Self-assembling polymeric dendritic peptide as functional osteogenic matrix for periodontal regeneration scaffolds-an in vitro study.

OBJECTIVE: Regeneration of periodontal defects is challenging as it necessitates the formation of complex tissue structure with cementum, periodontal ligament, and alveolar bone. Rather than the conventional barrier membranes, scaffolds mimicking extracellular matrix (ECM) can achieve faster healing as they promote migration, adhesion, and differentiation of native progenitor cells. This work explores the possibility of a functional osteogenic matrix based on self-assembling peptide appended dendritic polydiacetylene in regenerating diseased periodontia. METHOD: The amino acid lysine was appended onto a diacetylene core, which was converted to a polymeric dendritic lysine matrix (Lys-PDA) through photopolymerization. This bioactive matrix was evaluated in vitro for the viability, adhesion, spreading, and differentiation of cultured human periodontal ligament (hPDL) progenitor cells. Its osteogenic differentiation was analysed by histologic staining and expression of osteogenic markers (alkaline phosphatase and Osteonectin). Electrospun polycaprolactone (PCL) mat, a candidate barrier material, was fabricated and functionalized with Lys-PDA matrix, and the cell viability, adhesion, and spreading of hPDL cells were evaluated. RESULTS: The dendritic Lys-PDA matrix well supported the hPDL cell growth and differentiation. The cells were viable and showed good cytoskeletal organization. Early expression of osteogenic markers and mineralization was noted in vitro in the presence of Lys-PDA matrix. The electrospun PCL mat functionalized with Lys-PDA maintained the viability, morphology, and spreading of the hPDL cells. SIGNIFICANCE: The ECM mimetic dendritic peptide matrices are capable of hosting and differentiating cells which can lead to the regeneration of periodontal tissue architecture. They could be used in conjunction with barrier membranes for better results.

Drug loaded microbeads entrapped electrospun mat for wound dressing application.

A new design of antibiotic loaded wound dressing and its initial in vitro evaluation is described. Chitosan microbeads loaded with ampicillin were sandwiched within polycaprolactone electrospun mat (MbAPPCL). The morphology was analyzed by scanning electron microscopy and surface chemistry was characterized by Fourier Transform Infrared Spectroscopy. In vitro cytotoxicity using L-929 fibroblast cells by direct contact test and elution assay revealed non-cytotoxic nature of MbAPPCL. The cell adhesion and viability analysis further confirmed the cytocompatibility of MbAPPCL as a wound dressing material. Percentage hemolysis and platelet adhesion on the mat exposed to blood substantiated the hemocompatibility. The antibiotic susceptibility test analyzed on Staphylococcus aureus by agar plate method confirmed the drug release and antimicrobial property. The proposed wound dressing model explained with ampicillin as a candidate drug has the potential to include microbeads with different antibiotics for multi drug treatment.

Characterization and in vitro evaluation of electrospun chitosan/polycaprolactone blend fibrous mat for skin tissue engineering.

The electrospinning technique allows engineering biomimetic scaffolds within micro to nanoscale range mimicking natural extracellular matrix (ECM). Chitosan (CS) and polycaprolactone (PCL) were dissolved in a modified solvent mixture consisting of formic acid and acetone (3:7) and mixed in different weight ratios to get chitosan-polycaprolactone [CS-PCL] blend solutions. The CS-PCL blend polymer was electrospun in the same solvent system and compared with PCL. The physicochemical characterization of the electrospun fibrous mats was done using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), tensile test, swelling properties, water contact angle (WCA) analysis, surface profilometry and thermo gravimetric analysis (TGA). The CS-PCL fibrous mat showed decreased hydrophobicity. The CS-PCL mats also showed improved swelling property, tensile strength, thermal stability and surface roughness. The cytocompatibility of the CS-PCL and PCL fibrous mats were examined using mouse fibroblast (L-929) cell line by direct contact and cellular activity with extract of materials confirmed non-cytotoxic nature. The potential of CS-PCL and PCL fibrous mats as skin tissue engineering scaffolds were assessed by cell adhesion, viability, proliferation and actin distribution using human keratinocytes (HaCaT) and L-929 cell lines. Results indicate that CS-PCL is a better scaffold for attachment and proliferation of keratinocytes and is a potential material for skin tissue engineering.

Evaluation of allylated gelatin as a bioink supporting spontaneous spheroid formation of HepG2 cells.

The spheroid culture system has gained significant attention as an effective in vitro model to mimic the in vivo microenvironment. Even though numerous studies were focused on developing spheroids, the structural organization of encapsulated cells within hydrogels remains a challenge. Allylated gelatin or GelAGE is used as a bioink due to its excellent physicochemical properties. In this study, GelAGE was evaluated for its capacity to induce spontaneous spheroid formation in encapsulated HepG2 cells. GelAGE was synthesized and characterized using 1HNMR spectroscopy and ninhydrin assay. Then the physicochemical and biological attributes of GelAGE hydrogel was examined. The results demonstrate that GelAGE has remarkable ability to induce the encapsulated cells to self-organize into spheroids.

Silymarin enriched gelatin methacrylamide bioink imparts hepatoprotectivity to 3D bioprinted liver construct against carbon tetrachloride induced toxicity.

Three-dimensional liver bioprinting is an emerging technology in the field of regenerative medicine that aids in the creation of functional tissue constructs that can be used as transplantable organ substitutes. During transplantation, the bioprinted donor liver must be protected from the oxidative stress environment created by various factors during the transplantation procedure, as well as from drug-induced damage from medications taken as part of the post-surgery medication regimen following the procedure. In this study, Silymarin, a flavonoid with the hepatoprotective properties were introduced into the GelMA bioink formulation to protect the bioprinted liver against hepatotoxicity. The concentration of silymarin to be added in GelMA was optimised, bioink properties were evaluated, and HepG2 cells were used to bioprint liver tissue. Carbon tetrachloride (CCl4) was used to induce hepatotoxicity in bioprinted liver, and the effect of this chemical on the metabolic activities of HepG2 cells was studied. The results showed that Silymarin helps with albumin synthesis and shields liver tissue from the damaging effects of CCl4. According to gene expression analysis, CCl4 treatment increased TNF-α and the antioxidant enzyme SOD expression in HepG2 cells while the presence of silymarin protected the bioprinted construct from CCl4-induced damage. Thus, the outcomes demonstrate that the addition of silymarin in GelMA formulation protects liver function in toxic environments.

Liver tissue engineering and cell sources: issues and challenges.

Liver diseases are of major concern as they now account for millions of deaths annually. As a result of the increased incidence of liver disease, many patients die on the transplant waiting list, before a donor organ becomes available. To meet the huge demand for donor liver, alternative approaches using liver tissue engineering principles are being actively pursued. Even though adult hepatocytes, the primary cells of the liver are most preferred for tissue engineering of liver, their limited availability, isolation from diseased organs, lack of in vitro propagation and deterioration of function acts as a major drawback to their use. Various approaches have been taken to prevent the functional deterioration of hepatocytes including the provision of an adequate extracellular matrix and co-culture with non-parenchymal cells of liver. Great progress has also been made to differentiate human stem cells to hepatocytes and to use them for liver tissue engineering applications. This review provides an overview of recent challenges, issues and cell sources with regard to liver tissue engineering.

Peripheral Blood As a Source of Stem Cells for Regenerative Medicine: Emphasis Towards Corneal Epithelial Reconstruction-An In Vitro Study.

BACKGROUND: Mesenchymal stem cell-based treatments are now emerging as a therapy for corneal epithelial damage. Although bone marrow, adipose tissue and umbilical cord blood are the main sources of mesenchymal stem cells (MSCs), other tissues like the peripheral blood also harbor mesenchymal-like stem cells called peripheral blood-derived mononuclear cells (PBMNCs). These blood derived stem cells gained a lot of attention due to its minimally invasive collection and ease of isolation. In this study, the feasibility of using PBMNCs as an alternative cell source to corneal limbal stem cells envisaging corneal epithelial regeneration was evaluated. METHODS: Rabbit PBMNCs were isolated using density gradient centrifugation and was evaluated for mesenchymal cell properties including stemness. PBMNCs were differentiated to corneal epithelial lineage using rabbit limbal explant conditioned media and was evaluated by immuno-cytochemistry and gene expression analysis. Further, the differentiated PBMNCs were engineered into a cell sheet using an in-house developed thermo-responsive polymer. RESULTS: These blood derived cells were demonstrated to have similar properties to mesenchymal stem cells. Corneal epithelial lineage commitment of PBMNCs was confirmed by the positive expression of CK3/12 marker thereby demonstrating the aptness as an alternative to limbal stem cells. These differentiated cells effectively generated an in vitro cell sheet that was then demonstrated for cell sheet transfer on an ex vivo excised rabbit eye. CONCLUSION: PBMNCs as an alternative autologous cell source for limbal stem cells is envisaged as an effective therapeutic strategy for corneal surface reconstruction especially for patients with bilateral limbal stem cell deficiency.

Bioengineered corneal epithelial cell sheet from mesenchymal stem cells-A functional alternative to limbal stem cells for ocular surface reconstruction.

Limbal stem cell deficiency (LSCD) is the loss of limbal stem cells that reside in the corneoscleral junction resulting in vision loss or blindness. Bilateral LSCD is usually treated by allogeneic corneal transplantation, with instances of tissue rejection or failure in long-term follow-up. This study aims to use adipose mesenchymal stem cells (ASC) as an alternative autologous cell source for treating bilateral limbal deficiency conditions. ASCs derived from rabbit fat tissue were differentiated into corneal epithelial lineage using limbal explant condition media. Apart from transdifferentiation, ASC sheets were developed to facilitate effective delivery of these cells to the damage site. A thermoresponsive polymer N-isopropylacrylamide-co-glycidylmethacrylate (NGMA) was synthesized and characterized to demonstrate ASC sheet formation. Transdifferentiated ASCs showed positive expression of corneal epithelial marker CK3/12 on immunostaining, supported by gene expression studies. in vivo studies by transplanting cell sheet in rabbit models of corneal injury showed clear and smooth cornea in comparison to the sham models. Histology revealed a sheet of cells aligned and integrated on to the injured corneal surface, 1 month posttransplantation. Identifying ASCs as an alternative cell source along with cell sheet technology will be a novel step in the field of corneal surface therapies.

Calcium sulfate-based bioactive cement for periodontal regeneration: An In Vitro study.

BACKGROUND AND OBJECTIVE: Various types of osteoconductive graft materials are used for the management of alveolar bone defects arising out of periodontal disease. Inorganic, self-setting, bioactive bone cements are suggested to be most appropriate because they can conformally fill the bone defect and resorb progressively along with the regeneration of the host site. A new calcium sulfate-based bioactive bone cement (BioCaS) is developed, having simplicity and effectiveness for bone grafting applications. The response of primary human periodontal ligament (hPDL) cells to this material is investigated through in vitro cell culture model so as to qualify it for the repair of periodontal infrabony defects. METHOD: The BioCaS was designed as powder-liquid combination with in-house synthesized high purity calcium sulfate hemihydrate incorporating hydrogen orthophosphate ions. hPDL cells were isolated, cultured and characterized using optimized primary cell culture techniques. The cytotoxicity and cytocompatibility of the BioCaS samples were evaluated using the hPDL cells, with hydroxyapatite ceramic material as control. Osteogenic differentiation of the hPDL cells in presence of BioCaS was also evaluated using Alizarin red staining, Alizarin red assay, Von Kossa staining and Masson's trichrome staining. RESULTS: The primary cell culture techniques yielded a healthy population of periodontal ligament cells, with fibroblast morphology and characteristic marker expressions. The hPDL cells exhibited good viability, adhesion and spreading to the BioCaS cement in comparison to sintered hydroxyapatite. In addition, the cells differentiated to osteogenic lineage in the presence of the BioCaS cement, without extraneous osteogenic supplements, confirming the inherent bioactivity of the cement. CONCLUSION: The new BioCaS cement is a potential candidate for the repair of periodontal defects.

Direct cell imprint lithography in superconductive carbon black polymer composites: process optimization, characterization and in vitro toxicity analysis.

Cell imprint lithography (CIL) or cell replication plays a vital role in fields like biomimetic smart culture substrates, bone tissue engineering, cell guiding, cell adhesion, tissue engineering, cell microenvironments, tissue microenvironments, cell research, drug delivery, diagnostics, therapeutics and many other applications. Herein we report a new formulation of superconductive carbon black photopolymer composite and its characterization towards a CIL process technique. In this article, we demonstrated an approach of using a carbon nanoparticle-polymer composite (CPC) for patterning cells. It is observed that a 0.3 wt % load of carbon nanoparticles (CNPs) in a carbon polymer mixture (CPM) was optimal for cell-imprint replica fabrication. The electrical resistance of the 3-CPC (0.3 wt %) was reduced by 68% when compared to N-CPC (0 wt %). This method successfully replicated the single cell with sub-organelle scale. The shape of microvesicles, grooves, pores, blebs or microvilli on the cellular surface was patterned clearly. This technique delivers a free-standing cell feature substrate. In vitro evaluation of the polymer demonstrated it as an ideal candidate for biomimetic biomaterial applications. This approach also finds its application in study based on morphology, especially for drug delivery applications and for investigations based on molecular pathways.

A flexible thermoresponsive cell culture substrate for direct transfer of keratinocyte cell sheets.

Most cell sheet engineering systems require a support or carrier to handle the harvested cell sheets. In this study, polyethylene terephthalate-based overhead projection transparency sheets (OHPS) were subjected to surface hydrolysis by alkali treatment to increase pliability and hydrophilicity and enable poly(N-isopropylacrylamide-co-glycidylmethacrylate) copolymer (NGMA) coating to impart thermoresponsiveness. NGMA was applied on the modified OHPS by the technique of spin coating using an indigenously designed spin coater. The spin coating had the advantage of using low volumes of the polymer and a reduced coating time. The surface chemistry and thermoresponsive coating was analyzed by Fourier transform infrared spectroscopy and water contact angle. Human keratinocyte cells were cultured on the spin coated surface and scaffold-free cell sheets were successfully harvested by simple variation of temperature. These cell sheets were found to be viable, exhibited epithelial characteristic and cell-cell contact as confirmed by positive immunostaining for ZO-1. The integrity and morphology of the cell sheet was confirmed by stereomicroscopy and E-SEM. These results highlight the potential of the NGMA spin coated modified OHPS to serve as a thermoresponsive culture surface-cum-flexible transfer tool.

Differential expression of transcription factors NF-κB and STAT3 in periodontal ligament fibroblasts and gingiva of healthy and diseased individuals.

OBJECTIVE: Pathogens and host mediators can activate transcription factors in periodontal cells to bring about gene level alterations, thereby accentuating the periodontal disease process. Nuclear factor-kappa B (NF-κB) and signal transducers and activators of transcription 3 (STAT3) are two pivotal transcription factors implicated in chronic inflammatory diseases. But their importance in periodontal pathogenesis has not been investigated in detail. The aim of the present study was to evaluate the expression of activated transcription factors and their target genes in healthy and diseased periodontium. DESIGN: Primary culture of periodontal ligament fibroblasts (PDLF) were established from healthy and diseased periodontium using explant culture methods. NF-κB and STAT3 activation in these cells by Porphyromonas gingivalis LPS (lipopolysaccharide) was demonstrated using confocal microscopy and mRNA expression of target genes were evaluated by quantitative real time PCR. NF-κB and STAT3 expression in diseased and healthy gingival tissues were analyzed using immunohistochemistry. RESULTS: A basal upregulation of transcription factors and their target genes were noted in diseased PDLF compared to healthy ones. LPS challenge induced differential expression of NF-κB and STAT3 and their target genes in diseased PDLF compared to healthy ones. Immunohistochemical analysis revealed significant activation of transcription factors in diseased gingival tissues. CONCLUSION: The findings of the present study reveal the role of transcription factors NF-κB and STAT3 in periodontal pathogenesis and disease susceptibility of fibroblast subpopulations in periodontal disease could be mediated through activation of NF-κB and STAT3. Since genetic factors are nonmodifyable, transcription factors are promising targets for future host modulation therapy.

An ex vivo evaluation of the efficacy of andrographolide in modulating differential expression of transcription factors and target genes in periodontal cells and its potential role in treating periodontal diseases.

ETHANOPHARMACOLOGICAL RELEVANCE: Andrographolide is a herbal extract traditionally used in South Asian countries for treating inflammatory diseases. AIM OF THE STUDY: To evaluate the efficacy of andrographolide in management of periodontal disease which is a highly prevalent oral disease. MATERIALS AND METHODS: Periodontal ligament fibroblasts (PDLF) were cultured from healthy and diseased periodontium using explant culture methods. The safe dose of AG was determined using MTT assay. LPS (lipopolysaccharide) of the most important periodontopathogen, P gingivalis was used to activate NF-κB and STAT3 in PDLF. The efficacy of AG in inhibiting NF-κB and STAT3 was analyzed using immunofluorescence. Down regulation of expression of target genes of these transcription factors related to inflammation and bone resorption were analyzed using real time PCR. RESULTS: AG up to the concentration of 25µM was found to be safe as determined by MTT assay. Statistically significant activation of NF-κB and STAT3 in cultured PDLF was observed in diseased group compared to healthy controls before and after LPS challenge. 5µM AG pretreatment significantly inhibited activation of NF-κB and STAT3 and down regulated expression of inflammatory and bone resorptive genes in cultured PDLF. CONCLUSIONS: The findings of the present study propose the adjunctive use of a novel herbal drug andrographolide as a promising host modulation agent for periodontal therapy by inhibiting NF-κB and STAT3 activation and inhibition of inflammation and bone resorption related genes.

Rapid and complete cellularization of hydroxyapatite for bone tissue engineering.

Using a tissue construct generated by cells in a scaffold in reconstructive surgery, as a substitute for autografts, is still challenging. Routine methods of incorporating cells into scaffolds are either passive, i.e. by gravity, or forced, as in a bioreactor. Extensive use of these methods is obstructed by tissue formation around the scaffold, hindrance in cell penetration and time required for cell coverage within the scaffold. In this study, human osteoblast cells as cell sheet structures were seeded to porous and dense hydroxyapatite with the hypothesis that preservation of native extracellular structures and cell-cell contacts would facilitate the cellularization process. Cellularization was assessed by fluorescence, confocal and scanning electron microscopy at intervals of 1 h, 2 days and 7 days. Cell patches with intact cell-cell and cell-extra cellular matrix contact attached and adhered on a scaffold within 1 h. The patches formed a monolayer within 2 days and complete cellularization of the scaffold was attained in 7 days. Cell viability, proliferation and function were assessed to understand the application of cell patch transfer to bone substitute. This novel approach for application in bone tissue engineering was successful in uniform distribution of intact osteoblast cell sheet structures on to bone substitute materials for rapid and complete cellularization without altering material characteristics.

Galactosylated pullulan-curcumin conjugate micelles for site specific anticancer activity to hepatocarcinoma cells.

Galactosylated pullulan-curcumin conjugate (LANH2-Pu Ald-Cur SA) is developed for target specific delivery of curcumin to hepatocarcinoma cells by five step synthetic strategy, which includes oxidation of pullulan (Pu Ald), introduction of amino group to the targeting ligand (LANH2), grafting of the LANH2 to Pu Ald, modification of curcumin (Cur SA) and conjugation of Cur SA to pullulan. Nongalactosylated pullulan-curcumin conjugate (Pu-Cur SA) is also prepared to compare the enhancement in cytotoxicity offered by the targeting group. Both LANH2-Pu Ald-Cur SA and Pu-Cur SA conjugates could self assemble to micelle in water with hydrodynamic diameters of 355±9nm and 363±10nm, respectively. Both conjugates show spherical morphology and enhance stability of curcumin in physiological pH. Compared to Pu-Cur SA, LANH2-Pu Ald-Cur SA exhibits higher toxicity and internalization towards HepG2 cells. This indicates the enhanced uptake of LANH2-Pu Ald-Cur SA conjugate via ASGPR (asialoglycoprotein receptor) mediated endocytosis into HepG2 cells.

Nanogels based on alginic aldehyde and gelatin by inverse miniemulsion technique: synthesis and characterization.

Nanogels were developed from alginic aldehyde and gelatin by an inverse miniemulsion technique. Stable inverse miniemulsions were prepared by sonication of noncontinuous aqueous phase (mixture of alginic aldehyde and gelatin) in a continuous organic phase (Span 20 dissolved in cyclohexane). Cross-linking occurred between alginic aldehyde (AA) and gelatin (gel) in the presence of borax by Schiff's base reaction during the formation of inverse miniemulsion. The effects of surfactant (Span 20) concentration, volume of the aqueous phase and AA/gel weight ratio on the size of the alginic aldehyde-gelatin (AA-gel) nanoparticles were studied. Nanogels were characterized by DLS, FT-IR spectroscopy, TGA, SEM and TEM. DLS, TEM and SEM studies demonstrated nanosize and spherical morphology of the nanogels. Hemocompatibility and in vitro cytocompatibility analyses of the nanogels proved their nontoxicity. The results indicated the potential of the present nanogel system as a candidate for drug- and gene-delivery applications.

A non-adhesive hybrid scaffold from gelatin and gum Arabic as packed bed matrix for hepatocyte perfusion culture.

Development of liver support systems has become one of the most investigated areas for the last 50 years because of the shortage of donor organs for orthotopic liver transplantations. Bioartificial liver (BAL) device is one of the alternatives for liver failure which provides a curing method and support patients to recover from certain liver failure diseases. The biological compartment of BAL is called the bioreactor where functionally active hepatocytes are maintained to support the liver specific functions. We have developed a packed bed bioreactor with a cytocompatible, polysaccharide-protein hybrid scaffold. The scaffold prepared from gelatin and gum Arabic acts as a packed bed matrix for hepatocyte culture. Quantitative evaluation of the hepatocytes cultured using packed bed bioreactor demonstrated that cells maintained liver specific functions like albumin and urea synthesis for seven days. These results indicated that the system can be scaled up to form the biological component of a bioartificial liver.

In vitro cytocompatibility studies of Diamond Like Carbon coatings on titanium.

Diamond like carbon (DLC) films were deposited on to titanium (Ti) substrates by Plasma Enhanced Chemical Vapour Deposition (PECVD) process. The quality of the films were checked by Raman spectra and nano-hardness tests. The cytocompatibility of titanium and DLC coated titanium were studied using continuous cell lines of mouse fibroblast cells ( L-929), Human Osteoblast cells (HOS) and primary human umbilical cord vein endothelial cells (HUVEC). The cellular responses to the materials were assessed both quantitatively and qualitatively. The adhesion and spreading of cells on materials were compared using Ti as a control. Present study indicates an improved cytocompatibility of DLC coated Ti in comparison to bare Ti.

Molecular Characterization and Phylogenetic Analysis of a Histone-Derived Antimicrobial Peptide Teleostin from the Marine Teleost Fishes, Tachysurus jella and Cynoglossus semifasciatus.

Antimicrobial peptides (AMPs) are host defense peptides that are well conserved throughout the course of evolution. Histones are classical DNA-binding proteins, rich in cationic amino acids, and recently appreciated as precursors for various histone-derived AMPs. The present study deals with identification of the potential antimicrobial peptide sequence of teleostin from the histone H2A of marine teleost fishes, Cynoglossus semifasciatus and Tachysurus jella. A 245 bp amplicon coding for 81 amino acids was obtained from the cDNA transcripts of these fishes. The first 52 amino acids from the N terminal of the peptide were identical to previously characterized histone-derived antimicrobial peptides. Molecular and physicochemical characterizations of the sequence were found to be in agreement with previously reported histone H2A-derived AMPs, suggesting the possible role of histone H2A in innate defense mechanism in fishes.

Evaluation of polypropylene hollow-fiber prototype bioreactor for bioartificial liver.

Hepatocytes in high density are a requisite for the functional performance of complex devices such as bioartificial liver (BAL). In addition to high cell number, efficient mass transfer is also a key parameter in such devices. High-density culture of cells and efficient mass transfer can be achieved in BAL with hollow-fiber-based bioreactors. Even though different types of hollow fibers have been tried in a BAL, prospects of using polypropylene hollow fibers are not well evaluated. In this study, a prototype of bioreactor with polypropylene hollow fibers was fabricated and evaluated for cytotoxicity and hepatocyte function. High density of HepG2/adult hepatocyte cultures was used to evaluate polypropylene hollow fiber to support the biochemical activities (albumin and urea production), ammonia detoxification, and gene expression and to provide effective oxygenation. The results confirmed that a polypropylene hollow-fiber prototype bioreactor is able to provide efficient oxygenation and supported hepatocyte functions in a high-density culture.