Collagens are among the most ubiquitous and complex of the extracellular matrix (ECM) molecules of vertebrates and invertebrates. For most collagens, the majority of their sequence exists as a triple helix, which makes them unique among proteins. These domains are rigid, rope-like cylindrical structures that, depending on the collagen type, are sometimes interspersed between small flexible non-triple helical regions, or larger, sometimes globular non-collagenous domains. Type I collagen is the most abundant of the collagens and is a main focus of our research.

Type I collagen assembles with other collagens and non-collagenous extracellular matrix molecules into large cable-like fibrils that help dictate the structure and function of tissues including skin, tendon, bone and cornea. Collagen also has myriad uses in manufacturing, including leather. To help decipher the structure and biology of this ‘cornerstone of life’, we examined type I collagen fibrils in situ by X-ray diffraction, establishing the molecular conformation and packing topology of triple helices within the microfibril (fibril subunit) and fibril, and assembled and analysed a collagen interactome or ‘collagen road map’. The fibril was found to comprise two domains: one regulating cell interactions and fibril remodelling, and the other dictating structure, mediating proteoglycan binding, fibril cross-linking and biomineralisation.

Many questions remain in type I collagen biology and technology. How does the collagen fibril polymerise, and during fibril assembly, tissue-specific function(s), and remodelling, which sequences are on the outside, or inside of the fibril? Answering such questions may lead to the design of agents to inhibit pathological collagen deposition. Which collagen sequences nucleate hydroxyapatite crystal growth during bone mineralisation? Furthermore, in 1988, Dr Eckhardt Heidemann, for whom this lecture is named, constructed a collagen map, which illustrates the positions of chrome and aldehyde-reactive amino acids for the interest of leather chemists.

We updated Heidemann’s map and propose that such databases may complement experimental and theoretical studies on mechanisms of leather tanning; for example, fibril chemistry can be viewed before and after alkaline modification (liming), intermolecular crosslinking via chrome, glucosepane, or transglutaminase action, detergent fracture on accessibility or location of chrome-reactive groups. The collagen chemistry and biology interactomes may also be correlated to identify the structural features of collagen associated with its crucial biological functions.

Type III collagen

Our ongoing research also probes the biology and structure of type III collagen, which comprises a significant proportion of the hide protein of calf and other young animals. Type III collagen is a fibrillar collagen important in embryogenesis, haemostasis and wound healing, and is proposed to play a critical structural role in blood vessels and distensible organs, such as the large bowel and uterus.
Type III collagen has nearly twice the number of ‘atypical’ amino acid triplets than type I, which contributes to low triple helical stability and structural flexibility. We asked if the distribution of atypical triplets in type III collagen is random, and if not, where domains of greater and lower stability may exist on the protein. Their positions were binned into ten regions of equal size and we observed they predominate in different regions of the molecule. To further study the effect of atypical triplets on the stability of type III collagen, we used the Collagen Stability Calculator, observing three major regions of decreased stability.

Our ongoing research also probes the biology and structure of type III collagen, which comprises a significant proportion of the hide protein of calf and other young animals.

We thus propose type III collagen functions as a ‘flexi-rod’ in which a confluence of atypical triplets creates flexible domains, allowing focal expansion or deformation of several discrete fibril regions. The intervening rod-like domains may preserve the more rigid triple helical conformation to allow crucial functions like cell/ligand binding and proteolysis. Future work should determine if the flexibility inherent in type III collagen- based on its content and distribution of atypical triplets may functionally relate to its predominance in distensible tissues, and whether other collagens behave like flexi-rod or display less flexibility and structural heterogeneity.

Collagen-based medical devices: current reality, future dreams

Collagen-based medical devices comprise a multibillion-dollar market per year worldwide. Raw materials for such devices are most often derived from type I collagen-rich tissues like bovine or porcine skin, bone, tendon, bladder or intestinal submucosa. To satisfy regulatory agencies such as the US Food and Drug Administration requires stringent material sourcing and manufacturing controls to minimise the potential for contamination of the collagens, especially with pathogenic organisms. ‘Solid-phase’ collagen processing methods reminiscent of those of the leather industry are the most commonly used, and are simpler and less costly than ‘solution phase’ purification approaches.

Regarding the former, tissues may be manually trimmed of extraneous tissue, well rinsed and subjected to various treatments including liming/de-liming, organic solvent extraction protocols, heat denaturation, combined with physical processing like slicing, grinding, scaffolding, chemical cross-linking and lyophilisation. The resultant preparations are depleted of cellular material and other contaminants such as blood, and usually composed of partially denatured to near-native meshworks of cross-linked collagen fibrils, and in some cases additional ECM molecules and bioactive factors. Some materials instead are predominantly gelatin and/or its derivatives. Constructs are often sterilised by gamma irradiation. They may comprise durable, fibrous sheets of various thicknesses or grindates thereof used, for example, as haemostatic scaffolds, or bone regeneration formulations. In the future, techniques from the leather industry (cross-linking technology and enzymatic sculpting) could inspire novel methods to generate structurally robust collagen-based medical devices of virtually any architecture and dimensions. Acid or enzyme-extracted ‘solution phase’ collagen formulations now provide potentially the most biocompatible of collagenous materials for flowable medical devices like dermal fillers and surgical haemostats; yet, current technology cannot transform such collagens into robust load-bearing medical devices like tendon or bone substitutes. However, learning how to control fibrillogenesis and molecular alignment of soluble collagens via 3D bioprinting may help meet this challenge.

Measurement ranges

In native tissues like bone, tendon and cornea, collagen fibrils range in diameters from about fifteen to hundreds of microns, may be aligned, organised into layers, packed at high densities, and cross-linked together, or loosely packed and randomly oriented, depending on tissue region and function. In some tissues, fibrils may branch or exhibit structural heterogeneity, such as crimping, at about 100-micron intervals. In general, connective tissue strength positively correlates with the diameters of its collagen fibrils.

Someday, novel technologies may help create robust collagen scaffolds having physical properties and dimensions mimicking those of native tissues. Thus, methods and formulations must be developed to deposit aligned collagen fibrils of specific diameters, and to orchestrate when during scaffold fabrication the fibrils polymerise. Collagen alignment may best be achieved via 3D bioprinting, as it may achieve flow orientation of collagen solutions, which is one of four methods shown to achieve fibril alignment. To control polymerisation timing, microfibrillar collagen may be deposited layer upon layer to form a device, then polymerised by phosphate precipitation. Polymerisation rates and extents may be adjusted depending on whether pepsinised or non-pepsinised collagen is used or lanthanides included.

Another challenge is that typically the 3D printing of plastics and metals requires the material to be melt-extruded or laser sintered and then cooled.

Moreover, larger diameter fibrils may be obtained if collagen oligomers are kept at low concentrations relative to that of monomers. During scaffolding, removal of liquid from the collagen formulation could promote close fibril apposition to achieve target protein concentrations and chemical or enzymatic crosslinking applied when desired. However, 3D bioprinting presents technical challenges related to the delicate structure-function attributes of the print materials. As an example, the formulation of certain biomaterials requires them to remain extrudable and/or gel-like as well as compatible with living cells. Moreover, the bioink must assume its target structural complexity/architecture such as firmness, shape and porosity in the finished device.

Another challenge is that typically the 3D printing of plastics and metals requires the material to be melt-extruded or laser sintered and then cooled. This approach is not possible for biopolymers, since most biologics denature above physiologic temperatures. Thus, current attempts involving polymerisation/gelation of collagen, gelatin, or alginate, have yielded gels that are not very robust or stable, requiring the addition of curing agents.

Yet, collagens, if formulated appropriately, are potentially the most ideal components for 3D bioprinting owing to their biocompatibility, bioactivity, conformational flexibility, robust structure, availability in numerous processed forms and low cost. Thus, flowable solutions of denatured and/or hydrolysed collagen (gelatin), microfibrillar native collagen, particulate crosslinked or non-crosslinked gelatin, limed collagen or native fibrillar collagens may be formulated, each with distinctive physical and biological attributes. Native forms of collagens are more robust and stable than many other proteins and are insensitive to most proteases. Moreover, some collagens may be manipulated to remain soluble during 3D printing but undergo gelation or polymerisation afterwards. To date, 3D bioprinting of collagen has mainly been explored on a small-scale in labs for various non-commercial applications but holds significant promise for the manufacture of structurally robust and biocompatible collagen-based medical devices of virtually any architectures and dimensions.