Composite scaffolds of nano-hydroxyapatite and silk fibroin enhance mesenchymal stem cell-based bone regeneration via the interleukin 1 alpha autocrine/paracrine signaling loop
Introduction
As one of the three major elements of regenerative medicine, biomaterial scaffolds play an important role in orchestrating the interaction of cells with soluble bioactive factors during reconstruction of bone tissues. Among various biomaterials, hydroxyapatite (HAp) has attracted much attention for large bone defects regeneration [1], due to its excellent osteoconductivity and similarity to the mineral phase and crystalline structure of bone. However, its low compressive strength limits its application to non/low-load bearing bone repairs [2]. With the advancement of nano-technology, much progress has been made in fabricating functional scaffolds from natural polymers, which can be incorporated together with nano-HAp (nHAp) to achieve both controllable porosity and mechanical strength [3], [4]. Silk fibroin (SF) is such a natural biopolymer with remarkable mechanical strength, controllable biodegradability, excellent biocompatibility, and ease of processing [5]. Various different techniques have been utilized for SF-nHAp fabrication [6], [7], [8], [9]. Available data from the scientific literature suggest that SF-nHAp scaffolds can be an excellent source for bone regeneration [6], [10], [11]. However, the underlying mechanisms of osteoinduction by SF-nHAp are not completely understood.
It was believed that the major osteoinductive component of SF-nHAp scaffolds is nHAp [11]. To date, most theories about the osteoinductivity of HAp (including nHAp) focused on interaction of biomaterials with the surface molecules of osteo-progenitor cells i.e. integrin superfamily [12] and focal adhesion components [13]. These interactions subsequently trigger cytoskeletal rearrangement [13] and multiple intracellular signaling cascades, such as ERK/Sox9 [12], BMP/Smad [14], Wnt [15], TGF-β, MAPK, and Notch signaling pathways [16]. Lin and colleagues found that soluble factors produced by mesenchymal stem cells (MSCs) play an important role in osteoinduction with HAp scaffolds [17]. However, the authors did not elucidate the identity of these soluble factors. In this study, we attempted to rigorously investigate differential expression of genes involved in osteoinduction by rat bone marrow MSCs (BMSCs) cultured on SF-nHAp in comparison to SF, utilizing cDNA microarray technology. It is expected that this approach could lead to the identification of novel key regulators and mechanistic pathways of the osteoinduction process.
Section snippets
Materials and methods
For in vivo testing, twelve male/female Sprague–Dawley (SD) rats, two months old, were purchased and housed at the Center for Experimental Animals, Zhejiang University at constant room temperature (25 °C). All animal related experimental protocols have been approved by the institutional Animal Care and Use Committee of Zhejiang University. Unless otherwise stated, all reagents were purchased from Sigma-Aldrich Inc. (St. Loius, MO, USA), and all experimental data were collected in triplicates.
Characterization of BMSCs
The properties of BMSCs were confirmed by successful tri-lineage differentiation (Fig. 1A), CFU formation (Fig. 1B) and expression of characteristic surface markers (Fig. 1C). After osteogenic, chondrogenic and adipogenic induction, BMSCs were positive for alizarin red staining, SO staining and oil red staining respectively. Additionally crystal violet staining displayed that BMSCs colonies were intact and round. Expression of various MSC-associated surface markers were detected on BMSCs such
Discussion
In this study, nHAp was successfully incorporated within the SF scaffolds by a modified two-step mineralization method. In our previous work, the growth of nHAp on collagen scaffold was very slow and a pre-calcification step was developed to enhance the incorporation of nHAp into the collagen scaffold [19]. In this study, a modified two-step mineralization protocol was developed to directly assemble nHAp into the SF porous framework, which resulted in a SF-nHAp composite scaffold. The
Conclusion
In summary, SF-nHAp scaffolds have been demonstrated to be biocompatible and osteoinductive. The inhibition of adipogenesis and the acceleration of mitochondrial fatty acid oxidation through regulating the PPAR signaling pathway genes including Cpt1a, Angptl4, Scd and Pltp could be an alternative mechanism of the osteoinductive activity of nHAp. Cytokine–cytokine receptor interactions played a central role in linking the Wnt, MAPK and TGF-beta signaling pathways. The soluble factor IL-1α was
Acknowledgement
This work was supported by the National Basic Research Program of China (973 Program, 2012CB967904, 2012CB967900), the National Natural Science Foundation of China (31200739 and 31470948), the Doctoral Fund of Ministry of Education of China (20120101120003), the Science and Technology Department Program of Zhejiang Province (2013C33156), the International Science & Technology Cooperation Program of China (2011DFA32190), as well as sponsored by Regenerative Medicine in Innovative Medical
References (39)
- et al.
Advances in synthesis of calcium phosphate crystals with controlled size and shape
Acta Biomater
(2014) - et al.
Silk as a biocohesive sacrificial binder in the fabrication of hydroxyapatite load bearing scaffolds
Biomaterials
(2014) - et al.
Nanoscale hydroxyapatite particles for bone tissue engineering
Acta Biomater
(2011) - et al.
Silk fibroin biomaterials for tissue regenerations
Adv Drug Deliv Rev
(2013) - et al.
Development of nano-hydroxyapatite graft with silk fibroin scaffold as a new bone substitute
J Oral Maxillofac Surg Off J Am Assoc Oral Maxillofac Surg
(2011) - et al.
The promotion of bone regeneration by nanofibrous hydroxyapatite/chitosan scaffolds by effects on integrin-BMP/Smad signaling pathway in BMSCs
Biomaterials
(2013) - et al.
Hydroxyapatite coating affects the Wnt signaling pathway during peri-implant healing in vivo
Acta Biomater
(2014) - et al.
How useful is SBF in predicting in vivo bone bioactivity?
Biomaterials
(2006) - et al.
Hydroxyapatite fiber reinforced poly(alpha-hydroxy ester) foams for bone regeneration
Biomaterials
(1998) - et al.
Angptl4 protects against severe proinflammatory effects of saturated fat by inhibiting fatty acid uptake into mesenteric lymph node macrophages
Cell Metab
(2010)
The interleukin-1 receptor family
Seminars Immunol
Nano-scaled hydroxyapatite/polymer composite I. Coating of sintered hydroxyapatite particles on poly(gamma-methacryloxypropyl trimethoxysilane)grafted silk fibroin fibers through chemical bonding
J Mater Sci Mater Med
Fabrication of nano-hydroxyapatite on electrospun silk fibroin nanofiber and their effects in osteoblastic behavior
J Biomed Mater Res Part A
Nonwoven silk fibroin net/nano-hydroxyapatite scaffold: preparation and characterization
J Biomed Mater Res Part A
A novel approach to fabricate silk nanofibers containing hydroxyapatite nanoparticles using a three-way stopcock connector
Nanoscale Res Lett
Preparation and characterization of nano-hydroxyapatite/silk fibroin porous scaffolds
J Biomaterials Sci Polym Ed
Directing osteogenesis of stem cells with hydroxyapatite precipitated electrospun eri-tasar silk fibroin nanofibrous scaffold
J Biomaterials Sci Polym Ed
Signaling responses of osteoblast cells to hydroxyapatite: the activation of ERK and SOX9
J Bone Mineral Metabolism
Biological behavior of pre-osteoblasts on natural hydroxyapatite: a study of signaling molecules from attachment to differentiation
J Biomed Mater Res Part A
Cited by (139)
Effects of microenvironment and biological behavior on the paracrine function of stem cells
2024, Genes and DiseasesBiodegradable silk fibroin scaffold doped with mineralized collagen induces bone regeneration in rat cranial defects
2023, International Journal of Biological MacromoleculesSDF-1α and CTGF functionalized Gelatin methacryloyl (GelMA) hydrogels enhance fibroblast activation to promote wound healing
2023, Materials Today CommunicationsSilk scaffolds for tissue engineering in dentistry
2023, Silk-Based Biomaterials for Tissue Engineering, Regenerative, and Precision Medicine, 2nd EditionMechanical and morphological characterization of nano-hydroxyapatite (nHA) for bone regeneration: A mini review
2022, Biomedical Engineering Advances
- 1
Authors contribute equally to this paper.