3D Bio-printed Hydrogel Scaffold With Enhanced PRP For Spinal Cord Injury Repair

Background:Spinal cord injury(SCI)can cause locomotor dysfunction,and its repair is one of the most challenging conditions.Clinically,the main objective of repair is to remove the injury compression factors and restore the stability of spinal structure to the greatest extent,but there has been no appropriate method for nerve regeneration until now.Thus,scholars focus on nerve regeneration for SCI repair,including the direct transplantation of stem cells and cell-related factors,implantation of material tissue engineering scaffolds,etc.Neurons,astrocytes,and oligodendrocytes are some of the cells that can differentiate from NSCs when transplanted with cells and biological factors.NSCs injections into injured sites can have some repair effects,disordered growth and non-directed differentiation of cells must be addressed although.In academia,platelet-rich plasma(PRP)has always been regarded as a collection of growth factors,but the concentration of NGF,BDNF,NT-3and other nerve-related growth factors are low,which hampers nerve repair.On the other hand,the biocompatibility and predictable biodegradability of hydrogels make them popular in biological scaffolds for material tissue engineering;furthermore,most hydrogels contain specific cell binding sites that are necessary for cell attachment,proliferation,growth,and differentiation;it is also possible for their mechanical strength to adapt to the spinal cord tissue.It is important to note,however,that current research is primarily focused on injectable hydrogels,which means hydrogels are primarily used as cell carriers and to fill injured cavities.Current research focuses on SCI repair in varying degrees,but it is still unable to reach the ideal outcome.The development of 3D bioprinting technology makes it possible for researchers to manufacture complex 3D structures,and biological factors and hydrogels can be combined to manufacture tissue engineering scaffolds.With NSCs,growth factors and hydrogels,this technology can precisely arrange cells,so as to the orderliness of cell growth is achieved,and the potential for nerve cells to form neural networks is realized.In addition,specific growth factors can differentiate NSCs into specific types,which provides a new avenue for SCI research.Based on this,the bioprinted hydrogel scaffold was used as a base carrier in this study,we loaded NSCs,PRP and NGF into a cell-laden hydrogel scaffold for exploring the role of enhanced PRP in SCI repair.Objective:Based on the current research background on SCI repair,3D bioprinting combined with cell and related factor therapy will be explored in this study.1.A 3D gridded scaffold conforming to the elastic modulus of spinal cord was constructed by 3D bioprinting technology,and its related physicochemical properties were explored.2.Scaffolds containing NSCs and enhancing PRP were constructed,and the influence of scaffolds on the proliferation and differentiation ability of NSCs was explored in vitro,and the biocompatibility of scaffolds was comprehensively evaluated.3.The rat model of total spinal cord resection was constructed.After implantation of biological scaffolds,the functional and histological repair ability of scaffolds after SCI was investigated in vivo.Methods:1.NSCs were isolated from the hippocampus of fetal rat and identified.Their surface morphology during growth was observed by microscopy,and their purity and differentiation ability were identified by immunofluorescence method.In addition,PRP was isolated from whole blood of rats by the improved"two-step centrifugation"method,and the concentration of PRP was analyzed by blood count instrument.2.Prepare Gel MA hydrogel solution,and evaluate the gel preforming ability by inverted static experiment;3D gridded scaffolds were printed by bioprinter to evaluate printability of different concentrations and optimize printing parameters.In addition,the similarities and differences between the scaffold and pure Gel MA scaffold were observed after the PRP-loaded scaffold was printed.3.The mechanical strength of the scaffold was evaluated by rheometer,the microstructure of the scaffold was evaluated by microscope and SEM,the swelling ratio(SR),equilibrium water content(EWC)and degradation performance of the scaffold with different concentrations were measured,and the drug release of the scaffold with PRP was determined by BCA method.The proliferation of NSCs in biological scaffolds was evaluated by CCK-8,the survival rate of cells in scaffolds was evaluated by live/dead cell assay,and the targeted differentiation ability of NSCs in enhanced PRP-loaded scaffolds was evaluated by immunofluorescence.4.Complete transection SCI model of rat was constructed,BBB score and motor evoked potential(MEP)detection were used to evaluate the recovery ability of enhanced PRP scaffolds.HE staining was used to evaluate the toxicity of the scaffolds after implantation;meanwhile,the cavity recovery after SCI was observed.The tissue repair effect of enhanced PRP-loaded scaffolds was observed by immunofluorescence.Results:1.The NSCs isolated from the fetal rat hippocampus showed good proliferation and passage ability.Immunofluorescence staining showed positive expression of Nestin but no expression of GFAP,and positive expression of TUJ-1.The platelet concentration in whole blood was 504.2±30.54×10~9/L,while the platelet concentration in PRP was 2692.2±213.16×10~9/L,and the enrichment ratio was 5.34±0.62 times.2.After preheating,the Gel MA of different concentrations showed no obvious gelation trend at 5%and 8%concentrations within 25 minutes at room temperature,while the 10%concentrations showed mild gelation at 25 minutes.After 25min of refrigeration at 4℃and 5min at-20℃,there was no obvious deformation when standing upside down at room temperature for 25min.After refining the refrigeration time,under the condition of 4℃and the premise of stable printing model,5%concentration needs 20-25min,8%concentration needs 10-15min,10%concentration needs 10min;at-20℃,it takes 3-6min for 5%concentration,2-3min for 8%concentration and 1min for 10%concentration.When the refrigeration time is determined,the extrusion speed is 0.35-0.35mm3/s,and the printing speed is 3-4mm/s,the stable model can be printed with 5%concentration;0.45-0.75mm3/s,4-4.5mm/s,8%concentration can be stable printing;0.55-0.75mm3/s,4.5-5mm/s,10%concentration can be stable printing.PRP-loaded scaffolds can also be stably printed under this parameter.3.The scaffold of 8%concentration is about 1000Pa after 20 seconds of illumination after printing.The scaffold is a 3D gridded structure with thread diameter of about 450μm and thread gap of about 550μm;the micro-surface of the scaffold is multifold structure with diameter of about 50μm.The EWC of scaffolds with different concentrations was above 90%,and the SR was about 18.After 14days of natural degradation in vitro,the retention rate was still more than 80%,and more than 50%was retained after loading PRP.PRP stents consistently release growth factors,with an overall drug release rate of about 80%.Bioprinting did not cause cell damage and showed high cell survival rate(>90%),and 20%concentration of PRP effectively promoted cell proliferation.Enhanced PRP can promote neuronal orientation differentiation in vitro and reduce astrocyte differentiation accordingly.4.Compared with Control group,Gel MA group,Gel MA_Cgroup,Gel MA_C-PRP group and Gel MA_C-e PRP group can enhance the recovery of motor function in about 1 week,among which Gel MA_C-e PRP group has the best effect;at 3-4 weeks,the effect of Gel MA_C-PRP and Gel MA_C-e PRP groups were more obvious;and the Gel MA_C-e PRP group had a greater advantage after 5 weeks.Six weeks after injury,MEPs recovered in all treatment groups,and the Gel MA_C-e PRP group was the most obvious.All treatment groups had an effect on the reduction of spinal cavity,Gel MA_C-PRP group and Gel MA_C-e PRP group had the best effect,but there was no significant difference between them(p>0.05).Immunofluorescence results of TUJ-1and GFAP showed that the Gel MA_C-e PRP group had more TUJ-1 expression than the Gel MA_Cgroup(p<0.01),and had a certain effect compared with the Gel MA_C-PRP group(p<0.01).Gel MA_C-e PRP group also expressed less GFAP,but there was no significant difference between Gel MA_C-PRP group and Gel MA_C-e PRP group(p>0.05).Conclusion:1.The isolated NSCs have good growth,proliferation and differentiation abilities.The improved"two-step centrifugation"method can effectively isolate PRP from whole blood.2.The refrigeration condition at-20℃can make the Gel MA pregelation at about 5 times the speed,and can remain stable within 25 minutes at room temperature.The concentration,extrusion speed and printing speed restrict each other.With the increase of the concentration,extrusion speed also needs to be further increased.The printing speed has little influence on the molding effect,but the extrusion speed needs to be matched.The addition of PRP has no effect on the printing effect.3.Gel MA with 8%concentration has good mechanical strength,microenvironment,SR,EWC and degradation rate,which can provide a good microenvironment for the growth of NSCs,and it can continuously release growth factors within 60 hours.Bioprinting had little effect on the survival rate of cells.The survival rate of cells in scaffolds printed with 8%concentration was more than 90%.Only 20%concentration of PRP can effectively promote cell proliferation,and increasing the concentration has no effect on the proliferation effect.Enhanced PRP-loaded scaffolds can enable NSCs to have the ability of targeted differentiation.4.The implantation of scaffolds will not produce toxic effects in vivo;enhanced PRP-loaded scaffolds can enhance the repair effect of SCI both in function and histology.