ELSEVIER Materials and Design 201 (2021) 109484 Contents lists available at ScienceDirect Materials and Design journal homepage: www.elsevier.com/locate/matdes Physical and biological engineering of polymer scaffolds to potentiate repair of spinal cord injury a,1 Yiqian Luo ª‚¹, Fei Xue 1, Kai Liud, Baoqin Li ª, Changfeng Fu ª,*, Jianxun Dingb a Department of Spine Surgery, The First Hospital of Jilin University, 1 Xinmin Street, Changchun 130021, PR China b Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, PR China c School of Chemistry and Life Science, Advanced Institute of Materials Science, Changchun University of Technology, 2055 Yan'an Street, Changchun 130021, PR China Department of Hand and Foot Surgery, The First Hospital of Jilin University, 1 Xinmin Street, Changchun 130021, PR China d Check for updates GRAPHICAL ABSTRACT • HIGHLIGHTS Polymer scaffolds possess enormous po- tential in the repair of spinal cord injury. • The optimized physical properties of scaffolds provide a favorable microenvi- ronment for spinal cord injury repair. • The biologically functionalized scaffolds reverse the adverse factors during spinal cord injury repair. FN Physical support Mechanical performance Internal structure Biodegradation Conductivity Bioelectrical signal conduction ✓ Physical functions - Polymer scaffold Biological functions- Endogenous neurogenesis NSC differentiation Axon growth Antioxidant M1| Macrophage polarization M2 MO Microenvironment regulation materials DESIGN ARTICLE INFO Article history: Received 19 November 2020 Received in revised form 2 January 2021 Accepted 12 January 2021 Available online 16 January 2021 Keywords: Polymer scaffold Spinal cord injury repair Physical support Signal transduction Microenvironment regulation Endogenous neurogenesis ABSTRACT Although the mortality rates of patients suffering from spinal cord injury (SCI) have decreased as the modalities of clinical therapy have been improved, the recovery of motor and sensory functions remains a challenge, ulti- mately leading to paraplegia or quadriplegia. Recently, neural tissue engineering scaffolds with appropriate phys- ical and biological functions have been extensively developed to promote nerve regeneration and improve motor and sensory functions during SCI therapy. In this work, we summarized the physical support and bioelectrical sig- nal conduction of polymer scaffolds for SCI repair from the aspects of biocompatibility, biodegradation, internal structure, mechanical performance, and conductivity. In addition, the biological functions of the polymer scaf- folds were reviewed for the reversal of adverse pathophysiological factors to improve the microenvironments of the injured site and promote endogenous neurogenesis during SCI therapy. Moreover, the future development of these engineered scaffolds for potential clinical applications was predicted. © 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents 1. Introduction. 1.1. Overview of spinal cord injury * Corresponding author. E-mail address: fucf@jlu.edu.cn (C. Fu). 1 Y. Luo and F. Xue contributed equally to this work. https://doi.org/10.1016/j.matdes.2021.109484 0264-1275/Ⓒ 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2 2 materialstoday Y. Luo, F. Xue, K. Liu et al. 1.2. 1.3. 1.4. 2. 3. 4. 5. Microstructure of spinal cord. Pathophysiology of spinal cord injury Treatment strategies for spinal cord injury. Physical support of scaffolds for spinal cord injury repair. 2.1. Biocompatibility and biodegradability. 2.2. Internal structure. 2.3. Mechanical performance. Bioelectrical signal conduction of scaffolds for spinal cord injury repair Microenvironment regulation by scaffolds for spinal cord injury repair 4.1. Antioxidants 4.2. Regulation of macrophages. Endogenous neurogenesis induced by scaffolds for spinal cord injury repair 5.1. Promoting axon growth 5.2. Tuning endogenous neural stem cells 6. Concluding remarks and future perspectives Declaration of Competing Interest Acknowledgments References. Materials and Design 201 (2021) 109484 2 2 3 4 4 4 6 6 8 8 10 10 11 13 13 15 15 15 1. Introduction 1.1. Overview of spinal cord injury Spinal cord injury (SCI) can interrupt the connection between the brain and peripheral organs, leading to dysfunction, such as paraplegia or quadriplegia [1]. There are various causes of SCI, including traffic ac- cidents, industrial accidents, sports injuries, and so forth. Particularly, traffic accidents account for 45.4% of all SCI cases, falling accidents for 27.4%, heavy objects falling for 10.8%, and tumble for 6.5% [2]. More than 20 million patients are currently suffering from SCI worldwide, with an increase of approximately 700,000 people per year [3]. Such a difficult situation has brought considerable burden to the society, and the clinical therapeutics do not achieve satisfactory results. Currently, neural tissue engineering strategies are the most promising modality among the numerous treatment approaches being explored. In this review, we described the microstructure of the spinal cord, pathophysiology of SCI, clinical therapy strategies, and advantages of neural tissue engineering. After that, we summarized the biocompatibil- ity and biodegradability of polymer scaffolds and highlighted the signif- icance of internal structure, mechanical performance, and conductivity for SCI repair. In addition, we reviewed the past research results to show the development and advantages of neural tissue engineering scaffold-mediated SCI therapy through microenvironment regulation and promotion of endogenous neurogenesis (Fig. 1 and Table 1). This work provided a comprehensive review of the theoretical basis and ap- plication of neural tissue engineering scaffolds for SCI repair. 1.2. Microstructure of spinal cord The spinal cord is an essential part of the central nervous system. Its primary function is to conduct nerve signal transduction and process low-level non-conditioned reflexes [4]. In particular, the anatomical mi- crostructure of the spinal cord provides the basis for the orderly mainte- nance of physiological functions. From the cross-sectional view of the spinal cord, the center of the spinal cord is the longitudinal central canal that connects with the cerebral ventricle. The butterfly-shaped gray matter surrounds central canal, and the outer layer of the gray mat- ter is the white matter [5,6]. A small amount of cerebrospinal fluid in the central canal protects and supplies nutrients to the brain and spinal cord. The gray matter is composed of neurons, glial cells, and blood vessels. The aggregation of neurons forms a complex neural circuit called the processing center for low-level non-conditioned reflexes, such as myotatic, flexion, uri- nation, and defecation reflexes [7,8]. The white matter is composed of nerve fibers and fibrous astrocytes. Nerve fibers are longitudinally arranged into bundles, which are divided into ascending conduction bundles and descending conduction bundles. They transmit signals between the brain and peripheral organs. In brief, ascending conduc- tion bundles transmit the information that controls the sensation of the body, whereas descending conduction bundles mainly transmit motor information from the brain to skeletal muscles of the trunk and limbs [9,10]. 1.3. Pathophysiology of spinal cord injury The pathophysiological process of traumatic SCI can be classified into mechanical (primary) and secondary injuries [11,12]. Mechanical damages directly destroy specific structures of the spinal cord, such as gray matter, vascular system, blood-spinal cord barrier, and nerve con- duction bundles [13]. During a secondary injury, necrotic debris of the myelin sheath, aggregation of various active inflammatory cells, such Physical functions Mechanical Physical support Internal structure 2 performance Biodegradation FN NSC differentiation Endogenous neurogenesis Biological functions Axon growth M1 Polymer scaffold Conductivity Signal transduction MO M2 Antioxidant acrophage arization Microenvironment regulation Fig. 1. Physical and biological functionalities of polymer scaffolds for promotion of SCI repair. Y. Luo, F. Xue, K. Liu et al. Table 1 Preparations and functions of polymer scaffolds. Materials and Design 201 (2021) 109484 Material Carrying substance HA CNTF HA/MA NPCs CS/MA IFN-y and NSCs CS/Col Col CBD-PlexinB1-LBD Agarose Matrigel CBD-EphA4-LBD and Template method PEG Gel/PLLA Gel/MA NSLCs iNSCs PCL SCs and iNSCs PLLA/Poloxamer CAB HEMA/MA Heparin/Col PEDOT/Agarose РРУ/ТА NPCs and bFGF Electrospinning Chemical crosslinking Free radical cryopolymerization Electrodeposition Chemical crosslinking Fabrication technology Biological function Reference Mesh filtration Activate NSCs, and facilitate their migration to the lesion area and differentiation into mature neurons [37] Photocrosslinking Promot the differentiation of NPC into neurons [38] Click chemistry and UV crosslinking 3D printing Neurofilament fibers extend from the host tissue to the scaffold Decrease the formation of scar and cavity, and improve the regeneration of nerve fibers [39] [41] Chemical crosslinking Facilitate axonal regrowth and remyelination of the regenerated tissue [43] Enhance linearly organized axon regeneration, and guide the reconnection of [45] functional axons Water/oil emulsion method, free radical polymerization Inhibit the growth of glial scars, and provide an aligned structure to direct axonal regeneration [46] Co-electrospun Photocrosslinking Support the proliferation and differentiation of NSLSC into motor neurons Decrease inflammation and cavity formation, facilitate axonal and neuronal regeneration, and inhibit glial scar hyperplasia [47] [48] Increase tissue remodeling, and secrete neurotrophic factor Reduce fibrotic scarring, and boost nerve regeneration [52] [53] Promote the orientational regeneration of neurons, and bridge the spinal cord [55] stumps in the conduit Promote axonal growth [56] PANI/CS/Gel PCL/Melatonin OE-MSCs SCs Activate endogenous NSCs neurogenesis in the lesion area Promote the differentiation of OE-MSCs into motor neurons [57] [60] 3D multilayer molding Reduce post-traumatic inflammation and oxidative stress, and regulate impaired mitochondrial function [120] HA/MnO2 NP MSCs Improve the stem cell adhesive growth and nerve tissue bridging, and alleviate the oxidative microenvironments [121] Silk fibroin AECs Inhibit local inflammation, promote myelin regeneration, and enhance nerve fiber regeneration [123] PHEMA Melt casting Promote the polarization of macrophages to M2 phenotype, reduce fibrosis, and promote angiogenesis [129] PCL IL-10 Electrospinning Induce macrophage polarization toward the M2 activated state [132] PLGA Oil-in-water single emulsion solvent Induce macrophages to polarize to M2 phenotype, reduce scar formation, and [136] Fibrin HA evaporation Electrospinning and concurrent molecular self-assembling Enzyme crosslinking CS NT-3 and NSCs promote axonal regeneration Guide axon regeneration directionally, and promote directional invasion of host cells and reconstruction of vascular system [142] Promote axonal regeneration and protruding connection Support the growth and reproduction of NSCs, and promote the differentiation [162] of NSCs into neurons [144] Abbreviations: 3D, three-dimensional; AECs, amniotic epithelial cells; bFGF, basic fibroblast growth factor; CNTF, ciliary neurotrophic factor; Col, collagen; CS, chitosan; Gel, gelatin; HA, hyaluronic acid; IFN-y, interferon-y; IL-10, interleukin-10; iNSCs, induced pluripotent stem cells-derived neural stem cells; MA, methacrylic anhydride; MP, methylprednisolone; NPCs, neural precursor cells; NSCs, neural stem cells; NSLCs, neural stem-like cells; NT-3, neurotrophin-3; OE-MSCs, olfactory ecto-mesenchymal stem cells; PCL, poly(&-caprolactone); PEG, poly(ethylene glycol); PEGDA, poly(ethylene glycol) diacrylate; PHEMA, poly(2-hydroxyethylmethacrylate); PLGA, poly(lactic-co-glycolic acid); PLLA, poly(L-lactic acid); SCs, Schwann cells; TA, tannic acid; UV, ultraviolet. as granulocytes and macrophages, and active glial cell populations can be observed in the harsh local microenvironments [14]. According to the time after injury and pathological mechanism, the secondary injury can be divided into three stages: acute stage, subacute stage, and chronic stage [15]. The acute phase is defined as the 48 h period after SCI. The main pathological changes were bleeding due to vascular injury, resulting in tissue edema and ischemia. With the de- struction of microcirculation, the subsequent pathological changes, such as ion imbalance, excitatory toxins, excessive production of free radicals, and inflammatory reactions, lead to further damage of neurons and glial cells. The subacute phase is from 48 h to two weeks after in- jury. The main pathological processes include phagocytosis of the in- flammatory corpuscle and reactive proliferation of astrocytes [16]. In particular, phagocytosis is the characteristic of this stage. The reactive proliferation of astrocytes leads to glial scarring, which is a crucial barrier to axonal regeneration [17]. The definition of chronic stage is generally considered to be six months or more after SCI. The main path- ological features of chronic stage include the formation and develop- ment of scars [18,19]. The use of various therapy measures, such as regulating physical properties and endowing biological functions, to en- hance the regeneration of damaged axons and myelin sheath, is the pur- pose of treatment at this stage, while preventing the formation of scars is the current promising research direction. 1.4. Treatment strategies for spinal cord injury Clinically, SCI is usually accompanied by destruction of the vertebral column. Therefore, surgical therapy is an essential modality for SCI treatment. Surgical operations can reestablish the stability of the spine to relieve compression of the spinal cord, which creates an appropriate external microenvironment for recovery of the spinal cord. However, the approaches of surgery cannot provide further driving forces for SCI recovery [12,20]. In addition, non-surgical procedures, such as drugs, hyperbaric oxygen, and hypothermia, have shown specific efficacy in the treatment of SCI, but they have apparent shortcomings [21]. For ex- ample, the U.S. Food and Drug Administration has approved methyl- prednisolone (MP) for the treatment of SCI. MP can inhibit secondary injury by stabilizing cell membranes, inhibiting the release of endor- phins, and limiting inflammation. However, when used in high doses, it can also cause side effects, including abnormal autonomic reflexes, immunosuppression, deep vein thrombosis, and pulmonary embolism [22,23]. Furthermore, there has recently been a growing suspicion about the role of MP in promoting the recovery of nerve function during SCI therapy [24,25]. Both hypothermia and hyperbaric oxygen are aux- iliary therapy measures with limited efficacies and complicated pro- cesses [26,27]. Therefore, an effective strategy is urgently required for SCI therapy. 3 Y. Luo, F. Xue, K. Liu et al. Thus far, studies on SCI treatment have mainly focused on stem cell therapy, drug screening, gene therapy, tissue engineering, and their bi- ological mechanisms [28,29]. Particularly, tissue engineering has irre- placeable advantages in SCI therapy. The wound can be bridged, the formation of nerve scars can be inhibited, and axons at both ends of the injury can be physically guided to reconnect by tissue engineering scaffolds. Moreover, to improve the repair effect of injured spinal cord, tissue engineering scaffolds can be used as carriers of bioactive compo- nents or cells to form biofunctional scaffolds to inhibit secondary dam- age or provide the driving force for nerve regeneration [30,31]. As a result of advances in materials science and bioscience, re- searchers have developed various unique insights into the application of tissue engineering scaffolds for SCI repair and have predicted their clinical applications [32]. Several studies have confirmed the advan- tages of neural tissue engineering scaffolds for SCI therapy [33,34]. The internal structures of scaffolds not only provide adequate space for cell survival and material exchange in the nervous system, but they also offer physical guidance for axon growth to some extent. The mechanical performance of the scaffold can compensate for the lack of mechanical support caused by SCI. The conductivity of the scaffold can simulate the electrophysiological microenvironments of the nerve tissue and transmit endogenous bioelectrical signals [35]. Furthermore, the bio- logical function of the scaffold can specifically overcome adverse pathophysiological changes to regulate the microenvironments and en- dogenous neural stem cells (NSCs), promoting SCI repair. 2. Physical support of scaffolds for spinal cord injury repair Tissue engineering scaffolds provide a favorable growth microenvi- ronment and an instructional cue for nerve regeneration during SCI re- pair, which, to some extent, depends on the physical support of scaffolds [36]. Physical support suitable for SCI is a prerequisite of nerve repair, mainly depending on utilized materials and techniques during the prep- aration. Currently, various natural and synthetic polymers have been used in scaffold manufacturing. Natural polymers, including hyaluronic acid (HA) [37,38], chitosan (CS) [39-42], collagen (Col) [43,44], agarose [45], alginate [46], gelatin (Gel) [47,48], and acellular matrix [49], have been used to research nerve regeneration in SCI. Synthetic polymers in- clude poly(lactic-co-glycolic acid) (PLGA) [50,51], poly(ε-caprolactone) (PCL) [52], poly(lactic acid) (PLA) [47,53], poly(ethylene glycol) (PEG) [42,54], hydroxypropyl methacrylamide (HPMA) [55], poly(sialic acid) (PSA), methacrylate (MA) [48], poly(3,4-ethylene dioxythiophene) (PEDOT) [56], polypyrrole (PPy) [57–59], and polyaniline (PANI) [60]. The properties and applications of polymers have been discussed in de- tail in our previous review [61]. Current applications of polymers often involve combinations of mul- tiple polymers as it is challenging to provide the required physical sup- port for damaged spinal cords when using a single polymer. To prepare customized scaffolds with various forms, structures, and functions, many technologies, including electrospinning and three-dimensional (3D) printing, have been applied for the preparation [62,63]. In addition to biocompatibility and biodegradability, the internal structures, me- chanical properties, and electrical conductivities of scaffolds are also sig- nificant physical properties to ensure the best repair effect of SCI. 2.1. Biocompatibility and biodegradability Biocompatibility refers to the compatibility performance of living tissue in response to non-active materials. Implantation of foreign ma- terials into the body will affect the biological tissue microenvironments, and biological tissues will also affect the implanted materials [64]. The interaction between the implant and biological tissue will keep for a long time until a state of equilibrium is reached, or the implant is removed. Biocompatibility is necessary for the application of tissue en- gineering scaffolds in the medical field and a prerequisite for satisfac- tory repair effects because the physiological activities of cells, such as 4 Materials and Design 201 (2021) 109484 survival, proliferation, migration, and differentiation, are based on this characteristic [65,66]. Notably, during secondary injury following SCI, inflammatory cells accumulate at the site of injury and secrete large quantities of inflammatory factors, such as interleukin-1ẞ (IL-1ẞ), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-α), thus forming an inflammatory microenvironment [1,18]. In this case, scaffolds implanted into the damaged spinal cord need to have better biocompatibility. In order to meet this condition, both natural and synthetic polymers currently used to prepare scaffolds for SCI repair need to be tested for toxicity at the cell and tissue levels. In addition to polymers, the liquid used in the preparation process is also an essential factor affecting the biocompatibility of scaffolds. Toxic organic liquids, such as methylene chloride, trichloromethane, and tetrahydrofuran, are used as solvents or crosslinking agents to prepare scaffolds through electrospinning or 3D printing [67]. Although organic solvents can be removed by natural volatilization, vacuum drying, dialysis, freeze-drying or other methods, it is still quite challenging to remove them thoroughly. Therefore, re- searchers should make a long-term effort to explore strategies that can effectively remove toxic liquids. The biodegradation of scaffolds is another crucial factor in tissue re- pair. The ideal biodegradation rate can provide adaptive growth space and mechanical support for newborn tissues. In general, the perfect biodegradation rate of scaffolds mainly depends on the growth rate of tissues [68]. Currently, there is no specific standard for the degradation rates of scaffolds for SCI repair, with degradation times ranging from weeks to years. In the future, it is of great significance to explore the ac- curate degradation times of scaffolds for the best SCI repair effect. The degradation time of a scaffold can be controlled by using composite ma- terials. To prepare scaffolds with longer degradation times, CS and HA can be mixed to create satisfactory scaffolds. Similarly, mixtures of PLGA and poly(L-lysine) [51], CS CS and Col [41], and Gel and PLA [69] have been used to prepare scaffolds with appropriate degradation rates. Li et al. designed an injectable and biodegradable hybrid hydrogel for application in SCI models. Degradation tests in vivo and in vitro showed that the degradation times of scaffolds were at least two weeks (Fig. 2A, B, and D), and no abnormal inflammatory response was found in tissues around scaffolds (Fig. 2C). Besides, the hydrogel had no significant cytotoxicity in vitro (Fig. 2E and F) [53]. 2.2. Internal structure The internal structure of the scaffold can provide channels for the ex- change of metabolic substances. Additionally, it can guide the regenera- tion of nerve tissue through physical contact [70]. Researchers have attempted to create a scaffold with an internal structure similar to that of the spinal cord to provide a natural path for tissue growth [71,72]. Previously, the acellular matrix of neural tissue was considered as one of the most promising scaffolds because it can retain the major internal structure of nerve tissue. However, its application was signifi- cantly limited, owing to limited supply and immune rejection [73,74]. Many studies consider porosity, aperture, and specific morphology, such as orientation arrangement, to be the most essential factors of the internal structures for SCI repair. Particularly, scaffolds with a poros- ity of 90% and aperture of 10-100 μm are the most suitable scaffolds for cell growth, and the directional arrangement structure can guide the or- derly growth of nerve fiber to the maximum extent [75,76]. Currently, technologies, including freeze-drying, electrospinning [77], and 3D printing [62], are used to fabricate scaffolds with suitable internal struc- tures. Although freeze-drying can control the porosity and aperture of scaffolds, the prepared scaffolds usually have the disadvantages of in- sufficient mechanical integrity and non-orientation. Electrospinning is widely used to prepare oriented scaffolds by forming oriented fibers. However, electrospun scaffolds are mainly used to treat peripheral nerve injury and are rarely used for SCI repair. This may be due to the Y. Luo, F. Xue, K. Liu et al. A Day 1 Day 3 Day 7 Day 14 C Day 1 Materials and Design 201 (2021) 109484 Day 3 0.2 0.4 0.6 0.8 B Day 7 Day 14 Day 1 D 40 Weight loss (%) Day 3 Day 35 55 30 25 25 4°C 37 °C 20 20 15 ヨ Cell viability (%) Day 14 100 80 60 60 40 40 20 NIH-3T3 F Astrocyte Cell viability (%) 50 50 40 40 30 50 20 20 10 10 50 μm 10 2 3 4 Degradation time (week) 0 25 50 100 200 400 600 800 1000 Copolymer concentration (μg mL-1) 0 0 1 2 4 6 8 10 Concentration of CAB (nM) Fig. 2. Biodegradation and biocompatibility of cabazitaxel (CAB)-encapsulated hybrid hydrogel. (A) Representative fluorescence imaging of mice after subcutaneous injection of hydrogel. (B) A representative optical image of in vivo degradation time of mixed hydrogel. (C) Hematoxylin and eosin (H&E) staining was performed on the tissue around the injected hydrogel at a predetermined time point, and results demonstrated that the hydrogel had good biocompatibility. (D) Degradation rate of hydrogels at different temperatures. The degradation rate was faster at 37 °C compared to that at 4 °C. (E) and (F) The cytotoxicity of the polymer and CAB were tested. Reproduced with permission from [53]. A Lesion site Grey matter White matter B Cooing unit Heating unit C Rat spinal cord Top view Side view Growth Scaffold Cells A+B Cells B Cells A Fig. 3. Preparation of 3D printed scaffold with similar structure of spinal cord. (A) Schematic illustration of spinal cord illustrating grey matter and white matter boundaries and a design for a 3D bioprinted multichannel scaffold for modelingspinal cord. (B) Schematic diagram of 3D printing. (C) Exhibition of a transected rat spinal cord and design principle for scaffolds consisting of multiple and continuous channels. Top view and side view image of scaffold. Reproduced with permission from [81]. 5

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ELSEVIER Materials and Design 201 (2021) 109484 Contents lists available at ScienceDirect Materials and Design journal homepage: www.elsevier.com/locate/matdes Physical and biological engineering of polymer scaffolds to potentiate repair of spinal cord injury a,1 Yiqian Luo ª‚¹, Fei Xue 1, Kai Liud, Baoqin Li ª, Chang