Ab198228

After in vitro culture, the 3D constructs were fixed overnight in a 4% paraformaldehyde solution, before being embedded into paraffin. Histological sections (4.5 µm thickness) were stained with Hematoxylin-Eosin (#GHS116 and #HT110116, respectively from Sigma-Aldrich, Schaffausen, Switzerland), and Alizarin Red S (#A5533, Sigma-Aldrich, Schaffausen, Switzerland). The primary antibody for anti-OCN was purchased from Abcam (ab198228, Cambridge, UK) and used at 1:500 dilution, before the sections were stained with the secondary antibody (1:300 dilution) coupled to Alexa488 (Life Technologies, Carlsbad, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Schaffausen, Switzerland).

Triton X-100, tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTS), 1,6 hexanediol diacrylate, piperazine, sodium alginate, chlorhexidine diacetate, sodium acetate trihydrate, phosphate buffer solution (PBS) tablets, glutaraldehyde, Alizarin Red S, Calcein-AM, propidium iodide and “In Vitro Toxicology Assay Kit- LDH based” were purchased from Sigma-Aldrich, UK.
Cyclohexane, n-hexanol, ammonium hydroxide (35%), dichloromethane (DCM), diethyl ether, dimethyl-sulfoxide (DMSO), ethanol, methanol, glacial acetic acid and HPLC-grade acetonitrile were purchased from Fishers scientific, UK. Trihydrochloride Hoechst 33342 (Thermo Fisher Scientific, Eugene, OR, USA).
Cemex^® (Tecres^® SpA, Verona, Italy) bone cement was stored and used as from manufacturer’s guidelines.
Colorimetric ALP assay kit, picro-sirius red stain solution (ab150681), rabbit polyclonal antibodies against collagen I (ab21286), rabbit polyclonal antibodies against osteopontin (ab8448), rabbit polyclonal antibodies against osteocalcin (ab198228) and Alexa Fluor 488-labelled goat anti-rabbit IgG (ab150077) were purchased from Abcam, Cambridge, UK.
All reagents were stored according to manufacturer’s guidelines and used as received.

The cultures of PDLSCs and PSCs were incubated with antibodies to CD73 (MA5-15537, Invitrogen, Waltham, MA, USA); CD90 (ab133350, Abcam, Cambridge, UK); STRO-1 (39-8401, Invitrogen). Deparaffinized sections were incubated with antibodies to osteopontin (OPN, ab218237. Abcam), osteocalcin (OC, ab198228, Abcam), and dentin sialophosphoprotein (DSPP, ab216892, Abcam). Prior to immunohistochemical staining, antigen retrieval was performed using Dako PT Link (Dako, Denmark A/S) at 97 °C for 20 min. The unmasking was performed under low pH using EnVison FLEX Target Retrieval Solution (Dako, Glostrup, Denmark A/S). Endogenous peroxidase and host IgG were blocked, and the primary antibodies (dilution 1:200) incubated for 12 h at 4 °C. The EnVision FLEX Detection System (Dako, Denmark A/S) with the chromogen 2, 3-diamino-benzidine DAB (DAB Chromogen Solution, Dako) was used for imaging. Hematoxylin was used to counterstain nuclei. Incubation without primary antibodies was used as a negative control.

After fixation with 4% PFA for 24 h and decalcification with 10% (w/v) ethylenediaminetetraacetic acid (EDTA) for 3 weeks, tibia specimens were dehydrated with gradient ethanol, embedded in paraffin, and cut into sections (5 µm thick). For all staining, the sections that met the following criteria were used for analysis: consistent thickness, transparent appearance, complete tissue structures, no ruptures or folds, and clear contrast. Hematoxylin and eosin (H&E), Masson's trichrome, and Picrosirius red (PSR) staining were performed in the sections. For immunohistochemical staining, the sections first underwent incubation with primary antibodies overnight at 4°C, followed by incubation with anti-rabbit (1:200, ab6721, Abcam) or anti-mouse (1:200, ab6728, Abcam) HRP-conjugated secondary antibodies for 1 h at room temperature. An HRP-streptavidin system (Dako, Glostrup, Denmark) was employed to detect the positive area, followed by counterstaining with hematoxylin.
Primary antibodies against COL2 (1:100, #AF0135, Affinity Biosciences), OCN (1:200, ab198228, Abcam), p-ERK1/2 (1:200, #4370, Cell Signaling Technology), p-RUNX2 (1:100, #AF7379, Affinity Biosciences), and VEGFA (1:200, ab1316, Abcam) were used in this study.