Chitosan-coated and thymol-loaded polymeric semi-interpenetrating hydrogels: An effective platform for bioactive molecule delivery and bone regeneration in vivo.

Thymol (2-isopropyl-5-methylphenol; Thy) is a monoterpene phenolic phytocompound with medicinal properties; however, its impact on osteogenesis is yet to be thoroughly investigated. Its distribution is often hampered because of its intricate hydrophobic structure, which reduces its bioavailability. In this study, we synthesized a drug delivery vehicle using semi-interpenetrating polymer network (SIPN) hydrogels containing sodium alginate and poly(2-ethyl-2-oxazoline) (SA/Pox) loaded with Thy at varying concentrations (100, 150, and 200 µM). Subsequently, they were coated with chitosan (CS) to increase bioactivity and for sustained and prolonged release of Thy. Thy-loaded CS-coated SIPN hydrogels (SA/Pox/CS-Thy) were developed using ionic gelation and polyelectrolyte-complexation techniques. The addition of CS to hydrogels enhanced their physicochemical and material properties. These hydrogels were cytofriendly toward mouse mesenchymal stem cells (mMSCs). When mMSCs were cultured on hydrogels, Thy stimulated osteoblastic differentiation, as evidenced by calcium deposits at the cellular level. The expression of RUNX2, a key bone transcriptional factor, and other differentiation biomarkers was significantly enhanced in mMSCs cultured on SA/Pox/CS-Thy hydrogels. Notably, Thy in the SA/Pox/CS hydrogels significantly activated the TGF-ß/BMP signaling pathway, which is involved in osteogenesis. A rat tibial bone defect model system revealed that the incorporation of Thy into SA/Pox/CS hydrogels augmented bone regeneration. Thus, sustained and prolonged release of Thy from the SA/Pox/CS hydrogels promoted osteoblast differentiation in vitro and bone formation in vivo. These findings shed light on the effect of Thy bioavailability in fostering osteoblast differentiation and its prospective application in bone rejuvenation.

Orsellinic acid-loaded chitosan nanoparticles in gelatin/nanohydroxyapatite scaffolds for bone formation in vitro.

AIM: Orsellinic acid (2,4-Dimethoxy-6-methylbenzoic acid) (OA) is a hydrophobic polyphenolic compound with therapeutic potential, but its impact on actuating osteogenesis remains unknown. The bioavailability of OA is hampered by its hydrophobic nature. This study aimed to fabricate nano-drug delivery system-based scaffolds for OA and test its potential for osteogenesis in vitro. MATERIALS AND METHODS: OA was loaded into chitosan nanoparticles (nCS + OA) using the ionic gelation technique at different concentrations. nCS + OA were incorporated onto the scaffolds containing gelatin (Gel) and nanohydroxyapatite (nHAp) by the lyophilization method. Biocomposite scaffolds were examined for their physicochemical and material characteristic properties. The effect of OA in the scaffolds for osteoblast differentiation was determined by alizarin red and von Kossa staining at the cellular level and by reverse transcriptase-qPCR and western blot analysis at the molecular level. KEY FINDINGS: The scaffolds showed excellent physiochemical and material characteristics and remained cyto-friendly to mouse mesenchymal stem cells (mMSCs, C3H10T1/2). The release of OA from Gel/nHAp/nCS scaffolds enhanced the differentiation of mMSCs towards osteoblasts, as observed through cellular and molecular studies. Moreover, the osteogenic potential of OA was mediated by the activation of FAK and ERK signaling pathways through integrins. SIGNIFICANCE: The inclusion of OA into Gel/nHAp/nCS biocomposite scaffolds at 80 µM concentration promoted osteoblast differentiation via cell adhesion mediated signaling, compared with that shown by Gel/nHAp/nCS alone. Overall, this study identified the potential therapeutic OA containing Gel/nHAp/nCS scaffolds, accelerating its potential for clinical application towards bone regeneration.

Epigenetic modifications of histones during osteoblast differentiation.

In bone biology, epigenetics plays a key role in mesenchymal stem cells' (MSCs) commitment towards osteoblasts. It involves gene regulatory mechanisms governed by chromatin modulators. Predominant epigenetic mechanisms for efficient osteogenic differentiation include DNA methylation, histone modifications, and non-coding RNAs. Among these mechanisms, histone modifications critically contribute to altering chromatin configuration. Histone based epigenetic mechanisms are an essential mediator of gene expression during osteoblast differentiation as it directs the bivalency of the genome. Investigating the importance of histone modifications in osteogenesis may lead to the development of epigenetic-based remedies for genetic disorders of bone. Hence, in this review, we have highlighted the importance of epigenetic modifications such as post-translational modifications of histones, including methylation, acetylation, phosphorylation, ubiquitination, and their role in the activation or suppression of gene expression during osteoblast differentiation. Further, we have emphasized the future advancements in the field of epigenetics towards orthopaedical therapeutics.

Preparation and characterization of chitosan/carboxymethyl pullulan/bioglass composite films for wound healing.

Wound dressings play a vital role in the wound healing process. Although a variety of wound dressings have been developed so far, most of them still have many drawbacks such as rigidity, non-porosity, low mechanical strength, an affinity to stick onto the injury surface and less antimicrobial activity. To overcome these issues, a novel type of porous three-dimensional (3D) film was fabricated using chitosan/carboxymethyl pullulan polyelectrolyte complex (PEC) loaded with 45S5 bioglass (CCMPBG) by the freeze-drying method for wound healing application. The developed films were analysed by FTIR, XRD, EDS and SEM to confirm their chemical nature, microstructure and surface morphologies. The CCMPBG films exhibited rough surface morphology and well-interconnected micropores with an average size range of 101-74 µm. Compared to the control chitosan/carboxymethyl pullulan (CCMP) film, the CCMPBG films showed an enhanced mechanical strength and controlled rate of swelling and biodegradation behaviours due to the interaction of polymer matrix and 45S5 bioglass (BG). Furthermore, CCMPBG films presented the improved biocompatibility, antimicrobial activity and wound closure ability because of the synergistic effects of chitosan, carboxymethyl pullulan (CMP) and BG. The results demonstrated that CCMPBG films can be an effective dressing material for wound therapy.

Synthesis and characterization of magnesium diboride nanosheets in alginate/polyvinyl alcohol scaffolds for bone tissue engineering.

Boride, which belongs to the distinct category of ceramic materials, has attracted significant attention in tissue engineering applications. Magnesium diboride (MgB2) consists of a plane of magnesium atoms sandwiched between the layers of boron. Even though MgB2 showed its role in various applications, its effect on osteogenesis has not yet been investigated. In this study, we synthesized MgB2 nanosheets (MgB2NS), a new class of 2D-nanoscale structures, by the ultrasonication exfoliation method and incorporated them into a polymeric mixture of alginate (Alg) and polyvinyl alcohol (PVA) by the freeze-drying procedure. The synthesized scaffolds (Alg/PVA/MgB2NS) were characterized by SEM, XRD, FT-IR, protein adsorption, swelling, degradation, and biomineralization studies. These scaffolds were non-toxic to mouse mesenchymal stem cells (mMSCs). MgB2NS in the scaffolds enhanced osteoblast differentiation of mMSCs at the molecular level by the expression of Runx2 and osteoblast differentiation marker genes and at the cellular level by alkaline phosphatase, alizarin Red and von Kossa staining. Overall, our results showed that MgB2NS in Alg/PVA scaffolds have osteogenic potential, suggesting their possible use in bone tissue engineering applications.

Nanosheets-incorporated bio-composites containing natural and synthetic polymers/ceramics for bone tissue engineering.

Bone is a highly integrative and dynamic tissue of the human body that is regularly modeled and remodeled by bone cells such as osteoblasts and osteoclasts. When a fraction of a bone is damaged or deformed, stem cells and bone cells under the influence of several signaling pathways regulate bone regeneration at the particular locale, but in a dilatory manner. To overcome this problem, the field of bone tissue engineering (BTE) utilizes various bio-composite scaffolds that incorporate cells and appropriate growth factors to promote osteogenesis. Nanosheets are in two-dimensional (2D)-atomic structures, and metallic and non-metallic nanosheets play a keen role in biomedical applications, including BTE. Due to their intrinsic mechanical properties such as durability and flexibility, nanosheets strengthen the bio-composite scaffolds containing natural polymers (chitosan, gelatin, and collagen), synthetic polymers, bio-ceramics, and bio-glasses. Incorporating nanosheets into bio-composites promotes the bio-functionality of the cells, such as cell adhesion and osteoblast differentiation. Hence, this review was aimed to provide a detailed study on the nanosheets-incorporated bio-composites and their properties for BTE applications.

Polycaprolactone/polyvinylpyrrolidone coaxial electrospun fibers containing veratric acid-loaded chitosan nanoparticles for bone regeneration.

Veratric acid (3,4-dimethoxy benzoic acid) (VA) is a hydrophobic phenolic phytocompound possessing therapeutic potential, but it has not been reported as actuating bone regeneration to date. Furthermore, delivery of hydrophobic compounds is often impeded in the body, thus depreciating their bioavailability. In this study, VA was found to have osteogenic potential and its sustained delivery was facilitated through a nanoparticle-embedded coaxial electrospinning technique. Polycaprolactone/polyvinylpyrrolidone (PCL/PVP) coaxial fibers were electrospun, encasing VA-loaded chitosan nanoparticles (CHS-NP). The fibers showed commendable physiochemical and material properties and were biocompatible with mouse mesenchymal stem cells (mMSCs). When mMSCs were grown on coaxial fibers, VA promoted these cells towards osteoblast differentiation as was reflected by calcium deposits. The mRNA expression of Runx2, an important bone transcriptional regulator, and other differentiation markers such as alkaline phosphatase, collagen type I, and osteocalcin were found to be upregulated in mMSCs grown on the PCL/PVP/CHS-NP-VA fibers. Overall, the study portrays the delivery of the phytocompound, VA, in a sustained manner to promote bone regeneration.

Temperature- and pH-responsive chitosan-based injectable hydrogels for bone tissue engineering.

Spontaneous bone regeneration is heavily restricted because of bone defects, and external mediation is required to enhance repair and regeneration. Bone tissue engineering (BTE) is a multidisciplinary field that offers promising substitutes to traditional methods-namely, autografts, allografts, and xenografts. Amidst the various scaffolds for BTE applications, it has been demonstrated that hydrogels are promising templates for bone regeneration owing to their similarities to the natural extracellular matrix. Regardless of the development of a variety of biomaterials, chitosan (CS) as a natural biopolymer has drawn tremendous attention in recent years for its use as a valuable graft material to form thermo/pH-responsive injectable hydrogels. Formulations of CS-based injectable hydrogels are advantageous in terms of their high-water imbibing capability, minimal invasiveness, porous networks, and ability to mold perfectly into an irregular defect. In this review, the physicochemical properties and applications of thermo/pH-responsive CS-based hydrogels and their future perspectives in BTE are briefly outlined.

An osteoinductive effect of phytol on mouse mesenchymal stem cells (C3H10T1/2) towards osteoblasts.

In recent years, phytochemicals have been widely researched and utilized for the treatment of various medical conditions such as cancer, cardiovascular diseases, age-related problems and are also said to have bone regenerative effects. In this study, phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol), an acyclic unsaturated diterpene alcohol and a secondary metabolite derived from aromatic plants was investigated for its effect on osteogenesis. Phytol was found to be nontoxic in mouse mesenchymal stem cells (C3H10T1/2). At the cellular level, phytol-treatment promoted osteoblast differentiation, as seen by the increased calcium deposits. At the molecular level, phytol-treatment stimulated the expression of Runx2 (a bone-related transcription factor) and other osteogenic marker genes. MicroRNAs (miRNAs) play an essential role in controlling bone metabolism by targeting genes at the post-transcriptional level. Upon phytol-treatment in C3H10T1/2 cells, mir-21a and Smad7 levels were increased and decreased, respectively. It was previously reported that mir-21a targets Smad7 (an antagonist of TGF-beta1 signaling) and thus, protects Runx2 from its degradation. Thus, based on our results, we suggest that phytol-treatment promoted osteoblast differentiation in C3H10T1/2 cells via Runx2 due to downregulation of Smad7 by mir-21a. Henceforth, phytol was identified to bolster osteoblast differentiation, which in turn may be used for bone regeneration.

Nanocomposite chitosan film containing graphene oxide/hydroxyapatite/gold for bone tissue engineering.

Recently, polymer based biomaterials are utilized in medical fields including surgical sutures, drug delivery devices, tissue supports and implants for interior bone fixation. However, polymer based implants leads to the formation of bio-films that are highly susceptible to microbial adhesion. In this study, we have fabricated Chitosan/Polyvinyl alcohol/Graphene oxide/Hydroxyapatite/gold films for potential orthopedic application. Graphene oxide/Hydroxyapatite/gold nanocomposite (GO/HAP/Au) was synthesized by simple hydrothermal method and GO/HAP/Au nanocomposite incorporated polymeric film was fabricated using gel casting method. The morphology, phase composition, crystalline structure and chemical state of the nanocomposite were characterized using as XRD, HR-TEM, FE-SEM and FT-IR. The bio-films were found to be biocompatible with mouse mesenchymal cells and it enhanced osteoblast differentiation as evidenced by more alkaline phosphatase activity at the cellular level. Hence, these results suggested that the developed nanocomposites films are osteogenic potential for treating bone and bone-related diseases. Antibacterial analysis of the films shows high inhibition zones against Gram positive and Gram Negative bacteria (Escherichia coli, streptococcus mutans, Staphylococcus aureus and Pseudomonas aeruginosa). Thus, the obtained nanocomposites bio-films are highly biocompatible and it can be used for bone regeneration application.

Fabrication of PCL/PVP Electrospun Fibers loaded with Trans-anethole for Bone Regeneration in vitro.

Trans-anethole (TA) is a phenolic phytocompound widely used in the food and health sector because of its diverse biological properties. However, its role in the promotion of bone formation is not known. With the enhanced bioavailability of TA, we aimed to determine its effect on osteogenesis; TA at different concentrations (5, 10, and 20 µM) was loaded onto polycaprolactone (PCL)/polyvinylpyrrolidone (PVP) fibers by the electrospinning technique. The synthesized PCL/PVP + TA fibers were subjected to physiochemical and material characterization. The addition of TA did not have any effect on fiber thickness, swelling, protein adsorption, degradation, or biomineralization. The fibers were compatible with mouse mesenchymal stem cells (mMSCs). A sustained release of TA from the fibers promoted osteoblast differentiation at the cellular and molecular levels. Furthermore, the release of TA from fibers up-regulated the expression of Runx2, a bone transcription factor, and its co-activators, which are key molecules for osteoblast differentiation. Thus, these results provide insights into the bioavailability of TA in promoting in vitro osteoblast differentiation and the potential applications of TA in bone regeneration.

Sustained release of chrysin from chitosan-based scaffolds promotes mesenchymal stem cell proliferation and osteoblast differentiation.

Numerous phytochemical compounds have recently been reported to stimulate osteogenesis. In this study, the bioavailability and osteogenic effects of chrysin, a natural flavonoid, were investigated. Chrysin was incorporated at different concentrations into biocomposite scaffolds containing carboxymethyl cellulose, chitosan, and nano-hydroxyapatite, through the freeze-drying method. The physicochemical and material characteristics of chrysin-incorporated scaffolds were investigated, and chrysin had no effect on them. These chrysin-containing scaffolds were not cytotoxic to mouse mesenchymal stem cells (mMSCs). Chrysin released from scaffolds stimulated cell proliferation and promoted osteoblast differentiation. Osteoblast differentiation enhanced by chrysin from scaffolds could be due to downregulation of co-repressors of the osteoblast differentiation transcription factor Runx2 in these cells. Thus, chrysin release from scaffolds has potential effects on proliferation and differentiation of mMSCs; hence, it has potential application in bone tissue engineering.

Chitosan/nano-hydroxyapatite/nano-zirconium dioxide scaffolds with miR-590-5p for bone regeneration.

Bone tissue engineering (BTE) relies on biocomposite scaffolds and bioactive molecules for bone regeneration. The present study was aimed to synthesize and characterize biocomposite scaffolds containing chitosan (CS), nano-hydroxyapatite (nHAp) and nano­zirconium dioxide (nZrO2) along with microRNA (miRNA) for BTE applications. miRNAs act as post-transcriptional regulator of gene expression. The fabricated biocomposite scaffolds were characterized using SEM, FT-IR and XRD analyses. The effect of a bioactive molecule (miR-590-5p) with scaffolds was tested for osteoblast differentiation at the cellular and molecular levels using mouse mesenchymal stem cells (C3H10T1/2). The results showed that CS/nHAp/nZrO2 scaffolds promoted osteoblast differentiation, and this effect was further increased in the presence of miR-590-5p in C3H10T1/2 cells. Thus, we suggested that CS/nHAp/nZrO2 scaffolds with miR-590-5p would have potential towards the treatment of bone defects.

Syringic acid, a phenolic acid, promotes osteoblast differentiation by stimulation of Runx2 expression and targeting of Smad7 by miR-21 in mouse mesenchymal stem cells.

Syringic acid (SA), a phenolic acid, has been used in Chinese and Indian medicine for treating diabetes but its role in osteogenesis has not yet been investigated. In the present study, at the molecular and cellular levels, we evaluated the effects of SA on osteoblast differentiation. At the cellular level, there was increased alkaline phosphatase (ALP) activity and calcium deposition by SA treatment in mouse mesenchymal stem cells (mMSCs). At the molecular level, SA treatment of these cells stimulated expression of Runx2, a bone transcription factor, and of osteoblast differentiation marker genes such as ALP, type I collagen, and osteocalcin. It is known that Smad7 is an antagonist of TGF-ß/Smad signaling and is a negative regulator of Runx2. microRNAs (miRNAs) play a key role in the regulation of osteogenesis genes at the post-transcriptional level and studies have reported that Smad7 is one of the target genes of miR-21. We found that there was down regulation of Smad7 and up regulation of miR-21 in SA-treated mMSCs. We further identified that the 3'-untranslated region (UTR) of Smad7 was directly targeted by miR-21 in these cells. Thus, our results suggested that SA promotes osteoblast differentiation via increased expression of Runx2 by miR-21-mediated down regulation of Smad7. Hence, SA may have potential in orthopedic applications.

Natural and synthetic polymers/bioceramics/bioactive compounds-mediated cell signalling in bone tissue engineering.

Bone is a highly integrative and dynamic tissue of the human body. It is continually remodeled by bone cells such as osteoblasts, osteoclasts. When a fraction of a bone is damaged or deformed, stem cells and bone cells under the influence of several signaling pathways regulate bone regeneration at the particular locale. Effective therapies for bone defects can be met via bone tissue engineering which employs drug delivery systems with biomaterials to enhance cellular functions by acting on signaling pathways such as Wnt, BMP, TGF-ß, and Notch. This review provides the current understanding of polymers/bioceramics/bioactive compounds as scaffolds in activation of signaling pathways for the formation of bone.

Chitosan based nanofibers in bone tissue engineering.

Bone tissue engineering involves biomaterials, cells and regulatory factors to make biosynthetic bone grafts with efficient mineralization for regeneration of fractured or damaged bones. Out of all the techniques available for scaffold preparation, electrospinning is given priority as it can fabricate nanostructures. Also, electrospun nanofibers possess unique properties such as the high surface area to volume ratio, porosity, stability, permeability and morphological similarity to that of extra cellular matrix. Chitosan (CS) has a significant edge over other materials and as a graft material, CS can be used alone or in combination with other materials in the form of nanofibers to provide the structural and biochemical cues for acceleration of bone regeneration. Hence, this review was aimed to provide a detailed study available on CS and its composites prepared as nanofibers, and their associated properties found suitable for bone tissue engineering.

An investigation on the diversity of mosquitocidal bacteria and its relationship with incidence of vector borne diseases.

Control of mosquitoes is the most important aspect of public health, as mosquitoes transmit many human diseases, including the fatal infection, Japanese encephalitis. This paper addresses the isolation of new mosquitocidal bacteria from soil samples in the Union Territory of Pondicherry, India, where, no clinical cases of vector borne infections have been reported. Bacterial isolates from soil samples were screened for potential mosquitocidal strains and bioassays against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) were carried out. Genomic DNA of potential mosquitocidal isolates was amplified and species identification was carried out using BLASTn program (NCBI). Phylogenetic analysis of 16S rRNA sequences of mosquitocidal bacteria revealed seven potential isolates. SDS-PAGE results have shown that there was considerable difference in the protein profiles. Numerical analysis revealed 4 distinct groups at similarity level 25%. The relationship between VBDs and prevalence of soil mosquitocidal bacteria in the study sites has elicited considerable interest in the diversity of mosquitocidal bacteria and their application for mosquito borne diseases control.

Hexamerin a novel protein associated with Bacillus sphaericus resistance in Culex quinquefasciatus.

Bacterial insecticides like, Bacillus sphaericus and Bacillus thuringiensis serovar israelensis, have been used for the control of nuisance and vector mosquitoes for more than two decades. For many years, it was assumed that the use of microbial larvicides based on B. sphaericus would not lead to resistance in mosquitoes. However, recent reports have shown that B. sphaericus toxins are not free from this problem. Therefore, the resistance of mosquito populations to be will seriously threaten the sustainability of current mosquito control programme using these microbial insecticides. In the present study, we have characterised a novel protein responsible for resistance development in the filariasis vector of Culex quinquefasciatus. Laboratory selection experiments with B. sphaericus against the larvae were carried out up to 17 generations, and the occurrence of resistance was reported (resistance ratio (RR) at lethal concentration (LC)50 and LC90 = 1,987 and 2,051 folds, respectively). The protein profiles of B. sphaericus-resistant and susceptible population have confirmed with the expression of a new polypeptide (80 kDa) in the resistant strain only. Sequence result revealed that the newly expressed protein was 'hexamerin', and this factor might conceivably be responsible for the inheritance of resistance. This study is therefore valuable for comprehending the underlining factor and management of B. sphaericus resistance problem in mosquito population.

Identification and characterization of a novel marine Bacillus cereus for mosquito control.

Entomopathogenic bacteria to control mosquitoes are a promising environmentally friendly alternative to synthetic pesticides. In the present study, a novel mosquitocidal bacterium was isolated from marine soil collected from east coastal areas at Pondicherry (India). 16S rRNA gene sequence alignment depicted that this isolate belonged to Bacillus cereus VCRC-B520 (NCBI: KC-119192). Biochemical studies on bacterial growth, biomass, and toxin production have revealed that this strain could possibly be helpful in the production of a biopesticide in mosquito control. Toxicity assay with B. cereus against mosquito larvae has shown that the filariasis vector, Culex quinquefasciatus, is more susceptible than the other two species (Anopheles stephensi and Aedes aegypti). The LC50 and LC90 values for C. quinquefasciatus were 0.30 and 2.21 mg/L, respectively. No effect of B. cereus was found on nontargeted organisms. SDS-PAGE analysis and protein purification result from the cell mass of B. cereus have shown that a well-perceptible polypeptide was the dependable factor (85 kDa) for mosquitocidal action. Protein characterization (M/S MALDI-TOF) has shown that it is an endotoxin-specific insecticidal protein, namely "Cry4Aa". Phylogenetic analyses of 16S rDNA gene sequence from this marine isolate have revealed the presence of homology among closely related Bacillus strains. Therefore, considerable interest has been shown on the identification of a potential mosquitocidal bacterium from marine environment (B. cereus), which was not reported earlier in view of the current scenario of the rapid development of resistance to Bacillus sphaericus in mosquito vector control program.