A comparative dose-response study of cartilage-derived morphogenetic protein (CDMP)-1, -2 and -3 for tendon healing in rats.

Cartilage-derived morphogenetic proteins (CDMPs), belonging to the bone morphogenetic protein (BMP) family, are known to be cartilage and bone inducers as well as to induce tendon and ligament-like tissue. In this study we investigated the influence of CDMP-1, -2 or -3 at four different doses (0, 0.4, 2 and 10 microg) on tendon healing in a rat model, as well as differences in osteogenesis between the different CDMPs and doses.In 110 rats, a 3 mm segment of the Achilles tendon was removed via a 2 mm skin incision. CDMP-1, -2 or -3 was injected into the defect 6 h postoperative. The rats were killed 8 days after operation. The tendon regenerates were tested biomechanically. There was a significant dose-related increase in strength and stiffness with all three CDMPs, but no difference between the CDMPs was found.Another 50 rats were used to compare the highest dose of the CDMPs with controls and osteogenic protein 1 (OP-1), as regards cartilage or bone formation after 4 weeks. Cartilage occurred in all groups, including the controls. Some specimens in all groups contained bone, except the controls. No difference between the CDMPs could be demonstrated. The CDMP-1, CDMP-3 and OP-1 groups contained significantly more calcium than controls. Only the CDMP-2 group and the controls contained significantly less calcium than the OP-1 group.In conclusion, the three CDMPs appeared similar as regards improvement of tendon repair and osteogenicity in this setting.     

Synergistic effect of IGF-1 and OP-1 on matrix formation by normal and OA chondrocytes cultured in alginate beads

OBJECTIVE: Growth factor therapy may be useful for stimulation of cartilage matrix synthesis and repair. Thus, the purpose of our study was to further understand the effect of combined insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1) treatment on the matrix synthesized by human adult normal and osteoarthritic (OA) chondrocytes. DESIGN: Chondrocytes were isolated post-mortem from articular cartilage from tali of normal human donors and femoral condyles of OA patients undergoing knee replacement surgery. Cells were cultured in alginate beads for 21 days in four experimental groups: (1) "mini-ITS" control; (2) 100 ng/ml IGF-1; (3) 100 ng/ml OP-1; (4) IGF-1+OP-1, each at 100 ng/ml. Beads were processed for histological (Safranin O and fast green), morphometrical and immunohistochemical (aggrecan, decorin, type I, II, VI, and X collagens, and fibronectin accumulation) analyses. RESULTS: Histology showed that IGF-1 alone did not induce substantial matrix production. OP-1 alone caused a considerable matrix formation, but the highest matrix accumulation by normal and OA chondrocytes was found when OP-1 and IGF-1 were added together. Morphometrical analysis indicated larger matrices produced by OA chondrocytes than by normal cells under the combined treatment. All tested matrix proteins were more abundant in the combination group. Type X collagen was detected only under the combined OP-1 and IGF-1 treatment and was present at very low levels. Type I collagen was found only in OA chondrocytes. CONCLUSIONS: The results obtained in the current study suggest that combined therapy with IGF-1 and OP-1 may have a greater potential in treating cartilage defects seen in OA than use of either growth factor alone.     

CDMP-2 induces bone or tendon-like tissue depending on mechanical stimulation.

Cartilage derived morphogenetic proteins (CDMPs, also known as growth and differentiation factors, GDFs) are a subgroup of the bone morphogenetic protein (BMP) gene family. As most BMPs, they are known to induce cartilage or bone formation when implanted subcutaneously or intramuscularly on an appropriate carrier. However, similar implantation experiments with CDMPs have also reported the formation of a tendon-like tissue, without any cartilage or bone. A solution to this apparent contradiction might be offered by the mechanical tissue differentiation theory, suggesting that tissue differentiation depends on the mechanical environment. This study analyzes the response to CDMP-2 implants at different sites and under different loading conditions in the rat. Collagen sponges carrying CDMP-2 were implanted subcutaneously, intramuscularly or inside a freshly created defect in the achilles tendon. Large amounts of bone were induced subcutaneously, smaller amounts intramuscularly, and in the tendons, only small amounts of bone or cartilage were seen in few animals. Thus, the amount of bone appeared inversely related to the degree of mechanical stimulus. To confirm this, CDMP was also injected into tendon defects that were either loaded or partially unloaded. All the unloaded tendons showed bone induction after one CDMP-2 injection, whereas only 4 of 10 loaded ones showed any cartilage or bone (p = 0.0005). Single injections of a similar dose of CDMP-2 have previously been shown to augment tendon repair by increasing the size of the tendon callus. This study suggests that the response to CDMP-2 is dependent on the mechanical situation at the site where it is applied.     

Activation of annexin II and V expression, terminal differentiation, mineralization and apoptosis in human osteoarthritic cartilage

OBJECTIVE: To test the hypothesis that terminal differentiation of chondrocytes in human osteoarthritic cartilage might lead to the failure of repair mechanisms and might cause progressive loss of structure and function of articular cartilage. DESIGN: Markers for terminally differentiated chondrocytes, such as alkaline phosphatase, annexin II, annexin V and type X collagen, were detected by immunohistochemical analysis of human normal and osteoarthritic knee cartilage from medial and lateral femoral condyles. Apoptosis in these specimens was detected using the TUNEL labeling. Mineralization and matrix vesicles were detected by alizarin red S staining and electron microscopic analysis. RESULTS: Alkaline phosphatase, annexin II, annexin V and type X collagen were expressed by chondrocytes in the upper zone of early stage and late stage human osteoarthritic cartilage. However, these proteins, which are typically expressed in hypertrophic and calcifying growth plate cartilage, were not detectable in the upper, middle and deep zones of healthy human articular cartilage. TUNEL labeling of normal and osteoarthritic human cartilage sections provided evidence that chondrocytes in the upper zone of late stage osteoarthritic cartilage undergo apoptotic changes. In addition, mineral deposits were detected in the upper zone of late stage osteoarthritic cartilage. Needle-like mineral crystals were often associated with matrix vesicles in these areas, as seen in calcifying growth plate cartilage. CONCLUSION: Human osteoarthritic chondrocytes adjacent to the joint space undergo terminal differentiation, release alkaline phosphatase-, annexin II- and annexin V-containing matrix vesicles, which initiate mineral formation, and eventually die by apoptosis. Thus, these cells resume phenotypic changes similar to terminal differentiation of chondrocytes in growth plate cartilage culminating in the destruction of articular cartilage in osteoarthritis.     

Altered electrophysiological responses to mechanical stimulation and abnormal signalling through alpha5beta1 integrin in chondrocytes from osteoarthritic cartilage

OBJECTIVE: To establish whether chondrocytes from normal and osteoarthritic human articular cartilage recognize and respond to pressure induced mechanical strain in a similar manner.DESIGN: Chondrocytes, extracted from macroscopically normal and osteoarthritic human articular cartilage obtained from knee joints at autopsy, were grown in monolayer culture and subjected to cyclical pressure-induced strain (PIS) in the absence or presence of anti-integrin antibodies, agents known to block ion channels and inhibitors of key molecules involved in the integrin-associated signalling pathways. The response of the cells to mechanical stimulation was assessed by measuring changes in membrane potential. RESULTS: Unlike chondrocytes from normal articular cartilage, which showed a membrane hyperpolarization response to PIS, chondrocytes from osteoarthritic cartilage responded by membrane depolarization. The mechanotransduction pathway involves alpha5beta1 integrins, stretch-activated ion channels, tyrosine kinases and phospholipase C but the actin cytoskeleton and protein kinase C, which are important in the membrane hyperpolarization response in normal chondrocytes, are not necessary for membrane depolarization in osteoarthritic chondrocytes in response to PIS. CONCLUSION: Chondrocytes derived from osteoarthritic cartilage show a different signalling pathway via alpha5beta1 integrin in response to mechanical stimulation which may be of importance in the production of phenotypic changes recognized to be present in diseased cartilage.     

Activation and localization of cartilage-derived morphogenetic protein-1 at the site of ossification of the ligamentum flavum

Localization and expression of cartilage-derived morphogenetic protein (CDMP)-1 in tissues at the site of ossification of the ligamentum flavum (OLF) were examined by immunohistochemistry and in situ hybridization. The CDMP-1 protein and messenger ribonucleic acid (mRNA) were localized in spindle-shaped cells and chondrocytes in the OLF tissues. CDMP-1 was not detected in cells in non-ossified sites. These data indicate that CDMP-1 is locally activated and localized in spindle-shaped cells and chondrocytes at the site of OLE. Given the previously reported promoting action of CDMP-1 for chondrogenesis, the current results suggest that CDMP-1 may be involved in the progression of OLF, leading to the narrowing of spinal canal and thus causing severe clinical manifestations.     

OP-1/BMP-7 in cartilage repair

Three years ago we published a book chapter on the role of bone morphogenetic proteins (BMPs) in cartilage repair. Since that time our understanding of the function of osteogenic protein-1 (OP-1) or BMP-7 in cartilage homeostasis and repair has substantially improved and therefore we decided to devote a current review solely to this BMP. Here we summarise the information accumulated on OP-1 from in vitro and ex vivo studies with cartilage cells and tissues as well as from in vivo studies of cartilage repair in various animal models. The primary focus is on articular chondrocytes and cartilage, but data will also be presented on nonarticular cartilage, particularly from the intervertebral disc. The data show that OP-1 is a unique growth factor which, unlike other members of the same BMP family, exhibits in addition to its strong pro-anabolic activity very prominent anti-catabolic properties. Animal studies have demonstrated that OP-1 has the ability to repair cartilage in vivo in various models of articular cartilage degradation, including focal osteochondral and chondral defects and osteoarthritis, as well as models of degeneration in intervertebral disc cartilage. Together our findings indicate a significant promise for OP-1 as therapeutic in cartilage repair.     

Alterations in endogenous osteogenic protein-1 with degeneration of human articular cartilage.

A synchronized balance between synthesis and breakdown of extracellular matrix (ECM) molecules in normal articular cartilage is disturbed in osteoarthritis (OA). The focus of our study is the anabolic factor, osteogenic protein-1 (OP-1) that is expressed in articular cartilage and is able to induce the synthesis of ECM components. The major aim was to investigate both qualitatively and quantitatively endogenous OP-1 in normal, degenerative, and OA cartilage. Normal and degenerative cartilage was obtained at autopsies from femoral condyles of human organ donors with no documented history of joint disease; OA cartilage was obtained from patients undergoing joint arthroplasty. Appearance of donor cartilage was evaluated by Collins scale, where normal cartilage is assigned grades 0-1, and degenerated cartilage is assigned grades 2-4. OP-1 mRNA expression was assessed by RT-PCR; OP-1 protein (pro- and active forms) was qualitatively analyzed by Western blotting and quantified by OP-1 ELISA. The highest levels of OP-1 expression (mRNA and protein) were detected in normal cartilage of grade 0. The concentration of OP-1 protein was about 50 ng per gram cartilage dry weight. With the progression of cartilage degeneration (increased Collins grades and OA) OP-1 protein was down-regulated up to 9-fold. These changes affected primarily the active form of OP-1. OP-1 message also declined in cartilages with the increase of degenerative changes. In conclusion, an overall decrease in endogenous OP-1 in degenerated and OA tissue suggests that OP-1 could be one of the factors responsible for normal homeostasis and matrix integrity in cartilage.     

Experimental study on histological changes of articular cartilage of femoral head under nonweight-bearing conditions]

Histological changes of articular cartilage of the femoral head in the nonweight-bearing conditions were investigated using 10-week-old Wistar rats whose hind limbs had been unilaterally transected. At 2 weeks after limb transection, decreased DNA synthesis in chondrocytes and depletion of glycosaminoglycan in the cartilage matrix in the intermediate zone of the articular cartilage were observed. After 8 weeks, articular cartilage thickness decreased significantly, and at 16 weeks the number of chondrocytes diminished in comparison with those of a weight-bearing femoral head. At 20 weeks, chondrocyte death and reduced width of articular cartilage became evident. No osteoarthritic changes, however, were observed under nonweight-bearing conditions. These findings suggest that weight-bearing is very important in maintaining of both the structure and metabolism of the articular cartilage.     

软骨源形态发生蛋白对残耳软骨细胞体外增殖的实验研究

目的研究残耳软骨细胞在体外扩增时生物学特性的变化，以及软骨源形态发生蛋白（cartilage-derived morphogenetic protein-1，CDMP-1）对残耳软骨细胞生长分化的影响。方法在体外培养残耳软骨细胞，通过Ⅱ型胶原免疫组织化学染色和甲苯胺兰染色方法，研究CDMP-1对残耳软骨细胞分化的影响；通过MTT法在酶标仪上测定CDMP-1对残耳软骨细胞增殖的影响；使用流式细胞仪测定CDMP-1对残耳软骨细胞周期的影响。结果残耳软骨细胞在体外培养时随代数增加呈异染性的糖胺多糖减少，加入CDMP-1实验组Ⅱ型胶原阳性的比例明显高于对照组（P〈0．05），并且当CDMP-1的浓度为100ng／ml时促细胞增殖效应最大。结论CDMP-1对体外培养的残耳软骨细胞有促进增殖和维持细胞表型的作用。     

Expression of bone morphogenetic proteins, receptors, and tissue inhibitors in human fetal, adult, and osteoarthritic articular cartilage.

Coordinate expression of BMPs and their receptors and inhibitors is likely necessary for physiologic BMP regulation and activity. To characterize the expression of such factors in fetal, normal adult, and end-stage osteoarthritic articular cartilage, samples from these sources were analyzed. PCR-amplified sequences (BMPs 1-11), receptors (IA, IB, II), TGF-beta1, TGF-beta2, inhibitors noggin and follistatin, CDMP-1, COMP, and GAPDH from cDNAs generated from extracted total RNA were resolved by gel electrophoresis. Protein levels of BMPs 3, 7, and 8 were also analyzed by SDS-PAGE and Western blotting. RT-PCR revealed that BMPs 1, 2, 4-6, and 11, BMPR-IA and II, noggin, follistatin, CDMP-1, COMP, and GAPDH mRNAs were expressed in similar fashion in both fetal and adult (normal or osteoarthritic) cartilage. BMPs 9 and 10 mRNAs were not expressed in either group. BMPs 7, 8, and BMPR-IB mRNAs were consistently expressed in fetal but not in adult cartilage. BMP-3 mRNA was expressed in fetal and normal adult, but not in osteoarthritic samples. TGF-beta1 was expressed in both adult normal and osteoarthritic, but not fetal, samples. Similarly, Western blotting demonstrated BMPs 7 and 8 to be present in fetal but not in adult samples. BMP-3 protein was present in fetal and adult normal samples, to a lesser extent, but absent in osteoarthritic cartilage.     

Homeostasis of the extracellular matrix of normal and osteoarthritic human articular cartilage chondrocytes in vitro

OBJECTIVE: In normal articular cartilage cells, the IGFRI/insulin-like growth factor 1 (IGF-1) autocrine pathway was shown to overrule the catabolic effects of the IL-1/IL-1RI pathway by up-regulation of the IL-1RII decoy receptor. The activity of the IGF-1/IGFR1 and IL-1/IL-1R pathways, and of the IL-1RII control mechanism in the synthesis and turnover of the extracellular matrix (ECM) by chondrocytes from normal and osteoarthritic (OA) articular cartilage was compared in order to identify possible therapeutic targets of this disease. METHODS: Phenotypically stable human articular cartilage cells were obtained from normal and OA cartilage of the same knee showing focal OA. The cells were cultured in alginate beads over 1 week to re-establish the intracellular cytokine and growth factors, to reexpress the respective plasma membrane receptors and to reach equilibrium in accumulated cell-associated matrix (CAM) compounds. Following liberation of the cells from the alginate beads, the levels of cell-associated matrix (CAM) aggrecan, type II collagen and fibronectin, of intracellular IGF-1, IL-1alpha and beta and of their respective plasma membrane-bound receptors, IGFR1, IL-1RI and the decoy receptor IL-1RII, were assayed using flow cytometry. RESULTS: Coordinated production and accumulation of CAM aggrecan and type II collagen under the effect of the IGFR1/IGF-1 autocrine pathway-as documented for chondrocytes from healthy controls-was absent when the chondrocytes had been obtained from OA joints. When compared with cells obtained from normal tissues, chondrocytes from fibrillated OA cartilage expressed significantly higher intracellular IGF-1 levels and plasma membrane-bound IGFR1. At the same time, significantly higher intracellular IL-1alpha and beta levels and upregulated plasma membrane-bound IL-1RI were observed. Plasma membrane-bound IL-1RII decoy receptor was downregulated in OA chondrocytes. The levels of CAM aggrecan, type II collagen and fibronectin were significantly reduced in the chondrocytes obtained from pathological tissue. CONCLUSION: Paired analysis of normal and OA chondrocytes from the same knee joint has shown an enhanced capacity of chondrocytes from OA cartilage to produce ECM macromolecules. However, the same cells have increased catabolic signalling pathways. As a consequence of this increased IL-1 activity and the reduced amounts of IL-1RII decoy receptor, less of the produced ECM macromolecules may persist in the CAM of the OA chondrocytes.     

AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes

OBJECTIVE: AG-041R, a novel indolin-2-one derivative, has recently been demonstrated to induce systemic hyaline cartilage hyperplasia in rats. The aim of this study was to characterize its anabolic actions on chondrocytes. DESIGN: Chondrocytes were isolated from knee joints of 5-week-old SD rats. Effects of AG-041R on cartilage matrix synthesis were examined by measuring [(35)S]sulfate incorporation into proteoglycans, Alcian blue staining, and Northern blotting of cartilage matrix genes. ALP activity, mineral deposition and the expression of markers for hypertrophic chondrocytes, were assessed for terminal differentiation of chondrocytes. Roles of endogenous TGF-beta/BMPs and MEK1/Erk signaling in the action of AG-041R were investigated using the neutralizing soluble receptors and the MEK1 inhibitor. RESULTS: AG-041R accelerated proteoglycan synthesis assessed by both [(35)S]sulfate incorporation and Alcian blue stainable extracellular matrix accumulation. It also up-regulated the gene expression of type II collagen and aggrecan, as well as tenascin, a marker for articular cartilage. In contrast, AG-041R suppressed ALP activity, mineralization, and the gene expression of type X collagen and Cbfa1, indicating that AG-041R prevents chondrocyte terminal differentiation. AG-041R increased in BMP-2 mRNA, and the neutralizing soluble receptor for BMPs reversed the stimulatory effects of AG-041R on cartilage matrix synthesis. Moreover, AG-041R activated MEK1/Erk pathway, which was revealed to prevent chondrocyte terminal differentiation. CONCLUSION: AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes, which is mediated at least in part by endogenous BMPs and Erk. The data demonstrates that AG-041R has a potential to be a useful therapeutic agent for articular cartilage disorders.     

Stimulation of proteoglycan production by glucosamine sulfate in chondrocytes isolated from human osteoarthritic articular cartilage in vitro

OBJECTIVE: This study investigated the in-vitro effects of a crystalline glucosamine sulfate (GS) preparation on DNA synthesis and on proteoglycan (PG) and type II collagen (coll II) production by human articular chondrocytes isolated from human osteoarthritic articular cartilage in a 3-dimensional culture system for 4, 8, and 12 days. MATERIALS AND METHODS: Human articular chondrocytes from osteoarthritic femoral heads were isolated from their matrix by collagenase digestion and then cultured in suspension. Under constant agitation, cells aggregated and formed a cluster within a few days. The effects of GS (1-100 micrograms/ml) on chondrocytes were determined by quantifying DNA synthesis (by measurement of [3H]-thymidine uptake) as well as PG and coll II production using radiommunoassays (RIAs) specific for coll II and to human human cartilage PG. Cross-reaction with GS in the RIAs was not detected. Moreover, PG size distribution was determined by exclusion chromatography under associative conditions to determine the association of PG monomers with hyaluronic acid (HA) to form large molecular weight PG aggregates. RESULTS: Under the above conditions, PG production in culture media and chondrocyte clusters was increased by GS (10-100 micrograms/ml). DNA synthesis and coll II production were not modified by GS. In addition, GS did not modify the physico-chemical form of PG produced by cells during culture. CONCLUSIONS: Glucosamine sulfate did not affect DNA synthesis nor coll II production but caused a statistically significant stimulation of PG production by chondrocytes from human osteoarthritic cartilage cultured for up to 12 days in 3-dimensional cultures.     

Expression of NG2/human melanoma proteoglycan in human adult articular chondrocytes

OBJECTIVE: NG2 is a transmembrane chondroitin sulfate (CS) rich proteoglycan originally identified in rats. It has recently been shown to be identical to human melanoma proteoglycan (HMPG). In rats NG2 has a limited distribution in adult tissues, being expressed predominantly by neuronal and glial cells whereas during development it is also expressed in developing mesenchyme including cartilage. NG2/HMPG has putative roles in interactions between glial and melanoma cells with extracellular matrix (ECM) molecules. This study was undertaken to assess whether NG2/HMPG was expressed by normal and osteoarthritic human articular chondrocytes. DESIGN: Cryostat sections of human fetal knee joints and normal and osteoarthritic articular cartilage were immunostained with antibodies against rat NG2 (N143.8) and HMPG (M28B5, 9.2.27). Immunoprecipitation and Western blotting was carried out on protein extracts of chondrocytes from normal and osteoarthritic cartilage. Immunofluorescence of NG2 and potential ligands was carried out in vitro on cells from normal and osteoarthritic cartilage. RESULTS: Fetal and both normal and osteoarthritic adult cartilage showed strong immunoreactivity for NG2/HMPG. Western blotting showed a smeared component of molecular weight greater than 400 kDa and a faint band at 250 kDa which became predominant upon digestion with chondroitinase ABC. Immunofluorescence of chondrocytes in vitro showed NG2 to be distributed in a punctate pattern without co-localization of actin or several ECM proteins including fibronectin and type VI collagen. CONCLUSION: NG2/HMPG is expressed by human fetal and adult chondrocytes and in adult articular chondrocytes the core protein is chondroitin sulfated. The function of this molecular in human articular cartilage remains to be defined.