Metrics details Abstract Subtalar osteoarthritis STOA is often secondary to chronic ankle sprains, which seriously affects the quality of life of patients. Due to its etiology and pathogenesis was not studied equivocally yet, there is currently a lack of effective conservative treatments.
Although they have been used for tissue repair, platelet-rich plasma-derived exosomes PRP-Exo have the disadvantage of low retention and short-lived therapeutic effects. PRP-Exo incorporated Gel Exo-Gel system, composed of Poloxamer and mixture-based thermoresponsive hydrogel matrix in an optimal ratio, was determined by its release ability of Exo and rheology of Gel response to different temperature.
Exo released from Exo-Gel continuously for 28 days could promote the proliferation and migration of mouse bone mesenchymal stem cells mBMSCs and chondrocytes, at the same time enhance the chondrogenic differentiation of mBMSCs, and inhibit inflammation-induced chondrocyte degeneration.
In vivo experiments confirmed that Exo-Gel increased the local retention of Exo, inhibited subtaláris osteoarthritis apoptosis and hypertrophy of chondrocytes, enhanced their proliferation, and potentially played the role in stem cell recruitment to delay the development of STOA.
Therefore, it is a major public health problem and a major health care burden, although ankle sprain is often regarded as an inconsequential injury. Unfortunately, ankle sprain is not a onetime injury, which is one of the strongest risk factors for recurrent ankle sprain and chronic ankle instability CAI [ 34 ].
At present, the focus of attention has been on the talocrural joint degeneration caused by lateral ankle sprains. However, due to the similar trauma mechanisms and clinical manifestations of subtalar joint, subtaláris osteoarthritis leads to underdiagnosis or misdiagnosis of anklesubtalar joint complex injury [ 8 ]. To resolve these problems, pathophysiology hosszan tartó hátfájás ankle OA should be elucidated first.
Although such studies substantially promote the development of ankle joint research, the fájdalom jobb oldalon és hátul mechanisms of cartilage homeostasis in subtalar joint subtaláris osteoarthritis intervention for acute lateral ankle sprains are still rarely studied.
Most treatments for osteoarthritis, especially for ankle OA, are palliative and cannot prevent the progression of the disease, nor can it replace degenerated cartilage. At present, there is an urgent need for an effective conservative treatment method that can not only maintain joint function but also relieve pain, so as to improve the quality of life of patients.
Platelet-rich plasma PRP is an autologous blood product obtained by centrifugation of autologous blood. It contains a high concentration of platelets and a large number of growth factors. Various growth factors and cytokines are released from degranulated platelets and play a vital role in joint homeostasis and healing [ 14 ].
In recent years, intraarticular injection of PRP has become a subtaláris osteoarthritis option for knee [ 151617 ] and ankle [ 1819 ] OA. Despite these benefits, one defect that limits the clinical application of PRP is the requirement for autologous platelets. In addition to these growth factors, stimulation of platelets can also cause them to secrete a large number of extracellular vesicles, including exosomes Exo, 40— nm in diameter [ 20 ]. In the past few decades, the discovery of Exo is one of the most revolutionary contributions to cell biology [ 21 ].
They serve as unique carriers of biologically active proteins, mRNAs, and microRNAs, and participate in cell-cell communication. Importantly, recent studies have found that PRP-derived Exo PRP-Exo have great potential in the field of tissue repair and regeneration [ 2223 ], with antiinflammatory effect and positive regulation of cell biological activity [ 24 ].
Moreover, Exo are not immunogenic and have no species difference, which means that the signal carried by Exo can be transmitted across species [ 2526 ]. Due to their low immunogenicity and stability, Exo are the best carrier for clinical nanotherapy.
Considering this immediate subtaláris osteoarthritis of Exo, which will lead to a reduction in therapeutic exposure, scholars have conducted indepth studies on the use of injectable hydrogels Gel to increase the retention of therapeutic drugs [ 272829 ].
Incorporation of Exo in Gel can allow the controlled release of Exo for a longer period of time, which can even further increase the therapeutic exposure and maximize subtaláris osteoarthritis efficacy. Injectable Gel based on synthetic materials such as e. Besides, they may be less susceptible to batch-to-batch variations and the most promising candidate systems to increase the local retention of Exo [ 2730 ].
Previously, a thermoresponsive in situ gel drug delivery system was developed with biosafety poloxamers Poloxamers and , nonionic amphiphilic copolymers composed of a central hydrophobic block of polyoxypropylene PPO flanked by two hydrophilic blocks of polyoxyethylene PEO. Two hydrophilic polyethylene oxides PEO [ 3132 ]. The thermoresponsive property of Gel can be attributed to subtaláris osteoarthritis phase reverse thermal gelation of the poloxamers under certain condition.
However, whether PRP-Exo combined with thermosensitive Gel can prolong Exo retention and enhance therapeutic effects is unknown.
Here, we investigated the use of thermosensitive Gel with poloxamers as a subtaláris osteoarthritis for PRP-Exo delivery, aiming to prolong local delivery of Exo to targeted organs. In this study, PRP-Exo incorporated Gel Exo-Gel prolonged the release of Exo with biological activity, which promoted the proliferation and migration of mouse subtaláris osteoarthritis mesenchymal stem cells mBMSCs and chondrocytes, enhanced the chondrogenic differentiation of mBMSCs, and protected chondrocytes aganist apoptosis and degeneration induced by inflammation in vitro.
Animals All mice in this study were acquired from Gempharmatech China. All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals and protocols, which were approved by the Animal Care and Use Committee of Affiliated Drum Tower Hospital, Medical School of Nanjing University No. These reseeded cells were considered to be the first generation P1and so on as the second P2third P3subtaláris osteoarthritis.
Cell culture of chondrocytes To extract chondrocytes, 1 d-old pups were sacrificed for subtaláris osteoarthritis of cartilage from knees. First, cartilage was into small pieces after washing with PBS. Second, the samples were digested in 0. The released chondrocytes were seeded in T25 cell culture flasks. The culture medium subtaláris osteoarthritis refreshed every 3 days. The data was analyzed by Flowjo 7.
Adipogenic, Osteogenic, and chondrogenic differention of mBMSCs were performed according to standard culture methods, and measured via Oil red O staining, Alizarin Red staining, and Alcian blue AB staining, respectively. These images were obtained through bright-field microscope Nikon TS2, Japan. Chondrogenic differentiation of mBMSCs According to the instructions, induction medium Cyagen, China; containing: TGFβ3 1 mg per ml medium of chondrogenic differentiation was replaced every 3 days.
AB staining and COL II-immunofluorescence were conducted after 2 weeks of culture to evaluation of chondrogenic differentiation ability. Subtaláris osteoarthritis extraction Based on the methods of Tao et al. After centrifugation at g subtaláris osteoarthritis 10 min, platelet-containing plasma was carefully absorbed and transferred to a new centrifuge tube Beckman coulter, USA and centrifuged again at g alatti csípőfájdalom 15 min.
The supernatant plasma was discarded, before the platelet pellet was resuspended in the residual plasma to obtain 4 ml PRP. Then, the supernatant was filtered through a 0. The liquid was washed with PBS and ultrafiltered at g again.
After washing by PBS, Exo suspension was ultracentrifuged again at the same high speed for 70 min. Gel and Exo-Gel preparation The production methods of Gel have been described in our previous report [ 33 ], the total polymer concentration of blank Gel was set to Poloxamer and were added subtaláris osteoarthritis ddH2O according to the ratio to stir and mix evenly at 4 ºC. Then, the Gel in liquid state was fully mixed with extracted Exo, and this Exo incorporated Gel Exo-Gel system was freeze-dried by lyophilizer Christ Alpha, Germany.
After that, it was further detected by SEM Philips.
Liquid Gel was transferred to transwell inserts with 8 μm pore size Millipore and solidifed by raising temperature to The conditioned medium from Subtaláris osteoarthritis µg Exo incorporated in µl thermossensitive Gel incubation for 1 week was obtained by placing the transwell inserts in wells containing 1 ml of medium, and used to coculture with mBMSCs and chondrocytes for multiple functional tests, such as proliferation, migration, differentiation, and apoptosis.
Thermoresponsive release profile of Exo-Gel For release studies, the same amount of Exo-Gel was placed into transwell inserts as described above and incubated in medium at 25 and 37 °C. The supernatant was removed and fresh medium was added at different time-points. Nuclei was stained with Subtaláris osteoarthritis dihydrochloride. Fluorescent images were photographed by Cell imager Bio-tek Cytation1.
Osteoarthritis of the ankle: methods for joint-preservation
After 0, 12 h, 1 d, and 2 d, the wells were washed with PBS thrice, and CCK8 solution 10 µl; dilution subtaláris osteoarthritis fresh culture medium was added to wells and incubated for 2 h at 37 °C. The absorbance was measured at nm with a microplate reader ELx, Bio-tek.
The cell growth curve is hogyan kell kezelni a kéz ízületi gyulladását according to the measured OD value. At last, we added staining solution to each well followed by subtaláris osteoarthritis at room temperature for 30 min.
Nuclei was stained by Hoechst 33, and the images were taken under Cell imager Bio-tek Cytation1. Migration assay Transwell subtaláris osteoarthritis was used to analyze the migration ability of BMSCs and chondrocytes under different treatments.
Following incubation for 24 h, cells that migrated to the lower surface of the filter membrane were stained with 0. Migratory activity was assessed by counting the number of cells. A scratch wound assay was also performed to evaluate cell migration capability. We used a sterile µl pipette tip to scrape the cell layer.
After nyaki fájdalom osteochondrosissal with PBS, the images of each processing groups were recorded at 0 and 24 h after scratching by subtaláris osteoarthritis Nikon TS2. Then, samples were incubated with primary antibodies overnight at 4 °C, followed by Alexa conjugated-goat secondary antibody Jackson ImmunoResearch for 2 h at room temperature. Membranes were then incubated for 2 h at room temperature with the appropriate secondary gyulladáscsökkentő ízületi kezelés. Under anesthetic conditions, CFL, connecting from the apex of the fibular malleolus to the lateral surface of the calcaneus, and ATFL, subtaláris osteoarthritis from the distal anterior tip of the fibula to the lateral talar neck were both excised at its attachment sites.
The lateral ankle capsule, which connects from anterior aspects of fibula to the lateral side of the talus, subtaláris osteoarthritis incised after removal of CFL and ATFL. The entire operation was finished after the suture of subtaláris osteoarthritis incision. Mice were allowed to move freely in the cages and had free access to food and water.
The mice were sacrificed at 4 and 8 weeks after the surgery. Five-µm frontal sections were prepared and stained with safranin O and fast green, HE, toluidine blue TBand alcian blue ABrespectively, according to the standard protocol, and the morphology of the tissue was observed by microscopy Nikon TS2, Japan. Immunohistochemical analysis Ankle samples at 4 weeks postinjury were prepared according to standard dewaxing procedure.
The slices were soaked in citric acid buffer 10 mM citric acid, pH 6. Then, subtaláris osteoarthritis staining was performed using a previously reported protocol. After incubation with the primary antibodies at 4 °C overnigh, the slides were subsequently stained with horseradish peroxidase-conjugated secondary antibody Invitrogen.
- Térdfájdalomcsillapító kenőcs
- Hogyan lehet enyhíteni a térd fájdalmát
- Fájó lábízületek
- Select Page Subtalar Arthritis Subtalar arthritis is a type of arthritis which affects the subtalar joint, that is, the joint found below the ankle joint in the hindfoot.
- Az ízületek és a gerinc kezelésének módszerei
The presence of antigen in cartilage was determined by counting the number of positively stained chondrocytes by microscopy Nikon TS2. Nuclei was stained with DAPI.
Images were captured by using Cell imager Bio-tek Cytation1.
Citation, DOI & case data
Statistical analysis GraphPad 7. All independent experiments were performed in triplicate and all quantitative data were expressed means ± SEM. TEM shows that the diameter of round nanoparticles is between 40 and nm, which is consistent with the literature report [ 20 ] Fig.
The particle size distribution of NTA is similar Fig. In addition, subtaláris osteoarthritis blotting Fig. Since the main bioactive molecules in platelet pellet PPare encapsulated in the Exo [ 20 ], and released after activation, the surface markers of Exo and growth factors in the activated platelet pellet APP barely existed as compared with PRP-Exo Fig. Characterization of Exo-Gel As previously fájdalom a kéz ízületeiben kezelés by our team [ 33 ], the thermossensitive Gel was an ideal injectable carrier to deliver drugs, and the total polymer concentration of blank arthrosis komplexum was set to When Exo were mixed with Gel, Exo-Gel still was reversibly converted from a liquid polymer solution at room temperature into a hydrogel at 37 °C as blank gel, showing a thermoresponsiveness, which may highlight that the presence of Exo does not hamper Hatékony térdkezelés gelation process Fig.
The morphology of Exo loaded in Gel was examined by scanning electron microscopy SEM and the outline of ízületpusztító kezelés nanoparticles could be clearly observed Fig. To study the use of thermossensitive Gel for sustained Exo delivery, µl of Gel with kenőcs helyreállítja az ízületeket of Exo transferred to an insert of a transwell system containing medium in the bottom compartment at 25 and 37 subtaláris osteoarthritis Fig.
In the case of Exo-Gel, the vast majority of Exo released to the medium after 1 day at 25 °C. After increasing the temperature to 37 °C, Exo continually released from Gel to the medium, lasting for about 1 month.
Importantly, Exo have the ability to transfer their biological cargo, including proteins and RNA, between cells after Exo uptake by the recipient cell [ 34 ], which is key for biologically active of Exo. Therefore, to ensure that Exo maintain their integrity after release from Gel, we assessed Exo uptake by mouse bone marrow stromal cells mBMSCs and chondrocytes. S1A, Bflow cytometry Additional file 1 : Fig. S1C--F and three-lineage differentiation Additional file 1 : Fig.
These results indicated that Gel matrix had the ability to sustain the release of Exo without characteristic change for a long period of time at the body temperature.
Exo-Gel promoted proliferation subtaláris osteoarthritis migration of mBMSCs BMSCs have the ability to differentiate into chondrocytes and are a well-known cell subtaláris osteoarthritis for cartilage tissue engineering [ 3536 ]. Therefore, the key step is to stimulate the chemotaxis of a sufficient number of endogenous BMSCs to the injured site.
It has also been shown to induce the migration of BMSCs to other locations, such as the brain [ 42 ] and heart [ 43 ]. The proliferation of cells was examined by CCK-8 analysis Fig. The results revealed that conditioned medium after Exo-Gel incubation markedly increased proliferative capability of cells. Meanwhile, the conditioned medium could promote migration of mBMSCs evaluated using the transwell and scratch wound assays.
These results showed that Exo released by Exo-Gel after incubation for 1 week in vitro still had biological activity and could promote the proliferation and migration of mBMSCs. Exo-Gel promoted chondrogenic differentiation of mBMSCs Previous study has showed a higher amount of growth factors in PRP-Exo [ 20 ], which play an important role in chondrogenic differentiation.
Alcian blue AB staining Fig. In this subtaláris osteoarthritis, TGFβ3 was applied to induce chondrogenic differentiation, and our result csípőízület fáj kezelés that Exo contained a large amount of TGFβ1 protein Fig.
Exo-Gel promoted proliferation and migration of chondrocytes In order to further determine the potential therapeutic effect of Exo-Gel on cartilage repair, we evaluated the growth of chondrocytes by in vitro CCK8 test and EDU analysis Fig.
Transwell and scratch tests showed that Exo-containing culture after Exo-Gel incubation could markedly enhance the migration ability of chondrocytes Fig. In general, these findings indicated that compared with PBS and Gel treatment, Exo-Gel could subtaláris osteoarthritis the proliferation and migration to a greater extent in vitro.
Exo-Gel inhibited IL-1β-induced chondrocyte apoptosis and degeneration Among the cytokines believed to play a role in progression of OA, IL-1β and TNF-α are known to be the predominant mediators of inflammation [ 484950 ], which aggravate the catabolic imbalance in OA cartilage and exacerbate degradation and alterations of articular cartilage matrix.
Conditioned medium after Exo-Gel incubation remarkably decreased the rate of apoptosis of chondrocytes triggered by IL-1β. Western blotting Fig. These results indicated that Exo-Gel could attenuate inflammatory apoptosis during cartilage degeneration. Furthermore, inflammatory cytokines affect the physiological metabolism of chondrocytes by activating catabolic pathways, and promote the imbalance of catabolic and anabolic balance.
Single cell RNA-seq studies in OA, have shown that chondrocytes undergo phenotypic changes and become hypertrophy, leading to cartilage damage and aggravating diseases [ 545556 ].
In addition, hypertrophic chondrocytes produce alkaline phosphatase, leading to subtaláris osteoarthritis calcification and increase the expression of hypertrophic-related gene, type X Collagen COL X [ 57 ]. MMP13 as other main characteristic is also upregulated in hypertrophic chondrocytes [ 5556 ].
Immunofluorescence analysis Fig. The thermoresponsive Gel, due to its viscosity and hardness, provides a natural matrix barrier to lock Exo and prevents its rapid loss. In vitro, we had demonstrated that Exo-Gel served as a temporary repository for the continuous release of Exo without change biological characteristics.
The DiR-labeled Exo was intra-articularly injected with or without Gel in situ to subtalar joint, and then the retention was analyzed using IVIS imaging system in vivo Fig.