Localization of the expression of types I, III, and IV collagen, TGF-beta 1 and c-fos genes in developing human calvarial bones

Total RNA extracted from developing calvarial bones of 15- to 18-week human fetuses was studied by Northern hybridization: in addition to high levels of type I collagen mRNAs, the presence of mRNAs for type III and type IV collagen, TGF-beta and c-fos was observed. In situ hybridization of sections containing calvarial bone, overlying connective tissues, and skin was employed to identify the cells containing these mRNAs. Considerable variation was observed in the distribution of pro alpha 1(I) collagen mRNA in osteoblasts: the amount of the mRNA in cells at or near the upper surface of calvarial bone was distinctly greater than that in cells at the lower surface, indicating the direction of bone growth. High levels of type I collagen mRNAs were also detected in fibroblasts of periosteum, dura mater, and skin. Type III collagen mRNA revealed a considerably different distribution: the highest levels were detected in upper dermis, lower levels were seen in fibroblasts of the periosteum and the fibrous mesenchyme between bone spiculas, and none was seen in osteoblasts. Type IV collagen mRNAs were only observed in the endothelial cells of blood capillaries. Immunohistochemical localization of type III and IV collagens agreed well with these observations. The distribution of TGF-beta mRNA resembled that of type I collagen mRNA. In addition, high levels of TGF-beta mRNA were observed in osteoclasts of the calvarial bone. These cells, responsible for bone resorption, were also found to contain high levels of c-fos mRNA. Production of TGF-beta by osteoclasts and its activation by the acidic environment could form a link between bone resorption and new matrix formation.     

Regeneration of growth plates in the long bones of the neonatal rat hindlimb

Twenty-seven male albino rats underwent hindlimb amputations through the lower femur or the midshaft of the tibiofibula on the tenth to 12th day of life. Amputation stumps were examined grossly and histologically in order to assess the significance of level and angle of transaction as determinants of subsequent growth and regeneration and to ascertain whether growth plates can regenerate following their complete excision. Amputees survived for 17-73 days. In order to exclude limbs which had been severed at or distal to the level of the growth plate, amputated limb segments either were cleared to transparency and inspected under low magnification or were sectioned serially and examined by using a compound microscope. Following amputations through the femur, the predominant response involved repair of the skeletal defect and healing of adjacent soft tissues (ten of 17 rats). Among five other animals the skeletal terminus was covered with a plate of cartilage which, in three, included areas of growth-plate architecture. Two additional transfemoral amputees regenerated incomplete growth plates, each overlying a single epicondylar surface, and one provided with a regenerated hemiepiphysis. Five of ten transtibiofibular amputees formed cartilage plates which covered the skeletal terminus in whole or in part and one regenerated an entire growth plate restricted to the distal fibula. It is concluded that angle and level of transection are not pivotal modifiers of growth and regeneration processes, and that distal growth plates may regenerate entirely or in part following their complete removal from the hindlimb.     

Chondrocytes embedded in the epiphyseal growth plates of long bones undergo autophagy prior to the induction of osteogenesis

Bone growth takes place through the activities of chondrocytes embedded in the epiphyseal growth plate. Stress conditions in the plate can promote the autophagic response through the modulation of genes controlling metabolite utilization. mTOR plays a critical role in autophagy serving as the sensor that integrates metabolic and growth factor signals. Ongoing studies indicate that terminal chondrocytes exhibit autophagic characteristics. Morphologically, the arrested cells contain double membrane vacuoles; there is a loss of membrane structure, limited staining and organelle destruction. Since the life history of the growth plate chondrocyte is very short, even minor disturbances in the metabolic state can result in gross impairment of growth. We contend that the induction of the autophagic response, permits the terminally differentiated cells to survive the brief rigors of the harsh local microenvironment. Whether chondrocytes can recover from this state, and possibly participate in osteogenesis, is not known at this time.     

Division and death of cells in developing synovial joints and long bones

Articular chondrocytes are a unique set of cells from the time the cellular condensations that become the anlagen of the long bones develop in the embryo. In the presumptive joint the cells of the opposing bones are packed very closely together, but at cavitation, the central, flattened cells move apart to form the articular surfaces. As the articular cartilage develops the cells are pushed further apart by the cartilaginous matrix. To determine the contributions of cell proliferation and death to cavitation and the subsequent development and growth of articular cartilage, direct observations were made to identify mitotic cells and those with apoptotic bodies in haematoxylin-stained sections of developing joints, and growing and ageing articular cartilage of the rabbit knee. These observations were extended using antibodies to the proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP nick end labelling (TUNEL) on corresponding sections. Low levels of cell division do occur in the articular cartilage up to 6 weeks postnatally, but matrix formation makes the major contribution to the increase in size of the cartilage. Cell death is not observed during cavitation, nor during the development of the articular cartilage proper. Apoptosis is essential, however, for the removal of the epiphyseal cartilage during ossification of the epiphyses and in the growth plate.     

Synostosis dynamics in human long tubular bones

More than 8,000 roentgenograms and electroroentgenograms of brachial, ulnar, radiocarpal, coxofemoral, knee, talocrural joints have been studied in persons of both sex at the age from birth up to 25 years. The synostosis degree is appreciated by the six-mark system. The roentgenological data are corroborate histologically. The method of appreciation applied makes it possible to reveal cases of initially forming synostosis long before puberty. Three stages of synostosis process are defined. The first stage--slow increase of the synostosis mark. The second--quick synostosis formation. The third--final stage. According to the stages defined, a comparative analysis of the synostosis process in all metaepiphyseal zones of all long tubular bones, peculiarities of the process depending on the sex are demonstrated. The data on the time, when the points of ossification appear, on the beginning and completion of synostosis process in all metaepiphyseal zones of the long tubular bones are presented.     

Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.     

Perichondrial-mediated TGF-beta regulation of cartilage growth in avian long bone development

We previously observed using cultured tibiotarsal long-bone rudiments from which the perichondrium (PC) and periosteum (PO) was removed that the PC regulates cartilage growth by the secretion of soluble negative regulatory factors. This regulation is "precise" in that it compensates exactly for removal of the endogenous PC and is mediated through at least three independent mechanisms, one of which involves a response to TGF-beta. PC cell cultures treated with 2 ng/ml TGF-beta1 produced a conditioned medium which when added to PC/PO-free organ cultures effected precise regulation of cartilage growth. In the present study, we have investigated the possibility that TGF-beta itself might be the negative regulator which is produced by the PC cells in response to their treatment with TGF-beta1. Using a TGF-beta responsive reporter assay, we determined that PC cell cultures, when treated with 2 ng/ml or greater exogenous TGF-beta1, produce 300 pg/ml of active TGF-beta. Then we observed that this concentration (300 pg/ml) of active TGF-beta1, when added to PC/PO-free tibiotarsal organ cultures, effected precise regulation of cartilage growth, whereas concentrations of TGF-beta1 either greater or less than 300 pg/ml produced abnormally small cartilages. These results suggest that one mechanism by which the PC effects normal cartilage growth is through the production of a precisely regulated amount of TGF-beta which the PC produces in response to treatment with exogenous TGF-beta itself.     

Expression patterns of neurotrophic factor mRNAs in developing human teeth

Neurotrophic factors regulate survival, differentiation, growth and plasticity in the nervous system. In addition, based on their specific and shifting temporospatial expression patterns, neurotrophic factors have been implicated in morphogenetic events during tooth development in rodents. To determine whether these findings in rodents could be related to humans, we have now studied nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell-line derived neurotrophic factor (GDNF), and neurturin (NTN) mRNA expression patterns in developing human teeth during gestational weeks 6.5-11. Using in situ hybridization histochemistry, we found distinct and specific patterns of neurotrophin and GDNF mRNA expression in the developing human teeth. NGF mRNA labeling was weak and confined predominantly to the dental papilla. BDNF mRNA labeling was stronger than NGF mRNA and was seen in the mesenchyme located lateral to the dental organ, as well as in epithelial structures (inner dental epithelium and enamel knot). NT-3 mRNA was observed in the dental papilla and in the area of the cervical loop. NT-4 mRNA was expressed in both oral and dental epithelia in all stages studied. GDNF mRNA was found in the dental follicle and at different sites in the inner dental epithelium. Weak NTN mRNA labeling was also found in the developing teeth. Based on these findings, we suggest that neurotrophins, GDNF and NTN might be involved in morphogenetic events during early stages of tooth development in humans. Protein gene product (PGP) 9.5-immunoreactive nerve fibers were observed in the dental follicle by 11 weeks coinciding with the labeling for neurotrophic factor mRNAs in this structure. This suggests that these neurotrophic factors might be involved in the innervation of dental structures. The rich expression of neurotrophic factors in developing dental tissues suggests that developing, or possibly adult, dental tissue might be used as an allograft source of trophic support for diseases of the nervous system.     

The epiphyseal cartilage and growth of long bones in Rana catesbeiana.

The structure of the epiphyseal cartilage of the bullfrog Rana catesbeiana and its role in the growth of long bones were examined. The epiphyseal cartilage was inserted into the end of a tubular bone shaft, defining three regions: articular cartilage, lateral articular cartilage and growth cartilage. Joining the lateral cartilage to the bone was a fibrous layer of periosteum, rich in blood vessels. Osteoblasts with alkaline phosphatase activity were found on the surface of the periosteal bone, which presented a fibrous non-mineralised tip. The growth cartilage was inside the bone. The proliferative chondrocytes presented perpendicular separation of daughter cells and there was no columnar arrangement of the cells. Furthermore, chondrocyte hypertrophy was not associated with either calcification or endochondral ossification, in apparent contrast to the avian and mammalian models. Finally, there was no reinforcement system capable of directing cell volume increase into longitudinal growth. Since bone extension depends on the intramembranous ossification of the periosteum, the growth cartilage is inside and not at the end of the bone and the cells in the growth cartilage show no columnar arrangement and separate in a direction perpendicular to the long bone axis, we conclude that the growth cartilage mainly contributes to the radial expansion of the bone.     

Lipids at the stages of early osteogenesis in human long tubular bones

In 240 long tubular bone anlagen (LTBA) of extremities in 40 human embryos and prefetuses at the age of 6-12 weeks of the prenatal development the investigation has been performed concerning localization, dynamics of contents and spectrum of neutral lipids and phospholipids. Lipids are stained with Sudan III and IV, phospholipids-with Sudan black with corresponding control extractive methods and quantitative estimation of these substances values. The material for electron microscopical investigation of the lipids is treated according to the method, that preserves their safety. The spectrum of lipids and phospholipids in the LTBA is studied by means of the thin layered chromatography method. The data of the histochemical investigation and chromatography demonstrate decreasing contents of neutral lipids with the gradient from the zone of the preserved cartilage up to the ossification zone of the epiphyseal cartilage of the LTBA in the human embryos and prefetuses. Increased concentration of phospholipids and complication of their spectrum is noted in the areas of intensive deposits of calcium salts. An essential role of the substance of lipid origin is supposed in ossification and mineralization of the human LTBA.     

Response of the longitudinal growth of cranium and long bones to early weaning in the rabbit

To study the problem whether the growth rates of the cranium and of long bones are different from one another, in 13 age groups of 5 rabbits each three distances of the skull (SSO-SL, SSO-P and N-NSA) and the length of the femur, tibia and calcaneus were measured. Following the early weaning of the rabbits (41/2 weeks), the increase of all measurements was strongly delayed for about one week. Thereafter, during a two weeks' period a typical catch-up growth was observed. From these results the conclusion was drawn that the growth rates of cranium and long bones do not differ from each other, and that a temporary deficiency of food intake affects intramembranous bone growth and cartilaginous growth in a similar way.