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

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

Aims. Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism. Methods. Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN); COL1A1; COL10A1; SRY-box transcription factor 9 (SOX9); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); interleukin (IL)-1β; tumour necrosis factor alpha (TNFα); IL-8; and matrix metallopeptidase 13 (MMP-13) in chondrocytes at both messenger RNA (mRNA) and protein levels was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. In addition, the supplementation of suramin to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by biochemical analyses and immunofluorescence, as well as by immunohistochemistry. The expression of reactive oxygen species (ROS) and NOX activity were assessed by luciferase reporter gene assay, immunofluorescence analysis, and flow cytometry. Mutagenesis analysis, Alcian blue staining, reverse transcriptase polymerase chain reaction (RT-PCR), and western blot assay were used to determine whether p67. phox. was involved in suramin-enhanced chondrocyte phenotype maintenance. Results. Suramin enhanced the COL2A1 and ACAN expression and lowered COL1A1 synthesis. Also, in 3D pellet culture GAG and COL2A1 production was significantly higher in pellets consisting of chondrocytes expanded with suramin compared to controls. Surprisingly, suramin also increased ROS generation, which is largely caused by enhanced NOX (p67. phox. ) activity and membrane translocation. Overexpression of p67. phox. but not p67. phox. AD (deleting amino acid (a.a) 199 to 212) mutant, which does not support ROS production in chondrocytes, significantly enhanced chondrocyte phenotype maintenance, SOX9 expression, and AKT (S473) phosphorylation. Knockdown of p67. phox. with its specific short hairpin (sh) RNA (shRNA) abolished the suramin-induced effects. Moreover, when these cells were treated with the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor LY294002 or shRNA of AKT1, p67. phox. -induced COL2A1 and ACAN expression was significantly inhibited. Conclusion. Suramin could redifferentiate dedifferentiated chondrocytes dependent on p67. phox. activation, which is mediated by the PI3K/AKT/SOX9 signalling pathway. Cite this article: Bone Joint Res 2022;11(10):723–738

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

Aims. Gap junction intercellular communication (GJIC) in osteocytes is impaired by oxidative stress, which is associated with age-related bone loss. Ageing is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, it is still unknown whether AOPP accumulation is involved in the impairment of osteocytes’ GJIC. This study aims to investigate the effect of AOPP accumulation on osteocytes’ GJIC in aged male mice and its mechanism. Methods. Changes in AOPP levels, expression of connexin43 (Cx43), osteocyte network, and bone mass were detected in 18-month-old and three-month-old male mice. Cx43 expression, GJIC function, mitochondria membrane potential, reactive oxygen species (ROS) levels, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were detected in murine osteocyte-like cells (MLOY4 cells) treated with AOPPs. The Cx43 expression, osteocyte network, bone mass, and mechanical properties were detected in three-month-old mice treated with AOPPs for 12 weeks. Results. The AOPP levels were increased in aged mice and correlated with degeneration of osteocyte network, loss of bone mass, and decreased Cx43 expression. AOPP intervention induced NADPH oxidase activation and mitochondrial dysfunction, triggered ROS generation, reduced Cx43 expression, and ultimately impaired osteocytes’ GJIC, which were ameliorated by NADPH oxidase inhibitor apocynin, mitochondria-targeted superoxide dismutase mimetic (mito-TEMPO), and ROS scavenger N-acetyl cysteine. Chronic AOPP loading accelerated the degradation of osteocyte networks and decreased Cx43 expression, resulting in deterioration of bone mass and mechanical properties in vivo. Conclusion. Our study suggests that AOPP accumulation contributes to age-related impairment of GJIC in osteocytes of male mice, which may be part of the pathogenic mechanism responsible for bone loss during ageing. Cite this article: Bone Joint Res 2022;11(7):413–425

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

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

Juvenile bone cysts in children and adolescents are often discovered incidentally or in connection with a pathologic fracture. Although the diagnostic procedure in this type of lesion affecting the skeleton has become uniform, the treatment varies according to the principles established at different clinics. The aim of our study was to compare two Methods: applied in the treatment of juvenile bone cysts, i.e. the established method of a series of Methylprednisolone injections and a new mini-invasive method using a Tricalcium phosphate. In both groups of patients, we performed an evaluation of the number of required surgeries, general anaesthesias and subsequent hospitalizations (including the length of hospitalization), the treatment results and the interval between surgery and complete cyst healing using Neer’s evaluation criteria. The group of patients treated with Methylprednisolone consisted of 24 patients and the group of patients treated with Tricalcium phosphate comprised 20 patients. The outcome of the statistical analysis proves that in patients treated with Tricalcium phosphate significantly better results were obtained compared to patients where Methylprednisolone was applied. A subsequent surgery (additional application) was necessary only in two Tricalcium phosphate patients (10%) compared to nineteen Methylprednisolone patients (79%). The average length of hospitalization was 4 days in Tricalcium phosphate patients and 3.5 days in Methylprednisolone patients. Excellent and good results according to the Neer classification were documented in eighteen Tricalcium phosphate patients (19%) and in twelve Methylprednisolone patients (50%). The treatment of juvenile bone cysts with a biocompatible resorbable synthetic filler Tricalcium phosphate helps reduce the number of surgeries necessary for complete cyst healing and produces better results in terms of Neer’s evaluation criteria of bone cyst treatment results compared to the application of Methylprednisolone into the cyst. This work was supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic (NS9860-3/2008)

Objectives. Bioresorbable orthopaedic devices with calcium phosphate (CaP) fillers are commercially available on the assumption that increased calcium (Ca) locally drives new bone formation, but the clinical benefits are unknown. Electron beam (EB) irradiation of polymer devices has been shown to enhance the release of Ca. The aims of this study were to: 1) establish the biological safety of EB surface-modified bioresorbable devices; 2) test the release kinetics of CaP from a polymer device; and 3) establish any subsequent beneficial effects on bone repair in vivo. Methods. ActivaScrew Interference (Bioretec Ltd, Tampere, Finland) and poly(L-lactide-co-glycolide) (PLGA) orthopaedic screws containing 10 wt% β-tricalcium phosphate (β-TCP) underwent EB treatment. In vitro degradation over 36 weeks was investigated by recording mass loss, pH change, and Ca release. Implant performance was investigated in vivo over 36 weeks using a lapine femoral condyle model. Bone growth and osteoclast activity were assessed by histology and enzyme histochemistry. Results. Calcium release doubled in the EB-treated group before returning to a level seen in untreated samples at 28 weeks. Extensive bone growth was observed around the perimeter of all implant types, along with limited osteoclastic activity. No statistically significant differences between comparative groups was identified. Conclusion. The higher than normal dose of EB used for surface modification did not adversely affect tissue response around implants in vivo. Surprisingly, incorporation of β-TCP and the subsequent accelerated release of Ca had no significant effect on in vivo implant performance, calling into question the clinical evidence base for these commercially available devices. Cite this article: I. Palmer, S. A. Clarke, F. J Buchanan. Enhanced release of calcium phosphate additives from bioresorbable orthopaedic devices using irradiation technology is non-beneficial in a rabbit model: An animal study. Bone Joint Res 2019;8:266–274. DOI: 10.1302/2046-3758.86.BJR-2018-0224.R2

The recent development and use of Calcium Phosphate (CaP) based products as orthopaedic implants has increased the need for the development of a greater understanding of the starting materials inherent properties. Chemical composition and crystal phase greatly influence the final product’s behaviour in the body. By gaining a deeper understanding of these properties, greater control in making tailor-made products can be achieved. Calcium Phosphate based powders of varying CaO: P2O5 ratios were prepared by cold isostatic pressing into compact discs (13 mm diameter x 3.6 mm height) and subsequently sintered at temperatures ranging between 200 to 1140 degrees Celsius for a period of 8 hours. The starting powders had CaO:P2O5 ratios ranging from 1.165 to 1.22. Each composition was analysed for microstructural and associated crystal phase modifications with varying sintering temperatures, by scanning electron microscopy (SEM) and X-ray diffraction (XRD). All compositions were shown by XRD to have an initial starting composition of Hydroxyapatite (HA) and Dicalcium Phosphate Anhydrous (DCPA). With increasing sintering temperatures, it was found that the initial composition readily transformed to beta-tricalcium phosphate(B-TCP) for all of the CaO:P2O5 ratios. However, it was found that the higher CaO:P2O5 ratio materials tended to retain residual HA. Sintering of all powders was found to have a densification effect on the microstructure of these compositions. The sintering behaviour of calcium phosphates is extremely dependent on the CaO:P2O5 ratio of the material, influencing both the crystal as well as microstructural properties of the sintered components. This in-turn will have a strong influence on the behaviour of this biomaterial when placed in-vivo

Background. Osteoarthritis (OA) pain treatment has limitations in terms of serious adverse effects and low efficacy. We aimed to evaluate efficacy and safety of naproxen sodium/codeine phosphate combination in these patients. Methods. In this prospective, randomised, double blind, placebo controlled clinical trial, 135 patients with osteoarthritis, who were 40–65 years; applied to our institution's orthopaedics outpatient clinic; had grade 1, 2, or 3 primary osteoarthritis diagnosed in last 1 year; and had Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score≥40, and Visual Analogue Scale score≥40, were enrolled. Subjects were randomised (1:1) to placebo (n= 67) or combination (n= 68) arms, in which 550 mg naproxen sodium/30mg codeine phosphate was given orally twice a day for 7 days. Rescue medicine was 500 mg paracetamol (max= 6 tablets/day). Demographic characteristics, medical history, adverse events, VAS and WOMAC scores were collected in study visits performed within 10 days. The study was approved by local institutional ethics committee and written informed consents were obtained from all participants. Results. Patients were evenly matched in both arms. The mean age was 52.3±6.6 years (89.7% female). WOMAC and VAS scores decreased significantly within each group (p<0.001 for all). For the study group (n=43) the mean (+ SD) and median (min-max) VAS Score was −29.8 (+21.0) and −30 (−70−0), respectively. In contrast, for the control group (n=36) the mean and median (min-max) VAS Score was −16.1 (+24.4) and −12.5 (−65−40), respectively. WOMAC score was decreased more in combination group (p= 0.044). Changes in VAS scores between visits were significantly different (more in combination receivers) between groups (p= 0.018). Ratio of patients requiring no rescue medicine was higher among combination receivers than controls (64.8% vs. 35.2%, respectively). Most common adverse events were constipation, dyspepsia, and somnolence; no fatality was encountered. Conclusions. Naproxen sodium/codeine phosphate combination is effective and safe in osteoarthritis patients. Level of Evidence. Level 1

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

Aims: to develop a fast biomimetic method to deposit nanocrystalline calcium phosphate coatings on titanium substrates. Methods: Ti6Al4V disks (diameter = 15mm) were ultrasonically cleaned with acetone, ethanol and demineralized water, and then etched in a mixture of HF and HNO3. The disks were soaked in a supersaturated Ca/P solution at 37A1C, pH 7.2 for different periods of time up to 3 days. The structure and morphology of the obtained coatings were characterized using thin film X-ray diffraction, SEM and TEM investigation. Furthermore, coated and uncoated diskes were soaked in simulated body fluid (SBF) for different periods of time. Results: the first calcium phosphate deposits on the etched Ti6Al4V disks can be appreciated after 7 days of soaking in SBF solution. At variance, soaking of the Ti6Al4V disks in the supersaturated Ca/P solution, with a simpler composition than that of SBF, yields the deposition of a uniform calcium phosphate coating in a few hours. Thin film X-ray diffraction patterns show that the coating is constituted of a poor crystalline apatitic phase, and that the extent of deposition increases on increasing the soaking time in the supersaturated solution. TEM images recorded on the material detached from the coating by ultrasound vibration show that the apatitic deposition is constituted of thin nanocrystals. The results obtained on coated substrates submitted to interaction with SBF indicate that the presence of the coating reduces the time required to appreciate the deposition of apatite from SBF to just 24 hours. Conclusions: the method utilized in this work can be successfully applied to obtain fast deposition of uniform coatings of nanocrystalline hydroxyapatite on titanium substrates

The need for an artificial scaffold in very large bone defects is clear, not only to limit the risk of graft harvesting, but also to improve clinical success. The use of custom osteoconductive scaffolds made from biodegradable polyester and ceramics can be a valuable patient friendly option, especially in case of a concomitant infection. Multiple types of scaffolds for the Masquelet procedure (MP) are available, however these frequently demonstrate central graft involution when defects exceed a certain size and the complication rates remains high. This paper describes three infected tibial defect nonunions with a segmental defect over ten centimeters long treated with a customized 3D printed polycaprolactone-tricalcium phosphate (PCL-TCP) cage in combination with biological adjuncts. Three male patients, between the age of 37 and 47, were treated for an infected tibial defect nonunion after sustaining Gustilo grade 3 open fractures. All had a segmental midshaft bone defect of more than ten centimeters (range 11–15cm). First stage MPs consisted of extensive debridement, external fixation and placement of anterior lateral thigh flaps (ALT). Positive cultures were obtained from all patients during this first stage, that were treated with specific systemic antibiotics during 12 weeks. The second stage MP was carried out at least two months after the first stage. CT scans were obtained after the first stage to manufacture defect-specific cages. In the final procedure a custom 3D printed PCL-TCP cage (Osteopore, Singapore) was placed in the defect in combination with biological adjuncts (BMAC, RIA derived autograft, iFactor and BioActive Glass). Bridging of the defect, assessed at six months by CT, was achieved in all cases. SPECT-scans 6 months post-operatively demonstrated active bone regeneration, also involving the central part of the scaffold. All three patients regained function and reported less pain with full weight-bearing. This case report shows that 3D printed PCL-TCP cages in combination with biological adjuncts is a novel addition to the surgical treatment of very large bone defects in (infected) posttraumatic nonunion of the tibia. This combination could overcome some of the current drawbacks in this challenging indication

Infection is one of major problems in Total Knee Arthroplasty (TKA) or Bipolar Head Prosthesis (BHP). We have used Calcium Phosphate Paste (CPP) for treatment of infected TKA and BHP, and followed up for minimum one year. CPP is a mixture of alpha Tri-Calcium Phosphate, Tetra-Calcium Phosphate, Calcium Hydrogen Phosphate and Hydroxyapatite. CPP harden in 10 minutes and its stiffness increases to maximum in 3 days. Infected TKA were diagnosed in four osteoarthritis and four rheumatoid arthritis knees, and BHP infection were observed in two femoral neck fracture cases from 2001 to 2007. Two were male and eight were female, average age were 67.3 years old ranged 39 to 80. Follow-up period were one to 7 years. Six cases were MRSA infection, three MSSA, one was unidentified but diagnosed with clinical data. In TKA cases, CPP (10–12g) with vancomycin hydrochloride or tobramycin were filled on the back side of PMMA articulated surface spacers, and in BHP cases, CPP with antibiotics were filled in acetabulum. In all TKA cases, infection ceased in 2 to 4 month and revision TKA ware performed. One recurrence of infection was observed during follow up in BHP infection, and nine patients can walk with/without a cane. No venous thromboembolism were observed. CPP filled in the space between articulated spacer and bone is gradually crashed and can release antibiotics during walking and ROM exercise. CPP with antibiotics is useful for the treatment after infected TKA and BHP

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

The fixation of titanium or titanium alloy implants is related to their surface composition and topography. Osteoconductive calcium phosphate coatings promote bone healing and apposition, leading to the rapid biological fixation of implants. It’s no doubt that the addition of certain biologically active protein with biomaterial will improve the bioactivity of the material. Previously, we examined the biocompatibility of basic fibroblast growth factor (bFGF) incorporation with titanium implants. Now we investigate the effect of fibronectin (FN) incorporation with thin calcium phosphate film deposited on titanium by electron-beam evaporation since fibronectin is actively involved in cell adhesion, spreading, would healing, cytoskeletal reorganization, and bone tissue formation. A FN-apatite composite layer was formed on the surface of titanium by biomimetic process. The coating process was carried out by immersing thin calcium phosphate film coated Ti in Dulbecco’s Phosphate buffered saline containing FN (20 ug/ml). The surfaces of samples were examined with FESEM, Fourier transform infrared spectroscopy and X-ray diffraction. The quantity of FN taken up and the kinetics of protein release were monitored by BCA method and Elisa. The fibronectin was immobilized in the newly formed apatite layer. The adhesion of osteoblast cells to the FN-apatite composite layer was to show the biocompatibility of implants, and FN-apatite composite layer could enhance osseintegration of implants in vivo. This research was supported by a grant (code #: 08K1501-01220) from Center for Nanostructured Materials Technology under 21st Century Frontier R& D Program of the Ministry of Education, Science and Technology, Korea

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

Introduction: Filling bone defects with Polymethylmetaacrylate (PMMA) has been a easy, safe and reliable technique for past four decade. Newly developed Calcium Phosphate Paste (CPP) is a mixture of alpfa Tri Calcium Phsphate (TCP), Tetra Calcium Phosphate, Calcium Hydrogen Phosphate and Hydroxyapatite. This paste hardens in 10 minutes and its stffness increases to 80Mpa in seven days. It generates no heat, no gas and requires no organic solvents. In process of hardening, the TCP structure changes to Hydroxyapatite. Materials and methods: We have used CPP in two TKA cases associate with bone defect, and 14 fracture cases. In a MRSA infected revision TKA case, reconstruction was performed with PMMA-VCM articulated spacers, and they was fixed to bone with CPP-VCM. MRSA infection has been well controlled and weight bearing could be done in 10 days after surgery. In another TKA case, large bone necrosis in femoral condyle was filled with CPP and Cementless inplant were placed on it. Seven days later, this patient could walk with a cane. Results: CPP filled in bones were not absorbed for a year, and exess CPP in soft tissue were absorbed in several weeks. In 16 cases no side effects were observed during as long as one year. Conclusion: Handling CPP is much easier than Hydroxyapatite brick or granule. CPP can be useful for total joint arthroplasty, especially in large bone defect or infected cases. It can replace a part of PMMA as a bone cement for implants in the near future

Introduction: Incorporation of Silicon into the HA structure enhances the bioactivity of Hydroxyapatite (HA). Silicon substituted calcium phosphate (SiCaP/SiHA) has been introduced as an osteoconductive material for bone formation. However, the osseoinductive capacity of this biomaterial has not been assessed. A previous study by Hing et al shows that bioactivity of stoichiometric hydroxyapatite bone substitute materials is enhanced by increasing the level of porosity within the implant struts [1]. The aim of this study was to test the hypothesis that SiCaP bone graft results in superior osseoinduction compared to stoichiometric HA and osseoinduction enhancement using high microporosity materials. Methods: Implantation of 32 bone graft plugs (16 granular and 16 blocks) with 3 different strut porosities: 20% SiHA, 35% SiHA, 10% SiHA and 20% HA, all with matched 80% total porosity supplied by ApaTech Ltd into the paraspinalis muscle of 4 sheep for 12 weeks. HA and %SiHA locations were randomized at implant sites. Following euthanasia at 12 weeks histomorphometry was carried out to calculate Percentage of bone, soft tissue and implant area and Percentage of the amount of bone in contact with the calcium phosphate surface (% Bone attachment). Further evaluation of Calcium, Phosphate and Silicon levels within the implants and surrounding bone was carried out by Scanning Electron Microscopy (SEM) and EDAX. Results: Bone formation was observed within the pores of both granules and blocks of SiCaP and HA implants. Greater bone formation and attachment was detected in scaffolds with higher strut porosity (SiHA35) compared to implants of the same chemical composition but lower strut porosity (SiHA10, SiHA20. More bone formation and contact was observed in SiHA implants (SiHA20) compared to matched porosity HA implants where the amount of bone formed was minimal. Uniform distribution of Silicon (Si) was visible within the SiHA scaffold struts according to EDAX results. Greater quantities of Si existed in newly formed bone as compared to soft tissue adjacent to the SiHA implants. Silicon was not detected in either soft or hard tissues adjacent to HA implants. Conclusion: Both microporous HA and SiCaP promote bone ingrowth, as ectopic bone formation was observed in all four groups of synthetic materials. Matched porosity SiCaP is more osseoinductive than HA. Increasing strut porosity results in promotion of osseoinductivity. High strut porosity (> 10%) block environment contributes to greater osseoinductive behaviour. In conclusion we report that presence of silicon and the strut porosity influence the osseoinductive capacity of calcium phosphate bone substitute biomaterials