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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General discussion
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Although tissue engineering could be a panacea, only a limited number of tissues can be engineered after more than two decades of research: bladder, skin and cartilage1. While most other tissues depend on vascularization for the supply
of nutrients, oxygen and the removal of waste products, the vascularization of these tissues is limited. In general, the lack of vascularization in tissue engineered constructs (biomaterial scaffolds seeded with cells) of clinically relevant sizes will result in the death of the seeded cells within days of implantation. Because healing of the surgical wound in which the implant is placed, occurs in an initially anoxic and hostile environment, cells of well-vascularized tissues like bone and muscle cannot survive for long times without oxygen and nutrients. Only viable cells are expected to contribute to tissue repair.
It was hypothesized that use of an oxygen-delivering biomaterial would allow sufficient time for the vascularization to develop, while ensuring the viability of the implanted cells. While hypoxia is known to induce angiogenesis2, a too high extent
of hypoxia might induce cell death. Therefore, the amount of oxygen released from the oxygen-delivering composite biomaterials into the wound bed should be sufficient for the cells to stay metabolically active and at the same time not hinder angiogenesis. In the studies described in this thesis, steps were taken to create an oxygen-releasing biomaterial that enhances the viability of cells in a hypoxic environment.
First experiments were conducted using composite materials prepared from poly(lactide) and poly(lactide-co-glycolide) polymers and CaO2 particles. In
preliminary studies as well as literature data, the optimal amount of peroxide particles in the composite was found to be approximately 5% (w/w polymer). It was found using these materials that the period during which oxygen was released was relatively short, and that the rate of oxygen release was relatively high. These polymers degrade hydrolytically in a bulk degradation process, and the acidic
General discussion
When using poly(trimethylene carbonate) (PTMC) as a matrix in the oxygen-releasing composite, it was observed that oxygen was released at lower rates over prolonged periods of time. In contrast to the lactide polymers, PTMC is a polymer that degrades enzymatically by a surface erosion process3. The release rate of
oxygen was found to depend on the presence of cholesterol esterase, one of the enzymes that erodes PTMC. As the hydrophobic polymer matrix erodes at the surface, the CaO2 particles are exposed to water upon which the formed oxygen
is slowly released.
These PTMC/CaO2 composites were applied in the form of films and
microspheres. While (porous) films can be applied as tissue engineering scaffolds themselves, it should also be possible to prepare tissue engineering scaffolds from these oxygen-delivering composite materials by extrusion-based 3D printing. Then scaffolds with specific surface topographies that induce cell differentiation could be obtained4. Microspheres can be perfused into tissue engineering scaffolds prepared
from other materials to enhance cell survival upon their implantation. An interesting approach in bone tissue engineering would for example be to combine bone-inducing calcium phosphate scaffolds with these oxygen-delivering microspheres. As contact with water leads to a premature reaction with CaO2, the
preparation of oxygen-delivering composite microspheres required the development of a novel method that did not involve the use of water.
The oxygen-delivering PTMC/CaO2 microspheres themselves might prove
useful in in vivo situations. We made use of a mouse skin flap model to create an ischemic situation and investigated the effect of the presence of the composite microspheres on tissue necrosis. Upon injection of the oxygen-delivering microspheres, we could observe that significantly less necrosis had occurred in comparison to the control experiments that did not contain oxygen-delivering microspheres. The timespan of this experiment was relatively short (10 days) and revascularization was not observed. It would be interesting to extend the duration of the experiment, as revascularization of a tissue engineering scaffold of clinically useful size could require weeks5. Furthermore, as the release of oxygen depends
on the enzymatic induced degradation of PTMC, the site of implantation of the microspheres might affect their oxygen release behavior. Longer term studies to assess the degradation characteristics and erosion behavior of the composite at
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different implantation sites, as well as their biocompatibility, must be conducted before steps towards clinical models are taken.
While the viability of cells and tissues in vivo was enhanced upon implantation of the oxygen-delivering composites in the mouse skin flap model, the effect of the composites on hMSCs and Saos-2 cells in vitro using a hypoxic cell culture incubator was not that evident, due to the fact that cells in control conditions did not die in the induced ischemic conditions. This demonstrates the complexity of mimicking necrosis in the here used ischemic cell culture model. In vitro the effect of hypoxia appeared to be very limited while in vivo the detrimental effect of ischemia is high. This indicates that although lack of oxygen is an important factor in ischemia, it may not be the only direct reason for cell death.
Nutrients, other than oxygen, may play important roles as well. In most cases cell-scaffold constructs will be implanted in a wound bed filled with coagulating blood. In first instance the blood cloth is expected to provide sufficient nutrients. As inflammation during wound healing will proceed, the implanted cells will experience a hostile environment with many inflammatory cytokines. Waste products, which are not removed due to lack of vascularization, will toxify the cells. Such conditions were not present in the here-used cell culture model. Better in
vitro models are required and may include closed vessels without changes of culture
medium. The culture medium should also be supplemented with platelet-rich plasma instead of fetal bovine serum. Although these factors will make the culture model more complicated, it will also be like the in vivo situation. More research will be required to develop a functional ischemic cell culture model.
Finally, it would be interesting to study the effects of addition of oxygen to processes which normally proceed in anoxic conditions, such as wound healing. In the human being wound healing results in scar formation. This particularly becomes an issue in burn-wound patients, in whom scars result in severe contractions. The
General discussion
of oxygen ultimately will result in more or less fibrotic scar tissue may be an interesting research question.
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References
1. Orlando, G. et al. Regenerative medicine and organ transplantation: past, present, and future. Transplantation 91, 1310–7 (2011).
2. Kim, K.-R., Moon, H.-E. & Kim, K.-W. Hypoxia-induced angiogenesis in human hepatocellular carcinoma. J. Mol. Med. (Berl). 80, 703–14 (2002).
3. Zhang, Z., Grijpma, D. W. & Feijen, J. Triblock Copolymers Based on 1,3-Trimethylene Carbonate and Lactide as Biodegradable Thermoplastic Elastomers. Macromol. Chem. Phys.
205, 867–875 (2004).
4. Papenburg, B. J. et al. One-step fabrication of porous micropatterned scaffolds to control cell behavior. Biomaterials 28, 1998–2009 (2007).
5. Stokes, C. L., Lauffenburger, D. a & Williams, S. K. Migration of individual microvessel endothelial cells: stochastic model and parameter measurement. J Cell Sci 99 ( Pt 2), 419–30 (1991).
