Mohamed Eltohamy

Efficacy of mesoporous silica nanoparticles in delivering BMP-2 plasmid DNA for in vitro osteogenic stimulation of mesenchymal stem cells.

We report the ability of aminated mesoporous silica nanoparticles (MSN-NH2) with large mesopore space and positive-charged surface to deliver genes within rat mesenchymal stem cells (MSCs). The amine functionalized inorganic nanoparticles were complexed with bone morphogenetic protein-2 (BMP2) plasmid DNA (pDNA) to study their transfection efficiency in MSCs. Intracellular uptake of the complex BMP2 pDNA/MSN-NH2 occurred significantly, with a transfection efficiency of approximately 68%. Furthermore, over 66% of the transfected cells produced BMP2 protein. The osteogenic differentiation of the transfected MSCs was demonstrated by the expression of bone-related genes and proteins including bone sialoprotein, osteopontin, and osteocalcin. The MSN-NH2 delivery vehicle for BMP2 pDNA developed in this study may be a potential gene delivery system for bone tissue regeneration.

Therapeutic foam scaffolds incorporating biopolymer-shelled mesoporous nanospheres with growth factors.

A novel therapeutic scaffolding system of engineered nanocarriers within a foam matrix for the long-term and sequential delivery of growth factors is reported. Mesoporous silica nanospheres were first functionalized to have an enlarged mesopore size (12.2nm) and aminated surface, which was then shelled by a biopolymer, poly(lactic acid) (PLA) or poly(ethylene glycol) (PEG), via electrospraying. The hybrid nanocarrier was subsequently combined with collagen to produce foam scaffolds. Bovine serum albumin (BSA), used as a model protein, was effectively loaded within the enlarged nanospheres. The biopolymer shell substantially prolonged the release period of BSA (2-3weeks from shelled nanospheres vs. within 1week from bare nanospheres), and the release rate was highly dependent on the shell composition (PEG>PLA). Collagen foam scaffolding of the shelled nanocarrier further slowed down the protein release, while enabling the incorporation of a rapidly releasing protein, which is effective for sequential protein delivery. Acidic fibroblast growth factor (aFGF), loaded onto the shelled-nanocarrier scaffolds, was released over a month at a highly sustainable rate, profiling a release pattern similar to that of BSA. The biological activity of the aFGF was evidenced by the significant proliferation of osteoblastic precursor cells in the aFGF-releasing scaffolds. Furthermore, the aFGF-delivering scaffolds implanted in rat subcutaneous tissue for 2weeks showed a substantially enhanced invasion of fibroblasts with a homogeneous population. Taken together, it is concluded that the biopolymer encapsulation of mesoporous nanospheres effectively prolongs the release of growth factors over weeks to a month, providing a nanocarrier platform for a long-term growth factor delivery. Moreover, the foam scaffolding of the nanocarrier system is a potential therapeutic three-dimensional matrix for cell culture and tissue engineering.

Dynamic Mechanical and Nanofibrous Topological Combinatory Cues Designed for Periodontal Ligament Engineering

Complete reconstruction of damaged periodontal pockets, particularly regeneration of periodontal ligament (PDL) has been a significant challenge in dentistry. Tissue engineering approach utilizing PDL stem cells and scaffolding matrices offers great opportunity to this, and applying physical and mechanical cues mimicking native tissue conditions are of special importance. Here we approach to regenerate periodontal tissues by engineering PDL cells supported on a nanofibrous scaffold under a mechanical-stressed condition. PDL stem cells isolated from rats were seeded on an electrospun polycaprolactone/gelatin directionally-oriented nanofiber membrane and dynamic mechanical stress was applied to the cell/nanofiber construct, providing nanotopological and mechanical combined cues. Cells recognized the nanofiber orientation, aligning in parallel, and the mechanical stress increased the cell alignment. Importantly, the cells cultured on the oriented nanofiber combined with the mechanical stress produced significantly stimulated PDL specific markers, including periostin and tenascin with simultaneous down-regulation of osteogenesis, demonstrating the roles of topological and mechanical cues in altering phenotypic change in PDL cells. Tissue compatibility of the tissue-engineered constructs was confirmed in rat subcutaneous sites. Furthermore, in vivo regeneration of PDL and alveolar bone tissues was examined under the rat premaxillary periodontal defect models. The cell/nanofiber constructs engineered under mechanical stress showed sound integration into tissue defects and the regenerated bone volume and area were significantly improved. This study provides an effective tissue engineering approach for periodontal regeneration—culturing PDL stem cells with combinatory cues of oriented nanotopology and dynamic mechanical stretch.

Collagen gel combined with mesoporous nanoparticles loading nerve growth factor as a feasible therapeutic three-dimensional depot for neural tissue engineering

Three-dimensional matrices with controllable and sustainable delivery capacity of neurotrophic factors are promising platforms for neural tissue engineering. Here we developed a nerve growth factor (NGF) delivering cell culture system involving mesoporous silica nanoparticles (MSNs) combined with collagen hydrogel. Particularly for the loading of large protein molecules, the mesopores of MSNs were exploited to a size as large as about 16 nm with the help of an auxiliary surfactant. Loading of proteins was confirmed within the enlarged mesopores with a high loading capacity. The NGF-loaded MSNs were combined with collagen hydrogel by a temperature-mediated gelation which was used as the delivery system of NGF as well as the culture matrix for neural cells. The NGF loaded onto MSN was shown to release sustainably for over a week. When the NGF-loaded MSN was embedded within a collagen gel, the release amount of NGF was more sustained and followed the release pattern of NGF from MSN. A model study to examine the biological efficacy of the NGF release was performed using PC12 cells with culturing cells either outside or within the hydrogel. When cultured outside of the system, the neurite outgrowth of cells was significantly improved, confirming biological action of the NGF delivered from the MSN–NGF loaded collagen. Gene expression of growth-associated protein, GAP43, was significantly up-regulated, demonstrating the system is effective in delivering NGF to elicit biological activity of the growth factor. Furthermore, when cultured within the MSN–NGF–collagen hydrogel matrix, cells were stimulated to undergo neuritogenesis while preserving the cell viability, confirming the effectiveness for neural tissue engineering. The novel delivery system of NGF utilizing enlarged-pored MSNs in combination with collagen hydrogel may be potentially useful as therapeutic neural engineering matrices and the concept can also be extended to other growth factor delivering systems.

Electrospinning Technology in Tissue Regeneration

Electrospinning is one of the most versatile and effective tools to produce nanostructured fibers in the biomedical science fields. The nanofibrous structure with diameters from tens to hundreds of nanometers largely mimics the native extracellular matrix (ECM) of many tissues. Thus far, a range of compositions including polymers and ceramics and their composites/hybrids have been successfully applied for generating electrospun nanofibers. Different processing tools in electrospinning set-ups and assemblies are currently developed to tune the morphology and properties of nanofibers. Herein, we demonstrate the electrospinning process and the electrospun biomaterials for specific use in tissue regeneration with some examples, involving different material combinations and fiber morphologies.

Sol–gel-derived bioactive glass nanoparticle-incorporated glass ionomer cement with or without chitosan for enhanced mechanical and biomineralization properties

OBJECTIVE This study investigated the mechanical and in vitro biological properties (in immortalized human dental pulp stem cells (ihDPSCs)) of bioactive glass nanoparticle (BGN)-incorporated glass ionomer cement (GIC) with or without chitosan as a binder. METHODS After the BGNs were synthesized and characterized, three experimental GICs and a control (conventional GIC) that differed in the additive incorporated into a commercial GIC liquid (Hy-bond, Shofu, Japan) were produced: BG5 (5wt% of BGNs), CL0.5 (0.5wt% of chitosan), and BG5+CL0.5 (5wt% of BGNs and 0.5wt% of chitosan). After the net setting time was determined, weight change and bioactivity were analyzed in simulated body fluid (SBF) at 37°C. Mechanical properties (compressive strength, diametral tensile strength, flexural strength and modulus) were measured according to the incubation time (up to 28 days) in SBF. Cytotoxicity (1day) and biomineralization (14 days), assessed by alizarin red staining, were investigated using an extract from GIC and ihDPSCs. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukeys post hoc test; p<0.05. RESULTS BGNs were sol-gel synthesized to be approximately 42nm in diameter with a spherical morphology and amorphous structure. After the bioactivity and suspension ability of the BGNs were confirmed, all the experimental GIC groups had setting times of less than 6min and approximately 1% weight loss after 28days of incubation. In addition, BGNs incorporated into GIC (BG5 and BG5+CL0.5) exhibited surface bioactivity. The mechanical properties were increased in the BGN-incorporated GICs compared to those in the control (p<0.05). Without cytotoxicity, the biomineralization capacity was ranked in the order BG5, BG5+CL0.5, control, and CL0.5 (p<0.05). SIGNIFICANCE BGN-incorporated GIC showed enhanced mechanical properties such as compressive, diametral tensile and flexural strength as well as in vitro biomineralization properties in ihDPSCs without cytotoxicity. Therefore, the developed BGN-incorporated GIC is a promising restorative dental material, although further in vivo investigation is needed before clinical application.

Ionic and thermo-switchable polymer-masked mesoporous silica drug-nanocarrier: High drug loading capacity at 10 °C and fast drug release completion at 40 °C

The preparation of the ideal smart drug-delivery systems were successfully achieved by the in situ co-polymerization of a vinyl group-functionalized mesoporous silica nanoparticle (f-MSN) with 1-butyl-3-vinyl imidazolium bromide (BVIm) and N-isopropylacrylamide (NIPAAm) monomers. The thickness of the capping copolymer layer, poly(NIPAAm-co-BVIm) (p-NIBIm), was controlled at between 2.5nm and 5nm, depending on the monomers/f-MSN ratio in the reaction solution. The finally obtained smart drug-delivery systems are named as p-MSN2.5 and p-MSN5.0 (MSNs integrated by 2.5nm and 5nm p-NIBIm layer in thickness). The key roles of the mesoporous-silica-nanoparticle (MSN) core and the p-NIBIm shell are drug-carrying (or containing) and pore-capping, respectively, and the latter has an on/off function that operates in accordance with temperature changes. According to the swelling- or shrinking-responses of the smart capping copolymer to temperature changes between 10°C and 40°C, the loading and releasing patterns of the model drug cytochrome c were studied in vitro. The developed system showed interesting performances such as a cytochrome-c-loading profile (loading capacity for 3h=26.3% and 19.8% for p-MSN2.5 and p-MSN5.0, respectively) at 10°C and a cytochrome-c-releasing profile (releasing efficiency=>95% within 3 days and 4 days for p-MSN2.5 and p-MSN5.0, respectively) at 40°C. The cytotoxicity of the drug delivery systems, p-MSN2.5 and p-MSN5.0 (in the concentration range of <0.125mg/mL without drug), for human embryonic kidney (HEK 293) cells were minimal in vitro compared with that of a blank MSN. These results may be reasonably applied in the field of specified drug delivery.

Nanocomposite scaffolds incorporated with hydrophobically-functionalized mesoporous nanocarriers for the effective loading and long-term delivery of osteogenic drugs

Development of scaffolds with a delivery potential of drugs in a sustained and controlled manner is of special importance to achieve favorable tissue responses and to maximize tissue regeneration capacity. Here we design novel nanocomposite fibrous scaffolds made of polycaprolactone (PCL) biopolymer incorporating surface-functionalized mesoporous nanospheres which are to carry hydrophobic osteogenic drug for a long-term therapeutic purpose in bone regeneration. After tailoring the surface of nanospheres hydrophobically, the target drug dexamethasone (Dex) was shown to incorporate effectively (as high as ∼10 wt% loading efficiency), which was essentially released over a-week-period. When the Dex-nanoparticle complex was incorporated within PCL fiber matrix, the Dex release was continued over a month without showing an initial burst effect. The designed scaffolds sustainably delivering Dex drug demonstrated the therapeutic efficacy in vitro. Bone marrow mesenchymal stem cells cultured on the Dex-loaded scaffolds were substantially stimulated in the initial proliferation phase. Furthermore, osteogenic differentiation and cellular mineralization were significantly improved by the Dex delivery over a culture period of 21 days. Results demonstrated the novel nanocomposite fiber scaffolds are effective in loading hydrophobic Dex at large quantity and subsequently deliver over a long-term period, ultimately finding a potential therapeutic scaffold platform for bone regeneration.

Biomimetic Designing of Functional Silk Nanotopography Using Self-assembly

In nature inorganic-organic building units create multifunctional hierarchical architectures. Organic silk protein is particularly attractive in this respect because of its micro-nanoscale structural blocks that are attributed to sophisticated hierarchical assembly imparting flexibility and compressibility to designed biohybrid materials. In the present study, aqueous silk fibroin is assembled to form nano/microtopography on inorganic silica surface via a facile diffusion-limited aggregation process. This process is driven by electrostatic interaction and only possible at a specified aminated surface chemistry. The self-assembled topography depends on the age and concentration of protein solution as well as on the surface charge distribution of the template. The self-assembled silk trails closely resemble natural cypress leaf architecture, which is considered a structural analogue of neuronal cortex. This assembled surface significantly enhances anchorage of neuronal cell and cytoskeletal extensions, providing an effective nano/microtopographical cue for cellular recognition and guidance.