Purpose: To compare synovial fluid (SF) biomarker levels in samples from patients with rheumatoid (RA), osteoarthritis (OA), and normal organ donors for prognostic/diagnostic purposes. Methods: Synovial fluid (SF) was collected from the knees of 45 (6 males and 39 females) OA, 22 (14 females and 8 males) RA patients and 20 (15 females and 5 males) normal organ donors. Eight biomarkers analyzed by ELISA methods were evaluated: interleukin (IL)-1, Il-6, Il-8, Il-11, leukemia inflammatory factor (LIF), cartilage oligomeric protein (COMP), osteocalcin, and osteogenic protein-1 (OP-1). Multivariate analysis assessed the effects of gender and disease activity: WOMAC scores for OA samples, and SF WBC, ESR, CRP for RA samples. Multivariant KruskalWallis and Mann-Whitney Tests were used; p< 0.05 was considered significant. Results: The mean (±SD) age was: 53±9 years for OA, 54±11 for RA, and 52±7 for donors. No gender differences were identified between markers. In RA SF, the levels of 4 out of 5 tested cytokines/chemokines were higher than in OA and normal SF. The most significant differences were found for Il-6 and Il-8, where Il-6 concentration was 2.5-fold higher than in OA, 2587.59±6393.69 vs. 1045.68±5451.13 og/ml, respectively (p< 0.005), and 8-fold higher than in normal, 2587.59±6393.69 vs. 339.06±906.28, respectively (P< 0.002). The levels of Il-8 were 8-fold higher than in OA, 6490.04±16550.74 vs 849.48 og/ml, respectively (P< 0.0005) and normal, 6490.04±16550.74 vs 788.69±837.705 og/ml, respectively (P< 0.028). The differences for Il-11 were not as substantial, especially between RA and OA samples, while it was a 5-fold difference between OA and normal SF, 654.11±1411.844 og/ml, respectively (P< 0.003). The value of Il-1 was significantly higher in normal than OA, 9.81±2.72 vs 8.56±7.06 og/ml, respectively (P< 0.004); in the RA, it was a tendency for a higher concentration of Il-1 than in OA and normal, but it did not reach significance. Surprisingly, concentration of LIF was higher in normal samples than in RA (P< 0.044) and OA (P< 0.001). The highest levels of OP-1 were found in normal SF (541.92±28.61 ng/ml), which were almost 5-fold higher than in OA (112.40±124.64) (P< 0.001) and more than 2-fold higher than in RA SF (202.25±149.13 ng/ml, P< 0.001). No differences were detected in the levels of COMP or osteocalcin between the experimental groups. Although the differences between active and inactive states of OA or RA were insignificant, for cytokines and OP-1 each state was statistically different from normal. SF from both OA and RA had higher biomarker levels than normals, regardless of disease state, though there was a trend of higher chemokine levels in less active RA, and higher Il-1, Il-6, and Il-8 levels in active OA. Conclusions: Our findings suggest that the levels of pathophysiologically important biomarkers in SF of patients with OA and RA differ according to the mechanisms that drive each disease. Thus, Il-1, Il-11, LIF and OP-1 appear to be significant for OA; while Il-6, Il-8, and OP-1 may have significance for RA. As previously observed in cartilage, a strong negative correlation between the levels of OP-1 and Il-6 family of chemokines was seen. Larger studies are necessary to develop a biomarker algorithm that would be of diagnostic/prognostic use.

ObjectiveGrowth 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.DesignChondrocytes 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.ResultsHistology 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.ConclusionsThe 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.

Osteoarthritis and Cartilage Vol.14, Supplement B S81 up to 64 cells, Sox-9 was identified in all cells, along with c-Myc, nucleostemin and the proteolytic enzymes.RT-PCR confirmed expression of nucleostemin, c-myc and Sox-9.Conclusions: Early expression of c-myc may indicate temporary genomic instability, i.e. a gene expression pattern in flux.Because nucleostemin is found only in stem cells or cancer, the presence in clonal chondrocytes is surprising and suggests that chondrocytes undergo a de-differentiation to a progenitor-like intermediate.The re-activation of Sox-9 in late clones suggests reversion to an earlier chondrocytic differentiation stage.Overall, the findings suggest that OA chondrocytes, because they still retain some chondrocytic characteristics, display a complicated and mixed phenotype, with degradative chondrocytes arising from a progenitor-cell like intermediate stage, followed by re-differentiation.At the same time, the observations do not support the concept of continued differentiation to hypertrophic chondrocytes.

OBJECTIVE:To delineate the role of endogenous osteogenic protein 1 (OP-1) in human articular cartilage homeostasis via the inhibition of OP-1 gene expression by antisense oligonucleotides.METHODS:Human adult normal articular cartilage was obtained from the knee and ankle joints of 34 organ donors. Chondrocytes were cultured as tissue explants or isolated cells in alginate or high-density monolayers for 48 hours in the presence of OP-1 antisense or sense oligonucleotides. The effect of OP-1 antisense inhibition was evaluated by reverse transcription-polymerase chain reaction, (35)S incorporation, dimethylmethylene blue assay, histology with Safranin O staining, and immunohistochemistry with anti-proOP-1, anti-mature OP-1, and anti-aggrecan antibodies.RESULTS:Antisense treatment inhibited OP-1 gene expression by a mean +/- SD of 34 +/- 12% (P < 0.01) in chondrocytes cultured in monolayers and by 77 +/- 27% (P < 0.03) in alginate beads. The inhibition of autocrine OP-1 caused a striking decrease in aggrecan gene expression, in total proteoglycan content accumulated in cartilage matrix, and in the ability of chondrocytes to newly synthesize proteoglycans. OP-1 antisense reduced aggrecan messenger RNA expression by 42 +/- 17% (P < 0.05) and proteoglycan synthesis by 48 +/- 23% (P < 0.01). Histology and immunohistochemistry revealed a dramatic decrease in Safranin O staining and reduced anti-aggrecan staining (primarily in the superficial and middle cartilage layers) with OP-1 antisense treatment.CONCLUSION:Our results suggest that OP-1 is an important endogenous cartilage factor that regulates matrix integrity and possibly needs to be induced or up-regulated to maintain normal cartilage homeostasis. These findings confirm our hypothesis that a lack of autocrine OP-1 may lead to an elevated susceptibility of chondrocytes to the catabolic processes, thus contributing/promoting cartilage degeneration.

Objective. Osteoarthritis is characterized by dramatic changes in chondrocyte metabolism including the overexpression of catabolic enzymes, but also a lack of anabolic activity. In this respect, osteogenic protein 1 (OP-1) appears to be one of the most potent anabolic factors of chondrocytes. In this study, we were interested in: (1) whether recombinant human OP-1 exerts its anabolic effects also on osteoarthritic chondrocytes, (2) whether OP-1 modulates the expression of catabolic genes, and (3) whether the BMP effects are related to the expression levels of its intracellular mediators (R- and I-Smads).Methods. Chondrocytes were isolated from cartilage of either normal (n=5) or osteoarthritic (n=8) human knee joints and cultured in short-term high-density monolayer cultures with and without recombinant OP-1. RNA was isolated and analyzed for mRNA expression levels of anabolic (aggrecan, collagen type II), catabolic (MMP-1, 3, -13, ADAMTS-4), and intracellular signaling mediators (Smad 1, 4, 5, 6, 7, and 8) by quantitative online PCR.Results. After OP-1 stimulation, the anabolic genes were significantly upregulated in osteoarthritic chondrocytes in comparison to normal chondrocytes. Neither in normal nor osteoarthritic chondrocytes were significant changes observed for the matrix degrading enzymes. Smads were also expressed in both normal and osteoarthritic cells at roughly the same level with and without stimulation with OP-1.Conclusion. Osteoarthritic chondro( cytes are not hypo-responsive to anabolic stimulation by OP-1. Thus, human recombinant OP-1 could be a suitable anabolic activator of osteoarthritic chondrocytes. This might be of particular interest as chondrocytes themselves showed very low levels of OP-1 expression.

The aim of the present study was to evaluate the possible benefit of platelet-rich plasma (PRP) in sinus grafting as compared with recombinant human bone morphogenetic protein-7 (rhBMP-7). For this purpose, we performed a bilateral sinus augmentation with anorganic bovine bone and simultaneous insertion of a titanium screw implant in five miniature pigs. Six hundred microliters of PRP and 15%-vol. autologous bone, which was collected with a trap during preparation of the implant recipient site, were added to the right sinus and 420 microl rhBMP-7 to the left sinus. A polychrome sequential labeling was performed. The animals were sacrificed 6 weeks after surgery. Undecalcified ground sections were evaluated by microradiography, digitized histomorphometry and under fluorescent light. The mean bone-implant contact using rhBMP-7 was 45.8% and 5.7% under PRP (P=0.002). The mean height of newly mineralized bone in the augmented area using rhBMP-7 amounted to 8.3 mm as opposed to 3.6 mm under PRP (P=0.013). Using PRP, the mean area of the newly formed bone was enhanced (51.3%) as compared with rhBMP-7 (33.1%); however, this difference was not statistically significant (P=0.081). In conclusion, under the selected experimental conditions the use of rhBMP-7 led to superior outcomes with regard to the osseointegration of dental implants and the height of new bone as compared with the use of PRP.

Growth factors contained in platelet-rich plasma (PRP) have recently been proposed to enhance maturation of bone grafts and, in combination with anorganic bovine bone, to support repair in the treatment of small bone defects in maxillofacial surgery. Bone morphogenetic proteins (BMP) carried in a matrix may be able to replace the autologous bone graft in the treatment of critical size defects. However, no studies have compared the bone stimulating capacity of PRP and BMP. Likewise there is no data comparing the effects of PRP in either an autologous bone graft or in anorganic bovine bone. We augmented the mandible of Wistar rats (n = 28) on both sides with either anorganic bovine bone (Bio-Oss) or autologous rib bone. On the test side we applied either 20 microl of autologous PRP or 10 microl of rhBMP-7 (4 groups, n = 7). In addition, bone induction was evaluated in an extraskeletal site (n = 14). A polychrome sequential labeling was performed. The animals were sacrificed by intra-vital perfusion on day 50. Undecalcified ground sections were evaluated by microradiography, digitized histomorphometry and under fluorescent light. The qualitative analysis of fluorochrome labels suggested that PRP and rhBMP-7 accelerated bone growth. However, histomorphometric analysis revealed no significant differences in the area of newly mineralized bone under either the influence of PRP or rhBMP-7 on autologous bone graft. Likewise, the addition of PRP to anorganic bovine bone showed no statistical difference to the control group. The strongest bone stimulating effect was seen for the combination of rhBMP-7 with anorganic bovine bone (p = 0.028). In the extraskeletal model, newly formed bone was evident in the presence of rhBMP-7, but not of PRP. In conclusion, according to the histomorphometry, the addition of platelet-rich plasma failed to enhance bone formation on anorganic bovine bone and on autologous bone grafts.

Background: Articular cartilage injury has a poor prognosis for repair. Mesenchymal cells, when exposed to osteogenic proteins and other cytokines, can differentiate into cells that behave phenotypically as chondrocytes. In this study, we examined the ability of recombinant human osteogenic protein-1 (rhOP-1 or rhBMP-7) to elicit the repair of osteochondral defects in dogs. Methods: Bilateral osteochondral defects that were 5 mm in diameter by 6 mm deep were surgically created in the medial femoral condyles of sixty-five adult dogs. rhOP-1-treated (100 mg of a 3.5-mg rhOP-1/g bovine bone-derived Type-I collagen device) and control defects (untreated or treated with 100 mg bovine bone-derived collagen implants) were evaluated grossly and histologically at six, twelve, sixteen, twenty-six, and fifty-two weeks postoperatively. The influence of protected initial weight-bearing and surgical placement of periosteal flaps was also evaluated. Results: Gross and histologic grading of the defect repair indicated improvement in the rhOP-1-treated defects compared with that in the controls. Grossly, the repair tissue in the rhOP-1-treated defects was continuous with the adjacent intact cartilage and appeared translucent. By comparison, the repair tissue in the control defects was discontinuous and opaque or inhomogeneous in nature. Histologically, maturing cartilage similar in appearance to the intact articular cartilage was present in the rhOP-1-treated defects. Cartilage at the defect interface was minimally degraded. The control defects were filled primarily with fibrous tissue and fibrocartilage. Significant differences based upon treatment type were observed at twelve weeks, sixteen weeks, and for all time-periods combined (p = 0.0385, p = 0.0070, and p = 0.0026, respectively). Conclusion: rhOP-1 (rhBMP-7) induced hyaline cartilage-like repair of full-thickness osteochondral defects in a dog model. Differences in cartilage repair were maintained at fifty-two weeks postoperatively with no significant degradation of the rhOP-1-induced repair tissue. Clinical Relevance: The dog osteochondral defect model is a challenging one that reflects the difficulties of eliciting articular cartilage repair that are seen in the clinical setting. The results of this study indicate that rhOP-1 may improve the repair of articular cartilage, and they demonstrate the importance of further investigation to characterize the effects of growth factors on the cartilage repair process.

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.

BACKGROUND:Osteogenic protein-1 (OP-1, BMP-7) induces bone formation and cartilage growth. Since OP-1 is an anabolic factor expressed by human articular chondrocytes, we examined the response of endogenous OP-1 to interleukin-1beta (IL-1beta) in human articular cartilage. METHODS:Normal adult human articular cartilage explants were cultured for twenty-five days in the presence of medium only or were treated with a low dose (0.1 ng/mL) or high dose (1.0 ng/mL) of IL-1beta for forty-eight or ninety-six hours. Alternately, cartilage explants were cultured forty-eight hours with IL-1beta, followed by forty-eight hours in standard medium (recovery). Tissue was analyzed for OP-1 message (by means of the reverse transcriptase-polymerase chain reaction), protein (by means of enzyme-linked immunosorbent assay and Western blot analysis) and proteoglycan content. Medium was analyzed for released proteoglycans and OP-1. RESULTS:In the presence of medium, OP-1 maintained its steady state of mRNA and protein expression for as long as twenty-five days in culture. A low dose of IL-1beta led to some upregulation in message and a twofold (p < 0.02) increase in OP-1 protein characterized by enhanced processing and activation of OP-1. Removal of IL-1beta (recovery experiments) did not reverse its effect on OP-1 synthesis. A high dose of IL-1beta caused stronger upregulation of message and a twofold decrease in OP-1 protein content (p < 0.007) in the cartilage matrix. However, this decrease in the matrix was primarily due to a release of active OP-1 into the medium. After removal of the 1.0-ng/mL IL-1beta, the levels of OP-1 protein did not recover. CONCLUSION:The results of the present study indicate that human adult chondrocytes have an ability to respond anabolically to initial or early catabolic events through an upregulation of endogenous OP-1.

We assessed the distribution and relative immunohistochemical staining intensity of the bone morphogenetic protein-7, osteogenic protein-1 (OP-1), in its pro- and mature forms, and four of its receptors, type I (ALK-2, ALK-3, and ALK-6) and type II in normal adolescent New Zealand White rabbit articular cartilage. Expression of the protein and its receptors was also examined in cartilage from joints that had been previously subjected to cartilage matrix degradation. Pro-OP-1 was moderately expressed in chondrocytes of the superficial, middle, and deep cartilage zones and in the osteocytes. The expression of mature OP-1 was similar, with the exception of less staining in the superficial zone of cartilage. Expression of these two forms of OP-1 was enhanced in the middle and deep cartilage zones after catabolic challenge. The type I receptor, ALK-6, displayed the strongest staining of the receptors in both cartilage and bone, whereas ALK-2 displayed the weakest staining. No differences were observed in the receptor staining levels after catabolic challenge. This study shows that OP-1 and its receptors have been identified in rabbit articular cartilage and bone, suggesting a possible role for this pathway in cartilage and bone homeostasis.

Bone-derived bone morphogenetic/osteogenic proteins (BMPs/OPs) regenerate alveolar bone and cementum in surgically created furcation defects in the Chacma baboon (Papio ursinus). Recent studies showed the induction of cementogenesis by relatively low doses of human recombinant osteogenic protein-1 (hOP-1), suggesting that BMPs/OPs might possess a structure/activity profile that controls tissue morphogenesis and regeneration. More challenging is the demonstration of cementogenesis and bone regeneration in periodontally induced furcation defects with root surfaces exposed for a relatively long time to periodontal pathogens. Baboons experience naturally occurring site-specific gingivitis but periodontitis, as defined by loss of periodontal attachment, has not been found in any of twenty animals in our colony. A pathogenic human strain of Porphyromonas gingivalis was inoculated into the furcation areas of the 1 st and 2nd mandibular molars of four adult baboons twice a month for twelve months; follow-up examinations included sulcular probing depths and microbiological analyses of subgingival plaque. Chronic periodontitis was induced in all four animals as assessed by probing periodontal pocket depths, intra-oral radiographs, and microbiological analyses that confirmed the presence of P gingivalis. Two months after scaling, root planing and a plaque control regimen with clinical resolution of gingivitis, mucoperiosteal flaps were elevated to expose class 11 furcation defects of the affected molars filled with copious granulation tissue. After root planing and debridement, 12 furcation defects were implanted with 0.5 or 2.5 mg of gamma-irradiated hOP-1 per 1 g of xenogenic bovine insoluble collagenous bone matrix as carrier. Serial undecalcified sections prepared six months after surgery showed regeneration of alveolar bone and induction of cementogenesis with Sharpey's fibres uniting the regenerated bone to the newly induced cementum with both doses of hOP-1. These results indicate an additional specific use of hOP-1 for tissue morphogenesis in clinical contexts.

Twelve African green monkeys were implanted with recombinant human osteogenic protein-1 (rhOP-1) placed on a bovine bone-derived Type I collagen carrier to characterize healing in an ulna segmental bone defect model at 1, 3, 12, and 20 weeks postoperative. Defect healing was evaluated by plain film radiography, computed tomography (CT), magnetic resonance imaging (MRI), bone mineral density (BMD), and histologic analysis. Radiographically, new bone formation was observed as early as 3 weeks postoperative. By 6 weeks, new bone was visible in five of six defects. Increased quantity and mineralization of the new bone were apparent by 12 weeks. Reformation of the medullary cavity with appearance of marrow elements was demonstrated by CT and MRI at 20 weeks. BMD studies revealed a significant increase in the presence of bone with time. Histology at 1 week demonstrated that the implant material was well contained in the defect, and a proliferation of cells occurred at the defect borders. At 3 weeks cell proliferation continued and cell phenotype differentiation was recognized. By 12 weeks substantially less residual carrier was found in the defects, and calcifying tissues with plump chondrocytes, osteoblasts, and immature woven bone were observed. Areas of lamellar and woven bone were identified at 12 weeks, with advanced remodeling and revascularization observed at 20 weeks. The use of osteoinductive implants may provide an alternative to autologous and allogeneic bone tissue in the therapeutic approach to bone defects and promotion of fusion by eliminating the donor site morbidity associated with autogenous bone and the decreased efficacy and potential for disease transmission associated with allogeneic bone.

Articular cartilage has a poor reparative capacity. This feature is exacerbated with aging and during degenerative joint conditions, contributing to loss of motion and impairment of quality of life. This study focused on osteogenic protein-1 (OP-1) and its ability to serve as a repair-stimulating factor in articular cartilage. The purpose of this work was to develop a quantitative method for the assessment of the content of OP-1 protein in extracts from human articular cartilage and to investigate the changes in OP-1 mRNA expression and protein levels with aging of normal adult cartilage. Full thickness cartilage was dissected from femoral condyles of donors with no history of joint disease within 24 h of death. Levels of OP-1 mRNA expression were measured by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method; concentration of OP-1 protein was detected by new sandwich enzyme-linked immunosorbent assay (ELISA); qualitative changes in OP-1 forms were evaluated by Western blots with various anti-OP-1 antibodies. The sensitivity of the ELISA method allowed the detection of picogram quantities of OP-1 in cartilage extracts. We found that (1) concentration of OP-1 in normal cartilage is within the range of biological activity of OP-1 in vitro; and (2) during aging of human adult, articular cartilage, levels of OP-1 protein and message are dramatically reduced (more than 4-fold; p<0.02). The major qualitative changes affected primarily mature OP-1. The results of the current study suggest the possibility that OP-1 may be critical for chondrocytes to maintain their normal homeostasis and could also serve as a repair factor during joint disease or aging.

Native and recombinant human bone morphogenetic/osteogenic proteins (BMPs/ OPs) singly initiate bone induction in vivo. The finding of synchronous but spatially different BMPs/OPs expression during periodontal tissue morphogenesis suggests novel therapeutic approaches using morphogen combinations based on recapitulation of embryonic development. Twelve furcation defects prepared in the first and second mandibular molars of three adult baboons (Papio ursinus) were used to assess whether qualitative histological aspects of periodontal tissue regeneration could be enhanced and tissue morphogenesis modified by combined or single applications of recombinant hOP-1 and hBMP-2. Doses of BMPs/OPs were 100 microg of each protein per 1 g of insoluble collagenous bone matrix as carrier. Approximately 200 mg of carrier matrix was used per furcation defect. Undecalcified sections cut for histological analysis 60 d after healing of hOP-1-treated specimens showed substantial cementogenesis with scattered remnants of the collagenous carrier. hBMP-2 applied alone induced greater amounts of mineralized bone and osteoid when compared to hOP-1 alone or to combined morphogen applications. Combined applications of hOP-1 and hBMP-2 did not enhance alveolar bone regeneration or new attachment formation over and above the single applications of the morphogens. The results of this study, which is the first to attempt to address the structure-activity relationship amongst BMP/OP family members, indicate that tissue morphogenesis induced by hOP-1 and hBMP-2 is qualitatively different when the morphogens are applied singly, with hOP-1 inducing substantial cementogenesis. hBMP-2 treated defects, on the other hand, showed limited cementum formation but a temporal enhancement of alveolar bone regeneration and remodelling. The demonstration of therapeutic mosaicism in periodontal regeneration will require extensive testing of ratios and doses of recombinant morphogen combinations for optimal tissue engineering in clinical contexts.

A critical issue in tissue engineering and morphogenesis of bone is the development of novel biomimetic biomaterials that are capable of optimizing the biological activity of recombinant human bone morphogenetic and osteogenic proteins, which are molecules that initiate bone formation in vivo. From a therapeutic perspective, a carrier matrix is required for the local delivery of these proteins to evoke a desired osteogenic effect. In view of the affinity of these proteins for hydroxyapatite, which may reflect the in vivo supramolecular assembly of bone proteins bound to both the extracellular matrix and the mineral component of bone, we investigated the efficacy of single applications of different doses of human osteogenic protein-1 (hOP-1) adsorbed onto sintered porous hydroxyapatites for bone induction in orthotopic calvarial defects in 12 adult male baboons (Papio ursinus) and heterotopically in the rectus abdominis of four additional baboons. In orthotopic specimens, pretreatment of sintered porous hydroxyapatites with 100 microgram of hOP-1 in 500 microliter of 5 mM hydrochloric acid resulted in rapid and diffuse osteoinduction restricted within the porous spaces of the hydroxyapatite, as evaluated by histology and histomorphometry on day 30. Hydroxyapatites treated with 500 microgram of hOP-1 showed a different pattern of bone formation and distribution on day 30 as compared with the lower dose of the recombinant morphogen. Although bone formation was extensive with the higher dose, it was found on the endocranial and pericranial aspects of the specimens, enveloping the implanted hydroxyapatite carrier, and the internal porous spaces were occupied by a rich vascular network without any bone formation. By 90 and 365 days after the implantation of both doses of hOP-1, however, there was remodelling and complete penetration of the newly induced bone within the available porous spaces. The combination of hOP-1 and hydroxyapatite also showed extensive bone formation in heterotopic specimens harvested from the rectus abdominis muscle of the baboon using doses of 5, 25, and 45 microgram of hOP-1 per implant. These findings in the adult primate demonstrate extensive bone formation by hOP-1 adsorbed onto sintered porous hydroxyapatites and suggest that predictable osteogenesis in clinical contexts for treatment of craniofacial bone defects may be engineered using inorganic, nonimmunogenic, and carvable delivery systems that initiate osteogenesis with relatively low doses of recombinant osteogenic proteins, thus mimicking the macrostructure and microstructure of living bone.

To investigate the long-term efficacy of irradiated recombinant human osteogenic protein 1 (hOP-1) in bone regeneration and morphogenesis, hOP-1 was combined with a bovine collagenous matrix carrier (0, 0.1, 0.5, and 2.5 mg hOP-1/g of matrix), sterilized with 2.5 Mrads of y-irradiation, and implanted in 80 calvarial defects in 20 adult baboons (Papio ursinus). The relative efficacy of partially purified bone-derived baboon bone morphogenetic proteins (BMPs), known to contain several osteogenic proteins, was compared with the recombinant hOP-1 device in an additional four baboons. Histology and histomorphometry on serial undecalcified sections prepared from the specimens harvested on day 90 and day 365 showed that gamma-irradiated hOP-1 devices induced regeneration of the calvarial defects by day 90, although with reduced bone area compared with a previous published series of calvarial defects treated with nonirradiated hOP-1 devices. One year after application of the irradiated hOP-1 devices, bone and osteoid volumes and generated bone tissue areas were comparable with nonirradiated hOP-1 specimens. Moreover, 365 days after healing regenerates induced by 0.5 mg and 2.5 mg of irradiated hOP-1 devices showed greater amounts of bone and osteoid volumes when compared with those induced by nonirradiated hOP-1 devices. On day 90, defects treated with 0.1 mg and 0.5 mg of bone-derived baboon BMPs, combined with irradiated matrix, showed significantly less bone compared with defects receiving irradiated devices containing 0.1 mg and 0.5 mg hOP-1; 2.5 mg of partially purified BMPs induced bone and osteoid volumes comparable with the 0.1-mg and 0.5-mg hOP-1 devices. Control specimens of y-irradiated collagenous matrix without hOP-1 displayed a nearly 2-fold reduction in osteoconductive bone repair when compared with nonirradiated controls. These findings suggest that the reduction in bone volume and bone tissue area on day 90 may be caused by a reduced performance of the irradiated collagenous matrix substratum rather than to a reduction in the biological activity of the irradiated recombinant osteogenic protein. This is supported by the results of in vitro and in vivo studies performed to determine the structural integrity of the recovered gamma-irradiated hOP-1 before application in the baboon. Recoveries by high-performance liquid chromatography (HPLC) and sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (SDS/PAGE)/immunoblot analyses indicated that doses of 2.5-3 Mrads of gamma-irradiation did not significantly affect the structural integrity of the recovered hOP-1. Biological activity of the recovered hOP-1 was confirmed in vitro by showing induction of alkaline phosphatase activity in rat osteosarcoma cells (ROS) and in vivo by de novo endochondral bone formation in the subcutaneous space of the rat. These findings in the adult primate indicate that a single application of gamma-irradiated hOP-1 combined with the irradiated xenogeneic bovine collagenous matrix carrier is effective in regenerating and maintaining the architecture of the induced bone at doses of 0.5 mg/g and 2.5 mg/g of carrier matrix.

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.