Ispitivanje biokompatibilnosti nanokompozitnih hidrogelova srebro / polivinil-alkohol / grafen u potkožnom tkivu pacova, namenjenih biomedicinskoj primeni

This doctoral dissertation was designed in order to test the biocompatibility of two newly synthesized and previously in vitro characterized hydrogels based on poly(vinyl alcohol) polymers - Ag/PVA and Ag/PVA/Gr, which are intended for making wound dressings or soft tissue implants tissue. Poly(vinyl alcohol) (PVA) is a synthetic polymer commonly used in biomedicine due to its non-toxicity, solubility, biocompatibility, elasticity and the ability to produce reversible hydrogels with high sorption capacity. In the last few years, research has also focused on the incorporation of graphene (Gr) into hydrogel PVAs, in order to improve their mechanical strength and durability. In tissue engineering and regenerative medicine, it is particularly important that implants and bandages contain an antiinflammatory and antibacterial active ingredient to protect the sterility of the wound, or to prevent infection in the implant surrounding tissue. It is known that silver nanoparticles (AgNPs) have excellent antibacterial activity and are therefore often used as an active component of PVA-based hydrogel biomaterials. In order to test the biocompatibility of newly synthesized hydrogels, the soft tissue response to deep subcutaneously implanted Ag/PVA and Ag/PVA/Gr implants was compared to the reaction to the commercial calcium-alginate wound pad (Suprasorb ©, Lohmann & Rauscher GmbH & Co. KG, Neuwied, Germany), which is implanted in an identical manner. The tissue irritation index (IrI) was evaluated as the basic measure of tissue response to implanted hydrogels. In order to determine the degree of biocompatibility, the thickness of the connective tissue capsule around the implanted hydrogels, the number of inflammatory cells, including the Langerhans giant cells, the number of macrophages in the perimplant zone and their eneventual migration to the biomaterial, as well as the number of blood vessels around the implantation zone, were measured. The ratio of proliferating and apoptotic cellular skin and subcutaneous tissue cellular entities was observed, due to their responsibility for regeneration and reparation processes...Ova doktorska disertacija dizajnirana je u cilju da se ispita biokompatibilnost dva novosintetisana i prethodno in vitro okarakterisana hidrogela na bazi sintetskog polimera polivinil-alkohola − Ag/PVA i Ag/PVA/Gr, koji su namenjeni za izradu obloga za rane ili za implantate za meka tkiva. Polivinil-alkohol (PVA) je sintetički polimer koji se često koristi u biomedicini zbog svoje netoksičnosti, rastvorljivosti, biokompatibilnosti, elastičnosti i sposobnosti stvaranja reverzibilnih hidrogelova s visokom sposobnošću sorpcije. U poslednjih nekoliko godina, istraživanja se takođe fokusiraju na ugradnju grafena (Gr) u hidrogelove PVA kako bi se poboljšala njihova mehanička čvrstoća i trajnost. U inženjerstvu tkiva i regenerativnoj medicini posebno je važno da implantati i zavoji sadrže antiinflamatornu i antibakterijsku aktivnu komponentu kako bi zaštitili sterilnost rane ili sprečili infekciju u tkivu koje okružuje implantat. Poznato je da nanočestice srebra (eng. silver nanoparticles, AgNPs) poseduju odličnu antibakterijsku aktivnost, pa se zbog toga često koriste kao aktivna komponenta biomaterijala na bazi hidrogela od PVA. U cilju ispitivanja biokompatibilnosti novosintetisanih hidrogelova, praćena je reakcija mekog tkiva na duboko potkožno postavljene implantate od Ag/PVA i Ag/PVA/Gr i poređena je sa reakcijom na komercijalnu oblogu za rane na bazi kalcijum-alginata (Suprasorb©, Lohmann & Rauscher GmbH & Co. KG, Neuwied, Germany), koja je implantirana na identičan način. Procenjivan je tkivni iritacioni indeks (IrI) kao osnovna mera tkivnog odgovora na implantirane hidrogelove. Za određivanje stepena biokompatibilnosti merena je debljina vezivnotkivne kapsule oko implantiranih hidrogelova, broj inflamatornih ćelija, uključujući i Langerhansove džinovske ćelije, broj makrofaga u periimplantnoj zoni i njihova enevntualna migracija u biomaterijal, kao i brojnost krvnih sudova mikrovaskularnog korita oko zone implantacije. Posmatran je odnos proliferacije i apoptoze ćelijskih entiteta kože i potkožnog tkiva odgovornih za procese regeneracije i reparacije..

Biocompatibility of silver / poly(vinyl alcohol) / graphen nanocomposite hydrogels in rat subcutaneous tissues for biomedical applications

Ova doktorska disertacija dizajnirana je u cilju da se ispita biokompatibilnost dva novosintetisana i prethodno in vitro okarakterisana hidrogela na bazi sintetskog polimera polivinil-alkohola − Ag/PVA i Ag/PVA/Gr, koji su namenjeni za izradu obloga za rane ili za implantate za meka tkiva. Polivinil-alkohol (PVA) je sintetički polimer koji se često koristi u biomedicini zbog svoje netoksičnosti, rastvorljivosti, biokompatibilnosti, elastičnosti i sposobnosti stvaranja reverzibilnih hidrogelova s visokom sposobnošću sorpcije. U poslednjih nekoliko godina, istraživanja se takođe fokusiraju na ugradnju grafena (Gr) u hidrogelove PVA kako bi se poboljšala njihova mehanička čvrstoća i trajnost. U inženjerstvu tkiva i regenerativnoj medicini posebno je važno da implantati i zavoji sadrže antiinflamatornu i antibakterijsku aktivnu komponentu kako bi zaštitili sterilnost rane ili sprečili infekciju u tkivu koje okružuje implantat. Poznato je da nanočestice srebra (eng. silver nanoparticles, AgNPs) poseduju odličnu antibakterijsku aktivnost, pa se zbog toga često koriste kao aktivna komponenta biomaterijala na bazi hidrogela od PVA. U cilju ispitivanja biokompatibilnosti novosintetisanih hidrogelova, praćena je reakcija mekog tkiva na duboko potkožno postavljene implantate od Ag/PVA i Ag/PVA/Gr i poređena je sa reakcijom na komercijalnu oblogu za rane na bazi kalcijum-alginata (Suprasorb©, Lohmann & Rauscher GmbH & Co. KG, Neuwied, Germany), koja je implantirana na identičan način. Procenjivan je tkivni iritacioni indeks (IrI) kao osnovna mera tkivnog odgovora na implantirane hidrogelove. Za određivanje stepena biokompatibilnosti merena je debljina vezivnotkivne kapsule oko implantiranih hidrogelova, broj inflamatornih ćelija, uključujući i Langerhansove džinovske ćelije, broj makrofaga u periimplantnoj zoni i njihova enevntualna migracija u biomaterijal, kao i brojnost krvnih sudova mikrovaskularnog korita oko zone implantacije. Posmatran je odnos proliferacije i apoptoze ćelijskih entiteta kože i potkožnog tkiva odgovornih za procese regeneracije i reparacije...This doctoral dissertation was designed in order to test the biocompatibility of two newly synthesized and previously in vitro characterized hydrogels based on poly(vinyl alcohol) polymers - Ag/PVA and Ag/PVA/Gr, which are intended for making wound dressings or soft tissue implants tissue. Poly(vinyl alcohol) (PVA) is a synthetic polymer commonly used in biomedicine due to its non-toxicity, solubility, biocompatibility, elasticity and the ability to produce reversible hydrogels with high sorption capacity. In the last few years, research has also focused on the incorporation of graphene (Gr) into hydrogel PVAs, in order to improve their mechanical strength and durability. In tissue engineering and regenerative medicine, it is particularly important that implants and bandages contain an antiinflammatory and antibacterial active ingredient to protect the sterility of the wound, or to prevent infection in the implant surrounding tissue. It is known that silver nanoparticles (AgNPs) have excellent antibacterial activity and are therefore often used as an active component of PVA-based hydrogel biomaterials. In order to test the biocompatibility of newly synthesized hydrogels, the soft tissue response to deep subcutaneously implanted Ag/PVA and Ag/PVA/Gr implants was compared to the reaction to the commercial calcium-alginate wound pad (Suprasorb ©, Lohmann & Rauscher GmbH & Co. KG, Neuwied, Germany), which is implanted in an identical manner. The tissue irritation index (IrI) was evaluated as the basic measure of tissue response to implanted hydrogels. In order to determine the degree of biocompatibility, the thickness of the connective tissue capsule around the implanted hydrogels, the number of inflammatory cells, including the Langerhans giant cells, the number of macrophages in the perimplant zone and their eneventual migration to the biomaterial, as well as the number of blood vessels around the implantation zone, were measured. The ratio of proliferating and apoptotic cellular skin and subcutaneous tissue cellular entities was observed, due to their responsibility for regeneration and reparation processes..

Peripartal leukogram in cows with and without retained placenta

The aim of this study was to investigate whether prepartal leukogram in cows with retained placenta could indicate the presence of subclinical systemic inflammatory response before the onset of disease. After calving, sixteen highly pregnant Holstein cows, aged 3 to 9 years, without clinical signs of the disease prior to calving were divided into two groups: the first group (n=9) were animals without retained placenta, or any visible inflammation after birth; the second group (n=7) were cows with retained placenta. Blood was sampled three times before parturition, at intervals of one week, and once 24 hours after birth. The number of total leukocytes, segmented and non segmented neutrophilic granulocytes (NG), lymphocytes and monocytes were determined by standard laboratory techniques. The results have shown that in the group of cows with retained placenta the number of mature neutrophils was slightly elevated in the third, second and last week before calving, and equal number of non segmented neutrophils in regard to the group with no retention. The results have also shown that, in both groups of cows, 24 hours after calving, the number of total leukocytes and the number of segmented neutrophils decreased, but the number of the non segmented neutrophils increased. Based on this, we can conclude that cows with retained placenta had no systemic inflammatory response during three weeks prepartal period, but 24 hours after calving, systemic inflammatory response was documented in all the cows. Moreover, the intensity of inflammatory response in cows with retained placenta was not more pronounced in comparison to cows without retained placenta. [Projekat Ministarstva nauke Republike Srbije, br. 175061

Površinski epitel jajnika hipotireoidnih novorođenih i neonatalnih pacova - iz perspektive PCNA i kaspaze-3

Introduction. The ovarian surface epithelium (OSE) undergoes intensive regeneration and remodelling after each ovulation during the whole reproductive period. This process increases the risk of one of the most common ovarian tumors in women and the female dog. Considering the fact that maternal hypothyroidism highly impacts cell proliferation and cell death during folliculogenesis in the early neonatal period, we aimed to analyze its effect on OSE morphology and dynamics. Materials and Methods. The study was performed on newborn (24-h-old) and neonatal (4-day-old) female rats, a randomized trial between the control and hypothyroid groups, born under controlled circumstances and hypothyroid mothers, respectively. Their ovaries were analyzed histologically and processed to determine the OSE cell height as an average value of four measurement points. Also, the immunopositivity of the proliferating cell nuclear antigen (PCNA) and caspase-3 were assessed semiquantitatively. Results and Conclusions. No major structural differences of OSE were found between groups within the given ages except for a slight increment of OSE cell height and incompleteness of apical cell membrane with cytoplasmic projections in hypothyroid animals. PCNA immunopositivity of the OSE cells was higher in ovaries of hypothyroid animals of both ages in comparison to the controls. Moreover, only scarce OSE cells were caspase-3 positive in both groups and ages, with no difference in immunopositivity. Our study confirms the impact of hypothyroidism in the early postnatal period on morphology and proliferation rate of OSE cells, with no effect on caspase-3 dependent cell removal, which may serve as a premise for future investigation of potential carcinogenesis, in terms of prevention and treatment of ovarian cancer.Uvod. Tokom reproduktivnog perioda, nakon svake ovulacije, površinski epitel (PE) jajnika se intenzivno regeneriše i remodelira. Ovaj proces povećava rizik od nastajanja jednog od najčešćih tumora jajnika kod žena i kuja. Imajući u vidu činjenicu da hipotireoidizam majki u velikoj meri utiče na proliferaciju i ćelijsku smrt u toku folikulogeneze u ranom postnatalnom periodu, cilj rada je bio da analiziramo efekat hipotireoidizma majki na morfologiju i dinamiku PE jajnika potomaka. Materijal i metode. U eksperimentu su korišćeni tek rođeni (24 časa stari) i neonatalni (4 dana stari) mladunci koji potiču od kontrolnih i hipotireoidnih majki. Morfometrijskom analizom određivana je prosečna visina ćelija PE, izmerena na četiri mesta na svakom ispitivanom preseku jajnika. Takođe, imunopozitivnost proliferativnivnog ćelijskog nuklearnog antigena (PCNA) i kaspaze 3 je određivana semikvantitativno. Rezultati i zaključci. Veće promene u strukturi PE jajnika nisu zapažene između grupa, osim blagog povećanja visine ćelija i nepotpune apikalne membrane sa projekcijama citoplazme kod hipotireoidnih životinja. Imunopozitivnost PCNA je značajno povećana u jajnicima hipotireoidnih životinja obe starosne grupe u odnosu na kontrole, dok je imunopozitivnost na kaspazu 3 slabo izražena kod svih životinja. Naše istraživanje je pokazalo da hipotireoidizam u ranom postnatalnom periodu dovodi do promena u morfologiji i proliferaciji ćelija PE, bez efekta na uklanjanje ćelija delovanjem kaspaze 3. Ovaj model može da posluži u budućim istraživanjima potencijalne karcinogeneze u cilju prevencije i tretmana kancera jajnika

Z-cells and oogonia/oocytes in the advanced process of autophagy are the dominant altered cells in the ovaries of hypothyroid newborn rats

Induced prenatal hypothyroidism in rat pups leads to accelerated primordial follicle assembly and premature follicular atresia with ovary failure. This work investigates the influence of maternal hypothyroidism induced with 6-n-propyl-2-thyouracil (PTU) on the number and morphology of oogonia/oocytes in newborn rat pups with light and transmission electron microscopy. Expression of apoptosis and autophagy markers in oogonia/oocytes were examined using immunohistochemistry. Hypothyroid newborn pups had a decreased number of mitotic and resting oogonia, while the number of altered oogonia/oocytes was increased. Ultrastructural observations revealed the increased presence of degenerated pachytene oocytes (Z-cells) and oogonia/oocytes undergoing autophagy, apoptosis and combined apoptosis and autophagy, in this group. The most abundant altered oogonia/oocytes in the hypothyroid group were those with morphological features of advanced autophagy and Z-cells. The percentage of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) positive oogonia/ oocytes was significantly lower in the hypothyroid group. No significant difference was recorded in the expression of caspase-3, ATG7 and LC3 possibly reflecting that these proteins were not involved in the oogonia/ oocyte alteration process during prenatal rat hypothyroidism. The obtained results indicate that developmental hypothyroidism in the offspring enhances the number of Z-cells and oogonia/ oocytes altered with the advanced process of autophagy

Effect of subclinical and overt form of rat maternal hypothyroidism on offspring endochondral bone formation

Maternal hypothyroidism in its overt form affects skeletal development of the offspring, but these data are not available for the subclinical form which is becoming very frequent among pregnant women. We hypothesized that the subclinical form of hypothyroidism in rat dams, influences the process of offspring endochondral ossification affecting proliferation and differentiation of chondrocytes, osteoclasts and osteoblasts in pups. Seven-day-old male pups (n=18) derived from control dams and dams treated with a low dose (1.5 mg/L) or high dose (150 mg/L) of propylthiouracil in drinking water during pregnancy and lactation were used. Histomorphometric analysis of pups tibia proximal growth plate, expression of mRNA, immunohistochemical and histochemical visualization of extracellular matrix components was performed. The length of the tibia was reduced in hypothyroid pups. Secretion of type 2 and 10 collagens in the subclinical and overt form were lower while the amount of glycosaminoglycans was higher when compared with controls. Down-regulated tartrate resistant acid phosphatase mRNA indicated altered osteoclasts function while lower expression of dentin matrix acid protein-1 mRNA and reduced synthesis of type 1 collagen accentuated a compromised bone formation in the overt form of hypothyroidism. The subclinical form of maternal hypothyroidism had a negative effect on the differentiation of hypertrophic chondrocytes and calcified cartilage removal in 7-day-old pups. In addition, overt hypothyroidism had a negative effect on the proliferation of chondrocytes and deposition of osteoid. Both forms of hypothyroidism resulted in a decrease of tibia length due to changes in growth plate formation

Hypothyroidism in pregnant rats affects proliferation and cell death of growth plate chondrocytes in pups

Overt and subclinical maternal hypothyroidism affects cartilage and bone extracellular matrix formation during fetal endochondral bone development. Less is known about the effect maternal hypothyroidism has on cell proliferation and death of growth plate chondrocytes. This study aimed to evaluate the expression of proliferation (Ki67), autophagy (LC3) and apoptosis (caspase 3) markers in the epiphyseal cartilage of the tibia in pups from subclinical and overt hypothyroid does, in their early postnatal development.Hypothyroidism was induced with propylthiouracil using low (1.5 mg/L) for subclinical (H1) and high dose (150 mg/L) for overt (H2) form. Propylthiouracil was administered through drinking water in pregnant Albino Oxford does from the first day of gravidity and during lactation. Control (C) group was not treated. Six, seven-day-old male pups from each group were euthanized. Histological examinations were performed on paraffin sections of the proximal tibial growth plate. Immunohistochemistry was used to assess the expression of Ki67, LC3 and caspase 3. The number of Ki67 positive chondrocytes was evaluated in the resting and proliferating zone using Image J. The expression of LC3 was performed by counting the number of positive dots in chondrocytes.The number of Ki67 positive chondrocytes was higher in both hypothyroid groups, while the number of LC3 positive dots per cell was lower indicating that the chondrocyte cell cycle in hypothyroid animals is faster and that the basal level of autophagy is reduced due to frequent mitosis. Compared to controls, caspase 3 expression in the terminal hypertrophic chondrocytes was reduced in H1 and completely absent in H2 pups which might indicate impaired differentiation.We conclude that both forms of maternal hypothyroidism in rats lead to accelerated proliferation, slowed autophagy and compromised apoptosis of terminal hypertrophic chondrocytes in the early infantile period and a delay in cartilage to bone transition

Thyroid hormones affect the proliferation and/or mobilization of bulge stem cell population

Thyroid hormones (TH) play a pivotal role in the development of mammalian skin, and are necessary for both the initiation and maintenance of hair growth. Adult hypothyroid rats showed impaired epidermal proliferation, hair growth and wound healing. Stem cells (SCs) located at the bulges of the hair follicles are responsible for hair cycling, and contribute to the regeneration of the new epidermis after wounding. Therefore a reduction in the number or function of bulge stem cells could be a cause of disturbed skin reepithelization and hair follicle maintenance. We hypothesized that a decrease in TH during prenatal development would affect the bulge stem cell population. Subclinical hypothyroidism was induced with propylthiouracil through drinking water in a dose of 1.5 mg/l in pregnant Albino Oxford rats, from the first day of gravidity and during lactation. The control group was untreated. Five, seven-day-old pups were euthanized and skin samples were taken from the dorsal part of their bodies. The number of hair follicles was estimated on an area of 1mm2 of dermis. For the immunohistochemical study, proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology) and NANOG (Thermo Scientific) antibodies were used. The number of PCNA and NANOG positive cells was estimated on the area of 1mm2 of hair follicle. The number of hair follicles was reduced and the expression of PCNA was decreased in the cells of the inner and outer sheath of hair follicles in the hypothyroid pups compared to the controls. They also had an increased number of NANOG positive bulge cells which we presume are multipotent SCs. Decreased expression of PCNA demonstrates low proliferation and increased expression of NANOG, which may be explained in two ways: 1) the inhibited differentiation of putative SCs and/or 2) the reduction of mobilization of these cells to the epidermis or sebaceous gland. Our results confirm that the lack of TH influences the formation of hair follicles in early infantile rat pups

Ovarian surface epithelium of hypothyroid newborn and neonatal rats: From PCNA and caspase-3 perspective

Introduction. The ovarian surface epithelium (OSE) undergoes intensive regeneration and remodeling after each ovulation during the whole reproductive period. This process increases the risk of one of the most common ovarian tumors in women and the female dog. Considering the fact that maternal hypothyroidism highly impacts cell proliferation and cell death during folliculogenesis in the early neonatal period, we aimed to analyze its effect on OSE morphology and dynamics. Materials and Methods. The study was performed on newborn (24-h-old) and neonatal (4-day-old) female rats, a randomized trial between the control and hypothyroid groups, born under controlled circumstances and hypothyroid mothers, respectively. Their ovaries were analyzed histologically and processed to determine the OSE cell height as an average value of four measurement points. Also, the immunopositivity of the proliferating cell nuclear antigen (PCNA) and caspase-3 were assessed semiquantitatively. Results and Conclusions. No major structural differences of OSE were found between groups within the given ages except for a slight increment of OSE cell height and incompleteness of apical cell membrane with cytoplasmic projections in hypothyroid animals. PCNA immunopositivity of the OSE cells was higher in ovaries of hypothyroid animals of both ages in comparison to the controls. Moreover, only scarce OSE cells were caspase-3 positive in both groups and ages, with no difference in immunopositivity. Our study confirms the impact of hypothyroidism in the early postnatal period onmorphology and proliferation rate of OSE cells, with no effect on caspase-3 dependent cell removal, which may serve as a premise for future investigation of potential carcinogenesis, in terms of prevention and treatment of ovarian cancer

Karakterizacija matičnih ćelija izolovanih iz zubne pulpe mlečnih zuba dece

Background/Aim. The last decade has been profoundly marked by persistent attempts to use ex vivo expanded and manipulated mesenchymal stem cells (MSCs), as a tool in different types of regenerative therapy. In the present study we described immunophenotype and the proliferative and differentiation potential of cells isolated from pulp remnants of exfoliated deciduous teeth in the final phase of root resorption. Methods. The initial adherent cell population from five donors was obtained by the outgrowth method. Colony forming unit-fibroblast (CFU-F) assay was performed in passage one. Cell expansion was performed until passage three and all tests were done until passage eight. Cells were labeled for early mesenchymal stem cells markers and analysis have been done using flow cytometry. The proliferative potential was assessed by cell counting in defined time points and population doubling time was calculated. Commercial media were used to induce osteoblastic, chondrogenic and adipogenic differentiation. Cytology and histology methods were used for analysis of differentiated cell morphology and extracellular matrix characteristics. Results. According to immunophenotype analyses all undifferentiated cells were positive for the mesenchymal stem cell markers: CD29 and CD73. Some cells expressed CD146 and CD106. The hematopoietic cell marker, CD34, was not detected. In passage one, incidence of CFU-F was 4.7 ± 0.5/100. Population doubling time did not change significantly during cell subcultivation and was in average 25 h. After induction of differentiation, the multicolony derived cell population had a tri-lineage differentiation potential, since mineralized matrix, cartilage-like tissue and adipocytes were successfully formed after three weeks of incubation. Conclusion. Altogether, these data suggest that remnants of deciduous teeth dental pulp contained cell populations with mesenchymal stem cell-like features, with a high proliferation and tri- lineage differentiation potential and that these cultures are suitable for further in vitro evaluation of cell based therapies.Uvod/Cilj. Prošla dekada je bila posebno obeležena naporima na polju korišćenja ex vivo razvijenih i usmeravanih mezenhimskih matičnih ćelija (MSCs), kao sredstva za različite tipove regenerativne terapije. Cilj ove studije bio je da se utvrdi imunofenotip i potencijal za proliferaciju i diferencijaciju ćelija izolovanih iz zubne pulpe mlečnih zuba dece eksfoliranih u periodu kada je koren zuba bio u poslednjoj fazi resorpcije. Metode. Primarna adherentna populacija ćelija poreklom od pet donora dobijena je metodom eksplanta. Prisustvo progenitorskih ćelija koje obrazuju kolonije fibroblasta (CFU-F) pokazano je u prvoj pasaži. Do treće pasaže ćelije su ekspandirane, a potom korišćene za analiziranje. Imunofenotip je određen korišćenjem protočne citometrije. Proliferativni potencijal i vreme udvajanja ćelija (PDT) u kulturi je definisano na osnovu apsolutnog broja ćelija na početku i na kraju svake pasaže. Posle tronedeljne kultivacije ćelija u komercijalnim medijumima za stimulaciju osteogeneze, hondrogeneze i adipogeneze, citološkim i histološkim metodama je određena morfologija ćelija i karakteristike vanćelijskog matriksa. Rezultati. Antigeni koji karakterišu mezenhimske matične ćelije CD29 i CD73 su bili eksprimirani na svim nediferenciranim ćelijama, dok su antigeni CD146 i CD106 bili eksprimirani na ograničenom broju ćelija. Antigen CD34 (karakterističan za ćelije hematopoetske loze) nije bio eksprimiran. Incidencija CFU-F bila je 4,7 ± 0,5/100 ćelija. PDT se nije menjao tokom osam pasaža i u proseku je iznosio 25 h. Posle tronedeljne stimulacije diferencijacije u kulturama sa adipogenim medijumom došlo je do stvaranja ćelija sa masnim kapljicama, a u kulturama sa osteogenim medijumom došlo je do formiranja vanćelijskog matriksa sa deponovanim kalcijumovim solima. U kulturama sa hondrogenim medijumom došlo je do stvaranja tkiva sličnog hrskavici i vanćelijskog matriksa sa glikozaminoglikanima i kolagenom II. Zaključak. Zubna pulpa mlečnih zuba dece sadrži ćelijsku populaciju koja odgovara mezenhimskim matičnim ćelijama prema svojim karakteristikama, ima visok proliferativni potencijal i potencijal da se diferencira u tri ćelijske linije što je čini pogodnom za dalje in vitro analize i evaluaciju ćelijske terapije