Injectable hyperbranched poly(β-amino ester) hydrogels with on-demand degradation profiles to match wound healing processes

Abstract

Adjusting biomaterial degradation profiles to match tissue regeneration is a challenging issue. Herein, biodegradable hyperbranched poly(β-amino ester)s (HP-PBAEs) were designed and synthesized via "A2 + B4" Michael addition polymerization, and displayed fast gelation with thiolated hyaluronic acid (HA-SH) via a "click" thiol-ene reaction. HP-PBAE/HA-SH hydrogels showed tunable degradation profiles both in vitro and in vivo using diamines with different alkyl chain lengths and poly(ethylene glycol) diacrylates with varied PEG spacers. The hydrogels with optimized degradation profiles encapsulating ADSCs were used as injectable hydrogels to treat two different types of humanized excisional wounds - acute wounds with faster healing rates and diabetic wounds with slower healing and neo-tissue formation. The fast-degrading hydrogel showed accelerated wound closure in acute wounds, while the slow-degrading hydrogel showed better wound healing for diabetic wounds. The results demonstrate that the new HP-PBAE-based hydrogel in combination with ADSCs can be used as a well-controlled biodegradable skin substitute, which demonstrates a promising approach in the treatment of various types of skin wounds.

Fig. 1. A schematic of the proposed formation of HP-PBAEs and the function of HP-PBAE/HA-SH hydrogels for modulating wound healing. (A) HP-PBAEs are synthesized by the “A2 + B4” Michael addition reaction. Injectable hydrogels are formed by HP-PBAEs crosslinking with HA-SH. HP-PBAE/HA-SH/ADSCs hydrogels are mixed and injected onto full skin thickness humanized excisional wounds on the dorsum of the rats. (B) Non-diabetic wounds do not have pathophysiological abnormalities and undergo a quick healing process which correlates with the fast-degrading hydrogel. (C) Diabetic wounds possessing persistent pathophysiological defects show an impaired healing process which requires the slow-degrading hydrogel combined with therapeutic elements to optimize the healing conditions.

Fig. 2. The properties of injectable HP-PBAE/HA-SH hydrogels. (A) The formation of hydrogels using HA-SH to crosslink with the HP-PBAE polymer. (B) The gelation time of HP-PBAE/HA-SH hydrogels. (C) A typical gelation process of different concentrations of an injectable hydrogel (700-EDA/HA-SH) characterized by a rheometer. (D) The mechanical strength of HP-PBAE/HA-SH hydrogels.

Fig. 3. The degradation profile of HP-PBAE/HA-SH hydrogels. (A) The swelling ratio of the hydrogels in PBS at 37 °C (n = 4). (B) The in vivo degradation by subcutaneous implantation. The “X” represents the time at which the hydrogels totally disappeared (n = 4).

Fig. 4. Wound closure in humanized wound beds in nondiabetic rats. (A) Representative images of wound closure during 21-day in vivo experiments. (B) The quantification of the wound closure rate (%) over a 21-day period (n = 8). (C) Percentages of newly-formed tissue thickness at 21 days post-wounding. *p < 0.05; **p < 0.01.

Fig. 5. The immuno-histochemical analysis of wound sections in nondiabetic wound beds at scheduled time points. (A) Representative immunofluorescent images of tissue sections at 11 days post-wounding stained for TNF-α (green) and DAPI staining for nuclei (blue). (B) CD31 (for the endothelial marker, red), α-SMA (for the endothelial myofibroblast marker, green), and DAPI (for cell nuclei, blue) stained images (21 days post-wounding). The yellow triangles represent CD31+/α-SMA– vessels, and the yellow arrows represent CD31+/α-SMA+ vessels. Scale bar: 100 μm. (C)–(G) The quantification analysis of the positive stained inflammatory cytokines, which indicates total cellular infiltration into the constructs or wound areas as well as an immune response in the surrounding tissue. (B) and (C) The quantification analysis of blood vessels based on the CD31 and α-SMA positive stained vasculature. (D) The intensity of the VEGF expression in each experimental group. (E) The quantification analysis of the re-epithelialization rate (n = 16 wounds per treatment group). *p < 0.05; **p < 0.01.

Fig. 6. Wound closure in humanized wound beds in diabetic rats. (A) Representative images of wound closure during 21-day in vivo experiments. (B) The quantification of the wound closure rate (%) over a 21-day period. (C) Percentages of newly-formed tissue thickness at 21 days post-wounding. 700-EDA/HA-SH/ADSCs hydrogel treated wounds show significantly promoted healing compared to the PBS group (n = 8). *p < 0.05; **p < 0.01.

Fig. 7. The wounds treated with injectable 700-EDA/HA-SH/ADSCs hydrogels exhibit fewer inflammatory cells and more newly formed vasculature in diabetic rats. (A) Representative immunofluorescent images of tissue sections at 11 days post-wounding stained for TNF-α (green) and DAPI staining for nuclei (blue). (B) CD31 (for the endothelial marker, red), α-SMA (for the endothelial myofibroblast marker, green), and DAPI (for cell nuclei, blue) stained images (21 days post-wounding). The yellow triangles represent CD31+/α-SMA– vessels, and the yellow arrows represent CD31+/α-SMA+ vessels. Scale bar: 100 μm. (C)–(G) The immuno-histochemical analysis of wound sections in diabetic wound beds. (C) The quantification analysis of the positive stained inflammatory cytokines, which indicates total cellular infiltration into the constructs or wound areas as well as the immune response in the surrounding tissue. Less secretion of inflammatory factors is observed in injectable 700-EDA/HA-SH/ADSCs hydrogel treated wounds. (D) and (E) The quantification analysis of blood vessels based on CD31 and α-SMA positive stained vasculature. The injectable hydrogel treated groups exhibit significantly higher vessel density than the PBS group at 11 and 21 days post-wounding. (F) The intensity of VEGF expression in each experimental group. More secretion of VEGF is seen in 700-EDA/HA-SH/ADSCs hydrogel treated wounds. (G) The quantification analysis of the re-epithelialization rate at days 11 and 21 post-wounding. There is a much faster re-epithelialization rate in the injectable 700-EDA/HA-SH/ADSCs hydrogel groups than in the PBS group (n = 16 wounds per treatment group). *p < 0.05; **p < 0.01.