Towards in vivo application of oxygen-releasing microspheres for enhancing bone
regeneration
Buizer, Arina
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2018
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Buizer, A. (2018). Towards in vivo application of oxygen-releasing microspheres for enhancing bone regeneration. Rijksuniversiteit Groningen.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
Chapter 7
7
General discussion
Recapitulation
For reconstruction of bone tissue, the golden standard at this moment is the use of autograft bone material1–4. This is an osteogenic, osteoconductive and osteoinductive
material. However, harvesting autologous bone often results in donor site morbidity and it is available in limited amounts1–4. A possible solution for this problem is to
regenerate bone tissue on site. To regenerate bone tissue, at this moment usually a scaffold material seeded with cells that have the capacity to form bone, is used2,3.
Human mesenchymal stem cells (hMSCs) are easy to harvest in fair amounts and have the capacity to form bone, so therefore this type of cell is frequently used for regeneration of bone tissue5–7. At the moment of implantation of a cell-scaffold
complex, no vascularization is present within the scaffold. The maximum diffusion distance of oxygen is limited to about 200 micrometers, so mainly in large scaffolds, cells seeded in the center of the scaffold are exposed to very low oxygen circumstances8–10.
Therefore, the cells especially in the center of the scaffold do not survive and no bone tissue is formed. To support cell survival in large cell-scaffold complexes, we proposed the use of oxygen-releasing microspheres. The aim of the microspheres is to provide oxygen to scaffolded cells for a few weeks. This serves two goals. Firstly, oxygen provision leads to longer survival of scaffolded cells, and thus leads to more time for the development of a vascular network within the scaffold. Cell death in scaffolded cells due to ischemia may thus be postponed or even be prevented. Secondly, when cells are exposed to ischemia, they start producing angiogenic factors (AGF), which stimulate the ingrowth of blood vessels11. Whenever a vascular network has been
developed within the scaffold, sufficient oxygen can be transported to the cells and thus a sustainable oxygen supply is established. If the microspheres release enough oxygen for cells to survive, yet not so much that cells cease the production of AGF, improved cell survival in the initial phase after implantation and a the creation of a sustained oxygen supply in the weeks after implantation may be accomplished. To optimize bone growth within a scaffold, it is important that cells are seeded on a scaffold homogeneously without damaging the cells during the cell seeding process12,13. Tricalcium phosphate carrier materials are very frequently used materials
for bone regeneration at this moment. In chapter 2, we tested which cell seeding method is optimal for low porosity (45% porosity) as well as high porosity (90% porosity) tricalcium phosphate. For low porosity scaffolds, a vacuum cell seeding method resulted in higher numbers of adherent cells to the scaffold material than a static cell seeding method. However, on high porosity scaffolds, a static cell seeding
method resulted in more homogeneous cell seeding compared to the vacuum seeding method.
As previously mentioned, hMSCs are frequently used for seeding a scaffold meant to regenerate bone tissue. Especially, bone marrow-derived MSC (BM-MSC) have been studied extensively, as they are easy to harvest and they show high osteogenic capacity14. BM-MSC show low cell survival upon exposure to prolonged ischemic
circumstances15,16. It is known that these cells start producing AGF whenever they
reside under hypoxia11. These AGF stimulate the ingrowth of blood vessels, so that a
sustainable oxygen supply is accomplished. Ideally, exposure of hMSCs to an oxygen percentage that is high enough to sustain cell survival, yet induces AGF production at the same time, would contribute greatly to the optimization of bone regeneration. Our study showed that the oxygen range at which hMSCs in vitro have sufficient oxygen to survive yet produce AGF at the same time is 1-2% oxygen.
The next step was to develop a method to produce oxygen-releasing microspheres out of poly (1,3-trimethylene carbonate) (PTMC) and calcium peroxide (CaO2). An oil-in-oil solvent evaporation method was developed and is described in chapter 4. The microsphere oxygen release was tested in vitro. PTMC is metabolized through enzymatic degradation using cholesterol esterase17. Upon exposure to simulated body
fluid alone, very little oxygen was released. However, upon exposure to simulated body fluid supplemented with cholesterol esterase, PTMC- CaO2 microspheres could release oxygen for about 20 days. The microspheres were added to cell cultures of hMSCs and appeared not to be cytotoxic. The addition of PTMC-CaO2 microspheres to hMSCs cultured under hypoxic circumstances (0,1% oxygen) led to higher cell metabolic activity than the addition of PTMC microspheres that do not release oxygen. This finding suggests that the oxygen-releasing microspheres may support the survival of hMSCs cultured under hypoxic circumstances.
The following step was to explore the biocompatibility of the oxygen-releasing microspheres in vitro and in vivo. In biocompatibility tests in vitro, cell survival was decreased upon exposure of L929 cells to high dosages of oxygen-releasing microspheres, while exposure to lower dosages was tolerated well. In biocompatibility tests in vivo, the oxygen-releasing PTMC microspheres were tolerated well. In conclusion, the oxygen-releasing PTMC microspheres are biocompatible.
As a proof of concept the oxygen-releasing microspheres were implanted in mice in a random pattern skin flap model. In such a skin flap an ischemic gradient is created, with the most ischemic part being the distal part of the flap, while the basis of the flap is the least ischemic. Without intervention, the skin tissue becomes necrotic.
7
Initially, only the distal part of the skin flap will necrotize, but in a few days necrosis will progress towards the basis of the skin flap18,19. When PTMC-CaO
2 microspheres were
implanted under such a skin flap, the skin flap became less necrotic than skin flaps under which PTMC microspheres (not releasing oxygen) were implanted. Especially in the first 3-7 days after surgery this difference was striking. At 10 days after surgery the difference was still significant, but smaller than at 3 and 7 days. These findings may suggest that the application of oxygen-releasing microspheres may aid to cell survival in otherwise ischemic skin tissue. In vitro, the oxygen release from the oxygen-releasing microspheres gets exhausted after more than three weeks. However, the kinetics of oxygen release from the microspheres in vivo is yet unknown, and may be implantation site dependent. Nevertheless, the observation that the difference in amount of ischemia is smaller after 10 days may suggest that the oxygen release from the microspheres gets exhausted after 10 days.
In the following section, the research questions posed in the introduction of this thesis will be discussed.
Which method for seeding cells on ceramic biomaterials
provides optimal cell distribution throughout the scaffold,
cell seeding efficiency and cell proliferation after seeding?
An important factor in determining the success of bone tissue regeneration on a scaffold material is the technique that was used to seed cells on the scaffold material. Homogeneous distribution of the cells throughout the scaffold results in more equal tissue regeneration throughout the scaffold. Furthermore, an optimal cell seeding technique should not damage the cells and should be efficient, so that as many seeded cells as possible contribute to tissue regeneration12,13. Especially cell seeding
methods that are to be used in clinical practice should be easy to use, considering the broad range of skill of the people using these techniques. The ultimate use of the microspheres that were studied in this thesis is in a point-of-care setting, which implies that cells are harvested and seeded on a biomaterial in one operative session. In this case, the amount of time needed to seed cells is an important factor, too. Preferably, the costs of a cell seeding method should be low.
The presently available cell seeding techniques can be classified in two groups: static and dynamic cell seeding. For static cell seeding, no external force is used to seed cells on a scaffold, while dynamic cell seeding implies the use of an external force to seed cells on a scaffold. Examples of static cell seeding are mixing of a cell suspension with a scaffold material in a basin, or pipetting a cell suspension on top of a scaffold. Dynamic
cell seeding can be done for instance through the use of a vacuum force or a positive pressure source to force cells into a scaffold or through the use of a bioreactor in which a cell suspension flows through a scaffold constantly12. Each cell seeding method
has its own advantages and disadvantages. Generally speaking, passive cell seeding methods are easy to use, there is little damage to the cells during the seeding process and they are cheap. However, penetration of cells in the center of a scaffold may be less12. Active cell seeding methods often result in better penetration of cells within a
scaffold and result more often in homogeneous cell seeding than passive cell seeding methods do. Disadvantages of active cell seeding methods include their often lower ease of use and the higher time consumption12.
At this moment there is no consensus on which cell seeding method is the best. Which method is the best also depends on the setting in which it is used. For a point-of-care setting, as is intended for the oxygen-releasing microspheres, a method that takes a lot of time or that is not ‘fool proof’ is not feasible. In our study, it was shown that one method may be optimal for one type of scaffold material, while another method may be better for another type of scaffold material. Therefore, it is recommended to optimize the cell seeding method per scaffold material and per type of application of a scaffold material.
Which oxygen level is optimal for hMSC proliferation and
angiogenic factor production?
As stated before, upon implantation of a cell-seeded scaffold in the body, no adequate vascularization is present within the scaffold. Such scaffolds are implanted in an unfavorable environment, with diminished blood supply. The blood supply, and thus the supply of oxygen, glucose, and growth factors, to the cells seeded on the scaffold are marginal. Prolonged exposure of MSCs to a hypoxic environment results in lower cell proliferation11,15. Tissue regeneration through MSCs seeded on a scaffold and
exposed to a hypoxic environment will thus be disturbed.
A more important effect of the deprivation of blood supply to cells seeded on a scaffold is the diminished glucose supply20. Within cells, adenosine triphosphate (ATP) serves
as the main energy source and is essential for all kinds of metabolic processes. Without ATP, cells do not survive. Oxidative phosphorylation is the standard process of ATP generation within cells in the presence of oxygen. Metabolization of one molecule of glucose through oxidative phosphorylation yields 36 ATP, while anaerobic metabolism of one molecule of glucose, glycolysis, yields only 2 ATP per molecule of glucose21.
7
oxygen and under hypoxic circumstances the presence of glucose is essential to cell survival16,22,23.
A second effect of exposure of MSCs to a hypoxic environment is that the cells start transcribing angiogenic factors (AGF)24. AGF are substances that promote the ingrowth
of blood vessels, so that a sustainable blood supply to the cells is constructed. An endurable supply of oxygen and glucose, but also other factors of great importance to cell survival and cell proliferation, is thus created, as well as endurable waste removal. When MSCs are exposed to oxygen percentages that are normoxic or hyperoxic to them, they no longer have these proangiogenic properties24. In this thesis, the focus is
on oxygen supply for reasons of surveyability. However, other factors, such as glucose, are of great importance for effective tissue regeneration on scaffolds too.
MSCs have been used in this research project because of their osteogenic potential and their trophic effects, supporting the attraction of regenerative cells. Cell sources that have the same capacities could be stem cells derived from prenatal or natal tissues, or induced stem cells. However, in contrast to these types of cells, MSCs are readily available in several types of postnatal tissues. Since the purpose of the oxygen-releasing microspheres is to use them in a single operative session, in which bone forming cells are harvested, seeded on a scaffold and implanted in the body, a point-of care isolation method for bone-forming cells was demanded. At the moment the research project started, such point-of-care MSC isolation methods were only available for bone marrow-derived MSCs. Besides that, bone marrow-derived MSCs are well-studied and they naturally take part in the bone growth and regeneration process, which is why this MSC source was elected for these studies. Recently, point-of-care MSC isolation methods for adipose tissue have become available25. The higher
MSC yield from adipose tissue than from bone marrow26 and the availability of a
point-of-care MSC isolation method makes adipose tissue-derived MSCs an attractive alternative cell source for bone regeneration. Adipose-derived MSCs naturally reside in a different oxygen level than bone marrow-derived MSCs do27,28. The oxygen level
in which stem cells naturally reside can be of influence on their osteogenic capacities, but also on the expression of AGF and the oxygen level at which MSCs start expressing AGF29–32. This AGF expression profile is important to know, since it influences the
dosage of the oxygen-releasing microspheres if they are applied in a clinical situation. Therefore, before other MSC sources than bone marrow are eligible for clinical use, the AGF release profile should be investigated separately.
One of the aims of this research project was to create microspheres that release just enough oxygen for MSCs to survive prolonged exposure to hypoxia, yet still transcribe
AGF. If too much oxygen would be supplied by the microspheres, AGF transcription will cease. If a too low oxygen level is supplied to the cells, cell proliferation is lowered and may ultimately cease. Tissue regeneration is ineffective in such a situation. In in vitro tests, the oxygen ranges at which bone marrow-derived hMSCs proliferate yet transcribe AGF as well appeared to be 1-2% oxygen. This has not been tested in vivo, as it is difficult to tailor the oxygen supply to cells in an in vivo situation to research needs. It may very well be that the oxygen range at which MSCs proliferate and transcribe AGF is different in vivo than in vitro. In bone marrow, which is the natural habitat of the cells used in this research project, the oxygen level is around 1%33. It is
therefore not unexpected that bone marrow-derived MSCs thrive well at 1% oxygen. The bone marrow-derived hMSCs that were used for these experiments were isolated under exposure to 21% oxygen.
Oxygen level, among other factors, is of great influence of MSC proliferation and angiogenic profile. It is thus recommended to consider the oxygen circumstances in experiments investigating MSC proliferation and / or angiogenic profile. As exposure to 1% oxygen is natural to bone marrow-derived MSCs, this is a more rational choice of oxygen level than is 21%.
How can oxygen-releasing microspheres be produced out of
a polymer carrier material and a peroxide material?
Several methods for improving oxygen delivery in a scaffold material have been proposed. These methods are to be categorized in three groups34. The first group is
the application of hyperbaric oxygen. Upon exposure to hyperbaric oxygen tissue oxygenation rises, and the aim is thus to increase the oxygenation of cells seeded on a scaffold material. However, as vascularization is usually absent within a cell-seeded scaffold, increased oxygenation in the center of the scaffold is unlikely to occur. Furthermore, patients need intensive hyperbaric oxygen therapy after implantation of a cell-seeded scaffold, which costs a lot of time, resources, specialized equipment and money. Besides that, hyperbaric oxygen therapy may have serious side effects. The second method for improving oxygen delivery to scaffolded cells is the use of oxygen carriers. These are molecules to which oxygen is bound. The oxygen is released in tissues with a low oxygen concentration. Examples of these molecules are hemoglobin or perfluorocarbons. The great benefit of this type of oxygen delivery is that oxygen is delivered site-specifically, so only in ischemic areas. However, oxygen delivery using this type of materials is only short-lived34. Blood vessel ingrowth occurs at a growth
rate of less than a millimeter per day35–37, theoretically it takes about 10 days until
7
accomplish full vascularization. Mikos et al38 implanted 5 mm thick porous polymer
disks, measuring 13,5 mm diameter in well-vascularized rat mesentery. To reach high vascularization throughout the disks, it took up to 35 days. Clearly, for oxygen delivering biomaterials to be effective, several days to weeks of oxygen delivery is essential. The third method for improving oxygen delivery to scaffolded cells is the use of oxygen generating biomaterials. The application of peroxides in combination with a polymer is the most frequently studied39. Calcium peroxide, magnesium peroxide
and sodium percarbonate are among the most frequently studied peroxides used for oxygen generation40. Calcium peroxide reacts with water relatively quickly, it is
available in high purity, it is inexpensive, well studied, so there is a predilection for this peroxide. Peroxides exposed to water react and form hydrogen peroxide, as shown for calcium peroxide in the equation below40.
CaO2 + 2 H2O → Ca (OH)2 + H2O2
In the subsequent reaction of 2 molecules of hydrogen peroxide oxygen is generated, as shown in the following equation40:
2 H2O2 → O2 + 2 H2O
Gradual release of oxygen from peroxides is aimed for by incorporating peroxides in polymer materials that are degraded slowly39. Through consideration of the
degradation characteristics of the polymer carrier material, the oxygen-release profile of the thus created biomaterial can be customized to the specific needs. This will be elaborated further in this chapter. A major disadvantage of the use of peroxides for oxygen generation is that as an intermediate product of the reaction hydrogen peroxide is formed. This is a reactive oxygen species (ROS), which are cytotoxic34. The
enzyme catalase, which is available in most mammalian cells, catalyzes the degradation of hydrogen peroxide into oxygen and water39. Through implementation of catalase
in peroxide-based oxygen generating systems, the cytotoxic influence of hydrogen peroxide may be minimized34,39.
The application of small oxygen generating microspheres that are dispersed throughout a scaffold would theoretically result in oxygen generation that is evenly throughout a scaffold material. Furthermore, microspheres provide users with the benefit of being able to add them to any type of scaffold and location at will. Several methods for producing microspheres are available, such as emulsification, photolithography, microfluidic systems and micromolding41. For the production of
peroxide-based oxygen generating microspheres a production method that is not water-based is important. Exposure of peroxides to water during the production
process will lead to preliminary reaction of the peroxides with water. Thus, oxygen generation from the microspheres upon implantation will be reduced or even absent. By using an oil-in-oil solvent evaporation method, the exposure of calcium peroxide to water is avoided during the microsphere production process42. This production
method was used successfully for producing oxygen generating microspheres, as was shown in chapter four.
How is the oxygen release profile of the thus produced
oxygen-releasing microspheres?
Upon selection of a polymer carrier material for a peroxide, knowledge of its degradation behavior is important. Many factors influence the degradation behavior of a polymer, amongst which are its hydrophobic or hydrophilic nature or the enzymatic composition and temperature at the degradation site. A second factor to take into account upon selection of a polymer is the nature of its degradation products. For example, degradation products of poly (lactic acid) are acidic, which is unfavorable to cell survival. PTMC does not produce acidic degradation products43. Polymer
degradation products may also serve as nutrients to cells. Obviously, a polymer that produces degradation products that may serve as cell nutrients is preferable above a polymer that degrades into cytotoxic products.
PTMC is degraded enzymatically. The enzyme cholesterol esterase, present in macrophages, plays an important role in PTMC degradation17. This was confirmed
in our study. Because of this enzymatic degradation behavior, the polymer does not degrade before it is in contact with the enzyme, for example when it is still packaged. The type of polymer erosion is of influence on the oxygen release profile in polymer-based oxygen generating biomaterials. For example, if peroxide is dispersed in a polymer carrier that degrades through bulk erosion, oxygen will be released in bulk as well. PTMC is eroded through surface erosion, which means the material is degraded in a layer-by-layer fashion. As of this type of erosion, gradual exposure of calcium peroxide dispersed within the polymer is accomplished. Considering the PTMC degradation characteristics described here, PTMC seems a favorable peroxide carrier material. In in vitro tests, PTMC-CaO2microspheres released oxygen up to three weeks in the presence of cholesterol esterase. Several research groups have tested oxygen generating biomaterials composed of poly (lactic-co-glycolic acid) (PLGA) and peroxides in different combinations. Oxygen release varied from several hours up to at least two weeks44–48. Composites of poly (lactic acid) (PLA) and calcium peroxide
released oxygen only for a few days44. The combination of polycaprolactone with
7
composed disks, based on polydimethylsiloxane (PDMS) and calcium peroxide, that released oxygen for more than 6 weeks. However, PDMS is not biodegradable, so removal from the body may be needed after use of this biomaterial34.
To accomplish release of oxygen from a polymer carrier material according to a specific release profile, it is important to consider the polymer properties and degradation profile so that they are specific to the demands.
Are the oxygen-releasing microspheres biocompatible in
vitro and in vivo?
In the human body should only biocompatible materials be implanted. Biocompatibility has been defined as ‘The ability to be in contact with a living system without producing an adverse effects’51. The International Organization for Standardization (ISO) has
set ISO standard 10993 regarding the biological evaluation of medical devices. ISO standards 10993-5 and 10993-6 describe tests for in vitro biocompatibility and tests for local effects after implantation, respectively, and provide a worldwide consensus52,53.
The biocompatibility tests of PTMC-CaO2 microspheres were based on these standards. In in vitro tests, the microspheres appeared to be biocompatible if not dosed too high. In in vivo tests, the PTMC-CaO2 microspheres appeared to be biocompatible.
Does the application of oxygen-releasing microspheres
result in improved tissue survival in otherwise ischemic
tissue in vivo?
Several researchers investigated the effect of an oxygen delivering material on tissue survival using a devascularized skin flap model39. Harrison et al45 implanted
PLGA-sodium percarbonate (PLGA-SPO) films under random pattern devascularized skin flaps in mice. Three days after implantation, skin flaps under which the oxygen-releasing material had been implanted showed decreased tissue necrosis than skin flaps under which non-oxygen-releasing material had been implanted. However, at seven days after implantation, there was no difference between both materials. Chandra et al49 composed a four-layer wound dressing, of which the second layer
was composed of polycaprolactone, polyvinyl alcohol, and calcium peroxide and sodium percarbonate. This wound dressing was applied in a 10 x 10 cm skin defect in pigs. During the eight-week study period the wounds in which the oxygen-releasing dressing was applied showed significantly higher epithelization, better vascularization and smaller wound size than in wounds in which a non-oxygen-releasing dressing was applied. The PTMC-CaO2 microspheres studied in this thesis were implanted under a random pattern devascularized skin flap in mice. At 3, 7 and 10 days the skin flaps
under which oxygen-releasing microspheres had been implanted showed significantly less skin necrosis than the skin flaps under which non-oxygen-releasing microspheres had been implanted. This may indicate that the oxygen released by the PTMC-CaO2 microspheres supports tissue survival in otherwise ischemic tissue.
General remarks
In conclusion, PTMC-CaO2 microspheres that release oxygen for about three weeks in vitro have been produced using an oil-in-oil solvent evaporation method. These microspheres are biocompatible and they aid in tissue survival in otherwise ischemic tissue.
It is unknown if the supportive effect of the oxygen-releasing microspheres only applies to skin tissue. It would therefore be interesting to investigate if the effect also applies to other tissues, for example in a critical size bone defect.
There are many applications of oxygen-releasing microspheres that can be thought of. Diabetic or ischemic ulcers are wounds that are hard to treat. An important cause of this is diminished blood supply to these wounds. Perhaps oxygen-releasing biomaterials can support the healing of these types of wounds. In plastic surgery, skin flaps are frequently used to fill skin and subcutaneous tissue defects. A big part of the natural vascularization of these skin flaps needs to be removed to be able to place them in the defect. Flap necrosis is thus a frequently occurring complication, and maybe the addition of oxygen-releasing biomaterials may aid in skin flap survival. Cardiac muscle tissue is very sensitive to ischemia. For saving as much cardiac muscle tissue after a myocardial infarction the application of oxygen-releasing biomaterials may be supportive as well. Oxygen-releasing biomaterials may also be applied in the field of maxillofacial surgery; sinus floor augmentation frequently needs to be performed in patients with insufficient maxillar bone stock for implant placement. At this moment, autologous bone often needs to be harvested for sinus augmentation. This is a procedure that is not without complications, leads to patient discomfort, and costs extra time, resources and money. Artificial bone replacement materials are not very successful, partly due to diminished blood supply to bone precursor cells seeded on these materials. The PTMC-CaO2 microspheres may support cell survival on these materials.
In this research project, the focus was on oxygen suppletion. However, when cells are exposed to diminished blood supply, the supply of other nutrients and growth factors and the removal of waste products is also diminished. This is of great influence on cell metabolism as well and thus influences the tissue regeneration success as well.
7
There is a very complex interplay of nutrients, growth factors and signaling factors that decides cell fate, and perhaps it is an illusion to expect that all factors influencing cell fate will ever be completely understood. However, even unraveling part of the process may aid in improving tissue regeneration. The oxygen-releasing microspheres may provide ischemic cells with a ‘lunch box’ filled with oxygen, but in the future, this ‘lunch box’ may be filled with extra nutrients or growth factors.
The use of other carrier materials than PTMC could be considered in future research. For as far is known, the PTMC degradation products do not provide nutrition to the cells. Perhaps other carrier materials could provide more functional degradation products. For example, polysaccharides or starches may be an appropriate carrier material as well. Specific polysaccharides or starches may be metabolized into glucose within the body, thus providing a slow release as well as a nutrient system for the cells in one. Obviously, development of such materials will have to start from the lab bench again, studying the production process, the oxygen release profile, the in vitro and in vivo biocompatibility and the in vitro and in vivo applicability of the material.
Reference list
1. Calori GM, Mazza E, Colombo M, Ripamonti C. The use of bone-graft substitutes in large bone defects: Any specific needs? Injury 2011; 42: S56–S63.
2. Campana V, Milano G, Pagano E, Barba M, Cicione C, Salonna G, Lattanzi W, Logroscino G. Bone substitutes in orthopaedic surgery: from basic science to clinical practice. J Mater Sci Mater Med 2014; 25: 2445–2461.
3. Fillingham Y, Jacobs J. Bone grafts and their substitutes. Bone Joint J 2016; 98–B: 6–9.
4. García-Gareta E, Coathup MJ, Blunn GW. Osteoinduction of bone grafting materials for bone repair and regeneration. Bone 2015; 81: 112–121.
5. Chanda D, Kumar S, Ponnazhagan S. Therapeutic potential of adult bone marrow-derived mesenchymal stem cells in diseases of the skeleton. J Cell Biochem 2010; 111: 249–257.
6. Fishero BA, Kohli N, Das A, Cristophel JJ, Cui Q. Current concepts of bone tissue engineering for craniofacial bone defect repair. Craniomaxillofac Trauma Reconstr 2015; 8: 23–30.
7. Szpalski C, Barbaro M, Sagebin F, Warren SM. Bone tissue engineering: current strategies and techniques—Part II: Cell types. Tissue Eng Part B Rev 2012; 18: 258–269.
8. Ma J, Both SK, Yang F, Cui F-Z, Pan J, Meijer GJ, Jansen JA, van den Beucken JJJP. Concise review: cell-based strategies in bone tissue engineering and regenerative medicine. Stem Cells Transl Med 2014; 3: 98–107.
9. Jain RK, Au P, Tam J, Duda DG, Fukumura D. Engineering vascularized tissue. Nat Biotechnol 2005; 23: 821–823.
10. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000; 6: 389–395.
11. Potier E, Ferreira E, Andriamanalijaona R, Pujol J-P, Oudina K, Logeart-Avramoglou D, Petite H. Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression. Bone 2007; 40: 1078–1087.
12. Tan L, Ren Y, Kuijer R. A 1-min method for homogenous cell seeding in porous scaffolds. J Biomater Appl 2012; 26: 877–889.
13. Yoshii T, Sotome S, Torigoe I, Tsuchiya A, Maehara H, Ichinose S, Shinomiya K. Fresh bone marrow introduction into porous scaffolds using a simple low-pressure loading method for effective osteogenesis in a rabbit model. J Orthop Res 2009; 27: 1–7.
14. Yousefi AM, James PF, Akbarzadeh R, Subramanian A, Flavin C, Oudadesse H. Prospect of stem cells in bone tissue engineering: A review. Stem Cells Int 2016; epub ahead of print.
15. Holzwarth C, Vaegler M, Gieseke F, Pfister SM, Handgretinger R, Kerst G, Müller I. Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells. BMC Cell Biol 2010; 11: 11.
16. Potier E, Ferreira E, Meunier A, Sedel L, Logeart-Avramoglou D, Petite H. Prolonged hypoxia concomitant with serum deprivation induces massive human mesenchymal stem cell death. Tissue Eng 2007; 13: 1325–1331.
17. Bat E, van Kooten TG, Feijen J, Grijpma DW. Macrophage-mediated erosion of gamma irradiated poly (trimethylene carbonate) films. Biomaterials 2009; 30: 3652–3661.
18. Galla TJ, Saetzler RK-E, Hammersen F, Messmer K. Increase in skin-flap survival by the vasoactive drug Buflomedil. Plast Reconstr Surg 1991; 87: 130-136.
7
19. Park S, Tepper OM, Galiano RD, Capla JM, Baharestani S, Kleinman ME, Pelo CR, Levine JP, Gurtner GC. Selective recruitment of endothelial progenitor cells to ischemic tissues with increased neovascularization. Plast Reconstr Surg 2004; 113: 284–293.
20. Kim DS, Ko YJ, Lee MW, Park HJ, Park Yj, Kim D-I, Sung KW, Koo HH, Yoo KH. Effect of low oxygen tension on the biological characteristics of human bone marrow mesenchymal stem cells. Cell Stress Chaperones 2016; 21: 1089–1099.
21. Liu Y, Ma T. Metabolic regulation of mesenchymal stem cell in expansion and therapeutic application. Biotechnol Prog 2015; 31: 468–481.
22. Deschepper M, Manassero M, Oudina K, Paquet J, Monfoulet L-E, Bensidhoum M, Logeart-Avramoglou D, Petite H. Proangiogenic and prosurvival functions of glucose in human mesenchymal stem cells upon transplantation. Stem Cells 2013; 31: 526-535.
23. Zhu W, Chen J, Cong X, Hu S, Chen X. Hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells. Stem Cells 2006; 24: 416–425.
24. Paquet J, Deschepper M, Moya A, Logeart-Avramoglou D, Boisson-Vidal C, Petite H. Oxygen tension regulates human mesenchymal stem cell paracrine functions. Stem Cells Transl Med 2015; 4: 809–821. 25. van Dongen JA, Tuin AJ, Spiekman M, Jansma J, van der Lei B, Harmsen MC. Comparison of
intraoperative procedures for isolation of clinical grade stromal vascular fraction for regenerative purposes: a systematic review. J Tissue Eng Regen Med 2017; epub ahead of print.
26. Strioga M, Viswanathan S, Darinskas A, Slaby O, Michalek J. Same or not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells. Stem Cells Dev 2012; 21: 2724–2752.
27. Carreau A, Hafny-Rahbi B El, Matejuk A, Grillon C, Kieda C. Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J Cell Mol Med 2011; 15: 1239–1253. 28. Mohyeldin A, Garzón-Muvdi T, Quiñones-Hinojosa A. Oxygen in stem cell biology: A critical component
of the stem cell niche. Cell Stem Cell 2010; 7: 150–161.
29. Du WJ, Chi Y, Yang ZX, Li ZJ, Cui JJ, Song BQ, Li X, Yang SG, Han ZB, Han ZC. Heterogeneity of proangiogenic features in mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, and placenta. Stem Cell Res Ther 2016; 7: 163-173.
30. Hsiao ST-F, Asgari A, Lokmic Z, Sinclair R, Dusting GJ, Lim SY, Dilley RJ. Comparative analysis of paracrine factor expression in human adult mesenchymal stem cells derived from bone marrow, adipose, and dermal tissue. Stem Cells Dev 2012; 21: 2189–2203.
31. Dionigi B, Ahmed A, Pennington EC, Zurakowski D, Fauza DO. A comparative analysis of human mesenchymal stem cell response to hypoxia in vitro: Implications to translational strategies. J Pediatr Surg 2014; 49: 915–918.
32. Dmitrieva RI, Minullina IR, Bilibina AA, Tarasova OV, Anisimov SV, Zaritskey AY. Bone marrow- and subcutaneous adipose tissue-derived mesenchymal stem cells: differences and similarities. Cell Cycle 2012; 11: 377–383.
33. Heppenstall RB, Grislis G, Hunt TK. Tissue gas tensions and oxygen consumption in healing bone defects. Clin Orthop Relat Res 1975; (106): 357–365.
34. Farris A, Rindone A, Grayson W. Oxygen delivering biomaterials for tissue engineering. J Mater Chem B 2016; 4: 3422-3432.
35. Stokes CL, Lauffenburger DA, Williams SK. Migration of individual microvessel endothelial cells: stochastic model and parameter measurement. J Cell Sci 1991; 99: 419–430.
36. Santos MI, Reis RL. Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges. Macromol Biosci 2010; 10: 12–27.
37. Nomi M, Atala A, De Coppi P, Soker S. Principals of neovascularization for tissue engineering. Mol Aspects Med 2002; 23: 463–483.
38. Mikos AG, Sarakinos G, Lyman MD, Ingber DE, Vacanti JP, Langer R. Prevascularization of porous biodegradable polymers. Biotechnol Bioeng 1993; 42: 716–723.
39. Gholipourmalekabadi M, Zhao S, Harrison BS, Mozafari M, Seifalian AM. Oxygen-generating biomaterials: A new, viable paradigm for tissue engineering? Trends Biotechnol 2016; 34: 1010–1021. 40. Camci-Unal G, Alemdar N, Annabi N, Khademhosseini A. Oxygen releasing biomaterials for tissue
engineering. Polym Int 2013; 62: 843–848.
41. Khademhosseini A, Langer R. Microengineered hydrogels for tissue engineering. Biomaterials 2007; 28: 5087–5092.
42. Uchida T, Yagi A, Oda Y, Goto S. Microencapsulation of ovalbumin in poly (lactide-co-glycolide) by an oil-in-oil (o/o) solvent evaporation method. J Microencapsul 1996; 13: 509–518.
43. Zhang Z, Kuijer R, Bulstra SK, Grijpma DW, Feijen J. The in vivo and in vitro degradation behavior of poly (trimethylene carbonate). Biomaterials 2006; 27: 1741–1748.
44. Steg H, Buizer AT, Woudstra W, Veldhuizen AG, Bulstra SK, Grijpma DW, Kuijer R. Control of oxygen release from peroxides using polymers. J Mater Sci Mater Med 2015; 26: 207-210.
45. Harrison BS, Eberli D, Lee SJ, Atala A, Yoo JJ. Oxygen producing biomaterials for tissue regeneration. Biomaterials 2007; 28: 4628–4634.
46. Oh SH, Ward CL, Atala A, Yoo JJ, Harrison BS. Oxygen generating scaffolds for enhancing engineered tissue survival. Biomaterials 2009; 30: 757–762.
47. Li Z, Guo X, Guan J. An oxygen release system to augment cardiac progenitor cell survival and differentiation under hypoxic condition. Biomaterials 2012; 33: 5914–5923.
48. Ng SM, Choi JY, Han HS, Huh J-S, Lim JO. Novel microencapsulation of potential drugs with low molecular weight and high hydrophilicity: Hydrogen peroxide as a candidate compound. Int J Pharm 2010; 384: 120–127.
49. Chandra PK, Ross CL, Smith LC, Jeong SS, Kim J, Yoo JJ, Harrison BS. Peroxide-based oxygen generating topical wound dressing for enhancing healing of dermal wounds. Wound Repair Regen 2015; 23: 830– 841.
50. Pedraza E, Coronel MM, Fraker CA, Ricordi C, Stabler CL. Preventing hypoxia-induced cell death in beta cells and islets via hydrolytically activated, oxygen-generating biomaterials. Proc Natl Acad Sci U S A 2012; 109: 4245–4250.
51. Vert M, Doi Y, Hellwich KH, Hess M, Hodge P, Kubisa P, Rinaudo M, Schué F. Terminology for biorelated polymers and applications (IUPAC recommendations 2012). Pure Appl Chem 2012; 84: 377–410. 52. International Standard 10993-5 Biological evaluation of medical devices - Part 5: Tests for in vitro
cytotoxicity. Third edition, 2009. International Organisation for Standardisation, Geneva, Switzerland. 53. International Standard 10993-6 Biological evaluation of medical devices - Part 6: Tests for local effects
after implantation. Second edition, 2007. International Organisation for Standardisation, Geneva, Switzerland.
