Stimulation of wound healing and vascularization with calcium-releasing biomaterials

Chronic skin wounds are a major socioeconomic burden in developed societies, affecting specially elder and diabetic people. It is estimated that 1 to 2% of the population will suffer a chronic wound in their lifetime and, as global population ages and adopts a sedentary lifestyle, the incidence of these wounds will continue its upward trend. Chronic injuries are characterized for presenting a complicated and diverse pathophysiology that make them resistant to current therapies. For this reason, novel therapeutic strategies based on the release of growth factors and the use of tissue engineered constructs are being investigated and show promising results. However, very few biologically based products reach the market, mainly due to regulatory, economic and stability constraints, amplifying the need for easily translational novel treatments. Recently, inorganic biomaterials known as bioceramics have been acknowledged for their wound healing and vascularization capability, mainly due to their ion release. Based on this concept, the present thesis project was dedicated to investigate the potential application of novel bioceramics on wound healing and soft tissue regeneration. More specifically we focused on the role of the calcium ion and its release from newly designed bioceramics to stimulate wound healing and blood vessel formation in both in vitro and in vivo systems. Although it is known that calcium affects all the phases of wound healing, the concentrations and release profile that can improve the healing process has not been described. For this reason, we evaluated the effect of different concentrations of extracellular calcium in vitro on dermal fibroblast, a crucial cell type in the skin and the healing process, and found stimulation of relevant biological responses at specific concentrations. In addition, we compared whether similar effects could be obtained with the ion release of newly designed bioceramic particles containing equivalent calcium concentrations. Interestingly, while stimulating most of the effects, the ion release inhibited some responses triggered by calcium alone that are not desired in the context of chronic wound healing. Then, we investigated the cellular mechanism mediating some of the responses stimulated by calcium, focusing on the implication of the calcium-sensing receptor (CaSR). Several agonists of the receptor stimulated similar effects than calcium, suggesting the relevance of the CaSR on fibroblasts behavior, and opening a window to the design of novel bioceramics that release CaSR-agonists. In order to test the healing capability of the above mentioned bioceramic particles in vivo, they were incorporated in a mat of poly(lactic acid) (PLA) fibers. This novel dressing was applied on a model of chronic wounds in vivo, and compared with a mat of particle-free PLA and to a frequently used commercially available dressing. We found that the PLA-bioceramic mat accelerated wound closure and increased vasculature at the injured site at initial time-points. Thus, improved healing was achieved with the newly designed dressing. Finally, a different composite material was synthesized combining bioceramic particles and human mesenchymal stromal cells (hMSC) in a degradable hydrogel, and its vasculogenic potential was evaluated in soft tissue. This material supported hMSC survival and stimulated the release of the angiogenic factor IGF-1 from these cells in vitro. In addition, when implanted in soft tissue of immunocompromised mice, the composite construct improved hMSC survival and generated a more mature vasculature at the site of implantation. In conclusion, this thesis shows that calcium-releasing bioceramics can successfully contribute to the treatment of chronic wounds and soft tissue regeneration.Las heridas crónicas tienen un gran impacto socioeconómico sobre los países desarrollados, afectando especialmente a personas en edad avanzada y diabéticos. Se estima que entre el 1 y 2% de la población sufrirá una herida crónica a lo largo de su vida y, con el envejecimiento de la población y el aumento del sedentarismo, la incidencia de estas heridas seguirá una tendencia ascendente. Las heridas crónicas presentan una patofisiología complicada y diversa que las hace resistentes a las terapias actuales. Por esta razón, se están desarrollando nuevos productos mediante ingeniería de tejidos basados en el uso de factores de crecimiento y células. Sin embargo, la translación de estas terapias a la clínica es muy complicada por cuestiones regulatorias, económicas y de estabilidad del producto, por lo que hay una gran necesidad de nuevos tratamientos que puedan llegar más fácilmente al mercado. Recientemente, se ha descubierto que los biomateriales inorgánicos llamados biocerámicos pueden estimular la curación de heridas y la vascularización, principalmente a través del efecto de los iones que liberan. Partiendo de esta idea, este proyecto de tesis se ha centrado en investigar el uso potencial de nuevos biocerámicos en curación de heridas y la regeneración de tejido blando. Más concretamente, nos hemos centrado en el rol del ión calcio y su liberación de nuevos biocerámicos para estimular la curación de heridas y la formación de vasos sanguíneos in vitro e in vivo. A pesar de que el calcio afecta en todas las fases de la curación de una herida, las concentraciones y perfil de liberación que pueden mejorar el proceso de curación no han sido descritos. Por ello, evaluamos el efecto de diferentes concentraciones de calcio extracelular en fibroblastos dermales, un tipo celular esencial en el proceso de curación, y encontramos estimulación de diferentes respuestas biológicas a concentraciones específicas. Además, comparamos si se podían obtener efectos similares mediante el producto iónico liberado de unas nuevas partículas biocerámicas con concentraciones equivalentes de calcio. Curiosamente, el producto iónico inhibió algunos efectos estimulados por el calcio en solución que no son deseables en un contexto de tratamiento de heridas crónicas. Entonces, quisimos indagar en el mecanismo celular a través del cual el calcio estimula a los fibroblastos, centrándonos en la implicación del receptor sensor de calcio (CaSR). Varios agonistas de este receptor estimularon respuestas parecidas al calcio, mostrando la relevancia del CaSR sobre el comportamiento de los fibroblastos, y abriendo una ventana al diseño de nuevos biocerámicos con libración de agonistas del CaSR. Por otro lado, quisimos probar la capacidad curativa de las partículas biocerámicas usadas sobre los fibroblastos incorporándolas en fibras de ácido poliláctico. Este nuevo apósito generado se aplicó sobre un modelo de heridas crónicas in vivo, y su efecto se comparó con un apósito de PLA sin partículas y con un apósito comercial. El apósito de PLA-biocerámico aceleró la curación de las heridas además de estimular la formación de vasos sanguíneos a tiempos tempranos, con lo que se consiguió una mejora en la curación. Finalmente, se sintetizó un biomaterial implantable combinado partículas biocerámicas, células madre mesenquimales adultas (hMSC) y un hidrogel sintético degradable, con el objetivo de evaluar su capacitat vasculogénica en tejidos blandos. In vitro, el material mantuvo la superviencia de las células encapsuladas y se aumentó la liberación del factor angiogénico IGF-1. Además, al implantarse en tejido blando de ratones immunodeprimidos, el material con biocerámico mejoró la supervivencia de las hMSC y estimuló la maduración de la vasculatura en el sitio de implantació

Wound healing-promoting effects stimulated by extracellular calcium and calcium-releasing nanoparticles on dermal fibroblasts

Extracellular calcium has been proved to influence the healing process of injuries and could be used as a novel therapy for skin wound healing. However, a better understanding of its effect, together with a system to obtain a controlled release is needed. In this study, we examined whether the ionic dissolution of the calcium–phosphate-based ormoglass nanoparticles coded SG5 may produce a similar stimulating effect as extracellular calcium (from CaCl2) on rat dermal fibroblast in vitro. Cells were cultured in the presence of medium containing different calcium concentrations, normally ranging from 0.1 to 3.5 mM Ca2+. A concentration of 3.5 mM of CaCl2 increased metabolic activity, in vitro wound closure, matrix metalloproteinases (MMP) activity, collagen synthesis and cytokine expression, and reduced cell contraction capacity. Interestingly, the levels of migration and contraction capacity measured followed a dose-dependent behavior. In addition, media conditioned with SG5 stimulated the same activities as media conditioned with CaCl2, but undesired effects in chronic wound healing such as inflammatory factor expression and MMP activity were reduced compared to the equivalent CaCl2 concentration. In summary, calcium-releasing particles such as SG5 are potential biological-free biostimulators to be applied in dressings for chronic wound healing.Peer ReviewedPostprint (author's final draft

Instructive microenvironments in skin wound healing: biomaterials as signal releasing platforms

Skin wound healing aims to repair and restore tissue through a multistage process that involves different cells and signaling molecules that regulate the cellular response and the dynamic remodeling of the extracellular matrix. Nowadays, several therapies that combine biomolecule signals (growth factors and cytokines) and cells are being proposed. However, a lack of reliable evidence of their efficacy, together with associated issues such as high costs, a lack of standardization, no scalable processes, and storage and regulatory issues, are hampering their application. In situ tissue regeneration appears to be a feasible strategy that uses the body’s own capacity for regeneration by mobilizing host endogenous stem cells or tissue-specific progenitor cells to the wound site to promote repair and regeneration. The aim is to engineer instructive systems to regulate the spatio-temporal delivery of proper signaling based on the biological mechanisms of the different events that occur in the host microenvironment. This review describes the current state of the different signal cues used in wound healing and skin regeneration, and their combination with biomaterial supports to create instructive microenvironments for wound healing.Peer ReviewedPostprint (author's final draft

Wound healing-promoting effects stimulated by extracellular calcium and calcium-releasing nanoparticles on dermal fibroblasts

Extracellular calcium has been proved to influence the healing process of injuries and could be used as a novel therapy for skin wound healing. However, a better understanding of its effect, together with a system to obtain a controlled release is needed. In this study, we examined whether the ionic dissolution of the calcium-phosphate-based ormoglass nanoparticles coded SG5 may produce a similar stimulating effect as extracellular calcium (from CaCl2) on rat dermal fibroblast in vitro. Cells were cultured in the presence of medium containing different calcium concentrations, normally ranging from 0.1 to 3.5 mM Ca2+. A concentration of 3.5 mM of CaCl2 increased metabolic activity, in vitro wound closure, matrix metalloproteinases (MMP) activity, collagen synthesis and cytokine expression, and reduced cell contraction capacity. Interestingly, the levels of migration and contraction capacity measured followed a dose-dependent behavior. In addition, media conditioned with SG5 stimulated the same activities as media conditioned with CaCl2, but undesired effects in chronic wound healing such as inflammatory factor expression and MMP activity were reduced compared to the equivalent CaCl2 concentration. In summary, calcium-releasing particles such as SG5 are potential biological-free biostimulators to be applied in dressings for chronic wound healing

Wound healing-promoting effects stimulated by extracellular calcium and calcium-releasing nanoparticles on dermal fibroblasts

Extracellular calcium has been proved to influence the healing process of injuries and could be used as a novel therapy for skin wound healing. However, a better understanding of its effect, together with a system to obtain a controlled release is needed. In this study, we examined whether the ionic dissolution of the calcium-phosphate-based ormoglass nanoparticles coded SG5 may produce a similar stimulating effect as extracellular calcium (from CaCl2) on rat dermal fibroblast in vitro. Cells were cultured in the presence of medium containing different calcium concentrations, normally ranging from 0.1 to 3.5 mM Ca2+. A concentration of 3.5 mM of CaCl2 increased metabolic activity, in vitro wound closure, matrix metalloproteinases (MMP) activity, collagen synthesis and cytokine expression, and reduced cell contraction capacity. Interestingly, the levels of migration and contraction capacity measured followed a dose-dependent behavior. In addition, media conditioned with SG5 stimulated the same activities as media conditioned with CaCl2, but undesired effects in chronic wound healing such as inflammatory factor expression and MMP activity were reduced compared to the equivalent CaCl2 concentration. In summary, calcium-releasing particles such as SG5 are potential biological-free biostimulators to be applied in dressings for chronic wound healing

Wound healing-promoting effects stimulated by extracellular calcium and calcium-releasing nanoparticles on dermal fibroblasts

Extracellular calcium has been proved to influence the healing process of injuries and could be used as a novel therapy for skin wound healing. However, a better understanding of its effect, together with a system to obtain a controlled release is needed. In this study, we examined whether the ionic dissolution of the calcium-phosphate-based ormoglass nanoparticles coded SG5 may produce a similar stimulating effect as extracellular calcium (from CaCl2) on rat dermal fibroblast in vitro. Cells were cultured in the presence of medium containing different calcium concentrations, normally ranging from 0.1 to 3.5 mM Ca2+. A concentration of 3.5 mM of CaCl2 increased metabolic activity, in vitro wound closure, matrix metalloproteinases (MMP) activity, collagen synthesis and cytokine expression, and reduced cell contraction capacity. Interestingly, the levels of migration and contraction capacity measured followed a dose-dependent behavior. In addition, media conditioned with SG5 stimulated the same activities as media conditioned with CaCl2, but undesired effects in chronic wound healing such as inflammatory factor expression and MMP activity were reduced compared to the equivalent CaCl2 concentration. In summary, calcium-releasing particles such as SG5 are potential biological-free biostimulators to be applied in dressings for chronic wound healing

Wound healing-promoting effects stimulated by extracellular calcium and calcium-releasing nanoparticles on dermal fibroblasts

Extracellular calcium has been proved to influence the healing process of injuries and could be used as a novel therapy for skin wound healing. However, a better understanding of its effect, together with a system to obtain a controlled release is needed. In this study, we examined whether the ionic dissolution of the calcium–phosphate-based ormoglass nanoparticles coded SG5 may produce a similar stimulating effect as extracellular calcium (from CaCl2) on rat dermal fibroblast in vitro. Cells were cultured in the presence of medium containing different calcium concentrations, normally ranging from 0.1 to 3.5 mM Ca2+. A concentration of 3.5 mM of CaCl2 increased metabolic activity, in vitro wound closure, matrix metalloproteinases (MMP) activity, collagen synthesis and cytokine expression, and reduced cell contraction capacity. Interestingly, the levels of migration and contraction capacity measured followed a dose-dependent behavior. In addition, media conditioned with SG5 stimulated the same activities as media conditioned with CaCl2, but undesired effects in chronic wound healing such as inflammatory factor expression and MMP activity were reduced compared to the equivalent CaCl2 concentration. In summary, calcium-releasing particles such as SG5 are potential biological-free biostimulators to be applied in dressings for chronic wound healing.Peer Reviewe

Instructive microenvironments in skin wound healing: biomaterials as signal releasing platforms

Skin wound healing aims to repair and restore tissue through a multistage process that involves different cells and signaling molecules that regulate the cellular response and the dynamic remodeling of the extracellular matrix. Nowadays, several therapies that combine biomolecule signals (growth factors and cytokines) and cells are being proposed. However, a lack of reliable evidence of their efficacy, together with associated issues such as high costs, a lack of standardization, no scalable processes, and storage and regulatory issues, are hampering their application. In situ tissue regeneration appears to be a feasible strategy that uses the body’s own capacity for regeneration by mobilizing host endogenous stem cells or tissue-specific progenitor cells to the wound site to promote repair and regeneration. The aim is to engineer instructive systems to regulate the spatio-temporal delivery of proper signaling based on the biological mechanisms of the different events that occur in the host microenvironment. This review describes the current state of the different signal cues used in wound healing and skin regeneration, and their combination with biomaterial supports to create instructive microenvironments for wound healing.Peer Reviewe

Polymeric Composite Dressings Containing Calcium-Releasing Nanoparticles Accelerate Wound Healing in Diabetic Mice

[Objective] Wound healing is a complex process that involves the interaction between different cell types and bioactive factors. Impaired wound healing is characterized by a loss in synchronization of these interactions, resulting in nonhealing chronic wounds. Chronic wounds are a socioeconomic burden, one of the most prominent clinical manifestations of diabetes, however, they lack satisfactory treatment options. The objective of this study was to develop polymeric composites that deliver ions having wound healing properties and evaluate its performance using a pressure ulcer model in diabetic mice. [Approach] To develop a polymeric composite wound dressing containing ion-releasing nanoparticles for chronic wound healing. This composite was chemically and physically characterized and evaluated using a pressure ulcer wound model in diabetic (db/db) mice to explore their potential as novel wound dressing. [Results] This dressing exhibits a controlled ion release and a good in vitro bioactivity. The polymeric composite dressing treatment stimulates angiogenesis, collagen synthesis, granulation tissue formation, and accelerates wound closure of ischemic wounds created in diabetic mice. In addition, the performance of the newly designed composite is remarkably better than a commercially available dressing frequently used for the treatment of low-exuding chronic wounds. [Innovation] The developed nanoplatforms are cell- and growth factor free and control the host microenvironment resulting in enhanced wound healing. These nanoplatforms are available by cost-effective synthesis with a defined composition, offering an additional advantage in potential clinical application. [Conclusion] Based on the obtained results, these polymeric composites offer an optimum approach for chronic wound healing without adding cells or external biological factors.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO/FEDER) through the projects MAT2012-38793 and MAT2015-68906-R, the EuroNanoMed3 project nAngioDerm funded through the Spanish Ministry of Science and Innovation (ref. PCI2019-103648), the Spanish Ministry of Education, Culture, and Sports with the FPU grant (ref. AP-2012-5310), EIT Health (project EIT PoC-2016-SPAIN-03), La Caixa Banking Foundation through their CaixaImpulse Program and Caixaimpulse 2.0 Consolidate Program (Ref. LCF/TR/CN18/52210003)

PEG hydrogel containing calcium-releasing particles and mesenchymal stromal cells promote vessel maturation

The use of human mesenchymal stromal cells (hMSC) for treating diseased tissues with poor vascularization has received significant attention, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have also been suggested as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. In this study, calcium-releasing particles and hMSC were combined within a hydrogel to examine their vasculogenic potential in vitro and in vivo. The particles provided sustained calcium release and showed proangiogenic stimulation in a chorioallantoic membrane (CAM) assay. The number of hMSC encapsulated in a degradable RGD-functionalized PEG hydrogel containing particles remained constant over time and IGF-1 release was increased. When implanted in the epidydimal fat pad of immunocompromised mice, this composite material improved cell survival and stimulated vessel formation and maturation. Thus, the combination of hMSC and calcium-releasing glass-ceramics represents a new strategy to achieve vessel stabilization, a key factor in the revascularization of ischemic tissues. Statement of Significance: Increasing blood vessel formation in diseased tissues with poor vascularization is a current clinical challenge. Cell therapy using human mesenchymal stem cells has received considerable interest, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have been explored as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. By incorporating both human mesenchymal stem cells and glass-ceramic particles in an implantable hydrogel, this study provides insights into the vasculogenic potential in soft tissues of the combined strategies. Enhancement of vessel formation and maturation supports further investigation of this strategy.Peer Reviewe