Prepararea alogrefelor osoase pe pedicul vascular – studiu experimental: Teză de doctor în științe medicale: 321.18 Ortopedie şi traumatologie

Abstract

Topicality and importance of the subject studied Bone defects of different origins initiate natural regeneration mechanisms in the human body and most of the time sufficient for the recovery of the small bone defects [1]. The bone remodeling process is qualified as one with the most complex mechanisms. Bone cells, having a complex communication with each other, perfectly orchestrate the processes of bone healing (consolidation), the mechanical adaptation of the skeleton and of course regulate calcium homeostasis [2]. However, the treatment of bone defects becomes a real challenge when we talk about massive bone defects, especially those after severe trauma, massive tumor formations, localized and aggressive infectious processes or congenital bone defects. For such diagnoses, traditional treatment methods are used [3, 4, 5, 6, 7]. However, for bone defects exceeding 4-5 cm in length, the bone grafting procedures currently used may be insufficient [8, 9]. The increased demand for interventions to recover bone defects of different etiology, the improvement of technical possibilities, and of course the large-scale development of tissue engineering (TI) induces the approach of the given problem to another level [1, 10, 11]. The ideal material for bone construction should be a biocompatible, biodegradable, osteoconductive, osteoinductive, and with good mechanical properties. The advantages of using natural extracellular matrix (ECM) include primarily their similarity in morphology and 3D structure to that of native bone tissue [12, 13]. Medical engineering at the moment has a huge impact on organ and tissue transplantation, promising the development of obtaining and widely using ECMs of different origins that will offer the possibility of replacing any non-functional organ or organ segment [11]. The goal of bone TI is to regenerate/repair tissue with using a biocompatible and biodegradable graft (natural or synthetic). Taking into account that bone is a very well vascularized tissue, and all its stages of regeneration (inflamation, maturation, remodeling) depend on a strict regulation of blood supply, it is very important to develop strategies aimed at the rapid and efficient vascularization of the implanted graft [14, 15]. In the Republic of Moldova, the need for bone grafts exceeds 1.4 times the availability. Even more, according to a survey of 161 specialists, including 75 trauma orthopedic surgeons from 16 different medical institutions, there is an increased need for bone grafts processed by a method other than freezing [16, 17]. Purpose of the study Development of the universal protocol for decellularization of tissues with various degrees of mineralization in order to obtain vascularized composite bone grafts with minimal immunogenic qualities. Objectives of the study 1. Elaboration of the surgical protocol to procure the vascularized bone graft of the tibial bone in laboratory animals; 2. Elaboration of the protocol for decellularization of bone grafts (tibial bone) with preservation of the vascular pedicle; 3. To test the efficacy of the bone graft decellularization used method by qualitative (H&E, DAPI, SEM) and quantitative (DNA quantification) methods; 4. To test the biocompatibility of vascularized bone extracellular matrices using different cell types for in vitro recellularization. Methodology of scientific research In order to achieve the purpose of the work, we have started a preclinical experimental study that was based on 2 major elements: the increased demand for bone transplantation vs the evolution of the TI field. This experimental study was carried out on biological samples taken from laboratory animals (pigs) and is based on soft tissue and hard tissue samples (blood vessels of porcine origin of three different diameters, periosteum, avascular bone grafts, vascularized composite bone grafts). The objectives of the study were achieved by performing the following steps: (1) was developed the surgical protocol for vascularized bone grafts procurement; (2) a protocol described for the decellularization of small bone grafts was adjusted and tested on large, medium, and small caliber vascular grafts, plus on large avascular bone grafts (cortical and spongy bone grafts), and on vascularized composite bone grafts; (3) the effectiveness of the decellularization protocol was assessed through qualitative and quantitative tests; (4) the biocompatibility of vascular ECMs, avascular bone ECMs and vascularized bone ECM was tested performing in vitro recellularization using different cell types. Decellularization of soft and hard tissue grafts was possible using a protocol that included: an isotonic solution (0.1 % ethylenediaminetetraacetic acid, EDTA + phosphate saline buffer, PBS), a chelating agent (0.1 % EDTA + tris aminomethane buffer solution, TRis buffer), a detergent (0.5 % sodium dodecyl sulfate, SDS) and an enzyme solution (300 U/ml deoxyribonuclease, DNase). Static decellularization was used for vascular and avascular bone grafts, and the decellularization for vascularized composite bone grafts was performed by infusion of solutions using two infusion routes: (1) perfusion of the solutions through the vascular pedicle and (2) perfusion of the solutions through the bone diaphysis. Also, the efficacy of decellularization at increasing the detergent concentration (SDS) from 0.5% to 1% was tested. In order to assess the efficacy of the decellularization protocol, the qualitative analysis of soft and hard tissue samples (histological staining Hematoxylin and Eosin, H&E and 4', 6-diamidino-2-phenylindole, DAPI) was performed, and the quantitative analysis of the decellularization process was performed based on DNA quantification for avascular and vascularized bone grafts. In order to assess the microscopic changes that occurred at the level of the bone component of vascularized bone ECM, scanning electron microscopy (SEM) of native vs decellularized cortical and cancellous bone samples was performed. The most important moment of the study, the assessment of biocompatibility by in vitro recellularization of the obtained ECMs, was performed by using human umbilical venous endothelial cells (HUVEC) for vascular grafts, and the allogeneic mesenchymal stem cells from bone marrow – BM MSC (porcine origin) for avascular bone grafts and vascularized ones. The analysis of histological samples and cell cultures was performed using the optical microscope, fluorescent microscope and stereo microscope. The data were collected and processed with the help of the Excel program from Microsoft Office version 2017. Using the same program, continuous variables were expressed as mean + SD. The statistical processing was carried out with the help of the SPSS program (version 17.0). The comparison and assessment of the statistically significant differences between the mean values of the evaluated parameters was carried out with the help of the independent parametric test t-Test. The p-value was interpreted as statistically significant for p≤0.05. The study was approved by the Research Ethics Committee of the Nicolae Testemitanu State University of Medicine and Pharmacy of the Republic of Moldova, with the issuance of the favorable opinion no. 71 on 21.05.2018. This study was carried out at the Laboratory of Tissue Engineering and Cell Cultures of the Nicolae Testemitanu State University of Medicine and Pharmacy, Chisinau, Republic of Moldova and the Leibniz Research Laboratory of Biotechnology and Artificial Organs (LEBAO), Hannover School of Medicine (MHH), Germany, during 2017-2022. [...]