TEMPORAL EXPRESSION OF GROWTH FACTORS AND MATRIX MOLECULES IN HEALING TENDINITIS LESIONS
*Dahlgren, L A. (A-NIH, VOS, AHSA, Travers Research Fund); +*Nixon, A J.
+*Comparative Orthopaedics Laboratory, Cornell University, Ithaca, New York. 607-253-3050, Fax: 607-253-3497, [email protected]
Table 1: Glycosaminoglycan and DNA content of tendon tissue ∀ SD.
Letters indicate times significantly different from each other (p < 0.01).
Time (Weeks)
Glycosaminoglycan (::g/mg)
DNA (::g/mg)
0
27.09 ∀ 16.46ab
9.88 ∀ 0.23ab
1
6.58 ∀ 2.07b
7.34 ∀ 3.05b
b
2
6.71 ∀ 1.42
8.52 ∀ 1.56b
4
19.26 ∀ 1.70ab
14.45 ∀ 0.95a
8
34.72 ∀ 6.11a
12.67 ∀ 0.76ab
Message expression for TGF-∃ increased immediately and peaked at 1
week, then gradually decreased over the course of the study, remaining
slightly elevated at 8 weeks. Message expression for IGF-I increased more
slowly following injury, peaked at 4 weeks and remained elevated at the 8
week time point (Fig 1). There was a significant increase in IGF-I expression
between 1 and 4 weeks. There were no other significant differences in growth
4 .0
∃ / EF1∀
∀
IGF-I or TGF-∃
Introduction: Tendinitis is a common and debilitating injury affecting elite
and recreational athletes. Growth factors are known to have beneficial effects
on tendon healing. Little is known about the temporal expression of growth
factors in healing tendon and how the changes in expression of growth factors
may drive changes in expression of matrix molecules. This information is
essential for the accurate timing of administration of exogenous growth factor
therapy. The purpose of this study was to define the temporal and spatial
expression of insulin-like growth factor I (IGF-I) and transforming growth
factor ∃ (TGF-∃) and the matrix molecules collagen types I and III, and to
correlate the changes in expression of the matrix molecules with those of the
growth factors using a collagenase-induced model of flexor tendinitis in the
horse. We hypothesize that IGF-I and TGF-∃ will exhibit unique patterns of
expression in healing tendon coincident with the early inflammatory (TGF-∃)
and later regenerative (IGF-I) phases of healing. Expression of collagen types
I and III, and morphological changes within the tissue will be highly
correlated to these changes in growth factors.
Materials and Methods: Collagenase-induced lesions were created in the
tensile region of both flexor digitorum superficialis (FDS) tendons of 12 adult
horses. Horses were divided evenly into 4 groups and were euthanatized at 1,
2, 4, and 8 weeks post-injection. All protocols involving live animals were
approved by the IACUC. At the time of euthanasia, FDS tendons were
harvested under RNase free conditions, and a portion of each tendon was
snap-frozen in liquid nitrogen for molecular biology, rinsed in protease
inhibitors and snap-frozen in liquid nitrogen for biochemical analysis, or fixed
in 4% paraformaldehyde at 4ΕC for histology. Message expression for
collagen types I and III, IGF-I, and TGF-∃ was evaluated by in situ
hybridization, northern blot analysis (collagens), and semiquantitative RTPCR techniques using EF1∀ as an internal control (IGF-I and TGF-∃). Tissue
morphology was examined by H&E staining. DNA, GAG, and collagen type
were determined by fluorometry, spectrophotometry, and cyanogen bromide
cleavage respectively. Translation of mRNA into protein was confirmed by
immunohistochemical techniques for IGF-I, TGF-∃, collagen type I and
collagen type III. Data were analyzed by Kruskall-Wallis non-parametric
ANOVA with the appropriate post-hoc comparison for differences between
groups. Significance was set at p < 0.05.
Results: Discrete core lesions of moderate severity were created in all
tendons following collagenase injection. Grossly and microscopically, the
lesions progressed from a mass of amorphous, acellular tissue at 1 and 2
weeks post-injection to more organized scar tissue filled with collagen fibers
and fibroblasts arranged along lines of tension by 8 weeks. H&E stained
sections showed a gradual increase in cellularity from 1 to 8 weeks following
injection, and a change in cell morphology from round, fat cells, to more
typical spindle-shaped fibroblasts.
Glycosaminoglycan content of the tendon lesions was decreased
compared to normal tendon at 1, 2, and 4 weeks, and by 8 weeks postinjection had increased above baseline (Table 1). DNA content of the lesions
paralleled the histologic findings: DNA was decreased compared to normal
tendon at 1 and 2 weeks, and peaked at 4 weeks post-injection (Table 1).
IGF-I
TGF-∃
∃
3 .5
3 .0
2 .5
2 .0
1 .5
1 .0
0 .5
0 .0
0
1
2
3
4
5
6
7
8
Time (weeks)
Figure 1: IGF-I and TGF-∃ gene expression measured by semiquantitative
RT-PCR using EF1∀ as an internal control ∀ SD.
factor expression. TGF-∃ mRNA was documented at all time points by in situ
hybridization. Expression predominated in large round cells within the
endotenon; however, fibroblastic cells within the parenchyma were also
producing low levels of message for TGF-∃. Message expression for collagen
type I and collagen type III increased immediately following injury and
remained elevated throughout the course of the study (Table 2). There were
no significant differences in message expression over time. In situ
hybridization and immunohistochemistry for collagens type I and III
confirmed immediate and sustained increases in transcription and translation
of both proteins. Collagen type III was initially concentrated in the endotenon
at weeks 1 and 2 and both message and protein expression became more
diffusely expressed throughout the parenchyma at 4 and 8 weeks.
Table 2: Results of northern blot analysis of collagen type I and III gene
expression normalized by EF1∀. Ratio of pixel intensity ∀ SD.
Time(Weeks)
Collagen type I/EF1∀
∀
Collagen type III/EF1∀
∀
0
0.57 ∀ 0.04
0.48 ∀ 0.04
1
1.45 ∀ 0.71
1.00 ∀ 0.21
2
1.14 ∀ 0.18
0.90 ∀ 0.08
4
1.40 ∀ 0.19
0.97 ∀ 0.11
8
1.43 ∀ 0.33
1.01 ∀ 0.18
Discussion: The healing process began rapidly following injury, as evidenced
by immediate increases in growth factor and collagen expression. As
hypothesized, message expression for TGF-∃ increased acutely, followed by
IGF-I. Activated macrophages, fibroblasts, and other cells contained within
the inflammatory debris and hematoma in the early inflammatory phase of
wound healing as well as immature fibroblasts responding from the endotenon
are the likely source of the initial levels of growth factor mRNA. Initial
decreases in DNA and glycosaminoglycan content are consistent with the
degree of damage to the normal tendon architecture. In response to the
elevations in growth factor levels, and other chemotactic agents, healing
progressed by cell migration into the lesion, confirmed histologically, and by
increased DNA content of the tissues. Following migration into the damaged
area, production of glycosaminoglycan increased and message for the collagen
genes increased. As expected, growth factor expression peaked early in the
healing process, while collagen message expression remained high throughout
the course of the study. The increase in collagen mRNA expression and
protein translation was more immediate and robust than expected. The
immediate upregulation of collagen type I mRNA and protein production is
encouraging with respect to the feasibility of inducing a more robust early
cellular response following exogenous growth factor therapy.
Early
administration of exogenous growth factors, prior to the endogenous peak,
may be indicated to achieve an enhanced cellular response to tendon injury.
48th Annual Meeting of the Orthopaedic Research Society
Paper No: 0048