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New approaches in the development of Hypoallergenic implant material in Orthopaedics: steps to personalised medicine

Final Report Summary - HYPORTH (New approaches in the development of Hypoallergenic implant material in Orthopaedics: steps to personalised medicine)

Executive Summary:
Total joint arthroplasty is considered as one of the most successful procedures in orthopaedic surgery. Although many improvements were made, tissue reactions to biomaterials, infection and lacking fixation are still the main reasons for failure and revision surgery. Various materials - considered as “ideal” to wear resistance (e.g. CoCr-alloys) or “bioinert” (Ti-alloys) – are found to induce adverse tissue reactions or to support biofilms. Lymphocyte-mediated response to biomaterials and wear products have been shown to initiate inflammation and subsequent development of pseudotumours and osteolysis. Patients with a known metal allergy are at higher risk of developing adverse reactions to the biomaterial. Bone loss and soft tissue masses with necroses have detrimental effects on the health and quality of life of European citizens.
Local reactions to biomaterials involve cells from the monocytic lineage, lymphocytes, and bone cells. The activation/inhibition of the different cells may be caused by the bulk material, particulate debris or ionic wear released from implants.

The results of HypOrth have helped to understand local adverse reaction to total joint replacements and to improve the integration of potential hypoallergenic implants with improved biocompatibility. HypOrth further supported the necessary understanding of the underlying mechanisms of cellular reactions induced by wear. HypOrth used cell lines as culture models to test cell reactions to implant materials and wear products. 3 different patient cohorts from OVGU and UT were recruited to provide the possibility to transfer the cell line data to isolated material and cells from patients 1. with well-functioning implants, 2. receiving an implant, and 3. experiencing implant loosening.

HypOrth has completed patient recruitment after 36 months. A central patient database was successfully created by UT that securely guaranteed data transfer between OVGU and UT. Access was also granted to DTI and Progenika for microbiological and genomic/transcriptomic evaluation. Periprosthetic tissue samples from cohort 3 were thoroughly analysed for possible infection. Cell cultures from cell lines and PBMC’s from patients are also established. Extraction methods for DNA and RNA from revision tissues have been validated. The Affymetrix analyses for blood have been completed, those for periprosthetic tissue have been processed. A survey of different biomaterials and surfaces from different polymers, ceramics and metals have been included into the study analyses. Those biomaterials and surface preparations (potential prototype material as hypoallergenic implants and surface modifications) have been tested with PBMC and various cell lines. Wear particles and ionic wear chosen for cell studies reflect the present knowledge of agents that induce periprosthetic AIR.

Implant materials and surface coatings have been fabricated in sample size and were monitored. The surface preparation is durable and securely attached to the biomaterials. Wear and fretting corrosion tests are established and were validated. A wear test simulator has been established. Using the machine, final prototypes are currently being tested. All materials of new materials needed for biological assays were provided by Mathys, and all microbiological tests were performed, as well as all tests of prototype hip joint endoprosthesis were performed.
Besides, different implant prototypes for hip and knee joint were produced and biomechanical tests were performed with selected prototypes. For example, wear rates were investigated according to ISO 14242. Beside the biomechanical testing, Mathys produced and optimized the implant prototypes.

Overall, HypOrth helped to improve the diagnosis and prevention of AIR in total joint arthroplasty. New prototypes will provide options for potential new biomaterials or surface modifications for total joint arthroplasties that will prevent AIR. Further, profiling patients and assessing cytokine profiles will help to establish markers to prevent or to diagnose AIR.



Project Context and Objectives:
Background and Aims
A demographic challenge is obvious in Europe - Europe is confronted with a demographic change with an aging population as a decreasing work force has to support an increasing elderly population. The economic risk implied by this burden could be addressed by efforts to achieve an increase in Healthy Life Years. One key element would be to ensure unrestricted mobility for especially the elderly, allowing them to stay at work for longer. Irreversible joint deterioration often requires a joint replacement.
Implantation of artificial joints is one of the most successful orthopaedic interventions. However, an increasing number of patients receive revision surgery with these 10 % of these contracts an infection and 50 % develop an adverse immune reaction (AIR) to conventional implant material. At the moment the reasons for the development of AIR are inadequately understood. Our proposal contains innovative solutions concerning this problem.
A predictive approach using biomarkers will identify patients with risk to develop AIR. These patients will receive hypoallergenic endoprostheses, avoiding AIR to conventional material. Novel hypoallergenic material combinations will be developed in the frame of this consortium by material scientists and implant manufacturer.
Via immunological, microbiological and biocompatibility testing the development and production process will be improved constantly. The matching of implant material with the allergenic background of patients will avoid complicated and cost-intensive reverse reactions and is a step towards personalised medicine. A further approach is to achieve a better understanding of mechanisms of AIR, and its faster and easier diagnosis using sensitive diagnostic biomarkers for an accurate differentiation from low-grade infection. Additionally, mathematical modelling of results from different methods will show us the gene regulatory network that leads to an amplification of the adverse immune response triggered by prosthetic implants and will develop predictive models of AIR process.
Work strategy and general description
To determine the described tasks and aims, samples from more than 300 patients will be collected and analysed for infection and immunological parameters as well as genetic profiles. Transcriptomic, proteomics and exomics data are analysed for defined genetic profiles (Tartu and Barcelona). Immunocompetent cells will be isolated from different patient groups and used for cell culture experiment with particulate debris, ionic prosthetic constituents to examine cytokine expression profiles as well as live-cell imaging. Standard prosthetic materials as well as future or visionary materials will be developed, surfaces modified, and used in cell response experiments to these materials. New testing methods for the materials will be established. Test series are done. Prototypes will be developed with future / visionary biomaterials.
To fulfil these aims, HypOrth uses samples from patients in primary and revision cases in the Universities of Magdeburg and Tartu. Preoperative blood samples and intraoperative tissue samples are collected, clinical data and arthroplasty related information are pseudonomized and stored in a data base. Immune status of all patients is determined. Infection data are assessed by preop aspiration and intraoperative swabbing, tissue cultures and NextGenSequencing in Denmark. >10% false negative results by preop were considered as systemic reality. Culture analyses and histologies will be further support NGS analyses.
Various cytokine profiles are used to determine the immunogenic response to the biomaterials in vivo and in vitro. Therefore, primary cells derived from patients’ blood are isolated, amplified and cultured with degradation products from arthroplasties (CoCr, ionic Co, polyethylene, ceramics, titanium). Further, besides the cytokine expression profiles the migration activity of the cells during co-culture with wear debris is measured. Tests are performed in Magdeburg.
The results are introduced into a mathematical modelling to describe a vector analysis to predict factors associated with aseptic loosening.
New testing options including pin-on-disk, fretting corrosion and wear simulator are established to test the newly developed materials and the surface-modified existing materials. Surface modification are done by proprietary processes by Mathys in Switzerland and by Hungarian Institute of Academy of Sciences to cover materials with natural ceramics.
Prototypes are designed by Mathys in Switzerland including total hip arthroplasty and total knee arthroplasty designs.
Management structure and procedures
The Project Coordinator ensured the smooth operation of the project and guaranteed that all efforts were focused towards the objectives. He submitted all required progress reports, deliverables, financial statements to the European Commission, and, with the assistance of ARTTIC he was responsible for the proper use of funds and their transfers to participants. The Hyporth office was established by and based at the coordinator in Magdeburg and at ARTTIC in Munich. The Project Office at the Coordinator was concerned with the scientific management and the co-ordination of all research activities. The Project Office at ARTTIC was responsible for administrative, financial and contractual management and the organisational co-ordination of the project activities.

Objectives of Hyporth
The main goal of HypOrth project is to identify adverse immune reactions to orthopaedic implant material and to find remedial strategies against chronic adverse reactions and the development of an endoprosthetic prototype based on hypoallergenic and antibacterial materials and the definition of individual predictive and general diagnostic biomarkers for AIR.


Objective 1:
• Identification of adverse immune reactions (AIR) to implant material and differentiation of AIR from low-grade infection in the context of prosthesis loosening; inclusion of epidemiological and clinical expertise and the finding of diagnostic biomarkers for AIR

Objective 2:
• Understanding of mechanisms of adverse immune reaction and develop predictive computational models

Objective 3:
• Finding of predictive biomarkers for application in personalised medicine

Objective 4:
• Testing of conventional and new material combinations for implant and coating on different cell systems: bone cells and immune cells for biocompatibility, antibacterial properties and evoking immunological parameters including the newly identified biomarkers

Objective 5:
• Evaluation of data, production of prototypic hypoallergenic implant


Project Results:
WP1 Patient Pool, Clinical Experts & data Management

Standard operative procedures (SOP) were elaborated and general SOP was created. Also set for collection clinical material from operating room was created. Research Ethics Committee approvals are obtained for the beginning of the study (Tartu date 09.07.2013) and for amendment of the protocol, to receive blood 40 ml instead of 30 ml in primary version (Tartu date 14.05.2014)
The intention was, that patients will be recruited into three pools: I patients bearing endoprostheses without any complication; their immune system did not develop adverse immune reaction to implant material (100+ patients; blood), II patients receiving primary endoprotheses (100+ patients; blood and capsular tissue) and III patients with suspicion of adverse immune reaction receiving revision surgery (120+, patients; blood, periprosthetic tissue).
Recruitment period was extended to 30 months, as previously estimated time and number of patients into pool 3 was not achievable due to exclusion criteria of the study protocol. The primary number collection of patients into pool was 120. Patient number was reduced due to fact, that statistical power of further study will not harmed significantly, when collection to pool 3 remains with number 87, with potential number of infected patients ~20%. This was confirmed by leader of WP 6: Mathematical modelling of regulatory network of adverse immune reaction. Altogether were collected 104 patients into this pool. Microbiological molecular analysis of samples by WP3 demonstrated only 7 patients with non-detected infection in the hospital. Even the primary idea was form three patient pools 7 patients with infection do not fit into pool 3 and reasonable is to form subgroup for these patients. Into pools 1 and 2 were decided to recruite 150 patients, to have good quality samples. This is appropriate as some analyses e.g. DNA extractions outcome can be violated. All these corrections were topic of protocol amendment.
Into pool 2 were collected 159 patients, whom had drawn blood and collected tissue samples during joint replacement surgery. Into pool 1 were collected data from 156 patients. Altogether 419 patients were recruited.
Database is created. The first version of database was built up on the File Maker software. Later on, began in August 2016 and finalised in the beginning of 2017, biomedical database RedCap developed in Vanderbilt University in USA was taken into use. Data are kept, well firewalled, in the High-Performance Computing Center of University of Tartu. Transfer data from one database to another allowed to pass exact cross-check of data and find empty fields and find out some controversies.Data are anonymised and encrypted in the database, access to database is password protected and program allows follow log-ins into the database. All partners have received access to database.
Important is to fill in all patient data fields, if data are not applicable, then will be used n/a marking.
Cross-check of data is very important, this is done regularly using cross-check data as quality control.
Altogether 419 patients’ data until in 781 fields per patient are available.

WP 02: Immunological mechanisms of aseptic loosening

Total joint replacement is a common procedure with proven success in reducing pain and improving function. However, despite good survival of implants, reasons for loosening of implant components are a not fully understand complication. Reasons of implant loss may be aseptic and septic, but the distinctions are not always clear, and the basics of aseptic loosening are unclear. Many studies indicate the involvement of the immune system in aseptic loosening, especially in metal-on-metal prostheses. WP02 should therefore investigate immunological mechanisms that may lead to prosthetic loosening. Evidence of immunological reactions gave many previous studies. As signs of immune reactions in periprosthetic tissues of MoM (metal-on-metal) prostheses with aseptic implant loosening were interpreted: appearance of diffuse and perivascular infiltrates of T- and B- lymphocytes and plasma cells often called ALVAL (Aseptic lymphocyte-dominated vasculitis-associated lesion)1. Further were observed high endothelial venules, massive fibrin exudation and accumulation of macrophages. We therefore used patient blood and tissues collected in WP01 to study immunological mechanisms in aseptic loosening. For the analysis patients were divided into four analysis groups (1= patients with fixed prostheses, n= 182; 2= patients without endoprostheses, n=107; 3= patients with a loose prostheses without infection, n=97; 4= patients with loose prostheses, in which tissue infection has been demonstrated). Here we report our findings about immunological parameters comparing patients in different times of prosthesis loosening process. All data of WP02 were statistically analysed in collaboration with WP06. In our systemic approach, investigating blood parameters (immune cell status and activation of PBMCs) comparing analysis groups, we cannot suggest any typical specific cellular immune response in analysis group 3. One reason for this may be the low number of patients with MoM. In our cohort, AG 3 mainly contained MoPs. We further incubated PBMCs on material discs (WP10). After incubation, HLA-DR, CD25, CD69 were measured by flow cytometry. From the analysis it cannot be concluded that there are no differences between the analysis groups. The limited number of patients reduces the statistical power of the analysis. Another reason is the predominantly macrophage-MGC-FBGC-based reaction after adhesion of PBMCs to material discs (WP10) and not a particle- mediated such as phagocytosis.

In the local approach, looking at tissues of loosed endoprostheses we found typical foreign body reactions (nonspecific immune reactions). Foreign body responses mounted by the host against implants comprise a series of cell and tissue reactions. These responses have been shown to cause a variety of complications, including inflammation, infection, coagulation and fibrosis. In general, fibrosis (i.e. fibrous encapsulation) surrounds the biomaterial with its interface leading to a foreign body reaction, thereby isolating the implant and foreign body reaction from the local tissue environment. Also typical for a foreign body reaction is the formation of foreign body granulomas with FBGCs, consisting of macrophages, fibroblasts, capillaries. Depending upon the form and topography of the implanted material there are macrophage layers in different thicknesses. (Anderson, 2008)2. The adsorbed blood protein-modified material surface is the substrate with which the recruited monocytes/macrophages encounter and interact. Various proteins on the surface give cells means attaching via surface receptors. Integrins are a family of adhesion receptors that mediate cell-cell or cell-extracellular matrix interactions. They consist of α and a ß chain. Using antibody detecting αX subunit, we identified αXß2 (CD11c) as a marker for tissues with foreign bodies (Chamaon et.al 2016)3. αX is also the complement factor CR4 leading to of binding further ligands of the complement cascade C3bi and fibrinogen and is an important phagocytic receptor. In an in vitro IL-4 stimulated model, MGC expression of complement receptor 4 (CR4) was increased, but it functioned here primarily as an adhesion integrin (Milde 2015)4. We showed in revision tissues that different morphological cell types express αX. It is not clear whether the tissue expression of CD11c from different cells reflects different functions of CD11c including inter- and intracellular adhesion, adhesion to foreign material, phagocytosis of material, frustrated phagocytosis and fusion of MMM-C, MMM-M to MGCs and FBGCs, functions of complement cascade. Migration of cells is a further feature of inflammation process. We therefore stained the tissues for chemokine receptors CCR4, CCR6, CCR7. We found that MGCs and FBGCs highly express chemokine receptors CCR4, CCR6, CCR7. These findings indicate that increased migration into tissue of neocapsule is a hint for foreign body reaction and is restricted to multinuclear giant cells (unpublished data). CCR4 has a pro-inflammatory role and it was shown, that CCR4+ memory T-cells underwent CCL17 ligand triggered adhesion to ICAM-1, the ligand of CD11c (Campbell 1999)5. The role of CCR4 expression FBGCs is not clear. CCR6 has also a pro-inflammatory role, recruiting Th17 and Treg cells to site of inflammation and has a role in B-lymphocyte maturation (Hirota 2017)6. The role of CCR6 expression on FBGCs is not clear. CCR7 plays a role in T-cell migration (Kobayashi 2017) and it regulates cell adhesion via its ligand binding (Banas, 2004)7. However, the role of CCR7 expressed on FBGCs is unclear. The typical ALVAL reaction is the immigration of T- and B- lymphocytes into tissue. We therefore determined the presence of perivascular T- and B- lymphocytes in tissues of analysis groups 2 and 3. A typical ALVAL reaction as described for metal-on-metal sliding pairings could not be found in AG 3 compared to AG 2. Instead, arthritic tissues (AG 2) had significantly higher T- and B-lymphocytic counts than tissues after revision (neocapsule). Taking advantage of this knowledge, we started a HypOrth overarching pilot study with larger number of MoM prostheses. In a first evaluation, there was a shift in the ratio between T- and B- lymphocytes in MoM-containing prostheses with a significantly increased risk of B-cell deposits (paper in preparation).
References

1. Willert HG, Semlitsch M. Reactions of the articular capsule to wear products of artificial joint prostheses. J Biomed Mater Res 1977;11(2):157-64.
2. Anderson JM, Rodriguez A, Chang DT. Foreign body reaction to biomaterials. Semin Immunol 2008;20(2):86-100.
3. Chamaon K, Barber H, Awiszus F, Feuerstein B, Lohmann CH. Expression of CD11c in periprosthetic tissues from failed total hip arthroplasties. J Biomed Mater Res A 2016;104(1):136-44.
4. Milde R, Ritter J, Tennent GA, Loesch A, Martinez FO, Gordon S, Pepys MB, Verschoor A, Helming L. Multinucleated Giant Cells Are Specialized for Complement-Mediated Phagocytosis and Large Target Destruction. Cell Rep 2015;13(9):1937-48.
5. Campbell JJ, Pan J, Butcher EC. Cutting edge: developmental switches in chemokine responses during T cell maturation. J Immunol 1999;163(5):2353-7.
6. Hirota K, Yoshitomi H, Hashimoto M, Maeda S, Teradaira S, Sugimoto N, Yamaguchi T, Nomura T, Ito H, Nakamura T and others. Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model. J Exp Med 2007;204(12):2803-12.
7. Banas B, Wornle M, Merkle M, Gonzalez-Rubio M, Schmid H, Kretzler M, Pietrzyk MC, Fink M, Perez de Lema G, Schlondorff D. Binding of the chemokine SLC/CCL21 to its receptor CCR7 increases adhesive properties of human mesangial cells. Kidney Int 2004;66(6):2256-63.

WP 03: Differentiation of aseptic loosening from infection
Task 3.1. Initially, the Standard Operational Procedures (SOP) used for the collection, handling, storage and analysis of perioperationally obtained patient specimens for the diagnosis of joint infections were collected, both for conventional culture at the involved clinics in Tartu and Magdeburg and for the supplemental molecular biological analysis to be performed at DTI.
Task 3.2. A total of 104 patients were included in patient pool III and underwent revision surgery. For 97 patients, the preoperational diagnosis of ‘no infection’ was confirmed, and just seven patients were diagnosed as being infected based on the perioperationally obtained specimens. Six infections were diagnosed by conventional culture and four were diagnosed using molecular biological tools (next generation sequencing). Only one single patient was solely diagnosed with an infection by sequencing.

Table 1. Records on infected pool III patients in Magdeburg. JF: Joint fluid; SO: Sonicate; biops: Tissue biopsies; +: positive; -: negative.
ID OvGU DTI
HO077 3- biops., JF - JF Staphylococcus
HO141 4+, 1- biops., no JF S. epidermidis SO S. epidermidis
HO124 1+, 0- biops., no JF S. aureus Negative
HO224 1+, 1- biops., no JF S. capitis JF SO S. epidermidis
HO225 4+, 1- biops., no JF S. epidermidis (E. faecalis) E. faecalis

Table 2. Records on infected pool III patients in Tartu.
ID UT DTI
UT142 Staphylococcus aureus Negative
UT201 Arthrobacter Negative

Further investigation of the available patient data for pool III revealed that non-Metal-on-Polymer implant combinations were overrepresented among the seven infected cases. The significance of this finding is not clear as the HypOrth cohort excluded patients preoperative diagnosed for joint infection and as the number of infected cases is very low (seven). Initiation of register studies might allow to reveal the reasons behind this incidental finding.

Table 3. Overview of material combinations of the explanted prosthesis for 102 patients in pool III and the related occurrence of infections in these joints.
Material combination # patients # infected joints
Metal on polymer 78 3
Ceramic on polymer 16 4
Ceramic on ceramics 4
Metal on metal 4

Task 3.3. Despite the low number of infected cases included in pool III (seven out of 104), further analysis of relevant data was performed to identify possible biomarkers for differential diagnosis of aseptic joints and joints with low grade infections. Due to the unfavourable statistics, it would only be possible to identify markers with large effect size. Based on a prior knowledge of the involved partners a comprehensive list of candidate markers was collected. This list was compared to the collected data on microbiological status (WP03), clinical data (WP01), cytokine profiles (WP04) and transcriptomic data (WP05).
Unfortunately, no useful biomarker could be identified. This finding was not surprising, as other investigators have found it to be very difficult indeed to identify a useful biomarker for such low-grade infections in relation to artificial joints and as the analysed cohort was not specifically designed for this task. It is important to point out that more than 99% (103/104 patients in pool III) of our patients were seemingly diagnosed correctly using conventional culture and that the clinics were very competent in identifying infected patients preoperatively as evidenced by the low number of infected cases included into the study.

WP 04: Cytokine biomarkers

The implantation of an endoprosthesis causes a foreign body reaction that is accompanied by inflammatory processes. Polyethylen particle-induced chronic granulomatous inflammation is a non-immune nonspecific reaction, stimulating mainly macrophages. After activation, macrophages secrete potent proinflammatory cytokines and chemokines to systematically recruit more macrophages and inflammatory cells (Gibon, 2017)1. In this macrophage-rich areas were detected: MCP-1, MIP-1alpha, IL1α and ß, IL-8, TNFalpha, PDGF-alpha, TGF-ß, IL-8, MCP-1, TGF-ß1 and adhesion molecules such as sICAM-1 (Shanbhag, 2007)2
WP04 should help to find out which cytokines may play a role in the process of prosthetic loosening (comparing AG 1 and AG 3) and which cytokines play a role due to the presence of a prosthesis (comparing AG 2 and AG 3). For the analysis patients were divided into four analysis groups (1 = patients with fixed prostheses, 2 = patients without endoprosthesis, 3 = patients with a loose prostheses without infection, 4 = patients with loose prostheses, in which tissue infection has been demonstrated. In our approach, both systematic and local inflammatory processes should be recorded. Therefore, we analyzed the cytokine levels in blood and tissues. Local loosening processes were not recorded, as only tissues of AG 2 and AG 3 can be obtained. We determined in (neo)capsular tissues: EGF, FGF-2, Eotaxin, TGF-alpha, G-CSF, Flt-3L, GM-CSF, Fractalkine, INF-alpha2, INF-gamma, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, PDGF-AA, IL-13, PDGF-AB/BB, IL-15, sCD40L, IL-17A, IL-1RA, IL-1alpha, IL-9, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP10, MCP-1, MIP-1alpha, MIP-1beta, RANTES, TNF-alpha, TNF-beta, VEGF. We found that Fractalkine, sCD40L, IL-4, IL-7 IL-8 and VEGF allowed the rejection of the nullhypothesis that the samples from analysis group 1 and 3 were drawn from the same distribution.
To explore the systemic effects of endoprostheses, we determined Eotaxin, bFGF, G-CSF, GM-CSF, IFN-γ, IL-β, IL-1Rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, VEGF levels in blood sera. From the analysis it was found that, no cytokine has a significant difference between analysis groups in blood sera.
We focused on the IL-8, (CXCL8) chemokine released by macrophages that have been implicated in the site of implant debris-induced inflammation (Hallab, 2017)3. In in vitro experiments was shown that cobalt-alloy particles in monocytic cell line THP-1 caused up-regulation of IL-8 (Potnis, 2013)4. Cobalt ions induce IL-8 secretion in a variety of systemic cell lines as renal epithelial cells (Devitt 2010)5. There are contradictory reports about polymer (UHMWPE) stimulation of IL-8 e.g.: IL-8 is secreted by polymer particles (Goodman and Ma 2010)6, or UHMWPE did not elicit a significant increase in cytokine release of IL-8 (Kaufman 2008)7. Interestingly, nanometre-sized UHMWPE wear particles had no significant effect on IL-8 release, however micrometre-size UHMWPE wear particles (0.1-1.0μm) caused significantly elevated osteolytic cytokine release from PBMCs (Liu, 2015)8.
We determined the amount of IL-8 in the blood sera from 286 HypOrth patients using ELISA assay. For the analysis patients were further divided into joint groups hip (150 patients) or knee (136 patients). As shown for Bio-plex data, comparing AG 1 and AG 3 we couldn`t find difference in IL-8 mean concentration giving no hints for IL-8 as a loosening marker. It therefore remains unanswered whether IL-8 plays a role in the loosening process and whether its formation is induced by metals or polymers or both.
#We found a weak correlation of IL-8 blood concentration with implant age in knee group but not in hip group. Furthermore, IL-8 blood concentration of knee surgery group correlate significantly with IL-8 determined in tissue. From our data analysis it can be deduced that for tissue or systemic reactions joint types must be considered separately from each other. With regard to the inflammatory cytokine IL-8, inflammatory processes can be detected only in the hip joint both locally and systemically. This corresponds to several other studies with smaller patient numbers (Tanaka et al., 2005)9. The IL-8 cytokine is here a factor of the implant age. Contrary to the Tanaka study, in our study IL-8 is not a marker for the revision group compared to prosthesis-free tissue (AG 2). In the study by Tanaka, increased IL-8 is correlated with osteolysis. Classification of AG 3 after or without osteolysis was not carried out in our project. The assessment of osteolysis, in particular their degrees remains difficult (Howie et al., 2013)10 and there are studies that show no association of IL-8 expression with osteolysis. In contrast to the ALVAL reaction, which according to our observations is mainly restricted to MoM, IL-8 expression in our cohort is mainly due to MoP. It would be interesting to know if IL-8 expression is material dependent. Here, the determination of IL-8 from periprosthetic tissues of MoM or blood would be interesting.
Why IL-8 correlates with implant age of TKA (Total Knee Arthroplasty), but not in THA (Total Hip Arthroplasty), may be explained by different surgical procedures. While in a THA, the capsular tissue is almost always completely removed, the capsular tissue remains in largest parts in TKA. The IL-8 implant age correlation we found is relatively weak. It can be assumed that other factors influence this correlation, including metallic parts of MoPs. Further, in an unpublished pilot study we found the following: in tissues of THA there are no preferences of anatomical localization (neocapsule vs interface) of the metals Co, Cr, Ti. In contrast, in TKA significantly more titanium and chromium are concentrated in the (neo) capsule. It would be interesting to investigate if tissue IL-8 expression in the knee joint is dependent on the implantation duration and the anatomical locus. The titanium and chromium concentrations in the knee neocapsule vary widely, therefore, an association of metal concentration with IL-8 concentration cannot be ruled out.
We found a link between IL-8 in the tissue and in the blood. On what biological basis this dependency is based is unclear. Further investigations with “organ-crossing” cells would be here helpful.

References

1. Gibon E, Amanatullah DF, Loi F, Pajarinen J, Nabeshima A, Yao Z, Hamadouche M, Goodman SB. The biological response to orthopaedic implants for joint replacement: Part I: Metals. J Biomed Mater Res B Appl Biomater 2017;105(7):2162-2173.
2. Shanbhag AS, Kaufman AM, Hayata K, Rubash HE. Assessing osteolysis with use of high-throughput protein chips. J Bone Joint Surg Am 2007;89(5):1081-9.
3. Hallab NJ, Jacobs JJ. Chemokines Associated with Pathologic Responses to Orthopedic Implant Debris. Front Endocrinol (Lausanne) 2017;8:5.
4. Potnis PA, Dutta DK, Wood SC. Toll-like receptor 4 signaling pathway mediates proinflammatory immune response to cobalt-alloy particles. Cell Immunol 2013;282(1):53-65.
5. Devitt BM, Queally JM, Vioreanu M, Butler JS, Murray D, Doran PP, O'Byrne JM. Cobalt ions induce chemokine secretion in a variety of systemic cell lines. Acta Orthop 2010;81(6):756-64.
6. Goodman SB, Ma T. Cellular chemotaxis induced by wear particles from joint replacements. Biomaterials 2010;31(19):5045-50.
7. Kaufman AM, Alabre CI, Rubash HE, Shanbhag AS. Human macrophage response to UHMWPE, TiAlV, CoCr, and alumina particles: analysis of multiple cytokines using protein arrays. J Biomed Mater Res A 2008;84(2):464-74.
8. Liu A, Richards L, Bladen CL, Ingham E, Fisher J, Tipper JL. The biological response to nanometre-sized polymer particles. Acta Biomater 2015;23:38-51.
9. Tanaka R, Yasunaga Y, Hisatome T, Yamasaki T, Iwamori H, Ochi M. Serum interleukin 8 levels correlate with synovial fluid levels in patients with aseptic loosening of hip prosthesis. J Arthroplasty 2005;20(8):1049-54.
10. Howie DW, Neale SD, Haynes DR, Holubowycz OT, McGee MA, Solomon LB, Callary SA, Atkins GJ, Findlay DM. Periprosthetic osteolysis after total hip replacement: molecular pathology and clinical management. Inflammopharmacology 2013;21(6):389-96.
11. Weitkamp JH, Reinsberg J, Bartmann P. Interleukin-8 (IL-8) preferable to IL-6 as a marker for clinical infection. Clin Diagn Lab Immunol 2002;9(6):1401.

WP 05: Transcriptomic analysis

WP5 presents an integral transcriptomic analysis to AIR. Predictive biomarkers for AIR to orthopaedic implant material are still missing. We followed a transcriptomic approach to define parameters that can be quantified in patients and that are indicative of the underlying mechanism of AIR to implant material. Predictive biomarkers serve as instrument for clinician to choose the optimal prosthetic biomaterial with a minimized adverse potential for an individual patient -as a step to personalised medicine in orthopaedic arthroplasty- ultimately avoiding revision surgery
Tasks 5.1 and 5.2 were modified from the original proposal to accommodate several treated bone and immune cells with different wear particles from WP08. Affymetrix gene expression arrays and QPCR experiments were performed for a total 381 samples corresponding to different treatments and concentrations according to the following schema:

Standard validated protocols for sample hybridization were followed and well-known bioinformatic techniques were used for the analysis of all data obtained. Given the large amount of comparisons made, a priorization scheme was designed in order to select which genes were analysed in the QPCR validation experiments. This scheme was based on i) target genes were either pool-effect, treatment effect, and/or interaction-effect genes ii) Target genes should be “recommended” by some expert knowledge (HypOrth publications and other literature, Gene Ontology and KEGG pathways) and iii) genes with mathematical potential to separate treatment effects.
These tasks helped detecting a set of genetic markers that had a clear biological implication in the risk to develop an AIR to conventional implant material. Some of the biological processes affected by the alteration of these genes are shown below:
GO:0070887 cellular response to chemical stimulus
GO:0060348 bone development
GO:0042110 T cell activation
GO:0051716 cellular response to stimulus
GO:0030154 cell differentiation
GO:0030098 lymphocyte differentiation
GO:0006469 negative regulation of protein kinase activity
GO:0007275 multicellular organismal development
GO:0001775 cell activation
GO:0048870 cell motility
GO:0060349 bone morphogenesis
GO:0072091 regulation of stem cell proliferation
GO:0002521 leukocyte differentiation
GO:0030217 T cell differentiation

Tasks 5.3 and 5.4 aimed at comparing tissues without symptoms and tissues showing histologically AIR. To this end an RNA extraction protocol from fresh biopsies has been designed and validated between PGK and OVGU. Capsular and periprosthetic tissues were conserved for describing local events of AIR and for finding of biomarkers indicating the diagnosis of an adverse immune reaction. Collection of different pieces of tissues was as follows:
- Joint capsule – from primary replacements and from revisions
- Periprosthetic tissue – from revision surgery
- Bone from primary replacements (chippings from bone reaming)
Again, a standard functional genomics approach using Affymetrix arrays and a further step with QPCR for validation was followed on 328 clinical samples. The bioinformatics analysis detected genes and biological processes relevant to the clinical symptoms. Some of the most relevant biological findings include:

TermID Term
GO:0006955 immune response

GO:0006954 inflammatory response

GO:0071222 cellular response to lipopolysaccharide

GO:0006935 chemotaxis

GO:0070374 positive regulation of ERK1 and ERK2 cascade

GO:0043406 positive regulation of MAP kinase activity

GO:0030593 neutrophil chemotaxis

GO:0007267 cell-cell signaling

GO:0070098 chemokine-mediated signaling pathway

GO:0030316 osteoclast differentiation

GO:0045672 positive regulation of osteoclast differentiation

GO:0071356 cellular response to tumor necrosis factor

GO:0051092 positive regulation of NF-kappaB transcription factor activity

GO:0071347 cellular response to interleukin-1

GO:0050729 positive regulation of inflammatory response

GO:0010628 positive regulation of gene expression

GO:0002548 monocyte chemotaxis


Finally, from these results and following the selection criteria mentioned above, in Task 5.5 a final set of relevant genes was tested using QPCR on 261 samples treated with the new material composition from WP08 defined to be probable prototypes.
All the genes and biological processes identified in the scope of WP5 form the Deliverables of the work package. The genes and biological processes identified throughout the WP5 can throw new insights into the aetiology and the prediction of AIR and validate the transcriptomic approach as an appropriate methodology for detecting relevant biomarkers. Additionally, the full normalized data from WP5 without bioinformatics analysis was also shared with WP6 to perform an additional mathematical approach to identify relevant genes for a mathematical model of AIR.

WP6 Mathematical Modelling of regulatory Network of adverse Immune Reaction

Several tasks of WP02, WP04, and WP05 dealt with the attempt to find biological markers of the aseptic loosening process.
Specific mathematical analysis procedures (based on Monte-Carlo simulations) were developed to deal with 50,000 potential biomarkers within the HYPORTH cohort originating from WP02, WP04, and WP05.
None of the 50,000 candidates showed any convincing association with aseptic loosening.
This finding may be due to the fact that each individual patient has a specific biochemical network that is responsible for the aseptic loosening process.
In 2011 Wainwright et. al. (J Bone Jt Surg Ser B 2011; 93 B:1411-1415) established on registry data of 4,668 patients that patient age at primary joint replacement surgery is the most important risk factor for the observation of aseptic loosening. This finding was confirmed by Bayliss et al. (Lancet 2017; 389:1424-1430) on 117,434 patients.
In the HYPORTH cohort the odds ratio for the observation of aseptic loosening is 3.4 (95% confidence interval 2.1 – 5.5) for patients that are younger than sixty at primary TEP implantation compared to those older than sixty.
In a modelling attempt for a predictive model all variables (from WP01, WP02, WP04, and WP05) were considered. Only age at primary TEP implantation and type of joint (originating from the Redcap database of WP01) were retained for prediction of time to revision.
A simple model involving only type of joint and age at primary TEP implantation is able to explain almost 40% of the variance of the observed implant age at revision. We have not been able to detect a further molecular process to predict implant survival.

WP 07: Exome sequencing

During the project this WP was aimed to analyse a genetic variability and its relation to the development of AIR. Standard protocols for DNA collection were developed and DNA collection was successfully completed. For a genotyping we used Illumina chip version that allowed to genotype more than 500,000 SNPs. We have collected complex clinical information in order to model the relevant outcomes and association with genotypes.

During modelling we used two different approaches. First, we analysed what are these genetic variants that predict implant revision in patients (pool3). For this purpose, we performed case-control association analysis where pool3 patients (cases) were compared against pool 1 and pool 2 subjects (controls). This analysis identified the SNPs and genes that are important for the general risk to develop AIR. The genes that affect the risk for AIR are LOC107984671, CERK, SOS1, BNC2, ARHGEF38, PAPPA, SMYD2, VAV2, IFIT2, IFIT3, PGBD5, DCC, TNXB, FBP2, PALLD, CBR4, PRPSAP2, MGAT4C, TPRA1 and TOX. Variations in these genes predict development of AIR in patients with the joint implant and each of the SNP has different impact.
TNXB was considered to be functionally the most relevant gene in relation to the AIR susceptibility as there are already existing literature information supporting its role in the bone remodelling during implant adaptation. Other genes in this list are not so well known for their impact in the bone biology. However, some of them are related to the inflammatory response. Therefore, association analysis identified bone remodelling and inflammation regulatory genes involved in development of AIR.
Next step of our modelling involved the analysis of the impact of genetic variants on the survival of the implants. Survival of implants was defined as time from primary surgery to the revision. Survival of implants has a considerable variation and is clear measure to describe the pathological processes behind the implant rejection. Implant survival is clear end-point of the pathological process, it is easy to compare between patients and its use in analysis reduces or even avoids differences between surgeons. Moreover, it is easy and reliable to measure. Therefore, implant survival is excellent outcome measure to model and develop diagnostic test for AIR.
As a result of genotyping and modelling we identified four SNPs with very high predictive value for how long the implant survives in the body. We initially identified thirty-two SNPs with significant association with the implant survival. Additional modelling reduced this number into four (Figure 1). These SNPs showed statistically significant and strong effects in association study and in linear modelling for a predicting of implant survival. Additional modelling reduced the number of SNPs from thirty-two to four (Figures 2 and 3). These four SNPs had very strong statistical significance and they tolerated further modelling for the duration from the primary surgery to implant revision. All these four markers are in new genes without any known function or without any further information about their involvement in the bone remodelling and immune modulation. Therefore, all found genes are novel and further studies should identify their role in the implant survival and in the success of the joint replacement.

Figure 1. The most significant four SNPs regulating implant age in the Manhattan plot.

The DNA markers (SNPs) that are important for the predicting a personal risk for AIR are following: rs115871127, rs16823835, rs13275667 and rs2514486 (Figure 2). Each of these markers had independent strong effect for the persistence of the implants and clear genotype-related decline in the time between primary surgery and revision. All four SNPs had statistically significant differences in survival probabilities between different genotypes.

The individual effect of each of these markers to the development of AIR was remarkable. Therefore, analysis of these markers can predict the longevity of the implant and time from primary surgery to a revision (Figure 3). The identified SNPs are in different genetic loci and have potentially different roles. Marker rs115871127 is in the intron of a non-coding RNA gene LOC105378262. Persons with AA variant of rs115871127 had a mean implant survival for 9.6 years, with AG had 20.86 years implant survival.

Figure 2. Kaplan-Meier estimators for the four SNPs regulating implant age.

The SNP rs16823835 is an intronic polymorphism of the Long Intergenic Non-Protein Coding RNA 1412 (LINC01412) gene. Persons with the AA variant have mean implant survival for 8.3 years, with AG variant it is 12.2 years and with GG variant the implant survival is 15.5 years.

The third SNP, rs13275667, is intergenic variant between the LOC107986907 and LOC105377793. A person with AA genotype has 13.3 years of survival, with AG genotype the implant survival is 8.4 years and person with GG genotype has 8.0 years of implant survival.

The last SNP rs2514486 is another intergenic SNP. Persons with the AA genotype have 7.6 years of implant survival, with GA the implant survival is 9.5 years and GG has 13.2 years of survival. Taken together we identified four SNPs that predict development of AIR in patients with joint replacement.

Figure 3. Average difference in the implant durations depending on the genotype of four SNPs. The effect of individual SNPs is illustrated.

In conclusion, detection of these four SNPs would help to predict an implant loosening and development of AIR. Therefore, DNA genotyping for these SNPs could be the diagnostic tool for AIR. Further independent studies are needed to validate the markers for clinical use.

WP 08: Testing of implant material combinations and of prototype

Studies of tribological properties of new materials intended for elements of endoprostheses mere made to define that new materials have similar or greater wear resistance compared to those used in clinical practice.
The following testing methods were developed and used for tribological and corrosion characterisation of both reference and new materials:

- Standard Test Method for Ranking Resistance of Materials to Sliding Wear Using Block-on-Ring Wear Test according to ASTM F77-98. This method allows to get the following data on material tribological properties: friction coefficient against a distance, summary weight loss, wear rate, wear mechanism, roughness measurements (before and after the test), surface topography (before and after the test) – SEM
- Fretting corrosion characterisation using Pin-on-Plate testing machine. The tribo-system consists of a stationary test pin and the plate moving at the desired oscillatory frequency and amplitude. The test pin is pressed against the plate at the defined load. The liquid media is delivered to the contact zone allowing to study fretting corrosion phenomena
- Test of real implants (hip-joints) using simulator according to ISO 14242 - Standard implants for surgery - Wear of total hip-joint prostheses. To realise these tests the design and fabrication of original SBT-01.2 hip-joint simulator, that was done (patent No P.407894). The simulator allows measurement of friction forces (for three axes) in the tribological system of an endoprosthesis. Measurements, calculations and observations are followings: coefficient of friction, weight loss control and collecting of wear debris.


Standard and High Temperature Test Results
The following tests of reference materials defined by Mathys were performed: standard tribological tests using Block-on-ring and Pin-on-Disc testers; high temperature friction tests and fretting corrosion characterisation. The block-on-ring tests: The following material associations were tested: UHMWPE-CoCrMo and Vitamys-CoCrMo. The friction coefficient was fairly increasing with rising Sommerfeld number what may suggests mixed lubrication regime in both types of articulations. The film thickness was greater for UHMWPE-on-CoCrMo articulation, and this pair was characterized by lower friction coefficient values. The pin-on-disc tests showed that the average value of UHMWPE wear rate was comparable to the literature data. The obtained results show that the modification with vitamin E caused an increase in wear resistance. Based on the results of block-on-ring tests in different temperatures, the following conclusions were made: In the case of materials for which the surface of the ring was not modified, as the temperature rises, an increase in the weight of the ring was observed. It is probably caused by mass transfer from polymer blocks to rings made of PEEK. For rings with surface modification, the wear of rings made of PEEK was observed and it decrease as the temperature of the lubricating medium increased. The values of the coefficient of friction for both material combinations at all temperatures of the lubricating medium were comparable. The methodology proposed for fretting corrosion tests is correct and is reflected in studies carried out in accordance with ASTM F1875 standard.


The main results of the material tests are followings:
The use of polycarbonate-urethane elastomers for acetabular components in hip surgery is a viable option, based upon simulator wear testing of urethane polymers as bearing surfaces in joint replacement. The wear rates observed for the PCU bearing are considerable less than that for ultra-high molecular weight polyethylene materials, suggesting that there may be an advantage to PCU usage. The specific features of friction conditions (lubrication regimes) of PCU cups articulation as compared to those of UHMWPE ones were found due to use of new INOP simulator with simultaneous measurement of total friction factor. The simulator test results reveal about following mechanisms at the interface which need to be further studied:

• Polymer sorption mechanisms and its influence on the friction and wear parameters of HIPs
• Mechanisms of lubrication regime transformation during one cycle and the whole test
• Wear debris generation and compaction processes and their dependence on lubrication regime



Antibacterial properties:
A total of 21 reference implant materials and 9 novel implant materials were tested for their antibacterial properties in terms of the possible biofilm formation of these surfaces. Two test microorganisms (Staphylococcus epidermidis and Pseudomonas aeruginosa) were subjected to two test conditions (high shear and no shear) in 8 replicates each, resulting in more than 900 data points.
Grouping of the tested materials by their intended use (and thereby grouping them by surface roughness) showed that more biofilm was formed on the very rough osseointegrative surfaces, independent of the bacterium and the test condition used, exemplified by the biofilm formation data under static conditions shown in figure 1. Introduction of an antibiotic coating, did result in a significant reduction in biofilm formation (figure 2).




WP9 Ethics
In this work package all Ethical aspects of all work packages are overseen. The guidelines of FP7 (Charter of Fundamental Rights of the European union; European Group on Ethics in Science and New Technologies (EGE)) were followed.
The overall project and experiment plans were presented to the Institutional Review Boards of the Universities in Magdeburg and Tartu. Ethical votes were obtained from local IRB’s. Data protection plan in the database was described and approved by local IRB’s and supported by local computer information departments. Log-In to database required personal passwords, log files saved login footprints. Pseudonymisation of patients was described. Data management fulfilled criteria of ISO/IEC 27001:2005 standards. The study was registered in an ICMJE registry, the German Clinical Trials Registry (DRKS) (No. DRKS00010616). Over the whole funding period data protection and following ethical guidelines was continuously surveilled. Manuscripts that had been published to the end of the project period were placed in the trial registry.

WP 10: Development of hypoallergenic antibacterial implant prototype

Development of novel ceramic and polymer materials and coatings
For the development of new materials and surfaces, it is crucial to understand the state of the art (SOTA) even better, since so far there is no generally applicable guideline or standard for hypoallergenic implant materials when it comes to demonstrating effectiveness while taking account of potential risks. The tests are performed in cooperation with WP2 and WP8. Instead of testing the whole implant, simplified samples (“HypOrth Samples”) were used. To accommodate the needs of all participants who use the HypOrth Samples, a sample dimension with diameter of 12.7mm and height of 3.8mm was chosen. One surface of the samples imitates the implant surface (reference surface), the other is used for identification by marking.
As a result, a series of SOTA materials were produced in various surface qualities as well as proven coating systems in sample sizes. In total, 21 different materials (seven polymer, nine metals and five ceramics) and surfaces (coated, machined, corundum blasted or polished) were produced.
In addition to the SOTA materials, novel promising materials and coating systems are being developed taking into consideration regulatory requirements and are also being produced in sample sizes. The development of new materials is performed in cooperation with WP8. Based on materials with low allergenic potential (ceramic, polymers and Ti-alloys), MTA EK developed new coatings and materials. Complementary, Mathys assessed over 40 novel technologies regarding hypoallergenic antibacterial materials and coatings. Based on the assessment, the 9 most promising novel hypoallergenic materials and coatings were chosen: one polymer, three antibiotic coatings and five osseointegrative coatings.
To obtain adequate amounts of valid data within the planned tests of WP2 and WP8, 450 samples of each material were produced, cleaned, packaged and sterilized using implant manufacturing processes. In summary, 30 different materials and coatings (21 different reference (SOTA) HypOrth Samples and 9 different promising Novel HypOrth Samples) were manufactured. In total, more than 14000 HypOrth Samples made of polymers, metals, ceramics and different coatings were manufactured for all biological and microbiological tests at DTI and OVGU.

Production of implant prototypes for hip, knee and/or shoulder joint
Different implant prototypes for hip, knee and shoulder joint were produced and biomechanical tests were performed with selected prototypes.
Mathys developed a novel acetabular cup prototype for hip resurfacing. Clinical benefits of hip resurfacing compared to total hip arthroplasty is the higher functionality for young active patients, the high dislocation safety and the general benefit of bone preservation. Until today, hip resurfacing is prevailed with metal-on-metal (MoM) pairings. Revision rates are relatively high due to metallosis or pseudo-tumours caused by metal ions. Mathys decided to use new materials for hip resurfacing. One possible new combination is a thin vitamys cup against ceramic. vitamys is a vitamin E enhanced highly crosslinked polyethylene, also called VEPE. Wear resistance of vitamys vs. ceramic is better compared to MoM and without the risk of released metal ions. This combination contains of materials with no allergy potential. Novel thin vitamys cup prototypes were produced for the wear studies.
Demonstrators for unicondylar knee replacements were manufactured from the best hypo-allergenic hard-soft bearing combination (ceramys vs. vitamys) to avoid the potential of allergic reactions in knee replacement. The femural component is made of ceramys, the tibial component is made of vitamys. The material combination is new for this joint, but well tested and proven in other joints, e.g. hip. Manufacturing this kind of prototypes signified a technical challenge.
In anatomical shoulder joint replacements, short stem systems are spreading more and more. There are multiple reasons: less bone fixation is needed which offers enough healthy bone stock in case of a revision and a less invasive surgery which reduces the risk of infections. We combined this philosophy with biocompatible materials in a monolithic system instead of a modular one. Mathys developed a novel anatomical humeral component as shoulder prototype. The base material of the component is ceramic (ceramys). The preferred monolithic system was realized with a design change of the head and the application of Titanium plasma spray (TPS)-coating. Manufacturing these kinds of prototypes signified a technical challenge.
In summary different novel implant prototypes were manufactured for hip, knee and shoulder joint replacements. The application of new materials and designs were challenging, but their technical feasibility could be proven. Subsequently, selected prototypes were used for ddifferent biomechanical tests.

Biomechanical bench testing of implants and manufacturing of demonstrator
Implant samples made of reference materials and new promising material combinations were provided for joint simulation wear studies according to ISO 14242.
In addition, materials needed for specific testing e. g. pin-on-disc (fretting corrosion) and block-on-ring (sliding wear) at INOP were provided by Mathys to guarantee the required medical grade quality.

WP11 Training and Dissemination

Development of a corporate project identity (Logo, Website, Flyer, Poster, newsletters (2017 and 2018))
Launching of a web site - the website (www.hyporth.eu) is kept up to date and is continually updated with project news, including HypOrth related publications.
Within the HypOrth project young medical personnel from Germany as well from other countries (Singapore, China) was trained intensively. Parts of the HypOrth project were executed in form of PhD theses. In this context the PhD thesis of Qiang Ren, a Chinese Research fellow, entitled: “CXCR4 and TNF-alpha Expression by Osteoblasts and Lymphocytes treated with Metal Particles Is Regulated through the PLC/PKC- and not by the Erk-1/2 Pathway” should be emphasized. Parts of this thesis were published (see reference 6, significant results).
Dissemination activities mainly focused on:

• Major results, Publications on the HypOrth Web page
• HypOrth (results) poster for local and international meetings and conferences
• Presentation of results on institution web pages
• Press releases for local newspapers
• Summery on flyers for patients

WP12 Project Management

Ensures the proper overall management of the project to strengthen and support the Participants to achieve the objectives, complete the milestones in time and deliver the deliverables. Makes sure that the consortium’s contractual duties are carried out. Advise and guide the Participants to comply with the EU regulations and their contractual and legal requirements. Abide by the "good practice" of resources management as presented in the Financial Guidelines.
Set-up an effective communication infrastructure and foster the integrative process within the consortium and ensures that knowledge produced within the project is disseminated to the relevant target groups through publications and training.


Potential Impact:
Socio-economic impact and the wider societal implications of the project
WP 1 activities will help to find solution for adverse reactions after joint replacement. In WP2 Due to demographic change, we are confronted with an increasing need for endoprosthetic joint replacement. However, this is also associated with a steady number of necessary revision surgery. Why some patients lose their prosthesis in addition to infectious reasons, is still insufficiently clarified. WP02 served to better understand the aseptic prosthesis loss and the showed that when assessing tissue reactions, a clear distinction must be made between different materials. With CD11c we have found a reliable indication of the presence of foreign body material, especially abrasive particles. CD11c is formed by cells particularly strong at large abrasion particles (in the case of polyethylene) or a very large number of particles (pronounced metallosis). Due to the reliability of the CD11c staining, a quick assessment is possible with an (expert's) assessment of existing foreign body material in the tissue. Expensive and complex material analyzes of the tissues are averted, since CD11c indirectly shows foreign material
In WP3 no socio-economic impact and wider societal implications could be identified on the work performed and the results achieved and regarding WP04, contributions are only to be assessed in relation to the results of other WPs, e.g. WP03.Cytokines are part of established clinical diagnostics to characterize inflammatory processes. Il-8 has been shown to be a well-measurable cytokine, but the mechanisms of its regulation by materials are largely unclear. A differentiation from the infectious process is another open but interesting question. Maybe IL-8 could help to better understand differences of septic from aseptic processes, which would be crucial for therapy in the clinic.
In WP5 and in WP6 No socio-economic impact and wider societal implications could be identified on the work performed and the results achieved in both WPs.
WP7 markers could help to identify the patients who are in higher risk for the development of AIR and who require revision surgery much earlier than others. We have identified SNPs that predict even two times shorter implant survival periods, from 20 years to 10 years. This is substantial difference and the genotyping for these SNPs should be considered to all patients undergoing total joint arthroplasty.
In WP8 the hip arthroplasty procedure allows many people to improve the quality of life, function in society and often continue their professional work. The use of modern articulation is associated with an increase in the cost of the endoprosthesis. However, it gives the patient a better chance to return to full fitness by increasing the lifetime and biomechanical reliability of prosthesis. Improving the quality of endoprostheses through the use of modern materials with improved wear resistance and with biological properties reduces social costs resulting from the burden the state budget in the long term. These social costs are for example: the financial burden on institutions associated with the payment of disability benefits, costs related to incapacity to perform the current occupation (training and professional courses), the cost of loss of physical and mental health (professional advice, social services).
One aim of the HypOrth Project is the development of novel hypoallergenic implant prototypes to avoid adverse immune reactions in patients and consequently avoid stressful and expensive revision scenarios and within WP10, Mathys manufactured a large number of different samples and prototypes. The samples were used for biological, microbiological and biomechanical tests since so far there is no generally applicable guideline or standard for hypoallergenic implant materials when it comes to demonstrating effectiveness while taking account of potential risks.
Mathys developed different novel implant prototypes for hip, knee and shoulder with new promising material combination to the respective joint. Beside technical feasibility, an economical production (payable materials and technologies) of the implant prototypes were pursued. In the end, Mathys was able to prove the technical and economical feasibility of a number of implant prototypes without burdening the health care system with higher costs.


Contribution to Community and social objectives
No particular contribution from WP1 and overall, contributions of WP02 to community and social objectives are only to be assessed in relation to the results of other WPs. WST-1 assays to assess the metabolic activity of monocytic, lymphocytic and also osteoblasts have shown that the materials already used in endoprosthetics (WP10, reference materials) are well tolerated by all cells studied. The individual expression of wear found in the tissues was very different and could not be associated with a systemic immunological marker in the blood. These findings suggest that there is no pronounced systemic immune reactions (immune status) to the materials used so far, or that we are not able yet to demonstrate it with current measurement methods. No important community contribution was achieved within the framework of WP03.
For WP4 Il-8 is a marker for clinical infection (Weitkamp 2002)11. A faster diagnosis process in the distinction "aseptic or septic" is a benefit for the individual patient. Blood IL-8 may serve as surrogate marker for issue IL-8. This is a further important advantage, since only a systemic marker (blood sampling) is suitable for a simple and quick diagnosis.
In WP5 the cellular and biochemical processes running during adverse immune reaction are complex and not fully understood. The Genomic analysis here performed captures mechanisms of AIR for a better understanding and prediction of AIR. In the HypOrth project we tried to implement a model for the gene regulatory network underlying processes of AIR providing results from transcriptomics. Patients with osteoarthritis, whose therapeutic measure is to receive an artificial joint should after surgery symptom-free and integrated into their everyday life and work process as soon as possible. The inserted endoprosthesis should be functional as long as possible, without leading to infection or AIR. To avoid the development of AIR in advance, it is useful to identify patients with a risk of allergy. This transcriptomic approach can help to determine risk patients receiving then a prosthesis made of hypoallergenic material. Therefore, in the HypOrth project we searched for predictive biomarkers that provide clues to a risk of allergies as opposed to a reactive approach to treatment - after the signs and symptoms appear. Moreover, the determination of markers can help us to understand why some people develop this adverse reaction and others do not. For this purpose, a sample of blood for a valid test is a fast and easy method. Diagnostic and prognostic biomarkers are important tool for stratified medicine and they will take a leading role in in the practice of medicine. The current results validate the integral transcriptomic approach as a valid methodology of detection of biomarkers associated to the development of AIR.
No important community contribution was achieved within the framework of WP06. Work-package 7 contributed to Community by providing very useful and practical information to evaluate personalised risk for late implant rejection. This is considered to be autoimmune reaction and mainly caused by the long-term regulatory processes. What are these processes, we do not know, but we can use existing knowledge to predict the development of transplant rejection and to identify the persons with higher risk. No particular contribution is reported for WP8 and WP9.
For WP10 in the future (after regulatory approval), the community can benefit from novel hypoallergenic implants.
Beside the development of novel hypoallergenic implant prototypes, Mathys produced a large number of different samples for biological, microbiological and biomechanical testing. The results serve as basic information about the behaviour of state of the art (SOTA) as well as novel promising materials in interaction with human cells or bacteria. The test scope was extensive and has never been published in such a wide extent before. The acquired new knowledge will be important for all scientists and medical doctors in the field of orthopaedics.

Main dissemination activities and exploitation of results
WP 11 ensures dissemination of the HypOrth activities and results. In this framework publications play a key role.

List of HypOrth related publications (PUB Med listed):

1. Metsna V, Vorobjov S, Lepik K, Märtson A. Anterior knee pain following total knee replacement correlates with the OARSI score of the cartilage of the patella. Acta Orthop. 2014;85:427-432.
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2. Drynda A, Singh G, Buchhorn GH, Awiszus F, Ruetschi M, Feuerstein B, Kliche S, Lohmann CH. Metallic wear debris may regulate CXCR4 expression in vitro and in vivo. J Biomed Mater Res A. 2015;103:1940-1948.
3. Singh G, Nuechtern JV, Meyer H, Fiedler GM, Awiszus F, Junk-Jantsch S, Bruegel M, Pflueger G, Lohmann CH. Particle characterisation and cytokine expression in failed small-diameter metal-on-metal total hip arthroplasties. Bone Joint J. 2015;97-B(7):917-923.

4. Chamaon K, Barber H, Awiszus F, Feuerstein B, Lohmann CH. Expression of CD11c in periprosthetic tissues from failed total hip arthroplasties. J Biomed Mater Res A. 2016;104:136-144.

5. Singh G, Reichard T, Hameister R, Awiszus F, Schenk K, Feuerstein B, Roessner A, Lohmann C. Ballooning osteolysis in 71 failed total ankle arthroplasties. Is hydroxyapatite a risk facor? Acta Orthop. 2016; 87:401-405.

6. Drynda A, Ren Q, Buchhorn GH, Lohmann CH. The induction of CXCR4 expression in human osteoblast-like cells (MG63) by CoCr particles is regulated by the PLC-DAG-PKC pathway. J Biomed Mater Res B Appl Biomater 2016;105:2326-2332.

7. Zhytnik L, Maasalu K, Reimann E, Prans E, Kõks S, Märtson A. Mutational analysis of COL1A1 and COL1A2 genes among Estonian osteogenesis imperfecta patients. Hum Genomics 2017;11:19. doi: 10.1186/s40246-017-0115-5.

8. Tootsi K, Märtson A, Kals J, Paapstel K, Zilmer M. Metabolic factors and oxidative stress in osteoarthritis: a case-control study. Scand J Clin Lab Invest 2017;77:520-526.

9. Tootsi K, Kals J, Zilmer M, Paapstel K, Ottas A, Märtson A. Medium- and long-chain acylcarnitines are associated with osteoarthritis severity and arterial stiffness in end-stage osteoarthritis patients: a case-control study. Int J Rheum Dis. 2018 Jan 4. doi: 10.1111/1756-185X.13251. [Epub ahead of print]

10. Ho XD, Nguyen HG, Trinh LH, Reimann E, Prans E, Kõks G, Maasalu K, Le VQ, Nguyen VH, Le NTN, Phung P, Märtson A, Lattekivi F, Kõks S. Analysis of the Expression of Repetitive DNA Elements in Osteosarcoma. Front Genet. 2017 Nov 30; 8:193. doi: 10.3389/fgene.2017.00193. eCollection 2017.

11. Ho XD, Phung P, Q Le V, H Nguyen V, Reimann E, Prans E, Kõks G, Maasalu K, Le NT, H Trinh L, G Nguyen H, Märtson A, Kõks S. Whole transcriptome analysis identifies differentially regulated networks between osteosarcoma and normal bone samples. Exp Biol Med (Maywood) 2017; 242:1802-1811.

12. Drynda S, Drynda A, Feuerstein B, Kekow J, Lohmann CH, Bertrand J. The effects of cobalt and chromium ions and TGF-β patterns and mineralization in human osteoblast-like MG63 and SaOs-2 cells. J Biomed Mater Res A 2018;106: 2105-2115.

13. Drynda A, Drynda S, Kekow J, Lohmann CH, Bertrand J. Differential expression of cobalt and chromium ions as well as CoCr particles on the expression of osteogenic markers and osteoblast functions. IJMS, Major Revision.

All the HypOrthßs partners have been activiely involved in dissemination activites, here we present some presentations and and non-PUB Med listed publications:


1.) April 23 2014, Copenhagen Microbiology Center (CMC), Minisymposium, “Multi-species biofilms”, Wednesday, Oral presentation: “Detection of pathogens in multi-species biofilm in clinical settings”, Trine Rolighed Thomsen (Danish Technological Institute).

2.) HypOrth mentioned on slide:
August 23-30, ISME South Korea,
Oral presentation, Trine Rolighed Thomsen “Detection of pathogens in multispecies biofilm in clinical settings”,HypOrth mentioned on slide: (Danish Technological Institute)
3.) SICOT 19-22 Nov 2014, Polymicrobial biofilm - a challenge for diagnosis and treatment of infection,Trine Rolighed Thomsen invited speaker (Danish Technological Institute).

4.) Eurobiofilms 23-26 juni, 2015. Brno, Poster: Poly-microbial biofilms – prevalence and importance in infections. Trine R Thomsen. Price for best poster. HypOrth only mentioned @ poster visit, not mentioned on the actual poster (Danish Technological Institute).

5.) HypOrth mentioned on slide
EBJIS 2015 10-12 September 2015
Jan Lorenzen, oral presentation “Addressing Difficulties in Differential Diagnosis of Patients Presenting
with Joint Problems after THA and TKA” (Danish Technological Institute)

The following section is an overview of dissemination activities performed in different work packages:
In WP1 Articles about osteoarthritis and genomic research have been published and article describing database will be prepared. Results from WP02 were also successfully published and in WP7 a manuscript to address the major medical journal to present Hyporth´s results in under preparation.
For WP2 a poster prize was awarded on the 63. Jahrestagung der Norddeutschen Orthopäden und Unfallchirurgenvereinigung e. V. (NOUV) to Dr. Kathrin Chamaon for the work entitled: “Immunhistochemische Charakterisierung der CD11c (aXb2) Integrin Expression in periprothetischen Geweben nach Revision von Hüftendoprothesen.
In WP3 the diagnostic findings with respect to the differentiation of aseptic loosening from low grade infection and in particular the use of both conventional culture technique and molecular biological modalities were presented in a poster at the 35th Annual Meeting of the European Bone & Joint Infection Society (EBJIS), Oxford, UK, September 1-3, 2016.
• Detection of periprosthetic joint infections in presumed aseptic patients, 2016. Yijuan Xu, Jan Lorenzen, Trine Rolighed Thomsen, Kathrin Kluba, Kathrin Chamaon, Christoph Lohmann.

The general meetings which have been organised during these years represented a good opportunity for public local event as it was the case in Bilbao. The Bilbao’s General Assembly had the participation of relevant personalities from the health and research system from the Basque and Biscay Governments. In Magdeburg, during the Final Meeting a public session has been organized to present the main results and the Deputy Minister for research and digitalisation was also present.
For WP8 the biofilm data were presented by Daniel Delfosse@ the EFORT congress. Proper reference should be available in e.g. WP10.
For WP10 Mathys as industrial partner within the HypOrth Project has used the opportunity to inform the customers (subsidiaries, medical doctors and clinical staff) about the HypOrth Project, its aims and approach in the Mathys’ newsletter “move!”. The newsletter reached more than 12’000 readers worldwide in different languages (English, French and German). In addition to the distributed newsletter, a digital version is available online, too. In addition, Mathys held the talk “Material Science and Biocompatibility” at the Symposium “Adverse Immune Reactions In Patients With Knee And Hip Replacement” on 19th EFORT Congress 2018 in Barcelona, the biggest orthopaedic conference in Europe. Mathys provides support during preparation of publications. Peer-reviewed publications of pre-clinical data will be helpful for registration and clinical introduction.

Exploitation of results
The results obtained by the HypOrth project should be transferred in several ways: Publications: It is planned that all non-confidential results are published in high impact journals, if possible in open-access. The focus of the papers is on:
• Study design, patient pools
• Development of new hypo-allergic materials and surfaces
• Cellular interaction with new materials and surface coatings.
Further cooperation with appropriate companies within the project (Mathys) is planned for the development of implant prototypes from promising material specimen and testing of new designs in the Hip-simulator (Magdeburg). New cooperation with local companies is planned for the next period.
In Magdeburg the new developed materials are being tested in cell culture models. Findings concerning surface coating and surface modification obtained within this project have already impact towards metal-forming companies in Saxony-Anhalt. In connection with the certified joint-replacement centre of the clinic of orthopedic surgery and the in 2017 new founded competence centre “Orthopedics and Trauma Surgery” first cooperation with local companies have been initiated.
The HypOrth project could be a model for the generation of new interdisciplinary scientific clusters.
From Magdeburg´s perspective, the assignment of responsibility for the large and multinational HypOrth project indicates appreciation for Magdeburg as a location not only for orthopedic surgery but also for muscolo-scelettal research and immunology.We assume, that based on the results obtained from the HypOrth project new follow-up projects will be established in Magdeburg.
The results will be summarized to approach the following stakeholders:
• Lobby organizations
• Health insurances
• Decision makers
• Public health authorities
• Policy makers
• Regulation agencies (EMEA).
For patients and the lay public, the main results will be summarized on flyers which will be available in hospitals doctor´s surgeries and in pharmacies.

Outlook and future research

For WP1 a unique data of patients with osteoarthritis patients, who went to joint replacement surgery or had received artificial joint were collected and database is created, allowings further cooperation switching data into bigger databases or consortia and all partners have received database access through next address (https-//redcap.ut.ee/).
A significant insight of WP02 is, that the tissue reaction between MoM and MoP is fundamentally different. At the same time as the HypOrth project is running, other studies have also been published that confirm this knowledge (Gibon, Hallab 2017). The search for systemic blood markers for aseptic prosthetic loosening should be done with strict separation of materials used and those of the joint. In the case of MoP, the focus is on the monocytic-macrophagocytic reaction. Here it would be necessary to clarify the significance of the individual chemokine receptors on expressed from multinuclear cells. In addition, the functional importance of CD11c in periprosthetic tissues should be further investigated. For investigation of MoMs on lymphocytic reactions should be focused. First further investigations have already been carried out for this purpose.
For WP3 mainly, it is satisfying to conclude that preoperational diagnostics were very effective in excluding patients with infections in connection with their artificial joints. Furthermore, conventional culture diagnosed and identified all but one single infection using samples obtained during implant replacement surgery. The obtained data do indicate that only very few joint infections are overlooked using the standard diagnostic paradigm in place at the involved clinics. Therefore, implementation of supplemental molecular biological diagnostic modalities is not indicated based on the collected results. Interestingly, non-Metal-on-Polymer implants were overrepresented among the infected patients in the selected HypOrth cohort. Register studies are needed to investigate the significance of this incidental finding in our study.
In WP4 in a larger cohort of patients, IL-8 should be determined over time with clearly defined implant material or with / without infection. Further, IL-8 secreting should be isolated and further investigated.
In WP6 It is planned to test, if WP07 data (not part of WP06 DOW) may improve the model. Preliminary results show that some improvements may be possible due to the fact that there may be a genetic predisposal for young age at first endoprosthetic implant which results in an increased chance for these patients to observe aseptic loosening.
Related to the WP7 activities, UT would like to perform independent study with a new collection of patients and looking for additional international partners and cohorts.
Regarding WP8 the simulator test results reveals about following mechanisms at the interface which need to further be studied:
- Polymer sorption mechanisms and its influence on the friction and wear parameters of HIPs,
- Mechanisms of lubrication regime transformation during one cycle and the whole test;
- Wear debris generation and compaction processes and their dependence on lubrication regime.
The biofilm data may serve as a reference for antibacterial properties in relation to the development of new implant materials.
For WP 10 On the basis of the manufactured novel hypoallergenic implant prototypes, the next steps will be the development, production and registration of the novel endoprostheses and finally their introduction in a clinical investigation. We are confident that in the near future, a number of European patients with hip or knee osteoarthritis will benefit from these research results.


List of Websites:
www.hyporth.eu
Prof. Christoph Lohmann OTTO-VON-GUERICKE-UNIVERSITAET MAGDEBURG
Tel: 49 39167 14000
Fax: 49 391 67 13840
Email:christoph.lohmann@med.ovgu.de