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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 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 1. Donovan SM, Odle J. Growth factors in milk as mediators of infant development. Annu Rev Nutr. 1994;14:147–67.
Student’s t test; P , 0.05 was considered significant (SAS statistical 2. Braumrucker CR, Hadsell DJ, Blum JW. Insulin-like growth factors (IGFs) and IGF binding proteins in mammary secretions: origins and implications inneonatal physiology. In: Picciano MF, Lo¨nnerdal B, editors. Mechanisms regulatinglactation and infant nutrients utilization. New York: Wiley-Liss; 1992. p. 285–308.
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Isolated canine fibroblasts were incubated with BC at 3 4. Korhonen H, Marnila P, Gills HS. Bovine milk antibodies for health. Br J different concentrations (0.1, 0.3, and 1 mg/mL) for 24 and 48 h.
5. Mero A, Ka¨hko¨nen J, Nyka¨nen T, Parviainen T, Jokinen I, Takala T, Nikula Canine fibroblast growth, measured by the absorbance of the T, Rasi S, Leppa¨luoto J. IGF-I, IgA, and IgG responses to bovine colostrums different cultures (Table 1), was significantly increased in a dose- supplementation during training. J Appl Physiol. 2002;93:732–9.
dependent manner by the 3 different concentrations after 24 h of 6. Brinkworth GD, Buckley JD. Concentrated bovine colostrum protein supplementation reduces the incidence of self-reported symptoms of upper incubation (19 to 32% related to negative control, P , 0.05).
respiratory tract infection in adult males. Eur J Nutr. 2003;42:228–32.
7. Antonio J, Sanders MS, Van Gammeren D. The effects of bovine colos- 11. Budasz-Rwiderska M, Jank M, Motyl T. Transforming growth factor-beta 1 trum supplementation on body composition and exercise performance in active upregulates myostatin expression in mouse C2C12 myoblasts. J Physiol men and women. Nutrition. 2001;17:243–7.
Pharmacol. 2005;56: Suppl 2:195–214.
8. Saygin NE, Tokiyasu Y, Giannobile WV, Somerman MJ. Growth factors 12. Mosmann T. Rapid colorimetric assay for cellular growth and survival: regulate expression of mineral associated genes in cementoblasts. J Periodontol.
application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 9. Tanaka E, Aoyama J, Miyauchi M, Takata T, Hanaoka K, Iwabe T, Tanne 13. Hironaka T, Ohishi H, Masaki T. Identification and partial purification of K. Vascular endothelial growth factor plays an important autocrine/paracrine role in a basic fibroblast growth factor-like growth factor derived from bovine colostrum.
the progression of osteoarthritis. Histochem Cell Biol. 2005;123:275–81.
10. Tan Z, Gong P, Zhao Q. Influence of basic fibroblast growth factors on the 14. Takayama Y, Kitsunai K, Mizumachi K. Factors in bovine colostrum that migration, proliferation of osteoblast and periodontal ligament fibroblasts. Hua Xi enhance the migration of human fibroblasts in type I collagen gels. Biosci Kou Qiang Yi Xue Za Zhi. 2005;23:201–3.
Biotechnol Biochem. 2001;65:2776–9.



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