Delivery of Therapeutic miR-148b Mimic via Poly(ß Amino Ester) Polyplexes for Post-transcriptional Gene Regulation and Apoptosis of A549 Cells.

In this study, we utilized selectively modified, biodegradable polymer-based polyplexes to deliver custom, exogenous miR-148b mimics to induce apoptosis in human lung cancer (A549) cells. The gene regulatory effects of the payload miRNA mimics (miR-148b-3p) were first evaluated through bioinformatic analyses to uncover specific gene targets involved in critical carcinogenic pathways. Hyperbranched poly(ß amino ester) polyplexes (hPBAE) loaded with custom miR-148b mimics were then developed for targeted therapy. When evaluated in vitro, these hPBAE-based polyplexes sustained high intracellular uptake, low cytotoxicity, and efficient escape from endosomes to deliver functionally intact miRNA mimics to the cytosol. High-resolution confocal microscopy revealed successful intracellular uptake, cell viability was assessed through qualitative fluorescence microscopy and fluorescence-based DNA quantification, and successful cytosolic delivery of intact miRNA mimics was evaluated using real-time polymerase chain reaction (RT-PCR) to demonstrate target gene knockdown. The hPBAE-miRNA mimic polyplexes were shown to induce apoptosis among A549 cells through direct modulation of intracellular protein expression, targeting multiple potential carcinogenic pathways at the gene level. These results indicated that spatially controlled miR-148b mimic delivery can promote efficient cancer cell death in vitro and may lead to an enhanced therapeutic design for in vivo application.

Obesity inhibits the osteogenic differentiation of human adipose-derived stem cells.

BACKGROUND: Craniomaxillofacial defects secondary to trauma, tumor resection, or congenital malformations are frequent unmet challenges, due to suboptimal alloplastic options and limited autologous tissues such as bone. Significant advances have been made in the application of adipose-derived stem/stromal cells (ASCs) in the pre-clinical and clinical settings as a cell source for tissue engineering approaches. To fully realize the translational potential of ASCs, the identification of optimal donors for ASCs will ensure the successful implementation of these cells for tissue engineering approaches. In the current study, the impact of obesity on the osteogenic differentiation of ASCs was investigated. METHODS: ASCs isolated from lean donors (body mass index <25; lnASCs) and obese donors (body mass index >30; obASCs) were induced with osteogenic differentiation medium as monolayers in an estrogen-depleted culture system and on three-dimensional scaffolds. Critical size calvarial defects were generated in male nude mice and treated with scaffolds implanted with lnASCs or obASCs. RESULTS: lnASCs demonstrated enhanced osteogenic differentiation in monolayer culture system, on three-dimensional scaffolds, and for the treatment of calvarial defects, whereas obASCs were unable to induce similar levels of osteogenic differentiation in vitro and in vivo. Gene expression analysis of lnASCs and obASCs during osteogenic differentiation demonstrated higher levels of osteogenic genes in lnASCs compared to obASCs. CONCLUSION: Collectively, these results indicate that obesity reduces the osteogenic differentiation capacity of ASCs such that they may have a limited suitability as a cell source for tissue engineering.

Plasmon-Enhanced Photocleaving Dynamics in Colloidal MicroRNA-Functionalized Silver Nanoparticles Monitored with Second Harmonic Generation.

The photocleaving dynamics of colloidal microRNA-functionalized nanoparticles are studied using time-dependent second harmonic generation (SHG) measurements. Model drug-delivery systems composed of oligonucleotides attached to either silver nanoparticles or polystyrene nanoparticles using a nitrobenzyl photocleavable linker are prepared and characterized. The photoactivated controlled release is observed to be most efficient on resonance at 365 nm irradiation, with pseudo-first-order rate constants that are linearly proportional to irradiation powers. Additionally, silver nanoparticles show a 6-fold plasmon enhancement in photocleaving efficiency over corresponding polystyrene nanoparticle rates, while our previous measurements on gold nanoparticles show a 2-fold plasmon enhancement compared to polystyrene nanoparticles. Characterizations including extinction spectroscopy, electrophoretic mobility, and fluorimetry measurements confirm the analysis from the SHG results. The real-time SHG measurements are shown to be a highly sensitive method for investigating plasmon-enhanced photocleaving dynamics in model drug delivery systems.

Controlled Degradation of Polycaprolactone Polymers through Ultrasound Stimulation.

This study describes the development of an ultrasound-responsive polymer system that provides on-demand degradation when exposed to high-intensity focused ultrasound (HIFU). Diels-Alder cycloadducts were used to crosslink polycaprolactone (PCL) polymers and underwent a retro Diels-Alder reaction when stimulated with HIFU. Two Diels-Alder polymer compositions were explored to evaluate the link between reverse reaction energy barriers and polymer degradation rates. PCL crosslinked with isosorbide was also used as a non-Diels-Alder-based control polymer. An increase of HIFU exposure time and amplitude correlated with an increase of PCL degradation for Diels-Alder-based polymers. Ultrasound imaging during HIFU allowed for real-time visualization of the on-demand degradation through cavitation-based mechanisms. The temperature surrounding the sample was monitored with a thermocouple during HIFU stimulation; a minimal increase in temperature was observed. PCL polymers were characterized using Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), optical profilometry, and mechanical testing. PCL degradation byproducts were identified by mass spectrometry, and their cytocompatibility was evaluated in vitro. Overall, this study demonstrated that HIFU is an effective image-guided, external stimulus to control the degradation of Diels-Alder-based PCL polymers on-demand.

Hybrid adipose graft materials synthesized from chemically modified adipose extracellular matrix.

Decellularized extracellular matrix (ECM) from tissues is a promising biomaterial that can provide a complex 3D microenvironment capable of modulating cell response and tissue regeneration. In this study, we have integrated the decellularized thiolated adipose-derived ECM, at different concentrations, with polyethylene glycol (PEG) using Michael addition between thiol and acrylate moieties. The potential for this material to support adipogenic differentiation of human adipose-derived stem cells was evaluated by encapsulating cells in hydrogels with increasing concentrations of chemically modified ECM (mECM). Our results demonstrated a positive correlation between the ECM content in the hydrogels and cell proliferation, adipogenic marker expression, and lipid formation and accumulation. Furthermore, we have shown host cell infiltration and enhanced adipogenesis in vivo after implantation. These findings support the graft as a potential alternative for adipose tissue regeneration.

Clinical Translational Potential in Skin Wound Regeneration for Adipose-Derived, Blood-Derived, and Cellulose Materials: Cells, Exosomes, and Hydrogels.

Acute and chronic skin wounds due to burns, pressure injuries, and trauma represent a substantial challenge to healthcare delivery with particular impacts on geriatric, paraplegic, and quadriplegic demographics worldwide. Nevertheless, the current standard of care relies extensively on preventive measures to mitigate pressure injury, surgical debridement, skin flap procedures, and negative pressure wound vacuum measures. This article highlights the potential of adipose-, blood-, and cellulose-derived products (cells, decellularized matrices and scaffolds, and exosome and secretome factors) as a means to address this unmet medical need. The current status of this research area is evaluated and discussed in the context of promising avenues for future discovery.

Hybrid Synthetic-Biological Hydrogel System for Adipose Tissue Regeneration.

Hydrogels are promising scaffolds for adipose tissue regeneration. Currently, the incorporation of bioactive molecules in hydrogel system is used, which can increase the cell proliferation rate or improve adipogenic differentiation performance of stromal stem cells but often suffers from high expense or cytotoxicity because of light/thermal curing used for polymerization. In this study, decellularized adipose tissue is incorporated, at varying concentrations, with a thiol-acrylate fraction that is then polymerized to produce hydrogels via a Michael addition reaction. The results reveal that the major component of isolated adipose-derived extracellular matrix (ECM) is Collagen I. Mechanical properties of ECM polyethylene glycol (PEG) are not negatively affected by the incorporation of ECM. Additionally, human adipose-derived stem cells (hASCs) are encapsulated in ECM PEG hydrogel with ECM concentrations varying from 0% to 1%. The results indicate that hASCs maintained the highest viability and proliferation rate in 1% ECM PEG hydrogel with most lipids formation when cultured in adipogenic conditions. Furthermore, more adipose regeneration is observed in 1% ECM group with in vivo study by Day 14 compared to other ECM PEG hydrogels with lower ECM content. Taken together, these findings suggest the ECM PEG hydrogel is a promising substitute for adipose tissue regeneration applications.

In vitro evaluation of thermal frontally polymerized thiol-ene composites as bone augments.

Because of the large number of total knee replacement (TKR) surgeries conducted per year, and with projections of increased demand to almost a million primary TKR surgeries per year by 2030 in the United States alone, there is a need to discover more efficient working materials as alternatives to current bone cements. There is a need for surgeons and hospitals to become more efficient and better control over the operative environment. One area of inefficiency is the cement steps during TKR. Currently the surgeon has very little control over cement polymerization. This leads to an increase in time, waste, and procedural inefficiencies. There is a clear need to create an extended working time, moldable, osteoconductive, and osteoinductive bone augment as a substitution for the current clinically used bone cement where the surgeon has better control over the polymerization process. This study explored several compositions of pentaerythritol-co-trimethylolpropane tris-(3-mercaptopropionate) hydroxyapatite composite materials prepared via benzoyl peroxide-initiated thermal frontal polymerization. The 4:1 acrylate to thiol ratio containing augment material shows promise with a maximal propagation temperature of 160°C ± 10°C, with mechanical strength of 3.65 MPa, and 111% cytocompatibility, relative to the positive control. This frontally polymerized material may have application as an augment with controlled polymerization supporting cemented implants. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1152-1160, 2016.

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.

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.

In vitro human adipose-derived stromal/stem cells osteogenesis in akermanite:poly-ε-caprolactone scaffolds.

This study compared the metabolic activity, cell proliferation and osteogenic differentiation of human adipose-derived stromal/stem cells cultured on four different scaffolds (poly-ε-caprolactone, akermanite:poly-ε-caprolactone composites, akermanite and ß-tricalcium phosophate) with or without osteogenic media supplementation for up to 21 days. The hypothesis was that human adipose-derived stromal/stem cells osteogenesis in akermanite-containing scaffolds would be greater than the other scaffold types independent of the media supplementation. According to the results, human adipose-derived stromal/stem cells loaded on different scaffolds and cultured in both media conditions displayed significant changes in the metabolic activity and cell proliferation. After 21 days of culture in osteogenic medium, the human adipose-derived stromal/stem cells loaded onto akermanite-based scaffolds had greater calcium deposition and osteocalcin expression relative to human adipose-derived stromal/stem cells loaded onto ß-tricalcium phosophate and poly-ε-caprolactone. In vivo investigations are needed to further assess the bone tissue engineering potential of human adipose-derived stromal/stem cells loaded to akermanite:poly-ε-caprolactone composites.

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.

Human adipose-derived stem cells and three-dimensional scaffold constructs: a review of the biomaterials and models currently used for bone regeneration.

In the past decade, substantial strides have been taken toward the use of human adipose-derived stromal/stem cells (hASC) in the regeneration of bone. Since the discovery of the hASC osteogenic potential, many models have combined hASC with biodegradable scaffold materials. In general, rats and immunodeficient (nude) mice models for nonweight bearing bone formation have led the way to assess hASC osteogenic potential in vivo. The goal of this review is to present an overview of the recent literature describing hASC osteogenesis in conjunction with three-dimensional scaffolds for bone regeneration.

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.

Development of near infrared spectroscopy models for the quantitative prediction of the lignocellulosic components of wet Miscanthus samples.

Miscanthus samples were scanned over the visible and near infrared wavelengths at several stages of processing (wet-chopped, air-dried, dried and ground, and dried and sieved). Models were developed to predict lignocellulosic and elemental constituents based on these spectra. The dry and sieved scans gave the most accurate models; however the wet-chopped models for glucose, xylose, and Klason lignin provided excellent accuracies with root mean square error of predictions of 1.27%, 0.54%, and 0.93%, respectively. These models can be suitable for most applications. The wet models for arabinose, Klason lignin, acid soluble lignin, ash, extractives, rhamnose, acid insoluble residue, and nitrogen tended to have lower R(2) values (0.80+) for the validation sets and the wet models for galactose, mannose, and acid insoluble ash were less accurate, only having value for rough sample screening. This research shows the potential for online analysis at biorefineries for the major lignocellulosic constituents of interest.