Reagents
DONA® Crystalline Glucosamine Sulfate was provided by Mylan S.r.l (Italy), resuspended at 50 mg/mL concentration and stored at 4°C. Histopaque®-1077, Ascorbic acid‐2‐phosphate, β‐glycerophosphate, dexamethasone, 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (MTT), Alizarin Red S (ARS), paraformaldehyde, Triton X‐100, and Acid phosphatase, leukocyte (TRAP) Kit (no. 386), Fetal Calf Serum (FCS), L-Glutamine, antibiotics (penicillin and streptomycin) were purchased from Sigma‐Aldrich (Saint Louis, USA). High Capacity cDNA Reverse Transcription Kit, TaqMan Gene Expression assays, Universal Master mix II and Alexa Fluor 488 Phalloidin (#A12379) were purchased from ThermoFisher Scientific (Waltham, MA, USA). Antibodies for human Runx2 (#sc-10758), COL1a1 (#sc-28657), NFATc1 (#sc-13033), Cathepsin K (#sc-48353), were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and OPN (LF-123) is a generous gift from dr L. Fisher (NIH, Bethesda, USA). Dulbecco's Modified Eagle’s Medium High glucose (DMEM), Ham’s F12 and PBS were purchased from Euroclone (Milan, Italy).
Cell isolation and culture
Patients with osteoarthritis (OA patients, n=7, mean age 60 years, female) and healthy volunteers (n=4, mean age 45 years, female) were enrolled during routine medical check-up at “Centro di Medicina” (Ferrara, Italy) after informed consent, according to the Institution’s research protocol approved (n. 172201). Briefly, PBMCs (Peripheral Blood Mononuclear Cells) were obtained from 20 mL of peripheral blood and separated by Histopaque®-1077 as previously described [16]. Monocytes (hMCs) were purified from PBMCs by adhesion selection on polystyrene plates. 1 × 106 PBMCs/cm2 were plated, allowed to settle for 4 h at 37° and flasks were then rinsed to remove non-adherent cells. In order to confirm the ability of isolated hMCs to differentiate into mature osteoclasts (hOCs), M-CSF (25 ng/mL) and RANKL (30 ng/mL) (PeproTech EC Ltd, London, UK) were added to the culture medium and after 14 days, TRAP staining was carried out. The expression levels of the osteoclast-specific markers Cathepsin K, nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) were assessed by immunocytochemistry.
Human osteoblasts (hOBs) were obtained from vertebral lamina, discarded during spinal surgery. Bone fragments were obtained from 4 donors (mean age 45 years, 2 males and 2 females) using research protocol approved by Ethics Committee of the University of Ferrara and S. Anna Hospital (protocol approved on November 17, 2016). Briefly, the bone fragments were placed in sterile phosphate buffered solution (PBS) at 4°C and dissected within 16 hours after removal. Bone chips were minced into smaller pieces as previously reported [17], washed twice with PBS, plated in T-25 culture flasks (Sarstedt, Nümbrecht, Germany) and cultured in 50% DMEM high-glucose/50% Ham’s F12, supplemented with 10% FCS, 1 mM L-Glutamine, antibiotics (penicillin 100 μg/mL and streptomycin 10 μg/mL). Upon detection of a cell colony from the bone fragments (after 7 days), the cells were expanded until confluent (passage zero, P0). The cells were then harvested after treatment with 0.05 % trypsin ethylenediamine tetraacetic acid (Sigma-Aldrich), washed, counted by hemocytometric analysis, and used for further experiments (passage 1 to passage 3). During the culture period, cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and the medium was changed every 3 days. hOBs (P0) were characterized for the presence of the following specific bone markers: Osteopontin (OPN), RUNT related transcription factor 2 (Runx2) and collagen type 1 alpha (COL1a1) by immunostaining.
For osteogenic differentiation, hOBs were seeded at a density of 10000/cm2 in 12-well plates and cultured up to 14 days in osteogenic medium (OM) consisting in DMEM high-glucose 10% FCS, 10 mM β-glycerophosphate, 100 nM dexamethasone and 100 μM ascorbate, in the presence or absence of 200 µg/mL GLcN (treatment every 3 days).
Tartrate-resistant acid phosphatase (TRAP) staining
TRAP staining of the cells was performed as previously reported [18]. Briefly, the cells were fixed in 4% paraformaldehyde (PFA) with 0.1 M cacodilic buffer, pH 7.2 (0.1 M sodium cacodilate, 0.0025% CaCl2) for 15 min, extensively washed in the same buffer, and stained for TRAP (Acid Phosphatase Leukocyte Kit) according to the manufacturer's protocol. After washing with distilled water and drying, mature TRAP positive multinucleated cells containing more than three nuclei were counted as osteoclasts.
Immunocytochemistry
Immunocytochemical analysis was performed using an ImmPRESS Universal Reagent kit (Vector Laboratories, Inc., Burlingame, CA, USA). The hOCs or hOBs were seeded in 24-well plates, fixed in cold 100% methanol at room temperature (RT) for 10 min and permeabilized with 0.2% (vol/vol) Triton X-100, in Tris-buffered saline (TBS 1X). Then the cells were treated in 0.3% H2O2 in TBS 1X for 10 min at RT, and subsequently incubated with ready-to-use (2.5%) normal horse serum blocking solution (ImmPRESS reagent kit; Vector Laboratories, Inc.) for 15 min at room temperature.
After the incubation in blocking serum, the different primary antibodies were added and incubated at 4 °C overnight: rabbit anti-human polyclonal antibodies for Runx2 (1:200 dilution), COL1a1 (1:100 dilution), NFATc1 (1:300 dilution), Cathepsin K (1:200 dilution) and OPN (1:200 dilution). After rinsing in TBS 1X, the cells were incubated for 30 min at room temperature with ImmPRESS reagent (ImmPRESS reagent kit; Vector Laboratories, Inc.) and then stained with substrate/chromogen mix (ImmPACT™ DAB; Vector Laboratories, Inc.). After washing, the cells were mounted in glycerol/PBS 9:1 and observed with a Nikon Eclipse 50i optical microscope (Nikon Corporation, Tokyo, Japan).
MTT assay
The effect of GLcN on hOCs and hOBs viability was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The cells were seeded in 96-well plates and treated with increasing concentrations of GLcN (10-100-200 g/mL). After 72 hours of treatment, a solution of MTT in PBS was added to each well and the plate was incubated for 3 hours at 37°C. The MTT crystals were solubilized with 200 µL lysis buffer (10% Sodium dodecyl sulfate, SDS). Spectrophotometric absorbance of each sample was then measured at 570 nm by using a microplate reader (Sunrise™ Absorbance Reader, Tecan Group Ltd., Männedorf, Switzerland). Live cells were calculated as a percentage of control (untreated cells).
Apoptosis (TUNEL assay)
At the end of osteoclastogenic induction mature hOCs were treated with GLcN (100 and 200 µg/mL) for 72 hours. The cells were then rinsed twice with PBS and fixed for 25 min in 4% PFA at room temperature. Apoptotic cells were detected by the DeadEnd Colorimetric Apoptosis Detection System (Promega, Madison, WI, USA) according to the manufacturer's instructions. Moreover, all cells were subjected to hematoxylin staining, showing blue stained nuclei. The cells were mounted in glycerol/PBS 9:1 and observed under a Leica microscope (Leica Microsystems GmbH, Wetzlar, Germany). The measurement of apoptosis was calculated as a percentage of apoptotic nuclei (dark brown nuclei) vs total nuclei of multinucleated TRAP positive cells, evaluated in triplicate from each experimental sample.
Phalloidin staining
For analysis of F-actin organization, hOCs were fixed with 4% PFA for 10 min, permeabilized with 0.1 % Triton X-100 for 15 min and stained with Alexa Fluor 488 Phalloidin (1:500 dilution in PBS) for 30 min at room temperature. Nuclei were counterstained with 4′,6- diamidino-2-phenylindole (DAPI). Fluorescent Images were obtained with a fluorescence microscopy (Nikon Eclipse 50i).
RNA isolation and RT-qPCR
Total RNA was isolated from hOBs (2D cultured in OM in the presence or absence of GLcN 200 g/mL), by using the RNeasy Micro Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. RNA concentration and quality was measured using a NanoDrop ND1000 UV-VIS spectrophotometer (Isogen Life Science, de Meern, the Netherlands). cDNA was synthesized from total RNA in a 20 μL reaction volume using the High Capacity cDNA Reverse Transcription Kit. Finally 100 ng of cDNA was used for RT‐qPCR analysis. The TaqMan Universal Master Mix II and probes for human ALP (Hs01029144_m1), RUNX2 (Hs00231692_m1), OPN (Hs00959010_m1), COL1A1 (Hs00164004_m1), OCN (Hs01587813_g1), BSP (Hs00913377_m1). were used according to the manufacturer's instructions. RPL13a (Hs04194366_g1) was used for normalization of mRNA abundance. The gene expression was assessed using the CFX96TM PCR detection system (Bio-Rad, Hercules, CA, USA). Relative gene expression was calculated using the comparative 2-ΔΔCT method (expressed as fold change). All reactions were performed in triplicate, n=4.
hOBs/hMCs cultured in 3D dynamic system
The 3D dynamic culture condition was set up by using the RCCS-4TM bioreactor (Synthecon™, Inc., Houston, TX, USA), with a High Aspect Ratio Vessel (HARV™; Synthecon™, Inc., Houston, TX, USA). The HARV consists of a horizontally rotated culture chamber, where the cells are suspended, and a perfusion system with media continuously flowing through the culture chamber. The culture chamber can rotate in the X-axis at certain speeds (rpm): higher rpm is associated to lower gravity. The rotation speed applied for the experiments was 4 rpm corresponding to Ground Based dynamic culture in which aggregates were in continuous falling rotation close to the bottom of the vessel (3D-DycC condition).
hOBs/hMCs aggregates were generated in the absence of exogenous scaffolds as previously reported [15, 18]. Briefly, 1x106 cells/mL hOBs and 500x103 cells/mL hMCs (2:1 cell ratio) were inoculated in 2 mL HARV filled with DMEM high glucose supplemented with 10% FCS and all air bubbles were removed from the culture chamber. HARV was then inserted into the RCCS-4TM rotary bioreactor and placed in an incubator at 37 °C, in a humidified atmosphere with 5% CO2. After 24 hours, the presence of aggregates was observed, and the vessels were filled with osteogenic medium alone (OM) or in the presence of 200 µg/mL GlcN (OM/ GlcN). Osteogenic medium and treatment with GlcN was refreshed twice a week. After 14 days the aggregates were collected, fixed in 4% PFA, embedded in paraffin for further analysis.
Histology
Immunohistochemistry was performed employing the ImmPRESS (Vectorlabs, Burlingame, CA). Histological sections (5 μm) of aggregates were subjected to immunohistochemistry. Non-consecutive sections were deparaffinized, rehydrated and enzymatic treated with 1 mg/mL protease K (Sigma-Aldrich) for antigen retrieval and permeabilization. Slides were then immunostained overnight with the primary antibody against osteopontin (OPN; rabbit anti-human, 1:100 dilution), in a humid chamber at 4 °C, followed by treatment with Vecstain ABC reagent (Vectorlabs) for 30 min. The reaction were developed using DAB solution (Vectorlabs); the sections were counterstained with haematoxylin, mounted in glycerol and observed using the Nikon Esclipse 50i optical microscope.
For Alizarin Red Staining (ARS) the sections were deparaffinized and stained with 40 mM Alizarin Red S solution (pH 4.2) at room temperature for 20 min. TRAP staining was carried out with the Acid Phosphatase Leukocyte (TRAP) Kit according to the manufacturer's protocol. The stainings were quantified by a computerised video camera-based image analysis system (NIH, USA ImageJ software, public domain available at: http://rsb.info.nih.gov/nih-image/) under brightfield microscopy (Nikon Eclipse 50i; Nikon Corporation, Tokyo, Japan). Colour TIFF file images were converted to 32-bit images and inverted so that the background could be set to the lower threshold limit. After applying the image threshold, the background was removed and not counted toward mean pixel intensity. Mean pixel intensity per area was used for the histological images quantification of OPN, (five sections per sample; n= 3). The percentage of positive area was applied for the quantification of ARS and TRAP staining, considering the tears/holes within matrix of samples.
Statistical analysis
All data were presented as mean ± standard deviation (SD). Two-tailed Student’s t-tests were used to determine statistical significance. All tests were done using GraphPad Prism 5 program (La Jolla, CA, USA). p < 0.05 was considered statistically significant.