| BackgroundTendinopathy is an occupational disease,but also the most common sports injury disease.The incidence is high.It is one of the common injuries in foot and ankle surgery,sports medicine,rehabilitation medicine and other disciplines,which seriously affects people's daily work and life.The current treatment methods are mainly conservative treatment and surgery.The methods of conservative treatment include local braking rest,weight reduction training,oral steroidal anti-inflammatory drugs,physical therapy,traditional Chinese medicine,stem cell therapy and so on;Patients,ineffecting and getting worse,can be treated with surgery.These conservative treatment methods have many adverse reactions during the treatment process,and the treatment effect is poor.The pathogenesis of tendinopathy has not been clearly determined so far.The use of tendon overstretching and abnormal differentiation of tendon stem cells are the more recognized mechanisms of tendinopathy.Platelet-rich plasma(Platelet-rich plasma,PRP)local injection can effectively promote the repair of Achilles tendon tissue injury.But there is no clear definition of clinical application of PRP,so different patients and different doctors will produce different results,and even diametrically opposite results.Therefore,perfecting the clinical use of PRP is a scientific problem that we urgently need to solve.Three assumptions were proposed in the topic:First,PRP can promote the differentiation of TSCs into tendons under the conditions of normoxia and hypoxia,;Second,The repair effect of rabbit Achilles tendinopathy has a concentration correlation with the injection of PRP;Third,the repair effect of PRP on rabbit Achilles tendinopathy is related to the injection site(around the tendon or/and within the tendon tissue).Objective(1)Isolating and identifying rabbit tendon-derived stem cells,and verifying the effect of different concentrations of PRP and PRP on the differentiation of TSCs into tenocytes under hypoxic conditions;(2)To evaluate the therapeutic effect of different concentrations of PRP injected in the same site and the same concentration of PRP injected in different sites on the repair of rabbit Achilles tendinopathy.Methods1.Isolation,culture and identification of rabbit tendon stem cells: New Zealand white rabbits from 1 week to 1 month old were taken from both hindlimb Achilles tendon tissues to isolate and culture tendon stem cells with multi-directional differentiation potential.Immunofluorescence detection technique and induced differentiation to measure tendon stem cell self-renewal,multi-directional differentiation ability and expression of stem cell marker protein.2.The role of PRP in inducing tendon differentiation of TSCs under hypoxic environment: the experiment was divided into group ?-blank control group,group?-5% PRP co-culture group,group ?-10% PRP co-culture group,?-20% In the PRP co-culture group and the V-40% PRP co-culture group,complete media containing different concentrations of PRP was co-cultured with tendon stem cells for14 days,and their tendon formation,adipogenesis,osteogenesis and bone formation were detected by Western blot and RT-PCR technology.The relative expression of cartilage marker proteins and genes;under normoxic and hypoxic conditions,the experiment was divided into hypoxic group,hypoxic-PRP group,normoxic group,normoxic-PRP group,containing 20% concentration of PRP The complete medium was cultured with tendon stem cells for 72 hours.Flow cytometry was used to detect the apoptosis.Western blot and RT-PCR were used to detect the relative expression of the proteins and genes of tendon,adipogenesis,osteogenesis and chondrogenic markers.3.Evaluation of the therapeutic effect of different concentrations of PRP on the repair of rabbit Achilles tendinopathy model: A type I collagenase was used toestablish a model of rabbit Achilles tendinopathy,and the experimental rabbits were randomly divided into 6 groups: Group A-blank control group,Group B-model control group,Group C—5% PRP injection group,Group D—10% PRP injection group,Group E—20% PRP injection group,Group F—40% PRP injection group,blank control group and model control group were injected with the same amount of normal saline,Under the guidance of ultrasound,PRP of different concentrations inject accurately into the rabbit Achilles tendon for 4 weeks.The rabbit Achilles tendon tissue was taken through imaging,histology,western blot and RT-PCR technology to observe the arrangement and Morphology,detecting the relative expression of tendon,adipogenesis,osteogenesis and cartilage marker proteins and genes.4.Evaluation of the therapeutic effect of PRP injection on the repair of rabbit Achilles tendinopathy model in different parts: The experimental rabbits were randomly divided into 4 groups: group a-blank control group,group b-20% PRP intratendon injection group,group c-20% PRP peri-tendon injection group,group d—20% PRP intra-tendon combined with peri-tendon injection group,Under the guidance of ultrasound,the same concentration of PRP was injected accurately for 4weeks,and the rabbit Achilles tendon tissue was taken by imaging,histology,western Blot and RT-PCR techniques were used to observe the arrangement and morphology of the collagen fibers of the Achilles tendon,and to detect the relative expression levels of the marker proteins and genes of tendon formation,adipogenesis,osteogenesis and cartilage formation..Results1.Tendon stem cells derived from rabbit was isolated and extracted successfully.The isolated and extracted cells have a high value-added ability.After analysis by flow cytometry,the expression of TSCs surface markers CD29,CD44 and CD90 was positive,and the expression of CD34 and CD45 was negative.Immunofluorescence results showed that TSCs expressed SSEA-4,Oct-3,Nanog,Nucleostein positive,and the isolated cells had the ability to differentiate into osteoblasts and adipocytes.2.The cells of each group grew well along on the wall,and the cell morphology was long shuttle and polygonal,with strong adherence.When co-cultured with 20%concentration of PRP,the relative expression of Collagen ?,Collagen ?,TNC,TNMD proteins and genes was highest in tendon stem cells,and the relative expression of osteogenic,cartilage and adipogenic marker genes was basically the same at 40% concentration.The relative expression of Collagen ?,Collagen ?,TNC,TNMD proteins and genes of tendon stem cells did not show a concentration-dependent relationship when the PRP was co-cultured,but the growth ability of the cells decreased,the cell morphology changed,and apoptotic cells appeared in the culture medium.;The results show that 20% concentration of PRP is the most beneficial for TSCs to differentiate into tendon cells,and reduces the possibility of abnormal differentiation.3.Under hypoxic environment,the relative expression of Collagen ?,Collagen ?,TNC,TNMD proteins and genes of TSCs is reduced,the relative expression of osteogenic marker genes is increased,and the relative expression of cartilage and adipogenic marker genes is basically No difference.After co-cultivation with PRP,the relative expression of Collagen ?,Collagen ?,TNC,TNMD protein and genes increased under hypoxic environment,the relative expression of osteogenic marker genes decreased,and the relative expression of cartilage and adipogenic marker genes was basically No difference.The results show that PRP is more conducive to the differentiation of tendon stem cells towards tendon cells in a hypoxic environment and corrects the abnormal differentiation of tendon stem cells.4.After injecting the same amount of PRP with different concentrations in the same site,according to the imaging,histology,molecular biology and other test results,it shows that the collagen in the group E is parallel and ordered,and the Collagen?,Collagen?,The relative expression of TNC,TNMD proteins and genes increased;the repair effect of rabbit Achilles tendon in Groups C,D,and F was no different from that of the control group,indicating that 20% concentration of PRP had the best repair effect on rabbit Achilles tendinopathy.5.After injecting 20% PRP in different parts of the Achilles tendon,according to the imaging,histology,molecular biology and other test results,it shows that the collagen in the Achilles tendon is arranged in parallel and orderly in Group d,and Collagen?and Collagen?,The relative expression of TNC,TNMD protein and genes increased;the repair effect of rabbit Achilles tendon in groups b and c was no different from that of the control group,indicating that the combined injection of intra-tendon and peri-tendon was significantly better than injection.Conclusion1.TSCs was isolated and identificated successfully is helpful for us to understand the pathophysiology,pathogenesis and repair process of tendon at the cellular level,so that we can treat tendinopathy more accurately and effectively;2.In the case of co-cultivation of TSCs with platelet-rich plasma in vitro,it is conducive to the differentiation of tendon stem cells into tendon cells,and the best PRP induction effect at 20%;3.Platelet-rich plasma can effectively treat tendinopathy,and it is correlated with the concentration.At 20% PRP concentration,repairing of rabbits' tendon has the best effect;4.The effect of platelet-rich plasma treating tendinopathy is related to the injection site,and the effect combining intra-tendon and peri-tendon injection is significantly better than single-site injection. |
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