(460f) Injectable Dopamine-Functionalized Poly(oligoethylene glycol methacrylate) Hydrogels to Enhance Tissue Adhesion during Wound Healing

Background: Traditional wound closing systems such as tissue stapling or suturing can represent major challenges around inducing wound infections, damage to surrounding tissues, and/or hemorrhaging.¹ The knot tying procedure for sutures may cause tissue distortion, residual force, blocking of blood perfusion, and consequently impede wound healing.² Stapling can cause improper wound edge apposition or wound ischemia from applying staples too tightly, and scarring, while removal is uncomfortable for patients.³ In certain situations, primary closure of wounds using suturing or staples is not feasible because of the inadequacy of the skin flaps, especially after trauma or following excision of lesions.⁴ Bioadhesive sealants such as fibrin glue⁵ and cyanoacrylate⁵ offer several advantages over traditional mechanical approaches for wound closure but have their own safety risks and limitations.⁶ While fibrin-based glues are highly versatile hemostatic agents, they are limited by their mechanical properties, immunogenic responses and clotting stimulation.^6,7,8,5 Cyanoacrylate adhesives possess relatively strong tissue adhesion strength but raise concerns regarding toxicity of degradation products and tissue nectrosis.^9,5,10,11

As an alternative, herein we report injectable and degradable hydrazone crosslinked poly(oligoethylene glycol methacrylate) (POEGMA) hydrogels functionalized with dopamine (DA) methacrylamide for enabling both enhanced cytocompatibility and tissue adhesiveness bio-inspired by mussel adhesive proteins.¹² DA enables marine mussels to attach strongly to various surfaces in their turbulent, wet, and saline habitats through a combination of hydrogen bonding interactions, Ï€-Ï€ stacking, coordination, and covalent bonds between surface amines with its catechol moiety.¹³ We hypothesize that functionalizing our in situ-gelling, protein repellent, and tissue compatible POEGMA hydrogels with DA will preserve the controllable gelation, strong mechanical properties, and low tissue toxicity of POEGMA while enhancing the adhesive strength of the hydrogel at tissue interfaces, thus serving as an in situ gelling biological glue for wound closures.

Methods: Precursor POEGMA (n=8-9 ethylene oxide units per side chain) solutions functionalized with 30 mol% of aldehyde (A) (PO₁₀₀A₃₀D_x) or hydrazide (H) (PO₁₀₀H₃₀D_x) groups and 0, 10, 20 and 30 mol% of DA were synthesized through free radical polymerization. The precursor polymers were prepared in 10, 15 and 20 wt% solutions. The hydrogels were formed through the rapid A/H chemical crosslinking chemistry of PO₁₀₀A₃₀D_x and PO₁₀₀H₃₀D_x by simple mechanical mixing and/or co-injection through a double barrel syringe. The polymers were characterized using gel permeation chromatography for molecular weight determination, nuclear magnetic resonance for composition, and titration to establish the degree of functionalization. Gels were prepared using 10, 15 and 20 wt% precursor polymer concentrations functionalized with 0, 10, 20 and 30 mol% DA (PO₁₀₀, PO₁₀₀D₁₀, PO₁₀₀D₂₀, and PO₁₀₀D₃₀), with gelation and injectability tested by measuring the curing time via the vial inversion test. Rheological studies were performed with an 8 mm parallel plate at 25°C to obtain the storage (G’) and loss modulus (G”). The compression stress-strain and Young’s modulus of the POEGMA hydrogels with varying DA content were tested using micromechanical testing. Swelling and degradation studies were conducted daily over 23 days in 10 mM phosphate buffered solution (pH = 7.4) at 37°C. In vitro cell viability studies were conducted on C2C12 myoblast cells using a Presto Blue cell viability assay to demonstrate the non-cytotoxicity of the hydrogels. Lap shear testing on porcine skin were conducted to test the tissue adhesion strength of the DA hydrogels at a tissue interface. In vivo wound healing evaluation in Balb/c mice was conducted by administering two 4 mm diameter excisional wounds using biopsy punches through the layer of skin on the dorsal area and filling the wound site with POEGMA-DA hydrogels. Silicone rubber splints were applied around the wound site to provide mechanical support and prevent wound contraction. The wound sites were photographed on days 0, 3, 7 and 15, and wound areas were measured using ImageJ software. Histological tissue and blood samples were collected on days 0, 3, 7 and 14 for hematoxylin/eosin (H&E) and Masson’s trichrome staining as well as immunohistochemical testing for identification of immune markers.

Results and Discussion: 10, 15 and 20 wt% POEGMA precursor solutions functionalized with 10-30 mol% of DA (16 < M_n < 20 kDa) were synthesized, characterized, and formed into hydrogel discs using moulds. Based on the gelation properties of the DA hydrogels measured, the curing time for the highest DA content hydrogels, PO₁₀₀D₃₀, ranged from 22 s to 5 minutes while curing time for PO₁₀₀ ranged from 2.5 min to 1 hour depending on polymer concentration. The addition of DA significantly decreases the gelation time given that not only hydrazone bonding but also DA/DA interactions and oxidative self-polymerization are contributing to gelation. Due to these competing crosslinking mechanisms, the Young’s modulus and the shear storage modulus nearly doubled upon 10 mol% DA incorporation (to ~35 kPa in the 20 wt% hydrogels) but then decreasing slightly as the DA content was further increased (to 22 kPa for PO₁₀₀D₃₀ at 20 wt% polymer concentration); a similar trend was observed in the 10 wt% and 15 wt% POEGMA-DA hydrogels, although the base modulus value was lower. Based on swelling studies, the DA hydrogels reach maximum swelling within 24 hours and degrade slowly over 23 days, with faster degradation observed at lower polymer concentration. Cytotoxicity data demonstrated that >75% cell viability was maintained even at 2000 µg/mL precursor solution concentrations for C2C12 cells after 24 hours of exposure.

Incorporation of DA led to significant increases in the tissue adhesion as assessed using a pork skin lap shear test, with the 20 wt% PO₁₀₀D₃₀ hydrogels withstanding a maximum stress of ~1 kPa and the 15 wt% PO₁₀₀D₃₀ withstanding ~0.7 kPa of stress before delamination whereas the PO₁₀₀ hydrogels had functionally no adhesion with the tissue. Preliminary in vivo testing and histological analysis scores indicated a little to discrete presence of inflammatory cells at day 14 and regeneration of neoformed epidermis at the wound margins of the skin tissue for the 15 and 20 wt% PO₁₀₀ and PO₁₀₀D₃₀ hydrogels.

Conclusions: The bio-inspired hydrazone crosslinked POEGMA-DA hydrogels can mimic the adhesive properties of mussel adhesive proteins to enhance tissue adhesion while maintaining high cytocompatibility and promoting wound healing. Coupled with the avoidance of secondary procedures for removal of the adhesive and suppression of scarring achieved, these hydrogels offer potential for future use in bioadhesive would closure applications.

Acknowledgements: Funding from the Natural Sciences and Engineering Research Council of Canada Idea to Innovation grant program is gratefully acknowledged.

References:

(1) Zhou, F. et al. Bioinspired, injectable, tissue-adhesive and antibacterial hydrogel for multiple tissue regeneration by minimally invasive therapy. Applied Materials Today 2022, 26.

(2) van de Kuit et al. Surgical site infection after wound closure with staples versus sutures in elective knee and hip arthroplasty: a systematic review and meta-analysis. Arthroplasty 2022, 4 (1), 12.

(3) Brickman, K. R. et al. Evaluation of skin stapling for wound closure in the emergency department. Ann Emerg Med 1989, 18 (10), 1122-1125.

(4) Basha, S. L. et al. Randomized controlled trial of wound complication rates of subcuticular suture vs staples for skin closure at cesarean delivery. Am J Obstet Gynecol 2010, 203 (3), 285.e281-288.

(5) Mehdizadeh, M. et al. Design strategies and applications of tissue bioadhesives. Macromol Biosci 2013, 13 (3), 271-288.

(6) Duarte, A. P. et al. Surgical adhesives: Systematic review of the main types and development forecast. Prog Polym Sci 2012, 37 (8), 1031-1050. DOI: 10.1016/j.progpolymsci.2011.12.003.

(7) Spotnitz, W. D. Fibrin Sealant: The Only Approved Hemostat, Sealant, and Adhesive-a Laboratory and Clinical Perspective. ISRN Surg 2014, 2014, 203943.

(8) Spotnitz, W. D. Hemostats, Sealants, and Adhesives: A Practical Guide for the Surgeon. Am Surg 2012, 78.

(9) Bouten, P. J. M. et al. The chemistry of tissue adhesive materials. Prog Polym Sci 2014, 39 (7), 1375-1405.

(10) Barkan, Y. et al. Comparative evaluation of polycyanoacrylates. Acta Biomater 2017, 48, 390-400.

(11) Vinters, H. V. G. et al. The histotoxicity of cyanoacrylates. Neuroradiology 1985, 27.

(12) Kaushik, N. K. et al. Biomedical and Clinical Importance of Mussel-Inspired Polymers and Materials. Mar Drugs 2015, 13 (11), 6792-6817.

(13) Kord Forooshani, P et al. Recent approaches in designing bioadhesive materials inspired by mussel adhesive protein. J Polym Sci A Polym Chem 2017, 55 (1), 9-33.

(14) Sood, A. et al. Wound Dressings and Comparative Effectiveness Data. Adv Wound Care (New Rochelle) 2014, 3 (8), 511-529.

(15) Tran, N. Q. et al. In situ forming and rutin-releasing chitosan hydrogels as injectable dressings for dermal wound healing. Biomacromolecules 2011, 12 (8), 2872-2880.

(16) Wang, L. et al. A Novel Double‐Crosslinking‐Double‐Network Design for Injectable Hydrogels with Enhanced Tissue Adhesion and Antibacterial Capability for Wound Treatment. Adv Funct Mater 2019, 30 (1).

(17) Murphy, P. S. et al. Advances in wound healing: a review of current wound healing products. Plast Surg Int 2012, 2012, 190436.