Addressing bone defects is a complex medical challenge that involves dealing with various skeletal conditions, including fractures, osteoporosis (OP), bone tumours, and bone infection defects. Despite the availability of multiple conventional treatments for these skeletal conditions, numerous limitations and unresolved issues persist. As a solution, advancements in biomedical materials have recently resulted in novel therapeutic concepts. As an emerging biomaterial for bone defect treatment, graphene oxide (GO) in particular has gained substantial attention from researchers due to its potential applications and prospects. In other words, GO scaffolds have demonstrated remarkable potential for bone defect treatment. Furthermore, GO-loaded biomaterials can promote osteoblast adhesion, proliferation, and differentiation while stimulating bone matrix deposition and formation. Given their favourable biocompatibility and osteoinductive capabilities, these materials offer a novel therapeutic avenue for bone tissue regeneration and repair. This comprehensive review systematically outlines GO scaffolds’ diverse roles and potential applications in bone defect treatment. Cite this article: Bone Joint Res 2024;13(12):725–740

Objectives. This systematic review aimed to assess the in vivo and clinical effect of strontium (Sr)-enriched biomaterials in bone formation and/or remodelling. Methods. A systematic search was performed in Pubmed, followed by a two-step selection process. We included in vivo original studies on Sr-containing biomaterials used for bone support or regeneration, comparing at least two groups that only differ in Sr addition in the experimental group. Results. A total of 572 references were retrieved and 27 were included. Animal models were used in 26 articles, and one article described a human study. Osteoporotic models were included in 11 papers. All articles showed similar or increased effect of Sr in bone formation and/or regeneration, in both healthy and osteoporotic models. No study found a decreased effect. Adverse effects were assessed in 17 articles, 13 on local and four on systemic adverse effects. From these, only one reported a systemic impact from Sr addition. Data on gene and/or protein expression were available from seven studies. Conclusions. This review showed the safety and effectiveness of Sr-enriched biomaterials for stimulating bone formation and remodelling in animal models. The effect seems to increase over time and is impacted by the concentration used. However, included studies present a wide range of study methods. Future work should focus on consistent models and guidelines when developing a future clinical application of this element. Cite this article: N. Neves, D. Linhares, G. Costa, C. C. Ribeiro, M. A. Barbosa. In vivo and clinical application of strontium-enriched biomaterials for bone regeneration: A systematic review. Bone Joint Res 2017;6:366–375. DOI: 10.1302/2046-3758.66.BJR-2016-0311.R1

Aims. There is a lack of biomaterial-based carriers for the local delivery of rifampicin (RIF), one of the cornerstone second defence antibiotics for bone infections. RIF is also known for causing rapid development of antibiotic resistance when given as monotherapy. This in vitro study evaluated a clinically used biphasic calcium sulphate/hydroxyapatite (CaS/HA) biomaterial as a carrier for dual delivery of RIF with vancomycin (VAN) or gentamicin (GEN). Methods. The CaS/HA composites containing RIF/GEN/VAN, either alone or in combination, were first prepared and their injectability, setting time, and antibiotic elution profiles were assessed. Using a continuous disk diffusion assay, the antibacterial behaviour of the material was tested on both planktonic and biofilm-embedded forms of standard and clinical strains of Staphylococcus aureus for 28 days. Development of bacterial resistance to RIF was determined by exposing the biofilm-embedded bacteria continuously to released fractions of antibiotics from CaS/HA-antibiotic composites. Results. Following the addition of RIF to CaS/HA-VAN/GEN, adequate injectability and setting of the CaS/HA composites were noted. Sustained release of RIF above the minimum inhibitory concentrations of S. aureus was observed until study endpoint (day 35). Only combinations of CaS/HA-VAN/GEN + RIF exhibited antibacterial and antibiofilm effects yielding no viable bacteria at study endpoint. The S. aureus strains developed resistance to RIF when biofilms were subjected to CaS/HA-RIF alone but not with CaS/HA-VAN/GEN + RIF. Conclusion. Our in vitro results indicate that biphasic CaS/HA loaded with VAN or GEN could be used as a carrier for RIF for local delivery in clinically demanding bone infections. Cite this article: Bone Joint Res 2022;11(11):787–802

Objectives. We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells. Materials and Methods. We seeded mouse skeletal muscle cells C2C12 on the hydroxyapatite/calcium sulphate biomaterial and the phenotype of the cells was analysed. To mimic surgical conditions with leakage of extra cellular matrix (ECM) proteins and growth factors, we cultured rat bone cells ROS 17/2.8 in a bioreactor and harvested the secreted proteins. The secretome was added to rat muscle cells L6. The phenotype of the muscle cells after treatment with the media was assessed using immunostaining and light microscopy. Results. C2C12 cells differentiated into osteoblast-like cells expressing prominent bone markers after seeding on the biomaterial. The conditioned media of the ROS 17/2.8 contained bone morphogenetic protein-2 (BMP-2 8.4 ng/mg, standard deviation (. sd. ) 0.8) and BMP-7 (50.6 ng/mg, . sd. 2.2). In vitro, this secretome induced differentiation of skeletal muscle cells L6 towards an osteogenic lineage. Conclusion. Extra cellular matrix proteins and growth factors leaking from a bone cavity, along with a ceramic biomaterial, can synergistically enhance the process of ectopic ossification. The overlaying muscle acts as an osteoinductive niche, and provides the required cells for bone formation. Cite this article: D. B. Raina, A. Gupta, M. M. Petersen, W. Hettwer, M. McNally, M. Tägil, M-H. Zheng, A. Kumar, L. Lidgren. Muscle as an osteoinductive niche for local bone formation with the use of a biphasic calcium sulphate/hydroxyapatite biomaterial. Bone Joint Res 2016;5:500–511. DOI: 10.1302/2046-3758.510.BJR-2016-0133.R1

Intervertebral disc (IVD) degeneration is the most frequent cause of Low Back Pain (LBP) affecting nearly 80% of the population [1]. Current treatments fail to restore a functional IVD or to provide a long-term solution, so, there is an urgent need for novel therapeutic strategies. We have defined the IVD extracellular matrix (ECM) profile, showing that the pro-regenerative molecules Collagen type XII and XIV, are uniquely expressed during fetal stages [2]. Now we propose the first fetal injectable biomaterial to regenerate the IVD. Fetal decellularized IVD scaffolds were recellularized with adult IVD cells and further implanted in vivo to evaluate their anti-angiogenic potential. Young decellularized IVD scaffolds were used as controls. Finally, a large scale protocol to produce a stable, biocompatible and easily injectable fetal IVD-based hydrogel was developed. Fetal scaffolds were more effective at promoting Aggrecan and Collagen type II expression by IVD cells. In a Chorioallantoid membrane assay, only fetal matrices showed an anti-angiogenic potential. The same was observed in vivo when the angiogenesis was induced by human NP cells. In this context, human NP cells were more effective in GAG synthesis within a fetal microenvironment. Vaccum-assisted perfusion decellularized IVDs were obtained, with high DNA removal and sGAG retention. Hydrogel pre-solution passed through 21-30G needles. IVD cells seeded on the hydrogels initially decreased metabolic activity, but increased up to 70% at day 7, while LDH assay revealed cytotoxicity always below 30%. This study will open new avenues for the establishment of a disruptive treatment for IVD degeneration with a positive impact on the angiogenesis associated with LBP, and on the improvement of patients’ quality of life. Acknowledgements: Financial support was obtained from EUROSPINE, ON Foundation and FCT (Fundação para a Ciência e a Tecnologia)

Introduction. In specific conditions, infection may lead to bone loss and is difficult to treat. 1. Current clinical approaches rely on the introduction of antibiotics. While these may be effective, there are concerns regarding the rise of antimicrobial resistance. There is therefore interest in the development of antimicrobial bone graft substitutes for dental and trauma surgery. Aim & Objectives. The incorporation of zinc into biomaterials has been shown to confer broad spectrum antimicrobial activity, but this has not yet been applied to the development of a commercial bone graft substitute. The aim of this research was therefore to prepare and characterise a series of zinc-substituted nanoscale hydroxyapatite (nHA) materials, including evaluation of antimicrobial activity. Method. Zinc (Zn) substituted nHA materials were prepared (0, 5, 10, 15 & 20 mol.% Zn) using a wet chemical precipitation method with a rapid mixing. (2). The reaction was carried out using zinc hydroxide at pH 10. The suspension formed was washed and dried into both powder & paste forms. The resultant powders were characterized using transmission electron microscopy (TEM) and X-ray diffraction (XRD). The antimicrobial activity was evaluated against Staphylococcus aureus (S8650 strain - isolated from an osteomyelitis case), by two techniques. The Miles and Misra method was applied to determine the number of colony-forming units (CFUs) in bacterial suspensions incubated with pastes. Secondly, a biofilm initialization method was used to evaluate the capacity of the materials to prevent biofilm formation. One-way analysis of variance (ANOVA) was used for the statistical analysis and results with p-value < 0.05 were considered statistically significant. Results. XRD indicated the formation of pure hydroxyapatite with up to 10 mol.% Zn without any side products. However, when Zn was increased to 15 & 20 mol %, zinc oxide (ZnO) peaks were detected. The TEM showed nanoscale needle-like particles when Zn was increased compared to nHA particles. Regarding the antibacterial activity, ZnHA pastes at all concentrations caused a significant reduction in bacterial CFUs in a dose-dependent manner (50, 100 & 200 mg). Additionally, even the lowest zinc substitution (5 mol.%) significantly reduced biofilm formation. Conclusion. The results demonstrated a novel method to produce a Zn-substituted nHA that showed antimicrobial activity against a pathogen isolated from a bone infection

Background. Chronic low back pain is strongly linked to degeneration of the intervertebral disc (IVD), which currently lacks any targeted treatments. This study explores NPgel, a biomaterial combined with notochordal cells (NC), developmental precursor cells, as a potential solution. NCs, known for anti-catabolic effects on IVD cells, present a promising avenue for regenerating damaged IVD tissue. Methods. Bovine IVDs underwent enzymatic degeneration before NPgel (+/- NC) injection. Degenerated bovine IVDs were cultured under biomechanical loading for 21 days. Histology and immunohistochemistry assessed NC survival, phenotype, and matrix production. Within an in vivo sheep pilot study, NPgel (+/- NC) was injected into degenerated IVDs, blood was taken, and immune cell activation was monitored via flow cytometry over three months post-injection. Results. Within the ex vivo model, IVDs injected with NPgel (+/- NC) exhibited increased matrix expression and deposition. Viable NCs were detected post-culture, indicating survival and matrix production. In the in vivo model, NPgel injection into sheep IVDs did not significantly increase activation of immune cells compared to controls, suggesting no systemic inflammatory effects. Conclusion. NPgel, combined with NCs, shows promise for IVD regeneration. Ex vivo findings indicate NPgel supports NC survival and matrix production. Moreover, in vivo results demonstrate the absence of systemic immunogenic responses post-NPgel injection. This suggests NPgel's potential as a carrier for NCs in IVD regeneration therapy. These findings underscore NPgel's candidacy for further investigation in addressing chronic low back pain associated with IVD degeneration. Subsequent research, including long-term efficacy and safety evaluations, is imperative for clinical translation. Conflicts of interest. There are no conflicts of interest. Sources of funding. iPSpine, grant # 825925, Horizon 2020

Bone-regenerative and biocompatible materials are indicated for the regeneration of bone lost in periodontology and maxillofacial surgery. Bio-Oss is a natural bone mineral for bone grafting of bovine origin and the most common used in this kind of interventions. 1. Sil-Oss is a new synthetic nanostructured monetite-based material that is reabsorbed at the same time that is replaced by new bone tissue . 2. Bacterial infection is one of the complications related to this kind of material. Streptococcus oralis is the most associated oral infecting pathogen to oral surgery. 3. and Staphylococcus aureus is the most common infecting pathogen to maxillofacial non-oral interventions. 4. Here we evaluated bacterial adherence of two of the most common infecting bacteria of this kind of biomaterial: S. oralis and S. aureus, on Bio-Oss and Sil-Oss. S. oralis ATCC 9811 and S. aureus 15981 strains were used. Bacterial adherence was evaluated using the modified previously described protocol of Kinnari et al. 5. that was adapted to our biomaterial. The quantification was performed by the drop plate method. 6. The statistical data were analyzed by pairwise comparisons using the nonparametric Mann-Whitney test with a level of statistical significance of p<0.05. Values are cited and represented as medians. Bacterial adherence decreased significantly on Sil-Oss compared to Bio-Oss. S. oralis ATCC 9811 adherence was between 11 and 13-fold less on Sil-Oss compared to Bio-oss. In the case of S. aureus15981, the adherence was between 4 and 6-fold less on Sil-Oss compared to Bio-Oss. Sil-Oss nanostructured monetite-based biomaterial could be considered as a promising biomaterial to be used for the regeneration of bone defects since the bacterial adherence on it is lower than on another currently used material

Background. The doses of local rhBMP-2 in commercially available materials are high with known drawbacks such as inflammation and premature bone resorption. The latter can be prevented by adding bisphosphonates like zoledronic acid (ZA) but systemic ZA has side effects and patient adherence to treatment is low. In a recent study, we have shown that local co-delivery of rhBMP-2 and ZA via a calcium sulphate/hydroxyapatite (CS-HA) biomaterial can be used to regenerate both cortical and trabecular bone in a rat model of metaphyseal bone defect. Even low doses of local ZA in the biomaterial showed promising results and increased bone formation within the defect compared to the controls. A step before clinical translation of the local treatment regimen is to evaluate the in-vivo release kinetics of these additives and thus in this study, we aimed to investigate the in-vivo pharmacokinetics of rhBMP-2 and ZA from the CS-HA biomaterial in a rat abdominal muscle pouch model over a period of 4-weeks. Methods. In-vivo release kinetics of 125I labeled rhBMP-2 and 14C labeled ZA was performed using an abdominal muscle pouch model in rats (n=6). Both rhBMP-2 and ZA were labeled commercially with a radiochemical purity of >95%. The detection of 125I -rhBMP-2 release was performed by implanting pellets of the CS-HA biomaterial containing 125I -rhBMP-2 and ZA and the same animals followed over a period of 4-weeks (day 1, 3, 7, 14, 21& 28) using SPECT imaging. Similarly, the 14C-ZA was detected by implanting CS-HA pellets containing rhBMP-2 and 14C-ZA. Release was detected via scintillation counting and at each time point (Day 1, 7, 14& 28) 6-animals were sacrificed. Results. BMP Release. The CS-HA biomaterial retained 95±11% after 3-days, 88±12% after a week, 66±9% after 2 weeks, 51±5% after 3 weeks and 43±7% of 125I labeled rhBMP-2 after 4-weeks in-vivo (SPECT-CT). ZA Release. The CS-HA biomaterial retained 89±14% after a week, 84±8% after 2 weeks, 83±9% after 3 weeks and 77±3% of 14C labeled ZA after 4 weeks of in-vivo implantation. Discussion. Improved carriers and better knowledge of the release might improve the effect of bone active drugs in orthopedics. Our previous study shows that an off-the-shelf ceramic biomaterial combined with ZA alone or with both rhBMP-2 and ZA can be used to regenerate bone with potential for clinical translational. This study demonstrates long-term co-delivery of both rhBMP-2 and ZA in-vivo via the biomaterial. Constant availability of rhBMP-2 over a long period of time can give osteoinductive properties to the material while presence of local ZA prevents premature bone loss. The pharmacokinetic release pattern differs from what we have reported in vitro with less BMP and more ZA being released in vivo during the first 4 weeks. We speculate that rapid protein passivation of the ceramic material slows the release of BMP and partly preventing the ZA binding to apatite

Total knee arthroplasty with a rotating hinge knee with carbon-fibre-reinforced (CFR)-PEEK as an alternative bushing material with enhanced creep, wear and fatigue behaviour has been clinically established [1-4]. The objective of our study was to compare results from in vitro biotribological characterisation to ex vivo findings on a retrievals. A modified in vitro wear simulation based on ISO 14243-1 was performed for 5 million cycles on rotating hinge knee (RHK) designs (EnduRo®) out of cobalt-chromium and ZrN-multilayer ceramic coating. The rotational & flexion axles-bushings and the flanges are made of CFR-PEEK with 30% polyacrylonitrile fibre content. Analysis of 12 retrieved EnduRo® RHK systems in cobalt-chromium and ZrN-multilayer in regard to loosening torques, microscopic surface analysis, distinction between different wear modes and classification with a modified HOOD-score has been performed. For the RHK design with the polyethylene gliding surface and bushings and flanges made out of CFR-PEEK, a cumulative volumetric wear was measured to be 12.9±3.95 mm. 3. in articulation to cobalt-chromium and 1.3±0.21 mm. 3. to ZrN-multilayer coating - a significant 9.9-fold decrease (p=0.0072). For the CFR-PEEK flexion bushing and flanges the volumetric wear rates were 2.3±0.48 mm. 3. /million cycles (cobalt-chromium) and 0.21±0.02 mm. 3. /million cycles (ZrN-multilayer) (p=0.0016). The 5 million cycles of in vitro wear testing reflect a mean in vivo service life of 2.9 years, which is in accordance to the time in vivo of 12–60 months of the retrieved RHK components [5]. The main wear modes were comparable between retrievals and in vitro specimens, whereby the size of affected area on the retrieved components showed a higher variation. For the EnduRo® RHK design the findings on retrieved implants demonstrate the high suitability of CFR-PEEK as a biomaterial for highly loaded bearings, such as RHK bushings and flanges in articulation to cobalt-chromium and to a ZrN-multilayer coating

Introduction. Current cell-based treatments and marrow stimulating techniques to repair articular cartilage defects are limited in restoring the tissue in its native composition. Despite progress in cartilage tissue engineering and chondrogenesis in vitro, the main limitation of this approach is the progression towards hypertrophy during prolonged culture in pellets or embedded in biomaterials. The objectives of this study were (A) to compare human bone marrow-derived mesenchymal stromal cells (hMSC) chondrogenesis and hypertrophy in pellet culture from single cells or cell spheroids and (B) to investigate the effect of tyramine-modified hyaluronic acid (THA) and collagen I (Col) content in composite hydrogels on the chondrogenesis and hypertrophy of encapsulated hMSC spheroids. Materials and Methods. Pellet cultures were prepared either from hMSC single cells (250’000 cells/pellet) or hMSC spheroids (282 cells/spheroid) at the same final cell concentration (250’000 cells/pellet = 887 spheroids/pellet). The effect of polymer concentration on encapsulated hMSC spheroids (887 spheroids/hydrogel) was investigated in THA-Col hydrogels (50μl) at the following concentrations (THA-Col mg/ml): Group (1) 12.5–2.5, (2) 16.7–1.7, (3) 12.5–1.7, (4) 16.7–2.5 mg/ml. All samples were cultured for 21 days in standard chondrogenic differentiation medium containing 10ng/ml TGF-β1. Chondrogenic differentiation and hypertrophy of both pellet cultures and hMSCs spheroids encapsulated in THA-Col were analysed using gene expression analysis (Aggrecan (ACAN), COL1A1, COL2A1, COL10A1), dimethylmethylene-Blue assay to quantify glycosaminoglycans (GAGs) retained in the samples and (immuno-) histological staining (Safranin-O, collagen II, aggrecan) on day 1 and day 21 (n=3 donors). Results. The culture of hMSCs in pellets based on single cells or spheroids resulted in an increase in chondrogenic-associated markers COL2A1 (2’900–3’400-fold) and ACAN (45–47-fold) compared to respective samples on day 1 in both groups. GAGs increased in spheroid pellets to 21.2±3.4 mg/ml and in single cell pellets to 20.8±6.6 mg/ml on day 21. Comparing the levels of hypertrophic markers, single cell pellets showed 7-fold and 20-fold higher expression of COL1A1 and COL10A1 than spheroid pellets on day 21. The encapsulation of hMSC spheroids in THA-Col resulted in an upregulation of chondrogenic-associated markers and GAG content in all hydrogels with differences in cell differentiation related to the Col and THA polymer ratio, while level of hypertrophy was comparable in all groups with values similar to the spheroid pellet group. Spheroids embedded in hydrogels with lower THA content (group 1 and 3) resulted in more pronounced chondrogenic phenotype marked by upregulation of COL2A1 (3’200–4’500-fold) and ACAN (152–179-fold) relative to the respective samples on day 1. Spheroids embedded in higher THA content hydrogels (group 2 and 4) showed less pronounced chondrogenesis marked by lower upregulation of COL2A1 (980–1800-fold) and ACAN (25–68-fold, relative to day 1 samples). This was confirmed by quantification of GAGs, increasing from 2.5±1.9 and 2.5±1.7 mg/ml (day 1) to 11.4±2.5 and 9.9±3.8 mg/ml on day 21 for groups 1 and 4, respectively. (Immuno-) histological stainings resulted in a more homogenous staining in lower THA content hydrogels compared to a more local matrix deposition in samples with higher THA content. Conclusion. The reduced level of hypertrophy in hMSC pellets prepared from cell spheroids compared to single cell pellets at same cell count might be related to the packing density of the cells with cells being more densely packed in single cell pellets compared to pellets from spheroids. Investigating the effect of polymer ratios on chondrogenesis, it seems that the THA content is the driving factor influencing hMSC chondrogenesis rather than Col content in THA-Col composites at comparable mechanical properties. This study highlights the feasibility to use hMSC spheroids as alternative approach to study in vitro chondrogenic differentiation and the suitability to investigate the effect of biomaterial composition on chondrogenesis and hMSC hypertrophy

Introduction. Trabecular Titanium is a biomaterial characterized by a regular three-dimensional hexagonal cell structure imitating trabecular bone morphology. Components are built via Electron Beam Melting technology in aone- step additive manufacturing process. This biomaterial combines the proven mechanical properties of Titanium with the elastic modulus provided by its cellular solid structure (Regis 2015 MRS Bulletin). Several in vitro studies reported promising outcomes on its osteoinductive and osteoconductive properties: Trabecular Titanium showed to significantly affect osteoblast attachment and proliferation while inhibiting osteoclastogenesis (Gastaldi 2010 J Biomed Mater Res A, Sollazzo 2011 ISRN Mater Sci); human adipose stem cells were able to adhere, proliferate and differentiate into an osteoblast-like phenotype in absence of osteogenic factors (Benazzo 2014 J Biomed Mater Res A). Furthermore, in vivo histological and histomorphometric analysis in a sheep model indicated that it provided bone in-growth in cancellous (+68%) and cortical bone (+87%) (Devine 2012 JBJS). A multicentre prospective study was performed to assess mid-term outcomes of acetabular cups in Trabecular Titanium after Total Hip Arthroplasty (THA). Methods. 89 patients (91 hips) underwent primary cementless THA. There were 46 (52%) men and 43 (48%) women, with a median (IQR) age and BMI of 67 (57–70) years and 26 (24–29) kg/m2, respectively. Diagnosis was mostly primary osteoarthritis in 80 (88%) cases. Radiographic and clinical evaluations (Harris Hip Score [HHS], SF-36) were performed preoperatively and at 7 days, 3, 6, 12, 24 and 60 months. Bone Mineral Density (BMD) was determined by dual-emission X-ray absorptiometry (DEXA) according to DeLee &Charnley 3 Regions of Interest (ROI) postoperatively at the same time-points using as baseline the measureat 1 week. Statistical analysis was carried out using Wilcoxon test. Results. Median (IQR) HHS and SF-36 improved significantly from 48 (39–61) and 49 (37–62) preoperatively to 99 (96–100) and 76 (60–85) at 60 mo. (p≤0.0001). Radiographic analysis showed evident signs of bone remodelling and biological fixation, with presence of superolateral and inferomedial bone buttress, and radial trabeculae in ROI I/II. All cups resulted radiographically stable without any radiolucent lines. The macro-porous structure of this biomaterial generates a high coefficient of friction (Marin 2012 Hip Int), promoting a firm mechanical interlocking at the implant-bone interface which could be already observed in the operating room. BMD initially declined from baseline at 7 days to 6 months. Then, BMD slightly increased or stabilized in all ROIs up to 24 months, while showing evidence of partial decline over time with increasing patient' age at 60 months, although without any clinical significance in terms of patients health status or implant stability. Statistical significant correlations in terms of bone remodeling were observed between groups of patients on the basis of gender and age (p≤0.05). No revision or implant failure was reported. Conclusions. All patients reported significant improvements in quality of life, pain relief and functional recovery. Radiographic evaluation confirmed good implant stability at 60 months. These outcomes corroborate the evidence reported on these cups by orthopaedic registries and literature (Perticarini 2015 BMC Musculoskelet Disord; Bistolfi 2014 Min Ortop)

Introduction: It is well known that the fate of biomaterials is determined by the distribution of proteins attached to the surface from the initial contact with blood or serum. This profile determines wether a material is inert, creates a foreign body response or is bioactive. Bioinert materials, such as polyethylene completely denature surface proteins, whilst materials inducing inflammatory responses are predisposed to complement protein attachment. Bioactive materials such autologous tissue grafts adsorb, but do not denature serum proteins such as fibronectin and Von Willebrand’s factor. This does not interfere with the healing cascade. This aim of this study is to prepare a synthetic bone graft substitute that activates the body’s autologous healing cascade by activating platelets, without activating a complement response through the controlled adsorption of serum proteins. Methods: Polymers composed of varied concentration of acrylic acid (AA) and comonomers (methyl, ethyl and butyl methacrylates (MMA, EMA, BMA)) were prepared in glass vials by free radical polymerisation. Fresh blood was collected from a healthy donor and pipetted immediately into each chamber. Glass was used as a control. The chambers were incubated at 37o C for 2 hours. The surface morphology was examined using Scanning Electron Microscopy (SEM). Concentration of complement protein C5a and prothrombin fragments 1 and 2 were determined using commercial ELISA kits. Foreign body reaction (FBR) initiated by the biomaterial was estimated by counting leukocytes on clot sections using immunofluorescence. Results: Extent of coagulation was correlated with plasma concentrations of Prothrombin fragments 1 and 2. These measurements show blood incubated with various polymers composed of different comonomers all promoted the formation of blood clots. It was found that the leukocyte population towards the interface of clot and polymer (AA:MMA) decreased with increasing surface acid concentration (65%AA:MMA 30 leukocytes/0.25mm2, glass 70 leukocytes/0.25mm2 (p< 0.05)). FBR is induced by the activation of complement system. The percentage of C5a concentration detected in blood incubated with various polymers composed of different comonomers relative to normal serum level of C5a (35ng/mL). No significant elevations of C5a were measured from polymer 65% AA:MMA and 65% AA:EMA. Glass induced vigorous complement response as expected. The synergistic combination of surface acid concentration and comonomers had a significant effect on extent of FBR. Increased acid concentration resulted in decreased C5a level with MMA and ET but increased level with BMA. Discussion: The functional groups exposed on the surface of a material influence whether leukocyte or platelet activation is responsible for the subsequent physiological response. By modifying the combinations of surface acid concentrations and comonomers, we show that a biomaterial with an appropriate surface chemistry promotes the platelet plug formation and coagulation but down regulated foreign body reaction. This study shows that that a biomaterial with the appropriate surface chemistry to evoke the same coagulation response as damaged tissue, mediated through platelet activation and intrinsic and extrinsic coagulation, initiates the initial pathways of the bone healing cascade. This material is a realistic candidate for biomaterial induced bone regeneration

Despite its intrinsic ability to regenerate form and function after injury, bone tissue can be challenged by a multitude of pathological conditions. While innovative approaches have helped to unravel the cascades of bone healing, this knowledge has so far not improved the clinical outcomes of bone defect treatment. Recent findings have allowed us to gain in-depth knowledge about the physiological conditions and biological principles of bone regeneration. Now it is time to transfer the lessons learned from bone healing to the challenging scenarios in defects and employ innovative technologies to enable biomaterial-based strategies for bone defect healing. This review aims to provide an overview on endogenous cascades of bone material formation and how these are transferred to new perspectives in biomaterial-driven approaches in bone regeneration. Cite this article: T. Winkler, F. A. Sass, G. N. Duda, K. Schmidt-Bleek. A review of biomaterials in bone defect healing, remaining shortcomings and future opportunities for bone tissue engineering: The unsolved challenge. Bone Joint Res 2018;7:232–243. DOI: 10.1302/2046-3758.73.BJR-2017-0270.R1

Purpose: Filling bone defects is a major challenge in orthopaedic surgery. One of the therapeutical alternatives to combined autologous bone grafts and bone substitutes is to use a biomaterial carrying bone stem cells. The purpose of this study was to test a hybrid biomaterial in a major bone loss model in the rabbit. Material and methods: The study material was the AN 69 hydrogel (Hospal). Twenty-four rabbits were divided into four identical groups of six individuals. Each animal underwent a unilateral resection measuring 2 cm in the mid third of the cubitus: group 1 simple resection; group 2 resection and centromedullary pin; group 3 resection, centromedullary pin and biomaterial; group 4 resection, centromedullary pin, biomaterial with bone marrow stem cells. Animal were sacrificed at six weeks. A radiograph was obtained immediately after surgery and at sacrifice. The study parameters were: new bone formation, bone healing, bone remodelling. Each criteria was assessed with a mean score (Werntz score). A pathology examination was performed in all cases to study new bone formation, polylmere degradation and inflammation. Results: The overall radiographic score was group 1 = 2, group 2 = 8, group 3 = 24, group 4 = 42 for a maximum 62 points. Histologically, there was nonunion after simple pinning with formation of a defective callus. The nonunion persisted after pinning and hydrogel without cell seeding. New bone formation was moderate and predominated on the borders of the bone resection. After pinning associated with cell seeded hydrogel, an osteogenic lamina arose from the hydrogel network. This osteogenesis was continuous with osteogenesis originating from the bone section cut. Discussion: These findings demonstrate that associated a hydrogel with bone stem cells can produce more significant bone formation than in controls, confirming the animal model. Treatment of major bone loss and aseptic osteonecrosis after curettage could be proposed with this new biomaterial combining a hydrogel and CD34+ stem cells in humans

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 considered but no successful candidate has emerged thus far. The many preclinical and few clinical attempts include fibrin glue, mussel adhesive and even “Kryptonite” (US bone void filler). The most promising recent attempts are based on phosphorylating amino acids, part of a common cellular adhesion mechanism linking mussels, caddis fly larvae, and mammals. Rapid high bond strength development in the wetted fatty environment of fractured bone, that is sustained during biological healing, is challenging to prove both safety and efficacy. Additionally, there are no “predicate” preclinical animal and human models which led the authors to develop novel evaluations for an adhesive candidate “OsStic. tm. ” based on calcium salts and amino acids. Adhesive formulations were evaluated in both soft (6/12 weeks) and hard tissue (3,7,10,14 & 42 days) safety studies in murine models. The feasibility of a novel adhesiveness test, initially proven in murine cadaver femoral bone, is being assessed in-vivo (3,7,10,14 & 42 days) in bilateral implantations with a standard tissue glue as the control. In parallel an ex-vivo human bone model using freshly harvested human donor bone is under development to underwrite the eventual clinical application of such an adhesive. This is part of a risk mitigation project bridging between laboratory biomaterial characterisation and a commercial biomaterial development where safety and effectiveness have to meet today´s new medical device requirements

Oxide ceramics, such as alumina and zirconia have been used extensively in arthroplasty bearings to address bearing wear and mitigate its delayed, undesirable consequences. In contrast to oxide ceramics that are well-known to orthopaedic surgeons, silicon nitride (Si. 3. N. 4. ) is a non-oxide ceramic that has been investigated extensively in very demanding industrial applications, such as precision bearings, cutting tools, turbo-machinery, and electronics. For the past four years, Si. 3. N. 4. has also been used as a biomaterial in the human body; specifically in spinal fusion surgery. As a implantable biomaterial, Si. 3. N. 4. has unique properties, such as high strength and fracture toughness, inherent chemical and phase stability, low wear, proven biocompatibility, excellent radiographic imaging, antibacterial advantages, and superior osteointegration. This property combination has proven beneficial and desirable in orthopaedic implants made for spinal fusion, spinal disc reconstruction, hip and knee arthroplasty, and other total joints (Fig. 1). The physical properties, shapes, sizes and surface features of Si. 3. N. 4. can be engineered for each application – ranging from dense, finely polished articulation components, to highly porous scaffolds that promote osteointegration. Both porous and polished surfaces can be incorporated in the same implant, opening a number of opportunities for arthroplasty implant design. Crack propagation modes for in situ toughened Si. 3. N. 4. differ favorably from those of conventional ceramics, rendering Si. 3. N. 4. extremely resistant to catastrophic failure in vivo (Fig. 2). Most significantly, our recent work has shown that Si. 3. N. 4. is resistant to bacterial biofilm formation, colonization and growth, when compared to medical-grade PEEK and titanium. These anti-infective characteristics are particularly valuable for in vivo implantation. We will present the unique properties and characteristics of Si. 3. N. 4. , and compare these to other ceramic and non-ceramic biomaterials. Si. 3. N. 4. was once used only in industrial applications, but early data show that this novel biomaterial is positively impacting orthopaedic care and will continue to do so into the future

Abstract. Objectives. Additive manufacturing (AM) enables fine control over the architecture of porous lattice structures, and the resulting mechanical performance. Orthopaedic implants may benefit from the tailored stiffness/elastic modulus of these AM biomaterials, as the stiffness can be made to closer match the properties of the replaced trabecular bone. Methods. This study used laser powder bed fusion (PBF) to create stochastic porous lattice structures in stainless steel (SS316L) and titanium alloy (Ti6Al4V), with modifications that aimed to overcome PBF manufacturing limitations of build angles. The structures were tested in uni-axial compression (n = 5) in 10 load orientations relative to the structure, including the three orthogonal axes. Results. The testing verified that no hidden peaks in elastic modulus existed in the stochastic structure. The standard deviation of the 10 elastic modulus values in the final structure decreased from 249 MPa to 101 MPa when made in SS316L and from 95.9 MPa to 52.5 MPa for Ti6Al4V, indicating the structures were more isotropic. Conclusions. These modified stochastic lattices have similar stiffness to cancellous bone and have controllable anisotropy, giving them the potential to be used within implants which match the stiffness of trabecular bone. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Introduction and Aims: Treatment of rotator cuff tendon tear presents a significant therapeutic challenge to surgeons. Porcine small intestinal submucosa (SIS) is a biomaterial approved by TGA and FDA for the repair of rotator cuff tendon tear. The aims of this study are to evaluate the safety and efficacy of SIS. Method: SIS purchased from DePuy Johnson & Johnson was examined by histology and PCR technique. The material was also implanted into mice and rabbits for the evaluation of biological reaction and inflammatory response. Porcine immunoreceptor DAP12 gene was used to examine if the material contained porcine DNA. Results: Fresh SIS membrane before implantation contains multiple layers of spindle-shaped cells mixed with a small population of round-shaped cells. Chloro-acetate esterase staining showed that the round-shaped cells are positive, indicating that they are mast cells. The tissue architecture of SIS mimics to tendon structure as evidenced by H& E staining. To further confirm if cells present in SIS material were porcine origin, nested PCR for the amplification of DAP12 gene was used. The result demonstrated that SIS membrane contain porcine DNA materials. Conclusion: SIS contains porcine cells and nuclei acid, which contradicts with current views that SIS is a cell-free biomaterial. Although no foreign body reaction of SIS was observed, SIS implant may cause chronic inflammation. Further studies should be conducted to confirm the clinical efficacy of SIS implant

Sufficient vascularization is essential for osseointegration of biomaterials and their substitution by new bone. Angiogenic growth factors such as VEGF are promising agents to promote the vascularization of bone substitutes. To optimize the efficacy of VEGF delivery a continuous administration of low concentrations of VEGF seems to be beneficial. We hypothesized that a long-term release of VEGF from calcium phosphate ceramics may induce a sustained angiogenic response and sufficiently promote biomaterial vascularization in vivo. Vascular endothelial growth factor (VEGF, Genentech Inc., South San Francisco, USA.) was co-precipitated onto biphasic calcium phosphate ceramics (BCP, 80% HA, 20% β-TCP) at a concentration of 1μg/ml and 5μg/ml. The passive release and the cell-mediated release of VEGF were analyzed over 19 days by ELISA. For in vivo investigations BCP ceramics were implanted into a cranial window preparation in Balb/c mice. Angiogenesis and vascularization were investigated over 28 days by means of intravital microscopy. Functional capillary density (FCD, mm/mm. 2. ) served as parameter of biomaterial vascularization. Co-precipitation of VEGF onto BCP ceramics resulted in a significant improvement of protein retention as compared to conventional adsorption of the growth factor [Cumulative VEGF release: Adsorption: 320 ± 2.6 ng/ml, Co-precipitation 116 ± 14.6 ng/ml (p< 0.05)]. Murine bone marrow cells differentiated towards osteoclasts mediated a sustained release of co-precipitated VEGF. Preliminary in vivo results showed a significant increase of functional capillary density after implantation of BCP ceramics co-precipitated with VEGF as compared to negative controls [day 7: 1.7 ± 0.2 mm/mm. 2. vs. 0.9 ± 0.5 mm/mm. 2. ; day 14: 6.1 ± 0.3 mm/mm. 2. vs. 2.1 ± 0.6 mm/mm. 2. ; day 28: 8.7 ± 0.3 mm/mm. 2. vs. 3.9 ± 0.7 mm/mm. 2. , p< 0.05]. At 14 and 28 days after implantation, FCD induced by BCP ceramics co-precipitated with VEGF was significantly higher as compared to FCD induced by ceramics adsorbed with the VEGF [day 14: 6.1 ± 0.3 mm/mm. 2. vs. 4.0 ± 1.4 mm/mm. 2. ; day 28: 8.7 ± 0.3 mm/mm. 2. vs. 5.9 ± 0.7 mm/mm. 2. , p< 0.05]. The release kinetics critically influences the efficacy and the risks of local VEGF administration. By applying a co-precipitation technique the initial high liberation rate of VEGF was reduced and a sustained cell-mediated release at low concentrations was achieved. In vivo, VEGF promoted angiogenesis and vascularization of BCP ceramics. Vessel formation was more pronounced if VEGF was co-precipitated onto ceramics as compared to superficial adsorption of the growth factor, indicating that VEGF delivery at later stages of the healing process is beneficial. The present study provides evidence that, by delivering VEGF in a sustained manner at low local concentrations biomaterial vascularization can be markedly enhanced