A Composite Deferoxamine/Black Phosphorus Nanosheet/Gelatin Hydrogel Scaffold for Ischemic Tibial Bone Repair

Figures & data

Figure 1 (A) cut the skin and created 3mm diameter of tibial bone defects. (B) placed the BPN-DFO hydrogel in the defect. (C) hydrogels photo-crosslinking using 365 nm ultraviolet lamp irradiation.

Table 1 The Macroscopic and Histological Evaluation of Regeneration Bone Tissue

Macroscopic Evaluation		Points
Defect coverage
	>75%	4
	50~75%	3
	25~50%	2
	<25%	1
	0%	0
Defect surface
	Intact bone	3
	Small fissure	2
	Large fissure	1
	Necrosis	0
Integration with bone
	Complete	3
	>50%	2
	<50%	1
	Demarcation>1mm	0
Histological evaluation
Cell distribution
	Uniform	4
	Uniform/clusters	3
	Clusters	2
	Individual cells	1
	Disorganized	0
Surface
	Continuous smooth	2
	Continuous irregular	1
	Discontinuous	0
Matrix of new bone
	Mature	2
	Partial mature	1
	Immature	0
Tissue of new bone
	Normal	3
	Increased remodeling	2
	Necrosis or granulation tissue	1
	Fracture or callus at base	0

Figure 2 (A) Swelling rate of the BPN-DFO hydrogel and hydrogel in ultrapure water at room temperature. (B) Degradation rate of the BPN-DFO hydrogel and hydrogel.

Figure 3 Vitro phosphorus and ferric ion release cumulative percentage of BPN-DFO hydrogel. (A) The release cumulative percentage of phosphorus ion showed a sustained release during 21 days. (B) The release cumulative percentage of ferric ion are burst during the initial 24 h, then a constantly slow release after 48 h.

Figure 4 The live/dead stain fluorescence image of hBMSCs incubated at 1 and 3 days in two groups, respectively, and there were no dead cells (red) in two group. (A, B) The live/dead stain fluorescence image of BPN-DFO group. (C, D) The live/dead stain fluorescence image of hydrogel group.

Figure 5 Summarized data showing the effect of BPN-DFO hydrogel and hydrogel on mRNA expression of osteogenic differentiation related genes including Col-I, BMP4 and Runx2 after culturing for 7 and 14 days in vitro. (A) The expression of Col I in BPN-DFO group showed significantly improved than control group and hydrogel group at 7 and 14 days. (B) The expression of BMP4 in BPN-DFO group showed significantly improved than control group and hydrogel group at 7 and 14 days. (C) The expression of Runx 2 in BPN-DFO group showed significantly improved than control group and hydrogel group at 7 and 14 days. (*, p<0.05).

Table 2 Comparison of Macroscopic and Histology Scale Between Two Group

Group	Macroscopic	Histology
2 weeks
BPN-DFO hydrogel	6.24±0.68	7.41±0.73
Hydrogel	2.49±0.51	3.52±0.51
P value	<0.05	<0.05
4 weeks
BPN-DFO hydrogel	9.36±0.48	10.21±0.69
Hydrogel	5.82±0.69	7.02±0.59
P value	<0.05	<0.05

Figure 6 The macroscopic presence of bone defect site in tibial. 2 and 4 weeks in BPN-DFO hydrogel group and hydrogel group.

Figure 7 Bone defect repair in the tibial model. H&E staining and Masson staining of histological sections were performed 2 and 4 weeks after implantation. The left part is H&E staining and right part is Masson staining.

Figure 8 Bone defect repair in the tibial model. Immunohistochemistry analysis (COL I, CD31) of sections were performed 2 and 4 weeks after implantation. The left part is IHC of COL I and right part is IHC of CD 31. And in these two parts, tawny color meaning the positive, which present COL I and CD 31, respectively.

Figure 9 Summarized data showing the effect of BPN-DFO hydrogel and hydrogel on mRNA expression of BMP4 and Runx2 after culturing for 14 and 28 days in vivo. (A) The expression of Runx 2 in BPN-DFO group showed significantly improved than Hydrogel group at 14 and 28 days. (B) The expression of BPM 4 in BPN-DFO group showed significantly improved than Hydrogel group at 14 and 28 days. (*, p<0.05).