Biomedical subjects

B D Boyan

Publications and source records attributed to B D Boyan.

At least 109 records · Page 6Linked to original sources

PubMed1999

Woven bone formation around implants and the effect of bacterial infection.

Several implant materials used in dental and orthopedic surgery were placed in rat tibial bones to study their effects on mineralization. The implants consisted of bone bonding and non-bonding materials. Changes in mineralization were defined by morphometric analysis of matrix vesicle distribution at the implant interface and in normal bone healing following marrow injury. Bone-bonding materials induced an increase in matrix vesicle activity. This finding was supported by study of the biochemical changes in the same model that manifested high correlations to the morphometrical observations with regard to enhancement or delay of primary mineralization. In addition, the study of healing using nuclear methods indicated that implants alter bone healing as shown by the different uptakes of 99mTc and 32P in the different bone compartments. Decreased 32P uptake by the organic phase in the presence of bone-bonding implants suggested that cleavage of 99mTc-MD32P into its technetium and methylene diphosphonate moieties was inhibited by administration of implants. Further studies on the effect of bacterial infection on the peri-implant tissues revealed a decrease in woven bone formation due to infection.

Animals↗

PubMed1999

Bone and cartilage tissue engineering.

Tissue engineering of musculoskeletal tissues, particularly bone and cartilage, is a rapidly advancing field. In bone, technology has centered on bone graft substitute materials and the development of biodegradable scaffolds. Recently, tissue engineering strategies have included cell and gene therapy. The availability of growth factors and the expanding knowledge base concerning the genetics and regulation of bone formation have generated new materials for tissue-engineering applications. This information base also has benefited cartilage tissue engineering. The problems are more complex, however, and the solutions appear more elusive. Advances in scaffold design and cell culture have improved the prognosis for success.

Animals↗

PubMed1998

Prostaglandins mediate the effects of titanium surface roughness on MG63 osteoblast-like cells and alter cell responsiveness to 1 alpha,25-(OH)2D3.

Surface roughness affects proliferation, differentiation (alkaline phosphatase and osteocalcin), local factor production (transforming growth factor (TGF beta) and prostaglandin E2 (PGE2)], and response to 1,25-(OH)2D3 (1,25) of MG63 osteoblast-like cells. In this study, we examined whether the effect of surface roughness on MG63 cells is mediated by prostaglandins produced by the cells. Unalloyed titanium (Ti) disks were pretreated with HF/HNO3 (PT) and then machined and acid-etched (MA). Disks were also coarse grit-sandblasted (SB), coarse grit-sandblasted and acid-etched (CA), or plasma-sprayed with Ti particles (PS). The surfaces, from smoothest to roughest, were PT, MA, CA, SB, and PS. MG63 cells were cultured to confluence on the Ti disks in the presence or absence of 10(-7) M indomethacin (Indo), a specific inhibitor of cyclooxygenase activity, resulting in decreased prostaglandin production. When the cells reached confluence, cell number, cell layer alkaline phosphatase specific activity (ALPase), and osteocalcin (OC) and latent TGF beta (LTGF beta) production were determined. In addition, confluent cultures which had been grown in the absence of Indo were exposed to 10(-7) M 1,25, 10(-7) M Indo, or a combination of the two for 24 h. On the rougher surfaces, cell number was decreased and ALPase, OC, and LTGF beta were increased. When indomethacin was present throughout the culture period, the effect of surface roughness on cell number, OC, and LTGF beta was abolished. ALPase was reduced, but surface roughness-dependent effects were still observed. Addition of indomethacin to confluent cultures for 24 h had no effect on any of the parameters examined, with one exception: Cells cultured on MA surfaces exhibited a more differentiated phenotype. 1,25 increased all parameters examined on SB, CA, and PS surfaces. When indomethacin was added with 1,25, the 1,25-dependent effects on cell number and OC and LTGF beta production were abolished; however, ALPase was unaffected. This indicates that bone cell response to systemic hormones may be modified by implant surface roughness. This effect may be mediated, at least in part, by prostaglandins produced by the same cells.

Alkaline Phosphatase↗

PubMed1998

Titanium surface roughness alters responsiveness of MG63 osteoblast-like cells to 1 alpha,25-(OH)2D3.

Surface roughness has been shown to affect differentiation and local factor production of MG63 osteoblast-like cells. This study examined whether surface roughness alters cellular response to circulating hormones such as 1 alpha,25-(OH)2D3. Unalloyed titanium (Ti) disks were pretreated with HF/HNO3 (PT) and then were machined and acid-etched (MA). Ti disks also were sandblasted (SB), sandblasted and acid etched (CA), or plasma sprayed with Ti particles (PS). The surfaces, from smoothest to roughest, were: PT, MA, CA, SB, and PS. MG63 cells were cultured to confluence on standard tissue culture polystyrene (plastic) or the Ti surfaces and then treated for 24 h with either 10(-8) M or 10(-7) M 1 alpha,25-(OH)2D3 or vehicle (control). Cellular response was measured by assaying cell number, cell layer alkaline phosphatase specific-activity, and the production of osteocalcin, latent (L) TGF beta, and PGE2. Alkaline phosphatase activity was affected by surface roughness; as the surface became rougher, the cells showed a significant increase in alkaline phosphatase activity. Addition of 1 alpha,25-(OH)2D3 to the cultures caused a dose-dependent stimulation of alkaline phosphatase activity that was synergistic with the effect caused by surface roughness alone. 1 alpha,25-(OH)2D3 also caused a synergistic increase in osteocalcin production as well as local factor (LTGF beta and PGE2) production on the rougher CA, SB, and PS surfaces, but it had no effect on the production on smooth surfaces. The inhibitory effect of surface roughness on cell number was not affected by 1 alpha,25-(OH)2D3 except on the SB surface. 1 alpha,25-(OH)2D3 decreased cell number, increased alkaline phosphatase activity and osteocalcin production, and had no effect on LTGF beta or PGE2 production by MG63 cells grown on tissue culture polystyrene. These data suggest that bone cell response to systemic hormones is modified by surface roughness and that surface roughness increases the responsiveness of MG63 cells to 1 alpha,25-(OH)2D3. They also suggest that the endocrine system is actively involved in normal bone healing around implants.

Alkaline Phosphatase↗

PubMed1998

17beta-estradiol regulation of protein kinase C activity in chondrocytes is sex-dependent and involves nongenomic mechanisms.

17Beta-estradiol (E2) regulates growth plate chondrocyte differentiation in both a sex- and cell maturation-dependent manner, and the sex-specific effects of E2 appear to be mediated in part by membrane events. In this study, we examined whether E2 regulates protein kinase C (PKC) in a cell-maturation and sex-specific manner and whether E2 uses a nongenomic mechanism in regulating this enzyme. In addition, we determined if PKC mediates the E2-dependent stimulation of alkaline phosphatase activity seen in chondrocytes. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from male and female rat costochondral cartilage were treated with 10(-10) to 10(-7) M E2. E2 caused a dose-dependent increase in PKC in RC and GC cells from female rats. Peak stimulation was at 90 min. Increased PKC was evident by 3 min in both RC and GC and was still evident in RC cells at 720 min, but in GC cells activity returned to baseline by 270 min. Actinomycin D had no effect at 9, 90, 270, or 720 min, but there was a small decrease in E2-stimulated PKC in RC treated with cycloheximide at 90 and 270 min and in GC treated for 90 min. E2 increased cytosolic and membrane PKC at 9 min and by 90 min promoted translocation of PKC activity from the cytosol to the membranous compartment of female RC cells. Antibodies specific for the alpha, beta, delta, epsilon, and zeta isoforms of PKC revealed that PKCalpha in female GC and RC cells is activated by E2. There was a small, but statistically significant, increase in PKC in male RC cells in response to E2, but it was not dose-dependent, and no effect of E2 was noted in male GC cells. 17Alpha-estradiol, an inactive isomer of E2, did not affect PKC specific activity in RC or GC cells from either female or male rats. Chelerythrine, a specific inhibitor of PKC, inhibited E2-dependent alkaline phosphatase activity, indicating that E2 mediates its rapid effects on alkaline phosphatase via PKC.

Alkaline Phosphatase↗

PubMed1998

Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes.

Prior studies have shown that 24,25-(OH)2D3 and 1,25-(OH)2D3 regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)2D3 affecting primarily growth zone (GC) cells and 24,25-(OH)2D3 affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)2D3 has been shown to increase phospholipase A2 activity in GC, while 24,25-(OH)2D3 has been shown to decrease phospholipase A2 activity in RC. Stimulation of phospholipase A2 in GC caused an increase in PKC, whereas inhibition of phospholipase A2 activity in RC cultures increased both basal and 24,25-(OH)2D3-induced PKC activity, suggesting that phospholipase A2 may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A2 inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A2 activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A2 inhibitors decreased both basal and 1,25-(OH)2D3-induced PKC activity in GC. When phospholipase A2 activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)2D3-induced PKC activity in RC and increased 1,25-(OH)2D3-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A2 activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A2 activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A2. These effects are cell maturation- and time-dependent and metabolite-specific.

24,25-Dihydroxyvitamin D 3↗

PubMed1998

Growth plate chondrocytes store latent transforming growth factor (TGF)-beta 1 in their matrix through latent TGF-beta 1 binding protein-1.

Osteoblasts produce a 100 kDa soluble form of latent transforming growth factor beta (TGF-beta) as well as a 290 kDa form containing latent TGF-beta binding protein-1 (LTBP1), which targets the latent complex to the matrix for storage. The nature of the soluble and stored forms of latent TGF-beta in chondrocytes, however, is not known. In the present study, resting zone and growth zone chondrocytes from rat costochondral cartilage were cultured to fourth passage and then examined for the presence of mRNA coding for LTBP1 protein. In addition, the matrix and media were examined for LTBP1 protein and latent TGF-beta. Northern blots, RT-PCR, and in situ hybridization showed that growth zone cells expressed higher levels of LTBP1 mRNA in vitro than resting zone cells. Immunohistochemical staining for LTBP1 revealed fine fibrillar structures around the cells and in the cell matrix. When the extracellular matrix of these cultures was digested with plasmin, LTBP1 was released, as determined by immunoprecipitation. Both active and latent TGF-beta1 were found in these digests by TGF-beta1 ELISA and Western blotting. Immunoprecipitation demonstrated that the cells also secrete LTBP1 which is not associated with latent TGF-beta, in addition to LTBP1 that is associated with the 100 kDa latent TGF-beta complex. These studies show for the first time that latent TGF-beta is present in the matrix of costochondral chondrocytes and that LTBP1 is responsible for storage of this complex in the matrix. The data suggest that chondrocytes are able to regulate both the temporal and spatial activation of latent TGF-beta, even at sites distant from the cell, in a relatively avascular environment.

Animals↗

PubMed1998

Purification, amino acid sequence, and cDNA sequence of a novel calcium-precipitating proteolipid involved in calcification of corynebacterium matruchotii.

Corynebacterium matruchotii is a microbial inhabitant of the oral cavity associated with dental calculus formation. It produces membrane-associated proteolipid capable of inducing hydroxyapatite formation in vitro. This proteolipid was purified from chloroform:methanol extracts by chromatography on Sephadex LH-20 and migrated on SDS-polyacrylamide gel electrophoresis at 6-9 kDa. Removal of covalently attached acyl moieties by methanolic KOH decreased its molecular mass to approximately 5.5 kDa. The amino acid sequence of the apoproteolipid indicated a peptide of 50 amino acids, a calculated molecular weight of 5354 Da, and an isoelectric point of 4.28. Sequence analysis revealed an 8 amino acid sequence with homology to human phosphoprotein phosphatase 2A as well as several potential acylation sites and one phosphorylation site. The purified proteolipid induced calcium precipitation in vitro. Deacylation of the proteolipid by hydroxylamine treatment resulted in >50% loss of calcium-precipitating activity, suggesting that covalently attached lipids are required. Degenerate oligonucleotide primers, based on the amino acid sequence, were used to amplify the gene for the 5.5 kDa proteolipid from total chromosomal DNA of C. matruchotii by PCR. A 166 bp cDNA was isolated and sequenced, confirming the amino acid sequence of the proteolipid. Thus, we have sequenced a unique bacterial proteolipid that is involved in the formation of dental calculus by precipitating Ca2+ and possibly in transport of inorganic phosphate, necessary for hydroxyapatite formation.

Amino Acid Sequence↗

PubMed1998

Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition.

The success of an implant is determined by its integration into the tissue surrounding the biomaterial. Surface roughness and composition are considered to influence the properties of adherent cells. The aim of this study was to determine the effect of chemical composition and surface roughness of commercially pure titanium (Ti) and Ti-6A1-4V alloy (Ti-A) on MG63 osteoblast-like cells. Unalloyed and alloyed Ti disks were machined and either fine-polished or wet-ground, resulting in smooth (S) and rough (R) finishes, respectively. Standard tissue culture plastic was used as a control. Surface topography and profile were evaluated by cold field emission scanning electron microscopy and profilometry, while chemical composition was determined using Auger electron spectroscopy and Fourier transform infrared spectroscopy. The effect on the cells was evaluated 24 h postconfluence by measuring cell number, [3H]-thymidine incorporation into DNA, cell and cell layer alkaline phosphatase specific activity (ALPase), osteocalcin and collagen production, [35S]-sulfate incorporation into proteoglycan, and prostaglandin E2 (PGE2) and transforming growth factor-beta (TGF-beta) production. When compared to plastic, the number of cells was reduced on the pure Ti surfaces, while it was equivalent on the Ti-A surfaces; [3H]-thymidine incorporation was reduced on all surfaces. The stimulatory effect of surface roughness on ALPase in isolated cells and the cell layer was more pronounced on the rougher surfaces, with enzyme activity on Ti-R being greater than on Ti-A-R. Osteocalcin production was increased only on the Ti-R surface. Collagen production was decreased on Ti surfaces except Ti-R; [35S]-sulfate incorporation was reduced on all surfaces. Surface roughness affected local factor production (TGF-beta, PGE2). The stimulatory effect of the rougher surfaces on PGE2 and TGF-beta was greater on Ti than Ti-A. In summary, cell proliferation, differentiation, protein synthesis and local factor production were affected by surface roughness and composition. Enhanced differentiation of cells grown on rough vs. smooth surfaces for both Ti and Ti-A surfaces was indicated by decreased proliferation and increased ALPase and osteocalcin production. Local factor production was also enhanced on rough surfaces, supporting the contention that these cells are more differentiated. Surface composition also played a role in cell differentiation, since cells cultured on Ti-R surfaces produced more ALPase than those cultured on Ti-A-R. While it is still unknown which material properties induce which cellular responses, this study suggests that surface roughness and composition may play a major role and that the best design for an orthopaedic implant is a pure titanium surface with a rough microtopography.

Alkaline Phosphatase↗

PubMed1998

Treatment of resting zone chondrocytes with transforming growth factor-beta 1 induces differentiation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25-(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3.

To determine if transforming growth factor-beta 1 (TGF-beta 1) can induce the differentiation of resting zone (RC) chondrocytes, confluent, fourth passage cultures of these cells were pretreated for 24, 36, 48, 72, and 120 h with TGF-beta 1. At the end of pretreatment, the media were replaced with new media containing 10(-10)-10(-8) mol/L 1,25-(OH)2D3 and the cells incubated for an additional 24 h. This second treatment was chosen because prior studies had shown that only the more mature growth zone (GC) chondrocytes respond to this vitamin D3 metabolite. The effect of TGF-beta pretreatment on cell maturation was assessed by measuring alkaline phosphatase (ALPase)-specific activity. In addition, changes in matrix protein synthesis were assessed by measuring collagen synthesis, as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 120 h with TGF-beta 1, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase-specific activity and collagen synthesis, with no effect on proteoglycan production. RC cells pretreated with 1,25(OH)2D3 responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)2D3; however, RC cultures pretreated for 120 h with TGF-beta 1 lost their responsiveness to 24,25-(OH)2D3. These results indicate that TGF-beta 1 directly regulates the maturation of RC chondrocytes into GC chondrocytes and support the hypothesis that this growth factor may play a significant role in regulating chondrocyte maturation during endochondral ossification.

24,25-Dihydroxyvitamin D 3↗

PubMed1998

The synergistic effects of vitamin D metabolites and transforming growth factor-beta on costochondral chondrocytes are mediated by increases in protein kinase C activity involving two separate pathways.

Transforming growth factor-beta (TGFbeta), as well as the vitamin D3 metabolites 1,25-dihydroxyvitamin D3 (1,25) and 24,25-dihydroxyvitamin D3 (24,25), regulate chondrocyte differentiation and maturation during endochondral bone formation. Both the growth factor and secosteroids also affect protein kinase C (PKC) activity, although each has its own unique time course of enzyme activation. Vitamin D3 metabolite effects are detected soon after addition to the media, whereas TGFbeta effects occur over a longer term. The present study examines the interrelation between the effects of 1,25, 24,25, and TGFbeta on chondrocyte differentiation, matrix production, and proliferation. We also examined whether the effect is hormone-specific and maturation-dependent and whether the effect of combining hormone and growth factor is mediated by PKC. This study used a chondrocyte culture model developed in our laboratory that allows comparison of chondrocytes at two stages of differentiation: the more mature growth zone (GC) cells and the less mature resting zone chondrocyte (RC) cells. Only the addition of 24,25 with TGFbeta showed synergistic effects on RC alkaline phosphatase-specific activity (ALPase). No similar effect was found when 24,25 plus TGFbeta was added to GC cells or when 1,25 plus TGFbeta were added to GC or RC cells. The addition of 1,25 plus TGFbeta and 24,25 plus TGFbeta to GC and RC cells, respectively, produced a synergistic increase in [35S]sulfate incorporation and had an additive effect on [3H]thymidine incorporation. To examine the signal transduction pathway involved in producing the synergistic effect of 24,25 and TGFbeta on RC cells, the level of PKC activity was examined. Addition of 24,25 and TGFbeta for 12 h produced a synergistic increase in PKC activity. Moreover, a similar effect was found when 24,25 was added for only the last 90 min of a 12-h incubation. However, a synergistic effect could not be found when 24,25 was added for the last 9 min or the first 90 min of incubation. To further understand how 24,25 and TGFbeta may mediate the observed synergistic increase in PKC activity, the pathways potentially leading to activation of PKC were examined. It was found that 24,25 affects PKC activity through production of diacylglycerol, not through activation of G protein, whereas TGFbeta only affected PKC activity through G protein. The results of the present study indicate that vitamin D metabolites and TGFbeta produced a synergistic effect that is maturation-dependent and hormone-specific. Moreover, the synergistic effect between 24,25 and TGFbeta was mediated by activation of PKC through two parallel pathways: 24,25 through diacylglycerol production and TGFbeta through G protein activation.

24,25-Dihydroxyvitamin D 3↗

PubMed1998

The effect of prostaglandin E2 on costochondral chondrocyte differentiation is mediated by cyclic adenosine 3',5'-monophosphate and protein kinase C.

Recent studies indicate that vitamin D metabolites exert rapid effects on growth plate chondrocytes via changes in PG production and protein kinase C (PKC) activity. This suggests that these two products of vitamin D action may be interrelated. To test this hypothesis, we examined the effect of PGE2 on rat costochondral resting zone and growth zone cartilage cells and determined whether the effects of PGE2 are mediated by changes in the level of cAMP and/or PKC activity, whether there is a relationship between cAMP production and PKC activity, and whether cell maturation-specific effects are involved. Confluent, fourth passage resting zone and growth zone cartilage cell cultures were incubated in DMEM containing 10% FBS, 50 microg/ml vitamin C, and 1% antibiotics. The PGE2 concentration was varied from 0.007-15 ng/ml. Low concentrations of PGE2 caused a dose-dependent increase in cell number and [3H]thymidine incorporation and stimulated alkaline phosphatase specific activity. These effects were comparable in resting zone and growth zone cartilage cells at the same PGE2 concentrations. At higher concentrations, PGE2 caused a general increase in the synthesis of collagenase-digestible protein and noncollagenase-digestible protein in resting zone cartilage cells and of collagenase-digestible protein in growth zone cartilage cells, resulting in a net increase in the percent collagen synthesis for both cell types. cAMP production was increased over the entire range of chondrocyte response. Prevention of cAMP metabolism with the protein kinase A inhibitors H-8 and H-89 blocked the PGE2-dependent inhibition of PKC in resting zone cartilage cells in a dose-dependent manner. H-8 alone had no effect on PKC in resting zone cartilage cells, but stimulated PKC activity in growth zone cartilage cells; H-89 alone stimulated PKC activity in resting zone cartilage cells. These results suggest that low levels of PGE2 promote differentiation, whereas high doses promote an anabolic response; PGE2 increases cAMP production and PKC activity in a cell maturation-dependent manner; PGE2 exerts its effects via cAMP production and PKC activity; and regulation of PGE2-dependent PKC is via cAMP.

Alkaline Phosphatase↗

PubMed1998

1,25(OH)2D3 regulates protein kinase C activity through two phospholipid-dependent pathways involving phospholipase A2 and phospholipase C in growth zone chondrocytes.

We have previously shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 1,25(OH)2D3 to stimulate PKC activation. Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of GC cultures with 1,25(OH)2D3 elicited a dose-dependent increase in both inositol-1,4,5-trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D. Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity. Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH)2D3-treated GCs, indicating that neither Gi nor Gs proteins were involved. Phospholipase A2 inhibitors, quinacrine, OEPC (selective for secretory phospholipase A2), and AACOCF3 (selective for cytosolic phospholipase A2), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A2 activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E2, two downstream products of phospholipase A2 action, also increased PKC activity. These results indicate that 1,25(OH)2D3-dependent stimulation of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A2.

Animals↗

PubMed1998

Identification of a membrane receptor for 1,25-dihydroxyvitamin D3 which mediates rapid activation of protein kinase C.

This paper is the first definitive report demonstrating a unique membrane receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) which mediates the rapid and nongenomic regulation of protein kinase C (PKC). Previous studies have shown that 1,25(OH)2D3 exerts rapid effects on chondrocyte membranes which are cell maturation-specific, do not require new gene expression, and do not appear to act via the traditional vitamin D receptor. We used antiserum generated to a [3H]1,25(OH)2D3 binding protein isolated from the basal lateral membrane of chick intestinal epithelium (Ab99) to determine if rat costochondral resting zone (RC) or growth zone (GC) cartilage cells contain a similar protein and if cell maturation-dependent differences exist. Immunohistochemistry demonstrated that both RC and GC cells express the protein, but levels are highest in GC. The binding protein is present in both plasma membranes and matrix vesicles and has a molecular weight of 66,000 Da. The 66 kDa protein in GC matrix vesicles has a Kd of 17.2 fmol/ml and Bmax of 124 fmol/mg of protein for [3H]1,25(OH)2D3. In contrast, the 66 kDa protein in RC matrix vesicles has a Kd of 27.7 fmol/ml and a Bmax of 100 fmol/mg of protein. Ab99 blocks the 1,25(OH)2D3-dependent increase in PKC activity in GC chondrocytes, indicating that the 1,25(OH)2D3-binding protein is indeed a receptor, linking ligand recognition to biologic function.

Animals↗

PubMed1998

Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3.

To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2. At the end of pretreatment, the media were replaced with new media containing 10(-10)-10(-8) M 1,25-(OH)2D3 or 10(-9)-10(-7) M 24,25-(OH2)D3 and the cells incubated for an additional 24 h. This second treatment was chosen, because prior studies had shown that the more mature growth zone (GC) chondrocytes and RC cells respond to 1,25-(OH)2D3 and 24,25-(OH)2D3 in distinctly different ways with respect to the parameters examined. The effect of BMP-2 pretreatment on cell maturation was assessed by measuring alkaline phosphatase specific activity (ALPase). In addition, changes in matrix protein production were assessed by measuring collagen synthesis, as well as [35S]-sulfate incorporation into proteoglycans. When RC cells were pretreated for 72 or 120 h with BMP-2, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase specific activity and collagen synthesis, with no effect on proteoglycan sulfation. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)2D3; however, RC cultures pretreated for 72 or 120 h with BMP-2 lost their responsiveness to 24,25-(OH)2D3. These results indicate that BMP-2 directly regulates the differentiation and maturation of RC chondrocytes into GC chondrocytes. These observations support the hypothesis that BMP-2 plays a significant role in regulating chondrocyte maturation during endochondral ossification.

24,25-Dihydroxyvitamin D 3↗

PubMed1998

Addition of human recombinant bone morphogenetic protein-2 to inactive commercial human demineralized freeze-dried bone allograft makes an effective composite bone inductive implant material.

COMMERCIAL PREPARATIONS OF HUMAN DEMINERALIZED freeze-dried bone allograft (DFDBA) vary in their ability to induce new bone formation. This study tested the hypothesis that inactive DFDBA can be used as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2). Two batches of active DFDBA were used as controls. Two batches of DFDBA, previously shown to be inactive, were treated with vehicle or with 5 or 20 microg rhBMP-2 and implanted into the calf muscle of male Nu/Nu (nude) mice. Each mouse received one implant in each hind limb, both of which were of the same formulation, resulting in 8 groups of 4 mice per group: active DFDBA batch A, active DFDBA batch B, inactive DFDBA batch A, inactive DFDBA batch B, inactive DFDBA batch A plus 5 microg rhBMP-2, inactive DFDBA batch A plus 20 microg rhBMP-2, inactive DFDBA batch B plus 5 microg rhBMP-2, and inactive DFDBA batch B plus 20 microg rhBMP-2. After 56 days, the implants were removed and histologically examined. A semiquantitative bone induction index was calculated based on the amount of new bone covering each histological section. Histomorphometry was also used to evaluate the area of new bone formed and the area of residual implant material. The results showed that active DFDBA induces new bone formation, whereas inactive DFDBA does not. Addition of rhBMP-2 to inactive DFDBA results in new bone formation with a bone induction index comparable to that of active DFDBA. Histomorphometric analysis, however, revealed that the rhBMP-2-containing implants caused a dose-dependent increase in new bone area that exceeded that induced by active DFDBA. At the highest concentration of rhBMP-2, bone formation was exuberant. rhBMP-2 also caused the resorption of residual implant material to levels comparable to that seen in sites treated with active DFDBA, suggesting that this growth factor may regulate resorptive cells either directly or indirectly. This study shows that addition of rhBMP-2 to inactive DFDBA provides reproducible, consistent bone induction, and suggests that inactive commercial preparations may contain inadequate amounts of BMP to cause bone induction compared to active preparations.

Animals↗

PubMed1998

Ability of commercial demineralized freeze-dried bone allograft to induce new bone formation is dependent on donor age but not gender.

Demineralized freeze-dried bone allografts (DFDBA) have been used extensively in periodontal therapy. DFDBA is used because it contains bone morphogenetic protein (BMP), which induces new bone formation during the healing process. Most commercial bone banks do not verify the presence or activity of BMP in DFDBA nor the ability of DFDBA to induce new bone. Recently, we showed that different bone bank preparations of DFDBA, even from the same bank, varied considerably in their ability to induce new bone, suggesting inherent differences in the quality of the material. Therefore, we examined whether donor age or gender contributed to the variability seen with these preparations. Twenty-seven batches of DFDBA from different donors were donated by one bone bank which had been shown previously to supply DFDBA that was consistently able to induce new bone formation. Each batch was implanted bilaterally in the thigh muscle of nude mice. After 56 days, the implants were excised and examined by light microscopy and histomorphometry. Seventy percent of the preparations tested induced new bone formation. Most of these preparations produced ossicles containing cortical bone surrounding bone marrow-like tissue. The ability to induce bone appears to be age-dependent, with DFDBA from older donors being less likely to have strong bone-inducing activity. By contrast, no difference in ability to induce new bone was noticed between male or female donors. The results of this study confirm that commercial preparations of DFDBA differ in their ability to induce new bone formation. In fact, some of the batches had no activity at all. The ability of DFDBA to induce new bone formation is suggested to be age-dependent, but not gender-dependent by our study. These results indicate that commercial bone banks need to verify the ability of DFDBA to induce new bone formation and should reconsider the advisability of using bone from older donors.

Adolescent↗

PubMed1997

Bacterial lipopolysaccharide induces early and late activation of protein kinase C in inflammatory macrophages by selective activation of PKC-epsilon.

Experiments from our and other laboratories have shown that specific inhibitors of protein kinase C (PKC) inhibited the secretion of nitric oxide, TNF alpha, and IL-1 beta from lipopolysaccharide (LPS)-stimulated macrophages, suggesting an important role for PKC in the inflammatory response. The present study was designed to investigate the mechanism whereby LPS stimulates PKC activity in inflammatory macrophages. Mouse macrophages were stimulated with 0-1 microgram/ml LPS for 0-18 hours, and PKC activity was detected in cell lysates. PKC isoform specificity was determined by blocking PKC activity with isoform-specific antibodies. Treatment of macrophages with 1 microgram/ml LPS induced a two-fold increase in PKC activity within 15 minutes and an additional more significant peak of PKC activity appeared 3 hours post-LPS stimulation. A lower dose of LPS (10 ng/ml) induced the later peak only. The enhancement in PKC activity induced by LPS occurred in both the cytosol and membrane fractions, but the enhancement in the membrane fraction was significantly greater than in the cytosol. The increase in PKC activity in both peaks was abolished only by the addition of anti-PKC-epsilon antibody. The present experiments suggest that PKC activation is an important pathway in the LPS-induced secretory response of macrophages and that PKC-epsilon is the major isoform involved.

Animals↗