Production and function of IL-12 in islets and beta cells.

AIMS/HYPOTHESIS: IL-12 is an important cytokine in early inflammatory responses and is implicated in the immune-mediated pathogenesis of pancreatic islets in diabetes. However, little is known about the direct effects of IL-12 on islets and beta cells. METHODS: In this study, beta cell function, gene expression and protein production were assessed in primary human donor islets and murine beta cell lines in response to stimulation with IL-12 or a pro-inflammatory cytokine cocktail (TNF-α, IL-1ß and IFN-γ). RESULTS: The pro-inflammatory cytokine cocktail induced islet dysfunction and potently increased the expression and production of IL-12 ligand and IL-12 receptor in human islets. In human islets, the receptor for IL-12 co-localised to the cell surface of insulin-producing cells. Both IL-12 ligand and IL-12 receptor are expressed in the homogeneous beta cell line INS-1. IL-12 induced changes in gene expression, including a dose-dependent upregulation of IFNγ (also known as IFNG), in INS-1 cells. A neutralising antibody to IL-12 directly inhibited IFNγ gene expression in human donor islets induced by either IL-12 or pro-inflammatory cytokine stimulation. Functionally, IL-12 impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells and human donor islets. A neutralising antibody to IL-12 reversed the beta cell dysfunction (uncoupling of GSIS or induction of caspase-3 activity) induced by pro-inflammatory cytokines. CONCLUSIONS/INTERPRETATION: These data identify beta cells as a local source of IL-12 ligand and suggest a direct role of IL-12 in mediating beta cell pathology.

Targeting and immobilization of bioactive peptides on dentin matrix.

The regeneration of structurally/functionally competent tooth root cementum is a critical step for the successful restoration of periodontal attachment. In this study, we tested whether a poly-glutamic acid-rich domain and glutamine-containing transglutaminase substrate can be used to target biologically active peptides to the mineralized root matrix and to bind such peptides covalently to the organic matrix. As a biologically active model molecule, the integrin-binding motif, RGD, was used. The effects of immobilization of such synthetic peptides to the dentin matrix on cementoblastic adhesion in vitro and cementogenesis in vivo were studied. In vitro, cementoblastic adhesion improved significantly when the dentin surface contained covalently bound peptides. In vivo, this bound peptide significantly increased cementum formation compared with that attained in control conditions. Transglutaminase-catalyzed covalent binding of bioactive peptides targeted to mineralized collagenous dentin matrix via the poly-glutamate domain can be readily achieved. This approach offers potential for clinical use in periodontal regeneration.

Primary care reform: a pilot study to test the evaluative potential of the Patient Enablement Instrument in Poland.

BACKGROUND: Primary health care reform is underpinned by a move towards patient-centred holistic care. This pilot study uses the Patient Enablement Instrument (PEI) to assess outcome at a fundamental level: that of the patient and their doctor at consultation. OBJECTIVES: Our aim was to assess the evaluative potential of the PEI in relation to a reform programme in Poland by (i) comparing the outcomes of consultations (using the PEI) carried out by nine doctors (three diploma GPs who had participated in the training programme, three GPs who had not participated in the training programme and three polyclinic internists); and (ii) relating PEI scores to a proxy quality process measure (consultation length). METHODS: A cross-sectional quantitative questionnaire survey was carried out using the PEI. The subjects were patients consulting with nine doctors distributed within a single region around Gdansk. RESULTS: The overall results with the PEI and consultation length reflected UK experience. In addition, there were significant differences between groups in this pilot study. Patients seen by diploma GPs achieved higher patient enablement scores (mean 4.33, 95% confidence interval 4.09-4.58) relative to GPs (mean 3.44, 3.21-3.67) and polyclinic doctors (mean 3.23, 2.99-3.47). However, there is evidence of appreciable between-doctor variation in PEI scores within groups. The difference in patient enablement between groups was not affected by patient case mix, in contrast to the duration of consultation, which was. Holistically trained diploma GPs spent longer with patients with psychological problems. Patients seen by diploma GPs received longer consultations (mean 12.65 min, 95% confidence interval 12.18-13.13) relative to their colleagues (the GPs' mean was 10.11, 9.82-10.41 min; that of the polyclinic internists was 10.16, 9.81-10.50 min). The duration of consultation was positively correlated with patient enablement. CONCLUSION: The results of such training courses should be examined from the perspective of both the patient and their doctor. Significant differences were found in both patient enablement and consultation length between patients attending groups of doctors delivering primary care, but working from different paradigms. This pilot shows promising results which, if repeated in a larger study, would provide an objective means of evaluating such reform programmes.

Cementum-forming cells are phenotypically distinct from bone-forming cells.

Normal human cementum-derived cells (HCDCs), expanded in vitro, formed mineralized matrix when attached to a ceramic carrier and transplanted subcutaneously into immunodeficient mice. The mineralized matrix elaborated by transplanted HCDC exhibited several features identical to cementum in situ and was significantly different from bone deposited by similarly transplanted human bone marrow stromal cells (BMSCs). No bone marrow formation and very few or no tartrate-resistant acid phosphatase (TRAP)-positive cells (osteoclasts and osteoclastic precursors) were found in HCDC transplants. In contrast, in BMSC transplants both hematopoiesis and TRAP-positive cells were routinely observed. Furthermore, compared with BMSC-derived matrix, HCDC-derived matrix was less cellular, numerous empty lacunae were present, and fewer cells were found on the cementum matrix/ceramic carrier interface. The organization of collagen fibers in HCDC-derived matrix, as visualized by using the Picrosirus red staining method, was similar to cementum, with typical unorganized bundles of collagen fibers. In contrast, bone matrix elaborated by transplanted BMSC had lamellar structure, identical to mature bone in situ. Finally, cementocytes embedded in the cementum-like matrix were immunopositive for fibromodulin and lumican, whereas osteocytes within the bonelike matrix were negative. This pattern is consistent with the cementum and bone in situ, respectively. These results indicate that human cementum cells are phenotypically distinct from bone cells and provide further validation of the combined in vitro/in vivo model of human cementogenesis recently developed in our laboratory.

Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro.

The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen alpha chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of alpha chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross-linking pattern.

The natriuretic peptide clearance receptor locally modulates the physiological effects of the natriuretic peptide system.

Natriuretic peptides (NPs), mainly produced in heart [atrial (ANP) and B-type (BNP)], brain (CNP), and kidney (urodilatin), decrease blood pressure and increase salt excretion. These functions are mediated by natriuretic peptide receptors A and B (NPRA and NPRB) having cytoplasmic guanylyl cyclase domains that are stimulated when the receptors bind ligand. A more abundantly expressed receptor (NPRC or C-type) has a short cytoplasmic domain without guanylyl cyclase activity. NPRC is thought to act as a clearance receptor, although it may have additional functions. To test how NPRC affects the cardiovascular and renal systems, we inactivated its gene (Npr3) in mice by homologous recombination. The half life of [125I]ANP in the circulation of homozygotes lacking NPRC is two-thirds longer than in the wild type, although plasma levels of ANP and BNP in heterozygotes and homozygotes are close to the wild type. Heterozygotes and homozygotes have a progressively reduced ability to concentrate urine, exhibit mild diuresis, and tend to be blood volume depleted. Blood pressure in the homozygotes is 8 mmHg (1 mmHg = 133 Pa) below normal. These results are consistent with the sole cardiovascular/renal function of NPRC being to clear natriuretic peptides, thereby modulating local effects of the natriuretic peptide system. Unexpectedly, Npr3 -/- homozygotes have skeletal deformities associated with a considerable increase in bone turnover. The phenotype is consistent with the bone function of NPRC being to clear locally synthesized CNP and modulate its effects. We conclude that NPRC modulates the availability of the natriuretic peptides at their target organs, thereby allowing the activity of the natriuretic peptide system to be tailored to specific local needs.

Normal human cementum-derived cells: isolation, clonal expansion, and in vitro and in vivo characterization.

Cultures of primary human cementum-derived cells (HCDCs) were established from healthy premolar teeth extracted for orthodontic reasons. Cementum was manually dissected, fragmented, and digested twice with collagenase. Following a thorough wash to remove liberated cells, the remaining cementum fragments were plated in Dulbecco's modified Eagle's medium/F12 medium containing 10% fetal bovine serum. Discrete colonies that contained cells exhibiting fibroblast-like morphology were visible after 14-21 days of culture. When the colonies became sufficiently large, cells from individual colonies were isolated and subcultured. Cementum-derived cells exhibited low levels or no alkaline phosphatase activity and mineralized in vitro to a lesser degree than human periodontal ligament (PDL) cells and human bone marrow stromal cell (BMSC) cultures. To study differentiation capacities of HCDCs, cells were attached to hydroxyapatite/tricalcium phosphate ceramic and transplanted subcutaneously into immunodeficient mice. The transplants were harvested 3, 6, and 8 weeks after transplantation and evaluated histologically. In human BMSC transplants, new bone tissue was formed with a prominent osteoblastic layer and osteocytes embedded in mineralized bone matrix. No osseous tissue was formed by PDL cells. Of six single colony-derived strains of HCDCs tested, three formed a bone-like tissue that featured osteocyte/cementocyte-like cells embedded within a mineralized matrix and which was lined with a layer of cells, although they were somewhat more elongated than osteoblasts. These results show that cells from normal human cementum can be isolated and expanded in vitro. Furthermore, these cells are capable of differentiating and forming mineralized tissue when transplanted into immunodeficient mice.

Synthetic integrin-binding peptides promote adhesion and proliferation of human periodontal ligament cells in vitro.

Periodontal ligament (PDL) cells have been shown to express several integrins (alphav, alpha5, beta1, beta3) that use RGD (arginine-glycine-aspartic Acid)-dependent mechanisms for the recognition and binding of their ligands. The objective of this study was to evaluate the effects of certain integrin-binding cyclic and linear synthetic RGD-containing peptides on PDL cells' adhesion, proliferation, and de novo protein synthesis in vitro. Fifth passages of normal human PDL cells established from teeth extracted from patients (ages 12 to 14) for orthodontic reasons were used for all experiments. Synthetic peptides containing the EPRGDNYR sequence in two different spatial conformations (linear and cyclic) were covalently attached to bovine serum albumin (BSA). Type I collagen, EPRGDNYR-BSA conjugates, 1:1 mixtures of type I collagen and conjugates, as well as BSA (a negative control) were coated on bacteriological plastic and evaluated for their attachment-promoting activities. In addition, the effects of these substrates on cell proliferation were evaluated by [3H]thymidine incorporation by the PDL cells. For attachment and spreading, the cyclic forms of EPRGDNYR-BSA conjugate and type I collagen were most potent, followed by linear EPRGDNYR-BSA conjugate. The effects of all collagen/conjugate mixtures were equivalent to that of type I collagen except for the collagen/linear EPRGDNYR-BSA mixture, which was less potent. The cyclic EPRGDNYR-BSA conjugate was the most effective substrate to stimulate cell proliferation, and it was followed in potency by the linear peptide-BSA conjugate. Collagen alone did not stimulate [3H]thymidine incorporation above the control level. Mixtures of collagen with all of the conjugates showed stimulatory effects similar to that of the cyclic peptide-BSA conjugate. No significant differences in de novo protein synthesis were detected. These results suggest that the synthetic RGD-containing peptides attached to a carrier are potent ligands for the human PDL cells, and that they could provide a basis for the development of new strategies aimed at the regeneration of the periodontium.

The human bone sialoprotein gene contains an NF-E1/YY1 cis-acting sequence with putative regulatory activity.

Bone sialoprotein (BSP) is a noncollagenous matrix glycoprotein localized predominantly in mineralized tissues but also detected in extraskeletal sites undergoing focal mineralization. We have previously characterized the human BSP gene and have shown that the upstream sequence contains inverted TATA and CCAAT motifs at the expected locations from the transcriptional start site (J. M. Kerr et al. [13]) and a potential YY1 binding motif located within the first 30 bp of intron 1 of the human gene. Deletion analyses of the human BSP promoter/exon 1 sequence fused to a CAT reporter gene indicate that CCAAT enhances basal transcription of BSP in transiently transfected rat UMR106-01 BSP osteosarcoma and rat skin fibroblasts. Though this enhancing activity was lost with inclusion of 68 bp of intron containing a YY1 motif in these constructs, reporter activity in the UMR106-01-BSP cells was elevated four- to seven-fold relative to that of rat fibroblasts. Gel electrophoretic mobility shift, UV-crosslinking, and south-western experiments indicate that YY1 is present only in the extracts of nuclei isolated from the UMR cells and may contribute to the elevated transcriptional activity of the human BSP promoter construct in UMR106-01-BSP.

Integrins and bone--cell adhesion and beyond.

Integrins are a superfamily of cell surface receptors involved in cell-cell and cell-matrix adhesion. Integrin-mediated interactions are involved in the regulation of numerous cellular functions such as fertilization, implantation, cell differentiation and migration during embryonic development, maintaining tissue architecture, blood clot formation and retraction programmed cell death, tumor growth and metastasis formation, lymphocyte homing and response of cells to mechanical stresses. This broad spectrum of activity is achieved by combining the ability to create mechanically functional junctions (cell-matrix and cell-cell) and signal-transducing capabilities. Osteoblasts and osteoclasts express specific integrin receptors and the pattern of expression varies depending on the stage of cell differentiation. Interactions of integrins with bone-matrix adhesive proteins are thought to be important for regulating the tissue integrity and may provide a local, responsive regulatory system of osteoblastic differentiation as well. Osteoclasts most likely attach to osteopontin exposed on the bone surface via the classic vitronectin receptor alpha v beta 3 and this binding may be crucial to their bone resorption activity.

Attachment characteristics and involvement of integrins in adhesion of breast cancer cell lines to extracellular bone matrix components.

Evidence is mounting that changes in the ability of cancer cells to adhere to extracellular matrices play a decisive role in metastatic spread. The mechanism underlying the preference of breast cancer cells to metastasize to bone is, however, poorly understood. We investigated the expression and involvement of integrin adhesion receptors in the adhesion of breast cancer cells to bone matrix (constituents) in two in vitro attachment assays using RGD peptides and anti-integrin antibodies. Breast cancer cells adhered rapidly to extracellular bone matrix. Adhesion of most cells to vitronectin, fibronectin, thrombospondin, osteopontin, and the fairly bone-specific bone sialoprotein was inhibited by the 200 micrograms/ml GRGDS peptide. These data suggest that integrin adhesion receptors can modulate the attachment of breast cancer cells to bone matrix molecules. In accordance with these findings, we found that alpha 1-alpha 5(beta 1) and alpha v(beta 3) integrins were expressed by mammary carcinoma cells. Highly tumorigenic MDA-MB-231 cells, which form osteolytic metastases in vivo, expressed relatively high levels of alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3 integrins, when compared to MCF-7, T47D, and ZR75-1 breast cancer cells. Addition of function-blocking anti-alpha 2 beta 1, -alpha 3 beta 1, -alpha 5 beta 1, and -alpha v beta 3 antibodies significantly inhibited the adhesion of MDA-MB-231 breast cancer cells to bone matrices. In conclusion, our data suggest a possible role for beta 1 and beta 3 integrin subfamily members in the establishment of skeletal metastases in advanced breast cancer patients. Clearly, functional evidence is required to understand the mechanisms involved in the development of skeletal metastases in breast cancer patients.

Bone sialoprotein peptides are potent inhibitors of breast cancer cell adhesion to bone.

Bone and bone marrow are important sites of metastasis formation in breast cancer. Extracellular matrix proteins with attachment properties are generally believed to play a key role in tumorigenesis and metastasis formation. We have investigated whether mammary carcinoma cells (MDA-MB-231) can recognize constructs of the fairly bone-specific human bone sialoprotein, which encompass the RGD sequence (EPRGD-NYR). Exogenously added bone sialoprotein peptides with this amino acid sequence in their backbone structure, but not the more common fibronectin-derived GRGDS peptide, strongly inhibited breast cancer cell adhesion to extracellular bone matrix at micromolar concentrations. Most cyclic derivatives with the EPRGDNYR sequence were more effective inhibitors of tumor cell adhesion to bone than their linear equivalents. Furthermore, changes in the RGD-tripeptide of the backbone structure of the constructs, removal of the NYR flanking sequence, or a different tertiary cyclic structure significantly decreased their inhibitory potencies. In addition, the RGE-analogue EPRGENYR was capable of inhibiting breast cancer cell adhesion to bone, albeit to a lesser extent. We conclude therefore, that the inhibitory potency of the bone sialoprotein-derived peptides on breast cancer cell adhesion to bone is not solely due to a properly positioned RGD-motif alone but is also determined by its flanking regions, together with the tertiary structure of the EPRGDNYR peptide. Synthetic cyclic constructs with the EPRGDNYR sequence may, therefore, be potentially useful as antiadhesive agents for cancer cells to bone in vivo.

Synthesis and use of a new bromoacetyl-derivatized heterotrifunctional amino acid for conjugation of cyclic RGD-containing peptides derived from human bone sialoprotein.

A new amino acid derivative, N alpha-(tert-butyloxycarbonyl)-N beta-(bromoacetyl)diaminopropionic acid (BBDap), has been synthesized as a reagent for introducing side-chain bromaocetyl groups into any position of a peptide sequence during solid-phase peptide synthesis. By using minor modifications to the protocol of the automated peptide synthesizer and a two-step in situ neutralization procedure, the syntheses of (bromoacetyl)diaminopropionic acid (BDap) in Arg-Gly-Asp-containing peptides from human bone sialoprotein were optimized and completed. Following HPLC purification, the BDap-derivatized peptides were cyclized or/and conjugated to carrier protein or to glass cover slips. In addition, a new procedure for site-specific conjugation of cyclic peptides to protein carriers or to glass was developed. The cell attachment activity of the peptide derivatives and conjugates was tested in cell adhesion assays with human osteoblasts, and the specificity of the binding was confirmed by competition with linear and/or cyclic forms of GRGDS. The results show that conjugates containing the linear and cyclic derivatives of the peptide EPRGDNYR supported cell attachment and spreading in a dose-dependent manner when the peptides were immobilized as described. Cell attachment to the intact bone sialoprotein and to conjugates containing the linear peptides was abolished by competition with linear and cyclic RGD-containing peptides, whereas the attachment to conjugates containing the cyclic peptide was inhibited only partially, and the cell spreading was preserved even in the presence of RGD-peptides.

Bone matrix RGD glycoproteins: immunolocalization and interaction with human primary osteoblastic bone cells in vitro.

The interaction of cells with extracellular matrix is essential for their anchorage, proliferation, migration, and differentiation. In bone matrix there are multiple glycoproteins that contain the integrin-binding RGD sequence: fibronectin (FN), thrombospondin (TSP), osteopontin (OPN), bone sialoprotein (BSP), type I collagen (COLL I), and vitronectin (VN). In this study, the localization of TSP, FN, VN, and several integrins within developing human long bone using immunohistochemical methods was examined, as was the effect of all bone RGD proteins on the adhesion of human osteoblastic cells. Thrombospondin, fibronectin, and vitronectin showed distinct localization patterns within bone tissue. TSP was found mainly in osteoid and the periosteum; VN appeared to be present mainly in mature bone matrix. FN was present in the periosteum as well as within both mature and immature bone matrix. Using a panel of antiintegrin antibodies we found that bone cells in vivo and in vitro express alpha 4, alpha v, alpha 5 beta 1, alpha v beta 3, and beta 3/beta 5 integrins, and these receptors are for the most part expressed on all bone cells at different stages of maturation with quantitative rather than qualitative variations, with the exception of alpha 4, which is expressed mainly by osteoblasts. Cell attachment assays were performed using primary human cells of the osteoblastic lineage under serum-free conditions. COLL I, TSP, VN, FN, OPN, and BSP promoted bone cell attachment in a dose-dependent manner and were equivalent in action when used in equimolar concentrations. In the presence of GRGDS peptide in the medium, the adhesion to BSP, OPN, and VN was almost completely blocked (10, 10, and 15% of control, respectively), and attachment to FN, COLL I, and TSP was only slightly decreased (80, 75, and 55%, respectively). These results suggest that human bone cells may use RGD-independent mechanisms for attachment to the latter glycoproteins.

Chlorate-induced inhibition of tyrosine sulfation on bone sialoprotein synthesized by a rat osteoblast-like cell line (UMR 106-01 BSP).

Bone sialoprotein (BSP) is a major noncollagenous, RGD-containing glycoprotein found in the extracellular matrix of bone. The RGD sequence is flanked by two tyrosine-rich regions, which fit the established consensus requirements for tyrosine sulfation. Tyrosine sulfation is suggested to be important in the regulation of protein secretion and function. The role of this post-translational modification on the cell attachment activity and secretion of a highly sulfated form of BSP isolated from a rat osteoblast-like cell line (UMR 106-01 BSP) was investigated by inhibiting sulfation with chlorate. [35S]Sulfate, [3H]glucosamine, and [3H]tyrosine were used as metabolic precursors to monitor biosynthetic products. Chlorate was effective in inhibiting total [35S]sulfate incorporation by 90% without altering overall protein synthesis and secretion in cultures up to 72 h under serum-free conditions. Isolated proteoglycans and purified BSP were analyzed for sulfate incorporation. Proteoglycans isolated from the medium of cells treated with chlorate displayed a difference in the hydrodynamic properties of the molecules as compared with control cultures. An increase in the specific activity of proteoglycans labeled with [3H]glucosamine isolated from chlorate-treated cells was also observed suggesting a change in hexosamine metabolism induced by chlorate. BSP purified from the medium of chlorate-treated cells contained approximately 7% of the 35S incorporation as compared with nontreated control cultures. Quantification of sulfate incorporation into glycoconjugates versus tyrosine sulfate of BSP indicates that the amount of sulfate associated with N- and O-linked oligosaccharides was reduced by approximately 97%, while that on tyrosine residues was reduced by approximately 90%. Using normal human bone cells, the cell attachment activity of the reduced sulfate form of BSP was nearly equivalent to that of the fully sulfated product.

Structure and molecular regulation of bone matrix proteins.

The organic matrix of bone contains several protein families, including collagens, proteoglycans, and glycoproteins, all of which may be extensively modified by posttranslational events, such as phosphorylation and sulfation. Many of the glycoproteins contain Arg-Gly-Asp (RGD), the integrin-binding sequence, within their structure, whereas other constituent proteins contain gamma-carboxyglutamic acid. The deposition of bone matrix by cells in the osteoblastic lineage is regulated by extrinsic factors, such as systemic and local growth factors and physical forces, and factors that are intrinsic to the cell, such as position in the cell cycle, maturational stage, and developmental age of the donor. Recent studies of several bone matrix gene promoters have identified cis- and trans-acting elements that are responsible for gene activity, although the precise sequence of regulatory events is not known. Development of in vitro assays, coupled with studies of the appearance of these proteins during development in vivo, provides insight into the functions of these proteins during the various stages of bone metabolism. Potential roles for these proteins include proliferation and maturation of stem cells, formation of matrix scaffolding elaborated by bone-forming cells, modeling, and remodeling. Changes in the functional properties of the extracellular matrix may be involved in a variety of disease processes, including osteoporosis and oral bone loss.

Purification and fragmentation of nondenatured bone sialoprotein: evidence for a cryptic, RGD-resistant cell attachment domain.

Bone sialoprotein (BSP), a small (approximately 80,000 M(r)) integrin binding, RGD-containing bone matrix glycoprotein, has been purified in milligram quantities from the serum-free medium of the rat osteosarcoma cell line UMR-106-BSP using nondenaturing conditions. Routine protein purification without serine protease inhibitors or reducing agents consistently resulted in three major fragments. The largest fragment (E1) started at amino acid 117 and did not bind to antibodies made to the RGD region of the protein. Furthermore, the smallest fragment (E3), was shown by sequencing to contain the RGD region of the protein. Digestion of intact BSP with highly purified chymotrypsin also resulted in a large fragment (C1) with properties nearly identical to those of E1. The large, non-RGD-containing fragments, E1 and C1, as well as the intact BSP, supported attachment by normal human bone cells and human skin fibroblasts in vitro. Attachment to the intact BSP was totally blocked by 0.4 mM GRGDS peptide. Both preparations of skin fibroblasts and approximately half of the preparations of normal human bone cells, however, also would not attach to the E1 and C1 fragments in the presence of 0.4 mM GRGDS peptide. In contrast, half of the bone cell preparations had significant attachment activity to E1 (> 50%) and C1 (> 25%) in the presence of 0.4 mM GRGDS peptide. These data suggest that cleavage of the BSP results in either (1) the exposure of a previously unavailable or cryptic cell attachment site or (2) a conformational change that increases the affinity of the complex between a non-RGD-encoded binding region of the E1 and C1 fragments and at least one receptor. The possible homology of the second, non-RGD-suppressible site of BSP with the second cell attachment site on the gamma chain of fibrinogen is discussed.

Effect of radiation sterilization on the osteoinductive properties and the rate of remodeling of bone implants preserved by lyophilization and deep-freezing.

The effect of various doses of ionizing radiation on the osteoinductive properties of decalcified bone matrices implanted heterotopically and on the rate of remodeling of nondecalcified bone grafts implanted orthotopically in allogeneic systems was studied. Decalcified bone matrices and nondecalcified bone grafts were preserved by lyophilization or by deep-freezing and were subsequently irradiated with appropriate doses at room temperature or at -72 degrees. Lyophilized matrices irradiated at room temperature with 35 and 50 kGy, respectively, were completely resorbed five weeks after heterotopic implantation into the muscles and did not induce osteogenesis, whereas the resorption of deep-frozen ones irradiated with the same doses at -72 degrees was slower and new bone formation was induced. The preservation of the osteoinductive capacity of irradiated, deep-frozen matrices may depend on two factors: reduction of radiation damage on the inducing agents and collagen irradiated in the presence of water, which may diminish the rate of matrix resorption. The rate of remodeling of undecalcified deep-frozen bone implants irradiated at -72 degrees and grafted orthotopically was higher than that of lyophilized ones irradiated at room temperature. It is possible that the temperature during irradiation plays a critical role in protection against radiation damage.

Early and transient osteopetrosis in microphthalmic MIB-rats.

Microphtalmic blanc mutation (mib/mib) displays a very mild form of osteopetrosis in rats. The autosomal recessive mib mutation shows pleiotropic expressions in homozygotes. Microphtalmia, absence of eye and skin pigmentation, retardation in the tooth eruption were observed in the mutants. Most bone abnormalities occurred in newborns. An increased radiological opacity of long bones, persistence of primitive bone in medullary cavities, reduced number of poorly differentiated osteoclasts in mandibulae, reduced number of mononuclear peritoneal cells as well as reduced number of mononuclear osteoclast precursors in peritoneal cell population were found. In 3 weeks old and in adult mutants, both bone structure and the number of mandible osteoclasts appear normal, but the number of blood monocytes, peritoneal cells and mononuclear osteoclast precursors in peritoneal cell population remain significantly lower than in the healthy littermates. These observations indicate that the early failure of osteoclast differentiation and maturation is transient in the mib/mib form of osteopetrosis.

Spreading of cells on various substrates evaluated by Fourier analysis of shape.

In order to evaluate in mathematical terms the morphological changes occurring in the course of cell spreading, Fourier analysis of shape was applied. Human urothelial Hu 961 b cells plated on type IV collagen, fibronectin, laminin, glass and bovine serum albumin (BSA) were studied. Fourier parameters describing cell shape as well as surface areas covered by the cells on the substrate were subjected to statistical analysis. Using analysis of variance and discriminant analysis it was found that parameters describing cell shape (both gross shape of cells and their fine scale contour foldings) possessed a higher power of discrimination between the cells spread on various substrates than the differences in cell surface areas. In the course of observation (75 and 150 min) the highest number of attached cells and highest degree of spreading were found when cells were plated on type IV collagen. Moderate alterations in cell shape and moderate increase of surface area were seen in the group of cells seeded on fibronectin, whereas the cells plated on laminin, glass and BSA revealed a moderate increase of surface area, but no changes in their shape were observed. The differences in attachment of cells and in the degree of their spreading might be due to the variation in expression of plasma membrane receptors for various substrates. The Fourier analysis of cell shape coupled with measurement of surface area is a good tool for quantitative evaluation of cell spreading and can be used for discrimination between cells spread on different substrates.