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1.
FASEB J ; 34(1): 720-734, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31914611

RESUMO

Confusion persists over pathogenesis of spondylolysis. To confirm pathogenicity of the previously identified causative mutation of spondylolysis and investigate the genetic etiology, we generate a new mouse line harboring D673V mutation in the Slc26a2 gene. D673V mutation induces delayed endochondral ossification characterized by transiently reduced chondrocyte proliferation in mice at the early postnatal stage. Adult D673V homozygotes exhibit dysplastic isthmus and reduced bone volume of the dorsal vertebra resembling the detached vertebral bony structure when spondylolysis occurs, including the postzygopophysis, vertebral arch, and spinous process, which causes biomechanical alterations around the isthmic region of L4-5 vertebrae indicated by finite element analysis. Consistently, partial ablation of Slc26a2 in vertebral skeletal cells using Col1a1-Cre; Slc26a2 fl/fl mouse line recapitulates a similar but worsened vertebral phenotype featured by lamellar isthmus. In addition, when reaching late adulthood, D673V homozygotes develop an evident bone-loss phenotype and show impaired osteogenesis. These findings support a multifactorial etiology, involving congenitally predisposed isthmic conditions, altered biomechanics, and age-dependent bone loss, which leads to SLC26A2-related spondylolysis.

2.
Cell Death Dis ; 10(12): 887, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31767864

RESUMO

Chordoma is a malignant primary osseous spinal tumor with pronounced chemoresistance. However, the mechanisms of how chordoma cells develop chemoresistance are still not fully understood. Cytokeratin 8 (KRT8) is a molecular marker of notochordal cells, from which chordoma cells were believed to be originated. In this study, we showed that either doxorubicin or irinotecan promoted KRT8 expression in both CM319 and UCH1 cell lines, accompanied by an increased unfolded protein response and autophagy activity. Then, siRNA-mediated knockdown of KRT8 chemosensitized chordoma cells in vitro. Mechanistic studies showed that knockdown of KRT8 followed by chemotherapy aggravated endoplasmic reticulum stress through PERK/eIF2α arm of unfolded protein response and blocked late-stage autophagy. Moreover, suppression of the PERK/eIF2α arm of unfolded protein response using PERK inhibitor GSK2606414 partially rescued the apoptotic chordoma cells but did not reverse the blockage of the autophagy flux. Finally, tumor xenograft model further confirmed the chemosensitizing effects of siKRT8. This study represents the first systematic investigation into the role of KRT8 in chemoresistance of chordoma and our results highlight a possible strategy of targeting KRT8 to overcome chordoma chemoresistance.

4.
Theranostics ; 9(8): 2252-2267, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31149042

RESUMO

Progerin, a truncated unprocessed lamin A protein, causes tissue aging and degeneration. In this study we explored the role of progerin in the pathogenesis of intervertebral disc degeneration (IDD). We also examined the effect of sulforaphane (SFN) on progerin accumulation and mitochondrial dysfunction in IDD. Methods: The role of progerin in IDD was explored using human nucleus pulposus (NP) tissues, rat NP cells, and Lmna G609G knock-in mice. Immunostaining, X-ray imaging, and Western blotting were performed to assess the phenotypes of intervertebral discs. Alterations in senescence and apoptosis were evaluated by SA-ß-galactosidase, immunofluorescence, flow cytometry, and TUNEL assays. Mitochondrial function was investigated by JC-1 staining, transmission electron microscopy, and determination of the level of ATP and the activities of mitochondrial enzymes. Results: The progerin level was elevated in degenerated human NP tissues. Lmna G609G/G609G mice displayed IDD, as evidenced by increased matrix metalloproteinase-13 expression and decreased collagen II and aggrecan expression and disc height. Furthermore, progerin overexpression in rat NP cells induced mitochondrial dysfunction (decreased ATP synthesis, mitochondrial membrane potential, and activities of mitochondrial complex enzymes), morphologic abnormalities, and disrupted mitochondrial dynamic (abnormal expression of proteins involved in fission and fusion), resulting in apoptosis and senescence. SFN ameliorated the progerin-induced aging defects and mitochondrial dysfunction in NP cells and IDD in Lmna G609G/G609G mice. Conclusions: Progerin is involved in the pathogenesis of IDD. Also, SFN alleviates progerin­induced IDD, which is associated with amelioration of aging defects and mitochondrial dysfunction. Thus, SFN shows promise for the treatment of IDD.

5.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(5): 385-392, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31223106

RESUMO

Objective To investigate the effect of low-level laser on the polarization and secretory phenotype of primary cultured M1 bone marrow-derived macrophages (BMDMs) in neuronal axons of dorsal root ganglion (DRG). Methods BMDMs were isolated and cultured, and lipopolysaccharide (LPS) combined with IFN-γ were used to induce M1 phenotype polarization of BMDMs, and then F4/80 and CD16/32 expression was detected by flow cytometry. The mature M1 type BMDMs were randomly divided into low-level laser group and control group. The laser exposure group was subjected to the laser treatments of 0.4J, 4J and 10J, and no laser was used in the control group. After 24 hours of laser exposure, the mRNA level of inducible nitric oxide synthase (iNOS) of M1 type BMDMs was detected by reverse transcription PCR, and the protein level of iNOS was detected by Western blot analysis. The levels of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the supernatant of cultured cells were tested by ELISA. DRG neurons were cultured with the supernatant fluid of M1 type BMDMs, and immunofluorescence cytochemistry was employed to detect neuronal nuclei (NeuN) and ß-tubulin III expression of DRG neurons for determining the influence on the growth of DRG neuronal axons. Results Compared with the control group, the mRNA level of iNOS in M1 type BMDMs dramatically increased after 24 hours of low-level laser exposure. Among the 3 groups with different energy levels, the decrease of iNOS mRNA level was the most obvious in the group with 4J laser exposure. The protein levels of iNOS in the groups with 0.4J- and 4J- laser exposure were reduced more significantly than that in the control group, and the down-regulation was more prominent in the group with 4J laser exposure than that with 0.4J laser exposure. In addition, the secretion of TNF-α from M1 type BMDMs was reduced more significantly in the groups of 4J- and 10J- laser exposure than that in the control group. With regard to IL-1ß, its secretion was inhibited in all the laser exposure groups compared with the control group, and the suppression was more prominent in the groups of 0.4J- and 4J-laser exposure than that in the group of 10J-laser exposure. Furthermore, 4J-laser exposure significantly potentiated the secretion of BDNF and NGF in M1 type BMDMs compared with the control group. Moreover, co-culture with the supernatants from 4J- and 10J-laser exposure groups could significantly promote the growth of axons of DRG neurons. Conclusion Low-level laser exposure can inhibit the polarization of M1 type BMDM and the secretion of pro-inflammatory factor including TNF-α and IL-1ß. Besides, low-level laser exposure could contribute to the secretion of neurotrophic factors including BDNF and NGF, and promote the growth of DRG axon, and this effect is dose-dependent.


Assuntos
Axônios/fisiologia , Polaridade Celular , Gânglios Espinais/crescimento & desenvolvimento , Lasers , Macrófagos/citologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Humanos , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Macrófagos/efeitos da radiação , Fator de Crescimento Neural/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
6.
Biotechniques ; 67(1): 11-15, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31124698

RESUMO

A large number of animal experiments and clinical trials have confirmed that electrical stimulation can accelerate the growth of axons and recovery of motor function, all of which are inseparable from the formation of myelin. Therefore, establishment of a suitable electrical stimulation platform to study the effects of electrical stimulation on the myelin process of dorsal root ganglia and Schwann cells is of great significance for understanding the recovery of electrical stimulation. We designed a simple conductive glass cell culture system to overcome the shortcomings of direct contact of the electrode with the culture solution, and the number of culture chambers can be selected based on the purpose of the experiment in order to reduce experimental time and cost.

7.
Stem Cells Int ; 2019: 5037578, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31089329

RESUMO

Mesenchymal progenitors within bone marrow have multiple differentiation potential and play an essential role in the maintenance of adult skeleton homeostasis. Mesenchymal progenitors located in bone regions other than the bone marrow also display bone-forming properties. However, owing to the differences in each distinct microenvironment, the mesenchymal characteristics of skeletal progenitor cells within different regions of long bones may show some differences. In order to clearly elucidate these differences, we performed a comparative study on mesenchymal progenitors from different regions of long bones. Here, we isolated mesenchymal progenitors from the periosteum, endosteum, and bone marrow of rat long bones. The three groups exhibited similar cellular morphologies and expressed the typical surface markers associated with mesenchymal stem cells. Interestingly, after cell proliferation assays and bidirectional differentiation analysis, periosteal mesenchymal progenitors showed a higher proliferative ability and adipogenic differentiation potential. In contrast, endosteal mesenchymal progenitors were more prone to osteogenic differentiation. Using in vitro osteoclast culture systems, conditioned media from different mesenchymal progenitor cultures were used to induce osteoclastic differentiation. Osteoclast formation was found to be significantly promoted by the secretion of RANKL and IL-6 by endosteal progenitors. Overall, our results provide strong evidence for the importance of selecting the appropriate source of skeletal progenitors for applications in future skeleton regeneration therapies.

8.
FASEB J ; 33(7): 8600-8613, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30995417

RESUMO

Delivery of multiple neurotrophic factors (NTFs), especially with time-restricted release kinetics, holds great potential for nerve repair. In this study, we utilized the tetracycline-regulatable Tet-On 3G system to control the expression of c-Jun, which is a common regulator of multiple NTFs in Schwann cells (SCs). In vitro, Tet-On/c-Jun-modified SCs showed a tightly controllable secretion of multiple NTFs, including glial cell line-derived NTF, nerve growth factor, brain-derived NTF, and artemin, by the addition or removal of doxycycline (Dox). When Tet-On/c-Jun-transduced SCs were grafted in vivo, the expression of NTFs could also be regulated by oral administration or removal of Dox. Fluoro-Gold retrograde tracing results indicated that a biphasic NTF expression scheme (Dox+3/-9, NTFs were up-regulated for 3 wk and declined to physiologic levels for another 9 wk) achieved more axonal regeneration than continuous up-regulation of NTFs (Dox+12) or no NTF induction (Dox-12). More importantly, the Dox+3/-9-group animals showed much better functional recovery than the animals in the Dox+12 and Dox-12 groups. Our findings, for the first time, demonstrated drug-controllable expression of multiple NTFs in nerve repair cells both in vitro and in vivo. These findings provide new hope for developing an optimal therapeutic alternative for nerve repair through the time-restricted release of multiple NTFs using Tet-On/c-Jun-modified SCs.-Huang, L., Xia, B., Shi, X., Gao, J., Yang, Y., Xu, F., Qi, F., Liang, C., Huang, J., Luo, Z. Time-restricted release of multiple neurotrophic factors promotes axonal regeneration and functional recovery after peripheral nerve injury.

9.
J Clin Invest ; 129(6): 2578-2594, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30946695

RESUMO

The periosteum, a thin tissue that covers almost the entire bone surface, accounts for more than 80% of human bone mass and is essential for bone regeneration. Its osteogenic and bone regenerative abilities are well studied, but much is unknown about the periosteum. In this study, we found that macrophage-lineage cells recruit periosteum-derived cells (PDCs) for cortical bone formation. Knockout of colony stimulating factor-1 eliminated macrophage-lineage cells and resulted in loss of PDCs with impaired periosteal bone formation. Moreover, macrophage-lineage TRAP+ cells induced transcriptional expression of periostin and recruitment of PDCs to the periosteal surface through secretion of platelet-derived growth factor-BB (PDGF-BB), where the recruited PDCs underwent osteoblast differentiation coupled with type H vessel formation. We also found that subsets of Nestin+ and LepR+ PDCs possess multipotent and self-renewal abilities and contribute to cortical bone formation. Nestin+ PDCs are found primarily during bone development, whereas LepR+ PDCs are essential for bone homeostasis in adult mice. Importantly, conditional knockout of Pdgfrß (platelet-derived growth factor receptor beta) in LepR+ cells impaired periosteal bone formation and regeneration. These findings uncover the essential role of periosteal macrophage-lineage cells in regulating periosteum homeostasis and regeneration.

10.
Med Hypotheses ; 125: 5-7, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30902151

RESUMO

Osteoporosis and intervertebral disc degeneration (IDD) are both age-related diseases of the musculoskeletal system. With the average life expectancy longer than ever, the morbidity caused by these two diseases is increasing. Nowadays, treatment strategies for osteoporosis are mainly aimed at increasing the mineral density of the bone. Some of these therapies, including vitamin D, calcium, bisphosphonates, Wnt signal activators and parathyroid hormone regulators, have been suggested to be capable of causing calcification of the cartilage endplate in the intervertebral disc. This alteration could block nutrient and oxygen transportation to the center part of the disc, thus lead to intervertebral disc degeneration. Consequently, we hypothesize that osteoporosis therapies might be a potential risk for IDD. This assumption indicates that we should take the alterations of the cartilage endplate into consideration in further osteoporosis treatment to avoid IDD in the patient.


Assuntos
Conservadores da Densidade Óssea/efeitos adversos , Cartilagem/patologia , Degeneração do Disco Intervertebral/patologia , Osteoporose/tratamento farmacológico , Anel Fibroso/fisiologia , Densidade Óssea , Calcinose , Cálcio/metabolismo , Difosfonatos/efeitos adversos , Humanos , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/prevenção & controle , Núcleo Pulposo/fisiologia , Osteoporose/complicações , Hormônio Paratireóideo/metabolismo , Risco , Vitamina D/metabolismo , Proteínas Wnt/metabolismo
11.
EBioMedicine ; 40: 695-709, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30685387

RESUMO

BACKGROUND: Mutations in the SLC26A2 gene cause a spectrum of currently incurable human chondrodysplasias. However, genotype-phenotype relationships of SLC26A2-deficient chondrodysplasias are still perplexing and thus stunt therapeutic development. METHODS: To investigate the causative role of SLC26A2 deficiency in chondrodysplasias and confirm its skeleton-specific pathology, we generated and analyzed slc26a2-/- and Col2a1-Cre; slc26a2fl/fl mice. The therapeutic effect of NVP-BGJ398, an FGFR inhibitor, was tested with both explant cultures and timed pregnant females. FINDINGS: Two lethal forms of human SLC26A2-related chondrodysplasias, achondrogenesis type IB (ACG1B) and atelosteogenesis type II (AO2), are phenocopied by slc26a2-/- mice. Unexpectedly, slc26a2-/- chondrocytes are defective for collagen secretion, exhibiting intracellular retention and compromised extracellular deposition of ColII and ColIX. As a consequence, the ATF6 arm of the unfolded protein response (UPR) is preferentially triggered to overactivate FGFR3 signaling by inducing excessive FGFR3 in slc26a2-/- chondrocytes. Consistently, suppressing FGFR3 signaling by blocking either FGFR3 or phosphorylation of the downstream effector favors the recovery of slc26a2-/- cartilage cultures from impaired growth and unbalanced cell proliferation and apoptosis. Moreover, administration of an FGFR inhibitor to pregnant females shows therapeutic effects on pathological features in slc26a2-/- newborns. Finally, we confirm the skeleton-specific lethality and pathology of global SLC26A2 deletion through analyzing the Col2a1-Cre; slc26a2fl/fl mouse line. INTERPRETATION: Our study unveils a previously unrecognized pathogenic mechanism underlying ACG1B and AO2, and supports suppression of FGFR3 signaling as a promising therapeutic approach for SLC26A2-related chondrodysplasias. FUND: This work was supported by National Natural Science Foundation of China (81871743, 81730065 and 81772377).


Assuntos
Acondroplasia/genética , Acondroplasia/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Transportadores de Sulfato/deficiência , Resposta a Proteínas não Dobradas , Acondroplasia/patologia , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Cartilagem/metabolismo , Cartilagem/patologia , Diferenciação Celular/genética , Condrócitos/citologia , Condrócitos/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Regulação da Expressão Gênica no Desenvolvimento , Lâmina de Crescimento/embriologia , Lâmina de Crescimento/patologia , Humanos , Camundongos , Camundongos Knockout , Morfogênese/genética , Mutação , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Fenótipo , Resposta a Proteínas não Dobradas/genética
12.
Medicine (Baltimore) ; 97(42): e12691, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30334952

RESUMO

BACKGROUND: Alendronate has been used to prevent or treat glucocorticoid-induced osteoporosis (GIO), data regarding its efficacy are inconsistent. We conducted the current systematic review and meta-analysis to evaluate both efficacy and safety of alendronate in the treatment of GIO. METHODS: PubMed, Embase, the Cochrane Controlled Trials Registry, and the China Academic Journal Network Publishing Databases were searched up through March 1, 2018. Randomized controlled trials (RCTs) involving patients which received alendronate treatment were included. Outcome measures were bone mineral density (BMD) changes, bone fractures, and adverse reactions. Data from the individual studies were pooled using random or fixed effect models based on heterogeneity. Effect size was reported as standardized mean differences (SMD) for continuous outcomes and pooled odds ratios (OR) for dichotomous outcomes, with 95% confidence interval (CI). RESULTS: Overall, 10 studies involving 1002 patients were included in the present investigation. Alendronate treatment significantly increased BMD of the lumbar spine and femoral neck during 6 to 24 months. These beneficial effects were apparent at 12 months after treatment for the lumbar spine but not the femoral neck BMD. Alendronate treatment did not significantly change fracture risk nor induce significant differences in adverse gastrointestinal effects. CONCLUSION: Alendronate significantly increases BMD of the lumbar spine and femoral neck in patients with GIO, but does not appear to reduce the risk of fractures. As relatively insufficient data regarding the GIO fracture incidence has been reported, more RCTs need to be carried out to determine the efficacy of alendronate in the prevention of GIO fracture.


Assuntos
Alendronato/uso terapêutico , Conservadores da Densidade Óssea/uso terapêutico , Glucocorticoides/efeitos adversos , Osteoporose/induzido quimicamente , Alendronato/efeitos adversos , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/efeitos adversos , Feminino , Humanos , Masculino , Fraturas por Osteoporose/epidemiologia , Fraturas por Osteoporose/prevenção & controle , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do Tratamento
13.
Injury ; 49(8): 1477-1484, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29921534

RESUMO

Longitudinally oriented microstructures are essential for a nerve scaffold to promote the significant regeneration of injured peripheral axons across nerve gaps. In the current study, we present a novel nerve-guiding collagen-chitosan (CCH) scaffold that facilitated the repair of 30 mm-long sciatic nerve defects in beagles. The CCH scaffolds were observed with a scanning electron microscope. Eighteen beagles were equally divided into CCH group, autograft group and non-graft group. The posture and gait of each dog was recorded at 12 and 24 weeks after surgery. Electrophysiological tests, Fluoro-Gold retrograde tracing test, Histological assessment of gastrocnemius and immunofluorescent staining of nerve regeneration were performed. Our investigation of regenerated sciatic nerves indicated that a CCH scaffold strongly supported directed axon regeneration in a manner similar to that achieved by autologous nerve transplantation. In vivo animal experiments showed that the CCH scaffold achieved nerve regeneration and functional recovery equivalent to that achieved by an autograft but without requiring the exogenous delivery of regenerative agents or cell transplantation. We conclude that CCH nerve guides show great promise as a method for repairing peripheral nerve defects.


Assuntos
Regeneração Nervosa/fisiologia , Nervo Isquiático/fisiopatologia , Neuropatia Ciática/patologia , Animais , Materiais Biocompatíveis/química , Quitosana/química , Cães , Masculino , Modelos Animais , Próteses e Implantes , Recuperação de Função Fisiológica , Neuropatia Ciática/cirurgia , Engenharia Tecidual
14.
Sci Rep ; 8(1): 3867, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29497100

RESUMO

A fracture is the most dangerous complication of osteoporosis in patients because the associated disability and mortality rates are high. Osteoporosis impairs fracture healing and prognosis, but how intramembranous ossification (IO) or endochondral ossification (EO) during fracture healing are affected and whether these two kinds of ossification are different between glucocorticoid-induced osteoporosis (GIOP) and estrogen deficiency-induced osteoporosis (EDOP) are poorly understood. In this study, we established two bone repair models that exhibited repair via IO or EO and compared the pathological progress of each under GIOP and EDOP. In the cortical drill-hole model, which is repaired through IO, osteogenic differentiation was more seriously impaired in EDOP at the early stage than in GIOP. In the periosteum scratch model, in which EO is replicated, chondrocyte hypertrophy progression was delayed in both GIOP and EDOP. The in vitro results were consistent with the in vivo results. Our study is the first to establish bone repair models in which IO and EO occur separately, and the results strongly describe the differences in bone repair between GIOP and EDOP.


Assuntos
Consolidação da Fratura/fisiologia , Osteogênese/fisiologia , Osteoporose/patologia , Animais , Diferenciação Celular , Condrogênese/fisiologia , Modelos Animais de Doenças , Estrogênios/deficiência , Estrogênios/metabolismo , Feminino , Fraturas Ósseas/patologia , Glucocorticoides/efeitos adversos , Glucocorticoides/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Osteoporose/metabolismo , Periósteo/patologia
15.
Aging Cell ; 17(3): e12741, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29488314

RESUMO

Aging drives the accumulation of senescent cells (SnCs) including stem/progenitor cells in bone marrow, which contributes to aging-related bone degenerative pathologies. Local elimination of SnCs has been shown as potential treatment for degenerative diseases. As LepR+ mesenchymal stem/progenitor cells (MSPCs) in bone marrow are the major population for forming bone/cartilage and maintaining HSCs niche, whether local elimination of senescent LepR+ MSPCs delays aging-related pathologies and improves local microenvironment need to be well defined. In this study, we performed local delivery of tetramethylpyrazine (TMP) in bone marrow of aging mice, which previously showed to be used for the prevention and treatment of glucocorticoid-induced osteoporosis (GIOP). We found the increased accumulation of senescent LepR+ MSPCs in bone marrow of aging mice, and TMP significantly inhibited the cell senescent phenotype via modulating Ezh2-H3k27me3. Most importantly, local delivery of TMP improved bone marrow microenvironment and maintained bone homeostasis in aging mice by increasing metabolic and anti-inflammatory responses, inducing H-type vessel formation, and maintaining HSCs niche. These findings provide evidence on the mechanisms, characteristics and functions of local elimination of SnCs in bone marrow, as well as the use of TMP as a potential treatment to ameliorate human age-related skeletal diseases and to promote healthy lifespan.


Assuntos
Anti-Inflamatórios/uso terapêutico , Células da Medula Óssea/metabolismo , Células-Tronco Mesenquimais/metabolismo , Pirazinas/uso terapêutico , Vasodilatadores/uso terapêutico , Envelhecimento , Animais , Anti-Inflamatórios/farmacologia , Senescência Celular , Camundongos , Pirazinas/farmacologia , Vasodilatadores/farmacologia
16.
Acta Biomater ; 68: 223-236, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29274478

RESUMO

Scaffolds with inner fillers that convey directional guidance cues represent promising candidates for nerve repair. However, incorrect positioning or non-uniform distribution of intraluminal fillers might result in regeneration failure. In addition, proper porosity (to enhance nutrient and oxygen exchange but prevent fibroblast infiltration) and mechanical properties (to ensure fixation and to protect regenerating axons from compression) of the outer sheath are also highly important for constructing advanced nerve scaffolds. In this study, we constructed a compound scaffold using a stage-wise strategy, including directionally freezing orientated collagen-chitosan (O-CCH) filler, electrospinning poly(ε-caprolactone) (PCL) sheaths and assembling O-CCH/PCL scaffolds. Based on scanning electron microscopy (SEM) and mechanical tests, a blend of collagen/chitosan (1:1) was selected for filler fabrication, and a wall thickness of 400 µm was selected for PCL sheath production. SEM and three-dimensional (3D) reconstruction further revealed that the O-CCH filler exhibited a uniform, longitudinally oriented microstructure (over 85% of pores were 20-50 µm in diameter). The electrospun PCL porous sheath with pore sizes of 6.5 ±â€¯3.3 µm prevented fibroblast invasion. The PCL sheath exhibited comparable mechanical properties to commercially available nerve conduits, and the O-CCH filler showed a physiologically relevant substrate stiffness of 2.0 ±â€¯0.4 kPa. The differential degradation time of the filler and sheath allows the O-CCH/PCL scaffold to protect regenerating axons from compression stress while providing enough space for regenerating nerves. In vitro and in vivo studies indicated that the O-CCH/PCL scaffolds could promote axonal regeneration and Schwann cell migration. More importantly, functional results indicated that the CCH/PCL compound scaffold induced comparable functional recovery to that of the autograft group at the end of the study. Our findings demonstrated that the O-CCH/PCL scaffold with uniform longitudinal guidance filler and a porous sheath exhibits favorable properties for clinical use and promotes nerve regeneration and functional recovery. The O-CCH/PCL scaffold provides a promising new path for developing an optimal therapeutic alternative for peripheral nerve reconstruction. STATEMENT OF SIGNIFICANCE: Scaffolds with inner fillers displaying directional guidance cues represent a promising candidate for nerve repair. However, further clinical translation should pay attention to the problem of non-uniform distribution of inner fillers, the porosity and mechanical properties of the outer sheath and the morphological design facilitating operation. In this study, a stage-wise fabrication strategy was used, which made it possible to develop an O-CCH/PCL compound scaffold with a uniform longitudinally oriented inner filler and a porous outer sheath. The uniform distribution of the pores in the O-CCH/PCL scaffold provides a solution to resolve the problem of non-uniform distribution of inner fillers, which impede the clinical translation of scaffolds with longitudinal microstructured fillers, especially for aligned-fiber-based scaffolds. In vitro and in vivo studies indicated that the O-CCH/PCL scaffolds could provide topographical cues for axonal regeneration and SC migration, which were not found for random scaffolds (with random microstructure resemble sponge-based scaffolds). The electrospun porous PCL sheath of the O-CCH/PCL scaffold not only prevented fibroblast infiltration, but also satisfied the mechanical requirements for clinical use, paving the way for clinical translation. The differential degradation time of the O-CCH filler and the PCL sheath makes O-CCH/PCL scaffold able to provide long protection for regenerating axons from compression stress, but enough space for regenerating nerve. These findings highlight the possibility of developing an optimal therapeutic alternative for nerve defects using the O-CCH/PCL scaffold.


Assuntos
Regeneração Tecidual Guiada/métodos , Regeneração Nervosa , Nervos Periféricos/fisiologia , Tecidos Suporte/química , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Movimento Celular , Quitosana/química , Colágeno/química , Fibroblastos/citologia , Atrofia Muscular/patologia , Poliésteres/química , Porosidade , Células de Schwann/citologia , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , Fatores de Tempo
17.
J Tissue Eng Regen Med ; 12(1): e177-e189, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27592228

RESUMO

Local hypoxia in the early stages of peripheral nerve injury is a challenge for axonal regeneration. To address this issue, perfluorotributylamine (PFTBA)-based oxygen carrying fibrin hydrogel was prepared and injected into Schwann cell (SC)-seeded collagen-chitosan conduits to increase oxygen supply to SCs within the conduits. The conduit containing PFTBA-SC gel was then applied to bridge a 15-mm sciatic nerve defect in rats. It was observed that most of the GFP-labeled SCs initially seeded in the PFTBA hydrogel remained alive for approximately 28 days after their in vivo implantation. The number of SCs was significantly higher in the PFTBA-SC scaffold than that in the SC scaffold without PFTBA. In addition, nerve regeneration and functional recovery were examined after nerve injury repair. We found that the PFTBA-SC scaffold was capable of promoting axonal regeneration and remyelination of the regenerated axons. Further studies showed the PFTBA-SC scaffold was able to accelerate the recovery of motor and sensory function of the regenerating nerves. Electrophysiological analysis showed area under the curve of compound muscle action potential and nerve conduction velocity were also improved, and gastrocnemius muscle atrophy was partially reversed by PFTBA-SC scaffold. Furthermore, microvessel density analysis showed PFTBA-SC composites were beneficial for microvascular growth, which provided sustained oxygen for regenerating nerve in the later stages of nerve regeneration. In conclusion, enhanced survival of SCs by PFTBA is capable of promoting sciatic nerve regeneration and functional recovery, which provides a new avenue for achieving better functional recovery in the treatment of peripheral nerve injuries. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Regeneração Nervosa/efeitos dos fármacos , Oxigênio/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Células de Schwann/citologia , Nervo Isquiático/fisiologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Fluorcarbonetos/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Masculino , Microvasos/efeitos dos fármacos , Microvasos/crescimento & desenvolvimento , Ratos Sprague-Dawley , Células de Schwann/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos
18.
Spine (Phila Pa 1976) ; 43(7): E381-E389, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28767622

RESUMO

STUDY DESIGN: Given the results of previous in vitro investigations of the expression patterns of adiponectin and its receptors in healthy and degenerated intervertebral discs (IVDs), we studied the effects of adiponectin on tumor necrosis factor-alpha (TNF-α) production in degenerated nucleus pulposus (NP) cells and analyzed the association between adiponectin levels in IVD tissues and IVD Pfirrmann grades. OBJECTIVE: The aim of this study was to investigate the potential role of adiponectin in the pathogenesis of IVD degeneration. SUMMARY OF BACKGROUND DATA: Adiponectin has been reported to be involved in physiologic and pathologic processes associated with bone and cartilage diseases. However, the expression profiles of adiponectin and its receptors in human IVD tissues and the function of adiponectin in the pathogenesis of IVD degeneration remain unknown. METHODS: Real-time polymerase chain reaction, immunohistochemistry, and western blotting were performed to examine the expression levels of adiponectin, adiponectin receptors, and TNF-α in IVD tissues and isolated NP cells. The effects of adiponectin on TNF-α production in degenerated NP cells were detected by enzyme linked immunosorbent assay. RESULTS: Adiponectin expression levels were downregulated, while adiponectin receptor 1 (adipoR1) and adipoR2 expression levels were upregulated in degenerated IVD tissues and degenerated NP cells compared with those in healthy IVD tissues and healthy NP cells. Moreover, we confirmed that TNF-α production by degenerated NP cells was downregulated by adiponectin administration in a dose- and time-dependent manner. Furthermore, our data showed that adiponectin levels in degenerated IVD tissues were inversely correlated with IVD Pfirrmann grades. CONCLUSION: These results indicated that adiponectin may play an anti-inflammatory role with respect to the maintenance of IVD homeostasis by downregulating TNF-α production. LEVEL OF EVIDENCE: N/A.


Assuntos
Adiponectina/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Regulação para Baixo , Feminino , Humanos , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Masculino , Pessoa de Meia-Idade , Núcleo Pulposo/metabolismo , Receptores de Adiponectina/metabolismo , Regulação para Cima
19.
Int J Nanomedicine ; 12: 7815-7832, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29123395

RESUMO

Peripheral nerve repair is still challenging for surgeons. Autologous nerve transplantation is the acknowledged therapy; however, its application is limited by the scarcity of available donor nerves, donor area morbidity, and neuroma formation. Biomaterials for engineering artificial nerves, particularly materials combined with supportive cells, display remarkable promising prospects. Schwann cells (SCs) are the absorbing seeding cells in peripheral nerve engineering repair; however, the attenuated biologic activity restricts their application. In this study, a magnetic nanocomposite scaffold fabricated from magnetic nanoparticles and a biodegradable chitosan-glycerophosphate polymer was made. Its structure was evaluated and characterized. The combined effects of magnetic scaffold (MG) with an applied magnetic field (MF) on the viability of SCs and peripheral nerve injury repair were investigated. The magnetic nanocomposite scaffold showed tunable magnetization and degradation rate. The MGs synergized with the applied MF to enhance the viability of SCs after transplantation. Furthermore, nerve regeneration and functional recovery were promoted by the synergism of SCs-loaded MGs and MF. Based on the current findings, the combined application of MGs and SCs with applied MF is a promising therapy for the engineering of peripheral nerve regeneration.


Assuntos
Nanocompostos/química , Regeneração Nervosa/fisiologia , Células de Schwann/fisiologia , Nervo Isquiático/fisiologia , Animais , Quitosana/química , Glicerofosfatos/química , Campos Magnéticos , Masculino , Polímeros/metabolismo , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Nervo Isquiático/lesões , Tecidos Suporte , Transplante Autólogo
20.
Mol Med Rep ; 16(6): 8307-8314, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28983593

RESUMO

Long­term glucocorticoid therapy results in various side effects, including a high incidence of glucocorticoid­induced osteoporosis (GIOP), which is the most common form of secondary osteoporosis. Excess glucocorticoids reduce the viability of bone marrow­derived mesenchymal stem cells (BMSCs) and prolong osteoclast survival. These two types of cell are essential in the balance between bone formation and resorption. Tetramethylpyrazine (TMP), the pharmacologically active component extracted from Chuanxiong, has been reported to protect BMSCs from glucocorticoid­induced apoptosis. In the present study, the protective effects of TMP on BMSC differentiation and osteoclasts maturation in GIOP were investigated in vivo and in vitro. The immunostaining of osterix (OSX) and tartrate­resistant acid phosphatase (TRAP) staining indicated that TMP promoted osteogenesis and inhibited osteoclastogenesis in a rat model of GIOP. Treatment with 10­6 M dexamethasone (Dex) significantly inhibited BMSC differentiation and increased TRAP­positive cells in vitro. However, different concentrations of TMP (50, 100 and 200 µM) ameliorated the negative effects of Dex by promoting the activity of alkaline phosphatase (ALP) and the calcium mineralization of BMSCs following osteogenic induction, which increased the expression levels of osteogenic genes, including ALP, collagen type I α1, osteocalcin and OSX, and decreased osteoclastogenesis­related genes, including TRAP, nuclear factor of T­cells cytoplasmic 1 and cathepsin K. In addition, it was found that the inhibition of receptor activator of nuclear factor­κB ligand and intereleukin­6 in BMSCs may be a possible mechanism for the protective effects of TMP against glucocorticoid­induced osteoclastogenesis. These results are the first, to the best of our knowledge, to demonstrate that TMP promotes BMSC differentiation and inhibits osteoclastogenesis to ameliorate bone mass change in GIOP.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteogênese/efeitos dos fármacos , Pirazinas/farmacologia , Animais , Reabsorção Óssea , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Feminino , Interleucina-6/genética , Interleucina-6/metabolismo , Osteoclastos/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Ratos , Tomografia Computadorizada por Raios X
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