Biological performance of novel phosphate-based glass microspheres for mesenchymal stem cell therapy in osteoporotic patients

Abstract

In this study, degradable phosphate-based bulk or porous glass microspheres (BGMS or PGMS), with nominal molar compositions of P45-(45P2O5-16CaO-24MgO-11Na2O-4Fe2O3) and P40-(40P2O5-16CaO-24MgO-20Na2O), were evaluated for cytotoxicity, cytocompatibility and osteogenic potential for Mesenchymal stem cell (MSC)-based therapy in osteoporotic patients. Evaluations were performed using direct-contact and indirect-contact bone marrow derived human MSC (hMSC)-based experiments, in addition to material characterisations such as morphology, elemental composition and degradation behaviour, which were correlated to the hMSC experiments. Degradation of microspheres (MS) was measured using a novel method where Scanning Electron micrographs was used to assess the number of MS with surface damage (cracks and peeling effect), over 42 days of degradation in culture medium. Results showed that after 42 days, 2%, 46% and 29% of P45 BGMS, P40 BGMS and P40 PGMS, respectively, had cracks or peeling off surfaces. The results for direct-contact hMSC-experiments showed that P45 BGMS supported 1.4 times more hMSCs than P40 BGMS over 31 days of culture period. However, P45 BGMS were not osteoinductive, possibly due to hydrophobic nature of this glass and its slower dissolution rate. On the other hand, in comparison to P45 BGMS, hMSCs seeded on P40 BGMS showed up to 1.7 times higher alkaline phosphatase (ALP) activity on Day 7, up to 1.5 times more collagen and at least 6 times more Ca deposited in extracellular matrix, in addition to osteocalcin on Day 21 of culture, which strongly indicated the osteoinductive nature of P40 BGMS. This effect was also confirmed through indirect-contact experiments where there was higher collagen and Ca production by hMSCs was observed after 25 days of culture in P40 BGMS-conditioned medium as compared to control (no MS) or P45-conditioned medium. Elemental analysis using Energy Dispersive X-Ray Spectroscopic (EDS) analysis revealed that the Ca-based porogen used in the manufacturing of PGMS, may have been retained on the edges of the pores in PGMS. Therefore, an acid-washing step was introduced at the end of manufacturing process in order to remove the porogen and limit the possible cytotoxic effect of porogen and excess calcium. Characterisation results indicated that acid washing changed the physicality of these microspheres without changing their chemical composition. For example, mean and mode pore window sizes on the surface of PGMS increased from 2.63 μm to 2.73 μm and from 1.15 μm to 1.53 μm, respectively, and closed porosity decreased by 27%, as a result of acid washing. However, more detailed EDS analysis revealed that the Ca-based porogen was not being completely removed from PGMS even after acid washing and this may need further investigation. Cytotoxicity evaluations over 7 days of elution (indirect-contact hMSC experiments) suggested that there was marked improvement in hMSC membrane integrity and metabolic activity in PGMS neat extracts after acid washing. Moreover, direct-contact hMSC experiments also showed higher DNA content on acid washed (AW) P40 PGMS over 7 days of culture. Therefore, based on these results, it was hypothesised that acid washing may have opened up some of the pores and removed some of the glass fragments from PGMS surface, which may have been responsible for cytotoxicity in non-AW PGMS. Direct-contact experiments also showed that over 42-day culture period, there was up to 1.6 times higher hMSC numbers in AW P40 PGMS as compared to P40 BGMS. However, this increase was much lower than the expected range as there was more than 10-fold increase in surface area after the introduction of porosity. This was probably due to presence of <5 μm and <10 μm pore window sizes and interconnection sizes, respectively, in these microspheres, which allowed limited penetration of hMSCs into the porous structures. There was also evidence of at least 2 times more ALP activity up to day 42 of culture and up to 1.7 times more collagen production by day 21 of culture, in case of AW P40 PGMS as compared to P40 BGMS, which strongly indicated a positive effect of porosity on osteogenesis. Interestingly, there was also lower Ca and P deposited by hMSCs in porous microspheres, which was in line with the observations made through indirect-contact experiments, where there was lower collagen and Ca production by hMSCs in P40 PGMS-conditioned medium as compared to P40 BGMS-conditioned medium. This negative effect of PGMS was hypothesised due to excess release of glass fragments/particulates and calcium ions into the medium, possibly leading to cytotoxicity. Based on the results shown here, there is a potential of P40 BGMS and AW P40 PGMS for hMSC-based bone repair therapy. However, future work needs to be done in order to limit the delamination of glass surfaces and release of glass fragments/particulates from these MS, as a result of degradation