Fang, et al Chemical Engineering Journal 370(2019)573-586 Hep- Dopa NF-Ms(Fig 2A). As further confirmed through EDS ith the control group (TCP, normal tissue culture poly- shown in Table 4), the Nls contents were estimated at 0%, 6.50%, 3 days, no significant differences were observed in SHED and 5.24%, respectively, and the S2p contents were estimated at 0%, between control and PDA-NF-Ms groups. The PDA-NF-Ms 0%, and 1.33%, respectively gave much higher optical values than the pure NF- Ms group at 3 and The apparent WCa was further investigated to evaluate the surface 7 days of incubation (P 0.05 or P < 0.01) wettability of microspheres after modification with PDA. The surface Altogether, our results suggested that the nanofibrous microspheres topography of the scaffold had a great effect on its hydrophilicity; with PDA surface modifications of PDa were more favorable for SHED naterials possessing nano/micro- structural morphologies would be growth and proliferation than pure PLLA NF-Ms especially endowed with high hydrophobicity due to the entrapped air [26]. Hence, these two kinds of NF-Ms were compressed into a regular 3.4. The osteogenesis effects of rhBMP-2 released from Hep-Dopa NF-Ms cylinder to determine the effect of chemical ingredients on the surface properties of these microspheres As shown in Fig. 2(B and C), the WCa The bioactivity of rhBMP-2 released from Hep-Dopa NF-Ms was of bare PLLA NE-Ms was about 81.9. However, the WCA of NF-Ms assessed through ALP activity, ARS staining, and RT-PCR on BMSCs clearly decreased obviously after the surface modifications of PDA produced in the coating or Hep-Dopa conjugation, especially the WCA of PDA-NF-Ms early period of bone formation. As shown in Fig. 5(A), an obvious in. was down to56.18°. crease in ALP production by BMSCs in the medium containing rhBMP-2 was confirmed by ALP staining at 7 and 14 days. The relative ALP ac- 3. 2. Immobilization and release of rhBMP-2 from Hep-Dopa NF-Ms tivity expression of BMSCs was consistently with the staining results as mpared with blank group at 7 and 14 days(Fig 5B)(P 0.05). In The distribution of RBITC-labelled rhBMP-2 in microspheres was addition, ARS staining and quantification also suggested that groups observed by LSCM. The results shown in Fig. 3(A) were a clear in- containing rhBMP-2 exhibited more mineral deposits after sustained dication that red fluorescence labelled protein was uniformly dis- induction for 21 days(Fig. 5C and D)('p 0.05) tributed in Hep-Dopa NF-Ms on each cross section (every 20 um). In The mRNA expression levels of osteogenic differentiation-related comparison with NF-Ms without modification, the fluorescence in- genes(ALP, Runx2, and Coll) were analyzed to assess the os- tensity also confirmed the effective loading of rhBMP-2 that was in. teoinductivity of rhBMP-2 for BMSCs at 14 days (Fig. 5E). There was no tegrated with Hep-Dopa onto NF-Ms. significant difference in gene expression between the soluble BMP-2 The amount of rhBMP-2 loaded into microspheres was measured by group and the BMP-2/HD NE-Ms group of Aly emulated compared after osteogenic induction for evaluating the difference in weight between the supernatant solutions 14 days. However, the expression level enes and loading solutions using an ELISA kit. In contrast to pure PLLA NF. in both experimental groups were remarkably up-r Ms was remarkably enhanced from 16.7% to 38.2% after decorating medium without bioactive protein at 14 days (P 0.05 with Hep-Dopa(Fig 3B) P< 0.01). This result demonstrated that the rhBMP-2 released from The cumulative release profiles of rhBMP-2 from NF-Ms and Hep. the Hep-Dopa-conjugated microspheres was able to promote BMSCs Dopa NF-Ms over a period of 28 days are shown in Fig. 3(C). After NF- Ms being modified with Hep-Dopa conjugation, the initial burst release n conclusion, the osteogenic effects of rhBMP-2 obtained from Hep- of rhBMP-2 in 24h was cut down from 38.02 t 3.5% to Dopa NF-Ms was comparable to the effect of 100 ng/ml rhBMP-2 10.74+ 2.9%. And the protein release was significantly delayed and treatment, which proved that the rhBMP-2/HD NE-Ms effectively re- displayed a relatively stable release behavior in Hep-Dopa NF-Ms tained the protein bioactivity effectively and could be potently to in group. After 28 days, approximately 64.93% of the initially loaded duce osteogenic differentiation of BMSCs. rhBMP-2 was released and still kept releasing at a rate of 5.67 ng/day (see Sl-Fig. 1). 3.5. Ectopic bone formation 3.3. Growth and proliferation of SHED on PDA-NF-Ms The morphology of tissue constructs in various groups at 8 weeks ology was measured by SEM as shown in Fig. 4(A).Ac- fter subcutaneous implantation was shown in Fig. 7(A). The gross cording to the SEM images, SHED could adhere and spread well both on appearance of each composited tissue graft exhibited a similar volume, the nf. ms both with and without pda modification the cells were and all the harvested samples remained structurally intact. The white mineralized hard tissues had formed in BMP-2 and Dual groups, fully stretched when adhering on PDA-NF-Ms, showing a polygonal whereas the grafts in control and SHED groups were black soft tissue morphology with multiple stretched prolongations and extracellular matrix(ECM) on the scaffold surface. A ally, as shown in the magnified images in Fig 4(A), SHED extended more pseudopodia be. 3.5.1. Detection of GFP-SHED in vivo ween adjacent PDA-NF-Ms which could tightly cling to the surface of To evaluate the survival situation of shed growing in explant microspheres. This observation indicated a better integration of SHED constructs, cryo-sections of collected samples were processed for his with PDA-NF- Ms than pure NF-M tological analysis. From the histological results shown in Fig. 6(A-D),we The viability of SHED cultured on PDA-NF-Ms for 7 days was de- observed that the GFP-positive SHED (green) were identifiable in the termined by staining with calcein-AM/PI through LSCM. After the cell grafts at 7 days(Fig. 6B, C)and 4 weeks(Fig. 6E, F)after implantation and microspheres were co-cultured for 7 days, many green fluorescent in vivo. However, the expression of green fluorescence for implanted SHEd could be observed in three-dimensional reconstructed fluores- ence images. Moreover, the dead cells labelled with red fluorescence Table 4 ere observed in the PDA-NF-Ms group(Fig 4B). Cytoskeleton staining Surface elemental compositions of NF-MS, PDA-NF-Ms and Hep-Dopa NF-Ms as determined by EDS analysis. ormal, whereas PDA-NF-Ms adopted a stretched shape and formed the Cls Nls% cell clusters on the microspheres surfaces. The proliferation of SHEd grown on microspheres was determined NF-MS 24.44 71.69 by an MTT assay as shown in Fig 4(C). Both the PLLA NF-Ms and PDA- Hep-Dor NF-Ms were conducive to SHEd proliferation at different time points.Hep-Dopa NF-Ms (Fig. 2A). As further confirmed through EDS analysis (as shown in Table 4), the N1s contents were estimated at 0%, 6.50%, and 5.24%, respectively, and the S2p contents were estimated at 0%, 0%, and 1.33%, respectively. The apparent WCA was further investigated to evaluate the surface wettability of microspheres after modification with PDA. The surface topography of the scaffold had a great effect on its hydrophilicity; materials possessing nano-/micro- structural morphologies would be especially endowed with high hydrophobicity due to the entrapped air [26]. Hence, these two kinds of NF-Ms were compressed into a regular cylinder to determine the effect of chemical ingredients on the surface properties of these microspheres. As shown in Fig. 2(B and C), the WCA of bare PLLA NF-Ms was about 81.9°. However, the WCA of NF-Ms clearly decreased obviously after the surface modifications of PDA coating or Hep-Dopa conjugation, especially the WCA of PDA-NF-Ms was down to 56.18°. 3.2. Immobilization and release of rhBMP-2 from Hep-Dopa NF-Ms The distribution of RBITC-labelled rhBMP-2 in microspheres was observed by LSCM. The results shown in Fig. 3(A) were a clear in￾dication that red fluorescence labelled protein was uniformly dis￾tributed in Hep-Dopa NF-Ms on each cross section (every 20 μm). In comparison with NF-Ms without modification, the fluorescence in￾tensity also confirmed the effective loading of rhBMP-2 that was in￾tegrated with Hep-Dopa onto NF-Ms. The amount of rhBMP-2 loaded into microspheres was measured by evaluating the difference in weight between the supernatant solutions and loading solutions using an ELISA kit. In contrast to pure PLLA NF￾Ms, the loading efficiency of rhBMP-2 in per milligram of Hep-Dopa NF￾Ms was remarkably enhanced from 16.7% to 38.2% after decorating with Hep-Dopa (Fig. 3B). The cumulative release profiles of rhBMP-2 from NF-Ms and Hep￾Dopa NF-Ms over a period of 28 days are shown in Fig. 3(C). After NF￾Ms being modified with Hep-Dopa conjugation, the initial burst release of rhBMP-2 in 24 h was cut down from 38.02 ± 3.5% to 10.74 ± 2.9%. And the protein release was significantly delayed and displayed a relatively stable release behavior in Hep-Dopa NF-Ms group. After 28 days, approximately 64.93% of the initially loaded rhBMP-2 was released and still kept releasing at a rate of 5.67 ng/day (see SI-Fig. 1). 3.3. Growth and proliferation of SHED on PDA-NF-Ms Cell morphology was measured by SEM as shown in Fig. 4(A). Ac￾cording to the SEM images, SHED could adhere and spread well both on the NF-Ms both with and without PDA modification. The cells were fully stretched when adhering on PDA-NF-Ms, showing a polygonal morphology with multiple stretched prolongations and extracellular matrix (ECM) on the scaffold surface. Additionally, as shown in the magnified images in Fig. 4(A), SHED extended more pseudopodia be￾tween adjacent PDA-NF-Ms which could tightly cling to the surface of microspheres. This observation indicated a better integration of SHED with PDA-NF-Ms than pure NF-Ms. The viability of SHED cultured on PDA-NF-Ms for 7 days was de￾termined by staining with calcein-AM/PI through LSCM. After the cell and microspheres were co-cultured for 7 days, many green fluorescent SHED could be observed in three-dimensional reconstructed fluores￾cence images. Moreover, the dead cells labelled with red fluorescence were observed in the PDA-NF-Ms group (Fig. 4B). Cytoskeleton staining suggested that after co-culturing for 7 days, adhered SHED appeared normal, whereas PDA-NF-Ms adopted a stretched shape and formed the cell clusters on the microspheres surfaces. The proliferation of SHED grown on microspheres was determined by an MTT assay as shown in Fig. 4(C). Both the PLLA NF-Ms and PDA￾NF-Ms were conducive to SHED proliferation at different time points. Compared with the control group (TCP, normal tissue culture poly￾styrene) at 3 days, no significant differences were observed in SHED proliferation between control and PDA-NF-Ms groups. The PDA-NF-Ms gave much higher optical values than the pure NF-Ms group at 3 and 7 days of incubation (* P < 0.05 or **P < 0.01). Altogether, our results suggested that the nanofibrous microspheres with PDA surface modifications of PDA were more favorable for SHED growth and proliferation than pure PLLA NF-Ms. 3.4. The osteogenesis effects of rhBMP-2 released from Hep-Dopa NF-Ms The bioactivity of rhBMP-2 released from Hep-Dopa NF-Ms was assessed through ALP activity, ARS staining, and RT-PCR on BMSCs. Alkaline phosphatase (ALP) is a crucial protein that is produced in the early period of bone formation. As shown in Fig. 5(A), an obvious in￾crease in ALP production by BMSCs in the medium containing rhBMP-2 was confirmed by ALP staining at 7 and 14 days. The relative ALP ac￾tivity expression of BMSCs was consistently with the staining results as compared with blank group at 7 and 14 days (Fig. 5B) (* P < 0.05). In addition, ARS staining and quantification also suggested that groups containing rhBMP-2 exhibited more mineral deposits after sustained induction for 21 days (Fig. 5C and D) (* P < 0.05). The mRNA expression levels of osteogenic differentiation-related genes (ALP, Runx2, and Col1) were analyzed to assess the os￾teoinductivity of rhBMP-2 for BMSCs at 14 days (Fig. 5E). There was no significant difference in gene expression between the soluble BMP-2 group and the BMP-2/HD NF-Ms group after osteogenic induction for 14 days. However, the expression levels of ALP, Runx2, and Col1 genes in both experimental groups were remarkably up-regulated compared to levels in the blank group, which was only induced by induction medium without bioactive protein at 14 days (* P < 0.05 or * P < 0.01). This result demonstrated that the rhBMP-2 released from the Hep-Dopa-conjugated microspheres was able to promote BMSCs osteogenic differentiation. In conclusion, the osteogenic effects of rhBMP-2 obtained from Hep￾Dopa NF-Ms was comparable to the effect of 100 ng/ml rhBMP-2 treatment, which proved that the rhBMP-2/HD NF-Ms effectively re￾tained the protein bioactivity effectively and could be potently to in￾duce osteogenic differentiation of BMSCs. 3.5. Ectopic bone formation The morphology of tissue constructs in various groups at 8 weeks after subcutaneous implantation was shown in Fig. 7(A). The gross appearance of each composited tissue graft exhibited a similar volume, and all the harvested samples remained structurally intact. The white mineralized hard tissues had formed in BMP-2 and Dual groups, whereas the grafts in control and SHED groups were black soft tissue. 3.5.1. Detection of GFP-SHED in vivo To evaluate the survival situation of SHED growing in explant constructs, cryo-sections of collected samples were processed for his￾tological analysis. From the histological results shown in Fig. 6(A-I), we observed that the GFP-positive SHED (green) were identifiable in the grafts at 7 days (Fig. 6B, C) and 4 weeks (Fig. 6E, F) after implantation in vivo. However, the expression of green fluorescence for implanted Table 4 Surface elemental compositions of NF-MS, PDA-NF-Ms and Hep-Dopa NF-Ms as determined by EDS analysis. Samples C1s% O1s% N1s% S2p% NF-MS 75.56 24.44 0 0 PDA-NF-MS 71.69 21.81 6.50 0 Hep-Dopa NF-MS 67.72 25.71 5.24 1.33 T. Fang, et al. Chemical Engineering Journal 370 (2019) 573–586 579