Development of 3D in vitro platform technology to engineer mesenchymal stem cells

Figures & data

Figure 1 (A) Schematic illustration of nanoparticle formation, and (B) transmission electron microscopy image of dextran-spermine-plasmid DNA nanoparticles.

Note: The concentration of plasmid DNA is 10 μg/mL.

Table 1 Characterization of plasmid DNA, dextran-spermine, and DNA nanoparticles

Component	Charge ratio (N/P)	Apparent molecular size (nm)	Zeata potential (mV)
Free plasmid DNA	–	639 ± 120*	−4.6 ± 9.4*
Dextran	–	90 ± 10	−4.2 ± 2.3
Dextran-spermine	–	110 ± 12	19.2 ± 3.4
Dextran-DNA mixture	–	712 ± 12	−15.2 ± 3.4
DNA nanoparticles	1	490 ± 11	−11.3 ± 2.3
DNA nanoparticles	2	482 ± 12	−9.4 ± .22
DNA nanoparticles	3	405 ± 11	−3.4 ± 1.2
DNA nanoparticles	4	390 ± 16	1.1 ± 3.7
DNA nanoparticles	5	75 ± 14	5.2 ± 1.2
DNA nanoparticles	6	76 ± 19	5.1 ± 2.2
DNA nanoparticles	8	58 ± 12	6.2 ± 3.2
DNA nanoparticles	10	62 ± 13	8.2 ± 2.2

Notes:

* Mean ± standard deviation. The concentration of plasmid DNA is 0.25 mg/mL.

Figure 2 In vitro release profiles of plasmid DNA-BMP-2 from DNA nanoparticles incorporated into nanofibers (●). Naked plasmid DNA-BMP-2 incorporated into nanofibers (▴) was used as the control group.

Note: ^†P < 0.05; significant against the amount of plasmid DNA released from naked plasmid DNA incorporated into nanofibers.

Abbreviation: BMP-2, bone morphogenic protein-2.

Figure 3 Proliferation of mesenchymal stem cells on three-dimensional collagen-PGA nanofiber sheet (■) and two-dimensional tissue culture plate (●).

Note: ^†P < 0.05; significant relative to the two-dimensional culture system.

Abbreviation: PGA, poly (glycolic acid).

Table 2 BMP-2 activity of MSC 2 days after treatment of free plasmid DNA and nanoparticles of dextran-spermine-plasmid DNA

Experimental group	BMP-2 concentration (pg/mL)a
Naked DNA	82.35 ± 11.12b
Dextran-DNA mixture	79.24 ± 13.42
DNA nanoparticlesc	898.56 ± 42.12†

Notes:

a The activity was assayed 2 days after treatment of plasmid DNA;

b mean ± standard deviation

† P < 0.05; significant against the activity of MSC after treatment of naked plasmid DNA;

c The mixing charge ratio of dextran-spermine/DNA = 5.0.

Abbreviations: BMP-2, bone morphogenic protein-2; MSC, mesenchymal cells.

Figure 4 (A) Schematic illustration of electrospinning machine. (B) Cross-sectional scanning electron microscopy photographs of the structural morphology of PGA/collagen nanofibers fabricated by electrospinning.

Abbreviation: PGA, poly (glycolic acid).

Figure 5 Cross-sectional scanning electron microscopy photographs of the structural morphology of PGA/collagen nanofibers fabricated by electrospinning (A) 1 week, (B) 2 weeks, and (C) 3 weeks after culturing the mesenchymal stem cells on nanofibers incorporated with dextran-spermine-plasmid DNA nanoparticles.

Abbreviation: PGA, poly (glycolic acid).

Table 3 Biochemical analysis of the osteogenic differentiation of genetically engineered mesenchymal stem cells

Experimental group	ALP (mM/cells)			OCN (pg/cells)
	1 week	2 weeks	3 weeks	1 week	2 weeks	3 weeks
Naked DNA	6.4 ± 1.2a	7.1 ± 1.2	6.3 ± 1.4	7.1 ± 2.9	6.2 ± 1.3	5.6 ± 1.4
Dextran-DNA mixture	6.2 ± 1.2	6.8 ± 1.5	5.9 ± 2.3	6.9 ± 1.2	6.5 ± 1.6	5.9 ± 1.1
DNA nanoparticles	6.8 ± 1.3	49.1 ± 2.3‡	28.9 ± 4.3	6.2 ± 2.3	42.8 ± 5.9	68.5 ± 8.5‡

Notes:

a Mean ± standard deviation;

‡ P < 0.05; significant against naked DNA group.

Abbreviations: ALP, alkaline phosphatase; OCN, osteocalcin.