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Osteogenic protein-1 and its receptors in human articular cartilage
Abstract
Cartilage regeneration and repair is one of the major obstacles to treat arthritic diseases. The members of the bone morphogenetic protein (BMP) family of the transforming growth factor-β (TGFβ) superfamily received a high degree of attention among factors potentially capable of inducing and promoting anabolic processes in articular cartilage [1, 2]. BMPs were first isolated from demineralized bone matrix and shown to induce ectopic bone formation when implanted subcutaneously [3, 4]. The BMP family currently consists of more than 47 related molecules identified in different species [5,6]. Although BMPs were initially found in bone matrix, it has been shown that they are present in most tissues and organs. They are involved in numerous physiological and pathological processes: differentiation, embryogenesis, morphogenesis, skeletal patterning, development, tissue regeneration, organogenesis, apoptosis, etc.
... Osteogenic protein 1 (OP-1), a member of the bone morphogenetic protein (BMP) family, is expressed by human adult articular chondrocytes and plays a crucial role in the maintenance of cartilage matrix integrity and the promotion of repair processes [8,9]. OP-1 has a potent anabolic effect on articular cartilage and other connective tissues: it stimulates the synthesis of major cartilage matrix components [10-12], it promotes matrix assembly [13], and it serves as an antagonist to the deleterious effects of catabolic mediators [14-16] without inducing chondrocyte hypertrophy and proliferation [10,11]. OP-1 gene expression and protein expression have been detected in all of the connective tissues of the joint – cartilage, meniscus, synovium, ligament, and tendon [17] – and there appears to be a negative correlation between autocrine OP-1 production and degenerative articular processes [18,19]. ...
The measurement of body fluid levels of biochemical markers in joint tissues has begun to provide clinically useful information. Synovial fluid (SF) plays an important role in articular joint lubrication, nutrition, and metabolism of cartilage and other connective tissues within the joint. The purpose of our study was to identify and characterize osteogenic protein 1 (OP-1) in SF from patients with rheumatoid arthritis (RA) or with osteoarthritis (OA) and to correlate levels of OP-1 with those of hyaluronan (HA) and antigenic keratan sulfate (AgKS). SF was aspirated from the knees of patients with either RA or OA and from the knees of asymptomatic organ donors with no documented history of joint disease. The presence of detectable OP-1 in SF was demonstrated by western blots with specific anti-pro-OP-1 and anti-mature OP-1 antibodies. Measurement of levels of OP-1, HA and AgKS was performed using ELISAs. OP-1 was identified in human SF in two forms, pro-OP-1 and active (mature) OP-1--mature OP-1 being detected only in SF from OA patients and RA patients. Levels of OP-1 and HA were higher in RA patients than in OA patients and asymptomatic donors, while the level of AgKS was highest in SF from asymptomatic donors. Statistically significant differences were found between SF levels of OP-1 in RA and OA patients and between SF levels of AgKS among the three groups tested. The SF content of OP-1 tended to correlate positively with HA levels, but negatively with AgKS concentrations. In conclusion, the results of this study suggest that measurement of OP-1 in joint fluid may have value in the clinical evaluation of joint disease processes.
Cartilage regeneration and repair is one of the major obstacles in current orthopedics. The importance is enormous since osteoarthritis (OA) is a major cause of disability among the adult population in the United States. OA is considered a process of attempted, but gradually failing, repair of damaged cartilage extracellular matrix, as the balance between synthesis and breakdown of matrix components is disturbed and shifted towards catabolism. In recent times, members of the bone morphogenetic proteins (BMP) family of proteins have received the highest attention among potential anabolic factors for cartilage repair because of their ability to induce matrix synthesis and promote repair in different connective tissues, including cartilage. This Chapter will review the information accumulated on BMPs from in vitro studies as well as from studies of repair in various animal models. The data show significant promise for BMPs in cartilage repair and suggest that this indication could become the most important application for BMPs in orthopedics.
Cartilage repair and regeneration is a major obstacle in orthopedic medicine. The importance is enormous since osteoarthritis
(OA) is a major cause of disability among the adult population in the United States and degenerative disc disease (DDD) is
responsible for a significant amount of the chronic back pain. OA is considered a process of attempted, but gradually failing,
repair of damaged cartilage extracellular matrix, as the balance between synthesis and breakdown of matrix components is disturbed
and shifted towards catabolism. In recent times, members of the bone morphogenetic protein (BMP) family of proteins have demonstrated
a great potential as anabolic factors for cartilage repair because of their ability to induce matrix synthesis and promote
repair in cartilage.
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.
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.
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.
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.
Amino acid sequences of two tryptic peptides derived from enriched bovine osteogenic protein preparations revealed considerable homology to two members of the TGF-beta (transforming growth factor beta) supergene family, DPP (decapentaplegic protein) of Drosophila and Vg-1 (vegetal protein) of Xenopus. Building upon this information we constructed a synthetic consensus gene to use as a probe to screen human genomic libraries. This resulted in the isolation of three interrelated genes. Among these were BMP-2b and BMP-3 which have recently been described by others. The third gene, termed OP-1 (osteogenic protein one), is new and was subsequently shown to encode the human homolog of a major component of bovine osteogenic protein. The genomic clones were used to isolate the corresponding complementary DNA (cDNA) clones. Sequence analysis indicates that OP-1 is a relative of the murine Vgr-1 (Vg-1 related gene). This report describes the cDNA structure and putative amino acid sequence of OP-1.
The modulation of interleukin 1 (IL-1) effects on proteoglycan metabolism in intact murine patellar cartilage by transforming growth factor beta (TGF-beta) was investigated in vitro and in vivo. In vitro TGF-beta (400 pmol/l) had no effect on basal proteoglycan degradation. Proteoglycan degradation induced by IL-1, however, was suppressed by TGF-beta in serum free medium alone and in medium supplemented with 0.5 micrograms/ml insulin-like growth factor 1. This suggests a specific regulatory role for TGF-beta under pathological conditions. In contrast with the suppression of breakdown, synthesis of proteoglycans was stimulated by TGF-beta for both basal and IL-1 suppressed proteoglycan synthesis in cultures without insulin-like growth factor. In the presence of insulin-like growth factor no extra effect of TGF-beta on proteoglycan synthesis was observed. With insulin-like growth factor, however, TGF-beta potentiated the ex vivo recovery of IL-1 induced suppression of proteoglycan synthesis. Analogous to the in vitro effects, TGF-beta injected intraarticularly suppressed IL-1 induced proteoglycan degradation. Furthermore, TGF-beta injected into the joint counteracted IL-1 induced suppression of proteoglycan synthesis. This indicates that in vivo also TGF-beta can ameliorate the deleterious effects of IL-1 on the cartilage matrix.
The definitive mammalian kidney forms as the result of reciprocal interactions between the ureteric bud epithelium and metanephric mesenchyme. As osteogenic protein 1 (OP-1/bone morphogenetic protein 7), a member of the TGF-beta superfamily of proteins, is expressed predominantly in the kidney, we examined its involvement during metanephric induction and kidney differentiation. We found that OP-1 mRNA is expressed in the ureteric bud epithelium before mesenchymal condensation and is subsequently seen in the condensing mesenchyme and during glomerulogenesis. Mouse kidney metanephric rudiments cultured without ureteric bud epithelium failed to undergo mesenchymal condensation and further epithelialization, while exogenously added recombinant OP-1 was able to substitute for ureteric bud epithelium in restoring the induction of metanephric mesenchyme. This OP-1-induced nephrogenic mesenchyme differentiation follows a developmental pattern similar to that observed in the presence of the spinal cord, a metanephric inducer. Blocking OP-1 activity using either neutralizing antibodies or antisense oligonucleotides in mouse embryonic day 11.5 mesenchyme, cultured in the presence of metanephric inducers or in intact embryonic day 11.5 kidney rudiment, greatly reduced metanephric differentiation. These results demonstrate that OP-1 is required for metanephric mesenchyme differentiation and plays a functional role during kidney development.
Members of the Bone Morphogenetic Protein (BMP) family exhibit overlapping and dynamic expression patterns throughout embryogenesis. However, little is known about the upstream regulators of these important signaling molecules. There is some evidence that BMP signaling may be autoregulative as demonstrated for BMP4 during tooth development. Analysis of BMP7 expression during kidney development, in conjunction with studies analyzing the effect of recombinant BMP7 on isolated kidney mesenchyme, suggest that a similar mechanism may operate for BMP7. We have generated a beta-gal-expressing reporter allele at the BMP7 locus to closely monitor expression of BMP7 during embryonic kidney development. In contrast to other studies, our analysis of BMP7/lacZ homozygous mutant embryos, shows that BMP7 expression is not subject to autoregulation in any tissue. In addition, we have used this reporter allele to analyze the expression of BMP7 in response to several known survival factors (EGF, bFGF) and inducers of metanephric mesenchyme, including the ureteric bud, spinal cord and LiCl. These studies show that treatment of isolated mesenchyme with EGF or bFGF allows survival of the mesenchyme but neither factor is sufficient to maintain BMP7 expression in this population of cells. Rather, BMP7 expression in the mesenchyme is contingent on an inductive signal. Thus, the reporter allele provides a convenient marker for the induced mesenchyme. Interestingly LiCl has been shown to activate the Wnt signaling pathway, suggesting that BMP7 expression in the mesenchyme is regulated by a Wnt signal. Treatment of whole kidneys with sodium chlorate to disrupt proteoglycan synthesis results in the loss of BMP7 expression in the mesenchyme whereas expression in the epithelial components of the kidney are unaffected. Heterologous recombinations of ureteric bud with either limb or lung mesenchyme demonstrate that expression of BMP7 is maintained in this epithelial structure. Taken together, these data indicate that BMP7 expression in the epithelial components of the kidney is not dependent on cell-cell or cell-ECM interactions with the metanephric mesenchyme. By contrast, BMP7 expression in the metanephric mesenchyme is dependent on proteoglycans and possibly Wnt signaling.
This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms. (J Histochem Cytochem 48:239-250, 2000)
Cartilage matrix degradation generates collagen type II fragments. The objective of this study is to explore the possibility that these collagen fragments may be part of an endogenous metabolic feedback. Initially, collagen fragments were extracted from normal or osteoarthritic cartilage, as part of a matrix fragment preparation. Later, collagen fragments were generated by digestion of bovine collagen type II with bacterial collagenase (col2f). These fragments were added to cultures of isolated chondrocytes (bovine and human) and cartilage explants (human). In a dose-dependent manner, col2f caused inhibition of cell attachment to collagen, inhibition of collagen synthesis, and induction of matrix degradation. In addition, when col2f were added to human cartilage explants, an induction of gelatinase activity was detected in the media. These data sets present first evidence that degradation products of collagen may be directly involved in the regulation of cartilage homeostasis.
Osteogenic proteins, also referred to as BMPs, are a family of bone matrix polypeptides isolated from a variety of mammalian species. These proteins are members of the transforming growth factor-β superfamily of molecules that contain a highly conserved 7 cysteine transforming growth factor-β domain in their C-termini. Use of recombinantly produced human osteogenic protein-1, also referred to as BMP-7, implanted in conjunction with bovine bone derived Type 1 collagen or various non-proteinaceous biodegradable carriers into surgically created large diaphyseal segmental defects in animals leads to the regeneration of new bone that is fully functional biologically and biomechanically. Further study has shown that osteogenic protein-1 can be used as a bone graft substitute to promote spinal fusion and to aid in the incorporation of metal implants. Finally, osteogenic protein-1 shows promise as an agent for repair of osteochondral defects.
Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone
formation. The critical components of this extract, termed bone morphogenetic protein (BMP), that direct cartilage and bone
formation as well as the constitutive elements supplied by the animal during this process have long remained unclear. Amino
acid sequence has been derived from a highly purified preparation of BMP from bovine bone. Now, human complementary DNA clones
corresponding to three polypeptides present in this BMP preparation have been isolated, and expression of the recombinant
human proteins have been obtained. Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing
the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-beta supergene
family, while the third, BMP-1, appears to be a novel regulatory molecule.
Objective. Recombinant human osteogenic protein 1 (OP-1) is an effective stimulator of human cartilage 35S-proteoglycan synthesis. The present study was conducted to determine whether stimulation of human articular chondrocytes with OP-1 can help overcome interleukin-1β (IL-1β)-induced suppression of 35S-proteoglycan synthesis.
Methods. Human articular chondrocytes in alginate beads were maintained for 3 days in the absence (control) or presence of IL-1β at 0.1–100 pg/ml with or without OP-1 at 50 ng/ml, in medium containing 10% fetal bovine serum (FBS). Incorporation of 35S-sulfate into proteoglycans was quantified during the last 4 hours of culture and reported as counts per minute per μg DNA. Release of interleukin-1 receptor antagonist (IL-1Ra) and prostaglandin E2 into the medium was monitored by immunoassay.
Results. IL-1β at 10 pg/ml caused a 60% decrease in 35S-proteoglycan synthesis. This could be blocked by including 500 ng/ml IL-1Ra in the medium. The presence of 50 ng/ml OP-1 in the IL-1β-containing medium was effective in restoring 35S-proteoglycan synthesis to the level of that found in cultures not treated with IL-1β. The restorative effects of OP-1 and IL-1Ra were cumulative. The rate of release of prostaglandin E2 and IL-1Ra into the medium was not affected by the presence of OP-1.
Conclusion. Treatment of human articular chondrocytes with OP-1 cultured in the presence of FBS is effective in overcoming the down-regulation of proteoglycan synthesis induced by low doses of IL-1β.
Objective
To determine the effects of osteogenic protein 1 (OP-1) on hyaluronan (HA), CD44, and aggrecan biosynthesis as well as the contribution of these molecules in promoting matrix assembly by human articular chondrocytes.Methods
Normal human chondrocytes were cultured with or without OP-1 treatment. Changes in the relative expression of messenger RNA (mRNA) for HA synthases 2 and 3 (HAS-2 and HAS-3), CD44, and aggrecan were determined by competitive quantitative reverse transcriptase–polymerase chain reaction. Accumulation of HA was characterized by indirect staining, CD44 by flow cytometry, and aggrecan biosynthesis by 35SO4 incorporation.ResultsOP-1 stimulated the expression of HAS-2, CD44, and aggrecan mRNA in a time-dependent manner, resulting in increased expression of HA, CD44, and aggrecan. Prominent increases in HA-rich cell-associated matrices were also observed.ConclusionOP-1 stimulates not only the synthesis of matrix macromolecules such as aggrecan, but also the synthesis of other molecules required for matrix retention, namely, HA and CD44.
Objective . To study the effects of recombinant human osteogenic protein‐1 (rHuOP‐1; bone morphogenetic protein‐7) on proteoglycan and collagen synthesis by human articular chondrocytes.
Methods . Articular chondrocytes from fetal, adolescent, and adult human donors were cultured in alginate beads for 4 days in a mixture of Ham's F‐12, Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS), then for an additional 3‐10 days in the presence and absence of rHuOP‐1, with and without FBS. Chondrocyte synthetic activity was measured as the amount of incorporation of ³⁵ S‐sulfate into proteoglycans and ³ H‐proline into hydroxyproline. Sieve chromatography and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis were performed to identify specific proteoglycans and collagens.
Results . Recombinant human OP‐1 markedly stimulated the synthesis of proteoglycans (mostly aggrecan) and collagens (predominantly type II) by all chondrocyte preparations. This did not require the presence of FBS and was associated with continued expression of the chondrocyte phenotype.
Conclusion . Recombinant human OP‐1 is a more potent stimulator of the synthesis of cartilage‐specific molecules by human articular chondrocytes than are other factors tested for comparison, including TGFβ1 and activin A.
Morphogenesis is the developmental cascade of pattern formation and body plan establishment, culminating in the adult form. It has formed the basis for the emerging discipline of tissue engineering, which uses principles of molecular developmental biology and morphogenesis gleaned through studies on inductive signals, responding stem cells, and the extracellular matrix to design and construct spare parts that restore function to the human body. Among the many organs in the body, bone has considerable powers for regeneration and is a prototype model for tissue engineering. Implantation of demineralized bone matrix into subcutaneous sites results in local bone induction. This model mimics sequential limb morphogenesis and has permitted the isolation of bone morphogens, such as bone morphogenetic proteins (BMPs), from demineralized adult bone matrix. BMPs initiate, promote, and maintain chondrogenesis and osteogenesis, but are also involved in the morphogenesis of organs other than bone. The symbiosis of the mechanisms underlying bone induction and differentiation is critical for tissue engineering and is governed by both biomechanics (physical forces) and context (microenvironment/extracellular matrix), which can be duplicated by biomimetic biomaterials such as collagens, hydroxyapatite, proteoglycans, and cell adhesion glycoproteins, including fibronectins and laminin. Rules of tissue architecture elucidated in bone morphogenesis may provide insights into tissue engineering and be universally applicable for all organs/tissues, including bones and joints.
A bone morphogenetic protein (BMP) obtained in solution by digestion of demineralized rabbit cortical bone matrix with bacterial collagenase retains its biologically active conformation in a neutral salt/ethylene glycol mixture. BMP may be insolubilized by coprecipitation with calcium phosphate and resolubilized by chemical extraction with a neutral salt in the same solvent mixture. Upon concanavalin A-Sepharose chromatography, BMP is bound by hydrophobic interaction and carbohydrate recognition and is recovered by elution with either alpha-methyl mannoside or ethylene glycol solvent mixture. Implants of both eluates and the extracts of the coprecipitate in double-walled diffusion chambers induce transmembrane bone morphogenesis. BMP is not species specific; rabbit BMP induces new bone formation in the rat. The present observations indicate that BMP is a glycoprotein.
Subcutaneous implantation of demineralized diaphyseal bone matrix in allogeneic rats results in the local induction of endochondral bone differentiation. We have explored the potential of three dissociative extractants, 4 M guanidine hydrochloride (Gdn . HCl), 8 M urea/1 M NaCl, and 1% NaDodSO4 at pH 7.4, containing protease inhibitors to solubilize putative inductive molecules in the bone matrix. Extraction of bone matrix with any one of these extracts resulted in the loss of the bone inductive property. The solubilized extracts were then reconstituted with the residue by dialysis against water. The various reconstituted matrices were bioassayed for bone inductive potential by quantitation of alkaline phosphatase activity and 45Ca incorporation on day 12 after implantation. There was complete recovery of biological activity after reconstitution of the residues with each of the three extracts. Polyacrylamide gel electrophoresis of the extracts revealed similar protein profiles. Gel filtration of the 4 M Gdn. HCl extract on Sepharose CL-4B showed a heterogeneous broad peak. When fractions of that peak containing proteins less than 50,000 daltons were reconstituted with inactive 4 M Gdn . HCl-treated bone matrix and then implanted, new bone was induced. These observations demonstrate the dissociative extraction and successful biological reconstitution of bone inductive macromolecules in demineralized bone matrix.
Osteogenic proteins, also referred to as BMPs, are a family of bone matrix polypeptides isolated from a variety of mammalian species. These proteins are members of the transforming growth factor-beta superfamily of molecules that contain a highly conserved 7 cysteine transforming growth factor-beta domain in their C-termini. Use of recombinantly produced human osteogenic protein-1, also referred to as BMP-7, implanted in conjunction with bovine bone derived Type 1 collagen or various non-proteinaceous biodegradable carriers into surgically created large diaphyseal segmental defects in animals leads to the regeneration of new bone that is fully functional biologically and biomechanically. Further study has shown that osteogenic protein-1 can be used as a bone graft substitute to promote spinal fusion and to aid in the incorporation of metal implants. Finally, osteogenic protein-1 shows promise as an agent for repair of osteochondral defects.
A recurrent theme in embryonic development and tissue regeneration is that cells communicate with each other using just a handful of conserved families of signaling molecules. One of the largest of these multifunctional families is that of the bone morphogenetic proteins (BMPs), with >20 members identified in organisms ranging from sea urchin to mammals (Fig. II. The name BMP was first given to three proteins purified hom a demineralized bovine bone preparation that induced ectopic cartilage and endochondral bone when implanted in experimental animals (Wozney et al. 1988). One of these proteins (BMP1) was a putative protease of the astacin family, whereas the other two (BMP2 and BMP3) were related to human transforming growth factor beta (TGF beta). It is now clear that the name BMP is misleading because there is strong generic and experimental evidence that these molecules regulate biological processes as diverse as cell proliferation, apoptosis, differentiation, cell-fate determination, and morphogenesis. Moreover, the vertebrate BMPs are involved in the development of nearly all organs and tissues, including the nervous system, somites, lung, kidney, skin, and gonads, as well as in critical steps in the establishment of the basic embryonic body plan. This review focuses on recent studies related to BMP function in vertebrates, particularly those involving mutations of BMP-encoding genes in mice. Additional information about the larger TGF beta superfamily can be found in other reviews (Kingsley 1994a,b, McPherron and Lee 1996). The complexity of the numerous trans-membrane serine-threonine kinases that act as BMP signaling receptors precludes full discussion here, but again, a number of excellent reviews are available, in addition to those cited above (Massague et al. 1994, Massague and Weis-Garcia 1996).
Bone morphogenetic proteins have been shown to increase matrix synthesis by articular chondrocytes in short-term cultures. Members of this family of proteins have also been shown to induce endochondral ossification in vivo. The present study was performed to determine if the addition of human recombinant bone morphogenetic protein-2 to a long-term monolayer articular chondrocyte cell culture system affected the ability of the chondrocytes to divide in vitro, whether the cytokine altered expression of the articular chondrocyte phenotype and synthesis of matrix proteoglycans, and whether the cytokine was capable of inducing differentiation to a hypertrophic chondrocyte. Human recombinant bone morphogenetic protein-2 did not alter cell proliferation. It caused 3.5-6.2 times more proteoglycan synthesis by articular chondrocytes during each of the time points tested after 4 days in culture. Total proteoglycan accumulation in the extracellular matrix after 28 days in culture was 6.7 times as great in the treated cultures as in the control. Treatment with human recombinant bone morphogenetic protein-2 maintained the articular chondrocyte phenotype of cells in culture as demonstrated by Northern blot analysis: the expression of type-I collagen genes was increased and that of type-II collagen and aggrecan mRNA was lost in untreated chondrocyte cultures after 14-21 days in culture. In contrast, exposure to 100 ng/ml human recombinant bone morphogenetic protein-2 maintained expression of type-II collagen and increased expression of aggrecan compared with controls during the 28-day culture period. Northern blot analysis of the expression of type-X collagen and osteocalcin by chondrocytes treated with human recombinant bone morphogenetic protein-2 showed a lack of expression of these genes, indicating no alteration in phenotype. These experiments demonstrated the ability of human recombinant bone morphogenetic protein-2 to promote the articular chondrocyte phenotype and matrix synthesis in long-term culture. Characteristics of cell growth were not affected, and the cytokine did not induce differentiation to a hypertrophic chondrocyte.
A severe consequence of rheumatoid arthritis is depletion of proteoglycans (PGs) from articular cartilage leading to functional impairment of this tissue. We investigated whether local administration of anabolic factors (transforming growth factors-beta1 and -beta2 [TGF-beta1 and -beta2, respectively] and bone morphogenetic protein-2 (BMP-2) into joints could stimulate cartilage repair during arthritis. A unilateral arthritis was induced in mice by intra-articular injection of zymosan. Starting on Day 4 after the induction of arthritis, three injections of TGF-beta1 (200 ng) were given (Days 4, 6, and 8). On Day 11, articular cartilage PG synthesis was measured by 35S-sulfate incorporation, and histologic knee joint sections were prepared, which were used to analyze cartilage PG content by quantification of safranin O staining. Additionally, histologic sections were used to analyze inflammation and chondrophyte-formation. Local administration of TGF-beta1 did not modify inflammation but clearly stimulated PG synthesis and restored PG content of depleted cartilage. TGF-beta2 appeared to be as potent as TGF-beta1 in the stimulation of cartilage repair, and both TGF-beta isoforms also stimulated the formation of chondrophytes in this rodent model. In contrast to TGF-beta, three intra-articular injections with 200 ng BMP-2 did not stimulate the repair process. In summary, this study demonstrates for the first time that local administration of TGF-beta into arthritic joints stimulates the replenishment of PGs in depleted cartilage.
The related molecules bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta-1 (TGF-beta 1) have both been shown to stimulate chondrocyte proteoglycan (PG) synthesis in vitro. We investigated the in-vivo effects of these factors on articular cartilage PG metabolism.
Several doses of BMP-2 or TGF-beta 1 were injected into the murine knee joint, once or repeatedly. Patellar cartilage PG synthesis was measured by [35S]-sulfate incorporation and reverse transcriptase polymerase chain reaction (RT-PCR). PG content was analyzed by measuring safranin O staining intensity on histologic sections.
A single injection of 200 ng BMP-2 induced a much earlier and more impressive stimulation of articular cartilage PG synthesis, than 200 ng TGF-beta 1. RT-PCR revealed that both factors upregulated mRNA of aggrecan more than that of biglycan and decorin. However, 21 days after a single injection of 200 ng TGF-beta 1 PG synthesis still was significantly increased, while stimulation by BMP-2 only lasted for 3 to 4 days. Stimulation by BMP-2 could be prolonged to at least 2 weeks by triple injections of 200 ng each, at alternate days. Remarkably, even after this intense exposure to BMP-2, stimulation of PG synthesis was not reflected in long-lasting enhancement of PG content of articular cartilage. In contrast, even a single injection with 200 ng of TGF-beta 1 induced prolonged enhancement of PG content. After repeated injections, both BMP-2 and TGF-beta 1 induced chondrogenesis at specific sites. 'Chondrophytes' induced by BMP-2 were found predominantly in the region where the growth plates meet the joint space, while those triggered by TGF-beta 1 originated from the periosteum also at sites remote from the growth plates.
BMP-2 and TGF-beta stimulate PG synthesis and PG content with different kinetics, and these factors have different chondro-inductive properties.
The abilities of osteogenic protein-1 (OP-1) and TGF-beta1 to affect cartilage damage caused by fibronectin fragments (Fn-fs) that are known to greatly enhance cartilage proteoglycan (PG) degradation were compared.
Articular cartilage was obtained from 18 month old bovines.
To test blocking of damage, cartilage was cultured with or without OP-1 or TGF-beta in the presence of 100 nM Fn-fs. To test restoration of PG, cartilage was first cultured with Fn-fs and the cartilage then treated with factors.
Cartilage PG content was measured in papain digests using the dimethylmethylene blue assay. PG synthesis was measured by incorporation of 35S labeled sulfate.
OP-1 blocked damage and restored PG in damaged cartilage, apparently due to enhanced PG synthesis. However, TGF-beta1 alone decreased PG content.
These results clearly demonstrate differences between OP-1 and TGF-beta1, both members of the TGF-beta superfamily and illustrate the efficacy of OP- in blocking Fn-f mediated damage.
The bone morphogenetic proteins (BMPs) are a family of pleiotropic morphogens isolated and cloned from the demineralized extracellular matrix of bone. BMPs and related cartilage-derived morphogenetic proteins (CDMPs) initiate, promote and maintain bone and cartilage. The pleiotropic effects of BMPs are based on concentration-dependent thresholds. Targeted disruption of gene action by homologous recombination has demonstrated the role of BMP 7 in kidney, eye and skeletal development. BMP 7 is critical for kidney tubulogenesis, retinal pigmented epithelium differentiation and skeletal pattern. BMP 7 is also synthesized by the kidney and is detectable in serum; hence BMP 7 is both an autocrine and endocrine morphogen. It is likely renal BMP 7 may influence skeletal development and growth in children although there may be sources of other BMPs with skeletogenic actions. In conclusion, we are beginning to unravel the mysteries of kidney-bone connection with special reference to pediatric nephrology.
Structure, function, and orthopedic applications of osteogenic protein (OP-1)
- T K Sampath
- D C Rueger
- TK Sampath
BMP-7 prevents tubulointerstitial nephritis associated with chronic renal disease
- K A Hruska
- KA Hruska
BMPs - brain morphogenetic proteins - potential therapeutic utility in treating neurological damage and disease
- P L Kaplan
- PL Kaplan
BMP receptors specific for OP-1 are identified in human normal, degenerative and OA cartilage
- S Chubinskaya
- C Merrihew
- R Mikhail
- P Ten Dijke
- D Rueger
- Ke Kuettner
Increased matrix synthesis and matrix gene expression
- C Hidaka
- M Quitoriano
- E Attia
- J Hannafin
- R Warren