Materials and Methods•C3H 10T1/2 murine embryonic fibroblasts (cell line purchased from American Type Culture Collection)•Chondrogenic Medium: Dulbecco’s Modified Eagle Medium, 10% fetal bovine serum,

1% antibiotics/Antimycotics, 25 ng/ml human recombinant bone morphogenetic protein-2, 100μg/ml L-ascorbic acid (added daily)

•Microcentrifuge with 20 place rotor for 1.5-2ml microcentrifuge tubes (Jouan® A14) •Conical-ended, polypropylene microcentrifuge tubes (1.5 mL)

A. Borazjani1; S.H. Elder1, Ph.D.1 Department of Agricultural and Biological Engineering, Mississippi State University, Starkville, MS

ResultsReal-time RT-PCR results demonstrated that aggrecan, but not collagen type II, mRNA was upregulated by centrifugal loading (Fig. 5, Table 1). Histology confirmed that experimental pellets were substantially larger than controls and showed that they contained much more immunodetectable collagen type II. (Fig. 6a&b). Experimental pellets also exhibited more metachromatic toluidine blue staining, indicating a higher concentration of proteoglycans (Fig.6c&d).

DiscussionThe goal of this study was to determine whether murine embryonic fibroblasts can be induced to undergo chondrogenic differentiation in pellet culture under centrifugal loading. The results of this study support our hypothesis that centrifugal loading enhances chondroinduction of C3H10T1/2 cells above the baseline achieved with BMP-2 alone. Enhanced chondrogenesis was reflected by increased aggrecan mRNA expression and higher collagen type II and proteoglycan contents in centrifugally loaded pellets. The lack of loading-induced Col2a1 upregulation is not surprising, as other investigators have shown that certain magnitudes of mechanical stimulation (e.g. hydrostatic pressure) are sufficient to upregulate aggrecan mRNA but have negligible effect on collagen type II [Miyanishi et al., 2006]. The development of an effective means of chondroinduction is the gateway to creating a durable tissue engineered articular cartilage, and this study suggests that a bioreactor capable of applying intermittent centrifugal loading may be a useful tool in this endeavor.

ReferencesHunziker, E. B. OsteoArthritis and Cartilage 10 (2001): 432-63.Maeda et al. OsteoArthritis and Cartilage 13 (2005): 154-61.Miyanishi et al. Tissue Engineering 12.8 (2006): 2253-62.Tuli et al. Arthritis Res & Ther. Aug (2003): 235-38.

Effect of Centrifugal Loading on Chondroinduction of Fibroblasts with Multilineage Potential

Fig. 1 Mesenchymal Stem Cell Differentiation (Caplan and Bruder,2001)MSCs are progenitors of all connective tissue cells in the body. The have the potential to differentiate into a wide variety of cells.

C3H cells were thawed and grown in monolayer using standard tissue culture procedures until sub-confluent.

The cells were then trypsinized, spun, and re-suspended at a density of ≈170,000 cells / mL.

The suspension was aliquoted (1 ml) to eight 1.5ml microcentrifuge tubes, spun at 150g for 5 minutes, and allowed to incubate for 48 h for pellet consolidation.

The pellets were separated into control and experimental groups. The experimental group was spun 3 times per day at 14,000 RPM for five days. The control group was also spun 3 times per day but using only 100RPM.

After a 5 day loading period, RNA was isolated from 3 control and 3 experimental pellets and analyzed using RT-PCR for collagen type II and aggrecan mRNA expression (Refer to “RNA isolation and RT-

PCR”).

The remaining 2 pellets were embedded in plastic and stained with toluidine blue for detection of proteoglycans and immunostained (II-II6B3 monoclonal antibody from Developmental Studies Hybridoma Bank) for detection of collagen type II using the Zymed Histostain® SP Kit.

Calculation of Centrifugal Pressure

Diameter of tubes: 4.5 mmVolume of tubes: 1.5 mLRPM used: 14,000Acceleration =17,746 x g

If acceleration is a, thena=17,746 • 9.81=1.741 x 105 [m/s2]

If the force acting of the bottom of the tube is F, thenF = ma = 1.5 x 10-3[kg] • 1.741 x 105 = 261.1 [N]

Consequently, P ,centrifugal pressure, can be considered asP = 261.1/((π • (4.5 x 10-3)2) [Pa] = 4.101 [MPa]

RNA isolation and RT- PCR

RNA was isolated from the pellets using RNeasy® Spin columns (Qiagen). Real time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was carried out using the iScriptTM One-Step RT-PCR Kit with SYBR® Green (Bio-Rad) in the iCycler iQ™ Real-Time PCR Detection System (Bio-Rad). Loading induced changes in gene expression were calculated by the ΔΔCT method with GAPDH as the housekeeping gene. Gene-specific primers were as follows:

Col2a1 (collagen type II)Upstream Primer: 5’ GCC AAG ACC TGA AAC TCT GC 3’Downstream Primer: 5’ GCC ATA GCT CAA GTG GAA GC 3’

Aggrecan Upstream Primer: 5’ CTC AGT GGC TTT CCT TCT GG 3’Downstream Primer: 5’ CTG CTC CCA GTC TCA ACT CC 3’

GAPDHUpstream Primer: 5’ CTG AGG ACC AGG TTG TCT CC 3”Downstream Primer: 5’ GCC TCT CTT GCT CAG TGT CC 3’

Fig.5 Real time RT-PCR curvesRelative fluorescence intensity vs. cycle number. Arrow points to threshold used for determining CT values.

IntroductionToday, over 20 million Americans age 25 years or older suffer from cartilage

degenerative diseases. By 2030, this number is expected to increase to 72 million people [National Institute of Arthritis and Musculoskeletal and Skin Diseases]. Consequently, treatment for cartilage degenerative diseases or defects is a key concern in the field of orthopaedic research. Given the limited intrinsic healing capacity of cartilaginous tissue due to the lack of blood supply, damage to cartilage frequently results in an inadequate repair attempt by neighboring chondrocytes or an inferior fibrocartilage formation [Tuli, et al.] . Currently, the most popular methods for replacing areas of damaged cartilage are autografts and allografts which use tissue from either the same individual or a donor to replace cartilage defects. However, clinical experience with human patients has not yet yielded complete restoration and long-term stability of repaired tissue [Hunziker].

The advancing field of tissue engineering hold great promises to the area of cartilage degeneration by allowing tissue engineered constructs to sufficiently replace damaged areas of cartilage. Chondroinduction of mesenchymal stem cells is a valuable area in the development of tissue engineered cartilage. In this study, the effects of centrifugal pressure on murine fibroblasts were studied. It has already been demonstrated that centrifugal pressure stimulates chondrogenesis in fully mature chondrocytes [Maeda, et al.]. Our study was designed to test the hypothesis that centrifugal loading enhances BMP-2-induced chondroinduction of C3H10T1/2 murine embryonic fibroblasts in three-dimensional culture.

Image of cartilage defect (© 2001 by LeadingMD, Inc.)

AcknowledgementsMSU Life Sciences and Biotechnology InstituteThe II-II6B3 monoclonal antibody developed by T.F. Linsenmayer was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.

Table 1. Gene Expression ResultsCalculated Threshold for target genes and fold change in expression.

Fig. 2A. Embryonic Fibroblasts [ATCC]

Fig. 2B. Mature Chondrocytes [UC-Clermont College]

Fig. 6 Histological and Immunohistological Analyses. Presence of type II collagen indicated by brown deposits (a&b). Proteoglycan concentration proportional to intensity of metachromatic (purple/violet) staining with toluidine blue (c&d).

[a] Immunostaining for type II collagen control (40x)

[b] Immunostaining for type II collagenexperimental (40x)

[c] Toluidine blue staining for proteoglycans control (400x)

[d] Toluidine blue staining for proteoglycansexperimental (400x)

a b

dc