Optimization of a tunable process for rapid production of calcium phosphate microparticles using a droplet-based microfluidic platform.

Calcium phosphate (CaP) biomaterials are amongst the most widely used synthetic bone graft substitutes, owing to their chemical similarities to the mineral part of bone matrix and off-the-shelf availability. However, their ability to regenerate bone in critical-sized bone defects has remained inferior to the gold standard autologous bone. Hence, there is a need for methods that can be employed to efficiently produce CaPs with different properties, enabling the screening and consequent fine-tuning of the properties of CaPs towards effective bone regeneration. To this end, we propose the use of droplet microfluidics for rapid production of a variety of CaP microparticles. Particularly, this study aims to optimize the steps of a droplet microfluidic-based production process, including droplet generation, in-droplet CaP synthesis, purification and sintering, in order to obtain a library of CaP microparticles with fine-tuned properties. The results showed that size-controlled, monodisperse water-in-oil microdroplets containing calcium- and phosphate-rich solutions can be produced using a flow-focusing droplet-generator microfluidic chip. We optimized synthesis protocols based on in-droplet mineralization to obtain a range of CaP microparticles without and with inorganic additives. This was achieved by adjusting synthesis parameters, such as precursor concentration, pH value, and aging time, and applying heat treatment. In addition, our results indicated that the synthesis and fabrication parameters of CaPs in this method can alter the microstructure and the degradation behavior of CaPs. Overall, the results highlight the potential of the droplet microfluidic platform for engineering CaP microparticle biomaterials with fine-tuned properties.

Effect of minor amounts of ß-calcium pyrophosphate and hydroxyapatite on the physico-chemical properties and osteoclastic resorption of ß-tricalcium phosphate cylinders.

ß-Tricalcium Phosphate (ß-TCP), one of the most used bone graft substitutes, may contain up to 5 wt% foreign phase according to standards. Typical foreign phases include ß-calcium pyrophosphate (ß-CPP) and hydroxyapatite (HA). Currently, the effect of small amounts of impurities on ß-TCP resorption is unknown. This is surprising since pyrophosphate is a very potent osteoclast inhibitor. The main aim of this study was to assess the effect of small ß-CPP fractions (<1 wt%) on the in vitro osteoclastic resorption of ß-TCP. A minor aim was to examine the effect of ß-CPP and HA impurities on the physico-chemical properties of ß-TCP powders and sintered cylinders. Twenty-six batches of ß-TCP powder were produced with a Ca/P molar ratio varying between 1.440 and 1.550. Fifteen were further processed to obtain dense and polished ß-TCP cylinders. Finally, six of them, with a Ca/P molar ratio varying between 1.496 (1 wt% ß-CPP) and 1.502 (1 wt% HA), were incubated in the presence of osteoclasts. Resorption was quantified by white-light interferometry. Osteoclastic resorption was significantly inhibited by ß-CPP fraction in a linear manner. The presence of 1% ß-CPP reduced ß-TCP resorption by 40%, which underlines the importance of controlling ß-CPP content when assessing ß-TCP biological performance.

Innovating in the medical device industry - challenges & opportunities ESB 2015 translational research symposium.

The European Society for Biomaterials 2015 Translational Research Symposium focused on 'Innovating in the Medical Device Industry - Challenges & Opportunities' from different perspectives, i.e., from a non-profit research organisation to a syndicate of small and medium-sized companies and large companies. Lecturers from regulatory consultants, industry and research institutions described the innovation process and regulatory processes (e.g., 510K, PMA, combination product) towards market approval. The aim of the present article is to summarise and explain the main statements made during the symposium, in terms of challenges and opportunities for medical device industries, in a constantly changing customer and regulatory environment.

Progressing innovation in biomaterials. From the bench to the bed of patients.

A Translational Research Symposium was organized at the 2014 annual meeting of the European society for biomaterials. This brought together leading Tier one companies in clinical biomaterials and medical device markets, small and medium enterprises and entrepreneurial academics who shared their experiences on taking biomaterials technologies to commercial endpoints, in the clinics. The symposium focused on "Progressing Innovation in Biomaterials. From the Bench to the Bed of Patients". The aim of the present document is to illustrate the content of the symposium and to highlight the key lessons from selected lectures.

Effect of grain size and microporosity on the in vivo behaviour of ß-tricalcium phosphate scaffolds.

Defining the most adequate architecture of a bone substitute scaffold is a topic that has received much attention over the last 40 years. However, contradictory results exist on the effect of grain size and microporosity. Therefore, the aim of this study was to determine the effect of these two factors on the in vivo behaviour of ß-tricalcium phosphate (ß-TCP) scaffolds. For that purpose, ß-TCP scaffolds were produced with roughly the same macropore size (≈ 150 µm), and porosity (≈ 80 %), but two levels of microporosity (low: 10 % / high: ≈ 25 %) and grain size (small: 1.3 µm /large: ≈ 3.3 µm). The sample architecture was characterised extensively using materialography, Hg porosimetry, micro-computed tomography (µCT), and nitrogen adsorption. The scaffolds were implanted for 2, 4 and 8 weeks in a cylindrical 5-wall cancellous bone defect in sheep. The histological, histomorphometrical and µCT analysis of the samples revealed that all four scaffold types were almost completely resorbed within 8 weeks and replaced by new bone. Despite the three-fold difference in microporosity and grain size, very few biological differences were observed. The only significant effect at p < 0.01 was a slightly faster resorption rate and soft tissue formation between 4 and 8 weeks of implantation when microporosity was increased. Past and present results suggest that the biological response of this particular defect is not very sensitive towards physico-chemical differences of resorbable bone graft substitutes. As bone formed not only in the macropores but also in the micropores, a closer study at the microscopic and localised effects is necessary.

Influence of physico-chemical material characteristics on staphylococcal biofilm formation--a qualitative and quantitative in vitro analysis of five different calcium phosphate bone grafts.

Various compositions of synthetic calcium phosphates (CaP) have been proposed and their use has considerably increased over the past decades. Besides differences in physico-chemical properties, resorption and osseointegration, artificial CaP bone graft might differ in their resistance against biofilm formation. We investigated standardised cylinders of 5 different CaP bone grafts (cyclOS, chronOS (both ß-TCP (tricalcium phosphate)), dicalcium phosphate (DCP), calcium-deficient hydroxyapatite (CDHA) and α-TCP). Various physico-chemical characterisations e.g., geometrical density, porosity, and specific surface area were investigated. Biofilm formation was carried out in tryptic soy broth (TSB) and human serum (SE) using Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984). The amount of biofilm was analysed by an established protocol using sonication and microcalorimetry. Physico-chemical characterisation showed marked differences concerning macro- and micropore size, specific surface area and porosity accessible to bacteria between the 5 scaffolds. Biofilm formation was found on all scaffolds and was comparable for α-TCP, chronOS, CDHA and DCP at corresponding time points when the scaffolds were incubated with the same germ and/or growth media, but much lower for cyclOS. This is peculiar because cyclOS had an intermediate porosity, mean pore size, specific surface area, and porosity accessible to bacteria. Our results suggest that biofilm formation is not influenced by a single physico-chemical parameter alone but is a multi-step process influenced by several factors in parallel. Transfer from in vitro data to clinical situations is difficult; thus, advocating the use of cyclOS scaffolds over the four other CaP bone grafts in clinical situations with a high risk of infection cannot be clearly supported based on our data.

The relevance of biomaterials to the prevention and treatment of osteoporosis.

Osteoporosis is a worldwide disease with a very high prevalence in humans older than 50. The main clinical consequences are bone fractures, which often lead to patient disability or even death. A number of commercial biomaterials are currently used to treat osteoporotic bone fractures, but most of these have not been specifically designed for that purpose. Many drug- or cell-loaded biomaterials have been proposed in research laboratories, but very few have received approval for commercial use. In order to analyze this scenario and propose alternatives to overcome it, the Spanish and European Network of Excellence for the Prevention and Treatment of Osteoporotic Fractures, "Ageing", was created. This network integrates three communities, e.g. clinicians, materials scientists and industrial advisors, tackling the same problem from three different points of view. Keeping in mind the premise "living longer, living better", this commentary is the result of the thoughts, proposals and conclusions obtained after one year working in the framework of this network.

Phase and size separations occurring during the injection of model pastes composed of ß-tricalcium phosphate powder, glass beads and aqueous solutions.

Glass beads a few hundred micrometers in size were added to aqueous ß-tricalcium phosphate pastes to simulate the effect of porogens and drug-loaded microspheres on the injectability of calcium phosphate cements and putties. The composition of the pastes was monitored during the injection process to assess the effect of glass bead content, glass bead size and paste composition on the paste injectability. The results revealed that the injection process led to both liquid and glass bead segregations: the liquid flowed faster than the glass beads, which themselves flowed faster than the ß-tricalcium phosphate microparticles. In fact, even the particle size distribution of the glass beads was modified during injection. These results reveal that a good design of multiphasic injectable pastes is essential to prevent phase separation.

New depowdering-friendly designs for three-dimensional printing of calcium phosphate bone substitutes.

Powder-based three-dimensional printing (3DP) is a versatile method that allows creating synthetic calcium phosphate (CaP) scaffolds of complex shapes and structures. However, one major drawback is the difficulty of removing all remnants of loose powder from the printed scaffolds, the so-called depowdering step. In this study, a new design approach was proposed to solve this problem. Specifically, the design of the printed scaffolds consisted of a cage with windows large enough to enable depowdering while still trapping loose fillers placed inside the cage. To demonstrate the potential of this new approach, two filler geometries were used: sandglass and cheese segment. The distance between the fillers was varied and they were either glued to the cage or free to move after successful depowdering. Depowdering efficiency was quantified by microstructural morphometry. The results showed that the use of mobile fillers significantly improved depowdering. Based on this study, large 3DP scaffolds can be realized, which might be a step towards a broader clinical use of 3D printed CaP scaffolds.

Microporous calcium phosphate ceramics as tissue engineering scaffolds for the repair of osteochondral defects: Histological results.

Treatment of defects in joint cartilage aims to re-establish normal joint function. In vitro experiments have shown that the application of synthetic scaffolds is a promising alternative to existing therapeutic options. A sheep study was conducted to test the suitability of microporous pure ß-tricalcium phosphate (TCP) ceramics as tissue engineering scaffolds for the repair of osteochondral defects. Cylindrical plugs of microporous ß-TCP (diameter: 7mm; length: 25mm; porosity: 43.5±2.4%; pore diameter: ~5µm) with interconnecting pores were used. Scaffolds were seeded with autologous chondrocytes in vitro and cultured for 4weeks. A drill hole (diameter 7mm) was placed in both medial femoral condyles of sheep. For the left knee the defect was filled with a TCP plug and for the right knee the defect was left empty. After 6, 12, 26 and 52weeks, seven animals from each group were killed and studied. The samples were examined employing histological, histomorphometric and immunohistological methods as well as various imaging techniques (X-ray, microcomputer tomography and scanning electron microscopy). After explantation the cartilage defects were first assessed macroscopically. There were no signs of infection or inflammation. Histological grading scales were used for assessment of bony integration and cartilage repair. An increasing degradation (81% after 52weeks) of the ceramic with concomitant bone formation was observed. The original structure of cancellous bone was almost completely restored. After 26 and 52weeks, collagen II-positive hyaline cartilage was detected in several samples. New subchondral bone had formed. The formation of cartilage began at the outer edge and proceeded to the middle. According to the O'Driscoll score, values corresponding to healthy cartilage were not reached after 1year. Integration of the newly formed cartilage tissue into the surrounding native cartilage was found. The formation of biomechanical stable cartilage began at the edge and progressed towards the centre of the defect. After 1year this process was still not completed. Microporous ß-TCP scaffolds seeded with chondrocytes are suitable for the treatment of osteochondral defects.

Moisture based three-dimensional printing of calcium phosphate structures for scaffold engineering.

Powder based three-dimensional printing (3DP) allows great versatility in material and geometry. These characteristics make 3DP an interesting method for the production of tissue engineering scaffolds. However, 3DP has major limitations, such as limited resolution and accuracy, hence preventing the widespread application of this method within scaffold engineering [corrected].In order to reduce these limitations deeper understanding of the complex interactions between powder, binder and roller during 3DP is needed. In the past a lot of effort has been invested to optimize the powder properties for 3DP for a certain layer thickness. Using a powder optimized for an 88 µm layer thickness, this study systematically quantifies the surface roughness and geometrical accuracy in printed specimens and assesses their variation upon changes of different critical parameters such as the moisture application time (0, 5, 10 and 20s), layer thickness (44 and 88 µm) and the number of specimens printed per batch (6 and 12). A best surface roughness value of 25 µm was measured with a moisture application time (using a custom made moisture application device mounted on a linear stage carrying the print head) of 5s and a layer thickness of 44 µm. Geometrical accuracy was generally higher for the 88 µm thick layer, due to a less critical powder bed stability. Moisture application enabled 3DP of a 44 µm thick layer and improved the accuracy even for a powder initially optimized for 88 µm. Moreover, recycling of the humidified powder was not only possible but, in terms of reactivity, even beneficial. In conclusion, moisture-based 3DP is a promising approach for high resolution 3DP of scaffolds.

Microporous calcium phosphate ceramics as tissue engineering scaffolds for the repair of osteochondral defects: biomechanical results.

This work investigated the suitability of microporous ß-tricalcium phosphate (TCP) scaffolds pre-seeded with autologous chondrocytes for treatment of osteochondral defects in a large animal model. Microporous ß-TCP cylinders (Ø 7 mm; length 25 mm) were seeded with autologous chondrocytes and cultured for 4 weeks in vitro. Only the upper end of the cylinder was seeded with chondrocytes. Chondrocytes formed a multilayer on the top. The implants were then implanted in defects (diameter 7 mm) created in the left medial femoral condyle of ovine knees. The implants were covered with synovial membrane from the superior recess of the same joint. For the right knees, an empty defect with the same dimensions served as control. Twenty-eight sheep were split into 6-, 12-, 26- and 52 week groups of seven animals. Indentation tests with a spherical (Ø 3mm) indenter were used to determine the biomechanical properties of regenerated tissue. A software-based limit switch was implemented to ensure a maximal penetration depth of 200 µm and maximal load of 1.5 N. The achieved load, the absorbed energy and the contact stiffness were measured. Newly formed cartilage was assessed with the International Cartilage Repair Society Visual Assessment Scale (ICRS score) and histomorphometric analysis. Results were analysed statistically using the t-test, Mann-Whitney U-test and Wilcoxon test. Statistical significance was set at p<0.05. After 6 weeks of implantation, the transplanted area tolerated an indentation load of 0.05±0.20 N. This value increased to 0.10±0.06 N after 12 weeks, to 0.27±0.18 N after 26 weeks, and 0.27±0.11 N after 52 weeks. The increase in the tolerated load was highly significant (p<0.0001), but the final value was not significantly different from that of intact cartilage (0.30±0.12 N). Similarly, the increase in contact stiffness from 0.87±0.29 N mm-(1) after 6 weeks to 3.14±0.86 N mm(-1) after 52 weeks was highly significant (p<0.0001). The absorbed energy increased significantly (p=0.02) from 0.74×10(-6)±0.38×10(-6) Nm after 6 weeks to 2.83×10(-6)±1.35×10(-6) Nm after 52 weeks. At 52 weeks, the International Cartilage Repair Society (ICRS) scores for the central area of the transplanted area and untreated defects were comparable. In contrast, the score for the area from the edge to the centre of the transplanted area was significantly higher (p=0.001) than the score for the unfilled defects. A biomechanically stable cartilage was built outside the centre of defect. After 52 weeks, all but one empty control defect were covered by bone and a very thin layer of cartilage (ICRS 7 points). The empty hole could still be demonstrated beneath the bone. The histomorphometric evaluation revealed that 81.0±10.6% of TCP was resorbed after 52 weeks. The increase in TCP resorption and replacement by spongy bone during the observation period was highly significant (p<0.0001). In this sheep trial, the mechanical properties of microporous TCP scaffolds seeded with transplanted autologous chondrocytes were similar to those of natural cartilage after 52 weeks of implantation. However, the central area of the implants had a lower ICRS score than healthy cartilage. Microporous TCP was almost fully resorbed at 52 weeks and replaced by bone.

Effect of subvoxel processes on non-destructive characterization of ß-tricalcium phosphate bone graft substitutes.

The geometric features of bone graft substitutes, such as the pore and pore interconnection sizes, are of paramount importance for their biological performance. Such features are generally characterized by micro-computed tomography (µCT). Unfortunately, the resolution of µCT is often too limited. The aim of this study was to look at the effect of µCT resolution on the geometric characterization of four different bone graft substitutes. An attempt was also made to improve the characterization of these materials by applying a subvoxelization algorithm. The results revealed that both approaches increased the accuracy of the geometric characterization. They also showed that the interconnection size in particular was affected. Comparing the results obtained from the scanned and numerical subvoxelization datasets revealed a minor difference of less than 2.5% for the porosity values. The difference for the pore sizes was up to 10%. Considerable differences of up to 35-50% were found for the interconnection sizes. The present study demonstrates how complex geometric characterization is and how important it is for biomaterial researchers to be aware of the impact of µCT resolution on the pore and pore interconnection sizes.

The effect of ball milling grinding pathways on the bulk and reactivity properties of calcium phosphate cements.

Calcium phosphate cements (CPCs) are significant alternatives to autologous bone grafting. CPCs can be composed of biphasic or multiphase calcium phosphate (CaP) compounds. A common way to process CPCs is by ball milling. Ball milling can be used for grinding or mechanosynthesis. The aim of this study was to determine the effect of well-defined ball milling grinding parameters, applied via different milling pathways, on the properties of CPCs. Starting CaP compounds used included α-tricalcium phosphate, dicalcium phosphate anhydrous and precipitated hydroxyapatite. Scanning electron microscopy showed changes in the powder morphology, which were related to the behavior of the starting CaP materials. Specific surface area (SSA) and particle size (PS) measurements exposed the effect of ball milling on the CaP compounds and CPC powders. X-ray diffraction revealed no effect of ball milling pathways or milling time on the composition of CPCs or the starting materials, but affected their crystallographic properties. No contamination of the milling media or transformation into an amorphous calcium phosphate compound was found. The milling pathways affected setting and cohesion. Fourier transform infrared spectroscopy (FTIR) revealed differences on the CPC v4-PO4³â» bands according to the interaction, created between the CaP compounds by the milling pathways. FTIR confirmed that the milling pathways changed the crystallographic properties. This study demonstrates that the pathways used for milling grinding modify the PS, SSA, and crystallographic properties of the powders, without affecting their composition. These modifications affected the bulk and reactivity properties of the CPCs by creating different setting and cohesion behaviors.

Commentary: Deciphering the link between architecture and biological response of a bone graft substitute.

Hundreds of studies have been devoted to the search for the ideal architecture for bone scaffold. Despite these efforts, results are often contradictory, and rules derived from these studies are accordingly vague. In fact, there is enough evidence to postulate that ideal scaffold architecture does not exist. The aim of this document is to explain this statement and review new approaches to decipher the existing but complex link between scaffold architecture and in vivo response.

Structural and material approaches to bone tissue engineering in powder-based three-dimensional printing.

This article reviews the current state of knowledge concerning the use of powder-based three-dimensional printing (3DP) for the synthesis of bone tissue engineering scaffolds. 3DP is a solid free-form fabrication (SFF) technique building up complex open porous 3D structures layer by layer (a bottom-up approach). In contrast to traditional fabrication techniques generally subtracting material step by step (a top-down approach), SFF approaches allow nearly unlimited designs and a large variety of materials to be used for scaffold engineering. Today's state of the art materials, as well as the mechanical and structural requirements for bone scaffolds, are summarized and discussed in relation to the technical feasibility of their use in 3DP. Advances in the field of 3DP are presented and compared with other SFF methods. Existing strategies on material and design control of scaffolds are reviewed. Finally, the possibilities and limiting factors are addressed and potential strategies to improve 3DP for scaffold engineering are proposed.

Design of ceramic-based cements and putties for bone graft substitution.

In the last 15 years, a large number of commercial ceramic-based cements and putties have been introduced as bone graft substitutes. As a result, large efforts have been made to improve our understanding of the specific properties of these materials, such as injectability, cohesion, setting time (for cements), and in vivo properties. The aim of this manuscript is to summarize our present knowledge in the field. Instead of just looking at scientific aspects, industrial needs are also considered, including mixing and delivery, sterilization, and shelf-life.

Aqueous impregnation of porous beta-tricalcium phosphate scaffolds.

The ability of a porous bone graft substitute to be impregnated with an aqueous solution is of great importance for tissue engineering and in vivo applications. This study presents an impregnation test setup and assesses the effect of various synthesis parameters such as sintering temperature, composition, macroporosity and macropore size on the impregnation properties of porous beta-tricalcium phosphate scaffolds dipped in water. Among those parameters, the macropore size had by far the largest effect; generally, the bigger the macropore size, the lower the saturation level. The results also showed that impregnation was less complete when the samples were fully dipped in water than when they were only partially dipped, owing to the requirement for the system to create air bubbles under water.

Geometric analysis of porous bone substitutes using micro-computed tomography and fuzzy distance transform.

There is increased interest in resorbable bone substitutes for skeletal reconstruction. Important geometric design measures of bone substitute include pore size, interconnection size, porosity, permeability and surface area of the substitute. In this study, four substitute groups with variable geometric features but constant porosity were scanned using micro-computed tomography (microCT) and their geometric measures were determined using an advanced image-processing algorithm based on fuzzy distance transform and new pore size definition. The substitutes were produced using the calcium phosphate emulsion method. The geometric analysis revealed that the reproducibility of the emulsion method was high, within 5%. The average porosity of the four groups was 52.3 + or - 1.5. The pore diameter of the four bone substitute groups was measured to be 170 + or - 1.7, 217 + or - 5.2, 416 + or - 19, and 972 + or - 11 microm. Despite this significant change in pore size, the interconnection size only increased slightly with an increase of pore size. The specific surface decreased with increasing pore size. The permeability increased with the pore size and was inversely proportional to the specific surface. The combination of microCT and the fuzzy image-processing tool enables accurate geometric analysis, even if pore size and image resolution are in the same range, such as in the case of the smallest pore size. Moreover, it is an exciting tool to understand the structure of the substitute with the hope of designing better bone substitutes.

Mechanisms underlying the limited injectability of hydraulic calcium phosphate paste. Part II: particle separation study.

Calcium phosphate cements (CPCs) are of great interest for bone augmentation procedures. In these a hydraulic calcium phosphate paste is injected through a small bore needle into the bone. The injectability of these pastes is relatively poor, resulting into partial injection only. In earlier studies we have shown that phase separation brings the injection process to a halt. Phase separation is characterized by a faster flow of the liquid than of the solid during paste extrusion. So far it is unclear whether or not particle separation contributes to the poor injectability of such hydraulic pastes. It is hypothesized that fine particles behave like a liquid and thus separate under the injection pressure, leaving larger particles behind. A factorial experimental design was used to examine this hypothesis. The particle size distribution (PSD) of the extrudate was measured over the course of each injection experiment using laser diffraction. The solid content of the paste was further inspected using scanning electron microscopy. A total of 48 experiments covering four factors at two levels each were performed. One factor was the ultrasound exposure duration, to ensure the dispersion quality of the particles during the PSD measurements. Another factor was the location of the samples over the course of the injection, so as to compare the extrudate with the PSDs remaining in the syringe. The liquid:powder ratio (LPR) in the injected paste was another factor investigated. Specifically, two different pastes with 40% and 50% LPR were examined. The dispersal medium was a fourth factor investigated, to ensure adequate dispersion of the particles during the PSD measurements. Analysis of variance showed that sample location did not significantly affect PSD. No apparent PSD change for the extruded paste and the paste remaining in the syringe could be detected by scanning electron microscopy. In conclusion, the present study did not show any evidence suggesting that particle separation occurred over the course of injection and thus that phase separation remains the main phenomenon leading to the poor injectability of CPCs.