Osteoarthritis (OA) is a leading cause of chronic pain and disability, for which there is no cure. Mesenchymal stromal cells (MSCs) have been used in clinical trials for treating OA due to their unique ability to generate paracrine anti-inflammatory and trophic signals. Interestingly, these studies have shown mainly short-term effects of MSCs in improving pain and joint function, rather than sustained and consistent benefits. This may reflect a change or loss in the therapeutic effects of MSCs after intra-articular injection. The present study aimed to unravel the reasons behind the variable efficacy of MSC injections for OA using an in vitro co-culture model. Osteoarthritic human synovial fibroblasts (OA-HSFs) were co-cultured with MSCs to investigate their reciprocal effects on cell responses and whether a short-term exposure of OA cells to MSCs was sufficient for reducing their diseased characteristics in a sustained manner. Gene expression and histological analyses were performed. OA-HSFs exposed to MSCs showed short-term downregulation of inflammatory markers. However, the MSCs showed upregulation of inflammatory markers and impaired ability to undergo osteogenesis and chondrogenesis in the presence of OA-HSFs. Moreover, short-term exposure of OA-HSFs to MSCs was found to be insufficient for inducing sustained changes to their diseased behaviour. These findings suggested that MSCs may not provide long-term effects in correcting the OA joint environment due to them adopting the diseased phenotype of the surrounding tissues, which has important implications for the future development of effective stem-cell-based OA treatments with long-term therapeutic efficacy.

Extracellular matrix (ECM) biomaterials have shown promise for treating small artucular-joint defetcs. However, ECM-based biomaterials generally lack appropriate mechanical properties to support physiological loads and are prone to delamination in larger cartilage defects. To overcome these common mechanical limitations, a collagen hyaluronic-acid (CHyA) matrix, with proven regenerative potential, was reinforced with a bioabsorbable 3D-printed framework to support physiological loads. Polycaprolactone (PCL) was 3D-printed in two configurations, rectilinear and gyroid designs, that were extensively mechanically characterised. Both scaffold designs increased the compressive modulus of the CHyA matrices by three orders of magnitude, mimicking the physiological range (0.5-2.0 MPa) of healthy cartilage. The gyroid scaffold proved to be more flexible compared to the rectilinear scaffold, thus better contouring to the curvature of a femoral condyle. Additionally, PCL reinforcement of the CHyA matrix increased the tensile modulus and allowed for suture fixation of the scaffold to the subchondral bone, thus addressing the major challenge of biomaterial fixation to articular joint surfaces in shallow defects. In vitro evaluation confirmed successful infiltration of human mesenchymal stromal cells (MSCs) within the PCL-CHyA scaffolds, which resulted in increased production of sulphated glycosaminoglycans (sGAG/DNA; p = 0.0308) compared to non-reinforced CHyA matrices. Histological staining using alcian blue confirmed these results, while also indicating greater spatial distribution of sGAG throughout the PCL-CHyA scaffold. These findings have a great clinical importance as they provide evidence that reinforced PCL-CHyA scaffolds, with their increased chondroinductive potential and compatibility with joint fixation techniques, could be used to repair large-area chondral defects that currently lack effective treatment options.

Post-traumatic osteoarthritis in the temporomandibular joint (TMJ OA) is associated dysfunctional cellmatrix mediated signalling resulting from changes in the pericellular microenvironment after injury. Matrix metalloproteinase (MMP)-13 is a critical enzyme in biomineralisation and the progression of OA that can both degrade the extracellular matrix and modify extracellular receptors. This study focused on MMP-13 mediated changes in a transmembrane proteoglycan, Neuron Glial antigen 2 (NG2/CSPG4). NG2/CSPG4 is a receptor for type VI collagen and a known substrate for MMP-13. In healthy articular layer chondrocytes, NG2/CSPG4 is membrane bound but becomes internalised during TMJ OA. The objective of this study was to determine if MMP-13 contributed to the cleavage and internalisation of NG2/CSPG4 during mechanical loading and OA progression. Using preclinical and clinical samples, it was shown that MMP-13 was present in a spatiotemporally consistent pattern with NG2/CSPG4 internalisation during TMJ OA. In vitro, it was illustrated that inhibiting MMP-13 prevented retention of the NG2/CSPG4 ectodomain in the extracellular matrix. Inhibiting MMP-13 promoted the accumulation of membrane-associated NG2/CSPG4 but did not affect the formation of mechanical-loading dependent variant specific fragments of the ectodomain. MMP- 13 mediated cleavage of NG2/CSPG4 is necessary to initiate clathrin-mediated internalisation of the NG2/ CSPG4 intracellular domain following mechanical loading. This mechanically sensitive MMP-13-NG2/CSPG4 axis affected the expression of key mineralisation and OA genes including bone morphogenetic protein 2, and parathyroid hormone-related protein. Together, these findings implicated MMP-13 mediated cleavage of NG2/CSPG4 in the mechanical homeostasis of mandibular condylar cartilage during the progression of degenerative arthropathies such as OA.

Intervertebral disc degeneration (IDD) involves cellular changes in the nucleus pulposus (NP) characterised by a decline of the large vacuolated notochordal cells (vNCs) and a rise of smaller vacuole-free mature chondrocyte-like NP cells. An increasing number of studies demonstrate that notochordal cells (NCs) exert disease-modifying effects, establishing that NC-secreted factors are essential for the maintenance of a healthy intervertebral disc (IVD). However, understanding the role of the NCs is hampered by a restricted reserve of native cells and the lack of robust ex vivo cell model. A precise dissection enabled the isolation of NP cells from 4 d post-natal stage mouse spines and their culture into self-organised micromasses. The maintenance of cells' phenotypic characteristics was demonstrated by the presence of intracytoplasmic vacuoles and the immuno-colocalisation of the NC-markers (brachyury; SOX9) after 9 d of culture both in hypoxic and normoxic conditions. A significant increase of the size of the micromass was observed under hypoxia, consistent with a higher level of Ki-67+ immunostained proliferative cells. Furthermore, several proteins of interest for the study of vNCs phenotype (CD44; caveolin-1; aquaporin 2; patched-1) were successfully detected at the plasma membrane of NP-cells cultured in micromasses under hypoxic condition. IHC was performed on mouse IVD sections as control staining. An innovative 3D culture model of vNCs derived from mouse postnatal NP is proposed, allowing future ex vivo exploration of their basic biology and of the signalling pathways involved in IVD homeostasis that may be relevant for disc repair.

Because low back pain is frequently a result of intervertebral disc degeneration (IVDD), strategies to regenerate or repair the IVD are currently being investigated. Often, ex vivo disc cultures of non-human IVD organs or tissue explants are used that usually do not exhibit natural IVDD. Therefore, degenerative changes mimicking those reported in human IVDD need to be induced. To support researchers in selecting ex vivo disc cultures, a systematic search was performed for them and their potential use for studying human IVDD reviewed. Five degeneration induction categories (proinflammatory cytokines, injury/damage, degenerative loading, enzyme, and other) were identified in 129 studies across 7 species. Methods to induce degeneration are diverse and can induce mild to severe degenerative changes that progress over time, as described for human IVDD. The induced degenerative changes are model-specific and there is no "one-fits-all" IVDD induction method. Nevertheless, specific aspects of human IVDD can be well mimicked. Currently, spontaneously degenerated disc cultures from large animals capture human IVDD in most aspects. Combinatorial approaches of several induction methods using discs derived from large animals are promising to recapitulate pathological changes on several levels, such as cellular behaviour, extracellular matrix composition, and biomechanical function, and therefore better mimic human IVDD. Future disc culture setups might increase in complexity, and mimic human IVDD even better. As ex vivo disc cultures have the potential to reduce and even replace animal trials, especially during preclinical development, advancement of such models is highly relevant for more efficient and cost-effective clinical translation from bench-to-bedside.

The pathogenesis of posterior longitudinal ligament ossification (OPLL) remains inadequately understood. Mechanical stimulation is one of the important pathogenic factors in OPLL. As one of the mechanical stimulation transduction signals, the yes-associated protein (YAP) interacts with the Wnt/β-catenin signalling pathway, which plays an important role in osteogenic differentiation. This study aimed to demonstrate the role of YAP-Wnt/β-catenin axis in cell differentiation induced by mechanical stress. Primary cells extracted from posterior longitudinal ligament tissues from OPLL or non-OPLL patients were subjected to sinusoidal uniaxial cyclic stretch (5 %, 0.5 Hz, 3 d). The expression of runt-related transcription factor 2, collagen I, osterix, osteocalcin and alkaline phosphatase were compared between the static and the experimental groups. In addition, the cytoskeleton was detected using phalloidin staining while YAP phosphorylation states and nuclear location were identified using immunofluorescence. The results showed that mechanical stretching loading increased the expression of osteogenic genes and proteins in the OPLL group, while it had no significant effect on the control group. When OPLL cells were stretched, YAP exhibited an obvious nuclear translocation and the Wnt/β-catenin pathway was activated. Knocking down YAP or β-catenin could weaken the impact upon osteogenic differentiation induced by mechanical stimulation. YAP-mediated mechanical stimulation promoted osteogenic differentiation of OPLL cells through Wnt/β-catenin pathway and this progress was independent of the Hippo pathway.

The objective was to compare different dental splint models and materials for inducing abnormal loading on the gross morphology and histological appearance of the mandibular condylar processes of Sprague Dawley rats. Three different types of dental splints (resin molar, aluminum incisor, stainless-steel incisor) were placed unilaterally to induce occlusal perturbation for 4 weeks. At that time, mandibular condylar processes were assessed by gross appearance and histology. Quantitative measurements were also conducted on the hematoxylin and eosin images for condyle shape. The results showed that although the condylar cartilage was affected by all splint types, the resin molar splint was associated with the most extensive mandibular condylar process remodeling, which was primarily a slant (skewness) of the lateral aspect of the condylar process. Additionally, quantitative measurements on the histological specimens demonstrated that the split and tilt angle of the left (ipsilateral) condylar processes in the resin molar group (124.8 ± 12.7° and 104.1 ± 12.7°, respectively) increased significantly (p < 0.05) when compared to right (contralateral) condylar processes (104.7 ± 5.8°and 91.6 ± 4.4°, respectively). However, no changes were noted on the thickness of the fibrocartilage layer at medial, central, and lateral regions of the condylar process. Another major finding is the high variability of morphology of the naïve animals. Future studies will assess the impact of longer durations of splinting, age, and sex on the remodeling of the mandibular condylar process, allowing for the development of diagnostics and therapies.

Recent studies highlighted the crucial contribution of subchondral bone to OA development. Yet, only limited data have been reported on the relation between alteration to cartilage morphology, structural properties of the subchondral bone plate (SBP) and underlying subchondral trabecular bone (STB). Furthermore, the relationship between the morphometry of the cartilage and bone in the tibial plateau and the OA-induced changes in the joint's mechanical axis remains unexplored. Therefore, a visualisation and quantification of cartilage and subchondral bone microstructure in the medial tibial plateau was performed. End stage knee-OA patients with varus alignment and scheduled for total knee arthroplasty (TKA) underwent preoperative fulllength radiography to measure the hip-knee-ankle angle (HKA) and the mechanical-axis deviation (MAD). 18 tibial plateaux were μ-CT scanned (20.1 μm/voxel). Cartilage thickness, SBP, and STB microarchitecture were quantified in 10 volumes of interest (VOIs) in each medial tibial plateau. Significant differences (p < 0.001) were found for cartilage thickness, SBP, and STB microarchitecture parameters among the VOIs. Closer to the mechanical axis, cartilage thickness was consistently smaller, while SBP thickness and STB bone volume fraction (BV/TV) were higher. Moreover, trabeculae were also more superior-inferiorly oriented, i.e. perpendicular to the transverse plane of the tibial plateau. As cartilage and subchondral bone changes reflect responses to local mechanical loading patterns in the joint, the results suggested that region-specific subchondral bone adaptations were related to the degree of varus deformity. More specifically, subchondral sclerosis appeared to be most pronounced closer to the mechanical axis of the knee.

A critical component of the temporomandibular joint (TMJ) is the fibrocartilage articular disc (AD). Researchers have attempted to regenerate the AD to alleviate TMJ osteoarthritis but alternative cell sources for use in AD regenerative approaches are needed due to insufficient extracellular matrix (ECM) production by total articular disc cells (TACs). Tissue-specific progenitor cells have been identified in many tissues. The aim of the present study was to identify adult multipotent progenitor cells within the AD suitable for regenerative medicine applications. A novel AD progenitor cell population was identified in rhesus macaques. Clonally derived articular disc progenitor cells (ADPs) were isolated using fibronectin differential cell adhesion. ADPs represent between 1 and 3 % of the TAC population and are capable of in vitro expansion beyond 60 population doublings. ADPs were characterized using osteogenic, adipogenic, and fibrochondrogenesis differentiation assays. Clones exhibited phenotypic plasticity, differentiating into osteocytes, adipocytes, and fibrochondrocytes. ECM secretion profiles following fibrochondrogenic differentiation were assessed using immunohistochemistry (IHC), fluorescently activated cell sorting (FACS), total collagen, and glycosaminoglycan (GAG) assays and compared with TACs, articular cartilage progenitor cells (ACPs), tendon progenitor cells (TPCs) and bone-marrow-derived mesenchymal stem cells (BMMSCs). ADP pellet cultures produced a biochemical phenotype similar to native AD tissue, with production of versican (VCAN) and collagen types I, II, III, and VI (COL1, COL2, COL3, COL6). However, clonally derived ADP cell lines produced different amounts of ECM and exhibited different expansion potentials. These findings indicated flexibility in clone selection for potential regenerative strategies to recapitulate native anisotropy.

Periodontitis is a progressive disease that ultimately leads to bone and tooth loss. A major consequence of periodontal disease is the inability to regain lost bone in the periodontium. The importance was demonstrated of glucose-regulated protein-78 (GRP78) in the osteogenic differentiation of periodontal ligament stem cells and their potential use for regeneration of the periodontium. Previous studies have shown the relationship between GRP78 and dentine matrix protein-1 (DMP1). The importance of this receptor-ligand complex in supporting the process of osteogenesis and angiogenesis was confirmed in this study. To show the function of GRP78 in mineralised tissues, transgenic periodontal ligament stem cells (PDLSCs) were generated in which GRP78 was either overexpressed or silenced. Gene expression analysis of the cells cultured under osteogenic conditions showed an increase in key osteogenic genes with the overexpression of GRP78. RNA-Seq analysis was also performed to understand the transcriptome profile associated with genotype changes. Using the database for annotation, visualisation, and integration discovery (DAVID) for the functional enrichment analysis of differentially expressed genes, the upregulation of genes promoting osteogenesis and angiogenesis with GRP78 overexpression was demonstrated. Alizarin red staining and scanning electron microscopy analysis revealed matrix mineralisation with increased calcium deposition in GRP78 overexpressing cells. The in vivo osteogenic and angiogenic function of GRP78 was shown using a subcutaneous implantation rodent model. The results suggested that GRP78 in PDLSCs can regulate the expression of both osteogenesis and angiogenesis. Therefore, GRP78 could be considered as a therapeutic target for repair of diseased periodontium.

Skeletal muscle contractions are critical for normal skeletal growth and morphogenesis but it is unclear how the detrimental effects of absent muscle on the bones and joints change over time. Joint shape and cavitation as well as rudiment length and mineralisation were assessed in multiple rudiments at two developmental stages [Theiler stage (TS)24 and TS27] in the splotch-delayed "muscle-less limb" mouse model and littermate controls. Chondrocyte morphology was quantified in 3D in the distal humerus at the same stages. As development progressed, the effects of absent muscle on all parameters except for cavitation become less severe. All major joints in muscle-less limbs were abnormally shaped at TS24, while, by TS27, most muscle-less limb joint shapes were normal or nearly normal. In contrast, any joints that were fused at TS24 did not cavitate by TS27. At TS24, chondrocytes in the distal humerus were significantly smaller in the muscle-less limbs than in controls, while by TS27, chondrocyte volume was similar between the two groups, offering a cell-level mechanism for the partial recovery in shape of muscle-less limbs. Mineralisation showed the most pronounced changes over gestation. At TS24, all muscle-less rudiments studied had less mineralisation than the controls, while at TS27, muscle-less limb rudiments had mineralisation extents equivalent to controls. In conclusion, the effects of muscle absence on prenatal murine skeletogenesis reduced in severity over gestation. Understanding how mammalian bones and joints continue to develop in an environment with abnormal fetal movements provides insights into conditions including hip dysplasia and arthrogryposis.

The acetabular labrum is a fibrocartilaginous ring surrounding the acetabulum and is important for hip stability and contact pressure dissipation through a sealing function. Injury of the labrum may contribute to hip-joint degeneration and development of secondary osteoarthritis. Understanding how extracellular matrix (ECM) production and remodelling is regulated is of key importance for successful tissue restoration. The present study hypothesised that physiological stretching enhanced the metabolic activity and altered the ECM gene expression in labrum cells. Primary bovine labrum cells were physiologically stretched for up to 5 d. 24 h after the last stretch cycle, changes in metabolic activity were measured using the PrestoBlue™ HS Cell Viability Reagent and ECM gene expression was examined using the quantitative polymerase chain reaction method. Targets of interest were further investigated using immunofluorescence and enzyme-linked immunosorbent assay. Metabolic activity was not affected by the stretching (0.9746 ± 0.0614, p > 0.05). Physiological stretching upregulated decorin (DCN) (1.8548 ± 0.4883, p = 0.002) as well as proteoglycan 4 (PRG4) (1.7714 ± 0.6600, p = 0.029) and downregulated biglycan (BGN) (0.7018 + 0.1567, p = 0.008), cartilage oligomeric matrix protein (COMP) (0.5747 ± 0.2650, p = 0.029), fibronectin (FN1) (0.5832 ± 0.0996, p < 0.001) and spondin 1 (SPON1) (0.6282 ± 0.3624, p = 0.044) gene expression. No difference in PRG4 and DCN abundance or release could be measured. The here identified mechanosensitive targets are known to play relevant roles in tissue organisation. Therefore, physiological stretching might play a role in labrum tissue homeostasis and regeneration.

Extensive extracellular matrix production and increased cell-matrix adhesion by bone marrow stromal cells (BMSCs) are hallmarks of fibrotic alterations in the vertebral bone marrow known as Modic type 1 changes (MC1). MC1 are associated with non-specific chronic low-back pain. To identify treatment targets for MC1, in vitro studies using patient BMSCs are important to reveal pathological mechanisms. For the culture of BMSCs, fibroblast growth factor 2 (FGF2) is widely used. However, FGF2 has been shown to suppress matrix synthesis in various stromal cell populations. The aim of the present study was to investigate whether FGF2 affected the in vitro study of the fibrotic pathomechanisms of MC1-derived BMSCs. Transcriptomic changes and changes in cell-matrix adhesion of MC1-derived BMSCs were compared to intra-patient control BMSCs in response to FGF2. RNA sequencing and quantitative real-time polymerase chain reaction revealed that pro-fibrotic genes and pathways were not detectable in MC1-derived BMSCs when cultured in the presence of FGF2. In addition, significantly increased cell-matrix adhesion of MC1-derived BMSCs was abolished in the presence of FGF2. In conclusion, the data demonstrated that FGF2 overrides key pro-fibrotic features of MC1 BMSCs in vitro. Usage of FGF2-supplemented media in studies of fibrotic mechanisms should be critically evaluated as it could override normally dominant biological and biophysical cues.

Diarthrodial joint diseases, affecting hundreds of millions of people worldwide, mainly include osteoarthritis and cartilage injuries. No consensus on joint disease models has been achieved so far owing to the complex aetiologies, pathophysiological mechanisms and heterogeneity of disorders. The disease models established using isolated chondrocytes or small animals have the weaknesses of lacking native extracellular matrix and inter-species differences in anatomical and biomechanical cartilage properties. Osteochondral explants (OCEs) from large-animal or human joints present characteristics of native articular cartilage, showing promising potential for application in research on joint diseases. The present review focuses on OCEs and highlights the OCE sources, harvesting techniques, culture systems, applications and future developments. The OCE-centred ex vivo system has the potential to develop into preclinical models mimicking human joint diseases to help elucidate disease mechanisms, prompt therapeutic strategies and facilitate the clinical translation of findings in basic research.

Chronic tendinopathy represents a growing healthcare burden in the ageing global population. Curative therapies remain elusive as the mechanisms that underlie chronic inflammation in tendon disease remain unclear. Identifying and isolating key pathogenic and reparative cells is essential in developing precision therapies and implantable materials for improved tendon healing. Multiple discrete human tendon cell populations have been previously described ex vivo. To determine if these populations persist in vitro, healthy human hamstring tenocytes were cultured for 8 d on either tissue culture plastic or aligned electrospun fibres of absorbable polydioxanone. Novel single-cell surface proteomics combined with unbiased single-cell transcriptomics (CITE-Seq) was used to identify discrete tenocyte populations. 6 cell populations were found, 4 of which shared key gene expression determinants with ex vivo human cell clusters: PTX3_PAPPA, POSTN_SCX, DCN_LUM and ITGA7_NES. Surface proteomics found that PTX3_PAPPA cells were CD10+CD26+CD54+. ITGA7_NES cells were CD146+ and POSTN_SCX cells were CD90+CD95+CD10+. Culture on the aligned electrospun fibres favoured 3 cell subtypes (DCN_LUM, POSTN_SCX and PTX3_ PAPPA), promoting high expression of tendon-matrix-associated genes and upregulating gene sets enriched for TNF-a and IL-6/STAT3 signalling. Discrete human tendon cell subpopulations persisted in in vitro culture and could be recognised by specific gene and surface-protein signatures. Aligned polydioxanone fibres promoted the survival of 3 clusters, including pro-inflammatory PTX3-expressing CD10+CD26+CD54+ cells found in chronic tendon disease. These results improved the understanding of preferred culture conditions for different tenocyte subpopulations and informed the development of in vitro models of tendon disease.

The anterior cruciate ligament (ACL) is the most frequently injured ligament in the knee. The current method to treat the injured ligament is reconstruction using autografts and allografts. Reconstruction requires the regeneration of ligament, bone and their interface to ensure proper recovery. Recently, researchers have focused on using tissue-engineered scaffolds made of synthetic materials and biomaterials -such as collagen, decellularised tissues, silk and synthetic polymers produced following different manufacturing methods - for ACL reconstruction,. Different materials can be easily processed using various fabrication methods for mimicking the mechanical properties of the ACL. The advances in technologies play an important role in the production of constructions that can mimic native ACL.. The present review addresses integrative scaffold design, different challenges in the potential materials and manufacturing methods as well as future strategies for ACL repair. Furthermore, the review provides a road map to 3D printing combined with organ-on-chip technology to demonstrate the potential for cost-effective and user-friendly fabrication methods for ACL engineering. Finally, it underlines the potential of 3D bioprinting and organ-on-chip technologies for micro-engineering of ligaments and their associated environment.

The interphase between tendon and bone consists of a highly specialised tissue called enthesis. Typically, the enthesis is described as a succession of four different zones: tendon, non-mineralised fibrocartilage, mineralised fibrocartilage and bone. However, the microstructure of the entheses, cellular composition and mechanical properties vary depending on their anatomical location. The present study aimed to characterise three of the most relevant sites of enthesis injury in a rat model: the patellar tendon, the Achilles tendon and the supraspinatus enthesis, in terms of biomechanics, histology and genetic expression. The patellar enthesis presented the highest ultimate load and lowest stiffness of the three, while the supraspinatus was the weakest and stiffest. The histological characterisation revealed key differences at the insertion site for each enthesis. The patellar enthesis showed a large cartilaginous area at the tendon-to-bone interphase whilst this interphase was smaller in the supraspinatus entheses samples. Furthermore, the Achilles tendon enthesis displayed a more abrupt transition from tendon to bone. Additionally, each enthesis exhibited a particular and distinct pattern of expression of tenogenic, chondrogenic and osteogenic markers. This study provided valuable insights for a better understanding of the three entheses at relevant anatomical sites. Moreover, the larger cross-sectional area of the patellar enthesis, the strong mechanical properties and the easier surgical access to this location led to the conclusion that the patellar tendon enthesis site could be most suitable for the development of a preclinical model for general enthesis regeneration studies in rats.

Bone mechanobiology is the study of the physical, biological and mechanical processes that continuously affect the multiscale multicellular system of the bone from the organ to the molecular scale. Current knowledge derives from experimental studies, which are often limited to gathering qualitative data in a cross-sectional manner, up to a restricted number of time points. Moreover, the simultaneous collection of information about 3D bone microarchitecture, cell activity as well as protein distribution and level is still a challenge. In silico models can expand qualitative information with hypothetical quantitative systems, which allow quantification, testing and comparison to existing quantifiable experimental data. An overview of multiscale, multiphysics, agent-based and hybrid techniques and their applications to bone mechanobiology is provided in the present review. The study analysed how mechanical signals, cells and proteins can be modelled in silico to represent bone remodelling and adaptation. Hybrid modelling of bone mechanobiology could combine the methods used in multiscale, multiphysics and agent-based models into a single model, leading to a unified and comprehensive understanding of bone mechanobiology. Numerical simulations of in vivo multicellular systems aided in hypothesis testing of such in silico models. Recently, in silico trials have been used to illustrate the mechanobiology of cells and signalling pathways in clinical biopsies and animal bones, including the effects of drugs on single cells and signalling pathways up to the organ level. This improved understanding may lead to the identification of novel therapies for degenerative diseases such as osteoporosis.

Implant infection impairs osseointegration of orthopaedic implants by inducing inflammation. Acinetobacter spp. are increasingly prevalent multi-drug resistant bacteria that can cause osteomyelitis. Acinetobacter spp. can also cause inflammation and thereby inhibit osseointegration in mice. The purpose of the present study was to investigate the role of quorum sensing in this context. Therefore, wild-type bacteria were compared with an isogenic abaI mutant defective in quorum sensing in a murine osseointegration model. The abaI quorum- sensing mutant affected significantly less osseointegration and interleukin (IL) 1β levels, without detectably altering other pro-inflammatory cytokines. Wild-type bacteria had fewer effects on IL1 receptor (IL1R)-/- mice. These results indicated that quorum sensing in Acinetobacter spp. contributed to IL1β induction and the resultant inhibition of osseointegration in mice. Moreover, targeting the Gram-negative acyl-homoserine lactone quorum sensing may be particularly effective for patients with Acinetobacter spp. infections.

Biochemical and biophysical factors need consideration when modelling in vivo cellular behaviour using in vitro cell culture systems. One underappreciated factor is the high concentration of macromolecules present in vivo, which is typically not simulated under standard cell culture conditions. This disparity is especially relevant when studying biochemical processes that govern extracellular matrix (ECM) deposition, which may be altered due to dilution of secreted macromolecules by the relatively large volumes of culture medium required for cell maintenance in vitro. Macromolecular crowding (MMC) utilises the addition of inert macromolecules to cell culture medium to mimic such high concentration environments found in vivo. The present study induced MMC using the sucrose polymer Ficoll and examined whether fibrillin-1 deposition by human lung fibroblasts could be augmented. Fibrillin-1 forms extracellular microfibrils, which are versatile scaffolds required for elastic fibre formation, deposition of other ECM proteins and growth factor regulation. Pathogenic variants in the fibrillin-1 gene (FBN1) cause Marfan syndrome, where ECM deposition of fibrillin-1 can be compromised. Using immunocytochemistry, significantly enhanced fibrillin-1 deposition was observed when lung fibroblasts were cultured under MMC conditions. MMC also augmented fibrillin-1 deposition in Marfan syndrome patient-derived skin fibroblasts in a cell line- and likely FBN1 variant-specific manner. The ability of MMC to increase fibrillin-1 deposition suggested potential applications for tissue-engineering approaches, e.g. to generate tendon or vascular tissues, where fibrillin-1 microfibrils and elastic fibres are key determinants of their biomechanical properties. Moreover, it suggested the potency of MMC to better mimic in vivo ECM environments in cell culture studies.

Prior studies have outlined C-reactive protein (CRP) within the first 5 d following total hip arthroplasty (THA) as an inappropriate indicator of an early periprosthetic joint infection (PJI). Recently, interleukin-6 (IL-6), as a potential inflammatory marker following total joint arthroplasty (TJA), has gained increasing interest, particularly due to its considerably shorter half-life. The aim of the present study was to assess IL-6 measured on postoperative day 3 following TJA as a prediction marker of early onset PJI. 7,661 patients, who underwent total hip or knee arthroplasty (THA, TKA) at a single institution between 2016 and 2019, were evaluated. Serum IL-6 values were measured on postoperative day 3 and compared between patients, with and without early onset PJI in the postoperative follow-up, matched for age, gender, Surgical Site Infection Risk Score and Charlson comorbidity index. Overall (n = 7,661), there was no statistically significant difference in serum IL-6 levels comparing patients with and without early onset PJI following THA [38.9 pg/ mL vs. 32.0 pg/mL, p = 0.116] and TKA [30.6 pg/mL vs. 28.2 pg/mL, p = 0.718]. Male gender and high body mass index were associated with an increased risk of early onset PJI following THA (p = 0.027, p = 0.002). Matched cohort analysis (n = 86) showed no statistically significant difference in serum IL-6 levels between patients with and without early onset PJI following THA (p = 0.680) and TKA (p = 0.910). Serum IL-6 values on postoperative day 3 following THA or TKA could not predict early onset PJIs.