Studying the role of spatial cell distribution and substrate stiffness in inflammatory and fibrotic responses in human lung using bioengineered platforms

The extracellular matrix (ECM) has emerged as a major regulator of cell behaviours. Changes in extracellular matrix, especially its composition, organization/ dimensionality, and rigidity have been implicated in various aspects of cellular functions including cell growth, migration, and differentiation. In my thesis, I have focused on the effect of two biophysical properties of the extracellular matrix namely dimensionality and rigidity in the inflammatory and fibrotic pathologies of human lung. To study the role of matrix dimensionality, firstly electrospun scaffold based three-dimensional (3D) culture with similar architecture of human lung was developed. By applying this 3D model, inflammatory response was studied in an in vivo like environment by using NF-κB transcription factor activation as a tool for probing inflammatory response in human lung fibroblasts. According to my observations, it was confirmed that the matrix dimensionality together with spatial organisation of cells is crucial in lung inflammatory response, evidenced by the observation of the differences in the level and pattern of inflammatory response between 2D and 3D culture systems. To study the role of matrix rigidity in progression of lung fibrosis, we developed the ECM-based hydrogel platform with tuneable stiffness level relevant to normal and fibrotic lung. By using this disease relevant platform, I have shown that stiff matrix but not soft matrix can induce the myofibroblast differentiation and fibroblast proliferation, the two major features of lung fibrosis. To date, the molecular mechanisms underpinning this cellular mechanosensing process in response to matrix stiffening remains unknown. To achieve this, I further investigated the involvement of two potential mechanosensitive signalling pathways namely, Rho associated coiled coil forming kinase (ROCK) signalling and talin- (focal adhesion adaptor) signalling in this process. Interestingly, my data show that ROCK signalling differentially regulated stiffness induced myofibroblast differentiation between soft normal and stiff fibrotic matrix. Moreover, both ROCK isoforms 1 and 2 are synergistically important in myofibroblast differentiation driven by rigid matrix and the absence of one ROCK isoform can exaggerate myofibroblast differentiation on stiff fibrotic matrix. Regarding talin signalling, my preliminary data confirms that talin1 can control both stiffness induced fibroblast proliferation and myofibroblasts differentiation on stiff matrix. In contrast to talin1, talin2 showed a protective role in controlling myofibroblast differentiation. In conclusion, we have successfully developed two in vitro lung models for studying the effect of matrix dimensionality and rigidity in lung inflammation and fibrosis. Overall my PhD work has elucidated the significant contribution of biophysical cues of external cellular environment in lung inflammation and fibrosis

Effect of Natural Pozzolan on Carbonation and Chloride Penetration in Sustainable Concrete

A study on green concrete as well as its durability is essential to promote the sustainable development of the concrete industry. Since the majority of concrete deterioration is connected to corrosion of reinforcement due to carbonation or chloride penetration into concrete, the influence of pozzolan through them must be investigated for concrete durability. This paper mainly focuses on the experimental study of the influence of Popa volcanic natural pozzolan on carbonation and chloride penetration process. The experimental tests are made on replacement of natural pozzolan 10%, 20%, 30% instead of cement and analysis show that optimized pozzolan replacement can promote on the mechanical strength of concrete along with time than reference concrete. According to the results, in the CO2 chamber and the natural condition, it contributes negatively to carbonation because the rate of carbonation increased as the amount of pozzolan increased. However, increasing the pozzolan content led to higher resistance of the concrete in chloride ion penetration

Domain-Specific Sentiment Lexicon for Classification

Nowadays people express their opinions about products, government policies, schemes and programs over social media sites using web or mobile. At the present time, in our country, government changes policies in every sector and people follow with the eyes or the mind on these policies and express their opinion by writing comments on social media especially using Facebook news media pages. Therefore, our research group intends to do sentiment analysis on new articles. Domain-specific sentiment lexicon has played an important role in opinion mining system. Due to the ubiquitous domain diversity and absence of domain-specific prior knowledge, construction of domain-specific lexicon has become a challenging research topic in recent year. In this paper, lexicon construction for sentiment analysis is described. In this work, there are two main steps: (1) pre-processing on raw data comments that are extracted from Facebook news media pages and (2) constructing lexicon for coming classification work. The word correlation and chi-square statistic are applied to construct lexicon as desired. Experimental results on comments datasets demonstrate that proposed approach is suitable for construction the domain-specific lexicon

A paper-based in vitro model for on-chip investigation of the human respiratory system

Culturing cells at the air–liquid interface (ALI) is essential for creating functional in vitro models of lung tissues. We present the use of direct-patterned laser-treated hydrophobic paper as an effective semi-permeable membrane, ideal for ALI cell culture. The surface properties of the paper are modified through a selective CO2 laser-assisted treatment to create a unique porous substrate with hydrophilic regions that regulate fluid diffusion and cell attachment. To select the appropriate model, four promising hydrophobic films were compared with each other in terms of gas permeability and long-term strength in an aqueous environment (wet-strength). Among the investigated substrates, parchment paper showed the fastest rate of oxygen permeability (3 times more than conventional transwell cell culture membranes), with the least variation in its dry and wet tensile strengths (124 MPa and 58 MPa, remaining unchanged after 7 days of submersion in PBS).The final paper-based platform provides an ideal, robust, and inexpensive device for generating monolayers of lung epithelial cells on-chip in a high-throughput fashion for disease modelling and in vitro drug testing

Preliminary Field Observation of Cultivated Crops and Useful Plants in Northeast India and adjacent northern Sagaing Region of Myanmar

This is a preliminary report of a field observation of cultivated crops and useful plants in marketplaces and of farmers in Meghalaya State, Assam State, and Nagaland State of India in 2013, and in northern part of Sagaing Region where the Naga people inhabit in 2014. The field observation team focused on the areas, because they are located adjacent to the places where series of field studies on plant genetic resources were made recently, e.g. Kachin State and Chin State of Myanmar, northern Lao PDR and northern Thailand. The plants used traditionally for food and agriculture in the target areas might provide valuable information for strategic conservation of plant genetic resources for the future and to understand agriculture basic complex in Southeast Asia and South Asia. Both non-glutinous and glutinous rice were sold in the marketplaces of Assam, Meghalaya and Nagaland States of India and in Sagaing Region of Myanmar. Glutinous rice is specific to Southeast Asia and East Asia of which Lao PDR has its abundant cultivation area, while it has not been cultivated mostly in countries west of South Asia. Worldwide crops such as radish, broccoli, cabbage, carrot, common pea, eggplant, ginger, and others were also common there. In addition to such popular crops, various shallot plants and other different Allium spp., rice bean with various surface colors, Centella asiatica, Elsholtzia blanda, Zanthoxylum sp., and perilla seed were sold in a certain quantity at marketplaces in Dimapur and Kohima, Nagaland and in Homalin, Sagaing. Indian gooseberry, foxtail millet, hog plum and holy basil were also seen in Nagaland. Through interviews, various vernacular names were recorded in Northeast India and in Sagaing Region of Myanmar for certain crops, which were different from area to area in most cases, and from Hindi words widely used in North India. Traditional uses of rice bean, Centella asiatica, Elsholtzia blanda, Zanthoxylum sp., and perilla and fermented soybean suggested cultural similarity in the range including Nagaland of India and mountainous parts of Myanmar and Lao PDR. The field observation team concluded that minor crop varieties of those areas need to be studied, particularly in scattered Naga villages in Sagaing Region of Myanmar as soon as possible.本報は，2013年にインドのメガラヤ州，アッサム州，ナガランド州，ならびに2014年にミャンマーのザガイン地方域の地方市場と農家を対象に実施した現地観察記録の予備的報告である．これらの地域を対象に選んだのは，今までに現地調査を行なってきたミャンマー，ラオス，タイなどを含む東南アジアの丘陵および山間部の一部であり，東南アジアや南アジアの作物遺伝資源の多様性を考える上で調査が十分とはいえないためである．さらに作物遺伝資源を通じて地域の伝統的な農耕文化複合を理解することは，調査地点の選定や現地における農業生物多様性の保存戦略の策定にも有意義である．アッサム州，メガラヤ州およびザガイン地方域で訪れた市場ではウルチイネとモチイネの両者が売られていた．モチイネは東南アジアや東アジアに特異的であり，南アジアではめったに見ることができない．それらの市場ではダイコン，ブロッコリー，キャベツ，ニンジン，エンドウ，ナス，ショウガなどの世界中で見られる作物も一般的であった．それらに加えてさまざまな色のタケアズキ（ツルアズキ），ツボクサ，Elsholtzia blanda（ナギナタコウジュの仲間），サンショウの仲間，エゴマなどが相当量売られていた．さらにユカン，Spondias cytherea（ホグプラム），ホーリーバジルなども散見された．いくつかの作物についてはインタビューを通じて作物の方名を記録したが多くの場合に地域ごとに異なり，また，東北インドでは北インドで広く使われているヒンディー語の単語とも異なっていた．ザガイン地域の大きな町ではミャンマーの他の都市と同様であるが，山村では全く異なる呼称で呼ばれていた．ツルアズキ，ナギナタコウジュ属のElsholtzia blanda，サンショウ類，エゴマ，ダイズ等の発酵食品などの伝統的な利用はこれらの山地における農耕文化複合の共通性を暗示している．近代化は急速に進みつつあり，東北インドから東南アジア山地，とくにミャンマー・ザガイン地方域に点在するナガ族村落の伝統的な作物の調査を急ぐ必要がある

Role of ROCK isoforms in regulation of stiffness induced myofibroblast differentiation in lung fibrosis

Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho associated coiled-coil forming kinase (ROCK) has shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in fibrotic state. There are two known ROCK isoforms in human, ROCK1 (ROKβ) and ROCK2 (ROKα), but specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a Gelatin methacryloyl (GelMA) hydrogel based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix and not soft matrix, can induce myofibroblast differentiation with high αSMA expression. Furthermore, our data confirm that the inhibition of ROCK signalling by a pharmacological inhibitor (i.e. Y27632) attenuates stiffness induced αSMA expression and fibre assembly in myofibroblasts. To assess the role of ROCK isoforms in this process we used siRNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix as opposed to soft matrix where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fibre assembly. Moreover complete loss of αSMA fibre assembly was seen only in the absence of both ROCK isoforms suggesting that both isoforms are implicated in this process. Overall our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices

A foreign body response-on-a-chip platform

Understanding the foreign body response (FBR) and desiging strategies to modulate such a response represent a grand challenge for implant devices and biomaterials. Here, the development of a microfluidic platform is reported, i.e., the FBR?on?a?chip (FBROC) for modeling the cascade of events during immune cell response to implants. The platform models the native implant microenvironment where the implants are interfaced directly with surrounding tissues, as well as vasculature with circulating immune cells. The study demonstrates that the release of cytokines such as monocyte chemoattractant protein 1 (MCP?1) from the extracellular matrix (ECM)?like hydrogels in the bottom tissue chamber induces trans?endothelial migration of circulating monocytes in the vascular channel toward the hydrogels, thus mimicking implant?induced inflammation. Data using patient?derived peripheral blood mononuclear cells further reveal inter?patient differences in FBR, highlighting the potential of this platform for monitoring FBR in a personalized manner. The prototype FBROC platform provides an enabling strategy to interrogate FBR on various implants, including biomaterials and engineered tissue constructs, in a physiologically relevant and individual?specific manner

Unbiased analysis of the impact of micropatterned biomaterials on macrophage behaviour provides insights beyond pre-defined polarisation states

Macrophages are master regulators of immune responses towards implanted biomaterials. The activation state adopted by macrophages in response to biomaterials determines their own phenotype and function as well as those of other resident and infiltrating immune and non-immune cells in the area. A wide spectrum of macrophage activation states exists, with M1 (pro-inflammatory) and M2 (anti-inflammatory) representing either ends of the spectrum. In biomaterials research, cellinstructive surfaces that favour or induce M2 macrophages have been considered as beneficial due to the anti-inflammatory and pro-regenerative properties of these cells. In this study, we used a gelatin methacryloyl (GelMA) hydrogel platform to determine whether micropatterned surfaces can modulate the phenotype and function of human macrophages. The effect of microgrooves/ridges and micropillars on macrophage phenotype, function, and gene expression profile were assessed using conventional methods (morphology, cytokine profile, surface marker expression, phagocytosis) and gene microarrays. Our results demonstrated that micropatterns did induce distinct gene expression profiles in human macrophages cultured on microgrooves/ridges and micropillars. Significant changes were observed in genes related to primary metabolic processes such as transcription, translation, protein trafficking, DNA repair and cell survival. However, interestingly conventional phenotyping methods, relying on surface marker expression and cytokine profile, were not able to distinguish between the different conditions, and indicated no clear shift in cell activation towards an M1 or M2 phenotypes. This highlights the limitations of studying the effect of different physicochemical conditions on macrophages by solely relying on conventional markers that are primarily developed to differentiate between cytokine polarised M1 and M2 macrophages. We therefore, propose the adoption of unbiased screening methods in determining macrophage responses to biomaterials. Our data clearly shows that the exclusive use of conventional markers and methods for determining macrophage activation status could lead to missed opportunities for understanding and exploiting macrophage responses to biomaterials

Immunomodulation with self-crosslinked polyelectrolyte multilayer-based coatings

This study aims to design an optimal polyelectrolyte multilayer film of poly-L-lysine (PLL) and hyaluronic acid(HA) as an anti-inflammatory cytokine release system in order to decrease the implant failure due to any immune reactions. The chemical modification of the HA with aldehyde moieties allows self-cross-linking of the film and an improvement in the mechanical properties of the film. The cross-linking of the film and the release of immunomodulatory cytokine (IL-4) stimulate the differentiation of primary human monocytes seeded on the films into pro-healing macrophages phenotype. This induces the production of antiinflammatory cytokines (IL1-RA and CCL18) and the decrease of proinflammatory cytokines secreted (IL-12, TNF-α, and IL-1β). Moreover, we demonstrate that cross-linking PLL/HA film using HA-aldehyde is already effective by itself to limit inflammatory processes. Finally, this functionalized self-cross-linked PLL/HA-aldehyde films constitutes an innovative and efficient candidate for immunomodulation of any kind of implants of various architecture and properties

Generation of anti-inflammatory macrophages for implants and regenerative medicine using self-standing release systems with a phenotype-fixing cytokine cocktail formulation

The immediate tissue microenvironment of implanted biomedical devices and engineered tissues is highly influential on their long term fate and efficacy. The creation of a long-term anti-inflammatory microenvironment around implants and artificial tissues can facilitate their integration. Macrophages are highly plastic cells that define the tissue reactions on the implanted material. Local control of macrophage phenotype by long-term fixation of their healing activities and suppression of inflammatory reactions are required to improve implant acceptance. Herein, we describe the development of a cytokine cocktail (M2Ct) that induces stable M2-like macrophage phenotype with significantly decreased pro-inflammatory cytokine and increased anti-inflammatory cytokine secretion profile. The positive effect of the M2Ct was shown in an in vitro wound healing model; where M2Ct facilitated wound closure by human fibroblasts in co-culture conditions. Using a model for induction of inflammation by LPS we have shown that the M2Ct phenotype is stable for 12 days. However, in the absence of M2Ct in the medium macrophages underwent rapid pro-inflammatory re-programming upon IFNg stimulation. Therefore, loading and release of the cytokine cocktail from a self-standing, transferable gelatin/tyraminated hyaluronic acid based release system was developed to stabilize macrophage phenotype for in vivo applications in implantation and tissue engineering. The M2Ct cytokine cocktail retained its anti-inflammatory activity in controlled release conditions. Our data indicate that the direct application of a potent M2 inducing cytokine cocktail in a transferable release system can significantly improve the long term functionality of biomedical devices by decreasing pro-inflammatory cytokine secretion and increasing the rate of wound healing