A DNA controlled-release coating for gene transfer: Transfection in skeletal and cardiac muscle

In this paper we report a novel technique of DNA-polymer coating for gene transfer. A proprietary DNA polymer solution was used for thin-layer coating on a chromic gut suture as a model study. The coated sutures were characterized for physical properties such as coating thickness, mass of the DNA deposited on the suture, surface characteristics as determined by scanning electron microscopy, and in vitro DNA release characteristics under simulated physiologic conditions. The in vivo gene transfection using DNA-coated sutures was demonstrated in rat skeletal muscle and in canine atrial myocardium. A heat-stable human placental alkaline phosphatase (AP) plasmid was used as a marker gene. Incisions of 1 to 1.5 cm were made in the rat skeletal muscles or the canine atrial myocardium. The sites were closed with either the DNA-coated sutures or control sutures. Two weeks after the surgery, the tissue samples adjacent to the suture lines were retrieved and analyzed for AP activity. The DNA-coated sutures demonstrated a sustained release of the DNA under in vitro conditions, with an ∼84% cumulative DNA release occurring in 26 days. An agarose gel electrophoresis of the DNA samples released from the suture demonstrated two bands, with the lower band corresponding to the input DNA (supercoiled). It seems that there was a partial transformation of the DNA from a supercoiled to an open circular form due to the polymer coating. The tissue sites, which received the DNA-coated sutures, demonstrated a significantly higher AP activity compared with the tissue sites that received control sutures. In the rat studies, the mean AP activity (square root of cpm/ µg protein) was 43.6 ± 3.3 vs 20.6 ± 2.1 ( p = 0.001) at the control sites. Similarly, in the canine studies, the AP activity was 73.6 ± 7.4 Vs 21.6 ± 1.4 ( p = 0.0009) at the control sites. Thus, our studies demonstrated a successful gene transfer using our DNA-polymer coating technique. This technique could be useful for coating sutures used in vascular and general surgery, and also for coating medical devices, such as stents, catheters, or orthopedic devices, to achieve a site-specific gene delivery.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34492/1/14_ftp.pd

A non-clotting and non-instrumental method for sensing heparin and monitoring of extracorporeal anticoagulation.

A non-clotting and non-instrumentally based heparin assay was developed. This was achieved by studying the immobilization of protamine (a heparin antidote) on a porous filter paper strip; and the subsequent migration of a heparin sample through the paper in a descending manner. The area of paper to which heparin was adsorbed would be proportional to the heparin level in the sample. The bound heparin was visualized by spraying Methylene Blue NNX (a dye that interacts with heparin causing a metachromatic shift of the dye's absorption maximum from blue to purple) solution onto the paper strip upon the exhaustion of the sample reservoir. Heparin levels in the samples could be estimated according to the length of the purple region on the paper strip. The sensitivity, resolution and time requirement could be properly adjusted by varying the amount of protamine immobilized on paper, the sample volume applied and the type of paper used. This system was shown to be capable of accurately detecting heparin, at all relevant levels in aqueous sample in a simple and time efficient manner. Interference and long assay times were encountered when this system was applied to untreated plasma heparin samples; the accuracy of the assay was also significantly decreased. Subsequent investigations indicated that the plasma components sodium chloride and globulin were primarily responsible for the interference; whereas the high viscosity albumin imparted to plasma was accountable for the long assay time needed. The types of filter papers used (Whatman grade 541 and 113) contributed to difficulties in applying the sensing system to detect heparin in plasma, particularly at low levels. A modified "standard addition" method of sample treatment was devised in which plasma specimens were diluted with aqueous heparin solution of known concentration in order to lessen the interferences of plasma components. The heparin levels determined by applying treated plasma samples to the grade 31ET Chr paper strips were in agreement with their counterparts derived by plasma APTT clotting assay.Ph.D.PharmaceuticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105502/1/9135567.pdfDescription of 9135567.pdf : Restricted to UM users only

Synergistic inhibition of calcification of porcine aortic root with preincubation in FeCl 3 and α-amino oleic acid in a rat subdermal model

Postimplant calcific degeneration is a frequent cause of clinical failure of glutaraldehyde crosslinked porcine aortic valve bioprostheses. We demonstrated previously in rat subdermal and circulatory implants that α-amino oleic acid used as a bioprosthesis pretreatment was highly effective in mitigating aortic valve cusp but not aortic wall calcification. In this study we investigated the feasibility of synergistically applying two proven anticalcification agents (α-amino oleic acid and FeCl 3 ) as pretreatments for mitigating both bioprosthetic cusp and aortic wall calcification. α-Amino oleic acid is hypothesized to prevent calcification by disrupting calcium phosphate formation kinetics, whereas suppression of alkaline phosphatase activity and ferric-phosphate complexation at cellular membrane initiation sites may be important factors in ferric ion's inhibition of calcification. In vivo implant studies (21-day rat subdermal model) indicated that individually FeCl 3 (0.01 or 0.1 M for 24 h) or α-amino oleic acid (saturated solution) treatments were equally effective in mitigating cuspal calcification (tissue calcium levels: 30.2 ± 10.2, 29.8 ± 2.7, and 31.6 ± 7.8 μg/mg tissue, respectively). However, sequential application of first α-amino oleic acid and then FeCl 3 synergistically reduced aortic wall calcification more effectively than either of the agents alone. The benefit of a synergistic application of two anticalcification treatments, α-amino oleic acid and FeCl 3 , was demonstrated. However, the synergistic effect was observed on aortic wall only at a higher FeCl 3 concentration (i.e., 0.1 M ). © 1997 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 38: 43–48, 1997Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38015/1/6_ftp.pd

Mesenchymal stem cell therapy and delivery systems in nonhealing wounds

The objective of the study was to inform wound care practitioners of mesenchymal stem cell application for nonhealing wounds. Recent advances in delivery systems are also discussed in order to highlight potential improvements toward clinical application of stem cell therapy for chronic wounds. MEDLINE and PubMed Central were searched for scientific studies regarding the use of mesenchymal stem cells and delivery systems in wound healing. Preclinical studies using stem cells as therapeutic modality for chronic wounds were selected for this review. Information on study design, sample size and characteristics, stem cell source, type of delivery systems, and rate and time of wound closure was abstracted. Application of mesenchymal stem cells improved wound healing in experimental and clinical settings. Advances in stem cell therapy and delivery vehicles offer promising alternatives to current limited therapeutic modalities for chronic wounds. Stem cell therapy has recently emerged as a promising therapeutic strategy for nonhealing wounds. Further research is needed to evaluate the relationship between the various delivery systems and stem cells in order to maximize their therapeutic effects. Development of novel delivery vehicles for stem cells can open new opportunities for more effective cell therapy of chronic wounds

The role of stem cells in the treatment of diabetic foot ulcers

Diabetic foot ulcers (DFUs) are a significant and rapidly growing complication of diabetes and its effects on wound healing. Over half of diabetic patients who develop a single ulcer will subsequently develop another ulcer of which the majority will become chronic non-healing ulcers. One-third will progress to lower extremity amputation. Over the past decade, the outcomes for patients with DFUs ulcers have not improved, despite advances in wound care. Successful treatment of diabetic foot ulcers is hindered by the lack of targeted therapy that hones in on the healing processes dysregulated by diabetes. Stem cells are a promising treatment for DFUs as they are capable of targeting, as well as bypassing, the underlying abnormal healing mechanisms and deranged cell signaling in diabetic wounds and promote healing. This review will focus on existing stem cell technologies and their application in the treatment of DFUs