bluuebunny/crossref_metadata_2025 huggingface dataset:
CLINICAL TRIAL QUALITY ASSESSMENT IN INTERVERTEBRAL DISC REGENERATION: INSIGHTS FROM PUBLICATION STATUS AND FUNDING SOURCES
Authors: Ambrosio L. , Vadalà G. , Petrucci G. , Russo F. , Papalia R. , Denaro V.
Published: January 2024

Low back pain (LBP) is the main cause of disability worldwide and is primarily triggered by intervertebral disc degeneration (IDD). Although several treatment options exist, no therapeutic tool has demonstrated to halt the progressive course of IDD. Therefore, several clinical trials are being conducted to investigate different strategies to regenerate the intervertebral disc, with numerous studies not reaching completion nor being published. The aim of this study was to analyze the publication status of clinical trials on novel regenerative treatments for IDD by funding source and identify critical obstacles preventing their conclusion.

Prospective clinical trials investigating regenerative treatments for IDD and registered on ClinicalTrials.gov were included. Primary outcomes were publication status and investigational treatment funding. Fisher's exact test was utilized to test the association for categorical variables between groups.

25 clinical trials were identified. Among these, only 6 (24%) have been published. The most common source of funding was university (52%), followed by industry (36%) and private companies (12%). Investigational treatments included autologous (56%) or allogeneic (12%) products alone or in combination with a carrier or delivery system (32%). The latter were more likely utilized in industry or privately funded studies (Fig. 1, p=0.0112). No significant difference was found in terms of funding regarding the publication status of included trials (Table 1, p=0.9104).

Most clinical trials investigating regenerative approaches for the treatment of IDD were never completed nor published. This is likely due to multiple factors, including difficult enrollment, high dropout rate, and publication bias3. More accurate design and technical support from stakeholders and clinical research organization (CROs) may likely increase the quality of future clinical trials in the field.

For any figures or tables, please contact the authors directly.
FABRICATION OF A HIGH-THROUGHPUT 3D-PRINTED OSTEOGENIC CORAL-CONTAINING SCAFFOLD
Authors: Doyle S. , Winrow D. , Aregbesola T. , Martin J. , Pernevik E. , Kuzmenko V. , Howard L. , Thompson K. , Johnson M. , Coleman C.
Published: January 2024

In 2021 the bone grafting market was worth €2.72 billion globally. As allograft bone has a limited supply and risk of disease transmission, the demand for synthetic grafting substitutes (BGS) continues to grow while allograft bone grafts steadily decrease. Synthetic BGS are low in mechanical strength and bioactivity, inspiring the development of novel grafting materials, a traditionally laborious and expensive process. Here a novel BGS derived from sustainably grown coral was evaluated. Coral-derived scaffolds are a natural calcium carbonate bio-ceramic, which induces osteogenesis in bone marrow mesenchymal stem cells (MSCs), the cells responsible for maintaining bone homeostasis and orchestrating fracture repair. By 3D printing MSCs in coral-laden bioinks we utilise high throughput (HT) fabrication and evaluation of osteogenesis, overcoming the limitations of traditional screening methods.

MSC and coral-laden GelXA (CELLINK) bioinks were 3D printed in square bottom 96 well plates using a CELLINK BIO X printer with pneumatic adapter Samples were non-destructively monitored during the culture period, evaluating both the sample and the culture media for metabolism (PrestoBlue), cytotoxicity (lactose dehydrogenase (LDH)) and osteogenic differentiation (alkaline phosphatase (ALP)). Endpoint, destructive assays used included qRT-PCR and SEM imaging.

The inclusion of coral in the printed bioink was biocompatable with the MSCs, as reflected by maintained metabolism and low LDH release. The inclusion of coral induced osteogenic differentiation in the MSCs as seen by ALP secretion and increased RUNX2, collagen I and osteocalcin transcription.

Sustainably grown coral was successfully incorporated into bioinks, reproducibly 3D printed, non-destructively monitored throughout culture and induced osteogenic differentiation in MSCs. This HT fabrication and monitoring workflow offers a faster, less labour-intensive system for the translation of bone substitute materials to clinic.

Acknowledgements: This work was co-funded by Enterprise Ireland and Zoan Biomed through Innovation Partnership IP20221024.
EXPLORING THE EXTRACELLULAR MATRIX FROM YOUNG DONORS TO ENHANCE THE IMPAIRED OSTEOGENIC PROPERTIES OF AGED CELLS
Authors: Carvalho M.S. , Cabral J.M.S. , da Silva C.L.
Published: January 2024

Mesenchymal stromal cells (MSC) have been proposed as an emerging cell therapy for bone tissue engineering applications. However, the healing capacity of the bone tissue is often compromised by patient's age and comorbidities, such as osteoporosis. In this context, it is important to understand the impact of donor age on the therapeutic potential of MSC. Importantly, the impact on donor age is not restricted to cells themselves but also to their microenvironment that is known to affect cell function.

The extracellular matrix (ECM) has an important role in stem cell microenvironment, being able to modulate cell proliferation, self-renewal and differentiation. Decellularized cell-derived ECM (dECM) has been explored for regenerative medicine applications due to its bioactivity and its resemblance to the in vivo microenvironment. Thus, dECM offers the opportunity not only to develop microenvironments with customizable properties for improvement of cellular functions but also as a platform to study cellular niches in health and disease. In this study, we investigated the capacity of the microenvironment to rescue the impaired proliferative and osteogenic potential of aged MSC. The goal of this work was to understand if the osteogenic capacity of MSC could be modulated by exposure to a dECM derived from cells obtained from young donors. When aged MSC were cultured on dECM derived from young MSC, their in vitro proliferative and osteogenic capacities were enhanced. Our results suggest that the microenvironment, specifically the ECM, plays a crucial role in the osteogenic differentiation capacity of MSC. dECM might be a valuable clinical strategy to overcome the age-related decline in the osteogenic potential of MSC by recapitulating a younger microenvironment, attenuating the effects of aging on the stem cell niche. Overall, this study opens new possibilities for developing clinical strategies for elderly patients with limited bone formation capacity who currently lack effective treatments.

Acknowledgements: The authors thank FCT for funding through the project DentalBioMatrix (PTDC/BTM-MAT/3538/2020) and to the research institutions iBB (UIDB/04565/2020 and UIDP/04565/2020) and Associate Laboratory i4HB (LA/P/0140/2020).
ALTERNATION OF THE 3D SUBTALAR JOINT ALIGNMENT AFTER INFRAMALLEOLAR OSTEOTOMY IN PROGRESSIVE COLLAPSING FLATFOOT DEFORMITY
Authors: Raes L. , Peiffer M. , Kvarda P. , Leenders Tim , Audenaert Emanuel A. , Burssens Arne
Published: January 2024

A medializing calcaneal osteotomy (MCO) is one of the key inframalleolar osteotomies to correct progressive collapsing foot deformity (PCFD). While many studies were able to determine the hind- and midfoot alignment after PCFD correction, the subtalar joint remained obscured by superposition on plain radiography. Therefore, we aimed to perform a 3D measurement assessment of the hind- and subtalar joint alignment pre- compared to post-operatively using weightbearing CT (WBCT) imaging.

Fifteen patients with a mean age of 44,3 years (range 17-65yrs) were retrospectively analyzed in a pre-post study design. Inclusion criteria consisted of PCFD deformity correct by MCO and imaged by WBCT. Exclusion criteria were patients who had concomitant midfoot fusions or hindfoot coalitions. Image data were used to generate 3D models and compute the hindfoot - and talocalcaneal angle as well as distance maps.

Pre-operative radiographic parameters of the hindfoot and subtalar joint alignment improved significantly relative to the post-operative position (HA, MASa, and MACo). The post-operative talus showed significant inversion, abduction, and dorsiflexion of the talus (2.79° ±1.72, 1.32° ±1.98, 2.11°±1.47) compared to the pre-operative position. The talus shifted significantly different from 0 in the posterior and superior direction (0.62mm ±0.52 and 0.35mm ±0.32). The distance between the talus and calcaneum at the sinus tarsi increased significantly (0.64mm ±0.44).

This study found pre-dominantly changes in the sagittal, axial and coronal plane alignment of the subtalar joint, which corresponded to a decompression of the sinus tarsi. These findings demonstrate the amount of alternation in the subtalar joint alignment that can be expected after MCO. However, further studies are needed to determine at what stage a calcaneal lengthening osteotomy or corrective arthrodesis is indicated to obtain a higher degree of subtalar joint alignment correction.
GABAPENTINOIDS IN THE SYMPTOMATIC MANAGEMENT OF CANAL STENOSIS: A COMPARATIVE STUDY ON PAIN RELIEF, AMBULATION, AND SAFETY PROFILE
Authors: Martínez T. , Mariscal G. , Hevia E. , Barrios C.
Published: January 2024

The multimodal management of canal stenosis is increasing, and inhibitors of central sensitization are playing a crucial role in central sensitization processes. Pregabalin and gabapentin are antiepileptic drugs that reduce presynaptic excitability. The objective of this study was to investigate whether the use of pregabalin and gabapentin is effective in the symptomatic management of canal stenosis.

A literature search was conducted in four databases. The inclusion criteria were studies that compared pregabalin or gabapentin with a control group in lumbar canal stenosis. Randomized clinical trials and a comparative retrospective cohort study were included. The main clinical endpoints were VAS/NRS, ODI, and RDQ (Roland Morris Disability Questionnaire) at 2, 4, 8 weeks, and 3 months, adverse events, and walking distance were also collected. Data were combined using Review Manager 5.4 software.

Six studies and 392 patients were included. The mean age was 60.25. No significant differences were observed in VAS at 2, 4, and 8 weeks: (MD: 0.23; 95% CI: −0.63-1.09), (MD: −0.04; 95% CI: −0.64 to −0.57), and (MD: −0.6; 95% CI: −1.22 to 0.02). Significant differences were observed in favor of pregabalin with respect to VAS at three months: (MD: −2.97; 95% CI: −3.43 to −2.51). No significant differences were observed in ODI (MD: −3.47; 95% CI: −7.15 to −0.21). Adverse events were significantly higher in the pregabalin/gabapentin group (OR 5.88, 95%CI 1.28-27.05). Walking distance and RDQ could not be compared, although the results were controversial.

Gabapentinoids have not been shown to be superior to other drugs used in the treatment of LSS or to placebo. However, they have shown a higher incidence of adverse effects, improved results in VAS at 3 months, and a slight improvement in ambulation at 4 months in combination with NSAIDs compared to NSAIDs in monotherapy.
NOVEL STEM CELL STRATEGIES FOR TENDON REGENERATIVE MEDICINE
Authors: Montes A.A.H. , Mauro A. , Cerveró-Varona A. , Prencipe G. , El Khatib M. , Tosi U. , Wouters G. , Stöckl J. , Russo V. , Barboni B.
Published: January 2024

Adipose-derived stem cells (ADSCs) are an effective alternative for Teno-regeneration. Despite their applications in tendon engineering, the mechanisms promoting tendon healing still need to be understood. Since there is scattered information on ovine ADSCs, this research aims to investigate in vitro their teno-differentiation for potential use in preclinical tendon regeneration models.

Ovine ADSCs were isolated from the tail region according to FAT-STEM laboratories, expanded until passage six (P6), and characterized in terms of stemness, adhesion and MHC markers by Flow Cytometry (FCM) and immunocytochemistry (ICC). Cell proliferation and senescence were evaluated with MTT and Beta-galactosidase assays, respectively. P1 ADSCs’ teno-differentiation was assessed by culturing them with teno-inductive Conditioned Media (CM) or engineering them on tendon-mimetic PLGA scaffolds. ADSCs teno-differentiation was evaluated by morphological, molecular (qRT-PCR), and biochemical (WesternBlot) approaches.

ADSCs exhibited mesenchymal phenotype, positive for stemness (SOX2, NANOG, OCT4), adhesion (CD29, CD44, CD90, CD166) and MHC-I markers, while negative for hematopoietic (CD31, CD45) and MHC-II markers, showing no difference between passages. ICC staining confirmed these results, where ADSCs showed nuclear positivity for SOX2 (≅ 56%) and NANOG (≅ 67%), with high proliferation capacity without senescence until P6. Interestingly, ADSCs cultured with the teno-inductive CM did not express tenomodulin (TNMD) protein or gene. Conversely, ADSCs seeded on scaffolds teno-differentiated, acquiring a spindle shape supported by TNMD protein expression at 48h (p<0.05 vs. ADSCs 48h) with a significant increase at 14 days of culture (p<0.05 vs. ADSCs + fleece 48h).

Ovine ADSCs respond differently upon distinct teno-inductive strategies. While the molecules on the CM could not trigger a teno-differentiation in the cells, the scaffold's topological stimulus did, resulting in the best strategy to apply. More insights are requested to better understand ovine ADSCs’ tenogenic commitment before using them in vivo for tendon regeneration.

Acknowledgements: This research is part of the P4FIT project ESR5, under the H2020MSCA-ITN-EJD-P4 FIT-Grant Agreement ID:955685.
MECHANICALLY INSTRUCTIVE SCAFFOLDS TO STEER TISSUE REGENERATION: MERGING MECHANOBIOLOGY WITH BIOFABRICATION
Authors: Moroni L.
Published: January 2024

Regenerative medicine (RM) promises to restore both the mechanical functionality and the biological composition of tissues after damage. Three-dimensional scaffolds are used in RM to host cells and let them produce proteins that are the building blocks of the native tissues. While regenerating tissues evolve over time through dynamic biomechanical and biochemical changes, current scaffolds’ generation are passive causing mechanical mismatch, suboptimal growth, and pain. Furthermore, current scaffolds ignore the complexity of the reciprocal bio-mechanics regulation, hindering the design of the next-gen scaffolds. To regenerate tissues and organs, biofabrication strategies that impart spatiotemporal control over cell-cell and cell-extracellular matrix communication, often through control over cell and material deposition and placement, are being developed. To achieve these targets, the spatiotemporal control over biological signals at the interface between cells and materials is often aimed for. Alternatively, biological activity can be triggered through the control of mechanical cues, harnessing more fundamental know-how in mechanobiology that could be combined with biofabrication strategies. Here, I present some of our most recent advancements in merging mechanobiology with biofabrication that enabled the control of cell activity, moving towards enhanced tissue regeneration as well as the possibility to create more complex 3D in vitro models to study biological processes.
3D WRITING OF MULTICELLULAR TENDON-ON-CNC-CHIP MODELS
Authors: Monteiro R.F. , Bakht S.M. , Gomez-Florit M. , Reis R.L. , Gomes M.E. , Domingues R.M.A.
Published: January 2024

Relevant in vitro models emulating tendinopathies are highly needed to study these diseases and develop better treatments. We have recently proposed a new strategy that allows the automated 3D writing of microphysiological systems (MPS) embedded into its own biomimetic fibrillar support platform based on the self-assembling of cellulose nanocrystals (CNCs). Here, we explored this CNC platform for writing humanized in vitro tendon models using tendon decellularized extracellular matrix (dECM)-based bioinks to closely recapitulate the biophysical and biochemical cues of tendon cell niche and self-induce the tenogenic differentiation of stem cells. The proposed concept was further explored to study the crosstalk between the tendon core and vascular compartment.

Porcine flexor tendons were decellularized to produce the dECM bioink hydrogel. hASCs were used as cell source and the bioink was directly printed within the CNC fluid gel. Tendon constructs were co-printed with compartmentalized microvascular structures to evaluate the cellular crosstalk with endothelial cells. The tendon-on-chip models showed high cell viability and proliferation during culture up to 21 days, and the synergy between dECM cues and printed patterns induced anisotropic cell organization similar to tendon tissues. Gene and protein analysis showed upregulation of the most important tendon related markers on tendon constructs, demonstrating that the biophysical and biochemical cues of dECM induced hASCs commitment toward tenogenic phenotype. In co-culture system, chemotaxis induced endothelial cells migration toward the tendon compartment, but without significant infiltration. Gene and protein expression results suggest that the cellular crosstalk established in this MPS with endothelial cells boosted hASCs tenogenesis, emulating tendon development stages.

Overall, the proposed system might be promising for the automated fabrication of organotypic tendon-on-chip models that will be a valuable new tool to study tendon physiology, pathology, or the effect of drugs for the treatment of tendinopathy.

Acknowledgments: EU H2020 for ERC-2017-CoG-772817; ERC-PoC-BioCHIPs-101069302; FCT/MCTES for 2022.05526.PTDC, 2020.03410.CEECIND, and PD/BD/129403/2017
MULTILAYER DUAL-POROSITY 3D-PRINTED SCAFFOLDS TO RECREATE THE ANISOTROPIC MICROENVIRONMENT OF THE CARTILAGE
Authors: Ramos-Díez S. , Camarero-Espinosa S.
Published: January 2024

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.
AO FRACTURE MONITOR: CONTINUOUS SENSOR MONITORING FOR PERSONALIZED FRACTURE CARE
Authors: Ernst M. , Windolf M. , Varjas V. , Gehweiler D. , Gueorguiev-Rüegg B. , Richards R.G.
Published: January 2024

In absence of available quantitative measures, the assessment of fracture healing based on clinical examination and X-rays remains a subjective matter. Lacking reliable information on the state of healing, rehabilitation is hardly individualized and mostly follows non evidence-based protocols building on common guidelines and personal experience. Measurement of fracture stiffness has been demonstrated as a valid outcome measure for the maturity of the repair tissue but so far has not found its way to clinical application outside the research space. However, with the recent technological advancements and trends towards digital health care, this seems about to change with new generations of instrumented implants – often unfortunately termed “smart implants” – being developed as medical devices.

The AO Fracture Monitor is a novel, active, implantable sensor system designed to provide an objective measure for the assessment of fracture healing progression (1). It consists of an implantable sensor that is attached to conventional locking plates and continuously measures implant load during physiological weight bearing. Data is recorded and processed in real-time on the implant, from where it is wirelessly transmitted to a cloud application via the patient's smartphone. Thus, the system allows for timely, remote and X-ray free provision of feedback upon the mechanical competence of the repair tissue to support therapeutic decision making and individualized aftercare.

The device has been developed according to medical device standards and underwent extensive verification and validation, including an in-vivo study in an ovine tibial osteotomy model, that confirmed the device's capability to depict the course of fracture healing as well as its long-term technical performance. Currently a multi-center clinical investigation is underway to demonstrate clinical safety of the novel implant system. Rendering the progression of bone fracture healing assessable, the AO Fracture Monitor carries potential to enhance today's postoperative care of fracture patients.
INFLUENCE OF FLUID UPTAKE ON THE MECHANICAL AND THERMAL PROPERTIES OF PMMA-BASED BONE CEMENT
Authors: Emonde C. , Reulbach M. , Evers P. , Behnsen H. , Nürnberger F. , Jakubowitz E. , Windhagen H.
Published: January 2024

According to the latest report from the German Arthroplasty Registry, aseptic loosening is the primary cause of implant failure following primary hip arthroplasty. Osteolysis of the proximal femur due to the stress-shielding of the bone by the implant causes loss of fixation of the proximal femoral stem, while the distal stem remains fixed.

Removing a fixed stem is a challenging process. Current removal methods rely on manual tools such as chisels, burrs, osteotomes, drills and mills, which pose the risk of bone fracture and cortical perforation. Others such as ultrasound and laser, generate temperatures that could cause thermal injury to the surrounding tissues and bone. It is crucial to develop techniques that preserve the host bone, as its quality after implant removal affects the outcome of a revision surgery.

A gentler removal method based on the transcutaneous heating of the implant by induction is proposed. By reaching the glass transition temperature (TG) of the periprosthetic cement, the cement is expected to soften, enabling the implant to be gently pulled out. The in-vivo environment comprises body fluids and elevated temperatures, which deteriorate the inherent mechanical properties of bone cement, including its TG. We aimed to investigate the effect of fluid absorption on the TG (ASTM E2716-09) and Vicat softening temperature (VST) (ISO 306) of Palacos R cement (Heraeus Medical GmbH) when dry and after storage in Ringer's solution for up to 8 weeks.

Samples stored in Ringer's solution exhibited lower TG and VST than those stored in air. After 8 weeks, the TG decreased from 95.2°C to 81.5°C in the Ringer's group, while the VST decreased from 104.4°C to 91.9°C. These findings will be useful in the ultimate goal of this project which is to design an induction-based system for implant removal.

Acknowledgements: Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB/TRR-298-SIIRI – Project-ID 426335750
STABILITY OF BIOFUNCTIONALIZED HIGH-PERFORMANCE OXIDE CERAMICS AGAINST RADIATION STERILIZATION
Authors: Schräder P. , Montoya A.G. , Labude-Weber N. , Eschweiler J. , Neuss S. , Fischer H.
Published: January 2024

While high-performance ceramics like alumina and zirconia exhibit excellent wear resistance, they provide poor osseointegration capacity. As osseointegration is crucial for non-cemented joint prostheses, new techniques have been successfully developed for biofunctionalizing high-performance ceramic surfaces. Stable cell adhesion can be achieved by covalently bound specific peptides. In this study we investigate the effect of sterilization processes on organo-chemically functionalized surfaces.

To enhance the performance of alumina-toughened zirconia ceramics (ATZ), a 3-aminopropyldiisopropylethoxysilane (APDS) monolayer was applied and coupled with cyclo-RGD peptides (cRGD) by using bifunctional crosslinker bis(sulfosuccinimidyl)suberat (BS³). The samples were sterilized using e-beam or gamma-sterilization at 25 kGy, either before or after biofunctionalization with cRGD. Functionalization stability was investigated by contact angle measurements. The functionality of cRGD after sterilization was demonstrated using proliferation tests and cytotoxicity assays. Immunofluorescence staining (pFAK, Actin, DAPI) was conducted to evaluate the adhesion potential between the samples and human mesenchymal stem cells (hMSCs).

Functionalized samples before and after sterilization showed no significant difference regarding their contact angles. A proliferation test demonstrated that the cells on functionalized samples proliferate significantly more than on untreated samples before and after sterilization. hMSCs showed a significant higher proliferation on gamma sterilized samples compared to all other groups after 14 days. It was confirmed that the samples did not exhibit cytotoxic behavior before or after sterilization. Fluorescence microscopy demonstrated that both, cells on sterilized and on non-sterilized samples, expressed high levels of pFAK-Y397.

The investigated functionalization enables improved adhesion and proliferation of hMSCs and is stable against the investigated sterilization processes. This is of importance as the option of having a sterile product enables the start of the translation of this biofunctional coating towards preclinical and subsequently first-in-man applications.

Acknowledgments: We acknowledge the financial support of the Federal Ministry of Education and Research, BMBF (13GW0452A-C).
EVS-BASED THERAPIES FOR INTERVERTEBRAL DISC REGENERATION: WHERE DO WE STAND?
Authors: Tryfonidou M.
Published: January 2024

Within the field of disc degeneration-related low back pain, the spine community has been increasingly acknowledging the regenerative potential of extracellular vesicles (EVs). EVs are small lipid bilayer-delimited particles naturally released by cells, involved in intercellular signaling. They do so by interacting with recipient cells and releasing their biological cargo (e.g., mRNA, miRNA, DNA, protein, lipid)

EVs derived from mesenchymal stromal cells and, more recently, also EVs from notochordal cells, the cells residing within the core of the juvenile human disc, are being actively studied. In general, they have been proposed to mitigate inflammation/catabolic processes, reduce apoptosis, stimulate proliferation and even improve the matrix producing capacity of the treated cells. Within this context, appropriate characterization of EVs is essential to increase the level of evidence that the reported effects are indeed EV-associated. To analyze the purity and biochemical composition of EV preparations the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins co-isolated/recovered with EVs. Alongside, to prove that the effects are EV-associated and not due to co-isolated factors from the tissue or cells used to derive the EVs, appropriate technical controls need to be taken along (during cell/tissue culture). As such the question arises: “what is the evidence so far?”

While from a fundamental perspective EVs are very appealing, the use of natural EVs in clinical applications is challenging. It comes with drawbacks, including biologic variability, yield, cumbersome isolation, and challenging upscaling and storage to achieve industrial levels. To date there is no FDA-approved EV-based therapy for disc-related lower back pain.

Nonetheless, EV-based therapeutic approaches have unique advantages over the use of (pluripotent) stem cell-based therapies, such as a high biologic, but low immunogenic and tumorigenic potential.

Acknowledgements: This talk is based on experiences from part of the project NC-CHOICE [no. 19251] of the research talent programme VICI financed by the Dutch Research Council (NWO) and the iPSpine project that receives funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 825925.
ARTICULAR CHONDROCYTES FROM DIABETIC AND NON-DIABETIC RATS EXPOSED TO NORMO- OR HYPERGLYCAEMIA RESPOND DIFFERENTIALLY TO THE COMPLEMENT SPLIT FRAGMENT ANAPHYLATOXIN C5A AND TNFA
Authors: Fleischmann N. , Braun T. , Reinhardt A. , Schotte T. , Wehrmann J. , Rüdig V. , Gögele C. , Kokozidou M. , Werner C. , Schulze-Tanzil G.
Published: January 2024

Osteoarthritis (OA) and diabetis mellitus type 2 (DMT2) are pathogenetically linked. Complement dysregulation contributes to OA and could be involved in DMT2. The inflammatory anaphylatoxin C5a is released during complement activation. This study aims to understand the specific responses of chondrocytes isolated from diabetic and non-diabetic rats exposed to C5a and/or the proinflammatory cytokine TNFα in vitro dependent on the glucose supply. Articular chondrocytes of adult Zucker Diabetic Fatty (ZDF) rats (homozygous: fa/fa, diabetic, heterozygous: fa/+, lean controls) were exposed to 10 ng/mL TNFα and 25 ng/mL C5a alone or in combination, both, under normo- (NG, 1 g/L glucose) and hyperglycemic (HG, 4.5 g/L glucose) conditions (4 or 24 h). Chondrocyte survival, metabolic activity and gene expression of collagen type 2, suppressors of cytokine signaling (SOCS)1, −3 and anti-oxidative hemoxygenase-1 (HMOX1) were assessed. The complement regulatory protein CD46 and cell nuclei sizes were analyzed. Chondrocyte vitality remained unaffected by the treatment. Metabolic activity was impaired in chondrocytes of non-diabetic rats under HG conditions. Collagen type 2 transcription was suppressed by TNFα under HG condition in chondrocytes from nondiabetic donors and under both conditions in those of DMT2 rats (24 h)

Except for DMT2 chondrocytes under HG (4 h), HMOX1 was generally induced by TNFα +/- C5a (NG, HG). C5a elevated HMOX1 only in chondrocytes of controls. The SOCS1/3 genes were increased by TNFα (NG, diabetic, non diabetic, 4 and 24 h). This could also be observed in chondrocytes of diabetic, but not of lean rats (24 h, HG). At 4 h, C5a induced SOCS1 only in non diabetic chondrocytes (NG, HG). Cytoprotective CD46 protein was suppressed by TNFα under NG condition. Nuclear volumes of chondrocyte were lower in chondrocytes from DMT2 rats compared to those from controls. The differential response suggests that chondrocytes are irreversibly compromised by DMT2.

Achnowledgement: The authors are grateful for the support by the “Stiftung Edoprothetik (S 04/21)”
MOVEMENT DATA ANALYSIS AS A NOVEL PREDICTIVE TECHNOLOGY FOR TENDON REPAIR
Authors: Faydaver M. , Russo V. , Di Giacinto O. , El Khatib M. , Rigamonti M. , Rosati G. , Raspa M. , Scavizzi F. , Santos H. , Mauro A. , Barboni B.
Published: January 2024

Digital Ventilated Cages (DVC) offer an innovative technology to obtain accurate movement data from a single mouse over time [1]. Thus, they could be used to determine the occurrence of a tendon damage event as well as inform on tissue regeneration [2,3]. Therefore, using the mouse model of tendon experimental damage, in this study it has been tested whether the recovery of tissue microarchitecture and of extracellular matrix (ECM) correlates with the motion data collected through this technology.

Mice models were used to induce acute injury in Achilles tendons (ATs), while healthy ones were used as control. During the healing process, the mice were housed in DVC cages (Tecniplast) to monitor animal welfare and to study biomechanics assessing movement activity, an indicator of the recovery of tendon tissue functionality. After 28 days, the AT were harvested and assessed for their histological and immunohistochemical properties to obtain a total histological score (TSH) that was then correlated to the movement data.

DVC cages showed the capacity to distinguish activity patterns in groups from the two different conditions. The data collected showed that the mice with access to the mouse wheel had a higher activity as compared to the blocked wheel group, which suggests that the extra movement during tendon healing improved motion ability. The histological results showed a clear difference between different analyzed groups. The bilateral free wheel group showed the best histological recovery, offering the highest TSH score, thus confirming the results of the DVC cages and the correlation between movement activity and structural recovery.

Data obtained showed a correlation between TSH and the DVC cages, displaying structural and movement differences between the tested groups. This successful correlation allows the usage of DVC type cages as a non-invasive method to predict tissue regeneration and recovery.

Acknowledgements: This research is part of the P4FIT project ESR13, funded by the H2020-ITN-EJD MSCA grant agreement No.955685.
POST-PRINTING CHONDROGENIC DIFFERENTIATION OF STEM CELL SPHEROID CONSTRUCTS FOR CARTILAGE TISSUE ENGINEERING
Authors: Decarli M.C. , Seijas-Gamardo A. , Morgan F.L.C. , Wieringa P. , Baker M.B. , Silva J.V.L , Moraes A.M. , Lorenzo M. , Mota C.
Published: January 2024

Cartilage lesions often undergo irreversible progression due to low self-repair capability of this tissue. Tissue engineered approaches based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for the treatment of cartilage lesions. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. In this study we aimed at the development of a reproducible bioprinting process followed by post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs were bioprinted, ionically crosslinked, and chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV were observed. After 56 days in culture, the bioprinted constructs were still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results showed a promising procedure to obtain 3D cartilage-like constructs that could be potential use as stable chondral tissue implants for future therapies.

Acknowledgments: The National Council for Scientific and Technological Development (CNPq, Brazil – Grants # 314 724/2021-4, 307 829/2018-9, 430 860/2018-8, 142 050/2018-0 and 465 656/2014-5), the Coordination for the Improvement of Higher Educational Personnel (CAPES, Brazil – PrInt 88 887.364849/2019-00 and PrInt 88 887.310405/2018-00), the Fund for Support to Teaching, Research and Extension from the University of Campinas (FAEPEX/UNICAMP, Brazil – Grants # 2921/18, 2324/21), and the European Union's Horizon 2020 JointPromise project – Precision manufacturing of microengineered complex joint implants, under grant agreement 874 837 are acknowledged for the financial support of this study.
TRADITIONAL AND NOVEL 3D MEASUREMENTS USING CONE-BEAM CT IN WEIGHT-BEARING CONDITIONS
Authors: Belvedere C.
Published: January 2024

3D accurate measurements of the skeletal structures of the foot, in physiological and impaired subjects, are now possible using Cone-Beam CT (CBCT) under real-world loading conditions. In detail, this feature allows a more realistic representation of the relative bone-bone interactions of the foot as they occur under patient-specific body weight conditions. In this context, varus/valgus of the hindfoot under altered conditions or the thinning of plantar tissues that occurs with advancing age are among the most complex and interesting to represent, and numerous measurement proposals have been proposed. This study aims to analyze and compare these measurements from CBCT in weight-bearing scans in a clinical population. Sixteen feet of diabetic patients and ten feet with severe adult flatfoot acquired before/after corrective surgery underwent CBCT scans (Carestream, USA) while standing on the leg of interest. Corresponding 3D shapes of each bone of the shank and hindfoot were reconstructed (Materialise, Belgium). Six different techniques found in the literature were used to calculate the varus/valgus deformity, i.e., the inclination of the hindfoot in the frontal plane of the shank, and the distance between the ground and the metatarsal heads was calculated along with different solutions for the identification of possible calcifications. Starting with an accurate 3D reconstruction of the skeletal structures of the foot, a wide range of measurements representing the same angle of hindfoot alignment were found, some of them very different from each other. Interesting correlations were found between metatarsal height and subject age, significant in diabetic feet for the fourth and fifth metatarsal bones. Finally, CBCT allows 3D assessment of foot deformities under loaded conditions. The observed traditional measurement differences and new measurement solutions suggest that clinicians should consider carefully the anatomical and functional concepts underlying measurement techniques when drawing clinical and surgical conclusions.
PATHOGENETIC PATHWAYS AND POSSIBLE TREATMENT OPTIONS FOR NONUNION FRACTURES
Authors: van Griensven M.
Published: January 2024

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.
THE CORRELATION BETWEEN ELECTRICAL IMPEDANCE AND CALLUS QUALITY: AN IN VIVO STUDY OF TIBIAL FRACTURES IN RABBITS
Authors: Frost M.W. , Tirta M. , Rahbek O. , Rytoft L.A. , Ding M. , Shen M. , Duch K. , Kold S.
Published: January 2024

Healing after bone fracture is assessed by frequent radiographs, which expose patients to radiation and lacks behind biological healing. This study aimed to investigate whether the electrical impedance using electrical impedance spectroscopy correlated to quantitative scores of bone healing obtained from micro-CT and mechanical bending test.

Eighteen rabbits were subjected to tibial fracture that was stabilized with external fixator. Two electrodes were positioned, one electrode placed within the medullary cavity and the other on the lateral cortex, both three millimeters from the fracture site. Impedance was measured daily across the fracture site at a frequency range of 5 Hz to 1 MHz. The animals were divided into three groups with different follow-up time: 1, 3 and 6 weeks for micro-CT (Bone volume/tissue volume (BV/TV, %)) and mechanical testing (maximum stress (MPa), failure energy (kJ/cm3), young modulus (Mpa)).

There was a statistically significant correlation between last measured impedance at 5 Hz frequency immediately prior to euthanasia and BV/TV of callus (−0.68, 95%CI: (−0.87; −0.31)). Considering the mechanical testing with three-point bending, no significant correlation was found between last measured impedance at 5 Hz frequency immediately prior to euthanasia and maximum stress (−0.35, 95%CI: (−0.70; 0.14)), failure energy (−0.23, 95%CI: (−0.63; 0.26)), or young modulus (−0.28, 95%CI: (−0.66; 0.22)).

The significant negative correlation between impedance and BV/TV might indicate that impedances correlate with the relative bone volume in the callus site. The lack of correlation between impedance and mechanical parameters when at the same time observing a correlation between impedance and days since operation (0-42 days), might indicate that the impedance can measure biological changes at an earlier time point than rough mechanical testing.
SAFE USE OF INTRAOPERATIVE TOURNIQUET: DOES ORTHOPAEDIC PRACTICE NEED GUIDANCE TO PREVENT RARE COMPLICATIONS?
Authors: Elbahi A. , Wasim M. , Yusuf K. , Thilagarajah M.
Published: January 2024

Tourniquet is a commonly used tool in orthopaedic practice. Incidence of complications is low but if any develops, it is devastating. Transient nerve damage, ischemia or skin burns are the possible tourniquet related complications. There is big variation in practice regarding the limb occlusion pressure.

51 procedures in 50 patients were reviewed retrospectively in our district general hospital. We looked at quality of documentation guided by the BOAST standard (The Safe Use of Intraoperative Tourniquets, published in October 2021). Limb occlusion pressure and ischemic time were analysed. Intra-operative and post-operative notes were reviewed to assess quality of documentation and post-operative complications.

Although limb occlusion pressure was above the recommended range in more than 75% of cases, there were no significant complications observed. Two cases only developed transient neuropraxia in common peroneal nerve and median nerve following tibial plateau ORIF and trapeziectomy simultaneously. Tibial ORIF fixation case had prolonged ischemic time (more than 120 minutes) and the limb occlusion pressure for the hand case was above the recommended range. Both have recovered within few days with no long-term consequences. Minimum documentation threshold was not met with regarding tourniquet site condition, method of skin isolation and padding, and exsanguination method.

This relatively new standard with no previous similar guidance needs time until it is followed by the health care professionals especially when there is no high incidence of complications related to the use of the tourniquet. However, it is crucial to increase the theatre staff awareness of such standards. This will prevent devastating complications specifically in vulnerable patients. Adjustments to theatre checklist have been suggested to improved documentation. Additionally, local teaching sessions will be delivered to theatre personnel aiming at improving our compliance to this standard.
THE INDUCED MEMBRANE TECHNIQUE IMPROVES THE HEALTH-RELATED QUALITY OF LIFE IN PATIENTS WITH A POST-TRAUMATIC LONG BONE NONUNION
Authors: der Broeck L.V. , Geurts J. , Qiu S. , Poeze M. , Blokhuis T.J.
Published: January 2024

The optimal treatment strategy for post-traumatic long bone non-unions is subject of an ongoing discussion. At the Maastricht University Medical Center (MUMC+) the induced membrane technique is used to treat post-traumatic long bone non-unions. This technique uses a multimodal treatment algorithm involving bone marrow aspirate concentrate (BMAC), the reamer-irrigator-aspirator (RIA) and P-15 bioactive peptide (iFactor, Cerapedics). Bioactive glass (S53P4 BAG, Bonalive) is added when infection is suspected. This study aims to objectify the effect of this treatment algorithm on the health-related quality of life (HRQoL) of patients with post-traumatic long bone non-unions. We hypothesized that HRQoL would improve after treatment.

From January 2020 to March 2023, consecutive patients who were referred to a multidisciplinary (trauma, orthopaedic and plastic surgery) non-union clinic at the MUMC+, The Netherlands, were evaluated using the Non-Union Scoring System (NUSS). The EQ-5D-5L questionnaire and the Lower Extremity Functional Scale (LEFS) were employed to obtain HRQoL outcomes both prior to and subsequent to surgery, with a follow-up at 6, 18 and 35 weeks.

Seventy-six patients were assessed at baseline (T0), with a mean NUSS of 40 (± 13 SD). Thirty-eight patients had their first follow-up, six weeks after surgery (T1). Thirty-one patients had a second follow-up at 18 weeks (T2), and twenty patients had the third follow-up at 35 weeks (T3). The EQ-5D index mean at baseline was 0.480, followed by an index of 0.618 at T1, 0.636 at T2, and 0.702 at T3. A significant difference was found in the HRQoL score between T0 and T1, as well as T2 and T3 (p<0.001; p=0.011). The mean LEFS significantly increased from 26 before intervention to 34, 39, and 43 after treatment (p<0.001; p=0.033; p=0.016).

This study demonstrated a significant improvement in the health-related quality of life of patients with post-traumatic long bone non-unions after the standardized treatment algorithm following the induced membrane technique.