Transcript Centrifugal_Loading_Poster

Effect of Centrifugal Loading on Chondroinduction of
Fibroblasts with Multilineage Potential
A. Borazjani1; S.H. Elder1, Ph.D.
1 Department
of Agricultural and Biological Engineering, Mississippi State University, Starkville, MS
Introduction
Today, 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
Image of cartilage defect frequently results in an inadequate repair attempt by neighboring chondrocytes or
(© 2001 by LeadingMD, Inc.)
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-2induced chondroinduction of C3H10T1/2 murine embryonic fibroblasts in three-dimensional culture.
Results
Real-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).
Table 1. Gene Expression Results
Calculated Threshold for target genes and fold
change in expression.
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.
Fig.5 Real time RT-PCR curves
Relative fluorescence intensity vs. cycle number. Arrow
points to threshold used for determining CT values.
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 Lascorbic acid (added daily)
•Microcentrifuge with 20 place rotor for 1.5-2ml microcentrifuge tubes (Jouan® A14)
•Conical-ended, polypropylene microcentrifuge tubes (1.5 mL)
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).
Fig. 2A. Embryonic Fibroblasts Fig. 2B. Mature Chondrocytes
[ATCC]
[UC-Clermont College]
a
b
[b] Immunostaining for type II collagen
experimental (40x)
Calculation of Centrifugal Pressure
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.
Diameter of tubes: 4.5 mm
Volume of tubes: 1.5 mL
RPM used: 14,000
Acceleration =17,746 x g
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 RTPCR”).
The remaining 2 pellets were
embedded in plastic and stained with
toluidine blue for detection of
proteoglycans and immunostained (IIII6B3 monoclonal antibody from
Developmental Studies Hybridoma
Bank) for detection of collagen type II
using the Zymed Histostain® SP Kit.
[a] Immunostaining for type II collagen
control (40x)
If acceleration is a, then
a=17,746 • 9.81=1.741 x 105 [m/s2]
If the force acting of the bottom of the tube is F, then
F = ma = 1.5 x 10-3[kg] • 1.741 x 105 = 261.1 [N]
Consequently, P ,centrifugal pressure, can be considered as
P = 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’
GAPDH
Upstream Primer: 5’ CTG AGG ACC AGG TTG TCT CC 3”
Downstream Primer: 5’ GCC TCT CTT GCT CAG TGT CC 3’
[c] Toluidine blue staining for proteoglycans
control (400x)
[d] Toluidine blue staining for proteoglycans
experimental (400x)
c
d
Discussion
The 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.
References
Hunziker, 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.
Acknowledgements
MSU Life Sciences and Biotechnology Institute
The 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.