Methacrylated human recombinant collagen peptide as a hydrogel for manipulating and monitoring stiffness-related cardiac cell behavior.

Environmental stiffness is a crucial determinant of cell function. There is a long-standing quest for reproducible and (human matrix) bio-mimicking biomaterials with controllable mechanical properties to unravel the relationship between stiffness and cell behavior. Here, we evaluate methacrylated human recombinant collagen peptide (RCPhC1-MA) hydrogels as a matrix to control 3D microenvironmental stiffness and monitor cardiac cell response. We show that RCPhC1-MA can form hydrogels with reproducible stiffness in the range of human developmental and adult myocardium. Cardiomyocytes (hPSC-CMs) and cardiac fibroblasts (cFBs) remain viable for up to 14 days inside RCPhC1-MA hydrogels while the effect of hydrogel stiffness on extracellular matrix production and hPSC-CM contractility can be monitored in real-time. Interestingly, whereas the beating behavior of the hPSC-CM monocultures is affected by environmental stiffness, this effect ceases when cFBs are present. Together, we demonstrate RCPhC1-MA to be a promising candidate to mimic and control the 3D biomechanical environment of cardiac cells.

Human pluripotent stem cell-derived cardiomyocytes align under cyclic strain when guided by cardiac fibroblasts.

The myocardium is a mechanically active tissue typified by anisotropy of the resident cells [cardiomyocytes (CMs) and cardiac fibroblasts (cFBs)] and the extracellular matrix (ECM). Upon ischemic injury, the anisotropic tissue is replaced by disorganized scar tissue, resulting in loss of coordinated contraction. Efforts to re-establish tissue anisotropy in the injured myocardium are hampered by a lack of understanding of how CM and/or cFB structural organization is affected by the two major physical cues inherent in the myocardium: ECM organization and cyclic mechanical strain. Herein, we investigate the singular and combined effect of ECM (dis)organization and cyclic strain in a two-dimensional human in vitro co-culture model of the myocardial microenvironment. We show that (an)isotropic ECM protein patterning can guide the orientation of CMs and cFBs, both in mono- and co-culture. Subsequent application of uniaxial cyclic strain-mimicking the local anisotropic deformation of beating myocardium-causes no effect when applied parallel to the anisotropic ECM. However, when cultured on isotropic substrates, cFBs, but not CMs, orient away from the direction of cyclic uniaxial strain (strain avoidance). In contrast, CMs show strain avoidance via active remodeling of their sarcomeres only when co-cultured with at least 30% cFBs. Paracrine signaling or N-cadherin-mediated communication between CMs and cFBs was no contributing factor. Our findings suggest that the mechanoresponsive cFBs provide structural guidance for CM orientation and elongation. Our study, therefore, highlights a synergistic mechanobiological interplay between CMs and cFBs in shaping tissue organization, which is of relevance for regenerating functionally organized myocardium.

Neurocognitive and executive functioning in adult survivors of congenital heart disease.

OBJECTIVE: Congenital heart disease (CHD) can affect the developing central nervous system, resulting in neurocognitive and behavioral deficits. Preoperative neurological abnormalities as well as sequelae of the open heart operations required to correct structural abnormalities of the heart contribute to these deficits. There are few studies examining the neurocognitive functioning of adults with CHD. This study sought to investigate multiple domains of neurocognitive functioning in adult survivors of CHD who had childhood cardiac surgery with either moderate or severe disease complexity. DESIGN: A total of 48 adults (18-49 years of age) who had undergone cardiac surgery for CHD prior to five years of age participated in the study. CHD severity was classified as moderate or severe according to the 32nd Bethesda Guidelines. A computerized battery of standardized neurocognitive tests (CNS-Vital Signs), a validated rating scale of executive functioning, and demographic questionnaires were administered. RESULTS: There were no significant differences between the moderate CHD group and normative data on any cognitive measure. In contrast, the severe CHD group differed from norms in multiple domains: psychomotor speed, processing speed, complex attention, reaction time, and on the overall neurocognitive index. Number of surgeries was strongly related to worse executive functioning. There was no association between age at first surgery or time since last surgery and neuropsychological functioning. Number of surgeries was also unrelated to neurocognitive test performance. CONCLUSIONS: Patients with severe CHD performed significantly worse on measures of processing speed, attention, and executive functioning. These findings may be useful in the long-term care of adults with congenital heart disease.

Thermoresponsive hydrogels in biomedical applications: A seven-year update.

Thermally responsive hydrogels modulate their gelation behavior upon temperature change. Aqueous solutions solidify into hydrogels when a critical temperature is reached. In biomedical applications, the change from ambient temperature to physiological temperature can be employed. Their potential as in situ forming biomaterials has rendered these hydrogels very attractive. Advances in drug delivery, tissue engineering and cell sheet engineering have been made in recent years with the use of thermoresponsive hydrogels. The scope of this article is to review the literature on thermosensitive hydrogels published over the past seven years. The article concentrates on natural polymers as well as synthetic polymers, including systems based on N-isopropylacrylamide (NIPAAm), poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO), poly(ethylene glycol) (PEG)-biodegradable polyester copolymers, poly(organophosphazenes) and 2-(dimethylamino) ethyl methacrylate (DMAEMA).

Prescribing errors in adult congenital heart disease patients admitted to a pediatric cardiovascular intensive care unit.

BACKGROUND: Adults with congenital heart disease (CHD) are often cared for at pediatric hospitals. There are no data describing the incidence or type of medication prescribing errors in adult patients admitted to a pediatric cardiovascular intensive care unit (CVICU). METHODS: A review of patients >18 years of age admitted to the pediatric CVICU at our institution from 2009 to 2011 occurred. A comparator group <18 years of age but >70 kg (a typical adult weight) was identified. Medication prescribing errors were determined according to a commonly used adult drug reference. An independent panel consisting of a physician specializing in the care of adult CHD patients, a nurse, and a pharmacist evaluated all errors. Medication prescribing orders were classified as appropriate, underdose, overdose, or nonstandard (dosing per weight instead of standard adult dosing), and severity of error was classified. RESULTS: Eighty-five adult (74 patients) and 33 pediatric admissions (32 patients) met study criteria (mean age 27.5 ± 9.4 years, 53% male vs. 14.9 ± 1.8 years, 63% male). A cardiothoracic surgical procedure occurred in 81.4% of admissions. Adult admissions weighed less than pediatric admissions (72.8 ± 22.4 kg vs. 85.6 ± 14.9 kg, P < .01) but hospital length of stay was similar. (Adult 6 days [range 1-216 days]; pediatric 5 days [Range 2-123 days], P = .52.) A total of 112 prescribing errors were identified and they occurred less often in adults (42.4% of admissions vs. 66.7% of admissions, P = .02). Adults had a lower mean number of errors (0.7 errors per adult admission vs. 1.7 errors per pediatric admission, P < .01). Prescribing errors occurred most commonly with antimicrobials (n = 27). Underdosing was the most common category of prescribing error. Most prescribing errors were determined to have not caused harm to the patient. CONCLUSIONS: Prescribing errors occur frequently in adult patients admitted to a pediatric CVICU but occur more often in pediatric patients of adult weight.

Thermoresponsive, in situ cross-linkable hydrogels based on N-isopropylacrylamide: fabrication, characterization and mesenchymal stem cell encapsulation.

Hydrogels that solidify in response to a dual, physical and chemical, mechanism upon temperature increase were fabricated and characterized. The hydrogels were based on N-isopropylacrylamide, which renders them thermoresponsive, and contained covalently cross-linkable moieties in the macromers. The effects of the macromer end group, acrylate or methacrylate, and the fabrication conditions on the degradative and swelling properties of the hydrogels were investigated. The hydrogels exhibited higher swelling below their lower critical solution temperature (LCST). When immersed in cell culture medium at physiological temperature, which was above their LCST, hydrogels showed constant swelling and no degradation over 8 weeks, with the methacrylated hydrogels showing greater swelling than their acrylated analogs. In addition, hydrogels immersed in cell culture medium under the same conditions showed lower swelling compared with phosphate-buffered saline. The interplay between chemical cross-linking and thermally induced phase separation affected the swelling characteristics of the hydrogels in different media. Mesenchymal stem cells encapsulated in the hydrogels in vitro were viable over 3 weeks and markers of osteogenic differentiation were detected when the cells were cultured with osteogenic supplements. Hydrogel mineralization in the absence of cells was observed in cell culture medium with the addition of fetal bovine serum and ß-glycerol phosphate. The results suggest that these hydrogels may be suitable as carriers for cell delivery in tissue engineering.

Synthesis and characterization of dual stimuli responsive macromers based on poly(N-isopropylacrylamide) and poly(vinylphosphonic acid).

Stimulus responsive materials hold great promise in biological applications as they can react to changes in physiological stimuli to produce a desired effect. Stimulus responsive macromers designed to respond to temperature changes at or around 37 degrees C and the presence of divalent cations were synthesized from N-isopropylacrylamide, pentaerythritol diacrylate monostearate, 2-hydroxyethyl acrylate, and vinylphosphonic acid by free radical polymerization. Monomers were incorporated into the macromers in ratios approximating the molar feed ratios, and macromers underwent thermogelation around normal body temperature (36.2-40.5 degrees C) as determined by rheology and differential scanning calorimetry. Macromers containing vinylphosphonic acid interacted with calcium ions in solution, displaying decreased sol-gel transition temperatures (27.6-34.4 degrees C in 100 mM CaCl(2)), with decreases of greater magnitude observed for macromers with higher relative vinylphosphonic acid content. Critical micellar concentrations also decreased in a dose-dependent manner with increased vinylphosphonic acid incorporation in solutions with CaCl(2) but not in solutions with NaCl. These dually responsive macromers allow examination of the effect of increasing vinylphosphonic acid content in a macromer, which holds promise in biological applications such as drug and cell delivery or tissue engineering due to the macromer responsiveness at physiological temperatures and concentrations of calcium.

Injectable biomaterials for regenerating complex craniofacial tissues.

Engineering complex tissues requires a precisely formulated combination of cells, spatiotemporally released bioactive factors, and a specialized scaffold support system. Injectable materials, particularly those delivered in aqueous solution, are considered ideal delivery vehicles for cells and bioactive factors and can also be delivered through minimally invasive methods and fill complex 3D shapes. In this review, we examine injectable materials that form scaffolds or networks capable of both replacing tissue function early after delivery and supporting tissue regeneration over a time period of weeks to months. The use of these materials for tissue engineering within the craniofacial complex is challenging but ideal as many highly specialized and functional tissues reside within a small volume in the craniofacial structures and the need for minimally invasive interventions is desirable due to aesthetic considerations. Current biomaterials and strategies used to treat craniofacial defects are examined, followed by a review of craniofacial tissue engineering, and finally an examination of current technologies used for injectable scaffold development and drug and cell delivery using these materials.

Cytocompatibility evaluation of amphiphilic, thermally responsive and chemically crosslinkable macromers for in situ forming hydrogels.

The cytocompatibility of amphiphilic, thermoresponsive and chemically crosslinkable macromers was examined in vitro. Macromers synthesized from pentaerythritol diacrylate monostearate, N-isopropylacrylamide, acrylamide and hydroxyethyl acrylate in different molar ratios and with varying molecular weights and lower critical solution temperatures were evaluated for cytocompatibility with rat fibroblasts. Cell viabilities of over 60% for all and over 80% for most formulations were observed after 24-h incubation with macromers with molecular weights in the range of approximately 1500-3000 Da. The chemical modification of the macromers with a (meth)acrylate group was shown to have a time- and dose-dependent effect on cell viability. Uncrosslinked macromers with lower degrees of (meth)acrylation allowed for cell viability of over 60% for up to 6 h. (Meth)acrylated macromers with lower critical solution temperature (LCST) closer to physiological temperature allowed for higher cell viabilities as opposed to those with lower LCST. The data suggest that when the (meth)acrylated macromers are assembled into a physical gel, their cytotoxicity is diminished. After gel phase separation, cytotoxicity increased. This study gives information on the parameters that enable viable cell encapsulation for in situ forming hydrogel systems.

Synthesis and characterization of injectable, thermally and chemically gelable, amphiphilic poly(N-isopropylacrylamide)-based macromers.

In this study, we synthesized and characterized a series of macromers based on poly( N-isopropylacrylamide) that undergo thermally induced physical gelation and, following chemical modification, can be chemically cross-linked. Macromers with number average molecular weights typically ranging from 2000-3500 Da were synthesized via free radical polymerization from, in addition to N-isopropylacrylamide, pentaerythritol diacrylate monostearate, a bifunctional monomer containing a long hydrophobic chain, acrylamide, a hydrophilic monomer, and hydroxyethyl acrylate, a hydrophilic monomer used to provide hydroxyl groups for further chemical modification. Results indicated that the hydrophobic-hydrophilic balance achieved by varying the relative concentrations of comonomers used during synthesis was an important parameter in controlling the transition temperature of the macromers in solution and stability of the resultant gels. Storage moduli of the macromers increased over 4 orders of magnitude once gelation occurred above the transition temperature. Furthermore, chemical cross-linking of these macromers resulted in gels with increased stability compared to uncross-linked controls. These results demonstrate the feasibility of synthesizing poly( N-isopropylacrylamide)-based macromers that undergo tandem gelation and establish key criteria relating to the transition temperature and stability of these materials. The data suggest that these materials may be attractive substrates for tissue engineering and cellular delivery applications as the combination of mechanistically independent gelation techniques used in tandem may offer superior materials with regard to gelation kinetics and stability.

Effect of biomimetic conditions on mechanical and structural integrity of PGA/P4HB and electrospun PCL scaffolds.

The selection of an appropriate scaffold represents one major key to success in tissue engineering. In cardiovascular applications, where a load-bearing structure is required, scaffolds need to demonstrate sufficient mechanical properties and importantly, reliable retention of these properties during the developmental phase of the tissue engineered construct. The effect of in vitro culture conditions, time and mechanical loading on the retention of mechanical properties of two scaffold types was investigated. First candidate tested was a poly-glycolic acid non-woven fiber mesh, coated with poly-4-hydroxybutyrate (PGA/P4HB), the standard scaffold used successfully in cardiovascular tissue engineering applications. As an alternative, an electrospun poly-epsilon-caprolactone (PCL) scaffold was used. A 15-day dynamic loading protocol was applied to the scaffolds. Additionally, control scaffolds were incubated statically. All studies were performed in a simulated physiological environment (phosphate-buffered saline solution, T=37 degrees C). PGA/P4HB scaffolds showed a dramatic decrease in mechanical properties as a function of incubation time and straining. Mechanical loading had a significant effect on PCL scaffold properties. Degradation as well as fiber fatigue caused by loading promote loss of mechanical properties in PGA/P4HB scaffolds. For PCL, fiber reorganization due to straining seems to be the main reason behind the brittle behavior that was pronounced in these scaffolds. It is suggested that those changes in scaffolds' mechanical properties must be considered at the application of in vitro tissue engineering protocols and should ideally be taken over by tissue formation to maintain mechanically stable tissue constructs.

Thermoresponsive hydrogels in biomedical applications.

Environmentally responsive hydrogels have the ability to turn from solution to gel when a specific stimulus is applied. Thermoresponsive hydrogels utilize temperature change as the trigger that determines their gelling behavior without any additional external factor. These hydrogels have been interesting for biomedical uses as they can swell in situ under physiological conditions and provide the advantage of convenient administration. The scope of this paper is to review the aqueous polymer solutions that exhibit transition to gel upon temperature change. Typically, aqueous solutions of hydrogels used in biomedical applications are liquid at ambient temperature and gel at physiological temperature. The review focuses mainly on hydrogels based on natural polymers, N-isopropylacrylamide polymers, poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) polymers as well as poly(ethylene glycol)-biodegradable polyester copolymers.

Injectable matrices and scaffolds for drug delivery in tissue engineering.

Injectable matrices and depots have been the subject of much research in the field of drug delivery. The classical tissue engineering paradigm includes a matrix or scaffold to facilitate tissue growth and provide structural support, cells, and the delivery of bioactive molecules. As both tissue engineering and drug delivery techniques benefit from the use of injectable materials due to the minimal invasiveness of an injection, significant crossover should be observed between injectable materials in both fields. This review aims to outline injectable materials and processing techniques used in both tissue engineering and drug delivery and to describe methods by which current injectable materials in the field of drug delivery can be adapted for use as injectable scaffolds for tissue engineering.