| BackgroundCurrently,injured hyaline cartilage is the culprit behind knee pain experienced by millions of people young and old.Resulting from acute trauma,general wear,aging,or disease,cartilage lesions cause intermittent or chronic pain and can be accompanied by swelling,joint locking,the weakening of surrounding muscles,and/or reduced range of motion.Hyalouronic acid(HA)is a glycosaminoglycan which consists of repeating disaccharide units of(1-?-4)D-glucuronic acid and(1-?-3)N-acetyl-D-glucosamine.HA is an important component of the extracellular matrix in the tissues such as skin,corpus vitreum,synovial fluid and cartilage,having unique physicochemical properties and biological functions.Commercial HA is supplied in the form of its sodium salt(sodium hyaluronate,SH)prepared by microbial fermentation or animal-tissue extraction,and now widely used in the area of food,cosmetics and medicine.HA as a substrate for the growth of chondrocytes is an important component of the cartilage matrix,which can promote the metabolism of chondrocytes and stimulate the formation of cartilage matrix as well as maintaining the phenotype of chondrocytes.It is considered to be one of the most suitable materials when constructing a chondrocyte scaffold.The raw HAs with hygroscopicity and high water solubility are due to the polymers with long linear chains.They are easy to be distributed and digested by inherent enzymes or free radicals in the tissues of the body.Injectable HA in its natural form lasts only 1-2 days when applied to a local tissue of the body.The mechanical strength of non-cross-linked HA cannot meet the requirement of a scaffold for tissue engineering.A stable net work of the polymer is formed by cross-linking.Biodegradable and porous material can be prepared by controlling the degree of cross-linking and the process parameters,which would be a potential scaffold for tissue engineering of cartilage.An injectable gel of a cross-linked HA(CHA)with 1,4-butanediol diglycidyl ether(BDDE)as cross-linker was used to treat osteoarthritis(OA),soft-tissue defect and filling derma tissue.The efficacy and safety of those products have been confirmed in clinical trials.However,the value of CHA in tissue engineering hasn't been clarified yet until now.Tissue engineering provides an exciting alternative approach for treating articular cartilage diseases via the development of biological substitutes.Recent reports demonstrated that human stem cells,especially mesenchymal stem cells(MSCs),produced positive outcomes in the treatment of articular cartilage disease.Human MSCs can be obtained from a variety of adult tissues,expanded with ease,and subsequently differentiated into matrix-producing chondrocytes,eventually leading to the formation of hyaline articular cartilage.Due to the potential teratoma formation of pluripotent stem cells(e.g.,induced pluripotent stem cells or embryonic stem cells),they are less preferable compared to MSCs in cartilage tissue engineering.However,cartilage tissue engineering has yet to be proven effective in clinical use.The structural and functional properties of native articular cartilage have not been fully adopted by the tissue-engineered cartilage.Therefore,to date,there is no reliable long-term therapeutic strategy for articular cartilage repair.Conventional therapies(e.g.,microfracture,mosaicplasty and ACI)or traditional therapies(e.g.,joint surgery)possess several shortcomings.In joint surgery,implantation of a prosthetic device is performed to replace the living cartilage tissue,which can marginally rescue joint functions but for merely 10-15 years.This procedure also poses additional risks of post-surgery complications,including infection and inflammation.Thus,an efficient treatment to successfully repair or regenerate articular cartilage tissues is urgently needed.Hesperidin is a natural flavonoid that possesses anti-inflammatory properties in many disease models.For example,hesperidin has been shown in rodent model to reduce inflammation as well as inflammatory pain through suppression of cytokine production,NF-?B activity,and oxidative stress.In a similarly manner,in a mouse model of skin damage induced by ultraviolet B irradiation,hesperidin was demonstrated to inhibit oxidative stress and inflammation,by down-regulation of cytokine production including TNF-?,IL-1?.IL-6 and IL-10.However.the effect of hesperidin on the immune responses during chondrogenesis of MSCs has not yet been reported.PurposeIn this paper,the samples of CHA scaffold were prepared by a method of twice freeze-drying process with BDDE as cross-linker.Physicochemical properties of the scaffold were determined.The effect of the scaffold on cartilage repair was evaluated in a minipig model of full-thickness cartilage defect with microfracture surgery.The study will be the foundation for the application of the CHA scaffold in the area of cartilage engineering.In the future study.we isolated human MSCs and aimed to investigate the effect of hesperidin on chondrogenesis of MSCs for cartilage tissue repair.MethodsThree consecutive parts were included in the study.The experimental procedures were as the following:Part one:Experimental study on preparation of tissue engineering cartilage scaffolds by modified sodium hyaluronate and its physicochemical properties1.1 Preparation of CHA scaffoldThe cross-linked sodium hyaluronate(CHA)scaffold was made of sodium hyaluronate(SH)with BDDE as cross-linker by a method of twice freeze-drying process.CHA scaffold was made into a small disc with a punch of 5 mm in diameter for following surgery.1.2 Detections of physicochemical properties CHA scaffoldThe cross-linked sodium hyaluronate cell scaffold was observed under an optical microscope.The cross-linked sodium hyaluronate cell scaffold was cut into 0.5 cm×0.5 cm pieces with a thin blade,distilled water was added,the scaffold was fully extended and expanded,placed in a freeze dryer,pre-frozen at-20? for 1 hour,and vacuumed for drying.The dried sample is sprayed with gold and observed under a scanning electron microscope.Other physicochemical properties were detected:including the degree of modification of the CHA scaffold;the compression strength for dry samples and wet samples;the water absorption;the expansion ratio;and the porosity.Part two:Application of modified sodium hyaluronate tissue engineered cartilage scaffold in cartilage defects animal model of minipigs2.1 Animal model of full-thickness cartilage defectThe animals used in the present study were 12 common grade minipigs,weighted 20±2 kg,aged 4?5 months,which were provided by Shanghai Nanhui Laogang Huaxing Special Animal Breeding Center.After surgery,right knees were implanted by CHA scaffold,and then restored the normal structure of the joints and closed the wound.While the left knee joints acted as controls,which only restored the structure surgically and closed the wound.2.2 CHA scaffold implantationThe animal's right knee joint cartilage defect was coated with a thin layer of fibrin glue(referring to instructions for use of the product)on the bottom of the defect and around the defect cartilage edge.And then CHA scaffold disc was pressed into the defect,wetting by dripping saline,while using the surgical knife handle pressed lightly,to complete the adhesive fixation.The restoration of the patella with epidermis covering the defects department and knee bending action would further compact the implantation.And then open the defect under the epidermis,displace patellar,check the implant and the edge of the defect adhesion,the joint restoration and suture followed.After the animals were allowed to move freely,standard animal feed,clean drinking water,room temperature maintained at 15-26?,daily UV disinfection and postoperative intramuscular injection of penicillin with 500 million units every morning and afternoon for 3 days to prevent postoperative infection.2.3 Postoperative observationOutside cage observation was carried out once a day in 7days after operation,and once a week later,with a detailed recording of the appearance of signs,behavioral activities,salivation,respiratory conditions,urination and administration of local reactions.2.4 Hematological examinationBlood samples were taken from the veins of animals at 6 months and 12 months after surgery.The blood samples were collected using a Sysmex XT-2000iv automatic hematology analyzer.Centrifuged at 3500r/min for 15min,and then Hitachi 7180 automatic biochemical analyzer was used for serum biochemical detection.2.5 Pathological examinationMacroscopically observation was conducted at 6 months and 12 months after surgery.Three animals were anesthetized with 1%sodium pentobarbital solution,the common carotid arteries were removed and the entire hind legs were removed to reveal the knee joint to observe the defect repair,including the degree of defect repair,surface roughness as well as color change.With the surrounding normal cartilage integration,the border was clear,to make sure weather it with cracks or not.Synovial synovium proliferation was also be detected.After macroscopically photographing,the normal cartilage and subchondral bone of the defect repair site and adjacent undamaged sites were cut with a 8 mm trephine.After the samples were taken,the remaining were stored at-80?.1.5 cm defect-centered cartilage surface tissue and subchondral bone were cut off and fixed in 10%neutral formalin for more than 24h.Decalcified with 10%EDTA decalcification solution for 30 days,and then ethanol gradient dehydration,xylene transparent,vertical paraffin embedding.5?m sections were made and stained by safranin-O according to the conventional method.O'Driscoll's articular cartilage histological score was calculated according to the reported creteria showd in Tablel.2.6 Biomechanical TestingCartilage and normal cartilage samples frozen at-80? were removed,and indentation test was carried out after thawing at room temperature.Young's modulus(MPa),stress relaxation time(s)and creep time(s).2.7 Type II Collagen AssayThe repaired cartilage tissues were completely cutted off with a 6 mm diameter trephine,and also equal amounts of normal cartilage tissues.A small part of these tissues were used for the quantitative determination of type II collagen,meanwhile the rest were used for determination of glycosaminoglycans content.After that,the collagen content was detected by ELISA assay according to the procedure of Porcine collagen II(Col II)Elisa Kit(Elixir Canada Medicine Company Ltd.Hermes Criterion Biotechnology).2.8 Determination of glycosaminoglycan(GAG)contentFresh cartilage samples were lyophilized and the lyophilized tissue was then cut into small pieces,which would be soaked in 95%alcohol for 2-3 hours,then acetone for 2 hours,to be wrapped in filter paper to defat for 16 hours in a Soxhlet apparatus.The degreased sample was dried at 80? for 4 hours and ground to a fine powder.GAG and alcian blue could rapidly generate soluble GAG-Alcian blue complex.The light absorption of the complex was different from alcian blue.Thus the content of GAG could be calculated by colorimetric determination of the content of GAG-alcian blue complex.The content of GAG in the sample was determined by ELISA assay according to the procedure provided by the Porcine glycosaminoglycan(GAG)Elisa Kit(Elixir Canada Medicine Company Ltd.Hermes Criterion Biotechnology).Part three:Study on methods and mechanisms for promoting tissue engineering cartilage scaffold for cartilage repair3.1 Human MSC culturingThe protocol for the use of human cells was approved by the committee of the Second Hospital of Shandong University,and bone marrow cells were harvested from bone fragments of patients from the Second Hospital of Shandong University,with written consent forms acquired from all patients.3.2 Colony formation assayA total of 100 MSCs were plated into a 10-cm petri dish and continuously cultured for up to 21 days.Crystal violet(0.5%,SIGMA,MO,USA)was used to stain the formed colonies for 15 mins followed by counting under light microscope.Colonies larger than 2mm in diameter were counted.3.3 Proliferation assayThe proliferation of cells was assessed by commercial CCK-8 kit(Dojindo,Kumamoto,Japan).In brief,MSCs were plated into 6-well plate followed by various treatments.10ul CCK-8 solution was then added into each well and the chromogenic reaction was carried out at 37? for 15 mins.Microplate reader(Molecular Devices,Sunnyvale,CA,USA)was used to record the absorption at 450nm and relative cell viability was calculated.3.4 Chondrogenesis assaysMSCs were cultured in chondrogenic induction medium(DMEM,0.2 mM ascorbic 2-phosphate,20%FBS,and 10 mM glycerol 2-phosphate)for 14 days,with fresh medium exchanged every 2 days.Chondrogenesis was evaluated using Alcian Blue(Millipore,Billerica,MA,USA)staining.3.5 mRNA extraction and real-time PCRTotal mRNA was extracted using Trizol(Invitrogen,Carlsbad,CA,USA),and reverse transcribed to complementary cDNAs with Superscript II following manufacturer's instructions(Biorad,Hercules,CA,USA).Triplicate PCR reactions were conducted using cyber green-based system(Applied Biosystems,Waltham,MA,USA)with the following conditions:15 seconds at 95?,1 minute at 60? for 40 times.The relative expression levels were calculated using GAPDH as the internal control.3.6 Western blotCell resuspension was prepared in the lysis buffer containing 150 mM NaCl,50 mM Tris-HCl,10 mM HEPES,0.1%NP-40 alternative,0.5 mM NaF,0.25%Na-deoxycholate,1 mM Na3VO4,pH 7.4(Protease Inhibitor Cocktail,Roche,1 tablet/10 ml).Cell lysates were quantitated using BCA protein assays,and 30?g total protein was then run on SDS-P AGE followed by transfer to PVDF membranes.The membranes were subsequently blocked with 1%BSA(bovine serum albumin,Sigma,USA),and incubated with primary antibodies at 4? overnight.Primary antibodies for p65 and GAPDH were both purchased from Abcam.HRP conjugated secondary antibodies were utilized to visualize bands in an ECL-based imaging system.3.7 Enzyme-linked immunosorbent assay(ELISA)The MSCs were completely removed by centrifugation and clear medium was collected for ELISA analysis.The levels of IFN-?,IL-2,IL-4 and IL-10 were measured with the commercially available ELISA kits(Abcam,MA,USA)following the manufacturer's instructions.4 Statistical analysisAll data was analyzed using SPSS 22.0 system(IBM,Armonk,NY,USA),and presented as mean ± standard deviation(SD).The differences between groups were determined by Student's T tests and single factor variance analysis(ANOVA).P values less than 0.05 were considered statistical significant.Results1 Properties of CHA scaffoldsDetections of physicochemical properties show CHA scaffold is a porous spongy appearance with a pore size of 80-150?m under SEM(Fig.1,Fig.2).As shown in Table 2,the degree of modification of the CHA scaffold is(3.10±0.27)%.The compression strength is(14.80±4.06)kPas for dry samples and(0.5762±0,11)kPas for wet samples(Fig.3);the water absorption is(58.6±3.5)%,the expansion ratio is(100.7±4.8)%,and the porosity is(96.5±2.6)%.2.1 General postoperative observationThe animals were basically recovered from anesthesia 3-5 hours after operation.After 24 hours,the animals could stand up,with left hindlimb claudication,while food intake and drinking water were basically normal.Within 15 days after operation,the surgical sites were almost recovered,with the fur was shiny.Meanwhile,there was no secretion around the eyes,nose and ears,as well as o trauma and inflammation in other body parts.In addition,their behavior and gait were nearly normal.without obvious clinical adverse reactions found.2.2 Hematological examination after operationThe hematological factors were evaluated at 6 and 12 months after operation to make clear the influence of the surgery as well as the CHA scaffold implantation.As shown in Table 3,most of the results of routine blood test,hepatorenal function test and biochemical ion test had no significant difference when compared the CHA scaffold implantation group with the control,except for some biochemical ions and lipid factors.What's more,these routine hematological factors tested in all enrolled animals were within normal physiological fluctuations.2.3 Pathological characteristicsThe defected cartilage in the animal knee joint began to repair after operation for 6 months,gradually from the central to the surrounding under macroscopic observation.As shown in Fig.4,we found that after 6 months the cartilage defect was repaired in CHA scaffold implantation group,and junction of the defect and the repaired areas was clear and tight without cracks,but the repaired area showed slightly paler compared to normal cartilage.However,the repaired surface of the defect in control group was snatchy,and the junction of the defect and the repaired areas was not clear.After operation for 12 months,the surface of cartilage in the repair area of the CHA scaffold implantation knee was smooth,and the color was similar to that of normal cartilage.The margin of the repaired area was basically merged with the normal cartilage.There was no obvious boundaries between normal and the repaired areas and the degree of repairation was obviously improved compared with that of 6 months.Whereas there was no significant improvement in cartilage surface and margin after operation for 12 months compared to 6 months in control group.Furthermore,microscopic examination of HE staining on tissues acquired at 6 and 12 months after surgery showed that some chondrocytes were visible on the cartilage surface of the CHA implanted animals.Meanwhile,HE staining showed a dark blue nucleus and a red matrix around the cartilage,with varying degrees of Fibrous tissue repaired(Fig.5).Stain with safranin O,we discovered that the superficial and even deep layers in the repaired cartilage tissue were dyed lightly and unevenly in the implantation group after 6 months while the control group was more shallow and uneven.What's more,the matrix of the repaired area of the implantation group was stained much deeper at 12 months postoperation,but in the control group,only the superficial layer of cartilage and even the middle layer had a slight staining and uneven coloring.(Fig.5)According to the O'Driscoll Articular Cartilage Histology Scoring Criteria,the major histological types,structural features,degeneration of cells and the degenerative changes of adjacent cartilage were evaluated.The results of the composite scores of cartilage repair of the left and right knees are shown in Table 4.The degeneration of the cells and the degeneration of the adjacent cartilage in the implantantion group were higher than those in the control group at both 6 and 12 months postoperation,suggesting that CHA scaffold could reduce the cartilage degeneration induced by injury and might promote the cartilage defect repair ability.2.4 Biomechanical property of the repaired cartilageThe biomechanical property was evaluated by Young modulus,Stress relaxation time and creep time,whose tests were conducted at 6 as well as 12 months after the operation.As shown in Table 5,there was significant difference in biomechanical property among groups in Young modulus,Stress relaxation time as well as creep time both at 6 months and 12 months postoperatively.Our results suggested that the measured values of the repair area of the implanted stent were higher than that of control group means the left knee repair area(Fig.6),which were closer to the measured values of the normal area cartilage,indicating that the implantation of CHA scaffold cartilage was better than the operation control in biomechanical property of the repaired cartilage.Implantation with the CHA scaffold matrix was conducive to promot cartilage repair and improve its compression capacity.2.5 Type II collagen expressionThe content of type II collagen was an indicator for the ability of cartilage repaire.Thus type II collagen expression was detected in repaired cartilage in our study.As shown in Table 6,there was difference in type II collagen expression in in repaired cartilage among groups.The level of type II collagen in CHA scaffold implantation group was higher than that in the control group at 6 months and 12 months,but the values in both groups were significantly lower than normal cartilage at 6 months,while there was no significant difference among groups at 12 months after surgery.Our results suggested that CHA is beneficial to the accumulation of type II collagen in the cartilage tissue.However,the repair of cartilage is still different from normal cartilage within a short time.2.6 Glycosaminoglycan expressionIn order to make clear the level of repaired cartilage,Glycosaminoglycan was evaluated.We found that there were significantly difference among groups in GAG expression evaluated at 6 and 12 months after surgery,indicating that the expression of GAG in repaired cartilage was lower than that of noramal cartilage.Besides,we discovered that GAG content in cartilage of the control group was significantly lower than that of the experimental group(p<0.05).Furthermore,there was no significantly difference in GAG expression of the CHA scaffold implantation group and the normal group evaluated both at 6 and 12 months after surgery.In other words,the content of GAG in repaired cartilage after implanted with CHA scaffold almostly reached the normal level(Table 7).Which suggested that CHA was beneficial to stimulate the accumulation of GAG at the repair site,and the accumulation of GAG increased with time.3.1 Hesperidin improves self-renewal ability of MSCsThe chemical structure of hesperidin was identified as shown in Figure 1A.Here MSCs cells were challenged with different doses of hesperidin(0,1,5 and 10?M),and the self-renewal capacity was assessed by colony formation and proliferation assays.Both the relative number and average size of colonies were significantly increased following hesperidin treatments up to?M(Figure IB,C).Similarly,the cell viability determined using CCK-8 method clearly demonstrated that hesperidin markedly stimulated cell proliferation(Figure 1D).However,high dose of hesperidin(10?M in our system)induced slight inhibition on both colony formation and cell proliferation(Figure 1B,C,D).Thus,5?M of hesperidin was chosen as the optimal dosage for the subsequent experiments in the current study,and to our best knowledge,these findings provided the first evidence that hesperidin improved sternness of patient-derived MSCs.3.2 Hesperidin enhances chondrogenesis of MSCsNext,we sought to evaluate the possible effects of hesperidin on chondrogenesis potential of MSCs.Chondrogenesis was induced in the MSCs for 14 days upon hesperidin treatment.As shown in Figure 2A,Alcian Blue staining showed significant increase of chondrogenesis in hesperidin-treated MSCs.These phenotypic obser-vations were further confirmed at the molecular level by measuring specific chondrogenic marker Sox9,where hesperidin treatment induced evident up-regulation of Sox-9(Figure 2B).Our results clearly demonstrated that,besides self-renewal ability,hesperidin also enhanced chondrogenesis of MSCs.3.3 Hesperidin suppresses secretion of pro-inflammatory cytokinesPro-inflammatory cytokines are essential players in both innate and acquired immune responses.Therefore,we subjected the MSCs in the absence or presence of 5?M hesperidin,and then measured the levels of pro-inflammatory cytokines IFN-y,IL-2,IL-4 and IL-10 in the medium using ELISA.Results clearly indicated that hesperidin treatment inhibited the secretion of all of abovementioned cytokines compared with those of control(Figure 3).3.4 Hesperidin inhibits the expression of nuclear factor kappa B(NF-?B)subunit p65To determine the extent of inflammation,we examined the expression of biomarkers in inflammatory responses,such as NF-?B subunit p65.We treated the MSCs in the absence or presence of 5?M hesperidin,and then examined the effect on expression of p65.We found that both mRNA and protein levels of p65 were significantly reduced by hesperidin treatment(Figure 4A and 4B),indicating that hesperidin was able to inhibit the expression of NF-?B subunit p65.3.5 p65 is required for inhibition of cytokine secretions and enhancement of chondrogenesis by hesperidinWe then questioned whether the inhibitory effect of hesperidin on p65 contributed to the earlier observed suppression on cytokine secretions.To this end,we overexpressed p65 in MSCs,and verified that both mRNA and protein levels of p65 were greatly elevated(Figure 5A and 5B).Overexpression of p65 was able to reverse the hesperidin inhibited secretions of pro-inflammatory cytokines IFN-?,IL-2,IL-4 and IL-10(Figure 6),suggesting that p65 was indeed required for inhibition of hesperidin on cytokine secretions from MSCs.Next,we further examined the effect of p65 on enhancement of chondrogenesis by hesperidin.MSCs were transfected with either empty vector control or p65 plasmid,at day 14 after chondrogenesis induction in the absence(control)or presence of 5?M hesperidin.Overexpression of p65 was able to reverse the enhancing effect of hesperidin in terms of Alcian Blue staining(Figure 7A)and Sox9 expression(Figure 7B),indicating that p65 was also required for enhancement of hesperidin on chondrogenesis of MSCs.ConclusionAll in all,the result of effect of CHA scaffold on cartilage repair shows it may help to promote the repair of defect cartilage,the CHA scaffold implants promote joint repair of bone repair with safety.Implantation of CHA scaffold could improve the compressive capacity of repairing cartilage to a certain extent.In conclusion,the present experimental results preliminarily demonstrated that the implantation of CHA scaffold during microfracture is beneficial for the repair of cartilage and the reduction of cartilage degenerative changes caused by the injury,but more animals still need to be further tested to confirm the effect of cartilage repair.The CHA scaffold may promote cartilage repair when applied in microfracture surgery,and is promising for the application in the area of cartilage tissue engineering.our current study demonstrates that hesperidin serves as a therapeutic agent to effectively enhance chondrogenesis of human MSCs by inhibiting inflammation to facilitate cartilage tissue repair. |
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