As arthroplasty demand grows worldwide, the need for a novel cost-effective treatment option for articular cartilage (AC) defects tailored to individual patients has never been greater. 3D bioprinting can deposit patient cells and other biomaterials in user-defined patterns to build tissue constructs from the “bottom-up,” potentially offering a new treatment for AC defects. The aim of this research was to create bioinks that can be injected or 3D bioprinted to aid osteochondral defect repair using human cells.

Novel composite bioinks were created by mixing different ratios of methacrylated alginate (AlgMA) with methacrylated gelatin (GelMA). Chondrocytes or mesenchymal stem cells (MSCs) were then encapsulated in the bioinks and 3D bioprinted using a custom-built extrusion bioprinter. UV and double-ionic (BaCl2 and CaCl2) crosslinking was deployed following bioprinting to strengthen bioink stability in culture. Chondrocyte and MSC spheroids were also bioprinted to accelerate cell growth and development of ECM in bioprinted constructs.

Excellent viability of chondrocytes and MSCs was seen following bioprinting (>95%) and maintained in culture over 28 days, with accelerated cell growth seen with inclusion of MSC or chondrocyte spheroids in bioinks (p<0.05). Bioprinted 10mm diameter constructs maintained shape in culture over 28 days, whilst construct degradation rates and mechanical properties were improved with addition of AlgMA (p<0.05). Composite bioinks were also injected into in vitro osteochondral defects (OCDs) and crosslinked in situ, with maintained cell viability and repair of osteochondral defects seen over a 14-day period.

In conclusion we developed novel composite AlgMA/GelMA bioinks that can be triple-crosslinked, facilitating dense chondrocyte and MSC growth in constructs following 3D bioprinting. The bioink can be injected or 3D bioprinted to successfully repair in vitro OCDs, offering hope for a new approach to treating AC defects.

Patients with Paget's Disease of Bone (PDB) more frequently require total hip arthroplasty (THA) and total knee arthroplasty (TKA). However, controversy remains regarding their outcome. This project aims to evaluate the current literature regarding outcomes following THA and TKA in PDB patients.

MEDLINE, EMBASE and Cochrane databases were searched on February 15th, 2022. Inclusion criteria comprised studies evaluating outcomes following THA/TKA in PDB patients. Quality of included studies was assessed using the Newcastle-Ottawa Scale.

19 articles (published between 1976–2022) were included, comprising 58,695 patients (48,766 controls and 10,018 PDB patients), from 209 potentially relevant titles. No study was of high quality. PDB patient pooled mortality was 32.5% at mean 7.8(0.1-20) years following THA and 31.0% at mean 8.5(2-20) years following TKA. PDB patient revision rate was 4.4% at mean 7.2(0-20) years following THA and 2.2% at mean 7.4(2-20) years following TKA. Renal complications, respiratory complications, heterotopic ossification, and surgical site infection were the most common medical and surgical complications.

The largest systematic review, to date, evaluating outcomes following THA and TKA in PDB patients. All functional outcome scores improved. PDB patient revision rate was comparable to UK National Joint Registry. However, there is a significant need for prospective matched case-control studies to robustly compare outcomes in PDB patients with unaffected counterparts.

The aim of this study is to print 3D polycaprolactone (PCL) scaffolds at high and low temperature (HT/LT) combined with salt leaching to induced porosity/larger pore size and improve material degradation without compromising cellular activity of printed scaffolds. PCL solutions with sodium chloride (NaCl) particles either directly printed in LT or were casted, dried, and printed in HT followed by washing in deionized water (DI) to leach out the salt. Micro-Computed tomography (Micro-CT) and scanning electron microscope (SEM) were performed for morphological analysis. The effect of the porosity on the mechanical properties and degradation was evaluated by a tensile test and etching with NaOH, respectively. To evaluate cellular responses, human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) were cultured on the scaffolds and their viability, attachment, morphology, proliferation, and osteogenic differentiation were assessed. Micro-CT and SEM analysis showed that porosity induced by the salt leaching increased with increasing the salt content in HT, however no change was observed in LT. Structure thickness reduced with elevating NaCl content. Mass loss of scaffolds dramatically increased with elevated porosity in HT. Dog bone-shaped specimens with induced porosity exhibited higher ductility and toughness but less strength and stiffness under the tension in HT whereas they showed decrease in all mechanical properties in LT. All scaffolds showed excellent cytocompatibility. Cells were able to attach on the surface of the scaffolds and grow up to 14 days. Microscopy images of the seeded scaffolds showed substantial increase in the formation of extracellular matrix (ECM) network and elongation of the cells. The study demonstrated the ability of combining 3D printing and particulate leaching together to fabricate porous PCL scaffolds. The scaffolds were successfully printed with various salt content without negatively affecting cell responses. Printing porous thermoplastic polymer could be of great importance for temporary biocompatible implants in bone tissue engineering applications.

Articular cartilage is a multi-zonal tissue that coats the epiphysis of long bones and avoids its wear during motion. An unusual friction could micro-fracture this connective membrane and progress into an osteochondral defect (OD), where the affected cartilage suffers inflammation, fibrillation, and forfeiture of its anisotropic structure.

Clinical treatment for ODs has been focused on micro-fracture techniques, where the defect area is removed and small incisions are performed in the subchondral bone, which allows the exudation of mesenchymal stem cells (hMSCs) to the abraded zone. However, hMSCs represent less than 0.01% of the total cell population and are not able to self-organise coherently, so the treatments fail in the long term. To select, support and steer hMSCs from the bone marrow into a specific differentiation stage, and recreate the cartilage anisotropic microenvironment, multilayer dual-porosity 3D-printed scaffolds were developed.

Dual-porosity scaffolds were printed using prepared inks, containing specific ratios of poly-(d,l)lactide-co-caprolactone copolymer and gelatine microspheres of different diameters, which acted as sacrificial micro-pore templates and were leached after printing. The cell adhesion capability was investigated showing an increased cell number in dual-porosity scaffolds as compared to non-porous ones. To mimic the stiffness of the three cartilage zones, several patterns were designed, printed, and checked by dynamic-mechanical analysis under compression at 37 ºC. Three patterns with specific formulations were chosen as candidates to recreate the mechanical properties of the cartilage layers. Differentiation studies in the selected scaffolds showed the formation of mature cartilage by gene expression, protein deposition and biomolecular analysis. Given the obtained results, designed scaffolds were able to guide hMSC behaviour.

In conclusion, biocompatible, multilayer and dual-porosity scaffolds with cell entrapment capability were manufactured. These anisotropic scaffolds were able to recreate the physical microenvironment of the natural cartilage, which in turn stimulated cell differentiation and the formation of mature cartilage.

Acknowledgments: This work was supported by the EMAKIKER grant.

Numerous papers present in-vivo knee kinematics data following total knee arthroplasty (TKA) from fluoroscopic testing. Comparing data is challenging given the large number of factors that potentially affect the reported kinematics. This paper aims at understanding the effect of following three different factors: implant geometry, performed activity and analysis method.

A total of 30 patients who underwent TKA were included in this study. This group was subdivided in three equal groups: each group receiving a different type of posterior stabilized total knee prosthesis. During single-plane fluoroscopic analysis, each patient performed three activities: open chain flexion extension, closed chain squatting and chair-rising. The 2D fluoroscopic data were subsequently converted to 3D implant positions and used to evaluate the tibiofemoral contact points and landmark-based kinematic parameters.

Significantly different anteroposterior translations and internal-external rotations were observed between the considered implants. In the lateral compartment, these differences only appeared after post-cam engagement. Comparing the activities, a significant more posterior position was observed for both the medial and lateral compartment in the closed chain activities during mid-flexion. A strong and significant correlation was found between the contact-points and landmarks-based analyses method. However, large individual variations were also observed, yielding a difference of up to 25% in anteroposterior position between both methods.

In conclusion, all three evaluated factors significantly affect the obtained tibiofemoral kinematics. The individual implant design significantly affects the anteroposterior tibiofemoral position, internal-external rotation and timing of post-cam engagement. Both kinematics and post-cam engagement additionally depend on the activity investigated, with a more posterior position and associated higher patella lever arm for the closed chain activities. Attention should also be paid to the considered analysis method and associated kinematics definition: analyzing the tibiofemoral contact points potentially yields significantly different results compared to a landmark-based approach.

Functionalization of biomimetic nanomaterials allows to reproduce the composition of native bone, permitting better regeneration, while nanoscale surface morphologies provide cues for cell adhesion, proliferation and differentiation. Functionalization of 3D printed and bioprinted constructs, by plasma-assisted deposition of calcium phosphates-based (CaP) nanostructured coatings and by nanoparticles, respectively, will be presented. Stoichiometric and ion doped CaP- based nanocoatings, including green materials (mussel seashells and cuttlefish bone), will be introduced to guide tissue regeneration. We will show interactions between biomimetic surfaces and MSCs to address bone regeneration and SAOS-2 cells for bone tumor models. Our results show that combining AM and nanostructured biomimetic films permits to reproduce the architecture and the mechanical and compositional characteristics of bone. Stability behavior of the coatings, as well as MSCs behavior strongly depend on the starting CaP material, with more soluble CaPs and ion-doped ones showing better biological behavior. Green materials appear promising, as biomimetic films can be successfully obtained upon conversion of the marine precursors into hydroxyapatite. Last-not-least, nanoparticles-loaded scaffolds could be bioprinting without loss of cell viability, but ink characteristics depend on ion-doping as demonstrated for SAOS-2 cells over 14 days of culture. Biomimetic nanomaterials for functionalization in AM is a promising approach for bone modelling and regeneration.

Biofabrication is a popular technique to produce personalized constructs for tissue engineering. In this study we combined laponite (Lap), gellan gum (GG) with platelet-rich plasma (PRP) aiming to enhance the endothelial regeneration through the synergistic effects of their individual properties. Laponite has the ability to form porous three-dimensional networks mimicking the extracellular matrix structure, and PRP delivery of growth factors stimulates the endothelial cell proliferation and migration, offering a composite bioink for cell growth and support. The sustained release of these growth factors from the GG-laponite-PRP composite material over time provides a continuous source of stimulation for the cells, leading to more effective tissue engineering strategies for endothelial tissue regeneration. Four blend compositions comprising 1% w/v GG and 0.5 or 1% w/v Lap and 25% v/v PRP were combined with Wharton jelly mesenchymal stem cells (WJ-MSCs) and bioprinted into vessel-like structures with an inner diameter of 3 mm and a wall thickness of 1 mm. Stress/strain analysis revealed the elastomeric properties of the hydrogels with Young modulus values of 10 MPa. Increasing the Lap concentration led to a non-significant decrease of swelling ratio from 93 to 91%. Live/dead assay revealed cell viability of at least 76%, with the 0.5%Lap-GG viability exceeding 99% on day 21. Gradual increase of glycosaminoglycans accumulation and collagen production indicate promotion of ECM formation. The expression and membranous localization of PECAM-1 from day 7 and the granular intracellular localization of vWF after 2 weeks demonstrate in vitro endothelial functionality. In vivo subcutaneous implantation indicated the absence of any adverse immunological reactions. The results reveal the expression of both vWF and PECAM-1 by WJ-MSCs entrapped in all four construct compositions with significantly higher expression of vWF in the presence of PRP.

The primary aim was to assess the reliability of ultrasound in the assessment of humeral shaft fracture healing. The secondary aim was to estimate the accuracy of ultrasound assessment in predicting humeral shaft nonunion.

Twelve patients (mean age 54yrs [20–81], 58% [n=7/12] female) with a non-operatively managed humeral diaphyseal fracture were prospectively recruited and underwent ultrasound scanning at six and 12wks post-injury. Scans were reviewed by seven blinded observers to evaluate the presence of sonographic callus. Intra- and inter-observer reliability were determined using the weighted kappa and intraclass correlation coefficient (ICC). Accuracy of ultrasound assessment in nonunion prediction was estimated by comparing scans for patients that united (n=10/12) with those that developed a nonunion (n=2/12).

At both six and 12wks, sonographic callus was present in 11 patients (10 united, one developed a nonunion) and sonographic bridging callus (SBC) was present in seven patients (all united). Ultrasound assessment demonstrated substantial intra- (6wk kappa 0.75, 95% CI 0.47-1.03; 12wk kappa 0.75, 95% CI 0.46-1.04) and inter-observer reliability (6wk ICC 0.60, 95% CI 0.38-0.83; 12wk ICC 0.76, 95% CI 0.58-0.91). Absence of sonographic callus demonstrated a sensitivity of 50%, specificity 100%, positive predictive value (PPV) 100% and negative predictive value (NPV) 91% in nonunion prediction (accuracy 92%). Absence of SBC demonstrated a sensitivity of 100%, specificity 70%, PPV 40% and NPV 100% (accuracy 75%). Of three patients at risk of nonunion based on reduced radiographic callus formation (Radiographic Union Score for HUmeral fractures <8), one had SBC on 6wk ultrasound (and united) and the other two had non-bridging or absent sonographic callus (both developed a nonunion).

Ultrasound assessment of humeral shaft fracture healing was reliable and predictive of nonunion, and may be a useful tool in defining the risk of nonunion among patients with reduced radiographic callus formation.

Developments in the field of additive manufacturing have allowed significant improvements in the design and production of scaffolds with biologically relevant features to treat bone defects. Unfortunately, the workflow to generate personalized scaffolds is source of inaccuracies leading to a poor fit between the implant and patients' bone defects. In addition, scaffolds are often brittle and fragile, uneasing their handling by surgeons, with significant risks of fracture during their insertion in the defect. Consequently, we developed organo-mineral cementitious scaffolds displaying evolutive mechanical properties which are currently being evaluated to treat maxillofacial bone deformities in veterinary clinics. Treatment of dog patients was approved by ethic and welfare committees (CERVO-2022-14-V). To date, 8 puppies with cleft palate/lip deformities received the following treatment. Two weeks prior surgery, CT-scan of patient's skull was performed to allow for surgical planning and scaffold designing. Organo-mineral printable pastes were formulated by mixing an inorganic cement precursor (α-Ca3(PO4)2) to a self-reticulating hydrogel (silanized hyaluronic acid) supplemented with a viscosifier (hydroxymethylpropylcellulose). Scaffolds were produced by robocasting of these pastes. Surgical interventions included the reconstruction of soft tissues, and the insertion of the scaffold soaked with autologous bone marrow. Bone formation was monitored 3 and 6 months after reconstruction, and a biopsy at 6 months was performed for more detailed analyses. Scaffolds displayed great handling properties and were inserted within bone defects without significant issue with a relevant bone edges/scaffold contact. Osteointegration of the scaffolds was observed after 3 months, and regeneration of the defect at 6 months seemed quite promising. Preliminary results have demonstrated a potential of the set-up strategy to treat cleft lip/palate deformities in real, spontaneous clinical setting. Translation of these innovative scaffolds to orthopedics is planned for a near future.

In osteoarthritis, chondrocytes acquire a hypertrophic phenotype that contributes to matrix degradation. Inflammation is proposed as trigger for the shift to a hypertrophic phenotype. Using in vitro culture of human chondrocytes and cartilage explants we could not find evidence for a role of inflammatory signalling activation. We found, however, that tissue repair macrophages may contribute to the onset of hypertrophy (doi: 10.1177/19476035211021907) Intra-articularly injected triamcinolone acetonide to inhibit inflammation in a murine model of collagenase-induced osteoarthritis, increased synovial macrophage numbers and osteophytosis, confirming the role of macrophages in chondrocyte hypertrophy occurring in osteophyte formation (doi: 10.1111/bph.15780).

In search of targets to inhibit chondrocyte hypertrophy, we combined existing microarray data of different cartilage layers of murine growth plate and murine articular cartilage after induction of collagenase-induced osteoarthritis. We identified common differentially expressed genes and selected those known to be associated to inflammation. This revealed EPHA2, a tyrosine kinase receptor, as a new target. Using in silico, in vitro and in vivo models we demonstrated that inhibition of EPHA2 might be a promising treatment for osteoarthritis.

Recently, single cell RNA-seq. has revealed detailed information about different populations of chondrocytes in articular cartilage during osteoarthritis. We re-analysed a published scRNA-seq data set of healthy and osteoarthritic cartilage to obtain the differentially expressed genes in the population of hypertrophic chondrocytes compared to the other chondrocytes, applied pathway analyses and then used drug databases to search for upstream inhibitors of these pathways. This drug repurposing approach led to the selection of 6 drugs that were screened and tested using several in vitro models with human chondrocytes and cartilage explants.

In this lecture I will present this sequence of studies to highlight different approaches and models that can be used in the quest for a disease modifying drug for osteoarthritis.

Artificial Intelligence (AI) is becoming more powerful but is barely used to counter the growth in health care burden. AI applications to increase efficiency in orthopedics are rare. We questioned if (1) we could train machine learning (ML) algorithms, based on answers from digitalized history taking questionnaires, to predict treatment of hip osteoartritis (either conservative or surgical); (2) such an algorithm could streamline clinical consultation.

Multiple ML models were trained on 600 annotated (80% training, 20% test) digital history taking questionnaires, acquired before consultation. Best performing models, based on balanced accuracy and optimized automated hyperparameter tuning, were build into our daily clinical orthopedic practice. Fifty patients with hip complaints (>45 years) were prospectively predicted and planned (partly blinded, partly unblinded) for consultation with the physician assistant (conservative) or orthopedic surgeon (operative). Tailored patient information based on the prediction was automatically sent to a smartphone app. Level of evidence: IV.

Random Forest and BernoulliNB were the most accurate ML models (0.75 balanced accuracy). Treatment prediction was correct in 45 out of 50 consultations (90%), p<0.0001 (sign and binomial test). Specialized consultations where conservatively predicted patients were seen by the physician assistant and surgical patients by the orthopedic surgeon were highly appreciated and effective.

Treatment strategy of hip osteoartritis based on answers from digital history taking questionnaires was accurately predicted before patients entered the hospital. This can make outpatient consultation scheduling more efficient and tailor pre-consultation patient education.

Chondral defects in the knee have cartilage biomechanical differences due to defect size and orientation. This study examines how the tibiofemoral contact pressure is affected by increasing full-thickness chondral defect size on the medial and lateral condyle at full extension.

Isolated full-thickness, square chondral defects increasing from 0.09cm² to 1.0cm² were created sequentially on the medial and lateral femoral condyles of six human cadaveric knees with intact ligaments and menisci. Chondral defects were created 1.0cm from the femoral notch posteriorly. The knees were fixed to a uniaxial load frame and loaded from 0N to 600N at full extension. Contact pressures between the femoral and tibial condyles were measured using pressure mapping sensors. The peak contact pressure was defined as the highest value in the 2.54mm² area around the defect. The location of the peak contact pressure was determined relative to the centre of the defect.

Peak contact pressure was significantly different between (4.30MPa) 0.09cm² and (6.91MPa) 1.0cm² defects (p=0.04) on the medial condyle. On the lateral condyle, post-hoc analysis showed differences in contact pressures between (3.63MPa) 0.09cm² and (5.81MPa) 1.0cm² defect sizes (p=0.02).

The location of the stress point shifted from being posteromedial (67% of knees) to anterolateral (83%) after reaching a 0.49cm² defect size (p < 0.01) in the medial condyle. Conversely, the location of the peak contact pressure point moved from being anterolateral (50%) to a posterolateral (67%) location in defect sizes greater than 0.49cm² (p < 0.01).

Changes in contact area redistribution and cartilage stress from 0.49cm² to 1.0cm² impact adjacent cartilage integrity. The location of the maximum stress point also varied with larger defects. This study suggests that size cutoffs exist earlier in the natural history of chondral defects, as small as 0.49cm², than previously studied, suggesting a lower threshold for intervention.

In March 2020, COVID-19 was declared a pandemic by the World Health Organization. The pandemic imposed drastic changes in our social and professional routine. Professionally at all levels our hospital tasks were changed and prioritized. Surgeons and residents were deployed on rotations to fields other than their expertise in orthopaedics. Health-care education received major changes in these challenging times, and students did face difficulties in receiving education, as well as training due to limited clinical and surgical exposure.

In response to the WHO regulations, most of the teaching centres and hospitals worldwide have adopted the web-based teaching and learning model to continue the education and training of orthopaedic residents. These results brought significant changes to the training experience in orthopaedic surgery in combination with the fact that clinical duty hours and case volume were substantially reduced.

In what concerns orthopaedic journal publications, the Covid-19 pandemic resulted in a decline in the annual publication rate for the first time in over 20 years. Although not uniform, the reduction was most likely due to multifactorial causes.

Regarding the appraisal at the end of training, at the Orthopaedic European Board Examination we were able to verify that the outcome at the written part 1 exam was good, equivalent to the outcome prior to the pandemic. However the oral viva was much worse, probably due to the fact that residents skipped much of the clinical and surgical teaching and exposure during 2020 and 2021. At the end of training, theoretical/factual knowledge was good but poor from the clinical practical experience.

Lower back pain (LBP) is a worldwide clinical problem and a prominent area for research. Numerous in vitro biomechanical studies on spine specimens have been undertaken, attempting to understand spinal response to loading and possible factors contributing to LBP. However, despite employing similar testing protocols, there are challenges in replicating in vivo conditions and significant variations in published results. The aim of this study was to use the University of Bath (UoB) spine simulator to perform tests to highlight the major limitations associated with six degree of freedom (DOF) dynamic spine testing.

A steel helical spring was used as a validation model and was potted in Wood's metal. Six porcine lumbar spinal motion segments were harvested and dissected to produce isolated spinal disc specimens. These were potted in Wood's metal, ensuring the midplane of the disc remained horizontal and then sprayed with 0.9% saline and wrapped in saline-soaked tissue and plastic wrap to prevent dehydration. A 400N axial preload was used for spinal specimens. Specimens were tested under the stiffness and flexibility protocols.

Tests were performed using the UoB custom 6-axis spine simulator with coordinate axes. Tests comprised five cycles with data acquired at 100Hz. Stiffness and flexibility matrices were evaluated from the last three motion cycles using the linear least squares method.

According to theory, inverted flexibility matrices should equal stiffness matrices. In the case of the spring, the matrices matched analytical solutions and inverted flexibility matrices were equivalent to stiffness matrices. Matrices from the spinal tests demonstrated some symmetry, with similarities between inverted flexibility- and stiffness matrices, though these were unequal overall. Matrix element values were significantly affected by displacements assumed to occur at disc centre.

Spring tests proved that for linear, elastic specimens, the spine simulator functioned as expected. However, multiple factors limit the confidence in spine test results. Centre of rotation, displacement assumptions and rigid body transformations are known to impact the results from spinal testing, and these should be addressed going forward to improve the replication of in vivo conditions.

Injury of the intervertebral disc (IVD) can occur for many reasons including structural weakness due to disc degeneration. A common disc injury is herniation. A herniated nucleus can compress spinal nerves, causing pain, and nucleus depressurisation changes mechanical behaviour. Many studies have investigated in vitro IVD injuries including endplate fracture, incisions, and nucleotomy. There is, however, a lack of consensus on how the biomechanical behaviour of spinal motion segments is affected. The aim of this study was to induce defined changes to IVDs of spine specimens in vitro and apply 6 degree of freedom testing to evaluate the effect of these changes.

Six porcine lumbar spinal motion segments were harvested from organically farmed pigs. Posterior structures were removed to produce isolated spinal disc specimens. Specimens were potted in Wood's metal, ensuring the midplane of the IVD remained horizontal. After potting, specimens were sprayed with 0.9% saline, wrapped in saline-soaked tissue and plastic wrap to prevent dehydration. A 400N axial preload was equilibrated for 30 minutes before testing. Specimens were tested intact and after a partial nucleotomy removing ~0.34g of nuclear material with a curette through an annular incision.

Stiffness tests were performed using the University of Bath's custom 6-axis spine simulator with coordinate axes and displacement amplitudes. Tests comprised five cycles with data acquired at 100Hz. Stiffness matrices were evaluated from the last three motion cycles using the linear least squares method.

Stiffness matrices for intact and nucleotomy tests were compared. No significant differences in shear, axial or torsional stiffnesses were noted. Nucleotomy caused significantly higher stiffness in lateral bending and flexion-extension with increased linearity and the load-displacement behaviour in these axes displayed no neutral zone (NZ).

Induced changes were designed to replicate posterolaterally herniated discs. Unaffected shear, axial and torsional stiffnesses suggest the annulus is crucial in these axes. However, reduced ROM and NZ after nucleotomy suggests bending is most affected by herniation. Increased linearity and lack of defined NZ in these axes demonstrates herniation causes major changes to the viscoelastic behaviour of spine specimens in response to loading.

Osteosarcoma is a highly malignant primary tumor of bone tissue. The 5-year survival rate of patients with metastasis is below 20% and this scenario is unchanged in the last two decades, despite great efforts in pre-clinical and clinical research. Traditional preclinical models of osteosarcoma do not consider the whole complexity of its microenvironment, leading to poor correlation between in vitro/in vivo results and clinical outcomes. Spheroids are a promising in vitro model to mimic osteosarcoma and perform drug-screening tests, as they (i) reproduce the microarchitecture of the tumor, (ii) are characterized by hypoxic regions and necrotic core as the in vivo tumor, (iii) and recapitulate the chemo-resistance phenomena. However, to date, the spheroid model is scarcely used in osteosarcoma research.

Our aim is to develop a customized culture dish to grow and characterize spheroids and to perform advanced drug-screening tests. The resulting platform must be adapted to automated image acquisition systems, to overcome the drawbacks of commercial spheroids platforms.

To this purpose, we designed and developed a micro-patterned culture dish by casting agarose on a 3D printed mold from a CAD design. We successfully obtained viable and reproducible homotypic osteosarcoma spheroids, with two different cells lines from osteosarcoma (i.e., 143b and MG-63). Using the platform, we performed viability assays and live fluorescent stainings (e.g., Calcein AM) with low reagent consumption. Moreover, the culture dish was validated as drug screening platform, administrating Doxorubicin at different doses, and evaluating its effect on OS spheroids, in terms of morphology and viability. This platform can be considered an attractive alternative to the highly expensive commercial spheroid platforms to obtain homogeneous and reproducible spheroids in a high-throughput and cost effective mode.

One-fourth of all ankle trauma involve injury to the syndesmotic ankle complex, which may lead to syndesmotic instability and/or posttraumatic ankle osteoarthritis in the long term if left untreated. The diagnosis of these injuries still poses a deceitful challenge, as MRI scans lack physiologic weightbearing and plain weightbearing radiographs are subject to beam rotation and lack 3D information. Weightbearing cone-beam CT (WBCT) overcomes these challenges by imaging both ankles during bipedal stance, but ongoingdebate remains whether these should be taken under weightbearing conditions and/or during application of external rotation stress. The aim of this study is study therefore to compare both conditions in the assessment of syndesmotic ankle injuries using WBCT imaging combined with 3D measurement techniques.

In this retrospective study, 21 patients with an acute ankle injury were analyzed using a WBCT. Patients with confirmed syndesmotic ligament injury on MRI were included, while fracture associated syndesmotic injuries were excluded. WBCT imaging was performed in weightbearing and combined weightbearing-external rotation. In the latter, the patient was asked to internally rotate the shin until pain (VAS>8/10) or a maximal range of motion was encountered. 3D models were developed from the CT slices, whereafter. The following 3D measurements were calculated using a custom-made Matlab® script; Anterior tibiofibular distance (AFTD), Alpha angle, posterior Tibiofibular distance (PFTD) and Talar rotation (TR) in comparison to the contralateral non-injured ankle.

The difference in neutral-stressed Alpha angle and AFTD were significant between patients with a syndesmotic ankle lesion and contralateral control (P=0.046 and P=0.039, respectively). There was no significant difference in neutral-stressed PFTD and TR angle.

Combined weightbearing-external rotation during CT scanning revealed an increased AFTD in patients with syndesmotic ligament injuries. Based on this study, application of external rotation during WBCT scans could enhance the diagnostic accuracy of subtle syndesmotic instability.

A comprehensive understanding of the self-repair abilities of menisci and their overall function in the knee joint requires three-dimensional information. However, previous investigations of the meniscal blood supply have been limited to two-dimensional imaging methods, which fail to accurately capture tissue complexity. In this study, micro-CT was used to analyse the 3D microvascular structure of the meniscus, providing a detailed visualization and precise quantification of the vascular network.

A contrast agent (μAngiofil®) was injected directly into the femoral artery of cadaver legs to provide the proper contrast enhancement. First, the entire knee joint was analysed with micro-CT, then to increase the applicable resolution the lateral and medial menisci were excised and investigated with a maximum resolution of up to 4 μm. The resulting micro-CT datasets were analysed both qualitatively and quantitatively. Key parameters of the vascular network, such as vascular volume fraction, vessel radius, vessel length density, and tortuosity, were separately determined for the lateral and medial meniscus, and their four circumferential zones defined by Cooper.

In accordance with previous literature, the quantitative micro-CT data confirm a decrease in vascular volume fraction along the meniscal zones. The highest concentration of blood vessels was measured in the meniscocapsular region 0, which is characterized by vascular segments with a significantly larger average radius. Furthermore, the highest vessel length density observed in zone 0 suggests a more rapid delivery of oxygen and nutrients compared to other regions. Vascular tortuosity was detected in all circumferential regions, indicating the occurrence of vascular remodelling in all tissue areas.

In conclusion, micro-CT is a non-invasive imaging technique that allows for the visualization of the internal structure of an object in three dimensions. These advanced 3D vascular analyses have the potential to establish new surgical approaches that rely on the healing potential of specific areas of the meniscus.

Acknowledgements: The authors acknowledge R. Hlushchuk, S. Halm, and O. Khoma from the University of Bern for their help with contrast agent perfusions.

Fracture healing is a spatially controlled process involving crosstalk of multiple tissues. To precisely capture and understand molecular mechanism underlying impaired healing, there is a need to integrate spatially-resolved molecular analyses into preclinical fracture healing models. I will present our recent data obtained by spatial transcriptomics of musculoskeletal samples from fracture healing studies in mice. Subsequently, I will show how spatial transcriptomics can be integrated into multimodal approaches in preclinical fracture healing models. In combination with established in vivo imaging and emerging omics techniques, spatially-resolved analyses have the potential to elucidate the molecular mechanisms underlying impaired healing with optimization of treatments.

Recently, technologies to culture one or more cell types in three dimensions have attracted a great deal of attention in tissue engineering. Particularly, the improved viability, self-renewal capacity, and differentiation potential have been reported for stem cell spheroids. However, it is crucial to modulate spheroid functions with instructive signals to use multi-cellular spheroids in tissue engineering. We have been developing ECM-mimicking fibrous materials decorated with cell-instructive cues, which were incorporated within 3D stem cell spheroids to fine-tune their functions as modular building blocks for bottom-up tissue-engineering applications. In particular, we created composite spheroids of human adipose-derived stem cells (hADSCs) incorporating nanofibers coated with instructive signal of either transforming growth factor-β3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis of stem cells, respectively. The bilayer structure of osteochondral tissue was subsequently mimicked by cultivating each type of spheroid inside 3D-printed construct. The in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct under general media was locally regulated by each inductive component. More importantly, hADSCs from each spheroid proliferated and sprouted to form the integrated tissue with interface of bone and cartilage tissue. This approach may be applied to engineer complex tissue with hierarchically organized structure.

To determine the clinical efficacy of vitamin-D supplementation on pain intensity and functional disability in patients with chronic lower back pain.

This prospective cohort study was conducted from 20th March 2017 to 19th March 2019. The inclusion criteria were patients of CLBP aged between 15 to 55 years. Exclusion criteria included all the patients with Disc prolapse, Spinal stenosis, Any signs of neurological involvement, Metabolic bone disease (Hypo- or Hyperparathyroidism) and Chronic kidney disease/Chronic liver disease. Patients were supplemented with 50,000 IU of oral vitamin-D3 every week for 8 weeks (induction phase) and 50,000 IU of oral vitamin-D3 once monthly for 6 months (maintenance phase). Efficacy parameters included pain intensity and functional disability measured by VAS and modified Oswestry disability questionnaire (MODQ) scores at baseline, 2, 3 and 6 months post-supplementation. Vitamin-D3 levels were measured at baseline,2,3 and 6 months.

A total of 600 patients were included in the study. The mean age of patients was 44.2 ± 11.92 years. There were 337 (56.2%) male patients while 263 (43.8%) female patients. Baseline mean vitamin-D levels were 13.32 ± 6.10 ng/mL and increased to 37.18 ± 11.72 post supplementation (P < 0.0001). There was a significant decrease in the pain score after 2nd, 3rd& 6th months (61.7 ± 4.8, 45.2 ± 4.6 & 36.9 ± 7.9, respectively) than 81.2 ± 2.4 before supplementation (P < 0.001). The modified Oswestry disability score also showed significant improvement after 2nd, 3rd & 6th months (35.5, 30.2 & 25.8, respectively) as compared to baseline 46.4 (P < 0.001). About 418 (69.7%) patients attained normal levels after 6 months.

Vitamin-D supplementation in chronic lower back pain patients may lead to improvement in pain intensity and functional ability.

Total knee and hip arthroplasty (TKA and THA) are the most commonly performed surgical procedures, the costs of which constitute a significant healthcare burden. Improving access to care for THA/TKA requires better efficiency. It is hypothesized that this may be possible through a two-stage approach that utilizes prediction of surgical time to enable optimization of operating room (OR) schedules.

Data from 499,432 elective unilateral arthroplasty procedures, including 302,490 TKAs, and 196,942 THAs, performed from 2014-2019 was extracted from the American College of Surgeons (ACS) National Surgical and Quality Improvement (NSQIP) database. A deep multilayer perceptron model was trained to predict duration of surgery (DOS) based on pre-operative clinical and biochemical patient factors. A two-stage approach, utilizing predicted DOS from a held out “test” dataset, was utilized to inform the daily OR schedule. The objective function of the optimization was the total OR utilization, with a penalty for overtime. The scheduling problem and constraints were simulated based on a high-volume elective arthroplasty centre in Canada. This approach was compared to current patient scheduling based on mean procedure DOS. Approaches were compared by performing 1000 simulated OR schedules.

The predict then optimize approach achieved an 18% increase in OR utilization over the mean regressor. The two-stage approach reduced overtime by 25-minutes per OR day, however it created a 7-minute increase in underutilization. Better objective value was seen in 85.1% of the simulations.

With deep learning prediction and mathematical optimization of patient scheduling it is possible to improve overall OR utilization compared to typical scheduling practices. Maximizing utilization of existing healthcare resources can, in limited resource environments, improve patient's access to arthritis care by increasing patient throughput, reducing surgical wait times and in the immediate future, help clear the backlog associated with the COVID-19 pandemic.

Pelvic tilt can vary over time due to aging and the possible appearance of sagittal spine disorders. Cup position in total hip arthroplasty (THA) can be influenced due to these changes. We assessed the evolution of pelvic tilt and cup position after THA and the possible appearance of complications for a minimum follow-up of ten years.

343 patients received a THA between 2006 and 2009. All were diagnosed with primary osteoarthritis and their mean age was 63.3 years (range, 56 to 80). 168 were women and 175 men. 250 had no significant lumbar pathology, 76 had significant lumbar pathology and 16 had lumbar fusion. Radiological analysis included sacro-femoral-pubic (SFP), acetabular abduction (AA) and anteversion cup (AV) angles. Measurements were done pre-operatively and at 6 weeks, and at five and ten years post-operatively. Three measurements were recorded and the mean obtained at all intervals. All radiographs were evaluated by the same author, who was not involved in the surgery.

There were nine dislocations: six were solved with closed reduction, and three required cup revision. All the mean angles changed over time; the SFP angle from 59.2º to 60º (p=0.249), the AA angle from 44.5º to 46.8º (p=0.218), and the AV angle from 14.7º to 16.2º (p=0.002). The SFP angle was lower in older patients at all intervals (p<0.001). The SFP angle changed from 63.8 to 60.4º in women and from 59.4º to 59.3º in men, from 58.6º to 59.6º (p=0.012). The SFP angle changed from 62.7º to 60.9º in patients without lumbar pathology, from 58.6º to 57.4º in patients with lumbar pathology, and from 57.0º to 56.4º in patients with a lumbar fusion (p=0.919). The SFP cup angle was higher in patients without lumbar pathology than in the other groups (p<0.001), however, it changed more than in patients with lumbar pathology or fusion at ten years after THA (p=0.04).

Posterior pelvic tilt changed with aging, influencing the cup position in patients after a THA. Changes due to lumbar pathology could influence the appearance of complications long-term.

Lumbar diseases have become a major problem affecting human health worldwide. Conservative treatment of lumbar diseases is difficult to achieve ideal results, and surgical treatment of trauma, complications, it is imperative to develop a new treatment method. This study aims to explore the regulatory mechanism of cartilage endplate ossification caused by abnormal stress, and design intervention targets for this mechanism, so as to provide theoretical reference for the prevention and treatment of lumbar degeneration.

In vivo, we constructed spinal instability model in mice. In vitro, we used a mechanical tensile machine to simulate the abnormal stress conditions of the endplate cartilage cells. Through the high-throughput sequencing, we found the enrichment of Hippo signaling pathway. As YAP is a key protein in the Hippo signaling pathway, we then created cartilaginous YAP elimination mice (Col2::YAPfl/fl). The lumbar spine model was constructed again in these mice for H&E, SOFG and immunofluorescence staining. In vitro lentivirus was used to knock out YAP, immunofluorescence staining, WB and qPCR were performed. Finally, we conducted therapeutic experiments by using YAP agonist and AAV5 carrying YAP plasmids.

We collected 8w samples from C57/BL6 mice after modeling. We found ossification of the endplate in mice similar to human disc degeneration. High-throughput sequencing of stretched cells demonstrated high enrichment of the Hippo signaling pathway. By immunofluorescence staining, it was confirmed that Col-II decreased and Col-X gradually increased in the endplate cartilage of mice. This was also confirmed at 7 days after an in vitro stretch of 5% and 12%. Meanwhile, we found that cartilaginous YAP elimination mice developed very severe endplate degeneration. However, the endplate was well protected by intraperitoneal injection of YAP agonist or AAV5-YAP endplate injection, and the results in vitro were consistent with that.

In the process of cartilaginous ossification, abnormal stress regulates Col10a1 to promote cartilage endplate ossification through Hippo signaling pathway mediated YAP, and we expect to find potential drug targets for treatment through this mechanism.

Although bone morphogenetic protein 2 (BMP-2) has been FDA-approved for spinal fusion for decades, its disadvantages of promoting osteoclast-based bone resorption and suboptimal carrier (absorbable collagen sponge) leading to premature release of the protein limit its clinical applications. Our recent study showed an excellent effect on bone regeneration when BMP-2 and zoledronic acid (ZA) were co-delivered based on a calcium sulphate/hydroxyapatite (CaS/HA) scaffold in a rat critical-size femoral defect model. Therefore, the aim of this study was to evaluate whether local application of BMP-2 and ZA released from a CaS/HA scaffold is favorable for spinal fusion. We hypothesized that CaS/HA mediated controlled co-delivery of rhBMP-2 and ZA could show an improved effect in spinal fusion over BMP-2 alone. 120, 8-week-old male Wistar rats (protocol no. 25-5131/474/38) were randomly divided into six groups in this study (CaS/HA, CaS/HA + BMP-2, CaS/HA + systemic ZA, CaS/HA + local ZA, CaS/HA + BMP-2 + systemic ZA, CaS/HA + BMP-2 + local ZA). A posterolateral spinal fusion at L4 to L5 was performed bilaterally by implanting group-dependent scaffolds. At 3 weeks and 6 weeks, 10 animals per group were euthanized for µCT, histological staining, or mechanical testing. µCT and histological results showed that the CaS/HA + BMP-2 + local ZA group significantly promoted bone regeneration than other treated groups. Biomechanical testing showed breaking force in CaS/HA + BMP + local ZA group was significantly higher than other groups at 6 weeks. In conclusion, the CaS/HA-based biomaterial functionalized with bioactive molecules rhBMP-2 and ZA enhanced bone formation and concomitant spinal fusion outcome

Acknowledgements: Many thanks to Ulrike Heide, Anna-Maria Placht (assistance with surgeries) as well as Suzanne Manthey & Annett Wenke (histology).

We have developed a novel technique to analyse bone, using imaging mass cytometry (IMC) without the constraints of using immunofluorescent histochemistry. IMC can measure the expression of over 40 proteins simultaneously, without autofluorescence. We analysed mitochondrial respiratory chain (RC) protein deficiencies in human bone which are thought to contribute to osteoporosis with increasing age.

Osteoporosis is characterised by reduced bone mineral density (BMD) and fragility fractures. Humans accumulate mitochondrial mutations and RC deficiency with age and this has been linked to the changing phenotype in advancing age and age-related disease. Mitochondrial mutations are detectable from the age of 30 onwards, coincidently the age BMD begins to decline. Mitochondria contain their own genome which accumulates somatic variants at around 10 times the rate of nuclear DNA. Once these mutations exceed a threshold, RC deficiency and cellular dysfunction occur. The PolgD257A/D257A mouse model expresses a proof-reading deficient version of PolgA, a mtDNA polymerase. These mice accumulate mutations 3-5 times higher than wild-type mice showing enhanced levels of age-related osteoporosis and RC deficiency in osteoblasts.

Bone samples were analysed from young and old patients, developing a protocol and analysis framework for IMC in bone tissue sections to analyse osteoblasts in-situ for RC deficiency.

Samples from the femoral neck of 10 older healthy volunteers aged 40 – 85 were compared with samples from young patients aged 1-19. We have identified RC complex I defect in osteoblasts from 6 of the older volunteers, complex II defects in 2 of the older volunteers, complex IV defect in just 1 older volunteer, and complex V defect in 4 of the older volunteers.

These observations are consistent with the PolgD257A/D257A mouse-model and suggest that RC deficiency, due to age-related pathogenic mitochondrial DNA mutations, may play a significant role in the pathogenesis of human age-related osteoporosis.

Nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is key in maintaining redox homeostasis and the pathogenesis of osteoarthritis (OA) involves oxidative distress. We thus investigated whether Nrf2/ARE signaling may control expression of key chondrogenic differentiation and hyaline cartilage maintenance factor SOX9.

In human C-28/I2 chondrocytes SOX9 expression was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap “n” collar homology-associated protein 1 (Keap1). Putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays to verify whether Nrf2 transcriptionally regulates SOX9. SOX9 promoter activity without and with Nrf2-inducer methysticin were analyzed. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Data were analyzed by one-way ANOVA, a Student's t-test, or Wilcoxon rank-sum test, according to the normal distribution. Statistical significance was set to p < 0.05.

While Keap1-specific RNAi increased SOX9 expression, Nrf2-specific RNAi significantly decreased it. Putative ARE sites (ARE₁, ARE₂) were identified in the SOX9 promoter region. ARE₂ mutagenesis significantly reduced SOX9 promoter activity, while truncation of ARE₁ did not. A functional ARE₂ site was thus essential for methysticin-mediated induction of SOX9 promoter activity. Knee cartilage of young Nrf2-knockout mice further revealed significantly fewer Sox9-positive chondrocytes as compared to old Nrf2-knockout animals, which further showed thinner cartilage and more severe cartilage erosion.

Our data suggest that SOX9 expression in articular cartilage is directly Nrf2-dependent and that pharmacological Nrf2 activation may hold potential to diminish age-dependent osteoarthritic changes in knee cartilage through improving protective SOX9 expression.

Electrospinning is an advantageous technique for cartilage tissue engineering (CTE) applications due to its ability to produce nanofibers recapitulating the size and alignment of the collagen fibers present within the articular cartilage superficial zone. Moreover, coaxial electrospinning allows the fabrication of core-shell fibers able to encapsulate and release bioactive molecules in a sustained manner. Kartogenin (KTG) is a small heterocyclic molecule, which was demonstrated to promote the chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells(hBMSCs)[1].

In this work, we developed and evaluated the biological performance of core-shell poly(glycerol sebacate)(PGS)/poly(caprolactone)(PCL) aligned nanofibers (core:PGS/shell:PCL) mimicking the native articular cartilage extracellular matrix(ECM) and able to promote the sustained release of the chondroinductive drug KTG[2].

The produced coaxial aligned PGS/PCL scaffolds were characterized in terms of their structure and fiber diameter, chemical composition, thermal properties, mechanical performance under tensile testing and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. KTG was incorporated into the core PGS solution to generate core-shell PGS-KTG/PCL nanofibers and its release kinetics was studied by HPLC analysis. KTG-loaded electrospun aligned scaffolds capacity to promote hBMSCs chondrogenic differentiation was evaluated by assessing cell proliferation, typical cartilage-ECM production (sulfated glycosaminiglycans(sGAG)) and chondrogenic marker genes expression in comparison to non-loaded controls. All the scaffolds fabricated showed average fiber diameters within the nanometer-scale and the core-shell structure of the fibers was clearly confirmed by TEM. The coaxial PGS-KTG/PCL nanofibers evidenced a more sustained drug release over 21 days. Remarkably, in the absence of the chondrogenic cytokine TGF-β3, KTG-loaded nanofibers promoted significantly the proliferation and chondrogenic differentiation of hBMSCs, as suggested by the increased cell numbers, higher sGAG amounts and up-regulation of the chondrogenic genes COL2A1, Sox9, ACAN and PRG4 expression. Overall, our results highlight the potential of core-shell PGS-KTG/PCL aligned nanofibers for the development of novel MSC-based CTE strategies.

Acknowledgements: The authors thank FCT for funding through the project InSilico4OCReg (PTDC/EME-SIS/0838/2021) and to institutions iBB (UID/BIO/04565/2020) and Associate Laboratory I4HB (LA/P/0140/2020).

Conventional proximal tibial osteotomy is a widely successful joint-preserving treatment for osteoarthritis; however, conventional procedures do not adequately control the posterior tibial slope (PTS). Alterations to PTS can affect knee instability, ligament tensioning, knee kinematics, muscle and joint contact forces as well as range of motion.

This study primarily aimed to provide a comprehensive investigation of the variables influencing PTS during high tibial osteotomy using a 3D surgical simulation approach. Secondly, it aimed to provide a simple means of implementing the findings in future 3D pre-operative planning and /or clinically.

The influence of two key variables: the gap opening angle and the hinge axis orientation on PTS was investigated using three independent approaches: (1) 3D computational simulation using CAD software to perform virtual osteotomy surgery and simulate the post-operative outcome. (2) Derivation of a closed-form mathematical solution using a generalised vector rotation approach (3) Clinical assessment of synthetically generated x-rays of osteoarthritis patients (n=28; REC reference: 17/HRA/0033, RD&E NHS, UK) for comparison against the theoretical/computational approaches.

The results from the computational and analytical assessments agreed precisely. For three different opening angles (6°, 9° and 12°) and 7 different hinge axis orientations (from −30° to 30°), the results obtained were identical. A simple analytical solution for the change in PTS, ΔP_s, based on the hinge axis angle, α, and the osteotomy opening angle, θ, was derived:

ΔP_s=sin^-1(sin α sin θ)

The clinical assessment demonstrated that the absolute values of PTS, and changes resulting from various osteotomies, matched the results from the two relative prediction methods.

This study has demonstrated that PTS is impacted by the hinge axis angle and the extent of the osteotomy opening angle and provided computational evidence and analytical formula for general use.

Titanium alloys are one of the most used for orthopaedic implants and the fabrication of them by 3D printing technology is a raising technology, which could effectively resolve existing challenges. Surface modification of Ti surfaces is often necessary to improve biocorrosion resistance, especially in inflammatory conditions. Such modification can be made by coatings based on hydrogels, like alginate (Alg) - a naturally occurring anionic polymer. The properties of the hydrogel can be further enhanced with calcium phosphates like octacalcium phosphate (OCP) as a precursor of biologically formed hydroxyapatite. Formed Alg-OCP matrices have a high potential in wound healing, delivery of bioactive agents etc. but their effect on 3D printed Ti alloys performance was not well known.

In this work, Alg-OCP coated 3D printed samples were studied with electrochemical measurements and revealed significant variations of corrosion resistance vs. composition of the coating. The potentiodynamic polarization test showed that the Alg-OCP-coated samples had lower corrosion current density than simple Alg-coated samples. Electrochemical impedance spectroscopy indicated that OCP incorporated hydrogels had also a high value of the Bode modulus and phase angle. Hence Alg-OCP hydrogels could be highly beneficial in protecting 3D printed Ti alloys especially when the host conditions for the implant placement are inflammatory.

AcThis work was supported by the European Union Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Actions GA860462 (PREMUROSA). The authors also acknowledge the access to the infrastructure and expertise of the BBCE – Baltic Biomaterials Centre of Excellence (European Union Horizon 2020 programme under GA857287).

Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms.

Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2^-/-). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR.

Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized callus during fracture repair. The shift from chondrocytes over hypertrophic chondrocytes to bone-forming osteoblasts was accelerated. Collagen production was increased in mutant fracture callus. Serum levels of BMP-2 were increased in healing KO mice. Isolated integrin deficient osteoblast presented an earlier expression and production of active BMP-2 during the differentiation, which led to earlier mineralisation. Biomechanical testing showed no differences between wild-type and mutant bones.

Knockout of integrin α2β1 leads to a beneficial outcome for fracture repair. Callus maturation is accelerated, leading to faster recovery, accompanied by an increased generation of extra-cellular matrix material. Biomechanical properties are not diminished by this accelerated healing. The underlying mechanism is driven by an earlier availability of BMP-2, one main effectors for bone development. Local inhibition of integrin α2β1 is therefore a promising target to accelerate fracture repair, especially in patients with retarded healing.

In the native articular cartilage microenvironment, chondrocytes are constantly subjected to dynamic physical stimuli that maintains tissue homeostasis. They produce extra cellular matrix (ECM) components such as collagens (type II mainly, 50-75%), proteoglycans (10-30%) and other type of proteins¹ . While collagen offers a large resistance in tension, proteoglycans are the responsible of the viscoelastic response under compression due to the negative charge they confer to the ECM allowing it to entrap a large amount of interstitial fluid. In pathologic states (e.g. osteoarthritis), this ECM is degenerated and the negative charge becomes unbalanced, losing the chondroprotective properties and resulting on an overloaded chondrocytes that further degenerate the matrix.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) has been used to generate acoustic (pressure) waves that create bubbles that collapse with cells, inducing a stimulus that can modulate cell response². This mechanical stimulation promotes the expression of type II collagen, type X collagen, aggrecan and TGF-β, appearing as a great strategy to regenerate cartilage. However, current strategies make use of extrinsic forces to stimulate cartilage formation overlooking the physico-chemical properties of the degenerated cartilage, resulting in an excessive load-transfer to chondrocytes and the consequent hypertrophy and degeneration.

Here, interpenetrated networks (IPNs) with different compositions were created using methacrylated gelatin (GelMA), to mimic the collagen, and alginate functionalized with tyramine (Alg-tyr) to mimic glycosaminoglycans and to introduce a negative charge in the model. Within the matrix chondrocytes where encapsulated and stimulated under different conditions to identify the ultrasound parameters that enhance tissue formation. Samples with and without stimulation were compared analysing the expression and deposition of collagen II, aggrecan, collagen X and TGF-β. The results suggested that the chondrogenic marker expression of the samples stimulated for 10 minutes per day for 28 days, was two times higher overall in all of the cases, which was correlated to the tissue formation detected.

Acknowledgments: The authors would like to thank the Basque Government for the “Predoctoral Training Program for Non-Doctoral Research Staff 2021-2022” (Grant ref.: PRE_2021_1_0403). This work was supported by the RETOS grant PID2020-114901RA-I00 of the Ministry of Science and Innovation (MICINN).

Orthopedic Device-Related Infections (ODRIs) are a major medical challenge, particularly due to the involvement of biofilm-encased and multidrug-resistant bacteria. Current treatments, based on antibiotic administration, have proven to be ineffective. Consequently, there is a need for antibiotic-free alternatives. Antimicrobial peptides (AMPs) are a promising solution due to their broad-spectrum of activity, high efficacy at very low concentrations, and low propensity to induce resistance. We aim to develop a new AMP-based chitosan nanogel to be injected during orthopedic device implantation to prevent ODRIs. Chitosan was functionalized with norbornenes (NorChit) through the reaction with carbic anhydride and then, a cysteine-modified AMP, Dhvar5, a peptide with potent antibacterial activity, even against methicillin-resistant Staphylococcus aureus (MRSA), was covalently conjugated to NorChit (NorChit- Dhvar5), through a thiol-norbornene photoclick chemistry (UV= 365 nm). For NorChit-Dhvar5 nanogels production, the NorChit-Dhvar5 solution (0.15% w/v) and Milli-Q water were injected separately into microfluidic system. The nanogels were characterized regarding size, concentration, and shape, using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and Dynamic light scattering (DLS). The nanogels antibacterial properties were assessed in Phosphate Buffer (PBS) for 6 h, against four relevant microorganisms (Pseudomonas aeruginosa, S. aureus and MRSA, and in Muller- Hinton Broth (MHB), 50% (v/v) in PBS, supplemented with human plasma (1% (v/v)), for 6 and 24 h against MRSA. The obtained NorChit-Dhvar5 nanogels, presented a round-shaped and ∼100 nm. NorChit- Dhvar5 nanogels in a concentration of 10¹⁰ nanogels/mL in PBS were capable of reducing the initial inoculum of P. aeruginosa by 99%, S. aureus by 99%, and MRSA by 90%. These results were corroborated by a 99% MRSA reduction, after 24 h in medium. Furthermore, NorChit-Dhvar5 nanogels do not demonstrate signs of cytotoxicity against MC3T3-E1 cells (a pre-osteoblast cell line) after 14 days, having high potential to prevent antibiotic-resistant infection in the context of ODRIs.

Virtual mechanical testing is a method for measuring bone healing using finite element models built from computed tomography (CT) scans. Previously, we validated a dual-zone material model for ovine fracture callus that differentiates between mineralized woven bone and soft tissue based on radiodensity.¹ The objective of this study was to translate the dual-zone material model from sheep to two important clinical scenarios: human tibial fractures in early-stage healing and late-stage nonunions.

CT scans for N = 19 tibial shaft fractures were obtained prospectively at 12 weeks post-op. A second group of N = 33 tibial nonunions with CT scans were retrospectively identified. The modeling techniques were based on our published method.² The dual-zone material model was implemented for humans by performing a cutoff sweep for both the 12-week and nonunion groups. Virtual torsional rigidity (VTR) was calculated as VTR = ML/φ [N-m²/°], where M is the moment reaction, L is the diaphyseal segment length, and φ is the angle of twist.

As the soft tissue cutoff was increased, the rigidity of the clinical fractures decreased and soft tissue located within the fracture gaps produced higher strains that are not predicted without the dual zone approach. The structural integrity of the nonunions varied, ranging from very low rigidities in atrophic cases to very high rigidities in highly calcified hypertrophic cases, even with dual-zone material modeling.

Human fracture calluses are heterogeneous, comprising of woven bone and interstitial soft tissue. Use of a dual-zone callus material model may be instrumental in identifying delayed unions during early healing when callus formation is minimal and/or predominantly fibrous with little mineralization.

Bone regeneration is pivotal for the healing of fractures. In case this process is disturbed a non-union can occur. This can be induced by environmental factors such as smoking, overloading etc. Co-morbidities such as diabetes, osteoporosis etc. may be more intrinsic factors besides other disturbances in the process. Those pathways negatively influence the bone regeneration process. Several intrinsic signal transduction pathways (WNT, BMP etc.) can be affected. Furthermore, on the transcriptional level, important mRNA expression can be obstructed by deregulated miRNA levels. For instance, several miRNAs have been shown to be upregulated during osteoporotic fractures. They are detrimental for osteogenesis as they block bone formation and accelerate bone resorption. Modulating those miRNAs may revert the physiological homeostasis. Indeed, physiological fracture healing has a typical miRNA signature. Besides using molecular pathways for possible treatment of non-union fractures, providing osteogenic cells is another solution. In 5 clinical cases with non-union fractures with defects larger than 10 cm, successful administration of a 3D printed PCL-TCP scaffold with autologous bone marrow aspirate concentrate and a modulator of the pathogenetic pathway has been achieved. All patients recovered well and showed a complete union of their fractures within one year after start of the regenerative treatment.

Thus, non-union fractures are a diverse entity. Nevertheless, there seem to be common pathogenetic disturbances. Those can be counteracted at several levels from molecular to cell. Compositions of those may be the best option for future therapies. They can also be used in a more personalized fashion in case more specific measurements such as miRNA signature and stem cell activity are applied.

Tendon injuries present a major clinical challenge, as they necessitate surgical intervention and are prone to fibrotic progression. Despite advances in physical therapy and surgical technique, tendons fail to return to full native functioning, underlining the need for a biological therapeutic to improve tendon healing. Myofibroblasts are activated fibroblasts that participate in the proliferative and remodeling phases of wound healing, and while these matrix-producing cells are essential for proper healing, they are also linked to fibrotic initiation. A subset of tenocytes has been shown to give rise to the myofibroblast fate, and potentially contribute to fibrotic tendon healing. A viable anti-fibrotic therapy in other tissues has been reprogramming the fibroblast-myofibroblast differentiation route, avoiding a more pro-fibrotic myofibroblast phenotype. Thus, defining the molecular programs that underlie both physiological and pathological tendon healing is critical for the development of potential pharmacologic treatments. Towards that end, we have taken advantage of spatial transcriptomics, using the tenocyte marker Scleraxis as a tool, and have outlined three major spatiotemporally distinct tenocyte differentiation trajectories (synthetic, proliferative, and reactive) following acute tendon injury in mouse FDL. We have further outlined key transcriptional controls that may be manipulated to alter the differentiation process and influence the resulting myofibroblast phenotype, thereby promoting regenerative tendon healing.

Chronic inflammatory events have been associated to almost every chronic disease, including cardiovascular-, neurodegenerative- and autoimmune- diseases, cancer, and host-implant rejection. Given the toll of chronic inflammation in healthcare and socioeconomical costs developing strategies to resolve and control chronic states of inflammation remain a priority for the significant benefit of patients.

Macrophages (Mφ) hold a central role both in the initiation and resolution of inflammatory events, assuming different functional profiles. The outstanding features of Mφ counting with the easy access to tissues, and the extended networking make Mφ excellent candidates for precision therapy. Moreover, sophisticated macrophage-oriented systems could offer innovative immune-regulatory alternatives to effectively regulate chronic environments that traditional pharmacological agents cannot provide.

We propose magnetically assisted systems for balancing Mφ functions at the injury site. This platform combines polymers, inflammatory miRNA antagonists and magnetically responsive nanoparticles to stimulate Mφ functions towards pro-regenerative phenotypes. Strategies with magnetically assisted systems include contactless presentation of immune-modulatory molecules, cell internalization of regulatory agents for functional programming via magnetofection, and multiple payload delivery and release.

Overall, Mφ-oriented systems stimulated pro-regenerative functions of Mφ supporting magnetically assisted theranostic nanoplatforms for precision therapies, envisioning safer and more effective control over the distribution of sensitive nanotherapeutics for the treatments of chronical inflammatory conditions.

Acknowledgements: ERC CoG MagTendon No.772817; FCT Doctoral Grant SFRD/BD/144816/2019, and TERM

RES Hub (Norte-01-0145-FEDER-022190).

The regenerative capacity of hyaline cartilage is greatly limited. To prevent the onset of osteoarthritis, cartilage defects have to be properly treated. Cartilage, tissue engineered by mean of bioactive glass (BG) scaffolds presents a promising approach. Until now, conventional BGs have been used mostly for bone regeneration, as they are able to form a hydroxyapatite (HA) layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to compare two BGs based on a novel BG composition tailored specifically for cartilage (CAR12N) and patented by us with conventional BG (BG1393) with a similar topology. The highly porous scaffolds consisting of 100% BG (CAR12N, CAR12N with low Ca2+/Mg2+ and BG1393) were characterized and dynamically seeded with primary porcine articular chondrocytes (pACs) or primary human mesenchymal stem cells (hMSCs) for up to 21 days. Subsequently, cell viability, DNA and glycosaminoglycan contents, cartilage-specific gene and protein expression were evaluated. The manufacturing process led to a comparable high (over 80%) porosity in all scaffold variants. Ion release and pH profiles confirmed bioactivity for them. After both, 7 and 21 days, more than 60% of the total surfaces of all three glass scaffold variants was densely colonized by cells with a vitality rate of more than 80%. The GAG content was significantly higher in BG1393 colonized with pACs. In general, the GAG content was higher in pAC colonized scaffolds in comparison to those seeded with hMSCs. The gene expression of cartilage-specific collagen type II, aggrecan, SOX9 and FOXO1 could be detected in all scaffold variants, irrespectively whether seeded with pACs or hMSCs. Cartilage-specific ECM components could also be detected at the protein level. In conclusion, all three BGs allow the maintenance of the chondrogenic phenotype or chondrogenic differentiation of hMSCs and thus, they present a high potential for cartilage regeneration.

Total shoulder arthroplasty (TSA) and Reverse Total shoulder arthroplasty (RSA) are two of the most performed shoulder operations today. Traditionally postoperative rehabilitation included a period of immobilisation, protecting the joint and allowing time for soft tissue healing. This immobilisation period may significantly impact a patient's quality of life (Qol)and ability to perform activities of daily living (ADL's). This period of immobilisation could be safely avoided, accelerating return to function and improving postoperative QoL.

This systematic review examines the safety of early mobilisation compared to immobilisation after shoulder arthroplasty focusing on outcomes at one year.

Achilles tendon mechanical properties depend on a complex hierarchical design, with collagen being the smallest load-bearing unit. At the nanoscale, collagen molecules are organized into fibrils, which at the microscale are assembled into fibers, followed by larger structures such as sub-tendons or fascicles. Degree of in vivo loading affects the collagen content, and organization and consequently the tissue's mechanical response. We aim to unravel how composition, structural organization, and mechanical response are affected by degree of in vivo loading at each length scale. The presentation will outline the results to date about to the use of high-resolution synchrotron-based tissue characterisation methods on several length scales in combination with in situ mechanical tests. We use a rat model, where the tendons are subjected to varying loading in vivo. To characterize the tissue microstructure, phase-contrast enhanced synchrotron micro-tomography is performed. The 3D fiber organization in fully loaded tendons is highly aligned, whereas the fibers in unloaded tendons are significantly more heterogeneously arranged and crimped. To characterize the collagen fibril response, Small Angle X-ray Scattering is performed. Two types of fibril organizations are found; a single population oriented towards the main load direction and two fibril subpopulations with clearly distinct orientations. Scattering during loading showed that the fibrils in unloaded tendons did not strain as much in fully loaded. In situ loading concurrently with high resolution synchrotron experiments show the complex tendon response to in situ load and its relation to in vivo loading and tendon hierarchical structure. Unloading seems to alter the organization of the fibrils and fibers, e.g. increased crimping and more pronounced sub-tendon twists.

Acknowledgements: Funding from Knut and Alice Wallenberg Foundation and European Research Council (101002516). Paul Scherrer Institut, Switzerland for beamtime at cSAXS and TOMCAT.

Despite promising results in attempting intervertebral disc regeneration, intradiscal cell transplantation is affected by several drawbacks, including poor viability in the harsh disc environment, low cost-effectiveness, and immunogenic/tumorigenic concerns. Recently, the development of cell-free approaches is gaining increasing interest in the field, with a particular regard towards extracellular vesicles (EVs). Nucleus pulposus cell (NPC) progenitors characterized by Tie2 expression have shown a higher chondrogenic differentiation potential compared to MSCs. The aim of this study was to investigate the putative regenerative effects of EVs isolated from Tie2-overexpressing NPC progenitors on degenerative NPCs.

NPCs were isolated from young donors and underwent an optimized culture protocol to maximize Tie2 expression (NPCs^Tie2+) or a standard protocol (NPCs^STD). Following EV characterization, NPC isolated from patients affected by intervertebral disc degeneration (IDD) were treated with either NPCs^Tie2+-EVs or NPCs^STD-EVs. Cell proliferation and viability were assessed with the CCK-8 assay. Cell apoptosis and necrosis were evaluated with the Annexin V/PI assay. Cell senescence was investigated with b-galactosidase staining. EV uptake was assessed with PKH26 staining of EVs under confocal microscopy.

Treatment with EVs isolated from young NPC donors significantly increased degenerative NPC viability, especially in samples treated with NPCs^Tie2+-EVs. Likewise, NPCs^Tie2+-EVs significantly reduced cell senescence and did not show to exert necrotic nor apoptotic effects on recipient cells. Furthermore, EV uptake was successfully observed in all treated cells.

NPCs^Tie2+-EVs demonstrated to significantly enhance degenerative NPC viability, senescence and apoptosis. The use of committed progenitors naturally residing the in the nucleus pulposus may optimize EV regenerative properties and constitute the basis for a new therapy for IDD.

Osteoarthritis (OA) affects the whole joint and leads to chronic pain. The sympathetic nervous system (SNS) seems to be involved in OA pathogenesis, as indicated by in vitro studies as well as by our latest work demonstrating that sympathectomy in mice results in increased subchondral bone volume in the OA knee joint. We assume that chronic stress may lead to opposite effects, such as an increased bone loss in OA due to an elevated sympathetic tone. Therefore, we analyzed experimental OA progression in mice exposed to chronic stress. OA was induced in male C57BL/6J mice by surgical destabilization of the medial meniscus (DMM) and Sham as well as non-operated mice served as controls. Half of these groups were exposed to chronic unpredictable mild stress (CUMS). After 12 weeks, chronic stress efficiency was assessed using behavioral tests. In addition to measuring body weight and length, changes in subchondral bone were analyzed by μCT. Dynamic Weight Bearing system was used to monitor OA-related pain. Histological scoring will be conducted to investigate the severity cartilage degeneration and synovial inflammation. CUMS resulted in increased anxiety and significant decrease in body weight gain in all CUMS groups compared to non-CUMS groups. CUMS also increased serum corticosterone in healthy mice, with even higher levels in CUMS mice after DMM surgery. CUMS had no significant effect on subchondral bone, but subarticular bone mineral density and trabecular thickness were increased. Moreover, CUMS resulted in significant potentiation of DMM-associated pain. Our results suggest that the autonomic imbalance with increased sympathetic nervous activity induced by chronic stress exacerbates the severity of OA pain perception. We expect significantly increased cartilage degeneration as well as more severe synovial inflammation in CUMS DMM mice compared to DMM mice.

Mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) have great promise in the field of orthopaedic nanomedicine due to their regenerative, as well as immunomodulatory and anti-inflammatory properties. Researchers are interested in harnessing these biologically sourced nanovesicles as powerful therapeutic tools with intrinsic bioactivity to help treat various orthopaedic diseases and defects. Recently, a new class of EV mimetics has emerged known as nanoghosts (NGs). These vesicles are derived from the plasma membrane of ghost cells, thus inheriting the surface functionalities and characteristics of the parent cell while at the same time allowing for a more standardized and reproducible production and significantly greater yield when compared to EVs. This study aims to investigate and compare the osteoinductive potential of MSC-EVs and MSC-NGs in vitro as novel tools in the field of bone tissue engineering and nanomedicine. To carry out this investigation, MSC-EVs were isolated from serum-free MSC conditioned media through differential ultracentrifugation. The remaining cells were treated with hypotonic buffer to produce MSC-ghosts that were then homogenized and serially extruded through 400 and 200 nm polycarbonate membranes to form the MSC-NGs. The concentration, size distribution, zeta potential, and protein content of the isolated nanoparticles were assessed. Afterwards, MSCs were treated with either MSC-EVs or MSC-NGs under osteogenic conditions, and their differentiation was assessed through secreted ALP assay, qPCR, and Alizarin Red mineralization staining. Isolation of MSC-EVs and MSC-NGs was successful, with relatively similar mean diameter size and colloidal stability. No effect on MSC viability and metabolic activity was observed with either treatment. Both MSC-EV and MSC-NG groups had enhanced osteogenic outcomes compared to the control; however, a trend was observed that suggests MSC-NGs as better osteoinductive mediators compared to MSC-EVs.

Acknowledgements: The authors would like to acknowledge Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, CHRP, and McGill's Faculty of Dental Medicine and Oral Health Sciences for their financial support.

The Cochrane Collaboration has produced five new reviews relevant to bone and joint surgery since the publication of the last Cochrane Corner These reviews are relevant to a wide range of musculoskeletal specialists, and include reviews in Morton’s neuroma, scoliosis, vertebral fractures, carpal tunnel syndrome, and lower limb arthroplasty.

Abstract

Abstract

Abstract

Abstract

Abstract