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1.
Nature ; 567(7747): 234-238, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30814736

RESUMO

Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification1. However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth1,2, but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period.


Assuntos
Condrócitos/citologia , Células Clonais/citologia , Lâmina de Crescimento/citologia , Nicho de Células-Tronco/fisiologia , Envelhecimento , Animais , Cartilagem/citologia , Autorrenovação Celular , Células Clonais/metabolismo , Feminino , Lâmina de Crescimento/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos
2.
PLoS Genet ; 14(4): e1007346, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29659575

RESUMO

The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a Growth Plate Differential Gene Expression Library (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors.


Assuntos
Condrócitos/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fatores de Transcrição SOX9/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Desenvolvimento Ósseo/genética , Desenvolvimento Ósseo/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Condrócitos/citologia , Condrogênese/genética , Condrogênese/fisiologia , Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Feminino , Redes Reguladoras de Genes , Lâmina de Crescimento/citologia , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Modelos Biológicos , Fatores de Transcrição SOX9/genética , Transdução de Sinais , Ativação Transcricional , Proteína GLI1 em Dedos de Zinco/genética
3.
Biochem Biophys Res Commun ; 500(3): 525-529, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29626467

RESUMO

Rac1 and Cdc42, Rho family low molecular weight G proteins, are intracellular signaling factors that transmit various information from outside to inside cells. Primarily, they are known to control various biological activities mediated by actin cytoskeleton reorganization, such as cell proliferation, differentiation, and apoptosis. In order to investigate the functions of Rac1 and Cdc42 in bone formation, we prepared cartilage-specific double conditional knockout mice, Rac1fl/fl; Cdc42fl/fl; Col2-Cre (Rac1: Cdc42 dcKO mice), which died just after birth, similar to Cdc42fl/fl; Col2-Cre mice (Cdc42 cKO mice). Our findings showed that the long tubule bone in Rac1: Cdc42 dcKO mice was shorter than that in Rac1fl/fl; Col2-Cre mice (Rac1 cKO mice) and Cdc42 cKO mice. Abnormal skeleton formation was also observed and disordered columnar formation in the growth plate of the Rac1: Cdc42 dcKO mice was more severe as compared to the Rac1 cKO and Cdc42 cKO mice. Together, these results suggest that Rac1 and Cdc42 have cooperating roles in regulation of bone development.


Assuntos
Calcificação Fisiológica , Cartilagem/embriologia , Cartilagem/metabolismo , Condrogênese , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Fêmur/citologia , Lâmina de Crescimento/citologia , Camundongos Knockout , Fenótipo
4.
Histochem Cell Biol ; 149(4): 365-373, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29356962

RESUMO

Longitudinal bone growth progresses by continuous bone replacement of epiphyseal cartilaginous tissue, known as "growth plate", produced by columnar proliferated- and differentiated-epiphyseal chondrocytes. The endochondral ossification process at the growth plate is governed by paracrine signals secreted from terminally differentiated chondrocytes (hypertrophic chondrocytes), and hedgehog signaling is one of the best known regulatory signaling pathways in this process. Here, to investigate the developmental relationship between longitudinal endochondral bone formation and osteogenic progenitors under the influence of hedgehog signaling at the growth plate, genetic lineage tracing was carried out with the use of Gli1CreERT2 mice line to follow the fate of hedgehog-signal-responsive cells during endochondral bone formation. Gli1CreERT2 genetically labeled cells are detected in hypertrophic chondrocytes and osteo-progenitors at the chondro-osseous junction (COJ); these progeny then commit to the osteogenic lineage in periosteum, trabecular and cortical bone along the developing longitudinal axis. Furthermore, in ageing bone, where longitudinal bone growth ceases, hedgehog-signal responsiveness and its implication in osteogenic lineage commitment is significantly weakened. These results show, for the first time, evidence of the developmental contribution of endochondral progenitors under the influence of epiphyseal chondrocyte-derived secretory signals in longitudinally growing bone. This study provides a precise outline for assessing the skeletal lineage commitment of osteo-progenitors in response to growth-plate-derived regulatory signals during endochondral bone formation.


Assuntos
Desenvolvimento Ósseo , Osso e Ossos/metabolismo , Lâmina de Crescimento/metabolismo , Proteínas Hedgehog/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais , Animais , Osso e Ossos/citologia , Lâmina de Crescimento/citologia , Masculino , Camundongos , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
5.
Tissue Eng Part A ; 24(1-2): 94-105, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28525313

RESUMO

Defining the final size and geometry of engineered tissues through precise control of the scalar and vector components of tissue growth is a necessary benchmark for regenerative medicine, but it has proved to be a significant challenge for tissue engineers. The growth plate cartilage that promotes elongation of the long bones is a good model system for studying morphogenetic mechanisms because cartilage is composed of a single cell type, the chondrocyte; chondrocytes are readily maintained in culture; and growth trajectory is predominately in a single vector. In this cartilage, growth is generated via a differentiation program that is spatially and temporally regulated by an interconnected network composed of long- and short-range signaling mechanisms that together result in the formation of functionally distinct cellular zones. To facilitate investigation of the mechanisms underlying anisotropic growth, we developed an in vitro model of the growth plate cartilage by using neonatal mouse growth plate chondrocytes encapsulated in alginate hydrogel beads. In bead cultures, encapsulated chondrocytes showed high viability, cartilage matrix deposition, low levels of chondrocyte hypertrophy, and a progressive increase in cell proliferation over 7 days in culture. Exogenous factors were used to test functionality of the parathyroid-related protein-Indian hedgehog (PTHrP-IHH) signaling interaction, which is a crucial feedback loop for regulation of growth. Consistent with in vivo observations, exogenous PTHrP stimulated cell proliferation and inhibited hypertrophy, whereas IHH signaling stimulated chondrocyte hypertrophy. Importantly, the treatment of alginate bead cultures with IHH or thyroxine resulted in formation of a discrete domain of hypertrophic cells that mimics tissue architecture of native growth plate cartilage. Together, these studies are the first demonstration of a tunable in vitro system to model the signaling network interactions that are required to induce zonal architecture in growth plate chondrocytes, which could also potentially be used to grow cartilage cultures of specific geometries to meet personalized patient needs.


Assuntos
Alginatos/química , Cartilagem/citologia , Lâmina de Crescimento/citologia , Tecidos Suporte/química , Animais , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Camundongos , Transdução de Sinais
6.
Cell Biol Int ; 42(2): 169-179, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28980745

RESUMO

Longitudinal bone growth is governed by a complex network of endocrine signals including leptin. In mouse, leptin deficiency leads to distinct phenotypes in bones of the limb and spine, suggesting the appendicular and axial skeletons are subject to differential regulation by leptin. We established primary cultures for the chondrocytes from tibial and vertebral epiphyseal plates. Cellular proliferation and apoptosis were analyzed for the chondrocytes that had been treated with various concentrations of leptin. Crucial factors for chondrocyte proliferation and differentiation, such as BMP7 and Wnt3, were measured in the cells treated with leptin alone or in combination with pharmacological inhibitors of STAT and ERK signaling pathways. Primary culture of tibial epiphyseal plate chondrocytes has greater proliferating capability compared with that of vertebral epiphyseal plate chondrocytes. Leptin could promote the proliferation of tibial epiphyseal plate chondrocytes, while its effect on vertebral epiphyseal plate chondrocytes was inhibitory. Consistently, apoptosis is inhibited in tibial but promoted in vertebral epiphyseal plate chondrocytes by leptin. Importantly, leptin differentially modulates chondrogenic signaling pathways in tibial and vertebral epiphyseal chondrocytes through STAT and ERK pathways. Leptin differentially regulates chondrogenic proliferation and differentiation in appendicular and axial regions of the skeletons. The signaling pathways in these two regions are also distinct and subject to differential regulation by leptin through the STAT pathway in tibial epiphyseal plate chondrocytes but through the ERK pathway in vertebral epiphyseal plate chondrocytes. Therefore, the regulation of leptin is multi-faceted in the distinct anatomical regions of the skeleton. Knowledge gained from this system will provide insights into the pathophysiological causes for the diseases related to bone development and metabolism.


Assuntos
Lâmina de Crescimento/crescimento & desenvolvimento , Leptina/fisiologia , Osteogênese , Coluna Vertebral/crescimento & desenvolvimento , Tíbia/crescimento & desenvolvimento , Animais , Apoptose , Proliferação de Células , Condrócitos/citologia , Condrócitos/metabolismo , Feminino , Lâmina de Crescimento/citologia , Lâmina de Crescimento/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais
7.
Biomed Res Int ; 2018: 6348171, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30687754

RESUMO

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.


Assuntos
Lâmina de Crescimento , Procedimentos Ortopédicos , Animais , Apoptose , Fenômenos Biomecânicos/fisiologia , Doenças Ósseas , Modelos Animais de Doenças , Lâmina de Crescimento/química , Lâmina de Crescimento/citologia , Lâmina de Crescimento/patologia , Lâmina de Crescimento/cirurgia , Imuno-Histoquímica , Masculino , Distribuição Aleatória , Recidiva , Suínos , Porco Miniatura , Tíbia/química , Tíbia/citologia , Tíbia/patologia , Tíbia/cirurgia
8.
Elife ; 62017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-29205154

RESUMO

GWAS have identified hundreds of height-associated loci. However, determining causal mechanisms is challenging, especially since height-relevant tissues (e.g. growth plates) are difficult to study. To uncover mechanisms by which height GWAS variants function, we performed epigenetic profiling of murine femoral growth plates. The profiled open chromatin regions recapitulate known chondrocyte and skeletal biology, are enriched at height GWAS loci, particularly near differentially expressed growth plate genes, and enriched for binding motifs of transcription factors with roles in chondrocyte biology. At specific loci, our analyses identified compelling mechanisms for GWAS variants. For example, at CHSY1, we identified a candidate causal variant (rs9920291) overlapping an open chromatin region. Reporter assays demonstrated that rs9920291 shows allelic regulatory activity, and CRISPR/Cas9 targeting of human chondrocytes demonstrates that the region regulates CHSY1 expression. Thus, integrating biologically relevant epigenetic information (here, from growth plates) with genetic association results can identify biological mechanisms important for human growth.


Assuntos
Estatura , Condrócitos/fisiologia , Epigênese Genética , Variação Genética , Lâmina de Crescimento/citologia , Animais , Cromatina/metabolismo , Loci Gênicos , Humanos , Camundongos
9.
BMC Genomics ; 18(1): 983, 2017 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-29262782

RESUMO

BACKGROUND: Chondrogenesis is the earliest stage of skeletal development and is a highly dynamic process, integrating the activities and functions of transcription factors, cell signaling molecules and extracellular matrix proteins. The molecular mechanisms underlying chondrogenesis have been extensively studied and multiple key regulators of this process have been identified. However, a genome-wide overview of the gene regulatory network in chondrogenesis has not been achieved. RESULTS: In this study, employing RNA sequencing, we identified 332 protein coding genes and 34 long non-coding RNA (lncRNA) genes that are highly selectively expressed in human fetal growth plate chondrocytes. Among the protein coding genes, 32 genes were associated with 62 distinct human skeletal disorders and 153 genes were associated with skeletal defects in knockout mice, confirming their essential roles in skeletal formation. These gene products formed a comprehensive physical interaction network and participated in multiple cellular processes regulating skeletal development. The data also revealed 34 transcription factors and 11,334 distal enhancers that were uniquely active in chondrocytes, functioning as transcriptional regulators for the cartilage-selective genes. CONCLUSIONS: Our findings revealed a complex gene regulatory network controlling skeletal development whereby transcription factors, enhancers and lncRNAs participate in chondrogenesis by transcriptional regulation of key genes. Additionally, the cartilage-selective genes represent candidate genes for unsolved human skeletal disorders.


Assuntos
Condrócitos/metabolismo , Condrogênese/genética , Redes Reguladoras de Genes , Lâmina de Crescimento/metabolismo , Cartilagem/embriologia , Cartilagem/metabolismo , Elementos Facilitadores Genéticos , Feto , Perfilação da Expressão Gênica , Lâmina de Crescimento/citologia , Humanos , Mapas de Interação de Proteínas , RNA Longo não Codificante/metabolismo
10.
Mol Med Rep ; 16(6): 8019-8028, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28983600

RESUMO

Skeletal stem cells (SSCs) are a population of progenitor cells which give rise to postnatal skeletal tissues including bone, cartilage and bone marrow stroma, however not to adipose, haematopoietic or muscle tissue. Growth plate chondrocytes exhibit the ability of continuous proliferation and differentiation, which contributes to the continuous physiological growth. The growth plate has been hypothesized to contain SSCs which exhibit a desirable differentiation capacity to generate bone and cartilage. Due to the heterogeneity of the growth plate chondrocytes, SSCs in the growth plate are not well studied. The present study used cluster of differentiation (CD)146 and CD105 as markers to isolate purified SSCs. CD105+ SSCs and CD146+ SSCs were isolated using a magnetic activated cell sorting method. To quantitatively investigate the proliferation and differentiation ability, the colony-forming efficiency (CFE) and multi­lineage differentiation capacity of CD105+ SSCs and CD146+ SSCs were compared with unsorted cells and adipose-derived stem cells (ASCs). It was revealed that CD105+ and CD146+ subpopulations represented subsets of SSCs which generated chondrocytes and osteocytes, however not adipocytes. Compared with CD105+ subpopulations and ASCs, the CD146+ subpopulation exhibited a greater CFE and continuous high chondrogenic differentiation capacity in vitro. Therefore, the present study suggested that the CD146+ subpopulation represented a chondrolineage­restricted subpopulation of SSCs and may therefore act as a valuable cell source for cartilage regeneration.


Assuntos
Antígeno CD146/metabolismo , Diferenciação Celular , Condrogênese , Lâmina de Crescimento/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Adipogenia , Animais , Biomarcadores , Linhagem da Célula , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Citometria de Fluxo , Imunofenotipagem , Ratos
11.
J Clin Invest ; 127(11): 4136-4147, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28990933

RESUMO

Although peptides are safe and useful as therapeutics, they are often easily degraded or metabolized. Dampening the clearance system for peptide ligands is a promising strategy for increasing the efficacy of peptide therapies. Natriuretic peptide receptor B (NPR-B) and its naturally occurring ligand, C-type natriuretic peptide (CNP), are potent stimulators of endochondral bone growth, and activating the CNP/NPR-B system is expected to be a powerful strategy for treating impaired skeletal growth. CNP is cleared by natriuretic peptide clearance receptor (NPR-C); therefore, we investigated the effect of reducing the rate of CNP clearance on skeletal growth by limiting the interaction between CNP and NPR-C. Specifically, we generated transgenic mice with increased circulating levels of osteocrin (OSTN) protein, a natural NPR-C ligand without natriuretic activity, and observed a dose-dependent skeletal overgrowth phenotype in these animals. Skeletal overgrowth in OSTN-transgenic mice was diminished in either CNP- or NPR-C-depleted backgrounds, confirming that CNP and NPR-C are indispensable for the bone growth-stimulating effect of OSTN. Interestingly, double-transgenic mice of CNP and OSTN had even higher levels of circulating CNP and additional increases in bone length, as compared with mice with elevated CNP alone. Together, these results support OSTN administration as an adjuvant agent for CNP therapy and provide a potential therapeutic approach for diseases with impaired skeletal growth.


Assuntos
Proteínas Musculares/sangue , Peptídeo Natriurético Tipo C/sangue , Osteogênese , Fatores de Transcrição/sangue , Animais , GMP Cíclico/metabolismo , Feminino , Expressão Gênica , Lâmina de Crescimento/citologia , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Humanos , Vértebras Lombares/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Receptores do Fator Natriurético Atrial/metabolismo , Componente Amiloide P Sérico/metabolismo , Transdução de Sinais
12.
Elife ; 62017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-28994649

RESUMO

Both oriented cell divisions and cell rearrangements are critical for proper embryogenesis and organogenesis. However, little is known about how these two cellular events are integrated. Here we examine the linkage between these processes in chick limb cartilage. By combining retroviral-based multicolor clonal analysis with live imaging, the results show that single chondrocyte precursors can generate both single-column and multi-column clones through oriented division followed by cell rearrangements. Focusing on single column formation, we show that this stereotypical tissue architecture is established by a pivot-like process between sister cells. After mediolateral cell division, N-cadherin is enriched in the post-cleavage furrow; then one cell pivots around the other, resulting in stacking into a column. Perturbation analyses demonstrate that planar cell polarity signaling enables cells to pivot in the direction of limb elongation via this N-cadherin-mediated coupling. Our work provides new insights into the mechanisms generating appropriate tissue architecture of limb skeleton.


Assuntos
Cartilagem/citologia , Divisão Celular , Polaridade Celular , Extremidades/embriologia , Lâmina de Crescimento/citologia , Transdução de Sinais , Animais , Embrião de Galinha
13.
J Appl Physiol (1985) ; 123(5): 1101-1109, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28798204

RESUMO

Bones elongate through endochondral ossification in cartilaginous growth plates located at ends of primary long bones. Linear growth ensues from a cascade of biochemical signals initiated by actions of systemic and local regulators on growth plate chondrocytes. Although cellular processes are well defined, there is a fundamental gap in understanding how growth regulators are physically transported from surrounding blood vessels into and through dense, avascular cartilage matrix. Intravital imaging using in vivo multiphoton microscopy is one promising strategy to overcome this barrier by quantitatively tracking molecular delivery to cartilage from the vasculature in real time. We previously used in vivo multiphoton imaging to show that hindlimb heating increases vascular access of large molecules to growth plates using 10-, 40-, and 70-kDa dextran tracers. To comparatively evaluate transport of similarly sized physiological regulators, we developed and validated methods for measuring uptake of biologically active IGF-I into proximal tibial growth plates of live 5-wk-old mice. We demonstrate that fluorescently labeled IGF-I (8.2 kDa) is readily taken up in the growth plate and localizes to chondrocytes. Bioactivity tests performed on cultured metatarsal bones confirmed that the labeled protein is functional, assessed by phosphorylation of its signaling kinase, Akt. This methodology, which can be broadly applied to many different proteins and tissues, is relevant for understanding factors that affect delivery of biologically relevant molecules to the skeleton in real time. Results may lead to the development of drug-targeting strategies to treat a wide range of bone and cartilage pathologies.NEW & NOTEWORTHY This paper describes and validates a novel method for imaging transport of biologically active, fluorescently labeled IGF-I into skeletal growth plates of live mice using multiphoton microscopy. Cellular patterns of fluorescence in the growth plate were completely distinct from our prior publications using biologically inert probes, demonstrating for the first time in vivo localization of IGF-I in chondrocytes and perichondrium. These results form important groundwork for future studies aimed at targeting therapeutics into growth plates.


Assuntos
Cartilagem/metabolismo , Condrócitos/metabolismo , Lâmina de Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Ossos do Metatarso/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica , Tíbia/metabolismo , Animais , Transporte Biológico , Cartilagem/citologia , Feminino , Corantes Fluorescentes/metabolismo , Lâmina de Crescimento/citologia , Humanos , Cinética , Masculino , Ossos do Metatarso/citologia , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reprodutibilidade dos Testes , Tíbia/citologia , Técnicas de Cultura de Tecidos
15.
Spine J ; 17(11): 1674-1684, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28645676

RESUMO

BACKGROUND CONTEXT: The process of linear growth is driven by axial elongation of both long bones and vertebral bodies and is accomplished by enchondral ossification. Differences in regulation between the two skeletal sites are mirrored clinically by the age course in body proportions. Whereas long bone growth plates (GPs) can easily be discriminated, vertebral GPs are part of the cartilaginous end plate, which typically shows important species differences. PURPOSE: The objective of this study was to describe and compare histologic, histomorphometric, and regulatory characteristics in the GPs of the spine and the long bones in a porcine model. MATERIALS AND METHODS: Two- and six-week-old piglet GPs of three vertebral segments (cervical, thoracic, and lumbar) and eight long bones (proximal and distal radius, humerus, tibia, and femur) were analyzed morphometrically. Further, estrogen receptors, proliferation markers, and growth factor expressions were examined by immunohistochemistry. RESULTS: Individual vertebral GPs were smaller in width and contained fewer chondrocytes than long bone GPs, although their proliferation activity was similar. Whereas the expression pattern of growth hormone-associated factors such as insulin-like growth factor (IGF)-1 and IGF-1 receptor (IGF-1R) was similar, estrogen receptor (ER)-ß and IGF-2 were distinctly expressed in the vertebral samples. CONCLUSIONS: Vertebral GPs display differential growth, with measurements similar to the slowest-growing GPs of long bones. Further investigation is needed to decipher the molecular basis of the differential growth of the spine and the long bones. Knowledge on the distinct mechanism will ultimately improve the assessment of clinically essential characteristics of spinal growth, such as vertebral elongation potential and GP fusion.


Assuntos
Fêmur/crescimento & desenvolvimento , Lâmina de Crescimento/crescimento & desenvolvimento , Coluna Vertebral/crescimento & desenvolvimento , Animais , Condrócitos/metabolismo , Fêmur/citologia , Fêmur/metabolismo , Lâmina de Crescimento/citologia , Lâmina de Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Osteogênese , Coluna Vertebral/citologia , Coluna Vertebral/metabolismo , Suínos
16.
Free Radic Biol Med ; 110: 63-71, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28559051

RESUMO

In endochondral ossification, growth of bones occurs at their growth plate cartilage. While it is known that nitric oxide (NO) synthases are required for proliferation of chondrocytes in growth plate cartilage and growth of bones, the precise mechanism by which NO facilitates these process has not been clarified yet. C-type natriuretic peptide (CNP) also positively regulate elongation of bones through expansion of the growth plate cartilage. Both NO and CNP are known to use cGMP as the second messenger. Recently, 8-nitro-cGMP was identified as a signaling molecule produced in the presence of NO in various types of cells. Here, we found that 8-nitro-cGMP is produced in proliferating chondrocytes in the growth plates, which was enhanced by CNP, in bones cultured ex vivo. In addition, 8-nitro-cGMP promoted bone growth with expansion of the proliferating zone as well as increase in the number of proliferating cells in the growth plates. 8-Nitro-cGMP also promoted the proliferation of chondrocytes in vitro. On the other hand, 8-bromo-cGMP enhanced the growth of bones with expansion of hypertrophic zone of the growth plates without affecting either the width of proliferating zone or proliferation of chondrocytes. These results indicate that 8-nitro-cGMP formed in growth plate cartilage accelerates chondrocyte proliferation and bone growth as a downstream molecule of NO.


Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , GMP Cíclico/análogos & derivados , Lâmina de Crescimento/efeitos dos fármacos , Tíbia/efeitos dos fármacos , Animais , Cartilagem/citologia , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , GMP Cíclico/farmacologia , Feto , Lâmina de Crescimento/citologia , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Peptídeo Natriurético Tipo C/farmacologia , Óxido Nítrico/metabolismo , Cultura Primária de Células , Tíbia/citologia , Tíbia/crescimento & desenvolvimento , Tíbia/metabolismo , Técnicas de Cultura de Tecidos
17.
BMC Musculoskelet Disord ; 18(1): 235, 2017 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-28569158

RESUMO

BACKGROUND: Leptin plays an important role in mediating chondrogenesis of limb growth plate. Previous studies suggest that bone structures and development of spine and limb are different. The expression of Ob-Rb, the gene that encodes leptin receptors, is vertebral and appendicular region-specific, suggesting the regulation of leptin on VGP and TGP chondrogenesis may be very different. The aim of the present study was to investigate the differential regulation of leptin on the chondrogenesis of vertebral growth plate (VGP) and tibial growth plate (TGP). METHODS: We compared the VGP and TGP from wild type (C57BL/6) and leptin-deficient (ob/ob) mice. We then generated primary cultures of TGP and VGP chondrocytes. By treating the primary cells with different concentrations of leptin in vitro, we analyzed proliferation and apoptosis of the primary chondrocytes from TGP and VGP. We further measured expression of chondrogenic-related genes in these cells that had been incubated with different doses of leptin. RESULTS: Leptin-deficient mice of 8-week-old had shorter tibial and longer vertebral lengths than the wide type mice. Disturbed columnar structure was observed for TGP but not for VGP. In primary chondrocyte cultures, leptin inhibited VGP chondrocyte proliferation but promoted their apoptosis. Collagen IIA and aggrecan mRNA, and the protein levels of proliferation- and chondrogenesis-related markers, including PCNA, Sox9, and Smad4, were downregulated by leptin in a dose-dependent manner. In contrast, leptin stimulated the proliferation and chondrogenic differentiation of TGP chondrocytes at physiological levels (i.e., 10 and 50 ng/mL) but not at high levels (i.e., 100 and 1000 ng/mL). CONCLUSION: Leptin exerts a stimulatory effect on the proliferation and chondrogenic differentiation of the long bone growth plate but an inhibitory effect on the spine growth plate. The ongoing study will shed light on the regulatory mechanisms of leptin in bone development and metabolism.


Assuntos
Condrócitos/fisiologia , Condrogênese/fisiologia , Lâmina de Crescimento/crescimento & desenvolvimento , Leptina/farmacologia , Coluna Vertebral/crescimento & desenvolvimento , Tíbia/crescimento & desenvolvimento , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Relação Dose-Resposta a Droga , Lâmina de Crescimento/citologia , Lâmina de Crescimento/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Coluna Vertebral/citologia , Coluna Vertebral/efeitos dos fármacos , Tíbia/citologia , Tíbia/efeitos dos fármacos
18.
Histochem Cell Biol ; 148(3): 229-238, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28500502

RESUMO

Septoclasts, which are mononuclear and spindle-shaped cells with many processes, have been considered to resorb the transverse septa of the growth plate (GP) cartilage at the chondro-osseous junction (COJ). We previously reported the expression of epidermal-type fatty acid-binding protein (E-FABP, FABP5) and localization of peroxisome proliferator-activated receptor (PPAR)ß/δ, which mediates the cell survival or proliferation, in septoclasts. On the other hand, retinoic acid (RA) can bind to E-FABP and is stored abundantly in the GP cartilage. From these information, it is possible to hypothesize that RA in the GP is incorporated into septoclasts during the cartilage resorption and regulates the growth and/or death of septoclasts. To clarify the mechanism of the cartilage resorption induced by RA, we administered an overdose of RA or its precursor vitamin A (VA)-deficient diet to young mice. In mice of both RA excess and VA deficiency, septoclasts decreased in the number and cell size in association with shorter and lesser processes than those in normal mice, suggesting a substantial suppression of resorption by septoclasts in the GP cartilage. Lack of PPARß/δ-expression, TUNEL reaction, RA receptor (RAR)ß, and cellular retinoic acid-binding protein (CRABP)-II were induced in E-FABP-positive septoclasts under RA excess, suggesting the growth arrest/cell-death of septoclasts, whereas cartilage-derived retinoic acid-sensitive protein (CD-RAP) inducing the cell growth arrest or morphological changes was induced in septoclasts under VA deficiency. These results support and do not conflict with our hypothesis, suggesting that endogenous RA in the GP is possibly incorporated in septoclasts and utilized to regulate the activity of septoclasts resorbing the GP cartilage.


Assuntos
Cartilagem/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Proteínas de Ligação a Ácido Graxo/análise , Proteínas de Ligação a Ácido Graxo/metabolismo , Lâmina de Crescimento/efeitos dos fármacos , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/metabolismo , Pericitos/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Cartilagem/citologia , Morte Celular/efeitos dos fármacos , Proteínas de Ligação a Ácido Graxo/imunologia , Lâmina de Crescimento/citologia , Masculino , Camundongos , Proteínas de Neoplasias/imunologia , Pericitos/imunologia , Tretinoína/administração & dosagem , Vitamina A/metabolismo
19.
J Biomech ; 56: 76-82, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28365062

RESUMO

Longitudinal bone growth in children/adolescents occurs through endochondral ossification at growth plates and is influenced by mechanical loading, where increased compression decreases growth (i.e., Hueter-Volkmann Law). Past in vivo studies on static vs dynamic compression of growth plates indicate that factors modulating growth rate might lie at the cellular level. Here, in situ viscoelastic deformation of hypertrophic chondrocytes in growth plate explants undergoing stress-controlled static vs dynamic loading conditions was investigated. Growth plate explants from the proximal tibia of pre-pubertal rats were subjected to static vs dynamic stress-controlled mechanical tests. Stained hypertrophic chondrocytes were tracked before and after mechanical testing with a confocal microscope to derive volumetric, axial and lateral cellular strains. Axial strain in hypertrophic chondrocytes was similar for all groups, supporting the mean applied compressive stress's correlation with bone growth rate and hypertrophic chondrocyte height in past studies. However, static conditions resulted in significantly higher lateral (p<0.001) and volumetric cellular strains (p≤0.015) than dynamic conditions, presumably due to the growth plate's viscoelastic nature. Sustained compression in stress-controlled static loading results in continued time-dependent cellular deformation; conversely, dynamic groups have less volumetric strain because the cyclically varying stress limits time-dependent deformation. Furthermore, high frequency dynamic tests showed significantly lower volumetric strain (p=0.002) than low frequency conditions. Mechanical loading protocols could be translated into treatments to correct or halt progression of bone deformities in children/adolescents. Mimicking physiological stress-controlled dynamic conditions may have beneficial effects at the cellular level as dynamic tests are associated with limited lateral and volumetric cellular deformation.


Assuntos
Condrócitos/fisiologia , Lâmina de Crescimento/citologia , Animais , Desenvolvimento Ósseo , Masculino , Pressão , Ratos Sprague-Dawley , Estresse Mecânico , Tíbia
20.
J Bone Miner Res ; 32(6): 1320-1331, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28277610

RESUMO

Notch signaling is critical for osteoblastic differentiation; however, the specific contribution of individual Notch ligands is unknown. Parathyroid hormone (PTH) regulates the Notch ligand Jagged1 in osteoblastic cells. To determine if osteolineage Jagged1 contributes to bone homeostasis, selective deletion of Jagged1 in osteolineage cells was achieved through the presence of Prx1 promoter-driven Cre recombinase expression, targeting mesenchymal stem cells (MSCs) and their progeny (PJag1 mice). PJag1 mice were viable and fertile and did not exhibit any skeletal abnormalities at 2 weeks of age. At 2 months of age, however, PJag1 mice had increased trabecular bone mass compared to wild-type (WT) littermates. Dynamic histomorphometric analysis showed increased osteoblastic activity and increased mineral apposition rate. Immunohistochemical analysis showed increased numbers of osteocalcin-positive mature osteoblasts in PJag1 mice. Also increased phenotypically defined Lin- /CD45- /CD31- /Sca1- /CD51+ osteoblastic cells were measured by flow cytometric analysis. Surprisingly, phenotypically defined Lin- /CD45- /CD31- /Sca1+ /CD51+ MSCs were unchanged in PJag1 mice as measured by flow cytometric analysis. However, functional osteoprogenitor (OP) cell frequency, measured by Von Kossa+ colony formation, was decreased, suggesting that osteolineage Jagged1 contributes to maintenance of the OP pool. The trabecular bone increases were not due to osteoclastic defects, because PJag1 mice had increased bone resorption. Because PTH increases osteoblastic Jagged1, we sought to understand if osteolineage Jagged1 modulates PTH-mediated bone anabolism. Intermittent PTH treatment resulted in a significantly greater increase in BV/TV in PJag1 hind limbs compared to WT. These findings demonstrate a critical role of osteolineage Jagged1 in bone homeostasis, where Jagged1 maintains the transition of OP to maturing osteoblasts. This novel role of Jagged1 not only identifies a regulatory loop maintaining appropriate populations of osteolineage cells, but also provides a novel approach to increase trabecular bone mass, particularly in combination with PTH, through modulation of Jagged1. © 2017 American Society for Bone and Mineral Research.


Assuntos
Linhagem da Célula , Proteína Jagged-1/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osso Esponjoso/citologia , Osso Esponjoso/diagnóstico por imagem , Osso Esponjoso/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Lâmina de Crescimento/citologia , Lâmina de Crescimento/diagnóstico por imagem , Lâmina de Crescimento/metabolismo , Ligantes , Camundongos , Modelos Biológicos , Osteoblastos/efeitos dos fármacos , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Células-Tronco/efeitos dos fármacos , Microtomografia por Raio-X
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