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The purpose of this study was to develop a microplate assay for quantifying denatured collagen by measuring the fluorescence of carboxyfluorescein bound collagen hybridizing peptides (F-CHP). We have shown that F-CHP binds selectively with denatured collagen, and that mechanical overload of tendon fascicles causes collagen denaturation. Proteinase K was used to homogenize tissue samples after F-CHP staining, allowing fluorescence measurement using a microplate reader. We compared our new assay to our previous image analysis method and the trypsin-hydroxyproline assay, which is the only other available method to directly quantify denatured collagen. Relative quantification of denatured collagen was performed in rat tail tendon fascicles subjected to incremental tensile overload, and normal and ostoeoarthritic guinea pig cartilage. In addition, the absolute amount of denatured collagen was determined in rat tail tendon by correlating F-CHP fluorescence with percent denatured collagen as determined by the trypsin-hydroxyproline assay. Rat tail tendon fascicles stretched to low strains (7.5%) exhibited minimal denatured collagen, but values rapidly increased at medium strains (7.5-10.5%) and plateaued at high strains (≥12%). Osteoarthritic cartilage had higher F-CHP fluorescence than healthy cartilage. Both of these outcomes are consistent with previous studies. With the calibration curve, the microplate assay was able to absolutely quantify denatured collagen in mechanically damaged rat tail tendon fascicles as reliably as the trypsin-hydroxyproline assay. Further, we achieved these results more efficiently than current methods in a rapid, high-throughput manner, with multiple types of collagenous tissue while maintaining accuracy. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:431-438, 2019.

Purpose: The purpose of this study was intended to investigate the effect on the body composition of the dormitory-type middle school students by implementing the complexed exercise program. Method: As subjects of the study, the dormitory-type middle school students in Chungbuk, divided into 2 grade experimental group(N=46), 3 grade control group(N=49). Group-specific arbitration method, was applied to complexed exercise program (experimental group) and running group (control group). Each training courses 30 minutes 5times weekly, examined the changes in body composition to examine a total of 6-week course effectively. Result: The results were compared with change of Height, Muscle mass, Fat mass, Lean body mass and BMI. Experimental group were significant(p .05). Also there were significant difference(p

A new type of synthetic hydrogel scaffold that mimics certain aspects of structure and function of natural extracellular matrix (ECM) has been developed. We previously reported the conjugation of collagen mimetic peptide (CMP) to poly(ethylene oxide) diacrylate (PEODA) to create a polymer-peptide hybrid scaffold for a suitable cell microenvironment. In this study, we showed that the CMP-mediated microenvironment enhances the chondrogenic differentiation of mesenchymal stem cells (MSCs). MSCs were harvested and photo-encapsulated in CMP-conjugated PEODA (CMP/PEODA). After 3 weeks, the histological and biochemical analysis of the CMP/PEODA gel revealed twice as much glycosaminoglycan and collagen contents as in control PEODA hydrogels. Moreover, MSCs cultured in CMP/PEODA hydrogel exhibited a lower level of hypertrophic markers, core binding factor alpha 1, and type X collagen than MSCs in PEODA hydrogel as revealed by gene expression and immunohistochemisty. These results indicate that CMP/PEODA hydrogel provides a favorable microenvironment for encapsulated MSCs and regulates their downstream chondrogenic differentiation.

A new class of molecular composite piezoelectric material was produced by simultaneous poling and curing of a homogeneous solution comprising poly(γ-benzyl α, l -glutamate) and methylmethacrylate via corona discharge methods. This film exhibited high piezoelectricity ( d 33 = 23 pC N −1 ), and its mechanical characteristics (modulus = 450 MPa) were similar to those of low molecular weight poly(methylmethacrylate). As it is produced via solution-based fabrication processes, the composite film is conducive to miniaturization for small sensors with integrated electronics, and could also potentially be used in piezoelectric coating applications.

We report studies of the structure and packing of Langmuirmonolayers (LMs) of polypeptide poly(γ-4-(n-hexadecyloxy)benzyl α,L-glutamate) (C16–O–PBLG) on the surface of water. The molecule is a “hairy rod” and consists of side attachments of hexadecyloxy chains (–O–C16) to the rigid rod-like core made up of α-helical poly(γ-benzyl L-glutamate) (PBLG). Measurements include surface pressure (Π) versus area/monomer (A) isotherms, x-ray specular reflectivity (XR), and grazing incidence diffraction(GID). In contrast to the LM of bare PBLG on water, which undergoes a monolayer/bilayer transition with increasing Π, monolayers of C16–O–PBLG remain stable up to the highest densities. On the basis of XR and GID results, the structure of the C16–O–PBLG monolayer is characterized by the following main features. First, hydrophobicity causes the –O–C16 chains to segregate towards the film/gas interface and away from water and the PBLG cores, which sit parallel to and near the water/film interface. Since the attachment position of some of the side chains is at the core/water interface, the segregation forces these chains into the space between neighboring core rods. Compression associated with increasing Π thickens the film but the internally segregated structure is maintained for all Π (i.e., >∼30 dyne/cm). Second, the C16–O–PBLG rods form domains in which the rods are aligned parallel to each other and to the interface. The correlation length for the interhelix positional order of the rods is short and typically comparable to or less than the length of the rods. With increasing Π the spacing d between nearest-neighbor rods decreases linearly with A at high Π, indicating a direct correspondence between the macroscopic compressibility and the microscopic interhelix compressibility. Third, as Π increases past ∼5 dyne/cm, the local packing of tethered –O–C16 chains displays the same herringbone (HB) order that is common for high-density bulk and monolayer phases of alkyl chains. Various features of the observed GID peaks also imply that the HB order of –O–C16 chains is oriented with respect to the helical axes of aligned PBLG cores. We propose that the HB order is established initially by one-dimensionally confined chains between aligned rods at low Π and grows laterally with compression.

Macromolecules with precisely defined architectures on the nanometer scale are ideal building blocks for self-assembled materials and nanometer-scale devices. By utilizing recombinant DNA technology and bacterial protein expression, we have prepared a set of rodlike artificial proteins, Glu(OBzl)Asp(OBzl)[Glu(OBzl)_(17)Asp(OBzl)]_X-Glu(OBzl)Glu(OBzl) (PBLG-X, X = 3−6, 1), which serve as monodisperse analogues of poly(γ-benzyl α,l-glutamate) (PBLG), with variation of rod length from 8.7 to 17 nm. Synthesis was accomplished by (i) bacterial production of a precursor polypeptide GluAsp(Glu_(17)Asp)_XGluGlu (PLGA-X, X = 3−6), as a fusion to mouse dihydrofolate reductase (DHFR), (ii) CNBr digestion of the expressed protein to liberate PLGA-X, and (iii) side chain benzylation of PLGA-X via treatment with phenyldiazomethane. The growth rates of cultures transformed with the recombinant genes, and the yields of protein expressed therein, depended on the number of Glu_(17)Asp repeats appended to the fusion partner, decreasing as the number of repeats increased. PBLG-3, with the lowest aspect ratio (6.9), did not exhibit liquid crystalline (LC) behavior while each of the other polymers formed lyotropic LC phases. The phase transition behavior of PBLG-X was distinctly different from that of conventional polydisperse PBLG; when the concentration of the polymer solution was gradually increased, the transition from the isotropic to the liquid crystalline state occurred uniformly throughout the sample without formation of distinct birefringent droplets. Solution-cast films of PBLG-4 and PBLG-5 formed smectic supramolecular architectures with layer spacings (11.4 and 14.0 nm, respectively) precisely determined by the length of the monodisperse rods. Smectic ordering was observed only in films cast from mixtures of chloroform (97%) and trifluoroacetic acid (3%). In films prepared from dioxane, both polydisperse and monodisperse PBLGs adopted columnar order without formation of smectic layers.

Intrinsic membrane proteins represent a large fraction of the proteins produced by living organisms and perform many crucial functions. Structural and functional characterization of membrane proteins generally requires that they be extracted from the native lipid bilayer and solubilized with a small synthetic amphiphile, for example, a detergent. We describe the development of a small molecule with a distinctive amphiphilic architecture, a "tripod amphiphile," that solubilizes both bacteriorhodopsin (BR) and bovine rhodopsin (Rho). The polar portion of this amphiphile contains an amide and an amine-oxide; small variations in this polar segment are found to have profound effects on protein solubilization properties. The optimal tripod amphiphile extracts both BR and Rho from the native membrane environments and maintains each protein in a monomeric native-like form for several weeks after delipidation. Tripod amphiphiles are designed to display greater conformational rigidity than conventional detergents, with the long-range goal of promoting membrane protein crystallization. The results reported here represent an important step toward that ultimate goal.

This paper reports Brewster angle microscopy (BAM), x-ray specular reflectivity (XR), grazing incidence diffraction(GID) and off-specular diffuse scattering (XOSDS) measurements of Langmuir monolayers formed on water by both mono- and polydisperse samples of α-helical poly(γ-benzyl L-glutamate) (PBLG) as a function of area/monomer A. The microscopic behavior does not exhibit any discernible effects due to differing dispersity. At low surface densities (A>∼21Å^2/monomer,surface pressure Π=0), BAM images reveal partial surface coverage by solidlike monolayer islands. GID measurements show an interhelix peak corresponding to a local parallel alignment of rodlike PBLG molecules, indicating their tendency to aggregate laterally without external pressure. Compression to A

Functionalized collagen that incorporates exogenous compounds may offer new and improved biomaterials applications, especially in drug-delivery, multifunctional implants, and tissue engineering. To that end, we developed a specific and reversible collagen modification technique utilizing associative chain interactions between synthetic collagen mimetic peptide (CMP) [(ProHypGly) chi; Hyp = hydroxyproline] and type I collagen. Here we show temperature-dependent collagen binding and subsequent release of a series of CMPs with varying chain lengths indicating a triple helical propensity driven binding mechanism. The binding took place when melted, single-strand CMPs were allowed to fold while in contact with reconstituted type I collagens. The binding affinity is highly specific to collagen as labeled CMP bound to nanometer scale periodic positions on type I collagen fibers and could be used to selectively image collagens in ex vivo human liver tissue. When heated to physiological temperature, bound CMPs discharged from the collagen at a sustained rate that correlated with CMP's triple helical propensity, suggesting that sustainability is mediated by dynamic collagen-CMP interactions. We also report on the spatially defined modification of collagen film with linear and multi-arm poly(ethylene glycol)-CMP conjugates; at 37 degrees C, these PEG-CMP conjugates exhibited temporary cell repelling activity lasting up to 9 days. These results demonstrate new opportunities for targeting pathologic collagens for diagnostic or therapeutic applications and for fabricating multifunctional collagen coatings and scaffolds that can temporally and spatially control the behavior of cells associated with the collagen matrices.

Smectic ordering has been observed in perfectly monodisperse poly(γ-benzyl α,L-glutamate) (PBLG) which was synthesized using recombinant DNA technology. These PBLG molecules form rigid α-helical rods 11.45 nm in length. In the present study, transmission electron microscopy (TEM) and electron diffraction reveals a banded morphology with an approximately 120 nm period which provides strong evidence for helical rotation of the director field as in a cholesteric or twisted smectic. Detailed examination of the relative orientation of the banding in the morphology images and the reflections in the electron diffraction patterns, indicating interchain and intrachain correlations, leads to the conclusion that the structure observed is a twisted smectic phase. The relationship between the twist and the layering is found to be that of the twist grain boundary (TGB) phase. However, our data does not allow us to determine whether the monodisperse PBLG structure is blocky with discrete twist boundaries as in a true TGB or is a more continuously twisting structure. Thus, we will refer to the phase as TGB-like. Conventional, polydisperse PBLG is well-known to form cholesteric phases as a result of the chirality of the helical rod. The formation of a TGB-like phase in monodisperse PBLG is consistent with the superposition of a smectic-A layering resulting from the uniform rod length on the twisted texture present in the cholesteric.

Collagen is the principal tensile element of the extra-cellular matrix in animals and is the basic scaffold for cells and tissues. Abnormalities in its structure are known to result in a number of debilitating human diseases. Collagen mimetic peptides (CMPs) with repeat unit of (Pro-Hyp-Gly) are capable of forming right-handed triple-helical structures similar to that of the collagen triple helices. Recently, our group has shown that CMPs exhibit specific binding affinity to natural collagen under controlled thermal conditions. Using solid phase peptide synthesis, we have prepared a CMP cysteine derivative that was used to modify gold nanoparticles. Transmission electron microscopy (TEM) shows that the Cys-CMP functionalized gold nanoparticles have affinity to collagen fibers. We are investigating the interactions between Cys-CMP functionalized gold nanoparticles and collagen fibers. The Cys-CMP conjugated nanoparticles can potentially be used as a tool to visualize and understand unstable domains of collagen fibers which are related to a number of pathological conditions of extra cellular matrices.

Packing of tethered alkyl chains in Langmuir monolayers of a hairy-rod polypeptide poly[gamma-4-(n-hexadecyloxy)benzyl alpha,L-glutamate] on water has been studied by x-ray scattering measurements at room temperature. The rods lie parallel to the surface while the alkyl side chains segregate toward the vapor. Results indicate that the herringbone order of the alkyl chains is established initially by one-dimensionally confined chains between aligned rods and grows laterally with compression.

There are manifest scientific and technological interests in organizing nanocrystals (NC) into controlled architectures and work is rapidly expanding in exploiting opportunities through bio-assembly processes. In this research, we have applied biologically assembled nano-scale template to direct formation of precisely defined 2D nanocrystal arrays. We explored purple membrane (PM), a naturally occurring membrane protein (bacteriorhodopsin, BR) crystal patch from halobacteria, as a precisely structured nanometer-scale template. We have developed genetically engineered PMs that display unique functional group (cystein) on the membrane surfaces with precisely defined nano-scale symmetry. These reactive functional groups were used as specific anchoring sites for NC immobilization. We show that the cystein mutants of BR mutated at the surface of the PM are capable of forming stable PM-like hexagonal crystal lattices in the host cell membrane. These crystal patches are easily purified by ultracentrifugation and can be used to react with functionallized gold nanoclusters. We also present the variation of crystal lattice by detergent treatment and recrystallization techniques. The PM has been known to posses good materials properties and is expected to allow formation of robust nanocrystal array that can withstand variety of optical and electrical characterization conditions.

Medium chain length polyhydroxyalkanoates, MCL-PHAs, produced by bacteria as inclusion bodies or granules were analyzed in situ by differential scanning calorimetry (DSC) without isolation from the cells. The kinetic DSC study of PHA granules, which contained mostly 3-hydroxyoctanoate units (PHO), in Pseudomonas putida BM01 cells showed that the polymer within the granules existed in an amorphous state, but it crystallized after dehydration of the cells under freeze-drying condition (below -50 degrees C) followed by annealing at ambient temperature. In this manner, PHO within the cells readily crystallized to the maximum degree of crystallinity within 24 h at room temperature, which was much faster than for the same polymer isolated by solvent extraction. This observation suggests that the polymer within the cellular granules may be well organized. The DSC endothermic melting peak areas for the room-temperature annealed polymers within the cells were directly proportional to the amount of polymer in the cell, and the results from this type of quantitative analysis were essentially identical to those obtained by gas chromatographic and gravimetric analysis of the polymers. X-Ray diffraction analysis of the polymer in the freeze-dried, whole cells and of the isolated, fully crystallized polymer showed that the two types of PHO samples had similar crystal structures, but the polymer in the granules exhibited better side-chain packing and higher crystallinity.

Solutions and melts of stiff ('rod-like') macromolecules often exhibit nematic liquid crystalline phases characterized by orientational, but not positional, molecular order. Smectic phases, in which macromolecular rods are organized into layers roughly perpendicular to the direction of molecular orientation, are rare, owing at least in part to the polydisperse nature (distribution of chain lengths) of polymers prepared by conventional polymerization processes. Bacterial methods for polypeptide synthesis, in which artificial genes encoding the polymer are expressed in bacterial vectors, offer the opportunity to make macromolecules with very well defined chain lengths. Here we show that a monodisperse derivative of poly(gamma-benzyl alpha,L-glutamate) prepared in this way shows smectic ordering in solution and in films. This result suggests that methods for preparing monodisperse polymers might provide access to new smectic phases with layer spacings that are susceptible to precise control on the scale of tens of nanometres.

Recent widespread interest in the development of engineered tissue and organ replacement therapies has prompted demand for new approaches to immobilize exogenous components to natural collagen. Chemical coupling of synthetic moieties to amino acid side chains has been commonly practiced for such purposes; however, such coupling reactions are difficult to control on large proteins and are generally not conducive to modifying integrated collagen scaffolds that contain live cells and tissues. As an alternative to the conventional "covalent" modification method, we have developed a novel "physical" modification technique that is based on collagen's native ability to associate into a triple-helical molecular architecture. Here, we present a finding that collagen mimetic peptides (CMPs) of sequence -(Pro-Hyp-Gly)x- exhibit strong affinity to both native and gelatinized type I collagen under controlled thermal conditions. We also show that the cell adhesion characteristics of collagen can be readily altered by applying a poly(ethylene glycol)-CMP conjugate to a prefabricated collagen film.

Poly‐g‐benzyl‐L‐glutamety Polymethyl methacrylate (PBLG‐PMMA) composite polymer, a new piezoelectric transduction material, was invented by Yu et al. In this study a method is developed to fabricate a disk using detachable molding. Unlike film‐type piezoelectric polymers such as the polyvinylidenefluoride (PVDF), this new material can be used to form a body of various shapes and sizes such as conventional piezoelectric ceramics. The piezoelectric characteristics mainly come from the state of alignment of polygamma‐benzyl alpha, L‐glutamate molecules (PBLG), which have large electric dipole moment. The PBLG can be melted with methyl methacrylate by heating, and mixture with applying electric field can be polymerized as a piezoelectric PBLG‐PMMA composite in the mold. The fabricated specimens have the dimensions of 20 mm in diameter and 3 mm in height. The piezoelectric coefficient (d33) depends on the concentration of PBLG. Measured values were 0.45 and 1.9 pC/N with concentrations of 10 and 30 wt %, respe...

A new class of polymer composite piezoelectric materials based on piezoactive biopolymer, poly(‐benzyl,L‐glutamate) (PBLG), and a matrix polymer, poly(methylmethacrylate) (PMMA), is presented. The composite polymer offers the possibility of decoupling the electrical and mechanical properties, therefore improving the overall system efficiency. By simultaneously poling and curing (at ambient conditions) PBLG and methylmethacrylate (MMA) mixture solutions, we fabricated a flexible composite film with a significant portion of the PBLG molecules oriented normal to the film surface. This film exhibited moderate piezoelectricity (d33=20 pC/N), and its mechanical characteristics were similar to those of low molecular weight PMMA. Present samples are about 40 μm thick with tensile strength of 13 MPa and Young’s modules of 450 MPa. The frequency response is ± 3 dB from 0.1 to 7 kHz and at 1 kHz is linear with acceleration more than a 10 dB range.

Off-specular diffuse x-ray scattering measurements on both pure water and a homogeneous Langmuir monolayer of poly- γ-benzyl- L-glutamate (PBLG) on water establish the validity of a proposed sum rule for scattering from capillary fluctuations on liquid surfaces. Excess scattering above the predicted capillary contribution is observed when the PBLG monolayer is compressed beyond its elastic limit. This is interpreted in terms of a second-layer inhomogeneity with a surface correlation length of ∼1000Å. Excess off-specular scattering can be used to probe interface correlation lengths from 100 Å to 1μm.