Elsevier

Tissue and Cell

Volume 58, June 2019, Pages 84-92
Tissue and Cell

A biomimetic cartilage gradient hybrid scaffold for functional tissue engineering of cartilage

https://doi.org/10.1016/j.tice.2019.05.001Get rights and content

Highlights

  • Cs and Gel can provide natural binding sites for cells, and produces no harmful products after degradation.

  • It provides an important basis for the development of new gradient composite biomedical osteochondral repair materials.

  • The growth of the cells on the gradient scaffold was superior to that of the control cells.

Abstract

Osteochondral tissue has a complex layered structure that is not self-repairing after a cartilage defect. Therefore, constructing a biomimetic gradient scaffold that meets the specific structural requirements of osteochondral tissue is a major challenge in the field of cartilage tissue engineering. In this study, chitosan/Sodium β-glycerophosphate/Gelatin (Cs/GP/Gel) biomimetic gradient scaffolds were prepared by regulating the mass ratio of single layer raw materials. The same ratio of Cs/GP/Gel hybrid scaffold material was used as the control. Physical properties such as water absorption, porosity and the degradation rate of the material were compared to optimize the proportion of scaffold materials. P3 Bone Mesenchymal Stem Cells (BMSCs) were inoculated on the gradient and the control scaffolds to investigate its biocompatibility. Scanning electron microscopy (SEM) results show that 3:1:2, 6:1:3.5, 9:1:5, 12:1:6.5, 15:1:8 Cs/GP/Gel gradient scaffolds had excellent three-dimensional porous structures. Channels were also shown to have been interconnected, and the walls of the pores were folded. In the longitudinal dimension, gradient scaffolds had an obvious stratified structure and pore gradient gradualism, that effectively simulated the natural physiological stratified structure of real cartilage. The diameter of the pores in the control scaffold was uniform and without any pore gradient. Gradient scaffolds had good water absorption (584.24 ± 3.79˜677.47 ± 1.70%), porosity (86.34 ± 5.10˜95.20 ± 2.86%) and degradation (86.09 ± 2.46˜92.48 ± 3.86%). After considering the physical properties assessed, the Cs/GP/Gel gradient scaffold with a ratio of 9:1:5 was found to be the most suitable material to support osteochondral tissue. BMSCs were subsequently inoculated on the proportional gradient and hybrid scaffolds culture. These cells survived, distributed and extended well on the gradient and hybrid scaffold material. The biomimetic gradient scaffold designed and prepared in this study provides an important foundation for the development of new gradient composite biomedical materials for osteochondral repair.

Introduction

Tissue engineering aims to create constructs with the same biological and properties of a particular tissue [Kim et al., 2017; Scaffaro et al., 2016a,b; Liao et al., 2017]. As researchers gain a better understanding of the composition and role of extracellular matrices in natural tissue, more synthetic tissue-specific biomimetic materials have been developed. The natural microenvironment provides factors that influence the phenotypic expression of multifunctional cells. In recent years, many researchers have shown growing interest in the stratification/gradation/gradient/multiphase of osteochondral interface tissue engineering scaffold materials. Gradient scaffolds have been successfully prepared, and the biocompatibility, proliferation and differentiation properties of these scaffolds have also been characterized [Zhang et al., 2013; Yin et al., 2014; Mimura et al., 2008; Zhou et al., 2011; Di et al., 2016; Scaffaro et al., 2016a,b; Yucekul et al., 2017]. Bi et al. (2011) prepared a double-layered scaffold material with collagen/Cs for the cartilage phase and bioactive glass-collagen for the osteogenic phase. The two phases connected well and the BMSCs distributed evenly throughout the scaffold. Zhu et al. [Zhu et al., 2014] prepared Cs/polycaprolactone/type II collagen scaffold, and found that the pore size, porosity and mechanical properties of the scaffold showed gradient graduality that supported chondrocyte growth. Han et al. (2015) used the Cs/Gelatin (Gel) scaffold with transform growth factors a cartilage phase, and the L-polylactic acid scaffold of the composite concentration gradient bone morphogenetic protein for the osteogenic phase. The osteogenic and the cartilage phase scaffold was found to promote BMSCs differentiation into osteoblast and chondrocytes, respectively. Rowland et al. (2016) applied a freeze-drying technique to natural cartilage matrix to obtain gradual gradient pore sizes, and found that the scaffold was effective for cartilage formation after 28 days. Guo et al. (2017) used silk protein/biomineralized silicon as a scaffold, and complex selective peptides on the bottom of the scaffold as a calcification layer. In the osteoinductive environment, the scaffold promoted osteogenic differentiation of BMSCs in vitro.

In addition to testing the performance of gradient scaffolds in vitro, gradient scaffolds have been used in animal models to further characterise its potential applications in vivo. Liu et al. (2015) prepared cartilage phase material with L-polylactic acid composite polycaprolactone/type I collagen/hyaluronic acid fiber, and osteogenic phase material with β-tricalcium phosphate/hyaluronic acid fiber. After implanting the double-layer scaffold into the damaged area of rabbit bone cartilage, the repair effect was promising. Levingstone et al. (2016) prepared a gradient scaffold with type I collagen/type II collagen/hyaluronic acid for the cartilage phase, type I collagen/type II collagen/hydroxyapatite for the intermediate layer, and type I collagen/hydroxyapatite for the osteogenic phase. Regenerated subchondral bone was completely formed within 12 months after implantation in the injured area, 3 months earlier than the control group. The subchondral bone and hyaline cartilage were well integrated in the scaffold implantation area. Du et al. (2017) used a laser firing technique to prepare a multilayer gradient scaffold with polycaprolactone microspheres and hydroxyapatite/polycaprolactone microspheres. The scaffold was implanted into the injured area of the rabbit and induced new cartilage formation through early bone regeneration. The new cartilage was well integrated with the surrounding tissue. Shim et al. (2016) prepared a multilayer 3D structure containing human mesenchymal stromal cells using full-ring acrylic acid and hyaluronic acid as raw materials. This structure showed good regeneration ability in the reconstruction of osteocartilage tissue of rabbit knee joint.

As Cs and Gel are natural materials with good biocompatibility and degradability in vivo, and due to the gel properties of β-Sodium Glycerophosphate (GP), these raw materials were used in this study. Based on the concept of multi-coupling bionics; the functional advantages of each material were brought into play. The ratio of each material was regulated, and the composite functional scaffold materials with gradient gradual structure were prepared.

To test the biocompatibility of the scaffold, bone marrow mesenchymal stem cells (BMSCs) of SD rats with multidirectional differentiation potential were inoculated on the scaffold to form a cell-scaffold. The distribution, adhesion, and the extracellular matrix secretion of the cells on the scaffold were investigated. This study lays the foundation for the construction of natural cartilage gradient scaffold materials in vitro.

Section snippets

Experimental design

An overview of the experimental design is illustrated in Fig. 1. A bottle contains the mixture of Chitosan solution, Sodium β-glycerophosphate and gelatin, and another bottle contains 2% acetic acid solution. The preparation process is continuous, and the volume of liquid in the two bottles is always equal by using the law of connected vessels. After acetic acid is added to the mixture bottle, the solution is stirred evenly before it is poured into a mold for the preparation of the Cs/GP/Gel

Scanning electron microscopy of the scaffold materials

Cartilage tissue has a distinct gradient stratification structure, and a representative diagram of the cross-section of bone cartilage tissue (Fig. 2F) demonstrates that cartilage tissue is divided into a surface layer, a middle layer and a bottom layer [Seo et al., 2014]. We are committed to preparing similar stratification structure. Through scanning electron microscopy, the structural differences between the CS/GP/Gel gradient scaffolds were observed (Fig. 2). In the longitudinal dimension

Conclusion

Cs/GP/Gel gradient hybrid scaffolds were successfully prepared in this study to simulate the physiological structure of natural cartilage. The gradient scaffolds had suitable water absorption (584.24 ± 3.79 ˜ 677.47 ± 1.70%), porosity (86.34 ± 5.10 ˜ 95.20 ± 2.86%) and degradation rate (86.09 ± 2.46 ˜ 92.48 ± 3.86%). After further optimization, a Cs/GP/Gel ratio of 9:1:5 was found to have even better water absorption and porosity, and was identified to be the most suitable for the preparation

Conflict of interest

The authors have no conflicts of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31670978/31370991/21676041), the Fok Ying Tung Education Foundation (132027), the State Key Laboratory of Fine Chemicals (KF1111) and the Natural Science Foundation of Liaoning (20180510028).

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