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Bacterial collagen-like proteins differ from vertebrate collagens in that they do not contain hydroxyproline, which is seen as a characteristic of the vertebrate collagens, and which provides a significant contribution to the stability of the collagen triple-helix at body temperature. Despite this difference, the bacterial collagens are stable at around body temperature through inclusion of other stabilising sequence elements. Another difference is the lack of aggregation, and certain vertebrate collagen binding domains that can be introduced into the bacterial sequence lack full function when hydroxyproline is absent. In the present study we have demonstrated that a simple method utilising co-translational incorporation during fermentation can be used to incorporate hydroxyproline into the recombinant bacterial collagen. The presence and amount of hydroxyproline incorporation was shown by amino acid analysis and by mass spectrometry. A small increase in thermal stability was observed using circular dichroism spectroscopy. STATEMENT OF SIGNIFICANCE: Recombinant bacterial collagens provide a new opportunity for biomedical materials as they are readily produced in large quantity in E. coli. Unlike animal collagens, they are stable without the need for inclusion of a secondary modification system for hydroxyproline incorporation. In animal collagens, however, introduction of hydroxyproline is essential for stability and is also important for functional molecular interactions within the mammalian extracellular matrix. The present study has shown that hydroxyproline can be readily introduced into recombinant S. pyogenes bacterial collagen through direct co-translational incorporation of this modified imino acid during expression using the codons for proline in the introduced gene construct. This hydroxylation further improves the stability of the collagen and is available to enhance any introduced molecular functions.

Mineralization of bone is a dynamic process, involving a complex interplay between cells, secreted macromolecules, signaling pathways, and enzymatic reactions; the dysregulation of bone mineralization may lead to serious skeletal disorders, including hypophosphatemic rickets, osteoporosis, and rheumatoid arthritis. Very few studies have reported the role of osteocytes - the most abundant bone cells in the skeletal system and the major orchestrators of bone remodeling in bone mineralization, which is owed to their nature of being deeply embedded in the mineralized bone matrix. The Wnt/β-catenin signaling pathway is actively involved in various life processes including osteogenesis; however, the role of Wnt/β-catenin signaling in the terminal mineralization of bone, especially in the regulation of osteocytes, is largely unknown. This research demonstrates that during the terminal mineralization process, the Wnt/β-catenin pathway is downregulated, and when Wnt/β-catenin signaling is activated in osteocytes, dendrite development is suppressed and the expression of dentin matrix protein 1 (DMP1) is inhibited. Aberrant activation of Wnt/β-catenin signaling in osteocytes leads to the spontaneous deposition of extra-large mineralized nodules on the surface of collagen fibrils. The altered mineral crystal structure and decreased bonding force between minerals and the organic matrix indicate the inferior integration of minerals and collagen. In conclusion, Wnt/β-catenin signaling plays a critical role in the terminal differentiation of osteocytes and as such, targeting Wnt/β-catenin signaling in osteocytes may serve as a potential therapeutic approach for the management of bone-related diseases.

Focused Ion Beams such as the Helium Ion Microscope (HIM) as well as FIB/SEMs have sparked great interest within the biological sciences in recent years. The HIM allows high resolution imaging of uncoated non-conductive samples while the FIB/SEM (FIB/SEMs combine a focused ion beam with a Scanning Electron Microscope (SEM)) allows to prepare TEM lamellae, 3D reconstruct the sample or reveal sub surface structures with nanometre precision. FIB/SEMs have become the “go to” tool in the materials sciences and semiconductor industry. Despite these unique capabilities, it is not yet fully established in the biological sciences [1-5]. This is predominantly due to the heat-induced damage from the ion beam when processing soft materials including biological samples. This presentation shows how the HIM as well as FIB/SEMs can be used in biological sciences to reveal nature’s tiniest structures (Figure 1). The presented work then focuses on the underlying ion-solid interactions and the effect of processing parameters on heating induced by ion beams. The work presented here deals with gallium ion solid interactions, however the broader results are applicable to any type of FIB including the helium ion microscope (HIM) and plasma FIBs. The interactions of gallium ions in skin were simulated using Monte Carlo methods. The program SRIM [6] was used to obtain theoretical results which permit estimation of the ion beam induced temperature increases, using the physical principles of Fourier’s law of conductive heat transfer. The model suggests that the ion beam induced increase in temperature can be reduced by: 1. Reducing the local dose rate (smaller aperture/ion beam current) 2. Reducing the local dose (smaller aperture/ion beam current, introducing beam blur, reducing the beam overlap) The technique was tested on collagen, a soft biological material which is commonly used in biomedical applications. Collagen was chosen as a suitable test sample as it loses its fibrillary structure when denaturated by heat, permitting damage to easily be recognized. Cross-sections and TEM lamellas were prepared from non-embedded collagen with conventional FIB processing parameters (see Figure 2 left) as well as heat reducing FIB parameters (see Figure 1 right). The results also show that heat damage can be prevented by reducing the local dose rate and area underneath the ion beam. A TEM comparison of a microtome prepared lamella and a FIB prepared lamella (using heat reducing parameters) shows that the fibrillar structures can be maintained, and heat damage avoided. The approach described here can be used to determine suitable parameters for other soft materials. The authors acknowledge scientific and technical assistance of Peter Hines, Jamie Riches, Rachel Hancock, and Ning Liu and the facilities at the Australian Microscopy & Microanalysis Research Facility (AMMRF) at the Central Analytical Research Facility (CARF), Queensland University of Technology, Brisbane, Australia. References: [1] Drobne et al, ‘Surface Damage Induced by FIB Milling and Imaging of Biological Samples is Controllable’; Microscopy Research and Technique 70; 895-903 (2007) [2] Earl et al, ‘Characterization of dentine structure in three dimensions using FIB-SEM’; Journal of Microscopy 240, Pt 1, 1-5 (2010) [3] Schneider et al; ‘Serial FIB/SEM imaging for quantitative 3D assessment of the osteocyte lacuna-canalicular network’; Bone 49, 304-311 (2011) [4] Stokes et al; ‘A New Approach to Study Biological and Soft Materials Using Focused Ion Beam Scanning Electron Microscopy (FIB/SEM)’; Journal of Physics: Conference Series 26; 50-53 (2006) [5] Bandara et al.; ‘Bactericidal Effects of Natural Nanotopography of Dragonfly Wing on Escherichia coli’ ACS Applied Materials & Interfaces 2017 9 (8), 6746-6760 [6] Ziegler et al, www.srim.org

Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017.

A range of non-animal collagens has been described, derived from bacterial species, which form stable triple-helical structures without the need for secondary modification to include hydroxyproline in the sequence. The non-animal collagens studied to date are typically smaller than animal interstitial collagens, around one quarter the length and do not pack into large fibrillar aggregates like those that are formed by the major animal interstitial collagens. A consequence of this for biomedical products is that fabricated items, such as collagen sponges, are not as mechanically and dimensionally stable as those of animal collagens. In the present study, we examined the production of larger, polymeric forms of non-animal collagens through introduction of tyrosine and cysteine residues that can form selective crosslinks through oxidation. These modifications allow the formation of larger aggregates of the non-animal collagens. When Tyr residues were incorporated, gels were obtained. And with Cys soluble aggregates were formed. These materials can be formed into sponges that are more stable than those formed without these modifications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2369-2376, 2016.

Regenerative medicine strategies for restoring articular cartilage face significant challenges to recreate the complex and dynamic biochemical and biomechanical functions of native tissues. As an approach to recapitulate the complexity of the extracellular matrix, collagen-mimetic proteins offer a modular template to incorporate bioactive and biodegradable moieties into a single construct. We modified a Streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate (CS)-binding peptides and then cross-linked with a matrix metalloproteinase 7 (MMP7)-sensitive peptide to form biodegradable hydrogels. Human mesenchymal stem cells (hMSCs) encapsulated in these hydrogels exhibited improved viability and significantly enhanced chondrogenic differentiation compared to controls that were not functionalized with glycosaminoglycan-binding peptides. Hydrogels functionalized with CS-binding peptides also led to significantly higher MMP7 gene expression and activity while the HA-binding peptides significantly increased chondrogenic differentiation of the hMSCs. Our results highlight the potential of this novel biomaterial to modulate cell-mediated processes and create functional tissue engineered constructs for regenerative medicine applications.

Cuvierian tubules are expelled as a defence mechanism against predators by various species within the family Holothuridae. When the tubules are expelled, they become sticky almost immediately and ensnare the predator. The mechanism of this rapid adhesion is not clear, but proteins on the surface of the expelled tubules are widely believed to be involved. This study has examined such proteins from Holothuria dofleinii, sourced from adhesive prints left on glass after the removal of adhered tubules. Gel electrophoresis showed that seven strongly staining protein bands were consistently present in all samples, with molecular masses ranging from 89 to 17 kDa. N-terminal sequence data was obtained from two bands, while others seemed blocked. Tandem mass spectrometry-based sequencing of tryptic peptides derived from individual protein bands indicated that the proteins were unlikely to be homopolymers. PCR primers designed using the peptide sequences enabled us to amplify, clone and sequence cDNA segments relating to four gel bands; for each, the predicted translation product contained other peptide sequences observed for that band that had not been used in primer design. Database searches using the peptide and cDNA-encoded sequences suggest that two of the seven proteins are novel and one is a C-type lectin, while-surprisingly-at least three of the other four are closely related to enzymes associated with the pentose phosphate cycle and glycolysis. We discuss precedents in which lectins and metabolic enzymes are involved in attachment and adhesion phenomena.

Highly mesoporous titanium carbide (TiC) based composites were synthesized through a simple carbothermal reduction of homogenous monoliths which were prepared by a one-pot sol–gel process. Their application properties as a counter electrode of dye-sensitized solar cells (DSSC) were evaluated in this paper. The surface area (116–368 m 2 /g), average particles size (14–87 nm) and pore size (4–9.2 nm) of the mesoporous composites can be easily tuned by altering chemical compositions of the precursor. Introduction of silicon into the Ti–C–O system significantly reduced the average carbide grain size and also enhanced the mesoporousity of TiC composites. The dye-sensitized solar cell using a PEDOT:PSS doped TiC–SiC–C composite as the counter electrode showed a notable efficiency of 5.7%, which doubled that of pure PEDOT:PSS cell. Potentially, the reported method could be a convenient route to fabricate porous TiC based composites for other functional applications.

Bacterially derived triple-helical, collagen-like proteins are attractive as potential biomedical materials. The collagen-like domain of the Scl2 protein from S. pyogenes lacks any specific binding sites for mammalian cells yet possesses the inherent structural integrity of the collagen triple-helix of animal collagens. It can, therefore, be considered as a structurally-stable "blank slate" into which various defined, biological sequences, derived from animal collagens, can be added by substitutions or insertions, to enable production of novel designed materials to fit specific functional requirements. In the present study, we have used site directed mutagenesis to substitute two functional sequences, one for heparin binding and the other for integrin binding, into different locations in the triple-helical structure. This provided three new constructs, two containing the single substitutions and one containing both substitutions. The stability of these constructs was marginally reduced when compared to the unmodified sequence. When compared to the unmodified bacterial collagen, both the modified collagens that contain the heparin binding site showed marked binding of fluorescently labeled heparin. Similarly, the modified collagens from both constructs containing the integrin binding site showed significant adhesion of L929 cells that are known to possess the appropriate integrin receptor. C2C12 cells that lack any appropriate integrins did not bind. These data show that bacterial collagen-like sequences can be modified to act like natural extracellular matrix collagens by inserting one or more unique biological domains with defined function.

Graphical abstract, Recapitulation of the articular cartilage microenvironment for regenerative medicine applications faces significant challenges due to the complex and dynamic biochemical and biomechanical nature of native tissue. Towards the goal of biomaterial designs that enable the temporal presentation of bioactive sequences, recombinant bacterial collagens such as Streptococcal collagen-like 2 (Scl2) proteins can be employed to incorporate multiple specific bioactive and biodegradable peptide motifs into a single construct. Here, we first modified the backbone of Scl2 with glycosaminoglycan-binding peptides and cross-linked the modified Scl2 into hydrogels via matrix metalloproteinase 7 (MMP7)-cleavable or non-cleavable scrambled peptides. The cross-linkers were further functionalized with a tethered RGDS peptide creating a system whereby the release from an MMP7-cleavable hydrogel could be compared to a system where release is not possible. The release of the RGDS peptide from the degradable hydrogels led to significantly enhanced expression of collagen type II (3.9-fold increase), aggrecan (7.6-fold increase), and SOX9 (5.2-fold increase) by human mesenchymal stem cells (hMSCs) undergoing chondrogenesis, as well as greater extracellular matrix accumulation compared to non-degradable hydrogels (collagen type II; 3.2-fold increase, aggrecan; 4-fold increase, SOX9; 2.8-fold increase). Hydrogels containing a low concentration of the RGDS peptide displayed significantly decreased collagen type I and X gene expression profiles, suggesting a major advantage over either hydrogels functionalized with a higher RGDS peptide concentration, or non-degradable hydrogels, in promoting an articular cartilage phenotype. These highly versatile Scl2 hydrogels can be further manipulated to improve specific elements of the chondrogenic response by hMSCs, through the introduction of additional bioactive and/or biodegradable motifs. As such, these hydrogels have the possibility to be used for other applications in tissue engineering. Statement of Significance Recapitulating aspects of the native tissue biochemical microenvironment faces significant challenges in regenerative medicine and tissue engineering due to the complex and dynamic nature of the tissue. The ability to take advantage of, mimic, and modulate cell-mediated processes within novel naturally-derived hydrogels is of great interest in the field of biomaterials to generate constructs that more closely resemble the biochemical microenvironment and functions of native biological tissues such as articular cartilage. Towards this goal, the temporal presentation of bioactive sequences such as RGDS on the chondrogenic differentiation of human mesenchymal stem cells is considered important as it has been shown to influence the chondrogenic phenotype. Here, a novel and versatile platform to recreate a high degree of biological complexity is proposed, which could also be applicable to other tissue engineering and regenerative medicine applications.

The use of biomacromolecular therapeutics has revolutionized disease treatment, but frequent injections are required owing to their short half-life in vivo. Thus there is a need for a drug delivery system that acts as a reservoir and releases the drug remotely "on demand". Here we demonstrate a simple light-triggered local drug delivery system through photo-thermal interactions of polymer-coated gold nanoparticles (AuNPs) inside an agarose hydrogel as therapeutic depot. Localized temperature increase induced by the visible light exposure caused reversible softening of the hydrogel matrix to release the pre-loaded therapeutics. The release profile can be adjusted by AuNPs and agarose concentrations, light intensity and exposure time. Importantly, the biological activity of the released bevacizumab was highly retained. In this study we demonstrate the potential application of this facile AuNPs/hydrogel system for ocular therapeutics delivery through its versatility to release multiple biologics, compatibility to ocular cells and spatiotemporal control using visible light.

Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017.

A range of non-animal collagens has been described, derived from bacterial species, which form stable triple-helical structures without the need for secondary modification to include hydroxyproline in the sequence. The non-animal collagens studied to date are typically smaller than animal interstitial collagens, around one quarter the length and do not pack into large fibrillar aggregates like those that are formed by the major animal interstitial collagens. A consequence of this for biomedical products is that fabricated items, such as collagen sponges, are not as mechanically and dimensionally stable as those of animal collagens. In the present study, we examined the production of larger, polymeric forms of non-animal collagens through introduction of tyrosine and cysteine residues that can form selective crosslinks through oxidation. These modifications allow the formation of larger aggregates of the non-animal collagens. When Tyr residues were incorporated, gels were obtained. And with Cys soluble aggregates were formed. These materials can be formed into sponges that are more stable than those formed without these modifications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2369-2376, 2016.

Tissue engineering strategies for repairing and regenerating articular cartilage face critical challenges to recapitulate the dynamic and complex biochemical microenvironment of native tissues. One approach to mimic the biochemical complexity of articular cartilage is through the use of recombinant bacterial collagens as they provide a well–defined biological ‘blank template’ that can be modified to incorporate bioactive and biodegradable peptide sequences within a precisely defined three–dimensional system. We customized the backbone of a Streptococcal collagen–like 2 (Scl2) protein with heparin–binding, integrin–binding, and hyaluronic acid–binding peptide sequences previously shown to modulate chondrogenesis and then cross–linked the recombinant Scl2 protein with a combination of matrix metalloproteinase 7 (MMP7)– and aggrecanase (ADAMTS4)–cleavable peptides at varying ratios to form biodegradable hydrogels with degradation characteristics matching the temporal expression pattern of these enzymes in human mesenchymal stem cells (hMSCs) during chondrogenesis. hMSCs encapsulated within the hydrogels cross–linked with both degradable peptides exhibited enhanced chondrogenic characteristics as demonstrated by gene expression and extracellular matrix deposition compared to the hydrogels cross–linked with a single peptide. Additionally, these combined peptide hydrogels displayed increased MMP7 and ADAMTS4 activities and yet increased compression moduli after 6 weeks, suggesting a positive correlation between the degradation of the hydrogels and the accumulation of matrix by hMSCs undergoing chondrogenesis. Our results suggest that including dual degradation motifs designed to respond to enzymatic activity of hMSCs going through chondrogenic differentiation led to improvements in chondrogenesis. Our hydrogel system demonstrates a bimodal enzymatically degradable biological platform that can mimic native cellular processes in a temporal manner. As such, this novel collagen–mimetic protein, cross–linked via multiple enzymatically degradable peptides, provides a highly adaptable and well defined platform to recapitulate a high degree of biological complexity, which could be applicable to numerous tissue engineering and regenerative medicine applications.

Collagen I foams are used in the clinic as scaffolds to promote articular cartilage repair as they provide a bioactive environment for cells with chondrogenic potential. However, collagen I as a base material does not allow for precise control over bioactivity. Alternatively, recombinant bacterial collagens can be used as “blank slate” collagen molecules to offer a versatile platform for incorporation of selected bioactive sequences and fabrication into 3D scaffolds. Here, we show the potential of Streptococcal collagen-like 2 (Scl2) protein foams modified with peptides designed to specifically and noncovalently bind hyaluronic acid and chondroitin sulfate to improve chondrogenesis of human mesenchymal stem cells (hMSCs) compared to collagen I foams. Specific compositions of functionalized Scl2 foams lead to improved chondrogenesis compared to both nonfunctionalized Scl2 and collagen I foams, as indicated by gene expression, extracellular matrix accumulation, and compression moduli. hMSCs cultured in functionalized Scl2 foams exhibit decreased collagens I and X gene and protein expression, suggesting an advantage over collagen I foams in promoting a chondrocytic phenotype. These highly modular foams can be further modified to improve specific aspects chondrogenesis. As such, these scaffolds also have the potential to be tailored for other regenerative medicine applications.

This paper reviews the structure, function and applications of collagens as biomaterials. The various formats for collagens, either as tissue-based devices or as reconstituted soluble collagens are discussed. The major emphasis is on the new technologies that are emerging that will lead to new and improved collagen-based medical devices. In particular, the development of recombinant collagens, especially using microorganism systems, is allowing the development of safe and reproducible collagen products. These systems also allow for the development of novel, non-natural structures, for example collagen like structures containing repeats of key functional domains or as chimeric structures where a collagen domain is covalently linked to another biologically active component.

The collagen like domain Scl2 from Streptococcus pyogenes has been proposed as a potential biomedical material. It is non-cytotoxic and non-immunogenic and can be prepared in good yield in fermentation. The Scl2 collagen domain is about a quarter of the length, 234 residues, of the main collagen type, mammalian type I collagen (1014 residues) that is currently used in biomedical devices. In the present study we have made constructs comprising 1 to 4 copies of the Scl2 collagen domain, plus these same constructs with a CysCys sequence at the C-terminal, analogous to that found in mammalian type III collagens. The yields of these constructs were examined from 2 L fermentation studies. The yields of both series declined with increasing size. Circular dichroism showed that the addition of further collagen domains did not lead to a change in the melting temperature compared to the monomer domain. Addition of the CysCys sequence led to a small additional stabilization of about 2-3°C for the monomer construct when the folding (V) domain was present.

Recently, a different class of collagen-like molecules has been identified in numerous bacteria. Initial studies have shown that these collagens are readily produced in Escherichia coli and they have been isolated and purified by various small-scale chromatography approaches. These collagens are non-cytotoxic, are non-immunogenic, and can be produced in much higher yields than mammalian collagens, making them potential new collagens for biomedical materials. One of the major drawbacks with large-scale fermentation of collagens has been appropriate scalable down-stream processing technologies. Like other collagens, the triple helical domains of bacterial collagens are particularly resistant to proteolysis. The present study describes the development and optimization of a simple, scalable procedure using a combination of acid precipitation of the E. coli host proteins, followed by proteolysis of residual host proteins to produce purified collagens in large scale without the use of chromatographic methods.

Bacterially derived triple-helical, collagen-like proteins are attractive as potential biomedical materials. The collagen-like domain of the Scl2 protein from S. pyogenes lacks any specific binding sites for mammalian cells yet possesses the inherent structural integrity of the collagen triple-helix of animal collagens. It can, therefore, be considered as a structurally-stable "blank slate" into which various defined, biological sequences, derived from animal collagens, can be added by substitutions or insertions, to enable production of novel designed materials to fit specific functional requirements. In the present study, we have used site directed mutagenesis to substitute two functional sequences, one for heparin binding and the other for integrin binding, into different locations in the triple-helical structure. This provided three new constructs, two containing the single substitutions and one containing both substitutions. The stability of these constructs was marginally reduced when compared to the unmodified sequence. When compared to the unmodified bacterial collagen, both the modified collagens that contain the heparin binding site showed marked binding of fluorescently labeled heparin. Similarly, the modified collagens from both constructs containing the integrin binding site showed significant adhesion of L929 cells that are known to possess the appropriate integrin receptor. C2C12 cells that lack any appropriate integrins did not bind. These data show that bacterial collagen-like sequences can be modified to act like natural extracellular matrix collagens by inserting one or more unique biological domains with defined function.

Novel, distinct adhesive systems have been described for a wide range of marine species (Kamino, 2008). These highly effective, natural materials provide a link between biological science and material science, and can serve as models on which new, bioinspired synthetic materials could be based. These various adhesive systems have developed independently, on many occasions, and provide a wide range of opportunities for the development of new, biologically-inspired adhesives. The natural adhesives include, for example, the marine mussel (Mytilus sp.) (Lin et al., 2007), barnacle (Nakano et al., 2007) and stickleback (Jones et al., 2001) adhesives, which are protein-based, as well as sponge, certain algal and marine bacterial adhesives (Mancuso-Nichols et al., 2009) that are polysaccharide-based. In the present paper, we examine the adhesive system found associated with the Cuvierian tubules of a holothurian species (sea cucumber), Holothuria dofleinii. This is an example of the particularly rapid marine adhesive that is found on the surface of Cuvierian tubules when they are expelled (DeMoor et al., 2003; Muller et al., 1972; VandenSpiegel & Jangoux, 1987). The unique nature of this natural adhesive system, especially its rapid action under water, has suggested that if the mechanism can be understood, then it may prove to be possible to mimic the adhesive through biotechnology and/or synthetic chemistry. An adhesive that functions readily in an aqueous environment would be particularly valuable, especially in medical applications, as the majority of existing adhesives bind to dry surfaces more strongly than the same surfaces when wet. Cuvierian tubules provide a host defence mechanism for certain species of holothurians (Lawrence, 2001; VandenSpiegel & Jangoux, 1987). It has long been known that, on expulsion, the Cuvierian tubules fill with liquid and lengthen, become sticky and rapidly

A range of bacteria have been shown to contain collagen-like sequences that form triple-helical structures. Some of these proteins have been shown to form triple-helical motifs that are stable around body temperature without the inclusion of hydroxyproline or other secondary modifications to the protein sequence. This makes these collagen-like proteins particularly suitable for recombinant production as only a single gene product and no additional enzyme needs to be expressed. In the present study, we have examined the cytotoxicity and immunogenicity of the collagen-like domain from Streptococcus pyogenes Scl2 protein. These data show that the purified, recombinant collagen-like protein is not cytotoxic to fibroblasts and does not elicit an immune response in SJL/J and Arc mice. The freeze dried protein can be stabilised by glutaraldehyde cross-linking giving a material that is stable at >37 degrees C and which supports cell attachment while not causing loss of viability. These data suggest that bacterial collagen-like proteins, which can be modified to include specific functional domains, could be a useful material for medical applications and as a scaffold for tissue engineering.

The development of novel scaffolds will be an important aspect in future success of tissue engineering. Scaffolds will preferably contain information that directs the cellular content of constructs so that the new tissue that is formed is closely aligned in structure, composition and function to the target natural tissue. One way of approaching this will be the development of novel protein-based constructs that contain one or more repeats of functional elements derived from various proteins. In the present case, we describe a strategy to make synthetic, recombinant triple-helical constructs that contain repeat segments of biologically relevant domains. Copies of a DNA fragment prepared by PCR from human type III collagen have been inserted in a co-linear contiguous fashion into the yeast expression vector YEpFlag-1, using sequential addition between selected restriction sites. Constructs containing 1, 2 and 3 repeats were designed to maintain the (Gly-X-Y) repeat, which is essential for the formation of an extended triple helix. All constructs gave expressed protein, with the best being the 3-repeat construct which was readily secreted. This material had the expected composition and N-terminal sequence. Incubation of the product at low temperature led to triple-helix formation, shown by reaction with a conformation dependent monoclonal antibody.

When provoked, the Australian fossorial frog Notaden bennettii secretes from its dorsal skin an exudate which rapidly forms a tacky elastic hydrogel. This protein-based material acts as a promiscuous pressure-sensitive adhesive which works even in wet conditions. The largest protein, Nb-1R, is rich in Gly, Pro/Hyp and Glx and appears to be the key structural component; it probably contains extensive segments of intrinsic disorder along with some well-folded domains. Indeed, the material properties of adhesive secretions from both amphibians (vertebrates) and onychophorans (invertebrates) may rely upon large proteins containing long and intrinsically unstructured regions composed of imperfect tandem repeats. Although the N. bennettii secretion contains sterols, carotenoids and other undesirable metabolites, in vitro, ex vivo and in vivo studies suggest that the structural matrix of the set glue is highly biocompatible. Its open porous structure is likely to encourage cell infiltration, and glue pellets implanted subcutaneously in mice were fully resorbed by the surrounding tissues within two months. Ex vivo studies in sheep showed that the frog glue bonded meniscal tears more strongly than traditional protein-based medical glues, while conventional tendon attachment repairs were approximately doubled in strength when augmented with the frog adhesive. Overall, the properties of the frog glue suggest that a recombinant mimic would have great potential for medical applications.

Background Collagen has proved valuable as biomedical materials for a range of clinical applications, particularly in wound healing. It is normally produced from animal sources, such as from bovines, but concerns have emerged over transmission of diseases. Recombinant collagens would be preferable, but are difficult to produce. Recently, studies have shown that ‘collagens’ from bacteria, including Streptococcus pyogenes, can be produced in the laboratory as recombinant products, and that these are biocompatible. In the present study we have established that examples of bacterial collagens can be produced in a bioreactor with high yields providing proof of manufacture of this important group of proteins. Results Production trials in shake flask cultures gave low yields of recombinant product, < 1 g/L. Increased yields, of around 1 g/L, were obtained when the shake flask process was transferred to a stirred tank bioreactor, and the yield was further enhanced to around 10 g/L by implementation of a high cell density fed-batch process and the use of suitably formulated fully defined media. Similar yields were obtained with 2 different constructs, one containing an introduced heparin binding domain. The best yields, of up to 19 g/L were obtained using this high cell density strategy, with an extended 24 h production time. Conclusions These data have shown that recombinant bacterial collagen from S. pyogenes, can be produced in sufficient yield by a scalable microbial production process to give commercially acceptable yields for broad use in biomedical applications.