Simultaneous regulation of AGE/RAGE signaling and MMP-9 expression by an immunomodulating hydrogel accelerates healing in diabetic wounds.

PURPOSE: In chronic hyperglycemia, the advanced glycation end product (AGE) interacts with its receptor (RAGE) and contributes to impaired wound healing by inducing oxidative stress, generating dysfunctional macrophages, and prolonging the inflammatory response. Additionally, uncontrolled levels of proteases, including metallomatrix protease-9 (MMP-9), in the diabetic wound bed degrade the extracellular matrix (ECM) and biological cues that augment healing. A multifunctional antimicrobial hydrogel (Immuno-gel) containing RAGE and MMP-9 inhibitors can regulate the wound microenvironment and promote scar-free healing. RESULTS: Immuno-gel was characterized and the wound healing efficacy was determined in vitro cell culture and in vivo diabetic Wistar rat wound model using ELISA, Western blot, and Immunofluorescence staining. The Immuno-gel exhibited a highly porous morphology with excellent in vitro cytocompatibility. AGE-stimulated macrophages treated with the Immuno-gel released higher levels of pro-healing cytokines in vitro. In the hydrogel-wound interface of diabetic Wistar rats, Immuno-gel treatment significantly reduced MMP-9 and NF-κB expression and enhanced pro-healing (M2) macrophage population and pro-healing cytokines. CONCLUSION: Altogether, this study suggests that Immuno-gel simultaneously attenuates macrophage dysfunction through the inhibition of AGE/RAGE signaling and reduces MMP-9 overexpression, both of which favor scar-free healing. The combinatorial treatment with RAGE and MMP-9 inhibitors via Immuno-gel simultaneously modulates the diabetic wound microenvironment, making it a promising novel treatment to accelerate diabetic wound healing.

A multifunctional silk-hyaluronic acid self-healing hydrogel laden with alternatively activated macrophage-derived exosomes reshape microenvironment of diabetic wound and accelerate healing.

The impairment of phenotype switching of pro-inflammatory M1 to pro-healing M2 macrophage induced by hyperglycemic microenvironment often elevates oxidative stress, impairs angiogenesis, and leads to chronic non-healing wounds in diabetic patients. Administration of M2 macrophage-derived exosomes (M2Exo) at wound site is known to polarize M1 to M2 macrophage and can accelerate wound healing by enhancing collagen deposition, angiogenesis, and re-epithelialization. In the present study, M2Exo were conjugated with oxidized hyaluronic acid and mixed with PEGylated silk fibroin to develop self-healing Exo-gel to achieve an efficient therapy for diabetic wounds. Exo-gel depicted porous networked morphology with self-healing and excellent water retention behaviour. Fibroblast cells treated with Exo-gel showed significant uptake of M2Exo that increased their proliferation and migration in vitro. Interestingly, in a diabetic wound model of wistar rats, Exo-gel treatment induced 75 % wound closure within 7 days with complete epithelial layer regeneration by modulating cytokine levels, stimulating fibroblast-keratinocyte interaction and migration, angiogenesis, and organized collagen deposition. Taken together, this study suggests that Exo-gel depict properties of an excellent wound healing matrix and can be used as a therapeutic alternative to treat chronic non-healing diabetic wounds.

Laser-assisted synthesis of nano-hydroxyapatite and functionalization with bone active molecules for bone regeneration.

The main goal of bone tissue engineering research is to replace the allogenic and autologous bone graft substitutes that can promote bone repair. Owing to excellent biocompatibility and osteoconductivity, hydroxyapatite is in extensive research and high demand for both medical and non-medical applications. Although various methods have been developed for the synthesis of hydroxyapatite, in the present study we have shown the use of nanosecond laser energy in the wet precipitation method of nano-hydroxyapatite (nHAP) synthesis without using ammonium solution or any other chemicals for pH maintenance. Here, the present study aimed to fabricate the nanohydroxyapatite using a nanosecond laser. The X-ray diffraction and Fourier transform infrared spectroscopy have confirmed the hydroxyapatite formation under laser irradiation in less time without aging. A transmission electron microscopy confirmed the nano size of synthesized nHAP, which is comparable to conventional nHAP. The length and width of the laser-assisted nHAP were found to be in the range of 50-200 nm and 15-20 nm, respectively, at various laser parameters. The crystallite size obtained by Debye Scherrer formulae was found to be in the range of ∼ 16-36 nm. In addition, laser-assisted nHAP based composite cryogel (nanohydroxyapatite/gelatin/collagen I) was synthesized and impregnated with bioactive molecules (bone morphogenic protein and zoledronic acid) that demonstrated significant osteogenic potential both in vitro in cell experiment and in vivo rat muscle pouch model (abdomen and tibia muscles). Dual-energy X-ray analysis, micro-CT, and histological analysis confirmed ectopic bone regeneration. Micro-CT based histomorphometry showed a higher amount (more than 10-fold) of mineralization for animal groups implanted with composite cryogels loaded with bioactive molecules compared to only composite cryogels groups. Our findings thus demonstrate a controlled and rapid synthetic method for the synthesis of nHAP with various physical, chemical, and biological properties exhibited as comparable to conventionally synthesized nHAP.

Peptide-Based Scaffold for Nitric Oxide Induced Differentiation of Neuroblastoma Cells.

Nitric oxide is a gaseous messenger involved in neuronal differentiation, development and synaptogenesis, in addition to many other physiological functions. Therefore, it is imperative to maintain an optimal nitric oxide concentration to ensure its biochemical function. A sustained nitric oxide releasing scaffold, which supports neuronal cell differentiation, as determined by morphometric analysis of neurite outgrowth, is described. Moreover, the effect of nitric oxide on the neuroblastoma cell line was also confirmed by immunofluorescent analysis of neuronal nuclear protein (NeuN), specific neuronal marker and neurofilament (NF) protein, which revealed a significant increase in their expression levels, in comparison with undifferentiated cells.