WO2017039435A1 - Means and methods for sustained oxygen release in situ - Google Patents

Abstract

The invention relates to the field of tissue engineering and cell-based therapy. More in particular, it relates to an oxygen-releasing biomaterial that can enhance and support cell survival in vivo. Provided is an oxygen-delivering microsphere based on a biocompatible polymer, the microsphere comprising an agent capable of generating oxygen in situ encapsulated in a polymer matrix comprising poly (1,3-trimethylene carbonate) (PTMC). Also provided is an implantable device, like a scaffold for tissue engineering, comprising a plurality of oxygen-delivering microparticles according to the invention.

Description

Title: Means and methods for sustained oxygen release in situ. The invention relates to the field of tissue engineering and cell -based therapy. More in particular, it relates to an oxygen-releasing biomaterial that can enhance and support cell survival in vivo.

In 1993 Langer and Vacanti introduced the concept of tissue engineering. From that time on, increasing efforts have been made to implement tissue engineering into clinical practice in order to restore damaged or lost tissues. The concept of tissue engineering is to combine biomaterials, cells and growth factors to create 'tissue' in the laboratory. This tissue can subsequently be implanted at the defect site. Many combinations of biomaterials, cells and growth factors have been developed and evaluated in animal models. Thus far, a limited number of cell based tissue engineering applications has really made it to the clinic. Different

applications of autologous cell-laden constructs failed after implantation in orthotopic locations: After an in vitro period of culturing in a controlled environment, cells are introduced into the body in a wound bed, consisting of a blood clot and damaged tissue and the local vasculature. Closing of the wound soon creates a completely anoxic implantation site. The lack of capillaries in this initial phase after implantation, and thus the lack of oxygen and nutrients result in cell death, which is a major limitation of cell therapy for tissue restoration(2, 3). The supply of oxygen to meet the need of cells to stay viable is a major challenge for cell-based tissue engineering.

Furthermore, dying cells will not contribute to tissue repair other than by a 'trophic' effect(4). Although the trophic effect appears to be useful, the cells used in cell therapy are selected for their ability to contribute to tissue restoration, and not for their trophic effect.

In order to decrease the above-mentioned nutrient and oxygen deprivation, many strategies have been employed to provide cells with oxygen and nutrients. These strategies include pre-vascularization(5), artificial vascularization with membranes(6, 7) and controlled release of angiogenesis promoting factors, such as vascular endothelial growth factor(8). From bone lengthening studies it can be deduced that vasculature is able to keep up with bone lengthening at 1 mm a day, but not at 2 mm a day(9). So, in bone blood vessels grow at a rate of approximately 40 μιη/h. Bridging larger bone defects (1 to 2 cm) will take 10 to 20 days.

Recently, oxygen-releasing biomaterials were introduced with the aim to provide tissue near the wound bed with sufficient oxygen to limit necrosis, and instead initiate healing and revascularization. Harrison et al. (10) discloses a biomaterial comprising an oxygen rich compound of sodium percarbonate incorporated into films of Poly(D,L-lactide-co-glycolide) (PLGA) was manufactured with the aim to give the tissue time to heal and vascularize, while preventing the cells from dying.

Different oxygen- delivering materials, based on inorganic peroxides dispersed in poly(lactic-co- glycolic acid) polymer, showed promising results in in vitro experiments(l l, 12). Decreased cell death was observed during the first few hours to days.

WO2011/011131 discloses a composition comprising (i) at least one biocompatible polymer suitable for use in tissue-engineering scaffolds and (ii) at least one metal peroxide, preferably Ca02, Mg02 or a combination thereof. Upon reaction with water, the metal peroxide generates oxygen. Preferred polymers of WO2011/011131 are those which, upon hydrolysis, yields an acid degradation product, such as lactic acid or glycolic acid. Specifically disclosed are compositions comprising poly-(D,L-lactic acid) (PDLLA) or poly-(D,L-lactic-co-glycolic acid) (PLGA), preferably PDLLA. However, the sustained release of oxygen from these materials was limited which resulted in only transient effects.

The present inventors indeed observed that composites consisting of

Ca02 powder dispersed in poly(lactic acid) and poly(lactic-co-glycolic acid) polymers, both degrading by bulk hydrolysis, release all oxygen in approximately 24 hours (13).

Thus, although initial results of composites of oxygen releasing peroxide embedded in lactide-based polymers were promising, the period of oxygen delivery was very short and the burst release of oxygen is considered to be far from optimal for use in tissue engineering. Therefore, they set out to develop new materials that have potential in cell therapy. In particular, they aimed at providing biomaterial that can provide sustained release of oxygen to keep (transplanted) cells alive and metabolically active for a longer period of time, enabling these cells to contribute to tissue repair. More specifically, a slow oxygen-releasing biomaterial was sought for that allows application as a "sidekick system", e.g. such that surgeons have the possibility to adjust the amount of oxygen- delivering material per patient and case.

It was surprisingly found that at least some of these goals could be met by the provision of a microsphere based on a polymer matrix of poly (1,3- trimethylene carbonate) (PTMC) comprising an agent capable of generating oxygen in situ, such as CaC , being encapsulated in, or encased by said polymer matrix. As is shown herein below, oxygen-releasing PTMC-based microspheres can be produced in a water-free system and show long-term oxygen-release. Cells cultured near or on the materials show an increased mitochondrial activity probably caused by an increase in cell number with oxygen-releasing materials compared to with non-oxygen releasing materials. The microspheres did not show any cytotoxicity making them ideal oxygen-releasing vehicle for tissue engineering. Moreover, oxygen releasing PTMC-Ca02 microspheres implanted underneath a random pattern devascularised skin flap in mice provided a proof of concept of the in vivo working potential of these microspheres.

Accordingly, the invention relates to an oxygen- delivering microsphere based on a biocompatible polymer, the microsphere comprising an agent capable of generating oxygen in situ encapsulated in a polymer matrix comprising poly(l,3- trimethylene carbonate) (PTMC).

PTMC has been used in biomedical applications for drug delivery systems and soft tissue engineering(14). It is an amorphous polymer, with high flexibility and it is degraded by surface erosion(15). The surface erosion of PTMC is regulated and involves enzymes produced by macrophages in the body(16). It is suspected that lipase and cholesterol esterase (CE) are involved in the degradation of PTMC(17, 18). The resulting metabolites are non-acidic, and are expected to be less detrimental for bone regeneration than the acidic metabolites of poly-lactides. A microsphere of the invention thus effectively provides an oxygen- releasing active agent, as a time-stable but releasable agent in a PTMC-based polymeric encapsulating matrix, wherein the encapsulated or encased agent is essentially distributed in the polymer matrix as a microdispersed phase. The encapsulated or encased oxygen-releasing agent is gradually released from the enveloping matrix at a rate dependent on the rate of surface erosion and

degradation of the PTMC-based matrix.

Microp articles comprising PTMC are known per se in the art. Dinarvand et al. (19) reported the preparation, characterization and in vitro drug release properties of polytrimethylene carbonate/polyadipic anhydride blend microspheres. Disclosed are microspheres with different ratios of PTMC-PAA (85/15, 70/30, and 55/45) containing 5% buprenorphine HC1.

WO2012/094679 relates to compositions and methods for synthesis and delivery of high- affinity oxygen binding agents to tumors to increase intratumoral partial pressures of oxygen. The agents can be encapsulated in biodegradable polymer vesicles. The list of exemplary polymers includes pure or blends of multiblock copolymer, wherein the copolymer includes at least one of poly(ethylene oxide) (PEO), poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-co- glycolic acid) (PLGA), poly(e-caprolactone) (PCL), and poly (trimethylene carbonate)

(PTMC), poly(lactic acid), poly(methyl ε-caprolactone). The list of preferred high- affinity oxygen binding agents includes unmodified human myoglobin, unmodified myoglobin from another biological species, chemically or genetically modified myoglobin from humans or from another biological species, unmodified hemoglobin from another biological species, a biological agent including a small molecule, a metal-chelator complex, a peptide, a protein, a nucleic acid, a polysaccharide, and a polymer of a small molecule, a metal-chelator complex, a peptide, a protein, a nucleic acid, or a polysaccharide. Nothing is mentioned about agents capable of generating oxygen in situ and no preference is expressed for using PTMC.

EP1872806 relates to an implant comprising a source of oxygen capable of releasing oxygen, such as magnesium peroxide or calcium peroxide, and a material selected from the group consisting of biodegradable and/or bioactive glass, sol-gel produced silica and mixtures thereof. Whereas it is generally mentioned that the implant may additionally contain a biocompatible polymer, none of the examples include polymers. Importantly, EP1872806 fails to teach or suggest to prepare microspheres which contain a PTMC-based polymer matrix, let alone that the metal peroxide is encapsulated in the polymer matrix. WO2010/121024 relates to a composite for delivering extended-release of oxygen and discloses a biocompatible polymeric support having a plurality of solid peroxide particles suspended therein. The polymers must be highly stable, hydrophobic and of low diffusivity to ensure that reaction of water with the embedded peroxide is delayed. WO2010/121024 explicitly teaches the use of a stable polymer which does not suffer from degradation, and silicone is the only polymer used in the examples. In contrast, the microspheres of the present invention are based on PTMC, which is known in the art as being susceptible to surface erosion and degradation into non-acidic metabolites

A microsphere allowing for sustained oxygen according to the present invention is characterized by a PTMC-based polymer matrix encapsulating an agent capable of generating oxygen in situ. In one embodiment, the agent capable of generating oxygen in situ is a metal peroxide. The metal ion of the metal peroxide is preferably a divalent, biocompatible cation. Suitable biocompatible metal peroxides include zinc peroxide (Zn02), strontium peroxide (SrCte), calcium peroxide (CaCte), magnesium peroxide (MgC ) and mixed calcium/magnesium peroxide (Ca,Mg02). Preferred metal peroxides are Ca02 and Mg02. Oxygen release starts upon contact with water. For a divalent metal (Me) peroxide, the reaction is as follows:

2 Me0₂ +2H₂0 <—► 2Me(OH)₂ + O2

As the polymer is degraded by surface erosion, the peroxide molecules are gradually exposed to water and release oxygen. The PTMC-metal 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. Thus, as used herein, the term "oxygen- delivering microsphere" refers to a microsphere that can release oxygen under the appropriate conditions, depending on the type of oxygen-releasing agent that is encapsulated in the polymer matrix. Hence, the microsphere may not always have the status of being oxygen-releasing. In fact, a tunable release capacity (e.g. by exposure to water in case of a metal peroxide) is most desired.

Several types of materials can be produced out of polymer-peroxide composites, each optimized for their final application. In one embodiment, the agent consists of Ca02 or MgC . Calcium and magnesium phosphates are natural components of the body and play important roles in the formation and mineralization of bone.

Preferably, the agent is CaC .Ca02 has been used for its oxygen releasing capacities before(l l, 20-23). CaC is preferred for its favourable oxygen release profile combined with its low cytotocity and availability in high purity(l l). Ca02 reacts with water forming both Ca(OH)2 and H2O2. Catalase reduces the cytotoxic H2O2 to H2O and O2 according to the following equations:

Ca0₂ + 2H₂0 ≠ Ca{OH)₂ + H₂0₂

2H₂0₂ ≠2H₂0 + 0₂

In another aspect, a composition comprises a combination of CaC and Mg02, for example in a relative amount of between 10: 1 and 1: 10 by weight, preferably between 5: 1 and 1:5. In one embodiment, about equal amounts of both oxygen sources are used. In another embodiment, MgC is present in excess of

Ca02. For example, the relative weight ratio between CaC and MgC ranges from between 1: 1.1 to about 1: 10, preferably from about 1:2 to about 1: 8, such as 1: 3, 1:4 or 1:5. In yet another embodiment, CaC is present in excess of MgC . For example, the relative weight ratio between Ca02 and Mg02 ranges from between 1.1: 1 to about 10: 1, preferably from about 2: 1 to about 8: 1, such as 3: 1, 4: 1 or 5: 1.

The total amount of oxygen- delivering agent in a composition of the invention can vary, again depending on the desired oxygen- delivering

characteristics. The total amount of agent, e.g. metal peroxide(s), will generally be at least 1 weight%, preferably at least 2 weight%, more preferably at least 3 weight% based on the weight of the PTMC-based polymer matrix. In this context it is to be noted that oxygen can be very toxic to cells and tissue at high

concentrations. In addition, as is described herein below, oxygen release by peroxides is accompanied with the formation of hydroxides which cause a (local) increase in pH. Therefore, very high (initial) levels of oxygen release are preferably avoided. In one embodiment, the amount of metal peroxides does not exceed 20 weight%, preferably 15 weight%. For instance, the metal peroxides are present in an amount of 1-20 weight%, preferably 3-20 weight%, such as 3-17 weight%, 3-10 weight%, like 3 weight%, 4 weight%, 5 weight%, 10 weight%. Very good results were obtained with about 3-6 weight% metal peroxide, for example PTMC microspheres comprising 5 weight% Ca02.

A microsphere of the invention comprises a PTMC-based polymer matrix, i.e. its polymer component consists largely or completely of PTMC. Degradation of PTMC in vivo is thought to be a surface erosion process mediated by cells, probably involving enzymes like lipase (CE 3.1.1.3) or cholesterol esterase (CE 3.1.1.13) (16, 17). In in vitro studies, the degradation of PMTC could be controlled by applying these enzymes. PTMC incubated without these added enzymes and without cells can be considered to be a non-degradable polymer(24). Degradation products of PTMC are non- acidic, which makes this material a good candidate for tissue engineering. The glass transition temperature (Tg) of PTMC is approximately - 20°C, making PTMC an amorphous, rubber-like material at body temperature without potential side effects of crystallinity(25). The water uptake of PTMC is approximately 1%(18), similar to the water uptake capacity of poly(lactic acid)(26).

Without wishing to be bound be any theory, a direct contact between implanted cells and oxygen- delivering agent present in a PTMC-based microsphere of the invention is avoided such that the benefits of the delivery of oxygen are kept, but the toxic effects of H2O2 are suppressed. Preferably, the PTMC content of the polymer matrix encapsulating the oxygen- delivering agent is at least 50wt%, preferably at least 70wt%, more preferably at least 80wt%. In one embodiment, PTMC represents at least 85%, preferably at least 90%, more preferably at least 95% of the polymer in the matrix. For example, the polymer matrix comprises a blend of PTMC with one or more other polymers, such as lactides and/or ε- caprolacton. Exemplary co-polymers for use in a polymer matrix in a microsphere of the invention include mPEG-PTMC and PTMC-PCL-PTMC. In one aspect, the matrix consists of a trimethylene carbonate (TMC) based material having tuneable low erosion rates. In a specific aspect, the polymer matrix consists of PTMC, i.e. PTMC is the sole polymer.

PTMC can be prepared according to methods known in the art, see for example Pego et al. (15). In one embodiment, the PTMC is a cross-linked high molecular weight PTMC. For example, the invention provides a microsphere comprising high molecular weight PTMC having a number average molar mass (Mn) of at least 220 x 10³ g/mol, In one embodiment, the M_n is at least 250 x 10³ g/mol preferably at least 300 x 10³ g/mol, more preferably at least 500 x 10³ g/mol. In a specific aspect, the Mn is in the range of about 250 to about 700 x 10³ g/mol. See for example Bat et al. disclosing a method of photocrosslinking high molecular weight PTMC by UV irradiating PTMC films containing pentaerythritol triacrylate (PETA) and a photoinitiator (27).

Preferably, the diameter of the microsphere is between 50 and 250 micrometer. In an embodiment, the microsphere is less than 200 micrometers in diameter. In another embodiment, the diameter is less than 100 micrometers.

A further aspect of the invention relates to a method for preparing an oxygen- delivering microsphere according to the invention. The method is based on an oil- in-oil solvent evaporation process. In one embodiment, it comprises the steps of

(i) providing a polymer solution by dissolving PTMC in a suitable solvent; (ii) dispersing the agent capable of generating oxygen in said polymer solution to obtain a dispersion;

(iii) emulsifying said dispersion by introducing it under stirring in a continuous phase comprising a mineral oil and a surfactant;

(iv) heating the emulsion to evaporate the solvent and induce microsphere precipitation; (v) washing the microspheres to remove residual mineral oil and surfactant.

The polymer solution is typically prepared by dissolving PTMC (e.g. Mn 220,000 or Mn 320,000 g/mol) in an appropriate solvent. The amount of polymer to be added typically lies in the range of about 2- 15% (w/v), for example 3- 10% (w/v). Suitable solvents for dissolving PTMC are known in the art and include acetonitrile.

The agent capable of generating oxygen is then dispersed in said polymer solution to obtain a dispersion. For example, particles of a metal peroxide are added in an amount of about generally be at least 1 weight%, preferably at least 2 weight%, more preferably at least 3 weight% based on the weight of the polymer. As indicated above, the amount of metal peroxides does not exceed 20 weight%, preferably 15 weight%. For instance, the metal peroxides are present in an amount of 1-20% (w/w), preferably 3-20% (w/w), such as 3-17%, 3- 10% (w/w). Very good results were obtained when about 3-6 weight% metal peroxide, preferably Ca02, was added to a polymer solution comprising 2- 10% (w/v) PTMC. Thereafter, the dispersion comprising polymer and oxygen-releasing agent is emulsified by introducing it under stirring in a continuous phase comprising a mineral oil and a surfactant. For example, the dispersion is pipetted in a jacketed beaker containing the continuous phase at a temperature of about 10°C while stirring at 300-400 rpm. Suitable surfactants for use in a method of the invention include non-ionic surfactants that are soluble in mineral oil. For example, sorbitan fatty acid esters such as SPAN 80™, 83™, 85™, or 120™ are suitably used. In one aspect, the surfactant is sorbitan monooleate (HLB 4.3), also known as SPAN™80. In another aspect, the surfactant is sorbitan sesquioleate (HLB 3.7), also known as SPAN™ 83. In yet another aspect, the surfactant is sorbitan trioleate (HLB 1.8), also known as SPAN™ 85, or sorbitan Isostearate, also known as SPAN™ 120.

After emulsification, the emulsion is heated to evaporate the solvent and thereby induce microsphere precipitation. The temperature may be gradually elevated, for example to about 35°C, and maintained for an additional period of several hours, and then to even higher temperatures, like 50-70°C, depending on the solvent(s) used. For example, acetonitrile may be evaporated by keeping the emulsion for 3-6 days at about 65°C.

After solvent removal, the microspheres can be allowed to precipitate by gravity and they are suitably collected by filtering. Any residual mineral oil and surfactant is removed by washing the microspheres with a suitable solvent, for example n-hexane. The washed microspheres are typically dried (e.g. in a fume hood and/or under reduced pressure) at room temperature for at least 24-48 hours. The dried microspheres are readily stored below zero, preferably at -20°C, until further use.

As is exemplified herein below, a microsphere according to the invention are biocompatible and capable of releasing oxygen over a prolonged period of time, like several weeks or even up to 2 months. These properties make them

particularly suitable for cell therapy applications. Therefore, a further aspect relates to the use of an oxygen- delivering microsphere according to the invention in a method for tissue engineering. As used herein, the term tissue engineering refers to the use of a combination of cells, engineering and materials methods, and suitable biochemical and physicochemical factors to improve or replace biological functions, typically in a mammalian or human subject. Tissue engineering covers a broad range of applications, in practice the term is closely associated with applications that repair or replace portions of or whole structural tissues i.e., bone, cartilage, blood vessels, bladder, skin, muscle etc.

In one embodiment, the microsphere is used in a method comprising cell therapy, e.g. using autologous cells, for replacing malfunctioning tissue. Autologous cells are obtained from the same individual to which they will be re-implanted. Autologous cells have the fewest problems with rejection and pathogen

transmission, however in some cases might not be available. For example in genetic disease suitable autologous cells are not available. Recently there has been a trend towards the use of mesenchymal stem cells from bone marrow and fat. These cells can differentiate into a variety of tissue types, including bone, cartilage, fat, and nerve. A large number of cells can be easily and quickly isolated from fat, thus opening the potential for large numbers of cells to be quickly and easily obtained. Reconstructing tissues or organs by a simple cell injection is possible only in few cases. In order to form tissues with distinct three-dimensional shapes, support is usually needed. Biomaterial scaffolds provide this support by

functioning similarly to natural ECM and thus promoting cell proliferation and differentiation. When it comes to scaffolds, there are two main strategies in tissue engineering. The first approach involves using scaffolds as supporting constructs upon which cells are seeded in vitro. Secondly, they can be used as devices for growth factor/drug delivery. These two strategies can also be combined. The scaffold should degrade over a period of time that would allow tissue formation concurrently - ideally the scaffold disappears leaving behind regenerated tissue.

Tissue engineering field thus relies extensively on the use of porous 3D scaffolds to provide the appropriate environment for the regeneration of tissues and organs. These scaffolds essentially act as a template for tissue formation and are typically seeded with cells and occasionally growth factors, or subjected to biophysical stimuli in the form of a bioreactor. The cell-seeded scaffold is either cultured in vitro to synthesize tissues which can then be implanted into an injured site, or are implanted directly into the injured site, using the body's own systems, where regeneration of tissues or organs is induced in vivo. This combination of cells, signals and scaffold is often referred to as a tissue engineering triad. Because of their sustained oxygen-releasing capacity, the PTMC-based microspheres of the invention oxygen-releasing can contribute to cell survival of seeded cells prior to, during and/or after implantation of the scaffold. It is believed that the polymer matrix acts as a barrier for both inflow of water and outflow of active agent, slowing down the reaction and reducing cytotoxicity. Accordingly, in a preferred embodiment, a microsphere of the invention is used in a method comprising the implantation of a scaffold, preferably a scaffold onto which cells are seeded.

In a specific aspect, the invention finds it use in bone tissue engineering. Currently, autografts are the golden standard for bone repair due to their osteoconductive and osteoinductive properties and thus dominate the bone grafting business that has sales of over 2.5 billion dollars per year. The problems with autografts are related to their limited availability, donor-site morbidity and cost. Bone tissue engineering as a leading field in multidisciplinary tissue engineering can provide a functional biological substitute to bone grafts. The different cell types related to bone maintenance include osteocytes that are terminally differentiated and entrapped in the bone ECM, mesenchymal stem cells found in the bone marrow, bone-lining cells covering all bone surfaces, osteoblasts that are able to synthesize organic non-mineralized bone matrix, and finally osteoclasts being capable of resorbing bone tissue which is the first step of bone remodeling. The most promising strategy in bone tissue engineering involves seeding adult stem cells or osteoblasts into a 3D scaffold, culturing the construct in vitro and implanting it into the defect site.

In bone tissue engineering, osteoconductive biomaterials are often used as filler for non-union bone defects. These biomaterials give good results on the long term, but healing is slow due to slow infiltration of cells into the material. Application of autologous cells in the defect might solve this problem. However, difficulties with cell-survival after implantation thus far limited introduction of such therapies in the clinic. The lack of surviving cells is mainly seen in the center of the implant which supports the notion that the limitation of diffusion of oxygen is the major reason for this. Application of oxygen-releasing microspheres of the present invention can improve oxygen supply to the cells, thus enhancing survival of the implanted cells in vivo. Microspheres can be added to all kinds of scaffolds in any desired dosage, which is easily adapted to individual needs e.g. the condition of the tissue (highly vascularised or poorly vascularized), and to the size of the defect. This makes the microspheres highly suitable as a 'side kick' as an on the shelf product that is very easy applicable. A further aspect of the invention relates to an implantable device comprising a plurality of oxygen- delivering micr op articles according to the invention. For instance, the implantable device is a scaffold for tissue engineering. Preferably, the scaffold is a porous scaffold. The scaffold can be made of any suitable material. Typically, three individual groups of biomaterials, ceramics, synthetic polymers and natural polymers, are used in the fabrication of scaffolds for tissue

engineering. Each of these individual biomaterial groups has specific advantages and, needless to say, disadvantages so the use of composite scaffolds comprised of different phases is becoming increasingly common. Although not generally used for soft tissue regeneration, there has been widespread use of ceramic scaffolds, such as hydroxy apatite (HA) and tri-calcium phosphate (TCP), for bone regeneration applications. Ceramic scaffolds are typically characterized by high mechanical stiffness (Young's modulus), very low elasticity, and a hard brittle surface. From a bone perspective, they exhibit excellent biocompatibility due to their chemical and structural similarity to the mineral phase of native bone. The interactions of osteogenic cells with ceramics are important for bone regeneration as ceramics are known to enhance osteoblast differentiation and proliferation. Various ceramics have been used in dental and orthopedic surgery to fill bone defects and to coat metallic implant surfaces to improve implant integration with the host bone.

Scaffolds for tissue engineering can be designed according to specific needs and requirements using standard technology. Typically, the size and shape of said scaffold is a function of the tissue into which the scaffold is to be implanted. Other important and well known parameters include porosity, mechanical properties, pore size and interconnectivity of the pores. Ideally, the size of the scaffold is easy to adjust, either by cutting with a scalpel or by adding smaller pieces together. Furthermore, the filling of the tissue lesion should in the long term result in a normal tissue architecture. Also, the implanted scaffold should not have adverse effect on the newly generated tissue. It is furthermore desirable that the implant is completely remodelled resulting in a scarless regeneration of the defected tissue.

Generally speaking, the three-dimensional scaffold according to the present invention should be able to accommodate cells that will aid in the repair of the damaged tissue. In order for cells to be cultured inside and on the scaffold the three-dimensional scaffold should comprise cavities suitable in size in which cells should be able to live, multiply, differentiate and form tissue. In the following such cavities will be referred to as pores. The cell scaffold therefore typically has pores. The presence of pores may also allow for the population of the scaffold by cells originating from surrounding tissues by invasion. Thus, in general the size of the pores will range from about one to ten times the diameter of the cells to be seeded in the scaffold. The size of the pores is thus adapted to the type cell to be accommodated within the three dimensional scaffold considering which type of tissue is to be regenerated or repaired. It is important for the pores to be of a sufficiently large size (sufficient pore volume) so as to allow cells (i.e., living cells) to maintain their shape within the structure. Furthermore, a large pore volume is desirable in order to allow a cell suspension to fully penetrate the structure and thus permit cell seeding and/or cell migration throughout the material. Also, the pores should be interconnected and the interconnections should be of sufficient size. In relation to access to nutrients and efficient removal of waste products following cellular metabolism a sufficient pore volume is needed. According to the present invention the pores of the cell scaffold have a pore size in the range of from about μηι to 1000 μηι, such as 50 μηι to 1000 μηι, for example 100 μηι to 900 μηι, such as 200 μηι to 600 μηι.

The pores of the three-dimensional cell scaffold should be relatively uniform in size, which ensures that the pores are large enough to accommodate the living cells in a uniform manner throughout the three-dimensional scaffold. Thus, in one embodiment of the invention the pores of three-dimensional cell scaffold are uniform in size.

The device can be provided with oxygen-releasing microspheres using various techniques. However, the inventors developed a specific procedure to introduce the microspheres into a porous device, e.g. a ceramic scaffold. The procedure involves a seeding (or impregnation) method applying low pressure in a syringe system. Accordingly, the invention also provides a method for providing a porous scaffold into which a plurality of oxygen- delivering micr op articles is seeded, comprising

- providing a suspension of oxygen-releasing microspheres according to the invention in a suitable solvent, preferably n-hexane;

- setting the scaffold into a syringe

- drawing the suspension of microspheres and some air into the syringe, and closing the syringe is closed with a cap

- creating a low pressure within the syringe to obtain a homogenous microsphere distribution in the scaffold

- removing excess solvent .

The low pressure is readily obtained by retracting the plunger in the syringe to create a vacuum, similar to what was described by Tan et al. (2012), relating to a method for homogenous cell seeding in porous scaffolds (28). In a specific aspect, a vacuum is created by pulling back the plunger and maintaining the vacuum for several seconds, which process is repeated 2-6 times. In one embodiment, the device is furthermore provided with living cells, preferably mammalian cells, most preferably human cells. For example, the device is provided with bone marrow cells, osteoblasts, mesenchymal stem cells, cartilage cells, embryonic stem cells, gene transfected cells, endothelial cells and combinations thereof.

Genetically modified cells may be used, which have been created to be particularly useful for the regeneration of tissue, such as bone, tendon, ligament and/or cartilage. The cells may be genetically engineered to produce gene products beneficial to transplantation, e.g. anti-inflammatory factors, e.g., anti-GM-CSF, anti-TNF, anti-IL-1, anti-IL-2, etc. Alternatively, the cells may be genetically engineered to "knock out" expression of native gene products that promote inflammation, e.g., GM-CSF, TNFa, IL- 1 , IL-2, or "knock out" expression of MHC in order to lower the risk of rejection. In addition, the cells may be genetically engineered for use in gene therapy to adjust the level of gene activity in a patient to assist or improve the results of the cartilage transplantation by use of the three- dimensional cell scaffold according to the present invention.

Cell seeding on a scaffold should ideally result in a uniform distribution of cells inside the scaffold - otherwise the functionality and mechanical properties of the engineered construct can be compromised. Also, a high seeding efficiency is appreciated to avoid wasting valuable cells and to enable faster tissue formation. There are many different cell seeding methods and various ways to classify different seeding strategies. For example, the method can involve active or passive, surface or bulk seeding, or static or dynamic seeding. In a preferred aspect, the cell seeding method comprises a vacuum seeding method, e.g. as described by Tan et al (28).

An implantable device of the invention may contain one or more further useful additive(s). In one embodiment, it comprises at least one additive that contributes to cell survival, proliferation and/or differentiation. Also encompassed are additives which can neutralize, scavenge or absorb a component that would otherwise affect cell growth or survival in a negative fashion. Exemplary useful additives are nutrients, e.g. fermentable sugars such as glucose, etc., and biologically active agents e.g. cytokines, growth factors, hormones, inflammatory stimuli, angiogenic factors. A number of growth factors exist that are involved in inducing a variety of cellular responses in connection with a variety of cell functions. Some growth factors are for example osteoinductive, whereas other growth factors have inductive effect on articular cartilage.

The growth factor may be selected from the group consisting of platelet derived growth factor (PDGF) AA, PDGF BB, insulin-like growth factors, fibroblast growth factors (FGF), β-endothelial cell growth factor,; transforming growth factors (TGF), such as TGF-P1 , TGF l.2, TGF- 2, TGF- 3, TGF-βδ; bone morphogenic protein (BMP) 1 , BMP2, BMP 3, BMP 4, BMP 7, vascular endothelial growth factor (VEGF), placenta growth factor; epidermal growth factor (EGF),

amphiregulin, betacellulin, heparin binding EGF, interleukins (IL) - 1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL- 11 , IL- 12, IL- 13, IL- 14, IL- 15- 18, colony stimulating factor (CSF)-G, CSF-GM, CSF-M, erythropoietin, nerve growth factor (NGF), ciliary neurotropic factor, stem cell factor, and hepatocyte growth factor.

Still a further aspect relates to a method of forming tissue (in vitro or in vivo), the method comprising (a) providing an implantable device comprising oxygen- releasing PTMC-based microspheres according to the invention, (b) covering at least part of the surface of the device with living cells capable of forming tissue; and (c) culturing the device under conditions suitable to grow tissue on and/or in the device. Of course, another aspect relates to the therapeutic uses of an implantable device of the invention. In one embodiment there is provided a method for regenerating tissue in a mammal in need thereof, comprising implanting the three-dimensional cell scaffold of the present invention. In another embodiment, the invention provides a method for regenerating tissue in a mammal in need thereof, comprising implanting the three-dimensional cell scaffold of the present invention. The mammal is for example a goat, mouse, rabbit, rat, pig, dog, horse, cat, cow or a human. In a preferred embodiment, the mammal is a human. Also encompassed is a method for treating a tissue pathology in a subject, the method comprising (a) providing an implantable device according to the invention, (b) covering at least part of the surface of the scaffold with living cells capable of forming tissue; and (c) culturing the scaffold under conditions suitable to grow tissue on and/or in the scaffold, and (d) introducing the scaffold into the subject, and wherein the cells used in step (b) are capable of treating the tissue pathology. The tissue pathology for instance comprises loss, damage, injury, or combinations thereof to the tissue. The treatment comprises for example tissue remodeling, repair, regrowth, resurfacing, regeneration, or combinations thereof.

In another embodiment, the oxygen-releasing microspheres are used "as such" to increase the oxygen level at a site (prone to) suffering from hypoxia or anoxia. Areas of injured bone, cartilage and/or tissue compromise blood circulation, reducing the oxygen available to the surrounding tissue. Injuries may commonly result from a traumatic accident, surgery to correct trauma or degenerative processes. Ironically, the surgery that is often performed to correct an earlier traumatic injury can also perpetuate the injury. Both the traumatic injury and the resulting surgery inevitably cut through capillaries, arterioles and vennules. The reduced blood flow results in insufficient oxygen to fully support the metabolic needs of the tissues. Cell death, atrophy and osteonecrosis are induced by lack of oxygen. Paradoxically, injured tissue has a particularly high need for oxygen to support the healing process. The early stages in bone healing involve lymphocytes and osteoclasts which use considerable oxygen as they resorb damaged or un- needed tissues in preparation for the growth of new bone and associated tissues. Lack of oxygen delays the onset of the healing and bone formation process and slows healing once it is in progress. Additionally, low oxygen levels may increase the potential for infection or prolong existing infection.

As will be appreciated by the skilled person, the biocompatible microspheres of the present invention showing sustained oxygen release in situ are advantageously used to increase the amount of available oxygen in (the

surroundings of) an injured tissue. For example, the microspheres are used in a method for promoting the healing of a surface wound, comprising applying to the wound surface a therapeutically effective amount of (a suspension of ) oxygen- releasing microspheres of the invention. The term "wound" includes, but is not limited to, chronic, traumatic, and surgically created wounds. Optimal metabolic function of these cells to repopulate the wound requires that oxygen be available for all phases of wound healing. The more layers of tissue that are damaged the greater the risk for complications to occur in the wound healing process. Thus, the invention also provides the use of PMTC-based oxygen-releasing microspheres in a method for controlling tissue oxygenation for wound healing and promoting tissue viability. In a specific aspect, the method involves improving oxygenation of a surface wound created by (skin) transplantation.

Thus, the potential applications of oxygen delivering microspheres of the invention are manifold. Plastic surgeons frequently use skin flaps in clinical practice. A frequently occurring complication is necrosis of a skin flap [25]. As has been shown in Example 2 herein below, oxygen releasing microspheres can 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 infarction(29, 30). 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 ischemia(30). Oxygen delivering biomaterials may aid in cell survival and may have the potential to make heart tissue regeneration 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 day(31), it would take about 10 days to get a one centimetre long scaffold entirely vascularised, whenever blood vessels can grow in from two opposite sides. Cells seeded on a scaffold material 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-Ca02 microspheres produced oxygen for at least 21 days (data not shown). In vivo however, the oxygen releasing microspheres had a significant positive effect until at least ten days after implantation of the material. 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. Incorporation of a more hydrophobic carrier copolymer, which is degraded more slowly in the body, may also lengthen oxygen release from a polymer-peroxide construct. By adjusting the peroxide component, for example increasing the amount of peroxide or changing the type of peroxide, the oxygen delivery profile as well as its kinetics can be modified. The tissue to be treated according to the present invention may be bone, cartilage, tendon, ligament, nerve, skin, vascular, cardiac, pericardial, muscle, ocular, periodontal, breast, pancreatic, esophageal, stomach, kidney, hepatic, mammary, adrenal, urological, and intestinal tissue. Thus, in one embodiment of the invention the tissue that is to be regenerated or treated may be selected form the group consisting of bone, cartilage, tendon, ligament, nerve, skin, vascular, cardiac, pericardial, muscle, ocular, periodontal, breast, pancreatic, esophageal, stomach, kidney, hepatic, mammary, adrenal, urological, and intestinal tissue. In another embodiment the tissue may be selected form the group consisting of bone, cartilage, tendon and ligament. In a particular embodiment the tissue is cartilage. Alternatively, the tissue is bone.

Mammals in need of regenerating tissue comprise mammals wherein injury to tissue has occurred. Surgical intervention is often required to repair the damage. Such surgical repairs can include suturing or otherwise repairing the damaged tissue with known medical devices, augmenting the damaged tissue with other tissue, using an implant, a graft or any combination of these techniques. According to the present invention tissue may be regenerated in a mammal by implanting the three-dimensional scaffold of the present invention. In one embodiment of the invention the mammal may be suffering from a defect to tissue selected from the group consisting of articular cartilage defects, meniscal defects, discus intervertebralis defects, bone defects, vertebral body fractures, skin wounds, fascial defects, tendon ruptures, ligament ruptures, nerve injuries, spinal cord injuries, blood vessel defects, ear substitution, nasal cartilage defects, muscle defects, heart muscle defects, muscle degeneration, adipose defects, tooth injuries, bladder wall defects, gastric wall defects, intestinal wall defects, pancreatic island transplantation, and eye injuries.

The invention is exemplified by the Examples below.

LEGEND TO THE FIGURES

Figure 1: SEM pictures of non-oxygen releasing and oxygen releasing

microspheres. Panel A; PTMC. Panel B: PTMC/Ca0₂. Panel C: Ca0₂. The non- oxygen releasing microspheres showed a smooth surface and were visually observed to be white to transparent.

Figure 2: PTMC/Ca02 microspheres oxygen release without the addition of cholesterol esterase show no oxygen release after the initial bulk release.

Figure 3: PTMC/Ca02 microspheres oxygen release. Triangles (Δ) indicate the addition of cholesterol esterase (CE) to the measurement volume to degrade the PTMC-based polymer matrix and thereby release oxygen. After addition of the CE small increases in oxygen release were observed.

Figure 4: hMSC stained with MTT after being cultured in close proximity to oxygen releasing composite microspheres. Pictures were taken after 4 days of hypoxic (0.1%) culturing. Most MTT staining is localized around the microspheres indicating that the cells adhere to the PTMC/Ca02 microspheres and stay viable(A). The non-oxygen-releasing microspheres (B) show a similar image as does the empty TCPS(C).

Figure 5: Viability of hMSC cultured with PTMC/Ca02 microspheres. Cells were cultured in hypoxic (0.1%O2) environment. The cells are cultured without (A) and with cholesterol esterase (B). Since the addition of catalase in these circumstances did not change the outcome, only the results without catalase are shown. The differences between the materials were all significant except for the difference between PTMC and TCPS on day 4 and with CE on day 1. Addition of CE, results in significant differences in all situations except for PTMC on day 4 and 7 and for PTMC/Ca0₂ on day 1. On TCPS CE is not significant.

Figure 6: Representative pictures of random pattern devascularized skin flap after implantation of microspheres. 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-CaC 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.

Figure 7: 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-Ca02 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 μπι. 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.

EXPERIMENTAL SECTION

EXAMPLE 1: Preparation and in vitro characterization of oxygen- releasing microspheres.

Materials and Methods

1.1 Materials

Poly(l,3-trimethylene carbonate) (Mn 220,000g/mol) was synthesized according to the protocol used by Pego et al.[22]. Mineral oil, Span 80, CaC>2 (75%), NaN₃ , DMSO, catalase (bovine liver), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) and cholesterol esterase (porcine pancreas) were from Sigma-Aldrich bv (Zwijndrecht, the Netherlands). Hexane and acetonitrile were purchased from Merck (Darmstadt, Germany).

1.2 Microsphere production

The microspheres were produced as follows: 3.5 % (w/v) PTMC was dissolved in acetonitrile (Merck, Darmstadt, Germany), followed by the addition of 5 % (w/w PTMC) Ca0₂ (Sigma Aldrich, Zwijndrecht, the Netherlands). The PTMC-Ca0₂ suspension was pipetted in a jacketed beaker, containing mineral oil (Sigma Aldrich, Zwijndrecht, the Netherlands) supplemented with 0.05 % (v/v) Span 80 (Sigma Aldrich, Zwijndrecht, the Netherlands) at a temperature of 10°C while stirring at 350 rpm. Subsequently, the temperature was raised to 35°C for four hours and then to 65°C for five days, to evaporate all acetonitrile. After this five days period, the PTMC-Ca02 microspheres were allowed to precipitate by gravity and subsequently they were washed three times five minutes using hexane to remove traces of mineral oil and Span 80. After washing, the hexane was allowed to evaporate from the microspheres under a fume hood for 48 hours followed by 16 hours in vacuum at room temperature. The PTMC-Ca02 microspheres were stored in a sealed jar at -20°C until use. Control microspheres were made out of a 3.5 % (w/v) PTMC solution, not containing Ca02, using the same process. 1.3 SEM

Scanning electron microscopy PHENOM PURE DESKTOP SEM (Eindhoven the Netherlands) was used to determine the size and the surface morphology. 1.4 Oxygen release of microspheres

The amount of dissolved oxygen was monitored using a WTW cellox 325 3310 (Weilheim, Germany) oxygen probe. Oxygen release was measured in an anoxic cabinet, achieved by continuously flushing with N2 gas at 0.2 bar. lOOmg microspheres were placed in a jar and 35ml deoxygenated simulated body fluid (SBF) was added. Polymer microspheres without Ca02 were used as negative control. All data are represented relative to the negative control. To mimic the oxygen release of the microspheres in cell cultures in the presence of catalase also lOOU/ml catalase was added. Cholesterol esterase was supplied at 0.63U/ml cholesterol esterase where indicated. To prevent bacterial growth in this measurement the SBF also contained 0.02% (w/v) NaN3.

1.5 Cell culture

Human Mesenchymal Stem Cells (hMSC) were obtained from bone marrow aspirates during total hip or knee surgery from patients with osteoarthritis of rheumatoid arthritis, as described by Buizer et al(25). hMSCs were cultures in a- Modification of Eagle's Medium (a-MEM), supplemented with 10% (v/v) heat- inactivated fetal bovine serum, 2% Antibiotic-Antimycotic (10,000U/mL of penicillin, 10,000μg/mL of streptomycin, and 25μg/mL of Fungizone) (all from LifeTechnologies) and 0.2 mM ascorbic acid-2-phosphate in a cell culture incubator at 37°C and 5% CO2 at 100% humidity. Hypoxic cell cultures (0.1% O2) were performed using a Ruskinn Invivo2 200 incubator (LED Techno, Den Bosch, the Netherlands) under the same conditions.

For experiments 10.000 hMSC were seeded per well in 24-wells plates with lOmg PTMC or PTMC/Ca02 microspheres . Cells were cultured for 1, 4, or 7 days in hypoxic conditions (0.1% O2). Cells were cultured in medium supplemented with 10% FBS-Heat Inactivated, 0.2mM 2-phospho-L- ascorbic acid trisodium salt and. The medium was changed twice a week; deoxygenated medium was used. lOOU/ml catalase was used to catalyze the reaction from H2O2 to O2. Cholesterol esterase was used in a 2C^g/mL concentration and added every day from a lOOOx concentrated stock solution.

1.6 Viability staining

Cell viability was assessed using the MTT assay. Culture medium was replaced with MTT (0.5mg/ml) containing medium, after which cells were incubated for 2.5 hours at 37°C. Then the reaction medium was aspirated and samples were gently washed with PBS. The formazan formed by the cells was dissolved in DMSO. The DMSO mixtures were then transferred to 96-wells plates and absorbance was read at 575nm using a Fluostar optima microplate reader (BMG labtech, De Meern, the Netherlands). Catalase was not added to medium containing MTT, since catalase interferes with formazan formation and therefore with absorbance readings.

1.7 Statistical analysis

MTT data of oxygen delivering materials were statistical assayed using a

Univariate Anova in SPSS 20.0.0.2. Time, material, cholesterol esterase and catalase were the assessed variables.

Results

The oxygen-releasing microspheres were analyzed using SEM, results are shown in Figure 1. The microspheres were polydisperse, and the sizes of oxygen releasing microspheres and non-oxygen releasing microspheres were comparable.. Size of the microspheres was <200μηι. The non-oxygen releasing microspheres showed a smooth surface, where as oxygen releasing microspheres showed a slightly less smooth surface. This is most likely caused by the irregular forms of the Ca02 crystals inside.

Oxygen release of the microspheres was characterized in simulated body fluid. The PTMC/Ca02 microspheres show a very different oxygen release profile with and without CE (see Figures 2 and 3).

Without CE (Figure 2), PTMC/Ca02 microspheres release oxygen only in the beginning of the measurement. After this small bulk release there was no more oxygen-release observed, probably due to the low hydrolytic degradation rate of the material[25] . When CE was added to induce degradation of the polymer matrix we observed small peaks of oxygen-release (Figure 3). The microspheres showed oxygen-release for up to 20 days when induced by CE. CE is stable at 37°C only for a very short period in simulated body fluid (Sigma- Aldrich, product information C1403). Therefore, the enzyme had to be added repeatedly. The oxygen delivery of the PTMC/Ca02 microspheres shows to be directly related to and regulated by CE. Differences between the degrading and non-degrading microspheres concern not only the release rate, also the total amount of released oxygen. However, most oxygen-release was still observed during the first day of the measurement, with or without addition of CE.

After implantation of microspheres, monocytes will adhere to the implant. These monocytes differentiate towards macrophages that release CE(32). Since oxygen release is regulated by CE, a precise prediction to the precise oxygen release in the body can therefore not be made. The degradation of and thereby oxygen release from PTMC/Ca02 microspheres is dependent on the amount of PTMC-degrading cells, which may differ between different sites in the body.

Although we observed in earlier studies an increase of viability when catalase was added to the medium, using these composite microspheres we could not observe a difference. This was probably due to the low level of oxygen-release and thereby the low, non-toxic level of H2O2 from the PTMC-based composite microspheres thereby creating a material that can be used in cell culture without the addition of catalase.

Overall, we found a better cell viability and therefore higher absorbance value for cells cultured with oxygen-releasing microspheres compared to the ones without. The most significant effect of oxygen- delivery was found at day seven with CE (P=0.00) on which the difference in cell viability was the largest.

On day four and seven the CE showed to be significantly beneficial for the cells cultured on PTMC/Ca02. However, this effect is very little compared to the effect the oxygen-releasing material has in general. This indicates that cells are able to free oxygen from the microspheres without help of added CE from the medium. Since cells are observed to attach to the microspheres or in very close proximity to microspheres there is a possibility that a very low, non- traceable amount of oxygen find its way to the cells. Even when the water uptake by the PTMC is very low(18), it is significant and it may be of great influence for the cells. We expect that composite microspheres without CE will take up water and release oxygen for the cells. Therefore, the amount of CE used in the cell culture might be too low to change the oxygen concentration relevant. Furthermore, it might be possible that hMSC can play a role in degrading PTMC thereby also freeing Ca02 crystals.

In this study, we did not observe an increase in cell-death of cells cultured in hypoxia. Therefore, the difference between cells cultured in hypoxia with and without oxygen releasing material was little. hMSC are known to live at low oxygen tensions between 1% and 7% in bone marrow(33, 34) which may explain this result.

Based on the long-term oxygen-release from PTMC/Ca02 microspheres, they have a great potential for use in tissue engineering and cell therapy. Seeded cells might be able to survive until a new vascular system has been developed. Application and dosing of these microspheres is much simpler than pre-vascularization, thereby making tissue engineering cheaper and more accessible for less invasive

procedures.

In conclusion, this Example demonstrates that oxygen-releasing PTMC-based microspheres can be produced in a water-free system and show long-term oxygen- release. Oxygen release of these microspheres is related to the enzymatic degradation of the PTMC. Cells cultured near or on the materials show an increased mitochondrial activity probably caused by an increase in cell number with oxygen-releasing materials compared to with non-oxygen releasing materials. The PTMC/Ca02 microspheres did not show any cytotoxicity making them ideal oxygen-releasing vehicle for tissue engineering. As will be appreciated by the skilled person, these microspheres can be used in regenerative medicine in a broader sense. Currently regenerative medicine is only used in a very limited number of cases since larger 3D defects of vascularized tissue cannot be

reconstructed using cells due to massive cell death. Oxygen- delivering PTMC microspheres may overcome this problem, without having to compromise the choice of scaffold. EXAMPLE 2: Oxygen-releasing microspheres delay tissue necrosis 2.1 Material preparation

PTMC was dissolved 3.5 % (w/v) in acetonitrile (Merck, Darmstadt, Germany), followed by the addition of 5 % (w/w PTMC) Ca02 (Sigma Aldrich, Zwijndrecht, the Netherlands). The PTMC-CaC suspension was pipetted in a jacketed beaker, containing mineral oil (Sigma Aldrich, Zwijndrecht, the Netherlands)

supplemented with 0.05 % (v/v) Span 80 (Sigma Aldrich, Zwijndrecht, the

Netherlands) at a temperature of 10°C while stirring at 350 rpm. Subsequently, the temperature was raised to 35°C for four hours and then to 65°C for five days, to evaporate all acetonitrile. After this five days period, the PTMC-Ca02 microspheres were allowed to precipitate by gravity and subsequently they were washed three times five minutes using hexane to remove traces of mineral oil and Span 80. After washing, the hexane was allowed to evaporate from the microspheres under a fume hood for 48 hours followed by 16 hours in vacuum at room temperature. The

PTMC-CaO"2 microspheres were stored in a sealed jar at -20°C until use. Control microspheres were made out of a 3.5 % (w/v) PTMC solution, not containing Ca02, using the same process. 2.2 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. (10) 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-Ca02 microspheres. The operative procedure was performed under anaesthesia 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 centimetres long running parallel to the spine, 0.5 cm of the midline of the animal. Both incisions were connected with a transverse one- centimetre 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 anaesthesia 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 anaesthesia. The skin flaps were excised in a standard manner and further processed for histological examination.

2.3 Photography and image analysis

At days 3, 7 and 10 after the surgery, the animals were anaesthetised 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 discolouration due to skin necrosis was assessed using Image J analysis software by three independent observers blinded for the applied treatment. Each skin flap was assessed three times by each observer. The amount of skin necrosis was expressed in percentage of the skin flap that showed necrosis.

2.4 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 μηι thick sections were cut using a microtome. The sections were mounted on Superfrost slides (Thermo Scientific, Braunschweig, Germany) and stained with hematoxylin (Merck, Darmstadt, Germany) and eosin (Merck, Darmstadt, Germany). Light microscopy was performed using a DMR microscope (Leica HC, Wetzlar, Germany) equipped with a Leica DFC 420C camera (Leica, Wetzlar, Germany). 2.5 Statistical analysis

Data were analysed using the SPSS 20.0.1 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). P < 0.05 was considered significant.

Results

3.1 Animals

All animals tolerated the operations well and no complications occurred. There were no early dropouts. Animal discomfort was estimated to be 3/6.

3.2 Skin necrosis

Comparison of the three independent observers of the amount of skin necrosis (Figure 6), revealed that the inter-observer reliability had an ICC of 0,803 (95% CI: 0,599-0,890). The intra-observer reliability had ICCs of 0,983, 0,971, and 0,996 for observers 1, 2, and 3, respectively. The amount of necrosis was variable within both the PTMC group and the PTMC-Ca02 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-Ca02 group. These results indicate that the PTMC-Ca02 microspheres did support cells under circumstances of disturbed vascularis ation in contrast to PTMC microspheres not releasing oxygen.

3.3 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 (Figure 7), with the characteristic dark coloured, several cell layers 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 organised. 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 1 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 (Figure 7). 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 disorganised, and the laminar structure of the skin could not always be recognised. 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 for necrotic cells (35). At the cranial end of the skin flap, tissue architecture looked healthy.

Conclusion

This example demonstrates the effect of implanted oxygen releasing microspheres on the process of necrosis in partly devascularised skin flaps. In comparison to PTMC microspheres, subcutaneous implantation of oxygen-releasing PTMC microspheres resulted in a significant reduction of amount of necrotic skin tissue at all three follow-up moments. These findings suggest that the release of oxygen by the PTMC-Ca02 microspheres delays necrosis in the otherwise ischaemic tissue. 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 ischaemic gradient within the skin flap, the most caudal area being the most ischaemic, while the most cranial area is less or not ischaemic.

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 al (10) implanted films made out of a composite of Poly(D,L-lactide-co-glycolide) (PLGA) and sodium percarbonate under random pattern devascularised 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 films that did not release oxygen.

Importantly, the PTMC-CaO^"2 microspheres of the present invention delayed the occurrence of necrosis in a devascularised skin flap for a significantly longer period. Even after ten days, skin necrosis was significantly lower after implantation of oxygen releasing microspheres.

References

1. Alexis, F. Polym Int 54, 36, 2005.

2. Novosel, E.C., Kleinhans, C, and Kluger, P.J. Adv Drug Deliv Rev 63, 300, 2011.

3. van Blitterswijk, C. Tissue Engineering. Amsterdam: Academic Press; 2008.

4. Caplan, A.I., and Dennis, J.E.. J Cell Biochem 98, 1076, 2006.

5. Rouwkema, J., Rivron, N.C., and van Blitterswijk, C.A.. Trends Biotechnol 26, 434, 2008.

6. Bettahalli, N.M., Arkesteijn, I.T., Wessling, M., Poot, A.A., and Stamatialis, D. Acta Biomat 9, 6928, 2013.

7. Stamatialis, D.F., Papenburg, B.J., Girones, M., Saiful, S., Bettahalli, S.N.M., Schmitmeier, S., and Wessling, M. J Membrane Sci 308, 1, 2008.

8. Silva, E.A., and Mooney, D.J. Biomaterials 31, 1235, 2010.

9. Ilizarov, G.A. Clin Orthop Relat Res, 263, 1989.

10. Harrison, B.S., Eberli, D., Lee, S.J., Atala, A., and Yoo, J.J. Biomaterials 28, 4628, 2007.

11. Oh, S.H., Ward, C.L., Atala, A., Yoo, J.J., and Harrison, B.S. Biomaterials 30, 757, 2009.

12. Pedraza, E., Coronel, M.M., Fraker, C.A., Ricordi, C, and Stabler, C.L. Proc Natl Acad Sci U S A 109, 4245, 2012.

13. Steg, H., Buizer, A.T., Woudstra, W., Veldhuizen, A.G., Bulstra, S.K., Grijpma, D.W., and Kuijer, R. J Mater Sci Mater Med 26, 5542, 2015.

14. Wach, R.A., Adamus, A., Olejnik, A.K., Dzierzawska, J., and Rosiak, J.M. J Appl Polym Sci 127, 2259, 2013.

15. Pego, A.P., Grijpma, D.W., and Feijen, J. Polymer 44, 6495, 2003.

16. Bat, E., van Kooten, T.G., Feijen, J., and Grijpma, D.W. Biomaterials 30, 3652, 2009.

17. Liu, F., Yang, J., Fan, Z., Li, S., Kasperczyk, J., and Dobrzynski, P. Journal of biomaterials science Polymer edition 2011.

18. Zhang, Z., Kuijer, R., Bulstra, S., Grijpma, D., and Feijen, J. Biomaterials 27, 1741, 2006.

19. Dinarvand, R., Alimorad, M.M., Amanlou, M., and Akbari, H. J Appl Polym Sci 101, 2377, 2006.

20. Chevalier, L., and McCann, CD. Int J Environ Waste Manag 2, 245, 2008.

21. Hanh, D.N., Rajbhandari, B.K., and Annachhatre, A.P. Environ Technol 26, 581, 2005. 22. Ma, Y., Zhang, B.T., Zhao, L.X., Guo, G.S., and Lin, J.M. Luminescence 22, 575, 2007.

23. Northrup, A., and Cassidy, D. J Hazard Mater 152, 1164, 2005.

24. Uchida, T., Yagi, A., Oda, Y., and Goto, S. J Microencapsul 13, 509, 1996. 25. Buizer, A.T., Veldhuizen, A.G., Bulstra, S.K., and Kuijer, R. J Biomater Appl 29, 3, 2013.

26. Bao, L., Dorgan, J.R., Knauss, D., Hait, S., Oliveira, N.S., and Maruccho, I.M. J Membrane Sci 285, 166, 2006.

27. Bat, E., Kothman, B.H., Higuera, G.A., van Blitterswijk, C.A., Feijen, J., and Grijpma, D.W. Biomaterials 31, 8696, 2010.

28. Tan, L., Ren, Y., and Kuijer, R. J Biomater Appl 26, 877, 2012.

29. Iyer, R.K., Radisic, M., Cannizzaro, C., and Vunjak-Novakovic, G. Artificial cells, blood substitutes, and immobilization biotechnology 35, 135, 2007.

30. Li, Z., Guo, X., and Guan, J. Biomaterials 33, 5914, 2012.

31. Stokes, C.L., Lauffenburger, D.A., and Williams, S.K. Journal of cell science 99 ( Pt 2), 419, 1991.

32. Labow, R.S., Meek, E., and Santerre, J.P. Journal of biomedical materials research 39, 469, 1998.

33. Hirao, M., Hashimoto, J., Yamasaki, N., Ando, W., Tsuboi, H., Myoui, A., and Yoshikawa, H. Journal of bone and mineral metabolism 25, 266, 2007.

34. Kofoed, H., Sjontoft, E., Siemssen, S.O., and Olesen, H.P. Acta orthopaedica Scandinavica 56, 400, 1985.

35. Mescher, A., and Junqueira, L.C. Basic Histology. 13th edition ed. Columbus, OH: McGrawJ^ill Medical; 2013.

Claims

Claims

1. An oxygen- delivering microsphere based on a biocompatible polymer, the microsphere comprising an agent capable of generating oxygen in situ, the agent being encapsulated in a polymer matrix comprising poly (1,3-trimethylene carbonate) (PTMC).

2. Microsphere according to claim 1, wherein the agent capable of generating oxygen in situ is a metal peroxide.

3. Microsphere according to claim 2, wherein said metal peroxide is CaC , Mg02, or a combination thereof.

4. Microsphere according to any one of the preceding claims, wherein said agent is present in an amount of 1-20 weight%, preferably 3-10 weight%, based on the total weight of the polymer matrix.

5. Microsphere according to any one of the preceding claims, wherein PTMC content of the polymer matrix is at least 50wt%, preferably at least 70wt%, more preferably at least 80wt%.

6. Microsphere according to claim 5, wherein the polymer matrix consists of PTMC.

7. Microsphere according to any one of the preceding claims, wherein said

PTMC is a high molecular weight PTMC.

8. Microsphere according to claim 7, wherein said high molecular weight

PTMC has a number average molar mass (Mn) of at least 220 x 10³ g/mol, preferably at least 250 x 10³ g/mol, more preferably at least 300 x 10³ g/mol.

9. Microsphere according to any one of the preceding claims, wherein the diameter of the microsphere is between 50 and 200 micrometer.

10. A method for preparing an oxygen- delivering microsphere according to any one of the preceding claims, comprising the steps of:

(i) providing a polymer solution by dissolving PTMC in a solvent;

(ii) dispersing the agent(s) capable of generating oxygen in said polymer solution;

(iii) emulsifying the dispersion in a continuous phase comprising a mineral oil and a surfactant; and

(iv) evaporating the solvent.

11. Method according to claim 10, wherein the surfactant is a non-ionic surfactant that is soluble in mineral oil, preferably a sorbitan fatty acid ester.

12. Method according to claim 11, wherein said surfactant is selected from the group consisting of sorbitan monooleate ( SPAN™ 80), sorbitan sesquioleate (SPAN™ 83), sorbitan trioleate (SPAN™ 85) and sorbitan isostearate ( SPAN™ 120).

13. Use of an oxygen- delivering microsphere according to any one of claims

1-9 in a method for tissue engineering, preferably wherein said method comprises cell therapy using autologous cells.

14. Use according to claim 13, wherein said method comprises implantation of a scaffold, preferably a scaffold onto which cells are seeded.

15. Use of an oxygen- delivering microsphere according to any one of claims 1-9 in a method for wound healing.

16. An implantable device comprising a plurality of oxygen- delivering microp articles according to any one of claims 1-9.

17. Implantable device according to claim 16, being a scaffold for tissue engineering.

18. Implantable device according to claim 17, wherein the scaffold is a ceramic or polymer scaffold.

19. Implantable device according to any one of claims 16- 18, the device being provided with living cells, preferably mammalian cells, most preferably human cells.

20. Implantable device according to claim 19, provided with bone marrow cells, osteoblasts, mesenchymal stem cells, cartilage cells, embryonic stem cells, gene transfected cells, endothelial cells and combinations thereof.

21. A method for providing a porous scaffold into which a plurality of oxygen- delivering microp articles is seeded, comprising

- providing a suspension of oxygen-releasing microspheres according to any one of claims 1-9 in a suitable solvent, preferably n-hexane;

- setting the scaffold into a syringe

- drawing the suspension of microspheres and some air into the syringe, and closing the syringe is closed with a cap

- creating a low pressure within the syringe to obtain a homogenous microsphere distribution in the scaffold

- removing excess solvent .

22. A method of forming tissue, the method comprising (a) providing an implantable device according to any one of claims 16 to 20, (b) covering at least part of the surface of the scaffold with living cells capable of forming tissue; and (c) culturing the scaffold under conditions suitable to grow tissue on and/or in the scaffold.

23. A method for the treatment of a tissue pathology in a subject, the method comprising steps (a), (b) and (c) of claim 22, followed by step (d) of introducing the scaffold into the subject, and wherein the cells used in step (b) are capable of treating the tissue pathology.

24. Method according to claim 23, wherein the tissue pathology comprises tissue loss, tissue damage, tissue injury, or combinations thereof.

25. Method according to claim 23 or 24, wherein the treatment comprises tissue remodeling, tissue repair, tissue regrowth, tissue resurfacing, tissue regeneration, or any combination thereof.