Osteoblast Surface Research Articles

Iron lactate-induced osteomalacia in association with osteoblast dynamics.

Osteomalacia was induced in rats fed a diet containing 50,000 ppm (5%) iron lactate for 13 weeks. The histopathology and histomorphometrical dynamics of osteoblasts under this condition were examined. Bone histomorphometry of the proximal tibial metaphysis revealed that the osteoblast surface, osteoid volume, osteoid surface and labeled surface ratio, which are the parameters of bone formation had increased. The blood chemistry revealed the greatest elevation in the osteocalcin level; however, the parathyroid hormone (PTH) secretion and inorganic phosphorus level were very low. From the serum biochemical, histopathological and histomorphometrical findings, the bone lesion in iron lactate-overloaded rats was considered to be similar to low turnover osteomalacia showing decreased trabeculae in secondary spongiosa and increased lamellar osteoid. Furthermore, an iron-positive reaction was detected at the interface between osteoid and mineralized bone. In the bone lesions induced by chronic iron overload, osteoblast recruitment exceeded that of mineralization or, alternatively, the iron within osteoblasts along the trabecular bone suppressed the remodeling and led to an increase in osteoid thickness.

Mechanisms responsible for longitudinal growth of the cortex: coalescence of trabecular bone into cortical bone.

The purpose of the present study was to determine whether longitudinal growth of the cortex occurs through intramembranous bone formation involving the periosteum or through endochondral bone formation involving the growth plate and to explore the cellular and biochemical mechanisms responsible for this process. Cortical bone formation was studied in the metaphyses of growing New Zealand White rabbits by means of (1) oxytetracycline labeling and fluorescence microscopy, (2) computer-assisted histomorphometry, (3) osteoblast culture and [(3) H]-thymidine incorporation in the presence of periosteum or periosteum-conditioned medium, and (4) surgical insertion of membranes between the periosteum and the underlying spongiosa. Within the metaphyseal cortex, oxytetracycline labeling produced fluorescent closed curves outlining enlarging trabeculae derived from coalescing endochondral trabecular bone. In this region of coalescing trabeculae close to the periosteum, osteoblast surface was increased compared with trabeculae farther from the periosteum (p < 0.001). The osteoclast surface did not differ. In vitro, osteoblast proliferation was increased in the presence of periosteum (p < 0.001) or periosteum-conditioned medium (p < 0.001). Surgical insertion of permeable or impermeable membranes between the periosteum and the spongiosa did not prevent cortex formation. These observations demonstrate that metaphyseal cortical bone is formed by coalescence of endochondral trabecular bone. This coalescence is associated with increased osteoblast surface in the peripheral spongiosa. The increased osteoblast surface could be due to inductive effects of periosteum; in the present study, periosteum stimulated osteoblast proliferation in vitro but was not required for metaphyseal cortical bone formation in vivo. Understanding metaphyseal cortical growth may help to elucidate the pathophysiology of osseous growth disorders in children.

Bone remodeling after renal transplantation

Several studies have indicated that bone alterations after transplantation are heterogeneous. Short-term studies after transplantation have shown that many patients exhibit a pattern consistent with adynamic bone disease. In contrast, patients with long-term renal transplantation show a more heterogeneous picture. Thus, while adynamic bone disease has also been described in these patients, most studies show decreased bone formation and prolonged mineralization lag-time faced with persisting bone resorption, and even clear evidence of generalized or focal osteomalacia in many patients. Thus, the main alterations in bone remodeling are a decrease in bone formation and mineralization up against persistent bone resorption, suggesting defective osteoblast function, decreased osteoblastogenesis, or increased osteoblast death rates. Indeed, recent studies from our laboratory have demonstrated that there is an early decrease in osteoblast number and surfaces, as well as in reduced bone formation rate and delayed mineralization after transplantation. These alterations are associated with an early increase in osteoblast apoptosis that correlates with low levels of serum phosphorus. These changes were more frequently observed in patients with low turnover bone disease. In contrast, PTH seemed to preserve osteoblast survival. The mechanisms of hypophosphatemia in these patients appear to be independent of PTH, suggesting that other phosphaturic factors may play a role. However, further studies are needed to determine the nature of a phosphaturic factor and its relationship to the alterations of bone remodeling after transplantation.

The pathogenesis of osteodystrophy after renal transplantation as detected by early alterations in bone remodeling

Loss of bone mass after transplantation begins in the early periods after transplantations and may persist for several years, even in patients with normal renal function. While the pathogenesis of these abnormalities is still unclear, several studies suggest that preexisting bone disease, glucocorticoid therapy, and alterations in phosphate metabolism may play important roles. Recent studies indicate that osteoblast apoptosis and impaired osteoblastogenesis play important roles in the pathogenesis of glucocorticoid-induced osteoporosis. To examine the early alterations in osteoblast number and surfaces during the period following renal transplantation. Twenty patients with a mean age of 36.5 +/- 12 years were subjected to bone biopsy 22 to 160 days after renal transplantation. In 12 patients, a control biopsy was performed on the day of transplantation. Bone sections were evaluated by histomorphometric analysis and cell DNA fragmentation by the methods of terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL), using immunoperoxidase and direct immunofluorescence techniques. The main alterations in posttransplant biopsies were a decrease in osteoid and osteoblast surfaces, adjusted bone formation rate, and prolonged mineralization lag time. Peritrabecular fibrosis was markedly decreased. None of the pretransplant biopsies revealed osteoblast apoptosis. In contrast, TUNEL-positive cells in the proximity of osteoid seams or in the medullary space were observed in nine posttransplant biopsies of which four had mixed bone disease, two had adynamic bone disease, one had osteomalacia, one had osteitis fibrosa, and one had mild hyperparathyroid bone disease. Osteoblast number in posttransplant biopsies with apoptosis was lower as compared with posttransplant biopsies without apoptosis. In addition, most of them showed a marked shift toward quiescence from the cuboidal morphology of active osteoblasts. Serum phosphorus levels were lower in patients showing osteoblast apoptosis and correlated positively with osteoblast number and negatively with the number of apoptotic osteoblasts. In addition, posttransplant osteoblast surface correlated positively with parathyroid hormone (PTH) levels and negatively with glucocorticoid cumulative dose. The data suggest that impaired osteoblastogenesis and early osteoblast apoptosis may play important roles in the pathogenesis of posttransplant osteoporosis. The possible mechanisms involved in the pathogenesis of theses alterations include posttransplant hypophosphatemia, the use of glucocorticoids, and the preexisting bone disease. PTH seems to have a protective effect by preserving osteoblast survival.

Skeletal overexpression of noggin results in osteopenia and reduced bone formation.

Skeletal cells synthesize bone morphogenetic proteins (BMPs) and BMP antagonists. Noggin is a glycoprotein that binds BMPs selectively and antagonizes BMP actions. Noggin expression in osteoblasts is induced by BMPs and noggin opposes the effects of BMPs on osteoblastic differentiation and function in vitro. However, its effects in vivo are not known. We investigated the direct in vivo effects of noggin on bone remodeling in transgenic mice overexpressing noggin under the control of the osteocalcin promoter. Noggin transgenics suffered long bone fractures in the first month of life. Total, vertebral, and femoral bone mineral densities were reduced by 23-29%. Static and dynamic histomorphometry of the femur revealed that noggin transgenic mice had decreased trabecular bone volume, number of trabeculae, and bone formation rate. Osteoblast surface and number of osteoblasts/trabecular area were not significantly decreased, indicating impaired osteoblastic function. Osteoclast surface and number were normal/decreased, there was no increase in bone resorption, and the tissue had the appearance of woven bone. Vertebral microcomputed tomography scanning confirmed decreased trabecular bone volume and trabecular number. In conclusion, transgenic mice overexpressing noggin in the bone microenvironment have decreased trabecular bone volume and impaired osteoblastic function, leading to osteopenia and fractures.

Effect of low-power GaAlAs laser (660 nm) on bone structure and cell activity: an experimental animal study.

Low-level laser therapy (LLLT) is increasingly being used in the regeneration of soft tissue. In the regeneration of hard tissue, it has already been shown that the biomodulation effect of lasers repairs bones more quickly. We studied the activity in bone cells after LLLT close to the site of the bone injury. The femurs of 48 rats were perforated (24 in the irradiated group and 24 in the control group) and the irradiated group was treated with a GaAlAs laser of 660 nm, 10 J/cm2 of radiant exposure on the 2nd, 4th, 6th and 8th days after surgery (DAS). We carried out histomorphometry analysis of the bone. We found that activity was higher in the irradiated group than in the control group: (a) bone volume at 5 DAS (p=0.035); (b) osteoblast surface at 15 DAS (p=0.0002); (c) mineral apposition rate at 15 and 25 DAS (p=0.0008 and 0.006); (d) osteoclast surface at 5 DAS and 25 DAS (p=0.049 and p=0.0028); and (e) eroded surface ( p=0.0032). We concluded that LLLT increases the activity in bone cells (resorption and formation) around the site of the repair without changing the bone structure.

MECHANICAL LOADING INITIATES CELL PROLIFERATION IN VIVO

The appearance of osteoblasts following a brief episode of increased mechanical loading has been documented, but the proliferation of osteoblast precursors in the cellular periosteum has not yet been investigated. In this study, a single bending load (36 cycles, 2 Hz, 2000 μ strain) was applied to the right tibia of skeletally mature female rats and the time course of pre-osteoblast proliferation in the periosteum was examined. A single injection of bromodeoxyuridine (40 mg/kg) was administered 1 h prior to collection times at 12, 24, 30, 36, 42, 48, 60, and 72 h post-load. Endpoints included surface type (based on the morphology of surface cells), pre-osteoblast labeling index, and the distance of labeled and unlabeled cells to the bone surface. Loaded bones showed a significant increase in preosteoblast proliferation from 36–72 h as compared to non-loaded bones. Osteoblast surface was increased during the same time span, indicating that at least some of the early osteoblasts were derived from non-proliferative sources. The distance of labeled and unlabeled cells increased with time, but the distance of the two cell populations did not differ, indicating that there was no partitioning of the proliferative cells. Collectively, these results indicate that a single load application is sufficient to initiate an osteogenic response that includes the proliferation of osteoblast precursors. At earlier post-load time-points, the primary response may be the conversion of surface cells to active osteoblasts; however, at later time points, it seems likely that the proliferating pre-osteoblats are contributing new osteoblasts to active osteogenic sites.

Bone anabolic effects of subcutaneous treatment with basic fibroblast growth factor alone and in combination with estrogen in osteopenic ovariectomized rats

Although basic fibroblast growth factor (bFGF) is a potent stimulator of bone formation when administered intravenously, less is known regarding the effects of this peptide on bone following subcutaneous (sc) administration. In addition, it is unknown whether coadministration of estrogen enhances the bone response to treatment with bFGF. Therefore, the purpose of this study was (1) to characterize the skeletal response to sc injection of a high dose of bFGF, and (2) to determine whether concurrent administration of estrogen affects the skeletal response to bFGF treatment. Female Sprague-Dawley rats were ovariectomized (ovx) or sham-operated (sham) at 3 months of age and left untreated for 2 months to establish cancellous osteopenia in the ovx group. The sham rats ( n = 10) and one group of ovx rats ( n = 9) were then injected sc with vehicle alone for 3 weeks. Two additional groups of ovx rats were injected sc with bFGF ( n = 10) or with bFGF + estrogen ( n = 10) for 3 weeks. bFGF was administered sc at a daily dose of 1 mg/kg/day and estrogen was administered sc 4 days per week at a dose of 10 μg/kg for the 3-week duration of treatment. Lumbar vertebrae were collected and processed undecalcified for quantitative bone histomorphometry. Cancellous bone volume was lower and cancellous bone turnover was higher in vehicle-treated ovx rats than in vehicle-treated sham rats. Subcutaneous treatment of ovx rats with bFGF for 3 weeks resulted in a 4-fold increase in osteoblast surface and an 8-fold increase in osteoid surface in comparison to vehicle treatment of ovx rats. Osteoid volume was also markedly increased in the bFGF-treated ovx rats (7 ± 4%) in comparison to vehicle-treated ovx rats (<0.1%). Osteoblast surface, osteoid surface, and osteoid volume were nearly identical in ovx rats treated with bFGF alone and with bFGF + estrogen. Although the majority of the osteoid in bFGF- and bFGF + estrogen-treated animals was deposited along mineralized bone surfaces, osteoid spicules without any connections to preexisting bone surfaces were also detected, providing definitive proof for bone formation within bone marrow in response to bFGF administration. Osteoclast surface, an index of bone resorption, was not affected by bFGF treatment. However, cotreatment of ovx rats with bFGF + estrogen resulted in lower osteoclast surface in comparison to treatment of ovx rats with either vehicle or bFGF alone. In summary, these findings indicate that administration of a high dose of bFGF via sc injection markedly increases bone formation and may be a useful treatment for cancellous osteopenia in the estrogen-deplete skeleton. The anabolic effects of bFGF on bone are not enhanced by concurrent treatment with estrogen at the replacement dose used in this study.

Aging does not lessen the effectiveness of TGFbeta2-enhanced bone regeneration.

Controversy exists over the potency of bone healing in the aged skeleton, and there is concern that enhancement of bone regeneration after use of bone-stimulating growth factors may not be effective in the aged. In this study, 30 skeletally mature beagles (1-2 or 10-12 years old) had titanium implants placed bilaterally in the proximal humerus for a period of 4 weeks in a model of intramembranous bone regeneration. A bony defect made at the time of surgery created a 3-mm gap between the implant surface and the host bone. Some of the implants were treated with recombinant human TGFbeta2 (rhTGFbeta2) at various does (0.32-35 microg per implant), and some served as paired controls. The dose response was similar in young and old animals. The most effective dose, 35 microg, led to a 3-fold increase in the volume fraction of new bone within the gap in both the young (p = 0.001) and old (p = 0.002) animals. At this dose, there was a 5-fold increase in osteoblast surface. While age did not significantly affect the quantity of new bone formed as assessed by backscatter scanning electron microscopy, the older animals had thinner regenerated trabeculae that tended to be spaced more closely than the younger animals. Coupled with the finding that the increase in osteoid was greater in the old animals compared with the young animals, these qualitative differences suggest that there may have been a slight delay in the rate or a defect of mineralization in the old animals.

Zoledronic acid to prevent bone loss in the first 6 months after renal transplantation

Bisphosphonates can prevent bone mineral density loss after renal transplantation, but their effect on trabecular mineralization and bone morphology, two key factors of bone stability, remains unknown. In a 6-month, randomized, placebo-controlled study, 20 kidney transplant recipients received either 4 mg zoledronic acid or placebo twice within 3 months after engraftment. At transplantation and after 6 months, mean trabecular calcium concentration and trabecular morphometry were measured in bone biopsies. Bone mineral density (BMD) of the femoral neck and the lumbar spine were evaluated by dual-energy x-ray absorptiometry, and serum biochemical markers of bone metabolism were determined monthly. Trabecular calcium content increased significantly in the zoledronic acid group, but remained unchanged in the placebo group. BMD at femoral neck showed no change in the zoledronic acid group, but decreased in the placebo group. BMD of the lumbar spine was increased in the zoledronic acid group without change in the placebo group. High-turnover bone disease resolved similarly in both groups, as evidenced by a significant decrease of eroded bone surface, osteoclast and osteoblast surface. Serologic markers of bone formation and resorption were significantly lower in zoledronic acid-treated patients throughout the study. Kidney transplant function was stable after zoledronic acid therapy. Our results show that administration of zoledronic acid improves the calcium content of cancellous bone after kidney transplantation. The beneficial effect of bisphosphonate therapy is further evidenced by an increase of lumbar spine BMD, and stabilization of femur BMD.

Multiple Signaling Pathways Converge on the Cbfa1/Runx2 Transcription Factor to Regulate Osteoblast Differentiation

The Cbfa1/Runx2 transcription factor is essential for osteoblast differentiation. However, levels of Runx2 are often not well correlated with its transcriptional activity suggesting that this factor must be activated either by covalent modification or through interactions with other nuclear components. Runx2 is phosphorylated and activated by the mitogen-activated protein kinase (MAPK) pathway. This pathway is stimulated in at least two ways: by binding of type I collagen to f 2 g 1 integrins on the osteoblast surface and by treatment of cells with the osteogenic growth factor, FGF2. Protein kinase A (PKA) also may phosphorylate/activate Runx2 under certain conditions. Runx2 activity also is enhanced by factors known to stimulate specific signal transduction pathways such as PTH/PTHrP (signals through PKA and PKC pathways) and BMPs (Signal through Smad proteins). Interactions with Runx2 are complex involving both binding of distinct components such as AP-1 factors and Smads to separate sites on DNA, direct interactions between Runx2 and AP-1/Smad factors and MAPK or PKA-dependent Runx2 phosphorylation. These findings suggest that Runx2 plays a central role in coordinating multiple signals involved in osteoblast differentiation.

Bone mineralization in polyostotic fibrous dysplasia: histomorphometric analysis.

Fibrous dysplasia (FD) of bone can be complicated by renal phosphate wasting. The effect of hypophosphatemia on normal and dysplastic bone of FD patients has not been well characterized. In this study, we compared serum phosphorus (sPi) levels to histomorphometric findings in 27 iliac bone samples from 23 children and adolescents (aged 4.2-16.4 years) with polyostotic FD. The samples were separated into two groups, based on the presence (n = 10) or absence (n = 17) of a dysplastic lesion within the specimen. Histomorphometric results were compared with those from 18 age-matched control subjects without metabolic bone disease. In dysplastic lesions, trabeculae were clearly thinner and increased in number. Osteoid indices, osteoblast surface per bone surface, and mineralization lag time were elevated in dysplastic areas, but there was no detectable effect of sPi concentrations on these indices. In nondysplastic bone tissue, low sPi levels were associated with mildly increased osteoid thickness and prolonged mineralization lag time. None of the 13 patients in whom hand X-rays were available at the time of biopsy had radiological signs of rickets. In conclusion, low sPi can cause a mild systemic mineralization defect in FD, but the more severe mineralization defect seen in dysplastic lesions is independent of sPi levels. It is debatable whether the mild systemic mineralization defect warrants treatment with oral phosphorus supplementation if signs of rickets are absent.

Peptide-modified p(AAm-co-EG/AAc) IPNs grafted to bulk titanium modulate osteoblast behavior in vitro.

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) (p(AAm-co-EG/AAc) applied to model surfaces prevent protein adsorption and cell adhesion. Subsequently, IPN surfaces functionalized with the RGD cell-binding domain from rat bone sialoprotein (BSP) modulated bone cell adhesion, proliferation, and matrix mineralization. The objective of this study was to utilize the same biomimetic modification strategy to produce functionally similar p(AAm-co-EG/AAc) IPNs on clinically relevant titanium surfaces. Contact angle goniometry and X-ray photoelectron spectroscopy (XPS) data were consistent with the presence of the intended surface modifications. Cellular response was gauged by challenging the surfaces with primary rat calvarial osteoblast (RCO) surfaces in serum-containing media. IPN modified titanium and negative control (RGE-IPN) surfaces inhibit cell adhesion and proliferation, while RGD-modified IPNs on titanium supported osteoblast attachment and spreading. Furthermore, the latter surfaces supported significant mineralization despite exhibiting lower levels of proliferation than positive control surfaces. These results suggest that with the appropriate optimization, this approach may be practical for surface engineering of osseous implants.

Osteoblast-specific Knockout of the Insulin-like Growth Factor (IGF) Receptor Gene Reveals an Essential Role of IGF Signaling in Bone Matrix Mineralization

To examine the local actions of IGF signaling in skeletal tissue in a physiological context, we have used Cre-mediated recombination to disrupt selectively in mouse osteoblasts the gene encoding the type 1 IGF receptor (Igf1r). Mice carrying this bone-specific mutation were of normal size and weight but, in comparison with normal siblings, demonstrated a striking decrease in cancellous bone volume, connectivity, and trabecular number, and an increase in trabecular spacing. These abnormalities correlated with a striking decrease in the rate of mineralization of osteoid that occurred despite an unexpected osteoblast and osteoclast hyperactivity, detected from the significant increments in both osteoblast and erosion surfaces. Our findings indicate that IGF1 is essential for coupling matrix biosynthesis to sustained mineralization. This action is likely to be particularly important during the pubertal growth spurt when rapid bone formation and consolidation are required.

Biochemical measurements in the prediction of histologic subtype of renal transplant bone disease in women

Renal transplant osteodystrophy encompasses several histologic subtypes. Bone histomorphometric examination reliably distinguishes these groups but is invasive, is time-consuming, and delays diagnosis. Establishing a noninvasive method of correctly predicting histologic subtype in an individual to direct management is an attractive proposition. We identified 19 female renal transplant recipients with histologic evidence of hyperparathyroid bone disease (HPTH) and 14 with adynamic bone (ADB). We evaluated serum osteocalcin and bone-specific alkaline phosphatase as bone formation markers and urinary hydroxyproline (Hypro) and deoxypyridinoline cross-links as bone resorption markers. Mean concentrations for all markers were higher in the HPTH group, reaching significance for Hypro (HPTH, 24.8 ± 4.2 μmol/mmol creatinine; ADB, 13.2 ± 5.0 μmol/mmol creatinine; P = 0.01). A cutoff of 16.4 μmol/mmol creatinine for Hypro (Youden's index, 0.65) gave a sensitivity of 93% and specificity and positive predictive value (PPV) of 72% in predicting HPTH. In combination, Hypro greater than 16.4 μmol/mmol creatinine and parathyroid hormone greater than 80 pg/mL gave a specificity of 100%, sensitivity of 32%, and PPV of 100%. Conversely, for predicting ADB, Hypro less than 15.1 μmol/mmol creatinine (Youden's index, 0.45) gave a specificity of 93%, sensitivity of 53%, and PPV of 91%. Hypro less than 15.1 μmol/mmol creatinine plus osteocalcin less than 6.8 μg/L gave a specificity of 84.2%, sensitivity of 64.3%, and PPV of 75%. Significant associations between markers and histomorphometry were evident only for Hypro and osteocalcin (with osteoblast surface) and all markers (except deoxypyridinoline cross-links) with cortical volume. Markers have limited utility in identifying histologic subtype (Hypro was most effective) and, with the exception of Hypro and osteocalcin, showed little association with cell surface markers of bone cell activity. © 2002 by the National Kidney Foundation, Inc.

Changes in gene expression associated with the bone anabolic effects of basic fibroblast growth factor in aged ovariectomized rats

Basic fibroblast growth factor (bFGF) stimulates bone formation in vitro and in vivo. The purpose of this study was to determine changes in gene expression for bone matrix proteins, growth factors, and cytokines associated with the stimulatory effects of bFGF on bone formation in aged ovariectomized (ovx) rats. At 3 months of age, female Sprague-Dawley rats were sham-operated (sham) or ovariectomized (ovx), then maintained untreated for 1 year. At 15 months of age, baseline (BSL) sham and ovx rats were killed. All other rats received daily intravenous injections of bFGF (200 μg/kg) or vehicle (veh) for 14 days. Lumbar vertebrae were processed for quantitative bone histomorphometry or molecular analyses. Ovariectomy decreased vertebral cancellous bone volume by approximately 33% and increased most indices of bone turnover. Treatment of aged ovx rats with bFGF for only 14 days significantly increased cancellous bone volume compared with vehicle treatment of ovx rats, but this variable remained lower than in sham + veh rats. Osteoid volume, osteoblast surface, and osteoid surface were markedly increased, and osteoclast surface was significantly decreased in ovx + bFGF rats compared with sham + veh and ovx + veh rats. Northern analyses revealed that mRNA levels for osteocalcin and type I collagen, relative to 18S RNA, were significantly higher in ovx + bFGF rats than in ovx + veh rats by a factor of >10. RNase protection assays revealed that insulin-like growth factor (IGF-I) mRNA levels, relative to L32 housekeeping gene, were also significantly higher, by nearly a factor of 3, in ovx + bFGF rats than in ovx + veh rats. Treatment of ovx rats with bFGF did not appear to affect message levels for transforming growth factor-β (TGF-β), interleukin-6 (IL-6), and interferon-γ (IFN-γ). These in vivo results suggest that bFGF treatment upregulates gene expression for IGF-I, which may mediate, at least in part, the increased gene expression for bone matrix proteins and the bone anabolic effects of bFGF in aged ovx rats.

Sequential treatment with basic fibroblast growth factor and PTH is more efficacious than treatment with PTH alone for increasing vertebral bone mass and strength in osteopenic ovariectomized rats.

The study was designed 1) to determine whether treatment with basic fibroblast growth factor (bFGF) and PTH is more efficacious than treatment with PTH alone for increasing bone mass and strength and improving trabecular microarchitecture in osteopenic ovariectomized rats, and 2) to assess whether prior and concurrent administration of the antiresorptive agents estrogen and risedronate suppresses the bone anabolic response to treatment with bFGF alone and sequential treatment with bFGF and PTH. Three-month-old female Sprague Dawley rats were ovariectomized (OVX) or sham-operated (sham) and maintained untreated for 1 yr. Baseline sham and OVX rats were killed at this time (15 months of age). Groups of rats were injected sc with estrogen (10 microg/kg, 4 d/wk), risedronate (5 microg/kg, 2 d/wk), or vehicle. At the end of the second week of antiresorptive treatment, catheters were inserted into the jugular veins of all rats, and vehicle or bFGF at a dose of 250 microg/kg was injected daily for 14 d. Three groups of rats were killed at the end of bFGF treatment. The remaining rats were continued on their respective antiresorptive therapy and injected sc with vehicle or synthetic human PTH-(1-34) at a dose of 80 microg/kg, 5 d/wk, for 8 wk. Lumbar vertebrae were processed for cancellous bone histomorphometry and biomechanical testing. Ovariectomy resulted in a decrease in vertebral bone mass and strength. Treatment of OVX rats for 14 d with bFGF markedly increased osteoblast surface, osteoid surface, and osteoid volume compared with vehicle treatment of sham and OVX rats. Furthermore, osteoid bridges were observed extending between preexisting trabeculae in bFGF-treated OVX rats. Prior and concurrent administration of estrogen and risedronate did not suppress these bone anabolic effects of bFGF. Treatment of OVX rats with PTH alone increased vertebral cancellous bone mass and strength to the level of vehicle-treated sham rats. Sequential treatment of OVX rats with bFGF and PTH further augmented vertebral bone mass and strength to a level above that observed in OVX rats treated with PTH alone. The improvements in bone mass and strength were associated with an increase in trabecular thickness in OVX rats treated with PTH alone and with an increase in trabecular thickness and node to terminus ratio, an index of trabecular connectivity, in OVX rats treated sequentially with bFGF and PTH. Cotreatment with estrogen and risedronate did not suppress the anabolic response of bone to bFGF and PTH. In fact, a trend for an even greater increase in cancellous bone mass and node to terminus ratio was observed in OVX rats treated with risedronate, bFGF, and PTH. These findings indicate that sequential treatment with bFGF and PTH is more efficacious than treatment with PTH alone for increasing bone mass and strength and improving trabecular microarchitecture in osteopenic OVX rats.

Treatment of a murine model of high-turnover renal osteodystrophy by exogenous BMP-7

The secondary hyperparathyroidism of chronic kidney disease (CKD) produces a high turnover osteodystrophy that is associated with peritrabecular fibrosis. The nature of the cells involved in the development of peritrabecular fibrosis may represent osteoprogenitors expressing a fibroblastic phenotype that are retarded from progressing through osteoblast differentiation. To test the hypothesis that osteoblast differentiation is retarded in secondary hyperparathyroidism due to CKD producing bone marrow fibrosis, we administered bone morphogenetic protein 7 (BMP-7), a physiologic regulator of osteoblast regulation, to C57BL6 mice that had CKD produced by electrocautery of one kidney followed by contralateral nephrectomy two weeks later. Following the second surgical procedure, a subgroup of mice received daily intraperitoneal injections of BMP-7 (10 microg/kg). Three to six weeks later, the animals were sacrificed, blood was obtained for measurements of blood urea nitrogen (BUN) and parathyroid hormone (PTH) levels, and the femora and tibiae were processed for histomorphometric analysis. The animals had significant renal insufficiency with BUN values of 77.79 +/- 22.68 mg/dL, and the level of renal impairment between the CKD untreated mice and the CKD mice treated with BMP-7 was the same in the two groups. PTH levels averaged 81.13 +/- 51.36 and 75.4 +/- 43.61 pg/mL in the CKD and BMP-7 treated groups, respectively. The animals with CKD developed significant peritrabecular fibrosis. In addition, there was an increase in osteoblast surface and osteoid accumulation as well as increased activation frequency and increased osteoclast surface consistent with high turnover renal osteodystrophy. Treatment with BMP-7 eliminated peritrabecular fibrosis, increased osteoblast number, osteoblast surface, mineralizing surface and single labeled surface. There was also a significant decrease in the eroded surface induced by treatment with BMP-7. These findings indicate that BMP-7 treatment in the setting of high turnover renal osteodystrophy prevents the development of peritrabecular fibrosis, affects the osteoblast phenotype and mineralizing surfaces, and decreases bone resorption. This is compatible with a role of osteoblast differentiation in the pathophysiology of osteitis fibrosa.

Parathyroid hormone-independent osteoclastic resorptive bone disease: a new variant of adynamic bone disease in haemodialysis patients.

Osteitis fibrosa cystica (OFC) caused by secondary hyperparathyroidism is the pre-eminent form of uraemic osteodystrophy. In recent years, however, new bone abnormalities have been described. Among them adynamic bone disease (ABD) has become a focus of growing interest. Marked suppression of dynamic bone measurements with normal or near-normal static bone-forming parameters are the hallmarks of this disorder. Depressed parathyroid hormone (PTH) levels, frequently evident in this entity, have been linked causally with low bone turnover. We reviewed bone biopsy specimens from 96 patients with end-stage renal disease undergoing chronic haemodialysis. We found OFC in 50% of our patients, 20% had mixed bone disease, 24% showed bone morphology of ABD and a minority (6%) had osteomalacia, mostly due to aluminium accumulation. In the patients that were affected by ABD there was a distinct subgroup with bone morphology featuring a striking increase in osteoclast number and osteoclast surface, whereas the osteoid volume, osteoid thickness, osteoblast surface, tetracycline uptake and bone formation rates were diminished as in ordinary ABD. Similarly the PTH levels in this subgroup were low or undetectable. We describe patients undergoing chronic haemodialysis with static and dynamic bone forming parameters, indistinguishable from that of ABD, but differing from the classic ABD by the presence of increased osteoclastic bone resorption. The suppressed PTH levels in this subgroup suggests that factors other than PTH activate osteoclasts in some patients on chronic haemodialysis. Uraemic cytokines and/or toxic metabolites, including beta-microglobulin, may be involved in this disorder. The precise nature of this bone abnormality remains to be defined by further studies.

The effect of hemorrhagic shock in a caprine tibial fracture model.

The purpose of this study was to assess effects of hemorrhagic shock on fracture healing in a large animal closed fracture model. Prospective randomized trial. University Medical Center. Eight skeletally mature neutered male goats. Standardized bilateral closed midshaft tibia fractures were created in all the goats. The goats were randomized to a hemorrhage, shock and resuscitation group (shock group), or a control group. Hemorrhagic shock was induced in the four goats in the shock group. The shock state was maintained for thirty minutes. The remaining four goats were used as a control group. All fractures were stabilized with a standardized external fixator. Swan-Ganz catheters were used to record cardiac output (CO). Arterial blood was sampled to determine base deficit values for the goats after hemorrhage. Radiographs were obtained weekly. After four weeks the tibias were harvested and nondestructively tested in torsion. Immediately after mechanical testing, the tibias were processed for histologic analysis. Standard histomorphologic indices, including total bone volume (TV/BV%), osteoblast surfaces (Ob/BS%), and osteoid surface (OS/BS%), were measured. One goat in the shock group became agitated upon emergence from anesthesia and dislodged two of his external fixator pins. This animal was killed and was not included in the analysis of results. One goat that would have been entered into the control group was then switched to the shock group, leaving four goats in the shock group and three in the control group. One goat in the shock group developed a nonunion of his left tibia fracture. This nonunion occurred because of loosening of pin fixation in the distal tibia. The nonunited bone was not included in the analysis of results. The animal's right tibia healed uneventfully. Hemorrhage uniformly resulted in shock. In the shock group goats, average CO on induction of anesthesia was 3.7 l/min. Average CO dropped to 0.7 after hemorrhage and rose to 6.6 after fluid resuscitation. The average base deficit for the shock group animals was -9.9. The control group animals had minor drops in average CO, from 4.5 on induction of anesthesia to 4.3 on emergence from anesthesia. Radiographic analysis showed no apparent differences in healing between groups when comparing callus area and density. With the exception of the nonunion, all tibiae were considered to be healing normally radiographically. Biomechanical testing showed no statistical difference between the shock and control groups in maximum torque (p = 0.95), stiffness (p = 0.64), and energy absorbed at failure (p = 0.91). Histomorphologic results indicated there were no differences between groups. Shock did not appear to influence bone formation rate or callus remodeling compared with controls (p = 0.6). No evidence of osteocyte necrosis was observed. In this study no significant difference was found between the shock and control groups in any of the parameters measured. Transient hemorrhagic shock does not appear to adversely affect closed tibia fracture healing in a goat model.