University of Groningen
Oxygen-releasing biomaterials
Steg, Hilde
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Publication date: 2018
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Steg, H. (2018). Oxygen-releasing biomaterials. Rijksuniversiteit Groningen.
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Summary
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In the studies described in this thesis, the challenge is addressed of enhancingthe survival of cells seeded in tissue engineering scaffolds upon their implantation. An overview of the literature on cell-based therapies that are being researched is given in Chapter 2. Attempts have been made to promote vascularization in engineered tissues, either prior to or upon implantation, but tissues with sizes suitable for transplantation have been difficult to obtain. Alternative approaches, such as the use of oxygen-delivering biomaterials, are being investigated. In general, the materials that have been investigated are adequate for cell culturing and have in some cases shown to delay massive cell death upon implantation. However, the ideal oxygen-releasing biomaterials have not been found yet.
As reviewed in Chapter 2, oxygen-delivery has been effectuated by preparing composites consisting of a polymeric matrix and peroxide salts. These latter will generate oxygen upon contact and reaction with water. Calcium peroxide (CaO2) particles have been used most often. Factors that influence the
oxygen-release characteristics from these composites are, amongst others, the properties of the polymer matrix (polymer hydrophilicity and degradability) and those of the environment (water content, temperature, pH). For application in tissue engineering, it would be beneficial to prepare tissue engineering scaffolds using biodegradable polymer and calcium peroxide composites. To increase the survival rates of the transplanted cells, such scaffolds should allow the release of oxygen for a period of 2 to 3 weeks which is sufficiently long to allow the formation of a new vasculature.
In Chapter 3 a first study is presented in which oxygen-releasing composites were prepared and characterized. Oxygen-releasing composites based on poly(D,L-lactide) and poly(lactide-co-glycolide) and calcium peroxide as source of oxygen were prepared and their release characteristics compared to those from calcium peroxide particles not embedded in a polymer matrix. It was found that the rate of release of oxygen from the composites was initially higher than that from the CaO2 particles. The overall duration of oxygen-release, however, was
Summary
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could be related to the formation of acidic degradation products of the polymers upon hydrolysis, which in turn can influence the rate of oxygen-formation and release from the peroxide. Furthermore, human mesenchymal stem cells (hMSC) cultured on these oxygen-releasing composites required the presence of catalase enzyme in the medium to be able to proliferate. This enzyme reduces the effect of hydrogen peroxide, a toxic intermediate reaction product in the formation of oxygen from the peroxide. It can therefore be concluded that these lactide-based polymers were sub-optimal as matrix materials in these oxygen-delivering composites.
Chapters 4, 5 and 6 describe the preparation and properties of improved
oxygen-delivering composites based on poly(trimethylene carbonate) (PTMC) and CaO2 particles. Like poly(D,L-lactide) and poly(lactide-co-glycolide), PTMC is a
hydrophobic polymer. This polymer, however, degrades enzymatically in vivo and
in vitro by surface erosion without the formation of acidic degradation products.
In Chapter 4 PTMC/CaO2 composite microspheres, which could be
incorporated into any tissue engineering scaffold, were prepared. For this a novel method was developed in which oxygen-delivering composite microspheres could be prepared without any contact with water. These microspheres were then evaluated for their capacity to release oxygen. The characteristics of the oxygen-release were found to be dependent on the presence of cholesterol esterase (an enzyme that ensures surface erosion of the PTMC polymer in vitro) was in the medium. Microspheres were then added to hMSCs cultured under hypoxic conditions (0.1% oxygen), which were found to proliferate well. Also, in the absence of cholesterol esterase. These composite microspheres were not cytotoxic, and addition of catalase was not required.
In in vivo experiments, the PTMC/CaO2 composite microspheres were
assessed for their ability to reduce necrosis of a devascularized skin flap in mice.
Chapter 5 describes the results of a series of experiments, in which PTMC and
PTMC/CaO2 composite microspheres were implanted under a devascularized skin
flap in 12 mice. Photographs of the skin flaps were taken at 3, 7, and 10 days after surgery, and skin necrosis was assessed. Histological examination of the skin flaps was performed after termination of the animals at day ten. It could be observed,
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that significantly less skin necrosis was seen in skin flaps sutured over PTMC/CaO2composite microspheres compared to skin flaps sutured over PTMC microspheres. These findings were confirmed by histologic examination of the samples. The implantation of oxygen-releasing PTMC/CaO2 composite microspheres support
skin tissue survival, and thus aids in diminishing or delaying tissue necrosis in a devascularized skin flap.
In Chapter 6, in vitro experiments were conducted to assess the efficacy of oxygen-releasing films prepared from on poly(trimethylene carbonate) and CaO2
composites. In these in vitro models, hMSC cells and osteosarcoma (SaOs-2) cells were cultured on the composite films in a hypoxic environment. Although, improved viability was observed in vivo, the viability of the cells cultured on the PTMC/CaO2 composite films under hypoxic conditions was not found to be better
than when the cells were cultured on a non-oxygen-releasing PTMC controls. This indicates, that besides oxygen, other factors are likely to be of importance in determining cell viability upon implantation. A small study was performed to assess the effect of the presence of several compounds in the medium (growth factors, glutamine, glucose) on cell viability during culturing under hypoxic conditions. Clear conclusions could not be drawn, and more research is needed, but it was hypothesised that the composition of the medium is of essential influence in reaching high viabilities of the cells, even under normoxic circumstances.
Summary
