Apigenin Self-Assembled Collagen Biomatrix for Reprogramming the Obese Wound Microenvironment for Its Management and Repair.

Wound management in obesity is complicated by excessive exudates from wounded areas, pressure ulcerations due to stacking of the fat layer, and vascular rarefaction. The current study explored the development of biomaterials for reprogramming the altered wound microenvironment under obese conditions. Self-assembled collagen biomatrix with trans and de novo browning activator, apigenin, was fabricated as a soft tissue regenerative wound dressing material. The as-synthesized self-assembled collagen biomatrix exhibited excellent thermal, mechanical, and biological stability with a superior wound exudate absorption capacity. The apigenin self-assembled collagen biomatrix exhibited porous 3-D microstructure that mimicked the extracellular matrix that promoted cell adhesion and proliferation. The apigenin self-assembled collagen multifunctional biomatrix triggered adaptive localized thermogenesis in the subcutaneous fat layer, resulting in the activation of angiogenesis and fibroblast spreading and migration. The in vivo wound healing assay performed in DIO-C57BL6 mice showed faster tissue regeneration within 9 days, with well-defined neo-epidermis, blood vessel formation, thick collagen deposition, minimal inflammation, and significant activation of browning in the subcutaneous adipose layer. This study paves the way forward for the development of specialized regenerative biomatrices that reprogram the obese wound bed for faster tissue regeneration.

Localized trans-browning and pro-angiogenesis inductive self-assembled collagen resveratrol bio-matrix for tissue repair and regeneration in obese conditions.

The present study probes into the complexities of wound management in obesity by proposing a novel biomaterial designed to reprogram the altered skin physiology prevalent in obese conditions. The strategy involves the development of a multifunctional biomaterial addressing issues such as excessive exudate, pressure ulcers, and reduced vascularity. The bio-matrix demonstrates the localized transformation of white adipocytes through trans-browning, coupled with the simultaneous induction of angiogenesis at obese wound sites, resulting in expedited wound closure. The collagen bio-matrices, stabilized with Resveratrol (Rsv), exhibit remarkable thermal, mechanical, and biological stability. The porous 3D microstructure of the Rsv-stabilized collagen bio-matrix closely resembled the natural extracellular matrix, facilitating effective cell adhesion. The bio-matrix exhibited the unique capability to induce localized thermogenesis in the subcutaneous fat layer while concurrently activating angiogenesis. In vivo wound healing studies conducted on DIO-C57BL6 mice demonstrated complete healing within 10 days, showcasing accelerated tissue regeneration, blood vessel formation, robust collagen deposition, and significant activation of browning in the subcutaneous adipose layer. This study introduces the concept of tailored regenerative biomaterials with the ability to reprogram the challenging wound environment associated with obesity. This innovative approach opens up new avenues for enhanced wound care strategies, particularly for bariatric patients.

Microwave-assisted synthesis of crosslinked ureido chitosan for hemostatic applications.

In this study, we present a facile method for introducing hydrophilic ureido groups (NH2-CO-NH-) into chitosan using a microwave-assisted reaction with molten urea, with the aim of enhancing chitosan's interaction with blood components for improved hemostasis. The formation of the ureido groups through nucleophilic addition reaction between the amine groups in chitosan and in situ generated isocyanic acid was confirmed by FTIR, CP/TOSS 13C NMR, and CP/MAS 15N NMR spectroscopic techniques. However, in stark contrast to the glucans, the said modification introduced extensive crosslinking in chitosan. Spectroscopic studies identified these crosslinks as carbamate bridges (-NH-COO-), which were likely formed by the reaction between the ureido groups and hydroxyl groups of adjacent chains through an isocyanate intermediate. These carbamate bridges improved ureido chitosan's environmental stability, making it particularly resistant to changes in pH and temperature. In comparison to chitosan, the crosslinked ureido chitosan synthesized here exhibited good biocompatibility and cell adhesion, rapidly arrested the bleeding in a punctured artery with minimal hemolysis, and induced early activation and aggregation of platelets. These properties render it an invaluable material for applications in hemostasis, particularly in scenarios that necessitate stability against pH variations and degradation.

In vivosoft tissue regenerative potential of flax seed mucilage self-assembled collagen aerogels.

The present study demonstrates thein vivosoft tissue regenerative potential of flax seed mucilage (FSM) reinforced collagen aerogels in Wistar rats. The physiochemical, mechanical, and thermal properties were significantly improved upon the incorporation of flax mucilage into collagen when compared to the native collagen scaffold. In addition, the functional group of flax mucilage notably contributed to a better anti-oxidative potential than the control collagen. The flax mucilage-reinforced collagen at 4 mg ml-1concentration showed a 2-fold increase in porosity compared to native collagen. The tensile strength of native collagen, 2 mg ml-1, and 4 mg ml-1FSM reinforced collagen was 5.22 MPa, 9.76 MPa, and 11.16 MPa, respectively, which indicated that 2 mg ml-1and 4 mg ml-1FSM showed an 87% and 113% percentage increase respectively in tensile strength compared to the native collagen control. FSM-reinforced biomatrix showed 97% wound closure on day 15 post-wounding, indicating faster healing than controls, where complete healing occurred only on day 21. The mechanical properties of skin treated with FSM-reinforced collagen scaffold post-healing were considerably better than native collagen. The histological and immunohistochemistry analysis also showed complete restoration of wounded tissue like intact normal skin. The findings paved the way for the development of collagen-polysaccharide mucilage wound dressing materials and their further application in skin tissue engineering.

Modulation of angiogenic switch in reprogramming browning and lipid metabolism in white adipocytes.

Thermogenic activation via trans-and de novo browning of white adipocytes is a promising strategy to accelerate lipid metabolism for regulating obesity-related disorders. In this study, we investigated the intricate interplay between angiogenic regulation and browning in white adipocytes using the bioactive compound, resveratrol (Rsv). Rsv has previously been documented for its regulatory influence on the trans and de novo browning of white adipocytes. Our findings revealed that concurrent activation of angiogenesis is prerequisite for inducing browning within the microenvironment of white adipocytes when exposed to browning activators. Additionally, we observed a significant browning effect on white adipocytes when the local adipose tissue environment was prompted to undergo angiogenesis, notably facilitated by a proangiogenic molecule known as Vascular endothelial growth factor (VEGF). Intriguingly, this effect was reversed when angiogenesis was inhibited by treatment with the antiangiogenic agent thalidomide. Furthermore, the study revealed the role of VEGF in paracrine activation of white adipocytes resulting in the induction of browning in both 3T3-L1 cell lines and primary mouse white adipocytes. The cross-talk between angiogenesis and browning was found to be initiated via the transcriptional activation of Estrogen receptor α (ERα) triggering the VEGF/VEGFR2 signaling pathway leading to browning and a reconfiguration of lipid metabolism within adipocytes. In conclusion, this study sheds light on the intricate cross-talk between angiogenesis and browning of white adipocytes. Notably, the findings underscore the reciprocal relationship between these processes, wherein inhibition of one process exerts discernible effects on the other.

Caffeine-reinforced Collagen as Localized Microenvironmental Trans-Browning Bio-Matrix for Soft Tissue Repair and Regeneration in Bariatric Condition.

The wound exudates, hypoperfusion of the subcutaneous fat layer, and poor vasculature worsen wound management in obese subjects. In the current study, a multifunctional Caffeine-reinforced collagen biomaterial is developed that can simultaneously modulate lipid metabolism and angiogenesis in obese wound microenvironments for faster tissue regeneration. The biomaterial is fabricated specialized for obese conditions to initiate simultaneous lipolysis and angiogenesis locally in the hypoxic subcutaneous fat in wound margins of obese subjects. Caffeine-reinforced collagen biomatrix shows better structural integrity, thermal stability, bio-compatibility, and lesser proteolytic susceptibility. Caffeine-collagen biomaterial promote angiogenesis, fibroblast migration, and localized browning of white adipocytes to activate thermogenesis in the subcutaneous fat layer at the wound site. Full-thickness excision wound healing studies performed in obese C57BL6 mice shows faster wound closure within day 9 when compare to control mice. The Caffeine-reinforced collagen biomaterial remodeled the wound site locally by activating fibroblast to secrete collagen, activate endothelial cells to promote angiogenesis, and induce browning in white adipocytes in subcutaneous fat. The study opens a new direction in bariatric tissue regenerative medicine by locally modulating lipid metabolism, angiogenesis, and trans-browning at the injured site for faster complete restoration of the damaged tissue.

Semantic Segmentation of Cell Painted Organelles using DeepLabv3plus Model.

Cell painting based high content fluorescence imaging technique offers deep insight into the functional and biological changes in subcellular structures. However, advanced instrumentation and the limited availability of suitable fluorescent dyes restricts the tool to comprehensively characterize the cell morphology. Therefore, generating fluorescent specific organelle images using transmitted light microscopy provides an alternative solution for clinical applications. In this work, the utility of semantic segmentation deep network for predicting the Endoplasmic Reticulum (ER), cytoplasm and nuclei from a composite image is investigated. To perform this study, a public dataset consisting of 3456 composite images are considered from Broad Bioimage Benchmark collection. The pixel wise labeling is carried out with the generated binary masks for ER, cytoplasm and nuclei. DeepLabv3plus architecture with Atrous Spatial Pyramid Pooling (ASPP) and depth wise separable convolution is used as a learning model to perform semantic segmentation. The accuracy and loss function at different learning rates are analyzed and the segmentation results are validated using Jaccard index, mean Boundary F (BF) score and dice index. The trained model achieved 97.86% accuracy with a loss of 0.07 at the learning rate of 0.01. Mean BF score, dice index and Jaccard index for nuclei, ER and cytoplasm are (0.98, 0.94, 0.88), (0.97, 0.82, 0.7) and (0.95, 0.88, 0.66) respectively. The obtained results indicate that the adopted methodology could delineate the subcellular structures by accurately detecting sharp object boundaries. Therefore, this study could be useful for predicting the cell painted images from transmitted light microscopy without the requirement of fluorescent labeling.

Lanthanum Oxide Nanoparticles Reinforced Collagen ƙ-Carrageenan Hydroxyapatite Biocomposite as Angio-Osteogenic Biomaterial for In Vivo Osseointegration and Bone Repair.

A composite biomatrix fabricated with collagen, ƙ-carrageenan, hydroxyapatite reinforced with lanthanum oxide nanoparticles is explored as proangiogenic and osteogenic bone tissue repair biomaterial. The biomatrix shows increased physical and biological stability as observed from proteolytic degradation and thermal stability studies. The addition of lanthanum oxide nanoparticles facilitates good osseointegration coupled with simultaneous activation of proangiogenic properties to act as a bone mimicking material. The minimal level of reactive oxygen species and superior cytocompatibility help the as-synthesized biomatrix in achieving capillary migration into the bone micro environment. The composite biomatrix upregulates the expression of VEGF, VEGF-R2 genes in endothelial cells and osteopontin, osteocalcin in osteoblasts cells, respectively. The in vivo hard tissue repair experiment conducted in a rat model shows complete healing of the bone defect by eight weeks with the application of collagen-ƙ-carrageenan-hydroxyapatite-lanthanum oxide nanoparticle biomaterial when compared to the biomaterial made out of individual constituents alone. The biomaterial matrix gets biointegrated into the bone tissue and exerts its therapeutic value in bringing a faster osseo repair process. The study shows the feasibility of using rare-earth metal nanoparticles in combination with protein-polysaccharide biopolymers for bone regeneration.

White to brown adipocyte transition mediated by Apigenin via VEGF-PRDM16 signaling.

The dysregulated energy metabolism in white adipose tissues results in derangement of biological signaling resulting in obesity. Lack of vascularization in these white adipose tissues is one of the major reasons for dysregulated energy metabolism. Not much work has been done in this direction to understand the role of angiogenesis in white adipose tissue metabolism. In the present study, we evaluated the effect of angiogenic modulator in the metabolism of white adipocyte (WAC). Bioactive Apigenin was selected and its angiogenic ability was studied. Apigenin was shown to be highly proangiogenic hence the effect of Apigenin on de novo and trans-differentiation of WAT was studied. Apigenin showed enhanced de novo differentiation and trans-differentiation of mouse WAC into brown-like phenotype. To understand the effect of Apigenin on adipose tissue vasculature, coculture studies were conducted. Cross talk between endothelial cell and adipocytes were observed in coculture studies. Gene expression studies of cocultured cells revealed that browning of WAC occurred by triggering the expression of Vascular endothelial growth factor A. The study provides a new insight for inducing metabolic shift in WACs by modulation of angiogenesis in WAC microenvironment by the upregulation of PRDM16 cascade to trigger browning for the treatment of obesity.

An Approach to Differentiate Cell Painted ER and Cytoplasm Using Zernike Moment Descriptor and Multilayer Perceptron.

Differentiation of cell organelle characteristics from microscopic images is a challenging task due to its intricate structural details. In this work, an attempt has been made to categorize Endoplasmic Reticulum (ER) and cytoplasm using orthogonal Zernike moments and Multilayer Perceptron (MLP). For this, Cell painted public source dataset comprising of ER and cytoplasm are considered. Zernike moments for different orders and repetition of the azimuthal angle are extracted to characterize the shape features. The extracted features are validated using MLP classifier for differentiating ER and cytoplasm. The prediction accuracy for variations in the number of hidden layers are evaluated. The experimental results show that the accuracy varies as the size of hidden layer increases. The extracted features with MLP achieved an accuracy of 85% with a hidden layer size of 5. The receiver operating characteristic curve (ROC) demonstrates the distinguishing power of MLP classifier with AUC=0.92. This study suggests that the proposed framework can be employed for analyzing the morphological variations of cell organelles due to chemical perturbations, genome variations and cytotoxic effects using the combination of Zernike shape descriptor and MLP. The orthogonality property of Zernike shape descriptor provides independent unique features which reduce redundancy and improve prediction accuracy for large datasets.

Differentiation of Cell Painted Organelles Using Non Local Texture Descriptor and Random Forest Approach.

Discriminating the cell organelles from microscopic images is a challenging task due to their high similarity in image appearance. In this work, an attempt has been made to differentiate nuclei, Endoplasmic Reticulum (ER) and cytoplasm using a texture pattern descriptor and Random Forest classifier. For this, Cell Painted public dataset from Broad Bioimage Benchmark collection are considered. Texture features are extracted from each image using Non Local Binary Pattern (NLBP) that captures the relationship between global pixels and sampling instances in a local neighborhood. Non local central pixels called anchors are derived from central pixels of image patches and compared with sampling instances. Binary string generated from this is encoded into 29 patterns. Statistical one-way analysis of variance (ANOVA) is performed to select significant features and are validated using Random Forest classifier. The dependency of classifier performance on the local patch radius (R) and the number of anchors (K) are also evaluated. The results indicate that 8 patterns out of 29 are showing strong inter class variability with high F value. Classification accuracy of 84% is achieved with R=3 and K=5. Experimental results demonstrate that the proposed work captures complex patterns in cell structure useful for differentiating cell components which can be employed for evaluating the cytotoxic effects in cell lines.

Nanotized praseodymium oxide collagen 3-D pro-vasculogenic biomatrix for soft tissue engineering.

The current study explores development of highly vascularizable biomatrix scaffold containing rare-earth metal praseodymium oxide nanoadditives for angiogenic and soft tissue regenerative applications. The therapeutic potential of praseodymium oxide nanoparticles rendered excellent endothelial cell differentiation for inducing pro angiogenic microenvironment by eliciting VE-Cadherin expression in the biomatrix scaffold. The nanoparticles were incorporated into bio-macromolecule collagen which aided in stabilization of collagen by maintaining the structural integrity of collagen and showed less susceptibility towards protease enzymes, high cyto-compatibility and high hemo-compatibility. The scaffold provided 3-dimensional micro-environments for the proliferation of endothelial cells and fibroblast cells promoting the wound healing process in an orchestrated fashion. Biological signal modulatory property of rare earth metal is the unexplored domains that can essentially bring significant therapeutic advancement in engineering advanced biological materials. This study opens potential use of nano-scaled rare earth metals in biomaterial application for tissue regeneration by modulating the pro-angiogenesis and anti-proteolysis properties.

Praseodymium-Cobaltite-Reinforced Collagen as Biomimetic Scaffolds for Angiogenesis and Stem Cell Differentiation for Cutaneous Wound Healing.

The present study describes the fabrication of collagen reinforced with praseodymium-cobaltite nanoparticles for wound healing applications. Praseodymium-cobaltite nanoparticles (PCNP) reinforced with collagen resulted in an increased thermal stability and decreased proteolytic susceptibility to collagen. Circular dichroism spectroscopy and ATR-FTIR (attenuated total reflection Fourier transform infrared) spectroscopy analyses confirm the intact structural integrity of the collagen sheets after cross-linking with praseodymium-cobaltite nanoparticles. Cross-linked collagen has shown to possess biocompatibility, less protein adsorption behavior, and hemocompatibility, which are the desirable properties of a wound dressing material. The nanoparticle cross-linked collagen sheets provided a proper matrix elasticity that promotes mesenchymal stem cell attachment and angiogenesis. Further, the scaffold promoted tube formation in endothelial cells. The enhancement of angiogenesis is considered to be brought about by the therapeutic potential of nanoparticle formulation. Praseodymium-cobaltite nanoparticle cross-linking increased the ductility of collagen sheets for the pro-angiogenic and stem cell differentiation ability. Also, the praseodymium-cobaltite cross-linked collagen sheets have been shown to induce a mild level of ROS (reactive oxygen species) generation in the DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) assay, which is beneficial for angiogenesis as well as wound healing. This study paves the way for exploring the therapeutic potential of rare-earth-based nanoparticles for tissue engineering applications as an alternative for traditional wound healing materials.