University of Groningen Oxygen-releasing biomaterials Steg, Hilde

Oxygen-releasing biomaterials

Steg, Hilde

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Review on increasing the viability

of seeded cells in implanted tissue

engineering scaffolds

Hilde Steg, Arina T Buizer, Willem Woudstra, Albert G Veldhuizen, Sjoerd K Bulstra, Dirk W Grijpma, Roel Kuijer

Manuscript in preparation

Abstract

Most tissues have limited regenerative capacity and repair through the formation of a fibrous scar, which impairs their function. To restore tissue defects with functional tissue, therapies involving the application of autologous, tissue-specific cells have long been considered to have promising prospects. However, thus far only a limited number of such therapies have reached clinical application. The major reason for the failure of the investigated applications is the difficulty to include a vascular system in the constructed tissue. Applications that have reached the clinic most often concern tissues that are non-vascular (cartilage) or poorly vascularized and thin (bladder). Attempts to promote vascularization, either before, in a 3D culture, or after implantation, have failed. The lack of oxygen and nutrients, as well as the hostile inflammatory environment immediately after implantation, in most applications result in necrosis of the newly synthesized tissue. One possible way to limit this necrotic process would be to apply oxygen to both the wound bed in which the newly synthesized tissue graft is placed, and to the graft itself. For this, a biomaterial which can deliver oxygen and serves as scaffold for the tissue producing cells, would be an option. Although, these materials have been developed, an optimal oxygen-releasing biomaterial has not been found yet. In this review, the different oxygen-releasing materials that have been investigated are reviewed, as well as the factors that influence the release of oxygen.

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Current treatment of large tissue defects

To a certain extent, the human body is capable of regeneration. When the damage is too large for the body to restore, donor tissue can be used for repair. This tissue can originate from the patient’s own body (autologous), from a human donor (allogeneic) or an animal (xeno-transplantation). Although all these tissues generally result in acceptable repair, they have their specific disadvantages. Autologous tissue is the most compatible with the patient’s body, but the amount of tissue that can be harvested is limited and donor site morbidity is a recurrent problem1_{. Allogeneic tissue is more readily available, but limited availability of}

tissue and problems with compatibility resulting in tissue rejection, are common2,3_{. Moreover, the transmission of disease and the existence of a foreign}

body reaction can be a danger. For xenogeneic transplants these risks are even more existent. Engineering of new tissue from autologous cells would circumvent the disadvantages of transplantation of donor tissue.

Tissue engineering

Tissue engineering is the field of research in which ways are explored to engineer new tissue using biomaterials, growth factors and (stem) cells. Both combinations of biomaterials, growth factors, biomaterials with cells and combinations of materials with growth factors and cells are being evaluated. Here, the focus is on applications in which autologous (stem) cells are being used. Autologous stem cells are very valuable tools in healing the human body, since they are capable of differentiation into different lineages. Therefore, they can become or form cells that are able to repair different tissues. Mesenchymal stem cells (MSC) can be harvested from bone marrow, but also adipose tissue is used as a cell source4_{. MSC are known to be able to differentiate into bone, cartilage,}

fat and muscle5,6_{, under the influence of outside stimuli like growth factors and}

surface adhesion7–9_{. To create a new functional tissue, human mesenchymal stem}

cells (hMSC) should be differentiated into the right lineage, either by the supply of growth factors or by induction from the surroundings.

To create a three-dimensional (3D) tissue, the cells need some form of support. In the body the cells are supported by extracellular matrix. For cell-based therapies, a biomaterial scaffold is mostly used to provide stability. Biomaterials of different origin have been used, and include bioceramics10_,

biopolymers11_{, and synthetic polymers}12_{. In most applications, it is desirable for a}

biomaterial scaffold to degrade over time in such a way that there are no remnants left in the body. Its function will be taken over by the newly formed extracellular matrix13,14_{. Apart from providing support, biomaterials have also}

been known to be able to function as an initiator of differentiation of the stem cells15_{. Both tissue integrating and -differentiating properties of biomaterials are}

known and used13,16–18_{. Moreover, in the manufacturing process of the}

biomaterial, the shape of the biomaterial can effectively be altered. McBeath et al.8 _{showed that the topography of the attachment spot for the cells can be a}

useful trigger for the cells to differentiate into the cell type of choice, and the surface chemistry of the biomaterial is known to influence cell behaviour by signalling through the integrin binding19_{. Furthermore, growth factors can be used}

to direct the cells into the right differentiation lineage. The biomaterials can be used to deliver growth factors, and different biomaterials have shown to be suitable as a slow release system to prolong the delivery as well as to control the local concentration of growth factors. This minimizes the number of injections required and avoids peak concentrations of growth factors in the body20,21_.

Challenges in cell-based therapies

Most clinically relevant sized three dimensional constructs containing cultured cells or tissues prove to be not viable after implantation22,23_{. Oxygen}

dependent tissues that have been implanted successfully are less than 100-200µm thick, which is a few cell layers and include skin or bladder tissue24_{. Tissues that}

are less dependent on the supply of oxygen and other nutrients for survival, such as articular cartilage, can also be repaired using tissue engineering methods involving cultured chondrocytes24_{. Most tissues, however, have well-developed}

vascular systems with inter-capillary distances of 60–100µm. Repair of these tissues with tissue-engineered constructs containing cells has not yet been possible22,24–26_{. Survival of the implanted cells can be directly correlated to the}

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place27_{. Radisic et al.}28 _{developed a biomimetic culture system in which cardiac}

fibroblasts and myocytes were co-cultured on scaffolds with an array of parallel channels that mimics the role of a capillary network. By increasing the amount of oxygen carrier, the oxygen concentration could be varied. It was found that increase in oxygen content enhanced cell density and DNA content, the amounts of cardiac proteins, and their contractile properties28_{, supporting the hypothesis}

that oxygen is of vital importance to an implanted tissue construct29_{. The fact that}

oxygen-dependent cell- or tissue constructs become necrotic after implantation and that low oxygen dependent tissues have been reasonably successful, confirm that for larger 3D structures containing cells or tissue the availability of oxygen is important for a successful outcome in the clinic.

In the body, oxygen is normally delivered through erythrocytes which take up oxygen in the lungs and distribute the oxygen during their journey through the vascular network in exchange for carbon dioxide. A proper vascularization is essential for the vitality of our tissues. It is expected that vascularisation is also key for the larger cultured 3D structures30_{. A suitable biomaterial scaffold seeded}

with autologous cells able to produce the required tissue, will not perform adequately when it is not vascularized. This immediately shows the challenge we are facing. Well-vascularized tissues like bone or muscle are built up from cells that cannot survive for a sufficiently long time without oxygen and nutrients, making cell-based therapies nearly impossible.

Tissues cultured in the absence of fluid flow are solely dependent on diffusion, which leads to the formation of a 100-200µm outer layer of tissue that receives oxygen by diffusion31_{. This thickness corresponds to the oxygen diffusion}

limit in tissue, which is characteristically 100-200µm26_{. During cell culture,}

oxygen- and nutrient levels can be carefully monitored in the culture medium. However, after transplantation of the cell-scaffold into a patient’s body, the cells in the construct will experience a hostile environment in which a wound healing reaction takes place that involves an inflammatory reaction32_{. The surgeon has}

disrupted the local vasculature1 _{and the tissue contains high amounts of fibrin due}

an anoxic environment. Additionally, the limited refreshment of tissue fluids results in acidic conditions, in the end resulting in cells dying by necrosis32,33_.

It has been found that these dying cells can contribute to the healing process. Wu et al.34 _{showed that dying cells in a co-culture increase the growth}

of the second cell type. Apparently dying cells release many growth- and chemotactic factors to the environment, resulting in a so-called trophic effect33_.

Growth- and chemotactic factors produced by hMSCs, are beneficial for the healing process; these factors attract both immune cells and stem cells to the wounded area and stimulate proliferation and vascularization35_{. However, the}

carefully harvested, isolated, and cultured cells were selected for their ability to build new, specialized tissue, and not for their ability to produce growth factors while dying. It is possible that the chosen cells are not the most suited cells to create a trophic effect. More easily accessible cells or cell components like blood platelets36_{, might be a better source of trophic factors.}

Several research groups have investigated the simultaneous production of a vascular system during the production of the tissue-scaffold construct25,26_{. This}

was found to be more difficult than expected. To date, a completely vascularized tissue has not been engineered26_{, and although different artificial vascularization}

strategies, based on permeable membranes37,38_{, showed promising results when}

implanted subcutaneously, until now these systems failed when evaluated in orthotopic implantation sites and are not ready to be used in clinical situations. Therefore, cell-based therapies are now facing a most challenging problem: how to maintain cells alive upon implantation when there is no vascularization in situ.

After implantation of a cultured tissue, natural angiogenesis in the operated area will start from the outer edges. The ingrowth of new vascularization is limited to several hundreds of micrometres a day39_{. This means that complete}

vascularization of an implant of several millimetres to centimetres is at least several weeks. During this period, oxygen and nutrient deficiencies will occur, resulting to a lower cell survival and effectivity of the implant.

When cells are chosen for their capacity to either differentiate into a specific tissue or to proliferate and divide into cell types needed to restore the

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micro-vascularization is disrupted during the surgical procedure and the ingrowth of new vasculature is slow, the supply of oxygen, nutrients and the removal of waste products should be ensured in another manner. Here we focus on supplying oxygen to the cells by means of an oxygen-delivering biomaterial.

Oxygen-delivering biomaterials

Based on the above-mentioned assumptions, a device able to release low but significant concentrations of oxygen for a period of 3-4 weeks throughout the newly synthesized tissue, would solve the difficulties in cell culturing mentioned above. If such a device would be the scaffold on which the cells were seeded before implantation, the seeded cells would no longer suffer from a lack of oxygen (Figure 1). The period of oxygen-release should suffice for the body to synthesize blood vessels throughout the entire implant. This will be necessary, since the delivery of oxygen as the sole nutrient is not enough for the cells or tissue to stay viable. Thereby, vascularization is also necessary for the removal of metabolites created by the viable cells.

Figure 1: A homogeneously seeded scaffold larger than 200µm3 _{will over time become hypoxic in} the centre, resulting in cell necrosis. A homogenously seeded oxygen-releasing scaffold will provide the oxygen necessary for the cells in the centre of the scaffold, thereby supporting cells during the period required for vascularization40_.

Fortunately, by providing the cells with sufficient oxygen the amount of waste products will also decrease. In 1861 Louis Pasteur found that cells cultured in hypoxic or even anoxic environments start ‘fermenting’ glucose41_{. This}

‘fermenting’ process, later called anaerobic glycolysis, is far less efficient than oxidative phosphorylation at normal oxygen concentration. Although anaerobic glycolysis is beneficial for short periods of hypoxia, after implantation of

engineered tissue it is unwanted because of resulting high concentrations of waste products like lactate in the tissue. Oxidative phosphorylation produces an 18-fold production of adenosine triphosphate (ATP) per glucose molecule compared to anaerobic glycolysis42 _{(Figure 2) and much lower concentrations of}

waste products.

Figure 2: Oxygen is an important nutrient to produce energy. Although a cell is capable of anaerobic glycolysis when oxygen is not available, the amount of ATP produced is much lower42_.

In several studies, inadequate levels of oxygen have shown to decrease cell viability and lead to necrosis or apoptosis31,40,43_{. Lewis et al.}43 _{showed a}

mathematical model that describes the relationship between oxygen profiles and distribution of cells within tissue-engineered constructs during the early stages of development. It was concluded that cell-scaffold constructs in which oxygen supply is driven by diffusion will always produce proliferation near the outer edge of the scaffold. Since the solubility of oxygen in water or cell culture medium is low (2.2mmol/L)44_{, other means are needed to keep the oxygen concentration}

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oxygen-generating substance could decrease apoptosis and necrosis in newly formed cell-based tissues, but also provide a new therapy for other ischemic tissues like cardiac muscle after a myocardial infarct and chronic wounds like foot ulcers45,46_.

In chronic wounds the pO2 is critical for the whole cycle from prevention,

towards treatment. Oxygen-delivery in situ can make the difference between amputation or a healthy limb40,47_{. Furthermore, Wang et al. showed the potential}

for oxygen-releasing materials to inhibit bacterial infections48_.

Key to an effective releasing biomaterial is the rate of oxygen-release. The release should be sustained and tuneable to be useful in different circumstances. Since an oxygen-releasing biomaterial will not release oxygen indefinitely, the tissue should be provided with enough oxygen to keep the cells viable, while on the other hand the oxygen concentration should be low enough to stimulate blood vessel formation. Some level of hypoxia is required to stabilize the hypoxia inducible factor-1 (HIF-1) complex in cells which leads to the formation angiogenesis-promoting growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2)40_.

Figure 3: Schematic overview of the effects of hypoxia40_{. Different hormones are upregulated by} hypoxia. The extent of hypoxia is very important: when hypoxic conditions become anoxic, the right part of the diagram becomes more dominant, resulting in dying cells.

Clark et al.39 _{studied the growth of endothelial sprouting in frog (Hyla}

pickeringii) larvae and showed that the speed of ingrowing vessels is limited to tenths of micrometres a day. In bone lengthening studies in patients, the rate of blood vessel growth appeared to be similar. Ideally, the biomaterial would allow for the preparation of a tissue engineering scaffold that is larger than approximately 1cm3 _{and releases oxygen in a controlled manner for up to 21}

days. The release of oxygen must be from several days to 3 weeks and allow for vascularization of a functional adequately-sized tissue. This should lead to a normal oxygen concentration in the tissue that ranges from 0.6–2.6kPa (4-20mmHg)41_.

Oxygen-generating compounds

An essential substance in an releasing biomaterial is the oxygen-generating compound. Different oxygen-releasing materials have been explored. The use of substances originating from the body like encapsulated bovine haemoglobin in amylose to form a nano-sized oxygen carrier49,50 _{and chemical}

blood replacers like perfluorocarbon (PFC) have been investigated. These compounds both have to be charged with oxygen before their application. Chemicals such as peroxides release oxygen upon contact with water and appear to be more suitable for sustained oxygen-release51–55_.

A few oxygen-delivering biomaterials showing promising results have already been manufactured. Mostly, a solid peroxide like sodium percarbonate (2Na2CO3·3H2O2), calcium peroxide (CaO2) or magnesium peroxide (MgO2)

have been used as oxygen-releasing compounds. The oxygen-release rate from these peroxides depends on a large number of variables, including pH, temperature, the presence of a catalyst and the encapsulating material56_.

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peroxides described above include an intermediate step in which hydrogen peroxide is formed, hydrogen peroxide then forms oxygen:

CaO2 (s) + 2H2O Ca(OH)2 (s) + H2O2

MgO2 (s) + 2H2O Mg(OH)2 (s) + H2O2

(Na2CO3)2• 3H2O2 4Na+ +2CO3- + 3H2O2

2H2O2 2H2O + O2

H2O2, CaO2, MgO2 and (Na2CO3)2•3H2O2 have already been used to

prepare oxygen-releasing biomaterials53,57–60,61_{. Although hydrogen peroxide}

seems an optimal oxygen-generating compound since it leaves no waste product after the reaction, it is also highly reactive. It forms oxygen rapidly, which can lead to high concentrations of oxygen. Besides, proper encapsulation of hydrogen peroxide is essential to prevent its release and the sudden release of high concentrations of oxygen as both are detrimental to the cells.

Although a composite of sodium percarbonate and PLGA has been studied

in vitro58 _{and in vivo}59_{, sodium percarbonate was not deemed suited as}

oxygen-delivering component. Its high reactivity results in a high oxygen-release rates over a relatively short time period. Of the other metal-peroxides, CaO2 appears

most suitable based on its low water solubility and high purity62_{. Encapsulation of}

CaO2 within a polymer matrix with limited swelling behaviour in water should

result in the sustained release of low amounts of oxygen. Its side reaction product, Ca(OH)2, is a substance that occurs naturally in bone and appears to

induce bone growth63_.

Polymer matrix component of the

oxygen-delivering biomaterial

Another essential component for the slow release of oxygen is a polymer matrix to encapsulate the oxygen-generating compound. Encapsulation of the peroxide particles in a hydrophobic polymer matrix can be used to tune the oxygen-release profile to reduce the initial release rate and prolong the release duration. By encapsulation of peroxides in a hydrophobic, non-swelling polymer

matrix, the peroxides are shielded from body fluids. When the polymer matrix degrades or dissolves, the solid peroxide particles become exposed to water, the oxygen generating reaction starts and oxygen is released.

The intermediate step in the reaction of CaO2 with water leads to the

formation of Ca(OH)2 and H2O2. Since calcium hydroxide is a base with a low

solubility in water, the effect of pH on the reaction towards this intermediate will be evident. For this reason, the degradation mechanism, acidity and degradation speed will be critical for the formation of a sustained oxygen-releasing composite biomaterial. On the other hand, the influence of Ca(OH)2 on the pH in the body

will be limited due to its low solubility.

In earlier work, the suitability of using polymers as a controlling barrier between an active ingredient and its surroundings58,60,64–66_{, as well as the release}

of oxygen from composite biomaterials48,58,60,67_{, were shown. Examples of}

biodegradable polymers that have been used in the preparation of such composite materials are: poly(lactide-co-glycolide) (PLGA), in combination with sodium percarbonate58_{, hydrogen peroxide}67 _{or calcium peroxide}60_,

poly(-caprolactone) (PCL) in combination with CaO248 and poly(dimethylsiloxane)

(PDMS) in combination with CaO257. It should be noted that of these polymers

PDMS is non-degradable68 _{and PCL only degrades in the body at a very slow rate}

(≥ 1 year)69_{. The degradable biopolymers PCL and PLGA are non-soluble in}

water, their degradation is based on the hydrolytic attack of the ester bond which leads to the formation of acidic compounds11_{. Furthermore, the}

degradation of PLGA proceeds as a bulk degradation process70 _{resulting in a}

sudden release of acidic degradation products and loss of mechanical stability. Of the above-mentioned oxygen-delivering composite biomaterials, the most prolonged oxygen-release was found for the non-degradable PDMS-based composite. Oxygen was released from this composite for a period of 40 days and bèta-cells showed better viability on these materials than on PDMS itself. However, since this composite is not biodegradable, a second operation to remove the material would be inevitable. Essential for the low and sustained oxygen release from this PDMS-based composite was the encapsulation of the

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swelling capacity in water.

Our studies involved a biodegradable polymer with a limited ability to absorb water, that is biodegradable via a surface erosion process involving cellular enzymes like which cholesterol-esterase, does not lead to the formation of acidic degradation products69 _{(pH is known to affect the reaction of metal peroxides}

with water56,71_).

n Figure 4: structure of PTMC

The polymer of choice for this application is poly(trimethylene carbonate) (PTMC), it is a hydrophobic polymer with very interesting properties: it has an equilibrium water uptake of 1%, it is amorphous polymer, it is rubber-like at room- and body temperature and is biodegradable72 _{by surface erosion. In vitro,}

cholesterol esterase can be used to induce surface erosion of the polymer. These properties make PTMC an interesting polymer for use as a matrix in oxygen-delivering composite biomaterials.

Conclusions

Cell-based therapies have been shown not to be less successful than expected. Implanted cells tend to die upon implantation because of the absence of an adequate vascularisation. Attempts to overcome the lack of vascularisation have thus far failed.

Oxygen-releasing biomaterials have shown to prolong cellular viability, but more research will be necessary to ensure their success. Most oxygen-releasing biomaterials have too short oxygen-release profiles or are non-biodegradable. To create a material that makes cell-based therapies possible, a slow oxygen-releasing biodegradable implant needs to be manufactured. Such materials and devices may not only influence cell-based therapies but may also help in treating and healing ulcer wounds and other problems related to ischaemia.

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