Objective To explore the possibilityof constructing tissue engineering muscles by combining allogeneic myoblasts with small instestinal submucosa(SIS) in rabbits.Methods A large number of purified myoblasts were obtained with multiprocedure digestion and repeated attachment method from skeletal muscles taken from extremities of immature rabbits which were born 7 days ago. The myoblasts were labeled with BrdU, and then combined with SIS to construct tissue engineering muscles. This kind of tissue engineering muscles were grafted into the gastrocnemius muscle defect (1.5 cm in length, 1.0 cmin width) of fifteen rabbits as the experimental group. The SIS was grafted into the same position in the control group. The rabbits were sacrificed 4, 6, 8 weeks after operation. The tissue engineering muscles were evaluated by macroscopic、histological and immunohistochemical observations, and by quantitative analysis of local immunocyte in the grafting site. Results Allogeneic myoblasts with SIS were combined perfectly in vitro. The SIS was connected tightly to surrounding skeletal muscles and inflammation response was obvious 4 weeks after grafting.The SIS began to break down and inflammation response became slight 6 and 8 weeks after operation. Compared with that of 8th week, the quantitative analysis oflocal immunocyte in 4th and 6th week in both experimental and control group hassignificance(Plt;0.05). Newly formed muscle tissues were found around SIS in the experimental group in 4th, 6th, and 8th week. Expression of BrdU and myosin immunohistochemical staining were positive in the experimental group and negative inthe control group.Conclusion Tissue engineering muscles of rabbits which are constructed by combining allogeneic myoblasts with SIS can survive and proliferate.
Objective To compare the reparative effects between the acellular small intestinal submucosa andthe acellular amnion as dressings for traumatic skin defects. Methods Three full-thickness skin defects, which wereclose to the vertebral column of the pig, were created on both sides of the dorsum. The skin defects were randomlydivided into three groups. In each group, the following different materials were used to cover the skin defects: theacellular amnion in Group A, the acellular small intestinal submucosa (SIS) in Group B, and the physiological saline aguze in Group C (the control group). The specimens from the skin defects were harvested for a histological evaluation and for determination of the hydroxyproline content at 10 (2 pigs), 20 (2 pigs), and 30 days (3 pigs). We observed the healing process of the wound and its healing rate, counted the inflammatory cells, vasecular endothelial cells, and proliferating cells, and determined the hydroxyproline content. Results The acellular amnion in Group A and acellular SIS in Group B adhered to the wound tightly, but they did not adhere to the dressing; when the dressing was changed, the wound did not bleed. The saline gauze in Group C adhred to the wound tightly, but when the dressing was changed, the wound bled until 22 days after operation. Under the microscope, the collagen in the tissue below the epithelium was arranged more regularly and there were fewer cells concerned with inflammation in Groups A and B than in Group C at 10, 20, and 30 days after operation. At 10, 20, and 30 days after operation, the wound healing rate was greater in Groups A and B than in Group C, The number of the inflammatory cells and the proliferating cells were greater in Groupo C than in Groups A and B. There was a statistically significant difference (P lt; 0.05),At 20 and 30 days after operatin, the content of hydroxyproline was greater in Group c than in Group A and B. There was a statistically significant difference (P lt; 0.05). However, there was no statistically significant difference between Group A and Group B in the wound healing rate, the numbers of the inflammatory cells, vascular endothelial cells and prokiferating cells, and the content of hydroxyproline(P gt; 0.050). There was no statistically significant difference among the three groups in the number of the vascular endothelial cells. Conclusion Compared with Group C........
Objective To investigate effects of the autologous bone mesenchymal stem cells (MSCs) enriched by the small intestinal submucosa (SIS) film implantation on the myocardial structure, cardiac function, and compensator y circulation after myocardial infarction in the goats. Methods Sixteen black goats were selected and divided randomly into the control group (n=8)and the experimental group (n=8). The chronic myocardial infarction models were made by the ligation of the far end of the left anterior desc ending coronary artery. At the same time, MSCs were aspired from the thigh bone of the goats in the experimental group. MSCs were isolated by the centrifu gation through a percoll step gradient and purified by the plating culture and depletion of the non-adherent cells. Primary MSCs were cultured in the DMEM me dium supplemented with the fetal bovine serum in vitro. After that, the cultures were labeled by 5- BrdU. The active cells were transplanted into the SIS film. Six weeks after the ligation, the MSCs-SIS film was implanted by its being sutured onto the infarction area; whereas, the control group underwent a shamoperation. In both groups, echocardiographic measurements were performed before infarction, 6 weeks after infarction and 6 weeks after the MSC-collagen mplantion, respectively, to assess the myocardial structure and ca rdiac function. The left coronary artery angiography was performed with the digi tal subtraction angiography. Results In an assessment of the left ventricular function, at 6 weeks after operation, t he stroke volume and the ejection fraction of the control group and the experim ental group were 42.81±4.91, 37.06±4.75 ml and 59.20%±5.41%, 44.56%±4.23%, respectively (Plt;0.05). The enddisatolic volume and the endsystolic volume of the control group and the experimental group were 72.55±8.13, 83.31±8.61 ml and 29.75±5.98, 46.25±6.68 ml, respectively (Plt;0.05). The maximal velocity of peak E of contral group and experimental group were 54.8 5±6.35 cm/s and 43.14±4.81cm/s (Plt;0.01); and the maximal velocity of peak A o f control group and experimental grouop were 52.33±6.65 cm/s and 56.91±6.34 cm/s (Pgt;0.05). Echocowdiogr aphy sho wing a distinctly dilatation of left ventricle with the ventricular dyskinesia i n contral group, but without the ventricular dyskinesia in experimental group. T he selective-coronary evngiography revealed that the obvious compensatory circu l ation established between the anterior descending branch and the left circumflex branch in the experimental group. Conclusion Implantation of the autologus MSCs enriched by the SIS film can prevent dilatation of the left ventricular chamber and can improve the contractile ability of the myocardium, cardiac function, and collateral perfusion.
ObjectiveTo evaluate the effect of tissue engineered periosteum on the repair of large diaphysis defect in rabbit radius, and the effect of deproteinized bone (DPB) as supporting scaffolds of tissue engineering periosteum. MethodsBone marrow mesenchymal stem cells (BMSCs) were cultured from 1-month-old New Zealand Rabbit and osteogenetically induced into osteoblasts. Porcine small intestinal submucosa (SIS) scaffold was produced by decellular and a series mechanical and physiochemical procedures. Then tissue engineered periosteum was constructed by combining osteogenic BMSCs and SIS, and then the adhesion of cells to scaffolds was observed by scanning electron microscope (SEM). Fresh allogeneic bone was drilled and deproteinized as DPB scaffold. Tissue engineered periosteum/DPB complex was constructed by tissue engineered periosteum and DPB. Tissue engineered periosteum was "coat-like" package the DPB, and bundled with absorbable sutures. Forty-eight New Zealand white rabbits (4-month-old) were randomly divided into 4 groups (groups A, B, C, and D, n=12). The bone defect model of 3.5 cm in length in the left radius was created. Defect was repaired with tissue engineered periosteum in group A, with DPB in group B, with tissue engineered periosteum/DPB in group C; defect was untreated in group D. At 4, 8, and 12 weeks after operation, 4 rabbits in each group were observed by X-ray. At 8 weeks after operation, 4 rabbits of each group were randomly sacrificed for histological examination. ResultsSEM observation showed that abundant seeding cells adhered to tissue engineered periosteum. At 4, 8, and 12 weeks after operation, X-ray films showed the newly formed bone was much more in groups A and C than groups B and D. The X-ray film score were significantly higher in groups A and C than in groups B and D, in group A than in group C, and in group B than in group D (P<0.05). Histological staining indicated that there was a lot of newly formed bone in the defect space in group A, with abundant newly formed vessels and medullary cavity. While in group B, the defect space filled with the DPB, the degradation of DPB was not obvious. In group C, there was a lot of newly formed bone in the defect space, island-like DPB and obvious DPB degradation were seen in newly formed bone. In group D, the defect space only replaced by some connective tissue. ConclusionTissue engineered periosteum constructed by SIS and BMSCs has the feasibility to repair the large diaphysis defect in rabbit. DPB isn't an ideal support scaffold of tissue engineering periosteum, the supporting scaffolds of tissue engineered periosteum need further exploration.
ObjectiveTo investigate the feasibility of tissue engineered periosteum (TEP) constructed by porcine small intestinal submucosa (SIS) and bone marrow mesenchymal stem cells (BMSCs) of rabbit to repair the large irregular bone defects in allogenic rabbits. MethodsThe BMSCs were cultivated from the bone marrow of New Zealand white rabbits (aged, 2 weeks-1 month). SIS was fabricated by porcine proximal jejunum. The TEP constructed by SIS scaffold and BMSCs was prepared in vitro. Eighteen 6-month-old New Zealand white rabbits whose scapula was incompletely resected to establish one side large irregular bone defects (3 cm×3 cm) model. The bone defects were repaired with TEP (experimental group,n=9) and SIS (control group,n=9), respectively. At 8 weeks after operation, the rabbits were sacrificed, and the implants were harvested. The general condition of the rabbits was observed; X-ray radiography and score according to Lane-Sandhu criteria, and histological examination (HE staining and Masson staining) were performed. ResultsAfter operation, all animals had normal behavior and diet; the incision healed normally. The X-ray results showed new bone formation with normal bone density in the defect area of experimental group; but no bone formation was observed in control group. The X-ray score was 6.67±0.32 in experimental group and was 0.32±0.04 in control group, showing significant difference (t=19.871,P=0.001). The general observation of the specimens showed bone healing at both ends of the defect, and the defect was filled by new bone in experimental group; no new bone formed in the control group. The histological staining showed new bone tissue where there were a lot of new vessels and medullary cavity, and no macrophages or lymphocytes infiltration was observed in the defect area of experimental group; only some connective tissue was found in the control group. ConclusionTEP constructed by porcine SIS and BMSCs of rabbit can form new bone in allogenic rabbit and has the feasibility to repair the large irregular bone defects.
Objective To review the development of researches on the stem cells and the tissue engineering technique used in the intestines. Methods We comprehensively reviewed the literature related to the stem cells and the tissue engineering technique used in the intestines, and summarized the conclusions made by the researches concerned. Results The researches on the stem cells and the tissue engineering technique used in the intestines were attractive topics in the recent years and obtained some developments, especially in the field dealing with the characteristics, proliferation and differentiation of the intestinal stem cells as well as the tissue engineering framework of the small intestinal submucosa in vivo. However, the markers for the differentiation of the intestinal stem cells were still a critical problem, which had not been solved yet, and besides, the researches on the intestinal tissue engineering were still in the initial stage. Conclusion There is a broad prospective application of the intestinal stem cells and the tissue engineering technique to the intestinal problem solution. Substantial achievements can be obtained in the treatment of the inflammatory bowel disease, inan exploration on the oncogenesis mechanism, and in the clinical application ofthe intestinal tissue engineering.
Objective To explore the possibility of small intestinal submucosa (SIS) for reconstruction of urethral defect. 〖WTHZ〗Methods Twenty-four male rabbits weredivided into 4 groups: group A (the tubulate SIS graft for urethral repair), group B (control group, urethral tubulate defect), group C (the SIS patch graft forurethral repairs), group D (control group, urethral part defect). Then the regenerative segment was studied with histological technique by hematoxylineosin straining and immunohistological straining for α-actin after 6 and 12 weeks postoperatively. The retrograde urethrography and urodynamics were used to evaluate the function of the regenerative urethra at 12 weeks after operation. Results In groups A and C, at 6 weeks after operation, the luminal surface of matrix was completely covered by urothelium, minimal SIS graft was observed in the extracellular matrix, new smooth-muscle cells was confirmed; however, more inflammatory cells were observed in the host-matrix anastomosis in group A than in group C. At 12 weeks postoperatively, the regenerative tissue was equivalent to the normal urethral tissue and SIS disappeared in group C, but some minimal SIS grafts were observed in group A. In groups B and D, urethral strictures and fibrous connective tissue were observed except ３ cases. The urethrography showed wide smooth urethral in group A and C, meawhile urodynamic evaluation didn’t demonstrat significant difference(P＞0.05) in the bladder volume and the maximum urethral pressure between preoperation and postoperation in group A or group C. Conclusion SIS can be a useful material for urethral repair in rabbits, the SIS patch graft is superior to the tubulate SIS graft in urethra reconstruction.
Objective To make a comparison between the effects of the small intestinal submucosa (SIS) graft and the insideout vein graft on repairing the peripheral nerve defects. Methods SIS was harvested from the fresh jejunum of the quarantined pig by curetting the musoca, the tunica serosa, and the myometrium; then, SIS was sterilized, dried and frozen before use. Thirty-six male SD rats were divided into 3 groups randomly, with 12 rats in each group. Firstly, the 10mm defects in the right sciatic nerves were madein the rats and were respectively repaired with the SIS graft (Group A), the insideout autologous vein graft (Group B), and the autonerve graft (Group C). At 6 weeks and 12 weeks after the operations, the right sciatic nerves were taken out, and the comparative evaluation was made on the repairing effects by the histological examination, the neural electrophysiological examination, the computerized imaging analysis, and the Trueblue retrograde fluorescence trace. Results The histological examination showed that the regenerated nerve fibers were seen across the defects in the three groups at 6 weeks after the operations. The nerve fibers were denser, the formed nerve myelin was more regular, and the fibrous tissue was less in Group A than in Group B; the nerve regeneration was more similar between Group A and Group C. At 12 weeks after the operations, the neural electrophysiological examination showed that the neural conductive rate was significantly lower in Group B than in Groups A and C (Plt;0.05),but no statistically significant difference was found between Group A and GroupC (Pgt;0.05); the component potential wave amplitude was not statistically different between Group A and Group B; however, the amplitude was significantly lower in Groups A and B than in Group C (Plt;0.05). At 6 weeks and 12 weeks after the operations, the computerized imaging analyses showed that the axiscylinder quantity per area and the nerve-tissue percentage were significantly greaterin Group A than in Group B (Plt;0.05); the average diameter of the regenerated axis cylinder, the axiscylinder quantity per area, and the nerve-tissue percentage were significantly lesser in Group B than in Group C (Plt;0.05). At 12 weeks after the operations, the Trueblue retrograde fluorescence trace revealed that the positivelylabeled neurons were found in the lumbar 3-6 dorsal root ganglion sections in the three groups. Conclusion The small intestinal submucosa graft is superior to the autologous inside-out vein graft in repairing the peripheral nerve defects and it is close to the autonerve graft in bridging the peripheral nerve defects. Therefore, the small intestinal submucosa is a promising biological material used to replace the autonerve graft.
Objective To investigate the effect of porcine small intestinal submucosa extracellular matrix (PSISM) on the vitality and gene regulation of hepatocyte so as to lay the experimental foundation for the application of PSISM in liver tissue engineering. Methods The experiment was divided into two parts: ① BRL cells were cultured with 50, 100, and 200 μg/mL PSISM-medium which were prepared by adding PSISM into the H-DMEM-medium containing 10%FBS in groups A1, B1, and C1, and simple H-DMEM-medium served as a control (group D1); ② BRL cells were seeded on 1%, 2%, and 3% PSISM hydrogel which were prepared by dissolving PSISM in sterile PBS solution containing 0.1 mol/L NaOH in groups A2, B2, and C2, and collagen type I gel served as a control (group D2). At 1, 3, and 5 days after culture, the morphology and survival of liver cells were detected by the Live/Dead fluorescent staining. The cell vitality was tested by cell counting kit-8 (CCK-8) assay. And the relative expressions of albumin (ALB), cytokeratin 18 (CK18), and alpha-fetoprotein (AFP) in hepatocytes were determined by real-time fluorescent quantitative PCR (RT-qPCR). Results The Live/Dead fluorescent staining showed the cells survived well in all groups. CCK-8 results displayed that the absorbance (A) value of group C1 was significantly higher than that of group D1 at 5 days after culture with PSISM-medium, and there was no significant difference between groups at other time points (P>0.05). After cultured with PSISM hydrogels, theA values of groups A2, B2, and C2 were significantly higher than those of group D2 at 3 and 5 days (P<0.05), theA value of group A2 was significantly higher than that of groups B2 and C2 at 5 days (P<0.05), but there was no significant difference between groups at other time points (P>0.05). RT-qPCR showed that the relative expressions of ALB and CK18 mRNA significantly increased and the relative expression of AFP mRNA significantly decreased in groups A1, B1, and C1 when compared with group D1 (P<0.05). The relative expression of CK18 mRNA in group C1 was significantly lower than that in groups A1 and B1 (P<0.05). The relative expressions of ALB and CK18 mRNA were significantly higher and the relative expression of AFP mRNA was significantly lower in groups A2, B2, and C2 than group D2 (P<0.05); the relative expression of CK18 mRNA in group A2 was significantly higher than that in group B2 (P<0.05), and the relative expression of AFP mRNA in group A2 was significantly lower than that in group C2 (P<0.05), but no significant difference was found between other groups (P>0.05). Conclusion PSISM has good compatibility with hepatocyte and can promote the vitality and functional gene expression of hepatocyte. PSISM is expected to be used as culture medium supplement or cell carrier for liver tissue engineering.