Finite element method (FEM), mechanobiology and biomimetic scaffolds in bone tissue engineering

Techniques of bone reconstructive surgery are largely based on conventional, non-cell-based therapies that rely on the use of durable materials from outside the patient's body. In contrast to conventional materials, bone tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve bone tissue function. Bone tissue engineering has led to great expectations for clinical surgery or various diseases that cannot be solved with traditional devices. For example, critical-sized defects in bone, whether induced by primary tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold standard treatment for bone repair. The primary purpose of bone tissue engineering is to apply engineering principles to incite and promote the natural healing process of bone which does not occur in critical-sized defects. The total market for bone tissue regeneration and repair was valued at $1.1 billion in 2007 and is projected to increase to nearly $1.6 billion by 2014.Usually, temporary biomimetic scaffolds are utilized for accommodating cell growth and bone tissue genesis. The scaffold has to promote biological processes such as the production of extra-cellular matrix and vascularisation, furthermore the scaffold has to withstand the mechanical loads acting on it and to transfer them to the natural tissues located in the vicinity. The design of a scaffold for the guided regeneration of a bony tissue requires a multidisciplinary approach. Finite element method and mechanobiology can be used in an integrated approach to find the optimal parameters governing bone scaffold performance.In this paper, a review of the studies that through a combined use of finite element method and mechano-regulation algorithms described the possible patterns of tissue differentiation in biomimetic scaffolds for bone tissue engineering is given. Firstly, the generalities of the finite element method of structural analysis are outlined; second, the issues related to the generation of a finite element model of a given anatomical site or of a bone scaffold are discussed; thirdly, the principles on which mechanobiology is based, the principal theories as well as the main applications of mechano-regulation models in bone tissue engineering are described; finally, the limitations of the mechanobiological models and the future perspectives are indicated.     

Individual-based modelling of angiogenesis inside three-dimensional porous biomaterials

This paper presents a simulation modelling framework to study the growth of blood vessels and cells through a porous tissue engineering scaffold. The model simulates the migration of capillaries and the formation of a vascular network through a single pore of a tissue engineering scaffold when it is embedded in living tissue. The model also describes how the flow of blood through the network changes as growth proceeds. Results are given for how the different strategies of seeding the pore with cells affects the extent of vascularisation. Also simulations are made to compare results where the values of different model parameters are varied such as the pore dimensions, the density of endothelial cells seeded into the pore, and the release rate of growth factor from the scaffold into the pore. The modelling framework described in this paper is useful for exploring experimental strategies for producing well-vascularised tissue engineered constructs, and is therefore potentially important to the field of regenerative medicine.     

A multi-scale mechanobiological model of in-stent restenosis: deciphering the role of matrix metalloproteinase and extracellular matrix changes

Since their first introduction, stents have revolutionised the treatment of atherosclerosis; however, the development of in-stent restenosis still remains the Achilles' heel of stent deployment procedures. Computational modelling can be used as a means to model the biological response of arteries to different stent designs using mechanobiological models, whereby the mechanical environment may be used to dictate the growth and remodelling of vascular cells. Changes occurring within the arterial wall due to stent-induced mechanical injury, specifically changes within the extracellular matrix, have been postulated to be a major cause of activation of vascular smooth muscle cells and the subsequent development of in-stent restenosis. In this study, a mechanistic multi-scale mechanobiological model of in-stent restenosis using finite element models and agent-based modelling is presented, which allows quantitative evaluation of the collagen matrix turnover following stent-induced arterial injury and the subsequent development of in-stent restenosis. The model is specifically used to study the influence of stent deployment diameter and stent strut thickness on the level of in-stent restenosis. The model demonstrates that there exists a direct correlation between the stent deployment diameter and the level of in-stent restenosis. In addition, investigating the influence of stent strut thickness using the mechanobiological model reveals that thicker strut stents induce a higher level of in-stent restenosis due to a higher extent of arterial injury. The presented mechanobiological modelling framework provides a robust platform for testing hypotheses on the mechanisms underlying the development of in-stent restenosis and lends itself for use as a tool for optimisation of the mechanical parameters involved in stent design.     

A complete categorization of multiscale models of infectious disease systems

Modelling of infectious disease systems has entered a new era in which disease modellers are increasingly turning to multiscale modelling to extend traditional modelling frameworks into new application areas and to achieve higher levels of detail and accuracy in characterizing infectious disease systems. In this paper we present a categorization framework for categorizing multiscale models of infectious disease systems. The categorization framework consists of five integration frameworks and five criteria. We use the categorization framework to give a complete categorization of host-level immuno-epidemiological models (HL-IEMs). This categorization framework is also shown to be applicable in categorizing other types of multiscale models of infectious diseases beyond HL-IEMs through modifying the initial categorization framework presented in this study. Categorization of multiscale models of infectious disease systems in this way is useful in bringing some order to the discussion on the structure of these multiscale models.     

Simvastatin coating of TiO2 scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells

Bone tissue engineering requires an osteoconductive scaffold, multipotent cells with regenerative capacity and bioactive molecules. In this study we investigated the osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) on titanium dioxide (TiO₂) scaffold coated with alginate hydrogel containing various concentrations of simvastatin (SIM). The mRNA expression of osteoblast-related genes such as collagen type I alpha 1 (COL1A1), alkaline phosphatase (ALPL), osteopontin (SPP1), osteocalcin (BGLAP) and vascular endothelial growth factor A (VEGFA) was enhanced in hAD-MSCs cultured on scaffolds with SIM in comparison to scaffolds without SIM. Furthermore, the secretion of osteoprotegerin (OPG), vascular endothelial growth factor A (VEGFA), osteopontin (OPN) and osteocalcin (OC) to the cell culture medium was higher from hAD-MSCs cultured on scaffolds with SIM compared to scaffolds without SIM. The TiO₂ scaffold coated with alginate hydrogel containing SIM promote osteogenic differentiation of hAD-MSCs in vitro, and demonstrate feasibility as scaffold for hAD-MSC based bone tissue engineering.     

Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering

For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL), those from PCL and hydroxyapatite (HA), and those from PCL/HA and with a shifted pattern structure (PCL/HA/SP scaffold). Shifted pattern structure was fabricated to increase the cell attachment/adhesion. The PCL/HA/SP scaffold had a lower compressive modulus than PCL and PCL/HA scaffold. However, it has a better cell attachment than the scaffolds without a shifted pattern. MTT assay and alkaline phosphatase activity results for the PCL/HA/SP scaffolds were significantly enhanced compared to the results for the PCL and PCL/HA scaffolds. According to their degree of cell proliferation/differentiation, the scaffolds were in the following order: PCL/HA/SP > PCL/HA > PCL. These 3D scaffolds will be applicable for tissue engineering based on unique plotting system.     

Heparinized collagen scaffolds with and without growth factors for the repair of diaphragmatic hernia

A regenerative medicine approach to restore the morphology and function of the diaphragm in congenital diaphragmatic hernia is especially challenging because of the position and flat nature of this organ, allowing cell ingrowth primarily from the perimeter. Use of porous collagen scaffolds for the closure of surgically created diaphragmatic defects in rats has been shown feasible, but better ingrowth of cells, specifically blood vessels and muscle cells, is warranted. To stimulate this process, heparin, a glycosaminoglycan involved in growth factor binding, was covalently bound to porous collagenous scaffolds (14%), with or without vascular endothelial growth factor (VEGF; 0.4 µg/mg scaffold), hepatocyte growth factor (HGF; 0.5 µg/mg scaffold) or a combination of VEGF + HGF (0.2 + 0.5 µg/mg scaffold). All components were located primarily at the outside of scaffolds. Scaffolds were implanted in the diaphragm of rats and evaluated after 2 and 12 weeks. No herniations or eventrations were observed, and in several cases, growth factor-substituted scaffolds showed macroscopically visible blood vessels at the lung site. The addition of heparin led to an accelerated ingrowth of blood vessels at 2 weeks. In all scaffold types, giant cells and immune cells were present primarily at the liver side of the scaffold, and immune cells and individual macrophages at the lung side; these cell types decreased in number from week 2 to week 12. The addition of growth factors did not influence cellular response to the scaffolds, indicating that further optimization with respect to dosage and release profile is needed.     

Cell culture in autologous fibrin scaffolds for applications in tissue engineering

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth.     

Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells

Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu²⁺-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu²⁺-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu²⁺-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu²⁺-doped BG scaffolds release Cu²⁺, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches.     

Use of monocyte/endothelial cell co-cultures (in vitro) and a subcutaneous implant mouse model (in vivo) to evaluate a degradable polar hydrophobic ionic polyurethane

Potential benefits of co-culturing monocytes (MC) with vascular smooth muscle cells have been reported on for tissue engineering applications with a degradable, polar, hydrophobic, and ionic polyurethane (D-PHI). Since the interaction of MC and endothelial cells (EC) within the blood vessel endothelium is also a process of wound repair it was of interest to investigate their function when cultured on the synthetic D-PHI materials, prior to considering the materials' use in vascular engineering. The co-culture (MC/EC) in vitro studies were carried out on films in 96 well plates and porous scaffold disks were prepared for implant studies in an in vivo subcutaneous mouse model. After 7 days in culture, the MC/EC condition was equal to EC growth but had lower esterase activity (a measure of degradative potential), no pro-inflammatory TNF-α and a relatively high anti-inflammatory IL-10 release while the ECs maintained their functional marker CD31. After explantation of the porous scaffolds, a live/dead stain showed that the cells infiltrating the scaffolds were viable and histological stains (May-Grunwald, Trichrome) demonstrated tissue in growth and extracellular matrix synthesis. Lysates from the implant scaffolds analyzed with a cytokine antibody array showed decreased pro-inflammatory cytokines (IL-6, TNF-α, GM-CSF), increased anti-inflammatory cytokines (IL-10, IL-13, TNF-RI), and increased chemotactic cytokines (MCP-1, MCP-5, RANTES). The low foreign body response elicited by D-PHI when implanted in vivo supported the in vitro studies (EC and MC co-culture), demonstrating that D-PHI promoted EC growth along with an anti-inflammatory MC, further demonstrating its potential as a tissue engineering scaffold for vascular applications.     

Composite scaffolds for the engineering of hollow organs and tissues

Several types of synthetic and naturally derived biomaterials have been used for augmenting hollow organs and tissues. However, each has desirable traits which were exclusive of the other. We fabricated a composite scaffold and tested its potential for the engineering of hollow organs in a bladder tissue model. The composite scaffolds were configured to accommodate a large number of cells on one side and were designed to serve as a barrier on the opposite side. The scaffolds were fabricated by bonding a collagen matrix to PGA polymers with threaded collagen fiber stitches. Urothelial and bladder smooth muscle cells were seeded on the composite scaffolds, and implanted in mice for up to 4 weeks and analyzed. Both cell types readily attached and proliferated on the scaffolds and formed bladder tissue-like structures in vivo. These structures consisted of a luminal urothelial layer, a collagen rich compartment and a peripheral smooth muscle layer. Biomechanical studies demonstrated that the tissues were readily elastic while maintaining their pre-configured structures. This study demonstrates that a composite scaffold can be fabricated with two completely different polymer systems for the engineering of hollow organs. The composite scaffolds are biocompatible, possess adequate physical and structural characteristics for bladder tissue engineering, and are able to form tissues in vivo. This scaffold system may be useful in patients requiring hollow organ replacement.     

Mathematical modelling of skeletal repair

Tissue engineering offers significant promise as a viable alternative to current clinical strategies for replacement of damaged tissue as a consequence of disease or trauma. Since mathematical modelling is a valuable tool in the analysis of complex systems, appropriate use of mathematical models has tremendous potential for advancing the understanding of the physical processes involved in such tissue reconstruction. In this review, the potential benefits, and limitations, of theoretical modelling in tissue engineering applications are examined with specific emphasis on tissue engineering of bone. A central tissue engineering approach is the in vivo implantation of a biomimetic scaffold seeded with an appropriate population of stem or progenitor cells. This review will therefore consider the theory behind a number of key factors affecting the success of such a strategy including: stem cell or progenitor population expansion and differentiation ex vivo; cell adhesion and migration, and the effective design of scaffolds; and delivery of nutrient to avascular structures. The focus will be on current work in this area, as well as on highlighting limitations and suggesting possible directions for future work to advance health-care for all.     

Morphological characterization of a novel scaffold for anterior cruciate ligament tissue engineering

Tissue engineering offers an interesting alternative to current anterior cruciate ligament (ACL) surgeries. Indeed, a tissue-engineered solution could ideally overcome the long-term complications due to actual ACL reconstruction by being gradually replaced by biological tissue. Key requirements concerning the ideal scaffold for ligament tissue engineering are numerous and concern its mechanical properties, biochemical nature, and morphology. This study is aimed at predicting the morphology of a novel scaffold for ligament tissue engineering, based on multilayer braided biodegradable copoly(lactic acid-co-(e-caprolactone)) (PLCL) fibers The process used to create the scaffold is briefly presented, and the degradations of the material before and after the scaffold processing are compared. The process offers varying parameters, such as the number of layers in the scaffold, the pitch length of the braid, and the fibers' diameter. The prediction of the morphology in terms of pore size distribution and pores interconnectivity as a function of these parameters is performed numerically using an original method based on a virtual scaffold. The virtual scaffold geometry and the prediction of pore size distribution are evaluated by comparison with experimental results. The presented process permits creation of a tailorable scaffold for ligament tissue engineering using basic equipment and from minimum amounts of raw material. The virtual scaffold geometry closely mimics the geometry of real scaffolds, and the prediction of the pore size distribution is found to be in good accordance with measurements on real scaffolds. The scaffold offers an interconnected network of pores the sizes of which are adjustable by playing on the process parameters and are able to match the ideal pore size reported for tissue ingrowth. The adjustability of the presented scaffold could permit its application in both classical ACL reconstructions and anatomical double-bundle reconstructions. The precise knowledge of the scaffold morphology using the virtual scaffold will be useful to interpret the activity of cells once it will be seeded into the scaffold. An interesting perspective of the present work is to perform a similar study aiming at predicting the mechanical response of the scaffold according to the same process parameters, by implanting the virtual scaffold into a finite element algorithm.     

Biological properties of solid free form designed ceramic scaffolds with BMP-2: in vitro and in vivo evaluation

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.     

可降解组织工程血管支架成功用于犬股动脉移植

目的采用可降解的聚己内酯接枝肝素材料,负荷b-FGF(碱性成纤维细胞生长因子),体外构建的小口径组织工程血管,完成犬的股动脉移植动物实验。方法利用可降解的聚己内酯接枝肝素材料,电纺丝技术制备组织工程血管支架,并对支架负荷b-FGF生长因子,并进行材料的内皮细胞粘附实验。将体外构建的小口径组织工程血管,完成犬的股动脉移植动物实验,观察通畅率和移植术后组织工程血管的改变。结果可降解聚己内酯接枝肝素材料支架,负荷细胞生长因子(b-FGF),利于内皮细胞粘附。构建的组织工程血管进行体外动物实验构建,3个月移植物通畅率好,移植后取材,有新生内膜迁移和胶原纤维浸入。结论利用可降解聚己内酯接枝肝素材料构建小口径支架,初步符合构建组织工程血管支架的要求。     

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.     

Scaffolds in tissue engineering of blood vessels

Tissue engineering of small diameter (<5?mm) blood vessels is a promising approach for developing viable alternatives to autologous vascular grafts. It involves in vitro seeding of cells onto a scaffold on which the cells attach, proliferate, and differentiate while secreting the components of extracellular matrix that are required for creating the tissue. The scaffold should provide the initial requisite mechanical strength to withstand in vivo hemodynamic forces until vascular smooth muscle cells and fibroblasts reinforce the extracellular matrix of the vessel wall. Hence, the choice of scaffold is crucial for providing guidance cues to the cells to behave in the required manner to produce tissues and organs of the desired shape and size. Several types of scaffolds have been used for the reconstruction of blood vessels. They can be broadly classified as biological scaffolds, decellularized matrices, and polymeric biodegradable scaffolds. This review focuses on the different types of scaffolds that have been designed, developed, and tested for tissue engineering of blood vessels, including use of stem cells in vascular tissue engineering.     

Computational model of the in vivo development of a tissue engineered vein from an implanted polymeric construct

Advances in vascular tissue engineering have been tremendous over the past 15 years, yet there remains a need to optimize current constructs to achieve vessels having true growth potential. Toward this end, it has been suggested that computational models may help hasten this process by enabling time-efficient parametric studies that can reduce the experimental search space. In this paper, we present a first generation computational model for describing the in vivo development of a tissue engineered vein from an implanted polymeric scaffold. The model was motivated by our recent data on the evolution of mechanical properties and microstructural composition over 24 weeks in a mouse inferior vena cava interposition graft. It is shown that these data can be captured well by including both an early inflammatory-mediated and a subsequent mechano-mediated production of extracellular matrix. There remains a pressing need, however, for more data to inform the development of next generation models, particularly the precise transition from the inflammatory to the mechanobiological dominated production of matrix having functional capability.     

Darcian permeability constant as indicator for shear stresses in regular scaffold systems for tissue engineering

The shear stresses in printed scaffold systems for tissue engineering depend on the flow properties and void volume in the scaffold. In this work, computational fluid dynamics (CFD) is used to simulate flow fields within porous scaffolds used for cell growth. From these models the shear stresses acting on the scaffold fibres are calculated. The results led to the conclusion that the Darcian (k ₁) permeability constant is a good predictor for the shear stresses in scaffold systems for tissue engineering. This permeability constant is easy to calculate from the distance between and thickness of the fibres used in a 3D printed scaffold. As a consequence computational effort and specialists for CFD can be circumvented by using this permeability constant to predict the shear stresses. If the permeability constant is below a critical value, cell growth within the specific scaffold design may cause a significant increase in shear stress. Such a design should therefore be avoided when the shear stress experienced by the cells should remain in the same order of magnitude.     

A multiscale computational fluid dynamics approach to simulate the micro-fluidic environment within a tissue engineering scaffold with highly irregular pore geometry

Mechanical stimulation can regulate cellular behavior, e.g., differentiation, proliferation, matrix production and mineralization. To apply fluid-induced wall shear stress (WSS) on cells, perfusion bioreactors have been commonly used in tissue engineering experiments. The WSS on cells depends on the nature of the micro-fluidic environment within scaffolds under medium perfusion. Simulating the fluidic environment within scaffolds will be important for gaining a better insight into the actual mechanical stimulation on cells in a tissue engineering experiment. However, biomaterial scaffolds used in tissue engineering experiments typically have highly irregular pore geometries. This complexity in scaffold geometry implies high computational costs for simulating the precise fluidic environment within the scaffolds. In this study, we propose a low-computational cost and feasible technique for quantifying the micro-fluidic environment within the scaffolds, which have highly irregular pore geometries. This technique is based on a multiscale computational fluid dynamics approach. It is demonstrated that this approach can capture the WSS distribution in most regions within the scaffold. Importantly, the central process unit time needed to run the model is considerably low.