Gene expression and immunohistochemical localization of osteonectin in association with early bone formation in the developing mandible

We have compared the expression of osteonectin with that of osteocalcin and bone sialoprotein during bone formation in the rat mandible, using in situ hybridization and immunohistochemistry. Expression of osteonectin, osteocalcin and bone sialoprotein mRNAs were first observed in newly differentiated osteoblasts of the developing mandible at embryonic day 15 (E15) and subsequently increased with the number of osteoblasts through E20. Definitive osteonectin immunostaining was observed in newly differentiated osteoblasts, but not in the intercellular unmineralized matrix. Immunostaining for osteocalcin and bone sialoprotein was visible in osteoblasts and unmineralized matrix. Concomitant with the initiation of matrix mineralization at E16, mineralized bone matrix showed osteocalcin and bone sialoprotein immunostaining, but lacked osteonectin immuno-staining. The same staining profile was observed during subsequent phases of bone formation at E17–20. However, sequential demineralization with ethanolic trimethylammonium EDTA and protease digestion of tissue sections demonstrated prominent osteonectin immunostaining of the mineralized bone matrix. Western blot analysis of osteonectin in extracts of fresh specimens at E18 and 20 revealed that an EDTA extract contains osteonectin having M _r approximately 50kDa. These results indicate that newly differentiated osteoblasts synthesize and secrete osteonectin, which is mainly incorporated into the mineralized bone matrix and becomes a specific component of developing manibula of foetal rats.     

Temporal and spatial gene expression of major bone extracellular matrix molecules during embryonic mandibular osteogenesis in rats

It is not known how gene expression of bone extracellular matrix molecules is controlled temporally and spatially, or how it is related with morphological differentiation of osteoblasts during embryonic osteogenesis in vivo. The present study was designed to examine gene expressions of type I collagen, osteonectin, bone sialoprotein, osteopontin, and osteocalcin during mandibular osteogenesis using in situ hybridization. Wistar rat embryos 13–20 days post coitum were used. The condensation of mesenchymal cells was formed in 14-day rat embryonic mandibles and expressed genes of pro-(I) collagen, osteonectin, bone sialoprotein and osteopontin. Cuboidal osteoblasts surrounding the uncalcified bone matrix were seen as early as in 15-day embryonic mandibles, while flat osteoblasts lining the surface of the calcified bone were seen from 16-day embryonic mandibles. Cuboidal osteoblasts expressed pro-1(I) collagen, osteonectin and bone sialoprotein intensely but osteopontin very weakly. In contrast, flat osteoblasts expressed osteopontin very strongly. Osteocytes expressed the extracellular matrix molecules actively, in particular, osteopontin. The present study demonstrated the distinct gene expression pattern of type I collagen, osteonectin, bone sialoprotein, osteopontin and osteocalcin during embryonic mandibular osteogenesis in vivo.     

Matrix proteins associated with bone calcification are present in human vascular smooth muscle cells grownin vitro

Summary Atherosclerotic lesions are composed of cellular elements that have migrated from the vessel lumen and wall to form the cellular component of the developing plaque. The cellular elements are influenced by various growth-regulatory molecules, cytokines, chemoattractants, and vasoregulatory molecules that regulate the synthesis of the extracellular matrix composing the plaque. Because vascular smooth muscle cells (VSMC) constitute the major cellular elements of the atherosclerotic plaque and are thought to be responsible for the extracellular matrix that becomes calcified in mature plaques, immunostaining for collagenous and noncollagenous proteins typically associated with bone matrix was conducted on VSMC grownin vitro. VSMC obtained from human aorta were grown in chambers on glass slides and immunostained for procollagen type I, bone sialoprotein, osteonectin, osteocalcin, osteopontin, decorin, and biglycan. VSMC demonstrated an intense staining for procollagen type I, and a moderately intense staining for the noncollagenous proteins, bone sialoprotein and osteonectin, two proteins closely associated with bone mineralization. Minimal immunostaining was noted for osteocalcin, osteopontin, decorin, and biglycan. The presence in VSMC of collagenous and noncollagenous proteins associated with bone mineralization suggest that the smooth muscle cells in the developing atherosclerotic plaque play an important role in the deposition of the extracellular matrix involved in calcification of developing lesions.     

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.     

Purification and partial characterization of small proteoglycans I and II, bone sialoproteins I and II, and osteonectin from the mineral compartment of developing human bone

Using nondegradative isolation procedures we purified and characterized five major noncollagenous proteins from developing human bone. Small bone proteoglycan I, Mr approximately 350,000 on sodium dodecyl sulfate (SDS), 4-20% gradient polyacrylamide gels has a different amino-terminal sequence of NH2-Asp-Glu-Glu-()-Gly-Ala-Asp-Thr and is not cross-reactive with the small bone proteoglycan II, Mr approximately 200,000 on SDS-gradient polyacrylamide gels. Bone proteoglycan II is 95% N terminally blocked and the small amount that can be sequenced has an amino-terminal sequence (NH2-Asp-Glu-Ala-()-Gly-Ile. . .) that is apparently similar but not identical to a small proteoglycan isolated by Brennan, M.J., Oldberg, A., Pierschbacher, M.D., and Ruoslahti, E. (1984) J. Biol. Chem. 259, 13742-13750 from human fetal placenta membrane. Two bone sialoproteins, each of which migrates at a Mr approximately 80,000 on SDS gels, have also been isolated. Bone sialoprotein I has an amino-terminal sequence of NH2-Ile-Pro-Val-Lys-Gln-Ala. . . which is different from that of bone sialoprotein II with an amino-terminal sequence of NH2-Phe-Ser-Met-Lys-Asn-Leu. . . The two bone sialoproteins do not cross-react on Western blot analysis. Human bone osteonectin contains a large number of cysteines, more than 90% of which appear to be in disulfide bonds. The N-terminal amino acid sequence of human bone osteonectin was nearly identical to bovine bone osteonectin and had many similarities to a protein found in mouse parietal endoderm (Mason, I.J., Taylor, A., Williams, J.G., Sage, H., and Hogan, B.L.M. (1986) EMBO J. 5, 1831-1837.     

Extracellular matrix proteins involved in bone induction are vitamin D dependent

Subcutaneous implantation of demineralized diaphyseal bone matrix into allogeneic rats results in local formation of cartilage and bone. However, implantation of demineralized bone matrix obtained from rachitic rats did not induce bone. Rachitic bone matrix was therefore dissociatively extracted with 4 M guanidine HCl and then reconstituted with an inactive collagenous residue of control as carrier. Such reconstituted materials also lacked bone inductive potential. On the other hand, reconstitution of guanidine HCl extracts of control bone matrix with inactive vitamin D deficient matrix did result in bone induction. Partial purification (fractions containing proteins (less than 50,000 daltons) of the guanidine HCl extract from rachitic rats on Sepharose CL-6B followed by reconstitution with inactive collagenous residues resulted in a weak (25% of control) inductive response. These observations imply that bone inductive proteins are vitamin D dependent and are reduced in matrix obtained from rachitic rats.     

Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro

Adipose tissue-derived stem cells (ADSCs), which are easily harvested and show excellent pluripotency potential, have generated considerable interest in regenerative medicine. In this study, the differentiation of ADSCs was assessed after treatment with dental follicle cell conditioned medium (DFCCM) containing dentin non-collagenous proteins (dNCPs). ADSCs exhibited a fibroblast-like morphology and high proliferative capacity. However, after treatment with dNCPs/DFCCM, ADSCs changed from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and in vitro mineralization behaviour of ADSCs were significantly enhanced. Mineralization-related markers including cementum attachment protein, bone sialoprotein, osteocalcin, osteopontin and osteonectin were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype. These results demonstrate that ADSCs acquired cementoblast features in vitro with dNCPs/DFCCM treatment and could be a potential source of cementogenic cells for periodontal regeneration.     

Confocal imaging and timing of secretion of matrix proteins by osteoblasts derived from avian long bone

Primary osteoblasts derived from avian long bone have been evaluated in terms of spatial and temporal expression of known osteoblastic marker proteins during the early phases of cell culture. Confocal imaging of matrix proteins revealed that osteocalcin, bone sialoprotein, osteopontin, and osteonectin were restricted to the cell interior at day 4 of culture; secretion and deposition into the extra-cellular matrix of bone sialoprotein and osteopontin was evident at 8 and 12 days of culture. Osteocalcin and osteonectin were not deposited in the matrix within the timeframe of the study. Total collagen levels produced and alkaline phosphatase activity were substantial by day 4 of culture, and increased from that point 4.0- and 5.5-fold, respectively, by culture day 12. The expression of type I collagen, PTHrP receptor, osteopontin, bone sialoprotein and osteocalcin was followed by Northern blot analysis. Type I collagen and osteopontin mRNA were expressed at constant levels throughout the culture period. Over the 12 days of culture both PTH/PTHrP receptor and bone sialoprotein mRNA expression were found to increase by 2.3- and 2.5-fold, respectively. In contrast, the expression of osteocalcin message decreased by 2.5-fold by day 8 of culture.     

Cells isolated from the endosteal bone surface of adult rats express differentiated osteoblastic characteristics in vitro

Endosteal bone surface cells were previously shown to be involved in the regulation of bone formation in humans. In this study, we have characterized the cells isolated from the endosteal bone surface in adult rats. Fragments of periosteum-free tibia were obtained from 4-, 6- and 9-month-old rats by collagenase digestion, and the phenotypic characteristics of the osteoblastic cells migrating from the endosteal bone surface were evaluated in culture. Endosteal bone surface cells present a strong alkaline phophatase (ALP) activity as shown by cytochemistry and measured biochemically. The cells synthesize high levels of osteocalcin as measured by radioimmunoassay. Osteocalcin production was increased after stimulation with 10 nM 1,25 dihydroxyvitamin D (1,25(OH)₂ D) and the response to 1,25(OH)2 D was similar at all ages. Endosteal cells from young adult rats (4 months old) but not from older rats (6 and 9 months old) showed increased cAMP production in response to 10 nM parathyroid hormone (PTH), suggesting an agerelated decrease in the PTH-responsiveness of the bone surface cells. Immunocytochemistry using specific antibodies showed that preconfluent endosteal bone cells from adult rats expressed collagen and noncollagenous bone proteins in culture in the absence of inducers. The cells synthesized mostly type-I collagen which remained localized intracellularly. Type-III collagen was only expressed at low levels. The bone surface cells also expressed osteocalcin and bone sialoprotein, two markers of differentiated osteoblasts, as well as osteonectin. Endosteal cells plated at high density and cultured for 21 days with 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate formed multiple calcified nodules, as evidenced by von Kossa staining. This study shows that cells isolated from the endosteal bone surface of adult rats express in vitro characteristics of differentiated osteoblasts. These cell cultures can be used to study the dysfunctions of endosteal bone cells in relation to disorders of bone formation in adult rats.     

WHY DOES BONE MATRIX CONTAIN NON-COLLAGENOUS PROTEINS? THE POSSIBLE ROLES OF OSTEOCALCIN,OSTEONECTIN, OSTEOPONTIN AND BONE SIALOPROTEIN IN BONE MINERALISATION AND RESORPTION

Four major non-collagenous bone proteins were localised by single and double immuno-histochemistry during de novo mineralisation and bone resorption. Both osteopontin and bone sialoprotein were localised ahead of the mineralisation front, suggesting that both proteins are necessary for the initiation of bone mineralisation. This supports previous suggestions that bone sialoprotein acts as a crystal nucleator. The role of osteopontin is less certain, but might be related to ensuring that only the right type of crystal is formed. Osteocalcin and osteonectin were not present in areas of first crystal formation, but were present in the fully mineralised matrix. Their role may be to control the size and speed of crystal formation. Osteopontin, bone sialoproteins and osteocalcin (but not osteonectin) were also present at reversal lines. Interpreting this localisation together with information from the literature, the following functions are suggested during resorption: Osteocalcin may act as a chemoattractant for osteoclasts, while both osteopontin and bone sialoprotein may facilitate the binding of osteoclasts via the arg-gly-asp motif.     

The effects of vitamin D deficiency on proteoglycan and hyaluronate constituents of chick bone

The effect of vitamin D deficiency on proteoglycan and hyaluronate constituents of cortical diaphyseal chick bone was studied. Proteoglycans in rachitic bone showed no significant change with respect to their size, composition, or amount relative to other extractable macromolecular components. In contrast, bone hyaluronate levels were raised in chicks fed on diets that were either vitamin D-deficient or depleted in calcium or phosphate, a 7-fold increase being seen in hypocalcaemic vitamin D-deficient chicks. This increase in hyaluronate was not directly related either to the absence of vitamin D or to abnormal levels of blood calcium or phosphate per se; hyaluronate levels are probably regulated by another factor, not yet identified, that is responsive to changes in vitamin D and mineral metabolism.     

Lysosomal proliferation in rachitic avian intestinal absorptive cells following 1,25-Dihydroxycholecalciferol

Lysosomes in chick intestinal absorptive cells from rachitic (vitamin D-deficient) and vitamin D-replete animals were studied utilizing transmission electron microscopic histochemistry and ultrastructural morphometry. Absorptive cells from rachitic animals, serum calcium = 7.3±0.3 mg%, contained an average of 4.0±0.3 supranuclear lysosomes. In rachitic chicks sacrificed 9 hr post-injection of 1,25-dihydroxycholecalciferol, the active metabolite of vitamin D, the values for both serum calcium, 9.8 ± 0.2 mg%, and the number of apical absorptive cell lysosomes, 12.9±0.6, were increased over non-injected or vehicle-only injected animals. Lysosomes in vitamin D-replete absorptive cells were characterized by their intense staining with pyroantimonate, indicative of their high calcium content. The same organelles also produced a positive reaction for acid phosphatase. Rachitic lysosomes, also acid phosphatase positive, were only lightly stained with pyroantimonate. The lysosomal proliferation apparently induced by 1,25-dihydroxycholecalciferol may be a further indication that these organelles play a role in intestinal calcium transport and/or intracellular calcium homeostasis within the absorptive cell.     

Encapsulation of foreign particles in vitro by separated blood cells from crayfish,Astacus leptodactylus

Summary Bone matrix consists of type-I collagen and noncollagenous proteins. The latter represent only 10% of its total protein content. Since type-I collagen is also present in various other connective tissue sites (e.g., skin) it cannot be considered as bone specific. Among the non-collagenous components osteonectin — a 32 kilodalton (KD) glycoprotein linking mineral to collagen fibrils — is thought to be bone specific due to its biochemical properties. In the present study various skeletal and non-skeletal tissues were investigated for the presence of osteonectin by means of immunocytochemical methods. Two polyclonal antibodies against human and bovine osteonectin were applied. Immunocytochemically, osteonectin could be demonstrated in active osteoblasts and osteoprogenitor cells as well as in young osteocytes, while aged, quiescent osteocytes did not contain the protein, suggesting that the protein is a marker of the osteoblastic functional differentiation of bone cells. Osteonectin was absent in all non-skeletal tissues with the exception of chondrocytes in so-called mineralizing chondroid bone.     

Effects of vitamin D2-fortified shiitake mushroom on bioavailability and bone structure

Bioavailability and bone loss inhibitory effects of vitamin D₂ derived from UV-irradiated shiitake mushroom were determined in vivo. The effect of the absence of ovaries on the bioavailability of vitamin D₂ and bone structure was also investigated. Sham operated (sham) and ovariectomized (OVX) rats were divided in 3 groups according to their diets, i.e. control: only vitamin D-deficient diets; UV(X): vitamin D-deficient diets with non-irradiated mushroom powder; UV(O): vitamin D-deficient diets with irradiated mushroom powder. The obtained results showed that vitamin D₂ from shiitake mushroom was able to increase bone mineral density and trabecular bone structure of femur bone as well as its bioavailability. The absence of estrogen induced adverse effects not only on bioavailability of vitamin D₂ but also on trabecular bone. In conclusion, vitamin D₂-fortified shiitake mushroom might help postmenopausal women increase vitamin D₂ bioavailability and retard trabecular bone loss.

Abbreviations: OVX: ovariectomized; 25(OH)D: 25-hydroxyvitamin D; 1,25(OH)2D: 1,25-dihydroxyvitamin D; BMD: bone mineral density; micro-CT: micro computed tomography; RSM: response surface methodology; RP-HPLC: Reverse phase-high performance liquid chromatography; MS/MS: tandem mass spectrometry; E2: estradiol; NTx: N-terminal telopeptide of type I collagen; BV/TV: bone volume/total volume; BS/BV: bone surface/bone volume; Tb.Th: trabecular thickness; Tb.Sp: trabecular separation.     

The phosphorylation of 5′-oligoadenylic acids by adenylate kinase and adenosine triphosphate

1. This paper reports studies on the metabolism of bone from normal chicks and from chicks with vitamin D-deficiency rickets. Both in vitro and in vivo there was an increased incorporation of [(14)C]proline into collagen hydroxyproline by rachitic bone. The proportion of the collagen that was soluble in cold salt solutions was greater with the rachitic bone. These results show that in rickets there is an increased synthesis of bone collagen, but they do not provide any evidence of a defect in the maturation of collagen. 2. Rachitic bone incubated aerobically in vitro consumed more glucose and released more lactate than normal bone. Bone from rachitic chicks treated with vitamin D 48hr. previously had rates of glycolysis that were nearly normal. Though we were unable to show any direct action of vitamin D in vitro, we consider that vitamin D probably has a direct action on bone, possibly related to matrix biosynthesis.     

Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

In the presentstudy, we evaluated the possibility that the abnormal bone matrixproduced during spaceflight may be associated with reduced expressionof bone matrix protein genes. To test this possibility, we investigatedthe effects of a 14-day spaceflight (SLS-2 experiment) on steady-statemRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH),osteocalcin, osteonectin, and prepro-(1) subunit of type I collagenin the major bone compartments of rat femur. There were pronouncedsite-specific differences in the steady-state levels of expression ofthe mRNAs for the three bone matrix proteins and GAPDH in normalweight-bearing rats, and these relationships were altered afterspaceflight. Specifically, spaceflight resulted in decreases in mRNAlevels for GAPDH (decreased in proximal metaphysis), osteocalcin(decreased in proximal metaphysis), osteonectin (decreased in proximaland distal metaphysis), and collagen (decreased in proximal and distalmetaphysis) compared with ground controls. There were no changes inmRNA levels for matrix proteins or GAPDH in the shaft and distalepiphysis. These results demonstrate that spaceflight leads to site-and gene-specific decreases in mRNA levels for bone matrix proteins.These findings are consistent with the hypothesis thatspaceflight-induced decreases in bone formation are caused byconcomitant decreases in expression of genes for bone matrix proteins.

     

In vitro production of proteoglycans in the articular-epiphyseal cartilage of growing pigs

The failure of cartilage mineralization in osteochondrotic cartilage may be due to an impaired proteoglycan production. Thein vitro production of proteoglycans was therefore studied in the joint cartilage of growing pigs, aged 9–18 weeks, after incubation of cartilage samples with³⁵S-sulfate. Cartilage was obtained from different areas of the femoral condyles and samples from these areas were further divided into three layers, where the superficial layer contains articular cartilage and the basal layers consist of growth cartilage. There was no significant difference in the overall amount of³⁵S-proteoglycans synthesized in different areas of the condyles. However, the total production of³⁵S-proteoglycans per mg tissue was highest in the basal layer in all areas. This was not due to a larger number of cells; the superficial layer contained more DNA per mg tissue than the basal layer. Gel chromatography on Sepharose CL-2B of the cartilage extracts, which resulted in the separation of large proteoglycans (K _av 0.4) from proteoglycans of small hydrodynamic size (K _av 0.8), showed that the relative amount of large proteoglycans increased with the distance from the articular surface. Again, no difference in the relative amounts of large and small proteoglycans were found when cartilage from different areas were compared. Osteochondrotic cartilage was detected in the pigs aged 12–18 weeks. In areas where osteochondrotic cartilage were present, the total production of³⁵S-proteoglycans was lowered and the relative amount of large proteoglycans was less than that found in the adjoining areas devoid of osteochondrotic lesions. The data available indicate that the higher relative amount of small proteoglycans in the osteochondrotic cartilage was partly caused by degradation of the large proteoglycans (aggrecan).