Uporedna analiza efekata holekalciferola i alfakalcidola kao sastavni delovi implantata na proces zarastanja defekata u femuru ovariektomisanih pacova

Abstract

Postmenopauzna osteoporoza je sistemska skeletna bolest koja se karakteriše smanjenom koštanom masom i narušenom mikroarhitekturom kosti koje su posledica nedostatka estrogena. Smanjena sinteza estrogena u menopauzi dovodi do narušenog balansa između razgradnje i izgradnje kosti, usled čega dolazi do gubitka koštanog tkiva u pojedinim delovima skeleta i nastanka fraktura. Osteoporoza predstavlja veliki problem u stomatologiji i ortopediji zbog smanjenog kapaciteta osteoporotične kosti da obezbedi neophodnu potporu implantatima i omogući adekvatnu fiksaciju preloma. Zarastanje fraktura u osteoporotičnim uslovima je otežano zbog čega je poželjno korišćenje lekova i koštanih zamenika koji bi unapredili regeneraciju kosti i ojačali prelomljeno mesto. Pokazano je da vitamin D i njegovi analozi, koji se koriste u prevenciji i tretmanu osteoporoze, povoljno utiču na zarastanje preloma. Međutim, visoke doze ovog vitamina koje su neophodne za ostvarivanje direktnih anaboličkih efekata na kost, dovode do pojave hiperkalcemije. Kako bi se izbegli neželjeni efekti sistemske administracije visokih doza vitamina D, jedna od mogućnosti je njihova lokalna primena u mesto defekta. Glavni cilj doktorske disertacije je ispitivanje uticaja lokalno primenjenih holekalciferola i alfakalcidola, kao sastavnih delova implantata sačinjenih od mineralnog matriksa kosti (MMK) i plazme obogaćene trombocitima (PRP), na proces zarastanja defekata u osteoporotičnoj kosti pacova. Uporedni pregled osteogenih efekata holekalciferola i alfakalcidola, kao i poređenje dobijenih rezultata sa spontanom regeneracijom osteoporotične kosti i regeneracijom uz pomoć implantata sastavljenih samo od mineralnog matriksa kosti i PRP, su takođe analizirani u okviru disertacije. S tim u vezi, cilj disertacije je i uspostavljanje i verifikovanje eksperimentalnog modela osteoporoze izazvane ovariektomijom ženki pacova Wistar soja koji bi bio pogodan za ovakvu vrstu istraživanja. Prema postavljenim ciljevima istraživanja urađene su dve eksperimentalne studije. U prvoj studiji eksperimentalna osteoporoza je izazvana bilateralnom ovariektomijom i vreme koje je potrebno za razvoj osteoporotičnih znakova i nastanak promena u femoralnoj kosti je procenjivano endokrinološkim, biohemijskim i radiološkim metodama. U okviru druge eksperimentalne studije, nakon dobijanja modela osteoporoze kod pacova, kreirani su defekti do kostne srži u distalnom delu oba femura. Defekti su ostavljeni da spontano zarastaju (SR grupa) ili su ispunjavani implantatima sačinjenim od mineralnog matriksa kosti i PRP, koji su Jelena Rajković Doktorska disertacija primenjeni sami (SC grupa), ili u kombinaciji sa ispitivanim formama vitamina D, holekalciferolom (HC grupa) i alfakalcidolom (AC grupa). Mineralni matriks kosti se inače koristi kao koštani zamenik i skafold za naseljavanje ćelija, a u ovim ispitivanjima je služio i kao nosač za PRP i ispitivane forme vitamina D. Za praćenje regeneracije osteoporotične kosti korišćene su biohemijske, histološke, imunohistohemijske, histomorfometrijske i skening-elektronomikroskopske metode i analizirani su periodi dve nedelje i osam nedelja od kreiranja defekata u femuru pacova. Rezultati prve studije pokazuju da je do značajnog opadanja koncentracije estradiola, nakon njegovih početnih fluktuacija, došlo u trećem mesecu nakon ovariektomije pacova Wistar soja. Aktivnosti enzima ALP i AP su povišene tokom ispitivanog perioda u grupi ovariektomisanih pacova, dok je koncentracija Ca u serumu niža u četvrtom, a koncentracija P u drugom mesecu nakon ovariektomije u odnosu na pacove iz kontrolne grupe. Radiološka analiza je pokazala smanjenje koštane gustine u distalnom delu femura nakon tri meseca od ovariektomije. Rezultati druge studije pokazuju da lokalno primenjeni holekalciferol i alfakalcidol, u korišćenom eksperimentalnom dizajnu i primenjenoj dozi, odlažu rani proces zarastanja kosti i sprečavaju nastanak fibroze kod ovariektomisanog modela pacova. Obe ispitivane forme vitamina D smanjuju resorpciju partikula MMK u mestu defekta, što predstavlja smetnju za formiranje nove kosti u grupi tretiranoj alfakalcidolom. Alfakalcidol dovodi do nastanka slabo mineralizovanog kalusa koji se karakteriše intenzivnom balansiranom remodelacijom, kompaktnošću i dobrom integracijom u okolnu osteoporotičnu kost. Pod uticajem holekalciferola razvija se velika količina kalusa koji se odlikuje dobrom mineralizacijom, ali takođe i poroznošću i slabom integracijom, što su karakteristike kalusa slične onim u grupi sa spontanom regeneracijom. Holekalciferol i alfakalcidol su pokazali brojne efekte u korišćenom eksperimentalnom dizajnu na osnovu kojih se može sugerisati njihova primena kao potencijalnih agenasa u rešavanju različitih problema u regeneraciji osteoporotične kosti. Izbor odgovarajuće forme vitamina D, kao i biomaterijala koji bi bio korišćen kao implantat za popunjavanje defekata u kosti, treba biti napravljen u skladu sa ciljem tretmana. Rezultati ove doktorske disertacije su doprinos boljem razumevanju mehanizama delovanja alfakalcidola i holekalciferola na regeneraciju osteoporotične kosti, sa praktičnom primenom u otvaranju novih terapeutskih pristupa lečenju defekata kosti u osteoporotičnim uslovima.

Postmenopausal osteoporosis is a systemic skeletal disease caused by lack of estrogen and characterized by low bone mass and impaired microarchitecture. Reduced synthesis of estrogen in menopause leads to impaired balance between bone resorption and bone formation, causing a loss of bone tissue in some parts of the skeleton and susceptibility to fractures. Osteoporosis is a major problem in dentistry and orthopedics due to reduced capacity of osteoporotic bone to provide the necessary support to implants and enable adequate fracture fixation. Osteoporotic fractures are very difficult to heal in osteoporotic conditions, therefore, it is favourable to use drugs and bone substitutes that will improve the bone regeneration and strengthen the site of the fracture. It has been shown that vitamin D and its analogues, which are widely used in the treatment of osteoporosis, have a positive effect on the healing of fractures. However, high doses of vitamin D, which are necessary for obtaining the direct anabolic effects on bone, lead to the appearance of hypercalcemia. To avoid side effects of systemic administration of high vitamin D doses, one of the possibilities is their local application into the site of defect. Тhe main objective of this PhD thesis is to examine the impact of cholecalciferol and alfacalcidol, as integral parts of the implants consisting of bone mineral matrix (BMM) supplemented with platelet-rich plasma (PRP), on femur defect healing in osteoporotic rat bone. Comparative overview of osteogenic effect of cholecalciferol and alfacalcidol as well as comparison of results with the spontaneous regeneration of osteoporotic bone and regeneration with the help of implants composed only of BMM and PRP, were also analyzed in the dissertation. In this regard, the aim of the dissertation is to establish and verify an experimental model of osteoporosis in Wistar rats that is suitable for this type of research. According to the objectives of the research, two experimental studies were conducted. In the first study, experimental osteoporosis was induced by bilateral ovariectomy and the time required for development of osteoporotic conditions and the occurrence of changes in femoral bone was evaluated by using endocrinological, biochemical and radiological analysis. In the second experimental study, after obtaining osteoporotic rat model, defects were created up to the bone marrow in the distal part of the both femoral diaphysis. The defects were left to heal spontaneously (SR group) or filled with implants composed of bone mineral matrix supplemented with PRP, applied alone (SC group), or as carriers for the examined forms of vitamin D, cholecalciferol (HC group) and alfacalcidol (AC group). Bone mineral matrix serves as a bone substitute and a scaffold for cell colonization, and in these studies it has also served as a carrier for the PRP and tested forms of vitamin D. Јелена Рајковић Докторска дисертација Healing of defects and the quality of the newly formed bone tissue were observed by biochemical, histological, immunohistochemical, histomorphometric and scanning electron microscopy analyses. Regeneration process was monitored two and eight weeks after the creation of defects in the femurs of rats. Results of the first study show that a significant decline in estradiol concentration, after its initial fluctuations, occurred in the third month after ovariectomy. The ALP and AP activity in serum were increased during the examined period, while the concentration of Ca was lower in the fourth and the concentration of P in the second month after ovariectomy compared to control rats. Radiographic analysis showed decrease in bone density in the distal part of the femur, three months after ovariectomy. Results of the second study show that locally applied cholecalciferol and alfacalcidol, in the used experimental design and applied doses, delay early bone healing and prevent fibrosis in ovariectomized rat model. Both of the examined vitamins decreased the resorption of BMM particles in defects which is a barrier to the formation of new bone in the group treated with alfacalcidol. Alfacalcidol leads to the formation of poorly mineralized callus which is characterized by intense balanced remodeling, compactness and good integration with the surrounding osteoporotic bone. A large amount of callus develops under the influence of cholecalciferol and it is characterized by good mineralization, but also porosity and weak integration, which are characteristics of callus similar to those in the group with spontaneous regeneration. Cholecalciferol and alfacalcidol showed many effects in the used experimental design on the basis of which their use as potential agents in solving various problems related to the osteoporotic bones regeneration can be assessed. The choice of the appropriate form of vitamin D, as well as biomaterials that would be used as an implant to fill bone defects, should be made in accordance with the objective of treatment. The results of this PhD thesis contribute to a better understanding of the mechanisms of alfacalcidol and cholecalciferol actions in osteoporotic bone regeneration, with practical application in opening new therapeutic approach to the treatment of bone defects in osteoporotic conditions.