| Low back pain is a common clinical disease.Up to 80%of adults have low back pain experience in their lives.It not only affects the lives and work of patients,but also brings a heavy burden on families and society.Many studies have shown that degeneration of the intervertebral disc is closely related to low back pain.Although there are a variety of clinical treatments that can alleviate low back pain,none of the current measures can fundamentally inhibit or reverse disc degeneration.In order to solve this major clinical problem,researchers have tried to explore effective biological treatment methods to achieve the purpose of repairing or regenerating the intervertebral disc for many years,so as to fundamentally avoid the occurrence of degenerative disc disease.Biological treatment measures for disc degeneration include growth factor injection therapy,cell therapy,tissue engineering material implantation,and gene therapy.Among them,growth factor therapy has been early developed and is widely used.It is currently an effective treatment strategy for repairing or regenerating intervertebral disc degeneration.Compared with several other treatment methods,growth factor therapy is more convenient to operate,more suitable for large-scale clinical application,and has a broad research prospect.It is known that a variety of growth factors can stimulate the proliferation of intervertebral disc cells,inhibit apoptosis,and promote the synthesis of extracellular matrix.Growth factors have a repairing effect on the degenerative intervertebral disc,but it has also been found that a single kind of growth factor injected into the intervertebral disc has a very limited repair function.In order to increase the effect and duration of growth factors,researchers have tried to apply combining multiple growth factors to prove that their repair effect can be enhanced.Platelet-rich plasma(PRP)products,as self-derived active substances,are rich in various soluble cytokines.Among them,many kinds of growth factors have the functions of stimulating cell proliferation,enhancing cell function,inhibiting cell apoptosis,and improving the internal environment of the intervertebral disc.Based on this background,this study explored the effect of autologous platelet-rich plasma releasate(PRPr)on degenerative intervertebral discs,with a view to establishing a treatment method that can effectively repair or regenerate discs.PART 1 Establishment and assessment of rabbit lumbar disc degeneration modelResearch BackgroundThe occurrence of low back pain is closely related to intervertebral disc degeneration.It is increasingly important to study the mechanism of disc degeneration and to find an effective,safe and economic treatment.Animal models are an important tool for exploring the pathogenesis of disease and verifying the effect of treatment,especially in the preclinical research.At present,the intervertebral disc degeneration animal model can be roughly divided into two categories:spontaneous degeneration model and experimental induction model.The ultimate goal of this study is to explore the repairing effect of platelet-rich plasma releasate(PRPr)on moderate to severe degenerative discs.Nucleus pulposus aspiration can cause greater damage to intervertebral discs and can induce relatively more severe disc degeneration.Therefore,in this study,nucleus pulposus aspiration was used to establish a rabbit disc degeneration model in an attempt to simulate the process and performance of human disc degeneration,which was prepared for the next experimental treatment research.PurposeThis part of the study explores the establishment of a rabbit degenerative disc model by nucleus pulposus aspiration,and evaluates its degeneration performance in order to make experimental preparations for subsequent therapeutic research.MethodThirty-six New Zealand white rabbits were randomly divided into an experimental group and a control group of 18 rabbits.The L4/5,L5/6 and L6/7 intervertebral discs of experimental group rabbit were revealed,and 21G needles connected to 10ml syringes were used to puncture into the intervertebral discs.The puncture depth was controlled at about 5mm,and a small amount of nucleus pulposus tissue was aspirated.After the operation,the skin was sutured.The same intervertebral disc of control group were revealed,and no operations such as puncture or nucleus pulposus aspiration were performed.The two groups of animals were taken lumbar X-ray before operation and 1w,2w,4w after operation.DHI%was calculated to represent the change in disc height before and after modeling.Samples were sacrificed at 1w,2w and 4w after operation Histological staining of HE,saffron O,and Masson was performed to evaluate histologically score the discs.ELISA was used to determine the contents of AGC and COL Ⅱ in the discs.ResultsX-ray results showed that the DHI%in the experimental group began to decrease at 1w after operation and continued until 4w after operation.And the decrease of DHI%in the experimental group gradually increased.Pathological section staining revealed that the nucleus pulposus of the experimental group shrank or arranged disorderly,the number of cells in the nucleus pulposus decreased,the extracellular matrix reduced,and cell metaplasia appeared.Histological scoring results showed that the scores of the experimental group increased at 1 w,2w,and 4 w after modeling,and the degree of disc degeneration in the experimental group gradually increased over time.The ELISA results showed that the levels of AGC and COL Ⅱ in the experimental group were significantly lower than those in the control group at 1 w,2 w,and 4 w after operation.ConclusionThe results of this study showed that disc degeneration of rabbits was successfully induced by nucleus pulposus aspiration.Postoperative observations revealed that the height of disc was decreased,histological characteristics of disc degeneration appeared,and levels of proteoglycan and collagen in the disc reduced.These evidences has proved that the nucleus pulposus aspiration method can successfully establish a rabbit disc degeneration model,and this model can be used in the next therapeutic study for disc degeneration.PART 2 Preparation and activity evaluation of autologous platelet-rich plasma releasateResearch BackgroundPRP is a blood concentrate obtained from autologous whole blood by centrifugation,whic contains high concentration of platelets.As an autologous biological agent,PRP has been confirmed to promote tissue repair and regeneration.The premise of its biological repair ability is activation by activators such as thrombin and calcium chloride,which induce it to release various growth factors.However,PRP immediately turned into a gel after activation.which is not convenient for injection.PRPr is a mixture of multiple growth factors extracted from activated PRP.It is in a liquid state and can be used for intravertebral disc injection.PurposeIn this study,PRPr was extracted by three-step centrifugation,and its activity was measured to prepare for the next experiment.MethodTen health New Zealand white rabbits were selected.14 ml of fresh arterial blood was taken,and 1 ml of citrate was drawn into the syringe in advance to prevent blood coagulation.The whole blood was divided into three parts:2ml blood were used to determine the whole blood platelet concentration and the whole blood TGF-β1 concentration,and the rest was placed in a centrifuge tube to prepare PRPr.The whole blood was centrifuged at 250g for 10 minutes.Next,the plasma layer,the white membrane layer,and the white membrane layer and the red blood cell layer at the interface about 2 mm below the blood component were divided into another centrifuge tube.The mixture was centrifuged again at 1000g for 10 minutes,then 3/4 of the supernatant(PPP)was removed.The concentrated platelets below and the remaining part of PPP was preserved.This mixture is identified as PRP.0.2ml PRP was taken for platelet count.Next,100 μl of activator(1000 U/mL thrombin and 100 mmCaCl2)was added to each 1 ml of PRP.The clotted PRP were allowed to rest at room temperature for 3 hours,followed by centrifugation at 1000g for 10 minutes.The resulting soluble releasate from the clot preparation of PRP(PRP-releasate)was isolated and kept.Platelets were counted in whole blood and inactivated PRP using an automatic blood cell analyzer.ELISA was used to determine the concentration of TGF-β1 in whole blood and PRPr.ResultsThe platelet count in normal rabbit whole blood was(217.40±51.86)×109/L,and the platelet count in PRP was(991.30±107.76)×109/L.The platelet recovery was 66.80%±11.76%,and the enrichment coefficient was 4.72±0.89.The platelet concentration of PRP was about 4.7 times that of normal whole blood.The concentration of TGF-β1 in normal rabbit whole blood was 297.86±70.66 ng/L,and the concentration of TGF-β1 in PRPr was 1020.95±219.08 ng/L,which was 3.57±0.98 times that of whole blood.Correlation analysis results showed that the concentration of TGF-β1 in PRPr was significantly positively correlated with whole blood platelet count and PRP platelet count.ConclusionPRPr containing high concentration of TGF-β1 can be prepared by three step centrifugation method,which can meet the requirements of animal experiments or clinical applications.The concentration of TGF-β1 in PRPr was significantly positively correlated with the whole blood platelet count and PRP platelet count.Increasing the platelet concentration in PRP was helpful to obtain higher concentrations of PRPr.PART 3 Effect of autologous platelet-rich plasma on treatment of rabbit intervertebral disc degenerationResearch BackgroundAt present,the treatment of disc degenerative diseases is divided into two categories:conservative treatment and surgical treatment.However,the current treatments can only relieve the patients pain symptom,but cannot prevent or reverse disc degeneration.Therefore,researchers have been working to explore effective measures to inhibit disc degeneration or promote disc repair.At present,the promising repair measures are the use of various biological agents,including platelet-rich plasma(PRP)products.Studies have shown that PRP has certain repair functions for bone defects,articular cartilage degeneration or injury,ligament injury,meniscus injury,tendon and soft tissue defects.In vitro experiments show that PRP can effectively promote the animal disc cell proliferation and extracellular matrix metabolism.In actual clinical work,severe disc degeneration is often encountered,and its biological treatment is more difficult.The degeneration model established by nucleus pulposus aspiration can simulate the situation of moderate to severe disc degeneration.There is no report about the repair effect of PRP releaseat on moderate to severe disc degeneration.PurposeIn this study,nucleus pulposus aspiration method was used to establish a model of severe degeneration in the rabbit intervertebral disc,and then PRPr and TGF-β1 were injected into the intervertebral disc to evaluate the effect by imaging,histology,and immunology methods.The repair effect of PRPr and TGF-β1 were compared to explore the better treatment option.MethodSeventy-two New Zealand white rabbits were divided into 4 groups:PRPr group,TGF-β1 group,PBS group and Sham group(n=18).Nucleus pulposus aspiration was used to establish a rabbit disc degeneration model.After 2 weeks of modeling,the second operation was performed,that the agent was injected into the degenerated disc.The first three groups were injected with 20 μl of PRPr,TGF-β(1ng/ml)and PBS.The Sham group was only exposed and punctured without any injection.Lumbar spine X-ray examinations were performed before injection and 1 w,2w,and 4w after injection,and DHI%was measured to indicate the change of the height of disc.The samples were sacrificed at 1w,2w,and 4w postoperatively,and stained with HE,safranin O,and Masson for histological observation.The contents of AGC and COL Ⅱ in the intervertebral disc were determined by ELISA.ResultsAt 2 weeks after the modeling,the DHI%of the intervertebral disc height in the 4 groups all decreased significantly,and the decrease was about 20%.There was no significant difference in DHI%between the 4 groups.Compared with the PBS group or Sham group,the DHI%of the PRPr group or the TGF-β1 group was significantly increased at 2w after injection(P<0.001),and the increase of DHI%continued until 4w(P<0.001).Further analysis revealed that the increase in DHI%in the PRPr group was more significant than the TGF-β1 group.In the PRPr group,the DHI%was further increased at 4w than at 2w after injection,but DHI%did not continue to improve in the TGF-β1 group at 4w and 2w after surgery.Histological observations showed that after PRPr or TGF-β1 was injected into the degenerative disc,the process of disc degeneration gradually slowed down.Both PRPr group and TGF-β1 group showed regeneration in discs.The nucleus pulposus gradually returned to a circular shape,and follicular cells gradually appeared inside again Safranin O staining results showed that proteoglycan content in the nucleus pulposus increased,clustered chondrocyte-like cells increased,and the distribution of collagen fibers in annulus fibrosus became more regular than before.Statistical analysis of histological scores shows that comparing with the PBS group or the sham group,the histological score of the PRPr group began to decline as early as 2 weeks after operation.By 4 weeks after injection,the decrease in scores was still significant.But the histological score of the TGF-β1 group was only statistically significant at 4-week point.Compared with the TGF-β1 group,the scores were lower in the 2w and 4w PRPr groups.ELISA results also confirmed that PRPr or TGF-β1 can promote the synthesis of AGC and COL Ⅱ in degenerated intervertebral discs.At 1w after injection,compared with the PBS group or Sham group,the contents of AGC and COL Ⅱ increased in the PRPr group.With the extension of time,the increase in content continued to be statistically significant.But the increase in COL Ⅱ content in TGF-β1 group was delayed to 2w after operation.The results also showed that the levels of AGC and COLⅡ in the PRPr group were higher than those in the TGF-β1 group from 2w postoperatively and continued to 4w postoperatively.And the content of PRPr group gradually increased after injection.However,there was no significant difference in the content of TGF-β1 at three time points.The AGC and COL Ⅱ content of the PBS group and the Sham group showed a gradual decline.ConclusionThis study confirmed the role of PRPr in repairing and regenerating degenerative discs.In the rabbit disc degeneration model induced by nucleus pulposus aspiration,after the injection of PRPr into the degenerated disc,the disc height,histological morphology,cell number,and extracellular matrix content were all significantly improved.In addition,the repairing effect of PRPr on rabbit degenerative discs is better than that of TGF-β1 alone,which suggests that multiple growth factors in PRPr have a repairing effect on degenerative discs.PRPr has the advantages of high safety,convenient preparation and strong effect,which provides a potential choice for the clinical treatment of disc degenerative diseases in the future. |
