No abstract

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.

No abstract

Leg ulcers present a common and recurring problem in older people creating discomfort and distress for the patient and a great cost to the health care services. Cultured keratinocyte grafts have been used by many investigators to stimulate healing of chronic venous ulcers. It has been proposed that they may do this by producing cytokines which modulate the healing process. However, the types and levels of cytokines in the leg ulcer fluid before and during healing are not known. Wound fluid was collected from venous leg ulcers in 18 patients beneath occlusive Tegaderm dressing for 4 to 6 h. The leg ulcers were divided on clinical criteria into 'healing' and 'non-healing'. PDGF-AB, GM-CSF, IL-1 alpha, IL-1 beta, IL-6 and bFGF were measured by ELISA and the levels of IL-1 alpha, IL-1 beta and IL-6 were also measured using biological assays. The effect of leg ulcer wound fluid on fibroblast and keratinocyte proliferation was measured indirectly by 3H-thymidine incorporation and MTT assay. Total protein, albumin levels, fibronectin degrading activity and collagenase activity, both active and latent were measured. No statistically significant differences in the levels of cytokines or collagenase were identified between healing and non-healing leg ulcers in the sample of leg ulcers studied. However, this study does give valuable information concerning the levels of cytokines and collagenase in chronic leg ulcer wound fluid.

A clinical need exists for an immunologically compatible surgical patch with a wide range of uses including soft tissue replacement, body wall repair, cardiovascular applications, and as a wound dressing. This study aimed to produce an acellular matrix from human amniotic membrane for future assessment as a surgical patch and a delivery system for epithelial cells. A novel detergent-based protocol was modified to remove all cellular components from amnion to render it non-immunogenic. Amnion was harvested within 24 h after elective caesarean section (n = 12). One sample group remained fresh, whereas the other was treated with 0.03% (w/v) sodium dodecyl sulphate, with hypotonic buffer and protease inhibitors, nuclease treatment, and terminal sterilization, using peracetic acid (0.1% v/v). Fresh and treated amnion was analyzed histologically for the presence of cells, deoxyribonucleic acid (DNA), collagen, glycosaminoglycans (GAGs), and elastin. Quantitative analysis was performed to determine levels of GAGs, elastin, hydroxyproline, denatured collagen, and DNA. The biomechanical properties of the membrane were determined using uniaxial tensile testing to failure. Histological analysis of treated human amnion showed complete removal of cellular components from the tissue; the histoarchitecture remained intact. All major structural components of the matrix were retained, including collagen type IV and I, laminin, and fibronectin. Differences were observed between fresh and decellularized amnion in matrix hydroxyproline (34.7 microg/mg vs 49.7 microg/mg), GAG (42.5 microg/mg vs 85.4 microg/mg), denatured collagen (2.2 microg/mg vs 1.7 microg/mg), and elastin (359.2 microg/mg vs 490.8 microg/mg) content. DNA content was diminished after treatment. Acellular matrices were biocompatible, cells grew in contact, and there was no decrease in cell viability after incubation with soluble tissue extracts. In addition, no significant reduction in ultimate tensile strength, extensibility, or elasticity was found after decellularization. Removal of the cellular components should eliminate immunological rejection. The resulting matrix was biocompatible in vitro and exhibited no adverse effects on cell morphology or viability.

Tendon allografts are commonly used to replace damaged anterior cruciate ligaments (ACL). Some of the sterilization and preservation techniques used by tissue banks with tendon allografts are thought to impair the mechanical properties of graft tissues. The tensile mechanical properties of porcine toe extensor tendons were measured using a dynamic testing machine following either freezing, freeze-drying, freezing then irradiation at 25 kGy (2.5 MRad), freeze-drying then irradiation, or freeze-drying then ethylene oxide gas sterilization. There was a small but significant difference in Young's modulus between the frozen group (0.88 GPa + 0.09 SD) and both the fresh group (0.98 GPa 1 0.12 SD) and the frozen irradiated group (0.97 GPa 1 0.08 SD). No values of Young's modulus were obtained for the freeze-dried irradiated tendons. The ultimate tensile stress (UTS) of the freeze-dried irradiated group (4.7 MPa 1 4.8 SD) was significantly different from both the fresh and the frozen irradiated groups, being reduced by approximately 90 percent. There were no significant changes in UTS or Young's modulus between any of the other groups. If irradiation is to be used to sterilize a tendon replacement for an ACL it must take place after freeze-drying to maintain mechanical properties.

The aim of this study was to determine the biocompatibility of an acellular human amniotic membrane biomaterial, which may have clinical utility for cell delivery. Human amniotic membrane was decellularized using 0.03% (w/v) sodium dodecyl sulfate (SDS), with hypotonic tris buffer and protease inhibitors and nuclease treatment. The membrane was terminally sterilized using an optimal concentration of peracetic acid. Residual SDS present within the acellular membrane was quantified using radio-labeled C14 SDS. In vivo biocompatibility was assessed by implantation of acellular human amniotic membrane subcutaneously into mice for 3 months and comparison with fresh and glutaraldehyde-fixed tissue. Cellular infiltrate into the explanted tissues was characterized using monoclonal antibodies against the following cell surface markers: CD3, CD4, CD34, and F4/80. Calcification was determined using the Von Kossa's stain. The potential of acellular human amniotic membrane to support the attachment and proliferation, and maintain viability of primary human dermal fibroblasts and primary human dermal keratinocytes was assessed in vitro, using a static culture system. Peracetic acid at a concentration of 0.1% (v/v) was sufficient for the sterilization of acellular amniotic membrane. Levels of SDS present within the acellular tissue were 0.62 +/- 0.13 microg/mg. Analysis of explanted samples from the mice indicated that acellular amniotic membrane contained low numbers of T-cells and high numbers of fibroblastic cells, macrophages, and endothelial cells, indicative of a wound-healing response. There was no evidence of calcification present within explanted acellular amniotic membrane compared to explanted glutaraldehyde-fixed amniotic membrane. Acellular amniotic membrane was shown to be capable of supporting the attachment and proliferation of primary human fibroblasts and keratinocytes. The viability of the cells was maintained for up to 4 weeks. Cell-seeded acellular amniotic membrane has the potential for delivering autologous or allogeneic cells to treat a variety of conditions, including diabetic foot ulcers, corneal defects, and severe skin burns.

No abstract