Icariin loaded-hollow bioglass/chitosan therapeutic scaffolds promote osteogenic differentiation and bone regeneration

Highlights

• We firstly fabricated the icariin loaded-hollow bioglass/chitosan therapeutic scaffolds.

• Bioglass microspheres possessed hollow core and mesoporous shell.

• Therapeutic scaffolds served as drug delivery systems of icariin.

• As-released icariin drugs from scaffolds promoted osteogenic differentiation.

• Therapeutic scaffolds loaded with icariin drugs accelerated bone regeneration.

Abstract

The design and fabrication of therapeutic bone scaffolds that combine bioactive materials with osteoinductive drugs is a promising strategy for bone regeneration. Herein, we firstly developed icariin loaded-hollow bioglass/chitosan (ICA/HBG/CS) therapeutic scaffolds to treat critical-sized bone defects. The HBG/CS hybrid scaffolds possessed three-dimensional (3D) interconnected macropores with sizes of approximately 200 μm, which not only promoted the adhesion and spreading of stem cells, but also accelerated the in-growth of bone tissues. The HBG microspheres with hollow core and mesoporous shell were uniformly distributed on the macropore walls. The ICA drugs could be controllably released from the therapeutic scaffolds because of the hierarchically porous structures and hydrogen-bonding interactions. The as-released ICA drugs from the scaffolds remarkably up-regulated the expression levels of osteogenic-related genes (COL1 and RUNX2) and osteogenic-related proteins (ALP and p-Smad1/5). The micro-CT images, Masson’s trichrome staining and double fluorochrome labelling results revealed that the ICA/HBG/CS therapeutic scaffolds remarkably accelerated the formation of new bone tissues compared to the scaffolds without ICA drugs. Hence, the ICA/HBG/CS therapeutic scaffolds including bioactive materials and osteogenic drugs can serve as a kind of novel and promising bone materials for enhanced osteogenic differentiation and new bone regeneration.

Introduction

Bone defects caused by bone disease, traffic accident, trauma and surgical resection become a threat to human health [1]. Autografts and allografts can effectively treat bone defects, but they show the following disadvantages such as donor-site morbidity and limited sources [2]. An alternative strategy is to develop bone tissue engineering (BET) materials that are combined with stem cells and grow factors/therapeutic drugs [3]. Especially, the design and fabrication of therapeutic scaffolds represents a promising method, in which both the bioactive porous scaffolds and osteogenic drugs contribute to new bone regeneration [4], [5].

Up to now, bioglass scaffolds have been widely used for bone tissue engineering materials because of their excellent bioactivity, biocompatibility and biodegradability [6], [7]. The biodegradation products including Ca, P and Si elements from bioglass scaffolds facilitate collagen deposition and new bone formation [6], [7]. Unfortunately, the traditional bioglass scaffolds only possess a limited osteoinductivity so that they cannot meet the clinical needs especially for the patients with osteoporosis or other bone diseases. The design of therapeutic bioglass scaffolds is a promising approach, in which the controlled release of osteogenic drugs can accelerate bone regeneration [4], [5]. As drug delivery systems, the hierarchically porous structures in bioglass scaffolds play a pivotal role in drug loading-release performances [8], [9]. Hollow bioglass (HBG) microspheres with mesoporous shells are fit for drug carriers [10]. The hollow core within the microspheres increases great pore volume for drug storage, and the mesoporous shell become drug delivery channels in/out the microspheres [11]. In addition, chitosan (CS) that includes 2-acetamido-2-deoxy-β-d-glucan and 2-amino-2-deoxy-β-d-glucan units is one of the most important natural biomaterials with good biocompatible and antibacterial properties [11]. The therapeutic drugs can easily be attracted by the OH or NH₂ groups in CS via hydrogen-bonding interaction [9], [11].

Herba Epimedii as a Chinese traditional medical plant has been widely employed to treat osteoporosis, fractures and joint disease [12], [13]. Icariin (ICA), 2-(4′-methoxylphenyl)-3-rhamnosido-5-hydroxyl-7-glucosido-8-(3′-methyl-2-butylenyl)-4-chromanone, is a prenylated flavonol glycoside extracted from Herba Epimedii [13]. It has been reported that ICA affects bone remodeling and accelerates the maturation of primary osteoblast [14]. ICA improves the expression levels of osteogenic-specific proteins by activating BMP-2/Smad4 signaling or Wnt/β-catenin signalling pathways [15], [16]. Moreover, ICA can effectively suppress osteoclastogenic differentiation, immune response and bone resorption activity, and thus inhibit bone loss [13]. Therefore, it is an ideal and attractive strategy to incorporate ICA drugs into bioactive scaffolds for the effective treatment of critical-sized bone defects.

As ideal therapeutic scaffolds for treating bone defects, they should effectively regulate the loading-release performances of various grow factors/therapeutic drugs [17]. In the present work, we for the first time constructed the ICA loaded-HBG/CS (ICA/HBG/CS) therapeutic scaffolds according to the following steps: (i) the synthesis of HBG microsphere with mesoporous shell by organic template method; (ii) the preparation of HBG/CS hybrid scaffolds by a freeze-drying method; and (iii) the formation of ICA/HBG/CS therapeutic scaffolds after loading ICA drugs in the above scaffolds. Interestingly, the bioglass microspheres in the scaffolds exhibited hollow core and mesoporous shell, which contributed to the controlled drug release property. The as-released ICA drugs from the therapeutic scaffolds significantly enhanced the osteogenic related gene expression, and rapidly accelerated in vivo new bone regeneration.