3D macroporous biocomposites with a microfibrous topographical cue enhance new bone formation through activation of the MAPK signaling pathways

https://doi.org/10.1016/j.jiec.2021.08.041Get rights and content

Highlights

  • 3D macroporous biocomposites were fabricated using an electrohydrodynamic process.

  • The biocomposites were composed of a fibrous matrix structure of PCL/CDHA/collagen.

  • Biocomposites with the microfibrous topographical cue significantly improved in vitro cellular responses.

  • The fibrous biocomposite encouraged new bone formation in vivo.

Abstract

The fabrication of biomedical composite materials with macroporous structures and unique topographical cues has been widely investigated to achieve successful bone regeneration. In this study, porous biocomposites consisting of microfibrous bundles fabricated using an electrohydrodynamic direct printing process were prepared. The fibrous composite structure was composed of a fibrous matrix structure of polycaprolactone/α-tricalcium phosphate and collagen coated in fibrous biocomposites. Various cellular activities, cell proliferation, and osteogenic differentiation in biocomposites have been investigated using preosteoblasts (MC3T3-E1). The in vitro results demonstrated that biocomposites with the microfibrous topographical cue significantly improved various cellular responses, including cell proliferation and mRNA expression levels of osteoblastic genes of MC3T3-E1 cells, compared to biocomposites without a fibrous topography surface that were fabricated through normal 3D printing. This phenomenon could be attributed to the fibrous structure of composites that stimulated cultured cells, thereby activating extracellular signal-related kinases and p38 signaling pathways. To observe the ability of biocomposites for bone regeneration, a rat calvarial defect model was used; the fibrous biocomposite showed significantly higher level of new bone formation in comparison with the 3D-printed control, a biocomposite without fibrous topographical cues.

Introduction

Calcium phosphate (CaP)-based bioceramics and collagen are the major components of bone [1], [2], [3]. Particularly, bioceramics, hydroxyapatite, α-tricalcium phosphate (TCP), β-TCP, and biphasic calcium phosphate (BCP) have been widely used as dispersed phases in composite structures for developing bone tissue engineering materials or osteointegration-inducing coating materials on the surface of metallic prostheses [4], [5], [6]. The use of CaP ceramics as reinforced composite biomaterials or typical osteoconductive coating materials can be attributed to their intrinsic drawbacks, including low material resistance to crack growth compared to others, such as Al2O3 and ZrO2, and poor processability to form three-dimensional (3D) porous complex structures; however, they demonstrate outstanding biocompatibility, osseointegration, and osteoconductive properties [7], [8], [9], [10]. Therefore, composite materials using various biodegradable polymer materials, such as poly(ε-caprolactone) (PCL), poly(lactic acid) (PLA), poly(3-hydroxybutyrate) (PHB), and poly(glycolic acid) (PGA), have remained the optimal choice for satisfying the critical demands for successful bone tissue engineering [11], [12].

Various porous biocomposite fabrication methods involve the use of bioceramic and biocompatible polymers [13], [14], [15], [16], [17]. A typical method, known as the 3D printing process, has been widely adopted to fabricate composite scaffolds for bone tissue engineering owing to its versatility and ease of application in manufacturing processes for the production of complex structures in a multi-layered manner [18], [19], [20]. However, even though the composite scaffolds fabricated using the melt-extrusion-based 3D printing method have markedly improved the low physical facture toughness of CaP bioceramics and low processability of ceramics for the formation of various 3D complex structures [21], [22], its low concentration (∼30 vol%) of biomimetic inorganic composition and poor biomimetic morphological structure have been considered limitations for application in the fabrication of appropriate bone-tissue engineering scaffolds; therefore, higher volume fractions (>60 vol%) of the CaP bioceramic component and physically cell-affordable morphological structure are required [1], [18], [21], [22].

Nano/microfibrous biocomposite scaffolds have provided the most appropriate physical structure for bone tissue engineering materials owing to their considerable biological features and structures, similar to the physical dimensions of the native extracellular matrix (ECM) [10], [23], [24], [25]. Additionally, porous scaffolds for bone regeneration applications have been recommended with a pore size of 300–500 μm, which can enhance diffusion rates to induce a high degree of metabolic activities in cells [26]. Furthermore, porous scaffolds with multiple deposited nano/microfibrous structures can encourage significantly higher cell proliferation and differentiation by introducing unique ECM-mimic structural morphologies, which can directly guide various cellular activities [27], [28], [29]. The electrospinning process is a typical method adopted for producing nano/microfibrous composite structures [30], [31], [32], [33]. Although application of the method has resulted in physically improved hydrophilic properties, mechanical properties, and in the obtainment of biologically active fibrous composite structures supplemented with dispersed bioceramics along with the incorporation of bioactive components, thus eventually promoting various aspects of cultured cell physiology [34], [35], [36], the low manageability of micro or macropore geometries and poor 3D shape ability are persistent issues that ought to be overcome [13], [14], [15], [16], [17]. To overcome the deficiencies of the electrospinning process, we successfully developed a method to fabricate 3D microfibrous structures using an electrohydrodynamic printing process [37]. Based on this method, multilayered fibrous bundle structures using polycaprolactone (PCL), cellulose, and its composites as well as bioceramics were successfully developed [37], [38], [39], [40]. The fabricated structures showed effective proficiency for osteogenic cells, demonstrating efficient cell attachment and growth in 3D structures. However, previous studies have mainly focused on the exertion of effects of fabrication parameters, such as applied electric field conditions (electric strength and nozzle-to-target distance), printing speed, and mixture concentration, on fibrous structural formation [39].

In this study, we proposed the establishment of a biomedical bone-mimetic platform, a 3D fibrous composite scaffold of osteoconductive PCL/bioceramic matrix structure, via addition of a cell-supporting collagen component for bone regeneration and evaluated the modified composite scaffold for realistic bone formation in vitro and in vivo. To achieve this goal, we used α-TCP bioceramic (∼70 wt%) for osteoconduction, PCL as a processing and mechanical supporting material, and collagen Type-I as a biologically supporting material. Additionally, to evaluate in vitro osteogenic properties, including various osteogenic gene expressions of the proposed biocomposite, we used a normally 3D-printed structure with similar compositions of bioceramic/PCL/collagen and similar 3D geometrical shapes. The fibrous composite structure provided a significantly favorable microenvironment to cultured preosteoblast cells for various cellular activities and continuously acted as an osteogenic niche for the cultured cells. Through the assessment of the critical-sized rat calvarial defect model, we believe that the modified fibrous composite structure could clearly enhance efficient bone formation.

Section snippets

Materials

PCL (Mw = 80,000 g mol−1) and α-TCP (Mw = 310.18 g mol−1) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Methylene chloride (MC) and dimethylformamide (DMF; Junsei Chemical Co., Tokyo, Japan) were used as solvents. Collagen type I (purity: collagen type I > 98%, MSBIO, Seongnam, South Korea) was extracted from porcine skin. To enable crosslinking of collagen, we used a 1-(3-dimethylaminopropyl)-3-carbodiimide hydrochloride (EDC, Tokyo Chemical Industry, Tokyo, Japan) solution.

Fabrication of the composite scaffold

A solvent

Preparation of fibrous composite scaffold using the EHD process and coating of collagen

Fig. 1 shows a schematic representation of the fabrication process of fibrous composites consisting of PCL/α-TCP and collagen. Unlike general electrospinning, the EHD process involves the use of an electrically charged linear jet. As the charged jet enters the ethanol bath, the jet can be split into several microscale fibers owing to the replacement of polymer-dissolving solvent with ethanol, which has a relatively lower surface tension than the solvent [44], [45]. A more detailed mechanism to

Conclusions

In this study, 3D mesh-structured biocomposites with microfibrous bundles and fabricated homogeneous internal macropores were prepared using PCL, α-TCP, and collagen through the application of an electrohydrodynamic direct printing process. The fibrous biocomposites displayed a well-arranged pore structure, which consisted of multilayered microfibrous bundle struts. Although the mechanical properties of the fibrous composites were lower than those of the control (a 3D-printed biocomposite)

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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.

Acknowledgements

This study was supported by the National Research Foundation of Korea (NRF) Grant funded by the Ministry of Science and ICT for Bioinspired Innovation Technology Development Project (NRF-2018M3C1B7021997 and NRF-2017M3A9E4048170).

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