Mechanically Robust Hydrogels Facilitating Bone Regeneration through Epigenetic Modulation

Abstract Development of artificial biomaterials by mimicking extracellular matrix of bone tissue is a promising strategy for bone regeneration. Hydrogel has emerged as a type of viable substitute, but its inhomogeneous networks and weak mechanics greatly impede clinical applications. Here, a dual crosslinked gelling system is developed with tunable architectures and mechanics to promote osteogenic capacity. Polyhedral oligomeric silsesquioxane (POSS) is designated as a rigid core surrounded by six disulfide‐linked PEG shells and two 2‐ureido‐4[1H]‐pyrimidinone (UPy) groups. Thiol‐disulfide exchange is employed to fabricate chemical network because of the pH‐responsive “on/off” function. While self‐complementary UPy motif is capable of optimizing local microstructure to enhance mechanical properties. Taking the merits of biocompatibility and high‐mechanics in periodontal ligament stem cells (PDLSCs) proliferation, attachment, and osteogenesis, hybrid hydrogel exhibits outstanding osteogenic potential both in vitro and in vivo. Importantly, it is the first time that a key epigenetic regulator of ten‐eleven translocation 2 (Tet2) is discovered to significantly elevate the continuously active the WNT/β‐catenin through Tet2/HDAC1/E‐cadherin/β‐catenin signaling cascade, thereby promoting PDLSCs osteogenesis. This work represents a general strategy to design the hydrogels with customized networks and biomimetic mechanics, and illustrates underlying osteogenic mechanisms that will extend the design rationales for high‐functional biomaterials in tissue engineering.

ALP staining was performed using BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China). ASR staining was performed using Alizarin red S (Sigma-Aldrich, USA).

Preparation of cross-linked hydrogels
50 mg of POSS-P 6 -U 2 or POSS-SS-(PEG) 8 was dissolved in 445 μL of deionized water with the addition of 5 mg of cysteamine hydrochloride. Then 5 μL of 5 M NaOH was mixed to trigger the reaction. After ultrasonic processing and standing about 1 min, the solution slowly became milky and turned into the loose hydrogel. The hydrogel further shrank into the compact hydrogel if aging longer.

Characterization
1 H NMR spectra were measured on a Bruker DRX-400 spectrometer using CDCl 3 and DMSO-d 6 as solvents. Scanning electron microscopy (SEM) images were obtained at acceleration voltage of 5 kV on a JSM-6700F microscope (JEOL, Japan). Rheological measurements were carried out on a Thermo Haake Rheometer equipped with cone-parallel plate geometry (35 mm of diameter) at a gap of 0.5 mm. The sample was measured at 25 °C with a constant strain of 0.05% in frequency range of 100-0.1 rad s -1 . Fourier transform infrared (FTIR) was taken on a Bruker TENSOR-27 spectrometer in the frequency range 4000-400 cm -1 .

Assessment of PDLSCs viability, proliferation and differentiation
The human PDLSCs (hPDLSCs) were acquired form healthy human teeth from orthodontic patients of 10-25 years old without any history of periodontitis. The protocol of hPDLSCs isolation and cultivation was followed according to a previous publication [2] . The hPDLSCs were used for all experiments at Passage 3. All the researches were approved by the Ethics Committee of Peking University (PKUSSIRB-201311103).
Cell Counting Kit-8 (CK04-100T, Dojindo, Japan) was applied to examine the cell viability under manufacturer's protocol. hPDLSCs were seeded in 24-well plates at a density of 4.0 × 6 10 4 mL -1 , with the hybrid hydrogels on the bottom of the plates. After 1, 3, and 5 days incubation, 400 uL of culture medium containing 40 uL of CCK-8 solution was added to incubate hPDLSCs for 2 h. The cell viability was measured in Bio-Rad microplate reader at 450 nm wavelength.
For the Live/Dead staining, hPDLSCs were incubated on the hybrid hydrogels for 2 days, the Live/Dead working solution (L3224, Invitrogen, USA) was added according to the manufacturer's instruction. Olympus IX53 fluorescence microscope was used to observe.
The 5-Bromo-2-deoxyuridine (BrdU) assay was used to detect the proliferations of hPDLSCs. After seeding cells on the hybrid hydrogels for 2 days, the BrdU labeling reagent (Thermal Fisher Scientific, USA) was mixed with the culture medium and cells were cultured at 37°C for 12h. Then cells were treated overnight with BrdU antibody (1:200 diluted, Invitrogen, USA). Next, the cells were stained by Alexafluoro 568 conjugated secondary antibody for 1 h at room temperature. Finally, the cells were treated with Vectasheild mounting medium containing DAPI (Solarbio). Zeiss Axio Observer Z1 was used to count percentage of BrdU-positive cells among all the cells.
To evaluate the microtopography of cells with these hydrogel groups substrata, hPDLSCs (1.0 × 10 4 cells) were seeded and cultivated on the hybrid hydrogels. The samples were fixed with 4% (W/V) paraformaldehyde (PFA); next, every sample was lyophilized by Labconco SPEX 6770 freeze drier, which were then sputtered with Au (99.99%) and observed with a scanning electron microscopy. To assess the microtopography of hPDLSCs influenced by different hydrogels, branching analysis of cells was evaluated based on outline of cells. The primary branching point was defined as the projections descending from the base of the soma (greater than 5 μm); the secondary branching point was determined as the projections from the primary branching (greater than 5 μm); and the tertiary branching points was defined as the projections from the secondary branching (greater than 5 μm) [3] .
For ALP staining and ARS staining, hPDLSCs were incubated with osteogenic inducing medium (0.01 μM dexamethasone, 1.8mM KH 2 PO 4 , 10 mM β-glycerol phosphate, 0.1mM ascorbic acid-2-phosphate in regular growth media), and the hydrogel degradation was added into the medium concurrently, reaching a final concentration of 0.03 wt.%. Alkaline phosphatase (ALP) staining was conducted after 7 days of induction. The cells were rinsed with phosphate buffer saline (PBS) 3 times before fixed in 4% PFA for 20 min at room temperature. Then, washed with PBS 3 times, the cells were incubated in NBT/BCIP solution for 30 min at room temperature, and excessive stain was removed with distilled water.
Alizarin red S staining was performed after induction for 14 days. Briefly, after being washed 3 times with PBS, the cells were fixed in 4% PFA for 20min, rinsed 3 times with PBS And then stained with 1% Alizarin red S solution. Excessive dye was removed. Image J (ver. 1.8.0; NIH, USA) were used to record the stained areas

Immunofluorescence assays
For immunofluorescence staining of cultured cells, the hPDLSCs (4 × 10 4 cells) were seeded and cultivated on the hybrid hydrogels above the chamber slides for 48 h. After fixation with 4% PFA, the cells were permeabilized with 0.01% Triton X-100 (Invitrogen, USA) and blocked with 3% PBS diluted Albumin Bovine V (BSA) for 45 min at room temperature.
After rinsed with PBS, the slides were treated with following primary antibodies in 5 wt.% BSA in PBS: anti-vinculin antibody overnight, followed with thorough rinsing. Next, the samples were incubated with the following secondary antibodies for 45 min in the dark: goat anti-rabbit IgG/RBITC, goat anti-mouse IgG/RBITC, and goat anti-mouse IgG/FITC. FITC-Phalloidin was used for cytoskeletal staining. Finally, the cells were mounted by mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI, Solarbio, China). The images were observed and constructed using the Zeiss laser scanning microscope LSM 510.

8
For immunofluorescence staining of tissue sections, the tissues were fixed in 4% PFA after harvested. And the rat cranial bone tissues were decalcified with ethylenediaminetetraacetic acid (EDTA, pH 7.2). Then both the bone and the skin tissues were embedded in paraffin. The paraffin-embedded sections were blocked with serum, and incubated with primary antibodies overnight at 4 °C. Then the sections were treated with FITC or Rhodamin-conjugated secondary antibodies and mounted by mounting medium containing DAPI (DAPI, Solarbio, China). The images were observed and constructed using Olympus BX53 microscope (Japan).

Quantitative polymerase chain reaction (qPCR)
The total RNA from hPDLSCs encapsulated in hydrogels was isolated by TRIzol reagent (Invitrogen), and reverse-transcribed to cDNA by a PCR thermal cycler (Takara, Japan).  Table S1.

Western blot analysis and the co-immunoprecipitation assay
hPDLSCs encapsulated in hydrogels were lysed using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific), containing a Halt™ protease, phosphatase inhibitor cocktail and a ethylene diamine tetraacetic acid (EDTA) solution. After centrifugation for 30 min (12,000 rpm, 4 °C), the supernatants were gathered. Protein concentration was measuredusing a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Then, the samples were mixed with 4× sodium dodecyl sulfate (SDS) loading buffer (Solarbio, China) at a 1:3 ratio and heated for 5 min at 90 °C for protein degradation. Twenty micrograms applied proteins were separated on 4-12% NuPAGE gel (Solarbio, China) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% BSA (diluted with TBST) for 1 h, incubated in primary antibodies overnight according to manufacturers' instructions, and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The SuperSignal® West Pico Chemiluminescent Substrate (ThermoFisher, USA) and BioMax film (Kodak, USA) were applied for the detection of immunoractive proteins. β-actin was used as the internal control.
For the co-immunoprecipitation assay, the immunoprecipitations were conducted with anti-TET2, anti-HDAC1 or normal rabbit IgG at 4 °C overnight, after trimming the protein concentration. And then, the immunoprecipitates were incubated with ProteinIso® Protein A/G Resin for 2h. After thoroughly wash with lysis buffer, the immune products were resolved by SDS-PAGE (Solarbio, China) and evaluated by western blot.

Evaluation of bone regeneration in rat cranial bone defect model
All the animal experiments were approved by Peking University Biomedical Ethics Committee (LA20190074). Healthy SD rats (aged 6-8 weeks, female) employed for calvaria bone defects and transplantation treatments were purchased from The VitalRiver. The rats were randomly divided into 5 groups: (1) Control; (2) PEG; (3) POSS-P 8 -0.5 h; (4) POSS-P 6 -U 2 0.5 h; (5) POSS-P 6 -U 2 12 h. There were 8 rats in each group. The group without any additives was used as control. The hydrogels were cut into a diameter of 5 mm and height of 1-2 mm before surgery and incubated with 2.0 × 10 5 mL -1 hPDLSCs for 48 h, then the PDLSCs-hydrogel complexes were washed by PBS before implantation [4] . The 5 mm calvaria bone defects were fabricated by stainless-steel trephine on both sides of the skull.
The defect areas were filled by pretreated hydrogel with hPDLSCs. At 8 weeks after operation, The rats were sacrificed with 1% sodium pentobarbital solution. The calvaria bones were isolated and fixed in 4% neutralbuffered formaldehyde and then subjected to microcomputed tomography (μCT, SkyScan 1174) scanning to detect bone formation. The index including bone mineral density (BMD) and bone volume to total volume (BV/TV) were also quantitatively assessed. Serial sections (5 μm thick) were stained through hematoxylin and eosin (H&E), Masson staining and immunofluorescence staining to assess regenerated bone and inflammation status.

Assessment of biocompatibility and degradability of subepidermal implanted hydrogels
Healthy C57BL/6J mice (aged 8 weeks, female) were purchased from The VitalRiver to evaluate the toxicity of the hybrid hydrogels. The mice were randomly assigned to 5 groups: (1) Control; (2) PEG; (3) POSS-P 8 -0.5 h; (4) POSS-P 6 -U 2 0.5 h; (5) POSS-P 6 -U 2 12 h. There were 8 mice in each group. The group without any additives was used as control. After 2 and 8 weeks of subcutaneous implantation of the hydrogels on the dorsum of the mice, the skin and implants were harvested and fixed for 2 days. Then the samples were dehydrated and embedded in PMMA, and sections (3 μm) were created and treated using H&E staining and immunofluorescence staining for toxicity and inflammation evaluation.

Knockdown assays
To knockdown Tet2 expression in hPDLSCs, the cells were treated with Tet2 siRNA or the vehicle siRNA control with lipofectamine reagent (13778158, thermo, USA) following the manufacturer's instructions. To inhibit HDAC1 expression, the hPDLSCs were treated with Entinostat (MS275) or DMSO after cultured under the reduced serum medium (Opti-MEM, Gibco, USA).

Statistical Analysis
For the cell viability analysis, CCK-8 assay, BrdU-positive cell percentage analysis, quantitative analysis of Vinculin-positive area percentage, analysis of number of branching points, qPCR assay, and evaluation of differentiation, the data were presented as mean ± standard deviation. Statistical comparisons between two groups were assessed using Student's 11 t-tests, and one-way ANOVA was carried out when more than two groups were compared by using Graphpad Prism 8 software. P values < 0.05 were designated as statistically significant. Table S1. Sequences of primers used for qPCR analysis.