Morphological and histochemical events during first bone formation in embryonic chick limbs

Staged embryos from White Leghorn chicken eggs were used to assemble a detailed morphological, cellular and molecular picture of the complex events of first-bone formation. To provide these details, light and electron microscopic, histochemical and immunocytochemical techniques were used to establish a temporal sequence for long bone development in chick wing and leg from Hamburger-Hamilton stage 29 through stage 35. Three distinctive cell regions can be morphologically identified by stage 28 (leg) or 29 (wing) at the middiaphysis. These regions are: 1. an outer grouping of loose mesenchymal and myogenic cells, 2. an osteoprogenitor layer which will later divide to maintain this progenitor layer in a brickwork or stacked configuration and to produce round, tightly packed osteoblasts, and 3. a core (rod) of cartilage. First bone is laid down just outside the cartilage core, initially as a layer of Type I collagen-rich osteoid which later becomes mineralized. Vascular elements then come to reside above this mineral layer, and osteoid is laid down between vascular elements and eventually above them to form a second layer of trabecular bone. As this radial formation of layers of bone is progressing, so too is the proximal and distal expansion of the first bone forming process. A model is presented which considers that chondrogenic and osteogenic cell commitment occur simultaneously in early limb development and that it is the expression of the osteogenic phenotype which governs the boundaries of cartilage development. Importantly, the vasculature plays a key role in the patterning of bone formation well before it enters the cartilaginous core at stage 35 and participates in the erosion of the core. While this report is restricted to events occurring through stage 35, it relies on data presented in a companion report detailing later bone development and remodeling (Pechak et al; Bone 1986) and emphasizes that the cartilage model does not provide the scaffolding for bone but rather defines the marrow space.     

Cellular events associated with the induction of bone by demineralized bone

Implantation of demineralized bone (DB) in the form of powder or intact segments in extra skeletal sites stimulates new bone formation. Urist and co-workers presented substantial evidence that there is a noncollagenous protein that has the ability to induce bone formation. One aim of this study was to trace the process of bone formation when DB, in the form of perforated rectangular plates, is implanted subcutaneously in 2-month-old rats. A second objective was to determine whether cartilage cells play a role in the formation of bone in this model. Various DB plates with 0.25 mm diameter holes were implanted subcutaneously for 1-4 weeks in rats. One week after implantation, DB plates were covered by vascularized connective tissue that invaded the perforations. Aggregates of chondrocytes were observed within the holes and on periosteal surfaces in only a few specimens. Further cartilage proliferation was not observed, and by the 2nd week there was no evidence of endochondral bone formation. Where these cartilage-like cells were present, a thin layer of mineral was deposited around them; resorption and fibrous tissue infiltration followed. This aborted form of endochondral calcification was not followed spatially by bone formation. Patent vascularized channels were invaded by alkaline phosphatase-positive mononuclear cells and fibroblasts, and became enlarged by the enzymatic action of macrophages. The next step involved the calcification of DB plates adjacent to the wide spaces. Osteoclasts now appeared leading to the resorption of this recalcified matrix. The eroded and now enlarged lacunar surfaces were lined by newly formed bone and osteoblasts. This process continued so that, at the end of 4 weeks following implantation, the original DB plates were replaced by trabecular bone. Biochemical data on calcium and alkaline phosphatase levels in the implants paralleled the morphological observations.     

Internal remodeling of periosteal new bone during fracture healing

A closed fracture model of the rat tibia was employed to study internal remodeling of periosteal new bone during fracture repair. Static histomorphometric parameters of osteoid surface (or perimeter) and eroded surface (resorption surface) were used as indicators of appositional bone formation and resorption of bone trabeculae, respectively. Intracortical remodeling at the fracture site was evaluated using quantitative tetracycline histology and microradiography. The extents of osteoid and eroded bone surfaces did not differ significantly in the periosteal woven new bone in the early phases of fracture healing. Later on, the periosteal new bone had significantly more osteoid surface than eroded surface (p less than 0.001). The number of osteoclasts also decreased significantly over time during fracture healing (p = 0.028). Cortical bone showed a continuous increase of porosity (p less than 0.01) between 1 and 6 weeks after fracture. These results suggest that there is a time-related change in the balance of periosteal bone formation and resorption during the progress of fracture repair. We hypothesize that this change was related to the restoration of bony continuity. Further studies are, however, needed to indicate the histomorphometric features of periosteal new bone in fracture nonunions.     

Flurbiprofen-induced stimulation of periosteal bone formation and inhibition of bone resorption in older rats

The skeletal effects of flurbiprofen (Fb), a nonsteroidal antiinflammatory drug, was studied by histomorphometry in 9-month-old retired female breeder, Sprague-Dawley rats. Flurbiprofen was given subcutaneously at 0, 0.2, 0.1, 0.5, 2.5, or 5 mg/kg/d for 21 days. Flurbiprofen had no effect on longitudinal growth, but stimulated radial growth (+200%) over controls. In the tibial shaft, Fb stimulated the mineral apposition rate (+25%), mineral bone formation rate (+100%), and periosteal labeling length (+64%) at the 2.5 and 5.0 mg Fb/kg dose levels, and had no effect on marrow cavity size compared to controls. However, these changes were insufficient to increase cortical bone mass. In the proximal tibial metaphysis, Fb suppressed osteoclasts/mm² of metaphyseal tissue (-47%), osteoclasts/mm of bone surface (-46%), and the osteoclast/osteoblast ratio (-50%), increased the calcified cartilage core population (+100%), and had no effect on osteoblast numbers at all dose levels. There was an insignificant increase in metaphyseal cancellous bone mass. The current study leads to the conclusion that flurbiprofen-stimulated periosteal bone growth was due to direct stimulation of osteoblast recruitment and activity independent of longitudinal bone growth. Further, it confirms early findings in young rats that flurbiprofen induced depressed bone resorption without lowering bone formation. However, because of insufficient treatment time, the older rat did not accumulate bone as the young rats did.     

Effect of recombinant human granulocyte colony-stimulating factor (rh G-CSF) on rat bone: Inhibition of bone formation at the endosteal surface of vertebra and tibia

The effect of recombinant human granulocyte colony-stimulating factor (rh G-CSF) on bone was evaluated by histomorphometry using Sprague-Dawley rats. rh G-CSF was injected at doses of 0, 50, 150, and 450 μg/kg for 6 weeks.In vivo double fluorochrome labeling was performed before sacrifice. No significant change in body weight was observed. Bone mineral density (BMD) of lumbar vertebrae and femora was significantly decreased in G-CSF-treated groups. In the lumbar vertebra, osteoid surface, osteoid thickness, trabecular thickness, and labeled surface in G-CSF-treated groups were also significantly lower. In addition, osteoclast number and osteoclast surface were significantly higher in the G-CSF-treated groups. The endocortical surface at the mid-tibia showed lower labeled surface and mineral apposition rate in G-CSF-treated groups, without significant changes at the periosteal surface. Furthermore, numerous granulocytes fully occupied the bone marrow area. We conclude that proliferating granulocytes in the bone marrow may inhibit bone-forming cells from contacting the bone surface, resulting in reduction of bone formation;and increased osteoclastic bone resorption induced by G-CSF treatment contributed to the reduction of BMD.     

Dynamic histomorphometric evaluation of human fetal bone formation

We have evaluated dynamic and static parameters of bone formation in femoral metaphyses collected from two human fetuses at 19 weeks of gestation. Tetracycline was administered to the mother at set intervals (2-5-2 day schedule) before interruption of pregnancy. Labels were distinct and sharply linear, suggesting a well organized calcification front at this early stage of mineralization. Mineral apposition rate (MAR) was fastest (4.1 ± 0.3 μm/d) in the periosteal (Ps) envelope, and about half that value in the endosteal envelopes (endocortical: 2.5 ± 0.1, cancellous 2.1 ± 0.1 μm/d). Because cellular activities may vary throughout the metaphyseal area, sections were arbitrarily separated in 0.75 mm layers starting from the growth plate. Three measured parameters decreased rapidly with increasing distance from the physis: Ps MAR: 4.9 to 2.3 μm/d, trabecular osteoid thickness: 5.9 to 1.2 μm, and cartilage volume (CgV/TV): 5.4% to 1.2%. Others did not vary significantly along the metaphysis. Comparison of several static parameters with those measured in five autopsy specimens from full-term infants showed that bone and cartilage volume, and trabecular thickness increased while osteoid thickness and parameters of resorption decreased in the second half of the gestation period. The study indicates that fetal bone matrix mineralization is already highly organized at mid-gestation, and validates the use of histomorphometry to assess bone maturation during early skeletal development.     

Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation

Connexin 43 (Cx43) mediates osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/osteocytes or only in osteocytes (Cx43(ΔOt) mice) exhibit increased osteocyte apoptosis, endocortical resorption, and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated. Anatomical mapping of apoptotic osteocytes, osteocytic protein expression, and resorption and formation suggests that Cx43 controls osteoclast and osteoblast activity by regulating osteoprotegerin and sclerostin levels, respectively, in osteocytes located in specific areas of the cortex. Whereas empty lacunae and living osteocytes lacking osteoprotegerin were distributed throughout cortical bone in Cx43(ΔOt) mice, apoptotic osteocytes were preferentially located in areas containing osteoclasts, suggesting that osteoclast recruitment requires active signaling from dying osteocytes. Furthermore, Cx43 deletion in cultured osteocytic cells resulted in increased apoptosis and decreased osteoprotegerin expression. Thus, Cx43 is essential in a cell-autonomous fashion in vivo and in vitro for osteocyte survival and for controlling the expression of osteocytic genes that affect osteoclast and osteoblast function.     

Inflammation-mediated osteopenia (IMO) during acute inflammation in rats is due to a transient inhibition of bone formation

Summary Local inflammation was induced in rats through the subcutaneous injection of magnesium silicate. Trabecular bone volume of the tibia decreased progressively during a 3 week observation period following the inflammatory stimulus. The trabecular bone surface covered with osteoblasts was strikingly reduced during the first week but had normalized by the end of the third week. Calcification rate in the cortical bone of the tibia was reduced with a parallel reduction in endosteal osteoid seam width. Both calcification rate and tetracycline double-labeled surface of vertebral trabecular bone were reduced during the first 2 weeks. Neither total bone resorption surface nor active bone resorption surface were increased. There was a decrease in osteoclast numbers/mm² bone tissue associated with decreasing bone volume. Our data demonstrate a transient inhibition of bone formation during acute inflammation in the rat and indicate that changes in osteoblast function are part of the acute phase response following local inflammation.     

Role of bone bark during growth in width of tubular bones. A study in human fetuses.

The morphologic features of bone bark, a structure surrounding the distal and proximal ends of long bones, were studied in the distal femur, proximal tibia, and proximal fibula of 77 spontaneously aborted human fetuses varying in gestational age from 10 to 20 weeks. Standard histologic techniques used in addition to in situ immunohistochemical staining allowed the examination of the structure of the bone bark and localization of Types 1, 2, and 3 collagens at different gestational ages. The bone bark was shaped like a cylindrical sheath of bone lamellae of varying thickness. The epiphyseal end of the bone bark, known as the groove of Ranvier, was covered outwardly by a fibrous layer and inwardly by the epiphyseal cartilage and contained mesenchymal cells, chondroblastic precursor cells, and densely packed cells differentiating into osteoblasts. Neither the cell density in the groove nor the thickness of the bone bark were identical circumferentially, indicating an unequal growth in width. In addition, the presence of periosteal apposition and endosteal resorption of the bone bark on one side and of endosteal bone deposition accompanied by periosteal resorption of the bone bark on the opposite side support the concept of a spatial drift of bones. These observations furnish histologic proof that groove and bone bark, although assuring an equal growth in length, contribute to an unequal and eccentric growth in width.     

Periosteal osteosarcoma with secondary bone marrow involvement: a case report

Periosteal osteosarcoma is an exceedingly rare type of chondroblastic osteosarcoma, showing a rather good prognosis, and secondary bone marrow involvement is unusual. However, there have been some reports describing periosteal sarcoma involving medullary bone. We encountered a patient, a 38-year-old man, who had a bone surface tumor in the left tibia. An X-ray showed an erosive cortical mass extraosseous portion, located in the diaphysis of the tibia. Other images revealed a thin cortex, periosteal reactions, coarse mineralization in the extraosseous portion, and bone marrow involvement. Grossly, surgical materials showed that the tumor mainly existed at the periosteal portion, only a part of the cortex was destroyed, and there was medullary involvement throughout. Histological examinations showed a predominantly chondroid component with malignant osteoid formation. On the basis of the histological macroscopic and microscopic findings, we made the diagnosis of periosteal osteosarcoma with secondary bone marrow involvement.     

Histological study of effect of bone morphogenetic protein derived from bovine tooth on periosteum in rats

Summary In this study, we examined histologically the effect of a bone morphogenetic protein (BMP) derived from bovine tooth on the periosteum. Supraperiosteal injection of crude BMP into femurs of Wistar rats (28 day old) resulted in periosteal cell proliferation with subsequent bone and cartilage formation. Moreover, proliferating periosteal cells migrated into injected BMP, and formed both cartilage and bone. These observations show that exogenous BMP stimulates mesenchymal cells of the periosteum to proliferate and differentiate into osteoblasts, and therefore BMP may be one of factors which are involved in differentiation of osteoblasts in the periosteum.     

The role of three genetically distinct collagen types in endochondral ossification and calcification of cartilage.

The role of three genetically distinct collagen types in the formation of endochondral bone and in calcification and resorption of cartilage has been assessed. Using antibodies specific to types I, II and III collagen we have demonstrated in the embryonic chick tibia that endochondral bone formation began with deposition of type III collagen in lacunae of hypertropic chondrocytes by invading bone-marrow-derived cells. This was followed by the deposition of type I collagen, which is the collagenous constituent of endochondral osteoid. At later stages of development endochondral osteoid was found in the epiphysial growth plate in apparently intact lacunae of hypertrophic chondrocytes; this indicated that the latter might contribute to the synthesis of osteoid type I collagen. Immuno-histological staining for collagen types, and von Kossa staining for calcium phosphate on parallel sections, demonstrated that type I and type II collagen matrices were substrates for calcification. Endochondral bone (with type I collagen) was found on scaffolding of both uncalcified and calcified cartilage (with type II collagen), indicating that calcification of endochondral osteoid and of the underlying cartilage occurred independentyl. Spicules of endochondral cancellous bone of a four-week-old chick contained a core of calcified type II collagen.     

Morphometric Analysis of Gastrectomy-Evoked Osteopenia

Gastrectomy leads to osteopenia in the rat. The present study describes the effects of gastrectomy on bone morphology. Rats were subjected to gastrectomy or sham operation. Four weeks after the operation the rats were killed and both tibiae were removed. Bone morphology of the left tibia was analyzed with quantitative computer tomography, the right tibia with histomorphometry. Bone length, bone mineral content, as well as indices of bone resorption and formation were measured in the metaphysis and the diaphysis. Gastrectomy had no effect on longitudinal bone growth but it led to a low bone mineral content at both sites. Bone resorption was increased by gastrectomy, as shown by an increase in the medullary cavity area in the diaphysis. Gastrectomy also reduced bone formation, as shown by a decreased periosteal circumference and a decrease in the mean periosteal bone apposition in the diaphysis. In conclusion, gastrectomy-evoked osteopenia reflects impaired formation and increased resorption of bone. Received: 31 March 1997 / Accepted: 26 June 1997     

Trabecular bone turnover, bone marrow cell development, and gene expression of bone matrix proteins after low calcium feeding in rats

Low-calcium-fed animals have been accepted as one of the experimental models showing a reduction in bone mass. However, the effects of short-term low-calcium feeding on bone turnover, the development of osteoprogenitor cells, and gene expression of bone matrix proteins have not been reported. In this study, we examined the effect of a low-calcium diet on rat tibia and analyzed the changes in the bone by histomorphometry, bone marrow cell culture, and in situ and Northern hybridization of the bone matrix proteins. Rats were fed either a low-calcium diet (0.05% Ca) or a normal calcium diet (0.5% Ca) using the pair feeding technique. They were killed at day 0, 12 h, and days 1, 2, and 3. In the low-calcium group, the serum parathyroid hormone (PTH) level was temporarily increased in 12 h after feeding the low-calcium diet. Bone mineral density in the trabecular bone was significantly decreased from 1 day after the low-calcium feeding, but cortical bone did not show any changes during the experimental period. The bone volume per tissue volume in the proximal tibia also decreased from day 1 in the low-calcium group. The number of osteoclasts and osteoblasts on the trabecular bone surface was increased in the low-calcium group compared with the normal-calcium group. An ex vivo study showed that the number of progenitors of osteoclasts and osteoblasts in bone marrow was also increased in the low-calcium group of rats. The localization of type I collagen mRNA was observed in osteoblasts in the low-calcium group. The Northern hybridization study showed that the gene expression of type I collagen, osteopontin, and osteocalcin was increased at day 3 in the low-calcium group. These results indicated that the trabecular bone surface quickly responded to the low-calcium feeding and that bone remodeling activity was activated probably by PTH. The changes in bone marrow cell populations and the gene expression of bone matrix proteins are closely associated with increased bone turnover induced by the low-calcium diet, resulting in rapid bone loss of the trabecular bone.     

The Subchondral Bone in Osteoarthritis and Rheumatoid Arthritis of the Knee: A Histological and Microradiographical Study

Specimens of the joint surfaces of the tibia from patients with OA and RA were examined for bone mineralization, bone formation, osteoid tissue and bone resorption. Judging from the appearance of the osteoblasts in OA the sclerotic changes are mainly focal with relatively little osteogenesis. No osteoclasia was seen, in the sclerotic areas. Breakdown of the mineralized cartilage is followed by the development of cysts with highly cellular connective tissue with high osteoblastic activity and osteoclasia. Osteoid tissue is relatively sparse. The changes in RA are more diffuse with a more active osteoblastic activity and widespread zones of osteoid tissue as well as resorption by osteoclasts. It appears as if the increased uptake of ⁸³Sr in OA is more dependent on the occurrence of relatively inert osteosclerosis than on a rapid turnover of the bone tissue.     

Bone morphogenetic proteins in bone tumors

Bone morphogenetic proteins (BMPs), inducers of ectopic bone formation in vivo, are present in a number of osteosarcomas. BMPs are responsible for reactive bone formation, including periosteal reactions by normal osteoblasts, rather than production of tumorous osteoid by tumor cells. Osteosarcomas producing BMPs contain less-differentiated mesenchymal cells, resulting in a poorer prognosis for those patients. BMPs are also expressed in malignant fibrous histiocytomas (MFHs) of bone and dedifferentiated chondrosarcomas exhibiting undifferentiated features. However, BMPs in MFH do not show any osteoinductive activity in vivo, suggesting that those BMPs may be inactive forms and have additional functions unrelated to bone formation. Among benign bone tumors, BMPs are expressed in osteoid osteomas or osteoblastomas and effect reactive bone formation such as a surrounding sclerosis. BMPs and a BMP receptor (BMPRIB) are also detected in the cartilage cap in osteochondroma, suggesting that BMP signaling via BMPRIB might be involved in the pathogenesis of osteochondroma. Clinically, BMPs have utility as diagnostic and prognostic markers for characterizing the stage of differentiation of mesenchymal cells and mesenchymal tumors, and they may be of value in predicting the prognosis of sarcoma patients. This article reviews the accumulated information on BMPs in bone tumors, including the most recent findings, and discusses the biological and clinical significance of BMPs in bone tumors.Presented at the 36th Annual Musculoskeletal Tumor Meeting of the Japanese Orthopaedic Association, Kobe, Japan, July 2003     

Histochemical examination of ectopic bone formation induced in rat bone marrow

We examined the rapid formation and subsequent resorption of woven bone induced by partial ablation of rat bone marrow. On the 1st day after ablation, masses of clots occupied the region from which marrow was eliminated. On the 3rd day, alkaline phosphatase-(ALPase-) positive osteoblastic cells appeared in the vicinity of the marrow-eliminated region, forming woven bone. Other ectopic woven bone extended from the endosteal surface toward the bone marrow. Therefore, the newly formed bone originated in two different sites, the endosteal bone surface and the marrow tissues near the marrow-eliminated region. On the 7th day, numerous tartrate-resistant acid phosphatase- (TRAPase-) positive osteoclasts and ALPase-positive osteoblasts expressing the osteonectin gene indicated high activity in both formation and resorption of ectopic woven bone. On the 10th day, the ectopic bone had been markedly resorbed and replaced by bone marrow tissue as the ectopically formed woven bone had not been dynamically maintained, probably because of reduced bone formation activity. Immunoreactivity for basic fibroblast growth factor (bFGF) was indistinctly observed on osteoblastic and preosteoblastic cells on the 1st day after ablation. The fibroblastic cells in the marrow-eliminated region on the 3rd day, and both osteoblasts and preosteoblasts in the woven bone on the 7th day, showed strong immunoreactivity for bFGF. Unlike fractured cortical bone, no chondrogenesis was observed. This model appears to provide convenient material and an important clue for investigation of imbalanced bone formation and subsequent resorption.     

Early mortality after elective hip surgery

Specimens of the joint surfaces of the tibia from patients with OA and RA were examined for bone mineralization, bone formation, osteoid tissue and bone resorption. Judging from the appearance of the osteoblasts in OA the sclerotic changes are mainly focal with relatively little osteogenesis. No osteoclasia was seen, in the sclerotic areas. Breakdown of the mineralized cartilage is followed by the development of cysts with highly cellular connective tissue with high osteoblastic activity and osteoclasia. Osteoid tissue is relatively sparse. The changes in RA are more diffuse with a more active osteoblastic activity and widespread zones of osteoid tissue as well as resorption by osteoclasts. It appears as if the increased uptake of ⁸³Sr in OA is more dependent on the occurrence of relatively inert osteosclerosis than on a rapid turnover of the bone tissue.     

Bone marrow plays a role in bone metabolism: Histomorphometry of iliac bone in postmenopausal women

Summary We conducted a histomorphometrical study on the role of the bone marrow in cancellous bone metabolism using iliac bone specimens from 79 postmenopausal women. A gradual decrease in hematopoietic tissue of the bone marrow was proportionate to the decrease in cancellous bone and the ratio of osteoid perimeter/bone perimeter regardless of age. On the other hand, the ratio of eroded perimeter/bone perimeter remained almost steady until hematopoietic tissue decreased significantly. These findings suggest that a change in the bone marrow, that is, a decrease in hematopoietic tissue, causes an imbalance in bone formation and resorption and leads to bone loss.     

Anabolic effect of prostaglandin E2 on cortical bone of aged male rats comes mainly from modeling-dependent bone gain

In this study, prostaglandin E₂ (3 mg/kg per day) was administered to 20-month-old male Wistar rats for 10 and 30 days. Histomorphometric analyses were performed on double-fluorescent-labeled undecalcified tibial shaft sections. Thirty days of prostaglandin E₂ (PGE₂) administration increased bone formation rate/total bone surface from undetectable levels to 0.6 μm/day at the periosteal surface and from 0.5 to 2.1 μm/day at the endocortical surface. Endocortical osteoid surface area increased from 2% to 67% at day 10 and decreased to 6% at day 30; woven and lamellar bone formation started at day 0, but was most obvious at day 30, resulting in a 12% increase of total bone mass. The red to yellow marrow ratio was 0.2 in pretreatment controls, and increased to 1.6 by day 10 and 2.4 by day 30 with PGE₂ administration. Intracortical cavity number and area increased after 10 days of PGE₂ treatment, but with forming osteon number and area far exceeding those of resorption cavities at day 30. Endocortical modeling surface/endocortical surface was only 1.5%, and remodeling was 11.1% in pretreatment controls. PGE₂ treatment increased modeling to 24.5% in the 10 day group and 93.7% in the 30 day group, whereas remodeling remained unchanged at 10 days, and decreased to 6.2% at 30 days. Osteoprogenitor cells and osteoblasts could not be detected in pretreatment controls, but increased by day 10, and returned almost to control levels by 30 days. Our data indicate that PGE₂ induced periosteal and endocortical bone formation mainly by modeling-dependent bone gain, accompanied by increases in intracortical remodeling and red bone marrow, and a transient increase in the osteoprogenitor cells adjacent to the endocortical surface. These findings suggest that 20-month-old male Wistar rats were very responsive to the anabolic action of PGE₂ in the tibial shaft, a site consisting mainly of cortical bone and yellow marrow.