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Calcium phosphates (CaPs) are among the most utilized synthetic biomaterials for bone regeneration, largely owing to their established osteoconductive and osteoinductive properties. While angiogenesis is a crucial prerequisite to bone formation, research and applications for CaPs have not appreciated its crucial role. This review discusses how CaPs influence angiogenesis, and highlights promising strategies that address this topic. The objective is to draw attention to the gap in the literature and to highlight the importance of angiogenesis in CaP research, development, and use. [ABSTRACT FROM AUTHOR]

Our populations are aging. Some experts predict that 30% of hospital beds will soon be occupied by osteoporosis patients. Statistics show that 20% of patients suffering from an osteoporotic hip fracture do not survive the first year after surgery, all this showing that there is a tremendous need for better therapies for diseased and damaged bone. Human bone consists for about 70% of calcium phosphate (CaP) mineral, therefore CaPs are the materials of choice to repair damaged bone. To do this successfully, the process of CaP biomineralization and the interaction of CaPs and biological environment in the body need to be fully understood. First commercial CaP bone graft substitutes were launched 40 years ago, and they are currently often regarded as ‘old biomaterials’ or even as an ‘obsolete’ research topic. Some even talk about ‘stones’. The aim of this manuscript is to highlight the tremendous improvements achieved in CaP materials research in the past 15 years, in particular in the field of biomineralization, as carrier for gene or ion delivery, as biologically active agent, and as bone graft substitute. Besides an outstanding biological performance, CaPs are easily and inexpensively produced, are safe, and can be relatively easily certified for clinical use. As such, CaP materials have won their spurs, but they also offer a great promise for the future. [ABSTRACT FROM AUTHOR]

Abstract: Osteoinductive biomaterials are promising for bone repair. There is no direct proof that bone marrow mesenchymal stem cells (BMSCs) home to non-osseous sites and participate in ectopic bone formation induced by osteoinductive bioceramics. The objective of this study was to use a sex-mismatched beagle dog model to investigate BMSC homing via blood circulation to participate in ectopic bone formation via osteoinductive biomaterial. BMSCs of male dogs were injected into female femoral marrow cavity. The survival and stable chimerism of donor BMSCs in recipients were confirmed with polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). Biphasic calcium phosphate (BCP) granules were implanted in dorsal muscles of female dogs. Y chromosomes were detected in samples harvested from female dogs which had received male BMSCs. At 4 weeks, cells with Y-chromosomes were distributed in the new bone matrix throughout the BCP granule implant. At 6 weeks, cells with Y chromosomes were present in newly mineralized woven bone. TRAP positive osteoclast-like cells were observed in 4-week implants, and the number of such cells decreased from 4 to 6 weeks. These results show that osteoprogenitors were recruited from bone marrow and homed to ectopic site to serve as a cell source for calcium phosphate-induced bone formation. In conclusion, BMSCs were demonstrated to migrate from bone marrow through blood circulation to non-osseous bioceramic implant site to contribute to ectopic bone formation in a canine model. BCP induced new bone in muscles without growth factor delivery, showing excellent osteoinductivity that could be useful for bone tissue engineering. [Copyright &y& Elsevier]

Abstract: Metal ions originating from mechanical debris and corrosive wear of prosthetic implant alloys accumulate in peri-implant soft tissues, bone mineral, and body fluids. Eventually, metal ions such as cobalt (II) (Co2+), which is a major component of cobalt–chromium-based implant alloys and a known activator of osteolysis, are incorporated into the mineral phase of bone. We hypothesize that the accumulation of Co2+ in the mineral could directly activate osteolysis by targeting osteoclasts. To test this hypothesis, we coated tissue culture plastic with a thin layer of calcium phosphate (CaP) containing added traces of Co2+, thereby mimicking the bone mineral accumulation of Co2+. Murine bone marrow osteoclasts formed in the presence of M-CSF and RANKL were cultured on these surfaces to examine the effects of Co2+ on osteoclast formation and resorptive activity. Treatment conditions with Co2+ involved incorporation into the CaP layer, adsorption to the mineral surface, or addition to culture media. Micromolar concentrations of Co2+ delivered to developing osteoclast precursors by all 3 routes increased both osteoclast differentiation and resorptive function. Compared to CaP layers without Co2+, we observed a maximal 75% increase in osteoclast numbers and a 2.3- to 2.7-fold increase in mineral resorption from the tissue culture wells containing 0.1μm Co2+ and 0.1–10μm Co2+, respectively. These concentrations are well within the range found in peri-implant tissues in vivo. This direct effect of Co2+ on osteoclasts appears to act independently of the particulate phagocytosis/inflammation-mediated pathways, thus enhancing osteolysis and aseptic implant loosening. [Copyright &y& Elsevier]

Improvement of synthetic bone graft substitutes as suitable alternatives to a patient's own bone graft remains a challenge in biomaterials research. Our goal was to answer the question of whether improved osteoinductivity of a material would also translate to better bone-healing orthotopically. Three porous biphasic calcium phosphate (BCP) ceramics (BCPA, BCPB, and BCPC), consisting of hydroxyapatite and β-tricalcium phosphate, a porous biphasic calcium phosphate ceramic reinforced with a bioresorbable polylactic acid to improve its mechanical properties (BCPC+), a pure hydroxyapatite ceramic (HA), and a carbonated apatite ceramic (CA) were implanted intramuscularly and orthotopically by using a transverse process model in 11 goats for 12 weeks. BCPA and BCPB had similar chemical composition but differed in their microstructure. BCPB was not osteoinductive at all, but BCPA induced ectopic bone formation in 9 of 11 animals. Orthotopically, BCPA performed better than BCPB in both the amount and rate of bone formation. BCPC, similar to BCPA structurally and physicochemically, showed comparable results ectopically and orthotopically. Addition of resorbable polymer to BCPC made the material less osteoinductive (4 of 11 animals) and delayed bone formation orthotopically. Neither HA nor CA were osteoinductive, and their orthotopic performance was inferior to the osteoinductive ceramics. The results of the present study showed that material-derived osteoinduction significantly enhanced bone healing orthotopically, and that this material property appeared more sensitive for predicting orthotopic performance than physicochemical and structural characteristics. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1363-1370, 2008 [ABSTRACT FROM AUTHOR]

Abstract: Rapid prototyping is a valuable implant production tool that enables the investigation of individual geometric parameters, such as shape, porosity, pore size and permeability, on the biological performance of synthetic bone graft substitutes. In the present study, we have employed low-temperature direct 3D printing to produce brushite and monetite implants with different geometries. Blocks predominantly consisting of brushite with channels either open or closed to the exterior were implanted on the decorticated lumbar transverse processes of goats for 12 weeks. In addition, similar blocks with closed channel geometry, consisting of either brushite or monetite were implanted intramuscularly. The design of the channels allowed investigation of the effect of macropore geometry (open and closed pores) and osteoinduction on bone formation orthotopically. Intramuscular implantation resulted in bone formation within the channels of both monetite and brushite, indicating osteoinductivity of these resorbable materials. Inside the blocks mounted on the transverse processes, initial channel shape did not seem to significantly influence the final amount of formed bone and osteoinduction was suggested to contribute to bone formation. [Copyright &y& Elsevier]

Abstract: In this study, porous 3D fiber deposition titanium (3DFT) and 3DFT combined with porous biphasic calcium phosphate ceramic (3DFT+BCP) implants, both bare and 1 week cultured with autologous bone marrow stromal cells (BMSCs), were implanted intramuscularly and orthotopically in 10 goats. To assess the dynamics of bone formation over time, fluorochrome markers were administered at 3, 6 and 9 weeks and the animals were sacrificed at 12 weeks after implantation. New bone in the implants was investigated by histology and histomorphometry of non-decalcified sections. Intramuscularly, no bone formation was found in any of the 3DFT implants, while a very limited amount of bone was observed in 2 BMSC 3DFT implants. 3DFT+BCP and BMSC 3DFT+BCP implants showed ectopic bone formation, in 8 and 10 animals, respectively. The amount of formed bone was significantly higher in BMSC 3DFT+BCP as compared to 3DFT+BCP implants. Implantation on transverse processes resulted in significantly more bone formation in composite structure as compared to titanium alloy alone, both with and without cells. Unlike intramuscularly, the presence of BMSC did not have a significant effect on the amount of new bone either in metallic or in composite structure. Although the 3DFT is inferior to BCP for bone growth, the reinforcement of the brittle BCP with a 3DFT cage did not negatively influence osteogenesis, osteoinduction and osteoconduction as previously shown for the BCP alone. The positive effect of BMSCs was observed ectopically, while it was not significant orthotopically. [Copyright &y& Elsevier]

Abstract: 3D fiber deposition is a technique that allows the development of metallic scaffolds with accurately controlled pore size, porosity and interconnecting pore size, which in turn permits a more precise investigation of the effect of structural properties on the in vivo behavior of biomaterials. This study analyzed the in vivo performance of titanium alloy scaffolds fabricated using 3D fiber deposition. The titanium alloy scaffolds with different structural properties, such as pore size, porosity and interconnecting pore size were implanted on the decorticated transverse processes of the posterior lumbar spine of 10 goats. Prior to implantation, implant structure and permeability were characterized. To monitor the bone formation over time, fluorochrome markers were administered at 3, 6 and 9 weeks and the animals were sacrificed at 12 weeks after implantation. Bone formation in the scaffolds was investigated by histology and histomorphometry of non-decalcified sections using traditional light- and epifluorescent microscopy. In vivo results showed that increase of porosity and pore size, and thus increase of permeability of titanium alloy implants positively influenced their osteoconductive properties. [Copyright &y& Elsevier]

Several publications have shown the phenomenon of osteoinduction by biomaterials to be real. However, whether the ability of a biomaterial to initiate bone formation in ectopic implantation sites improves the performance of such osteoinductive biomaterial in clinically relevant orthotopic sites remains unclear. No studies have been published in which osteoinductive potential of a biomaterial is directly related to its performance orthotopically. In this study, we compared osteoinductive and nonosteoinductive biphasic calcium-phosphate (BCP) ceramics ectopically and in a clinically relevant critical-sized orthotopic defect in goats. The two materials, BCP1150 and BCP1300, had similar chemical compositions, crystallinities, and macrostructures, but their microstructures differed significantly. BCP1150, sintered at a lower temperature, had a large amount of micropores, small average crystal size, and hence a high specific surface area. In contrast, BCP1300, with few micropores, had a significantly lower specific surface area as compared to BCP1150. Twelve-week intramuscular implantation in goats ( n = 10) showed that bone was induced in all BCP1150 implants, while no signs of bone formation were found in any of the BCP1300 implants. After 12 weeks of implantation in a bilateral critical-sized iliac wing defect in the same goats, BCP1150 showed significantly more bone than BCP1300. In addition, the analysis of fluorochrome markers, which were administered to the animals 4, 6, and 8 weeks after implantation to follow the bone growth dynamics, showed an earlier start of bone formation in BCP1150 as compared to BCP1300. Significantly better performance of an osteoinductive ceramic in a critical-sized orthotopic defect in a large animal model in comparison to a nonosteoinductive ceramic suggests osteoinduction to be clinically relevant. Further improvement of material osteoinductive properties is thus a significant step forward in the search for alternatives for autologous bone graft. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [ABSTRACT FROM AUTHOR]

Abstract: In order to unravel the mechanism of osteoinduction by biomaterials, in this study we investigated the influence of the specific surface area on osteoinductive properties of two types of calcium phosphate ceramics. Different surface areas of the ceramics were obtained by varying their sintering temperatures. Hydroxyapatite (HA) ceramic was sintered at 1150 and 1250°C. Biphasic calcium phosphate (BCP) ceramic, consisting of HA and beta-tricalcium phosphate (β-TCP), was sintered at 1100, 1150 and 1200°C. Changes in sintering temperature did not influence the chemistry of the ceramics; HA remained pure after sintering at different temperatures and the weight ratio of HA and β-TCP in the BCP was independent of the temperature as well. Similarly, macroporosity of the ceramics was unaffected by the changes of the sintering temperature. However, microporosity (pore diameter <10μm) significantly decreased with increasing sintering temperature. In addition to the decrease of the microporosity, the crystal size increased with increasing sintering temperature. These two effects resulted in a significant decrease of the specific surface area of the ceramics with increasing sintering temperatures. Samples of HA1150, HA1250, BCP1100, BCP1150 and BCP1200 were implanted in the back muscles of Dutch milk goats and harvested at 6 and 12 weeks post implantation. After explantation, histomorphometrical analysis was performed on all implants. All implanted materials except HA1250 induced bone. However, large variations in the amounts of induced bone were observed between different materials and between individual animals. Histomorphometrical results showed that the presence of micropores within macropore walls is necessary to make a material osteoinductive. We postulate that introduction of microporosity within macropores, and consequent increase of the specific surface area, affects the interface dynamics of the ceramic in such a way that relevant cells are triggered to differentiate into the osteogenic lineage. [Copyright &y& Elsevier]

The in vivo behavior of a porous Ti6Al4V material that was produced by a positive replica technique, with and without an octacalcium phosphate (OCP) coating, has been studied both in the back muscle and femur of goats. Macro- and microporous biphasic calcium phosphate (BCP) ceramic, known to be both osteoconductive and able to induce ectopic bone formation, was used for comparison purpose.The three groups of materials (Ti6Al4V, OCP Ti6Al4V and BCP) were implanted transcortically and intramuscularly for 6 and 12 weeks in 10 adult Dutch milk goats in order to study their osteointegration and osteoinductive potential.In femoral defects, both OCP Ti6Al4V and BCP were performing better than the uncoated Ti6Al4V, at both time points. BCP showed a higher bone amount than OCP Ti6Al4V after 6 weeks of implantation, while after 12 weeks, this difference was no longer significant.Ectopic bone formation was found in both OCP Ti6Al4V and BCP implants after 6 and 12 weeks. The quantity of ectopically formed bone was limited as was the amount of animals in which the bone was observed. Ectopic bone formation was not found in uncoated titanium alloy implants, suggesting that the presence of calcium phosphate (CaP) is important for bone induction.This study showed that CaPs in the form of coating on metal implants or in the form of bulk ceramic have a significantly positive effect on the bone healing process. [Copyright &y& Elsevier]

Focuses on the biomimetic hydroxyapatite coating on metal implants. Advantages of biomimetic approach; Solubility of calcium phosphate in acidic conditions; Chemical and structural characterization of the coatings.

Microbiome is an integral part of the gut and is essential for its proper function. Imbalances of the microbiota can be devastating and have been linked with several gastrointestinal conditions. Current gastrointestinal models do not fully reflect the in vivo situation. Thus, it is important to establish more advanced in vitro models to study host-microbiome/pathogen interactions. Here, we developed for the first time an apical-out human small intestinal organoid model in hypoxia, where the apical surface is directly accessible and exposed to a hypoxic environment. These organoids mimic the intestinal cell composition, structure and functions and provide easy access to the apical surface. Co-cultures with the anaerobic strains Lactobacillus casei and Bifidobacterium longum showed successful colonization and probiotic benefits on the organoids. These novel hypoxia-tolerant apical-out small intestinal organoids will pave the way for unraveling unknown mechanisms related to host-microbiome interactions and serve as a tool to develop microbiome-related probiotics and therapeutics. [ABSTRACT FROM AUTHOR]

We present a novel high throughput photocatalyst efficiency assessment method based on 96-well microplates and UV-vis spectroscopy. We demonstrate the reproducibility of the method using methyl orange (MO) decomposition and compare kinetic data obtained with those provided in the literature for larger conventional photoreactors. To demonstrate the capabilities of the method, we rapidly screened the effects of salts, potentially present in wastewater, on kinetic rates of MO decomposition and briefly discuss the obtained data on the basis of existing literature. [ABSTRACT FROM AUTHOR]

Biomaterials can be endowed with biologically instructive properties by changing basic parameters such as elasticity and surface texture. However, translation from in vitro proof of concept to clinical application is largely missing. Porous calcium phosphate ceramics are used to treat small bone defects but in general do not induce stem cell differentiation, which is essential for regenerating large bone defects. Here, we prepared calcium phosphate ceramics with varying physicochemical and structural characteristics. Microporosity correlated to their propensity to stimulate osteogenic differentiation of stem cells in vitro and bone induction in vivo. Implantation in a large bone defect in sheep unequivocally demonstrated that osteoinductive ceramics are equally efficient in bone repair as autologous bone grafts. Our results provide proof of concept for the clinical application of "smart" biomaterials. [ABSTRACT FROM AUTHOR]

Little is known about the mechanism by which autologous bone grafts are so successful. The relevance of viable osteogenic cells, which is a prominent difference between autologous bone graft and conventional alternatives, is especially controversial. With the emergence of bone tissue engineering, knowledge of the exact role of these cells has become crucial. The most obvious question to answer is whether viability of the graft has an effect on bone formation. In the current study, we investigated this effect of bone graft viability in a transverse process model that represents the initial bone formation in posterolateral spinal fusion. Eight goats received viable and devitalized autologous bone grafts in chambers mounted on the decorticated lumbar transverse processes. In addition, five goats received empty chambers. Histology and histomorphometry were performed after a 12-week implantation, and the dynamics of bone formation was monitored by sequential fluorochrome labeling. An obvious qualitative effect of viability was demonstrated by the presence of early onset osteogenesis distant from the transverse process bone in the viable grafts only. Quantitative analysis indicated about 30% more bone in the viable grafts, however, this difference was not statistically significant. In the empty chambers, bone was found in comparable quantities. We conclude that there is a qualitative advantage of graft viability in terms of early graft-derived osteogenesis. However, this advantage did not lead to significantly more bone formation in the viable bone grafts. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1055-1059, 2009 [ABSTRACT FROM AUTHOR]

Embryogenic developmental processes involve a tightly controlled regulation between mechanical forces and biochemical cues such as growth factors, matrix proteins, and cytokines. This interplay remains essential in the mature body, with aberrant pathway signaling leading to abnormalities such as atherosclerosis in the cardiovascular system, inflammation in tendon tissue, or osteoporosis in the bone. The aim of bone regenerative strategies is to develop tools and procedures that will harness the body's own self-repair ability in order to successfully regenerate even very large and complex bone defects and restore normal function. To achieve this, understanding pathways that govern processes of progenitor differentiation towards the osteogenic lineages, their phenotypical maintenance, and the construction of functional bone tissue is imperative to subsequently develop regenerative therapies that mimic these processes. While a body of literature exists that describes how biochemical stimuli guide cell behavior in the culture dish, due to the lack of an appropriate mechanical environment, these signals are often insufficient or inappropriate for achieving a desirable response in the body. Moreover, bone regenerative therapies rarely rely on a biochemical stimulus, such as a growth factor alone, and instead often comprise a carrier biomaterial that introduces a very different microenvironment from that of a cell culture dish. Therefore, in this review, we discuss which biomaterials elicit or influence pathways relevant for bone regeneration and describe mechanisms behind these effects, with the aim to inspire the development of novel, more effective bone regenerative therapies. [ABSTRACT FROM AUTHOR]

Biodegradable porous calcium phosphate (CaP) ceramics are widely used as synthetic graft substitutes for bone regeneration, owing to their chemical and structural similarity to bone and associated bioactivity in terms of bone‐bonding, osteoconductive, and even osteoinductive properties. Nevertheless, the intrinsic brittleness and poor processability of porous CaP ceramics strongly impair their clinical applicability. Herein, a biphasic calcium phosphate (BCP) sponge is developed that consists of a self‐supporting network of seamlessly interwoven hydroxyapatite nanowires and β‐tricalcium phosphate nanofibers and possesses a highly interconnected porous structure with open cell geometry and ultrahigh porosity. Owing to its unique properties, the ceramic sponge can be easily processed into various shapes and dimensions, such as cylindrical scaffolds and thin, flexible membranes. Moreover, the BCP sponge can be introduced into a bone defect in a compacted or folded state from a syringe and, upon wetting, expand to its original shape, thereby filling the cavity. The nanofibrous sponge gradually degrades in vitro and rapidly mineralizes when immersed in simulated body fluid. Moreover, it adsorbs significantly more proteins than a conventional porous BCP ceramic. Finally, the nanofibrous sponge supports the attachment, proliferation, and osteogenic differentiation of human mesenchymal stromal cells comparable to the conventional porous BCP ceramic. [ABSTRACT FROM AUTHOR]

In the past decade, calcium phosphate (CaP) ceramics have emerged as alternatives to autologous bone grafts for the treatment of large, critical-sized bone defects. In order to be effective in the regeneration of such defects, ceramics must show osteoinductive behaviour, defined as the ability to induce de novo heterotopic bone formation. While a set of osteoinductive CaP ceramics has been developed, the exact processes underlying osteoinduction, and the role of the physical and chemical properties of the ceramics, remain largely unknown. Previous studies have focused on the role of the transcriptome to shed light on the mechanism of osteoinduction at the mRNA level. To complement these studies, a proteomic analysis was performed to study the behaviour of hMSCs on osteoinductive and non-osteoinductive CaPs. The results of this analysis suggest that plasma cell glycoprotein 1 (PC-1), encoded by the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene, plays a key role in the process of osteoinduction by CaP ceramics. Validation experiments have confirmed that indeed, the mRNA expression of ENPP1 and the production of PC-1 are higher on osteoinductive than on non-osteoinductive CaP ceramics, a trend that was also observed for other osteogenic markers such as bone morphogenetic protein 2 (BMP2) and osteopontin (OPN), but not for alkaline phosphatase (ALP). Our results also showed that the expression of PC-1 is restricted to those cells which are in direct contact with the CaP ceramic surface, plausibly due to the localised depletion of calcium and inorganic phosphate ions from the supersaturated cell culture medium as CaP crystallises on the ceramic surface. Replicating the surface of the osteoinductive ceramic in polystyrene resulted in a significant decrease in ENPP1 expression, suggesting that surface structural properties alone are not sufficient to induce ENPP1 expression. Finally, knocking down ENPP1 expression in hMSCs resulted in increased BMP2 expression, both at the mRNA and protein level, suggesting that ENPP1 is a negative regulator of BMP-2 signalling. Taken together, this study shows, for the first time, that ENPP1 /PC-1 plays an important role in CaP-induced osteogenic differentiation of hMSCs and thus possibly osteoinduction by CaP ceramics. Furthermore, we have identified a crucial role for the interfacial (chemical) events occurring on the CaP ceramic surface in the process of osteoinduction. This knowledge can contribute to the development of new bone graft substitutes, with improved osteoinductive potential. [ABSTRACT FROM AUTHOR]

The role that biomaterials play in the clinical treatment of damaged organs and tissues is changing. While biomaterials used in permanent medical devices were required to passively take over the function of a damaged tissue in the long term, current biomaterials are expected to trigger and harness the self-regenerative potential of the body in situ and then to degrade, the foundation of regenerative medicine. To meet these different requirements, it is imperative to fully understand the interactions biomaterials have with biological systems, in space and in time. This knowledge will lead to a better understanding of the regenerative capabilities of biomaterials aiding their design with improved functionalities (e.g. biocompatibility, bioactivity). Proteins play a pivotal role in the interaction between biomaterials and cells or tissues. Protein adsorption on the material surface is the very first event of this interaction, which is determinant for the subsequent processes of cell growth, differentiation, and extracellular matrix formation. Against this background, the aim of the current review is to provide insight in the current knowledge of the role of proteins in cell–biomaterial and tissue–biomaterial interactions. In particular, the focus is on proteomics studies, mainly using mass spectrometry, and the knowledge they have generated on protein adsorption of biomaterials, protein production by cells cultured on materials, safety and efficacy of new materials based on nanoparticles and the analysis of extracellular matrices and extracellular matrix–derived products. In the outlook, the potential and limitations of this approach are discussed and mass spectrometry imaging is presented as a powerful technique that complements existing mass spectrometry techniques by providing spatial molecular information about the material-biological system interactions. [ABSTRACT FROM AUTHOR]

Surface topography is increasingly being recognized as an important factor to control the response of cells and tissues to biomaterials. In the current study, the aim was to obtain deeper understanding of the effect of microgrooves on shape and orientation of osteoblast-like cells and to relate this effect to their proliferation and osteogenic differentiation. To this end, two microgrooved polystyrene (PS) substrates, differing in the width of the grooves (about 2 μm and 4 μm) and distance between individual grooves (about 6 μm and 11 μm, respectively) were fabricated using a combination of photolithography and hot embossing. MG-63 human osteosarcoma cells were cultured on these microgrooved surfaces, with unpatterned hot-embossed PS substrate as a control. Scanning electron- and fluorescence microscopy analyses showed that on patterned surfaces, the cells aligned along the microgrooves. The cells cultured on 4 μm-grooves / 11 μm-ridges surface showed a more pronounced alignment and a somewhat smaller cell area and cell perimeter as compared to cells cultured on surface with 2 μm-grooves / 6 μm-ridges or unpatterned PS. PrestoBlue analysis and quantification of DNA amounts suggested that microgrooves used in this experiment did not have a strong effect on cell metabolic activity or proliferation. However, cell differentiation towards the osteogenic lineage was significantly enhanced when MG-63 cells were cultured on the 2/6 substrate, as compared to the 4/11 substrate or unpatterned PS. This effect on osteogenic differentiation may be related to differences in cell spreading between the substrates. [ABSTRACT FROM AUTHOR]

Calcium phosphate ceramics with specific physicochemical properties have been shown to induce de novo bone formation upon ectopic implantation in a number of animal models. In this study we explored the influence of physicochemical properties as well as the animal species on material-induced ectopic bone formation. Three bioceramics were used for the study: phase-pure hydroxyapatite (HA) sintered at 1200°C and two biphasic calcium phosphate (BCP) ceramics, consisting of 60 wt.% HA and 40 wt.% TCP (β-Tricalcium phosphate), sintered at either 1100°C or 1200°C. 108 samples of each ceramic were intramuscularly implanted in dogs, rabbits, and rats for 6, 12, and 24 weeks respectively. Histological and histomorphometrical analyses illustrated that ectopic bone and/or osteoid tissue formation was most pronounced in BCP sintered at 1100°C and most limited in HA, independent of the animal model. Concerning the effect of animal species, ectopic bone formation reproducibly occurred in dogs, while in rabbits and rats, new tissue formation was mainly limited to osteoid. The results of this study confirmed that the incidence and the extent of material-induced bone formation are related to both the physicochemical properties of calcium phosphate ceramics and the animal model. [ABSTRACT FROM AUTHOR]

Abstract: The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid–alkali (AcAl), alkali–acid–heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial. [Copyright &y& Elsevier]

We present a microtiter plate-sized standalone chip holder for precise control of physiological conditions inside closed microfluidic cell culture systems, made from gas-impermeable materials. Specifically, we demonstrate the suitability of the holder to support cell growth in a glass chip, to allow time-lapse imaging of live cells and the creation of a hypoxic environment, all relevant for applications in regenerative medicine research. [ABSTRACT FROM AUTHOR]

Abstract: With biomimetic biomaterials, like calcium phosphate cements (CPCs), non-invasive assessment of tissue regeneration is challenging. This study describes a theranostic agent (TA) to simultaneously enhance both imaging and osteogenic properties of such a bone substitute material. For this purpose, mesoporous silica beads were produced containing an iron oxide core to enhance bone magnetic resonance (MR) contrast. The same beads were functionalized with silane linkers to immobilize the osteoinductive protein BMP-2, and finally received a calcium phosphate coating, before being embedded in the CPC. Both in vitro and in vivo tests were performed. In vitro testing showed that the TA beads did not interfere with essential material properties like cement setting. Furthermore, bioactive BMP-2 could be efficiently released from the carrier-beads. In vivo testing in a femoral condyle defect rat model showed long-term MR contrast enhancement, as well as improved osteogenic capacity. Moreover, the TA was released during CPC degradation and was not incorporated into the newly formed bone. In conclusion, the described TA was shown to be suitable for longitudinal material degradation and bone healing studies. [Copyright &y& Elsevier]

Synthetic bone graft substitutes based on composites consisting of a polymer and a calcium-phosphate (Cap) ceramic are developed with the aim to satisfy both mechanical and bioactivity requirements for successful bone regeneration. In the present study, we have employed extrusion to produce a composite consisting of 50 wt.% poly(D,L-lactic acid) (pLA) and 50 wt.% nano-sized hydroxyapatite (HA) powder, achieving homogeneous distribution of the ceramic within the polymeric phase. In vitro, in both a simulated physiological saline (SpS) and a simulated body fluid (SBF), a greater weight loss was observed for pLA/HA than for pLA particles upon 12-week immersion. Furthermore, in SpS, a continuous release of calcium and phosphate from the composite was measured, whereas in SBF, decrease of the amount of the two ions in the solution was observed both for pLA and pLA/HA accompanied with the formation of a Cap layer on the surface. In vitro characterization of the composite bioactivity was performed by culturing human mesenchymal stromal cells (hMSCs) and assessing proliferation and osteogenic differentiation, with pLA as a control. Both pLA/HA composite and pLA control were shown to support hMSCs proliferation over a period of two weeks. In addition, the composite significantly enhanced alkaline phosphatase (ALp) activity of hMSCs in osteogenic medium as compared with the polymer control. A novel implant design was employed to develop implants from dense, extruded materials, suitable for testing osteoinductivity in vivo. In a preliminary study in dogs, pLA/HA composite implants induced heterotopic bone formation upon 12-week intramuscular implantation in all animals, in contrast to pLA control, which was not osteoinductive. Unlike in vitro, a more pronounced degradation of pLA was observed in vivo as compared with pLA/HA composite. [ABSTRACT FROM AUTHOR]

The aim of regenerative medicine is to restore or establish normal function of damaged tissues or organs. Tremendous efforts are placed into development of novel regenerative strategies, involving (stem) cells, soluble factors, biomaterials or combinations thereof, as a result of the growing need caused by continuous population aging. To satisfy this need, fast and reliable assessment of (biological) performance is sought, not only to select the potentially interesting candidates, but also to rule out poor ones at an early stage of development. Microfluidics may provide a new avenue to accelerate research and development in the field of regenerative medicine as it has proven its maturity for the realization of high-throughput screening platforms. In addition, microfluidic systems offer other advantages such as the possibility to create in vivo-like microenvironments. Besides the complexity of organs or tissues that need to be regenerated, regenerative medicine brings additional challenges of complex regeneration processes and strategies. The question therefore arises whether so much complexity can be integrated into microfluidic systems without compromising reliability and throughput of assays. With this review, we aim to investigate whether microfluidics can become widely applied in regenerative medicine research and/or strategies. [ABSTRACT FROM AUTHOR]

Combining technologies to engineer scaffolds that can offer physical and chemical cues to cells is an attractive approach in tissue engineering and regenerative medicine. In this study, we have fabricated polymer-ceramic hybrid scaffolds for bone regeneration by combining rapid prototyping (RP), electrospinning (ESP) and a biomimetic coating method in order to provide mechanical support and a physico-chemical environment mimicking both the organic and inorganic phases of bone extracellular matrix (ECM). poly(ethylene oxide terephthalate)-poly(buthylene terephthalate) (PEOT/PBT) block copolymer was used to produce three dimensional scaffolds by combining 3D fiber (3DF) deposition, and ESP, and these constructs were then coated with a Ca-p layer in a simulated physiological solution. Scaffold morphology and composition were studied using scanning electron microscopy (SEM) coupled to energy dispersive X-ray analyzer (EDX) and Fourier tranform Infrared Spectroscopy (FTIR). Bone marrow derived human mesenchymal stromal cells (HMSCS) were cultured on coated and uncoated 3DF and 3DF + ESP scaffolds for up to 21 d in basic and mineralization medium and cell attachment, proliferation, and expression of genes related to osteogenesis were assessed. Cells attached, proliferated and secreted ECM on all the scaffolds. there were no significant differences in metabolic activity among the different groups on days 7 and 21. Coated 3DF scaffolds showed a significantly higher DNA amount in basic medium at 21 d compared with the coated 3DF + ESP scaffolds, whereas in mineralization medium, the presence of coating in 3DF+ESP scaffolds led to a significant decrease in the amount of DNA. An effect of combining different scaffolding technologies and material types on expression of a number of osteogenic markers (cbfa1, BMP-2, OP, OC and ON) was observed, suggesting the potential use of this approach in bone tissue engineering. [ABSTRACT FROM AUTHOR]

Combining technologies to engineer scaffolds that can offer physical and chemical cues to cells is an attractive approach in tissue engineering and regenerative medicine. In this study, we have fabricated polymer-ceramic hybrid scaffolds for bone regeneration by combining rapid prototyping (RP), electrospinning (ESP) and a biomimetic coating method in order to provide mechanical support and a physico-chemical environment mimicking both the organic and inorganic phases of bone extracellular matrix (ECM). poly(ethylene oxide terephthalate)-poly(buthylene terephthalate) (PEOT/PBT) block copolymer was used to produce three dimensional scaffolds by combining 3D fiber (3DF) deposition, and ESP, and these constructs were then coated with a Ca-P layer in a simulated physiological solution. Scaffold morphology and composition were studied using scanning electron microscopy (SEM) coupled to energy dispersive X-ray analyzer (EDX) and Fourier tranform Infrared Spectroscopy (FTIR). Bone marrow derived human mesenchymal stromal cells (hMSCs) were cultured on coated and uncoated 3DF and 3DF + ESP scaffolds for up to 21 d in basic and mineralization medium and cell attachment, proliferation, and expression of genes related to osteogenesis were assessed. Cells attached, proliferated and secreted ECM on all the scaffolds. there were no significant differences in metabolic activity among the different groups on days 7 and 21. Coated 3DF scaffolds showed a significantly higher DNA amount in basic medium at 21 d compared with the coated 3DF + ESP scaffolds, whereas in mineralization medium, the presence of coating in 3DF+ESP scaffolds led to a significant decrease in the amount of DNA. An effect of combining different scaffolding technologies and material types on expression of a number of osteogenic markers (cbfa1, BMP-2, OP, OC and ON) was observed, suggesting the potential use of this approach in bone tissue engineering. [ABSTRACT FROM AUTHOR]

A simple method is reported for fabricating polystyrene disk inserts coated with biomimetic carbonated hydroxyapatite (cHA) to be used for culturing osteoprogenitor cells or other stem cells. Roughened disks cut from tissue-culture polystyrene (TCPS) were coated in simulated body fluid with 5 × normal physiologic ionic concentrations (SBFx5) by a 2-step, 2-day method. The coatings were rigorously characterized by various methods and assessed in cell culture. An adherent, nearly 10 mm thick, relatively uniform layer of single-phase cHA was formed in two days. MC3T3-E1 and mouse calvaria-derived osteoprogenitor cells (pCOBs) were cultured on the cHA for various time points. Despite less initial attachment of both cell types to the cHA, proliferation rates on cHA were similar to that on TCPS. Two-fold greater cell attachment (P < 0.05) of the MC3T3-E1 cells was observed relative to the pCOBs, on both the TCPS and the cHA. Importantly, the coatings were relatively smooth, without the extensive agglomerates observed in other studies and remained adherent and morphologically unchanged after 21 days of culture. This technique can be used to rapidly produce high-quality cHA-coated TCPS disks for cell-culture studies. [ABSTRACT FROM AUTHOR]

Abstract: This study describes a medium-throughput system based on deposition of calcium phosphate films in multi-well tissue culture plates that can be used to study the effect of inorganic additives on the behavior of osteoblasts and osteoclasts in a standardized manner. All tested elements, copper, zinc, strontium, fluoride and carbonate were homogenously deposited into calcium phosphate films in varying concentrations by using a biomimetic approach. The additives affected morphology and composition of calcium phosphate films to different extent, depending on the concentration used. The effect on proliferation and differentiation of MC3T3-E1 osteoblasts depended on the compound and concentration tested. In general, copper and zinc ions showed an inhibitory effect on osteoblast proliferation, the effect of strontium was concentration dependent, whereas films containing fluoride and carbonate, respectively, augmented osteoblast proliferation. Copper and zinc had no effect or were mild inhibitory on osteoblast differentiation, while strontium, fluoride and carbonate ions demonstrated a clear decrease in differentiation in comparison to the control films without additives. Primary osteoclasts cultured on calcium phosphate films containing additives showed a significantly decreased resorptive activity as compared to the control, independent on the element incorporated. No cytotoxic effect of the elements in the concentrations tested was observed. The system presented in this study mimics bone mineral containing trace elements, making it useful for studying fundamental processes of bone formation and turnover. The present results can be used for modifying bone graft substitutes by addition of inorganic additives in order to affect their performance in bone repair and regeneration. [Copyright &y& Elsevier]

Abstract: An ideal material for maxillofacial vertical bone augmentation procedures should not only be osteoconductive, biocompatible and mechanically strong, but should also be applied using minimally invasive procedures and remain stable with respect to the original bone surfaces. This way, implant exposure and infection might be reduced and good mechanical stability may be achieved. Calcium phosphate cements are proven biocompatible and osteoconductive materials that can be injected using minimally invasive procedures. Among these cements, brushite based cements have the added advantage of being biodegradable in vivo. Therefore, this material has the potential for use in the aforementioned procedures. An in vivo study was performed in rabbits to evaluate the potential use of brushite cements in minimally invasive maxillofacial vertical bone augmentation procedures. In this study, we injected self-setting brushite cements on the subperiosteal bone surface using a minimally invasive tunnelling technique. The cement pastes were stable on the bone surface and hardened soon after they were injected thereby negating the need for additional supports such as membranes or meshes. The animals were sacrificed 8 weeks after the intervention and histological observations revealed signs of successful vertical bone augmentation. Therefore, we have demonstrated a minimally invasive vertical bone augmentation procedure that is an attractive alternative to current surgical procedures in terms of increased simplicity, reduced trauma, and lower cost of surgery. [Copyright &y& Elsevier]