In situ forming biomaterials as muscle void fillers for the provisional treatment of volumetric muscle loss injuries

Abstract

Volumetric muscle loss (VML) represents a devastating extremity injury which leads to chronic functional deficits and disability and is unrecoverable through normal healing pathways. When left untreated, the VML pathophysiology creates many challenges towards successful treatment, such as altered residual muscle architecture, excessive fibrosis, and contracture(s). As such, innovative approaches and technologies are needed to prevent or reverse these adverse sequelae. Development of a rationally designed biomaterial technology which is intended to be acutely placed within a VML defect - i.e., to serve as a muscle void filler (MVF) by maintaining the VML defect - could address this clinical unmet need by preventing these adverse sequelae as well as enabling multi-staged treatment approaches. To that end, three biomaterials were evaluated for their ability to serve as a provisional MVF treatment intended to stabilize a VML defect in a rat model for an extended period (28 days): polyvinyl alcohol (PVA), hyaluronic acid and polyethylene glycol combination (HA + PEG), and silicone, a clinically used soft tissue void filler. HA + PEG biomaterial showed signs of deformation, while both PVA and silicone did not. There were no differences between treatment groups for their effects on adjacent muscle fiber count and size distribution. Not surprisingly, silicone elicited robust fibrotic response resulting in a fibrotic barrier with a large infiltration of macrophages, a response not seen with either the PVA or HA + PEG. Taken together, PVA was found to be the best material to be used as a provisional MVF for maintaining VML defect volume while minimizing adverse effects on the surrounding muscle.

Keywords: Biocompatibility; Extremities; Military medicine; Prolonged care; Trauma.

Graphical abstract

Fig. 1

(A) Digital and SEM images of swelled biomaterials formed in vitro. (B) Calculated swelling ratio of in vitro formed biomaterials 24 h after incubation in PBS at 37 °C. (C) G′ of in vitro formed biomaterials. (D) Young's Moduli of in vitro formed biomaterials. Data analyzed by ANOVA followed by Holm-Sidak post hoc test (B,D) or analyzed by unpaired t-test (C). p-values less than 0.05 are listed for all comparisons. All data represented by mean ± SD.

Fig. 2

Digital photographs taken of the MVF in situ (A) immediately after formation and (B) at time of harvest 28 days after implantation (photographs taken from deep side of tibialis anterior with superficial fascia intact).

Fig. 3

(A) Representative cross sections of H&E and picrosirius red stained tissue 1 day and 28 days after MVF implantation (scale = 500 μm). (B) Rectangularity measurements of implanted MVF. (C) Representative close-up image from WGA-stained muscles (scale = 50 μm). (D) Muscle fiber count of muscles with MVF implantation 1 day and 28 days after implantation. Dotted line and shaded region represents the mean ± SD for fiber number from healthy tibialis anterior muscles. (E) Kernel density estimations of the distribution of minimum Feret diameters 1 and 28 days after MVF implantation. Data in panels B and D were analyzed first via a 2-way ANOVA followed by Holm-Sidak post hoc test. Median data in panel E were analyzed via Kruskal-Wallis test. Post hoc p-values less than 0.05 are listed for all comparisons. Data represented as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

(A) Color deconvolution of picrosirius red stained sections to isolate stained collagen. Zones of 25 μm thickness were made radiating out from the MVF-muscle border, pseudo-colored, and percent area of zone that consisted of collagen was quantified. Graph's dotted line and shaded region represents the mean ± SD for area of collagen in a healthy tibialis anterior muscle. *p < 0.05 for silicone vs PVA and silicone vs HA + PEG. (Scale = 500 μm) (B) Nuclei stained with DAPI and zones of 25 μm thickness were pseudo-colored and percent area of zone that consisted of positive DAPI signal was quantified (Scale = 50 μm). (C) Pan-macrophage marker CD68 was stained to look at cellular infiltrate. Analysis of total CD68 signal in cross section was quantified (Scale = 50 μm). All data were analyzed first via a 1 or 2-way ANOVA followed by Holm-Sidak post hoc test. Post hoc p-values less than 0.05 are listed for all comparisons. Data represented as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)