An electrospun polycaprolactone–collagen membrane for the resurfacing of cartilage defects

A polycaprolactone (PCL)–collagen electrospun mesh is proposed as a novel alternative to the conventional periosteal graft in autologous chondrocyte implantation. This is the first known attempt in designing a cartilage resurfacing membrane using a mechanically resilient PCL mesh with a weight-average molecular weight of 139 300 that is enhanced with bioactive collagen. PCL–collagen 10, 20 and 40% electrospun meshes (Coll-10, Coll-20 and Coll-40) were evaluated and it was discovered that the retention of surface collagen could only be achieved in Coll-20 and Coll-40. Furthermore Coll-20 was stiffer and stronger than Coll-40 and it satisfied the mechanical demands at the cartilage implant site. When seeded with mesenchymal stem cells (MSCs), the cells adhered on the surface of the Coll-20 mesh and they remained viable over a period of 28 days; however, they were unable to infiltrate through the dense meshwork. Cell compatibility was also noted in the chondrogenic environment as the MSCs differentiated into chondrocytes with the expression of Sox9, aggrecan and collagen II. More importantly, the mesh did not induce a hypertrophic response from the cells. The current findings support the use of Coll-20 as a cartilage patch, and future implantation studies are anticipated.

Chlamydia muridarum major outer membrane protein-specific antibodies inhibit in vitro infection but enhance pathology in vivo

PROBLEM Chlamydia trachomatis is a significant worldwide health problem, and the often-asymptomatic disease can result in infertility. To develop a successful vaccine, a complete understanding of the immune response to chlamydial infection and development of genital tract pathology is required. METHOD OF STUDY We utilized the murine genital model of chlamydial infection. Mice were immunized with chlamydial major outer membrane protein, and vaginal lavage was assessed for the presence of neutralizing antibodies. These samples were then pre-incubated with Chlamydia muridarum and administered to the vaginal vaults of immune-competent female BALB/c mice to determine the effect on infection. RESULTS The administration of C. muridarum in conjunction with neutralizing antibodies reduced the numbers of mice infected, but a surprising finding was that this accelerated the development of severe oviduct pathology. CONCLUSION Antibodies play an under-recognized role in chlamydial infection and pathology development, which possibly involves interaction with Th1 immunity.

Modulation of depth-dependent properties in tissue-engineered cartilage with a semi-permeable membrane and perfusion : a continuum model of matrix metabolism and transport

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57-73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue-engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.

The waddlia genome: A window into chlamydial biology

Growing evidence suggests that a novel member of the Chlamydiales order, Waddlia chondrophila, is a potential agent of miscarriage in humans and abortion in ruminants. Due to the lack of genetic tools to manipulate chlamydia, genomic analysis is proving to be the most incisive tool in stimulating investigations into the biology of these obligate intracellular bacteria. 454/Roche and Solexa/Illumina technologies were thus used to sequence and assemble de novo the full genome of the first representative of the Waddliaceae family, W. chondrophila. The bacteria possesses a 2′116′312bp chromosome and a 15′593 bp low-copy number plasmid that might integrate into the bacterial chromosome. The Waddlia genome displays numerous repeated sequences indicating different genome dynamics from classical chlamydia which almost completely lack repetitive elements. Moreover, W. chondrophila exhibits many virulence factors also present in classical chlamydia, including a functional type III secretion system, but also a large complement of specific factors for resistance to host or environmental stresses. Large families of outer membrane proteins were identified indicating that these highly immunogenic proteins are not Chlamydiaceae specific and might have been present in their last common ancestor. Enhanced metabolic capability for the synthesis of nucleotides, amino acids, lipids and other co-factors suggests that the common ancestor of the modern Chlamydiales may have been less dependent on their eukaryotic host. The fine-detailed analysis of biosynthetic pathways brings us closer to possibly developing a synthetic medium to grow W. chondrophila, a critical step in the development of genetic tools. As a whole, the availability of the W. chondrophila genome opens new possibilities in Chlamydiales research, providing new insights into the evolution of members of the order Chlamydiales and the biology of the Waddliaceae.

Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia. Conclusion We describe an Australian origin for B. pseudomallei, characterized by a single introduction event into Southeast Asia during a recent glacial period, and variable levels of lateral gene transfer within populations. These patterns provide insights into mechanisms of genetic diversification in B. pseudomallei and its closest relatives, and provide a framework for integrating the traditionally separate fields of population genetics and phylogenetics for other bacterial species with high levels of lateral gene transfer.

Partial protection against chlamydial reproductive tract infection by a recombinant major outer membrane protein/CpG/cholera toxin intranasal vaccine in the guinea pig Chlamydia cavaie model

Chlamydia trachomatis is a major cause of sexually transmitted diseases worldwide. There currently is no vaccine to protect against chlamydial infection of the female reproductive tract. Vaccine development has predominantly involved using the murine model, however infection of female guinea pigs with Chlamydia caviae more closely resembles chlamydial infection of the human female reproductive tract, and presents a better model to assess potential human chlamydial vaccines. We immunised female guinea pigs intranasally with recombinant major outer membrane protein (r-MOMP) combined with CpG-10109 and cholera toxin adjuvants. Both systemic and mucosal immune responses were elicited in immunised animals. MOMP-specific IgG and IgA were present in the vaginal mucosae, and high levels of MOMP-specific IgG were detected in the serum of immunised animals. Antibodies from the vaginal mucosae were also shown to be capable of neutralising C. caviae in vitro. Following immunisation, animals were challenged intravaginally with a live C. caviae infection of 102 inclusion forming units. We observed a decrease in duration of infection and a significant (p<0.025) reduction in infection load in r-MOMP immunised animals, compared to animals immunised with adjuvant only. Importantly, we also observed a marked reduction in upper reproductive tract (URT) pathology in r-MOMP immunised animals. Intranasal immunisation of female guinea pigs with r-MOMP was able to provide partial protection against C. caviae infection, not only by reducing chlamydial burden but also URT pathology. This data demonstrates the value of using the guinea pig model to evaluate potential chlamydial vaccines for protection against infection and disease pathology caused by C. trachomatis in the female reproductive tract.

Occurrence of iron and associated bacterial populations in soils of a forested subtropical coastal catchment

Iron (Fe) biogeochemistry is potentially of environmental significance in plantation-forested, subtropical coastal ecosystems where soil disturbance and seasonal water logging may lead to elevation of Fe mobilization and associated water quality deterioration. Using wet-chemical extraction and laboratory cultivation, we examined the occurrence of Fe forms and associated bacterial populations in diverse soils of a representative subtropical Australian coastal catchment (Poona Creek). Total reactive Fe was abundant throughout 0e30 cm soil cores, consisting primarily of crystalline forms in well-drained sand soils and water-logged loam soils, whereas in water-logged, low clay soils, over half of total reactive Fe was present in poorly-crystalline forms due to organic and inorganic complexation, respectively. Forestry practices such as plantation clear-felling and replanting, seasonal water logging and mineral soil properties significantly impacted soil organic carbon (C), potentially-bioavailable Fe pools and densities of S-, but not Fe-, bacterial populations. Bacterial Fe(III) reduction and abiotic Fe(II) oxidation, as well as chemolithotrophic S oxidation and aerobic, heterotrophic respiration were integral to catchment terrestrial FeeC cycling. This work demonstrates bacterial involvement in terrestrial Fe cycling in a subtropical coastal circumneutral-pH ecosystem.