Function of osteocytes in bone

Although the structural design of cellular bone (i.e., bone containing osteocytes that are regularly spaced throughout the bone matrix) dates back to the first occurrence of bone as a tissue in evolution, and although osteocytes represent the most abundant cell type of bone, we know as yet little about the role of the osteocyte in bone metabolism. Osteocytes descend from osteoblasts. They are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes remain in contact with each other and with cells on the bone surface via gap junction–coupled cell processes passing through the matrix via small channels, the canaliculi, that connect the cell body–containing lacunae with each other and with the outside world. During differentiation from osteoblast to mature osteocyte the cells lose a large part of their cell organelles. Their cell processes are packed with microfilaments. In this review we discuss the various theories on osteocyte function that have taken in consideration these special features of osteocytes. These are (1) osteocytes are actively involved in bone turnover; (2) the osteocyte network is through its large cell-matrix contact surface involved in ion exchange; and (3) osteocytes are the mechanosensory cells of bone and play a pivotal role in functional adaptation of bone. In our opinion, especially the last theory offers an exciting concept for which some biomechanical, biochemical, and cell biological evidence is already available and which fully warrants further investigations. © 1994 Wiley-Liss, Inc.     

Structural differences in the osteocyte network between the calvaria and long bone revealed by three-dimensional fluorescence morphometry, possibly reflecting distinct mechano-adaptations and sensitivities

The structural features of osteocytes and their cellular process network are thought to allow for mechanotransduction from the bone tissue to these cells. This study applied three-dimensional fluorescence microscopy to fixed and decalcified bone specimens to quantitatively compare the osteocytes and their networks between mouse parietal bone and tibia that are physiologically enforced by distinct mechanical loads. The subsequent morphometric analysis by the surface rendering of osteocyte cell bodies revealed the tibia to have relatively enriched cytoplasm in the osteocyte cell body in comparison to the parietal bone. Furthermore, quantitative tracing of the cellular processes in silico demonstrated that the numbers of the cellular processes and their bifurcation points per osteocyte in the tibia were significantly higher than those in the parietal bone. Though the total length of the processes per osteocyte in the tibia was two times longer, its total surface area and total volume were smaller than those in the parietal bone, due to its thinner diameter. These architectural differences in the osteocytes and their networks are thus implicated in the adaptation to physiologically different loading, and may also induce distinct mechanosensitivities.     

Osteoblasts and osteocytes respond differently to oscillatory and unidirectional fluid flow profiles

Bone cells subjected to mechanical loading by fluid shear stress undergo significant architectural and biochemical changes. The models of shear stress used to analyze the effects of loading bone cells in vitro include both oscillatory and unidirectional fluid shear profiles. Although the fluid flow profile experienced by cells within bone is most likely oscillatory in nature, to date there have been few direct comparisons of how bone cells respond to these two fluid flow profiles. In this study we evaluated morphologic and biochemical responses to a time course of unidirectional and oscillatory fluid flow in two commonly used bone cell lines, MC3T3-E1 osteoblasts and MLO-Y4 osteocytes. We determined that stress fibers formed and aligned within osteoblasts after 1 h of unidirectional fluid flow, but this response was not observed until greater than 5 h of oscillatory fluid flow. Despite the delay in stress fiber formation, oscillatory and unidirectional fluid flow profiles elicited similar temporal effects on the induction of both cyclooxygenase-2 (Cox-2) and osteopontin protein expression in osteoblasts. Interestingly, MLO-Y4 osteocytes formed organized stress fibers after exposure to 24 h of unidirectional shear stress, while the number of dendritic processes per cell increased along with Cox-2 protein levels after 24 h of oscillatory shear stress. Despite these differences, both flow profiles significantly altered osteopontin levels in MLO-Y4 osteocytes. Together these results demonstrate that the profile of fluid shear can induce significantly different responses from osteoblasts and osteocytes.     

Osteocytes inhibit osteoclastic bone resorption through transforming growth factor-beta: enhancement by estrogen

Osteocytes are the most abundant cells in bone and distributed throughout the bone matrix. They are connected to the each other and to the cells on the bone surface. Thus, they may also secrete some regulatory factors controlling bone remodeling. Using a newly established osteocyte-like cell line MLO-Y4, we have studied the interactions between osteocytes and osteoclasts. We collected the conditioned medium (CM) from MLO-Y4 cells, and added it into the rat osteoclast cultures. The conditioned medium had no effect on osteoclast number in 24-h cultures, but it dramatically inhibited resorption. With 5, 10, and 20% CM, there was 25, 39, and 42% inhibition of resorption, respectively. Interestingly, the inhibitory effect was even more pronounced, when MLO-Y4 cells were pretreated with 10(-8) M 17-beta-estradiol. With 5, 10, and 20% CM, there was 46, 51, and 58% of inhibition. When the conditioned medium was treated with neutralizing antibody against transforming growth factor-beta (TGF-beta), the inhibitory effect was abolished. This suggests that osteocytes secrete significant amounts of TGF-beta, which inhibits bone resorption and is modulated by estrogen. RT-PCR and Western blot analysis show that in MLO-Y4 cells, the prevalent TGF-beta isoform is TGF-beta3. We conclude that osteocytes have an active, inhibitory role in the regulation of bone resorption. Our results further suggest a novel role for TGF-beta in the regulation of communication between different bone cells and suggest that at least part of the antiresorptive effect of estrogen in bone could be mediated via osteocytes.     

Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes

Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load‐bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β‐glycyrrhetinic acid, we also demonstrated that this osteocyte‐related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial‐derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain–related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity.     

Development of Implant/Interconnected Porous Hydroxyapatite Complex as New Concept Graft Material

Background

Dental implant has been successfully used to replace missing teeth. However, in some clinical situations, implant placement may be difficult because of a large bone defect. We designed novel complex biomaterial to simultaneously restore bone and place implant. This complex was incorporated implant into interconnected porous calcium hydroxyapatite (IP-CHA). We then tested this Implant/IP-CHA complex and evaluated its effect on subsequent bone regeneration and implant stability in vivo.

Methodology/Principal Findings

A cylinder-type IP-CHA was used in this study. After forming inside of the cylinder, an implant was placed inside to fabricate the Implant/IP-CHA complex. This complex was then placed into the prepared bone socket in the femur of four beagle-Labrador hybrid dogs. As a control, implants were placed directly into the femur without any bone substrate. Bone sockets were allowed to heal for 2, 3 and 6 months and implant stability quotients (ISQ) were measured. Finally, tissue blocks containing the Implant/IP-CHA complexes were harvested. Specimens were processed for histology and stained with toluidine blue and bone implant contact (BIC) was measured. The ISQs of complex groups was 77.8±2.9 in the 6-month, 72.0±5.7 in the 3-month and 47.4±11.0 in the 2-month. There was no significant difference between the 3- or 6-month complex groups and implant control groups. In the 2-month group, connective tissue, including capillary angiogenesis, was predominant around the implants, although newly formed bone could also be observed. While, in the 3 and 6-month groups, newly formed bone could be seen in contact to most of the implant surface. The BICs of complex groups was 2.18±3.77 in the 2-month, 44.03±29.58 in the 3-month, and 51.23±8.25 in the 6-month. Significant difference was detected between the 2 and 6-month.

Conclusions/Significance

Within the results of this study, the IP-CHA/implant complex might be able to achieve both bone reconstruction and implant stability.     

Identification of osteocytes in osteoblast-like cell cultures using a monoclonal antibody specifically directed against osteocytes

The development of a monoclonal antibody, OB 7.3, directed against a cell surface antigenic site on osteocytes is described. Osteoblast-like cells were enzymatically isolated from calvaria of chicken embryos after removal of the periostea. The cells were cultured for 6 days, harvested and used to immunize mice. One of the monoclonal antibodies obtained, OB 7.3, reacted specifically with the cell surface of osteocytes. In frozen sections of bone only osteocytes were stained, all other cells present, including mature osteoblasts, were negative. Liver, kidney, spleen, intestine, bloodvessel and skin were also completely negative. Using the monoclonal OB 7.3, positive cells could be demonstrated in sparse osteoblast-like cell cultures. The OB 7.3 positive cells had a stellate morphology and were therefore identified as osteocytes. They behaved in culture as osteocytes in bone tissue in that they formed a network of cell processes connecting osteocytes with each other or with other neighbouring cells. Monoclonal OB 7.3 offers the possibility of isolating osteocytes thereby providing the means for a detailed study of their biochemical properties.     

Micro-Brillouin scattering measurements in mature and newly formed bone tissue surrounding an implant

The evolution of implant stability in bone tissue remains difficult to assess because remodeling phenomena at the bone-implant interface are still poorly understood. The characterization of the biomechanical properties of newly formed bone tissue in the vicinity of implants at the microscopic scale is of importance in order to better understand the osseointegration process. The objective of this study is to investigate the potentiality of micro-Brillouin scattering techniques to differentiate mature and newly formed bone elastic properties following a multimodality approach using histological analysis. Coin-shaped Ti-6Al-4V implants were placed in vivo at a distance of 200?μm from rabbit tibia leveled cortical bone surface, leading to an initially empty cavity of 200?μm×4.4?mm. After 7 weeks of implantation, the bone samples were removed, fixed, dehydrated, embedded in methyl methacrylate, and sliced into 190?μm thick sections. Ultrasonic velocity measurements were performed using a micro-Brillouin scattering device within regions of interest (ROIs) of 10?μm diameter. The ROIs were located in newly formed bone tissue (within the 200?μm gap) and in mature bone tissue (in the cortical layer of the bone sample). The same section was then stained for histological analysis of the mineral content of the bone sample. The mean values of the ultrasonic velocities were equal to 4.97×10(-3)?m/s in newly formed bone tissue and 5.31×10(-3)?m/s in mature bone. Analysis of variance (p=2.42×10(-4)) tests revealed significant differences between the two groups of measurements. The standard deviation of the velocities was significantly higher in newly formed bone than in mature bone. Histological observations allow to confirm the accurate locations of the velocity measurements and showed a lower degree of mineralization in newly formed bone than in the mature cortical bone. The higher ultrasonic velocity measured in newly formed bone tissue compared with mature bone might be explained by the higher mineral content in mature bone, which was confirmed by histology. The heterogeneity of biomechanical properties of newly formed bone at the micrometer scale may explain the higher standard deviation of velocity measurements in newly formed bone compared with mature bone. The results demonstrate the feasibility of micro-Brillouin scattering technique to investigate the elastic properties of newly formed bone tissue.     

Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin

Mechanosensing bone osteocytes express large amounts of connexin (Cx)43, the component of gap junctions; yet, gap junctions are only active at the small tips of their dendritic processes, suggesting another function for Cx43. Both primary osteocytes and the osteocyte-like MLO-Y4 cells respond to fluid flow shear stress by releasing intracellular prostaglandin E2 (PGE2). Cells plated at lower densities release more PGE2 than cells plated at higher densities. This response was significantly reduced by antisense to Cx43 and by the gap junction and hemichannel inhibitors 18 beta-glycyrrhetinic acid and carbenoxolone, even in cells without physical contact, suggesting the involvement of Cx43-hemichannels. Inhibitors of other channels, such as the purinergic receptor P2X7 and the prostaglandin transporter PGT, had no effect on PGE2 release. Cell surface biotinylation analysis showed that surface expression of Cx43 was increased by shear stress. Together, these results suggest fluid flow shear stress induces the translocation of Cx43 to the membrane surface and that unapposed hemichannels formed by Cx43 serve as a novel portal for the release of PGE2 in response to mechanical strain.     

Identification of osteocytes in osteoblast-like cell cultures using a monoclonal antibody specifically directed against osteocytes

Summary The development of a monoclonal antibody, OB 7.3, directed against a cell surface antigenic site on osteocytes is described.Osteoblast-like cells were enzymatically isolated from calvaria of chicken embryos after removal of the periostea. The cells were cultured for 6 days, harvested and used to immunize mice. One of the monoclonal antibodies obtained, OB 7.3, reacted specifically with the cell surface of osteocytes. In frozen sections of bone only osteocytes were stained, all other cells present, including mature osteoblasts, were negative. Liver, kidney, spleen, intestine, bloodvessel and skin were also completely negative. Using the monoclonal OB 7.3, positive cells could be demonstrated in sparse osteoblast-like cell cultures. The OB 7.3 positive cells had a stellate morphology and were therefore identified as osteocytes. They behaved in culture as osteocytes in bone tissue in that they formed a network of cell processes connecting osteocytes with each other or with other neighbouring cells. Monoclonal OB 7.3 offers the possibility of isolating osteocytes thereby providing the means for a detailed study of their biochemical properties.In honour of Prof. P. van Duijn     

Comparative Characteristics of Porous Bioceramics for an Osteogenic Response In Vitro and In Vivo

Porous calcium phosphate ceramics are used in orthopedic and craniofacial applications to treat bone loss, or in dental applications to replace missing teeth. The implantation of these materials, however, does not induce stem cell differentiation, so suitable additional materials such as porous calcium phosphate discs are needed to influence physicochemical responses or structural changes. Rabbit adipose-derived stem cells (ADSC) and mouse osteoblastic cells (MC3T3-E1) were evaluated in vitro by the MTT assay, semi-quantitative RT-PCR, and immunoblotting using cells cultured in medium supplemented with extracts from bioceramics, including calcium metaphosphate (CMP), hydroxyapatite (HA) and collagen-grafted HA (HA-col). In vivo evaluation of the bone forming capacity of these bioceramics in rat models using femur defects and intramuscular implants for 12 weeks was performed. Histological analysis showed that newly formed stromal-rich tissues were observed in all the implanted regions and that the implants showed positive immunoreaction against type I collagen and alkaline phosphatase (ALP). The intramuscular implant region, in particular, showed strong positive immunoreactivity for both type I collagen and ALP, which was further confirmed by mRNA expression and immunoblotting results, indicating that each bioceramic material enhanced osteogenesis stimulation. These results support our hypothesis that smart bioceramics can induce osteoconduction and osteoinduction in vivo, although mature bone formation, including lacunae, osteocytes, and mineralization, was not prominent until 12 weeks after implantation.     

E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation

Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.     

Calcium response in single osteocytes to locally applied mechanical stimulus: Differences in cell process and cell body

It is proposed that osteocytes embedded in the bone matrix have the ability to sense deformation and/or damage to the matrix and to feed these mechanical signals back to the adaptive bone remodeling process. When osteoblasts differentiate into osteocytes during the bone formation process, they change their morphology to a stellate form with many slender processes. This characteristic cell shape may underlie the differences in mechanosensitivity between the cell processes and cell body. To elucidate the mechanism of cellular response to mechanical stimulus in osteocytes, we investigated the site-dependent response to quantitatively controlled local mechanical stimulus in single osteocytes isolated from chick embryos, using the technique of calcium imaging. A mechanical stimulus was applied to a single osteocyte using a glass microneedle targeting a microparticle adhered to the cell membrane by modification with a monoclonal antibody OB7.3. Application of the local deformation induced calcium transients in the vicinity of the stimulated point and caused diffusive wave propagation of the calcium transient to the entire intracellular region. The rate of cell response to the stimulus was higher when applied to the cell processes than when applied to the cell body. In addition, a large deformation was necessary at the cell body to induce calcium transients, whereas a relatively small deformation was sufficient at the cell processes, suggesting that the mechanosensitivity of the cell processes was higher than that of the cell body. These results suggest that the cell shape with slender processes contributes to the site-dependent mechanosensitivity in osteocytes.     

The reaction of osteoclasts when releasing osteocytes from osteocytic lacunae in the bone during bone modeling

Osteocytes are released from the osteocytic lacunae when osteoclasts resorb the bone matrix during bone modeling and remodeling. It remains unknown how osteoclasts react when releasing osteocytes during bone modeling, and the fate of these released osteocytes is also unclear. Femoral mid-shafts of 2-day-old kittens were sectioned into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and examined by light microscopy (LM) and transmission electron microscopy (TEM). The sections showed many osteoclasts at the endosteum but there were no osteoblasts. There were many half-released, fully released, half-exposed, and fully exposed osteocytes on the bone surfaces. Many cell-like structures were seen in the cell bodies of osteoclasts by LM, and some semithin sections were re-sectioned into ultrathin sections for re-observation by TEM. By TEM, these were determinated to be mononuclear cells. The serial ultrathin sections showed that the mononuclear cells appeared to be engulfed in osteoclasts on one section but that the cell was connected with the bone surface of the osteocytic lacuna on another section. These results show that the mononuclear cells in the osteoclasts were osteocytes. The present study suggests that osteoclasts engulf some osteocytes but do not engulf others when releasing osteocytes during bone modeling.     

Phenotypic plasticity and mechano‐transduction in the teleost skeleton

The proper formation, growth and maintenance of many bones depends on the mechanical loads generated by gravity and muscles. Mechanical loading by muscle forces does not only affect bone growth and maintenance in adult and juvenile vertebrates, but also affects larval and embryonic bone development. We have reviewed the current understanding of mechanotransduction in birds and mammals and compared it to teleosts. The major mechanosensing cells in the adult mammalian and avian skeleton are osteocytes. They are interconnected via cell processes and are contained within a canalicular network. Basal teleosts have osteocytes but their connectivity is questionable and the presence of a functional canalicular network is unlikely. Advanced teleosts have acellular bone and therefore lack osteocytes. Yet the skeleton of teleosts does show adaptive responses to changes in mechanical load. In these animals it is likely that osteoblasts, bone surface cells and chondrocytes act as mechanosensors. The factors expressed by osteocytes upon mechanical stimulation have been extensively investigated in vitro and in vivo in adult mammals and birds. Less is, however, known about the mechanotransduction pathway during embryonic bone development. The zebrafish presents new opportunities to analyze the mechanotransduction pathway during early (larval) bone formation due to the ex utero development and genetic analyses.     

Ultrastructure of the osteogenesis of acellular vertebral bone in the Japanese medaka, Oryzias latipes (Teleostei, Cyprinidontidae)

An ultrastructural study by transmission electron microscopy (TEM) of the vertebrae of embryonic, larval, juvenile and mature medaka shows that each vertebra consists of a core of notochordal cells surrounded by a sheath of bone. The vertebral bone lacks either fully or partially embedded cells in the matrix throughout development. Bone matrix is secreted by a layer of cells that lies over the outer surface of the vertebral bone. During the early stages of osteogenesis, these cells secrete bone matrix all around themselves. However, because of the gradual flow of the newly synthesized bone matrix through intercellular spaces, matrix-producing cells do not become trapped in their own secretion. In later stages of osteogenesis, these cells secrete matrix only toward the already-deposited bone. This polarized matrix secretion allows the osteoblasts to stay always on the bone surface and never to become trapped in the matrix as osteocytes.     

The turnover of mineralized growth plate cartilage into bone may be regulated by osteocytes

During endochondral ossification, growth plate cartilage is replaced with bone. Mineralized cartilage matrix is resorbed by osteoclasts, and new bone tissue is formed by osteoblasts. As mineralized cartilage does not contain any cells, it is unclear how this process is regulated. We hypothesize that, in analogy with bone remodeling, osteoclast and osteoblast activity are regulated by osteocytes, in response to mechanical loading. Since the cartilage does not contain osteocytes, this means that cartilage turnover during endochondral ossification would be regulated by the adjacent bone tissue. We investigated this hypothesis with an established computational bone adaptation model. In this model, osteocytes stimulate osteoblastic bone formation in response to the mechanical bone tissue loading. Osteoclasts resorb bone near randomly occurring microcracks that are assumed to block osteocyte signals. We used finite element modeling to evaluate our hypothesis in a 2D-domain representing part of the growth plate and adjacent bone. Cartilage was added at a constant physiological rate to simulate growth. Simulations showed that osteocyte signals from neighboring bone were sufficient for successful cartilage turnover, since equilibrium between cartilage remodeling and growth was obtained. Furthermore, there was good agreement between simulated bone structures and rat tibia histology, and the development of the trabecular architecture resembled that of infant long bones. Additionally, prohibiting osteoclast invasion resulted in thickened mineralized cartilage, similar to observations in a knock-out mouse model. We therefore conclude that it is well possible that osteocytes regulate the turnover of mineralized growth plate cartilage.     

Asymmetric intercellular communication between bone cells: Propagation of the calcium signaling

Bone functional adaptation by remodeling is achieved by harmonized activities of bone cells in which osteocytes in the bone matrix are believed to play critical roles in sensing mechanical stimuli and transmitting signals to osteoclasts/osteoblasts on the bone surface in order to regulate their bone remodeling activities through the lacuno-canalicular network with many slender osteocytic processes. In this study, we investigated the intercellular communication between bone cells, particularly focusing on its directionality, through in vitro observations of the calcium signaling response to mechanical stimulus and its propagation to neighboring cells (NCs). Direct mechanical stimulus was applied to isolated bone cells from chick calvariae, osteocytes (Ocys) and bone surface cells (BSCs) mainly containing osteoblasts, and the percentage of calcium signaling propagation from the stimulated cell to NCs was analyzed. The results revealed that, regardless of the type of stimulated cell, the signaling propagated to BSCs with a significantly higher percentage, implying that calcium signaling propagation between bone cells strongly depends on the type of receiver cell and not the transmitter cell. In addition, in terms of mutual communication between Ocys and BSCs, the percentage of propagation from Ocys to BSCs is significantly higher than that in the opposite direction, suggesting that the calcium signaling mainly propagates asymmetrically with a bias from Ocys in bone matrix to BSCs on bone surfaces. This asymmetric communication between Ocys and BSCs suggests that osteocytic mechanosensing and cellular communications, which significantly affect bone surface remodeling activities to achieve functional adaptation, seem to be well coordinated and active at the location of biologically suitable and mechanically sensitive regions close to the bone surfaces.     

Ultrastructure of ceramic-bone interface using hydroxyapatite and beta-tricalcium phosphate ceramics and replacement mechanism of beta-tricalcium phosphate in bone

Hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) are useful for grafting and augmentation of bone tissue. Observation by transmission electron microscopy (TEM) was done to investigate the ultrastructures at the interfaces between the biomaterials and the adjacent tissue, and osteogenesis around the biomaterials in the present study. HA and beta-TCP ceramics were used in disk forms which had macropores and micropores, and were implanted between the parietal bone and the cranial periosteum of rats. Specimens were prepared for observation at 4 and 8 weeks postoperatively. The microscopic results indicated that an intervening layer was present on the surface of HA, whereas it was not present on the surface of beta-TCP. A characteristic fibrillar structure was observed in the intervening layer between HA and bone under decalcification by HCl. In beta-TCP, in reticular structures observed close to the bone tissue by optical microscopy, calcification and sparse collagen fibers were interspersed among the granules of beta-TCP. In addition, close to the interface between beta-TCP and bone, many osteocytes with numerous processes were present. Some processes were elongated towards the interface. These results revealed the difference in the ultrastructures of the interfaces between HA and beta-TCP, and the dissolution mechanism of beta-TCP in bone.     

Calbindin-D9K immunolocalization and vitamin D-dependence in the bone of growing and adult rats

This report presents evidence for the presence of the vitamin D-dependent calcium-binding protein, calbindin-D9K, in bone cells and matrix. In undecalcified frozen sections of growing and adult rat bone, calbindin-D9K was immunohistochemically localized in trabecular bone of the epiphysis and metaphysis and in cortical bone of the diaphysis. It was found within the cytoplasm of osteocytes, of osteoblasts lining the osteoid, and osteoblasts inside the osteoid seams. It was also found in the osteoblast processes and the anastomosed reticulum of the processes connecting the osteocytes with each other. Extracellularly, calbindin-D9K immunoreactivity was present in compact cortical bone in the areas of the mineralized matrix surrounding the osteocyte lacunae, and in the pericanalicular walls containing the cell processes. Calbindin-D9K immunoreactivity was low or absent from the cytoplasm of osteocytes in trabecular bone from severely vitamin D-deficient rats and restored in vitamin D-deficient rats given a single dose of 1,25(OH)2-VitD3. Thus, the synthesis of immunoreactive calbindin-D9K by osteoblasts and osteocytes in trabecular bone is vitamin D-dependent. The presence of immunoreactive calbindin-D9K in the osteocytes and their cell processes suggests that this calcium-binding protein is involved in the calcium fluxes regulating bone calcium homeostasis. Its localization in osteoblasts involved in bone formation and in their cell processes suggests that it has a role in the calcium transport from these cells towards the sites of active bone mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)