ime quantitative RT-PCR For quantitative analysis of the selected mRNAs aliquots of total RNA were DNase-treated, reverse-transcribed to cDNA and analyzed using real-time quantitative RT-PCR with TaqManH technology and Applied Biosystem’s 7900HT equipment. For mouse neuropeptide Y mRNA, pre-designed TaqManH Gene Expression assay was used. For other transcripts, oligonucleotide primers and dual-labeled probes were designed using RealTimeDesignTM software . The levels of b-actin mRNA were used to normalize the results between the samples. The samples were analyzed in three technical replicates. To determine statistical significance of the results, the data were analyzed with independent samples Ttest. Delayed maturation of myofibroblasts in Mmp132/2 mouse granulation tissue The appearance of myofibroblasts in wound granulation tissue is important for wound contraction during epithelial repair. To assess the presence of myofibroblasts in mouse experimental granulation tissue, sections harvested at 7, 14 and 21 d, were Culture of mouse skin fibroblasts WT and Mmp132/2 mouse skin fibroblasts PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22212322 from three individual mice for each genotype were established by explantation from the skin of 3 weeks old male and female mice. MMP-13 in Wound Granulation Tissue stained for a-SMA by IHC. At 7 d, a-SMA positive cells were detected in the areas adjacent to implant surface in WT mouse tissue and the staining pattern was typically dense and oriented parallel to the surface in accordance with the contractile function of myofibroblasts. In Mmp132/2 mice the orientation of a-SMApositive myofibroblasts was more random than in WT mice and did not display unified assembly of myofibroblast masses at 7 d. A semi-quantitative evaluation of the staining revealed a significant difference in the collective parallel orientation at 7 d, suggesting altered function and delayed maturation of myofibroblasts. Analysis of the granulation tissue harvested at 14 d showed prominent staining pattern of a-SMA positive cells extending throughout the cellular area and showing strong parallel orientation of myofibroblasts 
especially in the areas close to VCS surface. No obvious difference was noted between Mmp132/2 and WT tissues, suggesting that although lack of MMP-13 results in delayed maturation of myofibroblasts in the granulation tissue, this 3544-24-9 effect is subsequently compensated by other factors. Interestingly, at 21 d the a-SMA staining pattern was characterized by markedly diminished number of a-SMA-positive cells in the areas close to VCS surface apparently representing the most mature granulation tissue, and the a-SMA staining was more emphasized in the inner parts of the implant. The shift in the expression pattern of a-SMA was clearly more evident in WT than in Mmp132/2 tissue and appeared to be in accordance with the intensive tissue ingrowth. Altered vascularization in the granulation tissue of Mmp132/2 mice To examine the role of MMP-13 in vascularization of the experimental granulation tissue, the tissue sections were stained for CD34 by IHC. The CD34-positive vessels were morphometrically analyzed and subdivided into three groups based on the diameter. The vessel structures with the diameter less than 10 mm were considered as microvessels, the vessels with the diameter 10 40 mm as medium sized blood vessels, and the vessels over 40 mm in diameter as large vessels. In general, CD34 positive blood vessels were abundantly present in WT and Mmp132/2 mouse granulation tis
