Ahmed El-Fiqi

Collagen hydrogels incorporated with surface-aminated mesoporous nanobioactive glass: Improvement of physicochemical stability and mechanical properties is effective for hard tissue engineering.

Collagen (Col) hydrogels have poor physicochemical and mechanical properties and are susceptible to substantial shrinkage during cell culture, which limits their potential applications in hard tissue engineering. Here, we developed novel nanocomposite hydrogels made of collagen and mesoporous bioactive glass nanoparticles (mBGns) with surface amination, and addressed the effects of mBGn addition (Col:mBG = 2:1, 1:1 and 1:2) and its surface amination on the physicochemical and mechanical properties of the hydrogels. The amination of mBGn was shown to enable chemical bonding with collagen molecules. As a result, the nanocomposite hydrogels exhibited a significantly improved physicochemical and mechanical stability. The hydrolytic and enzymatic degradation of the Col-mBGn hydrogels were slowed down due to the incorporation of mBGn and its surface amination. The mechanical properties of the hydrogels, specifically the resistance to loading as well as the stiffness, significantly increased with the addition of mBGn and its aminated form, as assessed by a dynamic mechanical analysis. Mesenchymal stem cells cultivated within the Col-mBGn hydrogels were highly viable, with enhanced cytoskeletal extensions, due to the addition of surface aminated mBGn. While the Col hydrogel showed extensive shrinkage (down to ∼20% of initial size) during a few days of culture, the shrinkage of the mBGn-added hydrogel was substantially reduced, and the aminated mBGn-added hydrogel had no observable shrinkage over 21 days. Results demonstrated the effective roles of aminated mBGn in significantly improving the physicochemical and mechanical properties of Col hydrogel, which are ultimately favorable for applications in stem cell culture for bone tissue engineering.

Osteoinductive fibrous scaffolds of biopolymer/mesoporous bioactive glass nanocarriers with excellent bioactivity and long-term delivery of osteogenic drug.

Designing scaffolds with bioactive composition and long-term drug delivery capacity is a promising method to improve the therapeutic efficacy in bone regeneration. Herein, electrospun fibrous scaffolds of polycaprolactone-gelatin incorporating mesoporous bioactive glass nanoparticles (mBGn) were proposed to be excellent matrix platforms for bone tissue engineering. In particular, the mBGn were loaded with osteogenic drug Dexamethasone (DEX) to elicit additional therapeutic potential. The mBGn-added fiber scaffolds demonstrated excellent properties, including improved mechanical tensile strength, elasticity, and hydrophilicity compared to pure biopolymer matrix. The scaffolds could release substantial amounts of calcium and silicate ions. The loading of DEX onto mBGn was as high as 63%, that is, 0.63 mg DEX loaded per 1 mg of mBGn, demonstrating an effective nanodepot role of the mBGn. The release of DEX from the mBGn-added fiber scaffolds was highly sustainable, profiling an almost linear release kinetics up to the test period of 28 days, after a rapid initial release of ∼30% within 24 h. The proliferation and osteogenic differentiation of stem cells derived from periodontal ligament were significantly improved by the mBGn incorporation and synergistically stimulated with DEX loading, as confirmed by both direct and indirect cultures. The effects on bone regeneration in vivo, as analyzed by microcomputed tomography and histological stains in a rat calvarium model over 6 weeks, were substantial with the mBGn incorporation and even better with DEX loading, evidencing the osteogenic effects of the drug-eluting nanocomposite fiber scaffolds in bone formation. The current scaffolds with bone-bioactive composition and drug delivery capacity may be potentially useful for bone regeneration as novel osteogenic matrices.

Therapeutic bioactive microcarriers: co-delivery of growth factors and stem cells for bone tissue engineering.

Novel microcarriers made of sol-gel-derived bioactive glasses were developed for delivering therapeutic molecules effectively while cultivating stem cells for bone tissue engineering. Silica sols with varying concentration of Ca (0-30 mol.%) were formulated into microspheres ranging from 200 to 300 μm under optimized conditions. A highly mesoporous structure was created, with mesopore sizes of 2.5-6.3 nm and specific surface areas of 420-710 m(2)g(-1), which was highly dependent on the Ca concentration. Therapeutic molecules could be effectively loaded within the mesoporous microcarriers during microsphere formulation. Cytochrome C (cyt C), used as a model protein for the release study, was released in a highly sustainable manner, with an almost zero-order kinetics over a period of months; the amount released was ~2% at 9 days, and 15% at 40 days. A slight increase in the release rate was observed in the microcarrier containing Ca, which was related to the dissolution rate and pore size. The presence of Ca accelerated the formation of hydroxyapatite on the surface of the microcarriers. Cells cultured on the bioactive microcarriers were well adhered and distributed, and proliferated actively, confirming the three-dimensional substrate role of the microcarriers. An in vivo study performed in a rat subcutaneous model demonstrated the satisfactory biocompatibility of the prepared microspheres. As a therapeutic target molecule, basic fibroblast growth factor (bFGF) was incorporated into the microcarriers. A slow release pattern similar to that of cyt C was observed for bFGF. Cells adhered and proliferated to significantly higher levels on the bFGF-loaded microcarriers, demonstrating the effective role of bFGF in cell proliferative potential. It is believed that the developed mesoporous bioactive glass microspheres represent a new class of therapeutic cell delivery carrier, potentially useful in the sustainable delivery of therapeutic molecules such as growth factors, as well as in the support of stem cell proliferation and osteogenesis for bone tissue engineering.

Chitosan–nanobioactive glass electrophoretic coatings with bone regenerative and drug delivering potential

Nanocomposites with bone-bioactivity and drug eluting capacity are considered as potentially valuable coating materials for metallic bone implants. Here, we developed composite coatings of chitosan (CH)–bioactive glass nanoparticles (BGn) via cathodic electrophoretic deposition (EPD). BGn 50–100 nm in size with aminated surface were suspended with CH molecules at different ratios (5–20 wt% BGn) in aqueous medium, and EPD was performed. Uniform coatings with thicknesses of a few to tens of micrometers were produced, which was controllable by the EPD parameters (voltage, pH and time). Thermogravimetric analysis revealed the quantity of BGn within the coatings that well corresponded to that initially incorporated. Apatite forming ability of the coatings, performed in simulated body fluid, was significantly improved by the addition of BGn. Degradation of the coatings increased with increasing BGn addition. Of note, the degradation profile was almost linear with time; degradation of 5–13 wt% during 1 week became 30–40 wt% after 7 weeks at almost a constant rate. The CH–BGn coatings showed favorable cell adhesion and growth, and stimulated osteogenic differentiation. Drug loading and release capacity of the CH–BGn coatings were performed using the ampicillin antibiotic as a model drug. Ampicillin, initially incorporated within the CH–BGn suspension, was eluted from the coatings continuously over 10–11 weeks, confirming long-term drug delivering capacity. Antibacterial tests also confirmed the effects of released ampicillin using agar diffusion assay against Streptococcus mutants. The CH–BGn may be potentially useful as a coating composition for metallic implants due to the excellent bone bioactivity and cell responses, as well as the capacity for long-term drug delivery.

Biointerface control of electrospun fiber scaffolds for bone regeneration: Engineered protein link to mineralized surface

Control over the interface of biomaterials that favors the initial adhesion and subsequent differentiation of stem cells is one of the key strategies in bone tissue engineering. Here we engineer the interface of biopolymer electrospun fiber matrices with a fusion protein of fibronectin 9-10 domain (FNIII9-10) and osteocalcin (OCN), aiming to stimulate mesenchymal stem cell (MSC) functions, including initial adhesion, growth and osteogenic differentiation. In particular, a specific tethering of FNIII9-10-OCN protein was facilitated by the hydroxyapatite (HA) mineralization of the biopolymer surface through a molecular recognition of OCN to the HA crystal lattice. The FNIII9-10-OCN anchorage to the HA-mineralized fiber was observed to be highly specific and tightly bound to preserve stability over a long period. Initial cell adhesion levels, as well as the spreading shape and process, of MSCs within 24h were strikingly different between the fibers linked with and without fusion protein. Significant up-regulations in the mRNA expression of adhesion signaling molecules occurred with the fusion protein link, as analyzed by the reverse transcriptase polymerase chain reaction. The expression of a series of osteogenic-related genes at later stages, over 2-3weeks, was significantly improved in the fusion protein-tailored fiber, and the osteogenic protein levels were highly stimulated, as confirmed by immunofluorescence imaging and fluorescence-activated cell sorting analyses. In vivo study in a rat calvarium model confirmed a higher quantity of new bone formation in the fiber linked with fusion protein, and a further increase was noticed when the MSCs were tissue-engineered with the fusion protein-linked fiber. Collectively, these results indicate that FN-OCN fusion protein links via HA mineralization is a facile tool to generate a biointerface with cell-attractive and osteogenic potential, and that the engineered fibrous matrix is a potential bone regenerative scaffold.

Mesoporous bioactive nanocarriers in electrospun biopolymer fibrous scaffolds designed for sequential drug delivery

Here we communicate a novel design to deliver multiple drugs from scaffolds which have special therapeutic efficacy for the repair and regeneration of hard tissues. A sequential release of multiple drugs (a rapid release of drug 1 accompanied by a slow release of drug 2) was enabled by pre-loading drug 2 within mesoporous bioactive glass nanospheres (mBGn) which were added up to 30% to a polymer (polycaprolactone–gelatin) fiber matrix that has also encapsulated drug 1. In particular, excellent bioactive properties of mBGn, i.e., induction of bone mineral-like apatite formation and release of therapeutic ions (calcium and silicon) potentiate the usefulness of the mBGn-added scaffolds for bone regeneration. Proof-of-concept study utilizing two model drugs within the mBGn-added fiber (procaine hydrochloride (PCH) in mBGn and tetracycline hydrochloride (TCH) in nanofiber) demonstrated a typical sequential release pattern of the drugs, i.e., a rapid release of TCH within 24 h while a sustainable and long-term release of PCH over weeks to a month. Although biological efficacy of the drug-delivering scaffolds warrants further study, this finding suggests the mBGn-added polymer fiber may be a potential therapeutic matrix for bone regeneration.

Odontogenic stimulation of human dental pulp cells with bioactive nanocomposite fiber

The aim of the present study was to investigate the effects of a composite nanofibrous matrix made of biopolymer blend polycaprolactone-gelatin (BP) and mesoporous bioactive glass nanoparticles (BGNs) on the odontogenic differentiation of human dental pulp cells (HDPCs). BGN-BP nanomatrices, with BGN content of up to 20 wt%, were produced via electrospinning. The differentiation of the HDPCs was evaluated by using an ALP activity assay, calcified nodule formation, and mRNA expression for markers. Integrin and its underlying signal pathways were assessed via reverse transcriptase-polymerase chain reaction and Western blot analysis. Although cell growth and attachment on the BGN-BP nanomatrix was similar to that on BP, ALP activity, mineralized nodule formation, and mRNA, expressions involving ALP, osteocalcin, osteopontin, dentin sialophosphoprotein, and dentin matrix protein-1 were greater on BGN-BP. BGN-BP upregulated the key adhesion receptors (integrin components α1, α2, α5, and β1) and activated integrin downstream pathways, such as phosphorylated-focal adhesion kinase (p-FAK), and p-paxillin. In addition, BGN-BP activated BMP receptors, BMP-2 mRNA, and p-Smad 1/5/8, and such activation was blocked by the BMP antagonist, noggin. Furthermore, BGN-BP induced phosphorylation of extracellular signal-regulated kinase, protein kinase 38, and c-Jun-N-terminal kinase mitogen-activated protein kinases and activated expression of the transcription factors Runx2 and Osterix in HDPCs. Collectively, the results indicated for the first time that a BGN-BP composite nanomatrix promoted odontogenic differentiation of HDPCs through the integrin, BMP, and mitogen-activated protein kinases signaling pathway. Moreover, the nanomatrix is considered to be promising scaffolds for the culture of HDPCs and dental tissue engineering.

Novel bioactive nanocomposite cement formulations with potential properties: incorporation of the nanoparticle form of mesoporous bioactive glass into calcium phosphate cements

Injectable calcium phosphate cements (CPCs) with strong mechanical properties and improved biological performance have the potential to be extensively used for bone regeneration. Although many additive materials have been incorporated into CPCs in order to achieve improvements in their mechanical and biological properties, somehow the results have not been fully satisfactory. Here we focus on using the nanoparticle form of mesoporous bioactive glasses (mBGn) as additive nano-components for alpha-tricalcium phosphate-based CPCs. The effects of mBGn incorporated up to 10 wt% into CPCs were examined in depth with respect to the setting time, morphology, injectability, wash-out properties, consistency, ionic release, pH change, and mechanical strength. The addition of mBGn significantly increased the surface area (for both the as-cemented and the hydrated compositions) and also significantly accelerated the setting reaction of CPCs. The injectability and the anti-washout property of CPCs were remarkably enhanced with the addition of mBGn. In striking contrast to the case of pure CPCs, the morphological changes observed in simulated body fluid (SBF) revealed a spherical development of apatite crystals, replicating the nanospherical morphology of the mBGn and consequently resulting in a nano-micro-roughened surface. The mechanical compressive strength substantially increased after SBF immersion and significantly higher values were recorded for mBGn/CPC as compared to pure CPCs. The ion release, including that of calcium, phosphate, and silicon, was recorded at substantial levels during the test period, and the addition of mBGn caused changes in the pH towards less acidic. The in vivo study of the mBGn/CPCs in rat subcutaneous tissue confirmed excellent tissue compatibility with little evidence of inflammatory reactions while exhibiting viable fibroblastic cells with a substantial presence of mature endothelial cells surrounding the cements. When implanted in a rat calvarium defect, a substantial degradation of the samples was noticed in the interfacial region. The proposed mBGn/CPC is a novel, promising cement formulation for the repair and regeneration of bone due to setting characteristics, physico-chemical and mechanical properties, and excellent in vivo tissue compatibility and bioactivity.

Effects of bioactive cements incorporating zinc-bioglass nanoparticles on odontogenic and angiogenic potential of human dental pulp cells

Background The objective of this study was to investigate the effects of bioactive calcium phosphate cements (CPC, α-tricalcium phosphate-based) incorporating zinc-bioglass (ZnBG) on the odontogenic differentiation and angiogenesis of human dental pulp cells (HDPCs). Methods BGs with varying concentrations of Zn (0, 2.5 and 5%) were produced via a sol-gel process. The proliferation of HDPCs on CPC/BGs was determined by MTS assay. Alizarin red staining, RT-PCR, and ALP activity were used to assess odontogenic differentiation, and western blot analysis was used to asses signaling pathways. In vitro angiogenesis was examined via mRNA expression of angiogenic genes and tubule formation. Results All cement formulations showed no cytotoxicity. The CPCs with ZnBG showed increased ALP activity, enhanced formation of mineralized nodules, and upregulated mRNA expression of DMP-1, DSPP, Runx2, and osterix in a time- and dose-dependent manner, relative to CPCs without Zn. ZnBG upregulated integrins α1, α2, β1, and β3 and activated integrin downstream signal pathways, such as p-FAK, p-Akt, p-paxillin, RhoA, MAPK, and NF-κB, as well as canonical and non-canonical Wnt signaling. In addition, ZnBG upregulated VEGF mRNA in HDPCs and increased the tubular structure in endothelial cells. Conclusions Our results demonstrate that ZnBG incorporated within CPCs activates odontogenic differentiation and promotes angiogenesis in vitro through integrin, Wnt, MAPK, and NF-κB pathways. Thus, CPCs incorporating ZnBG are promising matrices in tissue engineering to stimulate endodontic regeneration.

Development of long-term antimicrobial poly(methyl methacrylate) by incorporating mesoporous silica nanocarriers.

OBJECTIVE Poly(methyl methacrylate) (PMMA) used as removable denture bases or orthodontic appliances has relatively poor antimicrobial properties, which accelerate oral infection and induce unfavorable odors. Mesoporous silica nanoparticles (MSNs) have been highlighted as a potential additive to overcome this issue because of their drug-loading capacity. Here, we present the long-term antimicrobial effect of MSN-incorporated PMMA with drug-loading capacity. METHODS After the MSNs were characterized, MSN incorporation into chemically activated PMMA (0.5, 1, 2.5 or 5wt%) relative to the methyl methacrylate powder by mass was fabricated into a rectangular specimen (1.4×3.0×19.0mm) for a 3-point flexural test at a speed of 1mm/min or a disk (∅=11.5mm and d=1.5mm) for investigation of its antimicrobial effects. RESULTS A typical spherical morphology with a well-ordered mesoporous structure of the MSNs was visualized and is beneficial for loading drugs and combining in matrixes. Among the tested levels of MSN incorporation in PMMA (0.5, 1, 2.5 or 5wt%), only 5wt% decreased the flexural strength (p<0.05), whereas the flexural modulus was not significantly decreased (p>0.05). The surface roughness and surface energy were increased with 2.5wt% or 5wt% incorporation. An anti-adherent effect against Candida albicans and Streptococcus oralis after 1h of attachment was only observed with 2.5 and 5wt% incorporation compared to a lack of MSNs (p<0.05). A long-term antimicrobial effect was observed for 2 weeks with 2.5wt% MSN-incorporated PMMA when amphotericin B was loaded into the MSNs on the PMMA surface. SIGNIFICANCE The long-term antimicrobial performance after loading amphotericin B into the MSN-incorporated PMMA suggests the potential clinical usefulness of MSN-incorporated PMMA resin.