Fluorinated Methacrylamide Chitosan Hydrogels Enhance Cellular Wound Healing Processes

Low availability of oxygen can lead to stalled wound healing processes and chronic wounds. To address local hypoxia and to better understand direct cellular benefits, a perfluorocarbon conjugated chitosan (MACF) hydrogel that delivers oxygen was created and applied for the first time to in vitro cultures of human dermal fibroblasts and human epidermal keratinocytes under both normoxic (21% O₂) and hypoxic (1% O₂) environments. Results revealed that local application of MACF provided 233.8 ± 9.9 mmHg oxygen partial pressure at 2 h and maintained equilibrium oxygen levels that were approximately 17 mmHg partial pressure greater than untreated controls. Cell culture experiments showed that MACF oxygenating gels improved cellular functions involved in wound healing such as cell metabolism, total DNA synthesis and cell migration under hypoxia in both fibroblasts and keratinocytes. Adenosine triphosphate (ATP) quantification also revealed that MACF treatments improved cellular ATP levels significantly over controls under both normoxia and hypoxia (p < 0.005). In total, these studies provide new data to indicate that supplying local oxygen via MACF hydrogels under hypoxic environments improves key wound healing cellular functions.     

Fluorinated methacrylamide chitosan hydrogel systems as adaptable oxygen carriers for wound healing

In this study a series of novel, biocompatible hydrogels able to repeatedly takeup and deliver oxygen at beneficial levels have been developed by conjugating various perfluorocarbon (PFC) chains to methacrylamide chitosan via Schiff base nucleophilic substitution, followed by photopolymerization to form hydrogels. The synthesized fluorinated methacrylamide chitosan (MACF) hydrogels were confirmed by high resolution ¹⁹F NMR. Synthesized MACF hydrogels were tested for their ability to takeup and then release oxygen for future use in dermal wound healing. Depending on the PFC substitution type maximum O₂ uptake was observed within 2–6 h, followed by complete release to the surrounding environment (5% CO₂) within 12–120 h at oxygen partial pressures of 1–25 mm Hg h^?1, providing outstanding system tuning for wound healing and regenerative medicine. MACFs with the most fluorines per substitution showed the greatest uptake and release of oxygen. Interestingly, adding PFC chains with a fluorinated aromatic group considerably enhanced oxygen uptake and extended release compared with a linear PFC chain with the same number of fluorine molecules. MACF hydrogels proved to be readily reloaded with oxygen once release was complete, and regeneration could be performed as long as the hydrogel was intact. Fibroblasts were cultured on MACFs and assays confirmed that materials containing more fluorines per substitution supported the most cells with the greatest metabolic activity. This result was true, even without oxygenation, suggesting PFC-facilitated oxygen diffusion from the culture medium. Finally, MACF gradient hydrogels were created, demonstrating that these materials can control oxygen levels on a spatial scale of millimeters and greatly enhance cellular proliferative and metabolic responses.     

In vivo assessment of guided neural stem cell differentiation in growth factor immobilized chitosan-based hydrogel scaffolds

In this study, we demonstrate that a unique growth factor-biomaterial system can offer spatial control of growth factors with sustained signaling to guide the specific lineage commitment of neural stem/progenitor cells (NSPCs) in vivo. First, recombinant fusion proteins incorporating an N-terminal biotin tag and interferon-γ (IFN-γ), platelet derived growth factor-AA (PDGF-AA), or bone morphogenic protein-2 (BMP-2) were immobilized to a methacrylamide chitosan (MAC) based biopolymer via a streptavidin linker to specify NSPC differentiation into neurons, oligodendrocytes, or astrocytes, respectively. MAC was mixed with growth factors (immobilized or adsorbed), acrylated laminin, NSPCs, and crosslinked within chitosan conduits. This system mimics regenerative aspects of the central nervous system ECM, which is largely composed of a crosslinked polysaccharide matrix with cell-adhesive regions, and adds the new functionality of protein sequestration. We demonstrated that these growth factors are maintained at functionally significant levels for 28 d in vitro. In the main study, immobilized treatments were compared to absorbed and control treatments after 28 d in vivo (rat subcutaneous). Masson's Trichrome staining revealed that small collagen capsules formed around the chitosan conduits with an average acceptable thickness of 153.07 ± 6.02 μm for all groups. ED-1 staining showed mild macrophage clustering around the outside of chitosan conduits in all treatments with no macrophage invasion into hydrogel portions. Importantly, NSPC differentiation staining demonstrated that immobilized growth factors induced the majority of cells to differentiate into the desired cell types as compared with adsorbed growth factor treatments and controls by day 28. Interestingly, immobilized IFN-γ resulted in neural rosette-like arrangements and even structures resembling neural tubes, suggesting this treatment can lead to guided dedifferentiation and subsequent neurulation.     

The effect of substrate stiffness on adult neural stem cell behavior

Adult stem cells reside in unique niches that provide vital cues for their survival, self-renewal and differentiation. In order to better understand the contribution of substrate stiffness to neural stem/progenitor cell (NSPC) differentiation and proliferation, a photopolymerizable methacrylamide chitosan (MAC) biomaterial was developed. Photopolymerizable MAC is particularly compelling for the study of the central nervous system stem cell niche because Young's elastic modulus (E_Y) can be tuned from less than 1 kPa to greater than 30 kPa. Additionally, the numerous free amine functional groups enable inclusion of biochemical signaling molecules that, together with the mechanical environment, influence cell behavior. Herein, NSPCs proliferated on MAC substrates with Young's elastic moduli below 10 kPa and exhibited maximal proliferation on 3.5 kPa surfaces. Neuronal differentiation was favored on the softest surfaces with E_Y < 1 kPa as confirmed by both immunohistochemistry and qRT-PCR. Oligodendrocyte differentiation was favored on stiffer scaffolds (>7 kPa); however, myelin oligodendrocyte glycoprotein (MOG) gene expression suggested that oligodendrocyte maturation and myelination was best on <1 kPa scaffolds where more mature neurons were present. Astrocyte differentiation was only observed on <1 and 3.5 kPa surfaces and represented less than 2% of the total cell population. This work demonstrates the importance of substrate stiffness to the proliferation and differentiation of adult NSPCs and highlights the importance of mechanical properties to the success of scaffolds designed to engineer central nervous system tissue.     

Differentiation of neural stem cells in three-dimensional growth factor-immobilized chitosan hydrogel scaffolds

The adult central nervous system (CNS) contains adult neural stem/progenitor cells (NSPCs) that possess the ability to differentiate into the primary cell types found in the CNS and to regenerate lost or damaged tissue. The ability to specifically and spatially control differentiation is vital to enable cell-based CNS regenerative strategies. Here we describe the development of a protein-biomaterial system that allows rapid, stable and homogenous linking of a growth factor to a photocrosslinkable material. A bioactive recombinant fusion protein incorporating pro-neural rat interferon-γ (rIFN-γ) and the AviTag for biotinylation was successfully expressed in Escherichia coli and purified. The photocrosslinkable biopolymer, methacrylamide chitosan (MAC), was thiolated, allowing conjugation of maleimide-strepatavidin via Michael-type addition. We demonstrated that biotin-rIFN-γ binds specifically to MAC-streptavidin in stoichiometric yields at 100 and 200 ng/mL in photocrosslinked hydrogels. For cell studies, NSPCs were photo-encapsulated in 100 ng/mL biotin-rIFN-γ immobilized MAC based scaffolds and compared to similar NSPC-seeded scaffolds combining 100 ng/mL soluble biotin-rIFN-γ vs. no growth factor. Cells were cultured for 8 days after which differentiation was assayed using immunohistochemistry for lineage specific markers. Quantification showed that immobilized biotin-rIFN-γ promoted neuronal differentiation (72.8 ± 16.0%) similar to soluble biotin-rIFN-γ (71.8 ± 13.2%). The percentage of nestin-positive (stem/progenitor) cells as well as RIP-positive (oligodendrocyte) cells were significantly higher in scaffolds with soluble vs. immobilized biotin-rIFN-γ suggesting that 3-D immobilization results in a more committed lineage specification.     

Short Duration Electrical Stimulation to Enhance Neurite Outgrowth and Maturation of Adult Neural Stem Progenitor Cells

New therapies are desperately needed for human central nervous system (CNS) regeneration to circumvent the lack of innate regenerative ability following traumatic injuries. Previously attempted therapies have been stymied by barriers to CNS regeneration largely because of protective mechanisms such as the blood brain barrier, inhibitory molecules, and glial scar formation. The application of electric stimulation (ES) has shown promise for enhancing peripheral nervous system regeneration, but is in its infancy in CNS regeneration. The objective of this study is to better understand how short duration ES can be harnessed to direct adult neural stem progenitor cell (NSPC) neurogenesis, neurite extension, and maturation. Herein, NSPCs were exposed to physiological levels of electrical stimulation of 0.53 or 1.83 V/m (applied power supply setting of 1.2 and 2.5 V) of direct current (DC) for 10 min/days for 2 days with a total differentiation time of 3 days. Culturing conditions consisted of either mitogenic growth factors or the neuronal differentiation factor interferon-γ (IFN-γ). Stimulated NSPCs showed lengths that were over five times longer than unstimulated controls (112.0 ± 88.8 μm at 0.53 V/m vs. 21.3 ± 8.5 μm for 0 V/m with IFN-γ) with the longest neurites reaching up to 600 µm. Additionally, ES resulted in mature neuronal morphologies and signs of differentiation through positive βIII tubulin, neuronal nuclei (NeuN), and better organized filamentous-actin (f-actin) staining with growth cone formation. Additionally, the neurites and soma of stimulated NSPCs showed increases in intracellular Ca²⁺ during stimulation, signifying the presence of functional neurons capable of electrical conductance and communication with other cells. Our study demonstrates that short stimulation times (10 min/ day) result in significant neurite extension of stem cells in a quick time frame (3  days). This ES modality is potentially advantageous for promoting axon re-growth at an injury site using delivered adult stem cells; however, significant work still remains to understand both the delivery approach of cells as well as ES application in vivo.     

Synthetic polycations with controlled charge density and molecular weight as building blocks for biomaterials

A series of polycations prepared by RAFT copolymerization of N-(3-aminopropyl)methacrylamide hydrochloride (APM) and N-(2-hydroxypropyl)methacrylamide, with molecular weights of 15 and 40 kDa, and APM content of 10–75 mol%, were tested as building blocks for electrostatically assembled hydrogels such as those used for cell encapsulation. Complexation and distribution of these copolymers within anionic calcium alginate gels, as well as cytotoxicity, cell attachment, and cell proliferation on surfaces grafted with the copolymers were found to depend on composition and molecular weight. Copolymers with lower cationic charge density and lower molecular weight showed less cytotoxicity and cell adhesion, and were more mobile within alginate gels. These findings aid in designing improved polyelectrolyte complexes for use as biomaterials.     

Injectable in situ cross-linking hyaluronic acid/carboxymethyl cellulose based hydrogels for drug release

A series of injectable in situ cross-linking hyaluronic acid/carboxymethyl cellulose based hydrogels (HA/CMC) was prepared via disulfide bonds by the oxidation of dissolved oxygen. The results showed that HA/CMC hydrogels exhibited tunable gelling time, appropriate rheology properties, high swelling ratio, good stability, and sustained drug release ability. The gelling time of HA/CMC hydrogels ranged from 1.4 to 7.0 min, and the values of the storage modulus, complex shear modulus, dynamic viscosity, and yield stress of HA3/CMC3 hydrogel were about 5869 Pa, 5870 Pa, 587 Pa·s, and 1969 Pa, respectively. The degradation percentage of HA1/CMC1, HA2/CMC2, and HA3/CMC3 hydrogels were about 60, 49, and 41% after incubating 42 days, and the in vitro cumulative release percentage of BSA from HA1/CMC1, HA2/CMC2, and HA3/CMC3 drug-loaded hydrogels were about 99, 91, and 82% after 30 days. The series of injectable in situ cross-linking HA/CMC hydrogels exhibited good comprehensive performance, signifying that these hydrogels could be potentially used in the fields of short- and medium-term controlled drug release, cell encapsulation, regenerative medicine, and tissue engineering.     

Promotion of cardiac differentiation of brown adipose derived stem cells by chitosan hydrogel for repair after myocardial infarction

The ability to restore heart function by replacement of diseased myocardium is one of the great challenges in biomaterials and regenerative medicine. Brown adipose derived stem cells (BADSCs) present a new source of cardiomyocytes to regenerate the myocardium after infarction. In this study, we explored an injectable tissue engineering strategy to repair damaged myocardium, in which chitosan hydrogels were investigated as a carrier for BADSCs. In vitro, the effect and mechanism of chitosan components on the cardiac differentiation of BADSCs were investigated. In vivo, BADSCs carrying double-fusion reporter gene (firefly luciferase and monomeric red fluorescent protein (fluc-mRFP)) were transplanted into infarcted rat hearts with or without chitosan hydrogel. Multi-techniques were used to assess the effects of treatments. We observed that chitosan components significantly enhanced cardiac differentiation of BADSCs, which was assessed by percentages of cTnT⁺ cells and expression of cardiac-specific markers, including GATA-4, Nkx2.5, Myl7, Myh6, cTnI, and Cacna1a. Treatment with collagen synthesis inhibitors, cis-4-hydroxy-d-proline (CIS), significantly inhibited the chitosan-enhanced cardiac differentiation, indicating that the enhanced collagen synthesis by chitosan accounts for its promotive role in cardiac differentiation of BADSCs. Longitudinal in vivo bioluminescence imaging and histological staining revealed that chitosan enhanced the survival of engrafted BADSCs and significantly increased the differentiation rate of BADSCs into cardiomyocytes in vivo. Furthermore, BADSCs delivered by chitosan hydrogel prevented adverse matrix remodeling, increased angiogenesis, and preserved heart function. These results suggested that the injectable cardiac tissue engineering based on chitosan hydrogel and BADSCs is a useful strategy for myocardium regeneration.     

Combining electrospun nanofibers with cell-encapsulating hydrogel fibers for neural tissue engineering

A promising component of biomaterial constructs for neural tissue engineering are electrospun fibers, which differentiate stem cells and neurons as well as direct neurite growth. However, means of protecting neurons, glia, and stem cells seeded on electrospun fibers between lab and surgical suite have yet to be developed. Here we report an effort to accomplish this using cell-encapsulating hydrogel fibers made by interfacial polyelectrolyte complexation (IPC). IPC-hydrogel fibers were created by interfacing acid-soluble chitosan (AsC) and cell-containing alginate and spinning them on bundles of aligned electrospun fibers. Primary spinal astrocytes, cortical neurons, or L929 fibroblasts were mixed into alginate hydrogels prior to IPC-fiber spinning. The viability of each cell type was assessed at 30 min, 4 h, 1 d, and 7 d after encapsulation in IPC hydrogels. Some neurons were encapsulated in IPC-hydrogel fibers made from water-soluble chitosan (WsC). Neurons were also stained with Tuj1 and assessed for neurite extension. Neuron survival in AsC-fibers was worse than astrocytes in AsC-fibers (p < 0.05) and neurons in WsC-fibers (p < 0.05). As expected, neuron and glia survival was worse than L929 fibroblasts (p < 0.05). Neurons in IPC-hydrogel fibers fabricated with WsC extended neurites robustly, while none in AsC fibers did. Neurons remaining inside IPC-hydrogel fibers extended neurites inside them, while others de-encapsulated, extending neurites on electrospun fibers, which did not fully integrate with IPC-hydrogel fibers. This study demonstrates that primary neurons and astrocytes can be encapsulated in IPC-hydrogel fibers at good percentages of survival. IPC hydrogel technology may be a useful tool for encapsulating neural and other cells on electrospun fiber scaffolds.     

Decanoic acid-modified glycol chitosan hydrogels containing tightly adsorbed palmityl-acylated exendin-4 as a long-acting sustained-release anti-diabetic system

Decanoic acid-modified glycol chitosan (DA-GC) hydrogels containing tightly adsorbed palmitic acid-modified exendin-4 (Ex4-C16) were prepared, and their pharmaceutical abilities as a long-acting sustained-release exendin-4 system for the treatment of diabetes were evaluated. Glycol chitosan (GC) was conjugated with N-hydroxysuccinimide-activated decanoic acid (DA) in anhydrous 0.4% dimethylaminopyridine/dimethylsulfoxide at different feed ratios. DA-GC hydrogels formed by physical self-assembly during dialysis vs. deionized water, and their inner network structures, swelling or gel-forming abilities and release properties were examined. The hypoglycemia caused by Ex4-C16-loaded DA-GC hydrogels was evaluated by subcutaneous administration in type 2 diabetic db/db mice. The results obtained showed that GC prepared at a DA:GC feed ratio of 1:100 had optimal properties with respect to hydrogel swelling, stiffness and Ex4-C16 incorporation, whereas DA-GC hydrogels prepared at a feed ratio of greater than 1:100 formed gels that were too stiff. The in vitro and in vivo release of Ex4-C16 from DA-GC hydrogels was dramatically delayed compared with native Ex4 probably due to strong hydrophobic interactions. In particular, Ex4-C16 in DA-GC hydrogels was found to be present around the injection site up to 10 days after subcutaneous administration, whereas Ex4 in DA-GC hydrogels was cleared from injection sites in ∼2 days in ICR mice. Finally, the hypoglycemia induced by Ex4-C16 DA-GC hydrogels was maintained for >7 days. Our findings demonstrate that Ex4-C16 DA-GC hydrogels offer a potential delivery system for the long-term treatment of type 2 diabetes.     

Exertional dyspnea-related acidotic and sympathetic responses in patients with sequelae of pulmonary tuberculosis

The objective of this study was to investigate whether exertional dyspnea correlates with exercise responses, especially arterial blood pH and plasma norepinephrine (NE) changes, in patients with sequelae of tuberculosis (TBsq). Cardiopulmonary exercise testings were performed in 49 TBsq patients and 9 controls. Each group had a break point in the dyspnea, plasma lactate, and plasma NE changes during exercise, all of which occurred at a similar exercise point. In TBsq patients in both exercise phases before and after the dyspnea break point, the dyspnea-slope (?Borg scale/?minute ventilation) correlated with the pH-slope (?pH/?oxygen uptake) (r = ?0.616, p < 0.0001; r = ?0.629, p < 0.0001, respectively, before and after the break point) and with the NE-slope (?NE/?oxygen uptake) (r = 0.443, p = 0.0012; r = 0.643, p < 0.0001, respectively, before and after the break point). In TBsq patients during exercise, increases in circulating NE levels and exertional acidosis were correlated with exertional dyspnea.     

Development of gelatin-chitosan-hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength

Hydroxyapatite–chitosan/gelatin (HA:Chi:Gel) nanocomposite scaffold has potential to serve as a template matrix to regenerate extra cellular matrix of human bone. Scaffolds with varying composition of hydroxyapatite, chitosan, and gelatin were prepared using lyophilization technique where glutaraldehyde (GTA) acted as a cross-linking agent for biopolymers. First, phase pure hydroxyapatite–chitosan nanocrystals were in situ synthesized by coprecipitation method using a solution of 2% acetic acid dissolved chitosan and aqueous solution of calcium nitrate tetrahydrate [Ca(NO₃)₂,4H₂O] and diammonium hydrogen phosphate [(NH₄)₂H PO₄]. Keeping solid loading constant at 30 wt% and changing the composition of the original slurry of gelatin, HA–chitosan allowed control of the pore size, its distribution, and mechanical properties of the scaffolds. Microstructural investigation by scanning electron microscopy revealed the formation of a well interconnected porous scaffold with a pore size in the range of 35–150 μm. The HA granules were uniformly dispersed in the gelatin–chitosan network. An optimal composition in terms of pore size and mechanical properties was obtained from the scaffold with an HA:Chi:Gel ratio of 21:49:30. The composite scaffold having 70% porosity with pore size distribution of 35–150 μm exhibited a compressive strength of 3.3–3.5 MPa, which is within the range of that exhibited by cancellous bone. The bioactivity of the scaffold was evaluated after conducting mesenchymal stem cell (MSC) – materials interaction and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay using MSCs. The scaffold found to be conducive to MSC’s adhesion as evident from lamellipodia, filopodia extensions from cell cytoskeleton, proliferation, and differentiation up to 14 days of cell culture.     

A chitosan–glutathione based injectable hydrogel for suppression of oxidative stress damage in cardiomyocytes

Overproduction of reactive oxygen species (ROS) is closely associated with myocardial infarction. The oxidative stress damage caused by ROS in grafted cells and host cells presents a major obstacle for successful myocardial repairs in cardiac tissue engineering. Previous injectable biomaterials in use of myocardial repairs typically lack consideration of their antioxidant properties. In this work, a thermosensitive chitosan chloride–glutathione (CSCl–GSH) hydrogel was developed to suppress the oxidative stress injury in cardiomyocytes (CMs). Glutathione (GSH) was conjugated on the chitosan chloride (CSCl) chain via amide bonds between carboxylic acid moieties of GSH and amine groups of CSCl. Our data show that CSCl–GSH conjugates in vitro could effectively scavenge the superoxide anion, hydroxyl radical and DPPH radical even at high concentrations and its antioxidant capacity can be modulated via adjusting the grafted degree of CSCl–GSH conjugates. In addition, CSCl–GSH hydrogels have shown an excellent biocompatibility to support the adhesion and survival of CMs. Moreover, it can remove the excessive intracellular ROS and thus suppress the oxidative stress damage and apoptosis in CMs in the presence of high ROS. These results suggest CSCl–GSH hydrogels could effectively support the myocardial repair via attenuating the oxidative stress damage to cells.     

Long-term research of stem cells in monocrotaline-induced pulmonary arterial hypertension

Our previous studies have shown that bone marrow mesenchymal stem cells (BMSCs) can inhibit the progression of pulmonary artery hypertension (PAH) in the monocrotaline (MCT) model in the short term. The aim of this study was to further investigate the long-term effect of BMSCs on PAH and to explore the mechanism of the protective effect including the pulmonary vascular remodeling and cell differentiation. PAH model was established by subcutaneous injection of 50 mg/kg MCT as previously study. Postoperatively, the animals were randomly divided into three groups (n = 10 in each group): control, PAH group, and BMSCs implantation group. Six months after injection, immunology and immunohistochemistry analysis indicated the MCT-induced intima-media thickness in muscular arteries was reduced (P < 0.05); the area of collagen fibers in lung tissue was lower (P < 0.05), and the proliferating cell nuclear antigen level in pulmonary artery smooth muscle cells was decreased (P < 0.05). Immunofluorescence showed that the cells have the ability to differentiate between von Willebrand factor and vascular endothelial growth factor. Six months after intravenous injection, BMSCs could significantly improve pulmonary function by inhibiting the ventricular remodeling and the effect of cell differentiation.     

A microfabricated platform for establishing oxygen gradients in 3-D constructs

Oxygen gradients are increasingly implicated in a number of biological processes, including stem cell differentiation and cancer metastasis. Unfortunately, the current in vitro tools designed to mimic conditions found in vivo lack application flexibility, simplicity in operation, and precise spatial control that most researchers require for widespread dissemination. The novel microfluidic-based device presented here addresses all the above concerns, offering a simple platform for enhanced control over the oxygen microenvironment exposed to three-dimensional cell-seeded constructs. The device utilizes an oxygen diffusion membrane approach to establish a gradient across a construct sandwiched between two continually perfused microfluidic networks. The device is capable of forming steady-state gradients at both the conditions tested—0 % to 5 % O₂ and 0 % to 21 % O₂—but a wide variety of profiles within the construct are possible. Cell viability with two model cell lines was also tested, with no adverse effects relative to the control.     

Laboratory evaluation of different agar media for isolation of carbapenem-resistant Acinetobacter spp.

The optimal method for surveillance of carbapenem-resistant Acinetobacter spp. (CRAB) is unknown. A collection of CRAB strains (n?=?42), carbapenem-susceptible strains (CSAB), and non-Acinetobacter strains (n?=?18) was used to evaluate six laboratory surveillance methods: MacConkey (MAC), MAC?+?1 μg/ml imipenem (MAC-IPM), minimal salts agar?+?1 % acetate (MSA), MSA with IPM disk (MSA-IPM), CHROMagarKPC, and CHROMagar Acinetobacter with CR102 (CHROMAcineto). CHROMAcineto was 100 % sensitive and specific. CHROMagarKPC and MAC-IPM were highly sensitive (>95 %), but their specificity was substantially hampered by the breakthrough growth of CSAB. MSA was unsuitable for CRAB detection. CHROMAcineto is a promising medium for CRAB detection and warrants further clinical evaluation.     

Intrinsic cardiac adrenergic (ICA) cell density and MAO-A activity in failing rat hearts

The efficiency (work/oxygen consumption) of isolated papillary muscles from failing hearts is reduced. We investigated whether this can be due to an increase of intrinsic cardiac adrenergic (ICA) cell density. The number of ICA cells in the septum and both ventricular walls was determined by tyrosine hydroxylase immunohistochemistry in rats with monocrotaline-induced pulmonary hypertension. We found that the number of ICA cells is about 200,000 per rat heart. ICA cell density was significantly lower in right ventricular myocardium of hypertrophied hearts (P < 0.01). MAO-A enzyme histochemistry and inhibition experiments with clorgyline in papillary muscles were performed to localize the enzyme and to determine its oxygen consumption. Upregulation of MAO-A was found in the right ventricular wall and papillary muscles of failing hearts (P = 0.018). A positive correlation between ICA cell density and MAO-A activity was absent. Clorgyline (2 μM) decreased the basal rate of oxygen consumption of right ventricular papillary muscles by 65 μM O₂/s (P = 0.027). This rate can only be maintained for several seconds judging from the catecholamine content of the preparations reported previously. High ICA cell activity rather than density and/or recycling of oxidized catecholamines are discussed as alternative explanations for the low myocardial efficiency in experimental pulmonary hypertension.     

Chitosan-g-hematin: Enzyme-mimicking polymeric catalyst for adhesive hydrogels

Phenol derivative-containing adhesive hydrogels has been widely recognized as having potential for biomedical applications, but their conventional production methods, utilizing a moderate/strong base, alkaline buffers, the addition of oxidizing agents or the use of enzymes, require alternative approaches to improve their biocompatibility. In this study, we report a polymeric, enzyme-mimetic biocatalyst, hematin-grafted chitosan (chitosan-g-hem), which results in effective gelation without the use of alkaline buffers or enzymes. Furthermore, gelation occurs under mild physiological conditions. Chitosan-g-hem biocatalyst (0.01%, w/v) has excellent catalytic properties, forming chitosan–catechol hydrogels rapidly (within 5 min). In vivo adhesive force measurement demonstrated that the hydrogel formed by the chitosan-g-hem activity showed an increase in adhesion force (33.6 ± 5.9 kPa) compared with the same hydrogel formed by pH-induced catechol oxidation (20.6 ± 5.5 kPa) in mouse subcutaneous tissue. Using the chitosan-g-hem biocatalyst, other catechol-functionalized polymers (hyaluronic acid–catechol and poly(vinyl alcohol)–catechol) also formed hydrogels, indicating that chitosan-g-hem can be used as a general polymeric catalyst for preparing catechol-containing hydrogels.     

Estimating Cell Concentration in Three-Dimensional Engineered Tissues Using High Frequency Quantitative Ultrasound

Histology and biochemical assays are standard techniques for estimating cell concentration in engineered tissues. However, these techniques are destructive and cannot be used for longitudinal monitoring of engineered tissues during fabrication processes. The goal of this study was to develop high-frequency quantitative ultrasound techniques to nondestructively estimate cell concentration in three-dimensional (3-D) engineered tissue constructs. High-frequency ultrasound backscatter measurements were obtained from cell-embedded, 3-D agarose hydrogels. Two broadband single-element transducers (center frequencies of 30 and 38 MHz) were employed over the frequency range of 13–47 MHz. Agarose gels with cell concentrations ranging from 1 × 10⁴ to 1 × 10⁶ cells mL^?1 were investigated. The integrated backscatter coefficient (IBC), a quantitative ultrasound spectral parameter, was calculated and used to estimate cell concentration. Accuracy and precision of this technique were analyzed by calculating the percent error and coefficient of variation of cell concentration estimates. The IBC increased linearly with increasing cell concentration. Axial and lateral dimensions of regions of interest that resulted in errors of less than 20% were determined. Images of cell concentration estimates were employed to visualize quantitatively regional differences in cell concentrations. This ultrasound technique provides the capability to rapidly quantify cell concentration within 3-D tissue constructs noninvasively and nondestructively.