| Objective:To establish methods for the isolation, culture and identification of adipose-derived stem cells (ADSCs) and Schwann cells(SCs) in vitro, induce ADSCs toward Schwann-like cells and prepare acellular nerve scaffolds of SD rat, provide seed cells and scaffold material for further research.Method:Collagenase I was used to digest and isolate ADSCs from four-week-old SD rat inguinal fat pads. ADSCs were cultured and passaged in vitro, viewed under inverted microscope and were induced toward osteoblasts and adipocytes.Osteogenic differentiation was confirmed by alkaline phosphatase, alizarin red and von Kossa staining, adipogenic differentiation was confirmed by oil red O staining. Growth curve was drew and cell surface antigens were identified by flow cytometer. Subsequently, ADSCs were induced toward Schwann-like cells by using polylysine, Forskolin, all-trans retinoic acid(ATRA),(3-mercaptoethanol, basic fibroblast growth factor(bFGF), platelet-derived growth factor(PDGF) and Heregulin, and then, gene S100β and gene GFAP were detected by real-time PCR, protein S100β and protein GFAP were detected by immunohistochemistry staining. Sciatic nerves of four-week-old SD rat were cut down, then Collagenase I was used to digest and isolate SCs,after15minutes the explant was cultured in a culture flask. SCs were cultured and passaged in vitro, viewed under inverted phase contrast microscope. To identify, RT-PCR was used to detect gene S100β and gene GFAP, immunohistochemistry staining was used to detect protein S100B and protein GFAP of SCs. To prepare acellular nerve scaffolds, sciatic nerves of mature SD rats were cut down under sterile condition,then were treated by TritonX-200^SB-16and SB-20to remove cells and cell component. After that, acellular nerve scaffolds were observed by slice HE staining and scanning electron microscope (SEM)Result:ADSCs:In vitro cultured ADSCs were spindle-shaped and easily proliferated, well-proportioned after passage. Passaging many times, ADSCs could still maintain strong proliferative ability. The growth curve was "S" shaped. After osteogenic induction, ADSCs showed a positive reaction for alkaline phosphatase, alizarin red and von Kossa staining, while the adipogenetic induction group showed a positive reaction for oil red O staining. All of control groups showed negative reaction for every staining. ADSCs were positive for CD29, CD44andCD105, but negative for CD31and CD45. Schwann-like cells:After the passage3ADSCs were induced toward Schwann-like cells, bipolar cells appeared after one week, neurospheres appeared when induced for two weeks, and neurospheres increased in the follow two weeks. Most of cells showed bipolar or multipolar and the appearance characteristics of SCs. When induced for3weeks, Real-time PCR showed that gene S100β and gene GFAP reached the highest expression levels, and immunohistochemistry staining showed high positive rates of protein S100β and protein GFAP.SCs:Cells appeared around explants within24hours and formed cell colony after72hours, most of the cells were bipolar and spindle-shaped. The cells expressed S100β and GFAP at the levels of transcription and translation, and the purity of SCs was about92%. Acellular nerve scaffolds slice HE staining and scanning electron microscope (SEM) showed that cells were removed completely, but extracellular matrix(ECM) remained. SEM also showed the Pore structure of ECM.Conclusion:ADSCs isolated from SD rat inguinal fat pads can be isolated, cultured, passaged and proliferated easily in vitro, and can be induced into osteoblasts and adipocytes under certain. Further more, ADSCs expressed interrelated phenotypes of mesenchymal stem cells, and expression rates of CD29、CD44、CD105were greater than98%.So that, ADSCs isolated from SD rat inguinal fat pads can be used as seed cells. After three weeks’induction, gene S100β and gene GFAP of Schwann-like cells reached the highest transcription levels. Meantime, protein S100β and protein GFAP almost reached the highest translation levels. Thus, Schwann-like cells induced for three weeks can be used to repair the SD rat sciatic nerve defects. Single enzyme digestion composite explant can obtain high purity, high activity of SCs quickly, and SCs were identified at transcription and translation levels. Accordingly, the cultured SCs can provide high quality and high purity positive control cells for repair and reconstruction of nerve function. Acellular nerve scaffolds showed cells were removed completely, the remained ECM contained Pore structure. In other words, Acellular nerve scaffolds were ideal for the following experiment. Objective:To explore a feasible way to repair peripheral nerve defects by using Schwann-like cells combined acellular nerve scaffolds to repair the SD rat sciatic nerve defect and observe the effectiveness.Method:40SD rats (male and female) were randomly divided into four groups (N=10). Experimental group:acellular nerve scaffolds+Schwann-like cells; positive control group:acellular nerve scaffolds+Schwann cells (SCs); negative control group:acellular nerve scaffolds+adipose-derived stem cells (ADSCs); blank control group:acellular nerve scaffolds.The left side sciatic nerves of SD rats were chosen to be the operated side, manufacturing10mm sciatic nerve defect model, and then Schwann-like cells, SCs, ADSCs were injected into acellular nerve scaffolds under the microscope.After that, scaffolds were used to bridge sciatic nerve defects directly. Postoperative assessments included:postoperative foot ulcer and healing, the level of limb sensory function recovery, neurological behavior of gait recovery, nerve conduction velocity, the ratio of gastrocnemius wet weight recovery and nervous histology observation. SPSS13.0software package was used to analyse datas, compared the restorative effects of different groups by using LSD-t test.Result:All operation side foots showed dropped deformity after surgery, the operation side of toes autophagy appeared within one week.Three weeks after surgery all groups had foot ulcers, foot ulcers began to heal after6-8weeks, and healing began from the positive control group, followed by the experimental group.The blank control group healed most slowly.Operation side limb sensory function test results showed that1-3weeks after surgery, almost no response to heat stimulation,4-6weeks after Operation side limbs began reaction. The first reaction began in the positive control group, followed by the experimental group. Negative control group and blank control group showed response to thermal stimulation at last.12weeks after the determination of results showed that all groups got sensory function recovery, reaction time responded to heat stimulation from short to long was positive control group and experimental group, negative control group and blank control group. Blank control group compared to the other three groups, the differences were statistically significant(P<0.05); Negative control group compared to experimental group and positive control group, the differences were statistically significant (P<0.05); Experimental group compared to positive group, the difference was not statistically significant (P=0.717). Neurological behavior observation showed that operation side had foot drop and each toe flexion deformity and abnormal gait1-2weeks after surgery when walking.6-8weeks after surgery, all groups’toes opened gradually and operative limbs can touchdown.12weeks after surgery, Sciatic function index showed each group restored to a certain extent. Positive control group compared to experimental group recovery, the difference was not statistically significant (P=0.577), but while compared to negative control group and blank control group, the differences were statistically significant (P<0.05). After12weeks, the nerve electrophysiological detection of neural conduction velocity results show that positive control group had the fastest nerve conduction velocity, followed by the experimental group, blank control group had the slowest nerve conduction velocity, the blank control group compared with other three groups, the differences were statistically significant (P<0.05); negative control group compared to experimental group and positive control group, the differences were statistically significant (P<0.05); experimental group compared to positive group, the difference was not statistically significant (P=0.891). After12weeks, cut the gastrocnemius of both sides of all groups for comparison The general appearance of gastrocnemius of both sides showed the gastrocnemius of operative side significantly less than that of normal side in blank control group. Bilateral gastrocnemius muscles had Similar sizes of both sides in positive control group and in experimental group respectively Experimental group and positive control group got the best functional recovery, the difference between the two groups was not statistically significant (P=0.155). Blank control group compared to the other three groups, the differences were statistically significant (P<0.05).Nerve histology observation showed that bridging nerves of each group had smooth surfaces, well integrity, no obviously dilatate sutured ends but some accretions to adjacent tissues.HE staining of nerve biopsy showed many cells within nerves of experimental group, positive control group and negative control group, and the three groups had regular arrangement nerve internal tissues, while nerves of blank control group had less cells and irregular tissuesConclusion:Used Schwann-like cells combined acellular nerve scaffolds to repair the SD rat sciatic nerve10mm defect and observed the effectiveness. Postoperative evaluation indicators showed that all groups got recovery to some extent, all indices of experimental group and positive control group are better than that of control group and negative control group, indicating that the restoration effect is better than that of control group and negative control group. All indices showed no significant differences between experimental group and positive control group, indicated that Schwann-like cells and Schwann cells had quite restoration effects. Accordingly, Schwann-like cells induced from ADSCs combined acellular nerve scaffolds can repair the SD rat10mm sciatic nerve defect... |
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