ABSTRACT
A Study of EDC-crosslinked Collagen/Hyaluronic Acid
Matrix for Skin Tissue Replacement
Si-Nae Park
The Graduate Program in Biomedical Engineering
The Graduate School
Yonsei University
Skin defects caused by burns, venous ulcer, diabetic ulcer, or acute injury
induce water, electrolytes, and protein loss from the wound site and may
allow bacteria to invade. Some of these processes can be prevented if the
wound is covered or the lost skin is replaced by a dressing or skin substitute.
Collagen, the most abundant and ubiquitous protein in the human body, has
been widely investigated as a material used for the preparation of dressing for
its unique structural and functional characteristics. The major applications of
collagen-based dressings that have been introduced are collagen films, collagen
gels and collagen sponges. 3-dimensional collagen based sponges serve as a
wound dressing since the large pores or channels, interchannel communications
and combinations of macromolecules of the connective tissue enhance wound
tissue infiltration in vivo as well as cell growth in vitro. Hyaluronic acid (HA),
an important component of extracellular matrix, has been also used as
viscoelastic biomaterials for medical purposes because of its high water
retention capacity, and in drug delivery systems because of its biodegradability.
However, obtaining collagen and/or HA based biomaterials with sufficient
strength to withstand biomechanically stressful applications and to resist the
rapid biodegradation has been problematic. Their application for coverage of
open wounds has some limitations because collagen-based biomaterials are
particularly susceptible to bacterial colonization and enzymatic digestion. In
order to overcome these problems, a porous collagen/HA composite material
for tissue regeneration which has proper mechanical strength and the resistance
to microbial contamination was designed.
This research is divided into four categories. First, a porous collagen-HA
scaffold with a good mechanical property was developed by chemical
cross-linking. Second, the effect of HA crosslinked with collagen matrices by
1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) was investigated in
culture of dermal fibroblasts in vitro. Third, topical antibiotics were loaded in
the crosslinked collagen-HA matrices and their biological activity was
evaluated. Forth, the EDC-crosslinked collagen-HA matrix containing antibiotics
and growth factors was developed and evaluated in animal model.
Porous matrices containing collagen and HA were fabricated by freeze
drying at 20, -70 or -196 ℃. The fabricated porous membranes were
crosslinked by using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) in
a range of 1 to 100 mM concentrations for enhancing mechanical stability of
the composite matrix. SEM views of the matrices demonstrated that the
matrices obtained before cross-linking process had interconnected pores with
mean diameters of 40, 90 or 230 ㎛ and porosity of 58-66 % according to the
freezing temperature, and also the porous structures were retained after
cross-linking process. The swelling test and IR spectroscopic measurement of
different crosslinked membranes were carried out as a measure of the extent of
cross-linking. The swelling behavior of crosslinked membranes showed no
significant differences as cross-linking degree increased. FT-IR spectra showed
that the increase of the intensity of the absorbencies at amide bonds (1655,
1546, 1458 cm-1) compared to that of CH bond (2930 cm-1). In enzymatic
degradation test, EDC treated membranes showed significant enhancement of
the resistance to collagenase activity in comparison with 0.625 %
glutaraldehyde treated membranes. In cytotoxicity test using L929 fibroblastic
cells, the EDC-cross-linked membranes demonstrated no significant toxicity.
Porous collagen matrices crosslinked with various contents of hyaluronic
acid (HA) by EDC were prepared as scaffolds for dermal tissue regeneration.
The effect of HA on cells in accordance with HA concentrations in the
collagenous matrices was investigated through the culture of fetal human
dermal fibroblasts in vitro. SEM views of the matrices demonstrated that all of
the collagen-HA matrices had interconnected pores with mean diameters of
150-250 ㎛. In the HA retention test, HA concentration decreased slowly after
an initial fast decrease within 24 hours. Fetal human dermal fibroblasts
adhered well into all the collagen-based matrices in comparison with PU
matrix used as a control. MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium
bromide) based proliferation test and the hematoxilin & eosin staining of 2
week cultured matrix showed that proliferation of fibroblasts was enhanced on
9.6 % HA contained collagen matrix. There is no significant difference among
the migration of fibroblasts into the various types of scaffolds as HA content
was increased. In animal study, dermis with treatment of the collagen or
collagen-HA matrices was thicker than that of control, epithelial regeneration
was accelerated, and collagen synthesis was increased in comparison with
control. However, the significant effect of HA for wound size reduction was
not found.
The tobramycin, ciprofloxacin and polymyxin B as antimicrobial agents were
incorporated into EDC-crosslinked porous collagen-hyaluronic acid matrix for
control of wound contamination. The matrices containing antibiotics were
characterized with respect to morphology, mechanical strength, in vitro release,
antibacterial activity and cytotoxicity. The porous drug loaded
collagen-hyaluronic acid matrices was successfully prepared and the
incorporated antibiotics did not alter the porous structure. As the drug loading
content increased, the high release rate was induced for the early days. The
antibacterial capacity increased as the amount of loaded drug increased and the
samples of ciprofloxacin loaded matrix over 48 hours maintained the
antibacterial effect. The cell viability test revealed that the high concentration
(0.4 mg/ml) of ciprofloxacin had cytotoxic effect on the fetal human dermal
fibroblasts. Added tobramycin and polymyxin B, up to 1.6 mg/ml and 0.8
mg/ml of concentration respectively, did not influence the cell viability.
The bilayered collagen-HA matrices was fabricated containing tobramycin
and cytokines and to evaluate the effect of the matrices on the wound healing
by using an in vivo full thickness dermal defect model. The significant effect of
HA for wound size reduction in was not found. In the entire group, the
wound size reduction increased gradually to reach about 95 % during 2 weeks
after creation of the skin defect. On postoperative days 3 and 7, wound size
reduction in the collagen-HA matrix-treated group increased to 53.2 % and 73.6
% of the original area, respectively. On the other hand, wound contraction
increased to 53.2 % and 62.8 % in the collagen matrix-treated group and to
42.4 % and 60.3 % in control groups, respectively. The tobramycin-incorporated
in collagen-HA matrix had no significant effect on wound healing compared
with control and drug unloaded collagen-HA matrix. The PDGF-incorporated
matrix had the early effect on the wound size contraction in the comparison
with bFGF. With collagen matrix or collagen-HA matrix, the inflammatory
response tended to be pronounced severely on postoperative days 3 and
gradually subsided thereafter. On day 7 as shown in figure 12, capillaries
appeared and the epithelialization was initiated from the edge. After 14 days,
the proliferation of fibroblasts and collagen was accumulated, blood vessels
were decreased and the regeneration of epithelium was observed. Dermis with
treatment of regenerative matrices was thicker than that of control. Epithelial
regeneration was accelerated and collagen synthesis was increased compared
with control.
Therefore, these studies suggest the feasibility of EDC-crosslinked
collagen-HA matrix containing antibiotics and/or growth factors for the
replacement of defective skin tissue.
Key words: collagen; hyaluronic acid; porous scaffold; EDC cross-linking;
fetal dermal fibroblast; antibiotics; growth factors; wound healing;
