Objective To investigate the effects of allogenic transplantation of acellular muscle bioscaffolds (AMBS) seeded with bone marrow mesenchymal stem cells (BMSCs) on the repair of acute hemi-transection spinal cord injury (SCI) in rats. Methods AMBS were prepared by reformed chemical approach and sterilized by compound cold sterilization; BMSCs were harvested by density gradient centrifugation and cultured with adherent method. The 3rd generation BMSCs labeled by Hoechst 33342 were injected into AMBS to construct the BMSCs-AMBS composite scaffolds; the biocompatibility was observed under scanning electron microscope (SEM) and fluorescence microscope in vitro at 14 days. Forty-eight adult female Sprague Dawley rats were used to build SCI model by hemi-transecting at T9-11 level, then randomly divided into 4 groups (n=12). Defects were repaired with BMSCs-AMBS composite scaffolds, BMSCs, and AMBS in groups A, B, and C, respectively; group D was blank control by injecting PBS. At 1, 2, 3, and 4 weeks after surgery, the functional recovery of the hind limbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. At 4 weeks after surgery, HE staining and immunofluorescent assay were adopted. Results Masson staining and HE staining showed that AMBS was mainly of the collagen fibers in parallel arrange, without muscle fibers. After 14 days of BMSCs and AMBS co-culture, a large number of survival BMSCs labeled by Hoechst 33342 were seen under fluorescence microscope; SEM showed that BMSCs grew and attached to the inner surfaces of AMBS. At 2-4 weeks, the BBB score in group A was significantly higher than that in groups B, C, and D (P lt; 0.05), and it was significantly lower in group D than in the other 3 groups (P lt; 0.05); at 4 weeks, the BBB score in group B was significantly higher than that in group C (t=10.352, P=0.000). HE staining revealed that the area of spinal cord cavity after SCI was markedly smaller in group A than in the other 3 groups; immunofluorescent assay showed that more neurofilament 200 positive fibers and Nestin positive cells were detected in group A than in groups B, C, and D, but glial fibrillary acidic protein (GFAP) positive cells significantly decreased. The integral absorbance (IA) values of GFAP were 733.01 ± 202.04, 926.42 ± 59.46, 1 069.37 ± 33.42, and 1 469.46 ± 160.53 in groups A, B, C, and D, respectively; the IA value of group A was significantly lower than that of groups B, C, and D (P lt; 0.05), and it was significantly higher in group D than in groups A, B, and C (P lt; 0.05). Conclusion With relatively regular internal structures and good biocompatibility, AMBS can inhibit glial scar and enhance the survival, migration, and differentiation of BMSCs, so AMBS is the ideal nature vector for cell transplantation. Co-transplantation of AMBS and BMSCs has synergistic effect in treating SCI, it can promote rat motor function recovery.
Objective To investigate tissue engineered spinal cord which was constructed of bone marrow mesenchymal stem cells (BMSCs) seeded on the chitosan-alginate scaffolds bridging the both stumps of hemi-transection spinal cord injury (SCI) in rats to repair the acute SCI. Methods BMSCs were separated and cultured from adult male SD rat. Chitosan-alginate scaffold was produced via freeze drying, of which the structure was observed by scanning electron microscope (SEM) and the toxicity was determined through leaching l iquor test. Tissue engineered spinal cord was constructed by seeding second passage BMSCs on the chitosan-alginate scaffolds (1 × 106/mL) in vitro and its biocompatibil ity was observed under SEM at 1, 3, and 5 days. Moreover, 40 adult female SD rats were made SCI models by hemi-transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). Tissue engineered spinal cord or chitosan-alginate scaffolds or BMSCs were implanted in groups A, B, and C, respectively. Group D was blank control whose spinal dura mater was sutured directly. After 1, 2, 4, and 6 weeks of surgery, the functional recovery of the hindl imbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. Other indexes were tested by wheat germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing, HE staining and immunofluorescence staining after 6 weeks of surgery. Results Chitosan-alginate scaffold showed three-dimensional porous sponge structure under SEM. The cells adhered to and grew on the surface of scaffold, arranging in a directional manner after 3 days of co-culture. The cytotoxicity of chitosan-alginate scaffold was in grade 0-1. At 2, 4, and 6 weeks after operation, the BBB score was higher in group A than in other groups and was lower in group D than in other groups; showing significant differences (P lt; 0.05). At 4 and 6 weeks, the BBB score was higher in group B than in group C (P lt; 0.05). After 6 weeks of operation, WGA-HRP retrograde tracing indicated that there was no regenerated nerve fiber through the both stumps of SCI in each group. HE and immunofluorescence staining revealed that host spinal cord and tissue engineering spinal cord l inked much compactly, no scar tissue grew, and a large number of neurofilament 200 (NF-200) positive fibers and neuron specitic enolase (NSE) positive cells were detected in the lesioned area in group A. In group B, a small quantity of scar tissue intruded into non-degradative chitosan-alginate scaffold at the lesion area edge, and a few of NSE flourescence or NF-200 flourescence was observed at the junctional zone. The both stumps of SCI in group C or group D were filled with a large number of scar tissue, and NSE positive cells or NF-200 positive cells were not detected. Otherwise, there were obviously porosis at the SCI of group D. Conclusion The tissue engineered spinal cord constructed by multi-channel chitosan-alginate bioscaffolds and BMSCs would repair the acute SCI of rat. It would be widely appl ied as the matrix material in the future.
Objective To investigate the influence of different transplantating times on the survival and immigration of the bone marrow mesenchymal stem cells (BMSCs) in injured spinal cord by subarachnoid administration, and to evaluate the most optimal subarachnoid administration times for BMSCs. Methods Eight adult male rats (weighing 120 g) were used to isolate BMSCs that were cultured, purified and labeled with Hoechst 33342 in vitro. Another 75 adult Wistar rats (weighing 220 g) were made the spinal cord injury (SCI) models at T9,10 level according to the improved Allen’s method and were randomly divided into 5 groups (groups A, B, C, D, and E, n=15). The labeled BMSCs at 1 × 107/mL 0.1 mL were injected into subarachnoid space of the rats via a catheters under the subarachnoid space in groups A (one time at 1 week), B ( two times at 1 and 3 weeks), C (3 times at 1, 3, and 5 weeks) and D (5 times at 1, 3, 5, 7, and 9 weeks) and 0.2 mL phosphate-buffered sal ine (PBS) was injected in group E (5 times at 1, 3, 5, 7, and 9 weeks) as blank control. The neurological functions were evaluated using the Basso-Beattie-Bresnahan (BBB) scale 1, 3, 5, 7, 9, and 12 weeks after transplantation. The migration, survival, differentiation, and histomorphological changes of BMSCs were observed by HE, immunohistochemistry, and fluorescence microscopy. Results At 3 weeks after injury, there were significant differences in the BBB scores between group E and groups A, B, C, D (P lt; 0.01), and between groups A, B and groups C, D (P lt; 0.01). At 7, 9, and 12 weeks, the BBB scores were significantly higher in groups C and D than in groups A and B (P lt; 0.01), and in group B than in group A (P lt; 0.01). There were no significant differences in the BBB scores between groups C and D (P gt; 0.05). The fluorescence microscopy showed that the transplanted BMSCs survived and grew in the injured region at 3 weeks after injury and as time went on, the transplanted cells gradually decreased in group A; in groups B, C, and D, BMSCs count reached the peak values at 5 and 7 weeks and then gradually decreased. At 12 weeks, the survival BMSCs were significantly more in groups C and D than in groups A and B (P lt; 0.01). HE staining showed that the formation of cavity was observed in each group at 3 weeks after injury and the area of cavity gradually decreased in groups A, B, C, and D. At 12 weeks, the area of cavity was the miximal in groups C and D, moderate in groups A and B, and the maximal in group E. The immunohistochemistry staining indicated that the expression of NF-200 was more intense in groups C and D than in groups A and B. The expression of NF-200-positive fibers was more intense in group C. Conclusion Multiple administration of BMSCs promotes the restoration of injured spinal cord and improves neurological functions, and three times for BMSCs transplantation is best
Objective To investigate the mechanism of adenosine-tri phosphate (ATP) activated mammal ian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signal pathway in the physiology and pathology of spinal cord injury (SCI). Methods Ninety-six adult healthy female Sprague-Dawley rats were randomly divided into 4 groups (groups A, B, C and D, n=24). In groups A, B and C, the rats were made the SCI models at T8-10 levels by using a modified Allen’ s stall, and in group D, rats were given laminectomy without SCI. The rats were subjected to the administration of ATP (40 mg/kg) for 7 days in group A, to the administration of physiological sal ine (equal-volume) for 7 days in group B, to the administration of ATP (40 mg/kg) and rapamycin (3 mg/kg) for 7 days in group C, and to the administration of physiological sal ine (equal-volume) for 7 days in group D. Locomotor activity was evaluated using the Basso-Beattie-Bresnahan rating scale at the postoperative 1st, 2nd, 3rd, and 4th weeks. Then, the expressions of spinal cord cell marker [Nestin, neuron-specific enolase (NSE), gl ial fibrillary acidic protein (GFAP)] and the mTOR/STAT3 pathway factors (mTOR, STAT3) were detected at the postoperative 1st, 2nd, 3rd, and 4th weeks by immunohistochemistry analysis, Western blot assay, and real-time fluorescence PCR analysis. Results The BBB scores in group A showed a steady increase in the postoperative 1st-4th weeks and were significantly higher than those in groups B and C (P lt; 0.01), but were lower than that in group D (P lt; 0.01). Real-time fluorescence PCR results showed that the mRNA expressions of mTOR, STAT3, NSE of group A steadily increased, however, the Nestin mRNA expression gradually decreased in the postoperative 1st-4th weeks, which were all significantly higher than those of groups B, C, and D (P lt; 0.01). The mRNA expression of GFAP showed a steady increase in group A and was significantly less than those of groups B and C, but was higher than that of group D (P lt; 0.01). There were significant differences (Plt; 0.01) in all markers between groups B, C, and group D; there were significant differences in mTOR, P-mTOR, STAT3, and P-STAT3 mRNA between groups B and C at 1st-4th weeks (P lt; 0.05). The similar changes were found by Western blot assay. Conclusion ATP can activate the mTOR/STAT3 pathway to induce endogenic NSCs to prol iferate and differentiate into neurons in rats, it enhances the heal ing of SCI.
【Abstract】 Objective To investigate the effect of salidroside on rat bone marrow mesenchymal stem cells (BMSCs)differentiation into the chol inergic nerve cells, so as to provide the theory basis of the combination of salidroside and stem cellsfor cl inical therapy of nervous system diseases. Methods BMSCs were isolated from 2 Wistar rats (aged 4-6 weeks, weighing 120 g), which were identified by CD34, CD45, CD90, and CD106 with flow cytometry. According to inducing method, BMSCs at passage 2 were divided into 3 groups: In groups A and B, BMSCs were induced by salidroside (20 μg/mL) and retinoic acid (5 μmol/mL) respectively for 1, 3, 6, and 9 days, in group C, BMSCs were cultured with serum-free DMEM/F12 medium as control. MTT assay was used to detect the cellular prol iferation activity. The immunofluorescence chemical technology was used to detect the expressions of nerver growth factor (NGF) and relevant marker molecule of nerve cells, including neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), β-Tubulin III, gl ial fibrillary acidic protein (GFAP), and the marker of cholinergic neuron, such as Acetylcholine (Ach) and NGF. RT-PCR was used to detect mRNA expressions of NSE, β-Tubulin III, GFAP, brain derived neurotrophic factor (BDNF), and γ-aminobutyric acid (GABA). ELISA was used to detect the levels of BDNF and NGF, and the expression level of NGF protein was analyzed by Western blot. Results The results of the flow cytometry showed that the cultured cells were CD90 and CD106 positive, and CD34 and CD45 negative, which indicated that the cells were BMSCs. The cellular proliferation activity in groups A and B were significantly higher than that in group C at 6 days and 9 days (P lt; 0.05). RT-PCR results showed that the expression level of NSE, BDNF, β-Tubulin III, GFAP mRNA were increased in groupA at 6 days; In group B, that expression level of NSE mRNA was up-regulated at 6 days, that expression level of BDNF mRNA increased at 1 days and reached the peak at 6 days, and that expression level of β-Tubulin III mRNA was up-regulated at 3 days, which was significantly higher than that at the other time points, and than that in group C (P lt; 0.01). But no GABA mRNA expression was detected in each group. Immunofluorescence chemical technology staining showed that the positive rates of NSE, MAP2, β-Tubulin III, and GFAP were significantly higher in group A than those in group C at 3 days; the positive rates of Ach were significantly higher at 3, 6, and 9 days than those at 1 day in groups A and B, and in groups A and B than in group C (P lt; 0.01); the positive rates of NGF in groups A and B were significantly higher than those in group C (P lt; 0.01). The levels of BDNF and NGF in groups A and B were significantly higher than those in group C at 1, 3, 6, and 9 days (P lt; 0.01), but no significant difference of BDNF was found between groups A and B (P gt; 0.05). The expression level of NGF protein in groups A and B were significantly higher than that in group C (P lt; 0.01). The NGF expression reached the peak at 6 days in group A and at 3 days in group B. Conclusion Sal idroside could induce rat BMSCs differentiate into chol inergic nerve cells in vitro.