Preparation Of Degradable Peptide-based Tissue Adhesive And Its Application In Dural Defect

Dural defects are common in spinal surgery and neurosurgery.There are many causes for dural damage,which can be broadly divided into two categories:intentional reasons and accidents.Intentional dural defects are mainly based on therapeutic needs,such as diagnostic lumbar puncture,therapeutic puncture(anesthesia,labor analgesia,etc.),removal of intradural(including intramedullary)tumors or cysts,selective shunt,etc.Most dural defects are caused by accidents:such as trauma(bone mass pierces the dura mater when spinal fracture),surgical complications(inadvertent tear of the dura mater during laminectomy).Cerebrospinal fluid leakage after dural rupture causes a series of complications.To reducing and avoiding complications,it is necessary to actively develop treatment strategies.For dural defects,the current clinical treatment options include suture with surgical suture,fixation with non-penetrating titanium clip,plugging and covering with biomaterials,and some other adjuvant treatment strategies.However,according to the clinical follow-up results and literature reports,the effects of the current treatment strategies are not ideal.Therefore,according to the current literature results,it is believed that watertight closure is the basis for the treatment of dural defects,prevention of cerebrospinal fluid leakage and reduction of related complications.Due to the uncertainty of the location,size and shape of dural defect in clinic,tissue adhesives have natural advantages in this application scenario.They are in liquid or semi-liquid state before use,and can be well spread above the damaged area.At present,the adhesive used in clinics can not achieve the ideal sealing effect of dural defects,which is mainly due to the material itself:weak mechanical properties of the material,weak adhesion with the interface,unreasonable gelation time,excessive swelling of materials,mismatch of elastic modulus of the material,toxicity of the material or degradation products,etc.Therefore,the successful preparation of a safe,effective and reasonable adhesive is an effective strategy to block dural defect.In this study,we prepared two kinds of adhesives based on the chemical crosslinking of aldehyde amine condensation and supramolecular force,which are the crosslinking system of O-phthalic aldehyde(OPA)terminated four arm polyethylene glycol and gelatin(4a PEG-OPA/Gelatin)and the crosslinking system of glycyrrhizic acid(GA)and short peptide(Pep/GA).The relevant specific research contents and main conclusions are as follows.(1)Based on the strategy that phthalaldehyde can rapidly crosslink with amine groups,we prepared 4a PEG-OPA/Gelatin hydrogel adhesive.Firstly,4a PEG-OPA was successfully prepared by modifying 4a PEG and capping with OPA.Then the solutions of 4a PEG-OPA and gelatin are mixed and the concentrations of both components used were 10%(w/v).After successful preparation of the hydrogel,we characterized the 4a PEG-OPA/Gelatin and evaluated its biological properties.The results showed that 10%(w/v)of 4a PEG-OPA could form a very stable hydrogel with gelatin.The gelation time was about 2-3 min(room temperature)and 50 s(37?).This time window meets the clinical needs and the operation time of doctors.Rheological tests show that the material has good viscoelasticity.The mechanical properties and fatigue resistance of 4a PEG-OPA/Gelatin hydrogel were measured by the universal mechanical testing machine.The results showed that the compression modulus could reach 111.98±17.15 k Pa,the tensile modulus could reach 38.54±5.02k Pa,and the lap shear adhesion strength was 79.90±11.95 k Pa,which confirming that 4a PEG-OPA/Gelatin hydrogel had good mechanical properties,adhesion strength and excellent fatigue resistance.Based on the above results,we then preliminarily evaluate the effectiveness of the material in blocking the dura mater.The bursting pressure in vitro and in vivo were measured.The results showed that the bursting strength of 4a PEG-OPA/Gelatin gradually increased with time.Results in vitro show that at 5 min,the bursting pressure was 44.28±10.49 cm H₂O;After 30 min,the burst pressure reached 208.0±37.99 cm H₂O.At the same time,we tested the burst pressure in vivo.The results showed that the burst pressure of 4a PEG-OPA/Gelatin was 68.94±10.37 cm H₂O at 5 min,which was enough to resist the pressure fluctuation of cerebrospinal fluid under physiological conditions.Then,we carried out the verification of the biological properties of the materials.Before the animal experiments,the biocompatibility tests of the materials were performed,including cytotoxicity test,live-dead staining,hemolysis experiments,blood test and histological analysis,which confirmed that the prepared 4a PEG-OPA/Gelatin had good biocompatibility at the cellular and histological levels.As a material used in spinal canal and cranial cavity,its degradation and swelling are not negligible.The results confirm that the material degraded slowly in vivo.The swelling rate test in vitro confirmed that 4a PEG-OPA/Gelatin had a low swelling rate.After confirming the safety of the material,we used the dural defect models to verify the blocking and repair efficiency of 4a PEG-OPA/Gelatin for dural defects.The incision appearance,magnetic resonance imaging(MRI)of head,cerebrospinal fluid biochemistry and histological analysis confirmed that 4a PEG-OPA/Gelatin could effectively block the dural defects and promote the repairing of dural.Through lumbar laminectomy and dural damage disease model,we explore whether 4a PEG-OPA/Gelatin could prevent and treat epidural fibrosis.The results of MRI,inflammatory factor detection and histological analysis showed that 4a PEG-OPA/Gelatin could act as a physical barrier to separate the dural sac and scar,reducing nerve adhesions.In conclusion,the novel 4a PEG-OPA/Gelatin hydrogel adhesive prepared by this study has good mechanical properties,which can effectively seal the dura mater and avoid CSF leakage.At the same time,it can act as a barrier,reducing or avoding epidural fibrosis.(2)Based on supramolecular interactions,we designed a novel underwater adhesive Pep/GA,which was prepared by the interaction of glycyrrhizic acid and short peptide.The concentration of short peptide was 20 wt%and the molar ratio of glycyrrhizic acid to Pep was 1:1.After mixing and adjusting the p H value of the solution to be neutral,the guanidinium group of Pep is protonated and glycyrrhizic acid is deprotonated,both are electrostatic adsorbed and hydrogen-bonded to form a gel.According to the different groups of the designed short peptide structural formula,we prepared three different short peptides:Pep1,Pep2,and Pep3.In additon,the prepared adhesive can be prepared into powder adhesive after freeze-drying and grinding.The successfully prepared powder adhesive becomes sticky and restores its own adhesion characteristics immediately after encountering water.In the follow-up study,we showed the related characterization and biological properties of Pep1/GA underwater adhesive.Scanning electron microscopy(SEM)shows that Pep1/GA underwater adhesive is interconnected by dense stacked sheet structure,which is beneficial for dural plugging.Rheological test results confirmed that Pep1/GA underwater adhesive had self-repairing,injectability and good viscoelasticity.Compared with room temperature,the crosslinking density of the adhesive decreased at 37°C,and the storage modulus also decreased(the strength decreased from 1 MPa to 0.1 MPa),indicating that even with reduced crosslink density,the cohesion of the gel is sufficient to resist CSF pressure.At the same time,the decrease of crosslinking density enhances the fluidity of the adhesive,which is more conducive to the spreading on the dural surface.The adhesive strength of Pep1/GA was tested by universal testing machine.The results showed that the surface Pep1/GA underwater adhesive had good adhesion to different substrates(inorganic materials,organic materials and biological tissues)under water.At the same time,we measured that the adhesion of Pep1/GA underwater adhesive to pig skin in a37°C water tank up to 18.8±1.4 k Pa,which was higher than that of fibrin adhesive.We also measured the burst pressure of Pep1/GA adhesive in vitro and in vivo.The results were 66.9±6.3 cm H₂O and 59.3±6.1 cm H₂O respectively,which were enough to resist the fluctuation of cerebrospinal fluid pressure.At the same time,the prepared dry powder adhesive could quickly absorb water and form gel to block the damaged dura mater under the condition of continuous exudation of cerebrospinal fluid.The biological safety of the material was evaluated by cytotoxicity test,live-dead staining,hemolysis test,blood test and histological analysis.The results showed that Pep1/GA material had good biocompatibility.The degradation time of Pep1/GA adhesive is directly related to body fluid exchange.In this study,the degradation performance of Pep1/GA was evaluated by in vitro experiment and subcutaneous implantation.The degradation time is 1-2 weeks.Its degradation is matched with dural repair to avoid physical barrier affecting the repair process of dural.At the same time,the material has little swelling,which is beneficial for use in the spinal canal and intracranial space.By replicating the dural defect model to evaluate the adhesion efficiency of Pep1/GA underwater adhesive,the results of incision appearance,MRI and tissue analysis fully confirmed that the new Pep1/GA adhesive prepared in this study can effectively block the dural defect,prevent cerebrospinal fluid leakage and promote dural repair.