Development of a Porcine Bladder Acellular Matrix with Well-Preserved Extracellular Bioactive Factors for Tissue Engineering

Bin Yang, M.D., Ph.D.,Yifen Zhang, M.D.,Liuhua Zhou, M.D.,Zeyu Sun, M.D.,Junhua Zheng, M.D.,2 Yun Chen, M.D., Ph.D.,1 and Yutian Dai, M.D., Ph.D.1

Intro

Regenerative medicine builds upon the utilization of a specifically engineered scaffold for the growth of different cell types. These scaffolds’ functionality lies in providing an environment for cells similar to the endogenous environment of the tissue. This allows proper growth and protein expressions of these cells to ensure continued functionality over time once it is in the patient. There have been a number of various synthetically derived scaffolds however the complexity of the ECM is incredibly great making these approaches quite difficult. Another approach is the decellularization of xenogeneic tissues to use as a scaffold for tissue regeneration. However there is variability in methods to decellularize tissues and little research has been done on porcine urinary bladder decullarization. Yang et al explore a porcine bladder acellular matrix while considering the efficiency of decellularization and preservation of certain important biological factors within the matrix.

Methods

The decellurization of the porcine bladder tissue was done in 4 different ways for comparison to identify the best method. The first was Group A, which was cut into small pieces and the urothelium/suburothelium was removed surgically. They were then incubated overnight with ice-cold hypotonic Tris-EDTA buffer. Then they were washed with ice cold PBS and placed in a room temperature hypertonic Tris buffer containing Triton X-100 for 24 hours. Group B was similar to Group A except after the final incubation, this group was further incubated at room temperature in a hypertonic solution of Tris buffer containing SDS(sodium dodecyl sulfate). Group C was also similar in terms of reagents used for the incubation steps in Group A, except Group C was kept intact and distended for the duration of the incubation. Group D was similar to Group C except the Tris buffer contained less Triton X-100.

All of the groups were then treated with RNases and DNases and mechanically agitated in order to remove the cells. The tissues were subsequently tested for a number of biological factors including collagen, which was analyzed by the chloramine-T method. Total genomic DNA was also measure and compared with native porcine bladders using a fluorescence microplate reader. Then the active biological factors in the matrix were evaluated by using two different cell lines, HMSMC, and HUVEC. Both were cultured in DMEM with 10% FBS and maintained in a incubator with media exchange every day and were used at passage 2-4. The HBSMC was seeded at 4x10^3 cells/well and the HUVEC was seeded at 5x10^3 cells/well. Cell migration was also tested. VEGF and PDGF was also quantified through use of an ELISA kit in order to determine the extracellular bioactive molecules.

Results and conclusion

Histological specimens of the different groups were compared and after DAPI staining it became evident that in groups B and D there was total elimination of the cellular nuclei. Also since the amount of DNA detected was significantly lower for group D than A, it implies that the distension when preparing the acellular matrix caused improved efficiency in decellularization. In addition, it seemed that the group C scaffold better promoted the proliferation of the cells. Furthermore, total collagen was greater in group C than in group B. It seems the distension of the bladder thinned the walls and made removal of cell and other immune responsive components easier

. A combination of treatment with Triton X-100, distension, and buffer treatment seemed to be the best approach for effective decellularization. Also the total content of collagen was higher in the decellularized matrices compared to that of native porcine tissue. It seems that used of Triton X-100 left the proteins in the matrix intact allowing better cellular adhesion and proliferation.

Commentary

Overall the paper is interesting and applicable in the realm of tissue engineering. It provides insights into the difficulty of engineering matrices suitable for tissue regeneration since a number of factors can alter the state of the matrix. In addition, the matrix needs to be properly decellularized in order to remove immune responsive elements however; this must be done carefully to preserve bioactive factors that will promote cellular adhesion and proliferation when attempting to regenerate tissue. My main critique is questioning their ability to state that they preserved these necessary bioactive factors by looking at only a select few factors. They looked at collagen and sulfated GAG however these may not be the best representation of actual bioactive factors necessary for angiogenesis and tissue production. In addition, it is difficult to tell whether or not these bioactive factors supposedly remaining on the surface of this matrix will cause an immune response or not within a human model. Since it is a xenogeneic matrix, the proteins and molecules might be in a slightly different three-dimensional configuration, which could then be immune rejected by the body. However, it seems their work is decently compelling and provides better research into methods of decelluarization and it would be interesting it method similar could be developed to obtain similar results for other various tissue applications.