In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

Bilayered constructs aimed at osteochondral strategies: the influence of medium supplements in the osteogenic and chondrogenic differentiation of amniotic fluid-derived stem cells

The development of osteochondral tissue engineered interfaces would be a novel treatment for traumatic injuries and aging associated diseases that affect joints. This study reports the development of a bilayered scaffold, which consists of both bone and cartilage regions. On the other hand, amniotic fluid-derived stem cells (AFSCs) could be differentiated into either osteogenic or chondrogenic cells, respectively. In this study we have developed a bilayered scaffolding system, which includes a starch/polycaprolactone (SPCL) scaffold for osteogenesis and an agarose hydrogel for chondrogenesis. AFSC-seeded scaffolds were cultured for 1 or 2 weeks in an osteochondral-defined culture medium containing both osteogenic and chondrogenic differentiation factors. Additionally, the effect of the presence or absence of insulin-like growth factor-1 (IGF-1) in the culture medium was assessed. Cell viability and phenotypic expression were assessed within the constructs in order to determine the influence of the osteochondral differentiation medium. The results indicated that, after osteogenic differentiation, AFSCs that had been seeded onto SPCL scaffolds did not require osteochondral medium to maintain their phenotype, and they produced a protein-rich, mineralized extracellular matrix (ECM) for up to 2 weeks. However, AFSCs differentiated into chondrocyte-like cells appeared to require osteochondral medium, but not IGF-1, to synthesize ECM proteins and maintain the chondrogenic phenotype. Thus, although IGF-1 was not essential for creating osteochondral constructs with AFSCs in this study, the osteochondral supplements used appear to be important to generate cartilage in long-term tissue engineering approaches for osteochondral interfaces. In addition, constructs generated from agarose-SPCL bilayered scaffolds containing pre-differentiated AFSCs may be useful for potential applications in regeneration strategies for damaged or diseased joints.     

Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds

This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA-C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process. For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6-well plates for 3, 7, and 15 days. The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions. However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion. This observation was confirmed by Von Kossa-stained sections and tetracycline fluorescence. Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA-C scaffolds, which had limited pore interconnectivity, under flow perfusion conditions. In the scaffolds cultured under static conditions, only a surface layer of cells was observed. These results suggest that flow perfusion culture enhances the osteogenic differentiation of marrow stromal cells and improves their distribution in three-dimensional, starch-based scaffolds. They also indicate that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue.     

Evaluation of two biodegradable polymeric systems as substrates for bone tissue engineering

The aim of this study was to evaluate two biodegradable polymeric systems as scaffolds for bone tissue engineering. Rat bone marrow cells were seeded and cultured for 1 week on two biodegradable porous polymeric systems, one composed of poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) and the other composed of cornstarch blended with poly(epsilon-caprolactone) (SPCL). Porous hydroxyapatite granules were used as controls. The ability of cells to proliferate and form extracellular matrix on these scaffolds was assessed by a DNA quantification assay and by scanning electron microscopy examination; their osteogenic differentiation was screened by the expression of alkaline phosphatase. In addition, the in vivo osteogenic potential of the engineered constructs was evaluated through ectopic implantation in a nude mouse model. Results revealed that cells were able to proliferate, differentiate, and form extracellular matrix on all materials tested. Moreover, all constructs induced abundant formation of bone and bone marrow after 4 weeks of implantation. The extent of osteogenesis (approximately 30% of void volume) was similar in all types of implants. However, the amount of bone marrow and the degree of bone contact were higher on HA scaffolds, indicating that the polymers still need to be modulated for higher osteoconductive capacity. Nevertheless, the findings suggest that both PEGT/PBT and SPCL systems are excellent candidates to be used as scaffolds for a cell therapy approach in the treatment of bone defects.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

The role of lipase and alpha-amylase in the degradation of starch/poly(epsilon-caprolactone) fiber meshes and the osteogenic differentiation of cultured marrow stromal cells

The present work studies the influence of hydrolytic enzymes (alpha-amylase or lipase) on the degradation of fiber mesh scaffolds based on a blend of starch and poly(epsilon-caprolactone) (SPCL) and the osteogenic differentiation of osteogenic medium-expanded rat bone marrow stromal cells (MSCs) and subsequent formation of extracellular matrix on these scaffolds under static culture conditions. The biodegradation profile of SPCL fiber meshes was investigated using enzymes that are specifically responsible for the enzymatic hydrolysis of SPCL using concentrations similar to those found in human serum. These degradation studies were performed under static and dynamic conditions. After several degradation periods (3, 7, 14, 21, and 30 days), weight loss measurements and micro-computed tomography analysis (specifically porosity, interconnectivity, mean pore size, and fiber thickness) were performed. The SPCL scaffolds were seeded with rat MSCs and cultured for 8 and 16 days using complete osteogenic media with and without enzymes (alpha-amylase or lipase). Results indicate that culture medium supplemented with enzymes enhanced cell proliferation after 16 days of culture, whereas culture medium without enzymes did not. No calcium was detected in groups cultured with alpha-amylase or without enzymes after each time period, although groups cultured with lipase presented calcium deposition after the eighth day, showing a significant increase at the sixteenth day. Lipase appears to positively influence osteoblastic differentiation of rat MSCs and to enhance matrix mineralization. Furthermore, scanning electron microscopy images showed that the enzymes did not have a deleterious effect on the three-dimensional structure of SPCL fiber meshes, meaning that the scaffolds did not lose their structural integrity after 16 days. Confocal micrographs have shown cells to be evenly distributed and infiltrated within the SPCL fiber meshes up to 410 microm from the surface. This study demonstrates that supplementation of culture media with lipase holds great potential for the generation of bone tissue engineering constructs from MSCs seeded onto SPCL fiber meshes, because lipase enhances the osteoblastic differentiation of the seeded MSCs and promotes matrix mineralization without harming the structural integrity of the meshes over 16 days of culture.     

Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.     

Student Award for Outstanding Research Winner in the Ph.D. Category for the 9th World Biomaterials Congress, Chengdu, China, June 1-5, 2012: The interplay of bone-like extracellular matrix and TNF-α signaling on in vitro osteogenic differentiation of mesenchymal stem cells

As an initial step in the development of a bone tissue engineering strategy to rationally control inflammation, we investigated the interplay of bone-like extracellular matrix (ECM) and varying doses of the inflammatory cytokine tumor necrosis factor alpha (TNF-α) on osteogenically differentiating mesenchymal stem cells (MSCs) cultured in vitro on 3D poly(ε-caprolactone) (PCL) microfiber scaffolds containing pregenerated bone-like ECM. To generate the ECM, PCL scaffolds were seeded with MSCs and cultured in medium containing the typically required osteogenic supplement dexamethasone. However, since dexamethasone antagonizes TNF-α, the interplay of ECM and TNF-α was investigated by culturing na?ve MSCs on the decellularized scaffolds in the absence of dexamethasone. MSCs cultured on ECM-coated scaffolds continued to deposit mineralized matrix, a late stage marker of osteogenic differentiation. Mineralized matrix deposition was not adversely affected by exposure to TNF-α for 4-8 days, but was significantly reduced after continuous exposure to TNF-α over 16 days, which simulates the in vivo response, where brief TNF-α signaling stimulates bone regeneration, while prolonged exposure has damaging effects. This underscores the exciting potential of PCL/ECM constructs as a more clinically realistic in vitro culture model to facilitate the design of new bone tissue engineering strategies that rationally control inflammation to promote regeneration.     

Repair of cranial bone defects with adipose derived stem cells and coral scaffold in a canine model

Adipose derived stem cells (ASCs) with osteogenic differentiation potential have been documented as an alternative cell source for bone regeneration. However, most of previous in vivo studies were carried out on small animals along with relatively short-term follow-up. In this study, we investigated the feasibility of using ASCs and coral scaffolds to repair a cranial bone defect in a canine model, and followed up the outcome for up to 6 month. Autologous ASCs isolated from canine subcutaneous fat were expanded, osteogenically induced, and seeded on coral scaffolds. Bilateral full-thickness defects (20 mm×20 mm) of parietal bone were created. The defects were either repaired with ASC-coral constructs (experimental group) or with coral alone (control group). Three-dimensional CT scan showed that new bones were formed in the experimental group at 12 weeks post-implantation, while coral scaffolds were partially degraded in the control group. By radiographic analysis at 24 weeks post-transplantation, it was shown that an average of 84.19±6.45% of each defect volume had been repaired in experimental side, while the control side had only 25.04±18.82% of its volume filled. Histological examination revealed that the defect was repaired by typical bone tissue in experimental side, while only minimal bone formation with fibrous connection was observed in the control group. The successful repair of critical-sized bone defects via the current approach substantiates the potentiality of using ASCs with coral scaffolds for bone regeneration.     

Flow perfusion culture of human mesenchymal stem cells on silicate-substituted tricalcium phosphate scaffolds

Autologous bone grafts are currently the gold standard for treatment of large bone defects, but their availability is limited due to donor site morbidity. Different substitutes have been suggested to replace these grafts, and this study presents a bone tissue engineered alternative using silicate-substituted tricalcium phosphate (Si-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (hMSC). The cells were seeded onto the scaffolds and cultured either statically or in a perfusion bioreactor for up to 21 days and assessed for osteogenic differentiation by alkaline phosphatase activity assays and by quantitative real-time RT-PCR on bone markers. During culture, cells from the flow cultured constructs demonstrated improved proliferation and osteogenic differentiation verified by a more pronounced expression of several bone markers, e.g. alkaline phosphatase, osteopontin, Runx2, bone sialoprotein II, and bone morphogenetic protein 2. Cells and matrix were distributed homogeneously throughout the entire scaffold in flow culture, whereas only a peripheral layer was obtained after static culture. A viable and homogenous ex vivo bone construct with superior osteogenic properties was produced in dynamic culture and may provide a replacement for autologous grafts.     

Experimental repair of segmental bone defects in rabbits by angiopoietin-1 gene transfected MSCs seeded on porous β-TCP scaffolds

Segmental bone defect repair remains a clinical and experimental challenge in tissue engineering with increasing focus on angiogenesis in the bone substitutes. The objective of this study was to investigate the osteogenic effects of angiopoietin-1 (Ang-1) gene transfected bone marrow-derived mesenchymal stem cells (MSCs) seeded on porous β-TCP scaffolds. This bone substitute (experimental group) and MSCs/β-TCP compounds (control group) were implanted into 15 mm segmental bone defects of the radii of 30 New Zealand white rabbits, with platelet-rich plasma injected at the same time. Bone regeneration and angiogenesis were assessed by Scanning electron microscope (SEM), X-ray, histology, immunohistology, and biomechanical outcome measurements made on the 2nd, 4th, 8th, and 12th week after the operation. In vitro, the amount of proliferation and differentiation of Ang-1 gene transfected MSCs was found to be gross increased than that of the control groups. In vivo, a significantly increased amount of new bone formation accompanied by active capillary vasculature regeneration was observed in the pores of the scaffolds which had been seeded with Ang-1 gene transfected MSCs, as compared with the control groups. The biomechanical test confirmed the failure load of new born bone was close to normal bone. These results suggest that transfer of gene encoding Ang-1 to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation in segmental bone defects.     

In vivo bone formation from human embryonic stem cell-derived osteogenic cells in poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffolds

We have previously reported the efficient osteogenic differentiation of human embryonic stem cells (hESCs) by co-culture with primary human bone-derived cells (hPBDs) without the use of exogenous factors. In the present study, we explored whether osteogenic cells derived from hESCs (OC-hESCs) using the previously reported method would be capable of regenerating bone tissue in vivo. A three-dimensional porous poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffold was used as a cell delivery vehicle. In vivo implantation of OC-hESC-seeded scaffolds showed significant bone formation in the subcutaneous sites of immunodeficient mice at 4 and 8 weeks after implantation (n=5 for each time point). Meanwhile, implantation of the control no cell-seeded scaffolds or human dermal fibroblast-seeded scaffolds did not show any new bone formation. In addition, the presence of BMP-2 (1 microg/scaffold) enhanced new bone tissue formation in terms of mineralization and the expression of bone-specific genetic markers. According to FISH analysis, implanted OC-hESCs remained in the regeneration sites, which suggested that the implanted cells participated in the formation of new bone. In conclusion, OC-hESCs successfully regenerated bone tissue upon in vivo implantation, and this regeneration can be further enhanced by the administration of BMP-2. These results suggest the clinical feasibility of OC-hESCs as a good alternative source of cells for bone regeneration.     

Collagen I gel promotes homogenous osteogenic differentiation of adipose tissue-derived mesenchymal stem cells in serum-derived albumin scaffold

Repair of bone defects is a difficult clinical problem for reconstructive surgeons. Bone tissue engineering using an appropriate scaffold with cells is a new therapy for the repair of bone defects. The aim of this study was to evaluate the in vitro osteogenesis of canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) cultured in a combination of collagen I gel and a porous serum-derived albumin scaffold. A serum-derived albumin scaffold was prepared with canine serum by cross-linking and freeze-drying procedures. Ad-MSCs were seeded into serum-derived albumin scaffolds with or without collagen I gel, and were exposed to osteogenic differentiation conditions in vitro. After 28?days of in vitro culture, the distribution and osteogenic differentiation of Ad-MSCs cultured in the scaffold were evaluated by scanning electron microscopy, histology, immunohistochemistry, alkaline phosphatase (ALP) activity assay, and calcium colorimetric assay. Ad-MSCs showed more homogeneous distribution and osteogenic differentiation in the scaffold with collagen I gel than without collagen I gel. ALP activity and extracellular matrix mineralization in the construct with type I collagen were significantly higher than in the construct without type I collagen (p?<?0.05). In conclusion, the combination of collagen I gel and the serum-derived albumin scaffold enhanced osteogenic differentiation and homogenous distribution of Ad-MSCs.     

Preparation and characterization of a three-dimensional printed scaffold based on a functionalized polyester for bone tissue engineering applications

At present there is a strong need for suitable scaffolds that meet the requirements for bone tissue engineering applications. The objective of this study was to investigate the suitability of porous scaffolds based on a hydroxyl functionalized polymer, poly(hydroxymethylglycolide-co-ε-caprolactone) (pHMGCL), for tissue engineering. In a recent study this polymer was shown to be a promising material for bone regeneration. The scaffolds consisting of pHMGCL or poly(ε-caprolactone) (PCL) were produced by means of a rapid prototyping technique (three-dimensional plotting) and were shown to have a high porosity and an interconnected pore structure. The thermal and mechanical properties of both scaffolds were investigated and human mesenchymal stem cells were seeded onto the scaffolds to evaluate the cell attachment properties, as well as cell viability and differentiation. It was shown that the cells filled the pores of the pHMGCL scaffold within 7 days and displayed increased metabolic activity when compared with cells cultured in PCL scaffolds. Importantly, pHMGCL scaffolds supported osteogenic differentiation. Therefore, scaffolds based on pHMGCL are promising templates for bone tissue engineering applications.     

Bone marrow stromal cells on a three-dimensional bioactive fiber mesh undergo osteogenic differentiation in the absence of osteogenic media supplements: The effect of silanol groups

Osteogenic differentiation is a tightly regulated process dependent on the stimuli provided by the micro-environment. Silicon-substituted materials are known to have an influence on the osteogenic phenotype of undifferentiated and bone-derived cells. This study aims to investigate the bioactivity profile as well as the mechanical properties of a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds functionalized with silanol (Si–OH) groups as key features for bone tissue engineering strategies. The scaffolds were made from SPCL by a wet spinning technique. A calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si–OH groups in a single-step approach. We also explored the relevance of silicon incorporated in SPCL–Si scaffolds to the in vitro osteogenic process of goat bone marrow stromal cells (gBMSCs) with and without osteogenic supplements in the culture medium. We hypothesized that SPCL–Si scaffolds could act as physical and chemical millieus to induce per se the osteogenic differentiation of gBMSCs. Results show that osteogenic differentiation of gBMSCs and the production of a mineralized extracellular matrix on bioactive SPCL–Si scaffolds occur for up to 2 weeks, even in the absence of osteogenic supplements in the culture medium. The omission of media supplements to induce osteogenic differentiation is a promising feature towards simplified and cost-effective cell culturing procedures of a potential bioengineered product, and concomitant translation into the clinical field. Thus, the present work demonstrates that SPCL–Si scaffolds and their intrinsic properties sustain gBMSC osteogenic features in vitro, even in the absence of osteogenic supplements to the culture medium, and show great potential for bone regeneration strategies.     

Kinetics of in vivo bone deposition by bone marrow stromal cells into porous calcium phosphate scaffolds: an X-ray computed microtomography study

In a typical bone tissue engineering application, osteogenic cells are harvested and seeded on a three-dimensional (3D) synthetic scaffold that acts as guide and stimulus for tissue growth, creating a tissue engineering construct or living biocomposite. Despite the large number of performed experiments in different laboratories, information on the kinetics of bone growth into the scaffolds is still scarce. Highly porous hydroxyapatite scaffolds were investigated before the implantation and after they were seeded with in vitro expanded bone marrow stromal cells (BMSC) and implanted for 8, 16, or 24 weeks in immunodeficient mice. Synchrotron x-ray computed microtomography (microCT) was used for qualitative and quantitative 3D characterization of the scaffold material and 3D evaluation of tissue engineered bone growth kinetics after in vivo implantation. Experiments were performed taking advantage of a dedicated set up at the European Synchrotron Radiation Facility (ESRF, Grenoble, France), which allowed quantitative imaging at a spatial resolution of about 5 microm. A peculiarity of these experiments was the fact that at first the data were obtained on the different pure scaffolds, then the same scaffolds were seeded by BMSC, implanted, and brought again to ESRF for investigating the formation of new bone. The volume fraction, average thickness, and distribution of the newly formed bone were evaluated as a function of the implantation time. New bone thickness increased from week 8 to week 16, but deposition of new bone was arrested from week 16 to week 24. Instead, mineralization of the newly deposited bone matrix continued up to week 24.     

Ectopic bone regeneration by human bone marrow mononucleated cells, undifferentiated and osteogenically differentiated bone marrow mesenchymal stem cells in beta-tricalcium phosphate scaffolds

Tissue engineering approaches using the combination of porous ceramics and bone marrow mesenchymal stem cells (BMSCs) represent a promising bone substitute for repairing large bone defects. Nevertheless, optimal conditions for constructing tissue-engineered bone have yet to be determined. It remains unclear if transplantation of predifferentiated BMSCs is superior to undifferentiated BMSCs or freshly isolated bone marrow mononucleated cells (BMNCs) in terms of new bone formation in vivo. The aim of this study was to investigate the effect of in vitro osteogenic differentiation (β-glycerophosphate, dexamethasone, and l-ascorbic acid) of human BMSCs on the capability to form tissue-engineered bone in unloaded conditions after subcutaneous implantation in nude mice. After isolation from human bone marrow aspirates, BMNCs were divided into three parts: one part was seeded onto porous beta-tricalcium phosphate ceramics immediately and transplanted in a heterotopic nude mice model; two parts were expanded in vitro to passage 2 before cell seeding and in vivo transplantation, either under osteogenic conditions or not. Animals were sacrificed for micro-CT and histological evaluation at 4, 8, 12, 16, and 20 weeks postimplantation. The results showed that BMSCs differentiated into osteo-progenitor cells after induction, as evidenced by the altered cell morphology and elevated alkaline phosphatase activity and calcium deposition, but their clonogenicity, proliferating rate, and seeding efficacy were not significantly affected by osteogenic differentiation, compared with undifferentiated cells. Extensive new bone formed in the pores of all the scaffolds seeded with predifferentiated BMSCs at 4 weeks after implantation, and maintained for 20 weeks. On the contrary, scaffolds containing undifferentiated BMSCs revealed limited bone formation only in 1 out of 6 cases at 8 weeks, and maintained for 4 weeks. For scaffolds with BMNCs, woven bone was observed sporadically only in one case at 8 weeks. Overall, this study suggests that ectopic osteogenesis of cell/scaffold composites is more dependent on the in vitro expansion condition, and osteo-differentiated BMSCs hold the highest potential concerning in vivo bone regeneration.     

Novel composite hyaluronan/type I collagen/fibrin scaffold enhances repair of osteochondral defect in rabbit knee

A new composite scaffold containing type I collagen, hyaluronan, and fibrin was prepared with and without autologous chondrocytes and implanted into a rabbit femoral trochlea. The biophysical properties of the composite scaffold were similar to native cartilage. The macroscopic, histological, and immunohistochemical analysis of the regenerated tissue from cell-seeded scaffolds was performed 6 weeks after the implantation and predominantly showed formation of hyaline cartilage accompanied by production of glycosaminoglycans and type II collagen with minor fibro-cartilage production. Implanted scaffolds without cells healed predominantly as fibro-cartilage, although glycosaminoglycans and type II collagen, which form hyaline cartilage, were also observed. On the other hand, fibro-cartilage or fibrous tissue or both were only formed in the defects without scaffold. The new composite scaffold containing collagen type I, hyaluronan, and fibrin, seeded with autologous chondrocytes and implanted into rabbit femoral trochlea, was found to be highly effective in cartilage repair after only 6 weeks. The new composite scaffold can therefore enhance cartilage regeneration of osteochondral defects, by the supporting of the hyaline cartilage formation.