A novel composite scaffold of Cu-doped nano calcium-deficient hydroxyapatite/multi-(amino acid) copolymer for bone tissue regeneration

Figures & data

Figure 1 A flow chart about the preparation of scaffold.

Abbreviations: n-CDHA, nano calcium-defcient hydroxyapatite; n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer; PAA, polyamino acid.

Figure 2 Micro-CT images of 1% Cu-doped n-CDHA/MAC with porosity of 81%. (A) cross-section of scaffold; (B) three-dimensional reconstruction of scaffold. The red arrow represents the interconnected pores located in the walls of macropores.

Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.

Table 1 Total porosity, compressive strength and concentration of Cu in scaffolds of Cu-doped n-CDHA/MAC composite scaffolds

	0% Cu-doped n-CDHA/MAC	0.5% Cu-doped n-CDHA/MAC	1 % Cu-doped n-CDHA/MAC	2% Cu-doped n-CDHA/MAC	5% Cu-doped n-CDHA/MAC
Total porosity(%)	81±3	82±3	82±3	81±2	81±4
Compressive strength(MPa)	12±1	12±1	12±2	11±1	12±1
Cu in scaffolds	-	(0.49±0.01)%	(0.96±0.02)%	(1.92±0.01)%	(4.87±0.02)%

Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.

Figure 3 Weight loss of n-CDHA/MAC composite scaffolds with different concentration of Cu after immersing in PBS over time.

Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer; PBS, phosphatic buffer solution.

Figure 4 Cu release curve of different Cu-doped n-CDHA/MAC scaffolds in PBS.

Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer; PBS, phosphatic buffer solution.

Figure 5 SEM micrographs of rat BMSCs cells attachment on the surfaces of Cu-doped n-CDHA/MAC composite scaffolds after 1 days (A, C, E, G, I) and 4 days (B, D, F, H, J) of culture. (A and B), 0% Cu-doped n-CDHA/MAC; (C and D), 0.5% Cu-doped n-CDHA/MAC; (E and F), 1% Cu-doped n-CDHA/MAC; (G and H), 2% Cu-doped n-CDHA/MAC; (I and J), 5% Cu-doped n-CDHA/MAC.

Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer; SEM, scanning electron microscopy; BMSCs, bone marrow stromal cells.

Figure 6 CCK assay showing the cell growth of rat BMSCs cultured with different Cu-doped n-CDHA/MAC composite scaffolds at day1, day 4 and day 7. * means significant difference between Cu-doped groups and control group (TCP) (p<0.05).

Abbreviations: CCK, cell count kit; BMSCs, bone marrow stromal cells; TCP, tissue culture plate; n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.

Figure 7 ALP activity of rat BMSCs cultured on different Cu-doped n-CDHA/MAC composite scaffolds at day 1, day 4 and day 7. * means significant difference between Cu-doped groups and 0% Cu-doped group (p<0.05).

Abbreviations: ALP, alkaline phosphatase; BMSCs, bone marrow stromal cells; n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.

Figure 8 X-ray images revealing the repair of bone defects in blank group and 1% Cu-doped n-CDHA/MAC group at postoperative 4, 8, and 12 weeks: (A, B and C) blank group; (D, E and F) 1% Cu-doped n-CDHA/MAC group. (A and D), 4 weeks; (B and E), 8 weeks; (C and F), 12 weeks. The red arrow represents new bone formation in bone defect.

Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.

Figure 9 HE staining of n-CDHA/MAC composite scaffolds (A, B and C) and 1% Cu-doped n-CDHA/MAC composite scaffolds (D, E and F) harvested at postoperative 4, 8, and 12 weeks (magnification 40×). (A and D), 4 weeks; (B and E), 8 weeks; (C and F), 12 weeks. The red arrow represents inflammatory cells. The blue arrow represents blood vessel. And yellow arrow represents bone matrix in macropores.

Abbreviations: HE, hematoxylin and eosin; n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.