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PubMed2005

Immunological responses to bone soluble proteins in recipients of bone allografts.

The significance of an immune response in complications of bone allograft procedures is not well understood. This study evaluates the immunological reaction to bone allografts in either cortical or cancellous form. Serological responses from osteoarthritis (OA) patients and normal individuals to soluble proteins extracted from allografts were assessed using Western blotting and ELISA techniques. A large number of patients expressed antibodies to bone proteins extracted from the washed bone as compared with normal controls. Antibodies were present in patients without the use of bone allografts, indicating that OA patients develop antibodies to bone soluble proteins. However, patients receiving allografts exhibited an increased immune reactivity against multiple bone proteins when compared with non-grafted patients. Protein characterization of the immunoreactive proteins revealed that the majority of antigenic targets were fragments of various collagen molecules. The data suggests that OA patients develop antibodies to bone soluble proteins prior to surgery, and these antibodies increase after revision surgery utilizing bone allografts. These findings support the implications that various collagen molecules as well as their degraded fragments represent potential immunogenic proteins within bone allografts. The removal of these antigenic proteins from the allografts prior to surgery may alleviate this immunological reactivity and improve graft outcome.

Adult↗
PubMed1991

Isolation of a novel insulin-like growth factor (IGF) binding protein from human bone: a potential candidate for fixing IGF-II in human bone.

Insulin-like growth factor-II (IGF-II) is the most abundant growth factor stored in human bone. Upon release from this storage depot, IGF-II could act in bone repair and in the coupling of bone formation to bone resorption, a process inherent to bone which is a key regulatory process for maintenance of bone tissue. In this study, we report the isolation and characterization of a novel IGF binding protein (IGFBP) from human bone and describe how this IGFBP may be involved in the fixation of IGF-II in human bone. This new IGFBP has an apparent molecular weight of 29 kDa and has several fold higher affinity for IGF-II than IGF-I which could explain the much greater abundance of IGF-II than IGF-I in human bone. In terms of biological activity, this IGFBP was found to potentiate the proliferative actions of IGF-II on bone cells. This work raises the possibility that this IGFBP may participate in mediating some of the actions of IGF-II.

Amino Acid Sequence↗
PubMed2000

L-arginine prevents bone loss and bone collagen breakdown in cyclosporin A-treated rats.

Cyclosporin A is implicated in the pathogenesis of post-transplantation bone disease. Because of recent evidence that cyclosporin A may cause renal and cardiovascular toxicity by inhibiting nitric oxide (NO) activity, and that NO slows bone remodeling and bone loss in animal and human studies, we investigated a possible link between NO production and beneficial effects on bone health in cyclosporin A-treated rats. Thirty-six 10-week-old male rats were assigned to six groups of six animals each, and treated for 4 weeks with: vehicle; cyclosporin A; L-arginine; N(G)-nitro-L-arginine methylester (L-NAME, a general inhibitor of NO synthase activity); a combination of cyclosporin A+L-arginine; and a combination of cyclosporin A+L-NAME. Whole body and regional (spine and pelvis) bone mineral content of rats were measured under basal conditions and at the end of the treatment period by dual-energy X-ray absorptiometry (DXA) scanning. Femur weights and serum concentrations of pyridinoline, a reliable marker of bone resorption, were measured at the end of the study period. Cyclosporin A-, L-NAME-, and cyclosporin A+L-NAME-treated rats had significantly lower bone mineral content and femur weights, and significantly higher pyridinoline levels than did control animals. The administration of L-arginine appeared to prevent bone loss caused by cyclosporin A, suggesting that this amino acid, which can be converted to produce NO, might prove useful in preventing disturbed bone modeling and inhibition of bone growth associated with cyclosporin A therapy.

Amino Acids↗
PubMed1999

Protective effect of an aldose reductase inhibitor against bone loss in galactose-fed rats: possible involvement of the polyol pathway in bone metabolism.

Many patients with diabetes mellitus show a moderate reduction in bone mass. Our recent in vitro studies showed that sustained exposure of osteoblast-like MG-63 cells to high glucose by itself impairs their functions partly via the polyol pathway. To investigate the role of hyperglycemia in the etiology of diabetic osteopenia in vivo separately from insulin deficiency, we determined whether epalrestat, an aldose reductase (AR) inhibitor (ARI), lessens the abnormalities in calcium (Ca) metabolism in galactose-fed rats. Weight gain was impaired in the rats, which was not altered by epalrestat. Galactose feeding temporarily enhanced bone resorption as reflected by increased biochemical markers for bone resorption (urinary excretion of pyridinoline [PYR] and deoxypyridinoline [DPYR]) at 1 to 3 months, which were significantly decreased by epalrestat. Epalrestat also restored the positive correlation between a bone-formation marker (serum osteocalcin [OC]) and a bone-resorption marker (urinary DPYR excretion) at 6.5 months. Histomorphometric analysis of bone performed 6.5 months after galactose feeding showed that both the bone volume and osteoblast numbers in the tibia, which were significantly suppressed by galactose feeding, were partly restored to a significant extent by the simultaneous administration of epalrestat. In summary, epalrestat partially protected against the development of osteoblast dysfunction and reduced the temporary increase in biochemical markers for bone resorption induced by galactose feeding, with a resultant increase in bone volume, suggesting that the polyol pathway may be intimately involved in the development of abnormal bone metabolism in galactose-fed rats.

Aldehyde Reductase↗
PubMed1999

Mechanical compression of a fibrous membrane surrounding bone causes bone resorption.

Early micromovement and migration of a prosthesis of a hip or knee predicts late clinical loosening of the prosthesis. Such migration is likely to be associated with mechanical compression of the fibrous membrane interpositioned between bone and prosthesis during movement. Compression of the fibrous membrane by loading may lead to locally high fluid pressures reaching the underlying bone tissue. It has been established that high fluid pressures can lead to bone resorption. This resorption may eventually lead to clinical loosening of the prosthesis. We developed an experimental model to study the effects of compression of a soft tissue layer located between a titanium implant and cortical bone. In twelve rabbits, this device was implanted in the proximal tibia and allowed to osseointegrate. Next, a layer of soft tissue was allowed to form between titanium and bone. Subsequently, in six rabbits a cyclic load of 60 times in 2 min per day during 2 weeks was applied, leading to compression of the interpositioned soft tissue layer only. In the other six rabbits no load was applied. In all six loaded specimens, osteocyte death and bone resorption was observed underneath the area where compression of the fibrous membrane was exerted to a depth exceeding the amplitude of the loading device. Furthermore, formation of fibrocartilage was observed in the loaded areas. Formation of fibrocartilage, osteocyte death or bone resorption did not occur in the controls. Our results indicate that compression of a fibrous membrane surrounding bone can lead to resorption of the underlying bone primarily because of osteocyte death and subsequent resorption of dead bone tissue. This may explain the observation that early migration of a hip or knee prosthesis is predictive of clinical loosening of the prosthesis.

Animals↗
PubMed2003

Histological evaluation of the bone response to calcium phosphate cement implanted in cortical bone.

The aim of this study was to investigate the physicochemical and biological properties of a newly developed calcium phosphate cement (CaP cement) implanted in cortical bone. CaP cement was injected as a paste into tibia cortical bone defects in goats. Polymethylmethacrylate (PMMA) bone cement was used as a control. The animals were killed after 3 days, 2, 8, 16 and 24 weeks. X-ray diffraction and Fourier transform infrared spectroscopy performed at retrieved samples showed that the CaP cement had set as a carbonate apatite and remained stable over time. Light microscopic evaluation showed that after 2 weeks the cement was in tight contact with the bone without any inflammatory reaction or fibrous encapsulation. At later time points, the CaP cement implants were totally covered by a thin layer of bone and osteoclasts, present at the interface, which were clearly resorbing the cement. At locations where CaP cement was resorbed, new bone was deposited. Transmission electron microscopy revealed that indeed a seamless contact existed between CaP cement and bone, as characterized by the occurrence of an electron dense line of 50-60 nm thick that covered the CaP cement. Osteoblasts, in contact with the cement, were depositing new bone. Although the bulk of the material was still in situ after 24 weeks, the progressive osteoclast resorption of the cement followed by new bone formation suggests that all of the material may be replaced eventually. In contrast to the CaP cement, the PMMA reference cement was always surrounded by a thin fibrous capsule. The results indicate that the investigated CaP cement is biocompatible, osteoconductive as well as osteotransductive and is a candidate material for use as a bone substitute.

Animals↗
PubMed2001

Bone morphogenetic protein-2, but not bone morphogenetic protein-7, promotes dendritic growth and calbindin phenotype in cultured rat striatal neurons.

Bone morphogenetic proteins are members of the transforming growth factor-beta superfamily. They are widely expressed in the mammalian nervous system, where they exert trophic effects on several neuronal populations. We studied the neurotrophic activity of bone morphogenetic protein-2 and bone morphogenetic protein-7 (also called osteogenic protein-1) on cultured striatal cells, previously shown to express bone morphogenetic protein ligands and receptors. Our results indicate that only bone morphogenetic protein-2 promoted the differentiation of GABAergic neurons, especially of the calbindin-positive subpopulation, the subset of projecting striatal neurons that degenerates in Huntington's disease. Bone morphogenetic protein-2 increased the area, perimeter and degree of arborization of GABAergic neurons, promoting calbindin phenotype without altering proliferation or apoptosis. In contrast, neither bone morphogenetic protein-2 nor -7 affected striatal cholinergic interneurons. However, they both increased the number of glial fibrillary acidic protein-positive cells. Suppression of glial proliferation with 5-fluorodeoxyuridine did not abolish bone morphogenetic protein-2 effects on the differentiation of striatal neurons, ruling out an indirect mechanism through astrocytes. In conclusion, our results show that bone morphogenetic protein-2 promotes the differentiation of cultured GABAergic striatal neurons, suggesting that bone morphogenetic proteins are involved in the development of the striatum.

Animals↗
PubMed2003

Growth hormone substitution increases gene expression of members of the IGF family in cortical bone from women with adult onset growth hormone deficiency--relationship with bone turn-over.

OBJECTIVE: To investigate the effects of growth hormone (GH) replacement therapy on bone matrix gene expression of insulin-like growth factors (IGFs) and markers of bone metabolism in women with adult-onset GH deficiency (GHD). DESIGN AND METHODS: Nineteen women, mean age 45 (range 24-56) years, were included in a double-blind, placebo-controlled parallel group study for 12 months. Biochemical markers were measured at baseline, 6 and 12 months. Bone biopsies were obtained and BMD was measured at baseline and after 12 months. RESULTS: Maximum responses were observed after 6 and 12 months, for bone resorptive and bone formative markers respectively. GH therapy enhanced gene expression in cortical bone of IGFs, GH-and calcitonin-receptor (CR) and osteoprotegerin (OPG), however with the most pronounced effects on CR and IGF-I. Changes in IGF-I gene expression during longitudinal follow-up were significantly correlated with changes in both circulating IGF-I (r = 0.82, p < 0.05), changes in markers of enhanced osteoclastic activity, measured both locally in bone (CR, r = 0.87, p < 0.01) and in serum (CTX-I, r = 0.86, p < 0.05), as well as serum bone ALP (r = 0.96, p < 0.01). CONCLUSIONS: This study indicates that both liver- and bone-derived IGF-I may be significant in mediating the effects of GH on bone metabolism in humans.

Adult↗
PubMed2001

The CAG repeat polymorphism in the androgen receptor gene affects bone density and bone metabolism in healthy males.

OBJECTIVE: Bone metabolism and bone density (BD) are influenced by sex hormones. Testosterone (T) action is exerted through the androgen receptor (AR). We investigated the potential impact of the CAG repeat (CAGR) polymorphism within the AR gene on BD and bone metabolism in healthy younger males. PATIENTS AND MEASUREMENTS: The number of CAGRs in 110 healthy men aged 20-50 years was determined by sequence analysis. We assessed BD by the radiation-free method of quantitative ultrasound (QUS) of the phalanges. Serum levels of bone-specific alkaline phosphatase (BAP) and urine secretion of free deoxypyridinoline (DPD, corrected for creatinine), serum levels of sex hormones, body fat content and lifestyle factors were determined. RESULTS: In stepwise multiple regression models controlling for age, body fat content and lifestyle factors, the number of CAGRs was an independent negative predictor of BD (partial r = - 0.286, P = 0.001), whereas it was positively associated with markers of bone turnover (for BAP: partial r = 0.32, P= 0.001; for DPD: partial r = 0-241, P = 0.013). Levels of free T and oestradiol showed an independent and positive association with BD; age contributed significantly to lower BD. Age and free T were negatively associated with markers of bone turnover, whereas oestradiol showed a positive correlation with BAP and DPD. ANOVA in groups according to age and the CAGR length suggested an increased age-dependent bone loss in subjects with a CAGR length of 22-31 compared with 14-21 CAGRs (overall P < 0.01). CONCLUSIONS: A high number of CAG repeats within the androgen receptor gene attenuates testosterone effects on bone density and bone metabolism. This seems to be associated with accelerated age-dependent bone loss.

Adult↗
PubMed2006

The usefulness of bone metabolic indices for the prediction of changes in bone mineral density after parathyroidectomy in patients with primary hyperparathyroidism.

Patients with primary hyperparathyroidism (pHPT) have reduced bone mineral density (BMD). Although pHPT causes high bone turnover, the exact metabolic bone markers useful for predicting changes in BMD after parathyroidectomy (PTX) remain elusive. The present study was performed to examine the relationship between bone metabolic indices and BMD changes after PTX in 29 pHPT Japanese patients, which received PTX successfully. BMD values were measured by dual-energy X-ray absorptiometry in the lumbar spine and distal one third of radius. As for bone metabolic indices, serum bone-type alkaline phosphates (BAP), serum osteocalcin (OCN), urinary deoxypiridinoline (Dpd), and urinary type I collagen cross-linked N-telopeptides (NTX) were measured. The study included 10 male and 19 female patients (17 postmenopausal). Urinary Dpd, but not NTX was significantly correlated with serum BAP and OCN. Either bone formation or bone resorption indices were significantly and highly correlated with Z-score of BMD in the radius, but not at lumbar spine. Urinary Dpd was significantly correlated with BMD changes at both lumbar spine and radius and at all time points over the two years after PTX. These correlations were most potent among bone metabolic indices in this study. The measurement of urinary Dpd would be useful for predicting long-term changes in BMD at radial and lumbar spine after PTX than other bone metabolic indices.

Bone Density↗
PubMed1997

Bone density and bone metabolic markers in active collegiate athletes: findings in long-distance runners, judoists, and swimmers.

We investigated the bone metabolic system status of 103 male and female volunteer collegiate athletes, who were actively pursuing one of three different sports: Long-distance running (LR); judo (JU); and swimming (SW). The following parameters were evaluated: total body bone mineral density (TMBD); bone-forming metabolic markers; serum procollagen type I C-peptide (PICP) levels; bone alkaline phosphatase (B-ALP) content; bone resorption markers, urinary pyridinoline (Pyd) and deoxypyridinoline (Dpd) levels. We found that the TBMD and urinary Dpd values in JU athletes were significantly higher (p < 0.001) than in athletes of the same sex in the other two groups. The urinary Pyd level in male JU athletes was also higher (p < 0.001) than that in the other two groups, but that in females JU athletes was only higher (p < 0.01) than that in female LR athletes. The PICP levels were similar to the TBMD values in all groups. No differences in bone density or in bone metabolic markers were seen in LR and SW athletes of the same sex. We thus conclude that differences in bone mineral density are in part due to the demands of the specific sport, and that they are reflected in bone metabolic markers. In addition, the status of bone metabolic turnover in male JU athletes in training may be hypermetabolic and as well as that of female JU athletes with regular menses cycles.

Adult↗
PubMed1974

Formation of bone tissue in culture from isolated bone cells.

A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This tissue culture system starting from isolated bone cells is therefore suitable for studies on the structure and function of bone.

Alkaline Phosphatase↗
PubMed1984

Fetal bovine bone cells synthesize bone-specific matrix proteins.

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.

Alkaline Phosphatase↗
PubMed2001

Suppressive levothyroxine therapy has no significant influence on bone degradation in women with thyroid carcinoma: a comparison with other disorders affecting bone metabolism.

The aim of this study was to examine different influences on bone degradation (estrogen status, thyroid function, parathyroid function, bone metastases) with special interest focusing on the significance of suppressive levothyroxine therapy (LT4) on bone degradation in patients with differentiated thyroid carcinoma (DTC). Two markers of bone degradation (ELItest NTx = U-NTx; Serum CrossLaps = S-CTx) were used (1) to quantify the influence of different metabolic influences on bone degradation and (2) to compare these two markers with each other. One hundred forty samples of 98 female patients ages 23-86 years were analyzed. The correlation between the two assays of bone degradation was high (r = 0.825; p < 0.001). Both assays demonstrated that estrogen deficiency, hyperparathyroidism, and bone metastases caused significant increases of bone degradation. A suppressive LT4 therapy, as used for patients with DTC, led to no significant increases of S-CTx and U-NTx. The study indicates that a well-controlled suppressive LT4 therapy has only a minor effect on the degree of bone degradation and that a possible estrogen deficiency in patients with DTC has a greater impact on bone degradation. Thus, female patients with DTC on suppressive LT4 therapy and estrogen deficiency may benefit from hormone replacement therapy, as patients with DTC and normal estrogen levels presented similar results to euthyroid controls.

Adult↗
PubMed2002

Cranioplasty using allogeneic perforated demineralized bone matrix with autogenous bone paste.

The efficacy of allogeneic perforated demineralized bone matrix with autogenous bone paste in the treatment of full-thickness cranial defects was evaluated in 10 consecutive patients between June 1998 and December 1998. The skull defects resulted from trauma in 9 patients and removal of a cranial tumor in 1 patient. The size of the skull defects ranged from 8 x 6 cm to 11 x 12.5 cm. Follow-up averaged 33 months for all patients. Postimplantation evaluations included serial photographs, repeated physical examination, and three-dimensional computed tomography for all patients. Visual inspection of the implanted biomaterial 6 months later was possible in 1 patient. The contour of the reconstructed skull was acceptable aesthetically without any secondary depression noted during the follow-up period. Three-dimensional computed tomographic scans taken 2 years after implantation indicated that the allogeneic perforated demineralized bone matrix provided a matrix for new bone formation with remarkable osteoinductive potential for new bone formation. The autogenous bone paste was able to caulk the demineralized bone matrix and fill the contour irregularities and gaps of the reconstructed cranium. The results from this clinical study indicated that allogeneic perforated demineralized bone matrix with autogenous bone paste is a promising alternative to an autogenous bone graft and or alloplastic material for cranioplasty.

Adult↗
PubMed1995

Bone formation in cranial, mandibular, tibial and iliac bone grafts in rats.

Several studies have suggested that grafts from membranous derived bone (e.g., calvarial grafts) retain their volume better than those from endochondral derived bone (e.g., iliac bone grafts). Increased osteogenesis in grafts of the former type has been offered as the explanation. However, simple volume measurements of the recovered grafts do not differentiate between viable and dead bone. We studied fresh syngeneic full-thickness bone grafts from calvaria, mandibula, tibia diaphysis, and iliac bone implanted in the back muscles of young Lewis rats. Bone formation in grafts recovered 3 weeks postoperatively was quantitatively evaluated by strontium 85 uptake analyses. We found that the strontium 85 uptake was greater in calvarial and mandibular grafts than in tibial grafts. No difference was found among calvarial, mandibular, and iliac grafts or between tibial and iliac grafts. We conclude that the anatomical area of harvest is important regarding new bone formation in syngeneic bone grafts. However, the results do not support the contention that better maintenance of volume of calvarial grafts compared with iliac bone grafts is due to enhanced osteogenesis in the former.

Animals↗
PubMed1996

Comparison of ossification of demineralized bone, hydroxyapatite, Gelfoam, and bone wax in cranial defect repair.

Demineralized bone allografts in the repair of calvarial defects are compared with other common bone fillers. This study uses a video-digitizing radiographic analysis of calvarial defect ossification to determine calcification of bone defects and its relation to postoperative clinical examination and regional controls. The postoperative clinical results at 3 months demonstrated that bony healing was greatest in bur holes filled with demineralized bone and hydroxyapatite. Radiographic analysis demonstrated calcification of demineralized bone-filled defects compared to bone wax- and Gelfoam-filled regions. Hydroxyapatite granules are radiographically dense, thus not allowing accurate measurement of true bone healing. The results suggest that demineralized bone and hydroxyapatite provide better structural support via bone healing to defined calvarial defects than do Gelfoam and bone wax.

Bone Regeneration↗
PubMed2001

Tissue-engineered bone repair of sheep cranial defects with autologous bone marrow stromal cells.

Cranial bone defect remains a major challenge to craniofacial surgeons because of limited availability of autologous bone graft to repair the defects and the donor site defects secondary to tissue harvesting. In contrast, tissue-engineering technique can generate a large bone tissue using small amount of autologous cells and therefore avoid these problems. Bone Marrow Stromal Cells (MSCs) have the potential of multi-lineage (including osteogenic) differentiation. The objective of this study was to investigate the potential of using autologous MSCs to repair cranial bone defects by a tissue-engineering approach. Autologous MSCs were isolated from eight adult sheep respectively and were in vitro expanded and induced to become osteogenic cells. Bilateral full-thickness defects (20 mm in diameter) of parietal bones were created in animals and the bone defects were either repaired with the bone implants constituted with MSCs and calcium alginate at the experimental side (n = 8) or treated with calcium alginate only without MSCs (n = 4) or left unrepaired (n = 4) at the control side. New bone tissues were observed either grossly or histologically at the defects of experimental group as early as 6 weeks post-repairing, but not in control groups. The engineered bone tissue became more mature at 18 weeks post-repairing. Three-dimensional computerized tomography (CT) scan revealed an almost complete repair of the defect of experimental group at 18 weeks. This study may provide insight for future clinical repair of cranial defect.

Alginates↗