研究背景

软骨自我修复能力有限，组织工程软骨成为软骨再生修复的理想选择。软骨细胞在二维培养条件下，细胞增殖及表型维持受限，易发生去分化。多孔微球因具备高度连通的孔状结构、大比表面积等优势而被用作细胞支架材料，其不仅可为细胞提供三维培养环境以更好地模拟体内生长条件，还为细胞提供足够的黏附、生长空间以促进细胞大量扩增。合成高分子材料左旋聚乳酸（PLLA）微球因具备优异的机械性能和可塑性，适用于软骨组织工程研究，但合成高分子材料的低细胞亲和力限制了细胞黏附和组织形成。基质细胞衍生因子-1α（SDF-1α）作为一种强趋化效力的稳态细胞因子，能够有效促进细胞迁移与增殖。将SDF-1α修饰于支架以促进软骨细胞增殖及组织形成为SDF-1α的应用提供了一种新途径。

研究目的

明确SDF-1α对软骨细胞迁移、增殖及表型维持的影响，筛选SDF-1α最佳应用浓度，探究接枝SDF-1α的PLLA多孔微球对软骨细胞生物学特性及软骨组织形成的影响，为软骨组织工程提供新的细胞支架选择。

研究方法

通过CCK-8实验、Transwell实验、qPCR实验分别验证不同浓度SDF-1α对软骨细胞增殖、迁移、表型维持的影响，筛选最佳浓度以备后续研究。

乳液-溶剂挥发法制备PLLA多孔微球，利用碳二亚胺法将SDF-1α接枝于微球上，并通过酶联免疫吸附实验及孵育SDF-1α特异荧光抗体验证接枝情况。

通过CCK-8实验、细胞活死染色实验、Dil细胞膜染色共聚焦观察分别验证接枝SDF-1α的PLLA多孔微球的细胞相容性以及对细胞的存活、黏附、增殖的影响。

分别将PLLA多孔微球和接枝SDF-1α的多孔微球与软骨细胞、甲基丙烯酰胺基明胶（GelMA）混合注模，以单纯软骨细胞组为对照，植入裸鼠皮下，8周后取材，通过苏木精-伊红染色、阿利新蓝染色和Ⅱ型胶原组织化学染色检测体内成软骨情况。通过DNA定量试剂盒、羟脯氨酸测定试剂盒和糖胺聚糖测定试剂盒以及成软骨相关基因mRNA相对表达量，验证体内新生软骨组织胶原、糖胺聚糖分泌情况。

研究结果

（1）与未添加及添加1000 ng/mL SDF-1α的培养条件相比，500 ng/mL SDF-1α有利于软骨细胞增殖(P<0.0001)、迁移(P<0.01)及胶原合成(P<0.01)。

成功制备出具有相互连接且高度开放孔状结构的PLLA多孔微球。接枝SDF-1α的PLLA多孔微球在电镜下可见颗粒样SDF-1α附着，孵育SDF-1α特异荧光抗体后可激发特异绿色荧光，经酶联免疫吸附实验验证微球上SDF-1α接枝率约93.75%。

PLLA多孔微球修饰SDF-1α后细胞相容性显著改善,有效促进了细胞黏附(P<0.05)、存活(P<0.01)、增殖(P<0.01)。

细胞-微球-GelMA复合物植入裸鼠皮下8周后，多孔微球修饰组微球内有明显软骨陷窝结构成簇样分布，糖胺聚糖和Ⅱ型胶原沉积丰富。qPCR结果进一步证实多孔微球修饰组的弹性蛋白(ELN)、Ⅱ型胶原(COLⅡ)、增殖细胞核抗原(PCNA)、B淋巴细胞瘤-2(Bcl-2)的mRNA表达更高(P<0.01)。

研究结论

SDF-1α修饰的PLLA多孔微球细胞相容性显著改善，有利于软骨细胞黏附、增殖、表型维持。细胞-修饰微球-GelMA复合物可在体内促进软骨组织形成，胶原及糖胺聚糖沉积显著增加，可作为软骨修复或3D生物打印材料。但SDF-1α的缓释浓度、微球比例、细胞与微球预混方式等因素都是影响最终组织形成的关键，仍需进一步深入研究和探讨。

Background

The self-repair capacity of cartilage is limited, thus tissue engineering cartilage offers an optimal solution for the regeneration and repair of cartilage. In two-dimensional culture conditions, chondrocytes exhibit limited cellular proliferation and phenotypic maintenance, and are prone to dedifferentiation. The utilization of porous microspheres as cell scaffold materials is attributed to their highly interconnected pore structure and substantial specific surface area, which not only facilitates the creation of a three-dimensional culture environment that closely mimics in vivo growth conditions but also offers ample adhesion and growth space for promoting extensive cell proliferation. The exceptional mechanical properties and moldability of synthetic Poly(L-lactic acid) (PLLA) microspheres make them highly suitable for research in cartilage tissue engineering. However, the limited cell affinity of synthetic polymers hinders cell adhesion and tissue formation. Stromal cell-derived factor-1α (SDF-1α) is a homeostatic cytokine with potent chemotactic properties that effectively enhances cellular migration and proliferation. Modifying scaffolds with SDF-1α to enhance chondrocyte proliferation and tissue formation offers a novel approach for the application of SDF-1α.

Objective

To investigate the effects of SDF-1α on chondrocyte migration, proliferation, and phenotype maintenance, to screen the optimal concentration of SDF-1α for application, and to explore the effects of SDF-1α-grafted PLLA porous microspheres on chondrocyte biological characteristics and cartilage tissue formation, providing new choices for cell scaffolds in cartilage tissue engineering.

Methods

(1)Through the CCK-8 cell proliferation test, Transwell Migration Assay, and qPCR, we verified the effects of different concentrations of SDF-1α on chondrocyte proliferation, migration, and phenotype maintenance. And we selected the optimal concentration for further research.

(2)PLLA porous microspheres were prepared using the emulsion-solvent extraction method, and SDF-1 was grafted onto them through carbodiimide reaction. The successful grafting was confirmed by Enzyme-linked immunosorbent assay (ELISA)  and incubation with a fluorescent antibody specific to SDF-1α.

(3)The cell compatibility and effects of cell survival, adhesion, and proliferation on cells following grafting of SDF-1α onto PLLA porous microspheres were assessed using the CCK-8 cell proliferation test, live/dead cell viability assay, and Dil cell membrane staining confocal observation technique.

(4)PLLA porous microspheres and SDF-1α-grafted porous microspheres were separately combined with chondrocytes and GelMA, followed by injection into the module. Additionally, chondrocytes and GelMA were injected into the module as control. Subsequently, the modules were surgically implanted subcutaneously in nude mice, and tissue samples were collected after an 8-week duration. The formation of cartilage was identified through hematoxylin-eosin staining, alcian blue staining, and type II collagen histochemical staining. Collagen and glycosaminoglycan secretion were verified by quantitative DNA kit, hydroxyproline determination kit, chondroitin sulfate determination kit, and by measuring the relative mRNA expression levels of chondroblast-related genes.

Results

(1)Compared to the culture conditions without addition and with 1000 ng/mL SDF-1α, the inclusion of 500 ng/mL SDF-1α is advantageous for chondrocyte proliferation (P<0.0001), migration (P<0.01), and collagen synthesis (P<0.01).

(2)PLLA porous microspheres with interconnected and highly open porous structures were successfully fabricated. The SDF-1α-grafted PLLA porous microspheres were observed under the electron microscope, exhibiting granule-like structures with adhered SDF-1α. Following incubation with SDF-1α-specific fluorescent antibodies, a distinct green fluorescence was induced. ELISA results confirmed that the grafting rate of SDF-1α onto the microspheres was approximately 93.75%.

(3)The cell compatibility of SDF-1α-modified PLLA porous microspheres was significantly enhanced, effectively facilitating cell adhesion (P < 0.05), viability (P < 0.01), and proliferation (P < 0.01).

(4)After an 8-week subcutaneous implantation in nude mice, the results demonstrated that the group treated with porous microspheres grafted with SDF-1 mixed with chondrocytes and GelMA exhibited a distinct clustered distribution of cartilage lacunar structures, along with enhanced deposition of cartilage-specific matrix and type II collagen. The qPCR results further validated that the relative mRNA expression levels of elastin (ELN), type II collagen (COLⅡ), proliferating cell nuclear antigen (PCNA), and B-lymphocyte lymphoma-2 (Bcl-2) were significantly elevated in the group where had porous microspheres grafted with SDF-1α mixed with chondrocytes and GelMA (P < 0.01).

Conclusions

The cell compatibility of SDF-1α-modified PLLA porous microspheres was significantly enhanced, thereby promoting chondrocyte adhesion, proliferation, and maintenance of phenotype. The cell-modified microsphere complex demonstrates the ability to enhance in vivo cartilage tissue formation, leading to significant augmentation of collagen and glycosaminoglycan deposition. Consequently, it can serve as a promising material for cartilage repair or 3D bioprinting applications. However, the sustained release concentration of SDF-1α, the microsphere ratio, and the method of cell-microsphere mixing all constitute crucial factors that impact final tissue formation. Therefore, further comprehensive research and discussion are needed.