Optimizing Biomaterials for Tissue Engineering Human Bone Using Mesenchymal Stem Cells.

BACKGROUND: Adequate biomaterials for tissue engineering bone and replacement of bone in clinical settings are still being developed. Previously, the combination of mesenchymal stem cells in hydrogels and calcium-based biomaterials in both in vitro and in vivo experiments has shown promising results. However, results may be optimized by careful selection of the material combination. METHODS: ß-Tricalcium phosphate scaffolds were three-dimensionally printed with five different hydrogels: collagen I, gelatin, fibrin glue, alginate, and Pluronic F-127. The scaffolds had eight channels, running throughout the entire scaffold, and macropores. Mesenchymal stem cells (2 × 10) were mixed with each hydrogel, and cell/hydrogel mixes were dispersed onto the corresponding ß-tricalcium phosphate/hydrogel scaffold and cultured under dynamic-oscillating conditions for 6 weeks. Specimens were harvested at 1, 2, 4, and 6 weeks and evaluated histologically, radiologically, biomechanically and, at 6 weeks, for expression of bone-specific proteins by reverse-transcriptase polymerase chain reaction. Statistical correlation analysis was performed between radiologic densities in Hounsfield units and biomechanical stiffness. RESULTS: Collagen I samples had superior bone formation at 6 weeks as demonstrated by volume computed tomographic scanning, with densities of 300 HU, similar to native bone, and the highest compression values. Bone specificity of new tissue was confirmed histologically and by the expression of alkaline phosphatase, osteonectin, osteopontin, and osteocalcin. The bone density correlated closely with histologic and biomechanical testing results. CONCLUSION: Bone formation is supported best by ß-tricalcium phosphate/collagen I hydrogel and mesenchymal stem cells in collagen I hydrogel. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Branched vascular network architecture: a new approach to lung assist device technology.

OBJECTIVE: A lung assist device would serve an important clinical need as a bridge to transplant or destination therapy for patients with end-stage lung disease. A new lung assist device has been developed that incorporates a branched network of vascular channels adjacent to a gas chamber, separated by a thin, gas-permeable membrane. This study investigated 2 potential gas exchange membranes within this new architecture. METHODS: Oxygen and carbon dioxide exchange within the device was tested in vitro using 3 gas-permeable membranes. Two of the membranes, silicone only and silicone-coated microporous polymer, were plasma impermeable. The third, a microporous polymer, was used as a control. Gas exchange testing was done using anticoagulated porcine blood over a range of flow rates. RESULTS: Oxygen and carbon dioxide transfer was demonstrated in the device and increased nearly linearly from 0.6 to 8.0 mL/min blood flow for all of the membranes. There was no significant difference in the gas transfer between the silicone and the silicone-coated microporous polymer membranes. The transfer of oxygen and carbon dioxide in the device was similar to existing hollow fiber oxygenators controlling for surface area. CONCLUSIONS: The silicone and silicone-coated microporous polymer membranes both show promise as gas-permeable membranes in a new lung assist device design. Further optimization of the device by improving the membranes and reducing the channel diameter in the vascular network will improve gas transfer. The current device may be scaled up to function as an adult lung assist device.

Liver-assist device with a microfluidics-based vascular bed in an animal model.

OBJECTIVE: This study evaluates a novel liver-assist device platform with a microfluidics-modeled vascular network in a femoral arteriovenous shunt in rats. SUMMARY OF BACKGROUND DATA: Liver-assist devices in clinical trials that use pumps to force separated plasma through packed beds of parenchymal cells exhibited significant necrosis with a negative impact on function. METHODS: Microelectromechanical systems technology was used to design and fabricate a liver-assist device with a vascular network that supports a hepatic parenchymal compartment through a nanoporous membrane. Sixteen devices with rat primary hepatocytes and 12 with human HepG2/C3A cells were tested in athymic rats in a femoral arteriovenous shunt model. Several parenchymal tube configurations were evaluated for pressure profile and cell survival. The blood flow pattern and perfusion status of the devices was examined by laser Doppler scanning. Cell viability and serum protein secretion functions were assessed. RESULTS: Femoral arteriovenous shunt was successfully established in all animals. Blood flow was homogeneous through the vascular bed and replicated native flow patterns. Survival of seeded liver cells was highly dependent on parenchymal chamber pressures. The tube configuration that generated the lowest pressure supported excellent cell survival and function. CONCLUSIONS: This device is the first to incorporate a microfluidics network in the systemic circulatory system. The microvascular network supported viability and function of liver cells in a short-term ex vivo model. Parenchymal chamber pressure generated in an arteriovenous shunt model is a critical parameter that affects viability and must be considered in future designs. The microfluidics-based vascular network is a promising platform for generating a large-scale medical device capable of augmenting liver function in a clinical setting.

Hemodynamic environments from opposing sides of human aortic valve leaflets evoke distinct endothelial phenotypes in vitro.

The regulation of valvular endothelial phenotypes by the hemodynamic environments of the human aortic valve is poorly understood. The nodular lesions of calcific aortic stenosis (CAS) develop predominantly beneath the aortic surface of the valve leaflets in the valvular fibrosa layer. However, the mechanisms of this regional localization remain poorly characterized. In this study, we combine numerical simulation with in vitro experimentation to investigate the hypothesis that the previously documented differences between valve endothelial phenotypes are linked to distinct hemodynamic environments characteristic of these individual anatomical locations. A finite-element model of the aortic valve was created, describing the dynamic motion of the valve cusps and blood in the valve throughout the cardiac cycle. A fluid mesh with high resolution on the fluid boundary was used to allow accurate computation of the wall shear stresses. This model was used to compute two distinct shear stress waveforms, one for the ventricular surface and one for the aortic surface. These waveforms were then applied experimentally to cultured human endothelial cells and the expression of several pathophysiological relevant genes was assessed. Compared to endothelial cells subjected to shear stress waveforms representative of the aortic face, the endothelial cells subjected to the ventricular waveform showed significantly increased expression of the "atheroprotective" transcription factor Kruppel-like factor 2 (KLF2) and the matricellular protein Nephroblastoma overexpressed (NOV), and suppressed expression of chemokine Monocyte-chemotactic protein-1 (MCP-1). Our observations suggest that the difference in shear stress waveforms between the two sides of the aortic valve leaflet may contribute to the documented differential side-specific gene expression, and may be relevant for the development and progression of CAS and the potential role of endothelial mechanotransduction in this disease.

On the multiscale modeling of heart valve biomechanics in health and disease.

Theoretical models of the human heart valves are useful tools for understanding and characterizing the dynamics of healthy and diseased valves. Enabled by advances in numerical modeling and in a range of disciplines within experimental biomechanics, recent models of the heart valves have become increasingly comprehensive and accurate. In this paper, we first review the fundamentals of native heart valve physiology, composition and mechanics in health and disease. We will then furnish an overview of the development of theoretical and experimental methods in modeling heart valve biomechanics over the past three decades. Next, we will emphasize the necessity of using multiscale modeling approaches in order to provide a comprehensive description of heart valve biomechanics able to capture general heart valve behavior. Finally, we will offer an outlook for the future of valve multiscale modeling, the potential directions for further developments and the challenges involved.

Functional endothelialized microvascular networks with circular cross-sections in a tissue culture substrate.

Functional endothelialized networks constitute a critical building block for vascularized replacement tissues, organ assist devices, and laboratory tools for in vitro discovery and evaluation of new therapeutic compounds. Progress towards realization of these functional artificial vasculatures has been gated by limitations associated with the mechanical and surface chemical properties of commonly used microfluidic substrate materials and by the geometry of the microchannels produced using conventional fabrication techniques. Here we report on a method for constructing microvascular networks from polystyrene substrates commonly used for tissue culture, built with circular cross-sections and smooth transitions at bifurcations. Silicon master molds are constructed using an electroplating process that results in semi-circular channel cross-sections with smoothly varying radii. These master molds are used to emboss polystyrene sheets which are then joined to form closed bifurcated channel networks with circular cross-sections. The mechanical and surface chemical properties of these polystyrene microvascular network structures enable culture of endothelial cells along the inner lumen. Endothelial cell viability was assessed, documenting nearly confluent monolayers within 3D microfabricated channel networks with rounded cross-sections.

A computational model of aging and calcification in the aortic heart valve.

The aortic heart valve undergoes geometric and mechanical changes over time. The cusps of a normal, healthy valve thicken and become less extensible over time. In the disease calcific aortic stenosis (CAS), calcified nodules progressively stiffen the cusps. The local mechanical changes in the cusps, due to either normal aging or pathological processes, affect overall function of the valve. In this paper, we propose a computational model for the aging aortic valve that connects local changes to overall valve function. We extend a previous model for the healthy valve to describe aging. To model normal/uncomplicated aging, leaflet thickness and extensibility are varied versus age according to experimental data. To model calcification, initial sites are defined and a simple growth law is assumed. The nodules then grow over time, so that the area of calcification increases from one model to the next model representing greater age. Overall valve function is recorded for each individual model to yield a single simulation of valve function over time. This simulation is the first theoretical tool to describe the temporal behavior of aortic valve calcification. The ability to better understand and predict disease progression will aid in design and timing of patient treatments for CAS.

Toward regenerating a human thumb in situ.

Regenerative technology promises to alleviate the problem of limited donor supply for bone or organ transplants. Most expensive and time consuming is cell expansion in laboratories. We propose a method of magnetically enriched osteoprogenitor stem cells, dispersed in self-assembling hydrogels and applied onto new ultra-high resolution, jet-based, three-dimensional printing of living human bone in a single-step for in situ bone regeneration. Human bone marrow-derived mesenchymal stem cells (hBMSCs) were enriched with CD 117+ cells, dispersed in different collagen I and RAD 16I hydrogel mixes, and applied onto three-dimensional printed btricalcium phosphate=poly(lactic-co-glycolic acid) scaffolds, printed from ultra-high-resolution volumetric CT images of a human thumb. Constructs were directly implanted subcutaneously into nude mice for 6 weeks. In vivo radiographic volumetric CT scanning and histological evaluations were performed at 1, 2, 4, and 6 weeks, and expression of bone-specific genes and biomechanical compression testing at 6 weeks endpoint. Time-dependant accumulation of bone-like extracellular matrix was most evident in CD 117+ hBMSCs using collagen I=RAD 16I hydrogel mix. This was shown histologically by Toluidine blue, von Kossa, and alkaline phosphatase staining, paralleled by increased radiological densities within implants approximating that of human bone, and confirmed by high expression of bone-specific osteonectin and biomechanical stiffness at 6 weeks. Human origin of newly formed tissue was established by expression of human GAPDH using RT-PCR. Statistical analysis confirmed high correlations between biomechanical stiffness, radiological densities, and bone markers. Bone tissue can be successfully regenerated in vivo using a single-step procedure with constructs composed of RAD 16I=collagen I hydrogel, CD 117+-enriched hBMSCs, and porous b-tricalcium phosphate=poly(lactic-co-glycolic acid) scaffolds.

A multiscale computational comparison of the bicuspid and tricuspid aortic valves in relation to calcific aortic stenosis.

Patients with bicuspid aortic valve (BAV) are more likely to develop a calcific aortic stenosis (CAS), as well as a number of other ailments, as compared to their cohorts with normal tricuspid aortic valves (TAV). It is currently unknown whether the increase in risk of CAS is caused by the geometric differences between the tricuspid and bicuspid valves or whether the increase in risk is caused by the same underlying factors that produce the geometric difference. CAS progression is understood to be a multiscale process, mediated at the cell level. In this study, we employ multiscale finite-element simulations of the valves. We isolate the effect of one geometric factor, the number of cusps, in order to explore its effect on multiscale valve mechanics, particularly in relation to CAS. The BAV and TAV are modeled by a set of simulations describing the cell, tissue, and organ length scales. These simulations are linked across the length scales to create a coherent multiscale model. At each scale, the models are three-dimensional, dynamic, and incorporate accurate nonlinear constitutive models of the valve leaflet tissue. We compare results between the TAV and BAV at each length scale. At the cell-scale, our region of interest is the location where calcification develops, near the aortic-facing surface of the leaflet. Our simulations show the observed differences between the tricuspid and bicuspid valves at the organ scale: the bicuspid valve shows greater flexure in the solid phase and stronger jet formation in the fluid phase relative to the tricuspid. At the cell-scale, however, we show that the region of interest is shielded against strain by the wrinkling of the fibrosa. Thus, the cellular deformations are not significantly different between the TAV and BAV in the calcification-prone region. This result supports the assertion that the difference in calcification observed in the BAV versus TAV may be due primarily to factors other than the simple geometric difference between the two valves.

In vitro analysis of a hepatic device with intrinsic microvascular-based channels.

A novel microfluidics-based bilayer device with a discrete parenchymal chamber modeled upon hepatic organ architecture is described. The microfluidics network was designed using computational models to provide appropriate flow behavior based on physiological data from human microvasculature. Patterned silicon wafer molds were used to generate films with the vascular-based microfluidics network design and parenchymal chamber by soft lithography. The assembled device harbors hepatocytes behind a nanoporous membrane that permits transport of metabolites and small proteins while protecting them from the effects of shear stress. The device can sustain both human hepatoma cells and primary rat hepatocytes by continuous in vitro perfusion of medium, allowing proliferation and maintaining hepatic functions such as serum protein synthesis and metabolism. The design and fabrication processes are scalable, enabling the device concept to serve as both a platform technology for drug discovery and toxicity, and for the continuing development of an improved liver-assist device.

Transient, three-dimensional, multiscale simulations of the human aortic valve.

A set of multiscale simulations has been created to examine the dynamic behavior of the human aortic valve (AV) at the cell, tissue, and organ length scales. Each model is fully three-dimensional and includes appropriate nonlinear, anisotropic material models. The organ-scale model is a dynamic fluid-structure interaction that predicts the motion of the blood, cusps, and aortic root throughout the full cycle of opening and closing. The tissue-scale model simulates the behavior of the AV cusp tissue including the sub-millimeter features of multiple layers and undulated geometry. The cell-scale model predicts cellular deformations of individual cells within the cusps. Each simulation is verified against experimental data. The three simulations are linked: deformations from the organ-scale model are applied as boundary conditions to the tissue-scale model, and the same is done between the tissue and cell scales. This set of simulations is a major advance in the study of the AV as it allows analysis of transient, three-dimensional behavior of the AV over the range of length scales from cell to organ.

Microfabrication of three-dimensional engineered scaffolds.

One of the principal challenges facing the field of tissue engineering over the past 2 decades has been the requirement for large-scale engineered constructs comprising precisely organized cellular microenvironments. For vital organ assist and replacement devices, microfluidic-based systems such as the microcirculation, biliary, or renal filtration and resorption systems and other functional elements containing multiple cell types must be generated to provide for viable engineered tissues and clinical benefit. Over the last several years, microfabrication technology has emerged as a versatile and powerful approach for generating precisely engineered scaffolds for engineered tissues. Fabrication process tools such as photolithography, etching, molding, and lamination have been established for applications involving a range of biocompatible and biodegradable polymeric scaffolding materials. Computational fluid dynamic designs have been used to generate scaffold designs suitable for microvasculature and a number of organ-specific constructs; these designs have been translated into 3-dimensional scaffolding using microfabrication processes. Here a brief overview of the fundamental microfabrication technologies used for tissue engineering will be presented, along with a summary of progress in a number of applications, including the liver and kidney.

Human shaped thumb bone tissue engineered by hydrogel-beta-tricalciumphosphate/poly-epsilon-caprolactone scaffolds and magnetically sorted stem cells.

Traumatic amputation of a thumb with bone loss leaves a patient in severe disability. Reconstructive procedures are restricted by limited shape and have the disadvantage of severe donor-site morbidity. To overcome these limitations, we used a tissue engineering approach to create a distal thumb bone phalanx, combining magnetically sorted 133+ human mesenchymal stem cells (hMSCs) suspended in successful tested hydrogels for bone formation and porous 3-dimensionally printed scaffolds (3DP) in the shape of a distal thumb bone phalanx. Collagen I and fibrin glue hydrogels with suspended hMSCs were first histologically evaluated in vitro for bone formation after 6 weeks. Then 3DP scaffolds, made from a mix of osteoinductive and -conductive beta-tricalciumphosphate (beta-TCP) and poly-epsilon-caprolactone (PCL), with hydrogels and suspended hMSCs, were implanted into nude mice subcutaneously for 15 weeks. Histologic evaluation, high-resolution volumetric CT (VCT) scanning, and biomechanical testing confirmed formation of bonelike tissue. Both hydrogels with CD 133+ hMSCs on 3DP scaffolds supported bone formation. Collagen I resulted in radiologically better bone formation. Bone tissue can be successfully tissue engineered with CD 133+ hMSCs, collagen I hydrogels, and porous 3DP beta-TCP/PCL scaffolds.

Comparison of hydrogels in the in vivo formation of tissue-engineered bone using mesenchymal stem cells and beta-tricalcium phosphate.

Availability of grafts and morbidity at the donor site limit autologous transplantation in patients requiring bone reconstruction. A tissue-engineering approach can overcome these limitations by producing bone-like tissue of custom shape and size from isolated cells. Several hydrogels facilitate osteogenesis on porous scaffolds; however, the relative suitability of various hydrogels has not been rigorously assessed. Fibrin glue, alginate, and collagen I hydrogels were mixed with swine bone marrow-derived differentiated mesenchymal stem cells (MSCs), applied to 3-dimensionally printed porous beta-tricalcium phosphate (beta-TCP) scaffolds and implanted subcutaneously in nude mice. Although noninvasive assessment of osteogenesis in 3 dimensions is desirable for monitoring new bone formation in vivo, correlations with traditional histological and mechanical testing need to be established. High-resolution volumetric computed tomography (VCT) scanning, histological examination, biomechanical compression testing, and osteonectin (ON) expression were performed on excised scaffolds after 1, 2, 4, and 6 weeks of subcutaneous implantation in mice. Statistical correlation analyses were performed between radiological density, stiffness, and ON expression. Use of collagen I as a hydrogel carrier produced superior bone formation at 6 weeks, as demonstrated using VCT scanning with densities similar to native bone and the highest compression values. Continued contribution of the seeded MSCs was demonstrated using swine-specific messenger ribonucleic acid probes. Radiological density values correlated closely with the results of histological and biomechanical testing and ON expression. High-resolution VCT is a promising method for monitoring osteogenesis.

A finite shell element for heart mitral valve leaflet mechanics, with large deformations and 3D constitutive material model.

This paper presents a shell finite element formulation appropriate for simulating the heart valve leaflet mechanics, including three-dimensional (3D) stress and strain effects. A 4-node mixed-interpolation shell is formulated in convected coordinates. This shell model is made capable of handling arbitrary 3D material models by use of an algorithm that satisfies the shell stress assumption at every element integration point. A method for tracking the fiber direction is incorporated. The resulting shell element operates under the same conditions as a standard 4-node shell element with 5 degrees of freedom per node, but extends the modeling capabilities to handle large-deformation and anisotropic behavior.

A large-strain finite element formulation for biological tissues with application to mitral valve leaflet tissue mechanics.

This paper presents a finite element formulation suitable for large-strain modeling of biological tissues and uses this formulation to implement an accurate finite element model for mitral valve leaflet tissue. First, an experimentally derived strain energy function is obtained from literature. This function is implemented in finite elements using the mixed pressure-displacement formulation. A modification is made to aid in maintaining positive definiteness of the stiffness matrix at low strains. The numerical implementation is shown to be accurate in representing the analytical model of material behavior. The mixed formulation is useful for modeling of soft biological tissues in general, and the model presented here is applicable to finite element simulation of mitral valve mechanics.

Hydrogel-beta-TCP scaffolds and stem cells for tissue engineering bone.

Trabecular bone is a material of choice for reconstruction after trauma and tumor resection and for correction of congenital defects. Autologous bone grafts are available in limited shapes and sizes; significant donor site morbidity is another major disadvantage to this approach. To overcome these limitations, we used a tissue engineering approach to create bone replacements in vitro, combining bone-marrow-derived differentiated mesenchymal stem cells (MSCs) suspended in hydrogels and 3-dimensionally printed (3DP) porous scaffolds made of beta-tricalcium-phosphate (beta-TCP). The scaffolds provided support for the formation of bone tissue in collagen I, fibrin, alginate, and pluronic F127 hydrogels during culturing in oscillating and rotating dynamic conditions. Histological evaluation including toluidine blue, alkaline phosphatase, and von Kossa staining was done at 1, 2, 4, and 6 weeks. Radiographic evaluation and high-resolution volumetric CT (VCT) scanning, expression of bone-specific genes and biomechanical compression testing were performed at 6 weeks. Both culture conditions resulted in similar bone tissue formation. Histologically collagen I and fibrin hydrogels specimens had superior bone tissue, although radiopacities were detected only in collagen I samples. VCT scan revealed density values in all but the Pluronic F127 samples, with Houndsfield unit values comparable to native bone in collagen I and fibrin glue samples. Expression of bone-specific genes was significantly higher in the collagen I samples. Pluronic F127 hydrogel did not support formation of bone tissue. All samples cultured in dynamic oscillating conditions had slightly higher mechanical strength than under rotating conditions. Bone tissue can be successfully formed in vitro using constructs comprised of collagen I hydrogel, MSCs, and porous beta-TCP scaffolds.