Josep A. Planell

Biomaterials in orthopaedics

At present, strong requirements in orthopaedics are still to be met, both in bone and joint substitution and in the repair and regeneration of bone defects. In this framework, tremendous advances in the biomaterials field have been made in the last 50 years where materials intended for biomedical purposes have evolved through three different generations, namely first generation (bioinert materials), second generation (bioactive and biodegradable materials) and third generation (materials designed to stimulate specific responses at the molecular level). In this review, the evolution of different metals, ceramics and polymers most commonly used in orthopaedic applications is discussed, as well as the different approaches used to fulfil the challenges faced by this medical field.

Effects of artificial micro- and nano-structured surfaces on cell behaviour.

Substrate topography, independently of substrate chemistry, has been reported to have significant effects on cell behaviour. Based on the use of fabrication techniques developed by the silicon microtechnology industry, numerous studies can now be found in the literature analyzing cell behaviour as to various micro- and nano-features such as lines, wells, holes and more. Most of these works have been found to relate the micro- and nano-sized topographical features with cell orientation, migration, morphology and proliferation. In recent papers, even the influence of substrate nanotopography on cell gene expression and differentiation has been pointed out. However, despite the large number of papers published on this topic, significant general trends in cell behaviour are difficult to establish due to differences in cell type, substrate material, feature aspect-ratio, feature geometry and parameters measured. This paper intends to compile and review the relevant existing information on the behaviour of cells on micro- and nano-structured artificial substrates and analyze possible general behavioural trends.

Corrosion behaviour of commercially pure titanium shot blasted with different materials and sizes of shot particles for dental implant applications

It is well known that the osseointegration of the commercially pure titanium (c.p. Ti) dental implant is improved when the metal is shot blasted in order to increase its surface roughness. This roughness is colonised by bone, which improves implant fixation. However, shot blasting also changes the chemical composition of the implant surface because some shot particles remain adhered on the metal. The c.p. Ti surfaces shot blasted with different materials and sizes of shot particles were tested in order to determine their topographical features (surface roughness, real surface area and the percentage of surface covered by the adhered shot particles) and electrochemical behaviour (open circuit potential, electrochemical impedance spectroscopy and cyclic polarisation). The results demonstrate that the increased surface area of the material because of the increasing surface roughness is not the only cause for differences found in the electrochemical behaviour and corrosion resistance of the blasted c.p. Ti. Among other possible causes, those differences may be attributed to the compressive residual surface stresses induced by shot blasting. All the materials tested have an adequate corrosion and electrochemical behaviour in terms of its possible use as dental implant material.

High-resolution PLA-based composite scaffolds via 3-D printing technology.

Fabrication of new biodegradable scaffolds that guide and stimulate tissue regeneration is still a major issue in tissue engineering approaches. Scaffolds that possess adequate biodegradability, pore size, interconnectivity, bioactivity and mechanical properties in accordance with the injured tissue are required. This work aimed to develop and characterize three-dimensional (3-D) scaffolds that fulfill the aforementioned requirements. For this, a nozzle-based rapid prototyping system was used to combine polylactic acid and a bioactive CaP glass to fabricate 3-D biodegradable scaffolds with two patterns (orthogonal and displaced double layer). Scanning electron microscopy and micro-computer tomography showed that 3-D scaffolds had completely interconnected porosity, uniform distribution of the glass particles, and a controlled and repetitive architecture. Surface properties were also assessed, showing that the incorporation of glass particles increased both the roughness and the hydrophilicity of the scaffolds. Mechanical tests indicated that compression strength is dependent on the scaffold geometry and the presence of glass. Preliminary cell response was studied with primary mesenchymal stem cells (MSC) and revealed that CaP glass improved cell adhesion. Overall, the results showed the suitability of the technique/materials combination to develop 3-D porous scaffolds and their initial biocompatibility, both being valuable characteristics for tissue engineering applications.

A finite element study of mechanical stimuli in scaffolds for bone tissue engineering

Mechanical stimuli are one of the factors that affect cell proliferation and differentiation in the process of bone tissue regeneration. Knowledge on the specific deformation sensed by cells at a microscopic level when mechanical loads are applied is still missing in the development of biomaterials for bone tissue engineering. The objective of this study was to analyze the behavior of the mechanical stimuli within some calcium phosphate-based scaffolds in terms of stress and strain distributions in the solid material phase and fluid velocity, fluid pressure and fluid shear stress distributions in the pores filled of fluid, by means of micro computed tomographed (CT)-based finite element (FE) models. Two samples of porous materials, one of calcium phosphate-based cement and another of biodegradable glass, were used. Compressive loads equivalent to 0.5% of compression applied to the solid material phase and interstitial fluid flows with inlet velocities of 1, 10 and 100 microm/s applied to the interconnected pores were simulated, changing also the inlet side and the viscosity of the medium. Similar strain distributions for both materials were found, with compressive and tensile strain maximal values of 1.6% and 0.6%, respectively. Mean values were consistent with the applied deformation. When 10 microm/s of inlet fluid velocity and 1.45 Pas viscosity, maximal values of fluid velocity were 12.76 mm/s for CaP cement and 14.87 mm/s for glass. Mean values were consistent with the inlet ones applied, and mean values of shear stress were around 5 x 10(-5)Pa. Variations on inlet fluid velocity and fluid viscosity produce proportional and independent changes in fluid velocity, fluid shear stress and fluid pressure. This study has shown how mechanical loads and fluid flow applied on the scaffolds cause different levels of mechanical stimuli within the samples according to the morphology of the materials.

Finite element study of scaffold architecture design and culture conditions for tissue engineering

Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.

Biofabrication of tissue constructs by 3D bioprinting of cell-laden microcarriers

Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs.

Growth and differentiation of human bone marrow osteoprogenitors on novel calcium phosphate cements.

Materials that augment bone cell proliferation and osteogenic activity have important therapeutic implications for bone regeneration and for use in skeletal reconstruction and joint replacement. We have studied the growth and interactions of human bone marrow cells on a variety of new cement composites in vitro. These cement materials are composed of calcium-deficient hydroxyapatites, carbonated apatite and amorphous calcium phosphate. Cell proliferation was significantly reduced and cell differentiation increased in the presence of these cements compared with cells cultured on tissue culture plastic. Alkaline phosphatase, one of the markers of the osteoblast phenotype, was dramatically stimulated by 3 of the 4 cements examined between day 4 and day 10, above levels observed following culture of human osteoblasts on plastic alone. Photomicroscopic examination demonstrated growth and close integration of bone marrow cells and 3 of the composites. Longer term marrow cultures (15 day) on the cements confirmed the stimulation of cell differentiation over proliferation. From these studies, enhanced osteoblastic differentiation was observed on a 70% carbonated apatite, which has a composition similar to bone mineral, whereas, cell toxicity was observed on cells grown on amorphous calcium phosphate. This in vitro culture system demonstrates the use of human bone marrow cells for the potential evaluation of new biomaterials and the development of a novel carbonated apatite that may be of potential use in orthopaedic implants.

Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis

Tissue engineering aims to regenerate tissues and organs by using cell and biomaterial-based approaches. One of the current challenges in the field is to promote proper vascularization in the implant to prevent cell death and promote host integration. Bone marrow endothelial progenitor cells (BM-EPCs) and mesenchymal stem cells (MSCs) are bone marrow resident stem cells widely employed for proangiogenic applications. In vivo, they are likely to interact frequently both in the bone marrow and at sites of injury. In this study, the physical and biochemical interactions between BM-EPCs and MSCs in an in vitro co-culture system were investigated to further clarify their roles in vascularization. BM-EPC/MSC co-cultures established close cell-cell contacts soon after seeding and self-assembled to form elongated structures at 3days. Besides direct contact, cells also exhibited vesicle transport phenomena. When co-cultured in Matrigel, tube formation was greatly enhanced even in serum-starved, growth factor free medium. Both MSCs and BM-EPCs contributed to these tubes. However, cell proliferation was greatly reduced in co-culture and morphological differences were observed. Gene expression and cluster analysis for wide panel of angiogenesis-related transcripts demonstrated up-regulation of angiogenic markers but down-regulation of many other cytokines. These data suggest that cross-talk occurs in between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms leading to modulation of the angiogenic response.

A short review: Recent advances in electrospinning for bone tissue regeneration

Nanofibrous structures developed by electrospinning technology provide attractive extracellular matrix conditions for the anchorage, migration, and differentiation of tissue cells, including those responsible for the regeneration of hard tissues. Together with the ease of set up and cost-effectiveness, the possibility to produce nanofibers with a wide range of compositions and morphologies is the merit of electrospinning. Significant efforts have exploited the development of bone regenerative nanofibers, which includes tailoring of composite/hybrid compositions that are bone mimicking and the surface functionalization such as mineralization. Moreover, by utilizing bioactive molecules such as adhesive proteins, growth factors, and chemical drugs, in concert with the nanofibrous matrices, it is possible to provide artificial materials with improved cellular responses and therapeutic efficacy. These studies have mainly focused on the regulation of stem cell behaviors for use in regenerative medicine and tissue engineering. While there are some challenges in achieving controllable delivery of bioactive molecules and complex-shaped three-dimensional scaffolds for tissue engineering, the electrospun nanofibrous matrices can still have a beneficial impact in the area of hard-tissue regeneration.