The Impact Of Protein Solubilization-based Decellularization Methods On The Biological And Mechanical Properties Of Porcine Aortic Valve

Part One The effect of protein solubilization-based decellularization methods on the structure and mechanical property of porcine aortic valveObjective:To investigate three protein solubilization-based protocols for porcine aortic valves decellularization by studying the efficiency to remove cellular and nuclear materials, the effect on extracellular matrix (ECM) contents and mechanical properties, and compared with a previously reported decellularization method (Triton X-100, SDC, and IGEPAL CA-630, TSI).Methods:Porcine aortic valves were decellularized with hydrophile solubilization alone (HSA), lipophile solubilization alone (LSA), sequential hydrophile and lipophile solubilization (SHLS) and TSI. DAPI staining was used to assess decellularization effectiveness. Histologic analysis with HE, Masson and Elastica Van Gieson (EVG) stainings were performed to test the effect on the structure of ECM, collagen and elastin fiber. Microstructure of samples was viewed with scanning electron microscopy (SEM). Quantitative biochemistry of collagen, elastin and GAGs were used to examine the impact of decellularization methods on the ECM contents. The mechanical properties of the leaflets of native and treated valves were determined using a uniaxial materials testing system.Results:HSA failed to completely remove cells of porcine valve although it preserved the structure and ECM components best among the four decellularization groups. LSA removed all cells completely but caused severe alterations of the structure. Masson’s Trichrome staining revealed poor collagen staining, and elastin fibers were completely eliminated as indicated by EVG staining in the LSA-treated valves. SHLS resulted in a complete removal of cells and a well preservation of the structure. Masson’s Trichrome staining of the SHLS-treated valves showed good collagen preservation, and EVG staining of the valves demonstrated a mild decrease in elastin fiber compared to the native samples. In contrast, a previously reported method using TSI resulted in a complete removal of all cells but caused some alterations of the structure. Masson’s Trichrome staining of the TSI-treated valves indicated a reduction in collagen fibers, especially in the fiber layer compared to the native samples. There was also a strong decrease of elastin fiber as indicated by EVG staining. The water contents of porcine valves treated with all four decellularization protocols had increased significantly compared to native tissues. The collagen content of tissues treated with all four decellularization protocols was not observed to be significantly different from that in native valve. Only HSA decellularization protocol did not affect the elastin content of native tissue. A reduction in elastin content of was found after decellularization with LSA, SHLS or TSI. A reduction in GAGs content of valve leaflets was observed after any decellularization procedure. All four decellularization protocols induced an increase in ultimate tensile strain in both the circumferential and the radial directions. HSA, SHLS or TSI protocol did not significantly compromise the ultimate tensile strength and Young’s modulus of the leaflets. However, the Young’s modulus of the circumferential direction was significantly deceased in tissues decellularized with LSA.Conclusion:SHLS method can be considered as a promising decellularization method for the decellularization of porcine aortic valves by removing cell completely, substantially maintaining original structure and ECM compositions, and preserving mechanical properties.Part Two The impact of protein solubilization-based decellularization methods on the in vitro immunogenicity and biocompatibility of porcine aortic valveChapter one:The impact of protein solubilization-based decellularization methods on the in vitro immunogenicity of porcine aortic valveObjective:To investigate three protein solubilization-based protocols for porcine aortic valves decellularization by studying the efficiency to remove xenogeneic antigens and the effect on immunogenic potentials in vitro, and compared with a previously reported decellularization method-TSI.Methods:Porcine aortic valves were decellularized with HSA, LSA, SHLS and TSI. The a-Gal epitope and major histocompatibility complex class I (MHC I) were detected in native and decellularized porcine aortic valves by immunofluorescent stainings. Native and decellularized porcine aortic valves were incubated with human blood for immunogenicity testing. Leukocyte and complement activations were studied by determinations of PMN elastase and SC5b-9 with ELISA.Results:After treatment with HSA, the a-Gal epitope and MHC I were detected in the decellularized valves. Complete elimination of the a-Gal epitope and MHC I was found in the leaflets of LSA-or SHLS-treated porcine aortic valves. Although the leaflet of TSI-treated porcine valve was completely free of MHC I, the a-Gal epitope was still present in the decellularized valves. LSA-treated and SHLS-treated valves showed a decrease in PMN elastase levels compared to native valves and tissues decellularized with HSA or TSI. However, the PMN elastase values in tissue treated with the HSA or TSI was not observed to be significantly different from that in native valve. There were no significant differences in SC5b-9 levels among all groups.Conclusion:SHLS and LSA removed a-Gal epitope and MHC I completely from porcine aortic valves, and SHLS-treated and LSA-treated tissues reduced leukocyte activation in vitro.Chapter two:The impact of protein solubilization-based decellularization methods on the in vitro biocompatibility of porcine aortic valveObjective:To isolate and culture bone marrow mesenchymal stem cells (BMSCs) as a cell source for tissue engineering heart valve; To investigate three protein solubilization-based protocols for porcine aortic valves decellularization by studying the effect on cell adhesion, proliferation, cytotoxicity and hemocompatibility, and compared with a previously reported decellularization method-TSI.Methods:BMSCs were isolated and cultured from bone marrows of adult Sprague-Dawley rats by whole bone marrow adherence method. The morphology of the cells was viewed by the light microscope. Cells (passage 3) were fluorescence activated cell sorter analyzed for BMSCs cell surface markers. BMSCs were cultured in differentiation cultures to induce differentiation into adipocytes and osteocytes. Decellularized and native valves were reseeded with BMSCs. The density of adherent cells was quantified with the WST-8 assay at 2 h and 24 h after cell seeding to estimate the adhesion amount of BMSCs in different groups. After 2,4, and 8 days of culture, the WST-8 assay was used to quantify the density of viable cells on the tissues to evaluate the proliferation of BMSCs. The cytotoxicity was detected by measuring the LDH-specific activity in the medium in which the cells were reseeded on the tissues for 2,4, and 8 days. Haemolysis tests were carried out by measuring haemolysis ratio of decellularized tissues incubated with human blood.Results:BMSCs were isolated and cultured successfully. Cells (passage 3) were adherent cell layer and appear uniformly spindle shaped in culture. Flow cytometry showed that they stained positive for CD90 and CD29, and stained negative for CD45. After 2 weeks differentiation culture, the BMSCs-derived cells were positively stained by oil red O and alizarin red staining respectively. At the 2 h and 24 h after cell seeding, there were significantly higher percentages of adherent BMSCs on all 4 decellularized valves versus on native valves, and there was a significantly higher percentages of adherent BMSCs on SHLS-treated valves than HSA-, LSA-, and TSI-treated valves. At 2,4 and 8 days of culture, there were significantly higher cell densities of BMSCs grown on all 4 decellularized valves versus native valves. At 4 and 8 days of culture, there were significantly higher cell densities of BMSCs grown on SHLS-treated valves than HSA-, LSA-, and TSI-treated valves. The cytotoxicity assay showed that at the 2 days after cell seeding, the LDH-specific activity was higher in TSI-treated valves than other tissues. TSI-treated valves also have higher haemolysis ratio when incubated with human blood than HSA-, LSA-, and SHLS-treated valves and native valves. Conclusion:BMSCs can be considered as a promising cell resource for the tissue engineering heart valve. Reseeding of BMSCs on SHLS-decellularized valves yielded higher adhesion and proliferation of cell numbers with lower cytotoxicity and haemolysis ratio.Part Three The impact of protein solubilization-based decellularization methods on the in vivo tissue remodeling and calcification of porcine aortic valveObjective:To investigate three protein solubilization-based protocols for porcine aortic valves decellularization by studying the effect on in vivo inflammatory response, tissue remodeling and calcification, and compared with a previously reported method decellularized valves, native valves and GA-fixed valves.Methods:Seventy-two female SD rats were randomly divided into 6 separate groups. Each rat was subjected to the abdominal wall defect surgical procedure and one of the treated valves was implanted in each animal. The treatment groups were then subdivided into two groups of two each that were sacrificed and explanted at 2 or 6 weeks postoperatively. The explanted specimens were stained with H&E and Masson staining. Histologic sections were evaluated with quantitative criteria for aspects of inflammatory and tissue remodeling response. Von Kossa staining and quantitative calcium analyses were used for tissue calcification. Immunohistochemical staining was performed to comprehensively compare the macrophage phenotypic profile of implants. Pro-inflammatory cytokines (IL-12, TNF-a, IL-6, IL-1β), anti-inflammatory cytokines (IL-10, IL-lra), MMPs (MMP1, MMP2, MMP9), TIMPs (TIMP1, TIMP2), and macrophage phenotype markers (iNOS, ARG) expression at various times post implantation was determined by quantitative real-time PCR. MMPs activity was carried out by in situ zymography.Results:Histomorphologic analysis showed that LSA-and SHLS-decellularized valves were presented with a degradation of the majority of scaffold and were not well identifiable, and the remodeling site was composed of organized host connective tissue and islands of skeletal muscle within the center of the remodeling site, which was indicative of a constructive remodeling. Native and GA-fixed valves were surrounded by a dense infiltration of inflammatory cells, lack of muscle ingrowth, still largely intact, and encapsulated with dense fibrous capsule, which was indicative of a foreign body response. HSA-and TSI-decellularized valves were characterized by a decrease in cellularity, both within and surrounding the implant, limited device degradation, limited new ECM deposition, and limited muscle ingrowth, which was little evidence of constructive tissue remodeling. Von Kossa stainings showed that visible calcifications were only found in GA-fixed valves at 6 weeks. In HSA-, LSA-, SHLS-and TSI-treated samples retrieved after implantation, calcium concentration was significantly lower than that in the implanted native tissues and GA-fixed valves. The ratio of M2:M1 macrophages was higher in LSA-and SHLS-decellularized valves at 2 weeks and 6 weeks than that in the other four groups, while the ratio of M2:M1 macrophages was higher in HSA-and TSI-treated samples than that in native and GA-fixed valves at 2 weeks and 6 weeks. The expression of anti-inflammatory cytokines were higher in LSA-and SHLS-decellularized valves at 2 weeks and 6 weeks, while the expression of pro-inflammatory cytokines were higher in native and GA-fixed valves at 2 weeks and 6 weeks. The expression of MMP2, MMP9 and TIMP2 was lower in LSA-and SHLS-decellularized valves at 2 weeks, higher at 6 weeks. The expression of MMP2, MMP9 and TIMP2 was still at a high level in GA-fixed valve at 2 weeks and 6 weeks. The results were in agreement with MMPs activities indicated by in situ zymography.Conclusion:The porcine aortic valves treated with different methods elicited distinct host tissue remodeling responses in vivo. LSA-and SHLS-decellularized valves were characterized by a constructive remodeling. Macrophages undergo dynamic changes towards an M1 or an M2 phenotype and involve the tissue remodeling in vivo through the production of pro-inflammatory and anti-inflammatory cytokines, MMPs and TIMPs.