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. 2015 Dec 21:6:256.
doi: 10.1186/s13287-015-0256-1.

Combining mesenchymal stem cell sheets with platelet-rich plasma gel/calcium phosphate particles: a novel strategy to promote bone regeneration

Yiying Qi  1 , Lie Niu  2 , Tengfei Zhao  3 , Zhongli Shi  4 , Tuoyu Di  5 , Gang Feng  6 , Junhua Li  7 , Zhongming Huang  8
Affiliations

Combining mesenchymal stem cell sheets with platelet-rich plasma gel/calcium phosphate particles: a novel strategy to promote bone regeneration

Yiying Qi et al. Stem Cell Res Ther. .

Abstract

Background: Promotion of bone regeneration is important for successful repair of bony defects. This study aimed to investigate whether combining bone marrow-derived mesenchymal stem cell (BMSC) sheets with platelet-rich plasma (PRP) gel/calcium phosphate particles could promote bone formation in the femoral bone defects of rats.

Methods: The proliferation and differentiation of BMSCs or BMSC sheets cultured with calcium phosphate particles and/or PRP were investigated in in vitro. In vivo, 36 2.5 × 5 mm bone defects were randomly divided into groups and treated with either BMSCs/PRP gel, calcium phosphate particles, PRP gel/calcium phosphate particles, a BMSC sheet/calcium phosphate particles, a BMSC sheet/PRP gel/calcium phosphate particles, or were left untreated (n = 6/group). A further 15 bone defects were treated with chloromethyl-benzamidodialkylcarbocyanine (CM-Dil)-labelled BMSC sheet/PRP gel/calcium phosphate particles and observed using a small animal in vivo fluorescence imaging system to trace the implanted BMSCs at 1 day, 3 days, 7 days, 2 weeks, and 4 weeks after surgery.

Results: The expression of collagen type I and osteocalcin genes of BMSCs or BMSC sheets treated with PRP and calcium phosphate particles was significantly higher than that of BMSCs or BMSC sheets treated with calcium phosphate particles or the controls (P <0.05). PRP can promote gene expression of collagen III and tenomodulin by BMSCs and in BMSC sheets. The VEGF, collagen I and osteocalcin gene expression levels were higher in the BMSC sheet than in cultured BMSCs (P <0.05). Moreover, alizarin red staining quantification, ALP quantification and calcein blue fluorescence showed the osteogenic potential of BMSCs treated with PRP and calcium phosphate particles The implanted BMSCs were detectable at 1 day, 3 days, 7 days, 2 weeks and 4 weeks after surgery by a small animal in vivo fluorescence imaging system and were visualized in the defect zones by confocal microscopy. At 4 weeks after implantation, the defects treated with the BMSC sheet/PRP gel/calcium phosphate particles showed significantly more bone formation than the other five groups.

Conclusions: Incorporation of an BMSC sheet into the PRP gel/calcium phosphate particles greatly promoted bone regeneration. These BMSC sheet and tissue engineering strategies offer therapeutic opportunities for promoting bone defect repair clinically.

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Figures

Fig. 1
Fig. 1
a TEM images of CaP particles. b TEM of CaP particles after being ultrasonicated by ultrasonic cell crushing apparatus. c Particle size distribution of the CaP particles. d XRD patterns of CaP particles
Fig. 2
Fig. 2
a PRP was activated to form PRP gel. b, a BMSC sheet was harvested using a cell scraper. c The PRP gel/CaP particles composite was wrapped by BMSC sheet. d BMSCs were stained by CM-Dil. e Bone defects were prepared in the cortical bone of femurs. f BMSC sheet/PRP gel/CaP particle composite was implanted into bone defects
Fig. 3
Fig. 3
Flow cytometry results for BMSCs, expressing CD44, CD73, CD90 and CD105, but not CD45. Red histogram is the negative control. Left side of the blue histogram (in the negative control part) represents the negative expression of CD marker; right side represents the positive expression of CD marker
Fig. 4
Fig. 4
Scanning electron microscopy of CaP particles (a), PRP gel (b), PRP gel/CaP particles (c), BMSC sheet-wrapped CaP particles (d) and BMSC sheet-wrapped PRP gel/CaP particles composite (e)
Fig. 5
Fig. 5
a Dose effects of CaP particles on BMSC proliferation after culturing for 1, 3 and 7 days (mean ± SD) (*P <0.05). b Gene expressions of BMSC sheet cultured with CaP particles or/and PRP for 7 days (*P <0.05). c Gene expressions of BMSCs cultured with CaP particles or/and PRP for 7 days (*P <0.05). d Osteocalcin, collagen I and vascular epithelial growth factor (VEGF) gene expression of the BMSC sheet and cultured BMSCs (*P <0.05). e, f, g The alizarin red staining of BMSCs cultured with PRP/CaP particles, CaP particles or without treatment for 14 days. e The alizarin red staining in the controls was much weaker. f The alizarin red staining was increased when treated with CaP particles. g A calcification node formed in the BMSCs co-cultured with CaP particles/PRP. h ALP activity of BMSCs cultured with PRP/CaP particles, CaP particles or without treatment for 14 days; ALP activity was 3.01 ± 0.51 nmol/s/mg protein, 1.95 ± 0.36 nmol/s/mg protein and 0.79 ± 0.08 nmol/s/mg protein in the CaP particles/PRP group, CaP particles group and control group respectively. i: Quantification of alizarin red staining of BMSCs cultured with PRP/CaP particles, calcium phosphate particles or without treatment for 14 days; expression of BMSCs co-cultured with CaP particles/PRP was 0.84 ± 0.07, the cells with CaP particles was 0.4 ± 0.06 and the control was 0.09 ± 0.01. j, k, l Calcein blue staining of BMSCs without treatment (j) or cultured with CaP particles (k) or PRP/CaP particles (l) for 14 days
Fig. 6
Fig. 6
Fluorescence images obtained by a small animal in vivo fluorescence imaging system at 1 day (a), 3 days (b), 7 days (c), 2 weeks (d), 4 weeks (e) after the bone defects were treated with CM-Dil-labelled BMSC sheet/PRP gel/CaP particles. Red fluorescence in the bone defect zones was visualised using a small animal in vivo fluorescence imaging system, which confirmed the presence of the implanted BMSCs. After further frozen sectioning, red fluorescence was also visible in the defect zones by confocal microscopy at 1 day (f), 3 days (g), 7 days (h), 2 weeks (i) and 4 weeks (j). However, the fluorescence intensity gradually weakened. The red and the blue colours were stained by CM-Dil and Hoechst 33258, respectively
Fig. 7
Fig. 7
Gross observation and radiographic analysis of repaired bone defects at 4 weeks after surgery. a and g Control group. b and h BMSCs/PRP gel group. c and i CaP particles group. d and j PRP gel/CaP particles group. e and k BMSC sheet/CaP particles group. f and l BMSC sheet/PRP gel/CaP particles group
Fig. 8
Fig. 8
Histological examination of the repaired bone tissues at 4 weeks after implantation. a and b Control group. c and d BMSCs/PRP gel group. e and f CaP particles group. g and h PRP gel/CaP particles group. I and j BMSC sheet/CaP particles group. k and l BMSC sheet/PRP gel/CaP particles group. b, d, f, h, j, l higher magnifications of a, c, e, g, i, k respectively. w woven bone tissue, c cortical bone tissue, f fibrous tissue. Arrow residual particles. Scale bar 300 μm
Fig. 9
Fig. 9
Extent of bone formation was expressed as a percentage of bone tissue area within the original cortical bone defect area. Error bars represent means ± SD (n = 5); *P <0.05; #CaP/BMSC sheet vs CaP/PRP, CaP, BMSCs/PRP, Control, P <0.05; $CaP/PRP/BMSC sheet vs other five groups, P <0.05
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