Role of lipids in calcification of cartilage

This review addresses the role of lipids and membranes in biologic calcification and examines their regulation during endochondral ossification. The close association of lipids with mineral deposition has been well established. Early observations indicated that lipids, particularly phospholipids, can not be totally extracted from calcified tissues until the tissues are decalcified. Phospholipids associated with mineral are also enriched in extracellular membrane vesicles, called matrix vesicles. Numerous studies indicate that mineral deposits in calcifying cartilage are first seen in these phosphatidylserine and alkaline phosphatase enriched vesicles and that the process of endochondral calcification of epiphyseal growth plate is possibly mediated by them. Matrix vesicles, and the phospholipids present in them, appear to be involved in initial formation of calcium hydroxyapatite crystals via the interaction of calcium and phosphate ions with phosphatidylserine to form phospholipid:Ca:Pi complexes (CPLX). CPLX is present in tissues which are undergoing initial mineral deposition but are absent from nonmineralizing tissues. Evidence suggests that CPLX resides in the interior of matrix vesicles where the earliest mineral crystals are formed in association with the vesicle membrane. More recently, it has been determined that specific membrane proteins, called proteolipids, participate in CPLX formation and hydroxyapatite deposition, in part by structuring phosphatidylserine in an appropriate conformation. Phosphatidylserine involvement in the initiation of mineralization has been extensively investigated because of its extremely high binding affinity for Ca2+. In addition to structuring a specific phospholipid environment, proteolipids may also act as ionophores, promoting export of protons and import of calcium and phosphate, both requirements of biologic calcification.(ABSTRACT TRUNCATED AT 250 WORDS)     

The expression of bone matrix protein mRNAs around beta-TCP particles implanted into bone

Tissue response around beta-tricalcium phosphate (beta-TCP) particles (150-300 microm in diameter) implanted into rat tibiae was analyzed by in situ hybridization with digoxigenin-labeled procollagen alpha1(I) (COL), osteonectin, osteocalcin, and osteopontin (OPN) RNA probes. Specimens were collected at 3, 5, 7, and 10 days after the operation. Holes without implantation were used as control. In both the beta-TCP implanted and control groups, new bone was formed centripetally and all four kinds of mRNA were expressed in activated osteoblasts. A COL signal was expressed most strongly and widely, and was detected at the peripheral region of the hole at day 3. The other three mRNAs were also expressed in bone forming osteoblasts by day 7. However, in the earlier cell reaction stage, OPN expression in the beta-TCP implanted group was different than that in the control group: OPN mRNA was seen exclusively in the cells on the particles, and an OPN signal was detected not only in COL-positive cells, but also in COL-negative cells. The former cells may be osteoblasts and reflect the early process of bone formation on biomaterials. The latter cells may be macrophages and reflect foreign body reactions. Expression of these OPN mRNAs induced by implantation of beta-TCP may play a role in bone formation on the materials and in determining their biocompatibility.     

成骨诱导脂肪基质细胞在骨组织工程体内成骨中的作用

背景：以往研究认为,经过成骨诱导后的脂肪基质细胞通过转化为成骨细胞分泌骨基质进而修复骨缺损,然而并没有明确结论证实。 目的：将经过体外成骨诱导的脂肪基质细胞复合支架材料分别植入骨缺损区和非骨区,根据是否成骨,验证经过成骨诱导后的脂肪基质细胞是否转化为成骨细胞。 方法：取12月龄犬背部皮下脂肪,经胶原酶消化法获得单个核细胞,将培养的第3代细胞与双相磷酸钙陶瓷形成复合物。在犬下颌骨两侧制备长20 mm、高10 mm的箱状缺损,拔除术区牙齿,将细胞支架复合物植入一侧术区,空白侧留作对照;另外在犬背部皮下肌肉区植入细胞支架复合物及骨诱导性磷酸钙陶瓷材料,术后6周及12周经组织学检测骨缺损修复情况。 结果与结论：脂肪基质细胞复合双相磷酸钙陶瓷在骨缺损区成骨,在肌肉区未形成新骨;骨诱导性磷酸钙陶瓷在肌肉区形成新骨。提示成骨诱导并不能将脂肪基质细胞转化为成骨细胞,其确切机制有待进一步研究。     

A composite graft material containing bone particles and collagen in osteoinduction in mouse.

Demineralized allogenic bone matrices (DABM) and demineralized freeze-dried bone allograft (DFDBA) have been successfully used as bone-graft materials in the treatment of acquired and congenital cranio-maxillofacial defects and in some orthopedic surgery. However, these bone-graft "powders" have many shortcomings. For example, placement of particulate graft material in a hemorrhaging site can result in inadequacies or inaccurate attachment as well as loss of the graft materials. To minimize the inadequacies of powderlike graft materials, xenogenic collagen isolated from human tendon, skin, or bone was added to the bone-graft particles to form a composite spongelike implant. This material is commercially available and consists of 60% collagen and 40% DFDBA (DynaGraft, GenSci Co., Irvine, CA). The goal of this study was to evaluate the characteristics of composite graft implants in the mineralization process in an animal model in comparison with DFDBA powder and pure collagen. Seventy-two Swiss Webster mice were divided into three groups: an experimental group implanted with DynaGraft, two comparison groups implanted with either DFDBA or collagen only. All the graft materials were surgically implanted and inserted into the left thigh muscle. Mice were humanely killed at 1, 2, 3, 4, 6, 8, and 12 weeks. Then the muscle tissues in the vicinity of the implants were excised and processed for histology. Paraffin sections were stained with hematoxylin and eosin (H&E), the Von Kossa method, and Masson's trichrome. Some selected specimens were processed for transmission electron microscopic observation. After 1 week of implantation, the DynaGraft group showed calcium deposition on the collagen material and on the periphery of the DFDBA particles. Increased calcification and bone-forming cells were observed at 4-6 weeks. After 8 weeks, the implant formed a calcified nodule and only heavily mineralized connective tissue was observed at the implanted site. The group implanted with DFDBA powder showed calcification around the particulates. The collagen-sponge control group revealed no calcification or bone formation during the period of implantation. The light microscopic findings were confirmed by electron microscopy. Quantitative radiographic density DynaGraft and DFDBA graft followed sequentially over a period 120 days. It was concluded that a higher rate of calcification and bone formation was produced in the composite graft implant compared to the DFDBA implant. The composite graft material (DynaGraft), which contains both collagen and DFDBA, proved to be more effective for bone formation than particle components alone.     

脱细胞天然骨基质与诱导性成骨细胞的体外相容性

背景：前期工作表明TritonX-100处理的脱细胞骨基质已满足组织学和免疫学方面的修复要求。如果细胞能在材料表面很好地生长,将利于进一步进行体内动物实验。 目的：采用细胞培养法在体外评估脱细胞骨基质与诱导后成骨细胞的生物相容性。 方法：第3代骨髓基质干细胞经成骨诱导分化培养液诱导分化为成骨细胞,接种于TritonX-100处理的脱细胞骨基质及羟基磷灰石表面,检测成骨细胞的碱性磷酸酶表达并用扫描电镜观察材料表面的细胞生长情况。 结果与结论：碱性磷酸酶活性分析均表明,TritonX-100处理的脱细胞骨基质在培养48 h之后比羟基磷灰石更利于诱导成骨细胞生长;扫描电镜下可见,成骨细胞在脱细胞骨基质表面呈现立体生长方式,细胞呈球形,并且聚集成簇。体外实验结果显示成骨细胞与脱细胞天然骨基质有较好的生物相容性。     

Role of fibronectin during biological apatite crystal nucleation: ultrastructural characterization.

The role of adhesion molecules like osteopontin and bone sialoprotein, both containing the Arg-Gly-Asp sequence have been shown to have a role in mineral formation, whereas fibronectin (FN), another adhesive protein, was never studied during the mineralization processes. The formation and maturation of biological apatite crystals are under matrix control, and one of the roles of specific crystal proteins is to control the nucleation and growth of biological apatite during the mineralization process (promotion or inhibition). In the case of calcium phosphate ceramic used as a bone substitute, a dissolution-precipitation process occurs after implantation before the bone ingrowth and bone mineralization. The early precipitation consists of common biological apatite crystals. These crystals are the result of secondary nucleation and a heteroepitaxic growth process on synthetic residual crystals. In in vivo studies, hydroxyapatite crystals were implanted subcutaneously into mice for 1 or 2 weeks. Fibronectin immunogold labeling of the newly formed crystals on surfaces of high-resolution transmission electron microscopy sections of retrieved implants revealed the close association of these precipitated crystals with FN. In in vitro experiments using a solution of human FN incubated in the presence of calcium phosphate crystals, we obtained apatite crystal precipitation. The fibronectin network observed in high-resolution transmission electron microscopy showed numerous clusters of very small particles (1 nm in diameter and 2 nm in length), whereas the same experiment realized as control on albumin revealed no crystal precipitation. These results demonstrate for the first time the role of FN in early biological crystal nucleation. This process could have important biological significance in accounting for ectopic calcification, primary nucleation in calcified tissue, and bone ingrowth on calcium phosphate ceramics.     

Bone formation in polymeric scaffolds evaluated by proton magnetic resonance microscopy and X-ray microtomography

Magnetic resonance microscopy (MRM) and X-ray microtomography (XMT) were used to investigate de novo bone formation in porous poly(ethyl methacrylate) (PEMA) scaffolds, prepared by a novel co-extrusion process. PEMA scaffolds were seeded with primary chick calvarial osteoblasts and cultured under static conditions for up to 8 weeks. Bone formation within porous PEMA scaffolds was confirmed by the application of histologic stains to intact PEMA disks. Disks were treated with Alizarin red to visualize calcium deposits and with Sirius red to visualize regions of collagen deposition. DNA analysis confirmed that cells reached confluence on the scaffolds after 7 weeks in static culture. The formation of bone in PEMA scaffolds was investigated with water proton MRM. Quantitative MRM maps of the magnetization transfer ratio (MTR) yielded maps of protein deposition, and magnetic resonance (MR) relaxation times (T1 and T2) yielded maps of mineral deposition. The location of newly formed bone and local mineral concentrations were confirmed by XMT. By comparing MRM and XMT data from selected regions-of-interest in one sample, the inverse relationship between the MR relaxation times and mineral concentration was validated, and calibration curves for estimating the mineral content of cell-seeded PEMA scaffolds from quantitative MRM images were developed.     

Mechanism of mineral formation in bone

The mechanism of mineral formation in bone is seen best where active new bone formation is occurring, e.g., in newly forming subperiosteal bone of the embryo, in the growing bone of young animals, and in healing rickets where the calcification process in osteoid is reactivated. A large body of ultrastructural evidence, using conventional and anhydrous methods for tissue preparation, has shown convincingly that extracellular matrix vesicles are present at or near the mineralization front in all of the above, and that these vesicles are the initial site of apatite mineral deposition. Thus bone resembles growth plate cartilage, predentin, and turkey tendon in having calcification initiated by matrix vesicles. Once the calcification cascade is begun, matrix vesicles are no longer needed to support mineralization and are consumed by the advancing mineralization front in which performed crystals serve as nuclei for the formation of new crystals. The rate of crystal proliferation is promoted by the availability of Ca2+, PO4(3-), and the presence of collagen, and retarded by naturally occurring inhibitors of mineralization such as proteoglycans and several noncollagenous calcium-binding proteins of bone including bone-Gla protein (osteocalcin), phosphoproteins, osteonectin, and alpha-2HS-glycoproteins. New electron microscopic immunocytochemical findings in our laboratory suggest that the origin of alkaline phosphatase-positive bone matrix vesicles is polarized to the mineral-facing side of osteoblasts and may be concentrated near the intercellular junctions of human embryonic osteoblasts.     

High-porosity poly(epsilon-caprolactone)/mesoporous silicon scaffolds: calcium phosphate deposition and biological response to bone precursor cells

In this study, the fabrication and characterization of highly porous composites composed of poly(epsilon-caprolactone) and bioactive mesoporous silicon (BioSilicon) prepared using salt-leaching and microemulsion/freeze-drying methods are described. The role of silicon, along with porosity, in the scaffolds on calcium phosphate deposition was assessed using acellular in vitro calcification analyses. The presence of bioactive silicon in these scaffolds is essential for the deposition of calcium phosphate while the samples are immersed in simulated body fluid (SBF). Silicon-containing scaffolds produced using salt-leaching methods are more likely to calcify as a consequence of SBF exposure than those produced using microemulsion methods. In vitro proliferation and cell viability assays of these porous composites using human embryonic kidney fibroblast cells indicate that no cytotoxic effects are present in the scaffolds under the conditions used. Preliminary analyses of bone sialoprotein and alkaline phosphatase expression using orthopedically relevant mesenchymal cells derived from bone marrow suggest that such scaffolds are capable of mediating osteoblast differentiation. Overall, the results show that these porous silicon-containing polymer scaffolds enhance calcification, can be considered nontoxic to cells, and support the proliferation, viability, attachment, and differentiation of bone precursor cells.     

Effect of phosphate functional groups on the calcification capacity of acrylic hydrogels

The incorporation of negatively charged groups into the structure of synthetic polymers is frequently advocated as a method for enhancing their calcification capacity required in orthopedic and dental applications. However, the results reported by various research groups are rather contentious, since inhibitory effects have also been observed in some studies. In the present study, phosphate groups were introduced in poly(2-hydroxyethyl methacrylate) (PHEMA) by copolymerization with 10% mol of either mono(2-acryloyloxyethyl) phosphate (MAEP) or mono(2-methacryloyloxyethyl) phosphate (MMEP). Incubation of these hydrogels for determined durations (1-9 weeks) in a simulated body fluid (SBF) solution induced deposition of calcium phosphate (CaP) deposits of whitlockite type. After 9 weeks, the amount of calcium deposited on the phosphate-containing polymers was four times lower than that found on PHEMA, as determined by X-ray photoelectron spectroscopy (XPS). Samples of copolymer HEMA-MAEP were implanted subcutaneously in rats and evaluated after 9 weeks. No CaP deposits could be detected on the copolymer by XPS or energy dispersive X-ray spectroscopy, while PHEMA samples were massively calcified. It was concluded that the presence of phosphate groups decreased the calcification capacity of the hydrogels, and that in the conditions of this study, the phosphate groups had an inhibitory effect on the deposition of CaP phases on HEMA-based hydrogels.     

Bone formation by rat bone marrow cells cultured on titanium fiber mesh: effect of in vitro culture time

The objective of this study was to examine the effect of cell culture time on bone formation by rat bone marrow cells seeded in titanium fiber mesh. As a seeding technique, a high cell suspension was used (3 x 10(6) cells/mL). Therefore, 30 meshes were repeatedly rotated in a 10 mL tube (containing 30 x 10(6) cells) on a rotation plate (2 rpm) for 3 h. Osteogenic cells were cultured for 1, 4, and 8 days on titanium fiber mesh and finally implanted subcutaneously in rats. Meshes without cells were also implanted subcutaneously in rats. DNA and scanning electron microscopy (SEM) analyses and calcium measurements determined cellular proliferation and differentiation during the in vitro incubation period of the mesh implants. Four weeks after implant insertion, the animals were sacrificed. The implants, with their surrounding tissue, were retrieved and prepared for histologic evaluation and calcium measurements. DNA analysis of the in vitro experiment showed a lag phase from day 1 through day 4, but a 42% increase in DNA between days 4 and 8. SEM and calcium measurements indicated an increase in calcium from day 1 to day 4, yet only a small but significant increase from days 4 to 8. Histologic analysis demonstrated that bone was formed in all day 1 and day 4 implants, and that the bone-like tissue was present uniformly through the meshes. The bony tissue was morphologically characterized by osteocytes embedded in a mineralized matrix, with a layer of osteoid and osteoblasts at the surface. The day 8 implants showed only calcium phosphate deposition in the titanium fiber mesh. Calcium measurements of the implants revealed that calcification in day 1 implants was significantly higher (p < 0.05) compared to day 4 and day 8 implants. No significant difference in calcium content existed between day 4 and day 8 implants. On the basis of our results, we conclude that 1) bone formation was generated more effectively in osteogenic cells by a short culture time after seeding in titanium fiber mesh; 2) dynamic cell seeding is probably more effective than static cell seeding; and 3) selection of the right cells from the heterogenous bone marrow population remains a problem.     

Crystallization pathways in bone

Many biomineralization processes involve the sequestering of ions by cells and their translocation through the cells to the final deposition site. In many invertebrate crystallization pathways the cells deposit an initial highly disordered mineral phase with intracellular vesicles, and this mineral is subsequently transported into the final deposition site outside the cell. As this initial mineral phase is metastable, it can easily dissolve or crystallize during sample preparation and examination. A cryogenic electron microscopy study of the forming fin bone of a zebra fish strain with continuously growing fins shows that the cells responsible for bone tissue formation do have mineral-bearing intracellular vesicles and that the mineral phase is a highly disordered calcium phosphate. We also show that globules of disordered calcium phosphate are present in the extracellular collageneous matrix and that they are not membrane bound. Close to the mineralization front these globules appear to penetrate into the collagen fibrils where they crystallize to form mature bone. This crystallization pathway is similar to pathways observed in invertebrates, and it differs from the matrix vesicle pathway documented for a variety of vertebrate mineralizing tissues as the extracellular mineral globules are not membrane bound.     

肝素抗凝材料联合血管束植入修复骨缺损的血管化

背景：前期研究显示肝素-壳聚糖脱细胞骨基质材料植入骨缺损受区后,材料内部血液供应明显改善,将血管束植入大体积抗凝材料能否促进骨缺损处血液灌注和血管化进程？ 目的：观察局部抗凝的肝素-壳聚糖脱细胞骨基质材料联合血管束植入对骨缺损修复早期血液灌注及血管化的影响。 方法：取25只健康新西兰大白兔制作双侧桡骨中段20 mm长骨缺损模型,右侧植入局部抗凝的肝素-壳聚糖脱细胞骨基质材料联合自体血管束(实验组),左侧植入脱细胞骨基质材料联合自体血管束(对照组),术后1,3,7,14,28 d行CT灌注成像及组织学观察。 结果与结论：术后1 d开始,实验组血容量、血流量显著高于对照组(P < 0.05),且实验组材料中心部位有明显血液灌注,对照组植入物周围有血液渗透,此种趋势持续到术后28 d。术后1 d,实验组复合材料中有大量红细胞和有核细胞,对照组以渗透液体为主,偶见细胞;术后3-7 d,实验组复合材料网孔中有血管形成,之后逐渐增多,对照组见植入血管血栓形成、管腔闭塞,周围组织纤维化包裹,实验组各时间点材料内部血管数高于对照组(P < 0.05)。提示局部抗凝的肝素-壳聚糖脱细胞骨基质材料可促进血管束植入后材料内部的血液灌注和早期血管化进程。     

TRIP-1 in the extracellular matrix promotes nucleation of calcium phosphate polymorphs

ABSTRACT

In search for bone and dentin extracellular matrix (ECM) proteins, transforming growth factor beta receptor II interacting protein 1 (TRIP-1) was identified as a novel protein synthesized by osteoblasts and odontoblasts and exported to the ECM. TRIP-1 is a WD-40 (WD is Tryptophan-Aspartic acid dipeptide) protein that has been well recognized for its physiological role in the endoplasmic reticulum (ER). In the ER, TRIP-1 functions as an essential subunit of eukaryotic elongation initiation factor 3 and is involved in the protein translational machinery. Recently, we reported that TRIP-1 is localized in the ECM of bone and dentin. In this study, we demonstrate that varying concentrations of TRIP-1 can participate in the nucleation of calcium phosphate polymorphs. Nucleation studies performed with high calcium and phosphate concentration demonstrated that recombinant TRIP-1 could orchestrate the formation of hydroxyapatite crystals. Nucleation experiments performed on demineralized and deproteinized dentin wafer under physiological conditions and subsequent transmission electron microscope analysis of the deposits at the end of 7 and 14 days showed that TRIP-1 promoted the deposition of calcium phosphate mineral aggregates in the gap-overlap region of type I collagen. Taken together, we provide mechanistic insight into the role of this intracellular protein in matrix mineralization.     

Ultrastructural, cytochemical, and biophysical aspects of mechanisms of bone matrix calcification

Primary calcification in embryonic ossification occurs as follows: crystallization within matrix vesicles, formation of calcified nodules, and finally the establishment of expansive calcified matrix. However, the participation of the matrix vesicles in other types of bone calcification, such as bone formation during bone remodeling in adults has not been examined sufficiently. We introduce our recent observations on the presence of matrix vesicles in aged bones. In addition, although it is well known that the extracellular fluid supersaturates the calcification crystal, hydroxyapatite, the specific mechanisms by which bone matrix calcify remain unclear. In order to further approach the mechanisms of bone matrix calcification, we also review ultrastructural and localizational alterations of the matrix organics according to the progression of calcification, and an evaluation of mineral micro-environment in the calcifying sites by energy-filter transmission electron microscopy.     

Scanning electron microscopy, x-ray diffraction, and electron microprobe analysis of calcific deposits on intrauterine contraceptive devices

Deposits found intrauterine contraceptive devices (IUDs) were studied by scanning electron microscopy, x-ray diffraction, and energy dispersive x-ray microanalysis. All seven devices, including five plastic and two copper IUDs, were coated with a crust containing cellular, acellular, and fibrillar material. The cellular material was composed of erythrocytes, leukocytes, cells of epithelial origin, sperm, and bacteria. Some of the bacteria were filamentous, with acute-angle branching. The fibrillar material appeared to be fibrin. Most of the acellular material was amorphous; calcite was identified by x-ray diffraction, and x-ray microanalysis showed only calcium. Some of the acellular material, particularly that on the IUD side of the crust, was organized in spherulitic crystals and was identified as calcium phosphate by x-ray microanalysis. The crust was joined to the IUD surface by a layer of fibrillar and amorphous material. It is suggested that the initial event in the formation of calcific deposits on IUD surfaces is the deposition of an amorphous and fibrillar layer. Various types of cells present in the endometrial environment adhere to this layer and then calcify. Thus, the deposition of calcific material on the IUDs is a calcification phenomenon, not unlike the formation of plaque on teeth. Hum Pathol 16:732-738, 1985.     

Bone marrow stem cells and biological scaffold for bone repair in aging and disease

The loss of bone mass observed in aging enhances the risk of fractures. The process of bone repair in aging is slow and limited due to reduced activity of the osteoblasts. Bone marrow stem cells (MSCs) residing in the bone marrow are the progenitors for osteoblasts. The ability to enhance healing of bone defect in aging by MSCs can contribute in the prevention of the complications resulting from long-term immobilization that are especially fatal in old age. Our aim was to test the ability of MSCs inserted into a biological scaffold to enhance bone defect repair. Osteoprogenitor cells were selected from rat bone marrow stem cells cultured in DMEM medium supplemented with FCS, antibiotics, ascorbic acid, beta-glycerophosphate, and dexamethasone. The selected osteogenic subpopulation was identified by osteocalcin immunohistochemistry as well as Alizarin red S and von Kossa staining which are specific for bone matrix and mineral deposition. Committed osteoprogenitor cells cultured on the hydrogel scaffold were transplanted into the area of a rat tibia segmental bone defect and examined after 6 weeks. Radiology images revealed that 6 weeks post-implantaion, calcified material was present in the site of the defect, indicating new bone formation. It is concluded that committed osteogenic MSCs contained in a biocompatible scaffold can provide a promising surgical tool for enhancement of bone defect healing that will minimize the complications of bone repair in aging and disease.     

A histological comparison of the tissue interface of bioglass and silica glass

The objectives of the present article are to confirm the bone bonding phenomenon of Bioglass (BG) developed by Hench et al., and to observe the singularity of tissue reaction to it. BG and nonreactive silica glass (SG) were implanted in the femurs of rabbits and rats. Histological examination revealed that a relatively acellular zone with little inflammation was formed on BG surface at 1 day after implantation. Neither fibrous tissue nor a distinct boundary was observed between BG and bone after 7 days. On the contrary, a moderate postoperative inflammatory reaction was observed on SG at 1 day, and fibrous tissue was observed between SG and bone after 7 days. From these findings, it was confirmed that BG bonded directly with bone. As the relatively acellular zone observed on BG surface at 1 day was replaced by bone after 7 days, the formation of this zone might play an important role in bone-bonding process. Further research should be focused on the mechanism and biological meaning of bone bonding, for this phenomenon can not be explained by the conventional pathological theory of foreign body encapsulation.     

Toward cell therapy for vascular calcification: osteoclast-mediated demineralization of calcified elastin.

BACKGROUND: Elastin-oriented vascular calcification is a clinically significant feature, which involves formation of ectopic bone-like structures. Taking advantage of the similarities between arterial calcification and bone regulation, our hypothesis was that therapeutic approaches for limitation of vascular calcification could be developed using site-specific delivery of autologous osteoclasts. In the present paper, we tested the hypothesis that bone-marrow-derived osteoclasts have the ability to demineralize calcified elastin, without significant alterations in elastin integrity. METHODS: Active, multinucleated osteoclasts were obtained by in vitro maturation of rat bone-marrow-derived progenitor cells in the presence of vitamin D(3) and retinoic acid. Cell phenotype was validated by staining for tartrate-resistant acid phosphatase, formation of resorption pits on hydroxyapatite-coated disks, and RT-PCR for identification of cathepsin K gene expression. Calcified aortic elastin was seeded with osteoclasts and calcium, and phosphorous levels were monitored in gels and culture media to detect demineralization of elastin. Soluble elastin peptides were also monitored in culture media for elastin degradation. For in vivo experiments, pure aortic elastin was coimplanted with allogenic osteoclasts subdermally into rats, and the degree of elastin calcification and degradation was evaluated using mineral analysis and desmosine quantitation. RESULTS: Bone-marrow-derived osteoclasts reduced mineral content of calcified elastin in vitro by 80%. Moreover, in vivo implantation of allogenic osteoclasts in the vicinity of calcifying elastin limited elastin mineralization by almost 50%, in the absence of detectable elastin degradation. CONCLUSIONS: Osteoclasts have the ability to demineralize calcified elastin, without significant alterations in elastin integrity.     

Histomorphometric analysis of sclerotic bone from idiopathic myeloid metaplasia (nine cases)

The osteosclerosis of idiopathic myeloid metaplasia was investigated by a histodynamic study of undecalcified bone from nine patients. In all cases osteosclerosis resulted from woven bone formation. In moderate osteosclerosis only intrabecular woven bone was observed, with osteoclastic hyperresorption, hyperosteoidosis with many osteoblasts, and an increased calcification rate. This may represent an early stage of the disease, osteosclerosis resulting from increased bone remodelling, with woven bone formation by osteoblasts. However, in extensive osteosclerosis both intratrabecular and medullary woven bone was observed, with moderate osteoclastic hyperresorption, hyperosteoidosis without osteoblasts and a very low calcification rate. The latter pattern could correspond to an advanced stage, and could result from stromal bone formation without osteoblasts. The classical histodynamic profile of osteomalacia was sometimes observed. The link between bone pain and possible osteomalacia remains vague.