Biological activity of the compound scaffolds based on highly porous hydroxyapatite and their influence on bone tissue engineering

Abstract

Since the beginning of this century the bone tissue engineering has developed into one of the essential research fields in reconstructive medicine, as an alternative approach compared with the conventional bone transplants. The main objective of this kind of therapy is to repair, regenerate and reconstruct the damaged or disease-stricken tissue. Tissue engineering is based on scaffolds which are of various biomaterials capable of participating in the process of bone tissue regeneration. A great number of materials has been researched and analyzed so as to be used as a scaffold for cell seeding. Therefore, the scaffold would enable those cells to grow, proliferate and penetrate the bone defects, whereupon the new tissue would take over the structure and function of the damaged tissue. Hydroxyapatites and other calcium phosphate materials were widely used as bone replacements for more than two decades. However, the poor mechanical features of porous hydroxyapatites led to their limited use. In case when they are combined into the compound materials together with polymer/biopolymer thin films, the porous hydroxyapatites acquire optimal characteristics required by scaffolds, which can offer excellent conditions for cell infiltration, growth and activity which is necessary in bone tissue engineering. The main objective of the doctoral dissertation was to carry out the characterization, examine the biocompatibility and biofunctionality of porous hydroxyapatite created by the modified hydrothermal method (pHAP), porous hydroxyapatite created by the modified hydrothermal method in combination with PLGA (Phap + PLGA) and porous hydroxyapatite created by the modified hydrothermal method in combination with metformin (pHAP + metformin). The research included the characterization of the tested materials by means of Fourier transform infrared spectroscopy (FTIR), X-ray distraction method (XRD), atomic forse microscopy method (AFM), scanning electorn microscopy analysis(SEM) and BET method. Biocompatibility was examined by MTT test (indirect contact of the tested materials with the cell culture L 929 fibroblasts), LDH test (direct contact of the tested materilas with the cell culture L929 fribroblasts) and test of local effects on skin in rabbits. Biofunctionality of the examined materials was researched by their implatation into defects of critical size of 6mm in the region of parietal bone in rabbits. After 12 weeks the animals were sacrifised, and tissue samples were prepared for pathohistological analysis. By means of the optical microscopy the following parametres were determined both quantitatively and qualitatively: the size of the defect, the presence of giant cells, the presence of neoangiogenesis, the presence of basophils, the presence of nonspecific inflammation in the tissue, the appearance of the newly created bone, the presence of fibroplasia in the tissue and the percentage of mineralization...

Tkivno inženjerstvo koštanog tkiva se u ovom veku razvilo u jedno od glavnih polja istraživanja u regenerativnoj medicini. Ono predstavlja alternativni pristup u odnosu na konvencionalne koštane transplantate. Osnovni cilj ove vrste terapije je da popravi, regeneriše i rekonstruiše oštećeno ili bolešću zahvaćeno tkivo. Osnovu tkivnog inženjerstva čine ćelijski nosači koji predstavljaju razne vrste biomaterijala sposobnih da učestvuju u regeneraciji koštanog tkiva. Veliki broj materijala je istraživan i analiziran sa ciljem da se koristi kao ćelijski nosač na koji bi mogle da se zasejavaju ćelije. Ćelijski nosač koji bi tim ćelijama omogućio rast, proliferaciju i njihovo unošenje u defekte kosti nakon čega bi novostvoreno tkivo preuzimalo strukturu i funkciju obolelog dela tkiva. Hidroksiapatiti i drugi kalcijum fosfatni materijali imali su široku upotrebu kao koštani zamenici više od dve decenije. Zbog loših mehaničkih osobina poroznih hidroksiapatita došlo je do ograničenja njihove primene. U slučaju kada se kombinuju u kompozitne materijale sa odgovarajućim polimer/biopolimer tankim filmovima, porozni hidroksiapatiti dobijaju optimalne osobine potrebne ćelijskim nosačima, koje mogu pružiti odlične uslove za infiltraciju, rast i aktivnost ćelija neophodnih u tkivnom inženjerstvu kosti. Osnovni cilj doktorske disertacije bio je izvršiti karakterizaciju, ispitati biokompatibilnost i biofunkcionalnost poroznog hidroksiapatita dobijenog modifikovanom hidrotermalnom metodom(pHAP), poroznog hidroksiapatita dobijenog modifikovanom hidrotermalnom metodom u kombinaciji sa PLGA (pHAP+PLGA) i poroznog hidroksiapatita dobijenog modifikovanom hidrotermalnom metodom u kombinaciji sa metforminom (pHAP+metformin). Istraživanje je podrazumevalo karakterizaciju ispitivanih materijala uz pomoć Fourier transform infracrvene spektroskopije (FTIR), metodom rentgen difrakcije (XRD), metodom atomski forsirane mikroskopije (AFM), analizom skening elektronske mikroskopije (SEM) i BET metodom. Biokompatibilnost je ispitivana pomoću MTT testa (indirektni kontakt ispitivanih materijala sa ćelijskom kulturom L 929 fibroblasta), LDH testa (direktni kontakt ispitivanih materijala sa ćelijskom kulturom L 929 fibroblasta) i testom kutane iritacije. Biofunkcionalnost ispitivanih materijala bila je istraživana njihovom implantacijom u defekte kritične veličine od 6 mm u predelu kalvarije kunića. Nakon 12 nedelja životinje su bile žrtvovane, a uzorci tkiva su obrađeni za patohistološku analizu. Optičkom mikroskopijom kvantitativno i kvalitativno obrađivani su sledeći parametri: veličina defekta, prisustvo džinovskih ćelija, prisustvo neoangiogeneze, prisustvo bazofila, postojanje znakova nespecifičnog zapaljenja u tkivu, pojava novostvorene kosti, prisustvo fibroplazije u tkivu i procenat mineralizacije...