Chemical cross‐linking of xenopericardial biomeshes: A bottom‐up study of structural and functional correlations

Decellularized bovine pericardium (DBP)‐based biomeshes are the gold standard in reconstructive surgery. In order to prolong their stability after the transplantation, various chemical cross‐linking strategies are employed. However, structural and functional properties of the biomeshes differ in dependence on the cross‐linker used. Here, we performed a bottom‐up study of structural and functional alterations of DBP‐based biomeshes following cross‐linking with hexamethylene diisocyanate (HMDC), ethylene glycol diglycidyl ether (EGDE), 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC) and genipin. The in vitro cytotoxicity tests supported their clinical applicability. Their structural differences (eg roughness, fibre thickness, pore morphology) were evaluated using the two‐photon confocal laser scanning, atomic force, scanning electron and polarized light microscopies. HMDC and EDC samples appeared to be the roughest. Complex mechanical trials indicated the tendency to reduced Young’s Modulus and mechanical anisotropy values of DBP upon cross‐linking. The lowest mechanical anisotropy was found in EDC and genipin sample groups. In vitro collagenase susceptibility was the highest for EDC samples and the lowest for EGDE samples. The comparative analysis of the results allowed us to recognize the strengths and weaknesses of each cross‐linker in relation to a particular clinical application.     

Structural assessments in decellularized extracellular matrix of porcine semilunar heart valves: Evaluation of cell niches

Tissue‐engineered heart valves aim to reproduce the biological properties of natural valves with anatomically correct structure and physiological performance. The closest alternative to creating an ideal heart valve substitute is to use decellularized porcine heart valves, due to their anatomy and availability. However, the immunological barrier and the structural maintenance limit the long‐term physiological performance of decellularized porcine heart valves. This study investigated the extracellular matrix (ECM) structure of aortic and pulmonary porcine valves decellularized by a low concentration sodium dodecyl sulfate (SDS)‐based method in order to determine the ECM scaffold (ECMS) conditions related to remodeling potential. To assess the structures of the leaflets and conduits of the heart valves, ECM components and their organization were evaluated by histology, biochemical analysis (BC), scanning electron microscopy, multiphoton microscopy, tensile test, immunofluorescence labeling (IF), and Raman microspectroscopy used to draw a profile of the cell niches. Histology and multiphoton imaging of decellularized aortic and pulmonary leaflets and conduits revealed a collagen and elastin histoarchitecture with rearrangement, loosening fibers, and glycosaminoglycan depletion confirmed by biochemistry quantification. The potential cytotoxicity of SDS residues was eliminated after 10 wash cycles. The mechanical properties of the structure of the valve indicated a functional resistance of decellularized ECM. The IF demonstrated the presence of basement membrane, suggesting a potential structure for host cell attachment. The RM analysis showed evidence of molecular interactions, suggesting conservation of the chemical composition, particularly among the protein molecular structures. The structural analyses performed in the semilunar porcine heart valves demonstrate that decellularized ECMS has structural properties that support physiological performance and potential host tissue integration. In fact, decellularized leaflet scaffolds were prone to cell interaction after human adipose‐derived stromal cell seeding and culturing. Further analysis of biocompatibility, particularly the ECM‐cell interaction, can elucidate the remodeling process, in preserved decellularized heart valve scaffold.     

Toward acellular xenogeneic heart valve prostheses: Histological and biomechanical characterization of decellularized and enzymatically deglycosylated porcine pulmonary heart valve matrices

The use of decellularized xenogeneic heart valves might offer a solution to overcome the issue of human valve shortage. The aim of this study was to revise decellularization protocols in combination with enzymatic deglycosylation, in order to reduce the immunogenicity of porcine pulmonary heart valves, in means of cells, carbohydrates, and, primarily, Galα1-3Gal (α-Gal) epitope removal. In particular, the valves were decellularized with sodium dodecylsulfate/sodium deoxycholate (SDS/SD), Triton X-100 + SDS (Tx + SDS), or Trypsin + Triton X-100 (Tryp + Tx) followed by enzymatic digestion with PNGaseF, Endoglycosidase H, or O-glycosidase combined with Neuraminidase. Results showed that decellularization alone reduced carbohydrate structures only to a limited extent, and it did not result in an α-Gal free scaffold. Nevertheless, decellularization with Tryp + Tx represented the most effective decellularization protocol in means of carbohydrates reduction. Overall, carbohydrates and α-Gal removal could strongly be improved by applying PNGaseF, in particular in combination with Tryp + Tx treatment, contrary to Endoglycosidase H and O-glycosidase treatments. Furthermore, decellularization with PNGaseF did not affect biomechanical stability, in comparison with decellularization alone, as shown by burst pressure and uniaxial tensile tests. In conclusion, valves decellularized with Tryp + Tx and PNGaseF resulted in prostheses with potentially reduced immunogenicity and maintained mechanical stability.     

Flow-controlled fluoroscopic angiography for the assessment of vascular integrity in bioengineered kidneys

Perfusion decellularization has been proposed as a promising method for generating nonimmunogenic organs from allogeneic or xenogeneic donors. Several imaging modalities have been used to assess vascular integrity in bioengineered organs with no consistency in the methodology used. Here, we studied the use of fluoroscopic angiography performed under controlled flow conditions for vascular integrity assessment in bioengineered kidneys. Porcine kidneys underwent ex vivo angiography before and after perfusion decellularization. Arterial and venous patencies were defined as visualization of contrast medium (CM) in distal capillaries and renal vein, respectively. Changes in vascular permeability were visualized and quantified. No differences in patency were detected in decellularized kidneys compared with native kidneys. However, focal parenchymal opacities and significant delay in CM clearance were detected in decellularized kidneys, indicating increased permeability. Biopsy-induced leakage was visualized in both groups, with digital subtraction angiography revealing minimal CM leakage earlier than nonsubtracted fluoroscopy. In summary, quantitative assessment of vascular permeability should be coupled with patency when studying the effect of perfusion decellularization on kidney vasculature. Flow-controlled angiography should be considered as the method of choice for vascular assessment in bioengineered kidneys. Adopting this methodology for organs premodified ex vivo under normothermic machine perfusion settings is also suggested.     

Optimizing the decellularization process of an upper limb skeletal muscle; implications for muscle tissue engineering

Upper limb muscle reconstruction is required following cancer resection, trauma, and congenital deformities. Current surgical reconstruction of the muscle involves local, regional and free flaps. However, muscle reconstruction is not always possible due to the size of the defect and functional donor site morbidity. These challenges could be addressed with the production of scaffolds composed of an extracellular matrix (ECM) derived from decellularized human skeletal muscle. This study aimed to find an optimal technique to decellularize a flexor digitorum superficialis muscle. The first two protocols were based on a detergent only (DOT) and a detergent-enzymatic protocol (DET). The third protocol avoided the use of detergents and proteolytic enzymes (NDNET). The decellularized scaffolds were characterized using qualitative techniques including histological and immunofluorescent staining and quantitative techniques assessing deoxyribonucleic acid (DNA), glycosaminoglycan (GAG), and collagen content. The DOT protocol consisting of 2% SDS for 4 hours was successful at decellularizing human FDS, as shown by DNA content assay and nuclei immunofluorescence staining. The DOT protocol maintained the microstructure of the scaffolds as shown by Masson’s trichrome staining and collagen and GAG content. DET and NDNET protocols maintained the ECM, but were unsuccessful in removing all DNA content after two cycles of decellularization. Decellularization of skeletal muscle is a viable option for muscle reconstruction using a detergent only technique for upper limb defects. Further testing in vivo will assess the effectiveness of decellularized scaffolds for upper limb muscle skeletal tissue engineering.     

不同去细胞方法对兔肌腱力学性能影响的实验研究

目的探讨不同去细胞方法对兔肌腱组织生物力学性能的影响,选出肌腱最佳的去细胞方法。方法应用6种去细胞方法1%TritonX-100、0.5%SDS、1%TnBP、1%TritonX-100+0.5%SDS、1%TnBP+0.5%SDS及1%TnBP+1%TritonX-100对兔半腱肌、趾屈肌腱行去细胞处理24h后行拉伸力学测试,比较其对兔肌腱生物力学性能的影响;对照组为新鲜肌腱。结果各去细胞肌腱组的破坏荷载与对照组比较均有一定的降低,1%TritonX-100、0.5%SDS1、%TnBP及1%TnBP+0.5%SDS组间无明显差异,1%TritonX-100+0.5%SDS去细胞组拉伸强度降低显著,但与对照组无明显差异(P>0.05)。去细胞肌腱的延伸率1%Tn-BP组与对照组有明显差异(P<0.01),余各组间无明显差异。结论拉伸实验表明各种去细胞处理均可最大程度保留肌腱的生物力学特性,单纯的TnBP组对肌腱的拉伸有一定的影响。     

Comparative evaluation of decellularized porcine liver matrices crosslinked with different chemical and natural crosslinking agents

The natural liver extracellular matrix (ECM) achieved by decellularization holds great potential in the fields of tissue engineering and regenerative medicine. Additionally, the use of crosslinking agents on the ECM to stabilize its ultrastructure and enhance scaffold durability is gaining interest in tissue engineering. The objective of this study was to compare the scaffold properties of porcine liver ECM crosslinked with different agents (glutaraldehyde, genipin, and quercetin) to find the best strategy for producing a decellularized matrix with optimal and stable characteristics for transplantation and regeneration. The properties examined include mechanical properties, material stability, immunogenicity, and angiogenic capacity. Scaffolds were implanted into the greater omentum of rats, and their abilities to induce immune cell subpopulation invasion and neovascularization were evaluated. The results show that genipin crosslinking of decellularized liver matrices increased the mechanical and proangiogenic properties and reduced the inflammatory response in vivo.     

Decellularized Ear Tissues as Scaffolds for Stem Cell Differentiation

Permanent sensorineural hearing loss is a major medical problem and is due to the loss of hair cells and subsequently spiral ganglion neurons in the cochlea. Since these cells lack the capacity of renewal in mammals, their regeneration would be an optimal solution to reverse hearing loss. In other tissues, decellularized extracellular matrix (ECM) has been used as a mechanical and biochemical scaffold for the induction of stem and other cells toward a target tissue phenotype. Such induced cells have been used for tissue and organ transplants in preclinical animal and human clinical applications. This paper reports for the first time the decellularization of the cochlea and identification of remaining laminin and collagen type IV as a first step in preparing an ECM scaffold for directing stem cells toward an auditory lineage. Fresh ear tissues were removed from euthanized mice, a rat and a human and processed for decellularization using two different detergent extraction methods. Cochleas were imaged with scanning thin-sheet laser imaging microscopy (sTSLIM) and brightfield microscopy. Detergent treatment of fresh tissue removed all cells as evidenced by lack of H&E and DNA staining of the membranous labyrinth while preserving components of the ECM. The organ of Corti was completely removed, as were spiral ganglion neurons, which appeared as hollow sheaths and tubes of basal lamina (BL) material. Cells of the stria vascularis were removed and its only vestige left was its laterally linking network of capillary BL that appeared to “float” in the endolymphatic space. Laminin and type IV collagen were detected in the ECM after decellularization and were localized in vascular, neural and epithelial BL. Further work is necessary to attempt to seed neural and other stem cells into the decellularized ECM to hopefully induce differentiation and subsequent in vivo engraftment into damaged cochleas.     

Pilot study of a novel vacuum‐assisted method for decellularization of tracheae for clinical tissue engineering applications

Tissue engineered tracheae have been successfully implanted to treat a small number of patients on compassionate grounds. The treatment has not become mainstream due to the time taken to produce the scaffold and the resultant financial costs. We have developed a method for decellularization (DC) based on vacuum technology, which when combined with an enzyme/detergent protocol significantly reduces the time required to create clinically suitable scaffolds. We have applied this technology to prepare porcine tracheal scaffolds and compared the results to scaffolds produced under normal atmospheric pressures. The principal outcome measures were the reduction in time (9 days to prepare the scaffold) followed by a reduction in residual DNA levels (DC no‐vac: 137.8±48.82 ng/mg vs. DC vac 36.83±18.45 ng/mg, p<0.05.). Our approach did not impact on the collagen or glycosaminoglycan content or on the biomechanical properties of the scaffolds. We applied the vacuum technology to human tracheae, which, when implanted in vivo showed no significant adverse immunological response. The addition of a vacuum to a conventional decellularization protocol significantly reduces production time, whilst providing a suitable scaffold. This increases clinical utility and lowers production costs. To our knowledge this is the first time that vacuum assisted decellularization has been explored. Copyright © 2015 John Wiley & Sons, Ltd.     

Hear the beat: decellularized mouse heart regenerated with human induced pluripotent stem cells

Heart tissue engineering holds a great potential for human heart disease therapy. Regeneration of whole biofunctional human heart is the ultimate goal of tissue engineering. Recent advances take the first step towards whole heart regeneration. However, a substantial amount of challenges have to be overcome.