Enhanced chondrogenic differentiation of human mesenchymal stem cells in silk fibroin/chitosan/glycosaminoglycan scaffolds under dynamic culture condition.

Cartilage tissue damage and diseases are the most common clinical situation that occurs because of aging and injury, thereby causing pain and loss of mobility. The inability of cartilage tissue to self-repair is instrumental in developing tissue engineered substitutes. To this effect, the present study aims to engineer cartilage construct by culturing umbilical cord blood-derived human mesenchymal stem cells (hMSCs) on novel 3D porous scaffolds developed from natural biopolymers, silk fibroin (SF) and chitosan (CS), with addition of cartilage matrix components, glucosamine (Gl) and chondroitin sulfate (Ch). The presence of Gl and Ch is expected to enhance cartilage regeneration. The developed SF/CS-Gl-Ch scaffolds possess desired pore size in the range 56.55-168.15 µm, 88-92% porosity, 44.7-46.8̊ contact angle, controlled swelling and biodegradability. Upon culturing under dynamic condition in a spinner flask bioreactor, the scaffold supported hMSCs attachment, proliferation, and further promoted chondrogenic differentiation. Cartilage-specific matrix and gene (Collagen II, Sox9 and aggrecan) expression analyses by histology, immunophenotype, immunofluorescence and quantitative PCR studies showed superiority of cell-scaffold construct generated in dynamic culture towards cartilage tissue generation as compared to cell aggregates formed by pellet culture. This study demonstrates the potentiality of SF/CS-Gl-Ch porous scaffold for the development of tissue construct for cartilage regeneration under dynamic culture condition.

Silk fibroin coated TiO2 nanotubes for improved osteogenic property of Ti6Al4V bone implants.

Titanium and its alloys especially Ti6Al4V have long been used in biomedical implants. Although, Ti6Al4V is biocompatible, yet there has been consistent effort to improve its osteoconductive and osteogenic property to enhance the implant performance. In this regard, surface modification of Ti6Al4V implants with TiO2 nanotubes and subsequent application of biopolymeric coating has started emerging as a promising approach. Keeping this perspective in mind, here we have coated nano TiO2 modified Ti6Al4V surface with silk fibroin isolated from B. mori cocoons. The coating of silk fibroin was done on the implant using electrophoretic deposition technique at three different voltages. Topography analysis by AFM confirms the uniform coating of silk fibroin. A variation in contact angle from 89.7 ±â€¯2° to 83.6 ±â€¯2° was observed when tested for wettability using drop shape analyzer. The biocompatibility studies showed SF coated substrates support the adhesion of both MG63 bone cells and human mesenchymal stem cells (hMSCs). The formation of peripheral vinculin complexes on SF surface confirmed the adhesion through focal adhesion points. Consequently, SF coating improved the cellular expression of alkaline phosphatase by 1.1 times compared to the polished Ti6Al4V surface (PTi64). Increased expression of late osteogenic markers osterix and osteocalcin was also observed in hMSCs cultured on SF coated nanotubular surface compared to PTi64. These results together implied that coating of silk fibroin on TiO2 modified Ti6Al4V surface improve the osteogenic potential of the implant.

Chondrogenic differentiation of mesenchymal stem cells on silk fibroin:chitosan-glucosamine scaffold in dynamic culture.

AIM: Cartilage damage is a common age-related problem that leads to progressive proteoglycan loss. Glucosamine stimulates proteoglycan synthesis and, therefore, its effect on the cartilage extracellular matrix synthesis over silk fibroin:chitosan (SF:CS) tissue-engineered scaffold was investigated for cartilage construct generation. MATERIALS & METHODS: Human mesenchymal stem cells (hMSCs) were cultured and differentiated over SF:CS-glucosamine porous scaffold, under dynamic culture condition in spinner flask bioreactor. RESULTS: hMSCs-seeded scaffold in dynamic culture exhibited homogenous cell distribution, proliferation and higher cell density at the core than static culture. Glucosamine in scaffold promoted proteoglycan and collagenous matrix synthesis as revealed by histological and immunofluorescence studies. Quantitative-PCR analysis showed upregulation of cartilage-specific genes, thereby confirming the chondrogenic differentiation. CONCLUSION: The chondrogenic differentiation of hMSCs was enhanced by the synergistic effect of glucosamine incorporated in SF:CS scaffold and influence of 3D dynamic culture environment, thereby resulting in chondrogenic phenotype of the cells that promoted cartilage regeneration.

Evaluation and Optimization of Organic Acid Pretreatment of Cotton Gin Waste for Enzymatic Hydrolysis and Bioethanol Production.

This paper investigates the efficiency of the organic acids on the pretreatment of an industrially generated cotton gin waste for the removal of lignin, thereby releasing cellulose and hemicellulose as fermentable sugar components. Cotton gin waste was pretreated with various organic acids namely lactic acid, oxalic acid, citric acid, and maleic acid. Among these, maleic acid was found to be the most efficient producing maximum xylose sugar (126.05 ± 0.74 g/g) at the optimum pretreatment condition of 150 °C, 500 mM, and 45 min. The pretreatment efficiency was comparable to the conventional dilute sulfuric acid pretreatment. A lignin removal of 88% was achieved by treating maleic acid pretreated biomass in a mixture of sodium sulfite and sodium chlorite. The pretreated biomass was further evaluated for the release of sugar by enzymatic hydrolysis and subsequently bioethanol production from hydrolysates. The maximum 686.13 g/g saccharification yield was achieved with maleic acid pretreated biomass which was slightly higher than the sulfuric acid (675.26 g/g) pretreated waste. The fermentation of mixed hydrolysates(41.75 g/l) produced 18.74 g/l bioethanol concentration with 2.25 g/l/h ethanol productivity and 0.48 g/g ethanol yield using sequential use of Saccharomyces cerevisiae and Pichia stipitis yeast strains. The production of bioethanol was higher than the ethanol produced using co-culture in comparison to sequential culture. Thus, it has been demonstrated that the maleic acid pretreatment and fermentation using sequential use of yeast strains are efficient for bioethanol production from cotton gin waste.

Preparation and characterization of gelatin-chitosan-nanoß-TCP based scaffold for orthopaedic application.

The primary aim of this study was to fabricate gelatin/chitosan/ß-TCP (GCT) composite scaffold to improve its compressive mechanical behaviour and in-vivo biocompatibility with predictable degradation rate. Beta tricalcium phosphate (ß-TCP) powder was synthesized in size range between 70-100 nm using aqueous precipitation route at a fixed Ca/P molar ratio of 1.5:1 at pH 10 and after subsequent heat treatment of as precipitated powder at 800 °C for 4 hours. The composite scaffolds were fabricated using solid-liquid phase separation of the slurry containing gelatin, chitosan, ß-tricalcium phosphate in varying proportion and subsequent lyophilisation of the phase separated mixture. The prepared scaffolds exhibited high porosity (>80%) with pore sizes ranging between 78-382 µm as determined using Hg-porosimetry. SEM result revealed that incorporation of ß-TCP to the extent of 30 wt% resulted in well-shaped and uniformly distributed interconnected pores of average pore size of 120 ±â€¯18.6 µm in it. Compressive strength of the scaffolds was increased from 0.8 MPa to 2.45 MPa on increase in ß-TCP content from 10 wt%-30 wt% in the prepared scaffold. Human Umbilical Cord derived mesenchymal stem cells (MSCs) exhibited higher degree of lamellopodia and fillopodia extensions and better spreading behaviour onto GCT30 scaffold. MTT assay and immunocytochemistry studies with cultured MSCs revealed that GCT30 scaffolds were more conducive to MSC's proliferation and differentiation into osteoblast lineage. In vivo implantation of GCT30 scaffold subcutaneously into mice did not indicate any significant inflammatory reaction, but ongoing vascularization.

Enhanced chondrogenesis of mesenchymal stem cells over silk fibroin/chitosan-chondroitin sulfate three dimensional scaffold in dynamic culture condition.

Chondroitin sulfate (Ch) is one of the main structural components of cartilage tissue, therefore, its presence in tissue engineered scaffold is expected to enhance cartilage regeneration. Previously, silk fibroin/chitosan (SF/CS) blend was proven to be a potential biomaterial for tissue development. In this study, the effect of Ch on physicochemical and biological properties of SF/CS blend was investigated and scaffolds with 0.8 wt% Ch was found to be favorable. The scaffolds possess pore size of 37-212 µm, contact angle 46.2-50.3°, showed controlled swelling and biodegradation. The biocompatibility of scaffold was confirmed by subcutaneous implantation in mouse. Human mesenchymal stem cells (hMSCs) seeded scaffolds cultured under spinner flask bioreactor promoted cell attachment, proliferation, distribution, and metabolic activity in vitro. The histology and immunofluorescence studies revealed that combined effect of Ch and dynamic condition resulted in higher glycosaminoglycan secretion and native cartilage type matrix synthesis in comparison to SF/CS scaffolds used as control. Higher expression of collagen-II, Sox9, aggrecan and decrease in collagen-I expression represented by quantitative polymerase chain reaction study confirmed the progression of chondrogenic differentiation. This study successfully demonstrates the potentiality of SF/CS-Ch scaffold for hMSCs recruitment and redirecting cartilage tissue regeneration with enhanced chondrogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2576-2587, 2018.

In vitro cartilage construct generation from silk fibroin- chitosan porous scaffold and umbilical cord blood derived human mesenchymal stem cells in dynamic culture condition.

Cartilage construct generation includes a scaffold with appropriate composition to mimic matrix of the damaged tissue on which the stem cells grow and differentiate. In this study, umbilical cord blood (UCB) derived human mesenchymal stem cells (hMSCs) were seeded on freeze dried porous silk-fibroin (SF)/chitosan (CS) scaffolds. Influence of static and dynamic (spinner flask bioreactor) culture conditions on the developing cartilage construct were studied by in-vitro characterization for viability, proliferation, distribution, and chondrogenic differentiation of hMSCs over the scaffold. Constructs developed in spinner flask consisted of 62% live cells, and exhibited 543% more cell density at the core than constructs cultured in static system. Quantification of DNA and glycosaminoglycans accumulation after 21 days showed the progression of chondrogenic differentiation of hMSCs was higher in dynamic culture compared to static one. In constructs generated under dynamic condition, histology staining for proteoglycan matrix, and fluorescence staining for collagen-II and aggrecan showed positive correlation between early and late stage chondrogenic markers, which was further confirmed by quantitative PCR analysis, showing low collagen-I expression and highly expressed Sox9, collagen-II and aggrecan. The present study demonstrated that construct generated by combining 3D SF/CS scaffold with UCB-hMSCs under dynamic condition using spinner flask bioreactor can be used for cartilage tissue regeneration for future medical treatments. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 397-407, 2018.

Ethno-Herbal-Medico in Wound Repair: An Incisive Review.

Wound healing/cicatrization is a complex series of intricate processes that involve renewal of skin/epidermis after injury. A large number of ethno-medicinal plants/plant extracts are used by tribal and folklore traditions in developing world for the treatment of wounds, burns and cuts in distinct appearances. Moreover, plants/plant extracts have a significant history and successful clinical track record as indigenous drugs in wound repair systems. This review provides detailed information on molecular and cellular mechanism of plant/plant extracts on wound healing applications and further analyses the opportunities and scope with its future openings and prospects owing to the multifaceted challenges attached with neo-tissue regeneration. Copyright © 2017 John Wiley & Sons, Ltd.

Biological and mechanical evaluation of poly(lactic-co-glycolic acid)-based composites reinforced with 1D, 2D and 3D carbon biomaterials for bone tissue regeneration.

Considering the fact that life on Earth is carbon based, carbon materials are being introduced in biological systems. However, very limited information exists concerning the potential effects of different structures of carbon materials on biological systems. In the present study, poly(lactic-co-glycolic acid) (PLGA)-based carbonaceous composites were developed by reinforcing 1 wt% of three different carbon-based materials i.e. carbon nanotubes (CNTs-1D), graphene nanoplatelets (GNPs-2D), and activated carbon (AC-3D). The developed composites were characterized for physicochemical, biological, and mechanical properties. Along with their hemocompatible nature, the composites exhibited better swelling ratio, degradation percentage, bioactivity, and tensile strength. The improvement in hydrophilicity and protein adsorption resulted in the enhancement of cell proliferation and differentiation. Overall, sheet-like GNPs showed the strongest effect on the composite's properties due to their larger exposed area. These results demonstrate the potential of PLGA-based carbonaceous composites for accelerating bone tissue regeneration.

Photo-mediated green synthesis of silver and zinc oxide nanoparticles using aqueous extracts of two mangrove plant species, Heritiera fomes and Sonneratia apetala and investigation of their biomedical applications.

Green synthesis by using biological agents has been a simple and effective approach for the synthesis of various forms of nanoparticles. The present investigation was intended to synthesis Ag-NPs and ZnO-NPs under photo-condition using the aqueous extracts of two mangrove plants namely Heritiera fomes and Sonneratia apetala and evaluate their potential biomedical applications. The formation of nanoparticles in aqueous solution of H. fomes and S. apetala under exposure to sun light was validated by change in color and formation of monodispersed NPs with a narrow particle size distribution. Fourier transform infrared spectroscopy (FT-IR) reveals the presence of Oxime and other heterocyclic compounds to be the most probable compounds responsible for the reduction and stability of nanoparticles in the solutions. The synthesized NPs displayed moderate free radical scavenging properties. The anti-inflammatory potential of ZnO-NPs was recorded to be comparatively higher than that of Ag-NP with 79% and 69.1% respectively. The Ag-NPs with unique properties of inhibiting α-amylase (91.14% and 89.16%) were found to be significantly high indicating its antidiabetic property. The synthesized NPs showed varied zone of inhibition (9-16mm) against the tested microbial pathogens. The synthesized nanoparticles possess strong biological activities in terms of antioxidant, anti-inflammatory, antidiabetic and antibacterial, potentials which could be utilized in various biological applications by the cosmetic, food and biomedical industries.

Serum-free non-toxic freezing solution for cryopreservation of human adipose tissue-derived mesenchymal stem cells.

OBJECTIVE: To develop a cost-effective, non-toxic and xeno-free freezing solution for the preservation of adipose tissue-derived stem cells (hADSC) with a long shelf-life. RESULTS: The potential of various hydrocolloids and organic osmolytes as cryoprotectants and individual components of phosphate buffered saline (PBS) as carrier media were evaluated to formulate a freezing solution for the cryopreservation of hADSCs. Among the hydrocolloids, the highest viability, 55 %, was achieved with post-thawed (after 48 h storage at -80 °C) hADSCs cryopreserved in 10 % (v/v) polyvinylpyrrolidone (PVP) using PBS as carrier media. 0.9 % NaCl was a superior carrier medium resulting an enhanced cell viability (70 %) when used in 10 % PVP than other components of PBS. A higher cell viability (81 %) was achieved when 10 % PVP/0.9 % NaCl was supplemented with 60 mM ectoin. The cryopreserved cells retained normal cytoskeletal distribution pattern and adipogenic and osteogenic differentiation ability during 14 and 21 days of incubation. CONCLUSION: A serum-free and non-toxic 10 % PVP/0.9 % NaCl/60 mM ectoin freezing solution was developed for cryopreservation of hADSC for application in tissue engineering and regenerative medicine.

Optimization and evaluation of silk fibroin-chitosan freeze-dried porous scaffolds for cartilage tissue engineering application.

Silk fibroin/chitosan blend has been reported to be an attractive biomaterial that provides a 3D porous structure with controllable pore size and mechanical property suitable for tissue engineering applications. However, there is no systematic study for optimizing the ratio of silk fibroin (SF) and chitosan (CS) which seems to influence the scaffold property to a great extent. The present research, therefore, investigates the effect of blend ratio of SF and CS on scaffold property and establishes the optimum value of blend ratio. Among the various blends, the scaffolds with blend ratio of SF/CS (80:20) were found to be superior. The scaffold possesses pore size in the range 71-210 µm and porosity of 82.2 ± 1.3%. The compressive strength of the scaffold was measured as 190 ± 0.2 kPa. The cell supportive property of the scaffold in terms of cell attachment, cell viability, and proliferation was confirmed by cell culture study using mesenchymal stem cells derived from umbilical cord blood. Furthermore, the assessment of glycosaminoglycan secretion on the scaffolds indicates its potentiality toward cartilage tissue regeneration.

Preparation and Evaluation of Gelatin-Chitosan-Nanobioglass 3D Porous Scaffold for Bone Tissue Engineering.

The aim of the present study was to prepare and characterize bioglass-natural biopolymer based composite scaffold and evaluate its bone regeneration ability. Bioactive glass nanoparticles (58S) in the size range of 20-30 nm were synthesized using sol-gel method. Porous scaffolds with varying bioglass composition from 10 to 30 wt% in chitosan, gelatin matrix were fabricated using the method of freeze drying of its slurry at 40 wt% solids loading. Samples were cross-linked with glutaraldehyde to obtain interconnected porous 3D microstructure with improved mechanical strength. The prepared scaffolds exhibited >80% porosity with a mean pore size range between 100 and 300 microns. Scaffold containing 30 wt% bioglass (GCB 30) showed a maximum compressive strength of 2.2 ± 0.1 MPa. Swelling and degradation studies showed that the scaffold had excellent properties of hydrophilicity and biodegradability. GCB 30 scaffold was shown to be noncytotoxic and supported mesenchymal stem cell attachment, proliferation, and differentiation as indicated by MTT assay and RUNX-2 expression. Higher cellular activity was observed in GCB 30 scaffold as compared to GCB 0 scaffold suggesting the fact that 58S bioglass nanoparticles addition into the scaffold promoted better cell adhesion, proliferation, and differentiation. Thus, the study showed that the developed composite scaffolds are potential candidates for regenerating damaged bone tissue.

Cobalt doped proangiogenic hydroxyapatite for bone tissue engineering application.

The present study delineates the synthesis and characterization of cobalt doped proangiogenic-osteogenic hydroxyapatite. Hydroxyapatite samples, doped with varying concentrations of bivalent cobalt (Co(2+)) were prepared by the ammoniacal precipitation method and the extent of doping was measured by ICP-OES. The crystalline structure of the doped hydroxyapatite samples was confirmed by XRD and FTIR studies. Analysis pertaining to the effect of doped hydroxyapatite on cell cycle progression and proliferation of MG-63 cells revealed that the doping of cobalt supported the cell viability and proliferation up to a threshold limit. Furthermore, such level of doping also induced differentiation of the bone cells, which was evident from the higher expression of differentiation markers (Runx2 and Osterix) and better nodule formation (SEM study). Western blot analysis in conjugation with ELISA study confirmed that the doped HAp samples significantly increased the expression of HIF-1α and VEGF in MG-63 cells. The analysis described here confirms the proangiogenic-osteogenic properties of the cobalt doped hydroxyapatite and indicates its potential application in bone tissue engineering.

Chitosan-poly(vinyl alcohol) nanofibers by free surface electrospinning for tissue engineering applications.

Deformities in tissues and organs can be treated by using tissue engineering approach offering the development of biologically functionalized scaffolds from a variety of polymer blends which mimic the extracellular matrix and allow adjusting the material properties to meet the defect architecture. In recent years, research interest has been shown towards the development of chitosan (CS) based biomaterials for tissue engineering applications, because of its minimal foreign body reactions, intrinsic antibacterial property, biocompatibility, biodegradability and ability to be molded into various geometries and forms thereby making it suitable for cell ingrowth and conduction. The present work involves the fabrication of nanofibrous scaffold from CS and poly(vinyl alcohol) blends by free-surface electrospinning method. The morphology and functional characteristics of the developed scaffolds were assessed by field emission scanning electron microscopy and fourier transformed infra-red spectra analysis. The morphological analysis showed the average fiber diameter was 269 nm and thickness of the mat was 200-300 µm. X-ray diffraction study confirmed the crystalline nature of the prepared scaffolds, whereas hydrophilic characteristic of the prepared scaffolds was confirmed by measured contact angle. The scaffolds possess an adequate biodegradable, swelling and mechanical property that is found desirable for tissue engineering applications. The cell study using umbilical cord blood-derived mesenchymal stem cells has confirmed the in vitro biocompatibility and cell supportive property of the scaffold thereby depicting their potentiality for future clinical applications.

Antimicrobial activity of iron oxide nanoparticle upon modulation of nanoparticle-bacteria interface.

Investigating the interaction patterns at nano-bio interface is a key challenge for safe use of nanoparticles (NPs) to any biological system. The study intends to explore the role of interaction pattern at the iron oxide nanoparticle (IONP)-bacteria interface affecting antimicrobial propensity of IONP. To this end, IONP with magnetite like atomic arrangement and negative surface potential (n-IONP) was synthesized by co-precipitation method. Positively charged chitosan molecule coating was used to reverse the surface potential of n-IONP, i.e. positive surface potential IONP (p-IONP). The comparative data from fourier transform infrared spectroscope, XRD, and zeta potential analyzer indicated the successful coating of IONP surface with chitosan molecule. Additionally, the nanocrystals obtained were found to have spherical size with 10-20 nm diameter. The BacLight fluorescence assay, bacterial growth kinetic and colony forming unit studies indicated that n-IONP (<50 µM) has insignificant antimicrobial activity against Bacillus subtilis and Escherichia coli. However, coating with chitosan molecule resulted significant increase in antimicrobial propensity of IONP. Additionally, the assay to study reactive oxygen species (ROS) indicated relatively higher ROS production upon p-IONP treatment of the bacteria. The data, altogether, indicated that the chitosan coating of IONP result in interface that enhances ROS production, hence the antimicrobial activity.

Autophagy protein Ulk1 promotes mitochondrial apoptosis through reactive oxygen species.

Regardless of rapid progression in the field of autophagy, it remains a challenging task to understand the cross talk with apoptosis. In this study, we overexpressed Ulk1 in HeLa cells and evaluated the apoptosis-inducing potential of the Ulk1 gene in the presence of cisplatin. The gain of function of Ulk1 gene showed a decline in cell viability and colony formation in HeLa cells. The Ulk1-overexpressing cells showed higher apoptotic attributes by an increase in the percentage of annexin V, escalated expression of Bax/Bcl2 ratio, and caspase-9, -3/7 activities. Further, reactive oxygen species (ROS) generation was found to be much higher in HeLa-Ulk1 than in the mock group. Scavenging the ROS by N-acetyl-L-cysteine increased cell viability and colony number as well as mitochondrial membrane potential (MMP). Our data showed that Ulk1 on entering into mitochondria inhibits the manganese dismutase activity and intensifies the mitochondrial superoxide level. The Ulk1-triggered autophagy (particularly mitophagy) resulted in a fall in ATP; thus the nonmitophagic mitochondria overwork the electron-transport cycle to replenish energy demand and are inadvertently involved in ROS overproduction that led to apoptosis. In this present investigation, our results decipher a previously unrecognized perspective of apoptosis induction by a key autophagy protein Ulk1 that may contribute to identification of its tumor-suppressor properties through dissecting the connection among cellular bioenergetics, ROS, and MMP.