University of Groningen Oxygen-releasing biomaterials Steg, Hilde

Oxygen-releasing biomaterials

Steg, Hilde

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6

Culturing of hMSC and SaOs-2

cells on oxygen-releasing

poly(trimethylene carbonate) and

CaO

2

composites under hypoxic

conditions

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

Manuscript in preparation

Abstract

In tissue engineering, the lack of vasculature in a cell-material implant can result in the premature death of the implanted cells. The use of oxygen-releasing biomaterials could contribute to the survival of the cells. In this study, the efficacy of oxygen-releasing composite materials based on poly(trimethylene carbonate) and oxygen-releasing CaO2 particles in prolonging the survival of cells cultured in

vitro in a hypoxic environment was evaluated.

Human mesenchymal stem cells (hMSC) and osteosarcoma (SaOs-2) cells were cultured on the composite films for different time periods. It was found that under hypoxic conditions the viability of the cells cultured on oxygen-delivering PTMC composites was not better than the viability of cells cultured on PTMC films that do not deliver oxygen. This is in contrast to observations in vivo, where application of oxygen-delivering PTMC/CaO2 composite microspheres in a murine

skin flap model was shown to result in significantly less skin necrosis. This indicates that not only oxygen, but also other factors are likely to be important in determining the viability of cells upon implantation.

6

Introduction

Since the conception of tissue engineering1_{, increasing efforts have been}

made to implement tissue engineering into clinical practice and allow the restoration of damaged or lost tissues. Although a large variety of constructs have been prepared by combining (polymeric) biomaterials, (stem) cells and growth- and differentiation factors and have been evaluated in animal models, only a limited number of tissue engineered constructs has been introduced into the clinic2_.

Cell-laden constructs have often been found to fail after implantation in orthotopic locations3–5_{. After a period of culturing in a controlled environment in}

vitro6_{, the cell-laden construct is introduced into the body in a wound bed that}

consists of a blood clot, damaged tissue and local vasculature. Closing of the wound creates rapidly ischaemia in the implantation site. In this initial phase following implantation, the absence of capillaries and the lack of oxygen and nutrients can result in cell death2,7_{. This is a major limitation of cell-based therapies in the}

restoration of damaged tissues. Ensuring adequate supplies of oxygen to allow the cells to remain viable is an important objective in tissue engineering research8–13_.

Several strategies have been followed to provide the grafted cells with oxygen. These include pre-vascularization2 _{strategies, artificial vascularization}

approaches with membranes14,15 _{and controlled delivery of angiogenesis-promoting}

factors like vascular endothelial growth factor16_{. Oxygen-releasing biomaterials}

were recently developed to provide the tissue near a wound bed with sufficient oxygen, thereby limiting necrosis and initiating healing and revascularization responses13_{. The sustained release and delivery of oxygen should keep the cells}

alive and maintain their metabolical activity for sufficiently long times to allow the implanted cells to contribute to the repair of the tissue. Ideally, oxygen levels should be sufficiently high to allow the survival of the cells, while the production of angiogenesis-inducing factors by the cells should at the same time promote angiogenesis17,18_.

In an aqueous environment, peroxides such as calcium peroxide (CaO2) can

be used to produce oxygen. See equations 1 and 2. The (slow) reaction of CaO2

will subsequently decompose into water and oxygen (equation 2). The cells can be protected from this reactive and potentially toxic compound by accelerating the latter reaction using catalase19_{. Ca(OH)2 is a basic substance with a low solubility}

product that can induce mineralization in bone20_.

𝐶𝑎𝑂2+ 2𝐻2𝑂 ⇌ 𝐶𝑎(𝑂𝐻)2 + 𝐻2𝑂2 [Equation 1] 2𝐻2𝑂2 ⇌ 2𝐻2𝑂 + 𝑂2(Catalase) [Equation 2]

Several oxygen-delivering composite materials comprising inorganic peroxides dispersed in a polymeric matrix have been prepared. The polymer prevents direct contact between the CaO2 particles and water, and oxygen is

formed (and then released) upon the uptake of water by the polymer matrix. The choice of the polymer is essential in creating a slow release system with optimal oxygen-delivery characteristics to ensure cell survival. The polymer should be relatively hydrophobic, allowing for the slow diffusion of water into it (and of the generated O2 out of it). These mass transport characteristics are also determined

by the crystallinity of the polymer and its stability or degradability21,22

In early work by Pedraza et al., poly(dimethyl siloxane) (PDMS) and CaO2

were used to prepare oxygen-delivering composites11_{. Although the stability of}

PDMS makes these composites less suited for tissue engineering applications, oxygen-release from this composite material was adequate and well controlled. Biodegradable polymers based on lactic acid (co)polymers have also been employed to prepare oxygen-delivering composites with inorganic peroxide particles11–13_{. Composites with poly(lactic-co-glycolic acid) polymers showed}

encouraging results in in vitro experiments11,12_{. However, while decreased cell death}

was expected, the sustained release of oxygen from these materials was quite limited in these experiments and only led to a transient effect. We too found that in composites of poly(lactic acid) and poly(lactic-co-glycolic acid) polymers and CaO2 particles all oxygen was released in approximately 24 hours23.

The above-mentioned biodegradable polymers degrade by a bulk erosion process, a hydrophobic, biodegradable polymer matrix that slowly degrades by enzymatic surface erosion would allow the more sustained release of oxygen by

6

surface. This principle is illustrated in Figure 1.

Figure 1: Schematic diagram of an oxygen-delivering composite biomaterials comprising CaO2

particles embedded in a hydrophobic surface eroding polymer. Upon erosion of the polymer matrix (in grey) at the surface of the composite material (e.g. due to enzyme activity), the CaO2 particles (in

black) are exposed to the aqueous environment and react to form oxygen at the surface of the composite biomaterial.

Poly(trimethylene carbonate) (PTMC) is a flexible and amorphous polymer, with a glass transition temperature (Tg) of approximately -20°C24,25_{. The}

equilibrium water uptake of this hydrophobic polymer is approximately 1%26_,

similar to that of poly(lactic acid)27_{. This is a little higher compared to the water}

uptake of the very hydrophobic of PDMS which is close to 0.2%28_.

Interestingly, PTMC was found to degrade in vivo by a surface erosion process29_{. In vitro this process could be mimicked by incubating the PTMC polymer}

in aqueous media containing lipase or cholesterol esterase enzymes. In the absence of enzymes or macrophage cells, PTMC was not found to degrade significantly and can be considered a stable polymer29,30_.

In recent studies, we prepared composite microspheres comprising a poly(trimethylene carbonate) matrix and calcium peroxide particles (PTMC/CaO2),

and showed that these composites were non-cytotoxic and able to release oxygen for prolonged periods of time31_{. In a subsequent series of animal experiments, the}

oxygen-releasing microspheres were implanted subcutaneously in a murine devascularised skin flap model32_{. At days 3, 7, and 10 after surgery, significantly less}

skin necrosis was observed in skin flaps under which oxygen-releasing PTMC/CaO2

microspheres were implanted than in skin flaps under which PTMC microspheres that did not contain calcium peroxide were implanted. This result suggests that the oxygen released from the composite microspheres delayed tissue necrosis in the devascularised skin flaps and supported the survival of the skin cells.

In tissue engineering, autologous human mesenchymal stem cells (hMSCs) are mostly used to induce tissue formation in situ. These cells are able to differentiate into different mesenchymal tissues including adipose tissue, bone, cartilage and muscle. These primary adult cells can be isolated from bone marrow or from adipose tissue from a donor or from the patient him/herself. The usual oxygen tension in bone marrow, where hMSC divide but do not differentiate, is approximately 1-7%33,34_.

In this current study, we set out to evaluate the effect of oxygen-release from PTMC/CaO2 composite films on the viability of cells cultured in vitro under

hypoxic conditions. Experiments were conducted with hMSC and osteosarcoma (SaOs-2) cells, a well-known cell line that originates from osteoblasts. These cells reside in an environment with an oxygen tension of 6.6-12% O235.

Materials and Methods

Materials

Poly(trimethylene carbonate) (PTMC, Mn=220kg/mol) was synthesized as

previously described24_{. CaO2 powder (75% purity, sieved to particle sizes <74µm)}

and chloroform were purchased from Sigma Aldrich (The Netherlands).

Human Mesenchymal Stem Cells (hMSC) were obtained from bone marrow aspirates collected during total hip or knee surgery from patients with osteoarthritis of rheumatoid arthritis, and isolated and characterized as described by Buizer et al.36 _{after approval of the Medical Ethical Committee of the University}

Medical Center Groningen and informed consent of the patients. SaOs-2 cells, originally derived from the American Type Culture Collection (ATCC) number HTB-85,were kindly provided by Dr. J.P.M. Cleutjens, of the Maastricht University Medical Center in The Netherlands.

Alpha Minimal Essential Medium (α-MEM), Dulbecco's Modified Eagle’s Medium (DMEM), foetal bovine serum (FBS) and antibiotic-antimycotic solution

were obtained from Life Technologies, The Netherlands). 2-Phospho-L-ascorbic acid, dimethylsulfoxide (DMSO), catalase (from bovine liver), cholesterol esterase

6

5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich, The Netherlands.

Oxygen-releasing PTMC and CaO

composite films

PTMC and CaO2 composites were prepared by dissolving the PTMC in

chloroform at a concentration of 3.5% (w/v), followed by suspension of 5% (w/w) CaO2 (with respect to the polymer) in the polymer solution. The suspension was

stirred overnight at room temperature. For oxygen-release measurements, 3.3mL PTMC and CaO2 suspension in chloroform, corresponding to approximately

100mg of solid composite, was pipetted into a 50ml flask. The solvent was then evaporated by drying under ambient conditions for 48 hours, followed by overnight drying under vacuum. For cell culturing studies, glass coverslips with a diameter of 5mm were coated with 250µL of the same suspension. The coated coverslips were then dried at ambient conditions for 48 hours followed by overnight drying under vacuum. Before cell culture, the materials were sterilized by UV irradiation for a period of 10 minutes.

PTMC samples that did not contain CaO2 were produced in the same

manner and served as controls.

Oxygen-release measurements

Oxygen-delivery from the PTMC and CaO2 composite films (weighing

approximately 100mg) into 35mL de-oxygenated simulated body fluid (SBF)37

supplemented with 0.02% (w/v) NaN3 was determined in duplicate using an Oxi

3310 (WTW, Germany) dissolved oxygen meter. The oxygen-release experiments were conducted at 37o_{C in a Plexiglas® cabinet that was continuously purged with}

nitrogen to maintain an oxygen-free environment. Dissolved oxygen concentrations were measured at 30-minute intervals for periods of up to two weeks. Any evaporated water was replenished with ultra-pure water that was de-oxygenated by equilibration in the oxygen free cabinet for at least 48 hours. The experiments were repeated three times. The SBF was also de-oxygenated by equilibration in the oxygen-free PMMA cabinet for at least 48 hours. To reflect the cell culturing conditions, see below, catalase enzyme was added twice a week to the SBF release medium at a concentration of 100U/mL.

To achieve surface erosion of the polymer matrix, cholesterol esterase was daily added to the medium to reach final concentrations of 20µg/mL. To control PTMC samples that did not contain CaO2, these amounts were also added.

Cell culturing

Well-characterized hMSC were cultured in α-MEM supplemented with 10% heat-inactivated FBS, 2% antibiotic-antimycotic solution, and 0.2mM 2-phospho-L-ascorbic acid. Cells in the third passage were used in the experiments36_{. SaOs-2}

cells were cultured in DMEM (1 g/L glucose), supplemented with 10% heat-inactivated FBS, 2% antibiotic-antimycotic solution and 0.2mM 2-phospho-L-ascorbic acid.

The hMSC and SaOs-2 cells were seeded at a density of 5000 cells/cm2 _{in a}

TCPS 24-wells plate. The cells were cultured for 1, 4 and 7 days at 37°C, 100% humidity and 5% CO2 under hypoxic conditions (0.1% O2) using anInvivO2 200

incubator (Ruskinn Technology, UK). The media were refreshed twice a week. When applicable, cholesterol esterase was added daily to reach concentrations of 20µg/mL.

Cell viability assays

Cell viability of the cells cultured on the TCPS, PTMC/CaO2 and PTMC

substrates was assessed using MTT viability assays. The PTMC and the oxygen-delivering PTMC composites were evaluated using glass coverslips coated with PTMC or the PTMC/CaO2 composite that were placed in the 24-well plates.

Culture medium containing 0.5mg/mL of MTT was added to the cells, after which the cells were incubated for another 2.5 hours at 37°C in a hypoxic environment (0.1% O2). In the latter case, the media were de-oxygenated by allowing them to

reach equilibrium in an oxygen-free incubator for at least 48 hours. The culture medium was then discarded, and the (composite) films with the adhering cells were gently washed with PBS. The water-insoluble formazan was dissolved in dimethyl sulfoxide (DMSO), and the absorbance was determined at 575nm using a Fluostar Optima microplate reader (BMG Labtech, the Netherlands). The absorbance values are indicative of the cell viability and are related to cell numbers and

6

absorbance values were corrected for any dissolved polymeric material.

Statistical methods

The data from the cell viability experiments was evaluated statistically using a Univariate ANOVA in IBM SPSS Statistics (version 20.0.0.2, USA). The assessed variables were time, presence of CaO2, presence of catalase and presence of

cholesterol esterase.

Results and discussion

Oxygen-release from PTMC and CaO

composite films

Composite poly(trimethylene carbonate) (PTMC) and CaO2 films were

prepared by casting a suspension of CaO2 particles in a solution of PTMC in

chloroform. The stirred suspension was stable and could easily be pipetted onto glass coverslips. After drying to the air, the composite films were found to be quite homogeneous. The composites were somewhat more rigid than PTMC films not containing CaO2 particles prepared in the same manner.

As CaO2 reacts with water to yield O2 and Ca(OH)2, it is to be expected

that contact of the composite films will lead to the release of oxygen. The oxygen-release from PTMC and CaO2 composites into deoxygenated simulated body fluid

(SBF) was determined using an oxygen-free cabinet that was continuously flushed with N2 gas. In this dynamic system, the oxygen concentration in the SBF will reach

zero values when the rate of oxygen-release from the composite is lower than the rate at which it is flushed out.

Oxygen-release profiles were determined for composite PTMC and CaO2

films in the presence and absence of cholesterol esterase (CE), an enzyme that induces the surface erosion of PTMC. Control experiments using PTMC films not containing CaO2 were also conducted.

Figure 2: Oxygen-release from PTMC and CaO2 composite films in simulated body fluid (SBF).

Specimens were placed in SBF in a cabinet purged with N2 gas, and the concentration of dissolved

oxygen (mg/L) in the SBF was determined as a function of time. Oxygen released from specimens into SBF to which cholesterol esterase (CE) was daily added were determined as well. Control experiments were conducted with PTMC films that did not release oxygen.

In Figure 2, the concentration of oxygen in the SBF is shown as a function of time for the different cases. It is inevitable that oxygen enters the N2-purged

cabinet during initiation of the experiments, therefore the oxygen concentrations determined in the SBF in the first day do not reflect released oxygen from the films (this is evident from the data of the control experiment using PTMC films). The system reaches dynamic equilibrium after the first day, thereafter oxygen concentrations resulting from the release of oxygen from the PTMC and CaO2

composite films can be determined for periods of up to 30 days. The slowly decreasing oxygen concentrations in time indicate a decrease in the rate of oxygen-release from the composite films.

While significant amounts of oxygen are released (and then dissolved in the medium) when the PTMC and CaO2 composite films are incubated in the SBF, the

addition of cholesterol esterase leads to increased oxygen concentrations and thus to enhanced release rates of oxygen. Due to the enzymatic degradation and surface erosion of the PTMC matrix, an increase in the oxygen concentration can be determined in the few hours after addition of the cholesterol esterase. Large amounts of oxygen are released from the PTMC and CaO2 composite and

dissolved in the medium mainly upon the first addition of cholesterol esterase.

6

37°C38_{, the action of cholesterol esterase in increasing oxygen concentrations in}

the medium was short-lived. Upon implantation, the number of cells and cell types present in situ that can produce enzymes that erode PTMC at the surface will determine the rate of oxygen-release. This implies that in vivo the oxygen-release rate from these composite materials will likely depend on the site of implantation.

The sustained in vitro oxygen-release from the PTMC and CaO2 described

above is comparable to the observed release from non-degradable poly(dimethylsiloxane)(PDMS) and CaO2 composites developed earlier11. In both

cases, the release continued for approximately 40 days. In contrast, oxygen-releasing composite materials based on a poly(lactic-co-glycolic acid) matrix and Na2CO3.1.5H2O2 or CaO2 as the source of oxygen were found to release oxygen

for a period of only three days12,13_.

Culturing of hMSC under normoxic and hypoxic conditions

Human mesenchymal stem cells (hMSC) are a type of cells that are often used in tissue engineering for their ability to differentiate into a variety of different cell types39,40_{. It is known that these cells are significantly influenced by the oxygen}

concentration in the environment they are cultured in8,9_{. To illustrate this, the cells}

were cultured under normoxic (21% O2) and under hypoxic conditions (0.1% O2)

for up to 7 days using tissue culture polystyrene (TCPS) 24-wells plates as a cell culturing substrate. In Figure 3, the viability of the cells, as determined using an MTT cell viability assay, are presented after culturing the cells for 1, 4 and 7 days on TCPS under the different oxygen conditions.

Figure 3: The viability of hMSC after 1, 4 and 7 days of culturing on TCPS under normoxic conditions (21% O2) and under hypoxic conditions (0.1 % O2). The viability is determined using an MTT cell

viability assay, the absorbance values are indicative of the cell viability and are related to cell numbers and mitochondrial activity.

The data in Figure 3 shows that under normoxic conditions, the cell viability increases in time. This is likely due to the increase in cell numbers as the cells proliferate. In the hypoxic cultures, the cell viability increased at a much lower rate than under normoxic conditions. This indicates that proliferation rates of hMSC are lower than under normoxic conditions.

Culturing of hMSC under hypoxic conditions on oxygen-delivering

PTMC/CaO

composites

In earlier in vivo work, we have shown that oxygen-delivering PTMC/CaO2

composite biomaterials were effective in reducing tissue necrosis in a skin flap model in mice32_{. These composites could also have a positive effect on hMSC}

cultured under hypoxic conditions in vitro.

To assess the effectiveness of the O2 delivering composites to limit cell death

in hypoxic cultures of hMSC in vitro, the adhesion, proliferation and viability of hMSC cultured on PTMC films and on oxygen-delivering composite PTMC/CaO2

films were evaluated. Furthermore, as the PTMC matrix degrades enzymatically by surface erosion, cholesterol esterase (CE) was also added to the medium. Figure 2 illustrates the effect of CE on oxygen-release from the PTMC/CaO2 composite.

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 1 4 7 A bs or ba nc e (5 75 nm )

Time points (days)

normox hypox

6

films and on the PTMC films under hypoxic conditions for 1-, 4- and 7-days. The results of the hMSC cell viability assays are shown in Figure 4.

The figure shows that after one day of culturing, minor differences in cell viability were observed between hMSC seeded on oxygen-delivering PTMC/CaO2

composites or non-oxygen-delivering PTMC films. Also, no difference in cell viability was found between the viability of cells cultured in the presence or absence of cholesterol esterase in the medium. After four days of culturing, a small significant difference in cell viability was observed in cultures conducted in the presence of CE. However, this difference was not found after seven days of culture and is thus expected to be too small to draw conclusions.

The seeding of the hMSC was the first contact of the PTMC/CaO2 composite

with water (culture medium). During the first day, the cells experienced relatively high levels of O2 and therefore also of H2O2 (levels that were even higher when

CE was added to the medium, Figure 2). These levels of oxygen and H2O2 did not

interfere with the adhesion of the cells, as after the first day similar metabolic activities were assessed in the cultures of hMSC on PTMC and on PTMC/CaO2

CE - + - +

Figure 4: Boxplots representing the viability of hMSC after 1, 4 and 7 days of culturing on PTMC/CaO2

films (white boxes) and PTMC films (grey boxes) under hypoxic conditions (0.1 % O2). Cells were

cultured in the presence or absence of cholesterol esterase (CE) in the medium. The viability is determined using an MTT cell viability assay, the absorbance values are indicative of the cell viability and are related to cell numbers and mitochondrial activity.

* Statistically significant (p<0.001). 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 t = 1 day PTMC/CaO2 PTMC 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 t = 4 day * 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 t = 7 day

6

would have a positive influence on the viability of the cells under hypoxic conditions, a difference in cell viability between hMSC cultured on oxygen-releasing PTMC/CaO2 composites and non-oxygen-releasing PTMC films could not be

shown. Given the effectivity of the oxygen-delivering PTMC/CaO2 system our

earlier in vivo experiments32_{, we did not anticipate on the absence of a positive}

effect on hMSC viability in hypoxic culture can be understood when considering the metabolic state of the cells in the natural environment: hMSC are found in the bone marrow, where the oxygen tension is relatively low and between 1 and 7%34_.

A period of hypoxia may thus be easily overcome by these cells.

To be able to observe a positive effect of the oxygen-delivering PTMC/CaO2

system on cells in a hypoxic environment and mimic the positive results obtained

in vivo in an in vitro model, culturing experiments were conducted using SaOs-2

cells. These osteosarcoma cells are expected to be more dependent on oxygen than hMSC, as the oxygen tension in bone is only 6-12%35 _{and cancerous cells are}

known for their oxygen-dependency41,42_.

In Figure 5, the viability of SaOs-2 cells is shown after different periods of culturing on oxygen-releasing PTMC/CaO2 composites and on

non-oxygen-releasing PTMC films under hypoxic conditions. Also, in the presence and absence of cholesterol esterase.

Similar to the culturing experiments using hMSC, no difference in viability of the cells between the different culturing conditions was seen after 1 day of culturing. After 4 days a small increase in the viability of the cells was found for cells cultured on PTMC films (both for media containing CE and not-containing CE), but the differences between the oxygen-delivering and non-oxygen-delivering films was not significant. After 7 days, a small but statistically significant difference in cell viability was observed between SaOs-2 cells cultured on non-oxygen-delivering PTMC films and on oxygen-non-oxygen-delivering PTMC/CaO2 composites in the

absence of cholesterol esterase. Differences in cell viability between the different conditions were very small, as was the case with hMSC, and definite conclusions should not be drawn.

Although SaOs-2 cells were expected to be more oxygen-dependent than hMSC, the oxygen-delivering PTMC/CaO2 materials once again did not show a

6

CE - + - +

Figure 5: Boxplots representing the viability of SaOs-2 after 1, 4 and 7 days of culturing on PTMC/CaO2 films (white boxes) and PTMC films (grey boxes) under hypoxic conditions (0.1 % O2).

Cells were cultured in the presence or absence of cholesterol esterase (CE) in the medium. The viability is determined using an MTT cell viability assay, the absorbance values are indicative of the cell viability and are related to cell numbers and mitochondrial activity.

* Statistically significant (p<0.001). 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 t = 1 day PTMC/CaO2 PTMC 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 A bs or ba nc e (5 75 n m ) t = 4 days 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 t = 7 days *

The inability to reproduce the positive effects of the oxygen-releasing PTMC/CaO2 composites in preventing tissue necrosis in vivo with this hypoxic in

vitro cell culturing model using hMSC and SaOs-2 cells may be due to

oversimplification. In the murine in vivo model previously used32_{, ischaemia was}

induced which not only led to a lack of oxygen, but also deprived the tissue of growth factors, glucose and other nutrients. Furthermore, it could also have led to acidosis induced by the adaptation of the cells to a hypoxic environment.

Deschepper et al.43 _{have indicated that a variety of nutrients (like glucose)}

might be of interest in further investigating ischaemia. To develop a more complete ischaemic model in which cells are cultured in vitro under hypoxic conditions, we conducted first cell culturing experiments with hMSC and SaOs-2 cells under normoxic conditions on TCPS and varied the composition of the cell culturing media. See supplementary information. We found that even under normoxic cell culturing conditions, a complete medium supplemented with both foetal bovine serum (FBS) and glutamine is essential for high cell numbers and proliferation rates. However, more research will be required to create an adequate ischaemic in vitro model.

Conclusions

An oxygen-releasing composite biomaterial was prepared from poly(trimethylene carbonate) (PTMC) and CaO2 particles. Upon degradation of the

polymer and reaction of the peroxide with water, oxygen is released. Oxygen-release could be determined for periods of up to 30 days and was influenced by the presence of cholesterol in the medium. In in vitro cell culturing studies of hMSC and SaOs-2 cells under hypoxic conditions, it was shown that oxygen released from the PTMC/CaO2 composite did not have a beneficial effect on the cells when

compared to culturing on non-oxygen-releasing PTMC films. This contrasted with earlier results obtained in in vivo experiments, where it was shown that the PTMC/CaO2 composites were effective in reducing cell death in an ischaemic

murine skin flap model. Apparently, this in vitro cell culturing model of ischaemia is too limited. Preliminary data show that the composition of the medium is determining in reaching high cell viabilities, even under normoxic conditions, and

6

ischaemia.

Acknowledgements

This research was financially supported by the Dutch Technology Foundation (STW), grant 10595.

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Supplementary data

Initial study on the effect of the composition of the cell culturing

medium on hMSC and SaOs-2 cell proliferation

Compared to non-oxygen-delivering PTMC, oxygen-delivering composite PTMC/CaO2 materials have shown to be effective in an ischaemic murine skin flap

model in vivo13_{. However, the cell death observed in the model may not have been}

exclusively induced by hypoxia. The lack of growth factors, glucose and other nutrients is likely to be of importance too.

In an effort to create a better in vitro model of ischaemia, hMSC and SaOs-2 cells were cultured under normoxic conditions in cell culture media of different compositions in TCPS 24 wells plates. Starting with media that did not contain glucose (no glucose, NG), the foetal bovine serum (FBS), glutamax (a more stable form of glutamine, Glut) and glucose (G) at different concentrations were added.

The basic medium used in the culturing of hMSC was α-MEM Eagle without glucose (NG) (P032301S powder, PAN Biotech Gmbh) dissolved in milliQ water supplemented with 2.2g/L NaHCO3 (Sigma-Aldrich) and 0.2mM

2-phospho-L-ascorbic acid (Invitrogen). This medium was supplemented with 1g/L D-glucose, 10% heat inactivated FBS and 1% Glutamax (all Invitrogen) to obtain the different media. Well-characterized third passage hMSC were used in the experiments36_.

The basic medium used for the SaOs-2 cells was DMEM D5030 powder without L-glutamine, glucose, phenol red, sodium pyruvate and sodium bicarbonate (no glucose, NG) (Sigma-Aldrich) dissolved in filtered water using a milliQ machine and supplemented with 3.7 g/L NaHCO3 and 0.2 mM 2-phospho-L-ascorbic acid.

The other components were the same as described for the culturing of hMSC. The media were refreshed twice a week. The hMSC and SaOs-2 cells were seeded at a density of 5000 cells/cm2 _{in 24-well plates, the cells were cultured for}

1, 4, 7 and 14 days at 37°C, 100% humidity and 5% CO2.

A Cyquant cell proliferation kit was used to determine the amount of DNA after the different culturing times. At the desired time points, the 24 wells culture

plates were inverted, and the medium was discarded. The adherent cells were washed with PBS and frozen at -70°C. After thawing the plate, 200µl lysate buffer was added to each well, mixed and incubated for 2-5 minutes. The fluorescence was determined using a Fluostar Optima microplate reader (BMG Labtech) using 480nm filters for excitation and 520nm filters for emission measurements. To calculate the amount of DNA from the data, a standard curve was prepared.

Figure 6: The amount of DNA of adherent hMSC cultured under normoxic conditions in different cell culturing media for periods of up to 14 days. The different media were prepared from a basic medium that did not contain glucose (NG) to which different components were added: foetal bovine serum (FBS), Glutamax (Glut) and glucose (G).

Figure 6 shows that hMSC cultured in media of the different compositions either show normal adhesion and proliferation or massive cell death. By comparing the results of the different media, it can be seen that the presence of FBS and Glutamax in the medium are necessary for cell proliferation, even in the absence of glucose in the medium. Cells cultured in FBS-containing medium with or without glucose show higher cell adhesion and proliferation than cells cultured in glutamine-containing medium with or without glucose, or cells cultured in basic medium that does or does not contain glucose.

0 200 400 600 800 1000 1200 0 5 10 15 ng D N A / w ell Time (days) hMSC NG NG+FBS NG+Glut NG+FBS+Glut NG+G 4,5g/L NG+G 4,5g/L+FBS NG+G 4,5g/L+Glut NG+G 4,5g/L+FBS+Glut

6

Figure 7: The amount of DNA of adherent SaOs-2 cells cultured under normoxic conditions in different cell culturing media for periods of up to 14 days. The different media were prepared from a basic medium that did not contain glucose (NG) to which different components were added: foetal bovine serum (FBS), Glutamax (Glut) and glucose (G).

Figure 7 shows that similar effects can be observed when culturing SaOs-2 cells, although in this case the effect of the presence of glucose in the medium is somewhat more pronounced. This can be expected, as SaOs-2 is a cancerous cell-line of which it is known that the glucose uptake is high. Nevertheless, the lack of glucose did not lead to massive cell death as was the case for the lack of FBS and Glutamax.

These results indicate, that for both cell types the combined presence of FBS and glutamine is important for cell survival. This was not expected as Deschepper et al.43 _{emphasized the important role for glucose in his cell cultures. In his cultures,}

the cells were deprived of oxygen by creating an environment in which the Pasteur effect takes over, and the cells will be required to use more glucose for their metabolism. However, as the effect of the presence of glutamine in the medium was shown to be so prominent, this compound might be most useful in achieving good cell survival under ischaemic conditions.

0 200 400 600 800 1000 1200 1400 0 5 10 15 ng D N A/ w ell Time (days) SaOs-2 NG NG+FBS NG+Glut NG+FBS+Glut NG+G 4,5g/L NG+G 4,5g/L+FBS NG+G 4,5g/L+Glut NG+G 4,5g/L+FBS+Glut

Even under normoxic circumstances, hMSCs and SaOs-2 cells die when cultured in media deprived of FBS and glutamine. In a normoxic environment, the presence or absence of glucose shows to be of lesser importance. This implies that under ischaemic conditions, such as upon implantation or transplantation of cells, cells might not only need oxygen but might also benefit from other nutrients. To create a more complete in vitro model of an ischaemia, for example to better understand the role of oxygen-delivering biomaterials, it will be important to take the deprivation of other nutrients besides oxygen into account.