Comparison of four methods for generating decellularized equine synovial extracellular matrix.

The research aimed to assess four different methods for creating decellularized equine synovial extracellular matrix, finding that the most effective technique was double incubation in 0.1% peracetic acid (PAA).

Objective and Sample

• The objective of the research was to evaluate four different methods for generating decellularized equine synovial extracellular matrix. This is an important task as it can provide vital information for clinical analyses and treatments.
• The synovial samples used for the study were sourced from the femoropatellar and medial femorotibial joints of four healthy adult horses that were less than seven years old. The samples were later frozen at a temperature of -80°C for future use.

Procedures

• The synovial samples were thawed and either left untreated as a control, or were decellularized with one of four methods with 15 samples per horse per method. The four decellularization procedures were: incubation in 0.1% peracetic acid (PAA), double incubation in 0.1% PAA, incubation in 1% Triton X-100 followed by incubation in DNase, and finally, incubation in 2M NaCl also followed by DNase incubation.
• The decellularized and control samples were examined for residual cells, the integrity of the villous structure, and the structure and integrity of collagen. This was done through histologic examination and scanning electron microscopy. The viability of the cells was analyzed through culture and exclusion staining.
• The efficiency of decellularization was determined by testing for DNA content and measuring the size of the DNA fragments.

Results

• The results showed that a single PAA incubation preserved the structure of the synovial villous, but resulted in high DNA content and retention of large (over 25,000 base pair) DNA fragments.
• Both Triton and NaCl incubation resulted in low DNA content and small (under 200 base pair) DNA fragments, but led to the loss of the synovial villous structure.
• Double PAA incubation yielded low DNA content and small DNA fragments while maintaining the synovial villous structure. Therefore, of all the methods evaluated, double incubation in 0.1% peracetic acid (PAA) was found to be the most effective in generating decellularized equine synovial extracellular matrix.