Protein Kinase C Modulates the Synthesis of Nitric Oxide by Osteoblasts

To determine the involvement of protein kinase C (PKC) in nitric oxide (NO) synthesis of osteoblast, a combination of proinflammatory cytokines (tumor necrosis factor-α, interferon-γ, bacterial lipopolysaccharide) were added on rat osteoblast-like cells. Results show that these cytokines clearly enhanced the synthesis of NO. The activation of PKC with phorbol ester also resulted in the stimulation of NO synthesis in these cells. These cytokines activated PKC and increased the levels of intracellular Ca²⁺. In addition, the cytokine-induced synthesis of NO was blocked by PKC inhibitors. Findings suggest the involvement of PKC in the synthesis of NO by rat osteoblasts. Received: 29 April 1997 / Accepted: 14 November 1997     

Etidronate (EHDP) Inhibits Osteoclastic-Bone Resorption, Promotes Apoptosis and Disrupts Actin Rings in Isolate-Mature Osteoclasts

Bisphosphonates, therapeutic reagents against tumoral bone diseases (Paget's disease or osteoporosis), are potent inhibitors of bone resorption. The mechanisms by which they directly act on mature osteoclasts remain unclear. Using a recently developed technique for isolation of highly purified mammalian mature osteoclasts, we demonstrated that etidronate [ethane-1-hydroxy-1,1-diphosphonate (EHDP), 1-hydroxy-1,1-ethylidenebisphosphonate], inhibited directly osteoclastic bone-resorbing activity by pit assay. In addition, EHDP also directly induced apoptosis and disrupted actin rings in osteoclasts. The data support previous data on non-purified osteoclasts and results in vivo. Received: 26 June 1997 / Accepted: 27 July 1998     

Prevention of Osteoporosis in Heart Transplant Recipients: A Comparison of Calcitriol with Calcitonin and Pamidronate

Bone loss and osteoporotic fractures are common in cardiac transplant recipients. To compare two prophylactic medical regimens after heart transplantation, 26 consecutive heart transplant recipients were randomized to receive either continuous oral calcitriol (0.5 μg/day) combined with nasal salmon calcitonin (200 U/day) for the first 3 months (group A) or intermittent intravenous pamidronate (0.5 mg/kg body weight) every third month (group B). Bone mineral density (BMD) and biochemical indices of bone turnover were measured at baseline and 3, 6, 12, and 18 months after transplantation. The mean pretransplant BMD, measured by dual energy X-ray absorptiometry (DXA) was significantly lower in the patients compared with age-matched healthy controls. During the first year of treatment, rates of bone loss at the lumbar spine and femoral neck were slightly but significantly slower in the patients treated with pamidronate, but there was no longer a significant difference between the two groups after 18 months of heart transplantation. Irrespective of the mode of osteoporosis prevention, osteocalcin levels increased whereas urinary deoxypyridinoline decreased after transplantation, and significant bone loss was observed in both treatment groups. We found no relationship between initial BMD, markers of bone turnover, cumulative glucocorticoid dose, or cyclosporine levels and the rate of bone loss after cardiac transplantation. In summary, we found that the rapid and severe bone loss following heart transplantation could be attenuated by two preventive measures, pamidronate or calcitriol with calcitonin. Received: 26 December 1997 / Accepted: 10 February 2000     

Increase in the Potential of Osteoblasts to Support Bone Resorption by Osteoclasts In Vitro in Response to Roughness of Bone Surface

The development of the potential of osteoblasts to support bone resorption by osteoclasts in response to roughness on bone slices was examined in the co-incubation cell system of immature osteoclasts and osteoblastic cells. The immature osteoclasts, which need alkaline phospatase (ALP)-positive osteoblastic cells for bone resorption, were generated in mouse spleen cultures with 1, 25-dihydroxyvitamin D₃ and prostaglandin E₂. ALP-negative osteoblastic cells from mouse calvaria were incubated on rough surfaced bone slices for 3 days. The number of ALP-positive cells increased greatly on the rough surface, but little on the smooth surface. When immature osteoclasts were added and incubated for 1 more day, the resorption pit number and the total pit areas on the smooth surface were not much different from those before incubation but were approximately four times higher on the rough surface. Received: 21 July 1998 / Accepted: 12 March 1999     

Calcitonin Provides Complete Protection Against Cancellous Bone Loss in the Femoral Neck of Ovariectomized Rats

Calcitonin (CT) has been found to partially prevent cancellous bone loss in the proximal tibia of ovariectomized (OVX) rats. The current study was designed to determine whether CT has similar bone protective effects in the femoral neck, a skeletal site with a slower rate of bone loss after ovariectomy than the proximal tibia. Female Sprague Dawley rats were sham-operated or ovariectomized at 3 months of age. Groups of OVX rats were injected s.c. with vehicle or CT at a dose of 16 U/kg body weight on alternate days for 30, 60, or 90 days. Sham-operated control rats were treated with vehicle alone on alternate days. The proximal femur from each rat was processed undecalcified for quantitative bone histomorphometry. Cancellous bone volume in the femoral neck of vehicle-treated OVX rats was significantly less than that of vehicle-treated control rats at all time points. This cancellous osteopenia induced by ovariectomy was associated with increased indices of bone turnover such as osteoclast surface, osteoblast surface, and bone formation rate (tissue level, total surface referent). In contrast, cancellous bone volume in the femoral neck of CT-treated OVX rats was nearly identical of that of vehicle-treated control rats throughout the study. In addition, CT treatment of OVX rats decreased all indices of bone turnover to near the level of vehicle-treated control rats. The results indicate that CT treatment depresses bone turnover and provides complete protection against moderate cancellous osteopenia in the femoral neck of OVX rats. Since previous studies have shown that CT only partially protects against more pronounced cancellous bone loss in the proximal tibia of OVX rats, our findings suggest that CT has a greater bone protective effect at a skeletal site with a slower rate of cancellous bone loss (femoral neck) than at a skeletal site with a rapid rate of cancellous bone loss (proximal tibia). Received: 21 August 1996 / Accepted: 10 December 1996     

Effect of Salmon Calcitonin on Femoral Bone Quality in Adult Ovariectomized Ewes

The aim of this study was to evaluate the effect of intermittent calcitonin on femoral bone quality in adult ewes from the time of ovariectomy. Six months after the start of the experiment, bone density measurements and mechanical testing (torsion and resonant frequency analysis of the diaphysis and compression of an excised trabecular bone cylinder from the femoral neck) were performed in sham-control and ovariectomized (OVX) ewes treated with placebo or salmon calcitonin (50 or 100 units, 3 times/week). Crystallinity of bone was evaluated by measuring X-ray diffraction line broadening. After OVX, a nonsignificant bone loss was found at all measured sites in the femur (−3 to −9%) together with a decreased biomechanical competence in the trabecular bone (compressive strain −28%, P < 0.05). Treatment with salmon calcitonin, 50 or 100 IU subcutaneously three times a week from the time of ovariectomy, resulted in a significant dose-dependent preservation of bone strength in the trabecular bone of the femoral neck compared with OVX. No adverse effects of calcitonin were observed on bone crystal composition as assessed by diffractiometry. We conclude that in adult ewes intermittent calcitonin treatment from the time of OVX was associated with a significant preservation of cancellous bone strength and strain in trabecular bone of the femoral neck, without affecting crystalline properties of bone. Received: 20 October 1995 / Accepted: 19 February 1996     

Prostaglandin E2 Directly Inhibits Bone-Resorbing Activity of Isolated Mature Osteoclasts Mainly Through the EP4 Receptor

Prostaglandins (PGs) are well known to be important local factors in regulating bone formation and resorption. PGE₂ is a potent stimulator of bone resorption because of enhancing osteoclast formation by its indirect action through stromal cells. However, the direct action of PGE₂ on functionally mature osteoclasts is still controversial. In this study using highly purified rabbit mature osteoclasts, we examined the direct effect of PGE₂ on osteoclastic bone-resorbing activity and its mechanism. PGE₂ inhibited resorption pit formation on a dentine slice by the purified osteoclasts in a dose- and time-dependent manner. The inhibitory effect appeared as early as 4 hours after the PGE₂ addition. Forskolin and 12-0-tetradecanoyl phorbol-13-acetate (TPA), respective activators of adenylate cyclase and protein kinase C, also decreased the osteoclastic bone-resorbing activity. PGE₂ increased the content of intracellular cAMP in a dose range effective for the inhibition of bone resorption, whereas the prostanoid did not alter the intracellular level of inositol triphosphate. The inhibition of osteoclastic bone resorption by PGE₂ was amplified and diminished by a cAMP phosphodiesterase inhibitor (isobutyl methylxanthine) and a protein kinase A inhibitor (Rp-cAMP), respectively. Of four different subtypes of PGE₂ receptors (EPs), EP4 mRNA was predominantly expressed in isolated osteoclasts, whereas the other types of EP mRNA were detected in only small amounts. These results suggest that the PGE₂ inhibitory effect was mediated by an adenylate cyclase system coupled with EP4. This possible association of PGE₂ with EP4 in mature osteoclasts was supported by the finding that a specific agonist of EP4 (AE-604) inhibited the bone-resorbing activity and elevated the intracellular cAMP content. However, butaprost, a selective EP2 agonist, also mimicked the PGE₂ effects on isolated osteoclasts although EP2 mRNA expression was minimal. In conclusion, PGE₂ directly inhibits bone-resorbing activity of functionally mature osteoclasts by activation of the adenylate cyclase system, perhaps mainly through EP4. Received: 21 July 1999 / Accepted: 31 January 2000     

Pasteurella multocida Toxin Stimulates Bone Resorption by Osteoclasts via Interaction with Osteoblasts

In this study we used an in vitro assay system with osteoblast and osteoclast co-cultures to assess the effect of purified recombinant Pasteurella multocida toxin on bone resorption. Resorption was measured by the release of a telopeptide breakdown product of type I collagen. It was found that P. multocida did not stimulate bone resorption by osteoclasts directly and also did not stimulate bone breakdown via the release of collagenase or other proteases from osteoblasts. During co-culture of osteoblasts and osteoclasts, with cell-cell contact prevented, P. multocida toxin produced no significant effect. Osteoblast-conditioned media gave a biphasic effect; low concentrations of P. multocida toxin stimulated bone resorption, whereas 100 ng/ml inhibited resorption by osteoclasts. However, when both cell types were co-cultured with cell-cell contact permitted, P. multocida toxin induced a large concentration-dependent increase in bone resorption over a 7-day period. This suggested that P. multocida toxin causes bone breakdown via an osteoblast-dependent mechanism and that a membrane-bound receptor may be involved. Received: 8 July 1997 / Accepted: 8 April 1998     

Regulation of c-fos and c-jun Expression by Calcitonin in Human Breast Cancer Cells

Breast cancer cells (BCC) have calcitonin (CT) receptors, yet the action of the hormone on these cells is largely unknown. We found that CT produced a strong and transient time- and dose-dependent increase in c-fos mRNA in BCC lines. This event was prevented by a protein kinase A (PKA) inhibitor, H89. CT alone did not influence the expression of c-jun and of the tissue inhibitors of metalloproteases (timp) -1 and -2 mRNAs; however, it reduced the induction of these mRNAs by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), without apparent changes in the half-life of the mRNA (measured for c-jun). Along the same line, CT reduced the c-jun induction and T-47D growth stimulation by epidermal growth factor (EGF) and insulin. These effects were mimicked by forskolin and/or prevented by H89, suggesting that PKA activation was involved. These results indicate that CT modulates in BCC the mRNA levels of two important growth-related early response genes (c-fos and c-jun) and of two other genes (timp-1 and -2) involved in the control of metastatic events. Received: 2 May 1996 / Accepted: 10 December 1996     

Phorbol Myristate Acetate Downregulates the Binding Sites for Colony-Stimulating Factor-1 on Osteoclasts Isolated from Rats

Colony-stimulating factor-1 (CSF-1) is the growth factor for the cells of the mononuclear phagocytic system and for osteoclasts. We tested whether phorbol myristate acetate (PMA), a phorbol ester activating protein kinase C, modulates the number of binding sites for CSF-1 on isolated rat osteoclasts. PMA decreased binding of CSF-1 to osteoclasts within 60 minutes. The effect of PMA was dose dependent at concentrations between 10⁻⁹ M and 10⁻⁶ M. The inactive phorbol ester, 4α-phorbol 12,13-didecanoate, had only a slight effect. Since the osteoclast preparation was contaminated with other cells, the action of PMA on the osteoclasts might have been either direct or indirect. In pure osteoclasts harvested by a micropipette, the downregulation of CSF-1 binding by PMA reached only about three-quarters of that in nonpurified preparations. Addition of osteoblastic osteosarcoma UMR106 cells increased the effect of PMA. Antiserum against CSF-1, which was added to osteoclasts contaminated with other cells, mainly osteoblasts, partially inhibited the effect of PMA, but the antiserum had no effect in pure osteoclasts. These data suggest that the effect mediated by osteoblasts or other contaminating cells is due to the release of CSF-1, which is known to downregulate its binding sites on osteoclasts. The direct action of PMA on osteoclasts decreased the binding only to about 40%, in contrast to CSF-1 which completely downregulated the binding. The data also differ from the published results about macrophages. In these cells, PMA downregulates the binding of CSF-1 completely. The CSF-1 binding sites on osteoclasts recovered within 4 hours after removal of PMA, and cycloheximide, an inhibitor of protein synthesis, inhibited the recovery. Received: 16 January 1997 / Accepted: 21 May 1997     

Comparison of Calcitriol Treatment with Etidronate–Calcitriol and Calcitonin–Calcitriol Combinations in Turkish Women with Postmenopausal Osteoporosis: A Prospective Study

Calcitriol has been widely used in the management of osteoporosis, but its efficiency is a matter of controversy. It is not known whether combinations of calcitriol and antiresorptive agents such as etidronate and calcitonin are superior to calcitriol alone in the treatment of postmenopausal osteoporosis. To make this determination, 30 Turkish women with postmenopausal osteoporosis between 45 and 68 years of age were randomized to receive either intermittent cyclical etidronate (400 mg/day, for 14 days) followed by 60 days of cyclical calcitriol therapy 0.25 μg twice daily (group 1; n= 10), or calcitriol 0.25 μg twice daily (group 2; n= 10), or calcitriol 0.25 μg/day in combination with 100 IU intranasal salmon calcitonin taken every other day (group 3; n= 10) through a 1-year period. Bone mineral density (BMD) of lumbar spine (L2 to L4) was determined for each patient by dual-photon absorptiometry (¹⁵³Gd) at baseline, after 6 months, and at the end of the study. There was no significant difference among groups with respect to mean spinal BMD at baseline, after 6, and after 12 months. No significant spinal BMD changes occurred in any group from baseline, after 6 months, and after 12 months. Four patients in groups 1 and 2 and five patients in group 3 developed hypercalcemia at least once during therapy. Hypercalciuria occurred at least once in 9, 10, and 7 patients in groups 1, 2, and 3, respectively. One patient in group 2 developed a renal stone at the end of the study. Mean urine hydroxyproline levels did not change significantly in any group with respect to baseline. The data suggest that one-year treatment with calcitriol, given either alone or in combination with antiresorptive agents, does not improve spinal BMD in Turkish women with postmenopausal osteoporosis, and is associated with a high rate of adverse events. Received: 4 October 1996 / Accepted: 31 December 1996     

Mechanism of the Morphological Changes Induced by Staurosporine in Rat Osteoblasts

Protein kinase C (PKC) plays an important role in the differentiation of cells, however, little is known about its relationship to bone metabolism. We have previously demonstrated that staurosporine concentration dependently transformed the cultured rat osteoblasts into stellate cells. In this study, we further investigated the possible mechanisms and significance of the morphological changes of osteoblasts induced by staurosporine. The morphological changes induced by staurosporine were inhibited by microtubule depolymerizers or elevated intracellular calcium, however, actin depolymerizers enhanced the effects of staurosporine. Fluorescence labeling showed that staurosporine caused the dissolution of the actin microfilaments, but left the microtubules and vimentin filaments intact. PKC activators partially antagonized the morphological changes induced by staurosporine. Inhibition of protein kinase A or calmodulin-dependent kinase is less effective in the induction of stellate cell formation. These results suggest that the morphological changes of osteoblasts induced by staurosporine may be partly due to PKC inhibition, but other mechanisms may also be involved. Received: 23 January 1996 / Accepted: 11 October 1996     

Gastric Fundectomy in the Rat: Effects on Mineral and Bone Metabolism, with Emphasis on the Gastrin–Calcitonin–Parathyroid Hormone–Vitamin D Axis

In humans, gastric surgery results in in osteopenia via mechanisms that are insufficiently understood; surgery-induced changes in the hormonal axes involving the stomach, thyroid, and the parathyroids may play a role. To study this in more detail, we evaluated calcium (Ca), magnesium (Mg), and phosphorus (P) metabolism as well as physical, chemical, and histomorphometric bone parameters in rats rendered hypergastrinemic by fundectomy (FX). In independent experiments, the response to an oral Ca challenge was investigated in intact rats versus FX, and in thyroidectomized versus thyroid-intact FX rats. Sixteen weeks following FX, body weight was approximately 80% that of sham-operated controls. In urine, P excretion was elevated fivefold, the pH was significantly decreased, and cAMP excretion was elevated as compared with controls; serum parathyroid hormone (PTH), calcitonin, 25OHD, Ca, Mg, and P were normal; gastrin and 1,25(OH)₂D were elevated. On the basis of bone ash mineral content, FX rats developed significant osteopenia, and histomorphometry indicated only slightly elevated bone turnover and mineralization. Following oral Ca, thyroid-intact FX rats developed hypercalcemia, serum gastrin decreased, and calcitonin increased significantly; in thyroidectomized FX rats, calcitonin remained at baseline levels although there was a similar degree of hypercalcemia; PTH decreased during the hypercalcemic period in both groups. Serum gastrin did not correlate with calcitonin or PTH, and in multivariate regression analysis the only predictor of serum 1,25(OH)₂D was urinary phosphorus. It was concluded that in the FX rat (1) osteopenia is not caused by intestinal Ca malabsorption, vitamin D, Ca deficiency, or secondary hyperparathyroidism; (2) osteopenia may be related to PTH-independent urinary hyperexcretion of P, followed by a rise of serum 1,25(OH)₂D; (3) the existence of endocrine axes among gastrin, calcitonin, and PTH cannot be substantiated. FX osteopenia appears to be related to gastric acid abolition, and the reactive hypergastrinemia probably stabilizes the mass and turnover of bone. Received: 12 August 1997 / Accepted: 26 January 1998     

Chondroclasts and Osteoclasts in Bones of Young Rats: Comparison of Ultrastructural and Functional Features

The aim of the present study was to characterize cells involved in resorption during endochondral bone formation. We investigated whether the cells involved in cartilage breakdown at the epiphyseal/metaphyseal border, i.e., chondroclasts, share the characteristics of bone/cartilage-resorbing osteoclasts at the metaphyseal/diaphyseal border regarding ultrastructural features and functional activity. Morphometric evaluation showed that chondroclasts do not form ruffled borders and clear zones, i.e., well-known resorption characteristics, to the same extent as osteoclasts, present at the lower metaphysis. Instead, chondroclasts tend to express an undifferentiated surface adjacent to the matrix, not structurally different from the basolateral plasma membrane. Tartrate-resistant acid phosphatase (TRAP) was used as a marker for functional activity. Immunohistochemical staining by light microscopy was strong in both chondroclasts and in osteoclasts. Furthermore, in situ hybridization revealed large amounts of TRAP mRNA in chondroclasts as well as in osteoclasts. Ultrastructural immunohistochemistry suggests extensive secretion of the TRAP enzyme in the ruffled border area of both chondroclasts and osteoclasts. Intracellular accumulation was seen particularly in chondroclasts, possibly as a consequence of a relative disinclination to develop a ruffled border. Thus, semiquantitative estimation of TRAP distribution showed an inverse relationship between extracellular and intracellular TRAP in chondroclasts and osteoclasts. These results indicate that chondroclasts and osteoclasts differ, not only with respect to location but possibly also by mode of action. The observed differences may reflect the maturation sequence of these multinucleated cells when associated with different metaphyseal trabecular surfaces. Received: 22 January 1998 / Accepted 8 April 1998     

Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo ³H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3–5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage. Received: 30 November 1999 / Accepted: 21 March 2000     

24,25-(OH)2D3 Regulation of Matrix Vesicle Protein Kinase C Occurs Both During Biosynthesis and in the Extracellular Matrix

Plasma membranes and matrix vesicles isolated from rat costochondral resting zone chondrocyte cultures contain predominantly protein kinase C alpha (PKCα) and PKCζ, respectively, and the level of PKC specific activity in these membrane fractions is regulated by 24,25-(OH)₂D₃ [14]. In the present study, we examined whether the effect of 24,25-(OH)₂D₃ on membrane PKC is via genomic mechanisms during biogenesis and through a nongenomic mechanism after the matrix vesicles are resident in the matrix. There was a dose-dependent decrease in matrix vesicle PKC specific activity and a significant increase in plasma membrane enzyme activity in cultures treated for 90 minutes with 10⁻⁹–10⁻⁷ M 24,25-(OH)₂D₃. However, at 12 hours, matrix vesicle PKC was stimulated, but no effect was seen in the plasma membranes, suggesting that the effect seen at 90 minutes was due to a direct action of the hormone on PKC activity in the membrane, and that the effect seen at 12 hours was due to new matrix vesicle production with altered PKC content. Neither actinomycin D nor cycloheximide inhibited matrix vesicle PKC at 30, 60, or 90 minutes, but by 12 hours, these inhibitors blocked the effect of the hormone. 24,25-(OH)₂D₃-dependent plasma membrane PKC was sensitive to both actinomycin D and cycloheximide at early time points, but by 12 hours, no effect of the inhibitors was seen. Monensin did not alter basal plasma membrane PKC activity or the 24,25-(OH)₂D₃-dependent increase, suggesting that this increase was due to translocation of cytosolic PKC rather than new membrane synthesis. Monensin did not affect matrix vesicle PKC at early time points, but it decreased 24,25-(OH)₂D₃-dependent enzyme activity at later times, indicating that new matrix vesicle production was blocked. At least part of the effect of 24,25-(OH)₂D₃ on PKC involved phospholipase A₂ (PA₂). Quinacrine (a PA₂ inhibitor) alone had no effect on matrix vesicle PKC, but in cultures treated for 12 hours with quinacrine and 24,25-(OH)₂D₃, a synergistic increase in matrix vesicle PKC was observed. Quinacrine caused a time-dependent decrease in matrix vesicle PKC and a dose- and time-dependent increase in plasma membrane PKC when incubated directly with the membranes, supporting the hypothesis that PA₂ plays a role in the nongenomic regulation of PKC by 24,25-(OH)₂D₃. Experiments using anti-isoform specific antibodies showed that 24,25-(OH)₂D₃ modulated the distribution of PKCα, β, and ζ between the plasma membrane and matrix vesicle compartments via translocation and new PKC synthesis. Thus, the data support the hypothesis that 24,25-(OH)₂D₃ regulates matrix vesicles through two pathways: a genomic one at the stage of biosynthesis and packaging, and a second nongenomic mechanism acting directly upon matrix vesicles in the matrix. These data also indicate that matrix vesicle regulation consists of complex events with several different points of regulation. Received: 11 October 1996 / Accepted: 25 April 1997     

Characterization of Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: I. Differences in the Cellular Response to Demineralized Bone Matrix Implanted in Calvarial Defects and in Subcutaneous Sites

The cellular and biochemical sequences of osteogenesis induced by implanting demineralized bone matrix (DBM) in rat cranial defects and in subcutaneous sites have been studied by histological, histochemical, and biochemical techniques from days 2 to 28 after implantation. In subcutaneous sites, allogenic DBM induced cartilage cells and matrix for approximately the first 10 days which were subsequently resorbed and replaced by bone with little evidence for the classical endochondral sequence of ossification. In sharp contrast, the first cells that differentiated from the mesenchymal stem cells in the cranial defects were alkaline phosphatase (ALP) positively stained osteoblasts that appeared 3 days after implantation followed by synthesis of bone matrix which calcified shortly thereafter. A few clusters of cartilage cells were observed beginning at days 6–7 which were spatially distinct from the new bone and later resorbed. By day 28 the tissue induced in both the subcutaneous and cranial sites consisted almost solely of bone; however, the total amount of new bone in the subcutaneous implants was significantly less than the mass of bone formed in the calvarial defects. Bovine DBM induced bone formation in rat cranial defects to a very much lesser extent than allogenic DBM. A few cartilage cells were induced by bovine DBM in subcutaneous sites and rapidly resorbed and not replaced with bone. These results clearly indicate that the cellular sequence induced by allogenic and xenogenic DBM and the repair tissues synthesized are distinctly different in the cranial defects from those induced in the subcutaneous sites. Received: 1 September 1998 / Accepted: 15 January 1999     

Parathyroid Hormone (1-34) Receptor-Binding and Second-Messenger Response in Rat Incisor Odontoblasts

Even though indirect evidence indicates that PTH exerts an anabolic effect on dentinogenesis, the existence of PTH receptors and any second-messenger response in odontoblasts have not been demonstrated. The aim of this study was to investigate whether rat incisor odontoblasts express PTH receptors, and to identify which second messenger pathway the hormone may activate. Odontoblasts were dissected from rat incisors. Amino-terminal (1-34) fragment rat PTH [rPTH(1-34)] conjugated to fluorescein isothiocyanate visualized receptor sites on the cell surface. Upon incubation of odontoblasts with rPTH(1-34), cAMP formation was increased. However, no fluctuations in intracellular calcium activity were observed upon rPTH(1-34) stimulation when using Fura-2 as a Ca²⁺ probe. In long-time incubations, stimulation with PTH(1-34) upregulated APase activity. The results demonstrate that rPTH(1-34) evokes an anabolic response in dentinogenically active odontoblasts, and that this may be mediated through the protein kinase A/cAMP pathway, whereas no indications for Ca²⁺ as a second messenger were evident. Received: 12 March 1997 / Accepted: 26 June 1997     

Bone Response to In Vivo Mechanical Loading in C3H/HeJ Mice

Bone, being sensitive to mechanical stimulus, adapts to mechanical loads in response to bending or deformation. Although the signal/receptor mechanism for bone adaptation to deformation is still under investigation, the mechanical signal is related to the amount of bone deformation or strain. Adaptation to changes in physical activity depends on both the magnitude of increase in strain above average daily levels for maintaining current bone density and the Minimum Effective Strain (MES) for initiating adaptive bone formation. Given the variation of peak bone density that exists in any human population, it is likely that variation in levels for MES is, to a considerable degree, inherited and varies among animal species and breeds. This study showed a dose-related periosteal response to loading in C3H/HeJ mice. The extent of active formation surface, the rate of periosteal bone formation, and area of bone formation increased with increasing peak periosteal strain. In these mice, the loaded tibia consistently showed lower endocortical formation surface and mineral apposition rate than the nonloaded bones at every load level. Although periosteal expansion is the most efficient means of increasing moment of inertia in adaptation to bending, a dose response increase in endocortical formation would have been predicted. Our characterization of the mouse bone formation response to increasing bending loads will be useful in the design of experiments to study the tibial adaptive response to known loads in different mouse breeds. Received: 17 February 1998 / Accepted: 9 December 1998     

Parathyroid Hormone-Related Protein (107-139) Decreases Alkaline Phosphatase in Osteoblastic Osteosarcoma Cells UMR 106 by a Protein Kinase C-Dependent Pathway

The C-terminal (107-111) region of parathyroid hormone-related protein (PTHrP) appears to inhibit osteoclastic bone resorption, and to affect osteoblastic growth and differentiation. We tested the effect of human PTHrP (107-139) on alkaline phosphatase (ALP) activity in osteoblastic osteosarcoma UMR 106 cells. We found that this C-terminal PTHrP peptide, between 10 nM and 10 fM, inhibited ALP activity in these cells during the log phase of growth. Human PTHrP (1-34) amide and human PTHrP (1-141) were as potent as PTHrP (107-139) in growing UMR 106 cells. This inhibitory effect of 10 nM PTHrP (107-139) on ALP activity was also observed in serum-depleted cells, and in the presence of 10 nM dexamethasone, which increased ALP activity by 40% in these cells. In addition, this effect of PTHrP (107-139) was blunted by 25 nM bisindolylmaleimide I, a protein kinase C inhibitor. These results support a role for the C-terminal region of PTHrP as a modulator of bone formation. Received: 7 July 1998 / Accepted: 10 January 1999