Ablation of the miRNA Cluster 24 Has Profound Effects on Extracellular Matrix Protein Abundance in Cartilage.

MicroRNAs (miRNAs) regulate cartilage differentiation and contribute to the onset and progression of joint degeneration. These small RNA molecules may affect extracellular matrix organization (ECM) in cartilage, but for only a few miRNAs has this role been defined in vivo. Previously, we showed that cartilage-specific genetic ablation of the Mirc24 cluster in mice leads to impaired cartilage development due to increased RAF/MEK/ERK pathway activation. Here, we studied the expression of the cluster in cartilage by LacZ reporter gene assays and determined its role for extracellular matrix homeostasis by proteome and immunoblot analysis. The cluster is expressed in prehypertrophic/hypertrophic chondrocytes of the growth plate and we now show that the cluster is also highly expressed in articular cartilage. Cartilage-specific loss of the cluster leads to increased proteoglycan 4 and matrix metallopeptidase 13 levels and decreased aggrecan and collagen X levels in epiphyseal cartilage. Interestingly, these changes are linked to a decrease in SRY-related HMG box-containing (SOX) transcription factors 6 and 9, which regulate ECM production in chondrocytes. Our data suggests that the Mirc24 cluster is important for ECM homoeostasis and the expression of transcriptional regulators of matrix production in cartilage.

Tissue-specific extracellular matrix scaffolds for the regeneration of spatially complex musculoskeletal tissues.

Biological scaffolds generated from tissue-derived extracellular matrix (ECM) are commonly used clinically for soft tissue regeneration. Such biomaterials can enhance tissue-specific differentiation of adult stem cells, suggesting that structuring different ECMs into multi-layered scaffolds can form the basis of new strategies for regenerating damaged interfacial tissues such as the osteochondral unit. In this study, mass spectrometry is used to demonstrate that growth plate (GP) and articular cartilage (AC) ECMs contain a unique array of regulatory proteins that may be particularly suited to bone and cartilage repair respectively. Applying a novel iterative freeze-drying method, porous bi-phasic scaffolds composed of GP ECM overlaid by AC ECM are fabricated, which are capable of spatially directing stem cell differentiation in vitro, promoting the development of graded tissues transitioning from calcified cartilage to hyaline-like cartilage. Evaluating repair 12-months post-implantation into critically-sized caprine osteochondral defects reveals that these scaffolds promote regeneration in a manner distinct to commercial control-scaffolds. The GP layer supports endochondral bone formation, while the AC layer stimulates the formation of an overlying layer of hyaline cartilage with a collagen fiber architecture better recapitulating the native tissue. These findings support the use of a bi-layered, tissue-specific ECM derived scaffolds for regenerating spatially complex musculoskeletal tissues.

Effect of Hemiepiphysiodesis on the Growth Plate: The Histopathological Changes and Mechanism Exploration of Recurrence in Mini Pig Model.

PURPOSE: Hemiepiphysiodesis has been widely used to correct angular deformity of long bone in immature patients. However, there is a limited knowledge about the biomechanical effect of this technique on the histopathological changes of the growth plate and the mechanism of recurrence of malformation after implant removal. We aimed to evaluate the biomechanical effect of hemiepiphysiodesis on the histopathological changes of the growth plate and the mechanism of recurrence of malformation after implant removal in Bama miniature pigs, and to explore the role of asymmetric stress during this procedure. METHODS: Eight 3-month-old male Bama miniature pigs sustained surgeries on the bilateral medial hind leg proximal tibia as the intervention group (n=16), and four pigs sustained bilateral sham surgeries as the control (n=8). In the 18th week after surgeries, hardware was removed in the unilateral leg of each animal in the intervention group. In the 24th week of the study, all animals were euthanized. A total of 24 samples were obtained and stained with H&E, TUNEL, and immunohistochemistry. Sixteen samples in the intervention group were divided into two subgroups. The tibias without an implant were included in the implant removal group (IR group), while the tibias with an implant were included in the implant persist group (IP group). The proximal tibia specimens were divided into 3 equidistant parts from medial to lateral, named as area A, area B, and area C, respectively. The change of thickness of growth plates, chondral apoptosis index, and the expression of Caspase-3, Caspase-9, CHOP, and P65 were compared. RESULTS: H&E staining showed the thickness of growth plate to be varied in different areas. In the IP group, the thickness of growth plate in areas A and B was statistically significantly thinner than that in area C (p<0.05). In the IR group, the thickness of growth plate in areas A and B was statistically significantly thicker than that in area C (p<0.05). TUNEL staining showed that the apoptosis rate increased significantly after hemiepiphysiodesis and declined after implant removal (p<0.05). Immunohistochemical staining suggested that the expression of Caspase-3, Caspase-9, P65, and CHOP protein was upregulated in the experimental group and downregulated after implant removal. CONCLUSION: The thickness parameter of the growth plate changes with asymmetric pressure. When the pressure is relieved, the recurrence of malformation is related to the thickening of the growth plate.

The effect of tannic acid on bone mechanical and geometric properties, bone density, and trabecular histomorphometry as well as the morphology of articular and growth cartilages in rats co-exposed to cadmium and lead is dose dependent.

Cadmium (Cd) and lead (Pb) are toxic elements that accumulate to the largest extent in bones. Rats were used to investigate whether tannic acid (TA; 0.5%, 1.0%, 1.5%. 2.0%, or 2.5%) would have a protective effect on the structure and properties of bones in the case of exposure to Cd and Pb (diet: 7 mg Cd/kg and 50 mg Pb/kg) for 6 weeks. The effects of administration of TA in Cd- and Pb-poisoned rats on bone characteristics and the morphology of articular and growth cartilages were determined. All the rats administered Cd and Pb had an enhanced Cd and Pb concentration in blood plasma and bone and reduced bone Ca content irrespective of the TA administration. Cd and Pb alone reduced the mechanical endurance and histomorphometric parameters of trabecular bone and the thickness of the growth plate and articular cartilage. Tannic acid improved cancellous bone parameters in the rat exposed to Cd and Pb. A diet rich in TA improved articular cartilage constituents in heavy metal-poisoned rats. These results suggest that alimentary TA supplementation can counteract in a dose-dependent manner some of the destructive changes evoked by Cd and Pb possibly by reducing the exposure.

Histological and histochemical evaluations on the effects of high incubation temperature on the embryonic development of tibial growth plate in broiler chickens.

The effects of experimentally induced high incubation temperature on the embryonic development of growth plate of the chicken were investigated by means of histological and enzyme histochemical methods. In the experiments, 250 fertile eggs of Ross-308 broiler strain were divided into two groups, the control eggs were maintained under optimal conditions (37.8°C and 65% ± 2% relative humidity, rh) during the whole incubation period. Heat-stress imposed eggs were maintained under normal conditions (37.8°C and 65% ± 2% rh) until the 10th day of incubation, and then, continuously (24 h per day) exposed to high temperature (38.8°C and 65% ± 2% rh). Tissue samples were taken from 10 animals of each group at the 11th, 13th, 15th, 18th, 21st days of incubation. Tissue samples were processed by enzyme histochemical methods in addition to routine histological techniques. The relative tibia weights and tibia length were lower in the heat-stress group compared to the control group. The results of the measurements of the growth plate showed that the proliferative zone was narrowed whereas, the transitional and hypertrophic zone were thickened in the heat stress group. Alkaline phosphatase (ALP) density was significantly decreased in the heat-stress group compared to the control group. In conclusion, bone formation and growth plate formation are crucial for embryo development and 1°C higher from optimum may increase the incidence of skeletal disorders and leg problems in broiler chickens which is one of the major animal welfare concerns for the poultry industry.

Bone microstructural defects and osteopenia in hemizygous ßIVSII-654 knockin thalassemic mice: sex-dependent changes in bone density and osteoclast function.

ß-Thalassemia, a hereditary anemic disorder, is often associated with skeletal complications that can be found in both males and females. The present study aimed to investigate the age- and sex-dependent changes in bone mineral density (BMD) and trabecular microstructure in ß(IVSII-654) knockin thalassemic mice. Dual-energy X-ray absorptiometry and computer-assisted bone histomorphometry were employed to investigate temporal changes in BMD and histomorphometric parameters in male and female mice of a ß(IVSII-654) knockin mouse model of human ß-thalassemia, in which impaired splicing of ß-globin transcript was caused by hemizygous C→T mutation at nucleotide 654 of intron 2. Young, growing ß(IVSII-654) mice (1 mo old) manifested shorter bone length and lower BMD than their wild-type littermates, indicating possible growth retardation and osteopenia, the latter of which persisted until 8 mo of age (adult mice). Interestingly, two-way analysis of variance suggested an interaction between sex and ß(IVSII-654) genotype, i.e., more severe osteopenia in adult female mice. Bone histomorphometry further suggested that low trabecular bone volume in male ß(IVSII-654) mice, particularly during a growing period (1-2 mo), was primarily due to suppression of bone formation, whereas both a low bone formation rate and a marked increase in osteoclast surface were observed in female ß(IVSII-654) mice. In conclusion, osteopenia and trabecular microstructural defects were present in both male and female ß(IVSII-654) knockin thalassemic mice, but the severity, disease progression, and cellular mechanism differed between the sexes.

Effects of Inhaled Corticosteroids on the Growth Plates of Infant Rats.

The most significant adverse effect of inhaled steroid administration in children is suppression of hypothalamic-pituitary-adrenal axis responsiveness and suppression of growth. This study evaluates the effects of inhaled corticosteroids on the growth plates in infant rats. Rats aged 10 days were divided into five groups. Low and high doses of budesonide and fluticasone propionate (50-200-250 mcg/day) were applied with a modified spacer for 10 days. The rat's tibias were then removed and the effects of the steroids on the growth plates were compared. Growth cartilage chondrocyte proliferation and apoptosis rates; IGF-1 and glucocorticoid receptor levels; and resting, proliferative, hypertrophic, and total zone (TZ) measurements were compared using immunohistochemical-staining methods. With high doses of fluticasone, growth plates were affected much more than with high doses of budesonide (p = 0.01). Fluticasone, particularly at a dose of 250 mcg, inhibited the growth plate with an intensive negative impact on all parameters.

Immunolocalisation of fibrillin microfibrils in the calf metacarpal and vertebral growth plate.

Overgrowth of limbs and spinal deformities are typical clinical manifestations of Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), caused by mutations of the genes encoding fibrillin-1 (FBN1) and fibrillin-2 (FBN2), respectively. FBN1 mutations are also associated with acromicric (AD) and geleophysic dysplasias (GD), and with Weill-Marchesani syndrome (WMS), which is characterised by short stature. The mechanisms leading to such abnormal skeletal growth and the involvement of the fibrillins are not understood. Postnatal longitudinal bone growth mainly occurs in the epiphyseal growth plate. Here we investigated the organisation of fibrillin microfibrils in the growth plate of the long bone and vertebra immunohistochemically. Fibrillin-1 was dual-immunostained with elastin, with fibrillin-2 or with collagen X. We report that fibrillin microfibrils are distributed throughout all regions of the growth plate, and that fibrillin-1 and fibrillin-2 were differentially organised. Fibrillin-1 was more abundant in the extracellular matrix of the resting and proliferative zones of the growth plate than in the hypertrophic zone. More fibrillin-2 was found in the calcified region than in the other regions. No elastin fibres were observed in either the proliferative or hypertrophic zones. This study indicates that, as fibrillin microfibrils are involved in growth factor binding and may play a mechanical role, they could be directly involved in regulating bone growth. Hence, mutations of the fibrillins could affect their functional role in growth and lead to the growth disorders seen in patients with MFS, CCA, AD, GD and WMS.

Effects of suture choice on biomechanics and physeal status after bioenhanced anterior cruciate ligament repair in skeletally immature patients: a large-animal study.

PURPOSE: The objective of this study was to assess the effect of absorbable or nonabsorbable sutures in bioenhanced anterior cruciate ligament (ACL) repair in a skeletally immature pig model on suture tunnel and growth plate healing and biomechanical outcomes. METHODS: Sixteen female skeletally immature Yorkshire pigs were randomly allocated to receive unilateral, bioenhanced ACL repair with an absorbable (Vicryl) or nonabsorbable (Ethibond) suture augmented by an extracellular matrix-based scaffold (MIACH). After 15 weeks of healing, micro-computed tomography was used to measure residual tunnel diameters and growth plate status, and biomechanical outcomes were assessed. RESULTS: At 15 weeks postoperatively, there was a significant difference in tunnel diameter with significantly larger diameters in the nonabsorbable suture group (4.4 ± 0.3 mm; mean ± SD) than in the absorbable group (1.8 ± 0.5 mm; P < .001). The growth plate showed a significantly greater affected area in the nonabsorbable group (15.2 ± 3.4 mm(2)) than in the absorbable group (2.7 ± 0.8 mm(2), P < .001). There was no significant difference in the linear stiffness of the repairs (29.0 ± 14.8 N/mm for absorbable v 43.3 ± 28.3 N/mm for nonabsorbable sutures, P = .531), but load to failure was higher in the nonabsorbable suture group (211 ± 121.5 N) than in the absorbable suture group (173 ± 101.4 N, P = .002). There was no difference between the 2 groups in anteroposterior laxity at 30° (P = .5117), 60° (P = .3150), and 90° (P = .4297) of knee flexion. CONCLUSIONS: The use of absorbable sutures for ACL repair resulted in decreased physeal plate damage after 15 weeks of healing; however, use of nonabsorbable sutures resulted in 20% stronger repairs. CLINICAL RELEVANCE: Choice of suture type for ACL repair or repair of tibial avulsion fractures may depend on patient skeletal age and size, with absorbable sutures preferred in very young, small patients at higher risk with physeal damage and nonabsorbable sutures preferred in larger, prepubescent patients who may place higher loads on the repair.

Recognizing different tissues in human fetal femur cartilage by label-free Raman microspectroscopy.

Traditionally, the composition of bone and cartilage is determined by standard histological methods. We used Raman microscopy, which provides a molecular "fingerprint" of the investigated sample, to detect differences between the zones in human fetal femur cartilage without the need for additional staining or labeling. Raman area scans were made from the (pre)articular cartilage, resting, proliferative, and hypertrophic zones of growth plate and endochondral bone within human fetal femora. Multivariate data analysis was performed on Raman spectral datasets to construct cluster images with corresponding cluster averages. Cluster analysis resulted in detection of individual chondrocyte spectra that could be separated from cartilage extracellular matrix (ECM) spectra and was verified by comparing cluster images with intensity-based Raman images for the deoxyribonucleic acid/ribonucleic acid (DNA/RNA) band. Specific dendrograms were created using Ward's clustering method, and principal component analysis (PCA) was performed with the separated and averaged Raman spectra of cells and ECM of all measured zones. Overall (dis)similarities between measured zones were effectively visualized on the dendrograms and main spectral differences were revealed by PCA allowing for label-free detection of individual cartilaginous zones and for label-free evaluation of proper cartilaginous matrix formation for future tissue engineering and clinical purposes.

A method for proteomic analysis of equine subchondral bone and epiphyseal cartilage.

Proteomic analyses of cartilage and, to a lesser extent, of bone have long been impaired because of technical challenges related to their structure and biochemical properties. We have developed a unified method based on phenol extraction, 2DE, silver staining, and subsequent LC-MS/MS. This method proved to be efficient to characterize the proteome of equine cartilage and bone samples collected in vivo. Since proteins from several cellular compartments could be recovered, our procedure is mainly suitable for in situ molecular physiology studies focused on the cellular content of chondrocytes, osteoblasts, and osteoclasts as well as that of the extracellular matrix, with the exception of proteoglycans. Our method alleviates some drawbacks of cell culture that can mask physiological differences, as well as reduced reproducibility due to fractionation. Proteomic comparative studies between cartilage and bone samples from healthy and affected animals were thus achieved successfully. This achievement will contribute to increasing knowledge on the molecular mechanisms underlying the physiopathology of numerous osteoarticular diseases in horses and in humans.

Heparin-like heparan sulfate from rabbit cartilage.

Glycosaminoglycans were extracted from both young rabbit growth plate (GRP) and articular (ART) cartilage tissues and enzymatically treated to selectively eliminate chondroitin sulfates and hyaluronic acid. The procedure avoided any fractionation step that could enrich the extract with over- or under-sulfated species. Isolated heparan sulfate (HS) was characterized by mono- and bidimensional nuclear magnetic resonance (NMR) spectroscopy to quantify their specific structural features and/or by mass spectrometry to establish the disaccharide composition. Both GRP and ART HSs, despite differing in their yield (GRP at least 100 times greater than ART), exhibited a surprisingly high degree of sulfation. Quantitative two-dimensional heteronuclear single-quantum coherence-NMR analysis of GRP HS revealed unusually high N-sulfated glucosamine and 2-O-sulfated iduronic acid contents, similar to heparin. The unique pentasaccharide sequence of the binding site for antithrombin was also detected in a significant amount. High-performance liquid chromatography mass spectrometry analysis of the enzymatic digests with a cocktail of heparin lyases of both cartilaginous HSs confirmed the NMR results. As well as the discovery of an unusual HS structure in the two different types of rabbit cartilage, the feasibility of the analytical method adopted here has been demonstrated within this study. Such a method can be used to isolate and analyze HS from both normal and pathologic tissues. Characterization of healthy and pathological HS structures will contribute to improve the understanding of diseases related to malfunctions of HS biosynthesis and/or metabolism.

Primary cilia modulate Ihh signal transduction in response to hydrostatic loading of growth plate chondrocytes.

Indian hedgehog (Ihh) is a key component of the regulatory apparatus governing chondrocyte proliferation and differentiation in the growth plate. Recent studies have demonstrated that the primary cilium is the site of Ihh signaling within the cell, and that primary cilia are essential for bone and cartilage formation. Primary cilia are also postulated to act as mechanosensory organelles that transduce mechanical forces acting on the cell into biological signals. In this study, we used a hydrostatic compression system to examine Ihh signal transduction under the influence of mechanical load. Our results demonstrate that hydrostatic compression increased both Ihh gene expression and Ihh-responsive Gli-luciferase activity. These increases were aborted by disrupting the primary cilia structure with chloral hydrate. These results suggest that growth plate chondrocytes respond to hydrostatic loading by increasing Ihh signaling, and that the primary cilium is required for this mechano-biological signal transduction to occur.

Gender- and region-specific variations of estrogen receptor α and ß expression in the growth plate of spine and limb during development and adulthood.

Although estrogen action is indispensable for normal bone growth in both genders, the roles of estrogen receptors (ERs) in mediating bone growth are not fully understood. The effects of ER inactivation on bone growth are sex and age dependent, and may differ between the axial and appendicular regions. In this study, the spatial and temporal expression of ERα and ß in the tibial and spinal growth plates of the female and male rats during postnatal development was examined to explore the possible mechanisms. The level of mRNA was examined and compared with quantitative real-time PCR. The spatial location was determined by immunohistochemical analysis. The 1-, 4-, 7-, 12- and 16-week age stages correspond to early life, puberty and early adulthood after puberty, respectively. Gender- and region-specific differences in ERα and ß expression were shown in the growth plates. Mainly nuclear staining of ERα and ß immunoreactivity was demonstrated in the spinal and tibial growth plate chondrocytes for both genders. Moreover, our study indicated significant effect of gender on temporal ERα and ß expression and of region on temporal ERα/ERß expression ratio. However, spatial differences of region-related ERα and ß expression were not observed. Gender-related spatial changes were detected only at 16 weeks of both spine and limb growth plates. ERα and ß immunoreactivity was detected in the resting, proliferative and prehypertrophic chondrocytes in the early life stage and during puberty. After puberty, ERα expression was mainly located in the late proliferative and hypertrophic chondrocytes in female, whereas the expression still extended from the resting to hypertrophic chondrocytes in males. Gender- and region-specific expression patterns of ERα and ß gene might be one possible reason for differences in sex- and region-related body growth phenotypes. Gender, age and region differences should be taken into consideration when the roles of ERs in the growth plate are investigated.

Chondroitin sulphate sulphation motif expression in the ontogeny of the intervertebral disc.

Chondroitin sulphate chains on cell and extracellular matrix proteoglycans play important regulatory roles in developing systems. Specific, developmentally regulated, sulphation motifs within the chondroitin glycosaminoglycan structure may help bind, sequester or present bioactive signalling molecules to cells thus modulating their behaviour. Using monoclonal antibodies 3B3(-), 4C3, 6C3 and 7D4, we have mapped the distribution of different chondroitin sulphation epitopes in a rat intervertebral disc developmental series. The sulphation epitopes had complex, dynamic and specific distributions in the disc and vertebral tissues during their differentiation, growth and ageing. At embryonic day [E]15, prior to disc differentiation, 4C3 and 7D4 occurred within the cellular disc condensations whilst 6C3 was present in the notochordal sheath. At E17, post disc differentiation, 4C3 and 7D4 occurred within the nucleus pulposus, inner annulus and vertebral bodies; 3B3(-) in the nucleus, inner annulus, annulus/vertebral body interface and perichondrium; and 6C3, ventrally, within the perichondrium. At E19, 3B3(-), 4C3 and 7D4 became further restricted to the nucleus, inner annulus, annulus/vertebral body interface and perichondrium. Prior to birth, all four epitopes occurred within the inner annulus and nucleus, with 6C3 and 7D4 also occurring within the future end-plate. Postnatal expression of the sulphation epitopes was more widespread in the disc and also within the growth plate. At 4 months, the epitopes were associated with chondrocyte clusters within the nucleus; and at 24 months, with annular lesions. Overall, our data suggests that differential sulphation of chondroitin correlates with significant events in development, growth and aging of the rat intervertebral disc.

Expression of Runx2 and type X collagen in vertebral growth plate of patients with adolescent idiopathic scoliosis.

The different expression of type X collagen and Runx2 between the convex and concave side of vertebral growth plate in scoliosis may help to improve our understanding of the role that growth plate tissue play in the development or progression of idiopathic scoliosis. In this investigation, there were significant differences of the total expression of type X collagen, Runx2 protein, and Runx2 mRNA between convex side and concave side growth plates of the apex vertebrae (p < 0.05). The total expression of type X collagen in the concave side growth plates of the lower end vertebrae was higher than that in the same side growth plates of apex (p < 0.05). The total expression of Runx2 in the concave side growth plates in the upper and lower end vertebrae were higher than that in the concave side growth plates of apex (p < 0.05). The expression of type X collagen, Runx2, and Runx2 mRNA, the cell density of type X collagen and Runx2 positive chondrocytes, and histological changes between convex side and concave side of the vertebral growth plate indicated that the vertebral growth plate was affected by mechanical forces, which was a secondary change and could contribute to progression of adolescent idiopathic scoliosis.

Temporal and spatial expression of a growth-regulated network of imprinted genes in growth plate.

In mammals, the somatic growth rate is rapid during fetal and early postnatal life and then gradually declines and eventually stops. In search of the fundamental biological mechanism causing coordinated growth deceleration in multiple tissues, a network of imprinted genes was recently identified based on a coordinated decline in expression in several organs during postnatal growth. To explore a possible role in longitudinal bone growth, we characterized expression of the network during postnatal growth in microdissected metaphyseal bone and growth plate zones of 1-, 3-, and 9-week-old rats using real-time PCR. The expression pattern of the network is modified in growth plate. Similar to the coordinated decline previously observed in kidney, lung, liver, and heart, expression of all genes, except Gtl2, decreased with age in metaphyseal bone. On the contrary, Mest, Dlk1, H19, and Gtl2 decreased, and Cdkn1c, Grb10, and Slc38a4 increased with age in growth plate. During differentiation from resting to hypertrophic zone, Mest, Dlk1, Grb10, and Gtl2 expression decreased, whereas Slc38a4 expression increased. In particular, developmental changes in the expression of growth-promoting genes, Mest, Dlk1, Gtl2, and growth-inhibitory genes, Cdkn1c and Grb10, may contribute to the decline in longitudinal bone growth that occurs with age.

Temporal and spatial changes in element distribution in bone and cartilage.

BACKGROUND: Bone and cartilage act as reservoirs for a number of elements, and the perturbation of these elements is suggested to contribute to various diseases. Even so, little is known about their respective temporal and spatial distribution. METHODS: Three knee joints of three mice on the first day after birth, three knee joints of 3-week-old mice, and three knee joints of 20-week-old mice were prepared. We performed element mapping in the bone and cartilage of normal mouse knee joints. We measured element distribution in articular cartilage, trabecular bone, cortical bone, joint cartilage, growth plates, and joint cavities using energy dispersive X-ray spectrometry. RESULTS: The analysis revealed the following: (1) The main elements in articular cartilage of 1-day-old mice were Na, O, P, S, and Ca; and those in bone were Na, O, P, S, K, Ca, and Mg. (2) The main elements in the growth plate of 3-week-old mice were Na, N, O, P, S, Cl, K, and Ca; those in joint cartilage were Na, O, P, S, Cl, and K; and those in bone were Na, O, P, Ca, and Mg. (3) The main elements in the growth plate of 20-week-old mice were Na, O, P, S, Cl, K, and Ca; and those in bone were Na, O, P, S, K, Ca, and Mg. After corrections were made for the Na ratios of these elements, we investigated the temporal and spatial changes in the distribution of each element. On the first day after birth, a spatial change was seen in the growth plate cartilage: the more the cartilage matured toward hypertrophy, the more S and Ca it contained. Temporal changes in element distribution in the growth plate cartilage, articular cartilage, and bone were observed. Growth plate cartilage of the older mice contained more S and Ca than that of the younger mice. Bone of the older mice contained more Ca and Mg than that of the younger mice. Spatial changes in element distribution in the cortical bone were also seen; that is, the more the cortical bone matured toward diaphysis, the more Mg and the less S it contained. In contrast, no temporal or spatial changes in element distribution were observed in the joint space. No significant temporal or spatial changes in the distribution of P, Cl, or K were seen. CONCLUSIONS: These results suggest that element mapping may be useful for identifying the age and maturity of different skeletal tissues. In particular, it may help distinguish between immature cartilage and mature cartilage based on the Ca and S contents.

Amelogenin expression in long bone and cartilage cells and in bone marrow progenitor cells.

The amelogenin protein is considered as the major molecular marker of developing ectodermal enamel. Recent data suggest other roles for amelogenin beyond structural regulation of enamel mineral crystal growth. Here we describe our novel discovery of amelogenin expression in long bone cells, in cartilage cells, in cells of the epiphyseal growth plate, and in bone marrow stromal cells.

Distinct developmental changes in the distribution of calcium, phosphorus and sulphur during fetal growth-plate development.

Gradients in the concentrations of free phosphate (Pi) and calcium (Ca) exist in fully developed growth zones of long bones and ribs, with the highest concentrations closest to the site of mineralization. As high concentrations of Pi and Ca induce chondrocyte maturation and apoptosis, it has been hypothesized that Ca and Pi drive chondrocyte differentiation in growth plates. This study aimed to determine whether gradients in the important spectral elements phosphorus (P), Ca and sulphur (S) are already present in early stages of development, or whether they gradually develop with maturation of the growth zone. We quantified the concentration profiles of Ca, P, S, chloride and potassium at four different stages of early development of the distal growth plates of the porcine femurs, using particle-induced X-ray emission and forward- and backward-scattering spectrometry with a nuclear microprobe. A Ca concentration gradient towards the mineralized area and a stepwise increase in S was found to develop slowly with tissue maturation. The increase in S co-localizes with the onset of proliferation. A P gradient was not detected in the earliest developmental stages. High Ca levels, which may induce chondrocyte maturation, are present near the mineralization front. As total P concentrations do not correspond with former free Pi measurements, we hypothesize that the increase of free Pi towards the bone-forming site results from enzymatic cleavage of bound phosphate.