Joanne Ingram

Development and Characterization of an Acellular Porcine Medial Meniscus for Use in Tissue Engineering

The objectives of this study were to characterize fresh porcine menisci and develop a decellularization protocol with a view to the generation of a biocompatible and biomechanically functional scaffold for use in tissue engineering/regeneration of the meniscus. Menisci were decellularized by exposing the tissue to freeze-thaw cycles, incubation in hypotonic tris buffer, 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors, nucleases, hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic acid and final washing in phosphate-buffered saline. Histological, immunohistochemical, and biochemical analyses of the decellularized tissue confirmed the retention of the major structural proteins. There was, however, a 59.4% loss of glycosaminoglycans. The histoarchitecture was unchanged, and there was no evidence of the expression of the major xenogeneic epitope, galactose-alpha-1,3-galactose. Biocompatibility of the acellular scaffold was determined by using contact cytotoxicity and extract cytotoxicity tests. Decellularized tissue and extracts were not cytotoxic to cells. Biomechanical properties were determined by indentation and tensile tests, which confirmed the retention of biomechanical properties following decellularization. In conclusion, this study has generated data on the production of a biocompatible, biomechanically functional scaffold for use in meniscal repair.

Tissue engineering of vascular conduits

Autologous conduits are not available in up to 40 per cent of patients with arteriopathy who require coronary or lower limb revascularization, and access sites for renal dialysis may eventually become exhausted. Synthetic prostheses achieve a poor patency rate in small‐calibre anastomoses. This review examines how vascular tissue engineering may be used to address these issues.

Tissue Engineering Small-Diameter Vascular Grafts: Preparation of a Biocompatible Porcine Ureteric Scaffold

This study aimed to investigate a biocompatible, biomechanically functional, small-diameter (<6 mm) scaffold for tissue engineering a vascular graft using acellular porcine ureters. Porcine ureters were decellularized and sterilized using sequential treatment with hypotonic Tris buffer, sodium dodecyl sulphate 0.1% w/v (plus proteinase inhibitors), nuclease solution (RNase and DNase), and peracetic acid. The scaffold was compared with fresh ureter according to histology, immunocytochemistry, quantitative determination of alpha-galactosyl (alpha-Gal), and biochemistry. The biomechanical properties of the scaffold were compared with those of fresh ureters and human saphenous vein. The biocompatibility of decellularized ureters was assessed using in vitro contact and extract cytotoxicity tests. The in vivo biocompatibility was investigated using a mouse model. The histioarchitecture of the acellular ureteric scaffolds was preserved with some loss of basement membrane proteins while showing no evidence of cellularity. There was no evidence of residual alpha-Gal epitope present in acellular ureter. The ultimate tensile strength, compliance, and burst pressures of the acellular ureters were not compromised, compared with fresh tissues (p > 0.05), and the results compared favorably with fresh human saphenous vein samples (p > 0.05). The decellularized scaffolds were shown to be biocompatible with porcine smooth muscle and endothelial cells in vitro. One month after subcutaneous implantation in mice, explants were analyzed immunohistochemically using anti-CD3, Factor VIII, F4/80 (macrophage), and alpha-smooth muscle actin antibodies. The fresh tissue controls had a significantly thicker capsule (of inflammatory cells and fibrous tissue) than decellularized implants (p < 0.05). Decellularized explants were infiltrated with a combination of fibroblast-like cells and macrophages, indicating a healthy repair process. This study has demonstrated the potential of acellular porcine ureteric scaffolds in tissue engineering small-diameter living vascular grafts.

De novo designed positively charged tape-forming peptides: self-assembly and gelation in physiological solutions and their evaluation as 3D matrices for cell growth

Learning to control self-assembling nanostructures is currently one of the biggest challenges and promises in nanoscale science and nanotechnology. Nanostructured 3D matrices in particular are considered essential components in tissue engineering and regenerative medicine, e.g. as multifunctional scaffolds for cell encapsulation, growth and differentiation. Self-assembling peptide gels are a promising novel class of matrices for tissue engineering. Recently a new versatile family of negatively or positively charged tape-forming peptides have been de novo designed. These peptides were all found to self-assemble into nanostructured networks and gel cell transport medium in a simple, consistent and reproducible manner. Here we focus on the positively charged peptides of this family. We systematically changed the peptide to be amphiphilic or completely polar, or to be based on different polar uncharged amino acids (glutamine, serine, asparagine or threonine). The peptides were sterilised by γ-irradiation and were all found to be biocompatible using the contact cytotoxicity test. L929 murine fibroblast cells were encapsulated in 3D cell cultures inside 2% w/v gels and their proliferation was measured after 14 days using the ATP Lite assay. In this way a structure–function activity was established. Trifluoroacetic acid present in the peptide from the purification step was found to have a negative effect on cell proliferation. Peptide self-assembly in physiological conditions was studied extensively using spectroscopic and microscopic techniques, allowing rationalization of the observed biological structure–function activity. This detailed and systematic study enables us to develop refined criteria for the design of positively charged tape forming peptides and gels for biological and medical applications.

Decellularised porcine ureter (DURE) is a strong, biocompatible and compliance‐matched scaffold for tissue engineering of a novel small calibre cardiovascular graft

(0·82 [0·52–1·46] versus 1·62 [0·86–2·34], p < 0·001), yet soluble P-selectin was significantly increased (43·26 [13·88–86·7] versus 24·67 [13·41–43·32], p = 0·039). Stimulated P-selectin was similar in both groups. Markers of coagulation were significantly increased in patients on HD: TAT 4·59 (2·67–6·04) versus 2·84 (1·81–3·82), p < 0·001 and D-dimer 876·5 (434·2–1338·5) versus 265·5 (175·0–401·51), p < 0·001. Conclusion: Patients on HD have a pro-thrombotic state with chronically activated platelets and elevated markers of coagulation. Drug therapy to counteract this pro-thrombotic state should be considered with the aim of preventing both cardiac events and vascular access thrombosis.