Trauma disrupts the anatomical cohesion of tissue structures, most evident by bleeding which indicates breakage of blood vessels and disruption of their endothelial lining. This immediately induces a haemostatic response encompassing platelet aggregation, blood clot formation and accumulation of provisional ECM 30 . Damaged epithelia secrete cytokines, growth factors and chemoattractants for mononuclear cells to phagocytose cellular debris at the site of injury and for fibroblasts to deposit collagen and remodel it. Thus, a scar is formed that eventually matures. The origin of these fibroblasts is currently a matter of debate. They are either differentiating homing mesenchymal stem cells 31 , fibrocytes from the blood circulation 32 or are derived from epithelia via epithelial-mesenchymal transition (EMT) 33 . The fibroblasts involved in scarring have a myofibroblast phenotype characterised by α-smooth muscle actin (α-SMA) expression, increased secretion of collagen type I and III, and contractility 30 . These cells are believed to be responsible for the majority of collagen production in most organs.

The cellular effectors of fibrosis are activated and phenotypically modulated by humoral players, namely chemokines, growth factors and cytokines. Most notorious is transforming growth factor β1 (TGF-β1) which supports wound healing and repair. Under pathological conditions, TGF-β1 coordinates a cross-talk between parenchymal inflammatory and collagen-expressing cells, and plays a key role in fibrosis progression. TGF-β1 is often referred to as a 'soluble' factor. We use quotation marks here because TGF-β1 is stored in its latent form bound to TGF-β1 binding proteins in the matrix 34 , and can in its active form be scavenged and possibly neutralized by decorin-mediated binding into the ECM 35 36 (for review see 37 ).

Along with factors such as the epithelial growth factor, basic fibroblast growth factor and interleukin-1, TGF-β1 appears to play a key role in EMT 32 . The connective tissue growth factor 38 and platelet-derived growth factor 39 have also been reported to be involved in fibrosis (for a more in-depth review on cytokines and molecular mechanisms involved in fibrogenesis, refer to 40 ). Inflammation typically precedes fibrosis, although it has been demonstrated that fibrosis is not always driven by inflammation. This suggests that the mechanisms that regulate fibrogenesis are, to a certain extent, distinct from those regulating inflammation 41 . This may explain the lack of efficacy of anti-inflammatory compounds in the treatment of fibrotic disease 42 . Antifibrotic strategies at the upstream level aim to interfere with fibrotic growth factors and make use of interfering antibodies 43 44 45 46 , small molecules 39 47 , proteins 48 49 , antisense technology 50 51 or the use of human recombinant TGF-β3. TGF-β3 is an alleged TGF-β1 antagonist that has shown some promise in phase III clinical trials in a prophylactic setting of small skin wounds 52 .

Irrespective of upstream events that trigger and entertain fibrosis, the final product of cellular activity is the massive deposition of collagen which results in scar formation, organ or peri-implantational fibrosis. Therefore, we would like to turn now to the obvious target in fibrosis, namely the biosynthetic pathway of collagen itself.

The overall amount of collagen deposited by fibroblasts is a regulated balance between collagen synthesis and collagen catabolism, which is a carefully controlled process. During a pathological maturation and remodelling phase, collagen synthesized by fibroblasts exceeds the rate at which it is degraded such that the net amount of collagen continues to increase. There are several key points along the collagen biosynthesis pathway that can be targeted to effect a net reduction of collagen secretion and/or deposition (Figure 1). Transcription interference can be affected using histone deacetylase inhibitors 53 54 or substances like halofuginone 55 56 . At the post-transcriptional level, siRNA targeting growth factors and key players have been investigated 50 51 57 and, recently, we and others have suggested the use of microRNAs 58 59 . Interfering with post-translational modifications by inhibiting prolyl-4 hydroxylase renders collagen triple helices less thermostable and prevents their secretion 59 60 , down-regulation of collagen chaperone hsp47 does likewise 61 62 . At the extracellular level, inhibition of procollagen C-proteinase/BMP1 prevents the removal of the C-terminal propeptide from the procollagen I molecule and, thus, the supramolecular assembly of collagen into fibrils 47 59 . Inhibition of lysyl oxidase mediation of inter-molecular cross-links between collagen triple helices can reduce collagen content of the ECM, presumably by rendering collagen aggregates more susceptible to proteolytic remodelling 63 . Similarly, the administration of hepatocyte growth factor 64 and matrix metalloproteinase 1 (MMP1) 48 49 increases collagen turnover in the ECM. It becomes clear that a meaningful in vitro system for the testing and characterization of antifibrotics should be able to emulate the above described complete collagen matrix formation cascade, encompassing its biosynthesis and all post-translational (intra- and extra-cellular) modifications that give rise to a stable supramolecular assembly, and also ideally allow the study of remodelling/fibrolysis. For the sake of convenience and efficiency for screening purposes, quantitation of collagen and other proteins of interest should preferentially be in and from one well.

(1) The first challenge for in vitro fibrogenesis is the sufficient production of collagen and its subsequent incorporation into a pericellular matrix. The omission of ascorbic acid in cell culture 38 65 66 results in minimal production and deposition of collagen on the cell layer. Ascorbate is a crucial cosubstrate of the enzymes responsible for the post-translational hydroxylation of prolyl and lysyl residues necessary for rendering the collagen triple helix thermostable, as well as for the extracellular cross-linking of collagen fibers, respectively 67 68 . In other words, scurvy can exist in cell culture. However, like its counterpart in vivo, it can be easily treated in vitro by the administration of ascorbate. On the other hand, L-ascorbic acid has a short half-life in culture and completely oxidizes after 3 days, so use of the stable form of ascorbate, such as a magnesium salt of L-ascorbic acid 2-phosphate hexahydrate, is highly recommended 67 .

About 23% of the collagen molecule is composed of proline and hydroxyproline. As a non-essential amino acid, proline is synthesized from arginine/ornithine via the urea cycle and glutamate (directly or indirectly from glutamine via glutaminase) through the citric acid cycle. Clinical observations in burn patients suggest a drain of arginine, ornithine and glutamate 69 , while wound fluid proline levels are at least 50% higher than plasma levels, suggesting active import of proline into the wound 70 . Providing additional proline or glutamine in the diet to enhance collagen biosynthesis, however, does not result in increased collagen accumulation. In contrast, arginine, and ornithine supplementation are most effective in increasing collagen deposition 71 . As cell culture media contains L-arginine and are usually supplemented with L-glutamine, direct proline supplementation of cell cultures in static and bioreactor conditions is not necessary for increasing collagens synthesis, as also shown recently 72 . However, systematic studies of proline-precursor supplementation in vitro under conditions of increased synthesis and deposition have yet to be conducted.

(2) Even under ascorbate supplementation, fibroblasts deposit only minimal amounts of secreted collagen I into their matrices. The reason for this lies in the tardy procollagen C-proteinase/BMP1 activity under current aqueous culture conditions. This results in an accumulation of unprocessed procollagen in the cell culture medium 73 where it does not belong and is discarded with every medium change. Analytical methods have, therefore, mostly focused on the convenient measurement of procollagen secreted into culture medium. This may be sufficient to assess compounds that primarily influence biosynthesis and/or secretion, but it would not allow assessment of any later step in collagen matrix formation.

To improve collagen deposition in cell culture, a fibroplasia model was developed using hyperconfluent human dermal fibroblasts exposed to TGF-β1 for up to 1 month to allow the formation of a fibroplastic tissue 63 . In an abbreviated form (8 days TGF-β1 treatment) this model is in use at Pfizer Global R&D (Sandwich, Kent). This model moves closer to emulating the entire collagen biosynthesis pathway as it allows the assessment of procollagen C-proteinase inhibitors, which are predicted to interfere with collagen deposition 47 . Destructive analysis of deposited collagen by high-performance liquid chromatography of 4-hydroxyproline is performed on the insoluble culture fraction. Our laboratory has systematically developed technology to accelerate collagen deposition in vitro by introducing macromolecules into the culture medium. We have developed two deposition technologies that differ in terms of speed and morphology of the deposited collagen. The first approach employs charged macromolecules such as dextran sulphate 500 kDa (DxS) and polysodium-4-styrene sulfonate 61 . DxS leads to a granular deposition of collagen within 48-72 h, exceeding that of non-crowded cultures by 20- to 30-fold within the same time frame. The second approach using neutral macromolecules in a Ficoll cocktail (Fc) 59 increases collagen deposition 10-fold in 6 days and in a reticular deposition pattern (Figure 2B). Both approaches are based on the creation of the excluded volume effect as explained elsewhere 61 73 74 . Briefly macromolecules drive reaction partners into closer collaboration resulting in improved protein folding and protein-protein interactions. In the case of fibrogenic cell culture, the conversion of procollagen to collagen is sped up as well as the supramolecular assembly of collagen triple helices to form fibres.

(3) Determination of the amount of collagen produced in vitro is the next challenge, and this can be done in a variety of ways ranging from simple colorimetric assays to elaborate chromatographic procedures using radioactive and non-radioactive material. What most of these procedures have in common is the need to destroy the cell layer to obtain solubilized collagen from the pericellular matrix. The oldest established colorimetric method employs chloramine-T method to measure hydroxyproline 75 . The destructive pre-solubilization requirement of collagen for this assay, lack of specificity in discriminating between collagen types, as well as a lack of internal normalization within samples, are disadvantages in a screening setting. The Sirius dye has been used since 1964 to identify collagen in histology specimens 76 77 . It is based on the selective binding of Sirius Red F3BA to collagen. Subsequent elution with sodium hydroxide-methanol and read-out at 540 nm can be done in cuvettes, in microtiterplates with collagen extract adsorbed to the plastic and using microplate readers 66 78 79 . Biocolor Ltd (County Antrim, Northern Ireland, UK) made the Sircol™ Collagen Assay commercially available in 2007. Precipitation of soluble collagen with the Sirius Red dye is required prior to release of the dye with an alkali. In our hands, this assay grossly overestimates collagen and procollagen secreted into cell culture medium due to interference of non-collagenous serum proteins, so we would recommend prepurification (peptic digest and ultrafiltration) to improve both sensitivity and specificity (Ricky R Lareu, Dimitrios Zeugolis and Michael Raghunath, unpublished experiments). An interesting solution appears to be the adsorption of this dye onto collagen deposited on the cell layer in culture 80 . This was recently suggested in a modified version for the testing of antifibrotic agents 66 , although this screening system did not use or recommend the addition of ascorbic acid 66 . Various enzyme-linked immunoassays can be used to quantify specific collagen types such as coating collagen extracts onto multiwell plates followed by detection using antibodies, sandwich assays and competitive enzyme immunoassays. These assays are very suitable for assessing soluble collagen from culture media but would require extraction and dialysis procedures to release insoluble collagen from pericellular matrices. In this case, normalization for differences between protein amounts or cell numbers in samples is not possible. The determination of the hydroxyproline/proline ratio via HPLC is based on total hydrolysis of protein samples, separation of hydroxyproline from proline and back calculating the possible collagen content of the sample 81 . While this method allows for the analysis of any given biological material in experienced laboratories, it is not amenable to a screening setting and would not allow for normalisation or discrimination between different collagen types, the same holds true for metabolically labelled cell cultures with the attending issues of isotope handling 82 . Similar issues arise with gas chromatography/mass spectrometry requiring derivatization using trifluoroacetylation and methanol esterification of 4-hydroxyproline in collagen. Incorporation of the stable isotope of oxygen, ¹⁸O₂, into collagen is also possible with this method and enables the examination of collagen synthesis in vitro 83 . Polyacrylamide gel electrophoresis can be very collagen specific using metabolic labelling of cell cultures with radiolabelled amino acids glycine and proline 84 and subsequent detection of radioactive bands using fluorography 85 . Metabolic labelling can be replaced with safer and very sensitive silver staining or immunoblotting 61 . The latter has the advantage of differentiating between various collagen types and comparison with an internal standard like actin as a cell mass equivalent for normalization. Collagen antibodies tend to recognize protein conformation in addition to sequence specificity, which can pose a problem for the detection of denatured collagen α-chains in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), hence native PAGE might be an alternative option 86 . All gel electrophoresis-based approaches are excellent qualitative and quantitative back-up techniques. However, their laboriousness would exclude them as screening tools.

(4) Normalization of data to account for cell number variations is another important consideration. Most of the methods discussed above lack the option of normalization and the use of parallel cultures subjected to the separate measurement of total protein or DNA content. In any case, cell density influences the amount of collagen deposited: in fact, subconfluent cultures produce the most collagen 87 . Depending on the substance screened, inhibition or stimulation of proliferation, or cytotoxicity may occur. As this certainly will impact on the collagen amount secreted/deposited, well-to-well variances not withstanding, the acquired data must account for these changes in cell number/density. Data normalization methods involve the destruction of cell layers to quantify housekeeping proteins by Western blot or DNA 81 83 . This not only increases sample-processing steps, but also cumulates error.

(5) Fibrosis is not only characterized by an excess build-up of collagen I but, depending on the tissue context, other proteins might be of interest. Also, the transition of fibrogenic cells from a quiescent fibroblast stage to a fibrotic myofibroblast phenotype should be considered. Markers such as α-SMA or fibroblast activation protein-α 88 are up-regulated in stimulated fibroblasts and a survey of the production and deposition of non-collagenous extracellular matrix (ECM) proteins as an internal control for the action of antifibrotic agents would be desirable. As well as capturing the actual number of cells in a given well, options for a high content read-out covering as many of the above markers as possible in addition to collagen I would constitute an ideal antifibrotic screening system.

Besides the current use of fibroblasts, this system has the flexibility and potential to screen the effect of potential antifibrotic compounds on other organ specific culprits such as hepatic stellate cells. The optical accessibility of the Scar-in-a-Jar has room for the introduction of one or more additional cell types like monocytes or smooth muscle cells to augment the fibrotic context and tailor it for a dermal wound healing situation or an atherosclerotic plaque. This could be done in direct coculture or using inserts, with the fibrotic target cells adhering to the bottom of the well. Besides preliminary testing of exogenously added matrix metalloproteinases 59 , the current system has not yet been fathomed for its ability to study collagen matrix metalloproteinases turnover that would be relevant for analysing effects of collagen cross-link inhibitors and inducers of matrix metalloprtoteinases like hepatocyte growth factor. We envision longer observation times in this instance or challenging cell cultures after an initial ECM build-up, with additional cell types or substances that induce remodelling. A very promising line of research using this system could be non-enzymatic glycation studies to capture a diabetic situation and testing breakers of advanced glycation end products.