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Decellularized Tendon-Bone Composite Grafts are Less Immunogenic and Stronger than Untreated Grafts an In Vivo Experimental Study in Rat
Simon Farnebo, MD, PhD; Colin Woon, MD; Taliah Schmitt, MD; Hung Pham, BS; James Chang, MD; Stanford University Medical Center
Stanford University Medical Center, Stanford, CA, Palo Alto, CA, USA

ABSTRACT

Introduction:

The reconstruction of digital flexor Zone I tendon-bone insertion site injuries is challenging. We previously described tissue engineered Achilles-calcaneus tendon-bone composite graft reconstruction of the tendon-bone interface (TBI) in a rat model as a surrogate for direct tendon-to-bone suture. Compared with untreated grafts, physicochemically decellularized grafts demonstrated no difference in strength and structure.

In this paper, we aimed to show that decellularization does not adversely affect scaffold integrity or ultimate failure load in vivo. We hypothesized that (1) replacing immune-competent Sprague Dawley (SD) rat TBIs with decellularized composite tendon-bone allografts from Wistar rats would lead to less inflammation than reconstruction with untreated Wistar allografts and (2) as a result of reduced inflammatory destruction, decellularized grafts would display greater strength properties than untreated grafts after 2 and 4 weeks in vivo.

Materials and Methods:

Composite Achilles-calcaneus TBI grafts were harvested from Wistar rats and divided into 2 groups. Grafts in Group 1 were decellularized using EDTA, SDS, peracetic acid and targeted ultrasonication of the tendon-bone insertion in the manner previously described. Grafts in Group 2 were untreated controls. SD rats underwent Wistar TBI allograft reconstruction of bilateral hindlimb Achilles tendon-calcaneus bone tendon-bone insertion sites using a pair-matched design (left, decellularized; right, untreated). SD rats were killed at 2 or 4 weeks and the reconstructed hindlimbs were harvested. The extent of B-cells and macrophage infiltration was determined using immunohistochemistry (IHC). The explants were subjected to mechanical testing to determine the ultimate failure load. Statistical analysis was performed using a paired Student’s T-test.

Results:

At 2 weeks,, there was increased B-cell and macrophage infiltration in Group 2 (untreated) compared with Group 1 (decellularized), both in the capsule surrounding the TBI and the tendon substance. There was improved ultimate failure load (33.6±7.5N vs 24.0±9.8N, respectively, p=0.044) in Group 1 (decellularized).

At 4 weeks, there was persistent B-cell and macrophage infiltration in Group 2 (untreated) compared with Group 1 (decellularized). At 4 weeks, Group 1 (decellularized) demonstrated persistently greater ultimate failure load (46.5±17.7N vs 22.7±7.3N, respectively, p=0.042) compared with Group 2 (untreated).

Conclusions:

Targeted physicochemical decellularization of tendon-bone composite grafts removes cell surface antigens leading to a decreased immune response when used for allograft reconstruction. These grafts showed better biomechanical properties at 2 and 4 weeks when compared with control (untreated) tendons. Decellularization is an important step in the processing of tissue engineered tendon-bone composite grafts for upper extremity TBI reconstruction.


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