Cell culture

Human mesenchymal stem cells (hMSCs) were isolated from the proximal femur bone marrow of consenting patients undergoing total hip replacement surgery at Southmead hospital according to Bristol Southmead Hospital Research Ethics Committee guidelines (REC reference: 20/LO/0614). Cell culture work was undertaken using laminar flow hoods (SAFE 2020, ThermoScientific), with incubation at 37 °C under a 5% CO2 atmosphere (HERACELL 240i, ThermoScientific). “Basal media” was prepared from low glucose DMEM supplemented with fetal bovine serum (10 v/v.%, Sigma-Aldrich), GlutaMax (2 mol/L, Invitrogen), and penicillin/streptomycin (100 units per mL/100 μg/mL, Sigma Aldrich). hMSCs were expanded in an “expansion media” consisting of basal media further supplemented with basic fibroblast growth factor 2 (5 ng/mL, Peprotech). For growth factor-induced hMSC differentiation, an “osteoinductive media” was prepared from basal media further supplemented with ascorbic acid (200 μmol/L, Sigma-Aldrich), bone morphogenic protein 2 (25 ng/mL, Gibco), dexamethasone (10 nmol/L, Sigma-Aldrich), and β-glycerophosphate (10 mmol/L, Sigma). All media used for culture of bioprinted constructs was further supplemented with CaCl2 (5 mmol/L).

Ascorbic Acid stock solutions (80 mmol/L) were prepared by dissolution of ascorbic acid 2-phosphate (69.5 mg, A8960, Sigma-Aldrich) in deionized water (3 mL) and filter sterilization (0.22 μm, 16534, Sartorius). Basic fibroblast growth factor 2 (FGF-2) stock solutions (10 μg/mL) were prepared by dissolution of basic human FGF (50 μg, 100-18B, Peprotech) in tris-buffered deionized water (4 mmol/L, 5 mL, pH = 7.6, 0.22 μm filter sterilized). Bone morphogenic protein 2 (BMP-2) stock solutions (100 μg/mL) were prepared by dissolution of recombinant BMP-2 (50 μg, PHC7145, Gibco) in 0.5 mL of a filter-sterilized solution of bovine serum albumin (1 mg/mL) in hydrochloric acid (4 mmol/L). β-Glycerophosphate stock solutions (1 mol/L) were prepared by dissolution of β-glycerophosphate disodium salt hydrate (1.08 g, G9422, Sigma-Aldrich) in deionized water (5 mL) and filter sterilization (0.22 μm). Basal ITS media composed of high glucose DMEM supplemented with penicillin/streptomycin (100 units per mL/100 mg/mL, P0781, Sigma-Aldrich), GlutaMAX (2 mmol/L, 35050038, Life Technologies), sodium pyruvate (1 v/v.%, 11360, Gibco) and insulin-transferrin-selenium (1 v/v.%, 41400045, Gibco) was prepared. Dexamethasone stock solutions (100 μmol/L) were prepared by dissolution of dexamethasone (3.925 mg, D4902, Sigma-Aldrich) in ethanol (1 mL), addition to basal ITS media (30 μL in 2.97 mL) and filter sterilized (0.22 μm).

Substrate preparation

Acellular thin dentine disks were provided either by GlaxoSmithKline or the Tooth Tissue Bank (HTA license 12200, NHS REC reference, 16/NI/0192, Project 80, Bristol Dental School) for use in this work. The dentine disks were first subject to exfoliation using ascending grades of sandpaper (P400-P1200) with deionized water drop cast over the area; followed by ultrasonication (750 W, 20 kHz, amplitude 50%, 1 min duration, 2 s pulse on, 1 s pulse off, Sonics Vibra-Cell) in citric acid solution (0.5 w/v.% in deionized water, CO759, Sigma-Aldrich) and deionized water. To anchor the dentine slices within the well for 3D bioprinting, 3D printed inserts (PLA, Supplementary Fig. S13) were fabricated. βTCP (9.5 mm diameter, 3D BioTek, TCP48) disks were exfoliated using ascending grades of sandpaper (P400-P1200) with deionized water drop cast over the area to ensure reproducibility across samples. The dentine disks, βTCP disks and inserts were sterilized by incubation in 70% ethanol (overnight), wicked dry using lab roll and further sterilized by UV-C radiation on both faces (30 min each) in a laminar flow hood immediately before printing.

Bioink preparation

A Pluronic F-127 stock solution (40 w/v.% in PBS, P2442, Sigma-Aldrich) was prepared and sterilized by autoclaving (121 °C, 45 min). Sodium Alginate (6 w.t.%, W20150, Lot# MKCC4541, Sigma-Aldrich) was added to basal media (60 v/v.%) and was mixed by hand. The gel was further mixed using a dual-asymmetric centrifuge (DAC, 3 500 r/min, 5–10 min until fully mixed, DAC 150.1 FVZ, Speedmixer). The gel was then sterilized by UV irradiation (>1 h) in a laminar flow hood. Pluronic F-127 stock solution (13 w/v.% final) was added using a positive displacement pipette, followed by dual-asymmetric centrifugation (1 500 r/min, 10–20 s until mixed). For cellular printing, cells were resuspended (7.5% final volume) and added to the gel, followed by dual-asymmetric centrifugation (1 500 r/min, 10 s). For acellular bioink, basal media (7.5% final volume) was added to the gel, followed by mixing using the DAC (1 500 r/min, 10 s).

Bioprinting

A BioX (Cellink) 3D bioprinter was used for all bioprinting experiments in a temperature controlled lab (17 °C). A sterilization program on the printer (UV-C exposure within the printing enclosure) was run both before and after the printing process to ensure sterility within the printer, and the laminar flow hood sterilized by UV-C irradiation (1 h) prior to use. To load the cell-laden or acellular bioink into pneumatic syringes for 3D bioprinting, the ink was first loaded into a 5 mL syringe (Tenmo, SS*05SE1) using a sterile spatula and extruded into a 3 mL pneumatic syringe (7012074, Nordson) via a female-female Luer connector (11891120, Fisher-Scientific). The pneumatic syringe was then equipped with a needle (22 G, 2484538, Onecall), placed within the printhead and connected to the pneumatic tubing. STL files were generated using Autodesk Inventor and sliced using the BioX on-board slicer (100% infill density, rectilinear style pattern, 20 mm/s, 0.41 mm nozzle diameter). All constructs were printed into either a 24 or 48 well plate (Corning, 10732552 or 10065370 respectively) which was heated by the print bed (37 °C). Straight after printing, the samples were crosslinked in DMEM supplemented with CaCl2 (100 mmol/L, 10 min, RT, 349615000, Acros Organics, filter sterilized). The samples were then cultured under standard conditions, with media supplemented with CaCl2 (5 mM, filter sterilized).

Cell viability studies

Live/Dead staining was performed using a commercially available kit (L3224, ThermoFisher). Staining solution was prepared fresh by addition of Calcein AM (0.5 μL/mL) and ethidium homodimer-1 (2 μL/mL) to pre-warmed PBS and wrapped in foil to prevent photobleaching. Bioprinted constructs cultured in expansion media for 1 or 7 days were washed with PBS (pre-warmed to 37 °C) and incubated in staining solution (0.5 mL, 30 min, 37 °C, wrapped in foil). After incubation, the samples were imaged using a widefield microscope (Calcein AM filter = GFP, ethidium homodimer-1 filter = Texas Red, Leica DMI6000 inverted epifluorescence microscope, equipped with a Leica LASX live cell imaging workstation and a Photometrics Prime 95B sCMOS camera). Cell counting was performed on widefield image stacks using FIJI.

Cell distribution studies

3D iterative thresholding of the live channel from widefield stacks used for cell viability studies was performed using the 3D manager package for FIJI (3D iterative thresholding plugin: min_vol_pix = 100, max_vol_pix = 20,000, min_threshold = 10, min_contrast = 0, criteria_method = MSER, threshold_method = step, segment_results = All, value_method = 10).56 Image sets were frequently recorded with a volume above and below the gel in z to ensure that the maximal volume of gel was imaged. This often led to void regions in the z axis where no cells were present. These regions were first determined manually on the original image stacks, and the corresponding distances were removed from the beginning and end of the z data. Histograms along each axis were then plotted with the calculated xyz coordinates to yield live cell distributions (distance normalized to dimensions of imaged volume). Distributions are presented as the average of all cell positions taken over at least 34 stacks.

Immunofluorescence microscopy

A “wash buffer” (WB, 0.05 v/v.% Tween 20 in PBS, T2700, Sigma-Aldrich), a “permeabilizing buffer” (PB, 0.1 v/v.% Triton X-100 in PBS, T8787, Sigma-Aldrich) and a “blocking buffer” (BB, 1 w/v.% bovine serum albumin in PBS, A7638, Sigma-Aldrich) were prepared fresh and filter sterilized (0.22 μm). DAPI staining solution was prepared from a DAPI stock solution (0.1 v/v.% in PBS, Millipore, 90229) and was wrapped in foil. For actin staining, a dual-staining solution consisting of anti-phalloidin TRITC solution (4 v/v.% of a 60 μg/mL stock, 90228, Millipore) and DAPI staining solution in PBS was prepared fresh just before use. Samples were washed with WB (500 μL, 3 times, 10 min each) and permeabilized with PB (500 μL, 10 min). Samples were washed with WB (500 μL, 3 times, 10 min each) and incubated (overnight, 4 °C) in dual-staining solution. The samples were then washed with WB (500 μL, 3 times, 10 min each), transferred to a confocal dish flooded with PBS and imaged using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope.

For collagen staining, anti-collagen I primary antibody solution (0.05 v/v.% in BB, Invitrogen, MA1-26771), mouse IgG solution (0.3 v/v.% in BB, BioVision, 1265-100), FITC GoatxMouse IgG secondary antibody solution (0.4 v/v.% in PBS, Millipore, AP124F), and DAPI staining solution were prepared fresh before use. Samples were washed with WB (500 μL, 3 times, 10 min each) before incubation in BB (500 μL, 30 min). Samples were incubated in primary antibody solution (500 μL, 4 °C, overnight, wrapped in foil). Isotype controls were prepared by incubation in mouse IgG solution (500 μL, 4 °C, overnight) instead of primary antibody solution. No primary antibody controls were prepared by incubation in blocking buffer (500 μL, 4 °C, overnight) instead of primary antibody solution. The next day, samples were washed with WB (500 μL, 3 times, 10–15 min each) before incubation (45 min, RT, wrapped in foil) in secondary antibody solution (500 μL). No secondary antibody controls were prepared by incubation in PBS instead of secondary antibody solution. The samples were then washed with WB (500 μL, 3 times, 10–15 min) before incubation in DAPI staining solution (500 μL, 30 min, RT, wrapped in foil). The samples were then washed with WB (500 μL, 3 times, 10–15 min each). The samples were then placed in confocal dishes and submerged with PBS for imaging using a confocal microscope (Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope).

Gene expression analysis

Cetyltrimethyl ammonium bromide buffer solution (CTAB, Promega, MC1411) was freshly supplemented with 1 v/v.% β-mercaptoethanol and pre-warmed to 65 °C. Bioprinted constructs were washed with PBS and pooled into an RNase-free Eppendorf (AM12400, Fisher). The samples were then dissolved using EDTA (as described in section 2.9.2.2.1) to yield a cell pellet prior to addition of CTAB buffer (600 μL). The samples were then homogenized by passing through a 21 G needle (>20 passes, ThermoFisher, 10472204) and incubated (65 °C, 15 min) with frequent mixing by inversion. Debris was isolated by centrifugation (14 000 × g, 2 min, RT), and the supernatant transferred to a new Eppendorf. Chloroform extraction was performed by addition of chloroform (100 v/v.%, VWR, 22705.323) to the supernatant and mixed by inversion. The mixture was then centrifuged (14 000 × g, 2 min, RT) and the supernatant was carefully 62 aspirated without disturbing the interfacial region. Chloroform extraction was repeated one further time, resulting in a clear interfacial region.

Nucleic acid precipitation was performed by addition of either ethanol (2–2.5× volume) and sodium acetate (3 mol/L, 0.1× total volume, 15480217, Alfa Aesar) to the supernatant, mixed by inversion, and incubation (−80 °C, overnight). The sample was thawed on ice, and the white precipitate was isolated by centrifugation (14 000 × g, 30 min, 4 °C).

The precipitate was then washed with ethanol (70 v/v.% in RNase-free water, ice cold), re-isolated by centrifugation (14,000 × g, 15 min, 4 °C), and the supernatant was removed. This wash step was repeated two further times. The samples were then incubated (5–10 min, RT) to allow residual ethanol to evaporate. Samples were then dissolved in RNase-free water (750024, Fisher) were further purified using RNeasy Mini kit (Qiagen, 74104) following manufacturer’s instructions. Samples were eluted with DEPC-treated deionized water (46-2224, Intvitrogen). cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher) according to manufacturer’s instructions using a SensoQuest Labcycler (Geneflow). qPCR was performed using a Step One Plus RT- PCR system (Applied Biosystems) and the following TaqMan probes: ALPL (Hs01029144_m1), BGLAP (Hs01587814_g1), COL1A2 (Hs01028969_m1), ELF1 (Hs01111177_m1), RUNX2 (Hs01047973_m1), YWHAZ (Hs01122445_g1).

Samples were analyzed using the ΔΔCT method, with the geometric mean of ELF1 and YWHAZ used as the reference. Statistical analysis for autoinduction was performed on ΔCT values using paired t-tests, whilst statistical analysis for comparison between groups was performed on ΔΔCT values using a 2-way ANOVA.

Histological analysis

Samples were first fixed in PFA solution (4 w/v.% paraformaldehyde in PBS, 2 h, RT). The samples were then immersed in ascending grades of ethanol (30, 50, 70, 90, 100 v/v.% in deionized water, 20 min each) before incubation in London Resin white (LRW), 50 v/v.% in ethanol, overnight, L9774, Sigma-Aldrich). The samples were incubated in three further changes of LR white (100%, 2 h each) before transfer into a TAAB capsule. The capsule was sealed and incubated (60 °C, ~2 days) to cure. If the sample required reorientation to section along a desired axis, the block was cut parallel to the desired axis using a hacksaw and remounted onto the same block base using commercial superglue. The block was then sectioned using an ultramicrotome (1 μm, Leica EM UC6), floated onto glass slides using deionized water and placed on a heating block (~80 °C) until fully dry.

For calcium staining, sections were incubated (2.5 min, RT) with alizarin red solution (2 w/v.% in deionized water, pH = 4.25, filtered) dropcast to cover the section. Excess stain was then removed by careful blotting. For glycosaminoglycan staining, sections were incubated (20 min, RT) in acetic acid solution (1 v/v.% in deionized water, pH = 2.20). Sections were then incubated (10 min, RT) in safranin-O solution (0.1 w/v.% in deionized water, pH = 2.20), with excess stain removed by washing with deionized water. Sections were allowed to dry and imaged using a widefield microscope (Leica DMI6000 inverted epifluorescence microscope, equipped with a Leica LASX live cell imaging workstation and a Leica DFC420C color camera).

Coculture studies

For bacterial coculture studies, all cell culture was performed without penicillin/streptomycin. Fusobacterium periodonticum (ATCC33693, strain 2B3) were inoculated and grown (2 days, 37 C) on fastidious anaerobic agar plates under anaerobic conditions (Oxoid Anaerogen, ThermoScientific) in a sealed container. F. periodonticum were then suspended in PBS (pre-warmed), and the concentration determined by absorbance (OD 600 = 1 corresponding to 108 cells per mL, GENESYS 6, ThermoSpectronic). For inoculation in the bioink, a small volume of basal media was first omitted from the alginate-gel formation step prior to UV sterilization. F. periodonticum (3 × 107 cells per mL, MOI = 10) was suspended in an equivalent volume and added to the bioink either alongside hMSCs or the last volume of basal media for acellular ink. The bioink was then mixed by dual asymmetric centrifugation (1 500 r/min, 15 s), and bioprinted using an INKREDIBLE+ (Cellink). All samples were subsequently cultured under aerobic conditions.

Samples inoculated with F. periodonticum were stained using the BacLight Bacterial Viability kit (L7007, ThermoFisher) according to manufacturer’s instructions, and imaged using a confocal microscope (Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope). Semi-quantitative viability analysis of F. periodonticum was performed on image stacks via thresholding and pixel counting. Viability analysis of hMSCs was performed using the analyze particles plugin in FIJI.