15502-m-pleumeekers

Scoring categories Score (1) Uniformity and darkness of the stain No stain 0 Weak stain of poorly formed matrix 1 Moderately even stain 2 Even dark stain 3 (2) Distance between cells / amount of matrix accumulated High cell densities with no matrix in between (no spacing between cells) 0 High cell densities with little matrix in between (cells <1 cell-size apart) 1 Moderate cell density with matrix (cells approximately 1 cell-size apart) 2 Low cell density with moderate distance between cells (>1 cell) and an extensive matrix 3 (3) Cell morphologies represented Condensed/necrotic/pycnotic bodies 0 Spindle/fibrous 1 Mixed spindle/fibrous with rounded chondrogenic morphology 2 Majority rounded/chondrogenic 3 Maximum score 9 Table 2. The Bern Score: Histological evaluation of engineered cartilage constructs. Biomechanical analysis For mechanical characterization of engineered cartilage constructs after in-vitro and in-vivo cell culture, we used 2.5 mm thick and 5 mm diameter constructs. The samples were placed in a close-fitting Ø5 mm stainless steel cylindrical wells. Mechanical testing was performed with a materials testing machine (Zwick Z005, Ulm, Germany) equipped with a 10 N load cell, a built-in displacement control, and a cylindrical, plane ended, stainless steel indenter (Ø1.2 mm). During mechanical testing the samples were immersed in PBS. Stress-strain testing was performed: the samples were compressed to a final height of 0.5 mm at a loading rate of 5 mm per minute. An in-house Matlab® script was used to locate the sample surface and measure the sample thickness. The sample surface was identified by detecting the corresponding slope discontinuity of the force-displacement curve using its second derivative. Force-displacement curves were then converted to stress-strain curves. Compressive modulus at 40% strain (E40%), defined as the derivative of the stress-strain curve at 40% strain, was determined for every sample ( n =98). Statistical analysis All data were analyzed with PASW Statistics 20.0 (SPSS inc. Chicago, USA). The mean and standard deviations are presented. For statistical evaluation, a mixed linear model was used. Cell source, time point and treatment (TGFβ1) were defined as fixed factors in the model. Donor and sample number were treated as random factors. Values of p <0.05 were considered statistically significant. For histological scoring we used the Kruskal-Wallis followed by the 68 CHAPTER 4