Apoptosis during intramembranous ossification

This paper concerns the role of apoptosis during the onset of bone histogenesis. Previous investigations by us performed on intramembranous ossification revealed the existence of two types of osteogenesis: static (SBF) and dynamic bone formation (DBF). During SBF, the first to occur, stationary osteoblasts transform into osteocytes in the same location where they differentiated, forming the primary spongiosa. DBF takes place later, when movable osteoblastic laminae differentiate along the surface of the primary trabeculae. The main distinctive feature between SBF and DBF is that the latter involves the invasion of pre-existing adjacent tissue, whereas the former does not. To ascertain whether programmed cell death during the invasive DBF process determines the fate of surrounding pre-existing mesenchyme differently from that occurring during the non-invasive SBF process, we studied apoptosis in ossification centres of tibial diaphysis in chick embryos and newborn rabbits with TUNEL and TEM. It emerged that, in both SBF and DBF, apoptosis affects mesenchymal cells located between the forming trabeculae and capillaries. However, apoptotic cells were observed more frequently during DBF than during SBF. This suggests that, during bone histogenesis, apoptosis, which is mostly associated with the invasive process of DBF, is probably dedicated to making space for advancing bone growth.     

Static and dynamic osteogenesis: two different types of bone formation

The onset and development of intramembranous ossification centers in the cranial vault and around the shaft of long bones in five newborn rabbits and six chick embryos were studied by light (LM) and transmission electron microscopy (TEM). Two subsequent different types of bone formation were observed. We respectively named them static and dynamic osteogenesis, because the former is characterized by pluristratified cords of unexpectedly stationary osteoblasts, which differentiate at a fairly constant distance (28+/-0.4 microm) from the blood capillaries, and the latter by the well-known typical monostratified laminae of movable osteoblasts. No significant structural and ultrastructural differences were found between stationary and movable osteoblasts, all being polarized secretory cells joined by gap junctions. However, unlike in typical movable osteoblastic laminae, stationary osteoblasts inside the cords are irregularly arranged, variously polarized and transform into osteocytes, clustered within confluent lacunae, in the same place where they differentiate. Static osteogenesis is devoted to the building of the first trabecular bony framework having, with respect to the subsequent bone apposition by typical movable osteoblasts, the same supporting function as calcified trabeculae in endochondral ossification. In conclusion, it appears that while static osteogenesis increases the bone external size, dynamic osteogenesis is mainly involved in bone compaction, i.e., in filling primary haversian spaces with primary osteons.     

Does static precede dynamic osteogenesis in endochondral ossification as occurs in intramembranous ossification?

Endochondral ossification takes place with calcified cartilage cores providing a rigid scaffold for new bone formation. Intramembranous ossification begins in connective tissue and new bone formed by a process of static ossification (SO) followed by dynamic ossification (DO) as previously described. The aim of the present study was to determine if the process of endochondral ossification is similar to that of intramembranous ossification with both a static and a dynamic phase of osteogenesis. Endochondral ossification centers of the tibiae and humeri of newborn and young growing rabbits were studied by light and transmission electron microscopy. The observations clearly showed that in endochondral ossification, the calcified trabeculae appeared to be lined first by osteoclasts. The osteoclasts were then replaced by flattened cells (likely cells of the reversal phase) and finally by irregularly arranged osteoblastic laminae, typical of DO. This cellular sequence did not include osteoblasts seen in the phase of SO. These findings clearly support our working hypothesis that SO only forms in soft tissues to provide a rigid framework for DO, and that DO requires a rigid mineralized surface. The presence of osteocytes in contact with the calcified cartilage also suggests the existence of stationary osteoblasts in endochondral ossification. Stationary osteoblasts did not appear to be a unique feature of SO. The presence of stationary osteoblasts may appear to provide the initial osteocytes during osteogenesis that may function as mechanosensors throughout the bone tissue. If this is the case, then bone would be capable of sensing mechanical strains from its inception.     

Osteoblast‐osteocyte transformation. A SEM densitometric analysis of endosteal apposition in rabbit femur

Transformation of osteoblasts into osteocytes is marked by changes in volume and cell shape. The reduction of volume and the entrapment process are correlated with the synthesis activity of the cell which decreases consequently. This transformation process has been extensively investigated by transmission electron microscopy (TEM) but no data have yet been published regarding osteoblast-osteocyte dynamic histomorphometry. Scanning electron microscope (SEM) densitometric analysis was carried out to determine the osteoblast and open osteocyte lacunae density in corresponding areas of a rabbit femur endosteal surface. The lining cell density was 4900.1 ± 30.03 n mm⁻², the one of open osteocyte lacunae 72.89 ± 22.55 n mm⁻². This corresponds to an index of entrapment of one cell every 67.23 osteoblasts (approximated by defect). The entrapment sequence begins with flattening of the osteoblast and spreading of equatorial processes. At first these are covered by the new apposed matrix and then also the whole cellular body of the osteocyte undergoing entrapment. The dorsal aspect of the cell membrane suggests that closure of the osteocyte lacuna may be partially carried out by the same osteoblast-osteocyte which developed a dorsal secretory territory. A significant proportion of the endosteal surface was analysed by SEM, without observing any evidence of osteoblast mitotic figures. This indicates that recruitment of the pool of osteogenic cells in cortical bone lamellar systems occurs prior to the entrapment process. No further additions occurred once osteoblasts were positioned on the bone surface and began lamellar apposition. The number of active osteoblasts on the endosteal surface exceeded that of the cells which become incorporated as osteocytes (whose number was indicated by the number of osteocyte lacunae). Therefore such a balance must be equilibrated by the osteoblasts'' transformation in resting lining cells or by apoptosis. The current work characterised osteoblast shape changes throughout the entrapment process, allowing approximate calculation of an osteoblast entrapment index in the rabbit endosteal cortex.     

Buried alive: how osteoblasts become osteocytes.

During osteogenesis, osteoblasts lay down osteoid and transform into osteocytes embedded in mineralized bone matrix. Despite the fact that osteocytes are the most abundant cellular component of bone, little is known about the process of osteoblast-to-osteocyte transformation. What is known is that osteoblasts undergo a number of changes during this transformation, yet retain their connections to preosteoblasts and osteocytes. This review explores the osteoblast-to-osteocyte transformation during intramembranous ossification from both morphological and molecular perspectives. We investigate how these data support five schemes that describe how an osteoblast could become entrapped in the bone matrix (in mammals) and suggest one of the five scenarios that best fits as a model. Those osteoblasts on the bone surface that are destined for burial and destined to become osteocytes slow down matrix production compared to neighbouring osteoblasts, which continue to produce bone matrix. That is, cells that continue to produce matrix actively bury cells producing less or no new bone matrix (passive burial). We summarize which morphological and molecular changes could be used as characters (or markers) to follow the transformation process.     

Localization of tartrate-resistant acid phosphatase (TRAP), membrane type-1 matrix metalloproteinases (MT1-MMP) and macrophages during early endochondral bone formation

Endochondral bone formation, the process by which most parts of our skeleton evolve, leads to the establishment of the diaphyseal primary (POC) and epiphyseal secondary ossification centre (SOC) in long bones. An essential event for the development of the SOC is the early generation of vascularized cartilage canals that requires the proteolytic cleavage of the cartilaginous matrix. This in turn will allow the canals to grow into the epiphysis. In the present study we therefore initially investigated which enzymes and types of cells are involved in this process. We have chosen the mouse as an animal model and focused our studies on the distal part of the femur during early stages after birth. The formation of the cartilage canals was promoted by tartrate-resistant acid phosphatase (TRAP) and membrane type-1 matrix metalloproteinases (MT1-MMP). In addition, macrophages and cells containing numerous lysosomes contributed to the establishment of the canals and enabled their further advancement into the epiphysis. As development continued, the SOC was formed, and in mice aged 10 days a distinct layer of type I collagen (= osteoid) was laid down onto the cartilage scaffold. The events leading to the establishment of the SOC were compared with those of the POC. Basically these processes were quite similar, and in both ossification centers, TRAP-positive chondroclasts resorbed the cartilage matrix. However, occasionally co-expression of TRAP and MT1-MMP was noted in a small subpopulation of this cell type. Furthermore, numerous osteoblasts expressed MT1-MMP from the start of endochondral ossification, whereas others did not. In osteocytogenesis, MT1-MMP has been shown to be critical for the establishment of the cytoplasmic processes mediating the communication between osteocytes and bone-lining cells. Considering the well-known fact that not all osteoblasts transform into osteocytes, and in accordance with the present data, we suggest that MT1-MMP is needed at the very beginning of osteocytogenesis and may additionally determine whether an osteoblast further differentiates into an osteocyte.     

An autoradiographic study of chondrocyte transformation into chondroclasts and osteocytes during bone formation In vitro

Excised mouse pubic bone rudiments were exposed to H³-thymidine. Rudiments preserved immediately after exposure consisted of mesenchyme with a large number of cells showing intense radioactivity. Rudiments incubated on a filter membrane after exposure went through the developmental stages of complete chondrification of the pubic rami followed by periosteal and then endochondral bone formation. Only chondrocytes showed radioactivity in rami consisting of cartilage and periosteal bone that were preserved prior to endochondral ossification. Cell types showing radioactivity in rami preserved during endochondral ossification were chondrocytes, chondroclasts, and osteoblasts and osteocytes of endochondral bone. The results of the study demonstrated that hypertrophic chondrocytes of the calcified cartilage of a developing mammalian long bone not only survive dissolution of their matrix, but transform into chondroclasts and osteoprogenitor cells that give rise to osteoblasts and osteocytes which form endochondral bone in the absence of blood vessels.     

Apoptosis in membranous bone formation: role of fibroblast growth factor and bone morphogenetic protein signaling

Membranous ossification occurs by the condensation of mesenchymal cells followed by their progressive differentiation into osteoblasts that form a mineralized matrix in ossification centers. The balance between proliferating and differentiated osteogenic cells at the suture areas between calvarial bones is essential for the control of suture maintenance and membranous bone formation. The mechanisms of regulation of na apoptosis in suture areas begin to be understood. Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) are important regulators of mesenchymal, preosteoblast, and osteoblast apoptosis in suture areas. Perturbations in FGF or BMP signaling lead to alter the number of apoptotic osteogenic cells, resulting in premature or delayed suture closure. Recent data indicate that FGF signaling downregulates preosteoblast apoptosis, thereby preventing premature fusion of adjacent mineralizing extremities. In contrast, continuous FGF signaling or constitutive FGF receptor activation, as well as BMP signaling, upregulate osteoblast apoptosis. Additionally, multiple signaling mechanisms, including PI3K and PKC, appear to be involved in the control of calvarial osteoblast apoptosis by FGF and BMP. These mechanisms allow a fine control of the number of functional bone-forming cells and, thereby, the normal progression of membranous bone formation.     

A model of osteoblast-osteocyte kinetics in the development of secondary osteons in rabbits

The kinetics of osteogenic cells within secondary osteons have been examined within a 2-D model. The linear osteoblast density of the osteons and the osteocyte lacunae density were compared with other endosteal lamellar systems of different geometries. The cell density was significantly greater in the endosteal appositional zone and was always flatter than the central osteonal canals. Fully structured osteons compared with early structuring (cutting cones) did not show any significant differences in density. The osteoblast density may remain constant because some of them leave the row and become embedded within matrix. The overall shape of the Haversian system represented a geometrical restraint and it was thought to be related to osteoblast-osteocyte transformation. To test this hypothesis of an early differentiation and recruitment of the osteoblast pool which completes the lamellar structure of the osteon, the number and density of osteoblasts and osteocyte lacunae were evaluated. In the central canal area, the mean osteoblast linear density and the osteocyte lacunae planar density were not significantly different among sub-classes (with the exclusion of the osteocyte lacunae of the 300-1000 μm(2) sub-class). The mean number of osteoblasts compared with osteocyte lacunae resulted in significantly higher numbers in the two sub-classes, no significant difference was seen in the two middle sub-classes with the larger canals, and there were significantly lower levels in the smallest central canal sub-class. The TUNEL technique was used to identify the morphological features of apoptosis within osteoblasts. It was found that apoptosis occurred during the late phase of osteon formation but not in osteocytes. This suggests a regulatory role of apoptosis in balancing the osteoblast-osteocyte equilibrium within secondary osteon development. The position of the osteocytic lacunae did not correlate with the lamellar pattern and the lacunae density in osteonal radial sectors was not significantly different. These findings support the hypothesis of an early differentiation of the osteoblast pool and the independence of the fibrillar lamellation from osteoblast-osteocyte transformation.     

载荷作用下骨细胞分泌因子对成骨细胞和破骨细胞的调节

骨细胞是骨组织主要的力学感受及转导细胞,它们通过众多突触结构相互连接,形成庞大的骨稳态细胞调控网络,联系着成骨细胞、破骨细胞等骨基质表面细胞。骨细胞通过旁分泌途径影响成骨细胞骨形成和破骨细胞骨吸收来调节骨代谢,维持骨更新。针对骨细胞在受到力学刺激后分泌或释放的一些信号分子或蛋白因子对成骨细胞和破骨细胞生长分化的影响,本文综述近年来关于受力学刺激的骨细胞如何与成骨/破骨细胞进行通讯,为骨细胞生物力学研究提供新思路。     

Poorly ordered bone as an endogenous scaffold for the deposition of highly oriented lamellar tissue in rapidly growing ovine bone

The mechanical properties of bone are known to depend on its structure at all length scales. In large animals, such as sheep, cortical bone grows very quickly and it is known that this occurs in 2 stages whereby a poorly ordered (mostly woven) bone structure is initially deposited and later augmented and partially replaced by parallel fibered and lamellar bone with much improved mechanical properties, often called primary osteons. Most interestingly, a similar sequence of events has also recently been observed during callus formation in a sheep osteotomy model. This has prompted the idea that fast intramembranous bone formation requires an intermediate step where bone with a lower degree of collagen orientation is deposited first as a substrate for osteoblasts to coordinate the synthesis of lamellar tissue. Since some osteoblasts become embedded in the mineralizing collagen matrix which they synthesize, the resulting osteocyte network is a direct image of the location of osteoblasts during bone formation. Using 3-dimensional imaging of osteocyte networks as well as tissue characterization by polarized light microscopy and backscattered electron imaging, we revisit the structure of growing plexiform (fibrolamellar) bone and callus in sheep. We show that bone deposited initially is based on osteocytes without spatial correlation and encased in poorly ordered matrix. Bone deposited on top of this has lamellar collagen orientation as well as a layered arrangement of osteocytes, both parallel to the surfaces of the initial tissue. This supports the hypothesis that the initial bone constitutes an endogenous scaffold for the subsequent deposition of parallel fibered and lamellar bone.     

From bone lining cell to osteocyte--an SEM study

We describe the SEM appearance of the rat endosteal bone lining cell ( BLC ) population, and the sequence of morphological changes of these cells as they self-incorporate into unmineralized bone matrix (osteoid), establish intercellular connections, and construct lacunae. The osteoblast/nascent osteocyte series was progressively unsheathed by gentle digestion of the osteoid with 0.25% collagenase. The osteoblasts which leave the polygonally packed BLC compartment rapidly develop numerous complexly branched processes that contact the processes elaborated by previous generations of maturing and mature osteocytes. As osteoblasts mature and approach the mineralization front, they appear to lose processes. The mature cells begin to form osteocyte lacunae by depositing an asymmetric perimeter of woven collagen fibrils, such that as the cells roof-over, the lacunae appear as pocketlike constructions. The collagen fibrils on the perilacunar matrix are oriented in a tangential or circular pattern, while those in the more distal matrix are arranged in a parallel pattern. With the completion of a lacuna, its wall appears to mineralize quickly, for lacunae could be recognized only when they are forming.     

From bone lining cell to osteocyte—an SEM study

We describe the SEM appearance of the rat endosteal bone lining cell (BLC) population, and the sequence of morphological changes of these cells as they self-incorporate into unmineralized bone matrix (osteoid), establish intercellular connections, and construct lacunae. The osteoblast/nascent osteocyte series was progressively unsheathed by gentle digestion of the osteoid with 0.25% collagenase. The osteoblasts which leave the polygonally packed BLC compartment rapidly develop numerous complexly branched processes that contact the processes elaborated by previous generations of maturing and mature osteocytes. As osteoblasts mature and approach the mineralization front, they appear to lose processes. The mature cells begin to form osteocyte lacunae by depositing an asymmetric perimeter of woven collagen fibrils, such that as the cells roof-over, the lacunae appear as pocketlike constructions. The collagen fibrils on the perilacunar matrix are oriented in a tangential or circular pattern, while those in the more distal matrix are arranged in a parallel pattern. With the completion of a lacuna, its wall appears to mineralize quickly, for lacunae could be recognized only when they are forming.     

Preliminary report of impaired oestrogen receptor-alpha expression in bone, but no involvement of androgen receptor, in male idiopathic osteoporosis

In western countries, osteoporosis affects at least 1 in 12 of all adult males and a third of osteoporotic men have idiopathic disease (MIO). Both oestrogen and testosterone are now known to be important to the male skeleton. As normal oestrogen levels have been found in younger MIO cases, it is hypothesized that, in bone, their responses to gonadal steroids may be defective, through impaired receptor expression. This study therefore compared oestrogen receptor (ER)-alpha and androgen receptor (AR) expression, by indirect immunofluorescence and semi-quantitative image analysis, in undecalcified fresh frozen bone sections from MIO patients (33-56 years), age-matched control men (n=7), and, for reference, ovarian steroid-replete (n=7) and -deficient women (n=6). In normal men, 23%+/-SEM 6% osteoblasts and 14%+/-SEM 2% osteocytes expressed ERalpha protein, similar to hormone-replete women. Although receptor expression decreased in hormone-deficient women, loss of ERalpha protein in MIO patients was more severe (1%+/-SEM 0.5% osteocytes, 2%+/-SEM 1% osteoblasts expressed receptor). In all four groups, there was little osteocyte AR expression, but in the women, a proportion of osteoblasts were receptor-positive. Deficient osteoblast and osteocyte ERalpha protein expression could explain the bone loss in these MIO patients.     

Effects of donor age on osteogenic cells of rat bone marrow in vitro

The effect of donor age on the production of bone-like tissue and expression of cellular alkaline phosphatase was examined in cultures of cells obtained from rat bone marrow. Stromal cells were obtained from the bone marrow of young (5-6 weeks) and old (18 months) rats and cultured in vitro. After 28 days in first subculture, the following were quantified: (1) the total number of mineralised nodules and the size distribution of nodules and (2) the density of osteoblasts and osteocytes associated with nodules in histological sections. The doubling times of the cultures and the numbers of cells in cultures which expressed alkaline phosphatase activity were determined in separate experiments. Cells from young cultures produced three times more bone-like nodules than old cultures, although no differences were seen in the size distribution of nodules, or on osteoblast and osteocyte density. Doubling times for both groups were similar. The numbers of alkaline phosphatase (AP) positive cells was reduced by half in old cultures. These data show that this model may be useful for the study of the mechanisms of ageing on osteogenesis, and demonstrate a reduced osteogenic capacity in old cultures. The results suggest that this effect may be due to a reduction in the generation of cells of osteoblast lineage during ageing.     

The fine structure of bone in the nasal turbinates of young pigs

The three types of bone cells in the nasal turbinates had characteristic ultrastructural features. Osteoblasts were located in areas of new bone formation and had abundant endoplasmic reticulum, prominent Golgi apparatuses, numerous vesicles, and cytoplasmic processes that penetrated the adjacent osteoid. Osteocytes had variable ultrastructural characteristics. The predominant cell filled the lacuna, had few organelles, smooth plasma membranes, and was interpreted to be a mature resting osteocyte. Some osteocytes appeared to be transitional between osteoblasts and mature osteocytes. Evidence of matrix formation was seen near osteocytes with well developed organelles, whereas osteocytes with swollen mitochondria, dense bodies and irregular plasma membranes appeared to be involved with resorption of bone. Multinucleated osteo-clasts contained numerous mitochondria and had crystals or unmineralized collagen fibrils between folds and within vacuoles of the cytoplasmic projections forming the brush border.     

成骨细胞和骨细胞的力学信号转导

背景:骨骼具有功能适应性的特点,骨骼细胞是力学信号敏感细胞,但细胞的力学信号转导功能是如何实现的,对骨骼如何调控仍不明确。 目的：了解成骨细胞和骨细胞的力学信号转导途径,为利用力学信号改善骨骼功能提供理论依据。 方法：应用计算机检索PubMed数据库2000-01/2011-03相关文献。英文检索词为“osteoblast,osteocyte,bone cells,mechanical stress”, 根据纳入标准共69篇文章进行综述,以此对骨骼细胞力学信号转导相关内容进行总结。 结果与结论：骨骼具有功能适应性的特点,骨骼细胞是力学信号敏感细胞,但细胞的力学信号转导功能是如何实现的,对骨骼具有怎样的调控仍不明确。研究表明,由于骨骼的结构特点和细胞位置,成骨细胞和骨细胞是最重要的力学敏感性细胞。力学信号在骨骼内的转导过程分为4个阶段：①力学偶联。②生化偶联。③信号的传递。④效应性细胞的反应。通过这4个阶段的作用,作用在骨骼上的应力信号转导为生物化学信号,并影响细胞的功能,最终导致骨骼组织出现相应的结构变化以适应应力环境的需要。对于力学信号在骨髓间充质干细胞中的调控机制还有待继续深入探索。     

Trabecular bone remodelling simulated by a stochastic exchange of discrete bone packets from the surface

Human bone is constantly renewed through life via the process of bone remodelling, in which individual packets of bone are removed by osteoclasts and replaced by osteoblasts. Remodelling is mechanically controlled, where osteocytes embedded within the bone matrix are thought to act as mechanical sensors. In this computational work, a stochastic model for bone remodelling is used in which the renewal of bone material occurs by exchange of discrete bone packets. We tested different hypotheses of how the mechanical stimulus for bone remodelling is integrated by osteocytes and sent to actor cells on the bone's surface. A collective (summed) signal from multiple osteocytes as opposed to an individual (maximal) signal from a single osteocyte was found to lead to lower inner porosity and surface roughness of the simulated bone structure. This observation can be interpreted in that collective osteocyte signalling provides an effective surface tension to the remodelling process. Furthermore, the material heterogeneity due to remodelling was studied on a network of trabeculae. As the model is discrete, the age of individual bone packets can be monitored with time. The simulation results were compared with experimental data coming from quantitative back scattered electron imaging by transforming the information about the age of the bone packet into a mineral content. Discrepancies with experiments indicate that osteoclasts preferentially resorb low mineralized, i.e. young, bone at the bone's surface.     

Identification and location of bone-forming cells within cartilage canals on their course into the secondary ossification centre

Osteoblasts and osteocytes derive from the same precursors, and osteocytes are terminally differentiated osteoblasts. These two cell types are distinguishable by their morphology, localization and levels of expression of various bone cell-specific markers. In the present study on the chicken femur we investigated the properties of the mesenchymal cells within cartilage canals on their course into the secondary ossification centre (SOC). We examined several developmental stages after hatching by means of light microscopy, electron microscopy, immunohistochemistry and in situ hybridization. Cartilage canals appeared as extensions of the perichondrium into the developing distal epiphysis and they were arranged in a complex network. Within the epiphysis an SOC was formed and cartilage canals penetrated into it. In addition, they were successively incorporated into the SOC during its growth in the radial direction. Thus, the canals provided this centre with mesenchymal cells and vessels. It should be emphasized that regression of cartilage canals could never be observed in the growing bone. Outside the SOC the mesenchymal cells of the canals expressed type I collagen and periostin and thus these cells had the characteristics of preosteoblasts. Periostin was also expressed by numerous chondrocytes. Within the SOC the synthesis of periostin was down-regulated and the majority of osteoblasts were periostin negative. Furthermore, osteocytes did not secret this protein. Tissue-non-specific alkaline phosphatase (TNAP) staining was only detectable where matrix vesicles were present. These vesicles were found around the blind end of cartilage canals within the SOC where newly formed osteoid started to mineralize. The vesicles originated from osteoblasts as well as from late osteoblasts/preosteocytes and thus TNAP was only expressed by these cells. Our results provide evidence that the mesenchymal cells of cartilage canals express various bone cell-specific markers depending on their position. We suggest that these cells differentiate from preosteoblasts into osteocytes on their course into the SOC and consider that cartilage canals are essential for normal bone development within the epiphysis. Furthermore, we propose that the expression of periostin by preosteoblasts and several chondrocytes is required for adhesion of these cells to the extracellular matrix.