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INVESTIGATING THE MECHANISM OF INTRACELLULAR SIGNALING OF MAGNESIUM IN HUMAN BONE MARROW STROMAL CELLS (hBMSCs): POTENTIAL ROLE IN OSTEOGENESIS

Shehabeldin, Mostafa (2016) INVESTIGATING THE MECHANISM OF INTRACELLULAR SIGNALING OF MAGNESIUM IN HUMAN BONE MARROW STROMAL CELLS (hBMSCs): POTENTIAL ROLE IN OSTEOGENESIS. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

Magnesium metal and its alloys have been investigated as promising biomaterials for internal bone fixation devices. Previous reports have shown that magnesium fixation devices enhance fracture healing in animal models while exhibiting biocompatibility and biodegradability. Moreover, mechanistic studies have indicated that mesenchymal stem cells (MSCs) display an osteogenic response to increased magnesium (Mg2+) concentrations in culture medium. We sought to study the signaling pathways underlying Mg-mediated osteogenesis in human bone marrow stromal cells (hBMSCs). We hypothesized that Mg2+ could be inducing the osteogenic response of hBMSCs in a calcium sensing receptor (CaSR) dependent manner. In the present study, hBMSCs were stimulated with basal medium (BM) supplemented with 10 mM MgSO4 (10-Mg), 4 mM CaCl2 (4-Ca) or 10 mM CaCl2 (10-Ca) at different time points. Western blot results showed a similar trend of CaSR phosphorylation in response to 10-Mg and 4-Ca media. Although this phosphorylation peaked at 15 minutes with both 10-Mg and 4-Ca media, it was not significantly higher than with BM alone. PKC-δ and Erk 1/2 are two downstream kinases of the CaSR. Both kinases have been reported to play a pivotal role in the differentiation of osteoprogenitors by regulating the expression of key osteogenic markers. We showed that hBMSCs exposure to 10-Mg medium resulted in significant phosphorylation of PKC-δ relative to BM. In contrast, Erk1/2 did not exhibit significant changes in its activity with 10-Mg medium. In addition, qPCR data showed upregulation of Cx43, ALPL and Col10A1 following hBMSCs treatment with 10-Mg medium. Similarly, a trend of upregulation of Runx2 mRNA was seen with 10-Mg medium; however, this upregulation was not statistically significant. VEGFA, a key angiogenic marker, was downregulated at the mRNA level by 10-Mg medium at a late time point (3 weeks). Chemical blocking of CaSR by its selective antagonist NPS2143 only potentiated ALPL expression, but did not have any effect on VEGFA or Col10A1. In conclusion, we propose a potential mechanism by which PKC-δ and Cx43 could mediate the osteogenic response of hBMSCs to increased Mg2+ concentrations. The suggested functional activity of PKC-δ might be regulated by a CaSR upstream signaling mechanism.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Shehabeldin, Mostafamss124@pitt.eduMSS124
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSfeir, Charlescsfeir@pitt.eduCSFEIR
Committee MemberBeniash, Eliaebeniash@pitt.eduEBENIASH
Committee MemberSzabo Rogers, Heatherhsrogers@pitt.eduHSROGERS
Committee MemberSyed Picard, Fatimasyedpicard@pitt.eduFNS4
Date: 10 August 2016
Date Type: Publication
Defense Date: 1 July 2016
Approval Date: 10 August 2016
Submission Date: 3 August 2016
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 52
Institution: University of Pittsburgh
Schools and Programs: School of Dental Medicine > Dental Science
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Magnesium Signaling, PKC-δ, Calcium sensing receptor, Bone regeneration, fixation devices.
Date Deposited: 10 Aug 2016 20:48
Last Modified: 15 Nov 2016 14:35
URI: http://d-scholarship.pitt.edu/id/eprint/29106

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