Pearl Powder Hybrid Bioactive Scaffolds from Microfluidic 3D Printing for Bone Regeneration

Pearl Powder Hybrid Bioactive Scaffolds from Microfluidic 3D Printing for Bone Regeneration

  一种负载血管内皮生长因子(VEGF)的新型珍珠粉(PP)混合鱼明胶甲基丙烯酸明胶(GelMa)水凝胶支架,用于骨再生。

Introduction

  创伤、感染、肿瘤、骨髓炎等引起的骨缺损是骨科常见病,尤其是大面积骨缺损,缺损面积超过自体修复的临界状态,在机体刺激下不能自愈,可能导致骨吸收、骨不连和骨延迟愈合,为促进骨再生,往往需要进行骨科手术治疗。骨科手术中最常用的方法是骨移植,包括自体骨移植和异体骨移植。然而,骨移植仍存在骨源短缺、潜在免疫排斥和病毒传播的风险。 作为替代方案,基于不同材料的组织工程支架已被广泛用于骨再生,如羟基磷灰石(HAP)、生物活性玻璃(BS)、藻酸钠(ALG)和甲基丙烯酸明胶(GelMa)。虽然这些支架能有效支持细胞迁移,并在骨修复方面表现出良好的骨传导性,但其生物活性通常有限,而且其成分与天然骨组织有很大差异。此外,与骨组织结合不良会导致关节失效,降低治疗效果。因此,目前仍需一种生物活性骨组织模拟支架,既能表现出令人满意的生物活性,又能与骨组织完美结合,以满足骨再生的需求。

  在此,作者受天然矿化物成分(通常与骨组织成分相似)的启发,通过微流控辅助三维打印技术,提出了一种用于骨再生的生物活性珍珠粉(PP)支架,如图1所示。珍珠粉是从珍珠中提炼出来的一种著名中药。与陆生动物来源的猪皮GelMa相比,水生来源的鱼皮GelMa可广泛获得且价格低廉,同时还具有独特的功能特性。微流控技术具有广泛的流体兼容性,可精确控制流体成分和流速,因此是一种应用广泛的微纤维制造技术,已被用于药物释放、细胞培养和生物传感器等领域。然而,在微流控辅助三维打印骨工程支架中,聚丙烯和鱼皮凝胶马作为生物墨水的组合仍有待探索,它们在骨再生中的生物活性仍有待研究。此外,骨愈合骨愈合是一个复杂而动态的生物过程,涉及早期的血管生成和后期的骨生成。在骨修复早期补充血管生成物质,如血管内皮生长因子(VEGF,以血管生成著称),可有效促进血管重建,改善氧气和营养物质的运输,从而招募内源性干细胞重建骨组织。 因此,将血管内皮生长因子的血管生成作用和聚丙烯的成骨作用结合起来,开发一种能有效促进骨修复的新型支架是非常可取的。

  本文采用微流控三维打印技术,开发了负载血管内皮生长因子的聚丙烯混合水凝胶支架,用于骨再生(图1)。在这一过程中,作者使用PP、鱼皮GelMa和血管内皮生长因子的混合物作为生物墨水,并通过微流控辅助打印法和原位光聚合法实现支架的制作。微流控三维打印的高可控性赋予了支架可调的形状,以满足不同的应用目的;原位光聚合很好地保留了支架结构,实现了生物活性添加剂的封装,并保持了其生物活性。由于聚丙烯和鱼皮GelMa的固有特性,所制备的混合生物活性支架具有良好的生物相容性、细胞粘附性和成骨分化性。PP支架的负载和可控VEGF释放可促进受损区域的血管重建,从而改善骨修复早期的氧气和营养物质运输。随后,随着PP支架在骨修复后期的降解,PP支架释放的成骨物质可有效促进内源性干细胞分化为成骨细胞,从而促进骨修复。综上所述,这种复合支架既具有聚丙烯的成骨作用,又具有血管内皮生长因子的血管生成作用,这一点在体内骨再生模型中得到了进一步证实。这些特点表明,负载了血管内皮生长因子的聚丙烯混合生物活性支架在大面积骨缺损中具有适应性,可作为临床骨再生的理想候选材料。

  • (a)PP混合生物活性支架的组成和微流控三维打印技术。

  • (b)PP混合生物活性支架在骨再生中的应用。

Characterization of the Scaffold

  • (a)PP混合支架的照片。

  • (b)显示其堆叠结构的支架显微图像。

  • (c)独立脱水的PP混合支架的照片。

  • (d-f)PP支架(d)、纤维堆叠(e)和PP支架表面(f)的扫描电镜图像。显示了PP支架的多孔表面,这有利于养分和氧气的交换以及生物活性添加剂的释放。

  • (g-i)能量色散光谱分析显示PP支架中的(g)钙、(h)镁和(i)铁元素。支架中含有来自PP的微量元素钙、镁和铁,这表明聚丙烯成骨成分的成功负载。

In Vitro Biocompatibility and Angiogenesis of the Scaffold

  • (a)PBS中加载FITC-BSA的GelMa-PP支架0至8 d的荧光图像。具体而言,选择FITC-BSA作为模型药物,将其封装到支架中,然后将支架浸入PBS溶液中,观察支架的荧光情况。

  • (b)支架的荧光强度量化。光强度持续降低表明药物缓慢释放。

  • (c)与不同组共孵育3 d后骨髓间充质干细胞(BMSCs)的钙黄绿素染色。

  • (d)培养6 h后不同组HUVEC的管形成。与对照组相比,经过3 d的培养,支架组的骨髓间充质干细胞和成骨细胞MC3T3-E1都表现出理想的细胞密度和良好的形态。

  • (e)第1 d至第3 d不同组BMSCs细胞活性的统计分析。可能是因为PP中的微量元素,如Mg和Ca能促进BMSCs细胞增殖。

  • (f)共培养6 h后不同组成管的统计分析。结果表明,负载VEGF的PP混合支架表现出良好的细胞相容性,并且可以促进血管生成,这将有利于进一步的骨再生。

In Vitro Osteogenic Activity and Cell Migration Induction of the Scaffold

  • (a)诱导2 w后BMSCs的碱性磷酸酶(ALP)染色,用来表示早期骨形成。

  • (b)诱导2 w后BMSCs的茜素红(AR)染色,用来表示成骨诱导过程中较晚的钙沉积。

  • (c)划痕试验,处理24 h后不同组BMSCs的荧光图像。

  • (d-f)ALP活性、矿化度和细胞迁移面积的定量分析。结果表明,含PP的组(包括 GelMa-PP 和 GelMa-PP&VEGF 组)的ALP活性显著提高。此外,AR 染色结果表明诱导2 w后所有组中都有钙沉积。划痕试验也表明GelMa-PP组和GelMa-PP&VEGF组的细胞迁移明显加速。

In Vivo Bone Regeneration Ability

  • (a)治疗8 w后大鼠头骨的代表性三维重建显微CT图像。与对照组相比,GelMa组、GelMa-PP组和GelMa-PP&VEGF组均观察到骨组织再生。

  • (b)治疗8 w后不同组缺损区域的代表性H&E图像。其中,GelMa-PP&VEGF组的骨再生效果最好,这与显微CT的结果一致,表明它们在促进骨修复方面具有更强的能力。

  • (c-d)对缺损部位新形成骨组织的骨体积与组织体积比BV/TV(c)和骨矿物质密度BMD(d)进行定量分析。与其他组相比,GelMa-PP&VEGF组的骨再生速度更快。

In Vivo Osteogenesis and Angiogenesis Capabilities

  • (a)DAPI、骨生成素(OCN)、骨桥蛋白(OPN)及合并通道的免疫荧光染色。与其他组相比,GelMa-PP组和GelMa-PP&VEGF组的OPN和OCN阳性蛋白表达上调。GelMa-PP&VEGF组的阳性面积百分比明显高于其他组,表明其骨修复性能最佳。

  • (b)缺损区域血小板内皮细胞粘附分子-1(CD31)的免疫组化染色。GelMa-PP&VEGF组比GelMa-PP组和GelMa组有更多的血管,这证明PP混合生物活性支架释放的VEGF能有效促进血管再通,进一步促进骨再生。

  • (c-d)OCN(c) 和OPN(d)阳性面积百分比的定量分析。

  • (e)不同组骨缺损区域血管数量统计结果。以上结果表明,负载VEGF的PP混合生物活性支架可通过成骨和血管生成的协同效应有效促进骨再生。

Conclusion

  • 提出了一种新型的微流控三维打印PP混合生物活性支架,可用于骨再生。
  • 这种由PP、鱼皮GelMa和血管内皮生长因子组成的支架可通过可定制的微流控打印技术获得。得益于温和而快速的光聚合反应,VEGF被成功载入支架并保持了较高的生物活性。
  • PP的封装为支架提供了骨增强因子、钙、镁和铁;鱼皮GelMa基质提供了良好的细胞粘附能力;VEGF的负载使支架能够促进血管生成。所有这些都使混合支架能有效上调ALP、OCN/OPN的表达,诱导细胞外基质矿化,从而促进干细胞在体外和体内的成骨分化。

Reference

Yang L, Fan L, Lin X, et al. Pearl Powder Hybrid Bioactive Scaffolds from Microfluidic 3D Printing for Bone Regeneration[J]. Advanced Science, 2023, 2304190.

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