T. Fang, et al. Chemical Engineering Journal 370(2019)573-586 NF-MS Hep-Dopa NF-MS B hBMP-2 released from Hep-Dopa NF-Ms hBMP-2 released from NF-Ms 45 E30 NF-Ms Hep-Dopa NF-Ms 481216202428 Time(Days) Fig 3.(A)(LSCM) The distribution of rhBMP-2 in NF-Ms and Hep-Dopa NF-Ms was determined by labelling proteins with red fluorescence ( B) The amount of loaded rhBMP-2 in 1 mg PLLA microspheres(NF-Ms)and 1 mg Hep-Dopa NF-Ms, respectively (P 005).(C)Cumulative release of rhBMP-2 from NF-Ms and Hep-Dopa NF-Ms over a period of 28 days in vitro. Statistically analysis by using one-way analysis of variance(ANOVA)(P 0.05).( For interpretation of the references color in this figure legend, the reader is referred to the web version of this article. SHED was undetectable at 8 weeks(Fig. 6H, D. All specimens exhibited dense reddish blue bone tissue, indicating a greater maturity of re- large number of infiltrated host cells n(nuclei counterstained with generated bone tissues compared to that in the BMp. DAPD)at all time points. Our data demonstrated that the implanted morphology of new bone tissue and quantitative of vascular SHED had a long-term survival(about 4 weeks)after delivered by PDA. density(see Fig. SI-3), we could refer that SHED would function sy. NF-Ms in vivo, which could be contributed in the effective involvement nergistically with rhBMP-2 to improve the quality of neonatal bone of bone repair during the proce eration in ectopic location. 3.5.2. Histological observations by H&E and Masson's trichrome staining 3.6. Orthotopic bone regeneration As shown in Fig. 7, the PDA-NF-Ms and Hep-Dopa NF-Ms could b istinguished by color differences; the former contained brown micro- 3.6.1. microCT spheres that infiltrated with many cells, and the latter one contained The micro-CT images were reconstructed and analyzed to evaluate white microspheres that aggregated to form bigger irregular scaffolds the 3D structure and amount of the regenerated new bone in an or- (all microspheres are indicated by green arrows). In H&E staining, all thotopic bone defect model (Fig. 8A). At 4 and 8 weeks after the op- cell nuclei were stained in dark blue, the collagenous connective tissues eration, new bone had scarcely formed at the defective area in the blank and new bone matrix were highlighted in pink to varying degrees. In group, presenting a clearly identifiable outline, indicating a rare bone contrast to MS group, many neo-vessels formed in the SHED group, self-healing defect for the 4-mm cranial bone injury in nude mice. Few filling with abundant hematopoietic cells which were surrounded by new bone tissues were produced at the edge of the defect site in SHEI SHED-delivered PDA-NF-Ms at 4 and 8 weeks(indicated by red arrows). groups, while in BMP-2 group, the bulk of generated bone tissues were New bone formation was not detected in these two groups, suggesting located from the edge toward the center in the bone defect. However, that both the modified NF- Ms and SHED could not induce bone re. the newly formed bone volume was much larger in the Dual group than generation in ectopic sites. Large amounts of interconnected calcified in the other groups, and especially at 8 weeks, the defect area was al trabecular bones were stained dark blue by Masson' s trichrome staining most fully covered with new mineralized tissues in BMP-2 and Dual groups at predetermined time points. Although the Quantitative analysis was carried out by determining the BV/Tv in BMP-2 and Dual ratios and BMD values within a region of interest based on the micro-CT oocytes replaced the bone-marrow like tis- data As shown in Fig 8(B and C), the calculated percentages of BV/T indicated risks in the BMP-2 group which barely ap- were consistent with the corresponding BMD values in all groups at 4 in the due As described by a previous study, the red- and 8 weeks. Interestingly, the Shed group in the orthotopic bone re- aining osteoid matrixes represents the degree of maturity of bone generation experiment exhibited osteogenic activity, presenting higher ssues [27]. At 8 weeks, the Dual group induced the production of a BV/TV (%)and BMD values relative to those in the blank groupSHED was undetectable at 8 weeks (Fig. 6H, I). All specimens exhibited a large number of infiltrated host cells n (nuclei counterstained with DAPI) at all time points. Our data demonstrated that the implanted SHED had a long-term survival (about 4 weeks) after delivered by PDA￾NF-Ms in vivo, which could be contributed in the effective involvement of bone repair process. 3.5.2. Histological observations by H&E and Masson’s trichrome staining As shown in Fig. 7, the PDA-NF-Ms and Hep-Dopa NF-Ms could be distinguished by color differences; the former contained brown micro￾spheres that infiltrated with many cells, and the latter one contained white microspheres that aggregated to form bigger irregular scaffolds (all microspheres are indicated by green arrows). In H&E staining, all cell nuclei were stained in dark blue, the collagenous connective tissues and new bone matrix were highlighted in pink to varying degrees. In contrast to MS group, many neo-vessels formed in the SHED group, filling with abundant hematopoietic cells which were surrounded by SHED-delivered PDA-NF-Ms at 4 and 8 weeks (indicated by red arrows). New bone formation was not detected in these two groups, suggesting that both the modified NF-Ms and SHED could not induce bone re￾generation in ectopic sites. Large amounts of interconnected calcified trabecular bones were stained dark blue by Masson’s trichrome staining in BMP-2 and Dual groups at predetermined time points. Although the similar bone marrow-like tissues were produced in BMP-2 and Dual groups, however, some adipocytes replaced the bone-marrow like tis￾sues as indicated by asterisks in the BMP-2 group which barely ap￾peared in the Dual group. As described by a previous study, the red￾staining osteoid matrixes represents the degree of maturity of bone tissues [27]. At 8 weeks, the Dual group induced the production of a dense reddish blue bone tissue, indicating a greater maturity of re￾generated bone tissues compared to that in the BMP-2 group. From the morphology of new bone tissue and quantitative results of vascular density (see Fig. SI-3), we could refer that SHED would function sy￾nergistically with rhBMP-2 to improve the quality of neonatal bone during the process of bone regeneration in ectopic location. 3.6. Orthotopic bone regeneration 3.6.1. microCT The micro-CT images were reconstructed and analyzed to evaluate the 3D structure and amount of the regenerated new bone in an or￾thotopic bone defect model (Fig. 8A). At 4 and 8 weeks after the op￾eration, new bone had scarcely formed at the defective area in the blank group, presenting a clearly identifiable outline, indicating a rare bone self-healing defect for the 4-mm cranial bone injury in nude mice. Few new bone tissues were produced at the edge of the defect site in SHED groups, while in BMP-2 group, the bulk of generated bone tissues were located from the edge toward the center in the bone defect. However, the newly formed bone volume was much larger in the Dual group than in the other groups, and especially at 8 weeks, the defect area was al￾most fully covered with new mineralized tissues. Quantitative analysis was carried out by determining the BV/TV ratios and BMD values within a region of interest based on the micro-CT data. As shown in Fig. 8(B and C), the calculated percentages of BV/TV were consistent with the corresponding BMD values in all groups at 4 and 8 weeks. Interestingly, the SHED group in the orthotopic bone re￾generation experiment exhibited osteogenic activity, presenting higher BV/TV (%) and BMD values relative to those in the blank group 50ȝm A NF-MS Hep-Dopa NF-MS B C Fig. 3. (A) (LSCM) The distribution of rhBMP-2 in NF-Ms and Hep-Dopa NF-Ms was determined by labelling proteins with red fluorescence. (B) The amount of loaded rhBMP-2 in 1 mg PLLA microspheres (NF-Ms) and 1 mg Hep-Dopa NF-Ms, respectively (* P < 0.05). (C) Cumulative release of rhBMP-2 from NF-Ms and Hep-Dopa NF-Ms over a period of 28 days in vitro. Statistically analysis by using one-way analysis of variance (ANOVA) (* P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) T. Fang, et al. Chemical Engineering Journal 370 (2019) 573–586 580