A Bioengineering‐Regenerative Medicine Approach for Ocular Surface Reconstruction Using a Functionalized Native Cornea‐Derived Bio‐Scaffold
Mijeong ParkAlexander RichardsonNaomi C. DelicKim NguyễnJoanna BiazikRichard ZhangLina SprogyteLamia NureenJustin G. LeesAngélica FajardoUrsula KunickiStephanie L. WatsonJohn MalesNick Di Girolamo
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Abstract Limbal stem cell deficiency (LSCD) is characterized by the loss of limbal epithelial stem cells (LESCs) which compromises corneal transparency, leading to blindness. It cannot be treated with pharmacological or corneal transplantation interventions, instead a specialized stem cell (SC) therapy is needed to restore eye health and sight. Herein, a native cornea‐derived biomaterial, a by‐product of a laser refractive surgical procedure called small incision lenticule extraction is identified as a new cell delivery matrix. Culture conditions are optimized to facilitate LESC attachment, expansion and stratification, and their identity is immunophenotyped. Using electron microscopy, bio‐constructs display stratification, similar to the architectural and cellular organization of a native mammalian cornea with formation of a basement membrane and an orderly array of collagen fibrils. Neuronal growth and depleted CD45 + /CD14 + leukocytes on lenticules are also shown, suggesting that in transplantation experiments, they will re‐innervate and not trigger a host‐mediated immune response. Finally, human lenticules are geometrically customized to successfully fit them over a LSCD murine cornea ex vivo, during which they maintain curvature. The authors are poised to conduced similar studies in live mice using these and other carriers currently used in the clinic to compare SC therapy outcomes.Keywords:
Regenerative Medicine
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Considering the limited number of available lung donors, lung bioengineering using whole lung scaffolds has been proposed as an alternative approach to obtain lungs suitable for transplantation. However, some decellularization protocols can cause alterations on the structure, composition, or mechanical properties of the lung extracellular matrix. Therefore, the aim of this study was to compare the acellular lung mechanical properties when using two different routes through the trachea and pulmonary artery for the decellularization process. This study was performed by using the lungs excised from 30 healthy male C57BL/6 mice, which were divided into 3 groups: tracheal decellularization (TDG), perfusion decellularization (PDG), and control groups (CG). Both decellularized groups were subjected to decellularization protocol with a solution of 1% sodium dodecyl sulfate. The behaviour of mechanical properties of the acellular lungs was measured after decellularization process. Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. TDG and PDG showed reduced Est and Edyn elastances after lung decellularization. Scanning electron microscopy showed no structural changes after lung decellularization of the TDG and PDG. In conclusion, was demonstrated that there is no significant difference in the behaviour of mechanical properties and extracellular matrix of the decellularized lungs by using two different routes through the trachea and pulmonary artery.
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Decellularized tissues, in which the extracellular matrix is isolated, have broad applications as implantable biomaterials and/or biological scaffolds for tissue repair, and show good clinical performance. Decellularized tissue characteristics, such as their shape, structure, mechanical properties, and biological activity, are strongly affected by the decellularization protocol. The orthotopic implantation of decellularized tissues, a common procedure, typically induces cell infiltration and extracellular matrix (ECM) reconstruction resulting in tissues that resemble the source tissues. The ectopic implantation of decellularized tissues results in reconstruction that is either adapted to the implantation site or to the decellularized tissue source. In this review, the differences between methods are discussed. In addition, new methods aimed at extending the applications of decellularized tissues are discussed, particularly methods that confer novel functions to decellularized tissues, such as devices that link native tissues with artificial materials using decellularized tissue as an intermediate.
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Abstract Decellularized extracellular matrix (ECM) scaffolds have been broadly used in tissue engineering because of their versatile bioactive nature and biomimetic properties. The ECM can be derived from various tissues, organs and cultured cells. A variety of decellularization methods have been developed to maximize the decellularization effect while minimizing the effect on ECM structures and compositions. The methods can be categorized into chemical, biological, and physical methods and their combinations. The properties and applications of ECM scaffolds are dependent on decellularization methods. This article summarizes the decellularization methods for preparation of decellularized ECM scaffolds for tissue engineering applications.
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To study the changes of tissue composition and immunogenicity of porcine and human aortic valves after decellularization.Three cryopreserved human aortic valves and 4 porcine valves were decellularized with trypsin, and the leaflet tissue was homogenized for SDS-PAGE protein electrophoresis and U-937 migration assay.Trypsin effectively removed the cells from the valve. SDS-PAGE demonstrated an obvious difference in the tissue composition between porcine and human valves. Although decellularization significantly diminished the differences between the valves, decellularized procine aortic valve stilled contained more protein components (between 26 000 and 43 000) than human valve. U-937 migration assay showed an obvious decrease of cell migration in the valves by decellularization (from 832.7×10(3) to 152.4∓31.1×10(3) for porcine valves, P<0.01, and from 644.9×10(3) to 91.2×10(3) for the human valves, P<0.01). Decellularized porcine valves induced a significantly greater cell migration than decellularized human valves (P<0.05).Decellularization with trypsin can effectively decrease the immunogenicity of human or porcine heart valve, but can not completely eliminate the antigen, and decellularized porcine valve still retain strong immunogenicity.
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