Comparison of four methods for generating decellularized equine synovial extracellular matrix

Abstract

OBJECTIVE To evaluate 4 methods for generating decellularized equine synovial extracellular matrix. SAMPLE Villous synovium harvested from the femoropatellar and medial femorotibial joints of 4 healthy adult horses < 7 years of age. Synovial samples were frozen (-80°C) until used. PROCEDURES Synovial samples were thawed and left untreated (control) or decellularized with 1 of 4 methods (15 samples/horse/method): incubation in 0.1% peracetic acid (PAA), incubation in 0.1% PAA twice, incubation in 1% Triton X-100 followed by incubation in DNase, and incubation in 2M NaCl followed by incubation in DNase. Control and decellularized samples were examined for residual cells, villous integrity, and collagen structure and integrity by means of histologic examination and scanning electron microscopy; cell viability was evaluated by means of culture and exclusion staining. Decellularization efficiency was assessed by testing for DNA content and DNA fragment size. RESULTS Incubation in PAA once preserved the synovial villous architecture, but resulted in high DNA content and retention of large (> 25,000 base pair) DNA fragments. Incubation in Triton and incubation in NaCl resulted in low DNA content and short (< 200 base pair) DNA fragments, but destroyed the synovial villous architecture. Incubation in PAA twice resulted in low DNA content and short DNA fragments while retaining the synovial villous architecture. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that of the methods evaluated, incubation in 0.1% PAA twice was the best method for generating decellularized equine synovial extracellular matrix.

Synovium extra cellular matrices seeded with transduced mesenchymal stem cells stimulate chondrocyte maturation in vitro and cartilage healing in clinically-induced rat-knee lesions in vivo

“…Cell and tissue preparation began 30 days before the start of the in vitro study with the fresh harvest of synovium and cartilage tissues for preparation of the sECM, sMSC, and isolated chondrocytes (Table 1) [8,18]. Once prepared, all cells and scaffolds were frozen and thawed prior to the start of the experiments that started on Day 0 on the time line detailed in Table 1.…”

“…Equine villous synovium from the medial femoral patellar joint and articular cartilage from the femoral condyles were harvested [8] from knee joints of healthy adults (2–7 years old), euthanized for reasons unrelated to this study. Briefly, the joints were aseptically prepared with 2 5min scrubs using 4% chlorhexidine and sterile H 2 O. Sterile gloves and instruments were used to enter and harvest tissue.…”

“…No attempt here has been made to quantify nor classify the pMSCs from the tissue sMSCs. sECM was decellularized from harvested synovium by immediately dividing the tissue into 1x1 cm sheets using a dissecting microscope, then incubating them at 37°C in 0.1% peracetic acid (PAA) and 4% ethanol for 6 hours with mechanical agitation twice [8]. Samples of sECM were prepared using a 6 mm diameter biopsy punch, frozen wet at –80°C, surrounded by dry ice in ethanol.…”

“…The grafted injured site does not receive a continued, renewable source of bioactive anabolic mediators. We have suggested a different strategy to overcome this weakness [8]: Use of a microenvironment paracrine-like feedback loop via a bioactive transplant in the synovium nearby a cartilage injury. Such a transplant, made from synovium components (sConstructs) has not, to our knowledge, been reported in the literature.…”

“…Additionally, in vitro , ECMs have been shown to encourage the formation of tissue specific phenotypes [20,21]. We have reported our decellularization procedures to produce synovium-derived extra-cellular matrix (sECM) [8] and our sMSC seeding procedures to show that sMSC will retain viability, distribute, proliferate, differentiate and produce soluble biomediators, including inserted transgenes, such as the chondrogenic bone morphogenetic protein two (BMP-2) [18]. BMP-2 was chosen because of its strong anabolic properties and our laboratory’s access and more than a decade of work with these reagents [22].…”