RESUMO
This article captures a collective reflection on the successes and challenges we experienced when teaching physiology laboratories online during the COVID-19 pandemic. Physiology instructors from six institutions discussed their own efforts to redesign meaningful physiology laboratories that could be taught remotely, as the nation scrambled to respond to the sudden shift out of the classroom. Despite the complexity of this task, clear themes emerged as our courses transitioned to an online format in spring 2020 and were solidified in the fall of 2020. This article reflects on the history, features, benefits, and challenges of current laboratory teaching when applying a scientific teaching approach to facilitate the redesign process. We believe online networks like ours can facilitate information sharing, promote innovations, and provide support for instructors. The insights we gained through this collaboration will influence our thinking about the future of the physiology lab, whether online or in person.
Assuntos
COVID-19 , Educação a Distância , Humanos , Pandemias , SARS-CoV-2 , EstudantesRESUMO
The proteoglycan-containing pericellular matrix (PCM) controls both the biophysical and biochemical microenvironment of osteocytes, which are the most abundant cells embedded and dispersed in bones. As a molecular sieve, osteocytic PCMs not only regulate mass transport to and from osteocytes but also act as sensors of external mechanical environments. The turnover of osteocytic PCM remains largely unknown due to technical challenges. Here, we report a novel imaging technique based on metabolic labeling and "click-chemistry," which labels de novo PCM as "halos" surrounding osteocytes in vitro and in vivo. We then tested the method and showed different labeling patterns in young vs. old bones. Further "pulse-chase" experiments revealed dramatic difference in the "half-life" of PCM of cultured osteocytes (~70 h) and that of osteocytes in vivo (~75 d). When mice were subjected to either 3-week hindlimb unloading or 7-week tibial loading (5.1 N, 4 Hz, 3 d/week), PCM half-life was shortened (~20 d) and degradation accelerated. Matrix metallopeptidase MMP-14 was elevated in mechanically loaded osteocytes, which may contribute to PCM degradation. This study provides a detailed procedure that enables semi-quantitative study of the osteocytic PCM remodeling in vivo and in vitro.
Assuntos
Envelhecimento , Osso e Ossos/metabolismo , Matriz Extracelular/metabolismo , Elevação dos Membros Posteriores/métodos , Metaloproteinase 14 da Matriz/metabolismo , Mecanotransdução Celular , Osteócitos/metabolismo , Animais , Osso e Ossos/citologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteócitos/citologiaRESUMO
Perlecan/Hspg2, a large monomeric heparan sulfate proteoglycan, is found in the basement membrane and extracellular matrix, where it acts as a matrix scaffold, growth factor depot, and tissue barrier. Perlecan deficiency leads to skeletal dysplasia in Schwartz-Jampel Syndrome (SJS) and is a risk factor for osteoporosis. In the SJS-mimicking murine model (Hypo), inferior cortical bone quality and impaired mechanotransduction in osteocytes were reported. This study focused on trabecular bone, where perlecan deficiency was hypothesized to result in structural deficit and altered response to disuse and re-loading. We compared the Hypo versus WT trabecular bone in both axial and appendicular skeletons of 8-38-week-old male mice, and observed severe trabecular deficit in Hypo mice, approximately 50% reduction of Tb.BV/TV regardless of skeletal site and animal age. Defects in endochondral ossification (e.g., accelerated mineralization), increases in osteoclast activity, and altered differentiation of bone progenitor cells in marrow contributed to the Hypo phenotype. The Hypo trabecular bone deteriorated further under three-week hindlimb suspension as did the WT. Re-ambulation partially recovered the lost trabecular bone in Hypo, but not in WT mice. The novel finding that low-impact loading could counter detrimental disuse effects in the perlecan-deficient skeleton suggests a strategy to maintain skeletal health in SJS patients.
Assuntos
Osso Esponjoso/patologia , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/genética , Osteócitos/citologia , Animais , Fêmur/patologia , Células-Tronco Hematopoéticas/citologia , Proteoglicanas de Heparan Sulfato/fisiologia , Cifose , Masculino , Mecanotransdução Celular , Metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteogênese , Fenótipo , Fatores de Risco , Estresse Mecânico , Caminhada , Microtomografia por Raio-XRESUMO
Perlecan, a heparan sulfate proteoglycan, acts as a mechanical sensor for bone to detect external loading. Deficiency of perlecan increases the risk of osteoporosis in patients with Schwartz-Jampel Syndrome (SJS) and attenuates loading-induced bone formation in perlecan deficient mice (Hypo). Considering that intracellular calcium [Ca2+]i is an ubiquitous messenger controlling numerous cellular processes including mechanotransduction, we hypothesized that perlecan deficiency impairs bone's calcium signaling in response to loading. To test this, we performed real-time [Ca2+]i imaging on in situ osteocytes of adult murine tibiae under cyclic loading (8N). Relative to wild type (WT), Hypo osteocytes showed decreases in the overall [Ca2+]i response rate (-58%), calcium peaks (-33%), cells with multiple peaks (-53%), peak magnitude (-6.8%), and recovery speed to baseline (-23%). RNA sequencing and pathway analysis of tibiae from mice subjected to one or seven days of unilateral loading demonstrated that perlecan deficiency significantly suppressed the calcium signaling, ECM-receptor interaction, and focal adhesion pathways following repetitive loading. Defects in the endoplasmic reticulum (ER) calcium cycling regulators such as Ryr1/ryanodine receptors and Atp2a1/Serca1 calcium pumps were identified in Hypo bones. Taken together, impaired calcium signaling may contribute to bone's reduced anabolic response to loading, underlying the osteoporosis risk for the SJS patients.
Assuntos
Sinalização do Cálcio , Proteoglicanas de Heparan Sulfato , Animais , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Mecanotransdução Celular , Camundongos , Transcriptoma/genéticaRESUMO
Osteocytes, the most abundant bone cells, form an interconnected network in the lacunar-canalicular pore system (LCS) buried within the mineralized matrix, which allows osteocytes to obtain nutrients from the blood supply, sense external mechanical signals, and communicate among themselves and with other cells on bone surfaces. In this study, we examined key features of the LCS network including the topological parameter and the detailed structure of individual connections and their variations in cortical and cancellous compartments, at different ages, and in two disease conditions with altered mechanosensing (perlecan deficiency and diabetes). LCS network showed both topological stability, in terms of conservation of connectivity among osteocyte lacunae (similar to the "nodes" in a computer network), and considerable variability the pericellular annular fluid gap surrounding lacunae and canaliculi (similar to the "bandwidth" of individual links in a computer network). Age, in the range of our study (15-32 weeks), affected only the pericellular fluid annulus in cortical bone but not in cancellous bone. Diabetes impacted the spacing of the lacunae, while the perlecan deficiency had a profound influence on the pericellular fluid annulus. The LCS network features play important roles in osteocyte signaling and regulation of bone growth and adaptation.
RESUMO
Voltage-sensitive calcium channels (VSCC) regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1) and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM) obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration.
Assuntos
Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Canais de Cálcio Tipo T/metabolismo , Condrócitos/metabolismo , Condrócitos/fisiologia , Metabolismo/fisiologia , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Transdução de Sinais/fisiologia , Estresse MecânicoRESUMO
Perlecan/HSPG2 (Pln) is a large heparan sulfate proteoglycan abundant in the extracellular matrix of cartilage and the lacunocanalicular space of adult bones. Although Pln function during cartilage development is critical, evidenced by deficiency disorders including Schwartz-Jampel Syndrome and dyssegmental dysplasia Silverman-Handmaker type, little is known about its function in development of bone shape and quality. The purpose of this study was to understand the contribution of Pln to bone geometric and mechanical properties. We used hypomorph mutant mice that secrete negligible amount of Pln into skeletal tissues and analyzed their adult bone properties using micro-computed tomography and three-point-bending tests. Bone shortening and widening in Pln mutants was observed and could be attributed to loss of growth plate organization and accelerated osteogenesis that was reflected by elevated cortical thickness at older ages. This effect was more pronounced in Pln mutant females, indicating a sex-specific effect of Pln deficiency on bone geometry. Additionally, mutant females, and to a lesser extent mutant males, increased their elastic modulus and bone mineral densities to counteract changes in bone shape, but at the expense of increased brittleness. In summary, Pln deficiency alters cartilage matrix patterning and, as we now show, coordinately influences bone formation and calcification.
Assuntos
Desenvolvimento Ósseo/fisiologia , Proteoglicanas de Heparan Sulfato/deficiência , Osteogênese/fisiologia , Envelhecimento , Animais , Osso e Ossos , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Confocal , Microtomografia por Raio-XRESUMO
The pericellular matrix (PCM), a thin coating surrounding nearly all mammalian cells, plays a critical role in many cell-surface phenomena. In osteocytes, the PCM is believed to control both "outside-in" (mechanosensing) and "inside-out" (signaling molecule transport) processes. However, the osteocytic PCM is challenging to study in situ because it is thin (â¼100 nm) and enclosed in mineralized matrix. To this end, we recently developed a novel tracer velocimetry approach that combined fluorescence recovery after photobleaching (FRAP) imaging with hydrodynamic modeling to quantify the osteocytic PCM in young murine bone. In this study, we applied the technique to older mice expressing or deficient for perlecan/HSPG2, a large heparan-sulfate proteoglycan normally secreted in osteocytic PCM. The objectives were (1) to characterize transport within an altered PCM; (2) to test the sensitivity of our approach in detecting the PCM alterations; and (3) to dissect the roles of the PCM in osteocyte mechanosensing. We found that: (1) solute transport increases in the perlecan-deficient (hypomorphic [Hypo]) mice compared with control mice; (2) PCM fiber density decreases with aging and perlecan deficiency; (3) osteocytes in the Hypo bones are predicted to experience higher shear stress (+34%), but decreased fluid drag force (-35%) under 3-N peak tibial loading; and (4) when subjected to tibial loading in a preliminary in vivo experiment, the Hypo mice did not respond to the anabolic stimuli as the CTL mice did. These findings support the hypothesis that the PCM fibers act as osteocyte's sensing antennae, regulating load-induced cellular stimulations and thus bone's sensitivity and in vivo bone adaptation. If this hypothesis is further confirmed, osteocytic PCM could be new targets to develop osteoporosis treatments by modulating bone's intrinsic sensitivity to mechanical loading and be used to design patient-specific exercise regimens to promote bone formation.
Assuntos
Osso e Ossos/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Osteócitos/metabolismo , Animais , Recuperação de Fluorescência Após Fotodegradação , CamundongosRESUMO
Cellular ribosomal protein L29 (RPL29) is known to be important in protein synthesis, but its function during angiogenesis has never been described before. We have shown previously that mice lacking ß3-integrins support enhanced tumour angiogenesis and, therefore, deletion of endothelial αvß3 can provide a method for discovery of novel regulators of tumour angiogenesis. Here, we describe significant upregulation of RPL29 in ß3-null endothelial cells at both the mRNA and protein level. Ex vivo, we show that VEGF-stimulated microvessel sprouting was reduced significantly in Rpl29-heterozygous and Rpl29-null aortic ring assays compared with wild-type controls. Moreover, we provide in vivo evidence that RPL29 can regulate tumour angiogenesis. Tumour blood vessel density in subcutaneously grown Lewis lung carcinomas was reduced significantly in Rpl29-mutant mice. Additionally, depletion of Rpl29 using RNA interference inhibited VEGF-induced aortic ring sprouting, suggesting that anti-RPL29 strategies might have anti-angiogenic potential. Overall, our results identify that loss or depletion of RPL29 can reduce angiogenesis in vivo and ex vivo.
Assuntos
Neovascularização Fisiológica/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/fisiologia , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Células Endoteliais/metabolismo , Expressão Gênica , Integrina alfaVbeta3/deficiência , Integrina alfaVbeta3/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/deficiência , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
The goal of this study was to use bioengineered injectable microgels to enhance the action of bone morphogenetic protein 2 (BMP2) and stimulate cartilage matrix repair in a reversible animal model of osteoarthritis (OA). A module of perlecan (PlnD1) bearing heparan sulfate (HS) chains was covalently immobilized to hyaluronic acid (HA) microgels for the controlled release of BMP2 in vivo. Articular cartilage damage was induced in mice using a reversible model of experimental OA and was treated by intra-articular injection of PlnD1-HA particles with BMP2 bound to HS. Control injections consisted of BMP2-free PlnD1-HA particles, HA particles, free BMP2 or saline. Knees dissected following these injections were analyzed using histological, immunostaining and gene expression approaches. Our results show that knees treated with PlnD1-HA/BMP2 had lesser OA-like damage compared to control knees. In addition, the PlnD1-HA/BMP2-treated knees had higher mRNA levels encoding for type II collagen, proteoglycans and xylosyltransferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycan chains, relative to control knees (PlnD1-HA). This finding was paralleled by enhanced levels of aggrecan in the articular cartilage of PlnD1-HA/BMP2-treated knees. Additionally, decreases in the mRNA levels encoding for cartilage-degrading enzymes and type X collagen were seen relative to controls. In conclusion, PlnD1-HA microgels constitute a formulation improvement compared to HA for efficient in vivo delivery and stimulation of proteoglycan and cartilage matrix synthesis in mouse articular cartilage. Ultimately, PlnD1-HA/BMP2 may serve as an injectable therapeutic agent for slowing or inhibiting the onset of OA after knee injury.
Assuntos
Proteína Morfogenética Óssea 2/administração & dosagem , Cartilagem/efeitos dos fármacos , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/síntese química , Proteoglicanas de Heparan Sulfato/química , Ácido Hialurônico/química , Osteoartrite/tratamento farmacológico , Animais , Proteína Morfogenética Óssea 2/química , Cartilagem/patologia , Hidrogéis/síntese química , Injeções Intra-Articulares , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite/patologia , Resultado do TratamentoRESUMO
Increased proteoglycan (PG) synthesis is essential for the stimulation of cartilage repair processes that take place during the reversible phase of osteoarthritis (OA). In articular cartilage, xylosyltransferase 1 (Xylt1) is the key enzyme that initiates glycosaminoglycan (GAG) chain synthesis by transferring the first sugar residue to the PG core protein. Biological activity of PGs is closely linked to GAG biosynthesis since their polyanionic nature directly contributes to the proper hydration and elastic properties of the cartilage tissue present at the articular interface. The aim of this study was to investigate whether variations in the level of Xylt1 present in serum can be used to predict OA disease progression. The influence of bone forming activity on the systemic release of this enzyme was addressed by experimentally-inducing OA in mice of two different genetic backgrounds that were previously characterized for their distinct bone metabolism: C57BL/6J (B6, high bone remodelers) or C3H/HeJ (C3H, high bone formers). Serum was collected after medial meniscectomy or sham surgeries in young adult mice of these two strains over a period of 3.5months at which point knee histopathology was assessed. A significant increase in serum Xylt1 levels observed shortly after meniscectomy positively correlated with severe cartilage damage evaluated by histological assessment at later time points in mice of the C3H background. In contrast, no temporal regulation of Xylt1 level was found between meniscectomies and control surgeries in B6 mice, which developed OA at a slower rate. Additionally, longitudinal evaluation of the serum levels of other markers of cartilage/bone metabolism (C1,2C, osteocalcin) did not reveal any association with late knee damages. Our results strongly support the idea that serum Xylt1 has a clinical value for monitoring risk of OA progression in young adults with high bone forming potential. Ultimately, the understanding of posttraumatic mechanisms regulating PG synthesis and their modification by GAG will be essential so that interventions that stimulate cartilage regrowth can be undertaken prior to irreversible destruction of the joint tissue. This article is part of a Special Issue entitled "Osteoarthritis".
Assuntos
Osteoartrite/sangue , Osteoartrite/enzimologia , Osteogênese/fisiologia , Pentosiltransferases/sangue , Ferimentos e Lesões/sangue , Ferimentos e Lesões/enzimologia , Animais , Biomarcadores/sangue , Cartilagem Articular/patologia , Colágeno/metabolismo , Progressão da Doença , Epitopos/imunologia , Masculino , Meniscos Tibiais/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite/etiologia , Osteoartrite/fisiopatologia , Osteocalcina/sangue , Proteólise , Ferimentos e Lesões/complicações , UDP Xilose-Proteína XilosiltransferaseRESUMO
Diamond-Blackfan anemia is a congenital hypoproliferative macrocytic anemia and 5q- syndrome myelodysplastic syndrome is an acquired hypoproliferative macrocytic anemia. Their common erythroid phenotype reflects a shared pathophysiology-haploinsufficiency of one of many ribosomal proteins and somatic deletion of one allele of the ribosomal protein S14 gene, respectively. Although these abnormalities lead to defective ribosome biogenesis, why ribosomal protein hemizygosity results in anemia is not certain. Here, we characterize the hematopoietic phenotype of mice lacking one allele of the ribosomal protein S6 gene. The mice have an erythroid phenotype similar to both Diamond-Blackfan anemia and the 5q- syndrome and lenalidomide therapy improves their anemia.
Assuntos
Anemia Macrocítica/genética , Modelos Animais de Doenças , Eritropoese/genética , Proteína S6 Ribossômica/genética , Agranulocitose/genética , Alelos , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/genética , Anemia Macrocítica/sangue , Anemia Macrocítica/tratamento farmacológico , Anemia Macrocítica/etiologia , Animais , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Índices de Eritrócitos/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Hemoglobinas/análise , Heterozigoto , Lenalidomida , Linfopenia/genética , Camundongos , Camundongos Endogâmicos C57BL , Prednisona/uso terapêutico , Proteínas de Ligação a RNA/genética , Proteína S6 Ribossômica/deficiência , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/genética , Ribossomos/fisiologia , Talidomida/análogos & derivados , Talidomida/uso terapêutico , Trombocitose/genéticaRESUMO
Subcellular localization of mRNAs is regulated by RNA-protein interactions. Here, we show that introduction of a reporter mRNA with the 3'UTR of ß-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of ß-actin mRNA, and introducing GFP with the 3'UTR of ß-actin mRNA depletes axons of endogenous ß-actin and GAP-43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of ß-actin and GAP-43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.
Assuntos
Actinas/genética , Axônios/fisiologia , Glicoproteínas/metabolismo , Regeneração Nervosa/fisiologia , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , Actinas/metabolismo , Animais , Transporte Axonal/genética , Proliferação de Células , Células Cultivadas , Proteína GAP-43/deficiência , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Cones de Crescimento/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Transporte de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismoRESUMO
Axonal mRNA transport is robust in cultured neurons but there has been limited evidence for this in vivo. We have used a genetic approach to test for in vivo axonal transport of reporter mRNAs. We show that ß-actin's 3'-UTR can drive axonal localization of GFP mRNA in mature DRG neurons, but mice with γ-actin's 3'-UTR show no axonal GFP mRNA. Peripheral axotomy triggers transport of the ß-actin 3'-UTR containing transgene mRNA into axons. This GFP-3'-ß-actin mRNA accumulates in injured PNS axons before activation of the transgene promoter peaks in the DRG. Spinal cord injury also increases axonal GFP signals in mice carrying this transgene without any increase in transgene expression in the DRGs. These data show for the first time that the ß-actin 3'-UTR is sufficient for axonal localization in both PNS and CNS neurons in vivo.
Assuntos
Axônios/metabolismo , Gânglios Espinais/citologia , Neurônios/citologia , RNA Mensageiro/metabolismo , Medula Espinal/citologia , Regiões 3' não Traduzidas/genética , Actinas/genética , Actinas/metabolismo , Análise de Variância , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Dendritos/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Microscopia Confocal/métodos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/genética , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Células de Schwann/metabolismo , Neuropatia Ciática/metabolismo , Neuropatia Ciática/patologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Osteocytes project long, slender processes throughout the mineralized matrix of bone, where they connect and communicate with effector cells. The interconnected cellular projections form the functional lacunocanalicular system, allowing fluid to pass for cell-to-cell communication and nutrient and waste exchange. Prevention of mineralization in the pericellular space of the lacunocanalicular pericellular space is crucial for uninhibited interstitial fluid movement. Factors contributing to the ability of the pericellular space of the lacunocanalicular system to remain open and unmineralized are unclear. Immunofluorescence and immunogold localization by transmission electron microscopy demonstrated perlecan/Hspg2 signal localized to the osteocyte lacunocanalicular system of cortical bone, and this proteoglycan was found in the pericellular space of the lacunocanalicular system. In this study we examined osteocyte lacunocanalicular morphology in mice deficient in the large heparan sulfate proteoglycan perlecan/Hspg2 in this tissue. Ultrastructural measurements with electron microscopy of perlecan/Hspg2-deficient mice demonstrated diminished osteocyte canalicular pericellular area, resulting from a reduction in the total canalicular area. Additionally, perlecan/Hspg2-deficient mice showed decreased canalicular density and a reduced number of transverse tethering elements per canaliculus. These data indicated that perlecan/Hspg2 contributed to the integrity of the osteocyte lacunocanalicular system by maintaining the size of the pericellular space, an essential task to promote uninhibited interstitial fluid movement in this mechanosensitive environment. This work thus identified a new barrier function for perlecan/Hspg2 in murine cortical bone.
Assuntos
Osso e Ossos/metabolismo , Osso e Ossos/patologia , Proteoglicanas de Heparan Sulfato/deficiência , Espaço Intracelular/metabolismo , Osteócitos/metabolismo , Animais , Regulação da Expressão Gênica , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Camundongos , Peso Molecular , Osteócitos/ultraestrutura , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Mice lacking HIP/RPL29, a component of the ribosomal machinery, display increased bone fragility. To understand the effect of sub-efficient protein synthetic rates on mineralized tissue quality, we performed dynamic and static histomorphometry and examined the mineral properties of both bones and teeth in HIP/RPL29 knock-out mice using Fourier transform infrared imaging (FTIRI). While loss of HIP/RPL29 consistently reduced total bone size, decreased mineral apposition rates were not significant, indicating that short stature is not primarily due to impaired osteoblast function. Interestingly, our microspectroscopic studies showed that a significant decrease in collagen crosslinking during maturation of HIP/RPL29-null bone precedes an overall enhancement in the relative extent of mineralization of both trabecular and cortical adult bones. This report provides strong genetic evidence that ribosomal insufficiency induces subtle organic matrix deficiencies which elevates calcification. Consistent with the HIP/RPL29-null bone phenotype, HIP/RPL29-deficient teeth also showed reduced geometric properties accompanied with relative increased mineral densities of both dentin and enamel. Increased mineralization associated with enhanced tissue fragility related to imperfection in organic phase microstructure evokes defects seen in matrix protein-related bone and tooth diseases. Thus, HIP/RPL29 mice constitute a new genetic model for studying the contribution of global protein synthesis in the establishment of organic and inorganic phases in mineral tissues.
Assuntos
Osso e Ossos/metabolismo , Minerais/metabolismo , Proteínas Ribossômicas/deficiência , Dente/metabolismo , Animais , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Colágeno/metabolismo , Hipercementose/diagnóstico por imagem , Hipercementose/patologia , Camundongos , Dente Molar/diagnóstico por imagem , Dente Molar/metabolismo , Dente Molar/patologia , Proteínas Ribossômicas/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Dente/diagnóstico por imagem , Dente/patologia , Microtomografia por Raio-XRESUMO
We have developed a biomimetic growth factor delivery system that effectively stimulates the chondrogenic differentiation of the cultured mesenchymal stem cells via the controlled presentation of bone morphogenetic protein-2 (BMP-2). Hyaluronic acid (HA)-based, microscopic hydrogel particles (HGPs) with inherent nanopores and defined functional groups were synthesized by an inverse emulsion polymerization technique. Recombinantly produced, heparan sulfate (HS)-bearing perlecan domain I (PlnDI) was covalently immobilized to HA HGPs (HGP-P(1)) via a flexible poly(ethylene glycol) (PEG) linker through the lysine amines in the core protein of PlnDI employing reductive amination. Compared to HGP without PlnDI, HGP-P(1) exhibited significantly (p<0.05) higher BMP-2 binding capacity and distinctly different BMP-2 release kinetics. Heparitinase treatment increased the amount of BMP-2 released from HGP-P(1), confirming the HS-dependent BMP-2 binding. While BMP-2 was released from HGPs with a distinct burst release followed by a minimal cumulative release, its release from HGP-P(1) exhibited a minimal burst release followed by linear release kinetics over 15 days. The bioactivity of the hydrogel particles was evaluated using micromass culture of multipotent mesenchymal stem cells (MSCs), and the chondrogenic differentiation was assessed by the production of glycosaminoglycan, aggrecan and collagen type II. Our results revealed that BMP-2 loaded HGP-P(1) stimulates more robust cartilage specific ECM production as compared to BMP-2 loaded HGP, due to the ability of HGP-P(1) to potentiate BMP-2 and modulate its release with a near zero-order release kinetics. The PlnDI-conjugated, HA HGPs provide an improved BMP-2 delivery system for stimulating chondrogenic differentiation in vitro, with potential therapeutic application for cartilage repair and regeneration.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/fisiologia , Condrogênese/fisiologia , Sistemas de Liberação de Medicamentos , Proteoglicanas de Heparan Sulfato/metabolismo , Ácido Hialurônico/metabolismo , Hidrogéis , Animais , Biomimética , Proteína Morfogenética Óssea 2/química , Cartilagem/citologia , Cartilagem/fisiologia , Linhagem Celular , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Proteoglicanas de Heparan Sulfato/química , Humanos , Ácido Hialurônico/química , Hidrogéis/síntese química , Hidrogéis/química , Hidrogéis/metabolismo , Camundongos , Modelos Moleculares , Conformação Molecular , Tamanho da PartículaRESUMO
Mice lacking HIP/RPL29, a ribosomal modulator of protein synthesis rate, display a short stature phenotype. To understand the contribution of HIP/RPL29 to bone formation and adult whole bone mechanical properties, we examined both developing and adult bone in our knockout mice. Results indicated that bone shortening in HIP/RPL29-null mice is due to delayed entry of chondro-osteoprogenitors into the cell cycle. Structural properties of adult null bones were analyzed by micro-computed tomography. Interestingly, partial preservation of cortical thickness was observed in null males indicating a gender-specific effect of the genotype on cortical bone parameters. Null males, and to a lower extent null females, displayed increased bone material toughness to counteract decreased bone size. This elevation in a bone material property was associated with increased bone mineral density only in null males. Neither male nor female null animals could withstand the same maximum load as gender-matched controls in three-point bending tests, and smaller post-yield displacements (and thus increased bone brittleness) were found for null animals. These results suggest that HIP/RPL29-deficient mice exhibit increased bone fragility due to altered matrix protein synthesis rates as a consequence of ribosomal insufficiency. Thus, sub-efficient protein translation increased fracture risk in HIP/RPL29-null animals. Taken together, these studies provide strong genetic evidence that the ability to regulate and amplify protein synthesis rates, including those proteins that regulate the cell cycle entry during skeletal development, are important determinants for establishment of normal bone mass and quality.
Assuntos
Osso e Ossos/patologia , Osteogênese , Proteínas Ribossômicas/genética , Animais , Fenômenos Biomecânicos , Osso e Ossos/metabolismo , Proliferação de Células , Feminino , Consolidação da Fratura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Proteínas de Ligação a RNA , Proteínas Ribossômicas/fisiologia , Ribossomos/metabolismo , Tomografia Computadorizada por Raios XRESUMO
Complex interactions occur among embryonic, placental and maternal tissues during embryo implantation. Many of these interactions are controlled by growth factors, extracellular matrix and cell surface components that share the ability to bind heparan sulfate (HS) polysaccharides. HS is carried by several classes of cell surface and secreted proteins called HS proteoglycan that are expressed in restricted patterns during implantation and placentation. This review will discuss the expression of HS proteoglycans and various HS binding growth factors as well as extracellular matrix components and HS-modifying enzymes that can release HS-bound proteins in the context of implantation and placentation.