Ammar T. Qureshi

Electrospun Bio-Nanocomposite Scaffolds for Bone Tissue Engineering by Cellulose Nanocrystals Reinforcing Maleic Anhydride Grafted PLA

Electrospun fibrous bio-nanocomposite scaffolds reinforced with cellulose nanocrystals (CNCs) were fabricated by using maleic anhydride (MAH) grafted poly(lactic acid) (PLA) as matrix with improved interfacial adhesion between the two components. Morphological, thermal, mechanical, and in vitro degradation properties as well as basic cytocompatibility using human adult adipose derived mesenchymal stem cells (hASCs) of MAH grafted PLA/CNC (i.e., MPLA/CNC) scaffolds were characterized. Morphological investigation indicated that the diameter and polydispersity of electrospun MPLA/CNC nanofibers were reduced with the increased CNC content. The addition of CNCs improved both the thermal stability and mechanical properties of MPLA/CNC composites. The MPLA/CNC scaffolds at the 5 wt % CNC loading level showed not only superior tensile strength (more than 10 MPa), but also improved stability during in vitro degradation compared with the MPLA and PLA/CNC counterparts. Moreover, the fibrous MPLA/CNC composite scaffolds were non-toxic to hASCs and capable of supporting cell proliferation. This study demonstrates that fibrous MPLA/CNC bio-nanocomposite scaffolds are biodegradable, cytocompatible, and possess useful mechanical properties for bone tissue engineering.

Silver Nanoscale Antisense Drug Delivery System for Photoactivated Gene Silencing

The unique photophysical properties of noble metal nanoparticles contribute to their potential as photoactivated drug delivery vectors. Here we demonstrate the synthesis and characterization of 60-80 nm silver nanoparticles (SNPs) decorated with thiol-terminated photolabile DNA oligonucleotides. In vitro assays and fluorescent confocal microscopy of treated cell cultures show efficient UV-wavelength photoactivation of surface-tethered caged ISIS2302 antisense oligonucleotides possessing internal photocleavable linkers. As a demonstration of the advantages of these novel nanocarriers, we investigate properties including: enhanced stability to nucleases, increased hybridization activity upon photorelease, and efficient cellular uptake as compared to commercial transfection vectors. Their potential as multicomponent delivery agents for oligonucleotide therapeutics is shown through regulation of ICAM-1 (Intracellular Adhesion Molecule-1) silencing. Our results suggest a means to achieve light-triggered, spatiotemporally controlled gene silencing via nontoxic silver nanocarriers, which hold promise as tailorable platforms for nanomedicine, gene expression studies, and genetic therapies.

miR-148b-nanoparticle conjugates for light mediated osteogenesis of human adipose stromal/stem cells.

Delivery systems providing spatial and temporal control have the potential to improve outcomes in surgical reconstruction and regenerative medicine by precise modulation of wound healing and tissue repair processes. In this study we describe a synthesis and oligonucleotide functionalization process of silver nanoparticle complexes for photoactivated microRNA (miRNA) delivery. The activity of the PC-miR-148b-SNP construct is demonstrated by light mediated delivery of miR-148b mimic resulting in differentiation of human autologous adipose derived mesenchymal stromal/stem cells (hASCs) into an osteogenic linage. The conjugate, upon photoactivation, increases alkaline phosphatase (ALP) activity in the cell membrane and calcification (mineralization) of hASCs on days 7 and 14 respectively. Additionally, the expression of mRNA for the early, middle and late stage osteogenic markers; ALP, RunX2 and osteocalcin (OCN) respectively, was also significantly upregulated at days 7 and 28, respectively after photoactivation of PC-miR-148b-SNP and release of miR-148b mimics. Additionally, PC-miR-148b-SNP conjugate is readily delivered to the intracellular compartment without the use of transfection vectors commonly required for free oligonucleotides. This technology demonstrates photo-controlled, spatial and temporal modulation of osteogenesis in hASCs.

Photoactivated miR-148b–nanoparticle conjugates improve closure of critical size mouse calvarial defects

Inducible systems providing temporal control of differentiation have the potential to improve outcomes in surgical reconstruction and regenerative medicine by precise modulation of wound healing and tissue repair processes. The aim of this study was to demonstrate that nanoformulated microRNA (miRNA) conjugates activated via photo exposure can lead to the induced osteogenic differentiation of human adipose-derived stromal/stem cells (hASCs) in vivo. The conjugate PC-miR-148b-SNP, a mimic of miRNA-148b tethered to silver nanoparticles (SNPs) via a photolabile linker, was used to modulate gene expression for improved closure of a critical size defect drilled on the right parietal bone of male CD-1 nude homozygous mice. The PC-miR-148b-SNP conjugates added to hASCs and loaded to either Matrigel or polycaprolactone (PCL) scaffolds resulted in different levels of healing of the defect. After 4 and 12weeks, 3-D micro-computed tomography reconstructed images indicate statistically significant defect closure from 3.83±1.19% to 5.46±2.01% and 6.54±4.28% to 32.53±8.3% for non-photoactivated and photoactivated conjugates, respectively, in the PCL scaffolds. The results were confirmed with H&E and Massons Trichrome stains in the transverse sections of photoactivated conjugates. Collagen fiber staining was greatest at 12weeks when it reached approximately the same density and thickness as the native calvarium. This technology provides a platform that can be used with other miRNAs that actively govern the pathways responsible for regenerative and wound healing processes.

Antimicrobial biocompatible bioscaffolds for orthopaedic implants.

Nationally, nearly 1.5 million patients in the USA suffer from ailments requiring bone grafts and hip and other joint replacements. Infections following internal fixation in orthopaedic trauma can cause osteomyelitis in 22–66% of cases and, if uncontrolled, the mortality rate can be as high as 2%. We characterize a procedure for the synthesis of antimicrobial and biocompatible poly‐l‐lactic acid (PLLA) and poly‐ethyleneglycol (PEG) bioscaffolds designed to degrade and absorb at a controlled rate. The bioscaffold architecture aims to provide a suitable substrate for the controlled release of silver nanoparticles (SNPs) to reduce bacterial growth and to aid the proliferation of human adipose‐derived stem cells (hASCs) for tissue‐engineering applications. The fabricated bioscaffolds were characterized by scanning transmission microscope (SEM) and it showed that the addition of tncreasing concentrations of SNPs results in the formation of dendritic porous channels perpendicular to the axis of precipitation. The antimicrobial properties of these porous bioscaffolds were tested according to a modified ISO 22196 standard across varying concentrations of biomass‐mediated SNPs to determine an efficacious antimicrobial concentration. The bioscaffolds reduced the Staphylococcus aureus and Escherichia coli viable colony‐forming units by 98.85% and 99.9%, respectively, at an antimicrobial SNPs concentration of 2000 ppm. Human ASCs were seeded on bioscaffolds and cultured in vitro for 20 days to study the effect of SNPs concentration on the viability of cells. SEM analysis and the metabolic activity‐based fluorescent dye, AlamarBlue®, demonstrated the growth of cells on the efficacious antimicrobial bioscaffolds. The biocompatibility of in vitro leached silver, quantified by inductively coupled plasma optical emission spectroscopy (ICP‐OES), proved non‐cytotoxic when tested against hASCs, as evaluated by MTT assay. Copyright

Evaluation of bone regeneration potential of dental follicle stem cells for treatment of craniofacial defects

BACKGROUND AIMS Stem cell-based tissue regeneration offers potential for treatment of craniofacial bone defects. The dental follicle, a loose connective tissue surrounding the unerupted tooth, has been shown to contain progenitor/stem cells. Dental follicle stem cells (DFSCs) have strong osteogenesis capability, which makes them suitable for repairing skeletal defects. The objective of this study was to evaluate bone regeneration capability of DFSCs loaded into polycaprolactone (PCL) scaffold for treatment of craniofacial defects. METHODS DFSCs were isolated from the first mandibular molars of postnatal Sprague-Dawley rats and seeded into the PCL scaffold. Cell attachment and cell viability on the scaffold were examined with the use of scanning electron microscopy and alamar blue reduction assay. For in vivo transplantation, critical-size defects were created on the skulls of 5-month-old immunocompetent rats, and the cell-scaffold constructs were transplanted into the defects. RESULTS Skulls were collected at 4 and 8 weeks after transplantation, and bone regeneration in the defects was evaluated with the use of micro-computed tomography and histological analysis. Scanning electron microscopy and Alamar blue assay demonstrated attachment and proliferation of DFSCs in the PCL scaffold. Bone regeneration was observed in the defects treated with DFSC transplantation but not in the controls without DFSC transplant. Transplanting DFSC-PCL with or without osteogenic induction before transplantation achieved approximately 50% bone regeneration at 8 weeks. Formation of woven bone was observed in the DFSC-PCL treatment group. Similar results were seen when osteogenic-induced DFSC-PCL was transplanted to the critical-size defects. CONCLUSIONS This study demonstrated that transplantation of DFSCs seeded into PCL scaffolds can be used to repair craniofacial defects.

Maghemite, silver, ceragenin conjugate particles for selective binding and contrast of bacteria.

New synthesis techniques are providing increasing control over many inorganic nanoparticle characteristics, facilitating the creation of new multifunctional theranostics. This report proposes the synthesis and testing of a combination nanoparticle comprised of a maghemite core for enhanced T2 MRI contrast diagnostics, a colloidal silver shell acting as an antimicrobial and therapeutic vehicle, and a ceragenin (CSA-124) surfactant providing microbial adhesion. A polyacrylic acid functionalized maghemite nanoparticle is synthesized by a high temperature organic phase reduction followed by thiol functionalization and gold cluster seeding. A silver shell is formed through AgNO3 reduction, and an oriented monolayer of the thiolated ceragenin, is bound through a self-assembly process. The process and products are characterized throughout synthesis through TEM, DLS, FT-IR, UV-Vis, ICP-OES, HPLC-ESI-TOF-MS, DC magnetization and susceptibility, X-ray diffraction, and in vitro MRI. Synthesized Diagnostic Antimicrobial Nanoparticles (DANs) were found to have a spherical morphology with a diameter of 32.47±1.83 nm, hydrodynamic diameter of 53.05±1.20 nm, maximum magnetic moment of 12 emu/g NP (54 emu/g Fe) with little variation due to temperature, and are predominantly paramagnetic. In vitro MRI studies show that DANs contrast well at concentrations as low as 9 ppm, and successfully adhere to Staphylococcus aureus. DAN MIC was determined to be approximately 12 ppm and 24 ppm against S. aureus and Escherichia coli respectively.

Human Adipose-Derived Stromal/Stem Cell Isolation, Culture, and Osteogenic Differentiation

Annually, more than 200,000 elective liposuction procedures are performed in the United States and over a million worldwide. The ease of harvest and abundance make human adipose-derived stromal/stem cells (hASCs) isolated from lipoaspirates an attractive, readily available source of adult stem cells that have become increasingly popular for use in many studies. Here, we describe common methods for hASC culture, preservation, and osteogenic differentiation. We introduce methods of ceramic, polymer, and composite scaffold synthesis with a description of morphological, chemical, and mechanical characterization techniques. Techniques for scaffold loading are compared, and methods for determining cell loading efficiency and proliferation are described. Finally, we provide both qualitative and quantitative techniques for in vitro assessment of hASC osteogenic differentiation.

Ceragenin mediated selectivity of antimicrobial silver nanoparticles.

The understanding that common broad-spectrum antimicrobials disrupt natural microbial flora important in acquiring nutrients and preventing infection has resulted in a paradigm shift favoring more selective antimicrobials. This work explores silver nanoparticles conjugated with ceragenin, or cationic antimicrobials (CSA-SNPs), as a potential Gram-positive selective antimicrobial. Herein, CSA-SNPs are characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential, and high-performance liquid chromatography-electrospray time-of-flight mass spectrometry (HPLC-ESI-TOF-MS). The antimicrobial properties are determined through minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) and time-kill studies. Spatial selectivity of the conjugate nanoparticle was evaluated using confocal imaging, MATLAB statistical analysis, and video monitored interactions between bacteria and CSA-SNPs via laser trapping techniques. Cytotoxicity was also determined by live/dead staining and flow cytometry. Average particle size, as determined through TEM analysis, and hydrodynamic diameter, as determined via DLS, are 63.5 ± 38.8 and 102.23 ± 2.3 nm, respectively. The zeta potential of the SNP before and after CSA attachment is -18.23 and -8.34 mV, respectively. MIC/MBC data suggest that CSA-SNPs are 8 times more effective against Staphylococcus aureus than SNPs alone. Furthermore, MATLAB analysis of confocal imaging found that 70% of CSA-SNPs are within 2 μm of S. aureus, whereas this percentage falls to below 40% with respect to Escherichia coli. These results are bolstered further by laser trapping experiments demonstrating selective adherence of CSA-SNPs conjugates with bacterial strains. Cytotoxicity studies of CSA-SNPs against 3T3 fibroblasts indicate 50% cell viability at 50 ppm.

Can a novel silver nano coating reduce infections and maintain cell viability in vitro

Herein we report a facile layer-by-layer method for creating an antimicrobial coating composed of silver nanoparticles on medical grade titanium test discs. Nanoscale silver nanoparticle layers are attached to the titanium orthopedic implant material via aminopropyltriethoxy silane crosslinker that reacts with neighboring silane moieties to create an interconnected network. A monolayer of silane, followed by a monolayer of silver nanoparticles would form one self-assembled layer and this process can be repeated serially, resulting in increased silver nanoparticles deposition. The release rate of silver ion increases predictably with increasing numbers of layers and at appropriate thicknesses these coatings demonstrate 3–4 log reduction of viable Escherichia coli and Staphylococcus aureus bacteria. Increasing the thickness of the coatings resulted in reduced bacterial colonization as determined by fluorescent staining and image analysis. Interestingly, the cytotoxicity of murine 3T3 cells as quantified by fluorescent staining and flow cytometry, was minimal and did not vary significantly with the coating thickness. Additionally, these coatings are mechanically stable and resist delamination by orthogonal stress test. This simple layer-by-layer coating technique may provide a cost-effective and biocompatible method for reducing microbial colonization of implantable orthopedic devices.