The scenario
Our hypothetical company's acellular dermal allograft, used in breast reconstruction and hernia repair, is on backorder. The company wants to change its tissue processing parameters to make more of it.
The risk is that a processing change could affect how well cells are removed and damage the extracellular matrix (ECM) that gives the graft its strength and flexibility. Our job was to write the plan of studies senior management would need before approving the change.
Study design
We designed three studies: mechanical, biochemical and histological. Each one compares grafts made with the standard process (n = 10) to grafts made with the modified process (n = 10).
All three use tissue from matched donors, randomized assignment to groups, and coded samples so the analyst doesn't know which process a sample went through. Each sample is measured in triplicate, and the studies are powered at 80% with α = 0.05.
Mechanical testing
Graft samples are cut into standard dog-bone shapes following ASTM soft-tissue guidelines, hydrated in PBS for 30 minutes, and pulled to failure on an Instron with a 100 N load cell at 10 mm/min.
The test records ultimate tensile strength, Young's modulus and elongation at break. Each sample is photographed before and after to check for tearing, delamination and fiber disruption. The two groups are compared with a two-sample t-test.
Biochemical testing
Collagen type I and elastin are measured with ELISA kits and normalized to total protein from a BCA assay. Technical replicates must agree within 15%.
The statistics plan checks normality first (Shapiro-Wilk), switches to Welch's t-test or a Mann-Whitney U test when the assumptions fail, and applies a Bonferroni correction across the three measures.
Histology and cell repopulation
First, a PicoGreen DNA assay confirms both grafts are fully decellularized, meaning less than 50 ng of double-stranded DNA per mg of dry tissue.
Then dermal fibroblasts are seeded onto 1 cm grafts at 50,000 cells/cm² and grown for 14 days. Sections are stained with DAPI for nuclei and phalloidin for the cytoskeleton, and nuclei per mm² are counted in ImageJ across at least five fields per section.
Decision criteria
Each study defines in advance what counts as acceptable, ambiguous or unacceptable.
Acceptable. No significant difference between the groups, so the change can go ahead.
Ambiguous. A small but significant drop, such as less than 20% in collagen or elastin. The change goes to further testing, like cyclic fatigue testing or SEM imaging, before a decision.
Unacceptable. A large loss, such as more than a 20% drop in collagen or elastin, or a graft that fails the DNA threshold. The change is rejected.