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.     

Periostin, a novel marker of intramembranous ossification, is expressed in fibrous dysplasia and in c-Fos-overexpressing bone lesions

Fibrous dysplasia is a benign bone disease caused by a mutation in the gene for the stimulatory guanine nucleotide-binding protein Gs alpha, leading to high cyclic adenosine monophosphate levels. Histologically, fibrous dysplasia is characterized by the production of fibrous tissue accompanied by the deposition of ectopic type I collagen and other bone-associated extracellular matrix proteins, as well as by irregular woven intramembranous bone onto which type I collagen-containing Sharpey fibers are often attached. Fibrous dysplasia is also characterized by high expression of c-Fos/c-Jun, known targets for cyclic adenosine monophosphate signaling. In this study, we examined the expression of the bone-related extracellular matrix protein, periostin, and its known receptor, integrin alpha v beta 3 (CD51/61), in normal bones as well as in fibrous dysplasia. Immunohistochemistry and in situ hybridization studies revealed that periostin was expressed in the extracellular matrix during intramembranous but not endochondral ossification, as well as in the fibrous component of fibrous dysplasia; and all cells adjacent to periostin-positive regions expressed CD51/61. Importantly, periostin was abundantly localized to Sharpey fibers. To investigate the contribution of c-Fos, we examined transgenic mice overexpressing c-fos, which develop sclerotic lesions closely resembling those found in fibrous dysplasia. In all lesions, transformed osteoblasts expressed high levels of periostin, whereas normal osteoblasts did not. Our results show that periostin is a novel marker for intramembranous ossification, and is a good candidate as a diagnostic tool and/or a therapeutic target in fibrous dysplasia. Moreover, the Gs alpha-cyclic adenosine monophosphate-c-Fos pathway might represent one mechanism of periostin up-regulation in fibrous dysplasia, resulting in altered collagen fibrillogenesis characteristic of this disease.     

An electron microscopic study of initial intramembranous osteogenesis

Calvariae of fetal mice were investigated with electron microscopy in order to demonstrate initial stages of immature, membrane bone calcification. As a consequence, nucleation sites for initial calcification of hydroxyapatite crystals in woven bone were found in extrusions from osteoblasts within the osteoid. Growth of the crystals epitaxially from these initial calcification loci into the surrounding collagen was spherulitic, forming discrete spheres referred to as “bone nodules”. These nodules, when coalesced, formed seams of bone. Decalcified, the organic substructure of the bone nodules was revealed. It contained a centrally disposed initial calcification locus, a peripheral zone of decomplexing collagen and an intermediate zone of altered collagen. Fully formed collagen fibrils remained interposed between most of the coalesced nodules. Polysaccharides were localized within the initial calcification locus and in the peripheral zone of the bone nodules where collagen was shown to be decomplexing.     

Expression of NG2 proteoglycan during endochondral and intramembranous ossification.

We have used immunohistochemistry to study the distribution of the NG2 proteoglycan during bone development in the mouse. At embryonic day 15.5, NG2 was strongly detected in the immature cartilage of developing limbs. After transient down-regulation in mature chondrocytes, NG2 was up-regulated during primary ossification, colocalizing with alkaline phosphatase and tenascin C. In the epiphyseal growth plates of newborn mouse tibia, NG2 and alkaline phosphatase exhibited overlapping patterns of expression by hypertrophic chondrocytes and by osteoblasts surrounding newly formed bone trabeculae. NG2 was down-regulated after puberty, being only faintly detectable in the tibial growth plates of 3-month-old mice. In cranial sutures, NG2 was strongly labeled in osteogenic bone fronts and in the suture matrix. Our results indicate that NG2 expression is up-regulated during both endochondral and intramembranous ossification, but is down-regulated as ossification is completed.     

用多次酶消化法体外培养的大鼠成骨细胞及其鉴定

目的 针对目前体外培养成骨细胞方法的不足,建立一种理想的原代培养成骨细胞的方法,为骨替代材料的研究提供成骨细胞.方法 本实验用新生1～2天大鼠颅盖骨,采用多次酶消化法进行细胞体外培养.倒置显微镜观察细胞形态,并对其碱性磷酸酶（ALP）活性及矿化能力进行鉴定.结果 所培养细胞具有成骨细胞的形态学特征及体内成骨细胞的生物学行为.结论 本实验体外培养成骨细胞的方法切实可行,可为骨替代材料的研究提供种子细胞,也可为骨细胞生物学和骨组织工程的研究提供一种客观而有效的实验手段.     

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.     

Dense fibrillar collagen matrices sustain osteoblast phenotype in vitro and promote bone formation in rat calvaria defect

Two pure collagen materials were prepared from acidic collagen solutions at 5 and 40?mg/mL. Benefits of collagen concentration on bone repair were evaluated in vitro with human calvaria cells and in vivo in a rat cranial defect. Both materials exhibited specific structures, 5?mg/mL was soft with an open porous network of fibrils; 40?mg/mL was stiffer with a plugged surface and bundles of collagen fibrils. Osteoblasts seeded on 5?mg/mL formed an epithelioid layer with ultrastructural characteristics of mature osteoblasts and induced mineralization. Numerous osteoblasts migrated inside 5?mg/mL, triggering reorganization of their actin cytoskeleton, whereas on 40?mg/mL osteoblasts remained in a resting state. In rat calvaria defects, both materials induced active bone formation. Dual-energy X-ray absorption bone area measures after 4 weeks averaged 84.0% with 5?mg/mL, 88.4% with 40?mg/mL, and 36.7% in the controls (p?相似文献   

Fine structure of ossification in craniopharyngiomas

Three cases of adamantinomatous craniopharyngiomas were examined by light and electron microscopy and special attention was paid to the formation of ossified tissue. The tumors were composed of neoplastic epithelial cells with keratinized cell nests and fibrous connective tissue. Keratinized cell masses sometimes directly contacted fibrous connective tissue. In these border areas, multipotential mesenchymal cells in the latter may have differentiated into osteoblasts. Ultrastructurally, these osteoblastic mesenchymal cells were surrounded by amorphous ground matrix and collagen fibrils. Membrane-bound vesicles were occasionally seen among the spaces between the collagen fibrils. These vesicles were presumably derived from osteoblastic mesenchymal cells and were morphologically similar to matrix vesicles. Precipitation of hydroxyapatite crystals in these vesicles was considered to be the initial stage of ossification. Further mineralization of adjacent collagen fibrils resulted in the formation of small bone trabeculae. Then apositional growth of ossified tissue occurred in the surrounding keratinized cell masses.     

Fine Structure of Ossification in Craniopharyngiomas

Three cases of adamantinomatous craniopharyngiomas were examined by light and electron microscopy and special attention was paid to the formation of ossified tissue. The tumors were composed of neoplastic epithelial cells with keratinized cell nests and fibrous connective tissue. Keratinized cell masses sometimes directly contacted fibrous connective tissue. In these border areas, multipotential mesenchymal cells in the latter may have differentiated into osteoblasts. Ultrastructurally, these osteoblastic mesenchymal cells were surrounded by amorphous ground matrix and collagen fibrils. Membrane-bound vesicles were occasionally seen among the spaces between the collagen fibrils. These vesicles were presumably derived from osteoblastic mesenchymal cells and were morphologically similar to matrix vesicles. Precipitation of hydroxyapatite crystals in these vesicles was considered to be the initial stage of ossification. Further mineralization of adjacent collagen fibrils resulted in the formation of small bone trabeculae. Then apositional growth of ossified tissue occurred in the surrounding keratinized cell masses.     

A new lethal brittle bone syndrome with increased amount of type V collagen in a patient

A new lethal brittle bone disease is described in three patients with slender long bones, thin ribs, hypomineralized calvaria, and normal facial appearance. In spite of several limb fractures this syndrome can be differentiated from the lethal forms of osteogenesis imperfecta and is better related to the thin-bone group of lethal dysplasias. Biochemical investigation of collagen from one of the patients by the use of gel electrophoresis and high-pressure liquid chromatography analyses failed to demonstrate any evident defect in the structure of type I collagen chains. Nevertheless collagen extractability from the dermis was altered owing to an increase in the proportion of acid-soluble material. Tritium-proline labeling of cultured fibroblasts confirmed the reduction in total collagen synthesis. This was attributed to a lower type I and type III amount whereas type V collagen level was markedly increased in the cell layer. RNA analysis of the three collagen types with the appropriate cDNA probes confirmed the protein data. Electron microscopic examination of bone and skin showed morphologically abnormal fibroblasts and osteoblasts with an abundant distended rough endoplasmic reticulum and an altered plasma membrane. Unexpected thin fibrils with a banding pattern and surrounding the type I fibrils were observed. They might represent type V collagen. We suggest that, in this patient, the moderate decrease in type I collagen amount is insufficient to account for the radiological findings and that type V collagen overproduction could play a role in the bone brittleness by interfering with the process of mineralization.     

Influence of collagen-fibril-based coatings containing decorin and biglycan on osteoblast behavior

Collagen is used as a scaffold material for tissue engineering as well as a coating material for implants with a view to enhancing osseointegration by mimicry of the bone extracellular matrix in vivo. The biomimicry strategy can be taken further by incorporating the small leucine-rich proteoglycans (SLRPs) decorin and biglycan, which are expressed in bone. Both bind to fibrils during fibrillogenesis in vitro. In this study, the ability of collagen types I, II, and III to bind decorin and biglycan was compared. Collagen type II bound significantly more SLRPs in fibrils than collagen I and III, with more biglycan than decorin bound by all three collagen types. Therefore, type II fibrils with bound decorin or biglycan or neither were used to coat titanium surfaces. Bioavailability of SLRPs was confirmed by direct ELISA after SLRP biotinilation. The in vitro behavior of osteoblasts from rat calvaria (rOs) and human knee (hOs) cultured on different surfaces was compared. Proliferation and collagen synthesis were determined. Also, the influence of SLRPs on the formation of focal adhesions by rO was investigated. Biglycan enhanced the formation of focal adhesions after 2 and 24 h. Decorin and biglycan affected rO and hO proliferation and collagen synthesis differently. Biglycan stimulated hO proliferation significantly but had no effect on rO proliferation, and also inhibited rO collagen synthesis significantly while not affecting hO collagen synthesis. Decorin promoted hO proliferation slightly but did not influence rO proliferation. The results could be relevant when designing implant coatings or tissue engineering scaffolds.     

Expression of Collagen Types I,II, IX,and X in the Mineralizing Turkey Gastrocnemius Tendon

The turkey gastrocnemius tendon mineralizes by intramembranous ossification with a transient chondrogenic phase. The mineralizing zone has hypertrophic chondrocytes similar to endochondral bone formation. These similarities prompted the evaluation of this tendon for the presence of type X collagen in the mineralizing zone. Tendons were removed, radiographed, decalcified, and embedded for frozen sections. Seral sections were H&E stained and immunostained individually with antibodies specific collagens (types I, II, IX, and X). Type I collagen was distributed widely throughout the mineralized tendon extracellular matrix. Types II and IX collagen were at the mineralized/non-mineralized junction. Type X collagen was in the pericellular matrix of hypertrophic chondrocytes and in some calcified matrix. These data support the theory that the gastrocnemius tendon has fibrocartilage characteristics and that type X collagen has a role in the tissue's mineralization. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.     

An investigation of cellular dynamics during the development of intramembranous bones: the scleral ossicles

The development of intramembranous bone is a dynamic and complex process requiring highly coordinated cellular activities. Although the literature describes the detailed cellular dynamics of early mesoderm‐derived endochondral bone, studies regarding neural crest‐derived intramembranous bone have failed to keep pace. We analyzed the development of chick scleral ossicles from the onset of osteoid deposition to mineralization at morphological, histological, and ultrastructural levels. We find that the mesenchymal condensations from which ossicles develop change their shape from ellipsoidal to trapezoidal concurrent with an increase in size. Furthermore, the size of an ossicle is dependent upon its time of induction. Our histological analyses of condensation growth reveal cell migration and osteoid secretion as key cellular processes determining condensation size; these processes occur concomitantly to increase both the area and thickness of condensations. We also describe the formation of the zone of overlap between ossicles and conclude that the process is similar to that of cranial suture formation. Finally, transmission electron microscopy of early condensations demonstrates that early osteoblasts secrete collagen parallel to the long axis of the condensation. This study elucidates fundamental mechanisms of intramembranous bone development at the cellular level, furthering our knowledge of this important process among vertebrates.     

Effect of systemic calcium deficiency on the expression of transforming growth factor-beta in chick embryonic calvaria.

The developmental process of intramembranous ossification involves bone formation directly from mesenchymal differentiation without a cartilage intermediate. We have previously observed that systemic calcium deficiency in the developing chick embryo, produced by long-term shell-less culture, results in the appearance of chondrocyte-like cells in the calvarium, a parietal bone which normally develops via intramembranous ossification. This investigation aims to analyze the mechanism underlying this calcium deficiency-related, aberrant appearance of cartilage phenotype in the chick embryonic calvarium. In view of the reported involvement of transforming growth factor beta (TGF-beta) in osteogenesis and chondrogenesis, we have examined and compared here the expression of TGF-beta in the chick embryonic calvaria of normal (in ovo development, NL), shell-less (SL), and calcium-supplemented SL (SL+Ca) embryos. TGF-beta expression was analyzed at the mRNA level by blot and in situ cDNA hybridization, and at the protein level by immunohistochemistry and immunoblotting. The results presented here indicate that: 1) TGF-beta is expressed in the chick embryonic calvarium by both periosteal cells and osteocytes, as revealed by in situ hybridization and immunohistochemistry; 2) TGF-beta expression is significantly increased in SL calvarium compared to NL calvarium, at both protein and mRNA levels; 3) the number of TGF-beta expressing cells increases in the SL calvarium, particularly along the central, subcambial core region of the bone; and 4) exogenous calcium repletion to the SL embryo affects the expression of TGF-beta such that the pattern approaches that in the NL embryo. Taken together, these results indicate that altered TGF-beta expression accompanies the aberrant appearance of cartilage phenotype caused by systemic calcium deficiency. We postulate that normal cellular differentiation along the osteogenic pathway during embryonic intramembranous ossification is crucially dependent on regulated TGF-beta expression.     

Collagen-phagocytosing ability of periodontal osteoblasts at the bone surface

The collagen-phagocytosing activity of osteoblasts at the alveolar bone-ligament interface of rat mandibular first molars was investigated both histologically and histochemically. Alveolar bones of male Wistar rats (6 months old) were used in this study. Collagen-containing phagosomes appeared in cuboidal osteoblasts aligned on the bone surface. The 5.7% of the osteoblasts exhibiting alkaline phosphatase activity revealed collagen-containing phagosomes, and the collagen fibrils within the phagosomes were at various stages of degradation. In addition, acid phosphatase activity and the immunocytochemical distribution of cathepsin B were found in these collagen-containing phagosomes at similar locations. The presence of both enzymes in the phagosomes suggests that an intracellular degradation of collagen occurs. Therefore, in addition to the osteoblastic functions of synthesizing and secreting bone matrices, osteoblasts are also capable of phagocytosis and the intracellular disintegration of collagen. Our findings suggest that osteoblasts at the alveolar bone-periodontal ligament interface have a collagen-phagocytosing ability and play an important role in the physiological remodeling and metabolic breakdown of collagen fibrils of periodontal ligament without osteoclastic bone remodeling.     

Angiogenesis and mineralization during distraction osteogenesis

Distraction osteogenesis is currently a standard method of bone lengthening. It is a viable method for the treatment of short extremities as well as extensive bone defects, because large amounts of bone can be regenerated in the distraction gap. Mechanical stimulation by distraction induces biological responses of skeletal regeneration that is accomplished by a cascade of biologic processes that may include differentiation of pluripotential tissue, angiogenesis, mineralization, and remodeling. There are complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Regenerate bone forms by three modes of ossification, which include intramembranous, enchondral, and transchondroid ossifications, although intramembraneous bone formation is the predominant mechanism of ossification. In this review we discussed the coupling between angiogenesis and mineralization, the biological and mechanical factors affecting them, the cellular and molecular events occurring during distraction osteogenesis, and the emerging modalities to accelerate regenerate bone healing and remodeling.