Development of a Methodology for Obtaining Connective Tissue Bioequivalent for Rabbits

Introduction. The potential of cell therapy and tissue engineering technologies in veterinary medicine is quite high. However, the use of these technologies in the Russian Federation is currently limited due to the absence of standardized protocols for cell isolation, donor selection and creation of tissue equivalents. Development of a methodology for obtaining connective tissue bioequivalent is particularly relevant for clinical veterinary medicine, as connective tissue constitutes up to the half of the body weight and ensures the normal functioning of skin, mucous membranes, and internal organs of animals. The aim of this study is to develop a methodology for obtaining connective tissue equivalent for rabbits.
Materials and Мethods. The study was conducted at Don State Technical University (DSTU) from November 13, 2023 to March 17, 2025. The objects of the study were multipotent mesenchymal stem cells (MMSCs) and fibroblasts from adult male rabbits. Enzymatic methods were used to isolate MMSCs from the greater omentum and fibroblasts from the animal skin. Stable cell lines were obtained, and their differentiation potential was studied in vitro during myogenic and lipogenic induction. Connective tissue equivalents were created using 3D extrusion bioprinting, their morphological properties were studied by means of light, confocal, and electron microscopy.
Results. Application of the sets of factors during induction ensured the adipogenic and myogenic differentiation of MMSCs. Adipogenic differentiation came along with formation of lipid droplets, while myogenic differentiation — with formation of myotubes. 3D bioprinting enabled creation of connective tissue equivalents with maintained cell viability, developing intercellular contacts, and active secretion for at least 72 hours.
Discussion and Сonclusion. A new approach to obtaining connective tissue equivalents for rabbits was developed by optimizing MMSCs isolation and differentiation techniques. The resulting constructs demonstrated morphological and functional activity, thus, confirmed their potential for using in clinical veterinary medicine for regeneration of connective tissue and for experimental studies.

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