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 modifications on-demand to injectable NFMs. • Bio-inspired hydrophilic modification by PDA supported the cellular behavior. • Hep-Dopa conjugation improved performance of NF-Ms in loading and delivering rhBMP-2. • SHED engraftment contributes to angiogenesis in ectopic and orthotopic sites. • Conjunctive use of SHED and rhBMP-2 led to vascularized bone tissue regeneration. 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 engraftment showed angiogenesis activity. But, no effect of SHED/PDA-NF-Ms on osteogenesis was found in ectopic implantation, which is different from the result in cranial defect. The rhBMP-2 released from Hep-Dopa NFMs 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 conditions. 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 recently 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 prominent [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 antiapoptosis 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 biodegradability 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 scaffolds, 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 extracellular matrix (ECM)-like structure of nanofibers, injectable microspheres, 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 microspheres 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 endogenous stem cells to migrate onto the scaffold [14]. At present, dopaminergic surface modification has been widely applied to improve the hydrophilicity of materials, providing a beneficial microenvironment 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 polydopamine (PDA) could improve its biological properties to act as an appropriate micro-carrier for SHED. As an extensively studied recombinant growth factor, bone morphogenetic 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 spatiotemporal 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 microspheres 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 separation 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 dissolution. 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 allowed 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 microscope (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 hydrophilicity 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-labelled 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 HepDopa 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 microspheres 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-NFMs (10:1 ratio) and co-cultured for at least 4 h to promote cell attachment. 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 cocultured 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 previously 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 permeabilized 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 TRITCPhalloidin (Life Technologies, OR, USA) and 4′,6-diamidino-2-phenylindole (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 microspheres 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 sulfoxide (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 comparing 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 incubating 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 containing 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 osteogenic-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 microspheres and their corresponding composite scaffolds (loading SHED or rhBMP-2) in vivo was determined in ectopic subcutaneous engraftment 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 international 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 abbreviations 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 further 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 cryosections. The frozen sections were counterstained with DAPI and further 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 promote 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 software, 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/HepDopa 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 microspheres 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