Biomedical subjects

M A Horton

Publications and source records attributed to M A Horton.

At least 19 recordsLinked to original sources

PubMed2006

Integrated confocal and scanning probe microscopy for biomedical research.

Atomic force microscopy (AFM) continues to be developed, not only in design, but also in application. The new focus of using AFM is changing from pure material to biomedical studies. More frequently, it is being used in combination with other optical imaging methods, such as confocal laser scanning microscopy (CLSM) and fluorescent imaging, to provide a more comprehensive understanding of biological systems. To date, AFM has been used increasingly as a precise micromanipulator, probing and altering the mechanobiological characteristics of living cells and tissues, in order to examine specific, receptor-ligand interactions, material properties, and cell behavior. In this review, we discuss the development of this new hybrid AFM, current research, and potential applications in diagnosis and the detection of disease.

Animals↗

PubMed2006

Skeletal tissues as nanomaterials.

Collagen is the most abundant protein in the body and, though the fibre-forming collagens have a 'common' structure, it is adapted to perform a large range of functions-from the differing mechanical needs of tendon versus bone to forming a transparent support structure in the cornea. This perfidy also suggests that collagen could form a generic basis for a range of scaffold needs for tissue engineering or medical device coating applications. We at the London Centre for Nanotechnology--a joint venture between University College London and Imperial College--are taking a bottom-up approach having decided that many of the 'accepted dogmas' of collagen biology may not be quite as soundly based as currently held. We are using several of the tools of 'hard' nanotechnology--such as atomic force microscopy--to re-examine collagen structure with the longer term aim of using such information to design materials with appropriate physical attributes. Examples of our current research on mineralised and soft tissue collagens are presented.

Animals↗

PubMed2006

Hypoxia induces giant osteoclast formation and extensive bone resorption in the cat.

Dental disease due to osteoclast (OC) overactivity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline odontoclastic resorptive lesions (FORL) involve extensive resorption of the tooth, leaving it liable to root fracture and subsequent loss. The etiopathogenesis of FORL remains unclear. Here, we explore the hypothesis that FORL is associated with hypoxia in the oral microenvironment, leading to increased OC activity. To investigate this, we developed a method of generating OCs from cat blood. Reducing O2 from 20% to 2% increased the mean area of OC eightfold from 0.01 to 0.08 mm2. In hypoxic cultures, very large OCs containing several hundred nuclei were evident (reaching a maximum size of approximately 14 mm2). Cultures exposed to 2% O2 exhibited an increase of approximately 13-fold in the area of bone slices covered by resorption lacunae. In line with this finding, there was a significant increase in cells differentiating under hypoxic conditions, reflected in increased expression of cathepsin K and proton pump enzymes. In conclusion, these results demonstrate that oxygen tension is a major regulator of OC formation in the cat. However, in this species, hypoxia induces the formation of "giant" OCs, which can be so large as to be visible with the naked eye and yet also actively resorb. This suggests that local hypoxia is likely to play a key role in the pathogenesis of FORL and other inflammatory conditions that are associated with bone resorption in cats.

Animals↗

PubMed2006

Analysis of the surface characteristics and mineralization status of feline teeth using scanning electron microscopy.

External resorption of teeth by odontoclasts is a common condition of unknown origin affecting domestic cats. Odontoclastic resorptive lesions involve the enamel cementum junction (ECJ, cervix) and root surface, leading to extensive loss of enamel, dentine and cementum. This study was undertaken in order to determine whether features of the surface anatomy and mineralization of feline teeth could explain why odontoclastic resorptive lesions are so prevalent in this species. Backscattered electron scanning electron microscopy was used to study enamel, cementum and dentine in non-resorbed, undemineralized teeth from adult cats. Analysis of the ECJ revealed thin enamel and cementum and exposed dentine at this site. Furthermore, enamel mineralization decreased from the crown tip to the ECJ, and dentine mineralization was lowest at the ECJ and cervical root. Analysis of cementum revealed variations in the organization and composition of fibres between the cervical, mid- and apical root although no significant differences in mineralization of cementum were detected between different regions of the root. Reparative patches associated with resorption of cementum by odontoclasts and repair by cementoblasts were present on the root surface. In conclusion, results suggest that the ECJ and cervical dentine could be at a greater risk of destruction by odontoclasts compared with other regions of the tooth. The relationship of these features to the development and progression of resorption now requires further examination.

Animals↗

PubMed2005

Mineralised tissues as nanomaterials: analysis by atomic force microscopy.

Mineralised tissues, such as bone, consist of two material phases: collagen protein fibrils that form the structural models upon which the mineral, calcium hydroxyapatite, is subsequently deposited. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair, and matrix proteins are replaced by the synthetic activity of osteoblasts and then calcify. The resolution of atomic force microscopy and use of unmodified, fully calcified samples has enabled the imaging of the overall bone and dentine structure, including collagen and mineral phases. Mineral crystals, in the diameter size range of 225 nm up to 1.4 microm, were found in unmodified bone and dentine respectively. D-banded collagen is observed in dentine after acid treatment and in bone after osteoclast-mediated matrix resorption; axial periodicity values of approximately 67 and 69 nm are observed, respectively. These experimental approaches have enabled the structure of mineralised tissues to be examined in native samples and will facilitate the study of bone structure in important clinical disorders of the skeleton, such as osteoporosis.

Animals↗

PubMed2004

Recapitulation of the parathyroid hormone-related peptide-Indian hedgehog pathway in the regenerating deer antler.

Parathyroid hormone (PTH)-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR) play an essential role in controlling growth plate development. The aim of the present study was to use the deer antler as a model to determine whether PTHrP and PPR may also have a function in regulating cartilage and bone regeneration in an adult mammal. Antlers are the only mammalian appendages that are able to undergo repeated cycles of regeneration, and their growth from a blastema involves a modified endochondral process. Immunohistochemistry was used to establish sites of localization of PTHrP and PPR in antlers at different stages of development. The pattern of Indian Hedgehog (IHH) and transforming growth factor-beta1 (TGF beta1) distribution was also investigated, because PTHrP expression in the developing limb is regulated by IHH and during embryonic growth plate formation TGF beta1 acts upstream of PTHrP to regulate the rate of chondrocyte differentiation. In the antler blastema (<10 days of development), PTHrP, PPR, and TGF beta1 were localized in epidermis, dermis, regenerating epithelium, and in mesenchymal cells but IHH expression was not detected. In the rapidly growing antler (weeks 4-8 of development), PTHrP, PPR, and TGF beta1 were localized in skin, perichondrium, undifferentiated mesenchyme, recently differentiated chondrocytes, and in perivascular cells in cartilage but not in fully differentiated hyperytrophic chondrocytes. IHH was restricted to recently differentiated chondrocytes and to perivascular cells in cartilage. In mineralized cartilage and bone, PTHrP, PPR, IHH, and TGF beta1 were immunolocalized in perivascular cells and differentiated osteoblasts. PTHrP and PPR were also present in the periosteum. TGF beta1 in vitro stimulated PTHrP synthesis by cells from blastema, perichondrium, and cartilage. The findings of this study suggest that molecules which regulate embryonic skeletal development and postnatal epiphyseal growth may also control blastema formation, chondrogenesis, and bone formation in the regenerating deer antler. This finding is further evidence that developmental signaling pathways are recapitulated during adult mammalian bone regeneration.

Animals↗

PubMed2004

A comparison of methods for measuring serum and urinary markers of bone metabolism in cats.

Biochemical markers of bone cell activity have recently been shown to be useful for monitoring skeletal health in domestic animals, including dogs and horses. The aim of this study was to evaluate a number of biochemical assays, originally developed for use in humans, for their ability to measure indicators of bone cell activity in serum and urine of normal cats over a range of ages. Bone alkaline phosphatase (BAP), a marker of bone formation, was measured in serum using wheatgerm lectin precipitation (WGL) and by ELISA. The curve derived from serial dilution of feline serum was parallel with the ELISA standard curve, indicating species cross-reactivity, and there was a significant relationship between assays (rs = 0.97, P < 0.001). Deoxypyridinoline (DPD), a marker of bone resorption, was measured in its total form in urine by HPLC and ELISA, and in its free form in serum and urine by ELISA. The dilution curve for free DPD in urine showed parallelism with the assay standard curve; however, the curves for total DPD in urine and serum did not. A significant relationship was established between total urinary DPD (HPLC) with total serum DPD (rs = 0.69, P < 0.001), and with free urinary DPD (rs = 0.95, P < 0.001) concentrations. Carboxy-terminal telopeptide of type I collagen (CTX) concentration, another marker of bone resorption, was measured in serum and urine by ELISA, and there was a significant relationship between assays (rs = 0.82, P < 0.001). CTX could not be measured reliably using an auto-analysis method. A significant relationship was established between total urinary DPD (HPLC) with serum CTX (rs = 0.59, P < 0.05), and urinary CTX (rs = 0.65, P < 0.001) concentrations. BAP (ELISA and WGL), total urinary DPD (HPLC), urinary CTX (ELISA), and serum CTX (ELISA) concentrations were significantly inversely correlated with age (rs = -0.66, -0.88, -0.61, -0.70, and -0.51, P < 0.05 respectively). Cats under two years of age had significantly higher BAP, total urinary DPD (HPLC), and urinary CTX concentrations compared to older cats. In conclusion, this study has shown that a number of commercially available assays provide reliable methods for non-invasively monitoring bone cell activity in cats and has shown that bone turnover decreases within the first two years of life, until complete skeletal maturity is attained. Future studies can now be directed at evaluating the potential clinical application of these methods.

Alanine Transaminase↗

PubMed2002

A feline assay using osteoclasts generated in vitro from peripheral blood for screening anti-resorptive agents.

Musculo-skeletal diseases are a major cause of pain and suffering in cats and several conditions involve increased bone resorption by osteoclasts. However, little is known about the biology of these cells in the cat. In this study we established a method to generate feline osteoclasts from blood mononuclear cells stimulated by macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Cultured osteoclasts are multinucleated, express tartrate resistant acid phosphatase (TRAP), form F-actin rings and resorb bone. They express alpha(v)beta3 vitronectin receptor and osteoclast enzymes, cathepsin K and MMP9; the myeloid antigen, CD18, and the megakaryocyte/platelet integrin, CD41, are absent. This phenotype is typical of osteoclasts from other species. Three resorption inhibitors were examined for activity against feline osteoclasts. Calcitonin, bisphosphonate and RGD integrin inhibitory peptide all reduced bone resorption at doses similar to those efficacious in rabbit or human. We conclude that blood-derived osteoclast cultures are a suitable in vitro system for assessing the ability of drugs to inhibit bone resorption in domestic cats.

Acid Phosphatase↗

PubMed2002

Macrophage colony-stimulating factor and receptor activator NF-kappaB ligand fail to rescue osteoclast-poor human malignant infantile osteopetrosis in vitro.

Malignant infantile osteopetrosis (MIOP) is a disease characterized by failure in bone resorption, leading to dense fragile bones with a severely reduced bone marrow cavity. Normal or increased numbers of osteoclasts are present in the common variant of this disease; in such cases, the defect is likely to be inherent to the mature osteoclast and can be cured by bone marrow transplantation. However, MIOP also results from failure of osteoclast formation (osteoclast-poor MIOP). We report on two infants diagnosed with osteoclast-poor MIOP and utilize modern cell culture techniques to investigate the pathogenesis of disease. Peripheral blood mononuclear cells (PBMNCs) from these children were cultured in the presence of recombinant macrophage colony-stimulating factor and receptor activator NF-kappaB ligand for up to 3 weeks. Control cultures included PBMNCs from age-matched children, one of whom had an osteoclast-rich form of MIOP. Formation of osteoclasts (cells coexpressing vitronectin receptor and F-actin rings) occurred in all the control cultures. Significant bone resorption occurred in cultures from PBMNCs of the healthy individuals, whereas almost no bone resorption occurred in the osteoclast-rich MIOP cultures. In contrast, PBMNC cultures from the osteoclast-poor MIOP child formed only very occasional small F-actin ring-positive osteoclasts, which coexpressed vitronectin receptor and cathepsin K, and extremely rare foci of resorption. Because neither macrophage colony-stimulating factor nor receptor activator NF-kappaB ligand rescued the defect in osteoclast differentiation in the two cases of osteoclast-poor MIOP in vitro, there would be little benefit in treating these children with either of these recombinant proteins. Finally, these results demonstrate that this experimental culture model replicates the human osteopetrosis phenotype observed in vivo and should prove useful in analyzing the pathogenesis of the various forms of MIOP.

Actins↗

PubMed2002

Nuclear localization of type I parathyroid hormone/parathyroid hormone-related protein receptors in deer antler osteoclasts: evidence for parathyroid hormone-related protein and receptor activator of NF-kappaB-dependent effects on osteoclast formation in regenerating mammalian bone.

Parathyroid hormone-related protein (PTHrP) is not required for osteoclastogenesis during embryonic development; however, after birth it has been shown to regulate osteoclast formation during tooth eruption. Our study explores the hypothesis that PTHrP also may regulate osteoclast differentiation in the regenerating skeletal tissues of deer antlers, bones capable of complete regeneration. Osteoclast-like multinucleated cells (MNCs) formed spontaneously in micromass cultures derived from antler cartilage and these cells had the phenotypic characteristics of osteoclasts. PTHrP and receptor activator of NF-kappaB ligand (RANKL) stimulated antler osteoclast formation although the effect of RANKL was less marked than that of PTHrP. The addition of osteoprotegerin (OPG) only partially decreased (by approximately 65%) the number of osteoclasts in PTHrP-treated cultures. To determine whether PTHrP also potentially could have direct effects on antler osteoclasts, we studied, by confocal microscopy, the expression of the type I PTH/PTHrP receptor (PTH1R) in MNCs cultured on glass and found the receptor protein to have a nuclear localization. In situ hybridization showed that antler MNCs also expressed PTH1R and PTHrP messenger RNAs (mRNAs). PTHrP was immunolocalized in MNCs cultured on glass but was undetectable in cells resorbing a dentine substrate. In tissue sections of antler cartilage, PTHrP and PTH1R were expressed in vitronectin receptor-positive (VNR+) osteoclast-like cells localized in the perivascular stroma. Thus, these data show that PTHrP plays a role in the regulation of osteoclast differentiation in regenerating skeletal tissues and that PTHrP can have effects on osteoclastogenesis that are independent of RANKL synthesis. Ours is the first study to describe the expression of the type I PTH/PTHrP receptor in mammalian osteoclasts at a protein and mRNA level, which indicates that PTHrP also may have a direct effect on osteoclasts. This also is the first study to show a nuclear localization of the PTHIR in cells of the osteoclast lineage, although the functional significance of this observation has yet to be established.

Animals↗

PubMed2001

Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages.

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass approximately 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.

Alkyl and Aryl Transferases↗

PubMed2001

Atomic force microscopy can be used to mechanically stimulate osteoblasts and evaluate cellular strain distributions.

In this study, atomic force microscopy (AFM) was used to mechanically stimulate primary osteoblasts. In response to mechanical force applied by the AFM, the indented cell increased its intracellular calcium concentration. The material properties of the cell could be estimated and the membrane strains calculated. We proceeded to validate this technique experimentally and a 20% error was found between the predicted and the measured diameter of indentation. We also determined the strain distributions within the cell that result from AFM indentation using a simple finite element model. This enabled us to formulate hypotheses as to the mechanism through which cells may sense the applied mechanical strains. Finally, we report the effect of the Poisson ratio and the cell thickness on the strain distributions. Varying the Poisson ratio did not change the order of magnitude of the strains; whereas the cellular thickness dramatically changed the order of magnitude of the cellular strains. We conclude that AFM can be used for controlled mechanical stimulation of osteoblasts and that cellular strain distributions can be computed with a good accuracy when the cell is indented in its highest part.

Animals↗

PubMed2001

Transforming growth factor-beta1 (TGF-beta) stimulates the osteoclast-forming potential of peripheral blood hematopoietic precursors in a lymphocyte-rich microenvironment.

Osteoclasts are generated from peripheral blood mononuclear cells (PBMNCs) in the presence of soluble receptor activator of NFkappaB ligand (sRANKL) and macrophage colony-stimulating factor (M-CSF). We show that human osteoclast formation is enhanced when PBMNCs are cultured in the presence of transforming growth factor (TGF)-beta and M-CSF prior to the addition of sRANKL. The effect was only observed in the presence of a nonadherent lymphocyte PBMNC fraction. Osteoclast formation was enhanced to a level equivalent to that induced by TGF-beta when nonadherent PBMNC fraction was removed from the cultures, prior to RANKL treatment. These data suggest that TGF-beta enhances osteoclast formation by abrogating the suppressive effect of the nonadherent PBMNCs, thereby maintaining the osteoclast-forming potential of the osteoclast precursor population. TGF-beta was without effect on proliferation of the adherent PBMNCs and did not stimulate osteoclast size or modify their immunophenotype. The effect was not mediated through prostaglandin synthesis. These results indicate that the microenvironment encountered by the osteoclast precursor prior to RANKL exposure contributes significantly to the regulation of osteoclast formation. Furthermore, the data emphasize that the effect of TGF-beta is determined by the cytokine milieu of the microenvironment and/or the state of activation of the cell being targeted by TGF-beta; thus, the effect of TGF-beta is context-dependent.

Carrier Proteins↗

PubMed2001

Interleukin-4 and interleukin-13: bidirectional effects on human osteoclast formation.

Osteoclasts are cells that resorb bone; they derive from macrophage colony-stimulating factor (M-CSF)-dependent hematopoietic precursors in the presence of soluble activator of NFkappaB ligand (sRANKL). Because transforming growth factor (TGF)-beta, a macrophage deactivator, enhances osteoclast formation we hypothesized that interleukin (IL)-4 and IL-13, also macrophage deactivators, should exert a similar effect. However, IL-4 and IL-13 have been reported as suppressors of murine osteoclast formation. In contrast to the effect of these molecules on murine osteoclast formation, IL-4 and IL-13 were found to be powerful inducers of osteoclast formation and bone resorption when added to human peripheral blood mononuclear cell (PBMC) cultures for 4 days. This stimulatory effect was only observed in cultures containing nonadherent PBMCs. In contrast, both molecules significantly suppressed osteoclast formation in lymphocyte-depleted cultures. These data demonstrate that the cytokine milieu and/or state of cell activation determines how cells of the osteoclast precursor respond to IL-4 and IL-13.

Antineoplastic Agents↗

PubMed2001

Generation of human osteoclasts in stromal cell-free and stromal cell-rich cultures: differences in osteoclast CD11c/CD18 integrin expression.

Osteoclasts form in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of Nfkappab ligand (RANKL), a membrane-bound differentiation factor that is now available as a soluble recombinant molecule. Acquisition of the osteoclast phenotype [the alphavbeta3 subunit of the vitronectin receptor (VNR)-, calcitonin receptor (CTR)- and F-actin ring-positive cells] is associated with loss of monocyte/macrophage-associated integrins, specifically CD11b, CD11c and CD18. We hypothesized that differences in the osteoclast integrin adhesion molecule profile may exist in osteoclasts generated in stromal cell-rich and in stromal-free conditions. Unlike osteoclasts generated in vivo, F-actin ring-positive (resorbing) osteoclasts formed in soluble RANKL in vitro, in the absence of stromal cells, and co-expressed CD11c and CD18. However, when osteoclasts were generated from peripheral blood mononuclear cells (PBMNCs) in co-cultures with the murine bone marrow stromal cell line 218 (which does not produce membrane-bound RANKL) in the presence of soluble RANKL, CD11c and CD18 were not expressed by osteoclasts. These findings indicate that the persistent expression of CD11c and CD18 is not accounted for by RANKL being presented in a soluble form and that membrane-bound RANKL is not required for the normal integrin expression in resorbing osteoclasts. This study demonstrates that potentially misleading information may arise by using data obtained from osteoclasts generated in the absence of stromal cells as they do not completely reflect the situation in vivo.

Actins↗

PubMed2001

Integrin antagonists as inhibitors of bone resorption: implications for treatment.

Currently 'accepted' treatments for bone disease utilise drugs that inhibit osteoclastic bone resorption; these lead to a reduction in subsequent bone loss and thence, indirectly, to an increase in bone mass and fewer fractures. Three classes of compounds currently form the mainstay of therapy for osteoporosis: oestrogens (hormone-replacement therapy), 'selective oestrogen receptor modulators' and the bisphosphonates. Problems of patient compliance, real or theoretical long-term toxicological risks and the lack of bone anabolic agents of clinical utility suggest that there is a need for the development of further novel osteoclast resorption inhibitors. Recent biological and genetic findings in the area of bone cell function have led to the identification of new drug targets. These drugs include agents that (directly or indirectly): inhibit osteoclast adhesion to bone matrix; modify osteoclast differentiation; act on the proton pump and hence affect extracellullar acidification; antagonise extracellular enzymes that are involved in bone matrix protein degradation. Particular emphasis is placed in the present review on the evaluation of antagonists of alphavbeta3 integrin-mediated cell adhesion for use in bone disease. The wealth of new agents being developed suggests that resorption inhibition will be the best treatment for osteoporosis in the short to medium term, with the long-term aim still being toward developing anabolic drugs or cell therapeutics.

Bone Remodeling↗

PubMed2001

Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide receptor subtypes in mouse calvarial osteoblasts: presence of VIP-2 receptors and differentiation-induced expression of VIP-1 receptors.

Three distinct complementary DNAs for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors have been cloned and designated VIP-1 receptor (VIP-1R), VIP-2 receptor (VIP-2R), and PACAP receptor (PACAP-R). In the present study, we have characterized the binding sites on primary mouse calvarial osteoblasts for VIP and related peptides. By analyzing the cAMP response, the rank order of response observed was PACAP 38 > PACAP 27 > helodermin > VIP > helospectin > glucagon > PHI >>> secretin. The VIP-2R/PACAP-R antagonist, PACAP 6-38, inhibited both VIP- and PACAP-stimulated cAMP formation. Binding studies using an atomic force microscopy (AFM) technique showed high affinity binding for VIP and PACAP 38, but not for secretin. Radioligand binding studies using (125)I-VIP and (125)I-PACAP 38 demonstrated a more specific and higher affinity binding for PACAP 38 than for VIP. Secretin failed to inhibit both (125)I-VIP and (125)I-PACAP 38 binding. RT-PCR demonstrated that undifferentiated mouse calvarial osteoblasts express messenger RNA for VIP-2R, but not for VIP-1R or PACAP-R. When the osteoblasts were cultured for 20 days to induce bone noduli formation, VIP-1R, in addition to VIP-2R, were expressed when the nodules started to mineralize at 12 days. Taken together, these data demonstrate that mouse calvarial osteoblasts express functional VIP-2R with higher affinity binding for PACAP than for VIP and that the VIP-1R expression is induced during osteoblastic differentiation.

Animals↗

PubMed2001

Cells in regenerating deer antler cartilage provide a microenvironment that supports osteoclast differentiation.

Deer antlers are a rare example of mammalian epimorphic regeneration. Each year, the antlers re-grow by a modified endochondral ossification process that involves extensive remodelling of cartilage by osteoclasts. This study identified regenerating antler cartilage as a site of osteoclastogenesis in vivo. An in vitro model was then developed to study antler osteoclast differentiation. Cultured as a high-density micromass, cells from non-mineralised cartilage supported the differentiation of large numbers of osteoclast-like multinucleated cells (MNCs) in the absence of factors normally required for osteoclastogenesis. After 48 h of culture, tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells (osteoclast precursors) were visible, and by day 14 a large number of TRAP-positive MNCs had formed (783+/-200 per well, mean +/- s.e.m., N=4). Reverse transcriptase/polymerase chain reaction (RT-PCR) showed that receptor activator of NF &kgr; B ligand (RANKL) and macrophage colony stimulating factor (M-CSF) mRNAs were expressed in micromass cultures. Antler MNCs have the phenotype of osteoclasts from mammalian bone; they expressed TRAP, vitronectin and calcitonin receptors and, when cultured on dentine, formed F-actin rings and large resorption pits. When cultured on glass, antler MNCs appeared to digest the matrix of the micromass and endocytose type I collagen. Matrix metalloproteinase-9 (MMP-9) may play a role in the resorption of this non-mineralised matrix since it is highly expressed in 100 % of MNCs. In contrast, cathepsin K, another enzyme expressed in osteoclasts from bone, is only highly expressed in resorbing MNCs cultured on dentine. This study identifies the deer antler as a valuable model that can be used to study the differentiation and function of osteoclasts in adult regenerating mineralised tissues.

Acid Phosphatase↗