Application of Mesenchymal Stem Cells for repair and regeneration of cartilage and bone

Australian efforts to provide orthopaedic surgeons with living, load-bearing scaffolds suitable for current joint (knee and hip) replacement surgery, non-union fracture repair, and miniscal and growth plate cartilage regeneration are being lead by teams at the Institute for Medical and Veterinary Science and Women's and Children's Hospital in Adelaide; the Peter MacCallum and St Vincent's Medical Research Institutes in Melbourne; and the Mater Medical Research Institute and new Institute for Health and Biomedical Innovation at QUT, Brisbane. In each case multidisciplinary teams are attempting to develop autologous living tissue constructs, utilising mesenchymal stem cells (MSC), with the intention of effecting seamless repair and regeneration of skeletal trauma and defects. In this article we will briefly review current knowledge of the phenotypic properties of MSC and discuss the potential therapeutic applications of these cells as exemplified by their use in cartilage repair and tissue engineering based approaches to the treatment of skeletal defects.

Biomaterial scaffolds in cartilage-subchondral bone defects influencing the repair of autologous articular cartilage transplants

The repair of articular cartilage typically involves the repair of cartilage-subchondral bone tissue defects. Although various bioactive materials have been used to repair bone defects, how these bioactive materials in subchondral bone defects influence the repair of autologous cartilage transplant remains unclear. The aim of this study was to investigate the effects of different subchondral biomaterial scaffolds on the repair of autologous cartilage transplant in a sheep model. Cylindrical cartilage-subchondral bone defects were created in the right femoral knee joint of each sheep. The subchondral bone defects were implanted with hydroxyapatite-β-tricalcium phosphate (HA-TCP), poly lactic-glycolic acid (PLGA)-HA-TCP dual-layered composite scaffolds (PLGA/HA-TCP scaffolds), or autologous bone chips. The autologous cartilage layer was placed on top of the subchondral materials. After three months, the effect of different subchondral scaffolds on the repair of autologous cartilage transplant was systematically studied by investigating the mechanical strength, structural integration and histological responses. The results showed that the transplanted cartilage layer supported by HA-TCP scaffolds had better structural integration and higher mechanical strength than that supported by PLGA/HA-TCP scaffolds. Furthermore, HA-TCP supported cartilage showed higher expression of acid mucosubstances and glycol-amino-glycan (GAG) contents than that supported by PLGA/HA-TCP scaffolds. Our results suggested that the physicochemical properties, including the inherent mechanical strength and material chemistry of the scaffolds, play important roles in influencing the repair of autologous cartilage transplants. The study may provide useful information for the design and selection of proper subchondral biomaterials to support the repair of both subchondral bone and cartilage defects.

Clinical strategies for the alleviation of contractures from a predictive mathematical model of dermal repair

Hypertrophic scars arise when there is an overproduction of collagen during wound healing. These are often associated with poor regulation of the rate of programmed cell death(apoptosis) of the cells synthesizing the collagen or by an exuberant inflammatory response that prolongs collagen production and increases wound contraction. Severe contractures that occur, for example, after a deep burn can cause loss of function especially if the wound is over a joint such as the elbow or knee. Recently, we have developed a morphoelastic mathematical model for dermal repair that incorporates the chemical, cellular and mechanical aspects of dermal wound healing. Using this model, we examine pathological scarring in dermal repair by first assuming a smaller than usual apoptotic rate for myofibroblasts, and then considering a prolonged inflammatory response, in an attempt to determine a possible optimal intervention strategy to promote normal repair, or terminate the fibrotic scarring response. Our model predicts that in both cases it is best to apply the intervention strategy early in the wound healing response. Further, the earlier an intervention is made, the less aggressive the intervention required. Finally, if intervention is conducted at a late time during healing, a significant intervention is required; however, there is a threshold concentration of the drug or therapy applied, above which minimal further improvement to wound repair is obtained.

High-resolution DNA melt curve analysis of the clustered, regularly interspaced short-palindromic-repeat locus of Campylobacter jejuni

A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of Campylobacter jejuni is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing C. jejuni genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species

The duplicitous nature of inflammation in wound repair

Skin plays a key role in protecting the body from the onslaught of pathogens and toxins we meet during our lifetime; thus, out of necessity, we have developed a rapid repair mechanism that quickly plugs any holes in this vital organ. Upon injury, a series of highly coordinated overlapping events, that include inflammatory, proliferation and maturation phases, result in the hasty closure of the wound and restoration of skin integrity. Over the past decade it has become clear that a number of immune cells that regulate the inflammatory phase, whilst important for removal of invading pathogens, are not necessary for repair and in fact may be responsible for the subsequent scar formation that seems to have resulted from having such a rapid repair process. The magnitude and length of inflammation in the wound not only appears to dictate the extent of scar formation but also in some cases may even prevent wound closure. In this review we will explore the two sides of inflammation in wound healing and review current and future drug therapies that target inflammation to modulate the healing outcome.

Geologically ancient DNA: fact or artefact?

Studies continue to report ancient DNA sequences and viable microbial cells that are many millions of years old. In this paper we evaluate some of the most extravagant claims of geologically ancient DNA. We conclude that although exciting, the reports suffer from inadequate experimental setup and insufficient authentication of results. Consequently, it remains doubtful whether amplifiable DNA sequences and viable bacteria can survive over geological timescales. To enhance the credibility of future studies and assist in discarding false-positive results, we propose a rigorous set of authentication criteria for work with geologically ancient DNA.

Combined mitochondrial and nuclear DNA sequences resolve the interrelations of the major Australasian marsupial radiations

Australasian marsupials include three major radiations, the insectivorous/carnivorous Dasyuromorphia, the omnivorous bandicoots (Peramelemorphia), and the largely herbivorous diprotodontians. Morphologists have generally considered the bandicoots and diprotodontians to be closely related, most prominently because they are both syndactylous (with the 2nd and 3rd pedal digits being fused). Molecular studies have been unable to confirm or reject this Syndactyla hypothesis. Here we present new mitochondrial (mt) genomes from a spiny bandicoot (Echymipera rufescens) and two dasyurids, a fat-tailed dunnart (Sminthopsis crassicaudata) and a northern quoll (Dasyurus hallucatus). By comparing trees derived from pairwise base-frequency differences between taxa with standard (absolute, uncorrected) distance trees, we infer that composition bias among mt protein-coding and RNA sequences is sufficient to mislead tree reconstruction. This can explain incongruence between trees obtained from mt and nuclear data sets. However, after excluding major sources of compositional heterogeneity, both the “reduced-bias” mt and nuclear data sets clearly favor a bandicoot plus dasyuromorphian association, as well as a grouping of kangaroos and possums (Phalangeriformes) among diprotodontians. Notably, alternatives to these groupings could only be confidently rejected by combining the mt and nuclear data. Elsewhere on the tree, Dromiciops appears to be sister to the monophyletic Australasian marsupials, whereas the placement of the marsupial mole (Notoryctes) remains problematic. More generally, we contend that it is desirable to combine mt genome and nuclear sequences for inferring vertebrate phylogeny, but as separately modeled process partitions. This strategy depends on detecting and excluding (or accounting for) major sources of nonhistorical signal, such as from compositional nonstationarity.

Isolation and characterisation of novel microsatellite and mitochondrial DNA markers for the Eastern Water Dragon (Physignathus lesueurii)

Habitat fragmentation as a result of urbanisation is a growing problem for native lizard species. The Eastern Water Dragon (Physignathus lesueurii) is a social arboreal agamid lizard, native to Australia. This species represents an ideal model species to investigate the effect of urbanisation because of their prominent abundance in the urban landscape. Here we describe the isolation and characterisation of a novel set of 74 di-, tri-, and tetramicrosatellites from which 18 were selected and optimised into two multiplexes. The 18 microsatellites generated a total 148 alleles across the two populations. The number of alleles per locus varied from 2 to 18 alleles and measures of Ho and He varied from 0.395 to 0.877 and from 0.441 to 0.880, respectively. We also present primers for four novel mitochondrial DNA (mtDNA) markers. The combined length of the four mtDNA marker pairs was 2,528 bp which included 15 nucleotides changes. In comparison to threatened species, which are generally characterised by small population sizes, the Eastern Water Dragon represents an ideal model species to investigate the effect of urbanisation on their behavioural ecology and connectivity patterns among populations.

Identification of bifunctional GA and AG intrastrand crosslinks formed between cisplatin and DNA

A combination of enzymatic digestion and electrospray ionisation mass spectrometry (ESI-MS) was used to characterise bifunctional adducts in which cisplatin is bound to GA base sequences in 8mer and 16mer oligonucleotides that do not contain other, higher affinity binding sites. The extent of formation of bifunctional adducts with GA base sequences was significant, but less than that seen with similar oligonucleotides containing either AG or GG sequences.

Comparison of the binding stoichiometries of positively charged DNA-binding drugs using positive and negative ion electrospray ionization mass spectrometry

Positive and negative ion electrospray ionization (ESI) mass spectra of complexes of positively charged small molecules (distamycin, Hoechst 33258, [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2) have been compared. [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2 bind to DNA by intercalation. Negative ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA showed ions from DNA-ligand complexes consistent with solution studies. In contrast, only ions from freeDNAwere present in positive ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA, highlighting the need for obtaining ESI mass spectra of non-covalent complexes under a range of experimental conditions. Negative ion spectra of mixtures of the minor groove binder Hoechst 33258 with DNA containing a known minor groove binding sequence were dominated by ions from a 1:1 complex. In contrast, in positive ion spectra there were also ions present from a 2:1 (Hoechst 33258: DNA) complex, suggesting an alternative binding mode was possible either in solution or in the gas phase. When Hoechst 33258 was mixed with a DNA sequence lacking a high affinity minor groove binding site, the negative ion ESI mass spectra showed that 1:1 and 2:1 complexes were formed, consistent with existence of binding modes other than minor groove binding. The data presented suggest that comparison of positive and negative ion ESI-MS spectra might provide an insight into various binding modes in both solution and the gas phase.