Tissue Engineering

Cell Survival in Tissue Engineering: Strategies for the Enhancement of Cell Survival Under Oxygen Deprivation

Yiangos Psaras, Thanos Sofroniou

Abstract

Lack of blood flow in transplanted tissue engineered constructs has detrimental effects towards cellular survival, due to lack of oxygen, glucose and the accumulation of metabolic products such as ethanol. Although transplanted constructs eventually become perfused and connected to the systemic circulation within 8 days, their decreased cellular survival prior to inosculation compromises their therapeutic potential and retards progress in the field of tissue engineering. Experimental studies have shown that cell survival can be enhanced with the induction of cellular mechanisms. The strategies discussed in our review center on the suppression of pro-apoptotic proteins, enhanced transcription of genes that carry cytoprotective functions, decreased reactive oxygen species production and their subsequent detoxification, and inhibition of autophagy. Although some strategies may find limited applicability due to parallel induction of mechanisms which require substrates, such as glucose, that cannot be supplied during a non-perfused state in the host, we believe that a combined approach of these strategies with strategies that can address other cell survival-limiting issues can achieve a major increase in cellular survival under ischemic conditions and help deliver new therapies. In this review, we discuss current strategies to address this major issue in the pursuit of improving cellular survival in oxygen deprivation.  

Introduction

Tissue engineering is an interdisciplinary field which made significant progress over the last decade with various improvements in biomaterial property tuning and characterization in terms of their surface chemistry and bulk properties, characterization of tissue properties and laboratory techniques for the induction of differentiation of stem cell into specific cell lines. Despite this progress, the main goal of tissue engineering, which is to bring together biomaterials and cell biology to achieve in vivo replacement, repair, maintenance or enhancement of biological function or for in vitro tissue models for basic research and drug development, is far from realisation. Simple tissues such as skin and cartilage have been successfully developed and even used in the clinic, albeit without appropriate clinical trials but rather as proof-of-principle investigations. [1] However, complex organs face several obstacles that prevent them from being translated for patients.

A main challenge of engineering complex tissues and organs is the ability of cells to survive throughout tissue construction in vitro and in the early stages of in vivo transplantation. Cell survival in tissue engineering refers to the maintenance of cellular function and prevention of apoptosis or necrosis. Several problems impede cell survival in complex tissues; these are directly attributed to the lack of perfusion of constructs, physiologically served by vascularization and its perfusion effects.

Vascularization of tissues provides delivery of oxygen, nutrients and various chemical factors that are essential for cell survival and behavior. The accumulation of cellular products

such as ethanol also hinders cell viability significantly, which makes a vascular system essential for tissue formation. [2-4] In this review, we describe mechanisms that enhance cell survival in oxygen deprived conditions.

Challenges

In the past, production and even clinical application of engineered grafts has been successful. Indeed the biggest success is possibly that of engineered trachea being transplanted in a human. [1, 5] However, grafts of low complexity seem to belittle the challenges faced in producing successful grafts. It must be pointed out that successful production has so far concerned largely avascular grafts with low oxygen demands. O’Hara reports that skeletally mature articular cartilage, the major component of the trachea, is an avascular tissue. [6] While some allowance is made on the basis that tracheal tissue does contain a cellular population, albeit one of limited diversity and number as well as limited innervation, it remains a fact that low metabolic demands must be met in such a graft. [7, 8] Thus, successful production of equivalents for metabolically active tissues such as the heart, kidney or liver has so far been hindered.

Radisic et al. presents that cell survival in engineered cardiac constructs exhibits a linear correlation to oxygen concentration. [9] This is a universal problem, despite the different oxygen requirements of different cell types; cardiomyocytes require 27.6mmol of oxygen per mg of protein per minute, whereas hepatocytes require 18 mmol of oxygen per mg of protein per minute. [10, 11] These cannot be supplied by simple diffusion of oxygen across thick tissue, which is restricted to a distance of 150-200 nm from the source. [12] Simple oxygen diffusion limits are dependent on the type of tissue, particularly its cell density and that of the extracellular matrix and its components. In vitro viability of cardiomyocytes can in fact be maintained on sheets no thicker than 100 μm. [13]

The limitations presented by the diffusion limit of oxygen has been extensively investigated. Notable examples include perfusion bioreactors, which significantly increase cell survival. [14-16] Although procedures that imitate the vascular network present great potential for in vitro work, translation into a clinical setting must achieve a functional vascular system for the simple purpose of connecting the graft to the host’s systemic circulation. Although the production of vascular grafts has been researched for the past few decades, in vitro work has achieved results inferior to those that have been achieved by in vivo work; indeed current grafts are heavily dependent on post-implant vascularization. [17] Vascularisation and inosculation to the systemic circulation can take up to 8 days, a time span which is too long and causes ischemic damage. [18]

Much in the same way that oxygen deprivation confers decreased cell survival, deprivation of nutrients such as glucose and lack of clearance of products material such as ethanol, which is during respiration, have damaging effects via increased reactive oxygen species (ROS) production, pH change caused by increased ROS, and lack of substrate (3, 19, 20). This review focuses largely on strategies to increase cell survival with regards to countering oxygen deprivation.

Strategies

         Several strategies have been proposed and studied for the enhancement of cell survival. Although the literature is relatively poor in the term cell survival, it is apparent that positive evidence is represented in cytoprotective mechanisms relating to conditions imitating the lack of oxygen and nutrient deprivation. Cytoprotective mechanisms have been extensively explored in research and, under specific circumstances, they can be directly employed in enhancing cell survival in scaffolds both in vitro and in vivo.

         A common strategy frequently employed is the delivery of growth factors to scaffolds as a means of manipulating cells to divide, differentiate or secrete factors that affect the extracellular matrix (ECM) or initiate angiogenesis and in turn promote cell survival. [21-23] However, this approach presents several problems. The delivery of growth factors on scaffolds may sometimes be unattractive due to their rapid loss by degradation or diffusion and their effect in surrounding tissues. [24] Additionally, high levels of several growth factors cause disruption of normal tissue function. [25, 26] The complex mixture of different growth factors and their cellular effects in vivo is a complex feat to replicate, as seen by the so far unsuccessful attempts to elucidate ideal specific cellular response. [27] Therefore major cellular responses controlled by key regulatory mechanisms seem more promising than delivering selected individual growth factors in the prospect of manipulating cell response.

HIF-1 stabilization

Hypoxia-inducible factor-1 (HIF-1) is the key regulator of the hypoxia-inducible factor (HIF) pathway that is involved in cellular responses to hypoxia. [28] HIF-1 forms a heterodimeric transcription factor which is a member of the basic helix-loop-helix protein family. The heterodimer consists of an oxygen (O2)-regulated hypoxia-inducible factor-1α (HIF-1α) and a hypoxia-inducible factor-1β (HIF-1β) subunit that is constitutively expressed. [29].

During normoxic conditions, HIF-1α undergoes constant ubiquitination and proteosomal degradation by prolyl hydroxylase domain protein 2 (PHD2), thus disrupting the heterodimerization of HIF-1 and its subsequent gene expression. [30] Therefore, the functions of HIF-1α are completely suppressed in normoxic conditions.

Figure 1. HIF-1α is a key modulator of hypoxia.  Normoxia:  In the presence of adequate oxygen, HIF-1α undergoes hydroxylation by PHD2 which prompts attachment to Von Hippel–Lindau (VHL) factor bound to a protein complex consisting of the proteins Cullin-2 (CUL-2), Elongin-B, Elongin-C, E2 and RBX1 which facilitate the ubiquitination of HIF-1α. Ubiquitination allows HIF-1α proteasomal degradation, inhibiting its function. Hypoxia:  HIF-1α binds to HIF-1β in the nucleus, forming the HIF-1 dimer. This binds to CBP/ E1A binding protein (p300), c-Jun N-terminal kinases(cJUN) and RNA polymerase II (Pol II). On formation of this complex, binding to hypoxia responsive elements (HRE) triggers transcription of more than 100 genes.

In hypoxic conditions, PHD2 activity is inhibited, allowing HIF-1 to bind to a specific DNA region known collectively as that of hypoxia-responsive-elements, thereby transcribing more than a hundred genes with a range of functions (Figure 1). [31] Although HIF-1 activation expresses a host of different genes; proteins of general interest for cell survival are outlined in Table 1.

A key strategy to improve cell viability in pre-vascularized grafts upregulates the HIF pathway to induce an increased expression of all the proteins naturally expressed upon HIF stabilization. HIF activation can counter ischemic damage, thereby increasing cell survival. [32] In fact, at less than 3% O2, HIF-1 encourages cell survival through hypoxia-induced autophagy, although at severe hypoxic conditions (less than 0.1%), hypoxia-induced autophagy eventually results in cell death. [33] A great advantage of increasing HIF activity is that the complexity of providing the appropriate mixture of growth factors is effectively removed as this function is taken by the cells.

Table 1. Important proteins expressed upon HIF-1 activation and their functions. [28]

Abbreviations: TGF-β- Transforming Growth factor beta, IGF-2- Insulin-like growth factor 2,  TGF-α-Transforming growth factor alpha, ADM- Adrenomedulin, MMPs- Matrix metalloproteinases, PAIs -Plasminogen activator receptors and inhibitors,VEGF- Vascular endothelial growth factor, PDGF- platelet-derived growth factor, LEP- Leptin, TGF-β3- Transforming Growth factor beta 3, NOS2- Nitric Oxide Synthase 2, LDHA- Lactate dehydrogenase A, PKM- Pyruvate kinase M, HM 1,2- Hexokinase 1,2, ENO1 - Enolase 1,  GLU1,3 - Glucose transporter-1,3, GAPDH- glyceraldehyde-3-phospate dehydrogenase, PFKL- Phosphofructokinase L, PGK1- Phosphoglycerate kinase.

Factors upregulated in HIF-1 stabilization	Function
TGF-β, IGF-2, TGF-α, ADM	Cell proliferation/survival
MMPs, PAIs	ECM regulation
VEGF, PDGF, LEP, TGF-β3, NOS2	Induction of angiogenesis
Adenylate kinase-3, LDHA, PKM, (HK1,2), ENO1, GLU1,3, GAPDH, PFKL, PGK1	Glucose metabolism

It has been presented that hypoxia-mimetic compounds such as cobalt chloride (CoCl2) and iron (Fe) chelates such as deferoxamine (DFO) initiate transcriptional changes that mimic the hypoxic response in normal oxygen conditions. [34] These agents upregulate the expression of HIF-1α and HIF-1β, increasing the formation of HIF-1 which in turn binds to hypoxia-responsive genes. The agents may exert their effects by interacting with a ferroprotein oxygen sensor similar to what has been observed in nature where cold-tolerant animals endure chronic hypoxia during hibernation by cobalt-haem binding which mimics hypoxia, inducing the activation of HIF-1. [31, 35] Other factors noted to regulate HIF-1 in normoxic conditions include Nickel (Ni), Arsenite, Chromium (Cr), Vanadate, interleukin 1 (IL-1), insulin growth-like factor 1 (IGF-1), nitric oxide (NO) and transforming growth factor alpha (TGF-α). [28]

The applicability of CoCl2 and DFO in HIF-1 stabilization has been overshadowed recently by studies presenting reduced cell viability on their induction.  Zeng et al. found that both compounds inhibit the proliferation and modify the morphology of mesenchymal stem cells (MSCs). [36] High concentrations have also been reported to induce apoptosis, although the underlying mechanism is unrelated to HIF-1α. [37] Nonetheless, careful consideration of these effects can maximize the effectiveness of hypoxia-mimetic compounds while minimizing their side effects. A great such example was illustrated by the development of hypoxia-mimicking bioactive glass for bone regeneration. In this study the authors incorporated cobalt ions in resorbable bioactive glasses to activate the HIF-1 pathway and stimulate apatite formation in bone. The controlled release of cobalt ions from the biomaterial produced low/non-toxic concentrations, which can sustain the activation of HIF-1 pathway in a manner that should be exploited further in tissue engineering. [38]

An alternative approach to increase HIF-1α is through gene therapy. A plasmid vector of HIF-1α injected in diabetic mice was shown to improve wound healing significantly, similar to CoCl2 administration. [39] Using viruses as vectors would generally be undesirable for tissue engineering since the incorporation of viral DNA into the host’s DNA could have undesired  long-term effects.

Hypoxic preconditioning

Different cell types display extensively different levels of susceptibility to hypoxia-induced cell death. Hypoxia tolerance can be enhanced by hypoxic preconditioning (HP), a process by which recurrent exposures to short-term, non lethal hypoxia fosters protection against a later hypoxic event. [33]

HP yields its protective effects via two different mechanisms, providing early/immediate protection followed by a late and prolonged protection phase which is characterized by the expression of several genes. During the prolonged protection phase, cells upregulate the expression of genes that promote angiogenesis and anaerobic metabolism while reducing pro-apoptotic protein expression, ROS production and mitochondrial metabolism. [33, 40] Also, the expression of the GLUT-1, phosphofructokinase (PFK), and lactate dehydrogenase (LDH) genes is increased significantly, improving glucose uptake and glycolytic capacity allowing cells to improve metabolic efficiency and survive in low oxygen environment. It has been suggested that the critical component in the stimulation of these genes is the activation of HIF-1. [32, 33]

Theus et al. exposed embryonic stem cells (ESCs) in sublethal hypoxia (1% O2 for 8 hours) which significantly improved their hypoxic tolerance for 6 days. Treated ESCs were transplanted into ischemic rat brains and corresponded to a 30-40% reduction in cell death compared to untreated ESCs. [41] The beneficial effects of HP have been reported elsewhere in relation to cell survival, suggesting that the effectiveness of hypoxic preconditioning can be enhanced when combined with HIF-stabilization. [42-47]

Rather than increasing oxygen supply, a different strategy to improve cell survival in hypoxic conditions is to reduce oxygen consumption by downregulating cellular metabolism. This approach has been demonstrated by Kim et al. by treating murine C2C12 myoblasts which lack the machinery to self-survive with adenosine, known to reduce the adenosine triphosphate (ATP) demands of Na+/K+ ATPase. Adenosine treated cells sustained their viability and were able to recover when returning to normoxic conditions, presenting that induction of decreased metabolism can mediate cell survival. [48]

Oxytocin pre-treatment

The neurohypophyseal hormone oxytocin, which is primarily expressed in the hypothalamus is used in the induction of cardiac cell differentiation. Initial evidence by Kim et al., presented possible pro-survival properties of the hormone when it was showed that a relatively small number of cells (5x105) delivered their therapeutic load in a rat myocardial infarction model when pre-treated with oxytocin. Further investigation determined that the concentration of activated transcription regulators Akt and extracellular signal regulated kinases (ERK) was significantly increased within one hour of treating with oxytocin. [49] These modulate the PI3K/Akt and the ERK1/2 pathways, which transduce anti apoptotic signals, hence enhancing cell survival. Indeed, similar work has found upregulation of several genes with known angiogenic, anti-apoptotic and anti-remodeling properties. [50, 51] Although the Akt pathway has been extensively investigated, its complex interactions have not been fully elucidated. Observations by Noiseux et al., indicate that on oxytocin pre-treatment, activated Akt accumulates intracellularly close to the mitochondrial marker cytochrome c oxidase subunit 4, suggesting that at least one interaction of Akt which increases cell survival may be related to mitochondrial functions. In the same study, pre-treatment increased cell proliferation by approximately 8% in the senescent (S) phase of the cell cycle and resulted in increased cell survival under hypoxic conditions. [50] The study also reported that the paracrine potential of MSCs towards cardiomyocytes in coculture was increased, as well as their angiogenic potency. These findings are supported by Jankowski et al., who reported that delivery of oxytocin in a rat infarcted heart model reduced the inflammatory effect and, critically, cell death. [52]

A potentially problematic effect of oxytocin pre-treatment was observed by Noiseux et al., as an increase in glucose uptake. This observation, also reported in hypoxic preconditioning may be potentially limiting for cell survival. [32, 33] The impact of increased glucose demand on the cytoprotective effects of oxytocin pre-treatment remains to be elucidated, however, as discussed earlier in this review, in the context of transplantation of constructs, lack of perfusion results in both nutrient and oxygen deprivation, hence a high demand for glucose cannot be met. Should glucose prove to be a limiting factor in oxytocin-induced cell survival, further research must investigate how to address this.

Coculture and growth factor preconditioning

Research by Hahn et al., presented significant enhanced survival of mesenchymal stem cells (MSCs) on culturing under 0.5% hypoxic conditions for 30 hours upon pre-treatment with a mixture of growth factors [fibroblast growth factor 2 (FGF-2), IGF-1 and bone morphogenic protein 2 (BMP-2)].  Pre-treated cells presented 67% 11% survival against 47%14% survival observed for untreated cells. [53] In the same study, pre-treated MSCs were cocultured with cardiomyocytes and had a higher cytoprotective effect towards cardiomyocytes than pure cardiomyocyte culture and coculture of cardiomyocytes with untreated MSCs. Enhanced cytoprotection was attributed to activation of the Akt pathway, which controls several signaling cascades responsible for apoptotic pathways, and CREB transcription, which results in cell survival via gap junction communication. Both the Akt pathway and CREB transcription were enhanced in MSCs treated with growth factors. [51, 53, 54] For a detailed review of the cytoprotective mechanisms modulated by the Akt pathway, the reader is referred to the work of Downward. [51] These findings were consistent when cellular constructs were transplanted in rat models, resulting in significantly smaller fibrotic scars, increased cardiac functionality 8 weeks after transplantation, larger areas of surviving MSCs 2 weeks after transplantation and reduced apoptosis one week after transplantation. [53] Although enhanced, this effect is not fully attributed to MSC pre-treatment alone, since MSCs have previously been proven to have paracrine effects by releasing large amounts of angiogenic and anti apoptotic factors, including VEGF and HGF. [55] The pro-angiogenic effects of MSCs are particularly significant in the scope of tissue engineered construct transplant, as the encouragement of infiltrating vascularity and hence inosculation to the host vasculature may be accelerated in the presence of angiogenic factors.

Treatment with Propofol

A strategy to enhance cell survival has emerged with the use of the intravenous anesthetic, propofol. Propofol has been shown to increase cell survival of PG12 cells under oxygen and glucose deprivation in vitro and in vivo in a rat model of ischemia/reperfusion injury in a dose dependent manner. Evidence by Cui et al., attribute this to inhibition of autophagy, mediated via the class III phophoinositide 3 kinase (PI3K)-Beclin-1-B-cell lymphoma 2(Bcl-2) mechanism. Autophagy-related proteins Beclin-1 and class III PI3K were found to have significantly reduced expression, while cytoprotective Bcl-2 inhibition ceased, bringing about an induced state of survival. [56] In vitro models have shown that the action of propofol can reverse cell damage in neuron/astrocyte cocultures to a level equal to that of the neuroprotective drug MK-801. This was speculated to be caused by maintenance of cellular transport activity of glutamate, hence preventing its accumulation within the cell, blockade of the N-methyl-D-aspartate (NMDA) receptors, or the activation of protein kinase C. [57]

Doxycycline pre-treatment

Doxycycline is a lipophilic second generation tetracycline derivative with anti-inflammatory effects, used as an antibiotic drug. [58] It has an anti-apoptotic effect by inducing microglial activation and blocking apoptotic pathways in two ways. Reducing the transcription of the pro-inflammatory mediators caspase 1, inducible NOS and cyclogenase 2, causes inhibition of the function of interleukin 1β (IL-1β), nitric oxide and prostaglandin-E2 (PGE2). Alternatively, or simultaneously, it causes the upregulation of Bcl-2, which has an antagonistic effect to Bcl-2-associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak) and BH3 interacting-domain death agonist (Bid), factors which prompt cytochrome c release, a pro-apoptotic factor. [59] Malik et al. have also presented that doxycycline preconditioning upregulates the expression of the NF-E2-related factor-2 (Nrf-2) gene, a transcription factor regulating the production of anti-oxidant and cytoprotective genes, whose pathway can be seen in figure 3. [60, 61]

In vitro glucose/oxygen deprivation ischemia/ reperfusion models using multipotent stem cells, subjected to 8 hours of glucose and oxygen deprivation followed by 24 hours of re-oxygenation presented increased cell survival, decreased cell death and ROS production in cells pre-treated with doxycycline compared with untreated cells. [60] These findings support that doxycycline pre-treatment is a potential strategy to increase cell survival. Being a drug, doxycycline carries the benefits of a well-characterized profile, including adverse effects, dosage, safety, and the additional feature of being an antibiotic agent, hence it is not unreasonable to support that doxycycline may be the cell survival strategy closest to realization. [62]

Figure 3: The Nrf-2 pathway: under normal physiological conditions, Nrf-2 complexes to Kelch-like ECH-associated protein 1 (Keap1). This complex associates with Cullin-3 (Cul3) and Rbx, which leads to ubiqiunination of Nrf-2 and disruption of its functions. Stabilisation of Nrf-2 due to ROS, proteasomal degradation of the Keap1-Cul3 complex or dissociation of Nrf-2 from the complexed molecule allows its translocation into the nucleus, where it complexes with transcription factors such as the musculoaponeurotic fibrosarcoma (Maf) transcription factor. These complexes can then bind and enhance the transcription of antioxidant responsive elements (ARE), which encode for proteins with antioxidant and detoxifying functions. [63-67]

Carnosol pre-treatment

The high electrophilic activity of carnosol has been reported to activate Nrf-2, which regulates the action of several potent detoxifying agents, and enhanced cell survival in induction by carnosol. [67-70] One major anti-oxidant enzyme is glutamine-cysteine ligase (GCL), which has a rate-limiting effect on glutathione (GSH), known to exhibit pronounced cytoprotective effects under high oxidative stress brought about by anaerobic respiration. [68] Ischemia, which partially mimics the transplantation environment, presents lack of oxygen. In the absence of oxygen, cells shift towards anaerobic respiration to meet their energy demands. The subsequent accumulation of electrons in an environment of low oxygen produces ROS when oxygen takes up electrons, which bring about cellular damage. [10, 71] Cellular necrosis is caused either by oxidative damage to cellular mechanisms or by seizure of ATP production and consumption of phosphate-rich reserves. Although glucose deprivation cannot be addressed in this strategy, evidence suggested that one function of Nrf-2 is the modulation of anti-oxidant mechanisms by mediating the transcription of GCL, ultimately controlling GSH-mediated cytoprotection. When short interfering RNA (siRNA) for Nrf-2 were transfected into HepG2 cells, the rate-limiting effect of GCL was enhanced, showing that inhibition of Nrf-2 downregulates the pathway. [72]

This mechanism of action was investigated by Chen et al., who reported that transcription of both the heavy and light subunits of GCL boosts the production of functional GCL, which increases the concentration of GSH by 160% in HepG2 cells after 6 hours of treatment with carnosol. Increased GSH concentration persisted for 12 hours, showing prolonged effects after treatment, resulting in higher HepG2 cell viability under oxidative stress caused by H2O2. [68] As such, carnosol preconditioning may bring about a relatively short-term cytoprotective effect, enhancing cell survival via the prevention of oxidative stress-induced death in tissue engineered constructs.

Ischemic preconditioning and Nitric Oxide

The protective effect of HP was firstly shown in a human heart by Yellon et al. [73] Bolli and colleagues later presented the hypothesis that this protective effect is mediated by the effects of nitric oxide (NO), supported in later findings in rat and mice hearts, when the nitric oxide-producing enzyme neuronal nitric oxide synthase (nNOS) was found to be necessary to afford protection in ischemic preconditioning. [74-76] Lunderg et al, have theorized that nitrite and nitrate form reservoirs for NO and that their enzymatic conversion to NO is dependent on the lack of oxygen. [77]

This theory is supported by the observation that on exposure to nitrite in an acute or preconditioning manner, cardiac and hepatic reperfusion injury was decreased. [78] Separate studies have found that nitrite conferred protection in rat and human models for ischemia/reperfusion injury by decreasing infarct size from 47% to 17.5% in vitro. [79] Work by Baker and colleagues also presented that in vitro rat heart models had a 47% reduction in infarct size when pre-treated with nitrite and 32% reduction in infarct size in vivo. Coronary vasodilation was reported as an additional potential mechanism of cytoprotection, which confers an added benefit when considering the application of nitrite in transplanted grafts.

Murillo et al., present that one cytoprotective mechanism arising in HP stimulates NO production via NOS, which is then oxidized to nitrite in normoxia. During subsequent exposure to ischemic conditions, reduction of nitrite to NO enhances survival. [72] NO has been shown to modulate mitochondrial respiration, a system largely disrupted in ischemic conditions. Its action is presented in figure 2. [78]

It must be noted however that, although this strategy can reduce or prevent oxidative stress in cells, clearance of anaerobic respiration products such as ethanol, and lack of glucose in order to meet energy demands are yet to be addressed.

Figure 2: NO inhibits mitochondrial complex I causing a reduction in electron transfer and subsequently a decreased amount of ROS generation. This prevents oxidative inactivation of mitochondrial complexes I, II, III, IV and aconitase, which prevent the opening of the mitochondrial transition pore and subsequent release of cytochrome c. [78] Complex V maintains its function of proton transport through the membrane.

Conclusion

Despite increasing efforts over the last few years, cell survival of grafts remains a major problem in tissue engineering. Oxygen deprivation, glucose insufficiency and waste accumulation are the major underlying causes. As a result, several strategies have been proposed whereby manipulating the machinery of the cells can enhance their tolerance towards oxygen and glucose deprivation. Induction of hypoxic responses through activation of the HIF pathway using cobalt ions or other hypoxia-mimicking compounds induces complex mechanisms which increase cell survival. Hypoxic or ischemic preconditioning is another strategy that counters cellular death by oxygen deprivation which could be used in conjunction with HIF pathway activation to increase hypoxia-tolerance. In addition, pharmacological treatment of cells to inhibit pro-apoptotic proteins, decrease ROS production or downregulation of cellular metabolism is also highly promising. Although approaches present overlap of mechanisms, the  inability to address increased glucose uptake is a significant hurdle to overcome since perfusion to the cells is not available in engineered grafts. We suggest that future studies on cell survival focus their efforts on elucidating cellular mechanisms that promote cell survival in low glucose environment, and we focus our future outlook towards studying the effects of combined approaches both with regards to combining approaches to counter cell death brought about by lack of oxygen, as well as combining these with strategies to counter the effects of glucose deprivation and waste tolerance.

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