Critical-sized bone defects can result from trauma, inflammation, and tumor resection. Such bone defects, often have irregular shapes, resulting in the need for new technologies to produce suitable implants. Bioprinting is an additive manufacturing method to create complex and individualised bone constructs, which can already include vital cells. In this study, we established an extrusion-based printing technology to produce osteoinductive scaffolds based on polycaprolactone (PCL) combined with calcium phosphate, which is known to induce osteogenic differentiation of stem cells. The model was created in python based on the signed distance functions. The shape of the 3D model is a ring with a diameter of 20 mm and a height of 10 mm with a spongiosa-like structure. The interconnected irregular pores have a diameter of 2 mm +/− 0.2 mm standard deviation. Extrusion-based printing was performed using the BIO X6. To produce the bioink, PCL (80 kDa) was combined with calcium phosphate nanopowder (> 150 nm particle size) under heating. After printing, 5 × 10. 6. hMSC were seeded on the construct using a rotating incubator. We were able to print a highly accurate ring construct with an interconnected pore structure. The PCL combined with calcium phosphate particles resulted in a precise printed construct, which corresponded to the 3D model. The bioink containing calcium phosphate nanoparticles had a higher printing accuracy compared to PCL alone. We found that hMSC cultured on the construct settled in close proximity to the calcium phosphate particles. The hMSC were vital for 22 days on the construct as demonstrated by life/dead staining. The extrusion printing technology enables to print a mechanically stable construct with a spongiosa-like structure. The porous PCL ring could serve as an outer matrix for implants, providing the construct the stability of natural bone. To extend this technology and to improve the implant properties, a biologised inner structure will be integrated into the scaffold in the future

Summary Statement. Calcium phosphate (CaP) particles have attracted great interest as transfection reagents, yet little is known about their mechanism of internalisation. We report live cell time-course tracking of CaP particles during internalisation and the influence of Ca:P ratio on transfection efficiency. Introduction. Relatively recent work has seen calcium phosphate (CaP) salts used for the delivery of biological materials into cells in the form of peptides, polymers and DNA sequences. Calcium phosphate salts have a critical safety advantage over other vectors such as viruses in that they pose no risk of pathogenicity due to mutation and show no apparent cytotoxicity. Previous work within the group showed that Ca:P ratio influenced the transfection efficiency, but the fate of the particles on internalisation is yet unknown. The difficulty in tracking the particles can be related to the visual similarity to granulation within the cells. Using a surface modification method that enables the fluorescent labeling of silicon-substituted hydroxyapatite (SiHA) particles, we have tracked the internalisation of the particles to understand their mechanism of entry and how particle composition may influence transfection efficiency. Patients & Methods. SiHA particles were synthesised by the dropwise addition of an aqueous solution of diammonium hydrogen phosphate and silicon tetraacetate to an aqueous solution of calcium nitrate while under mixing and maintained at pH10. The particles were functionalised with thiol groups using (3-mercaptopropyl)trimethoxysilane and dye-labelled with fluorescein-5-maleimide. MC3T3 osteoblast precursor cells were incubated in cell culture media containing labelled particles at a concentration of 0.6μg/mL for 12 hours. Confocal images were obtained with a Zeiss LSM 710 ConfoCor 3 system based around a Zeiss AxioObserverZ1 microscope. Results. DNA binding efficiency between 79 to 94%, the lowest being the CaP sample of new CaP route at Ca/P ratio of 0.33 by SEDS processing, which was 79% and the highest was the HAp SEDS processed sample at 40°C, solvent flowrate of 1 ml/min and antisolvent flowrate of 60 g/min (particle size of 131 nm). From the fluorescence microscopy images, localised regions of particles measuring around 500–1000nm were detected. With a typical SiHA particle size of 50–70nm in length, these regions contain 10's of particles. Discussion/Conclusion. Thiol functionalisation enabled the internalised SiHA to be visually discriminated from the other cellular material with similar morphology and optical contrast as shown in the bright field image. HA particles (Ca:P of 1.67) showed a strong affinity for the cell membrane despite extensive washing with PBS and their higher calcium content may enhance the binding of the DNA to the particle surface, therefore improving transfection efficiency

Treatment of bone infection often includes a burdensome two-stage revision. After debridement, contaminated implants are removed and replaced with a non-absorbable cement spacer loaded with antibiotics. Weeks later, the spacer is exchanged with a bone graft aiding bone healing. However, even with this two-stage approach infection persists. In this study, we investigated whether a novel 3D-printed, antibiotic-loaded, osteoinductive calcium phosphate scaffold (CPS) is effective in single-stage revision of an infected non-union with segmental bone loss in rabbits. A 5 mm defect was created in the radius of female New Zealand White rabbits. The bone fragment was replaced, stabilized with cerclage wire and inoculated with Staphylococcus aureus (MSSA). After 4 weeks, the infected bone fragment was removed, the site debrided and a spacer implanted. Depending on group allocation, rabbits received: 1) PMMA spacer with gentamycin; 2) CPS loaded with rifampin and vancomycin and 3) Non-loaded CPS. These groups received systemic cefazolin for 4 weeks after revision. Group 4 received a loaded CPS without any adjunctive systemic therapy (n=12 group1-3, n=11 group 4). All animals were euthanized 8 weeks after revision and assessed by quantitative bacteriology or histology. Covariance analysis (ANCOVA) and multiple regression were performed. All animals were culture positive at revision surgery. Half of the animals in all groups had eliminated the infection by end of study. In a historical control group with empty defect and no systemic antibiotic treatment, all animals were infected at euthanasia. There was no significant difference in CFU counts between groups at euthanasia. Our results show that treating an osteomyelitis with segmental bone loss either with CPS or PMMA has a similar cure rate of infection. However, by not requiring a second surgery, the use of CPS may offer advantages over non-resorbable equivalents such as PMMA

Summary Statement. The present study demonstrates the beneficial effects of strontium (Sr) modified calcium phosphate cement to improve new bone formation in a metaphyseal osteoporotic fracture defects in rats compared to calcium phosphate cement and empty defects. Keywords: strontium, fracture, calcium phosphate, bone formation. Introduction. Impaired fracture healing with subsequent implant failure is a dramatic problem in osteoporotic fractures. Biomaterials are of interest to stimulate fracture healing in osteoporotic defects and the objective of the current study is to investigate the effects of Strontium modified calcium phosphate cement (SrCPC) in a critical-size metaphyseal fracture defect of osteoporotic rats compared to calcium phosphate (CPC) and empty defect control group. Methods. 45 female Sprague-Dawley rats were randomized into 3 groups: SrCPC, CPC and empty defect (n=15 for each). A combinatorial approach of multi-deficiency diet for 3 months after bilateral ovariectomy was used for induction of osteoporosis. Left femur of all animals underwent a 4mm wedge-shaped metaphyseal osteotomy that was internally fixed with a T-shaped plate. The defect was then either filled with CPC or SrCPC and internally stabilised with a T shaped mini-plate. Empty defect served as a control. After 6 weeks femora were harvested followed by histological, histomorphometrical, immunohistochemical (bone-morphogenic protein 2, osteocalcin and osteoprotegerin), and molecular biology analysis (alkaline phosphatase, collagen10a1 and osteocalcin) to demonstrate the effects of the biomaterials on new bone formation. Time of flight secondary ion mass spectrometry (TOF-SIMS) technology was used to assess the distribution of released strontium ions and calcium appearance of newly formed bone. Results. Histomorphometric analysis showed a statistically significant increase in the bone formation at the tissue-implant interface in the SrCPC group (p<0.001). A statistically significantly more cartilage and unmineralised bone formation was also seen in the SrCPC group in comparision to the CPC group alone (p<0.05) and also to the empty defect (p<0.05) in the former fracture defect zone. These data were confirmed by the immunohistochemistry results which revealed an increase in bone-morphogenic protein 2, osteocalcin and osteoprotegerin and an increase in expression of genes responsible for bone formation viz. alkaline phosphatase, collagen10a1 and osteocalcin. TOF-SIMs analysis showed a higher release of Sr from the SrCPC into the interface region and related to a higher calcium content in this area compared to CPC. Discussion/Conclusion. SrCPC treatment showed enhanced new bone formation in a metaphyseal osteoporotic fracture defect of rats after 6 weeks compared to CPC-filled and empty defects in histomorphometry, immunochemistry and gene expression analysis. Strontium ranelate is a well-known anti-osteoporotic drug increasing bone formation and reducing bone resorption. As revealed by TOF-SIMS release of Sr out of the the SrCPC cement is most likely attributable for new bone formation. Therefore, Sr seems to be a good candidate not only for systemic treatment in osteoporosis but also in Sr-modification of biomaterials for local stimulation of new bone formation in osteoporotic fracture defects

Over the last decades, biodegradable metals emerged as promising materials for various biomedical implant applications, aiming to reduce the use of permanent metallic implants and, therefore, to avoid additional surgeries for implant removal. However, among the important issue to be solved is their fast corrosion - too high to match the healing rate of the bone tissue. The most effective way to improve this characteristic is to coat biodegradable metals with substituted calcium phosphates. Tricalcium phosphate (β-TCP) is a resorbable bioceramic widely used as synthetic bone graft. In order to modulate and enhance its biological performance, the substitution of Ca2+ by various metal ions, such as strontium (Sr2+), magnesium (Mg2+), iron (Fe2+) etc., can be carried out. Among them, copper (Cu2+), manganese (Mn2+), zinc (Zn2+) etc. could add antimicrobial properties against implant-related infections. Double substitutions of TCP containing couples of Cu2+/Sr2+ or Mn2+/Sr2+ ions are considered to be the most perspective based on the results of our study. We established that single phase Ca3−2x(MˊMˊˊ)x(PO4)2 solid solutions are formed only at x ≤ 0.286, where Mˊ and Mˊˊ—divalent metal ions, such as Zn2+, Mg2+, Cu2+, Mn2+, and that in case of double substitutions, the incorporation of Sr2+ ions allows one to extend the limit of solid solution due to the enlargement of the unit cell structure. We also reported that antimicrobial properties depend on the substitution ion occupation of Ca2+ crystal sites in the β-TCP structure. The combination of two different ions in the Ca5 position, on one side, and in the Ca1, Ca2, Ca3, and Ca4 positions, on another side, significantly boosts antimicrobial properties. In the present work, zinc-lithium (Zn-Li) biodegradable alloys were coated with double substituted Mn2+/Sr2+ β-TCP and double substituted Cu2+/ Sr2+ β-TCP, with the scope to promote osteoinductive effect (due to the Sr2+ presence) and to impart antimicrobial properties (thanks to Cu2+ or Mn2+ ions). The Pulsed Laser Deposition (PLD) method was applied as the coating's preparation technique. It was shown that films deposited using PLD present good adhesion strength and hardness and are characterized by a nanostructured background with random microparticles on the surface. For coatings characterization, Fourier Transform Infrared Spectroscopy, X-ray Diffraction, and Scanning Electron Microscopy coupled with Energy Dispersive X-ray and X-ray Photoelectron Spectroscopy were applied. The microbiology tests on the prepared coated Zn-Li alloys were performed with the Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative (Salmonella typhimurium, Escherichia coli) bacteria strains and Candida albicans fungus. The antimicrobial activity tests showed that Mn2+/Sr2+ β-TCP -coated and Cu2+/Sr2+ β-TCP coated Zn-Li alloys were able to inhibit the growth of all five microorganisms. The prepared coatings are promising in improving the degradation behavior and biological properties of Zn-Li alloys, and further studies are necessary before a possible clinical translation

Background. Due to their tailored porous texture, breathability and flexibility, carbon cloths (CCs) are good scaffolds for biomedical application. However, biocompatibility of CCs depends on their physic-chemical properties. Calcium phosphate ceramics (CaP) are well known for their use in orthopaedic field. So, carbon cloth-reinforced CaP composites are promising bioceramic materials for bone regeneration. Methods. CaP coating are performed using sono-electrochemical deposition method. The electrolyte consisted in an aqueous solution of calcium and phosphates precursors. CC was used as work electrode in three-electrode system. SEM, TEM, XRD, 1H and 31P MAS NMR and FTIR spectroscopies were performed to characterise the deposits. In vitro biocompatibility of CCs with and without coatings is tested with human osteoblasts. Results. The current density influences the morphology and the chemical composition of deposit: it consists mainly in carbonated hydroxyapatite with plate-like shape for lower current densities and needle-like shape for the highest. A hydrophobic surface of CC with due to small amount of oxygenated functions leads to a poor biocompatibility. Conclusion. The wettability of CCs is an important parameter of biocompatibility. Biomimetic CaP deposits obtained by sono-electrodeposition present a microstructure and a chemical composition close to the mineral phase of natural bone. This work was supported by Region Centre project: bioactive hybrid materials for bone reconstruction. 2014–2016

Summary Statement. Repetitive concavities threaded on the surface of bone implants have been already demonstrated to be effective on ectopic bone formation in vivo. The aim of this study was to investigate the effect of concavity on the mineralization process in vitro. Introduction. The role of implant surface geometry in bone formation has been extensively investigated. Ripamonti and co. investigated the possibility to induce bone formation by threading concavities on the surface of calcium phosphate implants, without the need for exogenous osteogenic soluble factors. The underlying hypothesis was that this geometry, by resembling the hemi-osteon trench observable during osteoclastogenesis, was able to activate the ripple-like cascade of bone tissue induction and morphogenesis. Despite several studies indicating a positive effect of concavities on bone induction, so far no attempts have rationalised this phenomenon by means of in vitro tests. Consequently, this study aimed to evaluate the effect of surface concavities on the mineralization of hydroxyapatite (HA) and beta-tricalciumphosphate (b-TCP) ceramics in vitro. Our hypothesis was that concavities could effectively guide the mineralization process in vitro. Materials and Methods. Different-size concavities were threaded into the surface of HA and b-TCP semi-sintered disks: 1.8 (large concavity, LC), 0.8 (medium concavity, MC) and 0.4 mm (small concavity, SC). Disks were fully sintered at 1200 or 1100 °C and soaked in simulated body fluid (SBF) up to 28 days. Distinct experiments were carried out in order to assess the role of chemical composition, sintering temperature and concavity size on the extent of mineralization in vitro. The mineralization process was followed by SEM, EDS, XRD and Ca assay tests. Results and Discussion. Massive mineralization occurred exclusively at the surface of the HA disks treated at 1200 °C. Firstly, aggregates of spherical-like amorphous calcium phosphate nucleated specifically within concavities and not on the planar surface. At a later stage, a flake-like apatitic phase replaced the spherical-like apatite and spread out the concavities until covering the entire disk surface. Instead, a lower extent of mineralization occurred on HA disk treated at 1100 °C, with no formation of flake-like phase. In contrast, no significative mineralization was observed for b-TCP disks, irrespective of sintering temperature. Finally, the extent of mineralization on disks exhibiting SC on the surface was found to be about 120- and 10-fold higher than mineralization disks threaded with LC and MC, respectively. Conclusions. The main results of this study are: i) the in vitro mineralization process of CaP ceramics with surface concavities starts preferentially within the concavities and not on the planar surface; ii) concavity size is an extremely effective parameter for controlling the extent of mineralization in vitro. Finally, the results reported in this study suggest correlation between the positive effect of concavities on mineralization in vitro and on ectopic bone formation in vivo

Miniscrew implants (MSIs) are widely used to provide absolute anchorage for the orthodontic treatment. However, the application of MSIs is limited by the relatively high failure rate (22.86%). In this study, we wished to investigate the effects of amorphous and crystalline biomimetic calcium phosphate coating on the surfaces of MSIs with or without the incorporated BSA for the osteointegration process with an aim to facilitate the early loading of MSIs. Amorphous and crystalline coatings were prepared on titanium mini-pin implants. Characterizations of coatings were examined by Scanning electron microscopy (SEM), Confocal laser-scanning dual-channel-fluorescence microscopy (CLSM) and Fourier-transform infrared spectroscopy (FTIR). The loading and release kinetics of bovine serum albumin (BSA) were evaluated by Enzyme linked immunosorbent assay (ELISA). Activity of alkaline phosphate (ALP) was measured by using the primary osteoblasts. In vivo, a model of metaphyseal tibial implantation in rats was used (n=6 rats per group). We had 6 different groups: no coating no BSA, no coating but with surface adsorption of BSA and incorporation of BSA in the biomimetic coating in the amorphous and crystalline coatings. Time points were 3 days, 1, 2 and 4 weeks. Histological and histomorphometric analysis were performed and the bone to implant contact (BIC) of each group was compared. In vitro, the incorporation of BSA changed the crystalline coating from sharp plates into curly plates, and the crystalline coating showed slow-release profile. The incorporation of BSA in crystalline coating significantly decreased the activity of ALP in vitro. In vivo study, the earliest significant increase of BIC appeared in crystalline coating group at one week. The crystalline coating can serve as a carrier and slow release system for the bioactive agent and accelerate osteoconductivity at early stage in vivo. The presence of BSA is not favorable for the early establishment of osteointegration

Introduction and Objective. Calcium phosphates are among the most commonly used bone graft substitute materials. Compositions containing predominantly monetite (∼84.7%) with smaller additions of beta-tricalcium phosphate (β-TCP; ∼8.3%) and calcium pyrophosphate (Ca-PP; ∼6.8%) have previously been demonstrated to exhibit osteoinductive properties. Such a multi-component calcium phosphate bioceramic was fashioned in the form of hollowed-out, dome-shaped devices (15 mm diameter, 4 mm height), each reinforced with a 3D printed Ti6Al4V ELI frame. With the aim to induce bone formation beyond the skeletal envelope, these devices were investigated in vivo using a sheep (Ovis aries) occipital bone model. Materials and Methods. The bioceramic composition was prepared from a mixture of β-TCP/dicalcium pyrophosphate and monocalcium phosphate monohydrate powders mixed with glycerol. The Ti6Al4V ELI frame was positioned into a dome-shaped mould and bioceramic paste was poured over the frame and allowed to set, in sterile water, prior to removal from the mould. In adult female sheep (n=7), the devices were positioned directly over the bone and stabilised using self-drilling screws. After 52 weeks, the devices were retrieved, resin embedded, and used for X-ray micro-computed tomography (micro-CT), histology, backscattered electron scanning electron microscopy (BSE-SEM), energy dispersive X-ray spectroscopy (EDX), micro-Raman spectroscopy, and Fourier transform infrared spectroscopy (FTIR). Results. The bioceramic composition (Ca/P: ∼0.85 at. %) transforms to carbonated apatite (Ca/P: ∼1.2 at. %, Mg/Ca: ∼0.03 at. %), in vivo, largely at the expense of monetite and Ca-PP whereas β-TCP remains detectable. Discrete particles of Ca-PP are identified by correlative BSE-SEM and micro-Raman spectroscopy. Together with chemical transformation, physical degradation is evident within the bulk of the bioceramic. Beyond the confines of the skeletal envelope, de novo bone occupies ∼53–84% (∼73 ± 11%; mean ± standard deviation) of the hollowed-out space. Low porosity and the arrangement of remodelled bone into a concentric lamellar pattern is indicative of cortical-like structure. Such areas are typically surrounded by yet unremodelled, and microstructurally disordered, woven bone that stains intensely with blue cationic dyes, owing to relatively higher acid phosphate content. This pattern indicates a recurring sequence of woven bone formation followed by remodelling. Bone formation is also visible within the bioceramic. Recently remodelled and areas of ongoing remodelling are identified by relatively lower mineral density than the surrounding woven bone. Dendritic extensions of osteocytes appear to extend into the bioceramic surface. Both micro-Raman spectroscopy and FTIR reveal little, if any, detectable difference between the mineral and organic phases of the extracellular matrix, between de novo and native bone. Conclusions. The bioceramic composition undergoes physical degradation, but remains largely intact by 52 weeks in vivo, and only partially transforms to carbonated apatite. In addition to very high bone volume within the hollowed-out bioceramic device, the overall composition and microstructure of de novo bone are similar to native bone. Notably, the mineral phase of bone in response to, and in direct contact with the β-TCP, monetite, and Ca-PP, remains exclusively carbonated apatite

Although multifunctional delivery systems can potentially improve safety and efficacy of therapeutic protein delivery in the biological treatment of injured tissues, ability to track and manipulate protein delivery systems in vivo to ensure localization at the treatment site is still a concern. We hypothesized that incorporating superparamagnetic iron oxide (SPIO) into calcium phosphate (CaP) coated β-tricalcium phosphate (β-TCP) microparticles would allow for Magnetic Resonance Imaging (MRI) based tracking in vivo and SPIO incorporation would not impact the biological activity of proteins delivered with these microparticles. To address the efficacy and limitations in therapeutic protein delivery, a CaP coated microparticle which incorporates superparamagnetic iron oxide (SPIO-CaP-MP) was created and used in a rat knee medial collateral ligament. The system has trifunctional properties: (1) it is trackable using magnetic resonance imaging (MRI), (2) it can be manipulated with a magnetic field, (3) it can release active proteins in the injury site. SPIO-Ca-MPs were formed on β-tricalcium phosphate cores. Using MRI, SPIO-CaP-MPs were visible in T2 weighted sequences as an area of hypointesive signal. SPIO-CaP-MPs could be visualized and remained localized for at least 15 days after injection into the medial collateral ligament. Recombinant human basic fibroblast growth factor delivered with SPIO-CaP-MPs stimulated the proliferation of human dermal fibroblasts. Finally, SPIO-CaP-MPs could be localized to a bar magnet when suspended in solution. Taken together, these results suggest that SPIO-CaPMPs could be useful for protein delivery applications in the treatment of ligament injury that may benefit from externally controlled localization and MRI-based tracking

Biphasic calcium phosphates (BCP) are the most frequently used materials because of their mineral analogy with bio-mineral part of bones. Their chemical synthesis can be modulated by doping, in order to respond to the biological needs. We present here the biological responses induced by copper ions in solution, to characterize its cytotoxicity and antibacterial activity. We also investigate the antibacterial property of Cu-doped BCP (Ca10 Cu0.1 (PO4)6 (OH)1.8 O0.2) on a strain of clinical interest: S. aureus, compared to undoped BCP. The sol-gel route has been used to prepare the BCP ceramics. Human BMC (Bone Marrow Cells) were obtained from metaphysal cancellous bone collected during hip arthroplasty and used for cytotoxicity evaluations. A strain of Staphylococcus aureus isolated from an osteoarticular infection after total knee arthroplasty was used to evaluate antibacterial activities. Results indicate that 3 ppm of copper ions leads to the death of all cultured bacteria in 24 hours and 25 ppm caused the death of all cells in 15 days. Regarding BCP, the undoped bioceramics increased the bacterial growth compared to a control without bioceramic. After 16 hours of contact, the copper ions released by the Cu-doped BCP induced a significant decrease of the bacterial concentration, indeed no viable bacteria were found. These materials seem to be a promising alternative for the preparation of multifunctional bone substitutes

Bone regenerative medicine aims at designing biomimetic biomaterials able to guide stem cells fate towards osteoblast lineage and prevent orthopaedic common pathogen adhesion. Owing to bone inorganic/organic composition, we herein report, using a versatile process based on simultaneous spray coating of interacting species, a calcium phosphate (CaP) / chitosan (CHI) / hyaluronic acid (HA) functionalized collagen membrane as a new strategy for bone regenerative medicine. Physicochemical characterizations of CaP-CHI-HA coating were performed by scanning electron microscopy, X-ray photoelectron and infrared spectroscopies and high-resolution transmission electron microscopy, revealing the formation of a thin coating mainly composed of non-stoichiometric crystalline hydroxyapatite dispersed into polymorphic organic film. Biocompatibility of CaP-CHI-HA coated membrane, evaluated after 7 days in contact with human mesenchymal stem cells (MSCs), showed spread, elongated and aligned cells. Metabolic activity and DNA quantification studies showed an increase in MSCs proliferation on coated membrane compared to uncoated membrane over the study time. Similarly, cytokines (IL-6, IL-8, osteoprotegerin) and growth factors (VEGF, bFGF) release in supernatant, as well as endothelial cells recruitment, were significantly increased in presence of CaP-CHI-HA coated membrane. Thus, CaP-CHI-HA coated membrane provides a suitable environment for MSCs to induce bone healing. Moreover, pro-inflammatory cytokines (IL-1β and TNF-α) secretion by human monocytes was significantly reduced on CaP-CHI-HA coating compared to LPS stimulation. CaP-CHI-HA coating also reduced significantly Staphylococcus aureus and Pseudomonas aeruginosa adhesion on the membrane, conferring a bacterial anti-adhesive surface. Based on our results, CaP-CHI-HA functionalized collagen membrane provides an interesting material for bone regeneration

Pseudoarthrosis after spinal fusion is an important complication leading to revision spine surgeries. Iliac Crest Bone Graft is considered the gold standard, but with limited availability and associated co-morbidities, spine surgeons often utilize alternative bone grafts. Determine the non-inferiority of a novel submicron-sized needle-shaped surface biphasic calcium phosphate (BCP<µm) as compared to autograft in instrumented posterolateral spinal fusion. Adult patients indicated for instrumented posterolateral spinal fusion of one to six levels from T10-S2 were enrolled at five participating centers. After instrumentation and preparation of the bone bed, the randomized allocation side of the graft type was disclosed. One side was grafted with 10cc of autograft per level containing a minimum of 50% iliac crest bone. The other side was grafted with 10cc of BCP<µm granules standalone (without autograft or bone marrow aspirate). In total, 71 levels were treated. Prospective follow-up included adverse events, Oswestry Disability Index (ODI), and a fine-cut Computerized Tomography (CT) at one year. Fusion was systematically scored as fused or not fused per level per side by two spine surgeons blinded for the procedure. The first fifty patients enrolled are included in this analysis (mean age: 57 years; 60% female and 40% male). The diagnoses included deformity (56%), structural instability (28%), and instability from decompression (20%). The fusion rate determined by CT for BCP<μm was 76.1%, which compared favorably to the autograft fusion rate of 43.7%. Statistical analysis through binomial modeling showed that the odds of fusion of BCP<μm was 2.54 times higher than that of autograft. 14% of patients experienced a procedure or possible device-related severe adverse event and there were four reoperations. Oswestry Disability Index (ODI) score decreased from a mean of 46.0 (±15.0) to a mean of 31.7 (±16.9), and 52.4% of patients improved with at least 15-point decrease. This data, aiming to determine non-inferiority of standalone BCP<μm as compared to autograft for posterior spinal fusions, is promising. Ongoing studies to increase the power of the statistics with more patients are forthcoming

Background. Bone is a hierarchically structured hard tissue that consists of approximately 70 wt% low-crystallinity hydroxyapatite. Intricate tubular channels, such as Haversian canals, Volkman's canals, and canaliculi are a preserved feature of bone microstructure. These structures provide pathways for vasculature and facilitate cell-to-cell communication processes, together supporting viability of cellular components and aiding in remodeling processes. Unfortunately, many commercial bone augmentation materials consist of highly crystalline phases that are absent of the structuring present within the native tissue they are replacing. This work reports on a the development of a novel bone augmentation material that is able to generate biologically analogous tubular calcium phosphate mineral structures from hydrogel-based spheres that can be packed into defects similar to those encountered in vivo. Experimental. Calcium loaded spheres were made by adding 5 wt% agar powder to 1 M calcium nitrate solutions, before heating the mixture to 80–90 oC and feeding droplets of gel into a reservoir of liquid nitrogen. Deposition of tubular mineral was initiated by exposure to ammonium phosphate solutions at concentrations between 500 mM and 1 M, and was characterized by micro-XRF mapping, XRD and SEM techniques. For an ex vivo model, human bone tissue was collected from patients undergoing elective knee replacement surgery. The United Kingdom National Research Ethics Service (East of Scotland Research Ethics Service) provided ethical approval (11/ES/1044). The augmented defect of the model was characterised by micro-XRF mapping and micro-CT techniques. Results and Discussion. Immersion of calcium-loaded hydrogel spheres in physiological solutions rich in phosphate promotes the release of calcium rich streams from the sphere surface, resulting in the precipitation of tubule structures. Micro-XRF mapping, XRD and SEM, revealed tubules possessed hierarchically structuring and consisted of low-crystallinity hydroxyapatite, making them analogous in composition and structure to incritate features of bone microstructure. When brought into close proximity with one another, spheres become fused in a matter of minutes by the entanglement and subsequent interstitial mineralisation of the mineral tubules. Micro-XRF mapping and micro-CT analysis of an augmented ex vivo human tissue defect model demonstrated the extensive deposition of low-crystallinity tubular mineral throughout a tissue defect. Conclusions. This is possibly the first example of a bone augmentation material that is able to generate biologically analogous structures in situ, and therefore may serve as a better scaffold for bone formation over synthetic alternatives. Moreover, the formation of structured mineral aids in achieving rapid hardening of the augmenting calcium-loaded hydrogel shperes within the defect space

Calcium phosphate cements (CPC) are used as biocompatible and bioactive bone void fillers. Ideally, the mechanical properties of these cements should match those of the surrounding bone. The knowledge of the real mechanical properties of the material is important in the decision-making process regarding possible use of the CPCs in different anatomical sites. Although it is generally recognized that these cements are stiffer and more brittle than desired, there is a limited amount of data about the possible deformation of this class of material before failure. The focus of this study was to determine these properties of injectable CPCs. Two different types of self-setting CPCs were investigated in this study: i) hydroxyapatite (HA), that historically has been the most widely studied CPC; ii) brushite, that recently has attracted attention due to its faster resorption than that of HA in vivo. Specimens of both cement types were prepared by mixing a powder phase with a liquid phase that were left to harden in phosphate buffered saline at 37°C. Once set, the specimens underwent a quasi-static compressive test to determine the compressive strength, the elastic modulus and the maximum deformation of the two materials. The material testing machine was equipped with a digital image correlation system, which allows accurate measurement of material deformation directly on the specimen surface. Brushite was found to be significantly more stiff (+80%) and resistant (+84%) than HA. Similar findings were found for the energy needed to create a first crack on the specimen surface. However, the first crack appeared on the specimen surface at the same low deformation level (∼0.15%) independently of the type of material tested. Complete failure of both materials occurred, on average, before reaching 0.25%. It has been demonstrated that the compressive behaviour of CPCs depends on their composition and porosity [1]. One of the main reasons for the high strength and stiffness of the brushite studied here was its low porosity (∼12%). However, the maximum deformation is not positively affected by this decrease in porosity. In fact, both materials show the same brittle behaviour, i.e. they undergo comparably little deformation before they break. Under these conditions, increasing the compressive strength may not always be beneficial clinically, e.g. in the treatment of vertebral compression fractures, where the high stiffness of the bone cements used has been identified as a risk factor for adjacent-level fractures [2]. However, it is not clear whether a 20-fold higher stiffness than the trabecular bone would give a different clinical outcome than a 10-fold higher stiffness. These high-strength, high-stiffness cements may also be used as a basis for further biomaterial development, e.g. in the creation of macro-porous scaffolds, which is usually challenging due to the commonly low mechanical properties of the base CPC material

Critical-sized bone defects remain challenging in the clinical setting. Autologous bone grafting remains preferred by clinicians. However, the use of autologous tissue is associated with donor-site morbidity and limited accessibility to the graft tissue. Advances in the development of synthetic bone substitutes focus on improving their osteoinductive properties. Whereas osteoinductivity has been demonstrated with ceramics, it is still a challenge in case of polymeric composites. One of the approaches to improve the regenerative properties of biomaterials, without changing their synthetic character, is the addition of inorganic ions with known osteogenic and angiogenic properties. We have previously reported that the use of a bioactive composite with high ceramic content composed of poly(ethyleneoxide terephthalate)/poly(butylene terephthalate) (1000PEOT70PBT30, PolyActive, PA) and 50% beta-tricalcium phosphate (β-TCP) with the addition of zinc in a form of a coating of the TCP particles can enhance the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) (3). To further support the regenerative properties of these scaffolds, inorganic ions with known angiogenic properties, copper or cobalt, were added to the coating solution.

β-TCP particles were immersed in a zinc and copper or zinc and cobalt solution with a concentration of 15 or 45 mM. 3D porous scaffolds composed of 1000PEOT70PBT30 and pure or coated β-TCP were additively manufactured by 3D fibre deposition. The osteogenic and angiogenic properties of the fabricated scaffolds were tested in vitro through culture with hMSCs and human umbilical vein endothelial cells, respectively. The materials were further evaluated through ectopic implantation in an in vivo mini-pig model. The early expression of relevant osteogenic gene markers (collagen-1, osteocalcin) of hMSCs was upregulated in the presence of lower concentration of inorganic ions. Further analysis will focus on the evaluation of ectopic bone formation and vascularisation of these scaffolds after implantation in a mini-pig ectopic intramuscular model.

Long term, secondary implant fixation of Total Disc Replacements (TDR) can be enhanced by hydroxyapatite or similar osseo-conductive coatings. These coatings are routinely applied to metal substrates. The objective of this in vivo study was to investigate the early stability and subsequent bone response adjacent to an all polymer TDR implant over a period of six months in an animal model.

Six skeletally mature male baboons (Papio annubis) were followed for a period of 6 months. Using a transperitoneal exposure, a custom-sized Cadisc L device was implanted into the disc space one level above the lumbo-sacral junction in all subjects. Radiographs of the lumbar spine were acquired prior to surgery, and post-operatively at intervals up to 6 months to assess implant stability. Flourochrome markers (which contain molecules that bind to mineralization fronts) were injected at specified intervals in order to investigate bone remodeling with time.

Animals were humanely euthanized six months after index surgery. Test and control specimens were retrieved, fixed and subjected to histological processing to assess the bone-implant-bone interface. Fluorescence microscopy and confocal scanning laser microscopy were utilized with BioQuant image analysis to determine the bone mineral apposition rates and gross morphology.

Radiographic evaluation revealed no loss of disc height at the operative level or adjacent levels. No evidence of subsidence or significant migration of the implant up to 6 months. Heterotopic ossification was observed to varying degrees at the operated level.

Histology revealed the implant primary fixation features embedded within the adjacent vertebral endplates. Flourochrome distribution revealed active bone remodeling occurring adjacent to the polymeric end-plate with no evidence of adverse biological responses. Mineral apposition rates of between 0.7 and 1.7 microns / day are in keeping with literature values for hydroxyapatite coated implants in cancellous sites of various species.

Radiographic assessment demonstrates that the Cadisc L implant remains stable in vivo with no evidence of subsidence or significant migration. Histological analysis suggests the primary fixation features are engaged, and in close apposition with the adjacent vertebral bone. Flourochrome markers provide evidence of a positive bone remodelling response in the presence of the implant.

Orthopaedic and trauma implant related infection remains one of the major complications that negatively impact clinical outcome and significantly increase healthcare expenditure.

Hydroxyapatite has been used for many years to increase implant osseointegration. Silver has been introduced into hydroxyapatite as an antimicrobial coating for orthopedic implants. This surface coatings can both increase tissue compatibility and prevent implant-related infections.

We examined infection markers and blood silver values, liver and kidney function tests of 30 patients with of three groups of orthopedic implants, external fixators, intramedullary nails and hip replacements, coated with Ag + ion doped CaP based ceramic powder to determine safety and effectiveness of this dual-function coating.

During 1 year follow-up, the pin sites were observed at the external fixator group, and wound areas for the proximal femoral nail and hip arthroplasty group at regular intervals. In addition, liver and kidney function tests, infection markers and blood silver values were checked in patients. In the external fixator group, only 4 out of 91 pin sites (%4.39) were infected. The wound areas healed without any problem in patients with proximal femoral nails and hip arthroplasty. There was no side effect suggesting silver toxicity such as systemic toxic side effect or argyria in any patient and blood silver level did not increase.

Compared to similar patient groups in the literature, much lower infection rates were obtained (p = 0.001), and implant osseointegration was good. In patients with chronic infection, the implants were applied acutely after removing the primary implant and with simple debridement. Unlike other silver coating methods, silver was trapped in hydroxyapatite crystals in the ionic form, which is released from the coating during the process of osseointegration, thus, the silver was released into the systemic circulation gradually that showed antibacterial activity locally.

We conclude that the use of orthopedic implants with a silver ion added calcium phosphate-based special coating is a safe method to prevent the implant-related infection.

This work was supported by TUBİTAK Project Number 315S101

Prosthetic Joint Infection (PJI) is a devastating complication that can occur after total joint replacement surgery. With increasing antimicrobial resistance, there is a need for non-antibiotic approaches to treat and prevent PJI. Doping calcium phosphates with antimicrobial ions shows promise for these purposes. This systematic review aims to search and summarise the evidence-base for the potential of calcium phosphates doped with different antimicrobial ions. A systematic review was conducted on PubMed, Embase, Web-Of-Science, Cochrane Library and Emcare of in vitro and animal studies on the antimicrobial activity of (co)substituted calcium phosphates according to PRIMSA guidelines.. The research protocol, listing search terms and in/exclusion criteria, was registered a priori at . https://doi.org/10.7910/DVN/HEP18U. Data was extracted regarding ions, micro-organisms and antimicrobial activity. The search retrieved 1017 hits of which 148 papers were included. The substitution of 33 different ions was reported. Silver (n= 46), zinc (n=39), copper (n=18) and magnesium (n=14) were the most commonly doped ions. 36 different micro-organisms were studied of which E. coli (n=109), S. aureus (n=99), and C. albicans (n=22) were the most common. 6 different outcomes were reported, most commonly the K-ratio (n=53), the log CFU (n=41) and the bacterial inhibition zone (n=39). A validated outcome for the evaluation of biofilm prevention was lacking. There was considerable heterogeneity in studied ions, micro-organisms and reported outcomes. A lack of clearly defined reporting guidelines in the field of antimicrobial materials has led to the use of clinically irrelevant micro-organisms and a general lack of consistency of the methods used and the reported results. Currently, there is no universally accepted measure for the effectiveness required from biomaterials for treatment and prevention of PJI

Minimally invasive surgery for the restoration of bone tissues lost due to diseases and trauma is preferred by the health care system as the related costs are continuously increasing. Recently, efforts have been paid to optimize injectable calcium phosphate (CaP) cements which have been recognized as excellent alloplastic material for osseous augmentation because of their unique combination of osteoconductivity, biocompatibility and mouldability. The sol-gel synthesis approach appears to be the most suitable route towards performing injectable calcium phosphates. Different strategies used to prepare bioactive and osteoinductive injectable CaP are reported. CaP gels complexed with phosphoserine-tethered poly(ε-lysine) dendrons (G3-K PS) designed to interact with the ceramic phase and able to induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) is discussed. Recently, attention has been given to the modification of hydroxyapatite with Strontium (Sr) due to its dual mode of action, simultaneously increasing bone formation (stimulating osteoblast differentiation) while decreasing bone resorption (inhibiting osteoclast differentiation). The effect of systems based on strontium modified hydroxyapatite (Sr-HA) at different composition on proliferation and osteogenic differentiation of hMSC is described. One more approach is based on the use of antimicrobial injectable materials. It has been demonstrated that some imidazolium, pyridinium and quaternary ammonium ionic liquids (IL) have antimicrobial activity against some different clinically significant bacterial and fungal pathogens. Here, we report several systems based on IL at different alkyl-chain length incorporated in Hydroxyapatite (HA) through the sol-gel process to obtain an injectable material with simultaneous opposite responses toward osteoblasts and microbial proliferation