A bone morphogenetic system in residues of bone matrix in the mouse.
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In vitro re-mineralization of demineralized bone matrix in human serum.
The aim of this study was to develop an in vitro re-mineralization model in human serum. For this purpose a commercially produced demineralized human bone matrix (DBM) was incubated in samples of human serum pools obtained from two physiologically different groups. The first group consisted of young males and the second of older females. After incubation periods of 4 and 7 days at 37 degrees C, changes in the levels of calcium and inorganic phosphate content of the serum and DBM samples were measured. The results of the study showed that the change in mineral content of serum and DBM samples in both study groups was statistically significant. The decrease in serum calcium content and increase in DBM inorganic phosphate content were significant in the young group for longer incubation times. In the older group, both serum calcium and inorganic phosphate decreased and DBM mineral content increased for the same incubation time. When the two physiological groups were compared, statistically significant differences were identified for changes in mineral levels in both serum and the DBM samples. These data indicate that the mineral content of human serum decreases and that of DBM increases when these two materials are incubated together. These changes provide evidence for the re-mineralization of DBM. The model described here could also detect a difference in re-mineralization capability between two different groups of human sera.
Adult↗
Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins.
Cancer of the prostate commonly metastasizes to bony sites where cells acquire an aggressive, rapidly proliferating, androgen-independent phenotype. The interaction between bone and prostate, thus, becomes a key factor in disease progression. Fluctuations in intracellular ionized Ca2+ [Ca2+]i are rapid, regulated signal transduction events often associated with cell proliferation. Hence, Ca2+ signals provide a convenient measure of early events in cancer cell growth. This study developed single cell fluorescent imaging techniques to visualize Ca2+ signals in Fura-2 loaded prostatic cancer cell lines of various metastatic phenotypes. Solubilized bone fractions containing extracellular matrix and associated proteins were tested for the ability to trigger Ca2+ signals in prostate cancer cell lines. Fractions representing the complete repertoire of non-collagenous proteins present in mineralized bone were tested. Results demonstrated that two bone fractions termed D3b- and D4a-triggered Ca2+ signals in prostate cancer cells derived from bone (PC-3), but not brain (DU-145) metastases of prostate cancer. Lymph-node derived LNCaP cells also did not produce a Ca2+ signal in response to addition of soluble bone matrix. No other bone fractions produced a Ca2+ signal in PC-3 cells. It is of interest that bone fractions D3b and D4a contain a number of non-collagenous matrix proteins including osteonectin (SPARC) and osteopontin (OPN), as well as prothrombin. Moreover, antibody LM609 that recognizes the alpha v beta 3 integrin, blocks the ability of OPN to trigger a Ca2+ transient in PC-3 cells. These studies support a conclusion that bone-matrix proteins play a role in the growth and progression of metastatic prostate cancer, and that prior growth in bone may be associated with development of a bone-matrix-responsive phenotype.
Adenosine Triphosphate↗
A stereomorphologic study of bone matrix apposition in HA-implanted cavities observed with SEM, being prepared by a microvascular cast and freeze-fracture method.
In order to obtain further understanding of the relationship between hydroxyapatite (HA) with regard to its properties as an implantation bed, dense HA particles were implanted into the tibiae of dogs. Following the healing periods of 2 weeks, 1 month, 2 months, 3 months and 6 months, the specimens were prepared with a combination of a microvascular cast method and a freeze-fracture technique, allowing observations to be made with a scanning electron microscope (SEM). Under SEM, osteogenesis among the HA particles developed in a programmed sequence. The unfolding sequence revealed that the sinusoidal capillaries provided the initial evidence of vascularization preceding new bone formation, with microvessels creeping along the interparticular space among the HA particles. Having established an intimate contact existing between the microvessels, collagen fibres and the HA surface, the HA particles served as a supporting scaffold for the vessels to creep over and to connect with each other to form a vascular network. The way that the collagen fibres attached to the HA particles was either through globular depositions or via directly abutting themselves on to the HA surface. On closer inspection the osteoblasts with extracellular collagen fibrils were observed over the HA surface. By appositional growth, osteoblasts laid down a bone matrix in successive layers, forming a woven bone around the HA particles. As the implantation time increased, bony tissues gradually transformed into mature bone occupying all of the interparticular space. This study successfully revealed the spatial relationship between bone cells, collagen fibres and blood vessels in an osteogenetic sequence among HA particles, as revealed by a microvascular cast and the freeze-fracture method.
Animals↗
Transforming growth factor-beta 1 in a guanidine-extracted demineralized bone matrix carrier rapidly closes a rabbit critical calvarial defect.
Transforming growth factor beta 1 (TGF-beta 1) is a polyfunctional regulatory cytokine that has been shown to have roles in extracellular matrix interactions, soft tissue healing, and osteogenesis. Twenty-five microL of recombinant human TGF-beta 1 was added to guanidine-extracted demineralized bone matrix carrier and the implants were used to fill a 14-mm osteoperiosteal critical calvarial defect in New Zealand white rabbit model. The defects were allowed to heal over 4 weeks and the degree of new bone formation was assess by radiodensitometry and undecalcified bone histomorphometry techniques. Implants with TGF-beta 1 showed complete bridging of the gap with new bone in all cases, while the controls showed fibrous tissue repair of the gap with little or no new bone formation. These results demonstrate the ability of TGF-beta 1 to induce new bone in a brief time period in an inactive carrier.
Animals↗
Healing response to various forms of human demineralized bone matrix in athymic rat cranial defects.
PURPOSE: This study compared the ability of a bone autograft and four distinct forms of human demineralized bone (DBM) to elicit bone repair in a critical size cranial defect in athymic rats. MATERIALS AND METHODS: Cranial defects were created in athymic rats and then grafted with either an autograft, rat DBM particles in glycerol (rGel), or one of four forms of human DBM: 1) hGel; 2) Putty (DBM fibers in glycerol); 3) Sheet (sheet of DBM fibers); or 4) Flex (DBM fiber sheet with glycerol). Histology, histomorphometry, and radiographic density of the graft sites were evaluated at 8 weeks. RESULTS: Of the grafted defects, 29% to 58% were found to be filled with new bone. The rGel and human forms of DBM stimulated similar amounts of new bone growth in comparison with the autograft-filled defects. The fiber-based grafts produced the largest amounts of new bone. CONCLUSIONS: Human DBM in gel, putty and sheet forms were found to perform as well as an autograft in a critical size cranial defect in the athymic rat.
Analysis of Variance↗
Expression of metalloproteinase 2 in the cell response to porous demineralized bovine bone matrix.
The purpose of the study was to analyze the involvement of metalloproteinase 2 (MMP-2) and macrophages in the tissue and cell response to the organic graft material produced from bovine cancellous bone. Thirty adult male white Wistar rats (Rattus norvegicus) received implants of blocks of demineralized bovine bone matrix between the fasciae of the quadriceps muscle. The specimens collected at 3, 7, 14, 21 and 28 days after implantation (n = 6/period). Sections of 6 microm thick were stained with hematoxylin and eosin and immunolabeled with anti-MMP-2 and anti-CD68 using standard avidin-biotin-peroxidase method. The tissue response to the material was initially mediated by polymorphonuclear neutrophils, evolving to a mononuclear inflammatory infiltrate with macrophages and few lymphocytes and plasma cells and presence of inflammatory multinucleated giant cells (GC) in contact with the material that exhibited signs of resorption. The number of cells immunolabeled to MMP-2 was highest at day 7 (103.2 +/- 39.1), but significantly decreased (F = 3.67; p = 0.044) until day 28 (45.9 +/- 13.1). CD68 immunostaining also significantly decreased (F = 6.75; p = 0.007) from day 7 (49.5 +/- 10.4) to day 28 (19.5 +/- 8.9). A positive and statistically significant correlation was observed between the evolutions of these two variables. The material had been almost completely resorbed at day 28. Among cells present at the granuloma, anti-MMP-2 immunostaining was predominant and more intense in macrophages, yet lightly immunolabeled multinucleated giant cells were found in close contact with the material. Thus, considering the experimental limitations of this study, we concluded that MMP-2 produced by macrophages participates in the resorption of demineralized bovine bone.
Animals↗
Immunoelectron microscopy of osteonectin and type I collagen in osteoblasts and bone matrix.
The pathway of production, secretion, and extracellular deposition of type I collagen and osteonectin was studied by immunoelectron microscopy using respective polyclonal antibodies. Protein A gold and immunogold methods yielded to similar results in human callus tissue used as a model of bone formation. The intracellular distribution of osteonectin in active osteoblasts is found as a faint immunolabeling of vesicular Golgi fields and some lamellae of rough endoplasmic reticulum. A more intensive labeling occurs in opaque cytoplasmic vesicles pointing to the process of secretion as some of the vesicles are connected with the basal cell membrane. Our type I collagen antibody did not label the respective intracellular compartments. Extracellularly, the type I collagen antibody showed a continuous labeling from the immature subcellular osteoid to the mineralized bone. Osteonectin antibodies were bound first to the deeper layer of osteoid maturation with intensity increasing below the mineralization front. Osteonectin is thought to be associated with mineralization of bone matrix.
Antibodies↗
Bone matrix turnover and balance in vitro. I. The effects of parathyroid hormone and thyrocalcitonin.
Labeled proline from incubation media has been shown to be incorporated into living bone matrix collagen in vitro. Hydroxyproline is released from fresh bone slices in similar systems in a characteristic curve against time. This hydroxyproline is derived from three distinct sources, each of which may be separately quantitated. Part of the total represents passive solubilization of matrix collagen, part is derived from new synthesis of soluble collagen occurring in vitro, and the remainder is released by cell-mediated resorptive action. The latter two processes are linear with time up to 8 hr; the former decays to zero at about 2 hr. Consequently, rates of collagen synthesis and of new collagen deposition and resorption can be quantitated simultaneously in the same system. The ability to measure these parameters of bone collagen metabolism provides methods both for the accurate evaluation of organic matrix resorption in vitro and for the accurate measurement of rates of collagen synthesis and collagen deposition. The application of the method is illustrated using parathyroid hormone and thyrocalcitonin. Parathyroid hormone diminishes collagen synthesis and stimulates collagen resorption. It reduces slightly the deposition of newly formed collagen in stable matrix. The net effect of these changes is to produce a marked negative balance. It does not significantly affect the solubility of matrix collagen.Thyrocalcitonin does not affect collagen synthesis or its deposition. It causes a marked fall in resorption rate. It has no effect on matrix collagen solubility. The net effect is to produce a marked positive balance of matrix collagen.
Animals↗
Glycosaminoglycan and surface glycoprotein syntheses by rat muscle fibroblast monolayers: response to solubilized bone matrix and effect of serum.
Glycosaminoglycan synthesis by muscle fibroblasts derived from neonatal rats has been studied using a sensitive radioisotope method. Feeding the cultures with serum-containing medium supplemented with 80 micrograms/ml of a solubilized bone matrix (SBM) preparation results in an initial enhancement of net glycosaminoglycan secretion and a later additional response, not seen in controls. A pronounced dilution of the specific radioactivity of secreted chondroitin sulfate characterizes the latter response. SBM-treated cultures (100 micrograms/ml) also demonstrate increased incorporation of radioactive glucosamine into a large molecular weight cell surface glycoprotein. SBM suppresses the growth rate and final cell densities at all serum concentrations tested. However, at similar cell densities, higher serum concentrations tend to minimize the relative stimulation of incorporation of radioactivity into the secreted glycosaminoglycans by SBM-treated cultures, compared to controls.
Animals↗
Mineralization and pH relationships in healing skeletal defects grafted with demineralized bone matrix.
Early studies had indicated that tissue repair is initially associated with a lower than normal serum pH that later becomes more alkaline. To determine how tissue pH may affect skeletal healing and mineralization, we used a rat skeletal repair model consisting of a long bone segmental defect grafted with acid-demineralized bone matrix (DBM), a biomaterial possessing both osteoinductive and osteoconductive repair properties. In this study, femoral and tibial diaphyses from young adult Sprague Dawley rats were cut into cylinders approximately 0.5 cm in length, demineralized in acid, perforated to accommodate a needle-type combination pH microelectrode, and grafted around a 0.3-cm-long diaphyseal fibula defect. The pH of repair tissues was recorded at various time intervals up to 28 days postgrafting. Healing and mineralization were monitored histologically and by the ash and calcium content of repair tissues. During the early healing phase, tissue pH was lower than normal serum pH, presumably because of an accumulation of acidic metabolites in tissue fluids. Subsequent pH increases to more alkaline values were accompanied by a rapid mineral deposition phase and a later phase characterized by a slow, gradual increase in tissue calcium content. The results of this study support previous observations suggesting that the pH of repair tissue fluids may play a regulatory role in the healing and mineralization of bone.
Animals↗
Chondrosarcoma treated by reimplantation of resected bone after autoclaving and supplementation with allogeneic bone matrix. A case report.
An extensive intramedullary lesion of the femur was diagnosed as a Grade I chondrosarcoma in 1986. At definitive surgery, four fifths of the femoral diaphysis were resected, autoclaved, and reimplanted. The reconstruction, after stabilization with AO plates, was supplemented with allogeneic bone matrix to induce new bone formation. No autografts were used. Despite subsequent infection and sequestration of the autoclaved tumorous segment, the reconstructed area was gradually enveloped by new bone. Three years after primary surgery, the infection persisted. The AO-plated and autoclaved sequestered bone was excised. Six years after the operation, the patient walks bearing full weight and works full time. There are no signs of tumor recurrence.
Adult↗
Cancellous bone repair using bovine trabecular bone matrix particulates.
At 5 and 15 weeks post-surgery, biomechanical and histological analyses of cancellous bone defects filled with the bovine trabecular bone matrix (BBM) and hydroxyapatite (Hap) particulates of dimensions 106-150 microm were investigated. It was observed that at 5 weeks post-surgery the stiffness properties of the BBM filled defects were significantly higher than those observed in the Hap filled defects (p < 0.01) but comparable to those recorded in intact cancellous bone from the same anatomical position. Histologically, no significant differences were observed in the percentage of new bone contact with the particles. The biomechanical properties of the Hap filled defects mirrored those in intact cancellous bone only at 15 weeks post-surgery. BBM particles thus appeared to accelerate the early healing of osteotomies. It is therefore suggested that particles of this bioceramic be the subject of intense research for more usage in both periodontal osseous defects and orthopaedic fractures.
Animals↗
Organic mass distribution in bone matrix undergoing osteoclastic resorption. Preliminary observations.
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Bone Matrix↗
Lack of effect of thyrocalcitonin on formation of bone matrix in mice and rats.
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Animals↗
Rapid bone healing delayed by bone matrix implantation.
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Animals↗
Glucocorticoids and bone matrix formation.
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Animals↗
Expression of bone matrix proteins during the osseus healing of topical conditioned implants: an experimental study.
OBJECTIVES: Osseointegration of implants depends on time and local bone conditions regarding quality and quantity. This led to the bone classification by Lekholm et al. The aim of the present study was to follow the expression of bone matrix proteins during the phase of osseointegration after conditioning of the bone bed by means of immunohistochemistry. METHODS: In the porcine frontal skull, implant beds of identical size were created. Before placement of the implants (Ankylos 4 x 3.5 mm), the implant beds were conditioned using bone condensation (cond), an osteoinductive collagen (Co) and platelet-rich plasma (PRP). These conditioning methods were compared with standard procedure. The animals were sacrificed after 2, 4 and 8 weeks. The specimens were then analyzed by light microcopy and immunohistochemistry for expression of bone morphogenic proteins (BMP)2, procollagen I and osteocalcin (OC). RESULTS: Light microscopy revealed an initial effect of condensation and the bovine collagen at 2 weeks in comparison with the standard group. The PRP did not achieve a significant effect. At 8 weeks, the results of the standard, bone condensation and the bovine collagen group had aligned. The PRP group showed a significantly lower bone-implant contact (BIC) (P=0.003) compared with the standard group. BMP2 expression was significantly higher in all evaluated test groups at 4 and 8 weeks, as well as at 2 weeks in the condensation group. The procollagen I expression at 2 weeks was significantly increased for PRP and lower in the collagen and condensation group compared with standard procedure. Values for 4 and 8 weeks were slightly higher than in the standard group. No significant differences were obvious in the OC group at any time. CONCLUSIONS: During the initial healing phase, an effect of the evaluated methods of topical bone conditioning can be demonstrated by differences in the expression of BMP2 and procollagen I. These findings had leveled at 8 weeks and were, in contrast, not detectable in the expression of OC or by means of light microscopy.
Animals↗