Design and Fabrication of Complex Scaffolds for Bone Defect Healing: Combined 3D Plotting of a Calcium Phosphate Cement and a Growth Factor-Loaded Hydrogel

ArticleinAnnals of Biomedical Engineering 45(1):224-236 · January 2017with 127 Reads
Abstract
Additive manufacturing enables the fabrication of scaffolds with defined architecture. Versatile printing technologies such as extrusion-based 3D plotting allow in addition the incorporation of biological components increasing the capability to restore functional tissues. We have recently described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of an oil-based CPC paste under mild conditions. In the present study, we have developed a strategy for growth factor loading based on multichannel plotting: a biphasic scaffold design was realised combining CPC with VEGF-laden, highly concentrated hydrogel strands. As hydrogel component, alginate and an alginate–gellan gum blend were evaluated; the blend exhibited a more favourable VEGF release profile and was chosen for biphasic scaffold fabrication. After plotting, two-step post-processing was performed for both, hydrogel crosslinking and CPC setting, which was shown to be compatible with both materials. Finally, a scaffold was designed and fabricated which can be applied for testing in a rat critical size femur defect. Optimization of CPC plotting enabled the fabrication of highly resolved structures with strand diameters of only 200 µm. Micro-computed tomography revealed a precise strand arrangement and an interconnected pore space within the biphasic scaffold even in swollen state of the hydrogel strands.

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  • ... Intriguingly, the modified post-plotting treatment also prevented formation of microcracks, which occurred during immersion in water and resulted in impaired mechan- ical properties [34]. Later on, we used multichannel plotting to extrude highly concentrated hydrogels next to CPC which were loaded with proteins and growth factors extending the range of growth factor release with respect to quantity and kinetics [33,35]. ...
    ... For the first time, scaffolds consisting of a resorb- able CPC and a cell-laden bioink have been fabricated in the present study. Both materials, CPC and the bioink, were combined in a strand wise assembly, similarly as it was shown before for the combination of CPC and highly concentrated hydrogels, which con- tained growth factors, but were not suitable for cell encapsulation [33,35]. CPC can be plotted to scaffolds or implants of any dimension in the scale of relevant bone defects and any shape [54]; combined plotting of CPC and alg/mc with various needle sizes was success- fully performed (supplementary figure S1) indicating that the shape fidelity of plotted CPC structures is not restricted by combination with the bioink. ...
    ... The usage of more rigid bioinks with high shape fidelity like the alg/mc blend can be seen as advantageous for combination with CPC, because they allow fabrication of biphasic scaffolds with a minimum distance of 300 μm between CPC and cell-laden bioink strands ensuring cell survival in the initial phase. The pore size and shape of the biphasic CPC-alg/mc combination could be adapted by usage of smaller nozzle diameters and change of the layer orientations and strand sequences [35], to realize scaffold structures with sui- table mechanical properties and pore structures but without significant change of the cellular properties. In order to reduce the cytotoxic effect in direct contact to CPC during setting, caused by the drop of the pH value, bioinks should be designed to buffer the change to acidic milieu. ...
    Article
    Due to their characteristic resemblance of the mineral component of bone, calcium phosphates are widely accepted as optimal bone substitute materials. Recent research focused on the development of pasty calcium phosphate cement (CPC) formulations, which can be fabricated into various shapes by low-temperature extrusion based additive manufacturing, namely 3D plotting. While it could be demonstrated that sensitive substances like growth factors can be integrated in such printed CPC scaffolds without impairment of their biological activity live cells cannot be suspended in CPC as they may not be functional when enclosed in a solid and stiff matrix. In contrast, 3D bioprinting of soft cell-laden hydrogels (bioinks) enables the fabrication of constructs with spatially defined cell distribution, which has the potential to overcome problems of conventional cell seeding techniques – but such objects lack mechanical stability. Herein, we combine 3D plotting of CPC and bioprinting of a cell-laden bioink for the first time. As model bioink, an alginate-methylcellulose blend (alg/mc) was used, previously developed by us. Firstly, a fabrication regime was established, enabling optimal setting of CPC and cell survival inside the bioink. As the cells are exposed to the chemical changes of CPC precursors during setting, we studied the compatibility of the complex system of CPC and cell-laden alg/mc for a combined extrusion process and characterized the cellular behaviour of encapsulated human mesenchymal stroma cells within the bioink at the interface and in direct vicinity to the CPC. Furthermore, biphasic scaffolds were mechanically characterized and a model for osteochondral tissue grafts is proposed. The manuscript discusses possible impacts of the CPC setting reaction on cells within the bioink and illustrates the advantages of CPC in bioprinting as alternative to the commonly used thermoplasts for bone tissue engineering.
  • ... In contrast, extrusion of CPC is conducted in Gels 2018, 4, 68 9 of 14 mild conditions (at room or physiological temperature, without any organic solvents involved or energy-intensive post-processing steps etc.). This enables fabrication of CPC together with temperature sensitive substances even living cells [26,31,32,53]. Therefore, 3D plotting of CPC with support material does not only allow creation of complex shaped structures or implants, but also enables biofunctionalization of calcium phosphate implants in a spatially defined manner, which is superior to most other additive manufacturing methods. ...
    ... On the other hand, the strand distance and orientation, the used needle diameters for extrusion of CPC and mc and plotting properties like the layer thickness, applied air pressure and plotting velocity influence the outcome significantly. For CPC, these parameters were optimized in our group, achieving filigree strands and scaffolds with high inner complexity [53]. ...
    Article
    Full-text available
    3D plotting is an additive manufacturing technology enabling biofabrication, thus the integration of cells or biologically sensitive proteins or growth factors into the manufacturing process. However, most (bio-)inks developed for 3D plotting were not shown to be processed into clinically relevant geometries comprising critical overhangs and cavities, which would collapse without a sufficient support material. Herein, we have developed a support hydrogel ink based on methylcellulose (mc), which is able to act as support as long as the co-plotted main structure is not stable. Therefore, 6 w/v %, 8 w/v % and 10 w/v % mc were allowed to swell in water, resulting in viscous inks, which were characterized for their rheological and extrusion properties. The successful usage of 10 w/v % mc as support ink was proven by multichannel plotting of the support together with a plottable calcium phosphate cement (CPC) acting as main structure. CPC scaffolds displaying critical overhangs or a large central cavity could be plotted accurately with the newly developed mc support ink. The dissolution properties of mc allowed complete removal of the gel without residuals, once CPC setting was finished. Finally, we fabricated a scaphoid bone model by computed tomography data acquisition and co-extrusion of CPC and the mc support hydrogel.
  • ... Many strategies have been developed to incorporate biological cues such as growth factors during or after scaffold fabrication by 3D printing. Methods such as dip coating or solution immersion [6, 33], incorporation of drug encapsulated particles in starting materials [34][35][36][37], or simultaneous addition of the growth factors while printing [38, 39] have successfully demonstrated controlled release and retention of functionality of the growth factors. In case of c/s printing, the drug or growth factor loaded core can act as a reservoir and the shell acts as a protective sheath for the sensitive components in the core, and thus the released drug or growth factor maintains its potency. ...
    Article
    Three-dimensional extrusion of two different biomaterials in a core/shell (c/s)fashion has gained much interest in the last couple of years as it allows for fabricating constructs with novel and interesting properties. We now demonstrate that combining high concentrated (16.7 wt%) alginate hydrogels as shell material with low concentrated, soft biopolymer hydrogels as core leads to mechanically stable and robust 3D scaffolds. Alginate, chitosan, gellan gum, gelatin and collagen hydrogels were utilized successfully as core materials—hydrogels which are too soft for 3D plotting of open-porous structures without an additional mechanical support. The respective c/s scaffolds were characterized concerning their morphology, mechanical properties and swelling behavior. It could be shown that core as well as shell part can be loaded with growth factors and that the release depends on core composition and shell thickness. Neither the plotting process nor the crosslinking with 1M CaCl2 denatured the proteins. When core and shell were loaded with different growth factors(VEGF and BMP-2, respectively) a dual release was achieved. Finally, live human endothelial cells were integrated in the core material, demonstrating that this new strategy can be used for bioprinting purposes as well.
  • ... As it is a self-setting material, no other treatment than immersion in aqueous media or contact with humidity is required to form mechanically stable constructs. By varying needle diameter and strand dis- tance as well as strand orientation in neighboring lay- ers, a variety of geometries can easily be realized [4,7,12]. ...
    Article
    Additive manufacturing techniques can be applied to individually craft medical implants and biomaterial scaffolds. We present the combination of macroscopic scaffold fabrication by strand deposition and high-resolution dosing of liquids using the ‘BioScaffolder 2.1’ 3D plotter from GeSiM with an integrated piezoelectric nanoliter pipette. A fluorescein solution, used as model substance, was dispensed on calcium phosphate bone cement strands during scaffold production; high reproducibility of the alternating subprocesses was demonstrated. Moreover, the release kinetics of VEGF loaded onto flat calcium phosphate cement substrates was investigated. The presented approach opens up new and exciting possibilities for tissue engineering. Various biological components can be integrated precisely into 3D scaffolds according to a predefined pattern creating tissue equivalents of high complexity.
  • ... In addition, human mesenchymal stem cells showed better cell attachment and proliferation than pure alginate. Calcium phosphate scaffolds combined with vascular endothelial growth factor (VEGF)-laden alginate-gellan gum hydrogel strands have also been shown to be suitable as VEGF delivery systems in bone tissue regeneration [36]. Scaffold shape fidelity was assessed using stereomicroscopic analysis and micro-CT, and was not compromised in the fabrication process. ...
    Chapter
    Congenital or acquired tissue loss and dysfunction is an enormous socioeconomic burden for healthcare systems globally. Tissue engineering holds promise to repair or replace human tissues and organs in order to restore normal function. One facet of tissue engineering is three-dimensional (3D) bioprinting which has emerged as an attractive method for assembling living and nonliving biological materials into spatially controlled, precise, and well-defined structures. It is essential that bioinks have the desired functional and mechanical properties that closely match the tissue it is to replace. Therefore it is imperative to correlate process and print parameters and factors that affect printability with shape fidelity and print resolution of the final fabricated 3D construct.
  • Chapter
    Biofabrication refers to the automated generation of constructs that can closely mimic the complex heterogenous nature and geometry of tissues and organs more than the current regenerative therapies do. This chapter presents an overview about the possibilities of mainly extrusion‐based manufacturing technologies to create scaffolds for three‐dimensional (3D) tissue models and techniques to approach a simulated natively heterogenous tissue structure. Scaffolds are designed with the aim of mimicking the in vivo microenvironment of the cells where cell‐cell and cell‐extracellular matrix (ECM) interactions occur, triggering cellular behavior and tissue function. Macroarchitecture describes the three‐dimensional geometry of the scaffolds that has the ability to promote cell proliferation and differentiation, designed to mimic the native ECM of the cells in an optimal way. Currently, 3D bioscaffolds are mostly printed to study the cell growth or for performing the basic material and viability tests. Multimaterial printing describes the capability to print 3D objects from materials with very different properties.
  • Article
    Recent advances in bioprinting technology used to precisely dispense cell-laden biomaterials for the construction of complex 3D functional living tissues or artificial organs. Organ printing and biofabrication provide great potential for the freeform fabrication of 3D living organs using cellular spheroids, biocomposite nanofibers or bioinks as building blocks for regenerative therapy. Vascularization is often identified as a main technological barrier for building 3D organs in tissue engineering. 3D printing of living tissues starts with potential support of biomaterials to maintain structural integrity and degrade certain time periods after printing of the scaffolds. Biofabrication is the production of complex living and non-living biological products from raw materials such as cells, molecules, ECM and biomaterials. Generally, two basic methods used for the fabrication of scaffolds such as conventional/traditional fabrication process and advance fabrication process for engineering organs. A wide range of polymers and biomaterials are used for the fabrication of scaffolds in tissue engineering applications. 3D additive manufacturing is advancing day-by-day; however there are various critical challenging factors used for fabricating 3D scaffolds. This review aimed to understand the various scaffolds fabrication techniques, types of polymers and biomaterials used for the fabrication process, various fields of applications and different challenges faced in their fabrication of scaffolds in regenerative therapy.
  • Article
    Full-text available
    The structural and functional repair of lost bone is still one of the biggest challenges in regenerative medicine. In many cases, autologous bone is used for the reconstruction of bone tissue; however, the availability of autologous material is limited, which always means additional stress to the patient. Due to this, more and more frequently various biocompatible materials are being used instead for bone augmentation. In this context, in order to ensure the structural function of the bone, scaffolds are implanted and fixed into the bone defect, depending on the medical indication. Nevertheless, for the surgeon, every individual clinical condition in which standardized scaffolds have to be aligned is challenging, and in many cases the alignment is not possible without limitations. Therefore, in the last decades, 3D printing (3DP) or additive manufacturing (AM) of scaffolds has become one of the most innovative approaches in surgery to individualize and improve the treatment of patients. Numerous biocompatible materials are available for 3DP, and various printing techniques can be applied, depending on the process conditions of these materials. Besides these conventional printing techniques, another promising approach in the context of medical AM is 3D bioprinting, a technique which makes it possible to print human cells embedded in special carrier substances to generate functional tissues. Even the direct printing into bone defects or lesions becomes possible. 3DP is already improving the treatment of patients, and has the potential to revolutionize regenerative medicine in future. (Eine der größten Herausforderungen in der regenerativen Medizin ist nach wie vor die funktionelle und strukturelle Wiederherstellung von verloren gegangenem Knochen. In vielen Fällen wird für die Rekonstruktion von Knochengewebe autologer Knochen verwendet, allerdings ist dieser nur begrenzt verfügbar und der Eingriff bedeutet immer eine zusätzliche Belastung für den Patienten. Deshalb kommen immer häufiger alternative biokompatible Materialien bei der Knochenaugmentation zum Einsatz. Um in diesem Zusammenhang die strukturelle Funktion des Knochens zu gewährleisten, werden - je nach medizinischer Indikation - Gerüste verwendet, die in die Knochendefekte eingebracht und fixiert werden. Trotzdem stellt jede individuelle klinische Situation den Chirurgen vor die Herausforderung, standardisierte Gerüste an die jeweilige Patientensituation anpassen zu müssen, was in vielen Fällen nicht uneingeschränkt möglich ist. Deshalb entwickelte sich in den letzten Jahrzehnten besonders der 3-D-Druck oder die additive Fertigung von Gerüsten zu einem der innovativsten Ansätze in der Chirurgie, um Patienten individualisiert und besser behandeln zu können. Zahlreiche Materialien sind für den 3-D-Druck verfügbar und je nach Bearbeitungsbedingungen dieser Materialien auch verschiedene Druckverfahren. Neben den konventionellen Druckverfahren ist im Kontext der medizinischen additiven Fertigung das sogenannte 3-D-Bioprinting ein weiterer vielversprechender Ansatz. Mit diesem Verfahren ist es möglich, menschliche Zellen eingebettet in speziellen Trägersubstanzen zu drucken, um funktionelle Gewebe zu generieren. Sogar das direkte Drucken in Knochendefekte oder Läsionen ist möglich. Der 3-D-Druck verbessert schon jetzt die Behandlung von Patienten und verfügt über das Potenzial, die regenerative Medizin in Zukunft zu revolutionieren.)
  • Article
    Full-text available
    Abstract The structural and functional repair of lost bone is still one of the biggest challenges in regenerative medicine. In many cases, autologous bone is used for the reconstruction of bone tissue; however, the availability of autologous material is limited, which always means additional stress to the patient. Due to this, more and more frequently various biocompatible materials are being used instead for bone augmentation. In this context, in order to ensure the structural function of the bone, scaffolds are implanted and fixed into the bone defect, depending on the medical indication. Nevertheless, for the surgeon, every individual clinical condition in which standardized scaffolds have to be aligned is challenging, and in many cases the alignment is not possible without limitations. Therefore, in the last decades, 3D printing (3DP) or additive manufacturing (AM) of scaffolds has become one of the most innovative approaches in surgery to individualize and improve the treatment of patients. Numerous biocompatible materials are available for 3DP, and various printing techniques can be applied, depending on the process conditions of these materials. Besides these conventional printing techniques, another promising approach in the context of medical AM is 3D bioprinting, a technique which makes it possible to print human cells embedded in special carrier substances to generate functional tissues. Even the direct printing into bone defects or lesions becomes possible. 3DP is already improving the treatment of patients, and has the potential to revolutionize regenerative medicine in future.
  • Article
    This study summarizes the recent research status and development of three-dimensional (3D)-printed porous ceramic scaffolds in bone tissue engineering. Recent literature on 3D-printed porous ceramic scaffolds was reviewed. Compared with traditional processing and manufacturing technologies, 3D-printed porous ceramic scaffolds have obvious advantages, such as enhancing the controllability of the structure or improving production efficiency. More sophisticated scaffolds were fabricated by 3D printing technology. 3D-printed bioceramics have broad application prospects in bone tissue engineering. Through an understanding the advantages and limitations of the different 3D-printing approaches, new classes of bone graft substitutes can be developed. [Key words] 3D printing; Additive manufacturing technologies; Bone tissue engineering; Scaffold; Bioactive Bioceramic
  • Article
    Full-text available
    Calcium phosphate cements (CPCs) are frequently used to repair bone defects. Since their discovery in the 1980s, extensive research has been conducted to improve their properties, and emerging evidence supports their increased application in bone tissue engineering. Much effort has been made to enhance the biological performance of CPCs, including their biocompatibility, osteoconductivity, osteoinductivity, biodegradability, bioactivity, and interactions with cells. This review article focuses on the major recent developments in CPCs, including 3D printing, injectability, stem cell delivery, growth factor and drug delivery, and pre-vascularization of CPC scaffolds via co-culture and tri-culture techniques to enhance angiogenesis and osteogenesis.
  • Article
    Supramolecular chemistry continues to experience widespread growth, as fine-tuned chemical structures lead to well-defined bulk materials. Previous literature described the roles of hydrogen bonding, ionic aggregation, guest/host interactions and π-π stacking to tune mechanical, viscoelastic, and processing performance. The versatility of reversible interactions enables the more facile manufacturing of molded parts with tailored hierarchical structures such as tissue engineered scaffolds for biological applications. Recently, supramolecular polymers and additive manufacturing processes merged to provide parts with control of the molecular, macromolecular, and feature length scales. Additive manufacturing, or 3D printing, generates customizable constructs desirable for many applications, and the introduction of supramolecular interactions will potentially increase production speed, offer a tunable surface structure for controlling cell-scaffold interactions, and impart desired mechanical properties through reinforcing inter-layer adhesion and introducing gradients or self-assembled structures. This review details the synthesis and characterization of supramolecular polymers suitable for additive manufacture and biomedical applications as well as the use of supramolecular polymers in additive manufacturing for drug delivery and complex tissue scaffold formation. The effect of supramolecular assembly and its dynamic behavior offers potential for controlling anisotropy of the printed objects with exquisite geometrical control. The potential for supramolecular polymers to generate well-defined parts, hierarchical structures, and scaffolds with gradient properties/tuned surfaces provides an avenue for developing next-generation biomedical devices and tissue scaffolds.
  • Article
    Three-dimensional printing (3DP) technology allows the fabrication of 3D objects with various geometrics in a layer-by-layer process. Some advantages of 3DP methods over the conventional manufacturing processes include the customization of medicines with individually adjusted doses, the ability to fabricate the sophisticated and complex solid dosage forms, on-demand manufacturing, and cost-effectiveness. Furthermore, recent years have seen an increasing interest in applying 3DP technology to the pharmaceutical manufacturing of drug products and development of various drug delivery systems. However, although 3DP technology exhibits many potential medical and economic benefits, there are also some technical and regulatory challenges restricting the wide applications of 3DP technology to pharmaceutical products. Accordingly, continuous innovation and refinement in 3DP methods are needed to overcome the current limitations and facilitate patient-specific health care with on-demand tailored medications in the future. This review introduces some 3DP techniques suitable for pharmaceutical manufacturing and also their applications to the development of drug dosage forms, indicating the feasibility of this technology in regular commercial production.
  • Chapter
    Bone defects exceeding a critical size are commonly treated by using bioceramic implants and scaffolds to fill space and to avoid fibrous tissue ingrowth. The porous nature of most bioceramics based on calcium phosphates or bioactive glasses is beneficial for a further drug modification, whereas the drug is released by diffusion processes at the application site. This chapter gives an overview about both the properties of common bioceramic matrices used in drug delivery as well as the most frequently used drugs for treating bone diseases. In addition, pharmaceutical models describing the release mechanism and testing methods for measuring not only the release of drugs but also their pharmaceutical activity are presented.
  • Article
    The field of tissue engineering has produced new therapies for the repair of damaged tissues and organs, utilizing biomimetic scaffolds that mirror the mechanical and biological properties of host tissue. The emergence of three-dimensional printing (3DP) technologies has enabled the fabrication of highly complex scaffolds that offer a more accurate replication of native tissue properties and architecture than previously possible. Of strong interest to tissue engineers is the construction of multilayered scaffolds that target distinct regions of complex tissues. Musculoskeletal and dental tissues in particular, such as the osteochondral unit and periodontal complex, are composed of multiple interfacing tissue types, and thus benefit from the usage of multilayered scaffold fabrication. Traditional 3DP technologies such as extrusion printing and selective laser sintering have been used for the construction of scaffolds with gradient architectures and mixed material compositions. Additionally, emerging bioprinting strategies have been used for the direct printing and spatial patterning of cells and chemical factors, capturing the complex organization found in the body. To better replicate the varied and gradated properties of larger tissues, researchers have created scaffolds composed of multiple materials spanning natural polymers, synthetic polymers, and ceramics. By utilizing high-precision 3DP techniques and judicious material selection, scaffolds can thus be designed to address the regeneration of previously challenging musculoskeletal, dental, and other heterogeneous target tissues. These multilayered 3DP strategies show great promise in the future of tissue engineering.
  • Article
    The US Food and Drug Administration approval of the first 3D printed tablet in 2015 has ignited growing interest in 3D printing, or additive manufacturing (AM), for drug delivery and testing systems. Beyond just a novel method for rapid prototyping, AM provides key advantages over traditional manufacturing of drug delivery and testing systems. These includes the ability to fabricate complex geometries to achieve variable drug release kinetics; ease of personalising pharmacotherapy for patient and lowering the cost for fabricating personalised dosages. Furthermore, AM allows fabrication of complex and micron-sized tissue scaffolds and models for drug testing systems that closely resemble in vivo conditions. However, there are several limitations such as regulatory concerns that may impede the progression to market. Here, we provide an overview of the advantages of AM drug delivery and testing, as compared to traditional manufacturing techniques. Also, we discuss the key challenges and future directions for AM enabled pharmaceutical applications.
  • Article
    Drop-on-demand (DOD) printing is widely used in bioprinting for tissue engineering because of little damage to cell viability and cost-effectiveness. However, satellite droplets may be generated during printing, deviating cells from the desired position and affecting printing position accuracy. Current control on cell injection in DOD printing is primarily based on trial-and-error process, which is time-consuming and inflexible. In this paper, a novel machine learning technology based on Learning-based Cell Injection Control (LCIC) approach is demonstrated for effective DOD printing control while eliminating satellite droplets automatically. The LCIC approach includes a specific computational fluid dynamics (CFD) simulation model of piezoelectric DOD print-head considering inverse piezoelectric effect, which is used instead of repetitive experiments to collect data, and a multilayer perceptron (MLP) network trained by simulation data based on artificial neural network algorithm, using the well-known classification performance of MLP to optimize DOD printing parameters automatically. The test accuracy of the LCIC method was 90%. With the validation of LCIC method by experiments, satellite droplets from piezoelectric DOD printing are reduced significantly, improving the printing efficiency drastically to satisfy requirements of manufacturing precision for printing complex artificial tissues. The LCIC method can be further used to optimize the structure of DOD print-head and cell behaviors.
  • Thesis
    Tissue Engineering (TE) is “an interdisciplinary field that applies principles of engineering and the life sciences toward development of biological substitutes that restore, maintain, or improve tissue function”. The First application of TE is to replace damaged tissues by artificial cell-materials products of tissue engineering (TE). Another TE application is to produce 2 or 3 dimensional (2D and 3D) models for biological and pharmacological in vitro studies. These models or tissue replacements can be fabricated using a combination of different interdisciplinary methods of medicine, biology, chemistry, physics, informatics and mechanics, providing specific micro-environment with different cell types, growth factors and matrix.One of the major challenges of tissue engineering is related to limited cell penetration in the inner parts of porous biomaterials. Poor cell viability in the center of engineered tissue is a consequence of limited oxygen and nutrients diffusion due to insufficient vascular network within the entire construct. Layer-by-layer (LBL) BioAssembly is a new approach based on assembly of small cellularized constructs that may lead to homogenous cell distribution and more efficient three dimensional vascularization of large tissue engineering constructs.Our hypothesis is that LBL Bioassembly approach is more suitable for bone regeneration than conventional tissue engineering approach. The primary objective of this thesis was to evaluate the advantages of LBL Bioassembly approach using 3D-printed polymer membranes seeded with human primary cells. We have evaluated the efficiency of vascular network formation in vivo within entire 3D tissue engineering construct using LBL bioassembly approach and comparing it to the conventional approach based on seeding of cells on the surface of massive 3D scaffolds. There was no significant difference in number of formed blood vessels in 3D at the outer parts of constructs implanted subcutaneously in mice 8 weeks post-implantation. But in the inner parts of implants which were not in direct contact with a host tissue, we could observe statistically more blood vessel formation when LBL bioassembly approach was used. This vascular network formation was more important in the case of co-cultures than mono-vultures of HBMSCs.There were several secondary objectives in this work. The first was to fabricate cellularized 3D constructs for bone tissue engineering using poly(lactic) acid (PLA) membranes and human primary cells: human bone marrow stroma cells (HBMSCs) isolated from the bone marrow, and endothelial progenitor cells (EPCs) isolated from the umbilical cord blood. Then, we have compared different Additive manufacturing technologies to fabricate scaffolds: direct 3D printing (3DP) starting from PLA powder dissolved in chloroform and fused deposition modelling (FDM) using a commercial or a custom-made printer with different resolutions.The custom-made printer equipped with 100 μm nozzle allowed the highest level of printing resolution concerning pores shape and size. In the meantime we evaluated different stabilization systems for layer-by-layer assembling of PLA membranes with human primary cells: the use of 3D printed PLA clips provided the most efficient stabilization to stack PLA membranes in 3D. Another advantage of this stabilization system is that it could be implanted together with LBL constructs. Then we investigated the most suitable cell culture system for such constructs and we observed more efficient cell proliferation and differentiation when co-culture system is used, comparing to mono-cultures.LBL bioassembly approach seems to be suitable solution for efficient vascularization within entire large 3D tissue engineering constructs especially when co-cultures of mesenchymal and endothelial cells are used.
  • Article
    The introduction of additive manufacturing (AM), often referred to as three-dimensional (3D) printing, has initiated what some believe to be a manufacturing revolution, and has expedited the development of the field of biofabrication. Moreover, recent advances in AM have facilitated further development of patient-specific healthcare solutions. Customization of many healthcare products and services, such as implants, drug delivery devices, medical instruments, prosthetics, and in vitro models, would have been extremely challenging—if not impossible—without AM technologies. The current special issue of the Annals of Biomedical Engineering presents the latest trends in application of AM techniques to healthcare-related areas of research. As a prelude to this special issue, we review here the most important areas of biomedical research and clinical practice that have benefited from recent developments in additive manufacturing techniques. This editorial, therefore, aims to sketch the research landscape within which the other contributions of the special issue can be better understood and positioned. In what follows, we briefly review the application of additive manufacturing techniques in studies addressing biomaterials, (re)generation of tissues and organs, disease models, drug delivery systems, implants, medical instruments, prosthetics, orthotics, and AM objects used for medical visualization and communication.
  • Article
    Full-text available
    Powder based three-dimensional printing (3DP) allows great versatility in material and geometry. These characteristics make 3DP an interesting method for the production of tissue engineering scaffolds. However, 3DP has major limitations such as limited resolution and accuracy, hence preventing this method from wide-spread application within scaffold engineering. In order to reduce these limitations, deeper understanding of the complex interactions between powder, binder and roller during 3DP is needed. In the past, a lot of work has been invested to optimize the powder properties for 3DP for a certain layer thickness. Using a powder optimized for an 88 μm layer thickness, this study quantifies systematically surface roughness and geometrical accuracy in printed specimens and assesses their variation upon changes of different critical parameters such as moisture time (0, 5, 10, 20 s), layer thickness (44 & 88 μm) and number of specimens printed per batch (6 & 12). Best surface roughness value of 25 μm was measured with a water moisture application (using a custom made moisture device mounted on the linear stage carrying the printhead) of 5s and a layer thickness of 44 μm. Geometrical accuracy generally was higher for 88 μm, due to less critical powder bed stability. Moisture application enabled 3DP for 44 μm and improved its accuracy even for a powder initially optimized for 88 μm. Moreover, recycling of humidified powder was not only possible but in terms of reactivity even beneficial. In conclusion, moisture-based three-dimensional printing is a promising approach for high resolution 3DP of scaffolds.
  • Article
    Full-text available
    In tissue engineering, additive manufacturing (AM) technologies have brought considerable progress as they allow the fabrication of three-dimensional (3D) structures with defined architecture. 3D plotting is a versatile, extrusion-based AM technology suitable for processing a wide range of biomaterials including hydrogels. In this study, composites of highly concentrated alginate and gellan gum were prepared in order to combine the excellent printing properties of alginate with the favorable gelling characteristics of gellan gum. Mixtures of 16.7 wt % alginate and 2 or 3 wt % gellan gum were found applicable for 3D plotting. Characterization of the resulting composite scaffolds revealed an increased stiffness in the wet state (15%–20% higher Young’s modulus) and significantly lower volume swelling in cell culture medium compared to pure alginate scaffolds (~10% vs. ~23%). Cytocompatibility experiments with human mesenchymal stem cells (hMSC) revealed that cell attachment was improved—the seeding efficiency was ~2.5–3.5 times higher on the composites than on pure alginate. Additionally, the composites were shown to support hMSC proliferation and early osteogenic differentiation. In conclusion, print fidelity of highly concentrated alginate-gellan gum composites was comparable to those of pure alginate; after plotting and crosslinking, the scaffolds possessed improved qualities regarding shape fidelity, mechanical strength, and initial cell attachment making them attractive for tissue engineering applications.
  • Article
    Additive manufacturing allows to widely control the geometrical features of implants. Recently, we described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of a storable CPC paste based on water-immiscible carrier liquid. Plotting and hardening is conducted under mild conditions allowing the (precise and local) integration of biological components. In this study, we have developed a procedure for efficient loading of growth factors in the CPC scaffolds during plotting and demonstrated the feasibility of this approach. Bovine serum albumin (BSA) or vascular endothelial growth factor (VEGF), used as model proteins, were encapsulated in chitosan/dextran sulphate microparticles which could be easily mixed into the CPC paste in freeze-dried state. In order to prevent leaching of the proteins during cement setting, usually carried out by immersion in aqueous solutions, the plotted scaffolds were aged in water-saturated atmosphere (humidity). Setting in humidity avoided early loss of loaded proteins but provided sufficient amount of water to allow cement setting, as indicated by XRD analysis and mechanical testing in comparison to scaffolds set in water. Moreover, humidity-set scaffolds were characterised by altered, even improved properties: no swelling or crack formation was observed and accordingly, surface topography, total porosity and compressive modulus of the humidity-set scaffolds differed from those of the water-set counterparts. Direct cultivation of mesenchymal stem cells on the humidity-set scaffolds over 21 days revealed their cytocompatibility. Maintenance of the bioactivity of VEGF during the fabrication procedure was proven in indirect and direct culture experiments with endothelial cells. Copyright © 2015. Published by Elsevier Ltd.
  • Article
    In the present work, gelatin/alginate and gelatin/alginate/hydroxyapatite (HAP) composite scaffolds were fabricated by 3D plotting based on high concentration gelatin/alginate pastes. At temperatures of 37℃ or above, the developed pastes could be easily processed into designed 3D structures; sequential crosslinking with Ca2+ ions (effective for alginate) and the carbodiimide EDC (effective for gelatin) resulted in stable scaffolds. Mechanical testing demonstrated that those plotted composite scaffolds had significant higher strength and modulus compared to most of reported gelatin scaffolds prepared by conventional methods. Cell experiments with human bone marrow-derived mesenchymal stem cells (hBMSC) revealed that the gelatin/alginate composite scaffolds favor cell adhesion and support proliferation. Furthermore, the cells showed a homogeneous distribution and excellent migration in the inner regions of plotted composite scaffolds over 21 days. In conclusion, gelatin/alginate scaffolds, with or without HAP, fabricated by 3D plotting according to a predesigned CAD-model might be potential candidates for the repair of bony and chondral defects, especially in complex defect situations affecting the osteochondral tissue interface since biphasic scaffolds with stable connection of the two parts can be easily fabricated by multi-channel 3D plotting.
  • Article
    Biofabrication of tissue engineering constructs with tailored architecture and organized cell placement using rapid prototyping technologies is a major research focus in the field of regenerative therapies. This study describes a novel alginate-based material suitable for both cell embedding and fabrication of three-dimensional (3D) structures with predefined geometry by 3D plotting. The favourable printing properties of the material were achieved by using a simple strategy: addition of methylcellulose (MC) to a 3% alginate solution resulted in a strongly enhanced viscosity, which enabled accurate and easy deposition without high technical efforts. After scaffold plotting, the alginate chains were crosslinked with Ca(2+) ; MC did not contribute to the gelation and was released from the scaffolds during the following cultivation. The resulting constructs are characterized by high elasticity and stability, as well as an enhanced microporosity caused by the transient presence of MC. The suitability of the alginate/MC blend for cell embedding was evaluated by direct incorporation of mesenchymal stem cells during scaffold fabrication. The embedded cells showed high viability after 3 weeks of cultivation, which was similar to those of cells within pure alginate scaffolds which served as control. Maintenance of the differentiation potential of embedded cells, as an important requirement for the generation of functional tissue engineering constructs, was proven for adipogenic differentiation as a model for soft tissue formation. In conclusion, the temporary integration of MC in to a low-concentrated alginate solution allowed the generation of scaffolds with dimensions in the range of centimetres without loss of the positive properties of low-concentrated alginate hydrogels with regard to cell embedding. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
  • Article
    Low temperature self-setting ceramic inks have been scarcely investigated for solid freeform fabrication processes. This work deals with the robocasting of alpha-tricalcium phosphate/gelatine reactive slurries as a bioinspired self-setting ink for the production of biomimetic hydroxyapatite/gelatine scaffolds. A controlled and totally interconnected pore network of 300 μm was obtained after ink printing and setting, with the struts consisting of a micro/nanoporous matrix of needle-shaped calcium deficient hydroxyapatite crystals, with a high specific surface area. Gelatine was effectively retained by chemical crosslinking. The setting reaction of the ink resulted in a significant increase of both the elastic modulus and the compressive strength of the scaffolds, which were within the range of the human trabecular bone. In addition to delaying the onset of the setting reaction, thus providing enough time for printing, gelatine provided the viscoelastic properties to the strands to support their own weight, and additionally enhanced mesenchymal stem cell adhesion and proliferation on the surface of the scaffold. Altogether this new processing approach opens good perspectives for the design of hydroxyapatite scaffolds for bone tissue engineering with enhanced reactivity and resorption rate.
  • Article
    Full-text available
    Bone substitutes, like calcium phosphate, are implemented more frequently in orthopaedic surgery to reconstruct critical size defects, since autograft often results in donor site morbidity and allograft can transmit diseases. A novel bone cement, based on β -tricalcium phosphate, polyethylene glycol, and trisodium citrate, was developed to allow the rapid manufacturing of scaffolds, by extrusion freeform fabrication, at room temperature. The cement composition exhibits good resorption properties and serves as a basis for customised (e.g., drug or growth factor loaded) scaffolds for critical size bone defects. In vitro toxicity tests confirmed proliferation and differentiation of ATDC5 cells in scaffold-conditioned culture medium. Implantation of scaffolds in the iliac wing of sheep showed bone remodelling throughout the defects, outperforming the empty defects on both mineral volume and density present in the defect after 12 weeks. Both scaffolds outperformed the autograft filled defects on mineral density, while the mineral volume present in the scaffold treated defects was at least equal to the mineral volume present in the autograft treated defects. We conclude that the formulated bone cement composition is suitable for scaffold production at room temperature and that the established scaffold material can serve as a basis for future bone substitutes to enhance de novo bone formation in critical size defects.
  • Article
    Full-text available
    The 3D printing technique based on cement powders is an excellent method for the fabrication of individual and complex bone substitutes even in the case of large defects. The outstanding bone remodeling capacity of biphasic calcium phosphates (BCPs) containing hydroxyapatite (HA) as well as tricalcium phosphate (TCP) in varying ratios makes the adaption of powder systems resulting in BCP materials to this fabrication technique a desirable aim. This study presents the synthesis and characterization of a novel powder system for the 3D printing process, intended for the production of complexly shaped BCP scaffolds by a hydraulic setting reaction of calcium carbonate and TCP with phosphoric acid. The HA/TCP ratio in the specimens could be tailored by the calcium/phosphate ratio of the starting powder. The scaffolds could be fabricated with a dimensional accuracy of >96.5% and a minimal macro pore size of 300 µm. Independent of the phase composition the printed specimens showed a microporosity of approximately 68%, while the compressive strength strongly depended on the chemical composition and increased with rising TCP content in the scaffolds to a maximum of 1.81 MPa. Post-treatment of the scaffolds with a polylactic-co-glycolic acid-solution enhanced the mechanical properties by a factor of 8. In vitro studies showed that all BCP scaffolds were cytocompatible and enhanced the cell viability as well as the cell proliferation, as compared with pure TCP. Cell proliferation is even better on BCP when compared to HA and cell viability is in a similar range on these materials.
  • Article
    Recent studies have pointed towards a decisive role of inflammation in triggering tissue repair and regeneration, while at the same time it is accepted that an exacerbated inflammatory response may lead to rejection of an implant. Within this context, understanding and having the capacity to regulate the inflammatory response elicited by 3D scaffolds aimed for tissue regeneration is crucial. This work reports on the analysis of the cytokine profile of human monocytes/macrophages in contact with biodegradable 3D scaffolds with different surface properties, architecture and controlled pore geometry, fabricated by 3D printing technology. Fabrication processes were optimized to create four different 3D platforms based on PLA, PLA/CaP glass or chitosan. Cytokine secretion and cell morphology of human peripheral blood monocytes allowed to differentiate on the different matrices were analyzed. While all scaffolds supported monocyte/macrophage adhesion and stimulated cytokine production, striking differences between PLA-based and chitosan scaffolds were found, with chitosan eliciting increased secretion of TNF-α, while PLA-based scaffolds induced higher production of IL-6, IL-12/23 and IL-10. Even though the material itself induced the biggest differences, scaffold geometry also impacted on TNF-α and IL-12/23 production, with chitosan scaffolds having larger pores and wider angles leading to a higher secretion of these pro-inflammatory cytokines. These findings strengthen the appropriateness of these 3D platforms to study modulation of macrophage responses by specific parameters (chemistry, topography, scaffold architecture).
  • Article
    Bone regeneration by using tissue engineered constructs requires strategies to effectively stimulate vascularization within such a construct that is crucial for its supply and integration with the host tissue. In this work, porous scaffolds of a collagen/hydroxyapatite nanocomposite were modified with heparin to generate biomimetic bone matrices which are able to release angiogenic factors in a controlled manner. Heparin was either integrated during material synthesis (in situ) or added to the scaffolds after their fabrication (post). Both approaches resulted in stable incorporation of heparin into the matrix of mineralized collagen. Investigations of binding and release of the vascular endothelial growth factor (VEGF-A165 ) loaded onto the scaffolds revealed an enhanced binding capacity as well as a sustained and nearly constant delivery of VEGF as result of both heparin modification methods. The release rate could be controlled by varying the quantity of incorporated heparin and the modification method. Whereas the biological activity of VEGF released after 7 days from the unmodified scaffolds was reduced in comparison to control VEGF, it was maintained after release from post or even enhanced after release from in situ modified scaffolds. In conclusion, the heparin-modified scaffolds of mineralized collagen exhibited favorable growth factor binding and release properties and may be beneficial to stimulate vascularization.
  • Article
    With advances in tissue engineering, the possibility of regenerating injured tissue or failing organs has become a realistic prospect for the first time in medical history. Tissue engineering - the combination of bioactive materials with cells to generate engineered constructs that functionally replace lost and/or damaged tissue - is a major strategy to achieve this goal. One facet of tissue engineering is biofabrication, where three-dimensional tissue-like structures composed of biomaterials and cells in a single manufacturing procedure are generated. Cell-laden hydrogels are commonly used in biofabrication and are termed "bioinks". Hydrogels are particularly attractive for biofabrication as they recapitulate several features of the natural extracellular matrix and allow cell encapsulation in a highly hydrated mechanically supportive three-dimensional environment. Additionally, they allow for efficient and homogeneous cell seeding, can provide biologically-relevant chemical and physical signals, and can be formed in various shapes and biomechanical characteristics. However, despite the progress made in modifying hydrogels for enhanced bioactivation, cell survival and tissue formation, little attention has so far been paid to optimize hydrogels for the physico-chemical demands of the biofabrication process. The resulting lack of hydrogel bioinks have been identified as one major hurdle for a more rapid progress of the field. In this review we summarize and focus on the deposition process, the parameters and demands of hydrogels in biofabrication, with special attention to robotic dispensing as an approach that generates constructs of clinically relevant dimensions. We aim to highlight this current lack of effectual hydrogels within biofabrication and initiate new ideas and developments in the design and tailoring of hydrogels. The successful development of a "printable" hydrogel that supports cell adhesion, migration, and differentiation will significantly advance this exciting and promising approach for tissue engineering.
  • Article
    Full-text available
    The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2) influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs) was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs) and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.
  • Article
    Herein, we present a new type of biphasic organic/inorganic scaffold, which can be fabricated by multi-channel 3D plotting under mild conditions based on a highly concentrated alginate paste and a ready-to-use calcium phosphate cement (CPC) for bone and osteochondral tissue engineering. The structures of scaffolds were characterised by light and scanning electron microscopy (SEM). Results indicated that the concentrated alginate and CPC pastes had comparable plotting consistency, and therefore could be combined in one (biphasic) scaffold applying predesigned plotting parameters. After crosslinking of alginate and setting of CPC, the biphasic scaffold obtained mechanical and structural stability. Mechanical test data revealed that biphasic CPC/alginate scaffolds had significantly increased compressive strength and modulus compared to pure alginate as well as mixed calcium phosphate (CaP)/alginate scaffolds in wet state, and improved strength and toughness compared to pure CPC scaffolds in both dry and wet conditions. Culture of human mesenchymal stem cells (hMSC) on these scaffolds over 3 weeks demonstrated the good cytocompatibility of the selected materials. Because of the mild preparation conditions, bovine serum albumin (BSA) as a model protein was loaded in alginate and CPC pastes prior to plotting with high loading efficiency. Release studies in vitro showed that BSA released much faster from alginate strands than from CPC strands, which might allow amount-controlled protein release from biphasic CPC/alginate scaffolds. Furthermore, an upgraded bipartite osteochondral scaffold consisting of an alginate part for chondral and a biphasic CPC/alginate part for bony repair was fabricated based on this technique. This scaffold showed a strong organic/inorganic interface binding due to interlocking and crosslinking of the alginate strands.
  • Article
    Full-text available
    Additive manufacturing in the field of regenerative medicine aims to fabricate organized tissue-equivalents. However, the control over shape and composition of biofabricated constructs is still a challenge and needs to be improved. The current research aims to improve shape, by converging a number of biocompatible, quality construction materials into a single three-dimensional fiber deposition process. To demonstrate this, several models of complex anatomically shaped constructs were fabricated by combined deposition of poly(vinyl alcohol), poly(ε-caprolactone), gelatin methacrylamide/gellan gum and alginate hydrogel. Sacrificial components were co-deposited as temporary support for overhang geometries and were removed after fabrication by immersion in aqueous solutions. Embedding of chondrocytes in the gelatin methacrylamide/gellan component demonstrated that the fabrication and the sacrificing procedure did not affect cell viability. Further, it was shown that anatomically shaped constructs can be successfully fabricated, yielding advanced porous thermoplastic polymer scaffolds, layered porous hydrogel constructs, as well as reinforced cell-laden hydrogel structures. In conclusion, anatomically shaped tissue constructs of clinically relevant sizes can be generated when employing multiple building and sacrificial materials in a single biofabrication session. The current techniques offer improved control over both internal and external construct architecture underscoring its potential to generate customized implants for human tissue regeneration.
  • Article
    Gelatin-methacrylamide (gelMA) hydrogels are shown to support chondrocyte viability and differentiation and give wide ranging mechanical properties depending on several cross-linking parameters. Polymer concentration, UV exposure time, and thermal gelation prior to UV exposure allow for control over hydrogel stiffness and swelling properties. GelMA solutions have a low viscosity at 37 °C, which is incompatible with most biofabrication approaches. However, incorporation of hyaluronic acid (HA) and/or co-deposition with thermoplastics allows gelMA to be used in biofabrication processes. These attributes may allow engineered constructs to match the natural functional variations in cartilage mechanical and geometrical properties.
  • Article
    Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self-assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three-dimensional (3D) cell-printed scaffolds using layer-by-layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell-encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell-based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL-alginate gel constructs. PCL-alginate gels containing transforming growth factor-β (TGFβ) showed higher ECM formation. The 3D cell-printed scaffolds of PCL-alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL-alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell-printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.
  • Article
    Calcium phosphate cements (CPC) are highly valuable materials for filling bone defects and bone augmentation by minimal invasive application via percutaneous injection. In the present study some key features were significantly improved by developing a novel injectable ready-to-use calcium phosphate cement based on water-immiscible carrier liquids. A combination of two surfactants was identified to facilitate the targeted discontinuous exchange of the liquid for water after contact with aqueous solutions, enabling the setting reaction to take place at distinct ratios of cement components to water. This prolonged shelf life of the pre-mixed paste and enhanced reproducibility during application and setting reactions. The developed paste technology is applicable for different CPC formulations. Evaluations were performed for the formulation of an α-TCP-based CPC as a representative example for the preparation of injectable pastes with a powder to carrier liquid ratio of up to 85:15. We demonstrate that the resulting material retains the desirable properties of conventional CPC counterparts for fast setting, mechanical strength, biocompatibility, shows improved cohesion, and will most probably show a similar degree of resorbability due to identical mineral structure of the set products.
  • Article
    In this study, we explored the feasibility of fabrication bioactive mesoporous calcium silicate/calcium phosphate cements (MCS/CPC) scaffolds with high mechanical strength by Freeform Fabrication System with Micro-Droplet Jetting. After preparation of ordered mesoporous calcium silicate (MCS) powder, ready-to-use MCS/CPC paste was formed by mixing calcium phosphate cement (CPC) powder and MCS powder with the binder polyvinyl alcohol (PVA) aqueous solution at a certain ratio of powder to liquid. MCS/CPC scaffolds with various architectures, pore sizes, and interconnectivity were then directly printed at room temperature using MCS/CPC paste. The mechanical strength, apatite formation, degradation rate, and cytocompatibility of the composite scaffolds were systematically investigated. The results showed that MCS/CPC paste exhibited outstanding printability to form MCS/CPC scaffolds. The hybrid MCS/CPC scaffolds with predefined pore size of 350 μm showed fast degradation rate, high mechanical strength, and good cytocompatibility. It was indicated that the hybrid MCS/CPC scaffolds might be a promising candidate for critical bone defect repair.
  • Article
    The major advantage of hydroxyapatite (HA)-forming calcium phosphate cements (CPCs) used as bone replacement materials is their setting under physiological conditions without the necessity for thermal treatment that allows the incorporation of biological factors. In the present study, we have combined the biocompatible consolidation of CPCs with the potential of rapid prototyping (RP) techniques to generate calcium phosphate-based scaffolds with defined inner and outer morphology. We demonstrate the application of the RP technique three-dimensional (3D) plotting for the fabrication of HA cement scaffolds. This was realized by utilizing a paste-like CPC (P-CPC) which is stable as a malleable paste and whose setting reaction is initiated only after contact with aqueous solutions. The P-CPC showed good processability in the 3D plotting process and allowed the fabrication of stable 3D structures of different geometries with adequate mechanical stability and compressive strength. The cytocompatibility of the plotted P-CPC scaffolds was demonstrated in a cell culture experiment with human mesenchymal stem cells. The mild conditions during 3D plotting and post-processing and the realization of the whole procedure under sterile conditions make this approach highly attractive for fabrication of individualized implants with respect to patient-specific requirements by simultaneous plotting of biological components. Copyright © 2012 John Wiley & Sons, Ltd.
  • Article
    A study was conducted to transfer the 3-D printing technique to the fabrication of TCP and BCP ceramics and evaluate the influence of the granulate composition on the 3-D printed scaffolds. An aqueous solution of dextrin (20 wt.-%) and saccharose (2.5 wt.-%) were used as printing binder. The printing raster resolution and the thickness of the powder layers were selected as 0.29mm for the grid test parts and 0.26mm for the compression test parts. The granulates were characterized in terms of their particle size distribution using laser granulometry and their morphology. It was observed that spray-dried granulates could be fabricated from 'HA and TCP powders' and from a mixture of HA and TCP with the same composition of organic additives. The study also concluded that the mechanical properties of 3-D printed scaffolds can be adjusted by the design of the inner channel structure of the scaffolds.
  • Article
    Calcium phosphate cements are used as synthetic bone grafts, with several advantages, such as their osteoconductivity and injectability. Moreover, their low-temperature setting reaction and intrinsic porosity allow for the incorporation of drugs and active principles in the material. It is the aim of the present work to: a) provide an overview of the different approaches taken in the application of calcium phosphate cements for drug delivery in the skeletal system, and b) identify the most significant achievements. The drugs or active principles associated to calcium phosphate cements are classified in three groups, i) low molecular weight drugs; ii) high molecular weight biomolecules; and iii) ions.
  • Article
    A key challenge in tissue engineering is overcoming cell death in the scaffold interior due to the limited diffusion of oxygen and nutrients therein. We here hypothesize that immobilizing a gradient of a growth/survival factor from the periphery to the center of a porous scaffold would guide endothelial cells into the interior of the scaffold, thus overcoming a necrotic core. Proteins were immobilized by one of three methods on porous collagen scaffolds for cardiovascular tissue engineering. The proteins were first activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/sulfo N-hydroxysuccinimide and then applied to the scaffold by one of three methods to establish the gradient: perfusion (the flow method), use of a source and a sink (the source-sink method) or by injecting 5 μl of the solution at the center of the scaffold (point source method). Due to the high reproducibility and ease of application of the point source method it was further used for VEGF-165 gradient formation, where an ~2 ng ml(-1) mm(-1) gradient was formed in a radial direction across a scaffold, 12 mm in diameter and 2.5mm thick. More endothelial cells were guided by the VEGF-165 gradient deep into the center of the scaffold compared with both uniformly immobilized VEGF-165 (with the same total VEGF concentration) and VEGF-free controls. All scaffolds (including the controls) yielded the same number of cells, but notably the VEGF-165 gradient scaffolds demonstrated a higher cell density in the centre. Thus we concluded that the VEGF-165 gradient promoted the migration, but not proliferation, of cells into the scaffold. These gradient scaffolds provide the foundation for future in vivo tissue engineering studies.
  • Article
    New generation biomaterials for bone regeneration should be highly bioactive, resorbable and mechanically strong. Mesoporous bioactive glass (MBG), a novel bioactive material, has been used to study bone regeneration due to its excellent bioactivity, degradation and drug delivery ability, however, the construction of three-dimensional (3-D) MBG scaffolds (as for other bioactive inorganic scaffolds) for bone regeneration remains a significant challenge due to their inherent brittleness and low strength. In this brief communication we report a new facile method to prepare hierarchical and multifunctional MBG scaffolds with a controllable pore architecture, excellent mechanical strength and mineralization ability for application in bone regeneration by a modified 3-D printing technique using polyvinylalcohol (PVA) as a binder. The method provides a new way to solve commonly existing issues for inorganic scaffold materials, for example, uncontrollable pore architectures, low strength, high brittleness and the requirement for a second sintering at high temperature. The 3-D printed MBG scaffolds obtained possess a high mechanical strength about 200 times that of traditional polyurethane foam templated MBG scaffolds. They have a highly controllable pore architecture, excellent apatite mineralization ability and sustained drug delivery properties. Our study indicates that 3-D printed MBG scaffolds may be an excellent candidate for bone regeneration.
  • Article
    The key feature of calcium phosphate cements (CPCs) lies in the setting reaction triggered by mixing one or more solid calcium phosphate salts with an aqueous solution. Upon mixture, the reaction takes place through a dissolution-precipitation process which is macroscopically observed by a gradual hardening of the cement paste. The precipitation of hydroxyapatite nanocrystals at body or room temperature, and the fact that those materials can be used as self-setting pastes, have for many years been the most attractive features of CPCs. However, the need to develop materials able to sustain bone tissue ingrowth and be capable of delivering drugs and bioactive molecules, together with the continuous requirement from surgeons to develop more easily handling cements, has pushed the development of new processing routes that can accommodate all these requirements, taking advantage of the possibility of manipulating the self-setting CPC paste. It is the goal of this paper to provide a brief overview of the new processing developments in the area of CPCs and to identify the most significant achievements.
  • Article
    The development of materials to support bone regeneration requires flexible fabrication technologies able to tailor chemistry and architecture for specific applications. In this work we describe the preparation of ceramic-based inks for robotic-assisted deposition (robocasting) using Pluronic F-127 solutions. This approach allows the preparation of pseudoplastic inks with solid contents ranging between 30 and 50 vol.%, enabling them to flow through a narrow printing nozzle while supporting the weight of the printed structure. Ink formulation does not require manipulation of the pH or the use of highly volatile organic components. Therefore, the approach can be used to prepare materials with a wide range of compositions, and here we use it to build hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP) and biphasic (HA/beta-TCP) structures. The flow of the inks is controlled by the Pluronic content and the particle size distribution of the ceramic powders. The use of wide size distributions favors flow through the narrow printing nozzles and we have been able to use printing nozzles as narrow as 100 microm in diameter, applying relatively low printing pressures. The microporosity of the printed lines increases with increasing Pluronic content and lower sintering temperatures. Microporosity can play a key role in determining the biological response to the materials, but it also affects the strength of the structure.
  • Article
    Hydroxyapatite (HA) ceramic is a widely used synthetic bone substitute material for the regeneration of bone defects. We manufactured HA scaffolds with adjustable pore sizes and pore geometry by dispense-plotting. In addition, we attached peptides covalently onto the HA surface and are able to simultaneously quantify the amount of covalently attached and adsorbed peptide down to the picomolar range with a novel fluorescence-based detection method. In cell culture assays with stromal bone marrow cells, we observed a positive effect of biofunctionalization on cell differentiation after 21 days of culture when comparing the scaffold functionalized with the RGD motif containing adhesion peptide to an unmodified scaffold.
  • Article
    Designing a three-dimensional (3-D) ideal scaffold has been one of the main goals in biomaterials and tissue engineering, and various mechanical techniques have been applied to fabricate biomedical scaffolds used for soft and hard tissue regeneration. Scaffolds should be biodegradable and biocompatible, provide temporary support for cell growth to allow cell adhesion, and consist of a defined structure that can be formed into customized shapes by a computer-aided design system. This versatility in preparing scaffolds gives us the opportunity to use rapid prototyping devices to fabricate polymeric scaffolds. In this study, we fabricated polycaprolactone scaffolds with interconnecting pores using a 3-D melt plotting system and compared the plotted scaffolds to those made by salt leaching. Scanning electron microscopy, a laser scanning microscope, micro-computed tomography, and dynamic mechanical analysis were used to characterize the geometry and mechanical properties of the resulting scaffolds and morphology of attached cells. The plotted scaffolds had the obvious advantage that their mechanical properties could be easily manipulated by adjusting the scaffold geometry. In addition, the plotted scaffolds provided more opportunity for cells to expand between the strands of the scaffold compared to the salt-leached scaffold.
  • Article
    Bone cements with biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres have already been proven to provide a macroporous calcium phosphate cement (CPC) during in situ microsphere degradation. Furthermore, in vitro/in vivo release studies with these PLGA microsphere/CPC composites (PLGA/CPCs) showed a sustained release of osteo-inductive growth factor when drug was distributed inside/onto the microspheres. The goal of this study was to elucidate the mechanism behind drug release from PLGA/CPC. For this, in vitro release and degradation characteristics of a low-molecular-weight PLGA/CPC (M(w) = 5 kg/mol) were determined using bovine serum albumin (BSA) as a model protein. Two loading mechanisms were applied; BSA was either adsorbed onto the microspheres or incorporated inside the microspheres during double-emulsion. BSA release from PLGA microspheres and CPC was also measured and used as reference. Results show fast degrading polymer microspheres which produced a macroporous scaffold within 4 weeks, but also showed a concomitant release of acidic degradation products. BSA release from the PLGA/CPC was similar to the CPC samples and showed a pattern consisting of a small initial release, followed by a period of almost no sustained release. Separate PLGA microspheres exhibited a high burst release and release efficiency that was higher with the adsorbed samples. Combining degradation and release data we can conclude that for the PLGA/CPC samples BSA re-adsorbed to the cement surface after being released from the microspheres, which was mediated by the pH decrease during microsphere degradation.
  • Article
    beta-Tricalcium phosphate (beta-TCP) scaffolds with designed, three-dimensional (3-D) geometry and mesoscale porosity have been fabricated by direct-write assembly (robocasting) techniques. Concentrated beta-TCP inks with suitable viscoelastic properties were developed to enable the fabrication of the complex 3-D structures. A comprehensive study of the sintering behavior of TCP as a function of the calcium content in the starting powder was also carried out, and the optimal heat treatment for fabricating scaffolds with dense beta-TCP rods has been determined. Such analysis provides clues to controlling the microstructure of the fabricated structures and, therefore, enabling the fabrication by robocasting of TCP scaffolds with tailored performance for bone tissue engineering applications.
  • Article
    Since calcium phosphate cements were proposed, several formulations have been developed, some of them commercialised, and they have proven to be very efficient bone substitutes in different applications. Some of their properties, such as the injectability, or the low-temperature setting, which allows the incorporation of different drugs, make them very attractive candidates as drug carriers. In this article, the performance of calcium phosphate cements as carriers of different types of drugs, such as antibiotics, analgesics, anticancer, anti-inflammatory, as well as growth factors is reviewed.
  • Article
    Full-text available
    Alpha-modified minimum essential medium (alphaMEM) has been found to cross-link a 1% gellan gum solution, resulting in the formation of a self-supporting hydrogel in 1:1 and 5:1 ratios of polysaccharide: alphaMEM. Rheological data from temperature sweeps confirm that in addition to orders of magnitude differences in G' between 1% gellan and 1% gellan with alphaMEM, there is also a 20 degrees C increase in the temperature at which the onset of gelation takes place when alphaMEM is present. Frequency sweeps confirm the formation of a true gel; mechanical spectra for mixtures of gellan and alphaMEM clearly demonstrate G' to be independent of frequency. It is possible to immobilize cells within a three-dimensional (3D) gellan matrix that remain viable for up to 21 days in culture by adding a suspension of rat bone marrow cells (rBMC) in alphaMEM to 1% gellan solution. This extremely simple approach to cell immobilization within 3D constructs, made possible by the fact that gellan solutions cross-link in the presence of millimolar concentrations of cations, poses a very low risk to a cell population immobilized within a gellan matrix and thus indicates the potential of gellan for use as a tissue engineering scaffold.
  • Article
    Scaffold-based bone tissue engineering aims to repair/regenerate bone defects. Such a treatment concept involves seeding autologous osteogenic cells throughout a biodegradable scaffold to create a scaffold-cell hybrid that may be called a tissue-engineered construct (TEC). A variety of materials and scaffolding fabrication techniques for bone tissue engineering have been investigated over the past two decades. This review aims to discuss the advances in bone engineering from a scaffold material point of view. In the first part the reader is introduced to the basic principles of bone engineering. The important properties of the biomaterials and the scaffold design in the making of tissue engineered bone constructs are discussed in detail, with special emphasis placed on the new material developments, namely composites made of synthetic polymers and calcium phosphates. Advantages and limitations of these materials are analysed along with various architectural parameters of scaffolds important for bone tissue engineering, e.g. porosity, pore size, interconnectivity and pore-wall microstructures.
  • Article
    Rapid prototyping is a valuable implant production tool that enables the investigation of individual geometric parameters, such as shape, porosity, pore size and permeability, on the biological performance of synthetic bone graft substitutes. In the present study, we have employed low-temperature direct 3D printing to produce brushite and monetite implants with different geometries. Blocks predominantly consisting of brushite with channels either open or closed to the exterior were implanted on the decorticated lumbar transverse processes of goats for 12 weeks. In addition, similar blocks with closed channel geometry, consisting of either brushite or monetite were implanted intramuscularly. The design of the channels allowed investigation of the effect of macropore geometry (open and closed pores) and osteoinduction on bone formation orthotopically. Intramuscular implantation resulted in bone formation within the channels of both monetite and brushite, indicating osteoinductivity of these resorbable materials. Inside the blocks mounted on the transverse processes, initial channel shape did not seem to significantly influence the final amount of formed bone and osteoinduction was suggested to contribute to bone formation.
  • Article
    Organ or tissue printing, a novel approach in tissue engineering, creates layered, cell-laden hydrogel scaffolds with a defined three-dimensional (3D) structure and organized cell placement. In applying the concept of tissue printing for the development of vascularized bone grafts, the primary focus lies on combining endothelial progenitors and bone marrow stromal cells (BMSCs). Here we characterize the applicability of 3D fiber deposition with a plotting device, Bioplotter, for the fabrication of spatially organized, cell-laden hydrogel constructs. The viability of printed BMSCs was studied in time, in several hydrogels, and extruded from different needle diameters. Our findings indicate that cells survive the extrusion and that their subsequent viability was not different from that of unprinted cells. The applied extrusion conditions did not affect cell survival, and BMSCs could subsequently differentiate along the osteoblast lineage. Furthermore, we were able to combine two distinct cell populations within a single scaffold by exchanging the printing syringe during deposition, indicating that this 3D fiber deposition system is suited for the development of bone grafts containing multiple cell types.