Ruchi Mishra

RETRACTED: Microbial production of dihydroxyacetone

Dihydroxyacetone is extensively used in cosmetic industry as an artificial suntan besides having clinical and biological applications. Thus, it is important to meet the commercial demand of dihydroxyacetone at an economical and qualitative level. Microbial route of production is found to be more favorable for dihydroxyacetone as compared to chemical methods. This review gives detailed information about the microbial route of dihydroxyacetone production. Till date the microorganism which is most utilized for dihydroxyacetone production is Gluconobacter oxydans. Some limitations associated with dihydroxyacetone production by G. oxydans like substrate inhibition, product inhibition and oxygen limitation are discussed here. Various fermentation modes and culture conditions have been tried for their ability to overcome these limitations. It has been found that fed-batch mode of fermentation provides a better yield as compared to batch mode for dihydroxyacetone production. Two-stage repeated fed-batch mode of fermentation has been found to be the most optimized mode. Immobilization has also been recognized as a much better alternative for fermentation since it avoids the problem of substrate and product inhibition to a greater extent. Although these methods have increased the dihydroxyacetone production to a prominent level yet the production has not reached the level required to meet the commercial demand. One looks for future prospects of developing recombinant microbial method for dihydoxyacetone production.

Cryogels: Freezing unveiled by thawing

Cryogels are interconnected supermacroporous gels prepared at sub-zero temperatures having applications in various research fields. The process of cryogelation is ideally thought to take place via following steps: phase separation with ice-crystal formation, cross-linking and polymerization followed by thawing of ice-crystals to form an interconnected porous cryogel network. This phenomenon mostly thought as a theoretical concept has now been revealed here in practical terms via data generated by micro-computed tomography (Micro CT). Micro CT is mainly used for characterizing the gel materials in terms of their physical properties like pore size, porosity, strut size, etc., whereas this work has pioneered its role in elucidating the process of cryogel formation.

Biocomposite cryogels as tissue-engineered biomaterials for regeneration of critical-sized cranial bone defects.

Analysis of the in vivo regeneration capability of any tissue-engineered biomaterial is necessary once it shows potential characteristics during in vitro studies. Thus, we applied polyvinyl alcohol-tetraethylorthosilicate-alginate-calcium oxide (PTAC) biocomposite cryogel on critical-sized cranial bone defects in wistar rats for examining the comparative bone regeneration of cryogel-treated and nontreated defects over a period of 4 weeks. An in-depth analysis was performed from macroscopic level till the gene level. Bone regeneration in cryogel-treated defects was clearly evident from the results, whereas the nontreated group did not show any defect healing except at few peripheral areas. At the macroscopic level, micro-computed tomography analysis revealed new bone formation. This was further confirmed at the cellular level, wherein, new bone formation was demonstrated by hematoxylin and eosin staining. Osteoblastic differentiation was further validated by immunohistological staining of runt-related transcription factor-2 (Runx-2) protein and via calcium-phosphate crystal formation after 2 weeks through scanning electron microscopy and energy dispersive X-ray spectroscopy. Finally, at the gene level, real-time PCR analysis confirmed the mRNA expression of osteoblastic markers, that is, runx-2, collagen type I (Col I), alkaline phosphatase (ALP), and osteocalcin (OCN). Therefore, the results of in vivo cranial defect model studies suggest that PTAC biocomposite cryogels can show suitable potential for human bone regeneration.

Inorganic/organic biocomposite cryogels for regeneration of bony tissues.

The present work focuses on the physical, mechanical and in vitro properties of porous inorganic/organic biocomposite scaffolds of polyvinyl alcohol–tetraethylorthosilicate–alginate–calcium oxide (PTAC). These scaffolds are prepared by means of cryogelation technology and are intended for bone tissue engineering applications. The biocomposite cryogels have much more favorable physical and biological properties compared to the previous work of our group on the same composition in the form of pellets and foams. The optimized and heat-treated PTAC biocomposite cryogels show homogenous porosity and good mechanical properties and also exhibit the formation of a hydroxyapatite-like layer on their surface on coming in contact with simulated body fluid (SBF). Furthermore, the biocomposite cryogels showed good biocompatibility with L929 fibroblasts. Also, the influence of pre-soaking in SBF to that of non-soaked scaffolds was compared in terms of proliferation of MG-63 osteoblast-like osteosarcoma cells on these scaffolds and it was found that the pre-soaking caused a decrease in cell proliferation. Finally, the response of human osteoblasts on these scaffolds was analyzed by MTT assay, scanning electron microscopy, energy dispersive X-ray spectroscopy and micro X-ray computing tomography. The cells revealed good biocompatibility with the biocomposite cryogels and were mostly present as cell sheets on the surface with thick bundles of collagenous extracellular matrix during initial period of incubation. During later phases, the formation of calcium phosphate-like mineral deposits was observed on the surface of the cryogels suggesting a high potential of the biocomposite cryogels towards bone regeneration. Therefore, the PTAC biocomposite cryogels, due to their favorable properties and high biocompatibility with human osteoblasts can be suggested as potential scaffolds for bone tissue engineering applications.

Study of in Vitro and in Vivo Bone Formation in Composite Cryogels and the Influence of Electrical Stimulation.

This work studies osteoinduction and bone conduction in polyvinyl alcohol-tetraethylorthosilicate-alginate-calcium oxide (PTAC) biocomposite cryogels along with the synergistic effect of electrical stimulation. In vitro osteoinduction of C2C12 myoblast towards osteogenic lineage is demonstrated through alkaline phosphatase assay, scanning electron microscopy and energy dispersive X-ray spectroscopy. These results were followed by in vivo implantation studies of PTAC biocomposite cryogel scaffolds in the bone conduction chamber model depicting bone formation after 24 days based on immunohistological staining for osteogenic markers, i.e., collagen type I (Col I), osteocalcin (OCN), osteopontin (OPN) and bone sialoprotein (BSP). Further, osteogenic differentiation of murine mesenchymal stem cells was studied with and without electrical stimulation. The q-PCR analysis shows that the electrically stimulated cryogels exhibit ~ 6 folds higher collagen type I and ~ 10 folds higher osteopontin mRNA level, in comparison to the unstimulated cryogels. Thus, PTAC biocomposite cryogels present osteoinductive and osteoconductive properties during in vitro and in vivo studies and support osteogenic differentiation of mesenchymal stem cells under the influence of electrical stimulation.

Effect of plasma polymerization on physicochemical properties of biocomposite cryogels causing a differential behavior of human osteoblasts.

HYPOTHESIS Plasma polymerization has been vastly employed for bringing changes in the surface functionality of the biomaterials by changing the surface chemistry without affecting the bulk properties. Therefore, we wanted to analyze the effect of plasma polymerization on the cellular response towards biocomposite cryogel surface and expected a positive response as found with the previous biomaterials. EXPERIMENTS We performed experiments for the incorporation of polyallylamine plasma on the polyvinyl alcohol-tetraethylorthosilicate-alginate-calcium oxide (PTAC) biocomposite cryogel surface. After plasma modification, the plasma coated/modified surfaces were studied at physicochemical level via methods like X-ray photoelectron spectroscopy, scanning electron microscopy, contact angle and rheology analysis. At biological level, analysis was performed through scanning electron microscopy, energy dispersive X-ray spectroscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and alkaline phosphatase (ALP) assay. FINDINGS In this paper we highlight a different aspect of plasma modification where both physical and chemical characteristics of surface gets altered on modification of PTAC biocomposite cryogels with plasma polymerized allylamine (ppAAm). This further affected the response of human osteoblasts on these modified/coated cryogels when compared to the unmodified/uncoated cryogels, wherein, the human osteoblasts on coated cryogel surface show higher alkaline phosphatase production and delayed mineralization as compared to the uncoated cryogels.