A novel route in bone tissue engineering: Magnetic biomimetic scaffolds
Introduction
The use of scaffolds in tissue engineering (TE) is constantly increasing, since these materials have become fundamental tools to help the body rebuild damaged or diseased tissues. In bone TE, the complete histomorphological and biological maturation of tissues is only achieved if angiogenesis is permanently stimulated by various angiogenic proteins, such as growth factors (GF), which have to be readily available to the tissues in the vicinity of the scaffold [1], [2]. During the bone healing process, a long regeneration time is expected to re-establish the complete functionality of damaged tissues. The temporal control of the tissue regeneration process is very important to allow optimal clinical outcomes in the tissue–biomaterial system, and it involves different agents at different times [3], [4]. However, in bone graft substitution, such temporal control cannot be achieved with traditional scaffold approaches, where GF are usually seeded in the scaffold before implantation [5], [6]. In some cases, the pre-loading techniques reduce the delivery of localized, controllable and long-term biochemical stimuli, thus impairing the tissue regeneration potential in the scaffold [6], [7]. A controlled delivery that mimics endogenous GF production therefore remains a serious issue in the use of conventional scaffolds in TE [8], [9], [10].
This problem was addressed by fabricating a conceptually new type of bone graft substitute: a magnetic scaffold able to attract and take up the GF or other bio-agents in vivo via a driving magnetic force. This novel concept involves the use of magnetic nanoparticles (MNP) which are functionalized with GF or other bio-agents (such as stem cells) to be taken up by the magnetic scaffold (V. Dediu, A. Russo, M. Marcacci, unpublished). These functionalized MNP act as carriers transporting the bio-agents towards and inside the magnetic scaffold under the effect of a magnetic field. Once in the magnetic scaffold, the magnetic carriers successively release the bio-agents that they are transporting, so that the bio-agents can be taken up by the tissues during the regeneration process. The magnetic scaffold can be thought of as a fixed “station” that can thus be reloaded repeatedly after implantation, every time this action is required by the healing process, providing the unique possibility of adjusting the scaffold activity to the specific needs of the healing tissue. Such a finely tuned delivery will be used to mimic the real endogenous GF production of the human body and will consequently enhance the control of tissue regeneration and angiogenesis processes. The magnetic scaffolds could thus provide a better alternative to the conventional ones in the cases which require a constant and heavy supply in GF, providing considerable benefit to the patients.
The magnetic guiding process is already well known in nanomedicine, though it is not yet applied in the field of scaffolds. It has been developed mainly for drug delivery and hyperthermia treatment of tumors, and it is guided in these cases by external magnetic fields, in order to increase the spatial accuracy and thus the efficacy of therapies [11], [12], [13], [14]. While this concept holds also for the guiding process towards the magnetic scaffold, the presence of such a magnetic scaffold modifies the magnetic flux distribution and leads to a much higher concentration of magnetic flux near/inside the scaffold. As a result, the magnetic scaffolds produce higher magnetic field gradients able to generate significant magnetic attractive forces. Using a soft magnetic or even superparamagnetic material, the resulting magnetic scaffold will be able to reach very high magnetization values under the application of an external magnetic field, but it will also have the attractive possibility to be magnetically “turned off” upon removal of the magnetic field.
To the best of the authors’ knowledge, there is only one report on magnetic scaffolds [15]. Nevertheless, in that case the authors treated non-biocompatible materials. The possibility of magnetizing commercial biocompatible scaffolds (Finceramica, Faenza, Spa, Italy) up to magnetization values that are used in drug delivery processes is demonstrated here. These starting commercial scaffolds are synthetic composite materials made of hydroxyapatite (HA) and collagen, and they are regularly used as bone graft substitutes because of their excellent biocompatibility properties [16], [17]. These are mainly due to the properties of each component of the composite scaffold. HA presents high osteoinductivity with neither antigenicity nor cytotoxicity, despite a low degradation rate. Collagen is known to be biocompatible, osteoinductive and, in particular, it acts as an excellent delivery system for bone morphogenetic proteins. When it is used in association with HA to form a composite, collagen prevents the dispersion of HA particles, resulting in better integration of the biomaterial to its implantation site. In addition, involving collagen enhances the adhesion of mesenchymal stem cells (MSC) to nano-apatite [18]. A possible explanation is that MSC have higher levels of alpha 2 integrin subunits compared with other types of cells (such as human osteogenic sarcoma cell lines SaOS2 and MG-63), leading to enhanced adhesion of MSC on HA/collagen composite scaffolds [19].
However, investigations have already confirmed that MNP with adequate biocompatible coatings do not have cytotoxic effects on cell development either in vitro or in vivo [20], and some MNP coated with Arg–Gly–Asp (RGD) peptides presented good biocompatibility in contact with osteoblasts [21]. It was also shown that the change in the magnetic properties of MNP in the presence of a magnetic field had no influence on cellular toxicity [22]. This suggests that the magnetization of standard HA/collagen composite scaffolds with MNP has no adverse effects on cell viability and development.
Therefore, MNP with biocompatible coatings was chosen to magnetize conventional scaffolds and maintain the initial biocompatibility properties of both materials. In order to reach this objective, an innovative magnetization technique was developed, consisting in the infilling of the MNP in the scaffolds by a simple process of dip-coating in ferrofluid, which is not damaging to biological matter.
Therefore, this paper first presents an innovative, efficient, reproducible and non-damaging way of magnetizing bioactive materials by a dip-coating process using the capillarity of the material. Second, preliminary data are presented, indicating the ability of these scaffolds to support the adhesion and proliferation of human bone marrow stem cells (hBMSC) in vitro.
Section snippets
Preparation of ferrofluids
The ferrofluids referred to as FF-DXS, FF-PAA and FF-DP were aqueous dispersions of magnetite nanoparticles (200 nm in size) and were purchased as ferrofluids from Chemicell Gmbh (Berlin, Germany). The magnetite nanoparticles from FF-DXS were previously coated by Chemicell with dextransulfate and functionalized with sodium sulfate functional groups (R-OSO3−, Na+). The magnetite nanoparticles from FF-PAA were coated with poly-dl-aspartic acid and functionalized with sodium carboxylate (−COO−, Na+
Preparation of the magnetic scaffolds
Synthetic bone graft substitutes, made of collagen or biomineralized collagen (i.e., biomimetic hydroxyapatite/collagen composites) were used as starting materials for the fabrication of magnetic scaffolds. These materials are routinely used clinically to replace damaged or diseased cartilaginous or bone tissue [24]. HA is the most suitable bioactive ceramic to be used in orthopedic reconstruction, since it replicates the mineral component of the hard tissues, and it therefore has excellent
Conclusions
This study introduced the magnetic scaffold as a conceptually new type of bone graft substitute. In order to support this innovative concept, as a first step, a simple and inexpensive method of fabricating the magnetic scaffolds was elaborated. As a starting basis, conventional commercial scaffolds made of hydroxyapatite and collagen, which are currently used for bone graft substitution, were used. Simply, the novel method is based on a dip-coating process of these conventional scaffolds in
Acknowledgments
The authors acknowledge Professor Massimo Solzi for magnetic calibrations, Daniela Casino for constructive criticism and helpful discussion, and Federico Bona for his help in technical aspects. Finally, the financial support from EU project “MAGISTER” NMP3-LA-2008-21468 is acknowledged.
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