Annulus fibrosus(AF) tissue engineering has recently received increasing attention as a treatment for intervertebral disc(IVD) degeneration; however, such engineering remains challenging because of the remarkable comp...Annulus fibrosus(AF) tissue engineering has recently received increasing attention as a treatment for intervertebral disc(IVD) degeneration; however, such engineering remains challenging because of the remarkable complexity of AF tissue. In order to engineer a functional AF replacement, the fabrication of cell-scaffold constructs that mimic the cellular, biochemical and structural features of native AF tissue is critical.In this study, we fabricated aligned fibrous polyurethane scaffolds using an electrospinning technique and used them for culturing AF-derived stem/progenitor cells(AFSCs). Random fibrous scaffolds, also prepared via electrospinning, were used as a control. We compared the morphology, proliferation, gene expression and matrix production of AFSCs on aligned scaffolds and random scaffolds. There was no apparent difference in the attachment or proliferation of cells cultured on aligned scaffolds and random scaffolds. However, compared to cells on random scaffolds, the AFSCs on aligned scaffolds were more elongated and better aligned, and they exhibited higher gene expression and matrix production of collagen-I and aggrecan. The gene expression and protein production of collagen-II did not appear to differ between the two groups. Together, these findings indicate that aligned fibrous scaffolds may provide a favourable microenvironment for the differentiation of AFSCs into cells similar to outer AF cells, which predominantly produce collagen-I matrix.展开更多
Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades.However,efficient harvest and handling of cell sheets remain challe...Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades.However,efficient harvest and handling of cell sheets remain challenging,including insufficient extracellular matrix content and poor mechanical strength.Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types.However,currently,there are no effective ways to apply mechanical loading to cell sheets.In this study,we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide)(PNIPAAm)to poly(dimethylsiloxane)(PDMS)surfaces.The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting.Subsequently,MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates.Upon maturation,the cell sheets were harvested by lowering the temperature.We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning.Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated.After implantation into the critical-sized calvarial defects of mice,the mechanically conditioned cell sheets significantly promoted new bone formation.Findings from this study reveal that thermo-responsive elastomer,together with mechanical conditioning,can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.展开更多
The stiffness of the extracellular matrix(ECM)plays an important role in regulating the cellular programming.However,the mechanical characteristics of ECM affecting cell differentiation are still under investigated.He...The stiffness of the extracellular matrix(ECM)plays an important role in regulating the cellular programming.However,the mechanical characteristics of ECM affecting cell differentiation are still under investigated.Herein,we aimed to study the effect of ECM substrate stiffness on macrophage polarization.We prepared polyacrylamide hydrogels with different substrate stiffness,respectively.After the hydrogels were confirmed to have a good biocompatibility,the bone marrow-derived macrophages(BMMs)from mice were incubated on the hydrogels.With simulated by the low substrate stiffness,BMMs displayed an enhanced expression of CD86 on the cell surface and production of reactive oxygen species(ROS)in cells,and secreted more IL-1βand TNF-αin the supernatant.On the contrary,stressed by the medium stiffness,BMMs expressed more CD206,produced less ROS,and secreted more IL-4 and TGF-β.In vivo study by delivered the hydrogels subcutaneously in mice,more CD68+CD86+cells around the hydrogels with the low substrate stiffness were observed while more CD68+CD206+cells near by the middle stiffness hydrogels.In addition,the expressions of NIK,phosphorylated p65(pi-p65)and phosphorylated IκB(pi-IκB)were significantly increased after stimulation with low stiffness in BMMs.Taken together,these findings demonstrated that substrate stiffness could affect macrophages polarization.Low substrate stiffness promoted BMMs to shift to classically activated macrophages(M1)and the middle one to alternatively activated macrophages(M2),through modulating ROS-initiated NF-κB pathway.Therefore,we anticipated ECM-based substrate stiffness with immune modulation would be under consideration in the clinical applications if necessary.展开更多
Critical-sized bone defects caused by traumatic fractures,tumour resection and congenital malformation are unlikely to heal spontaneously.Bone tissue engineering is a promising strategy aimed at developing in vitro re...Critical-sized bone defects caused by traumatic fractures,tumour resection and congenital malformation are unlikely to heal spontaneously.Bone tissue engineering is a promising strategy aimed at developing in vitro replacements for bone transplantation and overcoming the limitations of natural bone grafts.In this study,we developed an innovative bone engineering scaffold based on gelatin methacrylate(GelMA)hydrogel,obtained via a two-step procedure:first,solid lipid nanoparticles(SLNs)were loaded with resveratrol(Res),a drug that can promote osteogenic differentiation and bone formation;these particles were then encapsulated at different concentrations(0.01%,0.02%,0.04%and 0.08%)in GelMA to obtain the final Res-SLNs/GelMA scaffolds.The effects of these scaffolds on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and bone regeneration in rat cranial defects were evaluated using various characterization assays.Our in vitro and in vivo investigations demonstrated that the different Res-SLNs/GelMA scaffolds improved the osteogenic differentiation of BMSCs,with the ideally slow and steady release of Res;the optimal scaffold was 0.02 Res-SLNs/GelMA.Therefore,the 0.02 Res-SLNs/GelMA hydrogel is an appropriate release system for Res with good biocompatibility,osteoconduction and osteoinduction,thereby showing potential for application in bone tissue engineering.展开更多
Microbial biotransformation and detoxification of biotoxic selenite into selenium nanoparticles(SeNPs)has emerged as an efficient technique for the utilization of selenium.SeNPs are characterized by high bioavailabili...Microbial biotransformation and detoxification of biotoxic selenite into selenium nanoparticles(SeNPs)has emerged as an efficient technique for the utilization of selenium.SeNPs are characterized by high bioavailability and have several therapeutic effects owing to their antioxidant,anti-inflammatory and neuroprotective activities.However,their influence onmicroenvironment disturbances and neuroprotection after spinal cord injury(SCI)is yet to be elucidated.This study aimed to assess the influence of SeNPs on SCI and explore the underlying protective mechanisms.Overall,the proliferation and differentiation of neural stem cells were facilitated by SeNPs derived from Proteus mirabilis YC801 via the Wnt/b-catenin signaling pathway.The SeNPs increased the number of neurons to a greater extent than astrocytes after differentiation and improved nerve regeneration.A therapeutic dose of SeNPs remarkably protected the integrity of the spinal cord to improve the motor function of the hind limbs after SCI and decreased the expression of several inflammatory factors such as tumor necrosis factor-a and interleukin-6 in vivo and enhanced the production of M2-type macrophages by regulating their polarization,indicating the suppressed inflammatory response.Besides,SeNPs reversed the SCI-mediated production of reactive oxygen species.In conclusion,SeNPs treatment holds the potential to improve the disturbed microenvironment and promote nerve regeneration,representing a promising therapeutic approach for SCI.展开更多
基金supported by the National Natural Science Foundation of China (81171479, 51303120, 81471790)the China Postdoctoral Science Foundation (2012M521121)+2 种基金the Natural Science Foundation of Jiangsu Province (BK20130335)the Jiangsu Provincial Special Program of Medical Science (BL2012004)the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘Annulus fibrosus(AF) tissue engineering has recently received increasing attention as a treatment for intervertebral disc(IVD) degeneration; however, such engineering remains challenging because of the remarkable complexity of AF tissue. In order to engineer a functional AF replacement, the fabrication of cell-scaffold constructs that mimic the cellular, biochemical and structural features of native AF tissue is critical.In this study, we fabricated aligned fibrous polyurethane scaffolds using an electrospinning technique and used them for culturing AF-derived stem/progenitor cells(AFSCs). Random fibrous scaffolds, also prepared via electrospinning, were used as a control. We compared the morphology, proliferation, gene expression and matrix production of AFSCs on aligned scaffolds and random scaffolds. There was no apparent difference in the attachment or proliferation of cells cultured on aligned scaffolds and random scaffolds. However, compared to cells on random scaffolds, the AFSCs on aligned scaffolds were more elongated and better aligned, and they exhibited higher gene expression and matrix production of collagen-I and aggrecan. The gene expression and protein production of collagen-II did not appear to differ between the two groups. Together, these findings indicate that aligned fibrous scaffolds may provide a favourable microenvironment for the differentiation of AFSCs into cells similar to outer AF cells, which predominantly produce collagen-I matrix.
基金National Natural Science Foundation of China(No.81925027)Priority Academic Program Development of Jiangsu Higher Education Institutions.
文摘Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades.However,efficient harvest and handling of cell sheets remain challenging,including insufficient extracellular matrix content and poor mechanical strength.Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types.However,currently,there are no effective ways to apply mechanical loading to cell sheets.In this study,we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide)(PNIPAAm)to poly(dimethylsiloxane)(PDMS)surfaces.The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting.Subsequently,MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates.Upon maturation,the cell sheets were harvested by lowering the temperature.We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning.Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated.After implantation into the critical-sized calvarial defects of mice,the mechanically conditioned cell sheets significantly promoted new bone formation.Findings from this study reveal that thermo-responsive elastomer,together with mechanical conditioning,can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.
基金supported by the National Natural Science Foundation of China(81772312,891972059,81772358)Research and Development of Biomedical Materials and Substitution of Tissue and Organ Repair under the National Key R&D Program(2016YFC1101502)+1 种基金the Natural Science Foundation of Jiangsu Province(BK2019668,BK20151210)Jiangsu Provincial Clinical Orthopedic Center,the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD).
文摘The stiffness of the extracellular matrix(ECM)plays an important role in regulating the cellular programming.However,the mechanical characteristics of ECM affecting cell differentiation are still under investigated.Herein,we aimed to study the effect of ECM substrate stiffness on macrophage polarization.We prepared polyacrylamide hydrogels with different substrate stiffness,respectively.After the hydrogels were confirmed to have a good biocompatibility,the bone marrow-derived macrophages(BMMs)from mice were incubated on the hydrogels.With simulated by the low substrate stiffness,BMMs displayed an enhanced expression of CD86 on the cell surface and production of reactive oxygen species(ROS)in cells,and secreted more IL-1βand TNF-αin the supernatant.On the contrary,stressed by the medium stiffness,BMMs expressed more CD206,produced less ROS,and secreted more IL-4 and TGF-β.In vivo study by delivered the hydrogels subcutaneously in mice,more CD68+CD86+cells around the hydrogels with the low substrate stiffness were observed while more CD68+CD206+cells near by the middle stiffness hydrogels.In addition,the expressions of NIK,phosphorylated p65(pi-p65)and phosphorylated IκB(pi-IκB)were significantly increased after stimulation with low stiffness in BMMs.Taken together,these findings demonstrated that substrate stiffness could affect macrophages polarization.Low substrate stiffness promoted BMMs to shift to classically activated macrophages(M1)and the middle one to alternatively activated macrophages(M2),through modulating ROS-initiated NF-κB pathway.Therefore,we anticipated ECM-based substrate stiffness with immune modulation would be under consideration in the clinical applications if necessary.
基金supported by the Natural Science Foundation of Anhui Province(Grant No.2008085QH362)Key Program of Anhui Educational Committee(Grant No.KJ2020ZD51)+2 种基金Translational Medicine Key Projects of Bengbu Medical College(Grant Nos.BYTM2019006 and BYTM 2019012)Scientific Research Innovation Team of Bengbu Medical College(Grant No.BYKC201910)512 Talents Development Project of Bengbu Medical College(Grant Nos.by51202302 and by51202309).
文摘Critical-sized bone defects caused by traumatic fractures,tumour resection and congenital malformation are unlikely to heal spontaneously.Bone tissue engineering is a promising strategy aimed at developing in vitro replacements for bone transplantation and overcoming the limitations of natural bone grafts.In this study,we developed an innovative bone engineering scaffold based on gelatin methacrylate(GelMA)hydrogel,obtained via a two-step procedure:first,solid lipid nanoparticles(SLNs)were loaded with resveratrol(Res),a drug that can promote osteogenic differentiation and bone formation;these particles were then encapsulated at different concentrations(0.01%,0.02%,0.04%and 0.08%)in GelMA to obtain the final Res-SLNs/GelMA scaffolds.The effects of these scaffolds on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and bone regeneration in rat cranial defects were evaluated using various characterization assays.Our in vitro and in vivo investigations demonstrated that the different Res-SLNs/GelMA scaffolds improved the osteogenic differentiation of BMSCs,with the ideally slow and steady release of Res;the optimal scaffold was 0.02 Res-SLNs/GelMA.Therefore,the 0.02 Res-SLNs/GelMA hydrogel is an appropriate release system for Res with good biocompatibility,osteoconduction and osteoinduction,thereby showing potential for application in bone tissue engineering.
基金supported by the Key Program of the Anhui Educational Committee(Grant No.KJ2020ZD51,KJ2021ZD0089)Innovation Fund of Spinal Deformity Clinical Research Center of Anhui Province(Grant No.AHJZJX-GG2022-001)+2 种基金512 Talents Development Project of Bengbu Medical College(Grant No.by51202302,by51202309)Distinguished Young Scholars of First Affiliated Hospital of Bengbu Medical College(Grant No.2021byyfyjq01)Science Research Project of Bengbu Medical College(Grant No.2021bypd006).
文摘Microbial biotransformation and detoxification of biotoxic selenite into selenium nanoparticles(SeNPs)has emerged as an efficient technique for the utilization of selenium.SeNPs are characterized by high bioavailability and have several therapeutic effects owing to their antioxidant,anti-inflammatory and neuroprotective activities.However,their influence onmicroenvironment disturbances and neuroprotection after spinal cord injury(SCI)is yet to be elucidated.This study aimed to assess the influence of SeNPs on SCI and explore the underlying protective mechanisms.Overall,the proliferation and differentiation of neural stem cells were facilitated by SeNPs derived from Proteus mirabilis YC801 via the Wnt/b-catenin signaling pathway.The SeNPs increased the number of neurons to a greater extent than astrocytes after differentiation and improved nerve regeneration.A therapeutic dose of SeNPs remarkably protected the integrity of the spinal cord to improve the motor function of the hind limbs after SCI and decreased the expression of several inflammatory factors such as tumor necrosis factor-a and interleukin-6 in vivo and enhanced the production of M2-type macrophages by regulating their polarization,indicating the suppressed inflammatory response.Besides,SeNPs reversed the SCI-mediated production of reactive oxygen species.In conclusion,SeNPs treatment holds the potential to improve the disturbed microenvironment and promote nerve regeneration,representing a promising therapeutic approach for SCI.
