Despite the ongoing political debate regarding the legality of medical marijuana, clinical
investigations of the therapeutic use of cannabinoids are now more prevalent than at any
time in history. Cannabinoids have been shown to have analgesic, anti-spasmodic, anticonvulsant,
anti-tremor, anti-psychotic, anti-inflammatory, anti-oxidant, anti-emetic and
appetite-stimulant properties. There are mainly two well-known cannabinoid receptors,
CB1 and CB2, located in the central (CB1) and peripheral (CB2) nervous systems as well
as the immune system. More recently, endocannabinoids (ligands) and their receptors
have also been found in the skeleton which appear as the main body system and
physiologically regulated by CB2.
This study aimed to examine the effect of both CB1 and CB2 receptor stimulation on
wound closure response of MG-63 osteoblast bone cell monolayers using different
treatments with cannabinoid such as Winn55,212-2, URB602 and HU308. Also, cell
adhesion, cell proliferation and cell length was investigated. The study also aimed to
examine the effect of HU308 treatments in combination with TGF-β3 (transforming
growth factor beta -3) on wound healing, cell adhesion and extracellular matrix up
regulation (collagen type I, fibronectin and protien S-100A6) as well as other biological
factors such as secretion of matrix metalloproteinase (MMP-2) and nitric oxide (NO).
Finally, this study investigated HU308/TGF-β3 combination treatment on the regulation
of extracellular matrix (collagen type I, fibronectin and protien S-100A6) in a 3D
multilayer system of MG-63 osteoblast bone cells.
Wound healing assays of MG-63 monolayers revealed accelerated wound repair as well
as increased cell proliferation mainly regulated through CB2 receptors, and that
treatments with HU308 and HU308/TGF-β3 achieved minimum closure timings
compared with control groups (P<0.05). Our finding suggested that proliferation rate
with 500nM HU308 was significantly higher than control and TGF-β3/HU308
combination groups (P<0.05). Interestingly, percentage of wound remained open after
15 hours for combination groups was 17.6%±1.32 whereas treatment with 500nM
HU308 had 20%±2.25 indicating that the combination groups took the lead throughout wound healing. It was also observed that bridge formation in all treatment groups was
taking place between 15 to 20 hour periods whereas within control treatments bridge
formation started to take place after 25 hours.
Cell surface attachment was examined via the trypsinization assay in which the time
taken to trypsinize cells from the surface provided a means of assessing the strength of
attachment. The results indicated that higher concentrations of HU308 (2μM), induced
significant force of cell attachment compared with control and concentrations of 500nM
and 1μM (P<0.05). However, groups treated with TGF-β3 and combination
HU308/TGF-β3 indicated reduced cell surface attachment compared with control
groups, indicating enhanced cell migration.
Immunofluorescence staining as well as Elisa based semi-quantification technique
indicated that both collagen type I and fibronectin were unregulated using higher
concentrations of HU308 with decreased cell proliferation compared to lower
concentrations. Nevertheless, protein S-100A6 was up-regulated in treatments with
HU308, TGF-β3 and their combination HU308/TGF-β3 (P<0.05), indicating the
positive role of these treatments in promoting cell differentiation. MMP-2 levels in the
current study were also shown to be concentration-dependent, i.e. higher concentrations
of HU308 significantly reduced MMP-2 secretion leading to decreased cell migration,
while HU308/TGF-β3 combination treatment increased MMP-2 levels, indicating an
increase in cell migration. The current study also examined levels of nitric oxide
synthesis in relation to different treatments with HU308, TGF-β3 and HU308/TGF-β3
combination. It was found that nitric oxide up-regulation influences rate of MG-63
osteoblast wound healing in a concentration dependent manner.
Lastly, UpCell culture dishes proved to have efficacy in obtaining a multilayer model of
MG-63 osteoblast system in-vitro through changes in cell morphology. It was also
found that treatments with HU308, TGF-β3 and HU308/TGF-β3 combination
influenced collagen type I, fibronecton and protein S-100A6 secretion. These findings
supported the earlier Elisa based semi-quantification results obtained for monolayer
cultures.
| Identifer | oai:union.ndltd.org:BRADFORD/oai:bradscholars.brad.ac.uk:10454/6319 |
| Date | January 2013 |
| Creators | Genedy, Mohamed A. |
| Contributors | Youseffi, Mansour, Denyer, Morgan C.T. |
| Publisher | University of Bradford, Division of Medical Engineering, School of Engineering, Design and Technology |
| Source Sets | Bradford Scholars |
| Language | English |
| Detected Language | English |
| Type | Thesis, doctoral, PhD |
| Rights | <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by-nc-nd/3.0/88x31.png" /></a><br />The University of Bradford theses are licenced under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/">Creative Commons Licence</a>. |
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