Wouter Habraken

The fluorescent dye DAPI is useful for its association with and consequent amplification of an ∼460 nm emission maximum upon binding to dsDNA. Labelling with higher DAPI concentrations is a technique used to reveal Pi polymers [polyphosphate (polyP)], with a red-shift to ∼520-550 nm fluorescence emission. DAPI-polyP emissions of ∼580 nm are also generated upon 415 nm excitation. Red-shifted DAPI emission has been associated with polyP and RNA and has more recently been reported with polyadenylic acid (polyA), specific inositol phosphates (IPs) and heparin. We find that amorphous calcium phosphate (ACP) also demonstrates red-shifted DAPI emission at high DAPI concentrations. This DAPI spectral shift has been attributed to DAPI-DAPI electrostatic interactions enabled by molecules with high negative charge density that increase the local DAPI concentration and favour DAPI molecular proximity, as observed by increasing the dye/phosphate ratio. Excitation of dry DAPI (∼360 nm) confirmed ...

Previous studies on pre-molt gastroliths have shown a typical onion-like morphology of layers of amorphous mineral (mostly calcium carbonate) and chitin, resulting from the continuous deposition and densification of amorphous mineral spheres on a chitin-matrix during time. To investigate the consequences of this layered growth on the local structure and composition of the gastrolith, we performed spatially-resolved Raman, X-ray and SEM-EDS analysis on complete pre-molt gastrolith cross-sections. Results show that especially the abundance of inorganic phosphate, phosphoenolpyruvate (PEP)/citrate and proteins is not uniform throughout the organ but changes from layer to layer. Based on these results we can conclude that ACC stabilization in the gastrolith takes place by more than one compound and not by only one of these additives.

In this study, we investigated the in vivo degradation properties and tissue response towards injectable calcium phosphate cement (CPC) with no further addition, or calcium phosphate composite cement containing approximately 50 vol.% of microspheres. Three types of spheres were assessed, i.e. poly(lactic-co-glycolic acid) (PLGA), gelatin (GEL) and poly(trimethylene carbonate) (PTMC). The cements were injected into 4.6 mm diameter and 6mm deep cylindrical defects in the femoral condyle of New Zealand white rabbits, hardened in situ and, after wound closure, left to heal for 4, 8 and 12 weeks (n=6 for each composition and time period). After retrieval, specimens were analyzed using histological and histomorphometrical methods. Results showed that non-modified CPCs showed excellent bone contact but only very limited erosion at the surface. The CPC/PLGA implant degraded almost completely, while tissue response significantly improved at each time period. CPC/PTMC showed slower degradation characteristics compared to CPC/PLGA. Finally, at all time periods, there was an evident inflammatory response to the CPC/GEL composite cement. In conclusion, the degradation properties of the CPC/PLGA microspheres composite and its bone response when implanted into the femoral condyles of rabbits were significantly better than those of CPC/gelatin and CPC/PTMC microspheres composites.

Scaffolds for bone tissue engineering preferably should be mechanically stable, osteoconductive, biodegradable and porous. To comply with these characteristics, calcium phosphate cements (CPCs) with porcine (type A) gelatin microspheres were formulated. In this experiment, in vitro degradation of 10 wt % gelatin type A microsphere CPCs (GELA CPCs) was followed for 12 weeks in proteolytic medium. Results showed a gradual decrease in mass, compression strength and E-modulus. Morphology investigation showed that degradation of the spheres started at the surface of the composite and gradually proceeded to the inner part. Overall, porcine gelatin microspheres can be used to generate in situ macroporosity into an injectable CPC.

To improve the in vivo resorption of an injectable calcium phosphate cement (CPC) for bone tissue engineering purposes, in previous experiments macroporosity was introduced by the in situ degradation of incorporated gelatin microspheres. Gelatin microspheres are also suitable carriers for osteoinductive drugs/growth factors, where release occurs concomitantly with degradation of the hydrogel. Introduction of these microspheres into CPC can alter the release pattern of the cement, which usually shows a marginal release of incorporated drugs. The goal of this study was to determine the in vitro release characteristics of gelatin microsphere CPC. For this, recombinant human TGF-beta1, bFGF, and BMP-2 were labeled with (125)I and loaded onto gelatin type A (porcine, pI = 7.0-9.0)/type B (bovine, pI = 4.5-5.0) microspheres for a short (instant) and longer (prolonged) time before mixing them with the cement. Radioactivity of the resulting 5 or 10 wt % gelatin microsphere CPC composites was monitored for 6 weeks when subjected to proteolytic medium. Drug-loaded CPC was used as control. Results showed that release pattern/efficiency of gelatin microsphere CPCs and CPC controls was highly dependent on the type of growth factor but unaffected by the amount of growth factor. With gelatin microsphere CPC, release was also dependent on the type of gelatin, total volume of incorporated microspheres, and loading method.

Calcium phosphate (CaP) cements show an excellent biocompatibility and often have a high mechanical strength, but in general degrade relatively slow. To increase degradation rates, macropores can be introduced into the cement, e.g., by the inclusion of biodegradable microspheres into the cement. The aim of this research is to develop an injectable PLGA microsphere/CaP cement with sufficient setting/cohesive properties and good mechanical and physical properties. PLGA microspheres were prepared using a water-in-oil-in-water double-emulsion technique. The CaP-cement used was Calcibon, a commercially available hydroxyapatite-based cement. 10:90 and 20:80 dry wt% PLGA microsphere/CaP cylindrical scaffolds were prepared as well as microporous cement (reference material). Injectability, setting time, cohesive properties and porosity were determined. Also, a 12-week degradation study in PBS (37 degree C) was performed. Results showed that injectability decreased with an increase in PLGA microsphere content. Initial and final setting time of the PLGA/CaP samples was higher than the microporous sample. Porosity of the different formulations was 40.8% (microporous), 60.2% (10:90) and 69.3% (20:80). The degradation study showed distinct mass loss and a pH decrease of the surrounding medium starting from week 6 with the 10:90 and 20:80 formulations, indicating PLGA erosion. Compression strength of the PLGA microsphere/CaP samples decreased siginificantly in time, the microporous sample remained constant. After 12 weeks both PLGA/CaP samples showed a structure of spherical micropores and had a compressive strength of 12.2 MPa (10:90) and 4.3 MPa (20:80). Signs of cement degradation were also found with the 20:80 formulation. In conclusion, all physical parameters were well within workable ranges with both 10:90 and 20:80 PLGA microsphere/CaP cements. After 12 weeks the PLGA was totally degraded and a highly porous, but strong scaffold remained.

Bone cements with biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres have already been proven to provide a macroporous calcium phosphate cement (CPC) during in situ microsphere degradation. Furthermore, in vitro/in vivo release studies with these PLGA microsphere/CPC composites (PLGA/CPCs) showed a sustained release of osteo-inductive growth factor when drug was distributed inside/onto the microspheres. The goal of this study was to elucidate the mechanism behind drug release from PLGA/CPC. For this, in vitro release and degradation characteristics of a low-molecular-weight PLGA/CPC (M(w) = 5 kg/mol) were determined using bovine serum albumin (BSA) as a model protein. Two loading mechanisms were applied; BSA was either adsorbed onto the microspheres or incorporated inside the microspheres during double-emulsion. BSA release from PLGA microspheres and CPC was also measured and used as reference. Results show fast degrading polymer microspheres which produced a macroporous scaffold within 4 weeks, but also showed a concomitant release of acidic degradation products. BSA release from the PLGA/CPC was similar to the CPC samples and showed a pattern consisting of a small initial release, followed by a period of almost no sustained release. Separate PLGA microspheres exhibited a high burst release and release efficiency that was higher with the adsorbed samples. Combining degradation and release data we can conclude that for the PLGA/CPC samples BSA re-adsorbed to the cement surface after being released from the microspheres, which was mediated by the pH decrease during microsphere degradation.

ABSTRACT An ugly duckling grows into a swan: Many organisms grow their crystalline mineral phases through the secondary nucleation of nanospheres made of an amorphous precursor phase. Stable amorphous calcium carbonate biominerals were used to induce a similar transformation in vitro. The amorphous nanospheres underwent a solid-phase transformation that resulted in highly ordered calcite crystals composed of aggregated particles.

Ceramic composites and scaffolds are popular implant materials in the field of dentistry, orthopedics and plastic surgery. For bone tissue engineering especially CaP-ceramics or cements and bioactive glass are suitable implant materials due to their osteoconductive properties. In this review the applicability of these ceramics but also of ceramic/polymer composites for bone tissue engineering is discussed, and in particular their use as drug delivery systems. Overall, the high density and slow biodegradability of ceramics is not beneficial for tissue engineering purposes. To address these issues, macroporosity can be introduced often in combination with osteoinductive growth factors and cells. Ceramics are good carriers for drugs, in which release patterns are strongly dependent on the chemical consistency of the ceramic, type of drug and drug loading. Biodegradable polymers like polylactic acid, gelatin or chitosan are used as matrices for ceramic particles or as adjuvant to calcium phosphate cements. The use of these polymers can introduce a tailored biodegradation/drug release to the ceramic material.

In this study we have investigated the influence of the mechanism of microsphere degradation or erosion on the in vivo degradation of microsphere/calcium phosphate cement composites (microsphere CPCs) used in tissue engineering. Microspheres composed of poly(lactic-co-glycolic acid) (PLGA), gelatin and poly(trimethylene carbonate) (PTMC) were used as the model and the resulting microsphere CPCs were implanted subcutaneously for 4, 8 or 12weeks in the back of New Zealand white rabbits. Besides degradation, the soft tissue response to these formulations was evaluated. After retrieval, specimens were analyzed by physicochemical characterization and histological analysis. The results showed that all microsphere CPCs exhibited microsphere degradation after 12weeks of subcutaneous implantation, which was accompanied by decreasing compression strength. The PLGA microspheres exhibited bulk erosion simultaneously throughout the whole composite, whereas the gelatin type B microspheres were degradated from the outside to the center of the composite. High molecular weight PTMC microspheres exhibited surface erosion resulting in decreasing microsphere size. Furthermore, all composites showed a similar tissue response, with decreasing capsule thickness over time and a persistent moderate inflammatory response at the implant interface. In conclusion, microsphere CPCs can be used to generate porous scaffolds in an in vivo environment after degradation of microspheres by various degradation/erosion mechanisms.

In this study, we investigated the in vivo degradation properties and tissue response towards injectable calcium phosphate cement (CPC) with no further addition, or calcium phosphate composite cement containing approximately 50 vol.% of microspheres. Three types of spheres were assessed, i.e. poly(lactic-co-glycolic acid) (PLGA), gelatin (GEL) and poly(trimethylene carbonate) (PTMC). The cements were injected into 4.6 mm diameter and 6mm deep cylindrical defects in the femoral condyle of New Zealand white rabbits, hardened in situ and, after wound closure, left to heal for 4, 8 and 12 weeks (n=6 for each composition and time period). After retrieval, specimens were analyzed using histological and histomorphometrical methods. Results showed that non-modified CPCs showed excellent bone contact but only very limited erosion at the surface. The CPC/PLGA implant degraded almost completely, while tissue response significantly improved at each time period. CPC/PTMC showed slower degradation characteristics compared to CPC/PLGA. Finally, at all time periods, there was an evident inflammatory response to the CPC/GEL composite cement. In conclusion, the degradation properties of the CPC/PLGA microspheres composite and its bone response when implanted into the femoral condyles of rabbits were significantly better than those of CPC/gelatin and CPC/PTMC microspheres composites.

ABSTRACT Calcium carbonate is a common constituent of many natural materials, such as shells and skeletons of marine animals. While it is well documented that additives (organic and inorganic) modulate the crystallization of amorphous calcium carbonate (ACC), the effects of the intrinsic physicochemical characteristics of ACC such as particle size, shape and water content on the transformation to crystalline polymorphs are still poorly understood. Here we investigated the effect of particle size by preparing ACC nanoparticles with an average size ranging from ~66 nm to ~196 nm using a high-resolution titration setup. Our results show that the particle size determined the polymorph selection in solution; an increasing proportion of vaterite to calcite was observed with decreasing particle size. The polymorph selection was ascribed to a higher apparent solubility of ACC with decreasing particle size, a parameter from which we could determine the surface energy of ACC to be ~0.33 J/m2. Upon heating, particle size showed the opposite effect, as smaller particles favored a higher crystallization temperature from ACC into (only) calcite. When the particle size was large enough, crystallization occurred concomitantly with the removal of bulk water at lower temperatures, where the smallest particles transformed at ~310 °C, only after losing the final (surface) water. Our results highlight the importance of particle size as well as the crystallization conditions on the stability and transformation mechanisms of ACC.