Gene expression during redifferentiation of human articular chondrocytes.
ABSTRACT The aim of the present study was to investigate gene expression during the in vitro redifferentiation process of human articular chondrocytes isolated from clinical samples from patient undergoing an autologous chondrocyte transplantation therapy (ACT).
Monolayer (ML) expanded human articular chondrocytes from four donors were cultured in a 3D pellet model and the redifferentiation was investigated by biochemistry, histology, immunohistochemistry and microarray analysis.
The culture expanded chondrocytes redifferentiated in the pellet model as seen by an increase in collagen type II immunoreactivity between day 7 and 14. The gene expression from ML to pellet at day 7 included an increase in cartilage matrix proteins like collagen type XI, tenascin C, dermatopontin, COMP and fibronectin. The late phase consisted of a strong downregulation of extracellular signal-regulated protein kinase (ERK-1) and an upregulation of p38 kinase and SOX-9, suggesting that the late phase mimicked parts of the signaling processes involved in the early chondrogenesis in limb bud cells. Other genes, which indicated a transition from proliferation to tissue formation, were the downregulated cell cycle genes GSPT1 and the upregulated growth-arrest-specific protein (gas). The maturation of the pellets included no signs of hypertrophy or apoptosis as seen by downregulation of collagen type X, Matrix Gla protein and increased expression of caspase 3.
Our data show that human articular chondrocytes taken from surplus cells of patient undergoing ACT treatment and expanded in ML, redifferentiate and form cartilage like matrix in vitro and that this dynamic process involves genes known to be expressed in early chondrogenesis.
[show abstract] [hide abstract]
ABSTRACT: Recent research in tissue engineering for the treatment of cartilage defects have demonstrated that matrix-biomaterial, cell culture conditions, and cytokine-related factors influence the chondrogenic differentiation pattern, especially for the expression of matrix genes. However, little is known about the impact of cell seeding density in a three-dimensional environment on the key chondrogenic transcription factor Sox9. Here we investigated, whether the cell concentration of alginate encapsulated chondrocytes influences the Sox9 expression. Dedifferentiated passage-4 porcine chondrocytes were encapsulated in alginate beads at two different concentrations (4 x 10(6) versus 7 x 10(7) cells/mL) and cultivated for up to 4 weeks under TGF-ss stimulation. The expression of Sox9, Collagen I, II, and X was assessed via quantitative RT-PCR and compared to those observed in the initial monolayer culture. Cellular viability, cell morphology, and the sulphated glycosaminoglycan-production were monitored. Interestingly Sox9 expression was significantly upregulated in the low-cell-density group, whereas no difference between high-cell-density and monolayer culture group could be observed. Furthermore, the cellular survival and the sulphated glycosaminoglycan production were higher in the low-cell-density group. Collagen I expression was downregulated in the low-cell-density group whereas it was upregulated in the high-cell-density one. Surprisingly, only the high-cell-density group showed the expression of Collagen II, although it appeared not significant. Collagen X expression was upregulated in the low-cell-density group. Taken together our data indicate that a low concentration of cell seeding in a three-dimensional environment is beneficial for the overall chondrogenic development. However, this article reveals discrepancies between Sox9 and the chondrogenic pathway in redifferentiating chondrocytes that should be addressed in further work.Journal of Biomedical Materials Research Part A 01/2009; 91(3):910-8. · 2.63 Impact Factor
Article: Osteogenic differentiation and ectopic bone formation of canine bone marrow-derived mesenchymal stem cells in injectable thermo-responsive polymer hydrogel.[show abstract] [hide abstract]
ABSTRACT: This study describes an injectable, thermo-responsive hyaluronic acid-g-chitosan-g-poly(N-isopropylacrylamide) (HA-CPN) copolymer for bone tissue engineering. The wettability, temperature-dependent change of water content, and volume of HA-CPN hydrogel were measured, together with its biocompatibility in vitro and in vivo. The dried hydrogel morphology shows a three-dimensional, porous structure with interconnected pores. Canine bone marrow-derived mesenchymal stem cells (cBMSCs) were encapsulated in HA-CPN hydrogel and osteoinduction was assessed by comparing samples with different osteogenic differentiation induction times but with the same total cell culture time. Cell proliferation and time-dependent osteogenic differentiation, evident from secretion of extracellular matrix and formation of mineral deposits, were observed. The cells showed better proliferation in HA-CPN hydrogel than on tissue culture polystyrene after osteo-induced for 21 days and higher alkaline phosphatase activity regardless of osteo-induction times. Mineralization extent of cBMSCs in HA-CPN followed by Alizarin red stains showed positive stained nodules after osteo-induced longer than 7 days. The cells/hydrogel construct also showed increased mechanical strength and elasticity after osteogenic differentiation, and the increase could be correlated with osteo-induction time. In vivo studies confirmed the biocompatibility and bioresorption of the HA-CPN hydrogel and ectopic bone formation when the hydrogel was used as a cell carrier for osteo-induced cBMSCs and implanted in nude mice subcutaneously. Taken together, the results indicate the feasibility and efficacy of HA-CPN hydrogel as an injectable bone tissue engineering scaffold with cBMSCs.Tissue Engineering Part C Methods 08/2011; 17(11):1139-49. · 4.64 Impact Factor
Article: Pellet culture elicits superior chondrogenic redifferentiation than alginate-based systems.[show abstract] [hide abstract]
ABSTRACT: Although pellet culture and encapsulation of chondrocytes into gel-like biomaterials have lead to major advances in cartilage tissue engineering, a quantitative comparative characterization of cellular differentiation behavior during those cultivation procedures has not yet been performed. Our study therefore aimed at answering the following question: is the redifferentiation pathway of chondrocytes altered by slight changes in the type of alginate biomaterial (pure alginate, alginate-fibrin, alginate-chitosan) and how do the cells behave in comparison to biomaterial-free (pellet) three-dimensional culturing? Monolayer-expanded chondrocytes from healthy adult porcine knee joints were cultivated in alginate, alginate-chitosan, alginate-fibrin beads and as pellets up to 4 weeks. Quantitative PCR and Immunohistology were used to assess chondrogenic markers. Alginate-fibrin-encapsulated chondrocytes behaved almost like monolayer chondrocytes. Alginate- and alginate-chitosan encapsulation lead to a low chondrogenic marker gene expression. Although all 3D-cultured chondrocytes showed a considerable amount of Sox9 expression, only pellet cultivation lead to a sufficient Collagen II expression. This puts the usage of alginate-cultivated cartilage tissue engineering constructs under question. Fibrin addition is not beneficial for chondrogenic differentiation. Sox9 and Collagen II behave differently, depending upon the surrounding 3D-environment.Biotechnology Progress 08/2009; 25(4):1146-52. · 2.34 Impact Factor