3D-Bioprinted Constructs that Breathe

Abstract

An old but still hot topic in tissue engineering (TE) is the establishment of efficient vascularization networks proving fine, controlled, and long-term distribution of oxygen and nutrients. Combining elegant three-dimensional (3D) fabrication techniques with unconventional living microorganisms, namely photosynthetic species, complex 3D-printed TE constructs are proposed by Maharjan et al. enabling to provide a continuous and on demand oxygen supply to mammalian tissues. An old but still hot topic in tissue engineering (TE) is the establishment of efficient vascularization networks proving fine, controlled, and long-term distribution of oxygen and nutrients. Combining elegant three-dimensional (3D) fabrication techniques with unconventional living microorganisms, namely photosynthetic species, complex 3D-printed TE constructs are proposed by Maharjan et al. enabling to provide a continuous and on demand oxygen supply to mammalian tissues. Following the implantation of three-dimensional (3D) bioengineered systems, a series of well-orchestrated and complex biological processes occur aiming towards tissue repair and regeneration.1Lee K. Silva E.A. Mooney D.J. Growth factor delivery-based tissue engineering: general approaches and a review of recent developments.J. R. Soc. Interface. 2011; 8: 153-170Crossref PubMed Scopus (955) Google Scholar While the in vitro delivery of essential biomolecules assuring cell survival can be more easily circumvented, using for example cell culture apparatus such as perfusion bioreactors, most tissue engineered strategies fail upon implantation. In vivo, tissue survival is dictated by a 100-200 μm distance from the nearest capillary, thus the implanted biomaterials have to rely on a vascular network to supply oxygen and nutrients. Deprived from that, hypoxic regions are generated, and consequently cell proliferation is significantly jeopardized, as well as other important cellular processes, such as gene expression and matrix deposition. Ultimately, either by the absence of a vascular network, or its delayed establishment, cell death and microtissue necrosis occur, specially at the inner regions of the TE constructs. Considering that situations demanding the intervention of TE approaches are most often those that include large defects, otherwise solved by the innate self-healing of the human body, the establishment of a proper vascular supply is even more critical, and challenging, in such clinical-relevant 3D artificial strategies. Different approaches have been proposed to overcome nutrient perfusion and mass transport limitations of TE constructs, ranging from (1) bioengineered systems functionalized with key binding sites (Figure 1i) to guide and facilitate angiogenesis (e.g. integrins, growth factors);2Li S. Nih L.R. Bachman H. Fei P. Li Y. Nam E. Dimatteo R. Carmichael S.T. Barker T.H. Segura T. Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability.Nat. Mater. 2017; 16: 953-961Crossref PubMed Scopus (101) Google Scholar (2) systems based on void-spaced or liquified structures to enhance diffusion and vascular ingrowth – those devices can be produced by sophisticated techniques, such as 3D bioprinting and electrospinning-based approaches, that enable high geometrical precision, and thus playing with bulk features (Figure 1ii-a), such as architecture and porosity,3Kapfer S.C. Hyde S.T. Mecke K. Arns C.H. Schröder-Turk G.E. Minimal surface scaffold designs for tissue engineering.Biomaterials. 2011; 32: 6875-6882Crossref PubMed Scopus (271) Google Scholar or by using bottom-up strategies (Figure 1ii-b), such as encapsulation strategies in liquefied environments;4Correia C.R. Nadine S. Mano J.F. Cell Encapsulation Systems Toward Modular Tissue Regeneration: From Immunoisolation to Multifunctional Devices.Adv. Funct. Mater. 2020; 30: 1908061Crossref Scopus (9) Google Scholar,5Correia C.R. Bjørge I.M. Zeng J. Matsusaki M. Mano J.F. Liquefied Microcapsules as Dual-Microcarriers for 3D+ 3D Bottom-Up Tissue Engineering.Adv. Healthc. Mater. 2019; 8: 1901221Crossref Scopus (10) Google Scholar and (3) in vitro prevascularized scaffolds (Figure 1iii).6Guo S. Redenski I. Landau S. Szklanny A. Merdler U. Levenberg S. Prevascularized Scaffolds Bearing Human Dental Pulp Stem Cells for Treating Complete Spinal Cord Injury.Adv. Healthc. Mater. 2020; 9: 2000974Crossref Scopus (17) Google Scholar However, the in vivo establishment of functional vascular networks still remains a major challenge. While decorated constructs rely on the timely arrival of specific cells that possess the inherent ability to self-organize and achieve this complex stage, thus being limited by a spatiotemporal feature, prefabricated vascular constructs still present limited capacity to anastomose with the host vascular network. Additionally, such TE approaches also present inherent physical barriers that block the ingrowth of host vessels, either resultant from the biomaterial content itself and from the deposited extracellular matrix. Ultimately, besides failing the healing process of the target tissue, the resultant gradients of oxygen that led to the generation of hypoxic regions following the implantation of TE constructs, can also trigger tumor microenvironments by affecting metabolic, inflammatory, and differentiation pathways of endogenous cell subsets. It is thus easily to understand why vascularization is one of the main challenges that need to be taken to bring tissue engineering into the clinical practice at a large scale. As such, vascularization is an old but still hot topic in the tissue engineering community. More recently, in an attempt to overcome the associated road blocks of poorly vascularized bioengineered systems, a plethora of materials able to release oxygen (Figure 1iv-a) have been proposed.7Camci-Unal G. Alemdar N. Annabi N. Khademhosseini A. Oxygen Releasing Biomaterials for Tissue Engineering.Polym. Int. 2013; 62: 843-848Crossref PubMed Scopus (85) Google Scholar Such oxygen-releasing materials are often composed of solid peroxides (e.g. solid calcium and magnesium peroxides), hydrogen peroxide, sodium percarbonate, and fluorinated compounds. However, the associated burst release of oxygen arises cytotoxic concerns, as well as short-term supply curves. To overcome these limitations, continuous and on demand living suppliers of oxygen have been proposed relying on the co-culture of mammalian cells with unconventional organisms, such as photosynthetic species (Figure 1iv-b), namely microalgae,8Chávez M.N. Schenck T.L. Hopfner U. Centeno-Cerdas C. Somlai-Schweiger I. Schwarz C. Machens H.-G. Heikenwalder M. Bono M.R. Allende M.L. et al.Towards autotrophic tissue engineering: Photosynthetic gene therapy for regeneration.Biomaterials. 2016; 75: 25-36Crossref Scopus (51) Google Scholar or bacteria.9Li W. Wang S. Zhong D. Du Z. Zhou M. A Bioactive Living Hydrogel: Photosynthetic Bacteria Mediated Hypoxia Elimination and Bacteria-Killing to Promote Infected Wound Healing.Adv. Ther. 2020; 2000107: 1Google Scholar Different applications have been proposed aiming towards wound healing, cancer therapy, cardiovascular related diseases, and cell transplantation. In this issue of Matter, Maharjan et al.10Maharjan S. Alva J. Cámara C. Rubio A.G. Hernández D. Delavaux C. et al.Bionic Photosynthetic Oxygenation within 3D-Bioprinted Vascularized Tissues.Matter. 2020; 4Google Scholar take a step further in the development of such living oxygen suppliers by embedding bioprinted photosynthetic algae (C. reinhardtii) with mammalian cells (HepG2) in a 3D gelatin methacryloyl-based hydrogel matrix to create honeycomb-shaped constructs mimicking the human liver. The bioprinted algae was able to release oxygen upon specific light conditions, and consequently enhanced cell proliferation and production of liver-specific proteins. Remarkably, the authors were also able to produce microchannels taking advantage of the enzymatic degradation of the cellulose-based bioink. Afterwards, vascular networks were created by repopulating the hollow microchannels with endothelial cells (C2C12). These findings further open the door to the prospect of next-generation of vascularization strategies relying on the use of unconventional living components such as algae. We preview that the organelles of such microorganisms may also be explored, such as chloroplasts, since they exhibit similar potential in the continuous and unlimited release of oxygen. We believe that such strategies may contribute to significantly improve the in vivo bioperformance of TE constructs or to act as light-responsive drug delivery systems. Detailed understanding of the living organisms or organelles to be used in combination with biomaterials, its oxygen producing pathways, and the establishment of the adequate balance of the co-culture conditions suitable for the survival of both algae and mammalian cells, are key prerequisites to be considered in the design of such strategies. In order to develop a fully functional living organism-based oxygen release system, it is essential that all the surrounding factors such as type of microalgae species, culture medium, temperature range, and light intensity are mutually accepted by both algae and mammalian cells, which have distinct optimal conditions. We truly believe that photosynthetic microorganisms-based strategies can be harnesses as a sustainable, eco-friendly, cost-effective source of oxygen for human cells, thus bring considerable advantages to the TE field. Of course, the translation of such systems into the clinics will be hindered by important regulatory constrains that should be addressed in the future. Funding from the Portuguese Foundation for Science and Technology ( CIRCUS-PTDC/BTM-MAT/31064/2017 , and CICECO -Aveiro Institute of Materials- UIDB/50011/2020 & UIDP/50011/2020 ), and the European Research Council ( ERC-2014-ADG-669858-ATLAS ). We also acknowledge Sara Nadine for the help with Figure 1. Symbiotic Photosynthetic Oxygenation within 3D-Bioprinted Vascularized TissuesMaharjan et al.MatterNovember 18, 2020In BriefThree-dimensional-bioprinted unicellular green algae, Chlamydomonas reinhardtii, was used as a sustainable bionic source of O2 in engineered tissue constructs. O2 photosynthetically produced by bioprinted algae significantly improved the viability and functionality of the human cells within surrounding matrices while reducing their hypoxic conditions. Fugitive patterns encapsulating the algae were enzymatically dissolved by cellulase digestion to create interconnected microchannels, which were subsequently endothelialized, generating vascularized tissues. Full-Text PDF Open Archive