Bone fractures represent a significant global healthcare burden, particularly in osteoporotic patients. While the use of metal implants for bone fixation is a current gold standard, their widespread use has several limitations. To address these issues, a phosphoserine and calcium phosphate-based adhesive (‘OsStic®’) has been developed. This adhesive is designed to bind fragmented bone together following fracture, facilitating rapid healing and repair. Phosphoserine addition to the adhesive significantly improved the handling and mechanical properties, producing an adhesive that meets clinical requirements. The focus of this research was to further investigate the OsStic® adhesive and carry out an in vitro evaluation of the material. OsStic® samples were fabricated according to a previously optimised composition. HydroSet™ was used as a proprietary comparator, in addition to phosphoserine-free alpha tricalcium phosphate (αTCP) cement. The biocompatibility and osteogenic potential of the OsStic® adhesive was successfully demonstrated. Cytotoxicity testing showed no material-related cytotoxicity (Fig. 1a). Cells readily adhered and became confluent, confirmed through SEM (Fig. 1b) and LIVE/DEAD™ staining (Fig.1c-d). Sustained cell proliferation was confirmed over 4 weeks (Fig. 1e), with superior proliferation kinetics on the OsStic® adhesive observed. Cell-cell interactions were observed using actin staining (Fig.1f). Alizarin red staining confirmed significant biomineralization in all groups, with greater levels on the OsStic® adhesive (Fig. 1g). Alkaline phosphatase (ALP) activity was confirmed via an enzymatic activity assay and corroborated by positive ALP staining (Fig. 1h). The expression and secretion of osteogenic differentiation-related factors (Fig. 1i-k), including osteopontin, osteocalcin, RUNX2 and BMP-2, were observed across the three materials groups highlighting OsStic's potential to actively support bone regeneration and healing processes, in addition to its benefits in stabilising bone fractures. Overall, these results further demonstrate the significant potential of OsStic® as a next-generation bone adhesive, not only in terms of its physical properties but also its excellent biocompatibility compared to proprietary technologies used in orthopaedics. For any figures or tables, please contact the authors directly.

Surgeons treating fractures with many small osteochondral fragments have often expressed the clinical need for an adhesive to join such fragments, as an adjunct to standard implants. If an adhesive would maintain alignment of the articular surfaces and subsequently heal it could result in improved clinical outcomes. However, there are no bone adhesives available for clinical indications and few pre-clinical models to assess safety and efficacy of adhesive biomaterial candidates. A bone adhesive candidate based on water, α-TCP and an amino acid phosphoserine was evaluated in-vivo in a novel murine bone core model (preliminary results presented EORS 2019) in which excised bone cores were glued back in place and harvested @ 0, 3, 7, 14, 28 and 42days. Adhesive pull-out strength was demonstrated 0–28 days, with a dip at 14 days increasing to 11.3N maximum. Histology 0–42 days showed the adhesive progressively remodelling to bone in both cancellous and cortical compartments with no signs of either undesirable inflammation or peripheral ectopic bone formation. These favourable results suggested translation to a large animal model.A porcine dental extraction socket model was subsequently developed where dental implants were affixed only with the adhesive. Biomechanical data was collected @ 1, 14, 28 and 56 days, and histology at 1,14,28 and 56 days. Adhesive strength assessed by implant pull-out force increased out to 28 days and maintained out to 56 days (282N maximum) with failure only occurring at the adhesive bone interface. Histology confirmed the adhesive's biocompatibility and osteoconductive behavior. Additionally, remodelling was demonstrated at the adhesive-bone interface with resorption by osteoclast-like cells and followed by new bone apposition and substitution by bone. Whilst the in-vivo dental implant data is encouraging, a large animal preclinical model is needed (under development) to confirm the adhesive is capable of healing, for example, loaded osteochondral bone fragments.Acknowledgements: The murine study was supported, in part, by the Swedish Foundation for Strategic Research (#RMA15-0110).

INTRODUCTION:The fixation of small intraarticular bone fragments is clinically challenging and an obvious first orthopaedic indication for an effective bone adhesive. In the present study the feasibility of bonding freshly harvested human trabecular bone with OsSticR, a novel phosphoserine modified cement, was evaluated using a bone cylinder model pull-out test and compared with a commercial fibrin tissue adhesive. METHODS:Femoral heads (n=13) were collected from hip fracture patients undergoing arthroplasty and stored refrigerated overnight in saline medium prior to testing. Cylindrical bone cores with a pre-inserted bone screw, were prepared using a coring tool. Each core was removed and glued back in place with either the bone adhesive (α-tricalcium phosphate, phosphoserine and 20% trisodium citrate solution) or the fibrin glue. All glued bones were stored in bone medium at 37°C. Tensile loading, using a universal testing machine (5 kN load cell), was applied to each core/head. For the bone adhesive, bone cores were tested at 2 (n=13) and 24 (n=11) hours. For the fibrin tissue adhesive control group (n=9), bone cores were tested exclusively at 2 hours. The femoral bone quality was evaluated with micro-CT. RESULTS:The ultimate pull-out load for the bone adhesive at 2 hours ranged from 36 to 171 N (mean 94 N, SD 42 N). At 24 hours the pull-out strength was similar, 47 to 198 N (mean 123 N, SD 43 N). The adhesive failure usually occurred through the adhesive layer, however in two samples, at 167 N and 198 N the screw pulled out of the bone core. The fibrin tissue adhesive group reached a peak force of 8 N maximally at 2 hours (range 2.8-8 N, mean 5.4 N, SD 1.6 N). The mean BV/TV for femoral heads was 0.15 and indicates poor bone quality. CONCLUSION:The bone adhesive successfully glued wet and fatty tissue of osteoporotic human bone cores. The mean ultimate pull-out force of 123 N at 24 hours corresponds to ∼ 300 kPa shear stress acting on the bone core. These first ex-vivo results in human bone are a promising step toward potential clinical application in osteochondral fragment fixation.

Osteoporotic fractures are a growing issue due to the increasing incidence of osteoporosis worldwide. High reoperation rates in osteoporotic fractures call for investigation into new methods in improving fixation of osteoporotic bones. In the present study, the strength of a recently developed bone bioadhesive, OsStictm, was evaluated in vivo using a novel bone core assay in a murine animal model at 0, 3, 7, 14, 28, and 42 days. Histology and micro-CT were obtained at all time points, and the mean peak pull-out force was assessed on days 0–28. The adhesive provided immediate fixation to the bone core. The mean peak bone core pull-out force gradually decreased from 6.09 N (σ 1.77 N) at day 0 to a minimum of 3.09 N (σ 1.08 N) at day 7, recovering to 6.37 N (σ 4.18 N) by day 28. The corresponding fibrin (Tisseel) control mean peak bone core pull-out characteristic was 0.27 N (σ 0.27 N) at day 0, with an abrupt increase from 0.37 N (σ 0.28) at day 3, 6.39 N (σ 5.09 N) at day 7, and continuing to increase to 11.34 N (σ 6.5 N) by day 28. The bone cores failed either through core pull-out or by the cancellous part of the core fracturing. Overall, the adhesive does not interrupt healing with pathological changes or rapid resorption. Initially, the adhesive bonded the bone core to the femur, and over time, the adhesive was replaced by a vascularised bone of equivalent quality and quantity to the original bone. At the 42 day time point, 70% of the adhesive in the cancellous compartment and 50% in the cortical compartment had been replaced. The adhesive outwith the bone shell was metabolized by cells that are only removing the material excess with no ectopic bone formation. It is concluded that the adhesive is not a physical and biochemical barrier as the bone heals through the adhesive and is replaced by a normal bone tissue. This adhesive composition meets many of the clinical unmet needs expressed in the literature, and may, after further preclinical assessments, have potential in the repair of bone and osteochondral fragments.

We report the synthesis of a library of phospho-amino acid analogues using a novel allyl-phosphoester deprotection strategy. By reacting phenols and alcohols with a combination of POCl3 and allyl alcohol we generate protected phosphoesters that can be subsequently deprotected via treatment with Pd(OAc)2. We go on to show that the generated phosphoserine and phosphotyrosine analogues can be used to generate adhesive calcium phosphate cements with potential applications in the treatment of bone defects. In particular, we use mechanical testing to demonstrate the importance of multiple calcium binding motifs and steric arrangement in promoting adhgesive strength.

A new class of materials, bone adhesives, could revolutionise the treatment of highly fragmented fractures. We present the first biological safety investigation of a bio-inspired bone adhesive. The formulation was based upon a modified calcium phosphate cement that included the amino acid phosphoserine. This material has recently been described as substantially stronger than other bioresorbable calcium phosphate cements. Four adhesive groups with the active substance (phosphoserine) and two control groups without phosphoserine were selected for in vitro and in vivo biocompatibility testing. The test groups were subject for cell viability assay and subcutaneous implantation in rats that was followed by gene expression analysis and histology assessment after 6 and 12 weeks. All adhesive groups supported the same rate of cell proliferation compared to the α-TCP control and had viability between 45–64% when compared to cell control. There was no evidence of an increased immune response or ectopic bone formation in vivo. To conclude, this bio-inspired bone adhesive has been proven to be safe, in the present study, without any harmful effects on the surrounding soft tissue.

Phosphoserine-modified cements (PMCs) are a family of wet-field tissue adhesives that bond strongly to bone and biomaterials. The present study evaluated variations in the adhesive strength using a scatter plot, failure mode, and a regression analysis of eleven factors. All single-factor, continuous-variable correlations were poor (R2 < 0.25). The linear regression model explained 31.6% of variation in adhesive strength (R2 = 0.316 p < 0.001), with bond thickness predicting an 8.5% reduction in strength per 100 μm increase. Interestingly, PMC adhesive strength was insensitive to surface roughness (Sa 1.27–2.17 μm) and the unevenness (skew) of the adhesive bond (p > 0.167, 0.171, ANOVA). Bone glued in conditions mimicking the operating theatre (e.g., the rapid fixation and minimal fixation force in fluids) produced comparable adhesive strength in laboratory conditions (2.44 vs. 1.96 MPa, p > 0.986). The failure mode correlated strongly with the adhesive strength; low strength PMCs (<1 MPa) failed cohesively, while high strength (>2 MPa) PMCs failed adhesively. Failure occurred at the interface between the amorphous surface layer and the PMC bulk. PMC bonding is sufficient for clinical application, allowing for a wide tolerance in performance conditions while maintaining a minimal bond strength of 1.5–2 MPa to cortical bone and metal surfaces.

Currently there are no standard models with which to evaluate the biomechanical performance of calcified tissue adhesives, in vivo. We present, herein, a pre-clinical murine distal femoral bone model for evaluating tissue adhesives intended for use in both osseous and osteochondral tissue reconstruction. Cylindrical cores (diameter (Ø) 2 mm (mm) × 2 mm depth), containing both cancellous and cortical bone, were fractured out from the distal femur and then reattached using one of two tissue adhesives. The adhesiveness of fibrin glue (Tisseeltm), and a novel, biocompatible, calcium phosphate-based tissue adhesive (OsStictm) were evaluated by pullout testing, in which glued cores were extracted and the peak force at failure recorded. The results show that Tisseel weakly bonded the metaphyseal bone cores, while OsStic produced > 30-fold higher mean peak forces at failure (7.64 Newtons (N) vs. 0.21 N). The failure modes were consistently disparate, with Tisseel failing gradually, while OsStic failed abruptly, as would be expected with a calcium-based material. Imaging of the bone/adhesive interface with microcomputed tomography revealed that, for OsStic, failure occurred more often within cancellous bone (75% of tested samples) rather than at the adhesive interface. Despite the challenges associated with biomechanical testing in small rodent models the preclinical ex-vivo test model presented herein is both sensitive and accurate. It enabled differences in tissue adhesive strength to be quantified even for very small osseous fragments (<Ø4mm). Importantly, this model can easily be scaled to larger animals and adapted to fracture fragment fixation in human bone. The present model is also compatible with other long-term in vivo evaluation methods (i.e. in vivo imaging, histological analysis, etc.).

The aim of the present study was to evaluate the soft tissue bond strength of a newly developed, monomeric, biomimetic, tissue adhesive called phosphoserine modified cement (PMC). Two types of PMCs were evaluated using lap shear strength (LSS) testing, on porcine skin: a calcium metasilicate (CS1), and alpha tricalcium phosphate (αTCP) PMC. CS1 PCM bonded strongly to skin, reaching a peak LSS of 84, 132, and 154 KPa after curing for 0.5, 1.5, and 4 h, respectively. Cyanoacrylate and fibrin glues reached an LSS of 207 kPa and 33 kPa, respectively. αTCP PMCs reached a final LSS of ≈110 kPa. In soft tissues, stronger bond strengths were obtained with αTCP PMCs containing large amounts of amino acid (70–90 mol%), in contrast to prior studies in calcified tissues (30–50 mol%). When αTCP particle size was reduced by wet milling, and for CS1 PMCs, the strongest bonding was obtained with mole ratios of 30–50% phosphoserine. While PM-CPCs behave like stiff ceramics after setting, they bond to soft tissues, and warrant further investigation as tissue adhesives, particularly at the interface between hard and soft tissues.

There are clinical situations in fracture repair, e.g. osteochondral fragments, where current implant hardware is insufficient. The proposition of an adhesive enabling fixation and healing has been...

Calcium phosphate cements (CPCs) are clinically effective void fillers that are capable of bridging calcified tissue defects and facilitating regeneration. However, CPCs are completely synthetic/inorganic, unlike the calcium phosphate that is found in calcified tissues, and they lack an architectural organization, controlled assembly mechanisms, and have moderate biomechanical strength, which limits their clinical effectiveness. Herein, we describe a new class of bioinspired CPCs that can glue tissues together and bond tissues to metallic and polymeric biomaterials. Surprisingly, alpha tricalcium phosphate cements that are modified with simple phosphorylated amino acid monomers of phosphoserine (PM-CPCs) bond tissues up to 40-fold stronger (2.5–4 MPa) than commercial cyanoacrylates (0.1 MPa), and 100-fold stronger than surgical fibrin glue (0.04 MPa), when cured in wet-field conditions. In addition to adhesion, phosphoserine creates other novel properties in bioceramics, including a nanoscale organic/inorganic composite microstructure, and templating of nanoscale amorphous calcium phosphate nucleation. PM-CPCs are made of the biocompatible precursors calcium, phosphate, and amino acid, and these represent the first amorphous nano-ceramic composites that are stable in liquids.

Premixed calcium phosphate cements (CPC's) are becoming the material of choice for injectable cements as a result of their effective delivery to the target implantation site. For orthopaedic use, it is of vital importance that the attributes of these CPC's are not compromised by irradiation sterilization. Therefore, the aim of this study is to determine the influence of irradiation sterilization on a range of premixed CPC's, with an emphasis on improving product shelf life through the use of optimal packaging configurations and annealing steps. Electron spin resonance (ESR) confirmed the presence of free radicals in the inorganic phase of the CPC paste following irradiation. The inclusion of a 24-h annealing step was the only successful method in reducing the degree of free radical formation. Based on the results of injectability force testing, it was revealed that an annealing step greater than 24-h significantly altered the viscosity, however; at 24-h the key attributes of the CPC paste were minimally effected. Overall, it was established that vacuum packing the CPC paste, placing the contents into a foil pouch, gamma irradiating at the minimal dose required and using an annealing step of ≤ 24-h, has the potential to extend the shelf life of the cement.

This article focuses on the understanding of the biochemistry and surgical application of bone substitute materials (BSMs) and particularly the newer calcium phosphate materials that can form a structural orthobiologic matrix within the metaphyseal components of the periarticular bone. Six characteristics of BSMs are detailed that can be used as a guide for the proper selection and application of the optimal BSM type for periarticular fracture repair. These 6 characteristics of BSMs are divided into 2 pillars. One pillar details the 3 biochemical features of BSMs and the other pillar details the 3 surgical application properties.

The crystal phase and morphology of calcium phosphate salts precipitated in a wet field environment at temperatures between 30 and 70 °C and pHs between 3 and 8 were examined. Dicalcium Phosphate Dihydrate (DCPD) was the most prevalent phase precipitated. Using accelerated ageing study techniques, precipitates studied were aged, under dry conditions at 50 °C for 8 and 16 days, before being re-examined using XRD, FTIR and SEM techniques. DCPD was found to be most stable when precipitated at 40 °C and 5 pH. Considerably more phase transformation to Octacalcium Phosphate (OCP), Amorphous Calcium Phosphate (ACP) and Hydroxyapatite (HA) was seen at high temperatures and high pHs, and a greater tendency to form anhydrous salts was seen at high temperatures and low pHs. Using techniques such as XRD, FTIR and SEM the transformation of the DCPD precipitate to OCP was analysed and appeared to occur without the presence of an intermediate amorphous phase. However, transformation from OCP to HA did result in the formation of an intermediate amorphous phase.

The aim of this work was to assess the mixing, transfer and handling, properties, injectability, set time, wash-out characteristics, acceptance of hardware (i.e. stainless screws), and their delivery/transfer systems of self hardening synthetic bone cements made from calcium phosphate and calcium sulphate. Surgical procedures (i.e. cranioplasty) were performed on adult Labrador dogs using seven different bone cements from various manufacturers. Direct comparison of the defined intraoperative properties were evaluated and recorded. There is considerable variability with respect to application properties among commercially available injectable synthetic bone cements. Only one product was rated good to excellent in seven of the eight categories evaluated (6-excellent, 1-good, 1-fair) based on in-vivo tests. This study Outlines the critical parameters required for successful implantation of cements that have to be understood when designing new injectable bone cements for the future.

Introduction: Self-hardening synthetic bone substitutes based on calcium phosphate (CaP) have been used clinically for over a decade to replace, repair and augment non load bearing defects in cancellous bone structure. Whilst important to understand and advance the integration of the bone substitute scaffold technology into the surrounding bony structure; the ease of application of these cements in the operating room cannot be overlooked. The aim of this work was twofold; firstly to assess the properties of commercially available bone substitutes during the time of surgery (mixing, handling, injectability, and in vivo setting characteristics) and secondly, to show how these bone substitutes are incorporated into the surrounding bony structure via histological analysis.