T. Fang, et al. Chemical Engineering Journal 370(2019)573-586 Table 1 sections. The frozen sections were counterstained with daPi and fur. Sample abbreviations used in the ectopic implantation experiment. ther examined using a fluorescent microscope 2.8.2. In situ bone regeneration of constructs to Ms group The ability of co-delivering SHED and rhBMP-2 on NF- Ms to pro- BMP-2 group rhBMP-2 chemically bonded to Hep-Dopa NF-Ms mote orthotopic bone regeneration was measured in a cranial bone Combination of SHED/PDA-NF-MS and BMP-2/Hep-Dopa defect model. After the mice were anesthetized, a non-healing, full NF-Ms (ratio is 1: 1) thickness of 4 mm in diameter defect was created in the central reg of the cranial bone by using a dental surgical bur. Defects were filled with the prepared SHED/PDA- NF-Ms constructs, rhBMP-2/Hep-Dopa abl Sample abbreviations used in the orthotop NF-Ms, or their combinations (n= 3). The empty defects without any Dic implantation experiment. fillings were served as a control group. The animals were sacrificed at 4 Orthotopic Implantation Description of grouping and 8 weeks by injecting a lethal dose of the anesthetic drug into the abdominal cavity. Harvested samples were fixed for at least 24h for Empty defect without any filling HED group BMP-2 group bonded to Hep-Dopa NF-Ms Dual group Combination of SHED/PDA-NF-MS and BMP-2/Hep- 2.8.3. micro-CT measurement Dopa NF-Ms (ratio is l: 1) At 4 and 8 weeks after implantation, the harvested calvaria bones of all groups were scanned using micro-CT (Skyscan 1076, Bruker, which were situated on both sides lateral to the midline. The implants Belgium). After standardized reconstruction using the Nr econ soft. ware, analysis was carried out using a cylindrical volume of interest vere retrieved at 4 and 8 weeks. At every predetermined time, the with a 4-mm diameter, which was placed over the defected area in the transplanted grafts were carefully separated from the surrounding soft tissue. Then, the samples were fixed in 10% neutral formalin and fur- xial plane. Bone volume(BV) and bone mineral density(BMD) of the ther processed for histologically analysi regenerated tissue were calculated according to the scans reconstructed To track the survival time of implanted SHEDs in vivo, the cells had using the CTAn software(Bruker micro-CT, Belgium). been pre-transfected with green fluorescent protein (GFP)-expression lentiviral(Gene Pharma, Shanghai, China) particles in advance, and 2.8.4. Histological analysis stably expressing cell lines were screened by puromycin selection. The After CT analysis, the fixed specimens were decalcified in 10% mice in the SHED group were sacrificed after 1, 4, or 8 weeks to collect ethylene diamine tetraacetic acid(EDTA) for 7 days, then dehydrated the ectopic explants and detect the GFP-expressing shed by cryo. and paraffin-embedded. Sections were cut into 4-mm thick slices and stained by hematoxylin and eosin(H&E) and Masson,s trichrome B 120um PDA Hep- E G H electron microscope images:(A, B, D, G) PLLA NF-Ms(controD); (E, DA-NF-Ms; (F, I) Hep-Dopa NF-Ms.(G, H, I show a local high magnification three kinds microspheres, respectively. Yellow arrows indicate PDA aggregates scattered on the surface of NF-Ms. (C)A gross inspection of PLLA NF-l without surface-modification. The size distribution of various micro- heres with a diameter ranging from 50 to 200 um.( For interpretation of the refer lor in this figure legend, the reader is referred to the web version of thiswhich were situated on both sides lateral to the midline. The implants were retrieved at 4 and 8 weeks. At every predetermined time, the transplanted grafts were carefully separated from the surrounding soft tissue. Then, the samples were fixed in 10% neutral formalin and fur￾ther processed for histologically analysis. To track the survival time of implanted SHEDs in vivo, the cells had been pre-transfected with green fluorescent protein (GFP)-expression lentiviral (Gene Pharma, Shanghai, China) particles in advance, and stably expressing cell lines were screened by puromycin selection. The mice in the SHED group were sacrificed after 1, 4, or 8 weeks to collect the ectopic explants and detect the GFP-expressing SHED by cryo￾sections. The frozen sections were counterstained with DAPI and fur￾ther examined using a fluorescent microscope. 2.8.2. In situ bone regeneration of constructs to repair bone defects The ability of co-delivering SHED and rhBMP-2 on NF-Ms to pro￾mote orthotopic bone regeneration was measured in a cranial bone defect model. After the mice were anesthetized, a non-healing, full thickness of 4 mm in diameter defect was created in the central region of the cranial bone by using a dental surgical bur. Defects were filled with the prepared SHED/PDA-NF-Ms constructs, rhBMP-2/Hep-Dopa NF-Ms, or their combinations (n = 3). The empty defects without any fillings were served as a control group. The animals were sacrificed at 4 and 8 weeks by injecting a lethal dose of the anesthetic drug into the abdominal cavity. Harvested samples were fixed for at least 24 h for further imaging and histological analysis. 2.8.3. micro-CT measurement At 4 and 8 weeks after implantation, the harvested calvaria bones of all groups were scanned using micro-CT (Skyscan 1076, Bruker, Belgium). After standardized reconstruction using the NR econ soft￾ware, analysis was carried out using a cylindrical volume of interest with a 4-mm diameter, which was placed over the defected area in the axial plane. Bone volume (BV) and bone mineral density (BMD) of the regenerated tissue were calculated according to the scans reconstructed using the CTAn software (Bruker micro-CT, Belgium). 2.8.4. Histological analysis After CT analysis, the fixed specimens were decalcified in 10% ethylene diamine tetraacetic acid (EDTA) for 7 days, then dehydrated and paraffin-embedded. Sections were cut into 4-mm thick slices and stained by hematoxylin and eosin (H&E) and Masson’s trichrome Table 1 Sample abbreviations used in the ectopic implantation experiment. Ectopic Implantation Description of grouping MS group PDA-NF-MS and Hep-Dopa NF-Ms (ratio is 1:1) SHED group SHED adhered on PDA-NF-MS BMP-2 group rhBMP-2 chemically bonded to Hep-Dopa NF-Ms Dual group Combination of SHED/PDA-NF-MS and BMP-2/Hep-Dopa NF-Ms (ratio is 1:1) Table 2 Sample abbreviations used in the orthotopic implantation experiment. Orthotopic Implantation Description of grouping Control group Empty defect without any filling SHED group SHED adhered on PDA-NF-MS BMP-2 group rhBMP-2 chemically bonded to Hep-Dopa NF-Ms Dual group Combination of SHED/PDA-NF-MS and BMP-2/Hep￾Dopa NF-Ms (ratio is 1:1) A 100 D 20ȝ G 2ȝP ȝP P B 20ȝP E 20ȝP H 2ȝP C F 20ȝP I 2ȝP Fig. 1. Morphological characterization and size distribution of various microspheres. Scanning electron microscope images: (A, B, D, G) PLLA NF-Ms (control); (E, H) PDA-NF-Ms; (F, I) Hep-Dopa NF-Ms. (G, H, I) show a local high magnification images of three kinds microspheres, respectively. Yellow arrows indicate PDA aggregates scattered on the surface of NF-Ms. (C) A gross inspection of PLLA NF-Ms with or without surface-modification. The size distribution of various micro￾spheres with a diameter ranging from 50 to 200 μm. (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) 573–586 577