M. Vairamani

A novel injectable temperature-sensitive zinc doped chitosan/β-glycerophosphate hydrogel for bone tissue engineering.

Hydrogels are hydrophilic polymers that have a wide range of biomedical applications including bone tissue engineering. In this study we report preparation and characterization of a thermosensitive hydrogel (Zn-CS/β-GP) containing zinc (Zn), chitosan (CS) and beta-glycerophosphate (β-GP) for bone tissue engineering. The prepared hydrogel exhibited a liquid state at room temperature and turned into a gel at body temperature. The hydrogel was characterized by SEM, EDX, XRD, FT-IR and swelling studies. The hydrogel enhanced antibacterial activity and promoted osteoblast differentiation. Thus, we suggest that the Zn-CS/β-GP hydrogel could have potential impact as an injectable in situ forming scaffold for bone tissue engineering applications.

Scaffolds containing chitosan/carboxymethyl cellulose/mesoporous wollastonite for bone tissue engineering

Scaffold based bone tissue engineering utilizes a variety of biopolymers in different combinations aiming to deliver optimal properties required for bone regeneration. In the current study, we fabricated bio-composite scaffolds containing chitosan (CS), carboxymethylcellulose (CMC) with varied concentrations of mesoporous wollastonite (m-WS) particles by the freeze drying method. The CS/CMC/m-WS scaffolds were characterized by the SEM, EDS and FT-IR studies. Addition of m-WS particles had no effect on altering the porosity of the scaffolds. m-WS particles at 0.5% concentration in the CS/CMC scaffolds showed significant improvement in the bio-mineralization and protein adsorption properties. Addition of m-WS particles in the CS/CMC scaffolds significantly reduced their swelling and degradation properties. The CS/CMC/m-WS scaffolds also showed cyto-friendly nature to human osteoblastic cells. The osteogenic potential of CS/CMC/m-WS scaffolds was confirmed by calcium deposition and expression of an osteoblast specific microRNA, pre-mir-15b. Thus, the current investigations support the use of CS/CMC/m-WS scaffolds for bone tissue engineering applications.

Scaffolds containing chitosan, gelatin and graphene oxide for bone tissue regeneration in vitro and in vivo

Critical-sized bone defects are augmented with cell free and cell loaded constructs to bridge bone defects. Improving the properties of three-dimensional scaffolds with multiple polymers and others is of growing interest in recent decades. Chitosan (CS), a natural biopolymer has limitations for its use in bone regeneration, and its properties can be enhanced with other materials. In the present study, the composite scaffolds containing CS, gelatin (Gn) and graphene oxide (GO) were fabricated through freeze-drying. These scaffolds (GO/CS/Gn) were characterized by the SEM, Raman spectra, FT-IR, EDS, swelling, biodegradation, protein adsorption and biomineralization studies. The inclusion of GO in the CS/Gn scaffolds showed better physico-chemical properties. The GO/CS/Gn scaffolds were cyto-friendly to rat osteoprogenitor cells, and they promoted differentiation of mouse mesenchymal stem cells into osteoblasts. The scaffolds also accelerated bridging of the rat tibial bone defect with increased collagen deposition in vivo. Hence, these results strongly suggested the potential nature of GO/CS/Gn scaffolds for their application in bone tissue regeneration.

Detection of phenolic endocrine disrupting chemicals (EDCs) from maternal blood plasma and amniotic fluid in Indian population.

There is a widespread exposure of general population, including pregnant women and developing fetuses, to the endocrine disrupting chemicals (EDCs). These chemicals have been reported to be present in urine, blood serum, breast milk and amniotic fluid. We aimed to investigate the association between the maternal exposure and in utero fetal exposure levels of these chemicals to study their transfer from maternal to fetal unit indicating prenatal exposure. Samples of maternal blood and amniotic fluid were collected as set from 53 pregnant women at full term. Nine phenolic EDCs, methyl paraben (MP; 20.92ng/mL and 18.92ng/mL), ethyl paraben (EP; 1.97ng/ mL and 1.89ng/mL), propyl paraben (PP; 19.22ng/mL and 18.82ng/mL), butyl paraben (BP; 1.11ng/mL and 1.37ng/mL), p-hydroxybenzoic acid (PHBA; 29.99ng/mL and 26.15ng/mL), bisphenol A (BPA; 7.43ng/mL and 7.75ng/mL), triclosan (TCS; 7.17ng/mL and 7.04ng/mL), octyl phenol (OP; 5.46ng/mL and 5.72ng/mL) and nonyl phenol (NP; 9.38ng/mL and 8.44ng/mL), were simultaneously detected in samples of maternal blood plasma and amniotic fluid respectively using Gas Chromatography-Mass Spectrometry (GC-MS). Highest positive correlation was found for total concentration of 4-nonyl phenol, NP (r=0.575, p<0.001), whereas the lowest positive correlation was found for free form of bisphenol A, BPA (r=0.343, p<0.05), when compared between the two matrices. Our results suggest that maternal exposure to several EDCs is positively associated with in utero exposure to the developing fetus. Future studies should focus on collection of amniotic fluid at different trimesters and the corresponding maternal samples to better characterize the pharmacokinetics and the associated disease etiologies of these EDCs during fetal development.

Effects of silica and calcium levels in nanobioglass ceramic particles on osteoblast proliferation

At nanoscale, bioglass ceramic (nBGC) particles containing calcium oxide (lime), silica and phosphorus pentoxide promote osteoblast proliferation. However, the role of varied amounts of calcium and silica present in nBGC particles on osteoblast proliferation is not yet completely known. Hence, the current work was aimed at synthesizing two different nBGC particles with varied amounts of calcium oxide and silica, nBGC-1: SiO2:CaO:P2O5; mol%~70:25:5 and nBGC-2: SiO2:CaO:P2O5; mol%~64:31:5, and investigating their role on osteoblast proliferation. The synthesized nBGC particles were characterized by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) studies. They exhibited their size at nanoscale and were non-toxic to human osteoblastic cells (MG-63). The nBGC-2 particles were found to have more effect on stimulation of osteoblast proliferation and promoted entering of more cells into G2/M cell cycle phase compared to nBGC-1 particles. There was a differential expression of cyclin proteins in MG-63 cells by nBGC-1 and nBGC-2 treatments, and the expression of cyclin B1 and E proteins was found to be more by nBGC-2 treatment. Thus, these results provide us a new insight in understanding the design of various nBGC particles by altering their ionic constituents with desirable biological properties thereby supporting bone augmentation.

A Combinatorial effect of carboxymethyl cellulose based scaffold and microRNA-15b on osteoblast differentiation

The present study was aimed to synthesize and characterize a bio-composite scaffold containing carboxymethyl cellulose (CMC), zinc doped nano-hydroxyapatite (Zn-nHAp) and ascorbic acid (AC) for bone tissue engineering applications. The fabricated bio-composite scaffold was characterized by SEM, FT-IR and XRD analyses. The ability of scaffold along with a bioactive molecule, microRNA-15b (miR-15b) for osteo-differentiation at cellular and molecular levels was determined using mouse mesenchymal stem cells (mMSCs). miR-15b acts as posttranscriptional gene regulator and regulates osteoblast differentiation. The scaffold and miR-15b were able to promote osteoblast differentiation; when these treatments were combined together on mMSCs, there was an additive effect on promotion of osteoblast differentiation. Thus, it appears that the combination of CMC/Zn-nHAp/AC scaffold with miR-15b would provide more efficient strategy for treating bone related defects and bone regeneration.

Alginate/Gelatin scaffolds incorporated with Silibinin-loaded Chitosan nanoparticles for bone formation in vitro

Silibinin is a plant derived flavonolignan known for its multiple biological properties, but its role in the promotion of bone formation has not yet been well studied. Moreover, the delivery of Silibinin is hindered by its complex hydrophobic nature, which limits its bioavailability. Hence, in this study, we fabricated a drug delivery system using chitosan nanoparticles loaded with Silibinin at different concentrations (20μM, 50μM, and 100μM). They were then incorporated into scaffolds containing Alginate and Gelatin (Alg/Gel) for the sustained and prolonged release of Silibinin. The Silibinin-loaded chitosan nanoparticles (SCN) were prepared using the ionic gelation technique, and the scaffolds (Alg/Gel-SCN) were synthesized by the conventional method of freeze drying. The scaffolds were subjected to physicochemical and material characterization studies. The addition of SCN did not affect the porosity of the scaffolds, yet increased the protein adsorption, degradation rates, and bio-mineralization. These scaffolds were biocompatible with mouse mesenchymal stem cells. The scaffolds loaded with 50μM Silibinin promoted osteoblast differentiation, which was determined at cellular and molecular levels. Recent studies indicated the role of microRNAs (miRNAs) in osteogenesis and we found that the Silibinin released from scaffolds regulated miRNAs that control the bone morphogenetic protein pathway. Hence, our results suggest the potential for sustained and prolonged release of Silibinin to promote bone formation and, thus, these Alg/Gel-SCN scaffolds may be candidates for bone tissue engineering applications.

Characterization of Runx2 Phosphorylation Sites Required for TGF-β1-Mediated stimulation of Matrix Metalloproteinase-13 Expression in Osteoblastic cells†

Transforming growth factor‐beta1 (TGF‐β1), a highly abundant growth factor in skeletal tissues, stimulates matrix metalloproteinase‐13 (MMP‐13) expression in osteoblastic cells. MMP‐13 plays a critical role in bone remodeling. Runx2, a bone transcription factor, is required for TGF‐β1‐mediated stimulation of MMP‐13 expression in osteoblastic cells. In this study, the molecular mechanism responsible for TGF‐β1‐stimulation of MMP‐13 expression via Runx2 in osteoblastic cells was elucidated. TGF‐β1 stimulated the phosphorylation of Runx2 at serine amino acids, and ERK inhibition blocked this effect in rat (UMR106‐01) and human (MG‐63) osteoblastic cells. Pretreatment with okadaic acid, a serine‐threonine phosphatase inhibitor, increased Runx2 serine phosphorylation in osteoblastic cells. When cells were pretreated with an ERK inhibitor, TGF‐β1‐mediated stimulation of MMP‐13 mRNA expression decreased. Nano‐ESI/LC/MS analysis identified that TGF‐β1 stimulates Runx2 phosphorylation at three serine amino acids. Transient transfection of mouse mesenchymal stem cells (C3H10T1/2) with Runx2 serine mutant constructs decreased TGF‐β1‐mediated Runx2 serine phosphorylation. A luciferase reporter assay identified that TGF‐β1 stimulated MMP‐13 promoter activity in these cells only in the presence of the wild Runx2 construct, and not with mutant Runx2. Thus, TGF‐β1 stimulates the phosphorylation of Runx2 at three serine amino acids, and this event is required for MMP‐13 expression in osteoblastic cells. Hence, this study contributes to the knowledge of events governing bone remodeling and bone‐related diseases.

Proliferation and differentiation of mesenchymal stem cells on scaffolds containing chitosan, calcium polyphosphate and pigeonite for bone tissue engineering

Treatment of critical‐sized bone defects with cells and biomaterials offers an efficient alternative to traditional bone grafts. Chitosan (CS) is a natural biopolymer that acts as a scaffold in bone tissue engineering (BTE). Polyphosphate (PolyP), recently identified as an inorganic polymer, acts as a potential bone morphogenetic material, whereas pigeonite (Pg) is a novel iron‐containing ceramic. In this study, we prepared and characterized scaffolds containing CS, calcium polyphosphate (CaPP) and Pg particles for bone formation in vitro and in vivo.

Establishment of pancreatic microenvironment model of ER stress: Quercetin attenuates β-cell apoptosis by invoking nitric oxide-cGMP signaling in endothelial cells

The involvement of endoplasmic reticulum (ER) stress in endothelial dysfunction and diabetes-associated complications has been well documented. Inhibition of ER stress represents a promising therapeutic strategy to attenuate endothelial dysfunction in diabetes. Recent attention has focused on the development of small molecule inhibitors of ER stress to maintain endothelial homeostasis in diabetes. Here we have developed a reliable, robust co-culture system that allows a study on the endothelial cells and pancreatic β-cells crosstalk under ER stress and validated using a known ER stress modulator, quercetin. Furthermore, sensitizing of endothelial cells by quercetin (25 μM) confers protection of pancreatic β-cells against ER stress through nitric oxide (NO∙) signaling. In addition, increased intracellular insulin and NO∙-mediated cyclic 3,5-guanosine monophosphate (cGMP) levels in pancreatic β-cells further confirmed the mechanism of protection under co-culture system. In addition, the potential protein targets of quercetin against ER stress in the endothelial cells were investigated through proteomic profiling and its phosphoprotein targets through Bioplex analysis. On the whole, the developed in vitro co-culture set up can serve as a platform to study the signaling network between the endothelial and pancreatic β-cells as well as provides a mechanistic insight for the validation of novel ER stress modulators.