<p>Supplemental Material</p><p>Decellularized Bone Extracellular Matrix and Human Dental Pulp Stem Cells as a Construct for Bone Regeneration Francesco Paduano1, Massimo Marrelli2, Noura Alom3, Mahetab Amer3, Lisa J White3, Kevin M Shakesheff*3 & Marco Tatullo*1</p><p>1 Tecnologica Research Institute, Biomedical Section, Crotone, Italy</p><p>2 Unit of Maxillofacial Surgery, Calabrodental, Crotone, Italy</p><p>3 School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK</p><p>*Correspondence should be addressed to: </p><p>Kevin M. Shakesheff or Marco Tatullo [email protected] Wolfson Centre for Stem Cells, Tissue Engineering, and Modelling (STEM) Centre for Biomolecular Sciences University of Nottingham University Park Nottingham NG7 2RD, UK Tel: +44 (0) 115 9515 104/121 [email protected] Research and Development in Biomedicine, Stem Cells Unit, TECNOLOGICA Research Institute, st. E. Fermi Loc. Passovecchio I. Z., 88900 Crotone (KR), Italy. Tel: 00393498742445</p><p>Supplemental Material:</p><p>Supplemental Materials and Methods (RNA isolation and quantitative real-time PCR)</p><p>Supplemental Table (Table S1)</p><p>Supplemental Figures (Figures S1-S5)</p><p>Page 1 of 6 Supplemental Materials and Methods</p><p>RNA isolation and quantitative real-time PCR (qRT-PCR)</p><p>DPSCs/bECM or DPSCs/Col-I hydrogel constructs were washed three times in PBS and digested with a solution of 3 mg/mL type I collagenase and 4 mg/mL dispase for 1 h at 37°C.</p><p>Recovered cells (0.5X106) were washed and subsequently total cellular RNA was isolated using the Rneasy Mini Kit following manufacturer’s instructions (Qiagen). The concentration of the obtained RNA (50-300 ng/μl) was quantified using a spectrophotometer</p><p>(Multiskan Go, Thermo Scientific). The RNA samples (250 ng) were reverse-transcribed by</p><p>MultiScribe MuLV reverse transcriptase following the instructions from the manufacturer</p><p>(Life Technologies). 25 ng of cDNA were amplified by real-time PCR with Applied</p><p>Biosystems SYBR green kit and 5 pmol primers in a total volume of 10 µL. The qPCR reactions were carried out in a Pikoreal 96 instrument (Thermo Scientific) apparatus under the following program: initial denaturation step at 95°C for 10 min, followed by 40 cycles of</p><p>10 s at 95 °C and 1 min at 60°C. In addition, expected amplification was confirmed by Tm value measured by melting curve analysis of qRT-PCR. Ct values of each gene were normalised against the hypoxanthine phosphoribosyltransferase (HRPT) and the relative gene expression levels were obtained using the ΔΔCt method. Real-time efficiencies were calculated from the cycle threshold (Ct) curves obtained for the amplification of the cDNA samples (serial ten-fold dilutions: 1:10, 1:100 and 1:1000). The primer efficiencies were in the recommended range (90–105%). </p><p>2 Supplemental Table </p><p>Table S1. Experimental design. Treatment conditions, assignment of DPSCs/hydrogel constructs and response measures.</p><p>Results section Number of constructs Response measures (assignment of constructs) Treatment conditions Sum of experiments=208 Results in section 3.2 n=6 Cell morphology and distribution (n=3*) TCPS (basal medium) Viability by cell counter (n=3*) bECM 3, 4, 6, 8 mg/ml n=9 * A-Cell morphology and distribution (n=3*) Col-I 3, 4, 6, 8 mg/ml (for each type of B-Viability by Dead/Live assay (n=3*) (basal medium) hydrogel/concentration) C-Viability by cell counter (n=3*) total constructs=72 (Figure 2) A(24)+B(24)+C(24) Results in section 3.3 part A n=4 Gene expression by qRT-PCR (n=3 for each condition) TCPS (basal medium and osteogenic (for each condition) Protein expression by Flow Cytometry (n=1 for each condition) medium) bECM 3, 4, 6, 8 mg/ml n=7 * A-Gene expression by qRT-PCR (n=3*) Col-I 3, 4, 6, 8 mg/ml (for each type of B-Protein expression by Flow Cytometry (n=1*) (basal medium) hydrogel/concentration) C-Protein expression by Immunofluorescence Microscopy (n=3*) total constructs=56 (Figure 3) A(24)+B(8)+C(24)</p><p>Results in section 3.3 part B n=6 * bECM 3, 4, 6, 8 mg/ml (for each type of A-Mineral deposition by Von kossa (n=3*) Col-I 3, 4, 6, 8 mg/ml hydrogel/concentration) B-Mineral deposition by Alizarin Red (n=3*) (basal medium) total (constructs)=48 (Figure 4) A(24)+B(24) Results in section 3.4 n=4 Gene expression by qRT-PCR (n=3) TCPS (osteogenic medium) Protein expression by Flow Cytometry (n=1) bECM 4 mg/ml, n=4** A-Gene expression by qRT-PCR (n=3**) Col-I 4 mg/ml (for each type of hydrogel B-Protein expression by Flow Cytometry (n=1**) (basal medium and osteogenic and each condition) (Figure 5) medium) total constructs=16 A(12)+B(4)</p><p>Results in section 3.5 n=12 Gene expression by qRT-PCR (n=9) (3 for each condition) TCPS (basal medium, GFs Protein expression by Flow Cytometry (n=1) (1 for each condition) supplemented medium and osteogenic medium) A-Gene expression by qRT-PCR (n=3***) bECM 4 mg/ml, n=4 *** B-Protein expression by Flow Cytometry (n=1***) Col-I 4 mg/ml (for each type of hydrogel (Figure 6) (basal medium and GFs and each condition) supplemented medium) total constructs=16 A(12)+B(4)</p><p>* for each type of hydrogel (bECM, Col-I)/each concentration (3, 4, 6, 8 mg/ml). ** for each type of hydrogel (bECM, Col-I) /each condition (basal medium, osteogenic medium). ** for each type of hydrogel (bECM, Col-I) /each condition (basal medium, GFs supplemented medium). TCPS, tissue culture polystyrene; GFs, growth factors, bECM, bone extracellular matrix; Col-I, collagen type-I. </p><p>Page 3 of 6 Supplemental Figures</p><p>Figure S1. Representative images of bECM scaffold (4 mg/mL) used in this study.</p><p>Figure S2. Macroscopic appearance of Col-I hydrogels. Scale bar: 1 cm.</p><p>4 Figure S3. Distribution of DPSCs cultured on bECM (4 and 8 mg/mL). Phase contrast, scale bar: 100 μm.</p><p>Figure S4. Distribution of DPSCs cultured on tissue culture polystyrene (TCPS) at 1, 3, 7 and 14 days. Scale bar: 200 μm.</p><p>Page 5 of 6 Figure S5. Live/dead analysis of cells/scaffold constructs 14 days after cell seeding in basal medium, GFs supplemented medium and osteogenic medium. Viable cells stained green and dead cells stained red after Calcein/AM-EthD-III staining. Cells were observed under fluorescence microscope, scale bar: 100 μm.</p><p>6</p>