具有定向孔结构的磷酸钙骨水泥复合支架的构建和性能
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摘要
采用定向冰晶-冷冻干燥法制备具有定向层状大孔结构的磷酸钙骨水泥（CPC）支架，通过灌注将聚乳酸羟基乙酸酯（PLGA）复合到CPC支架的孔壁表面后，获得了具有较高强度和柔韧性的PLGA/CPC复合支架。本文对所制备的PLGA/CPC支架进行了体外降解特性、细胞行为和体内成骨性能的研究，并对支架的组成和结构进行了优化，从而进一步改善其力学性能、细胞反应性和体内成骨性。
显微观察表明，PLGA/CPC支架的孔壁表面覆盖着PLGA膜，在体外降解过程中，PLGA膜首先降解，使骨水泥基体逐渐暴露，从而有利于提高材料与细胞和骨组织的反应。PLGA/CPC支架的定向层状大孔结构有利于细胞的长入和增殖。将PLGA/CPC支架植入兔子的股骨缺损后，新生的骨组织沿着定向的层状大孔长入到支架的内部。由于PLGA/CPC支架表面包覆着PLGA膜，导致细胞反应性较差，植入初期，骨水泥骨传导性好的优点得不到发挥。为了改善PLGA/CPC支架的细胞反应性，本研究在氨气气氛下对PLGA/CPC复合支架进行等离子体表面处理，将胶原固定在PLGA/CPC支架孔壁PLGA膜的表面，改善支架的生物活性，得到Col/PLGA/CPC复合支架。经胶原表面改性后的支架的细胞种植率、粘附、增殖和分化均得到显著的提高。然而，植入兔子体内后发现，胶原改性对提高PLGA/CPC支架的成骨能力帮助并不大。
为了适应一些对支架强度有更高要求的应用，本研究仿天然骨的皮质骨/松质骨双层结构制备了强度可控的核壳结构磷酸钙骨水泥基复合支架。通过设计模具，采用等静压处理的方法制备了致密的管状骨水泥壳层。在致密壳层的空腔内用定向冰晶-冷冻干燥法制备具有定向层状大孔结构的多孔磷酸钙骨水泥芯，并用PLGA进行增强，然后用胶原进行表面改性。外层致密、内层多孔的骨水泥复合支架的强度可以通过改变致密层和多孔层的厚度比例调节（5~90MPa），以满足不同骨缺损部位修复的要求。定向大孔主要在轴向上具有高度的连通性，横向连通性较低。因此，致密的骨水泥外层对支架多孔芯的孔隙连通性没有明显的影响。细胞实验表明，细胞在双层骨水泥复合支架上粘附和增殖良好。
通过复合明胶微球改性PLGA/CPC支架的三维多孔结构。PLGA/CPC支架经过明胶微球（0%~30%）改性后，强度和孔隙率都有所变化，但分别保持在3.5~5MPa和62%~72%之间。经明胶微球改性后，支架的定向层状大孔被分割为尺寸较小的层状大孔。此外，明胶微球溶解后原位留下80~200μm左右的等轴状大孔。用20%明胶微球改性的支架的细胞种植率、增殖和分化改善效果最明显。明胶微球的改性并没有明显影响支架的轴向连通性，细胞仍然可以顺利长入到支架的内部。
将富血小板血浆（PRP）灌注到PLGA/CPC支架内部，制备了PRP-PLGA/CPC复合体。结果表明，PRP的复合大大地改善了PLGA/CPC支架的细胞反应。将PRP-PLGA/CPC复合支架植入到新西兰大白兔的股骨缺损后发现，PRP的复合明显促进了骨组织长入到支架的内部，加快血管的生成和材料的降解。此外，本研究首次将具有定向层状大孔结构的PLGA/CPC支架用于修复新西兰大白兔的桡骨节段性骨缺损。新生的骨组织沿着定向的层状大孔长入支架的内部，而没有受到其三维连通性有限的影响。PRP的复合对PLGA/CPC支架修复桡骨的节段性骨缺损具有显著的促进作用。
A PLGA/CPC scaffold with unidirectional lamellar pore structure was fabricated by theunidirectional freeze casting of CPC slurry and the following infiltration ofpoly(lactic-co-glycolic acid)(PLGA) in this study. In vitro degradation, cell response and invivo osteogenesis of PLGA/CPC scaffold were conducted. Furthermore, the composition,three dimensional (3D) structure of PLGA/CPC scaffold were redesigned to optimize itsmechanical performance, cell response and in vivo osteogenesis.
The pore wall of PLGA/CPC scaffold was covered by PLGA film. The PLGA film firstlydegraded during immersion in PBS solution, which was accompanied with the exposure ofCPC matrix that was beneficial for cell response and osteogenesis. The unidirectional lamellarpore structure of PLGA/CPC scaffold was beneficial for growth of cells into inner macropores,but unfavorable for cell seeding and differentiation. After being implanted in vivo, new bonetissues grew along the unidirectional lamellar pores into the scaffold. The PLGA film on thepore wall of PLGA/CPC scaffold compromises the osteoconductivity of CPC due to poor cellresponse of PLGA. In order to improve cell response of PLGA/CPC scaffold, collagen wasimmobilized on the PLGA film of PLGA/CPC composite scaffold by plasma treatment underthe ammonia atmosphere. After immobilization of collagen, the cell seeding, adhesion,proliferation and differentiation on PLGA/CPC scaffold were significantly enhanced.However, the results of histological evaluation showed that the osteogenesis of PLGA/CPCscaffold was not obviously improved after immobilization of collagen.
To meet the requirements of bone defect repair at various sites, a core/shell bi-layeredCPC-based composite scaffold with adjustable compressive strength, which mimicked thenatural structure of cortical/cancellous bone, was fabricated. The dense tube-like CPC shellwas prepared by designing a mould and isostatic pressing process. A porous core withunidirectional lamellar pore structure was fabricated inside the cavity of dense tube-like CPCshell by aforementioned unidirectional freeze casting, followed by infiltration of PLGA andimmobilization of collagen. The compressive strength of bi-layered CPC-based compositescaffold can be controlled by varying thickness ratio of dense layer to porous layer (590MPa). Compared to the scaffold without dense shell, the pore interconnection of bi-layeredscaffold was not obviously compromised because of its high unidirectional interconnectivitybut poor3D interconnectivity. The cells adhered and proliferated well on the bi-layered CPCbased composite scaffold.
Gelatine microspheres (GM) were used to modify the3D pore structure of PLGA/CPC scaffold. After being modified by various amounts of GM (5%30%), the compressivestrength and porosity of the PLGA/CPC scaffold remained in the range of2.45MPa and62%72%, respectively. The unidirectional lamellar pores were divided into smaller pores.Macropores with the size of80200μm were left being generated by the dissolution of GM.The scaffold modified by20%GM exhibited the most excellent cell seeding, proliferation anddifferentiation. The pore interconnection of scaffold was not starkly compromised aftermodification by GM so that cells still penetrated into the inner pores of scaffolds smoothly.
PRP-PLGA/CPC construct was fabricated by infiltration of platelet-rich plasma (PRP)into PLGA/CPC scaffold. The incorporation of PRP significantly improved cell response ofPLGA/CPC scaffold. The PRP-PLGA/CPC composite scaffold was implanted in femoralbone defects of rabbits, the results showed that PRP boosted markedly bone ingrowth,angiogenesis and degradation of PLGA/CPC scaffold. The PLGA/CPC scaffold withunidirectional lamellar pore structure was originally used to reconstruct the segmental bonedefect in radius of rabbit. The new bone tissues grew well along the unidirectional lamellarpores of scaffold regardless of its poor3D pore interconnection. The incorporation of PRPshowed outstanding improvement in osteogenesis of PLGA/CPC scaffold as implanted in thesegmental bone defect in the radius of rabbit.
显微观察表明，PLGA/CPC支架的孔壁表面覆盖着PLGA膜，在体外降解过程中，PLGA膜首先降解，使骨水泥基体逐渐暴露，从而有利于提高材料与细胞和骨组织的反应。PLGA/CPC支架的定向层状大孔结构有利于细胞的长入和增殖。将PLGA/CPC支架植入兔子的股骨缺损后，新生的骨组织沿着定向的层状大孔长入到支架的内部。由于PLGA/CPC支架表面包覆着PLGA膜，导致细胞反应性较差，植入初期，骨水泥骨传导性好的优点得不到发挥。为了改善PLGA/CPC支架的细胞反应性，本研究在氨气气氛下对PLGA/CPC复合支架进行等离子体表面处理，将胶原固定在PLGA/CPC支架孔壁PLGA膜的表面，改善支架的生物活性，得到Col/PLGA/CPC复合支架。经胶原表面改性后的支架的细胞种植率、粘附、增殖和分化均得到显著的提高。然而，植入兔子体内后发现，胶原改性对提高PLGA/CPC支架的成骨能力帮助并不大。
为了适应一些对支架强度有更高要求的应用，本研究仿天然骨的皮质骨/松质骨双层结构制备了强度可控的核壳结构磷酸钙骨水泥基复合支架。通过设计模具，采用等静压处理的方法制备了致密的管状骨水泥壳层。在致密壳层的空腔内用定向冰晶-冷冻干燥法制备具有定向层状大孔结构的多孔磷酸钙骨水泥芯，并用PLGA进行增强，然后用胶原进行表面改性。外层致密、内层多孔的骨水泥复合支架的强度可以通过改变致密层和多孔层的厚度比例调节（5~90MPa），以满足不同骨缺损部位修复的要求。定向大孔主要在轴向上具有高度的连通性，横向连通性较低。因此，致密的骨水泥外层对支架多孔芯的孔隙连通性没有明显的影响。细胞实验表明，细胞在双层骨水泥复合支架上粘附和增殖良好。
通过复合明胶微球改性PLGA/CPC支架的三维多孔结构。PLGA/CPC支架经过明胶微球（0%~30%）改性后，强度和孔隙率都有所变化，但分别保持在3.5~5MPa和62%~72%之间。经明胶微球改性后，支架的定向层状大孔被分割为尺寸较小的层状大孔。此外，明胶微球溶解后原位留下80~200μm左右的等轴状大孔。用20%明胶微球改性的支架的细胞种植率、增殖和分化改善效果最明显。明胶微球的改性并没有明显影响支架的轴向连通性，细胞仍然可以顺利长入到支架的内部。
将富血小板血浆（PRP）灌注到PLGA/CPC支架内部，制备了PRP-PLGA/CPC复合体。结果表明，PRP的复合大大地改善了PLGA/CPC支架的细胞反应。将PRP-PLGA/CPC复合支架植入到新西兰大白兔的股骨缺损后发现，PRP的复合明显促进了骨组织长入到支架的内部，加快血管的生成和材料的降解。此外，本研究首次将具有定向层状大孔结构的PLGA/CPC支架用于修复新西兰大白兔的桡骨节段性骨缺损。新生的骨组织沿着定向的层状大孔长入支架的内部，而没有受到其三维连通性有限的影响。PRP的复合对PLGA/CPC支架修复桡骨的节段性骨缺损具有显著的促进作用。
A PLGA/CPC scaffold with unidirectional lamellar pore structure was fabricated by theunidirectional freeze casting of CPC slurry and the following infiltration ofpoly(lactic-co-glycolic acid)(PLGA) in this study. In vitro degradation, cell response and invivo osteogenesis of PLGA/CPC scaffold were conducted. Furthermore, the composition,three dimensional (3D) structure of PLGA/CPC scaffold were redesigned to optimize itsmechanical performance, cell response and in vivo osteogenesis.
The pore wall of PLGA/CPC scaffold was covered by PLGA film. The PLGA film firstlydegraded during immersion in PBS solution, which was accompanied with the exposure ofCPC matrix that was beneficial for cell response and osteogenesis. The unidirectional lamellarpore structure of PLGA/CPC scaffold was beneficial for growth of cells into inner macropores,but unfavorable for cell seeding and differentiation. After being implanted in vivo, new bonetissues grew along the unidirectional lamellar pores into the scaffold. The PLGA film on thepore wall of PLGA/CPC scaffold compromises the osteoconductivity of CPC due to poor cellresponse of PLGA. In order to improve cell response of PLGA/CPC scaffold, collagen wasimmobilized on the PLGA film of PLGA/CPC composite scaffold by plasma treatment underthe ammonia atmosphere. After immobilization of collagen, the cell seeding, adhesion,proliferation and differentiation on PLGA/CPC scaffold were significantly enhanced.However, the results of histological evaluation showed that the osteogenesis of PLGA/CPCscaffold was not obviously improved after immobilization of collagen.
To meet the requirements of bone defect repair at various sites, a core/shell bi-layeredCPC-based composite scaffold with adjustable compressive strength, which mimicked thenatural structure of cortical/cancellous bone, was fabricated. The dense tube-like CPC shellwas prepared by designing a mould and isostatic pressing process. A porous core withunidirectional lamellar pore structure was fabricated inside the cavity of dense tube-like CPCshell by aforementioned unidirectional freeze casting, followed by infiltration of PLGA andimmobilization of collagen. The compressive strength of bi-layered CPC-based compositescaffold can be controlled by varying thickness ratio of dense layer to porous layer (590MPa). Compared to the scaffold without dense shell, the pore interconnection of bi-layeredscaffold was not obviously compromised because of its high unidirectional interconnectivitybut poor3D interconnectivity. The cells adhered and proliferated well on the bi-layered CPCbased composite scaffold.
Gelatine microspheres (GM) were used to modify the3D pore structure of PLGA/CPC scaffold. After being modified by various amounts of GM (5%30%), the compressivestrength and porosity of the PLGA/CPC scaffold remained in the range of2.45MPa and62%72%, respectively. The unidirectional lamellar pores were divided into smaller pores.Macropores with the size of80200μm were left being generated by the dissolution of GM.The scaffold modified by20%GM exhibited the most excellent cell seeding, proliferation anddifferentiation. The pore interconnection of scaffold was not starkly compromised aftermodification by GM so that cells still penetrated into the inner pores of scaffolds smoothly.
PRP-PLGA/CPC construct was fabricated by infiltration of platelet-rich plasma (PRP)into PLGA/CPC scaffold. The incorporation of PRP significantly improved cell response ofPLGA/CPC scaffold. The PRP-PLGA/CPC composite scaffold was implanted in femoralbone defects of rabbits, the results showed that PRP boosted markedly bone ingrowth,angiogenesis and degradation of PLGA/CPC scaffold. The PLGA/CPC scaffold withunidirectional lamellar pore structure was originally used to reconstruct the segmental bonedefect in radius of rabbit. The new bone tissues grew well along the unidirectional lamellarpores of scaffold regardless of its poor3D pore interconnection. The incorporation of PRPshowed outstanding improvement in osteogenesis of PLGA/CPC scaffold as implanted in thesegmental bone defect in the radius of rabbit.
引文
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