Abstract
1518
Introduction: In vitro quantification of the chondrogenic potential of human mesenchymal stem cells (hMSCs) is still a challenging problem in musculo-skeletal tissue engineering while chondrogenic 2- and 3-dimensional differentiation of MSCs is a common procedure. To track and quantify the successful differentiation, the Alcian blue staining and the GAG (Gylcosaminoglycan)-analysis are the most common methods. There is a huge demand for new, sensitive and non-destructive methods to proof and quantify the chondrogenic pathway as new differentiation methods and procedures are continuously developed. This is especially important for 3-dimensional chondrogenic cell cultures as the GAG-content of these scaffolds can only be accessed by lysing the scaffold in an extensive procedure. Recently, 99mTc-MDP labeling was successfully employed to track and quantify the osteogenic potential of hMSCs in monolayer cultures as well as in 3-dimensional scaffold cultures as the tracer binds highly affine to newly produced hydroxylapatite. This method, known as bone scintigraphy, is used in clinical routine since the late 1970s. In this abstract we present the first-time quantification of the chondrogenic potential of hMSCs using 99mTechnetium-Hyaluronic acid (99mTc-HA). Materials and Methods: Human mesenchymal stem cells (P1) (n=6) were cultured in two groups towards the 1. osteogenic (OSM) and 2. chondrogenic (CHO) lineage in 3,5 cm petri dishes (3.000cells/cm2, 37°C, 5% CO2) for 21 days. DMEM LG with 10% fetal calf serum and 1% Penicillin/Streptomycin was used as basal cell culture media. Osteogenic differentiation was archived by adding the standard supplements dexamethason, ascorbic-acid-2-phosphate and ß-glycerolphosphat. Chondrogenic differentiation was archived by adding the standard supplements FGF-2, TGFß-1, IGF and OP-1. After the cell cultures were terminated, the dishes were washed three times using PBS. In parallel, the Technetium was attached to the hyaluronic acid by solving 1 mg SnCl2 in 0,3 M HCl (pH = 2). Subsequently, 2,5 mg hyaluronic acid was added and then heated until it solved completely followed by adding the 99mTc. The solution was continuously heated for one hour and eluted with PBS. 1ml, containing 5 MBq 99mTc-Hyaluronic acid was added to each dish and incubated at room temperature for 2h. After incubation, followed by intensive washing, the dishes were placed under a gamma camera using a one-detector setting to determine the amount of bound tracer in each dish. Counts were acquired for 180 seconds and the data was post-processed by placing regions of interest around each dish to determine the exact amount of bound activity. Blank dishes were labeled in parallel. SPSS 20 was used for data analyzation. Student’s t-Test was performed to detect significant differences regarding the uptake of the tracer. Statistical significance was set to p < 0,05
Results: While there was a donor specific variety, reflecting the individual differentiation capacity of the donors, the mean uptake within the osteogenic group (OSM) was 20.287 counts/180 seconds while in the chondrogenic group (CHO) the uptake was much higher with a mean value of 34.441 counts/180 sec.. The minimum uptake for the OSM group was 12.477 counts/180 sec. and the maximum 31.469. The SEM was 2.245. The minimum uptake for the CHO group was 32.802 counts/180 seconds and the maximum 37.275. The SEM was 619. Student’s t-Test revealed a high significant major uptake within the chondrogenic group versus the osteogenic group (p<0,01). Discussion: 99mTc-HA Labeling is a promising tool for the quantification of the chondrogenic potential of hMSCs as the HA binds to in vitro chondrogenic cell culture layers. The uptake of the osteogenic cultures might have occurred as the cells differentiate in the beginning in a conjunctly osteo-chondral pathway before they further differentiate into the osteogenic and chondrogenic lineage. Further studies are necessary to correlate the tracer uptake with the GAG-amount of the cell cultures to verify the method in a 2- and 3-dimensional cell culture.