In vivo structural and cellular remodeling of engineered bone–ligament–bone constructs used for anterior cruciate ligament reconstruction in sheep

Abstract

Anterior cruciate ligament (ACL) ruptures rank among the most prevalent and costly sports-related injuries. Current tendon grafts used for ACL reconstruction are limited by suboptimal biomechanical properties. We have addressed these issues by engineering multiphasic bone-ligament-bone (BLB) constructs which develop structural and mechanical properties similar to native ACL. The purpose of this study was to examine the acute remodeling process that occurs as the BLB grafts advance toward the adult ligament phenotype in vivo. Thus, we implanted BLB constructs fabricated from male cells into female host sheep and allowed 3,7,14 or 28 days (n=4 at each time point) for recovery. To address whether or not graft-derived cells were even necessary, a subset of BLB constructs (n=3) were acellularized, implanted and allowed 28 days for recovery. At each recovery time point, the following histological analyses were performed: Picrosirius red staining to assess collagen alignment and immunohistochemistry to assess both graft development and host immune response. PCR analysis, performed on every explanted BLB, was used to detect the presence of graft-derived male cells remaining in the constructs and/or migration into surrounding host tissue. The analysis of the PCR and histology samples revealed a rapid migration of host-derived macrophages and neutrophils into the graft at 3 days, followed by increased collagen density and alignment, vascularization, innervation and near complete repopulation of the graft with host cells within 28 days. This study provides a greater understanding of the processes of ligament regeneration in our BLB constructs as they remodel towards the adult ligament phenotype.

Additively Manufactured Multiphasic Bone–Ligament–Bone Scaffold for Scapholunate Interosseous Ligament Reconstruction

“…The implantation of cells has been shown to profoundly influence the regenerative outcome via complex and intertwined mechanisms involving the release of biochemical cues, the in vivo deposition of ECM, and the formation of a template to recruit progenitor cells . A recent insight into the biological events leading to tissue regeneration using cell sheets for ACL regeneration has been provided in an ovine model . This study showed a significant remodeling of the cell sheet in the early period of the implantation, featuring a cell death peak at 3 d post implantation concomitant with an increase in neutrophil infiltration followed by collagen densification at a later stage, consistent with tissue maturation.…”

“…This study showed a significant remodeling of the cell sheet in the early period of the implantation, featuring a cell death peak at 3 d post implantation concomitant with an increase in neutrophil infiltration followed by collagen densification at a later stage, consistent with tissue maturation. In addition, the presence of implanted cells gradually diminished in the tissue engineered graft while the total cell number increased at 28 d post surgery suggesting infiltration of new cells at this more advanced healing stage . This indicates that the cells participated actively in both the recruitment of host cells and the creation of a suitable ECM template necessary for graft maturation and healing to take place.…”

Nerve grafting for peripheral nerve injuries with extended defect sizes

“…There is no standard large animal model for nerve repair studies, but a number of species are used in other disciplines. The ovine animal model is established as a standard surgery model regarding orthopedic questions to investigate treatment options in arthrosis, osteoarthritis or damage of cruciate ligaments [ 57 – 59 ]. Porcine animals are predominantly used in trauma and intensive care research [ 60 – 62 ] and feline models are used as ear, nose and throat (ENT) surgical standard models [ 63 ].…”

Tissue‐engineered tendon constructs for rotator cuff repair in sheep

“…After 6 months in vivo, repair of a torn rotator cuff tendon with an ETG‐RC showed a significant increase in collagen alignment at the enthesis compared to suture‐only repaired tendons. Previous work in our sheep ACL studies indicate that the constructs are being decellularized during the early stages of remodeling and ultimately may serve as a scaffold for the infiltrating host cells . The histological analysis of the tendon‐to‐bone interface of suture‐only repairs was characterized by a layer of poorly organized, cellular, fibrovascular tissue with no evidence of a native‐like enthesis.…”

“…This difference in recovery time may account for the differences in gap length. Additionally, previous work using our technology in five sheep ACL studies indicate that at least a 6‐month recovery and up to 2 years is necessary to yield more optimal outcomes …”