Combined decelularization of vascularized bone allograft. In vivo experimental study stage

State University of Medicine and Pharmacy "Nicolae Testemitanu“, Laboratory of Tissue Engineering and Cellular Culture, Congresul consacrat aniversării a 75-a de la fondarea Universității de Stat de Medicină și Farmacie „Nicolae Testemițanu” din Republica Moldova, Ziua internațională a științei pentru pace și dezvoltareIntroduction •Massive bone defects is an actual real dilemma for reconstructive surgery to the locomotor system. • The vascularized bone allograft would be a successful alternative, with his osteoplastic properties of the vascularized autograft and the orthotopic characteristics of the allogenic bone. •Decellularization of organs, including bone, gives an acellular biological graft, which keeps their extracellular 3D structure. Objective of the study: To extract the cellular component from the vascularized bone allograft by the combined method, according to the algorithm, without injuring the extracellular structure and matrix. Material and methods: The study was performing on New Zealand White Rabbit. The femur was taken with the internal iliac artery(I), located between the upper part of the great trochanter and the distal 1/3 of the femoral shaft, respecting the vascular continuity. The grafts were stored at -84.1C for 14 days, subsequently disengaged by the gradual method. The graft was examined histologically (A. internal iliac art.B. proximal femur) The graft was connected to the closed-circuit peristaltic pump (II). After that was processed, gradually, with a series of solutions (during 7 days): 1. sol. NaCl 0,9%-100ml+sol. Heparin 500Me/ml-5 ml 2. 0,1g EDTA + sol. PBS 100ml 3. 0,1g EDTA + 150 ml sol. TRIS tampon 10 millimolar 4. 0,5g SDS + 100ml sol. TRIS tampon 10 millimolar 5. 1ml sol. TRITON-X 100 + 100ml ml sol. TRIS tampon 10 millimolar 1. 1mg ARNaze + 100ml dist. Water 2. 0,153 ml sol. PenicillinStreptomycin (10,000un.- 10 mg/mL) + 100 ml PBS. Conclusions: The combined process of decellularizing of vascularized bone tissue can generate bone grafts devoid of immunological agents. The decellularized of vessels need additional studies to evaluate the processes for keep its resistance, an imperative factor in subsequent grafting

Decelularizarea combinată a allogrefei osoase vascularizate. Etapă de studiu experimental in vivo

Laboratorul de inginerie tisulară şi culturi celulare, Catedra de ortopedie şi traumatologie, USMF „Nicolae Testemiţanu”, Congresul consacrat aniversării a 75-a de la fondarea Universității de Stat de Medicină și Farmacie „Nicolae Testemițanu” din Republica Moldova, Ziua internațională a științei pentru pace și dezvoltareBackground. Tissue transplantation is a successful approach to rebuild the osteoarticular defects. Critical bone defects remain a dilemma for reconstructive surgery. Decellularization of organs, including bone, gives an acellular biological graft, which keeps their extracellular three-dimensional structure. Objective of the study. To extract the cellular component from the vascularized bone allograft by the combined method, according to the algorithm, without injuring the extracellular structure and matrix. Material and Methods. The bone segment was taken from the domestic rabbit. The femur was taken with the internal iliac artery, located between the upper part of the great trochanter and the distal 1/3 of the femoral shaft, respecting the vascular continuity. The graft was processed, gradually, with a series of solutions, during mechanical agitation. Results. The decellularization process was applied according to the established protocol. Used decellularizing agents were physical, chemical, and biological. They assured the efficient removal of cellular content from the tissue, without damaging the three-dimensional structure of the extracellular matrix. Another point of the present process was the hierarchy in the elimination of components. The greatest part - the cells, were removed first, and then the protein and lipid residues. In the last step, the smallest compartments DNA and RNA, were eliminated. The grafts were examined radiologically and morphologically. Conclusion. The combined process of decellularizing of vascularized bone tissue can generate bone grafts devoid of immunological agents. The decellularized vessel needs additional studies to evaluate its resistance, an imperative factor in subsequent grafting. Introducere. Transplantul de țesut osos este o abordare de succes a defectelor osteoarticulare. Însă, cele masive reprezintă o dilemă pentru chirurgia reconstructivă. Decelularizarea organelor, inclusiv a osului, asigură schele biologice acelulare ce își păstrează structurile tridimensionale. Scopul lucrării. De a extrage componentul celular din allogrefa osoasă vascularizată prin metoda combinată, conform algoritmului, fără a leza structura și matricea extracelulară. Material și Metode. Segmentul osos a fost prelevat de la iepurele domestic, femurul cu artera nutricia (ramură a a. iliace internă), situat între partea superioară de trohanterul mare și 1/3 distală a diafizei femurale, cu respectarea continuității vasculare. Grefa a fost prelucrată, pe rând, cu un șir de soluții, în condiții de agitare mecanică ale acestora. Rezultate. Procesul de decelularizare a fost aplicat conform protocolului. Agenții de decelularizare au fost fizici, chimici și biologici, asigurând eliminarea eficientă a conținutului celular din țesut, fără a deteriora sau a afecta negativ structura tridimensională a matricei extracelulare. Un alt moment al prezentului proces este ierarhizarea în omiterea compartimentelor. De fapt, cele mai mari părți, celulele sunt îndepărtate primele, iar apoi reziduurile de proteine și lipide. În ultima etapă, s-au exlus cele mai mici compartimente care sunt ADN și ARN. Grefele au fost cercetate radiologic și morphologic. Concluzii. Procedeul combinat de decelularizare a țesutului osos este capabil de a genera grefe osoase lipsite de agenții imunologici. Vasul decelularizat necesită studii suplimentare pentru aprecierea rezistenței sale, factor imperativ în grefarea ulterioară