Osteoblastogenic differentiation of mesenchymal stem cells through nanoscale stimulation: the conception of a novel 3D osteogenic bioreactor

Pemberton, Gabriel Delsol (2015) Osteoblastogenic differentiation of mesenchymal stem cells through nanoscale stimulation: the conception of a novel 3D osteogenic bioreactor. PhD thesis, University of Glasgow.

Abstract

Throughout this body of work low amplitude high frequency (500 Hz – 5000 Hz) mechanical stimulation and its effect to induce osteogenesis on bone marrow derived MSCs has been investigated. Due to the nanolevel amplitudes of these high frequency vertical vibrations the term nanokicking appeared to be appropriate and was subsequently used throughout this thesis to refer to these high frequency sinusoidal stimulations provided by the bioreactor. In the first instance this work was performed in 2D and biological analyses to determine osteogenesis were carried out at a transcript (mRNA), protein and mineralisation level. Affirmative results for osteogensis were observed from genes and proteins (RUNX2, osteocalin, osteopontin) related to the osteoblast phenotype by qRT PCR, in cell western, and immunostaining. To determine the prescence of inorganic osseous minerals, more specific techniques such as Raman spectroscopy, micro computed tomography and histological stainings (Von Kossa/Alizarin Red) were further employed. The results observed remained in line with previously published material (Gentleman et al., 2009) drawing the conclusion that calcium phosphate (Ca10(PO4)6, through nanokicking,was formed in vitro.

The natural progession of this research meant that a novel vibrational bioreactor was conceived and designed, through the use of Lean and Six Sigma principles (Andrew Thomas, 2004; Caldwell, 2006), in order to assess the potential of nanokicking in 3D. Here collagen was employed as a biomimetic scaffold and affirmative results for osteogenesis were observed.

The bioreactor was unique in that long term (up to 46 days) sterile culture was achieved, it was easy to use and there was no requirement for osteogenic media, growth factors or complex chemistries (e.g. dexamethasone, rhBMP2) in order to induce osteogenesis. The cost of use and maintence was relatively cheap compared to available commercial bioreactors (Rauh et al., 2011b).

It is envisaged that this technology may one day have real world use for ossesous tissue regeneration and care in a GMP and clinical setting, or for the preparation of autologous tissue for medical testing in the burgeoning field of personalised medicine.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Bioreactor, osteogenesis, mesenchymal stem cells, regenerative medicine.
• Subjects: Q Science > Q Science (General); Q Science > QH Natural history > QH301 Biology; Q Science > QH Natural history > QH345 Biochemistry; Q Science > QH Natural history > QH426 Genetics; R Medicine > R Medicine (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Dalby, Professor Matthew J.
• Date of Award: 2015
• Depositing User: Dr. Gabriel Delsol Pemberton
• Unique ID: glathesis:2015-6956
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 07 Jan 2016 14:36
• Last Modified: 10 Feb 2016 15:40
• URI: https://theses.gla.ac.uk/id/eprint/6956

Related URLs

• http://www.futuremedicine.com/doi/abs/10...