Biocompatibility of acellular human pericardium.

BACKGROUND: Previous studies have shown successful decellularization of human pericardium without affecting the major structural components and strength of the matrix. The aim of this study was to assess the biocompatibility and reseeding potential of the acellular human pericardial scaffold. MATERIALS AND METHODS: Pericardia were treated sequentially with hypotonic buffer, sodium dodecyl sulfate, and a nuclease solution. The presence of cellular attachment factors after decellularization was evaluated using immunohistochemistry. The scaffold was seeded with dermal fibroblasts and cellular attachment to and numbers of cells penetrating were assessed over time. Biocompatibility was also evaluated following subcutaneous implantation into a mouse model for three months. RESULTS: After decellularization, the scaffold stained positively for fibronectin, but collagen IV and laminin staining was reduced. Seeded fibroblasts attached to the mesothelial surface and were visualized in the tissue within a week of seeding. The majority of fibroblasts in the tissue were viable and there was evidence of remodeling of the matrix. Analysis of the explanted tissues from mice showed that fresh/frozen and glutaraldehyde-fixed pericardia were encapsulated with a thick layer of inflammatory cells and fibrous tissue. In contrast, the decellularized scaffold was infiltrated with myofibroblasts, CD34+ cells and macrophages, indicating a healthy repair process. Compared with the glutaraldehyde-fixed tissue, the calcium content of the fresh/frozen and decellularized pericardia was negligible. CONCLUSIONS: The pericardial scaffold was biocompatible in vitro and in the mouse model in vivo.

Development and characterization of an acellular human pericardial matrix for tissue engineering.

This study aimed to produce an acellular human tissue scaffold with a view to recellularization with autologous cells to produce a tissue-engineered pericardium that can be used as a patch for cardiovascular repair. Human pericardia from cadaveric donors were treated sequentially with hypotonic buffer, SDS in hypotonic buffer, and a nuclease solution. Histological analysis of decellularized matrices showed that the human pericardial tissue retained its histioarchitecture and major structural proteins. There were no whole cells or cell fragments. There were no significant differences in the hydroxyproline (normal and denatured collagen) and glycosaminoglycan content of the tissue before and after decellularization (p > 0.05). There were no significant changes in the ultimate tensile strength after decellularization (p > 0.05). However, there was an increased extensibility when the tissue strips were cut parallel to the visualized collagen bundles (p = 0.005). No indication of contact or extract cytotoxicity was found when using human dermal fibroblasts and A549 cells. In summary, successful decellularization of the human pericardium was achieved producing a biocompatible matrix that retained the major structural components and strength of the native tissue.

Tissue engineering of cardiac valves: re-seeding of acellular porcine aortic valve matrices with human mesenchymal progenitor cells.

BACKGROUND AND AIM OF THE STUDY: Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study investigated the potential for re-seeding an acellular porcine heart valve matrix using human mesenchymal progenitor cells (MPC). METHODS: MPC were isolated from the bone marrow of patients undergoing hip replacement operations. Putative MPC were then cultured in several differentiation media in order to determine the multipotential differentiation capacity of the cells. The MPC were also characterized by FACS analysis. Cells at passage 8 were then seeded at between 1 x 10(4) and 1 x 10(5) cells/cm2 onto a decellularized porcine aortic valve matrix, and recellularization of the matrix was assessed. The phenotype of the re-seeded cells and re-seeded cell density was then determined by histology and immunohistochemistry. RESULTS: Putative MPC were successfully isolated and differentiated into cells of the adipogenic, neurogenic, and myogenic lineages. FACS analysis showed the cells to have a similar phenotype to those isolated by others (CD45-, CD13+, D7FIB+, CD105+, CD10+/-, LNGFR+/-, CD55+, BMP- and AP+/-). Cells seeded onto an acellular valve matrix penetrated the center of the tissue after four weeks to 2% of homograft cell density. Phenotypic analysis of the cells in the re-seeded matrix revealed the cells to have a similar phenotype to native valve interstitial cells (vimentin+, alpha-smooth muscle actin+, heavy chain myosin slow-, desmin-). However, re-seeded cells also expressed osteogenic markers (alkaline phosphatase, osteonectin, and osteopontin). CONCLUSION: This study has shown, for the first time, that human MPC have the capacity to infiltrate an acellular porcine valve matrix under static conditions in vitro. Future studies will comprise culture under pulsatile flow in a physiological heart valve bioreactor to maintain the desired cell phenotype and increase cell density.

In-vitro assessment of the functional performance of the decellularized intact porcine aortic root.

BACKGROUND AND AIMS OF THE STUDY: Tissue-engineered heart valves offer the potential to deliver a heart valve replacement that will develop with the young patient. The present authors' approach is to use decellularized aortic heart valves reseeded in vitro or in vivo with the patient's own cells. It has been reported that treatment of porcine aortic valve leaflets with 0.1% (w/v) sodium dodecyl sulfate (SDS) in hypotonic buffer produced complete leaflet acellularity without affecting tissue strength. The present study aim was to investigate the effect of an additional treatment incorporating 1.25% (w/v) trypsin and 0.1% (w/v) SDS on the biomechanics and hydrodynamics of the aortic root. This treatment has been shown to produce decellularization of both the aorta and valve leaflets. METHODS: Fresh porcine aortic roots were treated to reduce the thickness of their aortic wall, and incubated in hypotonic buffer for 24 h. The leaflets were masked with agarose gel, and the aorta was treated with 1.25% (w/v) trypsin for 4 h at 37 degrees C. The trypsin and agarose were removed and the roots incubated with 0.1% (w/v) SDS in hypotonic buffer for 24 h. Fresh and treated circumferential and axial aortic specimens were subjected to uniaxial tensile testing, while intact porcine aortic roots were subjected to dilation and pulsatile flow testing. RESULTS: Decellularized aortic wall specimens demonstrated significantly decreased elastin phase slope and increased transition strain compared to the fresh control. However, the treatment did not impair tissue strength. Decellularized intact roots presented complete leaflet competence under systemic pressures, increased dilation and effective orifice areas, reduced pressure gradients, physiological leaflet kinematics and reduced leaflet deformation. CONCLUSION: The excellent leaflet kinematics and hydrodynamic performance of the decellularized roots, coupled with the excellent biomechanical characteristics of their aortic wall, form a promising platform for the creation of an acellular valve scaffold with adequate mechanical strength and functionality to accommodate dynamic cell repopulation in vitro or in vivo. This approach can be used for both allogeneic and xenogeneic tissue matrices.

Biocompatibility and recellularization potential of an acellular porcine heart valve matrix.

BACKGROUND AND AIM OF THE STUDY: Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. The study aim was to investigate the biocompatibility and recellularization potential of an acellular porcine valve matrix. METHODS: Acellular porcine valve matrix contact and extract cytotoxicity was tested against porcine fibroblasts and smooth muscle cells (SMC). Porcine cells were incubated with decellularized aortic valve leaflets and aortic wall, and then assessed for changes in morphology and contact inhibition of growth. Soluble tissue extracts were prepared from decellularized leaflets and aortic wall, and assessed for their effect on the viability of cultured porcine cells. Acellular leaflets were seeded with either fibroblasts or SMC at 1 x 10(3) to 1 x 10(6) cells/cm2 for 24 h, or 5 x 10(4) cells/cm2 for 1-4 weeks. Cell attachment onto, and migration into, the acellular matrix was assessed by scanning electron microscopy and histology. RESULTS: No contact inhibition of growth, or changes in fibroblast or SMC morphology, were observed following contact with the acellular valve matrix. No soluble extract cytotoxicity was found. Intermediate cell-seeding densities (2.5 x 10(4) to 7.5 x 10(4) cells/cm2) of both cell types produced confluent cell attachment; at the lowest concentration (1 x 10(3) cells/cm2) cell attachment was sparse, and at the highest (1 x 10(6) cells/cm2) it was multilayered. The SMC migrated throughout the leaflet matrix over four weeks, but there was no fibroblast migration into the matrix. CONCLUSION: The absence of contact and extract cytotoxicity indicated that the acellular valve matrix was biocompatible in vitro. The failure of porcine fibroblasts to grow on, or infiltrate into, the matrix suggested that the SMC may be the preferred cell type for future leaflet recellularization studies in the development of a tissue-engineered heart valve replacement.

Tissue engineering of cardiac valve prostheses I: development and histological characterization of an acellular porcine scaffold.

BACKGROUND AND AIMS OF THE STUDY: Several deficiencies in current heart valve prostheses make them problematic for use in younger patients. Tissue valve substitutes are non-viable with a life expectancy of only 10-15 years, while mechanical valves require long-term anti-coagulation therapy. A solution to these problems would be to develop a tissue-engineered heart valve containing autologous cells, enabling the valve to maintain its biochemical and mechanical properties, yet grow with the patient. The study aim was to optimize a protocol to produce a porcine acellular matrix scaffold for use in developing a tissue-engineered heart valve. METHODS: Fresh porcine aortic valve leaflets were treated with Triton X-100, sodium dodecyl sulfate (SDS), sodium deoxycholate, MEGA 10, TnBP, CHAPS, and Tween 20, over a range of concentrations, in the presence of protease inhibitors for up to 72 h. Histological analysis was used to detect the major structural proteins of the heart valve, collagen I, elastin and glycosaminoglycans. RESULTS: After 72 h, most protocols resulted in the retention of large numbers of whole cells and cell fragments. Only SDS (0.03-1%) or sodium deoxycholate (0.5-2%) resulted in total decellularization at 24 h. Histological analysis of acellular matrices showed that the major structural proteins had been retained and appeared to be intact. CONCLUSION: Protocols utilizing SDS or sodium deoxycholate were successful for leaflet decellularization, and histological analysis showed that the major structural components of the valve matrix had been maintained. These methods are being developed further with a view to reseeding with autologous cells to produce tissue-engineered solutions for clinical implantation.

Tissue engineering of cardiac valve prostheses II: biomechanical characterization of decellularized porcine aortic heart valves.

BACKGROUND AND AIMS OF THE STUDY: For both young patients with congenital heart disease and young, growing adults there is a need for replacement heart valves that will develop with the patient. Tissue-engineered heart valves coupled with in-vitro recellularization have this potential. One approach is to use acellular tissue matrices, but the decellularization treatment must not affect the biomechanical integrity of the valvular matrix. This study investigated the effect of 0.03% (w/v) and 0.1% (w/v) sodium dodecyl sulfate (SDS) on the mechanical integrity of porcine aortic valve leaflets. METHODS: Left coronary porcine leaflets were treated with SDS (0.03% or 0.1%, w/v) in hypotonic or isotonic buffer and buffer alone. SDS in hypotonic buffer produced accellularity. Circumferential and radial specimens of treated leaflets were subjected to uniaxial tensile testing, and the effect of the buffer on leaflet morphology was assessed. Whole porcine aortic roots were also treated with 0.1% (w/v) SDS and subjected to function testing. RESULTS: SDS treatment significantly increased extensibility of the leaflet specimens, which was greater in the circumferential than radial direction. This was seen as a significantly decreased slope of both the elastic and collagen phases of the stress-strain behavior. The ultimate tensile strength and transition stress were not affected significantly; nor was there any significant difference between hypotonic buffer and hypotonic buffer + SDS treatments. Study of the leaflet morphology suggested that the increased extensibility was due to shrinkage as well as to increased hydration of the treated leaflets caused by the hypotonic buffer. CONCLUSION: SDS treatment produced a more extensible tissue with equal strength compared with the fresh aortic valve. Functionality experiments with SDS-treated whole aortic roots showed complete valve leaflet competence under physiological pressures (120 mmHg) as well as physiological leaflet kinematics.