CRISPR/Cas mediated disruption of BMPR-1B gene and introduction of FecB mutation into the Caprine embryos using Easi-CRISPR strategy.

Bone Morphogenetic Proteins play a significant role in ovarian physiology and contribute to the reproductive fitness of mammals. The BMPR-1B/FecB mutation, a loss of function mutation increases litter size by 1-2 with each number of mutated alleles in sheep. Considering demand-supply gap of the meat industry, and low replacement rate of indigenous caprine species, the conservative BMPR-1B locus can be explored, and FecB mutated goats can be produced. The experiment one produced CRISPR/Cas mediated KO transferable caprine embryos, and experiment two generated caprine embryos with desired FecB mutation using Easi-CRISPR strategy. In the KO experiment, Cas9 and BMPR-1B guide RNA (100:100ng/ul) were electroporated into single stage caprine zygotes at 750V, 10 ms and 1pulse using Neon transfection system. In the second experiment, phosphorothioate (PS) modified single-stranded oligodeoxynucleotide (ssODN) was used as an HDR template along with CRISPR components (100:100ng/ul, ssODN 100ng/ul). The precise time and method of electroporation, RNP format of CRISPR components and PS modified asymmetric ssODN were the factors that affected the production of mosaicism free BMPR-1B edited caprine embryos. The editing efficiency of KO and KI experiments was 68.52 and 63.16% respectively, and successful production of goats with higher mean ovulation rate can be realized with addition of embryo transfer technology to these experiments.

Mesenchymal stem cell tailored bioengineered scaffolds derived from bubaline diaphragm and aortic matrices for reconstruction of abdominal wall defects.

Bioengineered scaffolds derived from the decellularized extracellular matrix (ECM) obtained from discarded animal organs and tissues are attractive candidates for regenerative medicine applications. Tailoring these scaffolds with stem cells enhances their regeneration potential making them a suitable platform for regenerating damaged tissues. Thus, the study was designed to investigate the potential of mesenchymal stem cells tailored acellular bubaline diaphragm and aortic ECM for the repair of full-thickness abdominal wall defects in a rabbit model. Tissues obtained from bubaline diaphragm and aorta were decellularized and bioengineered by seeding with rabbit bone marrow derived mesenchymal stem cells (r-BMSC). Full-thickness abdominal wall defects of 3 cm × 4 cm size were created in a rabbit model and repaired using five different prostheses, namely, polypropylene sheet, nonseeded diaphragm ECM, nonseeded aorta ECM, r-BMSC bioengineered diaphragm ECM, and r-BMSC bioengineered aorta ECM. Results from the study revealed that biological scaffolds are superior in comparison to synthetic polymer mesh for regeneration in terms of collagen deposition, maturation, neovascularization, and lack of any significant (P > 0.05) adhesions with the abdominal viscera. Seeding with r-BMSC significantly increased (P < 0.05) the collagen deposition and biomechanical strength of the scaffolds. The bioengineered r-BMSC seeded acellular bubaline diaphragm showed even superior biomechanical strength as compared to synthetic polymer mesh. Tailoring of the scaffolds with the r-BMSC also resulted in significant reduction (P < 0.01) in antibody and cell mediated immune reactions to the xenogeneic scaffolds in rabbit model.

Selection of biological prosthesis for abdominal wall repair on the basis of in vitro biocompatibility determination.

Research on prostheses for repairing abdominal wall defects has progressed through past decades for developing an ideal prosthesis. The study was designed to compare different extracellular matrix (ECM) derived biological prostheses as alternate to conventional synthetic polymeric prostheses for the repair of full thickness abdominal wall defects. Five biological scaffolds derived from bovine diaphragm, bovine aorta, bovine gall bladder, porcine gall bladder, and rabbit skin were prepared and screened for their in vitro biocompatibility. Decellularized ECMs were subjected to various biocompatibility analyses, namely, water absorption potential, matrix degradation analysis, biomechanical testing, and cytocompatibility analysis. Though the rabbit skin displayed maximum biomechanical strength, due to its rapid degradation, it failed to fulfill the criteria of an ideal prosthesis. ECMs derived from bovine diaphragm and aorta were found to be superior than others based upon hydration and matrix degradation analysis, with best scores for bovine diaphragm followed by bovine aorta. The bovine diaphragm and aorta also displayed sufficient biomechanical strength, with diaphragm being the second highest (next to rabbit skin), in biomechanical strength followed by aorta. None of the biological prosthesis revealed any cytotoxicity. Thus, bovine diaphragm and aorta derived ECM fulfill the necessary criteria for their use as biological prosthesis. Because these prostheses are biocompatible, apart from their low cost, ease of availability, and simple preparation, they present a potential alternative to synthetic prosthesis for repair of abdominal wall defects, especially in veterinary patients.

Polypropylene mesh seeded with fibroblasts: A new approach for the repair of abdominal wall defects in rats.

PURPOSE: The purpose of study was to develop bioengineered scaffolds by seeding primary mouse embryo fibroblast cells (p-MEF) on polypropylene mesh and to test its efficacy for the repair of abdominal wall defects in rats. METHODS: The study was conducted on 18 clinically healthy adult Wistar rats of either sex. The animals were randomly divided into two equal groups having nine animals in each group. In both the groups a 20mm×20mm size full thickness muscle defect was created under xylazine and ketamine anesthesia in the mid-ventral abdominal wall. In group I the defect was repaired with polypropylene mesh alone and in group II it was repaired with p-MEF seeded polypropylene mesh. Matrices were implanted by synthetic absorbable suture material (polyglycolic acid) in continuous suture pattern. The efficacy of the bio-engineered matrices in the reconstruction of full thickness abdominal wall defects was evaluated on the basis of macro and histopathological observations. RESULTS: Macroscopic observations revealed that adhesions with skin and abdominal viscera were minimum in group II as compared to group I. Histopathological observations confirmed better fibroplasia and collagen fiber arrangement in group II. No recurrence of hernia was found in both the groups. CONCLUSION: Hernias are effectively repaired by implanting polypropylene mesh. However, this work demonstrates that in vitro seeding of mesh with fibroblasts resulted in earlier subsidization of pain, angiogenesis and deposition of collagen, increased thickness of matrices with lesser adhesions with underlying viscera. On the basis of the results p-MEF seeded mesh was better than non-seeded mesh for repair of abdominal wall defects in rats.

Bubaline Cholecyst Derived Extracellular Matrix for Reconstruction of Full Thickness Skin Wounds in Rats.

An acellular cholecyst derived extracellular matrix (b-CEM) of bubaline origin was prepared using anionic biological detergent. Healing potential of b-CEM was compared with commercially available collagen sheet (b-CS) and open wound (C) in full thickness skin wounds in rats. Thirty-six clinically healthy adult Sprague Dawley rats of either sex were randomly divided into three equal groups. Under general anesthesia, a full thickness skin wound (20 × 20 mm(2)) was created on the dorsum of each rat. The defect in group I was kept as open wound and was taken as control. In group II, the defect was repaired with commercially available collagen sheet (b-CS). In group III, the defect was repaired with cholecyst derived extracellular matrix of bovine origin (b-CEM). Planimetry, wound contracture, and immunological and histological observations were carried out to evaluate healing process. Significantly (P < 0.05) increased wound contraction was observed in b-CEM (III) as compared to control (I) and b-CS (II) on day 21. Histologically, improved epithelization, neovascularization, fibroplasia, and best arranged collagen fibers were observed in b-CEM (III) as early as on postimplantation day 21. These findings indicate that b-CEM have potential for biomedical applications for full thickness skin wound repair in rats.

Comparison of radiography and ultrasonography for diagnosis of diaphragmatic hernia in bovines.

The present study was conducted on 101 animals suffering from thoracoabdominal disorders; out of which twenty seven animals (twenty six buffaloes and one cow) were diagnosed with diaphragmatic hernia based on clinical signs, radiography, ultrasonography, and left flank laparorumenotomy. Radiography alone confirmed diaphragmatic hernia in 18 cases (66.67%) with a sac-like structure cranial to the diaphragm. In 15 animals the sac contained metallic densities while in three cases a sac-like structure with no metallic densities was present. Ultrasonography was helpful in confirming diaphragmatic hernia in 23 cases (85.18%) and ultrasonographically reticular motility was evident at the level of 4th/5th intercostal space in all the animals. B+M mode ultrasonography was used for the first time for diagnosis of diaphragmatic hernia in bovines and the results suggested that ultrasonography was a reliable diagnostic modality for diaphragmatic hernia in bovines.