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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.


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.


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. Results. Compared to BMSCs-only culture medium, the co-culture medium showed substantially decreased early and late apoptosis rates, attenuation of intrinsic and extrinsic apoptotic pathways, and enhanced proliferation of the hamstring tendons and tenocytes. In addition, the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes in the hamstring tendons and tenocytes significantly increased in the co-culture medium compared to that in the control medium. Conclusion. In the presence of ACLRCs and BMSCs, the hamstring tendons and tenocytes significantly attenuated apoptosis and enhanced the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes. This in vitro study suggests that the ACLRCs mixed with BMSCs could aid regeneration of the hamstring tendon graft during ACL reconstruction. Cite this article: Bone Joint Res 2023;12(1):9–21


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. 8, Issue 10 | Pages 472 - 480
1 Oct 2019
Hjorthaug GA Søreide E Nordsletten L Madsen JE Reinholt FP Niratisairak S Dimmen S

Objectives

Experimental studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) may have negative effects on fracture healing. This study aimed to assess the effect of immediate and delayed short-term administration of clinically relevant parecoxib doses and timing on fracture healing using an established animal fracture model.

Methods

A standardized closed tibia shaft fracture was induced and stabilized by reamed intramedullary nailing in 66 Wistar rats. A ‘parecoxib immediate’ (Pi) group received parecoxib (3.2 mg/kg bodyweight twice per day) on days 0, 1, and 2. A ‘parecoxib delayed’ (Pd) group received the same dose of parecoxib on days 3, 4, and 5. A control group received saline only. Fracture healing was evaluated by biomechanical tests, histomorphometry, and dual-energy x-ray absorptiometry (DXA) at four weeks.


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. 8, Issue 8 | Pages 397 - 404
1 Aug 2019
Osagie-Clouard L Sanghani-Kerai A Coathup M Meeson R Briggs T Blunn G

Objectives. Mesenchymal stem cells (MSCs) are of growing interest in terms of bone regeneration. Most preclinical trials utilize bone-marrow-derived mesenchymal stem cells (bMSCs), although this is not without isolation and expansion difficulties. The aim of this study was: to compare the characteristics of bMSCs and adipose-derived mesenchymal stem cells (AdMSCs) from juvenile, adult, and ovarectomized (OVX) rats; and to assess the effect of human parathyroid hormone (hPTH) 1-34 on their osteogenic potential and migration to stromal cell-derived factor-1 (SDF-1). Methods. Cells were isolated from the adipose and bone marrow of juvenile, adult, and previously OVX Wistar rats, and were characterized with flow cytometry, proliferation assays, osteogenic and adipogenic differentiation, and migration to SDF-1. Experiments were repeated with and without intermittent hPTH 1-34. Results. Juvenile and adult MSCs demonstrated significantly increased osteogenic and adipogenic differentiation and superior migration towards SDF-1 compared with OVX groups; this was the case for AdMSCs and bMSCs equally. Parathyroid hormone (PTH) increased parameters of osteogenic differentiation and migration to SDF-1. This was significant for all cell types, although it had the most significant effect on cells derived from OVX animals. bMSCs from all groups showed increased mineralization and migration to SDF-1 compared with AdMSCs. Conclusion. Juvenile MSCs showed significantly greater migration to SDF-1 and significantly greater osteogenic and adipogenic differentiation compared with cells from osteopenic rats; this was true for bMSCs and AdMSCs. The addition of PTH increased these characteristics, with the most significant effect on cells derived from OVX animals, further illustrating possible clinical application of both PTH and MSCs in bone regenerative therapies. Cite this article:L. Osagie-Clouard, A. Sanghani-Kerai, M. Coathup, R. Meeson, T. Briggs, G. Blunn. The influence of parathyroid hormone 1-34 on the osteogenic characteristics of adipose- and bone-marrow-derived mesenchymal stem cells from juvenile and ovarectomized rats. Bone Joint Res 2019;8:397–404. DOI: 10.1302/2046-3758.88.BJR-2019-0018.R1


Objectives

Osteoporosis is a systemic bone metabolic disease, which often occurs among the elderly. Angelica polysaccharide (AP) is the main component of angelica sinensis, and is widely used for treating various diseases. However, the effects of AP on osteoporosis have not been investigated. This study aimed to uncover the functions of AP in mesenchymal stem cell (MSC) proliferation and osteoblast differentiation.

Methods

MSCs were treated with different concentrations of AP, and then cell viability, Cyclin D1 protein level, and the osteogenic markers of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2) were examined by Cell Counting Kit-8 (CCK-8) and western blot assays, respectively. The effect of AP on the main signalling pathways of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin was determined by western blot. Following this, si-H19#1 and si-H19#2 were transfected into MSCs, and the effects of H19 on cell proliferation and osteoblast differentiation in MSCs were studied. Finally, in vivo experimentation explored bone mineral density, bone mineral content, and the ash weight and dry weight of femoral bone.


Bone & Joint Research
Vol. 8, Issue 3 | Pages 107 - 117
1 Mar 2019
Lim ZXH Rai B Tan TC Ramruttun AK Hui JH Nurcombe V Teoh SH Cool SM

Objectives

Long bone defects often require surgical intervention for functional restoration. The ‘gold standard’ treatment is autologous bone graft (ABG), usually from the patient’s iliac crest. However, autograft is plagued by complications including limited supply, donor site morbidity, and the need for an additional surgery. Thus, alternative therapies are being actively investigated. Autologous bone marrow (BM) is considered as a candidate due to the presence of both endogenous reparative cells and growth factors. We aimed to compare the therapeutic potentials of autologous bone marrow aspirate (BMA) and ABG, which has not previously been done.

Methods

We compared the efficacy of coagulated autologous BMA and ABG for the repair of ulnar defects in New Zealand White rabbits. Segmental defects (14 mm) were filled with autologous clotted BM or morcellized autograft, and healing was assessed four and 12 weeks postoperatively. Harvested ulnas were subjected to radiological, micro-CT, histological, and mechanical analyses.


Bone & Joint Research
Vol. 7, Issue 10 | Pages 548 - 560
1 Oct 2018
Qayoom I Raina DB Širka A Tarasevičius Š Tägil M Kumar A Lidgren L

During the last decades, several research groups have used bisphosphonates for local application to counteract secondary bone resorption after bone grafting, to improve implant fixation or to control bone resorption caused by bone morphogenetic proteins (BMPs). We focused on zoledronate (a bisphosphonate) due to its greater antiresorptive potential over other bisphosphonates. Recently, it has become obvious that the carrier is of importance to modulate the concentration and elution profile of the zoledronic acid locally. Incorporating one fifth of the recommended systemic dose of zoledronate with different apatite matrices and types of bone defects has been shown to enhance bone regeneration significantly in vivo. We expect the local delivery of zoledronate to overcome the limitations and side effects associated with systemic usage; however, we need to know more about the bioavailability and the biological effects. The local use of BMP-2 and zoledronate as a combination has a proven additional effect on bone regeneration. This review focuses primarily on the local use of zoledronate alone, or in combination with bone anabolic factors, in various preclinical models mimicking different orthopaedic conditions.

Cite this article: I. Qayoom, D. B. Raina, A. Širka, Š. Tarasevičius, M. Tägil, A. Kumar, L. Lidgren. Anabolic and antiresorptive actions of locally delivered bisphosphonates for bone repair: A review. Bone Joint Res 2018;7:548–560. DOI: 10.1302/2046-3758.710.BJR-2018-0015.R2.


Bone & Joint Research
Vol. 7, Issue 4 | Pages 289 - 297
1 Apr 2018
Sanghani-Kerai A Osagie-Clouard L Blunn G Coathup M

Objectives. This study aimed to assess the effect of age and osteoporosis on the proliferative and differentiating capacity of bone-marrow-derived mesenchymal stem cells (MSCs) in female rats. We also discuss the role of these factors on expression and migration of cells along the C-X-C chemokine receptor type 4 (CXCR-4) / stromal derived factor 1 (SDF-1) axis. Methods. Mesenchymal stem cells were harvested from the femora of young, adult, and osteopenic Wistar rats. Cluster of differentiation (CD) marker and CXCR-4 expression was measured using flow cytometry. Cellular proliferation was measured using Alamar Blue, osteogenic differentiation was measured using alkaline phosphatase expression and alizarin red production, and adipogenic differentiation was measured using Oil red O. Cells were incubated in Boyden chambers to quantify their migration towards SDF-1. Data was analyzed using a Student’s t-test, where p-values < 0.05 were considered significant. Results. CD marker expression and proliferation of the MSCs from the three groups was not significantly different. The young MSCs demonstrated significantly increased differentiation into bone and fat and superior migration towards SDF-1. The migration of SDF-1 doubled with young rats compared with the adult rats (p = 0.023) and it was four times higher when compared with cells isolated from ovariectomized (OVX) osteopenic rats (p = 0.013). Conclusion. Young rat MSCs are significantly more responsive to osteogenic differentiation, and, contrary to other studies, also demonstrated increased adipogenic differentiation compared with cells from adult and ostopenic rats. Young-rat-derived cells also showed superior migration towards SDF-1 compared with MSCs from OVX and adult control rats. Cite this article: A. Sanghani-Kerai, L. Osagie-Clouard, G. Blunn, M. Coathup. The influence of age and osteoporosis on bone marrow stem cells from rats. Bone Joint Res 2018;7:289–297. DOI: 10.1302/2046-3758.74.BJR-2017-0302.R1


Bone & Joint Research
Vol. 7, Issue 4 | Pages 263 - 273
1 Apr 2018
Ferreira E Porter RM

Large bone defects remain a tremendous clinical challenge. There is growing evidence in support of treatment strategies that direct defect repair through an endochondral route, involving a cartilage intermediate. While culture-expanded stem/progenitor cells are being evaluated for this purpose, these cells would compete with endogenous repair cells for limited oxygen and nutrients within ischaemic defects. Alternatively, it may be possible to employ extracellular vesicles (EVs) secreted by culture-expanded cells for overcoming key bottlenecks to endochondral repair, such as defect vascularization, chondrogenesis, and osseous remodelling. While mesenchymal stromal/stem cells are a promising source of therapeutic EVs, other donor cells should also be considered. The efficacy of an EV-based therapeutic will likely depend on the design of companion scaffolds for controlled delivery to specific target cells. Ultimately, the knowledge gained from studies of EVs could one day inform the long-term development of synthetic, engineered nanovesicles. In the meantime, EVs harnessed from in vitro cell culture have near-term promise for use in bone regenerative medicine. This narrative review presents a rationale for using EVs to improve the repair of large bone defects, highlights promising cell sources and likely therapeutic targets for directing repair through an endochondral pathway, and discusses current barriers to clinical translation.

Cite this article: E. Ferreira, R. M. Porter. Harnessing extracellular vesicles to direct endochondral repair of large bone defects. Bone Joint Res 2018;7:263–273. DOI: 10.1302/2046-3758.74.BJR-2018-0006.


Bone & Joint Research
Vol. 7, Issue 2 | Pages 187 - 195
1 Feb 2018
Ziebart J Fan S Schulze C Kämmerer PW Bader R Jonitz-Heincke A

Objectives

Enhanced micromotions between the implant and surrounding bone can impair osseointegration, resulting in fibrous encapsulation and aseptic loosening of the implant. Since the effect of micromotions on human bone cells is sparsely investigated, an in vitro system, which allows application of micromotions on bone cells and subsequent investigation of bone cell activity, was developed.

Methods

Micromotions ranging from 25 µm to 100 µm were applied as sine or triangle signal with 1 Hz frequency to human osteoblasts seeded on collagen scaffolds. Micromotions were applied for six hours per day over three days. During the micromotions, a static pressure of 527 Pa was exerted on the cells by Ti6Al4V cylinders. Osteoblasts loaded with Ti6Al4V cylinders and unloaded osteoblasts without micromotions served as controls. Subsequently, cell viability, expression of the osteogenic markers collagen type I, alkaline phosphatase, and osteocalcin, as well as gene expression of osteoprotegerin, receptor activator of NF-κB ligand, matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-1, were investigated.


Bone & Joint Research
Vol. 7, Issue 2 | Pages 139 - 147
1 Feb 2018
Takahara S Lee SY Iwakura T Oe K Fukui T Okumachi E Waki T Arakura M Sakai Y Nishida K Kuroda R Niikura T

Objectives

Diabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM.

Methods

Closed transverse fractures were created in the femurs of 116 rats, with half assigned to the DM group and half assigned to the control group. Rats with DM were induced by a single intraperitoneal injection of streptozotocin. At post-fracture days five, seven, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture days five and 11. For further analysis, real-time polymerase chain reaction (PCR) analysis was performed at each timepoint.