Publications by authors named "Stan Gronthos"

156 Publications

Craniomaxillofacial morphology in a murine model of ephrinB1 conditional deletion in osteoprogenitor cells.

Arch Oral Biol 2022 May 4;137:105389. Epub 2022 Mar 4.

Adelaide Dental School, The University of Adelaide, Adelaide, SA, Australia; South Australian Health and Medical Research Institute, Adelaide, SA, Australia; Mesenchymal Stem Cell Laboratory, School of Biomedicine, The University of Adelaide, Adelaide, SA, Australia; Cleft and Craniofacial SA, Women's and Children's Hospital, Adelaide, SA, Australia.

Objective: EFNB1 mutation causes craniofrontonasal dysplasia (CFND), a congenital syndrome associated with craniomaxillofacial anomalies characterised by coronal craniosynostosis, orbital hypertelorism, and midface dysplasia. The aim of this murine study was to investigate the effect of the EfnB1 conditional gene deletion in osteoprogenitor cells on the craniomaxillofacial skeletal morphology.

Design: The skulls of male and female mice, in which EfnB1 was deleted by Cre (a site-specific DNA recombinase) under the control of the Osterix (Osx) promoter (EfnB1), were compared to those without EfnB1 deletion (Osx:Cre control) at two ages (4 and 8 weeks; n = 6 per group). The three-dimensional micro-computed tomography reconstructions were prepared to calculate 17 linear measurements in the cranial vault (brain box), midface and mandible. Coronal and sagittal sutures from the 8-week-old mice were also subjected to histological examination.

Results: EfnB1 mice displayed significantly larger cranial height, larger interorbital and nasal widths, smaller maxillary width than controls by 8 weeks (p < 0.05), but mandibular size was not significantly different (p > 0.05). Binomial testing showed significantly smaller EfnB1 skulls at 4 weeks but larger at 8 weeks (p < 0.05). Histological examination revealed increased bony fusion and fibrous connective tissue deposition at the coronal suture of EfnB1-/- mice compared with controls.

Conclusions: Craniofacial phenotype of the murine model of EfnB1 deletion in osteoprogenitor cells partially represents the human CFND phenotype, with implications for better understanding mechanisms involved in skeletal morphogenesis and malocclusion.
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http://dx.doi.org/10.1016/j.archoralbio.2022.105389DOI Listing
May 2022

Generation of two multipotent mesenchymal progenitor cell lines capable of osteogenic, mature osteocyte, adipogenic, and chondrogenic differentiation.

Sci Rep 2021 11 19;11(1):22593. Epub 2021 Nov 19.

Indiana Center for Musculoskeletal Health, Indiana University, Indianapolis, IN, 46202, USA.

Mesenchymal progenitors differentiate into several tissues including bone, cartilage, and adipose. Targeting these cells in vivo is challenging, making mesenchymal progenitor cell lines valuable tools to study tissue development. Mesenchymal stem cells (MSCs) can be isolated from humans and animals; however, obtaining homogenous, responsive cells in a reproducible fashion is challenging. As such, we developed two mesenchymal progenitor cell (MPC) lines, MPC1 and MPC2, generated from bone marrow of male C57BL/6 mice. These cells were immortalized using the temperature sensitive large T-antigen, allowing for thermal control of proliferation and differentiation. Both MPC1 and MPC2 cells are capable of osteogenic, adipogenic, and chondrogenic differentiation. Under osteogenic conditions, both lines formed mineralized nodules, and stained for alizarin red and alkaline phosphatase, while expressing osteogenic genes including Sost, Fgf23, and Dmp1. Sost and Dmp1 mRNA levels were drastically reduced with addition of parathyroid hormone, thus recapitulating in vivo responses. MPC cells secreted intact (iFGF23) and C-terminal (cFGF23) forms of the endocrine hormone FGF23, which was upregulated by 1,25 dihydroxy vitamin D (1,25D). Both lines also rapidly entered the adipogenic lineage, expressing adipose markers after 4 days in adipogenic media. MPC cells were also capable of chondrogenic differentiation, displaying increased expression of cartilaginous genes including aggrecan, Sox9, and Comp. With the ability to differentiate into multiple mesenchymal lineages and mimic in vivo responses of key regulatory genes/proteins, MPC cells are a valuable model to study factors that regulate mesenchymal lineage allocation as well as the mechanisms that dictate transcription, protein modification, and secretion of these factors.
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http://dx.doi.org/10.1038/s41598-021-02060-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8605002PMC
November 2021

Biodegradable and biocompatible graphene-based scaffolds for functional neural tissue engineering: A strategy approach using dental pulp stem cells and biomaterials.

Biotechnol Bioeng 2021 11 28;118(11):4217-4230. Epub 2021 Jul 28.

Neil Sachse Centre for Spinal Cord Research, Lifelong Health Theme, South Australian Health and Medical Research Institute (SAHMRI), Adelaide, Australia.

Neural tissue engineering aims to restore the function of nervous system tissues using biocompatible cell-seeded scaffolds. Graphene-based scaffolds combined with stem cells deserve special attention to enhance tissue regeneration in a controlled manner. However, it is believed that minor changes in scaffold biomaterial composition, internal porous structure, and physicochemical properties can impact cellular growth and adhesion. The current work aims to investigate in vitro biological effects of three-dimensional (3D) graphene oxide (GO)/sodium alginate (GOSA) and reduced GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in terms of cell viability and cytotoxicity. Herein, the effects of the 3D scaffolds, coating conditions, and serum supplementation on DPSCs functions are explored extensively. Biodegradation analysis revealed that the addition of GO enhanced the degradation rate of composite scaffolds. Compared to the 2D surface, the cell viability of 3D scaffolds was higher (p < 0.0001), highlighting the optimal initial cell adhesion to the scaffold surface and cell migration through pores. Moreover, the cytotoxicity study indicated that the incorporation of graphene supported higher DPSCs viability. It is also shown that when the mean pore size of the scaffold increases, DPSCs activity decreases. In terms of coating conditions, poly- l-lysine was the most robust coating reagent that improved cell-scaffold adherence and DPSCs metabolism activity. The cytotoxicity of GO-based scaffolds showed that DPSCs can be seeded in serum-free media without cytotoxic effects. This is critical for human translation as cellular transplants are typically serum-free. These findings suggest that proposed 3D GO-based scaffolds have favorable effects on the biological responses of DPSCs.
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http://dx.doi.org/10.1002/bit.27891DOI Listing
November 2021

Plant-derived soybean peroxidase stimulates osteoblast collagen biosynthesis, matrix mineralization, and accelerates bone regeneration in a sheep model.

Bone Rep 2021 Jun 27;14:101096. Epub 2021 May 27.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, Australia.

Bone defects arising from fractures or disease represent a significant problem for surgeons to manage and are a substantial economic burden on the healthcare economy. Recent advances in the development of biomaterial substitutes provides an attractive alternative to the current "gold standard" autologous bone grafting. Despite on-going research, we are yet to identify cost effective biocompatible, osteo-inductive factors that stimulate controlled, accelerated bone regeneration.We have recently reported that enzymes with peroxidase activity possess previously unrecognised roles in extracellular matrix biosynthesis, angiogenesis and osteoclastogenesis, which are essential processes in bone remodelling and repair. Here, we report for the first time, that plant-derived soybean peroxidase (SBP) possesses pro-osteogenic ability by promoting collagen I biosynthesis and matrix mineralization of human osteoblasts . Mechanistically, SBP regulates osteogenic genes responsible for inflammation, extracellular matrix remodelling and ossification, which are necessary for normal bone healing. Furthermore, SBP was shown to have osteo-inductive properties, that when combined with commercially available biphasic calcium phosphate (BCP) granules can accelerate bone repair in a critical size long bone defect ovine model. Micro-CT analysis showed that SBP when combined with commercially available biphasic calcium phosphate (BCP) granules significantly increased bone formation within the defects as early as 4 weeks compared to BCP alone. Histomorphometric assessment demonstrated accelerated bone formation prominent at the defect margins and surrounding individual BCP granules, with evidence of intramembranous ossification. These results highlight the capacity of SBP to be an effective regulator of osteoblastic function and may be beneficial as a new and cost effective osteo-inductive agent to accelerate repair of large bone defects.
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http://dx.doi.org/10.1016/j.bonr.2021.101096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178086PMC
June 2021

Eph-Ephrin Signaling Mediates Cross-Talk Within the Bone Microenvironment.

Front Cell Dev Biol 2021 9;9:598612. Epub 2021 Feb 9.

Mesenchymal Stem Cell Laboratory, Faculty of Health and Medical Sciences, Adelaide Medical School, University of Adelaide, Adelaide, SA, Australia.

Skeletal integrity is maintained through the tightly regulated bone remodeling process that occurs continuously throughout postnatal life to replace old bone and to repair skeletal damage. This is maintained primarily through complex interactions between bone resorbing osteoclasts and bone forming osteoblasts. Other elements within the bone microenvironment, including stromal, osteogenic, hematopoietic, endothelial and neural cells, also contribute to maintaining skeletal integrity. Disruption of the dynamic interactions between these diverse cellular systems can lead to poor bone health and an increased susceptibility to skeletal diseases including osteopenia, osteoporosis, osteoarthritis, osteomalacia, and major fractures. Recent reports have implicated a direct role for the Eph tyrosine kinase receptors and their ephrin ligands during bone development, homeostasis and skeletal repair. These membrane-bound molecules mediate contact-dependent signaling through both the Eph receptors, termed , and through the ephrin ligands, referred to as . This review will focus on Eph/ ephrin cross-talk as mediators of hematopoietic and stromal cell communication, and how these interactions contribute to blood/ bone marrow function and skeletal integrity during normal steady state or pathological conditions.
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http://dx.doi.org/10.3389/fcell.2021.598612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902060PMC
February 2021

Clinical Application of Bone Marrow Mesenchymal Stem/Stromal Cells to Repair Skeletal Tissue.

Int J Mol Sci 2020 Dec 21;21(24). Epub 2020 Dec 21.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA 5001, Australia.

There has been an escalation in reports over the last decade examining the efficacy of bone marrow derived mesenchymal stem/stromal cells (BMSC) in bone tissue engineering and regenerative medicine-based applications. The multipotent differentiation potential, myelosupportive capacity, anti-inflammatory and immune-modulatory properties of BMSC underpins their versatile nature as therapeutic agents. This review addresses the current limitations and challenges of exogenous autologous and allogeneic BMSC based regenerative skeletal therapies in combination with bioactive molecules, cellular derivatives, genetic manipulation, biocompatible hydrogels, solid and composite scaffolds. The review highlights the current approaches and recent developments in utilizing endogenous BMSC activation or exogenous BMSC for the repair of long bone and vertebrae fractures due to osteoporosis or trauma. Current advances employing BMSC based therapies for bone regeneration of craniofacial defects is also discussed. Moreover, this review discusses the latest developments utilizing BMSC therapies in the preclinical and clinical settings, including the treatment of bone related diseases such as Osteogenesis Imperfecta.
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http://dx.doi.org/10.3390/ijms21249759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767389PMC
December 2020

Human gingival fibroblast secretome accelerates wound healing through anti-inflammatory and pro-angiogenic mechanisms.

NPJ Regen Med 2020 Dec 10;5(1):24. Epub 2020 Dec 10.

Future Industries Institute, University of South Australia, Adelaide, SA, 5000, Australia.

Healing of the skin and oral mucosa utilises similar mechanisms of tissue repair, however, scarring and the rate of wound closure is vastly superior in the oral cavity suggesting differences between these two environments. One key difference is the phenotype of dermal fibroblasts compared to fibroblasts of gingival tissues. Human gingival fibroblasts (hGFs) are undifferentiated cells with multi-differentiation and self-renewal capacities. This study aimed to examine if delivering hGFs or their secretome, contained in hGF-conditioned media (hGF-CM), would improve healing of the skin and recapitulate features of oral healing. Human fibroblasts, keratinocytes and endothelial cells were first treated with hGF-CM and showed improved migration, proliferation and angiogenic functions. A significant reduction in macroscopic wound area and histologic dermal wound width, as well as an increased rate of re-epithelialisation, were observed in both hGFs and hGF-CM treated murine excisional wounds. This improvement was associated with reduced inflammation, increased angiogenesis and elevated collagen deposition. These findings demonstrate that treatment of dermal wounds with either hGFs or hGF-CM may provide beneficial gingival-like properties to dermal wounds and may be a potential opportunity for improving healing of the skin.
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http://dx.doi.org/10.1038/s41536-020-00109-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728777PMC
December 2020

Pharmacological targeting of KDM6A and KDM6B, as a novel therapeutic strategy for treating craniosynostosis in Saethre-Chotzen syndrome.

Stem Cell Res Ther 2020 12 9;11(1):529. Epub 2020 Dec 9.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, South Australia, Australia.

Background: During development, excessive osteogenic differentiation of mesenchymal progenitor cells (MPC) within the cranial sutures can lead to premature suture fusion or craniosynostosis, leading to craniofacial and cognitive issues. Saethre-Chotzen syndrome (SCS) is a common form of craniosynostosis, caused by TWIST-1 gene mutations. Currently, the only treatment option for craniosynostosis involves multiple invasive cranial surgeries, which can lead to serious complications.

Methods: The present study utilized Twist-1 haploinsufficient (Twist-1) mice as SCS mouse model to investigate the inhibition of Kdm6a and Kdm6b activity using the pharmacological inhibitor, GSK-J4, on calvarial cell osteogenic potential.

Results: This study showed that the histone methyltransferase EZH2, an osteogenesis inhibitor, is downregulated in calvarial cells derived from Twist-1 mice, whereas the counter histone demethylases, Kdm6a and Kdm6b, known promoters of osteogenesis, were upregulated. In vitro studies confirmed that siRNA-mediated inhibition of Kdm6a and Kdm6b expression suppressed osteogenic differentiation of Twist-1 calvarial cells. Moreover, pharmacological targeting of Kdm6a and Kdm6b activity, with the inhibitor, GSK-J4, caused a dose-dependent suppression of osteogenic differentiation by Twist-1 calvarial cells in vitro and reduced mineralized bone formation in Twist-1 calvarial explant cultures. Chromatin immunoprecipitation and Western blot analyses found that GSK-J4 treatment elevated the levels of the Kdm6a and Kdm6b epigenetic target, the repressive mark of tri-methylated lysine 27 on histone 3, on osteogenic genes leading to repression of Runx2 and Alkaline Phosphatase expression. Pre-clinical in vivo studies showed that local administration of GSK-J4 to the calvaria of Twist-1 mice prevented premature suture fusion and kept the sutures open up to postnatal day 20.

Conclusion: The inhibition of Kdm6a and Kdm6b activity by GSK-J4 could be used as a potential non-invasive therapeutic strategy for preventing craniosynostosis in children with SCS. Pharmacological targeting of Kdm6a/b activity can alleviate craniosynostosis in Saethre-Chotzen syndrome. Aberrant osteogenesis by Twist-1 mutant cranial suture mesenchymal progenitor cells occurs via deregulation of epigenetic modifiers Ezh2 and Kdm6a/Kdm6b. Suppression of Kdm6a- and Kdm6b-mediated osteogenesis with GSK-J4 inhibitor can prevent prefusion of cranial sutures.
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http://dx.doi.org/10.1186/s13287-020-02051-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726873PMC
December 2020

Crosstalk between skeletal and neural tissues is critical for skeletal health.

Bone 2021 01 16;142:115645. Epub 2020 Sep 16.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia; Precision Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, SA, Australia. Electronic address:

Emerging evidence in the literature describes a physical and functional association between the neural and skeletal systems that forms a neuro-osteogenic network. This communication between bone cells and neural tissues within the skeleton is important in facilitating bone skeletal growth, homeostasis and repair. The growth and repair of the skeleton is dependent on correct neural innervation for correct skeletal developmental growth and fracture repair, while pathological conditions such as osteoporosis are accelerated by disruptions to sympathetic innervation. To date, different molecular mechanisms have been reported to mediate communication between bone and neural populations. This review highlights the important role of various cell surface receptors, cytokines and associated ligands as potential regulators of skeletal development, homeostasis, and repair, by mediating interactions between the skeletal and nervous systems. Specifically, this review describes how Bone Morphogenetic Proteins (BMPs), Eph/ephrin, Chemokine CXCL12, Calcitonin Gene-related Peptide (CGRP), Netrins, Neurotrophins (NTs), Slit/Robo and the Semaphorins (Semas) contribute to the cross talk between bone cells and peripheral nerves, and the importance of these interactions in maintaining skeletal health.
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http://dx.doi.org/10.1016/j.bone.2020.115645DOI Listing
January 2021

Epigenetic Regulators of Mesenchymal Stem/Stromal Cell Lineage Determination.

Curr Osteoporos Rep 2020 10;18(5):597-605

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia.

Purpose Of Review: Although many signalling pathways have been discovered to be essential in mesenchymal stem/stromal (MSC) differentiation, it has become increasingly clear in recent years that epigenetic regulation of gene transcription is a vital component of lineage determination, encompassing diet, lifestyle and parental influences on bone, fat and cartilage development.

Recent Findings: This review discusses how specific enzymes that modify histone methylation and acetylation or DNA methylation orchestrate the differentiation programs in lineage determination of MSC and the epigenetic changes that facilitate development of bone related diseases such as osteoporosis. The review also describes how environmental factors such as mechanical loading influence the epigenetic signatures of MSC, and how the use of chemical agents or small peptides can regulate epigenetic drift in MSC populations during ageing and disease. Epigenetic regulation of MSC lineage commitment is controlled through changes in enzyme activity, which modifies DNA and histone residues leading to alterations in chromatin structure. The co-ordinated epigenetic regulation of transcriptional activation and repression act to mediate skeletal tissue homeostasis, where deregulation of this process can lead to bone loss during ageing or osteoporosis.
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http://dx.doi.org/10.1007/s11914-020-00616-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532954PMC
October 2020

Ageing human bone marrow mesenchymal stem cells have depleted NAD(P)H and distinct multispectral autofluorescence.

Geroscience 2021 04 13;43(2):859-868. Epub 2020 Aug 13.

ARC Centre of Excellence in Nanoscale Biophotonics, Graduate School of Biomedical Engineering, The University of New South Wales, Sydney, New South Wales, 2052, Australia.

Stem cell exhaustion plays a major role in the ageing of different tissues. Similarly, in vitro cell ageing during expansion prior to their use in regenerative medicine can severely compromise stem cell quality through progressive declines in differentiation and growth capacity. We utilized non-destructive multispectral assessment of native cell autofluorescence to investigate the metabolic mechanisms of in vitro mesenchymal stem cell (MSC) ageing in human bone marrow MSCs over serial passages (P2-P10). The spectral signals for NAD(P)H, flavins and protein-bound NAD(P)H were successfully isolated using Robust Dependent Component Analysis (RoDECA). NAD(P)H decreased over the course of hMSC ageing in absolute terms as well as relative to flavins (optical redox ratio). Relative changes in other fluorophore levels (flavins, protein-bound NAD(P)H) suggested that this reduction was due to nicotinamide adenine dinucleotide depletion rather than a metabolic shift from glycolysis to oxidative phosphorylation. Using multispectral features, which are determined without cell fixation or fluorescent labelling, we developed and externally validated a reliable, linear model which could accurately categorize the age of culture-expanded hMSCs. The largest shift in spectral characteristics occurs early in hMSC ageing. These findings demonstrate the feasibility of applying multispectral technology for the non-invasive monitoring of MSC health in vitro.
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http://dx.doi.org/10.1007/s11357-020-00250-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110641PMC
April 2021

HOPX regulates bone marrow-derived mesenchymal stromal cell fate determination via suppression of adipogenic gene pathways.

Sci Rep 2020 07 9;10(1):11345. Epub 2020 Jul 9.

Mesenchymal Stem Cell Laboratory, South Australian Health and Medical Research Institute, North Terrace, Level 5, Adelaide, SA, 5001, Australia.

Previous studies of global binding patterns identified the epigenetic factor, EZH2, as a regulator of the homeodomain-only protein homeobox (HOPX) gene expression during bone marrow stromal cell (BMSC) differentiation, suggesting a potential role for HOPX in regulating BMSC lineage specification. In the present study, we confirmed that EZH2 direct binds to the HOPX promoter region, during normal growth and osteogenic differentiation but not under adipogenic inductive conditions. HOPX gene knockdown and overexpression studies demonstrated that HOPX is a promoter of BMSC proliferation and an inhibitor of adipogenesis. However, functional studies failed to observe any affect by HOPX on BMSC osteogenic differentiation. RNA-seq analysis of HOPX overexpressing BMSC during adipogenesis, found HOPX function to be acting through suppression of adipogenic pathways associated genes such as ADIPOQ, FABP4, PLIN1 and PLIN4. These findings suggest that HOPX gene target pathways are critical factors in the regulation of fat metabolism.
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http://dx.doi.org/10.1038/s41598-020-68261-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347885PMC
July 2020

The changing epigenetic landscape of Mesenchymal Stem/Stromal Cells during aging.

Bone 2020 08 20;137:115440. Epub 2020 May 20.

Mesenchymal Stem Cell Laboratory, School of Medical Sciences, Faculty of Health Sciences, The University of Adelaide, Adelaide, SA, Australia; South Australian Health and Medical Research Institute, Adelaide, SA, Australia. Electronic address:

There is mounting evidence in the literature that mesenchymal stromal/stem cell (MSC) like populations derived from different tissues, undergo epigenetic changes during aging, leading to compromised connective tissue integrity and function. This body of work has linked the biological aging of MSC to changes in their epigenetic signatures affecting growth, lifespan, self-renewal and multi-potential, due to deregulation of processes such as cellular senescence, oxidative stress, DNA damage, telomere shortening and DNA damage. This review addresses recent findings examining DNA methylation, histone modifications and miRNA changes in aging MSC populations. Moreover, we explore how epigenetic factors alter cellular pathways and associated biological networks, contributing to the MSC aging phenotype. Finally we discuss the crucial areas requiring a greater understanding of these processes, in order to piece together a global picture of the changing epigenetic landscape in MSC during aging.
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http://dx.doi.org/10.1016/j.bone.2020.115440DOI Listing
August 2020

CMTM8 Is a Suppressor of Human Mesenchymal Stem Cell Osteogenic Differentiation and Promoter of Proliferation Via EGFR Signaling.

Stem Cells Dev 2020 07 4;29(13):823-834. Epub 2020 May 4.

South Australian Health and Medical Research Institute (SAHMRI), Adelaide, Australia.

Multipotent bone marrow-derived mesenchymal stem/stromal cells (BMSCs) exhibit a finite life span after ex vivo expansion leading to cellular senescence. Many factors can contribute to this. Recently, our group has identified for the first time expression of the chemokine-like factor superfamily 8 (CMTM8) gene in cultured human BMSCs. In this study, we examine the role of CMTM8 in BMSC proliferation, migration, and differentiation. Functional studies using siRNA-mediated knockdown of in human BMSCs resulted in decreased capacity to undergo proliferation and migration and an increased capacity for osteogenic differentiation in vitro. Furthermore, reduced levels led to a decrease in the epidermal growth factor receptor (EGFR) signaling pathway during BMSC proliferation and migration, respectively. Supportive studies using retroviral mediated enforced expression of in BMSC resulted in an increased capacity for proliferation and migration but a decreased osteogenic differentiation potential. Collectively, these data suggest that CMTM8 promotes BMSC proliferation and BMSC migration through the EGFR/ERK1/2 pathway. This study provides insight into novel regulatory mechanisms of human BMSC growth and cell fate determination.
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http://dx.doi.org/10.1089/scd.2020.0007DOI Listing
July 2020

Twist-1 is upregulated by NSD2 and contributes to tumour dissemination and an epithelial-mesenchymal transition-like gene expression signature in t(4;14)-positive multiple myeloma.

Cancer Lett 2020 04 31;475:99-108. Epub 2020 Jan 31.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia; Precision Medicine Theme, South Australian Health & Medical Research Institute (SAHMRI), Adelaide, Australia. Electronic address:

Approximately 15% of patients with multiple myeloma (MM) harbour the t(4;14) chromosomal translocation, leading to the overexpression of the histone methyltransferase NSD2. Patients with this translocation display increased tumour dissemination, accelerated disease progression and rapid relapse. Using publicly available gene expression profile data from NSD2 (n = 135) and NSD2 (n = 878) MM patients, we identified 39 epithelial-mesenchymal transition (EMT)-associated genes which are overexpressed in NSD2 MM plasma cells. In addition, our analyses identified Twist-1 as a key transcription factor upregulated in NSD2 MM patients and t(4;14)-positive cell lines. Overexpression and knockdown studies confirmed that Twist-1 is involved in driving the expression of EMT-associated genes in the human MM cell line KMS11 and promoted the migration of myeloma cell lines in vitro. Notably, Twist-1 overexpression in the mouse MM cell line 5TGM1 significantly increased tumour dissemination in an intratibial tumour model. These findings demonstrate that Twist-1, downstream of NSD2, contributes to the induction of an EMT-like signature in t(4;14)-positive MM and enhances the dissemination of MM plasma cells in vivo, which may, in part, explain the aggressive disease features associated with t(4;14)-positive MM.
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http://dx.doi.org/10.1016/j.canlet.2020.01.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829063PMC
April 2020

Non-destructive, label free identification of cell cycle phase in cancer cells by multispectral microscopy of autofluorescence.

BMC Cancer 2019 Dec 21;19(1):1242. Epub 2019 Dec 21.

Department of Physics and Astronomy, Macquarie University, North Ryde, New South Wales, 2109, Australia.

Background: Cell cycle analysis is important for cancer research. However, available methodologies have drawbacks including limited categorisation and reliance on fixation, staining or transformation. Multispectral analysis of endogenous cell autofluorescence has been shown to be sensitive to changes in cell status and could be applied to the discrimination of cell cycle without these steps.

Methods: Cells from the MIA-PaCa-2, PANC-1, and HeLa cell lines were plated on gridded dishes and imaged using a multispectral fluorescence microscope. They were then stained for proliferating cell nuclear antigen (PCNA) and DNA intensity as a reference standard for their cell cycle position (G1, S, G2, M). The multispectral data was split into training and testing datasets and models were generated to discriminate between G1, S, and G2 + M phase cells. A standard decision tree classification approach was taken, and a two-step system was generated for each line.

Results: Across cancer cell lines accuracy ranged from 68.3% (MIA-PaCa-2) to 73.3% (HeLa) for distinguishing G1 from S and G2 + M, and 69.0% (MIA-PaCa-2) to 78.0% (PANC1) for distinguishing S from G2 + M. Unmixing the multispectral data showed that the autofluorophores NADH, FAD, and PPIX had significant differences between phases. Similarly, the redox ratio and the ratio of protein bound to free NADH were significantly affected.

Conclusions: These results demonstrate that multispectral microscopy could be used for the non-destructive, label free discrimination of cell cycle phase in cancer cells. They provide novel information on the mechanisms of cell-cycle progression and control, and have practical implications for oncology research.
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http://dx.doi.org/10.1186/s12885-019-6463-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925881PMC
December 2019

Conditional knockout of ephrinB1 in osteogenic progenitors delays the process of endochondral ossification during fracture repair.

Bone 2020 03 19;132:115189. Epub 2019 Dec 19.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia; Cancer Theme, South Australian Health and Medical Research Institute, Adelaide, SA, Australia. Electronic address:

The Eph receptor tyrosine kinase ligand, ephrinB1 (EfnB1) is important for correct skeletal and cartilage development, however, the role of EfnB1 in fracture repair is unknown. This study investigated the role of EfnB1 during fracture repair where EfnB1 expression increased significantly at 1 and 2 weeks post fracture in C57Bl/6 wildtype mice, coinciding with the haematoma, soft callus formation/remodelling stages, respectively. To investigate the specific role of EfnB1 within the osteogenic lineage during fracture repair, male mice with a conditional deletion of EfnB1 in the osteogenic lineage (EfnB1), driven by the Osterix (Osx) promoter, and their male Osx:Cre counterparts were subject to a femoral fracture with internal fixation. Two weeks post fracture micro computed tomography (μCT) analysis revealed that EfnB1 mice displayed a significant decrease in bone volume relative to tissue volume within the fracture callus. This was attributed to an alteration in the distribution of osteoclasts within the fracture site, a significant elevation in cartilaginous tissue and reduction in the osteoprogenitor population and calcein labelled bone within the fracture site of EfnB1 mice. Supportive in vitro studies demonstrated that under osteogenic conditions, cultured EfnB1 stromal cells derived from the 2 week fracture site exhibited a reduced capacity to produce mineral and decreased expression of the osteogenic gene, Osterix, when compared to Osx:Cre controls. These findings suggest that the loss of EfnB1 delays the fracture repair process. The present study confirmed that EFNB1 activation in human BMSC, following stimulation with soluble-EphB2 resulted in de-phosphorylation of TAZ, demonstrating similarities in EfnB1 signalling between human and mouse stromal populations. Overall, the present study provides evidence that loss of EfnB1 in the osteo/chondrogenic lineages delays the soft callus formation/remodelling stages of the fracture repair process.
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http://dx.doi.org/10.1016/j.bone.2019.115189DOI Listing
March 2020

Stem cell-directed therapies for osteoarthritis: The promise and the practice.

Stem Cells 2020 04 7;38(4):477-486. Epub 2020 Feb 7.

Precision Medicine, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia.

Osteoarthritis (OA) is a disease of an entire synovial joint characterized by clinical symptoms and distortion of joint tissues, including cartilage, muscles, ligaments, and bone. Although OA is a disease of all joint tissues, it is a defined accessible compartment and is thus amenable to topical surgical and regenerative therapies, including stem cells. All tissues arise from stem progenitor cells, and the relative capacity of different cellular compartments, and different individuals, to renew tissues into adulthood may be important in the onset of many different degenerative diseases. OA is driven by both mechanical and inflammatory factors, but how these factors affect the proliferation and differentiation of cells into cartilage in vivo is largely unknown. Indeed, our very basic understanding of the physiological cellular kinetics and biology of the stem-progenitor cell unit of the articular cartilage, and how this is influenced by mechano-inflammatory injury, is largely unknown. OA seems, rather deceptively, to be the low-hanging fruit for stem cell therapy. Without the basic understanding of the stem cell and progenitor unit that generate and maintain articular cartilage in vivo, we will continue to waste opportunities to both prevent and manage this disease. In this review, we discuss the biology of chondrogenesis, the stem cell populations that support articular cartilage in health and disease, and future opportunities afforded through the translation of basic articular chondrocyte stem cell biology into new clinical therapies.
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http://dx.doi.org/10.1002/stem.3139DOI Listing
April 2020

Using Dental Pulp Stem Cells for Stroke Therapy.

Front Neurol 2019 29;10:422. Epub 2019 Apr 29.

Stroke Research Programme Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia.

Stroke is a leading cause of permanent disability world-wide, but aside from rehabilitation, there is currently no clinically-proven pharmaceutical or biological agent to improve neurological disability. Cell-based therapies using stem cells, such as dental pulp stem cells, are a promising alternative for treatment of neurological diseases, including stroke. The ischaemic environment in stroke affects multiple cell populations, thus stem cells, which act through cellular and molecular mechanisms, are promising candidates. The most common stem cell population studied in the neurological setting has been mesenchymal stem cells due to their accessibility. However, it is believed that neural stem cells, the resident stem cell of the adult brain, would be most appropriate for brain repair. Using reprogramming strategies, alternative sources of neural stem and progenitor cells have been explored. We postulate that a cell of closer origin to the neural lineage would be a promising candidate for reprogramming and modification towards a neural stem or progenitor cell. One such candidate population is dental pulp stem cells, which reside in the root canal of teeth. This review will focus on the neural potential of dental pulp stem cells and their investigations in the stroke setting to date, and include an overview on the use of different sources of neural stem cells in preclinical studies and clinical trials of stroke.
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http://dx.doi.org/10.3389/fneur.2019.00422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501465PMC
April 2019

The Late Osteoblast/Preosteocyte Cell Line MLO-A5 Displays Mesenchymal Lineage Plasticity and .

Stem Cells Int 2019 17;2019:9838167. Epub 2019 Jan 17.

Biomedical Orthopaedic Research Group, Centre for Orthopaedic and Trauma Research, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA 5005, Australia.

The process of osteoblast switching to alternative mesenchymal phenotypes is incompletely understood. In this study, we tested the ability of the osteoblast/preosteocyte osteogenic cell line, MLO-A5, to also differentiate into either adipocytes or chondrocytes. MLO-A5 cells expressed a subset of skeletal stem cell markers, including Sca-1, CD44, CD73, CD146, and CD166. Confluent cultures of cells underwent differentiation within 3 days upon the addition of osteogenic medium. The same cultures were capable of undergoing adipogenic and chondrogenic differentiation under lineage-appropriate culture conditions, evidenced by lineage-specific gene expression analysis by real-time reverse-transcription-PCR, and by Oil Red O and alcian blue (pH 2.5) staining, respectively. Subcutaneous implantation of MLO-A5 cells in a gel foam into NOD SCID mice resulted in a woven bone-like structure containing embedded osteocytes and regions of cartilage-like tissue, which stained positive with both alcian blue (pH 2.5) and safranin O. Together, our findings show that MLO-A5 cells, despite being a strongly osteogenic cell line, exhibit characteristics of skeletal stem cells and display mesenchymal lineage plasticity and . These unique characteristics suggest that this cell line is a useful model with which to study aging and disease-related changes to the mesenchymal lineage composition of bone.
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http://dx.doi.org/10.1155/2019/9838167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360058PMC
January 2019

Epigenetic Regulation of Bone Marrow Stem Cell Aging: Revealing Epigenetic Signatures associated with Hematopoietic and Mesenchymal Stem Cell Aging.

Aging Dis 2019 Feb 1;10(1):174-189. Epub 2019 Feb 1.

1Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia.

In this review we explore the importance of epigenetics as a contributing factor for aging adult stem cells. We summarize the latest findings of epigenetic factors deregulated as adult stem cells age and the consequence on stem cell self-renewal and differentiation, with a focus on adult stem cells in the bone marrow. With the latest whole genome bisulphite sequencing and chromatin immunoprecipitations we are able to decipher an emerging pattern common for adult stem cells in the bone marrow niche and how this might correlate to epigenetic enzymes deregulated during aging. We begin by briefly discussing the initial observations in yeast, drosophila and Caenorhabditis elegans () that led to the breakthrough research that identified the role of epigenetic changes associated with lifespan and aging. We then focus on adult stem cells, specifically in the bone marrow, which lends strong support for the deregulation of DNA methyltransferases, histone deacetylases, acetylates, methyltransferases and demethylases in aging stem cells, and how their corresponding epigenetic modifications influence gene expression and the aging phenotype. Given the reversible nature of epigenetic modifications we envisage "epi" targeted therapy as a means to reprogram aged stem cells into their younger counterparts.
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http://dx.doi.org/10.14336/AD.2017.1213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345334PMC
February 2019

Mesenchymal stem cells and biologic factors leading to bone formation.

J Clin Periodontol 2019 06;46 Suppl 21:12-32

School of Dentistry, University of Queensland, Brisbane, Qld, Australia.

Background: Physiological bone formation and bone regeneration occurring during bone repair can be considered distinct but similar processes. Mesenchymal stem cells (MSC) and associated biologic factors are crucial to both bone formation and bone regeneration.

Aim: To perform a narrative review of the current literature regarding the role of MSC and biologic factors in bone formation with the aim of discussing the clinical relevance of in vitro and in vivo animal studies.

Methods: The literature was searched for studies on MSC and biologic factors associated with the formation of bone in the mandible and maxilla. The search specifically targeted studies on key aspects of how stem cells and biologic factors are important in bone formation and how this might be relevant to bone regeneration. The results are summarized in a narrative review format.

Results: Different types of MSC and many biologic factors are associated with bone formation in the maxilla and mandible.

Conclusion: Bone formation and regeneration involve very complex and highly regulated cellular and molecular processes. By studying these processes, new clinical opportunities will arise for therapeutic bone regenerative treatments.
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http://dx.doi.org/10.1111/jcpe.13053DOI Listing
June 2019

Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination.

Epigenetics Chromatin 2019 01 3;12(1). Epub 2019 Jan 3.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, 5000, Australia.

Background: The 5 hydroxymethylation (5hmC) mark and TET DNA dioxygenases play a pivotal role in embryonic stem cell differentiation and animal development. However, very little is known about TET enzymes in lineage determination of human bone marrow-derived mesenchymal stem/stromal cells (BMSC). We examined the function of all three TET DNA dioxygenases, responsible for DNA hydroxymethylation, in human BMSC cell osteogenic and adipogenic differentiation.

Results: We used siRNA knockdown and retroviral mediated enforced expression of TET molecules and discovered TET1 to be a repressor of both osteogenesis and adipogenesis. TET1 was found to recruit the co-repressor proteins, SIN3A and the histone lysine methyltransferase, EZH2 to osteogenic genes. Conversely, TET2 was found to be a promoter of both osteogenesis and adipogenesis. The data showed that TET2 was directly responsible for 5hmC levels on osteogenic and adipogenic lineage-associated genes, whereas TET1 also played a role in this process. Interestingly, TET3 showed no functional effect in BMSC osteo-/adipogenic differentiation. Finally, in a mouse model of ovariectomy-induced osteoporosis, the numbers of clonogenic BMSC were dramatically diminished corresponding to lower trabecular bone volume and reduced levels of TET1, TET2 and 5hmC.

Conclusion: The present study has discovered an epigenetic mechanism mediated through changes in DNA hydroxymethylation status regulating the activation of key genes involved in the lineage determination of skeletal stem cells, which may have implications in BMSC function during normal bone regulation. Targeting TET molecules or their downstream targets may offer new therapeutic strategies to help prevent bone loss and repair following trauma or disease.
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http://dx.doi.org/10.1186/s13072-018-0247-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317244PMC
January 2019

High-Throughput Assessment and Modeling of a Polymer Library Regulating Human Dental Pulp-Derived Stem Cell Behavior.

ACS Appl Mater Interfaces 2018 Nov 5;10(45):38739-38748. Epub 2018 Nov 5.

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Future Industries Institute , University of South Australia , Mawson Lakes , South Australia 5095 , Australia.

The identification of biomaterials that modulate cell responses is a crucial task for tissue engineering and cell therapy. The identification of novel materials is complicated by the immense number of synthesizable polymers and the time required for testing each material experimentally. In the current study, polymeric biomaterial-cell interactions were assessed rapidly using a microarray format. The attachment, proliferation, and differentiation of human dental pulp stem cells (hDPSCs) were investigated on 141 homopolymers and 400 diverse copolymers. The copolymer of isooctyl acrylate and 2-(methacryloyloxy)ethyl acetoacetate achieved the highest attachment and proliferation of hDPSC, whereas high cell attachment and differentiation of hDPSC were observed on the copolymer of isooctyl acrylate and trimethylolpropane ethoxylate triacrylate. Computational models were generated, relating polymer properties to cellular responses. These models could accurately predict cell behavior for up to 95% of materials within a test set. The models identified several functional groups as being important for supporting specific cell responses. In particular, oxygen-containing chemical moieties, including fragments from the acrylate/acrylamide backbone of the polymers, promoted cell attachment. Small hydrocarbon fragments originating from polymer pendant groups promoted cell proliferation and differentiation. These computational models constitute a key tool to direct the discovery of novel materials within the enormous chemical space available to researchers.
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http://dx.doi.org/10.1021/acsami.8b12473DOI Listing
November 2018

Loss of EfnB1 in the osteogenic lineage compromises their capacity to support hematopoietic stem/progenitor cell maintenance.

Exp Hematol 2019 01 13;69:43-53. Epub 2018 Oct 13.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, South Australia, Australia; Cancer Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia. Electronic address:

The bone marrow stromal microenvironment contributes to the maintenance and function of hematopoietic stem/progenitor cells (HSPCs). The Eph receptor tyrosine kinase family members have been implicated in bone homeostasis and stromal support of HSPCs. The present study examined the influence of EfnB1-expressing osteogenic lineage on HSPC function. Mice with conditional deletion of EfnB1 in the osteogenic lineage (EfnB1), driven by the Osterix promoter, exhibited a reduced prevalence of osteogenic progenitors and osteoblasts, correlating to lower numbers of HSPCs compared with Osx:Cre mice. Long-term culture-initiating cell (LTC-IC) assays confirmed that the loss of EfnB1 within bone cells hindered HSPC function, with a significant reduction in colony formation in EfnB1 mice compared with Osx:Cre mice. Human studies confirmed that activation of EPHB2 on CD34 HSPCs via EFNB1-Fc stimulation enhanced myeloid/erythroid colony formation, whereas functional blocking of either EPHB1 or EPHB2 inhibited the maintenance of LTC-ICs. Moreover, EFNB1 reverse signaling in human and mouse stromal cells was found to be required for the activation of the HSPC-promoting factor CXCL12. Collectively, the results of this study confirm that EfnB1 contributes to the stromal support of HSPC function and maintenance and may be an important factor in regulating the HSPC niche.
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http://dx.doi.org/10.1016/j.exphem.2018.10.004DOI Listing
January 2019

mTORC1 plays an important role in osteoblastic regulation of B-lymphopoiesis.

Sci Rep 2018 09 28;8(1):14501. Epub 2018 Sep 28.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, Australia.

Skeletal osteoblasts are important regulators of B-lymphopoiesis, serving as a rich source of factors such as CXCL12 and IL-7 which are crucial for B-cell development. Recent studies from our laboratory and others have shown that deletion of Rptor, a unique component of the mTORC1 nutrient-sensing complex, early in the osteoblast lineage development results in defective bone development in mice. In this study, we now demonstrate that mTORC1 signalling in pre-osteoblasts is required for normal B-lymphocyte development in mice. Targeted deletion of Rptor in osterix-expressing pre-osteoblasts (Rptor) leads to a significant reduction in the number of B-cells in the bone marrow, peripheral blood and spleen at 4 and 12 weeks of age. Rptor mice also exhibit a significant reduction in pre-B and immature B-cells in the BM, indicative of a block in B-cell development from the pro-B to pre-B cell stage. Circulating levels of IL-7 and CXCL12 are also significantly reduced in Rptor mice. Importantly, whilst Rptor-deficient osteoblasts are unable to support HSC differentiation to B-cells in co-culture, this can be rescued by the addition of exogenous IL-7 and CXCL12. Collectively, these findings demonstrate that mTORC1 plays an important role in extrinsic osteoblastic regulation of B-cell development.
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http://dx.doi.org/10.1038/s41598-018-32858-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162303PMC
September 2018

miRNA-376c-3p Mediates TWIST-1 Inhibition of Bone Marrow-Derived Stromal Cell Osteogenesis and Can Reduce Aberrant Bone Formation of TWIST-1 Haploinsufficient Calvarial Cells.

Stem Cells Dev 2018 12 23;27(23):1621-1633. Epub 2018 Oct 23.

1 Mesenchymal Stem Cell Laboratory, Faculty of Health and Medical Sciences, Adelaide Medical School, The University of Adelaide , Adelaide, Australia .

Key transcription factors, which activate or repress master gene regulators and signaling pathways, tightly regulate self-renewal and cell lineage differentiation of bone marrow-derived stromal cells (BMSC). Among these factors is the basic helix-loop-helix transcription factor Twist-related protein 1 (TWIST-1), which is important in BMSC self-renewal, life span, and differentiation. Another layer of gene regulation comes from microRNAs (miRNAs). miRNAs are short noncoding RNAs that interfere with translation of specific target mRNAs and thereby regulate diverse biological processes, including BMSC lineage commitment. However, little is known of how TWIST-1-regulated miRNAs control osteogenic commitment, and influence the fate of bone precursor cells. In this study, we have discovered a novel TWIST-1-regulated miRNA, miR-376c-3p. Reduced miR-376c-3p expression by a miR-376c-3p inhibitor or due to TWIST-1 haploinsufficiency promotes alkaline phosphatase (ALP) activity, mineral deposition, and expression of osteoblast-associated genes in BMSC and calvarial cells. Conversely, overexpression of miR-376c-3p using a miR-376c-3p mimic inhibited BMSC proliferation and the osteogenic potential of BMSC and TWIST-1 haploinsufficient calvarial cells. This was demonstrated by a decrease in insulin growth factor 1 receptor (IGF1R) levels, Akt signaling, ALP activity, mineral deposition, and expression of osteoblast-associated genes. Thus, miR-376c-3p reduces IGF1R/Akt signaling in BMSC and is one mechanism by which osteogenesis may be inhibited. Overall, we have identified miR-376c-3p as a TWIST-1-regulated miRNA, which plays an important role in the osteogenesis of bone precursor cells and can mediate TWIST-1 inhibition of osteogenesis. Furthermore, overexpression of miRNA-376c-3p in TWIST-1 haploinsufficient calvarial cells can decrease the aberrant osteogenesis of these cells, which contributes to increased calvarial bone volume and premature fusion of the coronal sutures.
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http://dx.doi.org/10.1089/scd.2018.0083DOI Listing
December 2018

The osteoprogenitor-specific loss of ephrinB1 results in an osteoporotic phenotype affecting the balance between bone formation and resorption.

Sci Rep 2018 08 24;8(1):12756. Epub 2018 Aug 24.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, 5005, SA, Australia.

The present study investigated the effects of conditional deletion of ephrinB1 in osteoprogenitor cells driven by the Osterix (Osx) promoter, on skeletal integrity in a murine model of ovariectomy-induced (OVX) osteoporosis. Histomorphometric and μCT analyses revealed that loss of ephrinB1 in sham Osx:cre-ephrinB1 mice caused a reduction in trabecular bone comparable to OVX Osx:Cre mice, which was associated with a significant reduction in bone formation rates and decrease in osteoblast numbers. Interestingly, these observations were not exacerbated in OVX Osx:cre-ephrinB1 mice. Furthermore, sham Osx:cre-ephrinB1 mice displayed significantly higher osteoclast numbers and circulating degraded collagen type 1 compared to OVX Osx:Cre mice. Confirmation studies found that cultured monocytes expressing EphB2 formed fewer TRAP multinucleated osteoclasts and exhibited lower resorption activity in the presence of soluble ephrinB1-Fc compared to IgG control. This inhibition of osteoclast formation and function induced by ephrinB1-Fc was reversed in the presence of an EphB2 chemical inhibitor. Collectively, these observations suggest that ephrinB1, expressed by osteoprogenitors, influences bone loss during the development of osteoporosis, by regulating both osteoblast and osteoclast formation and function, leading to a loss of skeletal integrity.
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http://dx.doi.org/10.1038/s41598-018-31190-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109077PMC
August 2018

Osteoblast derived-neurotrophin‑3 induces cartilage removal proteases and osteoclast-mediated function at injured growth plate in rats.

Bone 2018 11 17;116:232-247. Epub 2018 Aug 17.

School of Pharmacy and Medical Sciences, Sansom Institute for Health Research, University of South Australia, Adelaide, SA 5001, Australia. Electronic address:

Faulty bony repair causes dysrepair of injured growth plate cartilage and bone growth defects in children; however, the underlying mechanisms are unclear. Recently, we observed the prominent induction of neurotrophin‑3 (NT-3) and its important roles as an osteogenic and angiogenic factor promoting the bony repair. The current study investigated its roles in regulating injury site remodelling. In a rat tibial growth plate drill-hole injury repair model, NT-3 was expressed prominently in osteoblasts at the injury site. Recombinant NT-3 (rhNT-3) systemic treatment enhanced, but NT-3 immunoneutralization attenuated, expression of cartilage-removal proteases (MMP-9 and MMP-13), presence of bone-resorbing osteoclasts and expression of osteoclast protease cathepsin K, and remodelling at the injury site. NT-3 was also highly induced in cultured mineralizing rat bone marrow stromal cells, and the conditioned medium augmented osteoclast formation and resorptive activity, an ability that was blocked by presence of anti-NT-3 antibody. Moreover, NT-3 and receptor TrkC were induced during osteoclastogenesis, and rhNT-3 treatment activated TrkC downstream kinase Erk1/2 in differentiating osteoclasts although rhNT-3 alone did not affect activation of osteoclastogenic transcription factors NF-κB or NFAT in RAW osteoclast precursor cells. Furthermore, rhNT-3 treatment increased, but NT-3 neutralization reduced, expression of osteoclastogenic cytokines (RANKL, TNF-α, and IL-1) in mineralizing osteoblasts and in growth plate injury site, and rhNT-3 augmented the induction of these cytokines caused by RANKL treatment in RAW cells. Thus, injury site osteoblast-derived NT-3 is important in promoting growth plate injury site remodelling, as it induces cartilage proteases for cartilage removal and augments osteoclastogenesis and resorption both directly (involving activing Erk1/2 and substantiating RANKL-induced increased expression of osteoclastogenic signals in differentiating osteoclasts) and indirectly (inducing osteoclastogenic signals in osteoblasts).
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http://dx.doi.org/10.1016/j.bone.2018.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550307PMC
November 2018

Tyrosine kinase receptor c-ros-oncogene 1 inhibition alleviates aberrant bone formation of TWIST-1 haploinsufficient calvarial cells from Saethre-Chotzen syndrome patients.

J Cell Physiol 2018 09 16;233(9):7320-7332. Epub 2018 Apr 16.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, South Australia, Australia.

Saethre-Chotzen syndrome (SCS), associated with TWIST-1 mutations, is characterized by premature fusion of cranial sutures. TWIST-1 haploinsufficiency, leads to alterations in suture mesenchyme cellular gene expression patterns, resulting in aberrant osteogenesis and craniosynostosis. We analyzed the expression of the TWIST-1 target, Tyrosine kinase receptor c-ros-oncogene 1 (C-ROS-1) in TWIST-1 haploinsufficient calvarial cells derived from SCS patients and calvaria of Twist-1 mutant mice and found it to be highly expressed when compared to TWIST-1 wild-type controls. Knock-down of C-ROS-1 expression in TWIST-1 haploinsufficient calvarial cells derived from SCS patients was associated with decreased capacity for osteogenic differentiation in vitro. Furthermore, treatment of human SCS calvarial cells with the tyrosine kinase chemical inhibitor, Crizotinib, resulted in reduced C-ROS-1 activity and the osteogenic potential of human SCS calvarial cells with minor effects on cell viability or proliferation. Cultured human SCS calvarial cells treated with Crizotinib exhibited a dose-dependent decrease in alkaline phosphatase activity and mineral deposition, with an associated decrease in expression levels of Runt-related transcription factor 2 and OSTEOPONTIN, with reduced PI3K/Akt signalling in vitro. Furthermore, Crizotinib treatment resulted in reduced BMP-2 mediated bone formation potential of whole Twist-1 mutant mouse calvaria organotypic cultures. Collectively, these results suggest that C-ROS-1 promotes osteogenic differentiation of TWIST-1 haploinsufficient calvarial osteogenic progenitor cells. Furthermore, the aberrant osteogenic potential of these cells is inhibited by the reduction of C-ROS-1. Therefore, targeting C-ROS-1 with a pharmacological agent, such as Crizotinib, may serve as a novel therapeutic strategy to alleviate craniosynostosis associated with aberrant TWIST-1 function.
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http://dx.doi.org/10.1002/jcp.26563DOI Listing
September 2018
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