ARTICLE
Wound healing involves a complex series of events that ultimately lead
to the restoration of injured tissue. In the process of wound healing,
cell migration, neoangiogenesis, granulation tissue formation and stromal
remodeling are essential. One of the major features of wound healing is
the production by fibroblasts of extracellular matrices (ECM), such as
collagens and proteoglycans. Matrix metalloproteinases (MMPs), a group
of enzymes that are collectively capable of cleaving all the components
of ECM, are involved in these events [1-4]. Tissue inhibitors of metalloproteinases
(TIMPs) act as local inhibitors of MMPs and thus control the MMP-induced
breakdown of ECM, an excess of which may lead to chronic inflammation
and nonhealing ulcers by indiscriminate degradation of the matrix, cytokines,
and other components of the wound environment.
Recently, an in vitro dermis-like model has been developed using
supplementation of ascorbic acid to fibroblast cultures [5]. This model
is useful in the study of cell-matrix interactions because of its close
similarity to the dermis [6, 7]. In the present study, we employed this
model as an assay system to investigate the direct effect of experimentally
punched wounds on the mRNA expression and the production of type I collagen,
MMP and TIMP.
Materials and methods
Human fibroblast culture
After informed consent was given, normal human dermal fibroblasts obtained
from five young healthy volunteers were expanded by the explant culture
method. Skin specimens were cut into small pieces, and the outgrown fibroblasts
were trypsinized and grown in Dulbecco's modified Eagle medium (DMED;
Nihon Seiyaku, Tokyo, Japan) containing 10% fetal calf serum (FCS: Cytosystems,
Castle Hill, Australia) at 37° C in 5% CO2 humidified
air. The culture medium was changed every three days. Cells were expanded
through two or three passages before the experiments.
Three dimensional culture and making experimental
wounds
Fibroblasts were seeded onto a 10 cm tissue culture dish at a density
of 5 x 105 cells per dish, and cultured in DMEM supplemented
with 10% FCS in the presence of 1.0 mM magnesium salt of L-ascorbic acid
phosphate (Asc-2p; Wako Pure Chemical Industries, Ltd., Osaka) at 37°
C in 5% CO2 humidified air. The addition of Asc-2p allowed
the fibroblasts to organize a self-produced three dimensional structure
in vitro [5, 6]. The culture medium was changed every three days.
After 21 days incubation, the medium was then replaced with FCS-free medium.
After serum deprivation for 24 hrs, 20 round wounds per dish were made
by punching the three dimensional cell layer with a Dispopunch (6 mm;
Stiefel, Germany) (Fig. 1).
After washing with FCS-free medium, the cells were further incubated with
FCS-free medium at 37° C in 5% CO2 humidified air and
harvested at 3, 6, 24, 36, 48 and 72 hrs. After collecting the medium,
the cell layer was rinsed three times with cold PBS for RNA extraction.
The experiments were carried out three times in triplicate.
RNA extraction and Northern blot analysis
Total RNA from the cell layer was isolated by a single-step method using
an acid guanidinium thiocyanate-phenol extraction reagent (Isogen, Nippongene,
Toyama, Japan). Aliquots of 10 mug of total RNA denatured in formaldehyde
were electrophoresed in 1.2% agarose-1.1 mol/l formaldehyde gels. The
quality of RNA samples was monitored by staining with ethidium bromide
to visualize 18S and 28S ribosomal RNA subunits under UV light. The RNA
was then transferred onto a nylon membrane and cross-linked by exposure
to 120 mJ/cm2 of 312 nm UV radiation in a spectra UV cross-linker
(Spectronics Corporation, Westburg, NY, USA). Filters were hybridized
to specific probes labeled with [alpha-32P]dCTP by the random
priming method (Gibco BRL, Gaithersburg, MD, USA). Hybridization was performed
at 42š C in a specific activity of at least 2 x 107 cpm/mug.
After hybridization, the filters were washed twice in 2 x SSPE/0.1% sodium
dodecyl sulphate (SDS) for 10 min and twice at 65° C in 1 x SSPE/0.1%
SDS for 20 min, followed by two washes at 65° C in 0.1 x SSPE/0.1%
SDS for 20 min at room temperature. The filters were exposed to Kodak
X-Omat films. The data were scanned with Bioimage Gel print 2000 i/VGA
(Genomic Solution Inc, USA) and the relative intensities of the bands
were quantified using Basic Quantifier (Genomic Solutions Inc, USA), a
computer software package. The intensity of each mRNA band was normalized
with the G3PDH mRNA band. The ratio of each mRNA to G3PDH mRNA at 3 hrs
without punched wounds was set at 100.
The following human sequence-specific cDNAs were used for hybridization:
a 1.4 kb-long cDNA, Hf 677-6, for proalpha1(I) collagen mRNA, a 0.7 kb-long
cDNA, K4 for MMP-1 (generously provided by Dr. A. Hatamochi, Chiba University,
Japan), and 0.6 kb-long cDNA, pBluescript, for TIMP-1 mRNA (generously
provided by Dr. H. Sato, Cancer Research Institute, Kanazawa University,
Japan), and a 1.1 kb-long cDNA for glyceraldehyde 3-phosphate dehydrogenase
(G3PDH) mRNA purchased from Clontech (Palo Alto, CA, USA).
Assay of carboxyterminal propeptide of type I
procollagen
Measurement of procollagen type I C-peptide in the supernatant is regarded
as a reasonable method to quantify type I collagen synthesis [8, 9]. Therefore,
type I collagen production was assessed by measuring the procollagen type
I C-peptide concentration using an enzyme-linked immunosorbent assay (ELISA),
a procollagen type I C-peptide kit (Takara Shuzo, Kyoto, Japan) [10, 11].
In each experiment, the procollagen type I C-peptide concentration was
calculated from the values of triplicate wells.
Assay of proMMP-1
The production of MMP-1 by fibroblasts was assessed by measuring the
proMMP-1 concentration using a one-step sandwich enzyme immunoassay, a
MMP-1 kit (Fuji Chemical Ind, Toyama, Japan). In each experiment, the
MMP-1 concentration was calculated from triplicate wells.
Assay of TIMP-1
Measurement of TIMP-1 in the supernatant was assessed by measuring the
TIMP-1 concentration using a one-step sandwich enzyme immunoassay, a TIMP-1
kit (Fuji Chemical Ind, Toyama, Japan). In each experiment, the TIMP-1
concentration was calculated from triplicate wells.
Statistical analysis
Data of concentration and expression of procollagen type I C-peptide,
proMMP-1 and TIMP-1 were expressed as mean ± SEM. Statistical analysis
of these experiments was carried out using Statview software (version
4.0; Abacus Concepts, Berkeley, CA, USA). The group data were analyzed
by variance testing to determine the overall impact of sample treatments
within the experiment. Additional post hoc testing using the Fisher
Protected Least Significant Difference (PLSD) test was carried out to
determine the statistical significance of individual sample treatments
on the parameters in question. The result of the analysis of variance
was considered as significant only if both the analyses of variance and
the Fisher PLSD test yielded a probability (P) value of 0.05 or lower.
Results
mRNA levels in three-dimensional culture system
with or without punched wounds
Changes in mRNA levels were examined at 3, 6, 12, 24, 48 and 72 hrs
after creating the wounds. All five samples demonstrated similar expression
patterns in proalpha1(I) collagen, MMP-1 and TIMP-1. There was no significant
change in the levels of mRNA expression of proalpha1(I) collagen and TIMP-1
regardless of the presence or absence of punched wounds during the incubation
period examined (data not shown). The levels of MMP-1 mRNA expression
increased to a maximum at 12 hrs and then gradually declined to a lower
level in the three-dimensional culture system in both punched and non-punched
tissues. However, in the case of the cell layer with wounds, the levels
of MMP-1 mRNA were significantly increased at 6, 12 and 24 hrs as compared
with the level at 3 hrs, and then decreased to the lower level. The levels
of MMP-1 mRNA expression were higher in the wounded cell layer than those
in the non-wounded cell layer at 6, 12 and 24 hrs (Fig.
2). The representative result of MMP-1 mRNA expression is shown
in Figure 3.
Effect of punched wounds on proMMP-1 production
The concentration of proMMP-1 gradually increased in both punched and
non-punched models. The concentrations of proMMP-1 with punched wounds,
however, were significantly elevated as compared with those without punched
wounds at all time points (p < 0.01, Fig.
4).
Effect of punched wounds on type I collagen production
Regardless of the presence or absence of punched wounds, the concentration
of the procollagen type I C-peptide gradually increased (Fig.
5). There was no significant difference between the supernatants
either with or without punched wounds.
Effect of punched wounds on TIMP-1 production
Regardless of the presence or absence of punched wounds, the concentration
of the TIMP-1 gradually increased (Fig.
6). There were no significant differences.
Discussion
The three-dimensional culture system supplemented with Asc-2p is a useful
model for investigating the functions of human dermal fibroblasts in
vitro [6, 7, 12]. The addition of Asc-2p to DMEM stimulates human
dermal fibroblasts to proliferate and produce ECM, which leads to the
formation of a three-dimensional structure. The composition of ECM in
this three-dimensional culture system was demonstrated to be similar to
that of the dermis [6, 7]. In our study, we employed this model to investigate
the mechanism of wound healing, focusing on the direct effect of wounds
on the connective tissue metabolism by dermal fibroblasts.
It is well recognized that various metalloenzymes are involved in the
early phase of wound repair [13]. Wound healing requires controlled synthesis
and degradation of extracellular matrix components, and it is possible
that a balanced synthesis of MMPs and their inhibitors is crucial in the
successful healing process [14]. In this study, the mRNA expression and
the protein production pattern of MMP-1 by fibroblasts with punched wounds
were significantly enhanced as compared with the non-punched control.
With a confocal microscope, we observed fibroblasts actively migrating
toward the punched area (data not shown). Although we did not examine
the proliferative activity of fibroblasts, fibroblasts do proliferate
and migrate in the early stage after wounding. Inoue et al. [15]
demonstrated that in in vitro wounds of skin organ-culture system,
MMP-1 began to rise in migrating keratinocytes 4-6 hrs after creation
of the wound, then peaked at 24-48 hrs, and gradually decreased during
the next few days, subsiding entirely upon re-epithelialization. The expression
of collagenase started significantly in late stage and persisted until
after complete re-epithelialization. Inoue et al. [15] speculated
that collagenase induction in keratinocytes did not require inflammation
and occurred as a rapid response to wounding, suggesting that interstitial
collagenase (MMP-1) was not only necessary for remodeling of the extracellular
matrix, but might have a role in initiating migration of keratinocytes
in wound healing. Similarly, we confirmed that the production of MMP-1
by fibroblasts was induced by acute injury to a dermis-like structure
without the presence of keratinocytes. We conclude that a rapid induction
of MMP-1 after wounding may be required to cleave the extracellular matrix
for initiating migration of fibroblasts into wounded sites. However, as
a wound healing process progresses under the control of many components,
further studies are required using other experimental models such as co-culture
system [16-19].
It is noteworthy that the levels of MMP-1 mRNA expression did peak at
12 hrs and then gradually declined to low levels in the three-dimensional
culture system even without punched wounds. We presume that the medium
exchange itself might have acted as a stimulatory signal for the production
of MMP-1 as well as type I collagen and TIMP-1. We could not determine
why the mRNA expression and production of type I collagen and TIMP-1 did
not show distinct alterations in response to punched wounds in this study.
It is possible that the production might have increased if the cells had
been incubated for a longer period. However, we could not examine the
changes of production of type I collagen and TIMP-1 because the cell layer
began to contract and finally detached from the plastic dish after a longer
incubation period [6], Saarialho suggests that the balance between MMPs
and their inhibitors may be altered in poorly healing wounds [20].
The function of MMPs during wound healing has not been adequately defined
principally because of limitations imposed by available wound-healing
models. In previous human studies, burn wound blister fluid and postsurgical
drainage fluids were used as models for the investigation of dermal wounds
[21-24]. Ours is the first study to show that the disruption of a three
dimensional structure upregulated MMP-1 synthesis by fibroblasts.
CONCLUSION
In conclusion, both MMP-1 from fibroblasts and keratinocytes may play
a pivotal role in acute wound healing. The three dimensional culture system
could be a new exprimental model to explore the direct effect of acute
injury on the connective tissue metabolism of human dermal fibroblasts.
Acknowledgements
This study was supported in part by the Lydia Oleary Memorial Foundation.
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