The use of human tissue was approved by the IRB at the University of Texas Health Science Center at Houston (UTHealth), and all animal experiments and procedures were approved by the IACUC at UTHealth (AWC-15-0072).
Cell isolation
Six populations of hMDSCs were isolated via a modified preplate technique, as previously described [26], from skeletal muscle biopsies purchased from the National Disease Research Interchange (NDRI). This pre-plate technique we used for isolation of MDSCs was modified from previously reported protocol [27,28,29]. Briefly, the muscle tissues purchased from NDRI were rinsed with Hanks Balanced Salt Solution (HBSS), and the fat and connective tissues were removed. Muscle tissues were cut into small pieces with scissors into slurry (1 mm in size) in HBSS solution. Then, the tissue slurry was centrifuged to remove HBSS, and pellets were weighed, and 1 ml of Collagenase XI was used for 0.1 g muscle. The tissues were digested with collagenase XI (0.2% weight/volume) (Sigma-Aldrich, Cat. #C7657), dispase (2.4 U/ml) (Invitrogen, Cat. #17105-041), and Trypsin-EDTA (0.1% weight/volume, Invitrogen, Cat. #15400-054) at 37 °C. The digestion time was 1 h for collagenase XI, 45 min for dispase, and 30 min for Trypsin-EDTA. The enzyme was removed by centrifugation at 2630 g for 5 min for each step. We added the dispase digestion step in our protocol compared to previous protocol. The cells were seeded in collagen I coated flasks (Sigma-Aldrich, Cat. #C9791) and incubated at 5% CO2 and 37 °C for 2 h. The un-adhered cells were transferred to another new collagen-coated flask. This step was repeated 5 more times until we obtained the preplate 6 (pp6). The pp6 cells eventually adhered and grew and were called hMDSCs [26]. The 6 populations of hMDSCs included three young populations (31-year-old female, hMDSC1; 21-year-old male, hMDSC2; 23-year-old male, hMDSC3) and three old populations (76-year-old female, hMDSC4; 78-year-old male, hMDSC5; 80-year-old male, hMDSC6) respectively. The hMDSCs were grown and maintained in proliferation medium (PM) that contained high glucose DMEM (Invitrogen) supplemented with 20% fetal bovine serum (FBS), 1% chicken embryo extract (CEE), and 1% penicillin/streptomycin.
Construction of the lenti-viral-BMP2/GFP vector
A lenti-viral vector encoding for human BMP2 gene under the control of the human cytomegalovirus (CMV) promoter with a green fluorescent protein (GFP) tag, which was separated by an internal ribosome entry site (IRES) from the target gene, was constructed in collaboration with Dr. Bing Wang’s Laboratory. The GFP tag allowed us to monitor transduction efficiency and use fluorescence-activated cell sorting (FACS) to select transduced cells. Lenti-GFP (LGFP) and lenti-BMP2/GFP (LBMP2/GFP) viral vectors were packaged using 293 T cells (ATCC) using established protocol.
Cell transduction
Human MDSCs were transduced with LBMP2/GFP or LGFP virus in the presence of polybrene (8 μg/ml) using 1:4 and 1:12 dilution with proliferation medium respectively at passage 8–10 for 16 h. At 24 h after transduction, transduction efficiency was observed under a fluorescent microscope. The transduction efficiency was about 50–60%. Cells were passaged 2 times after transduction and then subjected to GFP cell sorting (FACS) based on GFP fluorescence. After cell sorting, the cells were expanded in proliferation media. Supernatants were collected from different passages of each population and BMP2 secretion levels were measured using an ELISA (DBP200, R&D system). One population of young female cells (hMDSC1) was transduced with both LGFP and LBMP2/GFP for in vivo studies.
In vitro chondrogenesis
In vitro chondrogenesis assay was performed for non-transduced or LBMP2/GFP-transduced hMDSCs using a 3D pellet culture method, as previously described [30]. Briefly, 6 populations of non-transduced cells and 6 populations of LBMP2/GFP-transduced cells were cultured in proliferation medium and expanded. 1.25 × 105 cells from each population were aliquoted to 15 ml tubes in 4 replicates/population. The cells were centrifuged at 800g for 5 min and resuspended with complete chondrogenic medium (StemPro® Chondrogenesis Differentiation Kit, A1007101, ThermoFisher Scientific). The cells were then centrifuged at 500g for 5 min, and the lids of tubes were loosened ¼ turn to allow for oxygen exchange. Using this method, cells usually form pellets around 3 days of culture. Chondrogenic medium was changed every 2–3 days for 24 days. The pellets were fixed with neutral buffered formalin (NBF), rinsed once with PBS, then embedded in NEG freezing medium, snap frozen in liquid nitrogen, and stored at − 80 °C until sectioning, at which time 8-μm cryosections were cut. Pellets’ cultures were repeated three times for each population. Alcian blue staining was performed using online protocol (http://www.ihcworld.com/_protocols/special_stains/alcian_blue.htm.). Images were captured using a NIKON Cil microscope, and blue matrix was quantified using the NIKON NIS Element software. Collagen type II alpha 1 (Col2A1) immunohistochemistry (IHC) was also performed using goat anti-Col2A1 (SC7764, 1:50, Santa Cruz Biotechnology). In addition, Raman spectroscopy was utilized to quantitate sulfated cartilage matrix (proteoglycan aggrecan) at the Raman band ~ 1060 cm−1 (sulfate) and collagen at Raman band ~ 856 cm−1 (Proline) for the chondrogenic pellets derived from hMDSC5 and hMDSC6 before fixation by Dr. Xiaohong Bi’s laboratory.
Preparation of coacervate and binding of BMP2 and sFLT1
Preparation of coacervate and binding of BMP2 and sFLT1 was carried out as follows. BMP2 (120-02C, PeproTech) and sFLT1 (ab54346, Abcam) were purchased and resuspended according to the manufacturer’s protocol to a concentration of 100 ng/μl in phosphate-buffered saline (PBS). Coacervate was formed using heparin and PEAD, which was synthesized by Dr. Yadong Wang’s lab, as previously described [31, 32]. We engineered our controlled delivery system to be more stable than the typical coacervate after a selection process that paired the polycation, PEAD, with heparin. We have previously demonstrated that the coacervate was present after 4 weeks in an infarcted myocardium [33]. BMP2 (500 ng) alone, or BMP2 plus sFLT1 (500 ng each), was first mixed with 12.5 μl of heparin (2 mg/ml) to allow proteins to bind to heparin, followed by addition of 12.5 μl of PEAD (10 mg/ml), and the complex became turbid which indicated the formation of coacervate. Then, each coacervate mixture was ready for combining with the different cell populations, as described below.
In vivo articular cartilage (AC) repair using MIA-induced osteoarthritis model
In vivo cartilage repair was investigated using an MIA-induced global osteoarthritis model with delivery of BMP2 and hMDSCs using coacervate, and this delivery method was compared with LBMP2/GFP gene therapy. Thirty male nude rats (Taconic) of 12 weeks old were injected with 0.3 mg/150 g body weight MIA in 50 μl volume in the right knee (injured) according to our published paper [12], and the left knee (uninjured) was used as the normal control per our animal protocol approved by the IACUC at UTHealth. Two weeks after MIA injection, the rats were divided into 5 groups and injected in the injured knee joint with different combinations of cells/proteins or complexes (as stated below). Nude rats were divided into the following 5 groups (N = 6 each group).
PBS + coacervate group: PEAD (12.5 μl) was added to heparin (12.5 μl), and then PBS (25 μl) was added, followed by mixing and injection.
BMP2 + PBS + hMDSC-LGFP (1 × 106) group: BMP2 (500 ng in 5 μl) was added to PBS (25 μl) and then added to the hMDSC-LGFP cell suspension (1 × 106 cells in 20 μl PBS), followed by mixing prior to injection.
PBS + hMDSC-LBMP2/GFP (1 × 106) group: PBS (25 μl) was added to 25 μl of cell suspension, for a total of 50 μl, and mixed prior to injection.
BMP2 + coacervate + hMDSC-LGFP (1 × 106) group: BMP2 (500 ng in 5 μl) was mixed with heparin (12.5 μl), then PEAD was added (12.5 μl), and the complex was mixed with hMDSCs (1 × 106) in 20 μl PBS before injection.
BMP2 + sFLT1 + coacervate + hMDSC-LGFP group: BMP2 (500 ng in 5 μl) and sFLT1 (500 ng in 5 μl) were mixed with heparin (12.5 μl) and then PEAD (12.5 μl) was added, and then the protein-loaded complex was mixed with the hMDSC-LGFP cell suspension (1 × 106 in 15 μl PBS) just prior to knee joint injection. The nude rats were sacrificed at 12 weeks post-knee joint cell injection, and both the injured and uninjured knees were harvested and then fixed in NBF for 3 days for subsequent microCT and histology.
MicroCT scanning and analysis
After fixation, both injured and uninjured knees were scanned using microCT (Viva CT 40, Scanco Medical) without contrast using 70 kvp, 112 μA, and a 30-μm voxel size. 3D images of the whole knee joint were reconstructed using Gauss = 0.8, Sigma = 1, and a threshold of 200 using the same dimensions for subsequent analysis. After 3D reconstruction, the empty gap of each knee joint of 3D image was measured using image J software. The bigger gap indicated more severe the cartilage erosion. The knee joint gap differences were calculated using injured knee gap subtracted non-injured knee of the same rat and compared between groups.
Histology
After microCT, the knee joints were decalcified using 10% ethylenediaminetetraacetic acid disodium (EDTA) plus 1% sodium hydroxide for 3 months. Whole knee joints were cut in the middle sagittally, dehydrated, and then paraffin embedded so that the middle of the joint (groove level) and the edge of the joint (condyle level) could be viewed. Both levels of sections were used for quantification and histology score. Paraffin sections of 5 μm were cut. H&E staining and Alcian blue staining were performed per the following IHC WORLD protocols respectively: http://www.ihcworld.com/_protocols/special_stains/h&e_ellis.htm
http://www.ihcworld.com/_protocols/special_stains/alcian_blue.htm.
Toluidine blue staining was performed using IHC world protocol:
http://www.ihcworld.com/_protocols/special_stains/toluidine_blue.htm
Images were captured for entire area of cartilage surface of each animal at both middle (groove) and edge (condyle) at × 200 magnification. The summed area of each pathological change was measured with Image J for scoring. Histology score was given using Osteoarthritis Research Society International (OARSI) grading (1–6) and staging (1–4) criteria [34]. The grading and staging were performed blindly. If the score is higher, it indicates worse cartilage repair.
Immunohistochemistry
IHC staining of GFP-positive cells was used to reveal donor cells in the regenerated cartilage and Col2 staining was used to detect specific cartilage matrix collagen 2. Briefly, after deparaffinization, washing, and blocking with 5% donkey serum in PBS, sections were incubated with rabbit anti-GFP antibody (ab290, Abcam, 1:1000 dilution) and rabbit anti-Col2 (ab34712, Abcam: 1:400 dilution) in 5% donkey serum overnight. For Col2 staining, antigen retrieval was performed using 2% hyaluronidase (H3506-5G, Sigma,) in PBS at room temperature for 30 min, followed by washing with PBS three times before blocking and incubation with primary antibody. The following day, sections were treated with 0.5% H2O2 in PBS for 30 min at room temperature, washed in PBS, and then incubated with goat anti-rabbit biotin (BA 1000, Vector Laboratories, Burlingame, CA, USA, 1:200 dilution) for 2 h at room temperature. After three washes, each slide was incubated with ABC reagent (PK 7200, Elite ABC kits, Vector Laboratories) for 2 h at room temperature. After three washes with PBS, diaminobenzidine (DAB) staining (SK-4100, Vector Laboratories) was used to visualize the GFP-positive cells. Hematoxylin (H-3404, Vector laboratories) counterstaining was performed following the DAB color reaction.
Statistical analysis
One-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple test was used to analyze multiple quantitative data using GraphPad Prism 7. Two-side Student T test was used to compare the two groups. Wilcoxon rank-sum non-parametric test was used for microCT and histology score analysis due to high deviation of the parameters. In brief, the two comparison group values were ranked from small to large, and the numerical rank sum of each group was compared to the Wilcoxon rank-sum table; the P value was determined based on the upper tail and lower tails of the comparison groups. If the sum of one group was larger than the upper tail value, then we deemed P < 0.05 or P < 0.01 to be statistically different. Similarly, if the sum of one group was lower than lower tail probability value, we also deemed P < 0.05 or P < 0.01 was statistically different. Overall, a value of P < 0.05 was considered statistically significant for all statistical analysis method.