Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology.

3D bioprinting of tissues and organs

An overview of tissue and whole organ decellularization processes.

MUCKE aims to mine a large volume of images, to structure them conceptually and to use this conceptual structuring in order to improve large-scale image retrieval. The last decade witnessed important progress concerning low-level image representations. However, there are a number problems which need to be solved in order to unleash the full potential of image mining in applications. The central problem with low-level representations is the mismatch between them and the human interpretation of image content. This problem can be instantiated, for instance, by the incapability of existing descriptors to capture spatial relationships between the concepts represented or by their incapability to convey an explanation of why two images are similar in a content-based image retrieval framework. We start by assessing existing local descriptors for image classification and by proposing to use co-occurrence matrices to better capture spatial relationships in images. The main focus in MUCKE is on cleaning large scale Web image corpora and on proposing image representations which are closer to the human interpretation of images. Consequently, we introduce methods which tackle these two problems and compare results to state of the art methods. Note: some aspects of this deliverable are withheld at this time as they are pending review. Please contact the authors for a preview.

http://ieeexplore.ieee.org/iel2/4524/12820/00592460.pdf

Image Processing

Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure 1/28th the area of the starting gel in 24 hr. The rate of the process can be regulated by varying the protein content of the lattice, the cell number, or the con- centration of an inhibitor such as Colcemid. Fibroblasts of high population doubling level propagated in vitro, which have left the cell cycle, can carry out the contraction at least as efficiently as cycling cells. The potential uses of the system as an immu- nologically tolerated "tissue" for wound hea ing and as a model for studying fibroblast function are discussed.

Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative

The molecular and physical information coded within the extracellular milieu is informing the development of a new generation of biomaterials for tissue engineering. Several powerful extracellular influences have already found their way into cell-instructive scaffolds, while others remain largely unexplored. Yet for commercial success tissue engineering products must be not only efficacious but also cost-effective, introducing a potential dichotomy between the need for sophistication and ease of production. This is spurring interest in recreating extracellular influences in simplified forms, from the reduction of biopolymers into short functional domains, to the use of basic chemistries to manipulate cell fate. In the future these exciting developments are likely to help reconcile the clinical and commercial pressures on tissue engineering.

Complexity in biomaterials for tissue engineering

Understanding the organization and mechanical function of the extracellular matrix (ECM) is critical for the development of therapeutic strategies that regulate wound healing following disease or injury. However, these relationships are challenging to elucidate during remodeling following myocardial infarction (MI) due to rapid changes in cellularity and an inability to characterize both ECM microstructure and function non-destructively. In this study, we overcome those challenges through whole organ decellularization and non-linear optical microscopy to directly relate the microstructure and mechanical properties of myocardial ECM. We non-destructively quantify collagen organization, content, and cross-linking within decellularized healthy and infarcted myocardium using second harmonic generation (SHG) and two photon excited autofluorescence. Tensile mechanical testing and compositional analysis reveal that the cumulative SHG intensity within each image volume and the average collagen autofluorescence are significantly correlated with collagen content and elastic modulus of the ECM, respectively. Compared to healthy ECM, infarcted tissues demonstrate a significant increase in collagen content and fiber alignment, and a decrease in cross-linking and elastic modulus. These findings indicate that cross-linking plays a key role in stiffness at the collagen fiber level following infarction, and highlight how this non-destructive approach to assessing remodeling can be used to understand ECM structure-function relationships.

/pdf/optical-metrics-of-the-extracellular-matrix-predict-10jdh139r0.pdf

Optical metrics of the extracellular matrix predict compositional and mechanical changes after myocardial infarction.

Beta 1 integrin binding plays a role in the constant traction force generation in response to varying stiffness for cells grown on mature cardiac extracellular matrix

Enzyme activity plays an essential role in many physiological processes and diseases such as pulmonary emphysema. While the lung is constantly exposed to cyclic stretching, the effects of stretch o...

Differential effects of static and cyclic stretching during elastase digestion on the mechanical properties of extracellular matrices

A primary impediment to cardiac tissue engineering lies in the inability to adequately vascularize the constructs to optimize survival upon implantation. During normal angiogenesis, endothelial cells (ECs) require a support cell to form mature patent lumens and it has been demonstrated that pericytes, vascular smooth muscle cells and mesenchymal stem cells (MSCs) are all able to support the formation of mature vessels. In the heart, cardiac fibroblasts (CFs) provide important electrical and mechanical functions, but to date have not been sufficiently studied for their role in angiogenesis. To study CFs role in angiogenesis, we co-cultured different concentrations of various cell types in fibrin hemispheres with appropriate combinations of their specific media, to determine the optimal conditions for EC growth and sprout formation through DNA analysis, flow cytometry and immunohistology. ECs proliferated best when co-cultured with CFs and analysis of immunohistological images demonstrated that ECs formed the longest and most numerous sprouts with CFs as compared to MSCs. However, ECs were able to produce more multicellular sprouts when in culture with the MSCs. Moreover, these effects were dependent on the ratio of support cell to EC in co-culture. Overall, CFs provide a good support system for EC proliferation and sprout formation; however, MSCs allow for more multicellular sprouts, which is more indicative of the in vivo process.

/pdf/cardiac-fibroblasts-support-endothelial-cell-proliferation-3g6l4no454.pdf

Cardiac Fibroblasts Support Endothelial Cell Proliferation and Sprout Formation but not the Development of Multicellular Sprouts in a Fibrin Gel Co-Culture Model

Cardiomyocytes (CMs) undergo a rapid transition from hyperplastic to hypertrophic growth soon after birth, which is a major challenge to the development of engineered cardiac tissue for pediatric patients. Resting membrane potential (Vmem) has been shown to play an important role in cell differentiation and proliferation during development. We hypothesized that depolarization of neonatal CMs would stimulate or maintain CM proliferation in vitro. To test our hypothesis, we isolated postnatal day 3 neonatal rat CMs and subjected them to sustained depolarization via the addition of potassium gluconate or Ouabain to the culture medium. Cell density and CM percentage measurements demonstrated an increase in mitotic CMs along with a ~twofold increase in CM numbers with depolarization. In addition, depolarization led to an increase in cells in G2 and S phase, indicating increased proliferation, as measured by flow cytometry. Surprisingly depolarization of Vmem with either treatment led to inhibition of proliferation in cardiac fibroblasts. This effect is abrogated when the study was carried out on postnatal day 7 neonatal CMs, which are less proliferative, indicating that the likely mechanism of depolarization is the maintenance of the proliferating CM population. In summary, our findings suggest that depolarization maintains postnatal CM proliferation and may be a novel approach to encourage growth of engineered tissue and cardiac regeneration in pediatric patients.

/pdf/depolarization-of-cellular-resting-membrane-potential-vcazpf7e2x.pdf

Lauren D. Black

Papers

Optical metrics of the extracellular matrix predict compositional and mechanical changes after myocardial infarction.

Beta 1 integrin binding plays a role in the constant traction force generation in response to varying stiffness for cells grown on mature cardiac extracellular matrix

Differential effects of static and cyclic stretching during elastase digestion on the mechanical properties of extracellular matrices

Cardiac Fibroblasts Support Endothelial Cell Proliferation and Sprout Formation but not the Development of Multicellular Sprouts in a Fibrin Gel Co-Culture Model

Depolarization of Cellular Resting Membrane Potential Promotes Neonatal Cardiomyocyte Proliferation In Vitro