An injectable viscoelastic bioadhesive with strong tissue adhesion and rapid self-healing for regeneration of large muscle injury

Introduction: Traumatic muscle injuries, such as volumetric muscle loss (VML), significantly affect mobility and quality of life. Existing treatments, including scar tissue debridement and muscle flap positioning, often lead to suboptimal outcomes in aesthetics and function. With the increasing prevalence of muscle-related injuries, there is a growing need for more effective treatment strategies. In recent years, biomaterials placed in muscle defects have been demonstrated to enhance tissue regeneration by providing a supportive matrix for cellular infiltration and serving as a depot for therapeutics. Tissue adhesives, especially injectable ones, are becoming increasingly popular for their ability to seamlessly fill muscle defects, thereby reducing infection risks and surgical complexities. These adhesives must be mechanically robust enough to withstand muscle dynamics, such as contraction and relaxation, and ideally degrade over time to allow for replacement by regenerating tissue. However, finding adhesives that meet all these requirements remains a challenge. The objective of this study is to develop an injectable tissue adhesives that exhibit outstanding mechanical properties, sufficient to adapt to muscle dynamics and degrade at a rate that aligns with tissue healing. We hypothesize that this adhesive can remain secure in the defect site and serve as a matrix to promote muscle regeneration.

Materials and

Methods: The synthesis of PAA-BLA involves several key steps. Initially, PAA is dissolved, neutralized, and lyophilized to form a white powder. It is then functionalized with alkyne groups through carbodiimide coupling, adjusted to the correct pH, and stirred before being dialyzed and lyophilized again. This yields a PAA-alkyne, which is quantified for alkyne modification via 1H NMR spectroscopy. Subsequently, PAA-alkyne is further modified using copper-catalyzed click chemistry with azido-benzaldehyde in a phosphate buffered saline, stirred, filtered, and dialyzed to produce the final PAA-BLA product. To prepare the adhesive mixture, PAA-BLA was dissolved in HBSS without calcium and phenol red to a 10% concentration, with phenol red added for pH indication. Separately, gelatin was dissolved in HBSS to a 20% concentration. Both solutions were warmed to 40°C and loaded into syringes. They were then mixed using a Luer lock coupler at a 2:1 PAA-BLA to gelatin ratio, creating a uniform adhesive with a final concentration of 6.67%. This adhesive was quickly applied to molds or tissue samples for testing. For the animal experiments, female C57BL6/J mice aged 8-12 weeks were utilized. A surgical procedure involved making a lateral incision along the tibialis anterior muscle of the left hind limb. A sterile metal plate was placed between the tibialis anterior and the extensor digitorum longus muscle. Then, a 3mm biopsy punch was used to excise a full-thickness defect in the muscle against the previously inserted metal plate.

Results, Conclusions, and Discussions: The bioadhesive was prepared by combining benzaldehyde-modified poly(acrylic acid) (PAA-BLA) with gelatin (Gel). This resulted in a hydrogel matrix, crosslinked through reversible imine bonds between the BLA and gelatin's primary amine groups. Due to its dynamic nature, this hydrogel can be administered via needle injection, as shown in Fig. 1A. Remarkably, the adhesive exhibits superior mechanical properties; it can stretch up to three times its original length and demonstrates significant viscoelastic behavior, effectively relaxing stress upon deformation (Fig. 1B, C). Remarkably, the adhesive also features rapid self-healing capabilities—approximately 60% of its strength is recoverable within just one minute after damage (Fig. 1D). Moreover, it adheres strongly to various tissue types (Fig. 1E) and offers tunable biodegradability.

We then explored the potential of the adhesive to facilitate muscle regeneration. Volumetric loss was created in the tibialis anterior muscles of mice, and the mice were subsequently divided into three groups for treatment: one with gelatin alone, one with the PAA-BLA/Gel adhesive, and a control group that received no treatment. Using real-time high-resolution ultrasound imaging, we observed that all of the adhesive remained at the defect site on day 4 (Fig. 1F). In contrast, some mice treated with gelatin showed displacement of the gel. The therapeutic efficacy was next assessed. Histological analysis of muscle tissues showed that muscles treated with the adhesive exhibited levels of inflammation comparable to both untreated muscles and those treated with gelatin. Notably, the adhesive led to a decrease in iNOS expression, indicative of pro-inflammatory macrophages, while simultaneously increasing Arg1 expression, a characteristic marker of anti-inflammatory macrophages (Fig. 1G). The functional impact of treating VML with the adhesive was then analyzed. The gross size and weight of the muscles were observed to be greater in mice treated with the adhesive than in the control groups. Importantly, longitudinal muscle function analyses conducted over a 4-week period showed that muscles in the adhesive-treated group generated substantially higher tetanic forces compared to both the untreated group and those treated with gelatin (Fig. 1H). This work marks a significant advancement in the development of medical adhesives for tissue repair and regeneration.

Acknowledgements (Optional): S.N. and J.L. contributed equally to this work. This work was supported by the NIDCR/NIH (5R01DE013349). S.N. gratefully acknowledges funding support from the Wyss Technology Development Fellowship. Additionally, we thank Alexander Pauer from Wyss Institute for the help with GPC. We thank Dana-Farber/Harvard Cancer Center in Boston, Massachusetts, for the use of the Rodent Histopathology Core.