組織工程支架提供有利于細胞生長的微環境，是組織再生中的重要組成部分。各向異性支架模擬了神經和脊髓的細胞外基質結構，可以有效引導細胞軸突和雪旺細胞定向生長。該綜述首先介紹了外周神經和脊髓的解剖結構以及損傷的臨床和潛在治療方法，并從一維的表面特征、二維的纖維基材和三維的水凝膠三個方面綜述了目前各向異性神經/脊髓再生支架的制備和研究進展。

01、研究內容簡介

脊髓損傷(SCI)伴隨著一系列的細胞反應，在損傷發生部位，神經元和膠質細胞死亡，血管發生破裂(Figure 1A)。之后炎癥細胞聚集，膠質疤痕形成以保護損傷部位。隨著炎癥反應降低，脊髓通過軸突再生和新血管形成開始自我修復。但膠質疤痕的細胞外基質，包括硫酸軟骨素和蛋白聚糖阻止了神經元的有效再生。

外周神經損傷(PNI)后，遠端的軸突脫髓鞘化并且發生降解，這一過程也稱作Wallerian degeneration (Figure 1B)。之后近段形成Büngner bands促進神經再生，包括長度方向取向的雪旺細胞引導軸突向遠端生長，過程中也伴隨著炎癥反應。對于神經元沒有完全斷裂的損傷，神經可以通過自我修復完成再生。但對于大段神經損傷，炎癥細胞、基質的纖維化等使得神經損傷無法恢復。

Figure 1. Physiological changes after SCI (A) and PNI (B) at the injury site

各向異性的一維表面包括連續性和非連續性表面特征，其通過“接觸導向”作用影響著附著細胞的行為。制備通常以不可降解的硅、聚苯乙烯、PDMS和可降解的PLA，PCL等為基材，制備方法主要為激光刻蝕、光刻和軟光刻 (Figure 2)。凹槽和脊狀是典型的連續性表面特征，其寬度和深度影響著附著細胞的取向度。有研究表明，當凹槽的寬度和深度接近細胞尺寸時，雪旺細胞能更好的沿著凹槽方向生長，且上表達有關細胞骨架、髓鞘化等基因。作為非連續性的分布式柱狀和點狀特征，其各向異性排列，以及表面粗糙度也影響著DRG 和交感神經元的軸突生長。

Figure 2. A) Schematic of laser scanning. B) Schematic of photolithography. C) Four major soft lithographic techniques.

靜電紡絲纖維基支架較大的比表面積和孔隙率，使其廣泛應用于組織工程支架。作者總結了用于制備各向異性外周神經和脊髓再生支架的靜電紡絲接收裝置，包括旋轉的盤狀、柱狀、輪狀、桿狀以及平行電極等。為了彌補靜電紡絲支架力學性能等不足，一些纖維自組裝、剪切力/拉伸力、相分離等技術也被用于制備各向異性的外周神經和脊髓再生支架，作者在文中對其制備原理進行了綜述。各向異性纖維基材料可成膜狀，并進一步卷成管狀，或直接制備成管狀，以及制備成纖維束作為填充物，來連接損傷神經。雪旺細胞和軸突能沿著纖維方向生長和遷移，有效促進神經的再連接(Figure 3)。通過結合不同尺度的各向異性，如納米級和微米級，神經細胞的再生可以進一步被提高。

Figure 3. A)  i: Pictures of animal experiments. a: Image of the electrospun GelMA hydrogel fiber scaffold. b: 3 mm hemisection made in the right site of the T9 spinal cord. c: Scaffold implantation. d: Spinal cord specimens were collected 12 weeks after surgery. From top to bottom: control group, gelatin group, GelMA group. e–h: Animals at weeks 1,4,8, and12 after surgery. ii: Evaluation of the lower limb motor function of rats with a BBB score. iii–v: Immunofluorescence staining of neural stem cells and neural cells and the quantitative comparison of optical density among groups. Samples without implants were used as a control group. *p < 0.05. B) a–c: 40× fluorescent images of individual neurons seeded on astrocytes, which were cultured on aligned fiber scaffolds (a), PLLA films (b), or fiber/AFFT scaffolds (c). d–f: Isolated traces of individual neurons pictured in a, b, and c, respectively. g–i: Polar histograms showing total outgrowth and orientation of neurites seeded on astrocyte layers, which were cultured on the three scaffold types (g = aligned fibers, h = film, i = AFFT boundary). C) DRG extension (green: ß tubulin) on SAS fibers exhibiting smooth, porous, and grooved topography (i). Grooved fibers demonstrate a significantly longer neurite extension compared to smooth and porous fibers after DIV 7 (ii). Effect of fiber surface topography on DRG surface area aspect ratio after DIV 7 (iii). Neurite extension length from DRGs on fibers after DIV 1, DIV 4, and DIV 7 (iv). Scale bar is 1 mm. The white arrow shows fiber direction.

用于外周神經/脊髓的各向異性水凝膠具有巨大的應用潛力，作者總結了常用的制備方法，詳述了制備原理，其中包括單向冷凍法、3D打印、離子擴散法、磁場/電場，外力作用，以及模具法(Figure 4)。通過控制水凝膠支架的孔徑，可以得到曲向的微觀或宏觀的孔和通道。同樣的通過“接觸導向”作用，曲向的水凝膠孔或通道可以促進膠質細胞和軸突的定向生長，進而加速神經再生過程。

Figure 4. A) SEM characterization of the scaffold. a-f: Transverse section of the scaffold (a, d) with collagen/ chitosan filler (b, e) and PCL sheath (c, f). G–H: Longitudinal section of the collagen/ chitosan filler. i: Pore size distribution of the collagen/ chitosan filler. a, d, g: scale bars?=?1?mm; b, c and e: scale bars?=?50?μm; f: scale bar?=?200?μm; h: scale bar?=?100?μm. B) Scanning electron microscopy images of nerve guidance conduits showing the transverse section (dotted yellow line of the image on the right showing the plane where the samples were cut to acquire longitudinal images. C) Depth color‐coded images of magnetic fibers inside 3D fibrin hydrogels, prepared a: in the absence of an external magnetic field and b: in the presence of a 100 mT magnetic field. D) Multiscale anisotropy of biaxially compressed collagen scaffolds. The top row shows SEM images at the magnification specified in top right corner. The bottom row shows the corresponding polar plot from the image analysis, where the red lines represent the primary fiber axis, and the blue line is the magnitude of alignment at the specified angle.

最后，作者從生長錐和粘著斑兩個角度介紹了細胞和軸突在各向異性支架中定向生長的機理。生長錐是生長的軸突前端的感應性結構，由肌動蛋白纖維和微管組成(Figure 5A)，它們控制著生長錐的前進、后退等行為。束狀的微管在生長錐的中心部位，外周為肌動蛋白構成的板狀偽足和絲狀偽足。板狀偽足和絲狀偽足能感應、識別外界形貌和壓力的變化，并使自身沿著各向異性形貌曲向，以減少細胞骨架變形帶來的壓力。微管結構能劇烈的收縮和運動，進一步控制細胞的運動。粘著斑由大量的整合素組成(Figure 5B)，整合素是一種跨膜蛋白，連接著細胞骨架和基質，細胞可以通過整合素來根據外界機智改變細胞形態。

Figure 5. Schematic of cellular response to ECM by the growth cone (A) and focal adhesion (B)