Preparing Sr-containing nano-structures on micro-structured titanium alloy surface fabricated by additively manufacturing to enhance the anti-inflammation and osteogenesis

Highlights

• TC4 alloys with micro-structured surface were fabricated by SLM technique.

• The nano-structures of SrTiO₃ were fabricated by hydrothermal technology.

• Sr²⁺-containing micro/nano structure down-regulated the expression of pro-inflammatory genes.

• The osteogenic differentiation potential of macrophages was promoted on the micro/nano-structured surface.

• Micro/nano-structured surface boosted the proliferation and differentiation of osteoblast-like cells.

Abstract

The development of additive manufacturing technology has made it possible to customize joint implants. However, the fibrous tissue caused by long-term chronic inflammation delays bone regeneration. Moreover, the discovery of micro/nano-structure on the natural bone makes the study of implant surface morphology meaningful. In this study, a Sr-containing nano-structure on micro-structured titanium alloy surface was fabricated to enhanced the anti-inflammatory and osteogenic properties of implants. Ti6Al4V (TC4) alloys with micro-structured surface prepared by additive manufacturing were used as the material base model. Subsequently, spherical SrTiO₃ particles were fabricated on the TC4 surfaces by hydrothermal treatment. The anti-inflammatory and osteogenic performance of smooth surface, micro-structured surface, Sr-containing nano-structured surface and Sr-containing micro/nano-structured surface were investigated. In vitro results exhibited that the macrophages cultured on micro/nano-structured surface were polarized to anti-inflammatory M2 phenotype and enhanced the expression of osteogenic growth factors. The Sr-containing micro/nano-structured surface effectively upgraded the proliferation and differentiation of SaOS-2 cells compared with other surfaces. Sr²⁺ and micro/nano-structure effectively enhanced the anti-inflammatory and osteogenic properties of titanium alloys. This finding suggested that the micro/nano-structured surface doped with bioactive elements is expected to broaden the horizons of biomedical materials.

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

Introduction

With the globally increasing number of aging populations, the number of knee and hip joints replacement surgeries caused by cartilage wear and bone degeneration performed annually is increasing at a considerably high rate [1]. However, the demand for customization of joint implants is increasing due to the difference in height and weight of the patients. As a new way of producing joint implants, additive manufacturing (AM) technology is characterized by high precision and much greater design freedom compared with traditional methods [2], [3], [4]. More importantly, AM can easily realize the personalized design of parts, which has unique advantages in fabricating orthopedic implants of different shapes [5]. As the most popular AM technology for metal materials, selective laser melting (SLM) technology can process functional metal parts with complex geometric appearance and excellent mechanical properties [6], [7]. Therefore, it is increasingly used in the preparation of biomedical materials [8], [9], [10], [11]. In addition, the surfaces of the implant prepared by SLM are far from smooth and often have complex structures at micro-scale [12]. This micro-structure can promote the differentiation of stem cells into osteoblasts in vivo [13]. Nevertheless, as foreign bodies, the implants may induce the long-term chronic inflammation in vivo [14]. In our previous work, we noticed that the fibrous tissue was formed in the interface between Ti and nature bone due to the long-term chronic inflammation [15].

The natural micro/nano-structure on the surface of bone tissue was considered as a biological signal, which induces osteoblasts to migrate to the site of bone injury [16]. Therefore, from the perspective of bionics, constructing suitable micro/nano-structure on the surface of biomaterials is expected to improve the osteogenic properties. Shen et al. [17] found the micro/nano morphology of titanium surface can significantly promote the alkaline phosphatase activity, osteogenesis-related gene expression and in vitro mineralization of stem cells compared with smooth surfaces. Our research group found that micro/nano-structured surfaces of Ti implants regulated the immunomodulation of osteoblast by promoting the secretion of macrophage growth factor [18].

Macrophages, as a typical representative of immune cells, have a vital role in the inflammatory response after implant placement. According to the surface markers and cytokines, the phenotypes of macrophages are generally divided into pro-inflammatory M1 and anti-inflammatory M2 [19]. The transform of macrophages phenotypes was influenced by the surface morphology and functional ions released from the biomaterials [20], [21]. Murray et al. [22] found that M1 phenotype is conductive to clean bone debris at the injury site and form initial blood vessels during acute inflammation. However, uncontrollable and chronic inflammation is detrimental and causes delayed bone regeneration or fibrous encapsulation [15]. In contrast, M2 phenotype stimulates bone repairing by secreting growth factors and anti-inflammatory cytokines, and promotes the formation of blood vessels [23], [24]. Thus, it will be of great meaning to build nano-structures on the surface of additively manufactured implants with reduced inflammatory response and enhanced osteogenic properties.

Strontium (Sr), as a vital trace element in human body, has attracted increasing attention from researchers. It has been reported that Sr²⁺ reduces the early inflammatory response, reduces bone resorption and stimulates osteogenesis [25]. A small amount of Sr²⁺ can stabilize bone structure by inhibiting osteoclast activity and stimulating osteoblast maturation [26]. Thus, Sr has been investigated for a remedy and treatment for osteoporosis [27]. Romero-Gavilan et al. [28] found that the expression of marker of the early stages of inflammation was significantly reduced on the surfaces of Sr²⁺-containing samples, suggesting that Sr²⁺ might be able to reduce the pro-inflammatory effects of the implants. In general, the nano-structure containing Sr²⁺ were fabricated on the surface of Ti alloys by surface modification technology [29]. Therefore, we expect that the Sr-containing nano-structure built on the micro-structure surface of SLMed Ti alloys would improve anti-inflammatory and osteogenic properties.

Due to the excellent biocompatibility, resistance to fatigue loading and inherent high strength to weight ratio, the additively manufactured Ti6Al4V (TC4) alloys are very common biomaterials for joint replacement [30], [31]. In this study, TC4 alloy with micro-structured surface prepared by SLM technique was used as the material base model. Subsequently, nano-SrTiO₃ particles were fabricated on the surface of TC4 by hydrothermal treatment. The effect of smooth surface, micro-structured surface, Sr²⁺-containing nano-structured surface and Sr²⁺-containing micro/nano-structured surface on the behavior of macrophages was analyzed. Subsequently, the osteogenic differentiation potential of SaOS-2 cells in response to the surfaces with different characteristic was investigated.