Manish K. Jaiswal

Bioactive Nanoengineered Hydrogels for Bone Tissue Engineering: A Growth-Factor-Free Approach

Despite bones impressive ability to heal after traumatic injuries and fractures, a significant need still exists for developing strategies to promote healing of nonunion defects. To address this issue, we developed collagen-based hydrogels containing two-dimensional nanosilicates. Nanosilicates are ultrathin nanomaterials with a high degree of anisotropy and functionality that results in enhanced surface interactions with biological entities compared to their respective three-dimensional counterparts. The addition of nanosilicates resulted in a 4-fold increase in compressive modulus along with an increase in pore size compared to collagen-based hydrogels. In vitro evaluation indicated that the nanocomposite hydrogels are capable of promoting osteogenesis in the absence of any osteoinductive factors. A 3-fold increase in alkaline phosphatase activity and a 4-fold increase in the formation of a mineralized matrix were observed with the addition of the nanosilicates to the collagen-based hydrogels. Overall, these results demonstrate the multiple functions of nanosilicates conducive to the regeneration of bone in nonunion defects, including increased network stiffness and porosity, injectability, and enhanced mineralized matrix formation in a growth-factor-free microenvironment.

Thermal behavior of magnetically modalized poly(N-isopropylacrylamide)-chitosan based nanohydrogel

Poly(NIPAAm)-CS based nanohydrogels (NHGs) and iron oxide (Fe(3)O(4)) magnetic nanoparticles encapsulated magnetic nanohydrogels (MNHGs) were synthesized by free radical polymerization of N-isopropylacrylamide (NIPAAm) at 60 degrees C in presence of chitosan (CS) in different feed ratios. The polymerization of NIPAAm and the presence of CS as well as Fe(3)O(4) in hydrogels were confirmed from Fourier transform infrared (FTIR) spectra and X-ray diffraction (XRD), respectively. (13)C solid state nuclear magnetic resonance (NMR) spectra clearly revealed the grafting of CS into poly(NIPAAm). The scanning electron microscopy (SEM) and atomic force microscopy (AFM) images showed the formation of spherical shaped NHGs of different sizes ranging from 50 nm to 200 nm depending upon the feed ratios of CS and NIPAAm, which was further supported by mean hydrodynamic diameter measured by dynamic light scattering (DLS). It has been observed that CS not only served as a cross linker during polymerization but also plays a critical role in controlling the growth of NHG and enhancement in lower critical solution temperature (LCST). The encapsulation of Fe(3)O(4) nanoparticles (10-12 nm) into NHGs ( approximately 200 nm) was confirmed by transmission electron microscopy (TEM) and further corroborated with magnetic force microscopy (MFM) image. The LCST of poly(NIPAAm) was found to increase with increasing weight ratio of CS to NIPAAm. Furthermore, the encapsulation of iron oxide nanoparticles into hydrogels also caused an increment in LCST. Specifically, temperature optimized NHG and MNHG were fabricated having LCST close to 42 degrees C (hyperthermia temperature). The MNHG shows optimal magnetization, good specific absorption rate (under external AC magnetic field) and excellent cytocompatibility with L929 cell lines, which may find potential applications in hyperthermia treatment of cancer and targeted drug delivery.

Mechanically Stiff Nanocomposite Hydrogels at Ultralow Nanoparticle Content

Although hydrogels are able to mimic native tissue microenvironments, their utility for biomedical applications is severely hampered due to limited mechanical stiffness and low toughness. Despite recent progress in designing stiff and tough hydrogels, it is still challenging to achieve a cell-friendly, high modulus construct. Here, we report a highly efficient method to reinforce collagen-based hydrogels using extremely low concentrations of a nanoparticulate-reinforcing agent that acts as a cross-link epicenter. Extraordinarily, the addition of these nanoparticles at a 10 000-fold lower concentration relative to polymer resulted in a more than 10-fold increase in mechanical stiffness and a 20-fold increase in toughness. We attribute the high stiffness of the nanocomposite network to the chemical functionality of the nanoparticles, which enabled the cross-linking of multiple polymeric chains to the nanoparticle surface. The mechanical stiffness of the nanoengineered hydrogel can be tailored between 0.2 and 200 kPa simply by manipulating the size of the nanoparticles (4, 8, and 12 nm), as well as the concentrations of the nanoparticles and polymer. Moreover, cells can be easily encapsulated within the nanoparticulate-reinforced hydrogel network, showing high viability. In addition, encapsulated cells were able to sense and respond to matrix stiffness. Overall, these results demonstrate a facile approach to modulate the mechanical stiffness of collagen-based hydrogels and may have broad utility for various biomedical applications, including use as tissue-engineered scaffolds and cell/protein delivery vehicles.

Thermoresponsive magnetic hydrogels as theranostic nanoconstructs.

We report the development of thermoresponsive magnetic hydrogels based on poly(N-isopropylacrylamide) encapsulation of Fe3O4 magnetic nanostructures (MNS). In particular, we examined the effects of hydrogels encapsulated with poly-ethylene glycol (PEG) and polyhedral oligomeric silsesquioxane (POSS) surface modified Fe3O4 MNS on magnetic resonance (MR) T2 (transverse spin relaxation) contrast enhancement and associated delivery efficacy of absorbed therapeutic cargo. The microstructural characterization reveal the regular spherical shape and size (∼200 nm) of the hydrogels with elevated hydrophilic to hydrophobic transition temperature (∼40 °C) characterized by LCST (lower critical solution temperature) due to the presence of encapsulated MNS. The hydrogel-MNS (HGMNS) system encapsulated with PEG functionalized Fe3O4 of 12 nm size (HGMNS-PEG-12) exhibited relaxivity rate (r2) of 173 mM–1s–1 compared to 129 mM–1s–1 obtained for hydrogel-MNS system encapsulated with POSS functionalized Fe3O4 (HGMNS-POSS-12) of the same size. Further studies with HGMNS-PEG-12 with absorbed drug doxorubicin (DOX) reveals approximately two-fold enhance in release during 1 h RF (radio-frequency) field exposure followed by 24 h incubation at 37 °C. Quantitatively, it is 2.1 μg mg–1 (DOX/HGMNS) DOX release with RF exposure while only 0.9 μg mg–1 release without RF exposure for the same period of incubation. Such enhanced release of therapeutic cargo is attributed to micro-environmental heating in the surroundings of MNS as well as magneto-mechanical vibrations under high frequency RF inside hydrogels. Similarly, RF-induced in vitro localized drug delivery studies with HeLa cell lines for HGMNS-PEG-12 resulted in more than 80% cell death with RF field exposures for 1 h. We therefore believe that magnetic hydrogel system has in vivo theranostic potential given high MR contrast enhancement from encapsulated MNS and RF-induced localized therapeutic delivery in one nanoconstruct.

Biocompatibility, biodistribution and efficacy of magnetic nanohydrogels in inhibiting growth of tumors in experimental mice models

We report in vivo evaluation of a thermo-responsive poly(N-isopropylacrylamide)-chitosan based magnetic nanohydrogel (MNHG) incorporated with Fe3O4 nanoparticles (NPs) in mice models with expandible scope for use in localized delivery of chemotherapeutics. Biocompatibility and biodistribution of the MNHG are studied in normal Swiss mice while efficacy in tumor growth inhibition is studied in a subcutaneous fibrosarcoma tumor. The ex vivo time-dependent pattern of accumulated MNHG into vital organs; lung, liver, spleen, kidney and brain collected at 1 h, 48 h, 7 d and 14 d post intravenous administration are investigated using both a vibrating sample magnetometer (VSM) and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) method. The doses of MNHG (dose I ∼ 650 and dose II ∼ 325 μg g-1 body wt) used in the study are determined based on induced thermal activation of MNHG under an AC magnetic field (AMF). Fibrosarcoma tumor bearing mice are subjected to hyperthermia with a field of 325 Oe and 265 kHz for 30 min following intratumoral administration of dose I. Tumor size measured at an interval of 72 h for a period of 2 weeks reveals that the NPs mediated therapy decelerated the growth of the transplanted tumor by about three-fold (size, 1545 ± 720 mm3) as compared to the exponential growth of the tumor (size, 4510 ± 735 mm3) in control mice. These results suggest the feasibility of using poly(NIPAAm)-chitosan hydrogels loaded with NPs for combined thermo-chemotherapy where the efficacy may further be improved by temperature dependent release of the drugs from the magneto hydrogels.

A comparative study on thermoresponsive magnetic nanohydrogels: role of surface-engineered magnetic nanoparticles

A comparative study of thermoresponsive poly(N-isopropylacrylamide)(PNIPAAm)-chitosan (CS)-based magnetic nanohydrogels (MNHGs) encapsulating functionalized Fe3O4 nanoparticles (NPs) in terms of the parameters governing their suitability for real hyperthermia is reported. Iron oxide NPs functionalized with (a) citric acid (CA-Fe3O4), (b) ethylenediamine (Amine-Fe3O4) and (c) dimercaptosuccininc (DMSA-Fe3O4) have been synthesized and their encapsulation into MNHGs was obtained through physical encapsulation method. The structural characterizations of synthesized materials include X-ray diffraction, FT-IR, TGA, ICP-AES and X-ray photoelectron spectroscopy (XPS). Encapsulation of the functionalized NPs into MNHGs were observed in TEM micrographs, while SEM and AFM micrographs confirmed their spherical morphology (~250–300 nm). Lower critical solution temperature (LCST) variation was measured by UV–visible spectrophotometer and differential scanning calorimetry (DSC). MNHGs exhibited sufficient magnetization and heating ability for hyperthermia. Typically, hydrogels containing CA-Fe3O4 (50 mg/ml) raised the temperature of the medium to 43 °C, a suitable dose for in vivo application in tumor-bearing mice.

Photocrosslinkable and elastomeric hydrogels for bone regeneration

Nanocomposite biomaterials are extensively investigated for cell and tissue engineering applications due their unique physical, chemical and biological characteristics. Here, we investigated the mechanical, rheological, and degradation properties of photocrosslinkable and elastomeric nanocomposite hydrogels from nanohydroxyapatite (nHAp) and gelatin methacryloyl (GelMA). The addition of nHAp resulted in a significant increase in mechanical stiffness and physiological stability. Cells readily adhere and proliferate on the nanocomposite surfaces. Cyclic stretching of cells on the elastomeric nanocomposites revealed that nHAp elicited a stronger alignment response in the direction of strain. In vitro studies highlight enhanced bioactivity of nanocomposites as determined by alkaline phosphate (ALP) activity. Overall, the elastomeric and photocrosslinkable nanocomposite hydrogels can be used for minimally invasive therapy for bone regeneration.

Polymers for Bioprinting

Abstract Bioprinting is a process of precisely designed scaffolds using three-dimensional printing technologies for functional tissue engineering utilizing cell-laden biomaterials as bioink. A range of polymers can be used as bioink to stimulate favorable cellular interactions, leading to enhanced cell motility, proliferation, and subsequent differentiation. Both natural and synthetic polymers have been considered for various bioprinting applications, each with a corresponding set of advantages and limitations. Natural polymers more aptly mimic the native extracellular matrix, leading to more favorable cellular responses, while synthetic polymers can be more easily tailored for more efficient printing. Because many of these bioink materials are rooted in traditional tissue engineering scaffold design, bioprinting optimization remains a challenge; however, emerging trends in bioink development have begun to circumvent these issues, providing bioprinting research with a very promising future in regenerative medicine. Further investigation into the interplay of polymer type and fabrication technique will help to formulate new polymer bioinks that can expedite the process from printing to implantation.

Widespread changes in transcriptome profile of human mesenchymal stem cells induced by two-dimensional nanosilicates

Significance We demonstrate the use of next-generation sequencing technology (RNA-seq) to understand the effect of a two-dimensional nanomaterial on human stem cells at the whole-transcriptome level. Our results identify more than 4,000 genes that are significantly affected, and several biophysical and biochemical pathways are triggered by nanoparticle treatment. We expect that this systematic approach to understand widespread changes in gene expression due to nanomaterial exposure is key to develop new bioactive materials for biomedical applications. Two-dimensional nanomaterials, an ultrathin class of materials such as graphene, nanoclays, transition metal dichalcogenides (TMDs), and transition metal oxides (TMOs), have emerged as a new generation of materials due to their unique properties relative to macroscale counterparts. However, little is known about the transcriptome dynamics following exposure to these nanomaterials. Here, we investigate the interactions of 2D nanosilicates, a layered clay, with human mesenchymal stem cells (hMSCs) at the whole-transcriptome level by high-throughput sequencing (RNA-seq). Analysis of cell–nanosilicate interactions by monitoring changes in transcriptome profile uncovered key biophysical and biochemical cellular pathways triggered by nanosilicates. A widespread alteration of genes was observed due to nanosilicate exposure as more than 4,000 genes were differentially expressed. The change in mRNA expression levels revealed clathrin-mediated endocytosis of nanosilicates. Nanosilicate attachment to the cell membrane and subsequent cellular internalization activated stress-responsive pathways such as mitogen-activated protein kinase (MAPK), which subsequently directed hMSC differentiation toward osteogenic and chondrogenic lineages. This study provides transcriptomic insight on the role of surface-mediated cellular signaling triggered by nanomaterials and enables development of nanomaterials-based therapeutics for regenerative medicine. This approach in understanding nanomaterial–cell interactions illustrates how change in transcriptomic profile can predict downstream effects following nanomaterial treatment.

Vacancy-Driven Gelation Using Defect-Rich Nanoassemblies of 2D Transition Metal Dichalcogenides and Polymeric Binder for Biomedical Applications

A new approach of vacancy-driven gelation to obtain chemically crosslinked hydrogels from defect-rich 2D molybdenum disulfide (MoS2 ) nanoassemblies and polymeric binder is reported. This approach utilizes the planar and edge atomic defects available on the surface of the 2D MoS2 nanoassemblies to form mechanically resilient and elastomeric nanocomposite hydrogels. The atomic defects present on the lattice plane of 2D MoS2 nanoassemblies are due to atomic vacancies and can act as an active center for vacancy-driven gelation with a thiol-activated terminal such as four-arm poly(ethylene glycol)-thiol (PEG-SH) via chemisorption. By modulating the number of vacancies on the 2D MoS2 nanoassemblies, the physical and chemical properties of the hydrogel network can be controlled. This vacancy-driven gelation process does not require external stimuli such as UV exposure, chemical initiator, or thermal agitation for crosslinking and thus provides a nontoxic and facile approach to encapsulate cells and proteins. 2D MoS2 nanoassemblies are cytocompatible, and encapsulated cells in the nanocomposite hydrogels show high viability. Overall, the nanoengineered hydrogel obtained from vacancy-driven gelation is mechanically resilient and can be used for a range of biomedical applications including tissue engineering, regenerative medicine, and cell and therapeutic delivery.