Comparing the bone regeneration potential between a trabecular bone and a porous scaffold through osteoblast migration and differentiation: A multiscale approach.

Both cell migration and osteogenic differentiation are critical for successful bone regeneration. Therefore, understanding the mechanobiological aspects that govern these two processes is essential in designing effective scaffolds that promote faster bone regeneration. Studying these two factors at different locations is necessary to manage bone regeneration in various sections of a scaffold. Hence, a multiscale computational model was used to observe the mechanical responses of osteoblasts placed in different positions of the trabecular bone and gyroid scaffold. Fluid shear stresses in scaffolds at cell seeded locations (representing osteogenic differentiation) and strain energy densities in cells at cell substrate interface (representing cell migration) were observed as mechanical response parameters in this study. Comparison of these responses, as two critical factors for bone regeneration, between the trabecular bone and gyroid scaffold at different locations, is the overall goal of the study. This study reveals that the gyroid scaffold exhibits higher osteogenic differentiation and cell migration potential compared to the trabecular bone. However, the responses in the gyroid only mimic the trabecular bone in two out of nine positions. These findings can guide us in predicting the ideal cell seeded sites within a scaffold for better bone regeneration and in replicating a replaced bone condition by altering the physical parameters of a scaffold.

Bilayer regenerated cellulose/quaternized chitosan-hyaluronic acid/collagen electrospun scaffold for potential wound healing applications.

In this study, a bilayer electrospun scaffold has been prepared using regenerated cellulose (RC)/quaternized chitosan (CS) as the primary layer and collagen/hyaluronic acid (HA) as the second layer. An approximate 48 mol% substituted (estimated from 1H NMR) quaternized CS was used in this study. Both layers were crosslinked with EDC/NHS, reflecting an increase in UTS (2.29 MPa for the bilayer scaffold compared to 1.82 MPa for the RC scaffold). Initial cell viability, cell adhesion and proliferation, FDA staining for live cells, and hydroxyproline release rate from cells were evaluated with L929 mouse fibroblast cells. Also, detailed in vitro studies were performed using HADF cells, which include MTT Assay, Live/Dead imaging, DAPI staining, gene expression of PDGF, VEGF-A, and COL1 in RT-PCR, and cell cycle analysis. The collagen/HA-based bilayer scaffold depicted a 9.76-fold increase of VEGF-A compared to a 2.1-fold increase for the RC scaffold, indicating angiogenesis and vascularization potential. In vitro scratch assay was performed to observe the migration of cells in simulated wounds. Antimicrobial, antioxidant, and protease inhibitory activity were further performed, and overall, the primary results highlighted the potential usage of bilayer scaffold in wound healing applications.

Chirality-induced Lineage Enforcement of Mechanosensitive Mesenchymal Stem Cells Across Germ Layer Boundaries.

Mesenchymal to epithelial transition (MET) is instrumental in embryogenesis, tissue repair, and wound healing while the epithelial to mesenchymal transition (EMT) plays role in carcinogenesis. Alteration in microenvironment can modulate cellular signaling and induce EMT and MET. However, modulation of microenvironment to induce MET has been relatively less explored. In this work, effect of matrix stiffness in mediating MET in umbilical cord-derived mesenchymal stem cells (UCMSC) is investigated. Differential segregation of cell fate determinant proteins is one of the key factors in mediating altered stem cell fates through MET even though the genesis of apicobasal polarity remains ambiguous. Herein, it is also attempted to decipher if microenvironment-induced asymmetric cell division has a role to play in driving the cells toward MET. UCMSC cultured on stiffer PDMS matrices resulted in significantly (p < 0.05) higher expression of mechanotransduction proteins. It was also observed that stiffer matrices mediated significant (p < 0.05) upregulation of the polarity proteins and cell fate determinant protein, and epithelial marker proteins over lesser stiff substrates. On the contrary, expression of inflammatory and mesenchymal markers was reduced significantly (p < 0.05) on the stiffer matrices. Cell cycle analysis showed a significant increase in the G1 phase among the cells seeded on stiffer matrices. Transcriptomic studies validated higher expression of epithelial markers genes and lower expression of EMT markers. The transition from mesenchymal to epithelial phenotype depending on the gradation in matrix stiffness is successfully demonstrated. A computational machine learning model was developed to validate stiffness-MET correlation with 94% accuracy. The cross-boundary trans-lineage differentiation capability of MSC on bioengineered substrates can be used as a potential tool in tissue regeneration, organogenesis, and wound healing applications. In our present study, we deciphered the correlation between YAP/TAZ mechanotransduction pathway, EMT signaling pathway, and asymmetric cell division in mediating MET in MSC in a substrate stiffness-dependent manner. It is inferred that the stiffer PDMS matrices facilitate the transition from mesenchymal to epithelial state of MSC. Further, our study also proposed a scoring system to sort MSC from an intermediate hybrid E/M population while undergoing graded MET on matrices of different stiffnesses using a machine learning technique. This proposed scoring system can provide information regarding the E/M state of MSC on different bioengineered constructs based on their biophysical properties which may help in the proper choice of biomaterials in complex tissue-engineering applications.

Vitamin D3-incorporated chitosan/collagen/fibrinogen scaffolds promote angiogenesis and endothelial transition via HIF-1/IGF-1/VEGF pathways in dental pulp stem cells.

Effective vascularization during wound healing remains a critical challenge in the regeneration of skin tissue. On the other hand, mesenchymal stem cell (MSC) to endothelial phenotype transition (MEnDoT) is a potential phenomenon grossly underexplored in vascularized skin tissue engineering. Vitamin D3 has a proven role in promoting MEnDoT. Hence, a D3-incorporated scaffold made with biocompatible materials such as chitosan, collagen and fibrinogen should be able to promote endothelial lineage transition in vitro for tissue engineering purposes. In this study, we developed vitamin D3 incorporated chitosan-collagen-fibrinogen (CCF-D3) scaffolds physically crosslinked under UV and conducted thorough physicochemical and biological assays on it compared to a control scaffold without vitamin D3. Our study for the first time reports the potential vascularization property of the CCF-D3 scaffold by inducing the transitions of dental pulp MSC to endothelial lineage via the HIF-1/IGF-1/VEGF pathways. MSC seeded on UV-exposed CCF-D3 scaffolds had higher cell viability and transitioned towards endothelial lineage was observed by elevated proliferative and endothelial-specific gene expressions and flow cytometric analysis of SCA-1+ antibody. The difference in VEGF-A and α-SMA expressions was also observed in the D3-CCF scaffold compared to the scaffolds without D3.

3D Printing of Ti-6Al-4V-Based Porous-Channel Dental Implants: Computational, Biomechanical, and Cytocompatibility Analyses.

Objective: Loosening of dental implants due to resorption of the surrounding bone is one of the challenging clinical complications in prosthetic dentistry. Generally, stiffness mismatch between an implant and its surrounding bone is one of the major factors. In order to prevent such clinical consequences, it is essential to develop implants with customized stiffness. The present study investigates the computational and experimental biomechanical responses together with cytocompatibility studies of three-dimensional (3D)-printed Ti-6Al-4V-based porous dental implants with varied stiffness properties. Methods: Additive manufacturing (direct metal laser sintering, DMLS) was utilized to create Ti-6Al-4V implants having distinct porosities and pore sizes (650 and 1000 µm), along with a nonporous (solid) implant. To validate the compression testing of the constructed implants and to probe their biomechanical response, finite element models were employed. The cytocompatibility of the implants was assessed using MG-63 cells, in vitro. Results: Both X-ray microcomputed tomography (µ-CT) and scanning electron microscopy (SEM) studies illustrated the ability of DMLS to produce implants with the designed porosities. Biomechanical analysis results revealed that the porous implants had less stiffness and were suitable for providing the appropriate peri-implant bone strain. Although all of the manufactured implants demonstrated cell adhesion and proliferation, the porous implants in particular supported better bone cell growth and extracellular matrix deposition. Conclusions: 3D-printed porous implants showed tunable stiffness properties with clinical translational potential.

Evaluating the cell migration potential of TiO2 nanorods incorporated in a Ti6Al4V scaffold: A multiscale approach.

Improvement of cell migration by the nano-topographical modification of implant surface can directly or indirectly accelerate wound healing and osseointegration between bone and implant. Therefore, modification of the implant surface was done with TiO2 nanorod (NR) arrays to develop a more osseointegration-friendly implant in this study. Modulating the migration of a cell, adhered to a scaffold, by the variations of NR diameter, density and tip diameter in vitro is the primary objective of the study. The fluid structure interaction method was used, followed by the submodelling technique in this multiscale analysis. After completing a simulation over a global model, fluid structure interaction data was applied to the sub-scaffold finite element model to predict the mechanical response over cells at the cell-substrate interface. Special focus was given to strain energy density at the cell interface as a response parameter due to its direct correlation with the migration of an adherent cell. The results showed a huge rise in strain energy density after the addition of NRs on the scaffold surface. It also highlighted that variation in NR density plays a more effective role than the variation in NR diameter to control cell migration over a substrate. However, the effect of NR diameter becomes insignificant when the NR tip was considered. The findings of this study could be used to determine the best nanostructure parameters for better osseointegration.

FTIR microspectroscopy and multivariate analysis facilitate identification of dynamic changes in epithelial-to-mesenchymal transition induced by TGF-ß in prostate cancer cells.

The standard diagnosis of prostate cancer is accomplished by the identification of cytomorphological deviations in biopsied tissues while immunohistochemistry is used to resolve the equivocal cases. Accumulating evidence favors the concept that epithelial-to-mesenchymal transition (EMT) is a stochastic process composed of multiple intermediate states instead of a single binary switch. Despite its significant role in promoting cancer aggressiveness, the current tissue-based risk stratification tools do not include any of the EMT phenotypes as a metric. As a proof-of-concept, the present study analyzes the temporal progression of EMT in transforming growth factor-beta (TGF-ß) treated PC3 cells encompassing multifarious characteristics such as morphology, migration and invasion, gene expression, biochemical fingerprint, and metabolic activity. Our multimodal approach reinstates EMT plasticity in TGF-ß treated PC3 cells. Further, it highlights that mesenchymal transition is accompanied by discernible changes in cellular morphometry and molecular signatures particularly in the range of 1800-1600 cm-1 and 3100-2800 cm-1 of Fourier-transformed infrared (FTIR) spectra signifying Amide III and lipid, respectively. Investigation of attenuated total reflectance (ATR)-FTIR spectra of extracted lipids from PC3 cell populations undergoing EMT identifies changes in stretching vibration at FTIR peaks at 2852, 2870, 2920, 2931, 2954, and 3010 cm-1 characteristics of fatty acids and cholesterol. Chemometric analysis of these spectra indicates that the level of unsaturation and acyl chain length of fatty acid coregister with differential epithelial/mesenchymal states of TGF-ß treated PC3 cells. Observed changes in lipids also correlate with cellular nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide dihydrogen (FADH2) levels and mitochondrial oxygen consumption rate. In summary, our study establishes that morphological and phenotypic traits of epithelial/mesenchymal variants of PC3 cells concur with their respective biochemical and metabolic properties. It also underscores that spectroscopic histopathology has a definitive potential to refine the diagnosis of prostate cancer reckoning its molecular and biochemical heterogeneities.

Cerium oxide nanoparticles disseminated chitosan gelatin scaffold for bone tissue engineering applications.

Cell-free and cell-loaded constructs are used to bridge the critical-sized bone defect. Oxidative stress at the site of the bone defects is a major interference that slows bone healing. Recently, there has been an increase in interest in enhancing the properties of three-dimensional scaffolds with free radical scavenging materials. Cerium oxide nanoparticles (CNPs) can scavenge free radicals due to their redox-modulating property. In this study, freeze-drying was used to fabricate CG-CNPs nanocomposite scaffolds using gelatin (G), chitosan (C), and cerium oxide nanoparticles. Physico-chemical, mechanical, and biological characterization of CG-CNPs scaffolds were studied. CG-CNPs scaffolds demonstrated better results in terms of physicochemical, mechanical, and biological properties as compared to CG-scaffold. CG-CNPs scaffolds were cyto-friendly to MC3T3-E1 cells studied by performing in-vitro and in-ovo studies. The scaffold's antimicrobial study revealed high inhibition zones against Gram-positive and Gram-negative bacteria. With 79 % porosity, 45.99 % weight loss, 178.25 kPa compressive modulus, and 1.83 Ca/P ratio, the CG-CNP2 scaffold displays the best characteristics. As a result, the CG-CNP2 scaffolds are highly biocompatible and could be applied to repair bone defects.

Surgical cotton microfibers loaded with proteins and apatite: A potential platform for bone tissue engineering.

Tissue engineering has emerged as the best alternative to replacing damaged tissue/organs. However, the cost of scaffold materials continues to be a significant obstacle; thus, developing inexpensive scaffolds is strongly encouraged. In this study, cellulose microfibers (C), gelatin (G), egg white (EW), and nanohydroxyapatite (nHA) were assembled into a quaternary scaffold using EDC-NHS crosslinking, followed by freeze-drying method. Cellulose microfibers as a scaffold have only received a limited amount of research due to the absence of an intrinsic three-dimensional structure. Gelatin, more likely to interact chemically with collagen, was used to provide a stable structure to the cellulose microfibers. EW was supposed to provide the scaffold with numerous cell attachment sites. nHA was chosen to enhance the scaffold's bone-bonding properties. Physico-chemical, mechanical, and biological characterization of scaffolds were studied. In-vitro using MG-63 cells and in-ovo studies revealed that all scaffolds were biocompatible. The results of the DPPH assay demonstrate the ability of CGEWnHA to reduce free radicals. The CGEWnHA scaffold exhibits the best properties with 56.84 ± 28.45 µm average pore size, 75 ± 1.4 % porosity, 39.23 % weight loss, 109.19 ± 0.98 kPa compressive modulus, and 1.72 Ca/P ratio. As a result, the constructed CGEWnHA scaffold appears to be a viable choice for BTE applications.

Evaluation of Prebiotic Properties of Galactooligosaccharides Produced by Transgalactosylation Using Partially Purified ß-Galactosidase from Enterobacter aerogenes KCTC2190.

Transgalactosylation reaction is the penultimate step in the production of galactooligosaccharides (GOSs) which has prominent applications in the treatment of disorders. In the present study, partially purified ß-galactosidase from Enterobacter aerogenes KCTC2190 was used for the synthesis of prebiotic GOSs. GOSs were produced using lactose as substrate. Structural elucidation of collected fractions of GOSs by liquid chromatography electrospray ionization mass spectrometry exhibited the appearance of major peaks of produced GOSs at m/z 241.20, 481.39, 365.11, 527.17, and 701.51 respectively. GOSs facilitated the growth of potential probiotic strains (Lactobacillus delbrueckii ssp. helveticus, Bifidobacterium bifidum, and Lactiplantibacillus plantarum) and liberated propionate and butyrate as principal short-chain fatty acids which established its prebiotic potency. Synbiotic combinations exhibited good antioxidant activities. Synbiotic combinations also exhibited antimicrobial activities against pathogenic microorganisms namely Staphylococcus aureus and Escherichia coli. Synbiotic combinations of GOSs and the respective probiotic microorganisms were able to decrease viable human bone cancer cells (MG-63).

Chitosan-collagen-fibrinogen uncrosslinked scaffolds possessing skin regeneration and vascularization potential.

Clinical success of regenerative medicine for treating deep-tissue skin injuries depends on the availability of skin grafts. Though bioengineered constructs are tested clinically, lack of neovascularization provide only superficial healing. Thus constructs, which promotes wound healing and supports vascularization has gained priority in tissue engineering. In this study, chitosan-collagen-fibrinogen (CCF) scaffold was fabricated using freeze-drying method without using any chemical crosslinkers. CCF scaffolds proved cytocompatibility and faster healing in in vitro scratch assay of primary human adult dermal fibroblasts cells with progressively increasing vascular endothelial growth factor-A and reducing vascular endothelial growth factor receptor 1 expressions. Skin regeneration evaluated on in vivo full thickness wound model confirmed faster remodeling with angiogenic signatures in CCF scaffold-implanted mice. Histopathological observations corroborated with stereo-zoom and SS-optical coherence tomography images of wound sites to prove the maturation of healing-bed, after 12 days of CCF implantation. Therefore, it is concluded that CCF scaffolds are promising for skin tissue regeneration and demonstrates pro-angiogenic potential.

Reactive Oxygen Species in Modulating Intestinal Stem Cell Dynamics and Function.

Frequent exposure to mechanistic damages, pathological ingression, and chronic inflammation leads to recurrent cell death in the gut epithelium. Intestinal stem cells (ISCs) that reside in crypt-specific niches have an unprecedented role in gut epithelium renewal. ISC also facilitates the formation of mature intestinal epithelial cells (IECs) through regular differentiation and renewal in short turnover cycles. Interestingly, oxidative stress (OS) prevalent in the gut has a dominant role in the regulation of ISC proliferation and development. However, it is unclear, which axis OS controls the cellular signaling and underlying molecular mechanism to drive ISC turnover and regeneration cycle. Therefore, this review provides a comprehensive overview of the present understanding of OS generation in the gut, relatively directing the ISC development and regeneration under a conditional cellular environment. Additionally, the focus has been drawn on intestinal nutritional state and its related alteration on OS and its effect on ISCs. Moreover, recent findings and new approaches are emphasized herewith to enhance the present understanding of the mechanisms that direct universal ISC characteristics. Intestinal stem cells (ISC) form the basis of all repair mechanisms that help in the proliferation of the gut through their constant renewal and replacement. This activity is closely regulated in the ISC niche and is modulated by several extrinsic as well as intrinsic factors. Reactive Oxygen Species (ROS) form one of the major factors that influence ISC formation. The levels of ROS in the gut influence stem cell renewal ROS itself however is further influenced by several other factors such as the microbiota concerning the gut and immune cells which in turn also influence one another by various cross-talk mechanisms. Diet also forms an important part of this crosstalk. It also regulates the levels of ROS in the gut and helps in the proliferation of the ISC cells and their overall turnover rate.

Leveraging Substrate Stiffness to Promote Stem Cell Asymmetric Division via Mechanotransduction-Polarity Protein Axis and Its Bayesian Regression Analysis.

Asymmetric division of stem cells is an evolutionarily conserved process in multicellular organisms responsible for maintaining cellular fate diversity. Symmetric-asymmetric division pattern of mesenchymal stem cells (MSCs) is regulated by both biochemical and biophysical cues. However, modulation of mechanotransduction pathway by varying scaffold properties and their adaptation to control stem cell division fate is not widely established. In this study, we explored the interplay between the mechanotransduction pathway and polarity protein complex in stem cell asymmetry under varied biophysical stimuli. We hypothesize that variation of scaffold stiffness will impart mechanical stimulus and control the cytoskeleton assembly through RhoA, which will lead to further downstream activation of polarity-related cell signaling and asymmetric division of MSCs. To establish the hypothesis, umbilical cord-derived MSCs were cultured on polycaprolactone/collagen scaffolds with varied stiffness, and expression levels of several important genes (viz., Yes-associated protein [YAP], transcriptional coactivator with PDZ-binding motif [TAZ], LATS1, LATS2, Par3, Par6, PRKC1 [homolog of aPKC] and RhoA), and biomarkers (viz. YAP, TAZ, F-actin, Numb) were assessed. Support vector machine polarity index was employed to understand the polarization status of the MSCs cultured on varied scaffold stiffness. Furthermore, the Bayesian logistic regression model was employed for classifying the asymmetric division of MSCs cultured on different scaffold stiffnesses that showed 91% accuracy. This study emphasizes the vital role of scaffold properties in modulating the mechanotransduction signaling pathway of MSCs and provides mechanistic basis for adopting facile method to control stem cell division pattern toward improving tissue engineering outcome.

Stiffness prediction on elastography images and neuro-fuzzy based segmentation for thyroid cancer detection.

The elastography method detects metastatic changes by measuring the stiffness of tissues. Estimation of elasticities from elastography images facilitates more precise identification of the metastatic region and detection of the same. In this study, an automated segmentation algorithm is proposed that calculates pixel-wise elasticity values to detect thyroid cancer from elastography images. This intensity to elasticity conversion is achieved by constructing a fuzzy inference system using an adaptive neuro-fuzzy inference system supported by two meta-heuristic algorithms: genetic algorithm and particle swarm optimization. Pixels of the input color images (red, green, and blue) are replaced by equivalent elasticity values (in kilo Pascal) and are stored in a two-dimensional array to form an "elasticity matrix." The elasticity matrix is then segmented into three regions, namely, suspicious, near-suspicious, and non-suspicious, based on the elasticity measures, where the threshold limits are calculated using the fuzzy entropy maximization method optimized by the differential evolution algorithm. Segmentation performances are evaluated by Kappa and the dice similarity co-efficient, and average values achieved are 0.94±0.11 and 0.93±0.12, respectively. Sensitivity and specificity values achieved by the proposed method are 86.35±0.34% and 97.67±0.40%, respectively, showing an overall accuracy of 93.50±0.42%. Results justify the importance of pixel stiffness for segmentation of thyroid nodules in elastography images.

Comparative evaluation of proliferative potential and replicative senescence associated changes in mesenchymal stem cells derived from dental pulp and umbilical cord.

Mesenchymal stem cells (MSC) have been widely studied for tissue regeneration and cell-based therapy. MSC can be isolated from different body tissues while several biological waste sources like dental pulp, umbilical cord, cord derived blood, amniotic fluid or urine have also emerged as potential sources of MSCs. Specifically, isolation of MSCs from such non-conventional sources show promising outcomes due to the non-invasiveness of the extraction process and high proliferation capacity of the isolated MSC. However, these stem cells also exhibit the limitation of replicative senescence in long-term culture condition. Inter-cellular reactive oxygen species is an important contributor for inducing cellular senescence under long-term culture conditions. For translational application, it becomes imperative to compare the stem cells isolated from these sources for their senescence and proliferative properties. In this study, MSC were extracted from two different sources of biological waste materials-dental pulp and umbilical cord, and compared for their proliferation capacity and replicative senescence at different passage numbers (i.e. P2 and P6). Intracellular ROS production was significantly (p < 0.001) less in dental pulp stem cells culture in comparison to umbilical cord-derived stem cells at P6. The ß-gal expression also showed significantly (p < 0.001) low expression in DPSC culture compared to that of UCSC at P6. The study indicates the source of stem cells influences the proliferation capacity as well as replicative senescence of MSCs. This study will thus pave the path of future research in selecting appropriate stem cell source for regenerative medicine application.

Finite element analysis of the influence of cyclic strain on cells anchored to substrates with varying properties.

The response of cytoskeleton to mechanical cues plays a pivotal role in understanding several aspects of cellular growth, migration, and cell-cell and cell-matrix interactions under normal and diseased conditions. Finite element analysis (FEA) has become a powerful computational technique to study the response of cytoskeleton in the maintenance of overall cellular mechanics. With the revelation of role of external mechanical microenvironment on cell mechanics, FEA models have also been developed to simulate the effect of substrate stiffness on the mechanical properties of cancer cells. However, the models developed so far model cellular response under static mode, whereas in physiological condition, cells always experience dynamic loading conditions. To develop a more accurate model of cell-extracellular matrix (ECM) interactions, this paper models the cytoskeleton and other parts of the cell by beam and solid elements respectively, assuming spherical morphology of the cell. The stiffness and roughness of extracellular matrix were varied. Furthermore, static and dynamic sinusoidal loads were applied through a flat plate indenter on the cell along with providing sinusoidal strain at the substrate. It is observed that due to axial loading, cell reaches a plastic region, and when the sinusoidal loading is added to the axial load, the cell experiences permanent deformation. Degradation of the cytoskeleton elements and a physiologically more relevant spherical cap shape of the cell were also considered during the analysis. This study suggests that asperity topology of the substrate and indirect cyclic load can play a significant role in the shape alterations and motion of a cell.

Substrate-dependent control of the chiral orientation of mesenchymal stem cells: image-based quantitative profiling.

Stem-cell (SC) chirality or left-right (LR) asymmetry is an essential attribute, observed during tissue regeneration. The ability to control the LR orientation of cells by biophysical manipulation is a promising approach for recapitulating their inherent function. Despite remarkable progress in tissue engineering, the development of LR chirality in SCs has been largely unexplored. Here, we demonstrate the role of substrate stiffness on the LR asymmetry of cultured mesenchymal stem cells (MSCs). We found that MSCs acquired higher asymmetricity when cultured on stiffer PCL/collagen matrices. To confirm cellular asymmetry, different parameters such as the aspect ratio, orientation angle and intensity of polarized proteins (Par) were investigated. The results showed a significant (p < 0.01) difference in the average orientation angle, the cellular aspect ratio, and the expression of actin and Par proteins in MSCs cultured on matrices with different stiffnesses. Furthermore, a Gaussian support-vector machine was applied to classify cells cultured on both (2% and 10% PCL/Collagen) matrices, with a resulting accuracy of 96.2%. To the best of our knowledge, this study is the first that interrelates and quantifies MSC asymmetricity with matrix properties using a simple 2D model.

Improved Mesenchymal Stem Cell Proliferation, Differentiation, Epithelial Transition, and Restrained Senescence on Hierarchically Patterned Porous Honey Silk Fibroin Scaffolds.

We report a significant improvement of adipose-derived mesenchymal stem cells' (ADMSCs) biocompatibility and proliferation on hierarchically patterned porous honey-incorporated silk fibroin scaffolds fabricated using a combination of soft lithography and freeze-drying techniques. Parametric variations show enhanced surface roughness, swelling, and degradation rate with good pore interconnectivity, porosity, and mechanical strength for soft-lithographically fabricated biomimetic microdome arrays on the 2% honey silk fibroin scaffold (PHSF2) as compared to its other variants, which eventually made PHSF2 more comparable to the native environment required for stem cell adhesion and proliferation. PHSF2 also exhibits sustained honey release with remarkable antibacterial efficacy against methicillin-resistant Staphylococcus aureus (MRSA). Honey incorporation (biochemical cue) influences microdome structural features, that is, biophysical cues (height, width, and periodicity), which further allows ADMSCs pseudopods (filopodia) to grasp the microdomes for efficient cell-cell communication and cell-matrix interaction and regulates ADMSCs behavior by altering their cytoskeletal rearrangement and thereby increases the cellular spreading area and cell sheet formation. The synergistic effect of biochemical (honey) and biophysical (patterns) cues on ADMSCs studied by the nitro blue tetrazolium assay and DCFDA fluorescence spectroscopy reveals limited free radical generation within cells. Molecular expression studies show a decrease in p53 and p21 expressions validating ADMSCs senescence inhibition, which is further correlated with a decrease in cellular senescence-associated ß galactosidase activity. We also show that an increase in CDH1 and CK19 molecular expressions along with an increase in SOX9, RUNX2, and PPARγ molecular expressions supported by PHSF2 justify the substrate's efficacy of underpinning mesenchymal to epithelial transition and multilineage trans-differentiation. This work highlights the fabrication of a naturally healing nutraceutical (honey)-embedded patterned porous stand-alone tool with the potential to be used as smart stem cells delivering regenerative healing implant.

Dual cross-linked honey coupled 3D antimicrobial alginate hydrogels for cutaneous wound healing.

We report potentiation of healing efficacy of alginate by value addition at its structural level. Dual crosslinked (ionically and covalently) sodium alginate hydrogel coupled with honey (HSAG) brings about an intermediate stiffness in the fabric, confers consistent swelling property and limits erratic degradation of the polymer which ultimately provides conducive milieu to cellular growth and proliferation. In this work honey concentrations in HSAGs are varied from 2% to 10%. FTIR, XRD and nanoindentation studies on the HSAGs exhibited physicochemical integrity. In vitro degradation study provided the crucial finding on 4% HSAG having controlled degradation rate up to 12 days with a weight loss of 87.36 ± 1.14%. This particular substrate also has an ordered crystalline surface morphology with decent cellular viability (HaCaT and 3T3) and antimicrobial potential against Methicillin Resistant Staphylococcus aureus (MRSA) and Escherichia coli. The in vivo wound contraction kinetics on murine models (4% HSAG treated wound contraction: 94.56 ± 0.1%) has been monitored by both invasive (histopathology) and noninvasive (Swept Source Optical Coherence Tomography) imaging and upon corroborating them it evidenced that 4% HSAG treated wound closure achieved epithelial thickness resembling to that of unwounded skin. Thus, the work highlights structurally modified alginate hydrogel embedded with honey as a potential antimicrobial healing agent.

Surface Functionalization of Green-synthesized Reduced Graphene Oxide with PPIX Enhances Photosensitization of Cancer Cells.

Photosensitizer-functionalized reduced graphene oxide (rGO) nanoparticles are promising materials for photodynamic therapy in cancer management. In this study, rGO is synthesized by a green route employing glucose as the reducing agent and functionalized with photosensitizer, protoporphyrin IX (PPIX) in a convenient, single-step procedure. PPIX-functionalized rGO exhibits photodynamic effect against cancer cells (HeLa) at 0.001 mg mL-1 under visible light illumination (635 nm). A 50% elimination of HeLa cells after 5 min irradiation is observed while very low phototoxicity (80% cell viability) is noted against normal dermal fibroblast cells. A positive correlation with ROS accumulation and increased expression of caspase-3 in PPIX-functionalized rGO-treated cancer cells is also established. The results evidence a simple and cost-effective route for developing photosensitizer-functionalized rGO for effective and selective killing of cancer cells.