Modifications of Poly(Methyl Methacrylate) Cement for Application in Orthopedic Surgery.

Even with the emerging of newly-developed bone substitutes, poly(methyl methacrylate) (PMMA) cement is still a widely-used bone replacing biomaterial in orthopedic surgery with a long history. However, aseptic loosening, infection of the prosthesis and thermal necrosis to surrounding tissue are the common complications of PMMA. Therefore, additives have been incorporated in PMMA cement to target those problems. This chapter summarizes different additives to improve the performance of the PMMA cement, i.e.: (1) bioceramic additives; (2) filler additives; (3) antibacterial additives; (4) porogens; (5) biological agents, and (6) mixed additives. To improve the biological and mechanical performance of PMMA cement, mixed additives aiming to fabricate multifunctional PMMA seem the most suitable choice. Although in vivo animal studies have been conducted, long-term and clinical studies are still needed to evaluate the modifications of multifunctional PMMA cement for matching a specific clinical application.

Multimodal pore formation in calcium phosphate cements.

Calcium phosphate cements (CPCs) are commonly used as bone substitute materials. However, their slow degradation rate and lack of macroporosity hinders new bone formation. Poly(dl-lactic-co-glycolic acid) (PLGA) incorporation is of great interest as, upon degradation, produces acidic by-products that enhance CPC degradation. Yet, new bone formation is delayed until PLGA degradation occurs a few weeks after implantation. Therefore, the aim of this study was to accelerate the early stage pore formation within CPCs in vitro. With that purpose, we incorporated the water-soluble porogen sucrose at different weight percentages (10 or 20 wt %) to CPC and CPC/PLGA composites. The results revealed that incorporation of sucrose porogens increased mass loss within the first week of in vitro degradation in groups containing sucrose compared to control groups. After week 1, a further mass loss was observed related to PLGA and CPC degradation. Macroporosity analysis confirmed that macroporosity formation is influenced by the dissolution of sucrose at an early stage and by the degradation of PLGA and CPC at a later stage. We concluded that the combination of sucrose and PLGA porogens in CPC is a promising approach to promote early stage bone tissue ingrowth and complete replacement of CPC through multimodal pore formation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 500-509, 2018.

Bone Response to Porous Poly(methyl methacrylate) Cement Loaded with Hydroxyapatite Particles in a Rabbit Mandibular Model.

The aim of the current study was to evaluate bone formation and tissue response to porous poly(methyl methacrylate) (PMMA) cement with or without hydroxyapatite (HA) in a rabbit mandibular model. Therefore, 14 New Zealand White rabbits were randomly divided into two groups of seven according to the designed study end points of 4 and 12 weeks. For each rabbit, two decorticated defects (6 mm in height and 10 mm in width for each) were prepared at both sides of the mandible. Subsequently, the defects were filled with, respectively, porous PMMA and porous PMMA-HA cement. After reaching the designated implantation period, the rabbits were euthanized and the mandibles were retrieved for histological analysis. Results showed that both porous PMMA and porous PMMA-HA supported bone repair. Neither of the bone cements caused significant inflammation to nerve or other surrounding tissues. After implantation of 12 weeks, majority of the porosity was filled with newly formed bone for both cements, which supports the concept that a porous structure within PMMA can enhance bone ingrowth. Histomorphometrical evaluation, using histological grading scales, demonstrated that, at both implantation times, the presence of HA in the PMMA enhanced bone formation. Bone was always in direct contact with the HA particles, while intervening fibrous tissue was present at the PMMA-bone interface. On the basis of results, it was concluded that injectable porous PMMA-HA cement might be a good candidate for craniofacial bone repair, which should be further evaluated in a more clinically relevant large animal model.

Effect of calcium phosphate ceramic substrate geometry on mesenchymal stromal cell organization and osteogenic differentiation.

The composition of calcium phosphate (CaP) ceramics in combination with surface features have been shown to influence biological performance, and micro- and nano-scale topography is known to stimulate osteogenic differentiation of mesenchymal stromal cells (MSCs). In view of this, adipose tissue derived MSCs were cultured on CaP disks featuring hemispherical concavities of various sizes (440, 800 or 1800 µm diameter). It was hypothesized that (i) surface concavities would promote cell proliferation, cellular organization within the concavities, and osteogenic differentiation, as a result of a more pronounced 3D micro-environment and CaP nucleation in concavities, and (ii) MSC proliferation and osteogenic differentiation would increase with smaller concavity size due to more rapidly occurring 3D cell-cell interactions. We found that concavities indeed affect cell proliferation, with 440 µm concavities increasing cell proliferation to a larger extent compared to 800 and 1800 µm concavities as well as planar surfaces. Additionally, concavity size influenced 3D cellular organization within the concavity volume. Interestingly, concavity size promoted osteogenic differentiation of cells, as evidenced by increased osteocalcin gene expression in 440 µm concavities, and osteocalcin staining predominantly for 440 and 800 µm concavities, but not for 1800 µm concavities and only slightly for planar surface controls.

Physicochemical properties and mineralization assessment of porous polymethylmethacrylate cement loaded with hydroxyapatite in simulated body fluid.

The aim of this study was to evaluate the effect of carboxymethylcellulose (CMC) as a pore generator and hydroxyapatite (HA) as an osteoconductive agent on the physicochemical properties and in-vitro mineralization ability of porous polymethylmethacrylate (PMMA) cement. To this end, various compositions of PMMA cements, which differed in amount of millimeter-sized hydroxyapatite (HA) particles and CMC hydrogel, were prepared and immersed into simulated body fluid (SBF) for 0, 7, 14, 21 and 28 days. It was demonstrated that the incorporation of CMC hydrogel decreased the maximum temperature of cement to the normal body temperature and prolonged the handling time during polymerization. Further, the amount of CMC was responsible for the creation of porosity and interconnectivity, which in turn determined the final mechanical properties of cements. The loaded HA particles enhanced the potential bioactivity of cement for bone ingrowth. Albeit different amount of HA particles influenced their final exposures on the surface of cured cement, all of the three amounts of HA did not weaken the final mechanical properties of cements. The data here suggests that the HA particle loaded porous PMMA cement can serve as the promising candidate for bone reconstruction.

Influence of polymeric additives on the cohesion and mechanical properties of calcium phosphate cements.

To expand the clinical applicability of calcium phosphate cements (CPCs) to load-bearing anatomical sites, the mechanical and setting properties of CPCs need to be improved. Specifically, organic additives need to be developed that can overcome the disintegration and brittleness of CPCs. Hence, we compared two conventional polymeric additives (i.e. carboxylmethylcellulose (CMC) and hyaluronan (HA)) with a novel organic additive that was designed to bind to calcium phosphate, i.e. hyaluronan-bisphosphonate (HABP). The unmodified cement used in this study consisted of a powder phase of α-tricalcium phosphate (α-TCP) and liquid phase of 4% NaH2PO4·2H2O, while the modified cements were fabricated by adding 0.75 or 1.5 wt% of the polymeric additive to the cement. The cohesion of α-TCP was improved considerably by the addition of CMC and HABP. None of the additives improved the compression and bending strength of the cements, but the addition of 0.75% HABP resulted into a significantly increased cement toughness as compared to the other experimental groups. The stimulatory effects of HABP on the cohesion and toughness of the cements is hypothesized to derive from the strong affinity between the polymer-grafted bisphosphonate ligands and the calcium ions in the cement matrix.

Long-term evaluation of the degradation behavior of three apatite-forming calcium phosphate cements.

Calcium phosphate cements (CPCs) are injectable bone substitutes with a long clinical history because of their biocompatibility and osteoconductivity. Nevertheless, their cohesion upon injection into perfused bone defects as well as their long-term degradation behavior remain major clinical challenges. Therefore, the long-term degradation behavior of two types of α-tricalcium phosphate-based, apatite-forming CPCs was compared to a commercially available apatite-forming cement, that is HydroSet™ . Carboxyl methylcellulose (CMC) was used as cohesion promotor to improve handling properties of the two experimental cements, whereas poly (d, l-lactic-co-glycolic) acid (PLGA) microparticles were added to introduce macroporosity and stimulate CPC degradation. All three CPCs were injected into defects drilled into rabbit femoral condyles and explanted after 4, 12, or 26 weeks, after which the bone response was assessed both qualitatively and quantitatively. CPCs without PLGA microparticles degraded only at the periphery of the implants, while the residual CPC volume was close to 90%. On the contrary, bone ingrowth was observed not only at the periphery of the CPC, but also throughout the center of the implants after 26 weeks of implantation for the PLGA-containing CPCs with a residual CPC volume of approximately 55%. In conclusion, it was shown that CPC containing CMC and PLGA was able to induce partial degradation of apatite-forming CPCs and concomitant replacement by bone tissue.

Size matters: effects of PLGA-microsphere size in injectable CPC/PLGA on bone formation.

The aim of this study was to evaluate the effect of PLGA microsphere dimensions on bone formation after injection of calcium phosphate cement (CPC)/PLGA in a guinea pig tibial intramedullarly model. To this end, injectable CPC/PLGA formulations were prepared using PLGA microspheres with either a small (~25 µm) or large (~100 µm) diameter, which were incorporated at a 20:80 ratio (wt%) within apatite CPC. Both CPC/PLGA formulations were injected into a marrow-ablated tibial intramedullary cavity and, after an implantation period of 12 weeks, histology and histomorphometry were used to address bone formation. The results demonstrated bone ingrowth throughout the entire scaffold material for both CPC/PLGA formulations upon PLGA microsphere degradation. More importantly, bone formation within the CPC matrix was > two-fold higher for CPC-PLGA with 25 µm PLGA microspheres. Additionally, the pattern of bone and marrow formation showed distinct differences related to PLGA microsphere dimension. In general, this study demonstrates that PLGA microsphere dimensions of ~25 µm, leading to pores of ~25 µm within CPC, are sufficient for bone ingrowth and allow substantial bone formation. Further, the results demonstrate that PLGA microsphere dimensions provide a tool to control bone formation for injectable CPC/PLGA bone substitutes. Copyright © 2013 John Wiley & Sons, Ltd.

Effects of Stirring and Fluid Perfusion on the In Vitro Degradation of Calcium Phosphate Cement/PLGA Composites.

In vitro degradation rates of calcium phosphate bioceramics are investigated using a large variation of soaking protocols that do not all match the dynamic conditions of the perfused physiological environment. Therefore, we studied the effect of stirring and fluid perfusion on the in vitro degradation rate of apatitic calcium phosphate cements (CPC) containing poly(lactic-co-glycolic acid) (PLGA) microspheres. The composites were soaked in phosphate-buffered saline up to 6 weeks under unstirred, stirred, or perfused conditions followed by analysis of mass loss, compression strength, porosity, crystal phase composition, and morphology of the cement composites. The results showed that fluid perfusion reduced the decrease in pH and corresponding degradation rates, while nonperfused soaking conditions (i.e., stirred and unstirred conditions) resulted into more extensive acidification, the rate of which increased with stirring. After 2 weeks, the formation of a secondary brushite phase was observed for cement composites soaked under nonperfused (i.e., stirred and unstirred) conditions, whereas this phase was not detected in cements soaked under perfused conditions. The degradation rate of cement composites decreased in the order unstirred>stirred>perfused, as evidenced by quantification of mass loss, compression strength, and pore morphology. To summarize, we have demonstrated that soaking conditions strongly affected the in vitro degradation process of CPCs. As a consequence, it can be concluded that the experimental design of current in vitro degradation studies does not allow for correlation to (pre-)clinical studies.

Influence of ceramic disk material, surface hemispheres, and SBF volume on in vitro mineralization.

Calcium phosphate ceramics are the main mineral constituents of bone and teeth and have therefore been extensively investigated for bone regenerative applications. In the current study, the effect of disk material, surface geometry, and SBF volume on mineralization capacity was investigated. Hemispherical concavities were created on the surfaces of disks made of different materials (i.e., hydroxyapatite (HA), ß-tricalcium phosphate (ß-TCP), biphasic calcium phosphate (BCP) and titanium (Ti)) which were sintered at 1200 °C. Mineralization of CaP was assessed on disk surfaces after immersion of the samples in different volumes of simulated body fluid (SBF) up to 14 days by means of calcium assay and scanning electron microscopy (SEM). This study showed that different SBF volumes have different effects on mineralization, with an optimum material/liquid ratio of 5 mL of SBF per cm(2) . Additionally, at this volume, apparent differences based on disk material became obvious. Evidently, surface hemispherical concavities acted as initiator areas for nucleation and crystal growth.

Development of porous polyurethane/strontium-substituted hydroxyapatite composites for bone regeneration.

Polyurethane (PU) has been widely used for the biomedical applications but its potential for bone regeneration is limited due to its lack of osteoconductive properties. Strontium substituted hydroxyapatite (SrHA) particles, on the other hand, are known to exhibit a positive effect on bone formation. Therefore, the aim of this study was to (i) develop porous polyurethane scaffolds containing strontium SrHA nanoparticles (PU/SrHA) and (ii) compare their in vitro biological performance for applications in bone regeneration to PU scaffolds. SrHA and HA was synthesized using a conventional wet-chemical neutralization reaction at temperatures of 25, 50, and 80°C. Chemical analysis was performed by inductively coupled plasma-optical emission spectrometry. Synthesizing temperatures at 25 and at 50°C were selected for the composite preparation (abbreviated as HA-25, SrHA-25, HA-50, and SrHA-50, respectively). PU was synthesized from isophorone diisocyanate, polytetramethylene ether glycol, and 1,4-butanediol. Composite scaffolds were prepared by addition of HA or SrHA nanoparticles into PU scaffolds during polymer preparation. The results showed that the Sr content in HA nanoparticles increased with increasing synthesis temperature. The addition of nanoparticles decreased the elongation-at-break and tensile strength, but significantly increased the surface wettability of the PU scaffolds. In vitro degradation tests demonstrated that release of cations was significantly higher from PU/SrHA-50 composite scaffolds. Cell culture tests indicated that PU composites containing either HA or SrHA nanoparticles increased proliferation of bone marrow stem cells as compared to plain PU scaffolds, whereas osteogenic differentiation was not affected by the incorporation of HA nanoparticles irrespective of the incorporation of Sr.

Effects of calcium phosphate composition in sputter coatings on in vitro and in vivo performance.

Calcium phosphate (CaP) ceramic coatings have been used to enhance the biocompatibility and osteoconductive properties of metallic implants. The chemical composition of these ceramic coatings is an important parameter, which can influence the final bone performance of the implant. In this study, the effect of phase composition of CaP-sputtered coatings was investigated on in vitro dissolution behavior and in vivo bone response. Coatings were prepared by a radio frequency (RF) magnetron sputtering technique; three types of CaP target materials were used to obtain coatings with different stoichiometry and calcium to phosphate ratios (hydroxyapatite (HA), α-tricalciumphosphate (α-TCP), and tetracalciumphosphate (TTCP)) were compared with non-coated titanium controls. The applied ceramic coatings were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, and inductively coupled plasma optical emission spectroscopy. The in vitro dissolution/precipitation of the CaP coatings was evaluated using immersion tests in simulated body fluid (SBF). To mimic the in vivo situation, identical CaP coatings were also evaluated in a femoral condyle rabbit model. TCPH and TTCPH showed morphological changes during 4-week immersion in SBF. The results of bone implant contact (BIC) and peri-implant bone volume (BV) showed a similar response for all experimental coatings. An apparent increase in tartrate resistant acid phosphatase (TRAP) positive staining was observed in the peri-implant region with decreasing coating stability. In conclusion, the experimental groups showed different coating properties when tested in vitro and an apparent increase in bone remodeling with increasing coating dissolution in vivo.

Physicochemical properties and in vitro mineralization of porous polymethylmethacrylate cement loaded with calcium phosphate particles.

The main goal of this study was to evaluate the effects of incorporation of calcium phosphate (CaP) particles on the physicochemical properties and mineralization capacity of cements in vitro. Herein, two different types of CaP particles were loaded into polymethylmethacrylate (PMMA) cements exhibiting an interconnected porosity created by mixing with carboxymethylcellulose. The incorporation of CaP particles did not influence the maximum polymerization temperature of the porous PMMA, but reduced the porosity and the average pore size. Small CaP particles formed agglomerations within the PMMA pores, whereas big CaP particles were partially embedded in the PMMA matrix and partially exposed to the pores. Both types of CaP particles enhanced the mineralization capacity of PMMA cement without compromising their mechanical properties. The data presented herein suggest that porous PMMA/CaP cements hold strong promise for surgical application in bone reconstruction.

Tuning the degradation rate of calcium phosphate cements by incorporating mixtures of polylactic-co-glycolic acid microspheres and glucono-delta-lactone microparticles.

Calcium phosphate cements (CPCs) are frequently used as synthetic bone graft materials in view of their excellent osteocompatibility and clinical handling behavior. Hydroxyapatite-forming CPCs, however, degrade at very low rates, thereby limiting complete bone regeneration. The current study has investigated whether degradation of apatite-forming cements can be tuned by incorporating acid-producing slow-resorbing poly(D,L-lactic-co-glycolic) acid (PLGA) porogens, fast-resorbing glucono-delta-lactone (GDL) porogens, or mixtures thereof. The physicochemical, mechanical, and degradation characteristics of these CPC formulations were systematically analyzed upon soaking in phosphate-buffered saline (PBS). In parallel, various CPC formulations were implanted intramuscularly and orthotopically on top of the transverse process of goats followed by analysis of the soft tissue response and bone ingrowth after 12 weeks. In vitro degradation of GDL was almost completed after 2 weeks, as evidenced by characterization of the release of gluconic acid, while PLGA-containing CPCs released glycolic acid throughout the entire study (12 weeks), resulting in a decrease in compression strength of CPC. Extensive in vitro degradation of the CPC matrix was observed upon simultaneous incorporation of 30% PLGA-10% GDL. Histomorphometrical evaluation of the intramuscularly implanted samples revealed that all CPCs exhibited degradation, accompanied by an increase in capsule thickness. In the in vivo goat transverse process model, incorporation of 43% PLGA, 30% PLGA-5% GDL, and 30% PLGA-10% GDL in CPC significantly increased bone formation and resulted in higher bone height compared with both 10% GDL and 20% GDL-containing CPC samples.

Experimental evolution of external immune defences in the red flour beetle.

The red flour beetle, Tribolium castaneum, secretes quinones that control the microbial flora in the surrounding environment. These secretions act as an external immune defence that provides protection against pathogens. At high concentrations, however, these secretions are harmful to the host itself, and selection may thus have optimized the level of expression under natural conditions. Here, we show that the expression of external immunity responded to selection during experimental evolution within a few generations. At the same time, one component of internal immune defence (phenoloxidase activity) was compromised in beetles selected for either high or low external defences. Intriguingly, offspring protection against a natural pathogen was reduced in flour obtained from beetle lines selected for low amounts of secretions. Altogether, this suggests that external and internal immune defences work together efficiently under natural conditions, whereas every manipulation on the side of external immune defence comes with costs to the internal immune defence.

Substrate geometry directs the in vitro mineralization of calcium phosphate ceramics.

Repetitive concavities on the surface of bone implants have recently been demonstrated to foster bone formation when implanted at ectopic locations in vivo. The current study aimed to evaluate the effect of surface concavities on the surface mineralization of hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP) ceramics in vitro. Hemispherical concavities with different diameters were prepared at the surface of HA and ß-TCP sintered disks: 1.8mm (large concavity), 0.8mm (medium concavity) and 0.4mm (small concavity). HA and ß-TCP disks were sintered at 1100 or 1200°C and soaked in simulated body fluid for 28 days at 37°C; the mineralization process was followed by scanning electron microscopy, energy-dispersive spectroscopy, X-ray diffraction and calcium quantification analyses. The results showed that massive mineralization occurred exclusively at the surface of HA disks treated at 1200°C and that nucleation of large aggregates of calcium phosphate started specifically inside small concavities instead of on the planar surface of the disks. Regarding the effect of concavity diameter size on surface mineralization, it was observed that small concavities induce 124- and 10-fold increased mineralization compared to concavities of large or medium size, respectively. The results of this study demonstrated that (i) in vitro surface mineralization of calcium phosphate ceramics with surface concavities starts preferentially within the concavities and not on the planar surface, and (ii) concavity size is an effective parameter to control the spatial position and extent of mineralization in vitro.

Accelerated calcium phosphate cement degradation due to incorporation of glucono-delta-lactone microparticles.

Injectable calcium phosphate cements (CPC) are frequently used for filling of bone defects due to their excellent osteocompatibility. Their poor degradability, however, limits complete regeneration of bone defects. Organic additives that produce acid by-products are particularly attractive to create macroporosity in situ since CPC degrade by acid dissolution. The aim of the current study was to investigate whether glucono-delta-lactone (GDL) can be used as acid-producing microparticles for incorporation into CPC without compromising its osteocompatibility. Characterization studies confirmed that CPCs containing either low or high amounts of GDL were injectable and self-setting, while a considerable amount of porosity was formed already within 1 day of incubation in phosphate buffered saline due to dissolution of GDL. Histomorphometrical evaluation after 2 weeks of implantation revealed that CPC containing 10% of GDL degraded faster and was replaced by more bone tissue than CPCs containing either Poly (lactic-co-glycolic acid) (PLGA) or gelatin microspheres. Summarizing, the current study showed that CPCs containing appropriate amounts of GDL display accelerated degradation and new bone formation compared with CPCs containing microparticles made of conventional polymers such as PLGA or gelatin.

Injectable biphasic calcium phosphate cements as a potential bone substitute.

Apatitic calcium phosphate cements (CPCs) have been widely used as bone grafts due to their excellent osteoconductive properties, but the degradation properties are insufficient to stimulate bone healing in large bone defects. A novel approach to overcome the lack of degradability of apatitic CPC involves the development of biphasic CPCs (BCPC) based on tricalcium phosphate (TCP) in both α- and ß-polymorphs. The aim of the current study was to prepare and analyze the physicochemical properties of BCPCs based on dual phase α/ß-TCP as obtained by heat treatment of pure α-TCP. The handling and mechanical characteristics of the samples as well as the degradation behavior under in vitro condition were investigated and compared with a standard monophasic α-TCP-based CPC. The results showed that different heat treatments of commercially available α-TCP allowed the formation of biphasic calcium phosphate powder with a variety of α/ß-TCP ratios. The use of biphasic powder particles as a reactant for CPCs resulted into increased setting and injectability times of the final BCPCs. During hardening of the cements, the amount of apatite formation decreased with increasing ß-TCP content in the biphasic precursor powders. The morphology of the monophasic CPC consisted of plate-like crystals, whereas needle-like crystals were observed for BCPCs. In vitro degradation tests demonstrated that dissolution rate and corresponding calcium release from the set cements increased considerably with increasing ß-TCP content, suggesting that apatitic CPCs can be rendered degradable by using biphasic α/ß-TCP as powder precursor phase.

Shear resistance of fiber-reinforced composite and metal dentin pins.

PURPOSE: To assess whether dentin pins increase shear resistance of extensive composite restorations and to compare performance of mini fiber-reinforced composite (FRC) anchors with metal dentin pins in the laboratory. METHODS: 30 extracted sound molars were randomly divided into three groups. Occlusal surfaces were ground flat with a standard surface area and resin composite restorations were made in Group A. In Groups B and C similar restorations were made, with additionally four metal pins placed in Group B and four FRC pins in Group C. Specimens were statically loaded until failure occurred. Failure modes were characterized as intact remaining tooth substrate (adhesive or cohesive failure of restoration) or fractured remaining tooth substrate. RESULTS: Mean failure stresses were 6.5 MPa (SD 3.2 MPa) for Group A, 9.7 MPa (SD 2.6 MPa) for Group B and 9.2 MPa (SD 2.6 MPa) for Group C. Difference in mean failure stresses between Group A and Groups B and C was statistically significant (P = 0.01), while the difference between Groups B and C was not (P = 0.63). Failures of the restoration without fracture of tooth substrate were seen for 80% of specimens in Group A and 20% in Groups B and C (P = 0.04).

RANKL delivery from calcium phosphate containing PLGA microspheres.

Ideally, bone substitute materials would undergo cell-mediated degradation during the remodeling process of the host bone tissue while being replaced by newly formed bone. In an attempt to exploit the capacity of Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL) to stimulate osteoclast-like cells formation, this study explored different loading methods for RANKL in injectable calcium phosphate cement (CPC) and the effect on release and biological activity. RANKL was loaded via the liquid phase of CPC by adsorption onto or incorporation into poly(lactic-co-glycolic acid) (PLGA) microspheres with two different morphologies (i.e., hollow and dense), which were subsequently embedded in CPC. As controls nonembedded PLGA-microspheres were used as well as plain CPC scaffolds with RANKL adsorbed onto the surface. RANKL release and activity were evaluated by Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) and osteoclast-like cells formation in cell culture experiments. Results indicated that sustained release of active RANKL can be achieved upon RANKL adsorption to PLGA microspheres, whereas inactive RANKL was released from CPC-PLGA formulations with RANKL incorporated within the microspheres or within the liquid phase of the CPC. These results demonstrate that effective loading of RANKL in injectable CPC is only possible via adsorption to PLGA microspheres, which are subsequently embedded within the CPC-matrix.