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Mineralized Matrix (mineralized + matrix)

Distribution by Scientific Domains

Medical Sciences	68%
Life Sciences	25%

Selected Abstracts

Overexpression of Lysyl Hydroxylase-2b Leads to Defective Collagen Fibrillogenesis and Matrix Mineralization,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2005
Suchaya Pornprasertsuk
Abstract Several MC3T3-E1 cell-derived clones expressing higher levels of LH2b were analyzed for their abilities to form collagen fibrils and mineralization. The clones all exhibited smaller collagen fibrils and defective matrix mineralization in vitro and in vivo, indicating a critical role of LH2b-catalyzed post-translational modifications of collagen in bone matrix formation and mineralization. Introduction: We have recently shown that lysyl hydroxylase (LH) 2b, through its action on the telopeptidyl lysine residues of collagen, regulates collagen cross-linking pathway in the osteoblastic cell line, MC3T3-E1. To further elucidate the roles of LH2b in bone physiology, the effects of overexpression of LH2b on collagen fibrillogenesis and matrix mineralization were investigated. Materials and Methods: Several MC3T3-E1-derived osteoblastic cell clones expressing higher levels of LH2b (S clones) and two controls (i.e., MC3T3-E1 cells and those transfected with an empty vector) were cultured. MALDI-TOF mass spectrometry was used to identify the LH2b. The collagen fibrillogenesis in the cultures was characterized by transmission electron microscopy, and the ability of these clones and cells to form mineralized matrix was analyzed by both in vitro and in vivo mineralization assays. Results: The diameter of collagen fibrils in the S clone cultures was markedly smaller than that of the controls. The onset of matrix mineralization in the S clones was significantly delayed, and considerably fewer mineralized nodules were formed in their cultures in comparison with the controls. When transplanted into immunodeficient mice, the S clones failed to form mineralized matrices in vivo, whereas a bone-like mineralized matrix was well formed by the controls. The diameter of the collagen fibrils and the timing/extent of matrix mineralization in vitro were inversely correlated with the level of LH2b. In vitro cell differentiation was unaffected by the LH2b overexpression. Conclusions: These results indicate a critical role of LH2b catalyzed post-translational modification of collagen (i.e., telopeptidyl lysine hydroxylation and subsequent cross-linking) in collagen matrix formation and mineralization in bone. [source]

On the origin of intrinsic matrix of acellular extrinsic fiber cementum: Studies on growing cementum pearls of normal and bisphosphonate-affected guinea pig molars

EUROPEAN JOURNAL OF ORAL SCIENCES, Issue 3 2002
Chantha K. Jayawardena
Cementum pearls (CPs) belong to a type of acellular extrinsic fiber cementum (AEFC) that form on the maturing enamel of guinea pig molars. This study aimed to elucidate the forming process of intrinsic matrix of AEFC using the CPs of normal and bisphosphonate-affected guinea pig molars as experimental models. A group of guinea pigs were subjected to continuous administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) for 2 wk to inhibit mineralization of growing CPs. Fenestration of the enamel organ and migration of periodontal cells on to the exposed surface of maturing enamel appeared to be unaffected by HEBP, whereas de novo formation as well as growth of pre-existing CPs did not proceed under the same conditions. Immunoreactions for osteopontin were located exclusively on the mineralized matrix of preformed CPs, implying the absence of additional deposition or accumulation of putative intrinsic cementum matrix on the affected CPs, where the propagation of mineral phase had been arrested. In both normal and HEBP-treated groups, distinct enzymatic reactions for alkaline phosphatase appeared on the cells of the periodontal ligament associated closely with the sites of CP formation, and along the mineralization front of CPs. These observations suggest that the mineralization process per se plays a central role in the deposition of AEFC matrix and that alkaline phosphatase of periodontal cells penetrating through the enamel organ to the maturing enamel surface plays a key role in the mineralization process of CPs. [source]

Osteoconductive and Osteoinductive Properties of Zeolite MFI Coatings on Titanium Alloys

ADVANCED FUNCTIONAL MATERIALS, Issue 24 2009
Rajwant S. Bedi
Abstract The use of zeolite MFI-coated titanium alloy for bone cell growth and new bone formation in vitro is investigated. The corrosion-resistant MFI coating is shown to be osteoconductive and to promote proliferation of human fetal osteoblasts (hFOBs) as compared to bare titanium alloy, Ti6Al4V. The zeolite crystal microstructure appears to facilitate osteoblast adhesion and induces osteointegration, as evaluated with microscopy. In addition, the zeolite promotes the differentiation of hFOBs into mature osteoblasts, as well as the production of a mineralized matrix at earlier times in culture compared to Ti6Al4V, indicating higher osteoinductive properties of the MFI coating than titanium alone. A significant increase in the expression of the bone morphogenetic protein (BMP-2) gene is measured in hFOBs cultured on zeolite coatings compared to bare Ti6Al4V. This is the first report on highly corrosion-resistant zeolite MFI coatings on Ti6A14V alloys with the potential to be used as a material of improved osteointegration appropriate for bone tissue regeneration. [source]

Inhibition of Lamin A/C Attenuates Osteoblast Differentiation and Enhances RANKL-Dependent Osteoclastogenesis,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2009
Martina Rauner
Abstract Age-related osteoporosis is characterized by low bone mass, poor bone quality, and impaired osteoblastogenesis. Recently, the Hutchinson-Gilford progeria syndrome (HGPS), a disease of accelerated aging and premature osteoporosis, has been linked to mutations in the gene encoding for the nuclear lamina protein lamin A/C. Here, we tested the hypothesis that inhibition of lamin A/C in osteoblastic lineage cells impairs osteoblastogenesis and accelerates osteoclastogenesis. Lamin A/C was knocked-down with small interfering (si)RNA molecules in human bone marrow stromal cells (BMSCs) differentiating toward osteoblasts. Lamin A/C knockdown led to an inhibition of osteoblast proliferation by 26% and impaired osteoblast differentiation by 48% based on the formation of mineralized matrix. In mature osteoblasts, expression levels of runx2 and osteocalcin mRNA were decreased by lamin A/C knockdown by 44% and 78%, respectively. Furthermore, protein analysis showed that osteoblasts with diminished levels of lamin A/C also secreted less osteocalcin and expressed a lower alkaline phosphatase activity (,50%). Lamin A/C inhibition increased RANKL mRNA and protein levels, whereas osteoprotegerin (OPG) expression was decreased, resulting in an increased RANKL/OPG ratio and an enhanced ability to support osteoclastogenesis, as reflected by a 34% increase of TRACP+ multinucleated cells. Our data indicate that lamin A/C is essential for proper osteoblastogenesis. Moreover, lack of lamin A/C favors an osteoclastogenic milieu and contributes to enhanced osteoclastogenesis. [source]

Overexpression of Lysyl Hydroxylase-2b Leads to Defective Collagen Fibrillogenesis and Matrix Mineralization,

Regulation of Human Skeletal Stem Cells Differentiation by Dlk1/Pref-1

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2004
Basem M Abdallah
Abstract Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. Introduction: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. Materials and Methods: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. Results: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPAR,2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor ,1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected. Conclusion: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes. [source]

Purification of Matrix Gla Protein From a Marine Teleost Fish, Argyrosomus regius: Calcified Cartilage and Not Bone as the Primary Site of MGP Accumulation in Fish,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2003
DC Simes
Abstract Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius,, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5,rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted toregions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation. [source]

MLO-Y4 Osteocyte-Like Cells Support Osteoclast Formation and Activation,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2002
S. Zhao
Abstract Osteocytes are terminally differentiated cells of the osteoblast lineage that have become embedded in mineralized matrix and may send signals that regulate bone modeling and remodeling. The hypothesis to be tested in this study is that osteocytes can stimulate and support osteoclast formation and activation. To test this hypothesis, an osteocyte-like cell line called MLO-Y4 and primary murine osteocytes were used in coculture with spleen or marrow cells. MLO-Y4 cells support osteoclast formation in the absence of 1,25-dihydroxyvitamin D3 [1,25(OD)2D3] or any other exogenous osteotropic factor. These cells alone stimulate osteoclast formation to the same extent or greater than adding 1,25(OH)2D3. Coaddition of 1,25(OH)2D3 with MLO-Y4 cells synergistically increased osteoclast formation. Optimal osteoclast formation and pit formation on dentine was observed with 200,1000 MLO-Y4 cells per 0.75-cm2 well. No osteoclast formation was observed with 2T3, OCT-1, or MC3T3-E1 osteoblast cells (1000 cells/well). Conditioned media from the MLO-Y4 cells had no effect on osteoclast formation, indicating that cell contact is necessary. Serial digestions of 2-week-old mouse calvaria yielded populations of cells that support osteoclast formation when cocultured with 1,25(OH)2D3 and marrow, but the population that remained in the bone particles supported the greatest number of osteoclasts with or without 1,25(OH)2D3. To examine the mechanism whereby these cells support osteoclast formation, the MLO-Y4 cells were compared with a series of osteoblast and stromal cells for expression of macrophage colony-stimulating factor (M-CSF), RANKL, and osteoprotegerin (OPG). MLO-Y4 cells express and secrete large amounts of M-CSF. MLO-Y4 cells express RANKL on their surface and their dendritic processes. The ratio of RANKL to OPG mRNA is greatest in the MLO-Y4 cells compared with the other cell types. RANK-Fc and OPG-Fc blocked the formation of osteoclasts by MLO-Y4 cells. These studies suggest that both RANKL and OPG may play a role in osteocyte signaling, OPG and M-CSF as soluble factors and RANKL as a surface molecule that is functional in osteocytes or along their exposed dendritic processes. [source]

Inorganic phosphate as a signaling molecule in osteoblast differentiation,

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2003
George R. Beck Jr.
Abstract The spatial and temporal coordination of the many events required for osteogenic cells to create a mineralized matrix are only partially understood. The complexity of this process, and the nature of the final product, demand that these cells have mechanisms to carefully monitor events in the extracellular environment and have the ability to respond through cellular and molecular changes. The generation of inorganic phosphate during the process of differentiation may be one such signal. In addition to the requirement of inorganic phosphate as a component of hydroxyapatite mineral, Ca10(PO4)6(OH)2, a number of studies have also suggested it is required in the events preceding mineralization. However, contrasting results, physiological relevance, and the lack of a clear mechanism(s) have created some debate as to the significance of elevated phosphate in the differentiation process. More recently, a number of studies have begun to shed light on possible cellular and molecular consequences of elevated intracellular inorganic phosphate. These results suggest a model in which the generation of inorganic phosphate during osteoblast differentiation may in and of itself represent a signal capable of facilitating the temporal coordination of expression and regulation of multiple factors necessary for mineralization. The regulation of protein function and gene expression by elevated inorganic phosphate during osteoblast differentiation may represent a mechanism by which mineralizing cells monitor and respond to the changing extracellular environment. J. Cell. Biochem. 90: 234,243, 2003. Published 2003 Wiley-Liss, Inc. [source]

Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006
Akemichi Ueno
Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis. J. Cell. Physiol. 209: 322,332, 2006. © 2006 Wiley-Liss, Inc. [source]

Putative heterotopic ossification progenitor cells derived from traumatized muscle,

JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 12 2009
Wesley M. Jackson
Abstract Heterotopic ossification (HO) is a frequent complication following combat-related trauma, but the pathogenesis of traumatic HO is poorly understood. Building on our recent identification of mesenchymal progenitor cells (MPCs) in traumatically injured muscle, the goal of this study was to evaluate the osteogenic potential of the MPCs in order to assess the role of these cells in HO formation. Compared to bone marrow-derived mesenchymal stem cells (MSCs), a well-characterized population of osteoprogenitor cells, the MPCs exhibited several significant differences during osteogenic differentiation and in the expression of genes related to osteogenesis. Upon osteogenic induction, MPCs showed increased alkaline phosphatase activity, production of a mineralized matrix, and up-regulated expression of the osteoblast-associated genes CBFA1 and alkaline phosphatase. However, MPCs did not appear to reach terminal differentiation as the expression of osteocalcin was not substantially up-regulated. With the exception of a few genes, the osteogenic gene expression profile of traumatized muscle-derived MPCs was comparable to that of the MSCs after osteogenic induction. These findings indicate that traumatized muscle-derived MPCs have the potential to function as osteoprogenitor cells when exposed to the appropriate biochemical environment and are the putative osteoprogenitor cells that initiate ectopic bone formation in HO. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1645,1651, 2009 [source]

Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells

JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2002
Ulrich Nöth
Abstract Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast-related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase-pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow-derived mesenchymal progenitor cells. High-density pellet cultures of hOB maintained in chemically defined serum-free medium, supplemented with transforming growth factor-,1, were composed of morphologically distinct, chondrocyte-like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high-density pellet cultures were surrounded by a sulfated prote-oglycan-rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase-positive cells, that expressed osteoblast-related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, ,-glycerophosphate, and bone morphogenetic protein-2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte-specific genes, such as lipoprotein lipase and peroxisome proliferator-activated receptor ,2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3-isobutyl-l-methylxanthine. Taken together, these results show that cells derived from collagenase-treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source]

Bone differentiation of marrow-derived mesenchymal stem cells using ,-tricalcium phosphate,alginate,gelatin hybrid scaffolds

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 6 2007
Mohamadreza Baghaban Eslaminejad
Abstract The aim of the present study was to establish a 3D culture system for bone differentiation of mesenchymal stem cells (MSCs), using a new hybrid sponge. To manufacture the scaffold, a composite of ,-tricalcium phosphate,alginate,gelatin was prepared and cast as pellets of 1 cm diameter. The sponge was then fabricated by drying in freeze-dryer for 12 h. The porosity, mean pore size, compressive modulus and strength of the composite sponge fabricated in this study were 89.7%, 325.3 µm, 1.82 and 0.196 MPa, respectively. To establish a 3D culture system, the rat bone marrow-derived MSCs were suspended in 500 µl diluted collagen gel, loaded into the porous sponge and provided with medium with or without osteogenic supplements for 3 weeks. The day after loading, the cells appeared in the scaffold's internal spaces, where later some of them from either culture survived by anchoring on the surfaces. At the end of cultivation period, individually adhered cells from both cultures were observed to be replaced by cell aggregates, in which mineralized matrix was detected by alizarin red staining. Furthermore, RT-PCR analysis indicated that the bone-specific gene osteocalcin was expressed in cultures in both the presence and absence of the osteogenic supplements. Taken together, it seems that the studied scaffolds are cell-compatible and, more importantly, possess some osteo-inductive properties. Copyright © 2007 John Wiley & Sons, Ltd. [source]

Dentin matrix proteins and soluble factors: intrinsic regulatory signals for healing and resorption of dental and periodontal tissues?

ORAL DISEASES, Issue 2 2004
TA Silva
Dentin contains numerous polypeptides and signaling molecules sequestered in a mineralized matrix. The exposure and release of these molecules occur as a consequence of injury to the pulp and periodontal ligament, which may result from luxation, orthodontic movement or infections of tooth and periodontal structures. When released at these sites, dentin constituents have the potential to act on different surrounding cells, including periodontal cells, osteoblasts, osteoclasts and inflammatory cells, and to affect the course of dental disease. Experimental studies have highlighted the interactions between dentin and cells from tooth and periodontal tissues and reveal dentin to be a cell adhesive, signaling and migratory stimulus for various mesenchymal and inflammatory cells. These results support the hypothesis that dentin molecules might function as regulatory signals for the healing and resorption of dental and periodontal tissues. Data from recent and classical investigations are summarized, many open questions are discussed, and current hypotheses concerning the mechanisms of tooth resorption and periodontal healing are outlined. Many questions regarding the importance of dentin as a source of multifunctional molecules remain unanswered and provide important directions for future studies. [source]

In vitro spontaneous osteoclastogenesis of human peripheral blood mononuclear cells is not crucially dependent on T lymphocytes,

ARTHRITIS & RHEUMATISM, Issue 4 2009
Bernard Vandooren
Objective In vitro spontaneous osteoclastogenesis from peripheral blood mononuclear cells (PBMCs) is increased in diseases with excessive bone loss. The purpose of this study was to reassess the role of T lymphocytes in this process. Methods Fresh or cryopreserved PBMCs obtained from healthy subjects and from patients with rheumatoid arthritis, psoriatic arthritis, and non-psoriatic spondylarthritis were cultured at high density and stained for tartrate-resistant acid phosphatase (TRAP). Resorption of mineralized matrix was assessed by a dentin disc assay. CD14+ monocytes and CD3+ T cells were selected using magnetically labeled antibodies. Results Numerous multinucleated, TRAP+, dentin-resorbing osteoclasts developed spontaneously from fresh PBMCs from healthy individuals. This process was abrogated by T cell depletion and was restored by exogenous macrophage colony-stimulating factor (M-CSF) and RANKL, indicating the important role of T cells in spontaneous osteoclastogenesis in vitro. Using physiologic freezing and thawing as a model for the activation of PBMCs, spontaneous osteoclastogenesis was significantly increased in cryopreserved versus fresh cells. Under these conditions, spontaneous osteoclastogenesis was not dependent on T lymphocytes, since it was not influenced by T cell depletion and persisted in purified CD14+ cell cultures supplemented with M-CSF and RANKL. In contrast to studies with fresh PBMCs, spontaneous osteoclastogenesis under these conditions did not appear to be clearly different between healthy subjects and patients with arthritis. Conclusion Spontaneous osteoclastogenesis in vitro is dependent on T lymphocytes or on the direct activation of monocytic cells, depending on the test conditions. This variability warrants better validation of the relevance of this functional test for in vivo osteoclastogenesis. [source]