Towards in vivo application of oxygen-releasing microspheres for enhancing bone
regeneration
Buizer, Arina
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Chapter 5
Biocompatibility of poly
(trimethylene carbonate)-calcium
peroxide microspheres
A.T. Buizer, H. Steg, S.K. Bulstra, A.G. Veldhuizen,
H.W. de Haan-Visser, W. Woudstra, D.W. Grijpma, R. Kuijer
Abstract
In ischemic tissues, vascularization is usually diminished. Cells that are meant to repair ischemic tissues encounter therefore hypoxic circumstances with insufficient nutrient supply and waste removal. Due to this ischemic environment, repair cells do not function optimal or repair cell death even occurs. Tissue repair is therefore impaired. To resolve the lack of oxygen in ischemic tissues, microspheres have been developed that release oxygen for up to three weeks in vitro. Since the intermediate reaction product in the oxygen generating reaction is hydrogen peroxide, a well-known cytotoxic agent, the biocompatibility of the microspheres is a concern, and was tested following the ISO 10993 standards. Oxygen delivering microspheres based on Poly (1,3-Trimethylene Carbonate) (PTMC) and calcium peroxide were produced using an oil-in-oil solvent evaporation method. Cytotoxicity of these microspheres was tested in vitro by culturing L929 cells with an extract of the microspheres after which the metabolic activity of the cells was assessed with a MTT-assay. The microspheres were tested in vivo by implanting them in subcutaneous pockets in mice for 1 and 6 weeks, followed by histological and immunohistochemical assessment of local tissue reactions. In in vitro biocompatibility tests, the oxygen delivering microspheres appear to be biocompatible at low dosages. In in vivo tests, the oxygen delivering microspheres are biocompatible. In conclusion, PTMC-CaO2 microspheres are biocompatible.
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Introduction
One of the biggest challenges in regenerative medicine is the accomplishment of adequate oxygen supply to repair cells in ischemic tissues and to cells seeded on a scaffold material1. A frequently used method for tissue regeneration is to combine a
scaffold with cells that have to potential to grow into the type of tissue that needs to be regenerated. At the moment of implantation of a cell-seeded scaffold, blood supply within the scaffold is mostly absent1–3. Diffusion and hemoglobin-bound transport
of oxygen are the two main mechanisms of oxygen transport within the body4.
Considering that the maximum oxygen diffusion distance of oxygen within the body is around 200 µm5–7, hemoglobin-bound oxygen transport via the blood vessels is thus
essential for adequate oxygen supply throughout the tissues, but also throughout a cell-seeded scaffold. At the moment cells are exposed to ischemic circumstances for a prolonged period, ultimately cell death occurs8,9, leading to inadequate tissue
regeneration.
Several methods aiming at improvement of local vascularization have been proposed to increase repair cell survival under ischemic circumstances. Several researchers investigated the effect of hypoxic preconditioning of repair cells. The aim of hypoxic preconditioning is make repair cells produce more angiogenic factors (AGF), which are factors that stimulate ingrowth of blood vessels10,11
.
Other methods includemodification of cells through genetic engineering so that they produce more AGF, or application of exogenic AGF12,13. For improving vascularization in cell-seeded scaffolds,
co-culturing of endothelial cells and target cells on a scaffold prior to implantation, integration of host blood vessels into the scaffold at the moment of implantation or application of exogenously added angiogenic factors (AGF) have been studied2,14.
Another promising method for improving the oxygen supply to repair cells is the supply of oxygen via oxygen slow release systems. It is hypothesized that when MSCs are supplied with oxygen through such an oxygen release system, cell death due to prolonged ischemia will be prevented. In that way, there is extra time to achieve sufficient vascular ingrowth and a sustainable oxygen supply may thus be established. A slow release system for oxygen was developed, based on poly (1,3-trimethylene carbonate) (PTMC) and calcium peroxide (CaO2). PTMC degrades through a surface erosion mechanism, thus enabling for slow release of calcium peroxide integrated within the polymer15. CaO
2 reacts with water according to the following chemical
reactions16 (Equations 1 and 2):
Equation 2: 2H2O2 →O2 + 2H2O
Microspheres made of the composite of PTMC and CaO2 (PTMC-CaO2) release oxygen for up to 20 days in vitro17. Mesenchymal stem cells (MSCs) are frequently used for
tissue regeneration18–20. MSCs cultured in the presence of PTMC-CaO
2 microspheres
showed higher metabolic activity than MSCs cultured in the presence of PTMC microspheres, suggesting higher proliferation of the cells when exposed to PTMC-CaO2 microspheres17. The PTMC-CaO
2 microspheres could thus be a promising material
for long-term oxygen supply to MSCs, although the intermediate reaction product, hydrogen peroxide, remains a concern.
To take a first step towards application of this material in clinical practice, biocompatibility of the PTMC-CaO2 composite was tested in vitro using an extract test and in vivo using a subcutaneous pocket model in mice.
Materials and Methods
Biomaterial manufacture
The PTMC-CaO2 microspheres were produced according to the process described by Steg et al1, as is described in chapter 4. Briefly, microspheres were made out of a
suspension of 5% (w/w) CaO2 (Sigma-Aldrich, Steinheim, Germany) in a solution of 3,5 % (w/v) of poly (1,3-Trimethylene Carbonate) (PTMC) in acetonitrile (Merck, Darmstadt, Germany) using an oil-in-oil solvent evaporation method. As a control, PTMC microspheres not containing CaO2 were produced using the same manufacturing process. The spheres were stored at -20°C until use.
In vitro biocompatibility testing
In preparation of the experiment, extracts of PTMC and PTMC-CaO2 microspheres were made by adding 15 mg of either type of microspheres to one milliliter of standard medium, consisting of DMEM high glucose (Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% Glutamax (Life technologies, Eugene, OR, USA). Subsequently, the suspension was incubated for 24 hours in a shaking water bath at 37°C. After 24 hours, the extract was centrifuged at top speed for five minutes. Concentrated extracts (100%) and dilutions of 75%, 50%, and 25% of were made. A positive control was made by dissolving 1% sodium lauryl sulfate (SLS, Sigma-Aldrich) in medium. All extracts and the positive control were prepared in triplicate.
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L929 mouse fibroblast cells (Biosorb, the Netherlands) at passage 47 were seeded at a density of 10.000 cells per well in a 96 well plate (Greiner Bio-one, Alphen aan den Rijn, the Netherlands) and were allowed to adhere at 37°C and at 5% CO2 for 24 hours. 12 wells of each plate were filled with standard medium only, to serve as a blank. Then, the medium was replaced with 100 µl of extract. Each dilution of each extract was added to five wells. Subsequently, the cells were incubated at 37°C and at 5% CO2 for 24 hours. The MTT assay was performed by dissolving 0,5 mg/ml (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide / Thiazolyl Blue Tetrazolium Bromide (MTT) (Sigma-Aldrich) in full culture medium (MTT medium), replacing the medium of the cells with 50 µl MTT medium, and incubating the plate at 37°C for two hours in the dark. After two hours, MTT medium was carefully removed from the wells and 100 µl of 2-propanol (Merck) was added to each well. The plates were shaken for two minutes to dissolve the produced formazan, after which absorbance was read at 570 nm using a plate reader (Fluostar Optima, BMG labtech, Olfenburg, Germany). Background absorbance was read at 650 nm. Prior to viability calculations, background absorbance values were subtracted from the absorbance values read at 570 nm. The viability of the cells was calculated using the formula mentioned below.
Viability % =
(
Absorbance read at 570 nm of extract)
*100% Absorbance read at 570 nm of negative controlAll experiments were performed in triplicate.
Animals and surgical procedure
The Experimental Animal Committee of the University Medical Center Groningen approved all animal experiments, and Dutch national guidelines for animal care were followed.
In vivo biocompatibility testing
Twelve female Balb/c Ola/Hsd (Harlan, Horst, the Netherlands) mice of 7 weeks old were used for biocompatibility tests. The operations were carried out under aseptic conditions. General anesthesia was induced using isoflurane 4% and maintained using isoflurane 2%. Animals were administered buprenorphine 0,1 mg/kg sc once right before surgery. Two six millimeters long incisions were made on the backs of the mice on the midline and running parallel to the spine. Both incisions were one centimeter apart, the most cranial starting right caudal of the scapulae. From each incision, two subcutaneous pockets were created using blunt dissection, one left lateral to the incision and one right lateral to the incision. The pockets were ten millimeters deep.
On the bottom of each pocket, 25 mg of microspheres was placed. In each pair of pockets originating at one incision, one dose of PTMC microspheres was implanted on one side and one dose of PTMC-CaO2 microspheres was implanted on the opposite side. The type of material that was implanted on the left side of the incision was randomized. The wounds were closed using resorbable sutures. Half of the animals were terminated at one week after implantation of the biomaterials and half of the mice were terminated at six weeks after implantation by cervical dislocation. The biomaterial and near surroundings were excised after termination.
Processing of tissue samples
The tissue samples acquired in the biocompatibility tests were fixated in paraformaldehyde 3,7% (Boom, Meppel, the Netherlands) and cut in half after fixation. One half was embedded in paraffin and the other half was embedded in 2-hydroxyethyl methacrylate (HEMA) (Technovit® 8100, Heraeus-Kulzer, Wehrheim, Germany) according to the manufacturer’s instructions. A microtome was used to cut paraffin sections of 5 µm and HEMA sections of 4 µm. Three paraffin embedded slides were stained with Massons trichrome stain (Accustain® trichrome stains (Masson), Sigma-Aldrich) according to the manufacturer’s instructions. Three paraffin embedded slides were used for immunohistochemical staining with a CD3-antibody for identification of lymphocytes and with antibody F4/80 for identification of macrophages. The slides were dewaxed in xylene and rehydrated using phosphate buffered saline (PBS). The slides were preincubated with PBS supplemented with 10% serum from the species that produced the secondary antibody. Subsequently they were incubated with either rabbit-anti mouse CD3 monoclonal primary antibody (Abcam, Cambridge, United Kingdom) in a 1:500 dilution or rat-anti mouse F4/80 monoclonal primary antibody (BioRad, Veenendaal, the Netherlands) in a 1:1000 dilution. The slides were washed with PBS and endogenous peroxidases were blocked using H2O2. After washing the slides with PBS, the secondary antibodies, goat-anti rabbit-HRP (Agilent, Middelburg, the Netherlands) (dilution 1: 100) and goat-anti rat-HRP (BioRad) (dilution 1:200) respectively, were applied. Subsequently, the slides were washed with PBS and incubated with 3,3’-diaminobenzidine. After washing with PBS, the slides were counter-stained with hematoxylin (Merck).
Three plastic embedded slides were stained with hematoxylin and eosin (Merck). Quantification of the foreign body reaction was done according to an adapted system as proposed by Cohen21. All slides were photographed at a 400x magnification and
5
cells and giant cells and the amount of tissue necrosis and fibrosis. The results were recorded in tables as indicated in tables 1 and 2 (modified from ISO 10993-6: 2007). Three parts of each tissue sample were thus assessed by 2 observers. The numbers of lymphocytes and macrophages were counted using an automatic cell counting computer program. A score of the intervention tissue after subtraction of the control sample scores of 0,0 up to 2,9 was considered non-irritant, a score of 3,0 up to 8,9 was considered slight irritant, a score of 9,0 up to 14,9 was considered moderate irritant and a score of ≥15 was considered severe irritant.
Cell type/response Score
0 1 2 3 4 Polymorphonuclear cells 0 Rare, 1-5 phf* 5-10/phf Heavy infiltrate Packed Plasma cells 0 Rare, 1-5 phf 5-10/phf Heavy
infiltrate
Packed Giant cells 0 Rare, 1-5 phf 5-10/phf Heavy
infiltrate
Packed Necrosis 0 Minimal Mild Moderate Severe Lymphocytes 0 Rare, 1-5 phf 5-10/phf Heavy
infiltrate
Packed Macrophages 0 Rare, 1-5 phf 5-10/phf Heavy
infiltrate
Packed Fibrosis 0 Narrow band Moderately
thick band
Thick band Extensive band
Table 1 Histological evaluation system, explanation of scores. Phf is ‘per high magnification field’.
Biomaterial ID: Test sample Control sample
Slide type Scoring item A B C A B C
HE Polymorphonuclear cells Plasma cells Giant cells Necrosis CD3 stain Lymphocytes F4/80 stain Macrophages Sub-total (x2) Massonstrichrome Fibrosis Subtotal Total Test (-) control =
Statistical evaluation
The Statistical Package for Social Sciences (SPSS) was used for statistical evaluation of the results. Normality of the data was checked using a Shapiro-Wilk test. P values were calculated using a T-test. A p value of >0,05 was considered to be significant.
Results
In vitro biocompatibility testing
According to ISO standard 10993-5, an agent that causes the viability percentage in cytotoxicity assays to be lower than 70%, is considered to have cytotoxic potential. Cell viability percentages after in vitro cytotoxicity tests are indicated in table 3. At all tested conditions, cell viability was significantly lower than when cells were cultured with standard medium. When comparing cell viability of cells cultured with PTMC or with PTMC-CaO2 microspheres extract, cell viability was significantly lower in cells cultured with PTMC-CaO2 microspheres extract in 75% and 50% dilutions, but not at other dilutions. All extract dilutions of PTMC microsphere extract resulted in a cell viability higher than 70%, so PTMC microspheres are not cytotoxic at all tested concentrations of the extract (figure 1). After addition of 100% and 75% PTMC-CaO2 microspheres extract to the cells, cell viability was reduced to 62% and 65%, respectively. These microspheres thus have some cytotoxic effect in vitro. At lower dilutions of PTMC-CaO2 microspheres extract, a cytotoxic effect can no longer be observed.
Extract 100% Extract 75% Extract 50% Extract 25% Positive control
PTMC 78 73 78 83 0
PTMC-CaO2 62 65 80 91 0
Table 3 Viability percentages of cells after in vitro cytotoxicity tests.
Animals
No complications occurred during surgery or in the postoperative period. No preliminary termination of animals was needed. Animal discomfort was estimated to be 3/6, with 1 indicating slight discomfort and 6 indicating serious discomfort.
In vivo biocompatibility testing
Tissue samples of subcutaneous pockets containing either PTMC or PTMC-CaO2 microspheres were assessed for biocompatibility at 1 and at 6 weeks after implantation
5
and PTMC-CaO2 microspheres was -0,8 at one week and 0,6 at six weeks (figure 2). This indicates that the PTMC-CaO2 microspheres are non-irritants. The difference in biocompatibility score was not significant at both time points.
Figure 1 Bar chart indicating the results of the MTT assay. As a negative control, cells that were grown
without extract were used. A solution of 1% SLS in culture medium was used as a positive control. Means with standard deviations are indicated. Asterisks indicate a significant difference.
Figure 2 Bar chart indicating the average biocompatibility scores, given as described in the materials and
methods section. There was no significant difference between the PTMC and the PTMC-CaO2 groups at
In figure 3 representative pictures of histological specimens of the implants using four different stainings are shown. In all pictures, thin capsules surrounding the implants can be identified. These capsules are fibrotic as shown through the Masson’s trichrome stain. The F4/80 immunostaining shows these capsules contain many macrophages. On the hematoxylin-eosin stained slides only few foreign body giant cells could be identified, as well in PTMC implants as in PTMC-CaO2 implants. The numbers of polymorphonuclear cells, plasma cells and giant cells and the amount of necrosis are low in both types of material tested. The amount of fibrosis is somewhat higher, but still there is no difference between both groups. The numbers of macrophages and lymphocytes are larger, but again, there is no significant difference between both tested materials (table 4).
Figure 3 Representative pictures of histological specimens of PTMC (left column) and PTMC-CaO2
immu-5
Cell type/response Average score per type of microspheres
PTMC wk 1 PTMC-CaO2 wk 1 PTMC wk 6 PTMC-CaO2 wk 6 Polymorphonuclear cells 1,4 (0,6) 1,4 (0,6) 1,1 (0,6) 1,1 (0,6) Plasma cells 1,3 (0,6) 1,4 (0,6) 0,8 (0,5) 0,9 (0,7) Giant cells 0,6 (0,6) 0,7 (0,7) 0,8 (0,7) 0,7 (0,7) Necrosis 0,9 (0,8) 0,7 (0,6) 0,9 (0,6) 0,8 (0,6) Lymphocytes 2,9 (0,4) 2,2 (0,9)* 2,8 (0,6) 2,8 (0,4) Macrophages 3,8 (0,4) 3,7 (0,5) 3,8 (0,4) 3,9 (0,4) Fibrosis 1,8 (1,0) 2,0 (1,1)* 1,6 (0,9) 1,9 (0,9)
Table 4 Average histology scores per cell type and per type of material. Standard deviations are indicated
in brackets. A significant difference in scores between PTMC and PTMC-CaO2 microspheres is indicated with an asterisk.
Discussion and conclusion
The aim of this study was to evaluate the biocompatibility of PTMC-CaO2 microspheres
in vitro and in vivo. In in vitro tests with L929 mouse fibroblasts high concentrations
of an extract of PTMC-CaO2 microspheres resulted in cytotoxicity. However, at lower concentrations this effect disappeared. In tests in mice, the PTMC-CaO2 microspheres appeared to be biocompatible in vivo.
The lower cell viability of L929 cells upon exposure to 100% and 75% concentrations of the PTMC-CaO2 microsphere extract may be explained by the presence of hydrogen peroxide. Upon exposure of calcium peroxide to water, hydrogen peroxide is formed, as was shown in equation 116. A burst release of oxygen from PTMC-CaO
2
microspheres upon exposure to water was shown by Steg et al17. Hydrogen peroxide
is an intermediate product in the eventual reaction of calcium peroxide with water to produce oxygen. It could be that cytotoxic concentrations of hydrogen peroxide are present in the 100% and 75% concentrations of the PTMC-CaO2 microsphere extract, thus causing higher cell death than at lower concentrations of the extract. No hydrogen peroxide is produced during the preparation of the PTMC microsphere extract, which supports the hypothesis that the cytotoxic effect of high concentrations of PTMC-CaO2 microsphere extract is caused by excess hydrogen peroxide.
Based on these test results, no indication of the amount of microspheres that needs to be used in clinical practice can be given. A dose response curve should be created, preferably in in vivo tests, to be able to calculate the amount of microspheres that should be applied for adequate enhancement of tissue regeneration. For this experiment,
extracts of 15 micrograms of microspheres in one milliliter of medium were used. However, it is expected that in clinical practice dosages of 15 mg of microspheres per milliliter volume of tissue is too high to be used. The volume of the microspheres would in that case not allow adequate dispersion throughout the tissue. It is expected therefore, that the cytotoxic effect of the PTMC-CaO2 microsphere extract at high concentrations is not clinically relevant.
PTMC is a biocompatible polymer, which makes it a suitable blank to test the biocompatibility of PTMC-CaO2 microspheres22–24. The polymer is degraded
enzymatically in the presence of lipase or cholesterol esterase25,26. Macrophages
produce cholesterol esterase23,27. PTMC degradation occurs through a surface erosion
process22–25. In in vitro tests, it was shown that when macrophages were cultured in the
presence of PTMC films, no degradation of the PTMC films occurred. However, when the macrophages were cultured after being adhered to PTMC films, the material was eroded. Culturing fibroblasts on PTMC films did not result in PTMC degradation27. These
results suggest that macrophage contact with PTMC is important for PTMC degradation
in vitro. Upon implantation of PTMC specimens in vivo, initially, a fibrin layer is formed
around the implant. In this layer, macrophages and giant cells can be found. A couple of days after implantation, a fibrous capsule containing macrophages and foreign body giant cells can be identified22,23,28. The tissue reaction 5-7 days after implantation
remains discordant, maybe due to different implantation sites or differences between animal species that were used in different studies. Some authors describe layers of foreign body giant cells and macrophages containing phagocytosed PTMC particles around the implant. In the 2-52 weeks after implantation the material weight and volume decreases22,23,28. Others describe only multilayered fibrous capsules around
the implants, with only few macrophages or nodules of foreign body giant cells24,26.
As well around the PTMC microspheres as around the PTMC-CaO2 microspheres we found large macrophage infiltrates, which corroborates with the findings of Pêgo and Bat22,23,28. This supports the assumption that PTMC is degraded under influence of
contact with macrophages27.
Ischemia is a frequently occurring cause of tissue death. Therefore, many possible applications of oxygen-releasing microspheres can be proposed. Regeneration of bone tissue is often restrained due to a lack of adequate vascularization in a bone defect1–3.
Adding microspheres to a cell-scaffold complex in a bone regeneration site may aid cell survival on the scaffold and thus support bone regeneration. In maxillofacial surgery, a lack of bone stock in the mandibula presents a frequently occurring problem for placement of implants in the bone tissue29. Oxygen-releasing microspheres may aid
5
Another option is the application of microspheres in cardiac muscle. After myocardial infarction heart muscle tissue becomes ischemic30,31. In such situation, the application
of oxygen-releasing microspheres may support muscle cell survival, so that damage to the cardiac muscle may be limited. Furthermore, application of oxygen-releasing microspheres to support the healing of ischemic ulcers, or to support survival of malvascularized skin tissue flaps in plastic surgery applications can be thought of. In ISO standard 10993-5 it is advised to use L929 mouse fibroblasts for in vitro biocompatibility testing. For standardization purposes was chosen to use L929 cells in this study as well. However, in clinical applications, cell types other than mouse fibroblasts will be combined with the oxygen-releasing microspheres. The results from this study indicate that the PTMC-CaO2 microspheres are biocompatible in mice, but before clinical use in humans the material should be tested in combination with the cell types present on the intended location of implantation. In chapter 4 human mesenchymal stem cells (hMSC) were cultured in the presence of PTMC-CaO2 microspheres. hMSCs are present in human tissue of mesenchymal origin, including bone marrow. MSCs are able to differentiate into osteogenic cells and may play an important role in bone regeneration. When this cell type is cultured in the presence of PTMC microspheres or PTMC-CaO2 microspheres, the cells adhere to the microspheres rather than to tissue culture plastic, which indicates that the presence of PTMC and PTMC-CaO2 microspheres is tolerated well by hMSCs.
n the in vitro tests described in this study, the 100% and 75% concentrations of the PTMC-CaO2 extract appeared to be cytotoxic. In ISO standard 10993-5 no indication is given of the amount of material that should be used to prepare an extract of this material. Therefore, and as no comparison material is available, an amount of PTMC-CaO2 microspheres based on an educated guess was chosen to prepare the extract. Perhaps, if a lower amount of microspheres would have been chosen to prepare the extract, results could have been different. Possibly, the cytotoxic effect of the 100% and 75% concentrations of the PTMC-CaO2 microspheres extract was caused by an overdose of H2O2, which is a cytotoxic intermediate product in the reaction of CaO2 with water to produce oxygen. Catalase catalyzes the reaction stated in equation 2. The addition of catalase to the tissue culture wells in the in vitro biocompatibility tests may reduce the cytotoxic effect of H2O2.
In conclusion, the biocompatibility of PTMC-CaO2 microspheres was tested in vitro and
in vivo. In in vitro tests, 100% and 75% concentrations of a PTMC-CaO2 extract had a cytotoxic effect, whilst lower concentrations did not have a cytotoxic effect. In in vivo tests, the PTMC-CaO2 microspheres appeared to be biocompatible.
Acknowledgements
Authors would like to thank D.T.A. Ploeger, MSc. Ing., for assistance with immunostaining and Dr. S.M. van Putten for his assistance in the in vivo experiments.
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