Fabrication of Decellularized Amnion and Chorion Scaffolds to Develop Bioengineered Cell-Laden Constructs.

INTRODUCTION: Human mesenchymal stem cells (hMSCs) holds great promise for managing several clinical conditions. However, the low engraftment efficiency and obscurity to harvest these cells without compromising the cellular viability, structural and functional properties from the culture niche still remain major obstacles for preparing intact regenerative constructs. Although few studies have demonstrate different methods for generating cell-liberated amniotic scaffolds, a common method for producing completely cell-liberated amnion (D-HAM) and chorion (D-HCM) scaffolds and their cytocompatibility with hMSCs yet to be demonstrated. METHODS: A common process was developed for preparing D-HAM and D-HCM scaffolds for assessing hMSCs engraftment efficiency, proliferation and molecular shifts to generate cell-laden biological discs. The structural and functional integrity of D-HAM and D-HCM was evaluated using different parameters. The compatibility and proliferation efficiency of hMSCs with D-HAM and D-HCM was evaluated. RESULTS: Histological analysis revealed completely nucleic acid-free D-HAM and D-HCM scaffolds with intact extracellular matrix, mechanical and biological properties almost similar to the native membranes. Human MSCs were able to adhere and engraft on D-HCM better than D-HAM and expanded faster. Ultrastructural observations, crystal violet staining and expression studies showed better structural and functional integrity of hMSCs on D-HCM than D-HAM and control conditions. CONCLUSION: A common, simple and reliable process of decellularization can generate large number of cell-liberated amniotic scaffolds in lesser time. D-HCM has better efficiency for hMSCs engraftment and proliferation and can be utilized for preparing suitable cell-laden constructs for tissue engineering applications.

Biofabrication of allogenic bone grafts using cellularized amniotic scaffolds for application in efficient bone healing.

INTRODUCTION: The reconstruction/regeneration of human bone injuries/defects represents a crucial challenge due to the lack of suitable bio/immune compatible and implantable biological grafts. The available strategies represent implications of several types of grafting materials in the form of metals, synthetic, and various kinds of biological scaffolds; however, the lack of appropriate biological components required for activating and enhancing repair mechanisms at the lesion-site limits their wider applicability. METHODS: In this study, a unique approach for generating human osteogenic implantable grafts was developed using biofabrication technology. Using a gradient change of detergents and continuous agitation, developed a unique technique to generate completely cell-free amnion and chorion scaffolds. The absence of cellular components and integrity of biological and mechanical cues within decellularized human amnion (D-HAM) and chorion (D-HCM) were evaluated and compared with fresh membranes. Allogenic bone grafts were prepared through induction of human mesenchymal stem cells (hMSCs) into osteogenic cells on D-HAM and D-HCM and evaluated for their comparative behavior at the cellular, histological and molecular levels. RESULTS: The common decellularization process resulted in an efficient way to generate D-HAM and D-HCM while retaining their intact gross-anatomical architecture, surface morphology, extracellular matrix components, and mechanical properties. Both these scaffolds supported better growth of human umbilical cord blood derived MSCs as well as osteogenic differentiation. Comparative investigation revealed better growth rate and differentiation on D-HCM compared to D-HAM and control conditions. CONCLUSION: D-HCM could be used as a better choice for producing suitable allogenic bone grafts for efficient bone healing applications.

Intraperitoneal transplantation of bioengineered humanized liver grafts supports failing liver in acute condition.

Acute liver failure (ALF) is one of the most devastating fatal conditions which have posed crucial challenges to the clinicians and researchers for identifying permanent cure. Currently liver transplantation has been considered as the only managerial option. However it's wider applicability has been limited owing to non-availability of quality donor organs, cost-intensiveness, surgical hitches, life-long use of immunosuppressive drugs and long-term complications. Since last decades, several liver support systems have been developed for the management of failing liver in acute condition. However, the major limitation has been the lack of natural biological support and long-term survival of the grafts post-transplantation. Repopulation of decellularized xenogeneic organs is one of the emerging technologies for development of humanized neo-organs for demanding regenerative application. However, the earlier reported studies do not fulfil the insistence to provide immunologically tolerable humanized liver grafts for clinical applications. Here we demonstrate an efficient approach to generate transplantable humanized liver grafts which provides long-term support to the failing liver in Acute Liver Failure (ALF) animal models. These bioengineered humanized liver tissue grafts expresses several liver specific transcripts and performed crucial synthetic (albumin production) and detoxification (urea synthesis) functions at comparative level to normal liver. Intraperitoneal transplantation of these humanized liver grafts offered favourable microenvironment to exchange toxic substances across the barrier during ALF condition and provided long-term survival and function of the graft. In summary, the results of present study provide a first proof of concept in pre-clinical ALF animal model for the applicability of these bioengineered humanized livers in the management of failing liver on demand and may be considered as potential bridge to liver transplantation.

Molecular dynamics of pancreatic transcription factors in bioengineered humanized insulin producing neoorgan.

BACKGROUND: The present study has been aimed to identify molecular dynamics of pancreatic transcription factors (pTFs) during events of directed trans-differentiation of human hepatic progenitor cells (hHPCs) into insulin producing cells (InPCs) within bioengineered humanized neoorgan. The study demonstrates applicability of acellularized whole splenic scaffold (ASOS) to generate insulin producing humanized transplantable neoorgan through activation of pancreatic transcription factors. METHODS: An efficient acellularization process was developed for xenogeneic rat spleen using change in different gradients of reagents perfusion through splenic artery for varying time points. The acellularized xenogeneic spleen scaffold was characterized thoroughly for preservation of extra-cellular matrix and retention of organ specific vasculature and mechanical properties. Further scaffolds were sterilized and repopulated with hHPCs which were triggered using a stage wise induction with growth factors and hyperglycemic challenge for trans-differentiation into InPCs. Dynamics of pTFs alone or simultaneously during induction process was identified using gene expression analysis and immunological staining. RESULTS: The cells within the engineered neoorgan respond to growth factors and extrinsic hyperglycemic challenge and generate large number of InPCs under controlled dynamic regulation of pTFs. Highly controlled regulation of pTFs generates higher percentage of Nkx-6.1+/C-peptide+ cells within the engineered splenic scaffolds. Generation of high percentage of insulin and C-peptide positive cells in three-dimensional organ architecture responded better to hyperglycemic stimuli and produced higher quantity of insulin than 2D-culture system. CONCLUSION: The present study provides a novel platform for designing effective regenerative strategies using whole organ scaffolds to control hyperglycemia under tight regulation of pTFs using humanized neoorgan system.

Role of drug transporters and heat shock proteins during ethanol exposure to human neural precursor cells and its lineages.

INTRODUCTION: Ethanol exposure to developing brain may alter the growth and differentiation of neurological cells resulting in unfavorable pathologies. Earlier studies have provided very limited mechanistic insights of cellular and molecular mechanisms which do not mimic with human situation due to varying cell types and poses potential challenges for investigation. Therefore, the present study was undertaken to evaluate the role of ABC transporters and heat shock proteins mediated response in human neural precursor cells (NPCs) and its lineages during proliferation and lineage differentiation against ethanol exposure. METHODS: Effect of ethanol exposure was examined for neuronal cell survival and variation in cellular phenotype during neurospheres development and lineage differentiation. Generation of reactive oxygen species, and variation in cell cycle was identified along with transcriptional profiling for pluripotent markers (Nestin, NCAM, Sox-2, and Notch-2), drug transporters (ABCB1 and ABCG2) and stress protein (HSP70) during ethanol exposure. RESULTS: ABC transporters as well as HSP70 mRNA expression was higher during proliferation as compared to differentiation with chronic ethanol (1 M) exposure (p < 0.01). Ethanol exposure resulted in higher variability in size and shape of developing neurospheres and decreased ability to form new neurosphere colonies. Significant changes were observed in dendrite development due to late ethanol exposure (p < 0.0001). CONCLUSION: The present study demonstrated significant role of ABC transporters and HSP70 proteins in providing defense against ethanol-induced damage in human neurological cells. However, the over-expression of ABC transporter and HSP-70 proteins during such pathological conditions do not provide complete defense and additional strategies are required to repair the damage.

Protective Role of Hypothermia Against Heat Stress in Differentiated and Undifferentiated Human Neural Precursor Cells: A Differential Approach for the Treatment of Traumatic Brain Injury.

INTRODUCTION: The present study aimed to explore protective mechanisms of hypothermia against mild cold and heat stress on highly proliferative homogeneous human Neural Precursor Cells (NPCs) derived from Subventricular Zone (SVZ) of human fetal brain. METHODS: CD133+ve enriched undifferentiated and differentiated human NPCs were exposed to heat stress at 42°C. Then, Western-blot quantification was performed using Hsp-70 (70 kilodalton heat shock proteins) recombinant protein. Finally, changes in pluripotency and Hsp-70 expression were measured using immunofluorescence staining and RT-qPCR (Quantitative reverse transcription PCR) analysis, respectively. RESULTS: Heat stress resulted in abnormal neurospheres development. The apoptosis rate was enhanced during long-term in vitro culture of neurospheres. Neurogenic differentiation reduced and showed aberrent phenotypes during heat stress. After hypothermia treatment significant improvement in neurospheres and neuronal cell morphology was observed. CONCLUSION: Mild-hypothermia treatment induces attenuated heat shock response against heat stress resulting in induced HSP-70 expression that significantly improves structure and function of both undifferentiated human NPCs and differentiated neurons.