clock positions and 1 mm from the edge in the perilesional cartilage using a handheld digital durometer shore s1 m scale instron ltd uk a number between 0 100 would be given with an inbuilt calibrated error of u2212 5 these measurements were then repeated in the contralateral limb in the same anatomic sites histology following stiffness measurements the specimens were decalcified in formic acid sodium citrate over two weeks following complete decalcification the specimens were dehydrated through a series of ethanol exchanges of increasing concentrations and then embedded in paraffin wax sections of 10 u03bcm thickness were made through the central portion of the defect sections were stained with toluidine blue and safranin o fast green the histology sections were blindly scored by one investigator using a modified o 39 driscoll score table 4 the animals that scored the best and the median total modified o 39 driscoll scores within their experimental group were then identified and immunohistochemically performed on these sections in addition to the total modified o 39 driscoll score the individual components of the scoring system were analysed separately immunohistochemistry the following primary antibodies were used in this study monoclonal mouse anti human type i collagen mp biomedicals us monoclonal mouse anti human type ii collagen mp biomedicals us and monoclonal mouse anti rabbit type vi collagen abcam uk horseradish peroxidase conjugated secondary anti rabbit and mouse immunoglobulins were used as appropriate and the colour reaction developed with 0 1 3 u2032 3 diaminobenzidine tetrachloride dab 0 01 hydrogen peroxide normal species specific serum was used as a control in all experiments statistical analysis graphpad prism 5 statistical software package graphpad software inc la jolla calif was used for.