Fibroblasts from the Growing Margin of Keloid Scars Produce Higher Levels of Collagen I and III Compared With Intralesional and Extralesional Sites: Clinical Implications for Lesional Site-directed Therapy

F. Syed; E. Ahmadi; S.A. Iqbal; S. Singh; D.A. McGrouther; A. Bayat

Disclosures

The British Journal of Dermatology. 2011;164(1):83-96. 

In This Article

Materials and Methods

Patients and Tissue Samples

Keloid tissue biopsy samples from eight patients were included in this study (Table 1). At the time of surgical excision, the whole keloid tissue sample was carefully divided into four specific lesional sites: (i) extralesional (macroscopically like normal skin and not involving keloid, which was adjacent to the lesion in the same anatomical site), (ii) intralesional (top biopsy of the keloid), (iii) intralesional (middle or deep dermal biopsy of the centre of the keloid) and (iv) perilesional (growing margin of the periphery of the keloid) (Fig. 1). Intralesional tissue biopsies were divided into top and middle biopsies: the top biopsy included the epidermis as well as the dermis whereas the middle biopsy comprised reticular dermis alone. Perilesional site lesions refer to the periphery or growing margin of keloid scars abutting the normal skin. Only patients presenting with typical clinical features of keloid scarring (Fig. 1) who subsequently had confirmed histological diagnosis of keloid were recruited into this study. A patient was considered to have a typical keloid scar if the lesion had grown significantly beyond the boundary of the original lesion, had been present for a minimum of 1 year, had not responded to conventional treatment or had recurred following therapy such as surgery. This typical presentation is in contrast to other forms of scarring such as flat, stretched, wide spread or hypertrophic scars. Full ethical approval and consent were obtained from each subject prior to conducting the study. Complete clinical records, including detailed data on presenting history, cause, symptoms, duration, past medical history, previous treatment and follow up were obtained. The tissue biopsies from each lesional site were collected and bisected immediately into two portions: one hemibiopsy was preserved in 10% formalin buffer solution (Sigma-Aldrich, Poole, U.K.), and the other was placed in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories, Cölbe, Germany). The samples were processed for cell culture within 12 h and the tissue was processed within 24 h (Fig. S1; see Supporting Information).

Figure 1.

Schematic model of excised keloid and different lesional biopsy sites. Surgically excised tissue was divided into intralesional (top and middle sections), perilesional (growing margin of the keloid) and extralesional (normal skin adjacent to the keloid scar that was harvested as part of an elliptical excision of the lesion and was shown macroscopically and microscopically not to involve keloid disease).

Establishment of Keloid-derived and Normal Dermal Fibroblasts

The tissue was harvested carefully from different lesional sites of the keloid: extralesional, intralesional (top and middle biopsies) and perilesional site (growing margin of the keloid)[41,42] (Fig. 1) as described above. Primary human fibroblast cultures obtained from the fresh tissue biopsies (each measuring approximately 6 mm) of keloid scars were established from passage 0 (p0) to passage 3 (p3). The tissue was washed three times with phosphate-buffered saline (PBS) (PAA Laboratories) and incubated in freshly prepared Dispase II, 10 mg mL−1 (Roche, Burgess Hill, U.K.) for 3 h at 37 °C, and epidermis and fat were removed. Dermis was minced and incubated in a solution of collagenase type I, 0·5 mg mL−1 (Roche) and trypsin, 0·2 mg mL−1 (Roche) at 37 °C for 3 h. Cells were pelleted and then resuspended in complete media. The entire resuspended cell pellet, with residual tissue, was passed through a 100-μm cell strainer (BD Biosciences, Oxford, U.K.), and approximately 105 cells were transferred into T25 CellBind flasks (Nunc, Life Technologies Ltd, Wiesbaden, Germany) as p0 cells. p0 monolayer cultures were maintained in DMEM (PAA Laboratories) supplemented with 2 mmol L−1 L-glutamine, 100 U mL−1 penicillin and 100 U mL−1 streptomycin, 10% heat-inactivated FCS (PAA Laboratories) and 25 mmol L−1 HEPES (Cell Concepts, Umkirch, Germany). Cells were incubated at 37 °C in a 5% (v/v) CO2 humidified atmosphere. p0 cells were allowed to grow 90–100% confluent and then cells (104 cells mL−1) were collected by trypsinization as p0 and at the same time 105 cells from p0 were transferred to a new flask and subsequent subculture was called passage 1 (p1). Likewise, cells were collected from passages 0 to 3 from different keloid lesional sites and culture media (from T25 flasks after 90–100% confluence) from the same passages were used for all the experiments (Fig. S1; see Supporting Information). However, for in-cell Western blotting p0 cells obtained from T25 flasks were subsequently grown in Corning 96-well flat-bottom plates (Corning, NY, U.S.A.) for up to 48 h as instructed in the protocol, and hence called p1.

RNA Extraction, cDNA Synthesis and Quantitative Reverse Transcription–Polymerase Chain Reaction

Keloid tissue samples from different lesional sites and 10 000 cells from p0 to p3 (from extralesional, intralesional and perilesional sites) were collected in RNAlater (Applied Biosystems, Warrington, U.K.). Tissue samples were finely diced with a sterile scalp blade. Tissue samples and cells were placed in 2 mL round-bottom Eppendorf tubes each containing a flame-sterilized steel ballbearing and 0·5–1 mL Trizol (Invitrogen, Abingdon, U.K.). Qiagen Tissue Lyser (Qiagen, Crawley, U.K.) was used to homogenize the tissue and cells at 30 oscillations s−1 for 10 min. The homogenized cell suspension in each tube was transferred to a 1·5-mL Eppendorf tube and centrifuged at 18 000 g for 10 min. The resulting supernatant was transferred to a new Eppendorf tube, mixed well with 0·2 mL chloroform and left at room temperature for 2 min. The mixture was then spun at 18 000 g for 15 min. The upper aqueous layer was collected and mixed with an equal volume of 70% ethanol, which was then further processed with RNeasy kit (Qiagen) according to the manufacturer's instructions to extract total RNA. DNase treatment was then carried out using DNAfree kit (Ambion, Warrington, U.K.) according to the manufacturer's protocol. NanoDrop ND-1000 UV-visible spectrophotometer (Labtech International, Ringmer, U.K.) was used to estimate the total RNA concentration. RNA was normalized for all the tissue samples to 1 μg and for all the cell samples to 250 ng for the cDNA synthesis. qScript™, cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, U.S.A.) was used for cDNA synthesis. Quantitative polymerase chain reactions (PCRs) were done in real time using the LightCycler® 480 II platform (Roche). Each quantitative reverse transcription–PCR (qRT-PCR) was carried out in a final volume of 10 μL, consisting of 4 μL diluted template cDNA, 5 μL Light Cycler® 480 probes master mix (Roche Diagnostics, Burgess Hill, U.K.), 0·2 μmol L−1 of forward and reverse primer (Table S2; see Supporting Information) (Sigma-Aldrich), 1 μL probe from Universal Probe Library (Roche Diagnostics) and 0·5 μL nuclease-free water (Ambion). Each reaction was done in triplicate. White 96-well plates (Roche Diagnostics) were used for all the experiments. The qRT-PCR reactions were initiated at 95 °C for 10 min to activate the Hot Start Taq polymerase. Each of the 40 amplification cycles consisted of a 10-s denaturation step at 95 °C and a 30-s annealing and elongation step at 60 °C. The fluorescence intensity was recorded at the end of the annealing step and elongation step in each cycle. After the 40 cycles of amplification, a cooling step at 40 °C was carried out for 30 s. The gene expression levels were normalized with an average of two internal reference genes RPL32 and SDHA. PCR products were run on 1% agarose (Fisher Scientific, Loughborough, U.K.) gel in triplicate and visualized by ultraviolet radiation.

Development and Standardization of Capture Sandwich Enzyme-linked Immunosorbent Assay for Collagen I

An antibody-pair screening method was employed to select the optimum pairs of capturing and detecting antibodies for establishment of the capture sandwich ELISA to ensure a highly accurate and sensitive detection of collagen I. The microtitre plates (Nunc MaxiSorp™ flat-bottom 96-well plate; eBioscience Ltd, Hatfield, U.K.) were coated with various concentrations of antirabbit (ab59435; Abcam, Cambridge, U.K.) antibody against human collagen I in ELISA coating buffer (stock 5× concentration; AbD Serotec, Kidlington, U.K.). This polyclonal antibody served as the capture antibody, and the plates were incubated at 4 °C overnight. After three washes with PBS, the plates were blocked with 2% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS for 2 h at room temperature. Recombinant collagen I (ab7533; Abcam) was added at various concentrations and was incubated at 37 °C for 2 h, followed by washing with PBS (PAA Laboratories). The second primary antibody, mouse monoclonal human anticollagen I [(COL-1) ab6308; Abcam], was added at different concentrations and the microtitre plates were incubated at 37 °C for 2 h followed by washing with PBS. The detection antibody, goat antimouse IgG–horseradish peroxidase (HRP) (Bio-Rad, Hemel Hempstead, U.K.), was used and the plates were incubated at room temperature for 1 h. After three washes, the colour was developed using a ready to use, highly sensitive 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution (Thermo Scientific, Loughborough, U.K.) and the reaction was then stopped by 2 mol L−1 H2SO4. The optical density values were read at 450 nm using a microtitre plate reader (Fluostar Optima; BMG Labtech, Aylesbury, U.K.). Likewise, concentrations of the capture antibodies and detection antibody were optimized as described previously.[48,49] Additional methodological parameters were also optimized for sensitivity and specificity for collagen I detection. The precision and reproducibility of this assay was assessed with three different diluted positive collagen I samples, by comparing the test results from each of the 24 wells on the same microtitre plate for intra-assay variation. In addition, each of the 12 separate assays was measured for collagen I to test the same sample on different microtitre plates for interassay variation.

Enzyme-linked Immunosorbent Assay Procedure for Collagen I

Fibroblast cell growth medium and/or cells cultured up to p3 from different lesional sites of keloid biopsies were used for the measurement of collagen I. The cells were prepared by a traditional freeze/thaw method (four cycles of freezing and four cycles of thawing, each ~5 min). This technique involves freezing a cell suspension in a dry ice/ethanol bath and then thawing the material at 37 °C in a water bath. This method of lysis causes cells to swell, and ultimately break, as ice crystals form during the freezing process and then contract during thawing.[50] Ninety-six-well plates were coated with 5× coating buffer, made into a working dilution of 1× with PBS (PAA Laboratories). Rabbit polyclonal antihuman collagen I antibody (ab59435; Abcam) was added (1 : 4500 dilution) and the plates were incubated overnight at 4 °C. This antibody served as the first capture antibody. After three washes, plates were blocked with 2% BSA (Sigma-Aldrich) in PBS and the plates were incubated at room temperature for 2 h. The plates were then washed with PBS and incubated with the cell growth medium (100 μL) and/or cells (cells were lysed by freeze/thaw cycles) for 2 h, at 37 °C. The plates were then washed and a second capture antibody, mouse monoclonal antihuman collagen I [(COL-1) ab6308; Abcam], was used against the collagen at 1 : 1500 dilution. The plates were incubated at 37 °C for 2 h. Following the three washing steps with PBS, the detection antibody goat antimouse–HRP (Bio-Rad) was added at 1 : 2000 dilution. Incubation was carried out at room temperature for 2 h. After three washes, the colour was developed with TMB substrate (100 μL). The reaction was stopped with 50 μL of 2 mol L−1 H2SO4 after the desired colour was developed, prior to measuring the amount of collagen I at 450 nm in an ELISA plate reader.

Measurement of Collagen III by Indirect Enzyme-linked Immunosorbent Assay

For collagen III measurement, an indirect ELISA protocol was developed according to the manufacturer's instructions (Abcam). Briefly, recombinant collagen III protein (ab7535; Abcam) at different concentrations, cell lysates and/or media (50–100 μL), were coated with ELISA coating buffer (50 μL) and incubated overnight at 4 °C. Following three washes with PBS, capture antibody, mouse monoclonal antihuman collagen III (ab20702; Abcam) was added at 1 : 3000 dilution and incubated for 2 h at room temperature. Following the three washing steps with PBS, the detection antibody, goat antimouse–HRP, was added at 1 : 2000 dilution. Incubation was carried out at room temperature for 1 h. After three washes, the colour was developed with TMB substrate (100 μL). The reaction was stopped with 50 μL of 2 mol L−1 H2SO4 after the desired colour was developed, prior to measurement of the amount of collagen III at 450 nm in an ELISA plate reader. The sensitivity of the ELISA is 5–200 ng mL−1.

In-cell Western Blotting

p0–p3 keloid fibroblasts of all sites were grown to 95–100% confluence in T25 flasks. Before inoculating the cells in 96-well plates, the cells were starved in 0·5% serum DMEM for 16–18 h. Cells were trypsinized and counted using fluorescence-activated cell sorting (Accuri C6; Accuri Cytometers, Ann Arbor, MI, U.S.A.) and 10 000 cells were inoculated into each well of the 96-well plates (Corning) in triplicate and grown in DMEM containing 10% FCS for up to 48 h at 37 °C/5% CO2. The cells were then fixed in 4% formaldehyde for 1 h at room temperature. The wells were washed three times with PBS (150 μL per well), permeabilized with PBS/0·1% Triton X-100 (150 μL per well, three times, 5 min each), and blocked in Odyssey blocking buffer (LI-COR, Cambridge, U.K.) (150 μL per well) for 2 h at room temperature. The 96-well plates were then incubated with rabbit antihuman collagen I and mouse antihuman collagen III antibody (1 : 200 for optimal signal-to-noise ratio) in Odyssey blocking buffer (LI-COR) overnight at 4 °C (50 μL per well) and subsequently washed with PBS/0·1% Tween-20 (150 μL per well) three times. Infrared donkey antirabbit and goat antimouse IRDye 800CW secondary antibody (1 : 500) in PBS/0·5% Tween-20 were then added (50 μL per well). The plates were incubated for 1 h at room temperature, and the wells were washed with PBS/0·1% Tween-20 three times. The plates were covered with aluminium foil and imaged on an Odyssey infrared scanner (LI-COR) using the microplate 2 setting with sensitivity of 7 in the 800 nm wavelength channel. Data were acquired by using Odyssey software, exported and analysed in Excel (Microsoft, Reading, U.K.). Collagen I and III values were background subtracted from wells treated only with secondary antibody.

Haematoxylin and Eosin and Herovici Staining

Tissue specimens were fixed in formaldehyde, embedded in paraffin blocks, and sectioned to 5 μm thickness. The sections were then stained with haematoxylin and eosin (H&E) (Surgipath, Peterborough, U.K.) for histological evaluation. In 1963, Herovici[51] described a variant of a picropolychrome stain which contained three components, to stain selectively the nuclei, cytoplasm and connective tissues. Connective tissues stain both red and blue in a reproducible manner. This was confirmed by Lillie et al.[52] In 1986 Levame and Meyer,[53] using immunocytochemistry, demonstrated that the fibres which stained blue were type III collagen while the red-staining fibres were type I collagen. Herovici staining was therefore used to localize collagen I and III in all lesional sites of keloid samples. Tissue sections were first stained for nuclei with iron haematoxylin (Sigma-Aldrich) for 10 min, and washed with tap water. Subsequently, sections were submitted to van Gieson (Sigma-Aldrich) staining for 10 min, washed in 1% acetic acid for 1 min, dehydrated in graded ethanol (95% and 100%), cleared with xylene and mounted on slides.

Statistical Analysis

The collagen was measured in triplicate and the mean ± SD was calculated. The significance of the difference between the groups was analysed statistically by two-way ANOVA with repeated measures. A Tukey's post hoc analysis was used in the case of significant effects. The difference between the means for all conditions was considered statistically significant at P < 0·05. All statistical analyses were performed using the SPSS 13.0 software program (SPSS Inc., Chicago, IL, U.S.A.).

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