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