Summary
ACL remnant tissue is a potential viable option for a graft augmentation approach to improve healing following ACL reconstruction.
Abstract
Background
With re-injury following ACL reconstruction (ACLR) continuing to be a barrier for successful patient outcomes, orthobiologic interventions are being utilized to potentially augment tissue healing. ACL remnant tissue possesses progenitor cells that have potential to promote healing and tissue maturation; however, limited clinical data has elucidated specific composition or mechanisms through which this remnant tissue impacts graft healing. This novel study aimed to characterize remnant ACL tissue through quantification of cell populations and establish baseline gene expression levels that can influence the ligamentization and osseointegration processes of graft healing.
Methods
A pilot study was conducted between 2023-2024 within a single surgeon practice. Subjects were included if they met the following criteria: 14-45 years of age, undergoing primary ACLR with either bone-patellar tendon or quadriceps tendon autograft. Subjects were excluded from the study if: large meniscal tears or any injury that precludes early weight-bearing or a history of previous knee injury or surgery were present. Flow cytometry was utilized to quantify cell populations in harvested remnant tissue displaying markers (CD34+, CD90+, CD105+, CD146+ and alpha-SMA+) that have been demonstrated to influence ligamentization and osseointegration. qRT-PCR was utilized to assess baseline expression of genes involved in graft healing: ACAN, BGLAP, BMP-7, CCL-2, COL-2A1, CXCL8, IL-1, IL-6, GDF-5, PDGF PTGE-R, SOX-9, TGF-ß, TNC, and VEG-F. One-way repeated measures multivariate analyses of variance were conducted to determine if there were significant differences in cell counts and baseline gene expression levels with an a priori alpha = .05. Post-hoc comparisons with a Bonferroni adjustment were applied to evaluate significance between cell marker and expression profiles.
Results
A total of 38 patients were included the study. Baseline demographics are as follows: mean age (21.86 ± 8.15 yrs), BMI (24.57 ± 4.57 kg/ m2), time to surgery (48.32 ± 57.97 days), gender (37% female). Improved cell viability was noted in cases where time to surgery was <10 days. Mean quantification for each cell marker are as follows: CD34+(2674.97±6158.29), CD90+ (2789.45±5142.04), CD105+(2174.85±4555.55), CD146+(2099.97±4742.24) and alpha-SMA+(2798.91±5405.22). There was a significant difference in overall cell counts, F (4,29) = 5.734, p= .002. Post-hoc comparisons demonstrate a significant difference between CD90+ and CD146+ (mean difference = 689.49, p = .019). There were significant increases in expression of genes: ACAN (23.28 ± 1.93, p=0.018), COL-2A1 (23.17 ± 2.57 p= 0.021), VEG-F (27.68 ± 2.59 p=0.010 ), IL-6 (23.84 ± 1.74 p=0.014), IL-1 (20.83 ± 1.44 p= 0.023), CXCL8 (25.82 ± 3.24 p= 0.011), GDF-5( 26.76 ± 3.23 p=0.010), and TNC (34.59 ± 1.74 p=0.009).
Conclusion
While previous studies have emphasized the importance of CD34+ progenitors as the primary cellular influence in ACL remnant tissue, this study demonstrates an increased presence of CD90+ and alpha-SMA+ cells in harvested tissue. These results, with increased expression levels of ACAN, COL-2A1, TNC and VEGF, suggest a strong potential of ACL remnant tissue to impact the ligamentization process. While other studies have utilized mesenchymal cells from bone marrow , these data suggest remnant tissue as a potential viable option for graft augmentation approach to improve graft healing.