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Objective:To prepare the whole bladder acellular matrix (BAM) using the self-designed perfusion decellularization system, and evaluate the feasibility of constructing the tissue engineering bladder with the adipose-derived stem cells (ADSCs).Methods:This study was conducted from October 2020 to April 2021. The self-designed perfusion decellularization system was used, and four different decellularization protocols (group A, group B, group C and group D) were formulated, according to the flow direction of the perfusate and the action time of different decellularization solutions. Among them, the urethral orifice of the bladder tissue was used as the outflow tract of the perfusion fluid in groups A and B. The top of the bladder was cut off and used as the outflow tract of the perfusion fluid in groups C and D. In groups A and C, 1% Triton X-100 was treated for 6 h, and 1% sodium dodecyl sulfate (SDS) was treated for 2 h. In groups B and D, 1% Triton X-100 was treated for 7 h, and 1% sodium dodecyl sulfate (SDS) was treated for 1 h. In addition, the tissue in the normal bladder group was directly obtained from the natural bladder tissue, which did not require perfusion, cryopreservation and thawing. The fast and efficient decellularization protocol was screened out through HE, DAPI, Masson trichrome and Alcian Blue staining and quantitative analyses to prepare the whole bladder scaffold. The prepared BAM was used as the scaffold material, and the ADSCs were used as the seeding cells to construct the tissue engineering bladder. HE and DAPI staining were used to observe the distribution of ADSCs on the BAM.Results:HE and DAPI staining showed that there was no obvious nuclear residue in the group C. Masson trichrome and Alcian Blue staining showed that the collagen structure and glycosaminoglycan were well preserved in the group C. There was no significant difference in bladder wall thickness between the group C and the normal bladder group [(975.44±158.62)μm vs.(1 064.49±168.52)μm, P > 0.05]. The DNA content in the group C [(43.59 ±4.59) ng/mg] was lower than that in the normal bladder group, group A, group B and group D [(532.50±26.69), (135.17±6.99), (182.49±13.69) and(84.00±4.38)ng/mg], and the difference was statistically significant ( P<0.05). The collagen content [(10.98 ± 0.29)μg/mg] and glycosaminoglycan content [(2.30±0.18)μg/mg] in group C were not significantly different with those in the normal bladder group [(11.69±0.49) and (2.36±0.09)μg/mg, P>0.05]. Scanning electron microscopy showed that a large number of pore structures could be observed on the surface of the prepared BAM in groups A-D and were facilitated to cell adhesion. The isolated and cultured ADSCs were identified by flow cytometry to confirm the positive expression of CD90 and CD29, and the negative expression of CD45 and CD106. Live/dead staining and CCK-8 detection confirmed that the prepared BAM in the group C had no cytotoxicity. HE and DAPI staining showed that a large number of ADSCs were distributed on the surface and inside of the tissue engineering bladder. Conclusions:The whole bladder shape BAM prepared by the self-designed perfusion decellularization system could be used as the scaffold material for bladder tissue engineering, and the constructed tissue engineering bladder could be used for bladder repair and reconstruction.
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Objective:To study the correlation between the residual amounts of commonly used tissue decellularization reagents like sodium dodecyl sulfate (SDS), Triton X-100, sulfobetaine 10 (SB-10) and cytotoxicity, and to find the safety threshold for controlling the residual of the reagent.Methods:SDS, Triton X-100, SB-10 solutions of different concentrations were prepared, and the cytotoxicity of solutions of different concentrations was detected by L-929 cells and MC3T3-E1 cells according to the method of GB/T 16886.5. The mass concentration range of the above three decellularization reagents was refined, and the threshold for the residual non-cytotoxicity of a single reagent was determined. Mix the three reagents in pair or three blends, and determine the threshold of non-cytotoxicity after the reagents are mixed by cytotoxicity test.Results:SDS, Triton X-100 and SB-10 caused L-929 cells to produce cytotoxic concentration values of 4×10 -3, 2×10 -2 and 6×10 -1 mg/ml, and MC3T3-E1 cells 6×10 -3, 4×10 -2 and 2 mg/ml. The toxicity trend of the three reagents is SDS>Triton X-100>SB-10. When three reagents at critical concentrations are mixed in pairs or three, they have no cytotoxicity to L-929 cells and MC3T3-E1 cells. Conclusions:When using the above three decellularization reagents to prepare decellularized scaffolds, a variety of reagent cleaning procedures should be used to efficiently clean the tissues so that the residual amount of reagents is below the critical value to ensure the biological safety of the decellularized scaffolds.
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At present, acellular matrix is an effective replacement material for the treatment of skin damage, but there are few systematic evaluation studies on its performance. The experimental group of this study used two decellularization methods to prepare the matrix: one was the acellular matrix which sterilized with peracetic acid first (0.2% PAA/4% ethanol solution) and then treated with hypertonic saline (group A), the other was 0.05% trypsin/EDTA decellularization after γ irradiation (group B); and the control group was soaked in PBS (Group C). Then physical properties and chemical composition of the three groups were detected. Hematoxylin eosin (HE) staining showed that the acellular effect of group B was good. The porosity of group A and B were both above 84.9%. In group A, the compressive modulus of elasticity was (9.94 ± 3.81) MPa, and the compressive modulus of elasticity was (12.59 ± 5.50) MPa in group B. There was no significant difference between group A or B and group C. The total content of collagen in acellular matrix of group A and B was significantly lower than that of group C (1. 662 ± 0.229) mg/g, but there was no significant difference in the ratio of collagen Ⅰ/Ⅲ between group B and group C. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that there was no significant difference in microstructure. Qualitative detection of fibronectin and elastin in each group was basically consistent with that in group C. Therefore, acellular matrix of group B had better performance as scaffold material. The experimental results show that the acellular matrix prepared by γ-ray sterilization and decellularization of 0.05% Trypsin enzyme/EDTA could be used for the construction of tissue-engineered skin. It could also provide reference for the preparation and mounting of heterogeneous dermal acellular matrix. It was also could be used for electrostatic spinning or three-dimensional printed tissue engineered skin scaffold which could provide physical and chemical parameters for it.
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Acellular Dermis , Cells, Cultured , Extracellular Matrix , Porosity , Tissue Engineering , Tissue ScaffoldsABSTRACT
Objective To prepare biofilm scaffolds by using mesenteric acellular matrix, so as to investigate their physicochemical and biological characteristics. Methods The mesenteric tissues were subjected to trypsin digestion, and the mesenteric cells were removed after repeated freezing and thawing of mesenteric tissue. Mesenteries were divided into mesenteric matrix group (Group A) and acellular mesenteric matrix group (Group B). The physical and chemical properties of mesenteric matrix in two groups were tested by HE staining, electron microscopy, DNA detection, cytotoxicity test and tensile mechanics test. The blood flow of the vessels was detected by ultrasonography at 1st week, 1st month and 2nd month, and the vessels were observed by pathological examination. Results HE staining and electron microscopy showed that the mesentery of Group B was loose in acellular mesentery matrix, and the arrangement of fibers was neat, with no cells remaining. Compared with Group A, the expression level of DNA in Group B was lower, with more completely decellularized cells. CCK-8 cytotoxicity test showed that there was no cytotoxicity in Group A and Group B. FDA-PI fluorescence staining showed no cytotoxicity of cells in both groups. Cells in Croup A and Group B survived well, and no dead cells were found. Tensile mechanics test showed that there were no significant differences in maximum tensile force, maximum elongation, yield strength, yield point elongation between Group A and Group B. The early patency of acellular mesenteric stent implantation was good, and endothelial hyperplasia was obvious at 2nd month after stent implantation. Conclusion sMesenteric cells were removed by freeze-thaw and enzymatic digestion. Mesenteric stroma was completely removed without cytotoxicity, which showed good mechanical characteristics. Mesenteric stent implantation had good early patency and endothelial proliferation after 2 months.
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BACKGROUND: Bladder repair is currently one of the main treatments for bladder defects. Homologous tissue is less affected by various factors. Tissue engineered acellular bladder matrix has become an increasing area of interest. Porcine bladder acellular matrix has a wide range of sources and has a natural extracellular scaffold structure, which has become a hot topic in tissue engineering bladder substitute materials. OBJECTIVE: To explore the feasibility of acellular porcine bladder as a tissue engineering scaffold material. METHODS: The cell-free matrix of pig bladder was prepared by liquid nitrogen freezing and thawing, dodecyl sodium sulfate and trypsin decellularization method. According to different decellularization methods, pig bladders were divided into normal control group (without any treatment), experimental group (treated with 0.6% trypsin and 5% sodium lauryl sulfate (pH 8.0)) and acellular control group (treated with 0.75% trypsin (pH 8.0), 1% trypsin (pH 8.0), 5% sodium lauryl sulfate (pH 7.6) or 10% sodium dodecyl sulfate (pH 7.6)). The decellularization effect of pig bladder was observed by hematoxylin-eosin staining, van Gieson staining, DNA quantification, and α-Gal antigen detection. RESULTS AND CONCLUSION: Hematoxylin-eosin staining revealed that in the experimental group, the components of the bladder cells of the pigs were basically removed. van Gieson staining revealed that the DNA residues and α-Gal antigen residues in the cells were significantly lower than those in the control group (P < 0.05). These results suggest that treatment of pig bladder with 0.6% trypsin and 5% sodium dodecyl sulfate can effectively remove its cellular components while retaining the extracellular matrix of porcine bladder tissue. This provides a reference value for constructing accelular porcine bladder scaffolds.
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Objective: To study the vascularization of tubular graft of bone marrow mesenchymal stem cells (BMSCs) and bladder acellular matrix (BAM) wrapped up in omentum. Methods: We planted BMSCs on the BAM and made it into tubular grafts. Thirty healthy New Zealand white rabbits were randomly divided into three groups with 10 rabbits in each. Rabbits in experimental group received BMSCs-BAM tubular graft wrapped in omentum. Rabbits in control group received BAM tubular graft wrapped in omentum. Rabbits in blank group were planted with silicone catheter wrapped in omentum. Vascular endothelial growth factor (VEGF) protein, CD34 expression and microvessel density of the graft were measured at weeks 4 and 8, respectively. Results: ① The expression of VEGF protein was higher in the experimental group than in the control group and the blank group, and the expression of VEGF protein was higher in the control group than in the blank group; the differences among the three groups were statistically significant [F(2,15)=5.314, P=0.017]. ② The microvessels with positive CD34 expression were denser in the experimental group than in the other two groups, and the blood vessels were closely connected with each other. The microvessels with positive CD34 expression were also much denser in the control group than in the blank group. ③ The microvessel density was higher in the experimental group than in the control group and the blank group, and it was also higher in the control group than in the blank group. The differences among the three groups were statistically significant [F(2,12)=6.172, P=0.021]. Results: Omental wrapping promotes vascularization of BAM, and planting BMSCs on the BAM is more conducive to vascularization of BAM.
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Objective: To explore the effects of the microenvironment of rabbit bladder acellular matrix graft (BAMG) on proliferation, cell surface markers, and molecular protein level of human bone marrow mesenchymal stem cells (hBMSCs). Methods: We prepared BAMG immersion fluid medium and detected its effect on the proliferation of hBMSCs by MTT method. The expressions of CD44, CD45, CD73 and PDGFRβ were detected by flow cytometry. The expressions of PPAR, OCN and α-SMA were detected by RT-PCR, and the expression of OCT was detected by Western blot. Results: hBMSCs had good compatibility with BAMG. The MTT method showed that BAMG and BAMG immersion medium did not affect the proliferation capacity of hBMSCs. The surface of hBMSCs cells cultured with immersion fluid still expressed CD44, CD73 and PDGFRβ, but not CD45. RT-PCR showed that OCN, PPAR, and α-SMA were all expressed. Western blot test also showed the positive expression of OCT-4. Conclusion: hBMSCs can still keep their original biological characteristics in the microenvironment of rabbit BAMG. It can be the seed cells and combined substrate materials for urinary system tissue engineering.
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Objective To explore the feasibility of the application of yak pericardium as anti-calcified heart valve materials in transcatheter aortic valve replacement (TAVR). Cattle pericardium and commercial Sino products were used as controls. Methods Yak and cattle pericardium was decellularized, and then the acellular extent was qualitatively and quantitatively analyzed with hematoxylin-eosin (HE) staining and DNA detection kit respectively. Two cross-linking agents, i.e. glutaraldehyde and genipin, were used to cross-link the acellular pericardium, respectively. After testing the mechanical property, thermodynamic stability and biocompatibility of the pericardium, the cross-linked pericardium was subcutaneously implanted in juvenile Wistar rats. After 2～4 weeks of the implantation, histological staining and tissue calcification analysis were conducted. Results The nuclei of the yak pericardium were almost invisible after decellularized treatment, and the DNA content decreased from (0.90±0.13)μg/mg to (0.09± 0.02)μg/mg that was significantly lower than that of Sino product (P<0.001). The mechanical strength of the acellular yak pericardium, glutaraldehyde or genipin cross-linked yak pericardium was higher than that of the cattle pericardium (all P<0.05). The maximum stress value, obtained in the glutaraldehyde cross-linked yak pericardium, was (8.44±2.61) MPa, which was higher than (7.92±1.81) MPa of the Sino product (P<0.05). The shrinkage temperature of non- and cross-linked yak pericardium was slightly higher than that of cattle pericardium, but difference was not statistically significant (all P>0.05). For the two cross-linked yak pericardium, the hemolysis rate and cell proliferation ratio was similar with that of cattle pericardium (all P>0.05), the tissue regeneration ability of subcutaneous implantations was inferior, and the calcification level was higher than that of cattle pericardium with an no statistically significant difference (all P>0.05). After 4 weeks of the subcutaneously implantation, the calcium content of the glutaraldehyde cross-linked pericardium was 32.62～65.49μg/mg. Conclusions Yak pericardium has better mechanical properties and thermodynamic stability than cattle pericardium, and the biocompatibility can meet the requirements of transcatheter aortic valve replacement.
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Objective To evaluate the biochemical function of rat nature three-dimensional acellular pancreatic bioscaffold (3D-APB) in promoting cell proliferation and differentiation in vitro.Methods The fresh pancreas from 10 rats were perfused to prepare 3D-APB.The biocompatibility of 3D-APB was eva luated.The experiment was divided into 4 groups based on 4 kinds of three-dimensional media for AR42J culture,including blank control group,ECM group,PLGA group and 3D-APB group.We compared the proliferation and differentiation of AR42J pancreatic acinar cell at 3 days,5 days,7 days and 10 days after seeding among 4 groups.Results The 3D-APB could represent a biocompatible scaffold capable of integrating within host tissue.The proliferation rate of AR42J cells by MTT in 3D-APB was higher,while the apoptosis rate by flow cytometry was lower than those in other 3 media,which were all significantly different (P ＜ 0.05),respectively.The protein expression of PDX-1 and PTF-1 by western blot in 3D-APB group was greatly higher than those in other 3 groups (both P ＜ 0.05).The mRNA expression of PDX-1 and PTF-1 through qRT-PCR was significantly higher than those in other 3 groups (both P ＜ 0.05).Conclusion Compared with the commonly used chemical and natural scaffold at present,3D-APB could promote cell proliferation and differentiation,which was more appropriate for regenerative medicine.
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Objective · To investigate the micro/ nano structure of human vaginal wall acellular matrix, and provide parameters for bionic vagina.Methods · A total of 35 vaginal specimens were obtained from 35 postmenopausal patients who underwent cystocele repair and/or rectocele repair.Cells in specimens were entirely removed, and the extracellular matrix was maintained. Then the acellular matrix was observed by general observation,H-E staining, Masson staining, scanning electron microscope and Micro-CT. The external and internal structure properties were measured and analyzed.Results · All cells in 35 vaginal specimens were totally removed. The vaginal wall acellular matrix was a double-layer fibrous reticular structure composed of interlaced collagen fibers, which were further arranged into bundles. Pores were distributed among fiber bundles. Epithelial side structure of vaginal wall acellular matrix was dense and subepithelial tissue was loose. There was no significant difference in fiber diameter [(82.4±9.3) nm vs (87.5±10.2)nm, P=0.432] and fiber bundle thickness [(67.6±9.3) μm vs (65.3±5.2) μm, P=0.634] between the dense and loose layers. Fiber bundle separation [(180.1±24.5) μm vs (118.2±23.0) μm, P=0.003] and total porosity [(77.1±4.2)% vs (66.6±2.8)%, P=0.002] were higher in loose layer. There was no significant difference in the micro/nano structure parameters of the acellular matrix between the anterior vaginal wall and the posterior vaginal wall.Conclusion · There was no significant difference in the fiber diameter and the fiber bundle thickness between the dense layer and the loose layer of vaginal wall acellular matrix, but the porosity of the dense layer fiber bundle was smaller than that of the loose layer.
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Objective · To investigate the micro/ nano structure of human vaginal wall acellular matrix, and provide parameters for bionic vagina.Methods · A total of 35 vaginal specimens were obtained from 35 postmenopausal patients who underwent cystocele repair and/or rectocele repair.Cells in specimens were entirely removed, and the extracellular matrix was maintained. Then the acellular matrix was observed by general observation,H-E staining, Masson staining, scanning electron microscope and Micro-CT. The external and internal structure properties were measured and analyzed.Results · All cells in 35 vaginal specimens were totally removed. The vaginal wall acellular matrix was a double-layer fibrous reticular structure composed of interlaced collagen fibers, which were further arranged into bundles. Pores were distributed among fiber bundles. Epithelial side structure of vaginal wall acellular matrix was dense and subepithelial tissue was loose. There was no significant difference in fiber diameter [(82.4±9.3) nm vs (87.5±10.2)nm, P=0.432] and fiber bundle thickness [(67.6±9.3) μm vs (65.3±5.2) μm, P=0.634] between the dense and loose layers. Fiber bundle separation [(180.1±24.5) μm vs (118.2±23.0) μm, P=0.003] and total porosity [(77.1±4.2)% vs (66.6±2.8)%, P=0.002] were higher in loose layer. There was no significant difference in the micro/nano structure parameters of the acellular matrix between the anterior vaginal wall and the posterior vaginal wall.Conclusion · There was no significant difference in the fiber diameter and the fiber bundle thickness between the dense layer and the loose layer of vaginal wall acellular matrix, but the porosity of the dense layer fiber bundle was smaller than that of the loose layer.
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Objective To construct tissue engineering nucleus pulposus by culture of rabbit bone marrow mesenchymal stem cells (rBMSCs)-nucleus pulposus acellular matrix scaffold (NPAMS)complexes (rBMSCs-NPAMS).Methods Several NPAMS were prepared,and rBMSCs was seeded into NPAMS.The scaffolds and complex were detected by general observation,HE stai-ning,immunohistochemical,qRT-PCR,scanning electron microscopy.Results The scanning electron microscopy showed the seed cell in nucleus pulposus ECM-derived scaffold could adhesion and growth.The cell attachment and proliferation were observed by HE staining.Immunohistochemical examination with typeⅡ collagen showed positive results.qRT-PCR revealed the time-depend-ent of the mRNA expression of collagen Ⅱ,and which was smaller than positive control(P0.05).Con-clusion Natural nucleus pulposus acellular matrix scaffold composite allogeneic bone marrow mesenchymal stem cells can be suc-cessfully built into tissue engineering nucleus pulposus.
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Objective To explore the feasibility of human lung canccr cells grown in a decellularized rat lung matrix by perfusion.Methods Lungs were harvested from adult SD rats.Native cells of rat lungs were removed using 0.1％ sodium dodecyl sulfate (SDS) and 1％ Triton X-100 by perfusion to create a decellularized rat lung matrix.After decellularization,Human lung cancer H460 cells were implauted into the decellularized rat lung matrix and grown in a customized bioreactor with perfusion of oxygenated media for 1-2 weeks.Results Decellularized rat lung matrix showed preservation of matrix architecture devoid of all rat cells.H460 cells could grow in the bioreactor.Conclusions Human lung cancer H460 cells can grow in a customized bioreactor on a decellularized rat lung matrix.This ex vivo model can be used potentially to gain a deeper understanding of the biologic processes involved in human lung cancer.
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Objective To establish osteochondral scaffolds with remained calcified cartilage zone for finding an ideal scaffold for tissue engineered repair of osteochondral defect.Methods Cartilage zone was harvested from fresh adult porcine knee to fabricate type Ⅱ collagen hydrogel.Bone blocks measuring 8 mm in diameter with calcified cartilage zone were prepared by trephine and acellular treatment was performed.Histological staining was used to identify complete removal of cells.To fabricate the osteochondral models containing calcified cartilage zone,type Ⅱ collagen was seeded onto the acellular bone blocks with calcified cartilage zone,lyophilized,and cross-linked with 10 g/L of genipin ethanol solution.Then cell seeding and scanning electron microscope were performed after the models were established.Results HE staining,toluidine blue staining,solid green staining,safranin O staining,and DAPI staining showed cells were completely removed from bone blocks by decellularized process.Porosity of type Ⅱ collagen sponge was (91.1 ±3.8) ％ and pore size was (79.7 ± 17.1) μm.Porosity of acellular bone blocks was (73.5 ±2.6)％ and pore size was (470.2 ± 158.8) μm.Cells seeded onto osteochondral scaffolds grew well by scanning electron microscope.Conclusion Osteochondral scaffolds with calcified cartilage zone provide good biocompatibility and suitable pore size and porosity and may be an ideal material for repairing osteochondral defect in tissue engineering.
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ObjectiveTo investigate the clinical safety and validity of heterogeneous dermal acellular matrix graft (ACMG) for urethral reconstruction in patients.Methods Ten male patients aged 36( range,20 - 62),stricture length 6.9 cm ( range,3.0 - 15.0 cm),were treated with the ACMG material.During the operation,ACMG was sutured to a tubular graft or as a patch and replaced the defect urethra.Urinary catheter was inserted in the reconstructed urethra for 6 months.Then the outcomes of this operation method and whether there were rejections against the material were observed.Results All 10 patients could void through normal urethral,and no rejection was observed.Retrograde urethral visualization and urethroscope gave evidences of smooth urethral and continuous mucosa.The patients were followed up for 18months,and all patients voided normally except 2 cases needed urethral dilatation.ConclusionsHeterogeneous dermal ACMG for urethroplasty in men has the excellence of good biocompatibility,constructing the functional urethra.Additionally,this material could be manufactured with greater scale and advanced technology,which made it appropriate for operation of harder and longer urethral stricture diseases,serving as an ideal replacement material of urethra.
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ObjectiveTo assess the feasibility of seeding adipose-derived stem cells (ADSCs) onto bladder acellular matrix grafts (BAMGs) for bladder reconstruction in a rabbit model.MethodsAutologous ADSCs were isolated,expanded and identified by flow cytometry.In the experimental group,ADSCs were seeded onto BAMGS for reconstructing bladder defects in 12 male rabbits.Unseeded BAMGs were used for bladder reconstruction in the control group of 12 rabbits.Cystography was performed at 24 weeks after grafts implantation.Following cystography,the animals were scarified and grafts were harvested； H&E and immunohistochemical staining were performed with cytokeratin AE1/AE3,smooth muscle α-actin and S-100 markers.ResultsFlow cytometry demonstrated that the ADSCs expressed CD90,CD44,CD105,CD166 and CD34,but not CD45 or CD106.The cells demonstrated good biocompatibility with BAMGs.At 24 weeks,in the experimental group,the reconstructed bladders reached a mean volume of (94.68 ± 3.31 )％ of the precystectomy bladder capacity.Complete regeneration of smooth muscle and nerve tissue was evident.Regenerated SMCs,urothelium and nerve cells stained positively for α-smooth muscle actin,AE1/AE3 and S100.In the control group,the mean bladder volume was (69.33 ± 5.05 )％ of the pre-cystectomy volume.Histologically,the control group was characterized by multi-layered urothelium without evidence for organized muscle or nerve tissue.Conclusion The tissue engineering bladder constructed by ADSCs and BAMG can be used as an ideal biomaterial to replace and repair the bladder.
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Objective To investigate and assess the best seeding method for constructing three dimensional urethral tissue in vitro. Methods High speed agitation decellular method was used for preparing the porcine acellular corporous spongiosum matrix (ACSM). Before seeding, the matrix was sterilized via soaking compound iodine solution. Rabbit tongue epithelial cells and cavernosal smooth muscle cells were isolated and cultured. Three different groups of seeding method was used in this study. Group A (sandwich seeding group): The smooth muscle cells and epithelial cells were seeded onto the different side of ACSM by static method. Group B (injection seeding group): The smooth muscle cells were injected into the scaffold. Then, epithelial cells were seeded onto the urethral surface of ACSM by static method. Group C (agitation seeding group): The smooth muscle cells were seeded into the scaffold by agitation method. Then, epithelial cells were seeded onto the urethral surface of ACSM by static method. After being seeded, all matrixes were cultured in vitro for 14 d. HE and immunoassay staining were used to examine the results of seeding. Results Looser matrix was obtained after using high speed agitation decellular method. Compound iodine solution could not only sterilize efficiently but also reserve the original structure of biomaterial. An intact epithelial cellular layer onto the surface of scaffold could be observed in HE staining section after 14 d culturing in vitro.Few smooth muscle cells could be found in big space of biomaterial in group A. In group B, smooth muscle cells were restrained in some regions of the matrix. Smooth muscle cells were well distributed into the scaffold in group C. Conclusions After using high speed agitation decellular method, an ideal matrix with three dimensional structure can be obtained. Combined with agitated seeding method, three dimensional urethral tissue can be constructed.
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Objective To investigate the feasibility of replacing urinary epithelial cells with oral keratinocytes by being seeded on bladder acellular matrix graft(BAMG). Methods Twenty-four male rabbits were randomly divided into 2 groups:experimental group and control group.A length of 2.0 cm and width of 0.8 cm penile urethral mucosal defect was induced in the anterior urethra 2.0 cm awav from the urinary meatus in all the rabbits.Oral keratinocytes were isolated from a small buecal mucosa(1.0 cm×0.4 cm)of the 12 rabbits in experimental group and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3).Passage 2 oraI keratinocytes cuItured with i3T3 were expanded and seeded onto sterilized BAMG(2.2 cm×1.0 cm)to repair the de-fects of the urethra.Urethroplasty was performed with BAMG with no cell seeding in the controlgroup.Catheter examination and retrograde urethI ography was done in 1,2 and 6 months after graft-ing.The urethral grafts were harvested and analyzed by histological staining. Results Oral kerati-nocytes seeded onto a feeder layer of i3T3 had better morphous and amplification capability.The corn-patibility of the compound graft was assessed by HE staining and scanning electron microscope.Twelve rabbits in the experimental group could void without difficulty.Catheter examination and ret-rograde urethrogram revealed that a wide urethral caliber was maintained with no sign of strictures and fistula formation.Histological examination showed the grafted urethra was covered with smooth mu- cosa with no strictures at 1,2 and 6 months.The oral keratinocytes of the graft still existed even 6 months after grafting.On contrast,gross and urethrogram demonstrated urethral stricture in the con-trol group.And inflammatory reactions could be found in the area of the stricture through light micro-scope. Conclusions Oral keratinocytes have good compatibility with BAMG and the compound graft could be used for urethral reconstruction in the animal model.
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[Objective]To investigate the methods of preparing acellular cartilaginous matrix(ACM) and constructing the tissue engineered cartilage with adipose-derived stem cells(ADSCs)-seeded acellular cartilaginous matrix in vitro.[Method]The ADSCs were isolated from adult New Zealand albino rabbits by collagenase,cultured and amplified in vitro.Fresh cartilage isolated from adult New Zealand albino rabbits were freeze-dried for twelve hours and then treated with Triton X-100,Dnase and RNase so as to obtain the ACM.After sterilized with ultra-violet,the ADSCs were seeded in the acellular cartilaginous matrix at a final density of 2?107/L and cultured in chondrogenic differentiation medium for two weeks in order to construct tissue engineered cartilage.Histology,immunohistochemistry and transmission electron microscope(TEM) were applied to examine the fresh ACM and tissue engineered cartilage.[Result]The test of hematoxylin-eosin(HE) staining and TEM showed no cellular structure in the ACM with only recesses left by removed cells.The ACM had suitable interval porosity and aperture size.After integrated with ADSCs,cells migrated into the ACM and adhered to the surface of material and grew well.After cultured in chondrogenic differentiation medium for two weeks,immunohistochemical staining of type Ⅱ collagen showed part of the cells in the material had enhanced expression of type Ⅱ collagen.[Conclusion]ACM can be used as scaffold material in cartilage tissue engineering.If it was seeded with ADSCs and cultured in chondrogenic differentiation medium,ACM can be used to construct cartilage tissue successfully.
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Objective To find a new method to prepare the acellular matrix of the penile cavernous body. Methods Intact specimens of the penile cavernous body were collected from healty adult rabbits,treated with Triton-X100 and NH3?H2O,and then stained with HE.Light microscopy was employed to observe the acellular cells.Results Acellular matrix of the penile cavernous body was obtained after a 25-day treatment with Triton-X100 and NH3?H2O.The matrix was intact.HE staining showed that the matrix was composed of regularly arranged elastic fibers with wide intra-spaces without any cells inside.Conclusion Acellular matrix of the penile cavernous body can be prepared by using Triton-X100 and NH3?H2O.