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 that 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. Polymerase chain reaction (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 toward the adult ligament phenotype.

Keywords: ACL reconstruction; remodeling; scaffoldless; tissue engineering.

Figure 1

In Vitro BLB (A) Representative image of BLB. Each ACL BLB construct was fabricated with 2-5 smaller BLBs made of a 30 mm ligament portion flanked by two 15 mm bone ends. These constructs were placed side-by-side and allowed to fuse in vitro before implantation. (B) Representative longitudinal H&E section of a BLB in vitro prior to implantation. Scale bar indicates 200 μm.

Figure 2

Sectioning Procedure for Histological Analysis: Immunohistological quantification was achieved by first taking the middle 1 cm of the tissue spanning the intra-articular space and splitting this tissue longitudinally at the midline into two samples to expose the entire mid portion of the graft, including tissue from the periphery and core. The samples were sectioned at 12 μm starting from the midline and moving towards the superficial surface, taking sections for 6 separate stains.

Figure 3

Gross Morphology of BLB constructs at day of explanation. (A–E) Image of BLBs during explantation. White arrows indicate BLB within the intracapsular space. Blood clots were evident in the intracapsular space in 7 and 14 day samples (B,C). (F–J) Longitudinal H&E of BLB constructs at each time point.

Figure 4

Gross Morphology of 3-Day BLBs. BLBs at the 3-day time point showed the most variability in gross morphology. Two of the four 3-day BLBs looked very much like the graft prior to implantation (A,B), however, the other two 3-day grafts had already been encapsulated by blood clots and endogenous tissue, suggesting that the remodeling process had already begun (C–D).

Figure 5

Cell Density of BLB constructs. For total construct cell density, DAPI counts from the macrophage/neutrophil co-stain and M2 macrophage stain were averaged. There was a decrease in nuclei in the BLB at 7 and 14 days post implantation followed by an infiltration of new cells by 28 days. At 28 days, acellularized constructs reveal increased cell density by host cell infiltration. (*) indicates significance compared to the native ACL control. (#) indicates significance compared to the 14-day time point. (&) indicates significance compared to the 7-day time point.

Figure 6

Development of innervation and vasculature in BLB constructs. (A–E) Innervation, assessed with neurofilament first appeared in BLB construct by 7 days in vivo (B) and continued to form longer filament strands from 14 to 28 days (C,D, E) (F–J) Vasculature assessed with CD31first appeared in the BLB construct by 1 week in vivo (G). Vascularization increased with time in vivo (I–J).

Figure 7

Picrosirius Red Analysis of collagen alignment in BLB constructs. Picrosirius red staining showed a positive trend between time in vivo and brightness intensity. (A–F) Native ACL morphology of collagen alignment (n=4) (A) compared to BLB collagen alignment at 3 days (n=4) (B), 7 days (n=3) (C), 14 daysweeks (n=3) (D), 28 days (n=4) (E), and for the acellularized group (n=3) (F).

Figure 8

Picrosirius Red Quantification. The brightness analysis revealed an average greyscale brightness of 83.63 ±24.90 for native ACL, 12.88 ±8.08 brightness at the 3-day time point, 17.08 ±12.65 brightness at the 7-day time point, 21.60 ±6.82 brightness at the 14-day time point, 26.21 ± 7.20 brightness at the 28-day time point, and 24.64 ± 3.12 brightness for the acellularized group. Construct collagen alignment at all time points was significantly less than native ligament. (P<0.05).

Figure 9

Percentage Cell Death (Caspase-3) in BLB constructs. Percentage cell death in BLB constructs at 3 days (n=3), 7 days (n=4), 14 days (n=4), 28 days (n=4), and the acellularized group (n=3) compared to native ACL ligament (n=3). Quantification of caspase-3 staining showed an average 2.90 ± 3.01% cell death for native ACL ligament, an increase 12.34 ± 16.95% in cell death at 3 days, and a decrease back to baseline levels 2.486 ± 2.27% cell death at 7 days, 8.126 ± 10.46% cell death at 14 days, 1.98 ± 1.54 % cell death at 28 days, and 0.336 ± 0.19% cell death for the acellularized group. The percentage cell death was not significantly different compared to native ACL for any time points (p<0.05).

Figure 10

Neutrophil and Macrophage Percentages in BLB constructs. (A) Percentage neutrophils in BLB constructs at 3 days (n=3), 7 days (n=4), 14 days (n=3), 28 days (n=4), and acellularized group (n=3) compared to native ACL ligament (n=4). Neutrophil percentage at the 3-day time point is significantly higher than native ACL levels. Quantification of neutrophil staining showed an average 0.08 ± 0.09% of neutrophils within native ACL, 10.85 ±2.50% of neutrophils within the construct at 3 days, 0.46 ± 0.62% at 7 days, 0.46 ± 0.62% at 14 days, 0.17 ± 0.21% at 28 days, and 0.36 ± 0.40% in the acellularized group.(*) indicates significance compared to the native ACL control. (**) indicates significance compared to the 3-day time point. (B) Total macrophage percentage, marked by CD68, in BLB construct at 3 days (n=4), 7 days (n=4), 14 days (n=3), 28 days (n=4), and in the acellularized group (n=3) compared to native ACL (n=4). Total M2 macrophage percentage, marked by CD163, in BLB construct at 3 days (n=4), 7 days (n=3), 14 days (n=4), 28 days (n=4), and acellularized group (n=3) compared to native ACL (n=3). The average percentage of all macrophages (CD68) in the BLB construct was 7.40 ± 6.26% for the 3-day time point, 11.00 ± 4.39% in the 7-day time point, 8.15 ± 3.95% in the 14-day time point, 10.66 ± 2.30% in the 28-day time point, and 8.46 ± 2.40% for the acellularized group. For the total macrophage data (CD68), * indicates significance compared to the native ACL control. The average percent of M2 macrophage (CD163) in native ACL ligament is 0.01 ± 0.002%. The average percent of M2 macrophages (CD163) in the BLB construct was 3.00 ± 1.65% for the 3-day time point, 6.69 ± 3.99% for the 7-day time point, 3.00 ± 1.65% in the 14-day time point, 7.22± 1.39% for the 28-day time point, and 4.55 ± 0.86% for the acellularized group. For the M2 macrophage data (CD163), # indicates significance compared to the native ACL control. Total macrophage percentage was significantly higher at the 7-day time point. M2 macrophage percentage was significantly higher at both the 7-day and 28-day time points when compared to native ACL (p<0.05).