The Progress Report contains an overview of the scientific progress of the Preparation and сharacterization of reference materials used for prosthetic and regeneration treatment, Task 2.1 of the WP2 “Development of innovative in vitro and in silico models”. Task 2.1 of WP2 aims to collect basic data on biomaterials’ main properties (chemistry, surface, mechanical properties, etc.) and produce reference materials. The ESRs involved in the preparation of reference materials are ESRs 1,2,3,4. They prepared and characterized biomaterials with different tuned properties:

1) bulk, micro- and nano-powders, hierarchical scaffolds;

2) tailored compositions obtained by doping with therapeutic ions or by modulating the surface reactivity;

3) micro- and nano-textured surfaces personalized in terms of topography and chemistry with tailored levels of complexity and different physical-chemical properties,

4) surface functionalization with various moieties able to modulate the biological response with anti-inflammatory and antioxidant abilities.

Musculoskeletal diseases are increasing globally, particularly due to an ageing population, necessitating the development of efficient and economically sustainable therapies and biomedical systems. The PREMUROSA project addresses this need by advancing in vitro models for standardized testing and designing smart biomaterials with osteoconductive, angiogenic, and antibacterial properties.

Innovative 3D cell culture systems have been developed to better mimic bone tissue environments, such as ESR4’s bioreactor-based model using hydrogels and biomaterials. ESR5 focused on modelling osteosarcoma with a 3D porous scaffold, while ESR6 created a 3D co-culture model to study bone vascularization. ESR7 and ESR8 developed osteoarthritis models, including a microfluidic platform and studies on cartilage degeneration.

Smart biomaterials for musculoskeletal diseases have been advanced by various researchers. ESR1 and ESR3 focused on bioactive glasses with enhanced antimicrobial and cell response properties, while ESR9 investigated titanium alloys and extracellular vesicles to promote wound healing. ESR2 developed drug-releasing bioceramic powders for targeted cancer therapy, and ESR13 optimized an injectable hydrogel for treating low back pain.

This report represents the fulfilment of the requirements of PREMUROSA Deliverable 2.3, which aims to describe, by using conventional and “omic” approaches, how mesenchymal and immune cells from patients affected by musculoskeletal diseases react during early healing and regeneration phases in the presence of biomaterials. The current and first version of this deliverable was due in Month 28, but it was released with delay due to some technical problems in data generation, which was caused by the delay in the availability of some reagents. Furthermore, also COVID-19 pandemic affected the start-up of the research activities in the months between the end of 2021 and the beginning of 2022.

The main aims addressed in the present deliverable include:
✓ Test the immunobiocompatibility of bioactive glasses and titanium-based alloys;
✓ Evaluate the osteoinductive properties of the biomaterials in MSCs isolated from osteoporotic patients;
✓ Characterize EVs from the serum of healthy young and elderly subjects and patients with osteoporosis;
✓ Omically characterize EVs released by peripheral blood mononuclear cells (PBMC) and MSCs in contact
with biomaterials.

To sum up the results obtained, the assays performed until now have unravelled the potential of titanium-based discs – Ti-6Al-4V ELI polished and chemically treated (mimeTi) and the tellurium-doped bioactive glass to be considered safe for their potential implantation in the human body since these formulations do not induce detectable immune responses in vitro. Besides, mimeTi shows the best results in the metabolic activity tests performed on MSCs.

PREMUROSA mobilises 10 Beneficiaries and 6 Partner Organisations in representation of 10 countries, pursuing the ultimate goal of training a new generation of scientists with an integrated vision of the whole value chain in musculoskeletal regeneration technologies to make them capable of boosting the necessary innovations to achieve precision principles in developing innovative devices and optimized clinical applications. Deliverable D3.2 contains an overview of the scientific progress in validating 3D cocultures models to test biomaterials in comparison with in-vivo data regarding materials implantation, previously acquired. These activities have been carried out within the WP3 “Validation of the innovative in vitro and in silico models and development of decision support system” and they are still in progress.
The deliverable includes results regarding the validation with in vivo data of the co-cultures model and implanted biomaterials. Some other parts are still focused on advanced 3D cultures but are still in progress regarding the biomaterials testing that will be completed in the next few months. Immunocells treated with extracellular vesicles coming from mesenchymal stem cells (MSC) are presented as an alternative method to study the double play of MSC and immunosystem in regenerative treatment. Finally, co-culture models simulate clinical conditions after or post-treatment by assessing the similarity to natural healthy and diseased tissues as a current state of play of the PREMUROSA research activities. The ESRs involved in the preparation of the deliverable are ESRs 4-9. The report, according to each ESR-specific project, describes the advanced cell culture models, the effect of simulated biomaterial implantation or other clinical conditions and the comparison with that already described in vivo.

Deliverable D3.3 contains an overview of the scientific progress of the in-silico models developed so
far and their validation. These activities have been carried out within the WP3 “Validation of the
innovative in-vitro and in-silico models and development of decision support system” and are still ongoing. In-silico models, which are described in detail in paragraphs 2.2.1 and 2.2.2, were developed as part of a workflow that was divided into two phases, as described below:
Phase 1 – First Stage: during this stage, the PREMUROSA wet-lab data was not ready or available
for use. As a result, the in-silico models were built using historical datasets, which likely contained
relevant information and data that could be used to develop preliminary in-silico models. The methodologies used in this phase were designed to be exportable and adaptable to the PREMUROSA datasets, which were supposed to be used in the future.
Phase 2 – Second Stage: this phase, started in 2023, represents the later stage of the workflow. In
this stage, the PREMUROSA wet-lab data became available and was thus used to create and validate
more specific in-silico models. The in-silico models developed during this phase were likely more
tailored and accurate as they were based on the actual data from the PREMUROSA wet-lab
experiments. This phase allowed for the refinement and enhancement of the in-silico models by
incorporating real-world data from the experiments.
Overall, the two phases of the workflow demonstrate a progressive approach in the development of in-silico models. Phase 1 created general methodologies using historical datasets, while Phase 2 leveraged the availability of PREMUROSA wet-lab data to build and validate more specific and accurate in-silico models.

Deliverable D3.4 contains a report on the approach for the deployment of the risk and quality assessment (RAQA) and health technology assessment (HTA) features considering biomaterials combinations developed so far and their validation. These activities are part of the WP3 “Validation of the innovative in vitro and in silico models and development of decision support system”. The report does not include the PREMUROSA models description neither original data details, as they have been already reported in Deliverables D3.1-D3.3 in detail. Please note that this Deliverable is linked with Deliverable D4.1 “Report on in silico design of optimized biomaterials and on their risk assessment by advanced 3D in vitro models” which is submitted in parallel. Some herein referred data are therefore cited in both documents, for easier following up.

The objective of this Deliverable D3.4 was on analysis of the approach to the risks, safety and efficacy
assessment of new and improved biomaterials solutions for Class III medical devices. The data used in this analysis were based on original experimental designs and outcomes made within the PREMUROSA project and therefore are limited to the extent of the applicability of these experimental data (including their errors and uncertainties). The analysis has presented the methodology commonly deployed in medical device risk and quality assessment, as specified in regulative documents, manufacturing standards and practice as well as features of clinical efficacy and benefits which constitute key aspects of health technology assessment (HTA). Since most of the data analysed were only done in vitro, it is not possible to make any estimation of clinical efficacy and benefits/risks analysis on real-world data. However, this did not restrict on use of the best available estimation of the components important for risk assessment (such as complexity of the processing, potential risks related to failure or premature degradation, etc.), even with the limitations of such guess being yet subjective. The approach shown is therefore more a tool for future analysis of readouts, outcomes and endpoints for biomaterials solutions which enables early justification of whether a particular solution might have or not extra risks in practice which might not be easily spotted at the research phase.

In Deliverable D3.3 it was described how precision medicine integrates genotypes and other biological
data sources to produce personalized treatments, which is intended to overcome or improve the traditional cohort studies of evidence-based medicine. In the Deliverable D3.4 the datasets achieved within the PREMUROSA network have been analysed from the point of view of the practical medical devices (i.e. biomaterials combinations which are potentially usable for medical devices) and associated risk assessment methodology in manufacturing and use. Biomaterials are intended to interface with biological systems to evaluate, treat, augment, or replace any tissue, organ, or function of the body. Biomaterials can be synthetic or natural and can be manufactured from a variety of material types such as metals, polymers, and ceramics or a combination of these materials, including various surface treatment techniques. Biomaterials should cause no harm and no adverse effects with high efficacy and substantially added benefits/risks ratio. They should be biocompatible (e.g., no cytotoxicity) and have optimal chemical (e.g. corrosion resistance, elution, biodegradation) and physical (toughness, elasticity) properties.

Recent European regulation MDR 2017/745 defines a medical device as follows:
‘Medical device’ means any instrument, apparatus, appliance, software, implant, reagent, material
or other article intended by the manufacturer to be used, alone or in combination, for human beings
for one or more of the following specific medical purposes:

• diagnosis, prevention, monitoring, prediction, prognosis, treatment or alleviation of disease,

• diagnosis, monitoring, treatment, alleviation of, or compensation for, an injury or disability,

• investigation, replacement or modification of the anatomy or of a physiological or pathological process or state, providing information by means of in vitro examination of specimens derived from the human body, including organ, blood and tissue donations, and

• which does not achieve its principal intended action by pharmacological, immunological or metabolic means, in or on the human body, but which may be assisted in its function by such means.

In PREMUROSA applications, most of biomaterials solutions fall under Class III – the highest-risk medical devices, requiring an obligatory conformity assessment. With the PREMUROSA workflow, several in vitro and in silico models have been developed (Deliverables D3.3, D3.4) and were reported in conjunction with the integrated data sources. In this Deliverable, the focus is on the methodology of the selection of the optimal biomaterial solution based on the original experimental in vitro data (i.e. obtained within the project) by application of in silico methods based on the machine learning for the multi-criteria decision-making (MCDM).

In the framework of the PREMUROSA project, WP4 has been devoted to (i) design biomaterials and
medical devices for precise application and (ii) validate in vitro and in silico tools for design to optimize
procedures and protocols. The present Deliverable contains a report on the methodological activities
concerning the validation of in vitro and in silico models and the possible integration of omic signatures with clinical data.
Machine learning is crucial in translating omics data into clinical insights, bridging the gap between
molecular research and patient care. Omics data, such as genomics, transcriptomics, proteomics,
metabolomics, and others, provide a comprehensive view of biological systems at various molecular levels.
Integrating data from multiple omics layers (e.g., genomic, transcriptomic, and proteomic data) can provide a more comprehensive understanding of biological systems. Machine learning approaches, such as multi-omics data fusion and network-based methods, help integrate heterogeneous omics data and uncover complex interactions between molecular components. Machine learning in silico models can be trained to predict disease risk, prognosis, and treatment response at the individual level, enabling personalized medicine approaches. By analyzing omics data alongside clinical variables, these models can assist clinicians in making informed decisions about patient management and treatment strategies.

The recruitment of 13 ESRs was envisioned. The recruitment was meant to be accomplished by M4 (April 2020); however, the outburst of the COVID-19 pandemic and the consequent lock-down of most of the
Beneficiaries’ premises caused unavoidable delays in the selection procedure. Eventually the consortium managed to accomplish the ESRs’ selections by May 2020, although the appointment procedures were concluded only on 11/07/2020 (with the final appointment of ESR4), as some candidates initially selected, refused the positions, and the consortium had to contact the next ones in the ranking list, as better detailed in what follows. As a consequence of what said above, the recruitment procedure was delayed of roughly 4 months, as all ESRs are expected to sign an employment contract with their respective host institutions by September 2020 at the latest. Such a delay did not jeopardise the future course of the project, for the following reasons:
a) as regards research activities, late-recruited Fellows had the possibility to catch-up with the work and to finish their 36 months recruitment within the project duration;
b) secondment periods for ESRs were flexible and were mostly planned in project Y2; the few ones that were planned in September 2020 could be easily postponed for a few months;
c) for what concerns training activities, the consortium has decided to organise the Induction Meeting and the First Network School in October 2020 (instead of July): this delay had a minor impact on the project planning and was easily minimised with the planning of the subsequent project activities.
d) it is important to underline that, in case of unforeseen events or delays as regards the envisaged recruitment of some of the Fellows, it was be ensured that also those Fellows were able to follow with distance means of communication the First Network School in October and other relevant events, so as to ensure their full integration in PREMUROSA.

The present deliverable (D5.3) contains an overview of the training offered at the network level by the consortium to the ESRs during the first 24 months of the project implementation, namely 1st January 2020 – 31st December 2021, highlighting in detail the agenda and contents of the training programme and its evaluation. As further explained in the pages to follow, all the training activities planned in the first 24 months of the project duration have been successfully accomplished; it is to be noted that on top of the ordinary training activities initially foreseen in the project Annex 1, the PREMUROSA consortium organised a very diversified on-line weekly training programme, to allow the ESRs to catch up with the delay caused by the COVID-19 pandemic with respect to their training path.

The present deliverable (D5.5) contains an overview of the training offered at the network level by the consortium to the ESRs during the second reporting period of the project implementation, namely as of 01/01/2022 till the end of the project (30/09/2024), highlighting in detail the agenda (copy provided in the Appendix) and contents of the training programme and its evaluation. As further explained in the pages to follow, all the training activities planned in the period above mentioned have been successfully accomplished.

Within this context it is of utmost importance for the project success that PREMUROSA website allows not only the scientific community, but all relevant stakeholders (policymakers, potential customers, patients and society in general), to gather up-to-date information regarding project findings and its innovative approaches, by means of a user-friendly and attractive website, which is considered by the PREMUROSA consortium as a key means to maximise project impacts.
The PREMUROSA website has been operational as of 09/04/2020. Fully in line with the provisions of the project Annex 1, the website displays key information on the project, such as consortium members and involved ESRs; project’s objectives; project envisaged outcomes and results; training opportunities as well as information on main news and upcoming events.
Aim of the present deliverable is to provide an overview of the PREMUROSA website content as well as the ELearning platform, which is a key means to ensure that PREMUROSA ESRs as well as other cohorts of PhD students and relevant stakeholders can benefit from the vast variety of outcomes and results to be produced by the project.

D6.3 has been conceived as a Deliverable aimed at gathering the abstracts of scientific papers submitted by the PREMUROSA ESRs to peer-reviewed international journals and conferences, during the project lifetime. At the time of proposal writing the ambitious target of at least 30 abstracts was fixed for the present Deliverable. By the end of the project the PREMUROSA team managed to publish 49 papers in total, thus remarkably exceeding the initially envisaged target. The list of publications can be accessed on the page “Publications”.

PREMUROSA consortium has been fully committed to sharing project results and achievements along all the project duration: to this purpose, WP6 contained a set of tasks and activities designed to ensure a proper communication and dissemination of the new knowledge generated by the project. In particular, T6.3 envisaged the organisation of three main dissemination events, namely:

• Two Dissemination/Soft Skills Workshops, held during the project lifetime, to be served for dissemination purposes but also as a key mechanism to train ESRs to deliver presentations to large audiences and to communicate effectively research results;
• A Final Conference at the project end, conceived as the key final event for disseminating projects results.

The present deliverable (D6.4) contains an overview of the Dissemination/Soft Skills Workshops, which took place during the Third and Fourth Network Schools respectively, as well as of the project Final Conference. This report provides full details for each of the above-mentioned meetings, including the agenda and the contents of the events. The reports and photos of the mentioned Workshops and Conference can be found on the “News” page.

Deliverable D6.6 presents the results of the PREMUROSA project from the perspective of potential inputs for policymakers. Science-informed policymaking is crucial for advancing precision medicine in musculoskeletal regeneration through adaptive regulations, secure data-sharing, and stakeholder collaboration. This agile approach fosters innovation while ensuring safety, accelerating the development of targeted therapies and improving patient outcomes. In this deliverable, the results of the ESRs’ research projects are evaluated from the point of their potential use as evidence for advising the stakeholders in policy. Moreover, publicly available project data collected on the Zenodo platform is discussed, as well as the recent final event of PREMUROSA, where the main findings of the project were presented to the public. Finally, the present Deliverable is complemented with an open letter for policymakers (see below), highlighting the most important points described in D6.6.