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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.

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.

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.

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.

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.

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.

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.

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.

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.