Establishment of Singleton-Merten syndrome pulp cells: evidence of mineralization dysregulation.

Singleton-Merten syndrome (SMS) is a rare disease with a phenotype of dental dysplasia. Currently, the underlying mechanism of this disease is unknown. In order to investigate the functional mechanism of the SMS tooth phenotypes, we isolated dental pulp tissue and established SMS primary pulp cells. These cells exhibited normal morphology and could be maintained in culture. Their ability to express alkaline phosphatase and mineralize was confirmed by in vitro staining. A comparative osteogenesis polymerase chain reaction array analysis was performed revealing 22 genes up-regulated and 8 genes down-regulated greater than 2-fold in SMS versus unaffected pulp cells. Down-regulated genes included ALP, IGF2, TGFBR2 and COL1A1. Collagen type I was reduced in SMS cells as shown by Western blot analysis. Furthermore, matrix metallopeptidase 13 was found to be dramatically increased in SMS pulp cells. Our findings suggest that dentin mineralization is dysregulated in SMS and may contribute to the root phenotype found in this disease.

Improved bioengineered cartilage tissue formation following cyclic compression is dependent on upregulation of MT1-MMP.

The generation of bioengineered cartilage tissue suitable for transplantation is a potential therapy to treat damaged cartilage. We have shown previously that the physical and biomechanical properties of bioengineered cartilage can be improved by the application of 30 min of cyclic compression by a mechanism involving sequential upregulation of gene and protein levels of membrane type-1 matrix metalloproteinase (MT1-MMP) and MMP-13. In the current study, we demonstrated that MT1-MMP is critical to this response, as blocking the upregulation of MT1-MMP prevented the improvement in tissue formation. MT1-MMP seems to act by inducing tissue remodeling as evidenced by the presence of aggrecan degradation products by Western blot analysis and increased release of matrix molecules into the media. Release of these molecules was diminished when MT1-MMP upregulation was prevented. This matrix degradation was likely due to MT1-MMP, as under conditions where MMP-13 expression is maintained (stimulation in the presence of MT1-MMP siRNA) the release of these matrix molecules into the media was still prevented. It also appears that MT1-MMP does not regulate MMP-13 gene expression, as MT1-MMP-siRNA pretreatment had no effect on MMP-13 expression following mechanical stimulation. Further analysis of the anabolic genes and proteins involved in mechanically stimulated cartilage will lead to better understanding of the mechanism(s) underlying tissue formation yielding improved bioengineered cartilage.

Spatial distribution of osteocyte lacunae in equine radii and third metacarpals: considerations for cellular communication, microdamage detection and metabolism.

Osteocytes, which are embedded in bone matrix, are the most abundant cells in bone. Despite the ideal location of osteocytes to sense the local environment and influence bone remodeling, their functions, and the relative importance of these functions, remain controversial. In this study, we tested several hypotheses that address the possibilities that population densities of osteocyte lacunae (Ot.Lc.N/B.Ar) correlate with strain-, remodeling- or metabolism-related aspects of the local biomechanical environments of mid-third diaphyseal equine radii and third metacarpals from skeletally mature animals. Ot.Lc.N/B.Ar data, quantified in multiple cortical locations, were analyzed for possible correlations with (1) structural and material characteristics (e.g., cortical thickness, percent ash, secondary osteon population density, mean osteon cross-sectional area, and predominant collagen fiber orientation), (2) strain characteristics, including prevalent/predominant strain magnitude and mode (tension, compression, shear), (3) hypothesized strain-mode-related microdamage characteristics, which might be perceived by osteocyte 'operational' networks, and (4) variations in remodeling dynamics and/or metabolism (i.e. presumably higher in endocortical regions than in other transcortical locations). Results showed relatively uniform Ot.Lc.N/B.Ar between regions with highly non-uniform strain and strain-related environments and markedly heterogeneous structural and material organization. These results suggest that population densities of these cells are poorly correlated with mechanobiological characteristics, including local variations in metabolic rate and strain magnitude/mode. Although osteocytes hypothetically evolved both as strain sensors and fatigue damage sensors able to direct the removal of damage as needed, the mechanisms that govern the distribution of these cells remain unclear. The results of this study provide little or no evidence that the number of osteocyte lacunae has a functional role in mechanotransduction pathways that are typically considered in bone adaptation.

Osteocyte viability and regulation of osteoblast function in a 3D trabecular bone explant under dynamic hydrostatic pressure.

UNLABELLED: A new trabecular bone explant model was used to examine osteocyte-osteoblast interactions under DHP loading. DHP loading enhanced osteocyte viability as well as osteoblast function measured by osteoid formation. However, live osteocytes were necessary for osteoblasts to form osteoids in response to DHP, which directly show osteoblast-osteocyte interactions in this in vitro culture. INTRODUCTION: A trabecular bone explant model was characterized and used to examine the effect of osteocyte and osteoblast interactions and dynamic hydrostatic pressure (DHP) loading on osteocyte viability and osteoblast function in long-term culture. MATERIALS AND METHODS: Trabecular bone cores obtained from metacarpals of calves were cleaned of bone marrow and trabecular surface cells and divided into six groups, (1) live cores + dynamic hydrostatic pressure (DHP), (2) live cores + sham, (3) live cores + osteoblast + DHP, (4) live cores + osteoblast + sham, (5) devitalized cores + osteoblast + DHP, and (6) devitalized cores + osteoblast + sham, with four culture durations (2, 8, 15, and 22 days; n = 4/group). Cores from groups 3-6 were seeded with osteoblasts, and cores from groups 5 and 6 were devitalized before seeding. Groups 1, 3, and 5 were subjected to daily DHP loading. Bone histomorphometry was performed to quantify osteocyte viability based on morphology and to assess osteoblast function based on osteoid surface per bone surface (Os/Bs). TUNEL staining was performed to evaluate the mode of osteocyte death under various conditions. RESULTS: A portion of osteocytes remained viable for the duration of culture. DHP loading significantly enhanced osteocyte viability up to day 8, whereas the presence of seeded osteoblasts significantly decreased osteocyte viability. Cores with live osteocytes showed higher Os/Bs compared with devitalized cores, which reached significant levels over a greater range of time-points when combined with DHP loading. DHP loading did not increase Os/Bs in the absence of live osteocytes. The percentage of apoptotic cells remained the same regardless of treatment or culture duration. CONCLUSION: Enhanced osteocyte viability with DHP suggests the necessity of mechanical stimulation for osteocyte survival in vitro. Furthermore, osteocytes play a critical role in the transmission of signals from DHP loading to modulate osteoblast function. This explant culture model may be used for mechanotransduction studies in long-term cultures.

Measurement of bone mineral density via light scattering.

In this study we have investigated the potential of optical techniques to monitor changes in bone mineral density (BMD) via changes in scattering coefficient. For each of five bone samples, diffuse reflection and transmission coefficients were measured over the wavelength range 520-960 nm using an integrating sphere and CCD spectrometer. These were converted into optical absorption and scattering coefficients using a Monte Carlo inversion procedure. Measurements were made on samples immersed in formic acid solution for different lengths of time in order to investigate the effect of reduction in BMD on the optical properties. After full demineralization, the optical scattering coefficient fell by a factor 4. From the observed degree of fluctuation of the measurements, we estimate that BMD could be measured with an accuracy of 7% if optical scattering can be measured with an accuracy of 10%. We also report preliminary measurements of bone scattering using optical coherence tomography (OCT). An inter-side variability of 3% is obtained on dry samples with and without overlying periosteum. These results suggest that minimally invasive techniques for measuring optical scattering, such as OCT, may have a role in monitoring regional changes in BMD. This could be an important advance in our understanding of bone remodelling and its relationship to osteoarthritis. Both the integrating sphere and OCT measurements also suggest that light transport in bone is spatially anisotropic. OCT was used to assess probability of obtaining results in vivo.

Decreased bone resorption, osteoclast differentiation, and expression of vacuolar H+-ATPase in antisense DNA-treated mouse metacarpal and calvaria cultures ex vivo.

Expression and function of vacuolar H(+)-ATPase, a key enzyme in bone resorption, were monitored in antisense DNA-treated bone organ cultures ex vivo. A novel fluoroimmunoassay was used to quantitate mRNA levels after treatment with various antisense, sense, or random DNA oligonucleotides. Conventional slot blots and in vitro translation experiments were used to monitor the efficiency of the antisense molecules. In cell cultures, the used antisense molecules were transported into osteoclasts and a population of mononuclear cells. A significant decrease in bone resorption and in the expression of the 16 kDa, 31 kDa, 42 kDa, 60 kDa, 70 kDa, and 116 kDa subunits of V-ATPase was seen after antisense treatment. Also, osteoclast differentiation was decreased in antisense-treated mouse metacarpal cultures. These data show that the proper function of V-ATPase in osteoclasts requires expression of the 16 kDa, 31 kDa, 42 kDa, 60 kDa, 70 kDa, and 116 kDa subunits of V-ATPase. Antisense DNA molecules can be used to inhibit osteoclast differentiation and function in tissue cultures, in which the physical and chemical cellular environment resembles that in vivo. However, more studies are needed to learn if antisense DNA molecules can be used for inhibiting bone resorption also in vivo.

Ovariectomy and orchidectomy induce a transient increase in the osteoclastogenic potential of bone marrow cells in the mouse.

Withdrawal of gender steroids in both women and men is associated with an increase in bone turnover with bone resorption exceeding bone formation leading to bone loss. To further investigate this process, the osteoclastogenic potential of mouse bone marrow cells was assessed at different timepoints after ovariectomy (ovx) or orchidectomy (orx). Cocultures of osteoclast-free fetal mouse long bones together with bone marrow from ovariectomized or orchidectomized mice indicated that the withdrawal of gender steroids in female and male mice induces a transient increase in osteoclastogenesis. The osteoclastogenic potential of the bone marrow cells was increased 7 days after ovx or orx. However, osteoclastic resorption was not increased at 3 days after surgery and had normalized 30 days after either ovx or orx. These results suggest that the withdrawal of gender steroids induces a transient increase in osteoclastogenesis in mice of both genders, which is associated with the early phase of rapid bone loss.

Correlation between anatomic features and low-field magnetic resonance imaging of the equine metacarpophalangeal joint.

OBJECTIVE: To expand our current knowledge and to establish limits of correlation between signal intensities of the magnetic resonance (MR) image and actual macroscopic and microscopic anatomic features of the imaged structures of the equine metacarpophalangeal joint (MCPJ). SAMPLE POPULATION: The right MCPJ was obtained from 4 adult horses that were euthanatized for reasons unrelated to the musculoskeletal system. PROCEDURE: The distal portion of the right forelimbs was collected from 4 equine cadavers. The bones were drilled to provide fixed reference points and examined by MR imaging. After imaging, the joints were sectioned for gross and histologic inspection. The MR images were aligned and correlated with digitized gross and histologic images to identify tissue types. RESULTS: Comparison of the images resulted in identification of different bone types, articular cartilage, and soft tissue structures of the equine MCPJ. CONCLUSION: Results provided relevant information regarding the appearance of the imaged tissues of the equine MCPJ. CLINICAL RELEVANCE: Although MR imaging does not have current clinical applications for equine practitioners, its wide acceptance as the imaging modality used for most human musculoskeletal derangements may aid in developing more realistic applications in equine medicine.

Colony stimulating factor-1 plays a role in osteoclast formation and function in bone resorption induced by parathyroid hormone and parathyroid hormone-related protein.

Although colony stimulating factor-1 (CSF-1) plays a key role in osteoclast recruitment, studies examining the effect of CSF-1 on mature osteoclasts indicate that it may directly inhibit bone resorption by isolated rat osteoclasts. To define further CSF-1's role in bone remodeling, we examined the effect of neutralizing antisera to CSF-1 on basal and parathyroid hormone (PTH)-induced bone resorption using two organ culture assays designed to examine the recruitment of osteoclast precursors and the activation of mature osteoclasts, respectively. We first assessed whether PTH increases CSF-1 production from bone in organ culture by examining conditioned medium from 19-day-old fetal rat long bones in a mitogenesis assay employing a CSF-1-responsive cell line, CRX-1. Conditioned medium from untreated bones induced a titratable increase in CRX-1 cell proliferation, and treatment of bones with PTH for 72 h caused a significant increase in mitogenic activity. CSF-1 antiserum caused a significant decrease in mitogenic activity in conditioned medium, indicating that bone in organ culture produces CSF-1 constitutively and in response to PTH. To examine bone-derived CSF-1's role in bone resorption, we examined the effect of neutralizing antisera to CSF-1 on basal and PTH-induced bone resorption in the fetal rat long bone assay, which reflects activation of mature osteoclasts. Anti-CSF-1 caused a significant increase in unstimulated and PTH-induced bone resorption compared with control. By contrast, in the fetal mouse metacarpal assay, which examines proliferation and recruitment of osteoclast progenitors and precursors, anti-CSF-1 caused significant inhibition of PTH related protein (PTHrP)-induced bone resorption after 3 and 6 days of incubation. Consistent with these findings, histological examination of cultured 17-day-old fetal metacarpals demonstrated that anti-CSF-1 inhibits the formation of tartrate-resistant acid phosphatase-positive osteoclasts in PTHrP-treated explants, whereas it has no effect on unstimulated bones. We conclude that bone-derived CSF-1 may have a dual role in PTH/PTHrP-induced bone resorption by enhancing the appearance of osteoclast precursors while restraining the resorptive function of mature osteoclasts.

Nuragic bone collagen retains its molecular structure.

The molecular structure of bone collagen was found to be preserved in Nuragic bone samples several centuries after burial. Enhanced birefringence of bone and dentin collagen fibres observed in polarized light after Sirus red staining was used to demonstrate this fact.

Radionuclide, radiographic, and histomorphometric evaluation of healing of surgically created subchondral defects in equine bone.

In the present study, radionuclide scintigraphy and radiography were used to evaluate the rate and degree of healing that occurred in surgically created subchondral bone defects in horses. Following radionuclide scintigraphy and radiography the horses were killed, and histomorphometric analysis was performed on the defect sites. The histomorphometric results were compared to the radionuclide scintigraphic and radiographic results to determine which noninvasive technique provided the most accurate information concerning healing of the bone defects. It was concluded that radionuclide scintigraphy and radiographic evaluation seemed equally well suited for use in serial evaluation of healing of surgically created bone defects. However, it was felt that the most accurate assessment of healing of the bone defects and effects in the adjacent bone could be provided by combining the two methods of evaluation.

Functional adaptation of bone in response to sinusoidally varying controlled compressive loading of the ovine metacarpus.

To obtain carried load, quantitative data relating functional adaptation of bone to controlled, sinusoidally-varying, compressive loading of constant amplitude was applied to the right metacarpal bones via Steinmann's pins inserted through the metaphyses in 13 sheep. Loading was applied for two 1-hr periods/day at 24 cycle/minute, through-out a test period of 28 days. The amplitude of the applied loading was varied from test to test, giving peak stresses on the mid-diaphyseal cross-section that ranged from 2.2-8.3 N/mm2. In the mid-diaphyseal region, the bone responded by periosteal apposition, with maximum proliferation usually occurring on the medio- and laterovolar borders and relatively little new bone on the dorsal and volar aspects. The cross-sectional areas of new bone was roughly proportional to the applied stress, with a maximum increase of approximately 8% in the most highly stressed bones. There was also evidence that periosteal resorption had occurred, presumably as the first step in the apposition process. A significantly increased level of intracortical activity was found in the right metacarpus as compared with the contralateral bone.