Fibroblast phenotypes and their relevance for wound healing.
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Fibroblasts↗
Biomedical subjects
Esther Middelkoop
Publications and source records attributed to Esther Middelkoop.
Differential expression of CRABP-II in fibroblasts derived from dermis and subcutaneous fat.
We have shown previously that fibroblasts derived from fat or dermal tissue differ in their functional properties, such as proliferation rate and contractile properties. To study these differences further, two-dimensional electrophoresis (2D PAGE) was performed on proteins isolated from cultured subcutaneous fat and dermal fibroblasts. The 2D gels were screened for proteins that were differentially expressed in all donors (n = 5). Five protein spots were subjected to further analysis by mass spectrometry. Two proteins could be identified: brain acid soluble protein 1 (BASP1) and cellular retinoic acid binding protein-II (CRABP-II). CRABP-II is of interest in terms of re-epithelialisation and was clearly expressed in dermal fibroblasts but not in fat fibroblasts. Real time PCR was performed to confirm the 2D data on CRABP-II. The CRABP-II mRNA level was significantly increased in dermal tissue and cultured dermal fibroblasts compared to fat tissue and cultured fat-derived fibroblasts, respectively. The mode of action of CRABP-II in skin is to mediate retinoic acid activity. Retinoic acid is known to inhibit migration and to stimulate differentiation of keratinocytes. The expression of CRABP-II by dermal fibroblasts implicates a role for these fibroblasts in wound re-epithelialisation, in contrast to subcutaneous fat-derived fibroblasts.
Adipose Tissue↗
Skin elasticity meter or subjective evaluation in scars: a reliability assessment.
UNLABELLED: Various methods are available for evaluating the elasticity of scars. However, the reliability and validity of these methods have been sparsely examined. The aim of this study was to examine the reliability of the subjective evaluation of scar pliability, while at the same time testing the reliability of the measurements of a non-invasive suction device (Cutometer Skin Elasticity Meter 575) on scars. Four observers assessed 49 scar areas of 20 patients with a subjective assessment of pliability. Subsequently, each observer measured the scar areas with the Cutometer. The intraclass correlation coefficients (ICC) of the elasticity (Ue) and extension (Uf) parameters of the Cutometer were acceptable (r = 0.76 and 0.74, respectively) when a single observer carried out the measurements. The subjective assessment of pliability needs to be completed by two or more observers to make the evaluation reliable (r = 0.79). The concurrent validities between the subjective pliability-assessment and each of the Cutometer parameters were statistically significant and ranged from r = 0.29-0.53. The correlations between each of the Cutometer parameters were high and statistically significant (r > or = 0.71). CONCLUSION: A single observer can reliably use the Cutometer for the elasticity measurements of scars. Furthermore, either Ue or Uf, instead of all five elasticity values provided by the Cutometer, can be adequately used for the elasticity measurements of scars. The subjective assessment of pliability of scars can only be assessed reliably when completed by two or more observers. The concurrent validity showed that all Cutometer parameters, except for visco-elasticity (Uv), and the subjective assessment of pliability measured the same characteristic of a scar.
Adolescent↗
Colour evaluation in scars: tristimulus colorimeter, narrow-band simple reflectance meter or subjective evaluation?
The evaluation of scar colour is, at present, usually limited to an assessment according to a scar assessment scale. Although useful, these assessment scales only evaluate subjectively the degree of scar colour. In this study, the reliability of the subjective assessment of scar colour by observers is compared to the reliability of the measurements of two objective colour measurement instruments. Four independent observers subjectively assessed the vascularisation and pigmentation of 49 scar areas in 20 patients. The degree of vascularisation and pigmentation was scored according to a scale ranging from '1', when it appeared to be like healthy skin, to '10', which corresponds to the worst imaginable outcome of vascularisation or pigmentation. The observers also scored the pigmentation categories of the scar (hypopigmention, hyperpigmention or mixed pigmentation). Finally, each observer measured the scar areas with a tristimulus colorimeter (Minolta Chromameter) and a narrow-band simple reflectance meter (DermaSpectrometer). A single observer could reliably carry out measurements of the DermaSpectrometer and the Minolta Chromameter for the evaluation of scar colour (r = 0.72). The vascularisation of scars could also be assessed reliably with a single observer (r = 0.76) whereas for a reliable assessment of pigmentation at least three observers were necessary (r > or = 0.77). The agreement between the observers for the pigmentation categories also turned out to be unacceptably low (k = 0.349). This study shows that an overall evaluation of scar colour with the DermaSpectrometer and the Minolta Chromameter is more reliable than the evaluation of scar colour with observers. Of both instruments for measuring scar colour, we prefer, because of its feasibility, the DermaSpectrometer.
Adolescent↗
Increased formation of pyridinoline cross-links due to higher telopeptide lysyl hydroxylase levels is a general fibrotic phenomenon.
Fibrosis is characterized by an excessive accumulation of collagen which contains increased levels of pyridinoline cross-links. The occurrence of pyridinolines in the matrix is an important criterion in assessing the irreversibility of fibrosis, which suggests that collagen containing pyridinoline cross-links significantly contributes to the unwanted collagen accumulation. Pyridinoline cross-links are derived from hydroxylated lysine residues located within the collagen telopeptides (hydroxyallysine pathway). Here, we have investigated whether the increase in hydroxyallysine-derived cross-links in fibrotic conditions can be ascribed to an increased expression of one of the lysyl hydroxylases (LH1, LH2 with its splice variants LH2a and LH2b, or LH3) and/or to an increased expression of lysyl oxidase (LOX). In fibroblast cultures of hypertrophic scars, keloid and palmar fascia of Dupuytren's patients, as well as in activated hepatic stellate cells, increased levels of LH2b mRNA expression were observed. Only minor amounts of LH2a were present. In addition, no consistent increase in the mRNA expression levels of LH1, LH3 and LOX could be detected, suggesting that LH2b is responsible for the overhydroxylation of the collagen telopeptides and the concomitant formation of pyridinolines as found in the collagen matrix deposited in long-term cultures by the same fibrotic cells. This is consistent with our previous observation that LH2b is a telopeptide lysyl hydroxylase. We conclude that the increased expression of LH2b, leading to the increased formation of pyridinoline cross-links, is present in a wide variety of fibrotic disorders and thus represents a general fibrotic phenomenon.
Amino Acids↗
The patient and observer scar assessment scale: a reliable and feasible tool for scar evaluation.
At present, various scar assessment scales are available, but not one has been shown to be reliable, consistent, feasible, and valid at the same time. Furthermore, the existing scar assessment scales appear to attach little weight to the opinion of the patient. The newly developed Patient and Observer Scar Assessment Scale consists of two numeric scales: the Patient Scar Assessment Scale (patient scale) and the Observer Scar Assessment Scale (observer scale). The patient and observer scales have to be completed by the patient and the observer, respectively. The patient scale's consistency and the observer scale's consistency, reliability, and feasibility were tested. For the Vancouver Scar Scale, which is the most frequently used scar assessment scale at present, the same statistical measurements were examined and the results of the observer scale and the Vancouver scale were compared. The concurrent validity of the observer scale was tested with a correlation to the Vancouver scale. Furthermore, the authors examined which specific characteristics significantly influence the general opinion of the patient and the observers on the scar areas. Four independent observers have each used the observer scale and the Vancouver scale to assess 49 burn scar areas of 3 x 3 cm belonging to 20 different patients. Subsequently, the patients completed the patient scale for their scar areas. The (internal) consistency of both the patient and the observer scales was acceptable (Cronbach's alpha, 0.76 and 0.69, respectively), whereas the consistency of the Vancouver scale appeared not to be acceptable (alpha, 0.49). The reliability of the observer scale completed by a single observer was acceptable (r = 0.73). The reliability of the Vancouver scale completed by a single observer was lower (r = 0.69). The observer scale showed better agreement than the Vancouver scale because the coefficient of variation was lower (18 percent and 22 percent, respectively). The concurrent validity of the observer scale in relation to the Vancouver scale is high (r = 0.89, p < 0.001). Linear regression of the general opinions on scars of the observer and the patient showed that the observer's opinion is influenced by vascularization, thickness, pigmentation, and relief, whereas the patient's opinion is mainly influenced by itching and the thickness of the scar. Such an impact of itching and thickness of the scar on the patient's opinion is an important and novel finding. The Patient and Observer Scar Assessment Scale offers a suitable, reliable, and complete scar evaluation tool.
Adolescent↗
Reliability and accuracy of techniques for surface area measurements of wounds and scars.
The applicability of simple methods to measure the size of pathological skin lesions for management and research has been poorly studied to date. The interobserver reliability and accuracy (validity) was established for planimetry by photography and planimetry by tracing on a transparent sheet in this study. Drawings of 25, 50, and 75 cm(2) were created on 3 locations with increasing curvature (back, thigh, and forearm) in 20 healthy volunteers. Three investigators evaluated the drawings by both planimetry techniques. Both techniques showed a good reliability (r >or= 0.82, intraclass correlation) for 25 cm(2) areas. Planimetry by photography was more reliable than planimetry by tracings for the 50 -and 75-cm(2) areas and was more accurate than planimetry by tracing for all areas except for the area with the greatest curvature, the forearm. The study permits the conclusion that planimetry by photography is more suitable for surface area measurements than planimetry by tracing except for extremely curved body parts, where it is likely that effects of distortion supervene.
Journal Article↗
Collagen morphology in human skin and scar tissue: no adaptations in response to mechanical loading at joints.
Dermal collagen displays a random-like structure that has a major role in strength and function of the human integument. It is hypothesised that collagen bundles align in a parallel fashion in the direction of mechanical tension during scarring, which may explain the problematic scar formation that occurs specifically at joints. Scar tissue and normal skin were biopsied from joints and control areas and evaluated by the Fourier analysis. Collagen orientation was represented by an index ranging from 0 (perfectly random) to 1 (perfectly parallel). Collagen bundle packing signifies the average distance between the centres of collagen bundles. No differences were shown in collagen morphology of scar tissue and normal skin between joints and control areas. Normal skin had a significantly lower collagen orientation index than scar tissue (0.26 versus 0.44, P<0.001). The bundle packing of scar tissue differed significantly from normal skin (18.1 microm versus 23.7 microm, P<0.001). Collagen appeared less parallel orientated in deep dermis compared to superficial dermis especially for normal skin (0.27 versus 0.33, P=0.06). Normal skin had a less parallel organisation in sections that were cut parallel compared to those that were cut perpendicular to the epidermis (0.24 versus 0.30, P=0.02). Collagen orientation of scar tissue is more parallel compared to normal skin. Morphology differs with respect to superficial and deep dermal layers and parallel and perpendicular planes, but appears not to respond to mechanical tension.
Adult↗
Morphometry of dermal collagen orientation by Fourier analysis is superior to multi-observer assessment.
In human dermis, collagen bundle architecture appears randomly organized, whereas in pathological conditions, such as scar tissue and connective tissue disorders, collagen bundle architecture is arranged in a more parallel fashion. Histological examination by one or two observers using polarized light is the most common method to determine collagen orientation. The hypothesis on which this study is based is that an objective image analysis technique, Fourier analysis, would improve the reliability (are the measurements reproducible?) and the accuracy (does the method measure what it is supposed to measure?) of collagen orientation assessment, compared with observer ratings. Fourier analysis was applied to 271 images of scar tissue and normal skin that were acquired by confocal laser-scanning microscopy. Observers rated the same areas using polarized light as well as the confocal microscopy images. Computer images consisting of different types of ellipses were generated with a fixed orientation. Observers and Fourier analysis evaluated the images to evaluate accuracy. The inter-observer reliability was acceptable when at least three observers rated polarized light images (r > 0.69), whereas two observers were sufficient for rating confocal microscopy images (r > 0.71). Fourier analysis correlated better with observer ratings of confocal microscopy images (r = 0.69) than with polarized light microscopy images (r = 0.42). Fourier analysis was more accurate than four observers for the evaluation of the 'true' orientation for almost all types of computer-generated images. For the first time it is shown that Fourier image analysis is suitable for the morphometry of dermal collagen orientation and leads to a superior measurement of collagen orientation compared with subjective histological evaluation by several experts. If an evaluation is performed by conventional light microscopy, at least three observers are required to attain an acceptable inter-observer reliability.
Adult↗
Long-term results of a clinical trial on dermal substitution. A light microscopy and Fourier analysis based evaluation.
Although dermal substitution is a main topic of current wound healing research, there is a paucity of clinical trials with a long-term clinical and histopathological evaluation. A clinical trial was conducted to perform an intra-individual comparison of conventional treatment (split-thickness autograft) to a collagen/elastin dermal substitute in combination with an autograft. Promising results with this substitute were obtained with respect to dermal organisation and scar elasticity in animal studies and clinical trials with a short-term follow-up. Twenty-nine of the 42 pairs of the burn wounds and 28 of the 44 pairs of the scar reconstructions enrolled in the study were biopsied after 1 year. Promising but not statistically significant differences were found between substituted groups and control groups for epidermal thickness, basement membrane maturation, rete ridges (P=0.055), fibroblasts, myofibroblasts, inflammatory cells, vessels and extracellular matrix maturation. An objective and accurate technique, Fourier analysis, was used to evaluate collagen bundle orientation and packing. However, no statistically significant differences were obtained for these parameters. This microscopic evaluation provided no convincing evidence for a long-term effectiveness of a dermal substitute despite promising data over a short-term in in vitro and in vivo studies with the same material.
Adult↗
Fibroblasts derived from chronic diabetic ulcers differ in their response to stimulation with EGF, IGF-I, bFGF and PDGF-AB compared to controls.
Patients with diabetes mellitus experience impaired wound healing, often resulting in chronic foot ulcers. Healing can be accelerated by application of growth factors like platelet-derived growth factor (PDGF). We investigated the mitogenic responses, measured by 3[H]thymidine incorporation, of fibroblasts cultured from diabetic ulcers, non-diabetic ulcers, and non-lesional diabetic and age-matched controls, to recombinant human PDGF-AB, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF-I). We determined the optimal concentration of these factors and investigated which single factor, or combination of factors, added simultaneously or sequentially, induced the highest mitogenic response. For single growth factor additions, in all fibroblast populations significant differences in mitogenic response to different growth factors were observed, with PDGF-AB consistently inducing the highest response and IGF-I the lowest (p < 0.043). IGF-I produced only a 1.7-fold stimulation over control in diabetic ulcer fibroblasts, versus 2.95-fold for chronic ulcer, 3.2-fold for non-lesional (p = 0.007) and 5-fold for age-matched fibroblasts (p = 0.007). The highest mitogenic response induced by EGF was significantly less for chronic ulcer fibroblasts compared with age-matched and nonlesional controls (p < 0.03), chronic ulcer fibroblasts also needed significantly more EGF to reach this optimal stimulus (p < 0.02 versus age-matched and non-lesional controls). The simultaneous addition of FGF-IGF-I, PDGF-IGF-I and FGF-PDGF to diabetic ulcer fibroblasts always produced a higher stimulatory response than sequential additions (p < or = 0.05). Also the addition of bFGF, PDGF-AB and EGF prior to IGF-I induced a higher 3[H]thymidine uptake in all fibroblasts compared to the combination of each in reverse order. Significant differences were observed when comparing the combinations of growth factors with the highest stimulatory responses (PDGF-IGF-I, FGF-PDGF and EGF-PDGF added simultaneously) to a double dose of PDGF, with the highest mean rank for the combination PDGF-IGF-I (p = 0.018). In conclusion, combinations such as PDGF-AB and IGF-I may be more useful than PDGF-AB alone for application in chronic diabetic wounds.
Cell Division↗
Scar assessment tools: implications for current research.
Scarring is considered a major medical problem that leads to cosmetic and functional sequelae. Scar tissue is clinically distinguished from normal skin by an aberrant color, rough surface texture, increased thickness (hypertrophy), occurrence of contraction, and firmness. Marked histologic differences are the change in dermal architecture and the presence of cells such as the myofibroblast. Many assessment tools are available for analysis of pathologic conditions of the skin; however, there is no general agreement as to the most appropriate tools for evaluation of scar tissue. This review critically discusses currently available objective measurement tools, subjective assessment tools, and potential devices that may be available in the future for scar assessment.
Cicatrix↗
Allogeneic fibroblasts in dermal substitutes induce inflammation and scar formation.
In the present study, we compared the use of autologous versus allogeneic fibroblasts in dermal skin substitutes in a porcine wound model. The allogeneic fibroblast populations were isolated from female and a male pig (allo-1, - 2 and - 3) and the controls, autologous fibroblasts, from female graft-recipient pigs (control). The histocompatibility of the three donor pigs with the recipient pigs was determined with a mixed lymphocyte reaction. In two pigs, full-thickness wounds were treated with the fibroblast-seeded dermal substitutes (n = 5 per animal) and immediately overgrafted with meshed split-skin autografts. After 6 weeks, wound contraction was measured by planimetry and scar formation was scored. At 2, 4, and 6 weeks biopsies were taken and evaluated for the presence of inflammatory reactions, myofibroblasts, and scar formation. The mixed lymphocyte reaction of both recipient pigs showed the highest responses on peripheral blood mononuclear cells of the allo-3 donor pig, and was low or negative for allo-1 and allo-2. In all "allogeneic" wounds, more inflammatory cells were observed over time along with inflammatory foci consisting of a mix of lymphocytes and granulomatous cells. After 4 weeks, myofibroblasts were absent in the control wounds, whereas in "allogeneic" wounds, myofibroblasts colocalized with inflammation foci. The final scar tissue of the "allogeneic" wounds showed granulating areas with thin, immature collagen bundles. In contrast, the control wounds showed a dermal tissue with mature collagen bundles organized randomly like in normal skin. The wounds treated with allo-3 fibroblasts showed in both pigs a significant increase in scar formation and wound contraction when compared with control wounds. In conclusion, for optimal restoration of dermal skin function with minimal scar formation, skin substitutes containing autologous fibroblasts are preferred over skin substitutes with allogeneic fibroblasts.
Animals↗
Impact of modification of burn center referral criteria on primary patient outcome.
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Adolescent↗
The JCR:LA-cp rat: a novel model for impaired wound healing.
JCR:LA-cp/cp obese rats and their lean controls were evaluated as a type 2 diabetic wound healing model and the healing quality was characterized. This model of insulin resistance has been used extensively to study atherosclerosis but has not previously been used to study wound healing. Six circular excisional wounds were made on the dorsum of each rat and followed to day 21. Tracings of the wounds were made and used to assess the rate of wound closure. Planimetry showed a significantly diminished contraction of wounds in obese rats, but no significant difference in reepithelialization was observed. Collagen content was determined from the hydroxyproline content in wounded and unwounded skin. There were significantly lower levels of hydroxyproline in the wounds of obese compared to lean animals at day 21. Histology showed adipose tissue in place of dermal tissue in the JCR:LA-cp/cp rat in both unwounded tissue and in the wound at day 21. Active transforming growth factor-beta 1 (TGF-beta 1) was measured in the serum using the plasminogen activator inhibitor-1/luciferase assay and serum total TGF-beta was measured using an enzyme-linked immunosorbent assay. Active TGF-beta was significantly higher in the serum of obese animals compared with lean animals, while total TGF-beta 1 was not significantly different between the groups. Both active and total TGF-beta was measured in tissue sections using the plasminogen activator inhibitor-1/luciferase assay. There was no significant difference in active TGF-beta between genotypes, while obese rats had significantly higher levels of total TGF-beta at day 21. These results indicate a deficiency in wound healing in obese animals characterized by decreased wound contraction, decreased collagen production, and changes in histology. The JCR:LA-cp rat develops insulin resistance, atherosclerosis and early type 2 diabetes and may be a good model for impairment of wound healing in humans with metabolic syndrome.
Animals↗