New approaches in calcium phosphate cements and ceramics for bone regeneration.

Bone is among the most frequently transplanted tissues in the body. In Europe, about one million patients encounter a surgical bone reconstruction annually. The worldwide market of bone replacement materials is currently estimated at 5 billion Euros, with a 10% growth due to the ageing of the population. Natural grafts present several drawbacks which pushed scientists to investigate synthetic biomaterials. Although most synthetic bone substitutes available possess some of the positive properties of autografts, none yet have all the benefits of one's own bone. Among the available biomaterials, Calcium Phosphates (CaPs) are of great interest. Nonetheless, these materials can still be improved in several respects. The main aim of this PhD Thesis is to contribute to the improvement of the properties of CaPs for bone regeneration with primary regard to Calcium Phosphate Cements (CPCs). The Thesis is divided in three main parts: i) Biphasic Calcium Phosphate Cements (BCPCs) with modified solubility and ion release; ii) Fibre Reinforced Calcium Phosphate cements (FRCPCs) with improved mechanical properties; iii) Macroporous CaP scaffolds for simvastatin acid release. In the first part novel biphasic CDHA/ß-TCP cements were obtained by mixing two Tricalcium Phosphate (TCP) polymorphs with different solubility (a-TCP and ß-TCP) at different a-TCP/ß-TCP ratios, and characterised in terms of setting properties, mechanical properties, and degradation. In the second part of this manuscript, new FRCPCs were fabricated with a focus on improving the adhesion fibres/matrix, in order to enhance the load transfer and, thus, the toughness of the material and their physico-chemical properties were investigated. Different approaches were studied. The first approach was to increase the chemical affinity of the fibres towards the matrix, adding an element in the matrix with high affinity to the fibres. In the first approach, TryMethyl Chitosan (TMC) was introduced in the liquid phase of a matrix reinforced with chitosan fibres. In the same line, lactic acid (LA) was added in the liquid phase of cements reinforced with Poly-L-lactic acid (PLLA) yarns. The biological characterisation of FRCPCs was explored using human osteoblastic-like cells MG63 . Another approach was to investigate the potential of low temperature plasma surface modification of PLLA yarns for reinforcement of CPCs. Oxygen low pressure plasma was employed at different treatment times and the surface properties of the untreated and plasma-treated PLLA were evaluated. The third part of this Thesis consisted in producing low temperature (CDHA) or high temperature (ß-TCP) macroporous scaffolds as carriers for Simvastatin acid (SVA), an osteogenic and angiogenic promoter. In order to modulate the drug release beyond the intrinsic capacity of the material, plasma polymerisation with PCL:PEG copolymers was used to dry-coat the CaP scaffolds. The material properties, the plasma polymer layer and the drug release from the scaffold were characterised.El hueso es uno de los tejidos más trasplantados del cuerpo. Sólo en Europa, se cuentan alrededor de un millón de cirugías de reconstrucción ósea anualmente. La estimación del mercado global de los sustitutos óseos es aproximadamente de cinco billones de Euros por año, con un 10% de crecimiento anual debido al envejecimiento de la población. Debido a los problemas asociados a los injertos biológicos, la investigación y el desarrollo de materiales sintéticos y biocompatibles (Biomateriales) ha experimentado un gran auge. Aunque la mayoría de sustitutos sintéticos disponibles poseen algunas de las características de los autoinjertos, hasta el momento ninguno reúne todos los beneficios del hueso del propio individuo. Dentro de los biomateriales para regeneración ósea, los fosfatos de calcio han sido de gran interés debido a su composición química similar a la del hueso. Sin embargo, aún se requieren mejoras en distintos aspectos de estos materiales. El objetivo principal de esta Tesis Doctoral es contribuir a la mejora de las propiedades de los fosfatos de calcio para la regeneración ósea, con un interés especial en los cementos de fosfato de calcio. La Tesis investiga diferentes estrategias para el desarrollo de materiales para la sustitución ósea, novedosos y con propiedades mejoradas respecto a los actuales. La Tesis comprende tres partes principales: i) Cementos bifásicos de fosfato de calcio (BCPCs), constituidos por materiales con diferente solubilidad; ii) Fosfatos de calcio reforzados con fibras (FRCPCs), para la mejora de las propiedades mecánicas; iii) Andamios macroporosos para la liberación de una sal de simvastatina. En la primera parte de la Tesis, se describe el desarrollo de BCPCs compuestos por hidroxiapatita deficiente en calcio (CDHA) y fosfato tricálcico ß (ß-TCP). Estos materiales derivan de la reacción de las mezclas de dos polimorfos de fosfato tricalcico (TCP) con diferente solubilidad (a-TCP y ß-TCP) y, en esta tesis, se caracterizan su fraguado, sus propiedades mecánicas y degradación. En la segunda parte, se han desarrollado nuevos FRCPCs con especial atención hacia la mejora de la adhesión entre fibras y matriz, con el objetivo de mejorar la transferencia de carga entre ellos y por tanto, las propiedades mecánicas del compuesto. Se han investigado distintas estrategias. La primera de ellas basada en la investigación de materiales con una fase común (o con alta afinidad química) entre las fibras y la fase liquida del cemento; de esta manera se pretende crear un enlace más fuerte entre las fibras y la matriz. En un primer material se incorporó un 1 w/v% de Trimetilo de quitosán (TMC) en la fase líquida del cemento que a su vez se reforzó con fibras de quitosán. En un segundo grupo de materiales, se añadió un 10 v/v% de ácido láctico (LA) a la matriz del cemento junto con hilos discontinuos de ácido poliláctico (PLLA). Estos cementos también se caracterizaron biológicamente por medio de células osteoblásticas MG63. La segunda estrategia investigada en los FRCPCs se basa en la modificación superficial de las fibras de PLLA con plasma de baja temperatura con el fin de mejorar sus propiedades de mojado. Las fibras se trataron con plasma de oxigeno de baja presión a distintos tiempos y se incorporaron a la matriz de cemento, y se caracterizaron tanto las modificaciones superficiales de las fibras como las propiedades del cemento. La tercera parte ha consistido en el desarrollo de andamios macroporosos obtenidos a baja (CDHA) o alta (ß-TCP) temperatura para ser utilizados como formas de liberación de una sal de simvastatina (SVA), con propiedades osteogénicas y angiogénicas. Para conseguir modular la liberación del fármaco se recubrieron los andamios cargados con SVA con un copolímero de PCL:PEG mediante polimerización por plasma. Se caracterizaron las propiedades tanto del material como del recubrimiento y se evaluó la liberación del fármaco

A novel strategy to enhance interfacial adhesion in fiber-reinforced calcium phosphate cement

Calcium phosphate cements (CPCs) are extensively used as synthetic bone grafts, but their poor toughness limits their use to non-load-bearing applications. Reinforcement through introduction of fibers and yarns has been evaluated in various studies but always resulted in a decrease in elastic modulus or bending strength when compared to the CPC matrix. The aim of the present work was to improve the interfacial adhesion between fibers and matrix to obtain tougher biocompatible fiber-reinforced calcium phosphate cements (FRCPCs). This was done by adding a polymer solution to the matrix, with chemical affinity to the reinforcing chitosan fibers, namely trimethyl chitosan (TMC). The improved wettability and chemical affinity of the chitosan fibers with the TMC in the liquid phase led to an enhancement of the interfacial adhesion. This resulted in an increase of the work of fracture (several hundred-fold increase), while the elastic modulus and bending strength were maintained similar to the materials without additives. Additionally the TMC-modified CPCs showed suitable biocompatibility with an osteoblastic cell line.Preprin

Targeted ToF-SIMS Analysis of Macrophage Content from a Human Cranial Triphasic Calcium Phosphate Implant

Macrophages play a key role in determining the fate of implanted biomaterials, especially for biomaterials such as calcium phosphates (CaPs) where these cells play a vital role in material resorption and osteogenesis, as shown in different models, including clinical samples. Although substantial consideration is given to the design and validation of different CaPs, relatively little is known about their material-cell interaction. Specifically, the intracellular content of different CaP phases remains to be assessed, even though CaP-filled macrophages have been observed in several studies. In this study, 2D/3D ToF-SIMS imaging and multivariate analysis were directly applied on the histology samples of an explant to reveal the content of macrophages. The cellular content of the macrophages was analyzed to distinguish three CaP phases, monetite, beta-tricalcium phosphate, and pyrophosphate, which are all part of the monetite-based CaP implant composition under study. ToF-SIMS combined with histology revealed that the content of the identified macrophages was most similar to that of the pyrophosphate phase. This study is the first to uncover distinct CaP phases in macrophages from a human multiphasic CaP explant by targeted direct cell content analysis. The uncovering of pyrophosphate as the main phase found inside the macrophages is of great importance to understand the impact of the selected material in the process of biomaterial-instructed osteogenesis

Biomimetic versus sintered macroporous calcium phosphate scaffolds enhanced bone regeneration and human mesenchymal stromal cell engraftment in calvarial defects

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (ß-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic ß-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of ß-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to ß-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significance Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered ß-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to ß-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.Peer ReviewedPostprint (author's final draft

Design of calcium phosphate scaffolds with controlled simvastatin release by plasma polymerisation

Calcium Phosphates (CaPs) have excellent bone regeneration capacity, and their combination with specific drugs is of interest because it allows adding new functionalities. In CaPs, drug release is mainly driven by diffusion, which is strongly affected by the porosity of the matrix and the drug-material interaction. Therefore, it is very difficult to tune their drug release properties beyond their intrinsic properties. Furthermore, when the CaPs are designed as scaffolds, the increased complexity of the macrostructure further complicates the issue.; This work investigates for the first time the use of biocompatible plasma-polymers to provide a tool to control drug release from drug-loaded CaP scaffolds with complex surfaces and intricate 3D structure. Two different CaPs were selected displaying great differences in microstructure: low-temperature CaPs (Calcium-deficient hydroxyapatite cements, CDHA) and sintered CaP ceramics (beta-Tricalcium Phosphate, beta-TCP). The deposition of PCL-co-PEG (1: 4) copolymers on CaPs was achieved by a low pressure plasma process, which allowed coating the inner regions of the scaffolds up to a certain depth. The coating covered the micro and nanopores of the CaPs surface and produced complex geometries presenting a nano and micro rough morphology which lead to low wettability despite the hydrophilicity of the copolymer. Plasma coating with PCL-co-PEG on scaffolds loaded with Simvastatin acid (potentially osteogenic and angiogenic) allowed delaying and modulating the drug release from the bone scaffolds depending on the thickness of the layer deposited, which, in turn depends on the initial specific surface area of the CaP. (C) 2016 Elsevier Ltd. All rights reserved.Peer ReviewedPostprint (author's final draft

Gentamicin loading of calcium phosphate implants : implications for cranioplasty

BackgroundSurgical site infections (SSI) are a significant risk in cranioplasty, with reported rates of around 8-9%. The most common bacteria associated with these nosocomial infections are of the Staphylococcus species, which have the ability to form biofilm. The possibility to deliver antibiotics, such as gentamicin, locally rather than systemically could potentially lower the early postoperative SSI. Various antibiotic dosages are being applied clinically, without any true consensus on the effectiveness.MethodsDrug release from calcium phosphate (CaP), polyetheretherketone (PEEK), and titanium (Ti) samples was evaluated. Microbiological studies with Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) including strains from clinical infection were used to establish clinically relevant concentrations.ResultsThe CaP samples were able to retain and release gentamicin overtime, whereas the Ti and PEEK samples did not show any drug uptake or release. A gentamicin loading concentration of 400g/ml was shown to be effective in in vitro microbiological studies with both SA and SE.ConclusionsOut of the three materials studied, only CaP could be loaded with gentamicin. An initial loading concentration of 400g/ml appears to establish an effective gentamicin concentration, possibly translating into a clinical benefit in cranioplasty

Low-pressure plasma treatment of polylactide fibers for enhanced mechanical performance of fiber-reinforced calcium phosphate cements

Calcium phosphate cements (CPCs) are extensively used as synthetic bone grafts, but their poor mechanical properties limit their applicability to non-stress-bearing applications. The aim of the present work is to evaluate the potential of plasma surface modification of polylactide (PLA) fibers for reinforcement of CPCs. Oxygen low-pressure plasma was employed at different treatment times and the surface properties of the untreated and plasma-treated PLA were evaluated. Plasma treatment on the PLA fibers reduced the setting times of the PLA-CPC composites and improved their flexural properties.Peer ReviewedPostprint (published version

Development and characterization of biphasic hydroxyapatite/ß-TCP cements

Biphasic calcium phosphate bioceramics composed of hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP) have relevant properties as synthetic bone grafts, such as tunable resorption, bioactivity, and intrinsic osteoinduction. However, they have some limitations associated to their condition of high-temperature ceramics. In this work self-setting Biphasic Calcium Phosphate Cements (BCPCs) with different HA/ß-TCP ratios were obtained from self-setting a-TCP/ß-TCP pastes. The strategy used allowed synthesizing BCPCs with modulated composition, compressive strength, and specific surface area. Due to its higher solubility, a-TCP was fully hydrolyzed to a calcium-deficient HA (CDHA), whereas ß-TCP remained unreacted and completely embedded in the CDHA matrix. Increasing amounts of the non-reacting ß-TCP phase resulted in a linear decrease of the compressive strength, in association to the decreasing amount of precipitated HA crystals, which are responsible for the mechanical consolidation of apatitic cements. Ca2+ release and degradation in acidic medium was similar in all the BCPCs within the timeframe studied, although differences might be expected in longer term studies once ß-TCP, the more soluble phase was exposed to the surrounding mediaPeer Reviewe

A novel strategy to enhance interfacial adhesion in fiber-reinforced calcium phosphate cement

Calcium phosphate cements (CPCs) are extensively used as synthetic bone grafts, but their poor toughness limits their use to non-load-bearing applications. Reinforcement through introduction of fibers and yarns has been evaluated in various studies but always resulted in a decrease in elastic modulus or bending strength when compared to the CPC matrix. The aim of the present work was to improve the interfacial adhesion between fibers and matrix to obtain tougher biocompatible fiber-reinforced calcium phosphate cements (FRCPCs). This was done by adding a polymer solution to the matrix, with chemical affinity to the reinforcing chitosan fibers, namely trimethyl chitosan (TMC). The improved wettability and chemical affinity of the chitosan fibers with the TMC in the liquid phase led to an enhancement of the interfacial adhesion. This resulted in an increase of the work of fracture (several hundred-fold increase), while the elastic modulus and bending strength were maintained similar to the materials without additives. Additionally the TMC-modified CPCs showed suitable biocompatibility with an osteoblastic cell line

Plasma polymer coatings for modulation of drug release from bioceramics

Peer ReviewedPostprint (published version