Abstract used only recently in the 1980s, to employ

Abstract

Recent progress in the field of organ bioengineering based on decellularized organ scaffolds holds strong promise in addressing issues of donor organ shortage for transplantation in end-stage organ failure, biocompatibility of materials, continuous vascularisation and long-term functionality of regenerated tissues. This review highlights the key components in tissue engineering and approaches the perfusion-decellularization derived whole-organ scaffolds to serve as platforms in organ bioengineering. Important advances have occurred in small animal models, but the function exhibited by these designed organs has been rudimentary. In conclusion, this technology needs to be scaled up to human size to be of clinical relevance.

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Introduction

Tissue Engineering is defined as a multidisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve the biological function of a tissue or whole organ.1 Although the idea of regeneration of organs or whole individuals has been well known through historical arts and literature of various cultures, in scientific research, the term tissue engineering was first used only recently in the 1980s, to employ living tissues and theoretically combine them with prosthetic materials. In addition, the actual generation of new tissue utilizing biological components, either alone or in combination with appropriate scaffolding material was further described in an article published in 1991 titled “Functional Organ Replacement: The New Technology of Tissue Engineering”.2 It was decided that with the emergence of innovative biocompatible materials it would be possible to generate new tissues successfully. Over the years, several attempts have been made by scientists to grow new tissues by seeding specialised cells on appropriately configured scaffolds; chondrocytes seeded on bone spicules, a collagen matrix to support the growth of dermal fibroblasts and keratinocyte sheets onto burn patients etc. A landmark experiment in this emerging field was in 1997 when chondrocytes were effectively seeded onto a scaffold made from poly (glycolic acid) and poly (lactic acid) cast from plastic replica of a human ear and implanted subcutaneously on the back of a mouse by Vacanti et. al. Since then, researchers have increased their efforts in finding techniques necessary to develop functional tissue replacements of clinical relevance and complex organs such as the heart, lungs, kidney, liver and pancreas, remain the main challenge and goal.

Key Components in Tissue Engineering

The following image shows a simplified overview of the general methods used in tissue engineering:

Important requirements in the regeneration of a tissue can be classified into the cells, vascularization and scaffolds. Cells used in the process can be classified by their source; autologous, obtained from the same individual to which they will be implanted, Allogeneic, obtained from the body of a donor of the same species and xenogeneic, isolated from individuals of another species. The isolated cells are then layered on a biomaterial scaffolds to produce matrices resembling the native tissue. Cells extracted from a patient, called the primary cells, are most commonly used to reduce problems with rejection or pathogenic transmission.  However, this strategy has limitations, because of the invasive nature of cell collection and chances of genetic predisposition to certain unavoidable conditions.3 Hence, an alternate source of cells in the form of stem cells, including embryonic stem cells, pluripotent stem cells and multipotent stem cells lineages have arisen. After implantation of tissue constructs, the supply of oxygen and nutrients to the implant is important to maintain the survival and functionality of the implant. This is where vascularization plays and important role because the supply process is often limited by diffusion processes that can only supply blood and nutrients to cells near or slower. Different approaches like addition of proangiogenic factors, in vitro and in vivo pre-vascularization have been initiated.4

Scaffolds

Scaffolds form the framework for the growth of cells. They are materials that have been engineered to felicitate attachment, migration, cell-cell interaction, deliver and retain cells and biochemical factors, enable diffusion of vital cell nutrients and exert certain mechanical and biological influences to augment the formation of new functional tissues. Cells are seeded onto these structures which are engineered to support a three-dimensional growth. The properties that are critical for biomaterials to be used as scaffolds are high porosity, adequate pore size, biodegradability, injectability, non-immunogenicity and controlling the 3D environment to ensure reproducibility, to name a few. Scaffolds may be biodegradable such as poly lactic acid (PLA), natural matrix such as collagen or synthetic such as polyesters, high-pressure CO2 foamed scaffolds, injectable scaffolds and novel custom scaffolds.5

Decellularized whole organ scaffolds

The use of organ derived decellularized extra cellular matrix (ECM) scaffolds took centre stage in the 2010s when 3D bioprinting and biomimetic hydrogels also arose. The process of decellularization entails the isolation of the ECM from any given tissue with minimal loss, damage or disruption, while maximizing the removal of cellular material. A brief timeline of this line of work is described as follows:

Decellularization techniques aim to remove all the cells from a tissue or organ while not eliciting immunogenic responses and simultaneously preserving the native ECM composition, biological activity and mechanical properties. Perfusion or immersion are the most efficient ways of decellularization.6

Bioengineering of Heart

The first whole organ perfusion decellularization and seeding of neonatal cardiomyocytes in rat heart was performed successfully with almost no DNA content and complete recovery of ECM in 2008. Since then, the same procedure has been attempted in bigger animals and mechanical testing of decellularized heart tissue has shown increased stiffness consistent with densification, unchanged mechanical variations in tissue properties, functional cardiac activity (calcium metabolism) and expression of tissue specific markers.7 The most recent experiments have been in engineering in situ heart valves for replacement and disease modelling.8

Bioengineering of Lung

The first reports on lung bioengineering were published in 2009 using decellularized rat lung scaffolds obtained by means of perfusion-decellularization keeping the architecture of the airways and vasculature intact. Like the heart, DNA content was decreased, and immunostaining demonstrated preservation of collagen, elastin and laminin. But, decellularized lung scaffolds showed reduced oxygen absorption capacity.9 The latest research in this direction was published in 2017 with the development of an organ culture system to support recellularization of human paediatric lung scaffold.10

Bioengineering of Liver

Perfusion-decellularization of rat liver to obtain structure preserved, collagen type I and IV, fibronectin and laminin retained decellularized liver scaffolds was first reported in 2010.11 Liver scaffolds have since been studied in mice and pigs and repopulated with primary hepatocytes. Additionally, due to the regenerative ability of liver in most mammals, hepatic stem cells have been easily seeded into decellularized liver matrix resulting in high engraftment rates and markers of hepatocytic differentiation.12

Bioengineered Kidneys

Any acute injury to kidney leading to scar formation or maladaptive repair may result in kidney fibrosis and eventually renal failure. In addition to dialysis, the only permanent solution is kidney transplant which is met with limited supply of donor organs. Therefore, it is critical to find an alternative solution. A very important milestone was achieved in 2013 when the first full report on the regeneration of a rat kidney was published. Decellularized kidney scaffolds were obtained by perfusion-decellularization with a 1% SDS-based protocol, showing preservation of the microarchitecture, particularly the glomerular, Bowman’s capsule, and tubular basement membranes. The total number of glomeruli, glomerular diameter, Bowman’s space and glomerular capillary surface area were not different when compared to cadaveric kidneys using morphometric analysis.13

Other Bioengineered organs

Many other decellularized whole organ scaffolds from murine, porcine and even human samples have been used for seeding and repopulating the appropriate cells to recover the functional repair and regeneration of some tissues. There is published data of Decellularization of pancreas and reseeded with beta islet cells14, placenta15 and bladder16. All the experimental studies have resulted in successful decellularization using perfusion, maintaining the ECM intact, seeding with differentiated or adult stem cells, appropriate vascularization resulting in recellularization and establishing functionality.

Future Challenges and Applications

Decellularized whole-organ scaffolds can be obtained from virtually any organ in the body, while preserving the specific microarchitecture of the extracellular matrix. These scaffolds serve as a great platform for organ bioengineering as they preserve the organ’s 3D blueprint. The creation of a fully functional solid organ remains in the horizon of the tissue engineering and regenerative medicine fields. Despite scientific progress, there are few examples of the human application. Two potential explanations for this may be 1) problems associated with “scale up” and 2) cell death associated with implantation. Large numbers of cells are needed to generate relatively small volumes of tissue. To ultimately be effective in humans, it will be necessary to generate relatively large volumes, starting with very few cells. Mature cells, expanded in vitro, lose efficacy. Cell implantation and its associated vascular disruption results in a relatively hypoxic environment and cell death. The potential for different cell types to be expanded in vitro and survive a relatively hostile environment, at the time of implantation is now being explored. Research efforts in the coming years should focus on specific goals to standardize the decellularization process, manipulate stem cells as desired, preserve scaffolds and organs, evaluate the immune response and reach the next mile-stone: the transplantation of bioengineered organs in a large animal model.