In vivo evaluation of electrospun polycaprolactone graft for anterior cruciate ligament engineering

Abstract

The anterior cruciate ligament (ACL) is critical for the structural stability of the knee and its injury often requires surgical intervention. Because current reconstruction methods using autograft or allograft tissue suffer from donor-site morbidity and limited supply, there has been emerging interest in the use of bioengineered materials as a platform for ligament reconstruction. Here, we report the use of electrospun polycaprolactone (PCL) scaffolds as a candidate platform for ACL reconstruction in an in vivo rodent model. Electrospun PCL was fabricated and laser cut to facilitate induction of cells and collagen deposition and used to reconstruct the rat ACL. Histological analysis at 2, 6, and 12 weeks postimplantation revealed biological integration, minimal immune response, and the gradual infiltration of collagen in both the bone tunnel and intra-articular regions of the scaffold. Biomechanical testing demonstrated that the PCL graft failure load and stiffness at 12 weeks postimplantation (13.27±4.20N, 15.98±5.03 N/mm) increased compared to time zero testing (3.95±0.33N, 1.95±0.35 N/mm). Taken together, these results suggest that electrospun PCL serves as a biocompatible graft for ACL reconstruction with the capacity to facilitate collagen deposition.

FIG. 1.

Polycaprolactone (PCL) polymer design, fabrication, and three-dimensional rendering. PCL solution was electrospun and laser cut to yield a porous scaffold. (A) Low- and high-magnification scanning electron microscopy images show both (B) aligned fiber and (C) hole regions designed to facilitate cellular infiltration and collagen deposition in the scaffold. (D) Schematic of scaffold with dimensions of 1.5 mm×35 mm×900 μm and the eventual arrangement of the scaffolds into a stacked conformation. Color images available online at www.liebertpub.com/tea

FIG. 2.

Schematic demonstrates in vivo experimental design. Scaffolds are prepared, stacked, and coated with collagen before implantation into the left knee of model rodents. Replicates are designed such that four animals will be assessed for biological function at weeks 2, 6, and 12 postimplantation, and an additional six will undergo biomechanical testing (BMT) at week 12.

FIG. 3.

Gross and histological images of PCL implant are shown (A) intra-operatively and postoperatively with (B) hematoxylin and eosin (H&E) staining and (C) digital photomicroscopy. Tibial and femoral tunnels were drilled in the native footprint of the anterior cruciate ligament (ACL). The graft is passed and fixed extra-articularly with sutures to the periosteum. Images of PCL graft harvested at 12 weeks postimplantation and stained with (D) H&E and (E) Picrosirius red reveal resorption of graft (*) and replacement with densely aligned collagen. BT, bone tunnel; IA, intra-articular; F, femur; T, tibia. Color images available online at www.liebertpub.com/tea

FIG. 4.

Histology panel of PCL grafts stained with H&E at weeks 2, 6, and 12 postimplantation. Evidence of a modest host inflammatory response is visible beginning at week 2 in the BT and week 6 in the IA graft region. Aligned collagen and fibroblasts are present in the IA graft tissue beginning at week 6. BT, bone tunnel; IA, intra-articular regions, Fb, fibroblast; FBG, foreign body giant cell; M, macrophage. Color images available online at www.liebertpub.com/tea

FIG. 5.

Immunological staining and analysis of CD68 (ED1) macrophage lineage marker. (A) Graft sections were stained for expression of CD68 with representative images shown and (B) the number of CD68⁺ cells were counted in each high-power field (n=5) (HPF, 40× objective magnification) at 2, 6, and 12 weeks postimplantation, demonstrating that inflammatory responses peaked at 6 weeks postimplantation and decreased thereafter. *A p value of <0.01 between all time points. Color images available online at www.liebertpub.com/tea

FIG. 6.

Immunoflourescent staining of type 1 collagen was performed on graft samples harvested at weeks 2, 6, and 12 postimplantation demonstrated increased collagen deposition in the graft over time. Color images available online at www.liebertpub.com/tea

FIG. 7.

Quantitative assessment of in vivo collagen deposition. (A) Representative images of Picrosirius red staining are depicted for IA and BT regions at weeks 2, 6, and 12 and show gradual increases in fibrillar collagen type I and type III deposition. (B) ImageJ was used to analyze the percentage of field of view containing birefringent pixels for each HPF analyzed (n=5). *A p value of <0.01 between all groups. Color images available online at www.liebertpub.com/tea