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Recent advances have revived interest in the concept of osteocyte perilacunar/canalicular remodeling (PLR) and have motivated efforts to identify the mechanisms regulating this process in bone in the context of normal physiology and pathological conditions. Here, we describe several methods that are evaluating morphological changes associated with PLR function of osteocytes.

Purpose of Review: Osteocytes are the most abundant bone cells. They are completely encased in mineralized tissue, sitting inside lacunae that are connected by a multitude of canaliculi. In recent years, the osteocyte network has been shown to fulfill endocrine functions and to communicate with a number of other organs. This review addresses emerging knowledge on the connectome of the lacunocanalicular network in different types of bone tissue., Recent Findings: Recent advances in three-dimensional imaging technology started to reveal parameters that are well known from general theory to characterize the function of networks, such as network density, degree of nodes, or shortest path length through the network. The connectome of the lacunocanalicular network differs in some aspects between lamellar and woven bone and seems to change with age. More research is needed to relate network structure to function, such as intercellular transport or communication and its role in mechanosensation, as well as to understand the effect of diseases.

Purpose of Review: In perilacunar/canalicular remodeling (PLR), osteocytes dynamically resorb, and then replace, the organic and mineral components of the pericellular extracellular matrix. Given the enormous surface area of the osteocyte lacuna-canalicular network (LCN), PLR is important for maintaining homeostasis of the skeleton. The goal of this review is to examine the motivations and critical considerations for the analysis of PLR, in both in vitro and in vivo systems., Recent Findings: Morphological approaches alone are insufficient to elucidate the complex mechanisms regulating PLR in the healthy skeleton and in disease. Understanding the role and regulation of PLR will require the incorporation of standardized PLR outcomes as a routine part of skeletal phenotyping, as well as the development of improved molecular and cellular outcomes. Current PLR outcomes assess PLR enzyme expression, the LCN, and bone matrix composition and organization, among others. Here, we discuss current PLR outcomes and how they have been applied to study PLR induction and suppression in vitro and in vivo. Given the role of PLR in skeletal health and disease, integrated analysis of PLR has potential to elucidate new mechanisms by which osteocytes participate in skeletal health and disease.

In order for osteocytes to perceive mechanical information and regulate bone remodeling accordingly they must be anchored to their extracellular matrix (ECM). To date the nature of this attachment is not understood. Osteocytes are embedded in mineralized bone matrix, but maintain a pericellular space (50-80 nm) to facilitate fluid flow and transport of metabolites. This provides a spatial limit for their attachment to bone matrix. Integrins are cell adhesion proteins that may play a role in osteocyte attachment. However, integrin attachments require proximity between the ECM, cell membrane, and cytoskeleton, which conflicts with the osteocytes requirement for a pericellular fluid space. In this study, we hypothesize that the challenge for osteocytes to attach to surrounding bone matrix, while also maintaining fluid-filled pericellular space, requires different "engineering" solutions than in other tissues that are not similarly constrained. Using novel rapid fixation techniques, to improve cell membrane and matrix protein preservation, and transmission electron microscopy, the attachment of osteocyte processes to their canalicular boundaries are quantified. We report that the canalicular wall is wave-like with periodic conical protrusions extending into the pericellular space. By immunohistochemistry we identify that the integrin alphavbeta3 may play a role in attachment at these complexes; a punctate pattern of staining of beta3 along the canalicular wall was consistent with observations of periodic protrusions extending into the pericellular space. We propose that during osteocyte attachment the pericellular space is periodically interrupted by underlying collagen fibrils that attach directly to the cell process membrane via integrin-attachments., ((c) 2009 Wiley-Liss, Inc.)

In order to evaluate the influence of masticatory movement on cranial bone mineral density (BMD) and osteocyte and osteoblast micromorphology, we conducted a study in rats fed with solid feed (n=10) and powdered feed (n=10). Cranial BMD was measured by dual X-ray absorptiometry (DXA). Osteocyte morphology was evaluated by light microscopy. In addition, some of the tissue was treated with EDTA-KOH to digest the bone matrix and prepare osteocyte samples. Micromorphology of the osteocytes was examined by scanning electron microscopy (SEM). Bone mineral content (BMC) was significantly higher in the solid feed group (1.86 +/- 0.11 g) than in the powdered feed group (1.63 +/- 0.09 g) (p < 0.05). In the solid feed group, the maximum masseter muscle tension when crushing solid feed was 305 +/- 46.0 N. Immunohistochemical staining with DMP-1 showed greater positive localization of DMP-1 in bone lacunae in the solid feed group than in the powdered feed group. On examination of cranial bone sections by SEM, the lacunar area was significantly larger in the solid feed group (0.64 +/- 0.08 microm2) than in the powdered feed group (0.43 +/- 0.10 microm2) (p < 0.01). In the solid feed group, adjacent osteocytes were connected through cytoplasmic branches and reticular cell processes to form a 3-dimensional structure. In the powdered feed group, connecting osteocyte processes were sparser. Results suggest that masticatory movement stimulates mediator substances involved in dynamic interactions between osteocytes, increases cranial bone mass during the developmental period and influences osteoblast, osteocyte, and lacunar micromorphology.

The morphology of the osteocyte changes during the cell's lifetime. Shortly after becoming buried in the matrix, an osteocyte is plump with a rich rough endoplasmic reticulum and a well-developed Golgi complex. This "immature" osteocyte reduces its number of organelles to become a "mature" osteocyte when it comes to reside deeper in the bone matrix. We hypothesized that mineralization of the surrounding matrix is the trigger for osteocyte maturation. To verify this, we prevented mineralization of newly formed matrix by administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and then examined the morphological changes in the osteocytes in rats. In the HEBP group, matrix mineralization was disturbed, but matrix formation was not affected. The osteocytes found in the unmineralized matrix were immature. Mature osteocytes were seen in the corresponding mineralized matrix in the control group. The immature osteocytes in the unmineralized matrix failed to show immunoreactivity with anti-sclerostin antibody, whereas mature osteocytes in the mineralized matrix showed immunoreactivity in both control and HEBP groups. These findings suggest that mineralization of the matrix surrounding the osteocyte is the trigger for cytodifferentiation from a plump immature form to a mature osteocyte. The osteocyte appears to start secreting sclerostin only after it matures in the mineralized bone matrix.

Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.

In many multi-disciplinary fields of science, such as tissue engineering, where material and biological sciences are combined, there is a need for a tool that combines ultrastructural and chemical data analysis in a non-destructive manner at high resolution. We show that a combination of confocal Raman spectroscopy (CRS) and scanning electron microscopy (SEM) can be used for such analysis. Studies of atomic composition can be done by X-ray microanalysis in SEM, but this is only possible for atomic numbers greater than five and does not reveal molecular identity. Raman spectroscopy, however, can provide information on molecular composition and identity by detection of wavelength shifts caused by molecular vibrations. In this study, CRS-SEM revealed that early in vitro-formed bone extracellular matrix (ECM) produced by rat osteoprogenitor cells resembles mature bone chemically. We gained insight into the structure and chemical composition of the ECM, which was composed of mainly mineralized collagen type I fibres and areas of dense carbonated calcium phosphate related to the collagen fibre density, as revealed by Raman imaging of SEM samples. We found that CRS-SEM allows the study of specimens in a non-destructive manner and provides high-resolution structural and chemical information about inorganic and organic constituents by parallel measurements on the same sample.

Nucleobindin (Nuc), also known as CALNUC, is a Ca(2+)-binding protein, located in the nucleus, the Golgi apparatus and the endoplasmic reticulum (ER). The presence of a signal sequence in Nuc suggests secretion from the cell and it has been found in bone extracellular matrix. Within the present study, molecular biological and morphological methods were combined to evaluate the synthesis and distribution of Nuc in and around cells of rat metaphyseal and calvarial bone. Northern blot analysis and in situ hybridization of bone tissues confirmed that the protein was a product of bone cells. By electron microscopy, immunolabeling for Nuc was seen in osteoid of newly formed bone, on all surfaces facing the various bone cells and also in compact bone. Intracellularly, the gold particles were found in the rough ER of osteoblasts, which suggested synthesis of the protein by these cells. Compared to bone sialoprotein and osteopontin, Nuc demonstrated different localization pattern in bone trabeculae, with the majority of labeling restricted to nonmineralized osteoid. Moreover, the role of Nuc during the mineralization process was investigated in rat calvaria-derived primary osteoblasts grown under osteogenic conditions. Semiquantitative RT-PCR and Northern blot analysis showed Nuc expression to be low during cell proliferation, upregulated during differentiation and matrix maturation, but subsequently downregulated during mineralization. In summary, our data show that Nuc was synthesized by osteoblasts and osteocytes, and secreted into the osteoid, suggesting a role as a modulator of matrix maturation in the mineralization process in bone.

Osteocytes are believed to be the mechanical sensor cells in bone. One potential physical mechanism for the mechanosensing process is that osteocytes directly sense the deformation of the substrate to which they are attached. However, there is a fundamental paradox in this theory: tissue-level strains in whole bone are typically <0.2%, yet an extensive range of in vitro experiments show that dynamic substrate strains must be at least an order of magnitude larger in order for intracellular biochemical responses to occur. Recently, a theoretical model was developed (You et al. J. Biomech., 2001; 34:1375-1386) that provides a possible mechanism by which mechanical loading-induced fluid flow in the lacuno-canalicular system, under routine physical activity, can produce cellular-level strains on the osteocyte processes that are at least one order of magnitude larger than bone tissue deformations. This would resolve the fundamental paradox mentioned above. In this work we experimentally confirm and quantify the essential ultrastructural elements in this model: 1) the presence of the transverse elements that bridge the pericellular space surrounding the osteocyte process, which interact with the fluid flow and lead to an outward hoop tension on the process; and 2) the presence of bundled F-actin in the osteocyte processes, which resists the outward hoop tension and limits the cell process membrane deformation. Morphological data to support these assumptions are scant. Special staining techniques employing ruthenium III hexamine trichloride (RHT) were developed to elucidate these structures in the humeri of adult mice., (Copyright 2004 Wiley-Liss, Inc.)

Objective: To investigate the characteristics of ectopic chondroid/bone matrix and chondrogenic/osteogenic cells induced by recombinant human bone morphogenetic protein-2 (rhBMP-2)., Materials and Methods: rhBMP-2 (5 microg) combined with atelocollagen was implanted into calf muscles of rats and removed on days 7, 10, 14, 21, or 28. Tissue sections were examined using: (i) hematoxylin/Alcian blue/Sirius red stain, (ii) enzyme histochemistry for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase activity, (iii) immunohistochemistry for types I, II, and X collagen, and (iv) electron microscopy., Results: On day 7, numerous fibroblast-like cells with ALP activity were present on the pellet rim. On day 10, chondroid matrix (CM) had formed, contained both type I collagen and proteoglycans, and often continued into the BMP pellet. On day 14, bone-like matrix formed around hypertrophic chondrocytes simultaneously with endochondral ossification. Coexpression of types I and II collagen within chondrocytes and osteocytes was observed throughout the time course of the experiment., Conclusion: These results suggest that fibroblast-like cells invading the pellet differentiate into chondrocytes and form CM under the scaffold of the carrier component. It appears that some chondrocytes change their phenotype to produce the bone-like matrix and remain within the endochondral bone. This process enables rapid osteogenesis to occur.

We examined the histology and morphometry of biosamples (biopsies) of the iliac crest of monkeys, flown 14 days aboard the "Bion-11", using electron microscopy. We found, that some young osteocytes take part in the activation of collagen protein biosynthesis in the adaptive remodeling process of the bone tissue to microgravity conditions. Osteocyte lacunae filled with collagen fibrils; this correlates with fibrotic osteoblast reorganization in such zones. The osteolytic activity in mature osteocytes is intensified. As a result of osteocyte destruction, the quantity of empty osteocytic lacunae in the bone tissue increases., (c2002 COSPAR. Published by Elsevier Science Ltd. All rights reserved.)

Bone induction by different calcium phosphate biomaterials has been reported previously. With regard to (1) whether the induced bone would disappear with time due to the absence of mechanical stresses and (2) whether this heterotopically formed bone would give rise to uncontrolled growth, a long-time investigation of porous hydroxyapatite ceramic (HA), porous biphasic calcium phosphate ceramic (TCP/HA, BCP), porous alpha-tricalcium phosphate ceramic (alpha-TCP) and porous beta-tricalcium phosphate ceramic (beta-TCP) was performed in dorsal muscles of dog, for 2.5 years. Histological observation, backscattered scanning electron microscopy observation and histomorphometric analysis were made on thin un-decalcified sections of retrieved samples. Normal compact bone with bone marrow was found in all HA implants (n = 4) and in all BCP implants (n = 4), 48 +/- 4% pore area was filled with bone in HA implants and 41 +/- 2% in BCP implants. Bone-like tissue, which was a mineralised bone matrix with osteocytes but lacked osteoblasts and bone marrow, was found in all beta-TCP implants (n = 4) and in one of the four alpha-TCP implants. Both normal bone and bone-like tissues were confined inside the pores of the implants. The results show that calcium phosphate ceramics are osteoinductive in muscles of dogs. Although the quality and quantity varied among different ceramics, the induced bone in both HA and BCP ceramics did neither disappear nor grow uncontrollably during the period as long as 2.5 years.

Alveolar bone defects are conditions that impede the progress of orthodontic treatment. This study compared the mechanics of the healing of autogenous intramembranous (IM) bone grafts and grafts comprising a mixture of IM and demineralised bone matrix of autogenous intramembranous origin (DBMIM), in an attempt to determine the reliability of each material. Thirty-two New Zealand white rabbits had a single defect created in their skull. Sixteen were grafted with IM bone alone (Group I: autogenous IM), and the other 16 had a combined graft of composite IM sandwiched between two layers of DBMIM (Group II: composite IM-DBMIM). A third group (Group III) of eight rabbits each had two defects created in their skull; one defect was left empty (A: passive control) and the other filled with rabbit-skin collagen (B: active control). In Groups I and II, inflammatory cells were found to be present on Days 1 and 2 of tissue retrieval. The appearance of the mesenchymal cells and preosteoblasts, osteoblasts and osteocytes was earlier (Day 3) in Group II than in Group I (Day 5). In both groups, preosteoblasts, osteoblasts and osteocytes were observed with no cartilage at the intermediate stage. In conclusion, autogenous IM bone grafts and IM bone grafts in the presence of DBMIM healed through an osteogenic ossification route.

The tissue response of rat tibiae to the surgical placement of commercially pure titanium implants was examined at 2, 6, 10, and 28 days. The transcortical placement of 1.5-mm x 2-mm implants resulted in the apposition of threaded implant surfaces within cortical and cancellous regions of the tibia. In all regions, evidence of bone formation was obtained through pre-embedding fracture of the implant from the bone tissue interface. Scanning electron microscopy examination of early responses revealed a fibrin clot and rapid formation of a loosely organized collagenous matrix. Many extravasated blood cells contacted the implant surface. At day 6, a more organized matrix containing many blood vessels opposed the implant surfaces, and few extravasated blood cells remained in contact with the implant surface. By day 10, the surgical wound was filled with woven bone that approximated the contours of the threaded implant. Later, few cells were attached to the retrieved implants. The consolidation of the forming matrix was clearly evident at 28 days. The tissue interface was an amorphous matrix that revealed the surface characteristics of the machined implant. Light microscopic analysis of ground sections indicated that, from day 6 onward, cells morphologically consistent with the osteoblastic phenotype were predominant within the gap between the surgical margin and implant surface. Osteoblastic cells had achieved the formation of an osteoid seam upon which bone formation progressed. The matrix that had formed represented woven bone containing many osteocytes. At day 6, evidence of remodeling was observed at sites distant from the surgical site, and by day 28 osteoclastic activity was observed at trabecular sites adjacent to the implant surface. The rat tibia model provides evidence of rapid formation of bone at implant surfaces.

Osteocyte canalicular density (OCD) was evaluated at different levels of the wall of human secondary osteons, in subjects of different ages, to find out whether any correlation exists between the extension of the canalicular network and the exponential decrement of the appositional growth rate (AGR), which has been shown to occur during osteon formation. Scanning electron microscopy (SEM) was used to count the number of canalicular openings per unit surface on large Haversian canals of forming osteons as well as on small canals of completed osteons. Reflected polarized light microscopy (RPL) enabled the number per unit length of canaliculi to be counted at different concentric levels of the osteons. The results of both techniques agree in showing that, in the subjects examined, OCD does not change significantly throughout the osteon wall. Since no correlation exists between OCD and AGR, it follows that osteoblast flattening which was shown to occur in parallel to the decrement of the rate of concentric bone deposition, does not seem to depend primarily on the number of osteoblast-osteocyte contacts, but on other factors.

A monoclonal antibody raised against alkaline phosphatase (ALP) of human osteosarcoma was used to localize this enzyme in human fetal bone tissue. For light microscopy, the presence of alkaline phosphatase in osteoblasts and osteocytes was demonstrated by use of an avidin-biotin immunoperoxidase procedure. Electron microscopic immunolocalization was accomplished with an indirect immunoperoxidase method which revealed a concentration of the enzyme on matrix vesicle and osteoblast plasma membranes. In addition, many vesicular protrusions arising from areas of plasma membrane on the lateral surfaces of adjacent osteoblasts were strongly immunolabeled. Immunostaining for ALP was absent in vesicles which contained fine crystallites. Alkaline-phosphatase-rich matrix vesicles may play a significant role in the mineralization of the extracellular matrix.

This electron microscope study describes the subcellular occurrence and distribution of immunoreactive calbindin-D9K in the trabecular metaphyseal and compact cortical bone of normal rats, rachitic vitamin-D-deficient rats, and rachitic rats given 1,25-(OH)2D3. Undecalcified bones were embedded in Lowicryl K4M and calbindin-D9K antigenicity was detected by the protein A-gold method. Immunoreactive calbindin-D9K was localized in the cytoplasm and cell processes of osteoblasts and osteocytes. Immunoreactive calbindin-D9K was also found within matrix vesicles and calcifying matrix vesicles, where it lay over the needle-shaped crystallites, at the apparent site of initial crystal formation, but not along the whole crystallites. In fully mineralized bone it occurred at the same site, over the crystallites. Calibindin-D9K was vitamin-D-dependent in the osteoblasts and matrix vesicles, where its presence was correlated with the reappearance of crystallites in 1,25-(OH)2D3-treated vitamin-D-deficient rats. This suggests that immunoreactive calbindin-D9K is involved in mineral deposition in bone matrix vesicles. Abnormal intracellular calcification associated with calbindin-D9K antigenicity in the osteoblasts of 1,25-(OH)2D3-treated vitamin-D-deficient rats indicates that immunoreactive calbindin-D9K may also play a part in abnormal intracellular mineral deposition.

Tibiae of young rats were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in order to study the origin and structure of matrix vesicles in periosteal bone. SEM studies have shown that periosteal osteoblasts have elongated processes with globular structures of 0.1 micron in diameter attached to the cell surface and processes. Similar structures were found to cover the inner surface of osteoblastic lacunae. The SEM studies have further shown that in the periosteal surface (forming bone) the above-described globules once mineralized, aggregated to form larger, nonhomogeneous mineralized spherules in which, by proper treatment with NaOCl, hydroxyapatite crystals could be exposed. Endosteal osteoblasts had fewer processes, devoid of the globular structures. Similarly, osteocytic and osteoclastic processes, although elongated and numberous, were not covered by the globular structures. In the matrix, collagen fibers of forming bone were randomly orientated, while in the deeper areas of bone they formed bundles with a longitudinal orientation. TEM studies have shown that the structures found on the osteoblastic surface and in the matrix are membrane-bound matrix vesicles which seem to be formed by budding from cell processes. Preformed membrane-bound vesicles were also observed by TEM inside sections of osteoblastic processes. These vesicles resembled the extracellular matrix vesicles in size and shape, thus giving the impression that at least some of the matrix vesicles are preformed cellular structures. While comparing SEM with TEM, it can be conducted that in bone, as in cartilage, matrix vesicles which probably serve as the initial locus of calcification, are formed directly by osteoblasts.