Preservation of the chondrocyte's pericellular matrix improves cell-induced cartilage formation (original) (raw)
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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.
The aim of this study was to investigate the effects of serum and compressive dynamic loading on the cartilaginous matrix spatiotemporal distribution around chondrocytes in vitro. Murine chondrocytes suspended in agarose were cultured in serum-free media or in varying concentrations of serum with or without compressive dynamic loading. Gene expression was assayed by quantitative polymerase chain reaction. Immunohistochemistry was performed for type II collagen and type VI collagen, aggrecan, or cartilage oligomeric matrix protein (COMP) to study the effect of serum and dynamic loading on the spatiotemporal distribution of cartilage matrix components. Chondrocytes in serum-free culture exhibited negligible differences in type II collagen, aggrecan, and COMP mRNA expression levels over 15 days of cultivation. However, higher serum concentrations decreased matrix gene expression. Expression of the matrix metalloproteinases (MMP)-3 and MMP-13 mRNA increased over time in serum-free or reduced serum levels, but was significantly suppressed in 10% fetal bovine serum (FBS). Compressive loading significantly stimulated MMP-3 expression on days 7 and 15. Immunohistochemical analysis demonstrated that maximum pericellular matrix deposition was achieved in 10% FBS culture in the absence of compressive loading. The pericellular distribution of type II and VI collagens, aggrecan, and COMP proteins tended to be more co-localized in the pericellular region from day 9 to day 21; compressive loading helped promote this co-localization of matrix proteins. The results of this study suggest that the quantity, quality, and spatial distribution of cartilaginous matrix can be altered by serum concentrations and compressive loading.
Matrix changes during long-term cultivation of cartilage (organoid or high-density cultures)
Histology and histopathology, 1993
In high density (organoid or micromass) cultures of prechondrogenic mesenchymal cells from limb buds of 12-day-old mouse embryos typical cartilaginous tissue develops after 3 days. Immunomorphological investigations have shown that it contains the typical components of the cartilaginous matrix, such as collagen type II and cartilage-specific proteoglycans. After a 2-week cultivation period hypertrophic cartilage cells develop to an increasing extent. Many of these cells as well as normal chondroblasts detach from the matrix from the 2nd week in vitro onwards to assume a fibroblast-like appearance. At the same time thick (25-65 nm) collagenous fibrils occur at the surface of these cells. These thick fibrils contain collagen type I, as shown by immunomorphology. Hence, in these older cartilage cultures chondroblasts change their synthesis programme or direction of differentiation. Consequently, a model for the study of "dedifferentiation" of cartilage and possibly also trans...
Osteoarthritis and Cartilage, 2005
Objective: Clinical cartilage repair with transplantation of cultured chondrocytes, the first described technique introduced in 1994, includes a periosteal membrane but today cells are also implanted without the periosteal combination. The aim of this study was to see if the periosteum had more than a biomechanical function and if the periosteum had a biological effect on the seeded cells tested in an agarose system in which the clonal growth in agarose and the external growth stimulation could be analysed.
Importance of Floating Chondrons in Cartilage Tissue Engineering
World journal of plastic surgery, 2017
Dedifferentiation of chondrocytes remains a major problem for cartilage tissue engineering. Chondrocytes loss differentiated phenotype in in vitro culture that is undesired for repair strategies. The chondrocyte is surrounded by a pericellular matrix (PCM), together forming the chondron. PCM has a positive effect on the maintenance of chondrocyte phenotype during culture in comparison to uncovered chondrocyte. Studies suggest that the PCM influence on functional properties of the chondrocytes. However there is no study to show gene expression phenotype differences between round chondron and fibroblastic chondrocytes. We aimed to investigate the effect of pericellular matrix in maintaining of chondrogenic gene expression to solve dedifferentiation problem of chondrocyte. In this study enzymatically isolated chondrons were cultured for 7 days. Morphology of chondrons were assessed by microscopic examination. Chondrogenic gene expression of Sox9, aggrecan (AGG), cartilage oligomeric ma...
Response to the Removal of Extracellular Cartilage Matrix
Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunoflu- orescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin (CIG)) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with (3~S)methionine or (3H)glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface- associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear pre...
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.
Growth and differentiation factors for cartilage healing and repair
Injury, 2008
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Cartilage tissue enhances proteoglycan retention by nucleus pulposus cells in vitro
Arthritis & Rheumatism, 2010
Objective. To investigate the effect of cartilage on nucleus pulposus (NP) tissue in an in vitro model. Methods. Cells were isolated from bovine NP or articular cartilage and allowed to form tissue in vitro. The NP tissue was grown either alone or in the presence of cartilage tissue (coculture) for up to 4 weeks and examined for histologic appearance, gene expression, and biochemical composition. For selected experiments, NP tissue was grown in coculture with fragments of cartilage end-plate. Results. Coculture of in vitro-formed NP tissue with cartilage end-plate tissue resulted in a significant increase in proteoglycan content in the NP tissue by 2 weeks, compared with NP tissue grown alone. Substituting in vitro-formed cartilage tissue for cartilage endplate also had a positive effect on the NP tissue, suggesting that it was an appropriate substitute for cartilage end-plate. Coculture of NP with in vitroformed cartilage for 2 weeks increased aggrecan and collagen gene expression compared with that in NP tissue grown alone, and also reduced expression of matrix metalloproteinase 3 (MMP-3), MMP-13, and ADAMTS-5. NP cells from older and younger animals responded similarly to in vitro-formed cartilage. Expression of genes for tumor necrosis factor ␣ (TNF␣) and TACE in NP cells was higher when grown in the absence of cartilage. This corresponded with increased TNF␣ protein levels in the absence of cartilage. Conclusion. The data suggest that chondrocytes may secrete a factor(s) that positively enhances tissue growth, perhaps by inhibiting TNF␣ production. This could be a potential mechanism explaining how loss of the cartilage end-plate may contribute to the development of NP degenerative changes.