Engineering a microparticle-loaded rough membrane for guided bone regeneration modulating osteoblast response without inducing inflammation.

Guided bone regeneration (GBR) is a widely used procedure that prevents the fast in-growth of soft tissues into bone defect. Among the different types of membranes, the use of collagen membranes is the gold standard. However, these membranes are implanted in tissue location where a severe acute inflammation will occur and can be negatively affected. The aim of this study was to develop a collagen-based membrane for GBR that incorporated alginate-hydroxyapatite microparticles. Membranes were manufactured using collagen type I and gelatin and alginate-hydroxyapatite microparticles. Membranes were assessed in terms of topography by scanning electron microscopy and confocal microscopy; stability by swelling after an overnight incubation in saline and enzymatic degradation against collagenase and mechanical properties by tensile tests. Furthermore, the biological response was assessed with SaOs-2 cells and THP-1 macrophages to determine alkaline phosphatase activity and inflammatory cytokine release. Our results showed that the incorporation of different percentages of these microparticles could induce changes in the surface topography. When the biological response was analyzed, either membranes were not cytotoxic to THP-1 macrophages or to SaOs-2 cells and they did not induce the release of pro-inflammatory cytokines. However, the different surface topographies did not induce changes in the macrophage morphology and the release of pro- and anti-inflammatory cytokines, suggesting that the effect of surface roughness on macrophage behavior could be dependent on other factors such as substrate stiffness and composition. Collagen-gelatin membranes with embedded alginate-hydroxyapatite microparticles increased ALP activity, suggesting a positive effect of them on bone regeneration, remaining unaffected the release of pro- and anti-inflammatory cytokines.

Angiogenic and immunomodulation role of ions for initial stages of bone tissue regeneration.

It is widely known that bone has intrinsic capacity to self-regenerate after injury. However, the physiological regeneration process can be impaired when there is an extensive damage. One of the main reasons is due to the inability to establish a new vascular network that ensures oxygen and nutrient diffusion, leading to a necrotic core and non-junction of bone. Initially, bone tissue engineering (BTE) emerged to use inert biomaterials to just fill bone defects, but it eventually evolved to mimic bone extracellular matrix and even stimulate bone physiological regeneration process. In this regard, the stimulation of osteogenesis has gained a lot of attention especially in the proper stimulation of angiogenesis, being critical to achieve a successful osteogenesis for bone regeneration. Besides, the immunomodulation of a pro-inflammatory environment towards an anti-inflammatory one upon scaffold implantation has been considered another key process for a proper tissue restoration. To stimulate these phases, growth factors and cytokines have been extensively used. Nonetheless, they present some drawbacks such as low stability and safety concerns. Alternatively, the use of inorganic ions has attracted higher attention due to their higher stability and therapeutic effects with low side effects. This review will first focus in giving fundamental aspects of initial bone regeneration phases, focusing mainly on inflammatory and angiogenic ones. Then, it will describe the role of different inorganic ions in modulating the immune response upon biomaterial implantation towards a restorative environment and their ability to stimulate angiogenic response for a proper scaffold vascularization and successful bone tissue restoration. STATEMENT OF SIGNIFICANCE: The impairment of bone tissue regeneration when there is excessive damage has led to different tissue engineered strategies to promote bone healing. Significant importance has been given in the immunomodulation towards an anti-inflammatory environment together with proper angiogenesis stimulation in order to achieve successful bone regeneration rather than stimulating only the osteogenic differentiation. Ions have been considered potential candidates to stimulate these events due to their high stability and therapeutic effects with low side effects compared to growth factors. However, up to now, no review has been published assembling all this information together, describing individual effects of ions on immunomodulation and angiogenic stimulation, as well as their multifunctionality or synergistic effects when combined together.

Design of functional biomaterials as substrates for corneal endothelium tissue engineering.

Corneal endothelium defects are one of the leading causes of blindness worldwide. The actual treatment is transplantation, which requires the use of human cadaveric donors, but it faces several problems, such as global shortage of donors. Therefore, new alternatives are being developed and, among them, cell therapy has gained interest in the last years due to its promising results in tissue regeneration. Nevertheless, the direct administration of cells may sometimes have limited success due to the immune response, hence requiring the combination with extracellular mimicking materials. In this review, we present different methods to obtain corneal endothelial cells from diverse cell sources such as pluripotent or multipotent stem cells. Moreover, we discuss different substrates in order to allow a correct implantation as a cell sheet and to promote an enhanced cell behavior. For this reason, natural or synthetic matrixes that mimic the native environment have been developed. These matrixes have been optimized in terms of their physicochemical properties, such as stiffness, topography, composition and transparency. To further enhance the matrixes properties, these can be tuned by incorporating certain molecules that can be delivered in a sustained manner in order to enhance biological behavior. Finally, we elucidate future directions for corneal endothelial regeneration, such as 3D printing, in order to obtain patient-specific substrates.

Biofabrication of Collagen Tissue-Engineered Blood Vessels with Direct Co-Axial Extrusion.

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.

Multiple Ion Scaffold-Based Delivery Platform for Potential Application in Early Stages of Bone Regeneration.

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.

Assessing the potential role of copper and cobalt in stimulating angiogenesis for tissue regeneration.

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.

Evaluation of the immunomodulatory effects of cobalt, copper and magnesium ions in a pro inflammatory environment.

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.

Biofabrication of tissue engineering vascular systems.

Cardiovascular disease (CVD) is the leading cause of death among persons aged 65 and older in the United States and many other developed countries. Tissue engineered vascular systems (TEVS) can serve as grafts for CVD treatment and be used as in vitro model systems to examine the role of various genetic factors during the CVD progressions. Current focus in the field is to fabricate TEVS that more closely resembles the mechanical properties and extracellular matrix environment of native vessels, which depends heavily on the advance in biofabrication techniques and discovery of novel biomaterials. In this review, we outline the mechanical and biological design requirements of TEVS and explore the history and recent advances in biofabrication methods and biomaterials for tissue engineered blood vessels and microvascular systems with special focus on in vitro applications. In vitro applications of TEVS for disease modeling are discussed.

Biological Roles and Delivery Strategies for Ions to Promote Osteogenic Induction.

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.