Contraction-inducedMmp13and −14expression by goat articular chondrocytes in collagen type I but not type II gels (original) (raw)
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Chondrocytes and chondrons for tissue engineering of cartilage
OBJECTIVES. To investigate whether maintaining the chondrocyte's native pericellular matrix prevents collagen-induced up-regulation of collagenase-3 (MMP-13) and whether integrin α1 (ITGα1) and /or discoidin domain receptor 2 (DDR2) modulate MMP-13 expression and which signalling pathway plays a role in collagen-stimulated MMP-13 expression. METHODS. Goat articular chondrocytes and chondrons were cultured on collagen coatings. Small interfering RNA (siRNA) oligonucleotides targeted against Itgα1 and Ddr2 were transfected into primary chondrocytes. Chemical inhibitors for MEK1 (PD98059), FAK (FAK inhibitor 14), JNK (SP600125) and PKC (PKC412), and a calcium chelator (BAPTA-AM) were used in cell cultures. Real-time polymerase chain reaction was performed to examine gene expression levels of Mmp-13, Itgα1 and Ddr2 and collagenolytic activity was determined by measuring the amount of hydroxyproline released in the culture medium. RESULTS. Maintaining the chondrocyte's native pericellular matrix prevented Mmp-13 upregulation and collagenolytic activity when the cells were cultured on a collagen coating. Silencing of Itgα1 and Ddr2 reduced Mmp-13 gene expression and collagenolytic activity by primary chondrocytes cultured on collagen. Incubation with the PKC inhibitor strongly reduced Mmp-13 gene expression levels. Gene expression levels of Mmp-13 were also decreased by chondrocytes incubated with the MEK, FAK or JNK inhibitor. CONCLUSIONS. Maintaining the native pericellular matrix of chondrocytes prevents collagen-induced up-regulation of MMP-13. Both integrin α1 and discoidin domain receptor 2 modulate Mmp-13 expression after direct contact between chondrocytes and collagen. PKC, FAK, MEK and JNK are involved in collagen-stimulated expression of Mmp-13.
Tissue Engineering, 2000
Natural healing of articular cartilage defects generally does not occur, and untreated lesions may predispose the joint to osteoarthritis. To promote healing of cartilage defects, many researchers are turning toward a tissue engineering approach involving cultured cells and/or porous, resorbable matrices. This study investigated the contractile behavior of cultured canine chondrocytes seeded in a porous collagen-glycosaminoglycan (GAG) scaffold. Chondrocytes isolated from the knee joints of adult canines and expanded in monolayer culture were seeded into porous collagen-GAG scaffolds. Scaffolds were of two different compositions, with the predominant collagen being either type I or type II collagen, and of varying pore diameters. Over the 4-week culture period, the seeded cells contracted all of the type I and type II collagen-based matrices, despite a wide range of stiffness (145 6 23 Pa, for the type I scaffold, to 732 6 35 Pa, for the type II material). Pore diameter (25-85 m m, type I; and 53-257 m m, type II) did not affect cell-mediated contraction. Immunohistochemical staining revealed the presence of a-smooth muscle actin, an isoform responsible for contraction of smooth muscle cells and myofibroblasts, in the cytoplasm of the seeded cells and in chondrocytes in normal adult canine articular cartilage.
In vitro expansion affects the response of chondrocytes to mechanical stimulation
Osteoarthritis and Cartilage, 2008
Objective: Expansion of autologous chondrocytes is a common step in procedures for cartilage defect repair. Subsequent dedifferentiation can alter cellular response to mechanical loading, having major consequences for the cell's behavior in vivo after reimplantation. Therefore, we examined the response of primary and expanded human articular chondrocytes to mechanical loading.
195 Preservation of the Chondrocytes Pericellular Matrix Improves Cell-Induced Cartilage Formation
Osteoarthritis and Cartilage, 2009
Purpose: Chondrocytes are often used for cartilage tissue engineering. However, in native cartilage, the chondrocytes are surrounded by a pericellular matrix, together forming the chondron. Since cells are influenced by their surroundings, we hypothesized that retaining the pericellular microenvironment would influence the synthetic capacity of the chondrocytes. Therefore the aim of this study was to investigate whether the pericellular matrix has an effect on cell-induced cartilage formation. Methods: Chondrocytes and chondrons isolated from nucleus pulposus (NP), annulus fibrosus (AF), and articular cartilage (AC) from goats, were cultured for 25 days in alginate beads. After 7, 18 and 25 days of culture, the amount of proteoglycans present in the alginate beads was measured and collagen was extracted from the beads. Immunoblotting for type II collagen was performed on the collagen extracted from the alginate beads. Protein expression of matrix metalloproteinase 2 (Mmp2) and Mmp9 was analyzed by zymography and gene expression levels of Mmp13 were measured by real-time PCR. Results: Chondrons and chondrocytes were successfully isolated from AC, AF, and NP. The amount of proteoglycans found in the alginate beads was significantly higher in the chondrons from AC and NP compared to the chondrocytes, but no differences were found between chondrons and chondrocytes from AF. The type II collagen that was extracted from the alginate beads containing the chondrons from all the cartilage sources was cross-linked, whereas the type II collagen produced by the chondrocytes consisted only of non-crosslinked alpha1 (II) chains. Both Mmp2 and Mmp9 expression were higher by the chondrocytes from AC and NP compared to the chondrons, no differences were found with the AF cells. At day 0 the gene expression levels of MMP13 were low in both chondrocytes and chondrons. However, after 18 and 25 days of culture, there was a significant increased expression by the chondrocytes and not by the chondrons. Conclusions: This study shows that maintaining the native chondrocytes pericellular matrix affects both anabolic and catabolic activities. The cross-links present in the type II collagen produced by the chondrons isolated from all the different tissues suggests that the pericellular matrix has an effect on the expression or the activity of enzymes involved in collagen cross-linking. The type II collagen produced by the chondrons does more resemble the collagen found in the native tissues. It is also likely that the altered cell-ECM interactions caused by removal of the pericellular matrix plays a role in the increased expression of the matrix metalloproteinases. Taken together, our data suggest that the extracellular matrix surrounding the chondrocytes is essential for maintaining its proper composition and that preserving the thin matrix layer surrounding the chondrocytes improves cell-induced hyaline cartilage formation.
Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes
Tissue Engineering Part A, 2013
Introduction: Current cartilage repair procedures using autologous chondrocytes rely on a variety of carriers for implantation. Collagen types I and II are frequently used and valuable properties of both were shown earlier in vitro, although a preference for either was not demonstrated. Recently, however, fibrillar collagens were shown to promote cartilage degradation. The goal of this study was to evaluate the effects of collagen type I and type II coating on chondrogenic properties of in vitro cultured human chondrocytes, and to investigate if collagen-mediated cartilage degradation occurs. Methods: Human chondrocytes of eight healthy cartilage donors were isolated, expanded, and cultured on culture well inserts coated with either collagen type I, type II, or no coating (control). After 28 days of redifferentiation culture, safranin O and immunohistochemical staining for collagen types I, II, X, and Runx2/ Cbfa1 were performed and glycosaminoglycan (GAG) and DNA content and release were examined. Further, expression of collagen type I, type II, type X, MMP13, Runx2/Cbfa1, DDR2, a2 and b1 integrin were examined by reverse transcriptase-polymerase chain reaction. Results: The matrix, created by chondrocytes grown on collagen type I-and II-coated membranes, resembled cartilage more than when grown on noncoated membranes as reflected by histological scoring. Immunohistochemical staining did not differ between the conditions. GAG content as well as GAG/DNA were higher for collagen type II-coated cartilage constructs than control. GAG release was also higher on collagen type I-and IIcoated constructs. Expression of collagen type X was higher of chondrocytes grown on collagen type II compared to controls, but no collagen X protein could be demonstrated by immunohistochemistry. No effects of collagen coating on DDR2 nor MMP-13 gene expression were found. No differences were observed between collagen types I and II. Conclusion: Chondrocyte culture on collagen type I or II promotes more active matrix production and turnover. No significant differences between collagen types I and II were observed, nor were hypertrophic changes more evident in either condition. The use of collagen type I or II coating for in vitro models, thus, seems a sound basis for in vivo repair procedures.
Tissue Engineering, 2005
Autologous chondrocyte implantation is currently applied in clinics as an innovative tool for articular cartilage repair. Animal models have been and still are being used to validate and further improve the technique. However, in various species, the outcome varies from hyaline-like cartilage to fibrocartilage. This may be due partly to the spontaneous dedifferentiation of chondrocytes once cultured in vitro. Here we assessed whether the extent of dedifferentiation varies between species and we hypothesized that the level of chondrocyte phenotype stability during expansion may contribute to the maintenance of their chondrogenic commitment and redifferentiation potential. Condyle chondrocytes were harvested from sheep, dog, and human, and expanded for 1, 6, or 12 cell duplications. At each interval, cell phenotype was monitored (morphology and biosynthesis of cartilage markers) and redifferentiation was assessed by an in vitro assay of chondrogenesis in micromass pellet and an in vivo assay of ectopic cartilage formation in immunodeficient mice. Results indicate that, during culture, the sheep chondrocyte phenotype is maintained better than that of human chondrocytes, which in turn dedifferentiate to a lesser extent than dog chondrocytes Accordingly, after expansion, sheep chondrocytes spontaneously reform hyaline-like cartilage; human chondrocytes redifferentiate only under stimulation with chondrogenic inducers whereas, after a few passages, dog chondrocytes lose any capacity to redifferentiate regardless of the presence of inducers. Thus, conditions allowing cartilage formation in one species are not necessarily transposable to other species. Therefore, results with animal models should be cautiously applied to humans. In addition, for tissue-engineering purposes, the number of cell duplications must be, for each species, carefully monitored to remain in the range of amplification allowing redifferentiation and chondrogenesis.
Tissue Engineering, 2005
Autologous chondrocyte implantation is currently applied in clinics as an innovative tool for articular cartilage repair. Animal models have been and still are being used to validate and further improve the technique. However, in various species, the outcome varies from hyaline-like cartilage to fibrocartilage. This may be due partly to the spontaneous dedifferentiation of chondrocytes once cultured in vitro. Here we assessed whether the extent of dedifferentiation varies between species and we hypothesized that the level of chondrocyte phenotype stability during expansion may contribute to the maintenance of their chondrogenic commitment and redifferentiation potential. Condyle chondrocytes were harvested from sheep, dog, and human, and expanded for 1, 6, or 12 cell duplications. At each interval, cell phenotype was monitored (morphology and biosynthesis of cartilage markers) and redifferentiation was assessed by an in vitro assay of chondrogenesis in micromass pellet and an in vivo assay of ectopic cartilage formation in immunodeficient mice. Results indicate that, during culture, the sheep chondrocyte phenotype is maintained better than that of human chondrocytes, which in turn dedifferentiate to a lesser extent than dog chondrocytes Accordingly, after expansion, sheep chondrocytes spontaneously reform hyaline-like cartilage; human chondrocytes redifferentiate only under stimulation with chondrogenic inducers whereas, after a few passages, dog chondrocytes lose any capacity to redifferentiate regardless of the presence of inducers. Thus, conditions allowing cartilage formation in one species are not necessarily transposable to other species. Therefore, results with animal models should be cautiously applied to humans. In addition, for tissue-engineering purposes, the number of cell duplications must be, for each species, carefully monitored to remain in the range of amplification allowing redifferentiation and chondrogenesis.
Behavior of Human Articular Chondrocytes During In Vivo Culture in Closed, Permeable Chambers
Cell Medicine, 2012
The exact contribution of transplanted chondrocytes for cartilage tissue repair prior expansion in monolayer cultures remains undetermined. At our laboratory, we have created a new permeable chamber to study the chondrogenesis of dedifferentiated cells implanted ectopically in a closed and controlled environment. The behavior of chondrocytes has been studied in settings frequently used in clinical approaches during transplantation, namely injection of autologous chondrocyte cells in suspension (ACI), cells soaked in collagen membranes (MACI), and cells applied in a polymer gel (fibrin). As controls, we have tested the redifferentiation of chondrocytes in cell aggregates, and we have checked the proper functionality of chambers both in vitro and in vivo. After retrieval, firmed tissue-like shapes were recovered only from chambers containing cells seeded in membranes. Histomorphological, immunohistochemical, and ultrastructural analyses revealed synthesis of fibrous-like tissue, characterized by low-density collagen fibers, low collagen type II, abundant collagen type I, and low amounts of proteoglycans. Additionally, neither the collagen membranes nor the fibrin gel was reabsorbed by cells. In summary, our results show that the newly developed permeable chambers function correctly, allowing proper cell feeding and preventing cell leakage or host cell invasion. Additionally, our results suggest that, under these circumstances, chondrocytes are not able to orchestrate formation of hyaline cartilage and have little capacity to degrade artificial membranes or carrier gels such as fibrin. These are interesting observations that should be considered for understanding what role the transplanted chondrocytes play during restoration of articular cartilage after implantation.
Chondrocyte behaviour within different types of collagen gel in vitro
Biomaterials, 1995
In cartilage repair experiments chondrocytes are transplanted into osteochondral defects. Biological substances are used as cell vehicles and are likely to play an important role in the outcome of these studies. Collagen gel is formed by polymerization of type I collagen and is used in plastic surgery and for three-dimensional culture systems. To test collagen gel as a potential vehicle for transplantation, we evaluated chondrocyte behaviour in vitro in different collagen gels. Collagen type I was extracted and purified from rat tail tendon and fetal calf skin and compared with commercially available collagen type I. After suspension of bovine chondrocytes, five different collagen gels were cultured for 14 days and evaluated by light and electron microscopy. Cells proliferated within all gels and synthesized proteoglycans as assessed by 35S incorporation; 40-90% of cells maintained a chondrocyte-like morphology after 1 week in culture depending on the type of collagen gel. Synthetic and secretory activity was confirmed by electron microscopy. Based on these results, calf skin collagen is recommended for culturing chondrocytes for implantation.