Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

Figures & data

Figure 1 Fabrication of BMP-2-producing cell sheet and surgical schematic. (A) A schematic presentation of the fabrication procedure for the cell sheet and the surgical procedure. (B) Cultured BMSCs were lifted as a cell sheet using a scraper. (C) The cell sheet was folded to adapt the shape of the calvarial defect. (D) Two 5 mm diameter defects were prepared at the calvarial area of a rat, and the cell sheet was transplanted into the defect.

Abbreviations: BMSCs, bone marrow mesenchymal stem cells; PEI–al/pBMP-2, polyethylenimine–alginate/plasmid of bone morphogenetic protein 2; BMP-2, bone morphogenetic protein 2.

Table 1 Physical characteristics of PEI–al nanocomposites, BMP-2 plasmid, and PEI–al/pBMP-2 complexes

Sample	Average particle size (nm)	Zeta potential (mV)
PEI–al	73.8±5.1	+19.9±1.1
BMP-2 plasmid	1,097.0±33.8	−36.4±2.2
PEI–al/pBMP-2	149.6±13.0	+18.8±0.8

Notes: Data are expressed as mean ± standard deviation.

Abbreviations: PEI–al, polyethylenimine–alginate; BMP-2, bone morphogenetic protein 2; pBMP-2, plasmid of bone morphogenetic protein 2.

Figure 2 Physicochemical characterization of the PEI–al nanocomposites and PEI–al/pBMP-2 complexes. (A) SEM images of PEI–al nanocomposites. (B) SEM images of PEI–al/pBMP-2 complexes. Red arrows indicate PEI–al/pBMP-2 complexes. (C) TEM images of PEI–al nanocomposites. (D) TEM images of PEI–al/pBMP-2 complexes. Red arrows indicate PEI–al/pBMP-2 complexes. (E) FTIR spectra of PEI–al nanocomposites, PEI, and alginate. (F) Agarose gel electrophoresis retardation assay of PEI–al/pBMP-2 at various w/w ratios of 0.5:1, 1:1, 1.5:1, 2:1, 3:1, and 4:1.

Abbreviations: FTIR, Fourier transform infrared; PEI–al/pBMP-2, polyethylenimine–alginate/plasmid of bone morphogenetic protein 2; SEM, scanning electron microscopy; TEM, transmission electron microscopy; w/w ratios, weight ratios; PEI–al, polyethylenimine–alginate; PEI, polyethylenimine.

Figure 3 Evaluation of properties of PEI–al/pDNA on BMSCs in vitro. (A) Fluorescent microscopy images of BMSCs transfected with PEI/pEGFP and PEI–al/pEGFP complexes at different w/w ratios of nanocomposites/pEGFP. Bright field of BMSCs (A-1, A-2 and A-3) EGFP expression in BMSCs transfected by PEI/pEGFP (w/w =1:1) and PEI–al/pEGFP (w/w =2:1 and 3:1) complexes. Magnification of all images is 200×. (B) Cell viability of PEI/pBMP-2 and PEI–al/pBMP-2 complexes at various w/w ratios of 0, 0.5:1, 1:1, 2:1, and 3:1 transfected to BMSCs. (C) Release of human BMP-2 protein from PEI–al/pBMP-2 (w/w =3:1) transfected BMSCs on days 2, 4, 8, and 14.

Notes: *P<0.05; **P<0.01.

Abbreviations: BMSCs, bone marrow mesenchymal stem cells; pBMP-2, plasmid of bone morphogenetic protein 2; pEGFP, plasmid of enhanced green fluorescent protein; PEI–al/pDNA, polyethylenimine–alginate/plasmid DNA; w/w, weight ratios; EGFP, enhanced green fluorescent protein; PEI, polyethylenimine; BMP-2, bone morphogenetic protein 2.

Figure 4 Expression of specific osteogenic genes (COLL I, SP7, RUNX2, ALP) of BMSCs evaluated by qPCR at 3, 7, and 14 days post-transfection with PEI–al/pEGFP and PEI–al/pBMP-2 complexes. (A) 3 days post-transfection. (B) 7 days post-transfection. (C) 14 days post-transfection.

Notes: *P<0.05; **P<0.01.

Abbreviations: ALP, alkaline phosphatase; COLL I, I type collagen; pBMP-2, plasmid of bone morphogenetic protein 2; PEI–al/pEGFP, polyethylenimine–alginate/plasmid of enhanced green fluorescent protein; qPCR, real-time quantitative polymerase chain reaction; RUNX2, runt-related transcription factor 2; SP7, Osterix; BMSCs, bone marrow mesenchymal stem cells.

Figure 5 Microcomputed tomography images of rat cranial bone and quantification after 4 or 8 weeks post-implantation. (A) Blank control group after 4 weeks. (B) EGFP/CS group after 4 weeks. (C) BMP/CS group after 4 weeks. (D) Blank control group after 8 weeks. (E) EGFP/CS group after 8 weeks. (F) BMP/CS group after 8 weeks. (G) Quantification of new bone area.

Notes: *P<0.05; **P<0.01.

Abbreviations: BMP/CS, bone morphogenetic protein 2 gene-modified cell sheet; EGFP/CS, enhanced green fluorescent protein gene-modified cell sheet.

Figure 6 Hematoxylin and eosin staining of rat cranial defect after 4 or 8 weeks post-implantation. (A) Blank control group after 4 weeks (×20). (B) Blank control group after 8 weeks (×20). (C) EGFP/CS group after 4 weeks (×20). (D) EGFP/CS group after 8 weeks (×20). (E) BMP/CS group after 4 weeks (×20). (F) BMP/CS group after 8 weeks (×20). (G) Enlarged image of the edge of the defect in EGFP/CS group after 4 weeks (×400). (H) Enlarged image of the edge of the defect in BMP/CS group after 4 weeks (×400). Black arrows indicate the edge of the defect, and green arrows indicate osteoblasts.

Abbreviations: BMP/CS, bone morphogenetic protein 2 gene-modified cell sheet; EGFP/CS, enhanced green fluorescent protein gene-modified cell sheet.

Table 2 Hematology parameters of rats treated with CS and BMP/CS at 1, 3, and 7 days after surgery

Parameters	1 day		3 days		7 days
	CS	BMP/CS	CS	BMP/CS	CS	BMP/CS
WBC (10^9/L)	13.80±2.35	16.3±6.21	9.63±0.93	9.73±3.95	8.97±2.17	10.87±2.7
RBC (10^12/L)	7.61±0.50	6.71±0.94	7.46±0.20	7.03±0.45	6.74±0.44	6.16±0.21
Hb (g/L)	136.00±16.64	129.00±16.10	139.67±2.52	133.00±4.36	123.00±4.58	124.33±1.53
Plt (10^9/L)	888.67±162.28	903.67±145.50	873.00±212.64	1,049.00±24.58	1,162.33±11.72	1,102.67±37.00

Notes: Data are expressed as mean ± standard deviation.

Abbreviations: BMP/CS, bone morphogenetic protein 2 gene-modified cell sheet; CS, cell sheet; Hb, hemoglobin concentration; Plt, platelet; RBC, red blood cell number; WBC, white blood cell number.

Table 3 Serum biochemical parameters of rats treated with CS and BMP/CS at 1, 3, 7 days after surgery

Parameters	1 day		3 days		7 days
	CS	BMP/CS	CS	BMP/CS	CS	BMP/CS
TP (g/L)	54.33±2.08	54.33±1.15	54.30±1.81	55.40±0.98	57.00±3.60	55.00±2.52
ALT (U/L)	68.00±21.38	55.33±11.37	52.40±15.57	55.10±13.96	65.67±18.82	62.00±6.93
AST (U/L)	136.33±29.67	155.00±44.93	136.40±43.53	158.73±29.89	165.67±13.32	160.67±32.47
BUN (mmol/L)	7.31±0.99	6.52±0.13	5.92±0.40	5.85±0.49	6.55 ±1.29	6.10±1.09
Cre (μmol/L)	36.90±5.38	38.43±4.54	39.77±6.89	42.40±6.55	47.53±1.16	46.88±3.95

Notes: Data are expressed as mean ± standard deviation.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMP/CS, bone morphogenetic protein 2 gene-modified cell sheet; BUN, blood urea nitrogen; Cre, creatinine; CS, cell sheet; TP, total protein.

Figure 7 Hematoxylin and eosin staining of liver, spleen, and kidney from rats treated with CS and BMP/CS at 7 days after surgery. (A) Liver of CS group (×200). (B) Liver of BMP/CS group (×200). (C) Spleen of CS group (×200). (D) Spleen of BMP/CS group (×200). (E) Kidney of CS group (×200). (F) Kidney of BMP/CS group (×200).

Abbreviations: BMP/CS, bone morphogenetic protein 2 gene-modified cell sheet; CS, cell sheet.