BIOMATERIAL COMPRISING ADIPOSE-DERIVED STEM CELLS AND GELATIN AND METHOD FOR PRODUCING THE SAME

Abstract

The present invention relates to a biomaterial comprising adipose-derived stem cells (ASCs), an extracellular matrix and gelatin. The present invention also relates to methods for producing the biomaterial and uses thereof.

Claims

1. A biomaterial having a multi-dimensional structure comprising differentiated adipose-derived stem cells (ASCs), an extracellular matrix and gelatin.

2. The biomaterial according to claim 1, wherein said gelatin is porcine gelatin.

3. The biomaterial according to claim 1, wherein said gelatin is in the form of particles.

4. The biomaterial according to claim 3, wherein said particles have a mean diameter ranging from about 50 μm to about 1000 μm, have a mean diameter ranging from about 75 μm to about 750 μm, or have a mean diameter ranging from about 100 μm to about 500 μm.

5. The biomaterial according to claim 1, wherein said biomaterial is three-dimensional.

6. The biomaterial according to claim 1, wherein said ASCs are differentiated into cells selected from the group consisting of osteoblasts, chondrocytes, keratinocytes, myofibroblasts, endothelial cells and adipocytes.

7. (canceled)

8. A method for producing a biomaterial having a multi-dimensional structure comprising differentiated adipose-derived stem cells (ASCs), an extracellular matrix and gelatin comprising the steps of: adipose-derived stem cells (ASCs) proliferation, ASCs differentiation at the fourth passage, and multi-dimensional induction, optionally three-dimensional induction.

9. A multi-dimensional biomaterial obtainable by the method according to claim 8.

10. (canceled)

11. The method according to claim 16, wherein said tissue is selected from the group consisting of bone, cartilage, dermis, muscle, endothelium and adipose tissue.

12. The method according to claim 16, wherein said tissue defect is a dermis defect.

13. The method according to claim 16, wherein said biomaterial is for use for dermis reconstruction.

14. The method according to claim 16, wherein said biomaterial is for use for treating a dermis wound, optionally a diabetic dermis wound.

15. The method according to claim 16, wherein said biomaterial is for use for treating epidermolysis bullosa, giant congenital nevi, and/or aplasia cutis congenita.

16. A method for treating a tissue defect in a subject in need thereof, comprising administering to said subject a biomaterial having a multi-dimensional structure comprising differentiated adipose-derived stem cells (ASCs), an extracellular matrix and gelatin.

17. The method according to claim 16, wherein said gelatin is porcine gelatin.

18. The method according to claim 16, wherein said gelatin is in the form of particles.

19. The method according to claim 16, wherein said biomaterial is three-dimensional.

20. The method according to claim 16, wherein said ASCs are differentiated into cells selected from the group consisting of osteoblasts, chondrocytes, keratinocytes, myofibroblasts, endothelial cells and adipocytes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0188] FIGS. 1A-1B are photographs showing macroscopic views of a biomaterial. FIG. 1A: biomaterial formed with porcine gelatin (Cultispher G) and ASCs at 2.5 weeks of culture in osteodifferentiation medium. Fig. B: biomaterial formed with porcine gelatin (Cultispher G) and ASCs at 7.5 weeks of culture in osteodifferentiation medium.

[0189] FIGS. 2A-2B are photographs showing hematoxylin-eosin stainings of a biomaterial formed with porcine gelatin (Cultispher G) and ASCs at 7.5 weeks of culture in osteodifferentiation medium. FIG. 2A: Original magnification ×5. FIG. 2B: enlargement ×10.

[0190] FIGS. 3A-3B are photographs showing Von Kossa stainings of a biomaterial formed with porcine gelatin (Cultispher G) and ASCs at 7.5 weeks of culture in osteodifferentiation medium. FIG. 3A: Original magnification. FIG. 3B: enlargement ×10.

[0191] FIGS. 4A-4B are photographs showing osteocalcin expression of a biomaterial formed with porcine gelatin (Cultispher G) and ASCs at 7.5 weeks of culture in osteodifferentiation medium. FIG. 4A: Original magnification. FIG. 4B: enlargement ×10.

[0192] FIGS. 5A-5L are graphs showing expression of genes in the biomaterial of the invention formed with ASCs and Cultipher G (biomaterial) in osteodifferentiation medium compared to ASCs in MP (MP). FIG. 5A: ANG; FIG. 5B: ANGPT1; FIG. 5C: EPHB4; FIG. 5D: EDN1; FIG. 5E: THBS1; FIG. 5F: PTGS1; FIG. 5G: LEP; FIG. 5H: VEGFA; FIG. 5I: VEGFB; FIG. 5J: VEGFC; FIG. 5K: ID1; and FIG. 5L: TIMP1. *: p<0.05.

[0193] FIGS. 6A-6D are photographs showing the biomaterial of the invention formed with ASCs and Cultipher G at different maturation levels in osteodifferentiation medium. FIG. 6A: 4 weeks; FIG. 6B: 8 weeks; FIG. 6C: 12 weeks; and FIG. 6D: 25 weeks. Mineralization are displayed in yellow in the 3D matrix shown in transparent.

[0194] FIG. 7 is a photograph of radiographies of the “implant sites” of biomaterial formed with porcine gelatin (Cultispher G or S) and ASCs at 7.5 weeks of culture in osteodifferentiation medium in Nude rats at day 29 post-implantation.

[0195] FIG. 8 is a photograph of radiographies of the “implant sites” of biomaterial formed with porcine gelatin (Cultispher G or S) and ASCs at 7.5 weeks of culture in osteodifferentiation medium in Wistar rats at day 29 post-implantation.

[0196] FIG. 9 is a photograph showing Von Kossa staining of a biomaterial formed with porcine gelatin (Cultispher G or S) and ASCs at 7.5 weeks of culture in osteodifferentiation medium.

[0197] FIG. 10 is a photograph showing hematoxylin-eosin staining of a biomaterial formed with porcine gelatin (Cultispher S) and ASCs at 7.5 weeks of culture in osteodifferentiation medium.

[0198] FIG. 11 is a photograph showing Von Kossa staining 29 days after implantation in a Nude rat of a biomaterial formed with porcine gelatin (Cultispher S) and ASCs at 7.5 weeks of culture in osteodifferentiation medium.

[0199] FIGS. 12A-12B are photographs showing radiographies of the “implant sites” in Nude rats. FIG. 12A: at day 29 post-implantation of a biomaterial formed with porcine gelatin (Cultispher G or S) and ASCs at 7.5 weeks of culture in osteodifferentiation medium. FIG. 12B: at day 29 post-implantation of a biomaterial formed with porcine gelatin (Cultispher G or S) alone.

[0200] FIGS. 13A-13C are photographs showing wound healing of legs of rats at day 0 (D0), 15 (D15), 23 (D23) and 34 (D34). FIG. 13A: without implantation; FIG. 13B: after implantation of Cultispher S particles alone; and FIG. 13C: after implantation of a biomaterial formed with porcine gelatin (Cultispher S) and ASCs at 8 weeks of culture in osteodifferentiation medium (C). Left limbs: ischemic legs; right limbs: non-ischemic legs.

[0201] FIG. 14 is a histogram showing area under the curve (AUC) for the wound size in non-ischemic legs (black bars) and ischemic legs (white bars) not treated (sham) or treated with Cultispher S particles alone (Cultispher) or a biomaterial formed with porcine gelatin (Cultispher S) and ASCs at 8 weeks of culture in osteodifferentiation medium (biomaterial), evaluated in comparison with the sham, fixed at 100%.

[0202] FIGS. 15A-15B are graphs showing wound area in percentage from day 0 to day 34 after treatment with Cultispher S particles alone (squares) or a biomaterial formed with porcine gelatin (Cultispher S) and ASCs at 8 weeks of culture in osteodifferentiation medium (circles), or not treated (sham, triangles). FIG. 15A: on non-ischemic legs; FIG. 15B: on ischemic legs.

[0203] FIGS. 16A-16B are graphs showing days of complete wound closure after no treatment (sham, left), treatment with Cultispher S particles alone (middle) or a biomaterial of the invention (right). FIG. 16A: non-ischemic legs; FIG. 16B: ischemic legs.

[0204] FIGS. 17A-17C are graphs showing number of lymphocytes CD3 (black lines) and macrophages CD68 (gray lines) from day 0 to day 34 after treatment of an ischemic leg.

[0205] FIG. 17A: no treatment (sham control). FIG. 17B: with Cultispher S particles alone. FIG. 17C: with a biomaterial formed with porcine gelatin (Cultispher S) and ASCs at 8 weeks of culture in osteodifferentiation medium.

[0206] FIGS. 18A-18B are graphs showing the thickness of wound at day 15 and day 34 after no treatment (sham control), after implantation of Cultispher S particles alone (Cultisphers) and after implantation of a biomaterial formed with porcine gelatin (Cultispher S) and ASCs at 8 weeks of culture in osteodifferentiation medium. FIG. 18A: in an ischaemic model. FIG. 18B: in a non-ischaemic model.

[0207] FIGS. 19A-19D are histograms showing epidermal and dermal scores on non-ischemic legs at day 1, 5, 15 and 34 after treatment with Cultispher S particles alone (dotted histograms) or a biomaterial formed with porcine gelatin (Cultispher S) and ASCs at 8 weeks of culture in osteodifferentiation medium (black histograms), or not treated (sham, striped histograms). FIG. 19A: epidermal score of the core of non-ischemic leg. FIG. 19B: epidermal score of the periphery of non-ischemic leg. FIG. 19C: dermal score of the core of non-ischemic leg. FIG. 19D: dermal score of the periphery of non-ischemic leg.

[0208] FIGS. 20A-20D are photographs showing structures obtained with ASCs and particles in different medium. FIG. 20A: osteogenic medium; FIG. 20B: chondrogenic medium; FIG. 20C: myofibrogenic medium; and FIG. 20D: keratinogenic medium. Form of the structure (1.), grippability (2.), hematoxylin-eosin staining (3.) and tissue-specific stainings (4.), namely osteocalcin (OC) for osteogenic medium, alcian blue (AB) for chondrogenic medium, α-SMA for myofibrogenic medium, and CD34 for keratinogenic medium, were assessed.

EXAMPLES

[0209] The present invention is further illustrated by the following examples.

Example 1: Production of Biomaterials of the Invention

[0210] 1.1. Isolation of hASCs

[0211] Human subcutaneous adipose tissues were harvested by lipo-aspiration following Coleman technique in the abdominal region and after informed consent and serologic screening.

[0212] Human adipose-derived stem cells (hASCs) were promptly isolated from the incoming adipose tissue. Lipoaspirate can be stored at +4° C. for 24 hours or for a longer time at −80° C.

[0213] First, a fraction of the lipoaspirate was isolated for quality control purposes and the remaining volume of the lipoaspirate was measured. Then, the lipoaspirate was digested by a collagenase solution (NB 1, Serva Electrophoresis GmbH, Heidelberg, Germany) prepared in HBSS (with a final concentration of ˜8 U/mL). The volume of the enzyme solution used for the digestion was the double of the volume of the adipose tissue. The digestion was performed during 50-70 min at 37° C.±1° C. A first intermittent shaking was performed after 15-25 min and a second one after 35-45 min. The digestion was stopped by the addition of MP medium (proliferation medium, or growth medium). The MP medium comprised DMEM medium (4.5 g/L glucose and 4 mM Ala-Gln; Sartorius Stedim Biotech, Gottingen, Germany) supplemented with 5% human platelet lysate (hPL) (v/v). DMEM is a standard culture medium containing salts, amino acids, vitamins, pyruvate and glucose, buffered with a carbonate buffer and has a physiological pH (7.2-7.4). The DMEM used contained Ala-Gln. Human platelet lysate (hPL) is a rich source of growth factor used to stimulate in vitro growth of mesenchymal stem cells (such as hASCs).

[0214] The digested adipose tissue was centrifuged (500 g, 10 min, room temperature) and the supernatant was removed. The pelleted Stromal Vascular Fraction (SVF) was re-suspended into MP medium and passed through a 200-500 μm mesh filter. The filtered cell suspension was centrifuged a second time (500 g, 10 min, 20° C.). The pellet containing the hASCs was re-suspended into MP medium. A small fraction of the cell suspension can be kept for cells counting and the entire remaining cell suspension was used to seed one 75 cm.sup.2 T-flask (referred as Passage P0). Cells counting was performed (for information only) in order to estimate the number of seeded cells.

[0215] The day after the isolation step (day 1), the growth medium was removed from the 75 cm.sup.2 T-flask. Cells were rinsed three times with phosphate buffer and freshly prepared MP medium was then added to the flask.

[0216] 1.2. Growth and Expansion of Human Adipose-Derived Stem Cells

[0217] During the proliferation phase, hASCs were passaged 4 times (P1, P2, P3 and P4) in order to obtain a sufficient amount of cells for the subsequent steps of the process.

[0218] Between P0 and the fourth passage (P4), cells were cultivated on T-flasks and fed with fresh MP medium. Cells were passaged when reaching a confluence ≥70% and ≤100% (target confluence: 80-90%). All the cell culture recipients from 1 batch were passaged at the same time. At each passage, cells were detached from their culture vessel with TrypLE (Select 1×; 9 mL for 75 cm.sup.2 flasks or 12 mL for 150 cm.sup.2 flasks), a recombinant animal-free cell-dissociation enzyme. TrypLe digestion was performed for 5-15 min at 37° C.±2° C. and stopped by the addition of MP medium.

[0219] Cells were then centrifuged (500 g, 5 min, room temperature), and re-suspended in MP medium. Harvested cells were pooled in order to guaranty a homogenous cell suspension. After resuspension, cells were counted.

[0220] At passages P1, P2 and P3, the remaining cell suspension was then diluted to the appropriate cell density in MP medium and seeded on larger tissue culture surfaces. At these steps, 75 cm.sup.2 flasks were seeded with a cell suspension volume of 15 mL, while 150 cm.sup.2 flasks were seeded with a cell suspension volume of 30 mL. At each passage, cells were seeded between 0.5×10.sup.4 and 0.8×10.sup.4 cells/cm.sup.2. Between the different passages, culture medium was exchanged every 3-4 days. The cell behavior and growth rate from one donor to another could slightly differ. Hence the duration between two passages and the number of medium exchanges between passages may vary from one donor to another.

[0221] 1.3. Osteogenic Differentiation

[0222] At passage P4 (i.e. the fourth passage), cells were centrifuged a second time, and re-suspended in MD medium (differentiation medium). After resuspension, cells were counted a second time before being diluted to the appropriate cell density in MD medium, and a cell suspension volume of 70 mL was seeded on 150 cm.sup.2 flasks and fed with osteogenic MD medium. According to this method, cells were directly cultured in osteogenic MD medium after the fourth passage. Therefore, osteogenic MD medium was added while cells have not reached confluence.

[0223] The osteogenic MD medium was composed of proliferation medium (DMEM, Ala-Gln, hPL 5%) supplemented with dexamethasone (1 μM), ascorbic acid (0.25 mM) and sodium phosphate (2.93 mM).

[0224] The cell behavior and growth rate from one donor to another could slightly differ. Hence the duration of the osteogenic differentiation step and the number of medium exchanges between passages may vary from one donor to another.

[0225] 1.4. Multi-Dimensional Induction of Cells

[0226] The 3D induction was launched when cells reach a confluence and if a morphologic change appears and if at least one osteoid nodule (un-mineralized, organic portion of the bone matrix that forms prior to the maturation of bone tissue) was observed in the flasks.

[0227] After being exposed to the osteogenic MD medium, the culture vessels containing the confluent monolayer of adherent osteogenic cells were slowly and homogeneously sprinkled with gelatin particles (Cultispher-G and Cultispher-S, Percell Biolytica, Astorp, Sweden) at a concentration of 1, 1.5 and 2 cm.sup.3 for a 150 cm.sup.2 vessel.

[0228] Cells were maintained in MD medium. Regular medium exchanges were performed every 3 to 4 days during the multi-dimensional induction. Those medium exchanges were performed by carefully preventing removal of gelatin particles and developing structure(s).

Example 2: Characterization of the Biomaterials

[0229] 2.1. Materials and Methods

[0230] 2.1.1. Structure/Histology

[0231] The formation of a 3D structure obtained from ASCs and Cultispher G and S particles was tested. Particles of Cultispher were added on confluent ASCs at passage 4 from 6 different donors. Different volumes were tested: 1, 1.5, 2 cm.sup.3 particles per vessel of 150 cm.sup.2. The cells were maintained in differentiation medium (DMEM 4.5 g/L glucose with Ultraglutamine+1% penicillin/streptomycin+0.5% Amphotericin AB+dexamethasone (1 μM), ascorbic acid (0.25 mM) and sodium phosphate (2.93 mM)) with medium change every 3-4 days.

[0232] For the comparison of culture in MP and MD, biopsies of 3D structures in MD were taken at 5 days, 14 days and 8 weeks after addition of particles.

[0233] For the evaluation of the cellularity, biopsies of 3D structures were taken at 4 weeks, 8 weeks and 12 weeks after the addition of Cultispher particles.

[0234] They were fixed in formol and prepared for hematoxylin-eosin, Masson's Trichrome, Osteocalcin, and Von Kossa stainings.

[0235] The osteodifferentiation and the mineralization of the tissues were assessed on osteocalcin and Von Kossa-stained slides, respectively. The structure of the tissue, cellularity and the presence of extracellular matrix were assessed after hematoxylin-eosin and Masson's Trichrome staining.

[0236] 2.1.2. Biological Activity

[0237] The in vitro study of the bioactivity was assessed by (i) extraction and quantification of growth factors VEGF, IGF1, SDF-1α in the final product and (ii) the capacity of growth factors secretion/content of the biomaterial of the invention in hypoxia and hyperglycemia (conditions of diabetic wound healing for example). In addition, (iii) bioactive properties of the biomaterial of the invention were characterized in vitro at the molecular level by qRT-PCR.

[0238] Growth Factors Content

[0239] To assess the bioactivity of the tissue formed, biopsies were taken at 4 and 8 weeks post-addition of gelatin (1.5 cm.sup.3) for proteins extraction and quantification. The total protein and growth factors contents were quantified by colorimetry (BCA Protein Assay Kit, ThermoFisher Scientific) and ELISA for VEGF, SDF1α, IGF1 (Human Quantikine ELISA kits, RD Systems), according to suppliers' instructions.

[0240] Culture in Hypoxia and Hyperglycemia

[0241] To assess the bioactivity of the biomaterial of the invention and the impact of oxemia and glycemia on the bioactivity of this 3D structure, biopsies of the tissue formed with Cultispher G (1.5 cm.sup.3) and ASCs from 3 donors at 8 weeks were rinsed twice with PBS and placed in duplicate in 6 wells-plates in 10 mL of MD at 4.5 g/L (hyperglycemic condition) or 1 g/L (normoglycemic condition) glucose without HPL. Plates were placed in hypoxia (1% O2) or normoxia (21% O2), 5% CO2, 37° C., for 72 hours. Supernatants were then harvested for total protein and growth factors quantification by colorimetry (BCA Protein Assay Kit, ThermoFisher Scientific) and ELISA (BMP2, BMP7, VEGF, SDF-1α, IGF1, FGFb (Human Quantikine ELISA kits, RD Systems), respectively. The tissues were treated for proteins extractions, purification and total protein and growth factors contents quantification.

[0242] qRT-PCR

[0243] The pro-angiogenic potential of the biomaterial of the invention was investigated by the analysis of the expression of genes involved in the vasculogenesis and angiogenesis. Genes expression by adipose stem cells in different states was analyzed: adipose stem cells in proliferation media (without phenotype orientation, MP), adipose stem cells in classical osteogenic media without particles (MD) and finally the biomaterial of the invention (adipose stem cells with 1.5 cm.sup.3 of particles in view to induce the formation of the 3-dimension scaffold-free structure by the extracellular matrix).

[0244] Total RNA was extracted from >2000 ASCs cultured in proliferation medium (MP) (n=4 independent source of human adipose tissue) and from biopsies of ˜1 cm.sup.2 of the biomaterial of the invention (n=5) using the Qiazol lysis reagent (Qiagen, Hilden, Germany) and a Precellys homogenizer (Bertin instruments, Montigny-le-Bretonneux, France). RNAs were purified using Rneasy mini kit (Qiagen, Hilden, Germany) with an additional on column DNase digestion according to the manufacturer's instruction. Quality and quantity of RNA were determined using a spectrophotometer (Spectramax 190, Molecular Devices, California, USA). cDNA was synthesized from 0.5 μg of total RNA using RT.sup.2 RNA first strand kit (Qiagen, Hilden, Germany) for osteogenic and angiogenic genes expression profiles though commercially available PCR arrays (Human RT.sup.2 Profiler Assay—Angiogenesis). The ABI Quantstudio 5 system (Applied Biosystems) and SYBR Green ROX Mastermix (Qiagen, Hilden, Germany) were used for detection of the amplification product. Quantification was obtained according to the ΔΔCT method. The final result of each sample was normalized to the means of expression level of three Housekeeping genes (ACTB, B2M and GAPDH).

[0245] 2.1.3. Impact of the Maturation of the Biomaterial on its Properties

[0246] The impact of the maturation of the biomaterial (also referred as “tissue”) on its properties was assessed by the mineralization level evaluation, histological evaluation (cellularity determination) and bioactivity evaluation (extraction and quantification of growth factors VEGF, IGF1, SDF-1α). Maturation of the biomaterial means herein duration of culture of ASCs with Cultispher particles in differentiation medium.

[0247] Biopsies of 3D structures were taken at 4 weeks (one donor), 8 weeks (6 donors), 12 weeks (3 donors) and 25 weeks (1 donor) after the addition of Cultispher particles and fixed in formol for micro-CT scanner analysis. 3D structures mineralization was assessed using a peripheral quantitative CT machine (Skyscan 1172G, Bruker micro-CT NV, Kontich, Belgium).

[0248] In addition, biopsies of tissues (4 weeks (n=3), 8 weeks (n=8), 12 weeks (n=3) and 25 weeks (n=1)) were fixed in formol and prepared for hematoxylin-eosin, Masson's Trichrome, and Von Kossa stainings.

[0249] 2.2. Results

[0250] 2.2.1. Structure/Histology

[0251] No 3D structure was obtained when Cultispher particles were cultured with hASCs in proliferation medium. As no macroscopic 3D structure was found, no microscopic structure was formed.

[0252] In contrast to the proliferation medium, Cultispher cultured with ASCs in osteogenic differentiation medium showed the formation of a sheet-like 3D structure (FIG. 1A). Moreover, this structure was prehensile with forceps (FIG. 1B).

[0253] Histological examination of Cultispher cultured with ASCs in osteogenic differentiation medium revealed the presence of a cellularized interconnected tissue between particles. Moreover, extracellular matrix and cells were found in the pores of particles (FIGS. 2A and B). Von Kossa staining showed the presence of isolated mineralized particles. In contrast, the extracellular matrix was not stained by Von Kossa (FIGS. 3A and B). Finally, osteocalcine expression was found in the interconnective tissue (FIGS. 4A and B).

[0254] 2.2.2. Biological Activity

[0255] Growth Factors Content and Secretion

[0256] No protein content was found in Cultispher G and S alone. Only traces of IGF-1 were detected but below the lower limit of quantification of the ELISA method.

[0257] The levels IGF-1 and BMP7 detected in the supernatants of biopsies of Cultispher cultured with ASCs in osteogenic differentiation medium were below the lower limit of quantification of the ELISA methods while traces of BMP2 and FGFb were measured. In contrast, a significant secretion of VEGF and SDF-1α was found.

[0258] No significant impact of the culture conditions on the growth factors secretion were found (Table 1).

TABLE-US-00001 TABLE 1 Impact of culture conditions on VEGF and SDF-1α secretion by the biomaterial of the invention Secretion (ng/g) Oxemia Glycemia VEGF SDF-1α 21% O2   1 g/L  74 ± 24 19 ± 20 4.5 g/L  50 ± 28 27 ± 27 1% O2   1 g/L 130 ± 51 14 ± 10 4.5 g/L 106 ± 60 26 ± 10

[0259] The levels BMP2, BMP7 and FGFb detected in the protein extracts from the biopsies of Cultispher cultured with ASCs in osteogenic differentiation medium were below the lower limit of quantification of the ELISA methods. In contrast, a significant content in IGF-1, VEGF and SDF-1α was found.

[0260] No significant impact of the culture conditions on the VEGF content was found. However, a lower IGF-1 content in normoxia (21% O2) at 4.5 g/L glucose was found in comparison with other groups (p<0.05). A higher SDF-1α content was found in normoxia and normoglycemia vs hypoxia (1 and 4.5 g/L glucose) (p<0.05) (Table 2).

TABLE-US-00002 TABLE 2 Impact of culture conditions on VEGF, SDF-1α and IGF1 content of the biomaterial of the invention Secretion (ng/g) Oxemia Glycemia VEGF SDF-1α IGF1 21% O2   1 g/L 123 ± 47   117 ± 79** 53 ± 37 4.5 g/L 104 ± 61  139 ± 208  25 ± 22* 1% O2   1 g/L 152 ± 80  36 ± 29 109 ± 85  4.5 g/L 155 ± 101 36 ± 44 94 ± 78 *p <0.05 in comparison to other groups **p <0.05 in comparison to 1% O2 (1 and 4.5 g/L)

[0261] qRT-PCR Analysis

[0262] Over the 84 pro-angiogenic genes analyzed by qRT-PCR analysis, 13 mRNA were modulated between the different culture conditions. Ten genes were upregulated in the biomaterial of the invention in comparison to ASCs in proliferation medium (ANG, ANGPT1, EPHB4, EDN1, LEP, THBS1, PTGS1, VEGFA, VEGFB and VEGFC) and two genes were found to be down-regulated in the biomaterial of the invention in comparison to ASCs in MP (ID1, TIMP1) (FIG. 5).

[0263] A significant higher expression of angiopoietin (ANG and ANGPT1) mRNA was found in the biomaterial of the invention in comparison with ASCs in MP (FIGS. 5A and B). Angiopoietin signaling promotes angiogenesis, the process by which new arteries and veins form from preexisting blood vessels (Fagiani E et al, Cancer Lett, 2013).

[0264] EPHB4 (Ephrin receptor B4), a transmembrane protein, playing essential roles in vasculogenesis, Endothelin (EDN1), a potent vasoconstrictor (Wu M H, Nature, 2013), Thrombospondin 1 (THBS1), a vasodilatator and Cyclooxigenase 1 (PTGS1/COX-1), regulating endothelial cells were significantly up-regulated in the biomaterial of the invention compared to ASCs in MP (FIGS. 5C, D, E and F, respectively).

[0265] The expression of the Leptin (LEP) mRNA (an important enhancer of angiogenesis and inducer of the expression of VEGF; Bouloumie A et al, Circ. Res. 1998; Sierra-Honigmann M R et al, Science (New York, N.Y.) 1998) was also over-expressed in the biomaterial of the invention in comparison to ASCs in MP (FIG. 5G).

[0266] Finally, the expression of the vascular endothelial growth factor A, B and C mRNA (VEGFA/B/C) were also significantly improved for ASCs in the biomaterial of the invention in comparison to ASCs in MP (FIGS. 5H, I and J, respectively). VEGF is one of the most important growth factors for the regulation of vascular development and angiogenesis. Since bone is a highly vascularized organ (with the angiogenesis as an important regulator in the osteogenesis), the VEGF also positively impacts the skeletal development and postnatal bone repair (Hu K et al, Bone 2016).

[0267] In contrast, DNA-binding protein inhibitor (ID1) and Metallopeptidase inhibitor 1 (TIMP1), associated to reduced angiogenesis in vivo (Reed M J et al, Microvasc Res 2003) were down-regulated in the biomaterial of the invention in comparison to ASCs in MP (FIGS. 5K and L, respectively).

[0268] Overall, these molecular analyses show that the pro-angiogenic potential of ASCs is up-regulated when cells are embedded in their 3D matrix in the biomaterial of the invention.

[0269] 2.2.3. Impact of the Maturation of the Biomaterial on its Properties

[0270] Mineralization Level Evaluation

[0271] Photomacrographs of the 3D grafts at 4, 8, 12 and 25 weeks revealed the same macroscopic structure (FIGS. 6A and B) and were analyzed in micro-CT. Percentage of mineralization volume were determined: 0.07% at 4 weeks, 0.28%+/−0.33% at 8 weeks, 1.24%+/−0.35% at 12 weeks and 2.77% at 25 weeks (FIGS. 6C and D).

[0272] Therefore, the higher the maturation level, the higher the mineralization.

[0273] Histological Evaluation

[0274] No impact of the maturation of the tissue on the cellular content was found as similar cellularity was quantified in the different tissues analyzed (data not shown).

[0275] In contrast, the proportion of ECM in the tissue increased with the maturation level, with a significant lower proportion of ECM at 4 weeks and a higher proportion of ECM at 25 weeks (28±7 vs 33±11/34±11 vs 56±8% of ECM at 4, 8/12 and 25 weeks, respectively (p<0.05)) (Table 3).

TABLE-US-00003 TABLE 3 Histomorphological analysis of the biomaterial of the invention at different maturation times. Cells/mm.sup.2 ECM (%)  4 weeks 160 ± 104 28 ± 7*  8 weeks 175 ± 86  33 ± 11 12 weeks 177 ± 70  34 ± 11 25 weeks 191 ± 77  56 ± 8* *p <0.05 vs other groups

[0276] A higher mineralization degree was found at 12 and 25 weeks of maturation as shown by a more marked Von Kossa staining (data not shown).

[0277] Bioactivity Evaluation

[0278] The bioactivity of the biomaterial at 4, 8, 12 and 25 weeks of maturation was studied after proteins extraction, purification and growth factors (VEGF, IGF1, SDF-1α) quantification by ELISA (Table 4).

TABLE-US-00004 TABLE 4 Proteins and growth factors content in tissues at 4, 8, 12 and 25 weeks of maturation VEGF (ng/ml) IGF (ng/ml) SDF-1α (ng/ml)  4 weeks 117 ± 7  108 ± 17 105 ± 42   8 weeks 102 ± 91  50 ± 83 189 ± 180 12 weeks 181 ± 12 436 ± 18 663 ± 27  25 weeks 128 94 424

Example 3: In Vivo Study of the Angiogenic and Osteogenic Properties

[0279] 3.1. Materials and Methods

[0280] 3.1.1. In Vivo Experiment Using Nude Rats

[0281] Ten replicates of the biomaterial of the invention (ASCs cultured as described in Example 1, with 1.5 cm.sup.3 of Cultispher G or S during a maturation of 7.5 weeks) were sutured on cauterized lumbar muscle of nude rats at day 0. Twenty-nine days after implantation, biomaterials were harvested to be analyzed by imagery and histology.

[0282] 3.1.2. In Vivo Experiment Using Wistar Rats

[0283] Ten replicates of the biomaterial of the invention (ASCs cultured as described in Example 1, with 1.5 cm.sup.3 of Cultispher G or S during a maturation of 7.5 weeks) were sutured on cauterized lumbar muscle of Wistar rats at day 0. Twenty-nine days after implantation, biomaterials were harvested to be analyzed by imagery and histology.

[0284] The general clinical state of animals was checked daily over the course of the experimental period.

[0285] Analysis of mineralization of the 30 specimens was performed using the high-resolution X-ray micro-CT system for small-animal imaging SkyScan1076. Three-dimensional reconstructions of scans and analysis of mineralized tissue were performed using CTvol and CTan softwares (Skyscan).

[0286] Histological analyses were achieved on muscle samples in order to evaluate the in vivo angiogenic and osteoinductive properties of the products (hematoxylin-eosin, Masson's Trichrome, Von Kossa (to precise the location of the mineralization in the tissue), human tissue marker Ku80 (to confirm human origin of cells in animal tissue) and CD3 (to describe the repartition of CD3+ immune cells in the tissue) stainings.

[0287] 3.2. Results

[0288] 3.2.1. In Vivo Experiment Using Nude Rats

[0289] During the in vivo experiments, no sign of distress or significant lesion was noticed indicating that the product did not induce adverse effect on animals.

[0290] In Nude rats, presence of radiopaque structures suggesting mineralization was observed, on the radiographs performed at day 29 (FIG. 7).

[0291] The presence of human cells was highlighted in samples from Nude rats. When present, human cells represented on average half the cells of the implant sites, edge excluded, in the two groups. Cells from rat and human origins were homogeneously distributed in the implant sites, except at the edge, where only rat cells are present.

[0292] 3.2.2. In Vivo Experiment Using Wistar Rats

[0293] In Wistar rats, presence of radiopaque structures suggesting mineralization was observed, on the radiographs performed at day 29 (FIG. 8).

[0294] The analysis of the mineralization suggests the presence of mineralized tissue in each implant site.

[0295] Von Kossa staining indicates that the mineralization is localized on the particles (FIG. 9).

Example 4: In Vivo Bioactivity Study

[0296] 4.1. Materials and Methods

[0297] 4.1.1. Samples Preparation

[0298] Ten Samples of ˜0.5 g of biomaterial (ASCs cultured as described in Example 1, with 1.5 cm.sup.3 of Cultispher S during a maturation of 8 weeks) were prepared for implantation in paravertebral musculature of 10 nude rats. In addition, 2 samples of ˜0.5 g of Cultispher S particles were used as control.

[0299] In order to assess the growth factors content of the samples, a sample of biomaterial was prepared for proteins extraction and quantification (VEGF, IGF1, SDF-1α).

[0300] To evaluate the quality of the biomaterial, one sample was fixed in formol for hematoxylin-eosin (HE) and Von Kossa (VK) stainings. The assessment of the decellularization treatment efficacy was evaluated by counting the number of cells in the tissues after HE staining.

[0301] 4.1.2. Housing in Animal Facilities

[0302] Animals were housed in the animal facility “Centre Préclinique Atlanthera” approved by the veterinary services and used in all the experimental procedure in agreement with the at present current legislation (Decree N 2013-118, of Feb. 1, 2013, on animals used in experimental purposes). The animals were acclimatized for a minimum of 7 days prior to the beginning of the study during whom the general state of animals was daily followed. Animals were housed in an air-conditioned animal house in plastic boxes of standard dimensions. The artificial day/night light cycle was set to 12 hours light and 12 hours darkness. All animals had free access to water and were fed ad libitum with a commercial chow. Each animal was identified by an ear tag (ring).

[0303] 4.1.3. Experimental Protocol

[0304] At day 0, replicates of biomaterials were sutured on cauterized lumbar muscle of 10 nude rats while particles alone were implanted in muscular cauterized stalls realized in the lumbar muscle of 1 nude rat. Twenty-nine days after implantation, muscles containing biomaterials are harvested to be analyzed by imagery and histology.

[0305] Implantation into Lumbar Muscles

[0306] Animals were fully anaesthetized to perform the surgery under best conditions. An analgesia procedure was set up with injection of Buprenorphine almost 30 minutes before surgery followed by another injection the following day.

[0307] Surgery: for each animal, a longitudinal skin incision was made along the rachis at lumbar level. For 1 rat, muscular stalls were achieved at both sides of the skin incision (i.e. stalls were performed into the lumbar muscles). Stalls were cauterized. Particles alone were implanted into these stalls. For 10 rats, biomaterials were sutured on cauterized lumbar muscle. After the surgical procedure, the skin wounds were sutured using surgical staples.

[0308] Clinical Follow Up

[0309] The general clinical state of animals was checked daily over the course of the experimental period. Twice a week, a detailed clinical follow-up was achieved with focus on: Respiratory, eye, cardiovascular, gastrointestinal signs; Motor activity and behavior; Signs of seizure; Evaluation of the skin; Inflammation at the implantation site.

[0310] In addition, body weight was measured twice weekly at the same time of detailed clinical follow-up.

[0311] Terminal Procedures and Post-Mortem Analysis

[0312] At day 29, animals were sacrificed by exsanguination and macroscopic evaluation was achieved. During autopsy, the outside aspect of the corpse was observed and any pathological fluid loss, signing possible internal lesional anomalies, was recorded.

[0313] Thoracic and abdominal cavities were widely opened in order to evaluate any lesional modification of the intern organs, with focus on the heart, the kidneys, the spleen, the liver and the lung.

[0314] Macroscopic Evaluation at the Implant Site

[0315] Muscle implant site was exposed and a detailed macroscopic evaluation was achieved focusing on local tissue reaction and presence and localization of the implants (radiographic analysis).

[0316] Muscle implant sites were removed along. The explants were fixed in neutral-buffered formalin solution for 48 hours at room temperature.

[0317] 3D Histomorphometric Analysis

[0318] Analysis of mineralization of the specimens was performed using the high-resolution X-ray micro-CT system for small-animal imaging SkyScan1076.

[0319] Muscle samples were scanned at room temperature using the following parameters: Source Voltage: 50 kV; Rotation step: 0.5°; Pixel size: 18 μm; 1 frame per position.

[0320] Three-dimensional reconstructions of scans and analysis of mineralized tissue were performed using CTvol and CTan softwares (Skyscan).

[0321] In each sample, the quantity of signal similar to those of bone mineralized tissue (threshold 40/255) was determined (identified as bone volume: BV). The “Tissue Volume” values used are the volumes of implants formulated.

[0322] Histopathologic and 2D Histomorphometric Analyses

[0323] Histological analyses were achieved on muscle samples in order to evaluate the in vivo angiogenic and osteoinductive properties of the products.

[0324] Formalin fixed explants were decalcified 13 days in EDTA 15%. Then, the samples were dehydrated and embedded in paraffin. Sections of 4-5 μm were cut using a microtome and stretched on slides. The sections were performed at two different levels distant by 150 μm.

[0325] At these two sections areas, Hematoxyline-Eosine (HE), Masson's trichrome (MT) and Immunohistochemistry of CD146 were performed (using sections from the specimens embedded in paraffin or frozen).

[0326] Images of the complete stained sections were acquired using a digital slide scanner (Nanozoomer, Hamamatsu). The quantification of area occupied by blood vessels (Trichrome Masson, CD146) was performed using NDPview2 software: A region of interest was manually delineated on the basis of the tissue features to define the area of the “implant site” on the section. Each blood vessel was delineated manually to quantify the area occupied by blood vessels in the region of interest. The surface corresponding to vessels and the number of blood vessels were reported to the total area of the “implant site”.

[0327] 4.2. Results

[0328] 4.2.1. Histological Analyses

[0329] The number of cells in the tissues was determined after HE staining (FIG. 10): 146.5±50.4 cells/mm.sup.2.

[0330] Von Kossa staining of the tissue showed a weak mineralization localized on particles (FIG. 11).

[0331] 4.2.2. In Vivo Study of the Bioactivity of the Biomaterial

[0332] No sign of distress or significant lesion was noticed indicating that the product did not induce adverse effect on animal. The body weight of animals, recorded over the course of the experiment, indicated that all the animals did not present a gain of weight at day 2 and then showed a regular weight gain between day 2 and day 28. Lack of weight gain just after surgery is often observed and is not considered as a sign of any toxicity of the product tested. The regular weight gain observed between day 2 and day 28 confirms that the particles did not affect animal metabolism. At the end of in vivo experiment, the autopsy did not highlight any macroscopic organ lesion.

[0333] Mineral Content at the Implant Site

[0334] Presence of radiopaque structures suggesting mineralization was observed, on the radiographs performed at day 29, at all the sites implanted with the biomaterial (FIG. 12).

[0335] In order to quantify the percentage of formation of mineralized tissue into the muscle, analysis of mineralization of the “implant sites” was performed using the high-resolution X-ray micro-CT system for small-animal imaging SkyScan1076. The results are presented in the Table 5.

TABLE-US-00005 TABLE 5 Results of high-resolution X-ray micro-CT system for small-animal imaging SkyScan1076 Samples BV 40/255 (mm.sup.3) TV (mm.sup.3) BV/TV (%) NG-987  76.7677 514.6821 0.1492 NG-988  22.7560 518.1965 0.0439 NG-989 121.3495 470.9364 0.2577 NG-990 137.0365 724.1618 0.1892 NG-991  44.8830 519.4913 0.0864 NG-992  23.1673 560.8324 0.0413 NG-993  48.1291 496.7399 0.0969 NG-994  21.2821 791.3064 0.0269 NG-995 123.9947 638.3353 0.1942 NG-996  52.9368 561.4798 0.0943

[0336] The analysis suggests the presence of a noticeable content of mineralized tissue in each site implanted with the biomaterial, with a mean of BV/TV of 0.118.

[0337] Neovascularization of the Implant

[0338] The presence of capillaries in the fibrous connective tissue was examined in order to document the neovascularization.

[0339] The number of vessels/area and the vascular density in the implants and at the junction between muscle and implant site after Masson's Trichome staining were quantified.

[0340] The implants with the biomaterial were found vascularized by Masson's Trichome staining, with a number of 40.8±18.5 vessels/mm.sup.2.

Example 5: In Vivo Efficacy Study in a Hyperglycemic/Ischemic Xenogenic Rat Model

[0341] 5.1. Materials and Methods

[0342] 5.1.1 Animals

[0343] 56 female Wistar rats of 250-300 g received streptozotocin (50 mg/kg) intraperitonaly. Seven to ten days after streptozotocin administration, blood glucose levels were measured from tail venous blood by blood glucose test strips. Rats with glucose levels >11.1 mM were considered hyperglycemic and were included in the study (n−42 rats).

[0344] Ischemia was induced in the left limb of each rat as described in Levigné et al (Biomed Res Int 2013). Through a longitudinal incision in the inguinal region that was shaved, the external iliac and femoral arteries were dissected from the common iliac to the saphenous arteries. To provoke an ischemic condition, the dissected arteries were resected from the common iliac in the left limb while in the right limb arteries were conserved and limbs considered being nonischemic. All surgical procedures were performed under an operating microscope (Carl Zeiss, Jena, Germany), and animals were anesthetized by inhalation of isoflurane 5% for induction and 3% for maintenance of anesthesia.

[0345] Animals were randomly divided into 3 groups: [0346] Sham group (n=10 female Wistar rats); [0347] Cultispher group (n=10 female Wistar rats), i.e. particles alone; [0348] Biomaterial group (n=14 female Wistar rats), i.e. ASCs with gelatin particles forming a tissue.

[0349] 5.1.2 Test Items

[0350] 14 samples of ˜0.5 g of Cultispher particles were prepared, gamma-irradiated.

[0351] 14 Samples of ˜2 cm.sup.2 of biomaterial (ASCs cultured as described in Example 1, with 1.5 cm.sup.3 of Cultispher S during a maturation of 8 weeks) were prepared for implantation.

[0352] In order to assess the growth factors content of the samples, one sample of biomaterial was prepared for proteins extraction and quantification (VEGF, IGF1, SDF-1α).

[0353] To evaluate the quality of the biomaterial, a sample was fixed in formol for hematoxylin-eosin (HE) coloration. The assessment of the decellularization treatment efficacy was evaluated by counting the number of cells in the tissues after HE staining.

[0354] 5.1.3 Macroscopic Evaluation of Wound Healing

[0355] Pictures of legs were taken at days 0, 15, 24 and 34 after implantation.

[0356] To quantify the wound closure, the wound area was measured by image analysis using Image J software by two independent operators. The area under the curve was calculated on the wound area measured at each time point between D0 and D34 and were expressed in comparison to the sham group, fixed at 100%.

[0357] 5.1.4 Microscopic Evaluation of Wound Healing

[0358] Legs were dissected to remove the wound tissue and this latest was oriented transversally to have histological slides of the entire thickness of the tissue. Histological slides of 5 μm were prepared and stained with HE for epidermal (op 't Veld R C et al, Biomaterials 2018) and dermal scorings (Yates C et al, Biomaterials 2007):

[0359] Score epidermal healing in three representative sections of the wound (core and periphery): [0360] 0: no migration of epithelial cells, [0361] 1: partial migration, [0362] 2: complete migration with no/partial keratinization, [0363] 3: complete migration with complete keratinization, [0364] 4: Advanced hypertrophy.

[0365] Score dermal healing in three representative sections of the wound (core and periphery): [0366] 0: no healing, [0367] 1: inflammatory infiltrate, [0368] 2: granulation tissue present-fibroplasias and angiogenesis, [0369] 3: collagen deposition replacing granulation tissue >50%, [0370] 4: hypertrophic fibrotic response.

[0371] In addition, Masson's Trichome coloration was performed for the evaluation of the vascular area by histomorphometry and CD3, CD68 immunostaining for the evaluation of the immune and inflammatory responses. In addition, KU80 staining was performed to identify the presence of human cells after implantation.

[0372] 5.2. Results

[0373] On the 56 rats who received streptozotocin injection, 42 developed hyperglycemia and were selected for the study, while 14 presented low glycemia and developed surgical complications and were therefore excluded from the study.

[0374] 5.2.1. Macroscopic Evaluation of Wound Healing

[0375] Macroscopic pictures of wounds are presented in FIG. 13. A better wound healing can be observed from day 15 after surgery (D15) in the biomaterial group (FIG. 13C) in comparison to other groups (sham control (FIG. 13A) and particles alone, FIG. 13B). This difference is visible for both the ischemic (left limbs) and the non-ischemic wounds (right limbs).

[0376] Results of the areas under the curve for the non-ischemic wound are presented in FIG. 14. Implantation of Cultispher alone showed a decrease of wound healing in comparison to the non-treated animals by 23% respectively. In contrast, a better wound healing (25% was found in the group treated with the biomaterial of the invention.

[0377] The evolution of wound area for a non-ischemic wound and an ischemic wound between D0 and D34 is presented in FIG. 15 (A and B, respectively). Note that the wounds treated with the biomaterial of the invention present lower non-healed tissues from D21 to D34 in comparison with other groups. Complete closure of the wound is significantly faster when treated with the biomaterial of the invention, in non-ischemic and ischemic conditions (FIGS. 16A and B, respectively).

[0378] Results of histomorphometry for evaluating inflammatory reaction are presented in FIG. 17. These results show higher lymphocytes CD3 (black line) in border, core and total ischemic wounds treated with the biomaterial of the invention (FIG. 17C) compared to sham control (FIG. 17A) and Cultispher S alone (FIG. 17B). CD3 normally function to destroy infections and malfunctioning cells.

[0379] In addition, macrophages CD68 (gray line) reached a peak around D10 (FIG. 17C), like sham control (FIG. 17A) and Cultispher S alone (FIG. 17B). CD68 is characteristic of macrophages which are seen to infest tissue sites and remove cell debris and infections

[0380] These two observations confirm that implantation of the biomaterial of the invention leads to an increase of the wound closure kinetic by immune elicitation.

[0381] Wound thickness was also assessed (FIG. 18). In an ischemic model (FIG. 18A), the thickness of the wound decreased from D15 to D34 after implantation, showing a retractation. In a non-ischemic model (FIG. 18B), the thickness of the wound slightly decreased from D15 to D34 after implantation, but more importantly did not increase as in the case of sham control and Cultisphers alone. This result highlights the lack of hypertrophy when the biomaterial of the invention is implanted.

[0382] 5.2.2. Microscopic Evaluation of Wound Healing

[0383] Epidermal and dermal scores, evaluated on non-ischemic wounds at each time point, are presented in FIGS. 19A, B, C and D. Faster dermic and epidermic were found for biomaterials of the invention in comparison to other groups.

Example 6: Test of Different Differentiation Media

[0384] 6.1. Materials and Methods

[0385] The impact of the differentiation medium on the 3D structure formed was studied. ASCs were cultured with 1.5 cm.sup.3 of Cultispher S in different differentiation media for 4 weeks: osteogenic (same as in Example 1), chondrogenic (DMEM, 5% HPL, 100 μg/mL sodium pyruvate, ITS 1×, 40 μg/mL Proline, 10 ng/mL TGF-β1, 1 μM Dexamethazone), keratinogenic (DMEM, 5% HPL, 5 μg/mL insulin, 10 ng/mL KGF, 10 ng/mL hEGF, 0.5 μg/mL hydrocortisone, 1.5 mM CaCl2), and myofibrogenic (DMEM:F12, 100 μg/mL sodium pyruvate, 1×ITS, 1×RPMI 1640 vitamin, 1 ng/mL TGF-β1, 1 μg/mL Glutathione, 0.1 mM MEM). Cultures were maintained for 4 weeks with differentiation medium change every 3-4 days.

[0386] Biopsies of tissues at 4 weeks were fixed in formol for hematoxylin-eosin, Masson's Trichrome, and Von Kossa stainings. In addition, tissue-specific stainings were performed (osteocalcin, Alcian Blue, Pankeratin, CD34, α-SMA).

[0387] To assess the bioactivity of the tissue formed, biopsies were taken at 4 weeks post-addition of Cultispher for proteins extraction and quantification. The total protein and growth factors contents (VEGF and SDF-1α) were quantified by colorimetry (BCA Protein Assay Kit, ThermoFisher Scientific).

[0388] 6.2. Results

[0389] ASCs and Cultispher S in osteogenic medium serve as positive control for osteogenic differentiation. The formation of a large grippable 3D structure was observed. Histological analysis revealed integration of particles in the cellularized interconnective tissue and an osteocalcine positive staining of the matrix (FIG. 20A).

[0390] The culture in chondrogenic medium rapidly (only after a few days) showed the formation of a strength and thick 3D structure, easily grippable and resistant to mechanical forces. Histological analysis revealed integration of particles in the cellularized interconnective tissue and a matrix positive to alcian blue coloration (FIG. 20B).

[0391] The myofibrogenic differentiation medium allowed the formation of 3D structures. The structure formed were grippable, but fragile. Again, histological analysis revealed integration of particles in the cellularized interconnective tissue and α-SMA positive staining of the matrix (FIG. 20C).

[0392] ASCs and particles in keratinogenic medium formed a large, plane and thin 3D structure. This latest was very fragile and difficult to handle (FIG. 20D).

[0393] (Table 6).

TABLE-US-00006 TABLE 6 Characteristics of the structures formed in the differentiation media tested Differentiation Interconnective medium 3D structure Grippable Solidity tissue Osteogenic + + +/− + Chondrogenic + + + + Myofibrogenic + +/− +/− + Keratinogenic + +/− − +

[0394] Therefore, a 3D structure was observed in all the samples of biomaterial formed with ASCs and gelatin, with all the differentiation media tested.