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The WALTHAM International Nutritional Sciences Symposia
Bovine Colostrum Increases Proliferation of Canine Skin Fibroblasts1,2
Celina Torre,*3 Isabelle Jeusette,* Montserrat Serra,y Pilar Brazis,y and Anna Puigdemontz
* R&D Department, Afﬁnity Petcare, Barcelona, Spain; yUnivet, Cerdanyola, Spain by Univet, Barcelona,Spain; and zDepartment de Farmacologia, Universitat Auto`noma de Barcelona, Bellaterra, Spain
KEY WORDS: bovine colostrum dog ﬁbroblasts proliferation
Bovine colostrum (BC)4 is a milk secreted during the first
few days after calving and is a rich source of bioactivecomponents such as immunoglobulins, insulin-like growth
factor (I, II) (IGF I-II), transforming growth factor-b (TGF-
Skin samples were taken from fresh abdominal skin obtained from
b), platelet-derived growth factor, epidermal growth factor,
surgeries in 2 different public kennels in Barcelona (Arrabassada and
tumor necrosis factor, basic fibroblast growth factor, vaso-
Granollers). The skin (n ¼ 4) was always healthy skin from dogs be-
endothelial growth factor, and telomerase (1–3).
The importance of colostrum for the health of young animals
has been known for a long time (1–4), but other systemic effects
in adult humans fed BC orally have also been demonstrated,
Cultures of normal canine dermal fibroblasts were established from
such as increased salivary IgA and plasma IGF-I (5), reduced
canine skin samples taken from 4 dogs. The skin was well shaven and
symptoms of upper respiratory tract infection in humans (6),
cleaned with 70% EtOH/Betadine before the start of cell isolation. All
and changes in body composition of human athletes (7).
the fat tissue and blood vessels were removed from the skin with
Specific growth factors have been studied in different cul-
scissors, and then it was washed with 0.02 mol/L sodium phosphate
tured cells, such as cementoblasts (8), chondrocytes (9), fibro-
buffer with 0.15 mol/L sodium chloride, pH adjusted to 7.4, without
blasts (10), and myoblasts (11) to evaluate improved regenerative
therapies for periodontal tissues, osteoarthritis, and wound
Skin cells were enzymatically dispersed from skin biopsies. Briefly,
washed skin was chopped into 1-mm3 fragments and incubated for 140
The aim of this study was to evaluate the effects of a skim
min in 15 mL/g skin of Dulbecco’s Modified Eagle Medium (DMEM)
freeze-dried BC source (Colexan, Colostrum Technologies
(Gibco, Invitrogen) containing 30 mg collagenase, 18 mg hyaluronidase,12 mg pronase, 1.5 mg DNAse (all from Sigma) supplemented with 450 mg
GmbH) rich in bioactive components (immunoglobulins, natural
bovine albumin and antibiotics (100 U penicillin, 100 mg streptomycin)
growth factors, and hormones), as an in vitro stimulator of
(Gibco, Invitrogen). After digestion, cutaneous cells were washed with
canine skin fibroblast proliferation and activity. This in vitro
PBS-/-, and their viability was assessed. Cells were grown in DMEM
model could be used as a model for wound repair efficiency and
supplemented with 5% fetal calf serum and antibiotics (all from Gibco) in
periodontal tissue repair for topical applications.
100-mm culture dishes for the initial plating and passages. Fibroblasts grewin a humidified atmosphere at 378C with 5% CO2 for 2 d. Cells wereattached, and the medium was changed twice a week until the first passagewas necessary. Fibroblasts were used at passages 2–5.
1 Published in a supplement to The Journal of Nutrition. Presented as part of
The WALTHAM International Nutritional Sciences Symposium: Innovations inCompanion Animal Nutrition held in Washington, DC, September 15–18, 2005.
This conference was supported by The WALTHAM Centre for Pet Nutrition and
A freeze-dried skim BC source (Colexan, Colostrum Technologies
organized in collaboration with the University of California, Davis, and CornellUniversity. This publication was supported by The WALTHAM Centre for Pet
GmbH) with 85–90% total protein (85% IgG), ,1% fat, 1–5% lactose,
Nutrition. Guest editors for this symposium were D’Ann Finley, Francis A. Kallfelz,
5–7% ash, and natural growth factors and hormones (IGFI-II, 2.5–3.8
James G. Morris, and Quinton R. Rogers. Guest editor disclosure: expenses for
mg/g; TGF-b, 0.1–0.4 mg/g; cortisol, 170 mg/g; prolactin, 4 ng/g;
the editors to travel to the symposium and honoraria were paid by The WALTHAM
estradiol, 1.98 ng/g; testosterone, 33.6 ng/g; insulin, 1.98 mU/g, and
Author disclosure: This research has been supported by Afﬁnity Petcare.
3 To whom correspondence should be addressed. E-mail: ctorre@afﬁnity-
4 Abbreviations used: BC, bovine colostrum; IGF-I, IGF-II, insulin-like growth
factor (I, II); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;PBSÿ/ÿ, phosphate-buffered saline buffer without calcium or magnesium; TGF-b,
Activity that stimulated cell growth was assayed with the addition
of various concentrations of BC in a 96-well microplate, and the plate
0022-3166/06 $8.00 Ó
2006 American Society for Nutrition. J. Nutr. 136: 2058S–2060S, 2006.
COLOSTRUM EFFECTS ON DOG SKIN FIBROBLAST CULTURE
Effect on ﬁbroblast proliferation of different concentrations of bovine colostrum added to
1 Values are means 6 SEM. Cell proliferation was estimated by an MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] assay and is express as optical density units (OD).
2 The relative activity is calculated as 100 3 (absorbance of a culture/absorbance of the negative
3 SCF: stem cell factor. Triplicates were used for each treatment. Differences of absorbance were
assessed using Student’s t test. Means in a row without a common superscript letter differ, P , 0.05.
was incubated for 24 and 48 h at 378C in 5% CO2. The cell
Moreover, canine fibroblast growth was still significantly
proliferation in each well was estimated by a 3-(4,5-dimethylthiazol-
stimulated in a dose-dependent manner by the 2 highest jn.nutrition.org
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which was first
concentrations (stimulation of 12 and 32% related to negative
described by Mosmann in 1983 (12). This assay is based on the ability
control by 0.3 and 1 mg/mL, respectively, P , 0.05) after 48 h of
of a mitochondrial dehydrogenase enzyme from viable cells to cleave
the tetrazolium rings of the pale yellow MTT and form dark blueformazan crystals, which are largely impermeable to cell membranes,
The results of this trial showed that freeze-dried skim BC
thus resulting in its accumulation within healthy cells. Solubilization of
significantly (P , 0.05) stimulated dog fibroblasts at any dose
the cells by the addition of a detergent (dimethyl formamide) results in
used (0.1–1 mg/mL) at 24 h culture incubation, and the effect
the liberation of the crystals, which are solubilized. The number of
remained significant at 48 h for the dose of 0.3 and 1 mg/mL.
surviving cells is directly proportional to the level of the formazan
This activity of BC has already been shown on NIH 3T3 cells
product created. The color can then be quantified using a simple
(13) and in human fibroblasts (14). Hironaka et al. (13) ob-
colorimetric assay. The results can be read on a multiwell scanning
served a stimulation of NIH3T cell growth only with the cream
fraction of bovine colostrum and not with skim colostrum, but
Briefly, cells were plated at a density of 10,000 cells per well with the
in our case, skimmed BC was able to stimulate cell growth at dif-
different concentrations of colostrums to be tested (0.1, 0.3, and 1 mg/
mL by dry weight, respectively) or stem cell factor (SCF) (100 ng/mL,Amgen) as positive controls because it promotes cellular growth.
Further investigation would be useful to identify which
Negative controls (without any kind of factor that stimulates the cells)
components of BC are efficacious in improving canine fibroblast
were plated with the same water volumes as colostrums in the culture
medium. Cells were incubated for 24 and 48 h at 378C in 5% CO
The results of this trial showed that freeze-dried skim BC
humidified atmosphere, and 10 mL of MTT were added to 100 mL of
maintains activity of the bioactive substances that promote the
medium per well and incubated for 4 h until purple precipitate was
cellular growth, suggesting potential proliferative actions by direct
visible. Fifty microliters of dimethylformamide was added and incubated
contact with target cells. Further studies are needed to evaluate
at 378C in 5% CO2 humidified atmosphere in the dark for 18 h. Finally,
not only local effects but any systemic effect of an oral sup-
the absorbance was read at 570 nm. The number of surviving cells was
plementation of BC for regenerative therapies of different tissues.
directly proportional to the level of the formazan product created.
Results are presented as values of absorbance at 570 nm in optical
density units (OD). Differences of absorbance were assessed using
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The Model Formulation Count what is countable, measure what is measurable, and what is not measurable, make measurable. 4.1 The Overall Process In using any kind of analytical or modeling approach for attacking a problem, there are five major steps:1) Understanding the real problem. 2) Formulating a model of the problem. 3) Gathering and generating the input data for the model (e.g., pe
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