JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE J Tissue Eng Regen Med (2015) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.2061

REVIEW

Development of decellularized scaffolds for stem cell-driven tissue engineering Deepti Rana1, Hala Zreiqat2, Nadia Benkirane-Jessel3, Seeram Ramakrishna4 and Murugan Ramalingam1,5* 1 Centre for Stem Cell Research (CSCR), Institute for Stem Cell Biology and Regenerative Medicine (Bengaluru) Christian Medical College Campus, Vellore, India 2 Biomaterials and Tissue Engineering Research Unit, Faculty of Engineering and Bosch Institute, University of Sydney, NSW, Australia 3 INSERM, Osteoarticular and Dental Regenerative Nanomedicine Laboratory, UMR 1109, Faculté de Médecine, Strasbourg, France 4 Centre for Nanofibres and Nanotechnology, Department of Mechanical Engineering, National University of Singapore 5 WPI Advanced Institute for Materials Research, Tohoku University, Sendai, Japan

Abstract Organ transplantation is an effective treatment for chronic organ dysfunctioning conditions. However, a dearth of available donor organs for transplantation leads to the death of numerous patients waiting for a suitable organ donor. The potential of decellularized scaffolds, derived from native tissues or organs in the form of scaffolds has been evolved as a promising approach in tissue-regenerative medicine for translating functional organ replacements. In recent years, donor organs, such as heart, liver, lung and kidneys, have been reported to provide acellular extracellular matrix (ECM)-based scaffolds through the process called ‘decellularization’ and proved to show the potential of recellularization with selected cell populations, particularly with stem cells. In fact, decellularized stem cell matrix (DSCM) has also emerged as a potent biological scaffold for controlling stem cell fate and function during tissue organization. Despite the proven potential of decellularized scaffolds in tissue engineering, the molecular mechanism responsible for stem cell interactions with decellularized scaffolds is still unclear. Stem cells interact with, and respond to, various signals/cues emanating from their ECM. The ability to harness the regenerative potential of stem cells via decellularized ECM-based scaffolds has promising implications for tissue-regenerative medicine. Keeping these points in view, this article reviews the current status of decellularized scaffolds for stem cells, with particular focus on: (a) concept and various methods of decellularization; (b) interaction of stem cells with decellularized scaffolds; (c) current recellularization strategies, with associated challenges; and (iv) applications of the decellularized scaffolds in stem celldriven tissue engineering and regenerative medicine. Copyright © 2015 John Wiley & Sons, Ltd. Received 15 January 2015; Revised 22 April 2015; Accepted 4 May 2015

Keywords decellularization; scaffolds; stem cells; recellularization; tissue engineering; organ transplantation; regenerative medicine

1. Introduction Transplantation medicine is an emerging area for applied medical research. Organ transplantation is an approach for the repair and replacement of unhealthy tissues or organs, but is often hindered by dearth of donor tissues/organs and immunological problems *Correspondence to: Murugan Ramalingam, Centre for Stem Cell Research (CSCR), Unit of Institute for Stem Cell Biology and Regenerative Medicine (Bengaluru), Christian Medical College Campus, Vellore 632002, India. E-mail: [email protected] Copyright © 2015 John Wiley & Sons, Ltd.

associated with infectious diseases (Rana et al., 2014). According to a report from the US Organ Procurement and Transplantation Network, 19 426 transplants had been performed up to August 2014, whereas around 124 030 people needed a life-saving organ transplant, and this count still continues to grow (Health Resources and Services Administration, US Department of Health and Human Services, 2014). Although conventional methods, such as autografting and allografting, are clinically considered to be ’gold standard’ treatments, they have their own limitations. For example, the supply of autografts is limited and there is a possibility of

D. Rana et al.

pathogen transfer from allografts (Badylak et al., 2011). Therefore, the concept of tissue engineering has emerged as a unique and versatile approach in regenerative medicine, in which physiologically functional tissues or organs are created in the laboratory setting using cells and functional biomaterials called ‘scaffolds’ to repair or replace unhealthy tissues or organs that fail to regenerate spontaneously. A scaffold, also called a synthetic extracellular matrix (ECM), is a temporary physical support that helps to accommodate the cells and support their threedimensional (3D) growth during the tissue developmental stage. It facilitates the cells to secrete their own ECM required for tissue regeneration. A scaffold can be made from a variety of biomaterials, including polymers, bioceramics, proteins or their composites. Tissue or organ-derived biomaterials, through the process called ‘decellularization’ (also called ’decellularized biomaterials’ or ’decellularized scaffolds’) are among the best choices for the preparation of biomimetic scaffolds. The process of decellularization of a scaffold involves the discharge of cellular contents from the tissues or organs, retaining only the components of the native ECM. The decellularized scaffolds can later be recellularized with suitable cells for the purpose of creating tissue-engineered grafts suitable for tissue/organ transplantation (Figure 1). A decellularized scaffold possesses the nature-designed architecture from the whole organ, at the micro- and nanostructural level, to support cellular growth and tissue organization both in vitro and in vivo. In addition, it can be tailor-made with all the characteristic features of an ideal scaffold, such as biocompatibility, biodegradability, non-immunogenicity and the ability to provide structural, mechanical, biochemical and biological cues for

cell adhesion, proliferation, migration, differentiation and continued function, with a hope of potential clinical translation (Leor et al., 2005). Therefore, the decellularized scaffold can be considered to be an attractive system for tissue engineering. Stem cells have become an integral part of tissue engineering in general and provide a new dimension to decellularized scaffold-based tissue engineering in particular, either as a cell source for recellularization or as a source for generating ECM-based matrix. Stem cells are defined as undifferentiated cells that are able to differentiate into specific lineages (potency) and are able to divide by themselves to produce more stem cells (self-renewal). In native tissue, stem cells are housed in a 3D microenvironment (also called a niche) and their cellular behaviours are regulated or controlled by various biophysical and biochemical cues. It has been proved that the stem cell niche exerts control over the intrinsic genetic pathways regulating the multipotential and self-renewal ability via extrinsic signals from the surrounding microenvironment (Zhang and Li, 2008). Therefore, to understand the underlying mechanisms and matrix properties that can guide the stem cells towards a specific developmental lineage, it is important to look into the interactions of stem cells with the matrix. Armed with their native properties, decellularized scaffolds seeded with stem cells are attracting more attention in both basic research and clinical studies, and are currently being exploited for translating functional organs. However, the mechanisms behind the interaction of stem cells with decellularized scaffolds have still not been completely explored. Additionally, the methods of decellularization and the sources of cells used to repopulate the decellularized scaffolds are critical to the

Figure 1. The application of the decellularized scaffolds in the field of regenerative medicine. Decellularized tissue/organs can be recellularized with cells for generating bioengineered grafts suitable for tissue/organ transplantation Copyright © 2015 John Wiley & Sons, Ltd.

J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Decellularized scaffolds for tissue engineering

eventual functionality and clinical success of the engineered constructs, which paves a new avenue for further research in this fascinating field. Keeping these points in view, this article reviews the current status of decellularized scaffolds in the context of stem cell-driven tissue engineering. The article particularly focuses on: (a) the concept and various methods of decellularization; (b) interaction of the stem cells with decellularized scaffolds; (c) current strategies for recellularization and their associated challenges; and (d) applications of the decellularized scaffolds in stem cell-driven tissue engineering and regenerative medicine, with clinical examples.

2. Basics of stem cell-driven tissue engineering Tissue engineering is a multidisciplinary field that applies the principles of bioengineering and biosciences for the development of novel biological substitutes capable of restoring, maintaining or improving a tissue function that fails to regenerate or heal spontaneously (Ramakrishna et al., 2010). The tissue-engineering approach has the ability to develop patient-specific tissue grafts in order to mimic the functional properties of native tissues that could be transplanted back to the patient, with minimal surgical intervention and maximum host tissue integrity. The key factors responsible for the success of tissue engineering are the cells that create the tissue and the scaffold that provides structural support to the cells and cell– matrix (scaffold) interactions that direct the behaviour of the cells. Stem cells are considered to be a potent cell source for tissue-regenerative applications. Stem cells can be categorized broadly into embryonic and adult stem cells: adult stem cells are capable of differentiating into the particular germ layer lineage from which it has been derived, and also of undergoing transdifferentiation with greater plasticity (Ausim Azizi et al., 1998). Stem cells can also act as assisting cells that can promote tissue homeostasis, metabolism, growth and repair, emphasizing their importance for tissue/organ regeneration (Paschos et al., 2014). Scaffolds made up of decellularized scaffolds play a pivotal role in accommodating stem cells that can undergo proliferation, migration and differentiation, leading to the formation of a specific tissue while secreting the required ECM for tissue regeneration. For example, Kang et al. (2014) demonstrated the feasibility of decellularized scaffolds loaded with autologous adipose-derived stem cells (ADSCs) for cartilage defect repair in rabbits (Kang et al., 2014). The results of this study showed that the ADSC-loaded decellularized scaffolds induced cartilage tissue repair comparable to native cartilage in terms of mechanical properties and biochemical components. Therefore, the development of decellularized scaffolds, in combination with stem cells, with characteristics quite similar to native ECM, is an exciting area of research that opens a new arena in stem cell-driven tissue engineering (Kang et al., 2014). Copyright © 2015 John Wiley & Sons, Ltd.

3. Concept of decellularized tissues/organs Decellularization is a method that involves the removal of cellular components from any tissue/organ to generate ECM templates with a complex mixture of structural and functional proteins in order to preserve structurally organized entities, such as collagen, elastin, glycosaminoglycan (GAG) and fibronectin, to function as a biomimetic scaffold. The removal of cellular contents and antigens reduces the possibility of foreign body reaction, inflammation and potential immune rejection (Gilbert et al., 2006; Badylak et al., 1995). The concept of using decellularized scaffolds in tissue-regenerative applications relies on its structural and functional properties. This is because the use of these scaffolds warrants significant cell–matrix interactions that mediate favourable tissue organization and remodelling, as well as realizing cell–cell communications, to elucidate certain biological functionalities. It further provides the best cellular recognition amongst all scaffolds (Gui et al., 2009; Lopes et al., 2009) by preserving the overall structural composition and shape compatibility, adequate levels of mechanical integrity of the target tissue/organ, and bioactive molecules that trigger cell–cell communications, cell–matrix adhesion and new ECM formation (Baptista et al., 2012). It also facilitates constructive remodelling for a variety of tissues, in both preclinical (animal) studies and clinical applications. Various decellularized scaffolds, including small intestine submucosa (SIS), pericardium, skin, diabetic foot ulcers (Organogenesis Inc., MA, USA, 2014), cardiovascular tissues (CorMatrix ECM®, 2014), soft tissue repair (Kensey Nash Corp, 2012), thoracic and abdominal wall repair (Synovis Orthopedic and Woundcare Inc., Irvine, CA, USA, 2012) and heart valves, have been granted US Food and Drug Administration (FDA) approval (Fink, 2009) and have been successfully used in both preclinical animal studies (Lee et al., 2010) and human clinical applications (St. Jude Medical Inc., MO, USA, 2004). Additionally, decellularized scaffolds maintain a dynamic reciprocity relationship between the ECM and the resident cell population in a particular tissue/organ by maintaining the native composition and ultrastructure of the tissue’s or organ’s ECM and constantly changing it in response to the current metabolic activity of the resident cell population, the mechanical demands of the tissue and the prevailing microenvironment conditions (Bissell and Aggeler, 1987). Interestingly, it has been reported that decellularized scaffolds exhibit their organ-specific behaviour with regard to the source of the template tissue/organ ECM for organ restoration, e.g. decellularized scaffold harvested from heart tissue may be the preferred ECM substrate for cardiovascular progenitor cells (Sellaro et al., 2007). Table 1 lists commercially available decellularized scaffolds useful for tissueengineering and regenerative applications. Similar to the native ECM matrix, decellularized scaffold also shows the ability to modulate its interactions with the seeded cells in terms of their attachment and J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Copyright © 2015 John Wiley & Sons, Ltd.

Acellular porcine dermal implant Decellularized porcine heart valves

Stryker Corp., USA

CorMatrix® Cardiovascular Inc., USA Kensey Nash Corp., USA

IOP Inc., Costa Mesa, CA

Synovis Orthopedic and Woundcare Inc., Irvine, CA, USA Karocell Tissue Engineering AB, Sweden BiowelSciences, Islamabad, Pakistan

Covidien Inc., Dublin, Ireland

CryoLife Inc., USA

CorMatrix ECM™

Meso BioMatrix™

IOPatch™

OrthAdapt®, Unite®

Permacol Surgical Implant Synergraft®

SureDerm

Karoderm

Strattice™

Human acellular lyophilized dermis

Highly organized collagen scaffold derived from decellularized equine pericardium Acellular cadaver dermis

Fetal collagen scaffold from acellular fetal bovine dermis Acellular scaffold from porcine small intestine Acellular tissue graft derived from porcine peritoneum Acellular human sclera graft

Bioresorbable collagen scaffold derived from acellular porcine small intestine Acellular porcine dermis

TissueMend®

Acellular collagen and elastin scaffold derived from porcine dermal tissue Porcine acellular urinary bladder matrix Porcine small intestine submucosa (SIS) acellular grafts Porcine derived acellular SIS

FortaFlex®

Acell Inc., Columbia Cook Biotech Inc. USA

B. Braun Melsungen AG, Germany Zimmer Dental Inc., Warsaw, IN, USA Zimmer, UK

Bovine acellular pericardium valve leaflets Pure collagen implant obtained from lyophilized acellular bovine pericardium Bovine acellular pericardium membrane

Acellular porcine heart valve leaflets Porcine acellular valve Porcine acellular heart valve tissue

Human acellular premeshed dermis

Freeze-dried human acellular dermal matrix with preserved basement membrane Acellular human dermis derived from human allograft skin Acellular human dermis

Scaffolding materials

DePuy Orthopaedics Inc., IN, USA Organogenesis Inc., Boston, MA, USA LifeCell Corp., USA

Restore™

Zimmer Collagen Repair Patch™ MatriStem®, Acell Vet Oasis®, SurgiSIS®

CopiOs®

Lyoplant®

Epic™, SJM Biocor® Prima™ Plus Hancock® II, Mosaic®, Freestyle® Perimount®

Edwards Lifesciences LLC, USA

Musculoskeletal Transplant Foundation (MTF Sports Medicine), CO, USA Mentor Worldwide LLC, Santa Barbara, USA Wright Medical Technology Inc., Arlington, USA St. Jude Medical Inc., USA Edwards Lifesciences LLC, USA Medtronic Inc., USA

AlloPatch HD™, FlexHD® NeoForm™

GraftJacket®

LifeCell Corp., USA

Manufacturer

AlloDerm®

Commercial brand/name

Repair or replacement of damaged soft tissue Ventral hernia repair and abdominal wall reconstruction Cardiac tissue repair

Dermal construct

Pericardium and cardiac tissue regeneration Abdominal wall and breast reconstruction Repair and reinforcement of soft tissue Pericardial repair and reconstruction Soft tissue repair and reinforcement Ophthalmology, glaucoma implant/valve surgery Soft tissue, chronic wounds

Reinforcement of soft tissue

Soft tissue, acute and chronic wounds Soft tissue repair

Valve replacement Valve replacement Replace defective aortic and mitral valves Pericardial aortic bioprosthesis and valve replacement Repair of the dura mater in neurosurgery Guided bone regeneration and dentistry Soft tissue regeneration

Tendon augmentation and breast tissue engineering Breast, nose and other aesthetic and dermatological implants Soft tissues and chronic wounds

Hernia, abdominal wall and breast tissues reconstruction

Applications

Table 1. List of a few commercially available decellularized scaffolds used for tissue engineering and regenerative medicine applications

CryoLife Inc., Kennesaw, USA, 2014

Covidien Inc., Dublin, Ireland, 2014

BiowelSciences, Islamabad, Pakistan, 2014

IOP Ophthalmics, Costa Mesa, CA, USA, 2014 Synovis Orthopedic and Woundcare Inc., Irvine, CA, USA, 2012 Shevchenko, James and James, 2010

Kensey Nash Corp, 2012

CorMatrix ECM®, 2014

Stryker Corp., USA, 2007

DePuy Orthopaedics Inc., Warsaw, IN, USA, 2009 Organogenesis Inc., MA, USA, 2007 LifeCell Corp., Bridgewater, USA, 2014

ACell Inc., Columbia, MD, USA, 2014 Cook Biotech Inc., USA, 2014

AlloDerm® Regenerative Tissue Matrix, LifeCell Corp., USA, 2014 Mentor Worldwide LLC, Santa Barbara, CA, USA, 2008 Wright Medical Technology Inc., Arlington, USA, 2014 St. Jude Medical Brazil Ltd, USA, 2011 Edwards Lifesciences Corp., USA, 2014 Medtronic Life Line, Minneapolis, MN, USA, 2014 Edwards Lifesciences Corp., USA, 2003 B. Braun Melsungen AG, Germany, 2014 Zimmer Dental Inc., Warsaw, IN, USA, 2014 Zimmer Inc., UK, 2014

AlloDerm® Regenerative Tissue Matrix, LifeCell Corp., USA, 2014

Reference

D. Rana et al.

J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Decellularized scaffolds for tissue engineering

migration to specific locations within the scaffold (Brown et al., 2006) and modulation of tissue-specific phenotypic differentiation (Gong et al., 2008). Nevertheless, decellularized scaffolds are reported to affect the biological signalling activities of the host-remodelling response upon in vivo implantation through their degradation products, such as soluble bioactive cryptic peptides (Brennan et al., 2006). Therefore, the functional profile, the associated downstream host response and the remodelling process can be significantly modulated by inhibiting the degradation of the decellularized scaffold and the associated release of cryptic peptides. In addition to mechanical, structural and biological modulations, decellularized scaffolds can also affect the differentiation pathways of human-derived stem cells and selected progenitor cell populations (De Peppo et al., 2013; Marolt et al., 2012). Decellularized scaffolds have been shown to be suitable candidates for load-bearing functionalities, by providing the mechanical strength of collagen fibres/fibrils, the elasticity of elastin fibres and the hydration/cushion/binding functions of proteoglycans from their artificial ECM matrices (Fung, 1981). Also, decellularized scaffolds readily retain bioactive molecules for growth factor signalling and protein/biofactors that are important for cell–cell communication, cell–matrix interactions and new ECM formation (Pei et al., 2011). From the structure–function perspective, the above properties of decellularized scaffolds indicate their potential for recapitulation of the organization of target tissue/organs. Along with their superior properties over other scaffolding materials, decellularized scaffolds also possess some challenges, such as determining the optimum protocol for removing their cell/chromosome debris without disrupting the scaffold’s structural and mechanical integrity; then recellularization of the scaffolds, preventing non-homogeneous cell distribution and non-homogeneous ECM segregation. In vitro preservation, anticoagulation and endothelialization are some of the other critical factors that need to be improved further (Zhou et al., 2014a, 2014b). The characteristics of the scaffolding materials also vary depending on the method of decellularization. Therefore, all these factors should be carefully considered while designing decellularized scaffolds suitable for tissuespecific stem cell applications. The methods involved in decellularization are classified as physical, chemical, biological/enzymatic or a combination of these approaches (Gilbert, 2012; Badylak et al., 2012), which rely on a common basic principle of cell membrane disruption for leaching out all the cellular contents. Conventionally, in general, the tissue of interest is subjected to brief exposure to chemical or detergent solutions, followed by rinsing, accompanied by certain physical processes, such as freezing, thawing, agitation or manual scrapping to facilitate the easy removal of all the cellular contents. Nevertheless, these conventional techniques are only effective in tissues up to a few millimetres in thickness. Thicker tissues may require more extensive biochemical exposure, such as with Triton X-100, and longer rinse times, ranging from hours to several days. The most commonly used method of decellularization Copyright © 2015 John Wiley & Sons, Ltd.

includes the perfusion of chemical/enzymatic agents in combination with physical methods, such as sonication, freezing and thawing. The preparation of decellularized scaffolds from a pristine mammalian organ needs a variety of processing steps that can further notably affect the structure, composition and associated host response of the resultant scaffolds that these scaffolds will engender when treated as templates for organ reconstruction. The process involves exposure of the organ parenchymal cells to detergents, proteases and chemicals by perfusion into the native vasculature. It is often difficult to accomplish complete decellularization of any tissue/organ, as most of the decellularized scaffolding materials retain residual DNA and other cytoplasmic and nuclear material that can evoke problematic immune responses, such as chronic inflammation (Crapo et al., 2011). Besides, the efficacious removal of intracellular components and the antigenic epitopes related to the cell membranes of the tissues/organs have become a crucial matter because of the need to minimize the adverse immune responses generated by allogeneic or xenogeneic recipients of the decellularized scaffolds.

3.1. Physical route A physical route to decellularization means modulating the physical characteristics, e.g. temperature, force and pressure, of any tissue/organ in order to facilitate disruption of the cell membranes, promoting cell lysis. Physical methods include scraping, solution agitation, sonication, pressure gradients, snap-freezing, non-thermal irreversible electroporation and use of supercritical fluids. All these methods, along with their modes of action, effect on the ECM, advantages and disadvantages are briefly discussed in Table 2. Along with conventional physical methods, recently Hung et al. (2013) reported a combination of physical routes for larynx decellularization by combining freezedrying and sonication as an effective technique (Hung et al., 2013). Porcine larynges were made to undergo decellularization cycles that included overnight freezedrying, thawing in phosphate-buffered saline (PBS) for 30 min and washing in PBS for another 30 min. Sonication treatment was added during the thawing process. The study confirmed that use of the freeze-drying cycle’s modality has no effect on removing cellular components, whereas when sonication was added to the thawing process, a significant reduction in cellular growth was observed. However, this method may induce structural damage to the scaffold (Hung et al., 2013).

3.2. Chemical route For the removal of cellular components, the chemical route includes the use of acids and bases, hypotonic and hypertonic solutions, detergents, alcohols and other solvents. Acids and bases catalyse the hydrolytic degradation J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Copyright © 2015 John Wiley & Sons, Ltd.

Cells and tissue rupture by applying pressure

Ultrasonic cleaner removes cellular material Pressure gradients burst cells and aid in cell removal

Facilitate chemical exposure, leading to cell removal

Mechanically removes cells from the surface Micropore formation in cell membrane due to electricalpotential destabilization causes cell lysis Detaches cells from basement membrane

Mechanical force (agitation)

Sonication (ultraso und treatment) Pressure gradient system

Supercritical fluids

Scraping

Immersion and agitation

Electroporation (non-thermal, irreversible electroporation)

Intracellular ice crystals disrupt cell membranes

Mode of action

Snap-freezing

Method

ECM disruption is a function of tissue thickness/density, detergent used and intensity of agitation



Shows slow removal of cellular remnants from the tissues

Electric field oscillation can disrupt ECM



Uses inert gas (CO2) for cell removal and do not alter ECM’s mechanical properties –

Effective cell removal

Effective cell removal

Chemical exposure can be prevented

Effective cell removal

Advantages

Supercritical phase pressure can disrupt ECM

ECM ultrastructure can be disrupted



Mechanical force can damage ECM

ECM disruption during rapid freezing

Effects on ECM

Table 2. List of physical routes used for decellularization of tissues/organs

Effective processing for thin tissues and require long exposure time for thick tissues

Only small-sized tissues can be processed

Gives variable results

Not yet widely used

Parameters are not well standardized Not effective for densely organized ECM tissues

Ice crystals can disrupt ECM microstructures. Post-processing is required for scaffolds Controlling applied mechanical force is critical (no optimized standards)

Disadvantages

Heart valve, blood vessels, skeletal muscle/tendon, peripheral nerve, dermis, urinary bladder (Crapo et al., 2011)

Carotid artery (Phillips et al., 2010)

Heart tissues (Ott et al., 2008)

Umbilical veins (Montoya and McFetridge, 2009) Bladder tissue (Bolland et al., 2007) Aortic tissues (Sawada et al., 2008)

Tendinous and ligamentous tissues (Roberts et al., 1991) Nerve tissues (Gulati, 1988) Submucosal tissues (Freytes et al., 2004) Hepatic tissues (Lin et al., 2004) Heart valves (Schenke-Layland et al., 2003) Arteries (Dahl et al., 2003) Arteries (Azhim et al., 2011)

Reference

D. Rana et al.

J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Decellularized scaffolds for tissue engineering

of biomolecules, cytoplasmic components and nucleic acids; however, this could have adverse effects on GAG content. Hypertonic solutions dissociate DNA from proteins, and hypotonic solutions can readily cause cell lysis by a simple osmotic effect, with minimal changes in the matrix molecules and architecture. Detergents, ionic, non-ionic and zwitterionic, can solubilize cell membranes and dissociate the DNA from proteins, and thus effectively remove cellular material from tissue; but they may also disrupt and dissociate proteins in the ECM. The use of alcohols facilitates delipidation and causes tissue dehydration, leading to cell lysis (Crapo et al., 2011). An overview of commonly used chemical methods and their interactions with ECM, along with their advantages and disadvantages, is provided in Table 3.

(Crapo et al., 2011). α-Galactosidase is an enzyme that can be used in suppressing the immunogenic cell surface antigen galactose-α-1,3-galactose (Gal epitope) for decellularized xenogeneic tissues. It has been found that α-galactosidase can reduce ECM immunogenicity in vitro but does not adversely affect in vivo remodelling of xenogeneic ECM (Daly et al., 2009). Many researchers have utilized naturally occurring cytotoxic agents as toxins for decellularization. Toxins such as latrunculin, hyper/hypotonic solutions and enzymatic treatment combinations resulted in superior removal of DNA and retention of GAGs while preserving the mechanical properties of the scaffolds (Gillies et al., 2011). With a limitation for clinical applications, serum can also be used to aid the removal of nucleic acid fragments from the tissue; however, it does not facilitate the removal of xenogeneic constituents and may also introduce immunogenicity to the ECM (Crapo et al., 2011).

3.3. Biological route On the basis of the nature of the decellularization agents used, the biological route includes two types of agent, enzymatic and non-enzymatic (such as chelating agents). The commonly used decellularization enzymes include nucleases, trypsin, collagenases, lipases, dispases and thermolysin. These enzymes can provide high specificity for the removal of cell residues or undesirable ECM constituents, but there is a possibility that enzyme residues may impair recellularization or cause an immune response (Crapo et al., 2011). The commonly used enzymes, with their modes of action, effects on ECM, advantages and disadvantages are shown in Table 4. In addition to enzymatic agents, non-enzymatic agents are also being used in order to enhance the efficiency of decellularization. Chelating agents aid in cell dissociation from ECM proteins by disrupting cell attachment to collagen and fibronectin at the Arg–Gly–Asp receptor, by chelating divalent cations. Ethylene diamine tetra-acetic acid (EDTA) and ethylene glycol tetra-acetic acid are the commonly used chelating agents that contribute to subtle disruption in protein–protein interactions for cell removal from the ECM surface (Crapo et al., 2011). During cell lysis caused by decellularization agents, many intracellular proteases are released that may cause undesirable damage to the ECM. To protect the ECM from these proteases, serine protease inhibitors are commonly used to prevent ECM and intracellular protease interactions/long exposures and thus preserving the ECM ultrastructure. Commonly used serine protease inhibitors are phenyl methyl sulphonyl fluoride, aprotonin and leupeptin (Crapo et al., 2011). It has also been observed that application of streptokinase during the washing step facilitates decellularization, but the actual mechanisms behind this are still not clear (Kajbafzadeh et al., 2013). Microbial contamination during long-duration chemical decellularization has been prevented by utilizing antibiotics and antimycotics with the decellularized systems. Penicillin, streptomycin, amphotericin B and sodium azide are commonly being used to control microbial contamination, but this also encourages a regulatory hurdle for clinically approved decellularized scaffolds Copyright © 2015 John Wiley & Sons, Ltd.

3.4. Combinatorial methods Combinatorial methods are an approach to realizing efficient and effective decellularization of tissues and organs for successful tissue-regenerative applications. For instance, Jiang et al. (2014) have utilized a cryo-chemical decellularization method that combines physical and chemical approaches to generate acellular liver scaffolds from the whole liver; upon recellularization of the acellular scaffolds with mesenchymal stem cells, hepatic differentiation was promoted that could be transformed further into functional grafts for hepatic transplantation in vivo. In an interesting study, Evaristo et al. (2014) reported the development of decellularized tracheal scaffold which had been obtained by combining the traditional physical, chemical and enzymatic routes with light-emitting diodes (LEDs). It was found that cartilaginous tissue exposed to LED 630 nm and 475 nm irradiation showed a better number of gaps without cells, as compared to other traditional approaches, such as physical (agitation and LED irradiation), chemical (SDS and Triton X-100 detergents) and enzymatic (DNase and RNase). Cells surrounding the soft tissues were successfully removed after exposure of the tissue to LEDs, followed by chemical treatment.

4. Whole-organ decellularization Recent advancements in tissue engineering have established a foundation for the functional replacement of whole organs. Bioartificial whole-organ scaffolds could be developed by decellularization of the target organs; the resultant acellular scaffold could then be recellularized with autologous cells or stem cells and cultured in a physiologically suitable bioreactor to synthesize a functional organ in vitro. Recently, perfusion decellularization has been successfully developed to produce anatomically intact whole organs and has been widely adopted for tissue-regenerative medicine (Figure 2) (Ott et al., 2008). Whole-organ decellularization can be J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Copyright © 2015 John Wiley & Sons, Ltd.

Tributyl phosphate

Solvents Alcohols and acetone

Zwitterionic detergents CHAPS and SB-10/SB-16

Triton X-200

Sodium deoxycholate

Ionic detergents Sodium dodecyl sulphate (SDS)

Non-ionic detergents Triton X-100

Acids and bases (acetic acid, peracetic acid, hydrochloric acid, sulphuric acid, ammonium hydroxide) Hypotonic and hypertonic solutions

Method

Cell lysis by dehydration, solubilize and removing lipids Forms stable complexes with metals, disrupts protein–protein interactions

Exhibit properties of non-ionic and ionic detergents

Solubilizes cell and nucleic membranes

Effectively lyse cells, but does not remove cellular remnants

Cell lysis by osmotic shock, disrupt DNA–protein interactions Disrupts DNA–protein, lipid–lipid and lipid–protein interactions

Removes cells from dense tissues and inactivates pyrogens Results depends on tissue properties

Removes cells with greater disruption of ultrastructure Remove cells with mild disruption of ultrastructure

Results depend on tissue thickness

Removes nuclear remnants and cytoplasmic proteins from dense tissue

Depends on tissue properties (effective for thin tissues)

May damage collagen, GAG and growth factors

Effect on ECM

Solubilizes cytoplasmic components of cells, disrupts nucleic acid and tends to denature proteins

Mode of action

Table 3. List of chemical routes used for decellularization of tissues/organs

Minimal impact on mechanical properties of ECM



Preserves native ECM properties

Effective in removing cellular remnants



Effectively removes cells from the tissue

Leaves protein–protein interactions intact and retains sulphated GAG content

-

Disinfects material by entering inside microorganisms and oxidizing enzymes

Advantage

Crosslinks and precipitates proteins, including collagen Dense tissues may lose collagen

May denature proteins with a great tendency than non-ionic detergents

Disrupts native tissue architecture in comparison with SDS and removes GAG

Disrupts protein–protein interactions and causes decrease in GAG content and collagen integrity –

Disrupts ECM ultrastructure and lowers laminins/fibronectin content

Removal of DNA remnants can be difficult from tissue

May dissociate important molecules (GAGs, from collagenous tissue)

Disadvantage

Embryonic tissues (Cox and Emili, 2006) Tendons (Deeken et al., 2011)

Peripheral nerve tissue (Hudson et al., 2004) Umbilical artery (Gui et al., 2010)

Inferior epigastric artery (Henderson et al., 2010)

Kidney (Nakayama et al., 2010)

Heart valve, blood vessel, tendon, ligament (Crapo et al., 2011)

Lamina propria (Xu et al., 2007)

Submucosal layer, basement membrane plus tunica propria, layers of urinary bladder (Crapo et al., 2011)

Reference

D. Rana et al.

J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Amniotic membrane (Hopkinson et al., 2008)

Adipose tissues (Brown et al., 2011)

Adipose tissues (Flynn, 2010)

Superior decellularization than trypsin and exhibits cell infiltration Prolonged exposure to dispase can disrupt ECM ultrastructure and remove its components Dispase cleaves specific peptides (fibronectin and collagen IV) Dispase and thermolysin

– – Aids in delipidation

Copyright © 2015 John Wiley & Sons, Ltd.

Collagenases

Exonucleases

Lipase

– –

Do not support ultrastructure preservation and collagen retention Insufficient to remove all lipids when used alone Increased ECM disruption

Adipose tissues (Brown et al., 2011) – –

More effective than exonucleases Difficult to remove from the tissue

Catalyse the hydrolysis of the interior bonds of RNA and DNA Catalyse the hydrolysis of the terminal bonds of RNA and DNA – Endonucleases (Benzonase)



Pulmonary and vascular epithelium (Petersen et al., 2010)

Heart valves and other soft tissues (Yang et al., 2009)

Disruptive to elastin and collagen and needs lengthy incubation time Invokes immune response Better preservation of GAG content Prolonged exposure can disrupt ECM Cleaves peptide bonds on the C-side of Arg and Lys Trypsin

Effect on ECM Mode of action Method

Table 4. List of biological/enzymatic routes used for decellularization of tissues/organs

Advantages

Disadvantages

Reference

Decellularized scaffolds for tissue engineering

achieved by antegrade and retrograde perfusion of the decellularization agents through the vascular bed to remove cellular materials from the tissue while preserving its 3D architecture. Vascular networks within organs minimize the diffusion distance for oxygen and thus could be utilized to efficiently deliver decellularization agents to cells and for retrieving cellular debris, along with residual detergents, from the tissues. The end-product of whole-organ decellularization is biocompatible, non-toxic and directly suitable for use in downstream applications, including recellularization, ex vivo culture and regeneration. Ideal protocols are likely to be different for different solid organs, because of the inherent characteristics of the tissues. For instance, a higher concentration of detergents and high perfusion pressure would be required for heart, whereas a lower concentration of detergents and perfusion pressures would work for lungs. The protocol conditions would change according to the complexity of the organ and the density of the residing cells/tissues. For example, organs derived from large animals or humans would require considerably higher concentrations of detergent solutions and longer exposure times as compared those from smaller species. In more recent experiments it has been proved that, in comparison with different detergents, such as SDS, Triton X-100, peracetic acid and sodium deoxycholate, SDS-based perfusion decellularization protocols can generate acellular liver and kidney scaffolds from human-sized porcine organs that preserve vital ECM components and remove cellular material and xeno-immunogens (Wang et al., 2015). Additionally, Triton X-100 eliminates residual SDS from SDS-treated scaffolds to reduce their cytotoxicity (Wang et al., 2015). Pressure-controlled perfusion decellularization of whole organs for generating acellular 3D scaffolds, with preserved ECM protein content, ultrastructure and perfusable vascular conduits, has also been described; e.g. Guyette et al. (2014) demonstrated that by using appropriate modifications, pressure-controlled perfusion decellularization can be achieved in small-animal experimental models (rat organs, 4–5 days) and can be scaled to clinically relevant models (porcine and human organs, 12–14 days). To decellularize rat hearts, the retrograde perfusion of a 1% w/v SDS solution through the ascending aorta (for antegrade perfusion through the coronary arteries) demonstrated preservation of the cardiac architecture; however, a lower concentration of SDS, 0.1% w/v, was deemed optimal for the rat lung protocol, with sufficient decellularization without compromising the integrity of the membrane architecture in small vessels and alveolar septa. For hearts, the finally obtained acellular scaffold was then repopulated with harvested rat neonatal cardiomyocytes in order to form contractile tissue (Guyette et al., 2014). In an attempt to preserve the native matrix with a perfusion-based approach, the authors have suggested the use of constant-pressure perfusion at low physiological pressures. Vascular resistance is reported to change during the decellularization process; therefore, the persistent flow at the beginning of perfusion decellularization could cause damage to the basement J Tissue Eng Regen Med (2015) DOI: 10.1002/term

D. Rana et al.

Figure 2. Schematic diagram illustrating reported perfusion protocols for decellularization of tissues/organs: PBS/AA, phosphatebuffered saline with antibiotic/antimycotic; DI, deionized water; RT, room temperature; SDS, sodium dodecyl sulphate; EDTA, ethylene diamine tetra-acetic acid; NaN3, sodium azide

membrane of the native vasculature, owing to direct application of mechanical force. To limit the damage to the native vasculature and matrix, low or graduated flow rates could be employed in constant-flow decellularization methods. On the basis of the previous studies, the use of constant low physiological pressure would allow native matrix to adapt more gradually to the changes in vascular resistance and flow. In addition to conventional perfusion systems, an automated perfusion decellularization system has recently been reported to decellularize whole human-sized lungs by perfusing different detergent solutions through an automated software interfaced with a bioreactor that can control the selection, timings, volume and flow pressures of the decellularization fluids. In comparison to the manual method, automated perfusion resulted into more consistent acellular matrices and reduced process timings (Price et al., 2015). Besides the clinical feasibility of whole-organ decellularization methods, these scaffolds cannot be utilized for an expanded pool of donors with different disease histories and age groups. The effect of inherent organ damage due to disease or age may show adverse effects on decellularization or may negatively alter the ECM structure. These issues have not been widely investigated until now, thereby generating a need for further research regarding perfusion decellularization methods and their applicability to a wide variety of donors. Copyright © 2015 John Wiley & Sons, Ltd.

5. Evaluation and sterilization of decellularized scaffolds Evaluation of the residual materials and cellular components within the decellularized scaffolds is a critical step for translating them into clinically feasible implantable substrates. It is evidenced that cellular remnants within the decellularized scaffolds lead to in vitro cytotoxicity and evoke in vivo adverse host responses upon recellularization. Therefore, it is crucial to quantify the cellular remnants, such as double-stranded DNA (dsDNA), mitochondria or membrane-associated molecules, in a decellularized scaffold through commercially available assays (Crapo et al., 2011). For instance, dsDNA quantification can be done with commercially available dsDNA intercalators, such as Pico green, propium iodide or bisbenzimide, and by gel electrophoresis. Qualitative verification of the nuclear material can be evaluated by routine histological staining or immunofluorescent methods. However, the minimum concentration of cellular remnants responsible for evoking a negative remodelling response has yet to be studied in detail. Besides, it has been reported that the concentration of cellular remnants left behind after decellularization treatment may vary, depending upon tissue/organ source, tissue type into which the scaffold is being implanted and host immune function (Crapo et al., 2011). In general, some of the J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Decellularized scaffolds for tissue engineering

minimal criteria that have been observed and followed throughout the world are: (a) < 50 ng dsDNA/mg ECM dry weight; (b) < 200 bp DNA fragment length; (c) absence of visible nuclear material in tissue sections stained with 4,6-diamidino-2-phenylindole or haematoxylin and eosin (H&E). Use of these criteria has been shown to result in effective decellularized scaffolds for in vivo model systems (Crapo et al., 2011). Besides evaluating regular parameters, Schmitt et al. (2013) have studied the biomechanical behaviour and cell migration parameters of reseeded human adipose-derived stem cells onto decellularized human flexor tendon scaffolds. Biomechanical analysis confirmed no significant difference in ultimate load to failure and 2 mm gap force compared to native tissues. Cell migration had been evaluated through histological methods that revealed host cell invasion and proliferation of the seeded cells at the repair sites (Schmitt et al., 2013). Similarly, Lin et al. (2014) evaluated decellularized scaffolds in terms of biomechanical properties and biocompatibility. The biomechanical properties were evaluated through uniaxial tensile tests, which indicated properties comparable with those of native tissues. For biocompatibility, decellularized scaffolds were implanted in situ and analysed at days 10, 20 and 30 to evaluate the host–graft inflammatory reaction (Lin et al., 2014). Along with these, many other properties of the decellularized scaffolds, such as matrix biofunctionality, were also studied for better in situ functionality (Caralt et al., 2015). Clinical application of a decellularized organ scaffold would require donor scaffold sterilization. The sterilization process of decellularized scaffolds primarily eliminates endotoxins, intact viral and bacterial DNA that may induce unwanted inflammation upon scaffold implantation (Crapo et al., 2011). Decellularized scaffolds have been sterilized using simple treatments, such as incubation with acids or solvents, but these simple methods may not deliver adequate perforation or may vandalize the key ECM constituents (Crapo et al., 2011). Commonly practised sterilization methods, such as ethylene oxide exposure, γ-irradiation and electron beam irradiation are also known to alter ECM ultrastructure and mechanical properties (Freytes et al., 2008; Sun and Leung, 2008). Peracetic acid washing, or a combination of ethylene oxide followed by γ-irradiation, have been reported to completely sterilize human-sized liver decellularized scaffolds (Kajbafzadeh et al., 2013). Sterilization by γ-irradiation at a conventional dose can also modify acellular organ mechanics, with potential implications for organ bioengineering (Kajbafzadeh et al., 2013). Supercritical carbon dioxide and slightly acidic electrolysed water (SAEW) has recently been investigated as an alternative method for sterilizing decellularized scaffolds, with significant reductions in bacterial and viral contaminants within porcine dermal and liver scaffolds, followed by trivial changes in mechanical properties, relative to other sterilization methods. It has been found that SAEW-treated scaffolds support cell attachment and proliferation at a Copyright © 2015 John Wiley & Sons, Ltd.

higher rate than peracetic acid- and ethanol-sterilized scaffolds (Hussein et al., 2013). In an effort to minimize the multiple sequential stages during the processing of a decellularized scaffold, Anilkumar et al. (2014) performed pre-isolation ex situ incubation of the organ in a stabilizing agent that caused in situ crosslinking of tissue components and delamination of the collagen-rich ECM from the tissue layer beneath the mucosa. This scaffold satisfied preclinical safetytest procedures, such as cytotoxicity, local response and endotoxin load, thus providing an innovative approach to avoiding multiple sequential processing steps, including sterilization. With complete verification of the scaffold sterilization and cellular remnants removal, decellularized scaffolds can be taken up for cell reseeding, i.e. recellularization, which is discussed in next section.

6. Recellularization Recellularization is defined as renewed cellularization following decellularization of tissues/organs. The repopulation of the matrix to contain an appropriate cell composition allows the bioengineering of organs demonstrating functionality and responsiveness to the stimuli. After preparing tissue/organ-derived scaffolds using decellularization techniques, these decellularized scaffolds with intact vascular networks and 3D structures can then be readily recellularized with freshly isolated cells, and could be maintained in bioreactors for cell expansion, differentiation and continued function. Prior to recellularization, optimal cell sources and strategies for reseeding the cells need to be standardized to guarantee redistribution of the reseeded cells and, thus, a functional bioengineered organ. Cells and ECM have an inherently close and dependent relationship. Therefore, the types and sources of cells used to repopulate the organ-specific 3D scaffolds are critical to the eventual functionality and clinical success of the engineered construct. Engineering a complex tissue or organ requires rebuilding parenchyma, vasculature and underlying support structures, all of which differ in cell number and cell type, depending on the organ of interest. Additionally, the incorporation of certain growth factors or bioactive molecules into the decellularized scaffolds could also result in enhanced functionality of the recellularized tissue/organ constructs. For instance, Zhou et al. (2013) reported the potential of decellularized porcine aortic valve functionalized with Gly– Arg–Gly–Asp–Ser–Pro–Cys (GRGDSPC) peptides and vascular endothelial growth factor-165 (VEGF165) for enhanced cellular immobilization and proliferation of human umbilical vein endothelial cells (HUVECs). Various bioactive molecules that can be incorporated into the decellularized scaffolds to enhance the functionality are shown in Table 5. In the following sections, various cell sources for recellularization, methods of recellularization and the challenges associated with the recellularization are briefly discussed. J Tissue Eng Regen Med (2015) DOI: 10.1002/term

An et al., 2013 Bertanha et al., 2014 Sheridan et al., 2014 Corporal tissue engineering Vascular tissue engineering Vascular tissue engineering VEGF-expressing muscle-derived stem cells Mesenchymal stem cells Mesenchymal stem cells

Boyer et al., 2015 Neural tissue engineering N/A

Chondroitin sulphate proteoglycan-reduced acellular nerve grafts Acellular corporal collagen matrices Vein scaffolds Arterial scaffolds

Cardiac tissue engineering Cardiac tissue engineering HUVECs Bone marrow stem cells Porcine aortic valve Valvular conduits

Zhou et al., 2013 Zhou et al., 2014a, 2014b

6.1. Cell sources for recellularization

GRGDSPC peptide and VEGF165 Heparin–stromal cell-derived factor-1α multilayer Nerve growth factor and glial cell line-derived neurotropic factor Human VEGF (165) Endothelial inductor growth factor Hepatocyte growth factor

Cells Decellularized scaffold Bioactive molecules

Table 5. Various bioactive molecules incorporated within the decellularized scaffolds for tissue-engineering applications

Application

Reference

D. Rana et al.

Copyright © 2015 John Wiley & Sons, Ltd.

The selection of cell sources for the recellularization of decellularized scaffolds is of great importance in creating a physiologically functional tissue/organ. It is also essential to regenerate the specialized structures of the tissues along with vascular or ductal components and supporting structures, by repopulating them with stem or progenitor cells for organ maintenance. The bipotent differentiation potential of stem cells makes them an ideal source for recellularization, e.g. Oberwallner et al. (2014) demonstrated that human cardiac ECM scaffolds can direct proliferation and cell–matrix interactions of the seeded stem cells with higher viability, which can support the use of cardiac ECM for guided stem cell differentiation and myocardial repair. Stem or progenitor cells utilized for most tissue-engineering approaches can be broadly categorized as embryonic stem cells (ESCs), adult stem cells or recently introduced induced pluripotent stem cells (iPSCs) (Badylak et al., 2011). Non-stem or -progenitor cells used for organ engineering are usually: parenchymal cells, e.g. hepatocytes, cardiomyocytes or epithelium; vascular cells obtained from accessible sources, such as peripheral blood or bone marrow, e.g. endothelial cells; and supportive cells, e.g. fibroblasts, obtained from organ biopsies. Altogether, these parenchymal cell types contribute to the specific function of the organs and nonparenchymal cells enhance the functional phenotypes of the parenchymal cells and contribute to the organization of the cellular architecture of the tissue. For instance, endothelial cells provide a non-thrombotic barrier for the decellularized organ matrix and protect parenchymal cells from the shear stress generated by blood flow, while fibroblasts secrete and remodel the ECM to improve parenchymal cell function in co-cultures. ESCs, being pluripotent in nature, lack epigenetic modifications, which makes them more responsive to the in vitro environment than differentiated stem or progenitor cells. However, their ethical limitations and capability to support teratomas restrict their use in clinical trials. On the contrary, adult-derived stem cells have restricted proliferation and differentiation potential. Commonly used cell sources for adult-derived stem cells include bone marrow, peripheral blood, liver, lung, skeletal muscle, heart and adipose tissue. On the other hand, iPSCs include properties of embryonic as well as adult stem cells, which makes them a potential cell source for recellularization. iPSCs can be cultured in large numbers in vitro and can give rise to both parenchymal and supportive cells required for complex tissue formation. Recently, human mesenchymal stem cells have been reported to be isolated from fetal sources that can give rise to neuronal tissues and osteoblasts. It is evidenced that fetal stem cells contain multiple stem or progenitor cell types that can be reprogrammed to pluripotent lineages, such as osteogenic, adipogenic and neurogenic. Additionally, fetal hepatoblasts seeded onto bioscaffolds show the potential to differentiate into the biliary and hepatocytic lineages. Umbilical cord blood cells can also be considered as a rich source of stem or progenitor cells, J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Decellularized scaffolds for tissue engineering

as they are more primitive and exhibit potential to give rise to diverse cell types for organ regeneration. The sources of this vast variety of stem or progenitor cells could be autologous or allogeneic in nature, but their choice depends on different applications and reaction conditions, such as the number of cells required, the regeneration capacity of organs, ease of cell harvesting and expansion, in vitro differentiation abilities, regulatory issues, intellectual property constraints and cost-effectiveness. The choice of cell types is also interlinked with recellularization methods, e.g. to access the vasculature of an organ, perfusion of cells is an easy approach, whereas parenchymal targets would require local delivery of cells through injection. We therefore further emphasize the various available methods of recellularization with their biological significance.

6.2. Methods of recellularization The process of recellularization includes cell seeding to achieve a redistribution of cells quite similar to their native spatial configuration, followed by perfusion culture to prepare the seeded cells to suitability for in vivo use. There are mainly two types of cell-seeding approach that have been used to reseed scaffolds, static and dynamic. Static cell-seeding involves passive introduction of a dense cell suspension into a scaffold; although this provides ease of access, the low seeding efficiency of about 10–25% and minimal cell penetration within the scaffold walls limit its application (Aunins et al., 1989). The dynamic cellseeding technique provides an opportunity to overcome these drawbacks and has been proved to be more effective than static methods (Weinand et al., 2009). The commonly used approach for dynamic seeding involves direct addition of a high-concentration cell suspension into the vascular perfusion line, allowing cells to perfuse through the scaffold vasculature to an organ parenchyma. Alternative seeding approaches include magnetic, electrostatic, vacuum and centrifugal seeding. By employing the basic concepts of these approaches, many research groups have utilized different recellularization techniques, such as intramural injection of cells, infusion of cells into the vasculature followed by continuous perfusion, direct parenchymal injection, multistep infusion and continuous perfusion (Badylak et al., 2011). A high seeding efficiency of 86–96% has been reported for decellularized liver scaffolds by employing direct injection of cells into the portal vein, using a bioreactor system (Uygun et al., 2010). However, multiple injections of the cells have proved to be superior to single infusion of the cells (Soto-Gutierrez et al., 2011). Besides optimizing the method of recellularization, the selection of the route through which cells will traverse the organ vasculature is also important. For instance, it was demonstrated in liver scaffolds that portal vein-seeded endothelial cells were selectively deposited in the periportal area of the liver lobule, while vena cava-seeded endothelial cells were distributed in the pericentral area. Copyright © 2015 John Wiley & Sons, Ltd.

In order to compare different re-endothelialization strategies and their impact on decellularized scaffolds, Robertson et al. (2014) used three approaches to recellularize perfusion-decellularized rat heart vasculature with rat aortic endothelial cells (RAECs). The three approaches were retrograde aortic infusion, brachiocephalic artery (BA) infusion and a combination of inferior vena cava (IVC) plus BA infusion. The re-endothelialized scaffolds were examined in vitro for 7 days and assessed for thrombogenicity both in vitro and in vivo. The results confirmed that the combination (IVC + BA) delivery strategy resulted in enhanced scaffold vessel re-endothelialization compared to the single-route strategy. For the combination strategy, retrograde perfusion of media was stopped via the aorta and cannulated the IVC, which was followed by infusion of 2.0 × 107 RAECs with scaffold placing under retrograde perfusion of media via the aorta, and again infusion of 2.0 × 107 RAECs via the BA. Meanwhile, the scaffolds were continuously perfused with complete MCDB-131 via the aorta, with a progressively increasing flow rate of 1–3 ml/min over 3 days. After re-endothelialization, under retrograde perfusion, rat neonatal cardiac cells (1.3 × 108 cells) were injected into the left ventricular wall in three or four parallel injections, and the recellularization constructs were maintained by using retrograde Lagendorf perfusion. It was also proved that re-endothelialization reduced scaffold thrombogenicity and improved the contractility of left ventricular-recellularized constructs, thus promising great potential for whole-organ recellularization applications. There are many other factors that influence the efficiency of cell seeding, such as flow rate, cell type and cell number. Flow rate directly affects the reseeding efficiency in dynamic seeding approaches. For instance, Kim et al. (2000) stated a flow rate of 1 ml/min required for the initial cell mass survival, whereas in a subsequent study by Ji et al. (2012) the complete analysis of different flow rates of 0.5, 1, 2, 4 and 6 ml/min established that the 4 ml/min flow rate could ensure adequate oxygen and nutrition delivery for the seeded cells and also help in decreasing the shear stress on the seeded cells within decellularized scaffolds. Cell type is another important factor to consider while dealing with whole-organ scaffolds. The choice of cell types required for functional recellularization would vary from organ to organ. To reseed decellularized scaffold with a mixture of stem cells as well as supportive or vascular cells would require a proper seeding and culture protocol. The seeding efficiency is also directly controlled by the initial seeding density, which further depends on the organ type that is to be repaired or regenerated. For example, organs with specialized biomechanical functions, e.g. lung or heart, would require a high percentage of initial cell number, in contrast to organs that are responsible for basic metabolic functions, e.g. liver, pancreas or intestine. Organ parenchymal cells can be seeded together in a mixture with endothelial cells or via separate inoculations of unmixed cell populations, by utilizing different routes of the organ vasculature to populate the diverse cell types. J Tissue Eng Regen Med (2015) DOI: 10.1002/term

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Recently it has been reported that pretreatment of decellularized matrices with defined ECM proteins that match the repertoire of integrin receptors expressed by the cells to be seeded can increase the efficacy of the reseeding process (Lecht et al., 2014). This group determined the integrin receptor profile of mouse embryonic stem cells and performed reseeding in decellularized lung scaffolds. Surprisingly, the results confirmed enhanced reseeding of decellularized lung scaffold with mouse embryonic stem cells, by pretreating the scaffolds with medium conditioned by A549 human lung adenocarcinoma cells with a higher amount of laminin. Additionally, pretreatment also resulted in a significantly more uniform distribution of the seeded stem cells throughout the engineered organ. Cell seeding within bioreactors is a recent advancement in recellularization techniques. Bioreactors have a cylindrical configuration and spinner flask; the cylindrical configuration limits dead zones and promotes mixing, whereas a spinner flask is useful for cell seeding into bulk media. The continuous-flow stirred-tank reactor allows multiple passes of cell suspensions through the scaffold to maximize the retention of cells within the scaffold; extended periods of circulation should be avoided, because that may lead to anoikis. For recellularization, increased cell-seeding density maximizes the probability of cell aggregates, which can occlude vessels and form thick polylayers, leading to hypoxia and necrotic core formation. Further experimentation to determine the optimal medium content, growth factors and oxygen level are critically required.

6.3. Challenges associated with recellularization Major challenges for growth and redistribution of cells in their native spatial configuration within decellularized scaffolds include thorough, homogeneous repopulation with appropriate cell densities and cell phenotypes within the scaffolding microenvironment, to match the cell distribution that occurs in the native tissues/organs as much as possible. Another challenging task in recellularization is to achieve optimized cell alignment, interconnections, cell density and minimized risk of pore collapse within the reseeded scaffolds. In decellularized scaffolds, pore features (size, shape, alignment, interconnection, resistance to collapse) have been proved to influence the reseeding potential and permeability of the scaffold (Lu et al., 2004). For instance, large pore size in whole-organ decellularization scaffolds could not support cell infiltration into the centre of the scaffold, ceasing oxygen and nutrient delivery, which remained confined to the edges and peripheral range (Patnaik et al., 2014). During further tissue remodelling, the scaffold’s porosity decreases, due to new ECM deposition or the collapse of pores, which ultimately reduces the migration rate of cells and nutrient permeability, leading to cell death within the core regions. However, revascularization is necessary to provide oxygen and nutrients into the thick tissues and to establish a functional integration of the scaffold within the host. Copyright © 2015 John Wiley & Sons, Ltd.

As discussed previously, the choice of cell types and number of cells for recellularization depends purely upon the type of the organ that is to be repaired or regenerated, e.g. cardiomyocytes in heart, hepatocytes in liver, etc. Additionally, depending on the organ, various cell types are important for functional recellularization of whole-organ scaffolds. Therefore, a mixture of cell types can lead to a combinatorial problem, where serendipity may be necessary to find the proper seeding and culture protocol. The number of cells initially required for seeding the decellularized matrix is also crucial for recellularization, and depends on the type of organ to be regenerated. For example, a high percentage of native cells would be required for the repair of organs such as heart or lung because of their complex nature, which requires a functional whole organ transplant at the time of implantation, whereas organs such as liver can be implanted with only a small percentage of their native cell mass. The liver is estimated to contain approximately 4 × 109 cells/kg body weight in adults. Although some success has been achieved with the transplantation of 1–10% of native liver mass in pigs and humans (Fitzpatrick et al., 2009), optimization of the cell number require to support an animal model of hepatic failure is still difficult, and these numbers are quite large for stem/progenitor cell-based techniques and may raise practicality issues. Research in this direction is undergoing a dramatic revolution. Besides recellularization methodologies, culture conditions and preservation technologies also need to be improved for the realization of stem cell-based recellularization. For example, long-term in vitro culture is required for stem cellbased recellularization, in order to optimize cell proliferation and attachment. Conversely, long-term stem cells in in vitro preservation may lead to deterioration of the ECM components, affecting the applicability of the scaffold as well as the normal physiological functions of the seeded cells. Also, to minimize thrombogenicity and attain proper vascular functions, endothelial coverage of the vasculature lumens is essential. Endothelialized liver scaffolds have been used to identify the anticoagulant effect by perfusing fresh rat heparinized blood into seeded and control scaffolds. Recellularization with endothelial cells and increasing the anticoagulation efficiency are some of the rate-limiting issues that need to be overcome in the future. Despite much progress in the development of decellularized scaffolds, there are still concerns over the methodology of stem cell-based recellularization and studying the interaction of stem cells with decellularized scaffolds, which is essential to elucidate the cellular and other biological functions that are triggered when the cells are cultured in an artificial microenvironment.

7. Interaction of stem cells with decellularized scaffolds Stem cells have emerged as a promising cell source for tissue engineering and regenerative medicine, due to their unique self-renewal ability and the capacity to give rise J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Decellularized scaffolds for tissue engineering

to cells of all three germ layers. Stem cells secrete a large amount of endogenous ECM, which plays an important role in regulating self-renewal, lineage commitment and tissue morphogenesis. It is important to provide appropriate cues and signals to the cells from the simulated natural microenvironment of the tissue-derived decellularized scaffold in order to maintain the phenotype expression and differentiated function of stem cells. In support of this, it has been found that the use of decellularized scaffolds is an option that could provide biophysical and biochemical cues that are essential to mimic the growth of native tissues in terms of cell adhesion, proliferation and differentiation. Decellularized scaffolds are mainly comprised of 3D matrices of collagens and other structural proteins that are interlaced with proteoglycans, which together control the local microenvironment and contribute to the stem cell niche through their own signalling moieties and their ability to bind to growth factors, cytokines, enzymes and other diffusible molecules. Additionally, ECM scaffolds derived from embryonic stem cells have a broader signalling capacity compared to ECM scaffolds obtained from somatic cells, and are predicted to have a lower risk of tumour formation than is associated with embryonic stem cells (Sart et al., 2014). It has been demonstrated that the composition and function of cellular adhesions characterized in 3D matrices derived from tissues or cell culture are different from focal and fibrillar adhesions characterized on two-dimensional (2D) surfaces in terms of α5βl and αvβ3 integrins, paxillin other cytoskeletal components, and tyrosine phosphorylation of focal adhesion kinase (FAK) (Cukierman et al., 2001), therefore emphasizing on the fact that the changes in the feature size of the ECM substrate may influence the clustering of integrins and other cell adhesion molecules upon interaction of the substrate with stem cells, thus altering the number and distribution of focal adhesions (Cukierman et al., 2001). Additionally, Ross et al. (2012) demonstrated matrix–cell signalling in acellular wholeorgan scaffolds by inducing differentiation of pluripotent precursor cells to endothelial lineage. Production of mouse basement membrane was also found to support remodelling of host (rat)-derived scaffolds. These results were found to be in agreement with the hypothesis that decellularized scaffolds may retain signals for seeded pluripotent precursor cells to differentiate and recapitulate native structures and matrix–cell signalling, followed by cell–cell and cell–matrix interactions, thereby remodelling and replacing the original matrix. This would further reduce a decellularized scaffold’s antigenicity and enable xeno-allografts (Ross et al., 2012). Decellularized scaffolds exhibit the potential to mimic the native microenvironment of the seeded stem cells by managing their cellular behaviour in a dynamic fashion via exerting control over the intrinsic genetic pathway for regulating the multipotentiality and self-renewal ability of stem cells through extrinsic signals. This scaffold based cell–matrix interaction-induced signalling behaves as a critical determinant of cell behaviour. For instance, binding of ECM molecules to specific integrin receptors Copyright © 2015 John Wiley & Sons, Ltd.

may trigger the activation of a myriad of intracellular signalling. A wide variety of soluble growth factors, such as basic fibroblast growth factor-2, transforming growth factor-β, vascular endothelial growth factor and hepatocyte growth factor, bind to a component of ECM (such as heparin sulphate), which greatly slows their diffusion and therefore serves to fine-tune their local concentrations and gradients. In a recent study by Sart et al. (2014), ECMs from undifferentiated embryonic stem cells monolayers, undifferentiated aggregates or differentiated embryoid bodies at different developmental stages and lineage specifications were decellularized and reseeded with embryonic stem cells (Sart et al., 2014). It was found that embryonic stem cell-derived ECMs were able to influence the proliferation and differentiation of embryonic stem cells by direct interactions with the cells, and by influencing the signalling functions of the regulatory macromolecules, such as retinoic acid. This shows the potential to present regulatory signals to direct lineage- and development-specific cellular responses for in vitro applications (Sart et al., 2014). Decellularized stem cell matrix (DSCM) is another kind of biomimetic scaffold, which has been proved to act as a substratum to yield large-quantity and high-quality cells for tissue-engineering and regenerative applications (Figure 3). DSCM shows a rejuvenating and reprogramming effect on various stem and progenitor cell populations, which gives a new dimension to understanding the interaction between stem cells and decellularized scaffolds. Porcine synovium-derived stem cells (SDSCs) were seeded on DSCM deposited by SDSCs, which showed that the initially wide and flat SDSCs became thin and spindle-shaped and arranged themselves in a 3D spatial configuration with typical stem cell phenotypes (He et al., 2009). An increase in cell number and a greatly enhanced chondrogenic capacity were also observed without any concomitantly improved adipogenic or osteogenic potential. This finding proves that the DSCM can serve as niche for stem cell proliferation and lineage-specific differentiation in vitro (He et al., 2009). Therefore, DSCM can be considered as an important scaffolding system because of its biomimetic niche properties, which could provide binding sites to the tissue-specific stem cells for attachment, proliferation, migration and differentiation in a 3D fashion. Many investigations support the suitability of DSCM for stem cell-based tissue engineering. For instance, in a recent study by Pei et al. (2013), it was found that expansion of the human SDSCs (hSDSCs) onto DSCM upregulated antioxidative gene levels and chondrogenic potential, retarded the decrease in cell number and the increase in apoptosis and rendered SDSCs resistent to cell-cycle G1 arrest resulting from H2O2 treatment. In support of this study, Burns et al. (2011) demonstrated that decellularized human-derived mesenchymal stem cells (hMSCs) matrix had significant angiogenic potential, with at least 50 angiogenic cell surfaces and extracellular proteins implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC-BD11 surface galectin-1 expression was found to J Tissue Eng Regen Med (2015) DOI: 10.1002/term

D. Rana et al.

Figure 3. Stem cell-deposited ECM as a decellularized scaffold for lineage-specific differentiation

be responsible for the matrix–endothelial interactions and xenografted hMSC-BD11 cells were found to optimally recruit host vasculature. Similarly, He et al. (2013) reported that cell-deposited ECM can mimic the liver’s native stem cell microenvironment and facilitate hepatogenic maturation. Decellularization process preserved the fibrillar microstructure and a mix of matrix proteins in celldeposited ECM, such as collagen type I, collagen type III, fibronectin and laminins, that were identical to those found in native liver. Bone marrow-derived mesenchymal stem cells (BMSCs) cultured on cell-deposited ECM showed a spindle-like shape, a robust proliferative capacity and a suppressed level of intracellular reactive oxygen species, accompanied by upregulation of two superoxide dismutases. These experimental results are in agreement with the results obtained by other groups for DSCM. Hepatocyte-like cells differentiated from BMSCs on ECM scaffold were determined to have a more intensive staining of glycogen storage, an elevated level of urea biosynthesis and higher expression of hepatocyte-specific genes (He et al., 2013). The potential for in vivo colonization of decellularized heart valves by human-derived BMSCs (hBMSCs) towards the anisotropic layers ventricularis (VS) and fibrosa (FS) and in homo- vs heterotypic cell– ECM interactions were also investigated (Iop et al., 2009). The two valve layers were found to behave differently regarding hBMSCs repopulation potential, with a higher degree of 3D spreading and differentiation in VS than in FS, and with enhanced cell survival and colonization effects in the homotypic ventricularis matrix, suggesting that hBMSCs phenotypic conversion is strongly influenced in vitro by the anisotropic valve microstructure and species-specific matching between ECM and stem cells (Iop et al., 2009). Copyright © 2015 John Wiley & Sons, Ltd.

DSCM provides a stem cell microenvironment resulting in expanded cells with acquired enhanced proliferation ability and differentiation potential. These findings make cell-deposited ECM an important component for determining the regenerative potential of the stem cells seeded onto decellularized scaffolds. However, investigation of the mechanisms behind the interaction of stem cells with decellularized scaffolds is still in its infancy, and more research is needed to further unravel the mechanisms underlying the cell–matrix interactions and cell–cell communications during tissue organization.

8. Applications of decellularized scaffolds There are two sources of organs currently available for transplantation applications, living and decellularized. Decellularized tissues/organs in the form of scaffolds are widely used in tissue engineering and regenerative medicine. New sources of organs are urgently needed to solve the shortage of transplantable organs, and the development of regeneration medicine is poised to potentially change the paradigm of organ transplantation. Recent progress in tissue engineering has established a foundation for the functional replacement of whole organs. Bioartificial organ ECM (also called acellular organ scaffold) could be developed by decellularization of the target organs. The decellularized organ scaffold could then be recellularized with autologous cells and cultured in a physiologically appropriate bioreactor to create a functional organ in vitro. Development of a tissue-engineered organ would allow for a potential reduction in waiting list J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Decellularized scaffolds for tissue engineering

mortality, due to a decrease (or elimination) in waiting time if a bioartificial organ substitute could be produced ’on demand’. Over the last several years, many organs, including livers, lungs, kidneys and hearts, have been engineered in laboratory settings, implanted into rodent recipients and shown regenerative functions. Besides acellular scaffolds seeded with tissue-specific cells, decellularized scaffolds recellularized with stem cells have also shown potential use for clinical studies in different areas of tissueregenerative medicine. Some of the key areas of tissueengineering applications are briefly discussed below.

8.1. Cardiac tissue engineering Decellularized cardiac tissues are used as potential scaffolds for cardiac regenerative applications. In a notable study, Chamberland et al. (2014) hypothesized that matching the developmental stages of embryonic scaffold with primitive cardiac progenitors may be used to optimize the differentiation and maturation of bioengineered cardiac tissues. They further stated that embedding the stem cells within a tissue-specific environment matched to the developmental stage of the cardiac progenitors may offer a practical solution for stem cell-derived regenerative applications. In another study, Oberwallner et al. (2014) reported human cardiac ECM sheets embedded with mesenchymal stem cells, cardiomyocytes derived from induced pluripotent stem cells and natıve neonatal mouse cardiomyocytes as suitable scaffolds for cell– matrix interaction and cardiac tissues regeneration. For clinical applications, non-stem cells have also proved their capability for translating the laboratory research of cardiac tissues to next level. For instance, Dohmen et al. (2007) reported decellularized tissue-engineered heart valve implantation for reconstruction of the right ventricular outflow tract. A cryopreserved pulmonary allograft was decellularized, coated and seeded with autologous vascular endothelial cells, using a specially developed bioreactor that showed a viability of 93.2 ± 2.1%. Postoperatively, evaluation of tissue-engineered heart valve by transthoracic echocardiography showed a mean pressure gradient of 5.4 ± 2.0 mmHg at 10 years, with no calcification up to 10 years. This study demonstrated the impact of decellularized tissue-engineered heart valves, with an excellent haemodynamic performance and degeneration resistance during long-term follow-up for cardiac tissue engineering applications (Dohmen et al., 2007). Thus, decellularized scaffolds are significantly used in cardiac regenerative applications in conjunction with stem cells.

8.2. Bone tissue engineering Decellularized bone tissues are also used as a potential scaffold for bone regenerative applications. For example, Fröhlich et al. (2010) developed an in vitro 0.5 cm-sized bone construct using human adipose-derived stem cells, decellularized bone scaffolds and perfusion bioreactors. Copyright © 2015 John Wiley & Sons, Ltd.

After 5 weeks of cultivation, addition of osteogenic supplements to the culture medium significantly increased the construct cellularity and the amounts of bone matrix components, such as collagen, bone sialoprotein and bone osteopontin. The study also showed the effect of the medium perfusion on improving the distribution of cells and bone matrix within the engineered constructs, which can be further used to form compact and viable bone tissue constructs (Fröhlich et al., 2010). Other than the stem cells, many groups have proved the potential of autologous non-stem cells with decellularized scaffolds to form clinically suitable bone constructs. In support of this, Hesse et al. (2010) developed a novel multiple disc graft, made up of decellularized bovine trabecular bone discs seeded with autologous bone marrow cells to regenerate a segmental long bone defect in a patient. As regards being clinically significant, the multiple cell-seeded discs actively showed bone formation around the grafted defect as early as 6 weeks after surgery. The 2 year follow-up study proved the impact of multiple disc grafts in bone regenerative applications (Hesse et al., 2010). Thus, decellularized scaffolds have potential in bone tissue engineering.

8.3. Tracheal tissue engineering Decellularized scaffolds are commonly being used for the repair of trachea, owing to its simple structure and functions properties. For instance, Gray et al. (2012) constructed a tissue-engineered scaffold from xenologous decellularized leporine tracheal segment recellularized with autologous amniotic mesenchymal stem cells and compared it with decellularized scaffolds. The results of the animal study confirmed maximum survival with full epithelialization for the engineered scaffolds with increased elastin level after implantation, although collagen and GAG were normal. Additionally, many tracheal implants derived from decellularized tissues/organs seeded with stem cells have already been taken to the clinical level, thus confirming the potential of stem cellengineered airways for perinatal airway repair and regeneration (Gray et al., 2012).

8.4. Pulmonary tissue engineering In a further step forward for pulmonary tissue engineering, Nichols et al. (2013) expanded their limits from decellularizing small animal organs (rat lungs) to large (porcine and human) acellular trachea–lung scaffolds with potential clinical applicability. This group attempted to produce acellular pig or human trachea–lung scaffolds through decellularization techniques, and then recellularization with various stem and adult cells, such as murine embryonic stem cells, human fetal lung cells, pig bone marrow-derived mesenchymal stem cells and primary human alveolar epithelial type II cells. Although there were changes in the content of collagen type I and J Tissue Eng Regen Med (2015) DOI: 10.1002/term

D. Rana et al.

elastin, but they did not affect the mechanics of lung function. Examination of cells seeded onto pig and human acellular scaffolds showed cell attachment and viability (Nichols et al., 2013). Also, scaffolds produced using a variety of detergents indicated that the choice of detergents can influence the human immune response in terms of T cell activation and chemokine production. In view of the prospective safety and effectiveness from a tissue-engineering decellularized and recellularized scaffold, many clinical studies and follow-up observations have been done so far. Although decellularized scaffolds seeded with stem cells show great potential for tissue engineering and regenerative applications, further research is still required in this field to translate them into clinically feasible products. Some of the other regenerative applications of decellularized scaffolds recellularized with stem cells are shown in Table 6.

9. Clinical status In the last few years there has been considerable progress in the clinical translation of decellularized scaffolds from bench to bedside. However, so far no definitive standards have been developed for realizing the clinical success of decellularized scaffolds that can be recellularized with terminally differentiated progenitor cells or stem cells. In an effort to understand the current cutting-edge tissueengineering approaches, we highlight here some of the remarkable studies conducted to translate decellularized scaffolds into accessible products. Owing to its simple structure and function properties, trachea has been used as a starting organ to evaluate the possibility of retrieving clinically relevant respiratory organ scaffolds by employing decellularization methods. To assess the bioengineered tubular tracheal matrices, Macchiarini et al. (2008) demonstrated the synthesis of a human tissue-engineered trachea that had been treated for cells and MHC antigen removal, and then repopulated with autologous epithelial cells and mesenchymal stem cell-derived chondrocytes. This scaffold was then used to replace the left main bronchus of a 30 year-old woman suffering from end-stage bronchomalacia. The results showed an immediate functional airway and normal mechanical properties of the scaffold within 4 months, without any immunosuppressive medication (Macchiarini et al., 2008). In a 5-year follow up report, tracheal graft was found to be well vascularized and completely recellularized with respiratory epithelium, and showed normal ciliary function and mucus clearance ability. Also, no traces of stem cell-related teratoma and anti-donor antibody development had been found. Besides a cicatricle stenosis development as a post-transplantation effect in the native trachea in the vicinity of the bioengineered trachea anastomosis, the patient had a normal life (Otti et al., 2014). Although the clinical transplantation of this tissue-engineered trachea was successful, the graft Copyright © 2015 John Wiley & Sons, Ltd.

production time period was not feasible for a patient in need of urgent transplantation. To further reduce the production time period and improve the efficiency of the engineered trachea, Baiguera et al. (2010) reported an improved decellularization process to obtain human tracheal bioactive support in a short and clinically useful time of 3 weeks; the obtained bioengineered trachea was structurally and mechanically similar to native trachea and exhibited chemotactive and proangiogenic properties. In a another notable study carried out by Go et al. (2010), it was suggested that seeding of both epithelial and mesenchymal stem cell-derived chondrocytes is necessary for optimal tracheal graft survival. In order to accelerate the process of retrieving a suitable implantable scaffold, Elliott et al. (2012) reported replacement of an adult airway using stem cell bio-engineered decellularized tracheal scaffolds in a 12 year-old boy suffering from long-segment congenital tracheal stenosis and pulmonary sling. Scaffolds were decellularized, followed by a short course of granulocyte colony-stimulating factor, and then recellularized using bone marrow mesenchymal stem cells accompanied by autologous epithelium. Human recombinant erythropoietin and transforming growth factor-β were both applied topically to encourage angiogenesis and chondrogenesis, respectively. The graft was reported to be revascularized within 1 week after surgery, followed by a strong local neutrophil response for the first 8 weeks. Interestingly, epithelium restoration was evidenced after 1 year. In a 2 year follow-up study, a functional airway with normal CT scan results confirmed the suitability of the proposed tissue-engineering approach. To further investigate the potential of the given stem cell-based approach among a wide variety of patients, including old patients, Berg et al. (2014) transplanted a human-derived decellularized trachea with autologous stem cells into a 76 year-old patient with tracheal stenosis including the lower part of the larynx. Although the patient died after 23 days, due to cardiac arrest, the scaffold was found to be patent, open and stable, with intact anastomoses showing the presence of squamous epithelium, neovascularization, muscular cells, serous glands, nerve fibres and intact chondrocytes (Berg et al., 2014). Therefore, stem cell-seeded decellularized scaffolds exhibit an impressive potential to deal with a wide variety of donors. This approach promises the development of regenerative medicine applications that may meet clinical needs. Besides stem cell-based decellularized scaffolds, many acellular scaffolds with and without recellularization have also shown great significance in the regeneration of damaged tissues or organs in clinical trials. Some of the studies highlighting the success of decellularized scaffolds in clinical settings are listed in Table 7. These clinical results show that cell-based decellularized scaffolds can be attained and proved to be safe and accessible. However, several hurdles still need to be addressed with regard to cell types, cell numbers, recellularization technique, biomechanical long-term stability, physiological sustainability and surgical implementation. J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Copyright © 2015 John Wiley & Sons, Ltd.

Hepatic tissue engineering Cartilage tissue engineering

Neural tissue engineering

Vascular tissue engineering

Renal tissue engineering Tracheal tissue engineering

Pulmonary tissue engineering

Cardiac tissue engineering

Bone tissue engineering

Urethral tissue engineering

Skin tissue engineering

Applications

MatrACELL processed human acellular dermal matrix (Moore et al., 2015) 3D porous urinary bladder acellular matrix (Huang et al., 2014) Decellularized bone cylinders (De Peppo et al., 2013) Decellularized bone scaffolds (Fröhlich et al., 2010) Decellularized porcine pulmonary valves (Vincentelli et al., 2007) Acellular porcine and human trachea-lung scaffolds (Nichols et al., 2013) Decellularized rodent lung scaffolds (Lecht et al., 2014) Decellularized rat kidneys (Ross et al., 2009) Genepin cross-linked decellularized rat tracheal scaffold (Baiguera et al., 2014a, 2014b) Decellularized leporine tracheal scaffold (Gray et al., 2012) Decellularized vascular scaffold from rat abdominal arteries (Nagaoka et al., 2014) Decellularized inferior vena cava of rabbits (Bertanha et al., 2014) Genipin cross-linked gelatin electrospun scaffolds conditioned with decellularized rat brain extracellular matrix (Baiguera et al., 2014a, 2014b) Acellular whole liver scaffold (Jiang et al., 2014) Decellularized stem cell matrix (Pei et al., 2013)

Decellularized scaffolds

×

autologous amniotic mesenchymal stem cells

✓ × ×

✓ ×

× ✓ ✓

✓ ✓

N/A Adipose derived mesenchymal stem cells

Mesenchymal stem cells Human adult synovium-derived stem cells

Rat allogeneic mesenchymal stromal cells

× × ×

× ×

×

×

×

× × ×

✓ ✓ ✓

×



×



Murine embryonic stem cells; bone marrow-derived mesenchymal stem cells Mouse embryonic stem cells Pluripotent murine embryonic stem cells Bone marrow derived mesenchymal stromal cells

× × ×

×

✓ × ✓

Human induced pluripotent stem cells Human adipose-derived stem cells Autologous bone marrow mesenchymal stem cells

✓ ✓ ×







Clinical

In vivo

×

×

In vitro

Experimental stage

×

N/A

N/A

Cells

Table 6. Applications of decellularized scaffolds recellularized with stem cells in different areas of tissue engineering and regenerative medicine

Decellularized scaffolds for tissue engineering

J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Copyright © 2015 John Wiley & Sons, Ltd.

Decellularized porcine intestinal tissue hand-sewn into vagina-like structure

Decellularized nerve allografts of average length 2.23 cm

Vaginal engineering

Segmental nerve defect repair within hand and fingers

Autologous endothelial progenitor cells

Decellularized human pulmonary heart valve (PV) allografts

Decellularized bovine mesenteric veins using glutaraldehyde crosslinking

N/A

Decellularized porcine xenograft valve (Matrix P)

Vascular system engineering

N/A

Decellularized pulmonary homografts (DPHs)

Pulmonary valve replacement

N/A

Autologous patient-specific epithelial and muscle cells

N/A

N/A

Decellularized cryopreserved valve pulmonary allografts (DCAs) (CryoLife Inc.)

Right ventricular outflow tract reconstruction

N/A

Cells

Decellularized porcine submucosa (Surgisis Mesh, Wilson-Cook, NC, USA)

Scaffold

Oesophageal perforation

Application

Four patients with congenital vagina aplasia implanted with scaffolds using a perineal approach with a follow-up of 8 years Scaffold treats nerve gaps with sensory defect in seven patients with a short follow-up study of 5–12 months

Scaffolds implanted into two paediatric patients with congenital PV failure with a 3.5 year follow-up Scaffolds implanted in 183 patients on chronic haemodialysis with an earlier failed prosthetic graft

50 adult patients treated with Matrix P after the Ross operation

38 patients evaluated for transplanted DPHs in a 5 year follow-up

DCAs implanted into 47 patients between the right ventricle and pulmonary arteries

Scaffold used to treat cervical esophageal perforation in a 82 year-old patient

Study details

Table 7. Various clinically tested decellularized scaffolds for tissue-engineering applications with their clinical inference

No wound infections and improved sensations in nerve defects in the range 0.5–3 cm (Karabekmez et al., 2009)

No leakage upto day 6 allows oral intake; confirmed defect healing after 4 weeks with no residual defect (Clough et al., 2011) DCAs shows lower peak gradient and re-intervention; small valve sizes (≤18 mm) shows improved peak gradient but no advancement in valve insufficiency (Burch et al., 2010) DPHs valves improved freedom from explantation, low gradients in follow-up, and exhibited adaptive growth (Cebotari et al., 2011) No rise in right ventricular–pulmonary artery pressure gradients with normal valve functioning (Konertz et al., 2005) Grafts can remodel and grow according to the somatic growth of the child; no valve degeneration (Cebotari et al., 2006) No difference in 1 year primary patency rate and shows reduction in thrombosis, infection and interventions with the graft (Katzman et al., 2005) No abnormalities after surgery and exhibits normal structural and functional variables up to 8 years (Raya-Rivera et al., 2014)

Clinical inference

D. Rana et al.

J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Decellularized scaffolds for tissue engineering

10. Concluding remarks The burgeoning field of decellularization holds great promise for the creation of biomimetic scaffolds for stem cell engineering and tissue-regenerative medicine. It is clear that the decellularization treatments can have substantial effects on the composition, mechanical behaviour and host response of decellularized scaffolds derived from native tissue/organs, and could have significant implications for subsequent use in in vitro, in vivo and clinical applications. To date, with all the advancements in decellularization methods, it is critical to achieve the removal of maximal cell residues and the preservation of ECM components without damaging them. The choice of decellularization methods can be rationally selected if a thorough knowledge of the mechanism of disruptive action is contemplated and understood. The host tissue response following in vivo implantation of these scaffolding materials is dependent upon the efficacy of decellularization and the removal of cell remnants. Whole-organ transplant perfusion decellularization using bioreactors is currently receiving renewed interest. An extracorporeal organ perfusion device, in the form of a bioreactor, can provide a constant supply of nutrients and oxygen while simultaneously removing harmful wastes. Based on the experimental examples reviewed in this article and others, stem cells might be considered to be a promising cell source for the recellularization of decellularized scaffolds. However, a few challenges still remain. For example, the use of stem or progenitor cells for recellularization requires long-term culture in a suitable environment, such as 37 °C, 5% CO2, etc., for cellular proliferation, maturation and attachment, to guarantee the viability and function of the reseeded cells in the scaffold, but long-term preservation in this microenvironment may result in deterioration of the ECM components. Thus, improved preservation methods and technologies are urgently required. Reconstruction of

an in vitro microenvironment is the ultimate goal for organ regeneration applications, by utilizing the decellularized scaffold with seeded stem cells. DSCM has also been shown to provide a native tissue microenvironment, resulting in expanded cells with acquired enhanced proliferation ability and differentiation potential. In future, remodelling and repopulation of the organ could be tried to be made possible by simply transplanting the acellular organ scaffold in the patient and allowing it to grow with the patient’s own cells, thereby avoiding any immunological problems and long-term ex vivo culture in bioreactors (Badylak et al., 2011). In addition, vascularization is also a critical factor for the success of decellularized scaffolds. Surface modification of decellularized scaffolds with anticoagulant agents may be a good choice for the support of vascularization upon implantation, but research in this direction is still at the infant stage. To realize the potential benefits of decellularized scaffolds in clinical applications, some critical issues need to be resolved, such as preservation methods, anticoagulation, endothelialization and so on. Research in these directions is under way around the world. This is an exciting time to be involved in decellularized scaffolds in order to formulate them as a clinically ideal scaffolding system for stem cellbased tissue engineering and regenerative medicine, with great challenges and also great expectations ahead.

Conflict of interest The authors declare no conflicts of interest.

Acknowledgements This study was supported by CSCR. D.R. would like to thank the CSCR for the award of a Junior Research Fellowship.

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J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Development of decellularized scaffolds for stem cell-driven tissue engineering.

Organ transplantation is an effective treatment for chronic organ dysfunctioning conditions. However, a dearth of available donor organs for transplan...
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