FAK regulates tension transmission to the nucleus and endothelial transcriptome independent of kinase activity.

The mechanical environment generated through the adhesive interaction of endothelial cells (ECs) with the matrix controls nuclear tension, preventing aberrant gene synthesis and the transition from restrictive to leaky endothelium, a hallmark of acute lung injury (ALI). However, the mechanisms controlling tension transmission to the nucleus and EC-restrictive fate remain elusive. Here, we demonstrate that, in a kinase-independent manner, focal adhesion kinase (FAK) safeguards tension transmission to the nucleus to maintain EC-restrictive fate. In FAK-depleted ECs, robust activation of the RhoA-Rho-kinase pathway increased EC tension and phosphorylation of the nuclear envelope protein, emerin, activating DNMT3a. Activated DNMT3a methylates the KLF2 promoter, impairing the synthesis of KLF2 and its target S1PR1 to induce the leaky EC transcriptome. Repleting FAK (wild type or kinase dead) or inhibiting RhoA-emerin-DNMT3a activities in damaged lung ECs restored KLF2 transcription of the restrictive EC transcriptome. Thus, FAK sensing and control of tension transmission to the nucleus govern restrictive endothelium to maintain lung homeostasis.

Ruthenium complexes of 1,4-diazabutadiene ligands with a cis-RuCl2 moiety for catalytic acceptorless dehydrogenation of alcohols: DFT evidence of chemically non-innocent ligand participation.

The acceptorless dehydrogenative coupling (ADC) of primary alcohols to esters by diazabutadiene-coordinated ruthenium compounds is reported. Treatment of cis-Ru(dmso)4Cl2 in acetone at 56 °C with different 1,4-diazabutadienes [p-XC6H4N[double bond, length as m-dash]C(H)(H)C[double bond, length as m-dash]NC6H4X-p; X = H, CH3, OCH3, and Cl; abbreviated as DAB-X], gives trans-Ru[κ2-N,N-DAB-X]2Cl2 as the kinetic product of substitution. Heating these products in o-xylene at 144 °C gives the thermodynamically favored cis-Ru[κ2-N,N-DAB-X]2Cl2 isomers. Electronic structure calculations confirm the greater stability of the cis diastereomer. The molecular structures for each pair of geometric isomers have been determined by X-ray diffraction analyses. Cyclic voltammetry experiments on the complexes show an oxidative response and a reductive response within 0.50 to 0.93 V and -0.76 to -1.24 V vs. SCE respectively. The cis-Ru[κ2-N,N-DAB-X]2Cl2 complexes function as catalyst precursors for the acceptorless dehydrogenative coupling of primary alcohols to H2 and homo- and cross-coupled esters. When 1,4-butanediol and 1,5-pentanediol are employed as substrates, lactones and hydroxyaldehydes are produced as the major dehydrogenation products, while secondary alcohols afforded ketones in excellent yields. The mechanism for the dehydrogenation of benzyl alcohol to benzyl benzoate and H2 using cis-Ru[κ2-N,N-DAB-H]2Cl2 (cis-1) as a catalyst precursor was investigated by DFT calculations. The data support a catalytic cycle that involves the four-coordinate species Ru[κ2-N,N-DAB-H][κ1-N-DAB-H](κ1-OCH2Ph) whose protonated κ1-diazabutadiene moiety functions as a chemically non-innocent ligand that facilitates a ß-hydrogen elimination from the κ1-O-benzoxide ligand to give the corresponding hydride HRu[κ2-N,N-DAB-H][κ1-N-DAB-H](κ2-O,C-benzaldehyde). H2 production follows a Noyori-type elimination to give (H2)Ru[κ2-N,N-DAB-H][κ1-N-DAB-H](κ1-O-benzaldehyde) as an intermediate in the catalytic cycle.

Microsphere embedded hydrogel construct - binary delivery of alendronate and BMP-2 for superior bone regeneration.

Biomimetic delivery of osteoinductive growth factors via an osteoconductive matrix is an interesting approach for stimulating bone regeneration. In this context, the bone extracellular matrix (ECM) has been explored as an optimal delivery system, since it releases growth factors in a spatiotemporal manner from the matrix. However, a bone ECM hydrogel alone is weak, unstable, and prone to microbial contamination and also has been reported to have significantly reduced bone morphogenic protein-2 (BMP-2) post decellularization. In the present work, a microsphere embedded osteoinductive decellularized bone ECM/oleoyl chitosan based hydrogel construct (BOC) was developed as a matrix allowing dual delivery of an anti-resorptive drug (alendronate, ALN, via the microspheres) and BMP-2 (via the hydrogel) for a focal tibial defect in a rabbit model. The synthesized gelatin microspheres (GMs) were spherical in shape with diameter ∼32 µm as assessed by SEM analysis. The BOC construct showed sustained release of ALN and BMP-2 under the studied conditions. Interestingly, amniotic membrane-derived stem cells (HAMSCs) cultivated on the hydrogel construct demonstrated excellent biocompatibility, cell viability, and active proliferation potential. Additionally, cell differentiation on the constructs showed an elevated expression of osteogenic genes in an RT-PCR study along with enhanced mineralized matrix deposition as demonstrated by alkaline phosphatase (ALP) assay and alizarin red assay. The hydrogel construct was witnessed to have improved neo-vascularization potential in a chick chorioalantoic membrane (CAM) assay. Also, histological and computed tomographic findings evidenced enhanced bone regeneration in the group treated with the BOC/ALN/BMP hydrogel construct in a rabbit tibial defect model. To conclude, the developed multifunctional hydrogel construct acts as an osteoinductive and osteoconductive platform facilitating controlled delivery of ALN and BMP-2, essential for stimulating bone tissue regeneration.

Decellularized bone matrix/oleoyl chitosan derived supramolecular injectable hydrogel promotes efficient bone integration.

Hydrogels derived from decellularized extracellular matrix (ECM) have been widely used as a bioactive matrix for facilitating functional bone tissue regeneration. However, its poor mechanical strength and fast degradation restricts the extensive use for clinical application. Herein, we present a crosslinked decellularized bone ECM (DBM) and fatty acid modified chitosan (oleoyl chitosan, OC) based biohybrid hydrogel (DBM/OC) for delivering human amnion-derived stem cells (HAMSCs) for bone regeneration. DBM/OC hydrogel were benchmarked against collagen-I/OC (Col-I/OC) based hydrogel in terms of their morphological characteristics, rheological analysis, and biological performances. DBM/OC hydrogel with its endogenous growth factors recapitulates the nanofibrillar 3D tissue microenvironment with improved mechanical strength and also exhibited antimicrobial potential along with superior proliferation/differentiation ability. HAMSCs encapsulation potential of DBM/OC hydrogel was established by well spread cytoskeleton morphology post 14 days of cultivation. Further, ex-vivo chick chorioallantoic membrane (CAM) assay revealed excellent neovascularization potential of DBM/OC hydrogel. Subcutaneously implanted DBM/OC hydrogel did not trigger any severe immune response or infection in the host after 21 days. Also, DBM/OC hydrogels and HAMSCs encapsulated DBM/OC hydrogels were implanted at the tibial defect in a rabbit model to assess the bone regeneration ability. Quantitative micro-CT and histomorphological analysis demonstrated that HAMSCs encapsulated DBM/OC hydrogel can support more mature mineralized bone formation at the defect area compared to DBM/OC hydrogel or SHAM. These findings manifested the efficacy of DBM/OC hydrogel as a functional cell-delivery vehicle and osteoinductive template to accelerate bone regeneration.

Dual Functionalized Injectable Hybrid Extracellular Matrix Hydrogel for Burn Wounds.

Low strength and rapid biodegradability of acellular dermal matrix (ADM) restrict its wider clinical application as a rapid cell delivery platform in situ for management of burn wounds. Herein, the extracted ADM was modified by a dual cross-linking approach with ionic crosslinking using chitosan and covalent cross-linking using an iodine-modified 2,5-dihydro-2,5-dimethoxy-furan cross-linker, termed as CsADM-Cl. In addition, inherent growth factors and cytokines were found to be preserved in CsADM-Cl, irrespective of ionic/covalent crosslinking. CsADM-Cl demonstrated improvement in post crosslinking stiffness with a decreased biodegradation rate. This hybrid crosslinked hydrogel supported adhesion, proliferation, and migration of human foreskin-derived fibroblasts and keratinocytes. Also, the angiogenic potential of CsADM-Cl was manifested by chick chorioallantoic membrane assay. CsADM-Cl showed excellent antibacterial activity against Escherichia coli and Staphylococcus aureus. Moreover, CsADM-Cl treated full thickness burn wounds and demonstrated rapid healing marked with superior angiogenesis, well-defined dermal-epidermal junctions, mature basket weave collagen deposition, and development of more pronounced secondary appendages. Altogether, the bioactive CsADM-Cl hydrogel established significant clinical potential to support wound healing as an apt injectable antibacterial matrix to encounter unmet challenges concerning critical burn wounds.

Carbon nanodot decorated acellular dermal matrix hydrogel augments chronic wound closure.

Impaired skin regeneration in chronic wounds like in diabetes corresponds to high oxidative stress, poor angiogenesis and insufficient collagen hyperplasia. Therefore, a multifaceted strategy for treatment is required to address critical issues associated with chronic wound healing. Fascinating application of nanomaterials in chronic wounds is still limited; hence, in the present work bioactive solubilized decellularized dermal matrix (sADM) was employed to form a hydrogel with chitosan (CTS) at physiological pH/temperature and modified with reactive oxygen species (ROS) scavenging carbon nanodots (ND). A detailed in vitro investigation found that the ND modified bioactive hydrogel (CsADMND) is suitable for human amniotic membrane derived stem cell (hAMSC) delivery. Also, CsADMND was observed to possess a good ROS scavenging property, hemocompatibility and pro-angiogenic potential as demonstrated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), haemolysis and chick chorioallantoic membrane (CAM) assay, respectively. The hybrid hydrogel promoted migration of cells in vitro in scratch assay owing to its antioxidant potential and the presence of bioactive moieties. Further, its efficacy in healing full thickness (FT) chronic wounds was evaluated in a streptozotocin (STZ) induced diabetic model. The CsADMND hydrogel after association with hAMSCs led to stimulation of early angiogenesis, superior collagen deposition, rapid wound closure, complete reepithelialisation, and formation of distinct organized dermal epidermal junctions (DEJ) post 21 days of healing. These results suggest that the hAMSC laden CsADMND hydrogel may serve as a promising therapeutic strategy for the management of chronic wounds.

Bioinspired 3D porous human placental derived extracellular matrix/silk fibroin sponges for accelerated bone regeneration.

Critical bone defects arising from traumatic injury and diseases are of major health concern since they are unable to heal spontaneously without clinical intervention. In this context, bone tissue engineering provides an attractive approach to treat bone defects by providing a bioactive template which has the potential to guide osseous tissue regeneration. In this study, porous hybrid placental extracellular matrix sponge (PIMS) was fabricated by a combinatorial method using silk fibroin (SF)/placental derived extracellular matrix and subsequently evaluated its efficacy towards bone tissue regeneration. The presence of intrinsic growth factors was evidenced by immunoblotting of the extracted proteins derived from the placental derived extracellular matrix. This growth factor rich PIMS lends a unique bioactive scaffolding to human amniotic mesenchymal stem cells (HAMSCs) which supported enhanced proliferation as well as superior osteogenic differentiation. Gene expression studies demonstrated significant up-regulation of osteogenic related genes in the PIMS group. PIMS when implanted in the chick chorioallantoic membrane, significantly attracted allantoic vessels revealing its potential to stimulate angiogenesis ex vivo. Furthermore, no severe immune response to the host was observed on subcutaneous implantation of PIMS in vivo. Instead, it supported the formation of blood vessels, revealing its outstanding biocompatibility. Additionally, critical tibial defects treated with PIMS demonstrated higher bone volume after six weeks when analyzed by micro-CT, which was accompanied by high mineral density. Histological and immunofluorescence studies validated the results and revealed enhanced osseous tissue regeneration after six weeks of surgery. All these findings recapitulated that the growth factors incorporated bioactive PIMS could perform as an appropriate matrix for osteogenic differentiation and efficient bone regeneration.

Silk Sponges Ornamented with a Placenta-Derived Extracellular Matrix Augment Full-Thickness Cutaneous Wound Healing by Stimulating Neovascularization and Cellular Migration.

Regeneration of full-thickness wounds without scar formation is a multifaceted process, which depends on in situ dynamic interactions between the tissue-engineered skin substitutes and a newly formed reparative tissue. However, the majority of the tissue-engineered skin substitutes used so far in full-thickness wound healing cannot mimic the natural extracellular matrix (ECM) complexity and thus are incapable of providing a suitable niche for endogenous tissue repair. Herein, we demonstrated a simple approach to fabricate porous hybrid ECM sponges (HEMS) using a placental ECM and silk fibroin for full-thickness wound healing. HEMS with retained cytokines/growth factors provided a noncytotoxic environment in vitro for human foreskin fibroblasts (HFFs), human epidermal keratinocytes (HEKs), and human amniotic membrane-derived stem cells to adhere, infiltrate, and proliferate. Interestingly, HEMS-conditioned media accelerated the migration of HFFs and HEKs owing to the presence of cytokines/growth factors. Also, the ex vivo chick chorioallantoic membrane assay of HEMS demonstrated its excellent vascularization potential by inducing and supporting blood vessels. Additionally, HEMS when subcutaneously implanted demonstrated no severe immune response to the host. Furthermore, HEMS implanted in full-thickness wounds in a rat model showed augmented healing progression with well-organized epidermal-dermal junctions via pronounced angiogenesis, accelerated migration of HFFs/HEKs, enhanced granulation tissue formation, and early re-epithelialization. Taken together, these findings show that porous HEMS ornamented with cytokines/growth factors having superior physicomechanical properties may be an appropriate skin substitute for full-thickness cutaneous wounds.

Polycaprolactone nanofibers functionalized with placental derived extracellular matrix for stimulating wound healing activity.

Impaired wound healing is primarily associated with inadequate angiogenesis, repressed cell migration, deficient synthesis of extracellular matrix (ECM) component/growth factors, and altered inflammatory responses in the wound bed environment. Herein, we report a simple process for the fabrication of PCL nanofiber mats embedded with placental-derived bioactive molecules (PCL-sPEM) rich in growth factors for full-thickness cutaneous wound healing. The physicochemical attributes and biological composition of PCL-sPEM nanofiber mats delivered a nontoxic environment in vitro and significantly promoted the adhesion, infiltration, and proliferation of human fibroblasts/keratinocytes. Conditioned media extracted from PCL-sPEM nanofiber mats enhanced the migration potential of the cells (fibroblasts/keratinocytes) involved in wound healing due to the release of growth factors embedded in it. Further, PCL-sPEM nanofiber mats attracted, stimulated and supported vascularization as determined by the Chick Chorioallantoic Membrane (CAM) assay. Interestingly, critical skin wounds of rats treated with PCL-sPEM nanofiber mats facilitated improved wound closure with well-organized dermis and epidermis, which could be ascribed to prominent vascularization, augmented migration of human foreskin fibroblasts (HFFs) & human epidermal keratinocytes (HEKs), increased collagen synthesis and early re-epithelialization. Collectively, our results suggest that PCL-sPEM nanofiber mats embedded with growth factors could be a suitable matrix for treating critical full-thickness wounds.

Accelerated healing of full thickness dermal wounds by macroporous waterborne polyurethane-chitosan hydrogel scaffolds.

Wound healing is a dynamic process wherein cells, and macromolecules work in consonance to facilitate tissue regeneration and restore tissue integrity. In the case of full-thickness (FT) wounds, healing requires additional support from native or synthetic matrices to aid tissue regeneration. In particular, a matrix with optimum hydrophilic-hydrophobic balance which will undergo adequate swelling as well as reduce bacterial adhesion has remained elusive. In the present study, polyurethane diol dispersion (PUD) and the anti-bacterial chitosan (Chn) were blended in different ratios which self-organized to form macroporous hydrogel scaffolds (MHS) at room temperature on drying. SEM and AFM micrographs revealed the macroporosity on top and fracture surfaces of the MHS. FTIR spectra revealed the intermolecular as well as intra-molecular hydrogen bonding interactions between the two polymers responsible for phase separation, which was also observed by micrographs of blend solutions during the drying process. The effect of phase separation on mechanical properties and in vitro degradation (hydrolytic, enzymatic and pH dependent) of MHS were studied and found to be suitable for wound healing. In vitro cytocompatibility was demonstrated by the proliferation of primary rat fibroblast cells on MHS. Selected MHS was subjected to in vivo FT wound healing study in Wistar rats and compared with an analogous polyurethane containing commercial dressing i.e. Tegaderm™. The MHS-treated wounds demonstrated accelerated healing with increased wound contraction, higher collagen synthesis, and vascularization in wound area compared to Tegaderm™. Thus, it is concluded that the developed MHS is a promising candidate for application as FT wound healing dressings.

Oleoyl-Chitosan-Based Nanofiber Mats Impregnated with Amniotic Membrane Derived Stem Cells for Accelerated Full-Thickness Excisional Wound Healing.

Wound healing management is a major challenge for critical full-thickness skin wounds. Development of nanofibrous scaffolds with tunable wettability, degradation, and biocompatibility are highly desirable. Herein, we demonstrated synthesis of oleoyl chitosan (OC) by grafting monounsaturated fatty acid residue, C18 oleoyl chain, to the backbone of chitosan molecule and blending with gelatin to form the nanofiber mats. The physicochemical properties of the nanofiber mats revealed mechanical strength, moderate surface wettability, and suitable degradation rate. The nanofibrous mats showed excellent in vitro cytocompatibility with human amniotic membrane-derived stem cells (HAMSCs) in terms of enhanced adhesion and proliferation owing to biomimetic nanoarchitecture and chemical cues. Furthermore, the fabricated nanofiber was implanted with and without preseeded HAMSCs in the full-thickness wound to evaluate the skin wound healing efficacy in a rat model. Histological and immunohistochemical studies were conducted to evaluate the plausible changes of tissue architecture and expression of molecular markers involved in wound healing process. Both acellular and HAMSCs incorporated cellular nanofibers promoted wound contraction remarkably with superior skin tissue regeneration in terms of enhanced collagen synthesis, re-epithelialization and initiation of epithelial cells stratification compared to control group.

Onion derived carbon nanodots for live cell imaging and accelerated skin wound healing.

Nitrogen, sulfur, and phosphorous co-doped water-soluble carbon nanodots are synthesized from culinary waste onion peel powder (OPP) by a short microwave treatment. Onion Derived Carbon Nano Dots (OCND) that comprised hydrophilic group-decorated amorphous nano-dots exhibited bright, stable fluorescence at an excitation of 450 nm and emission wavelength at 520 nm along with a free radical scavenging property. The OCND exhibited excellent stability at different pH and UV exposure. Although extracted polyphenols degraded in the extract, interestingly it was shown to be cytocompatible and blood compatible as observed during cytotoxicity, fluorescence imaging of the cell and a hemolysis study. The present work not only focuses on the synthesis of OCND from the OPP extract but also provides an interesting fact that, even after the degradation of polyphenols in the extract, they are non-toxic to human cells (HFF & MG63) and RBCs. Moreover, OCND had no adverse effect on the migration rate of Human Foreskin-derived Fibroblasts (HFFs) as observed from a scratch assay. In addition to accelerating the migration rate of fibroblasts, the OCND altered intra- and extracellular reactive oxygen species (ROS) by enhancing the antioxidant mechanism of a fibroblast under oxidative stress. Further, OCND was observed to accelerate wound healing in a full thickness (FT) wound in a rat model for topical application, which can be attributed to its radical scavenging potential. In summary, this study leads to a new type of OCND synthesis route, which is inherently co-doped with phosphorous, sulfur and nitrogen and holds a great promise for a myriad of biological applications, including bio-imaging, free radical scavenging and wound healing.

Investigating the potential of human placenta-derived extracellular matrix sponges coupled with amniotic membrane-derived stem cells for osteochondral tissue engineering.

Osteochondral injuries are challenging to repair due to their complex tissue anatomy and restricted self-repairing ability associated with a limited blood supply. Osteochondral tissue engineering is an important clinical aspect of the management and treatment of cartilage and underlying bone. In the present study, we fabricated human placenta-derived extracellular matrix sponges (PEMS) for repair of osteochondral tissue through a decellularization process. There were no significant cellular components present in the PEMS; hematoxylin & eosin/DAPI staining, DNA quantification and agarose gel electrophoresis were used to evaluate the extent of decellularization. Moreover, no significant alteration to the collagen and glycosaminoglycan (native extracellular matrix) content of the PEMS was observed. PEMS in vitro provided a non-cytotoxic environment rich in bioactive cues for human amniotic membrane-derived stem cells (HAMSCs) to proliferate in and differentiate into chondrogenic and osteogenic lineages under induction. Histological analysis at 28 days after the PEMS were subcutaneously implanted demonstrated no severe immune response in the host and supported the formation of blood vessels. To assess the osteochondral tissue repair ability of PEMS, cell-free PEMS (CFP) and cell-seeded PEMS (CSP) were implanted at osteochondral defect sites in a rabbit model. Histological scores indicated that osteochondral regeneration was more successful in the defects filled with CSP compared to those filled with CFP and empty defects (ED) after 60 days of implantation. In summary, a naturally derived biocompatible scaffold composed of extracellular matrix from human placenta has been successfully developed for osteochondral tissue engineering.

Chitosan derivatives cross-linked with iodinated 2,5-dimethoxy-2,5-dihydrofuran for non-invasive imaging.

Radiopaque polymer derivatives were successfully prepared through surface diffusion mediated cross-linking of chitosan with iodinated 2,5-dimethoxy-2,5-dihydrofuran. The incorporation of iodine in 2,5-dimethoxy-2,5-dihydrofuran was validated by (1)H NMR and mass spectroscopy. The cross-linking of the glucosamine moieties of chitosan with the iodinated product was confirmed by (13)C NMR and energy-dispersive X-ray spectroscopy. Radiography analysis proved inherent opacity of the iodinated fibrous sheets and microspheres that were comparable to the X-ray visibility of aluminum hollow rings of equivalent thickness and commercially available radiopaque tape, respectively. Microscopic studies evidenced retention of the fiber/microsphere morphology after the iodination/cross-linking reactions. The effects of iodination/cross-linking on the mechanical and biodegradation properties of fibers were studied by nanoindentation and enzymatic assay, respectively. In vitro and in vivo studies established the nontoxic, biodegradable nature of radiopaque derivatives. Iodinated fiber mesh implanted in a rabbit model was significantly X-ray opaque compared to the uncross-linked fiber mesh and medical grade surgical swabs. Further, opacity of the iodinated mesh was evident even after 60 days, though the intensity was reduced, which indicates the biodegradable nature of the iodinated polymer. The opacity of the iodinated sutures was also established in the computed tomography images. Finally, the sufficient in vivo contrast property of the radiopaque microspheres in the gastrointestinal tract indicates its possible role in clinical diagnostics.