Biomedical subjects

Lloyd Wolfinbarger

Publications and source records attributed to Lloyd Wolfinbarger.

4 recordsLinked to original sources

PubMed2005

Extractable bone morphogenetic protein and correlation with induced new bone formation in an in vivo assay in the athymic mouse model.

A correlation between extractable bone morphogenetic proteins (BMPs) in demineralized bone matrix (DBM) and osteoinduction has been suggested. Extractable BMP-4 and osteoinductivity of DBM from 40 donors were assessed using enzyme-linked immunosorbent assay (ELISA) and in vivo athymic mouse assay, respectively. Extractable BMP-4 level averaged 3.7 +/- 0.21 ng/g of DBM and correlated with osteoinductivity of the DBM in an in vivo assessment of induced newbone formation.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

Ovine panel reactive antibody assay of HLA responsivity to allograft bioengineered vascular scaffolds.

BACKGROUND: Increasing evidence implicates immune response as a contributing factor in the failure of allograft valve transplants. Increases in panel reactive antibodies have been identified in human subjects. To correlate these responses with novel preimplantation processing methods to reduce cellularity, both a relevant panel reactive antibody assay and a chronic implantation animal model are necessary. We modified a human flow cytometric panel reactive antibody assay for ovine model use to detect antibody responses to residual antigen-loading decellularized scaffolds engineered from pulmonary artery tissue. METHODS: A clinical panel reactive antibody assay was modified with anti-sheep antibodies. Dimethyl sulfoxide cryopreserved (n = 4) and decellularized scaffolds (n = 8) fashioned as patches from pulmonary arteries were implanted for study. Fresh (nonprocessed) tissue implants were used as positive controls (n = 2), and sham-treated animals were used as negative controls (n = 2). Baseline, 10-week, and 20-week blood samples were assayed for panel reactive antibody levels. Immunohistochemistry with anti-major histocompatibility complex antibodies were performed on preimplantation scaffolds. RESULTS: Chronic implants of fresh tissue stimulated strong panel reactive antibody responses. Classically cryopreserved tissues provoked modest panel reactive antibody responses to major histocompatibility complex I antigen and no response to major histocompatibility complex II antigen. Decellularized tissue scaffolds provoked minimal to no panel reactive antibody responses to either major histocompatibility complex I or II antigen. Immunohistochemistry correlated with the panel reactive antibody results by identifying significant amounts of major histocompatibility complex I and II in fresh tissue, reduced antigen staining in cryopreserved control tissues, and minimal amounts in decellularized tissues. CONCLUSIONS: These studies with an ovine modified panel reactive antibody assay confirmed minimal immune allosensitization to transplanted decellularized tissue patches. Qualifying criteria for putative tissue-engineered scaffolds should include minimal recipient panel reactive antibody response.

Analysis of Variance↗

PubMed2004

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.

Animals↗