Effects of BMP-12-releasing sutures on Achilles tendon healing

Abstract

Tendon healing is a complex coordinated event orchestrated by numerous biologically active proteins. Unfortunately, tendons have limited regenerative potential and as a result, repair may be protracted months to years. Current treatment strategies do not offer localized delivery of biologically active proteins, which may result in reduced therapeutic efficacy. Surgical sutures coated with nanostructured minerals may provide a potentially universal tool to efficiently incorporate and deliver biologically active proteins directly to the wound. Additionally, previous reports indicated that treatment with bone morphogenetic protein-12 (BMP-12) improved tendon healing. Based on this information, we hypothesized that mineral-coated surgical sutures may be an effective platform for localized BMP-12 delivery to an injured tendon. The objective of this study was, therefore, to elucidate the healing effects of mineral-coated sutures releasing BMP-12 using a rat Achilles healing model. The effects of BMP-12-releasing sutures were also compared with standard BMP-12 delivery methods, including delivery of BMP-12 through collagen sponge or direct injection. Rat Achilles tendons were unilaterally transected and repaired using BMP-12-releasing suture (0, 0.15, 1.5, or 3.0 μg), collagen sponge (0 or 1.5 μg BMP-12), or direct injection (0 or 1.5 μg). By 14 days postinjury, repair with BMP-12-releasing sutures reduced the appearance of adhesions to the tendon and decreased total cell numbers. BMP-12 released from sutures and collagen sponge also tended to improve collagen organization when compared with BMP-12 delivered through injection. Based on these results, the release of a protein from sutures was able to elicit a biological response. Furthermore, BMP-12-releasing sutures modulated tendon healing, and the delivery method dictated the response of the healing tissue to BMP-12.

FIG. 1.

Formation of mineral coating on surgical sutures. (a) Scanning electron microscopy (SEM) images of as-received Mersilene, Ethibond Excel, Proline, and Polysorb (top panel), and mineral-coated (bottom panel) surgical sutures indicating successful mineral coating on various types of sutures. (b) SEM images of as-received Vicryl suture (top) and mineral-coated (bottom) Vicryl suture. (c) SEM image and energy dispersive spectroscopy spectrum (inset) of calcium phosphate (CaP) coating surface. X-ray diffraction spectroscopy (d, * indicates the characteristic peaks of CaP) and Fourier transform infrared (e, * and ^ denote the peaks from phosphate and carbonate, respectively) spectra of CaP coating created on coated Vicryl.

FIG. 2.

Protein binding on mineral-coated device. Micro-BCA results quantifying the amount of 100 or 500 μg/mL cytochrome c remaining in solution before (Control) and after the addition of the mineral-coated scaffold. Samples were collected at 1, 2, 3, and 4 h postincubation. At 100 μg/mL cytochrome c, protein concentration decreased significantly from solution (indicating increased binding to mineral coating) within 1 h of exposure to mineral coating. Incubation beyond 1 h was not different. At 500 μg/mL cytochrome c, the amount of unbound protein was significantly reduced by 4 h. If analysis of variance (ANOVA) results were p<0.10, Scheffe's post hoc pairwise analysis was performed. ^a,b Within the concentration indicates bars without a common superscript letter differ (p<0.05). Values are expressed as mean ranking±standard error of the mean (S.E.M.).

FIG. 3.

Spatial localization of mineral coating within the day 14 Achilles tendon. Graphs showing (a) Alizarin Red results between treatment groups after Achilles tendon repair with mineral-coated sutures with or without bone morphogenetic protein 12 (BMP-12) (a), the spatial distribution of mineral coating throughout the Achilles tendon (b); and representative micrographs of Alizarin Red staining within the tendon after repair with mineral coating sutures (c). Quantity of Alizarin Red, a stain for calcium, was similar among all treatment groups (a). Mineral coating was primarily localized to the suture area (b). Granulation tissue and the tendon ends (i.e., outside of the granulation tissue) also had measurable amounts of mineral, but were significantly lower than the suture area (b). Representative micrographs of mineral-coated (top left), BMP-12-releasing sutures (top right), sponge (bottom left), and BMP-12-injected and mineral-coated suture repair (bottom right), indicating Alizarin Red-stained mineral coating within the tendon (c). The sponge group represented a negative control for Alizarin Red staining. Arrows indicate areas positive for Alizarin Red staining. The p-value beneath the graph indicates ANOVA results. If ANOVA results were p<0.10, Scheffe's post hoc pairwise analysis was performed. ^a,b,c Within a graph indicates bars without a common superscript letter differ (p<0.05). Values are expressed as mean density±S.E.M. Color images available online at www.liebertpub.com/tea

FIG. 4.

Effect of BMP-12-releasing sutures on macroscopic results on the day 14 healing tendon. Graphs showing (a) surrounding area adhesions, and (b) surrounding area vascularity 14 days postinjury, and (c) total cells after varying doses of BMP-12 released from mineral-coated suture or blank suture. Doses of 1.5 and 3.0 μg BMP-12 significantly reduced adhesions to the tendon (a). Similarly, 1.5 μg BMP-12 significantly reduced the appearance of surrounding area adhesions (b). A dose of 3.0 μg BMP-12 reduced total cell number within the healing tendon (c). The p-value beneath the graph indicates ANOVA results. If ANOVA results were p<0.10, Scheffe's post hoc pairwise analysis was performed. ^a,b,c Within a graph indicates bars without a common superscript letter differ (p<0.05). Values are expressed as mean ranking±S.E.M. * Indicates p-value=0.0572.

FIG. 5.

Representative Hematoxylin and Eosin (H&E) micrographs of the granulation tissue after repair with the respective treatments. g.t., granulation tissue. Color images available online at www.liebertpub.com/tea

FIG. 6.

Effects of 1.5 μg BMP-12 using different delivery methods. Comparison of BMP-12 delivered through suture, sponge, and injection on (a) surrounding area adhesions, (b) surrounding area vascularity, (c) proliferating cells, (d) blood vessel lumen, (e) myofibroblasts, (f) type III collagen, (g) type I procollagen, and (h) collagen organization. The p-value beneath the graph indicates ANOVA results. If ANOVA results were p<0.10, Scheffe's post hoc pairwise analysis was performed. ^a,b,c Within a graph indicates bars without a common superscript letter differ (p<0.05). Values are expressed as mean density±S.E.M. *indicates p-value=0.08.

FIG. 7.

Use of H&E-stained samples for collagen quantification through fractal analysis. Graph showing collagen organization of the normal healing (without treatment) rat medial collateral ligament (MCL) (a). Intact tissue exhibits the greatest organization (i.e., lowest fractal dimension). Early ligament healing demonstrates significantly worse collagen organization (i.e., higher fractal dimension). By day 28, collagen organization has improved, but remains worse than the intact control. Graph demonstrating second-order polynomial curve fit of the normal healing results (b). Representative H&E images of the intact, and day 5, 9, 11, 14, and 28 postinjured healing MCL used for fractal analysis (c). The p-value beneath the graph indicates ANOVA results. If ANOVA results were p<0.10, Scheffe's post hoc pairwise analysis was performed. ^a,b,c,d,e within a graph indicates bars without a common superscript letter differ (p<0.05). Values are expressed as mean density±S.E.M. Color images available online at www.liebertpub.com/tea