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.

Bone morphogenetic proteins (BMPs) have been widely investigated for treating non-healing fractures. They participate in bone reconstruction by inducing osteoblast differentiation, and osteoid matrix production.¹ The human recombinant protein of BMP-7 was among the first growth factors approved for clinical use. Despite achieving comparable results to autologous bone grafting, severe side effects have been associated with its use.² Furthermore, BMP-7 was removed from the market.³ These complications are related to the high doses used (1.5-40 miligrams per surgery)² compared to the physiological concentration of BMP in fracture healing (in the nanogram to picogram per milliliter range).⁴ In this study, we use transcript therapy to deliver chemically modified mRNA (cmRNA) encoding BMP-7. Compared to direct use of proteins, transcript therapy allows the sustained synthesis of proteins with native conformation and true post-translational modifications using doses comparable to the physiological ones.⁵ Moreover, cmRNA technology overcomes the safety and affordability limitations of standard gene therapy i.e. pDNA.⁶ BMP-7 cmRNA was delivered using Lipofectamine™ MessengerMAX™ to human mesenchymal stromal cells (hMSCs). We assessed protein expression and osteogenic capacity of hMSCs in monolayer culture and in a house-made, collagen hydroxyapatite scaffold. Using fluorescently-labelled cmRNA we observed an even distribution after loading complexes into the scaffold and a complete release after 3 days. For both monolayer and 3D culture, BMP-7 production peaked at 24 hours post-transfection, however cells transfected in scaffolds showed a sustained expression. BMP-7 transfected hMSCs yielded significantly higher ALP activity and Alizarin red staining at later timepoints compared to the untransfected group. Interestingly, BMP-7 cmRNA treatment triggered expression of osteogenic genes like OSX, RUNX-2 and OPN, which was also reflected in immunostainings. This work highlights the relevance of cmRNA technology that may overcome the shortcomings of protein delivery while circumventing issues of traditional pDNA-based gene therapy for bone regeneration.

Acknowledgement: This work has been performed as part of the cmRNAbone project and has received funding from the European Union's Horizon 2020 research and innovation programme under the Grant Agreement No 874790.

Surgeons treating fractures with many small osteochondral fragments have often expressed the clinical need for an adhesive to join such fragments, as an adjunct to standard implants. If an adhesive would maintain alignment of the articular surfaces and subsequently heal it could result in improved clinical outcomes. However, there are no bone adhesives available for clinical indications and few pre-clinical models to assess safety and efficacy of adhesive biomaterial candidates. A bone adhesive candidate based on water, α-TCP and an amino acid phosphoserine was evaluated in-vivo in a novel murine bone core model (preliminary results presented EORS 2019) in which excised bone cores were glued back in place and harvested @ 0, 3, 7, 14, 28 and 42days. Adhesive pull-out strength was demonstrated 0–28 days, with a dip at 14 days increasing to 11.3N maximum. Histology 0–42 days showed the adhesive progressively remodelling to bone in both cancellous and cortical compartments with no signs of either undesirable inflammation or peripheral ectopic bone formation. These favourable results suggested translation to a large animal model.

A porcine dental extraction socket model was subsequently developed where dental implants were affixed only with the adhesive. Biomechanical data was collected @ 1, 14, 28 and 56 days, and histology at 1,14,28 and 56 days. Adhesive strength assessed by implant pull-out force increased out to 28 days and maintained out to 56 days (282N maximum) with failure only occurring at the adhesive bone interface. Histology confirmed the adhesive's biocompatibility and osteoconductive behavior. Additionally, remodelling was demonstrated at the adhesive-bone interface with resorption by osteoclast-like cells and followed by new bone apposition and substitution by bone. Whilst the in-vivo dental implant data is encouraging, a large animal preclinical model is needed (under development) to confirm the adhesive is capable of healing, for example, loaded osteochondral bone fragments.

Acknowledgements: The murine study was supported, in part, by the Swedish Foundation for Strategic Research (#RMA15-0110).

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).

Heterotopic ossification (HO) is defined as aberrant bone formation in extraskeletal locations. In this process, local stromal cells of mesenchymal origin abnormally differentiate, resulting in pathologic cartilage and bone matrix deposition. However, the specific cell type and mechanisms beyond this process are not well understood, in part due to the heterogeneity of progenitor cells involved. Here, a combination of single cell RNA sequencing (scRNA-Seq) and lineage tracing, defined the extent to which synovial / tendon sheath progenitor cells contribute to HO. For this purpose, a Tppp3 (tubulin polymerization-promoting protein family member 3) inducible reporter model was used, in combination with either Scx (Scleraxis) or Pdgfra (Platelet derived growth factor receptor alpha) reporter animals. Both arthroplasty-induced and tendon injury-mouse experimental HO models were utilized. ScRNA-Seq of tendon-induced traumatic HO suggested that Tppp3 is a progenitor cell marker for either osteochondral or tendon or cells. After HO induction, Tppp3 reporter+ cell population expanded in number and contributed to cartilage and bone formation in tendon and joint-associated HO. Using double reporter animals, we found that both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells produced HO-associated cartilage. Finally, the examination of human samples showed a significant population of TPPP3+ cells overlapping with osteogenic markers in areas of HO. Overall, these results provide novel observations that peritenon and synovial progenitor cells undergo abnormal osteochondral differentiation and contribute to heterotopic bone formation after trauma.

This study investigates the relationships between Intervertebral Disc (IVD) morphology and biomechanics using patient-specific (PS) finite element (FE) models and poromechanical simulations.

169 3D lumbar IVD shapes from the European project MySpine (FP7-269909), spanning healthy to Pfirrmann grade 4 degeneration, were obtained from MRIs. A Bayesian Coherent Point Drift algorithm aligned meshes to a previously validated structural FE mesh of the IVD. After mesh quality analyses and Hausdorff distance measurements, mechanical simulations were performed: 8 and 16 hours of sleep and daytime, respectively, applying 0.11 and 0.54 MPa of pressure on the upper cartilage endplate (CEP). Simulation results were extracted from the anterior (ATZ) and posterior regions (PTZ) and the center of the nucleus pulposus (CNP). Data mining was performed using Linear Regression, Support Vector Machine, and eXtreme Gradient Boosting techniques. Mechanical variables of interest in DD, such as pore fluid velocity (FLVEL), water content, and swelling pressure, were examined. The morphological variables of the simulated discs were used as input features.

Local morphological variables significantly impacted the local mechanical response. The local disc heights, respectively in the mid (mh), anterior (ah), and posterior (ph) regions, were key factors in general. Additionally, fluid transport, reflected by FLVEL, was greatly influenced (r2 0.69) by the shape of the upper and lower cartilage endplates (CEPs).

This study suggests that disc morphology affects Mechanical variables of interest in DD. Attention should be paid to the antero-posterior distribution and local effects of disc heights. Surprisingly, the CEP morphology remotely affected the fluid transport in NP volumes around mid-height, and mechanobiological implications shall be explored. In conclusion, patient-specific IVD modeling has strong potential to unravel important correlations between IVD phenotypes and local tissue regulation.

Acknowledgments: European Commission: Disc4All-MSCA-2020-ITN-ETN GA: 955735; O-Health-ERC-CoG-2021-101044828

Obesity is correlated with the development of osteoporotic diseases. Gut microbiota-derived metabolite trimethylamine-n-oxide (TMAO) accelerates obesity-mediated tissue deterioration. This study was aimed to investigate what role TMAO may play in osteoporosis development during obesity.

Mice were fed with high-fat diet (HFD; 60 kcal% fat) or chow diet (CD; 10 kcal% fat) or 0.2% TMAO in drinking water for 6 months. Body adiposis and bone microstructure were investigated using μCT imaging. Gut microbiome and serum metabolome were characterized using 16S rRNA sequencing and liquid chromatography-tandem mass spectrometry. Osteogenic differentiation of bone-marrow mesenchymal cells was quantified using RT-PCR and von Kossa staining. Cellular senescence was evaluated by key senescence markers p16, p21, p53, and senescence association β-galactosidase staining.

HFD-fed mice developed hyperglycemia, body adiposis and osteoporosis signs, including low bone mineral density, sparse trabecular microarchitecture, and decreased biomechanical strength. HFD consumption induced gut microbiota dysbiosis, which revealed a high Firmicutes/Bacteroidetes ratio and decreased α-diversity and abundances of beneficial microorganisms Akkermansiaceae, Lactobacillaceae, and Bifidobacteriaceae. Serum metabolome uncovered increased serum L-carnitine and TMAO levels in HFD-fed mice. Of note, transplantation of fecal microbiota from CD-fed mice compromised HFD consumption-induced TMAO overproduction and attenuated loss in bone mass, trabecular microstructure, and bone formation rate. TMAO treatment inhibited trabecular and cortical bone mass and biomechanical characteristics; and repressed osteogenic differentiation capacity of bone-marrow mesenchymal cells. Mechanistically, TMAO accelerated mitochondrial dysfunction and senescence program, interrupted mineralized matrix production in osteoblasts.

Gut microbial metabolite TMAO induced osteoblast dysfunction, accelerating the development of obesity-induced skeletal deterioration. This study, for the first time, conveys a productive insight into the catabolic role of gut microflora metabolite TMAO in regulating osteoblast activity and bone tissue integrity during obesity.

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases.

Infections are among the most diffused complications of the implantation of medical devices. In orthopedics, they pose severe societal and economic burden and interfere with the capability of the implants to integrate in the host bone, significantly increasing failure risk. Infection is particularly severe in the case of comorbidities and especially bone tumors, since oncologic patients are fragile, have higher infection rate and impaired osteoregenerative capabilities. For this reason, prevention of infection is to be preferred over treatment.

This is even more important in the case of spine surgery, since spine is among the main site for tumor metastases and because incidence of post operative surgical-site infections is significant (up to 15-20%) and surgical options are limited by the need of avoiding damaging the spinal cord.

Functionalization of the implant surfaces, so as to address infection and, possibly, co- adjuvate anti-tumor treatments, appears as a breakthrough innovation. Unmet clinical needs in infection and tumors is presented, with a specific focus on the spine, then, new perspectives are highlighted for their treatment.

Over the last decades, biodegradable metals emerged as promising materials for various biomedical implant applications, aiming to reduce the use of permanent metallic implants and, therefore, to avoid additional surgeries for implant removal. However, among the important issue to be solved is their fast corrosion - too high to match the healing rate of the bone tissue. The most effective way to improve this characteristic is to coat biodegradable metals with substituted calcium phosphates. Tricalcium phosphate (β-TCP) is a resorbable bioceramic widely used as synthetic bone graft. In order to modulate and enhance its biological performance, the substitution of Ca2+ by various metal ions, such as strontium (Sr2+), magnesium (Mg2+), iron (Fe2+) etc., can be carried out. Among them, copper (Cu2+), manganese (Mn2+), zinc (Zn2+) etc. could add antimicrobial properties against implant-related infections. Double substitutions of TCP containing couples of Cu2+/Sr2+ or Mn2+/Sr2+ ions are considered to be the most perspective based on the results of our study. We established that single phase Ca3−2x(MˊMˊˊ)x(PO4)2 solid solutions are formed only at x ≤ 0.286, where Mˊ and Mˊˊ—divalent metal ions, such as Zn2+, Mg2+, Cu2+, Mn2+, and that in case of double substitutions, the incorporation of Sr2+ ions allows one to extend the limit of solid solution due to the enlargement of the unit cell structure. We also reported that antimicrobial properties depend on the substitution ion occupation of Ca2+ crystal sites in the β-TCP structure. The combination of two different ions in the Ca5 position, on one side, and in the Ca1, Ca2, Ca3, and Ca4 positions, on another side, significantly boosts antimicrobial properties. In the present work, zinc-lithium (Zn-Li) biodegradable alloys were coated with double substituted Mn2+/Sr2+ β-TCP and double substituted Cu2+/ Sr2+ β-TCP, with the scope to promote osteoinductive effect (due to the Sr2+ presence) and to impart antimicrobial properties (thanks to Cu2+ or Mn2+ ions). The Pulsed Laser Deposition (PLD) method was applied as the coating's preparation technique. It was shown that films deposited using PLD present good adhesion strength and hardness and are characterized by a nanostructured background with random microparticles on the surface. For coatings characterization, Fourier Transform Infrared Spectroscopy, X-ray Diffraction, and Scanning Electron Microscopy coupled with Energy Dispersive X-ray and X-ray Photoelectron Spectroscopy were applied. The microbiology tests on the prepared coated Zn-Li alloys were performed with the Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative (Salmonella typhimurium, Escherichia coli) bacteria strains and Candida albicans fungus. The antimicrobial activity tests showed that Mn2+/Sr2+ β-TCP -coated and Cu2+/Sr2+ β-TCP coated Zn-Li alloys were able to inhibit the growth of all five microorganisms. The prepared coatings are promising in improving the degradation behavior and biological properties of Zn-Li alloys, and further studies are necessary before a possible clinical translation.

Decreasing the chance of local relapse or infection after surgical excision of bone metastases is a main goals in orthopedic oncology. Indeed, bone metastases have high incidence rate (up to 75%) and important cross-relations with infection and bone regeneration. Even in patients with advanced cancer, bone gaps resulting from tumor excision must be filled with bone substitutes. Functionalization of these substitutes with antitumor and antibacterial compounds could constitute a promising approach to overcome infection and tumor at one same time. Here, for the first time, we propose the use of nanostructured zinc-bone apatite coatings having antitumor and antimicrobial efficacy. The coatings are obtained by Ionized Jet Deposition from composite targets of zinc and bovine-derived bone apatite. Antibacterial and antibiofilm efficacy of the coatings is demonstrated in vitro against S. Aureus and E. Coli. Anti-tumor efficacy is investigated against MDA- MB-231 cells and biocompatibility is assessed on L929 and MSCs.

A microfluidic based approach is used to select the optimal concentration of zinc to be used to obtain antitumor efficacy and avoid cytotoxicity, exploiting a custom gradient generator microfluidic device, specifically designed for the experiments. Then, coatings capable of releasing the desired amount of active compounds are manufactured. Films morphology, composition and ion-release are studies by FEG- SEM/EDS, XRD and ICP. Efficacy and biocompatibility of the coatings are verified by investigating MDA, MSCs and L929 viability and morphology by Alamar Blue, Live/Dead Assay and FEG-SEM at different timepoints. Statistical analysis is performed by SPSS/PC + Statistics TM 25.0 software, one-way ANOVA and post-hoc Sheffe? test. Data are reported as Mean ± standard Deviation at a significance level of p <0.05.

Results and Discussion. Coatings have a nanostructured surface morphology and a composition mimicking the target. They permit sustained zinc release for over 14 days in medium. Thanks to these characteristics, they show high antibacterial ability (inhibition of bacteria viability and adhesion to substrate) against both the gram + and gram – strain.

The gradient generator microfluidic device permits a fine selection of the concentration of zinc to be used, with many potential perspectives for the design of biomaterials. For the first time, we show that zinc and zinc-based coatings have a selective efficacy against MDA cells. Upon mixing with bone apatite, the efficacy is maintained and cytotoxicity is avoided. For the first time, new antibacterial metal-based films are proposed for addressing bone metastases and infection at one same time. At the same time, a new approach is proposed for the design of the coatings, based on a microfluidic approach. We demonstrated the efficacy of Zn against the MDA-MB-231 cells, characterized for their ability to form bone metastases in vivo, and the possibility to use nanostructured metallic coatings against bone tumors. At the same time, we show that the gradient-generator approach is promising for the design of antitumor biomaterials. Efficacy of Zn films must be verified in vivo, but the dual-efficacy coatings appear promising for orthopedic applications.

Bone healing outcome is highly dependent on the initial mechanical fracture environment [1]. In vivo, direct bone healing requires absolute stability and an interfragmentary strain (IFS) below 2% [2]. In the majority of cases, however, endochondral ossification is engaged where frequency and amplitude of IFS are key factors. Still, at the cellular level, the influence of those parameters remains unknown. Understanding the regulation of naïve hMSC differentiation is essential for developing effective bone healing strategies.

Human bone-marrow-derived MSC (KEK-ZH-NR: 2010–0444/0) were embedded in 8% gelatin methacryol. Samples (5mm Ø x 4mm) were subjected to 0, 10 and 30% compressive strain (5sec compression, 2hrs pause sequence for 14 days) using a multi-well uniaxial bioreactor (RISystem) and in presence of chondro-permissive medium (CP, DMEM HG, 1% NEAA, 10 µM ITS, 50 µg/mL ascorbic acid, and 100 mM Dex). Cell differentiation was assessed by qRT-PCR and histo-/immunohistology staining. Experiments were repeated 5 times with cells from 5 donors in duplicate. ANOVA with Tukey post-hoc correction or Kurskal-Wallis test with Dunn's correction was used.

Data showed a strong upregulation of hypertrophic related genes COMP, MMP13 and Type 10 collagen upon stimulation when compared to chondrogenic SOX9, ACAN, Type 2 collagen or to osteoblastic related genes Type 1 Collagen, Runx2. When compared to chondrogenic control medium, cells in CP with or without stimulation showed low proteoglycan synthesis as shown by Safranine-O-green staining. In addition, the cells were significantly larger in 10% and 30% strain compared to control medium with 0% strain. Type 1 and 10 collagens immunostaining showed stronger Coll 10 expression in the samples subjected to strain compared to control.

Uniaxial deformation seems to mainly promote hypertrophic-like chondrocyte differentiation of MSC. Osteogenic or potentially late hypertrophic related genes are also induced by strain.

Acknowledgments: Funded by the AO Foundation, StrainBot sponsored by RISystemAG & PERRENS 101 GmbH

Despite the major advances in osteosynthesis after trauma, there remains a small proportion of patients (<10%) who exhibit delayed healing and/or eventual progression to non-union. While known risk factors exist, e.g. advanced age or diabetes, the exact molecular mechanism underlying the impaired healing is largely unknown and identifying which specific patient will develop healing complications is still not possible in clinical practice. The talk will cover our novel multimodal approaches in small animals, which have the potential to precisely capture and understand biological changes during fracture healing on an individual basis. Via combining emerging omics technologies with our recently developed femur defect loading equipment in mice, we provide a platform to precisely link mechanical and molecular analyses during fracture healing.

Biomaterials with mechanical or biological competence are ubiquitous in musculoskeletal disorders, and understanding the inflammatory response they trigger is key to guide tissue regeneration. While macrophage role has been widely investigated, immune response is regulated by other immune cells, including neutrophils, the most abundant leukocyte in human blood. As first responders to injury, infection or material implantation, neutrophils recruit other immune cells, and therefore influence the onset and resolution of chronic inflammation, and macrophage polarization. This response depends on the physical and chemical properties of the biomaterials, among other factors. In this study we report an in vitro culture model to describe the most important neutrophil functions in relation to tissue repair.

We identified neutrophil survival and death, neutrophils extracellular trap formation, release of reactive oxygen species and degranulation with cytokines release as key functions and introduced a corresponding array of assays. These tests were suitable to identify clear differences in the response by neutrophils that were cultured on material of different origin, stiffness and chemical composition. Overall, substrates from biopolymers of natural origin resulted in increased survival, less neutrophil extracellular trap formation, and more reactive oxygen species production than synthetic polymers. Within the range of mechanical properties explored (storage modulus below 5 k Pa), storage modulus of covalently crosslinked hyaluronic acid hydrogels did not significantly alter neutrophils response, whereas polyvinyl alcohol gels of matching mechanical properties displayed a response indicating increased activation.

Additionally, we present the effect of material stiffness, charge, coating and culture conditions in the measured neutrophils response. Further studies are needed to correlate the neutrophil response to tissue healing.

By deciphering how neutrophils initiate and modulate the immune response to material implantation, we aim at introducing new principles to design immunomodulatory biomaterials for musculoskeletal disorders.

Acknowledgments

This work was supported by the AO Foundation, AO CMF, grant AOCMF-21-04S.

Osteoarthritis (OA) is an inflammatory disease affecting the complete synovial joint including the cartilage layer and the subchondral bone plate. Due to the multifactorial causes and the not yet completely resolved molecular mechanisms, it lacks a gold standard treatment to mitigate OA. Hence, biomaterials capable of delaying or preventing OA are a promising alternative or supplement to antiphlogistic and surgical interventions. Magnesium (Mg) and its alloys are among the promising biomaterials with osteoinductive effects. This work investigated the impact of Mg micro cylinders (length ≈of 1.0 mm and width of 0.5 mm) in vitro, in favoring joint regeneration together with preventing OA progression. Therefore, a mesenchymal stem cell line (SCP-1) was applied in order to assess the compatibility of the degradable material. Furthermore, an in vitro OA model utilizing SCP-1 cells based on the supplementation of the cytokines; IL-1β, TNF-α was established and disclosed the capability of Mg microparticles in differentiating SCP-1 cells into chondrogenic and osteogenic lineages proven through extracellular matrix staining and gene marker analysis. A concentration above 10 mM revealed a reduction in the cell viability by 50 %. An increase in the expression of collagens especially and proteoglycans (COL2A1, Aggrecan) as extracellular matrix proteins as well as an increase in osteogenic marker (ALP, BMP2) favoring the mineralization process were observed. The inflammatory condition reduced the viability and productivity of the applied stem cell line. However, the application of Mg microparticles induced a cell recovery and reduction of inflammation marker such as MMP1 and IL6. The cytocompatible and the ability of Mg microparticles in supporting bone and cartilage repair mechanisms in vitro even under inflammatory conditions make biodegradable Mg microparticles a suitable implant material to treat OA therapy.

Acknowledgements: This project OAMag was funded by the German Research Foundation (project number 404534760). The author thank Dr. Björn Wiese (hereon) for the production of Mg based material and Prof. Böcker (MUM Musculoskeletal University Center Munich) for the provision of SCP-1 cell line.

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.

Invertebral disc degeneration (IDD) is a degenerative disease involving a variety of musculoskeletal and spinal disorders such as lower back pain (LBP). Secretome derived from mesenchymal stem cells (MSCs) have exerted beneficial effect on tissue regeneration. In this study, the goal was to investigate the paracrine and the anti-inflammatory effects of secretome from interleukin IL1β preconditioned Bone Marrow MSCs (BMSCs) on human nucleus pulposus cells (hNPCs) in a 3D in vitro model.

Secretome was collected from BMSCs (BMSCs-sec) after preconditioning with 10 ng/mL IL1β. hNPCs were isolated from surgical specimens, culture expanded in vitro, encapsulated in alginate beads and treated with: growth medium; IL1β 10 ng/mL; IL1β 10 ng/mL for 24 hours and then BMSCs-sec. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess assay) and ROS quantification (Immunofluorescence) iii) glycosaminoglycan (GAG) amount (DMBB) and iv) gene expression levels of extracellular matrix (ECM) components and inflammatory mediators (qPCR). One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D.

In vitro tests showed an enhancement of hNPCs proliferation after treatment with BMSCs-sec (p ≤ 0.05) compared to IL1β group. After 24 hours, the percentage of dead cells was higher in IL1β treated hNPCs compared to control group and decreased significantly in combined IL1β and BMSCs-sec sample group (p ≤ 0.01). Nitrite and ROS production were significantly mitigated and GAGs content was improved by preconditioned BMSCs-sec (p ≤ 0.05). Furthermore, gene expression levels were modulated by BMSCs-sec treatment compared to controls.

Our results supported the potential use of BMSCs' secretome as a cell-free strategy for IDD, overcoming the side effects of cell-therapy. Moreover, secretome derived from IL1β preconditioned BMSCs was able to reduce hNPCs death, attenuate ECM degradation and oxidative stress counteracting IDD progression.

Acknowledgements: Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects.

Metabolic bone diseases, such as osteoporosis and osteopetrosis, result from an imbalanced bone remodeling process. In vitro bone models are often used to investigate either bone formation or resorption independently, while in vivo, these processes are coupled. Combining these processes in a co-culture is challenging as it requires finding the right medium components to stimulate each cell type involved without interfering with the other cell type's differentiation. Furthermore, differentiation stimulating factors often comprise growth factors in supraphysiological concentrations, which can overshadow the cell-mediated crosstalk and coupling.

To address these challenges, we aimed to recreate the physiological bone remodeling process, which follows a specific sequence of events starting with cell activation and bone resorption by osteoclasts, reversal, followed by bone formation by osteoblasts. We used a mineralized silk fibroin scaffold as a bone-mimetic template, inspired by bone's extracellular matrix composition and organization. Our model supported osteoclastic resorption and osteoblastic mineralization in the specific sequence that represents physiological bone remodeling.

We also demonstrated how culture variables, such as different cell ratios, base media, and the use of osteogenic/osteoclast supplements, and the application of mechanical load, can be adjusted to represent either a high bone turnover system or a self-regulating system. The latter system did not require the addition of osteoclastic and osteogenic differentiation factors for remodeling, therefore avoiding growth factor use.

Our in vitro model for bone remodeling has the potential to reduce animal experiments and advance in vitro drug development for bone remodeling pathologies like osteoporosis. By recreating the physiological bone remodeling cycle, we can investigate cell-cell and cell-matrix interactions, which are essential for understanding bone physiology and pathology. Furthermore, by tuning the culture variables, we can investigate bone remodeling under various conditions, potentially providing insights into the mechanisms underlying different bone disorders.

Osteoarthritis (OA) leads to articular cartilage degradation, following complex dysregulation of chondrocyte's metabolism towards a catabolic state. Mechanical and biochemical signals are involved and need to be considered to understand the condition. Regulatory network-based models (RNM) successfully simulated the biological activity of the chondrocyte and the transduction of mechanical signals at the molecular and cell levels. However, the knowledge gap between single-cell regulation and intercellular communication in tissue volumes hinders the interpretability of such models at larger scales. Accordingly, a novel tissue-level biochemical model is proposed. We hypothesise that it is possible to simulate interacting network effects through the transport of diluted species in a finite-element model, to grasp relevant dynamics of cell and tissue regulation in OA. Chondrocyte RNM equations were translated into a reaction term of 18 multi-species diffusion model (e.g., 3 anti-inflammatory and 8 pro-inflammatory interleukins, 3 pro-anabolic and 1 pro-catabolic growth factors, 2 nociceptive factors and 2 pro-inflammatory cytokines). Elements with RNM reaction terms represented the chondrocytes and were distributed randomly through the model, according to known cellular density in the knee cartilage, and could both react to and produce diffusive entities through the pericellular matrix, associated with reduced diffusion coefficients. The model was constructed over a 2D square of 0.47 mm sides considered to be in the middle of the cartilage, so boundary conditions were settled as periodic. Different simulations were initialised with initial concentrations of either healthy or pro-OA mediators. Preliminary results showed that, independently of the initial conditions, the chondrocytes successfully evolved into anabolic states, in absence of sustained pro-catabolic external stimulations, in contrast to single-cell RNM [2]. Our intercellular model suggests that paracrine communication may increase robustness towards cartilage maintenance, and future tests shall reveal new OA dynamics.

Acknowledgements: Funding was provided by the European Commission (ERC-2021-CoG-O-Health-101044828).

Decreasing the bulk weight without losing the biomechanical properties of commercial pure titanium (Cp-Ti) medical implants is now possible by using Laser Powder Bed Fusion (L-PBF) technology. Gyroid lattice structures that have precious mechanical and biological advantages because of similarity to trabecular bone. The aim of the study was to design and develop L-PBF process parameter optimization for manufacturing gyroid lattice Cp-Ti structures. The cleaning process was then optimized to remove the non-melted powder from the gyroid surface without mechanical loss.

Gyroid cubic designs were created with various relative densities by nTopology. L-PBF process parameter optimization was progressed using with Cp-Ti (EOS TiCP Grade2) powder in the EOS M290 machine to achieve parts that have almost full dense and dimensional accuracy. The metallography method was made for density. Dimensional accuracy at gyroid wall thicknesses was investigated between designed and manufactured via stereomicroscope, also mechanical tests were applied with real time experiment and numerical analysis (ANSYS). Mass loss and strut thickness loss were investigated for chemical etching cleaning process.

Gyroid parts had 99,5% density. High dimensional accuracy was achieved during L-PBF process parameters optimization. Final L-PBF parameters gave the highest 19% elongation and 427 MPa yield strength values at tensile test. Mechanical properties of gyroid were controlled with changing relative density. A minute chemical etching provided to remove non-melted powders.

Compression test results of gyroids at numerical and real-time analysis gave unrelated while deformation behaviors were compatible with each other. Gyroid Cp-Ti osteosynthesis mini plates will be produced with final L-PBF process parameters. MTT cytotoxicity test will be characterized for cell viability.

Acknowledgements This project is granted by TUBITAK (120N943). Feza Korkusuz MD is a member of the Turkish Academy of Sciences (TÜBA).