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Background: Doxycycline (Doxy) has been shown to facilitate tendon healing by reducing on-site matrix metalloproteinase (MMP) activity, but its effect on graft healing after anterior cruciate ligament reconstruction (ACLR) has not been investigated, and the therapeutic effect of Doxy in preventing ACLR-induced posttraumatic osteoarthritis (PTOA) is unclear., Hypothesis: Doxy promotes graft healing and alleviates the progression of PTOA after ACLR., Study Design: Controlled laboratory study., Methods: Sprague Dawley rats (n = 74; age, 12-13 weeks; male) that underwent ACLR were divided into untreated control and Doxy treatment (50 mg/kg/d orally until sacrifice) groups. At 2 and 6 weeks after surgery, graft healing was assessed by biomechanical testing, histology, immunohistochemical staining, and micro-computed tomography (μCT). The progression of PTOA was evaluated at 6 weeks by histology, the Mankin score, and immunofluorescence staining of the tibial plateau, and osteophyte formation was evaluated by μCT. Hindlimb weight distribution was evaluated at 6 weeks, and gait patterns were evaluated at 2 and 6 weeks. Intra-articular MMP activity was evaluated at 6 weeks in vivo using an MMP-activatable near-infrared fluorescent probe., Results: Graft healing was enhanced by Doxy treatment, and the ultimate failure load ( P = .002) and stiffness of the graft ( P = .007) were significantly higher in the Doxy group at week 2. Bone mineral density and bone volume/total volume for both the tibial and the femoral tunnels at week 6 in the Doxy group were significantly higher compared with in the control group ( P < .05). The overall graft healing scores were significantly higher in the Doxy group. Doxy treatment enhanced graft integration, intratunnel graft integrity, and collagen birefringence; more collagen types 1 and 10 and less MMP-13 were found at the graft-bone interface. At week 6, the Doxy group had a lower modified Mankin score ( P = .033) and showed fewer MMP 13-positive chondrocytes at the articular cartilage surface ( P = .002), indicating moderate joint cartilage damage. μCT revealed less osteophyte formation, and gait analysis revealed more symmetric weightbearing and gait patterns, after Doxy treatment at week 6 ( P < .05). In vivo imaging with the near-infrared fluorescent probe identified significantly lower intra-articular MMP activity in the Doxy group at week 6 ( P = .016)., Conclusion: The oral administration of Doxy was able to synchronously promote graft healing and attenuate PTOA in an ACLR rat model., Clinical Relevance: Our results indicated that Doxy, a widely used drug, is potentially beneficial to patients after ACLR.

Background: Stem cell sheets provide a scaffold-free option for the promotion of graft healing after anterior cruciate ligament reconstruction (ACLR). However, cell viability, stability, and potential uncontrolled actions create challenges for clinical translation. The decellularization of cell sheets may overcome these problems as studies have shown that the natural extracellular matrix of stem cells is bioactive and can promote tissue repair., Hypothesis: The decellularized tendon-derived stem cell (dTDSC) sheet can promote graft healing after ACLR., Study Design: Controlled laboratory study., Methods: An optimized decellularization protocol was developed to decellularize the TDSC sheets. A total of 64 Sprague-Dawley rats underwent ACLR with or without the dTDSC sheet wrapping the tendon graft (n = 32/group). At 2 and 6 weeks after surgery, graft healing was assessed by micro-computed tomography, histology, and biomechanical testing. The accumulation of iNOS + and CD206 + cells and the expression of metalloproteinase 1 (MMP-1), MMP-13, and tissue inhibitor of metalloprotease 1 (TIMP-1) were assessed by immunohistochemistry., Results: The decellularization was successful, with the removal of 98.4% nucleic acid while preserving the collagenous proteins and bioactive factors. The expression of bone morphogenetic protein 2 (BMP-2) and VEGF in the dTDSC sheet was comparable with the TDSC sheet ( P > .05). Micro-computed tomography showed significantly more tunnel bone formation in the dTDSC sheet group. The dTDSC sheet group demonstrated better graft osteointegration and higher integrity of graft midsubstance with significantly higher ultimate failure load (16.58 ± 7.24 vs 8.93 ± 2.45 N; P = .002) and stiffness (11.97 ± 5.21 vs 6.73 ± 2.20 N/mm; P = .027). Significantly fewer iNOS + cells but more CD206 + cells, as well as lower MMP-1 and MMP-13 but higher TIMP-1 expression, were detected at the tendon-bone interface and graft midsubstance in the dTDSC sheet group., Conclusion: An optimized decellularization protocol for producing bioactive dTDSC sheets was developed. Wrapping tendon graft with a dTDSC sheet promoted graft healing after ACLR, likely via enhancing bone formation and angiogenesis by BMP-2 and VEGF, modulating macrophage polarization and MMP/TIMP expression, and physically protecting the tendon graft., Clinical Relevance: dTDSC sheets alleviate the quality control and safety concerns of cell transplantation and can be used as a cell-free alternative for the promotion of graft healing in ACLR.

Background: The anterior cruciate ligament (ACL) has poor regenerative capacity, and an injury leads to loss of function, limiting quality of life and increasing the incidence of osteoarthritis. Surgical interventions can stabilize the joint and improve functional recovery. The delivery of growth factors (GFs) enhances the healing process; however, this is complex in its regulation, is high in costs, has side effects, and can only be accomplished with supraphysiological concentrations and thus is currently not clinically feasible. However, the immobilization of a patient's endogenous GFs in biomaterials can overcome these problems., Purpose: To develop a method to capture endogenous bone morphogenetic protein-2 (BMP-2) and ultimately show enhanced ACL healing in vivo using this novel methodology., Study Design: Controlled laboratory study., Methods: BMP-2 binding peptides were synthetized, purified, and immobilized on polycaprolactone (PCL) films. The affinity between the peptide and human BMP-2 (hBMP-2) was confirmed with immunofluorescence and enzyme-linked immunosorbent assay. The C2C12 Luc reporter cell line was used to confirm the bioactivity of immobilized BMP-2. For in vivo experiments, the same functionalization technology was applied to the commercially available Polytape, and the functionalized tape was sutured together with the graft used for ACL reconstruction in rats. Each animal underwent reconstruction with either native Polytape (n = 3) or Polytape with BMP-2 binding peptides (n = 3). At 2 and 6 weeks after surgery, the graft was assessed by histology and micro-computed tomography., Results: The covalent immobilization of the peptide in PCL was successful, allowing the peptide to capture hBMP-2, which remained bioactive and led to the osteogenic differentiation of C2C12. In vivo experiments confirmed the potential of the Polytape functionalized with the BMP-2 binding peptide to capture endogenous BMP-2, leading to enhanced bone formation inside the femoral and tibial tunnels and ultimately improving the graft's quality., Conclusion: The incorporation of BMP-2 binding peptides into materials used for ACL reconstruction can capture endogenous hBMP-2, which enhances the healing process inside the bone tunnels., Clinical Relevance: These results demonstrate the potential of using synthetic peptides to endow biomaterials with novel biological functions, namely to capture and immobilize endogenous GFs.