In Situ Remodeling of Efferocytosis via Lesion‐Localized Microspheres to Reverse Cartilage Senescence

Abstract Efferocytosis, an intrinsic regulatory mechanism to eliminate apoptotic cells, will be suppressed due to the delayed apoptosis process in aging‐related diseases, such as osteoarthritis (OA). In this study, cartilage lesion‐localized hydrogel microspheres are developed to remodel the in situ efferocytosis to reverse cartilage senescence and recruit endogenous stem cells to accelerate cartilage repair. Specifically, aldehyde‐ and methacrylic anhydride (MA)‐modified hyaluronic acid hydrogel microspheres (AHM), loaded with pro‐apoptotic liposomes (liposomes encapsulating ABT263, A‐Lipo) and PDGF‐BB, namely A‐Lipo/PAHM, are prepared by microfluidic and photo‐cross‐linking techniques. By a degraded porcine cartilage explant OA model, the in situ cartilage lesion location experiment illustrated that aldehyde‐functionalized microspheres promote affinity for degraded cartilage. In vitro data showed that A‐Lipo induced apoptosis of senescent chondrocytes (Sn‐chondrocytes), which can then be phagocytosed by the efferocytosis of macrophages, and remodeling efferocytosis facilitated the protection of normal chondrocytes and maintained the chondrogenic differentiation capacity of MSCs. In vivo experiments confirmed that hydrogel microspheres localized to cartilage lesion reversed cartilage senescence and promoted cartilage repair in OA. It is believed this in situ efferocytosis remodeling strategy can be of great significance for tissue regeneration in aging‐related diseases.


Contents Experimental section
Preparation and characterization of liposomes

Preparation and characterization of liposomes
The thin film dispersion method was utilized for liposome preparation.Briefly, cholesterol (Aladdin, China), lecithin (Macklin, China), and ABT263 (Aladdin, China) were dissolved in dichloromethane at a mass ratio of 8:2:1.After vacuum rotary evaporation at 38 °C for half an hour, the dried lipid membranes were hydrated using PBS and sonicated for 10 minutes.Liposomes were obtained after repeated extrusion using polycarbonate membranes (0.45 and 0.22 μm, Millex, Ireland).To prepare fluorescently labeled liposomes, DSPE-PEG-FITC (Xi'an Ruixi Biology, China) or DiD (Beyotime, China) was added during the synthesis process.
To observe the liposome morphology, Lipo or A-Lipo (0.5 mg/mL) was dripped onto a TEM copper mesh (300 mesh) and allowed to dry completely.It was then negatively stained with 1% phosphotungstic acid (Solarbio, China) for 1 min and allowed to dry again for TEM (Hitachi-HT7800, Japan) observation.The size distribution, PDI, and zeta potential of Lipo and A-Lipo were determined by DLS (Zetasizer Nano-ZS, Malvern, UK).The stability of Lipo and A-Lipo in a physiological environment was assessed by dissolving them in 50% FBS (BioInd, Israel) at 37 °C and their size and PDI was measured by DLS at 1, 3, 5, 7, 14, 21, and 28 days.To calculate drug encapsulation (EE) and loading efficiency (LE), the liposome structure was disrupted using methanol and DMSO at a volume ratio of 1:9, and then the amount of ABT263 in A-Lipo was calculated using a concentration-OD value standard curve of ABT263.The formula to calculate the EE and LE of ABT-263 was as follows: (W: mass of ABT263 loaded on the liposomes; W0: total mass of drug added to the reaction)

LE (%)= W W+W Lipo ×100%
(WLipo mass of liposomes) Release behavior of ABT263 in vitro.A-Lipo was put in a dialysis bag (MW = points, 1 ml of the dialyzed sample was gathered, and an equivalent amount of fresh PBS with 1% Tween 80 was added.The OD values of the solutions collected at each time point were measured using a UV spectrophotometer (Jasco, Japan).The cumulative release rate was calculated, and a release curve was plotted.

Synthesis and characterization of HAMA and AHAMA hydrogels
AHA was synthesized using the previously reported method. [1]One gram of HA (MW = 74 kDa) was dissolved in 100 ml ultrapure water with mechanical stirring until complete dissolution.Then, 5 ml of 0.5 M NaIO3 (Macklin, China) was pipetted dropwise, and the reaction was carried out for two hours.Subsequently, ethylene glycol (Macklin, China) was added to terminate the reaction.AHA was obtained by lyophilization after three days of dialysis.
HAMA and AHAMA hydrogels were synthesized as described previously. [2]iefly, 10 g HA or AHA was dissolved in 500 ml ultrapure water to be completely transparent.Then, 20 ml MA (Sigma-Aldrich, USA) was added dropwise and mixed well.A NaOH solution (20 mL, 5 M) was added using a micro-syringe pump.The reaction was conducted at 4 °C while protected from light overnight.After the reaction, HAMA or AHAMA was obtained by lyophilization after three days of dialysis.
HA, AHA, HAMA, and AHAMA were characterized using a Fourier-transform infrared spectrometer (FTIR, Nicolet is50, Thermo Fisher, USA) and 1 H NMR (600 MHz, Bruker, Germany). [3]The grafting rate of MA is calculated by 1 H NMR. The MA grafting rate was calculated from the integral areas of proton peaks attributed to ethylenic bond groups of grafting MA side chain (S1, chemical shift 5.7 and 6.1 ppm) and methyl groups of MA side chain and HA or AHA backbone (S2, chemical shift 1.8 and 1.9 ppm) using the formula as follows: The grafting rate of MA (%) = S 1 2 ×3 S 2 -S 1 2 ×3 ×100% (S1: integral area of ethylenic bond proton peaks; S2: integral area of methyl proton peaks) The content of aldehyde groups in AHA and AHAMA was determined by hydroxylamine hydrochloride titration [4] and XPS.0. The monomer unit molar mass of HA is 376 g/mol, and the average monomer unit molar mass calculated from the grafting rate of MA was used as the monomer unit molar mass of HA grafted MA.

Synthesis and characterization of A-Lipo/PHM and A-Lipo/PAHM
Hydrogel microspheres were prepared by microfluidic technology and a photocrosslinking method. [5]Briefly, AHAMA or HAMA, A-Lipo, and LAP (Sigma-Aldrich, USA) (4%/0.2%/0.2%,mass ratio) were used as the dispersed phase, and the continuous phase used was liquid paraffin with 5% Span 80.Using an in-house microfluidic device, the dispersed phase was sheared into microdroplets by the continuous phase.After The cartilage explants were placed in a solution of 2 mg/mL microspheres, magnetically stirred for 5 min, and then left to stand for 1 min.The cartilage explants were taken out and rinsed with PBS several times.To observe adhered microspheres, digital photographs of the various groups were acquired.
Mouse BMSCs were isolated using a previously reported method.Briefly, the femur and tibia of 7-day-old C57BL/6 mice were removed, their ends were severed, and α-MEM medium (Hyclone, USA) containing 10% FBS and 1% penicillin/streptomycin was used to rinse the bone marrow cavity.The rinsed-out cells were cultured at 37 ℃ with 5% CO2.At 80% confluency, the cells were passaged at a 1:3 ratio.Passage 3 cells were used for subsequent experiments.
To isolate mouse BMDMs, [6] femurs and tibiae of 6-8-week-old C57BL/6 mice were extracted, and a cell suspension was collected from the bone marrow cavity as described above.After centrifugation, the cells were resuspended with α-MEM medium containing 20 ng/mL M-CSF (Proteintech, Wuhan, China), 10% FBS, and 1% penicillin/streptomycin, and cultured at 37 °C with 5% CO2, for which fresh complete medium was substituted on days 3 and 5. BMDMs were collected on day 7 for subsequent experiments.

Induction of chondrocyte senescence in vitro
Passage 2 chondrocytes were used to establish a chondrocyte senescence model.
Adherent chondrocytes were induced with Dox (100 ng/mL; Sigma-Aldrich, USA) for 14 days.qRT-PCR, WB, and IF were used to determine the successful establishment of the chondrocyte senescence model.

CCK-8 assays
The seeded chondrocytes were treated with various concentrations of ABT263 for 1 and 4 days, and then CCK-8 solution (Beyotime, China) was added.After incubation for 2 hours at 37 °C in the dark, the absorbance of the solution was measured at 450 nm using a microplate reader (Molecular Devices, FlexStation 3).

Live/dead cell staining
Chondrocyte viability was assessed on days 1 and 4 using a Calcein/PI Assay Kit (Beyotime, China).Briefly, washing cells gently, Calcein/PI working solution (300 μL) was added, with which cells were incubated for half an hour at 37 °C in the dark.The cells were observed by the fluorescence microscope, and chondrocyte viability was analyzed using ImageJ.

Lysosomal escape assay
After treating cells with FITC-labeled liposomes for 0.5 and 4 h, the cells were incubated with Lyso-Tracker Red working solution (Solarbio, China) at 37 °C for 10 minutes and Hoechst 33342 (Beyotime, China) was employed to stain nuclei.Images were randomly obtained under a laser confocal microscope.

SA-β-Gal staining
After fixation and washing, cells were incubated with the Senescence β-Galactosidase Staining Working Solution (Beyotimie, China) overnight at 37°C.
Synthesis and characterization of HAMA and AHAMA hydrogels Synthesis and characterization of A-Lipo/PHM and A-Lipo/PAHM Cell isolation and culture Induction of chondrocyte senescence in vitro CCK-8 assays Live/dead cell staining Lysosomal escape assay SA-β-Gal staining Efferocytosis assay Flow cytometry Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Figure S1 .
Figure S1.Stability of Lipo in vitro over 28 days.

Figure S2 .
Figure S2.Stability of A-Lipo in vitro over 28 days.

Figure S3 .
Figure S3.The cumulative release profile of ABT263 Figure S4.Schematic diagram of the chemical reaction between AHA and MA Figure S5.Schematic diagram of the chemical reaction between HA and MA Figure S6.(A) HM images acquired with a bright-field microscope.

Figure S10 .
Figure S10.The PDGF-BB loading efficiency of AHM and A-Lipo/AHM.

Figure S11 .
Figure S11.The PDGF-BB loading efficiency of HM and A-Lipo/HM.

Figure S12 .
Figure S12.Measuring the cumulative release rate of Lipo within Lipo/PHM under PBS or PBS containing HAase Figure S13.Measuring the cumulative release rate of PDGF-BB within Lipo/PHM under PBS or PBS containing HAase Figure S14.Representative fluorescence images of BMSCs cultured on HM, AHM, A-Lipo/AHM, and A-Lipo/PAHM.

Figure S16 .
Figure S16.Quantitative analysis and heatmap of mRNA expression after 14 days of Dox intervention.

Figure S17 .
Figure S17.Western blotting analysis after 14 days of Dox intervention.

Figure S18 .
Figure S18.Representative immunofluorescence images of P16 INK4a and P21 Figure S19.Cell viability analysis of chondrocytes after treatment with ABT263 Figure S20.Live/dead cell staining of chondrocytes Figure S21.IVIS images at different time points in vivo.

Figure S22 .
Figure S22.Assessment of the ability of microspheres to recruit stem cells in vivo.

Figure S23 .
Figure S23.Representative X-ray films of the mice knee joints Figure S24.To assess the biocompatibility of microspheres in vivo.
1 g of the sample was dissolved in ultrapure water until complete dissolution, and the pH value of the solution was adjusted to 5.0 with NaOH solution (0.1 M), then hydroxylamine hydrochloride solution (0.05 g/mL) was added for 4 h reaction at 40°C.At last, the pH value of the reaction solution was adjusted to 5.0 again with NaOH solution, and the volume of NaOH solution used in this step was recorded.The calculation formula is as follows: The oxidation level (%)= M×(V×n NaOH ) 2m ×100% (M (g/mol): the monomer unit molar mass of HA or HA grafted MA, V (L): the volume of NaOH solution recorded, nNaOH (mol/L): the concentration of the NaOH solution, m (g): mass of sample to be measured)

(
being collected at -20 °C and frozen overnight at -80 °C, the microdroplets were photocrosslinked by a 405 nm UV light for 10 min.Microspheres were collected, washed with diethyl ether and ultrapure water, and then lyophilized for use in subsequent experiments.To load PDGF-BB, 1 mg dried microspheres were co-incubated with PDGF-BB (300 ng/mL; Novoprotein, China) in a shaker at 4 °C overnight.Precipitates were collected to obtain A-Lipo/PHM or A-Lipo/PAHM.The size and morphology of microspheres were observed by optical microscopy and SEM (Hitachi Regulus8100, Japan).The size of microspheres was measured using ImageJ.The zeta potential of microspheres was measured by DLS.Microspheres loaded with FITC-labeled liposomes were observed by laser confocal microscopy (LSM800, ZEISS, Germany).To assess the swelling properties of microspheres, 10 mg of dry microspheres were added to 2 mL of PBS, and the mass of the microspheres was weighed after removing as much residual water as possible at various time points.The swelling ratio of microspheres was calculated as follows: Wwet: mass of wet microspheres after water absorption, Wdry: mass of dry microspheres) Evaluation of the degradation properties of microspheres.The lyophilized microspheres were resuspended in PBS containing hyaluronidase (1500 U/mL, Macklin, China), and the morphology of the microspheres was observed under an optical microscope at various time points.To assess the release behavior of liposomes in microspheres, LS release experiments were performed using microspheres loading with FITC-labeled liposomes.At the corresponding time points, supernatants were harvested, and the intensity of the fluorescence was quantified.To calculate the concentration of released liposomes, a standard curve of fluorescence intensity-concentration was constructed, and then the cumulative release curve of liposomes was plotted.To investigate the EE of PDGF-BB, an absorbance value-concentration standard curve of PDGF-BB was constructed.The PDGF-BB content in the supernatant was determined by ELISA, and the encapsulation efficiency of PDGF-BB was calculated using the encapsulation efficiency formula.To evaluate PDGF-BB release from microspheres, supernatants were collected at various time points, and the PDGF-BB concentration was determined using an ELISA to plot the cumulative release curve of PDGF-BB.Evaluation of injured cartilage adhesion properties of microspheres.BMSCs were cultured on AHM.Specifically, 1 mg of AHM and 1×10 6 /mL BMSCs were added to a low-adhesion plate and cultured for 3 days.Suspended cells were removed with a 70 μm cell sieve (Sangon Biotech, China).The growth of cells stained with Actin-Tracker Green-488 (Beyotime, China) and DAPI (Beyotime, China) on the microspheres was observed by laser confocal microscopy.An OA cartilage model was established by harvesting healthy full-depth cylindrical cartilage explants (7 mm diameter) and incubating them in 0.1% collagenase (Sigma-Aldrich, USA) for half an hour at 37 °C.

Figure S3 .
Figure S3.The cumulative release profile of ABT263 from A-Lipo in PBS containing 1% Tween 80. Data are presented as mean ± SD (n=3)

Figure S5 .
Figure S5.Schematic diagram of the chemical reaction between HA and MA, and 1 H NMR spectra of MA proton peak integrals.

Figure S8 .
Figure S8.In vitro degradation properties of AHM and HM observed under bright-field microscope.Scale bar: 50 μm.

Figure S10 .
Figure S10.The PDGF-BB loading efficiency of AHM and A-Lipo/AHM.Data are presented as mean ± SD(n=3).

Figure S11 .
Figure S11.The PDGF-BB loading efficiency of HM and A-Lipo/HM.Data are presented as mean ± SD(n=3).

Figure S12 .
Figure S12.Measuring the cumulative release rate of Lipo within Lipo/PHM under PBS or PBS containing HAase (1500 U/mL) by quantification of the fluorescence intensity of FITC-labeled Lipo.Data are presented as mean ± SD (n=3).

Figure S13 .
Figure S13.Measuring the cumulative release rate of PDGF-BB within Lipo/PHM under PBS or PBS containing HAase (1500 U/mL) using ELISA kit.Data are presented

Figure S23 .
Figure S23.Representative X-ray films of the mice knee joints in anterior-posterior (AP) and lateral (LAT).

Figure S24 .
Figure S24.To assess the biocompatibility of microspheres in vivo.Representative images of H&E staining of heart, liver, spleen, lung, and kidney.Scale bar: 50 μm.

Table S1 .
List of primers for qRT-PCR