hMSC response to augmented CaP nanotopography

Abstract

Calcium phosphate (CaP) biomaterials, such as hydroxyapatite (HA), are recognised as an important class of bioactive compounds that can be used as coatings on titanium (Ti) substrates to induce cell attachment, proliferation and differentiation. In this work, the response of human mesenchymal stem cells (hMSCs) to sputtered (amorphous) CaP coatings deposited onto titanium substrates with various topographies has been determined. This research focuses on the role that the specific forms of titanium nanotopography can play in controlling the dissolution of amorphous CaP and to determine the extent to which such surfaces can directly initiate and maintain hMSC differentiation to an osteogenic lineage. The conditions needed to elicit the necessary combination of stimuli from the exposed titanium nanotopography and the CaP chemistry has been determined. A key area of interest here is the difference in response between hMSCs cultured in normal media and those pre-conditioned in osteogenic media for 5 days prior to seeding on the surfaces of interest. This data tests the hypothesis that hMSCs that have a degree of induced osteoblastic characteristic are more likely to respond to the presence of the CaP when it is present in tandem with nanotopography. Polycrystalline titanium (Poly-Ti) surfaces were prepared by RF magnetron sputtering from 99.9% pure metal targets onto glass coupons followed by thermal annealing at 500oC for 2 hours. Titania Nanotube (TiNT) surfaces were created by electrochemical anodisation of titanium coupons at 20V for 120 minutes in a solution of ethylene glycol with 0.3%wt NH4F and 3%wt DI H20. Both types of titanium surface were coated with an amorphous CaP thin film by sputter deposition from hydroxyapatite (HA) targets at a power of 50W for 9 hrs. Dissolution studies of the CaP coatings on the titanium substrates were undertaken by immersion in 2ml of DMEM media at 37oC for up to 7 days Surfaces were characterised by XPS, XRD, AFM, SEM and optical profilometry to investigate how Poly-Ti and TiNT nanotopography affects the rate of CaP removal.

In vitro cell studies were carried out on the various surfaces, including samples that had been exposed to media for 7 days prior to testing. Both hMSCs cultured in normal media (NM) and those pre-conditioned in osteogenic media (OM) for 5 days prior to seeding were used in these experiments Analysis of cell viability, proliferation and preliminary signs of differentiation to an osteogenic lineage were undertaken using PicoGreen and ALP assays. qPCR analysis was carried out to examine the expression of genes associated with early and late stage osteogenic differentiation.
Both the Poly-Ti and TiNT surfaces were capable of controlling the release of the amorphous CaP from the attendant topography. As such, these features have the ability to entrap this bioactive material that can then act as reservoirs for its continuing release over the normal short time scales for normal “flat” surfaces. Furthermore, the combination of the residue from an amorphous CaP coating and exposed titanium nanotopography has been shown to encourage hMSC proliferation and direct differentiation to an osteogenic lineage over a 28 day culture period. Additionally, the work presented here reports for the first time that hMSCs that have been exposed to osteogenic media prior to culturing on TiNT-CaP surfaces show a statistical significant increase in late stage markers of hMSC differentiation to a osteogenic lineage as compared to those expanded only in normal media. Hence, this work shows that these pre-conditioned hMSCs undergo differentiation based on a combination of stimuli from nanotopography and amorphous CaP. These in vitro data suggest that such surfaces could have a role in creating and maintaining a form of early stage osteoprogenitor cells, derived from hMSCs for applications in tissue engineering (as cell sources to populate scaffolds) and regenerative medicine (as tissue forming cells in delivery systems).

Date of Award	May 2016
Original language	English
Sponsors	Department for Employment and Learning, Northern Ireland
Supervisor	Brian Meenan (Supervisor) & George Burke (Supervisor)