Osteogenic potential evaluation of biotin combined with magnesium-doped hydroxyapatite sustained-release film

Highlights

• BMP2, BSP and OPG proteins were significantly expressed in biotin film.

• The MgHA film facilitates the diffusion of the biotin to achieve sustainable biotin release.

• Multilayer sustained-release films were prepared layer by layer by PLD-LEBD coupling technique.

• Abundant bone-like and woven bones were formed around the implant with MgHA|biotin|MgHA|biotin film.

Abstract

Biotin is one of the water-soluble B-complex group of vitamins. Recent studies have found that the relative protein expression of BMP2, BSP and OPG in MC3T3-E1 cells is prominent after 14 days of co-culture with biotin film, especially for BMP2. It is also found that the rapid degradation of biotin film in vivo limits its application value. In this work, magnesium-doped hydroxyapatite (MgHA) film can form a porous network structure as a biological sustained-release film. Therefore, the multilayer (MgHA|biotin|MgHA|biotin) film was prepared by pulsed laser assisted electron beam deposition technique. The morphology, structure and properties of biotin film and multilayer film were analyzed and characterized. Also, the osteogenic effect of biotin film and multilayer film was evaluated after implantation into the femoral bone marrow cavity of SD rats. The results of micro-CT scan and 3D reconstruction showed that there were a large number of trabecular bones around the multilayer film, which was superior to biotin film in osteogenesis. Hematoxylin-eosin staining showed cancellous bone structure and intact bone marrow structure around the multilayer film, and the newly formed bone became lamellar. Masson-trichromatic staining revealed abundant osteoid and braided bone formation around the multilayer film. In conclusion, MgHA sustained release film can realize the continuous release of bioactive drugs, which provides a new route to accelerate the repair of bone defects.

Introduction

Biotin, also known as vitamin H, vitamin B₇, and coenzyme R, is one of the water-soluble B-complex group of vitamins, which is composed of a ureido ring and a tetrahydrothiophene ring [1]. Biotin plays a vital role in cell growth, fatty acid synthesis, and metabolism of fats and amino acids. Also, biotin not only helps rebuild tissues and accelerate cell growth, but also helps damaged muscle and tissue repair quickly [2]. Biotin is also involved in important processes such as skin regeneration and bone development [3]. It also reduces inflammation, which helps prevent joint pain and stiffness, as well as muscle soreness. The avidin-biotin binding system (ABBS) has been widely used in the field of biotechnology because of its strong non-covalent biological interaction, which guarantees the good coupling efficiency between biotin (or biotin derivative) and streptavidin [4], [5]. Furthermore, the ABBS has been shown to transform non-adherent Ehrlich ascites carcinoma cells into anchor-dependent cells [6]. The cell attachment to scaffolds is a key step in biomedical engineering applications. It has been reported that ABBS can increase cell attachment and migration on non-porous 2D and 3D scaffolds, and even on the surface of biodegradable biological scaffolds [7]. Therefore, biotin-immobilized surface can be a stable anchor for capturing target substances such as proteins, drugs and cells [8], [9].

Furthermore, magnesium (Mg) as a bone conduction and growth stimulator is essential for bone growth and strength. Also, it is a cofactor of a variety of enzymes, a stabilizer of DNA and RNA structure, and an activator of alkaline phosphatases that indirectly affect mineral metabolism [10], [11]. It is reported that Mg²⁺ can directly or indirectly stimulate the proliferation and migration of endothelial cells [12]. However, the main disadvantage of pure Mg as an orthopedic implant is its high degradation rate, which directly reduces its mechanical strength and even leads to excessive inflammatory response [13], [14]. Lai et al. [15] prepared porous PLGA/TCP/Mg (PTM) scaffolds, indicating that Mg in PTM scaffolds is the primary factor promoting neovascular formation and vascular structural remodeling. Yang et al. [16] developed a novel drug delivery system of Mg/PLLA composite microspheres, which proved the availability of using biodegradable Mg particles to manipulate drug release and reduce PLLA-induced inflammation. Moreover, Mg/PLLA composite microspheres have potential applications in various therapeutic agents for bone regeneration, especially in the controlled delivery of certain growth factors. In our previous studies [17], [18], four-layer films with a distribution of the ciprofloxacin hydrochloride (CIP) were obtained using the layered growth of CIP film and Mg-HA film by the pulsed laser assisted electron beam deposition technology. The results showed that Mg-HA film not only prolonged the CIP release period, but also enhanced the drug release in the middle and late stage. Importantly, MgHA film can rapidly form porous structure in physiological environment to provide channels for drug release, which is different from polymer-based sustained release systems. Polymer degradation is a slow process, which restricts the concentration of drug release.

At present, bone tissue engineering scaffolds are often used to fill bone defects, but it is difficult to achieve ideal repair effect by scaffolds themselves. Therefore, using protein (BMP2) and cells (BMSCs) to modify scaffolds can significantly improve the effect of bone repair [19], [20]. This method requires co-culture of the scaffold with the protein or cells to reach the optimal concentration for cultivation. However, it is difficult to control the dosage, preserve and lose the activity in the preparation process. Ideal materials can not only be absorbed and replaced by endogenous tissues, but also have obvious osteogenic effects. Therefore, the multilayer (MgHA|biotin|MgHA|biotin) film composed of MgHA film and biotin film was prepared layer by layer by pulsed laser assisted electron beam deposition technique for surface modification of scaffolds to achieve rapid bone repair. In view of the differences in the mechanism of action of Mg/Mg²⁺ and biotin on bone tissue repair, it is boldly envisaged to prepare multilayer film with a laminated structure to alternately release Mg/Mg²⁺ and biotin for bone tissue repair. The morphology, structure and properties of biotin film and multilayer film were studied and characterized. Also, the osteogenic effect of biotin film and multilayer film was evaluated after implantation into the femoral bone marrow cavity of SD rats.