fibrotic cartilage.

[0120] Specifically, hACs incorporated into GelMA and cultured under hypoxic and normoxic conditions produced a matrix containing similar amounts of type I collagen. Conversely, hACs cultured in the GelMA / GelOXA support showed a significant reduction in type I collagen production.

[0121] The GelMA / GelOXA support had a significant impact on the regulation of the hypoxia-driven HIF-1 a pathway. Indeed, hACs in the GelMA / GelOXA support (Figure 9C, F) induced HIF-1 a production at levels similar to those produced by hACs cultured in GelMA under hypoxic conditions (Figure 9A, D), and both showed significantly higher production (Figure 9G) compared to GelMA under normoxic conditions (Figure 9B, E).

[0122] These results surprisingly demonstrate that the GelMA / GelOXA support is capable of providing hypoxic signals in situ. MATERIALS AND METHODS

[0123] Magnetic Angle Spinning Solid-State Nuclear Magnetic Resonance Spectroscopy (MAS-ssNMR).

[0124] The19F MAS-NMR spectrum was obtained at room temperature using a Bruker Avance II 400 MHz (9.4 T) spectrometer operating at 376.49 MHz for the19F nucleus with a MAS rate of 25 kHz, a 90° pulse on the19F of 4 ps, a repetition delay of 4 s, and 32 scans. The sample (approximately 50 mg) was packed in a 2.5 mm zirconia rotor with VESPEL caps.

[0125] Collection and Expansion of Human Articular Chondrocytes (hACs)

[0126] Human chondrocytes (hACs) were harvested from healthy femoral condyles and tibial plateau cartilage obtained from donors undergoing total knee replacement (TKR).

[0127] In particular, tissues were collected from three female donors (64, 71 , and 82 years old), selected based on well-defined inclusion criteria (unicompartmental OA, no prior knee surgery, no significant comorbidities). Surgical waste materials were thoroughly washed with sterile PBS and placed in a sterile dish containing dissection medium (Diss-M: High Glucose DMEM - Gibco USA, Penicillin / Streptomycin / Amphotericin 1 % v / v - Gibco USA, Fetal Bovine Serum 10% v / v - Gibco USA).

[0128] Subseguently, the macroscopically intact cartilage was carefully removed using a sterile scalpel, avoiding the underlying subchondral bone, and transferred to a new sterile dish containing Diss-M, where it was minced into 1 cm2pieces.

[0129] The minced cartilage was then collected in a sterile 500 mL conical tube, and incubated with a solution of Collagenase A (Worthington, UK) at 5 mg / ml in Diss-M at 37°C. under mechanical stirring (250 rpm) overnight. Finally, the cells were recovered, filtered through a 40 pm cell sieve, counted, and seeded at 7.0 x 103cells / cm2in a T75 flask treated for tissue culture. Chondrocytes were allowed to grow to 80% confluence using chondro-FBS medium (cFBS-M: High Glucose DMEM - Gibco USA, Penicillin / Streptomycin / Amphotericin 1 % v / v - Gibco USA, Insulin / Transfemn / Selenium 1 % v / v - Thermofisher USA, Dexamethasone 0.1 pM - Sigma-Aldrich USA, L-Proline 40 pg / ml - Sigma-Aldrich USA, Fetal Bovine Serum 10% v / v - Gibco USA).

[0130] Biofabrication of GelMA-hACs and GelMA / GelOXA-hACs scaffolds hACs were gently detached by adding 5 mL of TryPLE (Gibco USA) to the T75 flask and holding at 37°C for 10 minutes. Cells were recovered by centrifugation at 1200 rpm for 7 minutes (rotor radius = 247 mm) and pooled. The cell pellet was resuspended in a 10% w / v GelMA solution (Cellink, USA) in a PBS solution containing 0.25% w / v lithium phenyl-2,4,6-trimethylbenzoin phosphinate (LAP) (Cellink, USA), avoiding the formation of air bubbles throughout the process.

[0131] For the GelMA / GelOXA-hACs scaffolds, 10% w / v GelOXA was added to the cell suspension. Finally, the cell suspension was distributed into a silicone mold, and UV-mediated crossover was induced by irradiating the scaffolds with a UV lamp equipped with a 395 nm LED (LEPRO, USA) for 3 minutes. Three independent replicates were performed for each condition.

[0132] Scaffold cultivation

[0133] The scaffolds were carefully removed from the silicone molds and transferred to an untreated 24-well tissue culture plate (day -1 , Figure 10). Subsequently, each scaffold was supplied with 1 mL of cFBS-M and incubated at 37 °C under an atmosphere containing 5% CO2 for 24 h, after which (day 0, Figure 10) cFBS-M was replaced with complete chondrocyte medium (cCM: High Glucose DMEM - Gibco USA, Penicillin / Streptomycin / Amphotericin 1 % v / v - Gibco USA, Insulin / Transfemn / Selenium 1 % v / v - Thermofisher USA, Dexamethasone 0.1 pM - Sigma-Aldrich USA, L-Proline 40 pg / ml - Sigma-Aldrich USA, Ascorbic Acid 50 pg / ml - Sigma-Aldrich USA, TGF-[33 10 ng / ml - Peprotech UK, FBS-free).

[0134] Viability Assays

[0135] Cells or cell-loaded scaffolds were washed twice in PBS to remove residual culture medium, then stained using a Live / Dead assay (Abeam, UK). Samples were stained with 4 pM calcein and 2 pM Ethd-1 (final concentration) in PBS for 30 minutes at 37°C under an atmosphere of 5% CO2. After staining, the samples were rinsed twice in PBS to remove excess dye and analyzed using an EVOS M5000 inverted epifluorescence microscope (Thermofisher, USA).

[0136] Proliferation Assay The Alamar blue assay (Thermofisher, USA) was used to assess chondrocyte proliferation rate. To build a calibration curve, hACs were seeded at decreasing density from 1 .0 X 105to 9.0 X 103cells / cm2. 2D cell culture samples were plated at 1 .5 X 105cells / cm2in a tissue culture-treated 96-well plate in cFBS-M (day -2). After 48 hours (day 0), cFBS-M was replaced with cCM for the cells to be used in the calibration curve and for the 2D culture test samples. All samples for the calibration curve were analyzed on day 0. For 2D cell culture, measurements were performed daily from day 0 to day 7 following the manufacturer’s instructions. Briefly, a 10% v / v dilution of Alamar Blue in cCM was used to incubate the cells for 3 hours at 37°C with 5% CO2, and fluorescence measurements (AExc = 560 nm, AEm = 590 nm) were performed using a Spark 10M plate reader (Tecan, Switzerland).

[0137] Histology

[0138] Cells cultured in 2D were fixed for 30 min at 4°C in 4% paraformaldehyde (Sigma, USA), washed twice with PBS, and stored in PBS for immunocytochemistry assays. Cellularized scaffolds were fixed overnight in 4% v / v paraformaldehyde (Sigma, USA) at 4°C with gentle agitation. The constructs were then dehydrated in an increasing series of ethanol (25%, 50%, 70%, 95%, and 100%) before being embedded in histology-grade paraffin (Leica Biosystems, Germany) using a HistoCore Arcadia H embedding station (Leica Biosystems, Germany). The embedded samples were then sectioned at 5 pm thickness using a Leica RM2265 motorized rotary microtome (Leica Biosystems, Germany), collected on Superfrost Plus adhesive slides (Thermofisher, USA), and stored at room temperature until immunostaining.

[0139] Immunocytochemistry

[0140] Immunological staining was performed in 24-well plates on days 0 and 7 for both 2D cell culture and cell-loaded scaffolds. Briefly, cells / scaffolds were washed twice with PBS and treated with 70% v / v EtOH for 15 minutes at room temperature to fix the cells. Suppression of nonspecific binding and cell membrane permeabilization were performed using a solution of 0.1 % w / v Triton X-100 (Sigma-Aldrich, USA) and 1 % w / v bovine serum albumin (BSA) (ITW, USA) in PBS for 45 minutes at room temperature. The samples were then washed twice and incubated with primary antibodies against type II collagen (ab34712, Abeam, UK) and type X collagen (ab49945, Abeam, UK) at a 1 / 50 v / v dilution overnight at 4°C.

[0141] Finally, the samples were washed three times with PBS and incubated with fluorescent secondary antibodies (Ab96919 and Ab98795, respectively, Abeam, UK) at a 1 / 50 dilution, along with 4’,6-diamidino-2-phenylindole (DAPI) as a nuclear stain. The samples were visualized using an Evos M5000 epifluorescence microscope (Thermofisher, USA).

[0142] Histochemistry and immunohistochemistry

[0143] Scaffolds were fixed overnight in 4% w / v paraformaldehyde (Sigma, USA) at 4°C with gentle shaking, followed by dehydration in an increasing series of ethanol (25%, 50%, 70%, 95%, and 100%).

[0144] Scaffolds were then immersed in xylene before being embedded in histology-grade paraffin (Leica Biosystems, Germany) using a HistoCore Arcadia H embedding station (Leica Biosystems, Germany).

[0145] The embedded specimens were sectioned at 5 pm thickness using a Leica RM2265 motorized rotary microtome (Leica Biosystems, Germany), collected on Superfrost Plus adhesive slides (Thermofisher, USA), and stored at room temperature in the dark.

[0146] For histochemical and immunohistochemical staining, the sections were deparaffinized with xylene (Sigma, USA), rehydrated by immersion in a decreasing series of ethanol (100%, 95%, 70%, and 50%), and gently washed in deionized water for 2 minutes.

[0147] Alcian Blue Staining

[0148] Slides were immersed in a 1 % w / v solution of Alcian blue in acetic acid (pH = 2.5) (Sigma, USA) for 1 hour at room temperature, and the staining solution was washed off with deionized water.

[0149] Subsequently, slides were dehydrated in an increasing series of ethanol (25%, 50%, 70%, 95%, and 100%), mounted with organic mounting medium / limonene, and stored at room temperature away from light.

[0150] Immunohistochemistry For immunological staining, unconjugated Ms-HIF1 a (1 :100 dilution, ab16066, Abeam, USA), Rb-COLI (1 :42 dilution v / v, ab233080, Abeam, USA), Rb-COLII (1 :50 dilution v / v, ab34712, Abeam, USA), and Ms-COLX (1 :1000 v / v dilution, ab49945, Abeam, USA) were used as primary antibodies.

[0151] The conjugated secondary antibodies Donkey anti-Mouse DL550 (1 :50 dilution v / v, Ab98795, Abeam, USA) and Donkey anti-Rabbit DL488 (1 :50 dilution v / v, Ab96919, Abeam, USA) were used for visualization.

[0152] Propidium iodide (250 pg / mL dilution, ab14083, Abeam, USA) and 4’,6-diamidino- 2-phenylindole dihydrochloride (DAPI) (Thermofisher, USA) were used as nuclear stains. Briefly, antigen retrieval was performed using an aqueous citrate buffer at pH 6.0 containing 0.294% w / v sodium citrate dihydrate (Sigma, USA), 5 pl of Tween 20 (Sigma, USA), and deionized water.

[0153] The slides were then incubated in a StainTray staining system (Merck, Germany) with primary antibodies overnight, at 4°C in the dark. Subsequently, the primary antibody was washed off by rinsing three times with PBS, and the samples were incubated with a mixture of secondary antibody and nuclear stain for 30 minutes at room temperature in the dark. Finally, the slides were mounted with Prolong™ Glass Antifade Mountant (Thermofisher, USA) and stored in the dark at 4°C.

[0154] Statistics

[0155] All analyses were performed using Prism 9.0 (GraphPad software, USA). Experimental results are expressed as mean±standard deviation (SD). The unpaired t-test with Welch’s correction, one-way ANOVA followed by Tukey’s post hoc test, or the Friedman test followed by Dunn’s post hoc tests were used, based on preliminary analyses of the data distribution. Differences were considered statistically significant if p < 0.05 (*p < 0.05, **p < 0.01 , and ***p < 0.001 ).

[0156] From the detailed description and the Examples reported above, the advantages achieved by the composition of the present invention are apparent. In particular, this composition has proven to be a surprisingly and advantageously promising scaffold for cartilage regeneration in the treatment of chondral diseases.

Claims

CLAIMS1. A composition comprising a polymeric scaffold and an unmodified gelatin functionalized with a fluorinated derivative, wherein:- said polymeric scaffold is selected from the group consisting of a hydrogel, an electrospun fiber, and chitosan; and- said fluorinated derivative of the unmodified gelatin is a fluorinated oxadiazole.

2. The composition according to claim 1 , wherein:- said polymeric scaffold is methacrylic gelatin (GelMA);- said fluorinated derivative is 3-pentadecafluoroeptyl-5-pentafluorophenyl-1 ,2,4- oxadiazole (OXA);- said unmodified gelatin is type A gelatin from porcine skin of formulaGelatin3. The composition according to any one of claims 1 to 2, wherein said composition is a GelMA / GelOXA mixture.

4. A method for the preparation of a GelMA / GelOXA composition comprising the steps of: a. reacting pentadecafluoroheptyl amidoximewith pentafluorobenzoyl chloride in the presence of pyridine, to obtain 0- pentafluorobenzoyl-pentadecafluoroheptyl amidoximeb. heating the O-pentafluorobenzoyl-pentadecafluoroheptyl amidoxime obtained from step a. at 160°C, allowing it to cool down, and dissolving the residue with hexane to obtain 3-pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4-oxadiazole (OXA); c. mixing the 3-pentadecafluoroheptyl-5-pentafluorophenyl-1 ,2,4-oxadiazole (OXA) with type A gelatin in triethylamine (TEA), treating with ethyl acetate (centrifuge / lyophilize) to obtain GelOXA; andd. mixing the GelOXA from step c. with methacrylic gelatin (GelMA) to obtain GelMA / GelOXA mixtures.

5. The method according to claim 4, wherein said pentadecafluoroheptyl amidoxime from step a. is suspended in toluene, and wherein said step b. of heating to 160°C is carried out for one hour, and the 3-pentadecafluoroheptyl-5-pentafluorophenyl- 1 ,2,4-oxadiazole (OXA) is then obtained by vacuum filtration.

6. The method according to claim 5, wherein said 3-pentadecafluoroheptyl-5- pentafluorophenyl-1 ,2,4-oxadiazole (OXA) and type A gelatin from step c. are dissolved in DMSO.

7. The method according to any one of claims 5 to 6, wherein said GelOXA from step c. is obtained after centrifugation and lyophilization.

8. The method according to any one of claims 5 to 7, wherein said method has an additional step e. of culturing cells on the GelMA / GelOXA support obtained from step d.

9. A product obtainable from the method according to any one of claims 5 to 8.

10. A GelMA / GelOXA composition according to any one of claims 1 to 3, for use as a medicament.11 . A GelMA / GelOXA composition according to any one of claims 1 to 3, for use in the treatment of chondral and osteochondral diseases or lesions.

12. The GelMA / GelOXA composition for use according to claim 1 1 , wherein said chondral and osteochondral diseases are selected from the group consisting of osteoarthritis, osteoarthrosis, arthrosis, arthritis, and the chondral and osteochondral lesions are selected from the group consisting of traumatic and post- traumatic lesions.

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