Comparison of Phenotypic Characterization between "Alginate Bead" and "Pellet" Culture Systems as Chondrogenic Differentiation Models for Human Mesenchymal Stem Cells

Chondrogenesis involves the recruitment of mesenchymal cells to differentiate into chondroblasts, and also the cells must synthesize a cartilage-specific extracellular matrix. There were two representative culture systems that promoted the chondrogenic differentiation of human mesenchymal stem cells. These systems were adaptations of the "pellet" culture system, which was originally described as a method for preventing the phenotypic modulation of chondrocytes, and the "alginate bead" culture system, which was used to maintain encapsulated cells at their differentiated phenotype over time, and also it was used to maintain the cells' proteoglycan synthesis at a rate similar to that of primary chondrocytes. We performed test on the differences of phenotypic characterization with the two methods of differentiating human mesenchymal stem cells into chondrocytes. The typical gene for articular cartilage, collagen type II, was more strongly expressed in the "alginate bead" system than in the "pellet" culture system, in addition, specific gene for hypertrophic cartilage, collagen type X, was more rapidly expressed in the "pellet" system than in "alginate bead" culture system. Therefore, the "alginate bead" culture system is a more phenotypical, practical and appropriate system to differentiate human mesenchymal stem cells into articular chondrocytes than the "pellet" culture system.

Chondrogenic Differentiation of Mesenchymal Stem Cells and Its Clinical Applications

Tissue engineering has the potential to provide cartilaginous constructs capable of restoring the normal function of native articular cartilage following joint injury or degradation. One approach to functional tissue engineering of cartilage involves the in vitro cultivation of tissue constructs by using: (i) chondrogenic cells that can be selected, expanded, and transfected to overexpress the genes of interest, (ii) scaffolds that provide a defined three-dimensional structure for tissue development and biodegrade at a controlled rate. Understanding the functional potential of the cells and the signaling mechanisms underlying their differentiation should lead to innovative protocols for clinical orthopaedic interventions. A large number of growth factors and hormones have been implicated in the regulation of chondrocyte biology, relatively little is known about the intracellular signaling pathways involved. We have tried to define the roles of specific TGF-betadependent signaling pathways involved in the regulation of chondrogenesis from human mesenchymal stem cells. Chondrogenesis induced by TGF-beta in alginate bead system was confirmed by examining cartilage specific type II collagen expression and aggrecan, whereas type I collagen expression was not affected by TGF-beta. Type II collagen mRNA expression was expressed strongly during chondrogenesis and MEK inhibition (U0126) resulted in complete down-regulation of type II collagen. In contrast, aggrecan expression was detected in same level by treatment of U0126. These results strongly suggest that the ERK signaling cascade is involved in TGF-beta induced-chondrogenesis signaling pathways and a role of its pathway is necessary over a longer period to promote type II collagen expression. However, their end product properties in vivo have not been well known. In this study, an articular cartilage from chondrogenic MSCs with PLGA scaffolds (75:25 and 65:35) were made and analyzed its biochemical, histological and mechanical properties in vitro and in vivo. And also, we evaluated the cartilage formation in vivo through the injection of cell-thermosensitive gel complex, a newly developed injectable material. At 12 weeks after PLGA scaffolds containing chondrogenic MSCs transplantation, the separated rabbit distal femur showed a good gross articular cartilage appearance in the transplanted site. In indentation test, compare to the native articular cartilage, the engineered cartilage from two types of (75:25 and 65:35) achieved up to 30-60% in mechanical stiffness. And also, a new model for cartilage formation in bladder, at 14 weeks after injection, we could find out mass formation in the submucosal area grossly. H&E staining, alcian blue staining and other special staining confirmed the chondrogenic differentiation in the mass. These cell therapy technologies can provide the possibility of clinical applications for vesicoureteral reflux and reflux esophagitis, and urinary incontinence as well as articular cartilage regeneration.

Multipotentiality of CD34- Cells Extracted from Human Cord Blood / 대한정형외과학회잡지

PURPOSE: We evaluated the differentiation potential of CD34-cells expanded from human cord blood into several differentiated cells, namely, osteoblasts, chondrocytes and adipocytes. MATERIALS AND METHODS: CD34- cells, extracted from cord blood and isolated using a MiniMACS system, were cultured. Cells labeled with appropriate antibodies were analyzed by FACScan. The phenotypes of adipogenic and osteogenic differentiation were evaluated by Oil Red O, alkaline phosphatase, and von Kossa staining. Chondrogenic differentiation was evaluated by RT-PCR using primers for aggrecan, collagen types I, II and X. RESULTS: CD34- cells showed a fibroblast-like morphology and expressed CD105, CD29, and CD44 antigens. These cells showed the deposition of a mineralized matrix and the expression of alkaline phosphatase in the osteogenic medium, and stained with Oil Red O in the adipogenic medium. In terms of chondrogenic differentiation, the expressions of aggrecan, and collagen types II and X showed a gradual increase, whereas the expression of type I collagen gradually decreased. CONCLUSION: Based on their differentiation potentials in at least three different tissue types, i.e., fat, bone, and cartilage, cord blood-derived CD34- cells can be visualized as attractive target cells for cellular or gene transfer therapeutic options.

Peroxisome Proliferator-Activated Receptor gammaActivation Promotes Adipogenesis in Human Mesenchymal Stem Cells / 대한정형외과연구학회지

PURPOSE: In this study, we determined that the troglitazones could induce uniform adipogenesis of human mesenchymal stem cells (MSCs) within a short time in a dose- and a time-dependent manners. MATERIALS AND METHODS: Human MSCs were isolated from bone marrow and cultured in basal or adipogenic medium in the presence of 0~50 micrometer troglitazone for 5 days. Then we performed flow cytometry, RT-PCR and western blot analysis. RESULTS: In FACS assay, troglitazone induced adipocyte differentiation in a dose-dependent manner. At concentration of 25 micrometer troglitazone in adipogenic medium, over 50% of the cells differentiated into adipocytes at day 5. This was accompanied by increased mRNA levels for the adipocyte gene markers (LPL, aP2 and PPAR gamma) in RT-PCR. In western blot analysis, we found that ERK phosphorylation was inhibited in the early stage of adipogenesis. CONCLUSION: Through the addition of troglitazone as a PPAR gamma agonist, we could get the uniform adipogenic differentiation within a short time. Thus, troglitazone directly regulates differentiation of human MSCs into adipocytes; induced PPAR gamma expression may play a key regulatory role in this process. And we suggest a role for ERK as a regulatory switch for these differentiation pathways.

Chemical, Structural & Osteoconductive Properties of Mineralized Porcine Cancellous Bone / 대한정형외과연구학회지

PURPOSE: The purpose of this study is to determine the efficacy of bioactive hydroxyapatite obtained from the porcine cancellous bone for the treatment of bone defect and nonunion. MATERIALS AND METHODS: Porcine cancellous bones were heat-treated at 1300degrees C for 2 hours. The chemical compositions, calcium to phosphate ratio and microstructures of mineralized porcine bone were examined. For in vivo implantation, bone defects were made on the anteromedial aspects of proximal tibia in 7 beagle dogs and theses artificial bones were inserted. Plain X-ray was taken at every 2 weeks interval for radiologic evaluation. At 12 weeks, specimens were evaluated histologyically with hematoxylin and eosin stain. RESULTS: The composition and morphology of mineralized porcine cancellous bone were similar to those of heat-treated human cancellous bone. Radiographs showed union at the host bone-bone block interfaces. At 12 weeks, all uniform and substantial new bone formation were observed. CONCLUSION: This mineralization technique has several advantages such as no disease transmission, no immune reaction, excellent biocompatibility, and cost-effectiveness. Consequently, mineralized porcine cancellous bone showed an effective osteoconductivity.

The Role of Cell Adhesion Molecules in the Modulation of Chondrocytes-extracellular Type II Collagen by Transforming Growth Factor-beta1 / 대한정형외과연구학회지

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