Thrombopoietic agents enhance bone healing in mice, rats, and pigs

Abstract

Achieving bone union remains a significant clinical dilemma. The use of osteoinductive agents, specifically bone morphogenetic proteins (BMPs), has gained wide attention. However, multiple side effects, including increased incidence of cancer, have renewed interest in investigating alternatives that provide safer, yet effective bone regeneration. Here we demonstrate the robust bone healing capabilities of the main megakaryocyte (MK) growth factor, thrombopoietin (TPO), and second-generation TPO agents using multiple animal models, including mice, rats, and pigs. This bone healing activity is shown in two fracture models (critical-sized defect [CSD] and closed fracture) and with local or systemic administration. Our transcriptomic analyses, cellular studies, and protein arrays demonstrate that TPO enhances multiple cellular processes important to fracture healing, particularly angiogenesis, which is required for bone union. Finally, the therapeutic potential of thrombopoietic agents is high since they are used in the clinic for other indications (eg, thrombocytopenia) with established safety profiles and act upon a narrowly defined population of cells.

Keywords: bone healing; bone regeneration; bone repair; fracture; thrombopoietin.

Graphical abstract

Figure 1

TPO promotes bone healing in a rat CSD model. Male Long Evans rats (450-500 g) underwent CSD surgeries for a pilot study. (A) Representative X-rays taken at 15-wk post-surgery with control (saline, n = 3), BMP-2 (10 μg, n = 3), or TPO (10 μg, n = 4) scaffolds. DCPD cement was used to carry the drugs. (B) Bridging X-ray scoring for rats at 15-wk post-surgery. 0 = no bony callus, 1 = partial healing across the gap, 2 = complete callus bridging across the gap, based on T-MG, by Chu et al. (C) μCT of rat midshaft femoral defects at 15-wk post-surgery. (D) Widest callus diameter measured in histological sections shown in panel E. (E) MacNeal’s staining of the fractured femur indicates presence of more bone in calluses of TPO-treated rats compared to BMP-2 or saline treated rodents. Arrows indicate mineralization at the boundary between the scaffold and pin in TPO group. (F and G) Quantitation of callus area and % bony callus, respectively. While callus area is not significantly different, the percentage of bone tissue in the calluses is, with TPO having greatest percentage of bony callus. (H) Rat CSD study flow chart conducted on Long Evan males. Data in panel B compared by Kruskal–Wallis test due to nonparametric data distribution. Data in panels D, F, and G are parametric and therefore were compared by one-way ANOVA with Tukey–Kramer post-hoc test, ^* = p < .05, ^** = <.01. “S” indicates scaffold in panel E.

Figure 2

Thrombopoietic agents promote bone healing in mouse CSD models. Ten-wk-old, male C57BL/6J mice underwent CSD surgeries (n = 5/group). (A) Representative X-rays of mouse femurs 2-, 4-, and 6-wk post-surgery with control (saline), BMP-2 (4 μg), or TPO (1 μg) scaffolds. DCPD cement was used to carry the drugs. Periosteal bridging evident in 4- vs 6-wk TPO femurs. Arrows: unhealed periosteum; brackets: bridged callus. (B) Representative μCT reconstructions of mouse midshaft femoral defects 15-wk post-surgery, with control (saline), BMP-2 (4 μg), or TPO (1 μg) scaffolds (green). DCPD cement was used to carry the drugs. (C) Representative X-rays of mouse femurs 10-wk post-surgery with control (saline), BMP-2 (1 μg), TPO (5 μg), or romiplostim (25 μg). A collagen sponge secured around the scaffold was used to carry the drugs. (D) Bridging X-ray scoring for mice at 10-wk post-surgery. 0 = no bony callus, 1 = partial healing across the gap, 2 = complete callus bridging across the gap, based on T-MG, by Chu et al. Each therapy induced more partial or full healing of fracture compared to control mice, as would be expected in a CSD model. (E) Experimental design flow chart for murine CSD experiments shown here in Figure 2A and B. (F) Experimental design flow chart for murine CSD figures shown here in Figure 2C and D. Nonparametric data compared by Kruskal–Wallis test. ^* = <.05, ^** = <.01, ^*** = <.001. n = 8 saline, n = 9 BMP-2 and TPO, n = 10 for Romiplostim.

Figure 3

TMP promotes bone healing in a closed fracture model. Female Swiss Webster mice were 12 wk of age when subjected to Einhorn fracture. (A) Structure of TMP. (B) Transverse μCT slice through fracture callus 3-wk post-surgery. (C-E) μCT analyses showed that treatment with 33 nmol/kg/d of TMP significantly increases multiple fracture callus properties compared to vehicle controls (saline). (F-H) Biomechanical testing demonstrated that treatment with 33 nmol/kg/d of TMP (n = 4) significantly increased the amount of energy the healed femur absorbed before it re-fractured (F), the maximum load to failure (G), and the stiffness (H) compared to saline controls (n = 5). (I) Experimental design flow chart for female Swiss Webster mouse data shown here in Figure 3 and Figure S4. All statistical comparisons performed via Student’s t-test.

Figure 4

TPO promotes bone healing in a pig CSD model. Skeletally mature Yucatán miniature pigs were approximately 20 mo of age at the time of CSD surgery. (A) X-ray of pig tibial CSD, 6 mo post-surgery with control (n = 7), 1.5 mg BMP-2 (n = 8), or 1.5 mg TPO treated scaffolds (n = 8 total; n = 4 healed and n = 4 not healed). A collagen sponge secured around the scaffold was used to carry the drugs. (B) Radiographic union score for tibial fractures (RUST score) indicates that all BMP-2 treated pigs healed, 4 of 8 TPO treated pigs healed, and none of the saline treated pigs healed. (C) Torsion stiffness of operated tibia as a percentage of contralateral tibia confirms healing/non-healing RUST scores. Significant differences determined by ANOVA with Tukey–Kramer post-hoc test. (D) Correlation between RUST score and torsional stiffness determined by Pearson’s correlation test demonstrates an R² value of 0.8396. (E) Experimental design flow chart for male Yucatán miniature pig CSD data shown here in Figure 4.

Figure 5

TPO promotes the upregulation of the angiogenesis pathway and growth factor activation. Bone/bone marrow cells were cultured with saline, BMP-2 (200 ng/mL), or TPO (100 ng/mL) for 48 hr and were processed for transcriptomic analysis (n = 4/group). (A) Functional network analysis found the angiogenesis pathway, as one of the key differential markers between BMP-2 and TPO treatment. The angiogenesis pathway was significantly activated in TPO treated samples (z-score = 1.15) but inhibited in BMP-2 treated samples (z-score = −1.86). Genes enriching the angiogenesis pathway are listed in Table S2. Network shown for TPO treatment only. Oval shaped nodes represent DEGs. (B) The color key with respect to genes log₂ (fold change) is shown at the bottom of the network for TPO and BMP-2; red and green colored genes were upregulated and down regulated, respectively. Genes are clustered into cellular compartments where the corresponding proteins are typically expressed. (C-E) Fracture callus was assessed 3-d post-surgery for mice treated with saline, BMP-2 (5 μg), or TPO (5 μg) and was then examined by multianalyte TGF-beta phospho-profiling arrays (n = 4/group). TPO increased activation of AKT1 (C), AKT2 (D), and PAK1 (E), whereas BMP-2 did not. Significant differences were determined by ANOVA with Tukey–Kramer post-hoc test.

Figure 6

TPO treatment promotes endothelial cell growth in vivo and in vitro. (A and B) Treatment of WT bone marrow endothelial cells (BMECs) with TPO (100 ng/mL) or BMP-2 (200 ng/mL) increased BMEC numbers (A) and vessel-like structure length or tube length (% relative to WT + saline control) (B). However, treatment of Mpl KO BMECs with TPO or BMP-2 did not increase BMEC numbers (A) nor tube length (B). Of note, cell number (A) and tube length (B) were significantly higher (or trending higher) in WT BMEC cultures compared to KO BMEC cultures with the same treatment (n = 3/group). Significant differences were determined by ANOVA with a Tukey–Kramer post-hoc test. (C) Working model as to how TPO stimulates angiogenesis and bone healing. TPO treatment upregulates the expression of PDGF, Angpt1, AKT, and MAPK, which can promote angiogenesis, a critical process of successful fracture healing. TPO also stimulates MKs, which increase osteoblast proliferation and bone formation. Further, TPO increases osteoclastogenesis, which is important to remove necrotic tissue and to remodel the fracture callus.