Towards in vivo application of oxygen-releasing microspheres for enhancing bone
regeneration
Buizer, Arina
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Chapter 6
Proof of principle of
oxygen-releasing poly (trimethylene
carbonate) 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
Tissue repair often needs to take place in poorly vascularized areas. Repair cells that are placed in ischemic tissues for their ability to repair the required tissue are exposed to severe and prolonged hypoxia. Most of the repair cells die before they contribute to new tissue formation. To support cell survival until a sustained oxygen supply through vascularization has been accomplished, oxygen-releasing microspheres were developed. As a carrier material, poly (1,3-Trimethylene Carbonate) (PTMC), a polymer that is enzymatically degraded within the body, was used. Calcium peroxide was dispersed into PTMC in order to create a slow-release system, as oxygen is a product of the reaction of calcium peroxide with water. Oxygen-releasing microspheres were implanted under a random pattern devascularized skin flap in 12 mice. Photographs were taken of the skin flaps at 3, 7, and 10 days after surgery, and skin necrosis was assessed by three independent observers using Image J. Histologic examination of the skin flaps was done after termination of the animals at day ten. Significantly less skin necrosis was measured in skin flaps under which oxygen-releasing microspheres were implanted than in skin flaps under which PTMC microspheres without calcium peroxide were implanted at day 3, 7, and 10 after surgery. These findings were confirmed by histologic examination. These results suggest that the oxygen release from these microspheres supports cell survival and may thus aid in delaying tissue necrosis in malvascularized tissues.
6
Introduction
For adequate tissue repair under ischemic circumstances the accomplishment of an adequate oxygen supply to repair cells remains a big challenge. The transport of oxygen within the body is most efficient when oxygen is bound to hemoglobin in the blood and delivered to the peripheral cells via the extensive vascular network that the body possesses1. Given that the maximum diffusion distance of oxygen within the
body is around 100-200 µm, the vascular network must be extensively branched to bridge this maximum oxygen diffusion distance2,3. In ischemic tissues vascularization
is often inadequate. Oxygen provision to repair cells is thus compromised2,4,5. The
ischemic conditions are an important cause of death of repair cells and they are an important contributor to the limited success rate of ischemic tissue recovery.
Several methods aiming at improvement of local vascularization have been proposed to increase repair cell survival under ischemic circumstances. A frequently studied option is the application of precursor cells, which are cells that have the capacity to differentiate into the desired type of tissue. An example is the application of adipose-derived mesenchymal stem cells (MSCs) in heart muscle tissue after myocardial infarction6. To improve the angiogenic potential of progenitor cells, cells may be
exposed to hypoxic circumstances prior to implantation, in order to increase the production of factors that increase vascular ingrowth7,8. This process is called hypoxic
preconditioning. A different option to increase vascular ingrowth is the application of exogenic angiogenic growth factors (AGF), which stimulate the ingrowth of blood vessels9. Also, the application of cells that are modified prior to implantation so that
they produce more AGF has been investigated10. In some situations, application of
cells alone may not be sufficient for tissue repair. A scaffold may be needed as a matrix for cells to grow upon. To improve vascularization in cell-scaffold complexes many techniques have been studied. Examples of these techniques include co-seeding of endothelial cells combined with several types of precursor cells on a scaffold11–13 and
the application of (AGF) in or near a scaffold14–17. The results were variable. A promising
option to increase repair cell survival is to supply repair cells with oxygen from an external source, so that they can survive until vascular ingrowth has occurred and thus sustained oxygen supply has been accomplished.
Oxygen release systems consisting of a polymer carrier material combined with a peroxide have been created by several research groups18–20. These usually comprise a
hydrolysable polymer combined with a peroxide material. In this study, a composite consisting of poly (1,3-Trimethylene Carbonate) (PTMC) as the polymer carrier
PTMC was chosen as it is degraded by means of surface erosion without production of acidic waste products. CaO2 was chosen for its favorable oxygen release profile combined with its availability in high purity21. Whenever the CaO
2 comes into contact
with water, the following chemical reactions occur, resulting in the release of oxygen (equations 1 and 2):
Eq 1: CaO2 + 2 H2O → Ca (OH)2 + H2O2 Eq 2: 2 H2O2 → O2 + 2 H2O
Incorporating peroxide into the polymer PTMC creates a slow release system of oxygen. As the polymer is degraded by surface erosion, the peroxide is are gradually exposed to water and oxygen is released gradually. The PTMC-calcium peroxide composite will thus react as a slow-release system for oxygen. By adjusting the dosage of the microspheres, or the type of polymer and the concentration of peroxide, oxygen release can be adapted to the oxygen demand. Several types of materials can be produced out of polymer-peroxide composites, each optimized for their final application. For application in bone, an important factor is the mechanical strength of the material. PTMC is an elastic polymer with insufficient mechanical properties for application in bone. Therefore, microspheres were produced than can be combined with porous bone replacement materials that, mostly when combined with materials for osteosynthesis, provide sufficient mechanical support. The microspheres produced oxygen for at least 20 days in vitro22. In in vitro and in vivo tests they were shown to
biocompatible (see Chapter 5).
In this study, oxygen-releasing PTMC-CaO2 microspheres were implanted under a
random pattern devascularized skin flap in mice as a proof of concept of the working potential of these microspheres.
Materials and methods
Material preparation
The microsphere production method is described in chapter 4. Briefly, a suspension 5% (w/w PTMC) of CaO2 (Sigma Aldrich, Zwijndrecht, the Netherlands) in a solution of 3,5% (w/v) PTMC in acetonitrile (Merck, Darmstadt, Germany) was made. An oil-in-oil solvent evaporation method was used to produce microspheres, based on the process described by Uchida et al 23, using mineral oil (Sigma Aldrich) supplemented
PTMC-6
CaO2 microspheres were allowed to precipitate by gravity and subsequently they were washed with hexane and extensively dried. The PTMC-CaO2 microspheres were stored at -20°C until use. Control microspheres did not contain CaO2.
Experimental animals and procedures
All animal experiments in this study were performed according to the national code of practice for laboratory animal care. The Laboratory Animal Committee of the University Medical Centre Groningen approved the experimental protocol. For this experiment, the model used by Harrison et al 18 was adapted to our needs. Twelve
female BALB/c mice (BALB/c OlaHsd, Harlan, Horst, the Netherlands) of 6-8 weeks old were randomly divided in a control group of 6 mice receiving PTMC microspheres and an intervention group of 6 mice receiving PTMC-CaO2 microspheres. The operative procedure was performed under anesthesia using isoflurane 2%. The animals were shaved and subsequently the stubbles were removed using depilation cream. A cranially based skin flap was created by making two incisions of three centimeters long running parallel to the spine, 0,5 cm of the midline of the animal. Both incisions were connected with a transverse one-centimeter long incision located at the caudal end of the longitudinal incisions. The skin was bluntly dissected from the muscular layer. Care was taken that no large vessels were included in the skin flap, so that blood supply would be limited to the cranial base of the flap. Then one longitudinal incision and the transverse incision were sutured using Monocryl 5.0 (Ethicon, Norderstedt, Germany) and interrupted sutures. Hundred milligrams of microspheres were applied on the muscular layer on the most caudal 2x1 cm area under the skin flap and spread evenly. The second longitudinal incision was sutured as well. Carprofen 5 mg/kg sc once per 24 hours was administered routinely under anesthesia using isoflurane 2% for the first three days after surgery. The animals had access to food and water ad
libitum and were housed in pairs in standard cages. Ten days after surgery the animals
were terminated by cervical dislocation under general anesthesia. The skin flaps were excised in a standard manner and further processed for histological examination.
Photography and image analysis
At days 3, 7, and 10 after the surgery, the animals were anesthetized using isoflurane 2% via a non-rebreathing face mask. The skin flap on their back was photographed using a digital camera and standard lighting. A ruler was included in each picture for calibration purposes. The area of brown discoloration due to skin necrosis was assessed three times on three separate days by three independent observers blinded for the applied treatment using Image J analysis software. The amount of skin necrosis was expressed as percentage of the total skin flap.
Histology
The skin flaps were cut in 4 equally sized longitudinal strips after excision from the animals and fixated in paraformaldehyde 3,7% (Boom, Meppel, the Netherlands). The strips were washed, dehydrated and then embedded in Technovit® 8100 (Heraeus-Kulzer, Wehrheim, Germany). Four µm thick sections were cut using a microtome. The sections were mounted on Superfrost slides (Thermo Scientific, Braunschweig, Germany) and stained with hematoxylin and eosin (Merck). Images were studied using a DMR microscope (Leica HC, Wetzlar, Germany) equipped with a Leica DFC 420C camera.
Statistical analysis
Data were analyzed using the SPSS 20.0 software package. Mann-Whitney-U-tests were used to compare both research groups. Intra rater and inter rater variability was indicated with an intraclass correlation coefficient (ICC). A p value lower than 0,05 was considered significant.
Results
Animals
All animals tolerated the operations well and no complications occurred. There were no early dropouts. Animal discomfort was estimated to be 3/6.
Skin necrosis
Comparison of the three independent evaluators of the amount of skin necrosis (Figure 1), revealed that the inter-rater reliability had an ICC of 0,803 (95% CI: 0,599-0,890). The intra-rater reliability had ICCs of 0,983, 0,971, and 0,996 for raters 1, 2, and 3 respectively. The amount of necrosis was variable within both the PTMC group and the PTMC-CaO2 group. Therefore, the results were not normally distributed; medians are given in table 1. At 3, 7, and 10 days post-surgery skin necrosis was significantly higher in the PTMC group than in the PTMC-CaO2 group. These results indicate that the PTMC-CaO2 microspheres did support cells under circumstances of disturbed vascularization in contrast to PTMC microspheres not releasing oxygen.
6
Day 3 Day 7 Day 10
PTMC PTMC-CaO2 PTMC PTMC-CaO2 PTMC PTMC-CaO2
Median 74 45 73,5 62,5 86 77,5
Table 1 Median percentage of skin necrosis at three time points in mice with PTMC microspheres
implant-ed and in mice with PTMC-CaO2 microspheres implantimplant-ed.
Figure 1 Representative pictures of a random pattern devascularized skin flap after implantation of
mi-crospheres. Under the skin flap in the mouse on pictures a, c, and e, PTMC microspheres were implanted. Under the skin flap in the mouse on pictures b, d, and f, PTMC-CaO2 microspheres were implanted. Pictures a and b were taken 3 days after surgery, pictures c and d were taken 7 days after surgery and pictures e and f were taken 10 days after surgery.
Histology
Gross histologic examination at a low magnification (25x) gave an indication of the course of skin necrosis along the full length of the skin flaps. At the cranial end of the skin flaps usually morphologically normal skin tissue was visible (see figure 2), with the characteristic dark colored, several cells thick, epidermal layer and the presence of typical papillary structures. Moving along to the caudal end of the skin flap, tissue morphology changed. The epidermal layer became thinner or even disappeared, and tissue architecture became less organized. The papillary structure of the epidermal layer became less evident or disappeared as well. At the caudal end of the skin flaps often a recurrence of normal skin architecture could be observed, as the skin flaps were excised including a rim of healthy skin tissue around the skin flaps. The histologic specimens shown in figure 2 are representative of the difference in tissue necrosis between control and experimental group skin flaps.
Histologic examination of the skin flaps at a higher magnification (400x) showed intact skin tissue at the cranial site of the skin flaps, with an epidermal layer including a clear stratum corneum, stratum granulosum and stratum spinosum. The cells were intact and the cell nuclei were clearly visible. When proceeding to the more caudal part of the skin flap, tissue architecture became disorganized, and the laminar structure of the skin could not always be recognized. The eosinophilia of the cytoplasm of the cells was striking and the cell nuclei were less sharply defined or could not be identified anymore. The combination of eosinophilia and deterioration of the cell nuclei is a clear indication that the cells were necrotic. At the cranial end of the skin flap, tissue architecture looked healthy.
6
Figure 2 Histologic specimens of skin flaps, HE staining. The specimens in figure A were taken from a skin
flap under which PTMC microspheres were implanted. The specimens in figure B were taken from a skin flap under which PTMC-CaO2 microspheres were implanted. The irregular shaped figures are pictures of the full skin flap at a magnification of 25x. The rectangular pictures represent detailed pictures of the skin tissue taken at a magnification of 400x. Scale bars in the rectangular pictures represent 50 µm. In the pictures of normal skin, the stratum corneum is indicated with a black arrow, the stratum granulosum is indicated with a white arrow and the stratum spinosum is indicated with a black asterisk.
Discussion and conclusion
The aim of this study was to explore the effect of implanted oxygen-releasing microspheres on the process of necrosis in partly devascularized skin flaps. In comparison to PTMC microspheres, subcutaneous implantation of oxygen-releasing PTMC microspheres resulted in a significant reduction of the amount of necrotic skin tissue at all three follow-up moments. These findings suggest that the release of oxygen by the PTMC-CaO2 microspheres may support survival of the otherwise ischemic skin cells and apparently aid in the delay of the process of necrosis. In the most cranial parts of the skin flaps, skin morphology was still intact 10 days after implantation of the microspheres. In the caudal regions, skin necrosis was clearly visible. These results suggest that there is an ischemic gradient within the skin flap, the most caudal area being the most ischemic, while the most cranial area is less or not ischemic.
To our knowledge, oxygen-releasing biomaterials existing of a polymer base and
a peroxide as oxygen donor have been tested in vivo only once. Harrison et al18
implanted films made out of a composite of poly (D,L-lactide-co-glycolide) (PLGA) and sodium percarbonate under random pattern devascularized flaps in mice and found that two and three days after implantation of the film, skin necrosis was significantly less in flaps under which oxygen-releasing PLGA films were implanted than in skin flaps under which control films not releasing oxygen were implanted. Seven days after implantation of the PLGA films, the amount of skin necrosis was similar in
oxygen-releasing films and in films that did not release oxygen. The PTMC-CaO2
microspheres that were tested in our study slowed down the occurrence of necrosis in a devascularized skin flap for a longer period, and even after ten days, skin necrosis was significantly lower after implantation of oxygen-releasing microspheres. An explanation for the shorter working time of the PLGA-sodium percarbonate composite could be the difference in degradation mechanism between PTMC and PLGA. PTMC is a polymer that degrades by surface erosion24,25, in contrast to PLGA, which degrades
by bulk degradation26. Surface erosion entails the breakdown of a material in a
layer-by-layer mode, so that the surface of the polymer degrades first, and the core of the material last. In bulk degradation, a polymer is penetrated with water throughout the polymer before the bonds between the monomers are disrupted, and ultimately the polymer breaks down through hydrolysis in bulk26. It is likely, that the surface erosion
degradation, as is applicable to PTMC, enables a more gradual and prolonged release of oxygen from the polymer carrier than does bulk degradation through hydrolysis, as is applicable to PLGA. The influence of the type of carrier polymer on oxygen release from a polymer-peroxide composite is confirmed by Pedraza et al20, who produced a
6
up to four weeks in vitro. Pancreatic islet cells grown under hypoxic circumstances in the presence of PDMS-calcium peroxide disks had higher metabolic activity, higher insulin production, less lactate dehydrogenase release and lower caspase activity than pancreatic islet cells grown without an oxygen-releasing disk. However, due to its hydrophobicity, PDMS shows hardly any degradation in vivo, a material property that is not always desirable in in vivo application20.
Although oxygen-releasing microspheres were implanted under the skin flap in this study, still skin necrosis occurred after a couple of days. Exhaustion of the oxygen supply may be a cause of the skin necrosis. It is also possible that not a lack of oxygen was the cause of the skin necrosis, but that a shortage of other essential nutrients that would otherwise be supplied via blood-borne transport, such as proteins or vitamins. The possible applications of oxygen delivering biomaterials are manifold. Plastic surgeons frequently use skin flaps in clinical practice. A frequently occurring complication is necrosis of a skin flap27. As has been shown in this study,
oxygen-releasing microspheres may aid in the prevention of necrosis in a skin flap. The use of oxygen-releasing materials has also been proposed for supporting regeneration of cardiac tissue, for example after myocardial infarction28,29. After myocardial infarction,
part of the heart muscle tissue is damaged and needs to be regenerated to regain optimal heart function. Regeneration of cardiac tissue by stem cell therapy has been inefficient until now. One of the leading causes of this inefficacy is cell death of the cells applied in heart tissue due to ischemia29. Oxygen delivering biomaterials may
aid cell survival and may have the potential to make heart tissue regeneration more successful. Other possible applications of oxygen producing biomaterials can be in regeneration of bone tissue in maxillofacial or orthopedic surgery, for treatment of large ischemic ulcers, and several other applications.
Assuming that vascular ingrowth takes place at an ingrowth rate of 0,5 mms per day30, it would take about 10 days to revascularize one centimeter of tissue, whenever
blood vessels can grow in from two opposite sides. Repair cells should thus survive for sometimes even several weeks until vascular ingrowth is completed, which means that oxygen delivering scaffold materials should provide oxygen for weeks as well.In vitro, PTMC-CaO2 microspheres produce oxygen for at least 20 days22. In vivo however,
the oxygen-releasing microspheres had a significant positive effect until at least ten days after implantation of the material. It is yet unknown how long the oxygen release keeps on supporting the skin cells and thus aids in preventing necrosis in the animal model that was used, but ten days are a good start. Perhaps the oxygen release from this type of materials could even be lengthened, by using a more
cross-linked type of PTMC, which is degraded slower and thus a prolonged oxygen release may be accomplished. Using a more hydrophobic carrier polymer, which is degraded more slowly in the body, may also lengthen oxygen release from a polymer-peroxide construct. Adjustment of the peroxide component, for example increasing the dosage of peroxide or using a more water-soluble peroxide than calcium peroxide, could also increase oxygen delivery by polymer-peroxide composites.
In conclusion, oxygen-releasing microspheres were produced that delayed skin necrosis in a random pattern devascularized skin flap model in mice significantly for at least 10 days. These microspheres may support cell survival in otherwise ischemic circumstances, and can aid in making the regeneration of tissue in ischemic environments more successful.
Acknowledgements
Authors would like to thank Annemieke Smit-van Oosten, Bianca Meijeringh, Michel Weij and André Zandvoort, micro surgeons in the Animal Laboratory Groningen, for their excellent support. Authors would like to thank Marja Stiemsma-Slomp for her excellent laboratory support.
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