MAS NMR measurements of intact articular bovine cartilage

Articular cartilage (AC), an avascular connective tissue lining articulating surfaces of the long bones, comprises extracellular biopolymers. In functionally compromised states such as osteoarthritis, thinned or lost AC causes reduced mobility and increased health-care costs. Understanding of the characteristics responsible for the load bearing efficiency of AC and the factors leading to its degradation are incomplete. DTI shows the structural alignment of collagen in AC [1] and T2 relaxation measurements suggest that the average director of reorientational motion of water molecules depends on the degree of alignment of collagen in AC [2]. Information on the nature of the chemical interactions involved in functional AC is lacking. The need for AC structural integrity makes solid state NMR an ideal tool to study this tissue. We examined the contribution of water in different functional ‘compartments’ using 1H-MAS, 13C-MAS and 13C-CPMAS NMR of bovine patellar cartilage incubated in D2O. 1H-MAS spectra signal intensity was reduced due to H/D exchange without a measureable redistribution of relative signal intensity. Chemical shift anisotropy was estimated by lineshape analysis of multiple peaks in the 1H-MAS spinning sidebands. These asymmetrical sidebands suggested the presence of multiple water species in AC. Therefore, water was added in small aliquots to D2O saturated AC and the influence of H2O and D2O on organic components was studied with 13C-MAS-NMR and 13C-CPMAS-NMR. Signal intensity in 13C-MAS spectra showed no change in relative signal intensity throughout the spectrum. In 13C-CPMAS spectra, displacement of water by D2O resulted in a loss of signal in the aliphatic region due to a reduction in proton availability for cross-polarization. These results complement dehydration studies of cartilage using osmotic manipulation [3] and demonstrate components of cartilage that are in contact with mobile water.

Solid-state MAS NMR measurements of intact articular bovine cartilage

Articular cartilage (AC), an avascular connective tissue lining articulating surfaces of the long bones, comprises extracellular biopolymers. In functionally compromised states such as osteoarthritis, thinned or lost AC causes reduced mobility and increased health-care costs. Understanding of the characteristics responsible for the load bearing efficiency of AC and the factors leading to its degradation are incomplete. DTI shows the structural alignment of collagen in AC [1] and T2 relaxation measurements suggest that the average director of reorientational motion of water molecules depends on the degree of alignment of collagen in AC [2]. Information on the nature of the chemical interactions involved in functional AC is lacking. The need for AC structural integrity makes solid state NMR an ideal tool to study this tissue. We examined the contribution of water in different functional ‘compartments’ using 1H-MAS, 13C-MAS and 13C-CPMAS NMR of bovine patellar cartilage incubated in D2O. 1H-MAS spectra signal intensity was reduced due to H/D exchange without a measureable redistribution of relative signal intensity. Chemical shift anisotropy was estimated by lineshape analysis of multiple peaks in the 1H-MAS spinning sidebands. These asymmetrical sidebands suggested the presence of multiple water species in AC. Therefore, water was added in small aliquots to D2O saturated AC and the influence of H2O and D2O on organic components was studied with 13C-MAS-NMR and 13C-CPMAS-NMR. Signal intensity in 13C-MAS spectra showed no change in relative signal intensity throughout the spectrum. In 13C-CPMAS spectra, displacement of water by D2O resulted in a loss of signal in the aliphatic region due to a reduction in proton availability for cross-polarization. These results complement dehydration studies of cartilage using osmotic manipulation [3] and demonstrate components of cartilage that are in contact with mobile water.

Preparation, characterization of surfactants modified clay minerals and nitrate adsorption

Organic surfactants modified clay minerals are usually used as adsorbents for hydrophobic organic contaminants remediation; this work however has shown organoclays can also work as adsorbents for hydrophilic anionic contaminant immobilization. Organoclays were prepared based on halloysite, kaolinite and bentonite and used for nitrate adsorption, which are significant for providing mechanism for the adsorption of anionic contaminants from waste water. XRD was used to characterize unmodified and surfactants modified clay minerals. Thermogravimetric analysis (TG) was used to determine the thermal stability and actual loading of surfactant molecules. Ion chromatography (IC) was used to determine changes of nitrate concentration before and after adsorption by these organoclays. These organoclays showed different removal capacities for anionic ions from water and adsorption mechanism was investigated.

Adsorption of the herbicide 2,4-D on organo-palygorskite

Among the clay minerals, montmorillonite is the most extensively studied material using as adsorbents, but palygorskite and its organically modified products have been least explored for their potential use in contaminated water remediation. In this study, an Australian palygorskite was modified with cationic surfactants octadecyl trimethylammonium bromide and dioctadecyl dimethylammonium bromide at different doses. A full structural characterization of prepared organo-palygorskite by X-ray diffraction, infrared spectroscopy, surface analysis and thermogravimetric analysis was performed. The morphological changes of palygorskite before and after modification were recorded using scanning electron microscopy, which showed the surfactant molecules can attach on the surface of rod-like crystals and thus can weaken the interactions between palygorskite single crystals. Real surfactants loadings on organo-palygorskites were also calculated based on thermogravimetric analysis. 1 CEC, 2 CEC octadecyl trimethylammonium bromide modified palygorskites, 1 CEC and 2 CEC dioctadecyl dimethylammonium bromide modified palygorskites absorbed as much as 12 mg/g, 42 mg/g, 9 mg/g and 25 mg/g of 2,4- dichlorophenoxyacetic acid respectively. This study has shown a potential on organo-palygorskites for organic herbicide adsorption especially anionic ones from waste water. In addition, equilibration time effects and the Langmuir and Freundlich models fitting were also investigated in details.

A comparative study of the proliferation and osteogenic differentiation of human periodontal ligament cells cultured on β-TCP ceramics and demineralized bone matrix with or without osteogenic inducers in vitro

The repair of bone defects that result from periodontal diseases remains a clinical challenge for periodontal therapy. β-tricalcium phosphate (β-TCP) ceramics are biodegradable inorganic bone substitutes with inorganic components that are similar to those of bone. Demineralized bone matrix (DBM) is an acid-extracted organic matrix derived from bone sources that consists of the collagen and matrix proteins of bone. A few studies have documented the effects of DBM on the proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs). The aim of the present study was to investigate the effects of inorganic and organic elements of bone on the proliferation and osteogenic differentiation of hPDLCs using three-dimensional porous β-TCP ceramics and DBM with or without osteogenic inducers. Primary hPDLCs were isolated from human periodontal ligaments. The proliferation of the hPDLCs on the scaffolds in the growth culture medium was examined using a Cell‑Counting kit‑8 (CCK-8) and scanning electron microscopy (SEM). Alkaline phosphatase (ALP) activity and the osteogenic differentiation of the hPDLCs cultured on the β-TCP ceramics and DBM were examined in both the growth culture medium and osteogenic culture medium. Specific osteogenic differentiation markers were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). SEM images revealed that the cells on the β-TCP were spindle-shaped and much more spread out compared with the cells on the DBM surfaces. There were no significant differences observed in cell proliferation between the β-TCP ceramics and the DBM scaffolds. Compared with the cells that were cultured on β-TCP ceramics, the ALP activity, as well as the Runx2 and osteocalcin (OCN) mRNA levels in the hPDLCs cultured on DBM were significantly enhanced both in the growth culture medium and the osteogenic culture medium. The organic elements of bone may exhibit greater osteogenic differentiation effects on hPDLCs than the inorganic elements.

Human amnion epithelial cell transplantation abrogates lung fibrosis and augments repair

Rationale: Chronic lung disease characterized by loss of lung tissue,inflammation, and fibrosis represents a major global health burden. Cellular therapies that could restore pneumocytes and reduce inflammation and fibrosis would be a major advance in management. Objectives: To determine whether human amnion epithelial cells (hAECs), isolated from term placenta and having stem cell–like and antiinflammatory properties, could adopt an alveolar epithelial phenotype and repair a murine model of bleomycin-induced lung injury. Methods: Primary hAECs were cultured in small airway growth medium to determine whether the cells could adopt an alveolar epithelial phenotype. Undifferentiated primary hAECs were also injected parenterally into SCID mice after bleomycin-induced lung injury and analyzed for production of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Mouse lungs were also analyzed for inflammation and collagen deposition. Measurements and Main Results: hAECs grown in small airway growth medium developed an alveolar epithelial phenotype with lamellar body formation, production of SPs A–D, and SP-D secretion. Although hAECs injected into mice lacked SPs, hAECs recovered from mouse lungs 2 weeks posttransplantation produced SPs. hAECs remained engrafted over the 4-week test period. hAEC administration reduced inflammation in association with decreased monocyte chemoattractant protein-1, tumor necrosis factor-a, IL-1 and -6, and profibrotic transforming growth factor-b in mouse lungs. In addition,lung collagen content was significantly reduced by hAEC treatment as a possible consequence of increased degradation by matrix metalloproteinase-2 and down-regulation of the tissue inhibitors f matrix metalloproteinase-1 and 2. Conclusions: hAECs offer promise as a cellular therapy for alveolar restitution and to reduce lung inflammation and fibrosis.

Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl4-treated mice

Chronic liver injury and inflammation lead to hepatic fibrosis, cirrhosis, and liver failure. Embryonic and mesenchymal stem cells have been shown to reduce experimental liver fibrosis but have potential limitations, including the formation of dysplastic precursors, tumors, and profibrogenic cells. Other stem-like cells may reduce hepatic inflammation and fibrosis without tumor and profibrogenic cell formation. To test this hypothesis we transplanted human amnion epithelial cells (hAEC), isolated from term delivered placenta, into immunocompetent C57/BL6 mice at week 2 of a 4-week regimen of carbon tetrachloride (CCl4) exposure to induce liver fibrosis. Two weeks following hAEC infusion, intact cells expressing the human-specific markers inner mitochondrial membrane protein and human leukocyte antigen-G were found in mouse liver without evidence of host rejection of the transplanted cells. Human albumin, known to be produced by hAEC, was detected in sera of hAEC-treated mice. Human DNA was detected in mouse liver and also spleen, lungs, and heart of some animals. Following hAEC transplantation, CCl4-treated animals showed decreased serum ALT levels and reduced hepatocyte apoptosis, compared to controls. hAEC-treated mouse liver had lower TNF-α and IL-6 protein levels and higher IL-10 compared to animals given CCl4 alone. Compared to CCl4 controls, hAEC-treated mice showed fewer activated collagen-producing hepatic stellate cells and less fibrosis area and collagen content. Reduced hepatic TGF-β levels in conjunction with a twofold increase in the active form of the collagen-degrading enzyme matrix metalloproteinase-2 in hAEC-treated mice compared to CCl4 controls may account for the reduction in fibrosis. hAEC transplantation into immunocompetent mice leads to cell engraftment, reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis.

Tendinopathy alters cumulative transverse strain in the patellar tendon postexercise.

Introduction This research evaluated the effect of tendinopathy on the cumulative transverse strain response of the patellar tendon to a bout of resistive quadriceps exercise. Methods Nine adults with unilateral patellar tendinopathy (age 18.2±0.7 years, height 1.92±0.06 m and weight 76.8±6.8 kg) and ten healthy adults free of knee pain (age 17.8±0.8 years, height 1.83±0.05 m and weight 73.2±7.6 kg) underwent standardised sagittal sonograms (7.2–14 MHz linear–array transducer) of both patellar tendons immediately prior and following 45 repetitions of a double–leg decline–squat exercise performed against a resistance of 145% bodyweight. Tendon thickness was determined 5–mm and 25–mm distal to the patellar pole. Transverse Hencky strain was calculated as the natural log of the ratio of post– to pre–exercise tendon thickness and expressed as a percentage. Measures of tendon echogenicity were calculated within the superficial and deep aspects of each tendon site from gray–scale profiles. Intratendinous microvessels were evaluated using power Doppler ultrasound. Results The cumulative transverse strain response to exercise in symptomatic tendinopathy was significantly lower than that of asymptomatic and healthy tendon (P<.05). There was also a significant reduction (57%) in the area of microvascularity immediately following exercise (P=.05), which was positively correlated (r=0.93, P<.05) with VISA-P score. Conclusions This study is the first to show that patellar tendinopathy is associated with an altered morphological and mechanical response of the tendon to exercise, which is manifest by a reduction in cumulative transverse strain and microvascularity, when present. Research directed toward identifying factors that influence the acute microvascular and transverse strain response of the patellar tendon to exercise in the various stages of tendinopathy is warranted.

Complementary surface-enhanced resonance raman spectroscopic biodetection of mixed protein solutions by chitosan- and silica-coated plasmon-tuned silver nanoparticles

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b5, whereas functionalization with SiO2 amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.

Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-L-lysine, poly-L-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-L-lysine and poly-L-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.

Effect of partial H2O-D2O replacement on the anisotropy of transverse proton spin relaxation in bovine articular cartilage

Anisotropy of transverse proton spin relaxation in collagen-rich tissues like cartilage and tendon is a well-known phenomenon that manifests itself as the "magic-angle" effect in magnetic resonance images of these tissues. It is usually attributed to the non-zero averaging of intra-molecular dipolar interactions in water molecules bound to oriented collagen fibers. One way to manipulate the contributions of these interactions to spin relaxation is by partially replacing the water in the cartilage sample with deuterium oxide. It is known that dipolar interactions in deuterated solutions are weaker, resulting in a decrease in proton relaxation rates. In this work, we investigate the effects of deuteration on the longitudinal and the isotropic and anisotropic contributions to transverse relaxation of water protons in bovine articular cartilage. We demonstrate that the anisotropy of transverse proton spin relaxation in articular cartilage is independent of the degree of deuteration, bringing into question some of the assumptions currently held over the origins of relaxation anisotropy in oriented tissues.

Chondrocyte redifferentiation and construct mechanical property development in single-component photocrosslinkable hydrogels

Hydrogels are promising materials for cartilage repair, but the properties required for optimal functional outcomes are not yet known. In this study, we functionalized four materials that are commonly used in cartilage tissue engineering and evaluated them using in vitro cultures. Gelatin, hyaluronic acid, polyethylene glycol, and alginate were functionalized with methacrylic anhydride to make them photocrosslinkable. We found that the responses of encapsulated human chondrocytes were highly dependent on hydrogel type. Gelatin hydrogels supported cell proliferation and the deposition of a glycosaminoglycan rich matrix with significant mechanical functionality. However, cells had a dedifferentiated phenotype, with high expression of collagen type I. Chondrocytes showed the best redifferentiation in hyaluronic acid hydrogels, but the newly formed matrix was highly localized to the pericellular regions, and these gels degraded rapidly. Polyethylene glycol hydrogels, as a bioinert control, did not promote any strong responses. Alginate hydrogels did not support the deposition of new matrix, and the stiffness decreased during culture. The markedly different response of chondrocytes to these four photocrosslinkable hydrogels demonstrates the importance of material properties for chondrogenesis and extracellular matrix production, which are critical for effective cartilage repair.

FGF-2 induces the proliferation of human periodontal ligament cells and modulates their osteoblastic phenotype by affecting Runx2 expression in the presence and absence of osteogenic inducers

The exact phenotype of human periodontal ligament cells (hPDLCs) remains a controversial area. Basic fibroblast growth factor (FGF‑2) exhibits various functions and its effect on hPDLCs is also controversial. Therefore, the present study examined the effect of FGF‑2 on the growth and osteoblastic phenotype of hPDLCs with or without osteogenic inducers (dexamethasone and β‑glycerophosphate). FGF‑2 was added to defined growth culture medium and osteogenic inductive culture medium. Cell proliferation, osteogenic differentiation and mineralization were measured. The selected differentiation markers, Runx2, collagen type Ⅰ, α1 (Col1a1), osteocalcin (OCN) and epidermal growth factor receptor (EGFR), were investigated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Runx2 and OCN protein expression was measured by western blotting. FGF‑2 significantly increased the proliferation of hPDLCs, but did not affect alkaline phosphatase activity. RT‑qPCR analysis revealed enhanced mRNA expression of Runx2, OCN and EGFR, but suppressed Col1a1 gene expression in the absence of osteogenic inducers, whereas all these gene levels had no clear trend in their presence. The Runx2 protein expression was clearly increased, but the OCN protein level showed no evident trend. The mineralization assay demonstrated that FGF‑2 inhibited mineralized matrix deposition with osteogenic inducers. These results suggested that FGF‑2 induces the growth of immature hPDLCs, which is a competitive inhibitor of epithelial downgrowth, and suppresses their differentiation into mineralized tissue by affecting Runx2 expression. Therefore, this may lead to the acceleration of periodontal regeneration.

Functional and mechanistic investigation of Shikonin in scarring

Scarring is a significant medical burden; financially to the health care system and physically and psychologically for patients. Importantly, there have been numerous case reports describing the occurrence of cancer in burn scars. Currently available therapies are not satisfactory due to their undesirable side-effects, complex delivery routes, requirements for long-term use and/or expense. Radix Arnebiae (Zi Cao), a perennial herb, has been clinically applied to treat burns and manage scars for thousands of years in Asia. Shikonin, an active component extracted from Radix Arnebiae, has been demonstrated to induce apoptosis in cancer cells. Apoptosis is an essential process during scar tissue remodelling. It was therefore hypothesized that Shikonin may induce apoptosis in scar-associated cells. This investigation presents the first detailed in vitro study examining the functional responses of scar-associated cells to Shikonin, and investigates the mechanisms underlying these responses. The data obtained suggests that Shikonin inhibits cell viability and proliferation and reduces detectable collagen in scar-derived fibroblasts. Further investigation revealed that Shikonin induces apoptosis in scar fibroblasts by differentially regulating the expression of caspase 3, Bcl-2, phospho-Erk1/2 and phospho-p38. In addition, Shikonin down-regulates the expression of collagen I, collagen III and alpha-smooth muscle actin genes hence attenuating collagen synthesis in scar-derived fibroblasts. In summary, it is demonstrated that Shikonin induces apoptosis and decreases collagen production in scar-associated fibroblasts and may therefore hold potential as a novel scar remediation therapy.