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Orthopedic and Rehabilitation Engineering

Orthopedic and Rehabilitation Engineering - Poster Session E

Poster AA8 - Tendon Allografts Modified with Genipin and Gold Nanoparticles (AuNPs) Enhance Healing post ACL Reconstruction

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Millions of people suffer from traumatic ligament ruptures every year. ACL tears are particularly common, often resulting from activity in popular team sports such as soccer, American football, volleyball, and lacrosse. Without surgical intervention, this type of injury can be very debilitating, painful, and athletic career-ending. Furthermore, damage to the ACL can lead to many troublesome, chronic complications such as accelerated progression of osteoarthritis. Most commonly, patients have their torn or ruptured ACL reconstructed by inserting a tendon graft in the anatomic location of the ACL, threaded through (and anchored to the periostea of) the distal femur and proximal tibia by way of freshly drilled bone tunnel that is similar diameter to the graft tissue.
These grafts can come from the patient's own tendons (autograft) if they are healthy enough, or from a limited supply of de-cellularized, lyophilized cadaver tissue (allograft). While bone-patellar tendon-bone (BPTB) or hamstring tendon autografts are preferred in healthier patients with less co-morbidities, many patients rely on cadaver tendon allografts for reconstruction of their torn ACL. Even if they are lucky enough to have access to this material (which is inherently in limited supply), many patients still suffer prolonged and painful healing courses due to the inherent limitations of the graft tissue, despite the best reconstructive efforts of highly-trained and skilled orthopedic surgeons. We hypothesize that modifying these grafts with genipin and 20nm gold nanoparticles (AuNPs) will result in enhanced cellular infiltration and remodeling, while reducing excessive levels of inflammation which can impede healing.

Materials and

Methods:  Lyophilized cadaver tendon donated from MTF Biologics was rehydrated in PBS, cut into 100mg strips, and incubated in a 3mM genipin solution for 15 minutes. This solution was then replaced with a 7*10¹¹AuNPs/mL solution, allowed to incubate for 5 or 10 more minutes, and PBS-rinsed twice more before use.
Scaffolds were first characterized according to their gold nanoparticle (AuNP) content. Conjugation efficiency was determined using Neutron Activation Analysis (NAA). The relative conjugation rates could also be grossly observed qualitatively due to the inherent staining properties of the MEA-capped AuNPs (figure 1). Release of AuNP’s from incubated grafts into solution over time was measured with UV-Vis spectroscopy. The absorption peak of pure 20nm AuNPs in suspension is ~521nm, but when conjugated to MEA and/or genipin the peak spreads and shifts towards 600nm.
In Vitro studies were performed to analyze the effects graft modifications have on key cells responsible for remodeling. A WST-1 metabolic assay was performed on fibroblasts and osteoblasts incubated in the graft supernatant per ISO-10993 standards. PicoGreen was used to fluorescently quantify the dsDNA content of the cells to measure their proliferation. ELISA kits were used to measure the quantity of extracellular IL-1,TNF-alpha, and IL-10 to assess effects on key cytokines in the inflammatory pathway.
The grafts were tested in vivo using a New Zealand White Rabbit model (n=12; 6f, 6m). A veterinary surgeon replaced their ACL’s with either the untreated or the AuNP-modified (xeno-)allografts, with histologic analysis at necropsy 12-weeks post-op.

Results, Conclusions, and Discussions: The binding of the AuNPs to the tissue via genipin cross-linker was proven to be effective according to the results of the NAA: grafts incubated in 1x AuNP for 10 minutes or 2x AuNP for 5 minutes provided sufficient decoration with AuNPs – even after a 24-hour incubation in PBS (17.8μg[Au]/g[tissue])-well within our target range of 15-25μg/g. Qualitative analysis of AuNP distribution on the surface of the grafts was performed via low-vacuum scanning electron microscopy (SEM) (figures 2, 3). The particulate matter was further confirmed to be our target compound (elemental gold) due to the characteristic, distinct peak noted at just over 2.1keV in the Energy Dispersive X-ray Spectroscopy (EDS) analysis focused on the region of interest containing the purported AuNPs (figure 4). The (expected) presence of several other atomic species were also noted (e.g. C, O, N, Na, Cl, etc.).
The in vitro analyses collectively displayed favorable biocompatibility regarding the graft modifications, as evidenced by similar proliferation and metabolic rates out to 10 days in the 1x/10min group and the untreated control group. The metabolic (WST-1) and proliferative (PicoGreen DS-DNA) activity only dropped 17 and 23 % respectively in the 2x/10min group compared to the standard. These results support the overall conclusion that our graft modifications result in a scaffold material that can reasonably be considered biocompatible. Although our OxiSelect ROS Assay was inconclusive due to the interfering nature of the AuNPs themselves, ELISA measuring the quantities of key cytokines of the inflammatory pathway revealed that conjugating AuNPs to the grafts via genipin cross-linking resulted in lower levels of inflammatory markers (IL-1, TNF-ɑ), while increasing the relative level anti-inflammatory marker IL-10.
The in vivo study in New Zealand White Rabbits confirmed our hypotheses that modifying allograft tissue with genipin and AuNP’s allows for an improved healing response 8 weeks post-op, per multivariate histologic analysis as performed by 3 separate blinded professionals. On average, recapitulated tendons grown from AuNP-genipin modified grafts displayed greater fibroblastic infiltration, enhanced vascularity, with mitigated inflammatory processes (monocytic/lymphocytic invasion). Overall, it can reasonably concluded that modifying the allografts with AuNPs and genipin enhanced the ligamentization process.

Acknowledgements (Optional):  -NIH-NIAMS
-SBIR/STTR
-MTF Biologics and their organ donors
-Coulter Translational Research Foundation
-Roy Blunt NextGen Precision Medicine Institute