Isolation plus culture of bone marrow-derived MSCs

The protocols to isolate hBMSCs were approved by the Institutional Ethics Committee at the South west Hospital of the Third Military Medical University. The volunteers who donated bone marrow signed informed consent types. The protocols to isolate and characterize hBMSCs had been described in our previous study [ 23 ]. The hBMSCs were isolated by density gradient centrifugation. hBMSCs had been cultured in DMEM/F12 (HyClone Laboratories, UT, USA) along with 10% fetal bovine serum (FBS; Gibco, USA), a hundred U/ml penicillin, and 0. 1  mg/ml streptomycin. The pv cells from passages 3-5 were used for the experiments defined in this study.

Culture of vascular smooth muscle cellular material and vascular endothelial cells

Human umbilical artery smooth muscle tissues (hUASMCs) and human umbilical vein endothelial cells (hUVECs) were from ScienCell (CA, USA). hUASMCs were classy in smooth muscle cell medium (ScienCell Inc., CALIFORNIA, USA), and hUVECs were cultured in endothelial cellular medium (ScienCell Inc., CA, USA).

Small interfering RNA transfection

Predesigned double-stranded small interfering RNAs (siRNAs) from Integrated GENETICS Technologies were used; 30 pmol of human Smad3 silencing RNA (siRNA) (Santa Cruz Biotechnology, TX, USA) or nonspecific control siRNA (Santa Cruz Biotechnology) had been transfected into hBMSCs using Lipofectamine RNAi (Thermo Fisher Scientific) according to the manufacturer’ s protocol. Then, after 48  hours of transfection, cells were treated with TGFβ 3 or more or vehicle control. FITC-conjugated control siRNA (Santa Cruz) was used to test for transfection efficiency, and around 80-90% of cells were transfected with siRNA.

Cellular migration

Single cell culture system

Approximately 5  ×   10 4 hBMSCs were seeded with DMEM/F12 medium on the upper Boyden chambers of 24-well dishes (8  μ m; Corning, Inc., USA) and the lifestyle medium with different concentrations of TGFβ 3 and 2% FBS was placed into the lower chambers. After incubating plates for 6  hours at 37  ° Chemical, the cells of the upper chamber were fixed, stained along with 0. 5% crystal violet dye, and removed having a cotton swab. The cells migrating to the lower surface had been photographed and counted under a microscope. For Tβ RI/II signaling pathway inhibition, hBMSCs were pretreated with SB431542 (15  μ M; Selleckchem Inc., TX, USA) pertaining to 1  hour prior to the growth factor administration.

Coculture approach to hBMSCs and vascular cells

Approximately 5  ×   10 4 hBMSCs were seeded with DMEM/F12 medium on the upper Boyden chambers of 24-well china (8  μ m; Corning, Inc. ) and around 5  ×   10 4 vascular cells (hUASMCs or hUVECs) were seeded using the culture medium with the different concentrations of TGFβ 3 or more and 2% FBS on the lower chambers. After incubating plates for 6  hours at 37  ° Chemical, hBMSC migration was assessed using the crystal violet coloring method.

TGFβ 3 delivery

In vitro, TGFβ three or more (Pepro Tech Inc., NJ, USA) was prepared from different concentrations (5-100  ng/ml) of culture medium in order to assess cell migration and protein levels. On the other hand, within vivo, 100  μ l TGFβ 3 at a dosage of 10  μ g/ml was adsorbed in absorbable gelatin sponges (Jinling Pharmaceutical Company, Jiangsu, China) to organize the scaffold loading TGFβ 3. TGFβ 3-free scaffold was used as a vehicle control [ 9 ].

Traditional western blotting analysis

To estimate the signaling pathway of vascular tissues (hUASMCs or hUVECs) stimulated with TGFβ 3 within the coculture system of hBMSCs and vascular cells, approximately 2  ×   10 5 hBMSCs were seeded on the upper Boyden chambers of six-well plates (0. 4  μ m; Corning, Inc. ), and 2  ×   10 5 vascular cells (hUASMCs or hUVECs) were seeded on the lower chambers. The cells of the coculture system had been incubated in culture medium containing 25  ng/ml TGFβ 3 for 6 or 24  hours at 37  ° C. Total protein was extracted with 100  μ l RIPA lysis buffer (P0013B; Beyotime, Jiangsu, China), subjected to SDS-PAGE, transferred onto nitrocellulose membranes (Millipore, Billerica, USA), and probed with specific primary Ab muscles against p-Smad3 (Cell Signaling Technology, USA), Smad3 (Santa Cruz Biotechnology), or GAPDH (Beyotime) at 1: five hundred dilution overnight at 4  ° C. Immunoreactive proteins bands were visualized using ECL chemiluminescence detection along with a western blot detection system (Bio-Rad, USA). The strength ratio was the relative expression of p-Smad3, Smad3, Tβ RI, and Tβ RII normalized to GAPDH.


Vascular tissue were treated by culture medium containing 25  ng/ml TGFβ 3 or PBS for 24  hours. The particular culture medium was collected and the concentrations of MCP1 and SDF1 were measured with the BCA protein assay kit, and the cytokine concentration was measured with ELISA kits (ELH-Human SDF1 alpha, ELH-Human MCP1; RayBiotech, USA).

Animal surgical procedure and experimental design

Eight-week-old FVB/N mice (weighing approximately 25– 30  g, from the Animal Experiment Center of Southwest Hospital of China) underwent a femoral osteotomy. The established surgical procedure has been reported previously [ 24 ]. Briefly, FVB/N mice were anesthetized and sits firmly with fixation plates. The unilateral 2-mm segmental problems with removal of the periosteum were created in every mouse. The different scaffolds were transplanted into the bone problems. The wounds were closed using a standard surgical procedure. Rodents were randomly assigned to two groups: the vehicle team ( n   =  24) and the TGFβ 3 group ( n   =  24). To test the particular host MSCs, scaffold samples were retrieved and employed for immunofluorescence colonization staining at 7  days postoperatively. To check vascularization of regenerated tissue, scaffold samples were gathered and used for immunohistochemical analysis at 7  days postoperatively. At 8  weeks post operation, the development of new bone tissue in the defects was monitored by micro-CT and the recovery capacity of different treatments was further confirmed by the histology assessment.

Quantitative real-time PCR

Scaffolds were retrieved at 3, seven, and 14  days. The total RNA of treated tissues was extracted with TRIzol reagent (TaKaRa, Shiga, Japan) and reverse transcribed with PrimeScript™ -RT reagent package (TaKaRa) according to the manufacturer’ s instructions. Real-time PCR had been performed using 2  ×   SYBR Green PCR Master Mix (Applied Biosystems, USA) on a Real-Time PCR System (Applied Biosystems 7500, USA). All of the primer sequences (Sangon Biotech Co., Ltd, Shanghai, China) were developed using primer 5. 0 software. The following primer units were used: MCP1 , forward 5′ -CTCGCCTCCAGCATGAAAGTCTC-3′ and reverse 5′ -TGGGGTCAGCACAGATCTCCTTG-3′; plus β -ACTIN , ahead 5′ -GCACAGAGCCTCGCCTTT-3′ and reverse 5′ -CGCCCACATAGGAATCCTTC-3′. The comparative expression of MCP1 was calculated using the 2 Δ Δ Ct method, with β -actin like a reference gene.

Immunofluorescent staining

The scaffolds retrieved at 7  days in vivo were embedded in optimal trimming temperature compound, and snap frozen at – 20  ° C. Sections (8-μ m thick) were kept overnight at 4  ° C with primary antibodies against Sca-1 (1: 500, 7 H4L3; Invitrogen, CALIFORNIA, USA) and PDGFR-α (1: 500; Invitrogen) [ 25 ]. As appropriate, secondary antibodies labeled with Alexa F (symbol) 488 (1: 100, donkey anti-rabbit) or Cy3 (1: 100, goat anti-rat; ZSGB-BIO, Beijing, China) were utilized, and DAPI was used to stain nuclei. Fluorescence pictures were acquired using a Two Photon Laser Scanning Program (LSM 510 NLO; Zeiss, Oberkochen, Germany). Endogenous cellular material and Sca-1 + PDGFR-α + MSCs migrating into the defect site had been quantified at day 7 based on immunofluorescent images. An overall total of three images per animal distributed within the problem area, with 800× magnification, were analyzed.


New bone fragments formation on weeks 4 and 8 was examined with micro-CT (Skyscan, Antwerp, Belgium). The regenerated femora with removal of muscle in 4% paraformaldehyde were scanned with the following settings: voxel size 10. 0  μ m, voltage 65  kV, current A, and direct exposure time 280  ms. The data were subsequently analyzed plus imaged using CT Analyser software (version 1 . sixteen. 1 . 0, Skyscan 1272; Bruker Microct, Kontich, Belgium). 3D pictures were made with CTvox software (version 3 or more. 2 . 0r1294, Skyscan 1272; Bruker Microct) [ 26 ]. In the zone of the regenerated bone with the defects, the particular elliptical region of interest (ROI) was setted as 80  ×   55  pixels, and the number of slices plus predetermined threshold was from 264 to 1500  magnesium HA/cm 3 . The relative bone quantity per tissue volume (BV/TV) and BMD of the regenerated bone within the defects were calculated using CTvox software program (version 3. 2 . 0r1294; Skyscan) [ 27 ].

Immunohistochemical analysis

The femur samples were retrieved. The muscle plus soft tissue were stripped off. The samples had been then fixed in 4% buffered paraformaldehyde, decalcified within 10% EDTA, embedded in paraffin, and sectioned on 4– 6  mm thickness. The slides were useful for immunohistochemistry of CD31 (the endothelial marker). The slideshow of deparaffinized and rehydrated tissue sections were incubated in 3% H 2 O 2 solution for 10  min in order to extinguish endogenous peroxidase activity and then washed with PBS. For antigen retrieval, the sections were irradiated inside a microwave oven for 5  min in pH  6. zero citrate buffer. The primary antibody for CD31 (1: twenty dilution; Santa Cruz) was applied overnight at 4  ° C followed by incubation with biotinylated anti-mouse IgG for 30  min. The sections were counterstained along with DAB for 3  min. Mayer′ s hematoxylin had been used for counterstaining [ 28 ].

Statistical analysis

One-way ANOVA followed by Tukey’ s test was utilized to determine the statistical importance of the differences in TGFβ 3-induced hBMSC migration and traditional western blot analysis. Two-way ANOVA followed by Sidak’ s several comparisons test was performed to determine the statistical significance from the hBMSC migration in the coculture system, quantitative real-time PCR (qRT-PCR), and micro-CT data. Data are expressed because the means  ±   SD. The results are displayed because the mean  ±   standard deviation for n   ≥   three or more samples per group in all cases, unless otherwise pointed out. For both the ANOVA and post-hoc tests, differences were regarded significant if P   <   0. 05.