Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc. (1/47)

Tissue engineering of intervertebral discs (IVD) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. However, since there is no unique marker characteristic of discs and since hyaline cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. This study was performed to compare the proteoglycan to collagen ratio (measured as GAG to hydroxyproline ratio) in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals and test the hypothesis that this ratio can be used for in vivo studies to distinguish between a normal NP and hyaline cartilage phenotype. Whole human lumbar spine specimens from fresh cadavers, ranging in age from 12 weeks to 79 years, were used to harvest the IVDs and adjacent endplates. The GAG to hydroxyproline ratio within the NP of young adults is approximately 27:1, whereas the ratio within the hyaline cartilage endplate of the same aged individuals is about 2:1. The production of an extracellular matrix with a high proteoglycan to collagen ratio can be used in vivo to distinguish NP cells from chondrocytes, and could help in identifying a NP-like phenotype in vivo as opposed to a chondrocyte when MSCs are induced to differentiate for tissue engineering of a disc.  (+info)

Insulin-transferrin-selenium prevent human chondrocyte dedifferentiation and promote the formation of high quality tissue engineered human hyaline cartilage. (2/47)

This study was to investigate the effects of insulin-transferrin-selenium (ITS) on the proliferation and quantitative gene expression of adult human nasal septum chondrocytes in monolayer culture expansion and the formation of tissue engineered hyaline cartilage. Effects of ITS on human nasal septum chondrocytes monolayer culture expansion and gene expression were evaluated in various culture media either added with 2% fetal bovine serum (FBS) or 1 ng/mL basic fibroblast growth factor plus 1 ng/mL transforming growth factor or both serum and growth factors supplementation in comparison with medium added with 10%FBS. Chondrocytes cultured in medium added with 2% fetal bovine serum and growth factors either supplemented with or without ITS were then mixed with pluronic F-127 hydrogel for in vivo tissue engineered cartilage formation in nude mice model. Engineered tissues were removed after 8 weeks of implantation and evaluated with histological staining, immunohistochemistry, transmission electron microscopy and quantitative gene expression analysis. ITS promoted human chondrocytes proliferation and reduced chondrocytes dedifferentiation in media supplemented with serum and growth factors. ITS with 2% FBS and growth factors provided 15-fold increased in chondrocytes number by the end of the culture period compared to the standard culture medium used in chondrocytes culture (medium added with 10% FBS). Engineered tissue resulted from ITS supplementation demonstrated higher quality of cartilage formation. In conclusion, our study has demonstrated the benefits of ITS supplementation in human chondrocytes monolayer culture and tissue engineering cartilage formation.  (+info)

Expression of the matrix receptor CD44v5 on chondrocytes changes with osteoarthritis: an experimental investigation in the rabbit. (3/47)

OBJECTIVE: To evaluate the expression of CD44v5 on chondrocytes of hyaline cartilage during the course of osteoarthritis (OA). METHODS: In 12 white New Zealand rabbits the anterior cruciate ligament (ACL) was resected to create an anterior instability of the knee. In 12 control rabbits only a sham operation without resection of the ACL was done. Four animals of each group were killed at 3, 6, and 12 weeks. The loadbearing area was evaluated histologically according to Mankin and by immunostaining for CD44v5. RESULTS: In the trial group, histological grades of OA showed a positive linear correlation with the time after surgery. Immunostaining showed an increased expression of CD44v5 in the control group after 3 and 6 weeks, which dropped to normal after 12 weeks. There was no difference between control and trial groups after 3 and 6 weeks, but a difference was seen after 12 weeks. A significant positive correlation between CD44v5 expression and the histological grade of OA was found (r = 0.314). CONCLUSIONS: An in vivo increase of expression of the hyaluronan receptor CD44v5 occurs during the course of OA. Further studies are needed to evaluate whether this pattern applies to man and whether new treatment approaches might evolve from this knowledge.  (+info)

The structure, location, and function of perlecan, a prominent pericellular proteoglycan of fetal, postnatal, and mature hyaline cartilages. (4/47)

The aim of this study was to immunolocalize perlecan in human fetal, postnatal, and mature hyaline cartilages and to determine information on the structure and function of chondrocyte perlecan. Perlecan is a prominent component of human fetal (12-14 week) finger, toe, knee, and elbow cartilages; it was localized diffusely in the interterritorial extracellular matrix, densely in the pericellular matrix around chondrocytes, and to small blood vessels in the joint capsules and perichondrium. Aggrecan had a more intense distribution in the marginal regions of the joint rudiments and in para-articular structures. Perlecan also had a strong pericellular localization pattern in postnatal (2-7 month) and mature (55-64 year) femoral cartilages, whereas aggrecan had a prominent extracellular matrix distribution in these tissues. Western blotting identified multiple perlecan core protein species in extracts of the postnatal and mature cartilages, some of which were substituted with heparan sulfate and/or chondroitin sulfate and some were devoid of glycosaminoglycan substitution. Some perlecan core proteins were smaller than intact perlecan, suggesting that proteolytic processing or alternative splicing had occurred. Surface plasmon resonance and quartz crystal microbalance with dissipation experiments demonstrated that chondrocyte perlecan bound fibroblast growth factor (FGF)-1 and -9 less efficiently than endothelial cell perlecan. The latter perlecan supported the proliferation of Baf-32 cells transfected with FGFR3c equally well with FGF-1 and -9, whereas chondrocyte perlecan only supported Baf-32 cell proliferation with FGF-9. The function of perlecan therefore may not be universal but may vary with its cellular origin and presumably its structure.  (+info)

Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. (5/47)

OBJECTIVE: To determine whether it is possible to engineer 3-dimensional hyaline cartilage using mesenchymal stem cells derived from the bone marrow (BMSCs) of patients with osteoarthritis (OA). METHODS: Expanded BMSCs derived from patients with hip OA were seeded onto polyglycolic acid scaffolds and differentiated using transforming growth factor beta3 in the presence or absence of parathyroid hormone-related protein (PTHrP) to regulate hypertrophy. Micromass pellet cultures were established using the same cells for comparison. At the end of culture, the constructs or pellets were processed for messenger RNA (mRNA) analysis by quantitative real-time reverse transcription-polymerase chain reaction. Matrix proteins were analyzed using specific assays. RESULTS: Cartilage constructs engineered from BMSCs were at least 5 times the weight of equivalent pellet cultures. Histologic, mRNA, and biochemical analyses of the constructs showed extensive synthesis of proteoglycan and type II collagen but only low levels of type I collagen. The protein content was almost identical to that of cartilage engineered from bovine nasal chondrocytes. Analysis of type X collagen mRNA revealed a high level of mRNA in chondrogenic constructs compared with that in undifferentiated BMSCs, indicating an increased risk of hypertrophy in the tissue-engineered cells. However, the inclusion of PTHrP at a dose of 1 microM or 10 microM during the culture period resulted in significant suppression of type X collagen mRNA expression and a significant decrease in alkaline phosphatase activity, without any loss of the cartilage-specific matrix proteins. CONCLUSION: Three-dimensional hyaline cartilage can be engineered using BMSCs from patients with OA. This method could thus be used for the repair of cartilage lesions.  (+info)

Parathyroid hormone/parathyroid hormone-related peptide modulates growth of avian sternal cartilage via chondrocytic proliferation. (6/47)

Parathyroid hormone (PTH; 10(-7) to 10(-15) M) decreased terminal chondrogenesis in the avian sterna. During the first half of an 8-day culture, 100 nM PTH (1-34) significantly increased sternal length and downregulated the deposition of type X collagen and its mRNA expression. However, it remains unclear how PTH increased cartilaginous growth. In this study, we examined growth by both cell proliferation and analysis of cyclin d1 and collagen mRNA. Types II, IX, and X collagens and cyclin d1 mRNA were quantified through real-time RT-PCR, while Ki-67 was used as an immunohistochemical proliferation marker. Extracellular matrix content was measured through mRNA quantification of types II, IX, and X collagen and observing deposition of the same collagens. PTH significantly increased the proliferation marker Ki-67 in the sternal cephalic region. There was less type II and X collagen in PTH-treated sterna with concomitant decreases in mRNA production, suggesting that proliferation was the major contributor to cartilage growth in the presence of PTH/PTH-related peptide receptor activation. In conclusion, these experiments demonstrated that PTH increased cartilage growth by upregulating cell proliferation or other extracellular matrix components.  (+info)

Composite hyaluronate-type I collagen-fibrin scaffold in the therapy of osteochondral defects in miniature pigs. (7/47)

The potential of novel scaffold containing sodium hyaluronate, type I collagen, and fibrin was investigated in the regeneration of osteochondral defects in miniature pigs. Both autologous chondrocyte-seeded scaffolds and non-seeded scaffolds were implanted into two defects located in the non-weight-bearing zone of the femoral trochlea (defect A was located more distally and medially, defect B was located more proximally and laterally). Control defects were left untreated. Twelve weeks after the operation, the knees were evaluated in vivo using MRI. Six months after the implantation, the defects were analyzed using MRI, histological, and immunohistochemical analysis. In the A defects of chondrocyte-seeded scaffold group, hyaline cartilage and fibrocartilage was formed, containing type II collagen, acidic and neutral glycosaminoglycans while the non-seeded scaffold group was predominantly filled with fibrocartilage. Defects in the control group were predominantly filled with fibrous tissue. Histomorphometric analysis of photomicrographs revealed a significantly higher amount of hyaline cartilage in the cell-seeded scaffold group in A defects than in other groups. Both scaffold groups in A defects showed significantly less fibrous tissue than cell-seeded defects B and the control group. Both histological and MRI analysis proved that the novel composite scaffold has a potential to regenerate osteochondral defects within six months.  (+info)

Stress-induced signaling pathways in hyalin chondrocytes: inhibition by Avocado-Soybean Unsaponifiables (ASU). (8/47)

OBJECTIVE: Avocado-Soybean Unsaponifiables (ASU) represent one of the most commonly used drugs for symptomatic osteoarthritis (OA). The mechanisms of its activities are still poorly understood. We investigate here the effects of ASU on signaling pathways in mouse or human chondrocytes. METHODS: Mouse or human chondrocytes stimulated with interleukin-1beta (IL1beta, 10 ng/ml) and cartilage submitted to a compressive mechanical stress (MS) were studied in the presence or absence of ASU (10 microg/ml). Nuclear factor kappaB (NF-kappaB) activation was assessed by immunoblot, using an I-kappa B alpha antibody, nuclear translocation of NF-kappaB using p65 antibody, and extra-cellular signal-regulated kinase (ERK)1/2 activation using phospho and ERK1/2 antibodies. The binding of the p50/p65 complex on DNA was studied by electrophoretic mobility shift assay. RESULTS: ASU decrease matrix metalloproteinases-3 and -13 expressions and Prostaglandin E(2) (PGE(2)) release in our model. The degradation of I-kappa B alpha is prevented in the presence of ASU as shown by the persistent expression of I-kappa B alpha protein in the cytosol when chondrocytes are stimulated by IL1beta or MS. Nuclear translocation of the NF-kappaB complex is shown by the decrease of the p65 protein from the cytosol, whereas p65 appears in the nucleus under IL1beta stimulation. This translocation is abolished in the presence of ASU. Moreover, bandshift experiments show an inhibition of the IL1beta-induced binding of p50/p65 complexes to NF-kappaB responsive elements in response to ASU. Finally, among the different mitogen-activated protein kinases known to be induced by IL1beta, ERK1/2 was the sole kinase inhibited by ASU. CONCLUSION: These results demonstrate that ASU express a unique range of activities, which could counteract deleterious processes involved in OA, such as inflammation.  (+info)