Physicochemical properties and angiogenesis-promoting effects of copper-containing calcium sulfate bone cement

Abstract

BACKGROUND: Calcium sulfate has been widely used as a bone graft for the treatment of alveolar bone loss, endodontic lesions, and periodontal disease. Copper plays an important role in various biological processes, including angiogenesis and cell migration. OBJECTIVE: To prepare copper-containing calcium sulfate bone cement and characterize its physicochemical properties and angiogenesis. METHODS: (1) Calcium sulfate hemihydrate was used as the solid phase and copper sulfate pentahydrate solutions of varying concentrations were used as the liquid phase. The solid and liquid phases were mixed at a ratio of 1.7 g/1 mL. The mass ratios of copper sulfate pentahydrate to calcium sulfate hemihydrate were 0.1%, 0.5%, 1%, and 2.5%, respectively. The prepared copper-containing calcium sulfate bone cements were designated 0.1%Cu-CS, 0.5%Cu-CS, 1%Cu-CS, and 2.5%Cu-CS, respectively. Pure calcium sulfate bone cement was also prepared. The micromorphology, compressive strength, and copper and calcium ion release in the in vitro degradation solution of the five cements were characterized. (2) Thirty SD rats selected and a single cortical bone defect model with a diameter of 3 mm and a length of 5 mm was established on the left tibia. These models were randomly divided into three groups: a blank group (n=10) received no intervention; a control group (n=10) received calcium sulfate bone cement implantation; an experimental group (n=10) received 0.5%Cu-CS bone cement implantation. At 6 weeks postoperatively, vascular Microfil perfusion followed by Micro-CT scanning was performed to observe angiogenesis at the tibial defect site, and CD31 immunohistochemical staining was used to observe angiogenesis. RESULTS AND CONCLUSION: (1) Scanning electron microscopy showed rod-like calcium sulfate crystals on the surface of calcium sulfate bone cement, while plate-like gypsum crystals were present on the surface of copper-containing calcium sulfate bone cement. With increasing copper sulfate pentahydrate content, the number of plate-like gypsum crystals increased. The compressive strength of 0.5%Cu-CS and 1%Cu-CS cements was higher than that of calcium sulfate cement and 0.1%Cu-CS cement (P < 0.05), and the compressive strength of 2.5%Cu-CS cement was higher than that of 0.5%Cu-CS and 1%Cu-CS cements (P < 0.05). After immersion in simulated body fluid for 6 weeks, calcium sulfate cement showed the highest calcium ion release concentration, while 2.5%Cu-CS cement showed the lowest. No copper ion release was detected from calcium sulfate cement; among copper-containing cements, 2.5%Cu-CS had the highest copper ion release, and 0.1%Cu-CS had the lowest. (2) Micro-CT scanning showed that the blank group had the least new blood vessel formation at the bone defect, while the experimental group had the most. CD31 immunohistochemical staining showed that the blank and control groups only exhibited punctate or linear new vascular structures, whereas the experimental group showed abundant and dense vascular formation. (3) These results indicate that copper-containing calcium sulfate bone cement possesses good mechanical properties and sustained copper ion release, and can promote angiogenesis.

1. Introduction

Calcium sulfate has been used clinically for over 100 years [1], and it possesses good biocompatibility and complete absorbability [2]. As a bone graft, calcium sulfate is used in the treatment of alveolar bone loss, endodontic lesions, and periodontal disease [3-4]. Studies have reported that calcium sulfate can serve as a carrier for antibiotics such as amoxicillin and moxifloxacin in the treatment of chronic osteomyelitis [5-7], and as an antibiotic carrier for prosthetic joint infections. Furthermore, calcium sulfate has some osteoregenerative effects; SHAH et al. [8] used calcium sulfate in surgeries for upper limb nonunion and malunion, and CEN et al. [9] found that poly-L-lactic acid/calcium sulfate composites exhibit significant immunomodulatory effects on bone. ZHANG et al. [10] showed that phosphosilicate bioactive glass and calcium sulfate composites can promote the proliferation and angiogenesis of human umbilical vein endothelial cells, indicating that calcium sulfate has certain pro-angiogenic effects.

Metal ions have advantages such as low cost, high stability, and clinical safety, making them attractive as bioactive factors for bioceramic materials [11]. Copper is an essential micronutrient for normal development in eukaryotes and plays key roles in many cellular and physiological activities, including enzyme activity, oxygen transport, and cell signaling [12]. FINNEY et al. [13] reported that copper is essential for angiogenesis and plays a critical role in skin regeneration and angiogenesis. BARRALET et al. [14] showed that low-dose copper-loaded calcium phosphate scaffolds can promote angiogenesis and wound healing. KONG et al. [15] confirmed that the synergistic effect of copper and silicon ions in copper-containing calcium silicate promotes angiogenesis. LI et al. [16] prepared a dual-network composite gel with self-regulating copper ion release to promote wound healing. LI et al. [17] studied the immunomodulatory and pro-angiogenic effects of copper-containing titanium alloy (Ti6Al4V-1.5Cu) and found that it promotes angiogenesis by releasing copper ions and inhibiting inflammation. In bioactive nanoparticles, copper regulates various cytokines and growth factors involved in different stages of wound healing [18]; additionally, copper has antibacterial activity and synergistic effects in promoting wound healing [19-20]. Therefore, the authors hypothesized that the addition of copper ions to calcium sulfate may have a synergistic biological effect, improving the properties of this bone substitute. In this study, we prepared copper-containing calcium sulfate cement and analyzed its in vivo pro-angiogenic effects.