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Supplemental Material
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
1 Tecnologica Research Institute, Biomedical Section, Crotone, Italy
2 Unit of Maxillofacial Surgery, Calabrodental, Crotone, Italy
3 School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK
*Correspondence should be addressed to:
Kevin M. Shakesheff or Marco Tatullo
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
Research and Development in Biomedicine, Stem Cells Unit, TECNOLOGICA Research Institute, st. E. Fermi
Loc. Passovecchio I. Z., 88900 Crotone (KR), Italy. Tel: 00393498742445
Supplemental Material:
Supplemental Materials and Methods (RNA isolation and quantitative real-time PCR)
Supplemental Table (Table S1)
Supplemental Figures (Figures S1-S5)
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Supplemental Materials and Methods
RNA isolation and quantitative real-time PCR (qRT-PCR)
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.
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
(Multiskan Go, Thermo Scientific). The RNA samples (250 ng) were reverse-transcribed by
MultiScribe MuLV reverse transcriptase following the instructions from the manufacturer
(Life Technologies). 25 ng of cDNA were amplified by real-time PCR with Applied
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
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%).
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Supplemental Table
Table S1. Experimental design. Treatment conditions, assignment of DPSCs/hydrogel constructs and response measures.
Results section Treatment conditions
Number of constructsSum of experiments=208
Response measures (assignment of constructs)
Results in section 3.2TCPS (basal medium)
bECM 3, 4, 6, 8 mg/ml Col-I 3, 4, 6, 8 mg/ml (basal medium)
n=6
n=9 * (for each type of hydrogel/concentration)total constructs=72 A(24)+B(24)+C(24)
Cell morphology and distribution (n=3*)Viability by cell counter (n=3*)
A-Cell morphology and distribution (n=3*)B-Viability by Dead/Live assay (n=3*) C-Viability by cell counter (n=3*)(Figure 2)
Results in section 3.3 part ATCPS (basal medium and osteogenic medium)
bECM 3, 4, 6, 8 mg/ml Col-I 3, 4, 6, 8 mg/ml (basal medium)
n=4(for each condition)
n=7 * (for each type of hydrogel/concentration)total constructs=56A(24)+B(8)+C(24)
Gene expression by qRT-PCR (n=3 for each condition)Protein expression by Flow Cytometry (n=1 for each condition)
A-Gene expression by qRT-PCR (n=3*)B-Protein expression by Flow Cytometry (n=1*)C-Protein expression by Immunofluorescence Microscopy (n=3*)(Figure 3)
Results in section 3.3 part BbECM 3, 4, 6, 8 mg/ml Col-I 3, 4, 6, 8 mg/ml (basal medium)
n=6 * (for each type of hydrogel/concentration)total (constructs)=48A(24)+B(24)
A-Mineral deposition by Von kossa (n=3*)B-Mineral deposition by Alizarin Red (n=3*)(Figure 4)
Results in section 3.4TCPS (osteogenic medium)
bECM 4 mg/ml, Col-I 4 mg/ml (basal medium and osteogenic medium)
n=4
n=4**(for each type of hydrogel and each condition)total constructs=16A(12)+B(4)
Gene expression by qRT-PCR (n=3) Protein expression by Flow Cytometry (n=1)
A-Gene expression by qRT-PCR (n=3**)B-Protein expression by Flow Cytometry (n=1**)(Figure 5)
Results in section 3.5TCPS (basal medium, GFs supplemented medium and osteogenic medium)
bECM 4 mg/ml, Col-I 4 mg/ml (basal medium and GFs supplemented medium)
n=12
n=4 ***(for each type of hydrogeland each condition)total constructs=16A(12)+B(4)
Gene expression by qRT-PCR (n=9) (3 for each condition)Protein expression by Flow Cytometry (n=1) (1 for each condition)
A-Gene expression by qRT-PCR (n=3***)B-Protein expression by Flow Cytometry (n=1***)(Figure 6)
* 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.
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Supplemental Figures
Figure S1. Representative images of bECM scaffold (4 mg/mL) used in this study.
Figure S2. Macroscopic appearance of Col-I hydrogels. Scale bar: 1 cm.
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Figure S3. Distribution of DPSCs cultured on bECM (4 and 8 mg/mL). Phase contrast, scale
bar: 100 μm.
Figure S4. Distribution of DPSCs cultured on tissue culture polystyrene (TCPS) at 1, 3, 7
and 14 days. Scale bar: 200 μm.
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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.
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