T. Fang, et al. Chemical Engineering Journal 370(2019)573-586 staining(Solarbio, Beijing, China) according to the manufacturer's Table 3 protocol. The histological staining images were collected with an in- Primer sequences of osteogenic-related genes. erted microscope (Olympus IX-70, NY, America). Then, these image ere further measured using Image J software for quantitative assess- ment of vascular density in new tissues. The number of blood vessels Forward)s'CATGTACGTTGCTATCCAGGC-3 was counted in 5 random fields (x 20) TCACGCACGAT-3 2.9. Statistical analysis Forward)5'-TGGTTACTGTCATGGCGGGTA-3 Forward)5'-GAGGGCCAAGACGAAGACATC-3 All quantitative data were statistically analyzed through analysis of (Reverse)5-CAGATCACGTCATOGCACAAC-3 ariance(ANOVA) with Tukey's test, and were expressed as mean t standard deviation (SD), n 2 3. Differences between various groups with P < 0.05 were considered statistically signif and method gave uniform spheres with an average diameter of the micro- P<0.01 was considered highly significant. structure of ultrafine fibers ranging from 50 to 200 um. The cross-sec- tional image in Fig. 1(B) clearly shows the nanofibrous network ar- 3. Results chitecture of PLLA NF- Ms. After the surface was coated with PDA, the diameter of each nanofiber became thicker. In addition, the self-poly 3.1. Characterization of surface-modified PLLA NF-Ms merized polydopamine particles were homogeneously scattered on the surface of nanofibers at the nanoscale. Both the PDA-coating and Hep- Dopa decorating modifications made the NF-Ms exhibit a relatively Visual observation(Fig. 1C)showed that PLLA NF-Ms and Hep- rough surface, but there was no obvious change on spheres size or Dopa NF-Ms were white powders, whereas the PDA modified NF-Ms nanofiber structure. tion of its monomer dopamine [25] EDS was used to detect the surface chemical properties of NF-Ms The morphologies of different PLLA NF-Ms were observed throu with PDA or Hep-Dopa modifications. Successful PDA or Hep-Dopa SEM(Fig. 1A, B, D-D)and the size distribution is presented in Fig. 1(D. immobilization on NF- MS was assessed by the appearance of nitrogen F). As shown in the SEM images, the traditional phase separation le)signals of PDA-NF-MS or nitrogen and sulfur (yellow) signals of 75.56% 24.44% 71.69% 21.81% 0% 67.72 257%524 I3% B C AFMs PD Hep-Dopu Fig. 2.(A)(A) Mapping images of elemental compositions(including C, o, N, and S elements distribution) of NF-MS by energy dispersive spectroscopy analysis(a), PDA-NF-Ms(b)and Hep-Dopa NF-Ms(c).(B)Water contact angle of (a) PLLA NF-Ms, (b)PDA-modified NF-Ms, and (c)Hep-Dopa NF-Ms. (C)Quantitative results of three kinds of NF-Ms Statistically analysis by using one-way analysis of variance(ANOVA)(P 0.05)staining (Solarbio, Beijing, China) according to the manufacturer’s protocol. The histological staining images were collected with an in￾verted microscope (Olympus IX-70, NY, America). Then, these images were further measured using Image J software for quantitative assess￾ment of vascular density in new tissues. The number of blood vessels was counted in 5 random fields (×20). 2.9. Statistical analysis All quantitative data were statistically analyzed through analysis of variance (ANOVA) with Tukey’s test, and were expressed as mean ± standard deviation (SD), n ≥ 3. Differences between various groups with * P < 0.05 were considered statistically significant and **P < 0.01 was considered highly significant. 3. Results 3.1. Characterization of surface-modified PLLA NF-Ms Visual observation (Fig. 1C) showed that PLLA NF-Ms and Hep￾Dopa NF-Ms were white powders, whereas the PDA modified NF-Ms were dark brown-black powders due to the produced by the autoxida￾tion of its monomer dopamine [25]. The morphologies of different PLLA NF-Ms were observed through SEM (Fig. 1A, B, D-I) and the size distribution is presented in Fig. 1(D￾F). As shown in the SEM images, the traditional phase separation method gave uniform spheres with an average diameter of the micro￾structure of ultrafine fibers ranging from 50 to 200 μm. The cross-sec￾tional image in Fig. 1(B) clearly shows the nanofibrous network ar￾chitecture of PLLA NF-Ms. After the surface was coated with PDA, the diameter of each nanofiber became thicker. In addition, the self-poly￾merized polydopamine particles were homogeneously scattered on the surface of nanofibers at the nanoscale. Both the PDA-coating and Hep￾Dopa decorating modifications made the NF-Ms exhibit a relatively rough surface, but there was no obvious change on spheres size or nanofiber structure. EDS was used to detect the surface chemical properties of NF-Ms with PDA or Hep-Dopa modifications. Successful PDA or Hep-Dopa immobilization on NF-MS was assessed by the appearance of nitrogen (blue) signals of PDA-NF-MS or nitrogen and sulfur (yellow) signals of a b c A B C O N S 21.81% 6.50% a 20ȝm CON S 75.56% 24.44% 0% 0% 0% c C O N S 67.72% 25.71% 5.24% 1.33% b 71.69% C Fig. 2. (A) (A) Mapping images of elemental compositions (including C, O, N, and S elements distribution) of NF-MS by energy dispersive spectroscopy analysis (a), PDA-NF-Ms (b) and Hep-Dopa NF-Ms (c). (B) Water contact angle of (a) PLLA NF-Ms, (b) PDA-modified NF-Ms, and (c) Hep-Dopa NF-Ms. (C) Quantitative results of three kinds of NF-Ms. Statistically analysis by using one-way analysis of variance (ANOVA) (* P < 0.05). Table 3 Primer sequences of osteogenic-related genes. Gene name Primer sequences β-actin (Forward)5′CATGTACGTTGCTATCCAGGC-3′ (Reverse) 5′-CTCCTTAATGTCACGCACGAT-3′ ALP (Forward) 5′-GTGAACCGCAACTGGTACTC-3′ (Reverse) 5′-GAGCTGCGTAGCGATGTCC-3′ Runx2 (Forward) 5′-TGGTTACTGTCATGGCGGGTA-3′ (Forward) 5′-TCTCAGATCGTTGAACCTTGCTA-3′ Coll I (Forward) 5′-GAGGGCCAAGACGAAGACATC-3′ (Reverse) 5′-CAGATCACGTCATCGCACAAC-3′ T. Fang, et al. Chemical Engineering Journal 370 (2019) 573–586 578