Anz Adam W, Cook Joshua J, Branch Eric A, Rahming Charlkesha A, Ostrander Roger V, Jordan Steve E
Andrews Institute Center for Regenerative Medicine, Department of Research, Andrews Research & Education Foundation (AREF), Gulf Breeze, Florida, U.S.A.
Arthrosc Sports Med Rehabil. 2024 Jan 12;6(1):100860. doi: 10.1016/j.asmr.2023.100860. eCollection 2024 Feb.
To investigate the viability of cells collected with an in-line-suction autologous tissue collector from the tissue byproducts of arthroscopic anterior cruciate ligament (ACL) reconstruction, to characterize cells from different tissue types, and to identify mesenchymal stem cells.
Patients aged 14 to 50 years with ACL injuries requiring arthroscopic reconstruction surgery were offered enrollment and screened for participation. In total, 12 patients were enrolled in the descriptive laboratory study. Arthroscopic byproduct tissue was collected with an in-line-suction autologous tissue collector from 4 intraoperative collection sites for each patient: ACL stump, ACL fat pad, notchplasty debris, and tunnel drilling debris. All tissue samples were digested using collagenase, and the derived cellular populations were analyzed in vitro, characterizing cellular viability, proliferative potential, qualitative multipotent differentiation capacity, and cell-surface marker presence.
An equivalent mass of arthroscopic byproduct tissue was taken from each of the 4 intraoperative collection sites (1.12-1.61 g, = .433), which all showed an average viability of at least 99.95% and high average total nucleated cells (≥1.37 × 10 cells/mL). No significant differences in collected mass ( = .433), cellular viability ( = .880), or total nucleated cells ( = .692) were observed between the 4 byproduct tissues. The byproduct tissues did exhibit significant differences in monocyte ( = .037) and red blood cell ( = .038) concentrations, specifically with greater values present in the ACL stump tissue. Cells from all byproduct tissues adhered to plastic cell culture flasks. Significant differences were found between colony-forming unit fibroblast counts of the 4 byproduct tissues when plated at 10 ( = .003) and 10 ( = .016) cells as the initial seeding density. There was a significant relationship found between both the starting concentration (χ = 32.7, < .001) and the byproduct tissue type (χ = 30.4, < .001) to the presence of ≥80% confluency status at 10 days. Cells obtained from all 4 byproduct tissues qualitatively showed positive tri-lineage (adipocyte, osteoblast, chondroblast) differentiation potential compared with negative controls under standardized in vitro differentiation conditions. Cells derived from all 4 byproduct tissues expressed cell-surface antigens CD105+, CD73+, CD90+, CD45-, CD14-, and CD19- (>75%), and did not express CD45 (<10%). There were no statistically significant differences in cell-surface antigens between the four byproduct tissues.
This descriptive laboratory study demonstrated that cells derived from arthroscopic byproduct tissues of ACL reconstruction remain viable when collected with an in-line-suction autologous tissue collector and these cells meet the ISCT criteria to qualify as mesenchymal stem cells.
It is known that viable mesenchymal stem cells reside in byproduct tissue of anterior cruciate ligament reconstruction surgery (ACLR). Practical methods to harvest these cells at the point of care require further development. This study validates the use of an in-line-suction autologous tissue collector for the harvest of viable mesenchymal stem cells after ACLR.
探讨使用在线抽吸式自体组织采集器从关节镜下前交叉韧带（ACL）重建的组织副产物中采集的细胞的活力，对不同组织类型的细胞进行特征分析，并鉴定间充质干细胞。
招募年龄在14至50岁之间、因ACL损伤需要进行关节镜重建手术的患者，并对其进行参与筛选。共有12名患者纳入了这项描述性实验室研究。使用在线抽吸式自体组织采集器从每位患者的4个术中采集部位收集关节镜下的副产物组织：ACL残端、ACL脂肪垫、切迹成形术碎片和隧道钻孔碎片。所有组织样本均用胶原酶消化，并对所得细胞群体进行体外分析，以确定细胞活力、增殖潜力、定性多能分化能力和细胞表面标志物的存在情况。
从4个术中采集部位的每个部位获取的关节镜下副产物组织质量相当（1.12 - 1.61 g，P = 0.433），所有组织的平均活力至少为99.95%，平均总核细胞数较高（≥1.37×10⁶细胞/mL）。在4种副产物组织之间，未观察到采集质量（P = 0.433）、细胞活力（P = 0.880）或总核细胞数（P = 0.692）的显著差异。副产物组织在单核细胞（P = 0.037）和红细胞（P = 0.038）浓度上确实存在显著差异，特别是ACL残端组织中的浓度更高。所有副产物组织的细胞均能附着于塑料细胞培养瓶。当以10⁴（P = 0.003）和10⁵（P = 0.016）个细胞作为初始接种密度接种时，4种副产物组织的集落形成单位成纤维细胞计数存在显著差异。在起始浓度（χ² = 32.7，P < 0.001）和副产物组织类型（χ² = 30.4，P < 0.001）与10天时≥80%汇合状态的存在之间均发现了显著关系。在标准化体外分化条件下，与阴性对照相比，从所有4种副产物组织获得的细胞在定性上均显示出阳性的三系（脂肪细胞、成骨细胞、成软骨细胞）分化潜能。从所有4种副产物组织衍生的细胞均表达细胞表面抗原CD105⁺、CD73⁺、CD90⁺、CD45⁻、CD14⁻和CD19⁻（>75%），且不表达CD45（<10%）。4种副产物组织之间的细胞表面抗原无统计学显著差异。
这项描述性实验室研究表明，使用在线抽吸式自体组织采集器从ACL重建的关节镜下副产物组织中采集的细胞在采集后仍具有活力，并且这些细胞符合国际细胞治疗协会（ISCT）的标准，有资格被认定为间充质干细胞。
已知在前交叉韧带重建手术（ACLR）的副产物组织中存在有活力的间充质干细胞。在临床现场采集这些细胞的实用方法需要进一步开发。本研究验证了使用在线抽吸式自体组织采集器在ACLR后采集有活力的间充质干细胞的可行性。
