bluuebunny/crossref_metadata_2025 huggingface dataset:
THE SECRETOME OF AEC: CHALLENGES AND OPPORTUNITIES IN CELL-FREE REGENERATIVE MEDICINE IN TENDON DISORDERS
Authors: Cerveró-Varona A. , Canciello A. , Prencipe G. , Peserico A. , Haidar-Montes A.A. , Santos H.A. , Russo V. , Barboni B.
Published: January 2024

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.
THERAPEUTIC POTENTIAL OF MESENCHYMAL STEM CELL-DERIVED BIOACTIVE FACTORS: IMPACT ON INTERVERTEBRAL DISC DEGENERATION
Authors: Teixeira G.
Published: January 2024

Back pain is a leading cause of disability worldwide and it is primarily considered to be triggered by intervertebral disc (IVD) degeneration (IVDD). Current treatments may improve pain and mobility, but carry high costs and fail to address IVD repair or regeneration. As no effective therapeutic approach has been proposed to restore inflamed and degenerated IVDs, there is the urgent need to clarify the key pathomechanism of IVDD, the involvement of inflammation, particularly complement activation in matrix catabolism, and how to target them towards tissue repair/regeneration. Mesenchymal stem cell (MSC)-based therapies have become the focus of several regenerative IVD studies. Although patients in clinical trials reported less pain after cell therapy, the long-term success of cell engraftment is unclear due to the hostile IVD environment. The mechanism-of-action of MSCs is mostly dependent on the secreted soluble factors. Moreover, priming of MSC with interleukin (IL)-1β modulates the secretome content, improving its anti-inflammatory and regenerative effect on IVDD organ culture models. MSC-derived extracellular vesicles (EVs) have also been shown to modulate human IVD cells towards a healthy IVD phenotype in vitro. However, the mechanisms involved in the effect of secretome and EVs, particularly with regard to immunomodulation and matrix metabolism, are not fully understood. Our work investigates the effects of secretome and EVs secreted by IL-1β-primed MSCs to impair IVD matrix degradation and/or improve matrix formation in IVDD.
BUILDING VASCULARIZED CONSTRUCTS FROM THE BOTTOM-UP: HARNESSING MICROSCALE LIVING MATERIALS IN TISSUE ENGINEERING
Authors: Barrias C.
Published: January 2024

Bottom-up tissue engineering (TE) strategies employing microscale living materials as building blocks provide a promising avenue for generating intricate 3D constructs resembling native tissues. These microtissue units exhibit high cell densities and a diverse extracellular matrix (ECM) composition, enhancing their biological relevance. By thoughtfully integrating different cell types, the establishment of vital cell-cell and cell-matrix interactions can be promoted, enabling the recreation of biomimetic micro-niches and the replication of complex morphogenetic processes. Notably, by co-assembling blood vessel-forming endothelial cells with supportive stromal cells, microtissues with stable capillary beds, referred to as vascular units (VUs), can be generated. Through a modular TE approach, these VUs can be further combined with other microtissues and biomaterials to construct large-scale vascularized tissues from the bottom up. Integration of VUs with technologies such as 3D bioprinting and microfluidics allows for the creation of structurally intricate and perfusable constructs. In this presentation, we will showcase examples of VUs and explore their applications in regenerative medicine and tissue modeling.

Acknowledgements: This work was supported by project EndoSWITCH (PTDC/BTM-ORG/5154/2020) funded by FCT (Portuguese Foundation for Science and Technology).
CONTROLLING CELL ORGANIZATION IN WAVY ELECTROSPUN SCAFFOLDS FOR THE REGENERATION OF THE ANTERIOR CRUCIATE LIGAMENT
Authors: Camarero-Espinosa S.
Published: January 2024

The anterior cruciate ligament (ACL) is the connective tissue located at the end of long bones providing stability to the knee joint. After tear or rupture clinical reconstruction of the tissue remains a challenge due to the particular mechanical properties required for proper functioning of the tissue. The outstanding mechanical properties of the ACL are characterized by a viscoelastic behavior responsible of the dissipation of the loads that are transmitted to the bone. These mechanical properties are the result of a very specialized graded extracellular matrix that transitions smoothly between the heterotypic cells, stiffness and composition of the ACL and the adjacent bone. Thus, mimicking the zonal biochemical composition, cellular phenotype and organization are key to reset the proper functioning of the ACL.

We have previously shown how the biochemical composition presented to cells in electrospun scaffolds results in haptokinesis, reverting contact-guidance effects.[1] Here, we demonstrate that contact guidance can also be disrupted by structural parameters in aligned wavy scaffolds. The presentation of a wavy fiber arrangement affected the cell organization and the deposition of a specific ECM characteristic of fibrocartilage. Cells cultured in wavy scaffolds grew in aggregates, deposited an abundant ECM rich in fibronectin and collagen II, and expressed higher amounts of collagen II, X and tenomodulin as compared to aligned scaffolds. In-vivo implantation in rabbits of triphasic scaffolds accounting for aligned-wavy-aligned zones showed a high cellular infiltration and the formation of an oriented ECM, as compared to traditional aligned scaffolds.[2]
APPLICATION OF WEIGHTBEARING CT IN CORRECTIVE OSTEOTOMIES
Authors: Burssens A.
Published: January 2024

Osteotomies in the musculoskeletal system are joint preserving procedures to correct the alignment of the patient. In the lower limb, most of the pre-operative planning is performed on full leg weightbearing radiographs. However, these images contain a 2-dimensional projection of a 3-dimensional deformity, lack a clear visualization of the joint surface and are prone to rotational errors during patient positioning. Weightbearing CT imaging has demonstrated to overcome these shortcomings during the first applications of this device at level of the foot and ankle. Recent advances allow to scan the entire lower limb and novel applications at the level of the knee and hip are on the rise. Here, we will demonstrated the current techniques and 3-dimensional measurements used in supra- and inframalleolar osteotomies around the ankle. Several of these techniques will be transposed to other parts in the lower limb to spark future studies in this field.
APPLICATIONS OF WEIGHTBEARING CT IN JOINT INSTABILITY
Authors: Peiffer M.
Published: January 2024

Acute syndesmotic ankle injuries continue to impose a diagnostic dilemma and it remains unclear whether weighbearing or external rotation should be exerted rotation during the imaging process. Therefore, we aimed to implement both axial load (weightbearing) and external rotation in the assessment of a clinical cohort of patients with syndesmotic ankle injuries syndesmotic using weightbearing CT imaging. In this retrospective comparative cohort study, patients with an acute syndesmotic ankle injury were analyzed using a WBCT (N= 20; Mean age= 31,64 years; SD= 14,07. Inclusion criteria were an MRI confirmed syndesmotic ankle injury imaged by a bilateral WBCT of the ankle during weightbearing and combined weightbearing-external rotation. Exclusion criteria consisted of fracture associated syndesmotic ankle injuries. Three-dimensional (3D) models were generated from the CT slices. Tibiofibular displacement and Talar Rotation was quantified using automated3D measurements (Anterior TibioFibular Distance (ATFD), Alpha Angle, Posterior TibioFibular Distance (PTFD) and Talar Rotation (TR) Angle) in comparison to a cohort of non-injured ankles.

Results

The difference in neutral-stressed Alpha° and ATFD showed a significant difference between patients with a syndesmotic ankle lesion and healthy ankles (P = 0.046 and P = 0.039, respectively) The difference in neutral-stressed PTFD and TR° did not show a significant difference between patients with a syndesmotic ankle lesion and healthy ankles (P = 0.492; P = 0.152, respectively).

Conclusion

Application of combined weightbearing-external rotation reveals a dynamic anterior tibiofibular widening in patients with syndesmotic ankle injuries. This study provides the first insights based on 3D measurements to support the potential relevance of applying external rotation during WBCT imaging. However, to what extent certain displacement patterns are associated with syndesmotic instability and thus require operative treatment strategies has yet to be determined in future studies.
A MICROPHYSIOLOGICAL SYSTEM OF INFLAMMATION AND FIBROSIS IN TENDON FIBROVASCULAR SCAR
Authors: Awad H.
Published: January 2024

Vascular inflammation and activation of myofibroblasts are significant contributors to the progression of fibrosis, which can severely impair tissue function. In various tissues, including tendons, Transforming growth factor beta 1 (TGF-β1) has been identified as a critical driver of adhesion and scar formation. Nevertheless, the mechanisms that underlie fibrotic peritendinous adhesions are still not well comprehended, and human microphysiological systems to help identify effective therapies remain scarce. To address this issue, we developed a novel human Tendon-on-a-Chip (hToC), comprised of an endothelialized vascular compartment harboring circulating monocytes and separated by a 5 μm/100 nm dual-scale ultrathin porous membrane from a type I/III collagen hydrogel with primary tendon fibroblasts and tissue-resident macrophages, all under defined serum-free conditions. The hToC models the crosstalk of the various cells in the system leading to the induction of inflammatory and fibrotic pathways including the activation of mTOR signaling. Consistent with phenotypes observed in vivo in mouse models and clinical human samples, we observed myofibroblast differentiation and senescence, tissue contraction, excessive extracellular matrix deposition, and monocytes’ transmigration and macrophages’ secretion of inflammatory cytokines, which were dependent on the presence of the endothelial barrier. This model offers novel insights on the role of vasculature in the pathophysiology of adhesions, which were previously underappreciated. Moreover, in testing whether the hToC could be used to evaluate efficacy of therapeutics, we were able to capture donor-specific variability in the response to Rapamycin treatment, which reduced myofibroblast activation regardless. Thus, our findings demonstrate the value of the hToC as a human microphysiological system for investigating the pathophysiology of fibrotic conditions in the context of peritendinous injury and similar fibrotic conditions, providing an alternative to animal testing.
ENHANCING CARTILAGE TISSUE FORMATION IN GELMA/ALGINATE-TYRAMINE INTERPENETRATED NETWORKS (IPNS) WITH LOW INTENSITY PULSE ULTRASOUND STIMULATION (LIPUS)
Authors: Larrañaga-Jaurrieta G. , Abarrategui A. , Camarero-Espinosa S.
Published: January 2024

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).
MICRORNAS AND THEIR RELEVANCE IN BONE REGENERATION AND DISEASE
Authors: Balmayor E.R. , Joris V. , van Griensven M.
Published: January 2024

Bone tissue is known to possess an intrinsic regeneration potential. However, in cases of major injury, trauma, and disease, bone loss is present, and the regeneration potential of the tissue is often impaired. The process of bone regeneration relies on a complex interaction of molecules. MicroRNAs (miRNA) are small, non-coding RNAs that inhibit messenger RNAs (mRNA). One miRNA can inhibit several mRNAs and one mRNA can be inhibited by several miRNAs. Functionally, miRNAs regulate the entire proteome via the local inhibition of translation. In fact, miRNA modulation has been shown to be involved in several musculoskeletal diseases1. In those pathologies, they modulate the transcriptional activity of mRNAs important for differentiation, tissue-specific activity, extracellular matrix production, etc. Because of their function in inhibiting translation, miRNAs are being researched in many diseases and are already being used for interventional treatment2. Bone tissue and its related conditions have been widely investigated up to this day1,3. This talk will focus on the relevancy of miRNAs to bone tissue, its homeostasis, and disease. After, examples will be given of how miRNAs can be used in bone regeneration and diseases such as osteoporosis and osteosarcoma. The use of miRNAs in both, detection and therapy will be discussed.
A NOVEL NON-CODING RNA-BASED APPROACH TO MODULATE OSTEOCLAST BEHAVIOUR
Authors: Moura S.R. , Olesen J.B. , Barbosa M.A. , Soe K. , Almeida M.I.
Published: January 2024

Osteoclasts (OCs) are multinucleated cells that play a pivotal role in skeletal development and bone remodeling. Abnormal activation of OCs contributes to the development of bone-related diseases, such as osteoporosis, bone metastasis and osteoarthritis. Restoring the normal function of OCs is crucial for bone homeostasis. Recently, RNA therapeutics emerged as a new field of research for osteoarticular diseases.

The aim of this study is to use non-coding RNAs (ncRNAs) to molecularly engineer OCs and modulate their function. Specifically, we investigated the role of the microRNAs (namely miR-16) and long ncRNAs (namely DLEU1) in OCs differentiation and fusion.

DLEU1/DLEU2 region, located at chromosome 13q14, also encodes miR-15 and miR-16. Our results show that levels of these ncRNA transcripts are differently expressed at distinct stages of the OCs differentiation. Specifically, silencing of DLEU1 by small interfering RNAs (siDLEU1) and overexpression of miR-16 by synthetic miRNA mimics (miR-16-mimics) led to a significant reduction in the number of OCs formed per field (OC/field), both at day 5 and 9 of the differentiation stage. Importantly, time-lapse analysis, used to track OCs behavior, revealed a significant decrease in fusion events after transfection with siDLEU1 or miR-16-mimics and an alteration in the fusion mode and partners. Next, we investigated the migration profile of these OCs, and the results show that only miR-16-mimics-OCs, but not siDLEU-OCs, have a lower percentage of immobile cells and an increase in cells with mobile regime, compared with controls. No differences in cell shape were found. Moreover, mass-spectrometry quantitative proteomic analysis revealed independent effects of siDLEU1 and miR-16-mimics at the protein levels. Importantly, DLEU1 and miR-16 act by distinct processes and pathways.

Collectively, our findings support the ncRNAs DLEU1 and miR-16 as therapeutic targets to modulate early stages of OCs differentiation and, consequently, to impair OC fusion, advancing ncRNA-therapeutics for bone-related diseases.

Acknowledgements: Authors would like to thank to AO CMF / AO Foundation (AOCMFS-21-23A). SRM and MIA are supported by FCT (SFRH/BD/147229/2019 and BiotechHealth Program; CEECINST/00091/2018/CP1500/CT0011, respectively).
MICRORNAS 125B, 199A-5P, AND 214 AS MODULATORS OF BONE HOMEOSTASIS
Authors: Joris V. , Balmayor E.R. , van Griensven M.
Published: January 2024

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network.

hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting.

Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics.

We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases.
PRECISION MAGNETOPLEXES FOR MICRORNA DELIVERY TARGETING TENDON INFLAMMATION
Authors: Almeida A.F. , Miranda M.S. , Crowe L.A.N. , Akbar M. , Rodrigues M.T. , Millar N.L. , Gomes M.E.
Published: January 2024

MicroRNA (miR) delivery to regulate chronic inflammation hold extraordinary promise, with new therapeutic possibilities emanating from their ability to fine-tune multiple target gene regulation pathways which is an important factor in controlling aberrant inflammatory reactions in complex multifactorial disease. However, several hurdles have prevented advancements in miR-based therapies. These include off-target effects of miRs, limited trafficking, and inefficient delivery. We propose a magnetically guided nanocarrier to transport therapeutically relevant miRs to assist self- resolving inflammation processes at injury sites and reduce the impact of chronic inflammation- related diseases such as tendinopathies. The high prevalence, significant socio-economic burden and increasing recognition of dysregulated immune mediated pathways in tendon disease provide a compelling rationale for exploring inflammation-targeting strategies as novel treatments in this condition. By combining cationic polymers, miR species (e.g., miR 29a, miR155 antagonist), and magnetic nanoparticles in the form of magnetoplexes with highly efficient magnetofection procedures, we developed inexpensive, easy-to-fabricate, and biocompatible systems with competent miR-binding and fast cellular uptake into different types of human cells, namely macrophages and tendon-derived cells. The system was shown to be cell-compatible and to successfully modulate the expression and production of inflammatory markers in tendon cells, with evidence of functional pro-healing changes in immune cell phenotypes. Hence, magnetoplexes represent a simple, safe, and non-viral nanoplatform that enables contactless miR delivery and high- precision control to reprogram cell profiles toward improved pro-regenerative environments.

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

RES Hub (Norte-01-0145-FEDER-022190).
DO LEUCOCYTE-RICH AND -POOR PLATELET-RICH PLASMA DIFFER BY THEIR METABOLITES?
Authors: Fidan B.B. , Demirdis I. , Çiftçi E. , Aydinli H. , Kaplan O. , Çelebier M. , Boyacioglu Ö. , Korkusuz P. , Karanfil Y. , Korkusuz F.
Published: January 2024

Platelet Rich Plasma (PRP), either rich (L-PRP) or poor (P-PRP) of leukocytes, is frequently used as an anti-inflammatory and regenerative tool in osteoarthritis (OA). PRP contains proteins but not genes as it is derived from megakaryocytes. Proteomics but not metabolomics of PRP was recently studied. Metabolomics is a field of ‘omics’ research involved in comprehensive portrayal of the small molecules, metabolites, in the metabolome. These small molecules can be endogenous metabolites or exogenous compounds found in an organism (1). Our aim was to determine the difference between L-PRP and P-PRP.

A cross-sectional clinical study was designed in six recreational male athletes between the ages of 18 and 35 years. 3 mL P-PRP and 3 mL -LPRP was prepared from 60 mL of venous blood after treating with 9 mL of sodium citrate and centrifugation at 2.700 rpm for 10 min. Half of the prepared PRP's were frozen at −20°C for a week. Fresh and frozen samples were analyzed at the Q-TOF LC/MS device after thawing to room temperature.

Untargeted metabolomic results revealed that the metabolomic profile of the L-PRP and P-PRP were significantly different from each other. A total of 33.438 peaks were found. Statistically significant (p<0.05) peaks were uploaded to the MetaboAnalyst 5.0 platform. Exogenous out of 2.308 metabolites were eliminated and metabolites found significant for our study were subjected to pathway analysis. Steroid biosynthesis, sphingolipid metabolism and metabolism of lipid pathways were affected. In the L-PRP samples, Nicotinamide riboside (FC: 2.2), MHPG (FC: 3.0), estrone sulfate (FC: 7.5), thiamine diphosphate (FC: 2.0), leukotriene E4 (FC: 7.5), PC(18:1 (9Z)e/2:0) (FC: 9.8) and Ap4A (FC: 2.1) were higher compared to P-PRP. C24 sulfatide (FC: −11.8), 3-hexaprenyl-4,5-dihydroxybenzoic acid (FC: −2.8) metabolites were furthermore lower in P-PRP. Clinical outcomes of PRP application should consider these metabolic pathways in future studies (2).
STAT3 ACTIVATION IN THE SYNOVIAL TISSUES FROM THE HIP JOINT IN THE EARLY STAGE OF RAPIDLY DESTRUCTIVE COXOPATHY
Authors: Yasuda T. , Hara S. , Yamashita S. , Mitsuzawa S. , Tsukamoto Y. , Takeuchi H. , Ota S. , Onishi E.
Published: January 2024

The interleukin-6/gp130-associated Janus Kinases/STAT3 axis is known to play an important role in mediating inflammatory signals, resulting in production of matrix metalloproteinase-3 (MMP-3). The hip joints with rapidly destructive coxopathy (RDC) demonstrate rapid chondrolysis, probably by increased production of MMP-3 observed in the early stage of RDC. In the recent study, no apparent activation of STAT3 has been shown in the synovial tissues obtained from the osteoarthritic joint at operation. However, no data are currently available on STAT3 activation in the synovial tissues in the early stage of RDC. This study aimed to elucidate STAT3 activation in the synovial tissues in the early stage of RDC. Synovial tissues within 7 months from the disease onset were obtained from four RDC patients with femoral head destruction and high serum levels of MMP-3. RDC synovial tissues showed the synovial lining hyperplasia with an increase of CD68-positive macrophages and CD3-positive T lymphocytes. STAT3 phosphorylation was found in the synovial tissues by immunohistochemistry using anti-phospho-STAT3 antibody. The majority of phospho-STAT3-positive cells were the synovial lining cells and exhibited negative expression of macrophage or T cell marker. Treatment with tofacitinib, a Janus Kinase inhibitor, resulted in a decrease in phospho-STAT3-positive cells, especially with high intensity, indicating effective suppression of STAT3 activation in RDC synovial tissues. Inhibitory effect of tofacitinib could act through the Janus Kinase/STAT3 axis in the synovial tissues in the early stage of RDC. Therefore, STAT3 may be a potential therapeutic target for prevention of joint structural damage in RDC.

Acknowledgements: This study was supported by Katakami Foundation for Clinical Research.
DUAL-MOBILITY ARTICULATION CONFERS LOWER DISLOCATION AND REVISION RATES: A STUDY USING REVIEW OF REVIEWS METHODOLOGY
Authors: Mehta S. , Goel A. , Mahajan U. , Reddy N.R. , Bhaskar D.
Published: January 2024

Dislocation post THA confers a higher risk of re-dislocation (Kotwal et al, 2009). The dual mobility (DM) cup design (1974) was aimed at improving the stability by increasing the femoral head to neck ratio (Cuthbert et al., 2019) combining the ideas of low friction arthroplasty with increased jump distance associated with a big head arthroplasty.

Understand the dislocation rates, rates of aseptic loosening, infection rate and revision rates between the 2 types of constructs to provide current and up-to date evidence.

Medline, pubmed, embase and Cochrane databases were used based on PRISMA guidelines. RevMan software was used for the meta-analysis. Studies (English literature) which used DM construct with atleast 6 months follow-up used as intervention and non DM construct as control were included. 2 independent reviewers conducted the review with a third reviewer in case of difference in opinion regarding eligibility. Primary outcome was dislocation rate and secondary outcome was rate of revision.

564 articles identified out of which 44 articles were screened for full texts and eventually 4 systematic review articles found eligible for the study. Thus, study became a review of systematic reviews. From the 4 systematic reviews, another 35 studies were identified for data extraction and 13 papers were used for meta-analysis. Systematic reviews evaluated, projected an average follow up of 6-8 years with significantly lower dislocation rates for DM cups. The total number of patients undergoing DM cup primary THA were 30,559 with an average age 71 years while the control group consisted of 218,834 patients with an average age of 69 years. DM group had lower rate of dislocation (p < 0.00001), total lower rate of cup revision (p < 0.00001, higher incidence of fracture (p>0.05).

DM THA is a viable alternative for conventional THA. The long-term results of DM cups in primary THA need to be further evaluated using high quality prospective studies and RCTs.
ORTHOPOD: PREDICTING INJURY PROPORTIONALITY FROM NECK OF FEMUR FRACTURES
Authors: Morris T. , Fouweather F. , Walshaw T. , Baldock T. , Wei N. , Eardley W.
Published: January 2024

The need to accurately forecast the injury burden has never been higher. With an aging, ever expanding trauma population and less than half of the beds available compared to 1990, the National Health Service (NHS) is stretched to breaking point1,2.

We utilised a dataset of 22,585 trauma patients across the four countries of the United Kingdom (UK) admitted to 83 hospitals between 22/08/22 – 16/10/22 to determine whether it is possible to predict the proportionality of injuries treated operatively within orthopaedic departments based on their number of Neck of Femur fracture (NOF) patients.

More operations were performed for elderly hip fractures alone than for the combined totals of the next four most common fractures: ankle, distal radius, tibial shaft and forearm (6387 vs 5922). Conversely, 10 out of the 13 fracture types were not encountered by at least one hospital and 93% of hospitals encountered less than 2 fractures of a certain type.

60% trauma is treated within Trauma Units (TUs) however, per unit, Major Trauma Centres (MTCs) treat approximately 43% more patients.

After excluding NOF, lower limb fractures accounted for approximately 57% of fractures in all countries and ankle and distal radius fracture combined comprised more than 50% in 74% of regions.

The number of hip fractures seen on average by an individual unit remains relatively consistent as does the regional variation of any given fracture; resultantly, it is possible to predict injury proportionality based off a unit's hip fracture numbers. This powerful tool could transform both resource allocation and recruitment.
TECHNICAL NOTE FOR THE NOVEL OSTEOPORE® WEDGE IN MEDIAL OPENING WEDGE HIGH TIBIAL OSTEOTOMY
Authors: Wong S.Y.W. , Lee K.H. , Razak H.R.B.A.
Published: January 2024

Medial opening wedge high tibial osteotomy (MOWHTO) is the workhorse procedure for correcting varus malalignment of the knee. There have been recent developments in the synthetic options to fill the osteotomy gap. The current gold standard for filling this osteotomy gap is autologous bone graft which is associated with donor site morbidity. We would like to introduce and describe the process of utilizing the novel Osteopore® 3D printed, honeycomb structured, Polycaprolactone and β-Tricalcium Phosphate wedge for filling the gap in MOWHTO. In the advent of additive manufacturing and the quest for more biocompatible materials, the usage of the Osteopore® bone wedge in MOWHTO is a promising technique that may improve the biomechanical stability as well the healing of the osteotomy gap.
COULD A DECELLULARIZATION PROTOCOL BE VALIDATED WITHOUT TALKING ABOUT HUMAN LEUKOCYTE ANTIGEN?
Authors: Manon J. , Evrard R. , Fievé L. , Xhema D. , Maistriaux L. , Schubert T. , Lengelé B. , Behets C. , Cornu O.
Published: January 2024

Decellularization techniques have advanced to reduce the risk of immune rejection in transplantation. Validation of these protocols typically relies on Crapo's criteria1, which include the absence of visible nuclei and low DNA content. In our study, five decellularization protocols were compared to determine the optimal approach for human fascia lata (HFL) samples. However, our findings raised questions as to why recipients can still develop immunity despite meeting validation criteria.

HFL samples were decellularized using four protocols with SDS-Triton X100-DNase (D1 to D4-HFL) and one protocol using solvent-detergent-based baths (D5-HFL). The decellularized samples (D-HFL) were compared to native samples (N-HFL) using histology, and DNA content was measured. The human leukocyte antigen (HLA) content within the matrix was assessed using western blot analysis. Both D-HFL and N-HFL samples, along with negative control patches, were implanted in the backs of 28 Wistar rats. Anti-human IgG serum levels were evaluated after one month.

H&E and Hoechst staining revealed the absence of residual cells in all decellularization protocols. DNA content was consistently below the critical threshold (p<0.05). All implanted D-HFL samples resulted in significantly lower anti-human IgG levels compared to N-HFL (p<0.01). However, 2.5 out of 4 rats developed immunity after being implanted with D1 to D4-HFL, with varying levels of anti-human IgG. Only rats implanted with D5-HFL showed undetectable levels of IgG and were considered non-immunized. Western blot analysis indicated that only D5-HFL had a residual HLA content below 1%.

The literature on decellularization has primarily relied on Crapo's criteria, which do not consider the role of HLA mismatch in acute immune rejection. Our results suggest that a residual HLA content below 1% should also be considered to prevent immunization, even if other validation criteria are met. Further research is needed to evaluate the impact of residual HLA levels on human allotransplantation outcomes.
TENOCYTES THROUGH SPACE AND TIME: DETERMINING THE FATE OF TENDON HEALING
Authors: Ackerman J.
Published: January 2024

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.
THE INTERPLAY OF ANGIOGENESIS AND OSTEOGENESIS FOR BONE REGENERATION
Authors: Banfi A.
Published: January 2024

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:

Controlling the distribution of VEGF protein in the microenvironment is key to recapitulate its physiologic function to couple angiogenesis and osteogenesis (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);

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.
FUNCTIONAL ACTIVITY OF MONOMERIC C-REACTIVE PROTEIN IN INTERVERTEBRAL DISC
Authors: Ruiz-Fernández C. , Eldjoudi D.A. , Gonzalez-Rodríguez M. , Barreal A.C. , Farrag Y. , Mobasheri A. , Pino J. , Sakai D. , Gualillo O.
Published: January 2024

Monomeric C reactive protein (mCRP) presents important proinflammatory effects in endothelial cells, leukocytes, or chondrocytes. However, CRP in its pentameric form exhibits weak anti-inflammatory activity. It is used as a biomarker to follow severity and progression in infectious or inflammatory diseases, such as intervertebral disc degeneration (IVDD). This work assesses for the first time the mCRP effects in human intervertebral disc cells, trying to verify the pathophysiological relevance and mechanism of action of mCRP in the etiology and progression of IVD degeneration.

We demonstrated that mCRP induces the expression of multiple proinflammatory and catabolic factors, like nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and lipocalin 2 (LCN2), in human annulus fibrosus (AF) and nucleus pulposus (NP) cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signaling of mCRP.

Our results indicate that the effect of mCRP is persistent and sustained, regardless of the proinflammatory environment, as it was similar in healthy and degenerative human primary AF cells. This is the first article that demonstrates the localization of mCRP in intravertebral disc cells of the AF and NP and that provides evidence for the functional activity of mCRP in healthy and degenerative human AF and NP disc cells.
CELLULAR AND MOLECULAR ENDOTYPES FOR OSTEOARTHRITIS
Authors: Pattappa G.
Published: January 2024

The biological understanding for the disease progression osteoarthritis (OA) has uncovered specific biomarkers from either synovial fluid, articular chondrocytes or synoviocytes that can be used to diagnose the disease. Examples of these biomarkers include interleukin-1β (IL-1β) or collagen II fragments (1, 2). In parallel, isolation of chondrocytes or bone marrow derived mesenchymal stromal cells (MSCs) has yielded cell-based strategies that have shown long- term beneficial effects in a specific cohort of patients, specifically in traumatic cartilage lesions (2). This latter finding shows that patient stratification of OA is an important tool to both match patients for a specific treatment and to develop novel therapies, especially disease modifying drugs. In order to create disease stage specific therapies, the use of next generation analysis tools such as RNAseq and metabolomics, has the potential to decipher specific cellular and molecular endotypes. Alongside greater understanding of the clinical phenotype (e.g. imaging, pain, co- morbidities), therapies can be designed to alleviate the symptoms of OA at specific points of the disease in patients. This talk will outline the current biological understanding of OA and discuss how patient stratification could assist in the design of innovative therapies for the disease.

Acknowledgements: This presentation was supported by the COST action, CA21110 – Building an open European Network on Osteoarthritis Research (NetwOArk)
A Qualitative Study on Korean Mothers’ Dysfunctional Beliefs and Meaning Structures Regarding Their Experiences With Infant Sleep Problems
Authors: Kang Solbi , Cheong Seockhoon , Suh Sooyeon
Published: January 2024

Objectives: One of the causes of infant sleep problems is excessive parental intervention at bedtime based on parental dysfunctional beliefs about infant sleep, which varies depending on the culture. This study aimed to identify the content of dysfunctional beliefs related to infant sleep problems among Korean mothers.Methods: Participants were 10 Korean mothers with infants aged 6 to 36 months. In-depth interviews were conducted to identify the infant sleep problems being experienced and the content of accompanying attitudes and dysfunctional beliefs. Individual in-depth interviews were conducted with the researcher based on a semi-structured manual. A qualitative analysis to identify dysfunctional beliefs was conducted based on phenomenological methods.Results: The meaning structures of dysfunctional beliefs held by Korean mothers who experienced infant sleep problems were divided into six categories, namely, “unable to fall asleep without the mother,” “various inferences about the cause of sleep problems,” “mother’s effort and sacrifice are required,” “concerns about child development,” “experience of fatigue and negative emotions,” and “mother’s experience of sleep disturbance.”Conclusions: Korean mothers reported similar results as previous studies in that they implemented strategies that interfered with infant sleep based on dysfunctional beliefs about their infant’s sleep problems. These behaviors subsequently increased the possibility of maintaining sleep problems for their infants and themselves.
Restless Sleep Disorder as a New Primary Sleep Disorder in Children and Adolescents
Authors: Kim Eun-Hee
Published: January 2024

Restless sleep is a frequent complaint clinically and often associated with specific sleep or medical conditions. Recently, restless sleep disorder (RSD) has been identified as a new pediatric sleep disorder with core features of restless sleep and frequent large muscle movements during sleep, which are not explained by other conditions. Based on the medical literature and expert clinical experience, the task force developed a consensus diagnostic criteria for RSD and scoring large muscle group movements in children aged 6–18 years. Eight essential criteria of RSD include a complaint of restless sleep, observed large body movements during sleep, video-polysomnographic documentation of ≥5 large body movements/hour, occurrence at least thrice weekly for at least 3 months, clinically significant impairment, and differentiation from other conditions that may secondarily cause restless sleep. There is evidence to support a pathophysiology based on iron deficiency, sleep instability, and increased sympathetic activation, and treatment options include oral or intravenous iron supplementation. This review describes the key features of RSD, potential pathophysiology, and current treatment options to help diagnose patients with RSD, which has been overlooked.
A Case of Intractable REM Sleep Behavior Disorder Improved With Nasal Continuous Positive Airway Pressure Therapy
Authors: Shin Hye-Rim
Published: January 2024

Rapid eye movement (REM) sleep behavior disorder (RBD) is characterized by dream enactment behavior and REM sleep without atonia and is traumatic for the patient and/or bedpartner. Obstructive sleep apnea (OSA) is a common comorbidity of RBD, and several reports have suggested that severe OSA may aggravate RBD symptoms. This case report discusses a patient with medically intractable RBD combined with OSA, confirmed by 10-year follow-up polysomnography (PSG) data, which improved with nasal continuous positive airway pressure (CPAP) therapy. The clinical symptoms of RBD markedly improved after CPAP treatment. Clinicians should raise the clinical suspicion of OSA in patients with intractable RBD and consider follow-up PSG and OSA treatments.
Paraxial sharp-edge diffraction of vortex beams by elliptic apertures
Authors: Borghi Riccardo
Published: January 2024

A semi-analytical computational algorithm to model the wave field generated by paraxial diffraction of a class of Laguerre–Gauss beams by sharp-edge elliptic apertures is here developed. Thanks to such a powerful computational tool, some basic aspects of an intriguing and still unexplored singular optics scenario can be studied, within a geometry as simple as possible, with arbitrarily high accuracies.