Biomaterials direct functional B cell response in a material specific manner

B cells are an adaptive immune target of biomaterials development in vaccine research but despite their role in wound healing have not been studied tissue engineering and regenerative medicine. We evaluated the B cell response to biomaterial scaffold materials implanted in a muscle wound; a biological extracellular matrix (ECM) and synthetic polyester polycaprolactone. In the local muscle tissue, small numbers of B cells are recruited in response to tissue injury and biomaterial implantation. ECM materials induced plasmablasts in lymph nodes and antigen presentation in the spleen while the synthetic PCL implants delayed B cell migration and induced an antigen presenting phenotype. In muMt- mice lacking B cells, the fibrotic response to the synthetic biomaterials decreased. Immunofluorescence confirmed antigen presenting B cells in fibrotic tissue surrounding silicone breast implants. In sum, the adaptive B cell immune response to biomaterial depends on composition and induces local, regional and systemic immunological changes. Overall design: Murine tissue samples (quadriceps femoris muscle and inguinal lymph nodes) were obtained for single cell flow cytometry and/or fluorescence activated cell-sorting. Quad tissue samples were finely diced and digested with 1.67 Wünsch U/mL Liberase TL (Roche Diagnostics) and deoxyribonuclease I (0.2 mg/ml; Roche Diagnostics) in RPMI-1640 medium (Gibco) for 45 min at 37°C. The digested quad tissues were ground through 70-µm cell strainers (Thermo Fisher Scientific), rinsed with RPMI-1640 + 5% fetal bovine serum. Percoll (GE Healthcare) density gradient centrifugation was used to remove debris from quad digestions and enrich the leukocyte fraction. Inguinal lymph node samples were ground through 70-µm cell strainers with excess RPMI-1640 + 10 mM HEPES buffer solution. The enriched single-cell suspensions were washed and stained following antibody panels, respective to the intended application. No differences in single-cell isolation from different material environments were observed.

B细胞（B cells）是疫苗研发领域生物材料开发的适应性免疫靶点，尽管其在伤口愈合中具有关键功能，但目前尚未在组织工程与再生医学领域得到相关研究。
我们评估了植入肌肉伤口的生物材料支架对B细胞应答的影响，测试材料包括生物源性细胞外基质（extracellular matrix, ECM）与合成聚酯聚己内酯（polycaprolactone, PCL）。
在局部肌肉组织中，少量B细胞会因组织损伤与生物材料植入而被招募。
细胞外基质材料可在淋巴结中诱导浆母细胞形成，并在脾脏中触发抗原呈递；而合成的PCL植入物则会延迟B细胞迁移，并诱导抗原呈递表型。
在muMt-缺陷小鼠（B细胞缺失型）中，针对合成生物材料的纤维化应答有所减弱。
免疫荧光实验证实，硅酮乳房植入物周围的纤维化组织中存在抗原呈递型B细胞。
综上，机体对生物材料的适应性B细胞免疫应答取决于材料组成，并可引发局部、区域及系统性的免疫变化。
整体实验设计：获取小鼠组织样本（股四头肌与腹股沟淋巴结）用于单细胞流式细胞术检测及/或荧光激活细胞分选。
将股四头肌组织样本剪碎后，于37℃下在含1.67 Wünsch U/mL Liberase TL（罗氏诊断）、0.2 mg/mL脱氧核糖核酸酶I（罗氏诊断）的RPMI-1640培养基（Gibco）中消化45分钟。
将消化后的股四头肌组织通过70 μm细胞筛（赛默飞世尔科技）研磨过滤，并用含5%胎牛血清的RPMI-1640培养基冲洗。
采用Percoll（GE医疗）密度梯度离心法去除股四头肌消化液中的细胞碎片，并富集白细胞组分。
将腹股沟淋巴结样本通过70 μm细胞筛研磨，并用过量的含10 mM HEPES缓冲液的RPMI-1640培养基冲洗。
将富集得到的单细胞悬液洗涤后，根据实验用途选择对应的抗体标记方案进行染色。
未观察到不同材料环境下的单细胞分离效果存在差异。