Hyaluronic acid-based hydrogels with tobacco mosaic virus containing cell adhesive peptide induce bone repair in normal and osteoporotic rats

doi:10.3877/cma.j.issn.2096-112X.2020.01.009

Biomaterials Translational ›› 2020, Vol. 1 ›› Issue (1): 89-98.doi: 10.3877/cma.j.issn.2096-112X.2020.01.009

Hyaluronic acid-based hydrogels with tobacco mosaic virus containing cell adhesive peptide induce bone repair in normal and osteoporotic rats

Jishan Yuan¹, Panita Maturavongsadit²^,³, Zhihui Zhou¹, Bin Lv¹, Yuan Lin⁴, Jia Yang², Jittima Amie Luckanagul⁵^,⁶^,^*()

1 Department of Orthopaedic Surgery, the Affiliated First People’s Hospital to Jiangsu University, Zhenjiang, Jiangsu Province, China
2 Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC, USA
3 Joint Department of Biomedical Engineering, North Carolina State University and The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
4 The State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin Province, China
5 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
6 Research Unit for Plant-produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand.

Received:2020-09-11 Revised:2020-10-13 Accepted:2020-10-14 Online:2020-12-28 Published:2020-12-28
Contact: Jittima Amie Luckanagul E-mail:jittima.luck@gmail.com

Abstract

Abstract:

Tobacco mosaic virus (TMV) has been studied as a multi-functional agent for bone tissue engineering. An osteo-inductive effect of wild-type TMV has been reported, as it can significantly enhance the bone differentiation potential of bone marrow stromal cells both on a two-dimensional substrate and in a three-dimensional (3D) hydrogel system. A TMV mutant (TMV-RGD1) was created which featured the adhesion peptide arginyl-glycyl-aspartic acid (RGD), the most common peptide motif responsible for cell adhesion to the extracellular matrix, on the surface of the virus particle to enhance the bio-functionality of the scaffold material. We hypothesised that the incorporation of either wild-type TMV or TMV-RGD1 in the 3D hydrogel scaffold would induce bone healing in critical size defects of the cranial segmental bone. We have previously tested the virus-functionalised scaffolds, in vitro, with a hyaluronic acid-based system as an in-situ hydrogel platform for 3D cell encapsulation, culture, and differentiation. The results of these experiments suggested the potential of the virus-functionalised hydrogel to promote in vitro stem cell differentiation. The hydrogel-forming system we employed was shown to be safe and biocompatible in vivo. Here, we further explored the physiological responses regarding bone regeneration of a calvarial defect in both normal and osteoporotic ovariectomized rat models. Our results, based on histological analysis in both animal models, suggested that both wild-type TMV and TMV-RGD1 functionalised hydrogels could accelerate bone regeneration, without systemic toxicity, evaluated by blood counts. New bone formation was intensified by the incorporation of the RGD-mutant viral particles. This finding increased the potential for use of the rod-shaped plant virus as a platform for the addition of powerful biofunctionality for tissue engineering applications. This study was approved by the Ethics Committee on Animal Use of the Zhenjiang Affiliated First People’s Hospital affiliated to Jiangsu University.

Key words: bone regeneration, calvarial defect, hydrogel, osteoporosis, tobacco mosaic virus

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Figures/Tables 5

Figure 1. Schematic illustration of our study design. Methacrylated hyaluronic acid (MeHA) was synthesised and used with incorporated virus particles as a hydrogel structural platform to fill skull defects in osteoporotic and normal Sprague-Dawley rats. The RGD mutant TMV graphic was generated from PyMol with coordinates 2TMV from the Protein Data Bank (www.rcsb.org) and created with BioRender.com. OVX: ovariectomised; RGD: arginyl-glycyl-aspartic acid; TMV: tobacco mosaic virus.

Figure 2. Haematological analysis of non-OVX animals treated with three different hydrogel materials (MeHA, MeHA + TMV, and MeHA + TMV-RGD) to fill their skull defects. Total blood counts were performed at five time points (4 days (d), 1, 2, 4, and 10 weeks (w)). Data are expressed as the mean ± SD (n = 3). *P < 0.05, vs. MeHA group; #P < 0.05, vs. MeHA + TMV group (one-way analysis of variance). MeHA: methacrylated hyaluronic acid; OVX: ovariectomised; RGD: arginyl-glycyl-aspartic acid; TMV: tobacco mosaic virus.

Figure 3. Haematological analysis of OVX animals treated with three different materials (MeHA, MeHA + TMV, and MeHA + TMV-RGD) to fill their skull defects. Total blood counts were performed at five time points (4 days (d), 1, 2, 4, and 10 weeks (w)). Data are expressed as the mean ± SD (n = 3). **P < 0.05, vs. MeHA group (one-way analysis of variance). MeHA: methacrylated hyaluronic acid; OVX: ovariectomised; RGD: arginyl-glycyl-aspartic acid; TMV: tobacco mosaic virus.

Figure 4. Inflammatory cell infiltration observed by hematoxylin and eosin staining. (A) Photomicrographs of hematoxylin and eosin-stained sections from skull defects implanted with each type of hydrogel (MeHA, MeHA + TMV, and MeHA + TMV-RGD) (original magnification, 40×). The new bone is visible as a compact structure with a pink colour. The connective tissue can be seen as a structured network of cells in a purple colour. (B) Hematoxylin and eosin histological scoring of the three types of hydrogels confirm the difference in proportion of bone healing area and degree of inflammation (arbitrary scoring). Data are expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, vs. sham group (one-way analysis of variance). MeHA: methacrylated hyaluronic acid; OVX: ovariectomised; RGD: arginyl-glycyl-aspartic acid; TMV: tobacco mosaic virus.

Figure 5. Histopathological analysis of bone substitute hydrogel implants stained with Masson’s trichrome. (A) Photomicrographs of corresponding Masson’s trichrome-stained sections from each type of hydrogel (original magnification, 40×). In general, Masson’s trichrome stains mature bone with osteoid formation red, whilst blue stain indicates developing calcified bone. (B) Histological scoring of the three types of hydrogels in tissue sections show the different amounts of mature bone area. Data are expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, vs. sham group (one-way analysis of variance). (C) Micro CT images of the cranial-defected bones dissected from non-OVX groups with different types of hydrogel implants at 10 weeks post-surgery. MeHA: methacrylated hyaluronic acid; OVX: ovariectomised; RGD: arginyl-glycyl-aspartic acid; TMV: tobacco mosaic virus.

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Hyaluronic acid-based hydrogels with tobacco mosaic virus containing cell adhesive peptide induce bone repair in normal and osteoporotic rats

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