Effects of various 3D biomaterials on osteoblastic differentiation of dental pulp mesenchymal cells

Milioni pacijenata širom sveta pate od oboljenja kostiju ili koštanih defekata kao što su osteosarkom, osteoporoza i frakture kostiju. U cilju rešavanja problema izgubljenog koštanog tkiva, inženjerstvo koštanog tkiva glavni akcenat stavlja na tkivnu regeneraciju a ne na zamenu tkiva, zbog čega postaje predmet sve većeg interesovanja. Matične ćelije i skafoldi imaju esencijalnu ulogu u stvaranju novog tkiva putem tkivnog inženjerstva. Ćelijska komponenta je neophodna zbog stvaranja i uspostavljanja ekstracelularnog matriksa u novom tkivu, dok su skafoldi zaduženi za obezbeđivanje mehaničke stabilnosti i temelja trodimenzionalne organizacije novog tkiva. Osnovni cilj doktorske disertacije bio je ispitati diferencijalni kapacitet izolovanih mezenhimalnih ćelija zubne pulpe u odnosu na 4 različita ćelijska nosača – skafolda: poroznog hidroksiapatita (pHAP) i poroznog hidroksiapatita u kombinaciji sa 3 polimera: polilaktid-koglikolnom kiselina (PLGA), alginatom i polimerom etilen vinil acetat/versatatom (EVA/EVV). Istraživanje je podrazumevalo izolovanje mezenhimalnih ćelija zubne pulpe poreklom iz mlečnih zuba u fiziološkoj smeni “outgrowth” metodom i njihovu karakterizaciju metodom protočne citometrije. Sintetisani biomaterijali su okarakterisani uz pomoć Fourier transform infracrvene spektroskopije (FTIR), rentgen difrakcije (XRD), mikroskopije atomskih sila (AFM), skening elektronske mikroskopije (SEM) i metode analize specifične površine čestice praha hidroksiapatita (BET metoda). Citotksičnost biomaterijala u prisustvu mezenhimalnih ćelija je ispitivana indirektnom metodom pomoću MTT testa i direktnom metodom pomoću LDH testa. Osteogena diferencijacija izolovanih mezenhimalnih ćelija u prisustvu različitih 3D biomaterijala praćena je bojenjem uzoraka alizarin crvenim, merenjem aktivnosti alkalne fosfataze i skening elektronskom mikroskopijom. Diferencijacijom ćelija u 3 različite vrste ćelija – adipocite, hondrocite i osteoblaste, i karakterizacijom CD29, CD73, CD90 i CD34 antitelima, pokazano je da su izolovane ćelije iz zubne pulpe mezenhimalne matične ćelije. Takođe, rezultati istraživanja pokazuju da su ispitivani skafoldi bili adekvatno dizajnirani i da su imali odgovarajuću morfologiju površine koja je neophodna za ćelijske nosače...Millions of patients worldwide suffer from bone disease or bone defects such as osteosarcoma, osteoporosis and bone fractures. In order to solve the problem of lost bone tissue, bone tissue engineering major emphasis is on tissue regeneration rather than tissue replacement, which is why it became the subject of growing interest. Stem cells and scaffolds have an essential role in the production of new tissue by tissue engineering. Cellular component is necessary for the generation and establishment of the extracellular matrix in new tissue, while scaffold is responsible for providing mechanical stability and the foundation of a new three-dimensional tissue organization. The main objective of the dissertation was to examine the differential capacity of isolated dental pulp mesenchymal cells in relation to 4 different cell carriers - scaffolds: porous hydroxyapatite (pHAP) and porous hydroxyapatite combined with 3 polymers: poly lactic-co-glycolic acid (PLGA), alginate and ethylene vinyl acetate / ethylene vinyl versatate (EVA / EVV). The research involved the isolation of dental pulp mesenchymal cells from human exfoliated deciduous teeth shift by "outgrowth" method and their characterization by flow cytometry. Characterization of synthesized biomaterials was performed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM) and methods of the specific surface area of hydroxyapatite powder (BET method). Cytotoxicity of biomaterials in the presence of mesenchymal cells was tested by an indirect method using the MTT test and by a direct method using the LDH test. Evaluation of osteogenic differentiation of isolated mesenchymal cells on 3D biomaterials samples was performed by alizarin red staining, alkaline phosphatase activity measurements and scanning electron microscopy. The differentiation of isolated dental pulp cells in 3 different cell types - adipocytes, chondrocytes and osteoblasts, and characterization of these cells by CD29, CD73, CD90 and CD34 antibodies, showed the isolated cells were mesenchymal stem cells..