Biomedical subjects

B D Boyan

Publications and source records attributed to B D Boyan.

At least 91 records · Page 5Linked to original sources

PubMed1999

Vitamin D3 metabolites regulate LTBP1 and latent TGF-beta1 expression and latent TGF-beta1 incorporation in the extracellular matrix of chondrocytes.

Growth plate chondrocytes make TGF-beta1 in latent form (LTGF-beta1) and store it in the extracellular matrix via LTGF-beta1 binding protein (LTBP1). 1,25-(OH)2D3 (1,25) regulates matrix protein production in growth zone (GC) chondrocyte cultures, whereas 24,25-(OH)2D3 (24,25) does so in resting zone (RC) cell cultures. The aim of this study was to determine if 24,25 and 1,25 regulate LTBP1 expression as well as the LTBP1 -mediated storage of TGF-beta1 in the extracellular matrix of RC and GC cells. Expression of LTBP1 and TGF-beta1 in the growth plate and in cultured RC and GC cells was determined by in situ hybridization using sense and antisense oligonucleotide probes based on the published rat LTBP1 and TGF-beta1 cDNA sequences. Fourth passage male rat costochondral RC and GC chondrocytes were treated for 24 h with 10(-7)-10(-9) M 24,25 and 10(-8)-10(-10) M 1,25, respectively. LTBP1 and TGF-beta1 mRNA levels were measured by in situ hybridization; production of LTGF-beta1, LTGF-beta2, and LTBP1 protein in the conditioned media was verified by immunoassays of FPLC-purified fractions. In addition, ELISA assays were used to measure the effect of 1,25 and 24,25 on the level of TGF-beta1 in the media and matrix of the cultures. Matrix-bound LTGF-beta1 was released by digesting isolated matrices with 1 U/ml plasmin for 3 h at 37 degrees C. LTBP1 and TGF-beta1 mRNAs are co-expressed throughout the growth plate, except in the lower hypertrophic area. Cultured GC cells express more LTBP1 and TGF-beta1 mRNAs than RC cells. FPLC purification of the conditioned media confirmed that RC cells produce LTGF-beta1, LTGF-beta2, and LTBP1. GC cells also produce LTGF-beta2, but at lower concentrations. 1,25 dose-dependently increased the number of GC cells with high LTBP1 expression, as seen by in situ hybridization. 24,25 had a similar, but less pronounced, effect on RC cells. 1,25 also caused a dose-dependent increase in the amount of TGF-beta1 protein found in the matrix, significant at 10(-8) and 10(-9) M, and a corresponding decrease in TGF-beta1 in the media. 24,25 had no effect on the level of TGF-beta1 in the matrix or media produced by RC cells. This indicates that 1,25 induces the production of LTBP1 by GC cells and suggests that the TGF-beta1 content of the media is reduced through the formation of latent TGF-beta1 -LTBP1 complexes which mediates storage in the matrix. Although 24,25 induced the expression of LTBP1 by RCs, TGF-beta1 incorporation into the matrix is not regulated by this vitamin D3 metabolite. Thus, vitamin D3 metabolites may play a role in regulating the availability of TGF-beta1 by modulating LTBP1 production.

24,25-Dihydroxyvitamin D 3↗

PubMed1999

Potential of porous poly-D,L-lactide-co-glycolide particles as a carrier for recombinant human bone morphogenetic protein-2 during osteoinduction in vivo.

Several different biodegradable bone graft materials are in clinical or preclinical use for the repair of bone defects in orthopedics, maxillofacial surgery, and periodontics. This study tested the hypothesis that poly-D,L-lactide-co-glycolide copolymer (PLG) can be used as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) and that the composite has osteoinductive ability. Porous PLG rods were shredded to a particle size ranging from 250 to 850 microm. Active and inactive demineralized freeze-dried bone allografts (DFDBA) with a comparable particle size were used as positive and negative controls, respectively. PLG particles were treated with vehicle or with 5 or 20 microg rhBMP-2. DFDBA and PLG particles were placed in gelatin capsules, mixed with vehicle or rhBMP-2, and implanted at intramuscular sites in male Nu/Nu (nude) mice. Each mouse underwent bilateral implantation with implants of the same formulation, resulting in five groups of four mice per group: active DFDBA, inactive DFDBA, PLG, PLG + 5 microg rhBMP-2, and PLG + 20 microg rhBMP-2. After 56 days, the implants were recovered and processed for histology. Bone induction was assessed by use of a semiquantitative scoring system based on the amount of new bone formed in representative histological sections. Histomorphometry was also used to measure the area of new bone formed and the area of residual implant material. The results showed that active DFDBA induced the formation of ossicles containing new bone with bone marrowlike tissue, whereas inactive DFDBA or PLG particles alone did not induce new bone. The addition of rhBMP-2 to PLG particles resulted in new bone formation that had a greater bone induction score than active DFDBA. Moreover, the histomorphometric analysis showed that the addition of rhBMP-2 to PLG particles induced the formation of a greater area of new bone and bone marrowlike tissue than active DFDBA. The resorption of the PLG particles was markedly increased with the addition of rhBMP-2, suggesting that rhBMP-2 may attract and regulate resorptive cells at the implantation site. The results of the present study indicate that PLG copolymers are good carriers for BMP and promote the induction of new bone formation. Further, the PLG copolymers with rhBMP-2 had a greater effect in inducing new bone formation and resorbing the implanted material than active DFDBA alone.

Animals↗

PubMed1999

Surface roughness modulates the response of MG63 osteoblast-like cells to 1,25-(OH)(2)D(3) through regulation of phospholipase A(2) activity and activation of protein kinase A.

Implant surface roughness influences osteoblast proliferation, differentiation, and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones such as 1, 25-(OH)(2)D(3) is altered by the effects of surface roughness; on the roughest Ti surfaces the effects of roughness and 1, 25-(OH)(2)D(3) are synergistic. Prostaglandin E(2) (PGE(2)) appears to be involved in mediating the effects of surface roughness on the cells, as well as in the response to 1,25-(OH)(2)D(3). However, it is not yet known through which signaling pathways surface roughness exerts its effects on the response of osteoblasts to 1, 25-(OH)(2)D(3). The present study examined the potential role of protein kinase A (PKA), phospholipase A(2)(PLA(2)), and protein kinase C (PKC) in this process. MG63 osteoblast-like human osteosarcoma cells were cultured on cpTi disks with R(a) values of 0. 54 microm (PT), 4.14 microm (SLA), or 4.92 microm (TPS). PKA was inhibited by adding H8 to the cultures; similarly, PLA(2) was inhibited with quinacrine or activated with melittin, and PKC was inhibited with chelerythrine. Inhibitors or activators were included in the culture media through the entire culture period or for the last 24 h of culture. In addition, cultures were treated for 24 h with inhibitors or activators in the presence of 1,25-(OH)(2)D(3). The effects on cell number and alkaline phosphatase specific activity were determined after 24 h; PKC activity was determined after 9 min and at 24 h. Cell number was reduced on rough surfaces, and alkaline phosphatase activity was increased. 1,25-(OH)(2)D(3) had a synergistic effect with surface roughness on alkaline phosphatase. However, neither surface roughness nor 1,25-(OH)(2)D(3) had an effect on PKC. H8 treatment for 24 h inhibited cell number and alkaline phosphatase on all surfaces; however, when it was present throughout the culture period, the PKA inhibitor had no effect on cell number, but decreased alkaline phosphatase-specific activity. H8 reduced the 1,25-(OH)(2)D(3)-mediated effect on cell number and alkaline phosphatase. Quinacrine inhibited cell proliferation and alkaline phosphatase on all surfaces and further reduced the 1,25-(OH)(2)D(3)-dependent decreases in both parameters. Melittin had no effect when applied for 24 h and did not modify the 1,25-(OH)(2)D(3) effect; however, when present throughout the culture period, it caused a decrease in proliferation and an increase in enzyme activity. Chelerythrine, the PKC inhibitor, only inhibited cell proliferation when it was present throughout the entire culture period. However, it decreased alkaline phosphatase in cultures treated for 24 h, but increased enzyme activity when it was present for the entire culture period. The results indicate that surface roughness and 1,25-(OH)(2)D(3) both mediate their effects through PLA(2) which catalyzes the rate-limiting step in PGE(2) production. Further downstream, PGE(2) activates PKA. Surface roughness-dependent effects are also mediated through PKC, but only after the cells have reached confluence and are undergoing phenotypic maturation. The effect of surface roughness on responsiveness to 1,25-(OH)(2)D(3) is mediated through PLA(2)/PKA and not through PKC.

Bioprosthesis↗

PubMed1999

Ultrahigh molecular weight polyethylene particles have direct effects on proliferation, differentiation, and local factor production of MG63 osteoblast-like cells.

Small particles of ultrahigh molecular weight polyethylene stimulate formation of foreign-body granulomas and bone resorption. Bone formation may also be affected by wear debris. To determine if wear debris directly affects osteoblasts, we characterized a commercial preparation of ultrahigh molecular weight polyethylene (GUR4150) particles and examined their effect on MG63 osteoblast-like cells. In aliquots of the culture medium containing ultrahigh molecular weight polyethylene, 79% of the particles were less than 1 microm in diameter, indicating that the cells were exposed to particles of less than 1 microm. MG63 cell response to the particles was measured by assaying cell number, [3H]thymidine incorporation, alkaline phosphatase specific activity, osteocalcin production, [35S]sulfate incorporation, and production of prostaglandin E2 and transforming growth factor-beta. Cell number and [3H]thymidine incorporation were increased in a dose-dependent manner. Alkaline phosphatase specific activity, a marker of cell differentiation for the cultures, was significantly decreased, but osteocalcin production was not affected. [35S]sulfate incorporation, a measure of extracellular matrix production, was reduced. Prostaglandin E2 release was increased, but transforming growth factor-beta production was decreased in a dose-dependent manner. This shows that ultrahigh molecular weight polyethylene particles affect MG63 proliferation, differentiation, extracellular matrix synthesis, and local factor production. These effects were direct and dose dependent. The findings suggest that ultrahigh molecular weight polyethylene wear debris particles with an average size of approximately 1 microm may inhibit bone formation by inhibiting cell differentiation and reducing transforming growth factor-beta production and matrix synthesis. In addition, increases in prostaglandin E2 production may not only affect osteoblasts by an autocrine pathway but may also stimulate the proliferation and activation of cells in the monocytic lineage. These changes favor decreased bone formation and increased bone resorption as occur in osteolysis.

Alkaline Phosphatase↗

PubMed1999

Osteochondral progenitor cells in acute and chronic canine nonunions.

This study examined the ability of cells isolated from early healing segmental defects and from tissue from chronic nonunions to support bone and cartilage formation in vivo and their response to transforming growth factor-beta1 in vitro. Ostectomies (3 mm) were created in the radial diaphysis of four dogs. The dogs were splinted 3-5 days postoperatively and then allowed to bear full weight. At 7 days, tissue in the defect was removed and any periosteum was discarded; cells in the defect tissue were released by enzymatic digestion. The dogs were splinted again and allowed to bear full weight for 12 weeks. Radiographs confirmed a persistent nonunion in each dog. Defect tissue was again removed, any periosteum was discarded, and cells were isolated. Cells were also obtained from the defect tissue by nonenzymatic means with use of explant cultures. One-half of the tissue and one-half of any preconfluent, first-passage cultures were shipped to Cleveland by overnight carrier. At second passage, cells were loaded into ceramic cubes and implanted into immunocompromised mice for 3 or 6 weeks. Harvested cubes were examined histologically for cartilage and bone with use of a semiquantitative scoring system. Confluent fourth-passage cultures of 7 and 84-day defect tissue cells were cultured with 0.03-0.88 ng/ml transforming growth factor-beta1 for 24 hours, and [3H]thymidine incorporation and alkaline phosphatase specific activity were determined. Donor-dependent differences were noted in the rate at which defect cells achieved confluence; in general, cells from 7-day tissue divided most rapidly. Seven-day defect cells formed less bone and at a slower rate than was seen in the ceramic cubes containing samples from day 84. Cells derived enzymatically behaved similarly to those from explant cultures. Ceramic cubes contained fibrous connective tissue, cartilage, bone, and fat, indicating that multipotent cells were present. Stimulation of [3H]thymidine incorporation in response to transforming growth factor-beta1 was donor dependent and variable; only two of six separate isolates of cells exposed to it had measurable alkaline phosphatase activity (which was relatively low), and none of the cultures exhibited an increase in response to transforming growth factor-beta1 for 24 hours. This indicates that mesenchymal progenitor cells are present in the healing defect tissue at 7 and 84 days and that the relative proportion of osteochondroprogenitor cells is greater at the later time. The response to transforming growth factor-beta1 is typical of multipotent mesenchymal cells but not of committed chondrocytes or osteoblasts, indicating that these committed and differentiated cells are not present in early stages of healing and suggesting that their differentiation is inhibited in chronic nonunion.

Alkaline Phosphatase↗

PubMed1999

Effect of surface roughness and composition on costochondral chondrocytes is dependent on cell maturation state.

During endochondral bone formation, as occurs in fracture healing, chondrocytes are one of the first cells to see an implant surface. We tested the hypothesis that chemical composition and surface roughness affect chondrocyte differentiation, matrix synthesis, and local factor production and that the nature of the response is dependent on the state of maturation of the cells. To do this, we harvested rat growth zone and resting zone chondrocytes and examined their response to smooth and rough disk surfaces manufactured from either commercially pure titanium or titanium alloy. Profilometry, scanning electron microscopy, Auger spectroscopy, and Fourier transform infrared spectroscopy were used to characterize the surfaces. Average roughness values were 0.22 microm for smooth titanium surfaces, 0.23 microm for smooth titanium alloy surfaces, 4.24 microm for rough titanium surfaces, and 3.20 microm for rough titanium alloy surfaces. Cells were grown on the different disk surfaces until the cultures had reached confluence on plastic. The effect of the surfaces was determined by assaying cell number and [3H]thymidine incorporation as measures of cell proliferation, cell layer and cell alkaline phosphatase specific activity as markers of differentiation, and collagen production and [35S]sulfate incorporation as indicators of extracellular matrix production. In addition, the synthesis of prostaglandin E2 and transforming growth factor-beta were examined to measure changes in local factor synthesis. In growth zone and resting zone cultures, cell number and [3H]thymidine incorporation were decreased on rough surfaces; however, this effect was greater on commercially pure titanium surfaces. Cell layer and cell alkaline phosphatase specific activity were decreased in resting zone cells grown on rough surfaces. Cell alkaline phosphatase specific activity in growth zone cells was decreased on rough surfaces, whereas cell layer alkaline phosphatase specific activity was increased only in growth zone cells grown on rough commercially pure titanium surfaces. Resting zone cell collagen production was decreased only on rough commercially pure titanium, whereas in growth zone cells, collagen production was increased. Increased prostaglandin E2 release into the media was found for growth zone and resting zone cell cultures on the disks with rough surfaces. The observed effect was greater on rough commercially pure titanium. Production of transforming growth factor-beta by resting zones was similarly affected, whereas an increase in its production by growth zone cells was measured only on rough commercially pure titanium. These results indicate that surface roughness affects chondrocyte proliferation, differentiation, matrix synthesis, and local factor production and that these parameters are also affected by chemical composition. Furthermore, the nature and extent of the cell response is dependent on cell maturation. The overriding variable in response to an implant material, however, appears to be roughness of the surface.

Alloys↗

PubMed1999

TGFbeta1 regulates 25-hydroxyvitamin D3 1alpha- and 24-hydroxylase activity in cultured growth plate chondrocytes in a maturation-dependent manner.

Chondrocytes metabolize 25-(OH)D3 to the two active dihydroxylated forms of the secosteroid, 1,25-(OH)2D3 and 24,25-(OH)2D3. The aim of the present study was to examine the activity of the enzymes responsible for this metabolism, 1alpha-hydroxylase and 24R-hydroxylase, and their regulation by TGFbeta1. Basal 1alpha- and 24R-hydroxylase activities were measured in homogenates of confluent, fourth passage rat costochondral resting zone and growth zone chondrocytes and mouse cortico-tubular cells (MCT) were used as a positive control. The cells were harvested and homogenized in buffer optimized to maintain the activity and stability of the hydroxylases. Homogenates were incubated for 90 minutes and 1alpha- and 24R-hydroxylase activities determined by measuring the conversion of [3H]-25-(OH)D3 to [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3 using an HPLC with an inline radioisotope detector. Resting zone cells were also treated with various concentrations of recombinant human TGFbeta1 for 24 hours, and enzyme activity in total cell homogenates as well as 24-hydroxylase mRNA levels were determined. In addition, [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3 released into the conditioned media by resting zone chondrocyte cultures in response to TGFbeta1 were measured. In culture, all three cell types were found to contain 1alpha- and 24R-hydroxylase activities. Basal 1alpha-hydroxylase specific activity was significantly higher than 24R-hydroxylase specific activity in all cells. RT-PCR confirmed that resting zone and growth zone cells expressed mRNA for 24R-hydroxylase. Treatment of resting zone cells with TGFbeta1 increased 24R-hydroxylase mRNA levels in a dose-dependent manner. TGFbeta1 also increased 24R-hydroxylase activity 2- to 5-fold and decreased 1alpha-hydroxylase activity by 20-30%. Similar changes were observed with MCT cells, but not growth zone cells. Production of [3H]-24,25-(OH)2D3 by resting zone cells increased with TGFbeta1 treatment, while [3H]-1,25-(OH)2D3 production decreased. The effect was time- and dose-dependent, correlating with hydroxylase activity and 24-hydroxylase gene expression. These results demonstrate that growth plate chondrocytes contain the necessary enzymes to produce 1, 25-(OH)2D3 and 24,25-(OH)2D3 from 25-(OH)D3. In addition, the activity of these enzymes in resting zone cells, but not growth zone cells, is regulated by TGFbeta1 by increasing gene transcription, indicating that cell maturation-dependent autocrine/paracrine pathways exist for regulating vitamin D metabolite production.

Animals↗

PubMed1999

Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2.

Earlier studies have shown that implant surface roughness influences osteoblast proliferation, differentiation, matrix synthesis and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones, such as 1,25-(OH)2D3, at the implant surface is also influenced by surface roughness and this effect is mediated by changes in prostaglandins. At present, it is not known which signaling pathways are involved in mediating cell response to surface roughness and how 1,25-(OH)2D3 treatment alters the activation of these pathways. This paper reviews a series of studies that have addressed this question. MG63 osteoblast-like cells were cultured on commercially pure titanium (cpTi) surfaces of two different roughnesses (Ra 0.54 and 4.92 microm) in the presence of control media or media containing 1,25-(OH)2D3 or 1,25-(OH)2D3 plus H8 (a protein kinase A inhibitor) or quinacrine (a phospholipase A2 inhibitor). At harvest, the effect of these treatments on cell number and alkaline phosphatase specific activity was measured. Compared to cultures grown on the smooth surface, cell number was reduced on the rough surface. 1,25-(OH)2D3 inhibited cell number on both surfaces and inhibition of protein kinase A in the presence of 1,25-(OH)2D3 restored cell number to that seen in the control cultures. Inhibition of phospholipase A2 in the presence of 1,25-(OH)2D3 caused a further reduction in cell number on the smooth surface, and partially reversed the inhibitory effects of 1,25-(OH)2D3 on the rough surface. Alkaline phosphatase specific activity was increased in cultures grown on the rough surface compared with those grown on the smooth surface; 1,25-(OH)2D3 treatment increased enzyme specific activity on both surfaces. Cultures treated with H8 and 1,25-(OH)2D3 displayed enzyme specific activity that approximated that seen in control cultures. Inhibition of phospholipase A2 also inhibited the 1,25-(OH)2D3-dependent effect on the smooth surface, but on the rough surface there was an inhibition of the 1,25-(OH)2D3 effect as well as a partial inhibition of the surface roughness-dependent effect. The results indicate that surface roughness and 1,25-(OH)2 D3 mediate their effects through phospholipase A2, which catalyzes one of the rate-limiting steps in prostaglandin E2 production. Further downstream, prostaglandin E2 activates protein kinase A.

Alkaline Phosphatase↗

PubMed1999

Prostaglandins mediate the effects of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes in a metabolite-specific and cell maturation-dependent manner.

Prior studies have shown that 1,25-(OH)2D3 stimulates alkaline phosphatase, phospholipase A2 (PLA2), and protein kinase C (PKC)-specific activities, and production of prostaglandin E2 (PGE2) in growth zone chondrocytes. In contrast, 24,25-(OH)2D3 stimulates alkaline phosphatase and PKC-specific activities but inhibits PLA2-specific activity and PGE2 production in resting zone cells. This indicates that different mechanisms are involved in the action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on their respective target cells. In this study, we examined the hypothesis that differential regulation of prostaglandin production modulates the activity of PKC and alkaline phosphatase. To do this, we examined the effect of the cyclooxygenase inhibitor indomethacin (Indo) on alkaline phosphatase, PLA2, and PKC-specific activities in growth plate chondrocytes treated with these two vitamin D metabolites. In addition, we examined whether inhibition of PKC altered PGE2 production. In growth zone cells, Indo inhibited basal alkaline phosphatase and blocked the 1,25-(OH)2D3-dependent increase in alkaline phosphatase. This effect was due to inhibition of both plasma membrane and matrix vesicle alkaline phosphatase. In resting zone cells, Indo increased basal alkaline phosphatase activity in a dose-dependent manner, but it did not further enhance the 24,25-(OH)2D3-dependent stimulation of this enzyme. The effect of Indo was found in both plasma membranes and matrix vesicles. These data indicate that 1,25-(OH)2D3-dependent increases in alkaline phosphatase-specific activity in growth zone cells are mediated through increased prostaglandin production, whereas 24,25-(OH)2D3-mediated changes in enzyme activity in resting zone cells are mediated through decreased prostaglandin production. Regulation of PLA2 by either 1,25-(OH)2D3 or 24,25-(OH)2D3 in their target cells was unaffected by Indo, indicating that the effect of the vitamin D metabolites on this enzyme is not dependent on changes in PGE2 production. The rapid increase in 1,25-(OH)2D3-dependent PKC-specific activity in growth zone cells was inhibited by Indo, whereas there was a potentiation of the effect of 24,25-(OH)2D3 on PKC activity in resting zone cells. In addition, inhibition of PKC blocked the 1,25-(OH)2D3-dependent increase in PGE2 production in growth zone cells and the 24,25-(OH)2D3-dependent decrease in PGE2 production by resting zone cells. These data indicate that prostaglandins are involved in mediating the rapid effects of 1,25-(OH)2D3 on growth zone cells, and contribute to the effects of 24,25-(OH)2D3 on resting zone cells; in both instances, the vitamin D metabolites exert their effects on PKC through changes in arachidonic acid via the action of PLA2. In addition, PKC by itself may mediate the production of PGE2.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

Implant surface characteristics modulate differentiation behavior of cells in the osteoblastic lineage.

This paper reviews the role of surface roughness in the osteogenic response to implant materials. Cells in the osteoblast lineage respond to roughness in cell-maturation-specific ways, exhibiting surface-dependent morphologies and growth characteristics. MG63 cells, a human osteoblast-like osteosarcoma cell line, respond to increasing surface roughness with decreased proliferation and increased osteoblastic differentiation. Alkaline phosphatase activity and osteocalcin production are increased. Local factor production is also affected; production of both TGF-beta 1 and PGE2 is increased. On rougher surfaces, MG63 cells exhibit enhanced responsiveness to 1,25-(OH)2D3. Prostaglandins mediate the effects of surface roughness, since indomethacin prevents the increased expression of differentiation markers in these cells.

Animals↗

PubMed1999

Potential mechanisms for the plasmin-mediated release and activation of latent transforming growth factor-beta1 from the extracellular matrix of growth plate chondrocytes.

Chondrocytes produce latent transforming growth factor-beta1 (TGF-beta1) in a small, circulating form of 100 kDa and also store latent TGF-beta1 in their matrix in a large form of 290 kDa containing the latent TGF-beta1 binding protein 1. As growth plate cartilage cells are exceptionally sensitive to TGF-beta1 and are known to produce plasminogen activator, the role of plasmin in the activation of soluble and matrix-bound latent TGF-beta1 was examined. As is true for other cell types, low-dose plasmin (0.01 U/ml) was found to release both active and latent TGF-beta1 from chondrocyte matrix in a time-dependent manner over 3 h. However, high-dose plasmin (1.0 U/ml) was found to release active TGF-beta1 more rapidly than low-dose plasmin, and this release ceased within 30 min; latent complex continued to be released over time (3 h). When high-dose plasmin was titrated against the serine protease inhibitors, aprotinin and alpha-(2-aminoethyl)benzenesulfonyl fluoride, results similar to low-dose plasmin were obtained, indicating that the effects of high-dose plasmin could be altered to mimic those of low-dose plasmin. No differences were observed on the effects of plasmin on the release of TGF-beta1 from the matrices of either growth zone or resting zone chondrocytes. We examined whether plasmin could further activate the truncated large latent TGF-beta1 complex of 230 kDa that was released into the media by plasmin. It is known that plasmin will activate the small latent complex, so this was compared with the truncated form. Plasmin completely activated the small latent complex, whereas a smaller, but significant, activation of the truncated form of latent TGF-beta1 also occurred. These studies may have relevance to normal physiological conditions, where plasminogen and/or plasmin is present in very small amounts in the cartilage and, therefore, small amounts of active TGF-beta1 would be present, and to pathological conditions such as fractures, where chondroprogenitor cells would be exposed to high concentrations of plasmin and, therefore, to short-term high concentrations of this potent chondrogenic growth factor.

Animals↗

PubMed1999

Arachidonic acid directly mediates the rapid effects of 24,25-dihydroxyvitamin D3 via protein kinase C and indirectly through prostaglandin production in resting zone chondrocytes.

Prior studies have shown that 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] plays a major role in resting zone chondrocyte differentiation and that this vitamin D metabolite regulates both phospholipase A2 and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipase A2 action and has been shown in other systems to affect a variety of cellular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH)2D3 on markers of proliferation, differentiation, and matrix production in resting zone chondrocytes and to characterize the mechanisms by which arachidonic acid regulates PKC, which was shown previously to mediate the rapid effects of 24,25-(OH)2D3 and arachidonic acid on these cells. Confluent, fourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [3H]thymidine incorporation and inhibited the activity of alkaline phosphatase and PKC, but had no effect on proteoglycan sulfation. In contrast, 24,25-(OH)2D3 inhibited [3H]thymidine incorporation and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC activity. In cultures treated with both agents, the effects of 24,25-(OH)2D3 were reversed by arachidonic acid. The PKC isoform affected by arachidonic acid was PKCalpha; cytosolic levels were decreased, but membrane levels were unaffected, indicating that translocation did not occur. Arachidonic acid had a direct effect on PKC in isolated plasma membranes and matrix vesicles, indicating a nongenomic mechanism. Plasma membrane PKCalpha was inhibited, and matrix vesicle PKCzeta was stimulated; these effects were blocked by 24,25-(OH)2D3. Studies using cyclooxygenase and lipoxygenase inhibitors indicate that the effects of arachidonic acid are due in part to PG production, but not to leukotriene production. This is supported by the fact that H8-dependent inhibition of protein kinase A, which mediates the effects of PGE2, had no effect on the direct action of arachidonic acid but did mediate the role of arachidonic acid in the cell response to 24,25-(OH)2D3. Diacylglycerol does not appear to be involved, indicating that phospholipase C and/or D do not play a role. Gamma-linolenic acid, an unsaturated precursor of arachidonic acid, elicited a similar response in matrix vesicles but not plasma membranes, whereas palmitic acid, a saturated fatty acid, had no effect. These data suggest that arachidonic acid may act as a negative regulator of 24,25-(OH)2D3 action in resting zone chondrocytes.

24,25-Dihydroxyvitamin D 3↗

PubMed1999

Altered expression of bone sialoproteins in vitamin D-deficient rBSP2.7Luc transgenic mice.

Bone sialoprotein (BSP) and osteopontin (OPN) are two major noncollagenous matrix proteins in mineralized connective tissue that have discrete roles in bone matrix formation, mineralization, and remodeling. The osteotropic secosteroid, 1,25-dihydroxyvitamin D3, a potent regulator of bone remodeling required for normal bone development, has been shown to exert differential effects on OPN and BSP expression by bone cells in vitro. To investigate these effects in vivo, we induced vitamin D3 deficiency in a transgenic mouse line (rBSP2.7Luc) that has a 2.7 kb rat BSP promoter linked to a luciferase reporter gene in its genome. Pregnant rBSP2.7Luc mice were fed vitamin D3-deficient food and demineralized water for 6 weeks. Their offspring were weaned at 3 weeks of age and then fed vitamin D-deficient food for an additional week. The control group were fed normal rodent pellets and water during the entire experimental procedure. Bone tissues from 40, 4-week-old offspring in each group were analyzed for BSP, OPN and luciferase expression. Vitamin D3-deficient mice displayed a rachitic phenotype that included reduced size and malformation of bones. Assays of the BSP promoter transgene in calvariae, mandibles, and tibiae of the rachitic mice showed increases in luciferase activity of 3.1-, 1.9-, and 4.6-fold, respectively, when compared with control littermates. Semiquantitative reverse transcriptase polymerase chain reaction assays of BSP mRNA revealed increases of 7-, 74-, and 66-fold, respectively, in the same rachitic bones, while OPN mRNA was reduced 12.5-fold in calvariae and 2-fold in tibiae and mandibles. In situ hybridization using mouse cRNA probes revealed that the increased BSP expression and decreased OPN expression in the vitamin D3-deficient mice was primarily in osteoblastic cells on the surface of calvariae and endosteal spaces of alveolar bone, on newly formed epiphyseal bone, and in cementoblasts and in hypertrophic chondrocytes. These studies are the first to show that BSP and OPN are differentially regulated by vitamin D3 in vivo, reflecting the diverse roles of these protein in bone remodeling. Moreover, the increased expression of the BSP transgene in the rachitic mice demonstrates that vitamin D3 regulation of BSP expression is mediated, in part, by element(s) within the 2.7 kb promoter region.

Animals↗

PubMed1999

Physiological importance of the 1,25(OH)2D3 membrane receptor and evidence for a membrane receptor specific for 24,25(OH)2D3.

We have recently identified a membrane vitamin D receptor (mVDR) specific for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and shown that it mediates the rapid activation of protein kinase C (PKC) in growth zone chondrocytes (GCs). In this study, we examine the role of the 1, 25(OH)2D3-mVDR in chondrocyte physiology and provide evidence for the existence of a specific membrane receptor for 24, 25-dihydroxyvitamin D3 (24,25(OH)2D3-mVDR). Fourth-passage cultures of growth plate chondrocytes at two distinct stages of endochondral development, resting zone (RC) and growth zone (GC) cells, were used to assess the role of the mVDR in cell proliferation, PKC activation, and proteoglycan sulfation. To preclude the involvement of the nuclear vitamin D receptor (nVDR), we used hybrid analogs of 1, 25(OH)2D3 with <0.1% affinity for the nVDR (2a, 1alpha-CH2OH-3beta-25D3; 3a, 1alpha-CH2OH-3beta-20-epi-22-oxa-25D3; and 3b, 1beta-CH2OH-3alpha-20-epi-22-oxa-25D3). To determine the involvement of the mVDR, we used an antibody generated against the highly purified 1,25(OH)2D3 binding protein from chick intestinal basolateral membranes (Ab99). Analog binding to the mVDR was demonstrated by competition with [3H]1,25(OH)2D3 using matrix vesicles (MVs) isolated from cultures of RC and GC cells. Specific recognition sites for 24,25(OH)2D3 in RC MVs were demonstrated by saturation binding analysis. Specific binding of 24,25(OH)2D3 was also investigated in plasma membranes (PMs) from RC and GC cells and GC MVs. In addition, we examined the ability of Ab99 to block the stimulation of PKC by analog 2a in isolated RC PMs as well as the inhibition of PKC by analog 2a in GC MVs. Like 1,25(OH)2D3, analogs 2a, 3a, and 3b inhibit RC and GC cell proliferation. The effect was dose dependent and could be blocked by Ab99. In GC cells, PKC activity was stimulated maximally by analogs 2a and 3a and very modestly by 3b. The effect of 2a and 3a was similar to that of 1, 25(OH)2D3 and was blocked by Ab99, whereas the effect of 3b was unaffected by antibody. In contrast, 2a was the only analog that increased PKC activity in RC cells, and this effect was unaffected by Ab99. Analog 2a had no effect on proteoglycan sulfation in RC cells, whereas analogs 3a and 3b stimulated it and this was not blocked by Ab99. Binding of [3H]1,25(OH)2D3 to GC MVs was displaced completely with 1,25(OH)2D3 and analogs 2a, 3a, and 3b, but 24, 25(OH)2D3 only displaced 51% of the bound ligand. 24,25(OH)2D3 displaced 50% of [3H]1,25(OH)2D3 bound to RC MVs, but 2a, 3a, and 3b displaced <50%. Scatchard analysis indicated specific binding of 24, 25(OH)2D3 to recognition sites in RC MVs with a Kd of 69.2 fmol/ml and a Bmax of 52.6 fmol/mg of protein. Specific binding for 24, 25(OH)2D3 was also found in RC and GC PMs and GC MVs. GC membranes exhibited lower specific binding than RC membranes; MVs had greater specific binding than PMs in both cell types. 2a caused a dose-dependent increase in PKC activity of RC PMs that was unaffected by Ab99; it inhibited PKC activity in GC MVs, and this effect was blocked by Ab99. The results indicate that the 1, 25(OH)2D3 mVDR mediates the antiproliferative effect of 1,25(OH)2D3 on chondrocytes. It also mediates the 1,25(OH)2D3-dependent stimulation of PKC in GC cells, but not the 2a-dependent increase in RC PKC activity, indicating that 24,25(OH)2D3 mediates its effects through a separate receptor. This is supported by the failure of Ab99 to block 2a-dependent stimulation of PKC in isolated PMs. The data demonstrate for the first time the presence of a specific 24, 25(OH)2D3 mVDR in endochondral chondrocytes and show that, although both cell types express mVDRs for 1,25(OH)2D3 and 24,25(OH)2D3, their relative distribution is cell maturation-dependent.

Animals↗

PubMed1999

Transforming growth factor-beta1 modulates chondrocyte responsiveness to 17beta-estradiol.

This study examined the interrelationship between transforming growth factor-beta1 (TGF-beta1) and 17beta-estradiol (E2) in the regulation of growth plate chondrocytes. To determine whether TGF-beta1 modulates chondrocyte response to E2, we used cells isolated from the resting zone (RC) and growth zone (GC) of costochondral cartilage. Confluent, fourth-passage cultures were pretreated with rhTGF-beta1 for 24 h, followed by treatment with E2 for 24 h. The effect of TGF-beta1 and E2 alone, or the sequential combination, were examined by measuring [3H]-thymidine incorporation (proliferation), alkaline phosphatase (AP) specific activity (differentiation), and [35S]-sulfate incorporation (matrix synthesis). TGF-beta1 alone increased [3H]-thymidine incorporation in both female and male RC and GC cells, but E2 affected this parameter only in RC cells, causing a dose-dependent decrease. At the highest concentration of TGF-beta1 and E2, [3H]-thymidine incorporation in female GC cells was the same as seen in untreated control cultures. In male GC cells, [3H]-thymidine incorporation in cultures treated with TGF-beta1 and E2 exhibited a comparable increase, as was seen in cultures treated with TGF-beta1 alone. TGF-beta1 caused a biphasic stimulation in AP that was maximal at 0.22 ng/mL, in both female and male RC and GC cells. E2, however, affected only female cells. Whereas the effect of TGF-beta1 predominated in RC and GC male cells, the biphasic stimulation caused by E2, maximal at 109 M, predominated in female RC cells. In female GC cells, however, TGF-beta1 caused a synergistic response, resulting in enhanced AP specific activity in cultures pretreated with 0.22 ng/mL of TGF-beta1 and 10(-8) M E2. TGF-beta1 alone caused dose-dependent increases in [35S]-sulfate incorporation in female RC and GC cells, as well as in male GC cells, but had no effect on male RC cells. E2 affected only female cells. TGF-beta1 potentiated the effect of E2 on this parameter, resulting in synergistic increases in the female cells. This is the first demonstration of a gender-specific response to TGF-beta1 in chondrocytes. These results suggest that chondrocyte response to a systemic hormone such as E2 can be modulated by local regulatory agents such as TGF-beta1.

Alkaline Phosphatase↗

PubMed1999

Evaluation of 2 novel approaches for assessing the ability of demineralized freeze-dried bone allograft to induce new bone formation.

BACKGROUND: Because of the wide variation in the ability of human demineralized freeze-dried bone allograft (DFDBA) to reproducibly induce new bone formation, there is a need for a reliable measure of bone induction activity. In this study we examined an immature osteoprogenitor cell line for its potential utility in measuring the activity of DFDBA in vitro. METHODS: We characterized the response of 2T9 cells, an immature osteoprogenitor cell line derived from the calvariae of transgenic mice containing the SV40 T-antigen driven by the mouse bone morphogenetic protein (BMP)-2 promoter, to recombinant human BMP-2 by measuring alkaline phosphatase specific activity, osteocalcin production, and matrix mineralization. Responses were compared to those obtained with 1,25-(OH)2D3. In addition, 2T9 cells were cultured with active or inactive human DFDBA in the presence or absence of BMP-2. We also tested the hypothesis that radio-opacity of tissue following implantation of DFDBA in vivo correlates with the ability of human DFDBA to induce new bone. DFDBA from 9 different donors, stratified by age, were implanted subcutaneously in the thorax of 18 nude (nu/nu) mice. Tissue was harvested at 36 days postoperatively and examined histologically and biochemically for calcium and phosphorus uptake. RESULTS: 2T9 cells exhibited a dose- and time-dependent response to soluble BMP-2. Proliferation was decreased and alkaline phosphatase activity, osteocalcin production, and mineralized nodule formation were increased. The effects were dose- and time-dependent. Peak effects on alkaline phosphatase and osteocalcin were noted on day 8, whereas mineral deposition did not begin to occur until day 12. 1,25-(OH)2D3 did not regulate these effects unless used with BMP-2. When the cells were exposed to active or inactive DFDBA in the presence or absence of BMP-2, no effect on 2T9 cell differentiation was observed. This indicated that DFDBA released no soluble factors with bone inductive ability and that if any active factors were adsorbed to the DFDBA, they were inactivated. When DFDBA was implanted subcutaneously in the thorax of nude mice, there was no histologic evidence of new bone formation. However, there was a donor age-dependent decrease in Ca and P uptake of the implanted tissue, reflecting a donor age-dependent decrease in remineralization of DFDBA. CONCLUSIONS: These data indicate that cell culture assays like the one used in this study may not be appropriate indicators of bone induction ability by DFDBA since soluble factors may not be responsible for bone induction in vivo. Nonetheless, in vitro assays are still needed. While Ca and P uptake by DFDBA-implanted tissue in the present study correlated with the age-dependent decrease in bone induction at intramuscular sites in a previously reported study, these data show that early x-rays may actually detect remineralization and not new bone formation. Thus, assessment of bone induction ability may still depend on histologic analysis of animal models.

Age Factors↗

PubMed1999

The effect of ultra-high molecular weight polyethylene wear debris on MG63 osteosarcoma cells in vitro.

BACKGROUND: Focal osteolysis due to ultra-high molecular weight polyethylene wear debris involves effects on both bone resorption and bone formation. METHODS: The response of MG63 osteoblast-like osteosarcoma cells to ultra-high molecular weight polyethylene wear debris isolated by enzymatic digestion of granulomatous tissue obtained from the sites of failed total hip arthroplasties was examined. Scanning electron microscopy, particle-size analysis, and Fourier transform infrared spectroscopy were used to characterize the number, morphology, size distribution, and chemical composition of the particles. Cell response was assessed by adding particles at varying dilutions to confluent cultures and measuring changes in cell proliferation (number of cells and [3H]-thymidine incorporation), osteoblast function (alkaline-phosphatase-specific activity and osteocalcin production), matrix production (collagen production and proteoglycan sulfation), and local cytokine production (prostaglandin-E2 production). RESULTS: The mean size of the particles was 0.60 micrometer, and 95 percent of the particles had a size of less than 1.5 micrometers. The number of particles per gram of tissue ranged from 1.39 to 3.38x10(9). Three of the four batches of particles were endotoxin-free. Exposure of the cells to particles of wear debris significantly increased the number of cells (p<0.05) and the [3H]-thymidine incorporation (p<0.05) in a dose-dependent manner. In contrast, the addition of particles decreased alkaline-phosphatase-specific activity and osteocalcin production. Collagen production and proteoglycan sulfation were also decreased, while prostaglandin-E2 synthesis was increased by the addition of particles. CONCLUSIONS: Ultra-high molecular weight polyethylene particles isolated from human tissue stimulated osteoblast proliferation and prostaglandin-E2 production and inhibited cell differentiation and matrix production. These results indicate that particles of wear debris inhibit cell functions associated with bone formation and that osteoblasts may produce factors in response to wear debris that influence neighboring cells, such as osteoclasts and macrophages. CLINICAL RELEVANCE: Particles of wear debris, especially ultra-high molecular weight polyethylene, have been implicated in the loosening of implants and the development of osteolysis. The present study shows that particles of ultra-high molecular weight polyethylene isolated from human tissue inhibit osteoblast functions associated with bone formation. In addition, particles of wear debris induced osteoblasts to secrete factors capable of influencing neighboring cells, such as osteoclasts and macrophages. These results suggest that osteoblasts may play a role in the cascade of events leading to granuloma formation, osteolysis, and failure of orthopaedic implants.

Adult↗

PubMed1999

Bone morphogenetic protein but not transforming growth factor-beta enhances bone formation in canine diaphyseal nonunions implanted with a biodegradable composite polymer.

BACKGROUND: The purpose of the present study was to create an effective bone-graft substitute for the treatment of a diaphyseal nonunion. METHODS: A standardized nonunion was established in the midportion of the radial diaphysis in thirty mongrel dogs by creating a three-millimeter segmental bone defect (at least 2 percent of the total length of the bone). The nonunion was treated with implantation of a carrier comprised of poly(DL-lactic acid) and polyglycolic acid copolymer (50:50 polylactic acid-polyglycolic acid [PLG50]) containing canine purified bone morphogenetic protein (BMP) or recombinant human transforming growth factor-beta (TGF-beta1), or both, or the carrier without BMP or TGF-beta1. Five groups, consisting of six dogs each, were treated with implantation of the carrier alone, implantation of the carrier with fifteen milligrams of BMP, implantation of the carrier with 1.5 milligrams of BMP, implantation of the carrier with fifteen milligrams of BMP and ten nanograms of TGF-beta1, or implantation of the carrier with ten nanograms of TGF-beta1. At twelve weeks after implantation, the radii were examined radiographically and the sites of nonunion were examined histomorphometrically. RESULTS: We found that implantation of the polylactic acid-polyglycolic acid carrier alone or in combination with ten nanograms of TGF-beta1 failed to induce significant radiographic or histomorphometric evidence of healing at the site of the nonunion. The radii treated with the carrier enriched with either 1.5 or fifteen milligrams of BMP showed significantly increased periosteal and endosteal bone formation on histomorphometric (p < 0.05) and radiographic (p < 0.02) analysis. CONCLUSIONS: Bone formation in a persistent osseous defect that is similar to an ununited diaphyseal fracture is increased when species-specific BMP incorporated into a polylactic acid-polyglycolic acid carrier is implanted at the site of the nonunion. TGF-beta1 at a dose of ten nanograms per implant did not induce a similar degree of bone formation or potentiate the effect of BMP in this model. CLINICAL RELEVANCE: The biodegradable implant containing BMP that was used in the present study to treat diaphyseal nonunion is an effective bone-graft substitute.

Absorbable Implants↗