Biomaterials with mechanical or biological competence are ubiquitous in musculoskeletal disorders, and understanding the inflammatory response they trigger is key to guide tissue regeneration. While macrophage role has been widely investigated, immune response is regulated by other immune cells, including neutrophils, the most abundant leukocyte in human blood. As first responders to injury, infection or material implantation, neutrophils recruit other immune cells, and therefore influence the onset and resolution of chronic inflammation, and macrophage polarization. This response depends on the physical and chemical properties of the biomaterials, among other factors. In this study we report an in vitro culture model to describe the most important neutrophil functions in relation to tissue repair. We identified neutrophil survival and death, neutrophils extracellular trap formation, release of reactive oxygen species and degranulation with cytokines release as key functions and introduced a corresponding array of assays. These tests were suitable to identify clear differences in the response by neutrophils that were cultured on material of different origin, stiffness and chemical composition. Overall, substrates from biopolymers of natural origin resulted in increased survival, less neutrophil extracellular trap formation, and more reactive oxygen species production than synthetic polymers. Within the range of mechanical properties explored (storage modulus below 5 k Pa), storage modulus of covalently crosslinked hyaluronic acid hydrogels did not significantly alter neutrophils response, whereas polyvinyl alcohol gels of matching mechanical properties displayed a response indicating increased activation. Additionally, we present the effect of material stiffness, charge, coating and culture conditions in the measured neutrophils response. Further studies are needed to correlate the neutrophil response to tissue healing. By deciphering how neutrophils initiate and modulate the immune response to material implantation, we aim at introducing new principles to design immunomodulatory biomaterials for musculoskeletal disorders. Acknowledgments. This work was supported by the AO Foundation, AO CMF, grant AOCMF-21-04S

Biomaterials are no longer considered innate structures and using functionalisation and biofabrication strategies to modulate a desired response whether it is a host or implant is currently an important focus in current research paradigms. Fundamentally, a thorough understanding of the host response will enable us to design appropriate strategies. The input from the host response needs to be weighed in depending on the host disease condition. Our current inputs have been through a thorough understanding of glyco-proteomics based tools which we are developing in our laboratory. In addition, biomaterials themselves provide immense therapeutic benefits which needs to be accounted in the design paradigm. Using functionalisation strategies such as enzymatic and hyperbranched linking systems, we have been able to link biomolecules to different structural moieties. The programmed assembly of biomolecules into higher-order self-organized systems is central to innumerable biological processes and development of the next generation of scaffolds. Recent design efforts have utilized a glycobiology and developmental biology approach toward both understanding and engineering supramolecular protein and sugar assemblies

For evaluation of orthopaedic biomaterials the closest hostile-like in vitro environments are desirable with relevant control of chemical, biological, mechanical etc. parameters. For faster screening and reduction of time and costs, combination of different critical key parameters in minimal tests is needed. New trends also favour minimisation of in vivo (2010/63/EC, towards replacement technology) and clinical tests (2001/20/EC, 2005/28/EC) for new products yet not compromising risks. Biomaterials manufacturers also are interested in shortening of the time-to-market keeping conformity to essential requirements and withstanding the simulated “worst case” conditions (2003/94/EC). Here we show the new approach of the creation of conditions closest to real life and applications, based on scientifically designed and optimised models, aiming on predictive outputs. With new device and designed protocols, several biomaterials for orthopaedic applications were analysed: titanium, biodegradable fibrous scaffolds and hydrogels. Creation of several favourable conditions for different tissues type formation took place on the surface of the porous titanium specimen. Such conditions could be designed for measurement of the cells proliferation and e.g. simultaneous bacterial adhesion with rather high precision. The method has been compared in independent laboratories for hydrogels with other measuring techniques and shown the benefits of the method especially in more precise control of biomechanical cues. It was observed that significant amount of data are containing in the recorded signals which underlines the importance of correct and holistic data post-processing. The protocols can be furthermore tailored to simulate different conditions, such as for specific positions in tibia, or humeral etc., and combined with patient-specific biomechanics (soft tissues) for customised implant design. The financial support from the Finnish Agency of Innovation (Tekes) is gratefully acknowledged. Author has no competing financial or conflicting interests

Scaffolds and Biomaterials used for skeletal tissue regeneration need to be biocompatible, osteo-inductive, osteo-conductive and mechanically compatible with bone to meet the requirements for bone tissue engineering. The aim of our research is to deliver. a new generation of stable, life-long orthopedic/dental implants that offer strong bone–implant anchorage. Novel smart scaffolds to permit greater control over the location and quality of bone regeneration, allowing faster healing. Currently available modalities for treating large bone defects, are limited in their success. Developing synthetic scaffolds that promote bone growth and adequate vascularization is vital in orthopaedic and maxillofacial surgeries. The current generation of synthetic scaffolds, does not combine the required posorsity, mechanical properties and bioactivity. This presentation will highlight some of our newly developed novel highly porous and mechanically strong scaffolds that promote the migration, proliferation and differentiation of bone and endothelial cells for effective skeletal tissue integration and vascularization. Despite major advances in prosthetic technologies, implants have a finite life of 1015 years, due to their premature failure. Novel micro-engineered surfaces are required to anchor prosthetic implants to the surrounding bony skeleton. Various surface chemical modifications have been applied to prosthetic devices to enhance osseointegration. To-date none have resulted in a stable interface strong enough to support functional loading for the lifetime of the implant. Our group demonstrated that surface chemisrty modification of biomaterials with bioactive molecules have the potential to provide a surface on a prosthesis that is conducive to normal bone metabolism

Menisci are crucial structures for knee homeostasis: they provide increase of congruence between the articular surfaces of the distal femur and tibial plateau, bear loading, shock absorption, lubrication, and proprioception. After a meniscal lesion, the golden rule, now, is to save as much meniscus as possible: only the meniscus tissue which is identified as unrepairable should be excised and meniscal sutures find more and more indications. Several different methods have been proposed to improve meniscal healing. They include very basic techniques, such as needling, abrasion, trephination and gluing, or more complex methods, such as synovial flaps, meniscal wrapping, or the application of fibrin clots. Basic research of meniscal substitutes has also become very active in the last decades. The features needed for a meniscal scaffold are: promotion of cell migration, it should be biomimetic and biocompatible, it should resist forces applied and transmitted by the knee, it should slowly biodegrade and should be easy to handle and implant. Several materials have been tested, that can be divided into synthetic and biological. The first have the advantage to be manufactured with the desired shapes and sizes and with precise porosity dimension and biomechanical characteristics. To date, the most common polymers are polylactic acid (PGA); poly-(L)-lactic acid (PLLA); poly- (lactic-co-glycolic acid) (PLGA); polyurethane (PU); polyester carbon and polycaprolactone (PCL). The possible complications, more common in synthetic than natural polymers are poor cell adhesion and the possibility of developing a foreign body reaction or aseptic inflammation, leading to alter the joint architecture and consequently to worsen the functional outcomes. The biological materials that have been used over time are the periosteal tissue, the perichondrium, the small intestine submucosa (SIS), acellular porcine meniscal tissue, bacterial cellulose. Although these have a very high biocompatibility, some components are not suitable for tissue engineering as their conformation and mechanical properties cannot be modified. Collagen or proteoglycans are excellent candidates for meniscal engineering, as they maintain a high biocompatibility, they allow for the modification of the porosity texture and size and the adaptation to the patient meniscus shape. On the other hand, they have poor biomechanical characteristics and a more rapid degradation rate, compared to others, which could interfere with the complete replacement by the host tissue. An interesting alternative is represented by hydrogel scaffolds. Their semi-liquid nature allows for the generation of scaffolds with very precise geometries obtained from diagnostic images (i.e. MRI).

Promising results have been reported with alginate and polyvinyl alcohol (PVA). Furthermore, hydrogel scaffolds can be enriched with growth factors, platelet-rich plasma (PRP) and Bone Marrow Aspirate Concentrate (BMAC). In recent years, several researchers have developed meniscal scaffolds combining different biomaterials, to optimize the mechanical and biological characteristics of each polymer. For example, biological polymers such as chitosan, collagen and gelatin allow for excellent cellular interactions, on the contrary synthetic polymers guarantee better biomechanical properties and greater reliability in the degradation time. Three-dimensional (3D) printing is a very interesting method for meniscus repair because it allows for a patient-specific customization of the scaffolds. The optimal scaffold should be characterized by many biophysical and biochemical properties as well as bioactivity to ensure an ECM-like microenvironment for cell survival and differentiation and restoration of the anatomical and mechanical properties of the native meniscus. The new technological advances in recent years, such as 3D bioprinting and mesenchymal stem cells management will probably lead to an acceleration in the design, development, and validation of new and effective meniscal substitutes.

The most common reason for revision surgery of total hip replacements is aseptic loosening of implants secondary to osteolysis, which is caused by immune-mediated reactions to implant debris. These debris can cause pseudotumour formation. As revision surgery is associated with higher mortality and infection, it is important to understand the pro-inflammatory process to improve implant survival. Toll-like receptor 4 (TLR4) has been shown to mediate immune responses to cobalt ions. Statin use in epidemiological studies has been associated with reduced risk of revision surgery. In-vitro studies have demonstrated the potential for statins to reduce orthopaedic debris-induced immune responses and there is evidence that statins can modulate TLR4 activity. This study investigates simvastatin's effect on orthopaedic biomaterial-mediated changes in protein expression of key inflammatory markers and soluble-ICAM-1 (sICAM-1), an angiogenic factor implicated in pseudotumour formation.

Human macrophage THP-1 cells were pre-incubated with 50µM simvastatin for 2-hours or a vehicle control (VC), before being exposed to 0.75mM cobalt chloride, 50μm3 per cell zirconium oxide or LPS as a positive control, in addition to a further 24-hour co-incubation with 50µM simvastatin or VC. Interleukin −8 (IL-8), sICAM-1, chemokine ligand 2 (CCL2), CCL3 and CCL4 protein secretion was measured by enzyme-linked immunosorbent assay (ELISA). GraphPad Prism 10 was used for statistical analysis including a one-way ANOVA.

Pre-treatment with simvastatin significantly reduced LPS and cobalt-mediated IL-8 secretion (n=3) and sICAM-1 protein secretion (n=2) in THP-1 cells. Pre-treatment with simvastatin significantly reduced LPS-mediated but not cobalt ion-mediated CCL2 (n=3) and CCL3 protein (n=3) secretion in THP-1 cells. Simvastatin significantly reduced zirconium oxide-mediated CCL4 secretion (n=3).

Simvastatin significantly reduced cobalt-ion mediated IL-8 and sICAM-1 protein secretion in THP-1 cells. This in-vitro finding demonstrates the potential for simvastatin to reduce recruitment of leukocytes which mediate the deleterious inflammatory processes driving implant failure.

Introduction and Objective

Total joint replacement (TJR) is indicated for patients with end-stage osteoarthritis (OA) where conservative treatment has failed. Approximately 1.3 million primary hip replacement surgeries have been recorded in the United Kingdom since 2003 and this number is set to rise due to an increase in obesity as well as an ageing population. Total hip replacement (THR) has a survival rate of 85% at 20 years; the most common reason for failure is aseptic loosening which often occurs secondary to osteolysis caused by immune-mediated inflammation responses to wear debris generated from the materials used in the THR implant. Therefore, by understanding the biological steps by which biomaterials cause immune-mediated reactions it should be possible to prevent them in the future thereby reducing the number of costly revision surgeries required.

Residual tumor cells left in the bone defect after malignant bone tumor resection can result in local tumor recurrence and high mortality. Therefore, ideal bone filling materials should not only aid bone reconstruction or regeneration, but also exert local chemotherapeutic efficacy. However, common bone substitutes used in clinics are barely studied in research for local delivery of chemotherapeutic drugs. Here, we aimed to use facile manufacturing methods to render polymethylmethacrylate (PMMA) cement and ceramic granules suitable for local delivery of cisplatin to limit bone tumor recurrence.

Porosity was introduced into PMMA cement by adding 1-4% carboxymethylcellulose (CMC) containing cisplatin, and chemotherapeutic activity was rendered to two types of granules via adsorption. Then, mechanical properties, porosity, morphology, drug release kinetics, ex vivo reconstructive properties of porous PMMA and in vitro anti-cancer efficacy against osteosarcoma cells were assessed. Morphologies, molecular structures, drug release profiles and in vitro cytostatic effects of two different drug-loaded granules on the proliferation of metastatic bone tumor cells were investigated.

The mechanical strengths of PMMA-based cements were sufficient for tibia reconstruction at CMC contents lower than 4% (≤3%). The concentrations of released cisplatin (12.1% and 16.6% from PMMA with 3% and 4% CMC, respectively) were sufficient for killing of osteosarcoma cells, and the fraction of dead cells increased to 91.3% within 7 days. Functionalized xenogeneic granules released 29.5% of cisplatin, but synthetic CaP granules only released 1.4% of cisplatin over 28 days. The immobilized and released cisplatin retained its anti-cancer efficacy and showed dose-dependent cytostatic effects on the viability of metastatic bone tumor cells.

Bone substitutes can be rendered therapeutically active for anticancer efficacy by functionalization with cisplatin. As such, our data suggest that multi-functional PMMA-based cements and cisplatin-loaded granules represent viable treatment options for filling bone defects after bone tumor resection.

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

Objectives

Low back pain is strongly associated with degeneration of the intervertebral disc (IVD). During degeneration, altered matrix synthesis and increased matrix degradation, together with accompanied cell loss is seen particularly in the nucleus pulposus (NP). It has been proposed that notochordal (NC) cells, embryonic precursors for the cells within the NP, could be utilized for mediating IVD regeneration. However, injectable biomaterials are likely to be required to support their phenotype and viability within the degenerate IVD. Therefore, viability and phenotype of NC cells were analysed and compared within biomaterial carriers subjected to physiological oxygen conditions over a four-week period were investigated.

Low back pain is strongly associated with degeneration of the intervertebral disc (IVD). During degeneration, altered matrix synthesis and increased matrix degradation, together with accompanied cell loss is seen particularly in the nucleus pulposus (NP). It has been proposed that notochordal (NC) cells, embryonic precursors for the cells within the NP, could be utilized for mediating IVD regeneration. However, injectable biomaterials are likely to be required to support their phenotype and viability within the degenerate IVD. Therefore, viability and phenotype of NC cells were analysed and compared within biomaterial carriers subjected to physiological oxygen conditions over a four-week period were investigated.

Porcine NC cells were incorporated into three injectable hydrogels: NPgel (a L-pNIPAM-co-DMAc hydrogel), NPgel with decellularized NC-matrix powder (dNCM) and Albugel (an albumin/ hyaluronan hydrogel). The NCs and biomaterials constructs were cultured for up to four weeks under 5% oxygen (n=3 biological repeats). Histological, immunohistochemical and glycosaminoglycans (GAG) analysis were performed to investigate NC viability, phenotype and extracellular matrix synthesis and deposition.

Histological analysis revealed that NCs survive in the biomaterials after four weeks and maintained cell clustering in NPgel, Albugel and dNCM/NPgel with maintenance of morphology and low caspase 3 staining. NPgel and Albugel maintained NC cell markers (brachyury and cytokeratin 8/18/19) and extracellular matrix (collagen type II and aggrecan). Whilst Brachyury and Cytokeratin were decreased in dNCM/NPgel biomaterials, Aggrecan and Collagen type II was seen in acellular and NC containing dNCM/NPgel materials. NC containing constructs excreted more GAGs over the four weeks than the acellular controls.

NC cells maintain their phenotype and characteristic features in vitro when encapsulated into biomaterials. NC cells and biomaterial construct could potentially become a therapy to treat and regenerate the IVD.

Novel biomaterials are being developed and studied, intended to be applied as bone graft substitute materials. Typically, these materials are being tested in in vitro setups, where among others their cytotoxicity and alkaline phosphatase activity (as a marker for osteoblastic differentiation) are being evaluated. However, it has been reported that in vitro tests correlate poorly with in vivo results and therefore many promising biomaterials may not reach the clinic as a bone graft substitute product. One of the reasons for the poor correlation, may be the minimal complexity of the in vitro tests, as compared to the in vivo environment. Ex vivo models, mimicking the natural tissue environment whilst maintaining control of culture parameters, may be a promising alternative to assess biomaterials for bone formation. Assess the possibility of an ex vivo culture platform to test biomaterials on their potential to stimulate new bone formation. Osteochondral plugs (cylinders n=10, Ø 10 mm, height 15 mm) were drilled from fresh porcine knees, from the slaughterhouse. A bone defect (Ø 6 mm) was created and which was filled with a biomaterial graft (S53P4 bioactive glass (n=3); collagen sponges loaded with BMP-2 (n=3, as positive control)) or kept empty (n=4). The explants were cultured in custom-made two-chamber bioreactors for six weeks (LifeTec Group BV). Cartilage and bone were physically separated, similar to the in vivo situation, by a sealing ring. The two tissues were cultured in separate compartments, allowing for specific culture medium for each tissue. Medium was changed every 2–3 days and weekly micro computed tomography (µCT) images were obtained to longitudinally monitor the formation of new bone. An MTT assay was performed on half of the samples after six weeks of culture. The other samples were fixed for histology, to determine which cells were present after six weeks. The MTT metabolic assay showed that a number of cells in the bone were viable after six weeks. The further away from the border, the fewer living cells were observed. The cells in the cartilage also survived. No significant bone formation was observed with µCT in either of groups, even though abundant bone formation was expected in the BMP-2 group. Explanations of the negative results of the positive group might be that too few viable cells remain after six weeks, or that the cells that are still present are not able to form bone. No significant bone formation was observed in the bone defects in osteochondral explants that were cultured with, or without, biomaterials for six weeks. However, the platform showed that it is capable to successfully culture osteochondral explants for six weeks.

Histology needs to be performed to evaluate which cells were present at the end of the culture and this will be compared to the cells present directly after drilling the explants.

Device-associated infection remains a serious clinical problem in orthopaedic and trauma surgery. The emergence of resistant organisms such as methicillin resistant Staphylococcus aureus (MRSA) has further exacerbated this problem by limiting the range of treatment options. Currently, systemic antibiotic therapy is the cornerstone of treatment, alongside surgical resection of infected tissues and implant removal. The potential for antibiotic loaded biomaterials to support the prevention and treatment of infection is significant, although the currently available options are limited in number and often re-purposed from other applications e.g. antibiotic loading of bone cement. The first part of the talk will cover the basic concepts involved in antibiotic treatment, with an emphasis on the ideal antibiotic release kinetics from biomaterials, and how bacterial biofilms and antibiotic resistance influence antimicrobial efficacy. The next generation of biomaterials for antibiotic delivery should be specifically designed with this knowledge in mind. Regulatory approval of antimicrobial combination devices, however, is an evolving process as regulatory bodies seek more robust and clinically relevant efficacy data. Approval will require preclinical efficacy using standardized animal models that recapitulate the key features of the clinical disease. The second part of this talk will cover best practice in this important stage of development.

By definition, a smart biomaterial is a material, such as a ceramic, alloy, gel or polymer, that can convert energy from one form into another by responding to a change in a stimulus in its environment. These stimuli may involve temperature, pH, moisture, or electric and magnetic fields. In particular, thermoresponsive biomaterials have been successfully employed to host mammalian cells with a view to musculoskeletal tissue engineering. The presentation provides an overview of the use of thermosensitive polymers for the non-enzymatic stem cell harvesting, cell sheet engineering, three-dimensional scaffolds fabrications and organ-printing materials.

Robust repair relies on blood flow. This vascularization is the major challenge faced by tissue engineering on the path to forming thick, implantable constructs. Without this vasculature, oxygen and nutrients cannot reach the cells located far from host blood vessels. To make viable constructs, tissue engineering takes advantage of the mechanical properties of synthetic materials, while combining them with extracellular matrix proteins to create a natural environment for the tissue- specific cells. Tropoelastin, the precursor of the elastin, is the extracellular matrix protein responsible for elasticity in diverse tissues, including robust blood vessels. We find that tropoelastin contributes a physical role in elasticity and also substantially to the biology of repairing tissue. The emerging model from a range of our in vivo studies is that tropoelastin encodes direct biological effects and has the versatility to promote repair. We have discovered that tropoelastin substantially improves healing by halving the time to repair bone in small animals and large animal preclinical models; tropoelastin elicits this response with early stage neo-angiogenesis, recruitment of endogenous cells with consistently accelerated repair. This potency is marked by the concerted appearance of blood vessels, tissue and phased cellular contributions that work together to accelerate repair.

Over the last 50 years, biomaterials, prostheses and implants saved and prolonged the life of millions of humans around the globe. The main clinical complications for current biomaterials and artificial organs still reside in an interfacial mismatch between the synthetic surface and the natural living tissue surrounding it. Today, nanotechnology, nanomaterials and surface modifications provides a new insight to the current problem of biomaterial complications, and even allows us to envisage strategies for the organ shortage. Advanced tools and new paths towards the development of functional solutions for cardiovascular clinical applications are now available. In this talk, the potential of nanostructured metallic degradable metals to provide innovative solutions at medium term for the cardiovascular field will be depicted. Focus will be on Fe-based biodegradable metals with exceptional resistance, ductility and elasticity, for pushing innovative vascular applications. The intrinsic goal of this talk is to present an extremely personal look at how biodegradable metals can impact materials, surfaces and interfaces, and how the resulting unique properties allowed biomedical functional applications to progress, from their introduction, to the promising future that biodegradable metals may or may not hold for improving the quality of the life of millions worldwide.

Biomimicry is defined as the design and production of materials, structures, and systems that are modelled on biological entities and processes. Within the medical device sector, biomimicry uses an ecological standard to judge the “rightness” of biomaterial components and devices. After 3.8 billion years of evolution, nature has learned what works, what is appropriate, and what lasts. Biomimicry is a new way of viewing and valuing nature, and it introduces an era based not on what we can extract from the natural world, but on what we can learn from it. Original design manufacturing biomaterial projects that leverage the practice of biomimicry will be discussed. Both natural and synthetic polymer platforms will be reviewed for soft tissue and hard tissue applications. Given the complexity of musculoskeletal tissue structures, the key challenge is identifying the most appropriate materials and forms for recapitulating the native function in a tissue scaffold design. The general field of biomimicry will be reviewed along with specific examples in the regenerative medicine sector.

Increased revision rates and early failure of Metal-on-Metal (MoM) hip replacements are often due to adverse reaction to metal debris (ARMD). Cobalt is a major component of MoM joints and can initiate an immune response via activation of the innate immune receptor Toll-like receptor 4 (TLR4). This leads to increased secretion of inflammatory cytokines/chemokines e.g. CCL3 and CCL4. The aim of this study was to evaluate whether TLR4-specific neutralising antibodies can prevent cobalt-mediated activation of TLR4.

MonoMac 6 (MM6) cells, a human macrophage cell line, were treated with two different TLR4-specific monoclonal antibodies followed by 0.75mM of cobalt chloride (CoCl2). Lipopolysaccharide (LPS), a known TLR4 agonist was used as a positive control. Enzyme-linked immunosorbent assay (ELISA) was used to assess CCL3/CCL4 protein secretion and real time- polymerase chain reaction (RT-PCR) allowed quantification of CCL3/CCL4 gene expression.

MM6 cells treated with cobalt and LPS up-regulate CCL3 and CCL4 gene expression and protein secretion. MM6 cells pre-treated with both monoclonal antibodies prior to stimulation with 0.75mM CoCl2 for 16 hours demonstrated significant inhibition of both CCL3 and CCL4 secretion as well as gene expression (both p=<0.0001). One of the antibodies failed to inhibit chemokine expression and secretion in LPS treated cells.

This study identifies for the first time the use of TLR4-specific monoclonal antibodies to prevent cobalt activation of TLR4 and subsequent inflammatory response. This finding demonstrates the potential to exploit TLR4 inhibition in the context of MoM joint replacements by contributing to the development of novel therapeutics designed to reduce the incidence of ARMD.

Evaluation of different biomaterials is being performed with various methods trying to simulate the closest hostile-like in vitro environments. However the complexity of the conditions usually limits practically feasible combination of most relevant chemical, biological, biomechanical parameters in one single test. Many biomaterials and tissue engineering developments rely on high-throughput screening to multiply number of specimens and thus to gather sufficient data. The price to be paid for these methods is limited number of physical readouts, increased inter-specimens scatter, and unavoidable spatial constrains driving the conditions away of the clinical scenarios. For orthopaedic biomaterials this is of a particular concern, as implantation site conditions cannot be squeezed too much without lost of natural-mimicking stimuli.

Here we are presenting another approach based on high-output screening of biomaterials, which is based on the strategy of raising the number of readouts obtainable from every specimen at more clinically-relevant conditions. On the contrary to common methods like ISO 10993 or simplified biomechanical tests, the biomaterials enhanced simulation testing (BEST) evaluates specimens without pre-selected biomaterial model, assessing the whole specimen as would happen in the implantation site. Besides reducing the risk of improper conclusions caused by wrong material model choice, the data processing with non-local method intrinsically includes the test history bypassing common challenges usually seen with hereditary integration. For properly designed experiment, readouts might include invariant moduli, viscous stiffness, fluidity, fluid permittivity and diffusivity (without need for pressure-driven separate tests), fluid source, effective channel size, and swelling pressure (if swelling is present) in addition to conventional biomechanical parameters.

New solutions in advanced and consistent evaluations for biomaterials allow better risks control, shorten lead development time and costs, and compliant with 3R-strategy (2010/63/EC) and new regulatory requirements (2012/0266/COD in EU and FY2017 regulatory priorities by FDA). The approach shown is able to combine scientifically based tests with multi-purpose protocols to secure patient safety by screening of biomaterials under proper conditions.

The authors thank Finnish Agency for Innovations (Tekes) for providing partial financial support.

Electromechanical coupling (piezoelectricity) is present in all living beings and provides basis for sense, thoughts and mechanisms of tissue regeneration. Herein, we ventured to assess the influence of MMC in mesenchymal stem cell culture. In this study, we fabricated piezoelectric regenerative scaffolds to assess the role of electromechamical stimulation on tendon regeneration. Tendon cells were selectively stimulated in vitro by mechanical or electromechanical cues using non-piezoelectric or piezoelectric scaffolds and optimal mechanical loading (4% deformation at 0.5 Hz). This was followed up with an in vivo study to assess tendon regeneration in a rat Achilles tendon injury model. P(VDF-TrFE), scaffolds were observed to mimic the fibrous structure of tendon tissue (figure 1) and were capable of producing electrical charges up to 17 pC/N when mechanically loaded (figure 1. Genes associated with tendon specific markers (Col.I/Col III, Scx and Mkx) and mechanosensitive ion channels such as PIEZO1, TRAAK and TRPV1 were significantly upregulated (figure 2). The upregulated genes were validated with individual real time Q-PCR and bioinformatics revealed a possible regulated function. Those results were further validated in vivo. Protein expression of repaired tendons showed a correlation between increase in expression of tendon related proteins SCX, TNMD, Decorin and expression of ion channels KCNK2, TRAAK and TRPV1. Collectively, these data clearly illustrate that scaffolds made of PVDF-TrFE can produce electrical charges when mechanically loaded. Moreover, gene and protein analyses showed a positive regulation of tendon specific markers through activation mechanosensitive voltage-gated genes.

For any figures or tables, please contact authors directly.

Introduction

Periprosthetic infections are leading causes of revision surgery resulting in significant increased patient comorbidities and costs. Considerable research has targeted development of biomaterials that may eliminate implant-related infections.¹ This in vitro study was developed to compare biofilm formation on three materials used in spinal fusion surgery – silicon nitride, PEEK, and titanium – using one gram-positive and one gram-negative bacterial species.

Spinal fusion is one of the most common surgical procedures in spine surgery, whose primary objective is the stabilization of the spine for the treatment of many degenerative, traumatic and oncological diseases of the spine. Autologous bone is still considered the “gold standard” technique for spinal fusion. However, biomaterials which are potentially osteogenic, osteoinductive and osteoconductive can be used to increase the process of spinal fusion. We evaluated two new bone substitutes as an alternative to autologous bone for spinal fusion, using an animal model of large size (adult sheep).

A preclinical study was designed to compare the efficacy of SINTlife® Putty and DBSINT® biomaterials with conventional bone autograft in an ovine model of lumbar spine fusion. SINTlife® is a biomaterial made from hydroxyapatite enriched with magnesium ions, resulting to be very similar to natural bone. DBSint® is a paste composite bone, osteo-inductive, pliable and conformable, consisting of demineralized bone matrix (DBM) carried by hydroxyapatite biomimetics. Eighteen adult female sheep were selected for two-levels spine surgical procedures. The animals were divided in two groups: in Group A, one fusion level was treated with SINTlife® Putty and the other level received cortical-cancellous bone autograft; in Group B, one fusion level was treated with DBSINT® and the other level received cortical-cancellous bone autograft. At the end of the experimental time, all the animals were euthanized. The spine segments were analyzed macroscopically, radiographically, microtomographically, histologically and histomorphometrically.

The SINT-Life® Putty shows a perfect osteointegration in all the histological specimens. A high percentage of newly formed bone tissue is detected, with lots of trabeculae having structure and morphology similar to the pre-existing bone. In all the specimens collected from DBSINT®-treated animals the presence of hydroxyapatite alone is reported but not the demineralized bone matrix. The presence of newly formed bone tissue can be detected in all the specimens but newly formed bone shows very thin and irregular trabeculae next to the cartilage zone, while away from the border of ossification there are thicker trabeculae similar to the pre-existing bone.

The use of the experimental biomaterial SINT-Life® Putty in an ovine model of spine fusion leads to the development of newly formed bone tissue without qualitative and quantitative differences with the one formed with autologous bone. The experimental material DBSINT® seems to lead to less deposition of newly formed bone with wider intertrabecular spaces. Following these results, we planned and submitted to the Ethical Committee a clinical study to evaluate the safety and efficacy of SINT-Life® product in comparison to autologous bone, as an alternative treatment for spine fusion procedures.

Extensive annulus fibrosus (AF) radial tears lead to intervertebral disc (IVD) herniation. While unrepaired defects in the AF are associated with postoperative reherniation and high IVD degeneration prevalence, current surgical strategies are limited to symptomatic treatment of pain and disregard the structural integrity of the AF. For all these reasons, this study is focused on i) designing polycaprolactone (PCL) electrospun implants that mimic the multi-lamellar fibrous structure of the native tissue and ii) assessing their ability to properly close and repair an AF defect in a sheep in vivo model. Oriented PCL mats were produced by electrospinning with average fiber diameters of 1.3µm and a tensile modulus (55±1MPa) matching the one of a native human AF lamella (∼47MPa). In vitro experiments demonstrated a spontaneous colonization of PCL mats by human and ovine AF cells. In vivo study was carried out on 6 sheep in which 5 lumbar discs were exposed using a left retroperitoneal approach. Defects (2×5mm, 2mm depth) were created in the outer annulus, with randomized distribution of conditions including 10-layer oriented or non-oriented mats, untreated and healthy groups. X-ray and MRI examinations were performed every month until explantations at 1, 3 and 6 months, followed by immuno-histological analysis. Data showed no dislocation of the implants, cell infiltration between the PCL mats and within the mats, and a continuous type I collagen tissue formation between the implants and the surrounding AF tissue. These results highlight that multi-layer PCL electrospun mat is a promising biomaterial for AF repair.

Total ankle replacement (TAR) is contraindicated in patients with significant talar collapse due to AVN and in these patients total talus body prosthesis has been proposed to restore ankle joint. To date, five studies have reported implantation of a custom-made talar body in patients with severely damaged talus, showing the limit of short-term damage of tibial and calcaneal thalamic joint surfaces. Four of this kind of implants have been performed. The first two realized with “traditional” technology CAD-CAM has been performed in active patients affected by “missing talus” and now presents a survival follow-up of 15 and 17 years. For the third patient affected by massive talus AVN we designed a 3D printed porous titanium custom talar body prosthesis fixed on the calcaneum and coupled with a TAR, first acquiring high-resolution 3D CT images of the contralateral healthy talus that was “mirroring” obtaining the volume of fractured talus in order to provide the optimal fit. Then the 3D printed implant was manufactured. The fourth concern a TAR septic mobilization with high bone loss of the talus. The “two-stage” reconstruction conducted with the implant of total tibio-talo-calcaneal prosthesis “custom made” built with the same technology 3D, entirely in titanium and using the “trabecular metal” technology for the calcaneous interface. Weightbearing has progressively allowed after 6 weeks. No complications were observed. All the implants are still in place with an overall joint mobility ranging from 40° to 60°. This treatment requires high demanding technical skills and experience with TAR and foot and ankle trauma. The 15 years survival of 2 total talar prosthesis coupled to a TAR manufactured by a CAD-CAM procedure encourages consider this 3D printed custom implant as a new alternative solution for massive AVN and traumatic missing talus in active patients.

The development of functional biomaterials scaffolds for bone tissue engineering applications includes the control of specific biological and mechanical parameters that are involved in the growth of bone tissue in a way that mimics the physiological process of healing bone defects. Here, we report on the development of composite scaffolds made from biodegradable natural and synthetic biomaterials with characteristic architectural features, functionalized with the osteoinductive growth factor bone morphogenetic protein BMP-2, and evaluating their osteogenic response in static and dynamic cell culture systems. The results show that scaffold designing with advanced technologies combined with appropriate biochemical and mechanical stimulating factors, results to an enhanced proliferative and osteogenic/chondrogenic differentiation response of cells cultured on the developed scaffolds, and thus controlling the new tissue formation and reconstruction.

Calcium phosphate ceramics and bioactive glasses are frequently used in orthopedic surgery to stimulate the regeneration of bone tissue due to their superior compatibility to bone tissue. Nevertheless, the brittleness and lack of self-healing behavior of bioceramics are still considered as serious drawbacks. Therefore, these bioceramics have been combined with organic biomaterials for several decades. Since the 1990s, the emergence of nanotechnology has accelerated the progress with respect to the development of organic-inorganic nanocomposites of improved functionality compared to conventional composite biomaterials. This presentation focuses on the development of injectable (nano)composites with self-healing and/or load-bearing capacity. To this end, the affinity between polymeric and inorganic components was tuned by modifying non-covalent interactions between both composite components. Specifically, we exploited reversible interactions between hydrogel matrices and inorganic nanoparticles (traditional nanocomposites), hydrogel nanoparticles and inorganic nanoparticles (colloidal nanocomposites), as well as fibers and bioceramic matrices (fiber-reinforced cement composites). The resulting composite biomaterials were mechanically strong and self-healing, which may open up new avenues of research on the applicability of self-healing and load-bearing composite biomaterials for regenerative medicine.

Introduction

The complex cellular mechanisms of the aseptic loosening of total joint arthroplasties still remain not completely understood in detail. Especially the role of adherent endotoxins in this process remains unclear, as lipopolysaccharides (LPS) are known to be very potent modulators of the cell response on wear particle debris. Contributing factors on the LPS affinity of used orthopedic biomaterials as their surface roughness have to be investigated. The aim of this study was to evaluate the affinity of LPS on the surface roughness of different biomaterials in vitro. The hypothesis of the study was that rough surfaces bind more LPS than smooth surfaces.

Background

Definitive proof is lacking on mesenchymal stem cell (MSCs) cellular therapy to regenerate bone if biological potential is insufficient. High number of MSCs after GMP expansion may solve the progenitor insufficiency at the injury but clinical trials are pending.

Although there is some clinical evidence of ceramic bearings being associated with a lower infection rate after total hip arthroplasty (THA), available data remains controversial since this surface is usually reserved for young, healthy patients. Therefore, we investigated the influence of five commonly-used biomaterials on the adhesion potential of four biofilm-producing bacteria usually detected in infected THAs.

In this in-vitro research, we evaluated the ability of S. aureus, S. epidermidis ATCC 35984, E. coli ATCC 25922 and P. aeruginosa to adhere to the surface of solid biomaterials, including a 28mm cobalt-chromium metal head, a 28mm fourth-generation ceramic head, a 48mm fourth-generation ceramic insert, a 48mm highly-crossed linked polyethylene insert and a 52mm titanium porous-coated acetabular component. After an initial vortex step, a bacterial separation from the surface of each specimen was done until no remaining attached bacteria were observed by digital optical microscope. The colony-forming units were counted to determine the number of viable adherent bacteria and the bacterial density.

We found no differences on global bacterial adhesion between the different surfaces. E. coli presented the least adherence potential among the analysed pathogens (p<0.001). The combination of E. coli and S. epidermidis generated an antagonist effect over the adherence potential of S. epidermidis individually (58±4% vs. 48±5%; p=0.007). The combination of P. aeruginosa and S. aureus presented a trend to an increased adherence of P. aeruginosa independently, suggesting an agonist effect (71% vs. 62%; p=0.07).

In this study, ceramic bearings appeared not to be related to a lower bacterial adhesion than other biomaterials. However, different adhesive potentials among bacteria may play a major role on infection's inception.

Cite this article: Bone Joint Res 2023;12(5):311–312.

Among plant derived molecules, polyphenols have antioxidant, anticancer and antibacterial ability [1,2]. Moreover, they can stimulate osteoblast differentiation and promote apoptosis of tumoral cells [3–4]. It's thus possible combine the properties of these molecules with those of bioactive materials trough surface functionalization.

A silica-based bioactive glass and chemically treated bioactive Ti6Al4V were used as substrates while gallic acid and polyphenols extracted from green tea or red grape skin as biomolecules for functionalization. The surface functionalization procedure was optimized in order to maximize the grafting and investigated by means of the Folin&Ciocalteu method and X-Ray Photoelectron Spectroscopy (XPS) analyses. The in vitrobioactivity was studied by means of Field Emission Scanning Electron Microscopy (FESEM) and Fourier Transform Infrared Spectroscopy (FTIR) after soaking in simulated body fluid (SBF).

Surface charge and isoelectric point were investigated by means of zeta potential measurements. Free radical scavenging activity evaluation was performed in order to investigate the antioxidant ability of glass samples. Finally, the functionalization selective killing activity towards osteosarcoma cells was in vitroassayed by the metabolic 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) test and compared with non-tumoral control bone cells.

The presence of polyphenols on the surfaces was confirmed by XPS analyses by the appearance of characteristic peaks (C-O and C=O bonds) in the carbon and oxygen regions. The Folin&Ciocalteu test demonstrated the presence and activity of polyphenols on all the substrates and evidenced a clear relation between surface reactivity and grafting ability. The bioactivity tests showed the deposition of hydroxyapatite on the functionalized samples and an influence of biomolecules on its amount and shape for glasses. Zeta potential measurements evidenced a shift of the isoelectric point of glass samples after functionalization. A certain antioxidant activity of bare glass has been evidenced and it is improved by the grafting of tea polyphenols. Accordingly, MTT results confirmed polyphenols selective killer activity towards osteosarcoma cells whose viability was significantly decreased in comparison with safe bone cells.

XPS analyses, zeta potential measurements and Folin&Ciocalteu tests showed the presence and the activity of the polyphenols on the surfaces. Bioactivity tests highlighted an improvement of the deposition of hydroxyapatite on the surface of the functionalized glass samples. Certain antioxidant ability has been evidenced for glass samples and was further improved by tea polyphenols. Moreover, a selective toxic activity towards tumor cells was in vitropreliminary confirmed.

In conclusions polyphenols were successfully grafted to the surface of glass and Ti6Al4V samples maintaining their activity. Polyphenols improve in vitro bioactivity, antioxidant and anticancer ability of glass. The surface functionalization seems to be a good way to combine the properties of bioactive materials for bone contact applications with those of polyphenols.

INTRODUCTION. A new class of soybean-based bio-materials has been presented to the scientific community (patent PCT/GB01/03464) which shows good mechanical properties and an intrinsic anti-inflammatory potential, probably related to the phyto-hormone Genistein. This plant isoflavone is also reported to inhibit osteo-clastic activity.

MATERIALS AND METHODS. De-fatted soybean curd was prepared into granules which were subsequently implanted in a cylindrical cavity drilled into the femural canal of New Zealand White rabbits. Retrieved femurs were embedded in poly-methyl-meta-acrylate and samples were analyzed by back-scattered electron microscopy (BSEM).

RESULTS. Retrieved operated femurs showed a macroscopic appearance similar to the non-operated controls. BSEM showed that granules were still present at the site of implantation after 8 weeks, but a clear progressive degradation took place from the periphery to the centre of the femural canal already after 3 weeks. The degradation of the granule was accompanied by the production of new trabeculae apposed to the surface of the material.

CONCLUSIONS. It can be hyothesised that the released Genistein shifts the metabolic balance towards bone production by inhibiting the macrophagic and osteo-clastic activities and that the material degrading surface supports the apposition and mineralization of the newly-formed bone.

The most challenging complications in orthopaedic trauma surgery are fracture-related infections (FRI). The incidence ranges from approximately 1% after closed fractures or joint replacement, to more than 30% in complex open limb fractures. Despite tremendous efforts with prolonged antibiotic therapy and multiple revision surgeries, these complications are associated with considerable rates of recurrent infections as well as permanent functional impairment. The primary aim for the clinician is to prevent infection, because once established, an infection is difficult to eradicate. The main reason for this is biofilm formation on the implanted device, which allows pathogens to protect themselves from host immune response and antimicrobial therapy. In open fractures with a considerable wound contamination and soft- tissue damage, systemically-delivered antibiotics may not reach sufficient local concentrations to eradicate the bacteria. Locally delivered antibiotics can overcome this problem by providing high local concentrations. Currently, several antibiotic loaded biomaterials for local infection prophylaxis and/or treatment are available. In this talk, next to the diagnostic challenges of FRIs, the currently available antimicrobial-loaded biomaterials will be described. Against a backdrop of increasing infection and antimicrobial resistance, the prudent use and availability of such materials will become even more important.

Aims: A new class of soybean-based biomaterials has been presented (patent PCT/GB01/03464) which shows good mechanical properties and an intrinsic anti-inflammatory potential, probably related to the phyto-hormone Genistein. This plant isoflavone is also reported to inhibit osteoclastic activity. Aim of this study is to evaluate in-vivo the bone response to such soybean-based biomaterials.

Methods: De-fatted soybean curd was prepared into granules which were subsequently implanted in a cylindrical cavity drilled into the femural canal of New Zealand White rabbits. Retrieved femurs were embedded in poly-methyl-methacrylate and samples were analyzed by back-scattered electron microscopy (BSEM).

Results: Retrieved operated femurs showed a macroscopic appearance similar to the non-operated controls. BSEM showed that granules were still present at the site of implantation after 8 weeks, but a clear progressive degradation took place from the periphery to the centre of the femural canal already after 3 weeks. The degradation of the granule was accompanied by the production of new trabeculae apposed to the surface of the material.

Conclusions: It can be hypothesised that the released Genistein shifts the metabolic balance towards bone production by inhibiting the macrophagic and osteo-clastic activities and that the material degrading surface supports the apposition and mineralization of newly-formed bone.

Resorbable porous ceramics derived from chemically converted corals have been used successfully as bone graft substitutes for many years. Converted corals provide a 3D porous architecture that resembles cancellous bone with a pore diameter of 200–700 μm. The success of these corals as a bone graft substitute relies on vascular ingrowth, differentiation of osteoprogenitor cells, remodelling and graft resorption occurring together with host bone ingrowth into the porous microstructure or voids left behind during resorption. The resorption rate of the coral can be controlled by partial conversion to provide a hydroxyapatite (HA) layer via thermal modification. This study examined the resorption rates and bone formation of partially converted corals in a bilateral metaphyseal defect model.

Bilateral defects (5 mm x 15 mm) were created 3 mm below the joint line in the proximal tibia of 41 skeletally mature New Zealand white rabbits following ethical approval. Two variations of a calcium carbonate–HA coral (Pro Osteon 200 R, Interpore-Cross International, Irvine, CA) were examined with different HA thickness (200R; 14% or 200 RT; 28%). Empty defects (negative control) or defects filled with morcellised bone autograft from the defect sites (positive control) were performed. The tibiae were harvested at 6, 12, 24, 36 or 52 weeks, radiographed (standard x-rays and faxitron) in the anteroposterior and lateral planes. Tibias were processed for torsional testing and quantitative histomorphometry using back scattering scanning electron microscopy. Four additional rabbits were killed at time zero to determine the mechanical properties of the intact tibia (n=6 tibias) and 2 for tibias for time zero histomorphometry. Data were analysed using a 3-way analysis of variance.

No clinical complications were encountered in this study. Radiographic assessment revealed a progression in healing, implant resorption and bone infiltration. Cortical closure in the 200 R and 200RT treated defects was noted by 24 weeks. All specimens failed in torsional testing with a spiral fracture initiating at the distal defect site and extending into the distal diaphysis. Torsional properties reached intact control tibia levels by 24 weeks in both groups. No significant differences were noted between 200 R and 200 RT based on torsional data. SEM revealed progressive resorption of the calcium carbonate core of the 200 R and 200 RT with time, infiltration of bone and ingrowth to the HA layers. Time and measurement site (cortical versus cancellous) were significant for implant resorption, bone, and void. The thinner HA layer (200 R) resorbed more quickly compared to the thicker layer (200 RT) in the canal as well as cortical sites. Increased bone and decreased void were noted at the cortex measurement sites in the 200 R group at 24 weeks and in the 200 RT group at 12 and 24 weeks (p< 0.05). Implants were nearly completely resorbed by 52 weeks with only a few percent of implant remaining.

Osteochondral lesions are frequent as a result of sport and daily activities.

The healing processes of these defects are prolonged and complicated and often leading to irreversible ostheo-arthritic changes. In this study, biotechanical and bioChemical approaches are being combined in an attempt to identify potential uses of biofabricated marine carbonate materials in biomedical applications, particularly as for remodeling cartilage and bone tissue. Biofabricated material was grafted into osteochondral induced defects in animals’ models during knee arthrotomy. Using histological sections, SEM, EDS studies it was revealed that the biofabricated, porous material is highly biocompatible. The graft was incorporated into the osteochondral defect area and followed by surface remodeling. After 4 months the interface and subchondral areas were been replaced by new cartilage and bone.

We believe that it is the first time that such biofabricated materials have been used for biomedical purposes. In face of the obvious environmental disadvantages of harvesting from limited natural resources, we propose the use of bio-engineered coralline and other materials such as those cultured by our group under field and laboratory conditions as a possible biomatrix for hard tissue remodeling.

Introduction

Due to its remarkable stoichiometric flexibility and surface chemistry, hydroxyapatite (HAp) is the fundamental structural material in all vertebrates. Natural HAp's properties inspired an investigation into silicon nitride (Si₃N₄) to see if similar functionality could be engineered into this bioceramic. Biological and in situ spectroscopic analyses were used to monitor the response of osteosarcoma cells (SaOS-2) to surface-modulated Si₃N₄ and a titanium alloy after long-term in vitro exposure.

Degeneration of intervertebral disc (IVD) Nucleus Pulposus (NP) is a major cause of low back pain (LBP). Healthy NP contains two cell types: notochordal cells (NTC) and nucleopulpocytes (NPCytes). While NTC are embryonic notochord derived cells that are regarded as the resident stem cells of NP, NPCytes are considered the mature NP cells responsible for extracellular matrix (ECM) synthesis. During IVD aging, some still unknown cues drive NTC disappearance. This loss of NTC alters their dialog with NPCytes thereby jeopardizing cell viability and ECM homeostasis, which in turn drives NP degeneration. In this context, NP regeneration by re-establishing this NTC/NPCytes dialog has been contemplated with clinical interest. We will first share our view of the mesenchymal stem cells (MSC)-based therapies that have been preclinically and clinically assessed in LBP. We will then comment on the biomaterial-assisted MSC therapies that recently enter the scene of IVD regeneration. Finally, we will present our REMEDIV project that aims at developing a NP substitute containing stem cells-derived NPCytes and NTC within an injectable hydrogel. We will share our results regarding the generation of NPCytes from adipose-derived MSC and our recent unpublished evidences that human induced-pluripotent stem cells can be differentiated into NTC. Finally, we will consider our ability to transplant these regenerative cells using hydrogels in various animal models. Whether this concept could open new therapeutic windows in the management of discogenic low back pain will finally be discussed.

Aims: The aim of the research is the functionalization of biosurfaces by anchoring on them biomolecules involved in the process of osteointegration (cellular adhesion, proliferation, differentiation, migration, matrix mineralization). Alkaline phosphatase (ALP) was used as model protein, because it is involved in the mineralization processes. The functionalized surfaces are biomimetic, because they show the biological signals triggering new tissue generation. A rapid osseointegration are the final goal and a good response and fast healing of bad quality bones is one of the main issues. The devices of interest for the research are dental or orthopaedic implants and substitutes of small bones.

Methods: Bioactive glasses of various compositions were employed as substrates. Bioactive glasses, when in contact with biological fluids, stimulate the precipitation of a hydroxyapatite layer on their surfaces, which in turn promotes effective osteointegration of the implant. Since bioactive glasses are prone to hydroxylation, they could be successfully functionalized and grafted by biomolecules. So the biomimetic materials considered will be bioactive both from a physicochemical (osteoconduction and apatite precipitation) and from a biochemical (osteoinduction) point of view. The research was focused first of all on the methods for developing active sites on the substrates. In the case of bioactive glasses the surface must be cleaned of any contaminants and the reactive hydroxyls activated.

Results: The immobilization of ALP was performed both with and without spacer molecules and a comparison among the different techniques will be presented. XPS was used for the analysis of the immobilized enzyme on titanium and bioglasses and specific signals for its identification were set. After the addition of the specific substrate, the ALP activity was evaluated by UV-VIS spectroscopy.

Conclusions: ALP was successfully grafted on the surface of bioactive glasses with and without the use of an intermediate layer of spacer molecules. The presence of ALP was determined on all the samples, as well as its enzymatic activity. Further analyses are necessary to evaluate the opportunity of using a spacer molecule. Cell adhesion and proliferation tests are in progress.

Although bovine serum is the lubricant recommended by several international standards for the wear testing of orthopedic biomaterials there are issues over its use. The inherent batch variation in protein content means that two bovine serum lubricants can give different wear rates. Due to degradation, the lubricant needs to be changed regularly, so that any third body wear particles are removed, thus potentially influencing wear regimes. There are also cost and safety issues with the use of bovine serum. For these reasons, alternative lubricants were investigated.

A 50-station wear test rig was used, which applied multi-directional motion to each ultra-high molecular weight polyethylene (UHMWPE) test pin. Each pin articulated against a cobalt chrome plate polished to better than 0.05 microns Ra. The following lubricants were used: 50% dilute bovine serum; soy protein; olive oil; wheatgerm oil; soya oil; albumin and globulin (AG) mix; albumin, globulin and chondroitin sulphate (AGC) mix; whole milk; Channel Island milk; 11 mg/ml protein egg white; 20 mg/ml egg white; and 40 mg/ml egg white. A minimum of 6 UHMWPE pins per lubricant were wear tested and the tests ran to 2.5 million cycles. Gravimetric measurements were taken throughout the test to determine the volume of wear and at the end of the test the samples were examined using a SEM.

The lubricants giving the closest results to bovine serum were 20 and 40 mg/ml egg white, with mean UHMWPE total wear volumes of 17.4 mm3 and 17.8 mm3 compared to bovine serum which gave 20.7 mm3. Surface topographies showed similar features too. The 11 mg/ml egg white lubricant and the AG and AGC lubricants were next closest in terms of wear. An UV absorbance assay found that all the protein based lubricants suffered from a high degradation rate, and the rate increased with increasing protein content.

Egg white may offer a less expensive alternative to dilute bovine serum as a test lubricant although it is likely that it too would need to be changed as regularly as bovine serum.

Replacement of damaged or diseased tissues with permanent metal implants based on stainless steel, cobalt chrome and titanium alloys has been at the forefront of classical biomaterials research and the orthopaedic medical device industry for decades. Biodegradable polymers have also reached the market but often have limited capacity in load bearing orthopaedic applications due to their low stiffness and poor mechanical properties. The development of biodegradable metals based on magnesium (Mg) could be heralded as a major breakthrough in the field of orthopaedic surgery. Degradable implants eliminate the time and cost associated with a secondary surgery to remove hardware, and reduces the period the implant is exposed to instability, fibrous encapsulation, stress shielding and inflammation. The metabolism of Mg and its excretion via the kidneys is a natural physiological process that is well understood, however, controlling the rapid degradation of Mg biomaterials in vivo is a major challenge yet to be resolved for the safe and effective use of Mg in orthopaedic implants.

In this study, we describe a novel manufacturing method for fabricating Mg/Mg alloy implants, as well as the development of an in vitro method for screening Mg/Mg alloy degradation rate by considering both their electrochemical corrosion behaviour and biological characteristics.

A range of Mg alloys with varying amounts of calcium (0.8–28%) and zinc (3–10%) were cast and then machined into Ø4mm and 15mm discs for biocompatibility (HETCAM) and parallel in vitro testing. Alloys were placed in various simulated body fluid (SBF) solutions in vitro (7–28 days) to determine effect of alloy composition on degradation rate. These potentiostatic and potentiodynamic tests were designed to simulate, to varying degrees, the in vivo environment, with the crucial factors (e.g. temperature, pH, serum proteins, CO2 level) controlled to ensure consistency across the test methods. The mechanisms of corrosion on the Mg/Mg alloy microstructure and the effect of protein adsorption all played key roles in dictating the corrosion of alloys in vitro. Specifically the inclusion of physiological levels of serum proteins decreased the corrosion rate up to 600% over more standard SBF solutions described in literature.

This work provides an improved understanding of the effects of corrosion variables on Mg alloys, while making major steps towards deciding the most appropriate screening tests for new alloys for their use as a biomedical material prior to moving to in vivo animal studies.

Aim

The primary aim of this in vitro study was to test the efficacy of daptomycin to eradicate staphylococcal biofilms on various orthopedic implant surfaces and materials. The secondary aim was to quantitatively estimate the formation of staphylococcal biofilm on various implant materials with different surface properties.

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.

A new class of soybean-based biomaterials has been presented to the scientific community (patent PCT/GB01/03464) that shows good mechanical properties and an intrinsic anti-inflammatory potential, probably related to the phyto-hormone Genistein. This plant iso-flavone is also reported to inhibit osteoclastic activity.

De-fatted soybean curd was prepared into granules which were subsequently implanted in a cylindrical cavity drilled into the femoral canal of New Zealand White rabbits. Retrieved femurs were embedded in polymethyl-meta-acrylate and samples were analysed by back-scattered electron microscopy (BSEM). Retrieved, operated femurs showed a macroscopic appearance similar to the non-operated controls. BSEM showed that granules were still present at the site of implantation after 8 weeks, but a clear progressive degradation took place from the periphery to the centre of the femural canal already after 3 weeks. The degradation of the granule was accompanied by the production of new trabeculae apposed to the surface of the material.

It can be hypothesised that the released Genistein shifts the metabolic balance towards bone production by inhibiting the macrophagic and osteoclastic activities and that the material degrading surface supports the apposition and mineralisation of the newly formed bone.

Biodegradable porous scaffolds play an important role in tissue engineering as the temporary templates for transplanted cells to guide the formation of the new organs. The most commonly used porous scaffolds are constructed from two classes of biomaterials. One class consists of synthetic biodegradable polymers such as poly (α-hydroxy acids), poly(glycolic acid), poly(lactic acid), and their copolymer of poly(DL-lactic-co-glycolic acid) (PLGA). The other class consists of naturally derived polymers such as collagen. These biomaterials have their respective advantages and drawbacks. Therefore, hybridization of these biomaterials has been expected to combine their advantages to provide excellent three-dimensional porous biomaterials for tissue engineering. Our group developed one such kind of hybrid biodegradable porous scaffolds by hybridizing synthetic poly (α-hydroxy acids) with collagen. Collagen microsponges were nested in the pores of poly (α-hydroxy acids) sponge to construct the poly (α-hydroxy acids)-collagen hybrid sponge.

Observation by scanning electron microscopy (SEM) showed that microsponges of collagen with interconnected pore structures were formed in the pores of poly (α-hydroxy acids) sponge. The mechanical strength of the hybrid sponge was higher than those of either poly (α-hydroxy acids) or collagen sponges both in dry and wet states. The wettability with water was improved by hybridization with collagen, which facilitated cell seeding in the hybrid sponge. Use of the poly (α-hydroxy acids) sponge as a skeleton facilitated formation of the hybrid sponge into the desired shapes with high mechanical strength, while collagen microsponges contributed good cell interaction and hydrophilicity. One of such kind of hybrids. Additionally, our group developed a hydrostatic pressure bioreactor for chondrocyte culture. And our study showed that hydrostatic pressure (0–3 MPa) had promotional effects on the production of proteoglycan and type II collagen by cultured chondrocytes. Therefore, it would be a promising pathway for reconstructing cartilage-like tissue to culture chondrocytes in this three-dimensional hybrid sponge under physiological hydrostatic pressure.

Introduction: The evolution of biomaterials has helped immensely the quality of reconstruction as well as the progress of medical specialties. The evolution of biomaterials for reconstruction has helped creating ones that are absorbable, look alike human tissues and contribute to daily activities of human body. Synthetic bone grafts, metal substitutes of bones and complex special tissues are the current therapeutic practice.

Material and methods: In this presentation maxillofascial reconstructions will be shown where Orthopaedic biomaterials have been successfully used.

Patients with large bone cysts that have been treated with or without grafting and titanium or stainless steel plates.

Patients with large bone defects of the mandible that were filled with bone grafts or absorbable membranes Patients with Ca of the mandible reconstruction with fibular graft and osteosynthesis.

Conclusion: The evolution of new biomaterials as well as the experience of Orthopaedic surgery has proved the essential step of contemporary Maxillofascial surgery.

Bone grafts are crucial for the treatment of bone defects caused by tumor excision. The gold standard is autograft but their availability is limited. Allografts are an alternative, but there is a risk of rejection by the immune system. The tissue engineering field is trying to develop vascularized bone grafts, using innovative biomaterials for surgery applications. While the gold standard in bone graft in dentistry is the use of decellularized bovine bone particles (Bio-Oss®), our work has produced a polysaccharide-based composite matrix (composed of PUllulan, DextraNand particles of HydroxyApatite (PUDNHA), as a new scaffold for promoting bone formation and vascularization of the tissue. In the context of bone tissue regeneration, the function of osteoblast and endothelial cells has been extensively studied, while the impact of osteocytes has been regarded as secondary. Nonetheless, the osteocytes represent 90–95% of bone cells and are responsible for orchestration of bone remodeling.

Here, we propose an original method to analyze the interaction between bone and biomaterials, after in vivo implantation of the matrix PUDNHA in an experimental sheep model. Our objectives are to analyze the network established by osteocytes in the newly formed tissue induced by the matrix, as well as their interactions with the blood vessels.

Sheep have been implanted with the Bio-Oss® or the PUDNHA using the sinus lift technique. After 3 (3M) and 6 months (6M), the animals were euthanazied and the explants were fixed, analyzed by X-ray, embedded in Methylmetacrylate/Buthylmetacrylate and analyzed histologically by Trichrome staining. Thereafter, the samples (n=3/group) were polished using different sand papers. A final polish was realized using a 1µm Diamond polishing compound. The blocks were incubated 10 or 30s with 37% phosphoric acid to remove the mineral on the surface, then dipped in 2.6% sodium hypochlorite to remove the collagen. The samples were air dried overnight, metallized with Gold palladium the following day, before being imaged with a SEM.

As expected, PUDNHA activates bone regeneration in this sinus lift model after 3M and 6M. X-ray analysis and histological data revealed more bone regeneration at 6M versus 3M in both groups. With this acid eching technique, we were able to visualize the interface of bone with the biomaterials. This treatment coupled with SEM analysis, confirmed the increase of bone formation with time of implantation in both groups. In addition, SEM images revealed that osteocyte alignment and their network were different in the new regenerated bone compared to the host bone. Moreover, images showed the direct contact of the osteocytes with the blood vessels formed in the new regenerated bone.

This acid eching technique can be useful in the field of biomaterials to see the relationship between cells, blood vessels and the material implanted and understand how the new bone is forming around the different biomaterials.

Bacterial infections related to orthopaedic implants is one of the serious types of complications. Recently, there has been a greater interest in antibacterial biomaterials. However, antibacterial evaluations of each material are inconsistant, so intercomparison of the antibacterial performance is difficult. This study focused on the Japanese Industrial Standards test (JIS Z2801), which is used for antibacterial evaluation of commodities. The study investigated a suitable evaluation method for in vitro antibacterial activity of biamaterials. In 2007, JIS Z2801 test was approved as international standard ISO 22196.

Hydroxyapatite (HA) powder containing 3 wt % of silver oxide (Ag) was sprayed on the surface of titanium disks with the thermal spraying method, using an acetylene torch. This coating has been proved to generate strong antibacterial activity in previous studies. The antibacterial activity was examined with the JIS Z 2801 test and modified JIS Z2801 test. The bacterial strains used in JIS Z2801 test were Escherichia coli (E.coli), Staphylococcus aureus (S.aureus). Bacterial culture medium was instilled onto the surface of the test disks (about 106 cells/ml) and covered with polystyrene films. After cultivation in 1/500 Nutrient Broth for 24 h at 35°C, the bacteria was washed out with the broth. The numbers of viable bacteria in the broth were counted with the agar plate culture method. Additionally, Modified JIS Z2801 test was performed. Modified points were added to the bacterial strain of biofilm-forming methicillin-resistant S.aureus (BF-MRSA), using Fetal Bovine Serum (FBS) as a culture medium, and cultivated at 37°C.

In the JIS Z2801 test, Antibacterial activity values of the HA-Ag disk were composed against E.coli 4.1 and S.aureus 5.0. In the modified JIS Z2801 test, antibacterial activity values against E.coli, S.aureus and BF-MRSA were 8.2, 5.5, and 7.1. When this value is greater than 2.0, it shows there is antibacterial activity. The titanium disk coated with HA-Ag showed antibacterial activity in both tests.

The JIS Z2801 test is designed to evaluate comodities in poor nutritional environment. However, the environment in the body is eutrophic. It is easy to make bacterial growth. For this reason, it is necessary to consider evaluating for biomaterials with suitable method considered in vivo. In this study, to examine the condition like that found in the body, we cultivated FBS at 37°C. In addition, the antibacterial activity against BF-MRSA was examined to consider the bacterial infection related to orthopaedic implants. The modified JIS Z2801 test showed that it is a suitable evaluation method for in vitro antibacterial activity of biomaterials.

Summary Statement

Description of an original in vitro protocol for assessing combined bacteria and cell competitive adherence on the surface of biomaterials of medical interest

Some different biodegradable vehicles have been tried in vitro and in vivo as possible methods of local antibiotic therapy.

The aim of this study is to evaluate the effectiveness of collagen-gentamicine (Collatamp^®) to eradicate bacterial colonisation of different biomaterials used in orthopaedic surgery in an in vitro study.

Three samples of similar shapes and dimensions of 4 different materials: stainless steel screw, titanium screw, titanium canulated screw and a cylinder of polyethylene were used. Three different solutions of 49 cc of thioglicolate plus 1cc of solution of methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis (MacFarland: 3) were prepared. Each solution received one sample of each material so that every material be tested in all 3 preparations. After incubation during one week and confirmation of bacterial colonisation of each sample by swabbing cultures, all of them were introduced in an individual receptacle containing 50 cc of thioglicolate and a piece of 5x5 cm of collagen-gentamicine (corresponding to 650 mg/ml of gentamicine). After incubation we analysed results by new swabbing cultures of all samples.

All samples were highly contaminated with different bacteria before introducing them in thioglicolate with the piece of collagen-gentamicine. After one week all samples were free of bacteria.

This in vitro study demonstrates the effectiveness of collagen-gentamicine in order to eliminate colonisation of different biomaterials used in orthopaedic surgery by most frequent bacteria.

Introduction: This study tested the hypothesis that the use of biomaterials in distraction osteogenesis (DO) would reduce the treatment time and enhance bone formation in bone defect management.

Methods: A 1.0cm tibial shaft was removed in the left tibia of 36 rabbits. Rabbits were divided into three groups: Group A, the defect gap was reduced with the tibia shortened for 1.0-cm; Group B, the defect gap was filled with 1.0-cm porous hydroxyapatite/tri-calcium phosphates cylindrical block (HA/TCP block, diameter 0.5-cm); Group C, The 1.0-cm defect gap was reduced 0.5cm and the remaining 0.5-cm defect gap was filled with 0.5-cm HA/TCP block. The tibia was then fixed with unilateral lengthener; for groups A and C, lengthening started 7 days after surgery at a rate of 1.0 mm/day, in two steps. Group A received lengthening for 10 days and Group C for 5 days, there was no lengthening for Group B. All animals were terminated at day 37 following surgery. The excised bone specimens were subject to micro-CT, mechanical testing and histological examinations.

Results: Bone mineral density and content and tissue mineral density and content, as well as the mechanical properties of the regenerates were significantly higher in Group C compared to Groups A and B. Micro CT and histological examinations also confirmed that the regenerates in Group C had most advanced bone formation, consolidation and remodeling compared to other groups.

Conclusion: The combined use of biomaterials and DO technique can reduce the treatment time and enhance bone consolidation in bone defect management.

Introduction and Objectives: In this study we attempted to analyze the biocompatibility of 3 different materials used in Orthopedic Surgery, Titanium, Tantalum and Hydroxyapatite, measuring their adsorption of fibrinogen by means of real time in vitro studies quantifying the mass of fibrinogen that becomes deposited on them.

Materials and Methods: We assessed fibrinogen adsorption on these biomaterials by means of quantitative studies using the QCM-D system and qualitative studies reviewing fibrinogen adhesion, by atomic microscopy. For the statistical analysis of the results of this study we used SSP 14.0.

Results: The results of the measurement of fibrinogen adsorption on the biomaterials used proved that when low concentrations of fibrinogen were used, 33 ng/ml, Ti and Ha adsorbed fibrinogen relatively rapidly, but Ta does not. However, when we used high concentrations of fibrinogen, 100 ng/ml, Ta adsorption increased markedly. When concentrations greater than 1000 ng/ml of fibrinogen were used, Ta was the biomaterial that showed most fibrinogen adsorption.

Discussion and Conclusions: The adsorption of proteins from body fluids is the first biological response to the implantation of a biomaterial, and this protein adhesion will guide cell adhesion on the implanted biomaterial. In our in vitro study, carrying out measurements in real time, we saw that Ta was the material with greatest biocompatibility.

Currently available calcium silicate based ceramics pseudowollostonite (CaSiO3) ceramics are regarded as a potential bioactive material for bone tissue regeneration due to their osseointegration properties. A drawback of CaSiO3 ceramics is that they possess high dissolution rate, leading to a high pH value in the surrounding environment thereby affecting the biological activity of bone cells. We hypothesize that chemical modification of CaSiO3 ceramics will improve their physical and biological properties. The coordinated activities of osteoblasts (OB) and osteoclasts (OC) are critical for proper bone remodelling. Moreover, growing evidence indicate that vascular endothelial cells are involved in bone development and remodelling.

Present study aims at Chemically modifying CaSiO3 by incorporating zinc (Zn) and titanium (Ti) into their structure to develop novel materials Hardystonite (HT, Ca2ZnSi2O7) and Sphene (CaTiSiO5), respectively and to determine their effect on bone cells OB & OC and on endothelial cells.

It is well known that cell behaviour in a culture system is influenced by the physiochemical characteristics of the substrate. Human bone derived cells (HBDC) cultured on HT and Sphene supported the HBDC attachment (cells exhibited well defined cytoskeletal structure) showed characteristic features of cellular proliferation and differentiation. In addition, Zn and Ti incorporation into CaSiO3 supported the formation of mature, active and functional OC. Moreover, the modified bio-materials were found to be conducive to Human micro-vascular dermal endothelial cell growth. Our results suggest that HT and Sphene possessed an improved physical characteristics and enhanced biological activities of bone cells (OB & OC) and endothelial cells thus rendering it a potential material for bone tissue regeneration and coatings onto commonly used orthopaedic and dental implants.

Aims

Calcium sulphate has traditionally been used as a filler of dead space arising during surgery. Various complications have been described following the use of Stimulan bio-absorbable calcium sulphate beads. This study is a prospective observational study to assess the safety profile of these beads when used in revision arthroplasty, comparing the complication rates with those reported in the literature.

Aims: Stribeck analyses were performed using both unimplanted and carbon (C) implanted heads of alumina, zirconia, zirconia-toughened-alumina and stainless steel so as to study the influence of C implantation on the frictional behaviour of these orthopaedic bearing materials. Methods: The selected biomaterials were implanted using an ion dose of 1 and 2.5 x 10¹⁷ C ions/cm² (75 keV). Friction testing was carried out on unimplanted and C implanted heads using a Hip Joint Friction Simulator with aqueous solutions of carboxy-methyl cellulose (CMC). Results: Both the unimplanted and C implanted bearing couples displayed a similar trend, i.e. by increasing the viscosity of the CMC fluid, the friction factor was found to decrease due to the formation of a fluid film between both bearing surfaces. However, the friction factor for the treated couples at low viscosities was lower than that of their unimplanted counterparts, with a drop of approximately 10% for the steel-on- UHMWPE and a drop of up to 85% being observed in the friction between the ceramic-on-ceramic bearing couples. This decrease can be explained by ion beam smoothening of the treated surface. Conclusions: The results from this study indicate a beneficial reduction in the friction factor of the C ion implanted surfaces. These results indicate that the use of C ion implantation to modify the bearing surfaces of present-day orthopaedic implants may be an effective means of reducing detrimental wear debris at the bearing interface.

Maggot therapy as an ancient method is succesfully used for treatment of acute and chronic wound infections in traumatology and orthopaedics. In this study, for the first time, the influence of sterile maggot excretions of Lucilia sericata on Pseudomonas aeruginosa (PAO1) biofilm formation on three common used orthopaedic materials was investigated.

Sterile maggot excretions were collected according to a standardized method and the protein concentration was measured. The influence of the excretions on PAO1-biofilm formation was tested on comb-like devices, especially designed for these experiments, made from polyethylene, titanium and stainless steel. These combs were made to fit into a flat-bottom 96-wells microtiter plate. In the wells a suspension of PAO1-bacteria, nutrient medium and maggot excretions were pipetted. In the control wells, no excretions were added. Combs were placed in the wells and incubated for 24 hours at 37°C. The formed biofilms were stained in crystal violet and eluted with ethanol. The Optical Density (OD 595 nm) was read to quantify biofilm formation. The experiments were conducted with excretions from young maggots (Instar-1 maggots) and full grown maggots (Instar-3 maggots). All experiments were done in quadruplicate.

The following can be concluded: PAO1-biofilm formation is the strongest on polyethylene and the weakest on stainless steel. Sterile maggot excretions are effective at preventing initial biofilm formation (p≤0.013) as well as preventing additional accumulation after its initiation (p≤0.038). The excretions even cause a significant breakdown of an existing biofilm (p≤0.028). Excretions from full grown maggots are more effective than those from young maggots.

This study shows for the first time that sterile maggot excretions of Lucilia sericata inhibit biofilm formation, prevent its further grow and break down existing biofilms. While biofilm formation on orthopaedic materials is a severe complication, this experimental study could indicate a new treatment for biofilm formation on infected biomaterials.

Purpose of the study: The efficacy of a new oestrogeneration biomaterial should be demonstrated by in vivo grafts in animal models. Critical filling of bone defects in the rat could be useful as a model before beginning studies in large animals such as the sheep, goat or dog. Creation of a critical defect in the rat femur has been described, but not standardized, leading to difficult comparison between series. In this work, we wanted to establish a detailed standardisable surgical protocol for the creation of a 6 mm femur defect in the rat.

Material and methods: We compared three anaesthesia protocols using 18 mal Wistar rats aged 21 weeks. We developed a surgical procedure enabling study of the advantages of the different commonly used surgical devices either in research or clinic to achieve osteosynthesis and a 6 mm bone defect. We also compared two types of fixation plates (and screws) available on the marker: a 1.2 mm thick titanium plate used for hand surgery and a 1.5 mm steel plate (veterinary medicine). Our postoperative clinical and radiographic follow-up was designed to validate our operative protocol and evaluate osteoregeneration.

Results: We demonstrated first that the use of multimodal anaesthesia radically improved the clinical outcome in the animals. We then demonstrated that the 1.2 mm titanium plates recommended in other studies were too fragile in our model and that the steel 1.5 mm veterinary plates were more adapted. We finally demonstrated the superiority of surgical devises to create a defect and for osteosynthesis. We described a postoperative protocol offering satisfactory evaluation, clinically and radiographically.

Discussion: This work is the first describing this protocol in detail. Improvements in feasibility and cost will make a readily exploitable model for other laboratories. The follow-up on this work should be aimed at improving the quality and pertinence of the analysis methods for the assessment of bone regeneration.

Conclusion: We propose a mode for the critical defect in rat femur bone as a reliable model for the study of osteogenic capacities of new biomaterials.

Objectives

This systematic review aimed to assess the in vivo and clinical effect of strontium (Sr)-enriched biomaterials in bone formation and/or remodelling.

Aims:. To test the effect of different surface roughness and fluorohydroxyapatite (FHA) coating on osteoblast-like cell (MG63) viability, proliferation, differentiation and synthetic activity, then to compare the various surfaces tested and try to identify an osteoblast parameter that can better explain the different behaviour of the tested surfaces observed in previous in vivo studies.

Methods: The tested materials were made of Ti6Al4V coated with Ti and with Ti plus FHA with different roughness; they can be divided into four groups: low roughness (LR; Ra: 5.9 B5m), low roughness plus FHA coating (LR+FHA; Ra: 5.6 B5m), high roughness (HR; Ra: 22.5 B5m), high roughness plus FHA coating (HR+FHA; Ra: 21.2 B5m). MG63 were cultivated on 6 samples of each group and on polystyrene as control; after 72 hours the proliferation assay (WST-1) was done, alkaline phosphatase activity (ALP) was determined and the synthesis of osteocalcin (OC), type 1 collagen (CICP), transforming growth factor α 1 (TGF-A71), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-a) were measured. Samples of each material were randomly processed for analysis with a scanning electron microscope (SEM).

Results: Cells proliferated on biomaterials more slowly than in the control group (p < 0.0001), the proliferation rate was higher on FHA-coated LR than uncoated HR (p = 0.037). CICP production was positively affected by the LR surface (p = 0.001) as compared to controls, while it was significantly lower (p = 0.0001) in the HR surfaces. Compared to controls, LR and HR surfaces led to enhanced production of TGF-A71, further improved by FHA (FHA-coated LR: p = 0.007; FHA-coated HR p < 0.0001 respectively). ALP, OC, IL-6 levels were not significantly different from the controls

Conclusions: Results suggest that CICP production could be useful in predicting the in vivo osteointegration rate of biocompatible biomaterials observed in previous studies.

Aims: Biosynthetic scaffolds made of degradable bio-materials enriched with cultured cells holds promise for peripheral nerve repair after complex traumatic injuries. In the perspective of future transplantation applications, the aim of this study was to investigate how cultures of olfactory ensheathing cells (OECs), in particular neonatal olfactory ensheathing cells (NOBECs), grow up in vitro on degradable polymeric films made with polycap-rolactone matrices and multi-block polyesterurethane respectively. In addition, since several transplantation studies use green fluorescent protein (GFP) positive cells so that they can be easily located in the receiving tissues, the cDNA encoding for GFP was cloned in expression vector and transfected in NOBECs.

Methods: To characterize NOBECs we employed electron microscopy, immunohistochemistry, RT-PCR and western blotting analyses. Moreover the proliferative ratio of NOBECs and the ability of the cells to migrate in a three dimensional environment were evaluated under basal and experimental culture conditions. Finally, the GFP-positive NOBEC were seeded on two types of synthetic films and their behaviour was analyzed to determine cell adhesion, survival and proliferation.

Results: We examined the expression of glial markers and NRG1/ErbB system in the NOBEC cell line at RNA and protein level. Results showed that NOBECs express both glial markers (GFAP and S-100), ErbB receptors (ErbB1, ErbB2 and ErB3) and different isoforms of NRG1. NOBECs exhibited a remarkable proliferation activity and a high basal migration activity. GFP positive NOBECs showed no significant difference in their behaviour as compared to untransfected parental cells. Finally, both normal and GFP-NOBECs showed good cell adhesion, survival and proliferation properties when seeded on both films employed in this study.

Conclusion: Taken together, results of our study showed that the glial cell line has similar biochemical properties as primary cultures of OECs. Moreover, we showed that NOBECs survive, proliferate and migrate on two different types of synthetic films that were prepared in the perspective of build up nerve scaffolds. Therefore, our results indicated that the NOBECs produce growth-promoting proteins and possess regeneration-promoting capabilities that make them a potentially good transplant material to enhance axonal regeneration inside synthetic tubes used to bridge nerve lesion with substance loss.

Objectives

Recently, the field of tissue engineering has made numerous advances towards achieving artificial tendon substitutes with excellent mechanical and histological properties, and has had some promising experimental results. The purpose of this systematic review is to assess the efficacy of tissue engineering in the treatment of tendon injuries.

Hip implant retrieval analysis is the most important source of insight into the performance of new materials and designs of hip arthroplasties. Even the most rigorous in vitro testing will not accurately simulate the behavior of implant materials and new designs of prosthetic arthroplasties. Retrieval analysis has revealed such factors as the effects of gamma-in-air sterilisation of polyethylene, fatigue failure mechanisms of polymethylmethacrylate bone cement, fretting corrosion of Morse taper junctions, third body wear effects of both hard-on-hard and hard-on-soft bearing couples, and the effects of impingement of components on the full spectrum of bearing surfaces, none of which was predicted by pre-implantation in vitro testing of these materials and combinations. The temporal sequence of the retrieval process is approximately six years from first implantation through retrieval analysis, laboratory investigation, and publication of results, and thus, in addition to rigorous clinical evaluation, represents the true development and insight cycle for new designs and materials.

Titanium alloys are one of the most used for orthopaedic implants and the fabrication of them by 3D printing technology is a raising technology, which could effectively resolve existing challenges. Surface modification of Ti surfaces is often necessary to improve biocorrosion resistance, especially in inflammatory conditions. Such modification can be made by coatings based on hydrogels, like alginate (Alg) - a naturally occurring anionic polymer. The properties of the hydrogel can be further enhanced with calcium phosphates like octacalcium phosphate (OCP) as a precursor of biologically formed hydroxyapatite. Formed Alg-OCP matrices have a high potential in wound healing, delivery of bioactive agents etc. but their effect on 3D printed Ti alloys performance was not well known. In this work, Alg-OCP coated 3D printed samples were studied with electrochemical measurements and revealed significant variations of corrosion resistance vs. composition of the coating. The potentiodynamic polarization test showed that the Alg-OCP-coated samples had lower corrosion current density than simple Alg-coated samples. Electrochemical impedance spectroscopy indicated that OCP incorporated hydrogels had also a high value of the Bode modulus and phase angle. Hence Alg-OCP hydrogels could be highly beneficial in protecting 3D printed Ti alloys especially when the host conditions for the implant placement are inflammatory. AcThis work was supported by the European Union Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Actions GA860462 (PREMUROSA). The authors also acknowledge the access to the infrastructure and expertise of the BBCE – Baltic Biomaterials Centre of Excellence (European Union Horizon 2020 programme under GA857287)

This study aimed to characterise the microarchitecture of bone in different species of animal leading to the development of a physiologically relevant 3D printed cellular model of trabecular (Tb) and cortical bone (CB). Using high resolution micro-computed tomography (μ-CT) bone samples from multiple species were scanned and analysed before creating in silico models for 3D printing. Biologically relevant printing materials with physical characteristics similar to that of in vivo bone will be selected and tested for printability. Porcine and murine bone samples were scanned using μ-CT, with a resolution of 4.60 μM for murine and 11 μM for porcine and reconstructed to determine the architectural properties of both Tb and CB independently. A region of interest, 1 mm in height, will be used to generate an in-silico 3D model with dimensions (10 mm. 3. ) and suitable resolution before being translated into printable G code using CAD assisted software. A 1 mm section of each bone was analysed, to determine the differences in the microarchitecture with the intent of setting a benchmark for the developmental 3D in vitro model to be comparable against. In contrast, porcine caudal vertebrae (PCV) have an increased volume due to the size of the bone sample. Interestingly, BV/TR for Tb is similar between species in all samples except murine femur. Murine tibia and PCV have a similar Tb. number and thickness, however different SMI shape and separation. μ-CT scanning and analysis permits tessellation of the 3D output which will lead to the generation of an in silico printable model. Biomaterials are currently under optimisation to allow printability and shape integrity to reflect the morphological and physiological properties of bone

Aim. The rise of multidrug-resistant bacteria and the decreasing efficacy of antibiotic therapy in successfully treating biofilm-associated infections are prompting the exploration of alternative treatment options. This study investigates the efficacy of different bioactive glass (BAG) formulations - alone or combined with vancomycin - to eradicate biofilm. Further, we study the influence of BAG on pH and osmotic pressure as important factors limiting bacterial growth. Method. Different BAG-S53P4 formulations were used for this study, including (a) BAG-powder (<45 μm), (b) BAG-granules (500–800 μm), (c) a cone-shaped BAG-scaffold and (d) two kinds of BAG-putty containing granules, with no powder (putty-A) or with additional powder (putty-B), and a synthetic binder. Inert glass beads were included as control. All formulations were tested in a concentration of 1750 g/ml in Müller-Hinton-Broth. Targeted bacteria included methicillin-resistant Staphylococcus aureus (MRSA) and epidermidis (MRSE). Vancomycin was tested at the minimum-inhibitory-concentration for each strain (1 µg/ml for MRSA; 2 μg/ml for MRSE). To investigate the antibiofilm effect of BAG alone or combined with vancomycin, 3 hour-old MRSA or MRSE biofilms were formed on porous glass beads and exposed to BAG ± vancomycin for 24h, 72h and 168h. After co-incubation, biofilm-beads were deep-washed in phosphate-buffered saline and placed in glass vials containing fresh medium. Recovering biofilm bacteria were detected by measuring growth-related heat production at 37°C for 24h by isothermal microcalorimetry. Changes in pH and osmotic pressure over time were assessed after co-incubation of each BAG formulation in Müller-Hinton-Broth for 0h, 24h, 72h and 168h. Results. All BAG formulations showed antibiofilm activity against MRSA and MRSE in a time-dependent manner, where longer incubation times revealed higher antibiofilm activity. BAG-powder and BAG-putty-B were the most effective formulations suppressing biofilm, followed by BAG-granules, BAG-scaffold and finally BAG-putty-A. The addition of vancomycin had no substantial impact on biofilm suppression. An increase in pH and osmotic pressure over time could be observed for all BAG formulations. BAG-powder reached the highest pH value of 12.5, whereas BAG-putty-A resulted in the lowest pH of 9. Both BAG-putty formulations displayed the greatest increase on osmotic pressure. Conclusions. BAG-S53P4 has demonstrated efficient biofilm suppression against MRSA and MRSE, especially in powder-containing formulations. Our data indicates no additional antibiofilm improvement with addition of vancomycin. Moreover, high pH appears to have a larger antimicrobial impact than high osmolarity. Acknowledgements. This work was supported by PRO-IMPLANT Foundation (Berlin, Germany). The tested materials were provided by Bonalive Biomaterials Ltd (Turku, Finland)

An ex vivo biomechanical test model for evaluating a novel bone adhesive has been developed. However, at day 1 in the in vivo pilot, high blood flow forced the study to halt until the solution presented here was developed. The profuse bleeding after bone core removal affected the bond strength and was reflected in the lower mean peak value 1.53N. After considering several options, we were successful in sealing the source of blood flow by pressing adhesive into place after bone core removal. After the initial adhesive had cured additional adhesive was used to secure the bone core in place. The animals were sacrificed after 24 h and a tensile test was undertaken on the bone core to failure. The ex vivo study produced mean peak tensile loads of 7.63N SD 2.39N (n=8, 4 rats 8 femurs). Whilst the mean peak tensile loads in the day 1 in vivo pilot were significantly lower 1.53N SD1.57 (n=8, 6 rats 8 femurs − 4 used for other tests). The subsequent layered adhesive bone cores showed a mean peak tensile force of 6.79N SD =3.13 (n=8, 4 rats 8 femurs). 7/8 failed at the bone to glue interface. This is the first successful demonstration of bonding bone in vivo for this class of adhesives. The development of a double adhesive method of fixing a bone core in the distal femur enabled mean peak tensile forces to be achieved in vivo at 24 hours that were comparable with the ex vivo results previously demonstrated. This method supports application in further animal series and over longer time scales. Biomaterials researchers that intend to use gel or paste like preparations in distal femur defects in the rat should be aware of the risks of biomaterial displacement by local blood flow

Biomaterials are no longer considered innate structures and using functionalisation strategies to modulate a desired response whether it is a host or implant is currently an important focus in current research paradigms. Fundamentally, a thorough understanding the host response will enable us to design proper functionalisation strategies. The input from the host response need to be weighed in depending on the host disease condition. In addition, biomaterials themselves provide immense therapeutic benefits which needs to be accounted for when using functionalisation strategies. Using strategies such as enzymatic and hyperbranched linking systems, we have been able to link biomolecules to different structural moieties. Our recent design efforts have harnessed the therapeutic effects of biomaterials and mapped the molecular fingerprint of this specific host response in a disease target. This approach allows us to rethink functionalisation strategies currently employed in the field. This talk will elucidate some of these ongoing strategies that have applications in the development of the next generation of orthopaedics devices

Introduction: We developed a signal inducing bone cement for surgical interventions under MR guidance. This cement is based on conventional polymethylmeth-acrylate (PMMA), which is mixed with 0.9% saline solution and a contrast agent (CA), or with a hydroxyapatite based bone-filler (Ostim. ®. , aap Biomaterials, Germany). This signal inducing cement should allow bone filling procedures, like vertebro- and kyphoplasty, under MR guidance in an open Highfield MR Scanner. As we added the signal inducing substances (saline solution, CA, bone substitute) to the PMMA, we changed the biomechanical properties of the cement. The purpose of this study was to evaluate the biomechanical properties of the signal inducing bone cement for vertebroplasty in a spine model. Materials/Methods: We placed cadaveric vertebral bodies (n=18, of 4 lumbar spines) between the crosshead and baseplate of a universal testing machine (Zwick. ®. , Germany) and compressed to failure. Then, we injected cements into the broken vertebral bodies through a transpedicular approach on both sides, under image intensifier control. The so treated vertebral bodies were then tested again in the testing machine. We injected three cements: a conventional PMMA cement (BonOs. ®. , aap Biomaterials, Germany, 12g PMMA, 5 ml MMA), an NaCl-cement compound (3 ml 0.9% saline solution, 12g PMMA, 5 ml MMA) and a bone substitute-cement compound (3 ml Ostim. ®. , 12g PMMA, 5 ml MMA). As the CA amount is negligible (< 9μl), it was neglected for these tests. Each cement type was injected in 6 vertebral bodies. We defined the initial strength (N) of the vertebral bodies as the load at failure, and the strength after treatment as the maximum load, which occurred within the first 6 mm of compression. Results: The initial strength of the vertebral bodies (n=18) was 4179 N (SD 497 N). The strength after treatment was 7433 N (SD 503 N) for the conventional cements (n=6), 5900 N (SD 376) for the NaCl-cements (n=6), and 7000 N (SD 413 N) for the Ostim. ®. -cements (n=6). Discussion: Although the PMMA cement is weakened by dilution with the signal inducing substances (saline solution, CA, bone substitute), the MRI-cements restored the initial strength of the vertebral bodies. The results suggest that these MRI-cements meet the biomechanical requirements for vertebroplasty, and can be used for MRI guided vertebroplasty

Aim. Infections in long bones can be divided in osteitis, osteomyelitis and septic non-unions. All are challenging situations for the orthopaedic surgeon. Treatment is a mix with debridement, radical resection of infected tissue, void filling with different types of products, and antibiotic therapy of different kinds. In cavitary bone defects, bioglasses such as BAG-S53P4 have given good results in early or mid-term follow-up. Results of such treatment in segmental bone defects remain unknown. The goal of our study was to evaluate efficacity of active bioglass BAG-S53P4 in septic segmental bone defects. Method. A retrospective cohort study has been done in a single specific orthopaedic center devoted to treatment of infected bony situations. All cases were a severe septic bone defect. We have compared the segmental bone defects to the cavitary ones. Results were analyzed on recurrence of infection, bone healing, functional result and complication rate. Results. 14 patients were included with a minimum follow-up of 1 year after treatment. 8 were in the group “cavitary”, 6 in the group “segmental”. The mean age was 54 years-old (30–76). Sex-ratio was 2.5. All patients have been treated with bone resection and debridement of infected bone and tissue, even if more than 1 surgery was necessary in some cases. After cleaning, 7 patients have needed a local flap, and 1 a free flap. Then, all bone defects were filled up by bioglass BAG-S53P4*. Additional antibiotherapy with specific molecules based of the results of bacterial analysis, was given for a minimum time-period of 6 weeks. In the “cavitary” group, the mean volume of BAG-S53P4 was de 21.25 ml (10–60). In the “segmental” group, it was of 12.5 ml (10–20). The healing rate was of 80% in the “cavitary” group and of 100% in the “segmental” one. No complication related to the bioglass insertion was noted. Conclusions. Different publications have been made using bioglass in the treatment of infected bone with a continuous bone such as osteitis or osteomyelitis. Our study is the first one to compare specifically the results obtained in a cavitary defect where the bone is still in continuity, and in a segmental defect. Active bioglass such as the BAG-S53P4 seems to be a good option in the treatment of segmental septic bone defects in the limb. *BonAlive Biomaterials Ltd, Turku, Finland

Aims

Refobacin Bone Cement R and Palacos R + G bone cement were introduced to replace the original cement Refobacin Palacos R in 2005. Both cements were assumed to behave in a biomechanically similar fashion to the original cement. The primary aim of this study was to compare the migration of a polished triple-tapered femoral stem fixed with either Refobacin Bone Cement R or Palacos R + G bone cement. Repeated radiostereometric analysis was used to measure migration of the femoral head centre. The secondary aims were evaluation of cement mantle, stem positioning, and patient-reported outcome measures.

Aims

The standard of wide tumour-like resection for chronic osteomyelitis (COM) has been challenged recently by adequate debridement. This paper reviews the evolution of surgical debridement for long bone COM, and presents the outcome of adequate debridement in a tertiary bone infection unit.

Purpose. Osteochondral lesions (OCL) of the talus remain a challenging therapeutic task to orthopaedic surgeons. Several operative techniques are available for treatment, e.g. autologous chondrocyte implantation (ACI), osteochondral autograft transfer system (OATS), matrix-induced autologous chondrocyte implantation (MACI). Good early results are reported; however, disadvantages are sacrifice of healthy cartilage of another joint or necessity of a two-stage procedure. This case describes a novel, one-step operative treatment of OCL of the talus utilizing the autologous matrix-induced chondrogenesis (AMIC) technique in combination with a collagen I/III membrane. Method. 20 patients (8 female, 12 male; mean age 36, range 17–55 years) were assessed in our outpatient clinic for unilateral OCL of the talus. Preoperative assessment included the AOFAS hindfoot scale, conventional radiography, magnetresonancetomography (MRI) and SPECT-CT. Surgical procedure consisted of debridement of the OCL, spongiosa plasty from the iliac crest and coverage with the I/III collagen membrane (Chondrogide, Geistlich Biomaterials, Wolhusen, Switzerland). Clinical and radiological followup was performed after one year. Results. The mean preoperative AOFAS hindfoot scale was poor with 63.1 points (SD 19.6). At one year followup the score improved significantly (p<0.01) to 86 points (SD 12). At one year followup conventional radiographs showed osseous integration of the graft in all cases. MRI at one year showed intact cartilage covering the lesions in all cases. Conclusion. The initial results of this ongoing study are encouraging. The clinical and radiological results at one year followup are comparable with the results of ACI, OATS and MACI. The AMIC procedure is a readily available, economically efficient, one step surgical procedure. No culturing after chondrocyte harvesting or destruction of viable cartilage is necessary

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

Aims

Biofilm-related infection is a major complication that occurs in orthopaedic surgery. Various treatments are available but efficacy to eradicate infections varies significantly. A systematic review was performed to evaluate therapeutic interventions combating biofilm-related infections on in vivo animal models.

Bioactive glasses (BAGs) are bone substitutes with bone bonding, angiogenesis promoting and antibacterial properties. The bioactive process leading to bone bonding has been described as a sequence of reactions in the glass and at its surface. Implantation of the glass is followed by a rapid exchange of Na+ in the glass with H+ and H3O+ from the surrounding tissue, leading to the formation of silanol (SiOH) groups at the glass surface. Due to migration of Ca2+ and PO43− groups to the surface and cystallization, a CaO-P2O5 hydroxyapatite (HA) layer is formed on top of the Si-rich layer. Finally, cell interactions with the HA layer subsequently initiate the bone forming pathway. The rapid increase in pH and the subsequent osmotic effect caused by dissolution of the glass have been suggested to partly explain the antibacterial properties observed for BAGs. Comparing bactericidal effects of different BAGs, BAG-S53P4 has been shown to be the most effective, with the fastest killing or growth inhibitory effect. This antibacterial effect has been observed in vitro for all pathogens tested, including the most important aerobic and anaerobic pathogens, as well as very resistant bacteria. In a multicentre study in 2007–2009, BAG-S53P4 was used as bone graft substitute in treatment of osteomyelitis. Eleven patients (nine males, two females) with a radiologically diagnosed osteomyelitis in the lower extremity (N=10) and in the spine (N-1) participated. In the operation, the infected bone and the soft tissue were removed, and the cavitary bone defects were filled with BAG-S53P4 (BonAlive™, Bonalive Biomaterials Ltd., Finland). In four patients, muscle flaps were used as part of the treatment. Eight patients were treated in a one-stage procedure. Kanamycin granules were used in one patient and Garamycin granules (Septocol ®) in two patients. Patient data were obtained from hospital patient' records until August 2010, resulting in a mean follow-up period of 29 months (range 15–43). BAG-S53P4 was well tolerated; no BAG-related adverse effects were seen in any patient. The use of BAG-S53P4 as a bone graft substitute resulted in a fast recovery. Long-term clinical outcome was good or excellent in ten of eleven patients. These primary results indicate that BAG-S53P4 can be considered as a good and usable material in treatment of osteomyelitis. After this study BAG-S53P4 has been used in several other patients with very promising results

Biomaterials used in regenerative medicine should be able to support and promote the growth and repair of natural tissues. Bioactive glasses (BGs) have a great potential for applications in bone tissue engineering [1, 2]. As it is well known BGs can bond to host bone and stimulate bone cells toward osteogenesis. Silicate BGs, e.g. 45S5 Bioglass® (composition in wt.%: 45 SiO. 2. , 6 P. 2. O. 5. , 24, 5 Na. 2. O and 24.5 CaO), exhibit positive characteristics for bone engineering applications considering that reactions on the material surface induce the release of critical concentrations of soluble Si, Ca, P and Na ions, which can lead to the up regulation of different genes in osteoblastic cells, which in turn promote rapid bone formation. BGs are also increasingly investigated for their angiogenic properties. This presentation is focused on cell behavior of osteoblast-like cells and osteoclast-like cells on BGs with varying sample geometry (including dense discs for material evaluation and coatings of highly porous Al. 2. O. 3. -scaffolds as an example of load-bearing implants). To obtain mechanically competent porous samples with trabecular architecture analogous to those of cancellous bone, in this study Al. 2. O. 3. scaffolds were fabricated by the well-known foam replication method and coated with Bioglass® by dip coating. The resulted geometry and porosity were proven by SEM and μCT. Originating from peripheral blood mononuclear cells formed multinucleated giant cells, i.e. osteoclast-like cells, after 3 weeks of stimulation with RANKL and M-CSF. Thus, the bioactive glass surface can be considered a promising material for bone healing, providing a surface for bone remodeling. Osteoblast-like cells and bone marrow stromal cells were seeded on dense bioactive glass substrates and coatings showing an initial inhibited cell attachment but later a strong osteogenic differentiation. Additionally, cell attachment and differentiation studies were carried out by staining cytoskeleton and measuring specific alkaline phosphatase activity. In this context, 45S5 bioactive glass surfaces can be considered a highly promising material for bone tissue regeneration, providing very fast kinetics for bone-like hydroxyapatite formation (mineralization). Our examinations revealed good results in vitro for cell seeding efficacy, cell attachment, viability, proliferation and cell penetration onto dense and porous Bioglass®-coated scaffolds. Recent in vivo investigations [3] have revealed also the angiogenic potential of bioactive glass both in particulate form and as 3D scaffolds confirming the high potential of BGs for bone regeneration strategies at different scales. Implant surfaces based on bioactive glasses offer new opportunities to develop these advanced biomaterials for the next generation of implantable devices and tissue scaffolds with desired tissue-implant interaction

The reconstruction of large bone segments is a major goal in orthopaedic surgery. Autologous cancellous bone is recognized as the most biologically active graft material, but autologous bone harvest is associated with significant morbidity and founds its limit in the available quantity. Biomaterials or allografts do not encounter these limitations, but have no osteogenic and limited osteinductive potential. In order to enhance tissue regeneration and healing we have tried to obtain a graft with osteconductive, inductive and osteogenic properties. The day before operation 350 cc of autologous blood is donated from the patient and centrifuged to obtain a platelet-rich plasma. Bone marrow is aspirated from the posterior iliac crests with the patient under spinal anaesthesia and is processed to increase its stem cell content. The structural scaffold used is morcellized cancellous bone provided from our Bone Bank. At operation bone is mixed with bone marrow buffy coat and Platelet Rich Plasma in a sterile glass becker with addition of CaCl2 till clot formation to produce a gel-like component that is handled easily. We have utilized this technique from November 2000 till January 2004 for 68 patients: 41 of these patients required healing of large bone defects: 22 males and 19 females. Fresh bone marrow alone was used for a percutaneous injection in 11 cases; open surgery with autologous growth factors, bone marrow buffy coat and allograft was used in 30 patients. The radiological and clinical results showed early healing of the defects treated with this technique and no complications related to the procedure at an average follow up of 23 months (3–40)

Introduction. Core decompression is used in precollapse lesions to forestall disease progression in avascular necrosis (AVN) of femoral head (FH). The author reports a new technique using reverse bone graft technique to effectuate core decompression. Aim. To prevent precollapse in Ficat Type 1&2 and revascularization using synthetic bone graft material. Methods. A 18 year female police trainee with Magnetic Resonance Imaging (MRI) confirming AVN Stage 2 Ficat, clinically painful hip not evident in x-rays consented to undergo this new technique. Reverse bone graft technique with a Coring reamer – Patent 5423823. A minimally invasive technique with lateral 2 cm incision introducing 8.5 mm core reamer to remove a core of bone up to the subchondral bone. The subchondral cyst decompressed and curetted under video recorded Image Intensifier (II). Demarcated avascular bone segment excised and bone graft reversed and inserted with cortical bone acting as a support to prevent collapse and the distal segment augmented using 5 grams of osteoconductive granular synthetic bone graft material based on calcium phosphate hydroxyapatite (HA 2500–5000 μm). Avascular segment histopathologically confirmed AVN. The metaphyseal entry was extrapoliated at the lateral cortex using the combined necrotic angle described by Kerboul in the anteroposterior and lateral views under II. Protected weight bearing for 2 months to prevent stress riser. Biomaterials. HA granules named as GranuMas™ developed under Intensified Research in Priority Areas (IRPA) Research Project (No. 03-01-03-0000-PR0026/05) and invented by the Advance Materials Research Centre (AMREC) and manufactured by GranuLab –Patent P1 20040748 fulfilling the criteria for American Society for Testing and Materials (ASTM) F1185-88(1993) Standards which is ‘Standard Specification for Composition of Ceramic Hydroxyapatite for Surgical Implants’. Derived from Malaysian limestone, ranging from 200–5000 μm gamma sterilized. Results. After 6 months, there was no collapse of subchondral bone and the FH showed revascularization along bone grafted site with viable graft and increased radiotracer activity using 99-Tc MDP Bone Planar Scintigraphy. Clinical analysis follow up at 2 years was descriptive rather than statistical with a x-ray evident incorporated graft and with pain free full range of movement. Discussion. Reduction in intraosseous pressure is achieved by using large bore 8.5 mm coupled with HA granules promoting revascularization. The core tract entering through the metaphyseal region reduces risk of subtrochanteric fracture a potential complication of vascularized fibular grafts and with less morbidity with other treatment methods for osteonecrosis of the femoral head. The concept can be extended in introducing stem cell and biologic material to treat AVN. Conclusion. This technique is minimally invasive and effective in young patients with early stage of FH AVN and has shown revascularization along the bone grafted site

It has been generally accepted that dynamic mechanical load is important for normal bone physiology, remodeling and fracture healing. Impacted morsellized grafts can be seen as healing of many small fractured bone parts, involving bone remodelling, apposition and formation of new bone. Therefore load may be stimulative for the incorporation of this type of graft. In a pilot study we observed a positive effect of load on the density of incorporated bone after 12 weeks. Based on these results we hypothesised that physiological loading has a stimulatory effect on the early stage of bone graft incorporation. To test this idea we implanted fresh frozen allograft bone chips in 12 goats and loaded these grafts with the newly developed subcutaneous pressure implant ( . Lamerigts et al., . Biomaterials. 2000. ; . 21. : . 741. –7. ). The goats were divided in two groups: non-loaded and loaded. The loaded group was subjected to a loading regime of 3 MPa for 5 days/week (1 Hz, one hour/day). After 5 weeks the bone mineral density was measured with quantitative CT scanning, followed by routine histology and histomorphometry. Bone mineral density was not affected by load. Histology revealed microscopic evidence of normal bone graft incorporation as seen in previous studies. The amount of active incorporating bone was higher under load (p< 0.05). The formation of a new bony structure was not affected by load in this early stage of bone graft incorporation. However, load resulted in a more active graft incorporation after 5 weeks. The difference between the loaded and non-loaded group might be partially obscured by a low level of physiological loading in the non-loaded group induced by the daily activity of the animals

Summary Statement. Magnesium has a number of qualities suitable for bioresorbable metallic implants. However, high corrosion rate and formation of hydrogen gas can compromise its performance. Combining magnesium with calcium phosphate improves magnesium's biocompatibility by decreasing gas formation and increasing bone remodeling. Introduction. Clinical problems like risk of postoperative infection and increased incidence of pediatric trauma requiring surgical intervention raised the need for temporary orthopedic implants that would resorb after the bone healing is complete. This would decrease high costs associated with repeated surgeries, minimise recovery times, decrease the risk of postoperative infections, and thus promote higher quality of life to the patients. The specific requirement for orthopedic implants, aside from being bioresorbable, is the ability to bear high loads. Magnesium was suggested as a suitable material for these purposes because it is biocompatible; has excellent mechanical properties; is natural for human body, and seems to stimulate new bone formation. However, an important problem with magnesium is high corrosion rate with consistent hydrogen gas formation on contact with fluids. This in vivo study focuses on investigation of new magnesium-based implants specifically designed to minimise hydrogen gas formation. Methods. Four types of degradable magnesium-based materials were tested for biocompatibility in this study: Magnesium-Hydroxyapatite implants (Mg-HA); Magnesium-Calcium Phosphate Cement (Mg-CPC); alloy of 96% Magnesium and 4% Yttrium (W4); and 99.95% pure magnesium which was a control group. Biomaterials were operated into 33 male New Zealand white rabbits. The animals were sacrificed after 6 and 12 weeks after which the samples were embedded into Epon, paraffin and Technovit resin. The staining was done with TRAP, hematoxylin eosin and toluidine blue. Additionally, TEM and immunohistochemical analysis were performed. The data was analysed both qualitatively and quantitatively by Statistical Package for the Social Sciences (SPSS, v18, SPSS Inc, Chicago, IL). Results. Mg-CPC showed the best performance in this study. New bone formation was significantly more prevalent in Mg-CPC group while gas formation was significantly less comparing to the other materials. Mg-HA had the worst properties due to extremely fast degradation already at 6 weeks, the least amount of new bone formation, and the lowest amount of osteoclasts and multinucleated cells in the implantation site. Pure magnesium and W4 had similar properties: both were surrounded with corrosion layer, and the gas volumes were significantly higher in these two groups compared to other materials. Discussion/Conclusion. New bone was seen forming either in direct contact to implants or around the gas bubbles. The later can be interpreted as body's reaction to protect from gas spreading. Mg-HA's degradation rate was far too fast and this is unacceptable for orthopedic fractures which often require several months to heal and that experience much load. Pure magnesium and W4 although maintained their integrity, were surrounded by corrosion layer and gas bubbles that were bigger in diameter than in the other groups. These findings could compromise implant stability. Mg-CPC was the most biocompatible; it showed significantly higher amount of osteoclasts which is a first sign of bone remodeling. It had also significantly less gas production than other groups. These results show that magnesium's biocompatibility could be improved by combining it with other suitable materials, such as calcium phosphate

Although osteochondral grafting techniques have nearly been perfected, donor site morbidity still causes concern. A synthetic β-tricalcium phospate cement was used in the attempt to obtain a primary closure of such osteochondral defects, while supplying a scaffold for tissue ingrowth. Twenty merino sheep underwent an osteochondral grafting procedure. The paste-like β-TCP cement was used to fill the ensuing cylindrical, full-thickness defect. Animals were sacrificed after 3 or 6 months. The macroscopic observations revealed neither osteophytes nor synovial proliferation, while demonstrating coverage of the defect with cartilage-like tissue. After 6 months, all defects were covered with a ”neo-cartilage” and the congruity of the joint surface was restored in 6 of 10 animals. A surface depression was found in the remaining cases. A demarkation of the defect border at the interface with the original cartilage could only be seen in 2 instances. The x-rays of the retrieved distal femurs revealed only traces of the dense β-TCP particles. Microradiographs demonstrated the incorporation of the implant. Fluorescent staining showed continuous bone ingrowth. Histologically, masses of unabsorbed TCP were irregularly distributed through-out the defect. Newly formed bone had filled much of the defect. The histological evaluation confirmed that the surface of the cement was covered with a cartilage-like tissue. This study showed, that the newly developed in-situ self-hardening resorbable β-tricalcium phosphate cement is easy to handle, hardens in a clinical-type setting, is bioactive and resorbable. Its osteoconductive effect lead to a restoration of biomechanically stable bone and allows for a normal remodeling process. Biomaterials made of β-TCP promise to play a role as a biodegradable scaffold, allowing osteo-blast ingrowth and cartilagenous resurfacing, while being fully resorbed during the process. The cement may also be used to deliver bioactive agents and cells for defect repair in the near future

Introduction: Bone-marrow stimulating techniques like microfracturing for focal chondral defects of the knee joint are widespread utilizing mesenchymal stem cells (MSC) for an autogenous reparation process. Microfracturing shows good results for smaller defects up to 2cm. 2. while larger defects tend to an early secondary degeneration. Autologous Matrix Induced Chondrogenesis (AMIC®) combines microfracturing with application of a porcine collagen type-I/III bilayer matrix to host the MSC and to stabilize the blood clot. Methods: 32 patients (25m, 7f, mean age 37.4y (18–52y)) with 35 focal chondral defects of the knee joint (ICRS III–IV°) of the condyle, trochlea and/or patella were treated by standardized microfracturing and application of a collagen matrix (Geistlich Biomaterials, Wolhusen, Switzerland). The outcome was evaluated prospectively by clinical scores and MRI with a follow-up of 6 to 24 months. The mean defect size was 3.86 cm. 2. (1.0 – 6.8 cm. 2. ). 22 patients (68%) had at least one operation (1–8) on the knee before. 9 defects were caused by trauma. All 7 patients with osteochondritis dissecans had an autologous bone grafting. In 5 patients an ACL stabilization was performed simultaneously. Results: All patients considered their knee as abnormal (ICRS III° (70%)) or severely abnormal (ICRS IV° (30%)) preoperatively according to the ICRS functional status. The Cincinnati-Score improved from 52.9 to 81.1 points while the Lysholm-Score rose from 60.4 to 85.9 points (each p< 0.001). Pain decreased significantly from 6.1 to 2.2 (10=max.) on the visual analogue scale. 4 biopsies (4–21 months) revealed reasonable results with regard to surface formation, filling and integration in the Brittberg score (∅10.25 pts., 12 pts.=max.) The MRI follow-ups showed an adequate filling of the defect, no prolonged effusion occured. Conclusion: Microfracturing in combination with a collagen matrix (AMIC®) is a minimal invasive, effective technique for the repair of focal cartilage defects of the knee joint. Not using cultured chondrocytes it can be performed cost-effectively as a single-step procedure. Both primary and secondary treatments are possible. The first results concerning clinical functional improvement, pain reduction and patients’ satisfaction as well as defect filling in MRI are promising

Statement: To present the early results of using new implants in the fixation of the hamstrings tendons for ACL reconstruction. Background and Aim of the study: PINN-ACL system (Conmed UK, Linvatec UK ltd.), is a recently developed implant designed for transverse femoral fixation of hamstrings grafts in ACL reconstruction, allowing for increased pull out strength. It consists of a Graft Harness composed of Poly L-Lactic Acid with a high strength polyethylene fibre loop and a Cross pin composed of Self-Reinforced PLLA. Tibial fixation is achieved by a bioabsorbable Matryx Interference Screw; composed of Self-Reinforced 96L/4D PLA and beta-Tri-Calcium Phosphate(Linvatec Biomaterials ltd.). We describe our early experience with this new system, the technique of fixation, short-term clinical results, functional outcome and MRI features of these implants. Materials and Methods: A prospective data collection was undertaken over the past 12 months. The operative steps:, four strand hamstring preparation, tensioning, femoral fixation of graft with graft harness and cross pin, tensioning the graft and tibial fixation with bio- absorbable interference screw. More than 80% of the cases were performed without tourniquet. The follow up were made at 2,12,24,36 weeks and further evaluation as needed for the purpose of the study. Outcomes were assessed with Lysholm, Tegner and IKDC scores. Results: 24 cases were performed in 23 cases. The mean age, gender and laterality were 34(17–51), 1.7M: 1F, 14L:10 R. The injury pattern: sports (77%) and RTA (11%). Tunnel view of the harness was excellent in 79%. Linvatec Tensioner was used in 60%. Graft was not detached in 20%. The mean follow up period was 7 months (2 –12). At last follow up Lachman and pivot shift were negative in 85% and grade 1 in 15%, The mean postoperative scores were Tegner-7 (5–10), Lysholm-7 (5–10) and IKDC-71 (57–93) respectively. 1 wound problem required washout. The tibial screw twisted off at final turn in 1 patient. The cross pin drill missed the guide in 1 patient. At 32 weeks MRI scan: the implants were still evident, However apart form 1 patient, there was no surrounding bone reaction and none showed tunnel widening. Conclusion: Early results are encouraging, both operative technique and fixation. However, harness size is limited to 8 and 9 mm only and the implants were still evident at a mean period of 32 weeks against the manufactures claim of 24 weeks

Introduction: Rheumatoid arthritis and osteoarthritis as well as other diseases can cause severely destruction of the finger joints. The treatment is surgical replacement with joint prosthesis manufactured from flexible silicone or other materials. Silicon prostheses (Swanson’s prostheses) are used worldwide already since 1974. However, the material used may not be strong enough on long term and several reports from breakdown of the prostheses have been published. The long-term results have also shown that bony resorption around the implant may occur. The known weaknesses of the current endoprostheses have lead researchers to look for new materials. Material: In the beginning of 1994 a fibrous cushion made of commercially available biodegradable fibres (Vicryl® and Ethisorb®) was introduced by the group of researchers from Tampere University Hospital in Finland. It was intended to act as the tendon in Vainio arthroplasty and the aim was to find a material that could work as a scaffold for the collagenous proliferation of connective tissue or fibrocartilage. However the resorption time on the material was too short, which led to the premature collapse of the joint space. Novel scaffolds were developed using a well-known poly-L/D-lactide copolymer with L/D-monomer ratio 96/4 (PLDLA) in collaboration with the Institute of Biomaterials at Tampere University of Technology and Tampere University Hospital. The PLDLA scaffolds are fibrous, porous cylinders enabling the in-growth of fibrous tissue, which then ideally forms a new, functional joint for the patient. Meltspun PLDLA scaffolds retain 50% of their strength at least 13 weeks in vitro. This enables to retain the shape and size of the scaffolds in situ long enough for tissue ingrowth. The scaffold will bioabsorb and be replaced with fibrous tissue in approximately 2–3 years. Method: Since January 2003 we take part in a prospective randomised international multicenter study, that is supported by the European Commission. The new bioreplaceable devices are implanted in hands (CMC, MCP, PIP und DIP) as well as feet (MTP I–V toe joints) and will be compared to the standard treatments (Swanson Prostheses, Arthrodeses). The study is surveyed by an Ethical Committee. Results: Up to now we implanted the bioreplaceable scaffolds in several joints of hands with good results. In future we will also use them for metatarso-phalangeal joints in feet. We will present in the meeting our experiences and outcomes so far

Introduction: We developed a new treatment option for localized articular cartilage defects: the matrix-induced, autologous chondrocyte transplantation (MACT) in which we seeded autologous chondrocytes on porcine porous matrices of type I/III collagen (Chondro-Gide®, Geistlich Biomaterials, Wolhusen, Switzerland) instead of a periosteum flap.The target of this clinical prospective study was to evaluate the outcome for a follow up period of five years after transplantation. Methods: Between 1998 and 2001 we treated 38 patients (19 male and 19 female) with localized cartilage defects (Outerbridge grade three to four). Within the follow up time of this study until October 2002 the patients were assessed clinically 3, 6, 12, 18, 24, 36 and 60 month after the transplantation using four different standard rating scales: the Meyers-score, the Tegner/Lysholm-score, the Lysholm/Gilquist-score and the ICRS-score as well as MRI. Results were documented and compared with the pre operatives. Furthermore histological stainings of four patients were assessed. Results: Mean patient age was 35 years (19 to 58 years). Average defect size was 5,6 qcm, 10 defects localized patellar, 16 femoral medial,3 femoral lateral and 9 combined. Two years after operation 66,7% (n=25) of the patients rated the function of their knee as much better or better than before in the subjective evaluation. After five years the percentage decreased to 57,1% (n=10). Up to a follow-up time of 24 month the clinical outcome of all four scores illustrated an significant improvement. Five years after transplantation two scores still showed significant improvement (Meyers-score: p= 0,02; Lysholm-Gilquist-Score: p=0,02). The other two scores showed improvement which turned out to be non significant (Tegner-Lysholm-Score: p=0,19; ICRS-Score: p=0,06) MRI scanning results after one year could not detect the quality of cartilage defect repair. Histological evaluation of four patients might not identify any association between the quality of the tissue and the clinical outcome. Conclusion: Five years results in two scores (Meyer- and Lysholm-Gilquist-Score) still showing significant improvement imply that MACT has turned out to be an acceptable alternative for the treatment of localized cartilage defects in the knee

Summary Statement. We have developed 3D combinatorial hydrogels containing cartilage extracellular matrix (ECM) proteins for modulating chondrogenesis of adipose-derived stromal cells. Our platform allows independently tunable biochemical and mechanical properties, which may provide a valuable tool for elucidating how ECM biochemical cues interact with matrix stiffness to regulate stem cell chondrogenesis. Introduction. Adipose-derived stromal cells (ADSC) hold great promise for cartilage repair given their relative abundance and ease of isolation. Biomaterials can serve as artificial niche to direct chondrogenesis of ADSCs, and extracellular matrix (ECM) protein-based scaffolds are highly biomimetic. However, incorporating ECM molecules into hydrogel network often lead to simultaneous changes in both biochemical ligand density and matrix stiffness. This makes it difficult to understand how various niche signals interact together to regulate ADSC fate. To overcome these limitations, the goal of this study is to develop an ECM-containing hydrogel platform with independently tunable biochemical and mechanical cues for modulating ADSC chondrogenesis in 3D. We hypothesise that decreasing the degree of crosslinking of ECM molecules may allow their incorporation without affecting the matrix stiffness. The effects of interactive signaling between ECM molecules and matrix stiffness on ADSC chondrogenesis in 3D was then examined using this platform. Methods. Three types of cartilage-specific extracellular matrix proteins (ECM) - chondroitin sulfate (CS), hyaluronic acid (HA) and heparan sulfate (HS) were chemically incorporated into hydrogel network via methacrylation. ECM proteins were modified with methacrylate end groups at varying degrees of methacrylation to allow minimal influence on matrix mechanical properties. To vary biochemical cues within the hydrogels, CS, HA and HS were incorporated at varying concentrations (0.5, 1.25, 2.5 and 5% w/v). To examine the stability of ECM molecules with varying degrees of methacrylation within the hydrogel network, ECM molecules are fluorescently labeled and incorporated within hydrogels. Leaching was monitored by measuring fluorescence over time. To vary mechanical property of the hydrogels, poly-(ethylene glycol) dimethylacrylate (PEGDMA) (4.6kDa) was incorporated at varying concentrations (5, 10, and 15% w/v). Based on the leaching test and mechanical test, ECM molecules with the optimal degree of methacrylation that allows stable interlocking without changing matrix stiffness were chosen for further studies. A total of 39 combinatorial hydrogel compositions were examined. Human ADSCs were encapsulated in combinatorial hydrogels and supplied chondrogenic medium for 21 days. Outcome was analyzed by quantifying chondrogenic gene expression (Aggrecan and Collagen II) and immunofluorescent staining. Results. Mechanical testing showed that hydrogel stiffness was controlled solely by varying the concentration of mechanical blocks (PEGDMA), and was not influenced by varying the ECM concentration within the range tested (up to 5% w/v). Increasing PEGDMA concentration from 5% to 15%(w/v) produced hydrogels with stiffness from 3–100kPa. Results from the leaching test confirmed that majority of the ECM molecules were stably crosslinked within the hydrogel network. Gene expression results showed that biochemical and mechanical signals interacted in a non-linear manner. At lower concentrations of biochemical cues, increasing mechanical stiffness promoted chondrogenesis. However, at higher concentrations of biochemical cues, biochemical cues played a more important role on regulating chondrogenesis. In soft matrix (5% PEGDMA), both HA and HS increased chondrogenesis in a dose-dependent manner. Discussion/ Conclusion. Here we report an ECM-containing hydrogel platform with independently tunable mechanical and biochemical cues. We identified optimised degree of methacrylation of ECM molecules that allowed their stable incorporation via covalent bonding, without affecting the matrix stiffness. This platform may provide a useful tool to facilitate elucidating how interactive niche signals regulate stem cells fate in 3D, and identify optimal scaffold compositions to promote musculoskeletal tissue differentiation

Autologous chondrocyte transplantation is a widely used technique for the treatment of cartilage lesions. This therapeutic strategy has been recently improved by the use of biocompatible scaffolds which allow a better fixation of the cells inside the defect together with the maintenance of their original phenotype. We have recently reported that human chondrocytes can efficiently grow on a hyaluronan acid derivative biomaterial (Hyaff-11, Fidia Advanced Biopolymers, Abano Terme, Italy) and are able to express and produce collagen type II and proteoglycans, molecules expressed by differentiated cells (Grigolo et al. Biomaterials 2002). However, from the histological evaluations of the grafted tissues there is not always evidence of hyaline cartilage neo-formation even in presence of good clinical symptoms. Only few studies deals with cellular, and biochemical processes that occur during the remodeling of the graft tissue after transplantation in humans. Biopsy samples harvested from the graft have been examined using a panel of specific antibodies. It was found that cell transplantation is followed not only by a process of cartilage repair but in some cases also by a regeneration achieved through the turnover of the initial fibrocartilagineous tissue via enzymatic degradation and synthesis of newly formed collagen type II. Therefore, we examined the expression of genes encoding extracellular matrix proteins and regulatory factors essential for cell differentiation in human cartilage biopsies of patients who underwent autologous chondrocyte transplantation. Human cartilage biopsies of patients treated by autologous chondrocyte transplantation and from a multi-organ donor were used. A Real-Time RT-PCR analysis was performed in isolated chondrocytes to evaluate the expression of collagen type I, II, X, aggrecan, cathepsin B, early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for ECM proteins and regulatory proteins was carried out on paraffin embedded sections. Real-time RT-PCR analysis showed that collagen type I mRNA was expressed in all the samples evaluated while collagen type II was present even if at lower levels compared to control. Collagen type X messenger was undetectable. Aggrecan mRNA was present in all the samples at lower levels compared to donor. Cathepsin B messenger was higher in the samples compared to control. Egr-1 and Sox-9 mRNAs were expressed at lower levels compared to donor. The immunohistochemical analysis showed a slight positivity for collagen type I in all the sections. Collagen type II was found in all the samples evaluated with a positivity confined inside the cells, while the control displayed a positivity which was diffuse in the ECM. Cathepsin B was slightly positive in all the samples while the control was negative. Egr-1 protein was particularly evident in the areas negative for collagen type II. Sox-9 was positive in all the samples, with evident localization in the superficial layer. Our results provide evidence that the remodelling of the graft tissue after autologous chondrocyte transplantation is regulated by a sophisticated gene expression machinery control addressed to new cartilage formation

Aims

The aim of this retrospective study was to determine if there are differences in short-term clinical outcomes among four different types of matrix-associated autologous chondrocyte transplantation (MACT).

Bone marrow is the tissue where hemopoiesis occurs in close contact with the stromal microenvironment which support hemopoietic stem cell growth and differentiation. The bone marrow stroma is composed of a variety of different cell types providing structural and functional support for hemopoiesis: endothelial cells, adipocytes, smooth muscle cells, reticular cells, osteoblasts and stromal fibroblasts. Among these cell types, stromal fibroblasts have a peculiar biologic relevance. They are in fact able to support hemopoiesis, to differentiate towards osteogenic, chondrogenic and adipogenic lineage and to form a bone structure complete of hemopoietic marrow in in vivo assays. Their in vitro clonogenic counterpart is represented by Colony Forming Units-fibroblasts (CFU-f), which in turn give rise to Bone Marrow Stromal Cells (BMSC). In vivo bone formation by BMSC has been strikingly demonstrated and therefore these cells are considered a progenitor compartment for osteoblasts, responsible for the maintenance of bone turnover throughout life. BMSC can be easily isolated from bone marrow aspirates. Nevertheless, given the low frequency of BMSC in a marrow sample, a step of extensive in vitro expansion is required to obtain a consistent number of cells available for both reconstruction and repair of mesodermally derived tissues. Moreover, their use for gene and cell therapy of skeletal diseases requires the long-lasting engraftment of BMSC endowed with a residual proliferation potential sufficient to sustain the low, but continuous, bone turnover in adulthood. The maintenance of BMSC stemness and the possibility to reprogram their commitment is therefore a field of primary interest given their potential use in regenerative medicine. Cell therapy of bone lesions by ex vivo expanded BMSC is passing from the phase of experimental animal model to the phase of clinical trials. Bone is repaired via local delivery of cells within a scaffold. Extremely appealing is the possibility of using mesenchymal progenitors in the therapy of genetic bone diseases via systemic infusion. Under some conditions where the local microenvironment is either altered (i.e. injury) or under important remodelling processes (i.e. fetal growth), engraftment of stem and progenitor cells seems to be enhanced. A better understanding of the mechanisms controlling BMSC differentiation and engraftment is required for their exploitation in therapy of human diseases. Furthermore, a better understanding of the interactions occurring between BMSC and biomaterials used to deliver cells in vivo will hopefully extend the field of therapeutic applications of mesenchymal progenitors. In this talk we will go through our experimental evidences on: a) influence of signaling molecule; b) transplantation route and engraftment; c) biomaterials. Growth factors are essential for a number of cellular functions. Our results show that FGF-2 supplemented BMSC primary cultures display better differentiation potential, a higher degree of osteogenicity and undergo an early increase in telomere size followed by a gradual decrease, whereas in control cultures telomere length decreases with increasing population doublings. In conjunction with clonogenic culture conditions, FGF-2 supplementation extends the life-span of BMSC to over 70 doublings and preserves their differentiation potential up to 50 doublings. All together, these data suggest that FGF-2 supplementation in vitro selects for the survival of a particular subset of cells enriched in pluripotent mesenchymal precursors and may be useful to obtain a large number of cells for mesenchymal tissue repair. BMSC intravenous infusion has been proposed as a means to support the hematopoiesis in Bone Marrow Transplants or as a vehicle for gene therapy. However, it seems that this route of injection leads to engraftment of a small proportion of BMSC. We have transplanted human BMSC transduced with the human erythropoietin gene, either intravenously or subcutaneously in NOD/SCID mice. Efficiency of engraftment was evaluated monitoring the hematocrit levels. Systemic infusion never increased hematocrit levels, whereas subcutaneous transplantation of the same number of cells induced an important increase of the hematocrit for at least two months. To determine whether the transient effect was due to cell loss or to reduction in expression, we recovered the cells implanted into a tridimensional scaffold, after the normalization of the hematocrit, expanded them in vitro, and re-implanted them in a new group of mice. Again the hematocrit levels rose one week after the transplantation. These results demonstrate that ex-vivo expanded human BMSC are not transplantable by systemic infusion, whereas the local implantation into a 3D scaffold allows their long term engraftment. Biomaterials for bone regeneration should have a suitable structure to allow cell adhesion and an ideal level of vascularisation, a key factor to achieve new bone formation. Furthermore, they have to be informative, driving the cells towards osteogenesis and allowing the deposition of bone extracellular matrix. Our results indicate that BMSC need a mineralized scaffold to initiate bone formation which will occur with an extent proportional to the availability of biomaterial surface

Aims

The main aims were to identify risk factors predictive of a radiolucent line (RLL) around the acetabular component with an interface bioactive bone cement (IBBC) technique in the first year after THA, and evaluate whether these risk factors influence the development of RLLs at five and ten years after THA.

Biomaterials improve the quality of life for an ever increasing number of people each year. The range of applications is vast and includes such things as joint and limb replacements, artificial arteries and skin, contact lenses and dentures. Ceramic biomaterials can be divided roughly into three main types governed by their in vivo behaviour and tissue response. In broad terms, there are the bioresorbable ceramics (b-tricalciumphosphate), bioreactive (hydroxyapatite, fluorapatite and bioglass) and bioinert (alumina, zirconia and pyrolytic carbon). The resorbable ones are incorporated into the surrounding tissue, or may even dissolve completely over a period of time. The bioreactive ones, like hydroxyapatite (used for coatings on metallic pins), encourage bonding to surrounding tissue with, for example, new bone growth being stimulated. The bionert ceramics are mostly used for structural components. Alumina and Zirconia are known for their general chemical inertness and hardness. These properties are exploited for implant purposes, where they are used as an articulating surface in hip and knee joints. Their ability to be polished to a high surface finish make them an ideal candidate for this wear application, where they operate against materials such as ultra high molecular weight polyethylene (UHMWPE). Alumina is a highly inert material and resistant to most corrosive environments, including the highly dynamic environment that is the human body. Under physiological conditions, it is classed as nearly inert, with evidence of any response from surrounding tissues and remaining essentially unchanged after many tyears of service. However, the body does recognise it as a foreign material and does attempt to isolate it by forming a layer of non adherent fibrous tissue around the implant where possible. Porous alumina may also be used to replace large sections of bone that have been removed for reasons such as cancer. Alumina has been used in dental applications. Specifically, it has been used for tooth replacements. The term high alumina ceramics is referred to materials that have the minimal content of 97% of alumina. If the percentage of minimal alumina is of 99% it is called high purity alumina ceramics. In its α phase (better famous like corundum), characterized from its particular structure and stability, the high purity alumina is used in orthopaedics, in the articulations of the hip and knee prostheses. From more than 30 years, the alumina has been successfully used. Today, more than 3,5 million of ball-heads e and 350 thousand of inserts of alumina BIOLOXA8 have been implanted confirming, in clinical use, the characteristics of low wear and biocompatibility that has allowed to reduce the problems of osteolisis induced by the polyethylene. The increase of the mechanical characteristics, the new shapes and the conical fixation have raised the reliability of the ball-heads and inserts of alumina. The BIOLOXA8forte (in commerce from 1994) is an high purity alumina (ca 99,7 %) with a small percentage of magnesium oxide (MgO). Approximately 50 years ago, magnesium oxide was introduced in the phase of sintering of the alumina, because it was discovered that a small amount of this additive prevented the increase of grains of alumina during the sintering process. It was therefore possible to have a more homogenous and dense microstructure, both characteristic directly correlated with the mechanical resistance. The suffix ‘forte’ derives from the increased mechanical characteristic caught up with the continuous optimization of the fabrication technology. Many laboratory tests and clinical cases have shown that the wear rate of alumina-alumina bearing coupling is extremely low (0.001 mm/year). If compared with metal-polyethylene (0,2 mm/year), it evidences the drastic reduction of particles of debris and therefore of the osteolysis problem. One of the main factors that the reduction of the wear rate involves is the characteristic molecular structure of alumina. Its superficial layer is composed of oxygen atoms that create a residual electric power which interacts with polarized molecules of the lubricant, binding it to the surface by strong Van der Waals ties. It is therefore guaranteed the presence of a fluid film that reduces the coefficient of clutch between the two surfaces involved during the articulation. The colour of alumina components is subjected to variations. Originally it is ivory, but it can easy stretch to the brown after sterilization with gamma beams that interact with the free valences introduced by the MgO. This change of colour does not induce changes of the mechanical characteristics. Currently the systems are completely modular and allow a wide choice of couplings. Ceramic acetabulum has been abandoned and replaced by ceramic inserts. In 1984 and subsequently in 1995, the introduction of ISO standards for the production of ceramics ball-heads and inserts and the concept of conical fixation has allowed to catch up higher reliability. The third generation of alumina has reduced the complications rates to values around 0.01% (for the 28 mm ball-heads and inserts), maintaining the excellent tribology and wear characteristics. Today, the alumina BIOLOXA8forte components are prepared in clean-room, sintered with high quality control processes, marked by laser and accurately inspected and tested. The dimension of grains of the microstructure, currently reduced to inferior values of 2 B5m, has allowed to raise the value of the mechanical resistance of about 45% (580 Mpa) of the value requested by ISO standard (400 Mpa). The tolerances between ceramics (ball-heads and inserts) and metallic parts (taper and metal shell) are fundamental for lengthening the implant reliability. It is important to control and certificate the stems and cups which the ceramic parts are applied on. Correct assembling and the respect of the compatibilities between parts (angle, material, producer) guarantee the longevity of the implants. Actually, in the orthopaedic field, the alumina application is mainly used in standard applications of the hip prostheses. Ball-heads of 22 milimeters of diameter, lengths of neck type XL, and the knee prostheses are not possible because of the mechanical characteristics of alumina not allowing to catch up the elevate stress values requested for these special applications. Between 1975 and 1977, the first studies issued that the strenght of alumina could be reinforced by the introduction of ceramic oxides. It was discovered that the strenght and toughness of alumina could endure a remarkable increment through the realization of composites with oxide of zirconium (zirconia). In the zirconia, during the phase of cooling from temperatures over 1170A1C, the grains endure a change of phase (from tetragonal to monoclinic), with an increase of 3% of volume. At ambient temperature the phase monoclina is stable. This transformation is martensitic, with energy absorption, and involves a heat-proof change of the simmetry of the structure. In the case of dispersed grains of zirconia in the alumina matrix, the transformation absorbs the energy of the crack and the tenacity of the ceramics increases. The Yttria (Y2O3) use, as stabilizing of the zirconia, has allowed to exceed the problem of the defects of the structure. It was introduced a percentage of zirconia stabilized with yttria (Y-TZP) in the alumina matrix and other mixed oxides to counterbalance the reduction of the hardness caused by particles of zirconia and to create lengthened particles during the sintering. All this studies have been used to create the new ceramics BIOLOXA8delta. Tests of biocompatibility in agreement with norms EN 30993 have been carried out allowing the implants of these new composite ceramics. The BIOLOXA8delta has a bending strenght around 1000 MPa, that is more than the double of the alumina standard (400 MPa). In the minimum fracture load test, ball-heads of 28 mm AF millimeter (neck L) have caught up values around 100 KN, very beyond the 46 KN requested by the FDA. Multiple cycles of sterilization in autoclaves have demonstrated that the BIOLOXA8delta does not endure alterations of the mechanical and tribological characteristics. On the basis of these results, BIOLOXA8delta will allow the realization of medical ceramics devices, already in study phase, like knee prosthesis, 22 mm ball-heads, thinner wallthickness of inserts, whose realization was not possible with the ceramic materials up to now available

During the last decades, several research groups have used bisphosphonates for local application to counteract secondary bone resorption after bone grafting, to improve implant fixation or to control bone resorption caused by bone morphogenetic proteins (BMPs). We focused on zoledronate (a bisphosphonate) due to its greater antiresorptive potential over other bisphosphonates. Recently, it has become obvious that the carrier is of importance to modulate the concentration and elution profile of the zoledronic acid locally. Incorporating one fifth of the recommended systemic dose of zoledronate with different apatite matrices and types of bone defects has been shown to enhance bone regeneration significantly in vivo. We expect the local delivery of zoledronate to overcome the limitations and side effects associated with systemic usage; however, we need to know more about the bioavailability and the biological effects. The local use of BMP-2 and zoledronate as a combination has a proven additional effect on bone regeneration. This review focuses primarily on the local use of zoledronate alone, or in combination with bone anabolic factors, in various preclinical models mimicking different orthopaedic conditions.

Cite this article: I. Qayoom, D. B. Raina, A. Širka, Š. Tarasevičius, M. Tägil, A. Kumar, L. Lidgren. Anabolic and antiresorptive actions of locally delivered bisphosphonates for bone repair: A review. Bone Joint Res 2018;7:548–560. DOI: 10.1302/2046-3758.710.BJR-2018-0015.R2.

Objectives

Bone tissue engineering is one of the fastest growing branches in modern bioscience. New methods are being developed to achieve higher grades of mineral deposition by osteogenically inducted mesenchymal stem cells. In addition to well established monolayer cell culture models, 3D cell cultures for stem cell-based osteogenic differentiation have become increasingly attractive to promote in vivo bone formation. One of the main problems of scaffold-based osteogenic cell cultures is the difficulty in quantifying the amount of newly produced extracellular mineral deposition, as a marker for new bone formation, without destroying the scaffold. In recent studies, we were able to show that ^99mTc-methylene diphosphonate (^99mTc-MDP), a gamma radiation-emitting radionuclide, can successfully be applied as a reliable quantitative marker for mineral deposition as this tracer binds with high affinity to newly produced hydroxyapatite (HA).

Aims

The success of anterior cruciate ligament reconstruction (ACLR) depends on osseointegration at the graft-tunnel interface and intra-articular ligamentization. Our aim was to conduct a systematic review of clinical and preclinical studies that evaluated biological augmentation of graft healing in ACLR.

Aims

We aimed to investigate factors related to the technique of medial opening wedge high tibial osteotomy which might predispose to the development of a lateral hinge fracture.

We have assessed the different adhesive properties of some of the most common bacteria associated with periprosthetic joint infection on various types of ultra high molecular Weight Polyethylene (UHMWPE). Quantitative in vitro analysis of the adhesion of biofilm producing strains of Staphylococcus aureus and Escherichia coli to physically and chemically characterised standard UHMWPE (PE), vitamin E blended UHMWPE (VE-PE) and oxidised UHMWPE (OX-PE) was performed using a sonication protocol. A significant decreased bacterial adhesion was registered for both strains on VE-PE, in comparison with that observed on PE, within 48 hours of observation (S. aureus p = 0.024 and E. coli p = 0.008). Since Vitamin E reduces bacterial adhesive ability, VE-stabilised UHMWPE could be valuable in joint replacement by presenting excellent mechanical properties, while reducing bacterial adhesiveness.

Cite this article: Bone Joint J 2014;96-B:497–501.