Oscillating Fluid Flow Activated Osteocyte Lysate‐Based Hydrogel for Regulating Osteoblast/Osteoclast Homeostasis to Enhance Bone Repair

Abstract As major regulators on bone formation/resorption in response to mechanical stimuli, osteocytes have shown great promise for restoring bone injury. However, due to the unmanageable and unabiding cell functions in unloading or diseased environments, the efficacy of osteogenic induction by osteocytes has been enormously limited. Herein, a facile method of oscillating fluid flow (OFF) loading for cell culture is reported, which enables osteocytes to initiate only osteogenesis and not the osteolysis process. After OFF loading, multiple and sufficient soluble mediators are produced in osteocytes, and the collected osteocyte lysates invariably induce robust osteoblastic differentiation and proliferation while restraining osteoclast generation and activity under unloading or pathological conditions. Mechanistic studies confirm that elevated glycolysis and activation of the ERK1/2 and Wnt/β‐catenin pathways are the major contributors to the initiation of osteoinduction functions induced by osteocytes. Moreover, an osteocyte lysate‐based hydrogel is designed to establish a stockpile of “active osteocytes” to sustainably deliver bioactive proteins, resulting in accelerated healing through regulation of endogenous osteoblast/osteoclast homeostasis.

MLO-Y4 cells, RAW 264.7 cells, 5TGM1 cells and MC3T3-E1 cells were obtained from the cell bank of the Chinese Academy of Science. Recombinant mouse Dkk-1 protein and receptor activator of nuclear factor κB ligand (RANKL) were purchased from R&D Systems (USA).

Effect of CLOO on osteoblast proliferation, morphology, migration and gene expression
First, the culture supernatant of murine myeloma cells (5TGM1) was collected and used as the conditioned culture medium (MM cm) to stimulate preosteoblast cells (MC3T3-E1). Then, CLOO was diluted with culture medium or a mixed solution of MM cm and normal culture medium (1:1) to a concentration of 300 μg mL -1 . Then, 2 × 10 3 MC3T3-E1 cells were seeded per well of a 96-well plate and cultured overnight. Subsequently, the culture medium was replaced with 200 μL of MM cm or CLOO, and after 24 h of stimulation, MC3T3-E1 proliferation was observed via a CCK-8 assay. To investigate whether CLOO can protect osteocytes from apoptosis in the MM microenvironment, 4 × 10 4 MC3T3-E1 cells were planted and cultured in 6-well plates (untreated surface) for 12 h and then cocultured with 2 × 10 5 5TGM1 cells under CLOO (total protein concentration = 300 μg mL -1 ) stimulation for 5 days.
After that, the suspension of 5TGM1 cells was removed, and MC3T3-E1 cells were collected for apoptosis analysis using a flow cytometer. The migration of MC3T3-E1 cells was evaluated by a Transwell assay. In brief, 400 μL of CLOO (total protein concentration = 300 μg mL -1 ) solution was added to the lower chambers, and 2 × 10 4 MC3T3-E1 cells were seeded in the upper chambers with 200 μL of medium containing 5% serum. After reaching the determined culturing time (24 or 48 h), the cells were fixed, stained and imaged by microscopy. The morphology of MC3T3-E1 cells during differentiation was visualized using an inverted fluorescence microscope after staining with DIO and DAPI after 24 h of CLOO stimulation.

Proteomic analysis of MLO-Y4 cells
LC-MS/MS was performed to analyze the differentially expressed proteins in MLO-Y4 cells with or without OFF loading. Briefly, the total cell lysates from the OFF loading group and untreated group were collected, and the protein content was quantified. Equal amounts of protein were loaded on a gel and separated by 12% SDS-PAGE, followed by Coomassie blue staining. The protein bands with a significant change in color intensity were excised from the gel and identified by LC-MS/MS technology (China). Subsequently, the cellular location and function of these differentially expressed proteins were classified.

Inhibitor Studies
A total of 4 × 10 6 MLO-Y4 cells were seeded in T75 cell culture flasks for 12 h, followed by treatment with culture medium containing different inhibitors (SP600125, SB352580, U126, DKK1) at working concentrations (Table S2). After being stimulated by the inhibitors for 1 h under static culture conditions, the cells were transferred into a CO2 oscillating incubator for another 24 h of OFF loading culture. Finally, the OFF-treated cells were collected and processed into cell lysates for future use.

Bone histological analyses
The bone tissue (tibia and parietal bone) was fixed in 4% PFA for 24-48 h at 4 °C and demineralized with EDTA decalcifying solution (Boster, China) at 37 °C with gentle shaking for up to 2 weeks. Paraffin-embedded bone tissue was sliced into 3-μm-thick sections and subjected to H&E and TRAP activity staining assays, which were used to assess the trabecular bone and osteoclast distribution in vivo. Masson's trichrome staining was used to evaluate 4 osteoblast and new bone formation in vivo. Images of tissue sections were captured by a microscope.

Rheological measurements
The temperature sweep oscillatory shear measurements were performed on an ARES/RFS Ⅱ rheometer (TA instruments, USA) using a 10 mm parallel-plate geometry between 15 and 55 °C and with a temperature interval of 2 °C . A shear stress of 10 Pa and an oscillatory frequency of 1 Hz were applied. Storage (G′) and loss (G″) moduli were determined using a frequency sweep test (0.1-10 rad s -1 frequency). The critical point at which the collapse of the hydrogel occurred was determined via the strain amplitude sweep test (0.1-1000%) at 1 rad s -1

Characterization of CLOO-MCH and HSOOL
To characterize the surface morphology and the microstructure of CLOO-MCH and HSOOL, the freeze-dried CLOO-MCH and HSOOL were sputter-coated with gold for 45 s and were then observed using a scanning electron microscope (SUPRA 60, Wavetest) at an acceleration voltage of 15 kV.

In vivo degradation and biocompatibility of HSOOL and CLOO-MCH
The HSOOL (Φ 3 mm × 0.1 mm) scaffold and 50 μL of CLOO-MCH were implanted into the subcutaneous tissues of BALB/c nude mice (~ 20 g). The mice were humanely sacrificed at 10, 20, and 30 days. The implantation regions on the back skin of the mice were carefully opened, and the remaining gels were photographed. The skin tissue was surgically removed from the area surrounding the implant and subjected to H&E staining for tissue response analysis.