Bone is the most studied tissue in the field of tissue regeneration. Even though it has intrinsic capability to regenerate upon injury, several pathologies and injuries could hamper the highly orchestrated bone formation and resorption process. Bone tissue engineering seeks to mimic the extracellular matrix of the tissue and the different biochemical pathways that lead to successful regeneration. For many years, the use of extrinsic factors (i.e., growth factors and drugs) to modulate these biological processes have been the preferred choice in the field. Even though it has been successful in some instances, this approach presents several drawbacks, such as safety-concerns, short release profile and half-time life of the compounds. On the other hand, the use of inorganic ions has attracted significant attention due to their therapeutic effects, stability and lower biological risks. Biomaterials play a key role in such strategies where they serve as a substrate for the incorporation and release of the ions. In this review, the methodologies used to incorporate ions in biomaterials is presented, highlighting the osteogenic properties of such ions and the roles of biomaterials in controlling their release.

Biomaterials and scaffolds for Tissue Engineering are widely used for an effective healing and regeneration. However, the implantation of these scaffolds causes an innate immune response in which the macrophage polarization from M1 (pro-inflammatory) to M2 (anti-inflammatory) phenotype is crucial to avoid chronic inflammation. Recent studies have showed that the use of bioactive ions such as cobalt (Co2+), copper (Cu2+) and magnesium (Mg2+) could improve tissue regeneration, although there is limited evidence on their effect on the macrophage response. Therefore, we investigated the immunomodulatory potential of Co2+, Cu2+ and Mg2+ in macrophage polarization. Our results indicate that Mg2+ and concentrations of Cu2+ lower than 10 μM promoted the expression of M2 related genes. However, higher concentrations of Cu2+ and Co2+ (100 μM) stimulated pro-inflammatory marker expression, indicating a concentration dependent effect of these ions. Furthermore, Mg2+ were able to decrease M1 marker expression in presence of a mild pro-inflammatory stimulus, showing that Mg2+ can be used to modulate the inflammatory response, even though their application can be limited in a strong pro-inflammatory environment.

Cardiovascular diseases are considered one of the worldwide causes of death, with atherosclerosis being the most predominant. Nowadays, the gold standard treatment is blood vessel replacement by bypass surgery; however, autologous source is not always possible. Thereby, tissue-engineered blood vessels (TEBVs) are emerging as a potential alternative source. In terms of composition, collagen has been selected in many occasions to develop TEBVs as it is one of the main extracellular matrix components of arteries. However, it requires specific support or additional processing to maintain the tubular structure and appropriate mechanical properties. Here, we present a method to develop support-free collagen TEBVs with co-axial extrusion in a one-step procedure with high concentrated collagen. The highest concentration of collagen of 20 mg/mL presented a burst pressure of 619.55 ± 48.77 mmHg, being able to withstand perfusion of 10 dynes/cm2. Viability results showed a high percentage of viability (86.1 and 85.8% with 10 and 20 mg/mL, respectively) of human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVEC) after 24 h extrusion. Additionally, HUVEC and HASMCs were mainly localized in their respective layers, mimicking the native distribution. All in all, this approach allows the direct extrusion of collagen TEBVs in a one-step procedure with enough mechanical properties to be perfused.

The use of copper (Cu2+) and cobalt (Co2+) has been described to stimulate blood vessel formation, a key process for the success of tissue regeneration. However, understanding how different concentrations of these ions affect cellular response is important to design scaffolds for their delivery to better fine tune the angiogenic response. On the one hand, gene expression analysis and the assessment of tubular formation structures with human umbilical vein endothelial cells (HUVEC) revealed that high concentrations (10μM) of Cu2+ in early times and lower concentrations (0.1 and 1μM) at later times (day 7) enhanced angiogenic response. On the other hand, higher concentrations (25μM) of Co2+ during all time course increased the angiogenic gene expression and 0.5, 5 and 25μM enhanced the ability to form tubular structures. To further explore synergistic effects combining both ions, the non-toxic concentrations were used simultaneously, although results showed an increased cell toxicity and no improvement of angiogenic response. These results provide useful information for the design of Cu2+ or Co2+ delivery scaffolds in order to release the appropriate concentration during time course for blood vessel stimulation.

Macrophages have been deemed crucial for correct tissue regeneration, which is a complex process with multiple overlapping phases, including inflammation. Previous studies have suggested that divalent ions are promising cues that can induce an anti-inflammatory response, since they are stable cues that can be released from biomaterials. However, their immunomodulatory potential is limited in a pro-inflammatory environment. Therefore, we investigated whether copper and magnesium ions combined with low concentrations of the anti-inflammatory drug, dexamethasone (dex), could have a synergistic effect in macrophage, with or without pro-inflammatory stimulus, in terms of morphology, metabolic activity and gene expression. Our results showed that the combination of copper and dex strongly decreased the expression of pro-inflammatory markers, while the combination with magnesium upregulated the expression of IL-10. Moreover, in the presence of a pro-inflammatory stimulus, the combination of copper and dex induced a strong TNF-α response, suggesting an impairment of the anti-inflammatory actions of dex. The combination of magnesium and dex in the presence of a pro-inflammatory stimulus did not promote any improvement in comparison to dex alone. The results obtained in this study could be relevant for tissue engineering applications and in the design of platforms with a dual release of divalent ions and small molecules.

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Implantoplasty is a mechanical decontamination technique that consists of removing the threads and polishing and smoothing the dental implant surface. During implantoplasty there is a large release of titanium metal particles that might provoke a proinflammatory response and reduce the viability of osteogenic cells. We analyze the inflammatory and osteogenic response induced by Ti6Al4V particles released during implantoplasty and by as-received commercially pure Ti particles. Macrophages stimulated with metal particles obtained by implantoplasty and with as-received Ti particles showed an increased proinflammatory expression of TNF-α and a decreased expression of TGF-β and CD206. Regarding cytokine release, there was an increase in IL-1β, while IL-10 decreased. The osteogenic response of Ti6Al4V extracts showed a significant decrease in Runx2 and OC expression compared to the controls and commercially pure Ti extracts. There were no relevant changes in ALP activity. Thus, implantoplasty releases metal particles that seems to induce a pro-inflammatory response and reduce the expression of osteogenic markers.

Bone has the intrinsic capacity to regenerate itself, as long as the damage is small, through the sequential stimulation of specific phases, such as angiogenesis followed by osteogenesis. However, when the damage is extensive it is unable to regenerate and bone tissue engineering is used as an alternative. In this study, we developed a platform to allow the triple ion delivery with sequential delivery capacity to potentially stimulate antibacterial, angiogenic and osteogenic processes. The scaffold-based platform consisted of alginate/hydroxyapatite (HA) microparticles embedded in alginate fibers. Firstly, microparticles were developed using different ratios of alginate:HA using the spraying method, resulting in a high reproducibility of the technique. Microparticle size between 100–300 µm and ratio 1:40 resulted in a more spherical morphology and were selected for their incorporation into alginate fiber. Different amounts of copper and cobalt were added with the microparticles and alginate fiber, respectively, were used as model ions which could eventually modulate and mimic antimicrobial and angiogenic processes. Moreover, calcium ion was also incorporated in both, in order to provide the system with potential osteogenic properties together with HA. The multiple delivery of copper, cobalt and calcium released were in the therapeutic range as measured by induced coupled plasma (ICP), providing a promising delivery strategy for tissue engineering.