A pharmacological model for studying mechanical properties of skin
Authors: Tsuboi R. 
Published: September 2010

The wound healing process consists of inflammation, followed by granulation tissue formation and then tissue remodeling [1]. Wound contraction following granulation tissue formation is crucial to the reduction of the size of the wound defect leading to eventual wound closure [1]. Although this phenomenon has been ascribed to the contraction of actin‐rich fibroblasts in granulation tissue, the mechanism and trigger for wound contraction have not yet been elucidated.

Age‐related changes in skin appearance, including dryness, wrinkling and laxity, are influenced by the behavior of keratinocytes and fibroblasts, and by the amount of matrices they produce. Aging is known to affect the wound contraction as well as the wound‐healing process. Quantitative analysis of wound contraction using an in vitro model may clarify certain aspects of skin aging and regeneration.

An in vitro model that is considered to simulate closely the wound contraction process was reported by Bell et al. in 1979 [2]. The model consisted of fibroblast‐incorporated collagen lattices. The assessment was quantitatively performed by measuring the diameter or area of the floating collagen gel. The rate of gel contraction in this model was found to vary depending on the phenotypic differences of the fibroblasts, and to be proportional to the number of incorporated fibroblasts and serum concentration, but inversely proportional to the concentration of collagen and the age of the fibroblast donor. Recent studies indicate that gel contraction is stimulated by some polypeptide cytokines such as transforming growth factor (TGF)‐β, platelet‐derived growth factor (PDGF) and endothelin‐1 (ET‐1).

In a series of studies, we demonstrated that cytokines and bioactive ligands work in combination to regulate fibroblast‐embedded collagen gel contraction. Our conclusions were as follows: 1) A combination of insulin‐like growth factor (IGF)‐I and IGF‐binding protein (BP)‐1 promotes the contraction of fibroblast‐embedded collagen gel [3]. 2) Endothelin‐1 secreted from keratinocytes stimulates fibroblast‐embedded collagen gel contraction [4]. 3) Sphingosylphosphorylcholine (SPC), a sphingolipid metabolite generated in atopic dermatitis, stimulates wound contraction by fibroblasts [5]. 4) Overexpression of Smad3, a major signal transducer of TGF‐β, enhances collagen gel contraction by fibroblasts, and TGF‐β1 mediated collagen gel contraction is suppressed by overexpression of Smad7, a major inhibitory regulator in the Smad family [6].

Materials and methods

Recombinant human insulin‐like growth factor‐I (IGF‐I), recombinant human IGF binding protein‐1(IGFBP‐1), and rabbit anti‐human IGFBP‐1 antibody were donated by Synergen Inc. (Boulder, CO, U.S.A.). Sphingosylphosphorylcholine (SPC, 99% purity) was purchased from Matreya Inc. (Pleasant Gap, PA, U.S.A.) and dissolved in absolute ethanol. Sphingosine (SP), sphingosine‐1‐phosphate (S1P), N‐acetyl‐D‐sphingosine (C2‐ceramide) and N‐hexanoyl‐D‐sphingosine (C6‐ceramide) were purchased from Sigma Chemical Co (St Louis, MO, U.S.A.), and synthetic human endothelin‐1 from Peptide Institute Inc. (Osaka, Japan). The recombinant E1‐deleted adenoviral vectors carrying mouse Smad3, Smad7 or lacZ cDNA under the cytomegalovirus promoters AdCMV‐Smad3, AdCMVSmad7 or AdCMV‐lacZ, were generated, purified and transfected as previously described.

Normal dermal fibroblasts isolated from infant foreskins were cultured using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 units mL‐1 penicillin and 100 μg mL‐1 streptomycin. The cells were cultured in a humidified atmosphere with 5% CO2, 95% air at 37 °C. The cells were harvested with 0.125% trypsin‐0.01% EDTA. All experiments were carried out using fibroblasts after several passages following primary culture. To prepare fibroblast‐embedded collagen gel, a collagen solution was prepared by mixing acid‐soluble porcine type I collagen (3 mg mL‐1), five‐fold concentrated DMEM and a buffer solution (0.05 N NaOH, 2.2% NaHCO3, 200 mM HEPES) (all from Nitta Gelatin Corp., Osaka, Japan) in the ratio of 7 : 2 : 1, respectively. A mixture of cell suspension in serum‐free DMEM and collagen solution was divided into 12‐well clusters each with a volume of 1 mL (Costar Corp., Cambridge, MA, U.S.A.), and then gelled at 37 °C for 30 min (final 1.0 × 105 cells mL‐1 and 2.1 mg mL‐1 collagen). Serum‐free DMEM (1 mL well‐1) was then poured on the gel to prevent the surface from drying up. After 12 h incubation, the gel was separated and floated from the well, and then given the various reagents. The diameter of the collagen gels during the specified incubation periods was measured using a dial caliper, and the surface area of each gel was calculated. The contraction of the gel was expressed as a percentage against the surface area of the un‐contracted state, defined at 100%.

Infection of recombinant adenoviruses was performed at a multiplicity of infection of 1 × 10 [2] plaque‐forming units per cell in 1 mL of serum‐free DMEM for 60 min. Dermal fibroblasts infected with the adenovirus solutions were cultured in DMEM supplemented with 10% FCS for 24 h and thereafter (usually 48–72 h after the infection) used for type I collagen gel contraction assay.

Results and discussion

Collagen gel contraction by fibroblasts has been ascribed to the contraction of actin filaments, which generate the cumulative traction force exerted by fibroblasts on collagen fibrils [2]. In the presence of serum, the degree of collagen gel contraction was found to be influenced by cell spreading, cell movement and reorganization of the collagen gels [4]. To avoid the influence of serum and cell‐spreading in collagen gel, we carried out our experiments in a serum‐free medium with 12 h pre‐incubation.

Neither IGF‐I nor IGFBP‐1, individually, produced any gel contraction except for 30 ng mL‐1 IGF‐I which produced a 6.8% gel contraction. However, a combination of IGF‐I and IGFBP‐1 at 1 ng mL‐1 of each reagent, a concentration at which gel contraction had not been observed when each reagent was tested individually, produced 14% gel contraction. The increased IGFBP‐I doses in combination with 1 ng mL‐1 IGF‐I tended to enhance gel contraction [2]. When either IGF‐I and anti‐IGF‐I antibody or IGFBP‐1 and anti‐IGFBP‐1 antibody were incubated prior to their addition to the assay medium, gel contraction induced by IGF‐I and IGFBP‐1 almost disappeared. The promoting effect exerted by the combination of IGF‐I and IGFBP‐1 was found to be especially significant during the initial 6 h incubation period, suggesting that collagen gel contraction occurred because of a quick response of the cytoskeleton through receptor‐mediated signal transduction [2].

Among the cytokines, TGF‐β and PDGF stimulated gel contraction, while bFGF, TGF‐β, IGF‐I, and IL‐1α failed to produce any affect. The gel contraction induced by PDGF was enhanced by the addition of TNF‐α, and then suppressed by further treatment with genistein [4]. The effect of conditioned media from keratinocytes and fibroblasts on gel contraction was then examined. The results indicated that only the conditioned media from keratinocytes showed a stimulatory effect on gel contraction. The contraction stimulant was later identified to be endothelin (ET)‐1 through the suppression of gel contraction by the addition of bosentan, an ET antagonist [4]. Recombinant ET‐1 showed potent gel contractile activity at the level of 1 pM or more. This effect was further stimulated by the addition of IGF‐I.

Among the sphingolipid metabolites, SPC and sphingosine‐1‐phosphate, but not sphingosine, C2‐ceramide and C6‐ceramide, stimulated collagen gel contraction [5]. Maximal gel contraction, observed at 10 μM of SPC, occurred as early as 1 h after the treatment and persisted for more than 48 h. The effect of SPC was not inhibited by pre‐treatment with anti‐TGF‐β or anti‐PDGF‐BB antibodies. Among the various signal transduction inhibitors, pertussis toxin, staurosporine and H7 were found to inhibit the action of SPC, whereas genistein and tyrphostin A47 were not, suggesting that fibroblast contraction induced by SPC is mediated by a trimeric GTP‐binding protein (G protein)‐coupled receptor and protein kinase [5]. Our findings imply that the effect of SPC as a healing stimulant might be due in part to the stimulation of fibroblast contraction in granulation tissue.

Adenoviruses carrying flag‐tagged Smad3, Smad7 or control lacZ cDNA were used for the introduction of the cDNAs into dermal fibroblasts. Transfection efficiency was at least over 90%, as judged by X‐gal staining of the fibroblasts at 48 h after infection with adenoviruses carrying control lacZ cDNA [6]. Expression of exogenous Smad3 or Smad7 was confirmed by Western blotting with anti‐Flag antibody at 72 h post infection. Over‐expression of Smad3, a major signal transducer in the Smad family, enhanced collagen gel contraction by fibroblasts when compared with fibroblasts over‐expressing a control lacZ. The addition of a very low concentration of TGF‐β1, which did not affect collagen gel contraction by itself, enhanced the contraction by fibroblasts by over‐expressing Smad3 [6]. In contrast, TGF‐β1‐mediated collagen gel contraction in fibroblasts was suppressed by over‐expression of Smad7, a major inhibitory regulator in the Smad family [6]. Modulation of Smad3 or Smad7 expression in dermal fibroblasts affected the ability of these substances to contract collagen gels, possibly by regulating TGF‐β signaling in fibroblasts.

These results suggest that fibroblast‐embedded collagen gel contraction assay is an ideal quantitative and sensitive in vitro assay for measuring the contractile activity of fibroblasts and for examining the various factors which affect the phenomenon.

References

1. Clark, R.A.F. Mechanisms of cutaneous wound repair. In: Dermatology in General Medicine, 4th edn. pp. 473–486. McGraw‐Hill, New York (1993).

2. 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. USA76, 1274–1278 (1979).

3. Lee, Y.‐R., Oshita, Y., Tsuboi, R. and Ogawa, H. Combination of insulin‐like growth factor (IGF)‐I and IGF‐binding protein‐1 promotes fibroblast‐embedded collagen gel contraction. Endocrinology137, 1–6 (1996).

4. Oshita, Y., Lee, Y.R. and Tsuboi, R. The effects of various factors on fibroblast‐embedded collagen gel contraction. Jpn J. Dermatol.108, 217–224 (1998).

5. Suhr, K.B., Tsuboi R, Ogawa H. Sphingosylphoshorylcholine stimulates contraction of fibroblast‐embedded collagen gel. Br. J. Dermatol.143, 66–71 (2000).

6. Sumiyoshi, K., Nakao, A., Setoguchi, Y., Okumura, K., Tsuboi, R. and Ogawa, H. Smads regulate collagen gel contraction by human dermal fibroblasts. Br. J. Dermatol.149, 464–470 (2003).