Synergistic interaction of platelet derived growth factor (PDGF) with the surface of PLLA/Col/HA and PLLA/HA scaffolds produces rapid osteogenic differentiation

https://doi.org/10.1016/j.colsurfb.2015.11.053Get rights and content

Highlights

  • PLLA based with or without collagen and HA fabricated using Electron spinning technique.

  • Cell viability, osteocalcin, Alizarin staining, ECP protein, Gene expression tested with PDGF-BB.

  • Results confirmed PDGF-BB treatment induced rapid differentiation.

Abstract

Scaffolds with structural features similar to the extracellular matrix stimulate rapid osteogenic differentiation in favorable microenvironment and with growth factor supplementation. In this study, the osteogenic potential of electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA, PLLA/HA and PLLA/Col) scaffolds were tested in vitro with the supplementation of platelet derived growth factor-BB (PDGF-BB). Cell attachment and topography, mineralization, extracellular matrix protein localization, and gene expression of the human mesenchymal stromal cells were compared between the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA. The levels of osteocalcin, calcium, and mineralization were significantly greater in the PLLA/Col/HA and PLLA/HA compared with PLLA/Col. High expression of fibronectin, intracellular adhesion molecule, cadherin, and collagen 1 (Col1) suggests that PLLA/Col/HA and PLLA/HA scaffolds had superior osteoinductivity than PLLA/Col. Additionally, osteopontin, osteocalcin, osterix, Runt-related transcription factor 2 (Runx2), and bone morphogenic protein (BMP2) expression were higher in PLLA/Col/HA and PLLA/HA compared with PLLA/Col. In comparison with PLLA/Col, the PLLA/Col/HA and PLLA/HA scaffolds presented a significant upregulation of the genes Runx2, Col 1, Integrin, osteonectin (ON), bone gamma-carboxyglutamic acid-containing protein (BGALP), osteopontin (OPN), and BMP2. The upregulation of these genes was further increased with PDGF-BB supplementation. These results show that PDGF-BB acts synergistically with PLLA/Col/HA and PLLA/HA to enhance the osteogenic differentiation potential. Therefore, this combination can be used for the rapid expansion of bone marrow stromal cells into bone-forming cells for tissue engineering.

Introduction

Scaffolds provide biological substitutes for tissue engineering that improve the functions of damaged bone and cartilage [1]. Various natural and synthetic materials have been used for the transplantation of stem cells in defective areas to allow the differentiation of these cells into osteogenic or chondrogenic cells. The use of scaffolds for stem cell transplantation requires the addition of multiple growth factors or commercially available osteogenic media for early differentiation [2]. The use of these growth factors is expensive. Many studies, therefore, have explored the use of biomaterials with the addition of a single growth factor [3].

Mesenchymal stromal cells (MSCs) are used for tissue regeneration due to their ability to replicate and differentiate into various mesenchymal lineages, including chondrocytes, osteoblasts, and adipocytes. Substantial advancements have been made to the MSC-based strategies for bone repair and regeneration [4]. Although the use of in vitro 2D culture flasks has traditionally been advocated, the use of fibrous scaffolds has now become more common because of their similarity to the intrinsic extracellular matrix (ECM) of bone and cartilage. However, many aspects of the use of fibrous scaffolds remain unclear, including compatibility, choice of growth factor, material composition, and degradation rate. For example, an alloplastic material under mechanical strain may not perform in a manner similar to that of the neighboring host bone tissues and may lead to structural defects at the implant site or inflammatory responses [5]. However, in the nano-fibrous environment, the cell-to-cell interactions, transfer of nutrients and the presence of components like Col and HA provide suitable surfaces for cell attachment and enhanced mineralization with growth factor supplementation. The differentiation of MSCs into an osteogenic lineage is improved by the synergistic actions of the growth factors, scaffold, and extracellular matrix components [6]. Numerous soluble and insoluble agents, including dexamethasone [7], BMP 2 [8], and Col 1 [9], encourage MSC differentiation into osteogenic cells. The MSCs express osteoblast-related genes and differentiate into osteogenic cells in response to the ECM proteins, collagen I [10], and HA [11] incorporated scaffolds. This suggests that the ECM plays a vital role in the differentiation process. Human fetal osteoblast cells supplemented with osteogenic factors incorporated into the fibrous scaffold can induce osteogenic differentiation in approximately three weeks [12]. Similarly, the electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA) scaffolds using 293 T cells and rabbit bone marrow stem cells produce similar results [13]. Scaffolds using BMP2 stimulation also have osteoinduction properties. Similarly, the deposition of n-HA on the PLLA/Col nano-fibers has been reported as a promising strategy for early cell capture [14].

Platelet derived growth factor-BB (PDGF-BB) induces the proliferation, migration, and differentiation of stromal cells [15]. However, there is some controversy regarding the different types of PDGF, with reports suggesting that some isoforms may hamper and others may enhance differentiation [16]. PDGF also inhibits ALP, OC, and type I collagen marker protein of mature osteoblasts in pre-osteocytic cell lines [17]. Conversely, some studies have shown no effect of PDGF-BB on the marker activity and mineralization in human stromal cells. Imatinib mesylate-induced blockade of the PDGFR-beta reduces the differentiation of bone marrow stromal cells [18]. On the other hand, the combination of PDGF-BB and peptides increases the proliferation, differentiation, and early calcification of the osteoblasts [19]. A few previous studies have reported the use of different combinations of PLLA with Col and HA. One study showed that the encapsulation of PDGF-BB into a microsphere enhances tissue regeneration in vitro and wound healing in vivo [20]. Although this method has several advantages, some concerns need to be addressed, such as the initial burst release within a short time and the deposition of the growth factor on the degraded microsphere. These concerns limit the use of such methods for routine tissue engineering. Additionally, high calcium (Ca) content hinders the cellular osteogenic activities of fetal osteoblasts [21]. To overcome these limitations, a biocomposite nanofibrous scaffold using 12% nano-HA was fabricated with 8% Col, and blended with high-molecular–molecular weight PLLA. We hypothesized that this artificial ECM environment supplemented with the growth factor PDGF-BB will accelerate mineralization for the rapid differentiation of human bone marrow stromal cells in vitro .

Section snippets

Fabrication of electrospun scaffolds

Electrospun scaffold sheets were prepared using high molecular weight poly(L-lactide) (PL18, Purac, The Netherlands) solutions containing HA nanoparticles (Sigma–Aldrich, USA) and/or bovine Col (Type I) (Sigma–Aldrich, USA), with an average fiber diameter of 200–950 nm. The procedure used has been described elsewhere [10].

Bone marrow stromal cell culture

Human bone marrow stromal cells were isolated using our standard laboratory protocol. The isolated cells were cultured in the DMEM medium (Invitrogen, Carlsbad, CA, USA)

Results and discussion

Bone marrow stromal cells play a key role in bone homeostasis by proliferating, migrating, and differentiating in response to stimuli [1]. These cells may potentially be used to treat several bone disorders [22]. PDGF-BB-one of the many stimulants for bone marrow stromal cells—is a potent mitogen that induces the proliferation and migration of cells. In contrast to the effect of PDGF-BB on bone marrow stromal cells, previous studies have shown decreased expression of alkaline phosphatase and OC

Conclusions

PDGF-BB enhances the osteogenic potential of PLLA/Col/HA and PLLA/HA, but there was no effect on the osteogenic potential of PLLA/Col. Supplementation of PDGF-BB into the nano-fibrous scaffolds increases the osteogenic differentiation potential. This increase probably results from the synergistic actions of the PDGF-BB and the scaffold materials. Therefore, composite fibers incorporating mitogen-like PDGF-BB may be useful as rapid stem cell differentiation tissue for engineering applications.

Conflict of interest

The authors declare no competing interests exists.

Acknowledgments

This study was supported by a major grant support (Reference number -UM.C/625/1/HIR/MOHE/CHAN/03, account number - A000003-50001), University of Malaya. A part of the research was supported by BK-035 from the University of Malaya. Special thanks to the University of Malaya Bright Sparks for their sponsorship and the Institute of Post Graduate dual Ph.D. funding for supporting G. Krishnamurithy's research.

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