Thesis (DTech. degree in Biomedical Sciences)--Tshwane University of Technology, 2009. / Damaged articular cartilage requires tissue engineering based therapeutic methods
because of its minimal self-repair capacity. Articular cartilage is a heterogeneous
avascular, aneural tissue that provides a smooth bearing surface for movement. It
consisting of superficial, middle, and deep layers with varying matrix composition. The
superficial zone is the site of superficial zone protein synthesis that functions as a
boundary lubricant. The middle and deep layers abound in collagen II and proteoglycan
aggrecan.
Since articular cartilage consists of zones that are specific in their functions, the
replication of these zones may be important in obtaining a functional tissue-engineered
construct. Understanding the differences in gene expression between these layers is
necessary for tissue engineering.
The aim of the study is to use bone morphogenetic proteins (BMPs) and growth factor
morphogens that may help in the regeneration and repair of articular cartilage through
stimulation of chondrocyte proliferation and differentiation. The role of various
transforming growth factor beta (TGF-)/Bone morphogenetic proteins (BMPs)
superfamily on expression of the following proteins: cartilage oligomeric matrix protein
(COMP), superficial zone protein (SZP), cartilage intermediate layer protein (CILP),
SRY-related (sex determining region Y)-sox 9 (SOX 9), aggrecan (AGG) and collagen
type II from three distinct zones of cartilage (superficial, middle and deep) was
investigated.
It is hypothesized that the chondrocytes from the superficial, middle, and deep zone of
articular cartilage would have different rates of proliferation and biosynthetic activity,
and that these proteins would respond differently when treated with the (TGF-)/ bone
morphogenetic proteins (BMPs) superfamily.
Stifle joints (n=28) from three month-old calves were obtained from a local abattoir.
Articular chondrocytes were isolated from three distinct zones of cartilage and cultured
as monolayers in a serum-free chemically defined medium. The results are presented
as the mean and standard deviations for each experiment. A one way analysis of
variance (ANOVA) with Tukey’s HSD (Honestly Significant Differences) to account for
multiple comparisons was used to determine the effects TGF- _1, BMP-7, GDF-5, and
Activins A, B and AB isoforms on COMP, SZP, CILP, aggrecan, sox 9 and collagen type
II accumulation in different zones of articular cartilage. A significance level of p < 0.05
was used to determine the difference between the control and treated cells and p < 0.01
the dose-dependence of morphogens within the zones.
Accumulation of COMP, SZP, CILP, aggrecan, sox 9 and collagen type II in culture
medium in response to various members of the BMP/TGF-_ superfamily was
determined by enzyme-linked immunosorbent assay (ELISA) and messenger
ribonucleic acid (mRNA) by quantitative real-time PCR. TGF-_1 significantly stimulated
the expression of COMP and SZP at the mRNA and protein level in the superficial zone
in a time- and dosage- dependent manner, CILP at the mRNA in the middle zone,
aggrecan, sox 9 and collagen type II at the mRNA in the middle and deep zone. An
unexpected discovery was that the superficial chondrocytes were more responsive to
morphogens than the deeper layers. BMP-7 and growth differentiation factors (GDF-5)
also up-regulate COMP, SZP, CILP, aggrecan and collagen type II expression, although
the effects were not as potent as with TGF-_1. Activins A, B and AB demonstrated no
effects on all the genes accumulation in all zones. In conclusion, TGF-_, BMP-7 and
GDF-5 are important regulators of different genes in different zones of articular
cartilage.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:tut/oai:encore.tut.ac.za:d1001160 |
| Date | January 2009 |
| Creators | Motaung, Shirley Caroline Keolebogile |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Text |
| Format | |
| Rights | © 2009 Tshwane University of Technology |
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