Objective To investigate the effect of machine-enzyme digestion method on the residual quantity of small intestinal submucosa (SIS) cell and the content of growth factors. Methods Fresh jejunum of pig within 4 hours after harvesting was prepared into SIS after machine digestion (removing placenta percreta, mucosa, and muscular layer), degrease,trypsinization, abstergent processing, and freeze drying. Samples were kept after every preparation step serving as groups A, B, C, D, and E, respectively (n=4 per group). And the fresh jejunum served as control group (group F, n=4). The histological alteration in each preparation process was reviewed with HE staining and scanning electron microscope (SEM). Nest-polymerase chain reaction (PCR) was used to determine the content of death associated protein 12 (DAP12), and enzyme-linked immunosorbent assay (ELISA) was appl ied to detect the content of vascular endothel ial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), tumor necrosis factor α (TNF-α). Results HE staining and SEM observation showed that there were residual cells in groups A and B, and there were no residual cells in groups C, D, and E. Nest-PCR test revealed the occurrence of DAP12 in each group. The contents of DAP12 in groups A, B, C, D, E, and F were (18.01 ± 9.53), (11.87 ± 2.35), (0.59 ± 0.27), (0.29 ± 0.05), (0.19 ± 0.04), and (183.50 ± 120.13) copy × 106/cm2. The content of DAP12 in group F was significant higher than that of other groups (P lt; 0.05), groups A and B was higher than groups C, D, and E (P lt; 0.05), there were significantdifferences among groups C, D, and E (P lt; 0.05), and there was no significant difference between groups A and B (P gt; 0.05). The ELISA test showed the content of VEGF, bFGF, TGF-β, and TNF-α in group A was significantly higher than that of groups B, C, D, and E (P lt; 0.05), and there was no significant difference among groups B, C, D, and E (P gt; 0.05). Conclusion SIS prepared by simple mechanical method has more residual cells, while the machine-enzyme digestion method can effectively remove the cells and significantly reduce the DAP12 content. This approach can not obviously reduce the growth factor content in SIS.
Objective To explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa（SIS） and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques. Methods The enzymetreatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth musclecells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co cultured for a further observation. At 5,7 and 9 days of the co culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold. Results The primary bladder smooth muscle cells that had been harvested by the enzyme treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitrofor 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindleshaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the welldeveloped actin filaments inthe cytoplasm and the dense patches in the cell membrane under the transmissionelectron microscope. The immunohistochemical staining showed the canine bladdersmooth muscle cells with positive reacting α actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a singlelayer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were（16.85±0.79）×105，（39.74±2.16）×105 and （37.15±2.02）×105, respectively. Thecell counts in the control group were（19.43±0.54）×105，（34.50±1.85）×105 and （33.07±1.31）×105, respectively. There was a significant difference between the two groups at 5 days （P＜0.05）. ConclusionWith the enzyme treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.
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 review the research advance of the preparation and characteristics of small intestinal submucosa(SIS). METHODS: Recent original articles related to such aspects of small intestinal submucosa were reviewed extensively. RESULTS: Small intestinal submucosa was an easily obtained biomaterial. SIS was a bio-absorbable and degradable material. SIS had tissue specific regeneration properties. CONCLUSION: SIS is a suitable bio-derived material for tissue engineering of blood vessel, muscle tendon, urinary bladder and abdomen.
Objective To review the recent progress of the small intestinal submucosa (SIS) in application research of tissue repair and reconstruction. Methods The domestic and international articles on the SIS were reviewed and summarized. Results As a natural extracellular matrix, SIS has outstanding biological advantages, such as good mechanical property, tissue compatibility, and lower immunogenicity. SIS has been used to repair and reconstruct various types of tissue defects in animal models and clinical application, especially in the treatment of hernia, urinary system disease, and refractory skin trauma. The development of the tissue engineering technology expands the field of SIS repair and reconstruction and promotes the intensive study of SIS. However, the long-term effect of SIS in tissue repair and reconstruction still remains to be further observation, while the cell/SIS material construction by tissue engineering technology also needs more studies. Conclusion SIS has a widely promising application future in the tissue repair and reconstruction.
Objective To study an optimal ratio of small intestinal submucosa (SIS) and (hydroxyapatite-tricalcium phosphate,HA-TCP,SIS/HA-TCP) compositions according to the effect of SIS/HA-TCP compositions with different ratios on repairing rabbit femoral condyle defect. Methods Thirty-six rabbits were made into bone defect models of 6 mm in diameter and 10 mm in depth in both sides of femoral condyles. Three different ratios of SIS/HA-TCP compositions (w/w: 1, 0.5, 0.25) were implanted into rabbit femoral condyle defect. After 2, 4, 8 and 12 weeks of operation, the repair effect wasobserved grossly. The histological evaluations were performed by histological scoring system and computer imaging analysis system. Results The amount of new bone formation in SIS/HA-TCP(0.5) group was more than that in SIS/HA-TCP(1) and SIS/HA-TCP(0.25) groups. Histological observation: In SIS/HA-TCP(1) group, few new bone formation was seen and bone defect was repaired in the 12th week. In SIS/HA-TCP(0.5) group, immature woven bone was found in the defect in the 2nd week; more immature woven bone appeared and formed trabeculae in the 4th week; the regenerated bone was vigorously growing into the interspaces of the implanted materials in the 8th week; the implanted materials was basically replaced by bony structure and the lamellar bone appeared in the 12thweek. The results of SIS/HA-TCP (0.25) group were similar to that of SIS/HA-TCP(0.5) group. The histological scoring was higher in SIS/HA-TCP(0.5) and SIS／HA-TCP(0.25) groups than that in SIS/HA-TCP(1) group (Plt;0.05) in the 2nd, 4th, 8th, and 12th weeks. The scoring was higher in SIS/HA-TCP(0.5) roup than that in SIS/HA-TCP(0.25) group in the 2nd and 12th weeks(P＜0.05). In new bone formation and the degradation of HA-TCP, SIS/HA-TCP(0.5) and SIS／HA-TCPC(0.25) groups were superior to SIS/HA-TCP(1) group(Plt;0.05), SIS/HA-TCP(0.5) group was superior to SIS/HA-TCP(0.25) group (Plt;0.05). Conclusion SIS/HA-TCP(0.5) has better effects of repairing bone defect and it can be used as a reference ratio in constructing bone scaffolds.
Objective to determine the modulus of elasticity (E) of small intestinal submucosa (SIS), a new biological graft material. Methods The longitudinal tensile testing was performed on 21 specimens of canine jejunum with the electronic material test machine. Results Stress (σ)strain (ε) data were obtained. It was found that the stress (σ)strain (ε) data fitted the expressionσ＝Kεα very well, the mean correlation coefficients R2 was0.991 6.Then the expression of the modulus of elasticity (E) of SIS was E=K1/ασ1-1/α. The mean values of α and K were 3.966 9 and 374.55，so E=4.3992σ0.75. Conclusion The modulus of elasticity was found to increase with increasing stress. The variations law is similar to that of the vessels. Furthermore when σ is 001333 MPa(100 mmHg),E is about 0.16 MPa, which is similar to that of the vessels.
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 obtain highly purified and large amount of Schwann cells (SCs) by improved primary culture method, to investigate the biocompatibility of small intestinal submucosa (SIS) and SCs, and to make SIS load nerve growth factor (NGF) through co-culture with SCs. Methods Sciatic nerves were isolated from 2-3 days old Sprague Dawley rats and digested with collagenase II and trypsin. SCs were purified by differential adhesion method for 20 minutes and treated with G418 for 48 hours. Then the fibroblasts were further removed by reducing fetal bovine serum to 2.5% in H-DMEM. MTT assay was used to test the proliferation of SCs and the growth curve of SCs was drawn. The purity of SCs was calculated by immunofluorescence staining for S-100. SIS and SCs at passage 3 were co-cultured in vitro. And then the adhesion, proliferation, and differentiation of SCs were investigated by optical microscope and scanning electron microscope (SEM). The NGF content by SCs was also evaluated at 1, 2, 3, 4, 5, and 7 days by ELISA. SCs were removed from SIS by repeated freeze thawing after 3, 5, 7, 10, 13, and 15 days of co-culture. The NGF content in modified SIS was tested by ELISA. Results The purity of SCs was more than 98%. MTT assay showed that the SCs entered the logarithmic growth phase on the 3rd day, and reached the plateau phase on the 7th day. SCs well adhered to the surface of SIS by HE staining and SEM; SCs were fusiform in shape with obvious prominence and the protein granules secreted on cellular surface were also observed. Furthermore, ELISA measurement revealed that, co-culture with SIS, SCs secreted NGF prosperously without significant difference when compared with the control group (P gt; 0.05). The NGF content increased with increasing time. The concentration of NGF released from SIS which were cultured with SCs for 10 days was (414.29 ± 20.87) pg/cm2, while in simple SIS was (4.92 ± 2.06) pg/cm2, showing significant difference (P lt; 0.05). Conclusion A large number of highly purified SCs can be obtained by digestion with collagenase II and trypsin in combination with 20-minute differential adhesion and selection by G418. SIS possesses good biocompatibility with SCs, providing the basis for further study in vivo to fabricate the artificial nerve conduit.
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.