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Behavior and biocompatibility of rabbit bone marrow mesenchymal stem cells with bacterial cellulosic membrane

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247 days ago
Behavior and biocompatibility of rabbit bone marrow mesenchymal stem cells with bacterial cellulosic membrane https://t.co/xKhbt1nlNW
350 days ago
Behavior and biocompatibility of rabbits bone marrow mesenchymal stem cells with bacterial cellulosic membrane https://t.co/GbsKvipM9w
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Supplemental Information

Bone marrow mesenchymal stem cell (BM-MSC) culture and expansion photomicrography

2 Bone marrow mesenchymal stem cell (BM-MSC) culture and expansion photomicrography. (A) Rabbit cells newly isolated from the bone marrow in 12-well plates (objective 4×, bar: 50 μm), (B) cells in the adhesion process on day 5 of cell culture performed in 12-well plates (objective 20×, bar: 25 μm), (C) cells arranged in parallel with fibroblastoid morphology at 80% confluency on day 10 of cell culture in 12-well plates (objective 10×, bar: 50 μm), (D) and (E) cytoplasmic adhesion and expansion with 80% confluency in 25 cm2 bottles after trypsinization on day 15 of culture (objective 10×, bar: 50 μm), and (F) cells with fibroblastoid morphology arranged in parallel and in colonies at 80% confluency in 25 cm2 bottles after trypsinization on day 20 of culture (10× objective, bar: 50 μm).

DOI: 10.7287/peerj.preprints.3329v3/supp-2

2 Growth curve of stem cells derived from rabbit bone marrow

2 Growth curve of stem cells derived from rabbit bone marrow during 15 days of culture after thawing, at a concentration of 1 × 104 cells/mL. Phases identified: lag (days 1–4), log (days 5–11), and culture decline (days 12–15).

DOI: 10.7287/peerj.preprints.3329v3/supp-4

2 CFU-F assay

2 CFU-F assay in a 24-well plate: photomicrography of Giemsa-stained BM-MSC colonies after 2 days of cell culture at 80% confluency, and colonies with more than 30 cells per field (objective 20×, bar: 25 μm).

DOI: 10.7287/peerj.preprints.3329v3/supp-5

2 Photomicrographs showing BM-MSC differentiation

2 Photomicrographs showing BM-MSC differentiation. (A) BM-MSC chondrogenic differentiation (objective 20×, bar: 25 μm), and (B) negative control for 14 days of chondrogenic differentiation (objective 10×, bar: 25 μm).

DOI: 10.7287/peerj.preprints.3329v3/supp-6

2 Photomicrographs showing BM-MSC differentiation

2 Photomicrographs showing BM-MSC differentiation. (A) BM-MSC osteogenic differentiation showing calcium deposits in the extracellular matrix (objective 10×, bar: 25 μm), and (B) negative control for osteogenic differentiation for 21 days (objective 10×, bar: 25 μm).

DOI: 10.7287/peerj.preprints.3329v3/supp-7

Adipogenic differentiation

2 Photomicrograph showing the adipogenic differentiation of BM-MSCs, with lipid vacuoles present in the cytoplasm stained red with Oil Red (objective 40×, bar: 25 μm).

DOI: 10.7287/peerj.preprints.3329v3/supp-8

BM-MSCs adhered to the bacterial cellulosic membrane

2 Photomicrographs of BM-MSCs adhered to the bacterial cellulosic membrane (BCM). (A) BM-MSC adhesion after 7 days of cell culture, highlighting the formation of CFU-F on the BCM (objective 20×, bar: 25 μm), and (B) BM-MSC colonies after 14 days of culture (objective 10×, bar: 50 μm).

DOI: 10.7287/peerj.preprints.3329v3/supp-9

Scanning electron microscopy showing BM-MSC anchorage and biointegration with the BCM

2 Scanning electron microscopy showing BM-MSC anchorage and biointegration with the BCM. (A) and (B) analysis after 24 h of cell culture (10,000× and 20,000×, respectively), and (C) and (D) with after 7 (E) and (F) 14 days of culture (40,000× and 16,000×, respectively).

DOI: 10.7287/peerj.preprints.3329v3/supp-10

Zymosan particle phagocytosis by macrophages in the presence of the BCM

2 Zymosan particle phagocytosis by macrophages in the presence of the BCM. The graph represents the mean ± standard error of the mean of three independent experiments performed in triplicate (control: mean 0.28567, standard deviation 0.03161; BCM: mean 0.36100, standard deviation 0.03474). ABS: absorbance; C: control; BCM: bacterial cellulosic membrane; *p < 0.05.

DOI: 10.7287/peerj.preprints.3329v3/supp-11

Colorimetric nitrite dosage produced by macrophages treated with lipopolysaccharide (LPS) in the presence of the BCM

2 Colorimetric nitrite dosage produced by macrophages treated with lipopolysaccharide (LPS) in the presence of the BCM. The plot represents the mean ± standard error of the average of three independent experiments performed in triplicate (control: mean 100.0000, standard deviation 0.0000; LPS: mean 150.8889, standard deviation 1.0541; BCM: mean 109.6300, standard deviation 11.0047). Student’s t-test was performed for comparison between groups and the control (0.2% dimethyl sulfoxide [DMSO] in RPMI 1640 medium). C: control; LPS: lipopolysaccharide; BCM: bacterial cellulosic membrane; *p < 0.05.

DOI: 10.7287/peerj.preprints.3329v3/supp-12

Tetrazole salt (MTT) incubated with cells with full metabolic activity

2 Formazan crystals in BCM cultured with (A) BM-MSCs, and (B) peritoneal macrophages. Increasing view 40×.

DOI: 10.7287/peerj.preprints.3329v3/supp-13

Effect of the BCM on BM-MSCs and mammalian peritoneal macrophage viability

2 Effect of the BCM on BM-MSCs and mammalian peritoneal macrophage viability. (A) BM-MSC viability in the BCM (control: mean 100.0000, standard deviation 0.0000; BCM: mean 94.4533, standard deviation 1.1926), and (B) viability of murine macrophages in the BCM (control: mean 100.0000, standard deviation 0.0000; BCM: mean 97.7867, standard deviation 3.3200). The plot represents the mean ± standard error of the mean of three independent experiments performed in triplicate. Student’s t-test was performed to compare the groups with the control (0.2% DMSO in DMEM/RPMI medium). C: control; BCM: bacterial cellulosic membrane; *p < 0.05.

DOI: 10.7287/peerj.preprints.3329v3/supp-14

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Marcello Silva Alencar conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Yulla Klinger Carvalho performed the experiments, analyzed the data.

Camila Ernanda Carvalho performed the experiments, analyzed the data.

Matheus Tajra Feitosa conceived and designed the experiments, wrote the paper, reviewed drafts of the paper.

Michel Muálem Alves performed the experiments, analyzed the data.

Fernando Aécio de Amorim Carvalho contributed reagents/materials/analysis tools.

Bartolomeu Cruz Viana performed the experiments, contributed reagents/materials/analysis tools.

Maria Angélica Miglino wrote the paper, reviewed drafts of the paper.

Ângela Faustino Jozala conceived and designed the experiments, analyzed the data, wrote the paper, reviewed drafts of the paper.

Maria Acelina Carvalho conceived and designed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The study was carried out in accordance with the recommendations of the Guide for the Laboratory Animals Care and Use of the National Institute of Health. The protocol was approved by the Ethics Committee on Animal Use of the Federal University of Piauí (CEUA-UFPI, permit number: 268/16).

Funding

This work had the financial support of the National Council of Scientific and Technological Development- CNPq (Process: 427626/2016-1) Integrated Nucleus of Morphology and Stem Cell Research – NUPCelt. National Council of Scientific and Technological Development – CNPq (Process: 427626/2016-1) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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