Objective To explore an effective method to culture and purify porcine keratinocytes, to observe the morphological characteristics of porcine keratinocytes growing on acellular amnion and to offer the experimental basis for that the amnion is used for tissue engineering. Methods The primary porcine keratinocytes were cultivated with DKSFM(Defined keratinocyteSFM) containing 10% fetal bovine serum (FBS). The second passage porcine keratinocytes were cultivated with the medium of DKSFM containing different concentrations of FBS. Because of the speciality that keratinocytes stick to flask fast, we purified the keratinocytes by 0.02% EDTA and 005% trypsin step by step. The second passage keratinocytes were seeded on amnion, the keratinocytes/amnion composites were observed by dye directly, histopathology and immunohistochemical staining. Results The proliferation of the primry porcine keratinocytes cultured with the medium ofDKSFM containing 10% FBS was fast and the morphological characteristics were good. The cultivated porcine keratinocytes expanded to 60%70% of the total area of the bottle of the flask after 5 days. The proliferation of the second passage porcine keratinocytes cultivated with the medium that DKSFM containing 5% FBS was faster than the second porcine keratinocytes cultured with the medium of DKSFMcontaining 10% FBS, or DKSFM without FBS. The proliferation of the second passage porcine keratinocytes cultivated with DKSFM without FBS was the slowest one among the 3 medium. The porcine keratinocytes that were purified by 0.02% EDTA and 005% trypsin step by step were got with high pure. After the keratinocytes were cultivated on the surface of amnion 12 days, the keratinocytes form a single layer on the surface of amnion and the cells were polygong and arranged like slabstone. After 14 and 16 days,the cells contacted more closely. But at 16 days after the cells were seeded, some of the cells got aging. Conclusion To culture primary porcine keratinocytes with the medium that DKSFMcontaining 10% FBS and to cultivate the second passage with the medium containing 5% FBS, the proliferation of porcine keratinocytes are faster. The method that purify the porcine keratinocytes is effective. Acellular amnion offers excellent bioscafold to support keratinocytes to adhere and grow. After the porcine keratinocytes are cultivated on the surface of the acellular amnion 12 days, the morphologic characteristics are better than that of other groups.
Objective To study the integration of rat marrow stromal stem cells (MSCs) after transplantation into acellular extracellular matrix (AECM). Methods We got 16 femurs from 8 Kunming rats, the femurs were treated by Triton X100 toget AECM, MSCs were collected from femoral marrow of 20 Kunming rats about a mouth old by PBS 4ml, centrifugalized and primary cultured in bottles,then therat MSCs were transplanted into AECM at a concentration of 5×106/ml and culturedfor 7 days. The integration of the donor cells was observed using one phase contrast microscope, a light microscope and a scanning electron microscope (SEM).Results In AECM bone lacunas there were MSCs nucleuses stained blue. The nucleuses were unevenly distributed in AECM with more in the peripheral AECM than in the central AECM and with more in the layer anear culture medium than in the layer far away from culture medium.AECM possessed a good spatial scaffold structure, the marrow stromal stem cells were well integrated into AECM.Conclusion AECM can be usedas a good scaffold material for tissue engineered bone construction.
Objective To choose the best procedure on preparation of acellularbovine pericardium (ABP) guided bone regeneration (GBR) material. Methods The BP was decellularized with 0.25% Trypsin+0.5% Triton X-100. The acellular bovine pericardiums (ABPs) were treated with phosphatebuffered saline(PBS) (group A), 95% glycerol (group B), EDAC (group C), and EDAC and 95% glycerol (group D) respectively. The treated ABPs were implanted subcutaneously in the back of SD rats respectively at random and no material was implanted as control. Seven rats were sacrificed at 2 weeks, twelve at 4 weeks, twelve at 8 weeks, seven at 16 weeks. Local reaction was studied grossly. The amount of antigen presenting cell (APC) and the percentage of ABP degeneration were reckoned by images analysis system. Results The ABPs were replaced by fibroblasts completely in group A at 8 weeks， in group C at 16 weeks, but only less than 50% till 16 weeks in groups B and D. In all groups, the depth of surrounding fibres attenuated timedependingly. The APC amount of the groups B and D was higher than that of the control group, and the ABP of the groups B and D degraded partly at 16 weeks. Conclusion The ABP treated with EDAC can be replaced by the surrounding tissues and has good biocompatibility.
【Abstract】 Objective To introduce the cl inical appl ication of heterogeneity (cattle) acellular dermal matrix(ADM)in the repair of mucosa defect otolaryngology. Methods From October 2006 to March 2007, 12 cases of mucosa defect was repaired with heterogeneity ADM after the surgery. There were 10 males and 2 females, aged 18-76 years. Defect was caused by deflection of nasal septum in 1 case, melanoma of front and midst basal is (capillary hemangioma) in 1 case, nasal vestibule angioma (T2N2M0)in 1 case, cancer of hypopharynx (T2N1M0) in 1 case, cancer of amygdale in 3 cases (2 of T2N0M0 and 1 of T3N1M0),cervical segments esophageal carcinoma in 1 case, and cancer of larynx in 4 cases (3 of T2N0M0 and 1 of T3N1M0). Results All these 12 cases were followed up for 6 months. The results of endoscope showed that heterogeneity ADM mingled with mucosa within 3 months after operation and the function was recovered. Pharynx fistula occurred in 1 case of hypopharynx cancer afterthe operation. After treatment of dressing change and antibiotics for 10 days, the wound healed, but after 2 months tumor recurred. All the patients were treated by radiation treatment. One case of amygdala cancer recurred and transferred to the neck after 2 months of radiation treatment. But 1 case of hypopharynx cancer died of massive haemorrhage after radiation treatment for 3 months. Conclusion Heterogeneity ADM can be easily obtained and it is a new method to repair mucosa defect. Theoperative procedure is easy to perform and worthwhile to be appl ied to cl inical operation.
Objective To evaluate the cl inical effect and the pathological characteristics of acellular allogeneic dermal matrix in repairing unstable burn scar. Methods From January 2007 to June 2008, 19 cases of unstable burn scars (24 parts) were treated, including 16 males (20 parts) and 3 females (4 parts) with a median age of 27 years (range, 3-58 years). Theinjury was caused by flame (14 cases, 18 parts), electricity (4 cases, 5 parts), and hot water (1 case, 1 part). The unstable burn scars located on hands (8 cases), forearms (2 cases), thighs (3 cases), legs (2 cases), feet (2 cases), chest (1 case), and abdomen (1 case). Scar formed for 3 months to 1 year. The area of defect varied from 7 cm × 5 cm to 22 cm × 15 cm after scar removal. Defects were covered with acellular allogeneic dermal matrix and autogenous spl it-thickness skin graft. At 6-18 months after operation, the pathological observations of the epidermis, the basal membrane, and structural components of the dermis were done. Results All wounds healed by first intention. Scar ulcer disappeared completely in 18 cases and the composite skin grafts all survived. Some bl isters occurred in 1 case and were cured after dressing changing. All patients were followed up 10 months to 2 years (18 months on average). The grafted-skin was excellent in the appearance, texture, and elasticity. The function recovered well. Only superficial scar was observed at skin donor sites. Pathological observation showed that the epidermis and the basal membrane of the skin grafts were similar to that of normal skin, and no significant difference was found in newly capillaries between them. Collagen fibers arranged regularly, and there were few inflammatory cells in the matrix. Conclusion Acellular allogeneic dermal matrix with autogenous spl it-thickness skin graft may effectivly repair the wound after removing the unstable burn scar, and its structure is similar to that of normal skin.
Objective To observe the histomorphology and the biocompatibil ity of acellular nerve prepared by different methods, to provide the experimental evidence for the selection of preparation of acellular nerve scaffold. Methods Forty-eight adult Sprague Dawley rats, male or female, weighing 180-220 g, were selected. The sciatic nerves were obtained from 30 rats and were divided into groups A, B, and C (each group had 20 nerves). The acellular sciatic nerves were prepared by the chemical methods of Dumont (group A), Sondell (group B), and Haase (group C). The effect to remove cells was estimated by the degree of decellularization, degree of demyel ination, and intergrity of nerve fiber tube. The histocompatibil ity was observed by subcutaneous implant test in another 18 rats. Three points were selected along both sides of centre l ine on the back of rats, and the points were randomly divided into groups A1, B1, and C1; the acellular nerve of groups A, B, and C were implanted in the corresponding groups A1, B1, and C1. At 1, 2, and 4 weeks after operation, the rats were sacrificed to perform the general observation and histological observation. Results The histomorphology: apart of cells and the dissolved scraps of axon could be seen in acellular never in the group A, and part of Schwann cell basilar membrane was broken. In group B, the cells in the acellular never were not removed completely, the Schwann cell basilar membrane formed bigger irregular hollows, part of the Schwann cell basilar membrane was broken obviously. But in the group C, the cells were completely removed, the Schwann cell basilar membrane remained intactly. Group C was better than group A and group B in the degree of decellularization, degree of demyel ination, integrity of nerve fiber tube and total score, showing significant differences (P lt; 0.05). The subcutaneous implant test: there were neutrophils and lymphocytes around the acellular nerve in 3 groups at 1 week after implant. A few of lymphocytes were observed around the acellular nerve in 3 groups at 2 weeks after implant. The inflammation was less in groups A1, B1, and C1 at 4 weeks after implant, part of the cells grew into the acellular nerve and arranged along the Schwann cell basilar membrane. The reaction indexes of the inflammational cells in group A1 and group B1 were higher than that in group C1 at 1, 2, and 4 weeks after implant, showing significant differences (P lt; 0.01), but there was no significant difference between group A1 and group B1 (P gt; 0.05). Conclusion The acellular sciatic nerves prepared by Haase method has better acellular effect and the histocompatibil ity than those by the methods of Dumont and Sondell.
ObjectiveTo prepare a composite scaffold using bladder acellular matrix (BACM) and polyurethane (PU) for bladder repair and regeneration, and to evaluate its mechanical properties and biocompatibility. MethodsFresh bladder tissues were obtained from New Zealand rabbits and then treated with 1%SDS and 1%Triton X-100 to obtain BACM. The BACM was combined with PU to fabricate PU-BACM composite scaffold. The tensile strength and elongation at break of BACM and PU-BACM scaffolds were tested. Scaffolds and extracts of scaffolds were prepared to evaluate the biocompatibility. For cell-proliferation analysis, cell counting kit 8 method was used at 1, 3, 5, and 7 days after co-culture of human bladder smooth muscle cell (HBSMC) and scaffolds. The cell cycle was tested by flow cytometry after HBSMC co-cultured with extracts of scaffolds and DMEM culture medium (control group) for 24 hours. Finally, 12 New Zealand rabbits were used to establish the model of bladder repair and regeneration. Incision of 5 mm was made on the bladder, and PU-BACM scaffold was sutured with the incision. The rabbits were sacrificed at 10, 20, 40, and 60 days after surgery to observe the inflammatory cell infiltration, new tissues formation, and regeneration of epithelium by HE staining. ResultsThe tensile strength of BACM and PU-BACM composite scaffold was (5.78 ± 0.85) N and (11.88 ± 3.21) N, and elongation at break was 14.46%±3.21% and 23.14%±1.32% respectively, all showing significant diffeence (t=3.182, P=0.034；t=4.332, P=0.012). The cell-proliferation rates of controls, PU, BACM, and PU-BACM were 36.78%±1.21%, 30.49%±0.89%, 18.92%±0.84%, and 22.42%±1.55%, it was significantly higher in PU-BACM than BACM (P<0.05). In the bladder repair and regeneration experiment, inflammatory cell infiltration was observed at 10 days after operation, and reduced at 20 days after implantation. In the meanwhile, the degradation of scaffolds was observed in vivo. The regeneration of epithelium could be observed after 40 days of implantation. At 60 days after implantation, in situ bladder tissue formed. ConclusionPU-BACM composite scaffold has higher mechanical properties and better biocompatibility than BACM scaffold. PU-BACM composite scaffold will not lead to strong immune response, and new bladder tissue can form in the in vivo rabbit bladder repair experiment. These results can provide research basis and theoretical data for further study.
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 the possibility to bridge peripheral nerve defects by xenogeneic acellular nerve basal lamina scaffolds. METHODS: Thirty SD rats were randomly divided into 5 groups; in each group, the left sciatic nerves were bridged respectively by predegenerated or fresh xenogeneic acellular nerve basal lamina scaffolds, autogenous nerve grafting, fresh xenogeneic nerve grafting or without bridging. Two kinds of acellular nerve basal lamina scaffolds, extracted by 3% Triton X-100 and 4% deoxycholate sodium from either fresh rabbit tibial nerves or predegenerated ones for 2 weeks, were transplanted to bridge 15 mm rat sciatic nerve gaps. Six months after the grafting, the recovery of function was evaluated by gait analysis, pinch test, morphological and morphometric analysis. RESULTS: The sciatic nerve function indexes (SFI) were -30.7% +/- 6.8% in rats treated with xenogeneic acellular nerve, -36.2% +/- 9.7% with xenogeneic predegenerated acellular nerve, and -33.9% +/- 11.3% with autograft respectively (P gt; 0.05). The number of regenerative myelinated axons, diameter of myelinated fibers and thickness of myelin sheath in acellular xenograft were satisfactory when compared with that in autograft. Regenerated microfascicles distributed in the center of degenerated and acellular nerve group. The regenerated nerve fibers had normal morphological and structural characters under transmission electron microscope. The number and diameter of myelinated fibers in degenerated accellular nerve group was similar to that of autograft group (P gt; 0.05). Whereas the thickness of myelin sheath in degenerated accellular nerve group was significantly less than that of autograft group (P lt; 0.05). CONCLUSION: The above results indicate that xenogeneic acellular nerve basal lamina scaffolds extracted by chemical procedure can be successfully used to repair nerve defects without any immunosuppressants.
ObjectiveTo prepare human acellular adipose tissue matrix and to evaluate the cellular compatibility so as to explore a suitable bio-derived scaffold for adipose tissue engineering. MethodsThe adipose tissue was harvested from abdominal skin graft of breast cancer patients undergoing radical mastectomy or modified radical mastectomy, and then was treated with a series of decellularization processes including repeated freeze-thaw, enzyme digestion, and organic solvent extraction. The matrix was examined by histology, immunohistochemistry, DAPI fluorescence staining, and scanning electron microscopy to observe the the removal of cells and to analyze its composition of collagen type IV, laminin, and fibronectin, and microstructure. The 3rd passage human adipose-derived stem cells (hADSCs) were co-cultured with acellular adipose tissue matrix and different concentrations of extracted liquid (100%, 75%, 50%, and 25%). The cytotoxic effects of the matrix were tested by MTT. The biocompatibility of the matrix was detected by live/dead staining and scanning electron microscopy observation. ResultsThe acellular adipose tissue matrix basically maintains intrinsical morphology. The matrix after acellular treatment consisted of extracellular matrix without any cell components, but there were abundant collagen type I; neither DNA nor lipid residual was detected. Moreover, the collagen was the main component of the matrix which was rich in laminin and fibronectin. At 1, 3, and 5 days after co-cultured with hADSCs, the cytotoxic effect of matrix was grade 0-1. The matrix displayed good cell compatibility and proliferation. ConclusionThe acellular adipose tissue matrix prepared by repeated freeze-thaw, enzyme digestion, and organic solvent extraction method remains abundant extracellular matrix and has good cellular compatibility, so it is expected to be an ideal bio-derived scaffold for adipose tissue engineering.