| Background and objectivesPeripheral nerve injury is a common and frequent clinical disease with a high incidence worldwide.It can be caused by a variety of factors such as severe limb trauma,congenital malformation and tumor resection,and often results in patients with movement or sensory dysfunction or even disability,and the clinical repair effect is often unsatisfactory,which brings health problems to patients’ life and also causes great social pressure and economic burden.Currently,the clinical treatment of choice for peripheral nerve injury is tension-free suturing of the peripheral nerve membrane by microsurgical techniques,and autologous nerve grafts can be performed if the nerve defect is present.However,the donor nerve source is limited,and the nerve excision graft inevitably brings about sensory impairment in the donor area.The technique of using biomaterials to prepare nerve conduits to create a microenvironment for nerve growth inside the conduit to guide and induce nerve regeneration was developed in the late 1970 s.This technique has high requirements for the materials used to make the nerve conduits,and often the material used does not allow for satisfactory results in nerve injury repair.The materials used to make nerve conduits must first have good safety and biocompatibility.Collagen is widely found in mammals in nature and is the main component of extracellular matrix,with the advantages of good biocompatibility,weak immunogenicity and degradability,and is an ideal cell carrier material,but pure collagen has the disadvantages of poor mechanical strength,rapid degradation and difficulty in mass extraction,which cannot be used as scaffold material alone.Chitosan is widely found in the cell walls of marine arthropod shells and higher plants,and is a degradable natural polymer polysaccharide that can be absorbed by human body after degradation,and chitosan is non-antigenic,non-toxic and has strong antibacterial properties,which is a safe natural material.Based on the properties of the above two natural materials,this study aims to design a new nerve composite film material to provide a stable space for nerve regeneration;refine the orientation structure of the nerve composite film to guide the orderly and directional growth of axons,avoid ineffective growth,and finally achieve the purpose of good functional regeneration of nerves.Nerve regeneration requires nutrients and the construction of a good microenvironment for nerve regeneration.Platelet-rich plasma(PRP)has been reported to contain high concentrations of various proteins and growth factors(GFs),such as platelet-derived growth factor(PDGF),vascular endothelial growth factor(VEGF),transforming growth factor(TGF-β1),epidermal growth factor(EGF),insulin-like growth factor(IGF),and basic fibroblast growth factor(b FGF),which have a wide range of applications in various fields of biomedicine.Therefore,this study was designed to load platelet-rich plasma into a new nerve composite film to provide nutrients for nerve regeneration and create a good microenvironment for nerve regeneration,thus promoting the repair of peripheral nerve injury with good efficacy.The main contents were as follows:(1)The composite films of collagen and chitosan with different mass ratios were prepared and evaluated and selected the composite films(nerve conduit materials)with good mechanical properties,hydrophilicity,in vitro degradation rate,swelling and cytocompatibility favorable for nerve regeneration.(2)The effect of PRP and PRP-loaded composite films on cell growth were evaluated by an in vitro experimental system by selecting Schwann cells RSC96,which have an important role in peripheral nerve injury repair,and the characterization of composite film after PRP gel loading and the growth factor release in PRP was measured.(3)To construct an animal model of peripheral nerve injury-SD rat sciatic nerve transection injury model,five experimental groups were set up.The in vivo efficacy of the PRP-loaded collagen/chitosan composite film on the repair of peripheral nerve injury was evaluated through various technical means,including motor and sensory functions,pathological changes and molecular level,in order to provide a new effective treatment method for clinical peripheral nerve injury repair.Materials and MethodsThe rat Schwann cells RSC96 used in the experiments were purchased from the Typical Culture Conservation Center of Shanghai Academy of Sciences,China;the SPF-grade healthy adult male Sprague Dawley rats used in the experiments were provided by the Experimental Animal Center of Dalian Medical University.Type I collagen solution and chitosan solutions were mixed in different mass ratios to make collagen/chitosan composite films with collagen to chitosan mass ratios of 1:0,1:1,and 1:3;the collagen/chitosan composite films were observed by morphological microscopy,mechanical properties(stress,tensile strength,elastic modulus and elongation at break),swelling properties,in vitro degradation rate,contact angle and roughness,etc.,and the best collagen/chitosan composite membrane was selected for biocompatibility determination.DMEM culture medium(containing 10% fetal bovine serum and 1% penicillin)at 37℃ and 5% CO2,and the effect of collagen/chitosan composite film on neuronal cell growth and cell activity was examined by CCK-8 and Taipan Blue rejection assay.Secondly,platelet-rich plasma(PRP)was prepared from rat whole blood by gradient centrifugation,and the effects of different concentrations and time points of PRP and collagen-chitosan composite films loaded with PRP on the growth of RSC96 in Schwann cells were analyzed by CCK-8 technique.Third,an animal model of peripheral nerve injury was constructed by sciatic nerve dissection in SD rats,and five experimental groups were set up:(1)Untreated control(UC;with injury but without interventions);(2)Sham-operated as positive control(SC);(3)Never suture group(NS);(4)Collagen/chitosan composite film group(COL/CS);(5)Collagen/chitosan composite film group loaded with PRP(PRP-COL/CS).The recovery of motor function and sensory function after sciatic nerve injury was evaluated by measuring the sciatic nerve function index and hot plate latency test at week 8 postoperatively;the degree of gastrocnemius atrophy and recovery were measured by gastrocnemius weight loss rate and cross-sectional area to indirectly assess the effect of sciatic nerve injury and repair;paraffin sections were taken from the gastrocnemius and sciatic nerves,and the results were analyzed by HE and toluidine blue staining.We also examined the expression of S-100β and GFAP in sciatic nerve tissues by immunohistochemistry to evaluate the regeneration of sciatic nerve fibers at the molecular level,and demonstrated that the collagen/chitosan composite membrane loaded with PRP(PRP-COL/CS)can be used as a nerve conduit in vivo.The nerve conduits made from PRP-loaded collagen/chitosan composite films(PRP-COL/CS)were used for the repair of sciatic nerve injury in vivo.1.Results Preparation of collagen/chitosan,a nerve conduit material,and evaluation of its physical properties and biocompatibility1.1 Morphological differences of collagen/chitosan composite films with different mass ratiosFrom the surface morphology graphs of scanning electron microscopy and the results of surface roughness measurement of composite films by atomic force microscopy,it can be seen that the surface of collagen composite films with chitosan added became gradually rougher and the roughness of the surface increased with the increase of chitosan/collagen mass ratio.1.2 Swelling properties of collagen/chitosan composite films with different mass ratiosThe saturated water content of collagen/chitosan composite films with different mass ratios were examined,and the results showed that the saturated water content of the composite films showed a gradual decrease with increasing chitosan mass: COL(1:0):86.66%±0.77%,COL/CS(1:1): 90.86% ± 0.81%,COL/3CS(1:3): 66.92%±2.8%.1.3 Mechanical properties of collagen/chitosan composite films with different mass ratiosStress,tensile strength,modulus of elasticity,and elongation at break are common metrics used to evaluate the mechanical properties of materials.The measured data of these indexes showed that the tension-stress curve,tensile strength,elastic modulus and elongation at break of the collagen/chitosan composite film were changed to different degrees after compounding the chitosan material.Among them,when the collagen and chitosan composite ratio was 1:1,the collagen/chitosan composite film had the best tensile strength(7.59 ± 0.7 MPa),elastic modulus(15.00± 2.27 MPa)and lower elongation at break(38.29 ± 5.63%).1.4 In vitro degradation rates of collagen/chitosan composite films with different mass ratiosThe degradation rate of composite films was tested by degrading collagen/chitosan composite films in vitro with 5 U/ml of type I lysozgme solution:among the different ratios of composite films,COL/CS(1:1)had the slowest degradation rate(residual mass%: 82.12% ± 0.50%,which at 263 h),followed by COL/3CS(COL/3CS: 61.37 ± 0.22 %,which at 263h).The fastest degradation was COL group(41.10% ± 0.10,which at 263h).1.5 Hydrophobicity of collagen/chitosan composite films with different mass ratiosIn this study,the contact angle of water droplets on the surface of the sample film was tested by the seat-drop method which is the most commonly used method to measure the degree of wettability of the material surface.If the contact angle <90°,the material is wet or partially wet,that is hydrophilic;a surface with a contact angle >90° is called a hydrophobic surface: COL: 65.29o±4.27o,COL/CS:87.34o±3.91o,COL/3CS: 86.46o±6.94o.1.6 Effect of collagen/chitosan composite film on the proliferation of Schwann cellsIn this study,the effect of COL/CS composite film on their growth and morphology was evaluated by CCK-8,Taipan Blue rejection assay and immunofluorescence to reflect the biological(cytochemical)compatibility of the composite film.48 h OD of normal culture group was 1.463±0.046 and the percentage of viable cells was 98.10%±0.969%,while the OD of COL/CS group was1.320±0.017(P<0.05)and the percentage of viable cells was 80.28%±1.112%(P<0.05),the OD of COL group was 1.281±0.015(P<0.05)and the percentage of viable cells was 77.04%±1.75%(P<0.05),the OD of COL/3CS group was1.297±0.019(P<0.05)and the percentage of viable cells was 76.58%±2.13%(P<0.05).The results demonstrate that the COL/CS composite film has good bio(cytocompatibility).In view of the above parameters of swelling properties,mechanical properties,degradation rate and hydrophobicity of the composite films with different ratios,the composite membrane with a collagen to chitosan mass ratio of 1:1(COL/CS)had the best performance as a biofilm for nerve injury repair in vivo.2.Effect of platelet-rich plasma(PRP)and PRP-loaded collagen-chitosan composite films on the growth of Schwann cells2.1 The proliferation of cells in each group was examined using the CCK-8method,and it was found that compared to the control group(OD: 2.196±0.143,48h),0.5% PRP,1.0% PRP,1.5% PRP and 2.0% PRP all significantly promoted cell proliferation(0.5% PRP: 2.960±0.157,48 h,P=0.000 P=0.000;1.0% PRP:3.355±0.092,48 h,P=0.000;1.5% PRP: 3.235±0.182,48 h,P=0.000;2.0% PRP:2.939±0.139,48 h,P=0.000),with 1.0% and 1.5% PRP being the most pronounced.2.2 Characterization of PRP gels loaded with COL/CS composite filmsThe surface morphology of the COL/CS composite films loaded with PRP gel was determined using scanning electron microscopy,and the surface roughness was tested by atomic force microscopy.The results of scanning electron microscopy showed that the surfaces of the PRP/COL and PRP-COL/CS groups became rough after the loading of PRP gel,which indicated that the PRP gel adhered well to the surfaces of the above two films.From the results of surface roughness,it can be seen that the surface roughness values of each group of the two composite films after loading PRP gel increased than that without PRP gel(90.58±2.32 nm for the PRP-COL group,95.36±2.55 nm for the PRP-COL/CS group and 113.02±6.16 nm for the PRP-3COL/CS group),which is consistent with the SEM surface morphology map,but the surface flatness of the two groups of composite films was good,and this surface structure is consistent with the basic conditions for cell growth in vitro.2.3 Effect of collagen-chitosan composite films loaded with PRP on the growth of RSC96 in Schwann cellsThe growth of the cells in each group was examined by CCK-8 and Taipan Blue rejection assay,and the data showed that the OD value of the cells in the COL/CS group at 48 hours was 1.320±0.017 and in the PRP-COL/CS group was 1.467±0.012(P<0.05 compared with the COL/CS group).Analysis of the percentage of viable cells in each group by Taipan blue rejection assay showed 80.28%±1.112% in the COL/CS group and 86.64%±1.550% in the PRP-COL/CS group at 48 hours(P<0.05 compared with the COL/CS group).Meanwhile,the immunofluorescent results showed that the number of stained cells was higher in the PRP-COL/CS group compared with the COL/CS group,which was close to that of the Control group,indicating that PRP had the effect of promoting the growth of RSC96 cells.2.4 Enzyme-linked immunosorbent assay to detect the release of VEGF and IGF-1 after PRP gel loaded COL/CS composite filmIn this experiment,enzyme-linked immunosorbent assay was used to measure the release of VEGF and IGF-1 in the PRP gel-loaded composite films at fixed time periods(1st d,3d,5d,7d,14 d,21d,and 28d),and the results showed that the Control group had higher VEGF release on days 1-3,but the duration was shorter,which was related to the formation of PRP gel,a large amount of growth factors were rapid release;however,the decreasing trend of VEGF release was obvious at days 3-14,and it was micro-release at days 21-28;the PRP-COL/CS group showed a slow decreasing trend during the VEGF test,but the release was high and sustained at the time period of days 7-21 compared to the Control group,and this characteristic facilitated the continuous repair after peripheral nerve injury.The release of IGF-1 in the Control group decreased significantly within 14 days,while the release of IGF-1 in the PRP-COL/CS group decreased gradually from days 3-21,which had a slow-release effect compared with the Control group.3.In vivo study on the repair of sciatic nerve injury in rats by PRP-loaded collagen-chitosan composite film3.1 Evaluation of motor function recovery after sciatic nerve injury in each group of SD ratsThe sciatic nerve function index(SFI)is commonly used to evaluate the recovery of motor function after sciatic nerve injury in rats.SFI=0 indicates normal motor function and SFI=-100 indicates complete loss of motor function.The SFI of the UC group was-97.36±10.37,the SFI of the NS group was-77.58±4.97,the SFI of the COL/CS group was-70.51±3.89,and the SFI of the PRP-COL/CS group was-57.02±5.15 which was the highest value at the 4th postoperative week;The SFI values of the NS,COL/CS and PRP-COL/CS groups were all higher than those of the UC group,with statistically significant differences(P<0.05).At postoperative week 8,the SFI values were-25.57±7.03 in the PRP-COL/CS group;the SFI values in the NS,COL/CS,and PRP-COL/CS groups were all higher than those in the UC group,and the PRP-COL/CS group had the highest SFI values,with statistically significant differences(P<0.05).These data suggest that the collagen/chitosan films loaded with PRP wrapped around the injured nerve end developed in this study can significantly promote the recovery of neuromotor function after sciatic nerve injury,and can accelerate the process of nerve function recovery.3.2 Evaluation of the recovery of sensory nerve function after sciatic nerve injury in each group of SD ratsThe recovery of thermal nociceptive sensitivity in the injured limb of rats at week 8 postoperatively was evaluated using the hot plate latency assay.Compared with the NS,COL/CS and PRP-COL/CS groups,the latency periods of the NS,COL/CS and PRP-COL/CS groups had different degrees of shortening(P<0.05),and the PRP-COL/CS group had the shortest latency time.These data suggest that the PRP-COL/CS group can indeed significantly promote the recovery of sensory nerve function after sciatic nerve injury,and the effect is better than that of other groups.3.3 The changes of gastrocnemius muscle after sciatic nerve injury in each group of SD ratsWet weight and cross-sectional area of gastrocnemius muscle were measured in each group of rats at 8th postoperative week.The gastrocnemius muscle weight loss rate was in the order of UC group > NS group > COL/CS group > PRP-COL/CS group,and the difference between all groups was statistically significant(P < 0.05).Similarly,the trend of the transverse area of gastrocnemius muscle in each group was UC group < NS group < COL/CS group < PRP-COL/CS group < SC group(P < 0.05),which was consistent with the trend of the muscle weight loss rate in each group.Meanwhile,HE staining results of gastrocnemius muscle tissues showed that the gastrocnemius muscle in the UC group showed wrinkles and fractures,fibrosis and glassy degeneration of the muscle bundles;the NS group showed disorganized arrangement of myocytes and different degrees of atrophy;collagen fibers and regenerated myofibers were visible between the muscle fibers in the COL/CS group,and the muscle bundles were loose from each other with different degrees of myocyte atrophy;while the PRP-COL/CS group showed obvious regeneration of myofibers and The degree of fibrosis was the least,the degree of muscle atrophy was the lowest,and the myofibers were red and plump,most similar to the SC group.3.4 Histopathological changes after sciatic nerve injury in various groups of SD ratsThe results of HE staining showed that the nerve fibers in the UC group were disorganized,with swollen or absent axons and varying degrees of edema and vacuolization;the myelin sheath in the NS group showed varying degrees of edema and vacuolization,with mild edema around the nerve fibers;the COL/CS group showed myelin regeneration and fiber The COL/CS group showed significant improvement in myelin regeneration and fiber condition,and only mild vacuolization was seen,indicating that the symptoms of nerve injury were reduced;the PRP-COL/CS group showed intact myelin sheaths and neatly arranged fibers,similar to the sciatic nerve in the SC group,indicating significant improvement in myelin regeneration and fiber condition in this group.The results of toluidine blue staining showed that the number of myelinated nerve fibers in the PRP-COL/CS group was significantly more than that in the UC,NS and COL/CS groups,but less than that in the SC group;the number of myelinated nerve fibers in the COL/CS group was more than that in the UC and NS groups,indicating that both COL/CS and PRP-COL/CS were beneficial to the regeneration of myelinated nerve fibers,but the PRP-COL/CS effect was more obvious.3.5 Expression of S-100β and GFAP in sciatic nerve fibersS-100β protein and GFAP are marker proteins of glial cells,and detecting their expression changes after nerve injury can help to understand the repair of nerve fibers after injury.The results of immunohistochemical detection of S-100β protein and GFAP showed that S-100β and GFAP protein were positively expressed in the cross-section of sciatic nerve fibers in all groups of rats,and the number of S-100βand GFAP protein positive cells were significantly reduced in the UC group.However,in the COL/CS groups,the gray value of S-100β and GFAP were higher 19.96%,24.22% in cross sections than UC group.The PRP-COL/CS group had the highest gray value,the value of S-100β and GFAP were higher 3.55%,3.99% than COL/CS group,which demonstrated increased nerve repair.Conclusion:1.The composite films with collagen to chitosan mass ratio of 1:1(COL/CS)were demonstrated to have the best performance as biofilms for in vivo nerve injury repair in terms of swelling properties,mechanical properties,degradation rate and hydrophobicity,and to have good bio(cytocompatibility).2.The appropriate concentration of platelet-rich plasma(PRP)can promote the growth of Schwann cells,and the addition of PRP gel to the neural composite film(compared with the neural composite film alone)has the same effect of promoting the growth of Schwann cells,and the PRP gel has a slow-release growth factor effect after loading to the composite film.3.The in vivo study of the rat sciatic nerve transection injury model,an animal model of peripheral nerve injury,fully demonstrates that the collagen-chitosan composite films loaded with PRP(PRP-COL/CS)synthesized in this study are effective in repairing sciatic nerve injury in rats,which can provide a new effective treatment strategy for clinical peripheral nerve injury repair,and is of great significance for the improvement of clinical peripheral nerve injury repair treatment methods,and has important practical application in the field of translational medicine. |
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