Dual-Nozzle 3D Printed Nano-Hydroxyapatite Scaffold Loaded with Vancomycin Sustained-Release Microspheres for Enhancing Bone Regeneration

Figures & data

Table 1 Experimental Factors and Level

Level	Factor A	Factor B	Factor C	Factor D
	VAN (mg)	PLGA (mg)	PVA (%)	Rotational Speed (r/Min)
1	100	300	2	600
2	200	400	2.5	900
3	300	500	3	1200

Figure 1 Characterisation of VAN/PLGA microspheres. (A) The standard release curve of VAN. (B) Macroscopic images of successfully prepared microspheres. (C–F) SEM images of the microspheres under different magnifications (150×, 500×, 1000×, 2000×, respectively). (G) Size distribution of microspheres.

Table 2 Orthogonal Test Table

Test Number	Factor				Observation Index
	A (VAN)	B (PLGA)	C (PVA)	D (r)	DE%	EE%	80%DE+20%EE
1	1	1	1	1	21.731	86.923	34.7694
2	1	2	2	2	17.123	85.616	30.8216
3	1	3	3	3	13.322	79.93	26.6436
4	2	1	2	3	25.796	64.49	33.5348
5	2	2	3	1	15.768	47.304	22.0752
6	2	3	1	2	22.719	79.518	34.0788
7	3	1	3	2	23.221	46.442	27.8652
8	3	2	1	1	27.538	64.256	34.8816
9	3	3	2	3	27.132	72.352	36.176
I	92.235	96.169	103.73	91.726			T=280.846
II	89.689	87.778	100.532	92.766
III	98.923	96.898	76.584	96.354
K1	30.745	32.056	34.577	30.575
K2	29.896	29.259	33.511	30.922
K3	32.974	32.299	25.528	32.118
R	3.078	3.04	9.049	1.543

Figure 2 In vitro release and bacteriostatic activity of VAN/PLGA-MS. (A) Cumulative release curve of VAN/PLGA microspheres in 24 days. The picture on the left shows the cumulative release curve of 0–24 h, and the picture on the right shows the cumulative release curve of 0–24 days. (B) Bacteriostatic experiment of PLGA microspheres in vitro. (C) Bacteriostatic test of VAN/PLGA microspheres in vitro.

Figure 3 Characterisation of two groups of scaffolds. (A and B) General view and stress–strain curve of n-HA/PLA scaffold. (C and D) General view and stress–strain curve of VAN/MS-PLA/n-HA scaffold. (E–J) SEM images of n-HA/PLA scaffold under various magnifications. (K–P) SEM images of VAN/MS-PLA/n-HA scaffold under various magnification.

Figure 4 In vitro biological evaluation of the two groups of scaffolds. (A) 14 days after adipogenic induction of ASCs with Oil Red O staining under inverted microscope (100×). (B) 14 days after ASC osteogenic induction, alizarin red staining under inverted microscope (100×). (C)The effect of different scaffold on the proliferation of ASCs. (D) Bacteriostatic test of n-HA/PLA scaffold invitro. (E) Bacteriostatic experiment of VAN/MS-PLA/n-HA scaffold invitro.

Figure 5 Femur specimens of different scaffold groups observed at 4, 8, and 12 weeks.

Figure 6 Femur specimens of different scaffold groups observed by X-ray, CT, and three-dimensional reconstruction at 4, 8 and 12 weeks.

Figure 7 Histopathology of femoral defects in different scaffold groups observed at 4, 8, and 12 weeks.

Figure 8 In vivo toxicity of experimental animals in different scaffold groups evaluated at 4, 8, and 12 weeks (liver and kidney tissue).