Metallic ions as therapeutic agents in tissue engineering scaffolds: an overview of their biological applications and strategies for new developments

1A common tissue engineering approach involves the development of novel biomaterials to produce three-dimensional porous scaffolds, that encourage cell infiltration and proliferation for tissue regeneration. Several debilitating and deadly conditions such as osteoporosis, osteoarthritis, retinopathy, burns, myocardial infarction as well as tendon and ligament defects, among others have the potential to be treated by tissue engineering strategies. Scaffolds made from biodegradable polymers, ceramics or their composites are popular choices for tissue engineering applications and there are increasing investigations focusing on loading engineered scaffolds with therapeutic drugs, generating a dual function for the matrices: scaffolds for the growth of new tissue and carriers for controlled in situ drug delivery. In addition, there is growing interest in developing matrices with the capacity to induce specific interactions within cells in order to unlock the innate path for self-repair. Further, it is worthwhile noting the current expansion of the field of therapeutic tissue engineering (TTE), which considers the enhancement of the functionality of scaffolds by incorporating a drug delivery function with therapeutic effectiveness. In this context, in order to design and develop TTE scaffolds, several variables have to be taken into account. From a tissue engineering perspective, selection of suitable processing methods that provide the best mechanical and structural properties to the final porous scaffold is of highest relevance. From a pharmaceutical perspective, the scaffold fabrication method must be compatible with drug stability and sustained drug release; conditions such as high temperature, use of some organic solvents, pressure and free radicals that may lead to drug decomposition will restrict the selection of fabrication processes. There are several otherwise very convenient fabrication techniques for tissue engineering scaffolds, involving however, processes that are incompatible with the incorporation and stability of organic drugs. It is therefore attractive to explore the use of metallic ions as therapeutic agents (MITAs) within the scope of TTE. A wide range of MITA, the majority of them being essential cofactors of enzymes, can be considered in this regard, including cobalt, copper, gallium, iron, manganese, silver, strontium, vanadium and zinc, and will be discussed further in this article. The use of MITA does not pose the risk of decomposition or instability, which is intrinsic to organic molecules. Further, the unique properties of MITA with therapeutic significance (e.g. hydrolytic and redox activity, Lewis acidity, electrophilicity, valency, geometry, magnetic effect, spectroscopy, radiochemical properties) indicate the ability of these ions to interact with other ions that can alter cellular functions, cell metabolism or biological functions, by binding to macromolecules such as enzymes and nucleic acids, and/or activating ion channels or secondary signalling. These actions of MITA may provide effects that are different from those that can be achieved through other chemical, biochemical or genetic manipulations. In addition, an MITA approach is usually economic and stable under typical processing conditions for biomaterial scaffold production, which may involve the use of organic solvents, high temperatures, pressure and free radicals. Nevertheless, the potential toxicity of metallic ions when delivered locally has to be taken into account. From this perspective, the purpose of this review is to provide an overview of the advances in the expanding field of application of metallic ions in regenerative medicine and tissue engineering, focusing on their therapeutic applications. Particular emphasis is given to bone tissue engineering (TE), as this particular TE area seems to be the more developed regarding the use of MITA (also named bioinorganics). This article is not encyclopaedic; rather, selected examples have been chosen to illustrate and summarize the progress in the research field. In addition, some works that detail the use of MITA to regulate specific metabolic processes are included despite not yet being used in tissue engineering, but with the potential to be considered in future TE strategies. The article is organized in the following manner: §2 discusses the general local release of metallic ions and their interaction with metabolic processes, §3 focuses on the key variables needed to be taken into account when considering the inclusion of MITA in controlled drug delivery systems in general and in scaffolds for tissue engineering in particular. Finally, remaining challenges in the field and directions for future research efforts are highlighted in §4.

2In the body, various metallic ions act as cofactors of enzymes and stimulate a chain of reactions associated with cell signalling pathways towards tissue equilibrium. These properties, far from specific, are reflected in the very wide range of pathological conditions in which metallic ions are involved. Interactions with metallic ions play important roles in a variety of diseases and metabolic disorders such as cancer, central nervous system disorders, infectious diseases, perturbation of gastrointestinal activity and endocrine disorders; studies based on the effects of metallic ions in a wide range of pathologies are reviewed in the literature. Thus, the efficiency and selectivity of the therapeutic effect of metallic ions can be improved by controlling the precise level and/or location of them in the body. In addition, the ionic states of certain metallic ions are unstable, and they may have toxic effects when directly ingested. To overcome these disadvantages, extensive research has been conducted to develop matrices to control the local release of metallic ions. Current metallic-ion-based drugs are prone to lead to significant systemic toxicity; thus, the design of matrices for the local delivery of relatively high concentrations of metallic-ion-based drugs to target tissues with reduced systemic adverse effects is of high interest. The degree of metallic ion loading into matrices for local delivery and the controlled and sustained release of the loaded ions are undoubtedly important to ultimately optimize metal ion delivery for therapeutic use. In addition, it is imperative to control the release rate of loaded ions. Nevertheless, ascertaining the appropriate degree of metallic ion loading and the appropriate amount released in a determined period is difficult because therapeutic levels of most MITA are unknown. A strategy very often used to load metallic ions into matrices is to bind them to a suitable substrate (zeolites, hydroxyapatite, bioactive glass, silica, carbon fibres) so that the stability of ionic states is improved and the ions can be released over a long period of time with potential applications in many fields. Despite the efforts made in this respect, the achievements in controlling and sustaining the release of loaded therapeutic metal ions—in terms of obtaining constant therapeutic amount release of the ion over a period of time—have been very limited. Amorphous peroxititanates (APT) might also be used to bind a variety of metal compounds with high-affinity forming complexes to control the delivery of metal-based drugs to the target tissue avoiding systemic toxicity, or to capture metal ions from body tissues. Wataha et al. demonstrated that metal–APT complexes facilitate metal ion delivery (such as gold and platinum) to monocytes as well as fibroblasts. Despite the improvements made in controlling ion release from a variety of biomaterials, potential accumulation and toxicity require further research. In healthy systems, free metallic ion concentrations are maintained at very low levels, and the normal metal metabolism delivers them in a selective manner to their sites of action, while maintaining rigid control over their reactivity. However, anomalous metallic ion metabolism can contribute to pathological states such as haemochromatosis, Wilson disease and Menkes disease. Moreover, as mentioned above, the singular properties of metallic ions, such as Lewis acidity, hydrolytic and redox activity, electrophilicity and valency, can alter cellular activities supporting the cell metabolism or, in the worse case scenario, inducing toxic effects. For example, minimal shortages of certain metallic ions are involved in the pathogenesis of various chronic diseases such as diabetes mellitus, rheumatoid arthritis, coronary heart disease, epilepsy, nephropathy and a variety of bone-related pathologies. By contrast, the uncontrolled release of metal ions may produce adverse effects such as the case of corrosion of metal implants, which causes the release of significant amounts of metal ions into the tissues in close contact with the implant and the systemic circulation, often resulting in complications such as inflammatory and immune reactions. The actions of MITA loaded within matrices for local release in general, and within scaffolds for tissue engineering in particular, may be different from those that can be achieved through other chemical, biochemical or genetic interactions. The local actions of MITA within the environment in which they are released are presumed to differ in general from the actions of non-metallic agents, offering singular therapeutic opportunities. On the other hand, it is important to gain control over the potential toxicity of MITA, and the appropriate therapeutic concentrations for local release must be defined. In this context and considering the growing interest in the local release of metallic ions for therapeutic purposes, the following issues must be taken into consideration: (i) reactions of metallic ions with cellular constituents (e.g. proteins, nucleic acids—DNA and RNA—lipids, carbohydrates, redox substrates, signalling molecules); (ii) reactions of metallic ions in the local cellular environment; (iii) incorporation of metallic ions into cells and delivery to specific organelles and cellular structures; and (iv) interactions of metallic ions with specific receptors and enzymes and their involvement in metabolic pathways to alter cell functions.

3The interest in the application of MITA in the field of regenerative medicine and TE scaffold development is growing owing to the fact that MITA may offer therapeutic opportunities coupled with high flexibility to be incorporated in engineered biomaterial scaffolds by a broad range of processing methods. Moreover, MITA have lower cost, higher stability and potentially greater safety than recombinant proteins or genetic engineering approaches. Table 1 summarizes the most common processes reported to produce scaffolds for tissue engineering with the potential to be used for the incorporation of MITA in scaffolds. Processing techniques such as rapid prototyping, electrospinning, thermally induced phase separation and solid free form fabrication are attractive because they enable fabrication of engineered three-dimensional, porous structures of high uniformity and reproducibility. Additionally, organic/inorganic composite scaffolds, particularly for bone tissue engineering applications, made of bioceramics or bioactive glasses and biodegradable polymers, often include metallic ions as part of the bioceramic or bioactive glass structural composition. These inorganic materials enable metal ion release during their degradation in vitro or in vivo. For example, when bioactive glass (e.g. 45S5 Bioglass) is used in scaffolds for bone tissue engineering and introduced to fill a bone defect, critical concentrations of soluble Si, Ca, P and Na ions are released, with the capability to produce both intracellular and extracellular effects at the interface between the glass and the cellular environment. These ions are known to stimulate various processes; for example, several investigations have demonstrated that released ions from bioactive glasses are able to induce gene expression with known roles in processes related to bone metabolism by signal transduction, thereby enhancing cell differentiation and osteogenesis. The ionic dissolution products of bioactive glasses can also promote angiogenesis. It is, therefore, vital that the kinetics of ion release from any scaffold (or implant) made from bioceramics can be tailored and controlled. A comprehensive review about the biological response to ionic dissolution products from glass–ceramics and bioactive glasses in the context of bone tissue engineering has been recently published. Nevertheless, it is important to highlight that few studies are focused on developing ideal matrices for the control and sustained release of loaded ions within specific therapeutic levels, over a previously defined period of time. Several attempts to intentionally load therapeutic metal ions rely on ion substitution in ceramic systems, limiting the possibility to control and sustain the release of a specific therapeutic dose over a period of time. In this sense, the application of inorganic ions in the field of bone regeneration, with special emphasis on the lack of a controlled, sustained and localized release of both structural and non-structural ions from bioceramics, is discussed in a recent report of Habibovic & Barralet. Novel strategies are based on biodegradable metals, such as magnesium alloys and iron, which are dissolved in vivo when no longer needed. In this regard, research activities are underway to make biodegradable metals practical for tissue engineering. There are also new techniques to produce degradable metallic implants, innovative coating technologies to yield special surface functionalities, new biodegradable materials and methods to develop nano-devices for monitoring implants and sensing functions. In addition, the use of metallic ions as cross-linkers of polymers in the formation of hydrogels and as network formers or modifiers of bioactive glasses (silicate or phosphate systems) in the elaboration of bioactive scaffolds are being increasingly investigated. Figure 1 provides a summary of the most common specific targets of relevant metallic ions reviewed in the present work in their role as therapeutic agents. Table 2 summarizes relevant functions and biological effects of metallic ions with promising applications in tissue engineering. As indicated above, one of the obvious negative effects of the localized release of ions could be potential ion accumulation and toxicity. It is, therefore, vital that the kinetic of ion release from any scaffold is tailored. Several investigations have shown how the incorporation of specific metallic ions in different matrices could affect (usually improve) the physiology and metabolism of cells close to the release area; a summary of previous investigations is presented in table 3. The list is intended to be illustrative, not exhaustive. The number of specific investigations on effects of MITA intentionally added to scaffolds for therapeutic purposes, aimed at engineering a wide range of tissues, is continuously growing. Particularly, in the case of bone TE, there is increasing interest in the role of certain metallic ions (e.g. copper, strontium and zinc) in bone pathologic states because many of them are cofactors in metabolic processes involving bone, articular tissues and immune system functions. Further, the loading of MITA within scaffolds lacks the risk of drug decomposition or instability depending on the employed processes of fabrication, as explained above. Moreover, bacterial adhesion to biomaterials that causes biomaterial-centred infection and poor tissue integration are problems that could limit the viability of the scaffold, especially when it is designed to be applied in vivo (as opposed to applications in bioreactors, for example). As mentioned above, there is growing interest in exploring the possibility of using the device itself to deliver therapeutic drugs to prevent possible bacterial colonization of the device following implant surgery and/or pro-angiogenic agents to secure vascularization. In this context, bacteriostatic effect and pro-angiogenic potential seem to be the most common aims of the incorporation of metallic ions within scaffolds to date and the most common ions studied in this regard are copper, silver, strontium and zinc (table 3).

4Metallic ions are of interest in the fields of regenerative medicine and tissue engineering owing to the possibility of exploiting their unique advantages for therapeutic applications: reduced cost, increased stability and, in terms of safety, potentially lesser risk than techniques of recombinant proteins or genetic engineering. Several biomaterial-based strategies are being designed for the controlled-localized delivery of metallic ions and the field is continuously expanding. However, many challenges remain. First, there is a need to acquire a deep understanding of the roles of specific metals in cellular regulation and cell–cell signalling in both healthy and diseased tissue when they are released locally from scaffolds, implants or other releasing devices. Second, more in vivo evidence confirming that metallic ions can be released locally from scaffolds without systemic toxicity and carcinogenic effects is bound to follow. In addition, broader knowledge about mechanisms linking univocally the improved biological performance provided by TE scaffolds to the effect of metallic ions release is also needed. Much of the work is expected to involve collaborations, including biologists, material scientists, pharmaceutical technologists, tissue engineers and biomedical researchers. A great deal of further work is necessary but current investigations suggest that such work may be fruitful towards more effective tissue engineering strategies with improved MITA-releasing biomaterials. The final objective of this review has been thus to encourage research that bridges the areas at the interface between materials chemistry and medicine for developing new tissue engineering therapeutic strategies based on controlled metal ion release.