Preparing Sr-containing nano-structures on micro-structured titanium alloy surface fabricated by additively manufacturing to enhance the anti-inflammation and osteogenesis
Graphical Abstract
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.
Section snippets
TC4 alloys and Sr-containing coatings preparation
Pre-alloyed spherical TC4 powders in the size range of 30–40 µm were used for SLM processing. A SLM device (Farsoon FS271M, China) equipped with a fiber laser (laser spot size of 80 µm and maximal laser power of 500 W) was employed to manufacture the TC4 specimens. The hatch distance was 40 µm. The laser power was 400 W. The scanning speed was 1700 mm/s. The laser scanning spacing was 10 µm.
The SLM specimens were classed into two groups. The as-built samples were labelled as AMT, where the
Characterization of different surfaces
The surface morphology of different samples is exhibited in Fig. 1a-d. The PT and PT_Sr showed relatively smooth surfaces with a small amount scratch inherited from the grinding process. The molten pool boundaries which were formed by rapid cooling rate during SLM process were found on the surface of AMT and AMT_Sr. After hydrothermal treatment, uniform globular like nanoparticles were observed on the surface of PT_Sr and AMT_Sr. As presented in Fig. 1e and f, the surfaces of PT_Sr and AMT_Sr
The formation process of micro/nano-structures on surfaces
During SLM processing, the molten pool boundary induced by the pulsed laser was formed on the surface of samples, which lead to the formation of micro-structured surface on Ti alloys [32]. When the TC4 was exposed to air, a TiO2 layer was rapidly formed on the surface [33]. TiO2 film could react with strong alkali to form unstable [Ti(OH)6]2− monomers during hydrothermal reaction [34], [35]. The Na2Ti3O7 layer was formed by TiO2 layer reacting with Na+ and OH− within NaOH solution [36]. In the
Conclusion
In this study, TC4 alloys with micro-structured surface were fabricated by SLM technique. Subsequently, the nano-structures of SrTiO3 were successfully fabricated on TC4 surface by hydrothermal technology. In vitro experiments showed that Sr2+-containing micro/nano hierarchical structure stimulated macrophage to polarize to M2 phenotype, down-regulating the expression level of pro-inflammatory cytokine genes TNF-α and IL-6, and up-regulating the anti-inflammatory cytokine gene IL-10.
CRediT authorship contribution statement
Gen Li: Investigation, Sample preparation, Data analysis, Writing-original draft, Wentao Liu: Investigation, Writing-original draft, Luxin Liang: Physicochemical property tests, Investigation, Writing - review & editing, Tang Liu: Macrophage behavior tests, Yingtao Tian: Osteoblast-like behavior tests, Hong Wu: Funding acquisition, Investigation, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by National Natural Science Foundation of China (Grant No. 52071346, No. 52111530193), Changsha Municipal Natural Science Foundation (Grant No. kq2202417).
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