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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 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.


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 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 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.


Bone & Joint Research
Vol. 9, Issue 10 | Pages 667 - 674
1 Oct 2020
Antich-Rosselló M Forteza-Genestra MA Calvo J Gayà A Monjo M Ramis JM

Aims

Platelet concentrates, like platelet-rich plasma (PRP) and platelet lysate (PL), are widely used in regenerative medicine, especially in bone regeneration. However, the lack of standard procedures and controls leads to high variability in the obtained results, limiting their regular clinical use. Here, we propose the use of platelet-derived extracellular vesicles (EVs) as an off-the-shelf alternative for PRP and PL for bone regeneration. In this article, we evaluate the effect of PL-derived EVs on the biocompatibility and differentiation of mesenchymal stromal cells (MSCs).

Methods

EVs were obtained first by ultracentrifugation (UC) and then by size exclusion chromatography (SEC) from non-activated PL. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and the expression of CD9 and CD63 markers by western blot. The effect of the obtained EVs on osteoinduction was evaluated in vitro on human umbilical cord MSCs by messenger RNA (mRNA) expression analysis of bone markers, alkaline phosphatase activity (ALP), and calcium (Ca2+) content.


Bone & Joint Research
Vol. 8, Issue 10 | Pages 481 - 488
1 Oct 2019
Nathan K Lu LY Lin T Pajarinen J Jämsen E Huang J Romero-Lopez M Maruyama M Kohno Y Yao Z Goodman SB

Objectives

Up to 10% of fractures result in undesirable outcomes, for which female sex is a risk factor. Cellular sex differences have been implicated in these different healing processes. Better understanding of the mechanisms underlying bone healing and sex differences in this process is key to improved clinical outcomes. This study utilized a macrophage–mesenchymal stem cell (MSC) coculture system to determine: 1) the precise timing of proinflammatory (M1) to anti-inflammatory (M2) macrophage transition for optimal bone formation; and 2) how such immunomodulation was affected by male versus female cocultures.

Methods

A primary murine macrophage-MSC coculture system was used to demonstrate the optimal transition time from M1 to M2 (polarized from M1 with interleukin (IL)-4) macrophages to maximize matrix mineralization in male and female MSCs. Outcome variables included Alizarin Red staining, alkaline phosphatase (ALP) activity, and osteocalcin protein secretion.


Bone & Joint Research
Vol. 7, Issue 1 | Pages 105 - 110
1 Jan 2018
Abar O Dharmar S Tang SY

Objectives

Advanced glycation end-products (AGEs) are a post-translational modification of collagen that form spontaneously in the skeletal matrix due to the presence of reducing sugars, such as glucose. The accumulation of AGEs leads to collagen cross-linking, which adversely affects bone quality and has been shown to play a major role in fracture risk. Thus, intervening in the formation and accumulation of AGEs may be a viable means of protecting bone quality.

Methods

An in vitro model was used to examine the efficacy of two AGE-inhibitors, aminoguanidine (AG) and pyridoxamine (PM), on ageing human cortical bone. Mid-diaphyseal tibial cortical bone segments were obtained from female cadavers (n = 20, age range: 57 years to 97 years) and randomly subjected to one of four treatments: control; glucose only; glucose and AG; or glucose and PM. Following treatment, each specimen underwent mechanical testing under physiological conditions via reference point indentation, and AGEs were quantified by fluorescence.