Interleukin 17 enhances bone morphogenetic protein-2-induced ectopic bone formation

Abstract

Interleukin 17 (IL-17) stimulates the osteogenic differentiation of progenitor cells in vitro through a synergy with bone morphogenetic protein (BMP)-2. This study investigates whether the diverse responses mediated by IL-17 in vivo also lead to enhanced BMP-2-induced bone formation. Since IL-17 is known to induce osteoclastogenesis, we studied the interactions between IL-17 and BMP-2 in ceramic scaffolds either or not carrying a coating with the bisphosphonate zoledronic acid (ZOL). Histological evaluation revealed that IL-17 alone did not induce any osteoclasts at day 10. On the other hand, BMP-2 clearly stimulated early tissue ingrowth and osteoclastogenesis. Both of these processes were blocked in presence of ZOL. IL-17 signaling restored early vascularized connective tissue formation and osteoclastogenesis induced by BMP-2 in ZOL-coated scaffolds. After 12 weeks, the bone volume induced by co-delivery of BMP-2 and IL-17 was doubled as compared to that induced by BMP-2 alone. We conclude that IL-17 has osteo-stimulatory effects through a synergy with bone-inductive BMP-2. Although local and single application of IL-17 does not mediate osteoclast formation, it could promote other processes involved in bone formation such as connective tissue ingrowth. The use of IL-17 may contribute to the development of improved bone graft substitutes.

Figure 1

Quantification of bone by histomorphometry. (A) Schematic representation showing the predefined subcutaneous implantation locations in the rabbit dorsum. (B) The bone tissue was stained with methylene blue/basic fuchsin on two MMA midsections. (C) The bone and scaffold material were pseudo-colored for calculation of the bone contact% and bone area%.

Figure 2

In vitro release of zoledronic acid (ZOL) from BCP scaffolds. Scaffolds precoated with 25 μg ZOL were incubated in MilliQ at 37 °C. The released amount of ZOL was measured in the supernatant at the indicated time points and normalized to the input. The values represent the mean ± SD for duplicate measurements.

Figure 3

Inflammatory response after 24 hours and 10 days. (A) H&E staining was performed on an empty scaffold retrieved after 24 days. This sample is representative for all 24 h samples. (B) Calprotectin staining (in brown, arrowheads) was performed to demonstrate activated phagocytes in the constructs after 24 h or 10 days. (C) Calprotectin-positive cell count at day 10, represented as the mean ± SD (n = 3). (D) CD3 staining for T lymphocytes was performed on an empty scaffold retrieved after 10 days (left panel). This sample is representative for all day 10 samples. A rabbit lymph node was used as a positive control (right panel). The top right insets provide an overview. BCP, biphasic calcium phosphate; F, fibrin network; N, neutrophils; RBC, red blood cells; T, connective tissue.

Figure 4

Connective tissue ingrowth in the 10-day study. (A) Goldner’s trichrome staining was indicative of collagen in connective tissue (in green); note differences in collagen between the periphery and central regions in a BMP-2-loaded construct without ZOL. CD31-positive blood vessels (BV, in brown) were only found in regions where connective tissue (T) had formed. (B) Goldner’s trichrome staining showing differences in connective tissue formation between groups. A region of interest is shown which covers half of the construct’s total area from the periphery (left) to the center (right). The areas within the dotted lines indicate regions of vascularized connective tissue formation. All images are representative for the group. (C) Quantification of vascularized connective tissue ingrowth, represented as the mean ± SD (n = 3). BCP, biphasic calcium phosphate; T, connective tissue; RBC, red blood cells.

Figure 5

Day 10 H&E staining. Images are taken from the edge of the scaffold and are representative for the group. Arrowheads point to multinucleated cells indicative of osteoclasts. Asterisks indicate blood vessels. BCP, biphasic calcium phosphate.

Figure 6

Role of osteoclasts during the early tissue response. (A) Tartrate-resistant acid phosphatase (TRAP) staining shows day-10 presence of osteoclasts (OCL, in red) and mononucleated TRAP-positive cells (indicated by asterisks). ZOL-coated and non-coated biphasic calcium phosphate (BCP) scaffolds were left untreated (control), or were loaded with IL-17 (100 ng/construct), BMP-2 (3 μg/construct), or their combination. Representative images of each group are given, with the scaffold overview in insets. (B) Osteoclast counts are represented as the mean ± SD (n = 3). Mononucleated TRAP-positive cells were not included in the osteoclast counts.

Figure 7

Presence of macrophages at day 10. Zoledronic acid (ZOL)-coated and non-coated biphasic calcium phosphate (BCP) scaffolds were loaded with BMP-2 (3 μg/construct) and/or IL-17 (100 ng/construct) and implanted subcutaneously for 10 days. (A) Images taken from the edge of the constructs stained with the RAM11 antibody (brown) show the presence of connective tissue macrophages (M) and multinucleated cells resembling osteoclasts (OCL). (B) Consecutive sections showing the co-localization of multinucleated RAM11-positive and tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts (OCL). Mononucleated RAM11-positive cells indicate connective tissue macrophages (M). Images are representative for BMP-2-loaded constructs without ZOL.

Figure 8

Bone formation in scaffolds with suboptimal BMP-2 dose after 12-week implantation (A) Methylene blue/basic fuchsin staining of bone (B, pink), with the scaffold overview in insets (B,C) Quantification of bone formation as bone area% (B) and bone contact% (C). Data are represented as the mean ± SD (n = 8). *p < 0.05. BCP, biphasic calcium phosphate; A, adipose tissue; T, connective tissue.

Figure 9

Presence of osteoclasts in the newly formed bone after 12-week implantation. (A) Tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts was performed on empty biphasic calcium phosphate (BCP) scaffolds (control) or scaffolds loaded with IL-17 (100 ng/construct). (B) ZOL- coated and non-coated scaffolds were loaded with BMP-2 (3 μg/construct) with or without IL-17 (100 ng/construct) and implanted subcutaneously. TRAP staining shows the presence of bone (B)-lining osteoclasts (OCL, in red). The images for this IL-17 concentration are representative for all IL-17 concentrations. (C) Osteoclast counts are represented as the mean ± SD (n = 8).