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.     

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.     

Histological identification of osteocytes in the allegedly acellular bone of the sea breams Acanthopagrus australis,Pagrus auratus and Rhabdosargus sarba (Sparidae,Perciformes, Teleostei)

The bone of advanced teleost fishes such as those of the family Sparidae is said to lack osteocytes or to be acellular. Acellularity has been determined by apparent lack of osteocyte lacunae. This study questions the validity of this criterion. Scanning electron and light microscopy of paraffin and resin sections were used to show that the sides of sea bream mandibles consist of laminar parallel-fibred bone that we call tubular bone, because it contains tubules, and localised regions of Sharpey fibre bone. Osteocytes lie along the walls of tubules that also contain collagen fibril bundles (T-fibres), or in the lumens of tubules that do not contain T-fibres. We show that the osteocytes are derived from osteoblasts. The T-fibre system is different from other fibre systems that have been described. The tubules enclose wide T-fibres (lenticular in cross-section, maximum width about 8 μm) that taper at their ends and continue as thin T-fibres (round in crosssection, about 2 μm wide). The T-fibres originate in the periosteum. In mature tubular bone, spaces of increasing size develop around the osteocytes. Osteocytes are released from the bone matrix and become postosteocytes or bone-lining cells. Secondary bone lines the largest spaces. In Sharpey fibre bone, small osteocytes in small lacunae (about 2 μm wide) are found in columns parallel to the Sharpey fibres. Large osteocytes are found in large round spaces and are much larger than comparable osteocytes in lacunae in the bone of the salmon Salmo salar. We conclude that an absence of visible or conventional osteocyte lacunae does not mean that the cells themselves are absent. There are cells and two types of collagen fibre bundle in the tubules. The cells are osteocytes derived from osteoblasts, and these osteocytes apparently resorb bone with the result that large amounts of bone are destroyed. “Acellular” tubular and Sharpey fibre bone are types of cellular bone that differ from each other and from conventional cellular bone.     

Dentine matrix protein 1 (DMP-1) is a marker of bone-forming tumours

Dentin matrix protein 1 (DMP-1) is highly expressed by osteocytes and is a non-collagenous matrix protein found in dentin and bone. In this study, we determined the expression of DMP-1 in mature and immature human bone and examined whether DMP-1 is useful in distinguishing osteoid/bone-forming tumours from other primary and secondary bone tumours. DMP-1 expression was immunohistochemically determined in paraffin sections of a wide range of benign and malignant primary bone tumours and tumour-like lesions (n?=?353). DMP-1 mRNA expression was also examined in osteosarcoma and fibrosarcoma cell lines as well as bone tumour specimens (n?=?5) using real-time PCR. In lamellar and woven bone, DMP-1 was expressed in the matrix around osteocyte lacunae and canaliculi; osteoblasts and other cell types in the bone were negative. Matrix staining of the osteoid and bone was seen in bone-forming tumours including osteoma, osteoid osteoma, osteoblastoma and osteosarcoma. DMP-1 staining was also seen in fibrous dysplasia, osteofibrous dysplasia and chondroblastoma and in reactive bone in solitary bone cysts and aneurysmal bone cysts. DMP-1 was not expressed in the tumour component of other bone neoplasms including Ewing sarcoma, chondrosarcoma, leiomyosarcoma, fibrosarcoma, giant cell tumour of bone and metastatic carcinoma. DMP-1 mRNA was expressed in osteosarcoma cell lines and tumour samples. DMP-1 is a matrix marker expressed around osteocytes in human woven and lamellar bone and is useful in identifying osteosarcoma and other bone-forming tumours.     

Histological identification of osteocytes in the allegedly acellular bone of the sea breams Acanthopagrus australis, Pagrus auratus and Rhabdosargus sarba (Sparidae, Perciformes, Teleostei)

The bone of advanced teleost fishes such as those of the family Sparidae is said to lack osteocytes or to be acellular. Acellularity has been determined by apparent lack of osteocyte lacunae. This study questions the validity of this criterion. Scanning electron and light microscopy of paraffin and resin sections were used to show that the sides of sea bream mandibles consist of laminar parallel-fibred bone that we call tubular bone, because it contains tubules, and localised regions of Sharpey fibre bone. Osteocytes lie along the walls of tubules that also contain collagen fibril bundles (T-fibres), or in the lumens of tubules that do not contain T-fibres. We show that the osteocytes are derived from osteoblasts. The T-fibre system is different from other fibre systems that have been described. The tubules enclose wide T-fibres (lenticular in cross-section, maximum width about 8 m) that taper at their ends and continue as thin T-fibres (round in crosssection, about 2 m wide). The T-fibres originate in the periosteum. In mature tubular bone, spaces of increasing size develop around the osteocytes. Osteocytes are released from the bone matrix and become postosteocytes or bone-lining cells. Secondary bone lines the largest spaces. In Sharpey fibre bone, small osteocytes in small lacunae (about 2 m wide) are found in columns parallel to the Sharpey fibres. Large osteocytes are found in large round spaces and are much larger than comparable osteocytes in lacunae in the bone of the salmon Salmo salar. We conclude that an absence of visible or conventional osteocyte lacunae does not mean that the cells themselves are absent. There are cells and two types of collagen fibre bundle in the tubules. The cells are osteocytes derived from osteoblasts, and these osteocytes apparently resorb bone with the result that large amounts of bone are destroyed. Acellular tubular and Sharpey fibre bone are types of cellular bone that differ from each other and from conventional cellular bone.     

Ultrastructure of osteoid osteoma.

The ultrastructure in five cases of osteoid osteoma is described. The osteoblasts generally had a morphology similar to that of normal osteoblasts with a few differences. They contained irregular indented nuclei, glycogen particles, abundant fine intracytoplasmic fibrils, and rare iron containing lysosomes. In several osteoblasts in two cases there were atypical mitochondria with a lobulated or "honeycomb" appearance. These atypical mitochondria were also observed in two osteoclasts; otherwise the cells resembled normal osteoclasts. Other cells present in osteoid osteoma besides osteocytes included osteoprogenitor cells resembling Scott type A and B cells and cells in transitional stages of differentiation. The osteoblasts most likely originated from Scott type A cells of preosteoblasts. The areas of mineralized matrix conformed to the morphology of coarse woven bone. Varying amounts of osteoid were noted. In two cases the osteoid contained, in addition to collagen, fine granular material, which probably represents polysaccharides. No nerve fibers were identified in the material studied. It is thought that osteoid osteoma is a benign neoplastic lesion. A case of osteoblastoma was studied for comparison; the osteoblastoma cells were found to have basically the same morphology as the cells in osteoid osteoma, including the atypical mitochondria. Our ultrastructural observations support the idea that osteoid osteoma and osteoblastoma are closely related lesions.     

Histological evidence of the altered distribution of osteocytes and bone matrix synthesis in klotho-deficient mice

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.     

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.     

Morphometric study of collagen maturation in chick compact bone

An ultrastructural-morphometric study was carried out on the process of osteoid maturation in growing surfaces of parallel-fibered chick bone. The aim was to investigate the distribution, size and amount of collagen fibrils (CFs), as well as the proteoglycan (PG) content, throughout the osteoid seam and in the adjacent bone. The results show that the organic components secreted by osteoblasts undergo complete maturation inside the osteoid seam only. Proceeding from the secreting plasma membrane of osteoblasts (osteoidogenic surface) towards the mineralizing surface, we found that CFs gradually increase in diameter but not in number per surface unit. As a consequence, the proportion of osteoid seam occupied by CF increases too, at the expense of the interfibrillar substance. PG content also decreases inversely in this direction. In the adjacent bone, CF size and density do not change significantly with respect to the mature osteoid close to the mineralizing surface.     

Two types of bone resorption lacunae in the mouse parietal bones as revealed by scanning electron microscopy and histochemistry

To understand the bone resorption process on the basis of the morphology of bone resorption lacunae, the inner surface of parietal bones in juvenile mice was exposed with a treatment of ultrasonic waves or NaOCl treatment and examined by scanning electron microscopy (SEM). The bone resorption lacunae were divided into two types (I and II) according to differences in morphological features of their walls; the wall of type I lacunae was covered with loose collagen fibrils, while that of type II lacunae was smooth with almost no fibrillar structures. Collagen fibrils in type I lacunae treated with ultrasonic waves differed in appearance from those treated with NaOCl; the collagen fibrils were thin and displayed a smooth surface in type I lacunae treated with ultrasonic waves, while they were thick and showed a rough surface in those treated with NaOCl-probably because superficial uncalcified collagen fibrils were digested with the chemical. The results indicated that type I lacunae occupied 77% of all of the bone resorption lacunae treated with ultrasonic waves, but 51% of those treated with NaOCl. This finding led to the idea that type I lacunae can be subdivided into two: lacunae (Ia), covered with partially calcified fibrils as well as superficial uncalcified fibrils; and lacunae (Ib), covered only with uncalcified fibrils. The presence of uncalcified fibrils in the bone resorption lacunae was further confirmed by backscattered electron (BSE) imaging of SEM. Histochemistry for acid phosphatase or immuno-histochemistry for cathepsin B or carbonic anhydrase in combination with SEM revealed that type I lacunae were located under osteoclasts but type II lacunae were not. These findings indicate that type I lacunae are in the process of bone resorption by osteoclasts, while type II lacunae are in the final stage of bone resorption and free from osteoclasts. Bone resorption may thus proceed in the order of Ia, Ib, and II.     

Histomorphometric study on the osteocyte lacuno-canalicular network in animals of different species. II. Parallel-fibered and lamellar bones

The shape, size and density of osteocyte lacunae in parallel-fibered and lamellar bone were histomorphometrically analyzed in relation to the organization of the collagen fiber texture and the animal species (frog, sheep, dog, bovine, horse and man). The following parameters were measured under the light microscope (LM) by a computer-assisted image analyzer: 1) shape, size and distribution of osteocyte lacunae; 2) osteocyte lacuno-canalicular density. In close agreement with our previous studies, which includes woven bone, it resulted that in all animals (even in frog) osteocyte lacunae have a rounded globous shape in woven bone and an oval shape in both parallel-fibered and lamellar bone; in the latter, however, they are more flattened, only located in loose lamellae and thus regularly distributed in rows. Osteocyte lacunar density is higher in woven-fibered, intermediate in parallel-fibered and lower in lamellar bone, whereas no correlation seems to exist with the animal species. In conclusion, these results suggest that osteocyte shape, size and density seem to depend mainly on collagen fiber texture rather than on the animal species. The role of osteocyte-recruitment on the spatial organization of collagen fibers in bone tissues is discussed.     

Osteocyte dendrogenesis in static and dynamic bone formation: an ultrastructural study

The present ultrastructural investigation into osteocyte dendrogenesis represents a continuation of a previous study (Ferretti et al., Anat. Embryol., 2002; 206:21-29), in which we pointed out that, during intramembranous ossification, the well-known dynamic bone formation (DBF), performed by migrating osteoblast laminae, is preceded by static bone formation (SBF), in which cords of stationary osteoblasts transform into osteocytes in the same site where they differentiated. The research was carried out on the perichondral center of ossification surrounding the mid shaft level of various long bones of chick embryos and newborn rabbits. Transmission electron microscope observations showed that the formation of osteocyte dendrites is quite different in the two types of osteogenesis, mainly depending on whether or not osteoblast movement occurs. In DBF, osteoblasts transform into small ovoidal/ellipsoidal osteocytes and their dendrites form in an asynchronous and asymmetrical manner in concomitance with, and depending on, the advancing mineralizing surface and the receding osteogenic laminae. In SBF, stationary osteoblasts give rise to big globous osteocytes, located inside confluent lacunae, with short and symmetrical dendrites that can radiate simultaneously all around their cell body because they are completely surrounded by unmineralized matrix. Contacts and gap junctions were observed between all osteocytes (both SBF- and DBF-derived) and between osteocytes and osteoblasts. Finally, a continuous osteocyte network extends throughout the bone, regardless of its static or dynamic origin. This network has the characteristic of a functional syncytium, potentially capable of modulating, by wiring transmission, the cells of the osteogenic lineage covering the bone surfaces.     

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.     

The role of cartilage canals in endochondral and perichondral bone formation: are there similarities between these two processes?

We investigated the development of cartilage canals to clarify their function in the process of bone formation. Cartilage canals are tubes containing vessels that are found in the hyaline cartilage prior to the formation of a secondary ossification centre (SOC). Their exact role is still controversial and it is unclear whether they contribute to endochondral bone formation when an SOC appears. We examined the cartilage canals of the chicken femur in different developmental stages (E20, D2, 5, 7, 8, 10 and 13). To obtain a detailed picture of the cellular and molecular events within and around the canals the femur was investigated by means of three-dimensional reconstruction, light microscopy, electron microscopy, histochemistry and immunohistochemistry [vascular endothelial growth factor (VEGF), type I and II collagen]. An SOC was visible for the first time on the last embryonic day (E20). Cartilage canals were an extension of the vascularized perichondrium and its mesenchymal stem cell layers into the hyaline cartilage. The canals formed a complex network within the epiphysis and some of them penetrated into the SOC were they ended blind. The growth of the canals into the SOC was promoted by VEGF. As the development progressed the SOC increased in size and adjacent canals were incorporated into it. The canals contained chondroclasts, which opened the lacunae of hypertrophic chondrocytes, and this was followed by invasion of mesenchymal cells into the empty lacunae and formation of an osteoid layer. In older stages this layer mineralized and increased in thickness by addition of further cells. Outside the SOC cartilage canals are surrounded by osteoid, which is formed by the process of perichondral bone formation. We conclude that cartilage canals contribute to both perichondral and endochondral bone formation and that osteoblasts have the same origin in both processes.     

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.     

Immunolocalization of keratan sulfate proteoglycan in rat calvaria

We investigate, by the immunogold method, the localization of keratan sulfate (KS) proteoglycan in rat calvaria in order to clarify the detailed process of intramembranous ossification. KS was localized in bone nodules corresponding to calcified nodules, close to the saggital suture of calvaria. The immunoreactivity decreased in fully calcified regions distant from the suture. Electron microscopic observation revealed that KS was distributed in and around matrix vesicles, among collagen fibrils at the initial crystal deposition stage, and then concentrated in bone nodules. According to the progress of mineralization, KS tended to be localized in the peripheral region of the nodules. In addition, these nodules came in contact with collagen fibrils which also showed KS-positive reactivity. In cell organelles of osteoblasts, KS was detected in the Golgi apparatus. These findings suggest that osteoblasts in intramembranous ossification sites actively synthesize KS. KS in the calcified nodules, as well as other glycosaminoglycans in osteoid, may play an important role in additional and/or collagenous calcification by trapping calcium ions through its negative charge.     

Collagen texture and osteocyte distribution in lamellar bone

A comparative scanning and transmission electron microscopy study was carried out on collagen fiber texture and osteocyte lacunae distribution in human lamellar bone. The results show that bony lamellae are not made up of parallel-arranged collagen fibers, as classically maintained. They are instead made up of highly interlaced fibers, and the lamellation appears to be due to the alternation of collagen-rich and collagen-poor layers, namely of dense and loose lamellae. The present study additionally shows that osteocyte lacunae are only located inside loose lamellae. Such structural organization of lamellar bone is briefly discussed in terms of bone biomechanics and osteogenesis.     

Attachment of Osteocyte Cell Processes to the Bone Matrix

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 αvβ3 may play a role in attachment at these complexes; a punctate pattern of staining of β3 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. Anat Rec, 292:355–363, 2009. © 2009 Wiley‐Liss, Inc.     

Ultrastructural study of osteosarcomas

The ultrastructural examination of four osteosarcomas (osteogenic, undifferentiated, and pleomorphic) is described. There are three types of tumor cells. Most of the cells are held in contact by desmosome-like tight junctions; they are atypical osteoblasts with cytoplasmic processes, dilated rough endoplasmic reticulum, mitochondria carrying calcific inclusions, lipid droplets surrounded by glycogen, and intracellular fine filamentous fibers. Other cells exhibiting varying degrees of osteoblastic maturity are also seen with multilobed nuclei, a clear cytoplasm, and straight bordered membranes. The last type is chondroid with abundant deposits of glycogen, lipid droplets, and undilated rough endoplasmic reticulum. The matrix is composed of fibrils without periodicity, scattered and deteriorated collagen fibers, and focal calcium deposits of hydroxyapatite crystals as in embryonal bone, dentine, or callus bone.     

以带血管蒂筋膜瓣与人脐带间充质干细胞和重组人骨形成蛋白2构建活性组织工程骨

背景：构建的组织工程骨块植入体内后的存活是骨组织工程面临的一个重大课题,临床上尤其缺少可行性强、可以不经体外长时间构建及预血管化而可一期应用的组织工程骨。 目的：探讨以带血管蒂的筋膜瓣作为膜包裹材料、以人脐带间充质干细胞作为种子细胞,以β-磷酸三钙生物陶瓷作为支架材构建组织工程骨的可行性及加入重组人骨形成蛋白2作为细胞活性因子、Ⅰ型胶原作为细胞活性因子缓释材料后成骨能力的变化。 方法：Wistar大鼠左侧L1~6背部带血管蒂的筋膜瓣包绕由β-磷酸三钙生物陶瓷、人脐带间充质干细胞、重组人骨形成蛋白2、Ⅰ型胶原构建的组织工程骨作为实验侧,右侧带血管蒂的筋膜瓣包绕接种了人脐带间充质干细胞的β-磷酸三钙生物陶瓷作为对照侧。 结果与结论：大鼠实验侧4周时幼稚骨组织连接形成原始层板骨样结构,形成的原始骨组织钙化程度低。8周时,大鼠两侧骨组织均基本成熟。成骨细胞位于骨陷窝中,周围有大量骨基质呈淡紫色,局部可见Ⅰ型胶原存在,有骨髓腔结构出现,但实验侧骨组织成熟度明显高于对照侧。实验侧骨组织哈夫氏小管清晰可见,形成多个骨化中心,骨小梁、骨岛遍布其中,可见成熟板层骨、立方状排列整齐的活性成骨细胞。8周时实验侧骨小梁成熟度高、典型、清晰可见,对照侧骨小梁成熟度稍差。但2组植入物的新骨形成面积接近。提示以带血管蒂的筋膜瓣作为膜包裹材料构建组织工程骨时,重组人骨形成蛋白2及缓释剂Ⅰ型胶原可促进其骨成熟度。