Evaluation of the growth environment of a hydrostatic force bioreactor for preconditioning of tissue-engineered constructs.

Bioreactors have been widely acknowledged as valuable tools to provide a growth environment for engineering tissues and to investigate the effect of physical forces on cells and cell-scaffold constructs. However, evaluation of the bioreactor environment during culture is critical to defining outcomes. In this study, the performance of a hydrostatic force bioreactor was examined by experimental measurements of changes in dissolved oxygen (O2), carbon dioxide (CO2), and pH after mechanical stimulation and the determination of physical forces (pressure and stress) in the bioreactor through mathematical modeling and numerical simulation. To determine the effect of hydrostatic pressure on bone formation, chick femur skeletal cell-seeded hydrogels were subjected to cyclic hydrostatic pressure at 0-270 kPa and 1 Hz for 1 h daily (5 days per week) over a period of 14 days. At the start of mechanical stimulation, dissolved O2 and CO2 in the medium increased and the pH of the medium decreased, but remained within human physiological ranges. Changes in physiological parameters (O2, CO2, and pH) were reversible when medium samples were placed in a standard cell culture incubator. In addition, computational modeling showed that the distribution and magnitude of physical forces depends on the shape and position of the cell-hydrogel constructs in the tissue culture format. Finally, hydrostatic pressure was seen to enhance mineralization of chick femur skeletal cell-seeded hydrogels.

Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and glial-derived neurotrophic factor enhance angiogenesis in a tissue-engineered in vitro model.

Skin is a major source of secretion of the neurotrophic factors nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial-derived neurotrophic factor (GDNF) controlling cutaneous sensory innervation. Beside their neuronal contribution, we hypothesized that neurotrophic factors also modulate the cutaneous microvascular network. First, we showed that NGF, BDNF, NT-3, and GDNF were all expressed in the epidermis, while only NGF and NT-3 were expressed by cultured fibroblasts, and BDNF by human endothelial cells. We demonstrated that these peptides are highly potent angiogenic factors using a human tissue-engineered angiogenesis model. A 40% to 80% increase in the number of capillary-like tubes was observed after the addition of 10 ng/mL of NGF, 0.1 ng/mL of BDNF, 15 ng/mL of NT-3, and 50 ng/mL of GDNF. This is the first characterization of the direct angiogenic effect of NT-3 and GDNF. This angiogenic effect was mediated directly through binding with the neurotrophic factor receptors tropomyosin-receptor kinase A (TrkA), TrkB, GFRα-1 and c-ret that were all expressed by human endothelial cells, while this effect was blocked by addition of the Trk inhibitor K252a. Thus, if NGF, BDNF, NT-3, and GDNF may only moderately regulate the microvascular network in normal skin, they might have the potential to greatly increase angiogenesis in pathological situations.

Human keratinocytes respond to direct current stimulation by increasing intracellular calcium: preferential response of poorly differentiated cells.

A direct current (DC) endogenous electric field (EF) is induced in the wound following skin injury. It is potentially implicated in the wound healing process by attracting cells and altering their phenotypes as indicated by the response to an EF of keratinocytes cultured as individual cells. To better define the signalization induced by a direct current electric field (DCEF) in human keratinocytes, we took advantage of an in vitro model more representative of the in vivo situation since it promotes cell-cell interactions and stratification. Human keratinocytes were grown into colonies. Their exposure to a DCEF of physiological intensity induced an increase of intracellular calcium. This variation of intracellular calcium resulted from an extracellular calcium influx and was mediated, at least in part, by the L-type voltage-gated calcium channel. The increase in intracellular calcium in response to a DCEF was however not observed in all the cells composing the colonies. The intracellular calcium increase was only detected in keratinocytes that didn't express involucrin, a marker of differentiated cells. These results indicate that DCEF is able to induce a specific calcium response in poorly differentiated keratinocytes. This study brings a new perspective for the understanding of the signaling mechanism of endogenous EF in reepithelialization, a critical process during skin wound healing.

Electric Potential Across Epidermis and Its Role During Wound Healing Can Be Studied by Using an In Vitro Reconstructed Human Skin.

BACKGROUND: After human epidermis wounding, transepithelial potential (TEP) present in nonlesional epidermis decreases and induces an endogenous direct current epithelial electric field (EEF) that could be implicated in the wound re-epithelialization. Some studies suggest that exogenous electric stimulation of wounds can stimulate healing, although the mechanisms remain to be determined. THE PROBLEM: Little is known concerning the exact action of the EEF during healing. The mechanism responsible for TEP and EEF is unknown due to the lack of an in vitro model to study this phenomenon. BASIC SCIENCE ADVANCES: We carried out studies by using a wound created in a human tissue-engineered skin and determined that TEP undergoes ascending and decreasing phases during the epithelium formation. The in vitro TEP measurements over time in the wound were corroborated with histological changes and with in vivo TEP variations during porcine skin wound healing. The expression of a crucial element implicated in Na+ transport, Na+/K+ ATPase pumps, was also evaluated at the same time points during the re-epithelialization process. The ascending and decreasing TEP values were correlated with changes in the expression of these pumps. The distribution of Na+/K+ ATPase pumps also varied according to epidermal differentiation. Further, inhibition of the pump activity induced a significant decrease of the TEP and of the re-epithelization rate. CLINICAL CARE RELEVANCE: A better comprehension of the role of EEF could have important future medical applications regarding the treatment of chronic wound healing. CONCLUSION: This study brings a new perspective to understand the formation and restoration of TEP during the cutaneous wound healing process.

Restoration of the transepithelial potential within tissue-engineered human skin in vitro and during the wound healing process in vivo.

Normal human epidermis possesses a transepithelial potential (TEP) that varies in different parts of the body (10­60mV). The role of TEP in normal epidermis is not yet identified; but after skin injury, TEP disruption induces an endogenous direct current electric field (100­200mV/mm) directed toward the middle of the wound. This endogenous electric field could be implicated in the wound healing process by attracting cells, thus facilitating reepithelialization. However, little is known on the restoration of the TEP during human skin formation and wound healing. In this study, the variations in TEP and Na+/K+ ATPase pump expression during the formation of the epithelium were investigated in vitro using human tissue-engineered human skin (TES) reconstituted by tissue engineering and in vivo with a porcine wound healing model. Results showed that TEP undergoes ascending and decreasing phases during epithelium formation in TES as well as during wound repair within TES. Similar results were observed during in vivo reepithelialization of wounds. The ascending and decreasing TEP values were correlated with changes in the expression of Na+/K+ ATPase pump. The distribution of Na+/K+ ATPase pumps also varied according to epidermal differentiation. Taken together, these results suggest that the variations in the expression of Na+/K+ ATPase pump over time and across epidermis would be a determinant parameter of the TEP, dictating a cationic transport during the formation and restoration of the epidermis. Therefore, this study brings a new perspective to understand the formation and restoration of TEP during the cutaneous wound healing process. This might have important future medical applications regarding the treatment of chronic wound healing.

Novel low-power ultrasound digital preprocessing architecture for wireless display.

A complete hardware-based ultrasound preprocessing unit (PPU) is presented as an alternative to available power-hungry devices. Intended to expand the ultrasonic applications, the proposed unit allows replacement of the cable of the ultrasonic probe by a wireless link to transfer data from the probe to a remote monitor. The digital back-end architecture of this PPU is fully pipelined, which permits sampling of ultrasonic signals at a frequency equal to the field-programmable gate array-based system clock, up to 100 MHz. Experimental results show that the proposed processing unit has an excellent performance, an equivalent 53.15 Dhrystone 2.1 MIPS/ MHz (DMIPS/MHz), compared with other software-based architectures that allow a maximum of 1.6 DMIPS/MHz. In addition, an adaptive subsampling method is proposed to operate the pixel compressor, which allows real-time image zooming and, by removing high-frequency noise, the lateral and axial resolutions are enhanced by 25% and 33%, respectively. Realtime images, acquired from a reference phantom, validated the feasibility of the proposed architecture. For a display rate of 15 frames per second, and a 5-MHz single-element piezoelectric transducer, the proposed digital PPU requires a dynamic power of only 242 mW, which represents around 20% of the best-available software-based system. Furthermore, composed by the ultrasound processor and the image interpolation unit, the digital processing core of the PPU presents good power-performance ratios of 26 DMIPS/mW and 43.9 DMIPS/mW at a 20-MHz and 100-MHz sample frequency, respectively.

A novel single-step self-assembly approach for the fabrication of tissue-engineered vascular constructs.

There is a clinical need for a functional tissue-engineered blood vessel because small-caliber arterial graft (<5 mm) applications are limited by the availability of suitable autologous vessels and suboptimal performances of synthetic grafts. This study presents an analysis of the mechanical properties of tissue-engineered vascular constructs produced using a novel single-step self-assembly approach. Briefly, the tissue-engineered vascular media were produced by culturing smooth muscle cell in the presence of sodium l-ascorbate until the formation of a cohesive tissue sheet. This sheet was then rolled around a tubular support to create a media construct. Alternatively, the tissue-engineered vascular adventitia was produced by rolling a tissue sheet obtained from dermal fibroblasts or saphenous vein fibroblasts. The standard self-assembly approach to obtain the two-layer tissue-engineered vascular constructs comprising both media and adventitia constructs consists of two steps in which tissue-engineered vascular media were first rolled on a tubular support and a tissue-engineered vascular adventitia was then rolled on top of the first layer. This study reports an original alternative method for assembling tissue-engineered vascular constructs comprising both media and an adventitia in a single step by rolling a continuous tissue sheet containing both cell types contiguously. This tissue sheet was produced by growing smooth muscle cells alongside fibroblasts (saphenous vein fibroblasts or dermal fibroblasts) in the same culture dish separated by a spacer, which is removed later in the culture period. The mechanical strength assessed by uniaxial tensile testing, burst pressure measurements, and viscoelastic behavior evaluated by stepwise stress relaxation tests reveals that the new single-step fabrication method significantly improves the mechanical properties of tissue-engineered vascular construct for both ultimate tensile strength and all the viscoelastic moduli.

Real-time hand-held ultrasound medical-imaging device based on a new digital quadrature demodulation processor.

A fully hardware-based real-time digital wideband quadrature demodulation processor based on the Hilbert transform is proposed to process ultrasound radio frequency signals. The presented architecture combines 2 finite impulse response (FIR) filters to process in-phase and quadrature signals and includes a piecewise linear approximation architecture that performs the required square root operations. The proposed implementation enables flexibility to support different transducers with its ability to load on-the-fly different FIR filter coefficient sets. The complexity and accuracy of the demodulator processor are analyzed with simulated RF data; a normalized residual sum-of-squares cost function is used for comparison with the Matlab Hilbert function. Three implementations are integrated into a hand-held ultrasound system for experimental accuracy and performance evaluation. Real-time images were acquired from a reference phantom, demonstrating the feasibility of using the presented architecture to perform real-time digital quadrature demodulation of ultrasonic signal echoes. Experimental results show that the implementation, using only 2942 slices and 3 dedicated digital multipliers of a low-cost and low-power field-programmable gate array (FPGA) is accurate relative to a comparable software- based system; axial and lateral resolution of 1 mm and 2 mm, respectively, were obtained with a 12-mm piezoelectric transducer without postprocessing. Because the processing and sampling rates are the same, high-frequency ultrasound signals can be processed as well. For a 15-frame-per-second display, the hand-held ultrasonic imaging-processing core (FPGA, memory) requires only 45 mW (dynamic) when using a 5-MHz single-element piezoelectric transducer.

Tissue engineering of a genitourinary tubular tissue graft resistant to suturing and high internal pressures.

The aim of this study was to evaluate the possibility of constructing a fully autologous tissue-engineered tubular genitourinary graft (TTGG) and to determine its mechanical and physiological properties. Dermal fibroblasts (DFs) were expanded and cultured in vitro with sodium ascorbate to form fibroblast sheets. The sheets were then wrapped around a tubular support to form a cylinder. After maturation, urothelial cells (UCs) were seeded inside the DF tubes, and the constructs were placed in a bioreactor. The TTGGs were then characterized according to histology, immuno-histochemistry, Western blot, cell viability, resistance to suture, and burst pressure. Results obtained were encouraging on all levels. All layers of the TTGGs had merged, and a pluristratified urothelium coated the luminal surface of the tubes. The burst pressure of non-sutured TTGGs was measured and found to be, on average, three times as resistant as that of porcine urethras. Suturing was accomplished without difficulty. Results have shown that our construct can sustain an entire week of pulsatile stimulation without loss of mechanical or histological integrity. The tissue-engineering technique used to produce this model seems promising for bioengineering a urethra or ureter graft and could open a doorway to new possibilities for their reconstruction.