INTRODUCTION: Stem cells have become promising treatments for osteoarthritis due to the cells’ ability to regenerate cartilage and availability from bone marrow. Various studies have established the chondrogenic potential of human marrow stromal cells (hMSCs) upon treatment with transforming growth factor β1 (TGF-β1), yet the difference in potential between cells derived from young subjects and those derived from elder subjects has not been confirmed. OBJECTIVES: This study seeks to establish whether the chondrogenic potential of hMSCs changes with age and sex. This study used a high-density 2D model to measure the acute response of hMSCs to chondrogenic induction over a short time course and various treatment levels. The experiments investigated the expression of chondrogenic genes and expression of TGF-β1 receptors (ALK5) in hMSCs after TGF-β1 treatment to determine whether pediatric hMSCs have more potential for chondrocyte differentiation than adult hMSCs. METHODS: With IRB approval, nine bone marrow samples were obtained from discarded tissue of adults undergoing total hip replacement and juveniles requiring bone graft for alveolar cleft repair. Subject ages ranged from age 8 to 66. Low-density mononucleated cells were cultured in plastic tissue culture dishes. Adherent hMSCs were expanded in monolayer culture with phenol red-free α-MEM medium with 10% fetal bovine serum. After 48 hours of treatment with TGF-β1, cells were collected for RNA extraction and RT-PCR analysis of chondrogenic genes and TGF-β1 receptor levels. Alcian blue staining in 24-well plates of hMSCs was performed after 10 days to compare the effects of different concentrations of TGF-β1, and the effects of another inducer of chondrogenesis, kartogenin (KGN) on matrix accumulation. RESULTS: Gel electrophoresis of PCR products revealed no consistent trend in chondrogenic mRNA expression in pediatric cells compared to adult cells, or female cells compared to male cells. The data indicate that the change in chondrogenic potential of hMSCs with age and sex is inconsistent. KGN showed no consistent effect on hMSCs. Cells with high baseline levels of TGF-β1 receptor (ALK5) showed no upregulation of ALK5 after TGF-β1 treatment, while samples with low basal expression of TGF-β1 receptors showed upregulation after TGF-β1 treatment. CONCLUSIONS: There is still much debate in the literature regarding the potential of adult hMSC chondrogenesis compared to juveniles. This study confirms the irreproducibility of displaying differences between young and adult hMSCs. A larger sample size is needed to establish a correlation between age and chondrogenic potential. Further in vitro studies will consider the optimum time course and concentration of TGF-β1 to observe differences in gene expression of cells, and will identify other clinical determinants of differentiation potential.

Cartilage has limited intrinsic healing potential upon injury, due to the low cell density and the lack of blood supply. Degenerative disease of the cartilage, such as osteoarthritis (OA), is challenging to treat without clear mechanistic understandings of cartilage development. With over 90% of the cartilage tissue occupied by extracellular matrix (ECM), understanding the cellular and molecular effects of cartilage ECM on chondrogenesis and chondrocyte behavior is crucial for therapeutic development. The focus of this work is to study the regulation of chondrogenesis and hypertrophic maturation of human mesenchymal stem cells (MSCs) by cartilage ECM in the context of potential therapeutic applications. To study the cartilage ECM, we created a decellularized ECM digest from native porcine cartilage and examined its effects on MSCs. Since native cartilage ECM maintains chondrocyte homeostasis without progressing to hypertrophic degeneration, we hypothesized that the decellularized ECM would promote MSC chondrogenesis and inhibit hypertrophy. Indeed, we showed that ECM promoted MSC chondrogenesis and matrix production, and inhibited hypertrophy and endochondral ossification. The chondrogenic effect was shown to potentially involve the PI3K-Akt-Foxo1 and Hif1 pathways. By recapitulating the activated Hif1 pathway, roxadustat, a small molecule stabilizer of Hif, was able to reproduce the chondrogenic and anti-hypertrophic effects of the cartilage ECM. It also reduced the expression of matrix metalloproteases (MMPs) in MSCs, healthy chondrocytes, and OA chondrocytes, and alleviated matrix degradation in bovine cartilage explants. We also attempted to identify ECM components that display chondrogenic properties. Collagen XI, a minor component of articular cartilage, was shown to promote cartilage matrix formation in MSCs and healthy chondrocytes, and to reduce matrix degradation in bovine cartilage explants. Taken together, this study reveals the dual roles of cartilage ECM in promoting chondrogenesis and matrix production and inhibiting cartilage hypertrophy. Importantly, small molecule drugs that recapitulate the signaling pathways of ECM regulation, and collagen XI, a component of the ECM, may serve as leads for further therapeutic development for cartilage injury and degenerative disease.

The Hedgehog (Hh) signaling pathway has received attention regarding its important role in embryonic development, however the mechanism by which pathway regulators, such as Suppressor of fused (Sufu), Kinesin family member 7 (Kif7), and primary cilium, mediate Hh signaling transduction is not entirely understood. The work presented here examines the roles of Sufu and Kif7 in regulating Hh signaling in growth plate chondrocytes, as well as how they mediate parathyroid hormone-like hormone (Pthlh) signaling during chondrocyte development. I show here that Sufu and Kif7 are essential regulators of Indian hedgehog (Ihh) signaling. While Sufu negatively regulates Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in Sufu protein degradation and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dosage of Sufu restores normal Hh pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory function of Sufu. Furthermore, Kif7 exerts inhibitory function on Gli transcriptional activity in chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and Sufu-independent mechanisms. I show that Sufu is crucial for mediating the negative effect of Pthlh on Gli transcriptional activity and chondrocyte hypertrophic differentiation, whereas Kif7 and primary cilium are dispensable in this process. Although primary cilium is required for Hh ligand-mediated activation of Gli transcription, Pthlh negatively controls Gli transcriptional activity in a cilia-independent manner. The results of this work provide insight into how Hh signaling is regulated by Sufu and Kif7 in the context of primary cilium, but also suggest Sufu serves as an important link between Ihh and Pthlh signaling during growth plate chondrocyte development.

The Hedgehog (Hh) signaling pathway has received attention regarding its important role in embryonic development, however the mechanism by which pathway regulators, such as Suppressor of fused (Sufu), Kinesin family member 7 (Kif7), and primary cilium, mediate Hh signaling transduction is not entirely understood. The work presented here examines the roles of Sufu and Kif7 in regulating Hh signaling in growth plate chondrocytes, as well as how they mediate parathyroid hormone-like hormone (Pthlh) signaling during chondrocyte development. I show here that Sufu and Kif7 are essential regulators of Indian hedgehog (Ihh) signaling. While Sufu negatively regulates Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in Sufu protein degradation and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dosage of Sufu restores normal Hh pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory function of Sufu. Furthermore, Kif7 exerts inhibitory function on Gli transcriptional activity in chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and Sufu-independent mechanisms. I show that Sufu is crucial for mediating the negative effect of Pthlh on Gli transcriptional activity and chondrocyte hypertrophic differentiation, whereas Kif7 and primary cilium are dispensable in this process. Although primary cilium is required for Hh ligand-mediated activation of Gli transcription, Pthlh negatively controls Gli transcriptional activity in a cilia-independent manner. The results of this work provide insight into how Hh signaling is regulated by Sufu and Kif7 in the context of primary cilium, but also suggest Sufu serves as an important link between Ihh and Pthlh signaling during growth plate chondrocyte development.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M.S.)--University of Michigan, 1986.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 162-215). Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 154-162). Also available in print.

Thesis (M.S.)--University of Michigan, 1986.

Due to mechanical loading, cartilage experiences distortional change, volumetric change, and fluid flow. Research has shown cells to be responsive to unconfined compression, a load that produces all three conditions. To isolate the factor(s) responsible for chondrogenesis, the first goal of this research was to design and implement a device for the application of shear deformation to cells. Secondly, using this device, Stage 23/24 chick limb bud cells were suspended in 2% alginate and subjected to 20% shear deformation at 1 Hz. for two hours daily for three days. Gene expression, DNA content, sGAG content, and cartilage nodule formation were determined after eight days in culture and compared to results obtained for non-loaded cells. Results indicated that shear deformation at the applied level did not have a significant effect on chondrogenesis in Stage 23/24 chick limb bud cells, suggesting that this cell type is not extremely sensitive to distortional change.