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組織工程支架提供有利于細(xì)胞生長(zhǎng)的微環(huán)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(jīng)和脊髓的細(xì)胞外基質(zhì)結(jié)構(gòu)，可以有效引導(dǎo)細(xì)胞軸突和雪旺細(xì)胞定向生長(zhǎng)。該綜述首先介紹了外周神經(jīng)和脊髓的解剖結(jié)構(gòu)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(gè)方面綜述了目前各向異性神經(jīng)/脊髓再生支架的制備和研究進(jìn)展。

01、研究?jī)?nèi)容簡(jiǎn)介

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

外周神經(jīng)損傷(PNI)后，遠(yuǎn)端的軸突脫髓鞘化并且發(fā)生降解，這一過(guò)程也稱(chēng)作Wallerian degeneration (Figure 1B)。之后近段形成Büngner bands促進(jìn)神經(jīng)再生，包括長(zhǎng)度方向取向的雪旺細(xì)胞引導(dǎo)軸突向遠(yuǎn)端生長(zhǎng)，過(guò)程中也伴隨著炎癥反應(yīng)。對(duì)于神經(jīng)元沒(méi)有完全斷裂的損傷，神經(jīng)可以通過(guò)自我修復(fù)完成再生。但對(duì)于大段神經(jīng)損傷，炎癥細(xì)胞、基質(zhì)的纖維化等使得神經(jīng)損傷無(wú)法恢復(fù)。

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(xù)性和非連續(xù)性表面特征，其通過(guò)“接觸導(dǎo)向”作用影響著附著細(xì)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?fàn)钍堑湫偷倪B續(xù)性表面特征，其寬度和深度影響著附著細(xì)胞的取向度。有研究表明，當(dāng)凹槽的寬度和深度接近細(xì)胞尺寸時(shí)，雪旺細(xì)胞能更好的沿著凹槽方向生長(zhǎng)，且上表達(dá)有關(guān)細(xì)胞骨架、髓鞘化等基因。作為非連續(xù)性的分布式柱狀和點(diǎn)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(jīng)元的軸突生長(zhǎng)。

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(yīng)用于組織工程支架。作者總結(jié)了用于制備各向異性外周神經(jīng)和脊髓再生支架的靜電紡絲接收裝置，包括旋轉(zhuǎn)的盤(pán)狀、柱狀、輪狀、桿狀以及平行電極等。為了彌補(bǔ)靜電紡絲支架力學(xué)性能等不足，一些纖維自組裝、剪切力/拉伸力、相分離等技術(shù)也被用于制備各向異性的外周神經(jīng)和脊髓再生支架，作者在文中對(duì)其制備原理進(jìn)行了綜述。各向異性纖維基材料可成膜狀，并進(jìn)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(lái)連接損傷神經(jīng)。雪旺細(xì)胞和軸突能沿著纖維方向生長(zhǎng)和遷移，有效促進(jìn)神經(jīng)的再連接(Figure 3)。通過(guò)結(jié)合不同尺度的各向異性，如納米級(jí)和微米級(jí)，神經(jīng)細(xì)胞的再生可以進(jìn)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(jīng)/脊髓的各向異性水凝膠具有巨大的應(yīng)用潛力，作者總結(jié)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(kuò)散法、磁場(chǎng)/電場(chǎng)，外力作用，以及模具法(Figure 4)。通過(guò)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(guò)“接觸導(dǎo)向”作用，曲向的水凝膠孔或通道可以促進(jìn)膠質(zhì)細(xì)胞和軸突的定向生長(zhǎng)，進(jìn)而加速神經(jīng)再生過(guò)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(zhǎng)錐和粘著斑兩個(gè)角度介紹了細(xì)胞和軸突在各向異性支架中定向生長(zhǎng)的機(jī)理。生長(zhǎng)錐是生長(zhǎng)的軸突前端的感應(yīng)性結(jié)構(gòu)，由肌動(dòng)蛋白纖維和微管組成(Figure 5A)，它們控制著生長(zhǎng)錐的前進(jìn)、后退等行為。束狀的微管在生長(zhǎng)錐的中心部位，外周為肌動(dòng)蛋白構(gòu)成的板狀偽足和絲狀偽足。板狀偽足和絲狀偽足能感應(yīng)、識(shí)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(xì)胞骨架變形帶來(lái)的壓力。微管結(jié)構(gòu)能劇烈的收縮和運(yùn)動(dòng)，進(jìn)一步控制細(xì)胞的運(yùn)動(dòng)。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(xì)胞骨架和基質(zhì)，細(xì)胞可以通過(guò)整合素來(lái)根據(jù)外界機(jī)智改變細(xì)胞形態(tài)。

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