The Influence of Specific Bioactive Collagen Peptides on Knee Joint Discomfort in Young Physically Active Adults: A Randomized Controlled Trial.

First evidence indicates that the supplementation of specific collagen peptides is associated with a significant reduction in activity-related joint pain in young adults. The purpose of the current investigation was to confirm the efficacy of the same collagen peptides in a comparable study population. In total, 180 active men and women aged between 18 and 30 years with exercise-related knee pain but no diagnosed joint disease completed the trial over a period of 12 weeks. Participants were randomly assigned to the group receiving 5 g of specific collagen peptides (CP-G) or to the placebo group (P-G). For the primary outcome, changes in pain during or after exercise from pre- to post-intervention were assessed by the participants using the Visual Analog Scale (VAS). These changes were additionally evaluated by the examining physician by means of anamnesis and physical examination of the affected knee joint. As secondary outcomes, pain under resting conditions and after 20 squats were compared between the study groups. In addition, the mobility of the knee joint and the use of alternative therapies (e.g., ointments or physiotherapy) were recorded. The supplementation of specific collagen peptides derived from type I collagen with a mean molecular weight of 3 kDa led to a significantly (p = 0.024) higher reduction of exercise-induced knee pain (-21.9 ± 18.3 mm) compared with the placebo group (-15.6 ± 18.5 mm). These findings were consistent with the physician's evaluation (-23.0 ± 19.2 mm vs. -14.6 ± 17.9 mm, p = 0.003). The decrease in pain under resting conditions and after squats did not significantly differ between the groups, as only a small number of participants suffered from pain under these conditions. Due to the clinically unremarkable baseline values, the mobility of the knee joint did not change significantly after the intervention. In conclusion, the current investigation confirmed that the oral intake of bioactive collagen peptides used in the current investigation led to a statistically significant reduction of activity-related joint pain in young active adults suffering from knee joint discomfort.

Evaluation of collagen type I scaffolds including gelatin-collagen microparticles and Aloe vera in a model of full-thickness skin wound.

Research on collagen type I scaffolds with Aloe vera is sparse. The aim of this work was to develop collagen type I scaffolds with gelatin-collagen microparticles and loaded with a dispersion of A. vera, to assess their performance as grafting material for healing of skin wounds. Scaffolds were evaluated in a Cavia porcellus model with full-thickness skin wound and compared with wounds healed by secondary intention (controls). Animals grafted with scaffolds without A. vera and their control wounds were also included in the study. Evaluation of enzymatic degradation and percentage of the scaffolds' free amino groups-as an indirect assessment of their cross-linking-were also carried out because A. vera contains compounds which affect their stability. We found that dispersions of lyophilized A. vera extract loaded on scaffolds do not have cytotoxic potential, and they decrease collagenase degradation of scaffolds in the range of 0.1 to 0.3% w/v in a dose-dependent manner. Only the A. vera dispersion with the highest concentration (0.3% w/v) decreased the percentage of free amino groups, which are the ones involved in the cross-link of collagen fibers. This finding suggests that cross-linking is not the mechanism by which the tested dispersions stabilize the scaffolds. Preclinical, histochemical, and histomorphometric analyses of repaired wound tissue indicate that loading collagen type I scaffolds, including microparticles of gelatin-collagen, with A. vera in the concentrations tested does not improve wound healing. Low biodegradability of the tested scaffolds caused by the inhibition of collagenase activity might account for these results.

Collagen from Cartilaginous Fish By-Products for a Potential Application in Bioactive Film Composite.

The acid solubilised collagen (ASC) and pepsin solubilised collagen (PSC) were extracted from the by-products (skin) of a cartilaginous fish (Mustelus mustelus). The ASC and PSC yields were 23.07% and 35.27% dry weight, respectively and were identified as collagen Type I with the presence of α, β and γ chains. As revealed by the Fourier Transform Infrared (FTIR) spectra analysis, pepsin did not alter the PSC triple helix structure. Based on the various type of collagen yield, only PSC was used in combination with chitosan to produce a composite film. Such film had lower tensile strength but higher elongation at break when compared to chitosan film; and lower water solubility and lightness when compared to collagen film. Equally, FTIR spectra analysis of film composite showed the occurrence of collagen-chitosan interaction resulting in a modification of the secondary structure of collagen. Collagen-chitosan-based biofilm showed a potential UV barrier properties and antioxidant activity, which might be used as green bioactive films to preserve nutraceutical products.

A novel variant of osteogenesis imperfecta type IV and low serum phosphorus level caused by a Val94Asp mutation in COL1A1.

Osteogenesis imperfecta (OI) is a rare congenital disorder characterized by bone fragility and fractures, and associated with bone deformity, short stature, dentin, ligament and blue­gray eye sclera. OI is caused by a heterozygous mutation in collagen α­1(I) chain (COL1A1) or collagen α­2(I) chain (COL1A2) genes that encode α chains of type I collagen. Collagen α chain peptide contains an N­propeptide, which has a role in assembly and processing of collagen. Point mutations in the N­propeptide domain appear to trigger OI. In the present study, a novel heterozygous missense mutation, c.281T>A (p.Val94Asp), was identified in the von Willebrand C domain of N­terminal of type I collagen in an individual with type IV OI. The majority of N­terminal mutations are associated with OI/Ehlers­Danlos syndrome (EDS); however, in the present study, the affected individual did not suffer from EDS and the level of serum phosphorus of the patient was low (0.67 mmol/l). A number of clinical phenotypes were observed at the same variation site or in the same region on the polypeptide chain of COL1A, which suggests that additional genetic and environmental factors may influence the severity of OI. The present study may provide insight into the phenotype­genotype association in collagen-associated diseases and improve clinical diagnosis of OI.

Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment.

Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

Regulation of matrix metalloproteinase secretion by green tea catechins in a three-dimensional co-culture model of macrophages and gingival fibroblasts.

OBJECTIVES: Elevated levels of matrix metalloproteinases (MMPs) have been associated with the active phases of tissue and bone destruction in periodontitis, an inflammatory disease characterized by a significant breakdown of tooth support. In the present study, we used a three-dimensional (3D) co-culture model of macrophages and gingival fibroblasts to investigate the ability of a green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) to regulate the secretion of MMP-3, -8, and -9. METHODS: The 3D co-culture model was composed of gingival fibroblasts embedded in a type I collagen matrix overlaid with macrophages. Two arbitrary ratios were tested. The ratio composed of 1 macrophage to 10 fibroblasts was used to mimic a slightly inflamed periodontal site while the ratio composed of 10 macrophages to 1 fibroblast was used to mimic a severely inflamed periodontal site. The 3D co-culture model was pre-treated for 2h with either the green tea extract or EGCG. It was then stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). The model was also first stimulated with LPS for 2h and then incubated with the green tea extract or EGCG. The concentrations of secreted MMP-3, -8, and -9 were quantified by enzyme-linked immunoassays. RESULTS: When the 3D co-culture model was stimulated with A. actinomycetemcomitans LPS, the 10:1 ratio of macrophages to gingival fibroblasts was associated with a highest secretion of MMP-3 and -9 and, to a lesser extent, MMP-8, than the 1:10 ratio. Non-cytotoxic concentrations of the green tea extract or EGCG reduced the basal secretion levels of all three MMPs. A 2-h treatment with the green tea extract or EGCG prior to the stimulation with LPS resulted in a dose-dependent decrease in MMP secretion, with MMP-9 showing the most significant decrease. A decrease in MMP secretion was also observed when the green tea extract or EGCG was added following a 2-h stimulation with LPS. CONCLUSIONS: Our results suggested that green tea catechins, and more specifically EGCG, offer promising prospects for the development of a novel adjunctive treatment for periodontitis because of their ability to decrease the secretion of MMPs, which are important tissue-destructive enzymes produced by mucosal and immune cells.

The effect of SDF-1α on low dose BMP-2 mediated bone regeneration by release from heparinized mineralized collagen type I matrix scaffolds in a murine critical size bone defect model.

The treatment of critical size bone defects represents a challenge. The growth factor bone morphogenetic protein 2 (BMP-2) is clinically established but has potentially adverse effects when used at high doses. The aim of this study was to evaluate if stromal derived factor-1 alpha (SDF-1α) and BMP-2 released from heparinized mineralized collagen type I matrix (MCM) scaffolds have a cumulative effect on bone regeneration. MCM scaffolds were functionalized with heparin, loaded with BMP-2 and/or SDF-1α and implanted into a murine critical size femoral bone defect (control group, low dose BMP-2 group, low dose BMP-2 + SDF-1α group, and high dose BMP-2 group). After 6 weeks, both the low dose BMP-2 + SDF-1α group (5.8 ± 0.6 mm³, p = 0.0479) and the high dose BMP-2 group (6.5 ± 0.7 mm³, p = 0.008) had a significantly increased regenerated bone volume compared to the control group (4.2 ± 0.5 mm³). There was a higher healing score in the low dose BMP-2 + SDF-1α group (median grade 8; Q1-Q3 7-9; p = 0.0357) than in the low dose BMP-2 group (7; Q1-Q3 5-9) histologically. This study showed that release of BMP-2 and SDF-1α from heparinized MCM scaffolds allows for the reduction of the applied BMP-2 concentration since SDF-1α seems to enhance the osteoinductive potential of BMP-2. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2126-2134, 2016.

Odontogenic Differentiation of Human Dental Pulp Stem Cells on Hydrogel Scaffolds Derived from Decellularized Bone Extracellular Matrix and Collagen Type I.

OBJECTIVES: The aim of this study was to evaluate the level of odontogenic differentiation of dental pulp stem cells (DPSCs) on hydrogel scaffolds derived from bone extracellular matrix (bECM) in comparison to those seeded on collagen I (Col-I), one of the main components of dental pulp ECM. METHODS: DPSCs isolated from human third molars were characterized for surface marker expression and odontogenic potential prior to seeding into bECM or Col-I hydrogel scaffolds. The cells were then seeded onto bECM and Col-I hydrogel scaffolds and cultured under basal conditions or with odontogenic and growth factor (GF) supplements. DPSCs cultivated on tissue culture polystyrene (TCPS) with and without supplements were used as controls. Gene expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE) was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and mineral deposition was observed by Von Kossa staining. RESULTS: When DPSCs were cultured on bECM hydrogels, the mRNA expression levels of DSPP, DMP-1 and MEPE genes were significantly upregulated with respect to those cultured on Col-I scaffolds or TCPS in the absence of extra odontogenic inducers. In addition, more mineral deposition was observed on bECM hydrogel scaffolds as demonstrated by Von Kossa staining. Moreover, DSPP, DMP-1 and MEPE mRNA expressions of DPSCs cultured on bECM hydrogels were further upregulated by the addition of GFs or osteo/odontogenic medium compared to Col-I treated cells in the same culture conditions. SIGNIFICANCE: These results demonstrate the potential of the bECM hydrogel scaffolds to stimulate odontogenic differentiation of DPSCs.

Comparative study on acid-soluble and pepsin-soluble collagens from skin and swim bladder of grass carp (Ctenopharyngodon idella).

BACKGROUND: Collagen has a wide range of applications in food, biomedical and pharmaceutical products. RESULTS: The collagens in grass carp (Ctenopharyngodon idella) skin and swim bladder were extracted using acetic acid and pepsin. Higher yield of pepsin-soluble collagen (PSC) was obtained from skin (178 g kg(-1)) than from swim bladder (114 g kg(-1)). Not surprisingly, yields of PSC from skin and swim bladder were also higher than those of acid-soluble collagen (ASC) from the same organs (89 and 51 g kg(-1)). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that ASC and PSC were type I collagens, with PSC containing a higher proportion of α components than ASC. Fourier transform infrared spectra revealed that ASC and PSC were very similar in their protein secondary structures. Scanning electron micrographs showed that the collagens had a spongy structure, with more pores being obtained in swim bladder than in skin collagens. The collagens showed high solubility in the acidic pH range. However, their solubility decreased in the presence of NaCl at concentrations above 20 g kg(-1). CONCLUSION: Collagens were successfully extracted from the skin and swim bladder of grass carp. These fish by-products could serve as an alternative source of collagens for a wide variety of applications in the food and nutraceutical industries.

The use of collagen-based scaffolds to simulate prostate cancer bone metastases with potential for evaluating delivery of nanoparticulate gene therapeutics.

Prostate cancer bone metastases are a leading cause of cancer-related death in men with current treatments offering only marginally improved rates of survival. Advances in the understanding of the genetic basis of prostate cancer provide the opportunity to develop gene-based medicines capable of treating metastatic disease. The aim of this work was to establish a 3D cell culture model of prostate cancer bone metastasis using collagen-based scaffolds, to characterise this model, and to assess the potential of the model to evaluate delivery of gene therapeutics designed to target bone metastases. Two prostate cancer cell lines (PC3 and LNCaP) were cultured in 2D standard culture and compared to 3D cell growth on three different collagen-based scaffolds (collagen and composites of collagen containing either glycosaminoglycan or nanohydroxyapatite). The 3D model was characterised for cell proliferation, viability and for matrix metalloproteinase (MMP) enzyme and Prostate Specific Antigen (PSA) secretion. Chemosensitivity to docetaxel treatment was assessed in 2D in comparison to 3D. Nanoparticles (NPs) containing siRNA formulated using a modified cyclodextrin were delivered to the cells on the scaffolds and gene silencing was quantified. Both prostate cancer cell lines actively infiltrated and proliferated on the scaffolds. Cell culture in 3D resulted in reduced levels of MMP1 and MMP9 secretion in PC3 cells. In contrast, LNCaP cells grown in 3D secreted elevated levels of PSA, particularly on the scaffold composed of collagen and glycosaminoglycans. Both cell lines grown in 3D displayed increased resistance to docetaxel treatment. The cyclodextrin.siRNA nanoparticles achieved cellular uptake and knocked down the endogenous GAPDH gene in the 3D model. In conclusion, development of a novel 3D cell culture model of prostate cancer bone metastasis has been initiated resulting, for the first time, in the successful delivery of gene therapeutics in a 3D in vitro model. Further enhancement of this model will help elucidate the pathogenesis of prostate cancer and also accelerate the design of effective therapies which can penetrate into the bone microenvironment for prostate cancer therapy.

Controlled release of an extract of Calendula officinalis flowers from a system based on the incorporation of gelatin-collagen microparticles into collagen I scaffolds: design and in vitro performance.

Aiming to develop biological skin dresses with improved performance in the treatment of skin wounds, acellular collagen I scaffolds were modified with polymeric microparticles and the subsequent loading of a hydroglycolic extract of Calendula officinalis flowers. Microparticles made of gelatin-collagen were produced by a water-in-oil emulsion/cross-linking method. Thereafter, these microparticles were mixed with collagen suspensions at three increasing concentrations and the resulting mixtures lyophilized to make microparticle-loaded porous collagen scaffolds. Resistance to enzymatic degradation, ability to associate with the C. officinalis extract, and the extract release profile of the three gelatin-collagen microparticle-scaffold prototypes were assessed in vitro and compared to collagen scaffolds without microparticles used as control. Data indicated that the incorporation of gelatin-collagen microparticles increased the resistance of the scaffolds to in vitro enzymatic degradation, as well as their association with the C. officinalis flower extract. In addition, a sharp decrease in cytotoxicity, as well as more prolonged release of the extract, was attained. Overall results support the potential of these systems to develop innovative dermal substitutes with improved features. Furthermore, the gelatin-collagen mixture represents a low-cost and scalable alternative with high clinical transferability, especially appealing in developing countries.

Collagen-based silver nanoparticles for biological applications: synthesis and characterization.

BACKGROUND: Type I collagen is an abundant natural polymer with several applications in medicine as matrix to regenerate tissues. Silver nanoparticles is an important nanotechnology material with many utilities in some areas such as medicine, biology and chemistry. The present study focused on the synthesis of silver nanoparticles (AgNPs) stabilized with type I collagen (AgNPcol) to build a nanomaterial with biological utility. Three formulations of AgNPcol were physicochemical characterized, antibacterial activity in vitro and cell viability assays were analyzed. AgNPcol was characterized by means of the following: ultraviolet-visible spectroscopy, dynamic light scattering analysis, Fourier transform infrared spectroscopy, atomic absorption analysis, transmission electron microscopy and of X-ray diffraction analysis. RESULTS: All AgNPcol showed spherical and positive zeta potential. The AgNPcol at a molar ratio of 1:6 showed better characteristics, smaller hydrodynamic diameter (64.34 ± 16.05) and polydispersity index (0.40 ± 0.05), and higher absorbance and silver reduction efficiency (0.645 mM), when compared with the particles prepared in other mixing ratios. Furthermore, these particles showed antimicrobial activity against both Staphylococcus aureus and Escherichia coli and no toxicity to the cells at the examined concentrations. CONCLUSIONS: The resulted particles exhibited favorable characteristics, including the spherical shape, diameter between 64.34 nm and 81.76 nm, positive zeta potential, antibacterial activity, and non-toxicity to the tested cells (OSCC).

Characterization of acid-and pepsin-soluble collagens from spines and skulls of skipjack tuna (Katsuwonus pelamis).

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the spine (ASC-SP and PSC-SP) and skull (ASC-SK and PSC-SK) of the skipjack tuna, Katsuwonus pelamis, were successfully isolated and characterized. The yields of ASC-SP, PSC-SP, ASC-SK and PSC-SK were (2.47 ± 0.39)%, (5.62 ± 0.82)%, (3.57 ± 0.40)%, and (6.71 ± 0.81)%, respectively, on the basis of dry weight. The four collagens contained Gly (330.2-339.1 residues/1 000 residues) as the major amino acid, and their imino acid contents were between 168.8 and 178.2 residues/1 000 residues. Amino acid composition, SDS-PAGE, and FTIR investigations confirmed that ASC-SP and ASC-SK were mainly composed of type I collagen, and had higher contents of high-molecular weight cross-links than those of PSC-SK and PSC-SP. The FTIR investigation also certified all the collagens had triple helical structure. The denaturation temperatures of ASC-SK, PSC-SK, ASC-SP, and PSC-SP were 17.8, 16.6, 17.6, and 16.5 °C, respectively. All isolated collagens were soluble at acidic pH (1-5) and lost their solubilities when the NaCl concentration was above 2% (W/V). The isolated collagens from the spines and skulls of skipjack tuna could serve as an alternative source of collagens for further application in food, cosmetic, biomedical, and pharmaceutical industries.

Biomimetic nanocomposites to control osteogenic differentiation of human mesenchymal stem cells.

The design of biomimetic nanomaterials that can directly influence the behavior of cells and facilitate the regeneration of tissues and organs has become an active area of research. Here, the production of materials based on nano-hydroxyapatite composites in scaffolds with nanofibrous and nanoporous topographies, designed to mimic the native bone matrix for applications in bone tissue engineering, is reported. Human mesenchymal stem cells grown on these nanocomposites are stimulated to rapidly produce bone minerals in situ, even in the absence of osteogenic supplements in the cell-culture medium. Nanocomposites comprising type I collagen and nano-hydroxyapatite are found to be especially efficient at inducing mineralization. When subcutaneously implanted into nude mice, this biomimetic nanocomposite is able to form a new bone matrix within only two weeks. Furthermore, when the nanocomposite is enriched with human mesenchymal stem cells before implantation, development of the bone matrix is accelerated to within one week. To the best of the authors' knowledge, this study provides the first clear in vitro and in vivo demonstration of osteoinduction controlled by the material characteristics of a biomimetic nanocomposite. This approach can potentially facilitate the translation of de novo bone-formation technologies to the clinic.

Effects of nanoparticle size and charge on interactions with self-assembled collagen.

HYPOTHESIS: Insights into bone formation have suggested that the critical first step in the biomineralization process is the integration of small (nanometer dimension) mineral clusters into collagen fibers. Not only is such behavior of interest for understanding biomineralization but also should be important to nanotoxicology because collagen is a major component of structural tissues in the human body and accounts for more than 25% of the whole body protein content. Here, utilizing the current insights from biomineralization, we hypothesize that the binding affinity of nanoparticles to self-assembled collagen fibers is size and surface charge dependent. EXPERIMENTS: We developed a self-assembled collagen substrate compatible with Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), which is very sensitive to mechanical changes of the substrate as a consequence of nanoparticle binding. QCM-D experiments were conducted with both positively and negatively charged gold nanoparticles between 2 and 10 nm in size. Complementary ex situ imaging Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) were used to confirm the QCM-D results. FINDINGS: We find that both positively and negatively charged nanoparticles of all sizes exhibited binding affinity for self-assembled collagen fibers. Furthermore, the smallest particles (2 nm) mechanically integrated with collagen fibers.

Alternate dipping preparation of biomimetic apatite layers in the presence of carbonate ions.

The classical simulated body fluids method cannot be employed to prepare biomimetic apatites encompassing metallic ions that lead to very stable phosphates. This is the case for heavy metals such as uranium, whose presence in bone mineral after contamination deserves toxicological study. We have demonstrated that existing methods, based on alternate dipping into calcium and phosphate ions solutions, can be adapted to achieve this aim. We have also especially studied the impact of the presence of carbonate ions in the medium as these are necessary to avoid hydrolysis of the contaminating metallic cations. Both the apatite-collagen complex method and a standard chemical (STD) method employing only mineral solutions lead to biomimetic apatites when calcium and carbonate ions are introduced simultaneously. The obtained materials were fully characterized and we established that the STD method tolerates the presence of carbonate ions much better, and this leads to homogeneous samples. Emphasis was set on the repeatability of the method to ensure the relevancy of further work performed on series of samples. Finally, osteoblasts cultured on these samples also proved a similar yield and standard-deviation in their adenosine triphosphate content when compared to commercially available substrates designed to study of such cell cultures.

Hydroxyapatite incorporated into collagen gels for mesenchymal stem cell culture.

Collagen gels could be used as carriers in tissue engineering to improve cell retention and distribution in the defect. In other respect hydroxyapatite could be added to gels to improve mechanical properties and regulate gel contraction. The aim of this work was to analyze the feasibility to incorporate hydroxyapatite into collagen gels and culture mesenchymal stem cells inside it. Human bone marrow mesenchymal stem cells (hMSC-BM) were used in this study. Gels were prepared by mixing rat tail type I collagen, hydroxyapatite microparticles and MSCs. After polymerization gels were kept in culture while gel contraction and mechanical properties were studied. In parallel, cell viability and morphology were analyzed. Gels became free-floating gels contracted from day 3, only in the presence of cells. A linear rapid contraction phase was observed until day 7, then a very slow contraction phase took place. The incorporation of hydroxyapatite improved gel stability and mechanical properties. Cells were randomly distributed on the gel and a few dead cells were observed all over the experiment. This study shows the feasibility and biocompatibility of hydroxyapatite supplemented collagen gels for the culture of mesenchymal stem cells that could be used as scaffolds for cell delivery in osteoarticular regenerative medicine.

Ex vivo killing of Enterococcus faecalis and mixed plaque bacteria in planktonic and biofilm culture by modified photoactivated disinfection.

AIM: To compare the efficacy of conventional and modified photoactivated disinfection (PAD) against Enterococcus faecalis and mixed plaque bacteria in suspension and biofilms. METHODOLOGY: Enterococcus faecalis (four strains) and mixed plaque bacteria from three adult volunteers were suspended in water, added to methylene blue (MB, 15 µmol L⁻¹), MB mixed with 0.5% hydrogen peroxide and 0.05% chlorhexidine (CHX), MB mixed with 0.5% hydrogen peroxide and 0.05% EDTA or MB mixed with 0.05% EDTA and 0.05% CHX and exposed to laser irradiation from 10 s to 5 min. After exposure, samples were taken, serially diluted and grown aerobically and anaerobically on Tryptic Soy Agar plates or on blood agar plates for 24 and 72 h, respectively. For biofilm experiments, E. faecalis and mixed plaque biofilms were grown on sterile hydroxyapatite (HA) discs coated overnight with bovine dermal collagen type I for 3 weeks. After exposure to MB or MB and low concentration of EDTA with either hydrogen peroxide or CHX, the percentage of killed bacteria by PAD was evaluated using viability staining and confocal laser scanning microscope. For statistical analysis, one-way analysis of variance was performed. RESULTS: Conventional PAD killed from 90.76% to 100% E. faecalis for 3 min, but failed to kill all plaque bacteria even after 5 min of laser irradiation. In modified PAD, up to 100% of suspended E. faecalis and mixed plaque bacteria were killed after 1 min and 30 s of irradiation. Up to twenty times more biofilm bacteria were killed by modified PAD than by conventional PAD with 15 µmol L⁻¹ MB (P < 0.001) and up to eight times more than 2% CHX (P < 0.001) and 1% sodium hypochlorite (P < 0.001). CONCLUSION: Modified PAD was superior to conventional PAD against planktonic and biofilm bacteria.

Collagen type II, alpha 1 protein: a bioactive component of shark cartilage.

Previous studies have shown that extracts of shark cartilage induce a cytokine response in human leukocytes, but the nature of the bioactive component(s) is unknown. Extracts treated with proteases lost 80% of their cytokine-inducing property, suggesting that the active component(s) was likely a complex protein. The aim of the present study was to determine the nature of the bioactive molecule(s). Solid phase extraction followed by ion exchange chromatography and electrophoretic separation were used to partially purify a bioactive preparation from commercial shark cartilage that has been identified as a small glycoprotein. LC-MS analysis yielded peptides with 100% molecular identity with collagen type II, alpha I protein from the lesser spotted catshark, Scyliorhinus canicula. The implications for the consumption of shark cartilage as a dietary supplement are discussed given the presence of collagen type II, alpha 1 protein in extracts.

Examining dietary variability of the earliest farmers of south-eastern Italy.

Stable isotope analysis of human remains has been used to address long-standing debates regarding the speed and degree to which the introduction of farming transformed diet. In Europe, this debate has centered on northern and Atlantic regions with much less attention devoted to the arrival of farming across the Mediterranean. This study presents carbon and nitrogen stable isotope analyses of collagen from 19 human and 37 faunal remains from eight sites in the Apulia and Marche regions of south-eastern and central Italy, dating to the early phases of agricultural adoption during the first half of the 6th Millennium BC. Where collagen preservation permitted, sulfur stable isotope analysis was also performed. Overall, there was significant isotopic variation between the different geographic regions, although there was also considerable uncertainty in interpreting these data, especially given heterogeneous isotope values for fauna from site to site. By considering isotope data from each region separately, it was noticeable that the degree of carbon isotope enrichment in humans compared to fauna was higher for individuals buried near the coast, consistent with increased marine consumption. Coastal individuals also had higher sulfur isotope values. Nitrogen isotope values were very variable between individuals and regions and, in some cases, were consistent with very high plant food consumption. Overall, early "farmers" in south-east and central Italy consumed a wide range of foods, including marine, and had much more variable stable isotope values than those observed in central and northern Europe during this period, perhaps indicating a different mode for agricultural adoption.