Growth Plates Of Long Bones Research Articles

Growth plate closure and therapeutic interventions.

Height gains result from longitudinal bone growth, which is largely dependent on chondrocyte differentiation and proliferation within the growth plates of long bones. The growth plate, that is, the epiphyseal plate, is divided into resting, proliferative, and hypertrophic zones according to chondrocyte characteristics. The differentiation poten-tial of progenitor cells in the resting zone, continuous capacity for chondrocyte differentiation and proliferation within the proliferative zone, timely replacement by osteocytes, and calcification in the hypertrophic zone are the 3 main factors controlling longitudinal bone growth. Upon ade quate longitudinal bone growth, growth plate senescence limits human body height. During growth plate senescence, progenitor cells within the resting zone are deplet ed, proliferative chondrocyte numbers de crease, and hypertrophic chondrocyte number and size decrease. After senescence, hypertrophic chondrocytes are replaced by osteocytes, the extracellular matrix is calcified and va-scularized, the growth plate is closed, and longitudinal bone growth is complete. To date, go nadotropin-releasing hormone analogs, aromatase inhi bitors, C-type natriuretic peptide analogs, and fibroblast growth factor receptor 3 inhibitors have been studied or used as therapeutic interv-entions to delay growth plate closure. Complex networks of cellular, genetic, paracrine, and endocrine signals are involved in growth plate closure. However, the detailed mechanisms of this process remain unclear. Further eluci-dation of these mechanisms will enable the development of new thera peutic modalities for the treatment of short stature, precocious puberty, and skeletal dysplasia.

Electron-Beam-Killed Staphylococcus Vaccine Reduced Lameness in Broiler Chickens

Broiler chicken lameness caused by bacterial chondronecrosis with osteomyelitis (BCO) is presently amongst the most important economic and animal welfare issues faced by the poultry industry, and the estimated economic loss is around USD 150 million. BCO lameness is associated with multiple opportunistic bacterial pathogens inhabiting the respiratory and gastrointestinal tracts. In cases of immune deficiency resulting from stress, injury, or inflammation of the tissue, opportunistic pathogens, mainly Staphylococcus spp., can infiltrate the respiratory or gastrointestinal mucosa and migrate through the bloodstream to eventually colonize the growth plates of long bones, causing necrosis that leads to lameness. This is the first report of developing a Staphylococcus vaccine against BCO lameness disease in broiler chickens. Electron beam (eBeam) technology causes irreparable DNA damage, preventing bacterial multiplication, while keeping the epitopes of the cell membrane intact, helping the immune system generate a more effective response. Our results show a 50% reduction of lameness incidence in the eBeam-vaccinated chicken group compared to the control. Additionally, the eBeam-vaccinated chickens present higher titer of anti-Staphylococcus IgA, signifying the development of an efficient and more specific humoral immune response. Our data establish the eBeam-killed Staphylococcus vaccine as an effective approach to reducing the incidence of lameness in broiler chickens.

Deoxynivalenol and fumonisin predispose broilers to bacterial chondronecrosis with osteomyelitis lameness

Bacterial chondronecrosis with osteomyelitis (BCO) lameness is the most critical animal health and welfare issue facing the broiler industry worldwide. It is estimated that 1 to 2% of bird condemnation at marketing age is caused by BCO lameness, resulting in tens of millions of dollars in annual losses. Fast-growing broilers are prone to mechanical stress that triggers bacterial translocation across epithelial barriers into the bloodstream, followed by bacterial colonization in the growth plate of long bones, and eventually, bone necrosis and lameness. Mycotoxins (MTX) are secondary metabolites produced naturally by microfungi, of which deoxynivalenol (DON), fumonisin (FUM), and zearalenone are the most prevalent in corn and soybean-meal-based diets. The presence of these mycotoxins in feed has been proven to reduce the barrier strength of the intestinal tracts and trigger immunosuppressive effects. In this study, we investigated the effects of the DON and FUM-contaminated feeds on the incidence of BCO lameness in broilers reared in both wire- and litter-floors. 720 one-day-old broiler chicks were assigned to the 2 × 2 factorial design: 2 MTX diets containing DON and FUM on wire flooring (MTX-W) and litter flooring (MTX-L), and 2 diets without MTX contamination on control wire flooring (CW) and control litter flooring (CL). Throughout the trial, the cumulative incidence of lameness per treatment was assessed by necropsying the lame birds. Birds in the MTX-W group had a higher incidence of lameness compared to those in CW (73.3% vs. 62.0%) (P < 0.05), and birds in the MTX-L group had a higher incidence of lameness compared to birds in CL (54.0% vs. 34.0%) (P < 0.05). MTX elicited net increases in BCO to a greater degree on litter (+20%) than on wire flooring (+12%). The increased incidence of BCO lameness in the MTX-W coincided with increased intestinal permeability supporting a correlation between intestinal barrier integrity and BCO lameness. To conclude, DON and FUM are predisposing factors for increasing BCO. However, no significant interaction exists between the diet and floor types in inducing lameness in broilers.

Atypical cell death and insufficient matrix organization in long-bone growth plates from Tric-b-knockout mice

TRIC-A and TRIC-B proteins form homotrimeric cation-permeable channels in the endoplasmic reticulum (ER) and nuclear membranes and are thought to contribute to counterionic flux coupled with store Ca2+ release in various cell types. Serious mutations in the TRIC-B (also referred to as TMEM38B) locus cause autosomal recessive osteogenesis imperfecta (OI), which is characterized by insufficient bone mineralization. We have reported that Tric-b-knockout mice can be used as an OI model; Tric-b deficiency deranges ER Ca2+ handling and thus reduces extracellular matrix (ECM) synthesis in osteoblasts, leading to poor mineralization. Here we report irregular cell death and insufficient ECM in long-bone growth plates from Tric-b-knockout embryos. In the knockout growth plate chondrocytes, excess pro-collagen fibers were occasionally accumulated in severely dilated ER elements. Of the major ER stress pathways, activated PERK/eIF2α (PKR-like ER kinase/ eukaryotic initiation factor 2α) signaling seemed to inordinately alter gene expression to induce apoptosis-related proteins including CHOP (CCAAT/enhancer binding protein homologous protein) and caspase 12 in the knockout chondrocytes. Ca2+ imaging detected aberrant Ca2+ handling in the knockout chondrocytes; ER Ca2+ release was impaired, while cytoplasmic Ca2+ level was elevated. Our observations suggest that Tric-b deficiency directs growth plate chondrocytes to pro-apoptotic states by compromising cellular Ca2+-handling and exacerbating ER stress response, leading to impaired ECM synthesis and accidental cell death.

Effect of Excess Fluoride Exposure on Radiographic and Histopathological Changes in Long Bones of Rabbit.

The present study aimed to investigate the effects of excess fluoride exposure on long bones in young rabbits (Oryctolagus cuniculus). New Zealand White rabbits (n = 30) were randomly divided into five equal groups and were provided drinking water containing 0, 50, 100, 200, and 400µg added fluoride per ml ad lib for a period of 90days. Blood samples were collected on days 0, 45, and 90 of the experiment, and femur samples were collected for fluoride estimation on day 90 after radiography of long bone before sacrifice. Study revealed significant increase in serum fluoride concentration following oral intake of excess fluoride. Alterations in activities of alkaline phosphatase, aspartate transaminase, alanine transaminase, and concentrations of creatinine and urea nitrogen in blood plasma were also recorded in animals receiving excess fluoride, though changes revealed inconsistent pattern. Radiographic changes in long bones in fluoride exposed rabbits included widening of metaphysis, thinning of cortical region, and a variety of osteopenic changes like osteoporosis and osteomalacia that were more prominent in animals receiving 200ppm or more than 200ppm fluoride in drinking water. Important changes in histomorphology of growth plate in long bones recorded in excess fluoride (> 100ppm) exposed rabbits included irregular thickening of epiphyseal growth plate with haphazard orientation of chondrocytes forming nodular protrusion into metaphysis. Fluoride exposure induced both osteogenesis and osteoporosis to a degree varying with dose of fluoride exposure.

Single-cell transcriptomic analysis identifies a highly replicating Cd168+ skeletal stem/progenitor cell population in mouse long bones

Skeletal stem/progenitor cells (SSPCs) are tissue-specific stem/progenitor cells localized within skeletons and contribute to bone development, homeostasis, and regeneration. However, the heterogeneity of SSPC populations in mouse long bones and their respective regenerative capacity remain to be further clarified. In this study, we perform integrated analysis using single-cell RNA sequencing (scRNA-seq) datasets of mouse hindlimb buds, postnatal long bones, and fractured long bones. Our analyses reveal the heterogeneity of osteochondrogenic lineage cells and recapitulate the developmental trajectories during mouse long bone growth. In addition, we identify a novel Cd168+ SSPC population with highly replicating capacity and osteochondrogenic potential in embryonic and postnatal long bones. Moreover, the Cd168+ SSPCs can contribute to newly formed skeletal tissues during fracture healing. Furthermore, the results of multicolor immunofluorescence show that Cd168+ SSPCs reside in the superficial zone of articular cartilage as well as in growth plates of postnatal mouse long bones. In summary, we identify a novel Cd168+ SSPC population with regenerative potential in mouse long bones, which adds to the knowledge of the tissue-specific stem cells in skeletons.

Haploinsufficiency of EXT1 and Heparan Sulphate Deficiency Associated with Hereditary Multiple Exostoses in a Pakistani Family.

Background and Objectives: Hereditary multiple exostoses (HME) is a disease characterized by cartilage-capped bony protuberances at the site of growth plates of long bones. Functional mutations in the exostosin genes (EXT1 and EXT2) are reported to affect the hedgehog signalling pathways leading to multiple enchondromatosis. However, the exact role of each EXT protein in the regulation of heparan sulphate (HS) chain elongation is still an enigma. In this study, a Pakistani family with HME is investigated to find out the genetic basis of the disease. Materials and Methods: Genotyping of eight members of the family by amplifying microsatellite markers, tightly linked to the EXT1 and EXT2 genes. Results: The study revealed linkage of the HME family to the EXT1 locus 8q24.1. Sanger sequencing identified a heterozygous deletion (c.247Cdel) in exon 1 of EXT1, segregating with the disease phenotype in the family. In silico analysis predicted a shift in the frame causing an early stop codon (p.R83GfsX52). The predicted dwarf protein constituting 134 amino acids was functionally aberrant with a complete loss of the catalytic domain at the C-terminus. Interestingly, an alternative open reading frame 3 (ORF3) caused by the frame shift is predicted to encode a protein sequence, identical to the wild type and containing the catalytic domain, but lacking the first 100 amino acids of the wild-type EXT1 protein. Conclusion: Consequently, haploinsufficiency could be the cause of HME in the investigated family as the mutated copy of EXT1 is ineffective for EXT-1/2 complex formation. The predicted ORF3 protein could be of great significance in understanding several aspects of HME pathogenesis.

Zebrafish endochondral growth zones as they relate to human bone size, shape and disease.

Research on the genetic mechanisms underlying human skeletal development and disease have largely relied on studies in mice. However, recently the zebrafish has emerged as a popular model for skeletal research. Despite anatomical differences such as a lack of long bones in their limbs and no hematopoietic bone marrow, both the cell types in cartilage and bone as well as the genetic pathways that regulate their development are remarkably conserved between teleost fish and humans. Here we review recent studies that highlight this conservation, focusing specifically on the cartilaginous growth zones (GZs) of endochondral bones. GZs can be unidirectional such as the growth plates (GPs) of long bones in tetrapod limbs or bidirectional, such as in the synchondroses of the mammalian skull base. In addition to endochondral growth, GZs play key roles in cartilage maturation and replacement by bone. Recent studies in zebrafish suggest key roles for cartilage polarity in GZ function, surprisingly early establishment of signaling systems that regulate cartilage during embryonic development, and important roles for cartilage proliferation rather than hypertrophy in bone size. Despite anatomical differences, there are now many zebrafish models for human skeletal disorders including mutations in genes that cause defects in cartilage associated with endochondral GZs. These point to conserved developmental mechanisms, some of which operate both in cranial GZs and limb GPs, as well as others that act earlier or in parallel to known GP regulators. Experimental advantages of zebrafish for genetic screens, high resolution live imaging and drug screens, set the stage for many novel insights into causes and potential therapies for human endochondral bone diseases.

Regulation of Molecular Targets in Osteosarcoma Treatment.

The most prevalent malignant bone tumor, osteosarcoma, affects the growth plates of long bones in adolescents and young adults. Standard chemotherapeutic methods showed poor response rates in patients with recurrent and metastatic phases. Therefore, it is critical to develop novel and efficient targeted therapies to address relapse cases. In this regard, RNA interference technologies are encouraging options in cancer treatment, in which small interfering RNAs regulate the gene expression following RNA interference pathways. The determination of target tissue is as important as the selection of tissue-specific promoters. Moreover, small interfering RNAs should be delivered effectively into the cytoplasm. Lentiviral vectors could encapsulate and deliver the desired gene into the cell and integrate it into the genome, providing long-term regulation of targeted genes. Silencing overexpressed genes promote the tumor cells to lose invasiveness, prevents their proliferation, and triggers their apoptosis. The uniqueness of cancer cells among patients requires novel therapeutic methods that treat patients based on their unique mutations. Several studies showed the effectiveness of different approaches such as microRNA, drug- or chemotherapy-related methods in treating the disease; however, identifying various targets was challenging to understanding disease progression. In this regard, the patient-specific abnormal gene might be targeted using genomics and molecular advancements such as RNA interference approaches. Here, we review potential therapeutic targets for the RNA interference approach, which is applicable as a therapeutic option for osteosarcoma patients, and we point out how the small interfering RNA method becomes a promising approach for the unmet challenge.

The Shape of the Jaw-Zebrafish Col11a1a Regulates Meckel's Cartilage Morphogenesis and Mineralization.

The expression of the col11a1a gene is essential for normal skeletal development, affecting both cartilage and bone. Loss of function mutations have been shown to cause abnormalities in the growth plate of long bones, as well as in craniofacial development. However, the specific effects on Meckel’s cartilage have not been well studied. To further understand the effect of col11a1a gene function, we analyzed the developing jaw in zebrafish using gene knockdown by the injection of an antisense morpholino oligonucleotide using transgenic Tg(sp7:EGFP) and Tg(Fli1a:EGFP) EGFP reporter fish, as well as wildtype AB zebrafish. Our results demonstrate that zebrafish col11a1a knockdown impairs the cellular organization of Meckel’s cartilage in the developing jaw and alters the bone formation that occurs adjacent to the Meckel’s cartilage. These results suggest roles for Col11a1a protein in cartilage intermediates of bone development, the subsequent mineralization of the bony collar of long bones, and that which occurs adjacent to Meckel’s cartilage in the developing jaw.

Sex, gender and stature: From biology to culture - and back

The origin of sexual dimorphism of stature (SSD) in the human species is a subject of debate, likely to have a sociocultural impact. Stature is optimally expressed in good environmental conditions, notably good food, with a strong hereditary determinism. The common academic interpretation, already proposed by Darwin, is that SSD results from sexual selection of stronger males, in most species of mammals, including humans. An alternative hypothesis proposes that it might result from alimentary gender coercion in humans. There is practically no SSD until female growth stops, by ossification of cartilage in the growth plates of long bones, largely under the action of estrogens. The mechanism is the same in males, with a delay due to a lesser and/or later concentration of estrogens. This explanation for SSD has the advantage of being valid for most mammalian species, including those like Pan paniscus where females are dominant. The fitness resulting from high estrogen levels would explain the relatively small stature of women, in spite of obstetric difficulties inversely correlated with height. If patriarchy is involved, it would be by the injunction of fertility rather than by alimentary coercion.

Pthlha and mechanical force control early patterning of growth zones in the zebrafish craniofacial skeleton.

ABSTRACTSecreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative-feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers, such as ihha, whereas local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative-feedback loop that later maintains GPs.

Uncovering pathways regulating chondrogenic differentiation of CHH fibroblasts

Mutations in the non-coding snoRNA component of mitochondrial RNA processing endoribonuclease (RMRP) are the cause of cartilage-hair hypoplasia (CHH). CHH is a rare form of metaphyseal chondrodysplasia characterized by disproportionate short stature and abnormal growth plate development. The process of chondrogenic differentiation within growth plates of long bones is vital for longitudinal bone growth. However, molecular mechanisms behind impaired skeletal development in CHH patients remain unclear. We employed a transdifferentiation model (FDC) combined with whole transcriptome analysis to investigate the chondrogenic transdifferentiation capacity of CHH fibroblasts and to examine pathway regulation in CHH cells during chondrogenic differentiation. We established that the FDC transdifferentiation model is a relevant in vitro model of chondrogenic differentiation, with an emphasis on the terminal differentiation phase, which is crucial for longitudinal bone growth. We demonstrated that CHH fibroblasts are capable of transdifferentiating into chondrocyte-like cells, and show a reduced commitment to terminal differentiation. We also found a number of key factors of BMP, FGF, and IGF-1 signalling axes to be significantly upregulated in CHH cells during the chondrogenic transdifferentiation. Our results support postulated conclusions that RMRP has pleiotropic functions and profoundly affects multiple aspects of cell fate and signalling. Our findings shed light on the consequences of pathological CHH mutations in snoRNA RMRP during chondrogenic differentiation and the relevance and roles of non-coding RNAs in genetic diseases in general.

Hereditary Multiple Exostoses with Ulnar Hemimelia

Hereditary Multiple Exostoses is a skeletal dysplasia that is very rare and defined by formation of numerous cartilage capped benign tumours either pedunculated or sessile known as osteochondromas throughout skeleton especially around the growth plates of ribs, vertebrae, pelvis and long bones. Rarely it can present forearm problems such bowing deformity of radius, ulnar shortening and radiocapitellar dislocation or subluxation. We are presenting a case of 20 year old female who presented with left distal ulnar exostosis resulting in ulnar shortening and radial bowing with restricted supination and pronation range of movement. Other complaint was of multiple non tender bony hard lumps in both upper and lower limbs. Excision of distal ulnar exostosis was done which resulted in marked improvement in pronation and supination range of movement. Hereditary multiple exostoses with forearm deformities though very rare but can present and the treatment is conservative except if any bony swelling manifests any complications such as pain or associated deformity.

Systemic Jak1 activation causes extrarenal calcitriol production and skeletal alterations provoking stunted growth.

Mineral homeostasis is regulated by a complex network involving endocrine actions by calcitriol, parathyroid hormone (PTH), and FGF23 on several organs including kidney, intestine, and bone. Alterations of mineral homeostasis are found in chronic kidney disease and other systemic disorders. The interplay between the immune system and the skeletal system is not fully understood, but cytokines play a major role in modulating calcitriol production and function. One of the main cellular signaling pathways mediating cytokine function is the Janus kinase (JAK)--signal transducer and activator of transcription (STAT) pathway. Here, we used a mouse model (Jak1S645P+/- ) that resembles a constitutive activating mutation of the Jak1/Stat3 signaling pathway in humans, and shows altered mineral metabolism, with higher fibroblast growth factor 23 (FGF23) levels, lower PTH levels, and higher calcitriol levels. The higher calcitriol levels are probably due to extrarenal calcitriol production. Furthermore, systemic Jak1/Stat3 activation led to growth impairment and skeletal alterations. The growth plate in long bones showed decreased chondrocyte proliferation rates and reduced height of terminal chondrocytes. Furthermore, we demonstrate that Jak1 is also involved in bone remodeling early in life. Jak1S645P+/- animals have decreased bone and cortical volume, imbalanced bone remodeling, reduced MAP kinase signaling, and local inflammation. In conclusion, Jak1 plays a major role in bone health probably both, directly and systemically by regulating mineral homeostasis. Understanding the role of this signaling pathway will contribute to a better knowledge in bone growth and in mineral physiology, and to the development of selective Jak inhibitors as osteoprotective agents.

Diagnosis and management of X-linked hypophosphatemia in children and adolescent in the Gulf Cooperation Council countries

IntroductionX-linked hypophosphatemia (XLH) is a rare inherited cause of hypophosphatemic rickets and osteomalacia. It is caused by mutations in the phosphate-regulating endopeptidase homolog, X-linked (PHEX). This results in increased plasma fibroblast growth factor-23 (FGF23), which leads to loss of renal sodium-phosphate co-transporter expression leading to chronic renal phosphate excretion. It also leads to low serum 1,25-dihydroxyvitamin D (1,25(OH)2D), resulting in impaired intestinal phosphate absorption. Chronic hypophosphatemia in XLH leads to impaired endochondral mineralization of the growth plates of long bones with bony deformities. XLH in children and adolescents also causes impaired growth, myopathy, bone pain, and dental abscesses. XLH is the most frequent inherited cause of phosphopenic rickets/osteomalacia. Hypophosphatemia is also found in calcipenic rickets/osteomalacia as a result of secondary hyperparathyroidism. Thus, chronic hypophosphatemia is a common etiologic factor in all types of rickets.ResultsThere is considerable overlap between symptoms and signs of phosphopenic and calcipenic rickets/osteomalacia. Wrong diagnosis leads to inappropriate treatment of rickets/osteomalacia. Nutritional rickets and osteomalacia are common in the Gulf Cooperation Council countries which include Saudi Arabia, United Arab Emirates, Kuwait, Qatar, Bahrain, and Oman. Due to high levels of consanguinity in the region, genetic causes of phosphopenic and calcipenic rickets/osteomalacia are also common.ConclusionThis guideline was developed to provide an approach to the diagnosis of XLH, especially where there is no family history of the disease, and that other related conditions are not mistaken for XLH. We also guide the medical management of XLH with conventional treatment and with burosumab, a recombinant human IgG1 monoclonal antibody to FGF23.

GR/Sp3/HDAC1/UGDH signaling participated in the maternal dexamethasone-induced dysplasia of the rat fetal growth plate.

Maternal dexamethasone decreases the body length of the newborn. However, whether dexamethasone inhibits the development of the growth plate of the fetal long bone is still unknown. Here, we found that lengths of fetal femur and growth plate were both shorter in the fetuses with maternal dexamethasone (0.2mg/kg.d from gestation day 9 to 20), with a decreased proteoglycan content of the growth plate in the fetal rat. Notable decreases in both the gene expression and H3K9 acetylation of UDP-glucose dehydrogenase (Ugdh) gene, which codes a key enzyme in the proteoglycan biosynthesis in the chondrocyte, were also observed. Meanwhile, up-regulation of glucocorticoid receptor (GR), specific protein 3 (Sp3), and histone deacetylase 1 (Hdac1) gene expression were detected in the fetal growth plate. Similar changes were also observed in the chondrogenic rat bone marrow stromal cells (BMSCs) with excessive exogenous dexamethasone. However, antagonizing GR with RU486 and silencing Hdac1 or Sp3 with specific siRNAs could all stimulate the H3K9 acetylation and gene expression of Ugdh previously inhibited by dexamethasone. Meanwhile, dexamethasone also induced the nuclear translocation of GR, which further directly bound to the Ugdh promoter and interacted with HDAC1 and Sp3, respectively. Collectively, our study revealed that maternal dexamethasone induced the direct binding of GR to the Ugdh promoter of the chondrocyte in the rat fetal growth plate, which recruited HDAC1 and Sp3, induced deacetylation of the H3K9, and subsequently inhibited Ugdh gene expression. Such changes further led to attenuated proteoglycan synthesis in the developing chondrocyte and therefore disrupted the development of growth plate and fetal long bone.

Innovative Three-Dimensional Microscopic Analysis of Uremic Growth Plate Discloses Alterations in the Process of Chondrocyte Hypertrophy: Effects of Growth Hormone Treatment.

Chronic kidney disease (CKD) alters the morphology and function of the growth plate (GP) of long bones by disturbing chondrocyte maturation. GP chondrocytes were analyzed in growth-retarded young rats with CKD induced by adenine intake (AD), control rats fed ad libitum (C) or pair-fed with the AD group (PF), and CKD rats treated with growth hormone (ADGH). In order to study the alterations in the process of GP maturation, we applied a procedure recently described by our group to obtain high-quality three-dimensional images of whole chondrocytes that can be used to analyze quantitative parameters like cytoplasm density, cell volume, and shape. The final chondrocyte volume was found to be decreased in AD rats, but GH treatment was able to normalize it. The pattern of variation in the cell cytoplasm density suggests that uremia could be causing a delay to the beginning of the chondrocyte hypertrophy process. Growth hormone treatment appears to be able to compensate for this disturbance by triggering an early chondrocyte enlargement that may be mediated by Nkcc1 action, an important membrane cotransporter in the GP chondrocyte enlargement.

The Roles of Indian Hedgehog Signaling in TMJ Formation.

The temporomandibular joint (TMJ) is an intricate structure composed of the mandibular condyle, articular disc, and glenoid fossa in the temporal bone. Apical condylar cartilage is classified as a secondary cartilage, is fibrocartilaginous in nature, and is structurally distinct from growth plate and articular cartilage in long bones. Condylar cartilage is organized in distinct cellular layers that include a superficial layer that produces lubricants, a polymorphic/progenitor layer that contains stem/progenitor cells, and underlying layers of flattened and hypertrophic chondrocytes. Uniquely, progenitor cells reside near the articular surface, proliferate, undergo chondrogenesis, and mature into hypertrophic chondrocytes. During the past decades, there has been a growing interest in the molecular mechanisms by which the TMJ develops and acquires its unique structural and functional features. Indian hedgehog (Ihh), which regulates skeletal development including synovial joint formation, also plays pivotal roles in TMJ development and postnatal maintenance. This review provides a description of the many important recent advances in Hedgehog (Hh) signaling in TMJ biology. These include studies that used conventional approaches and those that analyzed the phenotype of tissue-specific mouse mutants lacking Ihh or associated molecules. The recent advances in understanding the molecular mechanism regulating TMJ development are impressive and these findings will have major implications for future translational medicine tools to repair and regenerate TMJ congenital anomalies and acquired diseases, such as degenerative damage in TMJ osteoarthritic conditions.

Mineralization pathways in the active murine epiphyseal growth plate

Endochondral ossification in the growth plate of long bones involves cartilage mineralization, bone formation and the budding vasculature. Many of these processes take place in a complex and dynamic zone, the provisional ossification zone, of the growth plate. Here we investigate aspects of mineralization in 2D and 3D in the provisional ossification zone at different length scales using samples preserved under cryogenic or fully hydrated conditions. We use confocal light microscopy, cryo-SEM and micro-CT in the phase contrast mode. We show in 9 week old BALB/c mice the presence of vesicles containing mineral particles in the blood serum, as well as mineral particles without membranes integrated with the blood vessel walls. We also observe labeled mineral particles within cells associated with bone formation, but not in the hypertrophic cartilage cells that are involved with cartilage mineralization. High resolution micro-CT images of fresh hydrated tibiae, show that there are open continuous pathways between the blood vessel extremities and the hypertrophic chondrocyte zone. As the blood vessel extremities, the mineralizing cartilage and the forming bone are all closely associated within this narrow zone, we raise the possibility that in addition to ion transport, mineral necessary for both cartilage and bone formation is also transported through the vasculature.