The Feasibility Study Of Adipose-derived Stem Cells Combined With Biodegradable PLLA/PCL Nanofiber Scaffold To Reconstruct Functional Urethra

Smooth muscle cells (SMCs) are the main functional components of urethra. For the limited ability of proliferation, they are hard to recover after damage, this produce great difficulties for urethral reconstruction. Several groups reported the creation of tissue engineered muscle graft with mechanical and contractile functions using in vitro cultured SMCs isolated from vascular tissue biopsies. However, such mature differentiated SMCs have limited ability of proliferation and usually lose their contractile phenotype followed by switching to synthetic ones during in vitro expansion. What's more, the cultured SMCs always in irregular alignment and hard to achieve unitary contractile function. Thus, generating a functional smooth muscle layer through tissue engineering techniques is a prerequisite for successful reconstruction of urethra, which make it necessary to explore alternative cell source and scaffolds for urethral reconstruction since large numbers of functional cells are usually involved.Stem cells exist in an undifferentiated state, and exhibit both the capacity to self-renew, and the capability to differentiate into more than one type of cells. Although embryonic stem cells seem to exhibit unlimited differentiation potential, they are subject to ethical, legal and political concerns. Stem cells from adult tissue, on the other hand, suffer from few such restrictions. A potential source of stem cells for transplantation is adipose tissue. Adipose-derived stem cells(ASCs) are of mesenchymal origin, can easily be harvested in large quantities, show high proliferation rates in culture and have the capacity to differentiate into diverse cell types, such as dipogenic, osteogenic, chondrogenic, and myogenic lineages.ASCs located in the widespread fatty tissue of the body, with the advantages of plenty of storage and easily obtained, might to be the excellent "seed cells" for tissue engineering of urethra reconstruct. Favourable biological scaffold can stay in harmony with surrounding cells and tissues of the body, so as to contribute to the recoverage of the damaged tissue. Cocultured the seed cells with scaffolds in vitro to make a manmade graft, then the graft can be implant to the damanged tissue and repair large tissue defects.Biomaterial can be divided into two general groups:degradable material and non-degradable material.Non biodegradable materials can't be absorbed by the body and stay in the tissue, thus can not substitute the function of biological tissues, such as self-renew, metabolism and self-optimization ability. Degradable materials can repair the damaged tissue in morphology, structure and function,they degraded into non-toxic molecules such as CO2 and water in the body, left the loaded cells fused in the tissue.In this study, we used the coploymers of PLLA and PCL to synthesize our novel scaffolds,through adjust the ratio of PLLA and PCL, we can make grafts with different flexibility, stretching and degradation time.This study used PLLA and PCL to make directional nanofiber scaffolds through electrospinning technique, cocultured autologous ASCs with the scaffolds and used various ECM to improve the adhesive ability of the scaffolds. Investigated the cell and tissue biocompatibility of the scaffolds and the influence of the ECM to ASCs on its leiomyogenic differentiation.After been successfully induce to AD-SMCs, the cells loaded scaffolds were implanted in the rabbits with urethral defect.We detected the change of pressure of urethra of the rabbits after the scaffolds were implanted, these might give some messages for the feasibility of reconstruct funcitonal urethra through tissue engineering method.Materials and methodsI. Isolate, culture, identification of ASCs and selecting of PLLA/PCL nanofiber scaffolds.1.Isolate and culture of ASCs from New Zealand white rabbits (the same nunmber of male and female) inguinal subcutaneous fat area, subcutaneous fat of neck, fat arround the kidney. Using Zuk's method:wash the fresh adipose tissue (about 1.5g) with sterile PBS solution in vivo and cut it into pieces about 1-2mm in size in DMEM(10%FBS contained); sterile PBS repeatedly washed to remove blood cells and fat cells mixed; Enzyme digest the pieces with 0.1% collagenase under 37℃in the oscillator or 2 hours;equal volume of DMEM containing 10% fetal bovine serum medium for suspension of collagenase reaction; 250g centrifugation for 10 minutes, collected the lower cell mass; containing 10% FBS DMEM, scattered cell mass, adjusted the cell concentration to 1*106/ml, moved into sterile culture flasks after; cultured in 37℃,5% CO2 humidified cell incubator.2. Identification of ASCs Flow cytometry detected the surface cell marker CD106, CD 166 in vitro.3.Ability of the proliferation of different areas derived ASCs in vitro MTT assay detected the different areas derived ASCs(P2) OD varation on the 1th,2nd,3rd,4th,5th,6th,7th day when cultured in vivo.4. Preparation of PLLA/PCL nanofiber scaffolds by electrospinning method. Reference to Ma's method [2], the basic steps are as follows:Take 10ml of 20% of the PLLA/PCL chloroform solution, add a 20ml syringe, syringe pump by a uniform injection (0.5ml/h), the solution through a 20G (diameter 0.21mm) needles. And receiving the needle between the nano-fibers of aluminum (from 20-40cm) plus one high voltage (15kv), so that PLLA/ PCL chloroform solution will be ejected from the needle high-speed, in its movement to the receiving device in the process of rapid evaporation of the solvent chloroform, collected aluminum falling PLLA/PCL nanofibers. When receiving aluminum nanofibers into a uniform rotation of the receiver (aluminum plate), the nano-fibers can arrange a certain direction in the aluminum plate (Figure-1).Collected the PLLA/PCL nanofibers on the Aluminum plant when the nanofibers accumulated to a certain thickness (0.2-0.4mm). Scanning electron microscopy will be used to detect the morphology of nanofibers.5.Cytotoxicity of material ASCs from inguinal area from cocultured in different proportions of PLLA/PCL nanofiber scaffold, immunofluorescence living and dead cell staining (Live/Dead Stain) detected cytotoxicity of material, immunofluorescence staining and scanning electron microscopy cells can also detected the adhesion and proliferation of ASCs in different materials.6. Biocompatibility of materials and degradation of material in vivo implanted the scaffolds with different proportion of PLLA/PCL under the dorsal subcutaneous of SD rats, immunohistochemistry detected the expression of inflammatory markers CD31, CD45, CD68 at different times to assess Biocompatibility of materials and degradation of material.Ⅱ. ECM (extracellular matrix, ECM) modified PLLA/PCL nanofiber scaffolds.1. Different ECM (gelatin, collagen, laminin, fibronectin, high concentration of polylysine, low concentration of polylysine) pretreated flask and PLLA/PCL nanofiber scaffolds, MTT assay detected the change of the ability of cell proliferation before and after the pretreated with ECM in the culture dish and scaffolds. Immunofluorescence staining and scanning electron microscopy also used to detect the change of cell proliferation ability.2. Different ECM (gelatin, collagen, LN, FN, high concentration of polylysine, low concentration of polylysine)pretreated flask and PLLA/PCL nanofiber scaffolds, then cocultured the ASCs in SMIM. RT-PCR and Realtime-PCR detected the change of mRNA expression of the smooth muscle-specific marker genes (Calponin, SM22,α-SMA, MHC); Western blot detection the expression levels of according proteins.Ⅲ. Leiomyogenic differentiation of ASCs on dirrectional PLLA/PCL nanofiber scaffolds and urethral reconstruction of rabbits.1.Synthesize of directional PLLA/PCL nanofiber scaffolds, scanning electron microscopy detect. 2.ASCs cocultured with the directional PLLA/PCL nanofiber scaffolds in normal medium (DMEM), immune fluorescence staining, scanning electron microscopy and MTT assay detect the cell adhesion and proliferation ability.3. ASCs cocultured with the directional PLLA/PCL nanofiber scaffolds with/without LN pretreated in the smooth muscle induction medium (SMIM). Scanning electron microscopy detected the adhesion, proliferation changes and the arrangement of smooth muscle (AD-SMCs).4. Urethral stricture model of rabbit and urethral reconstruct with cells-loaded scaffolds Using the YAG laser distory the urethra of rabbits under direct vision(ureteroscopy),3 to 4 weeks after injured, the urethra can be strictured for scar forming. Retrograde imaging approved it. Isolated ASCs from the inguinal area of the damaged rabbit and cocultured them in directional nanofiber scaffolds with DMEM in vitro. When the ASCs proliferated in a high density, changed the medium with SMIM. After cultured in the smooth muscle induced medium for 6 weeks, ASCs differentiated into smooth muscle cells. Implanted the AD-SMCs loaded scaffolds to the restructed part of ASCs host rabit, detected the urethral pressure change at different time (2 weeks,8 weeks after graft implanted), compared the urethral pressure changes before and after the operation. Investigated the effect of AD-SMCs loaded directional nanofiber scaffolds.Results1. Three different areas derived rabbit adipose stem cells have the typical triangular or spindle-shaped appearance, flow cytometry detection show the cell surface marker of CD 105, CD 166 expression the same with other groups reported.2.MTT assay found different areas derived ASCs had various ability of proliferation in vitro(P<0.05). The most powerful proliferation area was the neck, followed by the inguinal area, retroperitoneal fat sources (kidney around) proliferation of the weakest.3.Electron microscope scan found the directional nanofiber scaffolds were successfully prepared with Electrospinning method.4. Immunofluorescence of living dead staining (Live/Dead Stain) showed the nanofiber scaffolds with different components ratio of PLLA/PCL had no significant cytotoxicity. Immunofluorescence staining and scanning electron microscopy detected the cell numbers on various scaffolds at different time points (cells scaffold cocultured for 1,2,3 days) of fixed area of vision, data show 1:1 scaffold had the strongest cell adhesion ability, cell proliferation had no significant difference on different materials (P>0.05).5.Different scaffolds implanted subcutaneously of the SD rats back, the 5th day, immunohistochemistry found the positive expression of inflammatory factors CD31, CD45, CD68. On the next 2nd,3rd,4th,6th,8th,12th weeks, immunohistochemistry found did not found the expression of such inflammatory factors. IPCM observed the gradual degradation of scaffolds. It almost can not see the of material at 12th week, and there were no granulation tissue appeared around the scaffolds.6. Compared the number of cells in the same size of area of materials under IPCM in different ECM pretreated materials (1:1 material) with the same area of untreated scaffolds, there were significant differences between the two group (P<0.05).7. MTT assay was used to detect the infulence of ECM to cell proliferation. Pre treated the dishes with different ECM, cell proliferation ability in the dish treated with PLL of low/high concentration and FN was enhanced, significantly different to the control groups (P<0.05); gelatin, collagen, LN treatment group and control group had no significant difference (P> 0.05).8. ASCs cultured in induction medium in different ECM pretreated dishes for 6 weeks, RT-PCR, Realtime-PCR and Western blot analysis showed that LN, gelatin, collagen pretreated group detected calponin after been induced for 2w; FN, PLL (high and low concentration), did not detected the expression calponin. Gelatin, collagen, LN and control group dectected the expression of SM22 after been induced for 3w; PLL (high and low concentration), FN group did not detected the expression of SM22. PLL (high and low concentration), FN, collagen, LN, gelatin group detected the positive expression ofα-actin after been induced for 4w. PLL (high and low concentration), gelatin, collagen, LN, FN group detected the positive expression of MHC after been induced for 6w.9. Electrospinning combined with electroporation prepared 1:1 PLLA/PCL nanofiber scaffolds, scanning electron microscopy found the nanofibers on the scaffold arranged in the same direction.10. Immunofluorescence staining and scanning electron microscopy detected the cell proliferated on the 1:1 directional PLLA/PCL nanofiber scaffolds gradually when ASCs cultured in DMEM for 1 w.11.Cocultured ASCs with 1:1 directional PLLA/PCL nanofiber scaffolds in SMIM for 6 weeks, scanning electron microscopy analysis showed that the induced cells arranged in the same direction. The long axis of cells paralleled to the nano-fibers of the scaffold.12. YAG laser distory the urethra of rabbits under direct vision(ureteroscopy),3 to 4 weeks after injured, the urethra can be strictured for scar forming. Retrograde imaging approved it. The successful induced cells loaded scaffolds implanted into the host rabits urethra, detected the urethral pressure of the rabbits at the first 2 weeks and the first 8 weeks, the urethral pressure showed the presence of urethral stricture at the 2nd week, data showed 2 of 4 rabbits implanted with AD-SMCs loaded scaffolds reached to normal urethral pressure at the 8th week.Conclusion1.ASCs derived from inguinal area is the appropriate source of ASCs.2.1:1 PLLA/PCL nanofiber scaffolds with no cytotoxicity, minimal inflammatory reaction and good cell adhesion character, is the appropriate choice of nanofiber scaffold.3.ECM can modify the adhesion character of nanofiber scaffolds.what's more, LN, gelatin and collagen can promote the ASCs to differatiate into SMCs.4.1:1 directional PLLA/PCL nanofiber scaffold can induce the smooth muscle cells arranged in the direction direction.5.Smooth muscle cells kept alive and accomplished overall contractile function after implanted the AD-SMCs loaded directional scaffold in the rabits urethra.6.Directional PLLA/PCL nanofiber scaffold loaded with AD-SMCs for functional urethra reconstruction is feasible.