Summary. The ankle X-ray has moderate diagnostic power to identify syndesmotic instability, showing large sensitivity ranges between observers. Classification systems and radiographic measurements showed moderate to high interobserver agreement, with extended classifications performing worse. Introduction. There is no consensus regarding the diagnosis and treatment of ankle fractures with respect to syndesmotic injury. The diagnosis of syndesmotic injury is currently based on intraoperative findings. Surgical indication is mainly made by ankle X-ray assessment, by several classification systems and radiographic measurements. Misdiagnosis of the injury results in suboptimal treatment, which may lead to chronic complaints, like instability and osteoarthritis. This study investigates the diagnostic power and interobserver agreement of three classification methods and radiographic measures, currently used to assess X-ankles and to identify syndesmotic injury. Patients and Methods. Twenty patients (43.2 ± 15.3yrs) with an ankle fracture, indicated for surgery, were prospectively included. All patients received a preoperative ankle X-ray, which was assessed by several observers: two orthopaedic surgeons, one trauma surgeon and two radiologists. The ankle X-ray was assessed on syndesmotic injury/stability and presence of fractures (fibula, medial/tertius malleolus). Three classification systems were used: Weber, AO-Müller (short-version n=3 options; extended-version n=27 options), Lauge-Hansen (short-version n=5 options; extended-version n=17 options) and two radiographic measurements were done: tibiofibular overlap (TFO) and ratio medial clearspace/superior clear space (MCS/SCS). All observers were instructed about the assessments before the measurements. During surgery, a proper intraoperative description of the syndesmosis was noted. Agreement (%), Intraclass Correlation Coefficients (ICC) and Kappa were calculated to determine interobserver agreement. Kappa statistic was interpreted according to Landis and Koch. To test the diagnostic power of ankle X-rays to identify syndesmotic instability, sensitivity and specificity were calculated with intraoperative findings serving as golden standard. Results. Six of 20 ankles showed syndesmotic instability intraoperatively. An overall sensitivity of 43% (specificity: 78) was found for X-rays in identifying syndesmotic instability, showing a wide range in sensitivity between observers (17–83%), with radiologists performing better (range 50–83%) than surgeons (range: 17–33%). Overall, substantial to perfect interobserver agreement (range 70–100%) was found for all short classification systems, showing an average kappa ≥0.60. The agreement reduced for more extended classification systems. E.g. observer agreement for the AO-Muller classification with 3, 9 and 27 options was respectively 85% (kappa 0.66), 68% (kappa 0.57) and 55% (kappa 0.51). One observer deviated slightly from others in all classification assessments. Removing this observer resulted in excellent agreement for all classification systems (>90%). Radiographic measurements showed moderate to high interobserver agreement, with TFO performing best (avg. ICC 0.88). Discussion/Conclusion. In ankle fractures, a preoperative X-ray has low sensitivity in detecting syndesmotic instability, showing large sensitivity ranges between observers. Further study is needed to investigate the contribution of classification systems in determining the best treatment method for syndesmotic injury. Ankle X-ray assessment using the three classification systems and radiographic measures was consistent among observers. Disagreement between observers can be attributed to intrinsic differences among the systems (e.g. stepwise classification vs. single assessment). No preference for one specific classification was found, as all showed comparable interobserver agreement. However classification systems with few options are recommended, as the observer agreement reduced with more extending classifications

There is no consensus on how to evaluate and grade pin site infection. A precise, objective and reliable pin site infectious score is warranted. The literature was reviewed for pin site infection classification systems, The Modified Gordon Score (MGS) grade 0-6 was used. The aim was to test the reliability of The Modified Gordon Infection Score. The observed agreement and inter-rater reliability were investigated between nurse and doctors. MGS was performed in the outpatient clinic at Aalborg University Hospital, Denmark on 1472 pin sites in 119 patients by one nurse and one of three orthopaedic surgeons blinded to each other's judgement. The data was stored in a Red Cap Database for further statistical analysis. The observed agreement between the nurse and the 3 orthopaedic surgeons was evaluated with a one-way random-effect model with interclass correlation with absolute agreement. Furthermore the observed agreement for each of the 3 surgeons with the nurse was calculated. The distribution of MGS infection grade in the 1472 pin sites was: Grade 0; n=1372, Grade 1; n=32, Grade 2; n=39, Grade 3; n=24, Grade 4; n=5, Grade 5; n=0, Grade 6; n=0. The observed agreement between the nurse and the surgeons was calculated as 98%. The ICC estimated between nurse and the surgeons was 0,8943 (ICC >0,85 = reliable). The grading was done by three different doctors with an agreement with the nurse as follows. Rater1 (n=416) =99,5 %, Rater2 (n=1440) =97,4%, Rater3 (n=1440) =96,6%. A limitation to this study is that the dataset represents mostly clean pin sites with MGS 0. Only 100 pin sites had signs of superficial infection MGS 1-4 none above 4. We found that the MGS infection score is highly reliable for low grade infections but we cannot conclude on reliability in severe infections

Glenoid and humeral head bone defects have long been recognized as major determinants in recurrent shoulder instability as well as main predictors of outcomes after surgical stabilization. However, a universally accepted method to quantify them is not available yet. The purpose of the present study is to describe a new CT method to quantify bipolar bone defects volume on a virtually generated 3D model and to evaluate its reproducibility. A cross-sectional observational study has been conducted. Forty CT scans of both shoulders were randomly selected from a series of exams previously acquired on patients affected by anterior shoulder instability. Inclusion criterion was unilateral anterior shoulder instability with at least one episode of dislocation. Exclusion criteria were: bilateral shoulder instability; posterior or multidirectional instability, previous fractures and/or surgery to both shoulders; congenital or acquired inflammatory, neurological, or degenerative diseases. For all patients, CT exams of both shoulders were acquired at the same time following a standardized imaging protocol. The CT data sets were analysed on a standard desktop PC using the software 3D Slicer. Computer-based reconstruction of the Hill-Sachs and glenoid bone defect were performed through Boolean subtraction of the affected side from the contralateral one, resulting in a virtually generated bone fragment accurately fitting the defect. The volume of the bone fragments was then calculated. All measurements were conducted by two fellowship-trained orthopaedic shoulder surgeons. Each measurement was performed twice by one observer to assess intra-observer reliability. Inter and intra-observer reliability were calculated. Intraclass Correlation Coefficients (ICC) were calculated using a two-way random effect model and evaluation of absolute agreement. Confidence intervals (CI) were calculated at 95% confidence level for reliability coefficients. Reliability values range from 0 (no agreement) to 1 (maximum agreement). The study included 34 males and 6 females. Mean age (+ SD) of patients was 36.7 + 10.10 years (range: 25 – 73 years). A bipolar bone defect was observed in all cases. Reliability of humeral head bone fragment measurements showed excellent intra-observer agreement (ICC: 0.92, CI 95%: 0.85 – 0.96) and very good interobserver agreement (ICC: 0.89, CI 95%: 0.80 – 0.94). Similarly, glenoid bone loss measurement resulted in excellent intra-observer reliability (ICC: 0.92, CI 95%: 0.85 – 0.96) and very good inter-observer agreement (ICC: 0.84, CI 95%:0.72 – 0.91). In conclusion, matching affected and intact contralateral humeral head and glenoid by reconstruction on a computer-based virtual model allows identification of bipolar bone defects and enables quantitative determination of bone loss

µCT images are commonly analysed to assess changes in bone density and architecture in preclinical murine models. Several platforms provide automated analysis of bone architecture parameters from volumetric regions of interest (ROI). However, segmentation of the regions of subchondral bone to create the volumetric ROIs remains a manual and time-consuming task. This study aimed to develop and evaluate automated pipelines for trabecular bone architecture analysis of mouse proximal tibia subchondral bone. A segmented dataset involving 62 knees (healthy and arthritic) from 10-week male C57BL/6 mice were used to train a U-Net type architecture, with µCT scans (downsampled) input that output segmentation and bone volume density (BV/TV) of the subchondral trabecular bone. Segmentations were upsampled and used in tandem with the original scans (10µ) as input for architecture analysis along with the thresholded trabecular bone. The analysis considered the manually and U-Net segmented ROIs using two available pipelines: the ITKBoneMorphometry library and CTan (SKYSCAN). The analyses included: bone volume (BV), total volume (TV), BV/TV, trabecular number (TbN), trabecular thickness (TbTh), trabecular separation (TbSp), and bone surface density (BSBV). There was good agreement for bone measures between the manual and U-Net pipelines utilizing ITK (R=0.88-0.98) and CTan (R=0.91-0.98). ITK and CTan showed good agreement for BV, TV, BV/TV, TbTh and BSBV (R=0.9-0.98). However, a limited agreement was seen between TbN (R=0.73) and TbSb (R=0.59) due to methodological differences in how spacing is evaluated. This U-Net/ITK pipeline seamlessly automated both segmentation and quantification of the proximal tibia subchondral bone. This automated pipeline allows the analysis of large volumes of data, and its open-source nature may enable the standardization of stereologic analysis of trabecular bone across different research groups

Introduction. Accurate assessment of alignment in pre-operative and post-operative knee radiographs is important for planning and evaluating knee replacement surgery. Existing methods predominantly rely on manual measurements using long-leg radiographs, which are time-consuming to perform and are prone to reliability errors. In this study, we propose a machine-learning-based approach to automatically measure anatomical varus/valgus alignment in pre-operative and post-operative standard AP knee radiographs. Method. We collected a training dataset of 816 pre-operative and 457 one-year post-operative AP knee radiographs of patients who underwent knee replacement surgery. Further, we have collected a separate distinct test dataset with both pre-operative and one-year post-operative radiographs for 376 patients. We manually outlined the distal femur and the proximal tibia/fibula with points to capture the knee joint (including implants in the post-operative images). This included point positions used to permit calculation of the anatomical tibiofemoral angle. We defined varus/valgus as negative/positive deviations from zero. Ground truth measurements were obtained from the manually placed points. We used the training dataset to develop a machine-learning-based automatic system to locate the point positions and derive the automatic measurements. Agreement between the automatic and manual measurements for the test dataset was assessed by intra-class correlation coefficient (ICC), mean absolute difference (MAD) and Bland-Altman analysis. Result. Analysing the agreement between the manual and automated measurements, ICC values were excellent pre-/post-operatively (0.96, CI: 0.94-0.96) / (0.95, CI: 0.95-0.96). Pre-/post-operative MAD values were 1.3°±1.4°SD / 0.7°±0.6°SD. The Bland-Altman analysis showed a pre-/post-operative mean difference (bias) of 0.3°±1.9°SD/-0.02°±0.9°SD, with pre-/post-operative 95% limits of agreement of ±3.7°/±1.8°, respectively. Conclusion. The developed machine-learning-based system demonstrates high accuracy and reliability in automatically measuring anatomical varus/valgus alignment in pre-operative and post-operative knee radiographs. It provides a promising approach for automating the measurement of anatomical alignment without the need for long-leg radiographs. Acknowledgements. This research was funded by the Wellcome Trust [223267/Z/21/Z]

Prosthetic joint infection (PJI) is an important cause of arthroplasty failure. There is no method to disclose the presence or map the distribution of the in vivo biofilm on infected arthroplasty despite the recognition that such a tool would aid intraoperative decision making and improve novel implant design. The aim of this study was to test the efficacy of four dyes to disclose bacterial biofilm in an in vitro setting. Four dyes with known affinity to bacterial biofilm were assessed to determine their efficacy to disclose biofilms in an in vitro model of PJI. Three dyes (Methylene Blue, Indocyanine Green and Rose Bengal) have established clinical utility and the other, Thioflavin T, is known to fluoresce in the presence of amyloid a known biofilm constituent. The efficacy of the dyes to discriminate between biofilms of different mass and vitality (high, low or the non-inoculated control) was determined after three minutes exposure of the biofilm to the dyes by calculating the amount of dye bound to the biofilm via sonication and spectrophotometry, quantification of the dye through standardised photographic imaging of the stained biofilm and the calculation of inter-observer agreement. Each experiment was performed in triplicate for each dye and repeated three times. For each of the disclosure dyes assessed there was significant difference demonstrated between the amount of dye bound to the high and low mass biofilms (p<0.05) as well as in the amount of dye quantified in photographic and fluorescent image assessment between biofilms of differing mass (p<0.01). There was excellent agreement between three observers, for each disclosure dye, in determining the biofilm mass of each stained disc (Kappa>0.91). This study demonstrates the efficacy of biofilm disclosure dyes in an in vitro PJI model which could one day be used to disclose and map the clinical biofilm in vivo

In the field of hand surgery, physicians are working to improve patient satisfaction by offering several minor procedures in the physician's office via the WALANT method. We seek to investigate the degree of patient satisfaction, out of pocket cost, convenience and comfort experienced with in-office hand procedures. A ten question survey consisting of a ten-point Likert scale of agreement and questions asking for a numerical answer was administered via phone call to 33 patients treated with minor hand operations in the office setting in the United States. There were 18 male and 15 female respondents with an average age of 65.59±12.64 years. Respondents underwent procedures including trigger finger release (18), needle aponeurotomy (7), and other minor hand operations. Survey responses indicated strong agreement with questions 1-3 and 6–8, with responses averaging 9.60±0.23 in these positive metrics. Questions 4 and 5, which asked whether the surgery and recovery period were painful, respectively, averaged 2.65±0.49, indicating a mild level of disagreement that either was “painful”. Additionally, most patients responded that they did not take time off work (12) or are not currently employed (11). Other respondents (3) reported taking between one to five days off work post-operatively. 27 respondents also reported an out of pocket cost averaging $382±$976, depending on insurance coverage. Patients reported a small degree of pain in the operative and post-operative period, a high degree of comfort and convenience and a high degree of satisfaction. Likewise, the patient-reported out of pocket cost was far lower than comparable surgical costs in alternate settings. These results support the use of in-office procedures for minor hand surgeries from a patient perspective and indicate a nearly universal intent to repeat any future hand operations in the office setting

The objectives of this study are to evaluate the impact of the CoVID-19 pandemic on the development of relevant emerging digital healthcare trends and to explore which digital healthcare trend does the health industry need most to support HCPs. A web survey using 39 questions facilitating Five-Point Likert scales was performed from 1.8.2020 – 31.10.2020. Of 260 participants invited, 90 participants answered the questionnaire. The participants were located in the Hospital/HCP sector in 11.9%, in other healthcare sectors in 22.2%, in the pharmaceutical sector in 11.1%, in the medical device and equipment industry in 43.3%. The Five-Point Likert scales were in all cases fashioned as from 1 (strongly disagree) to 5 (strongly agree). As the top 3 most impacted digital health care trends strongly impacted by CoVID-19, respondents named:. - remote management of patients by telemedicine, mean answer 4.44. - shared data governance under patient control, mean answer 3.80. - new virtual interaction between HCP´s and medical industry, mean answer 3.76. Respondents were asked which level of readiness of the healthcare system currently possess to cope with the current trend impacted by CoVID-19. - Digital and efficient healthcare logistics, mean answer 1.54. - Integrated health care, mean answer 1.73. - Use of big data and artificial intelligence, mean answer 2.03. Asked if collaborative research in the form of digital data platforms for research data sharing and increasing collaboration with multi-centric consortia would have a positive impact on the healthcare sector, the agreement was high with a value of mean 4.10 on the scale. We can conclude that the impact of COVID-19 appears to be a high agreement of necessary advances in digitalization in the health care sector and in the collaboration of HCPs with the health care industry. Health care professional are unsure, in how far the national health care sector is capable of transformation in healthcare logistics and integrated health care

The term macromolecular crowding is used to describe equilibria and kinetics of biochemical reactions and biological processes that occur via mutual volume exclusion of macromolecules in a highly crowded structureless medium. In vivo, the extracellular space is heavily crowded by a diverse range of macromolecules and thus, biological processes occur rapidly, whilst in vitro, in the absence of macromolecules, the same processes occur very slowly, if they are initiated at all (1-3). This talk will discuss the concept of macromolecular crowding, alone or in combination with other in vitro microenvironment modulators, in tendon engineering context. Acknowledgements: This work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, grant agreement No. 866126. This publication has emanated from research supported by grants from Science Foundation Ireland (SFI) under grant number 19/FFP/6982

Messenger RNA (mRNA) is a new class of drug that can be used to express a therapeutic protein and, in contrast to DNA, is safer and inexpensive. Among its advantages, mRNA will immediately begin to express its encoded protein in the cell cytoplasm. The protein will be expressed for a period of time, after which the mRNA is degraded. There is no risk of genetic damage, one of the concerns with plasmid DNA (pDNA) used in traditional gene therapy approaches. Nevertheless, mRNA application in tissue regeneration and regenerative medicine remains limited. In this case, mRNA must overcome its main hurdles: immunogenicity, lack of stability, and intracellular delivery. Research has been done to overcome these limitations, and the future of mRNA seems promising for tissue repair. 1,2. This keynote talk will address questions including: What are the opportunities for mRNA to improve outcomes in musculoskeletal tissue repair, in particular bone and cartilage? What are the key factors and challenges to expediting this technology to patient treatment (beyond COVID-19 vaccination)?. Acknowledgements: E.R.B thanks the cmRNAbone project funded by the European Union's Horizon 2020 research and innovation program under the grant agreement no. 874790 and the NIH R01 AR074395 from NIAMS for funding her mRNA work

The application of immune regenerative strategies to deal with unsolved pathologies, such as tendinopathies, is getting attention in the field of tissue engineering exploiting the innate immunomodulatory potential of stem cells [1]. In this context, Amniotic Epithelial Cells (AECs) represent an innovative immune regenerative strategy due to their teno-inductive and immunomodulatory properties [2], and because of their high paracrine activity, become a potential stem cell source for a cell-free treatment to overcome the limitations of traditional cell-based therapies. Nevertheless, these immunomodulatory mechanisms on AECs are still not fully known to date. In these studies, we explored standardized protocols [3] to better comprehend the different phenotypic behavior between epithelial AECs (eAECs) and mesenchymal AECs (mAECs), and to further produce an enhanced immunomodulatory AECs-derived secretome by exposing cells to different stimuli. Hence, in order to fulfill these aims, eAECs and mAECs at third passage were silenced for CIITA and Nrf2, respectively, to understand the role of these molecules in an inflammatory response. Furthermore, AECs at first passage were seeded under normal or GO-coated coverslips to study the effect of GO on AECs, and further exposed to LPS and/or IL17 priming to increase the anti-inflammatory paracrine activity. The obtained results demonstrated how CIITA and Nrf2 control the immune response of eAECs and mAECs, respectively, under standard or immune-activated conditions (LPS priming). Additionally, GO exposition led to a faster activation of the Epithelial-Mesenchymal transition (EMT) through the TGFβ/SMAD signaling pathway with a change in the anti-inflammatory properties. Finally, the combinatory inflammatory stimuli of LPS+IL17 enhanced the paracrine activity and immunomodulatory properties of AECs. Therefore, AECs-derived secretome has emerged as a potential treatment option for inflammatory disorders such as tendinopathies. Acknowledgement: This research is part of the P4FIT project ESR1, funded under the H2020-ITN-EJD-Marie-Skłodowska-Curie grant agreement 955685

Autologous osteochondral grafting has demonstrated positive outcomes for treating articular cartilage defects by replacing the damaged region with a cylindrical graft consisting of bone with a layer of cartilage, taken from a non-loadbearing region of the knee. Despite positive clinical use, factors that cause graft subsidence or poor integration are relatively unknown. The aim of this study was to develop finite element (FE) models of osteochondral grafts within a tibiofemoral joint and to investigate parameters affecting osteochondral graft stability. Initial experimental tests on cadaveric femurs were performed to calibrate the bone properties and graft-bone frictional forces for use in corresponding FE models, generated from µCT scan data. The effects of cartilage defects and osteochondral graft repair were measured by examining contact pressure changes using in vitro tests on a single cadaveric human tibiofemoral joint. Six defects were created in the femoral condyles which were subsequently treated with osteochondral autografts or metal pins. Matching µCT scan-based FE models were created, and the contact patches were compared. Sensitivity to graft bone properties was investigated. The bone material properties and graft-bone frictional forces were successfully calibrated from the initial tests with good resulting levels of agreement (CCC=0.87). The tibiofemoral joint experiment provided a range of cases to model. These cases were well captured experimentally and represented accurately in the FE models. Graft properties relative to host bone had large effects on immediate graft stability despite limited changes to resultant cartilage contact pressure. Model confidence was built through extensive validation and sensitivity testing, and demonstrated that specimen-specific properties were required to accurately represent graft behaviour. The results indicate that graft bone properties affect the immediate stability, which is important for the selection of allografts and design of future synthetic grafts. Acknowledgements. Supported by the EPSRC-EP/P001076

Digital Ventilated Cages (DVC) offer an innovative technology to obtain accurate movement data from a single mouse over time [1]. Thus, they could be used to determine the occurrence of a tendon damage event as well as inform on tissue regeneration [2,3]. Therefore, using the mouse model of tendon experimental damage, in this study it has been tested whether the recovery of tissue microarchitecture and of extracellular matrix (ECM) correlates with the motion data collected through this technology. Mice models were used to induce acute injury in Achilles tendons (ATs), while healthy ones were used as control. During the healing process, the mice were housed in DVC cages (Tecniplast) to monitor animal welfare and to study biomechanics assessing movement activity, an indicator of the recovery of tendon tissue functionality. After 28 days, the AT were harvested and assessed for their histological and immunohistochemical properties to obtain a total histological score (TSH) that was then correlated to the movement data. DVC cages showed the capacity to distinguish activity patterns in groups from the two different conditions. The data collected showed that the mice with access to the mouse wheel had a higher activity as compared to the blocked wheel group, which suggests that the extra movement during tendon healing improved motion ability. The histological results showed a clear difference between different analyzed groups. The bilateral free wheel group showed the best histological recovery, offering the highest TSH score, thus confirming the results of the DVC cages and the correlation between movement activity and structural recovery. Data obtained showed a correlation between TSH and the DVC cages, displaying structural and movement differences between the tested groups. This successful correlation allows the usage of DVC type cages as a non-invasive method to predict tissue regeneration and recovery. Acknowledgements: This research is part of the P4FIT project ESR13, funded by the H2020-ITN-EJD MSCA grant agreement No.955685

Knee joint distraction (KJD) has been associated with clinical and structural improvement and synovial fluid (SF) marker changes. However, structural changes have not yet been shown satisfactorily in regular care, since radiographic acquisition was not fully standardized. AI-based modules have shown great potential to reduce reading time, increase inter-reader agreement and therefore function as a tool for treatment outcome assessment. The objective was to analyse structural changes after KJD in patients using this AI-based measurement method, and relate these changes to clinical outcome and SF markers. 20 knee OA patients (<65 years old) were included in this study. KJD treatment was performed using an external fixation device, providing 5 mm distraction for 6 weeks. SF was aspirated before, during and immediately after treatment. Weight-bearing antero-posterior knee radiographs and WOMAC questionnaires were collected before and ~one year after treatment. Radiographs were analysed with the Knee Osteoarthritis Labelling Assistant (KOALA, IB Lab GmbH, Vienna, Austria), and 10 pre-defined biomarker levels in SF were measured by immunoassay. Radiographic one-year changes were analysed and linear regression was used to calculate associations between changes in standardized joint space width (JSW) and WOMAC, and changes in JSW and SF markers. After treatment, radiographs showed an improvement in Kellgren-Lawrence grade in 7 of 16 patients that could be evaluated; 3 showed a worsening. Joint space narrowing scores and continuous JSW measures improved especially medially. A greater improvement in JSW was significantly associated with a greater improvement in WOMAC pain (β=0.64;p=0.020). A greater increase in MCP1 (β=0.67;p=0.033) and lower increase in TGFβ1 (β=-0.787;p=0.007) were associated with JSW improvement. Despite the small number of patients, also in regular care KJD treatment shows joint repair as measured automatically on radiographs, significantly associated with certain SF marker change and even with clinical outcome

Cartilage lesions often undergo irreversible progression due to low self-repair capability of this tissue. Tissue engineered approaches based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for the treatment of cartilage lesions. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. In this study we aimed at the development of a reproducible bioprinting process followed by post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs were bioprinted, ionically crosslinked, and chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV were observed. After 56 days in culture, the bioprinted constructs were still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results showed a promising procedure to obtain 3D cartilage-like constructs that could be potential use as stable chondral tissue implants for future therapies. Acknowledgments: The National Council for Scientific and Technological Development (CNPq, Brazil – Grants # 314 724/2021-4, 307 829/2018-9, 430 860/2018-8, 142 050/2018-0 and 465 656/2014-5), the Coordination for the Improvement of Higher Educational Personnel (CAPES, Brazil – PrInt 88 887.364849/2019-00 and PrInt 88 887.310405/2018-00), the Fund for Support to Teaching, Research and Extension from the University of Campinas (FAEPEX/UNICAMP, Brazil – Grants # 2921/18, 2324/21), and the European Union's Horizon 2020 JointPromise project – Precision manufacturing of microengineered complex joint implants, under grant agreement 874 837 are acknowledged for the financial support of this study

Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), a potential mechanism of MSC therapies has been attributed to paracrine activity, in which extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor compete with adult MSC in their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSC) appear to provide a promising source. In this study, MSCs and iMSC during long term-expansion using a serum free clinical grade condition, were characterized for surface expression pattern, proliferation and differentiation capacity, and senescence rate. Culture media were collected continuously during cell expansion, and EVs were isolated. Nanoparticle tracking analysis (NTA), transmission electron microscopy, western blots, and flow cytometry were used to identify EVs. We evaluated the biological effects of MSC and iMSC-derived EVs on human chondrocytes treated with IL-1α, to mimic the OA environment. In both cell types, from early to late passages, the amount of EVs detected by NTA increased significantly, EVs collected during cells expansion, retained tetraspanins (CD9, CD63 and CD81) expression. The anti-inflammatory activity of MSC-EVs was evaluated in vitro using OA chondrocytes, the expression of IL-6, IL-8 and COX-2 was significantly reduced after the treatment with hMSC-derived EVs isolated at early passage. The miRNA content of EVs was also investigated, we identify miRNA that are involved in specific biological function. At the same time, we defined the best culture conditions to maintain iMSC and define the best time window in which to isolate EVs with highest biological activity. In conclusion, a clinical grade serum-free medium was found to be suitable for the isolation and expansion of MSCs and iMSC with increased EVs production for therapeutic applications. Acknowledgments: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671

Introduction and Objective. Digital infra-red thermography may have the capability of identifying local inflammations. Nevertheless, the role of thermography in diagnosing pin site infection has not been explored yet and the reliability and validity of this method for pin site surveillance is in question. The purpose of this study was to explore the capability and intra-rater reliability of thermography in detecting pin site infection. Materials and Methods. This explorative proof of concept study follows GRRAS -guidelines for reporting reliability and agreement studies. After clinical assessment of pin sites by one examiner using Modified Gordon Pin Infection Classification (Grade 0 – 6), thermographic images of the pin sites were captured with a FLIR C3 camera and analyzed by the FLIR tools software package. The maximum skin temperature around the pin site and the maximum temperature for the whole thermographic picture was measured. Intra-rater agreement was established and test-retests were performed with different camera angles. Results. Thirteen (4 females) patients (age 9–72 years) were included. Indications for frames: 4 fracture, 2 deformity correction, 1 lengthening, 6 bone transport. Days from surgery to thermography ranged from 27 to 385 days. Overall, 231 pin sites were included. Eleven pin sites were diagnosed with early signs of infection: five grade 1, five grade 2, one grade 3. Mean pin site temperature was 33.9 °C (29.0–35.4). With 34 °C as cut-off value for infection, sensitivity was 73%, specificity 67%, positive predictive value 10% and negative predictive value 98%. Intra-rater reliability for thermography was ICC 0.85 (0.77–0.92). The temperature measured was influenced by the camera postioning in relation to pin site with a variance of 0.2. Conclusions. Measurements of pin sites using the handheld FLIR C3 infrared camera was a reliable method and the temperature was related to infection grading. This study demonstrates that digital thermography with a handheld camera might be used for monitoring the pin sites after operations to detect early infection, however, future larger prospective studies are necessary

Bone regeneration is an area of acute medical need, but its clinical success is hampered by the need to ensure rapid vascularization of osteogenic grafts. Vascular Endothelial Growth Factor (VEGF) is the master regulator of vascular growth and during bone development angiogenesis and osteogenesis are physiologically coupled through so-called angiocrine factors produced by blood vessels. However, how to exploit this process for therapeutic bone regeneration remains a challenge (1). Here we will describe recent work aiming at understanding the cross-talk between vascular growth and osteogenesis under conditions relevant for therapeutic bone regeneration. To this end we take advantage of a unique platform to generate controlled signalling microenvironments, by the covalent decoration of fibrin matrices with tunable doses and combinations of engineered growth factors. The combination of human osteoprogenitors and hydroxyapatite in these engineered fibrin matrices provides a controlled model to investigate how specific molecular signals regulate vascular invasion and bone formation in vivo. In particular, we found that:. 1). Controlling the distribution of VEGF protein in the microenvironment is key to recapitulate its physiologic function to couple angiogenesis and osteogenesis (2);. 2). Such coupling is exquisitely dependent on VEGF dose and on a delicate equilibrium between opposing effects. A narrow range of VEGF doses specifically activates Notch1 signaling in invading blood vessels, inducing a pro-osteogenic functional state called Type H endothelium, that promotes differentiation of surrounding mesenchymal progenitors. However, lower doses are ineffective and higher ones paradoxically inhibit both vascular invasion and bone formation (Figure 1) (3);. 3). Semaphorin3a (Sema3a) acts as a novel pro-osteogenic angiocrine factor downstream of VEGF and it mediates VEGF dose-dependent effects on both vascular invasion and osteogenic progenitor stimulation. In conclusion, vascularization of osteogenic grafts is not simply necessary in order to enable progenitor survival. Rather, blood vessels can actively stimulate bone regeneration in engineered grafts through specific molecular signals that can be harnessed for therapeutic purposes. Acknowledgements: This work was supported in part by the European Union Horizon 2020 Program (Grant agreement 874790 – cmRNAbone). For any figures and tables, please contact the authors directly

Even minor lesions in articular cartilage (AC) can cause underlying bone damage creating an osteochondral (OC) defect. OC defects can cause pain, impaired mobility and can develop to osteoarthritis (OA). OA is a disease that affects nearly 10% of the population worldwide. [1]. , and represents a significant economic burden to patients and society. [2]. While significant progress has been made in this field, realising an efficacious therapeutic option for unresolved OA remains elusive and is considered one of the greatest challenges in the field of orthopaedic regenerative medicine. [3]. Therefore, there is a societal need to develop new strategies for AC regeneration. In recent years there has been increased interest in the use of tissue-specific aligned porous freeze-dried extracellular matrix (ECM) scaffolds as an off-the-shelf approach for AC repair, as they allow for cell infiltration, provide biological cues to direct target-tissue repair and permit aligned tissue deposition, desired in AC repair. [4]. However, most ECM-scaffolds lack the appropriate mechanical properties to withstand the loads passing through the joint. [5]. One solution to this problem is to reinforce the ECM with a stiffer framework made of synthetic materials, such as polylactic acid (PLA). [6]. Such framework can be 3D printed to produce anatomically accurate implants. [7]. , attractive in personalized medicine. However, typical 3D prints are static, their design is not optimized for soft-hard interfaces (OC interface), and they may not adapt to the cyclic loading passing through our joints, thus risking implant failure. To tackle this limitation, more compliant or dynamic designs can be printed, such as coil-shaped structures. [8]. Thus, in this study we use finite element modelling to create different designs that mimic the mechanical properties of AC and prototype them in PLA, using polyvinyl alcohol as support. The optimal design will be combined with an ECM scaffold containing a tailored microarchitecture mimicking aspects of native AC. Acknowledgments: This project has received funding from the European Union's Horizon Europe research and innovation MSCA PF programme under grant agreement No. 101110000

Within the field of disc degeneration-related low back pain, the spine community has been increasingly acknowledging the regenerative potential of extracellular vesicles (EVs). EVs are small lipid bilayer-delimited particles naturally released by cells, involved in intercellular signaling. They do so by interacting with recipient cells and releasing their biological cargo (e.g., mRNA, miRNA, DNA, protein, lipid). EVs derived from mesenchymal stromal cells and, more recently, also EVs from notochordal cells, the cells residing within the core of the juvenile human disc, are being actively studied. In general, they have been proposed to mitigate inflammation/catabolic processes, reduce apoptosis, stimulate proliferation and even improve the matrix producing capacity of the treated cells. Within this context, appropriate characterization of EVs is essential to increase the level of evidence that the reported effects are indeed EV-associated. To analyze the purity and biochemical composition of EV preparations the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins co-isolated/recovered with EVs. Alongside, to prove that the effects are EV-associated and not due to co-isolated factors from the tissue or cells used to derive the EVs, appropriate technical controls need to be taken along (during cell/tissue culture). As such the question arises: “what is the evidence so far?”. While from a fundamental perspective EVs are very appealing, the use of natural EVs in clinical applications is challenging. It comes with drawbacks, including biologic variability, yield, cumbersome isolation, and challenging upscaling and storage to achieve industrial levels. To date there is no FDA-approved EV-based therapy for disc-related lower back pain. Nonetheless, EV-based therapeutic approaches have unique advantages over the use of (pluripotent) stem cell-based therapies, such as a high biologic, but low immunogenic and tumorigenic potential. Acknowledgements: This talk is based on experiences from part of the project NC-CHOICE [no. 19251] of the research talent programme VICI financed by the Dutch Research Council (NWO) and the iPSpine project that receives funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 825925