Review Article Biomedical applications of collagens John A. M. Ramshaw CSIRO Manufacturing, Parkville, Victoria 3052, Australia Received 28 March 2015; revised 31 August 2015; accepted 17 September 2015 Published online 7 October 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33541 Abstract: Collagen-based biomedical materials have developed into important, clinically effective materials used in a range of devices that have gained wide acceptance. These devices come with collagen in various formats, including those based on stabilized natural tissues, those that are based on extracted and purified collagens, and designed composite, biosynthetic materials. Further knowledge on the structure and function of collagens has led to on-going developments and improvements. Among these developments has been the production of recombinant collagen materials that are well defined and are disease free. Most recently, a group of

bacterial, non-animal collagens has emerged that may provide an excellent, novel source of collagen for use in biomaterials and other applications. These newer collagens are discussed in detail. They can be modified to direct their function, and they can be fabricated into various formats, including films and sponges, while solutions can also be adapted for use in C surface coating technologies. V 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 665–675, 2016.

Key Words: collagen, biomolecular engineering, cell therapy, microspheres, vascular prosthesis

How to cite this article: Ramshaw JAM. 2016. Biomedical applications of collagens. J Biomed Mater Res Part B 2016:104B:665–675.

INTRODUCTION

Collagen is among the most abundant protein in animals, where it plays a major structural role in the extracellular matrix, including in all vertebrates and invertebrates. It also plays critical roles in molecular and cellular interactions in the extracellular matrix, defining the shape and form of tissues. Thus, collagen is the major protein of many tissues, including, for example, skin, bone, ligament, tendon, and cartilage. Collagen is, therefore, ideally suited for development as a biomedical material. As such, collagen-based biomaterials have a long history, and in the last few decades have been proven safe and effective in a wide variety of medical products and in various clinical applications where they have become well accepted by clinicians and patients. Nevertheless, interesting new developments in collagenbased materials and their applications continue to emerge. This review, based on a previous presentation,1 will address a range of examples of collagen-based biomedical products, and will discuss structural and functional aspects of these products. Collagen for biomedical products has usually been obtained from animal sources, including bovines. If one concern has emerged for collagen products, it is the possible transfer of diseases from use of animal products, whether it is collagen in biomedical materials, or the use of bovine serum and other additions in cultured products. This

issue is being addressed in collagen materials, particularly though the development of recombinant non-animal collagens, and this opportunity will also be discussed. COLLAGEN STRUCTURE

The defining feature of a collagen is its molecular structure that is characterized by a unique conformation, the collagen triple-helix. This structure consists of a supercoiled triplehelix, which is made from three left-handed polyproline-like chains twisted together into a right-handed triple-helix.2 It is interesting to note that it is 60 years since the triple-helical structure for collagen was first proposed, by Ramachandran and Kartha.3,4 It was termed the Madras (now Chennai) helix, following on from the California helix, the polypeptide a-helix and the Cambridge helix, the DNA double helix. Ramachandran (1922–2001) went on to further refine the structure and make other significant contributions to collagen chemistry and to broad aspects of protein chemistry.5 Subsequently, his triple helical concept proved correct and was further refined,6 with structural parameters determined by a detailed fiber diffraction study.7 An interesting feature of the triple helical motif is that the tight packing of the polypeptide chains requires that every third residue in each chain of the primary sequence

Correspondence to: J. A. M. Ramshaw; e-mail: [email protected]

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be Gly, because there is no space for any larger amino acid in the interior axis of the triple-helix. This gives collagen a characteristic repetitive amino acid sequence pattern of (Gly-Xaa-Yaa)n.2 The non-Gly positions in this repeating sequence are often occupied by the imino acid proline (Pro). Further, the Pro residues in the Yaa position, about 10% of all residues, are normally post-translationally modified by the enzyme prolyl-4-hydroxylase (P4H), to give 4hydroxyproline (Hyp).8 This modification is very important for several reasons; it leads to a significant increase in thermal stability up to body temperature, through stereoelectronic effects9 and/or hydration;10 it appears essential for collagen self-association, especially in the fibrillar collagens;11 it also appears key to effectiveness of certain receptor interactions.12 When the triple-helical structure of collagen was first proposed,3 only one type of collagen was thought to exist. It was some 15 years later before biochemical evidence was presented showing that a second, distinct collagen type was present in cartilage,13,14 whence a whole range of other collagen types has steadily emerged, such that 29 distinct types are now described.15,16 These show a range of distinct and different functions, including the predominant fibril forming collagens, as well as fibril associated, network forming and link forming collagens. Nevertheless, the most abundant collagens are the interstitial, fibril forming collagens, particularly type I collagen, which are present in all the major connective tissues. In addition, a range of other proteins not involved in the extracellular matrix, for example, C1q and lung surfactant proteins, were also found to contain a significant triple-helical motif.2 All these collagens and other triple-helical motifs exemplify these various key structural characteristics. In addition to the importance of developments in collagen structure are the developments in knowledge of the biological properties of collagens, which have been discussed extensively elsewhere.17–20 It may be worth noting that for both biology and structure, the amount of knowledge is greatest at the smallest scale, for example, structural mutations, biosynthesis pathways, molecular interactions, and so forth, but relatively little is known at higher structural orders, such as packing of collagen into fiber bundles and tissues and the development and control of function. Yet it is these data that are of particular relevance to collagen-based biomaterial and tissue engineering scaffold performance. BIOMEDICAL APPLICATIONS OF COLLAGEN

Collagen has been an important commodity for millennia, providing the basis for gelatin and leather products. More recently its role in food and food products has been better understood. The first recorded use of collagen in a biomedical product goes back almost two millennia, when Galen was using collagen (catgut) sutures along with other materials, particularly for repair of wounds and severed tendons in gladiators,21 although other materials preceded the use of collagen, probably keratin and silks.22 High value

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biomedical applications for collagen have been studied now for just over a century, initially focusing on tissue-derived materials, for example, through use of demineralized chicken bone as surgical conduits23 and fetal membranes in wound repair.24 However, more innovative manufactured articles started to emerge in the early 1940s,25,26 enabled in part from the understanding of soluble collagen and collagen fibril reformation that had been established.27 Subsequently, principally in the last 50 years, numerous successful collagen-based devices have emerged28,29 and these have proved safe and effective in medical products, and have achieved clinical and consumer acceptance. As a consequence, most recently, applications in the development of tissue engineering and cell therapies are providing new areas where collagen is an important biomedical material. Collagen for biomedical applications can be prepared in a variety of formats.28,29 In some cases, the natural architecture of the tissue is maintained and stabilized. In others, the natural fibrous composition, such as found in skin or tendon, is maintained when the tissue is comminuted into a fibrous powder, while alternatively, tissue is solubilized, typically through an enzyme treatment to produce a purified collagen solution which can be used directly or after fabrication into devices.28,29 Collagen is also used in a variety of composite materials of which integrated “biosynthetic” materials have particularly favorable properties. Typically, all these products are based on the use of animal collagens.28,29 This has led to an ongoing concern regarding the transmission of diseases, especially spongiform encephalopathies (“mad cow disease”). One proposal to limit this risk has been to use collagens from more primitive animal species, including avian,30 fish31 and jellyfish32 sources. The alternative, disease-free source of collagen is via recombinant production systems. A selection of examples of these various collagen formats and systems follows, typically based on examples from work at CSIRO, generally along with important university and commercial collaborations. TISSUE-BASED BIOMATERIALS

The characteristics of tissue-based collagen biomaterials include the retention of natural strength and defined shape which, after stabilization, frequently using glutaraldehyde (GA), gives a material with long-term durability. The essential stabilization process typically masks or substantially reduces any immunogenicity. Despite the stabilization, the material may retain some cell matrix interactions. However, as with any naturally derived material, batch to batch variation and risks of infectious agents are issues that can be of concern. While a range of tissue-based materials have been described,28,29 the best known examples of tissue based biomaterials are bioprosthetic heart valves, where a variety of tissues and configurations have emerged.33,34 The two most frequently used and studied types have been stabilized porcine aortic valves and stabilized and formed pericardial tissue.34–37 The stabilization is typically by GA, which

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introduces its own problem, that of tissue calcification.38 But without the stabilization, xenogenic tissues generally lack the required durability.33 It has been suggested that tissue engineering products would be a solution, but progress has been slower than predicted.39 Various other products have been noted,29 and recently tissues have been developed as acellular matrix biomaterials.40 Recently, various collagen materials, have been examined for cell delivery, especially to damaged heart tissues.41,42 Repair of cardiac tissue with cells delivered by an appropriate bioscaffold is expected to offer a superior, long-lasting treatment strategy. An example of this approach, using stabilized pericardium, has emerged from the work of Neethling et al.43–45 They have developed a new process, the AdaptTM process. This provides more suitable stabilization and has been incorporated in the CardiocelTM products (Admedus P/L) derived from bovine pericardium. The key aspects of the AdaptTM process include a low GA content with a low GA polymer content, through use of processing at elevated temperatures, which leads to little if any calcification. The process provides a material that can have controlled properties, including high porosity along with high flexibility and controlled turnover.43–45 Another key aspect is that it still provides a cell friendly environment.46 Thus, a CardiocelTM product has been examined as a natural extracellular matrix (ECM) patch for the delivery and retention of mesenchymal stem cells (MSC) for potential cardiac repair, using human bone marrow MSC at various cell densities under standard, static cell culture conditions.46 This study showed good cell attachment and viability followed by good proliferation. The seeded stem cells showed the capacity to differentiate into collagen-producing cells necessary to repair damaged ECM. R scaffold is an approThese data indicate that the CardioCelV priate substrate for stem cells and has the potential to both retain seeded stem cells and to act as a template for cell propagation and new tissue formation.46 PURIFIED, RECONSTITUTED COLLAGEN MATERIALS

Purified and especially solubilized collagen materials have the advantage of biochemical purity and low immunogenicity.28,29 Typically they are stabilized, for example, by GA as part of the manufacturing process. However, a range of other chemical methods are also available, including the use of other bis-aldehydes, bis-isocyanates, aliphatic epoxides, water soluble bis-carbodiimides, and non-enzymatic glycosylation.29 In addition, a range of physical stabilization methods have been used, including use of radiation and dehydrothermal crosslinking.29 The extent of stabilization allows control of turnover, and the extent of fibril reconstitution. The level of stabilization is frequently low, such that cell matrix interactions are retained. However, like all extracted natural materials, batch to batch variation and risks of transmission of infectious agents are both present. Purified and reconstituted collagen products, either made entirely from collagen or where collagen is a key component, have found extensive applications in wound and

burn treatments, where they are often the materials of choice.47,48 A variety of other areas, including membranes for dentistry, hemostats, nerve repair conduits, meniscal cartilage substitutes, tympanic membrane repair membranes, and drug or growth factor delivery materials have been examined as laboratory and clinical applications.29 Of these, the use of collagen as a dermal filler for tissue augmentation has been a significant commercial application.49 A more recent and quite distinct application is in fabrications of collagen/gelatin beads for use in cell therapy and tissue engineering applications.50,51 For these applications, expansion of cells, such as chondrocytes, on beads in spinner culture can provide advantages compared with monolayer culture. For example, in monolayer culture, chondrocytes rapidly lose their phenotype and production of cartilage components and revert to production of type I collagen. On the other hand, in spinner culture, chondrocytes retain their phenotype for an extended period.52 Further, resorbable beads can be included as an integral part of a construct or implant, minimizing the extent of cell handling and eliminating a final trypsin treatment to detach cells from the bead.51 Varying the extent of bead stabilization also allows control over the rate of resorption.51 A further, and useful advantage is that when small numbers of cells from a biopsy need amplifying, the proliferation in spinner culture is significantly better. For example, the proliferation of chondrocytes on collagen/gelatin beads can be >5 times greater than that on monolayer culture.53 The utility of this technology has been shown in a functional, load-bearing mini-pig model for cartilage repair.54 Articular cartilage is known to have poor healing capacity after injury, with autologous chondral grafting typically used to treat well-defined, full-thickness cartilage defects. In this animal study, cells from a biopsy were expanded over 3 weeks on these beads or in monolayer culture. Full-thickness cartilage defects were surgically created in the weightbearing surface of the femoral condyles and covered by periosteal patches taken from proximal tibia, and sealed with a fibrin glue. Two experimental groups and one control group with no further manipulation were examined. In one experimental group, cells from monolayer culture in culture medium were injected below the periosteal seal. In the other group, cell-laden beads were mixed with collagen type I gel and injected below the periosteal seal. The repair was examined 6 months post-surgery on the basis of macroscopic appearance and histological scores based on the International Cartilage Repair Society Scale.54 The cell/bead/gel experimental group scored best, higher than both the monolayer cells and the control, null group (Table I). In this case, the transplanted autologous chondrocytes survived and could yield hyaline-like cartilage. The application of beads and gel for transplantation helped to retain the transferred cells in situ and maintain a better chondrocyte phenotype.54 BIOSYNTHETIC MATERIALS

Soluble collagen has been used in combination with a range of synthetic polymeric materials to form composite

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TABLE I. Evaluation of Cartilage Repair after 6 Months in a Mini-Pig Model using Two Different Cell-Based Experimental Approaches

Morphology (Hyaline-Like) Total Score

Mean (n 5 16) Mean (n 5 16)

Cells/Beads/Gel

Monolayer Cells

Control

3.2 17.0

1.4 10.2

1.3 9.9

Scores are based on the International Cartilage Repair Society Scale.54

structures.55,56 However, these may not always behave as a single integral material, but rather the different phases degrade at different rates giving a complex and not always anticipated behavior. By the term “Biosynthetic materials” we mean highly integrated structures that are tissue based, and so have characteristics that are similar to tissue-based materials but enhanced by the synthetic component, and which stay in this form throughout the implant life. So these materials are fully integrated tissue-polymer composites, durable and strong like synthetic devices but also compatible like natural tissues. They can be made to a defined shape. They are normally stabilized, so are durable and have long turnover times. The stabilizing also masks any immunogenicity. But like all natural tissue derived materials, batch to batch variation will be present. The key to these materials is the integrated structure, so they are quite distinct from materials onto which a synthetic mesh or similar has been added as any additional, non-integrated component. The best characterized, and most extensively used biosynthetic device is the OmniflowTM Vascular Prosthesis that was developed and marketed by BioNova International (North Melbourne). This product is a biosynthetic vascular graft for femoral artery repair, but which is also useful for peripheral bypass and dialysis access. Briefly, the device is made by implant of a silicone mandrel, covered with polyester mesh, in sheep. When the mandrel-polymer implant is removed after about 12 weeks, the implant has been completely encapsulated by sheep tissue. Excess tissue is removed by trimming and the collagen stabilized. Removal of the silicone mandrel leaves a polymer mesh reinforced, stabilized collagen tube which is an integrated structure with the polyester mesh fully encapsulated by the collagen.57 Transmission electron microscopy has been used to examine the collagen ultrastructure (Figure 1).58 The inner, blood contact surface, had limited amounts of loose connective tissue and collagen that did not have a clear orientation [Figure 1(A)]. There was a large amount of collagen proximal to the polyester fiber, which was present as small defined fibrils [Figure 1(B)]. The main body of the wall, away from the polyester, consisted of larger well defined fibrils of a fairly uniform size that were typically highly oriented along the axis of the device [Figure 1(C)].58 This organization of the collagen gives the device good compliance. Prior to the now extensive clinical use, the devices were evaluated in dogs in an aorto-iliac model.57 These data showed that the devices had minimal intimal hyperplasia, unlike the much stiffer ePTFE based devices, and had good endothelialization in animal studies, although limited endo-

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thelialization is seen in clinical explants.59 They showed excellent patency in the dog model,57,60 and good patency has subsequently been reported from human clinical

FIGURE 1. Transmission electron microscopy of the wall of an OmniflowTM Vascular Prosthesis, using sections perpendicular to the length of the device. (A) The inner, luminal surface, (B) the collagen formed adjacent to the polyester fibers (p), and (C) the collagen of the main wall of the device. Adapted from White et al.58

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evalutation.61 Immunohistology data were also examined on these animal model explants. This required development of specialist monoclonal antibodies. These needed to be collagen type specific, but also for each collagen type they also needed species specificity so that the manufactured sheep collagen in the device could be distinguished from any new, host derived dog collagen (or human collagen in clinical samples).59,62 These specialist monoclonal antibodies showed, for example, that after 4 years the sheep type III collagen was still present in the device, while it was substantially augmented by host dog type III collagen.60 Other data showed that sheep type VI was present in the preimplant device, but not uniformly distributed; rather it was mainly associated with cells, particularly around the polyester mesh and silicone interfaces. Conversely, after 12 months in a dog model, new host type VI collagen was found predominantly in the wall of the device.59 The biosynthetic material technology can also be applied to clinical applications beyond vascular (e.g., femoral artery) replacement. The material can be fabricated into a U-shape for insert as an arterio-venous access shunt. Alternatively, various patches can be made, for example, as vascular repair patches. A further application of a patch is for abdominal repair.63 Examination in a rabbit model of two samples, one with an open collagen pore structure and one with a closed structure with few pores in the collagen component showed interesting results. The closed structure resulted in a totally non-integrated implant that induced a foreign-body capsule with minimal collagen and cell infiltration into the implant. Conversely, the open pore material promoted neovascularization, tissue integration, cellular infiltration, and neomatrix formation. This material was superior to a polypropylene mesh control implant, as the biological material gave significantly fewer adhesions.63 Monoclonal antibodies can also be used to track new collagen formation around or within an implant which is not collagen based, again providing useful information on the host response to the implant. While cellular events are generally examined and understood, tissue composition changes are much less studied and less well characterized. Porous materials are generally less often examined than non-porous materials. An example of tracking infiltration into a porous material was presented by following the changes in an ePTFE vascular tube sample implanted subcutaneously in sheep.64,65 These data showed that there was collagen deposition initially surrounding the implant at 6 days, and, with time, was seen to infiltrate within its pores. The deposition of different collagen types happened at different rates. The type V and VI collagens preceded the major interstitial collagens in the newly deposited tissue within the material’s open pores, although at longer time points, detection of type V collagen appeared to decrease. After disruption of the interstitial collagens with enzyme, the “masked” type V collagen was clearly still visible by immunohistochemistry. The “minor” collagens are all important for the development and function in particular tissues, not just the more obvious and abundant interstitial collagens.

FIGURE 2. Selected systems that have been used for the expression of various collagens.

In addition, application of monoclonal antibodies to the various collagen types present in a tissue could also potentially be important for tissue engineering. It is most probable that the more effective tissue engineering strategies will be those that produce tissue repair or regeneration that most resembles to original native tissue. Something better than a piece of scar tissue of the right shape and size is needed. An illustration could be the development of replacement heart valves. Immunohistology has allowed the complexity of the distribution of various collagens in the aortic, mitral, and pulmonary valve leaflets from animal sources to be examined.66 Thus each collagen type has its own distinct distribution, with minimal variation between heart valve anatomic sites and species. Of particular interest was type VI collagen, which had an asymmetric distribution that was principally localized along the outflow surface of the valve, while the distribution of type III collagen was not uniform through the thickness of the tissue.66 These data indicate the complexity of a tissue where tissue engineering may be an important repair solution. RECOMBINANT COLLAGENS

Recombinant collagens provide some clear advantages over naturally derived collagens, in that they are defined products and are virus and disease free. This approach allows the production of any collagen protein chain, even those that in nature are in very low amounts and not readily purified. Structures can be readily modified, to change interactions and turnover, or to make chimeric structures. The major difficulty to overcome is the need for secondary modifications, especially for inclusion of Hyp to give appropriate stability. A variety of approaches have been used to make recombinant constructs (Figure 2). In some, including mammalian and insect cell culture and transgenic systems, endogenous P4H in the host system is used. However, in plant and microbial systems P4H is absent and additional genetic engineering is needed to insert the two genes for

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TABLE II. Properties of Bacterial Collagens That Have Been Shown to Form a Stable Triple-Helix Bacterium/Gene S. pyogenes Scl1 S. pyogenes Scl2 Bacillus anthracis BclA Legionella pneumophila Lcl Clostridium perfringens Solibacter usitatus Rhodopseudomonas palustris Methylobacterium sp 4-46

N-Terminal Domain

Triple-Helix Domain

C-Terminal Domain

Calculated pI

Tm (8C) (Triple-Helix)

68 74 19 n.d. 53 42 9 102

150 237 228 105 189 246 117 147

93 100 134 n.d. 162 147 86 74

5.1 5.4 3.1 5.3 4.7 5.6 9.3 8.6

36.4 37.6 37.0 n.d. 38.8 38.5 37.0 35.0

this enzyme. This approach has led to some success, with recombinant hydroxylated collagens being produced in various yeast systems 67–69 as well as designed constructs, for example, with repetitive cell binding domains.70 However, obtaining competitive yields has proved difficult,71 with plant systems possibly now showing the best promise.72 Recently collagen-like sequences that form stable triplehelical structures have been characterized from bacteria.73,74 These novel, non-animal collagens provide potentially new, safe materials for biomedical and other applications. Initial studies, reported in 2000, indicated the presence of two, triple helical, collagen like proteins in the bacterium Streptococcus pyogenes.75,76 Both these proteins were subsequently confirmed as having triple-helical structures.77 Subsequently, a search of the emerging database of genome structures suggested that many other bacteria may also contain triple-helical proteins.73 These data showed that a minority of genomes (25 of 136) contained segments of (Gly-Xaa-Yaa)n, where n was >7. In some cases up to nine segments were present in a genome. The sequences often showed a preference for Pro in the Xaa position. How many may be present as triple helices? It is unlikely that segments with short n > 7 and n < 20 to 25 would be long enough to form stable triple-helices in the absence of Hyp.78 Nevertheless, the data search indicated that nav 5 76, suggesting that a good number of triple-helices may exist. The number of genomic sequences in databases has increased substantially since this study in 2003.73 A search with n > 30 revealed a wide range of structures.79 Although many gene sequences have been observed, in only a relatively few cases have triple-helical structures been confirmed, by resistance to proteolysis and by circular dichroism (CD) spectroscopy (Table II).74 These few examples, however, are still sufficient to show a variety of structures. The collagen-like domains vary from 117 to 246 amino acids, with calculated pI values from about 4.7 to 9.3. All have additional N- and C-terminal domains of various sizes. Of great interest is that all have high thermal stabilities, from 35.0 to 38.88C (Table II). In some cases, the unexpected stability, without Hyp, of these bacterial collagens can be explained in part from previous studies on the stability of short, synthetic collagen peptides.78,82 In these studies a range of “host-guest” peptides had been examined for stability using CD.82 These

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Ref. 77 77 80 81 79 79 79 79

peptides were based on a host peptide of acetyl-(Gly-ProHyp)8-Gly-Glyamide with internal substitutions of one or two amino acids in single or adjacent triplets, such as: acetyl-(Gly-Pro-Hyp)3-Gly-Pro-Hyp-Gly-Pro-Hyp-(Gly-Pro-Hyp)3-Gly-Glyamide acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Hyp-Gly-Pro-Hyp-(Gly-Pro-Hyp)3-Gly-Glyamide acetyl-(Gly-Pro-Hyp)3-Gly-Pro-Yaa-Gly-Pro-Hyp-(Gly-Pro-Hyp)3-Gly-Glyamide acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-Gly-Pro-Hyp-(Gly-Pro-Hyp)3-Gly-Glyamide and acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-Gly-Xaa0 -Yaa0 -(Gly-Pro-Hyp)3-Gly-Glyamide

Previously it was known that Gly was essential as every third residue in the triple-helix, with >20% of the Xaa and Yaa positions being proline, with about half of these, in the Yaa position, being modified to Hyp. Also, structure and sequence data indicated that there were steric constraints in the Yaa position, such that certain amino acids, such as Phe, Tyr, or Leu are rarely, if ever, found in the Yaa position. Beyond this, little was known about how the amino acids in the Xaa and Yaa positions affected the triple-helical stability. Host-guest peptides provide a partial solution to this problem. Initial studies showed the validity of the host-guest approach, with different substitutions leading to different stabilities.82,83 Subsequent studies on all single substitutions TABLE III. Experimentally Observed Melting Temperatures for Host-Guest Peptides Containing Two Adjacent Triplet Substitutions Compared with Those Predicted on the Basis of Individual Triplet Contributions86 Hexapeptide Insert GPOGPO GAOGPO GAOGAO GPAGPO GPAGPA GAAGPO GAAGAA GLOGPO GLOGLO GEKGPO GKOGEO GPKGEO GPKGDO GPRGDO GPRGEO

Tmexp (8C) 47.3 41.7 36.9 40.9 35.8 32.9 20.0 39.0 38.1 35.0 37.9 47.8 47.1 39.6 42.8

Tmpred (8C)

DTm (8C)

36.1

10.8

34.5

11.3

18.5

11.5

30.7

17.4

37.1 32.4 29.6 40.0 42.8

10.8 115.4 117.5 20.4 0.0

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FIGURE 3. The amino acid sequence of the Scl2 protein from S. pyogenes, showing sequence elements that contribute to triple-helical stability in the absence of hydroxyproline. These include Pro, Arg in the Yaa position, hydrophobic runs and charge pairs.

The amino acid sequence of the collagen domain from S. pyogenes Scl2 (Figure 3) also shows the three segments that are present in the protein. Thus, there is an N-terminal nonhelical domain, the V-domain, which is considered to be a registration and triple helix folding initiation domain.77 This is followed by the contiguous collagen domain (CL). The sequence is terminated by a short binding domain allowing surface attachment to the bacterium.77 For experimental studies, a range of changes have often been introduced (Figure 4). These include addition of a His6-Tag to assist in small scale purification, although this tag can also be added via a vector during production.88–90 Also, a protease domain can be added between the V and CL domains to assist in removal of the V-domain, taking advantage of the typical stability of the triple helix to proteolysis.88–90 Finally the non-helical tail is deleted as it is not required (Figure 4).88–90 Further modifications are possible, as discussed below. EXAMINATION OF NON-ANIMAL COLLAGENS

in both the Xaa and also the Yaa positions84 gave a scale of amino acid propensities for the triple-helix. The rankings for both the Xaa and Yaa positions show the highly stabilizing nature of imino acids and the destabilizing effects of Gly and aromatic residues. Many residues show differing propensities in the Xaa versus Yaa position, related to the structural nonequivalence of these positions.84 Most unexpected was the excellent stabilization provided by single triplets with Arg residues in the Yaa position, where the stability was equivalent to that of Hyp.85 Some stability data was available for Gly-XaaYaa guest peptides with two substitutions that along with the single triplet data allowed an “additive model” to be developed that gave good predictions when tested against the available double substitution data (Table III). The “addition” is of the destabilizing effect of single substitutions compared to the destabilizing effect of a double substitution.87 Further examination of the additive model showed that sequences with hydrophobic residues in adjacent Xaa positions were noticeably more stable than predicted. Further, sequences Yaa-GlyXaa with adjacent charge pair residues, involving a Lys and Glu or Asp, in the Xaa and Yaa positions had a very significant increase in stability (Table III).86 These peptide data can be used to look at how amino acid sequence contributes to the stability of bacterial collagens, but can also be used to design stable collagen sequences. The collagen domain from S. pyogenes Scl2 (Figure 3), which has been the most frequently studied, shows features that can provide stability in the absence of Hyp include, a large number of Pro residues in the Xaa positions (mostly N-terminal) and in the Yaa positions (mostly C-terminal), and a large number of single Arg residues in Yaa positions. There is a three triplet region near the N-terminal with hydrophobic residues in the Xaa positions. Most notable, however, there are a large number of charge pairs involving Lys, particularly in repeats near the C-terminal repeating sequence (Figure 3). Together these all contribute toward the high thermal stability of this triple-helix.

The various triple-helix forming, non-animal collagens that have been studied (Table II) have all been readily produced by recombinant technology and expression in Escherichia coli. The most frequently used expression system has been the high yield cold-shock expression system pCold (Takara Bio)91 but other vectors including pET are also entirely suitable.88 Purification using the introduced His6-tag and immobilized metal ion affinity chromatography (IMAC) using nickel has proved very effective, with additional chromatography using gel permeation columns used for polishing the products.89 Enzyme digestion, for example, using trypsin, proved efficient for removal of the V-domain, if required.89 As noted above (Table II) characterization by CD spectroscopy showed that all form stable triple-helical structures, with melting temperatures around mammalian body temperatures (Table II). This may be possibly expected for pathogens, but would not be expected for other non-

FIGURE 4. A schematic showing the structural elements of the Scl2 protein from S. pyogenes and the additional elements and changes introduced for recombinant expression and processing. The various changes that have been made to provide functional sequences, either through substitution of residues or insertion of residues are also indicated, including that within a single CL domain and also those between two CL domains.

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pathogenic species.74 The collagens also showed high values for calorimetric enthalpy.89 Compared with the animal interstitial collagens, which show a strong tendency for fibril formation, the non-animal, bacterial collagens do not have a strong tendency to ordered aggregation.89 At neutral pH and high concentrations, the bacterial constructs all show some tendency toward aggregation. The isolated collagen domains, CL and CL-CL, form units of diameter 4–5 nm which bundle together and twist to make larger structures. Although the bacterial collagens are typically cell surface proteins that do not apparently participate in higher order structures, the triple-helix domain has some potential of forming fibrillar structures. That this tendency is not overly strong may well reflect the lack of Hyp.89 A consequence of this limited aggregation is that dried samples are difficult to handle and that vapor rather than solution crosslinking may be preferable.90 If these new group of non-animal collagens are to be useful as biomaterials they must be non-cytotoxic and not immunogenic. Examination by various assays, including a R viability assay and a phenol red assay, showed Live/DeadV that the Scl2 collagen from S. pyogenes was not cytotoxic.90 Compared with other natural and particularly recombinant proteins where there is often little immunological data, the Scl2 collagen CL domain has indeed been the subject of an immunological evaluation.90 This was done in 8–9 weeks mice, using 2 strains, SJL/J, an inbred strain [H-2s], which has been shown previously to be most responsive to collagens92 and Arc [Arc:Arc (s) (Swiss)], an outbred albino mouse. Both strains of mice were immunized without adjuvant as well as with Incomplete Freund’s Adjuvant (IFA) to assess the maximum immunogenic potential. Mouse sera at different time points were analyzed by a standard ELISA.90 These data showed that the total Ig response against the CL protein after three immunizations without adjuvant, with sera collection over 42 days, was negligible by ELISA, even without any significant dilution of the sera. In the absence of adjuvant, the CL protein was non-immunogenic in both strains. As the response was negligible, differentiation to specific antibody isotypes was not feasible. These data were comparable (possibly better) to the commercial bovine collagen (Zyderm), which had also been examined in these mice strains. These experiments, obtained with repeated injections, also did not lead to adverse reactions indicating that acute toxicity and sub-chronic toxicity were also absent. So these data, along with other experiments indicate the potential of non-animal collagens as biomaterials. APPLICATIONS OF NON-ANIMAL COLLAGENS

An interesting feature of the CL domain of some of the nonanimal collagens is that the domain seems to lack any noticeable interactions with mammalian cells, and so behaves like a “blank slate.”93,94 Genetic engineering, therefore, allows the introduction and study of specific interaction domains94,95 which also expands the range and functions of the potential biomaterials. These include for example, matrix metalloproteinase sites,95 integrin sites,93 a

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fibronectin binding site96 and a heparin binding site94 (Figure 4). These substitutions and insertions are not limited to one per molecule, but two or more can be introduced.94 This approach also allows dissection of sequence hot spots from animal sequences where several overlapping functional sites are present.95,96 Further one can make new molecules, such as dimers and multimers97 and play “mix and match” with sequences from different species.79 These various products can be potentially used singly, or in combination with other modified forms. The non-animal collagens can also be modified by non-collagenous sequences. For example, addition of a terminal silk like sequence to a collagen enables the collagen molecule to be readily bound to a silk film surface.98 PRODUCTION OF NON-ANIMAL COLLAGENS AS BIOMATERIALS

It is important that if bacterial, non-animal collagens are to be useful as biomaterials, they must be able to be produced in large scale with an efficient and effective downstream purification protocol. Production trials in shake flask cultures gave only low yields of recombinant product, < 1 g/L. Examples of these collagens and their derivatives have now been produced in E. coli by using a high cell density fed-batch process and the use of suitably formulated fully defined media using fermentation, typically in 2 L volumes99 but also in 10 L. The use of defined media specifically avoids any animal-derived components, potentially providing a fully animal free product. Also, the preference has been to use the pCold vector system91 although other vector systems such as pET are also suitable.99 These fermentation trials have shown that extracted yields of more than 19 g/L,99 which should be suitable for commercial applications. Apart from constructs modified to include biologically functional domains, this production approach has also been examined for larger constructs based on multiple copies of the CL domain.94 The Scl2 collagen domain from S. pyogenes 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. Constructs comprising 1–4 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 have been examined. The yields of these constructs were examined from 2 L fermentation studies and found to decline with increasing size.94 Therefore, the use of small constructs may be preferred depending on a balance with the physical properties of the products. Biochemically, CD 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 2–38C for the monomer construct when the folding (V) domain was present.94 These initial studies have shown that these collagens are readily produced in E. coli. However, the various small-scale chromatography approaches that have been reported in various laboratory scale studies89 are not suitable for large

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scale commercial production, and an appropriate scalable down-stream processing technology is needed. Like other collagens, the triple helical domains of bacterial collagens are particularly resistant to proteolysis. The property has led to the successful development 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.99 The proteolysis also removes the V-domain leaving the ideal CL only domain as the product.99

12.

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CONCLUSIONS

Collagen-based biomaterials have proved successful in a wide range of products that have proved effective in clinical application and which have achieved strong consumer acceptance. New research and knowledge on collagen structure and function is driving a wide range of new initiatives that will see new products and opportunities in the future. ACKNOWLEDGMENTS

The author wishes to thank The Society for Biomaterials and Artificial Organs: India for its kindness in the presentation of the Chandra P. Sharma Award 2015 to the author. The author also wishes to thank all his present and former colleagues at CSIRO and collaborators elsewhere, including those in industry, for their inputs to the various studies described in this review. In particular, Dr Geoff Dumsday, Veronica Glattauer, Alan Kirkpatrick, Yong Peng, Violet Stoichevska, Tracy Tebb, Dr Jerome Werkmeister and Jacinta White at CSIRO, Professor Glenn Edwards, now at Charles Sturt University, Dr Anton Persikov and Dr Zhuoxin Yu when at University of Medicine and Dentistry of New Jersey, and Professor Barbara Brodsky when at University of Medicine and Dentistry of New Jersey and now at Tufts University. REFERENCES 1. Ramshaw JAM. Biomedical applications of collagens. In: Chandra P Sharma Award Lecture at the Indo-Australian Conference on Biomaterials, Tissue Engineering, Drug Delivery Systems and Regenerative Medicine. Anna University, Chennai, February 5–7, 2015. pp 21–22. 2. Brodsky B, Ramshaw JAM. The collagen triple-helix structure. Matrix Biol 1997;15:545–554. 3. Ramachandran GN, Kartha G. Structure of collagen. Nature 1954; 174:269–270. 4. Ramachandran GN, Kartha G. Structure of collagen. Nature 1955; 176:593–595. 5. Sarma, R. Ramachandran. Schenectady. NY: Adenine Press; 1998. 6. Rich A, Crick FH. The molecular structure of collagen. J Mol Biol 1961;3:483–506. 7. Fraser RDB, MacRae TP, Suzuki E. Chain conformation in the collagen molecule. J Mol Biol 1979;129:463–481. 8. Myllyharju J. Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis. Matrix Biol 2003;22:15–24. 9. Bretscher LE, Jenkins CL, Taylor KM, DeRider ML, Raines RT. Conformational stability of collagen relies on a stereoelectronic effect. J Am Chem Soc 2001;123:777–778. 10. Bella J, Eaton M, Brodsky B, Berman HM. Crystal and molecular structure of a collagen-like peptide at 1.9 A˚ resolution. Science 1994;266:75–81. 11. Perret S, Merle C, Bernocco S, Berland P, Garrone R, Hulmes D.J, Theisen M, Ruggiero F. Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of

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