Presentation1

Human Turbinate Mesenchymal Stromal Cells as a Potential Option forCartilage Tissue Engineering

Se Hwan Hwang1†, Sung Won Kim1†, Su Young Kim2, Sun Hwa Park1, Hun-Jun Lee2,Mi Young Choi1, Hea Jin Oh1, Se Hyeun Kim1, Jong Won Rhie3, and Dong Il Sun1*

1Department of Otolaryngology-Head and Neck Surgery, The Catholic University of Korea, College of Medicine, Seoul, Korea2Department of Pathology, The Catholic University of Korea, College of Medicine, Seoul, Korea

3Department of Plastic Surgery, The Catholic University of Korea, College of Medicine, Seoul, Korea

Introduction

Evaluated the effects of co-culture with human septal chondrocytes (hSCs) on the chondroinduction of hTMSCs.

The efficiency of co-culture with hSCs were analyzed histologically by toluidine blue O staining.

Mesenchymal stem cell surface markers was assessed by fluorescence-activated cell sorting.

Cartilage-specific gene expression and matrix production were evaluated quantitatively by real-time PCR and Western blotting

A. Nasal septum of a cadaverB. Inferior turbinate tissues obtained during turbinate surgery.C. (hTMSCs) 2 weeks after primary explant culture

Materials and Methods

Cell IsolationInferior turbinate tissue was obtained from a patient who underwent septoplasty and a partial turbinectomy

washed at room temperature 3 times with antibiotic antimycotic solution

1mm3 tissue were cultured in Dulbecco's modified Eagle's medium 37°C in at 5% CO2

After 14 days Third passage

The chondrocytes and hTMSCs were cultured

Immunolabeling and Flow Cytometry for hTMSC Surface Markers

cells were then incubated with monoclonal antibodies 40 min

cells were centrifuged (1200 rpm, 5 min)

FITC- or PE-labeled secondary antibodies were added 30 min in the dark at 40°C.

Cell fluorescence was evaluated by flow cytometry

Cell Seeding and 3-Dimensional Culture

3D cultures in 24-well plates (Nunc) using open-cell polylactic acid

hTMSCs and chondrocytes were adjusted to 2×106 cells/mL (1:0, 1:1, and 0:1)

chondrogenic supplements TGF-β1 and IGF-1 were added

Medium was replaced every 2-3 days

Samples were collected after 7, 14, and 28 days

Histological Studies Cells were fixed in 4% paraformaldehyde

Stained using toluidine blue O

washed with distilled water, and air-dried

RNA Extraction and Real-Time RT-PCR of Chondroinduced hTMSCs

Total RNA was extracted from cells cultured on sponges (3per group) using TRIzol reagent

Immunoblot Analysis of Chondroinduced hTMSCs

Western blotting

Statistical AnalysesStatistical analyses were conducted using ‘R’ statistical softwarestatistical significance calculated using ANOVA tablep-value < 0.05 was considered to indicate significance.

Results and discussion

• Flow cytometric analysis reveled expression of several MSC markers, HLA antigens, andhematopoietic stem cell markers.

• A metachromatic matrix indicating chondroinduction of hTMSC produced a red shift when stained with toluidine blue O within the tissue mass (Histological assay)

• COL2A1 and aggrecan expression was also lower in the co-culture group than in the hSCs group on days 7, 14, and 28. (hTMSCs proliferate rapidly )

• Western blot analysis also detect the expression of low COL2A1.

• Co-culture of bone marrow-derived MSCs and articular chondrocytes enhanced

matrix deposition.