Fabrication of self-healing injectable hyaluronic acid hydrogel for promoting angiogenesis

Objective·To construct a self-healing injectable hyaluronic acid (HA)-based hydrogel (HAPD-Cu) and investigate the effects of different copper ions on the properties of the hydrogel and its vasogenic efficacy to evaluate its feasibility for clinical wound healing.Methods·Bisphosphonated hyaluronic acid (HAPD) was prepared via a blue-light mediated thiol-ene click reaction between thiolated hyaluronic acid (HASH) and acrylated bisphosphonate (Ac-PD) in the presence of photoinitiator 2959. Then, HAPD was further interacted with Cu2+ through metal coordination to prepare HAPD-Cu hydrogels with different Cu2+ concentrations, i.e. HAPD-Cu1, HAPD-Cu2, HAPD-Cu3 and HAPD-Cu4. The molecular structures of HASH, Ac-PD, HAPD and HAPD-Cu were verified with 1HNMR and FTIR. Microscopic morphology of HAPD-Cu was observed under SEM. The shear-thinning and self-healing properties of HAPD-Cu were verified by rheometer. The Cu2+ release from HAPD-Cu was determined with ICP. Live-dead staining and CCK-8 assay were applied to evaluate the biocompatibility of HAPD-Cu. The in vitro vasculogenic activity of HAPD-Cu was determined by a tubule-forming assay with human umbilical vein vascular endothelial cells and the in vivo vasculogenic activity of HAPD-Cu was assessed by CD31 tissue staining. A rat wound defect model was established in vitro to evaluate its actual repair effect.Results·The preparation of the materials was demonstrated through chemical qualitative and quantitative analytical means. In vitro studies showed that all HAPD-Cu with a loose porous internal structure exhibited outstanding self-healing, injectability and degradability, with a one-week degradation cycle and abrupt release behavior, which can meet the needs of wound healing cycle. All HAPD-Cu showed good biocompatibility except HAPD-Cu4, due to its high Cu2+ concentrations. Moreover, its angiogenic effect in vitro or in vivo was enhanced with increasing Cu2+ concentrations within the permissible Cu2+ concentration range. In vitro wound model experiments also showed that the HAPD-Cu hydrogel significantly promoted wound healing compared with the control group.Conclusion·HAPD-Cu hydrogel constructed via the metal coordination shows excellent shape plasticity, allowing the filling of defective sites in a minimally invasive form, and the release of Cu2+ greatly facilitates the establishment of early vascular networks, with giant potential for use in the repair of clinically irregular wounds

Effect of high hydrophilic electrospun short fibrous sponge on wound repair

Objective·To construct an electrospun short fibrous sponge (Sponge@GO) laden with graphene oxide (GO) for chronic wound healing.Methods·Two types of short fibrous sponges (Sponge and Sponge@GO) without and with GO were prepared by means of electrospinning, homogenizing, shaping and crosslinking with glutaraldehyde, respectively. The internal structures of the two sponges were observed with a scanning electron microscope (SEM), and their hydrophilic properties were observed via contact angle and water absorption rate. The biocompatibility of the sponge was verified by CCK-8 and live/dead staining. Twelve 6-week-old SD male rats were divided into control group, Sponge group and Sponge@GO group, with 4 rats in each group. The diabetes models were established by intraperitoneal injection of 1% streptozotocin solution, and three full-layer skin defects with a diameter of 1.0 cm were prepared on the back of each rat after modelling. Covering on the wound, the material was fixed with medical gauze. The control group was only covered with sterile gauze dressing. The wound healing rate was measured and calculated on Day 7 and 14, respectively, while hematoxylin-eosin (H-E) staining and Masson staining were performed on tissues within 0.5 cm around the wound to observe pathological changes. The angiogenesis was observed by α-smooth muscle actin (α-SMA) immunofluorescence staining on Day 14.Results·SEM observation showed that the fiber diameter of Sponge@GO was significantly thinner and the porosity increased. The two types of short fiber scaffolds basically reached the maximum water uptake within 10 min, but the Sponge@GO scaffold showed better water absorption performance. The water contact angle of Sponge@GO scaffold was significantly smaller than that of Sponge, and the difference was statistically significant (P=0.000). The results of CCK-8 method showed that on Day 3 and 5, the Sponge group had better cell proliferation compared with the control group (both P<0.05), while there was no statistical significance between Sponge@GO group and control group. The results of live/dead staining showed that all the three groups of cells showed good cell growth trend. SEM and fluorescence staining showed that there were more cells in the Sponge@GO scaffold. In vivo experiment, no infection was found on the wound surface of the three groups of rats. The wound healing rate of Sponge@GO and Sponge groups was significantly higher than that of control group on Day 7 (both P<0.05). On Day 14, the wound healing rate of the Sponge@GO group was still significantly higher than that of the control group (P=0.009), while the difference between the Sponge group and the control group was not statistically significant. On Day 14, H-E staining showed more mature granulation tissue and more uniform and dense structure in the Sponge@GO group; Masson staining showed more dense collagen and significant epithelialization in the Sponge@GO group; α-SMA immunofluorescence staining showed more neovascularization and higher density in the Sponge@GO group.Conclusion·Sponge@GO sponge can ensure micro-moist environment on the wound surface after absorbing exudate and has shown promising results in promoting wound healing

Effect of hydrogel stiffness on nucleus pulposus cell phenotypes in vitro and its repairment of intervertebral disc in vivo

Objective·To investigate the effect of hydrogel stiffness on nucleus pulposus cell phenotype and its function in repairing intervertebral disc degeneration in rats.Methods·Methacrylate gelatin (GelMA) hydrogels with different concentrations were constructed. The stiffness of the hydrogels was investigated by using rheological analysis and uniaxial compression test. The microstructure and morphology of the hydrogels were observed by scanning electron microscopy (SEM). Nucleus pulposus cells with normal phenotype were inoculated on the surface of GelMA hydrogels. The biocompatibility of the hydrogel was evaluated by live-dead cell staining and the growth pattern of nucleus pulposus cells on hydrogels with different stiffness was observed with phalloidin staining under microscope. Immunofluorescence staining was performed to examine the nuclear localization of Yes-associated protein (YAP) and real-time quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of nucleus pulposus cell-associated genes [neural cell adhesion molecule 1 (Ncam-1), aggrecan (Acan), sex-determing region of Y chromosome (SRY)-box transcription factor 9 (Sox9)]. A rat caudal acupuncture intervertebral disc degeneration model was established. Nucleus pulposus cells cultured on different hydrogels were harvested and injected into the degenerated discs separately. Four weeks after surgery, magnetic resonance imaging (MRI) was performed to analyze the water content of the intervertebral discs in each group. Histological tests were performed to examine the disc structure and proteoglycan levels.Results·The elastic modulus of the hydrogels was 1 kPa and 200 kPa when the concentration of GelMA prepolymerisation solution was at 4% and 15% respectively. SEM observation revealed that the hydrogels showed a loose and porous microstructure, and the porosity of hydrogels decreased significantly with the decrease of their stiffness. In vitro experiments demonstrated that both GelMA hydrogel mediums showed good biocompatibility and the ability to support cell proliferation. Nucleus pulposus cells cultured on the soft matrix (4%GelMA) had a lower elongation and spreading area than those cultured on the stiff matrix (15%GelMA), showing a tendency of YAP concentration in the cytoplasm. The gene expression of nucleus pulposus cells was examined and the levels of Sox9, Acan and Ncam-1 in the soft matrix hydrogel group were 23.7, 6.6 and 12.7 times of those in the control group respectively. In vivo experiments on rat disc degeneration showed that the soft hydrogel matrix group had higher disc water content and structural integrity than the stiff hydrogel matrix group.Conclusion·Compared to stiff GelMA hydrogels, hydrogels with low stiffness better maintain the growth phenotypes in the nucleus pulposus cells and have better therapeutic effect on disc degeneration in vivo

Advanced liposome-loaded scaffolds for therapeutic and tissue engineering applications

Liposome is one of the most commonly used drug delivery systems in the world, due to its excellent biocompatibility, satisfactory ability in controlling drug release, and passive targeting capability. However, some drawbacks limit the application of liposomes in clinical, such as problems in transporting, storing, and difficulties in maintaining the drug concentration in the local area. Scaffolds usually are used as implants to supply certain mechanical supporting to the defective area or utilized as diagnosis and imaging methods. But, in general, unmodified scaffolds show limited abilities in promoting tissue regeneration and treating diseases. Therefore, liposome-scaffold composite systems are designed to take advantages of both liposomes’ biocompatibility and scaffolds’ strength to provide a novel system that is more suitable for clinical applications. This review introduces and discusses different types of liposomes and scaffolds, and also the application of liposome-scaffold composite systems in different diseases, such as cancer, diabetes, skin-related diseases, infection and human immunodeficiency virus, and in tissue regeneration like bone, teeth, spinal cord and wound healing.Peer reviewe

Localized Controlled Delivery of Gemcitabine via Microsol Electrospun Fibers to Prevent Pancreatic Cancer Recurrence

The low radical surgery rate of pancreatic cancer leads to increased local recurrence and poor prognosis. Gemcitabine (GEM) is the preferred chemotherapeutic for pancreatic cancer. However, systemic chemotherapy with GEM has reached a bottleneck due to its serious side effects after frequent injections. In this study, GEM is successfully enwrapped into electrospun fibers via microsol electrospinning technology to form a stable core-shell fibrous structure. The GEM release rate can be adjusted by altering the thickness of the hyaluronan-sol inner fiber and the quantity of loaded GEM, and the release can be sustained for as long as three weeks. In vitro assays show that these electrospun fibers effectively inhibit pancreatic cancer cells and promote apoptosis. In vivo studies show that the fibrous membranes are better for inhibiting the growth of residual tumors than that of integrated tumors. Furthermore, immunohistochemistry results show that GEM-loaded fibers promote a higher cell apoptosis rate than does systemically injected GEM in residual tumors. In addition, the local delivery of GEM with fibers significantly reduces liver toxicity. In summary, a core-shell electrospun fiber for the controlled and localized delivery of GEM, which greatly improves the treatment of residual tumors and prevents pancreatic tumor recurrence, is developed.Peer reviewe

Regulating Inflammation Using Acid-Responsive Electrospun Fibrous Scaffolds for Skin Scarless Healing

Skin injury in adult mammals brings about a series of events and inflammation in the wounded area is initiated first and provides lots of inflammatory factors, which is critical for the final scar formation. While the postinjured skin of fetus and nude mice heals scarlessly owing to the absence of inflammation or immunodeficient, we designed a feasible acid-responsive ibuprofen-loaded poly(L-lactide) (PLLA) fibrous scaffolds via doping sodium bicarbonate to prevent excessive inflammation and achieve scarless healing finally. The morphological results of in vivo experiments revealed that animals treated with acid-responsive ibuprofen-loaded PLLA fibrous scaffolds exhibited alleviative inflammation, accelerated healing process, and regulated collagen deposition via interference in the collagen distribution, the α-smooth muscle actin (α-SMA), and the basic fibroblast growth factor (bFGF) expression. The lower ratios of collagen I/collagen III and TGF-β1/TGF-β3 and higher ratio of matrix metalloproteinase-1 (MMP-1)/tissue inhibitor of metalloproteinase-1 (TIMP-1) in acid-responsive ibuprofen-loaded PLLA fibrous scaffolds group were confirmed by real-time qPCR as well. These results suggest that inhibiting the excessive inflammation will result in regular collagen distribution and appropriate ratio between the factors, which promote or suppress the scar formation, then decrease the scar area, and finally achieve the scarless healing

Enhancing Apoptosome Assembly via Mito-Biomimetic Lipid Nanocarrier for Cancer Therapy

Apoptosis is the natural programmed cell death process, which is responsible for abnormal cell clearance. However, many cancer cells develop various mechanisms to escape apoptosis through interrupting apoptosome assembly, which is a key step to initiate apoptosis. This promotes tumorigenesis and drug resistance, and thus, poses a great challenge in cancer treatment. Herein, a biomimetic lipid nanocarrier mimicking mitochondrial Cytochrome C (Cyt C) binding is developed. Cardiolipin, the major phospholipid of mitochondrial inner membrane, is introduced as the main component in biomimetic liposomal formulation. With the help of cardiolipin, Cyt C is sufficiently loaded in liposome based on electrostatic and hydrophobic interaction with cardiolipin. Lonidamine (LND) is added in hydrophobic phase of liposome to modulate the metabolic activity within cancer cells and sensitize the cells to Cyt C-induced apoptosis. The results suggest that LND reduces ATP level and creates favorable environment for Cyt C induced apoptosome assembly, exhibiting higher apoptosis level and anti-tumor efficacy in vitro and in vivo. The conjugation of a tumor-homing peptide, LinTT1, on the nanovesicle, increases the efficacy due to enhanced tumor accumulation. Overall, this biomimetic lipid nanocarrier proves to be an efficient delivery system with great potential of pro-apoptosis cancer therapy