WO2012093939A1

WO2012093939A1 - Particles comprising calcium phosphate and use thereof

Info

Publication number: WO2012093939A1
Application number: PCT/NL2012/050008
Authority: WO
Inventors: Yuelian Liu; Daniel WISMEIJER
Original assignee: Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patiëntenzorg
Priority date: 2011-01-05
Filing date: 2012-01-05
Publication date: 2012-07-12

Abstract

The present invention relates to new calcium phosphate particles, use of said particles in the treatment and/or prevention of a bone defect, preferably a peri-implantitis induced bone defect, and a method for producing said particle. When applied to a bone defect, the particles according to the invention, optionally mixed with additional bone fillers, improve bone formation and thereby healing to the bone defect, in comparison to when the particles according to the invention are not applied to said bone defect. In addition, the present invention relates to a method for treating a medical device that is (to be) placed into bone, thereby improving osseointegration of said medical device, in comparison to a medical device not treated according to the method. The invention thus also entails a medical device comprising said particles.

Description

Title: Particles comprising calcium phosphate and use thereof Field of the invention

The present invention relates to particles comprising calcium phosphate, use of said particles in the treatment and/or prevention of a bone defect, for example, and preferably a bone defect induced by peri-implantitis, and a method for producing said particles. In addition, the present invention relates to a method for treating a medical device that is (to be) placed into bone. The invention thus also entails a medical device comprising said particles.

Description of the background art

In dental implantology, maxillofacial surgery and orthopedics the repair of critical-sized bone defects is a great challenge (Liu Y. et al. 2010). The widely known gold standard for the repair of bone defects is the use of autologous bone. This is an effective method because bone formation is stimulated by the presence of bone scaffolds, osteoinductive growth factors and osteogenic cells (Liu Y. et al. 2010, Sokolsky-Papkov M. et al. 2007).

However, several studies demonstrate the limitations of this method: limited bone supply, prolonged surgeries for recruiting the graft, donor-site pain and morbidity (Sokolsky-Papkov M. et al. 2007). These drawbacks made researchers to develop bone fillers which can be premixed with autologous bone grafts, so the bone filling volume can be enlarged.

In clinics, the widely applied bone filler to be premixed with autologous bone is deproteinized bovine bone (e.g. Bio-Oss®). Although this bone-filler is both biocompatible and

biodegradable (Lysiak-Drawl K. et al. 2008), the effectiveness is limited due to the absence of osteogenic cells and osteoinductive growth factors. This results in limited osteoconductive and osteoinductive effects (Liu Y. et al. 2010, Wu G. et al. 2010a). Therefore, deproteinized bovine bone can only be used by premixing with autologous bone (Lysiak-Drwal K. et al. 2008).

Moreover, there are many studies on the risks of the appliance of deproteinized bovine bone (Wenz B. et al. 2001). For example, it must be considered that there is a risk of transmitting diseases even if deproteinized. Due to the occurrence of Bovine Spongiform

Encephalopathy (BSE) in cattle and the new variant of Creutzfeldt-Jacob disease in humans it is important to keep this in mind (Muller W. et al. 2000). Another approach to repair bone defects is to inject osteoinductive agents locally to stimulate the bone formation (Liu Y. et al. 2010). To be successful these agents have to be delivered properly to the site of the bone defect, so that the osteoinductive effect is prolonged. However, this method is not satisfactorily due to the fact that the agents are depleted too fast (Liu Y. et al. 2010, Wu G. et al. 2010b). Also, it has been tried to stimulate bone formation by applying carriers or coatings incorporating osteogenic agents. This also resulted in unsatisfying results because of complete depletion in 13 days (Wu G. et al. 2010b, Sokolsky-Papkov M. et al. 2007 ). A higher dose of osteogenic agents will have no effect and will result in side-effects for the patient (Sokolsky-Papkov M. et al. 2007).

Description of the invention

The present invention overcomes at least one of the above-mentioned problems in the prior art by providing particles that can be used in a new method for treating and/or preventing a bone defect and/or for treating a medical device such as a bone implant (e.g. a dental implant or hip implant).

In a first aspect, the present invention relates to a particle that comprises a core comprising, or substantially consisting of, amorphous calcium phosphate and a first layer around said core, wherein said first layer comprises or substantially consists of crystalline calcium phosphate, and an osteoconductive or osteoinductive agent or both.

In other words, the present invention relates to a particle that comprises:

a) a core comprising, or substantially consisting of, amorphous calcium phosphate; b) around the core of a), a layer comprising, or substantially consisting of, crystalline calcium phosphate, wherein said layer further comprises at least one

osteoconductive and/or at least one osteoinductive agent or both. Preferably said layer comprises at least one osteoinductive agent, and preferably at least one antibiotic. The present inventors found that by applying particles according to the invention to a bone defect, e.g. as a bone filler material, surprising and particular advantageous effects can be achieved. A bone defect is a disturbance of normal functioning of a bone. Bone defects may relate to bones and/or joints, which are associated with impaired functioning. Examples of bone defects that may be treated by means of particles according to the present invention include diseased bone such as cancerous bone, impaired functioning of hip/knee/shoulder joints, peri-implantitis induced bone defects, and so on. Bone defects may be caused by apicoectomy, extirpation of cysts or tumors, tooth extraction, or surgical removal of retained teeth. Also encompassed by the term bone defect are cartilage defects, for example destruction of cartilage surfaces in joints. A bone filler is considered a material that may be used to fill up or narrow a bone defect (a gap in natural bone) and which material may be replaced by natural bone over time, e.g. over 1 , 2, 3, 4, 5, 6 months. Alternatively, the material can be used to repair undesired conditions of bone or to (temporarily) replace or substitute natural bone.

The particles according to the invention are particularly suitable for use in so-called critical- sized and larger bone defects. Critical-sized bone defects are to be defined as the smallest size intraosseous wound that will not spontaneously heal completely with bone tissue, or the defect will heal by connective tissue during the lifetime of the animal or human (see e.g. Liu, Y et al 2010).

For example, when the particles according to the invention are applied to a bone defect, prolonged release of osteoconductive and/or osteoinductive agents (and if applicable, of the at least one antibiotic), in particular of the osteoinductive agent(s) may be achieved which may improve and/or may prolong bone formation at the site of the bone defect, in comparison to if prior art methods are applied to the same bone defect. Bone formation at the site of a bone defect is important for proper healing of the bone defect. Accordingly, by applying particles according to the invention to a particular site of a bone defect, healing of the bone defect may be improved and thereby normal functioning of the bone wherein the bone defect is (was) located may be restored. As such, medical attention is (no longer) necessary with respect to the site where the bone defect was located. Without being bound to any theory, the present inventors believe that the particles according to the invention may, over an extended period of time, promote differentiation of bone marrow stromal cells into osteoblasts, as well as the ingrowth of osseous tissue. Moreover, adherence of osteoblasts may be promoted. These mechanisms typically stimulate bone formation and allow proper healing of a bone defect.

For example, when particles according to the invention are applied to a bone defect, osteoconductive and/or osteoinductive agents (particularly osteoinductive agent(s) and if applicable the at least one antibiotic) may be released to the site of the bone defect over a period extending 10, 15, 20, 25, 30, 50, 75 or 100 days or more, thereby stimulating bone formation at the site of a bone defect over an extended period of time, in comparison to if prior art methods are applied to the same bone defect. The particles according to the invention may be used alone, i.e. it may not be necessary that the particles are immobilized to or coated on a substrate, such as to a (surface of a) bone implant, such as a titanium implant. Preferably, the particles according to the invention are not used as a coating on a substrate, because this greatly improves flexibility in the use of the particles according to the invention, and it allows use of the particles for treating bone defects wherein no substrates (implants) are used. Also, it allows for using the particles according to the invention for making granules, noodles, cylinders, tablets, blocks, or any other type of 3D shape that can be custom made and used to treat a particular bone defect and that preferably fits a particular bone defect (e.g. a hole). This may be achieved using computer software and/or compression of the particles according to the invention in a suitable mould. Alternatively, the particles according to the invention may be applied as a paste, which is considered a malleable consistency, or as an injectable. The form in which the particles according to the invention are applied may strongly vary depending on the particular bone defect to be treated. For example, bone defects that are difficult to reach by a medical professional may be treated with an injectable form, whereas bone defects that are more easy to reach can be treated with a paste or granules, noodles, cylinders, tablets, blocks and the like. The invention may also allow to more closely mimic the 3D structure of natural bone that would normally be present at a particular site of a bone defect. For example, inner structure of bone, e.g. the marrow has a less dense structure that more outer structures of bone, e.g. the outer layer, such as the outer 0.3-0.8 cm. For this, a mould may be made comprising the particles according to the present invention that is less dense in its inner structure that it is in its more outer structure, in such a way to closely represent the natural bone. Use of the particles according to the invention may reduce the amount of autologous and/or animal derived bone necessary to treat a particular bone defect, in comparison to a situation wherein prior art methods are used to treat said particular bone defect. Use of the particles according to the invention may even obviate the need to use autologous and/or animal derived bone.

Alternatively, the particles according to the invention may be used as a coating on a bone implant and/or as a coating on natural bone, to stimulate bone formation at a site of a bone defect. Using the particles as a coating on a bone implant may also improve

biocompatibility/osteoconductivity/osteoinductivity of the bone implant, in comparison to if such bone implant is not coated with particles according to the invention. The particles according to the invention may also be applied mixed with other types of bone fillers. Such other types of bone fillers may include autologous bone (harvested from the patient's own body, e.g. from the iliac crest), allograft bone (cadaveric bone from another individual of the same species), xenograft bone (animal derived bone), and synthetic materials having similar mechanical and/or biological properties to natural bone (e.g.

hydroxyapatite).

Non limiting examples of suitable other types of bone fillers which may be mixed with the particles according to the invention include demineralized (bovine) bone matrix,

deproteinized (bovine) bone, hydroxyapatite, calcium phosphate injectable cement, calcium phosphate cement, calcium phosphate putty, synthetic tricalcium phosphate, beta-tricalcium phosphate, coralline hydroxyapatite, cancellous hydroxyapatite, bovine collagen,

hydroxyapatite-coated bovine collagen, and/or a combination of bovine collagen,

hydroxyapatite, and tricalcium phosphate (see for details on the different bone fillers and/or further bone fillers that may be mixed with the particles according to the invention e.g. de Long et al 2007). Deproteinized bovine bone has a chemical composition and architectural geometry that is similar to that of human bone.

Encompassed by the present invention is for example a mixture comprising at least 1 , 5, 10, 25, 50, 75, 90% or 99% by dry weight particles according to the invention with the

remaining, e.g. 1 , 5, 10, 25, 50, 75, 90% or 99% by dry weight of the mixture being other types of bone fillers such as those described above, preferably deproteinized bovine bone.

As such, a bone filler mixture can be obtained that derives part of its volume from one or more of the above-mentioned bone fillers, for example deproteinized bovine bone, and that, because it further contains particles according to the invention, at the same time has

(improved) osteoinductive properties, in comparison to if said mixture does not contain particles according to the invention. The core of the particle(s) according to the invention may consist of at least 1 , 10, 20, 30, 40, 50, 60, 75, 90, or 95% by dry weight of the core of amorphous calcium phosphate and the first layer of the particle(s) of the invention may consist of at least 1 , 10, 20, 30, 40, 50, 60, 75, 90, or 95% by dry weight of the layer of crystalline calcium phosphate. The remaining of the core and/or the first layer preferably consists of additives that are non-toxic and suitable for being applied to the human body, such as osteoinductive and/or

osteoconductive agents as described herein, binding agents, antibiotics, fillers, drugs, and/or the like. Typically, higher calcium phosphate content of the particles may improve osteoconductivity and/or osteoinductivity of the particles according to the invention, in particular if compared to the same content consisting of materials without substantial osteoconductive and/or osteoinductive properties. The term calcium phosphate encompasses compositions comprising calcium ions, and phosphate ions. Such compositions include but are not limited to calcium ions (Ca²⁺) together with orthophosphates (P0₄ ^3"), metaphosphates or pyrophosphates (P₂0₇ ^4") and optionally hydrogen or hydroxide ions. Further examples of such compositions are calcium dihydrogen phosphate (Ca(H₂P0₄)₂), Calcium hydrogen phosphate (CaHP0₄), tricalcium phosphate or tricalcic phosphate (Ca₃(P0₄)₂, sometimes referred to as calcium

orthophosphate), hydroxy apatite (Ca₅(P0₄)₃(OH)), apatite (Ca₁₀(PO₄)6(OH, F, CI, Br)₂), and octacalcium phosphate (Ca₈H₂(P0₄)₆.5H₂0). Other examples are biphasic calcium

phosphate (hydroxyapatite + tricalcium phosphate) and triphasic calcium phosphate

(hydroxy apatite + tricalcium phosphate + dicalcium phosphate). Hydroxyapatite is a naturally occurring mineral form of calcium apatite with the formula Ca₅(P04)₃(OH), but is usually written Ca10(PO4)6(OH)2 to denote that the crystal unit cell comprises two entities. Biphasic calcium phosphate consists of hydroxyapatite and tricalcium phosphate (Ca₃(P0₄)₂).

Octacalcium phosphate is calcium phosphate with a formula Ca₈H₂(P0₄)₆.5H₂0 (see e.g. Reacquel (1985). Other types of calcium phosphate that may be used in the present invention are described in WO 02/100331 , WO 03/043673, WO 2006/016807, and WO 2008/1 19053. Preferably, the crystalline calcium phosphate according to the invention is octacalcium phosphate.

As used herein, "amorphous" means a non-crystalline material with significant amorphous character, for example an amorphous material content greater than 75%, preferable 90%, or 95% by weight, which may be characterized by for example X-ray diffraction as known by the skilled person (for example as described in the Example). Crystalline materials have a substantial part of their atoms placed in regular lattices that can form geometric shapes, which can also be verified upon X-ray diffraction, see for an example of a handbook for X- ray diffraction Azaroff et al. 1974.

The term "osteoinductive" refers to the capability of inducing transformation of mesenchymal stem cells into bone precursor cells such as osteoblasts and/or chondrocytes. Moreover, the term also encompasses capability of inducing transformation of osteoblasts into osteocytes, mature bone cells. Many different osteoinductive agents are known in the art including Bone Morphogenetic Proteins (see e.g. Wozney 2002), and Transforming Growth Factor beta (see e.g. Zhang et al 2009). An agent is osteoconductive if it allows or is conducive to bone formation (see e.g. Wozney 2002). The term osteogenic means being inductive to formation of new bone for example by osteoblasts (see for example Caetano-Lopes et al 2007). Bone formation as used herein thus refers to synthesis of new bone at the site of a particular bone defect, preferably wherein said bone formation heals the bone defect such that the bone regains its normal functioning.

Some patent documents describe calcium phosphate vehicles, devices, granules and the like to be used for treating bone defects, or regeneration in periodontology. None of them describe the (use of a) particle according to the present invention.

WO 02/100331 discloses calcium phosphate delivery vehicles for osteoinductive proteins for treating a bone defect (see abstract). A composition is described comprising a calcium phosphate material, an effective amount of an effervescent agent and a osteogenic protein like BMP.

WO 03/043673 discloses a device having osteoinductive and osteoconductive properties. The device comprises a carrier containing calcium phosphate which may be in the form of granules and an osteoinductive protein, wherein the carrier is homogeneously coated with said protein. The device may be used for bone augmentation, treating bone defects, and treating regeneration in periodontology.

WO2006/016807 discloses a biomimetic process for coating a substrate, medical device or implant. The process refers to immersing the substrate in a acidified biomimetic composition comprising calcium-, magnesium-, phosphate-, and bicarbonate ions and a bioactive substance.

WO 2008/1 19053 discloses a medical appliance useful for bone repair, regeneration, maintenance, or augmentation, comprising: a carrier matrix; an osteoinductive agent; and an osteoinductive enhancer for modulating the activity of the osteoinductive agent, wherein said osteoinductive agent and said osteoinductive enhancer are both integrated within the carrier matrix.

Liu, Y et al (2010) discloses biomaterial coated with a first layer of amorphous calcium phosphate and a second layer of crystalline calcium phosphate, wherein the crystalline layer is to be functionalized by the incorporation of bioactive agents such as bone morphogenetic protein-2 (BMP-2). In contrast, if particles according to the present invention are used as a coating for a device (such as a (dental) implant) or biomaterial (e.g. autologous or deproteinized bovine bone), the surface of the device will become coated with a layer of particles according to the invention, i.e. particles comprising an amorphous calcium phosphate core and a crystalline calcium phosphate layer surrounding the core. Upon analyzing such coating separate cores may be distinguished, in contrast to a prior art coating.

In comparison with prior art methods, use of particles according to the invention may sustain prolonged osteoconductivity, probably because of slower release of osteoinductive and/or osteoconductive agents. In addition, during normal use the particles according to the invention may not entail the risk of (iatrogenic) disease transmission, may be free of antigens, and their supply may not be limited to availability of human or animal donors.

The release rate of the osteoinductive and/or osteoconductive agents (particularly osteoinductive agent(s), and if applicable at least one antibiotic) may advantageously be further decreased by providing the particle according to the invention of a further layer (i.e. a surrounding), wherein said further layer comprises or substantially consists of amorphous calcium, preferably said further layer being positioned on the first layer of crystalline calcium phosphate. Said further layer may consist of at least 50, 60, 75, 90, or 95% by weight of the layer of amorphous calcium phosphate, with the remaining being materials that are preferably non-toxic to humans and for example being osteoconductive and/or

osteoinductive agens, anti-biotics, drugs, fillers, and/or the like. As will become more clear from Figure 1 (Type III particle), said further layer may cover (or incorporate) more than one, such as 2, 3, 4, 5, or more cores comprising amorphous calcium phosphate provided with a crystalline layer comprising an osteoconductive and/or osteoinductive agent or both, and/or at least one antibiotic.

The release rate of the osteoinductive and/or osteoconductive agents (particularly osteoinductive agent(s), and possibly at least one antibiotic) may advantageously even further be decreased by providing the particle according to the invention of, preferably being positioned on said further layer, an additional layer or layers comprising or substantially consisting of crystalline calcium phosphate, preferably comprising an osteoconductive or osteoinductive agent or both. Said further layer(s) may consist of at least 50, 60, 75, 90, or 95% by weight of the layer of crystalline calcium phosphate, with the remaining being materials that are preferably non-toxic to humans and for example being osteoconductive and/or osteoinductive agens, anti-biotics, drugs, fillers, and/or the like.

Accordingly, the particle according to the invention may comprise: a) a core comprising, or substantially consisting of, amorphous calcium phosphate, wherein the core may or may not comprise an osteoconductive or osteoinductive agent;

b) around the core of a), a layer comprising, or substantially consisting of, crystalline calcium phosphate, wherein said layer further comprises at least one

osteoconductive or at least one osteoinductive agent or both;

c) preferably, a layer comprising, or substantially consisting of, amorphous calcium

phosphate, preferably positioned on or around the layer of b), wherein the layer may or may not comprise an osteoconductive or osteoinductive agent;

d) preferably, a layer comprising, or substantially consisting of, crystalline calcium

phosphate, preferably comprising an osteoconductive or osteoinductive agent or both, preferably positioned on or around the layer of c).

The osteoinductive agent that may be comprised in any of the crystalline calcium phosphate layer(s) is preferably selected from the group consisting of Transforming Growth Factor beta protein family or Bone Morphology Protein family. Particular advantageous results may be obtained when the osteoconductive and/or osteoinductive agent is BMP-2, BMP-6, BMP-7, BMP-9, BMP-12, or BMP-13, wherein BMP-2 is most preferred because of good results (see e.g. Liu et al, Bone 36, 2005 P.745-757 for details on BMP). Alternatively or additionally the particles may comprise an antibiotic, which may prevent infection, or additional (bone) growth factors.

The different layers comprising crystalline calcium phosphate of the particle according to the invention may each comprise different osteoconductive agents, osteoinductive agents and/or other drugs such as antibiotics. For example, the most outer layer comprising crystalline calcium phosphate may comprise an antibiotic, while the second outer layer comprising crystalline calcium phosphate may comprise BMP-2.

It may also be possible to comprise an osteoconductive agent, osteoinductive agent and/or other drugs in any of the amorphous calcium phosphate layers, including the core, of the particle(s) according to the invention. It is however preferred that the osteoconductive and/or osteoinductive agents are comprised in the crystalline calcium phosphate layers, since these provide more room in their three dimensional structure to offer place to the said agents, in comparison to the more dense amorphous core/layer.

One may vary the diameter of the particle(s) according to the invention. A particle is considered to be an aggregation of sufficiently many atoms or molecules that it can be assigned macroscopic properties such as volume, density, and temperature. In general, it is preferred that the particles to be used in the method according to the invention have an average diameter of between 10 nm -10000 μηι, preferably 1 μι - 5000 μηι, more preferably 100 μηι - 2500 μηι, most preferably 250 μηι - 1000 μηι, because these diameters are useful in most applications. Average particle diameter may be measured by light microscopic examination, sieve analysis, and/or sedimentation (gravitational settling) techniques (see e.g. European Commission Directorate General 2002). Preferably a random sample of between 10-100000 particles, such as of 10, 50 or 100, or 1000 particles is taken, and the average particle diameter is determined by adding up the diameters of the individual particles and dividing the result by the number of particles. For bone defects, such as exceeding the critical size as described herein (see also Liu et al 2010) by more than 2, 5, or 10 times, or if the bone has been affected for example due to infection, e.g. wherein the bone has become more porous if compared to healthy bone, one may use relatively larger particles, such as particles exceeding 100, 500, or 1000 μηι in diameter.

Comparably, one may vary the ratio between the diameter of the core of the particle and the diameter of the whole particle. Preferably said ratio is between 1 : 100 and 99.9: 100, 10: 100 and 99.5: 100, more preferably between 20: 100 and 99: 100, even more preferably between 40: 100 and 98: 100, even more preferably between 60: 100 and 97: 100, most preferably between 80: 100 and 95: 100 (diameter core : diameter whole particle).

The particles preferably are biocompatible which may minimize host inflammatory reaction. As such, biocompatible means that no (substantial) detrimental response requiring medical attention is elicited in the host receiving the material that is biocompatible.

Preferably, the particles are biodegradable, which means that they disappear upon resorption over time when inside a subject's body and preferable the particles are replaced by natural bone over time, with no loss in the volume or integrity. This may be effected through the action of body fluids, enzymes or cells. Improved biodegradability generally refers to resorption in vivo reduced in time. Proper biodegradability may eliminate the need for a second operation within a certain time. For example, particles may be used that completely resorb after placement in a subject's body relatively fast as in less than 5 years, more preferably less than 2 years, more preferably less than 1 year, most preferably less than 6 months. Alternatively, particles may be used that resorb relatively slow, i.e. that do not completely resorb upon placement in a subject's body after the above-mentioned periods, e.g. particles that completely resorb upon placement in a subject's body over a period between 5 and 20 years, more preferably between 10 and 15 years. Biphasic calcium phosphate, triphasic calcium phosphate and octa calcium phosphate are examples of calcium phosphate types that resorb relatively fast, hydroxyapatite is an example of a calcium phosphate type that resorbs relatively slow. Depending on the condition of the bone defect and/or the surrounding bone, one may choose for different types of calcium phosphate that differ in biodegradability for use in the particle(s) according to the invention. For bone defects that are relatively large, for example 5, or 10 times the size of a critical-sized bone defect, or bone defects that have been affected for example due to infection, e.g. wherein the bone has become more porous if compared to healthy bone, one may choose to use a type of calcium phosphate that biodegrades relatively slowly when implanted, for example over 10, 15, or 20 years, which applies to for example hydroxyapatite. For smaller bone defects, for example a critical-sized bone defect or one of 2, or times said critical size, one may choose to use a type of calcium phosphate that biodegrades more quickly, for example over 1 , 2, or 3 months, such as is the case for biphasic calcium phosphate, triphasic calcium phosphate and octa calcium phosphate. The skilled person thus knows how to make appropriate choices for the type of calcium phosphate based on resorption speed and type of bone defect.

Different types of calcium phosphate may be used in the particle according to the invention. For example, the crystalline calcium phosphate of the particle preferably comprises or substantially consists of octocalcium phosphate. The particles preferably have a calcium to phosphate ratio that is comparable to naturally occurring bone.

Also encompassed is a composition comprising particles according to the invention, preferably wherein the composition comprises at least 50% particles, more preferably at least 60, 70, or 80%, most preferably at least 90% by weight of the composition. In addition to the particles according to the invention, the composition may further comprise

supplementary materials that are biocompatible, such as pharmaceutically acceptable salts, water, polysaccharides, proteins, synthetic polymers, natural polymers such as collagen, glycogen, chitin, celluloses, starch, keratins, silk, surfactants, solid structures such as sponges, meshes, films, fibers, gels, filaments, autologous bone and/or demineralized and/or deproteinized (bove) bone. Preferably the composition is decontaminated from contaminants such as micro-organisms.

The particle and/or the composition according to the invention may be used in the treatment and/or prevention of a bone defect such as cavities in bones, disorders accompanied with loss of bone tissue, and/or peri-implantitis, preferably a peri-implantitis induced bone defect. Peri-implantitis can be defined as an inflammatory reaction typically resulting in the loss of supporting bone in the tissues surrounding (a) functioning implant(s) (Bobia and Pop 2010). Under certain circumstances, implant bone grafting may be necessary to repair peri- implantitis induced bone defects so that the jawbone can hold the (dental) implant secure. The particle(s) of the current invention may be used for said repair.

Also encompassed is a particle or composition according to the invention, for use in the treatment of a bone defect, and/or peri-implantitis, preferably a peri-implantitis induced bone defect, preferably wherein the treatment comprises the steps of

a. optionally, decontaminating a medical device, such as a bone implant,

preferably a dental implant;

b. applying to said optionally decontaminated medical device particles and/or a composition according to the invention. It has been surprisingly found that by the above-described use of the particles according to the invention improved osseointegration of the medical device can be achieved, in comparison to if the particles according to the invention are not used to treat the same medical device. Osseointegration is considered the direct structural and functional connection between living bone and the surface of an (artificial) implant (see e.g. McCutchen, J.W. et al. 1990). A well- osseointegrated implant cannot be moved relative to the surrounding bone into which it is anchored. The method may also reduce fibrous encapsulation of the treated implant, which may be beneficial to osseointegration.

It is important for clinical performance that, if a medical device is to be anchored into bone, the medical device effectively osseointegrates into the bone. However, effective

osseointegration of the implant into the surrounding bone is not always sufficiently achieved, which may lead to infection, inflammation, resorption of the surrounding bone, and/or destabilization of the anchorage of the implant to the surrounding bone (Karoussis et al 2003; Tillander et al 2010).

For example, for long-term integration and durability of hip replacements the state of the bone-implant interface is crucial (Song et al 1999). This also applies to attempts to anchor prosthetic limbs with transcutaneous implants, where poor osseointegration of the implant into the surrounding bone may cause subsequent infection of the peri-implant site (Tillander et al 2010). Typically such infection is caused by staphylococci, with coagulase-negative staphylococci being the most common (Tillander et al 2010).

In the case of dental implants, poor osseointegration of the implant may result in a condition know in the field as peri-implantitis (see e.g. Galnut et al 2001), which is associated with Gram-negative anaerobe micro-organisms, including Spirochetes, like Prevotella intermedia, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacterioides forsythus, Treponema denticola, Prevotella nigrescens, Peptostreptococcus micros and Fusobacterium nucleatum (Bobia and Pop, 2010).

Therefore, the use of a particle or composition according to the invention in the treatment of a bone defect, and/or peri-implantitis, preferably a peri-implantitis induced bone defect according to the invention can particularly be applied to bone implants that are destined to be placed, i.e. anchored, for at least a part, e.g. for 5, 10, 25, 40, 50, 75, or 90%, or more of their volume, into bone of a subject, such as into the jaw at an edentulous site.

Alternatively, said use according to the invention may be applied to implants that are already in the subject's body, such as a dental implant already present at an edentulous site in a subject's mouth (oral cavity). The medical device according to the invention may thus be already present in an animal body, preferably a human, for example at a particular bone defect site. Alternatively, the medical device may, after step b) be applied to a bone defect site.

Suitable medical devices (implants) may include titanium implants, stainless steel implants, titanium alloy implants, or zirconiumor implants, or calcium phosphate implants, wherein calcium phosphate may be in the form of a ceramic (sintered calcium phosphate) and/or a cement (see for further examples of suitable implants e.g. Yuehuei and Draughn (2000). A suitable bone implant may also be a (hardened) bone filler material, and may already comprise particles according to the invention, but typically it does not comprise particles according to the invention.

As a result of the above-mentioned use, the invention also relates to a medical device, preferably a bone implant or a dental implant, more preferably a dental implant,

characterized in that said medical device comprises a particle and/or composition according to the invention. The degree of osseointegration may be verified using impulse testing (Dario et al 2002). For impulse testing, an accelerometer may be attached to the implant of which osseointegration is to be verified. The accelerometer measures acceleration of the implant when percussed with a calibrated hammer, and the acceleration time history, or ATH, is recorded. The ATH is a sine wave of decreasing amplitude, and the rate of the decrease is related to the damping characteristics of the implant. The greater the rate of decreasing amplitude, the higher the degree of osseointegration of the implant (see for further details on the method (Dario et al 2002). Osseointegration may be measured in time, for example 1 , 2, 3, 4, 5, or 6 months after placement of the implant, comparing osseointegration of an implant treated according to the invention, and osseointegration of an implant not treated according to the invention. Osseointegration is considered improved if said rate of decreasing amplitude after a particular time period, e.g. 1 , 2, 3, 4, 5, or 6 months after placement of the implant treated according to the invention, is decreased in comparison to the implant not treated according to the invention.

The present invention also entails a method for improving osseointegration of a medical device, the method comprising the steps of

a. optionally, decontaminating said medical device;

b. applying to said optionally decontaminated medical device particles or a

composition according to the invention.

The method may particularly be applied to medical devices (implants) that are destined to be placed, i.e. anchored, for at least a part, e.g. for 5, 10, 25, 40, 50, 75, or 90%, or more of their volume, into bone of a subject, such as into the jaw at an edentulous site. Alternatively, the method may be applied to implants that are already in the subject's body, such as a dental implant already present at an edentulous site in a subject's mouth (oral cavity).

Alternatively, the medical device may, after step b) be applied to a bone defect site.

Suitable medical devices (implants) may include titanium implants, stainless steel implants, titanium alloy implants, or zirconiumor implants, or calcium phosphate implants, wherein calcium phosphate may be in the form of a ceramic (sintered calcium phosphate) and/or a cement (see for further examples of suitable implants e.g. Yuehuei and Draughn (2000).

The method may result in improved osseointegration and/or more stable anchorage of the treated medical device such as a (bone) implant into the surrounding bone, if compared to a medical device not treated according to the method. The method may be applied in vivo as well as ex vivo. In the latter case, it may be necessary that the implant to be treated is made accessible prior to applying the method, for example, in the case of peri-implantitis, by applying surgical flap access as known by the skilled person (see e.g. Klinge et al. (2005). The method preferably is for treating medical devices that are (made) accessible without treatment of a human or animal body by surgery or therapy. The method thus preferably does not encompass treatment of a human or animal body by surgery or therapy.

The particle and/or the composition according to the invention may be produced by following the following steps:

a) incubating an aqueous solution comprising sodium chloride, calcium chloride, sodium hydrogen phosphate, sodium bicarbonate, in the presence of magnesium chloride, such that particles of amorphous calcium phosphate are formed;

b) optionally, immersing the particles of step a) in an aqueous solution comprising calcium chloride, sodium chloride, sodium hydrogen phosphate, potassium chloride, magnesium chloride, and sodium hydrogen carbonate, such that a layer of amorphous calcium phosphate is formed on the particles;

c) immersing the particles obtained in step a), or step b), in an aqueous solution comprising calcium chloride, sodium chloride, sodium hydrogen phosphate, in the presence of an osteoconductive and/or osteoinductive agent, such that a layer of crystalline calcium phosphate comprising an osteoconductive and/or osteoinductive agent is formed on the particle;

d) optionally, mixing the particles obtained in step c) with amorphous calcium phosphate precipitate, such that a layer of amorphous calcium phosphate is formed on the particles, wherein the calcium phosphate precipitate is obtained by incubating an aqueous solution comprising sodium chloride, calcium chloride, sodium hydrogen phosphate, sodium bicarbonate, in the presence of magnesium chloride, such that amorphous calcium phosphate precipitate is formed;

e) optionally, repeating steps c) and d),

wherein step a) preferably is performed at an acidic pH, such as a pH below 6.5, preferably below pH 6. A particle according to the invention may thus be obtained by the above-described method.

More preferably, the particle and/or the composition according to the invention may be produced as follows.

An amorphous calcium phosphate core may be produced by incubating a supersaturated (e.g. 2-10, preferably about 5 -fold) calcium phosphate solution containing:

150-250 mM (preferably about 200 mM) HCI; 15-25 mM (preferably about 20 mM)

CaCI₂ ^«2H₂0; 600-800 mM (preferably about 680 mM) NaCI; 5-15 (preferably about 10 mM) Na₂HP0₄; 200-300 mM (preferably about 250 mM) TRIS base (such that the pH is 6-9, preferably about 7.4). Preferably the solution is incubated by shaking it at 20-100, preferably 50 agitations/min at a temperature of 30-45°C, preferably at about 37°C. This procedure is known to the skilled person and further details may for example be found in Liu et al 2010 by referring to the standard procedure for producing biomimetic CaP coating as described therein. After 15-35 hours, such as after about 24 hours, calcium phosphate precipitations (gel-like) may be filtered or otherwise collected from the solution, which may be dried for examples at room temperature. To obtain a desired particle size, the particles may be ground into different sizes before drying. If the supersaturated calcium phosphate solution also comprises an osteoinductive and/or osteoconductive agent, such agent will be incorporated in the particle.

A fine, dense layer of amorphous calcium phosphate to be deposited on a amorphous calcium phosphate core or amorphous calcium phosphate layer may be produced by immersion in 2-10, preferably 5-fold concentrated body fluid being 10-15 mM (preferably about 12.5 mM) CaCI₂ ^«2H₂0, 600-800 mM (preferably about 684 mM) NaCI, 2-8 mM

(preferably about 5 mM) Na₂HP0₄, 2-8 mM (preferably about 5 mM) KCI, 5-10 mM

(preferably about 7.5 mM) MgCI₂ ^« 6H₂0, and 15-25 mM (preferably about 21 mM) NaHC0₃) for 10-35, preferably about 24 hours at 25-45°C, preferably about 37°C (granule/liquid ratio: 0.2-1 , or about 0.5 g/100 ml). Under these conditions, a fine dense layer of amorphous CaP can be formed, which may serve as a seeding substratum for the subsequent deposition of a more substantial crystalline layer, although the crystalline layer may also be formed directly on a amorphous calcium phosphate core/layer.

A crystalline calcium phosphate layer to be deposited on an amorphous calcium phosphate core or amorphous calcium phosphate layer may be produced by immersion in a

supersaturated calcium phosphate solution, such as one containing 30-50mM (preferably about 40 mM) HCI, 1-7 mM (preferably about 4 mM) CaCI₂ ^«2H₂0, 100-150 mM (preferably about 136 mM) NaCI, 1-4 mM (preferably about 2 mM) Na₂HP0₄, and 30-70 mM (preferably about 50 mM) Tris) for 24-96 hours (preferably about 48 hours) at 25-45°C, (preferably about 37°C) (granule/liquid ratio: 0.1-1 , or preferably about 0.5 g/100 ml). To incorporate an osteoinductive or osteoconductive agent, such agent should be added to supersaturated calcium phosphate solution. After drying at room temperature, or any other way, the layer is deposited. This protocol for forming a crystalline calcium phosphate layer is known to the skilled person for example from Liu et al 2004.

An amorpous calcium phosphate layer to be deposited on particles with an outer crystalline calcium phosphate layer may be produced for example by mixing the particles with the calcium phosphate precipitations (gel-like) as described above. After drying, the particles may be ground and filtered to obtain a desired particle size, such as of 0.25-1 mm.

Brief descriptions of drawings

Fig. 1. Schematic illustration of three types of calcium phosphate particles for protein encapsulation. Type I particles comprise amorphous calcium phosphate and contain protein in the whole volume (not according to the invention). Type II particles have a core comprising amorphous calcium phosphate, and on said core a layer comprising crystalline calcium phosphate, which layer comprises protein. Type III particles are an assembly of small-sized Type II particles contained in a further layer (surrounding) of amorphous calcium phosphate.

Fig. 2. Protein encapsulation efficiency (A) and Cumulative protein release kinetics from the above-mentioned three types of particles (FITC-BSA: 20 μg/ml) soaked in PBS at pH of 7.4 and at 37°C (B). Time points: hour 3, 6, day 1 , 2, 3, 5, 7, 10, 13, 17, 22, 28.

Fig. 3. Osteoclast-mediated Ca2+ release in cell culture medium at each time point of day 3, 7, 10, 14, 17, 21 and 24. Fig. 4. Cumulative Ca2+ release from the above-mentioned three types of calcium phosphate particles (FITC-BSA: 20 g/ml) soaked in PBS at pH of 4.5, 6.0, and 7.4 and at 37°C. Time points: hour 3, 6, day 1 , 2, 3, 5, 7, 10, 13, 17, 22, 28 and 35.

Fig. 5. Total osteoblast DNA content in calcium phosphate particles and deproteinized bovine bone groups (A); and Alkaline phosphatase activity (ALP), normalized to DNA concentration at each time point of day 1 , 3, 7 and 14 (B). EXAMPLE

We have developed and characterised bone graft substitutes, (biomimetic) calcium phosphate (BioCP) granules (particles), using a coprecipitation technique for controlled protein delivery (release). As explained earlier herein, the high initial burst release of protein is a main drawback of existing bone graft substitutes.

We produced 3-dimensional calcium phosphate constructs, calcium phosphate granules, in/on which bioactive agents are coprecipitated. The physicochemical and biological properties of calcium phosphate granules were characterised. Three protein loading modalities were designed for the granules for long-term controlled protein delivery (see Figure 1):

Type I granules comprise amorphous calcium phosphate and contain protein in the whole volume;

- Type II granules have a core comprising amorphous calcium phosphate, and on said core a layer comprising crystalline calcium phosphate, which layer comprises the protein; and

Type III granules are an assembly of small-sized Type II granules contained in a further layer (surrounding) of amorphous calcium phosphate.

Fluorescein-isothiocyanate labelled bovine serum albumin (FITC-BSA) was used as a model protein to study the protein distribution in the granules and its release kinetics.

The present example shows that Type II granules release protein more slowly than Type I granules, and Type III granules release protein even more slowly than Type II granules (see Figure 2B). In addition, it is shown that osteoclasts exhibit attachment to the different types of granules, and resorption pits were frequently found on BioCP. This observation as well as the increased Ca²⁺ release in the medium (Figure 3) indicates that BioCP granules are degraded by osteoclasts. Moreover, as a bone graft substitute, BioCP was able to support and promote proliferation and differentiation of murine osteoblasts better than deproteinized bovine bone DDB (Bio-Oss®), which is a well-established bone graft material.

Materials and methods

Fabrication of BioCP material and design of protein loading

A five-fold supersaturated CaP solution [200 mM HCI, 20 mM CaCI₂*2H₂0, 680 mM NaCI, 10 mM Na₂HP0₄, 250 mM TRIS base (pH 7.4)] was incubated in a shaking water bath (50 agitations/min) at 37°C according to the standard procedure for producing biomimetic CaP coating (Liu et al 2001). After 24 hours, CaP precipitations (gel-like) were filtered. After drying at room temperature, BioCP was ground into different sizes of granules or printed into tablets before drying. Three types of BioCP granules (size: 0.25-1.0 mm) were produced with different protein loading modalities: Type I granules contained protein equally in their whole volume (Figure 1); Type II granules contained protein in a surface layer of biomimetic CaP coating (Figure 1); and Type III granules, an assembly of small BioCP granules (size < 0.25 mm) which also contained protein in the biomimetic calcium phosphate coating layer (Figure 1).

Coprecipitation of bovine serum albumin in/on BioCP granules

Fluorescein-isothiocyanate labelled bovine serum albumin (FITC-BSA, Sigma, St. Louis, MO, USA) was employed as a model protein to study its distribution and release from BioCP granules (Vallet-Regi et al 2008). Based on the characterization of the three modalities of protein encapsulation, three different concentrations (10, 20, and 50 μg/ml) of FITC-BSA were used for Type I and Type II to study the encapsulation efficiency, while 20 μg/ml of FITC-BSA was used for Type III. Furthermore, the same concentration of FITC-BSA (20 μg/ml) was used to produce the three types of granules for determination of the protein and Ca²⁺ release kinetics.

Type I BioCP granule fabrication

FITC-BSA was present in the five-fold supersaturated CaP solution (see above) and coprecipitated equally into the BioCP granules during the procedure (Figure 1). After drying, the BioCP block was ground and filtered using metallic mesh filters to obtain Type I granules with a size of 0.25-1.0 mm.

Type II BioCP granule fabrication

Prefabricated BioCP granules (size: 0.25-1.0 mm, see above) were coated (biomimetically) with a layer of calcium phosphate in the presence of a protein according to a well- established biphasic protocol (Liu et al 2004). Briefly, BioCP granules were immersed in fivefold-concentrated simulated body fluid (12.5 mM CaCI₂ ^«2H₂0, 684 mM NaCI, 5 mM

Na₂HP0₄, 5 mM KCI, 7.5 mM MgCI₂ ^« 6H₂0, and 21 mM NaHC0₃) for 24 hours at 37°C (granule/liquid ratio: 0.5 g/100 ml). Under these conditions, a fine dense layer of amorphous CaP was formed on the BioCP granules, which served as a seeding substratum for the subsequent deposition of a more substantial crystalline layer. Although the crystalline layer may also be formed directly on the BioCP granules, it is preferred to apply a seeding layer first as to obtain a more uniform surface of the BioCP granule. The latter crystalline layer was produced by immersing the granules in another supersaturated CaP solution (40 mM HCI, 4 mM CaCI₂ ^«2H₂0, 136 mM NaCI, 2 mM Na₂HP0₄, and 50 mM Tris) for 48 hours at 37°C (granule/liquid ratio: 0.5 g/100 ml). FITC-BSA was present in the latter solution and was subsequently coprecipitated into the biomimetic CaP coating. After drying at room temperature, Type II BioCP granules were retrieved.

Type III BioCP granule fabrication

Small-sized Type II granules were prefabricated (size < 0.25 mm) (see above, for Type II). Afterwards, on average, 0.25g of these small-sized Type II granules was mixed uniformly with the CaP precipitations that had been filtered from 200 ml of the five-fold supersaturated CaP solution (see above). After drying, the mixture was ground and filtered to obtain granules with the size of 0.25-1 mm; thus, Type III granules were retrieved.

Characterization of BioCP

The mechanical strength of BioCP was evaluated by assessing its compressive strength. BioCP was placed in 6 cylinders (diameter: 5mm; height: 8 mm). Each cylinder was crushed at a crosshead speed of 1 mm/min, using a compressive strength machine (Instron 6022, High Wycombe, Bucks, U.K). The compressive strength value was obtained by calculating the average of 6 cylinders.

The BioCP granules were ground into a fine powder and used for powder X-ray diffraction (XRD) analysis. XRD patterns of the samples were recorded with a vertically mounted diffractometer system (Bruker D8 Advance, Bruker AXS, Germany), using Ni-filtered Cu Ka radiation generated at 40 kV and 40 mA. Specimens were scanned from 5° to 60° 2Θ (where Θ is the Bragg angle) in continuous mode. The biomimetic CaP coating has been already analysed by XRD in a previous study (Wu et al 2010).

The morphology of the BioCP granules with and without incorporated FITC-BSA was visualised using a scanning electron microscope (SEM, XL20, FEI Company, the

Netherlands), under an accelerating voltage of 10 kV after being sputter-coated with gold. An energy dispersive X-ray (Voyager, Eindhoven, The Netherlands) source was attached to the apparatus for the chemical composition analysis.

The presence and distribution of FITC-BSA in the three types of BioCP granules were analysed by cross section. The BioCP granules were embedded in methylmethacrylate. The blocks with granules were sectioned, and the sections (600 μηι thick) were glued to

Plexiglass holders and ground down to a thickness of 80 μηι. A series of 80^m-thick sections were obtained for analysis by fluorescence microscopy. Micrographs were taken with a digital camera (Leica, Wetzlar, Germany) mounted on an inverted light microscope (Leica) equipped with a fluorescence lamp. The coating thicknesses on BioCP granules (n=6) were also measured.

Protein encapsulation efficiency

To estimate the encapsulation efficiency of FITC-BSA in the three types of BioCP granules, 6 samples of each type of BioCP granule with different BSA concentrations were fabricated. Each sample (0.05 g) was immersed in 20 ml of 0.5 M ethylene diaminetetraacetic acid (EDTA, pH 8.0) and vortexed twice for 5 min. After dissolution, the triplicate 200-μΙ_ aliquots of this medium were collected and used for spectrophotometric analysis in a Fluorimeter (Spectramax M2, Molecular Devices, CA, USA), using 490 nm excitation and 504 nm emission wavelengths. Fluorescence readings were converted to amounts of protein using a standard curve that was generated from a dilution series of FITC-BSA prepared in 2 ml PBS.

Protein and Ca²⁺ release from BioCP granules

Ca²⁺ release from BioCP granules represents their degradation or dissolution due to the solubility of BioCP itself. The Ca²⁺ release from the three types of BioCP granules was investigated by soaking them in phosphate-buffered saline (PBS) with three different pH values (4.5, 6, and 7.4). Meanwhile, in vitro FITC-BSA release kinetics of the three types of BioCP granules were investigated in PBS at pH 7.4. All the samples were prepared using the same FITC-BSA concentration of 20 μg/ml. Each sample (0.05 g granules per sample, n = 6 for each type of BioCP granules) was placed in a 2-ml sealed Eppendorf tube containing 2 ml PBS. The tubes were incubated for up to 35 days in a shaking water bath (50 agitations/min) at 37°C. The 2 ml PBS was refreshed at each time point (hour 3, 6, day 1 , 2, 3, 5, 7, 10, 13, 17, 22, 28, and 35) and triplicate 200-μΙ_ aliquots of the PBS were withdrawn for spectrophotometric analysis and Ca²⁺ analysis. Ca²⁺ release was monitored by measuring Ca²⁺ concentration in PBS using atomic adsorption spectrometry (Analyst 100, PerkinElmer, USA). At the end of the release experiments, the residual FITC-BSA of BioCP was determined by dissolving the materials in EDTA as described above. The percentage of FITC-BSA released from the BioCP was calculated according to the formula [amount of the released fraction/ (amount of the released fraction + amount of the residual FITC-BSA of BioCP) x 100].

Osteoclast culture: cell-mediated degradation of BioCP

All cell culture in vitro experiments were performed at least three times. Human peripheral blood mononuclear cells (PBMCs) isolated from whole blood with Ficoll-Paque density gradient were in 96-well plates at a density of 106 cells per well on 800^m-thick BioCP tablets. The PBMCs were cultured with RANKL and M-CSF as described by precious studies (Olivier et al 2008; Hodge et al 2007). The cultures were incubated in a humidified 5% C02 atmosphere at 37°C. Cells were cultured in duplicate, and the culture media were refreshed twice a week. Ca2+ release was monitored from day 3 until day 24, and BioCP resorption was evaluated after 24 days (Olivier et al 2008; Hodge et al 2007).

Tartrate-resistant acid phosphatase (TRACP) is a marker enzyme of osteoclasts. The formation of osteoclasts was assessed by TRACP and SEM after 21 days of culturing on BioCP tablets (Olivier et al 2008; Faust et al 1999). The cultured cells were washed with PBS and fixed in 4% PBS-buffered formaldehyde for 5 min and stained for TRACP activity using the leucocyte acid phosphatase kit (Sigma). The nuclei were visualised by incubating the cell cultures with diamidino-2-phenylindole-dihydrochloride (DAPI) in PBS. The cells were fixed, dried, and sputter-coated for SEM investigation. The formation of lacunae was detected by SEM after the removal of cells from the samples after 24 days using

demineralised water. Osteoblast culture: biocompatibility of BioCP

Osteoblasts were isolated from murine long bones (6-week-old) according to method as described by Bakker et al. 2003. BioCP particles (test group) and deproteinized bovine bone (DBB, as control group) particles (Bio-Oss®, Geistlich) were deposited respectively in the 48-well plate (particle size < 0.1 mm; 5 mg/well). Cells were added on the layer of particles and at 1 x 104 cells/well. The cells were cultured under the following conditions: D-MEM

(Gibco BRL) + 10% foetal calf serum + 1 % antibiotics (PSF: 100 U/ml penicillin, 100 mg/ml streptomycin and 250 ng/ml amphotericin B).The cultures were incubated in a humidified 5% C02 atmosphere at 37°C. The medium was refreshed gently every 3 days.

To study osteoblast proliferation and differentiation, total DNA content and alkaline phosphatase (ALP) activity assay were performed on day 1 , 3, 7 and 14 of the culture. ALP activity was measured according to the method as described by Lowry (lowry 1957). Total DNA content was quantified using the Cyquant Cell Proliferation Assay (Molecular Probes, Eugene, OR, USA) according to the manufacturer's protocol. Finally, the ALP activity was normalized to DNA concentration.

Results

Characterization of BioCP

On an average, 200 ml of the five-fold supersaturated CaP solution produced approximately 0.5 g of BioCP granules with a size of 0.25-1.0 mm after grinding and filtration using metallic mesh filter. The compressive strength value of BioCP was 4.58 ± 0.31 MPa, compared with human trabecular bone which was ranged from 0.22 to 10.44 MPa, with a mean value of 3.9 MPa (Misch et al 1999). According to the EDX analysis, the Ca/P molar ratio of BioCP was 1.48; it also included 1.67 ± 0.29% sodium and 2.34 ± 1.1 1 % chloride. The BioCP powder without FITC-BSA exhibited a unique diffraction peak at 2Θ = 26°, 32°, and 46° in the XRD spectra, indicating analogies to bone (Vallet-Regi et al 2004; Habibovic et al 2002; Boskey 1997). These bands are characteristic of calcium-deficient apatite with low crystallinity (Dorozhkin 2010). The presence of incorporated FITC-BSA elicited no profound change in the major diffraction spectrum when the FITC-BSA concentrations were 10 or 20 μg/ml. When the FITC-BSA concentration was 50 μg/ml, there was no peak at 2Θ = 46°.

Scanning electron microscopy revealed that the surface of Type I granules in the absence of FITC-BSA was amorphous with tiny pores. The presence of FITC-BSA resulted in more numerous and larger pores than those found in its absence. There were no obvious differences in surface morphology among the three different FITC-BSA concentrations. A layer composed of densely packed crystal plates was present on Type II granules either in the absence or presence of FITC-BSA. This (biomimetic) coating had smaller crystal plates in the presence of FITC-BSA than in its absence. The surface of Type III granules was also amorphous with more pores than Type I. After being incubated in PBS (pH 7.4, 37°C) for 35 days, the crystal plates (approximately 0.5 μηι) of Type II granules were shorter than the initial crystal plates which had a length of approximately 2.5 μηι. The surface morphology of Type I and III granules did not change obviously after 35 days of incubation.

Cross sections of the three types of BioCP granules with FITC-BSA (20 μg/ml) were analysed by fluorescence microscopy. There was no obvious difference in the FITC-BSA distribution in BioCP granules among the three different concentrations. The distribution of FITC-BSA in Type I granules was nearly homogeneous. In Type II granules, the crystalline structure shown with the incorporated FITC-BSA had a fan-like shape. The crystalline coating layer of Type II granules had a thickness of 21.02 ± 7.53 μηι. As expected, Type III granules contained positively labelled small-sized Type II granules. Small Type II granules had small crystalline layer with a thickness of 10.42 ± 4.31 μηι. Ca²⁺ release from BioCP granules

The Cumulative Ca²⁺ release from the different types of BioCP granules in PBS at three different pH values (4.5, 6.0, and 7.4) are shown in Figure 4. All types of BioCP had a sustained and slow Ca²⁺ release pattern at pH 7.4. Furthermore, pH 4.5 and 6.0 resulted in a high burst release and subsequently a higher Ca²⁺ release than at pH 7.4. After 35 days of incubation at pH 7.4, the total amount of released Ca²⁺ from Type II granules was the lowest among the three types of BioCP granules. Protein encapsulation efficiency of BioCP

In Type I as well as Type II granules, the encapsulation efficiency of 10 μg/ml FITC-BSA was the highest (89.09 ± 1.91 %; and 45.57 ± 1.28%; respectively) among the three different concentrations (Figure 2A). Lower concentrations of FITC-BSA resulted in higher encapsulation efficiency in Types I and II (Fig. 2A). Among three types of granules, Type I had the highest encapsulation efficiency, when the same FITC-BSA concentration (20 μg/ml) were used. Small Type II granules (size < 0.25mm) had a higher encapsulation efficiency (67.32 ± 6.43%) than larger ones (29.74 ± 2.06%; size: 0.25-1 mm; 2-tailed t- test), when the FITC-BSA concentration was 20 μg/ml. After these small-sized Type II granules assembled in Type III, the encapsulation efficiency of Type III granules was 62.54 ± 5.22%.

Protein sustained release kinetics

The FITC-BSA release kinetics of the different types of BioCP granules are shown in Figure 2B. After the initial burst in the first 24 hours, the FITC-BSA release slowed down and exhibited a sustained release until the end of the study period (35 days) in all three types of BioCP granules. Type II granules show lower burst release if compared to Type I granules, and Type III granules had the lowest burst release among the three types of BioCP granules. After 35 days, there was still about 79%, 74%, and 88% of incorporated FITC-BSA retained in Type I, II, and III granules, respectively. The lowest total FITC-BSA release percentage was found in Type III granules (about 12%), being significantly different compared to Types I and II. Multiple proteins may also be delivered by BioCP granules by combination of Type I and Type II modalities. For example, one protein could be

incorporated into the body of BioCP granules, and meanwhile another protein could be carried by the biomimetic CaP coating layer; thus, these proteins may release in different time periods with different release kinetics. /PCT

Biological properties of BioCP

Human peripheral blood mononuclear cells were cultured on BioCP tablets. Figure 3A shows TRACP-positive multinucleated osteoclasts formed on BioCP tablets on day 21. Attachment of an osteoclast to the BioCP surface was shown, and resorption pits after 24 days of culture. Up to 14 days of culture no significant differences were found in Ca²⁺ concentration in the conditioned media collected from the cultures with or without cells, but at the later time points (17 to 24 days) the Ca²⁺ concentration was significantly higher in the conditioned media of the cultures with cells. These time points coincided with the formation of osteoclasts (Olivier et al 2008; Hodge et al 2007). The total DNA content of osteoblasts increased over time on BioCP and on DBB (Figure 5A). There were no significant differences in the DNA content between BioCP and DBB groups at day 1 , 3 and 7, but at days 14 the DNA content of BioCP group was significantly higher than DBB group. At all examined time points of culturing, the ALP activity of the BioCP group was significantly higher than the DBB group (Figure 5B). Moreover, an increase of ALP activity for BioCP was found from day 1 to 7, and it was significantly highest at day 7 among the four time points.

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Protection

Unit: Toxicology and Chemical Substances European Chemicals Bureau.

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Claims

1. Particles that comprise a core comprising amorphous calcium phosphate and a first layer around said core, wherein said first layer comprises crystalline calcium phosphate and an osteoconductive or osteoinductive agent or both.

2. Particles according to claim 1 , wherein the particles comprise a further layer, wherein said further layer comprises amorphous calcium phosphate, preferably said further layer being positioned on said first layer.

3. Particles according to claim 2 wherein the particles comprise an additional layer or layers comprising crystalline calcium phosphate, preferably comprising an osteoconductive or osteoinductive agent or both, preferably said additional layer(s) being positioned on said further layer comprising amorphous calcium phosphate.

4. Particles according to any of the previous claims, wherein the osteoinductive agent is selected from the group consisting of Transforming Growth Factor beta protein family or Bone Morphogenetic Protein family, preferably BMP-2, BMP-6, BMP-7, BMP-9, BMP- 12, or BMP- 13, more preferably BMP-2.

5. Particles according to any of the previous claims, wherein the particles have an average diameter of between 10 nm -10000 μηι, preferably 1 μι - 5000 μηι, more preferably 100 μηι - 2500 μηι, most preferably 250 μηι - 1000 μηι.

6. Particles according to any of the previous claims wherein the particles are

biodegradable.

7. Particles according to any of the previous claims wherein the core comprises at least 90% by weight of amorphous calcium phosphate.

8. Particles according to any of the previous claims wherein said first layer comprises at least 90% by weight crystalline calcium phosphate.

9. Particles according to any of the previous claims wherein the crystalline calcium phosphate is octacalcium phosphate.

10. Composition comprising particles according to any of the previous claims, preferably wherein the composition comprises at least 50% particles, more preferably at least 60, 70, or 80%, most preferably at least 90% by weight of the composition.

1 1. Particles according to any of claims 1 - 9, or a composition according to claim 10, for use in the treatment and/or prevention of a bone defect, and/or peri-implantitis, preferably a peri-implantitis induced bone defect.

12. Particles according to any of claims 1 - 9, or a composition according to claim 10, for use in the treatment of a bone defect, and/or peri-implantitis, preferably a peri-implantitis induced bone defect, wherein the treatment comprises the steps of

a. optionally, decontaminating a medical device;

b. applying to said optionally decontaminated medical device particles according to any of claims 1-9, or a composition according to claim 10.

13. Particles according to claim 12, wherein the medical device is a bone implant and/or a dental implant, preferably a dental implant.

14. Particles according to any of claims 12-13, wherein the medical device is present in an animal body, preferably a human.

15. Particles according to any of claims 12-14 for use in the treatment and/or prevention of peri-implantitis and/or treatment of a bone defect, preferably a peri-implantitis induced bone defect.

16. Medical device, preferably a bone implant or a dental implant, more preferably a dental implant, characterized in that said medical device comprises a particle according to any of claim 1-9, or a composition according to claim 10.

17. Method for improving osseointegration of a medical device, the method comprising the steps of

a. optionally, decontaminating said medical device;

18. Method for producing particles according to any of claims 1 -9, or a composition according to claim 10, the method comprising: a) incubating an aqueous solution comprising sodium chloride, calcium chloride, sodium hydrogen phosphate, sodium bicarbonate, in the presence of magnesium chloride, such that particles of amorphous calcium phosphate are formed;

e) optionally, repeating steps c) and d),

wherein step a) preferably is performed at an acidic pH, such as a pH below 6.5, preferably below pH 6.

19. Particles obtained by the method of claim 18.

20. Use of the particles according to any of claims 1-9, 1 1-16, or the composition according to claim 10, as bone filler, or as bone replacement.