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

Study design

Bone marrow samples were collected from three adult rabbits and used for isolation and cryopreservation of MSC. A Mus musculus mouse was used as a source of peritoneal macrophages. To determine cellular viability, Trypan Blue staining and growth curve analysis were performed. For the fibroblastoid colony-forming unit assay, cells collected from the bone marrow (BM) cultured in 24-well plates at passage 6 were used. Chondrogenic, osteogenic, and adipogenic induction were used to assess the potential for differentiation into mesenchymal lineages. To verify BM-MSC biointegration with the BCM, inverted light microscopy and scanning electron microscopy (SEM) were used to analyze the phagocytic capacity, toxicity, and cytotoxicity of the BCM. This study was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Ethics Committee on the Use of Animals of the Federal University of Piauí (permit number: 268/16).

Anesthetic protocol for bone marrow collection

After solid anesthetic fasting of 4 h, and 2 h of liquids, the rabbit was chemically restrained with a combination of 35 mg/kg of ketamine hydrochloride and 3 mg/kg of midazolam maleate. Trichotomy of the major trochanter region was performed, followed by antisepsis by femoral puncture with a 5 mL syringe; a heparinized 40 × 12 mm needle was used to obtain a BM sample. For antibacterial prophylaxis, 10 mg/kg of enrofloxacin was given twice daily for 7 days, and 25 mg/kg of sodium dipyrone plus 3 mg/kg of tramadol was administered twice daily for 3 days for pain control (Ninu et al., 2017).

BM-MSC isolation, cultivation, and expansion

The methodology presented was adapted from Argôlo Neto et al. (2016). Medullary aspirate (1.5 mL) was diluted in phosphate-buffered saline (PBS) at a ratio of 1:1 in 15 mL conical tubes. The resulting contents were filtered through 100 µm mesh, deposited in a 15 mL conical tube containing Ficoll Histopaque at a ratio of 1:1 (Ficoll:BM), and centrifuged at 2,000 rpm for 30 min at 20 °C to separate the cellular constituents by density gradient. The whitish halo, rich in mononuclear cells, was aspirated with an automatic pipettor (Houston; Swiftpro, HTL Lab solution, Warsaw, Poland), immediately diluted in sterile PBS with 1% antibiotic (100 U/mL penicillin and 100 µg/mL streptomycin) for cell lavage, and re-centrifuged at 1,500 rpm for 10 min at 20 °C. BM samples were resuspended in complete Dulbecco’s modified Eagle’s medium (DMEM) containing 3.7 g/L sodium bicarbonate and 10–15 mM HEPES (Invitrogen, no. 15630080; Invitrogen, Carlsbad, CA, USA), pH 7.5, 15% fetal bovine serum (Invitrogen), 1% penicillin–streptomycin, 1% L-glutamine (Invitrogen), and 1% non-essential amino acids (Sigma, St. Louis, MO, USA), and cell viability was assessed. For this purpose, a 50 µL aliquot of each sample was diluted in 50 µL 0.2% Trypan Blue dye, and mixed in a sterilized glass vial for cell counting in a Neubauer chamber.

Cells were seeded in a six-well cell culture plate (TPP) at a density of 10⁶ cells/well in 2.0 mL of low-glucose DMEM, and kept in an incubator (Thermo Scientific Series II Water Jacket; Thermo Scientific, Waltham, MA, USA) at 37 °C in 5% CO₂ and 95% humidity. The wells were washed twice every 3 days with PBS solution containing 1% antibiotic (100 U/mL penicillin and 100 µg/mL streptomycin), followed by exchange of the culture medium until the cultures reached 80% confluency. Subsequently, the wells were subjected to trypsinization with 2.0 mL 1× trypsin (Invitrogen, no. 25200-114, 10× Trypsin–EDTA solution), and incubated at 37 °C for 5 min. Following this, trypsin was inactivated with the addition of 4.0 mL low-glucose DMEM. The solution was transferred to a 15 mL conical bottom tube, and centrifuged (FANEM refrigerated Cytocentrifuge MOD.280R Excelsa 4) at 20 °C and 1,500 rpm for 10 min.

The supernatant was discarded, the pellet was resuspended in 1.0 mL of DMEM, and a new cell count was performed. The cells in suspension were used for expansion. To do this, 10⁶ cells/mL in 25 cm² tissue culture bottles with 3.0 mL of supplemented DMEM were incubated at 37 °C in 5% CO₂ and 95% humidity. The cultures were expanded and photographed with an inverted phase-contrast microscope (COLEMAN NIB-100), and peaked with twice the original area; cell concentration was verified at each passage.

Fibroblastoid colony-forming unit assay

After plating 1 ×10⁴ cells/mL of the BM-MSC rabbit fraction in 24-well plates, plates were observed daily to monitor the establishment of colonies with more than 30 cells. Cells were then fixed with 4% paraformaldehyde for 30 min, and stained with Giemsa for 10 min at room temperature. Any excess stain was washed away with distilled water. The colonies were observed and macroscopically counted on the 24-well plates (Paramasivam et al., 2016).

Cell viability

Cell count, which determines concentration and viability, was performed using the Trypan Blue exclusion method. After mixing 30 µL of the cell suspension with 30 µL Trypan Blue solution (50 µL of 4.25% sodium chloride in 200 µL of Trypan Blue), a 10 µL aliquot was observed in a Neubauer chamber under an optical microscope (10× objective). The BM-MSC growth curve was performed in duplicate by plating 1 ×10⁴ cells/mL in five six-well plates, and counting two wells every 24 h over the course of 15 days. The culture medium of the plates was changed every 3 days to maintain nutrient availability (Sangeetha et al., 2017).

Cell differentiation

For cell differentiation assays, we use the protocols provided by Stem Pro^®. Analysis of cell differentiation potential was performed with sixth-passage BM-MSCs cryopreserved in liquid nitrogen for 12 months. They were thawed and grown in 25 cm² bottles for cell expansion until 80% confluency was reached. Cultures were then trypsinized and seeded at the concentration according to the manufacturer’s instructions, for chondrogenic, osteogenic, and adipogenic differentiation.

For chondrogenic differentiation, 3 ×10⁵ cells per well were seeded in a 96-well plate. After 48 h, formation of spheroid bodies was observed, and the culture medium was replaced with that from a Stem Pro^® Chondrogenesis Differentiation Kit. Exchange of the medium was performed every 3 days during a 21-day period. Analysis was performed with histological sections stained with Alcian Blue.

For osteogenic differentiation, 6 ×10⁴ cells were seeded in a 24-well plate. Initially, the supplemented culture medium was removed and replaced with the osteogenic induction medium, and changed every 3 days during a 21-day period. During this period, morphological characteristics of the cells were evaluated. After osteogenic differentiation, cells were stained with Alizarin Red, which identifies the calcium-rich extracellular matrix, and is characteristic of the presence of osteoblasts. To do this, the cell monolayer was washed with PBS, and fixed with 10% alkaline phosphatase (AP) for 30 min at room temperature. The AP was then removed, the cell monolayer was washed with distilled water, and Alizarin Red was added for 5 min. Subsequently, the dye was removed, and five washes were performed with distilled water; the calcium-rich extracellular matrix and the amount of calcium deposits were recorded with an inverted light microscope.

For adipogenic differentiation, 2 × 10⁴ cells per well were seeded in a 24-well plate, and Stem Pro Adipogenesis Differentiation Kit induction medium was added once the cells reached 80% confluency. The culture medium was exchanged every 3 days over a period of 10 days. Once differentiation occurred, the culture was stained with Oil Red to visualize lipid vacuoles.

BM-MSC biointegration with the BCM

The bacterial cellulose membrane used in this study was developed in partnership with the pharmacy department of Sorocaba University - UNISO (Sorocaba, São Paulo-Brazil). MCB was obtained from the culture of G. xylinus ATCC 53582 prepared using 100 ml of the Hestrin and Schramm medium at 30 °C for 48 h under agitation of 150 rpm. After that, 10⁶ cells/mL-1 were withdrawn from the culture medium. For the production of MCB, 24 well plates were used. Each well was filled with 1mL of inoculated culture medium. Plates were maintained at 30 °C in a static culture for 0, 24, 48, 72 and 96 h. MSCs were seeded onto the membrane (Jozala et al., 2015).

To study BM-MSC expansion and biointegration with the BCM, 2 ×10⁴ cells were cultured in 12-well plates on BCM for three distinct periods (1, 7, and 14 days). The BM-MSCs were fixed to the BCM using 3% glutaraldehyde, washed once with PBS, and dehydrated by slow water exchange using a series of ethanol dilutions (30%, 55%, 70%, 88%, 96%, and 100%) for 20 min at each concentration. For analysis by SEM (FEI Quanta FEG 250), samples were fixed to the stub with double-sided carbon tape, placed in a dehumidifier for 2 h, and metalized with gold.

Phagocytic activation

Phagocytic activity was assessed by collecting resident macrophages from the mouse peritoneum. The animal was euthanized by cervical dislocation after being reassured and sedated by intraperitoneal injection of a combination of xylazine hydrochloride and ketamine hydrochloride (10 and 80 mg/kg body weight, respectively). Macrophage removal was performed in a laminar flow hood with the animal affixed in the dorsal decubitus position by administering 8 mL of sterile PBS at 4 °C into the abdominal cavity. The abdominal region was softly massaged, and aspiration was performed using a needle coupled to a sterile syringe. The cells were counted in a Neubauer chamber by the Trypan Blue exclusion colorimetric method, and a minimum of 95% of living cells was obtained. The cells were counted using Neutral Red to obtain the desired concentration of macrophages (2 ×10⁵ cells/mL). Peritoneal macrophages were plated in each well, and incubated on the BCM. After 48 h of incubation at 37 °C and 5% CO₂, 10 µL of stained zymosan solution was added, and incubation continued for 30 min at 37 °C. Following this, 100 µL of Baker’s fixative was added to paralyze the phagocytic process, and after 30 min the plate was washed with 0.9% saline solution to remove the zymosan and Neutral Red that were not phagocytized by macrophages. The supernatant was removed, 100 µL of extraction solution was added, and after solubilization on a Kline shaker, absorbance was measured at 550 nm in a BioTek plate reader (model ELx800) (Souza et al., 2017).

Toxicity

To assess toxicity, the nitric oxide (NO) induction test was performed. Peritoneal macrophages (2 × 10⁵ per well) were plated and incubated with the BCM after 24 h of incubation at 37 °C and 5% CO₂. Cell supernatants were transferred to another 96-well plate for nitrite dosing. The standard curve was prepared with sodium nitrite diluted in Milli-Q water at 1, 5, 10, 25, 50, 75, 100, and 150 µM in the appropriate culture medium. At the different timepoints, the standard curve was determined with the same volume of Griess reagent (1% sulfanilamide in 10% H₃PO₄ [v:v] in Milli-Q water, added in equal parts to 0.1% naphthylenediamine in Milli-Q water), and the absorbance was read on a BioTek plate reader (model ELx800) at 550 nm. Lipopolysaccharide (LPS) was used as a positive control (Sundaram et al., 2016).

BCM cytotoxicity

The basis of cytotoxicity assays is the evaluation of biomaterial-induced interference in cellular metabolic processes, and the investigation of processes that may intervene in cell growth/multiplication, or even culminate in cell death (Ávila et al., 2014).

According to Boersema et al. (2016), cytotoxicity can be evaluated by different methods according to the type of cell damage: alterations in plasma membranes can be evaluated by means of dyes such as Trypan Blue and alamarBlue; alterations in the metabolic functions of mitochondria can be measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric method.

The experiments were performed separately in 24-well plates. In the first plate, 2 × 10⁵ macrophages per well were plated, and 500 µL of supplemented RPMI 1640 medium was added. In the second plate, 1 × 10⁵ BM-MSCs in low-glucose DMEM were added. The plates were incubated at 37 °C and 5% CO₂ for 4 h to allow for cell adhesion. Two washes were performed with their respective media for removal of nonadherent cells. Subsequently, 500 µL of each medium was added, and the BCM (diameter 15.4 mm) was added. Macrophages were incubated for 48 h, and BM-MSCs for 7 days, followed by the addition of 10% 5 mg/mL MTT (diluted in medium). The macrophages and BM-MSCs were incubated for another 4 h in an incubator at 37 °C with 5% CO₂. The supernatant was discarded, and 100 µL of dimethyl sulfoxide (DMSO) was added to all wells. The BCM was removed, and the plate was shaken for 30 min on a Kline shaker (model AK 0506) at room temperature for complete dissolution of the formazan. The colorimetric reading was performed in a spectrophotometer at 550 nm in a BioTek plate reader (model ELx800). In the control group, the same conditions were applied to the culture media and the respective cultured cells (Barud et al., 2015).

Statistical analysis

For analysis of phagocytic capacity, Student’s t-test was used for independent samples of the cytotoxicity (MTT) and NO induction assays. GraphPad Prism version 5.0 was used to generate the graphs. These tests were performed in triplicate.

Differentiation into BM-MSC mesodermal lineages

The cell differentiation assay showed the potential of BM-MSCs to differentiate into chondrogenic, osteogenic and adipogenic lineages. Following chondrogenic differentiation, cells were stained vibrant blue by Alcian Blue, and control cells presented some spontaneous differentiation. During osteogenic induction, the culture demonstrated increased deposition of calcium in the extracellular matrix from day 13 of culture. On day 21 of induction, the culture exhibited osteogenic characteristics, which were confirmed with Alizarin Red staining. The negative control showed adhered cells with morphology indicative of spontaneous differentiation foci. During adipogenic differentiation, the cells gradually changed to a fibroblastoid morphology, and the cytoplasmic lipid vacuoles became bulky (Fig. 2).

BM-MSC biointegration with the BCM

In the BCM-associated cell culture, BM-MSCs with a fibroblastoid shape integrated with the biomaterial, and proliferation of the colonies was evident at 14 days of culture (Figs. 3A, 3B).

Using SEM, it was possible to observe that the rounded shape of the cells after 24 h of culture was maintained after being subtly anchored to the randomly arranged fibers of the BCM. After 7 days of culture, the cells presented themselves in groups, forming colonies with several fixation points, generating greater adhesion to the biomaterial. Micrographs recorded after 14 days of cell culture show BM-MSCs with their cytoplasm fully adhered to the BCM (Fig. 3).

Macrophage activation and BCM cytotoxicity

In the phagocytic activity assay, Student’s t-test was performed to determine the difference between the absorbance resulting from the association of macrophages with cellulose, and the control group (macrophages in the presence of 0.2% DMSO in RPMI 1640 medium). In the presence of the BCM, macrophage activity was significantly increased (Fig. 4A).

The colorimetric reading of NO release showed that the levels remained at a non-cytotoxic concentration for the cells in the presence of the BCM (Fig. 4B). The difference in NO release between the control and BCM was statistically significant at p < 0.05 (p-value 0.0184, t_0.05-critical: 2.6252), as was that between LPS and BCM (p-value: 0.0001; t_0.05-critical: 11.1963).

The tetrazole salt (MTT), incubated with cells with full metabolic activity, showed intense mitochondrial activity (Figs. 4C, 4D). In this trial, the metabolism of MTT by BM-MSCs showed a statistically significant difference (p-value: 0.0001; t_0.05-critical: 2.6252) but there was no statistically significant difference (p-value: 0.0628; t_0.05-critical: 2,000) between the BCM associated with murine macrophages and with the control. In both conditions, cell viability was greater than 94% (Figs. 4E, 4F).