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PubMed1994

Mechanical loading stimulates rapid changes in periosteal gene expression.

Although mechanical forces regulate bone mass and morphology, little is known about the signals involved in that regulation. External force application increases periosteal bone formation by increasing surface activation and formation rate. In this study, the early tibial periosteal response to external loads was compared between loaded and nonloaded contralateral tibia by examining the results of blot hybridization analyses of total RNA. To study the impact of external load on gene expression, RNA blots were sequentially hybridized to cDNAs encoding the protooncogene c-fos, cytoskeletal protein beta-actin, bone matrix proteins alkaline phosphatase (ALP), osteopontin (Op), and osteocalcin (Oc), and growth factors insulin-like growth factor I (IGF-I) and transforming growth factor-beta (TGF-beta). The rapid yet transient increase in levels of c-fos mRNA seen within 2 hours after load application indirectly suggests that the initial periosteal response to mechanical loading is cell proliferation. This is also supported by the concomitant decline in levels of mRNAs encoding bone matrix proteins ALP, Op, and Oc, which are typically produced by mature osteoblasts. Another early periosteal response to mechanical load appeared to be the rapid induction of growth factor synthesis as TGF-beta and IGF-I mRNA levels were increased in the loaded limb with peak levels being observed 4 hours after loading. These data indicate that the acute periosteal response to external mechanical loading was a change in the pattern of gene expression which may signal cell proliferation. The altered pattern of gene expression observed in the present study supports previous evidence of increased periosteal cell proliferation seen both in vivo and in vitro following mechanical loading.

Actins↗
PubMed1999

Alterations of matrix- and cell-associated proteoglycans inhibit osteogenesis and growth response to fibroblast growth factor-2 in cultured rat mandibular condyle and calvaria.

Matrix and cell surface proteoglycans (PGs) may play important roles in the control of cellular actions of heparan-binding growth factors such as fibroblast growth factor (FGF) during chondrogenesis and osteogenesis. In this study, we used 4-methylumbelliferyl-beta-d-xyloside, an inhibitor of PG synthesis, and sodium chlorate, a competitive inhibitor of glycoconjugate sulfation, to determine the functional consequences of alterations of PG metabolism on osteogenesis and on FGF actions in neonatal rat condyle and calvaria in vitro. Biochemical analysis showed that beta-d-xyloside (1 mM) or chlorate (15 mM) treatment for 1-8 days inhibited cellular PG synthesis by 60-80% in condyle and calvaria, as evaluated by [35S]sulfate incorporation. Histochemistry and immunohistochemistry showed that the inhibition of PG synthesis by beta-d-xyloside resulted in reduced incorporation of chondroitin sulfate into cartilage and bone matrix. This was associated with a 75% reduction in cell growth in condyle, determined by DNA synthesis, and in collagenous matrix synthesis in condyle and calvaria, evaluated by tritiated proline incorporation and type I collagen immunohistochemistry. Morphological and quantitative autoradiographic analyses also showed that inhibition of PG synthesis by beta-d-xyloside blocked bone matrix formation by perichondral progenitor cells in condyles and by osteoblasts in calvaria. In addition, alteration of PG metabolism blocked the mitogenic response to rhFGF-2 in calvaria. The data show that functional proteoglycans are essential for osteogenesis and for the growth response to FGF-2 during osteogenic differentiation in vitro.

Animals↗
PubMed1989

Histologic evaluation of bone inductive proteins complexed with coralline hydroxylapatite in an extraskeletal site of the rat.

The purpose of this histologic and biochemical study was to assess the osteogenic potential of bone inductive proteins complexed with coralline hydroxylapatite as the carrier vehicle after implantation in an extraskeletal site of the rat. Inductive proteins were extracted from bovine demineralized bone. Implants were placed in 16 male, 3-month old Long-Evans rats (200-300 grams), using paired subcutaneous sites overlying the ventral thorax. There were four experimental groups, with eight implants per group. These included hydroxylapatite alone (HA), hydroxylapatite with inductive protein (HA + P), inactive demineralized bone matrix with (IBM + P), and without inductive protein (IBM). All implants were harvested at 21 days. Findings indicate a lack of osteogenic potential in groups HA, HA + P, and IBM. However, when HA and HA + P were compared, there was a 79% increase in standardized field mean nuclear point counts in the HA + P group. Also, compared to the other three implant groups, controls of IBM + P histomorphometrically showed chondroid bone formation and increased alkaline phosphatase activity. In this model system it may be concluded that with a composite system of coralline hydroxylapatite and bovine-derived inductive protein, bone formation was not seen; positive controls consisting of IBM + P demonstrated a statistically significant increase in AP activity with corresponding histologic evidence of bone formation.

Alkaline Phosphatase↗
PubMed1989

Thrombospondin is an osteoblast-derived component of mineralized extracellular matrix.

Thrombospondin, the most abundant protein of platelet alpha granules, is a biosynthetic product of a variety of connective tissue cells and a component of many extracellular matrices. In this study, thrombospondin distribution in bone was investigated using a monoclonal antibody specific for the human protein. Thrombospondin was localized in osteoid of undemineralized, frozen sections of fetal subperiosteal bone, and identified as a component of mineralized bone matrix of neonatal and/or young (growing) bone of many animal species by Western blot analysis. Adult human bone cells were demonstrated to contain mRNA for thrombospondin by hybridization of a cDNA thrombospondin probe to a 6.1 kb mRNA. Pulse-chase experiments indicated that the protein was synthesized and the majority was secreted from osteoblastic cells. Treatment of the cells with TGF-beta (0.01-10 ng/ml) slightly decreased total thrombospondin synthesis, but caused an increase in the retention on newly synthesized thrombospondin in the cell layer/matrix fraction. In cell attachment assays, thrombospondin mediated adhesion, but not spreading of adult human bone cells.

Animals↗
PubMed1986

Parathyroid hormone-stimulated development of osteoclasts in cultures of cells from neonatal murine calvaria.

Evidence was sought for the presence of osteoclasts or preosteoclasts in cells prepared from neonatal murine calvaria by sequential enzymatic digestion. Freshly isolated cells released late in the digestion process resorbed both living and devitalized calvarial bone matrix in response to PTH, accompanied by the development of osteoclasts. Light and scanning electron microscopy of these cells after 1 to 2 days in culture revealed the presence of round cells (10 to 15 mu in diameter) with minimal surface microvilli in addition to the larger osteoblastic cells. Few cells contained tartrate-resistant acid phosphatase (TRAPase). If initially seeded at confluent density, more cells positive for TRAPase developed during subsequent culture, and these cells retained the ability to resorb calvarial bone matrix upon treatment with parathyroid hormone (PTH). These cultured cells responded to PTH with increased secretion of TRAPase into the medium and increased numbers of TRAPase positive cells. These were 20 to 50 mu in diameter, sometimes multinucleated, and some were spread 100 to 200 mu in length. Observations of living cells that took up neutral red showed that, upon treatment with calcitonin (CT), surface filopodia of some but not all of the labeled cells retracted within 30 minutes. Loss of resorptive response to PTH, as well as PTH-stimulated development of TRAPase-positive cells, occurred if the cells were initially seeded at low density and grown to confluence before exposure to hormone. This correlated with the loss of most of the 10 to 15 mu diameter round cells. These observations suggest that preosteoclasts are present among these small cells that can give rise to osteoclasts upon treatment with PTH.

Acid Phosphatase↗
PubMed1987

Immunofluorescence localization of type I and type III collagen and fibronectin in mouse dental tissues in late development and during molar eruption.

Affinity-purified antibodies produced intense staining for type I collagen in alveolar bone matrix and predentine, and moderate staining in the dentine matrix, lamina propria, connective tissue invaginating into papillary layer of the enamel organ, dental sac and periodontal ligament. No staining occurred in oral epithelium, stellate reticulum, stratum intermedium, ameloblasts and odontoblasts. Fibronectin was distributed similarly except at the interface between the epithelial diaphragm and pre-odontoblasts where type I collagen was absent but fibronectin was present. In contrast, type III collagen showed strong staining in the periodontal ligament and lamina propria but no staining in bone matrix, predentine, dentine and at the interface between the epithelial diaphragm and pre-odontoblasts. The staining pattern for type III collagen was similar to that of type I and fibronectin in other tissues including endosteal reticular tissue, the connective tissue invaginating into papillary layer and the extracellular matrix of the pulp.

Alveolar Process↗
PubMed1990

The occurrence of neuropeptides at different stages of DBM-induced heterotopic bone formation.

In developing heterotopic bone in the rat, induced by allogeneic bone matrix, we immunohistochemically detected nerves containing substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase (TH). After 10 days they were dicernible amidst differentiating chondroblastoid cells in fibrous tissue around and within the implants. Over the next 3 weeks, the nerves increased in number and gradually attained a shape and distribution resembling normal osseal nerves; varicose fibres frequently occurred in periosteum-like fibrous tissue and bone marrow adjacent to newly formed bone. At 8 weeks, NPY-fibres increased, particularly in the marrow and this abundance of NPY fibres remained at 16 weeks. VIP-immunoreactive fibres were only observed in the surrounding periosteum-like fibrous tissue 4-6 weeks after implantation. These observations, in combination with recent findings of receptors to neuropeptides on bone cells, suggest a neurogenic influence on physiological processes in bone tissue.

Animals↗
PubMed2004

The osteoinductive activity of bone morphogenetic protein (BMP) purified by repeated extracts of bovine bone.

Native bone morphogenetic proteins (BMPs) extracted from bone have been used clinically to stimulate bone regeneration and repair. However, preparation of purified BMP is a laborious process. This study investigated the yield, activity and cost effectiveness of repeatedly extracting the same bone matrix to produce purified BMP. While repeated extraction was able to increase the yield 62% the activity of the partially purified BMP in later extracts decreased both in vitro and in vivo. This decline in activity appears to be due to an increase in non-BMP contaminants, such as collagen, in the extracts. When the first three extracts were combined and processed together activity was equivalent to that of the first extract. A simple analysis based on the cost of reagents used and the time required for purification indicates that separate processing of the extracts is inefficient while combining the first and second extracts and processing them together would result in a small cost saving. Based on this study we would recommend that the demineralized bone matrix be extracted no more than twice and that the extracts be combined for further processing.

Alkaline Phosphatase↗
PubMed1989

[Osteoplasty in wound infection].

The outcome of a surgical intervention in the purulent wound together with other common clinical factors depends to a determining degree on the quality of the plastic material and its resistance to infection. The authors used allogenic transplants and demineralized bone matrix which had been conserved in solutions of aldehydes and was characterized by high bacteriostatic activity with reduced antigen properties and preservation of its biologic value. This allowed to perform reconstructive operations in 68 patients and to obtain positive results in 89.2% of the cases.

Adolescent↗
PubMed1987

A tissue-specific protein in rat osteogenic tissues.

A tissue-specific protein fraction has been detected in rat osteogenic tissue. Dissociative extraction of adult rat bone matrix with 4 M guanidinium chloride solution was followed sequentially by gel chromatography and polyacrylamide gel electrophoresis. By the latter procedure a prominent protein component of molecular weight 19,000 was isolated from the low molecular weight fraction, and antibodies directed against this protein were raised in rabbits. The antibodies were mainly against antigenic sites on this protein, as shown by protein blotting techniques. By embedding rat tissues in hydrophilic plastic and by using immunohistochemical procedures the presence of this protein was demonstrated specifically in bone matrix in vivo, in osteogenic tissue developing in diffusion chamber culture, and in a malignant osteoblast cell line (UMR 106). Soft tissues (liver, kidney, spleen, gut, skin, thymus, eye) showed no reactivity with the antiserum and in vitro a further malignant osteoblast cell line (ROS 17/2.8) did not synthesize the 19,000 molecular weight protein. This protein appears to be expressed solely by osteogenic tissue and may be used as a biochemical criterion of osteogenic differentiation.

Animals↗
PubMed1993

Skeletal dysplasia in perinatal lethal osteogenesis imperfecta. A complex disorder of endochondral and intramembranous ossification.

Osteogenesis imperfecta (OI) Type II is a rare heritable disorder of bone matrix that results in catastrophic congenital skeletal dysplasia. Two cases of OI Type II had symmetric rhizomelic skeletal dysplasia apparent on ultrasound at 16 and 20 weeks' gestation. Histologic and histochemical studies performed on skeletal tissue from fetal autopsies showed the following: (1) abnormal growth plate tissue characterized by failure of formation of primary bony spongiosa; (2) persistence of calcified cartilage bars in the diaphysis; (3) metaphyseal microfractures; (4) abundant cartilaginous fracture callus; (5) absence of bony callus; (6) failure of formation of intramembranous cortical diaphyseal bone; (7) angulation of long bones in portions of the metadiaphyses bordered by fracture callus; and (8) mechanical failure of the perichondral ring of LaCroix with a normal fibrous ossification groove of Ranvier. These findings suggest that skeletal dysplasia in OI Type II results from the action of muscular forces on a skeleton weakened by a complex disorder of endochondral and intramembranous ossification. The paucity of primary metaphyseal trabeculae and subperiosteal cortical bone leads to pathologic fractures of the immature fiber bone and an imperfect attempt at fracture repair. Angulation and shortening of long bones occurs between numerous sites of focal endochondral fracture callus. Mechanical failure of the fibrous perichondral ring leads to further collapse and shortening without obvious functional impairment of the fibrous ossification groove. Perinatal lethal OI provides insight into how a molecular disorder predominantly of Type I collagen metabolism results in pathology of numerous tissues, leading to severe skeletal dysplasia without primarily affecting chondrogenesis.

Bone Diseases, Developmental↗
PubMed2003

Requisite roles of Runx2 and Cbfb in skeletal development.

Each Runx (runt-related gene) protein exerts a fundamental role in different cell lineages. Runx2 is essential for osteoblast differentiation and plays an important role in chondrocyte maturation. Runx2 determines the lineage of osteoblastic cells from multipotent mesenchymal cells, enhances osteoblast differentiation at an early stage, and inhibits osteoblast differentiation at a late stage. In addition, Runx2 is involved in the production of bone matrix proteins. Further, Runx2 is a positive regulator of chondrocyte maturation and is involved in vascular invasion into the cartilage. Core binding factor beta (Cbfb) is a cotranscription factor which forms a heterodimer with Runx proteins. Cbfb is required for the functions of Runx1 and Runx2. Thus, Runx2/Cbfb heterodimers play essential roles in skeletal development.

Animals↗
PubMed2005

Histomorphometric analysis of glucocorticoid-induced osteoporosis.

Bone histomorphometry or quantitative histology consists of counting or measuring tissue components: cells, extracellular constituents and microarchitecture. Bone histomorphometry is the only method that allows the measurement of mineralization rate and the study of bone formation at three levels: cell, remodeling unit and tissue levels. It is a useful tool to explain the pathogenesis and cellular mechanisms of different metabolic bone diseases such as glucocorticoid-induced osteoporosis (GIO). Glucocorticoids (GC) affect calcium and bone metabolism at every level, but the main effect is the osteoblastic dysfunction. Concerning the bone formation, some histomorphometric studies have shown a depressed osteoblastic activity at a cell, bone remodeling unit, and tissue levels. In addition, there is evidence of a shortening of the period in which the osteoblasts work actively forming the bone matrix. This latter effect seems to occur after high cumulative doses of GC. With regard to the resorption, the results are still debated, but histomorphometric parameters seem to be increased in the majority of studies, at least in the first period of the GC treatment. From a structural point of view, GC seem to induce a thinning of the trabeculae without their perforation, which occurs only after high cumulative doses. Anti-resorptive treatments, such as bisphosphonates, are able to counteract the negative effects of GC on bone. In particular, along with their active working period, they prolong the lifespan of osteoblasts and osteocytes. In addition, the anti-resorptive treatments seem to extend the time for secondary mineralization through a reduction of the Activation Frequency. The latter is an intriguing mechanism of bisphosphonates in GIO that needs further ad hoc investigations.

Animals↗
PubMed1991

Mandibular reconstruction with a recombinant bone-inducing factor. Functional, histologic, and biomechanical evaluation.

Bone morphogenetic protein-2 (BMP-2) is a human recombinant bone-inducing factor that stimulates bone formation within 14 days. Twenty-six dogs underwent reconstruction of 3-cm full-thickness mandibular defects. After stabilizing the defects with stainless steel reconstruction plates, test implants composed of inactive dog bone matrix carrier and human recombinant BMP-2 were placed in defects of 12 animals (group 1). Control implants (carrier without BMP-2) were used in 10 animals (group 2), and no implants were placed in mandibular defects of four animals (group 3). Animals were killed at 3 and 6 months. The reconstructed segments were evaluated by roentgenography, analysis of functional stability, histology, histomorphometry, and analysis of biomechanical strength using three-point bend testing. In group 1, reconstruction plates were removed at 10 weeks because stiff, noncompressible mineralized bone formed across the defects, allowing the animals to chew a solid diet. The defects from groups 2 and 3 showed minimal, if any, bone formation and remained grossly unstable, prohibiting plate removal or advancement to a solid diet. Histomorphometric analysis at 6 months revealed that 68% of the group 1 implants were replaced by mineralized bone, whereas mineralized bone occupied less than 4% of the implants in groups 2 and 3. Biomechanical testing at 6 months revealed that the average bending strength of the reconstructed hemimandibles (expressed as a percentage of the contralateral hemimandible) was 27% for group 1 and 0% for group 2. The biomechanical strength of the defects reconstructed with BMP-2 increased significantly from 3 to 6 months and was related to degree of mineralization and thickness of bone bridging the defect.

Animals↗
PubMed1995

Interleukin-1 immunoreactive nerves in heterotopic bone induced by DBM.

The occurrence of interleukin-1-positive nerves was investigated by immunohistochemistry in developing heterotopic bone, induced by demineralized allogeneic bone matrix (DBM) in the rat. Interleukin-1 immunoreactivity was observed 1 week after implantation and remained until the end of the experiment at 12 weeks. Immunoreactive material was first identified in mononuclear cells at day 7. Interleukin-1 immunoreactive nerve fibers were first observed in the fibrous tissue at 2 weeks after implantation. A maximum density of fibers was reached at 8 weeks. Abundant immunofluorescent fibers were observed in the marrow tissue of the ossicles, and also in the surrounding fibrous tissue. A substantial number were vascular, but in the bone marrow most of the nerve fibers appeared as irregularly arranged, non-vascular terminals with ramifications and varicosities, intermingled between the marrow cells. No fibers could be detected in the proper bone tissue. The distribution of interleukin-1-positive nerves in the ossicles strongly resembled that previously observed in rat long bones. Moreover, the shape and distribution of the fibers exhibited a striking similarity to that of noradrenergic fibers identified previously both in ossicles and normal rat long bones. The late occurrence and predominant distribution in marrow tissue would seem to imply that neuronal interleukin-1 does not participate in the early differentiation of bone cells. The most important finding seems to be the presence of interleukin-1-positive nerve terminals in blood vessel walls and amidst marrow cells.

Animals↗
PubMed1990

Osteogenic enhancement of diaphyseal reconstruction. Comparison of bone grafts in the rabbit.

We investigated incorporation of autoclaved autografts in segmental defects of rabbit humeri for comparison with a previous study on similar grafts supplemented with demineralized allogeneic bone matrix (DABM). We also made similar reconstructions with frozen allografts--both DABM and nonsupplemented allografts. Before the animals were killed at 8 months, they underwent scintigraphy, showing that all 28 humeral reconstructions were metabolically active. Faxitrone radiography showed nonunion in three of nine with autoclaved autografts and in two of eight with frozen allografts, whereas all 11 DABM-supplemented frozen allografts had incorporated. Taking into account only the 23 healed reconstructions, the mean torsional strength in relation to the contralateral nonoperated on humeri was 0.81 for all three groups. Histologically, new bone enveloping, partly replacing, the implants was more abundant in DABM-supplemented reconstructions. Our study shows that osteogenic enhancement is more important than the type of nonviable bone chosen for diaphyseal repair. However, if healing is obtained, osteogenic enhancement per se does not increase the strength.

Animals↗
PubMed1983

Osteoclast changes induced by lead poisoning (saturnism).

This paper reports the results of histological, histochemical and electron microscopic investigations, and of electron probe analysis, carried out on bone cells of tibiae and femurs of lead-poisoned collie puppies. The most important changes were found in osteoclasts. They were increased in number, showed intranuclear and intracytoplasmic inclusion bodies, and frequently had shrunk, pyknotic nuclei and were detached from bone matrix. Organic components were histochemically demonstrable in the inclusion bodies, which consisted of a central core, peripheral filaments and intrinsically electron-dense granules. Lead was detected in the inclusion bodies by electron probe elemental analysis: Pb-L alpha pulses in the regions with inclusions are four times higher than those in the cytoplasm. Osteoblasts and osteocytes did not contain inclusion bodies, probably because in them lead does not reach a concentration sufficiently high to induce inclusion formation. On the contrary, this concentration can be reached in the osteoclasts when, during bone resorption, they accumulate the lead salts previously incorporated in the bone matrix.

Animals↗
PubMed2007

Augmentation of osseous phenotypes in vivo with a synthetic peptide.

The synthetic peptide B2A2-K-NS augmented the in vitro expression of osseous phenotypes when cells were stimulated with BMP-2, an osteoinductive growth factor. B2A2-K-NS significantly enhanced the effects of BMP-2-induced alkaline phosphatase activity and mineralization. In the absence of BMP-2, B2A2-K-NS did not have an effect on these endpoints. Based on these observations, in vivo studies were conducted to evaluate if B2A2-K-NS could augment osseous phenotypes in an osteoinductive environment in which BMP-2 should be present. In one study, human demineralized bone matrix (DBM) was used to generate an osteoinductive environment and the effects of B2A2-K-NS on ectopic mineralization of subcutaneous implants evaluated. In the second study, a noncritical sized defect in rabbit ulnas with inherent reparative capacity was used as the osteoinductive environment and was treated with or without B2A2-K-NS. In the DBM studies, B2A2-K-NS augmented mineralization as determined using a combination of radiographic analysis and von Kossa staining at 4 weeks postimplant. In the rabbit ulna model, B2A2-K-NS significantly increased the radiographic bone density in the defects compared to carrier-only or no-treatment controls after 6 weeks. Histological staining confirmed that B2A2-K-NS generated a pronounced bone repair response. The results are consistent with the hypothesis that B2A2-K-NS augments osseous phenotypes in an osteoinductive environment, and suggests that B2A2-K-NS may have clinical utility.

Alkaline Phosphatase↗