Hücre Tabaka Mühendisliği ile Göbek Kordonu Perisitlerinden Üç Boyutlu Damar Grefti Eldesi

In the case of limited accessibility of the autologous vessels and the disadvantages of synthetic scaffolds, small-diameter vascular grafts (SDVG) made from cellularized natural scaffolds are needed for the treatment of coronary artery diseases. The aim of this thesis is to generate three dimensional SDVG model. For this purpose, CD146+ pericytes were isolated from human umbilical cord vein (HUCV) and differentiated into smooth muscle cells (SMCs) and fibroblasts. They were combined with collagen type I/elastin/dermatan sulfate and collagen type I/fibrin to form tunica media and adventitia respectively. To generate tunica intima-like layer, HUCV endothelial cells (ECs) were seeded on the construct by cell sheet engineering method after fibronectin and heparin coating. Anti-thrombogenic property of intima layer was shown by adhesion test with thrombocyte differentiated from human cord blood CD34+ cells. Characterization of the vascular construct was performed by immunolabeling, histochemical staining and scanning and transmission electron microscopy (SEM and TEM). SEM and TEM analysis of vascular construct revealed the presence of three histologic tunicae. The VEGFR-1 expressing ECs in tunica intima; α-SMA expressing SMCs in tunica media and the tenascin expressing fibroblasts in tunica adventitia were detected by immunolabeling. This thesis will provide a preliminary blood vessel model by combining natural scaffolds and vascular cells differentiated from pericytes for generation of fully natural SDVG.Otolog damarlara erişilebilirliğin sınırlı olması ve sentetik iskelelerin sahip oldukları dezavantajlardan dolayı, koroner arter hastalıklarının tedavisi için, hücrelerin ve doğal malzemelerin birleştirilmesiyle oluşturulmuş küçük çaplı damar greftlerine (KÇDG) ihtiyaç duyulmaktadır. Bu tez çalışmasının amacı, üç boyutlu KÇDG modeli oluşturmaktır. Bu amaç doğrultusunda, CD146+ perisitler, insan göbek kordonu veninden izole edilerek düz kas hücrelerine ve fibroblastlara farklılaştırılmıştır. Bu hücreler, kollajen tip I/elastin/dermatan sülfat ve kollajen tip I/fibrin ile birleştirilerek sırasıyla tunika medya ve tunika adventisya-benzeri tabakalar oluşturulmuştur. Fibronektin ve heparin kaplaması sonrası, hücre tabaka mühendisliği yöntemiyle insan göbek kordon veni endotel hücreleri bu yapı üzerine ekilmiş ve tunika-intima benzeri tabaka oluşturulmuştur. İnsan göbek kordon kanı CD34+ hücreleri, trombosit yönünde farklılaştırılarak adezyon testi ile intima tabakasının anti-trombojenik özelliği gösterilmiştir. İmmün işaretleme, histokimyasal boyama, taramalı ve geçirimli elektron mikroskobu (SEM ve TEM) kullanılarak damar yapısı karakterize edilmiştir. SEM ve TEM analiziyle damar yapısında bulunan üç tabakanın varlığı, immün işaretleme ile bu tabakalarda yer alan düz kas hücreleri (α-SMA), fibroblastlar (Tenascin-C) ve endotel hücreleri (VEGFR1) gösterilmiştir. Bu tez çalışması, oluşturulacak olan doğal KÇVG’ler için, perisitlerden farklılaştırılan damar hücreleri ve doğal malzemeleri birleştirerek bir damar modeli meydana getirmesi yönünden önem taşımaktadır

Expansion Of Human Umbilical Cord Blood Hematopoietic Progenitors With Cord Vein Pericytes

The vascular niche is a site rich in blood vessels, whereas endothelial cells, pericytes, and smooth muscle cells create a microenvironment that recruits mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), which is important for stem cell mobilization, proliferation, and differentiation. In this study, CD146 + pericytes were purified and enriched from the human umbilical cord vein. In order to define their direct role in hematopoiesis, we tested the CD146 + pericytes as compared with osteoblasts derived from umbilical cord blood (UCB) MSCs to sustain human UCB hematopoietic progenitor cells in noncontact coculture settings or in culture media previously conditioned (CM) by these cells. The growth of UCB cells was the greatest in pericyte cocultures (2.8-fold vs. the control). The increased growth in pericyte and pericyte CM cultures was largely the result of increased frequency of CD34 + and CD38 + hematopoietic progenitors, CD34 + CD41 + megakaryocyte progenitors, and CD235 + erythroblasts. A total of 29 factors were found to be secreted by pericytes higher than by osteoblasts. The most secreted growth factor by pericytes was vascular endothelial growth factor (1.3-fold). We demonstrate for the first time that human CD146 + perivascular cell coculture and CM are able to directly support the ex vivo maintenance of human hematopoietic progenitor cells.WoSScopu

Effect of Ocimum basilicum on mesenchymal stem cell proliferation and differentiation: Does the effect change according to niches?

It is a big issue that reduced bone density and large fractıres in dentistry and orthopedics. Side effects caused by synthetic drugs lead to medical and ethical problems. Thus, plants and medicinal plant research take attention. Aim of this preliminary in vitro study is to investigate the effect of Ocimum basilicum extract on dental pulp (DP) and bone marrow (BM) derived mesenchymal stem cell (MSC) proliferation, osteogenic differentiation and immunological response to TNF-α. Human dental pulp tissue was obtained from patients (15-20 years of age) who were undergoing extraction of third molars for orthodontic reasons at the Department of Oral and Maxillofacial Surgery, University of Gazi University*. xCELLigence system was used to determine prolfieration of DP- and BM-MSCs. Adipogenic and osteogenic differentiation was shown and calcium concentration, osteocalcin and osteonectin levels were examined. Inflammatory environment was mimiced through TNF-α stimulation and IL-6 and IL-10 levels were defined by ELISA. Doubling time mwith O. basilicum was found in DP- MSCs (38 h) and BM-MSCs (76 h). IC50 value was shown as 148 µg/mL in DP-MSCs and 178 µg/mL in BM-MSCs. Calcium concentration of BM-MSCs was found decreased in O. basilicum treated groups. Level of ostoenectin was reduced in O. basilicum treated cells suggesting that the Extract accelerated the osteogenic differentiation. We suggest that O. basilicum could be a smart ostoeinductive agent where BM-MSCs should be investigated further. Rich flora of Turkey is an opportunity for us and encourangement can easily give inside to medicinal plant investigations. *B.30.2.GÜN.0.20-122 Ethics Committee Repor

Thymbra Spicata Var. Intricata Induces Mesenchymal Stem Cell Proliferation and Osteogenic Differentiation

A natural agent that maintains mesenchymal stem cell (MSCs) viability, promotes osteogenic differentiation while modulating the immunological response could achieve success in regeneration during healing and may also prevent bone resorption and improve regeneration. We aimed to demonstrate that a Thymbra spicata var. intricata extract could induce proliferation, differentiation, and modulate the immune responses of mesenchymal stem cells (MSCs). Using xCELLigence, a real-time monitoring system, we obtained a growth curve of MSCs. A dose of 10 mu g/mL was the most efficient concentration for vitality. Osteogenic differentiation and antiinflammatory activities were determined by using an ELISA Kit to detect early and late markers of differentiation. The Osteonectin (ON, early osteogenic marker) level decreased while the Osteocalcin (OCN, late osteogenic marker) level increased in the T. spicata var. intricata treated group, suggesting that T. spicata var. intricata may accelerate osteogenic differentiation. Reduced level of the IL-6 cytokine in repsonse to TNF-alpha was evident. T. spicata var. intricata could be a promising osteogenic inducer in dentistry and could be used safely in biocomposites or scaffold fabrications.WoSScopu