Timothy P. Quinn

Nondestructive evaluation of a new hydrolytically degradable and photo-clickable PEG hydrogel for cartilage tissue engineering.

UNLABELLED Photopolymerizable and hydrolytically labile poly(ethylene glycol) (PEG) hydrogels formed from photo-clickable reactions were investigated as cell delivery platforms for cartilage tissue engineering (TE). PEG hydrogels were formed from thiol-norbornene PEG macromers whereby the crosslinks contained caprolactone segments with hydrolytically labile ester linkages. Juvenile bovine chondrocytes encapsulated in the hydrogels were cultured for up to four weeks and assessed biochemically and histologically, using standard destructive assays, and for mechanical and ultrasound properties, as nondestructive assays. Bulk degradation of acellular hydrogels was confirmed by a decrease in compressive modulus and an increase in mass swelling ratio over time. Chondrocytes deposited increasing amounts of sulfated glycosaminoglycans and collagens in the hydrogels with time. Spatially, collagen type II and aggrecan were present in the neotissue with formation of a territorial matrix beginning at day 21. Nondestructive measurements revealed an 8-fold increase in compressive modulus from days 7 to 28, which correlated with total collagen content. Ultrasound measurements revealed changes in the constructs over time, which differed from the mechanical properties, and appeared to correlate with ECM structure and organization shown by immunohistochemical analysis. Overall, non-destructive and destructive measurements show that this new hydrolytically degradable PEG hydrogel is promising for cartilage TE. STATEMENT OF SIGNIFICANCE Designing synthetic hydrogels whose degradation matches tissue growth is critical to maintaining mechanical integrity as the hydrogel degrades and new tissue forms, but is challenging due to the nature of the hydrogel crosslinks that inhibit diffusion of tissue matrix molecules. This study details a promising, new, photo-clickable and synthetic hydrogel whose degradation supports cartilaginous tissue matrix growth leading to the formation of a territorial matrix, concomitant with an increase in mechanical properties. Nondestructive assays based on mechanical and ultrasonic properties were also investigated using a novel instrument and found to correlate with matrix deposition and evolution. In sum, this study presents a new hydrogel platform combined with nondestructive assessments, which together have potential for in vitro cartilage tissue engineering.

Pulsed-high intensity focused ultrasound (HIFU) exposures for enhanced delivery of therapeutics: Mechanisms and applications

The majority of focused ultrasound applications today involve long, continuous exposures that produce significant temperature elevations for tissue ablation and irreversible coagulative necrosis. Comparatively little has been done with non‐continuous (or, pulsed) exposures that can produce primarily mechanical effects with only minimal heat. Our investigations have shown that pulsed‐HIFU exposures can non‐invasively and non‐destructively enhance the delivery of both systemically and locally injected materials (e.g. imaging agents, optical probes, and plasmid DNA) in both normal and cancerous tissues. It is hypothesized that the enhancing effects are directly linked to tissue displacement from locally‐generated radiation forces. In normal tissue, it is thought that shear forces are produced between adjacent tissue regions experiencing non‐uniform displacement. The resulting strain opens cellular junctions in both the vasculature and the parenchyma, increasing extravasation and interstitial diffusion, respe...

An Instrumented Bioreactor for Mechanical Stimulation and Real-Time, Nondestructive Evaluation of Engineered Cartilage Tissue

Functional tissue engineering involves the application of physical loads to promote the development of tissue constructs that can withstand the mechanical demands encountered in vivo [1]. Specifically, the goal of functional tissue engineering of articular cartilage is to develop an engineered cartilage construct that exhibits structure and function sufficient to replace or repair damaged articular cartilage. To accomplish this goal, bioreactors have been developed to apply mechanical stimulation to cell-laden constructs. Design strategies may impart various types of load including hydrostatic pressure, compression, or shear [2–5]. However, few bioreactors include instrumentation that allow for continuous monitoring of tissue development. The successful in vitro development of functional tissue-engineered constructs could benefit from a method of assessment that allows for continuous evaluation of tissue while not compromising construct integrity, preserving the construct for continuous development and eventual implantation. Current methods for evaluating extracellular matrix (ECM) development and mechanical properties are time consuming and destructive to the construct, and require numerous replicates to obtain a comprehensive overview of construct quality. Nondestructive, continuous evaluation of a tissue construct during development can be useful not only for final clinical use, but also for determining appropriate bioreactor parameters to achieve sufficient structure and function. Nondestructive measurement systems have been developed to assess construct mechanical properties as well as bulk-tissue development [6,7]. Preiss-Bloom et al. developed a bioreactor to mechanically stimulate tissue-engineered cartilage and measure real-time force response [6]. The bioreactor was outfitted with load sensors to continuously log construct resistance to deformation by the bioreactor. Such measurements give insight into the change in construct stiffness during stimulation and development in the bioreactor. Hagenmuller et al. developed a bioreactor that combines mechanical loading and online microcomputed tomography (μCT) for monitoring the development of engineered bone tissue [7]. Cartridge-like culture chambers were designed to allow for sterile mechanical stimulation and μCT monitoring of mineral deposition without removing the constructs. Another potential method for nondestructive assessment of tissue formation is ultrasound. Ultrasonic techniques are sensitive to mechanical and biochemical properties of cartilage [8–10] and have the potential to nondestructively assess the quality of tissue-engineered cartilage during development. Ultrasonic waves are utilized to acquire acoustic images and make localized quantitative measurements of tissue properties. Propagation and scattering of ultrasonic waves depend on tissue composition and structure [11]. Specifically, the reflection coefficient, the fraction of ultrasound reflected from an interface with different acoustic impedances, is one parameter commonly used to evaluate tissue characteristics [12–16]. A number of studies have been conducted to examine the feasibility of ultrasound as a tool for diagnosis of osteoarthritis by measuring changes in ultrasonic parameters after spontaneous and selective enzymatic degradation of cartilage tissue [17–20]. Ultrasound has also been used as a tool for monitoring in vivo cartilage tissue development and repair [21–23]. However, ultrasound has only recently been used as a measurement tool for the evaluation of tissue-engineered cartilage [8,24] and has yet to be implemented for real-time evaluation of tissue development. The objective of this work was to develop an instrumented bioreactor that could be utilized to stimulate and nondestructively evaluate tissue-engineered cartilage. Our dynamic compression bioreactor is instrumented with an ultrasonic transducer, load cells, and a video microscope for assessing ECM development and mechanical properties of tissue-engineered cartilage. Chondrocyte-laden hydrogel constructs were placed in the bioreactor and subjected to a three-part loading regime including: (1) a ramp, (2) sinusoidal compression, and (3) no load. This regime was repeated twice per day for 7 days. Constructs were nondestructively evaluated with ultrasound on days 0 and 7. Constructs were also evaluated on days 0 and 7 for cell viability, cell number, sulfated glycosaminoglycan (sGAG), and collagen content. Histological sections were stained for sGAG and collagen with safranin O and Massons trichrome, respectively.

A Multi-Phasic, Continuum Damage Mechanics Model of Mechanically Induced Increased Permeability in Tissues

Recently, we have reported enhanced permeability of tissues due to in vivo treatment with pulsed high intensity focused ultrasound (pHIFU). This new therapy has shown promise as a way of increasing the penetration of large drug molecules, both out of the vasculature and through the tissue. To date, no clear physical model of tissue exists that can account for these effects. A new model is proposed that clearly establishes the link between tissue structure and fluid flow properties on one hand, and the history of applied mechanical forces on the other. The model draws inspiration from two different theoretical fields of materials science, multi-phase theory and continuum damage mechanics. The theory differs from the traditional bi-phasic solid-fluid model of tissues in that the fluid part here is broken into trapped (moving with the solid) and free (moving through the solid) parts. A damage-like variable links the effective elasticity of the tissue to the ratio of the trapped to free fluids. As the damage increases, the tissue becomes, in effect, less stiff and more permeable. Release of elastic energy drives the process. A distribution of energy barriers opposes the process and governs how the fluid is released as damage increases.

2A-2 Investigations into the Potential Contribution of a Thermal Mechanism for Pulsed High Intensity Focused Ultrasound Mediated Delivery

The mechanism behind pulsed high intensity focused ultrasound (pHIFU) effects leading to increased drug delivery is currently poorly understood. In this work, the thermal dose and peak temperatures associated with a typical pHIFU treatment were measured in mouse muscle. A non-ultrasonic hyperthermia (HT) treatment was then applied, designed to mimic the thermal component of the pHIFU treatment. The delivery of 200 nm fluorescent nanoparticles was measured as a surrogate marker for drug delivery by pHIFU and HT treatments. Only the pHIFU treatment showed a significant increase in particle delivery.

A Confined Compression Technique for Hydraulic Conductivity Measurement in Soft Tissues

Multiphasic tissue models have been used extensively to predict the behavior of cartilaginous tissues [1]. Their application to other soft tissues, however, has often been overlooked. Unlike the more commonly used continuum model of the viscoelastic solid [2], multiphasic models allow us to infer the behaviors and properties of tissue subcomponents by observing the behavior of the tissue whole. As a great deal of tissue function and structure is related to the control and transport of fluids and fluid-borne agents, there is clearly a need for this insight in all tissues. For example, there has been a great deal of interest recently in the possibility of modifying the flow properties of solid tumors and other tissues to allow the targeted delivery of large molecular weight drugs, such as chemotherapeutic or genetic agents [3–4]. It is well known that the high interstitial fluid pressures, confused vasculature, and lack of a lymphatic system prevent the effective distribution of directly injected or systemically administered drugs into tumors [3]. Increasing the effective permeability of these tumors can ameliorate these issues and allow for more effective treatment. A handful of studies have found that the biphasic model, along with some basic experimental tools, can reasonably represent the flow properties of tumors [4–5]. In this paper, we describe a technique using a simple confined compression experiment with the biphasic model to measure the hydraulic conductivity of samples of cardiac tissue.© 2007 ASME