Effects on Tissue Integration of Collagen Scaffolds Used for Local Delivery of Gentamicin in a Rat Mandible Defect Model

Abstract

Surgical site infections (SSIs) are a common complication following orthopedic surgery. SSIs may occur secondary to traumatic or contaminated wounds or may result from invasive procedures. The development of biofilms is often associated with implanted materials used to stabilize injuries and to facilitate healing. Regardless of the source, SSIs can be challenging to treat. This has led to the development of devices that act simultaneously as local antibiotic delivery vehicles and as scaffolds for tissue regeneration. The goal for the aforementioned devices is to increase local drug concentration in order to enhance bactericidal activity while reducing the risk of systemic side effects and toxicity from the administered drug. The aims of this study were to assess the effect of antibiotic loading of a collagen matrix on the tissue integration of the matrix using a rat mandibular defect model. We hypothesized that the collagen matrix could load and elute gentamicin, that the collagen matrix would be cytocompatible in vitro, and that the local delivery of a high dose of gentamicin via loaded collagen matrix would negatively impact the tissue-scaffold interface. The results indicate that the collagen matrix could load and elute the antimicrobial gentamicin and that it was cytocompatible in vitro with or without the presence of gentamicin and found no significant impact on the tissue-scaffold interface when the device was loaded with a high dose of gentamicin.

Keywords: biocompatibility; collagen matrix; drug elution; gentamicin; local drug delivery; mandibular model; surgical site infections; tissue regeneration.

Figure 1

Median gentamicin elution from Fibro-Gide^® cylinders in PBS over a 14-day period. Log transformation applied to best visualize elution curve, including both the initial burst release and sustained lower-level release of gentamicin.

Figure 2

MTS assay to measure cellular proliferation. Left-side panel (cells) demonstrates mean cellular proliferation, measured through absorbance, of MC3T3-E1 cells in cell culture exposed to standard media, or a low or high dose of gentamicin, through time. Right-side panel (Sc. Cells) demonstrates mean cellular proliferation, measured through absorbance, of MC3T3-E1 cells on Fibro-Gide^® wafers exposed to standard media, or a low or high dose of gentamicin, through time. Cells in cell culture had a significantly higher proliferation than cells on collagen matrix (p < 0.0001). Sc. Cells, cells on Fibro-Gide^® wafers.

Figure 3

Images of calcein-AM staining of MC3T3-E1 cells on Fibro-Gide^® wafers with inset positive control images of MC3T3-E1 cells in cell culture. (1A–1C) Cells on matrix on days 3 (A), 5 (B), 7 (C) in standard media. (1a–1c) Cells in culture on days 3 (a), 5 (b), 7 (c), standard media. (2A–2C) Cells on matrix on days 3 (A), 5 (B), 7 (C) exposed to low dose gentamicin. (2a–2c) Cells in culture on days 3 (a), 5 (b), 7 (c), low-dose gentamicin. (3A–3C) Cells on matrix on days 3 (A), 5 (B), 7 (C), exposed to high-dose gentamicin. (3a–3c): Cells in culture on days 3 (a), 5 (b), 7 (c), high-dose gentamicin.

Figure 4

SEM and H&E-stained histology images of Fibro-Gide^® collagen matrix. (A) SEM image of Fibro-Gide^® cylinder. (B–D) Histology images taken at 40× magnification of Fibro-Gide^® seeded with MC3T3-E1 cells on days 3 (B), 5 (C), 7 (D) of cell culture. Dark pink material is collagen matrix, dark purple material is cell nuclei, denoted by black arrows.

Figure 5

Three-dimensional CT scan renderings of mandibular defect. Image (A) displays the ideal placement of the critical-sized (5 mm diameter), full-thickness bone defect on rodent hemimandible. Image (B) demonstrates defect placement that is too far caudal on the hemimandible. Both images provide appreciation for the difficulty of completing quantitative analysis due to the small defect and animal size.

Figure 6

Histology images from control and experimental animals. (1A) 5× magnification, demonstrating cellular and connective tissue infiltration into the native collagen matrix (control animal, rat #7). (1B) 20× magnification, demonstrating cellular and connective tissue infiltration into native collagen matrix (control animal, rat #7). (2A) 5× magnification, demonstrating cellular and connective tissue infiltration into antibiotic-loaded collagen matrix (experimental animal, rat #4). (2B) 20× magnification, demonstrating cellular and connective tissue infiltration into antibiotic-loaded collagen matrix (experimental animal, rat #4). (3A) 5× magnification, demonstrating thick ring of fibrous tissue separating bone from native collagen matrix and surrounding suppurative inflammation (control animal, rat #9). (3B) 40× magnification, demonstrating dense population of neutrophils (left side of image), and necrotic cellular infiltration into native collagen matrix (control animal, rat #9). (4A) 10× magnification, demonstrating cellular and connective tissue infiltration into antibiotic-loaded collagen matrix (experimental animal, rat #12). (4B) 40× magnification, demonstrating presence of multinucleated giant cells, as well as blood vessels, throughout antibiotic-loaded collagen matrix (experimental animal, rat #12). Boxes are surrounding select blood vessels to highlight angiogenesis throughout the collagen matrix. Stars denote bone. Double-sided arrow highlights the thick rim of fibrous connective tissue. Thick arrows point at select multi-nucleated giant cells.