Am J Physiol Regulatory Integrative Comp Physiol

278: R1651–R1660, 2000.

Mitogenic whey extract stimulates wound repair activity

in vitro and promotes healing of rat incisional wounds
TIMOTHY E. RAYNER,1 ALLISON J. COWIN,1 J. GRAY ROBERTSON,1
RODNEY D. COOTER,2 RICHARD C. HARRIES,2 GEOFFREY O. REGESTER,1
GEOFFREY W. SMITHERS,3 CHRIS GODDARD,4 AND DAVID A. BELFORD4
1Cooperative Research Centre for Tissue Growth and Repair, Child Health Research Institute,

Women’s and Children’s Hospital, North Adelaide 5006; 2Plastic and Reconstructive Surgery,
University of Adelaide, Department of Surgery, The Queen Elizabeth Hospital, Woodville 5011;
4Commonwealth Scientific and Industrial Research Organization Division of Human Nutrition,

Adelaide BC 5000; and 3Food Science Australia, Highett, Victoria 3190, Australia

Rayner, Timothy E., Allison J. Cowin, J. Gray Robert-
son, Rodney D. Cooter, Richard C. Harries, Geoffrey O.
Regester, Geoffrey W. Smithers, Chris Goddard, and
David A. Belford. Mitogenic whey extract stimulates wound
repair activity in vitro and promotes healing of rat incisional
wounds. Am J Physiol Regulatory Integrative Comp Physiol
278: R1651–R1660, 2000.—The ability of single growth fac-
tors to promote healing of normal and compromised wounds
has been well described, but wound healing is a process
requiring the coordinated action of multiple growth factors.
Only the synergistic effect on wound healing of combinations
containing at most two individual growth factors has been
reported. We sought to assess the ability of a novel milk-
derived growth factor-enriched preparation [mitogenic bovine
whey extract (MBWE)], which contains six known growth
factors, to promote repair processes in organotypic in vitro
models and incisional wounds in vivo. MBWE stimulated the
contraction of fibroblast-populated collagen lattices in a dose-
dependent fashion and promoted the closure of excisional
wounds in embryonic day 17 fetal rat skin. Application of
MBWE increased incisional wound strength in normal ani-
mals on days 3, 5, 7, and 10 and reversed the decrease in
wound strength observed following steroid treatment. Wound
histology showed increased fibroblast numbers in wounds
from normal and steroid-compromised animals. These data
suggest the mixture of factors present in bovine milk exerts a
direct action on the cells of cutaneous wound repair to
enhance both normal and compromised healing.
growth factor; wound healing; skin; fetal wound healing;
fibroblast-populated collagen lattice
THE TOPICAL APPLICATION OF several classes of growth
factors has been shown to enhance all aspects of wound
repair, including inflammation, matrix synthesis, wound
contraction, epithelialization, and tissue remodelling
(6). Studies to date have largely focussed on the effects
of single recombinant growth factors, including mem-
bers of the fibroblast growth factor (FGF) (17, 30),
insulin-like growth factor (IGF) (22, 48), transforming
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
http://www.ajpregu.org 0363-6119/00 $5.00 Copyright r 2000 the American Physiological Society
growth factor (TGF)-b (34, 38, 39), platelet-derived
growth factor (PDGF) (17, 18, 39), and epidermal
growth factor (EGF) (19) families, the interleukins (26),
and colony stimulating factor (24). Importantly, these
factors have been shown to reverse the healing deficit
associated with diabetes (17), steroid administration
(38), cytotoxic therapy (25), infection (24), radiation (9,
35), and ischemia (49). However, the activity of these
factors in promoting wound repair on the experimental
animal has not been reproduced in clinical studies of
chronic wound healing (43). Whereas there are several
possible reasons for this discrepancy, it now seems
likely that the proinflammatory environment of the
chronic wound adversely affects the stability and activ-
ity of the applied growth factors (29, 50). Thus the
ability of any single factor to enhance the multiple
aspects of wound repair to produce a clinically signifi-
cant outcome may be limited.
Several studies have now clearly shown that wound
repair is the result of the temporally coordinated local
expression of multiple families of growth factors and
their receptors (5). It would therefore seem reasonable
to hypothesize that the application of mixtures of
growth factors to the healing wound may be more
effective than the addition of a single factor. Indeed,
several studies have shown this to be the case. In
particular, PDGF interacts with IGF-I (27), IGF-II (16),
EGF, and TGF-b (25), insulin (18), and TGF-a (7) to
promote the repair of a variety of wounds. EGF and
insulin act synergistically to promote collagen synthe-
sis in the diabetic rat (19). Similarly, coapplication of
IGF-I and its binding proteins is more effective at
promoting wound repair than application of IGF-I
alone (22, 33).
Bovine milk is a rich source of several classes of
growth factors, including PDGF (46), IGF-I and -II (11),
and TGF-b (21). We have reported that cation-exchange
fractionation of bovine whey results in a 100- to 200-
fold enrichment of the growth factor activity present in
milk (10). The resultant fraction, mitogenic bovine
whey extract (MBWE), was shown to stimulate the
growth of cells of mesodermal origin, although, due to
its TGF-b content, was found to be inhibitory to the
division of epithelial cells (3, 45). This fraction also
R1651

Downloaded from journals.physiology.org/journal/ajpregu (114.125.248.089) on December 13, 2020.

R1652 MITOGENIC WHEY EXTRACT STIMULATES WOUND REPAIR
contains significant concentrations of IGF-I and -II,
IGF binding proteins 2 and 3, PDGF, and FGF-1 and -2,
although together these known growth factors account
for only 50% of growth activity present (2, 44). Maximal
growth of fibroblast lines in response to MBWE signifi-
cantly exceeded that in the presence of several indi-
vidual recombinant factors including PDGF, IGF-I,
TGF-b, FGF-2, or EGF (3).
The aim of the current study was to investigate the
wound repair activity of the milk-derived growth factor
mixture. In vitro organotypic models initially were
used to characterize the tissue repair activity of MBWE,
and an in vivo model of incisional wound healing was
used to assess the ability of MBWE to promote cutane-
ous repair in normal animals and reverse the deficit in
healing associated with steroid administration.
MATERIALS AND METHODS
Materials
Human diploid skin fibroblasts (SF) were provided by the
Women’s and Children’s Hospital (North Adelaide, Australia)
and DMEM obtained from Flow Laboratories (Irvine, Scot-
land). Fetal bovine serum (FBS) was obtained from Cytosys-
tems (Castle Hill, Australia) and [3H]inulin (1.18 Ci/mmol)
from Amersham (Buckinghamshire, UK).
Male Sprague-Dawley rats (250–300 g) were purchased
from the University of Adelaide. All experiments were ap-
proved by the appropriate institutional animal care and
ethics committee of the Women’s and Children’s Hospital, The
Queen Elizabeth Hospital, or CSIRO Human Nutrition follow-
ing the Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes. Methylprednisolone (Depo-
Medrol, 20 mg/ml) was purchased from Upjohn (Rydalmere,
New South Wales, Australia). Fluothane anesthetic (Halo-
thane) was obtained from ICI Australia (Victoria, Australia)
and used in combination with oxygen and nitrous oxide (BOC
Gases, South Australia, Australia). Collagen was extracted
from rat tail tendons in a solution of 0.1% acetic acid
according to the method of Bell et al. (4). Before use as a
vehicle matrix, the collagen was diluted in sterile water to a
final concentration of 1 mg/ml and brought to neutrality with
phosphate-buffered saline and NaOH.
Production of MBWE
Fractionation of whey was carried out as previously de-
scribed (10). Briefly, pasteurized whey obtained as a byprod-
uct of cheese manufacture was clarified by passage through a
0.8-µm ceramic filter (Membralox, Bajet, France). The ultra-
filtrate was adjusted to pH 6.5 and applied to a 30-cm
diameter column (Moduline, Beverly, MA) packed with 5 l of
S-Sepharose Fast Flow cation-exchange resin (AMRAD Phar-
macia Biotech, Victoria, Australia) equilibrated with 50 mM
sodium citrate buffer at pH 6.5. After washing the column
with the same buffer, the absorbed material was eluted with
0.5 M NaCl. This eluate was desalted, concentrated, and
freeze dried. Growth activity was confirmed using a dye-
binding cell growth assay as previously described (3). TGF-b
concentration was determined using an Mv1Lu cell bioassay
(45). IGF-I concentration was measured by radioimmunoas-
say after removal of IGF binding proteins by acid gel-
permeation HPLC (37). MBWE was screened for bovine
diarrhea virus, infectious bovine rhinotracheitis, parainflu-
enza type 3 and coliforms. Endotoxin levels were also mea-
Downloaded from journals.physiology.org/journal/ajpregu (114.125.248.089) on December 13, 2020.
sured (Limulus Amebocyte Lysate assay, BioWhittaker, Walk-
ersville, MD).
Fibroblast-Populated Collagen Lattice Contraction
Human diploid SF were maintained in DMEM supple-
mented with penicillin (60 mg/l), streptomycin (100 mg/l),
fungizone (1 mg/l), and 10% FBS. Stock cultures were main-
tained in 75-cm2 flasks (Corning, NY) in 5% CO2-95% air at
37°C and routinely passaged after suspension in trypsin
(0.125%)/EDTA (0.5 mM) in Dulbecco’s phosphate buffered
saline.
A solution of rat tail collagen (2 mg/ml) was mixed with an
equal volume of 23 DMEM at 4°C before addition of human
SFs (200,000 cells/ml final concentration) and [3H]inulin (to
30,000–40,000 counts · min21 · ml21). Five-hundred microli-
ters of this solution were poured into each well of a 48-place
tissue culture plate (Costar, Cambridge, MA) and allowed to
set by incubation at 37°C for 30 min. Gels were freed from the
surrounding tissue culture plastic by rimming the margin
with a 25-gauge needle. Dilutions of MBWE were applied to
the top of the gels, which were then maintained in a humidi-
fied atmosphere of 5%CO2-95% air at 37°C. Control wells
containing either serum-free or FBS-supplemented medium
were incorporated on each plate. Fibroblast-induced contrac-
tion was assessed by counting the radioactivity remaining in
the gel after 24-h culture. Data were fit to a four-parameter
equation with the aid of a nonlinear curve-fitting program
(Tablecurve, Jandel Scientific, San Rafael, CA).
Fetal Skin Organ Culture
Wounding and culture of fetal rat skin were undertaken as
previously described (1). Pregnant rats were killed by CO2
asphyxiation, the fetuses (E16–E17) removed from the uterus,
weighed, and placed in Hank’s balanced salt solution. A 1 3
1-cm piece of skin was dissected from the back of each fetus
and a 1-mm diameter wound created in each explant using a
squared-off, sharpened 19-gauge needle. The wound margin
was marked by dipping the cutting needle into Indian ink
immediately before wounding. The wounded skin was then
mounted on a six-pin cradle and placed in a 12-well tissue-
culture dish (Costar). After three washes with medium,
serum-free DMEM or DMEM/MBWE (2.5 mg/ml) was added
in a final volume of 2 ml. Wounds were photographed using a
standard focal length jig at the time of culture and at 24, 48,
and 72 h. Wound area was calculated by tracing the wound
margin onto acetate sheets which were then scanned into a
computer (Apple Macintosh Onscanner connected to an Apple
Macintosh LCIII) equipped with an image analysis program
(Prismview, Dapple Systems, Sunnyvale, CA). For histology,
cultures were fixed in methacarn for 2 h before storage in 70%
alcohol and processing by graded dehydration. Skin pieces
were embedded vertically in wax blocks and 3-µm sections
taken through the wound. Sections were stained with hema-
toxylin and eosin, viewed, and photographed.
Incisional Wound Healing Model
Ability to promote repair of an incisional wound was
investigated as previously described (34, 38). After induction
of general anesthesia by inhalation of halothane, paired
6-cm incisions were created through the panniculus carnosis
either side of the midline on the dorsal surface of the rat using
a standard template. MBWE was dissolved in the collagen
vehicle at the indicated concentrations and a single 200-µl
dose of this formulation applied to one wound. The same
volume of collagen vehicle was applied to the contralateral
wound. The collagen was noted to cover the wound surface
 MITOGENIC WHEY EXTRACT STIMULATES WOUND REPAIR
and gelled within 1–2 min. Left- and right-sided wounds were
randomized with respect to treatment and were closed using
five sutures (4/O silk, Dyneck, South Australia, Australia).
Care was taken during suturing to evert the wound margins
to ensure dermal-dermal contact along the length of the
wound. All rats were housed individually after surgery and
were weighed the day after surgery and then every 2 days. At
various time points after wounding, rats were killed by CO2
asphyxiation and the pelts carefully removed. A standard
template was used to cut four strips of 0.5-cm width through
each wound (i.e., 1 strip through each of the wound segments
bordered by two sutures, excluding the top and bottom wound
margins) and either peak breaking strength measured using
a M1000E (Mecmesin, West Sussex, UK) tensiometer (time-
course study) or a breaking profile of each strip generated
using a PCM2500EL tensiometer (Mecmesin) connected to a
personal computer (dose-response study). The latter allowed
calculation of peak load, yield load (defined as the point at
which the gradient change in load over a set displacement
interval becomes ,20% of the calculated start gradient), force
absorbed to yield load, and force absorbed to peak load.
Histology
Samples of MBWE and collagen-treated wounds were
collected and placed in 10% Formalin fixative immediately
after harvesting and processed by graded dehydration. Three
sections (3 µm) were cut through each wound, stained with
hematoxylin and eosin, and scored by two independent observ-
ers (blinded). Each section was scored for mononuclear cell
and fibroblast infiltration. Scores of zero to four were recorded
for each section, and the average of three sections from each
scorer was used to obtain an average score for the wound,
which was used to calculate overall group means.
Data Analysis
Five percent of strips in the steroid-treated group showed
evidence of hemorrhage or dehiscence before dissection and
were removed from the wound strength data set. Similarly,
spontaneous separation of the dermal margins of the wound
during processing for tensiometry (i.e., once they had been
removed from the support of the sutures) was recorded as
dehiscence, and the strip was excluded from wound-strength
data analysis. All results are presented as means 6 SE with
wound-strength data analyzed using Student’s t-test (dose-
response study) or paired t-test (time-course study). Histology
scores were analyzed by Kruskall-Wallis one-way analysis of
variance on ranks with multiple comparisons versus the
control group (combined data) performed using Dunn’s
method. P , 0.05 was considered significant.
RESULTS
Activity of MBWE
Cell growth activity of MBWE was confirmed using
Balb/c 3T3 fibroblasts and SFs as previously described
(3). Growth activity corresponding to that observed in
10% FBS for Balb/c 3T3 fibroblasts and SFs was
achieved with 150 and 610 µg/ml MBWE, respectively.
The batch of MBWE used in the current study con-
tained 1.6 ng/mg active TGF-b and 61 ng/mg total
TGF-b bioactivity as determined by Mv1Lu cell bioas-
say before and after acid activation, respectively. The
batch of MBWE also contained 32 ng/mg IGF-I. The
concentrations of other growth factors known to be
Downloaded from journals.physiology.org/journal/ajpregu (114.125.248.089) on December 13, 2020.
R1653
present in MBWE, including PDGF and FGF, were not
routinely determined. In addition, MBWE was shown
to be free of all microbiological contaminants tested and
contained ,1 ng/ml endotoxin.
Effect of MBWE on Fibroblast-Induced Gel Contraction
The ability of MBWE to stimulate growth of several
fibroblast cell lines (3) led us to further examine its
repair activity using organotypic models of wound
healing. MBWE markedly stimulated fibroblast-in-
duced gel contraction in a dose-responsive manner
when applied to fibroblast-populated collagen lattices
(FPCL; Fig. 1). The dose that exerts half-maximal effect
above baseline in this assay over a series of similar
experiments was 195 6 44 µg/ml (mean 6 SE; n 5 3).
Effect of MBWE on Wound Closure in Organ Cultured
Fetal Rat Skin
The effect of MBWE on wound repair was further
examined using a fetal skin organ culture model. Skin
harvested from E16–E17 rat embryos retains the abil-
ity to heal an excisional wound in serum-supplemented
suspension culture (1) and allows the effect of agents
with potential wound-healing activity to be evaluated
in an organ culture model isolated from blood-borne or
environmental factors. Wounded E16–17 rat skin cul-
tures were established as described and cultured in
DMEM alone or DMEM plus MBWE (2.5 mg/ml). In
DMEM alone, wounds contracted to 35–40% of the
original wounded area over 72 h, although they never
completely closed (Fig. 2A, a and c) (1). Addition of
MBWE to the medium promoted complete closure over
Fig. 1. Fibroblast populated collagen lattice (FPCL) contraction.
FPCLs were prepared such that each 500 µl gel contained 1 mg/ml
collagen plus 105 skin fibroblasts and [3H]inulin [30–40,000 counts/
min (cpm)]. Gels were freed from side of well and cultured in a
dilution series of mitogenic bovine whey extract (MBWE). Control
wells containing either serum-free or fetal bovine serum (FBS)-
supplemented medium were incorporated on each plate. At 24 h,
culture gels were placed in scintillation fluid and the radioactivity
remaining in gel determined in a b-counter. Points represent means 6
SE of triplicate gels. Control values were: serum-free DMEM 23,140 6
840 cpm; DMEM/10% FBS 7,990 6 640 cpm (means 6 SE, n 5 4).
R1654 MITOGENIC WHEY EXTRACT STIMULATES WOUND REPAIR

epidermal layer over the wound edge to form a ‘‘plug’’ of

epithelium between the dermal margins (Fig. 3B).
Time Course of Response to MBWE
To examine the ability of MBWE to stimulate wound
healing in normal rats and determine the time course of
this response, MBWE was formulated at 2.5 mg/ml,
corresponding to a total dose of 0.5 mg/wound, and
applied to linear incisions on the dorsum of adult rats.
This concentration of MBWE has previously been shown
to stimulate maximal growth of human SFs in vitro (3),
although concentrations up to 5 mg/ml were tolerated
by these cells without a significant decrease in the
maximal growth response. Wound strength was as-
sessed at days 3, 5, 7, 10, 14, 21, and 42 after wounding.
The peak load tolerated by MBWE- and vehicle-
treated wounds is shown in Table 1. Highly significant
increases in wound strength in response to topical
MBWE were seen on days 5, 7, and 10, corresponding to
average increases of 44%, 28%, and 20% over control
wounds, respectively. The strength of MBWE-treated
wounds on days 3 and 21 were increased an average of
21% and 10% over control values, respectively. Break-

Fig. 2. Fetal excisional wound closure. Fetal skin was dissected from
dorsum of E17 rats and wounded using a squared off 19-gauge needle.
Wound margin was marked by dipping cutting needle into Indian ink
immediately before wounding. A: wounds are shown at time of
culture (a and b) and after 72 h in serum-free DMEM (c) or DMEM 1
2.5 mg/ml MBWE (d ). B: contraction curves from triplicate experi-
ments after measurement of wound areas at times shown for
wounded skin incubated in serum-free DMEM (s) or DMEM 1 2.5
mg/ml MBWE (r).

the 72-h culture period (Fig. 2A, b and d). The contrac-
tion of wounds in skin explants treated with MBWE
was greatly accelerated compared with skin incubated
in DMEM alone (Fig. 2B). The wound areas of MBWE-
treated explants at 24 and 48 h were 20 6 10% and
2.8 6 2.2% their original area, respectively (Fig. 2B,
means 6 SE of triplicate cultures). In agreement with a Fig. 3. Histology of excisional wounds in fetal rat skin after 48 h
previous study (1), histology through the wound after culture in MBWE. Experimental details are given in Fig. 2 legend. At
48 h in DMEM alone showed no evidence of epithelial or 48 h culture, skin explants were fixed in methacarn, processed for
mesenchymal movement into the wound defect (Fig. wax embedding, and 3-µm sections were taken through wound that
was identified by concentration of ink particles. Sections were stained
3A). In contrast, histology through healed wounds after with hematoxylin and eosin. A: wound margin after 48 h in serum-
48 h in the presence of MBWE showed that final wound free DMEM. B: healed wound after 48 h in DMEM 1 2.5 mg/ml
closure had been achieved by the movement of the MBWE.

Downloaded from journals.physiology.org/journal/ajpregu (114.125.248.089) on December 13, 2020.

 MITOGENIC WHEY EXTRACT STIMULATES WOUND REPAIR R1655

Table 1. Effect of MBWE on wound strength tensiometer. This degree of wound breakdown in the
in normal rats steroid-treated group was reduced to 13% in wounds
treated with 0.5 mg/wound MBWE, whereas the dehis-
Breaking Strength, g cence rate in wounds treated with 0.25 and 2.0 mg/
Day Collagen MBWE P wound MBWE was consistent at 21%. Wounds treated
with the 0.5 mg/wound dose of MBWE also exhibited a
3 27.8 6 2.5 33.5 6 2.4 0.02
5 45.4 6 3.6 65.4 6 4.8 0.0003 significant increase in both the force absorbed to yield
7 85.5 6 6.5 109.4 6 6.0 0.0003 load (not shown) and force absorbed to peak load
10 169.8 6 10.8 203.5 6 10.6 0.0003 compared with vehicle-treated wounds (Fig. 4B). In-
14 277.3 6 16 297.4 6 13.8 0.10 deed, 0.5 mg/wound MBWE increased the wound
21 958.6 6 50.1 1,053 6 59.2 0.02
42 3,300 6 110 3,289 6 80.4 0.9
strength to levels recorded from normal control wounds
Values are means 6 SE for 16 wounds (64 strips) pooled from 2
separate experiments. Paired linear incisions were created on the
dorsal surface of adult male rats as described in MATERIALS AND
METHODS. Mitogenic bovine whey extract (MBWE; 2.5 mg/ml of
collagen) was applied to one wound and collagen vehicle was only
applied to the contralateral wound. On the indicated days after
wounding 1 group was killed and the maximal load (grams) tolerated
by 4 strips of 0.5-cm width were taken through each wound deter-
mined using a tensiometer. Data were analyzed by paired t-test.

ing strengths on days 14 and 42 were not significantly

different from vehicle-treated controls.
Dose Response to MBWE
A series of studies aimed at determining the optimal
dose and assessing the histological picture of the
MBWE-treated wounds was undertaken using both
normal and steroid-compromised animals. Steroid-
treated rats received an intramuscular injection of
methylprednisolone (15 mg/kg) immediately before sur-
gery. MBWE was formulated at 10, 2.5, and 1.25 mg/ml,
each wound therefore receiving a total of 2, 0.5, and
0.25 mg of MBWE, respectively. As the maximal re-
sponse to MBWE in normal animals occurred 5 days
after wounding and application of the extract, all
wounds were harvested at this time point.
Normal animals. The force absorbed by each strip
harvested from normal animals 5 days after wounding
is shown in Fig. 4A. Both force absorbed to yield load
(not shown) and force absorbed to peak load were
significantly increased in wounds treated with 0.5
mg/wound MBWE compared with vehicle-treated
wounds. In addition, the yield load and peak load in the
MBWE-treated wounds were increased an average of
62% and 25% over control wounds, respectively (data
not shown). Application of either 0.25 or 2.0 mg/wound
MBWE did not significantly alter wound strength.
Steroid-treated animals. Administration of methyl-
prednisolone on the day of wounding induced an aver- Fig. 4. Wound strength in normal (A) and steroid-treated animals (B)
age 15% weight loss compared with an 8% weight gain in response to MBWE. Paired linear incisions were created on the
in the normal group over the course of the experiment dorsal surface of adult male rats. MBWE was applied to one wound at
and significantly reduced all indices of wound strength. indicated doses and collagen vehicle was only applied to the contralat-
Yield load, peak load, force absorbed to yield load, and eral wound. Animals in the steroid group were given an intramuscu-
lar injection of 15 mg/kg methylprednisolone at time of surgery.
force absorbed to peak load of vehicle-treated wounds Animals were killed 5 days after wounding and 4 strips of 0.5-cm
were decreased an average of 44%, 39%, 42%, and 39%, width were taken through each wound and placed in a tensiometer.
respectively, in the steroid animals (P , 0.0001 for all). Force absorbed to peak load was calculated from load-displacement
Whereas only ,1–2% of all strips taken through wounds curves. Bars represent means 6 SE of indicated number of strips (in
parentheses) pooled from 2 experiments for both MBWE (filled bars)-
on normal animals showed any signs of dehiscence, and matched vehicle-treated (open bars) wounds. Different number of
28% of strips cut through vehicle-treated wounds on strips analyzed reflects incidence of wound strip dehiscence in
steroid-treated rats disrupted before placement in the respective treatment groups. * P , 0.05 vs. vehicle-treated wounds.

Downloaded from journals.physiology.org/journal/ajpregu (114.125.248.089) on December 13, 2020.

R1656 MITOGENIC WHEY EXTRACT STIMULATES WOUND REPAIR

(Fig. 4A). Furthermore, the values for both the yield
load and peak load were increased an average of 60%
above their vehicle-treated controls (data not shown).
As observed with wounds from normal animals, applica-
tion of either 0.25 or 2.0 mg of MBWE did not signifi-
cantly affect the force absorbed by the wound compared
with their vehicle-treated controls (Fig. 4B).
Wound histology. Representative sections taken
through wound samples from normal and steroid rats
are shown in Fig. 5 with semiquantitative histology
scores of sections from all treatment groups presented
in Fig. 6. Comparison of Fig. 5, A and C, shows the
difference in wound histology expected between normal
and steroid-treated rats, which is characterized by the
marked absence of cellular infiltrate and granulation
tissue. Administration of methylprednisolone signifi-
cantly reduced the scores, reflecting fibroblast and
inflammatory cell infiltration by 46% and 34%, respec-
tively (Fig. 6, A and B). Treatment with MBWE in-
creased the cellular infiltrate in wounds on both normal
and steroid-treated rats (Fig. 5, B and D). This response
to MBWE was most noticeable in sections from wounds

Fig. 5. Wound histology. Photomicro-

graphs show cross sections through day
5 incisional wounds from normal (A and
B) and steroid-treated (C and D) rats
that received MBWE (B and D) or colla-
gen vehicle only (A and C) at time of
wounding. Sections were stained with
Masson’s trichrome and photographed
at 3250 magnification.

Downloaded from journals.physiology.org/journal/ajpregu (114.125.248.089) on December 13, 2020.

 MITOGENIC WHEY EXTRACT STIMULATES WOUND REPAIR
Fig. 6. Semiquantitative assessment of wound histology. Sections
through wounds on both normal (open bars) and steroid-treated
(filled bars) rats were scored for degree of fibroblast (A) or inflamma-
tory cell infiltration (B). Wounds received indicated dose of MBWE or
collagen vehicle only at time of wounding. Data show mean score for
each treatment group (6 SE), which was derived from triplicate
sections through each wound sample with number of wounds as-
sessed in each group shown in parentheses. * P , 0.05 vs. vehicle-
treated wounds (combined across all groups).
on steroid-treated animals, the degree of wound cellu-
larity comparable with sections from normal control
wounds (compare Fig. 5, A and D). This increase in
cellularity was due to an influx of fibroblasts into the
wound, which was not accompanied by a heightened
inflammatory response (Fig. 6, A and B). Within the
steroid-treated group, all doses of MBWE significantly
increased fibroblast numbers compared with their ve-
hicle-treated controls. In normal rats, wounds treated
Downloaded from journals.physiology.org/journal/ajpregu (114.125.248.089) on December 13, 2020.
R1657
with either 0.25 or 0.5 mg/wound MBWE showed a
significant increase in fibroblast score. MBWE had no
significant effect on the inflammatory score in wounds
on both normal and steroid-treated animals (Fig. 6B).
DISCUSSION
We previously showed that cation-exchange chroma-
tography results in a 100- to 200-fold enrichment of
the growth factor component of bovine milk. Although
consisting of only 0.5% of whey protein, it contains the
bulk of growth factor activity present in bovine milk
(10). Importantly, this fraction contains several growth
factors and their binding proteins (2) that have been
shown by others to act in synergistic fashion in the
wound, including IGF-I and PDGF (27), IGF-II and
PDGF (16), and IGF-I and IGF binding protein 3 (33).
In addition, MBWE contains significant concentrations
of members of the TGF-b family, with TGF-b1, a factor
known to significantly enhance the healing strength
of an incisional wound (34). Approximately 85% of the
TGF-b activity in MBWE is accounted for by TGF-b2,
which exists as an acid labile 80-kDa species consistent
with the small latent complex of TGF-b (45). In this
regard, it is interesting to note that some 30–40% of
platelet-derived TGF-b is retained in the clot as
the small latent complex, where it may act as a slow
release form of the molecule during the wound repair
process (15).
The activity of the growth factor-enriched milk-
derived fraction in promoting different aspects of tissue
repair was investigated in the first instance using in
vitro organotypic models. The ability of fibroblasts
incorporated in a collagen gel to reorganize the collagen
fibers and cause gel contraction is considered the in
vitro counterpart of wound contraction in the adult
mammal (4, 32). This phenomenon has been found to be
dependent on the trophic activity of FBS (14). However,
in the present study, MBWE was able to substitute for
serum in stimulating fibroblast-mediated collagen lat-
tice contraction (Fig. 1). MBWE-induced contraction
was dose dependent, with the maximal effect over 24 h
found to be at least equivalent to that observed in
serum-supplemented culture (not shown). Given that
the individual growth factors EGF, PDGF, and basic
FGF (bFGF) failed to stimulate FPCL contraction (32),
it would appear that the novel mixture of growth
factors contained within MBWE may account for the
response observed in this assay, with IGF-I or TGF-b
likely to be at least partly responsible for the activity
evident in MBWE (13, 41).
The assertion that a mixture of individual growth
factors such as that present in MBWE may be effective
at stimulating tissue repair is further supported by the
ability of MBWE to promote wound closure in organ-
cultured fetal skin. Skin harvested from the E16–17
embryo is able to heal a wound in vitro by a combined
process of mesenchymal contraction and substrate-
independent movement of the epithelium over the
dermal margins of the wound to effect final closure (1).
This repair response is dependent on the presence of
serum but is not promoted by the addition of single
R1658 MITOGENIC WHEY EXTRACT STIMULATES WOUND REPAIR
recombinant factors such as IGF-I, EGF, bFGF, TGF-b,
or PDGF. Although wound closure is likely to result
from the coordinated action of epithelial cells to mobi-
lize contractile proteins and close the wound by a
purse-string mechanism (28), the trophic requirement
for this form of epithelial repair is unknown. MBWE
was able to substitute for serum in promoting wound
closure, histology clearly revealing the presence of
epithelium spanning the dermal margins of the wound
(Fig. 3). Nevertheless, the dermal margins were consis-
tently more approximated after culture in MBWE than
in serum-supplemented cultures compared with previ-
ously published results (1), possibly reflecting the ac-
tion of the factors in MBWE on the mesenchymal layer
to enhance wound contraction. Thus the mixture of
factors contained in MBWE has the capacity to induce
novel effects on fetal wound closure that are not ob-
served after application of individual factors, suggest-
ing that appropriate combinations of growth factors
may act in a coordinated fashion to stimulate the
sequence of events required for successful healing. We
cannot, however, rule out the possibility that an as yet
unidentified molecule(s) may be present in MBWE that
has unique and singular effects on the repair process.
All surface wounds heal by a process of wound
contraction, matrix synthesis and remodelling, and
epithelialization. Apposition of the wound margins
allows healing by primary intention, which is mainly
achieved by matrix synthesis. Thus the ability of a
topical agent to enhance the strength of an incisional
wound reflects a direct or indirect ability to enhance
wound fibroblast division, migration into the wound, or
biosynthetic capacity. The ability of MBWE to promote
wound repair was most evident in steroid-treated ani-
mals, suggesting a direct action of the growth factors in
MBWE on the cells of wound repair. Steroid administra-
tion profoundly inhibits both the inflammatory re-
sponse to wounding and the expression of procollagen
genes by SFs (12). A single dose of methylprednisolone
at or before wounding inhibits wound contraction (23),
synthesis of granulation tissue, and angiogenesis and
decreases wound strength (38). In the current study, all
indices of wound strength were reduced by ,40% of
control values 5 days after administration of methyl-
prednisolone. The most effective dose of MBWE (0.5
mg/wound) returned the force absorbed by wounds on
steroid-treated animals to levels observed in normal
animals and resulted in a histological picture similar to
that seen on sections from control wounds on normal
animals.
The ability of MBWE to promote wound repair was
concentration dependent. Higher concentrations of
MBWE promoted fibroblast influx but did not increase
the strength of incisional wounds on either the normal
or steroid-treated animals. Whereas we cannot rule out
the presence of some inhibitory activity in MBWE, it is
possible this phenomenon is due to the bimodal action
of some individual growth factors in MBWE. Both
TGF-b and bFGF have been shown to have apparent
concentration-dependent bimodal effects on the strength
of incisional wounds in vivo (36, 39). Similarly, a
Downloaded from journals.physiology.org/journal/ajpregu (114.125.248.089) on December 13, 2020.
bimodal effect of TGF-b and bFGF on the volume of new
granulation tissue detected in rabbit ear excisional
wounds has also been reported (40).
The possible complex interactions among the known
growth factors in MBWE render any discussion of its
mechanism of action in the wound somewhat specula-
tive. Furthermore, it is impossible to rule out effects on
wound healing by components of MBWE that are not
growth factors. Moreover, although the activity of
MBWE in steroid-treated rats would suggest a direct
action on the wound, we cannot exclude indirect effects
of the components of MBWE on the cells of wound
repair. Nevertheless, comparative studies using single
growth factors may provide some clues. PDGF, TGF-b,
and FGF all stimulate granulation tissue deposition.
TGF-b1 in particular has been shown to act directly on
the cells of cutaneous healing to induce the fibrotic and
angiogenic components of repair in vivo (42). Thus
TGF-b1 is effective in promoting repair of wounds on
animals rendered monocytopenic by steroid administra-
tion (38) or total body irradiation (9), although it is
ineffective in rats whose dermal fibroblasts have been
damaged by surface irradiation (9). In contrast, PDGF
does not directly stimulate collagen synthesis (42) and
cannot reverse the wound healing deficit associated
with circulating and wound monocytopenia as a result
of steroid treatment (38), although it was able to
improve wound repair in animals whose dermal fibro-
blasts had been damaged by surface irradiation by
recruiting macrophages to the wound (35).
The degree of improvement in wound strength ob-
served in response to MBWE 5 days after wounding
steroid-treated rats is greater than that reported follow-
ing administration of recombinant TGF-b1 in a compa-
rable study (38), suggesting the potential synergistic
action of factors present in MBWE. In this regard,
IGF-I and -II are also known to promote fibroblast
division (8), protein, collagen, and fibronectin synthesis
(20), and local administration of IGF-I has been shown
to reverse the effects of steroids on inhibition of granu-
lation tissue ingrowth to a subcutaneous chamber (48).
Coadministration of IGF-I with its binding proteins
increases wound strength (22). Similarly, both acidic
and basic FGF have been shown to increase wound
strength (30, 31). Indeed, Lynch et al. (27) found a
combination of PDGF-2 and IGF-I to be the most potent
inducer for healing partial-thickness porcine wounds
compared with various other individual growth factors
or combinations tested.
Although some studies have shown the maximum
wound strength achieved by the application of single
growth factors (principally TGF-b) to be greater than
that observed in our experiments, it should be noted
that comparative studies have generally added micro-
gram quantities of single recombinant growth factors to
wounds (27, 34, 36, 39, 40). The amount of individual
growth factors applied to wounds treated with MBWE
was in the low nanogram range. It is possible further
purification of MBWE designed to concentrate specific
growth factor components of the extract may enhance
the wound healing activity of these fractions.
 MITOGENIC WHEY EXTRACT STIMULATES WOUND REPAIR
The current report represents the first to describe the
ability of a mitogenic extract containing the comple-
ment of growth factors present in milk to stimulate a
healing response in organotypic wound models and
promote healing of the incisional wound in vivo. To-
gether with in vitro data showing significant activity in
assays of cell growth (3, 10), our results would suggest
the unique mix of agents in MBWE may confer an
ability to promote wound repair over and above that
obtained with single growth factors and that is particu-
larly relevant to rectifying the healing deficits associ-
ated with compromised conditions resulting in chronic
cutaneous wounds (47).
Perspectives
Wound healing is a complex series of events aimed at
restoring the integrity of damaged tissue. Included in
these events are the actions of growth factors, which
play an essential role in directing wound repair. The
action of such mediators, however, is disturbed in
tissue compromised by lack of blood flow or systemic
diseases such as diabetes, leading to failure of the
cellular processes of healing. Whereas growth factors
have been shown to enhance all aspects of wound repair
in experimental models, the effectiveness of individual
growth factors has generally been difficult to replicate
when applied to clinical studies in chronic wounds. As
wound repair results from the temporally coordinated
local expression of multiple families of growth factors
and their receptors, it would seem reasonable that the
application of mixtures of growth factors to the healing
wound may be more effective than a single factor.
Bovine milk is a rich source of several classes of growth
factors, and cation-exchange fractionation of bovine
whey results in a mitogenic extract that contains a 100-
to 200-fold enrichment of the growth factor activity
present in milk. Our results demonstrate that this
mitogenic extract derived from milk has considerable
wound healing activity, and, given the mixture of
growth factors present, we speculate that it may have
potential as a clinical treatment to stimulate healing of
chronic ulcers and other problem wounds. The socioeco-
nomic benefits of such an agent that can effectively heal
chronic wounds is likely to be substantial, given the
morbidity and high cost of treatment of this often
age-related problem.
We thank Kaylene Pickering, Xenia Georgiou, Anna Seamark,
Caroline Payne, and Sue Millard for technical assistance. Microbio-
logical screening of MBWE was undertaken by the Victorian Institute
of Animal Sciences.
This work was supported by a grant from the Dairy Research and
Development Corporation of Australia and the Cooperative Research
Centre Scheme, Commonwealth Government of Australia.
Address for reprint requests and other correspondence: T. E.
Rayner, Child Health Research Institute, Women’s and Children’s
Hospital, 72 King William Rd., North Adelaide 5006, SA, Australia
(E-mail: trayner@medicine.adelaide.edu.au).
Received 3 June 1999; accepted in final form 3 December 1999.
REFERENCES
1. Belford DA. The mechanism of excisional fetal wound repair in
vitro is responsive to growth factors. Endocrinology 138: 3987–
3996, 1997.
Downloaded from journals.physiology.org/journal/ajpregu (114.125.248.089) on December 13, 2020.
R1659
2. Belford DA, Rogers ML, Francis GL, Payne C, Ballard FJ,
and Goddard C. Platelet-derived growth factor, insulin-like
growth factors, fibroblast growth factors and transforming growth
factor b do not account for the cell growth activity present in
bovine milk. J Endocrinol 154: 45–55, 1997.
3. Belford DA, Rogers ML, Regester GO, Francis GL, Smith-
ers GW, Liepe IJ, Priebe IK, and Ballard FJ. Milk-derived
growth factors as serum supplements for the growth of fibroblast
and epithelial cells. In Vitro Cell Develop Biol 31: 752–760, 1995.
4. Bell E, Ivarsson B, and Merrill C. Production of a tissue-like
structure by contraction of collagen lattices by human fibroblasts
of different proliferative potential in vitro. Proc Natl Acad Sci
USA 76: 1274–1278, 1979.
5. Bennett NT and Schultz GS. Growth factors and wound
healing: biochemical properties of growth factors and their
receptors. Am J Surg 165: 728–737, 1993.
6. Bennett NT and Schultz GS. Growth factors and wound
healing: II. Role in normal and chronic wound healing. Am J
Surg 166: 74–81, 1993.
7. Brown RL, Breeden MP, and Greenhalgh DG. PDGF and
TGF-a act synergistically to improve wound healing in the
genetically diabetic mouse. J Surg Res 56: 562–570, 1994.
8. Cook JJ, Haynes KM, and Werther GA. Mitogenic effects of
growth hormone in cultured human fibroblasts; evidence for
action via local insulin-like growth factor I production. J Clin
Invest 81: 206–212, 1988.
9. Cromack DT, Porras-Reyes B, Purdy JA, Pierce GF, and
Mustoe TA. Acceleration of tissue repair by transforming growth
factor b1: identification of in vivo mechanism of action with
radiotherapy-induced specific healing deficits. Surgery 113: 36–
42, 1993.
10. Francis GL, Regester GO, Webb HA, and Ballard FJ.
Extraction from cheese whey by cation-exchange chromatogra-
phy of factors that stimulate the growth of mammalian cells. J
Dairy Res 78: 1209–1218, 1995.
11. Francis GL, Upton FM, Ballard FJ, McNeil KA, and Wal-
lace JC. Insulin-like growth factors 1 and 2 in bovine colostrum:
sequences and biological activities compared with those of a
potent truncated form. Biochem J 251: 95–103, 1988.
12. Fuller GC and Cutroneo KR. Pharmacological interventions.
In: Wound Healing Biochemical and Clinical Aspects, edited by
Cohen IK, Diegelmann RF, and Lindblad WJ. Philadelphia, PA:
Saunders, 1992, p. 305–315.
13. Gillery P, Leperre A, Maquart FX, and Borel JP. Insulin-like
growth factor-I (IGF-I) stimulates protein synthesis and collagen
gene expression in monolayer and lattice cultures of fibroblasts.
J Cell Physiol 152: 389–396, 1992.
14. Gillery P, Maquart FX, and Borel JP. Fibronectin dependence
of the contraction of collagen lattices by human skin fibroblasts.
Exp Cell Res 167: 29–37, 1986.
15. Grainger DJ, Wakefield L, Bethell HW, Farndale RW, and
Metcalf JC. Release and activation of platelet latent TGF-beta
in blood clots during dissolution with plasmin. Nat Med 1:
932–937, 1995.
16. Greenhalgh DG, Hummel RP III, Albertson S, and Bree-
don MP. Synergistic actions of platelet-derived growth factor
and the insulin-like growth factors in vivo. Wound Rep Reg 1:
69–81, 1993.
17. Greenhalgh DG, Sprugel KH, Murray MJ, and Ross R.
PDGF and FGF stimulate wound healing in the genetically
diabetic mouse. Am J Pathol 136: 1235–1246, 1990.
18. Grotendorst GR, Martin GR, Pencev D, Sodek J, and
Harvey AK. Stimulation of granulation tissue formation by
platelet-derived growth factor in normal and diabetic rats. J Clin
Invest 76: 2323–2329, 1985.
19. Hennessey PJ, Black CT, and Andrassy RJ. Epidermal
growth factor and insulin act synergistically during diabetic
healing. Arch Surg 125: 926–929, 1990.
20. Ignotz RA and Massagué J. Transforming growth factor-b
stimulates the expression of fibronectin and collagen and their
incorporation into the extracellular matrix. J Biol Chem 261:
4337–4345, 1986.
21. Jin Y, Cox DA, Knecht R, Raschdorf F, and Cerletti N.
Separation, purification, and sequence identification of TGF-beta
R1660 MITOGENIC WHEY EXTRACT STIMULATES WOUND REPAIR
1 and TGF-beta 2 from bovine milk. J Protein Chem 10: 565–575,
1991.
22. Jyung RW, Mustoe TA, Busby WH, and Clemmons DR.
Increased wound-breaking strength induced by insulin-like
growth factor I in combination with insulin-like growth factor
binding protein-1. Surgery 115: 233–239, 1994.
23. Karr BP, Bubak PJ, Sprugel KH, Pavlin EG, and Engrav
LH. Platelet-derived growth factor and wound contraction in the
rat. J Surg Res 59: 739–742, 1995.
24. Kucukcelebi A, Carp SS, Hayward PG, Hui P, Cowan WT,
Ko F, Cooper DM, and Robson MC. Granulocyte-macrophage
colony stimulating factor reverses the inhibition of wound contrac-
tion caused by bacterial contamination. Wounds 4: 241–247,
1992.
25. Lawrence WT, Sporn MB, Gorschboth C, Norton JA, and
Grotendorst GR. The reversal of an adriamycin induced heal-
ing impairment with chemoattractants and growth factors. Ann
Surg 203: 142–147, 1985.
26. LeGrand EK, Senter LH, Gamelli RL, and Kiorpes TC.
Evaluation of PDGF-BB, PDGF-AA, bFGF, IL-1, and EGF dose
responses in polyvinyl alcohol sponge implants assessed by a
rapid histologic method. Growth Factors 8: 315–329, 1993.
27. Lynch SE, Colvin RB, and Antoniades HN. Growth factors in
wound healing: single and synergistic effects on partial thickness
porcine skin wounds. J Clin Invest 84: 640–646, 1989.
28. Martin P and Lewis J. Actin cables and epidermal movement
in embryonic wound healing. Nature 360: 179–183, 1992.
29. Mast BA and Schultz GS. Interactions of cytokines, growth
factors and proteases in acute and chronic wounds. Wound Rep
Reg 4: 411–420, 1996.
30. McGee GS, Davidson JM, Buckley A, Sommer A, Wood-
ward SC, Aquino AM, Barbour R, and Demetriou AA.
Recombinant basic fibroblast growth factor accelerates wound
healing. J Surg Res 45: 145–153, 1988.
31. Mellin TN, Mennie RJ, Cashen DE, Ronan JJ, Capparella
J, James ML, DiSalvo J, Frank J, Linemeyer D, Gimenez-
Gallego G, and Thomas KA. Acidic fibroblast growth factor
accelerates dermal wound healing. Growth Factors 7: 1–14,
1992.
32. Montesano R and Orci L. Transforming growth factor beta
stimulates collagen-matrix contraction by fibroblasts: implica-
tions for wound healing. Proc Natl Acad Sci USA 85: 4894–4897,
1988.
33. Mueller RV, Spencer EM, Sommer A, Maack CA, Suh D,
and Hunt TK. The role of IGF-I and IGFBP-3 in wound healing.
In: Modern Concepts of Insulin-like Growth Factors, edited by
Spencer EM. New York: Elsevier, 1991, p. 185–192.
34. Mustoe TA, Pierce GF, Thomason A, Gramates P, Sporn
MB, and Deuel TF. Accelerated healing of incisional wounds in
rats induced by transforming growth factor-b. Science 237:
1333–1336, 1987.
35. Mustoe TA, Purdy J, Gramates P, Deuel TF, Thomason A,
and Pierce GF. Reversal of impaired wound healing in irradi-
ated rats by platelet-derived growth factor-BB. Am J Surg 158:
345–350, 1989.
36. Okumura M, Okuda T, Nakamura T, and Yajima M. Effect of
basic fibroblast growth factor on wound healing in healing-
impaired animal models. Drug Res 46: 547–551, 1996.
Downloaded from journals.physiology.org/journal/ajpregu (114.125.248.089) on December 13, 2020.
37. Owens PC, Johnson RJ, Campbell RG, and Ballard FJ.
Growth hormone increases insulin-like growth factor-I (IGF-I)
and decreases IGF-II in plasma of growing pigs. J Endocrinol
124: 269–275, 1990.
38. Pierce GF, Mustoe TA, Lingelbach J, Masakowski VR,
Gramates P, and Deuel TF. Transforming growth factor b
reverses the glucocorticoid-induced wound-healing deficit in
rats: possible regulation in macrophages by platelet-derived
growth factor. Proc Natl Acad Sci USA 86: 2229–2233, 1989.
39. Pierce GF, Mustoe TA, Lingelbach J, Masakowski VR,
Griffin GL, Senior RM, and Deuel TF. Platelet-derived growth
factor and transforming growth factor-b enhance tissue repair
activities by unique mechanisms. J Cell Biol 109: 429–440, 1989.
40. Pierce GF, Tarpley JE, Yanagihara D, Mustoe TA, Fox GM,
and Thomason A. Platelet-derived growth factor (BB ho-
modimer), transforming growth factor-b1, and basic fibroblast
growth factor in dermal wound healing. Am J Pathol 140:
1375–1388, 1992.
41. Piscatelli SJ, Michaels BM, Gregory P, Jennings RW,
Longaker MT, Harrison MR, and Siebert JW. Fetal fibro-
blast contraction of collagen matrices in vitro: the effects of
epidermal growth factor and transforming growth factor-beta.
Ann Plast Surg 33: 38–45, 1994.
42. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS,
Wakefield LM, Heine UI, Liotta LA, Falanga V, Kehrl JH,
and Fauci AS. Transforming growth factor type b: rapid induc-
tion of fibrosis and angiogenesis in vivo and stimulation of
collagen formation in vitro. Proc Natl Acad Sci USA 83: 4167–
4171, 1986.
43. Robson MC. The role of growth factors in the healing of chronic
wounds. Wound Rep Reg 5: 12–17, 1997.
44. Rogers ML, Belford DA, Francis GL, and Ballard FJ.
Identification of fibroblast growth factors in bovine cheese whey.
J Dairy Res 62: 501–507, 1995.
45. Rogers ML, Goddard C, Regester GO, Ballard FJ, and
Belford DA. Transforming growth factor b in bovine milk:
concentration, stability and molecular mass forms. J Endocrinol
151: 77–86, 1996.
46. Shing Y and Klagsbrun M. Purification and characterization of
a bovine colostrum-derived growth factor. Mol Endocrinol 1:
335–338, 1987.
47. Stadelmann WK, Digenis AG, and Tobin GR. Physiology and
healing dynamics of chronic cutaneous wounds. Am J Surg 176,
Suppl 2A: 26S-38S, 1998.
48. Suh DY, Hunt TK, and Spencer EM. Insulin-like growth
factor-I reverses the impairment of wound healing induced by
corticosteroids in rats. Endocrinology 131: 2399–2403, 1992.
49. Uhl E, Barker JH, Bondàr I, Galla TJ, Leiderer R, Lehr HA,
and Messmer K. Basic fibroblast growth factor accelerates
wound healing in chronically ischaemic tissue. Br J Surg 80:
977–980, 1993.
50. Yager DR, Chen SM, Ward SI, Olutoye OO, Diegelmann RF,
and Cohen IK. Ability of chronic wound fluids to degrade
peptide growth factors is associated with increased levels of
elastase activity and diminished levels of proteinase inhibitors.
Wound Rep Reg 5: 23–32, 1997.