Decellularized Scaffolds

The use of decellularised matrices as scaffolds offers the advantage of great similarity with the tissue to be replaced. Moreover, decellularised tissues and organs can be repopulated with the patient’s own cells to produce bespoke therapies. Great progress has been made in research and development of decellularised scaffolds, and more recently, these materials are being used in exciting new areas like hydrogels and bioinks. However, much effort is still needed towards preserving the original extracellular matrix composition, especially its minor components, assessing its functionality and scaling up for large tissues and organs. Emphasis should also be placed on developing new decellularisation methods and establishing minimal criteria for assessing the success of the decellularisation process. The aim of this review is to critically review the existing literature on decellularised scaffolds, especially on the preparation of these matrices, and point out areas for improvement, fini...

Tissue engineering is a newly emerging biomedical technology, which aids and increases the repair and regeneration of deficient and injured tissues. It employs the principles from the fields of materials science, cell biology, transplantation, and engineering in an effort to treat or replace damaged tissues. Tissue engineering and development of complex tissues or organs, such as heart, muscle, kidney, liver, and lung, are still a distant milestone in twenty-first century. Generally, there are four main challenges in tissue engineering which need optimization. These include biomaterials, cell sources, vascularization of engineered tissues, and design of drug delivery systems. Biomaterials and cell sources should be specific for the engineering of each tissue or organ. On the other hand, angiogenesis is required not only for the treatment of a variety of ischemic conditions, but it is also a critical component of virtually all tissue-engineering strategies. Therefore, controlling the dose, location, and duration of releasing angiogenic factors via polymeric delivery systems, in order to ultimately better mimic the stem cell niche through scaffolds, will dictate the utility of a variety of biomaterials in tissue regeneration. This review focuses on the use of polymeric vehicles that are made of synthetic and/or natural biomaterials as scaffolds for three-dimensional cell cultures and for locally delivering the inductive growth factors in various formats to provide a method of controlled, localized delivery for the desired time frame and for vascularized tissue-engineering therapies.

Biomaterials based on seeding of cells on decellularized scaffolds have gained increasing interest in the last few years and suggested to serve as an alternative approach to bioengineer artificial organs and tissues for transplan-tation. The reaction of the host toward the decellularized scaffold and transplanted cells depends on the biocom-patibility of the construct. Before proceeding to the clinical application step of decellularized scaffolds, it is greatly important to apply a number of biocompatibility tests in vitro and in vivo. This review describes the different methodology involved in cytotoxicity, pathogenicity, immunogenicity and biodegradability testing for evaluating the biocompatibility of various decellularized matrices obtained from human or animals.

ABSTRACT
No ideal cross-linking agent has been identified for decellularized livers (DLs) yet. In this study, we evaluated structural improvements and biocompatibility of porcine DLs after cross-linking with silver nanoparticles (AgNPs). Porcine liver slices were decellularized and then loaded with AgNPs (100 nm) after
optimization of the highest non-toxic concentration (5 mg/mL) using Human hepatocellular carcinoma
(HepG2) and EAhy926 human endothelial cell lines. The cross-linking effect of AgNPs was evaluated
and compared to that of glutaraldehyde and ethyl carbodiimide hydrochloride and N-hydroxysuccinimide. The results indicated that AgNPs improved the ultra-structure of DLs’ collagen fibres with good
porosity and increased DLs’ resistance against in vitro degradation with good cytocompatibility. AgNPs
decreased the host inflammatory reaction against implanted porcine DL slices in vivo and increased the
polarization of M2 macrophages. Thus, structural and functional improvements of Porcine DLs could be
achieved using AgNPs.

"The development of cardiovascular prostheses derived of biological tissue in recent years has allowed to extend the time and improve the quality of life of patients requiring bioprostheses such as heart valves and vascular grafts. However, despite relevant advances, some complications have been found in these bioprostheses such as biological matrix deterioration and tissue degeneration associated to calcification. Therefore, in order to overcome the limitations of these clinical and experimental medical devices in addition to socio-economic constraints in accessing such devices, various research projects have provided alternatives to address these disadvantages. Sereval strategies have been designed to answer the various complications of bioprostheses. These strategies can be grouped into (i) tissue decellularization procedures to remove cellular antigens, (ii) tissue fixation procedures to suppress inmunogenecity and increase durability and (iii) functionalization to reduce citotoxicity and calcification.
This book chapter has the purpose to consider the research that has been performed with pericardial tissue and other collagen-rich biological tissues used as biomaterials in the construction of bioprostheses such as heart valves and vascular grafts. With this en mind, the chapter objetives are:
• To describe the composition and structure of bovine pericardium
• To provide a review on the chemical, physical and enzimatic modifications for collagenous tissues used as biomaterials
• To understand how different treatments affect biological, physical and biomechanical properties of pericardial biomaterial
• To describe the relationship tissue decellularization treatments- acellular tissue properties for bovine pericardial tissue
• To describe the influence of several crosslinking methods on tissue degeneration, calfication and biocompatibility
• To demostrate the use of polyurethane prepolymer as crosslinking agent effective for pericardial tissue (own research)
• To describe the functionalization of pericardial tissue with a thiol compound (own research)"

Decellularization is an attractive method for scaffold designing in regenerative medicine. The resulting extracellular matrix (ECM) consists of structural proteins such as collagen and elastin, growth factors, and glycosaminoglycans, which can direct site-appropriate remodeling after in vivo implantation. Mainly, collagen and elastin of ECM
are exposed to the enzymatic biodegradation in the host. To control the biodegradation process, treatment of
decellularized tissue by a cross-linking agent is required. Cross-linking also reduces antigenicity and increases the
storage properties. Cross-linkers should be nontoxic, with the ability to preserve the ECM components, especially
glycosaminoglycans and associated growth factors for retention of scaffold bioactivity. In this review, we describe
the different cross-linking agents and methods of evaluation of cross-linking efficiency.

FIELD: medicine; biology.

SUBSTANCE: invention refers to biology and medicine, particularly to regenerative medicine and tissue engineering, and can be used for rapid production of extracellular matrixes of organs and tissues used in reconstructive surgery for replacement and prosthetics of the involved organs and tissues. Method for decellularization of biological tissue or organ involves removing cell elements by washing biological tissue or organ with a solution containing tris(hydroxymethyl)aminomethane, sodium chloride, sodium dodecyl sulphate, sodium deoxycholate, triton X-100, and deionized water with the following content of components, wt%: tris(hydroxymethyl)aminomethane 1.7–4.1, sodium chloride 4.62–11.62, sodium dodecyl sulphate 0.08–0.16, sodium deoxycholate 0.25–0.65, triton X-100 0.65 –1.25, deionized water up to 100.

EFFECT: disclosed is a method for declerularisation of biological tissue.

1 cl, 3 dwg, 1 tbl, 2 ex

Decellularized corneas obtained from other species have gained intense popularity in the field of tissue engineering due to its role to serve as an alternative to the limited availability of high-quality donor tissues. However, the decellularized cornea is found to evoke an immune response inspite of the removal of the cellular contents and antigens due to the distortion of the collagen fibrils that exposes certain antigenic sites, which often lead to graft rejection. Therefore, in this study we tested the hypothesis that cross-linking the decellularized corneas with chondroitin sulfate may help in restoring the distorted conformationation changes of fibrous matrix and thus help in reducing the occurrence of graft rejection. Cross-linking of the decellularized cornea with oxidized chondroitin sulfate was validated by ATR-FTIR analysis. An in vitro immune response study involving healthy monocytes and differentiated macrophages with their surface marker analysis by pHrodo red, Lysotracker red, ER tracker, and CD63, LAMP-2 antibodies confirmed that the cross-linked decellularized matrices elicited the least immune response compared to the decellularized ones. We implanted three sets of corneal scaffolds obtained from goat, i.e., native, decellularized, and decellularized corneas conjugated with chondroitin sulfate into the rabbit stroma. Histology analysis, three months after implantation into the rabbit corneal stromal region, confirmed the restoration of the collagen fibril conformation and the migration of cells to the implanted constructs, affirming proper graft integration. Hence we conclude that the chondroitin sulfate cross-linked decellularized corneal matrix may serve as an efficient alternative to the allograft and human cadaveric corneas.

Three-dimensional in vitro tumor models are needed to obtain more information about drug behavior in tumors. The aim of this study is to establish a new model for hepatocellular carcinoma (HCC) using decellularized rat livers. After generating the rat liver scaffolds, HepG2 liver cancer cells were perfused via the portal vein and placed in a bioreactor for 10 days. Histology was performed to analyze cell distribution within the scaffolds. Function and tumor-related gene expression were examined by polymerase chain reaction (PCR). We evaluated the function of HepG2 cells grown on scaffolds in the presence of a well-known anti-cancer drug to investigate the potential application of our system for drug screening. The scaffolds were devoid of cellular materials and preserved extracellular matrix components. HepG2 cells grew well on the scaffolds. The PCR results showed that the cells maintained function and invasion ability at significantly higher levels than cells grown on two-dimensional (2-D) dishes or spheroids on Matrigel. Unlike the 2-D cultures, albumin secretion and alpha-fetoprotein expression in three-dimensional cultures were less susceptible to lower concentrations of the drug. Cells grown in scaffolds seemed to respond to the drug in an analogous manner to its known activity in vivo. These findings strengthen the potential use of rat liver scaffolds for screening HCC drugs. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015.

Whole kidney decellularization is a promising approach in regenerative medicine for engineering a functional organ. The reaction of the potential host depends on the biocompatibility of these decellularized constructs. Despite the proven ability of decellularized kidney scaffolds to guide cell attachment and growth, little is known about biocompatibility and hemocompatibility of these scaffolds. Our aim is to prepare decellularized kidneys of a clinically relevant size and evaluate its biocompatibility and hemocom-patibility. Porcine kidneys were cannulated via the renal artery, and then perfused with 0.1% sodium dodecyl sulfate solution. Hematoxylin and eosin as well as DAPI staining confirmed cellular clearance from native kidneys in addition to preservation of the microstructure. SEM confirmed the absence of any cellular content within the scaffold, which is maintained in a well-organized 3D architecture. Decellularized kidneys retained the intact renal vasculature upon examination with contrast radiography. The essential structural extracellular matrix molecules were well-preserved. Scaffolds were susceptible to enzymatic degradation upon collagenase treatment. Scaffolds showed a good hemocompatibility when exposed to porcine blood. Decellularization was efficient to remove 97.7% of DNA from native kidneys in addition to the immunogenic and pathogenic antigens. Scaffolds did not induce the human immune response in vitro. Decellularized kidneys were non-cytotoxic to pig kidney cells (PKs). PKs were able to grow and proliferate within the decellularized renal scaffolds with maintaining a higher function than cells grown as monolayers. Thus, we have developed a rapid decellularization technique for generating biocompatible kidney scaffolds that represents a step toward development of a transplantable organ.

FIELD: medicine. SUBSTANCE: invention refers to medicine, namely to experimental surgery, and can be used to assess viability of a tissue engineering structure when closing a critical tracheal defect on an experimental animal model. Part of the tracheal wall of the experimental animal is excised between 2nd and 7th rings to form a critical tracheal wall defect of at least 2×4 mm, fixation of tissue engineering structure in defect area. At end of 3 months after fixation of the tissue engineering, the trachea is examined by the radial technique with construction of 3D-model for evaluation of a degree of constriction of the tracheal lumen. After 6 months post- mortal histological and immunohistochemical examination of the explanted tissue engineering structure is performed, diagnostic test results are used as following: percent of tracheal lumen narrowing in area of fixation of tissue engineering structure (Fz), at narrowing by 75 % and more are assigned 0 points, less than 75 % – 1 point; evaluation of epithelisation of internal surface of tissue engineering (Se), with complete epithelisation – 1 point, with no epithelialization or partial epithelisation – 0 points; density of microvessels in a submucosal layer of a tissue engineering structure (Bc), with density of 0–25 mm^-2 is assigned 0 points, at 26^–50 mm – 1 point, more than 50 mm^-2 – 2 points. Value Lcb corresponding to the product of points is calculated by formula: Lcb=Fz*Se*Bc. In the case of the calculated value Lcb<1, the absence of viability of the tissue engineering structure is stated. At Lcb≥1 is concluded on viability of tissue engineering structure. EFFECT: method provides obtaining reliable data on biological and physiological compatibility of tissue engineering structures based on matrices of different origin when the tracheal critical defect is closed on the model of the experimental animal by creating a reliable method for evaluating the viability of the tissue engineering structure when closing the critical airway defect, allowing to assess the biological and physiological compatibility of the tracheal implant. 1 cl, 1 tbl, 2 ex, 5 dwg

Decellularization of tissues can significantly improve regenerative medicine and tissue engineering by producing natural,
less immunogenic, three-dimensional, acellular matrices with high biological activity for transplantation. Decellularized
matrices retain specific critical components of native tissues such as stem cell niche, various growth factors, and the ability
to regenerate in vivo. However, recellularization and functionalization of these matrices remain limited, highlighting the
need to improve the characteristics of decellularized matrices. Incorporating nanoparticles into decellularized tissues can
overcome these limitations because nanoparticles possess unique properties such as multifunctionality and can modify the
surface of decellularized matrices with additional growth factors, which can be loaded onto the nanoparticles. Therefore,
in this minireview, we highlight the various approaches used to improve decellularized matrices with incorporation of
nanoparticles and the challenges present in these applications.

Liver fibrosis results from excessive accumulation of extracellular matrix (ECM) proteins that distort the hepatic architecture. Progression of liver fibrosis results in cirrhosis and liver failure, and often, liver transplantation is required. The decellularized liver tissue contains different components that mimic the natural hepatic environment. We hypothesized that a decellularized liver hydrogel can be used to replace the necrotic hepatocytes and damaged ECM. Therefore, our aim in this study is to develop a therapy for treating liver fibrosis. Mice livers were decellularized and processed to form a hepatic hydrogel. We evaluated the biocompatibility and bioactivity of the hydrogel. The ability of the hydrogel to enhance the migration of hepatocytes and endothelial cells was investigated. Human hepatic stellate cell line (LX-2) activated by transforming growth factor-β1 (TGF-β1) was used as in vitro model for fibrogenesis. Then, the hydrogel was injected into the liver parenchyma of mice after the induction of liver fibrosis using thioacetamide. The resulting hydrogel maintained a complex composition, which included glycosaminoglycans, collagen, elastin, and growth factors. Hepatocytes and endothelial cells were shown to migrate toward the hydrogel in vitro. Liver hydrogel improved TGF-β1-induced LX-2 cells activation via blocking the TGF-β1/Smad pathway. The matrix was delivered successfully in vivo and enhanced the reduction of fibrosis and recovery to a nearly normal structure. In conclusion, we have demonstrated that the liver hydrogel can be utilized as an injectable biomaterial for liver tissue engineering in order to reduce the degree of fibrosis.

Liver transplantation is the last resort for liver failure patients. However, due to the shortage of donor organs, bioengineered liver generated from decellularized whole liver scaffolds and induced pluripotent stem cell (iPSC)-derived hepatocytes (iPSC-Heps) is being studied as an alternative approach to treat liver disease. Nevertheless, there has been no report on both the interaction of iPSC-Heps with a liver extracellular matrix (ECM) and the analysis of recellularized iPSC-Heps into the whole liver scaffolds. In this study, we produced porcine iPSC-Heps, which strongly expressed the hepatic markers a-fetoprotein and albumin and exhibited hepatic functionalities, including glycogen storage, lipid accumulation, low-density lipoprotein uptake, and indocyanine green metabolism. Supplementation of ECM from porcine decellularized liver containing liver-derived growth factors stimulated the albumin expression of porcine iPSC-Heps during differentiation procedures. The iPSC-Heps were reseeded into decellularized liver scaffolds, and the recellularized liver was cultured using a continuous perfusion system. The recellularized liver scaffolds were transplanted into rats for a short term, and the grafts expressed hepatocyte markers and did not rupture. These results provide a foundation for development of bioengineered liver using stem cell and decellularized scaffolds.

Whole organ decellularization is a cell removal process that creates a natural extracellular matrix for use in transplantation. A lack of an intact endothelial layer in the vascular network of decellularized organs results in blood clotting even with anti-coagulation treatment. Furthermore, shear stress caused by blood flow may affect reseeded parenchymal cells. We hypothesized that a heparin-gelatin mixture (HG) can act as an antithrombotic coating reagent and induce attachment and migration of endothelial cells (ECs) on vascular wall surfaces within decellularized livers, with subsequent parenchymal cell function enhancement. Portal vein (PV) perfusion was performed for right lateral lobe decellularization of porcine livers. We tested if HG-precoating of isolated decellularized PV could increase EC attachment and migration. Additionally, we coated PV and hepatic artery walls in decellularized liver with HG, and then repop-ulated it with ECs and maintained it under vascular flow in a bioreactor for 10 days. Re-endothelialized scaffolds were perfused with porcine blood for thrombogenicity evaluation. We then co-cultured hepa-tocellular carcinoma (HepG2) cells and ECs to evaluate the effect of endothelialization on parenchymal cells. Finally, we transplanted these scaffolds heterotopically in pigs. HG improved ECs' ability to migrate and adhere to vessel discs. ECs efficiently covered the vascular compartments within decellularized scaffolds and maintained function and proliferation after HG-precoating. No thrombosis was observed after 24 h blood perfusion in HG-precoated scaffolds, indicating an efficiently endothelialized vascular tree. HepG2 cells displayed a higher function in scaffolds endothe-lialized after HG-precoating compared to uncoated scaffolds in vitro and after in vivo transplantation. Our results lay the groundwork for engineering human-sized whole-liver scaffolds for clinical applications. Statement of Significance A major obstacle to successful organ bioengineering is vasculature reconstruction to avoid thrombosis and deliver nutrients through blood to the whole scaffold after in vivo transplantation. Although many attempts have been made to construct endothelial cell layers on the vascular network within decellular-ized organs, complete coverage has not be achieved. Here, we describe an effective approach for endothe-lial cell seeding to reconstruct a patent vascular tree within decellularized livers by coating the vasculature using heparin-gelatin mixture. Our results have demonstrate that enhancement of endothe-lial cell attachment by heparin-gelatin treatment could improve vascular patency and parenchymal cell function in vitro and in vivo. These results represent a significant advancement toward bioengineering functional liver tissue that maintains vascular patency for transplantation.

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ｔｈａｔ Ｈｕｍｂｏｌｄｔ ｃｈｉｔｉｎ ｉｓ ｍｏｒｅ ｅｆｆｅｃｔｉｖｅ ｉｎ ｓｅｒｖｉｎｇ ａｓ ａｎｔｉｂａｃｔｅｒｉａｌ ｍａｔｅｒｉａｌ ａｎｄ ｃａｎ ｂｅ ｕｔｉｌｉｚｅｄ ｆｏｒ ｗｏｕｎｄ ｃａｒｅ． Ｄｉｆｆｅｒｅｎｃｅｓ ｉｎ ｓｔｅｒｉｏｃｈｅｍｉｃａｌ ｓｔｒｕｃｔｕｒｅ ｗｅｒｅ ｏｂｓｅｒｖｅｄ ａｍｏｎｇ β - ｃｈｉｔｉｎ ｓｔｒｕｃｔｕｒｅｓ ｏｂｔａｉｎｅｄ ａｎｄ ａｍｉｎｅ ｇｒｏｕｐｓ ｐｒｅｓｅｎｃｅｓ ｗｅｒｅ ｆｏｕｎｄ ａｌｏｎｇ ｗｉｔｈ ａｂｉｌｉｔｙ ｏｆ ｍａｔｅｒｉａｌｓ ｔｏ ｓｗｅｌｌ．

To provide mechanical augmentation for rotator cuff repair, it is necessary (though perhaps not sufficient) that scaffolds have tendon-like material and suture retention properties, be applied to the repair in a surgically appropriate manner, and maintain their mechanical properties for an acceptable period of time following surgery. While allograft fascia lata has material, structural, and biochemical properties similar to tendon tissue, its poor suture retention properties abrogates its potential as an augmentation device. The goal of this work was to design a novel reinforced fascia patch with suture retention and stiffness properties adequate to provide mechanical augmentation for rotator cuff repair. Fascia was reinforced by stitching with PLLA or PLLA/PGA polymer braids. Reinforced fascia patches had a maximum construct load greater than (or equal to) the suture retention properties of human rotator cuff tendon (∼250N) at time zero and after in vivo implantation for 12 weeks in a rat subcutaneous model. The patches were able to withstand the 2500 loading cycles projected for the early post-operative period. The patches also demonstrated biocompatibility with the host using a rat abdominal wall defect model. These studies suggest the potential use of reinforced fascia patches to provide mechanical augmentation, minimize tendon retraction and possibly reduce the incidence of rotator cuff repair failure. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

Decellularized corneas obtained from other species have gained intense popularity in the field of tissue engineering due to its role to serve as an alternative to the limited availability of high-quality donor tissues. However, the decellularized cornea is found to evoke an immune response inspite of the removal of the cellular contents and antigens due to the distortion of the collagen fibrils that exposes certain antigenic sites, which often lead to graft rejection. Therefore, in this study we tested the hypothesis that cross-linking the decellularized corneas with chondroitin sulfate may help in restoring the distorted conformationation changes of fibrous matrix and thus help in reducing the occurrence of graft rejection. Cross-linking of the decellularized cornea with oxidized chondroitin sulfate was validated by ATR-FTIR analysis. An in vitro immune response study involving healthy monocytes and differentiated macrophages with their surface marker analysis by pHrodo red, Lysotr...

No ideal cross-linking agent has been identified for decellularized livers (DLs) yet. In this study, we evaluated structural improvements and biocompatibility of porcine DLs after cross-linking with silver nanoparticles (AgNPs). Porcine liver slices were decellularized and then loaded with AgNPs (100 nm) after optimization of the highest non-toxic concentration (5 µg/mL) using Human hepatocellular carcinoma (HepG2) and EAhy926 human endothelial cell lines. The cross-linking effect of AgNPs was evaluated and compared to that of glutaraldehyde and ethyl carbodiimide hydrochloride and N-hydroxysuccinimide. The results indicated that AgNPs improved the ultra-structure of DLs&#39; collagen fibres with good porosity and increased DLs&#39; resistance against in vitro degradation with good cytocompatibility. AgNPs decreased the host inflammatory reaction against implanted porcine DL slices in vivo and increased the polarization of M2 macrophages. Thus, structural and functional improvements...

The use of decellularised matrices as scaffolds offers the advantage of great similarity with the tissue to be replaced. Moreover, decellularised tissues and organs can be repopulated with the patient’s own cells to produce bespoke therapies. Great progress has been made in research and development of decellularised scaffolds, and more recently, these materials are being used in exciting new areas like hydrogels and bioinks. However, much effort is still needed towards preserving the original extracellular matrix composition, especially its minor components, assessing its functionality and scaling up for large tissues and organs. Emphasis should also be placed on developing new decellularisation methods and establishing minimal criteria for assessing the success of the decellularisation process. The aim of this review is to critically review the existing literature on decellularised scaffolds, especially on the preparation of these matrices, and point out areas for improvement, finishing with alternative uses of decellularised scaffolds other than tissue and organ reconstruction. Such uses include three-dimensional ex vivo platforms for idiopathic diseases and cancer modelling.

The development of decellularized small‐diameter vascular grafts is a potential solution for patients requiring vascular reconstructive procedures. However, there is a limitation for acellular scaffolds due to incomplete recellularization and exposure of extracellular matrix components to whole blood resulting in platelet adhesion. To address this issue, a perfusion decellularization method was developed using a custom‐designed set up which completely removed cell nuclei and preserved three‐dimensional structure and mechanical properties of native tissue (sheep carotid arteries). Afterwards, carboxymethyl kappa carrageenan (CKC) was introduced as a novel anticoagulant in vascular tissue engineering which can inhibit thrombosis formation. The method enabled uniform immobilization of CKC on decellularized arteries as a result of interaction between amine functional groups of decellularized arteries and carboxyl groups of CKC. The CKC modified graft significantly reduced platelet adhesion from 44.53 ± 2.05% (control) to 19.57 ± 1.37% (modified) and supported endothelial cells viability, proliferation, and nitric oxide production. Overall, the novel CKC modified scaffold provides a promising solution for thrombosis formation of small‐diameter vessels and could be a potent graft for future in vivo applications in vascular bypass procedures.