Paul F. Gratzer

Control of pH alters the type of cross‐linking produced by 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide (EDC) treatment of acellular matrix vascular grafts

Carbodiimide cross-linking of bioprosthetic materials has been shown to provide tissue stabilization equivalent to that of glutaraldehyde cross-linking, but without the risk of the release of unreacted or depolymerized cytotoxic reagent after implantation. In this study, the effects of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) treatment on acellularized ovine carotid arteries were studied under two different pH conditions: (i) pH controlled at an optimal value of 5.5; and (ii) a simpler, but industrially significant, uncontrolled pH system. A multimode approach was employed involving biochemical assays, thermomechanical, tensile, and shear mechanical testing, and in vitro enzyme degradation analyses. EDC treatment decreased the hoop tangent modulus of acellular matrix (ACM) arterial grafts measured at 20 kPa of stress regardless of pH control. Extensibility of ACM arterial grafts measured at 20 kPa of stress was reduced after EDC treatment with pH control only. In contrast, shear stiffness of ACM arterial grafts increased to a greater degree under cross-linking without pH control (21 x compared to 14 x with pH control). Thermomechanical analyses revealed that EDC cross-linking with pH control also increased the collagen denaturation temperature of ACM arteries to a greater degree (a rise of 24.3 +/- 0.6 degrees C vs. 21.7 +/- 0.7 degrees C for no pH control), whereas cross-linking without pH control consumed a larger amount of lysine residues after 3 h of treatment. Most interestingly, both EDC treatments were equally effective in stabilizing ACM arteries against multiple degradative enzymes in vitro. The observed differences between EDC treatments under different pH conditions are attributed to differences in the location and types of the exogenous cross-links formed. The absence of pH control may have favored the formation of interfibrillar or intermolecular cross-links in collagen as well as involvement of other extracellular matrix components (proteoglycans and glycosaminoglycans). Furthermore, it may be emphasized that the location or type of cross-links differentially affected the mechanical behavior of treated materials without affecting the increase in resistance to enzymatic degradation.

Repair of tympanic membrane perforation using novel adjuvant therapies: A contemporary review of experimental and tissue engineering studies

OBJECTIVE To perform a contemporary review of experimental studies to describe the effects of various novel adjuvant therapies in enhancing tympanic membrane (TM) perforation healing. METHODS A PubMed search for articles from January 2000 to June 2012 related to TM perforation, along with the references of those articles, was performed. Inclusion and exclusion criteria were applied to all experimental studies assessing adjuvant therapies to TM healing. RESULTS Many studies have assessed the efficacy of biomolecules or growth factors, such as epidermal growth factors and basic fibroblast growth factors, in TM regeneration with significant success. More recent strategies in TM tissue engineering have involved utilizing bioengineered scaffold materials, such as silk fibroin, chitosan, calcium alginate, and decellularized extracellular matrices. Most scaffold materials demonstrated biocompatibility and faster TM perforation healing rates. CONCLUSION Although several studies have demonstrated promising results, many questions still remain, such as the adequacy of animal models and long-term biocompatibility of adjuvant materials. As well, further studies comparing various adjuvant substances and bioscaffolds are required prior to clinical application.

Effect of basic fibroblast growth factor on the cellular repopulation of decellularized anterior cruciate ligament allografts.

The use of decellularized anterior cruciate ligament (ACL) allografts in ACL replacement surgery may allow for the native structure of the ligament to be retained, thereby recapturing the function of the ligament post‐injury. Our previous work has focused on repopulating decellularized allograft ACL tissue with ACL fibroblasts in order to prevent destructive remodelling of the implanted tissue by extrinsic host cells. In this study, the use of basic fibroblast growth factor (bFGF) to improve the cellular repopulation of decellularized ACL tissue was assessed. A concentration of 6 ng/ml bFGF was demonstrated to be effective in increasing cellular growth in the absence of tissue; however, this concentration, as well as reduced and increased levels of bFGF (0.1 and 60 ng/ml, respectively), failed to increase cellular repopulation of ACL fibroblast‐seeded decellularized tissue after 28 days of culture. Mean repopulation levels of 11–19% of fresh tissue [3200–5300 cells/mg dry weight (dwt) tissue] were achieved after 28 days in culture. Qualitative observation of histological samples suggested that different repopulation characteristics exist at various concentrations of bFGF and, in particular, that bFGF may be stimulating a catabolic pathway resulting in matrix destruction. Significant differences in the effects of bFGF observed between cell‐only and cell‐and‐tissue studies serve to reinforce the concept that cells respond to stimuli in a different manner, depending on the surrounding environment. As a result, caution should be used when information obtained from studies utilizing cells alone is applied to the development of tissue‐engineered constructs. Copyright

Development and characterization of decellularized human nasoseptal cartilage matrix for use in tissue engineering.

Reconstruction of cartilage defects in the head and neck can require harvesting of autologous cartilage grafts, which can be associated with donor site morbidity. To overcome this limitation, tissue‐engineering approaches may be used to generate cartilage grafts. The objective of this study was to decellularize and characterize human nasoseptal cartilage with the aim of generating a biological scaffold for cartilage tissue engineering.

Efficient decellularization of rabbit trachea to generate a tissue engineering scaffold biomatrix

OBJECTIVES Most tracheal decellularization protocols are lengthy and can lead to reduced biomechanical stability. The objectives of this study were: 1) to generate a tracheal extracellular matrix scaffold using an efficient decellularization process and 2) to characterize the decellularized scaffold to assess its suitability for tissue engineering applications. METHODS Twelve rabbit tracheae underwent a decellularization process that involved enzymatic-detergent treatments. For characterization, fresh (control) and decellularized tissues underwent histological, immunohistochemical, and biochemical analyses. Tensile testing, scanning electron microscopy, and biocompatibility assay were also conducted. RESULTS Post-decellularization, the tracheal tissue had significantly less genetic material while the structural integrity was maintained. Specifically, the deoxyribonucleic acid content was significantly reduced and the glycosaminoglycan content was unchanged. Cell and cellular components were largely removed; at the same time the tensile properties and surface ultrastructural characteristics were unaltered. Biocompatibility was confirmed by contact cytotoxicity assay. CONCLUSIONS Overall, an efficient decellularization process was used to treat rabbit tracheal tissue. The effectiveness of the decellularization process was demonstrated and at the same time there was preservation of the underlying extracellular matrix structure. This decellularized material may serve as a potential scaffold for tracheal tissue engineering.