Construction Of Engineered Corpus Cavernosum With Primary Mesenchymal Stem Cells In Vitro

Penis is an important reproductive organ in men.Conditions such as congenital malformations,trauma,cancer,or other deformities of the penis often lead to the loss of penile function or even penile loss.This will not only affect the quality of life of patients,but also affect the mental health of patients.Therefore,these patients often require penile reconstruction.There are two methods for the obtainment of penis including penis reconstruction and penile transplantation.Many surgical reconstructive procedures,such as using different flap types or implanting cartilages or prosthetic components,have been attempted to recreate penile function,but the main obstacles to successful reconstruction are surgical complications,including infection,necrosis and imperfect functions.The procedure of penile transplantation is complicated and it is limited by shortage of native tissues.Besides,patients are prone to physical and psychological rejection.To eliminate these malpractices,the use of tissue engineered corpus cavernosum with small trauma,no immune rejection and abundant sources to solve the problem of penis obtainment has attracted wide attentions.Therefore,we will reconstruct the engineered corpus cavemosum with primary mesenchymal stem cells(MSCs)in vitro in a rabbit model in order to provide theoretical basis for penis regeneration.Acellular scaffolds are extracellular matrix after removal of cell components from normal tissues.It is a kind of scaffold with 3D structure and function.Its functional proteins and structure can interact with cytoskeleton and nuclear components.Extracellular matrix is a part of cell microenvironment and important for cell survival.To regenerate the organs or tissues by recellularization of acellular scaffolds are the focus of tissue regeneration research in recent years.The construction of engineered corpus cavernosum is benefit from this technique.First of all,the acellular corporal matrices(ACMs)are prepared by an established method,then they are recellularized to gain the functional corpus cavernosum tissues.The reason why acellular scaffolds play such an important role in regenerative medicine is that their molecules and receptors participate in regeneration and repairment in the following ways:(1)providing 3D scaffolds;(2)secreting and storing growth factors and cytokines;(3)regulating signal transduction.The components of acellular scaffolds include collagen ?,?,? and ?,proteoglycan,hyaluronic acid,chondroitin sulfate,laminin.In addition,there are many active factors that induce cell differentiation,such as VEGF IGFsN EGF and so on.Acellular scaffolds possess essential substances for cell survival,matrix growth factors and cytokines,and they can induce progenitor cells to differentiate into organ-specific cells.Therefore they are appropriate for the organ regeneration.After successfully obtaining the ACMs,the next step is to select the appropriate seed cells to recellularize.At present,the selection of seed cells is the key to the research of organ regeneration by using the acellular scaffold.Anthony Atala used smooth muscle cells and endothelial cells as seeding cells.Song lujie selected umbilical artery endothelial cells as seeding cells.They tested the functions of the constructed tissue engineered corpus cavernosum by using cavernosometry,cavernosography,organ baths and mating experiments and dicovered that the engineered corpus cavernosum could restore part of functions of the penis.However,there's still a big gap between engineered corpus cavernosum and normal tissues.There are endothelial cells,smooth muscle cells,leukocytes,fibroblasts and myogenic stem cells in the corpus caverrnosum.Because endothelial cells,smooth muscle cells and umbilical artery endothelial cells can not be redifferentiated,they might restrict the function recovery of engineered corpus cavernosum.Besides,the culture of different cells are not the same.The using of multiple cells increases the risk of infection and technical difficulty.Therefore,stem cells have become the primary choice of seeding cells for tissue engineering.The tumorigenicity and ethical and religious problems of embryonic stem cells limit their clinical application.Bone marrow mesenchymal stem cells have the advantages of simple obtainment,multiple differentiation potential,no tumorigenesis and ethical problems,which make them a promising seeding cell for the construction of engineered corpus cavernosum.Based on the above analysis,we decided to construct the engineered corpus cavernosum with primary MSCs in vitro in a rabbit model.The success of our experiment will prove the possibility of construction the engineered corpus cavernosum with primary MSCs and represent another step towards the construction of engineered corpus cavernosum in vitro.Part ? The construction of acellular corporal matrices and the detection of its biological characteristicsPurpose:The goal of the our study was to establish an effective decellularization method to obtain the ACMs for the construction of engineered corpus cavernosum.Methods:1.The obtainment of ACMs:Experiments were carried out using New Zealand White male rabbits aged 4 months with weights ranging from 2.5-3 kg.male rabbits were killed via an air embolism.After sterilization,corpora cavernosa were dissected through longitudinal incisions in the tunica albugineas and cleared of extra soft tissues.2.The corpus cavernosum were then cut into 4-mm-thick slices.The corporal tissues were washed with distilled water in a closed flask,placed on a shaker in a super clean bench for 24 hours to allow partial cell lysis and then treated with 1%Triton X-100 and 0.1%ammonium hydroxide under continuous shaking for 14 days to remove the remaining cells.Finally,the matrices were washed with PBS for 24 hours and then processed with DMEM/F12 medium for another 24 hours.Samples of tissue fragments were stained with haematoxylin-eosin and DAPI to ensure that no remaining cellular components were detected.3.Characterization of the ACMs(1)Basic histological analysis.Haematoxylin-eosin staining and Masson staining were performed to estimate clearance of cells and the preservation of collagen fibrils.(2)Molecular biological detection.DNA assay kit was applied to detect the changes of DNA between ACMs and normal tissues.A Sircol soluble collagen assay was applied to detect the changes of soluble collagen between ACMs and normal tissues.VEGF and IGF-1 ELISA kits were applied to detect VEGF and IGF-1 between ACMs and normal tissues.(3)Microstructure detection.Transmission electron microscope was used to detect the microstructure of ACMs and normal tissues.Results:1.The corpus cavernosum became more whiter and softer after treating with Triton X-100 and PBS.2.Compared with normal tissues,haematoxylin-eosin staining,Masson trichrome staining and DAPI staining of ACMs revealed that no nuclear material was detected.In addition,most collagen fibres were preserved and remained arranged in a loosely packed and congruent order.3.Molecular biological detection.DNA quantification showed that little DNA remained in ACMs compared with normal tissues and fulfilled the criteria of less than 50 ng/mg residual DNA,thus confirming that the native cellular materials had been removed.Collagen quantification revealed that collagen concentration per milligram of ACMs did not significantly differ from that in normal tissues.ACMs still contained some cytokines,including VEGF and IGF-1,but the concentration of these cytokines was reduced compared with normal tissues.4.Transmission electron microscopy of ACMs confirmed that the ACMs contained no residual cellular components in contrast to normal tissues.The microstructure of collagen didn't changed much between ACMs and normal tissues.Conclusion:We successfully obtained the ACMs by treating with Triton X-100.Part ? Construction of engineered corpus cavernosum with primary mesenchymal stem cells in vitroPurpose:The goal of our study was to construct the engineered corpus cavernosum with primary MSCs in vitro and detect the differentiation of mesenchymal stem cell.Methods:1.The MSCs were prepared and cultured according to a modified method of the Percoll density gradient centrifugation method.The percentages of cells positive for CD44,CD90,CD105,CD34,CD45 and CD14 were analysed in third-generation MSCs using flow cytometry.Third-generation MSCs were treated with osteogenetic induction medium,adipogenic induction medium or chondrogenic induction medium.2.The MSCs were digested,centrifuged,resuspended and were injected into the ACMs at the concentration of 30*106/ml to ensure that a sufficient number of cells were injected into the ACMs to construct the engineered corpus cavernosum.The seeded ACMs were then preserved in a 37 ? incubator for 6 hours to allow the cells to aggregate and attach to the ACMs.3.The seeded ACMs were transferred to 12-well plates containing 3 mL DMEM/F12 supplemented with 10%FBS and 1%penicillin-streptomycin,which were changed daily.The 12-well plates were placed on a shaker spinning at 40 RPM for 14 days in a 37 ? incubator.The seeded ACM samples were examined by haematoxylin-eosin staining using paraffin-embedded sections every 2 days to observe the state of the seeded cells.4.Haematoxylin-eosin staining was used to observe the state of the seeded cells after 14 days.Besides,immunofluorescence,WB and RT-PCR were used to detect CD31?vWF?myosin and a-SMA.At last,transmission electron microscope was used to observe the cell type directly.5.A Click-iTTM EdU Colorimetric IHC Detection Kit was used to monitor the proliferation of cells in the engineered corpus cavernosum.Results:1.Phenotype detection by flow cytometry showed that the percentages of cells expressing CD44,CD90 and CD 105 were greater than 95%and the percentages of cells expressing CD34,CD 14 and CD45 were less than 2%.These results suggested a high purity level of MSCs.After adipogenic induction,oil red O staining revealed red lipid droplets in adipose cells.After osteogenetic induction,cells were positive for alkaline phosphatase staining.After chondrogenic induction,chondrogenic pellets were coloured blue with Alcian blue staining.2.MSCs were seeded on the ACMs and cultured for 2 weeks in vitro.The seeded ACMs were examined by HE staining after 2 weeks.The results showed that numerous MSCs had distributed and grown well3.Immunofluorescence revealed that some seeded cells stained positively for vWF and CD31 and that some cells stained positively for smooth muscle actin(SMA)and myosin.Immunofluorescence studies revealed the presence of endothelial cells and smooth muscle cells in the engineered corpus cavernosum.Results of RT-PCR and WB confirmed the results above.4.Transmission electron microscopy of the engineered corpus cavernosum confirmed the existence of endothelial cells and smooth muscle cells directly.5.MSCs seeded in the ACMs were detected by EdU absorption from the culture medium.The nuclei of MSCs that had proliferated were coloured brown,indicating cell proliferation in the ACMs.Conclusion:Engineered corpus cavernosum was successfully constructed with primary MSCs in vitro.MSCs seeded on ACMs differentiated into smooth muscle cells and endothelial cells.Our study have proved the possibility of construction the engineered corpus cavernosum with primary MSCs and represent another step towards the construction of engineered corpus cavernosum in vitro.