Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Apr;22(4):786-96.
doi: 10.1038/mt.2013.288. Epub 2014 Jan 9.

A collagen-based scaffold delivering exogenous microrna-29B to modulate extracellular matrix remodeling

Michael Monaghan  1 , Shane Browne  2 , Katja Schenke-Layland  3 , Abhay Pandit  2
Affiliations

A collagen-based scaffold delivering exogenous microrna-29B to modulate extracellular matrix remodeling

Michael Monaghan et al. Mol Ther. 2014 Apr.

Abstract

Directing appropriate extracellular matrix remodeling is a key aim of regenerative medicine strategies. Thus, antifibrotic interfering RNA (RNAi) therapy with exogenous microRNA (miR)-29B was proposed as a method to modulate extracellular matrix remodeling following cutaneous injury. It was hypothesized that delivery of miR-29B from a collagen scaffold will efficiently modulate the extracellular matrix remodeling response and reduce maladaptive remodeling such as aggressive deposition of collagen type I after injury. The release of RNA from the scaffold was assessed and its ability to silence collagen type I and collagen type III expression was evaluated in vitro. When primary fibroblasts were cultured with scaffolds doped with miR-29B, reduced levels of collagen type I and collagen type III mRNA expression were observed for up to 2 weeks of culture. When the scaffolds were applied to full thickness wounds in vivo, reduced wound contraction, improved collagen type III/I ratios and a significantly higher matrix metalloproteinase (MMP)-8: tissue inhibitor of metalloproteinase (TIMP)-1 ratio were detected when the scaffolds were functionalized with miR-29B. Furthermore, these effects were significantly influenced by the dose of miR-29B in the collagen scaffold (0.5 versus 5 μg). This study shows a potential of combining exogenous miRs with collagen scaffolds to improve extracellular matrix remodeling following injury.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Quantitative physico-chemical characteristics of RNA releasing tunable collagen scaffold. (a) Quantification of free amine groups after crosslinking with 4S-StarPEG. * indicates statistical significance between 1 mmol/l crosslinking compared with 0.25 and 0.125 mmol/l crosslinking, ** compared with all other groups as in the case of glutaraldehyde (GTA); percentage of remaining amine groups is statistically decreased when compared with all other groups. There exists an inversely proportional relationship between the concentration of 4S-StarPEG and the percentage of free amines remaining in scaffolds after crosslinking. (b) Degradation by collagenase quantified by mass remaining after 48 hours. Noncrosslinked control (0) has degraded after 48 hours and therefore no mass is represented. Glutaraldehyde (GTA) was used as a positive control. Asterisk indicates a statistically significant difference using one-way ANOVA compared with all other data sets presented in the graph (n = 3; P < 0.05). (c) Release profiles of Cy3 labeled RNA from scaffolds crosslinked with varying concentrations of 4S-StarPEG. Release of Cy3 labeled RNA was determined spectrophotometrically and extrapolating from standard curves. Data presented is the mean ± SD, n = 4. * indicates statistically significant release profiles using a 0.05 mmol/l 4S-StarPEG crosslinker concentration compared with concentrations of 1 and 0.5 mmol/l, P < 0.05.
Figure 2
Figure 2
qRT-PCR data demonstrating the effect of miR-29B in silencing collagen type I and collagen type III mRNA in rat cardiac fibroblasts when delivered from a 1 mmol/l 4S-StarPEG crosslinked collagen scaffold. Relative mRNA expression was determined by comparing the collagen type I and type III Ct values to the housekeeping GAPDH Ct values, and normalizing with that of rat cardiac fibroblasts treated with a 1 mmol/l 4S-StarPEG collagen scaffold alone (empty control). Asterisk indicates a statistically significant difference compared with the use of a 1 mmol/l 4S-StarPEG crosslinked collagen scaffold alone (all data relative to this group), P < 0.05. Data is presented as the mean ± SD, n = 4.
Figure 3
Figure 3
Quantitative effect of the RNA releasing collagen scaffolds on the healing wound in vivo. (a) Effect of treatments on wound contraction (normalized to wound width at day 0). Wound contraction was evaluated with stereological methods using pictures such as the representative images in Figure 4. * indicates statistical significance when compared with indicated groups; P < 0.05. # indicates statistical difference compared to all other groups presented P < 0.05. (b) Normalized wound contraction is the relative decrease in wound margin width at day 28 compared with day 0. Granulation volume fraction was evaluated stereologically using pictures such as the representative images in Figure 4. Granulation volume fraction is indicated by the cyan/blue granulation tissue presented centrally in the images of Figure 4 between the wound margin boundaries and the epithelium. * indicates statistical significance between the groups indicated. (c) Ratio of collagen type III-like fibers to collagen type I-like fibers, within the wound bed, determined from sections staining with picrosirius red (representative images in Figure 4). * indicates statistical significance when compared with healthy unwounded skin, P < 0.05. Data presented is the mean ± SD analyzed by one-way ANOVA and Tukey's post hoc test, P < 0.05.
Figure 4
Figure 4
Representative micrographs of wound beds following treatment with RNA releasing collagen scaffolds. Representative Russel-Movat's pentachrome staining of wound bed sections at day 28 with blue/cyan representing the field of granulation tissue and yellow being the original skin collagen that indicates the wound margins. Scale bar = 100 μm. Polarized light microscopy of picrosirius red stained wound bed sections. Green-stained fibers demonstrate the presence of collagen type III-like fibers and yellow and red-stained fibers demonstrate the presence of collagen type I-like fibers. Collagen type-I like fibers are highlighted with red arrows within some sections and collagen type-III like fibers are highlighted with green arrows. Sections are counterstained with Weigerts hematoxylin (purple). Scale bar = 100 μm. Micrographs of collagen type III immunohistochemical staining of wound bed sections, counterstained with hematoxylin. Scale bar = 100 μm.
Figure 5
Figure 5
Venn diagram summarizing the proteins that are upregulated (red) and downregulated (blue). Proteins are clustered into various pathways such as those indicative of remodeling, growth factors, inflammatory markers and ligands of apoptosis. Proteins that are dysregulated are presented relative to a nontreated excisional wound.
Figure 6
Figure 6
Quantitation of wound healing factors following treatments with RNA releasing collagen scaffolds in vivo. (a) Fold change of TGF-β1 expression in excised tissue at day 28, data normalized to healthy unwounded skin. (b) Fold change of TIMP-1 expression in excised tissue at day 28, data normalized to healthy unwounded skin. (c) Fold change of MMP-8 expression in excised tissue at day 28. (d) Ratios of MMP-8 to TIMP-1 expression in excised tissue at day 28, data normalized to healthy unwounded skin. NT indicates an excisional wound with no treatment applied (NT). Data presented is the mean of n = 4 ± SD analyzed by one-way ANOVA and Tukey's post hoc test. * indicates statistical significance between the groups indicated (P < 0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Stadelmann WK, Digenis AG, Tobin GR. Physiology and healing dynamics of chronic cutaneous wounds. Am J Surg. 1998;176:26S–38S. - PubMed
    1. Murphy PS, Evans GR. Advances in wound healing: a review of current wound healing products. Plast Surg Int. 2012;2012:190436. - PMC - PubMed
    1. Yannas IV, Burke JF, Orgill DP, Skrabut EM. Wound tissue can utilize a polymeric template to synthesize a functional extension of skin. Science. 1982;215:174–176. - PubMed
    1. Formiga FR, Pelacho B, Garbayo E, Abizanda G, Gavira JJ, Simon-Yarza T, et al. Sustained release of VEGF through PLGA microparticles improves vasculogenesis and tissue remodeling in an acute myocardial ischemia-reperfusion model. J Control Release. 2010;147:30–37. - PubMed
    1. Ghahary A, Tredget EE, Shen Q, Kilani RT, Scott PG, Takeuchi M. Liposome associated interferon-alpha-2b functions as an anti-fibrogenic factor in dermal wounds in the guinea pig. Mol Cell Biochem. 2000;208:129–137. - PubMed

Publication types

MeSH terms