| BackgroundNowadays,many patients suffer from low back pain,which not only reduces the quality of life of patients and makes them lose the ability to work,but also consumes too much limited medical and economic resources.Studies revealed that intervertebral disc degeneration(IDD)was one of the main causes of lower back pain.At present,there are two main methods for the treatment of lumbar intervertebral disc degeneration:conservative treatment and surgical treatment.However,conservative treatment based on drug therapy can only alleviate the pain of patients,but cannot stop the process of disc degeneration.Surgical treatment is the first choice after the failure of conservative treatment.Although the treatment is relatively lasting,the patients are troubled by the problems of postoperative recurrence and adjacent stage degeneration.It is one of the hotspots in the medical field to delay intervertebral disc degeneration by enhancing the repair ability of the cells in the intervertebral discBone morphogenetic protein7(bmp-7),also known as osteogenic protein-1(op-1),can promote the secretion of proteoglycan and type II collagen by nucleus pulposus cells,as well as the proliferation of nucleus pulposus cells and their differentiation into chondrocytes.However,the growth factor alone has a short half-life,requiring multiple injections to maintain the effect,and bmp-7 injection directly into the intervertebral disc also has the risk of osteogenesis.A new self-assembled peptide nanofiber scaffold material RADA16-I(Ac n-radaradaradadadada-cnh)biological activity is enhanced by the compound of short bioactive peptides with different functions at the c-terminal.This study will short peptide fragments with BMP 7 functions(KPSSAPTQLN)by way of chemical bonds in RADA16-i C end to construct functional self-assembly peptide nanofiber RADA-KPSS hydrogel scaffold material,and then put the RADA-KPSS and RADA16-I to 1:1 ratio mixed form a new type of functional self-assembly RADKPS peptide nanofiber hydrogel scaffold materials,research has shown that RADKPS can obviously promote the in vitro human degenerative nucleus pulposus cells of proteoglycan and type II collagen synthesis,The duration was longer than bmp-7.The previous experiments of our research group proved that RADKPS,a new functional self-assembled polypeptide nanofiber hydrogel modified with functional fragments of bmp-7,had good cellular,blood and histocompatibility.In order to study the effect of RADKPS in the repair of delayed intervertebral disc degeneration,RADKPS was injected into the animal model of disc nucleus pulposus defect degeneration at low temperature to detect the repair effect,and to provide experimental basis for further clinical application.Objective:1.To establish a model of intervertebral disc degeneration in dogs.2.The effect of RADKPS on delaying intervertebral disc degeneration was observed in animal experiments.Methods:1.A total of 16 healthy hybrid dogs(10-16 months old,10-15kg)of 1 year old age,male or female,were selected to evaluate the overall condition of intervertebral disc by X-ray and MRI.Animals with no obvious spinal deformity and good overall condition of intervertebral disc were selected as experimental objects by pfirrmann grading.2.16 adult hybrid dogs,each with 4 intervertebral discs,L3-4,L4-5,L5-6,L6-7.MRI examination was performed before the modeling to ensure that eachlumbar intervertebral disc was not degenerated before the modeling with good uniformity.The degenerative model of intervertebral disc was made by puncture3.Therapeutic intervention of canine degenerative intervertebral disc Four weeks after the modeling,an MRI was performed to confirm that the successful and well-homogenised dogs had the same pfirrmann for each disc.Different intervertebral discs of the same dog were included in the following groups:16 adult dogs,,each with a uniform disc after modeling,were divided into the normal control group(L7-S1),the RADKPS group(L5-6),the bone morphogenetic protein 7(bmp-7)control group(L6-7),the Rada16-I control group(L3-4)without compound bmp-7,and the PBS control group(L4-5).4.Before modeling,4 weeks after modeling,4 weeks after intervention,8 weeks and 12 weeks.X-ray examination and MRI examination were performed at these 5 time points.The time point of the intervention operation was set at 0,and the time was recorded as-4w,0w,4w,8w and 12w.5.Histological examinationIn 12w,dogs were euthanized by ketamine injection after the end of imaging examination,and the L3-S1 spinal column was separated together with the surrounding soft tissue.Then,each intervertebral disc and upper and lower vertebral bodies were separated.Paraffin-embedded sections were treated with HE staining,,and collagen immunohistochemical staining,proteoglycan immunohistochemical staining.6.Biochemical composition detectionThe experimental animals were euthanized after 12w.Rabbit L3 to S1 were separated from the soft tissue around the spinal union.Incised from intervertebral disc with a sharp knife,the amiable tissue was isolated,and then the content of proteoglycan and 1 type collagen was quantitatively detected by ELISA.Results:MRI:the manager software of the imaging workstation was used to determine the relative gray index(RGI)of the intervertebral disc nucleus pulposus and the relative parts of the cerebrospinal fluid.At-4w,the included intervertebral discs had good uniformity and no congenital degeneration.At Ow,the models of all intervertebral discs degenerated uniformly.When the 12 w.The RGI value of the normal control group was significantly higher than that of the other four groups.Meanwhile,the RGI values of the RADKPS experimental group were all higher than those of the bmp-7 control group,radal6-i control group and PBS control group.The RGI values of the Pbs control group were significantly lower than those of the other four groups.At 4w,8w and 12w,the RGI values of the three groups,bmp-7 control group,rada16-i control group and PBS control group,decreased inversely with time.In the RADKPS experimental group,there was no significant difference in RGI values at 4w,8w and 12w.X-ray:X-ray examination was performed at Ow and L2 hours.X-ray plain film results showed that the height of intervertebral discs decreased from L2-5.At Ow,the uniformity of mould making was good,which was consistent with the results of nuclear magnetic detection.At 12w,DHI%of the RADKPS experimental group was higher than that of the other three control groups,but lower than that of the normal control group.DHI%in Psb control group was lower than the other four groups.There was no significant difference between bmp-7 control group and radal6-i control group.There was no significant difference between the Ow and 12w RADKPS groups.T2 maping:at 0w,there was no significant difference in T2 value between the four groups.At 12w,RADKPS was the highest in the experimental group,but lower than in the normal control group.The Psb control group was lower than the other four groups.There was no significant difference between bmp-7 control group and radal6-i control group.There was no significant difference between the Ow and 12w RADKPS groups.HE staining:In the normal group,the nucleus pulposus tissue and the annulus fibrosus tissue had obvious boundaries.In the RADKPS group,the boundaries between the nucleus pulposus and the annulus fibrosus tissues were still obvious,and the staining was uniform,with a small number of clustered cells.In the bmp-7 control group and the Radal6-i control group,the boundaries between the nucleus pulposus and the annulus fibrosus tissue were not obvious,the clubb-like cells disappeared,and the structure of the nucleus pulposus was disorganized.In the Psb control group,the boundaries between the nucleus pulposus and the annulus fibrosus disappeared,and the number of cells was significantly reduced.The tissues were gradually replaced by collagen.Immunohistochemical staining:the protein polysaccharides in the nucleus pulposus of the RADKPS experimental group were deeply stained with type ii collagen,which was significantly stronger than the other three groups.ConclusionRADKPS polypeptide hydrogel can delay intervertebral disc degeneration in early stage.Compared with direct injection of rada-16 and bmp-7,RADKPS inherited the characteristics of slow metabolism and persistent action of rada-16 in intervertebral disc,and had good biological activity of bmp-7,but reduced the risk of osteogenesis.RADKPS role in May and it can promote the intervertebral disc nucleus pulposus cells secrete AGG and COLL II are concerned,there are may and it can promote BMSCs to move within the organization and to promote its to nucleus pulposus cell differentiation. |
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