BACKGROUND: The formation of an inhibitory inflammatory microenvironment after spinal cord injury (SCI) remains a great challenge for nerve regeneration. The poor local microenvironment exacerbates nerve cell death; therefore, the reconstruction of a favorable microenvironment through small-molecule drugs is a promising strategy for promoting nerve regeneration. METHODS: In the present study, we synthesized curcumin-loaded micelle nanoparticles (Cur-NPs) to increase curcumin bioavailability and analyzed the physical and chemical properties of Cur-NPs by characterization experiments. We established an in vivo SCI model in rats and examined the ability of hind limb motor recovery using Basso–Beattie– Bresnahan scoring and hind limb trajectory assays. We also analyzed neural regeneration after SCI using immunofluorescence staining. RESULTS: The nanoparticles achieved the intelligent responsive release of curcumin while improving curcumin bioavailability. Most importantly, the released curcumin attenuated local inflammation by modulating the polarization of macrophages from an M1 pro-inflammatory phenotype to an M2 anti-inflammatory phenotype. M2-type macrophages can promote cell differentiation, proliferation, matrix secretion, and reorganization by secreting or expressing pro-repair cytokines to reduce the inflammatory response. The enhanced inflammatory microenvironment supported neuronal regeneration, nerve remyelination, and reduced scar formation. These effects facilitated functional repair in rats, mainly in the form of improved hindlimb movements. CONCLUSION Here, we synthesized pH/temperature dual-sensitive Cur-NPs. While improving the bioavailability of the drug, they were also able to achieve a smart responsive release in the inflammatory microenvironment that develops after SCI. The Cur-NPs promoted the regeneration and functional recovery of nerves after SCI through anti-inflammatory effects, providing a promising strategy for the repair of SCIs.

BACKGROUND: Intervertebral disk (IVD) degeneration, which can cause lower back pain, is a major predisposing factor for disability and can be managed through multiple approaches. However, there is no satisfactory strategy currently available to reconstruct and recover the natural properties of IVDs after degeneration. As tissue engineering develops, scaffolds with embedded cell cultures have proved critical for the successful regeneration of IVDs. METHODS: In this study, an integrated scaffold for IVD replacement was developed. Through scanning electron microscopy and other mechanical measurements, we characterized the physical properties of different hydrogels. In addition, we simulated the physiological structure of natural IVDs. Nucleus pulposus (NP) cells and annulus fibrosusderived stem cells (AFSCs) were seeded in gelatin methacrylate (GelMA) hydrogel at different concentrations to evaluate cell viability and matrix expression. RESULTS: It was found that different concentrations of GelMA hydrogel can provide a suitable environment for cell survival. However, hydrogels with different mechanical properties influence cell adhesion and extracellular matrix component type I collagen, type II collagen, and aggrecan expression. CONCLUSION This tissue-engineered IVD implant had a similar structure and function as the native IVD, with the inner area mimicking the NP tissue and the outer area mimicking the stratified annulus fibrosus tissue. The new integrated scaffold demonstrated a good simulation of disc structure. The preparation of efficient and regeneration-promoting tissueengineered scaffolds is an important issue that needs to be explored in the future. It is hoped that this work will provide new ideas and methods for the further construction of functional tissue replacement discs.

BACKGROUND: In the intricate pathological milieu post-spinal cord injury (SCI), neural stem cells (NSCs) frequently differentiate into astrocytes rather than neurons, significantly limiting nerve repair. Hence, the utilization of biocompatible hydrogel scaffolds in conjunction with exogenous factors to foster the differentiation of NSCs into neurons has the potential for SCI repair. METHODS: In this study, we engineered a 3D-printed porous SilMA hydrogel scaffold (SM) supplemented with pH-/ temperature-responsive paclitaxel nanoparticles (PTX-NPs). We analyzed the biocompatibility of a specific concentration of PTX-NPs and its effect on NSC differentiation. We also established an SCI model to explore the ability of composite scaffolds for in vivo nerve repair. RESULTS: The physical adsorption of an optimal PTX-NPs dosage can simultaneously achieve pH/temperature-responsive release and commendable biocompatibility, primarily reflected in cell viability, morphology, and proliferation.An appropriate PTX-NPs concentration can steer NSC differentiation towards neurons over astrocytes, a phenomenon that is also efficacious in simulated injury settings. Immunoblotting analysis confirmed that PTX-NPs-induced NSC differentiation occurred via the MAPK/ERK signaling cascade. The repair of hemisected SCI in rats demonstrated that the composite scaffold augmented neuronal regeneration at the injury site, curtailed astrocyte and fibrotic scar production, and enhanced motor function recovery in rat hind limbs. CONCLUSION The scaffold’s porous architecture serves as a cellular and drug carrier, providing a favorable microenvironment for nerve regeneration. These findings corroborate that this strategy amplifies neuronal expression within the injury milieu, significantly aiding in SCI repair.

BACKGROUND: In the intricate pathological milieu post-spinal cord injury (SCI), neural stem cells (NSCs) frequently differentiate into astrocytes rather than neurons, significantly limiting nerve repair. Hence, the utilization of biocompatible hydrogel scaffolds in conjunction with exogenous factors to foster the differentiation of NSCs into neurons has the potential for SCI repair. METHODS: In this study, we engineered a 3D-printed porous SilMA hydrogel scaffold (SM) supplemented with pH-/ temperature-responsive paclitaxel nanoparticles (PTX-NPs). We analyzed the biocompatibility of a specific concentration of PTX-NPs and its effect on NSC differentiation. We also established an SCI model to explore the ability of composite scaffolds for in vivo nerve repair. RESULTS: The physical adsorption of an optimal PTX-NPs dosage can simultaneously achieve pH/temperature-responsive release and commendable biocompatibility, primarily reflected in cell viability, morphology, and proliferation.An appropriate PTX-NPs concentration can steer NSC differentiation towards neurons over astrocytes, a phenomenon that is also efficacious in simulated injury settings. Immunoblotting analysis confirmed that PTX-NPs-induced NSC differentiation occurred via the MAPK/ERK signaling cascade. The repair of hemisected SCI in rats demonstrated that the composite scaffold augmented neuronal regeneration at the injury site, curtailed astrocyte and fibrotic scar production, and enhanced motor function recovery in rat hind limbs. CONCLUSION The scaffold’s porous architecture serves as a cellular and drug carrier, providing a favorable microenvironment for nerve regeneration. These findings corroborate that this strategy amplifies neuronal expression within the injury milieu, significantly aiding in SCI repair.

BACKGROUND: In the intricate pathological milieu post-spinal cord injury (SCI), neural stem cells (NSCs) frequently differentiate into astrocytes rather than neurons, significantly limiting nerve repair. Hence, the utilization of biocompatible hydrogel scaffolds in conjunction with exogenous factors to foster the differentiation of NSCs into neurons has the potential for SCI repair. METHODS: In this study, we engineered a 3D-printed porous SilMA hydrogel scaffold (SM) supplemented with pH-/ temperature-responsive paclitaxel nanoparticles (PTX-NPs). We analyzed the biocompatibility of a specific concentration of PTX-NPs and its effect on NSC differentiation. We also established an SCI model to explore the ability of composite scaffolds for in vivo nerve repair. RESULTS: The physical adsorption of an optimal PTX-NPs dosage can simultaneously achieve pH/temperature-responsive release and commendable biocompatibility, primarily reflected in cell viability, morphology, and proliferation.An appropriate PTX-NPs concentration can steer NSC differentiation towards neurons over astrocytes, a phenomenon that is also efficacious in simulated injury settings. Immunoblotting analysis confirmed that PTX-NPs-induced NSC differentiation occurred via the MAPK/ERK signaling cascade. The repair of hemisected SCI in rats demonstrated that the composite scaffold augmented neuronal regeneration at the injury site, curtailed astrocyte and fibrotic scar production, and enhanced motor function recovery in rat hind limbs. CONCLUSION The scaffold’s porous architecture serves as a cellular and drug carrier, providing a favorable microenvironment for nerve regeneration. These findings corroborate that this strategy amplifies neuronal expression within the injury milieu, significantly aiding in SCI repair.

BACKGROUND: In the intricate pathological milieu post-spinal cord injury (SCI), neural stem cells (NSCs) frequently differentiate into astrocytes rather than neurons, significantly limiting nerve repair. Hence, the utilization of biocompatible hydrogel scaffolds in conjunction with exogenous factors to foster the differentiation of NSCs into neurons has the potential for SCI repair. METHODS: In this study, we engineered a 3D-printed porous SilMA hydrogel scaffold (SM) supplemented with pH-/ temperature-responsive paclitaxel nanoparticles (PTX-NPs). We analyzed the biocompatibility of a specific concentration of PTX-NPs and its effect on NSC differentiation. We also established an SCI model to explore the ability of composite scaffolds for in vivo nerve repair. RESULTS: The physical adsorption of an optimal PTX-NPs dosage can simultaneously achieve pH/temperature-responsive release and commendable biocompatibility, primarily reflected in cell viability, morphology, and proliferation.An appropriate PTX-NPs concentration can steer NSC differentiation towards neurons over astrocytes, a phenomenon that is also efficacious in simulated injury settings. Immunoblotting analysis confirmed that PTX-NPs-induced NSC differentiation occurred via the MAPK/ERK signaling cascade. The repair of hemisected SCI in rats demonstrated that the composite scaffold augmented neuronal regeneration at the injury site, curtailed astrocyte and fibrotic scar production, and enhanced motor function recovery in rat hind limbs. CONCLUSION The scaffold’s porous architecture serves as a cellular and drug carrier, providing a favorable microenvironment for nerve regeneration. These findings corroborate that this strategy amplifies neuronal expression within the injury milieu, significantly aiding in SCI repair.

BACKGROUND: Degeneration of the annulus fibrosus (AF), an important structure of the intervertebral disc, is one of the main causes of degenerative disc disease. Fabrication of scaffolds replicating the stratified microstructure of the AF is critical for the successful regeneration of AF. METHODS: In this study, we cultured rabbit AF-derived stem cells (AFSCs) using fabricated electrospun fibrous poly-Llactic acid scaffolds with different diameters. We applied cyclic tensile strain (CTS) on the scaffolds to regulate the differentiation of AFSCs into specific cell types that resided at the inner, middle, and outer zones of the AF. RESULTS: We found that the morphologies of AFSCs on the smaller-fiber-diameter scaffolds were nearly round, whereas spindle-like cells morphologies were observed on large-diameter scaffolds. CTS enhanced these phenomena and made the cells slender. The expression levels of collagen-I in cells increased as a function of the fiber diameter, whereas collagen-II and aggrecan exhibited opposite trends. Moreover, the application of CTS upregulated the gene expressions of collagen-I, collagen-II, and aggrecan. CONCLUSION Overlaying the scaffolds with different CTS-stimulated cells could eventually lead to engineered AF tissues with hierarchical structures that approximated the native AF tissue. Thus, the proposed methodologies could be potentially applied for AF regeneration.

BACKGROUND: Degeneration of the annulus fibrosus (AF), an important structure of the intervertebral disc, is one of the main causes of degenerative disc disease. Fabrication of scaffolds replicating the stratified microstructure of the AF is critical for the successful regeneration of AF. METHODS: In this study, we cultured rabbit AF-derived stem cells (AFSCs) using fabricated electrospun fibrous poly-Llactic acid scaffolds with different diameters. We applied cyclic tensile strain (CTS) on the scaffolds to regulate the differentiation of AFSCs into specific cell types that resided at the inner, middle, and outer zones of the AF. RESULTS: We found that the morphologies of AFSCs on the smaller-fiber-diameter scaffolds were nearly round, whereas spindle-like cells morphologies were observed on large-diameter scaffolds. CTS enhanced these phenomena and made the cells slender. The expression levels of collagen-I in cells increased as a function of the fiber diameter, whereas collagen-II and aggrecan exhibited opposite trends. Moreover, the application of CTS upregulated the gene expressions of collagen-I, collagen-II, and aggrecan. CONCLUSION Overlaying the scaffolds with different CTS-stimulated cells could eventually lead to engineered AF tissues with hierarchical structures that approximated the native AF tissue. Thus, the proposed methodologies could be potentially applied for AF regeneration.

Objective:To investigate the effects of oligodeoxynucleotide (ODN) MT01 on the morphology, proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs).Methods:The BMSCs of SD rat were isolated and cultured by direct adherence method . The extracted cells were identified by cell morphology of different generations, the expression of surface markers detected by flow cytometry and osteogenic differentiation potential. ODN MT01 group was set up in a gradient of concentrations (0.5, 1.0, 2.0, 4.0 μg/ml) and PBS group as control. Each group of experiments was repeated three times. The morphological changes of cell nucleus and cytoskeleton were fluorescent stained by DAPI and FITC-phalloidin, respectively. The proliferation activities of the BMSCs in different group were analyzed by CCK-8 assay at 1, 4 and 7 d. The degrees of osteogenic differentiation of BMSCs in different group were assessed via alkaline phosphatase (ALP) staining, ALP activity assay and alizarin red S staining respectively on the 7th and 21st days after cultured in osteogenic induction medium. Statistical differences between two groups and among groups were analyzed by t-test and one-way ANOVA, respectively. Differences were regarded as statistically significant when a P value of less than 0.05. Results:Flow cytometry showed that the BMSCs were positive for CD29 (99.8%) and CD44 (96.1%) while negative for CD11b (1.03%) and CD45 (1.74%). ALP staining and alizarin red S staining were positive at different stages of osteogenesis induction confirmed that BMSCs was able to differentiate into the osteoblast. The nucleus and cytoskeleton staining showed that BMSCs were shrunk and the extensibility was reduced when the concentration of ODN MT01 was 4.0 μg/ml. CCK-8 assay showed that the absorbance value of control group was 0.446±0.018, 1.0 μg/ml ODN MT01 was 0.505±0.019, 2.0 μg/ml ODN MT01 was 0.528±0.014 after cultured for 4 days. Compared with the control group, the difference is statistically significant ( t=2.954, 4.083, P=0.033, 0.008). The absorbance value of control group was 0.514±0.027, 1.0 μg/ml ODN MT01 was 0.607±0.007, and 2.0 μg/ml ODN MT01 was 0.636±0.023 after cultured for 7 days. Compared with the control group, the difference was statistically significant ( t=4.664, 6.091, P=0.009, 0.008). The proliferation ability of BMSCs was significantly higher than that of the control group. However, 4.0 μg/ml ODN MT01 (0.427±0.013) had an inhibitory effect on the proliferation ability of BMSCs ( t=4.332, P=0.0149). The blue mass and mineralized nodule improved significantly with the increase of ODN MT01 concentration during the induction of osteogenic differentiation of BMSCs. After cultured for 4 days, the result of ALP activity assay was similar to ALP staining. The activity value of ODN MT01 in the control group was 1.207±0.023, 0.5 μg/ml ODN MT01 was 1.747±0.095, 1.0 μg/ml ODN MT01 was 2.200±0.136, 2.0 μg/ml ODN MT01 was 3.560±0.088, 4.0 μg/ml ODN MT01 was 3.490±0.144. Compared with the control group, the difference was statistically significant ( t=4.313, 7.934, 18.800, 18.240; P=0.005, 0.001, <0.001, <0.001). But there was no difference between 2.0 and 4.0 μg/ml groups ( t=0.562, P=0.590). Conclusions:ODN MT01 with concentration of 2.0 μg/ml could significantly stimulate the proliferation and osteogenic differentiation of BMSCs without affecting the morphology of BMSCs.

Objective:To investigate the effects of oligodeoxynucleotide (ODN) MT01 on the morphology, proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs).Methods:The BMSCs of SD rat were isolated and cultured by direct adherence method . The extracted cells were identified by cell morphology of different generations, the expression of surface markers detected by flow cytometry and osteogenic differentiation potential. ODN MT01 group was set up in a gradient of concentrations (0.5, 1.0, 2.0, 4.0 μg/ml) and PBS group as control. Each group of experiments was repeated three times. The morphological changes of cell nucleus and cytoskeleton were fluorescent stained by DAPI and FITC-phalloidin, respectively. The proliferation activities of the BMSCs in different group were analyzed by CCK-8 assay at 1, 4 and 7 d. The degrees of osteogenic differentiation of BMSCs in different group were assessed via alkaline phosphatase (ALP) staining, ALP activity assay and alizarin red S staining respectively on the 7th and 21st days after cultured in osteogenic induction medium. Statistical differences between two groups and among groups were analyzed by t-test and one-way ANOVA, respectively. Differences were regarded as statistically significant when a P value of less than 0.05. Results:Flow cytometry showed that the BMSCs were positive for CD29 (99.8%) and CD44 (96.1%) while negative for CD11b (1.03%) and CD45 (1.74%). ALP staining and alizarin red S staining were positive at different stages of osteogenesis induction confirmed that BMSCs was able to differentiate into the osteoblast. The nucleus and cytoskeleton staining showed that BMSCs were shrunk and the extensibility was reduced when the concentration of ODN MT01 was 4.0 μg/ml. CCK-8 assay showed that the absorbance value of control group was 0.446±0.018, 1.0 μg/ml ODN MT01 was 0.505±0.019, 2.0 μg/ml ODN MT01 was 0.528±0.014 after cultured for 4 days. Compared with the control group, the difference is statistically significant ( t=2.954, 4.083, P=0.033, 0.008). The absorbance value of control group was 0.514±0.027, 1.0 μg/ml ODN MT01 was 0.607±0.007, and 2.0 μg/ml ODN MT01 was 0.636±0.023 after cultured for 7 days. Compared with the control group, the difference was statistically significant ( t=4.664, 6.091, P=0.009, 0.008). The proliferation ability of BMSCs was significantly higher than that of the control group. However, 4.0 μg/ml ODN MT01 (0.427±0.013) had an inhibitory effect on the proliferation ability of BMSCs ( t=4.332, P=0.0149). The blue mass and mineralized nodule improved significantly with the increase of ODN MT01 concentration during the induction of osteogenic differentiation of BMSCs. After cultured for 4 days, the result of ALP activity assay was similar to ALP staining. The activity value of ODN MT01 in the control group was 1.207±0.023, 0.5 μg/ml ODN MT01 was 1.747±0.095, 1.0 μg/ml ODN MT01 was 2.200±0.136, 2.0 μg/ml ODN MT01 was 3.560±0.088, 4.0 μg/ml ODN MT01 was 3.490±0.144. Compared with the control group, the difference was statistically significant ( t=4.313, 7.934, 18.800, 18.240; P=0.005, 0.001, <0.001, <0.001). But there was no difference between 2.0 and 4.0 μg/ml groups ( t=0.562, P=0.590). Conclusions:ODN MT01 with concentration of 2.0 μg/ml could significantly stimulate the proliferation and osteogenic differentiation of BMSCs without affecting the morphology of BMSCs.