Cell Sheets of Co-cultured Endothelial Progenitor Cells and Mesenchymal Stromal Cells Promote Osseointegration in Irradiated Rat Bone

Irradiated bone has a greater risk of implant failure than nonirradiated bone. The purpose of this study was to investigate the influence of cell sheets composed of co-cultured bone marrow mesenchymal stromal cells (BMSCs) and endothelial progenitor cells (EPCs) on implant osseointegration in irradiated bone. Cell sheets (EPCs, BMSCs or co-cultured EPCs and BMSCs) were wrapped around titanium implants to make cell sheet-implant complexes. The co-cultured group showed the highest osteogenic differentiation potential in vitro, as indicated by the extracellular matrix mineralization and the expression of osteogenesis related genes at both mRNA and protein levels. The co-cultured cells promoted ectopic bone formation as indicated by micro-computed tomography (Micro-CT) and histological analysis. In the irradiated tibias of rats, implants of the co-cultured group showed enhanced osseointegration by Micro-CT evaluation and histological observation. Co-cultured EPCs and BMSCs also up-regulated the expression of osteogenesis related genes in bone fragments in close contact with implants. In conclusion, cell sheets of co-cultured EPCs and BMSCs could promote osseous healing around implants and are potentially useful to improve osseointegration process for patients after radiotherapy.


Culture and characterization of BMSCs Isolation and culture of BMSCs
Rat BMSCs were isolated and cultured as it reported 1 . Briefly, tibias and femurs were removed after the rats were euthanized. Bone marrow was flushed out with αminimum essential medium (α-MEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. China) and 1% penicillin and streptomycin. Upon reaching 80% confluence, adherent cells were trypsinized and sub-cultured for further experiments.

Differentiation assays of BMSCs
For osteogenesis, cells were incubated in osteogenesis-inducing medium (10 mM β-glycerolphosphate, 50µg/ml Vc and 0.1mM dexamethasone, all from Sigma-Aldrich) for three weeks. The cells were stained with Alizarin Red S (Sigma-Aldrich, USA) after fixation. For adipogenesis, the cultured cells were incubated in adipogenic differentiation medium (50mM indomethacin, 100nM dexamethasone, 10 mM insulin, 0.5mM methylisobutylxanthine, all from Sigma-Aldrich) for two weeks. The cells stained with Oil Red O (Sigma-Aldrich) after fixation. For chondrogenesis, cells were centrifuged to form a pelleted micromass and were incubated in chondrogenic differentiation medium (Cyagen, China, RASMX-90042) for 28 days. Pellets were formalin fixed and paraffin embedded for Alcian blue stain (Cyagen, China).

Culture and characterization of EPCs Isolation and culture of EPCs
Rat EPCs were isolated and cultured as reported before 2 . The bone marrow suspension was fractionated by density gradient centrifugation (Histopaque-1083, Sigma, USA) for 30 min at 400g. The mononuclear cells were washed three times with PBS at 250g for 10 min. The cell pellet was then suspended in EBM-2 medium with EGM-2 MV SingleQuots (Lonza, USA) and plated on fibronectin-coated culture dishes.
After 48h the non-adherent cell population was aspirated and transferred to new fibronectin-coated dishes. The culture medium was changed every 2 days and cultured for a total of 13 days. The adherent cells were subcultured for further experiments.

Senescence-associated β-galactosidase staining
Senescence of cultivated BMSCs and EPCs at passages 3 were studied using senescence-associated β-Galactosidase Staining Kit (Beyotime, China) according to the manufacturer's protocol. At the end of staining procedure, five pictures were taken from random areas of each culture. The percentage of senescent cells was calculated (number of cells with intracellular blue deposits/ total number of cells ×100%).

In vitro osteogenesis of different cell sheet-complexes ALP production
Cell sheet-complexes were washed with PBS and fixed in 4% paraformaldehyde for 15min, stained with the BCIP/NBT ALP color development kit (Beyotime, China) for 10minutes at room temperature, and were observed and photographed by Stereo Microscope.

ECM mineralized nodule staining
After fixation, cell sheet-complexes were stained with 1wt% Alizarin Red S for 5 minutes at room temperature, and several washes with PBS were used to remove unbound dye. The stained calcium nodules were then observed and photographed by Stereo Microscope. For quantification, the stain was dissolved with 350ul of destain solution (10% (w/v) cetylpyridinium chloride in 10 mmol sodium phosphate) for 1h in 12-well plates. The absorbance was quantitatively measured at 620nm.

Quantitative Real-time Polymerase Chain Reaction
Total RNA was isolated using TriZol (Invitrogen, USA). Total RNA was transcribed into complementary DNA (cDNA) using a PrimeScript RT reagent kit (TaKaRa, Japan). The analysis was performed on the CFX96™Real-Time PCR System with SYBR PremixExTaq™II (TaKaRa, Japan). The following components were prepared:1μlcDNA(50ng/μl), 5ul SYBR PremixEx Taq™II, 0.4μl Forward Primer(5μM), 0.4μl Reverse Primer(5μM) and H2O was added to a final volume of 10μl. The PCR conditions were 95°C for 3m followed by 40 cycles of 95°C for 10s and 60°C for 30s. All of the reactions were run in triplicate and were normalized to Gapdh. The relative gene expression was determined using the ΔΔCt method. The primers were synthesized as shown in Supplemental Table S1.

Western blot analysis
The total proteins were extracted from the cell sheet-complexes by lysing in RIPA buffer with a protease inhibitor cocktail (Sigma, USA). Protein concentration was quantified using a coomassie brilliant blue (CBB) G250 protein assay kit (Beyotime, China). The proteins were separated by SDS-PAGE and transferred to the nitrocellulose membrane. After blocked with 5% BSA for 2 h, the membranes were then incubated with primary antibodies for rat RUNX2 (Santa Cruz Biotechnology, sc-10758), ALP (Abcam, ab65834), COL-1(Abcam, ab90395), BMP2 (Abcam, ab14933) and GAPDH (Abcam, ab8245).Then the membranes were incubated for 2 h with secondary antibodies (Cowin Biotech Co., Ltd, Beijing, China). Finally, the membrane was visualized with an enhanced chemiluminescent detection system Supplemental Table S1. Primers used for Real Time RT-PCR