Injectable Affinity and Remote Magnetothermal Effects of Bi-Based Alloy for Long-Term Bone Defect Repair and Analgesia

Abstract

As alternatives, metallic/nonmetallic bone graft materials play significant roles in bone defect surgery to treat external trauma or bone disease. However, to date, there are rather limited long-term implantable materials owning to in situ molding incapability of metallics and poor mechanical property of nonmetallics. Here, Bi-based low melting point alloy, with unique properties of injectability, solid-liquid phase transition, mechanical capability, and biocompatibility, present obvious long-lasting bone affinity as the excellent artificial bone-substitute. It is particularly necessary to point out that the targeted injected Bi alloy remains in its original position for up to 210 days without moving, as well as, displays good osseointegration ability to resolve repeated revision trauma caused by losing bone repair material. Additionally, with outstanding electrical and thermal conductivity, an unconventional way using Bi alloy to realize very beneficial hyperthermia analgesia via non-invasive wireless energy delivery is first proposed, which avoids adverse effects on bone remodeling inflicted by traditional drugs. The significantly decreased expression of pain sensitizing factor, such as, interleukin-6, neuropeptide substance, and transient receptor potential vanilloid 1 reveals the potential mechanism of hyperthermia analgesia. The present findings suggest the combination therapy of Bi alloy in bone repair and analgesia, which owns far-reaching clinical application value.

Keywords: bone defect repair; hyperthermia analgesia; injectable Bi alloy; metallic graft materials; non-invasive wireless energy delivery.

Scheme 1

Schematic illustration of Bi alloy‐based bone defect filling and analgesia. A) Bone defect model construction. B) Treatment of bone defect by filling Bi alloy with a heated syringe. Micro‐CT image shows metal filling position (the fillers in bone marked green represent Bi alloy). C) The temperature of Bi alloy was controlled remotely with AMF to suppress pain. D) Bone defect leads to increased expression of pain sensitizing factor (IL‐6, SP) and pain receptor (TRPV1), and enhanced pain signal transmission. E) After magnetic hyperthermia, sensitization factors and pain signals are inhibited and pain is released.

Figure 1

Characterization of Bi alloy (Bi₃₅In_48.6Sn₁₆Zn_0.4). A) The optical photo and B) SEM image of Bi alloy. C,D) are the EDS maps corresponding to (B). E) XRD patterns of Bi alloy. F) Stress–strain curves of Bi alloy under compression. G) Stress–strain curves of Bi alloy in tension. H) DSC curves of Bi alloy. I) Injectability of Bi alloy via heatable syringe. J) The temperature–time curve of Bi alloy injecting process. K) Thermal cycling by switching the AMF ON for 60 s and AMF OFF for 30 s (the test lasted for 6 min).

Figure 2

In vitro and in vivo repair of bone defects. A) Bone defect model of chicken legs (a₁ and a₂) and the implant of Bi alloy into the bone defect (a₃), as well as, the Micro‐CT imaging of bone repair (a₄). B) The binding force of Bi alloy embedded in bone tissue was tested through hanging weights and dynamometer. C) Bone defect model of rat legs and the implant of Bi alloy into the bone defect. D) Micro‐CT images of rat legs about non‐surgical group (control), bone defect group (sham), and Bi alloy implantation into bone defect group (Bi alloy) for 1, 15, 210 days. Arrows show the site of bone defect with Bi alloy or not.

Figure 3

Biocompatibility assessment of no material (control and sham) or Bi alloy in bone defect at 210 days after implantation. A) Unstained tissue sections show that the Bi alloy is intact and maintains a good filling effect at the defect. B) HE staining indicated a more complete structure around the filler Bi alloy and fewer inflammatory cells can be seen around it. C) TB staining shows that dark blue new regeneration bone tissue and pink new osteoblasts can be seen around the filling Bi alloy. D) VG staining shows pink new collagen fibers. All images were taken at 1× and 100× magnification.

Figure 4

Evaluation of the effects of magneto‐thermal analgesia and thermal stimulation on the physical condition of rats. A) The time course of changes in PWT values after operation was investigated (n = 5, ns p > 0.05, *p < 0.05; ^# p < 0.05, ^## p < 0.01). B) The time course of changes in PLL values after operation was investigated (n = 5, ns p > 0.05, *p < 0.05; ^## p < 0.01). C) The change of weight (n = 5, p > 0.05) and D) leg length (n = 5, p > 0.05) over time. The obtained data are expressed as mean ± SEM, p‐values are calculated using two‐tailed unpaired t‐test.

Figure 5

Immunohistochemistry for expression of IL‐6, SP, and TRPV1 in L4‐5 DRG after the treatment of pain by AMF or not among all groups. A) Light micrographs (×40) showing immunohistochemistry for IL‐6, SP, and TRPV1 in L4‐5 DRG. Arrows show some high expressions of them in neurons. Scale bar: 20 µm. Quantification of expression levels of IL‐6, SP, and TRPV1, as shown in (B, C and D), by the average optical density (AOD) of neurons in the staining sections. Results represent mean ± SEM in each group, n shown in histogram, p‐values are calculated using two‐tailed unpaired t‐test, ns p > 0.05, **p < 0.01, ****p < 0.0001, compared to naive control.

Figure 6

Toxicology evaluation of the Bi alloy in vitro and in vivo. A) The cell viability of BALB/c 3T3 cells incubated with the Bi alloy extract medium for 3 or 5 days (n = 7, p > 0.05). B) Live/dead cells image, scale bar 100 µm. C) The blood tests including (c₁) red blood cells counts (RBC), (c₂) white blood cells counts (WBC) and (c₃) percentage of lymphocytes (LYM) after surgery with or without implanting Bi alloy to the left leg for 0, 1, 2, 3, 5 weeks (n = 5, *p < 0.05). D) The blood tests including (d₁) RBC, (d₂) WBC, and (d₃) percentage of LYM after surgery with implanting Bi alloy to the left leg for 7 months (n = 5, p > 0.05). E) The biochemical tests about liver and kidney function including (e₁) alanine aminotransferase (ALT), (e₂) aspartate aminotransferase (AST), (e₃) creatinine (CREA), and (e₄) urea (UREA) levels in the blood after surgery with or without implanting Bi alloy to the left leg for 0, 1, 5, 10 weeks (n = 4, p > 0.05). F) The biochemical tests about liver function including (f₁) ALT and AST and kidney function including (f₂) CREA and UREA after implanting Bi alloy to the left leg for 7 months (n = 4, p > 0.05). G) Body weight (n = 5, p > 0.05) and H) leg length of rats after surgery with or without implanting Bi alloy to the left leg for 0, 1, 2, 3, 4, 5, 6 weeks (n = 5, p > 0.05). Results represent mean ± SEM in each group, p‐values are calculated using two‐tailed unpaired t‐test.