Zn-Loaded and Calcium Phosphate-Coated Degradable Silica Nanoparticles Can Effectively Promote Osteogenesis in Human Mesenchymal Stem Cells.

Nanoparticles such as mesoporous bioactive glasses (MBGs) and mesoporous silica nanoparticles (MSN) are promising for use in bone regeneration applications due to their inherent bioactivity. Doping silica nanoparticles with bioinorganic ions could further enhance their biological performance. For example, zinc (Zn) is often used as an additive because it plays an important role in bone formation and development. Local delivery and dose control are important aspects of its therapeutic application. In this work, we investigated how Zn incorporation in MSN and MBG nanoparticles impacts their ability to promote human mesenchymal stem cell (hMSC) osteogenesis and mineralization in vitro. Zn ions were incorporated in three different ways; within the matrix, on the surface or in the mesopores. The nanoparticles were further coated with a calcium phosphate (CaP) layer to allow pH-responsive delivery of the ions. We demonstrate that the Zn incorporation amount and ion release profile affect the nanoparticle's ability to stimulate osteogenesis in hMSCs. Specifically, we show that the nanoparticles that contain rapid Zn release profiles and a degradable silica matrix were most effective in inducing hMSC differentiation. Moreover, cells cultured in the presence of nanoparticle-containing media resulted in the highest induction of alkaline phosphate (ALP) activity, followed by culturing hMSC on nanoparticles immobilized on the surface as films. Exposure to nanoparticle-conditioned media did not increase ALP activity in hMSCs. In summary, Zn incorporation mode and nanoparticle application play an important role in determining the bioactivity of ion-doped silica nanoparticles.

Calcium Phosphate-Coated and Strontium-Incorporated Mesoporous Silica Nanoparticles Can Effectively Induce Osteogenic Stem Cell Differentiation.

Ceramic (nano)materials are promising materials for bone regeneration applications. The addition of bioinorganics such as strontium (Sr) and zinc (Zn) is a popular approach to further improve their biological performance. However, control over ion delivery is important to prevent off-target effects. Mesoporous silica nanoparticles (MSNs) are popular nanomaterials that can be designed to incorporate and controllably deliver multiple ions to steer specific regenerative processes. In this work, MSNs loaded with Sr (MSNSr ) and surface coated with a pH-sensitive calcium phosphate (MSNSr -CaP) or calcium phosphate zinc layer (MSNSr -CaZnP) are developed. The ability of the MSNs to promote osteogenesis in human mesenchymal stromal cells (hMSCs) under basic cell culture conditions is explored and compared to ion administration directly to the cell culture media. Here, it is shown that MSN-CaPs can effectively induce alkaline phosphatase (ALP) levels and osteogenic gene expression in the absence of other osteogenic stimulants, where an improved effect is observed for MSNs surface coated with multiple ions. Moreover, comparatively lower ion doses are needed when using MSNs as delivery vehicles compared to direct ion administration in the medium. In summary, the MSNs developed here represent promising vehicles to deliver (multiple) bioinorganics and promote hMSC osteogenesis in basic conditions.

The shape-effect of calcium phosphate nanoparticle based films on their osteogenic properties.

Calcium phosphates (CaPs) in the form of hydroxyapatite (HA) have been extensively studied in the context of bone regeneration due to their chemical similarity to natural bone mineral. While HA is known to promote osteogenic differentiation, the structural properties of the ceramic have been shown to affect the extent of this effect; several studies have suggested that nanostructured HA can improve the bioactivity. However, the role shape plays in the osteogenic potential is more elusive. Here we studied the effect of HA nanoparticle shape on the ability to induce osteogenesis in human mesenchymal stromal cells (hMSCs) by developing nanoparticle films using needle-, rice- and spherical-shaped HA. We showed that the HA films made from all three shapes of nanoparticles induced increased levels of osteogenic markers (i.e. runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN) on protein and gene level in comparison to hMSCs cultured on cover glass slides. Furthermore, their expression levels and profiles differed significantly as a function of nanoparticle shape. We also showed that nanoparticle films were more efficient in inducing osteogenic gene expression in hMSCs compared to adding nanoparticles to hMSCs in culture media. Finally, we demonstrated that hMSC morphology upon adhesion to the HA nanoparticle films is dependent on nanoparticle shape, with hMSCs exhibiting a more spread morphology on needle-shaped nanoparticle films compared to hMSCs seeded on rice- and spherical-shaped nanoparticle films. Our data suggests that HA nanoparticle films are efficient in inducing hMSC osteogenesis in basic cell culture conditions and that nanoparticle shape plays a vital role in cell adhesion and morphology and extent of induction of osteogenic differentiation.

Guiding mesenchymal stem cell differentiation using mesoporous silica nanoparticle-based films.

The development of smart interfaces that can guide tissue formation is of great importance in the field of regenerative medicine. Nanoparticles represent an interesting class of materials that can be used to enhance regenerative treatments by enabling close control over surface properties and directing cellular responses. Moreover, nanoparticles can be used to provide temporally controlled delivery of (multiple) biochemical compounds. Here, we exploited the cargo loading and surface functionalization properties of mesoporous silica nanoparticles (MSNs) to design films that can guide human mesenchymal stem cell (hMSC) differentiation towards the osteogenic lineage. We developed biocompatible MSN-based films that support stem cell adhesion and proliferation and demonstrated that these MSN films simultaneously allowed efficient local delivery of biomolecules without effecting film integrity. Films loaded with the osteogenesis-stimulating drug dexamethasone (Dex) were able to induce osteogenic differentiation of hMSCs in vitro. Dex delivery from the films led to increased alkaline phosphatase levels and matrix mineralization compared to directly supplementing Dex to the medium. Furthermore, we demonstrated that Dex release kinetics can be modulated using surface modifications with supported lipid bilayers. Together, these data demonstrate that MSN films represent an interesting approach to create biomaterial interfaces with controllable biomolecule release and surface properties to improve the bioactivity of biomaterials. STATEMENT OF SIGNIFICANCE: Engineering surfaces that can control cell and tissue responses is one of the major challenges in biomaterials-based regenerative therapies. Here, we demonstrate the potential of mesoporous silica nanoparticles (MSNs) as drug-delivering surface coatings. First, we show differentiation of mesenchymal stem cells towards the bone lineage when in contact with MSN films loaded with dexamethasone. Furthermore, we demonstrate that modification of MSNs with supported lipid bilayer allows control over drug release dynamics and cell shape. Given the range of loadable cargos and the tunability of release kinetics, MSN coatings can be used to mimic the sequential appearance of bioactive factors during tissue regeneration, which will ultimately lead to biomaterials with improved bioactivity.

Lipid surface modifications increase mesoporous silica nanoparticle labeling properties in mesenchymal stem cells.

BACKGROUND: Nanoparticles have emerged as promising cell-labeling tools, as they can be precisely tailored in terms of chemical and physical properties. Mesoporous silica nanoparticles (MSNs), in particular, are easily tunable with regard to surface and core chemistry, and are able to confine dyes and drug molecules efficiently. PURPOSE: The aim of this study was to investigate the effect of lipid and polyethylene glycol (PEG) surface modifications on MSN stem-cell-tracking abilities. METHODS: Lipid and PEG surface functionalized MSNs were synthesized and the effect of surface functionalization on cell internalization, proliferation, differentiation and cell proteomics was investigated in patient derived mesenchymal stem cells (MSCs). RESULTS: MSNs and lipid surface-modified MSNs were internalized by >80% of the MSC population, with the exception of nanoparticles modified with short PEG chains (molecular weight 750 [MSN-PEG750]). Lipid-modified MSNs had higher labeling efficiency with maximum uptake after 2 hours of exposure and were in addition internalized 17 times higher compared to unmodified MSNs, without negatively affecting differentiation capacity. Using a mass-spectrometry-based label-free quantitative proteomics approach, we show that MSN labeling leads to the up- and downregulation of proteins that were unique for the different surface-modified MSNs. In addition, functional enrichments were found in human MSCs labeled with MSNs, MSN-PEG750, and lipid-modified MSNs. SUMMARY: Here we show that organic modifications with lipids and PEGylation can be used as a promising strategy to improve MSN labeling capabilities. In particular, we show that lipid modifications can optimize such probes in three distinct ways: significantly improved signal strength, a barrier for sustained release of additional probes, and improved stem-cell-labeling efficiency.