Bioactive antibacterial silica-based nanocomposites hydrogel scaffolds with high angiogenesis for promoting diabetic wound healing and skin repair

Diabetic wound repair and skin regeneration remains a worldwide challenge due to the impaired functionality of re-vascularization. Methods: This study reports a bioactive self-healing antibacterial injectable dual-network silica-based nanocomposite hydrogel scaffolds that can significantly enhance the diabetic wound healing/skin tissue formation through promoting early angiogenesis without adding any bioactive factors. The nanocomposite scaffold comprises a main network of polyethylene glycol diacrylate (PEGDA) forming scaffolds, with an auxiliary dynamic network formed between bioactive glass nanoparticles containing copper (BGNC) and sodium alginate (ALG) (PABC scaffolds). Results: PABC scaffolds exhibit the biomimetic elastomeric mechanical properties, excellent injectabilities, self-healing behavior, as well as the robust broad-spectrum antibacterial activity. Importantly, PABC hydrogel significantly promoted the viability, proliferation and angiogenic ability of endothelial progenitor cells (EPCs) in vitro. In vivo, PABC hydrogel could efficiently restore blood vessels networks through enhancing HIF-1α/VEGF expression and collagen matrix deposition in the full-thickness diabetic wound, and significantly accelerate wound healing and skin tissue regeneration. Conclusion: The prominent multifunctional properties and angiogenic capacity of PABC hydrogel scaffolds enable their promising applications in angiogenesis-related regenerative medicine.


Synthesis of bioactive glass nanoparticles (BGN) and bioactive glass nanoparticles with copper (BGNC).
BGN was prepared by a traditional template method in our previous study, in which DDA was a catalyst and a template agent. In brief, DDA was dissolved in ethanol and deionized water with a volume ratio of 1:1, and TEOS, TEP, and CaN were added sequentially. After the reaction, the product was centrifuged, lyophilized and calcined in a muffle furnace to get the final product.
The synthesis method of BGNC was basically the same as the above, in which the original CaN was replaced with the CaN and CuN mixture (the molar ratio of CaN and CuN = 1:1).

Swelling behavior
To study the swelling behavior, PAB composite hydrogels were tested by a gravimetric method. Briefly, the PAB composite hydrogel was immersed in deionized water at room temperature. Samples were taken from the water at selected different time points and surface droplets were removed. Then, the sample was weighed and placed back in deionized water.
Swelling ratio Qw is calculated as follows: (1) where W0, Wt were the initial weight of the sample, and the weight of the sample after swelling at a certain time point, respectively.

Rheological and self-healing studies
Rheological measurements of PAB hydrogels were performed with a rheometer (DHR-2， TA) at 25°C. Using a parallel plate with a diameter of 20 mm, the shear frequency was increased from 0.1 to 100 Hz for stable shear rheological measurements. At different shear strains (1%-1000%), the change in storage modulus and loss modulus of different samples was measured. After the hydrogels were completely damaged, the storage modulus and loss modulus of the PAB hydrogel at different shear strains were recorded over time.

Mechanical properties Test
In this work, the mechanical test was mainly evaluated from the tension and compression of the material by the rheometer. The detailed method was as follows. Firstly, a cylindrical hydrogel was prepared with a 20 mm diameter (the same size as a parallel plate) and a 10 mm thickness.
Then, the hydrogel was vertically placed and completely in contact between the operator station and the parallel plate. At 25°C, records were collected by the rheometer for the changes in the material surface tension (the parallel plate moved up) or compression (the parallel plate moved down) with a constant rate of movement (100 μm/s).

Anti-bacterial activity
To evaluate the anti-bacterial activity of hydrogels, Gram-negative bacteria (Escherichia coli, E.coli) and Gram-positive bacteria (Staphylococcus aureus, S. aureus) were selected for the experiment. First, a cylindrical hydrogel (0.8 mL) was prepared in a centrifuge tube and sterilized at 55°C. Bacteria, grown in approximately 10 8 cells/mL of MHB broth were centrifuged out of the MHB broth, washed and diluted 100-fold in a sterile PBS solution. Then, the hydrogel was immersed in the 8 mL bacterial suspension. After incubation at 37 °C for 3 h, 6 h and 9 h on the shaker, the original solution was diluted to 100-fold and 10 μL of solution were prepared LBA plates to determine the amount of residual bacteria. Another option was to add the same volume of broth residually for 0 h, 3 h and 6 h. Then, after 3 h, 6 h, and 9 h, the original bacterial solution was diluted 100-fold and 10 μL of solution were prepared LBA plates to determine the amount of bacteria left PAB-0 and PAB-3 were the control group, and PABC-3 was the experimental group.
To test the minimum inhibitory concentration (MIC), PAB and PABC were evenly dispersed in PBS (4 mg/mL) to add to the 1st well , and diluted into the 2nd to 10th well plate in a doubling manner. Equal amounts of bacteria solution were added to the 1st to 11th wells, and sterile culture solution was added to the 12th well as a blank control. The 96-well plate with the uniform solutions was placed in a 37 °C microbial incubator for 8-10 h. Finally, we counted the number of transparent wells in the plate and calculated the minimum inhibitory concentration (MIC).

In vitro assessment of PABC hydrogel on endothelial progenitor cells (EPCs)
Endothelial progenitor cells (

Preparation of diabetic wound and implantation of hydrogels
After another week of observation, the mice were anesthetized with 4% chloral hydrate (150 mg/kg), shaved and sterilized. Sequentially, two circular full-thickness wounds were created on the back of the mice with a diameter of 8 mm (to a level of panniculus muscle). After saline irrigation, the prepared PA, PAB and PABC scaffolds was then used to cover the wounds. Saline treatment was used as control. All mice were kept individually in a specific pathogen free (SPF) animal room and fed with standard food and water with close observation.

Blood flow measurement
At day 7, 14 and 21, mice were anesthetized, shaved and the blood flow around the wound area was assessed by a laser Doppler imager (MoorLDI-2, Moor Instruments Limited, Devon, UK). The scanning parameters were set with a laser wavelength of 633 nm, scan distance of 55 cm and duration of 5 min. The obtained data was then analyzed by the MoorLDI Review V6.1 software.

Histological evaluation, immunofluorescence staining and western blotting analysis
Wound samples were collected at day 7, 14 and 21, fixed in 4% paraformaldehyde, After 7-day of treatment by PABC scaffold, wound samples were collected and homogenized in RIPA lysis buffer. The obtained protein extracts were then quantified with a BCA reagent. 65 μg tissue proteins were loaded with loading buffer and heated at 95 °C for 5 min. Then proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked by 5% non-fat milk for two hours and incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The bands were visualized by an electrochemiluminescence reagent