Assessment of safety, accuracy, and human CD34+ cell retention after intramyocardial injections with a helical needle catheter in a porcine model.

OBJECTIVES: Assess accuracy of Helix injections via fluoroscopic-mapping and evaluate delivery safety. BACKGROUND: Percutaneous intramyocardial-delivery of agents must be safe and accurate; retention is also important. A delivery system (Helical Infusion/Morph Guide-Catheter, Biocardia Inc) has been developed to improve maneuverability and stability of catheter-needle-myocardium intersection. METHODS: Accuracy and safety: 12 swine underwent LV and coronary angiography via 8F sheath. Targeted delivery was assigned into LAD, LCX, or RCA. System was advanced into LV and 6 targeted intramyocardial dye injections (5 mm apart) delivered using fluoroscopy. After euthanization, hearts underwent gross and histologic evaluation. Retention was assessed by iron-oxide and fluorochrome labeled CD34+ cells. Cells were injected into 6 swine using same techniques. Delivery system was advanced into LV, and injections delivered using fluoroscopy. Euthanization was performed at 2 hr and hearts formalin fixed. MRI was performed on 6 treated hearts and 4 untreated controls. Blinded analysis performed by 2 radiologists. Two treated hearts underwent immunohistological analysis. RESULTS: Accuracy and safety evaluation: 71/72 injections (98.6%) were within prespecified zone; 7/72 (9.7%) less than 5 mm apart. No adverse events occurred. MRI-presence of iron-oxide labeled CD34+ cells were correctly identified in 95% (19/20) of imaged injections. Anti-CD34+ antibody staining and fluorescence microscopy confirmed CD34+ cells in myocardium. Histology confirmed cell viability at fixation. CONCLUSIONS: Helix system was accurate and safe. Retention of CD34+ cells was confirmed by MRI and immunohistology. Further preclinical studies are needed to characterize retention over time and quantify efficiency. Studies are needed to confirm accuracy, safety, and retention in humans.

Mobilized human hematopoietic stem/progenitor cells promote kidney repair after ischemia/reperfusion injury.

BACKGROUND: Understanding the mechanisms of repair and regeneration of the kidney after injury is of great interest because there are currently no therapies that promote repair, and kidneys frequently do not repair adequately. We studied the capacity of human CD34(+) hematopoietic stem/progenitor cells (HSPCs) to promote kidney repair and regeneration using an established ischemia/reperfusion injury model in mice, with particular focus on the microvasculature. METHODS AND RESULTS: Human HSPCs administered systemically 24 hours after kidney injury were selectively recruited to injured kidneys of immunodeficient mice (Jackson Labs, Bar Harbor, Me) and localized prominently in and around vasculature. This recruitment was associated with enhanced repair of the kidney microvasculature, tubule epithelial cells, enhanced functional recovery, and increased survival. HSPCs recruited to kidney expressed markers consistent with circulating endothelial progenitors and synthesized high levels of proangiogenic cytokines, which promoted proliferation of both endothelial and epithelial cells. Although purified HSPCs acquired endothelial progenitor markers once recruited to the kidney, engraftment of human endothelial cells in the mouse capillary walls was an extremely rare event, indicating that human stem cell mediated renal repair is by paracrine mechanisms rather than replacement of vasculature. CONCLUSIONS: These studies advance human HSPCs as a promising therapeutic strategy for promoting renal repair after injury.

Hemocompatibility evaluation of poly(diol citrate) in vitro for vascular tissue engineering.

One of the ongoing challenges in tissue engineering is the synthesis of a hemocompatible vascular graft. Specifically, the material used in the construct should have antithrombogenic properties and support the growth of vascular cells. Our laboratory has designed a novel biodegradable, elastomeric copolymer, poly(1,8-octanediol citrate) (POC), with mechanical and degradation properties suitable for vascular tissue engineering. The hemocompatibility of POC in vitro and its ability to support the attachment and differentiation of human aortic endothelial cell (HAEC) was assessed. The thrombogenicity and inflammatory potential of POC were assessed relative to poly(l-lactide-co-glycolide) and expanded poly(tetrafluoroethylene), as they have been used in FDA-approved devices for blood contact. Specifically, platelet aggregation and activation, protein adsorption, plasma clotting, and hemolysis were investigated. To assess the inflammatory potential of POC, the release of IL-1beta and TNF-alpha from THP-1 cells was measured. The cell compatibility of POC was assessed by confirming HAEC differentiation and attachment under flow conditions. POC exhibited decreased platelet adhesion and clotting relative to control materials. Hemolysis was negligible and protein adsorption was comparable to reference materials. IL-1beta and TNF-alpha release from THP-1 cells was comparable among all materials tested, suggesting minimal inflammatory potential. POC supported HAEC differentiation and attachment without any premodification of the surface. The results described herein are encouraging and suggest that POC is hemocompatible and an adequate candidate biomaterial for in vivo vascular tissue engineering.

Hemocompatibility evaluation of poly(glycerol-sebacate) in vitro for vascular tissue engineering.

Poly(glycerol-sebacate) (PGS) is an elastomeric biodegradable polyester that could potentially be used to engineer blood vessels in vivo. However, its blood-material interactions are unknown. The objectives of this study were to: (a) fabricate PGS-based biphasic tubular scaffolds and (b) assess the blood compatibility of PGS in vitro in order to get some insight into its potential use in vivo. PGS was incorporated into biphasic scaffolds by dip-coating glass rods with PGS pre-polymer. The thrombogenicity (platelet adhesion and aggregation) and inflammatory potential (IL-1beta and TNFalpha expression) of PGS were evaluated using fresh human blood and a human monocyte cell line (THP-1). The activation of the clotting system was assessed via measurement of tissue factor expression on THP-1 cells, plasma recalcification times, and whole blood clotting times. Glass, tissue culture plastic (TCP), poly(l-lactide-co-glycolide) (PLGA), and expanded polytetrafluorethylene (ePTFE) were used as reference materials. Biphasic scaffolds with PGS as the blood-contacting surface were successfully fabricated. Relative to glass (100%), platelet attachment on ePTFE, PLGA and PGS was 61%, 100%, and 28%, respectively. PGS elicited a significantly lower release of IL-1beta and TNFalpha from THP-1 cells than ePTFE and PLGA. Similarly, relative to all reference materials, tissue factor expression by THP-1 cells was decreased when exposed to PGS. Plasma recalcification and whole blood clotting profiles of PGS were comparable to or better than those of the reference polymers tested.

Novel biphasic elastomeric scaffold for small-diameter blood vessel tissue engineering.

Compliance mismatch, thrombosis, and long culture times in vitro remain important challenges to the clinical implementation of a tissue-engineered small-diameter blood vessel (SDBV). To address these issues, we are developing an implantable elastomeric and biodegradable biphasic tubular scaffold. The scaffold design uses connected nonporous and porous phases as a basis to mimic, respectively, the intimal and medial layers of a blood vessel. Biphasic scaffolds were fabricated from poly(diol citrate), a novel class of biodegradable polyester elastomer. Scaffolds were characterized for tensile and compressive properties, burst pressure, compliance, foreign body reaction (via subcutaneous implantation in rats), and cell distribution and differentiation (via histology, scanning electron microscopy, and immunohistochemistry). Tensile tests, burst pressure, and compliance measurements confirm that the incorporation of a nonporous phase to create a "skin" connected to the porous phase of a scaffold can provide bulk mechanical properties that are similar to those of a native vessel. Compression tests confirm that the scaffolds are soft and recover from deformation. Subcutaneously implanted poly(diol citrate) porous scaffolds produce a thin fibrous capsule and allow for tissue ingrowth. In vitro culture of tubular biphasic scaffolds seeded with human aortic smooth muscle cells (HASMCs) and endothelial cells (HAECs) demonstrates the ability of this design to support cell compartmentalization, coculture, and cell differentiation. The newly formed HAEC monolayer stained positive for von Willebrand factor whereas collagen- and calponin-positive HASMCs were present in the porous phase.

Microfabricated grooves recapitulate neonatal myocyte connexin43 and N-cadherin expression and localization.

Our objective is to alter the surface topography on which cardiac myocytes are grown in culture so that they more closely resemble their in vivo counterparts. Microtextured silicone substrata were made using photolithography and microfabrication techniques and then coated with laminin. Primary cardiac myocytes from newborn rats were plated on microgrooved and nontextured substrata. Myocytes were highly oriented on 5 microm grooves (69.8 +/- 2.0%) and significantly different, p < 0.0001, compared with randomly oriented cells grown on nontextured surfaces (2.9 +/- 0.95%; n = 19). Cells on shallower, 2 microm, grooves were slightly less well oriented (46.9 +/- 4.3%, n = 5, p < 0.001). The lateral spacings of the grooves were altered to examine changes in cell-to-cell contact by confocal immunocytochemistry and quantitative protein analysis. Connexin43 and N-cadherin were distributed around the perimeter of the myocytes plated on 10 x 5 x 5 microgrooved surfaces, similar to the localization found in the neonate. Connexin43 expression in cultures on 5 microm deep grooved substrata was equal to the neonatal heart, whereas it differed in nontextured surfaces. We conclude that it is necessary to combine groove depth (5 microm) and lateral ridge dimensions between grooves (5 microm) in order to recapitulate connexin43 and N-cadherin expression levels and subcellular localization to that of the neonate.

Microtextured substrata alter gene expression, protein localization and the shape of cardiac myocytes.

Many of the experiments designed to understand fundamental principles in cardiac physiology are performed in vitro using myocytes isolated from adult or neonatal hearts. However, these cells have probably lost some of their original properties in culture prior to study. Our objective is to recapitulate cardiac myocyte structure and function by growing cells on microtextured silicone substrata produced by photolithography and microfabrication techniques. Myocytes are plated on nontextured, micropegged (5 microm high), microgrooved (parallel grooves with a depth of 5 microm) or combination (micropegged and grooved) substrata. Myocytes plated on microtextured surfaces display a change in cell shape with an increase in myofibrillar height and a decrease in cell area. This shape change did not affect the stoichiometry of the myofibrillar proteins but did elicit microenvironmental remodeling of proteins that mechanically attach the cell to its surroundings. Cells terminate in a sarcomeric striation on the vertical interface of the peg whereas on nontextured surfaces they end in long nonstriated cables. Vinculin, a focal adhesion protein, was found to decrease in expression on combination surfaces as compared to nontextured substrata. A three-dimensional microtextured substratum appears to reintroduce a more physiological microarchitecture for tissue culture that may have potential uses in biological research as well as in tissue engineering and diagnostic applications.

Sodium current modulation by a tubulin/GTP coupled process in rat neonatal cardiac myocytes.

Microtubule disassembly by colchicine increases spontaneous beating of neonatal cardiac myocytes by an unknown mechanism. Here, we measure drug effects on spontaneous calcium transients and whole cell ionic currents to define the route between microtubule depolymerization and the increase in the rate of contraction. Colchicine treatment disassembles microtubules resulting in free tubulin dimers, thereby increasing the spontaneous beating frequency and changing both the rates of rise and decay of calcium transients. In addition, colchicine treatment produces an increase of the sodium current (I(Na)) while I(Ca) is not modified. The colchicine-enhanced I(Na) was blocked by the addition of 10 microM TTX. In addition, the colchicine-induced increase of I(Na) was prevented when GTP was omitted from the patch pipette. Vinblastine also depolymerizes microtubules but re-aggregates tubulin into paracrystalline structures. Free tubulin dimers are not increased with vinblastine treatment. We found no modification in calcium transients or I(Na) in the presence of vinblastine. Action potential durations measured at 50 % and 90 % repolarization were shorter, and the dV/dt was larger, in colchicine-treated cells compared to untreated cells. The resting membrane potential and overshoot of the action potentials were comparable in both kinds of cells. Our data suggest that release of free tubulin dimers may activate G proteins, which in turn modulate the sodium channel. An increase in whole cell I(Na) changes the spontaneous firing rate and this may be the underlying cause of the increase in the frequency of contraction in neonatal cardiac myocytes. We suggest a new role for dimeric tubulin in regulating membrane excitability.