Synergistic effect of stem cells from human exfoliated deciduous teeth and rhBMP-2 delivered by injectable nanofibrous microspheres with different surface modifications on vascularized bone regeneration

Chemical Engineering Journal 370(2019)573-586 Contents lists available at ScienceDirect HEMICAL RING JOURNAL Chemical Engineering Journ ELSEVIER journalhomepagewww.elsevier.com/locate/cej Synergistic effect of stem cells from human exfoliated deciduous teeth and rhBMP-2 delivered by injectable nanofibrous microspheres with different surface modifications on vascularized bone regeneration Tengjiaozi Fang, Zuoying Yuan", Yuming Zhao, Xiaoxia Li, Yue Zhai, Jingzhi Li, Xiaotong Wang Nanquan Rao, Lihong Ge@, Qing Cai, omatology, and Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China b State Key Laboratory of Organic-Inorganic Composites, Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, China HIGHLIGHTS GRAPHICAL ABSTRACT We applied different surface mod- ifications on-demand to injectable NF- Bio-inspired hydro by PDa supported the cellular beha Ccaat cabo ance of NF-Ms in loading and delivering rhBMP-2. SHED engraftment contributes to an- giogenesis in ectopic and orthotopic Conjunctive use of SHED and rhBMP-2 led to vascularized bone tissue re. ARTICLE INFO ABSTRACT To provide a suitable 3D microenvironment for bone regeneration, we selected the injectable poly (l-lactic acid) anofibrous microspheres(PLLA NF-Ms) with different surface modifications to serve as cell micro-carriers or protein vehicles on-demand. Results showed that polydopamine(PDA)modified NF-Ms(PDA- NF-Ms) exhibited Stem cells from human exfoliated deciduous good affinity for stem cells from human exfoliated deciduous teeth(SHED), and Heparin-Dopamine(Hep-Dopa) teeth njugated with NF-Ms(Hep-Dopa NF-Ms)are able to immobilize and slowly release system morphogenic protein-2 (rhBMP-2)efficiently. In vivo evaluations were carried out in both ectopic subcutaneous plantation and orthotopic cranial bone defect nude mouse models (p= 4 mm). The results suggested that PDA-NF-Ms could support SHED survival over 4 weeks. All experimental groups with SHED/PDA-NF-Ms en- graftment showed angiogenesis activity. But, no effect of SHED/PDA-NF-Ms on osteogenesis was found in ec- topic implantation, which is different from the result in cranial defect. The rhBMP-2 released from Hep-Dopa NF. Ms could significantly guide bone tissue regeneration in both ectopic and orthotopic site. At 8 weeks, both BMP-2 group and dual group showed large amounts of bone formation in situ, despite the fact that quantitative results of micro-ct did not demonstrate significant difference between them. More blood vessels were observed in SHED and Dual groups, which verifies the quality improvement of regenerated osseous tissues. Re ascularized bone tissue was, thus, highly expected upon implanting SHED/PDA-NF-Ms and rhBMP-2-loaded Emst adress: gelihongos 0919@163.com(LGe),caiqing@mailbuct.edu.cn(Q.Cai https://doi.org/10.1016/j.cej.2019.03.151 Received 21 October 2018; Received in revised form 14 March 2019; Accepted 16 March 2019 ailable online 20 March 2019 1385.8947/C 2019 Elsevier B V. All rights reserved

Contents lists available at ScienceDirect Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej Synergistic effect of stem cells from human exfoliated deciduous teeth and rhBMP-2 delivered by injectable nanofibrous microspheres with different surface modifications on vascularized bone regeneration Tengjiaozi Fanga , Zuoying Yuanb , Yuming Zhaoa , Xiaoxia Lia , Yue Zhaia , Jingzhi Lia , Xiaotong Wanga , Nanquan Raoa , Lihong Gea,⁎ , Qing Caib,⁎ a Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology, National Engineering Laboratory for Digital and Material Technology of Stomatology, and Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China b State Key Laboratory of Organic-Inorganic Composites, Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, China HIGHLIGHTS • We applied different surface mod￾ifications on-demand to injectable NF￾Ms. • Bio-inspired hydrophilic modification by PDA supported the cellular beha￾vior. • Hep-Dopa conjugation improved per￾formance of NF-Ms in loading and delivering rhBMP-2. • SHED engraftment contributes to an￾giogenesis in ectopic and orthotopic sites. • Conjunctive use of SHED and rhBMP-2 led to vascularized bone tissue re￾generation. GRAPHICAL ABSTRACT ARTICLE INFO Keywords: Injectable nanofibrous microspheres Surface modification Stem cells from human exfoliated deciduous teeth Cell micro-carrier Sustained release system ABSTRACT To provide a suitable 3D microenvironment for bone regeneration, we selected the injectable poly (l-lactic acid) nanofibrous microspheres (PLLA NF-Ms) with different surface modifications to serve as cell micro-carriers or protein vehicles on-demand. Results showed that polydopamine (PDA) modified NF-Ms (PDA-NF-Ms) exhibited good affinity for stem cells from human exfoliated deciduous teeth (SHED), and Heparin-Dopamine (Hep-Dopa) conjugated with NF-Ms (Hep-Dopa NF-Ms) are able to immobilize and slowly release recombinant human bone morphogenic protein-2 (rhBMP-2) efficiently. In vivo evaluations were carried out in both ectopic subcutaneous implantation and orthotopic cranial bone defect nude mouse models (φ = 4 mm). The results suggested that PDA-NF-Ms could support SHED survival over 4 weeks. All experimental groups with SHED/PDA-NF-Ms en￾graftment showed angiogenesis activity. But, no effect of SHED/PDA-NF-Ms on osteogenesis was found in ec￾topic implantation, which is different from the result in cranial defect. The rhBMP-2 released from Hep-Dopa NF￾Ms could significantly guide bone tissue regeneration in both ectopic and orthotopic site. At 8 weeks, both BMP-2 group and dual group showed large amounts of bone formation in situ, despite the fact that quantitative results of micro-CT did not demonstrate significant difference between them. More blood vessels were observed in SHED and Dual groups, which verifies the quality improvement of regenerated osseous tissues. Regeneration of vascularized bone tissue was, thus, highly expected upon implanting SHED/PDA-NF-Ms and rhBMP-2-loaded https://doi.org/10.1016/j.cej.2019.03.151 Received 21 October 2018; Received in revised form 14 March 2019; Accepted 16 March 2019 ⁎ Corresponding authors. E-mail addresses: gelihong0919@163.com (L. Ge), caiqing@mail.buct.edu.cn (Q. Cai). Chemical Engineering Journal 370 (2019) 573–586 Available online 20 March 2019 1385-8947/ © 2019 Elsevier B.V. All rights reserved. T

T. Fang, et al. Chemical Engineering Journal 370(2019)573-586 Hep-Dopa NF-Ms together. The strategy developed in this study represents a promising method for satisfactorily promoting bone regeneration. 1. Introduction microenvironment to support cell migration, proliferation, and oriented differentiation [11]. In comparison with traditional pre-formed scaf Critical bone defects are generally caused by maxillofacial tumors, folds, injectable microspheres can be applied in a minimally invasive periodontitis, and congenital skeletal deformities, among other condi- manner to easily fill and repair irregularly shaped tissue defects via ons. The treatment of these defect is necessary to recover bone direct injection, which decreases the risk of infection from a surgical structure and function, which is a still a challenge worldwide. Tissue procedure [12, 13]. Due to the huge specific surface area and extra- engineering, as a novel strategy for bone reconstruction, has overcome cellular matrix (ECM)-like structure of nanofibers, injectable micro- many disadvantages of conventional autologous bone grafting, such as spheres, especially those with nanofibrous architecture, are suitable to limited bone sources, surgical trauma, and infection risks [1, 2 serve as cell micro- carriers to facilitate cell-material interactions [14] Tissue engineering often involves the use of stem cells which are Previous studies showed the ideal properties of PLLa nanofibrous mi cultured on the surface of scaffolds and induced to generate new bone crospheres to deliver dental pulp stem cells(DPSCs) and promote tissue by osteoinductive molecules [3]. Nevertheless, there is mounting dentin-pulp tissue regeneration [15]. However such synthetic scaffold evidences indicates a predominant paracrine trophic role of stem cells materials are "bio-inert, and therefore cannot effectively recruit en- in promoting tissue regeneration due to their enriched secretome in dogenous stem cells to migrate onto the scaffold [14]. At present, do- production of angiogenic and chemotactic factors, rather than their paminergic surface modification has been widely applied to improve direct replacement of damaged cells at the injury site [4]. Given that, it the hydrophilicity of materials, providing a beneficial microenviron- is necessary to improve the microenvironment of bone damage and ment for cell adhesion and migration without causing adverse effects on mobilize progenitor cells for better healing outcomes. Stem cells from cell biological behaviors [16]. Based on this information, we supposed human exfoliated deciduous teeth (SHED), which originate from the that coating PLLA nanofibrous microspheres (NF- Ms) with poly- neural crest and expressing mesenchymal stem cell markers, have re- dopamine(PDA) could improve its biological properties to act as an cently gained more attention than bone marrow-derived stem cells appropriate micro-carrier for SHED. (BMSCs) in regenerative medicine currently. Compared with BMSCs As an extensively studied recombinant growth factor, bone mor- SHED can be obtained noninvasively, and their ability of extensive phogenetic protein-2(BMP-2) has been successfully applied for treating proliferation and multiple differentiation capabilities are mon bone diseases or inducing osteogenesis in endogenous cells [17, 18 nent [5-7. Besides, SHEd have shown the multifaceted the There have been plenty of attempts to develop an effective carrier for nctions on many disease models, which can be attributed delivering BMP-2 into a defective area while considering its spatio paracrine effects on anti-inflammation, pr emporal distribution and optimal dosage to avoid complications and poptosis effects [8-10]. However, the inductive effect of rapid diffusion [19]. Although the PDA-assisted coating strategy has steogenesis and a suitable scaffold for their implantation in vivo have also been applied to modify scaffolds for protein delivery [20], the high been rarely reported affinity of Heparin to form covalent bonds with proteins makes it more Advanced tissue engineering have go effective as a sustained delivery system while maintaining proteins gradability and biocompatibility; ale bioactivity [21]. Kim et al. prepared BMP-2-immobilized porous mi structure that is similar to native re; and crospheres modified with Hep-Dopa, which provided optimal Dopamine rhBMP-2 ep- Tris-HCI pH8.0 Hep-Dopa NF-Ms rhBMP2/Hep-Dopa NF-Ms PLLA NF-Ms Dopamine Pre-culture 7 days Tris-HCI(pH 8.5) SHED/PDA NF-Ms NF-Ms HED Scheme 1. Schematic illustration for synthesis of NF-Ms with different surface modifications

Hep-Dopa NF-Ms together. The strategy developed in this study represents a promising method for satisfactorily promoting bone regeneration. 1. Introduction Critical bone defects are generally caused by maxillofacial tumors, periodontitis, and congenital skeletal deformities, among other condi￾tions. The treatment of these defect is necessary to recover bone structure and function, which is a still a challenge worldwide. Tissue engineering, as a novel strategy for bone reconstruction, has overcome many disadvantages of conventional autologous bone grafting, such as limited bone sources, surgical trauma, and infection risks [1,2]. Tissue engineering often involves the use of stem cells which are cultured on the surface of scaffolds and induced to generate new bone tissue by osteoinductive molecules [3]. Nevertheless, there is mounting evidences indicates a predominant paracrine trophic role of stem cells in promoting tissue regeneration due to their enriched secretome in production of angiogenic and chemotactic factors, rather than their direct replacement of damaged cells at the injury site [4]. Given that, it is necessary to improve the microenvironment of bone damage and mobilize progenitor cells for better healing outcomes. Stem cells from human exfoliated deciduous teeth (SHED), which originate from the neural crest and expressing mesenchymal stem cell markers, have re￾cently gained more attention than bone marrow-derived stem cells (BMSCs) in regenerative medicine currently. Compared with BMSCs, SHED can be obtained noninvasively, and their ability of extensive proliferation and multiple differentiation capabilities are more promi￾nent [5–7]. Besides, SHED have shown the multifaceted therapeutic functions on many disease models, which can be attributed to their paracrine effects on anti-inflammation, pro-angiogenesis, and anti￾apoptosis effects [8–10]. However, the inductive effect of SHED on osteogenesis and a suitable scaffold for their implantation in vivo have been rarely reported. Advanced tissue engineering scaffolds should have good biode￾gradability and biocompatibility; possess a nanoscale or macroscale structure that is similar to native bone architecture; and mimic the microenvironment to support cell migration, proliferation, and oriented differentiation [11]. In comparison with traditional pre-formed scaf￾folds, injectable microspheres can be applied in a minimally invasive manner to easily fill and repair irregularly shaped tissue defects via direct injection, which decreases the risk of infection from a surgical procedure [12,13]. Due to the huge specific surface area and extra￾cellular matrix (ECM)-like structure of nanofibers, injectable micro￾spheres, especially those with nanofibrous architecture, are suitable to serve as cell micro-carriers to facilitate cell-material interactions [14]. Previous studies showed the ideal properties of PLLA nanofibrous mi￾crospheres to deliver dental pulp stem cells (DPSCs) and promote dentin-pulp tissue regeneration [15]. However, such synthetic scaffold materials are ‘bio-inert’, and therefore cannot effectively recruit en￾dogenous stem cells to migrate onto the scaffold [14]. At present, do￾paminergic surface modification has been widely applied to improve the hydrophilicity of materials, providing a beneficial microenviron￾ment for cell adhesion and migration without causing adverse effects on cell biological behaviors [16]. Based on this information, we supposed that coating PLLA nanofibrous microspheres (NF-Ms) with poly￾dopamine (PDA) could improve its biological properties to act as an appropriate micro-carrier for SHED. As an extensively studied recombinant growth factor, bone mor￾phogenetic protein-2 (BMP-2) has been successfully applied for treating bone diseases or inducing osteogenesis in endogenous cells [17,18]. There have been plenty of attempts to develop an effective carrier for delivering BMP-2 into a defective area while considering its spatio￾temporal distribution and optimal dosage to avoid complications and rapid diffusion [19]. Although the PDA-assisted coating strategy has also been applied to modify scaffolds for protein delivery [20], the high affinity of Heparin to form covalent bonds with proteins makes it more effective as a sustained delivery system while maintaining proteins bioactivity [21]. Kim et al. prepared BMP-2-immobilized porous mi￾crospheres modified with Hep-Dopa, which provided optimal Scheme 1. Schematic illustration for synthesis of NF-Ms with different surface modifications. T. Fang, et al. Chemical Engineering Journal 370 (2019) 573–586 574

T. Fang, et al. Chemical Engineering Journal 370(2019)573-586 conditions for the long-lasting release of BMP-2 to induce osteogenic 2.3. Characterization of NF- Ms and modified NF-M differentiation of cells [22]. Nevertheless, the small size of nanofibrous structures has advantages over porous structures, which can provide The surface morphology and microstructure of NF-Ms, PDA-NF-Ms more binding sites for proteins [16]. Herein, we functionalized the and Hep-Dopa NF-Ms were characterized by a scanning electron mi- surface of PLLA NF-MS by Hep-Dopa conjugation to immobilize BMP-2 croscope(SEM, Hitachi, S-2400 Japan). Each specimen was dehydrated and promote its controlled release and evaluated the effectiveness of and immobilized on the surface of an aluminum stub via a double-sided this novel protein delivery system for bone tissue engineering adhesive carbon tape, then sputtered with gold (vacuum, 20 mA, 120 s) Multiple polymer scaffolds modified via single surface-modification using a sputter-coater(Polaron E5600, USA)and observed by utilizing rategies have been developed to improve the delivery of stem cells SEM at an accelerating voltage of 10 kV. The size distribution of the and biomolecules. However, most of these tissue engineering scaffold microspheres was determined by analyzing the SEM images with Nano are non-specific delivery systems, which cannot efficiently satisfy the Measurer (V1. 2.5)Software. The elemental analysis was performed by eds of both. Hence, the objective of this study was to design a energy dispersive spectroscopy(EDS)and elemental mapping(under 15 ioactive PLLA NF-MS scaffold that modified with either a PDa layer or KV as SEM observation with an exposure time of 180 s). The hydro Hep-Dopa, which further specifically targeted SHED to support their philicity of various microspheres was evaluated by testing the water long-time survival and deliver BMP-2 constantly to enhance bone tissue contact angle(WCA). PLLA NF-Ms, PDA-NF-Ms, and Hep-Dopa NF-Ms regeneration. We suppose that the involvement of SHed may promote were pressed into similar plate- like samples to estimate the effect of the bone tissue regeneration more than a single application of BMP-2, surface components on hydrophilicity which is not only achieved by the paracrine trophic effect of ShEd to prove the microenvironment, but also resulted from the synergistic 2. 4. BMP-2 immobilization and release assay To impregnate rhBMP-2 on NF-Ms, a 500 ng/ml rhBMP-2 solution 2. Materials and method (stabilized by BSA) was added to the Hep-Dopa modified NF-Ms(mass ratio of BMP-2/polymer 10 ug/mg) in 0.1 M MES buffer(pH 5.6). 2.1. Preparation of microspheres Then, this mixture was shaken overnight to fix positively charged rhBMP-2 on Hep-Dopa NF-Ms by using a magnetic stirrer (JOANLAB Nanofibrous microspheres(NF-Ms) were fabricated via a phase se- HS.17) at 150 rpm. NF- Ms without any modification were loaded with Gration method as previously described [23J. Briefly, 1 g PLLA rhBMP-2 via the same approach as the control. The encapsulation ef. IW= 50,000, Shandong College of Pharmacy, China) was dissolved ficiency of immobilized rhBMP-2 was determined as the difference in in 50 ml tetrahydrofuran (THF) at a concentration of 2.0%(wt/v)at values of the original rhBMP-2 solution and in the supernatant by an 50C and the solution was rigorously agitated to ensure adequate dis- enzyme-linked immunosorbent assay(ELISA) kit(Abcam, MA). Next, solution. Afterwards, heated glycerol was gradually poured into the two kinds of NF-Ms were washed with Di water to remove free rhBMP-2 PLLA solution at a ratio of 3: 1 under mechanical stirring for 10 min. and lyophilized [24]. The obtained rhBMP-2-loaded microspheres were This mixture was then quickly poured into liquid nitrogen and added lyophilized and stored at -80'C for later experiments into a water/ice mixture for 24 h to promote solvent exchange. To To visualize the distribution of rhBMP-2 in microspheres with or ensure that the glycerol residue was removed, NF-Ms washed with without Hep-Dopa conjugation, the RBITC-labelled rhBMP-2 distilled water every 3 h. Then, PLLA NF-Ms were lyophilized for 48 h (PeproTech, USA)was applied according to the procedure described and stored at room temperature for further use. above. Briefly, two kinds of microspheres were mixed with RBITC-la belled rhBMP-2 (at a concentration of 500 ng/ml) in MEs buffe (pH =5.6), and incubated for 3 h at room temperature in the dark. 2. 2. Modification of NF-MS with PDA and Hep-Dopa Thereafter, the samples were washed with phosphate buffered saline (PBS, PH =7. 4) for three times to remove unbound fluorescent dyes We modified injectable NF-Ms with either PDA-coating or Hep-Dopa before imaging through laser scanning confocal microscopy(LSCM, conjugation for different purpose. The schematic diagram clearly shows Nikon, Japan) the synthesis process of two kinds modification(see Scheme 1).For For the release study, rhBMP-2-loaded NF-Ms with or without Hep- coating with PDA, NF-Ms were directly soaked into a dopamine(Da) Dopa conjugation were immersed in 5 ml PBS (pH 7. 4)at 37C under (Sigma-Aldrich) solution (2 mg/mL in 10 mM Tris-HCl, pH 8.5) and continuous shaking. The in vitro release kinetics of proteins released evenly stirred for 24 h at room temperature. The obtained PDA-NF-Ms from two kinds of microspheres was examined on days 1, 3, 5, 7, 10, 14, that. rinsed with deionized water(DD water for several times to ensure 1, and 28. At specific time intervals, 1 ml supernatant was collected the ions and adsorbed polymers were removed completely; this was and replaced with an equal amount of fresh PBS for 4 weeks. The followed by freeze-drying overnight. quantity of rhBMP-2 released from microspheres was measured by To functionalize and immobilize heparin on the surface of NF-M using a rhBMP-2 ELISA Kit(n=5). heparin was first chemically conjugated with dopamine. 400mg heparin was reacted with 190.4 mg EDC (Thermal USA)and 114.8 mg NHS (Sigma, USA)in 10 ml of MES buffer m 2. 5. Cell culture and allowed to react for 10 min. Then, DA (102.2 mg)dissolved in 1 ml of MES (pH 4.5)was added to the activated heparin solution and al- Normal exfoliated human deciduous teeth and human bone marrow lowed to react overnight. The mixture was dialyzed (molecular weight stromal cells(BMScs)were a gift from the Oral Stem Cell Bank( China, cutoff (MWCO)= 2000, Spectrum)against acidified distilled water for Beijing). These cells were characterized by this public institution and 48 h and lyophilized at last. Then, 2 mg/ml Hep- Dopa was dissolved in stored in liquid nitrogen vapor for long-term cryopreservation. briefly, 10 mM Tris-HCl (pH 8.0). NF- Ms were added to the Hep-Dopa solution, after SHEDs and BMSCs were thawed, they were incubated in a-MEM nd this reaction was maintained overnight in the dark environment. (Life Technologies, CA, US)supplemented with 10% fetal bovine serum The resultant Hep-Dopa NF-Ms were washed with DI water and dried (FBS, Gibico, USA) and 1% penicillin-streptomycin (100 U/ml and nder nitrogen. All microspheres used in this study were sterilized by 100 ug/ml, Invitrogen, UK) in an atmosphere containing 5%CO2 and exposure to ultraviolet light for 6h. 95%O2 at 37C. Both SHED and BMSCs were passaged when con- fluence reached 80% and cells were used after three to five passages

conditions for the long-lasting release of BMP-2 to induce osteogenic differentiation of cells [22]. Nevertheless, the small size of nanofibrous structures has advantages over porous structures, which can provide more binding sites for proteins [16]. Herein, we functionalized the surface of PLLA NF-MS by Hep-Dopa conjugation to immobilize BMP-2 and promote its controlled release and evaluated the effectiveness of this novel protein delivery system for bone tissue engineering. Multiple polymer scaffolds modified via single surface-modification strategies have been developed to improve the delivery of stem cells and biomolecules. However, most of these tissue engineering scaffold are non-specific delivery systems, which cannot efficiently satisfy the needs of both. Hence, the objective of this study was to design a bioactive PLLA NF-MS scaffold that modified with either a PDA layer or Hep-Dopa, which further specifically targeted SHED to support their long-time survival and deliver BMP-2 constantly to enhance bone tissue regeneration. We suppose that the involvement of SHED may promote bone tissue regeneration more than a single application of BMP-2, which is not only achieved by the paracrine trophic effect of SHED to improve the microenvironment, but also resulted from the synergistic effect of their combination. 2. Materials and method 2.1. Preparation of microspheres Nanofibrous microspheres (NF-Ms) were fabricated via a phase se￾paration method as previously described [23]. Briefly, 1 g PLLA (MW = 50,000, Shandong College of Pharmacy, China) was dissolved in 50 ml tetrahydrofuran (THF) at a concentration of 2.0% (wt/v) at 50 °C and the solution was rigorously agitated to ensure adequate dis￾solution. Afterwards, heated glycerol was gradually poured into the PLLA solution at a ratio of 3:1 under mechanical stirring for 10 min. This mixture was then quickly poured into liquid nitrogen and added into a water/ice mixture for 24 h to promote solvent exchange. To ensure that the glycerol residue was removed, NF-Ms washed with distilled water every 3 h. Then, PLLA NF-Ms were lyophilized for 48 h and stored at room temperature for further use. 2.2. Modification of NF-MS with PDA and Hep-Dopa We modified injectable NF-Ms with either PDA-coating or Hep-Dopa conjugation for different purpose. The schematic diagram clearly shows the synthesis process of two kinds modification (see Scheme 1). For coating with PDA, NF-Ms were directly soaked into a dopamine (DA) (Sigma–Aldrich) solution (2 mg/mL in 10 mM Tris-HCl, pH 8.5) and evenly stirred for 24 h at room temperature. The obtained PDA-NF-Ms were rinsed with deionized water (DI) water for several times to ensure that the ions and adsorbed polymers were removed completely; this was followed by freeze-drying overnight. To functionalize and immobilize heparin on the surface of NF-Ms, heparin was first chemically conjugated with dopamine. Briefly, 400 mg heparin was reacted with 190.4 mg EDC (Thermal Scientific, USA) and 114.8 mg NHS (Sigma, USA) in 10 ml of MES buffer (pH 4.5) and allowed to react for 10 min. Then, DA (102.2 mg) dissolved in 1 ml of MES (pH 4.5) was added to the activated heparin solution and al￾lowed to react overnight. The mixture was dialyzed (molecular weight cutoff (MWCO) = 2000, Spectrum®) against acidified distilled water for 48 h and lyophilized at last. Then, 2 mg/ml Hep-Dopa was dissolved in 10 mM Tris-HCl (pH 8.0). NF-Ms were added to the Hep-Dopa solution, and this reaction was maintained overnight in the dark environment. The resultant Hep-Dopa NF-Ms were washed with DI water and dried under nitrogen. All microspheres used in this study were sterilized by exposure to ultraviolet light for 6 h. 2.3. Characterization of NF-Ms and modified NF-Ms The surface morphology and microstructure of NF-Ms, PDA-NF-Ms and Hep-Dopa NF-Ms were characterized by a scanning electron mi￾croscope (SEM, Hitachi, S-2400. Japan). Each specimen was dehydrated and immobilized on the surface of an aluminum stub via a double-sided adhesive carbon tape, then sputtered with gold (vacuum, 20 mA, 120 s) using a sputter-coater (Polaron E5600, USA) and observed by utilizing SEM at an accelerating voltage of 10 kV. The size distribution of the microspheres was determined by analyzing the SEM images with Nano Measurer (V1.2.5) Software. The elemental analysis was performed by energy dispersive spectroscopy (EDS) and elemental mapping (under 15 KV as SEM observation with an exposure time of 180 s). The hydro￾philicity of various microspheres was evaluated by testing the water contact angle (WCA). PLLA NF-Ms, PDA-NF-Ms, and Hep-Dopa NF-Ms were pressed into similar plate-like samples to estimate the effect of the surface components on hydrophilicity. 2.4. BMP-2 immobilization and release assay To impregnate rhBMP-2 on NF-Ms, a 500 ng/ml rhBMP-2 solution (stabilized by BSA) was added to the Hep-Dopa modified NF-Ms (mass ratio of BMP-2/polymer = 10 ug/mg) in 0.1 M MES buffer (pH 5.6). Then, this mixture was shaken overnight to fix positively charged rhBMP-2 on Hep-Dopa NF-Ms by using a magnetic stirrer (JOANLAB HS-17) at 150 rpm. NF-Ms without any modification were loaded with rhBMP-2 via the same approach as the control. The encapsulation ef- ficiency of immobilized rhBMP-2 was determined as the difference in values of the original rhBMP-2 solution and in the supernatant by an enzyme-linked immunosorbent assay (ELISA) kit (Abcam, MA). Next, two kinds of NF-Ms were washed with DI water to remove free rhBMP-2 and lyophilized [24]. The obtained rhBMP-2-loaded microspheres were lyophilized and stored at −80 °C for later experiments. To visualize the distribution of rhBMP-2 in microspheres with or without Hep-Dopa conjugation, the RBITC-labelled rhBMP-2 (PeproTech, USA) was applied according to the procedure described above. Briefly, two kinds of microspheres were mixed with RBITC-la￾belled rhBMP-2 (at a concentration of 500 ng/ml) in MES buffer (pH = 5.6), and incubated for 3 h at room temperature in the dark. Thereafter, the samples were washed with phosphate buffered saline (PBS, pH = 7.4) for three times to remove unbound fluorescent dyes before imaging through laser scanning confocal microscopy (LSCM, Nikon, Japan). For the release study, rhBMP-2-loaded NF-Ms with or without Hep￾Dopa conjugation were immersed in 5 ml PBS (pH 7.4) at 37 °C under continuous shaking. The in vitro release kinetics of proteins released from two kinds of microspheres was examined on days 1, 3, 5, 7, 10, 14, 21, and 28. At specific time intervals, 1 ml supernatant was collected and replaced with an equal amount of fresh PBS for 4 weeks. The quantity of rhBMP-2 released from microspheres was measured by using a rhBMP-2 ELISA Kit (n = 5). 2.5. Cell culture Normal exfoliated human deciduous teeth and human bone marrow stromal cells (BMSCs) were a gift from the Oral Stem Cell Bank (China, Beijing). These cells were characterized by this public institution and stored in liquid nitrogen vapor for long-term cryopreservation. Briefly, after SHEDs and BMSCs were thawed, they were incubated in α-MEM (Life Technologies, CA, US) supplemented with 10% fetal bovine serum (FBS, Gibico, USA) and 1% penicillin-streptomycin (100 U/ml and 100 μg/ml, Invitrogen, UK) in an atmosphere containing 5% CO2 and 95% O2 at 37 °C. Both SHED and BMSCs were passaged when con- fluence reached 80% and cells were used after three to five passages. T. Fang, et al. Chemical Engineering Journal 370 (2019) 573–586 575

T. Fang, et al. Chemical Engineering Journal 370(2019)573-586 2.6. The biological effects of PDA-NF-Ms on SHED 5 104 cells per well. The induction medium and the medium con- taining rhBMP-2 was changed every 2-3 days. At days 7 and 14, the Before being applied for in vitro cell culture, all the prepar BMSCs were fixed with chilled 4% paraformaldehyde for 20 min and crospheres were soaked in 75% ethanol for 1 h to pre-wet the rinsed with PBS; subsequently, each group was treated with ALP then washed with PBS three times to replace the residual ethanol staining solution(Beyotime, Shanghai, China) for 30 min. The general the microspheres were immersed in a a-medium with serum overnight images were captured using er(HP ScanJet 2400) to aid cell attachment. On the next day, SHED In addition, ALP activity was also quantified at the same time of 1x 106cells/ml onto the 0.5 mg(about 1 x 105 spheres)of PDA-NF. points. The cell lysates were prepared by adding 1 ml of distilled water Ms(10: 1 ratio) and co-cultured for at least 4 h to promote cell attach- followed by three freeze-thaw cycles. Then, the level of ALP activity ment. The obtained SHED/PDA-NF- Ms constructs were further used in was measured using an AKP/ALP assay kit(Jiancheng, Nanjing, China) all in vitro and in vivo at an ODs20nm and was normalized to the amount of total proteins(BCa Cells adherence and spreading on PLLA NF-Ms and PDA-NF-Ms were assay kit, Biyuntian Biotechnology, China) by using BSA as the standard observed by SEM. After SHED were seeded onto microspheres and co- protein. cultured for 7 days, the constructs were retrieved and fixed with 4% paraformaldehyde. For morphological characterization of SHED, the 2.7.2. Alizarin red S staining(ARS) samples were dehydrated rapidly by using a serious of graded ethanol When the BMSCs in each group were induced with osteogenic solutions. Then, the dried mixture was further viewed by SEM as pre- medium for 21 days, the cells were fixed and washed in the same viously described. fashion as that used for ALP staining. Furthermore, the solutions were The viability of SHED on the surface of NF- Ms was assessed by a replaced with 1% prepared alizarin red working solution to react with live/dead assay kit (Invitrogen, USA)at 7 days after cell seeding. calcium deposits for 20 min and were rinsed with PBS several times. Briefly, the SHED/PDA-NF-Ms were rinsed with PBS three times and The images were taken using phase contrast microscopy and a scanner. then incubated with a PBS solution containing 2 uM calcein AM and To quantify the calcium matrix content, the dye was dissolved in 4 HM ethidium homodimer-1 for 30 min at room temperature. The 10% cetylpyridinium chloride to release the calcium-bound alizarin red constructs were then washed with PBS several times, and the viable into solution. The absorbance of the supernatant was measured at cells emitting green fluorescence(dead cells were red-labelled) were 562 nm using a microplate reader observed by LSCM. Immunofluorescent staining was performed to characterize the 2.7.3. RT-PCR(ALP, Runx2, Coll D) morphology of the cells that attached to microspheres. After the cells For RT-PCR assays, BMSCs in each well were rinsed with PBS after bilized with 0. 1% Triton x-100 for 10 min and blocked with 1% BSA fo 14 days of osteogenic inductive culture, followed by the addition of a 1 ml Trizol reagent(Ambion, chnologies", USA) to extract the 1 h. Afterwards, the cells and microspheres were co-stained with TRITc- total RNA. The quantity and purity of RNAs were determined at an Phalloidin(Life Technologies, OR, USA) and 4, 6-diamidino-2-phen absorbance at 260/280 nm. Then, cDNA was reverse-transcribed using dole(DAPl, Sigma-Aldrich, USA) for 1 h at room temperature, which the PrimeScript RT reagent Kit (Takara, Japan) according to the ould specifically bind with actin filaments(F-actin) and cell nucleus, manufacturers protocol. RT-PCR was performed using SYBR Green respectively. The samples were then washed with PBS three times to Master (Roche, USA) via a Step One Plust real-time PCR system remove free fluorescent dyes, and the cells in the complexes were (Thermo Fisher, CA). A total of 45 cycles of RT-PCR were carried out The SHED proliferation on microspheres was confirmed by MTT exclude the influence of cell number. The specific primers of three.o photographed with LSCM. and data were normalized to levels of the housekeeper gene B-actin to ssay(Sigma-Aldrich). Briefly, the sterilized NF- nd PDA-NF-Ms teogenic-related genes, including those encoding ALP, Runx2, and (0. 1 mg/well) were respectively placed into 96-well plates to cover the Collagen I( Col D), were designed as listed in Table 3. bottom of plate, individually. SHEDs were then seeded onto micro- a 96-well plate directly without microspheres were used as a contro/ 2 spheres in each well at a density of 3 x 10 cells/well. Cells seeded into 8. In vivo experiments group. At the scheduled time points, 20 ul of prepared MTT solution The osteogenic regenerative capacity of two kinds of modified mi- (5 mg/ml) was added into each well and incubated for 3h at 3 crospheres and their corresponding composite scaffolds SHED After the resulting supernatant was discarded, 150 ul of dimethyl sulf- or rhBMP-2)in vivo was determined in ectopic subcutaneous engraft oxide(DMSO)was then added into per well to dissolve the formazan ment experiments and an orthotopic cranial bone defect model. All crystals that formed. The dissolved supernatant was moved to a new 96. experimental animal procedures were approved by the Animal Care and well plate and measured by a microplate reader(Bio-Rad 680, USA)at Use Committee of Peking University(China) in accordance with inter- avelength of 490 nn LA2018001) For the animal surgery, 6-8 weeks old BALB/c nude mice 2.7. In vitro osteogenesis study (Weitonglihua Biotechnology, Beijing, China) were anaesthetized by intraperitoneal injection of 4% chloral hydrate(8 ml/kg). A total of 48 The bioactivity of the released rhBMP-2 was examined nude mice were used and randomly divided into four groups in each paring its ability to induce osteogenic differentiation of BMs animal experiment, and the corresponding groupings and sample ab- that of directly applied rhBMP-2. The osteogenic induction breviations used in ectopic or orthotopic implantation was listed(see components included 50 mg/ml L-ascorbic acid and 10 mM B-glycerol Tables 1 and 2) (Sigma, USA). This study contained three groups in total: [1] Blank (BMSCs cultured only with induction medium) group; [2] 100 ng/ml 2.8.1. Ectopic bone formation upon constructs subcutaneous injection soluble rhBMP-2 group; and [3] rhBMP-2 loaded Hep-Dopa NF-Ms SHEDs were pre-cultured on PDA-NF-Ms for 7 days to allow for cell (0. 1 mg grafts) group. The extracted medium was collected by in roliferation, and this cells/PDA-NF- Ms mixture was then resuspended abating 1 mg BMP-2/Hep-Dopa NF-Ms in 10 ml of cultured medium in medium(in a ratio of 2 mg/ml) prior to implantation. Thereafter, the and was refreshed every 2-3 days. cells.protein microspheres grafts according to the groups above were injected subcutaneously under the dorsal surface of nude mice(n=3), 2.7.1. Alkaline phosphatase(ALP)activity respectively. About 1 mg of microspheres or constructs were injected BMSCs were seeded in a 12 well-plate at an initial density of per site, and each mouse accepted two injections of the same group

2.6. The biological effects of PDA-NF-Ms on SHED Before being applied for in vitro cell culture, all the prepared mi￾crospheres were soaked in 75% ethanol for 1 h to pre-wet the surface, then washed with PBS three times to replace the residual ethanol. Then, the microspheres were immersed in a α-medium with serum overnight to aid cell attachment. On the next day, SHED were seeded at a density of 1 × 106 cells/ml onto the 0.5 mg (about 1 × 105 spheres) of PDA-NF￾Ms (10:1 ratio) and co-cultured for at least 4 h to promote cell attach￾ment. The obtained SHED/PDA-NF-Ms constructs were further used in all in vitro and in vivo experiments. Cells adherence and spreading on PLLA NF-Ms and PDA-NF-Ms were observed by SEM. After SHED were seeded onto microspheres and co￾cultured for 7 days, the constructs were retrieved and fixed with 4% paraformaldehyde. For morphological characterization of SHED, the samples were dehydrated rapidly by using a serious of graded ethanol solutions. Then, the dried mixture was further viewed by SEM as pre￾viously described. The viability of SHED on the surface of NF-Ms was assessed by a live/dead assay kit (Invitrogen, USA) at 7 days after cell seeding. Briefly, the SHED/PDA-NF-Ms were rinsed with PBS three times and then incubated with a PBS solution containing 2 μM calcein AM and 4 μM ethidium homodimer-1 for 30 min at room temperature. The constructs were then washed with PBS several times, and the viable cells emitting green fluorescence (dead cells were red-labelled) were observed by LSCM. Immunofluorescent staining was performed to characterize the morphology of the cells that attached to microspheres. After the cells were fixed on NF-Ms and PDA-NF-Ms, the construct was then permea￾bilized with 0.1% Triton X-100 for 10 min and blocked with 1% BSA for 1 h. Afterwards, the cells and microspheres were co-stained with TRITC￾Phalloidin (Life Technologies, OR, USA) and 4′,6-diamidino-2-pheny￾lindole (DAPI, Sigma-Aldrich, USA) for 1 h at room temperature, which could specifically bind with actin filaments (F-actin) and cell nucleus, respectively. The samples were then washed with PBS three times to remove free fluorescent dyes, and the cells in the complexes were photographed with LSCM. The SHED proliferation on microspheres was confirmed by MTT assay (Sigma-Aldrich). Briefly, the sterilized NF-Ms and PDA-NF-Ms (0.1 mg/well) were respectively placed into 96-well plates to cover the bottom of plate, individually. SHEDs were then seeded onto micro￾spheres in each well at a density of 3 × 103 cells/well. Cells seeded into a 96-well plate directly without microspheres were used as a control group. At the scheduled time points, 20 μl of prepared MTT solution (5 mg/ml) was added into each well and incubated for 3 h at 37 °C. After the resulting supernatant was discarded, 150 μl of dimethyl sulf￾oxide (DMSO) was then added into per well to dissolve the formazan crystals that formed. The dissolved supernatant was moved to a new 96- well plate and measured by a microplate reader (Bio-Rad 680, USA) at wavelength of 490 nm. 2.7. In vitro osteogenesis study The bioactivity of the released rhBMP-2 was examined by com￾paring its ability to induce osteogenic differentiation of BMSCs with that of directly applied rhBMP-2. The osteogenic induction medium components included 50 mg/ml L-ascorbic acid and 10 mM β-glycerol (Sigma, USA). This study contained three groups in total: [1] Blank (BMSCs cultured only with induction medium) group; [2] 100 ng/ml soluble rhBMP-2 group; and [3] rhBMP-2 loaded Hep-Dopa NF-Ms (0.1 mg grafts) group. The extracted medium was collected by in￾cubating 1 mg BMP-2/Hep-Dopa NF-Ms in 10 ml of cultured medium and was refreshed every 2–3 days. 2.7.1. Alkaline phosphatase (ALP) activity BMSCs were seeded in a 12 well-plate at an initial density of 5 × 104 cells per well. The induction medium and the medium con￾taining rhBMP-2 was changed every 2–3 days. At days 7 and 14, the BMSCs were fixed with chilled 4% paraformaldehyde for 20 min and rinsed with PBS; subsequently, each group was treated with ALP staining solution (Beyotime, Shanghai, China) for 30 min. The general images were captured using a scanner (HP ScanJet 2400). In addition, ALP activity was also quantified at the same time points. The cell lysates were prepared by adding 1 ml of distilled water followed by three freeze-thaw cycles. Then, the level of ALP activity was measured using an AKP/ALP assay kit (Jiancheng, Nanjing, China) at an OD520nm and was normalized to the amount of total proteins (BCA assay kit, Biyuntian Biotechnology, China) by using BSA as the standard protein. 2.7.2. Alizarin red S staining (ARS) When the BMSCs in each group were induced with osteogenic medium for 21 days, the cells were fixed and washed in the same fashion as that used for ALP staining. Furthermore, the solutions were replaced with 1% prepared alizarin red working solution to react with calcium deposits for 20 min and were rinsed with PBS several times. The images were taken using phase contrast microscopy and a scanner. To quantify the calcium matrix content, the dye was dissolved in 10% cetylpyridinium chloride to release the calcium-bound alizarin red into solution. The absorbance of the supernatant was measured at 562 nm using a microplate reader. 2.7.3. RT-PCR (ALP, Runx2, Coll I) For RT-PCR assays, BMSCs in each well were rinsed with PBS after 14 days of osteogenic inductive culture, followed by the addition of a 1 ml Trizol reagent (Ambion®, Life Technologies™, USA) to extract the total RNA. The quantity and purity of RNAs were determined at an absorbance at 260/280 nm. Then, cDNA was reverse-transcribed using the PrimeScript® RT reagent Kit (Takara, Japan) according to the manufacturer’s protocol. RT-PCR was performed using SYBR Green Master (Roche, USA) via a Step One Plust real-time PCR system (Thermo Fisher, CA). A total of 45 cycles of RT-PCR were carried out and data were normalized to levels of the housekeeper gene β-actin to exclude the influence of cell number. The specific primers of three os￾teogenic-related genes, including those encoding ALP, Runx2, and Collagen I (Col I), were designed as listed in Table 3. 2.8. In vivo experiments The osteogenic regenerative capacity of two kinds of modified mi￾crospheres and their corresponding composite scaffolds (loading SHED or rhBMP-2) in vivo was determined in ectopic subcutaneous engraft￾ment experiments and an orthotopic cranial bone defect model. All experimental animal procedures were approved by the Animal Care and Use Committee of Peking University (China) in accordance with inter￾national standards on animal welfare (authorization number: LA2018001). For the animal surgery, 6–8 weeks old BALB/c nude mice (Weitonglihua Biotechnology, Beijing, China) were anaesthetized by intraperitoneal injection of 4% chloral hydrate (8 ml/kg). A total of 48 nude mice were used and randomly divided into four groups in each animal experiment, and the corresponding groupings and sample ab￾breviations used in ectopic or orthotopic implantation was listed (see Tables 1 and 2). 2.8.1. Ectopic bone formation upon constructs subcutaneous injection SHEDs were pre-cultured on PDA-NF-Ms for 7 days to allow for cell proliferation, and this cells/PDA-NF-Ms mixture was then resuspended in medium (in a ratio of 2 mg/ml) prior to implantation. Thereafter, the cells-protein microspheres grafts according to the groups above were injected subcutaneously under the dorsal surface of nude mice (n = 3), respectively. About 1 mg of microspheres or constructs were injected per site, and each mouse accepted two injections of the same group T. Fang, et al. Chemical Engineering Journal 370 (2019) 573–586 576

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 this

which 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

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

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

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 group

SHED 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

T. Fang, et al. Chemical Engineering Journal 370(2019)573-586 NF-MS PDA-NF-Ms 3 Days 7 Days NF-Ms PDA-NF-Ms B Live/Dead PDA-NF-Ms 0.6 b Phalloidin/Nuclei Fig.4.〔A)SEM of cells 3 and 7 days after seeding on NF-Ms and PDA-NF-Ms on 3 days and 7 of SHEDs adhered on NF-Ms or PDA-NF-Ms for the corresponding white column at different time points(the location of SHED is indicated by red arrows; the ECM is pointed out by yellow arrows). viability staining was performed with calcein AM/PI at 7 days (green representes live cells, red representes dead cells);(b) phalloidine(red fluorescence)and nuclei stained with DAPI (blue)of SHEDs on PDA-NF-Ms on 7 days. (C)MTT assay for the SHED proliferation on NF-Ms -Ms on 3 and 7 days. Statistically analysis by using one-way analysis of variance(ANOVA)(P 0.05, P 001).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (P <0.05). Although the amount of bone formation in the Dual group group. For both 4 and 8 weeks, the new vessels density in SHED group was higher than BMP-2 group, no statistically significant difference (4 week: 1.59+ 0.72%; 8 week: 3.48+ 1.28%)is the highest among between these two group. However, both of these two groups were all groups(P <0.05). Thus, all above results indicated that SHED/PDA presented potent osteoinductive ability than other groups (p< 0.05 NF-Ms had the potential to induce the angiogenesis and osteogenesis in orp<0.01) an orthotopic bone damage environment, and its combination with sustained release of rhBMP-2 by Hep-Dopa NF-Ms contributed to a stronger effect on bone tissue regenerati 3.6.2. Histological observations by H&E and Masson's trichrome staining The sagittal sections of calvarial bone were further stained with H& E and Masson's for histological analysis. High-magnification images 4 (200 x)were taken to examine vascularization or osteogenesis in al As shown in Fig. 9, a large amount of bone matrix and bone. Recent advances in bone tissue neering have suggested that like structures(as indicated by black arrows)were generated in biomimetic scaffolds based-stem cells regenerative therapy provides a defects in the BMP-2 and Dual groups. The conspicuous dif- promising strategy for treating bone defects. However, the performance between these two groups was that more red blood cells had features of both a scaffold and stem cells have far-reaching effects on infiltrated in the Dual Group at 4 and 8 weeks, with higher vascular bone regeneration outcomes. Therefore, the developed scaffold systems density of 1.56+ 0.59% and 2.53+ 0.81% respectively (see SI- should be continuously optimized and new applications of stem cells Fig. 4). In the blank group, there was only a thin-layer of connective should be explored in tissue engineering. The aim of this study was to tissue had formed in defect area. Meanwhile, a considerable amount of determine the osteoinductive capacity of SHED supported by PDA- collagenic fibrils dep wathet blue) could be observed in the modified PLLA NF-Ms in SHED group, and small fragmented tissues were scattered among col- 2, which was slowly released from Hep-Dopa conjugated NF-Ms, in gen fibers as pointed out with black arrows (Massons trichrome promoting bone regeneration. aining in Fig. 8D). Furthermore, new vessels dispersed near by the Mussel-inspired PDA-coating is readily available via a simple pro- SHED-delivered microspheres(marked with red arrows)in the SHED cedure of dopamine (DA)self-polymerization in situ, which has been

( * P < 0.05). Although the amount of bone formation in the Dual group was higher than BMP-2 group, no statistically significant difference between these two group. However, both of these two groups were presented potent osteoinductive ability than other groups (* P < 0.05 or **P < 0.01). 3.6.2. Histological observations by H&E and Masson’s trichrome staining The sagittal sections of calvarial bone were further stained with H& E and Masson’s for histological analysis. High-magnification images (200×) were taken to examine vascularization or osteogenesis in all groups. As shown in Fig. 9, a large amount of bone matrix and bone￾marrow like structures (as indicated by black arrows) were generated in calvarial defects in the BMP-2 and Dual groups. The conspicuous dif￾ferences between these two groups was that more red blood cells had infiltrated in the Dual Group at 4 and 8 weeks, with higher vascular density of 1.56 ± 0.59% and 2.53 ± 0.81% respectively (see SI￾Fig. 4). In the blank group, there was only a thin-layer of connective tissue had formed in defect area. Meanwhile, a considerable amount of collagenic fibrils deposition (wathet blue) could be observed in the SHED group, and small fragmented tissues were scattered among col￾lagen fibers as pointed out with black arrows (Masson’s trichrome staining in Fig. 8D). Furthermore, new vessels dispersed near by the SHED-delivered microspheres (marked with red arrows) in the SHED group. For both 4 and 8 weeks, the new vessels density in SHED group (4 week: 1.59 ± 0.72%; 8 week: 3.48 ± 1.28%) is the highest among all groups (P < 0.05). Thus, all above results indicated that SHED/PDA NF-Ms had the potential to induce the angiogenesis and osteogenesis in an orthotopic bone damage environment, and its combination with sustained release of rhBMP-2 by Hep-Dopa NF-Ms contributed to a stronger effect on bone tissue regeneration. 4. Discussion Recent advances in bone tissue engineering have suggested that biomimetic scaffolds based-stem cells regenerative therapy provides a promising strategy for treating bone defects. However, the performance features of both a scaffold and stem cells have far-reaching effects on bone regeneration outcomes. Therefore, the developed scaffold systems should be continuously optimized and new applications of stem cells should be explored in tissue engineering. The aim of this study was to determine the osteoinductive capacity of SHED supported by PDA￾modified PLLA NF-Ms in vivo, and their potential synergy with rhBMP- 2, which was slowly released from Hep-Dopa conjugated NF-Ms, in promoting bone regeneration. Mussel-inspired PDA-coating is readily available via a simple pro￾cedure of dopamine (DA) self-polymerization in situ, which has been A NF-Ms PDA-NF-Ms C 10ȝm 3 Days 7 Days 10ȝm B 10ȝm 20ȝm 20ȝm 20ȝm 10ȝm Live/Dead Phalloidin/Nuclei NF-Ms PDA-NF-Ms a b Fig. 4. (A) SEM images: The morphology of cells 3 and 7 days after seeding on NF-Ms and PDA-NF-Ms on 3 days and 7 days. Right panel: high-magnification images of SHEDs adhered on NF-Ms or PDA-NF-Ms for the corresponding white column at different time points (the location of SHED is indicated by red arrows; the ECM is pointed out by yellow arrows). (B) (a) Cell viability staining was performed with calcein AM/PI at 7 days (green representes live cells, red representes dead cells); (b) Observation of cytoskeleton stained with phalloidine (red fluorescence) and nuclei stained with DAPI (blue) of SHEDs on PDA-NF-Ms on 7 days. (C) MTT assay for the SHED proliferation on NF-Ms or PDA-NF-Ms on 3 and 7 days. Statistically analysis by using one-way analysis of variance (ANOVA) (* P < 0.05, **P < 0.01). (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) 573–586 581

T. Fang, et al. Chemical Engineering Journal 370(2019)573-586 Blank BMP-2 BMP-2/HD NF-Ms B a BMP-2/HD NF-Ms 7Days 14 Days 14 Days BMP-2/HD NF-\s >⌒ 21 Days E BMP-2/HD NF-Ms □BMP2/HDNF-Ms sss 3 14 Days Fig. 5. The osteogenesis effects of 100 ng/ml soluble rhBMP-2(BMP-2 group)and rhBMP-2 released from Hep-Dopa NF-Ms(BMP-2/HD NF-Ms)on BMSCs in vitro. (A) The ALP staining and(B)quantitative analysis of relative alP expression on 7 days and 14 days.(C)Alizarin Red s(ARS)staining and (D)qu the 21st day.(E) Real-time PCR analysis of osteogenic-related gene ALP, Runx2 and Coll expression of BMSCs in different groups after induced with rhBMP-2 at 14 days. Statistically analysis by using one-way analysis of variance(ANOVA)(P 0.05, P 0.01).( For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) DAPI GFP-SHED Merge 6. The survival of sheds in the constructs fter implantation in vivo at different time B cryosections collected at week 1 (A-C), week 4 7Days plantation. The GFP-expressing SHED(green; B were observed in ectopic tissue grafts at l and 4 weeks, but undetectable at 8 weeks (H). Scale bars (A-D): 100 um. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this D F 4 Week G 8 Weeks

21 Days C A 7 Days 14 Days Blank BMP-2 BMP-2/HD NF-Ms B D E Fig. 5. The osteogenesis effects of 100 ng/ml soluble rhBMP-2 (BMP-2 group) and rhBMP-2 released from Hep-Dopa NF-Ms (BMP-2/HD NF-Ms) on BMSCs in vitro. (A) The ALP staining and (B) quantitative analysis of relative ALP expression on 7 days and 14 days. (C) Alizarin Red S (ARS) staining and (D) quantitative results at the 21st day. (E) Real-time PCR analysis of osteogenic-related gene ALP, Runx2 and Col1 expression of BMSCs in different groups after induced with rhBMP-2 at 14 days. Statistically analysis by using one-way analysis of variance (ANOVA) (* P < 0.05, **P < 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) DAPI GFP-SHED Merge 7 Days 4 Weeks 8 Weeks ABC D E F GHI 100ȝm Fig. 6. The survival of SHEDs in the constructs after implantation in vivo at different time points. Fluorescence detection was performed in cryo-sections collected at week 1 (A-C), week 4 (D-F), and week 8 (G-I) after subcutaneous im￾plantation. The GFP-expressing SHED (green; B, E) were observed in ectopic tissue grafts at 1 and 4 weeks, but undetectable at 8 weeks (H). Scale bars (A-I): 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this ar￾ticle.) T. Fang, et al. Chemical Engineering Journal 370 (2019) 573–586 582