Low rate of detection of SARS-CoV-2 RNA in deceased tissue donors.

Given the possibility for disease transmission, this study was performed to determine whether there is detectable SARS-CoV-2 viral RNA in the blood of deceased tissue donors. A retrospective analysis of blood samples from eligible deceased tissue donors from Oct 2019 through June 2020 was performed. Plasma aliquots were initially tested with a SARS-CoV-2 NAT Assay; positive samples were further tested using an alternate NAT and an antibody assay. The proportion of donors with confirmed RNAemia and 95% confidence intervals were computed. Of donor samples collected in 2019, 894 yielded valid results, with 6 initially positive, none of which confirmed positive by alternate NAT. Of donor samples collected in 2020, 2562 yielded valid initial NAT results, with 21 (0.8%) initially positive. Among those, 3 were confirmed by alternate NAT, 17 were not confirmed, and 1 had an invalid alternate NAT result. The rate of SARS-CoV-2 RNAemia in deceased tissue donors is approximately 1 per 1000, and it is unknown whether this RNAemia reflects the presence of infectious virus. Given these results, the risk of transmission through tissue is thought likely to be low.

Testing of tissue specimens obtained from SARS-CoV-2 nasopharyngeal swab-positive donors.

Risk for transmission of SARS-CoV-2 through allogeneic human tissue transplantation is unknown. To further evaluate the risk of virus transmission, tissues were obtained from deceased donors who had tested positive for SARS-CoV-2 RNA via nasopharyngeal swab. This study evaluated an array of human tissues recovered for transplantation, including bone, tendon, skin, fascia lata, vascular tissues, and heart valves. Tissue samples and plasma or serum samples, if available, were tested for viral RNA (vRNA) using a real time PCR system for the presence of virus RNA. All samples were tested in quadruplicate for both subgenomic (sgRNA) and genomic (gRNA) RNA encoding the SARS-CoV-2 nucleocapsid gene. Amplification of a cellular housekeeping gene served as the positive control for every sample. A total of 47 tissue samples from 17 donors were tested for SARS-CoV-2 RNA. Four donors had plasma or serum available for paired testing. SARS-CoV-2 RNA was not detected from any tissue or plasma/serum sample tested. Based on these findings, risk of transmission through the transplantation of tissue types studied from SARS-CoV-2 infected donors is likely to be low.

Decellularization of human dermis using non-denaturing anionic detergent and endonuclease: a review.

Decellularized human dermis has been used for a number of clinical applications including wound healing, soft tissue reconstruction, and sports medicine procedures. A variety of methods exist to prepare this useful class of biomaterial. Here, we describe a decellularization technology (MatrACELL(®)) utilizing a non-denaturing anionic detergent, N-Lauroyl sarcosinate, and endonuclease, which was developed to remove potentially immunogenic material while retaining biomechanical properties. Effective decellularization was demonstrated by a residual DNA content of ≤4 ng/mg of wet weight which represented >97 % DNA removal compared to unprocessed dermis. Two millimeter thick MatrACELL processed human acellular dermal matrix (MH-ADM) exhibited average ultimate tensile load to failure of 635.4 ± 199.9 N and average suture retention strength of 134.9 ± 55.1 N. Using an in vivo mouse skin excisional model, MH-ADM was shown to be biocompatible and capable of supporting cellular and vascular in-growth. Finally, clinical studies of MH-ADM in variety of applications suggest it can be an appropriate scaffold for wound healing, soft tissue reconstruction, and soft tissue augmentation.

Prototype anionic detergent technique used to decellularize allograft valve conduits evaluated in the right ventricular outflow tract in sheep.

BACKGROUND AND AIM OF THE STUDY: Biodegradable polymeric materials or extracellular matrix scaffolds are used in tissue-engineered heart valve designs, with the expectation of replicating the anatomic, histological and biomechanical characteristics of semi-lunar valves. The study aim was to evaluate the extent of in-vivo recellularization and the explant pathology findings of a prototype anionic, non-denaturing detergent and endonuclease technique used to decellularize allograft (homograft) valve conduits implanted in the right ventricular outflow tract (RVOT) of sheep, and to identify possible risks associated with tissue-engineered heart valve conduits based on decellularized allograft semilunar valve scaffolds. METHODS: Valve conduits were decellularized using a solution of N-lauroylsarcosinate and endonucleases, rinsed in lactated Ringers solution, and stored in an antibiotic solution at 4 degrees C until implanted. Explanted valves and unimplanted controls were examined macroscopically, radiographically (for calcification) and histologically using immunohistochemistry (IHC), routine and special histological stains, transmission electron microscopy (TEM) and polarized light microscopy (evaluation of collagen crimp). RESULTS: Cells and cellular remnants were uniformly absent in the decellularized cusps, but occasional focal sites of arterial wall smooth muscle cells and to a greater extent subvalvular cardiac myocytes were variably retained. The trilaminar histological structure of the cusp was preserved. Valve conduit-related pathology consisted of intracuspal hematoma formation, collagen fraying, thinning of the conduit wall, and inflammatory cells associated with cardiac myocyte remnants. Cuspal calcification was not seen, but elastic fibers in the conduit wall and retained subvalvular cardiac myocyte remnants were liable to calcification. Fibrous sheath formation was present on the luminal surface of the conduit and extended over the cuspal surfaces to a variable extent. Myofibroblast-like cells repopulated the conduit wall and the basal region of the cusp. Re-endothelialization was variably present on the cuspal surfaces. CONCLUSION: Explant pathology findings showed that in-vivo recellularization occurred, but was focally limited to regions of the arterial wall and cusp base. Safety concerns related to detergent and endonuclease use were identified. Methods to eliminate the potential for structural deterioration and enhance the rate and extent of recellularization of valve conduit tissue are required. Pathology findings showed implantation of valve conduits in the RVOT of juvenile sheep for 20 weeks to be a reliable animal model for the initial in-vivo assessment of decellularized valves. A 20-week period may be insufficient however to evaluate the long-term safety and effectiveness of a tissue-engineered valve conduit, as these depend on effective and phenotypically appropriate recellularization accompanied by sustained cell viability and function.

Decellularization reduces calcification while improving both durability and 1-year functional results of pulmonary homograft valves in juvenile sheep.

OBJECTIVE: The juvenile sheep functional valve chronic implant calcification model was used to compare long-term calcification rates, functional performance, and durability for 3 types of right ventricular outflow tract implants: classically cryopreserved homografts and 2 decellularized pulmonary valved conduits. METHODS: Fifteen juvenile sheep were randomly assigned to one of 3 study arms and underwent pulmonary valve replacement. The arms included the following: (1) cryopreserved ovine pulmonary valves; (2) cryopreserved, decellularized, saline (1 degrees C-10 degrees C)-stored ovine pulmonary valves; and (3) cryopreserved, decellularized, glycerolized (-80 degrees C) stored ovine pulmonary valves. Animal growth, serial echocardiographic results (with valve performance assessment), dimensions, and tissue-specific calcification measurements were compared with pre-explant angiographic analysis and right ventricular outflow tract pressure measurements, cardiac magnetic resonance imaging, specimen radiographic analysis, gross explant pathology, and histopathology. Parametric and nonparametric statistical analysis were performed. RESULTS: All but 2 study animals receiving implants thrived postoperatively, with similar growth rates, explant valve dimensions, ventricular functions, cardiac output, and indices during the study. As determined by means of echocardiographic analysis, 3 animals in arm 1 (and one in arm 2) had leaflet dysfunction. Valve regurgitation was recognized in 1 survivor each from both arms 1 and 2. Although 1 arm 1 animal died with calcified subacute bacterial endocarditis, and the other 4 had leaflet and conduit wall calcification by the time of death, no arm 2 or arm 3 animals demonstrated leaflet calcium, and no arm 3 and only 1 arm 2 animals had calcium in the conduit wall over the entire year, as determined with any measurement method. All cryopreserved conduit walls had calcium by 20 weeks, whereas only 1 of 10 decellularized conduits (arms 2 plus 3) had wall calcium. CONCLUSION: Cryopreserved-decellularized-glycerolized valves retained normal valve function, with absent leaflet and minimal wall calcifications 1 year postoperatively, as opposed to classically cryopreserved allografts. These results might be predictive of the prolonged durability and functionality of a cryopreserved-decellularized-glycerolized allograft valve.

Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery.

We have previously reported the initial clinical feasibility with our small diameter tissue engineered blood vessel (TEBV). Here we present in vitro results of the mechanical properties of the TEBVs of the first 25 patients enrolled in an arterio-venous (A-V) shunt safety trial, and compare these properties with those of risk-matched human vein and artery. TEBV average burst pressures (3490+/-892 mmHg, n=230) were higher than native saphenous vein (SV) (1599+/-877 mmHg, n=7), and not significantly different from native internal mammary artery (IMA) (3196+/-1264 mmHg, n=16). Suture retention strength for the TEBVs (152+/-50 gmf) was also not significantly different than IMA (138+/-50 gmf). Compliance for the TEBVs prior to implantation (3.4+/-1.6%/100 mmHg) was lower than IMA (11.5+/-3.9%/100 mmHg). By 6 months post-implant, the TEBV compliance (8.8+/-4.2%/100 mmHg, n=5) had increased to values comparable to IMA, and showed no evidence of dilation or aneurysm formation. With clinical time points beyond 21 months as an A-V shunt without intervention, the mechanical tests and subsequent lot release criteria reported here would seem appropriate minimum standards for clinical use of tissue engineered vessels.

Recellularization of decellularized allograft scaffolds in ovine great vessel reconstructions.

BACKGROUND: Decellularized allograft tissues have been identified as a potential extracellular matrix (ECM) scaffold on which to base recellularized tissue-engineered vascular and valvular substitutes. Decreased antigenicity and the capacity to recellularize suggest that such constructs may have favorable durability. Detergent/enzyme decellularization methods remove cells and cellular debris while leaving intact structural protein "scaffolds." Allograft pulmonary artery tissues decellularized with an anionic detergent/enzyme methodology were tested in a long-term implantation model that used arterial wall repairs in the great vessels of juvenile sheep. METHODS: Twelve test sheep were implanted (n = 4) for each of three different scaffold protocols that compared traditional dimethylsulfoxide cryopreservation, cryopreservation followed by decellularization, and decellularization of fresh tissue. Four additional sheep served as controls (n = 2 sham, n = 2 fresh tissue). Patches were fashioned and implanted into pulmonary artery and aortic defects. Panel reactive antibodies (PRA) were measured over time (10 to 20 weeks). Explant histopathology determined recellularization morphology as well as calcium, collagen, and elastin distribution within explanted tissue. RESULTS: Unlike traditionally cryopreserved tissues, the decellularized tissues contained no residual cells or cellular debris before implantation, which correlated with measurable reductions in PRA. Decellularized explants demonstrated time-dependent migration of recipient cells through matrix, typically staining positive for alpha-smooth muscle actin with no calcification. CONCLUSIONS: The properties demonstrated seem consistent with characteristics necessary for implantable tissue-engineered scaffolds. The decellularization method described appears to create a biologically suitable ECM scaffold for in vivo migration of phenotypically appropriate cells while avoiding antigenicity and calcification.