Gustavo A. Higuera

Development and analysis of multi-layer scaffolds for tissue engineering

The development of 3D scaffolds consisting of stacked multi-layered porous sheets featuring microchannels is proposed and investigated in this work. In this concept, the inner-porosity of the sheets allows diffusion of nutrients and signalling products between the layers whereas the microchannels facilitate nutrient supply on all layers as they provide space for the culture medium to be perfused throughout the scaffold. Besides the above, these scaffolds have excellent distribution of the cells as seeding and attaching of the cells occurs on individual layers that are subsequently stacked. In addition, these scaffolds enable gaining local data from within the scaffolds as unstacking of the stacked layers allows for determination of various parameters per layer. Here, we show the proof of this concept by culturing C2C12 pre-myoblasts and A4-4 cells on stacked Poly(l-lactic acid) (PLLA) sheets featuring microchannels. The results obtained for culturing under static conditions clearly indicate that despite inhibited cell proliferation due to nutrient limitations, diffusion between the layers takes place and cells on various layers stay viable and also affect each other. Under dynamic conditions, medium flow through the channels improves nutrient availability to the cells on the various layers, drastically increasing cell proliferation on all layers.

Ultraviolet light crosslinking of poly(trimethylene carbonate) for elastomeric tissue engineering scaffolds

A practical method of photocrosslinking high molecular weight poly(trimethylene carbonate)(PTMC) is presented. Flexible, elastomeric and biodegradable networks could be readily prepared by UV irradiating PTMC films containing pentaerythritol triacrylate (PETA) and a photoinitiator. The network characteristics, mechanical properties, wettability, and in vitro enzymatic erosion of the photocrosslinked PTMC films were investigated. Densely crosslinked networks with gel contents up to 98% could be obtained in this manner. Upon photocrosslinking, flexible and tough networks with excellent elastomeric properties were obtained. To illustrate the ease with which the properties of the networks can be tailored, blends of PTMC with mPEG-PTMC or with PTMC-PCL-PTMC were also photocrosslinked. The wettability and the enzymatic erosion rate of the networks could be tuned by blending with block copolymers. Tissue engineering scaffolds were also fabricated using these flexible photocrosslinkable materials. After crosslinking, the fabricated PTMC-based scaffolds showed inter-connected pores and extensive microporosity. Human mesenchymal stem cell (hMSC) culturing studies showed that the photocrosslinked scaffolds prepared from PTMC and PTMC/PTMC-PCL-PTMC blends are well-suited for tissue engineering applications.

Integration of hollow fiber membranes improves nutrient supply in three-dimensional tissue constructs

Sufficient nutrient and oxygen transport is a potent modulator of cell proliferation in in vitro tissue-engineered constructs. The lack of oxygen and culture medium can create a potentially lethal environment and limit cellular metabolic activity and growth. Diffusion through scaffold and multi-cellular tissue typically limits transport in vitro, leading to potential hypoxic regions and reduction in the viable tissue thickness. For the in vitro generation of clinically relevant tissue-engineered grafts, current nutrient diffusion limitations should be addressed. Major approaches to overcoming these include culture with bioreactors, scaffolds with artificial microvasculature, oxygen carriers and pre-vascularization of the engineered tissues. This study focuses on the development and utilization of a new perfusion culture system to provide adequate nutrient delivery to cells within large three-dimensional (3D) scaffolds. Perfusion of oxygenated culture medium through porous hollow fiber (HF) integrated within 3D free form fabricated (FFF) scaffolds is proposed. Mouse pre-myoblast (C2C12) cells cultured on scaffolds of poly(ethylene-oxide-terephthalate)-poly(butylene-terephthalate) block copolymer (300PEOT55PBT45) integrated with porous HF membranes of modified poly(ether-sulfone) (mPES, Gambro GmbH) is used as a model system. Various parameters such as fiber transport properties, fiber spacing within a scaffold and medium flow conditions are optimized. The results show that four HF membranes integrated with the scaffold significantly improve the cell density and cell distribution. This study provides a basis for the development of a new HF perfusion culture methodology to overcome the limitations of nutrient diffusion in the culture of large 3D tissue constructs.

The effect of perfluorocarbon-based artificial oxygen carriers on tissue-engineered trachea.

The biological effect of the perfluorocarbon-based artificial oxygen carrier (Oxygent) was investigated in tissue-engineered trachea (TET) construction. Media supplemented with and without 10% Oxygent were compared in all assessments. Partial tissue oxygen tension (PtO(2)) was measured with polarographic microprobes; epithelial metabolism was monitored by microdialysis inside the TET epithelium perfused with the medium underneath. Chondrocyte-DegraPol constructs were cultured for 1 month with the medium before glycosaminoglycan assessment and histology. Tissue reaction of TET epithelial scaffolds immersed with the medium was evaluated on the chick embryo chorioallantoic membrane. Oxygent perfusion medium increased the TET epithelial PtO(2) (51.2 +/- 0.3 mm Hg vs. 33.4 +/- 0.3 mm Hg at 200 microm thickness; 12.5 +/- 0.1 mm Hg vs. 3.1 +/- 0.1 mm Hg at 400 microm thickness, p < 0.01) and decreased the lactate concentration (0.63 +/- 0.08 vs. 0.80 +/- 0.06 mmol/L, p < 0.05), lactate/pyruvate (1.87 +/- 0.26 vs. 3.36 +/- 10.13, p < 0.05), and lactate/glucose ratios (0.10 +/- 0.00 vs. 0.29 +/- 0.14, p < 0.05). Chondrocyte-DegraPol in Oxygent group presented lower glycosaminoglycan value (0.03 +/- 0.00 vs. 0.13 +/- 0.00, p < 0.05); histology slides showed poor acid mucopolysaccharides formation. Orthogonal polarization spectral imaging showed no difference in functional capillary density between the scaffolds cultured on chorioallantoic membranes. The foreign body reaction was similar in both groups. We conclude that Oxygent increases TET epithelial PtO(2), improves epithelial metabolism, does not impair angiogenesis, and tends to slow cartilage tissue formation.

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 (k1) 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.

The physics of tissue formation with mesenchymal stem cells

Cells react to various forms of physical phenomena that promote and maintain the formation of tissues. The best example of this are cells of musculoskeletal origin, such as mesenchymal stem cells (MSCs), which consistently proliferate or differentiate under cues from hydrostatic pressure, diffusive mass transport, shear stress, surface chemistry, mechanotransduction, and molecular kinetics. To date, no other cell type shows greater receptiveness to macroscopic and microscopic cues, highlighting the acute sensitivity of MSCs and the importance of physical principles in tissue homeostasis. In this review, we describe the literature that has shown how physical phenomena govern MSCs biology and provide insight into the mechanisms and strategies that can spur new biotechnological applications with tissue biology.

Supporting data of spatiotemporal proliferation of human stromal cells adjusts to nutrient availability and leads to stanniocalcin-1 expression in vitro and in vivo

This data article contains seven figures and two tables supporting the research article entitled: spatiotemporal proliferation of human stromal cells adjusts to nutrient availability and leads to stanniocalcin-1 expression in vitro and in vivo[1]. The data explain the culture of stromal cells in vitro in three culture systems: discs, scaffolds and scaffolds in a perfusion bioreactor system. Also, quantification of extracellular matrix components (ECM) in vitro and staining of ECM components in vivo can be found here. Finally the quantification of blood vessels dimensions from CD31 signals and representative histograms of stanniocalcin-1 fluorescent signals in negative controls and experimental conditions in vivo are presented.

420 A Platform of Porous Biomaterials as 3D Culture Systems for Cancer Biology

Background: The role of stem cells in tissue development and repair is beginning to be unravelled and will open remarkable opportunities to improve current medical treatments. Yet, the cascade of events that enable us to distinguish between abnormal and functional tissue morphogenesis is not well known and the potential involvement of stem cells in cancer initiation or tissue regeneration is still at an embryonic stage. Most biological studies rely on culturing cells onto two-dimensional (2D) substrates, which poorly reflect the three-dimensional (3D) environment that governs the physical, chemical, and biological processes at the heart of tissue development. Here, we introduce a library of 3D culture systems − scaffolds − with enhanced cell-material interactions. Materials and Methods: 3D scaffolds made of biodegradable synthetic polymers were fabricated by either rapid prototyping, eletrospinning and their combination. Mesenchymal stem cells derived from bone marrow were isolated from patients after informed consent, seeded on the scaffolds and cultured for up to 35 days. Cell morphology was observed by scanning electron microscopy. Cell number and metabolic activity were quantified by DNA and alamar blue assays. Differentiation was assessed by gene expression, while extrfacellular matrix (ECM) formation by biochemical assays. Results: 3D scaffolds with tailored mechanical and physichochemical properties could be fabricated by different processing technologies. While rapid prototyping resulted in the fabrication of scaffolds with controlled porosity at the macro scale, electrospinning enabled the creation of fibrillar meshes mimicking the physical micro and nano scale dimensions of native ECM. Nutrient availability had a profound effect on tissue formation in 2D and 3D. Despite steep nutrient concentration gradients in 3D scaffolds, stem cells proliferated while avoiding significant death. Cell migration into millimeter-size circular patterns in the scaffold’s pores was supported by ECM organization. Higher concentrations of nutrients controlled the rates of proliferation and did not induce differentiation markers. Furthermore, scaffolds with customized physicochemical and surface properties influenced stem cell morphology and activity. Conclusions: These 3D scaffolds offer a new platform to study the mechanisms behind stem cell driven tissue morphogenesis and may play a role in cancer biology research to create organotypic 3D models to study cancer initiation and development, as well as the potential involvement of stem cells in these processes.