Engineering three-dimensional constructs of the periodontal ligament in hyaluronan-gelatin hydrogel films and a mechanically active environment.

BACKGROUND AND OBJECTIVE: Periodontal ligament (PDL) cells in stationary two-dimensional culture systems are in a double default state. Our aim therefore was to engineer and characterize three-dimensional constructs, by seeding PDL cells into hyaluronan-gelatin hydrogel films (80-100 µm) in a format capable of being mechanically deformed. MATERIAL AND METHODS: Human PDL constructs were cultured with and without connective tissue growth factor (CTGF) and fibroblast growth factor (FGF)-2 in (i) stationary cultures, and (ii) mechanically active cultures subjected to cyclic strains of 12% at 0.2 Hz each min, 6 h/d, in a Flexercell FX-4000 Strain Unit. The following parameters were measured: cell number and viability by laser scanning confocal microscopy; cell proliferation with the MTS assay; the expression of a panel of 18 genes using real-time RT-PCR; matrix metalloproteinases (MMPs) 1-3, TIMP-1, CTGF and FGF-2 protein levels in supernatants from mechanically activated cultures with Enzyme-linked immunosorbent assays. Constructs from stationary cultures were also examined by scanning electron microscopy and immunostained for actin and vinculin. RESULTS: Although initially randomly distributed, the cells became organized into a bilayer by day 7; apoptotic cells remained constant at approximately 5% of the total. CTGF/FGF-2 stimulated cell proliferation in stationary cultures, but relative quantity values suggested modest effects on gene expression. Two transcription factors (RUNX2 and PPARG), two collagens (COL1A1, COL3A1), four MMPs (MMP-1-3, TIMP-1), TGFB1, RANKL, OPG and P4HB were detected by gel electrophoresis and Ct values < 35. In mechanically active cultures, with the exception of P4HB, TGFB1 and RANKL, each was upregulated at some point in the time scale, as was the synthesis of MMPs and TIMP-1. SOX9, MYOD, SP7, BMP2, BGLAP or COL2A1 were not detected in either stationary or mechanically active cultures. CONCLUSION: Three-dimensional tissue constructs provide additional complexity to monolayer culture systems, and suggest some of the assumptions regarding cell growth, differentiation and matrix turnover based on two-dimensional cultures may not apply to cells in three-dimensional matrices. Primarily developed as a transitional in vitro model for studying cell-cell and cell-matrix interactions in tooth support, the system is also suitable for investigating the pathogenesis of periodontal diseases, and importantly from the clinical point of view, in a mechanically active environment.

The effect of cyclic mechanical strain on the expression of adhesion-related genes by periodontal ligament cells in two-dimensional culture.

BACKGROUND AND OBJECTIVE: Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion-related genes by human periodontal ligament cells. MATERIAL AND METHODS: Cultured periodontal ligament cells were subjected to a cyclic in-plane tensile deformation of 12% for 5 s (0.2 Hz) every 90 s for 6-24 h in a Flexercell FX-4000 Strain Unit. The following parameters were measured: (i) cell viability by the MTT assay; (ii) caspase-3 and -7 activity; and (iii) the expression of 84 genes encoding adhesion-related molecules using real-time RT-PCR microarrays. RESULTS: Mechanical stress reduced the metabolic activity of deformed cells at 6 h, and caspase-3 and -7 activity at 6 and 12 h. Seventy-three genes were detected at critical threshold values < 35. Fifteen showed a significant change in relative expression: five cell adhesion molecules (ICAM1, ITGA3, ITGA6, ITGA8 and NCAM1), three collagen α-chains (COL6A1, COL8A1 and COL11A1), four MMPs (ADAMTS1, MMP8, MMP11 and MMP15), plus CTGF, SPP1 and VTN. Four genes were upregulated (ADAMTS1, CTGF, ICAM1 and SPP1) and 11 downregulated, with the range extending from a 1.76-fold induction of SPP1 at 12 h to a 2.49-fold downregulation of COL11A1 at 24 h. CONCLUSION: The study has identified several mechanoresponsive adhesion-related genes, and shown that onset of mechanical stress was followed by a transient reduction in overall cellular activity, including the expression of two apoptosis 'executioner' caspases.

Osteogenic gene expression by human periodontal ligament cells under cyclic tension.

The forces that orthodontic appliances apply to the teeth are transmitted through the periodontal ligament (PDL) to the supporting alveolar bone, leading to the deposition or resorption of bone, depending upon whether the tissues are exposed to a tensile or compressive mechanical strain. To evaluate the osteogenic potential of PDL cells, we applied a 12% uni-axial cyclic tensile strain to cultured human PDL cells and analyzed the differential expression of 78 genes implicated in osteoblast differentiation and bone metabolism by real-time RT-PCR array technology. Sixteen genes showed statistically significant changes in expression in response to alterations in their mechanical environment, including cell adhesion molecules and collagen fiber types. Genes linked to the osteoblast phenotype that were up-regulated included BMP2, BMP6, ALP, SOX9, MSX1, and VEGFA; those down-regulated included BMP4 and EGF. This study has expanded our knowledge of the transcriptional profile of PDL cells and identified several new mechanoresponsive genes.

On the transplantation, regeneration and induction of bone: the path to bone morphogenetic proteins and other skeletal growth factors.

The clinical and experimental transplantation of bone dates back to the seventeenth century and human allogeneic (homogeneic) bone has been successfully used as an alternative to autogenous bone since 1878, when Sir William Macewen reconstructed the right humerus of William Connell. This review describes how subsequent studies of bone transplantation led to the eventual discovery of a new family of secreted signalling molecules--the bone morphogenetic proteins (BMPs), and the realisation of the important role of polypeptide growth factors in mediating the growth, remodelling and regeneration of the skeleton. The development of suitable alternatives to both autogenous and allogeneic bone has been a goal of bone and biomaterials research for more than 30 years. The first requirement is a biocompatible, bioresorbable, osteoconductive framework supporting the ingrowth of host cells from the recipient bed. Many materials including collagen, calcium phosphate ceramics and synthetic polymers have been widely tested experimentally with varying success. The discovery of osteoinductive BMPs and their availability in recombinant human forms has given considerable impetus to the field. However, progress to date in engineering significant quantities of functional bone tissue in vivo has been disappointing; finding suitable carriers for BMPs has proven to be a greater challenge than expected. The dilemma for the clinician and the biotechnology industry, at present, is that, while recombinant human growth factors are readily available for clinical use, the lack of delivery systems that can adequately mimic both the physical properties and release kinetics of bone matrix remains a major handicap.

Remodeling the dentofacial skeleton: the biological basis of orthodontics and dentofacial orthopedics.

Orthodontic tooth movement is dependent upon the remodeling of the periodontal ligament and alveolar bone by mechanical means. Facial sutures are also fibrous articulations, and by remodeling these joints, one can alter the positional relationships of the bones of the facial skeleton. As might be expected from the structure and mobility of the temporomandibular joint (TMJ), this articulation is more resistant to mechanical deformation, and whether functional mandibular displacement can alter the growth of the condyle remains controversial. Clinical investigations of the effects of the Andresen activator and its variants on dentofacial growth suggest that the changes are essentially dento-alveolar. However, with the popularity of active functional appliances, such as the Herbst and twin-block based on 'jumping the bite', attention has focused on how they achieve dentofacial change. Animal experimentation enables informed decisions to be made regarding the effects of orthodontic treatment on the facial skeleton at the tissue, cellular, and molecular levels. Both rat and monkey models have been widely used, and the following conclusions can be drawn from such experimentation: (1) Facial sutures readily respond to changes in their mechanical environment; (2) anterior mandibular displacement in rat models does not increase the mitotic activity of cells within the condyle to be of clinical significance, and (3) mandibular displacement in non-human primates initiates remodeling activity within the TMJ and can alter condylar growth direction. This last conclusion may have clinical utility, particularly in an actively growing child.

The evolution of plastic and maxillofacial surgery in the twentieth century: the Dunedin connection.

At the outbreak of the Second World War there were four full-time plastic surgeons in the United Kingdom: Gillies, Kilner, McIndoe and Mowlem, known universally as the 'big four.' Three were from New Zealand, two had been born in Dunedin (Harold Gillies and Archibald McIndoe) and two (McIndoe and Rainsford Mowlem) had studied medicine in Dunedin at the University of Otago. The story of Gillies and McIndoe is well known to many. Perhaps less well known are the contributions of Mowlem and Henry Pickerill, another surgeon with Dunedin connections, and how the futures of these men were shaped by a small 19th century Scottish settlement at the bottom of the South Island of New Zealand.

Mechanical deformation inhibits IL-10 and stimulates IL-12 production by mouse calvarial osteoblasts in vitro.

The skeleton is continuously remodelled throughout life, a process that is orchestrated by cells of the osteoblast lineage. Remodelling involves a complex network of cell-cell signalling involving systemic hormones, locally produced cytokines, growth factors and the mechanical environment of the cells. Here, we report on the effect of mechanically-induced strain on the synthesis by mouse calvarial osteoblasts in monolayer culture of IL-10 and IL-12, two cytokines that inhibit osteoclast formation in bone marrow cultures; IL-10 also suppresses osteoblast differentiation suggesting a role for both cytokines in bone physiology. A tensile strain was applied to the cells intermittently for 6s, every 90s, for 2-96h. After 2-h culture, supernatants from deformed cells contained significantly less IL-10 than control cultures. In contrast, mechanical deformation had a stimulatory effect on IL-12 synthesis; however, by 48h both had returned to control levels. These data suggest that IL-10 and IL-12 can be added to the growing list of mechanical stress-responsive genes. The down-regulation of IL-10 and stimulation of IL-12 further suggests that the initial response of the cells to mechanical deformation was an osteogenic one.

The effects of serine proteinase inhibitors on bone resorption in vitro.

The aims of this study were to identify the role and sites of action of serine proteinases (SPs) in bone resorption, a process which involves a cascade of events, the central step of which is the removal of bone matrix by osteoclasts (OCs). This resorbing activity, however, is also determined by recruitment of new OCs to future resorption sites and removal of the osteoid layer by osteoblasts (OBs), which enables OCs to gain access to the underlying mineralized bone. The resorption systems we have studied consisted of (i) neonatal calvarial explants, (ii) isolated OCs cultured on ivory slices, (iii) mouse OBs cultured on either radiolabelled type I collagen films or bone-like matrix, (iv) bone marrow cultures to assess OC formation and (v) 17-day-old fetal mouse metatarsal bone rudiments to assess OC migration and fusion. Two separate SP inhibitors, aprotinin and alpha(2)-antiplasmin dose-dependently inhibited (45)Ca release from neonatal calvarial explants: aprotinin (10(-6) M) was the most effective SP inhibitor, producing a maximum inhibitory effect of 55.9%. Neither of the SP inhibitors influenced either OC formation or OC resorptive activity. In contrast, each SP inhibitor dose-dependently inhibited OB-mediated degradation of both type I collagen fibrils and non-mineralized bone matrix. In 17-day-old metatarsal explants aprotinin produced a 55% reduction in the migration of OCs from the periosteum to the mineralized matrix after 3 days in culture but after 6 days in culture aprotinin was without effect on OC migration. Primary mouse osteoblasts expressed mRNA for urokinase type plasminogen activator (uPA), tIssue type plasminogen activator (tPA), the type I receptor for uPA, plasminogen activator inhibitor types I and II and the broad spectrum serine proteinase inhibitor, protease nexin I. In situ hybridization demonstrated expression of tPA and uPA in osteoclasts disaggregated from 6-day-old mouse long bones. We propose that the regulation of these various enzyme systems within bone tIssue determines the sites where bone resorption will be initiated.

Human breast-cancer cells stimulate the fusion, migration and resorptive activity of osteoclasts in bone explants.

A central event in bone resorption is the recruitment of osteoclasts to future resorption sites. Breast-cancer cells invariably metastasise to the skeleton and induce extensive bone destruction by osteoclasts. However, our understanding of the mechanisms by which cancer cells interact with osteoclasts remains unclear. Consequently, we compared the effects of conditioned medium (CM) from 2 human breast-cancer cell lines, MB-MDA-231 and MCF-7, with those of a normal human breast epithelial cell line, HME, on osteoclastic fusion, resorptive activity and migration from the periosteum to the developing marrow cavity of fetal mouse metatarsals in culture. Osteoclastic resorptive activity was assessed by pre-labelling 17-day-old fetal metatarsal explants with 45Ca, whilst fusion and migration were monitored by histomorphometry and osteoclasts were identified by their tartrate-resistant acid phosphatase activity. CM from TPA-stimulated breast-cancer cell lines produced a significant increase in osteoclastic resorptive activity, whilst the normal breast cell line produced a minimal increase. The breast-cancer cell lines also stimulated osteoclastic fusion and migration in the metatarsal explants, but the normal breast cell line was without effect. The stimulatory effect of CM from MDA-MB-231 cells on osteoclastic fusion, but not migration, was partially inhibited by preventing prostaglandin and leukotriene synthesis by cells within the bone explants. In contrast, a synthetic matrix metalloproteinase (MMP) inhibitor, but not a cysteine proteinase inhibitor, prevented the migration of osteoclasts to the calcified centre of the metatarsal explants in response to CM from MDA-MB-231 cells. MDA-MB-231 cells also induced an increase in the expression of MMP-9 by migrating osteoclasts. Fractionation of the TPA-stimulated breast cancer cell CM established that the resorptive activity was associated with factors of m.w. >3 kDa. We determined by immuno-assay that human breast-cancer cells secrete parathyroid hormone-related protein (PTH-rP), tumour necrosis factor-alpha (TNF-alpha) and interleukins (ILs) 6 and 11. Neutralizing experiments with human antibodies to these cytokines established that PTH-rP and TNF-alpha production by MDA-MB-231 cells were responsible for mediating their effects on osteoclastic migration and ultimately bone resorption in the metatarsal explants.

Autocrine signals promote osteoblast survival in culture.

We have studied the survival requirements of osteoblasts to test the hypothesis that osteoblasts undergo programmed cell death (PCD) or apoptosis unless they are continuously signalled by other cells not to do so. Osteoblasts survived for 6 days in culture at high cell density in the absence of other cell types, serum or exogenous proteins, but they died with the morphological features of apoptosis in these conditions at low cell density. Osteoblast survival was enhanced during the first 2 days of culture by the addition of the sulphydryl compound, cysteine to the culture medium which was converted intracellularly to the antioxidant glutathione. Catalase, an enzyme decomposing hydrogen peroxide, also protected the cells, whereas superoxide dismutase had no effect. Therefore, osteoblasts in culture are sensitive to toxic compounds derived from molecular oxygen, i.e. hydroxyl radicals or hydrogen peroxide spontaneously generated in CMRL medium containing ascorbate and ferrous ions. Conditioned medium from high density cultures prevented osteoblast apoptosis in low density cultures, as long as antioxidants were also present. The enhancing effect of conditioned medium on osteoblast survival was prevented by neutralizing antibodies to insulin-like growth factor-I (IGF-I) and IGF-II but not by antibodies to either platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). These results suggest that in addition to regulating cell growth and differentiation, IGF-I and IGF-II also function as survival factors for osteoblasts. Our data also indicate that antioxidants are required for osteoblast survival and that they enhance growth factor mediated osteoblast survival.

Stromelysin (MMP-3) synthesis is up-regulated in estrogen-deficient mouse osteoblasts in vivo and in vitro.

Sex steroids are important regulators of bone cell function and osteoblast-derived matrix metalloproteinases (MMPs) are key mediators of bone resorption during the initial stage of osteoid removal prior to osteoclast attachment. To investigate the mechanism of bone loss following estrogen deficiency, we examined the effects of estrogen on osteoblast synthesis of MMPs and tissue inhibitor of metalloproteinases (TIMPs). Immunolocalization in mouse bone samples ex vivo and primary mouse osteoblast (MOB) cultures was used to document the synthesis of mouse interstitial collagenase (MMP-13), stromelysin-1 (MMP-3), gelatinase-A (MMP-2), and gelatinase-B (MMP-9). Endosteal bone lining cells from distal femoral head and lumbar vertebral body showed an increase in the pattern of synthesis of stromelysin-1 following ovariectomy, compared with sham-operated controls; the synthesis of other MMPs was unaffected. The expression of all classes of MMPs and TIMP-1 and TIMP-2 by MOB in culture was demonstrated by reverse transcriptase-polymerase chain reaction. Following the withdrawal of 17beta-estradiol, MOB cultures showed a significant increase in the number of cells synthesizing stromelysin-1; this effect was enhanced by stimulation with either interleukin-1 or interleukin-6. Northern blot analysis showed only a slight increase in stromelysin-1 mRNA message following the withdrawal of 17beta-estradiol. Our data show an unexpected up-regulation of stromelysin-1 synthesis by osteoblasts both in vivo and in vitro following estrogen withdrawal. Although this effect was not reflected in a significant change in stromelysin-1 mRNA expression in vitro, there is evidence to suggest a role for this enzyme in the early stages of bone loss during the pathogenesis of osteoporosis.

G-protein signalling pathways and oestrogen: a role of balanced maintenance in osteoblasts.

Oestrogen (E2) is an important regulator of bone cell function and alterations in oestrogen levels may cause abnormal bone metabolism in vivo. In this study we examined the long term effects of 17beta-oestradiol (17beta-E2) on G-proteins and the secondary signalling pathways of phospholipase C (PLC), cyclic adenosine monophosphate (cAMP), and 1,4,5-inositol triphosphate (IP3). Cells from neonatal mouse calvariae were cultured in phenol red-free RPMI 1640 medium supplemented with charcoal stripped foetal calf serum for 192 h with either oestrogen (10(-8) M), or oestrogen withdrawal after 48 h. Cultures were stimulated for the final 48 h with IL-6 (10(-10) M), or left unstimulated. Western blot analysis was undertaken on osteoblast membrane preparations obtained by 10 mM Tris-HCl, 0.1 mM EDTA pH 7.8 and centrifugation at 40,000 x g for 2 h. For cAMP study, cells were stimulated with IL-6 for either 15 min or 30 min. Intracellular cAMP was extracted from cells and measured by ELISA methodology. For the IP3 assay, cells were stimulated with IL-6 for 20 s and IP3 levels measured using radioimmunoassay. The blots revealed increased levels of Gialpha-, and Gqalpha-proteins with oestrogen withdrawal and IL-6 stimulation. This was in comparison to cells which were unstimulated, or stimulated with IL-6 with continuous 17beta-E2, or IL-6 alone. Gsalpha expression decreased with oestrogen withdrawal compared to the control. Limited amounts of Gialpha-, Gsalpha-, and Gqalpha-proteins were identified with continuous 17beta-E2. The levels of PLC isoforms PLCbeta1-2 were not affected by the differing oestrogen conditions. The cAMP production induced by IL-6 stimulation for 30 min and withdrawal of 17beta-E2 was lower and significantly different compared to the control study (P<0.05). Also IL-6 activation with continuous oestradiol increased cAMP levels and was significantly different from the control cells (P<0.01). However, 17beta-E2 had no effect on the formation of intracellular IP3, although IL-6 significantly lowered IP3 levels in all the groups compared to the control (P<0.01). These results suggest that oestrogen modulates the signal transduction pathways of G-protein molecules, and the secondary pathways of cAMP in mouse osteoblast-like cells.

An analysis of Ca2+ release by DGEA: mobilization of two functionally distinct internal stores in Saos-2 cells.

Osteoblasts can be activated by their collagen matrix and in particular the DGEA peptide motif. We have reported that DGEA is able to activate Ca2+ signaling pathways in the human osteoblast-like cell line, Saos-2, by a tyrosine kinase-dependent pathway (T. J. McCann, W. T. Mason, M. C. Meikle, and F. McDonald. Matrix Biol. 16: 271-280, 1997). In the present study, we show that this activity is due to coupling of the signal to intracellular Ca2+ stores, since the DGEA action is not blocked by La3+ but is lost when Ca2+ stores are depleted with 2 microM and blocked by 10 microM ryanodine. The activated stores also differ functionally from those activated by thrombin, as blockade with U-73122 obstructs only thrombin-activated Ca2+ release. We have shown that the DGEA activity was not due to its high-charge density, since the two acidic residues can be substituted with their uncharged homologues (asparagine and glutamine) without significant loss of activity. This was in turn measured by an adhesion assay that also demonstrated this level of specificity. Furthermore, by constructing DGEA bound to FITC, we have shown that DGEA binding was dependent on divalent cations. We have also demonstrated that an intact actin cytoskeleton is not required for Ca2+ activation by inhibiting actin polymerization with the addition of cytochalasin B. These data strengthen the argument that collagen has a significant role in regulating osteoblast function via this peptide motif.

The cellular actions of interleukin-11 on bone resorption in vitro.

The pleiotropic cytokine interleukin-11 (IL-11) stimulates osteoclast formation in vitro, but it is not known whether it influences other steps in the bone-resorptive cascade. Using a variety of in vitro model systems for studying bone resorption we have investigated the effects of IL-11 on 1) osteoclast formation, fusion, migration, and activity; and 2) osteoblast-mediated osteoid degradation. The involvement of matrix metalloproteinases (MMPs) and products of arachidonic acid metabolism in IL-11-mediated resorption were also assessed. We first examined the bone-resorptive effects of IL-11 by assessing 45Ca release from neonatal mouse calvarial bones. IL-11 dose-dependently stimulated bone resorption with an EC50 of 10(-10) M. The kinetics of IL-11-mediated 45Ca release demonstrated that it was without effect for the first 48 h of culture, but by 96 h, it stimulated 45Ca release to the same level as that produced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (a hormone that stimulates osteoclast formation and activity). IL-11 also produced a dose-dependent increase in osteoblast-mediated type I collagen degradation with a maximum of 58.0 +/- 6.2% at 5 x 10(-9) M; this effect of IL-11 was less than that produced by 1,25-(OH)2D3 (76.5 +/- 7.1%) and was prevented by an inhibitor of MMPs, but not those blocking arachidonic acid metabolism. We then tested the effects of IL-11 on isolated mouse osteoclasts cultured on ivory slices in the presence and absence of primary mouse osteoblasts. IL-11 had no effect on isolated osteoclast activity even in coculture with primary osteoblasts. We then examined the effects of IL-11 on the formation of osteoclast-like multinucleate cells in mouse bone marrow cultures and the resorptive activity of such cultures using ivory as a substrate. IL-11 dose-dependently increased 1) the number of tartrate-resistant acid phosphatase-positive osteoclast-like multinucleate cells and 2) the surface area of lacunar resorption, although the effects were less than that of 1,25-(OH)2D3. The effect of IL-11 on bone marrow lacunar resorption was prevented by a combination of inhibitors of 5-lipoxygenase and cyclooxygenase. In 17-day-old metatarsal bones, IL-11 prevented the migration of (pre)osteoclasts to future resorption sites, whereas their fusion was unaffected. These results provide strong evidence that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblast-mediated osteoid degradation rather than stimulating osteoclast migration and activity. Our data also suggest that the stimulatory effects of IL-11 involve both MMPs and products of arachidonic acid metabolism.

Multiple extracellular signals promote osteoblast survival and apoptosis.

Programed cell death (PCD) or apoptosis is a naturally occurring cell suicide pathway induced in a variety of cell types. In many cases, PCD apparently arises as a result of competition for limiting amounts of survival signals. In this study, we have investigated the potential role of growth factors (GF), cytokines, and osteotropic hormones on osteoblast survival in vitro. Our results indicate that in the absence of any of these factors, osteoblasts rapidly undergo PCD, as determined by cell morphology, mitochondrial function, and nuclei fragmentation. Osteoblast survival was promoted by insulin-like growth factor I (IGF-I), IGF-II, insulin, and basic fibroblast growth factor (bFGF). Platelet-derived growth factor had no effect on osteoblast survival, but this GF potentiated the survival-promoting effects of IGF-I, IGF-II, and insulin. A similar effect occurred when bFGF was added in combination with either of the IGFs or insulin. The effects of the IGFs were blocked by alphaIR-3, an antibody to the type I IGF receptor, whereas the effects of insulin were only partially blocked. This antibody blocked the potentiating effects of platelet-derived growth factor on IGF-I-mediated osteoblast survival, but only partially blocked those of bFGF. Although a 100% survival of osteoblasts was seen in the presence of 2% FCS, the highest level attained by any of the above GF combinations was approximately 75%. The monocyte-derived factor, tumor necrosis factor-alpha (TNF alpha) was the only agent that enhanced PCD in this study. These results suggest that osteoblast survival is promoted by those GFs sequestrated in bone matrix and that the type I, but not the type II, IGF receptor is involved in the response. Our data also indicate that other unidentified GFs or components of the extracellular matrix may be involved in promoting osteoblast survival and that TNF alpha may abrogate their effects in vivo. We propose that these GFs may be released from bone matrix during phases of bone resorption and promote osteoblast survival, thereby playing an important role in bone remodeling, and that PCD induced by TNF alpha may contribute to the bone loss in inflammatory bone disease.

The functional balance of metalloproteinases and inhibitors in tissue degradation: relevance to oral pathologies.

Members of the family of matrix metalloproteinases (MMPs) are key enzymes in normal and pathological tissue remodelling. They function at neutral pH and can digest synergistically all the macromolecules of the extracellular matrix. Biochemical and cloning studies indicate that there are three major groups: the specific collagenases cleave interstitial collagens; the gelatinases degrade other types of collagen and act synergistically with collagenases by degrading denatured collagens (gelatins); and the stromelysins which have broader specificity and can degrade basement membrane collagens as well as proteoglycans and matrix glycoproteins. Others in the family, but not in the major groups, are matrilysin, metallo-elastase, and several recently cloned membrane-bound metalloproteinases. Naturally occurring inhibitors, TIMPs (tissue inhibitors of metalloproteinases), are important controlling factors in the actions of MMPs, and tissue destruction in disease processes often correlates with an imbalance of MMPs over TIMPs. The relevance of recent molecular research to periodontal diseases is discussed.

A collagen peptide motif activates tyrosine kinase-dependent calcium signalling pathways in human osteoblast-like cells.

A collagen peptide motif (DGEA) which is a putative alpha 2 beta 1 integrin binding site was examined for its ability to activate Ca2+ signalling pathways in the human osteoblast-like cell line SaOS-2. We show that these cells express both alpha 2 beta 1 integrin subunits (by immunocytochemistry) and that an anti-beta 1 monoclonal antibody (DF5) mobilizes Ca2+ in these cells. DGEA elevated intracellular Ca2+ in fura-2-loaded cells, in a concentration- and sequence-dependent fashion, with an EC50 of 250 microM. The tyrosine kinase inhibitor herbimycin A reduced the number of cells responding to DGEA and to transforming growth factor alpha. Thrombin also stimulated a rise in intracellular Ca2+, but the number of cells responding was not reduced by herbimycin A. The DGEA response was dependent on extracellular Ca2+, but was not due to Ca2+ influx, since it was blocked by thapsigargin and not by lanthanum. Using three different anti-alpha 2 monoclonal antibodies, we were unable to show that the DGEA-induced Ca2+ signal was mediated by the alpha 2 beta 1 integrin. In summary, the DGEA collagen motif does appear to activate receptor-mediated Ca2+ signalling events in SaOS-2 cells, in a divalent cation-dependent manner, but we were unable to demonstrate a role for alpha 2 beta 1 integrin in this response.

Identification of matrix metalloproteinases and their inhibitor in the articular disc of the craniomandibular joint of the rabbit.

Connective tissue cells synthesize and secrete matrix metalloproteinases (MMPs), a family of matrix-degrading enzymes (comprising collagenases, gelatinases and stromelysins), which are capable of degrading all the constituent molecules of connective tissues at physiological pH. This investigation documents the synthesis and distribution of MMPs and their inhibitor TIMP-1 (tissue inhibitor of metalloproteinases-1) in the developing articular disc of the craniomandibular joint of the rabbit using indirect immunofluorescence microscopy. Cells of the disc synthesised all three classes of MMPs as well as TIMP-1 in all regions of the disc at all stages examined. MMPs and TIMP-1 were detected as bright intracellular accumulations probably within Golgi vesicles and as occasional diffuse, matrix-bound deposits. These results suggest that MMP-mediated matrix remodelling is a prominent feature of growth in craniomandibular joint disc.