Comparing the Endoscopic Sub Ureteral Injection of Lyophilized/Micronized Decellular Prepuce with Urodex: Histological Findings

Objectives: In the present study, a new engineered bulking agent called lyophilized micronized prepuce was examined as a natural scaffold to compare its safety and efficacy with the Urodex®. Methods: For an in vivo study, 12 rabbits were divided into two groups. In the first group (n = 6), 0.2 cc of lyophilized and micronized prepuce, and in the second group, 0.2 cc of Urodex® was injected into the seromuscular wall of the bladder. The biopsy was provided from all animals for histological evaluation in 3 and 6 months’ post-surgery and for each timeline 12 animals were assigned. The biopsies were stained with H&E and trichrome Masson. IHC staining was also performed with anti-LCA+, anti-CD34+, and anti-CD68+ antibodies. Resluts: Microscopic examination of acellular prepuce compared with normal tissue demonstrated the success of this process, and ECM and collagen fibers were preserved with no evidence of cellular remnants in the acellular tissue, Immunohistochemistry staining with CD68 and LCA revealed a higher inflammation grade in Urodex as compared with Prepuce. However, no significant difference was detected in CD34 staining between Prepuce and Urodex experimental groups SEM analysis detected the micronized particle size varying between 2-5 µm. MTT assay revealed that cell proliferation was similar in the presence of control group and acellular prepuce. Conclusion: The results of this study disclosed that lyophilized and micronized prepuce could be an operative alternative to Urodex® as a natural and non-synthetic bulking agent in the treatment of children with vesicoureteral reflux (VUR).

Application of Acellular Tissue Matrix for Enhancement of Weak Abdominal Wall in Animal Model

Background. Abdominal wall weakness occurs when the strength of muscle decreases due to physiological reason or iatrogenic injury. However, the treatment of this disease is complicated. Aim. To study the therapeutic effect of acellular tissue matrix (ACTM), compared with the polypropylene mesh. Methods. An abdominal wall weakness model was established in rabbits through motor nerves cutting. The polypropylene mesh and ACTM were implanted in the left and right abdomen sides, respectively. Mechanical testing of abdominal wall muscle and histology and scanning electron microscopy (SEM) evaluation of abdominal tissue explants were performed. Results. In animal model establishment, the abdominal length of healthy and weakened abdominal wall was 17.0 ± 0.7 cm and 19.0 ± 1.2 cm, respectively (P=0.022), and the weak abdominal wall group showed a significant decrease of 1.116 ± 0.221 MPa in tensile stress (P<0.001) and 9.126 ± 2.073% in tensile strain (P<0.001). In materials implantation experiment, compared with polypropylene group, ACTM group decreased 2.409 ± 0.806 MPa after 24 weeks (P<0.001) and 2.319 ± 0.486 MPa after 48 weeks (P<0.001) in tensile stress and increased 15.259% after 24 weeks (P<0.001) and 15.845% after 48 weeks (P<0.001) remarkably in tensile strain. Conclusion. The abdominal wall weakness model in rabbits was successfully established. ACTM is a promising biological material to be possibly further applied in clinical surgery in patients with abdominal wall weakness.

Development of a novel hybrid bioactive hydrogel for future clinical applications

Three-dimensional hydrogels are ideal for tissue engineering applications due to their structural integrity and similarity to native soft tissues; however, they can lack mechanical stability. Our objective was to develop a bioactive and mechanically stable hydrogel for clinical application. Auricular cartilage was decellularised using a combination of hypertonic and hypotonic solutions with and without enzymes to produce acellular tissue. Methacryloyl groups were crosslinked with alginate and PVA main chains via 2-aminoethylmathacrylate and the entire macromonomer further crosslinked with the acellular tissue. The resultant hydrogels were characterised for its physicochemical properties (using NMR), in vitro degradation (via GPC analysis), mechanical stability (compression tests) and in vitro biocompatibility (co-culture with bone marrow-derived mesenchymal stem cells). Following decellularisation, the cartilage tissue showed to be acellular at a significant level (DNA content 25.33 ng/mg vs. 351.46 ng/mg control tissue), with good structural and molecular integrity of the retained extra cellular matrix (s-GAG= 0.19 μg/mg vs. 0.65 μg/mg ±0.001 control tissue). Proteomic analysis showed that collagen subtypes and proteoglycans were retained, and SEM and TEM showed preserved matrix ultra-structure. The hybrid hydrogel was successfully cross-linked with biological and polymer components, and it was stable for 30 days in simulated body fluid (poly dispersal index for alginate with tissue was stable at 1.08 and for PVA with tissue was stable at 1.16). It was also mechanically stable (Young’s modulus of 0.46 ± 0.31 KPa) and biocompatible, as it was able to support the development of a multi-cellular feature with active cellular proliferation in vitro. We have shown that it is possible to successfully combine biological tissue with both a synthetic and natural polymer and create a hybrid bioactive hydrogel for clinical application.

Development of a composite acellular tissue graft for musculoskeletal tissue engineering

A composite acellular tissue graft comprised of decellularized tendon conjugated with nanomaterials has been developed for musculoskeletal tissue engineering applications. The focus of this dissertation is on the development of composite grafts derived from decellularized human tendon conjugated with gold nanoparticles and hydroxyapatite nanoparticles for use in anterior cruciate ligament (ACL) reconstruction. Gold nanoparticles are used to promote remodeling, cellularity, and biological incorporation of grafts. Hydroxyapatite nanoparticles are used to promote osseointegration, cellularity, and to enhance the graft/bone interface. These composite grafts along with several other variations, were characterized in vitro using a variety of cell-based assays including cell viability, cell proliferation, and cell migration assays. Two in vivo studies were conducted. A green fluorescent protein (GFP) porcine model was investigated as a new method to evaluate host tissue integration into soft tissue grafts as well as the in vivo biocompatibility of subcutaneously implanted composite grafts. Results demonstrate biocompatibility and remodeling of composite grafts and the value of using the GFP model as a qualitative method for assessing host tissue integration. A rabbit ACL reconstruction model was used to investigate graft remodeling in addition to the overall viability of using composite grafts to serve as a functional ACL replacement. Results demonstrate successful replacement of ACLs using composite grafts with enhanced remodeling from the addition of nanoparticles. Overall, studies demonstrate the success and potential further application of using composite grafts for musculoskeletal tissue engineering applications. Future studies will include expanding development of variations of these composite materials to address additional clinical needs.