Magnetic Nanoparticles in Bone Tissue Engineering: Comparison
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Large bone defects with limited intrinsic regenerative potential represent a major surgical challenge and are associated with a high socio-economic burden and severe reduction in the quality of life. Tissue engineering approaches offer the possibility to induce new functional bone regeneration, with the biomimetic scaffold serving as a bridge to create a microenvironment that enables a regenerative niche at the site of damage. Magnetic nanoparticles have emerged as a potential tool in bone tissue engineering that leverages the inherent magnetism of magnetic nano particles in cellular microenvironments providing direction in enhancing the osteoinductive, osteoconductive and angiogenic properties in the design of scaffolds. There are conflicting opinions and reports on the role of MNPs on these scaffolds, such as the true role of magnetism, the application of external magnetic fields in combination with MNPs, remote delivery of biomechanical stimuli in-vivo and magnetically controlled cell retention or bioactive agent delivery in promoting osteogenesis and angiogenesis. 

  • magnetic nanoparticles
  • SPIONs
  • bone tissue engineering
  • scaffolds for bone tissue engineering

1. Introduction

Bone tissue is capable of natural regeneration by harnessing intramembranous and endochondral ossification since postnatal bone can carry out self-repair and remodel at the site of damage to restore function [1]. However, this self-healing mechanism fails to occur in the case of critically sized defects [2]. Traumatic injury, degenerative disease, tumour resection, infection and congenital defects can all lead to critical-sized bone defects, which necessitate intervention to achieve complete healing [3]. Bone autografts and allografts as well as metallic implants are presently established treatment modalities for critical-sized defects. Autografts possess excellent osteoinductive, osteoconductive properties that are histocompatibile and non-immunogenic hence resulting in higher rates of success. Nevertheless, autografts can result in significant donor site morbidity and surgical risks such as infection, bleeding and pain, making them less feasible for defects that require large volumes of bone [4]. Using allografts can eliminate the issue of donor site harvesting, however, they have lower osteogenic capability along with concerns of immunoreactions and infection transmission. Both these grafting techniques incur substantially high costs to perform, and the grafting market is struggling to meet the current high demands [5]. Although a wide variety of synthetic biomaterials are used as alloplastic materials, the clinical outcomes are variable. Overall, none of the current treatment options have all the desired characteristics that possess good osteoinductive properties and exhibit angiogenic potential, biosafety, availability, reasonable cost, low patient morbidity and no size restrictions [6].

Bone Tissue Engineering

Bone tissue engineering (BTE) is a field that strives to outperform current treatments by providing potential alternatives to overcome the limitations of current approaches in bone regeneration [7]. Bone tissue engineering aims to induce new tissue repair and regeneration by the synergy of reparative cells, signalling molecules and scaffolds. Three-dimensional scaffolds that can mimic the extracellular matrix template, whilst offering mechanical support, provides an attractive environment for cell attachment, proliferation and differentiation. Hence, materials for scaffolds are selected based on their ability to display biomimicry, with regards to key parameters such as mechanical properties, porosity, osteoinduction and osteoconduction. Research over the last two decades has focused on scaffolds with and without biological components, however, the complex regulatory requirements, high costs and need for feasible clinical translational technologies have steered the direction of research towards acellular scaffolds that are designed to recruit cells from surrounding native tissue post-implantation to enable bone regeneration. The success of in-situ bone regeneration depends on the effective recruitment of host stem cells or progenitor cells into the scaffold consequently inducing the infiltrating cells into tissue-specific cell lineage for functional bone tissue regeneration. The use of appropriate bioactive agents or bioactive scaffolds that can recruit cells with osteogenic capability through the formation of mineralised matrices through the entire scaffold structure can help drive and accelerate the regenerative process. Crucially, vascularisation must occur alongside bone formation to support the needs of the growing tissue [8][9]. Many strategies have been explored towards enhancing the osteogenic and angiogenic capacity of scaffolds [7][8] with structural hallmarks close to the nanoscale composition of natural bone and modifications to enhance physicochemical interactions, biocompatibility, mechanical stability and cellular attachment/survival. While bone tissue engineering has provided promising results, it has become increasingly clear that the hierarchical integration of bone scaffolds and vascular networks to create constructs that support both osteogenic and angiogenic growth is crucial for success. Leveraging principles that can drive the in-situ body’s innate cellular populations to regenerate tissues is of high interest and with advances in nanomaterials and specifically magnetic nanoparticles (MNP), provide new opportunities in the development of more effective therapeutic efficacy.

2. Magnetic Nanoparticles in Scaffolds for Bone Tissue Engineering

Many different strategies have been investigated to combine scaffolds, cells and biologically active cues using a wide range of fabrication techniques, to provide innovative solutions for bone tissue biomimicry. Thus far there has been an emphasis on the microarchitecture of the scaffold that focuses on porosity, pore size and pore interconnectivity, to facilitate the proper mass transfer of nutrients and waste products, as well as vascularisation and tissue infiltration in addition to mechanical properties and bioactivity. Mechano-transduction is understood to facilitate osteogenesis and thus has been considered into a multitude of in vitro bone tissue engineering approaches to effectively control cell behaviour. More recently, magnetic nano actuation is being explored to remotely manipulate cell behaviour with much greater control and accuracy. MNPs can be integrated within scaffold matrices using a range of fabrication techniques such as electrospinning, covalent linkages and freeze-drying. The effect of MNPs containing scaffolds on osteogenesis is discussed first and then their role in promoting angiogenesis is examined.

2.1. Impact on Osteogenesis

A summary of the findings of selected recent studies that investigated the impact of incorporating MNPs into scaffolds on osteogenesis is presented in Table 1. It is important to note that the studies included in this table used different types of scaffolds with differing chemistries, variation in MNP content and magnetic intensity. Despite these differences, all studies concluded that the addition of MNPs significantly enhanced osteogenesis. There was also a substantial crossover in the mechanisms suggested for the enhanced osteogenic activity between the studies.
Most of the results summarised in Table 1 also indicated that bioactivity was enhanced in the presence of MNPs in scaffolds, specifically revealing greater cell adhesion and cell spreading, which displayed more stretched and spindle-like morphology [10].
One reason for these findings could be attributed to the greater hydrophilicity of the scaffolds since MNPs are inherently hydrophilic and their incorporation remarkably improves the wettability of the scaffold thereby enhancing the affinity for cells and proteins that mediate cell attachment [11]. The incorporation of MNPs was also reported to alter nano structural features of scaffolds, especially in calcium phosphates (CPC) wherein the crystal size shows a decrease leading to a greater surface area [12][13], consequently increasing adhesion of protein molecules that facilitate subsequent cell adhesion. Similar findings were observed on polycaprolactone (PCL) polymeric scaffolds that exhibited enhanced protein adsorption which increased with increasing MNP content [11]. Additionally, MNPs induce changes in nanostructure topography, such as greater surface roughness, which encourages cell adhesion and spreading. Overall, there is consensus on the relationship between MNP incorporation in scaffolds and enhanced cell adhesion mainly attributed to increased hydrophilicity [10][11][14][15][16] or the nanostructure [12][13][14][15][17].
The inclusion of MNPs and its concentration has a bearing on the mechanical properties of scaffolds. Several studies [13][15][16][17][18] have reported that improvement in mechanical properties is dependent on the amount of MNPs present and beyond certain concentrations show a steady decline in their mechanical properties. The improvement in mechanical properties [10][14] has been attributed to either chemical interactions or the influence on microarchitecture. For example, PCL-MNP scaffolds have been reported to exhibit an increase in stiffness [18] whilst a chitosan collagen scaffold showed a higher compressive modulus due to the interactions between the inorganic MNPs and organic chitosan collagen matrix [17]. In contrast, improvements in the mechanical properties of CPC-based scaffolds were mainly attributed to a reduction in pore size and pore volume fraction [12][14] although some studies have demonstrated that MNP incorporation has no significant impact on the porosity level of a scaffold [11][16][19][20]. However, it is prudent to consider the material type to understand the effect on porosity since MNPs have been found to generally reduce the porosity of PCL or CPC scaffolds, whilst having the opposite effect on natural scaffold materials such as chitosan or collagen. From the analysis of these studies, it can be deduced that the mechanical properties of scaffolds can be manipulated by incorporating MNPs; however, the optimisation of the content is imperative to achieve benefits.
A common feature of the different studies summarised in Table 1 was the reportedly remarkable improvement in cell proliferation and osteogenic differentiation irrespective of the type of scaffold. Most of the studies utilised SPIONs such as magnetite or maghemite as the MNP and their addition endowed the scaffolds with a superparamagnetic property. Each SPION within the scaffold behaves as a single magnetic domain and the combined effect of all the nanoparticles generates a magnetic microenvironment. The cells are stimulated by this microenvironment because of significant alterations to ion channels and receptors on the cell membrane that activate intracellular signalling pathways [11][15][16]. This effect is very similar to cell responses to mechanical stimuli whereby cells are transduced via the activation of mechanosensitive ion channels or receptors [21]. Magnetic induction could therefore explain the accelerated cell cycles and osteogenic differentiation, although the exact mechanism by which this occurs is yet to be elucidated. In contrast, Xia et al. used a demagnetised magnetic scaffold generated through high-temperature annealing and compared directly to magnetic scaffolds, which revealed no difference in cell behaviour [12]. Therefore, it he study excluded the effect of magnetism and instead concluded that the nanostructure was the main reason for improved cell performance. Hence, further work needs to be conducted to authenticate the role of magnetism in osteogenesis.
ThLiterature findings suggest that MNPs can induce a transmembrane effect in the form of an upregulated magneto-sensing receptor that promotes osteogenesis within ADSCs, however, the signalling cascade that mediates this is not clear. Chen et al. [10] found that the gene expression of an exogenous magnetoreceptor, iron-sulphur cluster assembly protein 1 (ISCA1), was upregulated because of the magnetic microenvironment in ADSCs. Moreover, the expression of ISCA1 was highly correlated with the upregulated expression of osteogenic genes ALP and RUNX2. Xia et al. [14] proposed that the WNT signalling pathway regulated the osteogenic differentiation of DPSCs. The upregulation of the transmembrane receptor WNT1 and intracellular protein β catenin indicates the role of this pathway in mediating osteogenic gene expression [14]. Alternatively, Lu et al. [18] observed that the BMP-2/Smad/RUNX2 pathway was activated within BMSCs upon magnetic stimulation, indicated by the greater expression of its components [18]. These contrasting findings may imply that the signalling pathway via which proliferation and osteogenic differentiation occur is dependent on the stem cell type being investigated. However, the studies did not rule out the involvement of other signalling pathways, meaning that multiple pathways could be working simultaneously. Hence a study that investigates the involvement of a range of signalling pathways in ADSCs, DPSCs and BMSCs is needed to elucidate how different stem cells mediate osteogenesis upon magnetic stimulation.
As noted earlier that the concentration of MNPs in a scaffold influences mechanical properties, it too has a profound effect on cell proliferation and osteogenic differentiation. Although the magnetic microenvironment can be intensified by increasing the MNP content in scaffolds [10] there is a limiting value after which cell activity markedly decreases. Studies that assessed different content of MNP noted that cell proliferation and osteogenic differentiation increased with increasing MNP content [11][15][18][22]; however, in both PCL and CPC scaffolds, cell performance, including proliferation and ALP activity, was found to be at its maximum at 3% and 15% MNP content, respectively, [15][18] after which there was rapid decline. These findings indicate that increasing the MNP content can improve cell performance, however, the optimum content must be carefully elucidated to avoid toxicity.
In vivo studies to validate the potential of MNP-loaded scaffolds, refs. [16][17][18][19][22][23][24] showed superior bone formation and mineralisation of defects filled with MNP-loaded scaffolds compared to controls. This was quantitatively represented with significantly greater bone mineral density and bone volume fraction. A study by Zhao et al. [17] showed that osteoblasts had greater adhesion and infiltration through the scaffold, supporting the hypothesis that MNPs improve the nano structural properties [17]. It was also reported that the new bone tissue was well fused and better integrated with the host bone than in control groups [18][22]. Overall, the subjects that received MNP-loaded scaffolds displayed better healing outcomes.
Table 1. A table summarising the various studies that investigated the impact of MNP-incorporated scaffolds on osteogenesis.
Scaffold Material MNP Composition MNP Content within Scaffold Magnetism Intensity (emu/g) Osteogenic Impact Mechanism
HA and Collagen [23] NI 2.65% NI Enhanced bone maturity in-vivo, identified by improved mechanical properties. Incongruous magnetic moment created by the distribution of MNPs within the scaffold.
PCL [21] Maghemite 7.9% NI Improved cell adhesion, proliferation and osteogenic differentiation (elevated ALP) of MSCs. MNP incorporation generates a magnetic microenvironment.
PCL [14] GdHA 2.67% NI Greater cell attachment, spreading, proliferation and osteogenic differentiation (higher ALP, RUNX2) of MSCs.

Improved mechanical properties.		 Gadolinium released entered cells and promoted cell cycle progression.

Greater hydrophilicity and surface area facilitate protein adsorption.

Reduced PCL fibre diameter increases scaffold strength.
PCL [24] FeHA 4.5% NI Improved cell growth.

Scaffold filled with new bone after just 4 weeks in-vivo.		 MNP incorporation generates a magnetic microenvironment.
PCL [11] Magnetite 5%

10%		 5%—1.6

10%—3.1		 Greater cell adhesion, proliferation and osteogenic differentiation (enhanced cellular mineralisation) of MSCs. Elevated hydrophilicity improved cell adhesion that facilitated proliferation and differentiation to follow.

MNP incorporation generates a magnetic microenvironment.
PCL [18] Magnetite 5%, 10%, 15%, 20% 5%—1.0

20%—11.2		 Better cell adhesion, spreading, penetration and osteogenic differentiation (ALP, COL-1, OPN, BSP) of MSCs.

Histology showed higher blood vessel

formation and better integration with the host tissue in-vivo.

Enhanced mechanical properties.		 MNP incorporation generates a magnetic microenvironment.

Controlled degradation rate allows ingrowth of cells and vascularisation. Strong chemical interaction between MNPs and polymer chains.
PCL and PLGA [10] Maghemite 16.4% 3.56 Improved cell adhesion, spreading and osteogenic differentiation (higher ALP, RUNX2, OCN, COL-1 and bone mineralisation) of ADSCs.

Better mechanical properties.		 Greater hydrophilicity and protein adsorptions facilitate cell attachment.

Higher gene expression of a transmembrane magnetoreceptor ISCA1-osteogenic enhancement as a result of transmembrane effect of MNPs.
PLLA and PGA [22] Magnetite 2.5%, 5%, 7.5%, 10% 2.5%—1.66

10%—8.51		 Greater cell adhesion, spreading, proliferation and osteogenic differentiation (ALP) of MG63 cells.

Improved mechanical properties.

Better BMD, BVF, fusion and blood vessel formation in-vivo.		 Improved hydrophilicity and magnetic microenvironments facilitate improved cellular activity.

MNPs resist deformation of the polymer chains.

Microenvironment promoted adhesion, migration and differentiation of osteocytes in-vivo.
PCL and Mesoporous Bioactive glass [20] Magnetite 5%, 10%, 15% 5%—3.1

10%—6.2

15%—9.3		 Increased cell adhesion, proliferation and osteogenic differentiation (elevated ALP, RUNX2, OCN, BMP-2 and COL-1) of MSCs. Improved hierarchal pore structure.

MNP incorporation generates a magnetic microenvironment.
CPC [13] Magnetite 0.05–5% 0.1%—0.05

1%—0.35		 Greater cell adhesion, spreading, proliferation and osteogenic differentiation (increased ALP) of BMSCs.

Improved mechanical properties.		 Altered surface morphology- change in crystal shape and reduced size increased the surface area for adhesion of proteins involved in cell adhesion.

MNP incorporation generates a magnetic microenvironment.
CPC [12] Maghemite NI NI Enhanced cell attachment, spreading, proliferation and osteogenic differentiation (increased ALP, RUNX2, OCN, COL-1) of DPSCs. Altered surface morphology-reduced crystal size increased the surface area for adhesion of proteins involved in cell adhesion.

MNPs released by the degrading scaffolds and interact with cells via membrane adsorption and internalisation.
CPC [15] Maghemite 1–6% NI Improved cell adhesion, spreading, proliferation and osteogenic differentiation (increased ALP, RUNX2, OCN, COL-1) of DPSCs.

Enhanced the mechanical properties.		 Greater hydrophilicity and improved nanostructure facilitated cell adhesion and spreading.

The WNT signalling pathway is activated and mediates proliferation osteogenic differentiation upon magnetic stimulation.

Cells internalise released MNPs.
Gelatin and Siloxane [16] Magnetite 1–3% 1%—0.24

3%—0.64		 Greater cell adhesion, proliferation and osteogenic differentiation (greater ALP and mineralisation) of MSCs.

Improved mechanical properties.		 Improved hydrophilicity allowed better cell adhesion.

MNP incorporation generates a magnetic microenvironment.
Bioglass and Chitosan [19] SrFe12O19 1:7, 1:3

(ratio of SrFe12O19 to Bioglass)		 1:7–4.44

1:3–7.68		 Enhanced cell adhesion, spreading, proliferation and osteogenic differentiation (increased ALP, RUNX2, OCN, COL-1, BMP-2) of BMSCs.

Greater bone mineralisation, BMD and BV/TV in-vivo.		 Proliferation and osteogenic differentiation are mediated by BMP-2/Smad/RUNX2 pathway upon magnetic stimulation.
Chitosan and Collagen [17] Magnetite NI 0.025 Improved cell adhesion, proliferation and osteogenic differentiation (better mineralisation) in pre-osteoblasts.

Enhanced bony ingrowth, BMD and BVF in-vivo.

Better mechanical properties.		 Improved hierarchical nanostructure- surface roughness and interconnected porosity. This can improve cell adhesion, cell penetration as well as nutrient transfer and flow transportation in the scaffold.
Abbreviations: NI—Not Included, HA—Hydroxyapatite, PCL—Polycaprolactone, PLGA—Poly(lactic co-glycolic acid), PLLA—Polylactic acid, PGA—Poly(glycolic acid), CPC—Calcium Phosphate cement, GdHA—Gadolinium-doped Hydroxyapatite nanoparticles, FeHA—Iron-doped Hydroxyapatite nanoparticles, BMD—Bone Mineral Density, BVF—Bone Volume Fraction, BV/TV—Bone Volume/Tissue Volume. Magnetite—Fe3O4; Maghemite—ΥFe2O3

2.2. Effect of MNPs on Angiogenesis

Angiogenesis alongside osteogenesis is pivotal for the survival of cells, especially in deeper regions of a scaffold where the nutrient exchange is even more challenging. This has currently limited BTE to small constructs that do not meet the clinical demands for repairing large bone defects, marking angiogenesis as a major challenge in BTE [25]. To combat this, various strategies to enhance the angiogenic capacity of a construct have been studied [26]. Examples include the co-delivery of growth factors VEGF and BMP-2 [27] or the addition of trace elements such as Mg2+ or Si4+ [28]. Recent evidence suggests that MNPs may not just influence osteogenesis but also promote angiogenesis implying that MNP-incorporated scaffolds could potentially have a dual function. The promotion of both processes is termed osteogenic-angiogenic coupling, which plays a major role in bone regeneration.
To elucidate the effect of MNPs on angiogenesis, several studies have been attempted to understand the effect. An in vivo study implanted gelatine sponges with SPIONS in incisor sockets of rats that exposed the SPIONS due to the rapid degradation of the gelatine [29]. The gelatine sponges carrying the SPIONS displayed higher bone mineral density and trabecular volume/tissue volume, supported by greater new bone formation on histology. Interestingly, histology also revealed enhanced blood vessel formation alongside bone development, and it was evident that osteoblasts and vascular endothelial cells had internalised the SPIONs leading to elevated osteogenic and angiogenic performances [29]. In support of these findings, another study reported neo blood vessel formation in addition to bone formation [18] when SPIONS were included in PCL scaffolds. In both these studies, the SPION containing constructs gave rise to substantially greater neovascularisation in comparison to the controls that contained no SPIONs implying potential pro-angiogenic effects. The results from these in-vivo studies are promising, however, the mechanisms by which MNPs promote angiogenesis are yet to be elucidated. Thus, in-vitro studies that investigate the impact of MNPs on endothelial cells are expected to provide insights into the understanding of the angiogenic effects of MNPs and how to best apply them in-vivo for more successful results. However, the pro-angiogenic effect of MNPs is still conflicted as researchers suggest that although it positively impacts osteogenesis, there is no angiogenic activity [22] and furthermore to refute the role of MNPs, a growing body of researchers have discovered the antiangiogenic effect of SPIONs. They are being used to inhibit the growth of tumours by impeding vascular growth. In brief, one in-vitro study reported that polyethyleneimine-coated SPIONs impaired the activation, migration and tube formation of primary human umbilical cord vein endothelial cells (HUVECs). The mechanisms underlying this were attributed to the SPIONs increased reactive oxygen species production that altered actin cytoskeleton activity in HUVECs [30]. Overall, the current research investigating the impact of SPIONs on angiogenesis is conflicting. Some studies have noted a pro-angiogenic impact in BTE, whilst others demonstrate an anti-angiogenic effect in an anti-tumoral therapy investigation, which clearly demonstrates that more exhaustive studies are required.

2.3. External Magnetic Stimulation

The application of an external magnetic field can work synergistically to enhance the magnetic stimulation of cells and this combined strategy has been experimented on in BTE, resulting in improved bone formation and an enhanced angiogenic impact than just MNPs alone [31]. The results of a study on a nanocomposite scaffold comprised of PCL/magnetic nanoparticles revealed that the stimulatory effect of the magnetic scaffold and the SMF was more significant than the magnetic scaffold alone [32].
ItThe isstudy showed that the magnetic stimulation equipped the osteoblasts with enhanced functional activity, so they could secrete molecules that have a positive impact on endothelial cell function. This effect was more profound in the combined stimulus group, implying that the addition of an external magnetic field can boost the angiogenic capacity of a magnetic bone scaffold. The effect of this combined strategy was examined to see the effect it had on macrophages [33] and reported that stimulated macrophages could secrete higher levels of angiogenic growth factors compared to unstimulated control cells, which could be attributed to the higher consumption of oxygen by the macrophages because of the more substantial stretching and bending forces within the scaffold from the combined stimulus.
Although studies demonstrate that the application of an external field can enhance osteogenesis and pro-angiogenic potential, the introduction of an external field introduces a handful of complexities. First and foremost, studies should begin to suggest how such a device can be applied clinically with regards to the length of use and follow up. The device should be reasonably practical for the patient, and this should consider parameters such as adherence and ease of manipulation. Importantly, the costs of the magnets should be affordable, given that some strategies in tissue engineering, such as the use of growth factors, have received criticism due to their high costs [26]. Given that all these conditions are met, an external magnetic field can be warranted for use. Alternatively, more research into the use of just MNPs can help determine if the external field is necessary or can be avoided because MNPs alone perform adequately for sufficient angiogenesis.

3. Conclusions and Future Perspectives

To conclude, herein discusses the diverse ways through which MNP can augment BTE, by offering novel solutions and enhancements to each of the three tissue engineering components. Cells experience a marked improvement in osteogenic differentiation and cell proliferation through direct interactions with SPIONs. This is supported by in-vivo findings, showing that osteoblasts and endothelial cells internalise the SPIONs, yielding superior bone regeneration accompanied by blood vessel formation. Moreover, the use of an external magnetic field offers control over MNP labelled cells, providing advances in minimally invasive cell-based regenerative therapy of bone defects and fabricating scaffold-free cell-based constructs for BTE. Furthermore, bioactive agents carrying MNPs can then be delivered to target sites under external magnetic control. Specifically, SPIONs can transport angiogenic plasmids to cells within a scaffold or precisely produce growth factor gradients for complex bone tissue interface engineering.
Finally, MNP-incorporated scaffolds hold a clear advantage over generic scaffolds attributed to their enhanced mechanical properties and cell performance in-vitro. However, the disagreement in the literature regarding how this occurs needs attention to elucidate the impact. More research investigating the impact of demagnetising MNP-loaded scaffolds on cell performance should be conducted. This will help clarify if the magnetic microenvironment genuinely plays a role in improving cell performance along with the justified effect of the nano structural properties of MNP-incorporated scaffolds. If magnetism does play a role, further research exploring the activation of intracellular pathways is of interest. This research should incorporate a range of stem cell sources to uncover any differences or similarities in the responses between stem cell types. Nevertheless, the enhanced in-vitro cell performances were strongly supported in-vivo, as the scaffolds displayed greater bone regeneration and host tissue integration. However, the literature has only lightly touched upon the enhanced angiogenic performance of the scaffolds in-vivo. Studies investigating the impact of MNP-loaded scaffolds and an external magnetic field on angiogenesis have been conducted, however more studies are required. Future research warrants the study of the impact of solely MNP loaded scaffolds. This could take shape with an in-vitro study, exploring the effect that the scaffold has on endothelial cell performances. What is also evident in the literature is that these enhancements are dose-dependent. Beyond a given dose, agglomeration and potential toxicity of the MNPs may hinder the mechanical properties and cell performance, respectively.
Leading on from this, herein aimed to explore the concerns of toxicity related to MNPs. Both in-vitro and in-vivo studies have evidenced the fact that toxicity is dose-dependent. It has been established that the toxic dose is cell-specific and the MNP features can directly influence the dose that is internalised by cells. Therefore, each specific MNP-cell interaction should be closely examined to prevent toxicity in each biomedical application.
Given these findings, herein collated data from the literature to examine the impact that a given dose of SPION had on the osteogenic differentiation of stem cells. Two studies reported that osteogenesis was impaired in human MSCs, however, toxicity played no role in this. The remainder of the studies observed no concern of toxicity in human stem cells with osteogenesis remaining unaffected or even promoted in some cases. It could be suggested that the toxic dose of SPION in human stem cells undergoing osteogenic differentiation should be determined with a future study. However, considering that osteogenesis was in fact promoted at very low doses (0.9 pg), it may be unnecessary to elucidate this toxic dose. What is a more pressing area of future research is the ultimate fate of SPIONs that avoid degradation. Considering that toxicity is caused by SPION degradation, SPION coatings that are degradation resistant can undoubtedly prevent toxicity. The fate of such SPIONs that remain intact and uncleared from the body for long periods needs to be thoroughly investigated in-vivo. A prospective study should monitor the subjects for the length of time required to finally discover the fate of the SPIONs. Given that this is a safe outcome, MNPs will gain further validation for application in biomedical applications.

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