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Bao H.-J.,Shanghai University | Zou J.,Shanghai University | Yin S.,Shanghai Tissue Engineering Research and Development Center | Cui L.,Shanghai Tissue Engineering Research and Development Center
Fudan University Journal of Medical Sciences | Year: 2010

Objective To explore the feasibility of using autologous rabbit adipose derived stem cells (rASCs) for corneal stromal defect repairing with biodegradable polylactic-co-glycolic acid (PLGA) as scaffold. Methods ASCs isolated from rabbit lipoaspirate were cultured to passage 4 and seeded on PLGA to fabricate cell-PLGA constructs. Scanning electron microscope was used to observe the adhesion and proliferation of cells on scaffold. After 1 week of cultivation in vitro, the construct was implanted into rabbit corneal stroma. The tissue engineered cornea was observed and harvested for histological evaluation and transmission electron microscopy (TEM) examination at 12 weeks and 24 weeks after transplantation. The rabbits implanted with PLGA scaffold alone without cells were served as control. Results rASCs attached firmly and grew well on the scaffold in vitro. The PLGA material was degraded and the cornea was nearly transparent at 12 weeks after transplantation in vivo. Histological structure of tissue engineered cornea was relatively similar to that of normal cornea at 24 weeks after surgery. The diameter of collagen fibrils observed by transmission electron microscopy showed no significant difference with that of normal stroma. Conclusions Autologous rabbit adipose derived stem cells (rASCs) could be seed cells for constructing tissue engineered corneal stroma.


Zhao L.,Shanghai Tissue Engineering Research and Development Center | Zhao L.,Shanghai JiaoTong University | He C.,Shanghai Tissue Engineering Research and Development Center | Cui L.,Shanghai JiaoTong University
Journal of Biomimetics, Biomaterials, and Tissue Engineering | Year: 2010

To investigate the influence of initial copolymer compositions of poly (lactic-co-glycolic acid) (PLGA) on mechanical properties, degradation behavior and biological properties of the scaffolds, porous PLGA scaffolds with different initial copolymer compositions (lactide/glycolide (PLA/PGA) molar ratio: 50:50, 70:30 and 80:20) were prepared by solvent casting/particulate leaching method. Mechanical properties were measured by testing the tensile strength and degradation rate was detected by soaking the scaffolds in phosphate buffered solution at 37 °C for various time points. Human dermal fibroblasts were seeded on PLGA scaffolds with different copolymer compositions. The morphology, adhesion efficiency, proliferation rate, and total collagen contents of cells on the scaffolds were analyzed. The results showed that the ratio of PLA/PGA is one important factor which influences the degradation of scaffolds. The mechanical strength of PLGA scaffolds with the ratio of 70:30 and 80:20, was higher than that of PLGA scaffolds with the ratio of 50:50.. Compared to 70:30 and 80:20 PLGA scaffolds, 50:50 PLGA had a quicker degradation. The three PLGA scaffolds had no obvious difference for cell response and all of them had excellent cytocompatibility, indicated by their high efficiency for human dermal fibroblast adhesion, fast proliferation rate and stretched cell morphology. A large amount of extracellular matrix was secreted and after 7 days of culture, and cell nearly covered the entire surface of the scaffolds. Overall, our results indicate that the copolymer compositions of PLGA have important effect on degradation and mechanical strength, but have no obvious effect on the biological properties of the scaffolds. © (2010) Trans Tech Publications, Switzerland.


Zhu W.-D.,Shanghai JiaoTong University | Xu Y.-M.,Shanghai JiaoTong University | Feng C.,Shanghai JiaoTong University | Fu Q.,Shanghai JiaoTong University | And 2 more authors.
World Journal of Urology | Year: 2010

Purpose: To assess the feasibility of seeding adipose-derived stem cells (ADSCs) onto bladder acellular matrix grafts (BAMGs) for bladder reconstruction in a rabbit model. Methods: Autologous 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 4, 12 and 24 weeks after grafts implantation. Following cystography, the animals were killed and grafts were harvested; H&E and immunohistochemical staining were performed with cytokeratin AE1/AE3, smooth muscle α-actin and S-100 markers. Results: Flow 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 pre-cystectomy 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 S-100. 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. Conclusions: These data demonstrate that seeding ADSCs onto BAMGs promote regeneration of smooth muscle and nervous tissue regeneration in a rabbit model. This compound graft was more suitable for bladder reconstruction than BAMG alone. © 2010 Springer-Verlag.


Feng C.,Shanghai JiaoTong University | Xu Y.-M.,Shanghai JiaoTong University | Fu Q.,Shanghai JiaoTong University | Zhu W.-D.,Shanghai JiaoTong University | And 2 more authors.
Journal of Biomedical Materials Research - Part A | Year: 2010

The aim of this study was to evaluate the mechanical properties and biocompatibility of biomaterials, including bladder submucosa (BAMG), small intestinal submucosa (SIS), acellular corpus spongiosum matrix (ACSM), and polyglycolic acid (PGA), to identify the optimal scaffold for urethral tissue engineering. Tensile mechanical testing was conducted to evaluate mechanical properties of each scaffold. Rabbit corporal smooth muscle cells were cultured with the extracts of biomaterials and mitochondrial metabolic activity assay was used to determine the cytotoxicity of scaffold. The pore sizes of each scaffold were measured. Additionally, smooth muscle cells were seeded on biomaterials. Cell infiltration was evaluated. Mechanical evaluation showed that Young modulus, stress at break in ACSM were prior to those in other biomaterials (p < 0.05). MTT assay confirmed that all scaffolds supported normal cellular mitochondrial metabolic without inducing cytotoxic events. SEM demonstrated that PGA has the largest pore size (>200 μm). The ACSM has different pore sizes in urethral (<5 μm) and cavernosal surfaces (>10 μm). Widespread distribution of cells could be observed in PGA 14 days after seeding. Multilayer cellular coverage developed in BAMG and urethral surface of ACSM without any sign of cellular invasion. Moderated cellular penetration could be found in SIS and cavernosal surface of ACSM. Although each scaffold demonstrated suitable mechanical properties, which is similar to normal urethra, ACSM showed better response in some parameters than those in other biomaterials. It suggested that this scaffold may be an alternative for urethral reconstruction in the future. © 2010 Wiley Periodicals, Inc.


Fu Q.,Shanghai JiaoTong University | Song X.-F.,Shanghai JiaoTong University | Liao G.-L.,Shanghai JiaoTong University | Deng C.-L.,Shanghai JiaoTong University | Cui L.,Shanghai Tissue Engineering Research and Development Center
Urology | Year: 2010

Objectives: To investigate the application of adipose-derived stem cell (ADSC) technology in the treatment of stress incontinence. Methods: The vaginal balloon dilatation method was used to establish an animal model of stress incontinence (in 20 female Sprague-Dawley rats), which was further examined by urodynamics and histology. Endogenous rat ADSCs were collected and induced into myoblasts with 5-Aza induction technology in vitro. The identity of myoblasts was confirmed through immunofluorescence labeling with desmin and myosin. Induced cells were injected into the posterior urethral muscularis in the bladder neck of animals with stress incontinence. The effects were examined after 1 and 3 months by urodynamics and histology. Untreated ADSCs were also implanted as a method of control. Results: Both maximal bladder capacity and leak point pressure significantly increased after 1 and 3 months postimplantation, compared with the control (P <.05). Increased thickness of inferior muscularis in urethral mucosa and a greater number of large longitudinal muscle bundles were observed. Increased numbers of myoblasts appeared under the mucosa, as demonstrated by the immunochemistry analysis of α-smooth actin. Conclusions: ADSCs have the ability of differentiating into multiple lineages, including myoblasts. This ability to induce myoblasts can be used to treat stress incontinence, with the advantages of minimal invasion and faster recovery. © 2010 Elsevier Inc. All rights reserved.


Feng C.,Shanghai JiaoTong University | Xu Y.-M.,Shanghai JiaoTong University | Fu Q.,Shanghai JiaoTong University | Zhu W.-D.,Shanghai JiaoTong University | Cui L.,Shanghai Tissue Engineering Research and Development Center
Tissue Engineering - Part A | Year: 2011

Tissue engineering technique seems to be the best strategy for treating the urethra disease in the near future. In present study, porcine acellular corpus spongiosum matrices (ACSMs) were combined with autologus corporal smooth muscle cells (CSMCs) and lingual keratinocytes by static-dynamic seeding method to form three-dimensional (3D) "neourethra" tissue for repairing the urethral defection. Six scaffolds with two kinds of cells (Group C), six scaffolds with only lingual keratinocytes (Group B), and six matrices alone (Group A) were used to repair the rabbit urethral defection. Retrograde urethrography and histological analyses were performed to evaluate the results of urethroplasty. In vitro, hematoxylin and eosin staining of seeded ACSM showed multiple epithelial layers and well-distributed CSMCs into the matrix. In vivo, the urethra kept a wide caliber in Group C. Strictures were observed in groups A and B. Histologically, the retrieved urethra in group A showed fibrosis and inflammation during 6 months. Simple epithelial layer regenerated in group B while there was no evidence of CSMCs growing into grafts during study period. Stratified epithelial layer and organized muscle fiber bundles were evident after 6 months in group C. Our study suggested that lingual keratinocytes and CSMCs could be used as a source of seed cells for urethral tissue engineering. Using the dynamic-static seeding method, 3D urethra could be constructed in vivo. © 2011, Mary Ann Liebert, Inc.


Feng C.,Shanghai JiaoTong University | Xu Y.-M.,Shanghai JiaoTong University | Zhu W.-D.,Shanghai JiaoTong University | Cui L.,Shanghai Tissue Engineering Research and Development Center | Chen J.,Shanghai JiaoTong University
Journal of Clinical Rehabilitative Tissue Engineering Research | Year: 2011

BACKGROUND: Recently debates on what kinds of urethral repair and reconstruction of tissue engineering scaffolds are more suitable occurred constantly, its evaluation of biocompatibility and mechanical properties are also poorly reported. OBJECTIVE: To evaluate the mechanical properties and biocompatibility applied to urethral repair and reconstruction of a variety of biomaterials. METHODS: Small intestinal submucosa (SIS), bladder acellular matrix graft (BAMG), and acellular corpus spongiosum matrix (ACSM) were prepared by decellularization method; polyglycolic acid (PGA) scaffold was prepared by knitting method. Uniaxial tensile mechanical testing was conducted to determine biomechanical properties of each scaffold; the surface pore size of scaffold was determined by light microscopy and scanning electron microscopy. Cytotoxicity of various biomaterials was detected by MTT assay. Corporal cavernosum smooth muscle cells (CCMSc) were inoculated in all scaffolds, cell permeability was further assessed after 13 days cultured in vitro. RESULTS AND CONCLUSION: Biomechanical evaluation showed that the detection results of ACSM in terms of elastic modulus and fracture strength was prior to other materials (P < 0.05). MTT assay showed that all the scaffolds were in support of normal cell growth and metabolism without obvious cytotoxicity. PGA showed the largest pore size in the scanning electron microscope (> 200 μm); meanwhile, the ACSM has different pore sizes in urethral (< 5 μm) and corpus cavernosum surfaces (> 10 μm). Widespread distribution of cells could be observed in PGA 14 days after seeding. There was no obvious sign of cell penetration in BAMG and ACSM urethral surfaces. However, significant cell penetration growth performances in cavernosal surfaces of SIS and ACSM were observed. It is indicated that all scaffolds demonstrated good biocompatibility, which is similar to normal urethral tissue in terms of mechanical properties. However, ACSM has certain advantages in many parameters of mechanics and histology.

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