SUMMARY It is widely believed that perinatal cardiomyocyte terminal differentiation blocks cytokinesis, thereby causing binucleation and limiting regenerative repair after injury. This suggests that heart growth should occur entirely by cardiomyocyte hypertrophy during preadolescence when, in mice, cardiac mass increases many-fold over a few weeks. Here we show thata thyroid hormone surge activates the IGF-1/IGF1-R/Akt pathway on postnatal day-15andinitiates a brief but intense proliferative burst of predominantly binuclear cardiomyocytes. This proliferation increases cardiomyocyte numbers by ~40%, causing a major disparity between heart and cardiomyocyte growth. Also, the response to cardiac injury at postnatal day15 is intermediate between that observed at postnatal day-2 and -21, further suggesting persistence of cardiomyocyte proliferative capacity beyond the perinatal period. If replicated in humans, this may allow novel regenerative therapies for heart diseases.

Surface immobilized poly(N-isopropyl acrylamide) (pNIPAM) is currently used for a wide variety of biosensor and biomaterial applications. A thorough characterization of the surface properties of pNIPAM thin films will benefit those applications. In this work, we present analysis of a plasma polymerized NIPAM (ppNIPAM) coating by multiple surface analytical techniques, including timeof-flight secondary ion mass spectrometry (ToF-SIMS), contact angle measurement, atomic force microscopy (AFM) and sum frequency generation (SFG) vibrational spectroscopy. ToF-SIMS data show that the plasma-deposited NIPAM polymer on the substrate is crosslinked with a good retention of the monomer integrity. Contact angle results confirm the thermoresponse of the film as observed by a change of surface wettability as a function of temperature. Topographic and force distance curve measurements by AFM further demonstrate that the grafted film shrinks or swells depending on the temperature of the aqueous environment. A clear transition of the elastic modulus is observed at 31-32°C. The change of the surface wettability and mechanical properties vs. temperature are attributed to different conformations taken by the polymer, which is reflected on the outmost surface as distinct side chain groups orienting outwards at different temperatures as measured by SFG. The results suggest that a ppNIPAM thin film on a substrate experiences similar mechanical and chemical changes to pNIPAM bulk polymers in solution. The SFG result provides evidence supporting the current theory of the lower critical solution temperature (LCST) behavior of pNIPAM.

Polyethylene oxide (PEO) surfaces reduce non-specific protein and cell interactions with implanted biomaterials and may improve their biocompatibility. PEO-like polymerized tetraglyme surfaces were made by glow discharge plasma deposition onto fluorinated ethylene propylene copolymer (FEP) substrates and were shown to adsorb less than 10 ng/cm2 of fibrinogen in vitro. The ability of the polymerized tetraglyme surfaces to resist leukocyte adhesion was studied in vitro and in vivo. Polymerized tetraglyme and FEP were implanted subcutaneously in mice and removed after 1 day or 4 weeks. Histological analysis showed a similar degree of fibrous encapsulation around all of the 4-week implants. Darkly stained wells were present in the fibrous tissues at the tissue-material interface of both FEP and tetraglyme. Scanning electron micrographs showed that in vivo macrophage adhesion to polymerized tetraglyme was much higher than to FEP. After 2-hour contact with heparinized whole blood, polymorphonuclear leukocyte (PMN) adhesion to polymerized tetraglyme was much higher than to FEP, while platelet adhesion to polymerized tetraglyme was lower than to FEP. When PMNs isolated from blood were suspended in 10% autologous plasma, cell adhesion to polymerized tetraglyme was higher than to FEP; however when the cells were suspended in heat inactivated serum, cell adhesion to FEP was higher than to polymerized tetraglyme. The surface chemistry of polymerized tetraglyme did not change after 2-hour blood contact, but displayed nitrogen functional groups after 1-day implantation and became slightly degraded after 4-week implantation. The surface chemistry of FEP did not change significantly after blood contact or implantation. Loosely bound proteins such as fibrinogen on polymerized tetraglyme may contribute to the adhesion of PMNs and macrophages and ultimately to fibrous encapsulation (the foreign body response) around the implants.

Adsorbed proteins on implanted biomedical devices mediate platelet and leukocyte adhesion. Radio frequency plasma deposited tetraglyme (CH3O(CH2CH2O)4CH3), which forms a PEO-like coating, has been shown to resist protein adsorption and monocyte adhesion in vitro. By using different plasma deposition powers (5-80 W), we produced a series of plasma-deposited tetraglyme surfaces that varied in surface chemistry as measured by electron spectroscopy for chemical analysis (ESCA) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Both fibrinogen and IgG adsorption were increased on surfaces made at high plasma power. Monocyte adhesion correlated linearly with the amount of adsorbed protein. To identify the surface chemical features that contributed to the nonfouling properties of plasma-deposited tetraglyme, multivariate analysis using partial least squares (PLS) regression was applied. A PLS calibration model based on deposited tetraglyme samples placed downstream in the plasma reactor successfully predicted fibrinogen adsorption to deposited tetraglyme samples placed midstream in the reactor. The model identified how each surface spectral variable from ESCA and ToF-SIMS contributed to protein adsorption. The fraction of carbon in ether carbon linkages as measured by ESCA and ToF-SIMS peaks at m/z 59 and 103 was higher on surfaces that exhibited ultralow fibrinogen adsorption (<10 ng/cm 2 ). The fraction of hydrocarbon-like carbons as measured by ESCA and low-mass ToF-SIMS peaks such as m/z 29 and 31 was greater on surfaces exhibiting high fibrinogen adsorption (>200 ng/cm 2 ). This study elucidated the surface chemical features of deposited tetraglyme that most affect its resistance to protein and cell uptake and provided guidelines for engineering improved nonfouling biomaterial surfaces.