Effect of Mesenchymal Stem Cell-Derived Secretome Injection on Functional Outcomes After Anterior Cruciate Ligament Reconstruction Using Peroneus Longus Tendon Autograft: A Double-Blind Randomized Controlled Trial

Technical Summary: Effect of Mesenchymal Stem Cell-Derived Secretome Injection on Functional Outcomes After ACL Reconstruction

Problem Statement
Anterior cruciate ligament (ACL) reconstruction, while common, faces significant challenges regarding graft healing. Inadequate ligamentization and poor tendon–bone integration can lead to graft failure, re-rupture, and persistent instability, with failure rates remaining as high as 10%. Although the peroneus longus tendon (PLT) offers favorable biomechanical properties and lower donor-site morbidity compared to conventional grafts, the biological quality of healing remains a critical determinant of long-term success. While preclinical studies suggest that mesenchymal stem cell (MSC)-derived secretome (a cell-free conditioned medium rich in bioactive factors) can enhance tendon–bone integration and graft remodeling, no clinical trial had previously evaluated its efficacy in human ACL reconstruction using a PLT autograft.

Methodology
This study was a prospective, double-blind, randomized, placebo-controlled trial (Level 1 evidence) conducted at a tertiary academic medical center in Indonesia between June 2025 and June 2026.

• Participants: 24 patients (aged 18–40) with confirmed unilateral, complete ACL ruptures were enrolled. Exclusion criteria included multi-ligament injuries, systemic inflammatory diseases, and recent corticosteroid injections.
• Intervention: All participants underwent arthroscopic single-bundle ACL reconstruction using a PLT autograft. They were randomly assigned (1:1 ratio) to:
  • Secretome Group (n=12): Received an intralesional injection of 1.5 mL of concentrated MSC-derived secretome (derived from human umbilical cord MSCs, USEPro®). The injection protocol involved 0.5 mL into each graft end (proximal and distal) prior to tunnel insertion, and 0.5 mL intra-articularly around the graft after fixation.
  • Control Group (n=12): Received an identical volume (1.5 mL) of sterile normal saline following the same injection protocol.
• Blinding: The study maintained double-blinding; the surgeon, participants, and outcome assessors were unaware of the treatment allocation.
• Outcome Measures: Functional outcomes were assessed preoperatively and at six months postoperatively using the International Knee Documentation Committee (IKDC) Subjective Knee Evaluation Form and the Tegner–Lysholm Knee Scoring Scale.
• Statistical Analysis: Between-group comparisons utilized independent-samples t-tests or Mann–Whitney U tests, with significance set at p < 0.05. A post hoc power analysis was conducted.

Key Results
All 24 participants completed the six-month follow-up with no dropouts.

• Baseline: Both groups showed comparable preoperative scores (IKDC: 48.57 vs. 47.51; Tegner–Lysholm: 53.42 vs. 52.02; p > 0.05).
• Six-Month Functional Outcomes:
  • IKDC Scores: The secretome group demonstrated significantly higher scores (86.89 ± 5.11) compared to the control group (78.27 ± 3.91), with a p-value < 0.01. The mean improvement in the secretome group was 38.32 points versus 30.76 points in the control group.
  • Tegner–Lysholm Scores: The secretome group achieved significantly higher scores (87.08 ± 5.16) compared to the control group (79.33 ± 2.67), with a p-value < 0.01. Based on classification, the secretome group's mean score fell into the "good" category (84–90), while the control group remained in the "fair" category (65–83).
• Safety: No major adverse events, infections, systemic reactions, or graft failures occurred. Minor, transient adverse events in the secretome group (localized swelling and mild pain) resolved spontaneously within 3–5 days.
• Statistical Power: Post hoc analysis confirmed adequate power (0.85 for IKDC; 0.89 for Tegner–Lysholm) to detect the observed differences.

Significance and Claims
The paper claims that this is the first randomized controlled trial to evaluate the clinical efficacy of MSC-derived secretome as an adjuvant in human ACL reconstruction using a PLT autograft. The primary significance lies in the demonstration that adjuvant secretome injection yields significantly superior six-month functional outcomes compared to standard reconstruction without biologic augmentation.

The authors posit that the secretome serves as a promising, safe, and effective cell-free biologic adjuvant. By leveraging bioactive factors (such as TGF-β1, VEGF, IGF-1, and PDGF) that modulate the local microenvironment, the treatment appears to enhance early graft healing and functional recovery. The study concludes that while the findings are encouraging, larger multicenter trials with longer follow-up durations and objective imaging endpoints are necessary to validate these preliminary results and establish optimal treatment protocols.

Limitations Acknowledged by the Authors
The paper modestly notes several limitations:

1. Sample Size: The relatively small cohort (n=24) limits generalizability and the ability to detect differences in rare secondary outcomes.
2. Follow-up Duration: The six-month period captures early healing but does not assess long-term graft survival, return-to-sport rates, or re-injury risks.
3. Single-Center Design: Conducted by a single surgeon at one institution, which may limit external validity despite enhancing internal consistency.
4. Lack of Objective Imaging: The study did not include MRI evaluation of graft signal intensity or second-look arthroscopy, preventing a direct correlation between functional scores and biological graft maturation.
5. Standardization: Variability in secretome composition between batches remains a challenge for broader clinical application.