| BackgroundsAbout 80%of the population will experience low back pain during adulthood, which is commonly in due to degenerative disc diseases(DDD).Staging therapies of symptomatic degenerative disc diseases should be tailored to grade of degeneration in principle,following with increasing cost,risk and collateral damage.Therefore,disc degeneration should be intervened by spine arthroplasty in the early stage to preserve and restore physical roles of intervertebral disc in the functional spinal uint(FSU). Nucleus pulposus replacement is a modality of spine arthroplasty for early DDD to restore height of disc and motion of FSU by reconstructing anterior column with prosthetic implant,and has become an attractive spot in spinal surgery studies.The Prosthetic Disc Nucleus(abbr.PDN) was the hydrophilic prosthesis of nucleus pulposus and early applied to clinic with good outcome in the short term follow-up,however there were considerable damages of cartilaginous endplates (60.0%) and subsidence of prosthesis(49.2%) in the four-year-follow-up.The stress concentration of cartilaginous endplate was responsible for damages and subsidence, which resulted from the higher elastic modulus of materials and a wedge shape or rectangle.Therefore,prosthetic nucleus pulposus should be made of the hydrogel with suitable elastic modulus,whose shape should match with cartilaginous endplate. Based on the poly(vinyl alcohol)(abbr.PVA) hydrogel,compounding pectin extracted from the cactus with PVA,this paper is to develop a new kind of composite hydrogel,the pectin/PVA composite(abbr.CoPP) hydrogel,whose elastic modulus will be suitable to make prosthetic nucleus pulposus.What's more,this paper is to optimally design the shape and size of prosthetic nucleus pulposus made of CoPP hydrogel in order to avoid stress concentration of cartilaginous endplate.Therefore,it is the new CoPP hydrogel and optimal design of prosthesis that is to provide basic study for development of prosthetic nucleus pulposus made of CoPP hydrogel.Objectives1.To explore preparation methods of pectin/poly(vinyl alcohol) composite(CoPP) hydrogel,characterize its microstructures and evaluate the swelling properties.2.To evaluate biomechanical properties of CoPP hydrogel compressed statically at the swelling equilibration.3.To evaluate biocompatibility of CoPP hydrogel for nucleus pulpous prosthesis, including in vitro cytotoxicity and in vivo inflammatory reaction.4.To explore reconstructive methods of three-dimensional nuclues pulposus derived from lamellar MR imaging,and to optimally design shape and size of prosthetic nucleus pulposus made of CoPP hydrogel for Chinese people.Methods1.The mixture of XPV-MC300 pectin,PVA-124 and distilled water with appropriate mass percentage was given swelling,heating by low frequency oscillation and freezing thawing to prepare cylindrical test pieces of CoPP hydrogel and PVA hydrogel.The PVA hydrogel tests(n=12) and CoPP hydrogel tests(n=24) were randomly divided into two groups and dehydrated by vacuum freeze-dry(abbr. FD) and anhydrous alcohol(abbr.AE),respectively,whose mass and volume dehydrant ratio were compared by variance analysis of repeated measure data and dehydrant ratio were compared by variance analysis of repeated measure data and between two dehydrant methods and two hydrogels compared by independent sample t test.The mierostructures of CoPP hydrogel were characterized by FTIR, DSC,XRD and SEM.Eight dehydrated test pieces of PVA and CoPP hydrogel were selected and soaked into normal saline and high glucose DMEM solution with 10%calf serum at 37℃and 0.10MPa(1 atm).The swelling ratio of mass and volume were observed.Furthermore,the diffusion characteristic index(n) and swelling velocity constant(k) of CoPP were calculated according to the equation: Ig(SRM) = 1g(k) + n 1g(t).2.Four cylindrical test pieces of CoPP hydrogel and PVA hydrogel were prepared and compressed lengthways statically by MTS-858 Mini Bionix material test machine at the swelling equilibration.After 5 circles of preload,whose maximal load was 500N with 5N/s loading rate,600N was applied and kept for 30min.The force,displacement and time were recorded at real time.The masses and sizes of test pieces were measured before loading and during 24 hours after unloading. Based on above data,these indexes were calculated:①elastic modulus was the coefficient of line fitting between stress and strain of 6th loading;②absorption ratio of power was the relative value of areas between hysteresis loop and under the buildup curve,calculated by Metlab7.0 software according to the stress-strain curve of the fifth loading and unloading,in the same X-axis strain(0～55%), Y-axis stress(0～2.0MPa) and 1.25 ratio of X-axis/Y-axis drawn by Origin7.5;③normalization creep was the ratio of creep between every other 5min with continous load(600N) and at the beginning of 600 N;④normalization mass and volume percentage were the ratio of mass and volume after unloading relative to them before loading.The elastic modulus and absorption ratio of power between CoPP and PVA hydrogels were compared by independent sample t test,and normalization mass and volume percentage were compared by variance analysis of repeated measure data and independent sample t test.3.The in vitro cytotoxicity of CoPP hydrogel was tested by NCTC L929 cell lines, which were divided into four groups:normal,negative control(PVA/FD and PVA/AE),experiment(CoPP/FD and CoPP/AE) and positive control(0.64% phenol),regularly intervened by each treatment.The growth status of cells were observed regularly,OD values of each group were measured at 2nd,4th and 7th day and then relative growth rates(abbr.RGR) were calculated.There were thirty SD rats(15 male and 15 female,body mass 182g±13g) for inflammatory reaction in vivo.CoPP and PVA hydrogels(10mm*3mm*3mm,Length*Width* Thickness) were implanted into left and right intramuscular of gluteus of SD rats,respectively. Postoperative wounds and general status of rats were observed,and local muscles containing with hydrogels were taken and sectioned at 1st,4th,and 12th week.The macroscopic specimen and microscopic reaction of inflammatory were observed and evaluated according to the standard of GB/ T 161752-1996,including the microstructure of CoPP hydrogel with light microscope.4.The lamellar lumbar MRI of normal Chinese subject was screened from subjects accepting lumbar standard scan by GE Signa Excite 3.0.Trauma,inflammation, deformity and tumor of anterior and middle column of spine were excluded.The criteria of inclusions were at age of 20～50 years old,disc degeneration not exceeding Pfirrmann 2,degeneration of endplate not exceeding Modic 1.The selected subject was scanned in T2 serials to get DICOM 3.0 MR imaging of lumbar in sagittal or coronal view,with 1mm～1.5mm thickness and 0 mm space. These DICOM images of lumbar MR were imported into the Mimics10.01 software.After accurate segmentation of nucleus pulposus in the imaging,the three-dimension(3D) models were reconstructed by Calculate 3D function of Mimics.The sagittal diameter,transverse diameter,height and slipping angle of nucleus pulposus were measured in MR imaging and 3D model,respectively.The shape of prosthetic nucleus pulposus made of CoPP hydrogel was designed according to the reconstructive 3D model of nucleus pulposus.According to equation of volume dehydrant ratio and volume swelling ratio of CoPP hydrogel, the volume conversion coefficents of preparation(Vpre) and dehydrated implant (Vimp) relative to human nucleus pulposus were calculated.The expected sizes of CoPP hydrogel prosthesis were multiplying results between the cube roots of volume conversion coefficents and anatomic sizes of human nucleus pulposus.On basis of expected sizes,shapes and sizes of prosthetic nucleus pulposus made of CoPP hydrogel were optimally designed.Results1.CoPP hydrogel was successfully prepared from the water solution of PVA(25%) and pectin(5%) by swelling,heating with low frequency oscillation and freezing thawing,whose cylindrical shapes can be maintained not by vacuum freeze-dry but by anhydrous alcohol in the dehydrating course.However,the dehydrant ratios of vacuum freeze-dry hydrogels were significantly superior to them of anhydrous alcohol hydrogels.The dehydrant ratios of PVA hydrogel were significantly superior to them of CoPP hydrogel in the same dehydrant course. The FTIR and XRD graph of CoPP hydrogel were superposition of PVA and pectin,respectively.There were two melt apexes in DSC graph of CoPP hydrogel, at the temperature of 182.6℃and 225.1℃,whose posterior apex was corresponding to the melt apex of PVA powder.The section of CoPP hydrogel were full of 10μm-50μm pores with granules;however,there was no pore in the section of PVA hydrogel.The diffusion characteristic index(n) of CoPP hydrogel was near to 0.5,and the swelling velocity constant(k),the swelling ratio of mass (197.38%～252.49%) and swelling ratio of volume(241.47%～308.93%) of CoPP hydrogel at the swelling equilibration were superior to those of PVA hydrogel.2.There was not any test pieces of CoPP and PVA hydrogels destroyed by loading and unloading.All curvatures of stress-strain consisted two parts:the first was mild starting,and the posterior was steep rise,which were nonlinear.The elastic modulus of CoPP hydrogel was(2.76±0.09) MPa,near to 3MPa which is optimal elastic modulus of preformed nucleus prosthesis,and was significantly inferior to that of PVA hydrogel(4.89±0.61) MPa.There was also significant difference of absorption ratio of power between CoPP hydrogel(12.50±1.47)%and PVA hydrogel(8.57±1.78)%.Normalization creeps of CoPP and PVA hydrogels under 30 min continuous loads were 1.076±0.005 and 1.105±0.030,respectively.The normalization mass percentage of CoPP hydrogel was(97.67%±0.98)%, significantly inferior to that of PVA hydrogel(99.15±0.18)%.At time of 24 hours after unloading,there was no significance of the normalization percentage of mass and volume between two kinds of hydrogels.3.The L929 cells of negative group(PVA/FD and PVA/AE) and experiment group (CoPP/FD and CoPP/AE) were long fusiform adhered to the wall at the same time relative to normal group,and occurred contact growth inhibiting.Less 10%of cells were circular with discrete grain in negative and experimental groups.RGRs of CoPP group were over 75%,RGRs of PVA group were over 80%,and cytotoxicity of both groups was grade 0～1.On the contrast,RGRs of positive group were inferior to 24%and cytotoxicity was over grade 4.All of experimental rats waked up and began walking during 2～4 hours after operation,without death, and wounds healed primarily without red swelling,exudation or suppuration.At week 1,PVA and CoPP hydrogels were surrounded by bright and red muscles without exudation or suppuration,movable and were larger than those of implants. The sections of CoPP hydrogel were basophilia,stained blue and porous,however, sections of PVA hydrogel were close to neutrophilia,stained undertint red,and pligoporous with smaller pore.The inflammatory reactions of local tissue were grade 4 of inflammatory cell infiltrate(ICI) without integrate cyst.At week 4,the PVA and CoPP hydrogels were also surrounded by normal muscle,however,were not movable.The inflammatory reactions of local tissue were grade 1 of ICI and grade 4 of cyst reaction.At week 12,volumes and shapes of PVA and CoPP hydrogels were similar to that at 1st and 4th week,and both ICI and cyst of the local tissue were grade 1.Sections of CoPP hydrogel were also basophilia,stained blue,whose edge were consistent without defect.There were full of pores with micrograins at 12th week,which were similar to that at 1st and 4th week.4.Lamellar lumbar MR images of 34 years old woman in this paper were scanned in coronal vies with 1.2mm thickness and 0 mm space by T2 serials(OCor fs, TE82.7ms,TR4000ms),including anterior and posterior margin of invertebral discs from L1 to S1.There were 44 sheets of DICOM3.0 images of MR,whose pixel size was 0.625mm.The disc degeneration of lumbar MRI was Pfirrmann 1～2,whose nucleus pulposus was segmentated well.3D models of nucleus pulposus were successfully reconstructed from lamellar lumbar MR images, whose lateral views were ellipse from fiat to rear fomix,and whose shapes were from discs-form to rugby-form.The sagittal diameter,transverse diameter,height were increasing from upper to lower,especially transverse diameter.The average of sagittal diameter,transverse diameter and height in MR images were (20.8±1.5)mm,(34.7±3.5)mm and(9.2±1.1)mm,which were similar to them of 3D models.The dehydrated CoPP hydrogel prosthesis would be implanted,whose volume was 25.90%～29.29%and whose size was 63.74%～66.41%of human nucleus pulposus.The CoPP hydrogel prosthesis were ellipsoid near to rugby,with four sizes(S,M1,M2 and L),whose sagittal diameters were 13mm～14mm, transverse diameters were 20mm～24mm and heights were 5.5mm～7.0mm.Conclusions1.It is successful for swelling,heating with low frequency oscillation and freezing thawing to prepare CoPP hydrogel,and it is proper to be dehydrated by anhydrous alcohol.CoPP hydrogel is porous and with determinate crystallization,including two kinds of crystallization zones.CoPP hydrogel has good rehydration with Fick model diffusion of water,and are faster and larger than those of PVA hydrogel.2.CoPP hydrogel is a kind of viscoelastic material,which is softer and more tenacious,has better absorption shock of load,whose elastic modulus is near to the optimal elastic modulus of preformed prosthetic nucleus pulposus,has better permeability of substance in contrast to PVA hydrogel,and is suitable to made preformed prosthesis of lumbar nucleus pulposus.3.The cytotoxicity of CoPP hydrogel in vitro is 0～1 grade,the inflammatory reaction of CoPP hydrogel in vivo is mild,and then the basic biocompatibility of CoPP hydrogel is good and there is no degradation of CoPP hydrogel.The genotoxicity,carcinogencity and reproductive toxicity of CoPP hydrogel need evaluating in the future.4.3D model of nucleus pulposus can be successfully reconstructed from lamellar lumbar MR images.The CoPP hydrogel prosthesis of nucleus pulposus is ellipsoid near to rugby,matching with the shape of cartilaginous endplate,which is helpful to reasonable transmission of load.It is smaller volumes that help CoPP hydrogel prosthesis to be held with ball-and-socket instrument and minimally invasively implanted by posterior diskoscope.
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