Multi-Dimensional Biomaterial and Method for Producing the Same

Abstract

Biomaterial having a multi-dimensional structure and comprising demineralized bone matrix dispersed within differentiated mesenchymal stem cells (MSCs) tissue, wherein MSCs are adipose tissue-derived stem cells. Method for producing this biomaterial comprising incubating MSCS in osteoblastic and/or chondrogenic medium in presence of demineralized bone matrix. Use of this biomaterial for alleviating or treating a bone or cartilage defect, supporting or correcting a congenital or acquired abnormality, supporting a bon or articular bone replacement following surgery or trauma, and/or supporting a musculoskeletal implant.

Claims

1.-17. (canceled)

18. Method for producing a multi-dimensional biomaterial having a multi-dimensional structure and comprising differentiated mesenchymal stem cells (MSCs) tissue and demineralized bone matrix, wherein said demineralized bone matrix is dispersed within the differentiated MSCs tissue, and wherein said MSCs are adipose tissue-derived stem cells, said method comprising the steps of: a. incubating MSCs in osteoblastic and/or chondrogenic medium during 15-25 days, b. adding demineralized bone matrix in said media, and c. maintaining incubation for an additional period of 15-30 days.

19. The method according to claim 18, further comprising the step of replacing the media every 2 days during the additional period, without removing the demineralized bone matrix.

20. The method according to claim 18, wherein the MSCs are late-passaged adipose tissue-derived stem cells.

21. The method according to claim 18, wherein the incubation of step c. is maintained for an additional period of 15-25 days.

22. The method according to claim 18, wherein the incubation of step c. is maintained for an additional period of 20 days.

23. The method according to claim 18, wherein the demineralized bone matrix is added in the media in an amount of 1 to 20 mg/ml.

24. The method according to claim 18, wherein the demineralized bone matrix is added in the media in an amount of 1 to 10 mg/ml.

25. The method according to claim 18, wherein the demineralized bone matrix is added in the media in an amount of 5 to 10 mg/ml.

26. The method according to claim 18, wherein the demineralized bone matrix is in the form of particles having a mean diameter of 50-2500 m.

27. The method according to claim 18, wherein the osteoblastic medium is a standard medium which is supplemented with Human Serum, dexamethasone, sodium ascorbate, sodium dihydrophosphate, penicillin, and streptomycin.

28. The method according to claim 18, wherein the chondrogenic medium is a standard medium which is supplemented with Human Serum, dexamethasone, TGF-B3, L-proline, sodium ascorbate, sodium dihydrophosphate, sodium pyruvate, Insulin-Transferrin-Selenium, penicillin, and streptomycin.

29. A multi-dimensional biomaterial obtainable by the method according to claim 18.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 shows human AMSCs differentiation in osteogenic medium supplemented with Human serum and DBM. A multi-layer structure (A) with tissue retraction and inter-connective tissue (B) was found by staining with 15 Trichrom Masson's staining (C).

[0082] FIG. 2 shows AMSCs differentiation in chondrogenic medium without (Left) and with (Right) DBM. A monolayer structure with confluent cell was developed in chondrogenic medium without DBM. In contrast, a multilayer structure obtained by cellular tissue retraction was composed by chondrogenic-like matrix stained by Alcian blue.

[0083] FIG. 3 shows human AMSCs differentiation in osteogenic medium without (A, B, C) and with (D, E, F) DBM. A monolayer structure (A) made of individual bone nodules (Alizarin Red staining, B) and intra-nodule collagen tissue (C) was developed in osteogenic medium without DBM. In contrast, cellular tissue retraction (D) with inter-connective tissue (E) made of mineralized collagen (F, Von Kossa staining in black: left rectangle) with osteocalcin-expressing cells (F, right rectangle).

[0084] FIG. 4 shows BM-MSCs and AMSCs viability alter 60 days post-implantation in para-vertebral muscles of nude rats. Mesenchymal stem cells were detected by immunohistochemistry for GFP and osteocalcin antibody. In case of bone alone implantation, no expression of GFP and osteocalcin staining cells were found. In case of composite grafts made of MSChuman bone graft, GFP and osteocalcin cells were detected.

[0085] FIG. 5 shows the development of multi-dimensional structures for AMSCs incubated in osteogenic (A-C) and chondrogenic (E-F) medium supplemented with DBM. Tissue retraction (A, D), DBM inter-connexion (B, E) and expression of Osteocalcin and proteoglycan (F) proteins.

[0086] FIG. 6 shows the angiogenic potential of AMSCs versus BM-MSCs. In vitro potential was found by incubation of both cells at different Oxygen concentrations (0.1, 3, 5 and 21% 02). AMSCs demonstrated, at each 02 concentrations, a significant higher release of VEGF than BM-MSCs.

EXAMPLES

Example 1

Preparation of the Multi-Dimensional Biomaterial According to the Invention

Animals Source for AMSCs in Pre-Clinical Model

[0087] Green Fluorescent transgenic pigs were used as donor for bone marrow and adipocytes stem cells (Brunetti D, Cloning Stem Cells, eupb 2008). Animals were housed according to the guidelines of the Belgian Ministry of Agriculture and Animal Care. All procedures were approved by the local Ethics Committee for Animal Care of the Universite catholique de Louvain.

Source for AMSCs of Animal Origin

[0088] The collagenase (0.075 g) is reconstituted in Hank's Balanced Salt Solution (with calcium ions) and stored at 2-8 C. prior digestion. Fatty tissues (a mean of 15 g) were washed three times with NaCl 0.009% and were cut in a Petri-dish to remove vessels and fibrous connective tissue. The fat is weighed prior to digestion and is transferred in a 50-ml Flacon tube containing the enzyme. The tissue is placed in a shaking water bath at 37 C. with continuous agitation for 60 minutes. After digestion, the collagenase is inactivated in DMEM (500 ml) supplemented with 50 ml of Human serum, L-glutamin (5 ml) and 5 ml antibiotics (penicillin/streptomycin). Collected tissue is centrifuged for 10 min at 1500 rpm at room temperature (20-25 C.). The supernatant, containing mature adipocytes, is aspirated. The pellet is re-suspended in 20 ml of proliferation medium (MP) made of DMEM supplemented with 10% of Human Serum and antibiotics (100 U/ml of penicillin and 100 g/ml of streptomycin) and filtered through a 500 m mesh-screen. The collected tissue (after filtration) is centrifuged for 10 min at 1500 rpm at room temperature (20-25 C.) and the pellet is re-suspended in the MP and is identified as the Stromal Vascular Fraction (SVF) cells. This initial passage of the primary cells was referred to as passage 0 (P0). After 24-48 hours of incubation at 37 C. at 5% CO.sub.2, the cultures were washed with PBS and maintained in the MP up to P4 (fourth passage) and then differentiated in specific media (see below).

Source for AMSCs of Human Origin

[0089] Human fatty tissue (small pieces of subcutaneous fatty tissue, 1-2 g, n=4) was removed during routine surgery (abdominal and orthopaedic surgery), preserved in a cold 4 C. physiological solution up to the laboratory for processing. Human fatty digestion was processed as previously described for pig fatty tissue processing. After digestion, human AMSCs were cultivated in MP and differentiated in specific media (see below for exact composition) supplemented with (i) Foetal Bovine Serum (10% v/v) or (ii) Human Serum (10% v/v).

[0090] It was observed that the AMSC differentiated in both differentiation media containing FBS and differentiation media containing Human Serum.

Source for Demineralized Bone Matrix

[0091] Human demineralized bone matrix was provided by the University Tissue Bank (Cliniques universitaires Saint-Luc, Brussels, Belgium), and produced from multi-organ human donors. Diaphysis of femoral or tibial bone is cut and pound in particles below 1000 m for demineralization treatment (see below).

[0092] Human DBM is performed by grinding cortical bones from selected human donors. Firstly, human bone tissue is defatted by Acetone (99%) bath during an overnight and followed by washing in demineralised water during 2 hours. Decalcification is performed by immersion in HCL 0.6 N during 3 hours (20 ml solution per gram of bone) under agitation at room temperature. Then, demineralized bone powder is rinsed with demineralised water during 2 hours and the pH is controlled. If the pH is too acid, DBM is buffered with Phosphate solution at 0.1 M under agitation. Finally, DBM is dried and weight. The DBM is sterilized with 25 kGray by Gamma irradiation at frozen temperature.

Stem Cells Differentiation and Characterization

[0093] Adipogenesis

[0094] Confluent cultures of AMSCs were induced to undergo adipogenesis by replacing the MP with adipocyte induction medium composed of Iscove modified Duibecco's Medium (IMDM) supplemented with 20% Human Serum, L-glutamine (5 ml), bovine insulin (5 g/ml), indomethacine (50 M), 3-isobutyl-1-methyl-xanthine (IBMX, 0.5 mM), dexamethasone (1 M) and penicillin 100 U/ml and streptomycin 100 g/ml (Mauney J R, Biomaterials 2005, vol 26: 6167). Cells were maintained in adipogenic culture with media replaced every 2 days. Cultures were rinsed with PBS and fixed in formalin solution and adipocyte differentiation was determined by staining of neutral lipids with oil red.

Osteogenesis

[0095] Confluent cultures of AMSCs were induced to undergo osteogenesis using a osteogenic medium obtained by supplementing DMEM with Human Serum (10% v/v), dexamethasone (1 M), sodium ascorbate (50 g/ml), sodium dihydrophosphate (36 mg/ml), penicillin (100 U/ml), and streptomycin (100 g/ml) (see FIG. 3-A, B, C). Cells were maintained in osteogenic culture with media replaced every 2 days. Cultures were rinsed with PBS and fixed in 70% ethanol and osteogenic differentiation was determined by staining for calcium phosphate with Alizarin red. In addition, immunohistochemistry for osteocalcin and von Kossa stainings were performed to confirm the bone phenotype (see FIG. 4).

Chondrogenesis

[0096] Confluent cultures of AMSCs were induced to undergo chondrogenesis using a chondrogenic medium obtained by supplementing DMEM with Human Serum (10% v/v), dexamethasone (1 M), TGF-B3 (10 ng), L-proline (40 g/ml), sodium ascorbate (50 g/ml), sodium dihydrophosphate (36 mg/ml), sodium pyruvate (100 g/ml), ITS (Insulin-Transferrin-Selenium, e.g. from insulin-transferrin-sodium selenite media supplement lyophilized powder available from Sigma) (100 g/ml), penicillin (100 U/ml), and streptomycin (100 g/ml) (Taipaleenmaki H, Experimental Cell Research 2008 vol 314: 2400). Cells were maintained in chondrogenic culture with media replaced every 2 days.

Impact of Differentiation Media (Osteogenic and Chondrogenic on AMSCs)

Growth.

Cell and Culture Conditions.

[0097] AMSCs were grown in proliferation medium (MP) and maintained at 37 C. (95% air and 5% CO.sub.2) until about 85-90% confluence. The medium was changed every 2 days. To suspend cells for cytotoxicity assay, cells were detached from the culture flask with a 0.25% trypsin-EDTA mixture for 10 min at 37 C. and were resuspended in the culture medium. Cells were seeded in 96-well microplates for MTS (3-[4,5dimethylthiazol-2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium bromide) at a density of 1104 cells/well. They were grown near confluency at 96 hours at 37 C. prior exposition to different tested media: (i) MP, (ii) osteogenic medium, and (iii) chondrogenic medium for 5 days.

[0098] MTS assay. After 24 hours of extract-cell contact, 20 l of Cell titer 96 AQ.sub.ueousOne Solution Cell Proliferation Assay (Promega, Madison, Wis.) was added directly to each well containing 100 l of extract medium. Cells were incubated for 3 hours at 37 C. The absorbance was measured at 492 nm using a microtiter plate spectrophotometer (Multiskan Ex, Labsystems, Brussels, Belgium). The reference wavelength was 690 nm. The optical density difference OD=OD.sub.492nm-OD.sub.690nm was estimated.

[0099] It was observed that differentiation occurred only when using specific differentiation media.

Development of Multi-Dimensional Structure

[0100] After 15-20 days of incubation of AMSC (subculture Passage 4) in specific media (osteogenic or chondrogenic), demineralized bone matrix (DBM) was supplemented to osteogenic and chondrogenic media resulting in a multi-dimensional structure (1 mg of DBM/ml of differentiated media) for an additional period of 20 days. The media is replaced every 2 days without removal of DBM.

[0101] At the end of differentiation, cultures were rinsed with PBS and fixed in formalin solution for bone and chondrogenic characterization by histology for osteocalcin, Alcian Blue and Giemsa stainings (FIG. 2, FIG. 3-D, E, F).

[0102] Cellular pellets were fixed in 4% paraformaldehyde for overnight. Serial sections (5 m thick) were mounted on glass slides with demineralized water, dried for 12 hours at 37 C. and processed by immuno-classical detection or histochemistry. Endogenous peroxidase activity was blocked by placing sections in hydrogen peroxide (0.3% H.sub.2O.sub.2) for 30 min. After washing in Tris-Triton buffered saline (TBS-0.05M, 0.05%, pH=7.4), slides were incubated for 30 minutes at room temperature with normal goat serum (1:10; BIOSYS, Boussens, France) and overnight with a primary antibody for staining osteocalcin (anti-osteocalcin monoclonal mouse antibody; ABCAM, Cambridge, UK) at a dilution of 1:100. After washing with Tris-TBS, the slides were incubated for 1 hour with secondary anti-mouse IgG for immunoperoxydase detection.

[0103] Collagen structure was studied by Masson's Trichrom on each sample for all types of un/differentiated cells. Fibroblastic cells incubated in proliferation and differentiation (osteogenic, chondrogenic and adipogenic) media served as negative control.

[0104] Proteoglycan-secreting chondrocytes were stained with Alcian Blue and Giemsa stainings.

[0105] AMSCs were stained with saturating amounts of monoclonal CD90 antibody conjugated with phycoerythrin (PE). At least 15,000 events were analyzed by flow cytometry (FACScan, BD Biosciences) with Celiquest software.

[0106] In both osteogenic and chondrogenic differentiation media, supplemented DBM induced the development of a three-dimensional structure by a cellular construction, collagen synthesis and regroupment of demineralized bone matrix particles (see FIG. 5 A-E). Microscopically, osteocalcin expression and proteoglycan secretion were revealed in osteogenic (FIG. 5C) and chondrogenic (FIG. 5F) conditions, respectively.

In Vivo Implantation and Histology Analysis

[0107] Osteoblastic differentiated GFP-pig AMSCs were seeded on a human treated/decellularized bone matrix (Dufrane D, Eur Cell Mater, vol 1:52, 2001) provided by the University Tissue Bank (University clinical hospital Saint-Luc, Brussels, Belgium). The composite graft was implanted subcutaneously in para-vertebral region of nude rats (2 implants/recipient and n=10) (male, 6-8 weeks old). After 60 days, animals were sacrificed and implants were explanted for immunohistochemistry processing. Implants were then decalcified in HCL, processed and embedded in parrafin and sectioned (5 m). Masson's Trichrom and immunohistochemistry for osteocalcin and Green Fluorescent Protein (monoclonal antibody) were then performed.

EXAMPLE

[0108] Potential of Multidimensional Bone-Like Graft from Adipose Mesenchymal Stem Cells

[0109] Materials and Methods

Pig and Human AMSC Isolations

[0110] For the purpose of those experiments, the pig and human AMSC isolations were carried out as disclosed in example 1.

Angiogenesis Potential of AMSCs

In Vitro

[0111] To assess in vitro the proangiogenic capacities of stem cells in normoxic and hypoxic conditions, porcine Bone Marrow-MSC and Adipose-MSC were placed in hypoxia chambers for 24, 48 and 72 hr at 0.1, 3, 5 and 21% 02 and the release of VEGF was quantified by ELISA testing.

Optimalization of Multi-Dimensional Structure with Demineralised Bone Matrix

[0112] AMSCs were induced to undergo osteogenesis by supplementing the MP with Foetal Bovine Serum (FBS, 10% v/v), dexamethasone (1 M), sodium ascorbate (50 g/ml), sodium dihydrophosphate and penicillin 100 U/ml and streptomycin 100 g/ml. Cells were maintained in osteogenic culture with media replaced every 2 days (Post et Al. Bone 2008, 43, 1; 32-39, Qu et Al In Vitro Cell. Dev. Biol. Anim. 2007; 43; 95-100). Cultures were rinsed with PBS and fixed in 70% ethanol and osteogenic differentiation was determined by staining for calcium phosphate with Alizarin red. In addition, immunohistochemistry for osteocalcin and von Kossa stainings were performed to confirm the bone phenotype.

[0113] A multi-dimensional structure with AMSC was performed with co-incubation in the presence of Demineralized Bone Matrix (DBM) procured by the University Tissue Bank (University clinical hospital St-Luc, Brussels, Belgium). The production of demineralized bone matrix is disclosed in example 1) Source for Demineralized Bone

Matrix

[0114] The efficacy of DBM is assessed by: (i) the measurement of residual calcium concentration after demineralization process (>97% of [calcium] reduction) and (ii) the in vivo (in nude rats) ostogenic potential at 1 month post-implantation. After an incubation of AMSC (subculture Passage 4) in osteoblastic media during a mean of 15-18 days, different concentration (0/1/5/10 and 20 mg/ml) of DBM is added in the specific differentiation media. The media is replaced every 2 days without removal of DBM. The degree of 3-D structure, the cellular structure, the osteocalcin expression and Von Kossa staining (for calcium deposition) are assessed to select the appropriate DBM concentration for 3-D bone-like structure.

Implantation Procedure and Follow Up of Multi-Dimensional Structure with Demineralised Bone Matrix and AMSCs.

[0115] AMSCs issued from the osteogenic cultures (at passage 4) with optimal DBM concentration (for multi-dimensional structure) were collected for implantation.

[0116] Nude rats. (Charles River Laboratories International, Inc., Wilmington, Mass., USA) were used as recipients. Cells were implanted in the paraspinal muscles. A longitudinal incision centred on the spine was performed and the sub cutaneous tissues were dissected to expose the fascia. Multi-dimensional structure were directly (University Tissue Bank, Universit Catholique de Louvain, Brussels, Belgium) implanted in the para-vertebral musculature. Control was ensured by implanting lyophilized cancellous bone alone in the control lateral para spinal muscles of the same rat. The fascia was closed using non resorbable suture to allow easy recovery of the implantation site. The animals were sacrificed and explantation was performed at day 30 post-transplantation by the mean of a T61 intracardiac injection (Intervet Int. GmbH-D, Germany) under general anesthesia. The implants were then harvested and processed for histology and micro computed tomography.

[0117] Pig recipient. The grafts are tested in two different surgical models. The inventors analyze: (i) their bridging capacity in a femoral cortical bone defect and (ii) their fusion potential in an anterior lumbar interbody fusion (ALIF).

[0118] (i) The cortical bone defect model is well known in experimental orthopedic surgery and in our laboratory mainly through the work of Professor C. Delloye. Pigs are used in similar models where a segmental diaphysial bone defect is created on a long bone and then filled with graft material or left empty. In our experiment, both femurs of the pig are operated on. A 1.5-cm cortical bone defect is made and stabilized by a standard 4.5-mm titanium locking compression plate. One femoral defect is left empty and the AMSC graft is implanted into the contralateral leg.

[0119] (ii) The ALIF model is well established in porcine experimental surgery. Our technique consists of a four-level ALIF procedure by a posterolateral approach. Fusion is obtained by means of an interbody polyethylethylketone (PEEK) cage. The intervertebral disk is opened and the nucleus pulposus removed, cartilage is reamed to expose the subchondral bone, and the PEEK cage is then inserted. These cages are designed empty but can be filled with various experimental materials. In this case, each level will receive a different graft tissue, creating four different groups: one cage is left empty as the negative control, one cage is filled with freeze-dried irradiated cancellous bone (as often practiced in the clinical field), one cage contains autologous cancellous bone graft (considered as gold standard in fusion procedure and therefore our positive control), and the last cage has the AMSC graft. The animals were sacrificed and explantation was performed at week 7 post-transplantation by the mean of a T61 intracardiac injection (Intervet Int. GmbH-D, Germany) under general anesthesia. The implants were then harvested and processed for histology and computed tomography.

Follow-Up Nude Rats:

[0120] Explanted implants were decalcified in HCl, processed, embedded in parrafin and sectioned (5 m). Standard coloration by Hemalun Eosin, Masson's Green Trichrom, Osteocalcin and Von Kossa staining were used for histologic staining. Osteocalcin staining were obtained by the means of a monoclonal antibody (OC4-30, Abcam, Cambrige) revealed by the Envision Rsystem monoclonal antibody (Dako, Denmark). The microstructure of the harvested implants was analysed using a pQCT (Peripheral quantitative computed tomography machine, model XCT Research SA, Stratee, Pforzheim, Germany). Cortical and total bone density was measured on multiple slides of each implant. Quantification of in vivo neo-angiogenesis was performed by the quantification of newly-formed vessels after von Willebrandt staining (see above).

Pigs:

[0121] The pigs are housed individually with free access to food and water. Postoperative care and analgesia are provided following standard protocols for experimental animal care. In the autograft model, biologic follow-up will consist of inflammation assessment by means of blood samples.

[0122] Radiologic follow-up allows us to compare in vivo bone formation by computed tomography (CT) scanning at 1, 5 and 7 weeks post-implantation. The high resolution and multiplanar reconstructions of clinical CT scans are so precise that the inventors can analyze the content of the PEEK cages in vivo, thus allowing evaluation of the fusion process.

[0123] The same procedure is used on the femurs, with all information gathered through one scan sweep. An even better resolution is obtained after euthanasia by processing the explanted grafts through micro-CT. Histologic and immunohistochemical investigations on explanted grafts are in progress to assess the new bone formation and the graft revascularization (vascular endothelial growth factor, CD51, von Willebrand factor), consolidation (osteocalcin), and mineralization (von Kossa) compared to the natural cortical bridging and fusion abilities.

[0124] The decalcification process allows histologic and immunohistochemical studies of the grafted material. Histology, histomorphometry, and microradiography require keeping the tissue calcified; thus, non-decalcified processing is also mandatory.

Statistics

[0125] The statistical significance of differences between groups was tested by one-way analysis of variance with a Bonferroni's post hoc test. The statistical tests were carried out with Systat version 8.0. Differences were considered to be significant at/p<0.05.

Results

[0126] Proof of Concept for Multi-Dimensional Structure with Optimal DBM Concentration: In Vitro Bone-Like Structure

[0127] In view to avoid a biological support for AMSC cell transplantation, a multi-dimensional structure was developed in combination with DBM. Demineralized Bone Matrix (with a mean diameter of 700 m) with an in vivo capacity to promote Osteogenic differentiation in nude rats (Quality control required prior using in contact with AMSCs), is required to achieve an in vitro cellular remodelling in co-incubation with AMSCs.

[0128] In both osteogenic differentiation media, supplemented-DBM induced the development of a tri-dimensional structure by a cellular constriction, collagen synthesis and regroupment of DBM particles in comparison with AMSCs alone without DBM. In view to optimise the development of multidimensional structure, gradual DBM doses were tested. Extreme concentrations (more than 20 mg/ml) were found to be not adapted. 1 mg/ml did not developed an optimal 3-D structure for graft handling in view to surgical application. In contrast, 5 and 10 mg/ml were found to develop an optimal tissue retraction for graft removal and surgical application. Microscopically, osteocalcin expression and mineralization process (Von Kossa staining) were revealed with these latest DBM concentration. A mean of 203 days of co-incubation with DBM is required to obtain a tri-dimensional structure with AMSCs in osteogenic conditions.

Proof of Concept for Multi-Dimensional Structure with Optimal DBM Concentration: In Vivo Osteogenesis

Nude Rats

[0129] After 30 days post-implantation in nude rats, AMSCs alone without DBM did not induced the formation a new bone-like structure. Only small bone nodules (osteocalcin+) as stained by Von Kossa were sparsely distributed in nude rats musculature. In contrast, the implantation of the multi-dimensional structure was easily performed with a good localization of implantation site. After 30 days post-implantation, a compact/strong bone-like structure was found into the muscle with a microscopically dense connective tissue within DBM particle. A bone-like structure was found with osteocalcin staining connective tissue and mineralization process.

Pig Recipients

[0130] After 5 weeks post-implantation, in pig, no spontaneous consolidation was found in bone femoral defect (with no-treatment) as revealed by CT-scan. It was easy to incorporate the multi-dimensional structure in the bone defect without any scaffold. This latest graft significantly improve the bone consolidation at early period post-implantation (5 weeks post-transplantation) with bone-like structure bridging the native cortical bone defect.

Proof of Concept for Multi-Dimensional Structure with Optimal DBM Concentration: In Vivo Angiogenesis

[0131] To assess in vitro the proangiogenic capacities of stem cells in normoxic and hypoxic conditions, pre-clinical porcine Bone Marrow-MSC and Adipose MSC were placed in hypoxia chambers for 24, 48 and 72 hr at 0.1, 3, 5 and 21% 02 and the release of VEGF was quantified by ELISA. BM-MSC released more VEGF in hypoxia than in normoxic conditions and maintained this secretion in the time with higher levels of VEGF at 48 and 72 hr than at 24 hr (p<0.05). In contrast, VEGF release from AMSC was similar in the different culture conditions but significant higher levels of VEGF were released by AMSC than BM-MSC (11274679 vs. 236494 pg/ml, respectively; p<0.05) (FIG. 6).

[0132] These results were confirmed in vivo after transplantation of BM-MSC and AMSC. A significant higher angiogenesis was found in case of osteogenic AMSCs transplantation in comparison to BM-MSCs (p<0.05).