Upregulation of Adipogenesis and Chondrogenesis in MSC Serum-Free Culture.

Serum-free media have been shown to be effective in the expansion of mesenchymal stem cells (MSCs). However, the effects may go beyond cell expansion as the differentiation potentials of the cells may be modified, thus influencing their efficacy for downstream applications. The latter is poorly understood, and this has prompted an evaluation of the influence of a serum-free formulation on the chondrogenic, adipogenic, and osteogenic potential of MSCs. The media consisted of Knockout™ Serum Replacement (KSR) with a cocktail of growth factors coupled with either collagen or fibronectin coatings. Collagen coating was selected as it promoted consistent cellular attachment. When compared against fetal bovine serum (FBS) controls, cell proliferation in the serum-free media was enhanced at passage 1. Similar levels of surface markers were observed in the two groups with a slight reduction in CD90 and CD73 in the serum-free culture at passage 3. The cultures were screened under differentiation conditions and a better maintenance of the chondrogenic potential was noted in the serum-free media with higher expressions of glycoaminoglycans (GAGs) and collagen II. Chondrogenesis was deficient in the FBS group and this was attributed to the inherent inconsistency of animal serum. Adipogenesis was enhanced in the serum-free group with a higher PPARG expression and lipid accumulation. Similar levels of osteogenic mineralization was noted in the FBS and serum-free groups but collagen I gene expression was suppressed in the latter. This was initially observed during expansion. These observations were attributed to the signaling cascades triggered by the cytokines presented in the serum-free formulation and the interaction with the collagen substrate. The serum-free media helps to maintain and enhance the chondrogenic and adipogenic potentials of the MSCs, respectively. This advantage can be exploited for therapeutic applications in cartilage and adipose tissue engineering.

The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model.

Conventional clinical therapies are unable to resolve osteochondral defects adequately; hence, tissue engineering solutions are sought to address the challenge. A biphasic implant that was seeded with mesenchymal stem cells (MSCs) and coupled with an electrospun membrane was evaluated as an alternative. This dual phase construct comprised of a polycaprolactone (PCL) cartilage scaffold and a PCL-tricalcium phosphate osseous matrix. Autologous MSCs were seeded into the entire implant via fibrin and the construct was inserted into critically sized osteochondral defects located at the medial condyle and patellar groove of pigs. The defect was resurfaced with a PCL-collagen electrospun mesh, which served as a substitute for periosteal flap in preventing cell leakage. Controls without either implanted MSCs or resurfacing membrane were included. After 6 months, cartilaginous repair was observed with a low occurrence of fibrocartilage at the medial condyle. Osteochondral repair was promoted and host cartilage degeneration was arrested as shown by superior glycosaminoglycan maintenance. This positive morphological outcome was supported by a higher relative Young's modulus, which indicated functional cartilage restoration. Bone ingrowth and remodeling occurred in all groups, with a higher degree of mineralization in the experimental group. Tissue repair was compromised in the absence of the implanted cells or the resurfacing membrane. Moreover, healing was inferior at the patellar groove when compared with the medial condyle and this was attributed to the native biomechanical features.

A serum free approach towards the conservation of chondrogenic phenotype during in vitro cell expansion.

OBJECTIVE: Functionally viable chondrocytes in sufficient quantity is crucial for the success of matrix associated autologous chondrocyte implantation. This is difficult with conventional methods as chondrocytes dedifferentiate during 2D expansion with the loss of their chondrogenic phenotype. Moreover, established protocols are dependent on the use of serum which is not without its drawbacks. This study sought to address the issue by evaluating the feasibility of serum free, growth factors supplemented chondrocyte media with extracellular matrix (ECM) coatings. DESIGN: Passage 2 human chondrocytes were cultured in serum supplemented media or serum free media with collagen I or fibronectin coatings. Cell attachment and proliferation were assessed in these conditions. The cells were redifferentiated via pellet cultures for 7 and 14 days before being subjected to histological and gene expression analysis. RESULTS: The serum-free, growth factor cocktail supplemented with ECM coating improved long-term chondrocyte proliferation with enhanced basal Sox 9 expression. Upon induction, the redifferentiated chondrocytes expressed aggrecan and collagen II especially so for the cells plated on collagen coated surfaces. The chondrocytic phenotype was better conserved under the serum free conditions but the loss of the hyaline cartilage characteristics was not completely halted given the expression of collagen I. These essential cartilage markers were, however, reduced or absented for cells expanded with serum. Moreover, serum cultures displayed a higher tendency of undergoing hypertrophy given the stronger collagen X gene expression. CONCLUSION: The advocated technique promoted cell expansion with respect to conventional serum supplemented cultures while reducing the loss of the chondrogenic phenotype. This demonstrates the feasibility and potential of the novel concomitant use of serum free media and ECM coatings in the expansion of chondrocytes for cartilage regenerative applications.

Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts.

In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone-calcium phosphate (mPCL-CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (5 x 5 x 4mm(3)) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10 x 10 x 4mm(3)) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.

Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits.

The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the defect (4 mm diameter x 5 mm depth) on medial femoral condyle of adult New Zealand White rabbits. Empty scaffolds were used at the control side. Cell survival was tracked via fluorescent labeling. The regeneration process was evaluated by several techniques at 3 and 6 months post-implantation. Mature trabecular bone regularly formed in the mPCL scaffold at both 3 and 6 months post-operation. Micro-Computed Tomography showed progression of mineralization from the host-tissue interface towards the inner region of the grafts. At 3 months time point, the specimens showed good cartilage repair. In contrast, the majority of 6 months specimens revealed poor remodeling and fissured integration with host cartilage while other samples could maintain good cartilage appearance. In vivo viability of the transplanted cells was demonstrated for the duration of 5 weeks. The results demonstrated that mPCL scaffold is a potential matrix for osteochondral bone regeneration and that fibrin glue does not inherit the physical properties to allow for cartilage regeneration in a large and high-load-bearing defect site.

A comparison of micro CT with other techniques used in the characterization of scaffolds.

The structure and architecture of scaffolds are crucial factors in scaffold-based tissue engineering as they affect the functionality of the tissue engineered constructs and the eventual application in health care. Therefore, effective scaffold assessment techniques are required right at the initial stages of research and development so as to select or design scaffolds with suitable properties. Various techniques have been developed in evaluating these important features and the outcome of the assessment is the eventual improvement on the subsequent design of the scaffold. An effective evaluation approach should be fast, accurate and non-destructive, while providing a comprehensive overview of the various morphological and architectural characteristics. Current assessment techniques would include theoretical calculation, scanning electron microscopy (SEM), mercury and flow porosimetry, gas pycnometry, gas adsorption and micro computed tomography (CT). Micro CT is a more recent method of examining the characteristics of scaffolds and this review aims to highlight this current approach while comparing it with other techniques.

Application of a polyelectrolyte complex coacervation method to improve seeding efficiency of bone marrow stromal cells in a 3D culture system.

High seeding efficiency with homogenous distribution of limited cell sources such as bone marrow stromal cells (BMSCs) are of clinical relevance in scaffold-based tissue engineering. Therefore, considerable research efforts have been invested to ameliorate the seeding efficiency in 3D scaffolds. Preliminary data demonstrated that indeed BMSCs were viable and were able to proliferate in a model 3D scaffold, i.e. Cytomatrix scaffold. However, the eventual practical application of BMSCs in such 3D scaffolds is limited by the low seeding efficiency of the cells within the scaffold. Here, we demonstrated that the cell seeding efficiency of BMSCs in the Cytomatrix scaffold can be improved significantly (t-test, p<0.05) by means of macroencapsulating the scaffold via the complex coacervation of a methylated collagen and terpolymer. The thickness and density of the polyeletrolyte complex can be modulated by the contact time between the methylated collagen and terpolymer to balance between cell entrapment efficacy and mass transfer impedance imparted by the complex. Porcine BMSCs were macroencapsulated in Cytomatrix scaffolds using various polyelectrolyte contact time and cultured under both static and dynamic conditions. Throughout the range of contact time investigated, macroencapsulation did not affect the viability of the porcine BMSCs in dynamic culture. However, the viability of the cells under static cultures was compromised with longer polyelectrolyte contact time. Therefore, this proposed method of macroencapsulation enables customization to achieve enhanced seeding efficiency without mass transfer impedance for different culture configurations.