How Degradation of Calcium Phosphate Bone Substitute Materials is influenced by Phase Composition and Porosity

The chemical composition of calcium phosphate (CaP) materials for the regenerative therapy of large bone defects is similar to that of bone. Additionally, calcium phosphates show an excellent biocompatibility. Besides the support of defect healing calcium phosphate implants should be completely degraded within an adequate time period to be replaced by newly formed bone. Although degradation of CaP‐implants occurs mainly by dissolution of the material, it is important to characterize the osteoclastic resorption as well, which is involved in native bone remodeling. The degradation of bone substitutes made of calcium phosphate ceramics is influenced by various parameters, such as defect size and localization, the general health situation, and age of the patient, but also material properties are important. Especially, the calcium phosphate composition is crucial for the degradation behavior of a calcium phosphate material. Additionally, at the cellular level the micro‐ and macroporosity, including interconnecting pores, influences both, the dissolution and the osteoclastic resorption. In our study, three different calcium phosphate materials (hydroxyapatite, tricalcium phosphate, and a biphasic calcium phosphate) and two different geometries (dense 2D samples and porous 3D scaffolds) are compared regarding their dissolution and resorption behavior. The results show, that the dissolution of CaP‐ceramics, as examined by the incubation in a degradation solution, depends mainly on the calcium phosphate phase but also on the porosity of the implant. Regarding the resorption, cell proliferation and differentiation of a monocytic cell line as well as the formation of resorption lacunas are analyzed. Cell proliferation is comparable on all phase compositions. Cell differentiation and resorption, however, are influenced by the calcium phosphate phase composition and by the implant porosity as well. By understanding these two mechanisms of degradation, bone substitute materials and, as a result, the bone regeneration of large bone defects using CaP‐ceramics can be improved.     

Current state of the art of biphasic calcium phosphate bioceramics

We have developed 15 years ago, with the collaboration of Lynch, Nery, and LeGeros in the USA, a bioactive concept based on biphasic calcium phosphate (BCP) ceramics. The concept is determined by an optimum balance of the more stable phase of HA and more soluble TCP. The material is soluble and gradually dissolves in the body, seeding new bone formation as it releases calcium and phosphate ions into the biological medium. The bioactive concept based on the dissolution/transformation processes of HA and TCP has been applied to both Bulk, Coating and Injectable Biomaterials. The events at the calcium phosphate (CaP) biomaterial/bone interface represent a dynamic process, including physico-chemical processes, crystal/proteins interactions, cells and tissue colonization, bone remodeling, finally contributing to the unique strength of such interfaces. An important literature and numerous techniques have been used for the evaluation of the fundamental physico chemical and biological performance of BCP concept. This type of artificial bone used from a long time in preclinical and in clinical trial, revealed the efficiency for bone filling, performance for bone reconstruction and efficacy for bone ingrowth at the expense of the micro macroporous BCP bioceramics.     

Morphological,mechanical, and biocompatibility characterization of macroporous alumina scaffolds coated with calcium phosphate/PVA

In bone tissue engineering, a highly porous artificial extracellular matrix or scaffold is required to accommodate cells and guide the tissue regeneration in three-dimension. Calcium phosphate (CaP) ceramics are widely used for bone substitution and repair due to their biocompatibility, bioactivity, and osteoconduction. However, compared to alumina ceramics, either in the dense or porous form, the mechanical strength achieved for calcium phosphates is generally lower. In the present work, the major goal was to develop a tri-dimensional macroporous alumina scaffold with a biocompatible PVA/calcium phosphate coating to be potentially used as bone tissue substitute. This approach aims to combine the high mechanical strength of the alumina scaffold with the biocompatibility of calcium phosphate based materials. Hence, the porous alumina scaffolds were produced by the polymer foam replication procedure. Then, these scaffolds were submitted to two different coating methods: the biomimetic and the immersion in a calcium phosphate/polyvinyl alcohol (CaP/PVA) slurry. The microstructure, morphology and crystallinity of the macroporous alumina scaffolds samples and coated with CaP/PVA were characterized by X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM/EDX) analysis. Also, specific surface area was assessed by BET nitrogen adsorption method and mechanical behavior was evaluated by axial compression tests. Finally, biocompatibility and cytotoxicity were evaluated by VERO cell spreading and attachment assays under SEM. The morphological analysis obtained from SEM photomicrograph results has indicated that 3D macroporous alumina scaffolds were successfully produced, with estimated porosity of over 65% in a highly interconnected network. In addition, the mechanical test results have indicated that porous alumina scaffolds with ultimate compressive strength of over 3.0 MPa were produced. Concerning to the calcium phosphate coatings, the results have showed that the biomimetic method was not efficient on producing a detectable layer onto the alumina scaffolds. On the other hand, a uniform and adherent inorganic–organic coating was effectively formed onto alumina macroporous scaffold by the immersion of the porous structure into the CaP/PVA suspension. Viable VERO cells were verified onto the surface of alumina porous scaffold samples coated with PVA–calcium phosphate. In conclusion, a new method was developed to produce alumina with tri-dimensional porous structure and uniformly covered with a biocompatible coating of calcium phosphate/PVA. Such system has high potential to be used in bone tissue engineering.     

Nanocrystalline calcium phosphate ceramics in biomedical engineering

Nanocrystalline calcium phosphate based bioceramics are the new rage in biomaterials research. Conventionally, calcium phosphates based materials are preferred as bone grafts in hard tissue engineering because of their superior biocompatibility and bioactivity. However, this group of bioceramics exhibits poor mechanical performance, which restricts their uses in load bearing applications. The recent trend in bioceramic research is mainly concentrated on bioactive and bioresorbable ceramics, i.e. hydroxyapatite, bioactive glasses, tricalcium phosphates and biphasic calcium phosphates as they exhibit superior biological properties over other materials. In recent times, the arena of nanotechnology has been extensively studied by various researchers to overcome the existing limitations of calcium phosphates, mainly hydroxyapatite, as well as to fabricate nanostructured scaffolds to mimic structural and dimensional details of natural bone. The bone mineral consists of tiny HAp crystals in the nano-regime. It is found that nanocrystalline HAp powders improve sinterability and densification due to greater surface area, which could improve the fracture toughness and other mechanical properties. Nano-HAp is also expected to have better bioactivity than coarser crystals. Nanocrystalline calcium phosphate has the potential to revolutionize the field of hard tissue engineering from bone repair and augmentation to controlled drug delivery devices. This paper reviews the current state of knowledge and recent developments of various nanocrystalline calcium phosphate based bioceramics from synthesis to characterization.     

Preparation and characterization of an electrodeposited calcium phosphate coating associated with a calcium alginate matrix

A new way of optimizing osteoconduction of biomaterials is to bring to them biological properties. In this work, we associated a novel release system with an electrodeposited calcium phosphate (CaP) coated titanium alloy Ti6Al4V. The characterization of this material was performed by means of light microscopy, scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM) and X-ray energy dispersive spectroscopy (EDXS). The electrodeposited CaP coating was a tricalcium phosphate, and the release system was composed of microcapsules entrapped in an alginate film. We observed that the alginate matrix had a close contact with the coating. An intermediate layer containing calcium and phosphorus appeared at the interface between the alginate matrix and the CaP coating. These results allowed us to conclude that the association of two techniques, i.e. electrodeposition followed by deposition of a calcium alginate matrix, led to the elaboration of a new biomaterial.     

Performance of evacuated calcium phosphate microcarriers loaded with mesenchymal stem cells within a rat calvarium defect

Tissue engineering of stem cells in concert with 3-dimensional (3D) scaffolds is a promising approach for regeneration of bone tissues. Bioactive ceramic microspheres are considered effective 3D stem cell carriers for bone tissue engineering. Here we used evacuated calcium phosphate (CaP) microspheres as the carrier of mesenchymal stem cells (MSCs) derived from rat bone marrow. The performance of the CaP-MSCs construct in bone formation within a rat calvarium defect was evaluated. MSCs were first cultured in combination with the evacuated microcarriers for 7?days in an osteogenic medium, which was then implanted in the 6?mm-diameter calvarium defect for 12?weeks. For comparison purposes, a control defect and cell-free CaP microspheres were also evaluated. The osteogenic differentiation of MSCs cultivated in the evacuated CaP microcarriers was confirmed by alkaline phosphatase staining and real time polymerase chain reaction. The in vivo results confirmed the highest bone formation was attained in the CaP microcarriers combined with MSCs, based on microcomputed tomography and histological assays. The results suggest that evacuated CaP microspheres have the potential to be useful as stem cell carriers for bone tissue engineering.     

Fourier-transform infrared spectroscopy study of an organic–mineral composite for bone and dental substitute materials

A new injectable biomaterial for bone and dental surgery is a composite consisting of a polymer as a matrix and bioactive calcium phosphate (CaP) ceramics as fillers. The stability of the polymer is essential in the production of a ready-to-use injectable sterilized biomaterial. The purpose of this study was to detect possible polymer degradation which may have been caused by the interaction with the fillers using Fourier transform infrared spectroscopy. Composites containing CaP fillers (biphasic calcium phosphate, hydroxyapatite and peroxidized hydroxyapatite) and polymer (hydroxypropyl methyl cellulose) were prepared. To investigate the properties of the polymer, the inorganic and organic phases of the composite were separated using several extraction methods. The difficulty in separating the organic (polymer) from the mineral (CaP fillers) phases in the composite investigated in this study suggested the presence of strong interactions between the two phases. Spectra of extracted polymers showed new absorption bands of low intensities and indications that some chemical modifications of the original polymers have occurred. Results also indicated that the filler composition has an effect on the integrity of the polymer.     

A Natural Bone Cement—A Laboratory Novelty Led to the Development of Revolutionary New Biomaterials

Research on calcium phosphate chemistry at NIST led to the discovery of the worlds first self-hardening calcium phosphate cements (CPC) in 1987. Laboratory, animal, and clinical studies were conducted to develop CPC into clinically useful biomaterials. The combination of self-hardening capability and high biocompatibility makes CPC a unique material for repairing bone defects. Near perfect adaptation of the cement to the tissue surfaces in a defect, and a gradual resorption followed by new bone formation are some of the other distinctive advantages of this biomaterial. In 1996 a CPC, consisting of tetracalcium phosphate and dicalcium phosphate anhydrous, was approved by the Food and Drug Administration (FDA) for repairing cranial defects in humans, thus becoming the first material of its kind available for clinical use. This paper will review the course of the development, the physical and chemical properties, and clinical applications of CPC.     

Calcium-phosphate ceramics and polysaccharide-based hydrogel scaffolds combined with mesenchymal stem cell differently support bone repair in rats

Research in bone tissue engineering is focused on the development of alternatives to autologous bone grafts for bone reconstruction. Although multiple stem cell-based products and biomaterials are currently being investigated, comparative studies are rarely achieved to evaluate the most appropriate approach in this context. Here, we aimed to compare different clinically relevant bone tissue engineering methods and evaluated the kinetic repair and the bone healing efficiency supported by mesenchymal stem cells and two different biomaterials, a new hydrogel scaffold and a commercial hydroxyapatite/tricalcium phosphate ceramic, alone or in combination.Syngeneic mesenchymal stem cells (5?×?10⁵) and macroporous biphasic calcium phosphate ceramic granules (Calciresorb C35^®, Ceraver) or porous pullulan/dextran-based hydrogel scaffold were implanted alone or combined in a drilled-hole bone defect in rats. Using quantitative microtomography measurements and qualitative histological examinations, their osteogenic properties were evaluated 7, 30, and 90 days after implantation. Three months after surgery, only minimal repair was evidenced in control rats while newly mineralized bone was massively observed in animals treated with either hydrogels (bone volume/tissue volume?=?20%) or ceramics (bone volume/tissue volume?=?26%). Repair mechanism and resorption kinetics were strikingly different: rapidly-resorbed hydrogels induced a dense bone mineralization from the edges of the defect while ceramics triggered newly woven bone formation in close contact with the ceramic surface that remained unresorbed. Delivery of mesenchymal stem cells in combination with these biomaterials enhanced both bone healing (>20%) and neovascularization after 1 month, mainly in hydrogel.Osteogenic and angiogenic properties combined with rapid resorption make hydrogels a promising alternative to ceramics for bone repair by cell therapy.     

Ionic Colloidal Molding as a Biomimetic Scaffolding Strategy for Uniform Bone Tissue Regeneration

Inspired by the highly ordered nanostructure of bone, nanodopant composite biomaterials are gaining special attention for their ability to guide bone tissue regeneration through structural and biological cues. However, bone malformation in orthopedic surgery is a lingering issue, partly due to the high surface energy of traditional nanoparticles contributing to aggregation and inhomogeneity. Recently, carboxyl‐functionalized synthetic polymers have been shown to mimic the carboxyl‐rich surface motifs of non‐collagenous proteins in stabilizing hydroxyapatite and directing intrafibrillar mineralization in‐vitro. Based on this biomimetic approach, it is herein demonstrated that carboxyl functionalization of poly(lactic‐co‐glycolic acid) can achieve great material homogeneity in nanocomposites. This ionic colloidal molding method stabilizes hydroxyapatite precursors to confer even nanodopant packing, improving therapeutic outcomes in bone repair by remarkably improving mechanical properties of nanocomposites and optimizing controlled drug release, resulting in better cell in‐growth and osteogenic differentiation. Lastly, better controlled biomaterial degradation significantly improved osteointegration, translating to highly regular bone formation with minimal fibrous tissue and increased bone density in rabbit radial defect models. Ionic colloidal molding is a simple yet effective approach of achieving materials homogeneity and modulating crystal nucleation, serving as an excellent biomimetic scaffolding strategy to rebuild natural bone integrity.     

Bonelike apatite formation on niobium metal treated in aqueous NaOH

The essential condition for a biomaterial to bond to the living bone is the formation of a biologically active bonelike apatite on its surface. In the present work, it has been demonstrated that chemical treatment can be used to create a calcium phosphate (CaP) surface layer, which might provide the alkali treated Nb metal with bone-bonding capability. Soaking Nb samples in 0.5 M NaOH, at 25 degrees C for 24 h produced a nano-porous approximately 40 nm thick amorphous sodium niobate hydrogel layer on their surface. Immersion in a simulated body fluid (SBF) lead to the deposition of an amorphous calcium phosphate layer on the alkali treated Nb. The formation of calcium phosphate is assumed to be a result of the local pH increase caused by the cathodic reaction of oxygen reduction on the finely porous surface of the alkali-treated metal. The local rise in pH increased the ionic activity product of hydroxyapatite and lead to the precipitation of CaP from SBF that was already supersaturated with respect to the apatite. The formation of a similar CaP layer upon implantation of alkali treated Nb into the human body should promote the bonding of the implant to the surrounding bone. This bone bonding capability could make Nb metal an attractive material for hard tissue replacements.     

Usage of polymer brushes as substrates of bone cells

Implant medical research and tissue engineering both target the design of novel biomaterials for the improvement of human health and clinical applications. In order to develop improved surface coatings for hard tissue (bone) replacement materials and implant devices, we are developing micropatterned coatings consisting of polymer brushes. These are used as organic templates for the mineralization of calcium phosphate in order to improve adhesion of bone cells. First, we give a short account of the current state-of-the-art in this particular field of biomaterial development, while in the second part the preliminary results of cell culture experiments are presented, in which the biocompatibility of polymer brushes are tested on human mesenchymal stem cells.     

3D Biomaterial Microarrays for Regenerative Medicine: Current State‐of‐the‐Art,Emerging Directions and Future Trends

Three dimensional (3D) biomaterial microarrays hold enormous promise for regenerative medicine because of their ability to accelerate the design and fabrication of biomimetic materials. Such tissue‐like biomaterials can provide an appropriate microenvironment for stimulating and controlling stem cell differentiation into tissue‐specific lineages. The use of 3D biomaterial microarrays can, if optimized correctly, result in a more than 1000‐fold reduction in biomaterials and cells consumption when engineering optimal materials combinations, which makes these miniaturized systems very attractive for tissue engineering and drug screening applications.     

A new insight into in vitro behaviour of poly(ε-caprolactone)/bioactive glass composites in biologically related fluids

In the present work, the role of content, size and chemical composition of gel-derived bioactive glass particles from the SiO₂–CaO–P₂O₅ system in modulating the in vitro bioactivity, osteoinductive properties and long-term (up to 15 months) degradation behaviour of poly(ε-caprolactone)-based composite films was investigated. Bioactivity was assessed in simulated body fluid (SBF) and HEPES-free Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% foetal bovine serum (FBS), while hydrolytic degradation tests were performed in phosphate buffer saline. Obtained composite films showed excellent calcium phosphate (CaP) layer forming ability in both SBF and DMEM-10% FBS. However, kinetics of bioactivity process strongly depended on the type of medium used. The layer of amino acids and proteins, derived from cell culture medium, on the surfaces of composites created barrier that inhibited release of the ions on the one hand, while increasing nucleation density of calcium phosphates, affecting the morphology of formed CaP layers on the other. The presence of bioactive glass fillers was shown to impart osteoinductive properties to obtained films, supporting osteoblast attachment and proliferation, as well as stimulating cell differentiation and also matrix mineralization process in vitro. We showed that kinetics of bioactivity process and also osteoinductive properties of composite films could be easily modulated with the use of different contents and chemical compositions of fillers. The results showed that modification of PCL matrix with bioactive glass particles accelerated its degradation. We proved that the degradation rate of composites could be controlled and optimized for bone regeneration, in particular by using bioactive fillers causing different calcium phosphate layer forming ability on the surfaces of composites, depending on particle size and chemical composition. We have presented new opportunities to design and obtain multifunctional composites with tunable degradation and bioactivity kinetics, as well as biological properties that can meet complex requirements of bone tissue engineering.     

Multifunctional Nanoengineered Hydrogels Consisting of Black Phosphorus Nanosheets Upregulate Bone Formation

Tissue‐engineered hydrogels have received extensive attention as their mechanical properties, chemical compositions, and biological signals can be dynamically modified for mimicking extracellular matrices (ECM). Herein, the synthesis of novel double network (DN) hydrogels with tunable mechanical properties using combinatorial screening methods is reported. Furthermore, nanoengineered (NE) hydrogels are constructed by addition of ultrathin 2D black phosphorus (BP) nanosheets to the DN hydrogels with multiple functions for mimicking the ECM microenvironment to induce tissue regeneration. Notably, it is found that the BP nanosheets exhibit intrinsic properties for induced CaP crystal particle formation and therefore improve the mineralization ability of NE hydrogels. Finally, in vitro and in vivo data demonstrate that the BP nanosheets, mineralized CaP crystal nanoparticles, and excellent mechanical properties provide a favorable ECM microenvironment to mediate greater osteogenic cell differentiation and bone regeneration. Consequently, the combination of bioactive chemical materials and excellent mechanical stimuli of NE hydrogels inspire novel engineering strategies for bone‐tissue regeneration.     

Cellular response to calcium phosphate ceramics implanted in rabbit bone

Two hydroxyapatite ceramics, synthesized by sintering from bovine bone and from a mixture of phosphate tricalcium and natural hydroxyapatite, were implanted in bone sites in rabbits. From day 7 after implantation, osteoblast-like cells were visible with thin layers of new bone on both biomaterials. Histomorphometry showed progressive increase in volume and surface of newly formed bone. Signs of cell-dependent resorption were visible at the surface of biomaterials and newly formed bone. There was a progressive decrease in relative volume and trabecular thickness of the biomaterials. Resorption of biomaterials appears to involve two cell types: multinucleated giant cells and osteoclast-like cells. The multinucleated giant cells observed had neither tartrate resistant acid phosphatase activity (TRAP) nor a ruffled border. Vesicles and vacuoles containing crystals observed in these cells suggest phagocytosis of biomaterials. The number of these cells decreased after day 14 following implantation. The osteoclast-like cells were TRAP positive. The structured modification and the TRAP activity demonstrated in the subjacent biomaterial suggest that the dissolution of the implant may be associated to an extracellular enzymatic activity of these cells. Electron microscopy revealed a clear zone and cytoplasmic membrane infolding in these cells, suggesting a ruffled border differentiation. The number of these cells increased with delay after implantation. It was concluded that the implantation of calcium phosphate ceramics in bone leads to new bone formation as well as to resorption of the biomaterials. The mechanism of resorption appears to associate crystal endocytosis by multinucleated giant cells and more classical resorption by osteoclast-like cells.     

Porous calcium phosphate ceramics prepared by coating polyurethane foams with calcium phosphate cements

Porous calcium phosphates have important biomedical applications such as bone defect fillers, tissue engineering scaffolds and drug delivery systems. While a number of methods to produce the porous calcium phosphate ceramics have been reported, this study aimed to develop a new fabrication method. The new method involved the use of polyurethane foams to produce highly porous calcium phosphate cements (CPCs). By firing the porous CPCs at 1200 °C, the polyurethane foams were burnt off and the CPCs prepared at room temperature were converted into sintered porous hydroxyapatite (HA)-based calcium phosphate ceramics. The sintered porous calcium phosphate ceramics could then be coated with a layer of the CPC at room temperature, resulting in high porosity, high pore interconnectivity and controlled pore size.     

Clinical applications of glass-ceramics in dentistry

Glass-ceramics featuring special properties can be used as a basis to develop biomaterials. It is generally differentiated between highly durable biomaterials for restorative dental applications and bioactive glass-ceramics for medical use, for example, bone replacements. In detail, this paper presents one biomaterial from each of these two groups of materials. In respect to the restorative dental biomaterials, the authors give an overview of the most important glass-ceramics for clinical applications. Leucite, leucite-apatite, lithium disilicate and apatite containing glass-ceramics represent biomaterials for these applications. In detail, the authors report on nucleation and crystallization mechanisms and properties of leucite-apatite glass-ceramics. The mechanism of apatite nucleation is characterized by a heterogeneous process. Primary crystal phases of α - and β -NaCaPO₄ were determined. Rhenanite glass-ceramics represent biomaterials with high surface reactivity in simulated body fluid, SBF, and exhibit reactive behaviour in tests with bone cells. Cell adhesion phenomena and cell growth were observed. Suitable colonization and proliferation and differentiation of cells as a preliminary stage in the development of a material for bone regeneration applications was established. The authors conclude that the processes of heterogeneous nucleation and crystallization are important for controlling the required reactions in both biomaterial groups.     

Scaffolds with a standardized macro-architecture fabricated from several calcium phosphate ceramics using an indirect rapid prototyping technique

Calcium phosphate ceramics, commonly applied as bone graft substitutes, are a natural choice of scaffolding material for bone tissue engineering. Evidence shows that the chemical composition, macroporosity and microporosity of these ceramics influences their behavior as bone graft substitutes and bone tissue engineering scaffolds but little has been done to optimize these parameters. One method of optimization is to place focus on a particular parameter by normalizing the influence, as much as possible, of confounding parameters. This is difficult to accomplish with traditional fabrication techniques. In this study we describe a design based rapid prototyping method of manufacturing scaffolds with virtually identical macroporous architectures from different calcium phosphate ceramic compositions. Beta-tricalcium phosphate, hydroxyapatite (at two sintering temperatures) and biphasic calcium phosphate scaffolds were manufactured. The macro- and micro-architectures of the scaffolds were characterized as well as the influence of the manufacturing method on the chemistries of the calcium phosphate compositions. The structural characteristics of the resulting scaffolds were remarkably similar. The manufacturing process had little influence on the composition of the materials except for the consistent but small addition of, or increase in, a beta-tricalcium phosphate phase. Among other applications, scaffolds produced by the method described provide a means of examining the influence of different calcium phosphate compositions while confidently excluding the influence of the macroporous structure of the scaffolds.     

The use of calcium phosphate-based biomaterials in implant dentistry

Since calcium phosphates (CaPs) were first proposed, a wide variety of formulations have been developed and continuously optimized, some of which (e.g. calcium phosphate cements, CPCs) have been successfully commercialized for clinical applications. These CaP-based biomaterials have been shown to be very attractive bone substitutes and efficient drug delivery vehicles across diverse biomedical applications. In this article, CaP biomaterials, principally CPCs, are addressed as alternatives/complements to autogenous bone for grafting in implant dentistry and as coating materials for enhancing the osteoinductivity of titanium implants, highlighting their performance benefits simultaneously as carriers for growth factors and as scaffolds for cell proliferation, differentiation and penetration. Different strategies for employing CaP biomaterials in dental implantology aim to ultimately reach the same goal, namely to enhance the osseointegration process for dental implants in the context of immediate loading and to augment the formation of surrounding bone to guarantee long-term success.