John M. Freund

Quantification of DNA in Biologic Scaffold Materials

Biological scaffold materials composed of extracellular matrix (ECM) are routinely used for a variety of clinical applications ranging from the treatment of chronic skin ulcers to hernia repair and orthopaedic soft tissue reconstruction. The tissues and species from which the ECM is harvested vary widely as do the methods used to remove the cellular component of the source tissues. The efficacy of decellularization procedures can be quantified by examination of the DNA that remains in the ECM. The objective of the present study was to determine the DNA content and fragment length in both laboratory produced and commercially available ECM scaffold materials. Results showed that the majority of DNA is removed from ECM devices but that small amounts remained in most tested materials.

The Effects of Processing Methods upon Mechanical and Biologic Properties of Porcine Dermal Extracellular Matrix Scaffolds

Biologic materials from various species and tissues are commonly used as surgical meshes or scaffolds for tissue reconstruction. Extracellular matrix (ECM) represents the secreted product of the cells comprising each tissue and organ, and therefore provides a unique biologic material for selected regenerative medicine applications. Minimal disruption of ECM ultrastructure and content during tissue processing is typically desirable. The objective of this study was to systematically evaluate effects of commonly used tissue processing steps upon porcine dermal ECM scaffold composition, mechanical properties, and cytocompatibility. Processing steps evaluated included liming and hot water sanitation, trypsin/SDS/TritonX-100 decellularization, and trypsin/TritonX-100 decellularization. Liming decreased the growth factor and glycosaminoglycan content, the mechanical strength, and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for all). Hot water sanitation treatment decreased only the growth factor content of the ECM (p ≤ 0.05). Trypsin/SDS/TritonX-100 decellularization decreased the growth factor content and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). Trypsin/Triton X-100 decellularization also decreased the growth factor content of the ECM but increased the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). We conclude that processing steps evaluated in the present study affect content, mechanical strength, and/or cytocompatibility of the resultant porcine dermal ECM, and therefore care must be taken in choosing appropriate processing steps to maintain the beneficial effects of ECM in biologic scaffolds.

Polypropylene Surgical Mesh Coated with Extracellular Matrix Mitigates the Host Foreign Body Response

Surgical mesh devices composed of synthetic materials are commonly used for ventral hernia repair. These materials provide robust mechanical strength and are quickly incorporated into host tissue; factors that contribute to reduced hernia recurrence rates. However, such mesh devices cause a foreign body response with the associated complications of fibrosis and patient discomfort. In contrast, surgical mesh devices composed of naturally occurring extracellular matrix (ECM) are associated with constructive tissue remodeling, but lack the mechanical strength of synthetic materials. A method for applying a porcine dermal ECM hydrogel coating to a polypropylene mesh is described herein with the associated effects upon the host tissue response and biaxial mechanical behavior. Uncoated and ECM coated heavy-weight BARD™ Mesh were compared to the light-weight ULTRAPRO™ and BARD™ Soft Mesh devices in a rat partial thickness abdominal defect overlay model. The ECM coated mesh attenuated the pro-inflammatory response compared to all other devices, with a reduced cell accumulation and fewer foreign body giant cells. The ECM coating degraded by 35 days, and was replaced with loose connective tissue compared to the dense collagenous tissue associated with the uncoated polypropylene mesh device. Biaxial mechanical characterization showed that all of the mesh devices were of similar isotropic stiffness. Upon explanation, the light-weight mesh devices were more compliant than the coated or uncoated heavy-weight devices. This study shows that an ECM coating alters the default host response to a polypropylene mesh, but not the mechanical properties in an acute in vivo abdominal repair model.

Refinement of Technique in Injection Lipolysis Based on Scientific Studies and Clinical Evaluation

The unusual evolution of the practice of injection lipolysis has generated doubt regarding its safety and efficacy among many physicians. During the early years of this decade, mesotherapy was practiced by a few physicians, but the practice was not widespread. Paramedical practitioners and business developers saw the market potential for nonsurgical fat reduction, and the practice of injection lipolysis was packaged and sold before the mechanism of action was understood. Because of the early lack of scientific research and understanding of the limitations of injection lipolysis, many unsuitable patients were treated with this modality. To better understand the way injection lipolysis works, the inclusion and exclusion criteria for patients desiring treatment, and an accurate clinical evaluation format for potential treatment regions, a series of scientific studies was performed in 2007 and early 2008. These studies included a serial histopathology evaluation of treated patients over time, a stem cell study performed with the McGowan Research Institute in Pittsburgh, an animal study performed in conjunction with the Colorado State University veterinary school, and a prospective multicenter clinical trial using injection lipolysis in the back roll region. The purpose of these studies was to determine the way injection lipolysis works, how modifications of the formula and technique change the outcome, the role of each constituent component of various formulas, and the degree of fat reduction and skin retraction that is attainable with these treatments. The influence of depth of injection, distance between injection points, volume of injection, and ratios of constituent components was studied. The degree of topographic contour correction and the amount of volume reduction were evaluated. Following a review of these recent studies, an updated recommendation for the clinical practice of injection lipolysis was formulated.

Protein kinase C inhibition by UCN-01 induces apoptosis in human glioma cells in a time-dependent fashion

Recent studies in our laboratory have shown that UCN-01 (7-hydroxystaurosporine), which is a derivative of the non-selective protein kinase inhibitor staurosporine that exhibits relative selectivity for protein kinase C (PKC), is a potent inhibitor of glioma growth in in vitro and in vivo models. This agent exhibits both cytotoxic and cytostatic effects, depending on the time period of drug exposure. In the present study, we examined whether UCN-01-induced cytotoxicity correlated with the induction of apoptosis, and characterized further the time course of this process as a prelude to application of UCN-01 in clinical trials. We first demonstrated that the cytotoxic effects of UCN-01 were associated with the induction of morphological features of apoptosis. Secondly. we identified electrophoretic features of apoptosis semiquantitatively at a series of time points using field inversion gel electrophoresis. These studies showed a peak in the induction of high-molecular-weight DNA fragmentation after 3–6 days of drug treatment. Thirdly, we measured the percentage of cells undergoing apoptosis at various time points using a terminal transferase-catalyzed in situ end-labeling technique, which confirmed a time- and concentration-dependent increase in apoptotic cell numbers. This correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. Cell killing peaked within 2–4 days after beginning UCN-01 treatment, but continued at a lower level in the ensuing days. Taken together, these studies demonstrated that extended periods of exposure to UCN-01 are needed for optimal manifestation of cytotoxic effects against glioma cells, a factor that must be taken into consideration in the design of future clinical trials with this agent for malignant gliomas.