Development of an Experimental Model of a Decellularized Kidney Scaffold by Perfusion Mode and Analyzing the Three-dimensional Extracellular Matrix Architecture by Edge Detection Method.

Renal transplant is treatment of choice for the patients with end stage renal disease. The kidney transplants are expensive and there are risks of immunological and infectious complications. We planned to develop an in vitro decellularized kidney scaffold model using sheep kidney. Kidney decellularization was carried out by perfusing chemical detergents such as sodium dodecyl sulfate (SDS), SDS and trypsin, and SDS and ethylenediaminetetraacetic acid solvent solution. Complete kidney was decellularized in 5 days by perfusing various chemical detergents in time-dependent intervals. Histological finding revealed the complete removal of cellular material in various regions of renal corpuscle, distal convoluted tubules, other cortex and medulla region. Details of interlobular veins and arteries were seen through naked eyes after trypan blue dye injection. We used edge detection technique for developing a three-dimensional (3-D) image (Image J software) for nephrological vasculature constructed of decellularized kidney scaffold specimen. This technique opens a gateway for the whole organ decellularization by perfusion technology and further imaging of its 3-D extracellular matrix texture by edge detection technique software.

Structural and thermal characterization of PHAs produced by Lysinibacillus sp. through submerged fermentation process.

In this investigation an attempt has been made to characterize and identify Lysinibacillus sp. 3HHX by 16S-rDNA sequencing. The bacterium exhibited occurrence of PHAs granules on an average 11±1 per cell of 1.0µm length and breadth 0.72µm, revealed from TEM studies. Under optimized condition, 4.006gm/L of PHAs was extracted using hypochlorite digestion and multi-solvent extraction process. PhaC gene of ∼540bp and higher PHA synthase activity was detected at 48h of cultivation. The extracted PHAs was structurally characterized by GC-MS and 1H NMR reported to be P(3HB-co-3HDD-co-3HTD) and amorphous in nature with 112°C melting point, -11.0°C glass transition point and 114.76°C decomposition temperature detected by DSC & TGA respectively. The C/O of biopolymer disc was 1:65 as revealed from C1s and O1s spectra of XPS, that was completely biodegradable within 30 days. This biopolymer was observed to be non-cytotoxic to NIH 3T3 mouse fibroblast cells. The report is of its kind in establishing the abilities of Lysinibacillus sp. 3HHX for non-growth associated PHA co-polymer production. Moreover the biocompatible and biodegradable nature of the biopolymer conferred to its substantial biomedical applications.

Induction of bone formation in biphasic calcium phosphate scaffolds by bone morphogenetic protein-2 and primary osteoblasts.

Bone tissue engineering strategies mainly depend on porous scaffold materials. In this study, novel biphasic calcium phosphate (BCP) matrices were generated by 3D-printing. High porosity was achieved by starch consolidation. This study aimed to characterise the porous BCP-scaffold properties and interactions of osteogenic cells and growth factors under in vivo conditions. Five differently treated constructs were implanted subcutaneously in syngeneic rats: plain BCP constructs (group A), constructs pre-treated with BMP-2 (group B; 1.6 µg BMP-2 per scaffold), seeded with primary osteoblasts (OB) (group C), seeded with OB and BMP-2 (group D) and constructs seeded with OB and pre-cultivated in a flow bioreactor for 6 weeks (group E). After 2, 4 and 6 weeks, specimens were explanted and subjected to histological and molecular biological analyses. Explanted scaffolds were invaded by fibrovascular tissue without significant foreign body reactions. Morphometric analysis demonstrated significantly increased bone formation in samples from group D (OB + BMP-2) compared to all other groups. Samples from groups B-E displayed significant mRNA expression of bone-specific genes after 6 weeks. Pre-cultivation in the flow bioreactor (group E) induced bone formation comparable with group B. In this study, differences in bone distribution between samples with BMP-2 or osteoblasts could be observed. In conclusion, combination of osteoblasts and BMP-2 synergistically enhanced bone formation in novel ceramic scaffolds. These results provide the basis for further experiments in orthotopic defect models with a focus on future applications in orthopaedic and reconstructive surgery.