Towards autotrophic tissue engineering: Photosynthetic gene therapy for regeneration
Introduction
Oxygen is an essential molecule for cell metabolism. It plays a key role in tissue survival and regeneration, yet paradoxically, its appropriate delivery is one of the major problems for the clinical translation of non-vascularized tissue engineering approaches [1]. As a result, cells within bioartificial tissue constructs are stringently dependent on oxygen diffusion, which barely reaches few hundred micrometers and limits the construction of artificial tissues to non-clinically relevant sizes [2]. Besides the basic oxygen requirements, tissue regeneration relies on several bioactive molecules, such as growth factors, that control key processes including inflammation, angiogenesis and tissue remodeling [3], [4], [5]. Scaffold bioactivation with growth factors has been intensively investigated as strategy to enhance the regenerative potential of engineered tissues [6]. However, direct growth factor administration has major limitations related to the short biological half-life of the molecules, and the consequent need for repeated administration of large and potentially toxic bulk doses [7], [8]. Conversely, traditional gene therapy represents a better strategy to provide a constant growth factor supply. Yet, this approach raises ethical and technical concerns that affect their translation into clinical settings. For instance, the delivery of target genes often creates risk of oncogenicity, insertional mutagenesis and viral vector-related immune reactions [9], [10], while target tissue-specificity and high transduction efficiency are still unrealized for non-viral gene delivery methods [11], [12]. Additionally, sustained transgene expression is not guaranteed if the transgenic cell survival depends of local oxygen tension at the injection site.
Recently, we have introduced HULK (from the German Hyperoxie Unter Licht Konditionierung) as a novel concept to deliver oxygen into biomaterials independently of blood vessel perfusion. The main idea behind the HULK approach is that, by incorporating photosynthetic microalgae such as Chlamydomonas reinhardtii (C. reinhardtii) into artificial constructs, the local induction of photosynthesis could be able to supply the metabolic needs of bioengineered tissues in vitro [13] and in vivo [14]. To take HULK one step forward, in this work we evaluated the feasibility of implanting photosynthetic scaffolds in fully immunocompetent mice. Then, we explored the idea of using genetically modified microalgae to engineer photosynthetic scaffolds that, in addition to oxygen, could provide other pro-regenerative molecules to the wounded tissue. As a proof of concept, we created a gene modified C. reinhardtii strain that constitutively secretes the human vascular endothelial growth factor VEGF-165 (VEGF).
In this work we propose that the activation of biomaterials with gene modified microalgae could be used to locally deliver oxygen and other pro-regenerative molecules into bioartificial tissue constructs.
Section snippets
Cell culture of C. reinhardtii
The cell-wall deficient, cw15-30-derived UVM4 C. reinhardtii strain [15] was grown photomixotrophically at 20 °C on either solid Tris Acetate Phosphate (TAP) medium or in liquid TAPS-medium supplemented with 1% (w/v) sorbitol [16]. For light stimulation, a lamp with the full spectrum of white light (Nano Light, 11 Watt, Dennerle, Vinningen, Germany) was used to provide constant illumination (2500 lux, eq. 72.5 μE/m2∙s1). Cell concentration in the culture was determined using a Casy Counter TT
Engraftment of photosynthetic scaffolds does not trigger a significant immune response
In our previous work, we showed that, after short periods of implantation, the use of scaffolds seeded with the microalgae C. reinhardtii, did not trigger a significant inflammatory response in an athymic immunodeficient nude mice model [14]. Here, as a further step to evaluate the safety of HULK in vivo, photosynthetic scaffolds for dermal regeneration were used to replace full-skin defects in fully immunocompetent mice [23] and the results were evaluated after fourteen days. No complications
Discussion
We previously proposed that the development of a photosynthetic biomaterial, capable of oxygen self-production, could offer an unlimited local source of oxygen supply for the regenerating tissue, which would merely depend on light stimulation [13]. In this approach, the green unicellular microalgae C. reinhardtii are incorporated into scaffolds and exposed to illumination to trigger photosynthesis and achieve oxygen production in situ. Moreover, we also demonstrated that C. reinhardtii
Conclusion
In order to overcome hypoxia, we have previously suggested that photosynthetic tissue engineering could be used as an alternative source of oxygen supply to blood vessel-perfusion. In this work, we demonstrated that photosynthetic scaffolds can be compatibly engrafted in fully immunocompetent mice without causing a significant immune response and improving tissue regeneration. In addition, we were able to genetically engineer a C. reinhardtii strain to secrete the human growth factor VEGF, and
Financial disclosure
This work was partially financed by a CIRM-BMBF Early Translational II Award to J.T.E., ICGEB (CRP/CHI11-01) and the FONDAP Center for Genome regulation (Nr. 15090007) to both M.A. and J.T.E., and a DFG grant (Ni390/7-1) to J.N. M.H. was supported by an ERC starting grant (LiverCancerMech), the SFBTR 36 and the Stiftung für Bio-medizinische Forschung. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interest
JTE is founder and VP of Technology at SymbiOx Inc. This startup did not provide any financial support to this work but is closely related to some topics of this manuscript.
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