Rifapentine Polylactic Acid Sustained-Release Microsphere Complex for Spinal Tuberculosis Therapy: Preparation, in vitro and in vivo Studies

Figures & data

Figure 1 Effects of different concentrations of rifapentine on the proliferation curve of BMSCs.

Notes: The values presented were the mean ± SD of three independent experiments. **vs 0 μg/mL, **P<0.01.

Abbreviation: BMSCs, bone marrow mesenchymal stem cells.

Figure 2 The properties of RPSMs in vitro and the growth of the BMSCs in the RPSMs complex.

Notes: (A) Representative scanning electron micrograph of RPSMs (300× magnification, scale bar =50 μm); (B) UV-vis spectra of rifapentine, RPSMs and blank PLA microspheres. (C) Representative scanning electron micrograph of BMSCs adhesive growth in the complex (1500× magnification, scale bar =10 μm).

Abbreviations: BMSCs, bone marrow mesenchymal stem cells; RPSMs, rifapentine polylactic acid sustained-release microspheres.

Figure 3 (A) In vitro rifapentine release curve. (B) In vitro cumulative percentage release curve of rifapentine.

Note: The values presented were the mean ± SD of three independent experiments.

Figure 4 The effects of drugs on osteogenic differentiation of BMSCs were conducted by the Alizarin Red and ALP staining after 21 days of culture.

Notes: (A) Representative Alizarin Red and ALP staining images of the cells (100× magnification, scale bar =50 μm). (B) Quantification of stained area of cells in each group. The stained area in cells was quantified using ImageJ software. Above results were obtained from three independent experiments. All data were presented as the mean ± SD. **vs the BMSCs group, **P<0.01.

Abbreviations: BMSCs, bone marrow mesenchymal stem cells; IBMSCs, induced bone marrow mesenchymal stem cells; RPSMs, rifapentine polylactic acid sustained-release microspheres; HA/β-TCP, hydroxyapatite/β- tricalcium phosphate; ALP, alkaline phosphatase.

Figure 5 Effects of complex on osteogenic marker collagen I and osteocalcin of four groups of cells were analyzed by Real-time PCR and Western blot in vitro.

Notes: (A) The expressions of osteogenic marker genes collagen I and osteocalcin were conducted by Real-time PCR. (B) The protein expressions of collagen I and osteocalcin in BMSCs were detected by Western blot. (C) The relative intensity of each band was quantitated using ImageJ software. GAPDH was employed as a loading control. Above results were obtained from three independent experiments. The data were presented as mean ± SD. **vs the BMSCs group, **P<0.01; ^{^^}vs the IBMSCs group, ^{^^}P<0.01; ^♯♯vs complex group, ^♯♯P<0.01.

Abbreviations: BMSCs, bone marrow mesenchymal stem cells; IBMSCs, induced bone marrow mesenchymal stem cells; RPSMs, rifapentine polylactic acid sustained-release microspheres; HA/β-TCP, hydroxyapatite/β- tricalcium phosphate.

Table 1 Serological Biochemical Markers in the Complex Group at Different Time Points (Mean ± SD, n=6)

Biomarkers	Preoperative	Week 1	Week 2	Week 3	Week4
AST (U/L)	26.00±4.71	25.50±5.46	27.11±6.54	23.98±5.33	25.05±5.38
ALT (U/L)	46.62±13.64	46.93±11.61	51.33±12.80	47.58±10.86	43.15±16.08
BUN (mmol/L)	5.63±3.34	6.31±3.17	6.00±2.88	5.82±2.20	5.74±2.31
Cr (μmol/L)	63.17±16.54	57.67±20.36	61.50±20.59	64.83±18.81	56.67±15.12

Abbreviations: AST, aspartate aminotransferase; ALT, alanine transaminase; BUN, blood urea nitrogen; Cr, creatinine.

Figure 6 The changes of serum ESR (A) and CRP (B) in each group at different time were observed.

Notes: The results are the mean ± SD of an experiment performed in triplicate (n=6). *, **vs pre-TB infection, *P<0.05, **P<0.01.

Abbreviations: ESR, the erythrocyte sedimentation rate; CRP, C-reactive protein; HA/β-TCP, hydroxyapatite/β- tricalcium phosphate.

Figure 7 Macroscopic observations of the vertebral body defect healing in the three groups at 6 weeks (A–C) and 12 weeks (D–F) after surgery. The white arrow of picture is pointing to the spine defect area.

Abbreviation: HA/β-TCP, hydroxyapatite/β- tricalcium phosphate.

Figure 8 The images of osteocalcin immunohistochemical staining from three groups at 6 weeks (A–C) and 12 weeks (D–F) after surgery (40× magnification, scale bar =200 μm). The white arrow of picture is pointing to the surface of the spine defect area.

Abbreviation: HA/β-TCP, hydroxyapatite/β- tricalcium phosphate.

Figure 9 Nilsson histological scores for the samples from the three groups at 6 and 12 weeks after surgery (n=6).

Notes: All data were presented as the mean ± SD. **vs the Control group, **P<0.01; ^♯♯vs HA/β-TCP group, ^♯♯P<0.01; ^{^, ^^}vs 6w, ^{^}P<0.05, ^{^^}P<0.01.

Abbreviation: HA/β-TCP, hydroxyapatite/β- tricalcium phosphate.