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Chronic Wound Healing with CollagenCollagen and Wound Healing -
After induction, all mice received preemptive analgesia buprenorphine, 0. Two full-thickness wounds through the panniculus carnosus were created on either side of the spine on the midback of mice using a 6-mm dermal punch Miltex and then covered with a sterile occlusive dressing Tegaderm; 3M, St.
Paul, Minn. Full-thickness wounds were also created over the crown of the skull using a 6-mm punch in 4 of the 10 aged mice of each genotype and covered with Tegaderm as described above. Wounds were photographed with an in-picture ruler for scale using a digital camera at the times indicated.
The images were imported into an image analysis software SigmaScan Pro; SPSS Science, Chicago, Ill. The average WA for each mouse was utilized for statistical analysis. Bisected wounds were paraffin embedded and processed as described previously [Volk et al.
Louis, Mo. To assess wound healing quantitatively, the epithelial gap EG , RE, and GT area were measured at 7 days using computer-assisted morphometric analysis SpotAdvanced Image Analysis Software 4. Two observers S. and E. As previously described [Badillo et al. RE was calculated by adding together the lengths of the newly formed epithelium from the lateral edge of the hyperproliferative epithelium lacking hair follicles and the epithelial tongue.
GT area was defined as the cellular area from the lateral transition zone from the normal epidermis to the hypertrophic epidermis, inferior to the level of the panniculus carnosus and superior to the epithelial basement membrane or open wound surface.
For indirect immunohistochemistry, evaluation of 5-µm serial sections from formalin-fixed paraffin-embedded tissues were mounted on negatively charged glass slides Superfrost Plus; Fisher Scientific, Pittsburgh, Pa. Slides with tissue sections were heated in a 60°C oven for 1 h to help the adherence of paraffin-embedded tissue sections to slides.
Deparaffinization and rehydration of the slides was obtained in several changes of PRO PAR Clearant Anatech Ltd. Endogenous peroxidase activity was inactivated by the application of hydrogen peroxide to tissue sections for 10 min at room temperature. The slides were incubated with the primary antibody directed against alpha-smooth muscle actin α-SMA; dilution; Abcam for 30 min at room temperature or the universal negative control antibody FLEX Dako North America, Inc.
The tissue sections were counterstained with hematoxylin for 1 min followed by progressive alcohol dehydration and coverslipping.
The utilization of dermal fibroblasts obtained from fetal mice E Skin from freshly harvested embryos was placed dermal side down and allowed to adhere to culture wells, allowing fibroblast outgrowth.
The genotype from harvested embryos was determined as described above. Fibroblasts were harvested from cultures using 0. Cells were replated at a density of 0. Cells were used prior to passage 8 for all experiments. To correlate the effect of Col3 expression with the contractile potential of fibroblasts, dermal fibroblasts were grown in attached collagen type I lattices [Grinnell et al.
Briefly, fibroblast-populated collagen lattices FPCLs were created by suspending fibroblasts in a neutralized type I collagen solution BD Biosciences; Bedford, Mass.
The final concentration of the collagen was 1. The resuspended fibroblasts, at a final concentration of 0.
The complete medium was carefully added to wells and replaced on day 2 or 3 in a manner so as to avoid gel detachment. All experiments were performed in triplicate using cells isolated from at least 3 individual embryos of each genotype. On day 5, gels were released and photographed after 30, 60, , and min.
The percent of gel contraction was determined by calculating the lattice area at the specified time relative to that prior to release. Cells were washed in PBS followed by 10 min in ice-cold methanol and then washed in PBS. To examine α-SMA-positive stress fibers, fixed cells within the collagen lattices were incubated overnight at room temperature with a polyclonal antibody directed against α-SMA Abcam; on a rocking platform.
To permit visualization of the antibody-antigen complex, FPCLs were incubated with an Alexa Fluor donkey anti-rabbit antibody Invitrogen, Carlsbad, Calif. Stained FPCLs were placed on glass slides and a coverslip was placed on top.
All slides were viewed with an Olympus Fluorescent microscope with appropriate filters and digital photographs obtained using a constant exposure threshold. The percentage of α-SMA-positive cells within gels was quantitated as previously described [Vaughan et al.
Values are expressed as means ± standard deviation SD in the text and figures unless otherwise stated. Study groups were compared utilizing SigmaPlot software Systat, Inc.
Nonparametric analyses utilized the Shapiro-Wilk test for normality and the Mann-Whitney rank sum test. The aged group of mice was examined due to the fact that human vascular EDS patients are often not symptomatic and remain undiagnosed until the mean age of 28 years [Oderich et al.
Wound healing was examined both grossly and histologically on day 7 postwounding. Digital photographs of the wounds were taken prior to tissue harvesting and the WA was calculated. The day 7 head WA in Col3-deficient heterozygous mice was also decreased compared to that in wild-type mice Each bar represents the mean ± SEM.
Wounds of a representative pair of mice from each genotype are shown. The original wound size was not significantly different between mice of the 2 genotypes data not shown. Significant differences in the percentage of wound closure were found between the 2 genotypes from 4 to 10 days postwounding, with the greatest differences between days 4 and 8 fig.
All mice in both groups had complete closure of 1 or both wounds by day Col3-deficient mice exhibit accelerated wound closure. Wound healing was expressed as a percent change in the WA relative to the original wound size mean ± SD. To assess the contribution of RE and wound contraction to wound closure in aged wild-type and Col3 heterozygous mice, histomorphometric analysis was performed.
The percentage of wound closure due to contraction, assessed histologically, was found to be increased more than 3-fold in Col3 heterozygous mice compared to wild-type mice fig.
Although the mean EG in the day 7 wounds of Col3-deficient heterozygotes was less than half of that found in control wounds, the difference between the 2 genotypes was not statistically significant table 1. The histomorphometric data show that increased wound closure in Col3-deficient mice is due to wound contraction rather than the formation of new tissue neoepithelialization.
Wounds were harvested at 7 days postoperatively. Arrows indicate the lateral margins of hyperproliferative epithelium lateral edge of the reepithelialized wound. The scale bar beneath the histologic sections is equal to the original wound diameter of 6 mm.
b Quantitative comparison of the percentage of wound closure due to contraction mean ± SD. To elucidate a cellular mechanism by which Col3 deficiency leads to an increase in wound contraction, we investigated whether differences in Col3 expression in the wound bed environment modulated myofibroblast differentiation.
Myofibroblasts are known to contribute to GT contraction and are identified by their expression of the procontractile protein α-SMA. To examine the effect of Col3 deficiency on α-SMA expression in vivo, immunostaining for α-SMA was performed in histological sections of day 7 wounds.
Wounds in aged Col3-deficient mice show increased wound contraction and GT α-SMA expression. Corresponding negative controls from adjacent serial sections are shown e and f , respectively. Dermal fibroblasts from Col3 mice were utilized in a stressed FPCL assay, an established model of in vitro wound contraction [Mochitate et al.
In comparison to free-floating FPCL assays, stressed FPCL assays more specifically investigate the myofibroblast activity of the seeded cells [Mochitate et al.
Dermal fibroblasts were isolated from E Wild-type and Col3-deficient fibroblasts were grown in attached collagen gels for 5 days. Stressed lattices were then released and the percentage of contraction calculated at 30, 60, , and min post-release.
Values represent means ± SEM for individual cell isolates from at least 3 embryos for each genotype with 3 lattices per individual cell isolate.
To examine whether the increased contraction in gels seeded with Col3-deficient fibroblasts was associated with increased myofibroblast differentiation, FPCLs were fixed and immunostaining for α-SMA was performed.
Incorporation of α-SMA into stress fibers was identified within cells of all 3 genotypes fig. These cell culture data support our in vivo results described above fig.
Cells from all 3 genotypes, utilized in the attached fibroblast-populated collagen gel assay, were immunostained for α-SMA green and counterstained with DAPI blue. Collagen lattices seeded with cell isolates from at least 3 embryos per genotype were analyzed.
a Myofibroblasts within collagen lattices were identified by α-SMA expression. α-SMA incorporation into stress fibers arrows could be visualized in cells from all 3 genotypes.
e Graphic representation of the percentage of α-SMA-positive cells quantitated as described in Materials and Methods, expressed as means ± SD. Col3 is often cited as an important ECM component of healing tissues, particularly cutaneous wounds, due to increased expression during the early repair phase of a variety of tissues [Merkel et al.
However, its role in tissue repair has yet to be defined. Liu et al. However, additional roles for Col3 in tissue maintenance and its specific role in repair have yet to be defined. Gross examination of healing wounds indicated that Col3 deficiency in aged mice accelerated wound closure fig.
Histomorphometric analysis confirmed that differences in wound closure between Col3-deficient mice and controls were due to an increase in wound contraction fig. Because differences in wound size and closure were found to be most significant between days 4 and 8, a time characterized by myofibroblast-mediated wound contraction, we examined the hypothesis that Col3 deficiency would promote myofibroblast differentiation.
Both the in vitro and in vivo experiments presented here support this hypothesis fig. Mutations in the Col3 gene lead to an inherited connective tissue disorder, the vascular form of EDS.
The murine model of Col3 deficiency utilized here shares phenotypic overlap with vascular EDS patients with respect to the development of vascular lesions in aged haploinsufficient individuals [Cooper et al.
Cutaneous wounds in vascular EDS patients have been described to heal with increased scar formation [Burk et al. The synergistic detrimental effects of advancing age and Col3 deficiency appear to be important in the development of pathology, as vascular EDS in human patients is often not diagnosed until the third decade of life [Oderich et al.
The number and cumulative severity of vascular lesions in haploinsufficient mice has also been found to correlate with age [Cooper et al.
It may be that at a young age wounds heal with such efficiency that differences cannot be detected with the available methodology. Histomorphometric analysis confirmed that this difference in healing was a result of increased wound contraction fig.
The key cellular mediators of wound contraction are myofibroblasts, identified by their de novo expression of the procontractile protein α-SMA in stress fibers [Arora and McCulloch, ; Hinz et al. The density of myofibroblasts in both GT and collagen gels has been associated with increased contraction in vivo and in vitro, respectively [McGrath and Hundah, ; Rungger-Brandle and Gabbiani, ; Hinz et al.
These data may seem to be in conflict with data by Ehrlich et al. However, our studies have used the stressed FPLC model rather than the free-floating FPLC model used previously and therefore may highlight differences in the ability of Col3 to regulate cellular contraction versus fibroblast migration and reorganization.
Secondly, differences in the biomechanical properties of lattices comprised of Col1 versus Col3, and therefore the ability of the fibroblasts to contract them, may further explain the differences seen in the contraction of matrices by fibroblasts in these 2 studies.
The persistence of myofibroblasts within wound tissue has been associated with increased scar tissue formation [Desmouliere et al. It is now widely accepted that ECM components contribute to tissue repair through the modulation of cell behavior beyond their ability to provide a structural framework for cells.
Previous mechanisms by which ECM has been shown to alter cell activity and fate include their ability to modulate integrin expression, regulate growth factor expression and bioavailability, and control matrix metalloproteinase production and activation. Although the detailed mechanism by which Col3 modulates myofibroblast differentiation is currently under investigation, data by Zoppi et al.
Fibroblasts isolated from individuals with vascular EDS express increased αv and decreased α5β1 and α2β1 integrins compared to those isolated from individuals unaffected by EDS. Both the αvβ3 receptor vitronectin receptor and the α5β1 receptor fibronectin receptor have been shown to be important in myofibroblast differentiation [Lygoe et al.
Differential integrin expression may alter the ability of fibroblasts to interact with the provisional matrix in an early wound and promote the differentiation of protomyofibroblasts to myofibroblasts.
Another potential mechanism currently under investigation is suggested by the phenotypic overlap of vascular EDS with Marfan syndrome, which has been shown to result from an increase in TGFβ bioavailability secondary to mutations in the ECM protein fibrillin [Neptune et al.
The N-propeptides of both type I and II collagen, which show close sequence homology with that of Col3, have previously been shown to modulate TGFβ family member signaling [Zhu et al.
Expression of TGFβ1 has been shown to correlate with a scarring phenotype [Lee et al. Furthermore, the addition of TGFβ1 to fetal wounds induces scar formation in a typically scar-free environment and inhibition of TGFβ1 and 2 reduces scar formation [Lin et al. Conversely, TGFβ3 is capable of diminishing scar formation [Shah et al.
The development of therapeutic strategies to prophylactically reduce scar formation, such as Avotermin human recombinant TGFβ3 , highlights the importance of elucidating the key players in the process of scar tissue formation [Occleston et al. Due to the profound effect of Col3 on wound contraction, we focused our investigation on the ability of Col3 to regulate the myofibroblast phenotype.
The effect of Col3 on other cell types within the wound is currently under investigation. Although this suggests that a Col3-rich environment positively influences RE and supports our hypothesis that Col3 provides an optimal regenerative niche for progenitor and other reparative cells, data must be interpreted cautiously in this model.
Studies which limit wound contraction, by stenting the surrounding skin, are under investigation and should address this question. Here we have described a role for Col3 in modulating myofibroblast differentiation and activities during cutaneous repair and have established a cellular mechanism for an increase in scar formation in wounded Col3-deficient skin.
Although previous work has identified the role of Col3 in maintaining the structural integrity of tissues, we have shown a novel role for Col3 in the early reparative niche of healing tissues. Our data support our overall hypothesis that healing in Col3-deficient environments occurs via a reduced regenerative response and provide support for the further investigation of Col3-rich biomaterials to improve tissue regeneration.
This work is supported by a grant from NIAMS S. We thank Keith Alcorn and Jason Combs for mouse colony care and maintenance. We also thank Antoneta Radu, Patrice Costello, Juliana Burns, and Jackie Ferracone for their technical assistance with histopathology. Sign In or Create an Account.
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Research Articles January 19 Diminished Type III Collagen Promotes Myofibroblast Differentiation and Increases Scar Deposition in Cutaneous Wound Healing Subject Area: Further Areas , Pathology and Cell Biology.
Volk ; Susan W. Departments of a Clinical Studies and Animal Biology and b Pathobiology, School of Veterinary Medicine, and c Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pa. This Site. Google Scholar. Yanjian Wang ; Yanjian Wang. Elizabeth A.
Mauldin ; Elizabeth A. Kenneth W. Liechty ; Kenneth W. Sherrill L. Adams Sherrill L. Cells Tissues Organs 1 : 25— Article history Accepted:. Cite Icon Cite. toolbar search Search Dropdown Menu. toolbar search search input Search input auto suggest. View large Download slide.
Table 1 Analysis of wound EG, RE, and GT area. View large. View Large. Arora, P. McCulloch Dependence of collagen remodelling on alpha-smooth muscle actin expression by fibroblasts. J Cell Physiol — Badillo, A. Redden, L. Zhang, E. Doolin, K. Liechty Treatment of diabetic wounds with fetal murine mesenchymal stromal cells enhances wound closure.
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McGillivray, P. MacLeod, R. Lowry Clinical and ultrastructural heterogeneity of type IV Ehlers-Danlos syndrome.
Hum Genet — Cass, D. Sylvester, E. Yang, T. Crombleholme, N. Adzick Myofibroblast persistence in fetal sheep wounds is associated with scar formation. J Pediatr Surg — Cook, H. Davies, K. Harding, D.
Thomas Defective extracellular matrix reorganization by chronic wound fibroblasts is associated with alterations in TIMP-1, TIMP-2, and MMP-2 activity.
J Invest Dermatol — Cooper, T. Zhong, M. Krawczyk, H. Tae, G. Müller, R. Schubert, L. Myers, H. Dietz, M. Talan, W. Briest The haploinsufficient Col3a1 mouse as a model for vascular Ehlers-Danlos syndrome. Vet Pathol — Cuttle, L.
Nataatmadja, J. Fraser, M. Kempf, R. Kimble, M. Hayes Collagen in the scarless fetal skin wound: detection with Picrosirius-polarization. Wound Repair Regen — Desmouliere, A. Geinoz, F. Gabbiani, G. Gabbiani Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in GT myofibroblasts and in quiescent and growing cultured fibroblasts.
J Cell Biol — Redard, I. Darby, G. Gabbiani Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am J Pathol 56— Ehrlich, H. Tissue Cell 47— J Pathol — Germain, D. Orphanet J Rare Dis 2: 32— Goldberg, S. Quirk, V.
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Ho, Y. Lin, G. Skuta a Differences in the regulation of fibroblast contraction of floating versus stressed collagen matrices. J Biol Chem 2: — Zhu, M. Carlson, J. Abrams b Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue.
Exp Cell Res — Hinz, B. Celetta, J. Tomasek, G. Gabbiani, C. Chaponnier Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell — Gabbiani Mechanisms of force generation and transmission by myofibroblasts. Curr Opin Biotechnol Hurme, T. Kalimo, M.
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There were no significant differences in weight, body mass index, dietary energy and protein intakes between the two groups.
Keywords: Burn; Collagen; Hospital stay; Pre-albumin; Wound healing. Copyright © Elsevier Ltd and ISBI. All rights reserved. Abstract Introduction: Burn is among the most severe forms of critical illness, associated with extensive and prolonged physical, metabolic and mental disorders.
Publication types Randomized Controlled Trial. Substances Dietary Sugars Prealbumin Gelatin Collagen.
WWound W. CollabenEnhance cognitive capabilities Collagen and Wound HealingElizabeth A. MauldinKenneth W. LiechtySherrill L. Adams; Diminished Type III Collagen Promotes Myofibroblast Differentiation and Increases Scar Deposition in Cutaneous Wound Healing. Cells Tissues Organs 1 June ; 1 : 25—
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