Glycosaminoglycans as a measure of outcome of cell-on-scaffold seeding (decellularization) technology

KEYWORDS:

• Extracellular matrix
• glycosaminoglycans
• decellularization
• scaffold

Within the field of tissue engineering, cell-on-scaffold seeding technology (CSST) [1] – whereby cells are seeded on supporting scaffolding materials – appears to offer the quickest route to clinical application. The literature reports that more than 250 patients have been the recipients of bioengineered organoids that were manufactured from autologous cellular material in combination with biomaterial scaffolds [2]. Crucially, immunosuppression was never needed at any time after implantation.

The rationale for using scaffolds lies on the striking evidence that the cell milieu represented by the extracellular matrix (ECM) is critical for cells to be viable and functional. Understood as sine qua non, ECM enables multicellular tissues and organs to maintain their anatomical and physiological identity and specificity [3,4]. Currently used scaffolds can be categorized in synthetic and natural. Natural scaffolds can be derived from the ECM of native organs of animal or human origin, through a process called decellularization whereby the cellular compartment of a given tissue or organ structure is dissolved and cleared [5,6]. Other forms of natural scaffolds include those generated from non-synthetic materials such as collagen and chitosan. Nevertheless, ECM scaffolds have become very popular in the field of whole organ bioengineering because they maintain their architecture and gross molecular composition following decellularization. Furthermore, they the innate vasculature that is subsequently preserved demonstrates the ability to sustain physiological blood pressure [7]. This ability is of paramount importance in view of the in vivo implantation of bioengineered whole organs, particularly when no other technology – 3D printing included – has been reported to be able to reproduce a vascular bed with physiological and anatomical characteristics comparable to their innate counterparts. Moreover, native ECM lacks immunogenic cell membrane proteins and retains numerous growth factors (GFs) that are stored in the 3D structure of the ECM. When multipotent stem cells are seeded on such scaffolds, they attach well and are able to migrate throughout the 3D framework of the scaffold. They subsequently remodel the ECM and mount inflammatory and angiogenic responses in the appropriate contexts [8,9].

The hurdles to overcome before CSST can deliver bioengineered solid organ for transplant purposes at a population-level scales are numerous. Contemporary investigators will need to elucidate the mechanisms of organ ontogenesis, development, and repair and regeneration intrinsic to adult tissues and organs. Furthermore, ad hoc bioreactors currently employed in bioengineering investigations must mimic the in vivo environment. Investigators must also identify the most appropriate cell type to use to regenerate the parenchymal compartment of a given tissue or organ. Potential complications must also be addressed including the intrinsic risk for cancerous degeneration. Crucially, the specific mechanisms through which cells interact with ECM at the biomolecular level must be decoded. Theoretically, ECM scaffolds should recapitulate the innate ECM before decellularization, in terms of molecular composition. For example, one of the most important components of the innate ECM is represented by glycosaminoglycans (GAGs), which are long unbranched polysaccharides comprehending a repeating disaccharide unit. The disaccharide units comprise either of two modified sugars, N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc), and an uronic acid as glucuronate (GlcA) or iduronate. GAGs differ from one another in the identity of their disaccharide unit, geometry of glycosidic linkage, extent of sulfation, and the nature of the core proteins that may be covalently bound to the polysaccharide chain. They are highly negatively charged molecules, with extended conformation that imparts high viscosity to the solution in which they reside, and are located primarily on the surface of cells or in the ECM but are also found in secretory vesicles in some types of cells. The majority of GAGs in the body are linked to core proteins that constitute proteoglycans, including heparin, chondroitin sulfate, dermatan sulfate, and heparin sulfate: exerting several functions fundamental at virtually all stages of life (http://themedicalbiochemistrypage.org/glycans.php#proteoglycans). GAGs provide not only a structural quality to the tissue but they also confer organ-specific functional properties with a direct involvement in cell migration, proliferation, and differentiation. Moreover, GAGs’ role in morphogenesis, angiogenesis, immune response, cellular homeostasis, structural resistance to tension, and compression has been studied in depth [10]. GAGs have the capacity to assemble protein-protein complexes including growth factor receptors (for instance, TGF-beta, VEGF, EGF family members, PDGF) both on extracellular environment and on cellular surface [11,12].

We infer that physiological amounts of GAGs are retained in the ideal ECM scaffold. As a corollary, we propose GAG preservation as metric of successful decellularization protocols. The implications of this approach are critical. For example, embryonic stem cells are often employed in CSST due to their plasticity and potential to generate all adult cell lines and ultimately tissues. It has been demonstrated that, during differentiation, the evolution of specific cell lineages is associated with particular patterns of GAGs expression, and that the addition of soluble GAGs saccharides to cells can influence the pace and outcome of differentiation, so highlighting specific pattern requirements for particular lineages [13]. Therefore, lack of adequate GAGs in ECM scaffolds may impair the ability to regenerate the cellular compartment.

Unfortunately, among the now numerous studies on CSST present in the literature, only few address the matter of GAG content within the ECM scaffold. Indeed, most studies do not perform any GAG quantification, while others quantify GAG content obtained through different protocols without relating these values to the GAG content of the corresponding innate organs before decellularization. Moreover, the most frequently reported method to quantitate GAGs was chemical, whereas only a handful of studies resorted to mass spectrometry which has shown great accuracy in studying the innate ECM and establishing the matrisome of tissues and organs [14]. Li et al. recently reported the quantitative proteomic results of investigations of four types of biological scaffolds – namely, rat-tail type I collagen, growth factor reduced-Matrigel, decellularized rat livers, and decellularized human lungs. Each was studied with ECM proteomics using filter-aided sample preparation as preparation strategies [15]. Their study provided the first insight into the complexity of the proteome of biological scaffolds and shed new light on the quantitative protein changes that occur with the decellularization process. Most of the remnant cellular proteins significantly decrease following decellularization, whereas the structural matrix proteins including GAGs remain and are well preserved.

To foster progress in tissue engineering as it is applied to organ transplantation, universal criteria and measure of outcomes must be defined and accepted by the scientific community. Given the many relevant functions exerted by GAGs, we propose GAGs preservation as measure of outcome of tissue engineering technologies whereby acellular ECM scaffolds are employed as cell-supporting biomaterial.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties

Additional information

Funding

This manuscript was funded by the Liberi Tutti Onlus Foundation.