JBJS | Stimulation of Proteoglycan Synthesis in Explants of Porcine Articular Cartilage by Recombinant Osteogenic Protein-1 (Bone Morphogenetic Protein-7)*

The Journal of Bone & Joint Surgery, Volume 79, Issue 8

Articles | August 01, 1997

Stimulation of Proteoglycan Synthesis in Explants of Porcine Articular Cartilage by Recombinant Osteogenic Protein-1 (Bone Morphogenetic Protein-7)*

STEVEN A. LIETMAN, M.D.†, BALTIMORE; MASAKI YANAGISHITA, M.D.‡, BETHESDA; T. K. SAMPATH, PH.D.§; A. HARI REDDI, PH.D.†, BALTIMORE, MARYLAND

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Investigation performed at the Laboratory of Musculoskeletal Cell Biology, Department of Orthopaedic Surgery, The Johns Hopkins University School of Medicine, Baltimore, and The National Institute of Dental Research, The National Institutes of Health, Bethesda

The Journal of Bone & Joint Surgery. 1997; 79:1132-7

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Abstract

Osteogenic protein-1 (also known as bone morphogenetic protein-7) is a member of the bone morphogenetic protein family. Bone morphogenetic proteins and related members of the TGF-ß (transforming growth factor-ß) superfamily are involved in the development and repair of bone. Recombinant bone morphogenetic proteins induce the formation of new cartilage and bone at heterotopic sites. We investigated the influence of recombinant osteogenic protein-1 (at doses of three, ten, thirty, or 100 nanograms per milliliter) on the synthesis and release of proteoglycans and the maintenance of a steady-state concentration of proteoglycans in explants of porcine articular cartilage that were maintained in chemically defined serum-free medium. We found a dose-dependent stimulation of proteoglycan synthesis and a concurrent decrease in the rate of release of proteoglycans from the explants. The size of the proteoglycan monomers and the composition of the glycosaminoglycan chains in the untreated articular cartilage were similar to those in the articular cartilage treated with osteogenic protein-1. The capacity of the newly synthesized proteoglycan monomers to form aggregates with exogenous hyaluronic acid was found to be similar to that of proteoglycans in bovine nasal cartilage. Our results demonstrated that osteogenic protein-1 stimulated the synthesis of proteoglycans and diminished the release of proteoglycans from explants of porcine articular cartilage.CLINICAL RELEVANCE: The maintenance and repair of articular cartilage is a formidable challenge in clinical orthopaedics. The stimulation of proteoglycan synthesis by osteogenic protein-1 (bone morphogenetic protein-7) in explants of cartilage maintained in chemically defined serum-free medium implies that recombinant osteogenic protein-1 may play a role in the maintenance of a steady-state concentration of proteoglycans in articular cartilage, a desirable prerequisite for optimum repair of cartilage. Osteogenic protein-1 can initiate the formation of cartilage from mesenchymal cells. Once new cartilage has formed at the site of repair, osteogenic protein-1 also may maintain the synthesis of proteoglycans.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

Bone morphogenetic proteins are a group of proteins found in demineralized bone matrix that have the capacity to induce the formation of cartilage and bone at heterotopic sites^21,29. Bone morphogenetic proteins are members of the TGF-ß (transforming growth factor-ß) superfamily and share seven highly conserved cysteines in the carboxyl terminal domain^21,33. More than a dozen members of the bone morphogenetic protein family have been identified²¹. There is increasing evidence that bone morphogenetic proteins may have a regulatory role in the initiation of the differentiation of cartilage and bone-forming cells from pluripotent mesenchymal stem cells^{2,4,6,9,19,20,23,27}. The synthesis of proteoglycans in explants of bovine articular cartilage has been shown to be increased by bone morphogenetic proteins 3 and 4 and by certain growth factors^2,13,14,24. In humans, osteogenic protein-1 is localized to embryonic hypertrophic chondrocytes³⁰ and perichondrium. Proteoglycans represent a major component of articular cartilage^17,18 and are responsible for the resistance of such cartilage to compressive forces. The compressive modulus (a measure of compressive stiffness) of articular cartilage is correlated with the proteoglycan content^1,3,18,22. In fact, when proteoglycans are removed from cartilage, there is a tenfold decrease in the compressive modulus¹⁸. A similar loss of proteoglycans from articular cartilage is discernible in association with the degenerative changes of osteoarthrosis^10,15,16. It may be possible to reverse some of these degenerative changes by increasing the synthesis of proteoglycans by articular chondrocytes¹⁸.

*One or more of the authors has received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article. In addition, benefits have been or will be directed to a research fund, foundation, educational institution, or other non-profit organization with which one or more of the authors is associated. Funds were received in total or partial support of the research or clinical study presented in this article. The funding source was Creative BioMolecules, Hopkinton, Massachusetts.

†Department of Orthopaedic Surgery, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 225, Baltimore, Maryland 21205.

‡The National Institute of Dental Research, The National Institutes of Health, Bethesda, Maryland 20892.

§Creative BioMolecules, 35 South Street, Hopkinton, Massachusetts 01748.

†Department of Orthopaedic Surgery, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 225, Baltimore, Maryland 21205.

‡The National Institute of Dental Research, The National Institutes of Health, Bethesda, Maryland 20892.

§Creative BioMolecules, 35 South Street, Hopkinton, Massachusetts 01748.

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Fig. 1 Graph demonstrating the effect of osteogenic protein-1 (OP-1) on the release of proteoglycans from explants of articular cartilage. Two sets of triplicate cultures of explants were labeled with ³⁵SO4 and were maintained in basal medium alone, in medium with osteogenic protein-1 (100 nanograms per milliliter), or in medium with 20 per cent fetal bovine serum (FBS). The release of ³⁵SO4 was determined daily, and the amount of ³⁵SO4 that was left in the tissue was calculated. The I-bars represent the standard error of the mean. An asterisk indicates a significant difference (p < 0.05) from the result for the explants that had been maintained in basal medium alone.

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Fig. 2 Graph demonstrating the effect of osteogenic protein-1 (OP-1) on proteoglycan synthesis in explants of articular cartilage. Six cultures of explants were maintained in basal medium or were treated with different concentrations of osteogenic protein-1 for three, seven, or ten days. The tissues were labeled with ³⁵SO4, and the effect of osteogenic protein-1 was expressed as the activity of ³⁵SO4 per microgram of hydroxyproline (Hyp). The hydroxyproline content of the explants that had been maintained in basal medium alone was similar to that of the explants that had been treated with osteogenic protein-1. The I-bars represent the standard error of the mean. An asterisk indicates a significant difference (p < 0.05) from the result for the explants that had been maintained in basal medium alone.

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Fig. 3 Graphs demonstrating the effect of osteogenic protein-1 (OP-1) on the size of proteoglycan monomers in explants of articular cartilage. Triplicate cultures of explants were maintained in basal medium alone or were treated with 100 nanograms of osteogenic protein-1 for three, seven, or ten days. The explants were labeled with ³⁵SO4, and the proteoglycan monomers were extracted with four-molar guanidine hydrochloride and were analyzed on Sephacryl S-500 columns (Pharmacia LKB Biotechnology). Treatment with osteogenic protein-1 had no marked effect on the size of proteoglycan monomers (aggrecan molecules). dpm = disintegrations per minute, and Kd = kilodaltons.

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Fig. 4 Graphs demonstrating the effect of osteogenic protein-1 on the elution profiles of glycosaminoglycans that were released in response to the treatment of proteoglycans with one-molar sodium borohydride (NaB). Explants were maintained for ten days in the absence or presence of 100 nanograms of osteogenic protein-1, and the glycosaminoglycan chains were analyzed with chromatography on a Superose 6 column (Pharmacia LKB Biotechnology). The glycosaminoglycan chains from the explants that had been treated with osteogenic protein-1 (B) were slightly larger than those from the explants that had been maintained in basal medium alone (A). Treatment with chondroitinase AC (chAC), chondroitinase ABC (chABC), or chondroitinase ABC with keratanase (chABC/ker) revealed no apparent differences in the sizes of the oligosaccharides that formed in the absence (C, E, and G) or presence (D, F, and H) of osteogenic protein-1. dpm = disintegrations per minute, and Kd = kilodaltons.

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Explant Cultures

The hindlimbs of eight-week-old male pigs were obtained immediately after death. The limbs were sterilized, and all procedures were performed under sterile conditions. The distal end of the femur was exposed, and the articular cartilage was dissected in one large piece from the underlying bone. The articular cartilage was soaked in phosphate-buffered saline solution (consisting of 0.15-molar NaCl, three-millimolar KCl, ten-millimolar Na₂HPO₄, and two-millimolar KH₂PO₄, pH 7.4) containing penicillin (0.05 milligram per milliliter), streptomycin (0.05 milligram per milliliter), and neomycin (0.1 milligram per milliliter). Explants were obtained from this large piece of cartilage with use of a biopsy punch; each explant was two millimeters in diameter and two to three millimeters thick, and each included all layers of articular cartilage. The explants were washed two times in the same phosphate-buffered saline solution containing antibiotics and then were placed in twenty-four-well cell-culture plates, with three explants per well. One milliliter of basal medium (Dulbecco modified Eagle medium and Ham F-12 medium [1:1] with 0.2 per cent bovine serum albumin, penicillin [0.05 milligram per milliliter], streptomycin [0.05 milligram per milliliter], and neomycin [0.1 milligram per milliliter]) was added to each well. The cultures were maintained at 37 degrees Celsius in 95 per cent air and 5 per cent carbon dioxide^12,14,24. The hydroxyproline content of the explants was determined with the chloramine-T method^26,31.

Treatment

The explants were kept in serum-free basal medium for a five-day period of equilibration. The medium was changed daily. The explants then were maintained either in basal medium alone or in basal medium with recombinant osteogenic protein-1 at a dose of three, ten, thirty, or 100 nanograms per milliliter. The medium was changed daily, and osteogenic protein-1 was added every day.

Proteoglycan Synthesis

Proteoglycan synthesis was determined as has been previously described^8,12,13,23. Briefly, on days 0, 3, 7, and 10, the explants were labeled with medium containing Na₂³⁵SO₄ (forty microcuries [1480 kilobecquerels] per milliliter) for four hours. The radioactive medium was removed, and the explants were washed with ice-cold buffer (ten-millimolar EDTA and 0.1-molar sodium phosphate, pH 6.5) and were digested in the twenty-four-well plates with proteinase K (one milligram per milliliter) for twenty hours at 37 degrees Celsius. No pieces of cartilage were visible at the end of this interval. Next, 100-microliter samples were subjected to chromatography on Sephadex G-25 (PD-10) columns (Pharmacia LKB Biotechnology, Piscataway, New Jersey) in four-molar guanidine hydrochloride to remove any unincorporated ³⁵SO₄. The radioactivity in the macromolecular fraction was determined with a scintillation counter. The rates of incorporation then were normalized according to the hydroxyproline content of each sample^{4,5,26,31,34,35}.

Proteoglycan Release

As the steady-state concentration of proteoglycans is the balance of synthesis and degradative release, we sought to determine the influence of osteogenic protein-1 on the release of proteoglycans. The explants were obtained and were kept in basal medium for a five-day period of equilibration, as already described. On the sixth day, the explants were labeled with ³⁵SO₄ (forty microcuries [1480 kilobecquerels] per milliliter) for four hours. Next, the explants were washed two times in basal medium and were kept in basal medium for an additional two days to remove any unincorporated radioisotope. The explants then were maintained in basal medium alone, in medium with osteogenic protein-1 (100 nanograms per milliliter), or in medium with 20 per cent fetal bovine serum. The media were collected daily and were stored separately. After seven days of treatment, the explants were digested with proteinase K. The amounts of radiolabeled proteoglycans in the media that were collected daily and in the final digest were determined as already described, and a release curve was derived from these data; this curve demonstrated the amount of incorporated radioisotope remaining in each explant as a function of the time in culture^{14,24,32,34,35}.

Analysis of the Size of Proteoglycan Monomers and the Capacity to Form Aggregates with Hyaluronic Acid

We compared the sizes of the intact proteoglycan monomers (aggrecan molecules) in explants that had been treated with and without osteogenic protein-1. After the explants had been labeled and washed, individual explants were extracted with four-molar guanidine hydrochloride with protease inhibitors (five-millimolar benzamidine hydrochloride, ten-millimolar EDTA, ten-millimolar N-ethylmaleimide, and one-millimolar phenylmethylsulfonyl fluoride) for twenty hours. Sephadex G-25 columns then were used to remove unincorporated radioisotope. Next, 250-microliter samples of macromolecular proteoglycan were analyzed on one by thirty-centimeter columns of Sephacryl S-500 (Pharmacia LKB Biotechnology) and were eluted with a solution of four-molar guanidine hydrochloride, 0.5 per cent Triton X-100, and 0.1-molar sodium acetate (pH 5.8). The radioactivity was determined in each fraction^5,34,35. The capacity of radiolabeled proteoglycan monomers to form aggregates with hyaluronic acid under associative conditions was examined as described previously⁷.

Analysis of the Size and Composition of Glycosaminoglycan Chains

One hundred microliters of the ³⁵SO₄-labeled macromolecular proteoglycan from each of the Sephadex G-25 columns was used to evaluate the size and composition of the glycosaminoglycan chains. The proteoglycan fractions were dialyzed extensively against water, dried with use of a Speed Vac concentrator (Savant Instruments, Farmington, New York), and dissolved overnight in a solution of one-molar sodium borohydride and fifty-millimolar sodium hydroxide at 45 degrees Celsius to cleave glycosaminoglycan chains from the protein core remaining after digestion with proteinase K. The samples were neutralized with acetic acid on ice. Portions of the samples then were subjected to chromatography on a Superose 6 column (Pharmacia LKB Biotechnology), either directly or after digestion with specific glycosaminoglycan-degrading enzymes (chondroitinase ABC, chondroitinase AC, and chondroitinase ABC with keratanase), and were eluted with a solution of four-molar guanidine hydrochloride, 0.5 per cent Triton X-100, and 0.1-molar sodium acetate (pH 5.8). The column had been calibrated with glycosaminoglycan standards of known molecular weights^5,34,35. Fractions were collected at one-minute intervals, and the radioactivity was determined.

Analysis of chondroitin sulfate disaccharides: The treatment of proteoglycans with one-molar sodium borohydride cleaved the glycosaminoglycan chains from their protein core, as already mentioned. Samples of the glycosaminoglycan chains were digested with chondroitinase ABC, a chondroitin sulfate-degrading enzyme. The resultant digests then were analyzed on a high-performance liquid chromatography column (PA-03-5; YMC Yamamura Chemical Laboratories, Kyoto, Japan) in order to separate chondroitin 6-sulfate from chondroitin 4-sulfate²⁸.

Results

Introduction | Materials and Methods | Results | Discussion | References

Effect of Osteogenic Protein-1 on Proteoglycan Synthesis

A comparison of the effects of the different doses of osteogenic protein-1 on proteoglycan synthesis revealed time and dose-dependent responses (Fig. 1). There was a significant increase (p < 0.05) in proteoglycan synthesis on day 7 in explants that had been treated with thirty or 100 nanograms of osteogenic protein-1 compared with explants that had been maintained in basal medium alone. There also was a significant increase (p < 0.05) in proteoglycan synthesis on day 10 in explants that had been treated with ten, thirty, or 100 nanograms of osteogenic protein-1 compared with explants that had been maintained in basal medium alone (Fig. 1).

Effect of Osteogenic Protein-1 on Proteoglycan Release

A significant decrease (p < 0.05) in the rate of release of proteoglycans was observed on days 3, 4, 5, and 7 when the explants that had been treated with osteogenic protein-1 were compared with those that had been maintained in basal medium alone (Fig. 2). Fetal bovine serum, which contains various growth factors that have been shown to decrease proteoglycan catabolism^13,24, was used as a positive control. The half-life of radiolabeled proteoglycans was determined to be 4.3 days in explants maintained in basal medium alone, 8.1 days in explants treated with 20 per cent fetal bovine serum, and 6.8 days in explants treated with osteogenic protein-1 (100 nanograms per milliliter). These findings indicate that treatment with osteogenic protein-1 was associated with an inhibition of degradation.

Analysis of Proteoglycans

Size: The hydrodynamic size of the intact proteoglycan monomers that were analyzed with Sephacryl S-500 did not change substantially in response to osteogenic protein-1, regardless of the dose or the duration of treatment (Fig. 3). The size of the glycosaminoglycan chains that were released as a result of treatment of the proteoglycans with one-molar sodium borohydride also did not change in response to osteogenic protein-1 (Fig. 4). The glycosaminoglycan chains from the explants that had been treated with osteogenic protein-1 were slightly larger than those from the explants that had been maintained in basal medium alone (Fig. 4).

Composition: Chondroitin sulfate accounted for more than 95 per cent of the glycosaminoglycans in all samples; there were smaller amounts (less than 5 per cent) of keratan sulfate and no detectable amounts of dermatan sulfate or heparan sulfate. The relative amounts of chondroitin 4-sulfate and chondroitin 6-sulfate did not change in response to different doses of osteogenic protein-1 at day 10.

Capacity to form aggregates with hyaluronic acid: One of the functional hallmarks of the cartilage proteoglycan, aggrecan, is its capacity to aggregate with hyaluronic acid. Therefore, we examined the capacity of radiolabeled proteoglycans to form proteoglycan aggregates. Radiolabeled proteoglycans extracted from explants treated with and without osteogenic protein-1 were able to form aggregates in the presence of excess amounts of unlabeled proteoglycan and hyaluronic acid. The capacity of the newly synthesized proteoglycan monomers to form aggregates with exogenous hyaluronic acid was found to be similar to that of proteoglycans in bovine nasal cartilage.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

Osteogenic protein-1 increased the synthesis of proteoglycans by 70 to 120 per cent after seven to ten days in culture without changing the size or composition of the glycosaminoglycan chains. Osteogenic protein-1 also decreased the metabolic release of proteoglycans from the explants of articular cartilage. Thus, there was a net increase in the proteoglycan content of explants that had been treated with osteogenic protein-1. The hydrodynamic size of the intact proteoglycan monomers did not appear to be altered by treatment with osteogenic protein-1. The finding that osteogenic protein-1 stimulated chondrocytes to produce proteoglycans normally seen in articular cartilage suggests that osteogenic protein-1 may have a role in the treatment of the decrease in proteoglycans that is observed in association with diseases such as osteoarthrosis. Additionally, the proteoglycan monomers in the explants that were treated with osteogenic protein-1 did not differ from those in the explants that were maintained in basal medium alone with regard to their ability to aggregate with hyaluronic acid. This finding is another indication that the increased proteoglycan synthesis in response to osteogenic protein-1 yielded normal, functional proteoglycans⁸. Thus, as with bone morphogenetic proteins 3 and 4, osteogenic protein-1 is able to increase the synthesis of functional proteoglycans^2,13,14,24.

Previous work with purified native and recombinant bone morphogenetic proteins has firmly established that these morphogens initiate the formation of cartilage in vivo by a process similar to the endochondral ossification that occurs in the growth plate^21,29 and during fracture repair. Bone morphogenetic proteins also can maintain proteoglycan synthesis in cultures of explants. It is noteworthy that, although TFG-ß1 and IGF-1 (insulin-like growth factor-1) can maintain proteoglycan synthesis in cultures of explants¹³, they cannot initiate the formation of cartilage at heterotopic sites²¹. However, osteogenic protein-1 may be used both to initiate the formation of cartilage and to maintain or stimulate the synthesis of proteoglycans in cartilage explants. It is well known that, during repair of articular cartilage, there is recruitment of mesenchymal cells into the site of the defect before differentiation of cartilage^11,25,31. The findings of the present investigation support the concept that the proteoglycan synthesis in newly formed cartilage might be maintained by osteogenic protein-1 and perhaps also by other bone morphogenetic proteins^13,14. Future studies are planned to evaluate further the relevance of the present in vitro data to the in vivo repair of damaged cartilage.

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