Single Cell RNA Sequencing of Synovial Tissue in Adolescents Undergoing ACL Reconstruction. Single Cell RNA Sequencing of Synovial Tissue in Adolescents Undergoing ACL Reconstruction

Background: Loss of motion and arthrofibrosis after anterior cruciate ligament reconstruction (ACLR) can be a devastating complication for athletes. The cellular and molecular pathogenesis of arthrofibrosis is poorly understood, limiting prevention and treatment options. Synovial inflammation may contribute to post-ACLR arthrofibrosis. Hypothesis/Purpose: We hypothesized that higher synovial immune cell infiltration and inflammatory/catabolic gene expression patterns at the time of ACLR would correlate with poorer motion-related outcomes. Study Design: Case Series Methods: Patients aged 10-18 undergoing primary ACLR were enrolled in a prospective pilot study, and synovial tissue biopsies were obtained during ACLR. Flow cytometry and single cell RNA-sequencing explored synovial cell types/frequencies and gene expression. Principle component analysis followed by clustering grouped patients into distinct immunophenotypes based on their synovial cell composition. Clinical follow-up with knee range of motion (ROM), need for lysis of adhesions, and patient reported outcome measures were collected and compared between immunophenotypes. Results: Enrolled patients (n = 17) underwent ACLR at a median of 37 days post-injury. Analysis revealed three distinct immunophenotypes. Type 1 comprised patients with the longest time between injury and surgery and the lowest hematopoietic and T cell infiltration. Types 2 and 3 had similar times between injury and surgery, and Type 2 had intermediate while Type 3 had the highest hematopoietic and T cell percentages. Type 3 was associated with worse ROM at 2- and 6-weeks post-op; T cell prevalence and ROM were inversely correlated at those time points. The only patient requiring lysis of adhesions for arthrofibrosis was in Type 3. Conclusion: Synovial immune infiltration after ACL injury shows variability between patients that clusters into three immunophenotypes correlating with early ROM and the risk of arthrofibrosis. T cell recruitment and infiltration was the strongest factor correlated with ROM outcomes and presents an exciting venue for future research on post-ACLR arthrofibrosis. Overall design: Synovial tissue biopsies were obtained during ACL reconstruction from six patients, enzymatically digested to obtain heterogenous single-cell suspensions, and analyzed by scRNA-seq. The six scRNA-seq samples as well as eleven other patient samples were categorized into three Immunotypes based on flow cytometry quantification of the immune cells present in the synovium tissue. The scRNA-seq output was analyzed for a more comprehensive characterization of the cell types present as well as the relative expression of select inflammatory markers and immunomodulatory genes.

背景：前交叉韧带重建术（anterior cruciate ligament reconstruction, ACLR）后出现的活动丧失与关节纤维化，对运动员而言可能是毁灭性并发症。目前对关节纤维化的细胞与分子发病机制尚不清楚，这限制了其预防与治疗手段的发展。滑膜炎症可能与前交叉韧带重建术后关节纤维化的发生相关。

假说/研究目的：我们假设，在前交叉韧带重建术时，滑膜免疫细胞浸润程度更高、炎症/分解代谢基因表达模式更显著的患者，其术后活动相关结局会更差。

研究设计：病例系列研究

方法：纳入10~18岁接受初次前交叉韧带重建术的患者，开展一项前瞻性预试验，术中获取滑膜组织活检样本。通过流式细胞术（flow cytometry）与单细胞RNA测序（single cell RNA-sequencing, scRNA-seq）分析滑膜细胞的类型、占比及基因表达情况。采用主成分分析结合聚类分析，根据患者滑膜细胞组成将其划分为不同免疫表型。收集患者术后随访数据，包括膝关节活动度（range of motion, ROM）、粘连松解术需求及患者报告结局指标，并在不同免疫表型组间进行比较。

结果：纳入的17例患者均接受了前交叉韧带重建术，受伤至手术的中位时间为37天。分析结果显示存在三种明确的免疫表型。1型患者的受伤至手术间隔时间最长，造血细胞与T细胞浸润程度最低；2型与3型患者的受伤至手术间隔时间相近，2型患者的造血细胞与T细胞占比处于中等水平，而3型患者占比最高。3型患者术后2周与6周的膝关节活动度更差；在这两个时间点，T细胞占比与活动度呈负相关。唯一因关节纤维化需要接受粘连松解术的患者属于3型组。

结论：前交叉韧带损伤后滑膜免疫浸润存在个体差异，可聚类为三种免疫表型，且与早期膝关节活动度及关节纤维化风险相关。T细胞募集与浸润是与活动度结局相关性最强的影响因素，为后续前交叉韧带重建术后关节纤维化的研究提供了极具前景的方向。

整体研究设计：从6例接受前交叉韧带重建术的患者术中获取滑膜组织活检样本，经酶解获得异质性单细胞悬液后，通过单细胞RNA测序进行分析。结合流式细胞术对滑膜组织中免疫细胞的定量结果，将这6例单细胞RNA测序样本与另外11例患者样本划分为三种免疫表型。对单细胞RNA测序结果进行分析，以更全面地表征滑膜组织中的细胞类型，以及选定的炎症标志物与免疫调节基因的相对表达水平。