Doxycycline restores the impaired osteogenic commitment of diabetic-derived bone marrow mesenchymal stromal cells by increasing the canonical WNT signaling
Introduction
Diabetes mellitus (DM) encompasses a group of metabolic disorders characterized by hyperglycemia due to the lack of insulin production, peripheral insulin resistance, or both. DM seems to affect the skeletal system and the bone metabolism, as a relation between an increased risk of fragility fractures and DM has been established, and associated with excess morbidity, mortality and health-care costs (Miao et al., 2005; Vestergaard et al., 2009; Vestergaard, 2007a). Evidences both from the bench and bedside reveal a strong correlation between DM and bone functional impairment, with type 1 diabetes mellitus (T1DM) affecting the bone tissue more severely, with established alterations not only at the cell functionality, but also at the extracellular matrix level, disturbing the organic collagen structure and the inorganic bone mineral (Pan et al., 2014; Vestergaard, 2007b). T1DM is further characterized by a diminished bone turnover, particularly at the expenses of the bone formation process (Starup-Linde, 2013), as sustained by the reduced biochemical markers of bone formation and histomorphometric indices at the trabecular and cortical bone of experimental diabetes animal models and diabetic patients (Silva et al., 2009; Massé et al., 2010; Gunczler et al., 2001; Keenan and Maddaloni, 2016; Cervino et al., 2019). Hence, at the cellular level, an impaired osteoblastogenesis has been reported, with an increased cell death and altered functional activity, impaired osteogenic priming and delayed osteoblastic differentiation (McCabe et al., 2011; Hie et al., 2011; Silva et al., 2015). This is corroborated by the downregulation of osteogenic transcription factors and downstream targets, in association with an increased adipogenic priming of precursor cell populations, in distinct T1DM models (Lu et al., 2003; Fowlkes et al., 2007; Nyman et al., 2017; Bortolin et al., 2017). Clinically, an increased fracture rate, at different bone locations, has been established for diabetic individuals, as well as an increased risk of complications during fracture healing – with significant higher rates of delayed union, nonunion and postoperative infection, comparing to nondiabetic individuals (Sundararaghavan et al., 2017). As a result, there is an urge of innovative therapeutic approaches, based on effective modulatory agents that are capable of restoring the osteogenic functionality in diabetic conditions and prime/accelerate tissue healing (Herford et al., 2013).
Tetracyclines embrace a group of broad-spectrum antibacterial compounds – highly effective against gram-positive and gram-negative bacteria, spirochetes and rickettsia – that show a high affinity to mineralized tissues, forming bioactive complexes with the inorganic mineral component (Chopra and Roberts, 2001), and an adequate cytocompatible profile (Kallala et al., 2012). Tetracyclines, and particularly the semi-synthetic minocycline and doxycycline at a low dosage regimen, display an effective modulatory activity within the bone tissue metabolism – tempering the remodeling and healing processes – through antibacterial-independent mechanisms (Golub et al., 2016; Griffin et al., 2011). Our group has previously shown that low concentrations of doxycycline increased the metabolic activity and the osteogenic potential of human osteoblastic populations, with increased expression of osteogenic markers, enhanced extracellular matrix mineralization, inducing no negative effects on calcium metabolism (Gomes and Fernandes, 1997). Bone regenerative biomaterials, releasing low levels of semi-synthetic tetracyclines, also showed an increased osteogenic activation, in vitro and in vivo (Walter et al., 2014; Silva et al., 2017, 2018). Tetracyclines were further found to normalize bone remodeling and enhance the bone formation process in compromised conditions (Zhou et al., 2010; Kinugawa et al., 2012), supporting their successful clinical application in the management of osteopenia/osteoporosis (Payne and Golub, 2011; Golub et al., 2010; de Figueiredo et al., 2019).
Minocycline and doxycycline have also been found to modulate the diabetic disease course, lessening diabetic-related complications, such as the peripheral and autonomic neuropathy (Syngle et al., 2014), retinopathy (Scott et al., 2014), nephropathy (Aggarwal et al., 2010), and periodontal disease (ŁKondzielnik et al., 2012), in a process presumably related with the management of the excessively activated inflammatory response, matrix metalloproteinases modulation and normalization of the altered oxidative stress balance (Garrido‐Mesa et al., 2013). Despite the evidence of the positive modulation of diabetic-related complications by tetracyclines, studies concerning the diabetic osteopenia and tetracyclines are still very scarce. These have been limited to the assessment, in the early 90's of the 20th century, of Golub et al. reporting that doxycycline (Golub et al., 1990) and minocycline (Sasaki et al., 1992) prevented osteopenia, in a rat diabetic model, based on the morphological characterization of the bone tissue. Therefore, and to the best of the authors' knowledge, no cellular or molecular data on the potential modulatory activity of these drugs regarding the diabetes-mediated impaired osteoblastic functionality have been addressed. In this frame, this study aims to detail the modulatory capacity and associated molecular signaling of a low dosage doxycycline regimen, in the functional activity of an osteoblastic precursor population, established from a translational T1DM experimental model.
Section snippets
Animals
This study was conducted in accordance with accepted standards for the humane animal care and manipulation. Procedures were approved by Direção Geral de Alimentação e Veterinária and encompassed the standards for the protection of experimental animals, according to the Portuguese and European legislations.
Experimental diabetic model
All animals of both SHAM and STZ groups survived throughout the duration of the experimental protocol. Whether no significant differences were found on the initial weight (SHAM, 339.5 g ± 23.1 and STZ, 317.0 g ± 33.8), 6 weeks following induction, animals of the SHAM group increased in weight (396.7 g ± 37.3), while animals of the STZ group significantly loss weight (228.1 g ± 26.41). Further, at euthanasia, all STZ-administered animals had glycemic levels over 300 mg/dl, while animals from the
Discussion
The strong correlation between DM and bone functional impairment reflects significant changes at the cellular and tissue level, namely concerning osteogenic priming, differentiation and functionality (McCabe et al., 2011; Silva et al., 2014). Underlying mechanisms include the downregulation of osteogenic transcription factors and downstream targets due to impaired molecular signaling pathways directing osteogenesis (Zhao et al., 2013; Al-Qarakhli et al., 2019) and the interconnected
Conclusions
In this work, we have shown that doxycycline, at a low dosage regimen (1 μg ml−1), enhanced the functional activity and osteogenic capability of MSCs derived from STZ-induced diabetic rats, to levels similar to those of control, i.e. MSCs cultures established from SHAM animals. Doxycycline normalized the cell proliferation and metabolic activity of the established cultures, further decreasing cell apoptosis. The addition of this drug to the culture environment increased the osteogenic
Funding
The work was supported by UIDB/50006/2020 with funding from FCT/MCTES through national funds, and by the Faculty of Dental Medicine, U. Porto.
Ethics approval
This study was conducted in compliance with Portuguese and European legislations (Directive, 2010/63/EU in Europe) with accepted standards of humane animal care and was approved by the local IACUC.
Consent to participate
Not applicable.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Code availability
Not applicable.
Authors' contributions
MHF was responsible for conception of the study, made substantial contributions for the interpretation of data and substantively revised the manuscript. PSG was responsible for the study design, data analysis, interpretation and draft the manuscript. MR was responsible for data acquisition, analysis and interpretation. All authors have approved the submitted version and have agreed both to be personally accountable for the their own contributions and to ensure that questions related to the
CRediT authorship contribution statement
Pedro Sousa Gomes: Conceptualization, Supervision, Writing - review & editing. Marta Resende: Investigation, Formal analysis, Writing - original draft. Maria Helena Fernandes: Resources, Methodology, Validation, Funding acquisition.
Declaration of competing interest
The authors declare that they have no competing interests.
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