Review

Oxygen-Generating
Biomaterials: A New, Viable
Paradigm for Tissue
Engineering?
Mazaher Gholipourmalekabadi,1,2 Susan Zhao,3
Benjamin S. Harrison,3 Masoud Mozafari,4,* and
Alexander M. Seifalian5,6,*
There have been many attempts to provide sufcient nutrients, especially
Trends
oxygen, to engineered large tissues to overcome the effects of hypoxia or poor
The successful outcome of organs
vascularization. Delivering sufcient oxygen to the transplanted cells is one made from tissue engineering princi-
of the most critical issues that affects cell survival and correct maturation of pally depends on delivery of sufcient
nutrients, especially oxygen to cells for
engineered tissues. An emerging approach is using 3D scaffolds made from the initial period of the formation of
oxygen-generating biomaterials to tackle transport limitations deep within the microvasculature in the construct.
engineered tissues. This class of biomaterials has opened a new window for
An emerging approach is using 3D
overcoming the challenges associated with ischemia occurring within large scaffolds made from oxygen-generat-
tissue constructs. This review critically assesses oxygen-generating reagents, ing biomaterials to tackle transport lim-
itations deep within the engineered
the main approaches for developing oxygen-generating biomaterials, and their
tissues.
potential as 3D scaffolds for regenerative medicine in a clinical setting.
This class of biomaterials has opened a
new window for overcoming the chal-
Oxygen Requirements in Engineered Tissues lenges associated with ischemia occur-
We are facing an ageing population with an increasing a number of damaged and failing organs. ring within large tissue constructs.

The greatest challenges facing the eld of organ transplantation today are a shortage of organs,
This review critically assesses oxygen-
the lack of available organ donors, high cost of transplantation, and morbidity and mortality of generating reagents, the main
consequent life-long immunosuppression [1]. Tissue engineering (TE) has opened a new approaches for developing oxygen-
window of opportunity for supplying organ substitutes [24] (Box 1). An example of this was generating biomaterials, and their
potential as 3D scaffolds for regenera-
the world's rst synthetic trachea [5], which used nanocomposite 3D scaffold and a patient's tive medicine in a clinical setting.
own stem cells (Figure 1).

Despite remarkable progress in the TE eld, some continued challenges remained unsolved [6].
One of the major problems associated with tissue transplantation is sufciently supplying oxygen
and nutrients to meet the demands of cells in 3D cellular engineered constructs following
1
transplantation. This drawback could critically limit the development of functional tissue for Cellular and Molecular Research
Center, Iran University of Medical
human TE. It has been reported that a TE 3D scaffold requires interconnected pores with a pore Sciences, Tehran, Iran
diameter of at least 100 mm to allow for sufcient nutrient transport [7]. Oxygen deciency occurs 2
Department of Tissue Engineering
when the distance between cell and blood vessels exceed 100200 mm [8]. and Regenerative Medicine, Faculty of
Advanced Technologies in Medicine,
Iran University of Medical Sciences,
Adequate and homogeneous nutrient distribution, especially oxygen, facilitates cell migration, Tehran, Iran
3
neovascularization (see Glossary) and tissue ingrowth [9]. The level of hypoxia in the central Wake Forest Institute for
Regenerative Medicine, Wake Forest
part of the scaffold is higher than that in the periphery. Therefore, cells that migrate into the School of Medicine, Winston-Salem,
depths of the scaffold are often exposed to hypoxic conditions [10]. While adequate oxygen NC, USA

1010 Trends in Biotechnology, December 2016, Vol. 34, No. 12 http://dx.doi.org/10.1016/j.tibtech.2016.05.012

2016 Elsevier Ltd. All rights reserved.
 Box 1. Tissue Engineering 4
Bioengineering Research Group,
The term tissue engineering (TE) was dened at a National Science Foundation meeting as the application of the Nanotechnology and Advanced
principles and methods of engineering and life sciences towards the fundamental understanding of structurefunction Materials Department, Materials and
relationships in normal and pathological mammalian tissues and the development of biologic substitutes that restore, Energy Research Center (MERC),
maintain, or improve tissue function. Some pivotal strategies underlying TE have been identied (Figure I): Tehran, Iran
5

Source and extraction of stem cells and other cells. Centre of Nanotechnology and

3D scaffolds, whether synthetic or biological, act as structure supporting frameworks.
Regenerative Medicine, University
College London Medical School,

Growth factors, peptides, and/or antibodies to enhance cell proliferation, differentiation, and integration to surround-
London, UK
ing tissue. 6
NanoRegMed Ltd, The London

Bioreactors and placement of TE organs to grow in vitro or in vivo.
Innovation BioScience Centre,
London, UK

*Correspondence:
mozafari.masoud@gmail.com
Epidermal Adipose-derived Adipose-derived MSCs / MSCs /
(M. Mozafari) and a.seifalian@gmail.com
stem cells stem cells stem cells Perichondrocytes Osteoblasts
(A.M. Seifalian).

Scaold Contoured Scaold Scaold Rigid scaold

scaold

Mineralized
Matrix Growth Mechano-electrical Chondrogenic substrates /
proteins factors Vasculogenesis smulaon / factors osteogenic
promong factors myogenic factors factors

Skin Fay ssue Heart ssue Carlage Bone

(e.g. breast)

Figure I. Tissue Engineering Strategies for Organ Development. (First row) Source and isolation of stem cells/
cells, usually autologous. Abbreviation: MSCs, mesenchymal stem cells. (Second row) 3D scaffolds, where oxygen-
generating biomaterials may enhance the formation of the microvasculature. (Third row) Growth factors, peptides, or
stimulation to enhance cell proliferation and integration to the surrounding tissue. (Fourth row) Constructs can be placed
in bioreactors in vitro or inside patients in vivo prior to transplantation [6].

diffusion facilitates graft maturation, its absence ultimately often results in poor extracellular
matrix deposition and/or cell death and tissue necrosis [11].

The cell behavior in hypoxic conditions and recent efforts employed to resupply oxygen
deciency in hypoxic conditions are described in Box 2.

Oxygen diffusion becomes more critical when cells or cell/scaffold constructs are implanted in
defects larger than a few millimeters [12].

Cells die in hypoxic conditions due to their inability to aerobically respire. Respiration allows for the
production of adenosine triphosphate (ATP). ATP molecules are necessary for survival because of
the numerous chemical reactions in cells in which they are involved. Tissue damage is a major
cause of tissue hypoxia because it destroys the network of vascularization, which prevents

Trends in Biotechnology, December 2016, Vol. 34, No. 12 1011

 Glossary
Catalase: a hemoprotein enzyme
that is found in almost all cells except
certain anaerobic bacteria; this
enzyme catalyzes the reduction and
decomposition of hydrogen peroxide.
Encapsulation: the act of
entrapping and enclosing materials in
a capsule.
Hydrogel: a colloid gel suspended in
water as its continuous phase.
Hydrophilic materials: materials
with strong afnity for water, tending
to dissolve in water.
Hydrophobic materials: materials
with no afnity for water, tending to
ward off water.
Ischemia: local inadequate supply of
blood to a body part due to the
mechanical obstruction of blood
vessels.
Necrosis: the pathologic death of
living cells or tissues resulting from
irreversible damage.
Neovascularization: formation of
new functional microvascular
networks in abnormal tissue or in
abnormal positions.
Oxygenation: the addition of oxygen
to any chemical or physical system,
Figure 1. The World's First Synthetic Trachea Made of Nanocomposite 3D Scaffold and Autologous Stem including the human body. A process
Cells, Implanted in a 36-year-old Patient in June 2011 [5]. of treating something with oxygen.
Scaffold: an articial 3D structure
that serves as a mimic of the
Box 2. Oxygen Requirements in Diabetes
extracellular matrix for cellular
Diabetes is a growing problem caused by the inability of the pancreas to regulate blood glucose levels. Islets are the ingrowth, adhesion, proliferation,
regions of the pancreas that contain endocrine hormone-producing cells such as / and b cells, which become either less migration, differentiation, and tissue
effective or absent as the disease progresses. Among the cells present in islets, the b cells are the main cells involved in regeneration in three dimensions.
regulating blood glucose due to their secretion of insulin.

Delivery of insulin-producing cells to the pancreas is one strategy for the treatment of diabetes. However, there are some
challenges associated with delivery of such cells, including an unfavorable immune response and inability in providing
sufcient oxygen supply. b Cells have a high oxygen demand for normal activity and functionality.

Thus, hypoxic conditions profoundly affect insulin production by these cells.

oxygen from reaching large areas of tissue, hence depriving the cells of oxygen. Environments of
low oxygen promote the production of free radicals, which have the potential to damage
deoxyribonucleic acid (DNA), cause mutations, and arrest the cell cycle. By contrast, some cells
such as b cells (in the pancreas), muscle cells, hepatocytes, and neurons are cells with high
oxygen demands. Therefore, such cells are very sensitive to hypoxic conditions. To prevent
further damage, it is imperative to provide damaged tissue with a sufcient oxygen supply.

To overcome the aforementioned challenges, oxygen should be released gradually in a consis-

tent manner over time to support the tissue until host neovascularization is achieved [11,13]. To
date, several attempts have been developed to supply sufcient oxygen for cells of damaged
tissue. Examples of such attempts are using oxygen-rich uids [such as peruorocarbons
(PFCs), silicone oils, and crosslinked hemoglobin] as oxygen carriers to facilitate the direct
delivery of oxygen to implanted tissue, utilization of angiogenic factors (such as endothelial cells

1012 Trends in Biotechnology, December 2016, Vol. 34, No. 12

and vascular endothelial growth factorsVEGFs), and implantation of the engineered tissue
adjacent to vascular-rich tissues, such as omentum, is another developed strategy in this regard.

Although recent strategies had partial successes in supplying oxygen, failure to supply sufcient
oxygen in a consistent manner over time to support the tissue until host neovascularization is
achieved has remained challenging for large tissue masses. Despite several attempts in this
regard, there is no efcient strategy for compensating low oxygen pressure in the vascular
destroyed areas. Recent efforts have focused on designing oxygen-releasing biomaterials
(ORBs) to provide a sustained and localized delivery of oxygen to engineered tissues [14]. In
this article, we critically review the development of ORBs and the mechanisms by which these
materials deliver oxygen. In addition, the applications of this emerging eld of study are
discussed in TE and in clinical settings. We also aim to introduce the salient features of this
class of biomaterials, the hurdles that must be overcome, the hopes associated with it, and
practical constraints to develop this strategy for different tissues.

Oxygen-Releasing Biomaterials
ORBs have a potential application in providing sufcient and prolonged oxygen supply for
engineered tissue both in vitro and in vivo in the clinical setting [15,16]. These materials release
oxygen by diffusion of entrapped, adsorbed oxygen or by chemical generation of oxygen.
Oxygen-generating biomaterials (OGBs) comprise a subclass of ORBs. These classes of
materials are designed to provide a gradual release of oxygen to cells as rapid or high
concentration of oxygen potentially causes cell death due to the formation of free radicals
[17]. The controlled gradual release is the key parameter for viable outcome of organs, especially
benecial to provide adequate time for early neovascularization and maturation of the engineered
tissue [18]. To prolong the release of oxygen, these materials have been incorporated into tissue
engineered constructs in various forms such as microspheres [17], lms [13], electrospun
nanobers [19], and scaffolds [11]. Sodium percarbonate (SPO) [13], calcium peroxide
(CaO2) [11,18], magnesium peroxide (MgO2) [13], hydrogen peroxide (H2O2) [20], and uori-
nated materials [21] are of the most common ORBs. It has also been suggested that encap-
sulation of such biomaterials in a hydrophobic polymer could remarkably slow down the release
of oxygen. Additionally, chemical components of the OGBs could affect the consistency of
oxygenation [13]. For example, OGBs produce oxygen when exposed to water. The rate and
speed of the oxygenation depends on factors such as pH, temperature, purity, and solubility of
peroxides, as well as presence of specic buffers or catalysts [16]. OGBs produce metal
hydroxides upon oxygen decomposition after exposure to water, which increases the pH of
the microenvironment and ultimately affects the rate of oxygen generation. Therefore, buffers are
commonly used to adjust the pH.

Some OGBs (such as calcium peroxide) produce reactive oxygen species as a byproduct during
oxygen decomposition. Accumulation of hydrogen peroxide (H2O2) as a residual reactive oxygen
species could remarkably increase the production of free radicals and reduce cell viability.
Therefore, catalase, an enzyme found in almost all living cells that is responsible for decom-
posing hydrogen peroxide to water and oxygen, can be introduced as an antioxidant to
accelerate decomposition of H2O2. After OGBwater interaction, the released H2O2 is decom-
posed into oxygen, as a useful and safe product for combatting hypoxia, by catalase present
within the engineered scaffold. For in vivo application of OGBs, catalase, as an antioxidant, can
also be incorporated into the engineered scaffolds to minimize the cytotoxicity effects of the
accumulated hydrogen peroxide (H2O2), an alternative product of OGBwater interaction [11].

In this regard, microencapsulated hydrogen peroxide in poly(D,L-lactide-co-glycolide) (PLGA)

acts as a core or intrinsic layer [20]. The core was then surrounded by a secondary layer of
alginate, with the catalase enzyme chemically bound to the alginate backbone. When hydrogen

Trends in Biotechnology, December 2016, Vol. 34, No. 12 1013

(A) O2 (B)
O2 Hydrogel
H2O2 PLGA PLGA
O2
O2 O2 H2O2
Release
H2O2
O2 O2
H2O2
PVP
O2 O2
Catalase conversion
O2
O2 Alginate immobilized
with catalase Cells O2 + PVP

Figure 2. (A) H2O2-PLGA-catalase/Alginate [20]. (B) H2O2 was xed to PVP, encapsulated in PLGA, and surrounded with
catalase-containing hydrogel [17]. Abbreviations: H2O2, hydrogen peroxide; PLGA, poly(D,L-lactide-co-glycolide); PVP, poly
(vinyl pyrrolidone).

peroxide was released from the PLGA (Figure 2A), a small amount of H2O2 degrades and
produces oxygen, while the majority of H2O2 is released into the secondary layer. The catalase
present in the alginate layer decomposes the remaining H2O2 into oxygen. It was also indicated
that decreasing the thickness of the alginate layer remarkably increased the rate of oxygen
diffusion across the construct. It has been noted that this nding was due to increased porosity
of the alginate when the concentration was decreased to 10% [20]. H2O2 has also been used in
an injectable form to design an oxygen supplier for augmentation of cell survival and cardio-
sphere-derived cell (CDC) differentiation in a simulated hypoxic condition (1% O2). First, H2O2
was bound to poly(vinyl pyrrolidone) (PVP). The PVP/H2O2 complex was encapsulated in PLGA
to form a core shell microsphere. The resultant microsphere was placed in an injectable and
thermo-sensitive, catalase-containing hydrogel (Figure 2B). The hydrogel was used to retain
stem cells and suspend the PVP/H2O2/PLGA microspheres in an injectable form. This construct
could mimic hypoxic conditions that arise during myocardial infarction. It has been reported that
hydrogels can protect polymers and prolong the release of oxygen up to 1 week [20]. Ultimately,
the duration of oxygen release time must be increased to a couple of weeks to allow for
neovascularization. As reported previously, the rate of release decreases as the molecular
weight of the polymer increases [17]. It has been noted that PVP can bind to H2O2 and form a
stable complex, leading to a more gradual release of oxygen. It was demonstrated that PVP/
H2O2/PLGA/catalase/hydrogel increased the durability of oxygen release for at least 14 days.

In our previously published study [13], SPO was incorporated into PLGA lms using a solvent
casting technique to engineer a material with sustained release of oxygen. A high rate of oxygen
release was observed during the rst day while total oxygen generation was completed over the
rst 70 h. The prolonged and consistent production of oxygen is of the utmost importance in the
elimination of hypoxic conditions following tissue injuries. This group carried out another study by
Oh et al. [11] to develop a method for slowing down the release of oxygen. For this purpose, they
encapsulated calcium peroxide (CPO) in PLGA and evaluated in vitro oxygen generation over the
rst 10 days. To assay the amount and consistency of oxygen release, the engineered construct
(PLGA/CPO) was embedded into a media under hypoxia conditions (0.5% O2, 5% CO2, 378C). It
was reported that small oxygen bubbles were observed upon embedding the engineered
construct in media. Furthermore, oxygen generation was prolonged over 10 days without much
change in pH, which remained between 7.2 and 7.6. In cell cultures, PLGA containing 5% CPO
had signicantly higher cell count up to 72 h among the 0%, 1%, and 10% CPOPLGA
constructs.

1014 Trends in Biotechnology, December 2016, Vol. 34, No. 12

CPO also has been granulated with hard fat, PLGA, and ethyl cellulose (EC) to develop an
oxygen supply system (W. Schlgl, PhD thesis, Ludwig Maximilan University Munich, 2012).
The initial burst of oxygen decreased with increasing particle size. Of the prepared con-
structs, the CPO/CE construct demonstrated a reduced initial burst, while retaining oxygen
release concentration at a sufcient level up to 72 h. Therefore, the CPO/CE was incorpo-
rated into a collagen/ceramic composite to augment consistency of the oxygen release.
Catalase was combined with the resultant composite to decompose the released hydrogen
peroxide. The development of an ideal construct requires further investigation; however, this
strategy could provide an oxygen-releasing system for manufacturing constructs for large
tissue regeneration.

The commonly used peroxides are in the form of solids; however, recently a liquid H2O2
oxygen-releasing system has been proposed, to see whether it is capable of sustainably
supplying oxygen to stem cells [17]. Their proposed oxygen-release system consisted of H2O2-
releasing PLGA microspheres, catalase, and an injectable thermo-sensitive hydrogel based
from N-isopropylacrylamide (NIPAAm), acrylic acid (AAc), and a macromer hydroxyethyl
methacrylateoligo(hydroxybutyrate) (HEMAoHB).

While most of the examples of OGBs involve inorganic peroxides, recently there have been
reports of organic-based oxygen-generating materials. Methylated pyridone-derived endoper-
oxides that are water-soluble can undergo reactions in an aqueous environment to release
oxygen in high yield and with half-lives of up to 13 h. These molecules, in combination with
vitamin C as singlet oxygen quencher, signicantly improved survival of 3T3 broblasts and rat
smooth muscle cells challenged with oxygen-depleted conditions. Singlet oxygen (1O2) is a high-
energy state of oxygen that can react readily with electron-rich double bonds and cause cellular
damage through oxidizing the cellular components.

Absorbed Oxygen Delivery Biomaterials

The sources of oxygen for cells, apart from peroxides, are PFCs, which are able to dissolve
large amounts of oxygen and carbon dioxide. It is expected that there will be a great
potential for the use of these materials under hypoxic conditions where tissues are not
oxygenated adequately. In a recent study [22], an effective diffusivity of oxygen in alginate-
based hydrogels containing stable PFC emulsions demonstrated that the increase in oxygen
transport is largely due to improved oxygen solubility in PFC-containing gels. Since
oxygen transport is a challenge in nearly all aqueous hydrogel biomaterials, this system
is expected to be a promising solution for many limitations of current soft biomaterials for TE
and cells.

In a study, a stable PFC-encapsulated alginate was synthesized, with consistent oxygen release
of more than 7 days in vitro [23]. The prepared construct was expected to be capable of
encapsulating islets for the growth and production of insulin, as well as encapsulation of stem
cells for proliferation and differentiation into cells of interest. Therefore, the investigation of such
constructs in vivo is worthy of future testing.

Another approach is functionalized hyaluronic acid derivatives with uorinated oxadiazole (OXA)
moieties, as a PFC, to develop a new oxygenating system [24]. The prepared system reacted
with a vinyl sulfone derivative of inulin to form an injectable hydrogel. The presence of PFC
positively affected the mechanical properties of the resultant hydrogel and signicantly promoted
metabolic activity and viability of broblasts up to 7 days. The system increased the solubility of
related gases [24] and mitigated the effects of hypoxia compared with analogous unuorinated
systems. A summary of recently conducted studies in vivo, as well as their ndings and
limitations, is listed in Table 1.

Trends in Biotechnology, December 2016, Vol. 34, No. 12 1015

Table 1. A List of Studies That Investigated Oxygen-Generating Biomaterials In Vitro as well as Their Findings
and Limitationsa
OGBs/ORBs Final Construct Findings Limitations Refs

H 2O2 H2O2PLGAAcatalase Slowed the release of O2. Low oxygenation [20]

construct: H2O2 was
microencapsulated in
PLGA and surrounded
with catalase immobilized
A.
Safe product.
Decrease in
concentration of A results
in increased O2 diffusion
across the scaffold.
consistency.
Needs to be incorporated
into an appropriate
scaffold.
Requires further in vivo
investigations.

PVPH2O2PLGA Binding H2O2 to PVP Needs to be incorporated [16]

catalasehydrogel
construct:
H2O2 was xed to PVP,
encapsulated in PLGA,
and surrounded with
catalase-containing
hydrogel.
increased O2 generation
time.
Resultant construct was
injectable.
Capable of oxygenation
over 14 days.
into an appropriate
scaffold.
Requires further in vivo
investigations.

H2O2PLGAcatalaseA Construct minimized its Still at early stage. [38]

construct: H2O2 was cytotoxicity against
encapsulated in PLGA. muscle cells.
The resultant Feasible application in
microsphere was muscle tissue
surrounded with a layer of engineering.
catalase-immobilized A.

CPO Four different constructs
including CPOhard fat,
CPOPLGA, CPOEC,
and CPOECcatalase CPOhard fat and CPO
collagenceramic
composite.
CPO was granulated in
hard fat, PLGA, and EC.
CPOEC granules were
incorporated in catalase-
containing collagen
ceramic composite.
The rate and initial burst of Low oxygenation (W. Schlgl,
O2 decreased with consistency. PhD thesis,
increased particle size. Ludwig
Needs to be tested for Maximilan
PLGA showed high rates oxygenation consistency University
of O2 initial burst. over longer periods of Munich,
CPOEC was able to time. 2012)
decrease the initial burst
of oxygenation, so that O2 Requires further in vivo
was produced up to 72 h. investigations.
The presence of catalase
in CPOcollagenceramic
composite decreased
cytotoxicity of the
released hydrogen
peroxide and increased
effectiveness of the
strategy.

CPOPLGA: A high amount of O2 was Low oxygenation [11]

CPO was incorporated released during the rst consistency.
into PLGA lms. day in exposure to water.
CPOPLGA was depleted Requires further in vivo
of O2 within 72 h. investigations.

PDMSCaO2 disc: CPO PDMSCaO2 sustained Requires further in vivo [17]

was encapsulated in
PDMS and then placed
inside an agarose gel.
release of O2 up to 42 investigations.
days, at a rate of
0.026 mM per day.
The construct reduced
the negative effects of
induced ischemia on b
cell line and rat pancreatic
islets in vitro for 14 days.

1016 Trends in Biotechnology, December 2016, Vol. 34, No. 12

Table 1. (continued)
OGBs/ORBs Final Construct Findings Limitations Refs

CPOPCLascorbic acid CPOPCLascorbic acid Cytotoxic effects of the [19]

electrospun mesh: electrospun meshes were construct were observed
different ratios of CPO successfully synthesized. on human cells.
were incorporated into Ascorbic acid
PCL and ascorbic acid. successfully protected Initial burst of CPO
The resulted compounds human osteoblast cells by decomposition
were electrospun to form minimizing the effects of byproducts.
nanobers with oxidative stress on cells.
antibacterial properties. Burst release of O2 Requires further in vivo
occurred on day 1. investigations.
Release of O2 continued
up to 4 days.
Feasible application of
such constructs includes
prevention of device-
related infections.

PFC HepG2PFCA Producing a novel and Requires further in vivo [23]

microsphere: efcient strategy for investigations.
HepG2 + encapsulated in encapsulating HepG2
PFCA emulsion. cells in PFCA. Needs to be incorporated
A stable peruorocarbon- into an appropriate
encapsulated alginate scaffold for large tissue
was synthesized. implants.
The resulted system
provided sustained
release of O2 over 7 days.

MACF hydrogel: Fabrication of a novel Requires some [34]

various PFC chains were injectable hydrogel modications for
incorporated into MACF. capable of repeatable prolonging the release of
uptake and release of O2. O2.
The synthesized hydrogel
was easily reloaded with Requires further in vivo
O2 upon complete O2 investigations.
depletion.
Maximum uptake and
completed release of O2
was found to be within 2
6 and 12120 h.
A feasible application of
such a hydrogel is in
wound dressings.

Abbreviations: A, alginate; CPO, calcium peroxide; EC, ethyl cellulose; H2O2, hydrogen peroxide; MACF, uorinated
a

methacrylamide chitosan; O2, oxygen; OGBs, oxygen-generating biomaterials; PFC, peruorocarbon; PCL, polycapro-
lactone; PDMS, polydimethylsiloxane; PLGA, poly(D,L-lactide-co-glycolide); PVP, poly(vinyl pyrrolidone).

Oxygen-Generating Mechanisms
In considering materials for TE applications, biocompatibility is key [25,26]. However, the rate of
oxygen release from these materials is actually the most important factor that directly inuences
the formation of new tissues. The oxygen release mechanism needs to be adjustable for
sustained release based on the needs of the target tissue. If there is a signicant burst effect
or very prolonged release behavior, the system cannot deliver a consistent, satisfactory amount
of oxygen for living cells to maintain healthy cellular function. To correctly adjust the oxygen
formation rate from peroxide compounds, a number of factors, such as temperature, pH,
amount of peroxide, and type of medium, must be considered [27].

Trends in Biotechnology, December 2016, Vol. 34, No. 12 1017

Over the past few years, solid inorganic peroxides have been frequently reported to be the most
promising OGBs for a wide range of TE applications. In aqueous environments, hydrogen
peroxide (H2O2) is rst formed, and then decomposes into oxygen [28]:
Calciumperoxide CaO2 s 2H2 O ! CaOH2 s H2 O2
Magnesiumperoxide MgO2 s 2H2 O ! MgOH2 s H2 O2

Sodiumpercarbonate Na2 CO3 2 3H2 O2 ! 4Na 2CO3 2 3H2 O2

2H2 O2 ! O2 2H2 O

All of these peroxides have been reported to be useful in TE [2931], but due to the availability of
higher purity commercial formulations (around 70% purity), CaO2 has been shown to be the
most promising oxygen generation [14,32]. In contrast, MgO2 has been shown to be the slowest
oxygen formation due to its lower solubility and consequently slower reaction rate. In the case of
adding peroxide compounds as llers in polymeric composites, the characteristics of the matrix
could also affect the oxygen release behavior. It has been shown that entrapment in more
hydrophobic materials may slow down the rate of oxygen release, while encapsulation in
more hydrophilic materials may increase the diffusion rate due to the rapid diffusion of water
into the structure [30].

The Applications of Oxygen-Generating Biomaterials in Tissue Engineering

Recently, efforts have been focused on the development of strategies for incorporating OGBs
into tissue-engineered constructs and using the resultant devices in vivo [13,16]. As mentioned
earlier, OGBs were blended with biomaterials and used for in vivo TE in various forms, such as
scaffolds and injectable hydrogels. The in vivo applications of the OGB-blended constructs are
summarized in Table 2.

Pancreas
Diabetes is a growing problem caused by the inability of the pancreas to regulate blood glucose
levels. The essential of oxygen requirements for pancreas tissue regeneration is summarized in
Box 2. Several studies have been conducted to provide a sufcient level of oxygen for islet cells
[18,33] with different ranges of success. Recently, researchers have adopted OGBs to explore a
way for overcoming the delivery of oxygen. Pedraza et al. [18] developed a novel construct made
of polydimethylsiloxane (PDMS)-encapsulated solid calcium peroxide (PDMSCaO2) to enhance
the durability of oxygen release. PDMS is a highly hydrophobic and biostable polymer. As
expected, the reduction in water uptake remarkably decreased the rate of decomposition of the
encapsulated OGBs and resulted in the enhanced consistency of oxygen release. PDMSCaO2
was fabricated in disc form and then embedded in b cell-laden agarose gels. The encapsulation

Table 2. A List of Studies That Evaluated Oxygen-Generating Biomaterials In Vivo as well as Their Findings
and Limitationsa
Tissue Final Construct Findings and Limitations Refs

Dermis SPOPLGA: various concentrations of SPO
were incorporated into PLGA to prepare an
SPO-containing PLGA lm.
The prepared lm delayed the onset of necrosis [13]
by up to 3 days when implanted in the skin ap
model.
Necrosis occurred after 7 days in an SPOPLGA
implanted animal, similar to a control.

Muscle SPO was injected in a rat model with partial Increased contractility of muscle cells in vivo was [16]
hindlimb hypoxia. observed.
Decreased oxidative stress, HIF1/
accumulation, and metabolic activity over 24 h
were observed.

a
Abbreviations: PLGA, poly(D,L-lactide-co-glycolide); SPO, sodium percarbonate; HIF1/, hypoxia-inducible factor 1/.

1018 Trends in Biotechnology, December 2016, Vol. 34, No. 12

of CPO in the hydrophobic PDMS profoundly increased the durability of oxygen delivery, so that
each single disc released 0.026 mM oxygen per day, for up to 42 days. The resultant construct
sustained release of oxygen through the decrease in water inux and delayed the formation of
hydrogen peroxide. In contrast to CPO-free PDMS, PDMSCaO2 interestingly minimized the
negative effects of induced hypoxic conditions on viability and functionality of the MIN6 b cell line
and rat pancreatic islets in vitro for 14 days. These results strongly imply a high potential
application of such strategies for oxygen delivery in a sustained manner within large implants.
However, further examinations should be done to evaluate the behavior and efcacy of the
PDMSCaO2 construct in vivo.

Skin Regeneration
Similar to other tissues, one of the major challenges in dermal wound TE is insufcient oxygen
level in the transplanted site due to loss of the vascular network during injury. Ischemia in the
defected site affects the wound healing response and causes scar formation. In a study, various
PFC chains were incorporated into the methacrylamide chitosan (MACF) to develop a novel
injectable hydrogel with the capability of repeatable uptake and release of oxygen for wound
healing applications [34]. The synthesized hydrogel demonstrated a maximum uptake and
completed release of O2 at 26 and 12120 h, respectively. The uorinated MACF hydrogel was
found to be easily reloaded with oxygen upon complete oxygen depletion. It has also been
shown that the positive effects of the synthesized hydrogels on viability and metabolic activity of
the broblasts increased with a corresponding increase of PFC concentration within the
construct. The ndings obtained from their study suggest the feasibility of implementing the
MACF hydrogel in wound TE. However, future use of the MACF requires further experiments to
prove the efcacy of such hydrogels in wound healing in vivo.

The roles of oxygen in tissue regeneration, especially in wound healing, are still not clearly
understood due to the differences in results obtained in vitro and in vivo [31,35,36]. A skin ap
model has been developed as the gold standard method to mimic ischemia conditions in vivo for
investigation of tissue survival after implantation (A.D. Murphy, PhD thesis, National University of
Ireland Galway, 2015) [37]. For example, Harrison et al. [13] implanted SPO/PLGA lms as a
polymeric oxygen-generating (POG) device in a skin ap model created in nude mice. Mice
treated with SPO-doped PLGA lms showed signicantly reduced necrotic areas compared
with those of mice implanted with PLGA-only lms after 23 days. However, at day 7 post-
surgery, necrosis in animals implanted with POG-containing PLGA was observed to be similar to
controls. Lactate level, a reliable measurement that increases in hypoxic and ischemic con-
ditions, decreased in SPO/PLGA-implanted animals compared with controls. Their strategy
successfully delayed the onset of necrosis during the rst 7 days. This therapy needs a few
modications to delay oxygen release over longer periods of time. A possible way to overcome
this obstacle may be to incorporate such materials into more hydrophobic polymers [11]. By
contrast, encapsulating OGBs in such hydrophobic materials may reduce cell integration and
cell adhesion. Attempts are currently underway to determine efcient strategies to extend
oxygen generation without negatively affecting cell adhesion and cell migration.

Muscle
Muscle cells are cells with high oxygen demand. Oxygen deciency after muscle tissue replace-
ment due to poor vascular networks, especially in early stage of transplantation, leads to
ischemia and necrosis [38]. Therefore, supplying sufcient oxygen during the rst 3 weeks is
crucial to prevent hypoxia within the damaged tissues. Utilization of oxygen-producing bio-
materials as an alternative strategy has been studied for providing adequate oxygen supply with
different grades of success [16,39]. One of the successful approaches synthesized a hydrogen
peroxide-encapsulated PLGA microsphere surrounded by a secondary layer of alginate [38]. For
this purpose, different concentrations of hydrogen peroxide, ranging from 0.5% to 30.0%, were

Trends in Biotechnology, December 2016, Vol. 34, No. 12 1019

encapsulated in the PLGA. The resultant H2O2PLGA construct was surrounded with a Outstanding Questions
secondary layer of alginate. Catalase was embedded in the alginate layer to decompose the What is the best way to eliminate the
released H2O2. Free H2O2 and H2O2-free PLGAalginate served as control samples. It was production of residual reactive oxygen
species and free radicals as byprod-
found that free H2O2 had a signicantly higher cytotoxicity on the L6 rat skeletal muscle cells ucts of OGB decomposition?
(SMCs) in all tested concentrations after 48 h of incubation under hypoxic conditions, in
comparison with encapsulated H2O2. Coating H2O2PLGA with catalase-embedded alginate What is the most appropriate strategy
was proven to signicantly reduce the cytotoxic effects of H2O2. Ischemia reduces metabolic to provide an optimal and sustained
release of oxygen from OGBs?
activity of muscle cells, leading to loss of contractile activity of cells. In vivo injection of SPO in a rat
model of partial hindlimb, hypoxia led to increased contractility as well as decreased oxidative
How can OGBs be incorporated into
stress, hypoxia-inducible factor 1/ (HIF1/) accumulation, and metabolic activity over 24 h [16]. TE scaffolds and injectable hydrogels
to enhance the durability of oxygen
A summary of the studies carried out using OGBs in muscle TE is listed in Table 2. release?

Some factors, such as the rate of

Concluding Remarks and Future Perspectives
hydration, the pH of the environment,
Many studies have demonstrated feasible utilization of OGBs for the consistent delivery of oxygen and catalysts present in the in vivo
to implanted engineered tissues in an effort to avoid hypoxia, especially in large tissue constructs. environment, can remarkably affect
Providing an optimal and sustained release of oxygen until the formation of early neovasculature the quality and quantity of oxygenation.
How can we optimize such parameters
can occur is one of the most critical issues in the preparation of an oxygen delivery system. To
to provide the best microenvironment
address this, OGBs have been incorporated into hydrophobic polymers in various forms such as for oxygenation?
cell scaffold, hydrogel, and electrospun mesh. It is expected that in addition to the organs tested
with OGBs, other important organs will benet from this class of biomaterials in the next few years.
For example, OGBs can be applied for the regeneration of the brain and the heart, two of the most
important organs. While the brain normally gets enough oxygen to survive, in some specic
situations when regeneration is needed, the brain may not be getting a sufcient amount oxygen, a
situation in which OGBs may be useful. Similarly, the heart muscles need their own supply of blood
because they need oxygen and other nutrients to stay healthy. The functionality of the heart is
increased when it pumps more oxygen-rich blood to its own muscle through the coronary arteries.

The main concern associated with using OGBs is the production of toxic agents such as
hydrogen peroxide, residual reactive oxygen species, and salts as decomposition byproducts
(see Outstanding Questions). Moreover, factors such as the rate of hydration, the pH of the
environment, and catalysts present in the in vivo environment can remarkably affect the quality
and quantity of oxygenation. Therefore, the negative effects of such agents should be minimized.
Current efforts are being undertaken to investigate various factors that affect the release of
oxygen as well as to develop novel strategies for fabricating an ideal oxygen delivery system.
Research is focused towards developing human organs using 3D scaffolds and autologous
stem cells. These scaffolds can either be made from biological or synthetic materials, but due to
rapid progress in smart synthetic materials, especially nanotechnology-based materials, we
believe that hollow organs will move towards clinical study over the next 5 years. OGBs will play a
considerable role in this development.

References
1. Saidi, R. and Kenari, S.H. (2014) Challenges of organ shortage for
transplantation: solutions and opportunities. Int. J. Organ Trans-
plant. Med. 5, 87
2. Gholipourmalekabadi, M. et al. (2015) Optimization of nanobrous
silk broin scaffold as a delivery system for bone marrow adherent
cells: in vitro and in vivo studies. Biotechnol. Appl. Biochem. 62,
785794
3. Yildirimer, L. et al. (2012) Skin regeneration scaffolds: a multimodal
bottom-up approach. Trends Biotechnol. 30, 638648
4. Reddy, N. et al. (2015) Crosslinking biopolymers for biomedical
applications. Trends Biotechnol. 33, 362369
5. Jungebluth, P. et al. (2011) Tracheobronchial transplantation with
a stem-cell-seeded bioarticial nanocomposite: a proof-of-con-
cept study. Lancet 10, 19972004
6. Yildirimer, L. and Seifalian, A. (2015) Tissue engineering. In Plastic
and Reconstructive Surgery: Approaches and Techniques (Far-
hadieh, R.D. et al., eds), pp. 6268, John Wiley & Sons
7. Dehghani, F. and Annabi, N. (2011) Engineering porous scaffolds
using gas-based techniques. Curr. Opin. Biotechnol. 22,
661666
8. Lim, J.O. et al. (2015) Functionalized biomaterials-oxygen releas-
ing scaffolds. J. Biotechnol. Biomater. 5, 1
9. Sarker, M. et al. (2015) Experimental approaches to vascularisa-
tion within tissue engineering constructs. J. Biomater. Sci. Polym.
Ed. 26, 683734
10. Becquart, P. et al. (2012) Ischemia is a prime but not the only
cause of hMSC death in tissue engineered constructs in vivo. Tiss.
Eng. A 18, 20842094

1020 Trends in Biotechnology, December 2016, Vol. 34, No. 12

11. Oh, S.H. et al. (2009) Oxygen generating scaffolds for enhancing
engineered tissue survival. Biomaterials 30, 757762
12. Stiers, P-J. et al. (2016) Targeting the hypoxic response in bone
tissue engineering: a balance between supply and consumption to
improve bone regeneration. Mol. Cell. Endocrinol 432, 96105
13. Harrison, B.S. et al. (2007) Oxygen producing biomaterials for
tissue regeneration. Biomaterials 28, 46284634
14. Camci-Unal, G. et al. (2013) Oxygen-releasing biomaterials for
tissue engineering. Polym. Int. 62, 843848
15. Lee, H.-Y. et al. (2015) Controlling oxygen release from hollow
microparticles for prolonged cell survival under hypoxic environ-
ment. Biomaterials 53, 583591
16. Ward, C.L. et al. (2013) Oxygen generating biomaterials preserve
skeletal muscle homeostasis under hypoxic and ischemic con-
ditions. PLoS ONE 8, e72485
17. Li, Z. et al. (2012) An oxygen release system to augment cardiac
progenitor cell survival and differentiation under hypoxic condition.
Biomaterials 33, 59145923
18. Pedraza, E. et al. (2012) Preventing hypoxia-induced cell death in
beta cells and islets via hydrolytically activated, oxygen-generating
biomaterials. Proc. Natl. Acad. Sci. U.S.A. 109, 42454250
19. Wang, J. et al. (2010) Oxygen-generating nanober cell scaffolds
with antimicrobial properties. ACS Appl. Mater. Interf. 3,
6773
20. Abdi, S.I.H. et al. (2011) An enzyme-modulated oxygen-producing
micro-system for regenerative therapeutics. Int. J. Pharm. 409,
203205
21. White, J.C. et al. (2013) Addition of peruorocarbons to alginate
hydrogels signicantly impacts molecular transport and fracture
stress. J. Biomed. Mater. Res. A 101, 438446
22. White, J.C. et al. (2014) Peruorocarbons enhance oxygen transport
in alginate-based hydrogels. Polym. Adv. Technol. 25, 12421246
23. Khattak, S.F. et al. (2007) Enhancing oxygen tension and cellular
function in alginate cell encapsulation devices through the use of
peruorocarbons. Biotechnol. Bioeng. 96, 156166
24. Palumbo, F.S. et al. (2014) Peruorocarbon functionalized hyalur-
onic acid derivatives as oxygenating systems for cell culture. RSC
Adv. 4, 2289422901
25. Gholipourmalekabadi, M. et al. (2015) Decellularized human amni-
otic membrane: more is needed for an efcient dressing for pro-
tection of burns against antibiotic-resistant bacteria isolated from
burn patients. Burns 41, 14881497
26. Gholipourmalekabadi, M. et al. (2015) In vitro and in vivo evalua-
tions of three-dimensional hydroxyapatite/silk broin nanocompo-
site scaffolds. Biotechnol. Appl. Biochem. 62, 441450
27. Soleymani, M. et al. (2011) Hydrogen peroxide decomposition
over nanosized La1xCaxMnO3 (0  x  0.6) perovskite oxides.
Chem. Eng. Technol. 34, 4955
28. Farris, A.L. et al. (2016) Oxygen delivering biomaterials for tissue
engineering. J. Mater. Chem. B 4, 34223432
29. Steg, H. et al. (2015) Control of oxygen release from peroxides
using polymers. J. Mater. Sci. Mater. Med. 26, 14
30. Fraker, C.A. et al. (2011) Complementary methods for the deter-
mination of dissolved oxygen content in peruorocarbon emul-
sions and other solutions. J. Phys. Chem. B 115, 1054710552
31. Chandra, P.K. et al. (2015) Peroxide-based oxygen generating
topical wound dressing for enhancing healing of dermal wounds.
Wound Repair Regen. 23, 830841
32. Wang, H. et al. (2015) Controlled-release oxygen using polymers.
In International Conference on Manufacturing Science and Engi-
neering (ICMSE 2015), pp. 16981701, Atlantis Press
33. Colton, C.K. (2014) Oxygen supply to encapsulated therapeutic
cells. Adv. Drug Deliv. Rev. 67, 93110
34. Wijekoon, A. et al. (2013) Fluorinated methacrylamide chitosan
hydrogel systems as adaptable oxygen carriers for wound healing.
Acta Biomater. 9, 56535664
35. Wilcox, J.R. and Covington, D.S. (2016) Wound healing and
hyperbaric oxygen therapy physiology: oxidative damage and
antioxidant imbalance. In Oxidative Stress and Antioxidant Pro-
tection: The Science of Free Radical Biology and Disease (Arm-
strong, D. and Stratton, R.D., eds), pp. 347356, Wiley Blackwell
36. Chen, B. et al. (2015) Burn wound care. In Chinese Burn Surgery,
pp. 113169, Springer
37. Zellner, S. et al. (2015) A dissolved oxygen dressing: a pilot study in
an ischemic skin ap model. J. Int. Med. Res. 43, 93103
38. Abdi, S.I.H. et al. (2013) Controlled release of oxygen from PLGA
alginate layered matrix and its in vitro characterization on the
viability of muscle cells under hypoxic environment. Tiss. Eng.
Regen. Med. 10, 131138
39. Seifu, D.G. et al. (2011) Tissue engineering scaffolds containing
embedded uorinated-zeolite oxygen vectors. Acta Biomater. 7,
36703678
Trends in Biotechnology, December 2016, Vol. 34, No. 12 1021