| Postoperative epidural fibrosis and adhesion remains a clinically challenging problem in spine surgery.Unfortunately,currently there are no effective and safe anti-fibrotic and anti-adhesion biomaterials that have been specifically developed for this complication in clinical practice.Therefore,novel biomaterials based on rational design are urgently required for effective and safe treatment of epidural fibrosis.Pathologically,epidural adhesion develops from scar tissue,in which fibroblasts play an important role.Surgery-induced inflammation and hematoma can promote adhesion by affecting fibroblasts.At laminectomy sites,a large number of fibroblasts propagate and produce collagen fibers to repair local vertebral defects after activation by inflammatory cytokines and growth factors,such as tumor necrosis factor(TNF)-?,interleukin(IL)-1,IL-6,and transforming growth factor(TGF)-?.Meanwhile,these molecular mediators are up-regulated by inflammatory cell infiltration related to oxidative stress via increasing vascular permeability to improve fibroblast proliferation,extracellular matrix synthesis,and eventual scar formation.In addition,oxidative stress-mediated release of reactive oxygen species(ROS)can induce cell injury,promote generation of inflammatory mediators,and exacerbate chronic inflammation.Consequently,combination of anti-fibrosis,anti-inflammation,and antioxidative stress should be promising strategy to prevent postoperative epidural adhesion.Herein we designed and engineered an advanced hydrogel with multiple functionalities,including temperature-responsive gelation,self-healing,tissue adhesiveness,anti-oxidation,anti-inflammation,and anti-fibrosis.This multifunctional supramolecular hydrogel can be facilely constructed by integrating three functional modules,i.e.,a thermosensitive triblock copolymer poloxamer 407(PX),a broad reactive oxygen species(ROS)-eliminating and anti-inflammatory nanotherapy(TPCD NP),and an adhesion-enhancing compound tannic acid(TA).Therapeutically,local treatment with PXNT hydrogel effectively prevented epidural fibrosis and adhesion after laminectomy in both rats and rabbits.Of note,PXNT hydrogel showed more beneficial efficacy than control thermosensitive hydrogels and a commercial barrier Interceed.Mechanistically,PXNT hydrogel can significantly attenuate local oxidative stress,inhibit inflammatory responses,and reduced fibrotic tissue formation.Moreover,treatment with PXNT hydrogel did not cause systemic adverse effects and neurological symptoms.Methods1.Clinical outcomes of MIS-TLIF via a tube and postoperative epidural fibrosis evaluationThe perioperative parameters and treatment efficacy of ZISTA-TLIF and Open TLIF were compared retrospectively.MRI was used to evaluate the fatty infiltration of paravertebral muscles and the postoperative epidural fibrosis.2.Synthesis and characterization of TPCD NPTpl and CDI were dissolved in anhydrous DCM.The obtained mixture was stirred at room temperature for 45 min to obtain CDI-activated Tpl.Then,?-CD and DMAP were added to DMSO solution containing CDI-activated Tpl and stirred at room temperature for 24 h.Thus obtained product(i.e.,TCD)was precipitated from a mixture solution of diethyl ether and methanol.In addition,PBAP and CDI were reacted in 30 mL of DCM to give rise to CDI-activated PBAP.Subsequently,CDI-activated PBAP,DMAP,and TCD were dissolved in 40 m L of anhydrous DMSO.After reaction at room temperature for 48 h,the final product TPCD was collected by centrifugation and lyophilization.TPCD nanoparticles(abbreviated as TPCD NP)were also prepared using following methods.First,lecithin and DSPE-PEG were dissolved in 7.5mL of deionized water at 65°C to obtain an aqueous phase.Then,25 mg TPCD dissolved in 5mL of methanol was added to the aqueous phase at room temperature.Methanol was removed under reduced pressure to obtain TPCD NP.TPCD NPs labeled with either Cy5 or Cy7.5 were fabricated through similar procedures.Finally,Transmission electron microscopy(TEM)and scanning electron microscopy(SEM)images were obtained.The size of TPCD NP were measured with a Malvern zetasizer instrument.3.Formulation screening for engineering a bio-adhesive,anti-inflammatory,and thermosensitive hydrogelPoloxamer 407(PX)solutions varying from 16,18,to 20 wt% were first examined.At these concentrations,rapid gelation was observed at 37°C.Then TPCD NP was added into PX solution.For different groups,PX concentrations varied from 16,18,to 20 wt%(defined as PX16,PX18,and PX20,respectively),while TPCD NP changed from 0.2,1,to 5 mg/m L(defined as NP0.2,NP1,and NP5,respectively).Subsequently,the effects of TPCD NP incorporation on biological functions were examined after local administration in rats subjected to lumbar laminectomy.Meanwhile,sol-gel transition capacity of different aqueous solutions also needs to be determined.Based on the above results,appropriate concentrations of TPCD NP were used for further screening experiments.Next,TA was introduced into formulations to afford bio-adhesive capability.Bio-adhesive test was performed both on a tilted plane test platform and a test bench material testing machine.In the end,the hydrogel with the optimal ratio was screened out.4.Characterization of PXNT hydrogelDetailed characterization was then conducted for the finally screened hydrogel,which is abbreviated as PXNT.(a)The test-tube inversion method was employed to determine the gelation performance of different solutions.(b)Fluorescence image of PXNT hydrogel containing FITC-labeled-PX and Cy5-labeled TPCD NP was obtained by confocal microscopy observation.(c)After different solution samples containing different materials were lyophilized,their Fourier transform infrared(FT-IR)spectra were recorded on a PerkinElmer FT-IR spectrometer.(d)For observation by scanning electron microscopy(SEM),different hydrogel samples were first lyophilized,and then they were fractured to show the internal structures.After coating with gold,morphology observation was performed by SEM.(e)Rheological experiments were performed for different aqueous solutions containing PX and other materials on a rheometer using a parallel plate(plate diameter,40 mm;gap,0.05 mm).The storage modulus(G')and loss modulus(G'')of different combinations were under different conditions.For the temperature dependent experiments,the heating rate was set at 2.0°C/min.The sol-gel transition temperature was determined as the intersection point of G' and G''.On the other hand,oscillation strain-dependent changes in G' and G'' were measured with the shear strain varying from 0.1% to 1000% at 37°C.The oscillation strain corresponding to the sol-gel transition was defined as the intersection point of G' and G''.Also,time dependent changes in G? and G? values were measured as a function of time at 37°C,with a strain rate of 2%.Frequency-dependent rheological measurements were conducted at 37°C,with a strain rate of 2%.Step-strain measurements were performed at high(200%)and low(2%)strains at 37°C,with an angular frequency of 10?rad?s-1.Shear-dependent changes in viscosity and step-shear measurements were carried out at 37°C.(f)For hydrolysis tests,2 mL of aqueous solutions containing either PXN or PXNT was introduced into glass vials that were incubated at 37°C for 10 min.Then 3 m L of 0.9% saline that was preheated to 37°C was added.At predetermined time points,the supernatant was removed.The weight of remaining gel was measured,followed by addition of fresh medium to the initial volume.These procedures were repeated until the hydrogels were completely hydrolyzed.(g)In vivo retention of thermosensitive hydrogels.Aqueous solutions containing PXN or PXNT,in which TPCD NP was labeled with Cy7.5,were injected into deep sacrospinalis with a volume of 0.5 mL.At predetermined time points,all animals were subjected to in vivo imaging with a spectrum living imaging system.The fluorescence intensity was analyzed by the Living Imaging software.(h)For direct assessment of TPCD NP released from PXNT hydrogel,Cy5-labeled TPCD NP was first dispersed in PXT solution with a final concentration of 0.5 mg/m L.Then 3 m L of saline was added to hydrogel gelatinized from 2 ml aqueous solution of PXNT at 37°C.The supernatant was replaced every 24 h.The fluorescence intensity of supernatant was measured on a fluorescence spectrometer.The excitation and emission wavelengths were set at 646 and 820 nm,respectively.The cumulative release percentage of TPCD NP was calculated and plotted against time.In addition,the size and size distribution profile of released TPCD NP in the supernatant were measured using a Malvern Zetasizer NanoZS instrument at 25°C.5.In vivo prevention of epidural adhesion after lumbar laminectomy(a)In vivo prevention of epidural fibrosis by PXNT was examined in rats.In this case,we used control thermosensitive hydrogels based on a triblock copolymer of PLGA-PEG-PLGA(defined as PL)or PL plus TPCD NP.Post laminectomy at the L4 lamina of rats,different formulations(PXNT,PXN,PXT,PLN,PL and PX)were directly applied on the dura mater.Eight weeks after treatment,magnetic resonance(MR)imaging was used to evaluate post-operative epidural fibrosis after intravenous injection of a contrast agent gadolinium-DTPA.Then,the experimental animals were euthanized,and the surgery sites were probed again for gross observation grading score.In addition,histological analyses of hematoxylin and eosin(H&E)and Masson's Trichrome stained section were conducted.(b)In a separate study,we compared the anti-adhesion effect of PXNT hydrogel with a commercially available product,i.e.,Interceed.MR imaging,macroscopic observation,and H&E or Masson staining was performed.(c)In vivo prevention of epidural adhesion after lumbar laminectomy in rabbits.MR imaging and macroscopic observation was performed.6.Mechanistic studies(a)To quantify the neutrophil counts and the levels of myeloperoxidase(MPO),hydrogen peroxide(H2O2),tumor necrosis factor(TNF)-?,interleukin(IL)-1?,IL-6,and transforming growth factor(TGF)-?1,a sterile plastic drainage tube was placed in the surgical area and fixed securely.The animals were subjected to restrictions on the movement in the next 7 days to prevent the tube from pulling out.The surgical region and drainage tubes were disinfected daily.At defined time points,PBS was injected into the wound via the drainage tube,and the lavage was collected immediately.Neutrophil cells were stained with APC-conjugated rat anti-rat CD11b/c and FITC-conjugated rat anti-rat HIS-48 for analysis by flow cytometry.The levels of TNF-?,IL-1?,IL-6,TGF-?1,MPO,and H2O2 in the supernatant were assessed by ELISA.(b)Immunofluorescence staining was used to detect the expression of CD31 and CD68 in epidural fibrosis tissues to examine neovascularization and macrophages,respectively.The sections were imaged by confocal laser scanning microscopy.For quantification,normalization by the tissue area from each specimen(n = 10)was used to determine the CD31+(vessels)and CD68+(macrophages)areas in each field.7.Safety studies(a)In vivo safety of PXNT.MTT method was used to determine the cytotoxicity of hydrogels with different formulations.After treatment with different formulations as aforementioned,rats in the model and PXNT groups were enrolled.At month 1,animals were euthanized,and blood samples were collected for biochemical analyses.The levels of TNF-?,IL-1?,and IL-6 in the blood serum were quantified by ELISA assay.In addition,major organs including heart,liver,spleen,lung,and kidney were isolated and histological sections were prepared and stained with hematoxylin and eosin(H&E).(b)Neural function safety of PXNT.Data of Basso,Beattie,and Bresnahan(BBB)grading scales were collected at week 8 after different treatments for nerve function assessment.Examination on myelin sheath morphology was performed at week 8 after different treatments.The myelin sheath of spinal nerves were examined via TEM.Motor evoked potentials(MEPs)were measured to examine the spinal motor conductions on a Nerve Monitoring System.The amplitude and latency data were collected pre-operation,4 weeks post-operation,and 8 weeks post-operation in the PXNT group.Results1.Clinical outcomes of MIS-TLIF via a tube and postoperative epidural fibrosis evaluationAt the last follow-up,ZISTA-TLIF group obtained the same efficacy as the Open-TLIF group,while slowing the degeneration of paraspinal muscles.There was no significant difference in the scores of epidural fibrosis adhesion between the two groups.2.Synthesis and characterization of TPCD NPThe anti-inflammatory and antioxidative stress nanoparticles were prepared using a broad-spectrum ROS-eliminating material(TPCD)derived from ?-cyclodextrin,giving rise to spherical nanoparticles(TPCD NP)with well-defined shape and relative narrow size distribution.3.Formulation screening for engineering a bio-adhesive,anti-inflammatory,and thermosensitive hydrogelCompared with the saline-treated model group,TPCD NP at 1 or 5 mg/m L significantly attenuated oxidative stress and inflammation,as implicated by remarkably decreased levels of peroxide hydrogen(H2O2)and IL-6 in fluid wound lavage,independent of PX concentrations.TPCD NP at 0.2 mg/mL,however,showed no significant effects.Furthermore,significant reduction of TGF-?1 was only found for the formulations with 18 wt% PX and 1 mg/mL TPCD NP(i.e.,PX18-NP1).Of note,abnormally elevated expressions of IL-6 and TGF-?1 are responsible for surgery-induced epidural fibrosis.Nevertheless,gelation occurred for PX solutions containing 5 mg/m L TPCD NP even at room temperature(20-25°C),regardless of PX concentrations examined(Figure S1),which is undesirable for injection due to high viscosity post gelation.For PX18-NP1 hydrogels,dura adhesion considerably increased when the TA content varied from 0,0.5,1,to 2 wt%(Figure 2h),which was independent of PX concentrations(Figure 2i).Further increase in TA(such as 3 wt% TA),however,led to formulations that cannot form hydrogel at 37°C(Figure S2).Furthermore,bioadhesive capacity of hydrogels was directly measured using rat muscles(Figure 2j).We observed notably increased adhesive force between two muscle tissues bound together with hydrogels derived from 18% PX and 1 mg/m L TPCD NP,when TA changed from 0.5 to 2 wt%(Figure 2k).Collectively,the finally screened hydrogel derived from 18 wt% PX,1 mg/mL TPCD NP,and 2 wt% TA,which is abbreviated as PXNT.4.Characterization of PXNT hydrogel(a)PXNT solution is a free-flowing transparent liquid at room temperature,clear sol-gel transition and hydrogel formation were observed at 37°C.(b)Confocal microscopy observation showed homogenous distribution of Cy5-labeled TPCD NP in FITC-labeled hydrogel.(c)The presence of different components was also confirmed by FT-IR spectrometry.(d)Scanning electron microscopy(SEM)observation of the fracture surface of lyophilized hydrogel revealed a porous network structure.Embedded nanoparticles were clearly found on the cross-section and sample surface.(e)Both storage modulus(G?)and loss modulus(G?)increased with temperature(Figure 3g).Below a specific temperature,G?? was remarkably larger than G?,indicating the liquid-state behavior in these cases.By contrast,G? > G?? was detected upon further increasing temperature,which is a typical characteristic of the solid-like behavior.Thus the sol-gel transition temperature,defined as the intersection point of G?/G?? curves,was determined to be 26.9°C.For aqueous solutions of 18 wt% PX,18 wt% PX plus 1 mg/m L TPCD NP(PXN),or 18 wt% PX plus 2 wt% TA(PXT),similar temperature-dependent rheological behaviors were observed(Figure S5),with the gelation temperature varied from 23.3,23.9,to 25.6°C.At 37°C,the sol-gel transition of PXNT occurred within 15 s(Figure 3h).Further angular frequent-dependent experiments revealed a nearly linear response for both G? and G?? within the examined viscoelastic region(Figure 3i).The ratio of G?? and G?(i.e.,tan?)was calculated to be varied from 0.48 to 0.37 for PXNT as the frequency changed from 0.1 to 100 rad s-1---(Figure S6),indicating typical elastic behavior under these conditions.Oscillatory rheology indicated PXNT hydrogel formed at 37°C was stable when strains varied from 1% to 60%,while it became liquid-like with additional increase in strains(Figure 3j),implying network rupture at high strains.In addition,step-strain experiments were conducted to characterize recovery performance of the dynamic PXNT hydrogel at 37°C,in which low strain was followed by high strain(Figure 3k).At a high strain of 200%,fluid-like behavior was observed for PXNT,while it rapidly recovered within 20 s when a low strain(2%)was applied.Notably,this recovery profile was repeatable.Additional steady shear measurement demonstrated shear-shinning behavior of PXNT hydrogel(Figure 3l),as implicated by gradually reduced viscosity when the shear rate extended from 0.1 to 1000 s-1.Also,step-shear experiments showed remarkable shear-shinning at high shear rates,while rapid recovery occurred at low shear rates(Figure 3m).(f)In vitro hydrolysis behaviors of PXNT were tested in 0.9% saline at 37°C.We found gradual erosion of PXNT hydrogel,with complete hydrolysis at day 7.In the absence of TA,PXN hydrogel was completely hydrolyzed within 3 days.(g)The hydrolysis result is consistent with the cumulative release profiles of hydrogels,in which sustained and slowed release of TPCD NP was also observed for PXNT hydrogel.Nanoparticles released from PXNT hydrogel exhibited a size distribution profile very similar to that of freshly prepared TPCD NP.(h)After local injection of aqueous solution of PXNT containing Cy7.5-labeled TPCD NP into deep sacrospinalis in rats,relatively long retention of Cy7.5 fluorescence was observed,and fluorescence remained at day 7.By contrast,PXN hydrogel only maintained for approximately 3 days in vivo.In addition,significantly stronger fluorescence signals were found for PXNT hydrogel at different time points examined,as compared to those of PXN hydrogel.5.In vivo prevention of epidural adhesion after lumbar laminectomy(a)Eight weeks after treatment,gross observation indicated that the dura mater around the laminectomy site was oppressed by severe epidural hardened scars in rats treated with PX,PL,or PLN,and it was difficult to dissect them.In the PXN group,limited scar tissue was formed,but the epidural adhesion was not severe.By contrast,the PXNT group displayed only soft or weak fibrosis tissue around the dura mater,with slight epidural adhesion that could be easily dissected by manual traction without injury to the dura mater.In addition,the dura surface was smooth after the adhesion was removed and the epidural fibrosis score was only 1.2 ± 0.4,the lowest one among all groups.For rats treated with PX,PXT,PL,or PLN,their spinal cords were connected with enhanced low signals overlying tissues without clear delineation in either axial or sagittal MR images at 8 weeks post-operation.Unexpectedly,both PL and PLN groups even regenerated more scar tissues around the dural sac than the model group.In the PXN group,incomplete peridural scar tissue was observed at the laminectomy site.As for the PXNT group,there was only a thin peripheral rim of enhancement detected around the dural sac,suggestive of scar granulation tissue but no direct adhesion on the dura.Consistently,quantitative analysis of T1-weighted axial MR images revealed the lowest EF area in the PXNT group.Similar results were found for MR imaging-based grading scores.At week 8,the outside dural canal was filled with a number of collagen and fibroblasts in the model group.Histological assessment scores revealed severe adhesion in untreated rats,and comparable results were found in animals treated with PX,PL,or PLN.The PL and PLN groups even exhibited more abundant fibroblasts than other groups.Whereas treatment with PXN and PXT resulted in less fibroblasts in epidural scar tissues,no significance was found compared to the model group.By contrast,the EF score of PXNT-treated rats significantly decreased when compared with other groups,indicating significantly less epidural adhesion in this group.Further,Masson staining revealed serried distribution of dense collagenous tissues around the dura mater in the model group.For rats treated with PXN,dura was slightly attached with the fibrous tissue,and some fibroblasts and sparse collagen fibers were visualized.In the case of the PXNT group,there was a clear interspace between the dura mater and scars,showing slight epidural scar.The lowest fibrotic area was detected for PXNT-treated rats.By contrast,both PL and PLN groups displayed higher fibrotic area than the model group,with significant difference between PL and model groups.(b)In a separate study,we compared the anti-adhesion effect of PXNT hydrogel with a commercially available product,i.e.,Interceed.According to results based on macroscopic observation,MR imaging,and H&E or Masson staining,PXNT-treated rats showed significantly less fibroblast infiltration and fibrotic tissue formation,as compared to those treated with Interceed.Although the Interceed group displayed a blurry gap between dura and fibrotic tissue in histological sections,the gap in sections corresponding to PXNT-treated animals was definitely more explicit.Consequently,our results implied that Interceed had very limited anti-adhesion capacity in the rat model of post-laminectomy epidural fibrosis.(c)In vivo efficacy of PXNT in a rabbit model.Three weeks postsurgery,re-probing the laminectomy site suggested that the PXNT-treated dura mater was easy to expose because of a lack of scar tissue.A thin unmatured fibrotic tissue surrounding the dura mater could be easily removed.In the model group,however,some compact scars were tightly adhered to the dura,which were difficult to separate.Moreover,a separation procedure led to dural sac tears.Macroscopic assessment revealed significantly decreased EF scores in the PXNT group,as compared to the model group.In addition,MR images revealed a region of low signals(scar tissues)at the laminectomy site in both T1-and T2-weighted axial images in the model group,which were remarkably reduced after PXNT treatment.From sagittal images,the spinal cord in the PXNT group exhibited a regular pattern and a linear homogeneous signal separating the dura from the posterior tissue.In the model group,however,the posterior scar tissue was adhered to the dura and invaded the margin of the spinal canal.Generally,PXNT treatment afforded desirable therapeutic outcomes.6.Mechanistic studiesSubsequently,mechanisms responsible for the desirable anti-fibrosis and anti-adhesion effects of PXNT were examined in rats.During the first 7 days,overall low levels of neutrophils and myeloperoxidase(MPO)in wound lavage fluid were detected for PXNT and PXN groups,particularly the PXNT group at day 7(Figure S12a-f).The expression levels of TNF-?,IL-1?,and IL-6 were also significantly reduced after treatment with nanotherapy-containing PX hydrogels.In addition,nanotherapy-containing formulations displayed remarkably decreased levels of H2O2,a typical and major component of ROS.Of note,PXNT resulted in significantly lower H2O2 levels after treatment for long periods of time,compared to the model group.These results substantiated that local treatment with PXN and PXNT effectively attenuated surgery-induced inflammatory responses and oxidative stress.Treatment with other control formulations,however,showed no or limited beneficial effects on reducing inflammatory cells/cytokines and oxidative mediators.Additionally aggravated inflammatory reactions were even observed for the PL group.For the undesirable effect of PL-based hydrogels,this may be attributed to prolonged exposure of PL near the surgery sites,which can maintain and exacerbate local inflammation by sustainably stimulating peripheral tissues as a foreign body throughout the entire healing process.Furthermore,the PLGA component in PL can be degraded into lactic acid and glycolic acid,thereby causing local inflammation.Actually,different anti-inflammatory drugs have been used to reduce PLGA-induced inflammatory responses and foreign body reactions for tissue generation.As for TA-containing PXT hydrogel,despite its antioxidant capability to a certain degree,the anti-inflammatory activity was extremely limited in this post-laminectomy rat model.We also quantified the expression of TGF-?1(a cytokine manly excreted by macrophages),in view of its important role in the pathogenesis of fibrosis in different organs/tissues.At all examined time points,notably low levels of TGF-?1 were detected in the PXNT group.Consistently,immunofluorescence analysis of tissues at laminectomy sites at week 8 post-operation revealed the lowest macrophage count in the PXNT group.These results demonstrated that the newly engineered anti-inflammatory and bio-adhesive PXNT hydrogel can also effectively suppress surgery-mediated fibrosis by preventing TGF-?1 production via macrophages.Further,these data confirmed the inhibited inflammatory cell infiltration by PXNT,because macrophages are also typical inflammatory cells.In addition,immunofluorescence analysis showed notably reduced CD31+ areas after 8 weeks of treatment with PXNT,indicating inhibited neovascularization at laminectomy sites.Previous studies demonstrated that angiogenesis is closely associated with the pathogenesis of fibrosis in different diseases.7.Safety studiesFinally,in vivo safety of PXNT was examined.During the first 7 days after operation and treated with various formulations in rats,all groups showed comparable white blood cell counts at different time points.After 8 weeks,PXNT-treated rats showed no abnormal increase in serum levels of typical biomarkers associated with hepatic and renal functions including alanine aminotransferase(ALT),aspartate aminotransferase(AST),and creatinine(CREA),as compared to the model control group.Also,the PXNT group exhibited no significant increase in inflammatory cytokines in blood serum.Furthermore,examination on H&E-stained histological sections of major organs from rats treated with different hydrogels revealed no distinguishable injuries and pathological changes.These preliminary results indicated that local treatment with PXNT did not cause systemic adverse effects in rats.At week 8 post-operation,various degrees of neurodegeneration could be clearly observed in the model group,characterized by delamination of the myelin sheath.Myelin sheaths in both PL and PLN groups were delaminated massively.Also,considerable delamination was found in PX and PXT groups.By contrast,the PXNT group showed negligible abnormalities.Notably,significant delamination was also observed after treatment with the commercial product Interceed.Subsequently,we evaluated the effects of different treatments on locomotor activity by an open field test based on Basso,Beattie,and Bresnahan(BBB)grading scales.Nearly no alteration in motor function was found for rats in the PXNT group.However,the results of PL and PLN groups were even worse than those in the model group.Interceed-treated rats also exhibited impaired locomoter activity.Further,electrophysiological tests showed comparable waveforms of motor-evoked potentials(MEPs)before operation(Pre-OP)and post operation and PXNT treatment(Post-OP).After either 4 or 8 weeks of treatment with PXNT,there were no significant differences in both average latency and amplitude.Together,these results demonstrated that local application of PXNT hydrogel did not cause adverse effects on nerve functions.Collectively,the above results demonstrated that local treatment with our multifunctional hydrogel effectively prevented epidural adhesion after lumbar laminectomy by attenuating inflammatory and oxidative responses as well as inhibiting fibrosis and neovascularization at injured sites.ConclusionIn summary,ZISTA-TLIF could not reduce the postoperative epidural fibrosis.Thus,we engineered a multifunctional hydrogel with anti-inflammatory,anti-oxidative,anti-fibrosis,bio-adhesive,self-healing,and thermosensitive properties by facile combination of three functional modules.The optimized hydrogel was developed by hierarchical screening based on in vitro properties and in vivo activities.In vivo studies demonstrated that the finally constructed functional hydrogel PXNT can effectively prevent epidural adhesion after lumbar laminectomy in both rats and rabbits.Compared to other biomaterials currently developed for prevention of post-laminectomy epidural adhesion,our multifunctional hydrogel has the following advantages: 1)Easy deployment/handling by clinically favorable methods to completely cover the laminectomy sites and simultaneously form an effective physical barrier;2)Appropriate bio-adhesive capability to prevent rapid detachment due to natural movement of the spinal cord;3)Suitable residence/retention time to achieve clinical benefits but avoid severe foreign body reactions owing to long period of exposure;4)Desirable viscoelasticity and softness to enable spine mobility and to avoid possible spinal cord/nerve compression;5)Beneficial anti-oxidative stress,anti-inflammatory,and anti-fibrotic activities to prevent epidural fibrosis;6)Good biocompatibility and no impairment to the spinal cord.In addition to lumbar laminectomy,PXNT hydrogel is highly promising for prevention of post-operative adhesions in other conditions,such as stapedectomy,pelvic and abdominal surgery,cardiac surgery,hepatectomy,splenectomy,and nephrectomy,in view of its good safety profile. |
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