Study Of Preparation And Properties Of Mussel Adhesive Proteins Bio-Inspired Medical Adhesive

In clinical surgery,hemostasis and wound closure are both essential core surgical techniques and issues that must be carefully considered.Traditional surgical sutures,staples and mechanical fixation have been used to deal with the wound closure.Years of clinical experience shows that these methods can solve most of the traumatic cases,but they also have disadvantages and shortcomings.Medical adhesive has the advantages of easy operation,non-invasive closure,no damage to the surrounding tissues during wound closure,reduction of pain,biodegradable,effectively preventing the leakage of air and body fluids,and so on,so it has catered to the concept of modern medicine and the requirement of surgery,the medical adhesive is regarded as an alternative to traditional wound closure.However,the moisture-resistant adhesive properties,Biosafety and therapeutic effects of current medical adhesive do not meet the requirements of clinical surgery.It has been found that mussel adhesive proteins have the properties of all-purpose adhesive that can be bonded in a variety of substrate surface,rapid curing ability,of high moisture resistance adhesive strength in seawater or humid environment.It provides a possibility and way for developing an ideal medical adhesive.In this paper,we used the biocompatible and biodegradable chitosan and polyethylene glycol as medical adhesive starting materials,and combined with the development and requirements of bioadhesive and mussel adhesive protein,we have prepared mussel inspired hydrogel adhesives by polymer biomimetic method,so that the hydrogel adhesives has the characteristics of mussel adhesive protein.Photocrosslinking technology and functionalization group reaction method was chose to enable the rapid cross-linking of mussel inspired adhesive.The gelation time,swelling,in vitro degradability,mechanical strength and cell compatibility of the hydrogel adhesive were tested and evaluated.Furthermore,since the hydrogel as a glue layer,the mechanical strength plays an important role in the adhesive strength,so we designed a double cross-linked mussel inspired hydrogel to study the effects of the mechanical strength to the adhesive properties.Biodegradability and biocompatibility of the double cross-linked hydrogels in vivo were also investigated.First,thiolated chitosan?CSS?and polyethylene glycol methacrylate?PEGDMA?were successfully synthesized by us.The prepolymer solutions were cross-linked by UV irradiation through Michael addition reaction and then the hydrogel was formed rapidly.Fourier transform infrared spectroscopy?FTIR?and proton nuclear magnetic resonance spectroscopy?¹HNMR?were used to confirm the molecular structure of CSS and PEGDMA,the substitution degree of PEGDMA was 87%according to ¹HNMR,and the content of thiol group?250?mol/g?in CSS was determined by UV-Vis absorption spectrometry.The gelation time,swelling,degradation,morphology,mechanical strength and adhesive strength of the CSS/PEGDMA hydrogels were characterized.Results showed that the gelation time,swelling and degradation behavior of CSS/PEGDMA hydrogels were controlled by the ratio of the components.Adhesive strength of the hydrogel adhesive was increased with the concentration of the prepolymer solution increasing,especially the thiol group in the thiolated chitosan played an important role.Compression test results showed that CSS/PEGDMA hydrogel has good mechanical properties,and the three-dimensional network porous structure was observed in the hydrogel by scanning electron microscopy?SEM?,which is conducive to cell adhesion and growth on the material.Furthermore,the mouse fibroblast cells?L929 cells?are cultured at hydrogel extract to evaluate the biocompatibility of the hydrogel,and MTT test results showed that the photocured hydrogel has excellent cytocompatibility.Based on the previous study of photocrosslinked CSS/PEGDMA hydrogel adhesives,next,a photocured mussel inspired terpolymer hydrogel adhesive?CPD?,which containing CSS,polyethylene glycol acrylate?PEGDAA?and mussel mimetic moieties?Dopa-AA?,was prepared.Three components of the terpolymer adhesive were synthesized and then characterized by FTIR and ¹HNMR.The substitution degree of PEGDAA?85%?and the content thiol group?350?mol/g?in CSS were measured.Hydrogels with different Dopa-AA contents was prepared,and the gelation time,equilibrium water content,degradability and morphological changes of the CPD hydrogels were characterized.Results showed that the gelation time of hydrogels was greatly affected by the inhibitory effects of catechol group to the free radical polymerization,so that it was increased with contents of Dopa-AA increasing.Due to the physical and chemical interactions of catechol group generated during the degradation of CPD hydrogels,it was degraded more slowly with catechol content increasing.The mechanical strength and adhesive strength of the prepared hydrogels were also measured,and the results showed that CPD hydogel possessed good mechanical properties and it may be cross-linked for the second time and the highest adhesive of CPD adhesive can reach up to 1.0 MPa.The viability of L929 cell that cultured in CPD hydrogel extract was evaluated by MTT assay,it showed the adhesives had low cytotoxicity due to the generation of reactive oxygen spices that inhibited the cell viability during the catechol groups oxidized.Then,due to the limitations of photocrosslinking technology in clinical application,we designed an in situ injectable cross-linked mussel inspired chitosan based hydrogel adhesive,the mussel inspired chitosan was a chitosan derivative?CSDS?that functionalized with thiol group and catechol groups.CSDS precursor solution would cross-link quickly to form hydrogels upon the addition of sodium periodate,meanwhile to bind the wounds.CSDS was prepared by carbodiimidyl method and characterized by FTIR and ¹HNMR.The content of thiol group and catechol group in CSDS was 400?mol/g and 75?mol/g respectively,determined by UV-Vis absorption spectroscopy.CSDS hydrogels was prepared with sodium periodate and CSDS at different molar ratios.The gelation time,equilibrium water content,degradation performance,morphology,mechanical properties and adhesive properties were investigated.Additionally,cytocompatibility of CSDS hydrogels was evaluated using both MTT assay and live/dead cell staining assay.It showed that CSDS hydrogels was cross-linked rapidly within 1 minute,and the equilibrium water content was more than 90%.It was a soft polymeric adhesive material with porous structure.The adhesive testing showed CSDS hydrogels have high adhesive strength to the porcine skin,and cytocompatibility experiments indicated that CSDS hydrogels was biocompatible.Therefore CSDS hydrogels have the potential application for soft tissue wound closure and repair.Finally,because the mussel inspired polymer would be cross-linked quickly to form hydrogel by the addition of sodium periodate or Fe³⁺,and the mechanical properties of hydrogel play an important role when act as the glue layer.We therefore designed a double cross-linked tissue adhesives containing mussel inspired chitosan CSDS and 4-arm polyethylene glycol succinimide carbonate PEG-4S.For the double cross-linked hydrogels,PEG-4S was act as the slow cross-linker,and sodium periodate and Fe³⁺was act as the fast cross-linker,respectively.PEG-4S was synthesized and then characterized by FTIR and ¹HNMR.The substitution degree of PEG-4S was 75%calculated by the ¹HNMR,and the content of thiol group and catechol groups in CSDS was 400?mol/g and 100?mol/g respectively,determined by UV-Vis spectra.It showed that the double cross-linked hydrogel can be rapidly cured to form.As the average cross-linking density of the hydrogels increased,the equilibrium water content and the degradation rate of the hydrogel were decreased,while the mechanical strength increased,and the adhesive strength of the hydrogel has been improved.MTT assay and CCK8 assay were used to determine the viability of L929 cells and endothelial cells that cultured in the hydrogel extractions,respectively.The results showed that the double cross-linked hydrogels has good cytocompatibility.In addition,double-cross-linked hydrogel can be degraded in vivo by subcutaneous implantation in mice model,though the double-cross-linked hydrogel caused minor acute inflammation by Histological analysis,it is an in vivo biocompatibility hydrogel adhesive.In summary,since the chemical bonding mechanism was the mainly adhesive mechanism for the medical adhesive.To enhance the adhesive strength,reactive groups,such as thiol group and catechol group,were introduced to the prepolymers.In this paper,we designed and prepared mussel inspired hydrogel adhesives based on chitosan and polyethylene glycol.Mussel inspired hydrogel adhesive was cross-linked rapidly by using photocrosslinking technology and chemical functional group reaction.The prepared mussel inspired hydrogels exhibit low cytotoxicity due to the oxidation of catechol groups,but the presence of catechol groups enhanced the hydrogel adhesion,meanwhile increased the mechanical strength of the hydrogel also contributes to promote adhesive strength.Our paper provides a new idea for the study and development of hydrogel adhesives with high moisture resistance and biocompatibility.It is also of great significance to the research of high adhesion and mechanical strength hydrogel adhesives.