Advertisement for orthosearch.org.uk
Results 1 - 20 of 187
Results per page:
Bone & Joint 360
Vol. 14, Issue 1 | Pages 7 - 10
1 Feb 2025
Ollivere B


Bone & Joint Research
Vol. 13, Issue 12 | Pages 764 - 778
12 Dec 2024
Huang Q Zhuo Y Duan Z Long Y Wang J Zhang Z Fan S Huang Y Deng K Xin H

Aims

Mesenchymal stem cells (MSCs) are usually cultured in a normoxic atmosphere (21%) in vitro, while the oxygen concentrations in human tissues and organs are 1% to 10% when the cells are transplanted in vivo. However, the impact of hypoxia on MSCs has not been deeply studied, especially its translational application.

Methods

In the present study, we investigated the characterizations of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in hypoxic (1%) and normoxic (21%) atmospheres with a long-term culture from primary to 30 generations, respectively. The comparison between both atmospheres systematically analyzed the biological functions of MSCs, mainly including stemness maintenance, immune regulation, and resistance to chondrocyte apoptosis, and studied their joint function and anti-inflammatory effects in osteoarthritis (OA) rats constructed by collagenase II.


Bone & Joint Research
Vol. 13, Issue 12 | Pages 695 - 702
1 Dec 2024
Cordero García-Galán E Medel-Plaza M Pozo-Kreilinger JJ Sarnago H Lucía Ó Rico-Nieto A Esteban J Gomez-Barrena E

Aims

Electromagnetic induction heating has demonstrated in vitro antibacterial efficacy over biofilms on metallic biomaterials, although no in vivo studies have been published. Assessment of side effects, including thermal necrosis of adjacent tissue, would determine transferability into clinical practice. Our goal was to assess bone necrosis and antibacterial efficacy of induction heating on biofilm-infected implants in an in vivo setting.

Methods

Titanium-aluminium-vanadium (Ti6Al4V) screws were implanted in medial condyle of New Zealand giant rabbit knee. Study intervention consisted of induction heating of the screw head up to 70°C for 3.5 minutes after implantation using a portable device. Both knees were implanted, and induction heating was applied unilaterally keeping contralateral knee as paired control. Sterile screws were implanted in six rabbits, while the other six received screws coated with Staphylococcus aureus biofilm. Sacrifice and sample collection were performed 24, 48, or 96 hours postoperatively. Retrieved screws were sonicated, and adhered bacteria were estimated via drop-plate. Width of bone necrosis in retrieved femora was assessed through microscopic examination. Analysis was performed using non-parametric tests with significance fixed at p ≤ 0.05.


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_18 | Pages 40 - 40
14 Nov 2024
Siverino C Sun Q Yang D Solomon B Moriarty F Atkins G
Full Access

Introduction. Bone and joint infection (BJI) is often characterized by severe inflammation and progressive bone destruction. Osteocytes are the most numerous and long-lived bone cell type, and therefore represent a potentially important long-term reservoir of bacterial infection. Staphylococcus aureus is known to establish stable intracellular osteocytic infections, however, little is known about the less virulent yet second most prevalent BJI pathogen, S. epidermidis, associated with late-diagnosed, chronic BJI. Thus, this study sought to establish an in vitro model to study the infection characteristics of S. epidermidis in human osteocyte-like cells. Methods. SaOS2 cells (1 ×10. 4. cells/cm. 2. ) were grown to confluence either without differentiation, representing an osteoblast-like (OB) state (SaOS2-OB) or differentiated to an osteocyte-like stage (SaOS2-OY), using established methods. Four S. epidermidis strains used (ATCC-12228, ATCC-14990, ATCC-35984 and a clinical osteomyelitis strain RAH-SE1) were tested to be Lysostaphin-resistant, necessitating antibiotic (Levofloxacin) control of extracellular bacteria. Infection of host cells (OB or OY) was tested at three multiplicities of infection (MOI: 10, 100 and 1000). Extracellular bacteria were controlled by overnight incubation at a 10X minimum inhibitory concentration (MIC) of Levofloxacin and thereafter at 1XMIC. At each time point (days 1, 3, 5) viable intra- and extracellular bacteria were quantified. Result. All strains displayed similar intracellular infection and persistence capabilities in SaOS2-OB and SaOS2-OY. Independent of MOI, intracellular bacteria in SaOS2-OB decreased over time, becoming non-culturable by day 5. In contrast, SaOs2-OY displayed enhanced intracellular bacterial persistence at each time point. In the presence of increased Levofloxacin concentration (10XMIC), S. epidermidis could persist intracellularly for at least 14 days. Conclusion. This study showed for the first time that S. epidermidis can infect human osteocytes and persist intracellularly. Additionally, even a 10xMIC antibiotic concentration failed to eradicate intracellular bacteria, suggesting that persistence within osteocytes could contribute to treatment failure and establishment of chronic BJI


Bone & Joint Research
Vol. 13, Issue 10 | Pages 559 - 572
8 Oct 2024
Wu W Zhao Z Wang Y Liu M Zhu G Li L

Aims

This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels.

Methods

A closed tibial fracture model was established using 12 male Japanese white rabbits, and divided into elastic and stiff fixation groups based on different fixation methods. Two weeks after the operation, a radiograph and pathological examination of callus tissue were used to evaluate fracture healing. Then, the differentially expressed proteins (DEPs) were examined in the callus using proteomics. Finally, in vitro cell experiments were conducted to investigate hub proteins involved in this process.


Bone & Joint Research
Vol. 13, Issue 3 | Pages 127 - 135
22 Mar 2024
Puetzler J Vallejo Diaz A Gosheger G Schulze M Arens D Zeiter S Siverino C Richards RG Moriarty TF

Aims

Fracture-related infection (FRI) is commonly classified based on the time of onset of symptoms. Early infections (< two weeks) are treated with debridement, antibiotics, and implant retention (DAIR). For late infections (> ten weeks), guidelines recommend implant removal due to tolerant biofilms. For delayed infections (two to ten weeks), recommendations are unclear. In this study we compared infection clearance and bone healing in early and delayed FRI treated with DAIR in a rabbit model.

Methods

Staphylococcus aureus was inoculated into a humeral osteotomy in 17 rabbits after plate osteosynthesis. Infection developed for one week (early group, n = 6) or four weeks (delayed group, n = 6) before DAIR (systemic antibiotics: two weeks, nafcillin + rifampin; four weeks, levofloxacin + rifampin). A control group (n = 5) received revision surgery after four weeks without antibiotics. Bacteriology of humerus, soft-tissue, and implants was performed seven weeks after revision surgery. Bone healing was assessed using a modified radiological union scale in tibial fractures (mRUST).


Bone & Joint Research
Vol. 13, Issue 2 | Pages 52 - 65
1 Feb 2024
Yao C Sun J Luo W Chen H Chen T Chen C Zhang B Zhang Y

Aims. To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism. Methods. In a series of in vitro experiments, we used tert-Butyl hydroperoxide (TBHP) to induce senescence of MLO-Y4 cells successfully, and collected conditioned medium (CM) and senescent MLO-Y4 cell-derived exosomes, which were then applied to MC3T3-E1 cells, separately, to evaluate their effects on osteogenic differentiation. Furthermore, we identified differentially expressed microRNAs (miRNAs) between exosomes from senescent and normal MLO-Y4 cells by high-throughput RNA sequencing. Based on the key miRNAs that were discovered, the underlying mechanism by which senescent osteocytes regulate osteogenic differentiation was explored. Lastly, in the in vivo experiments, the effects of senescent MLO-Y4 cell-derived exosomes on age-related bone loss were evaluated in male SAMP6 mice, which excluded the effects of oestrogen, and the underlying mechanism was confirmed. Results. The CM and exosomes collected from senescent MLO-Y4 cells inhibited osteogenic differentiation of MC3T3-E1 cells. RNA sequencing detected significantly lower expression of miR-494-3p in senescent MLO-Y4 cell-derived exosomes compared with normal exosomes. The upregulation of exosomal miR-494-3p by miRNA mimics attenuated the effects of senescent MLO-Y4 cell-derived exosomes on osteogenic differentiation. Luciferase reporter assay demonstrated that miR-494-3p targeted phosphatase and tensin homolog (PTEN), which is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overexpression of PTEN or inhibition of the PI3K/AKT pathway blocked the functions of exosomal miR-494-3p. In SAMP6 mice, senescent MLO-Y4 cell-derived exosomes accelerated bone loss, which was rescued by upregulation of exosomal miR-494-3p. Conclusion. Reduced expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenic differentiation and accelerates age-related bone loss via PTEN/PI3K/AKT pathway. Cite this article: Bone Joint Res 2024;13(2):52–65


Bone & Joint Research
Vol. 13, Issue 1 | Pages 4 - 18
2 Jan 2024
Wang Y Wu Z Yan G Li S Zhang Y Li G Wu C

Aims

cAMP response element binding protein (CREB1) is involved in the progression of osteoarthritis (OA). However, available findings about the role of CREB1 in OA are inconsistent. 666-15 is a potent and selective CREB1 inhibitor, but its role in OA is unclear. This study aimed to investigate the precise role of CREB1 in OA, and whether 666-15 exerts an anti-OA effect.

Methods

CREB1 activity and expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) in cells and tissues were measured by immunoblotting and immunohistochemical (IHC) staining. The effect of 666-15 on chondrocyte viability and apoptosis was examined by cell counting kit-8 (CCK-8) assay, JC-10, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) staining. The effect of 666-15 on the microstructure of subchondral bone, and the synthesis and catabolism of cartilage, in anterior cruciate ligament transection mice were detected by micro-CT, safranin O and fast green (S/F), immunohistochemical staining, and enzyme-linked immunosorbent assay (ELISA).


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_1 | Pages 16 - 16
2 Jan 2024
Lipreri M Pasquarelli A Scelfo D Baldini N Avnet S
Full Access

Osteoporosis is a progressive, chronic disease of bone metabolism, characterized by decreased bone mass and mineral density, predisposing individuals to an increased risk of fractures. The use of animal models, which is the gold standard for the screening of anti-osteoporosis drugs, raises numerous ethical concerns and is highly debated because the composition and structure of animal bones is very different from human bones. In addition, there is currently a poor translation of pre-clinical efficacy in animal models to human trials, meaning that there is a need for an alternative method of screening and evaluating new therapeutics for metabolic bone disorders, in vitro. The aim of this project is to develop a 3D Bone-On-A-Chip that summarizes the spatial orientation and mutual influences of the key cellular components of bone tissue, in a citrate and hydroxyapatite-enriched 3D matrix, acting as a 3D model of osteoporosis. To this purpose, a polydimethylsiloxane microfluidic device was developed by CAD modelling, stereolithography and replica molding. The device is composed by two layers: (i) a bottom layer for a 3D culture of osteocytes embedded in an osteomimetic collagen-enriched matrigel matrix with citrate-doped hydroxyapatite nanocrystals, and (ii) a upper layer for a 2D perfused co-culture of osteoblasts and osteoclasts seeded on a microporous PET membrane. Cell vitality was evaluated via live/dead assay. Bone deposition and bone resorption was analysed respectively with ALP, Alizarin RED and TRACP staining. Osteocytes dendrite expression was evaluated via immunofluorescence. Subsequently, the model was validated as drug screening platform inducing osteocytes apoptosis and administrating standard anti-osteoporotic drugs. This device has the potential to substitute or minimize animal models in pre-clinical studies of osteoporosis, contributing to pave the way for a more precise and punctual personalized treatment


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_16 | Pages 52 - 52
17 Nov 2023
Jones R Bowd J Gilbert S Wilson C Whatling G Jonkers I Holt C Mason D
Full Access

Abstract. OBJECTIVE. Knee varus malalignment increases medial knee compartment loading and is associated with knee osteoarthritis (OA) progression and severity. 1. Altered biomechanical loading and dysregulation of joint tissue biology drive OA progression, but mechanistic links between these factors are lacking. Subchondral bone structural changes are biomechanically driven, involve bone resorption, immune cell influx, angiogenesis, and sensory nerve invasion, and contribute to joint destruction and pain. 2. We have investigated mechanisms underlying this involving RANKL and alkaline phosphatase (ALP), which reflect bone resorption and mineralisation respectively. 3. and the axonal guidance factor Sema3A. Sema3A is osteotropic, expressed by mechanically sensitive osteocytes, and an inhibitor of sensory nerve, blood vessel and immune cell invasion. 4. Sema3A is also differentially expressed in human OA bone. 5. HYPOTHESIS: Medial knee compartment overloading in varus knee malalignment patients causes dysregulation of bone derived Sema3A signalling directly linking joint biomechanics to pathology and pain. METHODS. Synovial fluid obtained from 30 subjects with medial knee OA (KL grade II-IV) undergoing high tibial osteotomy surgery (HTO) was analysed by mesoscale discovery and ELISA analysis for inflammatory, neural and bone turnover markers. 11 of these patients had been previously analysed in a published patient-specific musculoskeletal model. 6. of gait estimating joint contact location, pressure, forces, and medial-lateral condyle load distribution in a published data set included in analyses. Data analysis was performed using Pearson's correlation matrices and principal component analyses. Principal Components (PCs) with eigenvalues greater than 1 were analysed. RESULTS. PC1 (32.94% of variation) and PC2 (25.79% of variation) from PCA analysis and correlation matrices separated patients according to correlated clusters of established inflammatory markers of OA pain and progression (IL6/IL8, r=0.754, p<0.001) and anti-inflammatory mediators (IL4/IL10, r=0.469, p=0.005). Bone turnover marker ALP was positively associated with KL grade (r=0.815, p=0.002) and negatively associated with IL10 (r=−0.402, p=0.018) and first peak knee loading pressures (r=−0.688, p=0.019). RANKL was positively associated with IL4 (r=0.489, p=0.003). Synovial fluid Sema3A concentrations showed separate clustering from all OA progression markers and was inversely correlated with TNF-α (r=−0.423, p=0.022) in HTO patients. Sema3A was significantly inversely correlated with total predicted force in the medial joint compartment (r=−0.621, p=0.041), mean (r=−0.63, p=0.038) and maximum (r=−0.613, p=0.045) calculated medial compartment joint pressures during the first phase and mean (r=−0.618, p=0.043) and maximum (r=−0.641, p=0.034) medial compartment joint pressures during midstance outputs of patient-specific musculoskeletal model. CONCLUSIONS. This study shows joint inflammatory status and mechanical overloading influence subchondral bone-remodelling. Synovial Sema3A concentrations are inversely correlated to patient-specific musculoskeletal model estimations of pathological medial overloading. This study reveals Sema3A as a biological mediator with capacity to induce OA pain and disease progression that is directly regulated by gait mechanical loading. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_16 | Pages 49 - 49
17 Nov 2023
Jones R Gilbert S Mason D
Full Access

Abstract. OBJECTIVE. Changes in subchondral bone are one of few disease characteristics to correlate with pain in OA. 1. Profound neuroplasticity and nociceptor sprouting is displayed within osteoarthritic (OA) subchondral bone and is associated with pain and pathology. 2. The cause of these neural changes remains unestablished. Correct innervation patterns are indispensable for bone growth, homeostasis, and repair. Axon guidance signalling factor, Sema3A is essential for the correct innervation patterning of bony tissues. 3. , expressed in osteocytes. 4. and known to be downregulated in bone OA mechanical loading. 5. Bioinformatic analysis has also shown Sema3a as a differentially expressed pathway by bone in human OA patients. 6. HYPOTHESIS: Pathological mechanical load and inflammation of bone causes dysregulation of Sema3A signalling leading to perturbed sensory nerve plasticity and pain. METHODS. Human KOLF2-C1 iPSC derived nociceptors were generated by TALEN-mediated insertion of transcription factors NGN2+Brn3A and modified chambers differentiation protocol to produce nociceptor-like cells. Nociceptor phenotype was confirmed by immunocytochemistry. Human Y201-MSC cells were embedded in 3D type-I collagen gels (0.05 × 106 cell/gel), in 48-well plates and silicone plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) and soluble IL-6 receptor (sIL-6r (40ng/ml), IL6/sIL6r and mechanical load mimetic Yoda1 (5μM) or unstimulated (n=5/group) (48-well plates) or were mechanically loaded in silicone plates (5000μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). Conditioned media transfer was performed from osteocyte to nociceptor cultures assessed by continuous 24-hour phase contrast confocal microscopy. 24-hours after stimulation RNA was quantified by RT-qPCR (IL6) or RNAseq whole transcriptome analysis/DEseq2 analysis (Load). Protein release was quantified by ELISA. Normally distributed data with homogenous variances was analysed by two-tailed t test. RESULTS. IPSC-derived nociceptor-like cells display elongated (>5mm) dendritic projections and nociceptive molecular markers such as TUJ1, PrPH and Neun and TrkA. Sema3A signalling ligands were expressed in 100% of osteocyte cultures. Mechanical loading regulated the Sema3 pathway; Sema3A (0.4-fold, p<0.001), Sema3B (13-fold, p<0.001), Sema3C (0.4-fold, p<0.001). Under inflammatory stimulation by IL6/IL6sR, SEMA3A (7-fold, p=0.01) and receptor Plexin1 (3-fold, p=0.03) show significant regulation. Sema3A protein release showed a significant downregulation of Sema3A release by IL6/sIL6r+Yoda1 (2-fold, p=0.02). Continuous 24-hour phase contrast confocal microscopy measuring the number of extending/retreating dendritic projections revealed that sensory nerve cultures exposed to media from osteocytes stimulated with IL-6/sIL-6R+Yoda1 displayed significantly more invading dendritic projections (p=0.0175, 12-fold±SEM 3.5) across 3 random fields of view within a single stimulated neural culture and significantly fewer retracting dendritic projections (p=0.0075, 2-fold±SEM 0.33) compared to controls. CONCLUSIONS. Here we show osteocytic regulation of Sema3A under pathological mechanical loading and the ability of media pathologically loaded osteocyte cultures to induce the branching and invasion of cultured nociceptor-like cells as displayed in OA subchondral bone. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project


Bone & Joint Research
Vol. 12, Issue 11 | Pages 691 - 701
3 Nov 2023
Dai Z Chen Y He E Wang H Guo W Wu Z Huang K Zhao Q

Aims

Osteoporosis is characterized by decreased trabecular bone volume, and microarchitectural deterioration in the medullary cavity. Interleukin-19 (IL-19), a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. The aim of our study was to investigate the effect of IL-19 on osteoporosis.

Methods

Blood and femoral bone marrow suspension IL-19 levels were first measured in the lipopolysaccharide (LPS)-induced bone loss model. Small interfering RNA (siRNA) was applied to knock down IL-19 for further validation. Thereafter, osteoclast production was stimulated with IL-19 in combination with mouse macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The effect of IL-19 was subsequently evaluated using tartrate-resistant acid phosphatase (TRAP) staining and quantitative real-time polymerase chain reaction (RT-qPCR). The effect of IL-19 on osteoprotegerin (OPG) was then assessed using in vitro recombinant IL-19 treatment of primary osteoblasts and MLO-Y4 osteoblast cell line. Finally, transient transfection experiments and chromatin immunoprecipitation (ChIP) experiments were used to examine the exact mechanism of action.


Bone & Joint Research
Vol. 12, Issue 10 | Pages 644 - 653
10 Oct 2023
Hinz N Butscheidt S Jandl NM Rohde H Keller J Beil FT Hubert J Rolvien T

Aims

The management of periprosthetic joint infection (PJI) remains a major challenge in orthopaedic surgery. In this study, we aimed to characterize the local bone microstructure and metabolism in a clinical cohort of patients with chronic PJI.

Methods

Periprosthetic femoral trabecular bone specimens were obtained from patients suffering from chronic PJI of the hip and knee (n = 20). Microbiological analysis was performed on preoperative joint aspirates and tissue specimens obtained during revision surgery. Microstructural and cellular bone parameters were analyzed in bone specimens by histomorphometry on undecalcified sections complemented by tartrate-resistant acid phosphatase immunohistochemistry. Data were compared with control specimens obtained during primary arthroplasty (n = 20) and aseptic revision (n = 20).


Bone & Joint Research
Vol. 12, Issue 9 | Pages 580 - 589
20 Sep 2023
Dai X Liu B Hou Q Dai Q Wang D Xie B Sun Y Wang B

Aims

The aim of this study was to investigate the global and local impact of fat on bone in obesity by using the diet-induced obese (DIO) mouse model.

Methods

In this study, we generated a diet-induced mouse model of obesity to conduct lipidomic and 3D imaging assessments of bone marrow fat, and evaluated the correlated bone adaptation indices and bone mechanical properties.


Bone & Joint Research
Vol. 12, Issue 9 | Pages 536 - 545
8 Sep 2023
Luo P Yuan Q Yang M Wan X Xu P

Osteoarthritis (OA) is mainly caused by ageing, strain, trauma, and congenital joint abnormalities, resulting in articular cartilage degeneration. During the pathogenesis of OA, the changes in subchondral bone (SB) are not only secondary manifestations of OA, but also an active part of the disease, and are closely associated with the severity of OA. In different stages of OA, there were microstructural changes in SB. Osteocytes, osteoblasts, and osteoclasts in SB are important in the pathogenesis of OA. The signal transduction mechanism in SB is necessary to maintain the balance of a stable phenotype, extracellular matrix (ECM) synthesis, and bone remodelling between articular cartilage and SB. An imbalance in signal transduction can lead to reduced cartilage quality and SB thickening, which leads to the progression of OA. By understanding changes in SB in OA, researchers are exploring drugs that can regulate these changes, which will help to provide new ideas for the treatment of OA.

Cite this article: Bone Joint Res 2023;12(9):536–545.


Bone & Joint Research
Vol. 12, Issue 7 | Pages 397 - 411
3 Jul 2023
Ruan X Gu J Chen M Zhao F Aili M Zhang D

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive cartilage degradation, synovial membrane inflammation, osteophyte formation, and subchondral bone sclerosis. Pathological changes in cartilage and subchondral bone are the main processes in OA. In recent decades, many studies have demonstrated that activin-like kinase 3 (ALK3), a bone morphogenetic protein receptor, is essential for cartilage formation, osteogenesis, and postnatal skeletal development. Although the role of bone morphogenetic protein (BMP) signalling in articular cartilage and bone has been extensively studied, many new discoveries have been made in recent years around ALK3 targets in articular cartilage, subchondral bone, and the interaction between the two, broadening the original knowledge of the relationship between ALK3 and OA. In this review, we focus on the roles of ALK3 in OA, including cartilage and subchondral bone and related cells. It may be helpful to seek more efficient drugs or treatments for OA based on ALK3 signalling in future.


Bone & Joint Research
Vol. 12, Issue 5 | Pages 339 - 351
23 May 2023
Tan J Liu X Zhou M Wang F Ma L Tang H He G Kang X Bian X Tang K

Aims

Mechanical stimulation is a key factor in the development and healing of tendon-bone insertion. Treadmill training is an important rehabilitation treatment. This study aims to investigate the benefits of treadmill training initiated on postoperative day 7 for tendon-bone insertion healing.

Methods

A tendon-bone insertion injury healing model was established in 92 C57BL/6 male mice. All mice were divided into control and training groups by random digital table method. The control group mice had full free activity in the cage, and the training group mice started the treadmill training on postoperative day 7. The quality of tendon-bone insertion healing was evaluated by histology, immunohistochemistry, reverse transcription quantitative polymerase chain reaction, Western blotting, micro-CT, micro-MRI, open field tests, and CatWalk gait and biomechanical assessments.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_8 | Pages 74 - 74
11 Apr 2023
Gilbert S Jones R White P Mason D
Full Access

Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and SNPs in the Piezo1 locus are associated with changes in fracture risk. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. The current study used a human, cell-based physiological, 3D in vitro model of bone to determine whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Human Y201 MSCs, embedded in type I collagen gels and differentiated to osteocytes for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and assessed by RNAseq analysis. To mimic mechanical load and activate Piezo1, cells were differentiated to osteocytes for 13 days and treated ± Yoda1 (5µM, 2- and 24-hs, n=4); vehicle treated cells served as controls (n=4). RNA was subjected to RT-qPCR and data normalised to the housekeeping gene, YWHAZ. Media was analysed for IL6 release by ELISA. Mechanical load upregulated Piezo1 gene expression (16.5-fold, p<0.001) and expression of the transcription factor NFATc1, and matricellular protein CYR61, known regulators of Piezo1 mechanotransduction (3-fold; p= 5.0E-5 and 6.8-fold; p= 6.0E-5, respectively). After 2-hrs, Yoda1 increased the expression of the early mechanical response gene, cFOS (11-fold; p=0.021), mean Piezo1 expression (2.3-fold) and IL-6 expression (103-fold, p<0.001). Yoda1 increased the release of IL6 protein after 24 hours (7.5-fold, p=0.001). This study confirms Piezo1 as an important mechanosensor in osteocytes. Piezo1 activation mediated an increase in IL6, a cytokine that drives inflammation and bone resorption providing a direct link between mechanical activation of Piezo1, bone remodeling and inflammation, which may contribute to mechanically induced joint degeneration in diseases such as osteoarthritis. Mechanistically, we hypothesize this may occur through promoting Ca2+ influx and activation of the NFATc1 signaling pathway


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_8 | Pages 39 - 39
11 Apr 2023
Jones R Gilbert S Mason D
Full Access

Osteoarthritis (OA) is a common cause of chronic pain. Subchondral bone is highly innervated, and bone structural changes directly correlate with pain in OA. Mechanisms underlying skeletal–neural interactions are under-investigated. Bone derived axon guidance molecules are known to regulate bone remodelling. Such signals in the nervous system regulate neural plasticity, branching and neural inflammation. Perturbation of these signals during OA disease progression may disrupt sensory afferents activity, affecting tissue integrity, nociception, and proprioception. Osteocyte mechanical loading and IL-6 stimulation alters axon guidance signalling influencing innervation, proprioception, and nociception. Human Y201 MSC cells, embedded in 3D type I collagen gels (0.05 × 106 cell/gel) in 48 well plastic or silicone (load) plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) with soluble IL-6 receptor (sIL-6r (40ng/ml) or unstimulated (n=5/group), or mechanically loaded (5000 μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). RNA extracted 1hr and 24hrs post load was quantified by RNAseq whole transcriptome analysis (NovaSeq S1 flow cell 2 × 100bp PE reads and differentially expressed neurotransmitters identified (>2-fold change in DEseq2 analysis on normalised count data with FDR p<0.05). After 24 hours, extracted IL-6 stimulated RNA was quantified by RT-qPCR for neurotrophic factors using 2–∆∆Ct method (efficiency=94-106%) normalised to reference gene GAPDH (stability = 1.12 REfinder). Normally distributed data with homogenous variances was analysed by two-tailed t test. All detected axonal guidance genes were regulated by mechanical load. Axonal guidance genes were both down-regulated (Netrin1 0.16-fold, p=0.001; Sema3A 0.4-fold, p<0.001; SEMA3C (0.4-fold, p<0.001), and up-regulated (SLIT2 2.3-fold, p<0.001; CXCL12 5-fold, p<0.001; SEMA3B 13-fold, p<0.001; SEMA4F 2-fold, p<0.001) by mechanical load. IL6 and IL6sR stimulation upregulated SEMA3A (7-fold, p=0.01), its receptor Plexin1 (3-fold, p=0.03). Neutrophins analysed in IL6 stimulated RNA did not show regulation. Here we show osteocytes regulate multiple factors which may influence innervation, nociception, and proprioception upon inflammatory or mechanical insult. Future studies will establish how these factors may combine and affect nerve activity during OA disease progression


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_8 | Pages 38 - 38
11 Apr 2023
Stewart M Nguyen J
Full Access

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity. This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes. PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity. This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions