Human knee cartilage specimen assortment
The pattern assortment process was authorized by the ethics committee of Shanghai Ninth Folks’s Hospital affiliated to Shanghai Jiaotong College College of Drugs (SH9H-2021-T401-4). Human cartilage samples have been collected from sufferers who supplied signed knowledgeable consent. Throughout whole knee arthroplasty (TKA) procedures, samples have been collected from human tibial plateaus. Osteochondral plugs with a dimension of 0.5 cm × 0.5 cm have been drilled from the broken (OA) or intact (comparatively wholesome) elements of the tibial plateau. These eliminated plugs have been then fastened in 4% paraformaldehyde for subsequent histological evaluation utilizing Safranine O/Quick Inexperienced staining and FGF18 immunohistochemistry.
Chemically modified mRNA (mRNA) synthesis and formulation
mRNA encoding FGF18, Fluc and EGFP gene have been synthesized with a T7 RNA Polymerase in vitro transcription (IVT) package purchased from Yeasen, China (10673ES). The UTR data was listed within the Supplementary Desk 2. Throughout mRNA transcription, uridine was changed by N1-methyl pseudouridine (m1ψ). IVT response particulars, capping and tailing of mRNA and subsequent purification of mRNA was carried out based on the protocols of kits purchased from Yeasen, China (10673ES) and Beyotime, China (R7075). Focus of mRNA have been quantified by Nanodrop spectrophotometers (Thermo Fisher Scientific, USA). A chimeric UTR is designed based mostly on the globin and gp130 related protein, which is called as synUTR.
Lipid nanoparticles manufacturing and characterization
Lipid nanoparticles have been composed of 4 completely different lipids together with SM-102, 1,2-distearoyl-sn-glycero-3-PC (1,2-DSPC), ldl cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) purchased from Cayman Chemical (LNP-102, USA) at lipid molar ratios of fifty:10:38.5:1.5 respectively, dissolved in absolute ethanol. mRNA was dissolved in 50mM sodium acetate, pH 5.0, and combined with ethanolic lipid combination with an ethanol: aqueous ratio of 1:3. Mixing process was carried out with a microfluidic machine with a Y-channel design. After combination, LNPs have been dialyzed in impartial buffer towards 1000 volumes of buffer in a single day. Encapsulation effectivity was greater than 90% for all LNP-mRNAs on this research decided by Ribogreen. Particle dimension and zeta potential of LNPs have been evaluated utilizing dynamic gentle scattering (DLS) instrument from Malvern Devices Ltd. (ZS-90) and Bettersize Instrument (BeNano 90). A specific amount of pattern was dispersed in deionized water (0.1% w/v), adopted by sonication for 3 min. The measurements have been carried out thrice, and the common values have been calculated.
Cryo-transmission electron microscopy (Cryo-TEM), TEM and scanning electron microscopy (SEM) analyses
For cryo-TEM, a polymer-coated carbon-reinforced copper grid was handled with a small drop (roughly 3 μl) of mRNA-LNPs resolution for 1 min, blotted for five s, and swiftly plunged into liquid ethane cooled by liquid nitrogen. This course of was carried out at 8 °C and 100% humidity utilizing a Vitrobot Mark IV from FEI, Thermo Fisher Scientific. The grids have been subsequently imaged with a Talos Arctica system at 200 kV, using a Falcon 3EC detector and EPU for knowledge assortment.
For TEM, the samples have been fastened with 2.5% glutaraldehyde and saved at 4 °C for 12–24 h. Subsequently, fastened samples have been dissolved in 1% osmium tetroxide for 1–2 h and washed thrice with PBS for 15 min every time. The samples have been then dehydrated and infiltrated with a gradient focus of acetone for embedding. The resin blocks have been minimize into 70–90 nm ultrathin sections utilizing an ultramicrotome, adopted by retrieval of the sections utilizing copper grids. Sections have been stained with uranyl acetate for 8–10 min and lead citrate for 8–10 min. After drying, the samples have been noticed and pictures have been captured for evaluation utilizing a transmission electron microscope. For SEM, samples have been fastened, dehydrated and coated with 10 nm of gold earlier than SEM scans (Gemini300, Carl Zeiss).
Cartilage infiltration assay
Recombinant FGF18 was labeled by Cy3 (BRC-15, Conflure Biology) with protocol “NHS Ester Labeling of Amino-Biomolecules, www.lumiprobe.com”. In brief, Cy3 NHS ester was combined with FGF18 in a 0.1 M sodium bicarbonate buffer and incubated in darkish for 8 h. LNP was labeled with Dil (MedChemExpress, HY-D0083) resolution with a focus of 100 μM and incubated for 8 h at nighttime. Cy3-FGF18 and Dil-LNPs have been purified utilizing dimension exclusion chromatography columns to take away free dyes (Thermo Fisher Scientific, 89882). For cartilage infiltration assay, Cy3-FGF18 and Dil-LNPs have been intra-articularly injected to younger (3 m) and outdated (18 m) mice. Knee joints have been collected after 24 h for subsequent frozen sections. Nuclei was stained with DAPI and pictures have been taken with a confocal microscopy.
Animals
All animal experiments have been performed in accordance with the rules authorized by the ethics committee of the Shanghai Ninth Folks’s Hospital affiliated to Shanghai Jiaotong College College of Drugs (SH9H-2023-A856-1). C57BL/6J mice of wild-type have been obtained from the animal middle of Shanghai Ninth Folks’s Hospital. The mice have been housed in particular pathogen-free (SPF) cages below commonplace situations, with a temperature of 24℃, a humidity of 60%, and a 12-hour light-dark cycle. They have been supplied with a normal food regimen.
For the DMM surgical procedure, 10-week-old C57BL/6J mice have been chosen and anesthetized with isoflurane. The precise knee joint was uncovered, and the medial meniscotibial ligament was transected to induce destabilization of the medial meniscus, following a beforehand established protocol [56]. 500ng FGF18 protein (20 μL, 25 μg/mL) or 6 μg LNP-FGF18 mRNA (20 μL, 300 μg/mL) was injected intraarticularly in the precise knee as soon as every week after surgical procedure. After eight weeks post-surgery, the mice have been euthanized, and the precise knee joint was collected. The joints have been fastened in 4% paraformaldehyde (PFA) for 48 h after which transferred to 75% ethanol for subsequent micro-computed tomography (micro-CT) and histological evaluation.
For the senile osteoarthritis (OA) mannequin, C57BL/6J mice have been fed a traditional food regimen and allowed to age till 24 months, with 3-month-old mice serving as controls. 500 ng FGF18 protein (20 μL, 25 μg/mL) or 6 μg LNP-FGF18 mRNA (20 μL, 300 μg/mL) was injected intra-articularly in knee joints of 18-month-old mouse as soon as every week for 2 months. Proper decrease extremities have been collected and glued for additional radiological and histological evaluation.
Gait analyses
Gait analyses was evaluated with the CatWalk gait evaluation system (Noldus Know-how, Netherlands) after eight weeks post-surgery or senile mice at 24-month-old [57]. Mice have been pretrained to get used to crossing the walkway for thirty minutes earlier than gait recording. The recordings have been carried out in a darkish surroundings. Gait evaluation was recorded utilizing a video-based system. The paw, upon contacting the glass plate, mirrored inexperienced gentle to a video digicam, which recorded all the run at a frequency of 100 Hz. The identical investigator performed all the gait evaluation experiments with blindness to grouping. Each hind and entrance paws have been assessed for knowledge evaluation. Compliant runs have been outlined with a most velocity variation of 60%. Runs with backing, sniffing, cleansing, or lengthy stops have been excluded. Three compliant runs have been required for statistical evaluation. CatWalk software program will mechanically label all areas and determine and assign them to the respective paw. The next parameters have been analyzed: max contact space, print space, imply depth, and obligation cycle (share of stance part in a step cycle).
Von-frey check and hotplate check
The Von Frey check was performed to measure the ache threshold within the hindlimbs of mice utilizing an digital Von Frey filament (Xinruan Know-how, China). Previous to the check, every mouse was allowed to acclimate to the check chamber for 30 min. A constructive response was recorded if the animal exhibited any nociceptive conduct, resembling paw withdrawal, shaking, or licking. The edge pressure exerted by the digital filament was recorded, with a minimum of three replicative forces measured for every mouse in every check. The Von Frey check was carried out each two weeks after the DMM surgical procedure.
For hotplate check, mice have been subjected to the hotplate check at a temperature of 55 °C. The response time was recorded because the length between the hindlimbs touching the plate and response behaviors resembling paw shaking, paw licking, or leaping. The hotplate ache assay was carried out each two weeks following the DMM surgical procedure. No less than three replicative response instances have been recorded for every mouse. Each Von Frey or hotplate checks have been performed by an observer who was blinded to the grouping of mice.
Micro-CT
Knee joints from mice have been fastened for 2 days after which scanned utilizing a high-resolution micro-CT scanner (Skyscan 1072, Belgium) with a pixel dimension of 9 μm, a 55 kVp supply, and a 145 μAmp present. Osteophyte quantity, bone quantity/tissue quantity (BV/TV), trabecular quantity (Tb. N), trabecular thickness (Tb. Th) and trabecular separation (Tb. Sp) of subchondral bone have been calculated with CTAn software program (Bruker, Germany). Three-dimensional reconstructions of subchondral bone sections have been produced with CT-Vox software program (Bruker, Germany), as described in our earlier research [58].
Histological evaluation
For histologic evaluation, knee joints and human specimens have been fastened, decalcified, dehydrated, and embedded into paraffin. Serial tissue sections of a thickness of 5 μm have been minimize in a sagittal or coronal airplane. Safranine O/Quick Inexperienced staining was carried out to evaluate cartilage degeneration of tibial and femur cartilage utilizing the Osteoarthritis Analysis Society Worldwide (OARSI) scoring system [59]. Hematoxylin and eosin (HE) staining was additionally carried out to judge synovial irritation utilizing the synovitis rating [60, 61].
For immunohistochemistry (IHC) staining, the sections have been incubated with a major antibody towards FGF18 (11495-1-AP, 1:200, Proteintech), Ki67 (1:200, ab16667, Abcam) and LC3 (14600-1-AP, 1:100, Proteintech). Photos have been captured utilizing a Zeiss DM4000B high-resolution microscope, and quantitative evaluation was carried out utilizing the ImageJ software program. Optimistic cells have been counted by two completely different researchers (KK and BL). Common variety of constructive cells calculated by the 2 researchers was used as the ultimate rely of constructive cells. This quantity was then divided by the entire variety of cells within the part to find out the proportion of constructive cells.
In vivo bioluminescence
On days 1, 2, 3, 4, and 6 after injecting LNP-Fluc mRNA into the precise knee joint cavity of C57BL/6J mice, bioluminescent imaging was carried out with an IVIS Spectrum system (PerkinElmer, USA). To stop interference from dark-colored fur, the hair on the decrease limbs of the mice was eliminated. D-luciferin was diluted in phosphate-buffered saline (PBS) and injected intraperitoneally at a dose of 150 mg/kg. After 20 min, the mice have been anesthetized utilizing isoflurane. Photon emissions from the precise decrease limb have been collected with a 1-minute publicity time. Luminescent sign was overlaid onto the mouse picture, and the areas of curiosity have been delineated utilizing Dwelling Picture software program (PerkinElmer, USA) to calculate the sign flux throughout the areas.
For assessing the metabolic pathway of LNP-mRNA, bioluminescent imaging was carried out on mice 24 and 48 h after injecting LNP-Fluc mRNA into the knee joint cavity of C57BL/6J mice. D-luciferin was injected intraperitoneally at a dose of 150 mg/kg. After 20 min, the mice have been euthanized, and their hearts, livers, spleens, lungs, and kidneys have been collected and imaged utilizing the IVIS imaging system with a 1-minute publicity time. Dwelling Picture software program was used to detect the sign flux throughout the areas of curiosity.
Cell tradition and reagents
Murine major chondrocytes have been remoted from new child C57BL/6J mice following a beforehand established protocol [62]. Major chondrocytes have been cultured in Dulbecco’s Modified Eagle Medium/Nutrient Combination F12 (DMEM/F12) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The ATDC5 cell line, an immortalized mouse chondrocyte cell line, and HEK293T cells have been obtained from the Cell Financial institution of the Chinese language Academy of Sciences (Shanghai, China). ATDC5 chondrocytes have been cultured in DMEM with 4.5 g glucose/L, 5% FBS, and 1% penicillin-streptomycin. 293T cells have been cultured in DMEM with 4.5 g glucose/L, 10% FBS, and 1% penicillin-streptomycin. IL-1β and TNF-α protein was bought from Genscript. Recombinant human FGF18 protein was bought from Sino Organic (13206-H08H). 3-Methyladenine (3-MA) was bought from TargetMol (T1879). Lipofectamine 3000 was bought from Thermo Fisher Scientific, United States. 10 ng/mL IL-1β, 10 ng/mL TNF-α and 5 mM 3-MA have been used to incubate chondrocytes.
Dot blot
Dot blot assay was used to judge the protein expression of FGF18 in cell tradition supernatants. After transfecting ATDC5 or 293T cells with LNP or plasmid for twenty-four h, cell tradition supernatants have been collected and centrifuged at 3000 rpm for 15 min to acquire the supernatant. Then, 30 μl of the supernatant was noticed onto a nitrocellulose membrane, and 10 μg and 1 μg of rhFGF18 have been noticed as constructive controls. After full absorption of the liquid on the membrane, it was blocked with 5% BSA (Bovine Serum Albumin) for 60 min. After washing thrice with TBST (tris-buffered saline and Tween 20), the membrane was incubated in a single day at 4 °C with the first antibody towards FGF18 (1:1000, 11495-1-AP, Proteintech). On the next day, after washing thrice with TBST, the membrane was incubated with the corresponding fluorescent secondary antibody at room temperature for two h. The membrane was then visualized utilizing the Odyssey V3.0 picture scanner (Li-COR. Inc., Lincoln, NE, USA). The fluorescence depth of the supernatant dots and the rhFGF18 dots have been measured utilizing ImageStudio Lite software program (Li-COR) to find out the expression degree of FGF18 protein within the supernatant.
Luciferase reporter gene assay
Cells have been seeded in a black-bottom 96-well plate (BS-MP-96B, Biosharp) and transfected with completely different concentrations of LNP-Fluc mRNA. At completely different time factors after transfection (24 h, 48 h, 72 h), the firefly luciferase exercise was measured utilizing the firefly luciferase reporter gene assay package (RG051, Beyotime). 100 μl of the assay reagent was added to every nicely of the 96-well plate and incubated at room temperature for five min. Subsequently, chemiluminescent indicators have been detected utilizing a multifunctional microplate reader.
Plasmid and lentivirus transfection
FGF18 plasmids with completely different UTRs have been synthesized by Genecefe Biotechnology Inc. China. Sh-FOXO3a lentivirus was bought from Obio expertise, China. Plasmid was transfected with the protocol of Lipofectamine 3000 and the protein expression was detected at 48 h after transfection. For lentiviral an infection, we added lentivirus at a multiplicity of an infection (MOI) of 10 and incubated the cells. After 48 h of an infection, the media was changed, and the efficiently transfected cells have been chosen utilizing puromycin to determine a steady FOXO3a knockdown cell line.
Micromass tradition
After digestion, centrifugation, and suspension, a suspension of ATDC5 chondrocytes was ready at a focus of 1.5 × 10^7 cells per mL. Then, 10 μL of the cell suspension was added to the middle of every nicely in a 24-well tradition plate. The plate was incubated in a cell incubator for 2 hours to permit cell attachment. 1 mL of cell medium containing 1% insulin-transferrin-selenium (ITS) (41400045, Thermo Fisher) and both 100 ng of FGF18 protein or 3 μg of LNP-FGF18 mRNA was gently added to every nicely. Cell medium was modified each two days. After completely different time durations, the cell lots have been fastened and stained with Alcian blue or toluidine blue dyes for 30 min. The built-in optical density (IOD) worth of every micromass was calculated utilizing Picture J software program.
Cell toxicity
To check the toxicity of the LNP-mRNA system on cells, we used cell counting kit-8 (cck8) (Dojindo, Japan) and calcein/pi staining package (C2015, Beyotime, China) to check the toxicity of LNP. Within the cck8 experiment, we handled cells with completely different concentrations of LNP system for twenty-four h, 48 h, and 72 h. Then, at completely different time factors, we added 10% cck8 reagent (Dojindo, Kumamoto, Japan) to the cells and incubated them in a cell incubator for two h. Lastly, an Infinite M200 professional multimode microplate reader (Tecan Life Sciences, Switzerland) was used to detect the absorbance at 450 nm of every nicely. For calcein/pi staining, we added calcein/pi working reagent to cells and incubated them at 37 °C at nighttime for 30 min. Then calcein and pi constructive cells have been noticed and quantified below a laser confocal microscope.
EdU assay
EdU assay was utilized to evaluate cell proliferation by incorporating the thymidine analogue, EdU (5-ethynyl-2’-deoxyuridine), into DNA replication. The EdU assay protocol adopted the directions supplied with the kits bought from Beyotime, China (C0075). Major chondrocytes have been incubated with a ten μM EdU working resolution at 37 °C for two h to label the cells with EdU. Following this, the cells have been fastened, washed, and permeabilized. To organize the press response resolution, we mixed elements resembling click on response buffer, CuSO4, azide 555, and click on additive resolution. The ready Click on response resolution was then added to the cells and incubated at nighttime for 30 min. Lastly, we captured consultant photographs utilizing a laser confocal microscope and additional quantitative evaluation was executed with Picture J.
Senescence-associated-β-galactosidase (SA-β-gal) staining
Mobile senescence was examined with a SA-β-gal staining package purchased from Beyotime, China (C0602). Major chondrocytes have been cultured with FGF18, LNP-FGF18 or IL-1β for 7 days. The SA-β-gal staining process was carried out following the directions supplied with the package. Random microscopy fields have been chosen for picture acquisition. ImageJ software program was utilized to rely the variety of β-gal staining-positive cells for subsequent quantitative evaluation.
Immunofluorescence (IF)
For cell immunofluorescence, we handled ATDC5 chondrocytes with completely different medicine for twenty-four h. Then, we fastened, permeabilized, blocked, and washed the cells. Subsequent, we incubated the cells with the corresponding major antibodies in a single day at 4 °C. After that, we incubated the cells with Alexa Fluor-conjugated secondary antibodies (1:1000) at room temperature for 1 h and stained the cell nuclei with DAPI. Lastly, we noticed and captured consultant photographs below a laser confocal microscope and quantified the variety of constructive cells utilizing ImageJ software program. COL2A1 antibody (1:100, ab34712), SOX9 antibody (1:100, ab185230), and Ki67 antibody (1:200, ab16667) have been all bought from Abcam.
Western blot
After treating cells with completely different situations for twenty-four h, we lysed the cells utilizing RIPA lysis buffer (P0013, Beyotime) supplemented with 1% protease/phosphatase inhibitor and purified and quantified cell proteins via centrifugation and bicinchoninic acid assay (BCA). 20 μg protein was loaded into every nicely of SDS-PAGE gel, and Tris-Glycine was used because the electrophoresis buffer. After electrophoresis at 140 V for 1 h, protein was then transferred to a PVDF membrane and blocked with a 5% BSA resolution. Subsequently, the membrane was incubated with the corresponding major and secondary antibodies and the protein bands have been imaged utilizing an Odyssey V3.0 picture scanner (Li-COR. Inc., Lincoln, NE, USA). COL2A1 antibody (1:1000, ab34712), SOX9 antibody (1:1000, ab185230), MMP3 antibody (1:1000, ab52915), and MMP13 antibody (1:1000, ab39012) have been bought from Abcam. β-actin antibody (4970, 1:2000), BECLIN1 antibody (3495, 1:1000), FOXO3a antibody (12829, 1:1000), and LC3B antibody (43566, 1:1000) have been bought from CST. FGF18 antibody (11495-1-AP, 1:1000) have been bought from Proteintech.
RNA sequencing
Chondrocytes have been seeded in six-well plates at a density of 4 × 106 cells per nicely. After 24 h of incubation with 10 ng/mL TNF-α (C600052, Sangon) or 3 μg/mL LNP-FGF18 mRNA, whole RNA was extracted utilizing the Complete RNA Extraction Package (R6812-01HP, Omega, United States). RNA sequencing was carried out by Wuhan Huada Gene Know-how Co., Ltd. (China). Gene Ontology (GO) enrichment evaluation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment evaluation, and gene set enrichment evaluation (GSEA) have been performed utilizing the Mybgi platform (Wuhan Huada Gene Know-how, https://mybgi.bgi.com/tech/login). Heatmap evaluation was carried out in GraphPad Prism 8.0 (GraphPad Software program Inc., San Diego, CA, USA) based mostly on the Z-score of TPM counts for every gene.
Quantitative real-time PCR (qRT-PCR)
Complete RNA was extracted from cells utilizing a Complete RNA Extraction Package (R6812-01HP, Omega, United States). 1000 ng cDNA was synthesized with the help of a cDNA synthesis package (RR036A, Takara, Japan), diluted in 200 μL of ddH2O. A complete of 10 μL of the qRT-PCR combine, consisting of three.6 μL of double distilled H2O, 1 μL of cDNA, 0.2 μL of upstream and downstream primers respectively, and 5 μL of SYBR premix (B21202, Selleck, USA), was added to 384-well plate. qRT-PCR course of was carried out on an ABI 7500 Sequencing Detection System (Utilized Biosystems, USA). The primer sequences used for the goal genes are listed in Supplementary Desk 1.
Statistical evaluation
Statistical analyses have been performed utilizing SPSS model 25.0 (SPSS Inc., Chicago, IL, USA). For knowledge that adopted a traditional distribution, comparisons between two teams have been carried out utilizing the unpaired Pupil’s t-test. One-way ANOVA adopted by Turkey’s publish hoc checks have been used for comparisons amongst three or extra teams. Non-normally distributed knowledge have been analyzed utilizing the Mann-Whitney and Kruskal-Wallis checks. GraphPad Prism 8.0 (GraphPad Software program Inc., San Diego, CA, USA) was utilized for the design and creation of statistical charts. All P values reported on this research are two-sided, and statistical significance was outlined as P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
