Michael R. Neidert

Fiber alignment imaging during mechanical testing of soft tissues

AbstractA method to image fiber alignment during mechanical testing of soft tissues was developed based on quantitative polarized light microscopy. Images were acquired after passing light through a rotating polarizer, a tissue sample, and an effective circular analyzer at multiple polarizer positions during uniaxial mechanical testing. The image set was analyzed off-line using harmonic analysis to generate an alignment image, which contains the direction and strength of alignment at each image pixel. Alignment images of the entire tissue sample were generated every 3–5 s during the mechanical test allowing stress-strain behavior to be correlated with fiber alignment. Loading of fresh tissue-equivalent samples in the direction normal to the initial direction of fiber alignment revealed a spatially inhomogeneous realignment into the loading direction, with most realignment occurring near the free edges undergoing maximum lateral contraction and prior to significant load developing. Glutaraldehyde-fixed samples, in contrast, showed little realignment until yielding occurred.

Enhanced fibrin remodeling in vitro with TGF-β1, insulin and plasmin for improved tissue-equivalents

The aim of this study was to better understand how to increase collagen content in and enhance mechanical properties of tissue-equivalents formed by entrapping tissue cells in fibrin gels, with the ultimate goal of developing mechanically robust artificial tissues. The two main areas of focus were cell culture medium composition and replacement frequency relative to a base case of incubating constructs in medium supplemented with just serum and replaced weekly. The optimal condition involved a combination of all three factors investigated-TGF-beta, insulin, plasmin-with medium replacement three times a week. Compared to a base case without these three factors, the combination case resulted in 20 times more collagen and a ten-fold increase in tensile strength. The high strain modulus and tensile strength were within an order of magnitude of cardiovascular tissues. This study indicates great potential for fibrin-based tissue-equivalents in soft connective tissue applications.

Cryopreservation of Collagen-Based Tissue Equivalents. I. Effect of Freezing in the Absence of Cryoprotective Agents

The effect of freezing on the viability and mechanical properties of tissue-equivalents (TEs) was determined under a variety of cooling conditions, with the ultimate aim of optimizing the cryopreservation process. TEs (a class of bioartificial tissues) were prepared by incubating entrapped human foreskin fibroblasts in collagen gels for a period of 2 weeks. TEs were detached from the substrate and frozen in phosphate-buffered saline using a controlled rate freezer (CRF) at various cooling rates (0.5, 2, 5, 20, and 40 degrees C/min to -80 or -160 degrees C) or in a directional solidification stage (DSS) (5 degrees C/min to -80 degrees C) or slam frozen (>1000 degrees C/min). Viability of the fibroblasts in the TEs was assessed by ethidium homodimer and Hoechst assays immediately after thawing. Uniaxial tension experiments were also performed on an MTS (Eden Prairie, MN) Micro Bionix system to assess the postthaw mechanical properties of the frozen-thawed TEs. Cooling rates of either 2 or 5 degrees C/min using the CRF were optimal for preserving both immediate cell viability and mechanical properties of the TEs, postthaw. By 72 h postthaw, TEs frozen in the CRF at 5 degrees C/min to -80 degrees C showed a slight decrease in cell viability, with a significant increase in tangent modulus and ultimate tensile stress suggesting a cell-mediated recovery mechanism. Both the postthaw mechanical properties and cell viability are adversely affected by freezing to the lower end temperature of -160 degrees C. Mechanical properties are adversely affected by freezing in the DSS.

Cryopreservation of Collagen-Based Tissue Equivalents. II. Improved Freezing in the Presence of Cryoprotective Agents

In Part I of this study we determined an optimal cooling rate for cryopreservation of collagen-based tissue equivalents (TEs) that preserves both the postthaw cell viability and mechanical properties, but results in tissue contraction and an overall loss of opacity. The empirically determined optimal cooling rate (5 degrees C/min) was obtained in a freezing medium consisting solely of phosphate-buffered saline (PBS) at physiological concentration (1x). In the present study we report the effect of freezing on TEs in the presence of PBS and two cryoprotective agents (CPAs) (glycerol and dimethyl sulfoxide [Me(2)SO]), at two different concentrations (0.5 and 1.0 M), to two different end temperatures (-80 and -160 degrees C), at a cooling rate of 5 degrees C/min. The controlled rate freezing experiments, postthaw cell viability, and mechanical property measurements were performed as described in Part I of this study. In addition to studying the effect of CPAs on the postthaw properties of TEs, we also investigated (1). the effect of freezing TEs attached to the substrate (as opposed to detached and floating in medium) to determine differences when freezing TEs subject to static mechanical stress via a mechanical constraint to contraction; (2). the effect of freezing glutaraldehyde-fixed TEs to determine differences in freezing-mediated damage to the microstructure; and (3). the effect of freezing more mature TEs that were incubated for 4 weeks in growth factor-supplemented medium as opposed to 2 weeks in basal medium. All TEs frozen at 5 degrees C/min to -80 degrees C in the presence of 0.5 M glycerol or Me(2)SO in PBS were found to be optimally cryopreserved in terms of maintaining opacity and structure as well as cell viability and mechanical properties as compared with unfrozen TEs. The postthaw mechanical properties were adversely affected by freezing to the lower end temperature of -160 degrees C in the presence of CPAs, with the samples frozen in the 1.0 M concentration of CPAs exhibiting a total loss of structural integrity on thawing. Furthermore, TEs frozen attached to the substrate showed decreased opacity and significant contraction as compared with TEs frozen detached from the substrate, as did cross-linked samples frozen without CPA.

Early patient experience with an electro-anatomic navigation system dedicated to device lead implantation: feasibility and safety.

Introduction:  Fluoroscopy‐guided pacing lead placement has well‐recognized limitations and risks. We studied the safety and feasibility of using a novel electromagnetic navigation system specifically designed to guide pacemaker and implantable cardioverter defibrillator lead placement.

Development and characterization of improved tissue engineered valve-equivalents using chemical and mechanical signaling

Tissue engineered valves hold considerable promise as replacement valves that avoid many of the problems present in current replacement valve technology. Furthermore, these valves, as a living construct, would be able to grow and remodel in vivo. We have developed a bileaflet biopolymer-scaffold based valve equivalent that possesses the correct geometry and underlying collagen fibril alignment. These valve-equivalents, however, have significantly worse mechanical properties as compared to healthy, native valves (in terms of ultimate tensile stress and tangent modulus). Furthermore, valve equivalents with initial collagen scaffolds show very little compositional remodeling leaving a predominantly collagen valve with little of the elastin and proteoglycan content present in native valves. We present work here aimed at improving the compositional and mechanical properties of valve-equivalents (VEs) by using a combination of chemical signaling by using a fibrin (as opposed to collagen) scaffold incubated with TGF-/spl beta/ and insulin and mechanical signaling achieved by VE incubation in a bioreactor.