An overview of decellularisation techniques of native tissues and tissue engineered products for bone, ligament and tendon regeneration
Introduction
Traumatic injuries and infective conditions affecting bone, tendons and ligaments are a significant health and economic burden and generally require surgical reconstruction using autologous or allogenic grafting materials. Autologous grafts have the advantages of being histocompatible and non-immunogenic [1] and have a low risk of disease transmission, but their availability is limited and donor site morbidity is a significant issue. While the use of allografts and xenografts eliminates donor site morbidity and decreases operating times, these materials have the potential risk of transmitting diseases, immune rejection and may require extended time periods for regeneration to occur [1].
One of the major concerns with the use of allografts and xenografts is the management of the subsequent immune response. The immunogenicity of xenografts depends largely on the presence of α-gal epitopes, expressed by most species with the notable exceptions of humans and some primates [2]. Another important mechanism, responsible for rejection of both allografts and xenografts is the major histocompatibility complex (MHC). The main function of surface proteins coded by the MHC is to bind foreign antigens, and present them to immune cells in order to initiate an immune response. In graft tissues, MHC coded surface proteins act as antigens, which initiates an immune response by the host [3]. However, extracellular matrix (ECM) components, probably because of their crucial roles in cell migration, proliferation, and differentiation [4], [5], are highly conserved across species and do not seem to trigger a major destructive inflammatory reaction [6]. Hence, the removal of cellular and MHC components i.e. ‘decellularisation’ of allograft and xenograft material has therefore emerged as a promising tool for tissue engineering and regenerative medicine applications, both preclinically and clinically. Decellularisation allows the preservation of naturally occurring biological components while significantly reducing the immunogenicity of grafts, providing a physical and possibly a biochemical niche for the homing of progenitor cells enabling subsequent tissue regeneration [7], [8]. Biological scaffolds composed of decellularised extracellular matrix (dECM) have been utilised in the repair of various tissues, including skin [9], bladder [10], [11] and heart valve [12], [13]. Several decellularised tissues have received FDA approval for clinical use in humans, including porcine heart valves (Synergraft®; Cryolife), dermis tissue (Alloderm®; LifeCell) and porcine urinary bladder (Urinary bladder matrix; ACell) [14], [15] and are commercially available.
The preparation of acellular matrices requires the thorough removal of cellular components while preserving the native ultrastructure and composition of the ECM during the process of tissue decellularisation [8], [16].
Bone is a hard tissue, composed of mineralised inorganic phase inserted in an organic phase, mainly collagen I, and some non-collagenous proteins such as osteocalcin, bone morphogenetic proteins (BMPs), proteoglycans [17]. Ligaments and tendons are soft tissues, which connect bone to bone and bone to muscle respectively. Collagen is the major protein in both tendons and ligaments, accounting for approximately 75% of the dry weight [18]. Other ECM components include non-collagenous matrix such as proteoglycans, composed of glycosaminoglycan chains (GAG) and proteins, which play an important structural role [18]. Indeed, GAGs are a major component of the soluble protein fraction of the ECM and are directly associated with mechanical loading capacity in a variety of tissues [19]. Entheses are the interfaces where tendon or ligament inserts into bone, allowing transmission of forces between two mechanically dissimilar tissues, hence avoiding mechanical mismatch [20]. They are highly hierarchical tissues, which exhibit gradients in tissue organisation with varying cellular and extracellular matrix compositions and arrangements [21].
Due to the unique characteristics of each tissue, (such as anatomical structure, dimensions, porosity, thickness, presence of soft tissue, etc.), a wide range of different decellularisation methods have been proposed encompassing a great variety of detergents, concentrations and incubation times [22], [23], [24] with the primary objective of achieving appropriate removal of the cellular components, while the preservation of the ECM being a secondary consideration.
There is a plethora of documented decellularisation techniques, many of which are tissue specific, and the establishment of a standardised protocol remains elusive if not impossible. As such, multiphasic tissues including bone-ligament grafts are even more challenging due to different tissue morphologies and architectures [20]. This review therefore provides an overview of the main decellularisation techniques for bone and ligament tissues, and their potential for application in regenerative medicine and tissue engineering. The second part of the review elaborates on the current limitations of decellularised matrices and how recent developments involving in vitro recellularisation prior to implantation can potentially enhance their regenerative potential. Ultimately, the recent concept of synthetic scaffold decoration by natural ECM protein is described and its in vitro and in vivo efficacy towards tissue regeneration is critically examined.
Section snippets
Decellularisation of native tissues: ligaments, tendons and bone
Decellularised matrices can be considered as biological scaffolds derived from native tissues, treated to remove the cellular components while preserving structural characteristics and functional ECM proteins [16]. Decellularisation is generally achieved by a combination of chemical, enzymatic and/or physical treatment. As stated above, the tissue structural properties determine the decellularisation protocol and a non-exhaustive list of protocols utilised for tendon/ligament and bone tissues
Decellularisation of native ECM for the formation of hydrogels
The use of hydrogels allows a localised delivery with minimal invasive surgical intervention.
In order to extract ECM protein from natural tissues, a decellularisation step is necessary in order to prevent immune reaction once implanted. This applies for natural ECM based hydrogel and scaffolds cultured in vitro prior to removing the cellular components leading to ECM decoration.
The native tissues are decellularised via one of the methods described in the previous section, and then further
Biologically enhanced synthetic scaffolds
Because of their complex structure and the essential mechanical properties of ligaments and tendons, decellularisation of whole tissue is the most suitable scaffold for ligament/tendon replacement, at least from a biomechanical aspect. In contrast, bone defects often require anatomically accurate grafts which may not be obtained by using native decellularised tissues. Therefore, the technology of 3D-printing enables the development of such personalised structures, although such scaffolds
Future directions
This review outlines the main decellularisation techniques for native tissues using either chemical, enzymatic or physical treatments for the removal of the cellular components and has highlighted their limitations. Although the utilisation of decellularised matrices for tissue regeneration is widespread, there is still limited knowledge on the extent of remaining cellular component that could trigger an immune rejection. Indeed, the characterisation of decellularised tissues is almost
Acknowledgments
This study has been financially supported by the Australian National Health Medical Research Council (grant number: APP1086181).
Author disclosure statement
No competing financial interests exist.
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