In Vivo Conditioning of Tissue-engineered Heart Muscle Improves Contractile Performance

The ability to engineer cardiac tissue in vitro is limited by the absence of a vasculature. In this study we describe an in vivo model which allows neovascularization of engineered cardiac tissue. Three-dimensional cardiac tissue, termed “cardioids,” was engineered in vitro from the spontaneous delamination of a confluent monolayer of cardiac cells. Cardioids were sutured onto a support framework and then implanted in a subcutaneous pocket in syngeneic recipient rats. Three weeks after implantation, cardioids were recovered for in vitro force testing and histological evaluation. Staining for hematoxylin and eosin demonstrated the presence of viable cells within explanted cardioids. Immunostaining with von Willebrand factor showed the presence of vascularization. Electron micrographs revealed the presence of large amounts of aligned contractile proteins and a high degree of intercellular connectivity. The peak active force increased from an average value of 57 µN for control cardioids to 447 µN for explanted cardioids. There was also a significant increase in the specific force. There was a significant decrease in the time to peak tension and half relaxation time. Explanted cardioids could be electrically paced at frequencies of 1–5 Hz. Explanted cardioids exhibited a sigmoidal response to calcium and positive chronotropy in response to epinephrine. As the field of cardiac tissue engineering progresses, it becomes desirable to engineer larger diameter tissue equivalents and to induce angiogenesis within tissue constructs. This study describes a relatively simple in vivo model, which promotes the neovascularization of tissue-engineered heart muscle and subsequent improvement in contractile performance.  Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74650/1/j.1525-1594.2005.00148.x.pd

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

BACKGROUND: Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. CONCLUSIONS/SIGNIFICANCE: Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria

Heart Valve Tissue Engineering: Concepts, Approaches, Progress, and Challenges

Potential applications of tissue engineering in regenerative medicine range from structural tissues to organs with complex function. This review focuses on the engineering of heart valve tissue, a goal which involves a unique combination of biological, engineering, and technological hurdles. We emphasize basic concepts, approaches and methods, progress made, and remaining challenges. To provide a framework for understanding the enabling scientific principles, we first examine the elements and features of normal heart valve functional structure, biomechanics, development, maturation, remodeling, and response to injury. Following a discussion of the fundamental principles of tissue engineering applicable to heart valves, we examine three approaches to achieving the goal of an engineered tissue heart valve: (1) cell seeding of biodegradable synthetic scaffolds, (2) cell seeding of processed tissue scaffolds, and (3) in-vivo repopulation by circulating endogenous cells of implanted substrates without prior in-vitro cell seeding. Lastly, we analyze challenges to the field and suggest future directions for both preclinical and translational (clinical) studies that will be needed to address key regulatory issues for safety and efficacy of the application of tissue engineering and regenerative approaches to heart valves. Although modest progress has been made toward the goal of a clinically useful tissue engineered heart valve, further success and ultimate human benefit will be dependent upon advances in biodegradable polymers and other scaffolds, cellular manipulation, strategies for rebuilding the extracellular matrix, and techniques to characterize and potentially non-invasively assess the speed and quality of tissue healing and remodeling

Myocardial Engineering in Vivo: Formation and Characterization of Contractile, Vascularized Three-Dimensional Cardiac Tissue

Engineering cardiac tissue in three dimensions is limited by the ability to supply nourishment to the cells in the center of the construct. This limits the radius of an in vitro engineered cardiac construct to approximately 40 µm. This study describes a method of engineering contractile three-dimensional cardiac tissue with the incorporation of an intrinsic vascular supply. Neonatal cardiac myocytes were cultured in vivo in silicone chambers, in close proximity to an intact vascular pedicle. Silicone tubes were filled with a suspension of cardiac myocytes in fibrin gel and surgically placed around the femoral artery and vein of adult rats. At 3 weeks, the tissues in the chambers were harvested for in vitro contractility evaluation and processed for histologic analysis. By 3 weeks, the chambers had become filled with living tissue. Hematoxylin and eosin staining showed large amounts of muscle tissue situated around the femoral vessels. Electron micrographs revealed well-organized intracellular contractile machinery and a high degree of intercellular connectivity. Immunostaining for von Willebrand factor demonstrated neovascularization throughout the constructs. With electrical stimulation, the constructs were able to generate an average active force of 263 µN with a maximum of 843 µN. Electrical pacing was successful at frequencies of 1 to 20 Hz. In addition, the constructs exhibited positive inotropy in response to ionic calcium and positive chronotropy in response to epinephrine. As engineering of cardiac replacement tissue proceeds, vascularization is an increasingly important component in the development of three-dimensional structures. This study demonstrates the in vivo survival, vascularization, organization, and functionality of transplanted myocardial cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63336/1/ten.2005.11.803.pd

Self-organization of rat cardiac cells into contractile 3-D cardiac tissue

The mammalian heart is not known to regenerate following injury. Therefore, there is great interest in developing viable tissue‐based models for cardiac assist. Recent years have brought numerous advances in the development of scaffold‐based models of cardiac tissue, but a self‐organizing model has yet to be described. Here, we report the development of an in vitro cardiac tissue without scaffolding materials in the contractile region. Using an optimal concentration of the adhesion molecule laminin, a confluent layer of neonatal rat cardiomyogenic cells can be induced to self‐organize into a cylindrical construct, resembling a papillary muscle, which we have termed a cardioid. Like endogenous heart tissue, cardioids contract spontaneously and can be electrically paced between 1 and 5 Hz indefinitely without fatigue. These engineered cardiac tissues also show an increased rate of spontaneous contraction (chronotropy), increased rate of relaxation (lusitropy), and increased force production (inotropy) in response to epinephrine. Cardioids have a developmental protein phenotype that expresses both α‐ and β‐tropomyosin, very low levels of SERCA2a, and very little of the mature isoform of cardiac troponin T.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154481/1/fsb2fj042034fje-sup-0001.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154481/2/fsb2fj042034fje.pd

Tissue Engineering of Recellularized Small-Diameter Vascular Grafts

A tissue-engineered small-diameter arterial graft would be of benefit to patients requiring vascular reconstructive procedures. Our objective was to produce a tissue-engineered vascular graft with a high patency rate that could withstand arterial pressures. Rat arteries were acellularized with a series of detergent solutions, recellularized by incubation with a primary culture of endothelial cells, and implanted as interposition grafts in the common femoral artery. Acellular grafts that had not been recellularized were implanted in a separate group of control animals. No systemic anticoagulants were administered. Grafts were explanted at 4 weeks for definitive patency evaluation and histologic examination; 89% of the recellularized grafts and 29% of the control grafts remained patent. Elastin staining demonstrated the preservation of elastic fibers within the media of the acellular grafts before implantation. Immunohistochemical staining of explanted grafts demonstrated a complete layer of endothelial cells on the lumenal surface in grafts that remained patent. Smooth muscle cells were observed to have repopulated the vessel walls. The mechanical properties of the matrix were comparable to native vessels. Such a strategy may present an alternative to autologous harvest of small vessels for use in vascular bypass procedures.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63381/1/ten.2005.11.778.pd

Rehabilitation post-distraction osteogenesis for brachymetacarpia: a case report

Abstract Distraction osteogenesis for brachymetacarpia has been described in several small case series and single case reports, but the rehabilitation required to optimize outcomes has not been reported. We present a case report describing the hand rehabilitation program of a 13-year-old girl with congenital brachymetacarpia who underwent distraction osteogenesis of the third metacarpal. Intense weekly hand therapy including desensitization, scar massage, range of motion exercises and splinting was essential up to 28 weeks postoperatively to address the progressive changes in the anatomical structures. At final follow-up, she had full active range of motion, no functional deficits in grasp or in-hand manipulation skills and resumed her participation in competitive baton twirling. Patient and family satisfaction with outcome was high. However, better education regarding the progressive symptoms with distraction and daily challenges of wearing an external fixator would have improved the overall experience. With a strong family commitment to rehabilitation and thorough patient education, distraction osteogenesis for brachymetacarpia has the potential to improve functional and aesthetic outcome in the hand. Level of evidence V