| 【Background】There has always been heavy task on military training and operating,the articular cartilage of soldiers will bear excessive pressure and friction in parachuting and marching,which can easily lead to articular cartilage injury,osteoarthritis and even cartilage defects.it has become an important cause of suspension of training and military disability during combat preparation and training.In the meantime,during flight,pilots for fighter aircrafts and combat aerial vehicles will sustain excessive positive acceleration on the stress of environmental load,and the diseases of temporomandibular joint cartilage affects up to33.43%,causing pain and masticatory difficulties,seriously interfering with training and combat missions.Therefore,eliminating or reducing the poor stress load during joint functional movement has become a key point in the treatment of joint diseases and cartilage regeneration and repairing.Based upon this,"braking" has become an important issue in the clinical treatment of joint diseases,it has even become a must especially under the serious situations such as repair of cartilage defect.Even though the gold standard protocol for the treatment of autologous chondrocyte transplantation requires weeks to months on joint immobilization,which will directly increase the pain of patients,the risk of complications,raise the cost of medical treatments and bring the serious burden to families and society.Therefore,the rapid recovery of the movement for patients under the condition of "non-braking" is not only a characteristic guarantee to enhance combat effectiveness in military medicine,but also an important requirement in the field of cartilage repair.In order to solve the above issues,we explore the structure and physiological characteristics of natural cartilage.Articular cartilage is mainly subjected to two aspects of stress during movement,that is vertical pressure and shear friction.First of all,the supporting network structure formed by type II collagen(Col-II)is filled with glycosaminoglycans with HA as the main body to realize the viscoelastic properties of cartilage and provide good compressive properties;at the same time,the internal HA is released and distributed on the surface of articular cartilage under pressure,providing good lubrication and anti-friction properties.After the pressure is relieved,HA will be inhaled back into the cartilage,since the lubrication of HA does not depend on its self-consumption,so that HA can be recycled,reduce HA lose with the losing synovial fluid,and further improve the stability of lubrication.Therefore,only for bionics from our point of view,we believe that it is expected to achieve articular cartilage regeneration with non-braking by constructing a new combination material that simulates the natural cartilage Col-II grid and HA,and establishing the new material on physiological HA cycle in load-bearing,will be benefit to balance the properties of anti-compression and lubrication anti-friction.【Design Concept】The study aims to achieve the regeneration of articular cartilage with non-braking.Based on the previous study:1.Replace Col-II with gelatin material which can be completely degraded in vivo and have excellent biocompatibility.After modification,the gelatin material is crosslinked to form a reticular structure to provide the main scaffold structure.2.Cyclodextrin was modified on HA to form a mechanical response mode of hostguest interaction with adamantane modified on gelatin scaffold,that is,the bond was broken under pressure,HA was detached from gelatin scaffold and released into the articular cavity,and due to negative pressure,HA was absorbed back into the gelatin scaffold as soon as the pressure was relieved,and meantime,the bond was reformed,so that HA was fixed inside the scaffold,thus simulating the physiological HA cycle in the load-bearing natural cartilage,and achieve long-term and stable lubrication.3.With the further use of liquid injection-photosensitive molding technology,the new material is expected to be combined with minimally invasive treatment techniques such as arthroscopy in the future,which is easy to be popularized by grass-roots units.【Purpose】The study intends to achieve the following four goals:1.Construct the above new hydrogels.2.Verify whether the new hydrogel accords with the innovative concepts of physiological HA cycle in biomimetic load-bearing cartilage.3.Evaluate whether the new hydrogel can repair articular cartilage under the condition of "non-braking" in animals.4.Evaluate whether the new hydrogel can quickly restore the ability of animals’ movement.【Methods】Four parts of study were carried out to achieve the aforementioned objectives:1.Material construction and basic performance characterization.The material preparation was carried out according to the design concept.First of all,through organic chemical modification and modification processing technology,gelatin modification,cyclodextrin-adamantane modification,photosensitizer doping and final material assembly and parameter optimization were carried out.Then,the required reaction synthesis pathway would be verified by nuclear magnetic resonance spectroscopy,and the cross-sectional structure of hydrogel under no stress,single impact load and repeated compression would be observed by scanning electron microscope.The injectionphotosensitive molding properties were verified by general observation,and the mechanical properties of hydrogels were evaluated by elastic modulus(including energy storage modulus and loss modulus),viscosity and shear cycle stability.The biological safety of hydrogel was evaluated by systemic toxicity test,stimulation hypersensitivity test and hemolysis test.Thus,the new hydrogel was constructed and assembled were done gradually,and its basic properties were systematically characterized.2.HA cycle and lubrication performance verification.Experiments that operate in vitro and in vivo were combined to verified the existence of HA cycle and stable lubrication and anti-friction properties of the new hydrogel were from different angles.First of all,the release effect of HA in hydrogel was qualitatively observed by repeated pressure experiments in simulated body fluids,then the release effect of HA in hydrogel was quantitatively detected by in vivo imaging fluorescence detection experiment of small animals.And finally,the hydrogel was implanted into animal joints,and the actual effect of hydrogel restoring joint lubrication and anti-abrasive at different time points was tested by friction and abrasive tester + toluidine blue staining.In addition,on the one hand,the effect of hydrogel on the adhesion and growth of chondrocytes was detected by cytological experiment in vitro.On the other hand,frozen section staining was performed after in vivo implantation,and CD44 and COLII were selected for immunofluorescence staining to test the cell recruitment effect of BMSCs.So as to verify whether the new hydrogel conforms to the innovative concept of imitating the physiological HA cycle in cartilage under pressure.3.Evaluation of articular cartilage regeneration under "non-immobilization" mild exercise conditions.Under the conditions of active movement and passive mild movement,the effect of the new hydrogel on the regeneration and repair of cartilage defects in the stress area of rabbit knee joint was evaluated.Firstly,to innovate the animal model,remove the meniscus to simulate the classic cause of joint instability,and make a model in the actual stress zone of the knee joint and the femur during the movement,so as to simulate the joint disease caused by heavy stress.At present,it has always been used in the actual clinic case treatment of cartilage,without removing the etiological factors.Based upon this,the effect of hydrogel on cartilage repair was evaluated qualitatively through general observation;the repair of articular cartilage and subchondral bone was quantitatively detected by MRI and Micro-CT imaging detection;the regeneration effect of hydrogel was quantitatively analyzed by histopathology study through chemical staining and immunohistochemical staining;the concentration of HA in synovial fluid was detected by ELISA to evaluate the recovery effect of HA physiological circulation of joint itself after hydrogel treatment.The effect of the new hydrogel on articular cartilage regeneration under non-immobilization conditions was systematically evaluated from the aspects of gross appearance,imaging,histopathology and joint fluid biochemistry.4.Evaluation of the effect of rapid recovery of exercise capacity of animals under extreme exercise conditions.Under the condition of passive extreme exercise,the speed and degree of movement function recovery of rats treated with the new hydrogel were evaluated.First of all,the animals with similar movement ability were selected for this part of the study.After modeling,the small animal running wheel system was used to carry out passive extreme exercise with 15 min twice a week and 0.12m/s rotational speed(this parameter was optimized by screening).The recovery effect of extremely running time and distance were detected after hydrogel treatment in first,second and the third months.Then,the gait analysis test was used to detect the gait difference between the model side and the healthy side,in order to eliminate the effect of compensatory exercise on the healthy side.In addition,the expression level of classical inflammatory factors in animals was detected by serological test to evaluate the general condition after hydrogel treatment.The effect of the new hydrogel on rapid recovery of animal exercise ability was evaluated from three aspects:extreme movement ability,gait analysis and serum biochemistry.Finally,the regeneration of articular cartilage under extreme exercise conditions was analyzed qualitatively and quantitatively by chemical staining and immunohistochemical staining histopathology【Result】1、(1)Detection of synthetic path and composition: The new hydrogel has the designed components such as modified gelatin,cyclodextrin-adamantane and so on,verified by nuclear magnetic resonance spectroscopy.(2)The observation of the structure under different forces shows that the new hydrogel has a uniform reticular porous structure without stress,and its cavity is uniformly filled with HA;the microstructure has no obvious change with good impact resistance under a single impact load;when repeated pressure,the reticular structure is maintained well,and the internal HA is released.(3)Injectablephotosensitive molding performance verification: the new hydrogel is liquid at room temperature with certain fluidity and good operation precision.It can be cured after 2min irradiation at 365 nm wavelength UV,and the effective curing depth is about 3mm.The overall operation parameters are similar to the filling process of dental resin.(4)Test of basic mechanical properties: the new hydrogel has the characteristics of viscoelastic resistance and good shear cycle stability.(5)Biosafety test: the new hydrogel will not lead to systemic toxicity,stimulation of hypersensitivity and hemolysis in vitro,and has high biological safety.2、(1)Detection of HA release in vitro: The fluorescence range can directly reflect the release of HA labeling with fluorescence.In the simulated body fluid,the initial fluorescence range is set at 100%.After continuous cyclic pressure,HA can overflow from the hydrogel surface,and the fluorescence distribution range spreads to 204%.After the pressure is removed,it falls back to 148%,and the overflow fluorescence is concentrated around the hydrogel and rarely dissolves in the simulated body fluid.(2)Detection of HA release in vivo: The new hydrogel was implanted into the rat joints,and quantitative analysis with in vivo fluorescence imaging technology of small animals showed that the fluorescence intensity also showed periodic reciprocal changes that increased during joint movement and fell back at rest.It also reflects the cyclic release performance of HA.(3)Detection of lubrication and anti-friction properties in vivo: The samples were taken 1,2 and 3 months after the hydrogel was implanted in vivo,and it was found that the new hydrogel had good lubrication and anti-friction properties in the first month,and was no different from normal cartilage in the second month.And it can still be maintained at 3 months,reflecting the lubrication and anti-friction properties of quick and long-lasting effect.(4)Detection of cell effect: The new hydrogel can promote the adhesion and growth of chondrocytes,and the trend is more significant in three-dimensional culture.Meanwhile,hydrogel was implanted into the femoral joint of SD rats,and mechanical stimulation was performed once every two days.After 2 weeks,the samples were taken for frozen section staining.It was found that CD44+ cells in the cyclic release group could penetrate deep into the material,while cells in the physical blend group mainly stayed on the surface of the material.At the same time,COLII expression was higher in the cyclic release group,indicating a better promotion of chondroblast differentiation.3、(1)The animal model is successfully constructed,and the conditions of active movement,passive and mild movement are given to provide a stable model basis for followup detection.(2)General observation: after treating with the new hydrogel,the cartilage in the modeling area was relatively flat in 1 month,but it could clearly distinguish between the hydrogel and the surrounding natural tissue;the boundary was blurred in 2 months;in 3months,the boundary could not be distinguished under the light microscope,there was smooth surface,and high glossiness,and no obvious abrasion and edema in the cartilage of lateral tibial plateau.(3)Imaging evaluation: continuous MRI examination in vivo showed that the new hydrogel could connect with the surrounding area at 2 months to form a smooth and continuous cartilage image;at the same time,Micro-CT showed that the supporting subchondral bone was flat and continuous at 3 months,and the trabecular structure was intact.It also reflects that the new hydrogel can repair articular cartilage under non-braking conditions.(4)Biochemical detection of articular fluid: the new hydrogel can quickly supplement HA at the initial stage,but it is different from the sudden release and depletion of HA after modeling in the control group.It can maintain the stability of HA content in synovial fluid for a long time,reflecting the physiological function of its regenerated cartilage,which is of great significance for the long-term effect.4、(1)Detection of the recovery of animals’ movement: exercise training was carried out according to the plan one week after operation(after the skin and joint capsule wounds healed).The extreme running time and distance of the animals were measured in 1,2 and 3months.It was found that the new hydrogel could lasting 30 min of with the distance of more than 200 m,which was up to 2 times that of the untreated group,and there was no significant difference between the new hydrogel and healthy rats.(2)Gait analysis: in order to exclude the recovery of motor ability caused by compensation of the healthy side,the gait of the treated side and the healthy side of each group was analyzed.It was found that the new hydrogel had a significant effect after one month of treatment,and the Achilles tendon function index approached to normal after 3 months.(3)Histopathological examination:Compared with the mild exercise condition,the results of this part of the study more highlighted the advantages of cartilage regeneration in the cyclic release group under nonimmobilization conditions.(4)Serological test: there was no significant difference in IL-1βand TGF-β1 between the new hydrogel and normal rats,and the general condition was stable,which was of positive importance for long-term recovery.【Conclusion】1.According to the design concept,the new hydrogel can be constructed and assembled gradually,which has a hierarchical structure of modified gelatin porous grid and internal uniform filling of HA,with the characteristics of viscoelastic pressure resistance,and has the advantages of injectable-photosensitive molding and high biosafety.2.The new hydrogel has the characteristics of releasing HA after compression,efficient lubrication,reabsorption of HA after force removal,and long-lasting efficacy,which accords with the innovative concept and design of physiological HA cycle in imitating cartilage load-bearing capacity.3.After treating with this new type of hydrogel,the articular cartilage of animals can be repaired under the condition of "non-braking" of active movement,passive and mild movement,clear organizational structure,and make the supporting subchondral bone flat and continuous.The HA metabolism of joint physiological state can be restored after treatment,which is of great significance for the long-term effect.4.After treating with this new type of hydrogel,animals can move in a short time,the exercise time and distance are basically restored,and there is no significant difference in motor ability between the treated side and the healthy side,that is,the recovery of motor ability does not depend on the compensation of healthy bodies.It is a real sense of rapid recovery of motor function.【Innovation】This study aims to achieve the regeneration of articular cartilage without immobilization.The innovations are as follows:1.Provided a new idea: a new bionic idea of self-lubrication to simulate the physiological HA cycle in the above natural cartilage load bearing process,and guide the design of materials.2.Developed a new material: self-lubricating hydrogel was designed according to the guidance of bionic thinking,which has the function of cyclic release and reabsorption under HA stress response.3.Achieved a new effect: the regeneration of articular cartilage can be realized under the condition of "non-braking" mild movement,and the motor function can be quickly restored under the condition of passive extreme movement,providing a new scheme for rapid repair in wartime. |
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