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PubMed1989

Dystrophic calcification and mineralization during bone induction: biochemical differences.

The calcification of implants of glutaraldehyde-cross linked collagenous tissues and collagen was studied in young and old rats and compared to bone induction by non-crosslinked osteogenically active demineralized bone matrix (DBM). Glutaraldehyde-crosslinked implants of DBM, tendon, and cartilage calcified in young but not in old animals and accumulated only trace amounts of BGP (Bone Gla protein, osteocalcin). Alkaline phosphatase activity and BGP was high in implants of DBM and undetectable in crosslinked implants. To try and understand why bone formation is so significantly reduced in older Fischer 344 rats, we developed a system which consists of cylinders of DBM sealed at the ends with a Millipore filter. Cells originating from 20 day old embryo donors were introduced into the chambers prior to subcutaneousmplantation. After 4 weeks of implantation in 26 month old rats, the cylinders containing embryonic calvaria or muscle cells were found to be full of bone and/or cartilage.

Animals↗
PubMed1990

Immunohistochemical observations on bone morphogenetic protein in normal and abnormal conditions.

Localization of bone morphogenetic protein (BMP) in human tissues and cells is important for investigating the mechanism of bone induction. A stable cell line secreting monoclonal antibody against bovine BMP (bBMP-McAb) was obtained by the hybridoma technique. The result of immunohistochemical staining (ABC method) showed that BMP is distributed along collagen fibers of normal bone, in periosteal cells, and in mesenchymal cells of marrow stroma. Little BMP can be found in bone cells of lamellar bone or in calcified bone matrix. BMP may be abundant in human tooth anlagen such as predentin, cells of the outer and inner enamel epithelium, and cells of dental sac generating bone. BMP is found in the cytoplasm of tumor cells of osteosarcoma and chondrosarcoma. Immunohistochemical staining showed that BMP plays a role in bone fracture healing. The ability of BMP-McAb to detect BMP and to inhibit the generation of new bone also makes it potentially useful in diagnosing, treating, and providing a prognosis for osteosarcoma and other bone diseases.

Animals↗
PubMed1989

Dystrophic calcification and mineralization during bone induction: biochemical differences.

The calcification of implants of glutaraldehyde-crosslinked collagenous tissues and collagen was studied in young and old rats and compared to bone induction by non-crosslinked osteogenically active demineralized bone matrix (DBM). Glutaraldehyde-crosslinked implants of DBM, tendon, and cartilage calcified in young but not in old animals and accumulated only trace amounts of BGP (Bone Gla protein, osteocalcin). Alkaline phosphatase activity was high in implants of DBM and undetectable in crosslinked implants. To try and understand why bone formation is so significantly reduced in older Fischer-344 rats, we developed a system which consists of cylinders of DBM sealed at the ends with a Millipore filter. Cells originating from 20-day-old embryo donors were introduced into the chambers prior to subcutaneous implantation. After 4 weeks of implantation in 26-month-old rats, the cylinders containing embryonic calvaria or muscle calls were found to be full of bone and/or cartilage.

Age Factors↗
PubMed1995

1995 Volvo Award in basic sciences. The use of an osteoinductive growth factor for lumbar spinal fusion. Part II: Study of dose, carrier, and species.

STUDY DESIGN: Efficacy of a bovine-derived osteoinductive growth factor was studied in a rabbit model and in a nonhuman primate model of posterolateral lumbar spinal fusion. OBJECTIVES: To determine the minimum effective dose of growth factor and the influence of different carrier material on the outcome of intertransverse process lumbar fusion. SUMMARY OF BACKGROUND DATA: Bone morphogenetic proteins and related growth factors are becoming increasingly available in purified extract or genetically engineered forms and are capable of inducing new bone formation in vivo. Osteoinductive growth factors to enhance lumbar spinal infusion have not been well studied in models of posterolateral intertransverse process fusion. Because of the diminished potential of bone regeneration in primates (including humans) compared with phylogenetically lower animals, extrapolations regarding dose and efficacy cannot be made directly from results obtained in experiments performed on phylogenetically lower animals. Experiments on non-human primates are a critical step before attempting to use these growth factors on humans. METHODS. One hundred fifteen adult New Zealand white rabbits and 10 adult rhesus macaques underwent single level posterolateral intertransverse process lumbar spinal arthrodesis to evaluate different doses and carrier materials for a bovine-derived osteoinductive bone protein extract. Rabbit fusion masses were evaluated 5 weeks after arthrodesis by manual palpation, radiography, biomechanical testing, and light microscopy. Monkey fusion masses were evaluated 12 weeks after arthrodesis by radiography and light microscopy. RESULTS: Successful posterolateral intertransverse process spinal fusions were achieved in the rabbit models using an osteoinductive growth factor with three different carriers (autogenous iliac bone, demineralized allogeneic bone matrix, and natural coral). There was a dose-dependent response to the osteoinductive growth factor in the rabbit model, indicating that a threshold must be overcome before bone formation is induced. The methodology for biologic enhancement of spinal fusion developed in the rabbit model transferred successfully to the rhesus monkey, where the use of the osteoinductive growth factor with a demineralized bone matrix carrier resulted in spinal fusion in 12 weeks. CONCLUSION: These experiments provide an essential building block in the understanding of the biology of spinal fusion and the use of osteoinductive growth factors to enhance a posterolateral intertransverse process spinal fusion. The achievement of posterolateral spinal fusion in the rhesus monkey using an osteoinductive growth factor is a significant step toward the biologic enhancement of spinal fusion in humans.

Animals↗
PubMed1989

The relative effects of collagen fiber orientation, porosity, density, and mineralization on bone strength.

This investigation determined the relative importance of collagen fiber orientation, porosity, density, and mineralization in determining the tensile strength of bovine cortical bone. Thirty-nine specimens were tested for failure stress and the values of eight histologic and compositional parameters: collagen fiber orientation, wet and dry apparent density, percent mineralization of the bone matrix, and several components of porosity (Haversian canals, Volkmann's canals, and plexiform vascular spaces). Linear regression analysis showed that collagen fiber orientation was consistently the single best predictor of strength. Mineralization of the bone matrix was generally a poor predictor of strength. Density and porosity ranked between these variables in importance. Multiple regression equations containing all significantly correlated variables achieved correlation coefficients of 0.607 for plexiform bone and 0.881 for osteonal bone. Also, separate analysis of plexiform and osteonal specimens showed that the latter group was weaker even though it was less porous, apparently because it had collagen fibers which were less longitudinally oriented. This study suggests it is feasible to develop better empirical formulae for the prediction of cortical bone strength than are currently available if a variety of variables is introduced. Additional data are needed to confirm these results.

Animals↗
PubMed1991

Transdifferentiation of hypertrophic chondrocytes into osteoblasts in murine fetal metatarsal bones, induced by co-cultured cerebrum.

The fate of hypertrophic chondrocytes in 17-day-old metatarsal bones of fetal mice was studied in a culture system in which these cells were kept confined to their lacunae. Because the periosteum had been stripped off, osteoclasts could not invade the long bone and resorb the lacunar walls. The majority of the hypertrophic chondrocytes stayed alive and dedifferentiated gradually into cells with the appearance of stromal cells. When the long bones were co-cultured with pieces of cerebrum, the chondrocytes transdifferentiated into osteoblasts. We followed this process from day to day. The cells produced bone matrix that immunostained for collagen type I and osteocalcin. To exclude with certainty the possibility that the intralacunar osteoblasts had derived from remaining periosteal osteoprogenitor cells that invaded the lacunae, the long bones were pre-cultured with cytochalasin D, which inhibits cell proliferation and migration. After removal of the drug this effect persisted until after transdifferentiation had occurred. This proved that the bone matrix producing osteoblasts inside the cartilage lacunae were transdifferentiated chondrocytes. The transdifferentiation stimulating factor from brain tissue is still unknown.

Animals↗
PubMed2006

Destination of titanium particles detached from titanium plasma sprayed implants.

Small titanium particles may detach from titanium plasma sprayed (TPS) implants during implant insertion, when no preliminary tapping is used, probably for the frictional force between titanium coating and host bone. Aim of this study was to investigate the destination of these titanium particles observed in the peri-implant environment. Twenty-four TPS screws were implanted in tibiae of two sheep. Fourteen and 90 days after implantation the implants with the surrounding bone were removed and processed to be analyzed by light microscope and scanning electron microscope (secondary electron and back-scattered electron probes). Small titanium particles detached from the unloaded TPS implants were observed both in the newly-formed bone matrix and in marrow tissue. Histomorphometric analysis showed that both at 14 and 90 days after implantation the titanium particles appeared more concentrated in marrow tissue than in calcified bone matrix, decreasing by 66.4% over time. In particular, smaller particles (<250 microm(2)) decreased by 81.5%, whereas the larger ones (250-2000 microm(2)) did not show any significant variations over time, suggesting that most of the smaller particles may undergo to ionic dissolution, probably migrating into the peri-implant marrow lacunae. A slight migration of titanium particles from the implant surface towards the more distant peri-implant tissues was also demonstrated over time.

Animals↗
PubMed1994

Use of osteoinductive implants in the treatment of bone defects.

Osteogenic proteins (OPs) are a family of bone matrix polypeptides isolated from a variety of mammalian species, including mouse, rat, bovine, monkey, and man. OPs initiate chondroblastic differentiation in pluripotent mesenchymal progenitor cells, followed by the synthesis of new bone by endochondral ossification. OPs have the ability to induce healing of osteoperiosteal defects in several animal models, supporting a possible therapeutic role in the reconstruction of bone defects. OPs are responsible for the osteoinductive capacity of demineralized bone matrix (DBM) implants, which may also prove to be clinically useful. Preliminary studies using purified, naturally occurring human osteogenic proteins in the clinical management of non-unions have reported promising results. A prospective, randomized clinical trial is currently underway, comparing recombinant human osteogenic protein-1 (rhOP-1) to autograft in the treatment of tibial non-unions. The use of osteogenic protein implants to augment or replace autogenous and allogenous bone grafts will reduce morbidity, and circumvent the risk of disease transmission associated with transplantation.

Animals↗
PubMed1998

Carrier-dependency of cellular differentiation induced by bone morphogenetic protein in ectopic sites.

Partially purified bone morphogenetic protein (BMP) was delivered into two different types of carriers, porous particles of hydroxyapatite (PPHAP) and particles of insoluble bone matrix (IBM), and the ossification process was examined after subcutaneous implantation of the BMP/PPHAP and BMP/IBM in rats. The ossification in the BMP/PPHAP system was predominantly direct through bone formation similar to intramembranous ossification, whereas in the BMP/IBM system it was predominantly endochondral. The differences observed between the BMP/PPHAP and the BMP/IBM indicate the importance of the structure and nature of the carrier in the process of bone induction. The findings suggest that bone and cartilage differentiation is controlled not only by the regulation factor (BMP), but also by its interaction with the carrier, and that the BMP-induced cell differentiation is dependent upon the microenvironment derived from the carrier.

Animals↗
PubMed1994

Bone substitute with osteoinductive biomaterials--current and future clinical applications.

In craniomaxillofacial surgery, possible indications for the use of osteo-inductive biomaterials are interposition in intraosseous defects, contour augmentation, and reconstruction of segmental defects. The experimental results in the field of bone morphogenetic protein (BMP) research within the last few years have shown that it is possible to combine osteoinductive proteins with suitable carrier materials to obtain new composite osteoinductive biomaterials. These carrier materials function as slow-delivery systems for BMP. By combination of BMP with different carrier materials such as various types of calcium phosphate ceramics, collagen or inactive collagenous bone matrix, and other organic and inorganic carriers, the biomaterial can be adapted to clinical demands in a wide range. In several experimental animal studies, we investigated nine different calcium phosphate ceramics and inactive rat bone matrix for their use as BMP carrier. All materials tested seem to be suitable carriers for BMP. The first clinical applications are discussed.

Animals↗
PubMed1991

Bone particles from gallium-treated rats are resistant to resorption in vivo.

Gallium nitrate is a clinically effective agent for the treatment of cancer related hypercalcemia. The mechanism of action of this agent was investigated following development of a quantitative in vivo bone resorption assay modified from the method of Glowacki. In a preliminary study, the time course of resorption of 50 mg subcutaneous implants of bone powder in growing rats was followed by chemical analysis of mineral (ash and Ca) contents, enzymatic and histochemical assay of tartrate resistant acid phosphatase (TRAP) activity, and image analysis of changes in particle size using von Kossa stained sections. Day 21 was chosen as a single time point for the comparison of the extent of resorption of gallium-containing and control bone particles. Resorption of bone particles containing 0.39 micrograms Ga/mg bone was significantly inhibited relative to control particles. Mineral content (6.7 vs. 3.6 mg), Ca content (1.72 vs. 1.37 mg), and the percentage of the field covered by bone particles (12 vs. 9%) were greater in the animals which received gallium-containing bone particles. Similarly, the number of osteoclast-like cells and the TRAP activity in the gallium-containing bone particle implants at 21 days were increased relative to controls. These data indicate that gallium incorporation into bone matrix confers resistance to resorption.

Acid Phosphatase↗
PubMed2004

Collagen, type V, alpha1 (COL5A1) is regulated by TGF-beta in osteoblasts.

Bone matrix contains high concentrations of growth factors that are known to play important regulatory roles during osteogenesis, particularly transforming growth factor-beta (TGF-beta). Divergent effects of TGF-beta on bone formation have been reported both in vitro and in vivo depending upon experimental conditions, cells employed and their stage of maturation. In this study, we have used a clonal osteoblastic cell line MC3T3-E1, derived from newborn mouse calvaria, as an in vitro model of bone development. These cells undergo an ordered, time-dependent developmental sequence characterized by three stages (proliferation, differentiation and mineralization), over a 30-35-day period. In this study, cDNA microarray technology was used to study the expression profile of 8470 genes, in the presence of TGF-beta1 during osteoblast development. Microarray analysis revealed 120 cDNAs to be differentially expressed in MC3T3-E1 osteoblasts that had been treated with TGF-beta1. From the 120 differentially expressed genes, we selected Collagen, type V, alpha1 (COL5A1) {differential expression=+4.9} for further studies since it represents a previously uncharacterized component of the bone matrix. Using Northern blotting, we found that, when MC3T3-E1 cells were treated with TGF-beta1, COL5A1 was up-regulated during the proliferation and differentiation phases of osteogenesis. Furthermore, by a combination of RNA in situ hybridization and Northern blotting, we found COL5A1 mRNA to be expressed in the calvaria and developing bone of the E17.5 mouse embryos. Lastly, significant COL5A1 protein expression was observed by immunohistochemistry in the developing bone of the E17.5 mouse embryos. In conclusion, by the use of in vitro and in vivo approaches, we have discovered that the COL5A1 gene is a target of TGF-beta during osteogenesis.

Alkaline Phosphatase↗
PubMed1990

Fibroblast growth factor in chick osteogenesis.

In vivo effects of FGF on osteogenesis in the chick embryo were evaluated. Day-11 embryos were injected with FGF followed by radiotracers. 3H-thymidine labelling of osteogenic cells was significantly higher following FGF administration; 3H-proline labelling over bone matrix was greater in the controls. Cartilage cells and matrix were sparsely labelled indicating a low level of metabolic activity. These data provide the first in vivo evidence that FGF stimulates osteogenic cell proliferation, while inhibiting production of bone matrix collagen. A role for FGF in embryonic osteogenesis and in fracture repair is suggested.

Animals↗
PubMed1991

Purification, characterization, and studies on biosynthesis of a 59-kDa bone sialic acid-containing protein (BSP) from rat mandible using a monoclonal antibody. Evidence that 59-kDa BSP may be the rat counterpart of human alpha 2-HS glycoprotein and is synthesized by both hepatocytes and osteoblasts.

A monoclonal antibody was raised against a mineralized tissue-specific sialoprotein containing no phosphorus using partially purified noncollagenous bone matrix proteins from rats as antigen. Then the sialoprotein was purified by high performance liquid chromatography from rat mandibulae using the monoclonal antibody as a marker. The sialoprotein (59-kDa bone sialoprotein (BSP)) with a molecular weight of 59,000 contained 1.4% sialic acid but no detectable phosphorus. Immunohistochemical studies with the antibody showed that the protein was specific to mineralized tissues such as bone and dentin, and present in osteoblasts, osteocytes, and bone matrix. No other soft tissues, such as the cartilage, liver, kidney, and periosteum, were stained. However, Western blot analysis showed that plasma contained immunoreactive 59-kDa BSP. The quantitative amino acid composition of 59-kDa BSP resembled that of human alpha 2-HS glycoprotein (alpha 2-HSG) (Lee, C.-C., Bowman, B.H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407; Kellermann, J., Haupt, H., Auerswald, E.-A., and Muller-Esterl, W. (1989) J. Biol. Chem. 264, 14121-14128) and rat 64-kDa protein (Franzén, A., and Heinegård, D. (1985) in The Chemistry and Biology of Mineralized Tissues (Butler, W.T., ed), p. 132, EBSCO Media, Birmingham, AL). Amino acid sequence analyses of the amino-terminal region and four peptide fragments of 59-kDa BSP revealed that about 50% of the amino acids were homologous with those of human alpha 2-HSG, which is known to be synthesized by the liver, transported in the bloodstream, and incorporated into calcified tissues. But when newborn rat calvaria, primary cultures of osteoblast-rich cells, and adult rat hepatocytes were incubated with radioactive leucine, immunoreactive 59-kDa BSP was detected in their conditioned medium by fluorography. Several characteristics, including the amino acid sequence, suggest that 59-kDa BSP may be the rat counterpart of human alpha 2-HSG. However, rat 59-kDa BSP is a single peptide and synthesized by both osteoblasts and hepatocytes, whereas human alpha 2-HSG is known to be a heterodimer and to be synthesized by the liver.

Amino Acid Sequence↗
PubMed1994

Bone turnover and calcium metabolism during 20 days bed rest in young healthy males and females.

Bone is a dynamic tissue that functions not only as a mechanical support, but also as a major component of the metabolic and endocrine systems maintaining mineral homeostasis. It has been shown that immobilization induces decalcification of bone. To evaluate the effect of immobilization on bone mineral density and calcium metabolism, we investigated 9 young healthy males and females during 20 days bed rest. Three methods for measuring bone mineral density were performed to quantify whole body and regional bone changes: 1) dual energy X-ray absorptiometry, 2) quantitative computed tomography, and 3) multiple scanning X-ray photodensitometry, respectively. Bone mineral density showed a rapid decreasing tendency, especially in both lumbar and metacarpal bones (mean +/- SE: 4.6 +/- 0.6% and 3.6 +/- 0.4%, respectively). Urinary daily excretion of deoxypyridinoline, a sensitive marker of bone matrix resorption, tended to increase by day 10, and to decline by day 20 (mean +/- SE: 42.2 +/- 1.4, 27.6 +/- 2.2 nmol day-1, respectively). However, neither alkaline phosphatase nor tartrate-resistant acid phosphatase, both markers of osteoclast and mature osteoblast function, changed. These results showed that in the early stage of immobilization, bone matrix might be resorbed without any activation of osteoclasts, resulting in rapid decalcification of vertebral and cortical bones without any discernible changes in anatomical structure.

Absorptiometry, Photon↗
PubMed2003

Posterior cervical fusion failure in three morbidly obese patients following circumferential surgery.

BACKGROUND: The stability of multilevel anterior corpectomy with fusion (ACF) is often enhanced by simultaneous posterior fusion (PF) which provides a "posterior tension band." Three morbidly obese patients undergoing circumferential surgery had posterior fusions performed without autogenous iliac crest graft to avoid donor site morbidity. METHODS: Three morbidly obese patients (300-350 lbs.), averaging 48 years of age, presented with rapidly progressive moderate/severe myelopathies. Magnetic resonance imaging (MRI) and computed tomography (CT) studies demonstrated severe ventral ossification of the posterior longitudinal ligament. Two to four level plated ACFs were performed utilizing fibula strut allograft and plates. Posterior spinous process wiring/fusion from C2-T1 were completed with braided titanium cables, fibula strut allografts, Inductive Conductive Matrix (a form of demineralized bone matrix), and allograft bone to avoid iliac crest donor site morbidity in such morbidly obese patients. Halo devices were utilized until fusion was documented on postoperative X-ray and 2D-CT studies subsequently obtained 3, 6, and up to 12 months postoperatively. Patients were followed an average of 3 years. RESULTS: Postoperatively, all 3 patients demonstrated mild residual myelopathy (Nurick Grade 0-I). Nevertheless, all 3 exhibited posterior pseudarthroses accompanied by anterior strut/plate extrusion (1 patient), partial anterior graft pseudarthrosis (1 patient), and a delayed strut fracture (1 patient). The first 2 patients required secondary posterior fusions performed with autogenous iliac crest graft, while the third fused with 6 months of additional bracing. CONCLUSIONS: Following circumferential cervical procedures, posterior fusions failed in 3 morbidly obese patients where iliac crest autograft was omitted in an attempt to avoid donor site morbidity.

Bone Plates↗
PubMed2001

Combination use of bovine porous bone mineral, enamel matrix proteins, and a bioabsorbable membrane in intrabony periodontal defects in humans.

BACKGROUND: The purpose of this study was to evaluate the effectiveness of a combination of enamel matrix proteins (EMP), bovine porous bone mineral (BPBM), and a bioabsorbable membrane for guided tissue regeneration (GTR) as regenerative therapy for intrabony defects in humans and compare it to an open flap debridement (OFD) technique. METHODS: Using a split-mouth design, 18 pairs of intrabony defects were treated and surgically reentered 6 months after the initial surgery. Experimental sites were treated with EMP, grafted with BPBM, and received a collagen/polylactic acid membrane for GTR. Control sites were treated with OFD. The primary outcomes evaluated in the study included probing depth resolution, clinical attachment gain, and bony defect fill. RESULTS: Preoperative probing depths, attachment levels, and intraoperative bone measurements were similar for the experimental and control groups. Postsurgical measurements taken at 6 months revealed a significantly greater reduction in probing depth in the experimental group (4.95+/-1.52 mm on buccal sites and 4.74+/-1.47 mm on lingual sites) when compared to the control group (2.83+/-0.83 mm on buccal sites and 2.90+/-0.91 mm on lingual sites). The experimental sites also presented with significantly more attachment gain (3.89+/-1.16 mm on buccal sites and 3.78+/-1.14 mm on lingual sites) than the control sites (1.52+/-0.83 mm on buccal sites and 1.48+/-0.78 mm on lingual sites). Surgical reentry of the treated defects revealed a significantly greater amount of defect fill in favor of the experimental group (4.76+/-1.36 mm on buccal sites and 4.81+/-1.37 mm on lingual sites) as compared to the control group (1.78+/-0.92 mm on buccal sites and 1.67+/-0.90 mm on lingual sites). CONCLUSIONS: The results of this study indicate that a combination technique including BPBM, EMP, and GTR results in better clinical resolution of intrabony defects than treatment with OFD. Differences observed were both statistically and clinically significant. The exact role of each of the 3 technique components in achieving the clinical improvement observed in this study remains to be determined.

Absorbable Implants↗
PubMed2003

Effects of intermittent parathyroid hormone administration on bone mineralization density in iliac crest biopsies from patients with osteoporosis: a paired study before and after treatment.

Anabolic effects of PTH have been observed at several skeletal sites in humans by dual x-ray absorptiometry without differentiating between an actual increase in bone volume and an increase in mineral content within already established bone. The present study addressed this issue by evaluating the bone mineralization density distribution of iliac crest bone biopsies before and after PTH treatment for 18-36 months in men and women with osteoporosis using quantitative backscattered electron imaging. In cortical bone, pairwise comparison of the two biopsies before and after treatment revealed a reduction in the typical calcium concentration in men (-3.32%; P = 0.02, by paired t test), but no change in women, and the heterogeneity of mineralization increased in both males and females [+18.80% (P = 0.09) and +18.14% (P = 0.005), respectively]. In cancellous bone, there was no change in the typical calcium concentration, but there was a greater heterogeneity of mineralization in both men and women [+19.65% (P = 0.02) and +21.59% (P = 0.056), respectively] due to newly formed bone matrix. Small angle x-ray scattering performed on a subgroup of subjects revealed normal collagen/mineral structure. The findings confirm the observations that PTH stimulates skeletal remodeling, resulting in an increased percentage of newly formed bone matrix of lower mineral density.

Absorptiometry, Photon↗