Combination Of Electrospun Nanofibrous Scaffold And Adipose-Derived Stem Cells For Urethra Tissue Engineering

Tissue engineering, an important component of regenerative medicine, is a comprehensive discipline combining many kinds of sciences and technologies. The approaches of tissue engineering may very, but most involve the essential strategies including seed-cells selection, scaffolds design and interaction of cells and scaffolds.Adipose tissue is a new source reservoir for adult stem cells. Because of convenient harvesting, minimal invasion and abundant quantity, adipose-derived stem cells (ASCs) have been regarded as forefront seed-cells in tissue engineering. Many studies have revealed a close relationship between anatomic site and cellular behavior. ASCs originate from adipose tissues. However, the impacts of anatomic site of harvesting on ASCs are obscure till now.The synthetic scaffold plays an increasing role in tissue engineering field. The polymers are preferable for various purposes due to stable and controllable properties. Poly (L-lactic acid) (PLLA) is brittle with high degradation and poly (ε-caprolactone) (PCL) is flexile with low degradation. The composites of PLLA and PCL of different weight ratios offer the scaffolds of different properties. The blends of PLLA and PCL, which we prepared from electrospinning method, are three-dimensional in weaving fibers in nanometer scale, indicating their replacement for extracellular matrix.In this study, we compared the biology of ASCs from three anatomic sites and discovered the subcutaneous inguinal region was the best site for cell harvesting. Moreover, we evaluated the effects of weight ratio of PLLA/PCL blend and confirmed the 1/1 synthesized scaffold had the highest performance. On the basis of the above data, we employed ASCs to differentiate towards smooth muscle cells on the electrospun PLLA/PCL blend nanofibers of 1/1 blending weight ratio to explore the feasibility of such construct for tissue engineered-urinary tract. Objectives To study and compare the impacts of harvesting anatomic site on the biological behaviors of adipose-derived stem cells (ASCs), and to provide theoretical support for the strategy of seed-cell selection in tissue engineering.Materials and Methods Five female New Zealand white rabbits born simultaneously were used in our study. They were raised in the identical environment until to 4 months age old and weighed averagely 2.4kg (ranging from 2.37 to 2.42kg). With the general anesthesia by auricular vein injection, the adipose tissues were resected under the asepsis condition from three different anatomic sites of each rabbit, including subcutaneous inguinal (SI), subcutaneous dorsocervical (SD) and retroperitoneal perinephric (RP) regions. The ASCs obtained from removed tissues were isolated and cultured conventionally and categorized into three experimental groups according to their region of origin. The ASCs of three groups were compared on the biological properties by the following methods:(1) ASCs were characterized by morphological observation under an inverted phase contrast microscope and the expression of surface markers, CD 105 and CD 166, by fluorescence-activated cell sorting (FACS). (2) Cell counting and MTT assay were employed to evaluate proliferation. (3) A known standard curve and Live/Dead cells staining were employed to evaluate viability. (4) Histochemistry staining (ALP staining and Oil red O staining), Real-time PCR and Western blot were employed to evaluate the multilineage differentiation towards to osteogenesis and adipogenesis.Results (1) Characterization of ASCs:①ASCs from three anatomic sites demonstrated typical forms of the mesenchymal stem cells (MSCs). No obvious difference was found by macroscopic observation.②The positive percentages of CD 105 and CD 166 were assessed by FACS and analyzed to nonsignificant difference. (2) Proliferation assay:①On day 4, the average numbers of the ASCs in SI group and SD groups were significantly higher than those in RP group (both p<0.05), with no significant difference between SI and SD group (p>0.05). On day 7, no significant difference was detected among the ASCs from three anatomic sites (p>0.05).②The one way ANOVA was used to compare the growth curves of three groups generated from MTT assay data. The subcutaneous (SI and SD) ASCs had both statistical significance to RP ASCs (both p< 0.05), while nonsignificance was determined between SI and SD ASCs (p>0.05). (3) Viability assay:①The three groups had significant differences in the number of living cells, with a decline from SI to SD to RP ASCs (p<0.05 for these comparisons).②The living cells were counted in five random views of Live/Dead staining. The differences between any two or all of the sites were not statistically significant (all p>0.05). (4) Differentiation capacity:①At the end of osteogenetic culture, the SI ASCs had higher ALP activity than SD or RP cells (both p<0.05). At the end of adipogenic culture, there were no significant differences among the three groups (p>0.05).②All of the gene markers were detected by Real-time PCR during osteogenesis and adipogenesis. In the evaluation for each gene, the expression levels of the subcutaneous (SI and SD) groups were significant higher than that of RP group (both p<0.05). In the comparison between two subcutaneous groups, SI ASCs acted better than SD ASCs (p<0.05).③The results of protein expression levels determined by Western blot were in agreement with Real-time PCR.Conclusions (1) ASCs are a typical type of MSCs, whose morphology and phenotype complied with the MSCs'characteristics; (2) The derived anatomic site has impacts on the proliferation, viability and differentiation capacity of ASCs. The subcutaneous ASCs are preferable than retroperitoneal ASCs, and the SI ASCs are better than SD ASCs. (3) The inguinal region may be a applicable resource reservoir for cell harvesting. (4) The ASCs are capable of multipotential to osteogenesis and adipogeneous, implying their application in tissue engineering. (5) The adipose tissue is highly heterogeneous, both among individuals and also within an individual. PartⅡ. Biocompatibility of electrospun PLLA/PCL blend nanofibrous scaffold and adipose-derived stem cellsObjectives To study the biocompatibility of PLLA/PCL blend fibers prepared from electrospinning method and to discuss the effects of blending weight ration on it.Materials and Methods PLLA and PCL blends with weight ratio of 3/1,2/1 and 1/1 were electrospun into nanofibrous scaffold (NFS). The morphology, porosity and degradation in vitro of such three NFS were compared. The histocompatibility was observed on the inflammatory responses of NFS implanted into subcutaneous pockets of rats. The cytocompatibility was evaluated by proliferation, attachment, viability and multipotential towards to osteogenesis, chondrogensis and adipogenesis.Results 1.Mophology:(1) Average diameter (AD):AD3/1=1.25±0.27μm, AD2/1= 870±45nm, AD1/1=452±24nm. The fibers of 1/1 NFS were significantly thinner than those of 3/1 and 2/1 NFS (both p<0.01); (2) Porosity: The porosity rates of three NFSs were respectively 78.3±3.4%,78.7±5.1% and 82.2±4.8%, with no significant difference among them; (3) Degradation:According to the degradation curve, the rate of degradation was accelerated with the increasing content of PLLA.2. Histocompatibility:The inflammatory responses were all observed during the NFS transplants. The response course was as long as about 2 weeks and vanished at the end of 4 weeks.3. Cytocompatibility:The ASCs were able to attach, grow and differentiate into multilineages on NFSs. The NFS of 1/1 blending ratio was the most suitable scaffold to be fabricated with ASCs.Conclusions (1) The blend composite of PLLA and PCL can be electrospun into nano-scale fibrous scaffold; (2) The weight ratio of PLLA and PCL blending has impacts on morphology, porosity and degradation of electrospun fibrous scaffold; (3) The electrospun PLLA/PCL blend nanofibrous scaffold has good biocompatibility and can be a candidate biomaterial for tissue engineering. PartⅢ. Urethra tissue engineering:leiomyogenic differentiation of adipose-derived stem cells on electrospun nanofibrous scaffoldObjectives To study the leiomyogenic differentiation of adipose-derived stem cells(ASCs) on electrospinning PLLA/PCL blend nanafibrous scaffold and to explore the feasibility of the application of such novel material in urethra tissue engineering.Materials and Methods ASCs obtained from subcutaneous adipose tissue in inguinal region were seeded on the electrospun PLLA/PCL blend nanofibrous scaffold (NFS), which was blended in 1:1 weight ratio, to fabricate ASCs-NFS constructs. The smooth muscle inductive medium was used for ASCs differentiating towards smooth muscle cells (SMCs). The difference between the ASCs-derived smooth muscle cells (AD-SMCs) with and without scaffolds were evaluated by morphological observation, immuno fluorescence, cell proliferation, gene and protein expression assay.Results After 6 weeks, the AD-SMCs demonstrated the typical morphology of SMCs and expressed specific markers, a-actin and myosin heavy chain (MHC). The contents of F-actin of the AD-SMCs were similar with the SMCs. During the proliferation assay, there was no statistical significance between the AD-SMCs with and without the scaffolds (p> 0.05) for 3-week induction. However, great significant difference was detected when it went to the 6th week. Both of the gene transcription and protein expression showed the AD-SMCs were positive to SMCs specific genes or proteins. No effect of the existence of scaffold was found in the course of differentiation from ASCs to SMCs. The AD-SMCs posed good contraction ability by using collagen gel assay.Conclusions (1) ASCs can differentiate into contractile SMCs on PLLA/PCL NFS prepared by electrospinning; (2) The constructs combined ASCs and NFS can be used for the researches of urethra reconstruction; (3) The electrospun PLLA/PCL blend nanofiber can be a candidate material for SMCs tissue engineering.