| Summary: | 博士 === 臺北醫學大學 === 醫學科學研究所 === 96 === In this thesis we studied the chondrogenesis mechanisms in intervertebral disc (IVD) and articular cartilage regenerations. Human IVD degeneration often initiated from the human nucleus pulposus (hNP) with aging leading to IVD destruction and extracellular matrix (ECM) depletion. Previously, we have successfully employed transforming growth factor-??1 (TGF-??1) to promote chondrogenesis of mesenchymal progenitor cells (MPCs) and immortalized human mesenchymal stem cells. In the first part of this thesis, we examined the role of TGF-??1 in platelet-rich plasma (PRP) on disc regeneration, including proliferation, redifferentiation, and the reconstitution of tissue-engineered NP. hNP cells were isolated from volunteers with different ages and cultured in the presence of PRP. We found that the most effective concentration for hNP proliferation was designated as 1 ng/ml TGF-??1 in PRP, which was further applied in the following experiments. hNP cell proliferation in all age groups were increased time-dependently by PRP and cell morphologies showed aggregation. The mRNA of Sox9, type II collagen, and aggrecan were all significantly upregulated by PRP through RT-PCR. Glycosaminoglycan (GAG) accumulation reached the highest value at day 7 and continued to day 9 culture. PRP promoted NP regeneration via the Smad pathway was also determined and highly activated p-Smad2/3 at 30 min and continuously sustained to 120 min. Immunostaining of type II collagen indicates that PRP participates in chondrogenesis of tissue-engineered NP with collagen scaffolds. We concluded that growth factors in PRP can effectively react as a growth factor cocktail to induce hNP proliferation and differentiation, and also promote tissue-engineered NP formation. These findings are the first to demonstrate that PRP might be a therapeutic candidate for prevention of disc degeneration.
We then investigated the chondrogenesis stages of MSCs committed by articular chondrocytes. Osteoarthritis (OA) is an imbalance in cartilage homeostasis which could potentially be corrected by mesenchymal stem cells (MSCs)-based therapies. However, unexpected results were examined in undifferentiated MSCs after in vivo implantation. To facilitate this, we created a co-culture preconditioning system to improved chondrogenic potentials and to study the detailed chondrogenesis stages of human MSCs (Kp-hMSCs) committed by human chondrocytes (hPi-GFP). We demonstrated that chondrocytes induced commitment to chondrocytic differentiation of hMSCs, based on expressions of chondrogenic (Sox9, type II collagen and aggrecan) but not osteogenic (ALP, osteopontin and osteocalcin) markers, as well as RUNX2. During chondrocytic differentiation of hMSCs, we have more accurately delineated the chondrogenesis stages and have defined the molecular markers. MSCs chondrogenesis stages initiated with highly activated chondrogenic adhesion molecules (ICAM-1) and integrins in committed Kp-hMSCs. Growth factors that promoting chondrogenesis, including TGF-?? superfamily and their downstream regulators SMADs as well as EGF, FGF, IGF and VEGF, were stimulated. An increased accumulation of Col II and glycosaminoglycan (GAG) in committed Kp-hMSCs was detected by immunofluorescent and Alcian blue staining. Furthermore, committed Kp-hMSCs acquired neocartilage forming potentials within the collagen scaffold. Our results first indicate that hMSCs were committed to chondroprogenitor stage by chondrocytes pass through detailed chondrogenesis stages. In addition, the in vitro chondrogenesis of MSCs represents an advance in cell-based transplantation for future clinical use.
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