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Advances in Controlled Oxygen Generating Biomaterials for Tissue Engineering and Regenerative Therapy Nureddin Ashammakhi, Mohammad Ali Darabi, Nermin Seda Kehr, Ahmet Erdem, Shu-kai Hu, Mehmet R. Dokmeci, Ali S. Nasr, and Ali Khademhosseini Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00546 • Publication Date (Web): 04 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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Advances in Controlled Oxygen Generating Biomaterials for Tissue Engineering and Regenerative Therapy

Nureddin Ashammakhi1,2,3,4,*, Mohammad Ali Darabi1,2,3,4, Nermin Seda Kehr1,3,4,5, Ahmet Erdem1,3,4,6,7, Shu-kai Hu1,3,4,5, Mehmet R Dokmeci1,2,3,4, Ali S. Nasr8, Ali Khademhosseini1,2,3,4,9,* 1Center

for Minimally Invasive Therapeutics (C-MIT), University of California - Los Angeles, Los Angeles, California, USA 2Department of Radiological Sciences, David Geffen School of Medicine, University of California - Los Angeles, Los Angeles, California, USA 3Department of Bioengineering, University of California - Los Angeles, Los Angeles, California, USA 4California NanoSystems Institute (CNSI), University of California - Los Angeles, Los Angeles, California, USA 5Physikalisches Institut and Center for Soft Nanoscience, Westfälische Wilhelms-Universität Münster, Busse-Peus-Strasse 10, 48149 Münster, Germany 6Department of Chemistry, Kocaeli University, Umuttepe Campus, 41380, Kocaeli, Turkey 7Department of Biomedical Engineering, Kocaeli University, Umuttepe Campus, 41380, Kocaeli, Turkey 8Division of Cardiothoracic Surgery, Department of Surgery, University of Iowa Hospitals and Clinics, Carver College of Medicine, University of Iowa 9Department of Chemical Engineering, University of California - Los Angeles, Los Angeles, California, USA (*) Corresponding authors California NanoSystems Institute (CNSI), University of California – Los Angeles, 570 Westwood Plaza, Building 114, Room 4523, Los Angeles CA 90095, USA, Tel. +1 310 794 5845, Email: [email protected], [email protected]

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2 KEYWORDS. biomaterials, ischemia, regenerative, oxygen, tissue engineering, wound healing.

ABSTRACT. Oxygen (O2) generating biomaterials are emerging as important compositions to improve our capabilities in supporting tissue engineering and regenerative therapeutics. Several in vitro studies demonstrated the usefulness of O2 releasing biomaterials in enhancing cell survival and differentiation. However, more efforts are needed to develop materials that can provide sustained O2 release for long-term. In this paper, we present different O2 generating sources, including hydrogen peroxide, sodium percarbonate, calcium peroxide and magnesium peroxide, also covered types of carriers and relevant methods of fabricating O2 generating systems. Then, the applications of O2 generating materials in supporting engineered constructs, supplying high O2 demanding cell transplants, and supporting ischemic tissues are discussed. Moreover, the challenges and future perspectives are highlighted.

1. INTRODUCTION Tissue defects may result from congenital or acquired disorders. The latter includes tissue damage by disease, trauma or removal by surgery 1, 2. Several methods have been developed to treat tissue defects including the use of autografts, allografts and xenografts. Although autografts are traditionally preferred 3, their retrieval leads to comorbidity of the donor site and the autografts are also short in supply 4. One of the main issues associated with the use of grafts is the survival of cells after transplantation 5. The use of allografts was explored such as in cases of bone 6, 7 and skin 8. The use of xenografts 9, 10 was also suggested but this may lead to major immune reactions and may carry the risk of transmission of infectious agents

11.

In

addition, ethical and cultural issues are limiting the use of animal-based sources as possible


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3 alternatives. Search for alternatives thus far, has continued including the use of alloplastic materials for reconstructive purposes

12,

cells for replacement therapy

13,

e.g. hormone

secreting cells 14 or biomolecules 15 that may help the regeneration of tissues such as those of the skeletal system 16 and skin 17.

Alternatively, tissue engineering approaches in which biomaterials are combined with cells were proposed 18. More recently, the method of three-dimensional (3D) bioprinting was used to develop living tissue constructs in vitro 19-21 and several bioinks were developed to construct 3D biomimicking models, incorporating tissue complexity in terms of mechanical, physical and biological variations 22. One major issue that limits the success of translating the engineered products to the clinic is the failure of their vascularization after implantation in the body 23, 24. Cells in engineered constructs obtain their nutrients and oxygen through diffusion 25 which is a limiting factor due to the far distance of cells from capillaries. Accordingly, new capillaries need to be rapidly formed (termed angiogenesis) within the implanted tissues to compensate the diffusion limit of oxygen 26. Since angiogenesis needs time to take place 27, 28, a strategy is needed to bridge this gap to ensure un-interrupted supply of nutrients and oxygen to implanted constructs and prevent cell death. Since O2 is one of the most important nutrients for cell survival, approaches for the delivery of oxygen to implanted tissues are explored in this paper.

In general, O2 can be supplied directly (e.g. using perfluorocarbon-based O2 releasing systems 29, 30),

or delivered by a biomaterial carrier

31.

Different biomaterials have been explored as

scaffolds, yet only very few have been filled with O2 32. One concern so far, has been the burst release of O2 which can be toxic to cells 33. Providing sustainable O2 release can support cells residing in the implanted construct before angiogenesis takes place (1-2 weeks

34)

and new

vessels take over to supply O2 to cells. This has been largely addressed by using more



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4 hydrophobic carrier materials that can release O2 over longer periods of time

35, 36

extending

for up to 10 days 30. The efficiency of these biomaterials and their impact on the survival of cells has been demonstrated in vitro 37.

In addition to the support of engineered constructs during the critical period of their immediate post-implantation stage, O2 generating materials can also be useful in the treatment of ischemic tissues such as chronic wounds and problems that follow the blockage of feeding vessels, e.g. myocardial infarction 38. Moreover, O2 generating materials can be used for supporting O2 to cells having high metabolic activity such as pancreatic cells, muscle cells, hepatocytes, and neurons39. So far, most of studies were focused on combining various carrier materials with an O2 source 32. Reported beneficial impact was demonstrated in cases of bone 40 and muscle 41 tissue engineering, cardiac ischemic problems 37, wound healing 42 and in support of pancreatic cells 43.

In this review, we discuss O2 source materials, carrier scaffolds, fabrication methods, mode of release, their characterization methods and their effect on cells and in vivo testing. We also highlight challenges, future outline and summarize recent advances in this dynamic and important area. It is expected that more developments and applications of O2 generating systems will emerge and will have an impact on the future of engineered tissue constructs and their clinical applications as well as the treatment of various ischemic conditions and cell replacement therapeutics.

2. OXYGEN GENERATING BIOMATERIALS


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5 In this section, we discuss O2 source materials, carrier materials, fabrication of O2 generating systems, their characterization and the mechanism of O2 release.

2.1.

O2 source materials

Therapeutic O2 can be delivered in gas form or via liquid or solid sources. In gas form, hyperbaric O2 was applied in the treatment of skin wounds

44.

However, this approach has

several complications such as pulmonary damage, and it needs repeated applications

45.

For

sustained prolonged delivery, the use of solid and liquid O2 sources was thus, explored. O2 sources, that have been used so far, include hydrogen peroxide (H2O2) 46, sodium percarbonate (SPO) ((Na2CO3)2•3H2O2) 47, calcium peroxide (CPO) (CaO2) 48 and magnesium peroxide (MPO) (MgO2)

49

(Table 1). Solid peroxides first dissociate to H2O2, which

decomposes to water and O2. O2 release kinetics from peroxide compounds can be affected by different factors such as pH 50, temperature 51, and the use of catalase 50, 52.

Purity and decomposing of solid peroxides are also other important factors in the rate of O2 release from their carriers 53. Similar to H2O2, SPO dissolves in water and provides rapid O2 release. MPO has the slowest O2 release kinetics because of its low decomposition rate 53. Commercially available MPO has a purity of 15-25% while CPO has a purity of 60–80% 54. For SPO, the available H2O2 is around 20-30%. CPO is considered as one of the safest peroxides and it has been preferred in O2 generating biomaterials

27.

In addition to these

O2 generating peroxides, other potential O2 sources include perfluorocarbon O2 carriers endoperoxides 56 and microtanks 57.

2.2. Carrier biomaterials


55,


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6 The use of biomaterials as carriers for O2 sources is required to avoid burst release of O2 58 and associated cytotoxicity 59, as well as ensuring sustained O2 generation and release

35, 36

for a

period of time sufficient to support cells during angiogenesis 27, 28. In general, various materials including polymers

60

and ceramics

61

has been used for drug release purposes 62. However,

due to the low biodegradability of ceramics, polymers are more commonly used in tissue engineering and wound healing applications. Polymers used for drug release include natural ones, synthetic ones and their mixtures 63.

So far, only few polymers were utilized to include O2 sources such as poly(lactide-coglycolide)

(PLGA)

31

poly--caprolactone

isopropylacrylamide (NIPAAm)

38,

(PCL)

64,

polyurethane

and poly(N-vinylpyrrolidone) (PVP)

31.

65

and

N-

Corresponding

results demonstrated the possibility to achieve sustained O2 release over several days. O2 generating sources are usually mixed with these carrier polymers and the mixture is processed using various methods that are discussed in the following section, to produce various forms of O2 generating materials. These polymers have the advantages of proven biocompatibility, tunable biodegradability and O2 source carrying capability. In addition to biocompatible polymer materials, hydrogels have also been explored, such and gelatin methacryloyl (GelMA) 37

and alginate 66, which include not only O2 generating sources but can also encapsulate cells

for 3D tissue engineering applications. Another material that was recently investigated is gellan gum hydrogel which was combined with CPO and it was shown to generate O2 for up to 64 hrs in hypoxic conditions 67.

It is worth noting that the mechanism of degradation and release time are the most important criteria for choosing materials as carriers or adsorbing function. For example, polymer hydrophilicity can be altered to change the degree of degradation and provide long-term


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7 sustained release of O2. In addition to biodegradable polymers, nondegradable ones such as polydimethylsiloxane (PDMS) was also explored 68 and these polymers may have their unique application or use where stability of the implant or device is favored. Moreover, the recent development and interest in stimuli responsive biomaterials 69, 70 offers new possibilities where local and external triggers can be used to control O2 generation. Accordingly, O2 generation can be achieved in response to local triggering factors by using polymers responsive to local changes in temperature magnetic field

74

71

or light

or pH 75.

72,

or to remote external factors such as electric field

73,

Apart from currently used carriers, other biomaterials such as

ceramics or ceramic polymer composites can also to be explored and may widen the applications of O2 generating biomaterials further.

2.3. Mechanism of O2 release When an O2 source is encapsulated, carrier polymer undergoes degradation in aqueous environment leading to the production of O2. In the first step, O2 generating material such as CPO or MPO will react with water to produce calcium dihydroxide (Ca (OH)2) or magnesium dihydroxide (Mg(OH)2), and H2O2. SPO directly decomposes to sodium ions, carbonate ion and H2O2. In the second step, formed H2O2 dissociates into water and O2. H2O2 not only produces O2, but also generates hydroxyl radicals which can damage cells. Therefore, the use of a catalyst (catalase) in the construct is useful to help the safe conversion of H2O2 to O2.

Catalase is an enzyme found in the blood and liver of mammals, and it can be used to reduce and decompose H2O2 into O2 and water with high efficiency 27. Without catalase, the cytotoxic byproduct H2O2 may lead to cell damage. Abdi et al. pre-immobilized catalase to alginate and encapsulated H2O2 in PLGA microspheres to enable safe decomposition of H2O2. According to in vitro studies, the use of catalase resulted in improved cell viability (Figure 1) 66. Also,



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8 Kang et al. studied the effect of catalase concentration (0-100 u/ml) on cell viability and release studies from alginate-based gels. Using catalase in concentrations of 50 U/mL or higher led to reduced amount of produced H2O2, and it was associated with prolonged O2 release of 48 h (Figure 2) 76.

Other cell protective measures that were developed include the addition of antioxidants such as ascorbic acid 77 which are released from the O2 generating biomaterials. In other studies, it was noted that slow release of O2 was associated with no detrimental cytotoxic effects even in the absence of antioxidants 37. It is clear that burst O2 release is the most important factor that may adversely affect cells and using a mechanism to avoid the burst release is critical in developing a safe O2 generating platform.

2.4. Fabrication methods of O2 generating biomaterials O2 generating biomaterials have been developed by the incorporation of O2 sources into different biomaterials with various geometries such as microparticles 41, 46, 49, 66, 78, fibers 31, 77, films

48, 64, 79, 80

and 3D scaffolds

37.

Scaffolds’ architecture can be an important factor for

adjusting the degradation rates of particles and hence the O2 release. For instance, while using highly porous O2 generating materials, e.g. nanofiber-based scaffolds, the surface area is very high, and this leads to more exposure of the material to aqueous environment resulting in faster material degradation and hence, faster oxygen release. Different fabrication techniques including solvent casting, freeze drying, electrospinning or spraying, emulsion and gelation methods have been developed, and they are discussed below (Figure 3).

2.4.1 Solvent casting and evaporation method


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9 In the solvent casting method, a particle containing polymer suspension is deposited on a substrate, and followed by solvent evaporation, resulting in the formation of a solid composite film. Using this method, Harrison et al. encapsulated SPO within a PLGA polymer that was dissolved in methylene chloride. The film was shown to generate O2 for up to 70 hrs 79. Using solvent casting method, PLGA or PLA dissolved in chloroform were also used to encapsulate the CPO along with catalase to reduce the risk of H2O2 related cytotoxicity 48. Although these films were shown to prolong the release of O2 as compared to the use of free CPO without any encapsulating polymer; they failed to provide a sustained slow O2 release most probably due to acidity resulting from the polymer degradation byproducts 48.

To develop a wound dressing with O2 generation capability, Chandra et al. used PCL 9.5% (w/v), polyvinyl alcohol (PVA) 0.5% (w/v) that were dissolved in hexafluoro-2-propanol to encapsulate CPO (4.44% w/v) and SPO (3.33% w/v) and produced a membrane which was then combined with other layers to confer flexibility and gas permeability and be used as a wound dressing 64. This dressing generated O2 continuously for more than three days 64. Lv et al. used solvent casting and evaporation method to develop a scaffold made of silk/keratin (60:40) and 2% gelatin dissolved in water. The material was used to encapsulate 20% CPO and the release of O2 for more than two weeks was demonstrated 80.

Solvent casting and evaporation method is a simple approach to make films. For preparation of O2 generating materials, this method is useful since it employs organic solvents, which do not trigger O2 release during processing 81. However, this process takes time, and thus uniform distribution of solid peroxide particles in the substance of the carrier film is difficult to achieve because they precipitate in the bottom of the film. This method can be affected by different factors such as polymer concentration which affects the thickness of the resulting films. Using



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10 this approach, one can build up structures in a layer by layer fashion which can be used to develop multiple layers of homogenous compositions, e.g. solid peroxide particles in a carrier matrix.

2.4.2. Freeze drying method In freeze-drying method, O2 sources can be used with solution of carrier material, and frozen solvent is eliminated by the sublimation process under low pressure. Example of studies that employed this method to develop O2 generating biomaterials include the one by Shiekh et al. who developed O2-generating and antioxidant releasing polyurethane (PUAO) cryogel. The cryogel was formed by dissolving PUAO in dimethyl sulfoxide (DMSO), followed by freeze and thawing of PUAO-CPO solution in ethanol or cold water to remove DMSO 65. This method of freeze drying is useful to produce porous scaffolds having homogenously distributed solid peroxide particles. However, only water and DMSO can be used for freezing and thawing which it means that neither of these two can be used to dissolve carrier material.

2.4.3. Emulsion solvent evaporation method The other alternative for the compound encapsulation is the emulsion solvent evaporation method. Using this method, a carrier material having continuous and dispersed phases can be processed. In this method, loaded particles such as those of CPO can be mixed with the dissolved carrier matrix and surfactant. After the solvent is evaporated, surfactant is removed to obtain pure encapsulated particles. This method has been commonly used for encapsulation of drugs 82.

The most commonly used protocol for the encapsulation of water-soluble compounds is double emulsion, in which water insoluble matrices can be used to encapsulate an aqueous phase


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11 containing dissolved compounds 82. To produce O2 generating materials, Ng et al. encapsulated H2O2 in PLGA microparticles by using a double emulsion solvent evaporation method 46. In this study, PLGA was dissolved in dichloromethane, and then aqueous H2O2 was emulsified using a homogenizer to form a water-oil emulsion. This mixture was added to an aqueous solution of PVA as a stabilizer and re-homogenized to obtain a water-oil-water emulsion. After the evaporation of the solvent, microparticles (25–250 µm) were collected, surfactant (PVA) was removed and they were dried using freeze-drying which led to a limited O2 generation time of 5 hrs. Abdi et al. embedded microspheres that were produced by Ng et al. 46 into a catalase containing alginate matrix 83, 84 to extend O2 generation time to more than 24 hrs 41, 84.

In another work, Mallepally et al. encapsulated H2O2 in polymethylmethacrylate (PMMA). In this study, H2O2 in water and PMMA in acetone and acetonitrile were emulsified into a surfactant containing mineral oil. After the evaporation of solvent, microparticles were separated in a centrifuge. O2 generation from produced microparticles (5 to 30 µm) was sustained for over 24 hrs 49. Also, Steg et al. produced composite microparticles composed of a poly(trimethylene carbonate) (PTMC) matrix and CPO by using an oil-in-oil emulsion method. In this method, CPO particles were dispersed in a polymer solution (%7 w/v PTMC in acetonitrile), and then the mixture was added slowly into a surfactant containing mineral oil. After the evaporation of acetonitrile, microparticles were collected and washed with n-hexane and dried. It was observed that O2 generation from obtained microparticles (200 μm particle size) continued for 20 days without showing any cell toxicity 78.

This method is useful to fabricate microspheres with encapsulated O2 source. However, it may not be used to produce monodispersed microparticles. Parameters to control microparticle size and morphology in emulsion solvent evaporation method include the type of surfactant, type



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12 of solution and solvent used, which in turn can indirectly affect the rate of O2 release from the resulting product. Solvent evaporation rate also influences the porosity of particles which increases the release rate of O2. Oil-in-oil and oil-in-water based emulsion were employed for producing O2 generating materials. In oil-in-water system, water is not only used for emulsion, but it is also used for the removal of the surfactant. During this process, peroxide can get in contact with water which may directly trigger its decomposition. To avoid this outcome, oilin-oil based emulsion system can be used.

2.4.4. Electrospraying and electrospinning methods Electrospinning and electrospraying methods are based on the principle of applying an electric field to a solution droplet, which is being ejected by a syringe to form a sphere or fiber 85. The method has been used to develop various drug releasing micro- and nanomaterials which have been utilized for tissue engineering applications 86-88. For developing O2 generating materials, Wang et al. used electrospinning to fabricate CPO-containing PCL nanofibers 77. They used hexafluoroisopropanol as a solvent and also added ascorbic acid to further protect the cells from possible oxidative stress. Using electrospraying, Li et al. developed microparticles using a mixture of H2O2 that was conjugated to a high molecular weight polymer PVP, and PVP was encapsulated in a PLGA shell. They found that O2 generation continued for up to 14 days due to the effect of PLGA on slowing O2 diffusion 31.

The process of electrospraying and electrospinning can be affected by several factors that can be tuned and manipulated to control the fiber/particle size, uniformity, etc. Important factors include electrical field, distance between the needle and the collector, solution concentration, and the type of collector which may indirectly affect O2 release 89-91. This method is useful for producing submicron fibers that can be used as scaffolds. Electrospraying is used to produce


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13 small submicron droplets. One limitation of electrospraying/electrospinning is the possible precipitation of solid peroxides at the syringe, which may affect the uniformity of produced fibers/droplets

92.

Also, this method does not provide a good control over droplet size

distribution.

2.4.5. Polydimethylsiloxane (PDMS) curing method To prolong O2 release time, O2 sources can be encapsulated in hydrophobic PDMS. This method involves mixing O2 source particles with a PDMS solution and the mixture is then heated to cure PDMS. In one study, PDMS was used by Pedraza et al. to encapsulate CPO with different ratios and then PDMS was cured at 40oC for 24h 68. It was found that the use of PDMS can extend O2 release time to more than seven weeks 68. In another study, McQuilling et al. fabricated O2 generating films by curing PDMS and SPO mixture. Using this approach, burst release was avoided and O2 release from these films continued for 4 days in vitro

93.

Ring

scaffolds were also produced by using CPO-containing PDMS by Lee et al. The scaffold provided sustained O2 release for more than 24 hrs in vitro 94.

Although PDMS-based systems can provide extended O2 release profile, their in vivo application is questionable since they are not biodegradable and would either remain in the body indefinitely or need to be removed surgically 94. This method is useful for producing thin constructs since water cannot penetrate deep into PDMS to help decomposing loaded peroxide. In the future, PDMS might be combined with other biomaterials to provide new composites with different release properties.

2.4.6. Gelation method



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14 In this method, a pregel is used and it is solidified using either physical, chemical or a combination of the methods 95. Cells, molecules or compounds can be included into the cured gel. For O2 source containing hydrogels, GelMA was explored. In this study, CPO was added to a GelMA prepolymer solution and UV was applied subsequently to solidify the pregel solution. It was found that O2 can be generated over 5 days in vitro, allowing for cell support, survival and function 37. In another study, Newland et al. developed an O2 generating hydrogel based on gellan gum by the addition of CPO into the gellan gum solution. Released calcium ions (dissociated from CPO) resulted in the crosslinking of the polymer and hydrogel formation. O2 release profile from encapsulated CPO in the hydrogel extended for up to 48 h and 64 h in normoxic and hypoxic conditions, respectively. This was found to be dependent on the concentration of embedded CPO 96.

This method can be applied further to include other materials and O2 sources and their combinations. This method is characterized to be relatively fast for preparing O2 generating materials. However, the O2 release can also be fast which can be a limiting factor. To improve the release duration, one can incubate O2 in a hydrophobic coating and combine it with the gel.

2.5. Characterization Characterization methods of O2 generating materials include chemical 37, 65, 77, physical 31, 65, 96, 97,

and biological methods such as in vitro cell studies 38 and in vivo experiments 65. In addition,

the role of in silico models and machine learning can be explored in future. For chemical characterization, alizarin red staining was used to compare the CPO content inside materials based on the calcium concentration. For physical characterization, porosity, particle size, viscosity of the material are the most important characteristics that should be evaluated. For biological studies, cell viability and function need to be evaluated.


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15

With the development of computational approaches and mathematical modeling, new algorithms and artificial intelligence algorithms can be used to study and characterize drug releasing biomaterials

98, 99.

The role of these models is to understand drug delivery systems

and optimize them by correlating results with available experimental data. These models can also be applied in studying and developing new O2 releasing biomaterials. Important variables such as cell viability, the amount of reactive O2 species (ROS) amount, carrier material degradation, O2 release profiles, foreign body reaction, regeneration, angiogenesis, and wound healing can be used as parameters in machine learning to come up with the optimum parameters that are required for a specific application and help define dosage of O2. For example, recently unsupervised machine-learning clustering algorithms (screened thousands of two-dimensional images) combined with fluorine magnetic resonance imaging were used for spatiotemporal tracking and O2 sensing of biomaterial implants or cells that change over time and move in the intraperitoneal space. This methodology can be used where the oxygenation is an important component for therapeutic efficacy 100.

3. APPLICATIONS In this section, the applications of O2 generating materials are discussed. These applications fall in three main categories, 1) supporting engineered constructs, 2) supporting high O2 demanding cell transplants, and 3) supporting ischemic tissues such as chronic wounds and myocardial infarction.

3.1 Tissue engineering applications Recent advances in science and technology enabled the engineering of tissues such as in cardiac, skin, bone, muscle, and pancreatic tissues. These advances include utilization of 3D



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16 biomaterials, hydrogels, stem cells and tissue culture methods. However, adequate supply of O2 to implanted tissue constructs is still considered to be one of the major challenges in tissue engineering 88, 101. Tissues require vascularization in order to survive. To achieve this, various approaches were used such as the use of vascularized flaps 102, 3D bioprinting methods to make heterogenous vascularized constructs

22, 103,

as well as other microfabrication approaches for

vascularizing engineered tissues 104, 102. Following implantation, cells may be deprived of O2 105

and die

30

before the formation of new vessels and will not be able to receive nutrients,

leading to the failure of implanted tissue constructs 88. To overcome this problem, controlled O2 releasing biomaterials have been developed to provide O2 to implanted tissue constructs. These were investigated to support various tissues and tissue constructs such as cardiac, skin, bone and muscle tissues.

O2 generating scaffolds may serve as an advanced system to provide 3D structural integrity and also maintain cell viability in the construct, and thus support vascularizationa and avoid cell death that is caused by the lack of O2. In this respect, Oh et al. reported CPO containing PLGA scaffolds. CPO particles were incorporated into PLGA at different concentrations (0, 1, 5 and 10 wt.%). Produced scaffolds showed elevated and sustained release of O2 over the course of 10 days, when tested under hypoxic conditions, and they also maintained their mechanical integrity. PLGA-CPO scaffolds demonstrated improved fibroblast cell viability and proliferation under hypoxic conditions. It was noted that the metabolic rate of cells in PLGA control samples was reduced, while in O2 generating PLGA-CPO scaffolds it was increased to over 10 days 30. This demonstrates that O2 generating scaffolds may support cell survival after implantation and help achieve successful tissue engineering.


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17 Even though there are promising results, O2 generating biomaterials were investigated in only limited number of in vitro 28, 30, 40, 43, 47, 83 and in vivo 38, 42, 64, 65, 79, 80 studies. Clinical translation of these materials is still under development.

3.1.1. Bone tissue engineering O2 generating scaffolds can be used in engineering large bone tissue implants suffering from hypoxic conditions to improve cell viability. Touri et al. developed an O2 generating biomaterial to enhance bone regeneration, employing calcium phosphates, beta-tricalcium phosphate (β-TCP), hydroxyapatite (HA) and biphasic calcium phosphates (BCPs)

40.

They

printed BCP scaffolds consisting of 60% HA and 40% β-TCP that were then, coated with PCL encapsulating CPO. The role of hydrophobic PCL was to slow down the decomposition rate of CPO and thus provide a sustained O2 release during the early stages of the construct postimplantation period under hypoxic condition. The results showed that depending on the CPO concentration O2 generation can be sustained over the 10-day testing period. Scaffolds promoted the metabolic activity, cell viability and proliferation of osteoblasts. It was found that 3% CPO-coated scaffolds provided the highest cell viability and proliferation as compared with the uncoated BCP and other scaffolds coated with other materials. Decreased cell viability was however noted in scaffolds that were coated with 5% CPO which was possibly due to the cytotoxicity resulting from the generation of higher levels of ROS 40 (Figure 4).

3.1.2. Muscle tissue engineering For engineering of smooth muscles for urethral tissue engineering, Lv et al. developed a silk fibroin/keratin/gelatin/CPO based O2 generating film, which showed good mechanical properties, non-cytotoxicity, anti-bacterial effect and steady release of O2 over two weeks. CPO incorporating films displayed improved rabbit smooth muscle cell growth and viability in comparison with non-CPO-containing films. In vivo experiments, employing urethral defect



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18 animal model demonstrated the formation of epithelial cell layers and organized muscle bundles in animals treated with CPO films 80.

3.2. Supporting high O2 demanding cells One of the treatment possibilities of insulin-dependent diabetes is the use of islet cell transplantation, which is usually performed by using micro-and macro-encapsulation techniques 106. However, cells in these products do not have enough O2 supply and thus islet survival and function cannot be maintained. Therefore, studies have been focused on the use of O2 generating biomaterials to enhance the viability and insulin production by transplanted islet cells.

Lee et al. reported on the effect of PDMS/CPO on the viability and glucose-stimulated insulin secretion by porcine neonatal pancreatic cells. PDMS/CPO scaffolds were associated with a sustained O2 release for more than 24 hours in vitro. Higher cell viability of neonatal pancreatic cell clusters grown in PDMS/CPO scaffolds was observed than of those grown in plain PDMS scaffolds or no scaffolds. Furthermore, PDMS/CPO scaffolds performed better than PDMS or control cells with respect to lower caspase-3 and caspase-7 activities, hypoxic cell expression, glucose- stimulated insulin secretion function and ROS levels

94.

Similarly, Petraza et al.

reported that CPO- encapsulating PDMS disks could support the viability of β cell line and rat pancreatic islets for more than three weeks in vitro under hypoxic conditions 68.

Silicone films containing SPO and CPO particles encapsulated in permselective alginate were also suggested by McQuilling et al. to serve as potential supplemental O2 sources for islets before and immediately after their transplantation. O2 release rate of SPO was increased with increasing temperature and catalase. Incorporation of SPO into silicone films slowed down the


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19 release rate of O2 from SPO. The addition of SPO during the islet isolation process provided a higher insulin secretion rates by islets and a higher metabolic activity and viability for islets that was SPO-concentration dependent. The use of higher concentrations of SPO led to damage of islets due to oxidative stress. Moreover, co-encapsulation of individual islets with CPO particles in alginate micro-beads enhanced islet viability and metabolic activity in a low-O2 environment compared to the control sample 93. These studies have further shown that SPO and CPO are excellent candidates to improve islet viability and functionality and, have the potential to provide adequate amount of O2 over extended periods of time.

Another approach to prevent inadequate oxygenation of cell transplants was reported by Coronel et al. They described the effects of an O2 generating biomaterial, namely OxySite, on islet viability, glycolysis, the production of inflammatory cytokines, and angiogenesis. OxySite was generated by incorporation of 25% w/w CPO into PDMS. OxySite provided higher graft efficiency and strong pancreatic intra-islet vascularization under hypoxic conditions. Hypoxic stress in both rat and nonhuman primate pancreatic islets was mitigated by the positive effect of OxySite on cell metabolic activity and viability, supporting aerobic metabolism, maintaining responsiveness to glucose, and reducing the generation of inflammatory cytokines

43

(Figure

5).

3.3. Treating hypoxia and preventing ischemic damage 3.3.1. Wound healing To achieve sustained release of O2, biomaterials are used to include O2 generating materials and hence, help to improve the healing of refractory wounds. In refractory wounds, the most challenging problem is that the lack of enough O2 leads to continuous cell death, failure of wounds to heal and chronicity. O2 plays a crucial role in wound healing as a metabolic substrate



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20 as well as a signaling molecule that can regulate cell viability, proliferation, migration, and differentiation

107.

O2 is considered as a key factor for achieving wound closure. Up to date,

various O2-based therapeutic approaches have been used for wound healing. However, most of them have found limited success. For example, hyperbaric O2 therapy is currently in clinical use, but it is associated with limited O2 diffusion and it leads to pulmonary damage 108.

Therefore, the development of advanced O2 delivery systems that overcome these limitations are of great importance. In this respect, Park et al. reported hyperbaric O2 generating (HOG) hydrogels 42. HOG hydrogels were prepared by mixing solutions of thiolated gelatin and CPO. It was found that HOG hydrogels could gradually generate O2 and maintain hyperoxic levels for a period of up to 12 days in vitro, and for 4 hrs in vivo. In vitro experiments showed enhanced human endothelial cell and dermal fibroblast proliferation activities. Moreover, HOG hydrogels were demonstrated to promote wound healing and lead to enhanced neovascularization in vivo, indicating the potential of the use of these new O2 generating biomaterials in the treatment of hypoxic conditions and ischemic tissues (Figure 6).

Topical O2 therapy (TOT) has been used as an alternate to hyperbaric O2 therapy to promote wound healing. In TOT, O2 is locally applied to wounds. The use of TOT is limited because most of the currently available TOT devices employ gaseous O2, and they fail to provide a constant supply of O2 over extended periods of time. In addition, the use of an externally attached O2 providing device to patients may limit their activity and quality of life, and also increases the overall cost of treatment. Moreover, the use of O2 releasing films for in situ production of O2 was developed by Harrison et al. 79. It was found that SPO containing PLGA films led to O2 release over 70 hrs. For in vivo studies a skin flap model in nude mice was used and films were implanted in the dorsal subcutaneous tissue in mice. SPO-free PLGA films were


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21 also used as control. Tissue were found to become necrotic when SPO-free PLGA films were used. On the other hand, a significantly reduced amount of visible necrosis at days 2 and 3 was observed when the SPO containing films were used. These results indicate that O2 producing biomaterials can reduce necrosis and extend the viability of tissues by releasing O2 into hypoxic tissues (Figure 7).

One limitation of the existing O2 generating scaffolds include the production of ROS. The accumulation of free radicals results in increased oxidative stress, and cell death. Recently, Shiekh et al. reported a new O2 releasing implantable polymeric biomaterial with the property of reduced risk of free radical formation for the use in a skin flap model in mice 65. PUAOCPO was fabricated by CPO incorporation into PUAO scaffolds. Sustained release of O2 by PUAO-CPO was observed over 10 days and it occurred in a dose-dependent manner. Consequently, it led to reduced oxidative stress-induced cell death. In vitro experiments demonstrated an increase in the metabolic activity of the cardiomyoblasts that were cultured on PUAO-CPO cryogels over time. The results were comparable to those obtained from cells that were grown under normoxic conditions. It was also noted that cells that were grown on 1 and 2% PUAO-CPO cryogels have maintained their viability. However, increased CPO concentration in PUAO resulted in reduced cell viability, which is most probably due to an increased release of O2 and subsequent production of toxic ROS. Therefore, it was thought that PUAO-CPO cryogels with 1% CPO can support optimal cell viability and metabolic activity and thus, they were used for further testing in skin flap model in mice. The results demonstrated that tissue necrosis was prevented by the use of PUAO-CPO O2 releasing scaffolds, while necrosis was increased in controls (Cryogels with no CPO). Accordingly, O2 releasing PUAOCPO cryogels may be clinically applicable for the treatment of ischemic conditions, such as those associated with chronic wounds.



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22

Chandra et al. investigated an in situ O2 generating wound dressing with sustained release of O2 in an animal model 64. The dressing was prepared by combining four distinct layers having different properties and purposes. They comprise a gelatin-based layer (Layer 1), a O2 generating layer (Layer 2), a silicone-based layer (Layer 3), and a polyvinylidene chloride (PVC)-based layer (Layer 4). Layer 1 was used for direct contact with the wound. Layer 2 was used to generate O2 from an SPO and CPO incorporating PCL. Layer 3 provided the dressing with mechanical stability and flexibility. Layer 4 has low permeability to vapor and gas, and it was utilized as the outermost covering of the dressing. It was shown that ~70% of O2 was released during the first 24 hrs, with release extending over 48 hrs. For in vivo studies, the dressing was applied on porcine skin wounds. When SPO and CPO incorporating dressing was used, the wound area re-epithelialization was found to be faster, vascularization was enhanced, and the edges of the wound were found to closing more uniformly as compared to wounds where control dressings were used. Furthermore, quantitative analysis of epidermal thickness and collagen content of dermis demonstrated the significant impact of using SPO/CPO dressing on the acceleration of the wound healing (Figure 8). This new topical O2 wound dressing can be useful in the treatment of acute and chronic wounds.

3.3.2. Skeletal muscle Failure to provide sufficient O2 supply to hypoxic skeletal muscle can lead to its necrosis. Currently limited treatment options are available to prevent ischemic death of muscle tissue. O2 generating biomaterials provide the possibility to develop injectable therapy to preserve skeletal muscle homeostasis under ischemic conditions. In one study, Ward et al. investigated the injectability of O2 generating biomaterial into soft tissue. Small homogenous SPO particles (>25 µm) were prepared using cryogenic grinding and sifting and they were added into a


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23 physiological solution of Dulbecco's Modified Eagle Medium. The peroxide content of the solution reached its highest value within 6 hrs (10 mM), then it decreased to baseline values within 24 hours. Subsequent cell experiments displayed an SPO concentration-dependent decrease in the metabolic activity of myoblasts. Low concentrations of SPO (0.001–0.1 mg/mL), resulted in reduced cell viability, while with higher concentrations (1 & 10 mg/mL) there were no viable cells. The optimal concentration of SPO was 1–2 mg/mL, and preparations with these concentrations were injected into the skeletal muscle of rats. They were found to maintain contractility and preserve homeostasis in skeletal muscle under hypoxic conditions 47.

In addition, Abdi et al. reported PLGA-Alginate based dual layer system for O2 release. The system was prepared by coating PLGA microspheres with a secondary catalase containing alginate layer. Catalase led to the decomposition of encapsulated H2O2 into water and O2. The dual layered architecture prevented the interaction of pure H2O2 with the surroundings and allowed control over the release of O2, and achieved optimal support required for the survival of muscle cells exposed to hypoxic condition. They demonstrated that very high or very low concentrations of H2O2 can compromise cell viability (Figure 1). Higher concentrations of encapsulated H2O2 (10%, 20% and 30%) were found to be toxic to the cells and led to a decrease in cell proliferation and viability, while low concentrations (0.5, 1, and 2%) had no significant effect on the cellular activity. It was observed that 4% of encapsulated H2O2 was the optimal concentration which enhanced the viability of skeletal muscle cells 41. These results provide a guide for the optimal amount of O2 that can be provided in future studies and clinical applications.

3.3.3. Cardiac muscle



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24 Insufficient O2 suppy results in ischemic heart disease, and in the long term in progressive heart failure. Currently, attempts to use cell based therapy for the regeneration of cardiac tissue are associated with conflicting outcomes and fail to attain satisfactory results and standards 106, 109. Thus, strategies utilizing O2 generating materials were explored. Alemdar et al. reported the use of CPO-laden GelMA as an O2 generating hydrogel. CPO-GelMA was prepared by incorporating of CPO at concentrations in the range from 0 to 3 wt.% in GelMA. CPO-GelMA hydrogels were found to release significant amounts of O2 over five days under hypoxic conditions (1% O2). This resulted in preserving survival of cells and enhanced the proliferation of cardiac side population cells (CSPs) and reduced cell death

37

(Figure 9). This hydrogel-

based O2 generating biomaterials approach can provide a new method for minimally-invasive and regenerative therapy of cardiac ischemic conditions 110.

The use of stimuli-responsive biomaterials represents a unique group of materials 69, 70 that may enable the development of innovative constructs

111.

In one study, Fan et al. developed an

injectable O2 microsphere delivery system using a thermosensitive, injectable hydrogel composed of NIPAAm/2-hydroxyethyl methacrylate (HEMA)/acrylate-oligolactide

38.

O2

release was maintained over a 4-week testing period. It was found that this release duration led to increased survival of cardiac cells that were tested under hypoxic conditions in vitro. The system was also implanted into infarcted hearts of rats for four weeks. Significant increase in cardiomyocyte survival and enhancement of cell proliferation in the infarcted area were observed (Figure 10). The study also demonstrated the feasibility of using acute myocardial infarction (MI) model to assess the efficacy of controlled O2 release and its tole in protecting cardiac cells and promoting their survival, leading to cardiac repair. Further developments of in vivo models that can mimic the native conditions are needed before translation of this approach to the clinic.


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25

Stem cell therapy has been also succesfully used to regenerate heart tissue following MI, eventhough it has low efficacy due to the low survival of cells under hypoxic condition found in infarcted hearts. Li et al. evaluated the capability of O2 releasing system on providing sustained O2 release to stem cells exposed to hypoxia, in order to improve their survival and differentiation. An O2 releasing system was developed by combining H2O2 releasing PLGA and PVP microspheres, catalase and an injectable, thermosensitive NIPAAm, acrylic acid, and a macromer HEMA-oligo(hydroxybutyrate) hydrogel. Sustained O2 release for over two weeks was observed. Cardiosphere-derived cells (CDCs) were encapsulated in the hydrogel and their differentiation into cardiomyocytes, under in hypoxic conditions simulating the infarcted hearts (1% O2), was evaluated. The experiments showed that the utilization of an O2 generating platform can improve the viability of CDCs and reestablish their differentiation. Accordingly, the potential of using O2 releasing system in improving the efficacy of stem cell therapy for the treatment of MI was demonstrated.

4. CHALLENGES Currently, O2 generating biomaterials have been used in a limited number of applications, which can be attributed to difficulty in dispersing peroxides, challenges in providing prolonged controlled release, cell compatibility and proper degradation. The degradation rates of carrier materials and O2 release profiles need to be adjusted according to the intended application. The approach must prevent sudden burst release 33 and provide sustained release 65, 68 and support cells but with minimal ROS production

65.

One possibility is to use a combination of

biomaterials, or different coatings of different carrier biomaterial layers having tunable degradation properties

112.

The use of 3D bioprinting and microfluidic chips can also help to



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26 develop materials having more controlled and uniform O2 delivery rates. Employing stimuliresponsive materials 69, 70 and remotely-controlled devices 113 can be useful for implementing on demand O2 release systems 114, 115. The use of smart materials such as those which can be triggered by changes in local pH can be useful for employing as O2 generating systems. For example, lower perfusion and ischemia are associated with lower pH levels in tissues and this can be employed as a trigger for O2 release from pH responsive materials 72, 116.

In vivo models need to be developed further to help better understand the dynamics of O2 therapeutic systems. Sensors can be utilized for in vivo monitoring of physiological response over the period of treatment and defining O2 releasing profiles

115.

Some cells have higher

demand for O2 supply which means higher levels of O2 delivery, where on the other hand large amount of O2 can be detrimental to other cells. Thus, defining and monitoring of appropriate O2 delivery requirements and targeted localized release is of utmost importance.

There are attempts to move towards the functionalization and/or preparation of targeted, e.g. injectable O2 releasing biomaterials to supply O2 in a controlled and site-specific manner 31, 38, 41, 97.

However, these studies are very limited. Moreover, the development of multifunctional

biomaterials is needed in order to provide appropriate amount of O2 in the right place for the right duration of time in a controlled and sustained manner that can be suitable for use in the clinic. Advances in the development of novel O2 generating biomaterials will help to solve a range of problems that are associated with the healing of refractory wounds, tissue engineering, and regenerative therapeutics. Scaling up need to be developed further to enable industrial production. The regulatory approval of active agent generating materials is another challenge and approvals may take longer times that for usual materials and devices. This may delay translation of research results to clinical practice. There is enough evidence for the benefit of


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27 O2 generating biomaterials but nevertheless there is a need for larger in vivo studies to provide substantial understanding and conclusions to help take the next steps for clinical applications.

5. CONCLUSIONS AND FUTURE PERSPECTIVES O2 generating biomaterials represent an advancing frontier in improving the outcome of tissue engineering, healing of ischemic tissues and tissue regeneration. Various O2 sources such as CPO and SPO have been used in generating O2 releasing materials. To achieve sustained release of O2, various biomaterials were used as a carrier such as PLGA, PCL and GelMA. Increasingly, the importance of these materials is being recognized, and research in this area is expected to expand further to improve our understanding and define biomaterial behavior and biocompatibility. Challenges include achieving a balanced sustained O2 release that promotes cell viability and prevent rapid O2 release which can be toxic to the cells. The choice of the appropriate O2 source, carrier material and release control method are required to establish successful oxygenation for multiple applications and help translate results into the clinical practice.

In the future, O2 sources can be combined with injectable hydrogels for use in minimally invasive and regenerative therapy, where O2 may help to enhance the survival of delivered cells. The use of non-invasive imaging along with computational modeling and machine learning can help provide feedback on the level of O2 and on required adjustments of other parameters to assess the needs for in vivo applications. Developing new testing methods for the generated O2 systems by employing organ-on-a-chip models can also help 1) to provide personalized disease models for effective therapy, 2) better imaging and monitoring, and 3) physiological mimicking conditions.



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28

ACKNOWLEGDGEMENT The authors also acknowledge funding from the National Institutes of Health (R01AR066193, R01AR057837, and R01EB021857), American Heart Association Transformational Project Award (18TPA34230036). Deutsche Forschungsgemeinschaft (German research funding organization), and TUBITAK-2219 (1059B191700093). Authors thank Mohammed Xohdy for drawing Figure 3.

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36 Table 1. Oxygen generating sources O2 generating source H2O2

CPO MPO SPO

Pros It can bind to high molecular weight polymer for encapsulation. High purity. More sustained release. Slowest oxygen formation. High solubility. Biocompatible byproducts.


Cons Difficulty in controlling oxygen release rate. Difficult for encapsulation because of its liquid form. Less solubility. Increase the pH. Less purity. Less solubility. Faster decomposition rate to control oxygen release.

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37

FIGURE LEGNDS Figure 1. A) (i and ii) Schematic illustration and optical image for the routes of H2O2 decomposition in PLGA micro systems immobilized in alginate-catalase capsules. B) effect of different polymer shells in encapsulating different concentrations of H2O2 in hypoxic conditions (1% O2, 5% CO2, and 94% N2) on cell viability. Reprinted from ref 66, Copyright 2011, with permission from Elsevier.

Figure 2. The effect of catalase concentration on cell viability and release studies. A) quantitative data for H2O2 released from O2 generating hydrogels containing different ratio of catalase, including 0, 50, and 100 U/mL. B) Human dermal fibroblasts viability. (n = 3-6). * P < 0.05, ** P < 0.01, and ***P < 0.001. A2C0, A2C0.45 and A2C0.75 contain 0, 0.45 wt% and 0.75 wt% CPO, respectively. Reprinted from ref

76,

Copyright 2018, with permission from

Elsevier.

Figure 3. Oxygen releasing biomaterial preparation methods. A) Solvent casting and evaporation method, B) Freeze drying method, C) Emulsion solvent evaporation method, D) Electrospraying and electrospinning method and E) Encapsulation in PDMS.

Figure 4. A) Schematic of the preparation of 3D HA/β-TCP scaffolds following by coating with PCL-CPO. B) (i) SEM image of PCL-CPO hybrid coating. (ii) Higher resolution SEM with arrows showing nanoscale CPO powders which are uniformly distributed. C) Laser confocal microscope images of osteoblast cells on (i) uncoated BCP and (ii-iv) BCP coated



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38 with PCL containing 1% (ii), 3% (iii) and 5% (iv) CPO. Reprinted from ref 40, Copyright 2017, with permission from Elsevier.

Figure 5. A) Schematic representation of O2 generating discs, fabricated by using CPO containing PDMS. The hydrophobicity of the PDMS hinders diffusion of water, whereas O2 generated with hydrolytic reaction with calcium peroxide rapidly diffuses out of the disc. B) O2 trace measurements of either a 25% wt/wt CaO2 containing PDMS disc (filled diamonds) or a PDMS-only disc (open diamonds) incubated in buffered saline (n = 3). C) Immunohistochemistry of grafts explanted 40 days after transplantation, stained with antismooth muscle actin (SMA, red), anti-insulin (green) and anti-glucagon (pink) (bottom row). Figure 5A and 5B are reproduced with permission from ref

28.

Figure 5C is reprinted

from ref 43, Copyright 2017, with permission from Elsevier.

Figure 6. Wound healing application. A) Schematic of the HOG hydrogel formation and photos of the sol-gel phase transition which can be injected and contains oxygen bubbles and calcium particles. B) Wound healing performance of HOG hydrogel compared to control group without oxygen at day 0, 7 and 14 (NG: oxygen-free) (HG: hyperbaric gel). C) Area of closed wound compared to the day 0. Reprinted from ref 42, Copyright 2018, with permission from Elsevier.

Figure 7. Wound healing application. A) optical images showing of representing flap necrosis of POG and control (PLGA only) groups at days 3 and 7. B) Percentage of flap necrosis measured at days 2, 3 and 7 for both groups (n=4; p=0.58). C) Apoptotic cell study of both groups on selective dermis sections after 3 days shows higher number of apoptosis positive


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39 cells with brown nuclei for control PLGA group and D) Quantitative data of apoptotic cells shows significantly lower apoptosis for O2 containing POG group (n=7; p=0.30). Reprinted from ref

79,

Copyright 2007, with permission from Elsevier.

Figure 8. Oxygen supplying wound dressing. A) and B) Schematic and photo image of oxygen generating wound dressing containing four layers; from bottom to top, the first layer in contact with wound is made of gelatin containing manganese chloride to provide hydrophilicity, oxygen permeability and comfort to the wound; second layer is made of contains SPO and CPO oxygen generating agents incubated in PVA and PCL solutions. Third layer with silicon protects the wound and promote elasticity and forth layer with polyvinylidene chloride covers the system and avoid leaking produced oxygen. C) Wound closure percentage over 8 weeks using control dressing (without SPO and CPO). *p ≤ 0.05 and #p ≤0.01 for the two groups during the same time (week). p ≤ 0.01 between 1st and 3rd week (***); p ≤ 0.05 between 5th and 8th weeks (**). D) Healing progress observed for surgical wounds (10 × 10 cm2) in a pig model. Reproduced from ref

64

with permission of John Wiley and Sons. Copyright 2015 by the

Wound Healing Society.

Figure 9: Schematic fabrication and structure of CPO-GelMA oxygen supplying hydrogels. A) GelMA was prepared and CPO and cells were incorporated, and system placed in a mold and crosslinked with UV light. B) Oxygen releasing profile for different concentrations of CPO. C) CPO-GelMA improves the metabolic activity of CSPs that were cultured under hypoxic conditions close to normoxic levels. D) Using CPO in GelMA improved CSPs survivability by avoiding hypoxia related necrosis. Confocal microphotographs and semi quantification of (i,ii) live (green)/dead (red) and (iii,iv) healthy (blue), apoptotic (green), and



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40 necrotic (red) CSPs that were cultured under hypoxic conditions in 0, 1, 2, or 3% CPO containing GelMA. (v) Extracellular LDH activity of CSPs that were cultured in CPO-GelMA. Scale bar equals 50 μm. Reproduced with permission from ref

37.

Copyright 2016 American

Chemical Society.

Figure 10. Oxygen supplying microparticles for cardiac regeneration. A) preparation of oxygen generating microspheres containing H2O2, PVP incubated in PLGA. B) dsDNA content of human umbilical vein endothelial cells (HUVECs), cardiac fibroblasts, and cardiomyocytes measured at day 2. Each type of Cells was seeded in 3D collagen gel and then injected into collagen hydrogels and cultured under normal and hypoxic conditions. Also, O2 generating microcapsules were injected into the collagen and cultured under hypoxic condition (*p

Advances in Controlled Oxygen Generating ... - ACS Publications

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