Chondrogenesis Of hMSCs Research Articles

Spatial regulation of human mesenchymal stem cell differentiation in engineered osteochondral constructs: effects of pre-differentiation, soluble factors and medium perfusion

The objective of the study was to investigate the combined effects of three sets of regulatory factors: cell pre-differentiation, soluble factors and medium perfusion on spatial control of human mesenchymal stem cell (hMSC) differentiation into cells forming the cartilaginous and bone regions in engineered osteochondral constructs. Bone-marrow derived hMSCs were expanded in their undifferentiated state (UD) or pre-differentiated (PD) in monolayer culture, seeded into biphasic constructs by interfacing agarose gels and bone scaffolds and cultured for 5 weeks either statically (S) or in a bioreactor (BR) with perfusion of medium through the bone region. Each culture system was operated with medium containing either chondrogenic supplements (C) or a cocktail (Ck) of chondrogenic and osteogenic supplements. The formation of engineered cartilage in the gel region was most enhanced by using undifferentiated cells and chondrogenic medium, whereas the cartilaginous properties were negatively affected by using pre-differentiated cells or the combination of perfusion and cocktail medium. The formation of engineered bone in the porous scaffold region was most enhanced by using pre-differentiated cells, perfusion and cocktail medium. Perfusion also enhanced the integration of bone and cartilage regions. (1) Pre-differentiation of hMSCs before seeding on scaffold was beneficial for bone but not for cartilage formation. (2) The combination of medium perfusion and cocktail medium inhibited chondrogenesis of hMSCs. (3) Perfusion improved the cell and matrix distribution in the bone region and augmented the integration at the bone-cartilage interface. (4) Osteochondral grafts can be engineered by differentially regulating the culture conditions in the two regions of the scaffold seeded with hMSCs (hydrogel for cartilage, perfused porous scaffold for bone).

Chondrogenesis of Human Bone Marrow Mesenchymal Stem Cells in Fibrin–Polyurethane Composites Is Modulated by Frequency and Amplitude of Dynamic Compression and Shear Stress

In this study, human bone marrow mesenchymal stem cell (hMSC)-seeded fibrin-polyurethane composite scaffolds were subjected to various mechanical load protocols to determine the effect of compression and surface rotation frequency and axial compression magnitude on the induction of chondrocyte-specific gene expression and protein synthesis. After 7 days of preculture and 7 days of load, application of dynamic compression and surface shear 1 h daily enhanced chondrogenesis of hMSCs compared with the nonloaded control samples. Higher load frequency and higher compression amplitude induced higher glycosaminoglycan synthesis, higher chondrocytic gene expression, higher chondrocytic:fibroblastic, chondrocytic:hypertrophic, and chondrocytic:osteoblastic gene expression ratios, as well as higher transforming growth factor beta1 (TGFB1) and TGFB3 gene expression. The chondrogenesis level of hMSCs was positively related to the TGFB1 and TGFB3 gene expression level, which was determined by various load regimes. In conclusion, chondrogenesis of human MSCs induced by mechanical stimulation can be further enhanced by modifying frequency and compression.

Cyclic Compression Maintains Viability and Induces Chondrogenesis of Human Mesenchymal Stem Cells in Fibrin Gel Scaffolds

Mechanical loading has long been shown to modulate cartilage-specific extracellular matrix synthesis. With joint motion, cartilage can experience mechanical loading in the form of compressive, tensile or shearing load, and hydrostatic pressure. Recent studies have demonstrated the capacity of unconfined cyclic compression to induce chondrogenic differentiation of human mesenchymal stem cell (hMSC) in agarose culture. However, the use of a nonbiodegradable material such as agarose limits the applicability of these constructs. Of the possible biocompatible materials available for tissue engineering, fibrin is a natural regenerative scaffold, which possesses several desired characteristics including a controllable degradation rate and low immunogenicity. The objective of the present study was to determine the capability of fibrin gels for supporting chondrogenesis of hMSCs under cyclic compression. To optimize the system, three concentrations of fibrin gel (40, 60, and 80 mg/mL) and three different stimulus frequencies (0.1, 0.5, and 1.0 Hz) were used to examine the effects of cyclic compression on viability, proliferation and chondrogenic differentiation of hMSCs. Our results show that cyclic compression (10% strain) at frequencies >0.5 Hz and gel concentration of 40 mg/mL fibrinogen appears to maintain cellular viability within scaffolds. Similarly, variations in gel component concentration and stimulus frequency can be modified such that a significant chondrogenic response can be achieved by hMSC in fibrin constructs after 8 h of compression spread out over 2 days. This study demonstrates the suitability of fibrin gel for supporting the cyclic compression-induced chondrogenesis of mesenchymal stem cells.

Chondrogenesis From Immortalized Human Mesenchymal Stem Cells: Comparison Between Collagen Gel and Pellet Culture Methods

Human mesenchymal stem cells (hMSCs) can differentiate into cells of connective tissue lineages, including cartilage. To overcome the limiting autogenous chondrocyte populations available in cartilage repair, various methods have been developed to maximize chondrogenesis of hMSCs in vitro, most of which use cells derived from primary culture. In this study, we compared chondrogenesis of immortalized hMSCs embedded in collagen gel to those grown in pellet culture. The hMSCs in collagen scaffolds expressed more glycosaminoglycan than those in pellet culture. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that the expression of genes encoding sox-9, aggrecan, and types I and II collagen increased in pellet culture over time. However, in the collagen cultures, only type II collagen and aggrecan expression increased over time, whereas sox-9 expression remained unchanged and type I collagen expression decreased. These results indicate that the immortalized hMSC line is a promising tool for further in vitro chondrogenic studies.

Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Three-Dimensional Alginate Gels

We characterized the temporal changes in chondrogenic genes and developed a staging scheme for in vitro chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in three-dimensional (3D) alginate gels. A time-dependent accumulation of glycosaminoglycans, aggrecan, and type II collagen was observed in chondrogenic but not in basal constructs over 24 days. qRT-PCR demonstrated a largely characteristic temporal pattern of chondrogenic markers and provided a basis for staging the cellular phenotype into four stages. Stage I (days 0-6) was defined by collagen types I and VI, Sox 4, and BMP-2 showing peak expression levels. In stage II (days 6-12), gene expression for cartilage oligomeric matrix protein, HAPLN1, collagen type XI, and Sox 9 reached peak levels, while gene expression of matrilin 3, Ihh, Homeobox 7, chondroadherin, and WNT 11 peaked at stage III (days 12-18). Finally, cells in stage IV (days 18-24) attained peak levels of aggrecan; collagen IX, II, and X; osteocalcin; fibromodulin; PTHrP; and alkaline phosphatase. Gene profiles at stages III and IV were analogous to those in juvenile articular and adult nucleus pulposus chondrocytes. Gene ontology analyses also demonstrated a specific expression pattern of several putative novel marker genes. These data provide comprehensive insights on chondrogenesis of hMSCs in 3D gels. The derivation of this staging scheme may aid in defining maximally responsive time points for mechanobiological modulation of constructs to produce optimally engineered tissues.

In Vitro Chondrogenesis of Mesenchymal Stem Cells in Recombinant Silk-elastinlike Hydrogels

In this study the chondrocytic differentiation and cartilage matrix accumulation of human mesenchymal stem cells (hMSCs) were investigated after encapsulation in a genetically engineered silk-elastinlike protein polymer SELP-47 K as an injectable matrix for delivery of cell-based therapeutics. hMSCs were encapsulated in SELP-47 K and cultured for 4 weeks in chondrogenic medium with or without transforming growth factor-beta3 (TGF). Chondrogenic differentiation was evaluated by histological, RNA and biochemical analyses for the expression of cartilage extracellular matrix components. Histological and immunohistochemical staining revealed that the cells acquired a rounded morphology and were embedded in significant amounts of chondrogenic extracellular matrix. Reverse transcriptase (RT)-PCR showed an up-regulation in aggrecan, type II and type X collagen and SOX9 in presence of TGF-beta3. By day 28, constructs cultured in the presence of TGF-beta3 exhibited significant increase in sulfated glycosaminoglycan and total collagen content up to 65 and 300%, respectively. This study demonstrates that SELP-47 K hydrogel can be used as a scaffold for encapsulation and chondrogenesis of hMSCs. The ability to use recombinant techniques to precisely control SELP structure enables the investigation of injectable protein polymer scaffolds for soft-tissue engineering with varied physicochemical properties.

Quantitative Study of Tissue-Engineered Cartilage With Human Bone Marrow Mesenchymal Stem Cells

To assess the possibility of cartilage tissue engineering using human mesenchymal stem cells (hMSCs) and to investigate the quantitative relationship between hMSCs and engineered cartilage. Human mesenchymal stem cells were cultured, cryopreserved, and expanded in vitro. Surface antigens were detected by flow cytometry. In vitro chondrogenesis of hMSCs and cryopreserved hMSCs was performed. The chondrogenesis-induced hMSCs were seeded onto polyglycolic acid scaffolds, cultured in vitro for 3 weeks in chondrogenic medium, and then implanted into nude mice. The implants were harvested after 10 weeks and examined with histologic and immunochemical staining. The construction of cartilages was identified grossly and histologically: 1.9 to 2.5 x 10(7) nucleated cells were obtained from 1 mL of bone marrow, and about 1 to 2 x 10(6) hMSCs were obtained from the primary culture. The number of hMSCs tripled at every passage and reached 1.4 to 2.8 x 10(12) at passage 15. The purity of hMSCs was 95% and 98% at the primary and the fourth passages, respectively. Twenty-one days was the optimal (induction rate, 95%) induction time, with no apparent differences in induction rates among different passages. Based on our findings, hMSCs from 0.07 to 0.14 mL of bone marrow, expanded during 4 passages and induced for 21 days, would be sufficient to engineer 1 cm(2) of cartilage, 3-mm thick. Quantitative standards of hMSCs as seed cells for cartilage tissue engineering were established and may have value for later clinical work.