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Bone & Joint Research
Vol. 9, Issue 11 | Pages 827 - 839
1 Nov 2020
Hameister R Lohmann CH Dheen ST Singh G Kaur C

Aims. This study aimed to examine the effects of tumour necrosis factor-alpha (TNF-α) on osteoblasts in metal wear-induced bone loss. Methods. TNF-α immunoexpression was examined in periprosthetic tissues of patients with failed metal-on-metal hip arthroplasties and also in myeloid MM6 cells after treatment with cobalt ions. Viability and function of human osteoblast-like SaOs-2 cells treated with recombinant TNF-α were studied by immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Results. Macrophages, lymphocytes, and endothelial cells displayed strong TNF-α immunoexpression in periprosthetic tissues containing metal wear debris. Colocalization of TNF-α with the macrophage marker CD68 and the pan-T cell marker CD3 confirmed TNF-α expression in these cells. Cobalt-treated MM6 cells secreted more TNF-α than control cells, reflecting the role of metal wear products in activating the TNF-α pathway in the myeloid cells. While TNF-α did not alter the immunoexpression of the TNF-receptor 1 (TNF-R1) in SaOs-2 cells, it increased the release of the soluble TNF-receptor 1 (sTNF-R1). There was also evidence for TNF-α-induced apoptosis. TNF-α further elicited the expression of the endoplasmic reticulum stress markers inositol-requiring enzyme (IRE)-1α, binding-immunoglobulin protein (BiP), and endoplasmic oxidoreductin1 (Ero1)-Lα. In addition, TNF-α decreased pro-collagen I α 1 secretion without diminishing its synthesis. TNF-α also induced an inflammatory response in SaOs-2 cells, as evidenced by the release of reactive oxygen and nitrogen species and the proinflammatory cytokine vascular endothelial growth factor. Conclusion. The results suggest a novel osteoblastic mechanism, which could be mediated by TNF-α and may be involved in metal wear debris-induced periprosthetic bone loss. Cite this article: Bone Joint Res 2020;9(11):827–839


Bone & Joint Research
Vol. 10, Issue 9 | Pages 611 - 618
27 Sep 2021
Ali E Birch M Hopper N Rushton N McCaskie AW Brooks RA

Aims. Accumulated evidence indicates that local cell origins may ingrain differences in the phenotypic activity of human osteoblasts. We hypothesized that these differences may also exist in osteoblasts harvested from the same bone type at periarticular sites, including those adjacent to the fixation sites for total joint implant components. Methods. Human osteoblasts were obtained from the acetabulum and femoral neck of seven patients undergoing total hip arthroplasty (THA) and from the femoral and tibial cuts of six patients undergoing total knee arthroplasty (TKA). Osteoblasts were extracted from the usually discarded bone via enzyme digestion, characterized by flow cytometry, and cultured to passage three before measurement of metabolic activity, collagen production, alkaline phosphatase (ALP) expression, and mineralization. Results. Osteoblasts from the acetabulum showed lower proliferation (p = 0.034), cumulative collagen release (p < 0.001), and ALP expression (p = 0.009), and produced less mineral (p = 0.006) than those from the femoral neck. Osteoblasts from the tibia produced significantly less collagen (p = 0.021) and showed lower ALP expression than those from the distal femur. Conclusion. We have demonstrated for the first time an anatomical regional variation in the biological behaviours of osteoblasts on either side of the hip and knee joint. The lower osteoblast proliferation, matrix production, and mineralization from the acetabulum compared to those from the proximal femur may be reflected in differences in bone formation and implant fixation at these sites. Cite this article: Bone Joint Res 2021;10(9):611–618


Bone & Joint Research
Vol. 13, Issue 9 | Pages 462 - 473
6 Sep 2024
Murayama M Chow SK Lee ML Young B Ergul YS Shinohara I Susuki Y Toya M Gao Q Goodman SB

Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes.

Cite this article: Bone Joint Res 2024;13(9):462–473.


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.


Bone & Joint Research
Vol. 12, Issue 1 | Pages 9 - 21
9 Jan 2023
Lu C Ho C Chen S Liu Z Chou PP Ho M Tien Y

Aims

The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction.

Methods

The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and tenogenic genes (scleraxis (SCX), tenascin C (TNC), and tenomodulin (TNMD)) of the hamstring tendons were compared between the co-culture medium (ACL remnant cells (ACLRCs) and BMSCs co-culture) and control medium (BMSCs-only culture). We also evaluated the apoptosis, cell proliferation, migration, and gene expression of hamstring tenocytes with exposure to co-culture and control media.


Bone & Joint Research
Vol. 11, Issue 12 | Pages 854 - 861
1 Dec 2022
Park TJ Park SY Cho W Oh H Lee HJ Abd El-Aty AM Bayram C Jeong JH Jung TW

Aims

Myokine developmental endothelial locus-1 (DEL-1) has been documented to alleviate inflammation and endoplasmic reticulum (ER) stress in various cell types. However, the effects of DEL-1 on inflammation, ER stress, and apoptosis in tenocytes remain unclear.

Methods

Human primary tenocytes were cultured in palmitate (400 μM) and palmitate plus DEL-1 (0 to 2 μg/ml) conditions for 24 hours. The expression levels of ER stress markers and cleaved caspase 3, as well as phosphorylated 5' adenosine monophosphate-activated protein kinase (AMPK) and autophagy markers, were assessed by Western blotting. Autophagosome formation was measured by staining with monodansylcadaverine, and apoptosis was determined by cell viability assay and caspase 3 activity assay.


Aims

Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation.

Methods

Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expression of microRNA (miR)-760 and ankyrin repeat and FYVE domain containing 1 (ANKFY1) in OP tissues and hBMSCs. Cell viability was measured using the Cell Counting Kit-8 assay. The expression of cyclin D1 and osteogenic marker genes (osteocalcin (OCN), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2)) was evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mineral deposits were detected through Alizarin Red S staining. In addition, Western blotting was performed to detect the ANKFY1 protein levels following the regulation of miR-760. The relationship between miR-760 and ANKFY1 was determined using a luciferase reporter assay.


Aims

This study examined whether systemic administration of melatonin would have different effects on osseointegration in ovariectomized (OVX) rats, depending on whether this was administered during the day or night.

Methods

In this study, a titanium rod was implanted in the medullary cavity of one femoral metaphysis in OVX rats, and then the rats were randomly divided into four groups: Sham group (Sham, n = 10), OVX rat group (OVX, n = 10), melatonin day treatment group (OVX + MD, n = 10), and melatonin night treatment group (OVX + MN, n = 10). The OVX + MD and OVX + MN rats were treated with 30 mg/kg/day melatonin at 9 am and 9 pm, respectively, for 12 weeks. At the end of the research, the rats were killed to obtain bilateral femora and blood samples for evaluation.


Bone & Joint Research
Vol. 13, Issue 3 | Pages 91 - 100
1 Mar 2024
Yamamoto Y Fukui T Sawauchi K Yoshikawa R Takase K Kumabe Y Maruo A Niikura T Kuroda R Oe K

Aims

Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 μg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells.

Methods

Cells were isolated from the bone tissue grafts collected from six patients using the Reamer-Irrigator-Aspirator system, and exposed to different gentamicin concentrations. Live cells rate, apoptosis rate, alkaline phosphatase (ALP) activity, expression of osteoblast-related genes, mineralization potential, and restoration of cell viability and ALP activity were examined by in vitro studies.


Bone & Joint Research
Vol. 12, Issue 6 | Pages 375 - 386
12 Jun 2023
Li Z

Aims

Long non-coding RNAs (lncRNAs) act as crucial regulators in osteoporosis (OP). Nonetheless, the effects and potential molecular mechanism of lncRNA PCBP1 Antisense RNA 1 (PCBP1-AS1) on OP remain largely unclear. The aim of this study was to explore the role of lncRNA PCBP1-AS1 in the pathogenesis of OP.

Methods

Using quantitative real-time polymerase chain reaction (qRT-PCR), osteogenesis-related genes (alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), and Runt-related transcription factor 2 (RUNX2)), PCBP1-AS1, microRNA (miR)-126-5p, group I Pak family member p21-activated kinase 2 (PAK2), and their relative expression levels were determined. Western blotting was used to examine the expression of PAK2 protein. Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. To examine the osteogenic differentiation, Alizarin red along with ALP staining was used. RNA immunoprecipitation assay and bioinformatics analysis, as well as a dual-luciferase reporter, were used to study the association between PCBP1-AS1, PAK2, and miR-126-5p.


Bone & Joint Research
Vol. 13, Issue 1 | Pages 28 - 39
10 Jan 2024
Toya M Kushioka J Shen H Utsunomiya T Hirata H Tsubosaka M Gao Q Chow SK Zhang N Goodman SB

Aims

Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice.

Methods

We used a murine model of chronic inflammation induced by continuous intramedullary delivery of lipopolysaccharide-contaminated polyethylene particles (cPE) using an osmotic pump. Specimens were evaluated using micro-CT and histomorphometric analyses. Sex-specific osteogenic and osteoclastic differentiation potentials were also investigated in vitro, including alkaline phosphatase, Alizarin Red, tartrate-resistant acid phosphatase staining, and gene expression using reverse transcription polymerase chain reaction (RT-PCR).


Bone & Joint Research
Vol. 12, Issue 3 | Pages 219 - 230
10 Mar 2023
Wang L Li S Xiao H Zhang T Liu Y Hu J Xu D Lu H

Aims

It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth.

Methods

C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs.


Bone & Joint Research
Vol. 11, Issue 11 | Pages 763 - 776
1 Nov 2022
Zhang Y Jiang B Zhang P Chiu SK Lee MH

Aims

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent matrix metalloproteinases (MMP) and A disintegrin and metalloproteinases (ADAM) involved in extracellular matrix modulation. The present study aims to develop the TIMPs as biologics for osteoclast-related disorders.

Methods

We examine the inhibitory effect of a high affinity, glycosyl-phosphatidylinositol-anchored TIMP variant named ‘T1PrαTACE’ on receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclast differentiation.


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. 11, Issue 11 | Pages 803 - 813
1 Nov 2022
Guan X Gong X Jiao ZY Cao HY Liu S Lin C Huang X Lan H Ma L Xu B

Aims

The involvement of cyclin D1 in the proliferation of microglia, and the generation and maintenance of bone cancer pain (BCP), have not yet been clarified. We investigated the expression of microglia and cyclin D1, and the influences of cyclin D1 on pain threshold.

Methods

Female Sprague Dawley (SD) rats were used to establish a rat model of BCP, and the messenger RNA (mRNA) and protein expression of ionized calcium binding adaptor molecule 1 (IBA1) and cyclin D1 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. The proliferation of spinal microglia was detected by immunohistochemistry. The pain behaviour test was assessed by quantification of spontaneous flinches, limb use, and guarding during forced ambulation, mechanical paw withdrawal threshold, and thermal paw withdrawal latency.


Aims

This study examined the relationship between obesity (OB) and osteoporosis (OP), aiming to identify shared genetic markers and molecular mechanisms to facilitate the development of therapies that target both conditions simultaneously.

Methods

Using weighted gene co-expression network analysis (WGCNA), we analyzed datasets from the Gene Expression Omnibus (GEO) database to identify co-expressed gene modules in OB and OP. These modules underwent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and protein-protein interaction analysis to discover Hub genes. Machine learning refined the gene selection, with further validation using additional datasets. Single-cell analysis emphasized specific cell subpopulations, and enzyme-linked immunosorbent assay (ELISA), protein blotting, and cellular staining were used to investigate key genes.


Bone & Joint Research
Vol. 11, Issue 8 | Pages 548 - 560
17 Aug 2022
Yuan W Yang M Zhu Y

Aims

We aimed to develop a gene signature that predicts the occurrence of postmenopausal osteoporosis (PMOP) by studying its genetic mechanism.

Methods

Five datasets were obtained from the Gene Expression Omnibus database. Unsupervised consensus cluster analysis was used to determine new PMOP subtypes. To determine the central genes and the core modules related to PMOP, the weighted gene co-expression network analysis (WCGNA) was applied. Gene Ontology enrichment analysis was used to explore the biological processes underlying key genes. Logistic regression univariate analysis was used to screen for statistically significant variables. Two algorithms were used to select important PMOP-related genes. A logistic regression model was used to construct the PMOP-related gene profile. The receiver operating characteristic area under the curve, Harrell’s concordance index, a calibration chart, and decision curve analysis were used to characterize PMOP-related genes. Then, quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression of the PMOP-related genes in the gene signature.


Bone & Joint Research
Vol. 11, Issue 5 | Pages 304 - 316
17 May 2022
Kim MH Choi LY Chung JY Kim E Yang WM

Aims

The association of auraptene (AUR), a 7-geranyloxycoumarin, on osteoporosis and its potential pathway was predicted by network pharmacology and confirmed in experimental osteoporotic mice.

Methods

The network of AUR was constructed and a potential pathway predicted by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) terms enrichment. Female ovariectomized (OVX) Institute of Cancer Research mice were intraperitoneally injected with 0.01, 0.1, and 1 mM AUR for four weeks. The bone mineral density (BMD) level was measured by dual-energy X-ray absorptiometry. The bone microstructure was determined by histomorphological changes in the femora. In addition, biochemical analysis of the serum and assessment of the messenger RNA (mRNA) levels of osteoclastic markers were performed.


Bone & Joint Research
Vol. 11, Issue 2 | Pages 121 - 133
22 Feb 2022
Hsu W Lin S Hung J Chen M Lin C Hsu W Hsu WR

Aims

The decrease in the number of satellite cells (SCs), contributing to myofibre formation and reconstitution, and their proliferative capacity, leads to muscle loss, a condition known as sarcopenia. Resistance training can prevent muscle loss; however, the underlying mechanisms of resistance training effects on SCs are not well understood. We therefore conducted a comprehensive transcriptome analysis of SCs in a mouse model.

Methods

We compared the differentially expressed genes of SCs in young mice (eight weeks old), middle-aged (48-week-old) mice with resistance training intervention (MID+ T), and mice without exercise (MID) using next-generation sequencing and bioinformatics.


Bone & Joint Research
Vol. 10, Issue 8 | Pages 488 - 497
10 Aug 2021
Cleemann R Sorensen M West A Soballe K Bechtold JE Baas J

Aims

We wanted to evaluate the effects of a bone anabolic agent (bone morphogenetic protein 2 (BMP-2)) on an anti-catabolic background (systemic or local zoledronate) on fixation of allografted revision implants.

Methods

An established allografted revision protocol was implemented bilaterally into the stifle joints of 24 canines. At revision surgery, each animal received one BMP-2 (5 µg) functionalized implant, and one raw implant. One group (12 animals) received bone graft impregnated with zoledronate (0.005 mg/ml) before impaction. The other group (12 animals) received untreated bone graft and systemic zoledronate (0.1 mg/kg) ten and 20 days after revision surgery. Animals were observed for an additional four weeks before euthanasia.