University of Groningen
Towards in vivo application of oxygen-releasing microspheres for enhancing bone
regeneration
Buizer, Arina
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Publication date: 2018
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Buizer, A. (2018). Towards in vivo application of oxygen-releasing microspheres for enhancing bone regeneration. Rijksuniversiteit Groningen.
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Summary
133 In this thesis, the steps taken to translate the use of oxygen-releasing microspheres for enhancing bone regeneration from lab bench to in vivo application are described. At this moment, the golden standard for repairing bone defects is the use of autologous bone. The main disadvantages of the use of autologous bone grafts are the occurrence of donor site morbidity, the (cost of an) extra surgical procedure that is needed for harvesting bone tissue and the limited availability of autologous bone. Therefore, bone regeneration options are explored increasingly. Especially, bone regeneration using scaffolds seeded with cells that have the capacity to form bone is frequently studied. Human mesenchymal stem cells (hMSC) have the capacity to form bone, they are easy to harvest and they show trophic effects. These properties make hMSCs a favorable cell type to seed on scaffolds for bone regeneration. The vascularization in cell-scaffold complexes is an important issue to be addressed. At the moment of implantation in the body, the seeded hMSCs are exposed to prolonged ischemic circumstances, because they are often implanted in sites with disrupted vascularization and because of an absence of vascularization within the scaffold. Especially cells seeded in the center of a scaffold do not receive enough oxygen to survive, resulting in poor bone regeneration. Whenever hMSCs encounter ischemia, they start producing angiogenic factors (AGF). These AGF enhance the ingrowth of blood vessels. Through establishment of a vascular network in a cell-scaffold complex, a sustainable oxygen supply is accomplished. Due to the presence of a durable oxygen supply, higher numbers of cells seeded on a scaffold may survive and thus optimal bone regeneration could be achieved. In these studies, microspheres that release oxygen gradually were developed, firstly to support hMSC survival in de first weeks after implantation in the body. Secondly, by releasing only small amounts of oxygen the AGF production of hMSCs is stimulated, so that the establishment of sustainable oxygen supply in the longer term is supported. The oxygen-releasing microspheres are prepared with poly (1,3-trimethylene carbonate) (PTMC) as a matrix material and calcium peroxide particles (CaO2) as oxygen donor. CaO2 reacts with water resulting in the release of oxygen according to equations below.
CaO2 + 2 H2O → Ca(OH)2 + H2O2 2 H2O2 → O2 + 2 H2O
PTMC is degraded through a surface erosion process, which implies that the CaO2
particles dispersed in the PTMC matrix are exposed to water gradually. Thus, a slow oxygen release system is acquired.
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The aims of this thesis are:
• To assess which method is optimal for seeding cells on low and on high porosity ceramic biomaterials;
• To evaluate which oxygen level is optimal for hMSC proliferation and angiogenic factor production;
• To establish a PTMC-CaO2 microsphere preparation method and evaluate the
oxygen release profile of these microspheres;
• To assess biocompatibility of the oxygen-releasing microspheres in vitro and in vivo; • To investigate if the application of oxygen-releasing microspheres results in
improved tissue survival in otherwise ischemic tissue in vivo.
In Chapter 2, a static and a vacuum cell seeding technique to seed hMSCs on high and low porosity tricalcium phosphate scaffolds are compared. Three different cell seeding densities are evaluated for use on high porosity tricalcium phosphate scaffolds. Cell viability, cell proliferation and cell seeding homogeneity are the end points in this study. hMSCs were isolated from reaming debris acquired during hip replacement surgery. Cells acquired using this method are used in all hMSC experiments described in this thesis. They were characterized according to the guidelines given by the International Society for Cellular Therapy (ISCT) and the cell profile complied with the ISCT requirements. Regardless of the cell seeding technique used, in low as well as high porosity scaffolds, more cells adhered to the outsides than to the insides of the scaffolds. In low porosity scaffolds, a vacuum cell seeding method resulted in the most homogeneous cell seeding and higher numbers of cells adherent to the scaffold than the static cell seeding technique. On high porosity scaffolds, however, the static cell seeding technique resulted in more homogeneous cell seeding and higher numbers of cells adherent to the scaffold than the vacuum cell seeding technique. The application of vacuum to the cells did not result in decreased hMSC proliferation. The absolute numbers of cells on the scaffolds decreased over time after seeding on both scaffold types and after use of both cell seeding techniques. Both cell seeding techniques tested resulted in a cell seeding efficiency of around 90%. On low porosity scaffolds the highest cell seeding density resulted in the highest number of cells adherent to the scaffold.
In Chapter 3, the oxygen range at which hMSC proliferate and are metabolically active, yet transcribe angiogenic factors, is investigated. hMSCs proliferation was found to be
Summary
135 the highest at 1% oxygen. At 21% oxygen, which is in fact a hyperoxic situation to bone marrow-derived hMSCs, the cells showed the highest corrected cell metabolic rate. Under hypoxic circumstances, the corrected cell metabolic rate was the highest at 2% oxygen. The transcription of AGF under hypoxic circumstances was related to the transcription of AGF when hMSCs were exposed to 21% oxygen. As HIF-1α is transcribed standard, it was expected that the transcription of HIF-1α did not differ significantly between hypoxic and normoxic conditions. However, at 2% O2, VEGF and ANG-1 transcription was significantly higher than at 21% oxygen. Therefore, it was concluded that the oxygen range at which bone marrow-derived hMSCs show high proliferation, are metabolically active and show high transcription of AGF is at 1-2% O2.
In Chapter 4 a method to prepare oxygen-releasing PTMC-CaO2 microspheres is
described. Furthermore, the oxygen release profile of the thus created microspheres is evaluated and the effect of the presence of the microspheres on the metabolic activity of hMSCs cultured under hypoxic circumstances is assessed. As the microspheres start releasing oxygen upon contact with water, contact with water should be avoided during the preparation process. Therefore, an oil-in-oil solvent evaporation method was used, and polydisperse microspheres of max 200 μm large were produced. In
vitro, in simulated body fluid complemented with cholesterol esterase, the
PTMC-CaO2 microspheres released oxygen for about 20 days. The presence of PTMC-CaO2 composite microspheres led to higher hMSC adherence to the microspheres than to tissue culture plastic, suggesting the spheres are not cytotoxic. When cultured under hypoxic circumstances, hMSCs showed higher metabolic activity in the presence of oxygen-releasing microspheres than in the presence of non-oxygen-releasing microspheres after 7 days of culturing, suggesting that the oxygen release from the microspheres increases hMSC survival after prolonged exposure to hypoxic circumstances.
The biocompatibility of the oxygen-releasing PTMC-CaO2 microspheres is assessed using in vitro and in vivo tests based on ISO standard 10993-5 in Chapter 5. The in vitro tests, where L929 mouse fibroblasts were cultured with several concentrations of a PTMC-CaO2 microsphere extracts, cytotoxicity of high concentrations of the extracts was shown. However, lower concentrations, which more resemble the expected in
vivo concentrations, did not lead to increased cell death. This was confirmed in in vivo
tests. Upon implantation of PTMC-CaO2 microspheres in subcutaneous pockets in
mice, no adverse effects were observed. It was concluded that the oxygen-releasing microspheres are biocompatible.
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As a proof of principle, the oxygen-releasing PTMC-CaO2 microspheres were implanted under a random pattern devascularized skin flap in mice. The results are described in
Chapter 6. Histologic analysis of the skin flaps indicated that the amount of necrosis in
the skin tissue increased from the cranial basis of the skin flap towards the caudal end of the flap. This implies that there is a gradient of ischemia in the skin flap, where the cranial basis of the skip flap is well-vascularized and not ischemic, and the caudal end of the flap is the least vascularized and thus is the most ischemic. Skin flaps under which non-oxygen-releasing PTMC microspheres had been implanted, showed significantly more necrosis than skin flaps under which oxygen-releasing PTMC-CaO2 microspheres had been implanted at 3, 7 and 10 days after implantation. These findings suggest that the release of oxygen by the PTMC-CaO2 microspheres may prevent cell death in cells exposed to ischemic circumstances, thereby promoting the survival of otherwise ischemic tissue.
The outcomes of this research project are discussed and suggestions for further research are given in Chapter 7, the general discussion. The oxygen-releasing PTMC-CaO2 microspheres that were studied in this thesis were tested in a skin tissue application in mice. However, it would be interesting to investigate the application in other tissues, such as bone tissue or cardiac muscle tissue as well. In malvascularized tissue, cells do not only suffer a lack of oxygen, but also of other nutrients, such as glucose, amino acids or vitamins. Supplementing more nutrients but only oxygen to ischemic cells may improve cell survival as well. This is worth further investigation. In this research project, microspheres that release oxygen for three weeks were prepared. Perhaps, through modification of the carrier material, an even more prolonged oxygen release may be accomplished.
In conclusion, oxygen-releasing PTMC-CaO2 microspheres were prepared that released oxygen in vitro for about 20 days. In in vivo tests, the microspheres were biocompatible and they significantly decreased skin necrosis in a random pattern devascularized skin flap model in mice for at least 10 days. This suggests that the oxygen-releasing PTMC-CaO2 microspheres may facilitate cell survival in ischemic tissues, and may enhance the regeneration of tissue under ischemic circumstances.
