An ultrastructural study of bone cells: The occurrence of microtubules,microfilaments and tight junctions

Bone cells of calvaria from young mice were studied at the ultrastructural level. Microtubules were demonstrated in both osteoblasts and osteocytes in the cell body, but not in the cell processes. Instead, the cytoplasm of cell processes is filled with bundles of 50 to 70 Å microfilaments, running parallel to the long axis of the process. Where two cell processes meet, the cell membranes form a tight junction. These junctions are found between osteocytes, between osteocytes and osteoblasts, and between bodies of osteoblasts on the cell surface. The cell processes usually meet side-to-side, thus forming an extended tight junction. The junctions between osteoblasts are short and are believed to be spot-like. Inside the bone scarcely any extracellular space is visible. The likelihood of intracellular transport is discussed.     

In situ imaging of the autonomous intracellular Ca(2+) oscillations of osteoblasts and osteocytes in bone

Bone cells form a complex three-dimensional network consisting of osteoblasts and osteocytes embedded in a mineralized extracellular matrix. Ca(2+) acts as a ubiquitous secondary messenger in various physiological cellular processes and transduces numerous signals to the cell interior and between cells. However, the intracellular Ca(2+) dynamics of bone cells have not been evaluated in living bone. In the present study, we developed a novel ex-vivo live Ca(2+) imaging system that allows the dynamic intracellular Ca(2+) concentration ([Ca(2+)](i)) responses of intact chick calvaria explants to be observed without damaging the bone network. Our live imaging analysis revealed for the first time that both osteoblasts and osteocytes display repetitive and autonomic [Ca(2+)](i) oscillations ex vivo. Thapsigargin, an inhibitor of the endoplasmic reticulum that induces the emptying of intracellular Ca(2+) stores, abolished these [Ca(2+)](i) responses in both osteoblasts and osteocytes, indicating that Ca(2+) release from intracellular stores plays a key role in the [Ca(2+)](i) oscillations of these bone cells in intact bone explants. Another possible [Ca(2+)](i) transient system to be considered is gap junctional communication through which Ca(2+) and other messenger molecules move, at least in part, across cell-cell junctions; therefore, we also investigated the role of gap junctions in the maintenance of the autonomic [Ca(2+)](i) oscillations observed in the intact bone. Treatment with three distinct gap junction inhibitors, 18α-glycyrrhetinic acid, oleamide, and octanol, significantly reduced the proportion of responsive osteocytes, indicating that gap junctions are important for the maintenance of [Ca(2+)](i) oscillations in osteocytes, but less in osteoblasts. Taken together, we found that the bone cells in intact bone explants showed autonomous [Ca(2+)](i) oscillations that required the release of intracellular Ca(2+) stores. In addition, osteocytes specifically modulated these oscillations via cell-cell communication through gap junctions, which maintains the observed [Ca(2+)](i) oscillations of bone cells.     

Biochemical and histological studies on various bone cell preparations

Summary Four different cell populations—designated PF, OB, OC, and PC—were isolated from calvaria of 18-day-old chick embryos for analysis of the effects of hormones on bone tissue. The cell populations were studied with histological and biochemical methods. Apart from the well-known cell types present in calvaria, a new cell type was found in the noncalcified organic matrix between the osteoblastic layer and the calcified matrix. These cells were provisionally called osteocytic osteoblasts. They represent the “transition state” between osteoblasts and osteocytes. On the basis of histological studies with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM), the PF population was considered to originate primarily from the periosteal fibroblasts, the OB population from the osteoblasts and osteocytic osteoblasts. The population of cells still present in calvaria after removal of periosteal fibroblasts and osteoblasts was called the OC population. This cell population was very much enriched with osteocytes. The fourth isolated population (PC) was a mixed population of fibroblasts, osteoblasts, and preosteoblasts. On exposure to parathyroid hormone (PTH), all four cell populations showed increased lactate production, but only the OB and OC populations displayed increased cAMP production. Prostaglandin E₁ (PGE₁) stimulated cAMP production in both OB and PF cells. From the results of this study it was concluded that PTH receptors are present on all of the cell types studied, but that occupancy of the receptor induces adenylate cyclase stimulation only in osteocytes and fully differentiated osteoblasts.     

Functional gap junctions between osteocytic and osteoblastic cells.

Morphological evidence shows that osteocytes, bone cells that exist enclosed within bone matrix, are connected to one another and to surface osteoblasts via gap junctions; however, it is unknown whether these gap junctions are functional. Using a newly established murine osteocytic cell line MLO-Y4, we have examined functional gap junctional intercellular communication (GJIC) between osteocytic cells and between osteocytic and osteoblastic cells. In our hands, MLO-Y4 cells express phenotypic characteristics of osteocytic cells including a stellate morphology, low alkaline phosphatase activity, and increased osteocalcin messenger RNA (mRNA) compared with osteoblastic cells. Northern and Western blot analysis revealed that MLO-Y4 cells express abundant connexin 43 (Cx43) mRNA and protein, respectively. Lucifer yellow dye transferred from injected to adjacent cells suggesting that osteocytic cells were functionally coupled via gap junctions. Functional GJIC between osteocytic and osteoblastic (MC3T3-E1) cells was determined by monitoring the passage of calcein dye between the two cell types using a double labeling technique. The ability of bone cells to communicate a mechanical signal was assessed by mechanically deforming the cell membrane of single MLO-Y4 cells, cocultured with MC3T3-E1 cells. Deformation induced calcium signals in MLO-Y4 cells and those elicited in neighboring MC3T3-E1 cells were monitored with the calcium sensitive dye Fura-2. Our results suggest that osteocytic MLO-Y4 cells express functional gap junctions most likely composed of Cx43. Furthermore, osteocytic and osteoblastic cells are functionally coupled to one another via gap junctions as shown by the ability of calcein to pass between cells and the ability of cells to communicate a mechanically induced calcium response.     

Formation of bone by isolated,cultured osteoblasts in millipore diffusion chambers

Summary Osteoblast-like and osteoclast-like cells freed from neonatal calvaria by sequential enzymatic digestion after 6–7 days in culture were placed in diffusion chambers and implanted in the peritoneal cavities of CD-1 mice. About half of the chambers also contained a dead calvarium to test for the need of an “inducer.” After 20 days, 11 of 18 chambers containing the osteoblast-like cells formed large foci of mineralized bone that corresponded to alkaline phosphatase activity throughout the chambers. Moreover, only type I (i.e., bone) collagen was formed. Occasional deposits of bone were found in only 3 of 22 chambers containing the osteoclast-like cells. The presence of dead bone did not affect any of the results. These data confirm the osteoblast-like nature of the isolated cell populations and demonstrate that these cells retain their differentiated function in culture.     

Prostaglandins change cell shape and increase intercellular gap junctions in osteoblasts cultured from rat fetal calvaria

Mounting experimental evidence indicates that osteoblasts may be cellular intermediaries in the local activation of bone remodeling. To elucidate the role of these cells in activation, we examined the effects of prostaglandins (PGs), known resorption stimulators, on cell shape and intercellular junctional relationships in osteoblasts cultured from rat fetal calvaria. Exposure to PGE2 and PGE1, promoters of bone resorption, rapidly (within 20 min) converted the osteoblasts from a flattened to a stellate shape (shape change), and markedly increased the appearance of intercellular (gap) junctions within 10 min. Both effects were directly related to the prostaglandin concentration, as little as 1 nM being effective. PGE1, but not PGB1, PGF1 alpha, PGD2, and PGF2 alpha, mimicked the substantial effect of PGE2 on shape change. Shape change and gap junction formation appear to arise independently. PTH, an inducer of shape change, did not affect the number of gap junctions appreciably. Colchicine, a microtubule polymerization inhibitor, and trifluoperazine, an inhibitor of calmodulin action, blunted PGE2-mediated shape change but not the effect of PGE2 on gap junctions. Shape change and gap junction formation may be important events in local activation, shape changes in surface osteoblasts serving to expose bone surfaces which are chemotactic for osteoclasts and gap junctions propagating locally initiated activation messages.     

Osteocyte: the unrecognized side of bone tissue

Introduction  

Osteocytes represent 95% of all bone cells. These cells are old osteoblasts that occupy the lacunar space and are surrounded by the bone matrix. They possess cytoplasmic dendrites that form a canalicular network for communication between osteocytes and the bone surface. They express some biomarkers (osteopontin, β3 integrin, CD44, dentin matrix protein 1, sclerostin, phosphate-regulating gene with homologies to endopeptidases on the X chromosome, matrix extracellular phosphoglycoprotein, or E11/gp38) and have a mechano-sensing role that is dependent upon the frequency, intensity, and duration of strain.     

Variation of increases in free cytosolic calcium ion induced by prostaglandin E2 in mouse osteoblast clone,MC3T3-E1—Single cell assay using microspectro-fluorometry

Osteoblasts exhibit multiple phenotypic expression in response to prostaglandin E₂ (PGE₂). Intracellular calcium concentration ([Ca²⁺] _i ) was elevated by PGE₂ treatment in the mouse osteoblast clone, MC3T3-E1, but the degree of elevation was varied by the day after subculturing. To study the different response to PGE₂, we have used microspectrofluorometry to measure [Ca²⁺] _i in a single MC3T3-E1 cell loaded with fura-2. In the presence of extracellular Ca²⁺, the increase in [Ca²⁺] _i in the osteoblast exhibited multiple patterns. The patterns were roughly classified into four groups by the time reached maximum level; “transient”, “gradual”, “transient and gradual” and “no response”. Within 2 days after subculturing, the cells showing “gradual” and “no response” were predominant, whereas after day 3 the cells showing “transient” and “transient and gradual” were predominant. We also investigated the daily change in the maximum level of [Ca²⁺] _i in the cells showed “transient” in response to PGE₂. The magnitude of [Ca²⁺] _i increase was also varied in cultivating period. These data suggest that there are phenotypic variations in a single cell even in a cloned cell line and this phenotype may change in the stage of cell proliferation.     

Passive accumulation of magnesium,sodium, and potassium by chick calvaria

Summary Four-day-old chick calvaria were used to determine the passive concentrations of magnesium, sodium, and potassium in metabolically poisoned bone. When incubated in buffers containing the blood levels of sodium and magnesium, these calvaria contained sodium and magnesium at the identical concentrations found in freshly dissected calvaria. Calvarial sodium and magnesium levels could be varied by altering the buffer concentrations of these cations. The potassium content of metabolically poisoned calvaria incubated in buffers containing 4 mM potassium was less than 20% of the content of freshly dissected calvaria. When the buffer concentrations of sodium and potassium were systematically varied, ouabain-poisoned calvaria concentrated these cations in the bone extracellular fluid by a factor of approximately two above buffer cation levels. Presumably, the hydroxyapatite crystal zeta potential is responsible for this concentrative phenomenon. These results are discussed in terms of the control of the ionic content of the bone extracellular fluid by the postulated “bone membrane.”     

On the measurement of water compartments,pH, and gradients in calvaria

Summary The concept of fluid compartmentalization in bone has emphasized the need for and lack of suitable methodology for the quantitation of water “spaces” in bone, in particular intracellular water, ICF, and extracellular water, BECF. Over a dozen commonly used marker substances were studied intensively. Small columns of hydroxyapatite crystals were employed to reveal physiochemical interactions with the mineral phase, and isotope distributions in live and dead calvaria (rat pup, and adult mice) were employed to evaluate interactions with the combined organic-inorganic matrix of bone itself. The results were most discouraging. For example, for total water space, only water itself (either by direct weight or by³H₂O exchange) is a reliable measure in all instances. All markers studied were passively concentrated or excluded to varying degrees. In the end, it was necessary to measure intracellular space “by difference.” An extracellular marker, polyethylene glycol (¹⁴C-, mol. wt. 4000), was incubated with viable calvariae and also with comparable calvariae having lysed cells (0.1% Triton X-100). The intracellular space thus determined by difference agreed well with correlative data obtained on mixed cell-isolates from similar specimens. In neonatal calvariae, the intracellular space was 30% of the total water present; that of adult mouse tibia, 18%; and that of adult mouse calvariae, 14.5%. Measurements of dimethyl-2,4-oxazolidinedione (DMO) distribution in live and lysed calvaria revealed an overall pH differential (inside bone vs. medium) of 0.1 unit or less. Using the best available data for ICF, BECF, and the passive concentrating effects of matrix-K⁺ interactions, there still remained an unexplained excess of K⁺ in the BECF.     

Twisted plywood architecture of collagen fibrils in human compact bone osteons

Summary Ultrathin sections of decalcified human compact bone, observed by transmission electron microscopy, reveal that collagen fibrils can be distributed in the form of a superimposed series of nested arcs. This characteristic pattern has never been interpreted in previous works on compact bone structure. We demonstrate, by goniometric observations at the ultrastructural level, that such series of nested arcs are a consequence of the “twisted plywood” architecture of collagen fibrils in the compact bone matrix. In the same specimens, an “orthogonal plywood” disposition of collagen fibrils is also observed; a transition exists between these two types of orders. We show that the “twisted plywood structure” accounts well for certain optical properties of osteons, observed in polarizing microscopy, described as “intermediate osteons.” The particular geometry of collagen fibrils, leading to nested arcs in oblique sections, is analogous to the distribution of molecules in certain liquid crystals (called cholesteric liquid crystals). The principle of a liquid crystalline self-assembly of the collagen matrix in bone is therefore discussed.     

Specialized Cell Contacts and the Blood-testis Barrier in the Seminiferous Tubules of the Domestic Fowl (Gallus domesticus)

The ultrastructure of Sertoli-Sertoli and Sertoli-germ cell surface specializations in the domestic fowl was studied in material fixed by vascular perfusion through the thoracic aorta. Three main types of surface specializations were found between adjacent Sertoli cells. These are focal tight junctions, desmosome-like devices, and a specialization characterized by the presence of long and dilated subsurface cisternae of rough endoplasmic reticulum. Typical inter-Sertoli cell junctions similar to those of mammals were absent. Germ cells were attached to Sertoli cells mainly by desmosome-like devices of varying appearance. The junctions between Sertoli cells and elongating or elongated spermatids, "the mantle", consisted of only slight condensations of filamentous material in the Sertoli cell. The tight junctions between adjacent Sertoli cells were efficient in preventing lanthanum from passing towards the lumen beyond the level of the spermatogonia.     

Bone Quality Parameters of the Distal Radius as Assessed by pQCT in Normal and Fractured Women

The aim of this study was to test the ability of some indicators of different aspects of bone quality (assessed by peripheral quantitative computed tomography in the distal radius) to discriminate between fractured and nonfractured individuals. The study compared 214 women aged 45–85 years, free of any bone-affecting treatment, of whom 107 had suffered a Colles” fracture in the previous 6 months and 107 did not. The determinations included bone tissue or mineral “mass” indicators (trabecular, cortical and total volumetric mineral content, cortical bone area); bone “density” estimates (trabecular, cortical and total volumetric mineral density), and the Cartesian (rectangular) and polar moments of inertia as influences of cross-sectional architecture on resistance to bending and torsional loads, respectively.  The influences of body height, weight and age on the tomographic indicators were minimized by adjusting the data according to the partial coefficients of multiple stepwise regressions. The adjusted values of all the indicators were lower in fractured than in nonfractured groups. The prevalence of fractures was directly related to the actual values of the indicators, rather than the age or body habitus of the individuals. The significance of these differences between the assessed indicators decreased in the following order: trabecular “mass” > trabecular “density” > cortical or total “mass” > cortical architecture > total or cortical “density” indicators. Within the same type of bone, the tissue or mineral “mass” indicators performed better than the “density” indicators. The cortical bone density did not give useful information, probably because of technical difficulties. Odds-ratios and receiver-operating characteristic (ROC) analyses confirmed those features. The selected “cut-off” values of the indicators as determined by the ROC curves (very close to those determined by the inflexion points of the logistic reression curves) may indicate reference limits to detect persons at risk of fracture according to the type of information provided by each variable. These results show that these tomographic indicators discriminate well between fractured and nonfractured individuals, and should be suitable to assess how total, cortical and trabecular bone strength in the distal radius could affect different kinds of strength regardless of the age or body habitus of the individual. Their ability to estimate fracture risk from different biomechanical points of view should be assessed by adequately designed, prospective studies. Received: June 2000 / Accepted: January 2001     

Evidence for capping of Fcγ receptors on osteoclasts

Summary Fc receptors, cell surface structures which bind the Fc portion of immunoglobulins, facilitate endocytosis and mediate triggered enzyme release in the phagocytic cells of the mononuclear phagocyte system (MPS). The osteoclast and the macrophage share many similarities, and it has been suggested that they are both derived from a common MPS precursor cell. Nevertheless, Fcγ receptors, found on both primitive and well-differentiated cells of the mononuclear phagocyte system, have yet to be demonstrated on the osteoclast cell membrane. The aim of the present study was first, to study the distribution of rat endogenous IgG around resorbing bone using immunohistochemistry and the avidin-biotin-peroxidase (ABC) technique; and second, to determine if rat osteoclasts express Fcγ receptors, using a recently described technique based on the ABC method. Observations that endogenous IgG accumulated at osteoclast/bone interfaces, and that Fcγ receptors, expressed by osteoclasts, “capped” in the same area, have important implications regarding the origin of this cell and its mechanisms of bone attachment and resorption.     

Osteoclasts,mononuclear phagocytes,and physiological bone resorption

Conclusions The concerns expressed here, as well as those noted by others in recent reviews, underscore the confusion that has entered the literature regarding the role of osteoclasts, as distinct from that of mononuclear phagocytes, in bone resorption. This confusion could be resolved if the following suggestions are adopted. First, the term “resorption” or “bone resorption” should be reserved for describing a process of bone removal in which osteoclasts are the primary function cell type. Second, the term osteoclast should be used to define a bone-resorbing cell characterized by the criteria presented earlier. Third, until unambiguous evidence is provided, it must not be assumed that cells of the mononuclear phagocyte series, such as peripheral blood monocytes or elicited peritoneal macrophages, are osteoclast precursors.     

Current Concepts in Scar Evolution and Control

The basic principles influencing scar expression and outcome have long been defined. Although these were relatively clear at the time, the exact events at a molecular level were poorly defined. The past decade has delineated the myriad of events that occur in the run-up to scar evolution far more clearly, although the intricate details have yet to be elucidated. What is clear is that a series of conversations and crosstalk takes place in the cell cytosol, in the cellular nucleus, and outside the cell within in the extracellular matrix. This interaction or “dynamic reciprocity” takes place via a series of signals, protein activation, ionic translocations, and receptor transactions. Marrying the previously defined principles with current described cellular/extracellular matrix (ECM) interactions enables us to describe more accurately the crosstalk occurring in scar evolution and possibly to influence the “wording” of that crosstalk to improve scar outcome. Thus, the principles of mechanostimulation and scar support, hydration occlusion, controlled inflammation, and collagen/extracellular remodeling are discussed with possible interventions in each category.     

Historical Evolution of Limb Amputation

The amputation of a limb is one of the oldest surgical procedures. In the course of medical history operative techniques and surgical instruments have been improved continuously. As early as the first century Celsus described an amputation. A major step in the development of the operative technique was the introduction of an artery forceps by Paré during the sixteenth century. Nevertheless, due to a lack of analgesics and narcotics the operation had to take only a few minutes. Therefore the amputation was completed in one cut (i.e., detachment of the skin, muscles, and bone at the same level). This technique, known as “classic circular cut,” was modified several times in the following period: to reduce suture tension Petit recommended that we transect the skin first and the muscles and bone more proximally (“two-stage circular cut,” 1718), and Bromfield approved that the skin be cut first, the muscles more proximally and the bone most proximal (“three-stage circular cut,” 1773). Lowdham (1679), Verduyn (1696), and Langenbeck (1810) changed the operative technique in that they used a soft-tissue flap to cover the bone without tension (“flap amputation”).