Biomaterials in regenerative medicine: engineering to recapitulate the natural.

The functional significance of the extracellular matrix (ECM) has generally been defined in terms of the provision of a structural support for cell adhesion and the establishment of tissue physical integrity. Recent evidence has, however, led to a paradigm shift according to which the ECM is increasingly recognised to exert a profound influence on cell behaviour, including gene expression, migration and the maintenance of functional homeostasis. The objective of this focussed review is to highlight selected observations underpinning this conclusion. Finally, we discuss the implications of these findings for regenerative medicine in the specific context of developing the 'next generation' of advanced wound care devices for the clinical management of recalcitrant chronic wounds.

Bistable switch in migration stimulating factor expression: regulation by the concerted signalling of transforming growth factor-ß1 and the extracellular matrix.

Migration stimulating factor (MSF) is an oncofetal motogenic/angiogenic cytokine constitutively expressed by epithelial and stromal cells in fetal and neoplastic tissues. Fibroblasts derived from healthy adult skin do not express MSF but can be induced to do so by treatment with transforming growth factor-ß1 (TGF-ß1). As the bioactivities of both MSF and TGF-ß1 are modulated by the extracellular matrix, we investigated whether the induction of MSF expression by TGF-ß1 is also matrix dependent. We now report that adult fibroblasts are induced to express MSF by a transient treatment with TGF-ß1 (as short as 2 hr) but only when the cells are adherent to a "wound" matrix, such as denatured type I collagen, fibrin or plastic tissue culture dishes. Unexpectedly, this induction of MSF expression persists unabated for the entire subsequent lifespan of the treated cells in the absence of further TGF-ß1 and irrespective of the substratum. Such "activated" MSF expression may, however, be persistently switched off again by a second transient exposure to TGF-ß1 but this time only when the cells are adherent to a "healthy" matrix of native type I collagen. Significantly, the constitutive expression of MSF by fetal and cancer patient fibroblasts could also be persistently switched off by this means. We conclude that TGF-ß1 may both switch on and switch off MSF expression in a manner critically determined by the nature of the matrix substratum and suggest that this may be a possible mechanism underlying the observed dual functionality of TGF-ß1 as both a tumour suppressor and tumour promoter.

Migration-stimulating factor as a novel biomarker in salivary gland tumours.

BACKGROUND: The identification of novel stratification biomarkers would benefit the clinical management of patients with salivary gland tumours. Migration-stimulating factor (MSF) is a potent stimulator of cell invasion, matrix remodelling and angiogenesis. The aim of this study was to determine whether MSF was expressed in salivary gland tumours and its potential value as a diagnostic biomarker. METHODS: Paraffin-embedded archival specimens of small salivary gland tumours were stained with an MSF-specific antibody. The specimens included 27 malignant and seven benign tumours; histologically normal salivary gland adjacent to the tumour was present in 16 specimens. MSF expression was assessed by consensus of 2-4 independent observers according to various indices, including 'overall MSF grade', 'percentage of area stained' and 'intensity of the staining'. The motogenic effect of MSF on a salivary gland tumour cell line, HSG, was examined in the transmembrane assay. RESULTS: Overall MSF expression increased significantly in a step-wise fashion from normal salivary gland to benign and malignant tumours (P = 0.04-0.0001); with moderate/strong positive specimens representing 6%, 33% and 74% of the normal, benign and malignant specimens, respectively. MSF was heterogeneously expressed in both carcinoma and stromal cell compartments, its expression being higher in malignant than benign tumours regarding various MSF indices. In tissue culture studies, exogenous MSF stimulated the migration of HSG cells. CONCLUSIONS: These immunohistochemical and functional studies suggest that MSF expression is a potentially useful biomarker of salivary gland tumour progression.

Migration Stimulating Factor (MSF) promotes fibroblast migration by inhibiting AKT.

The protein kinase AKT is activated strongly by many motogenic growth factors, yet has recently been shown capable of inhibiting migration in several cell types. Here we report that treatment with Migration Stimulating Factor (MSF), a truncated form of fibronectin that promotes the migration of many cell types, inhibits AKT activity in human fibroblasts and endothelial cells. In fibroblasts, treatment with either MSF or the AKT inhibitor, Akti-1/2, stimulated migration into 3D collagen gels to a similar extent and the effects of Akti-1/2 on migration could be blocked by the expression of an inhibitor-resistant mutant, AKT1 W80A. These data indicate that MSF promotes fibroblast migration, at least in part, by inhibiting the activity of AKT.

Multi-factorial modulation of IGD motogenic potential in MSF (migration stimulating factor).

Migration Stimulating Factor (MSF) is a genetically truncated isoform of fibronectin (Fn). MSF is a potent stimulator of fibroblast migration, whereas full length Fn is devoid of motogenic activity. MSF and Fn contain four IGD motifs, located in the 3rd, 5th, 7th and 9th type I modules; these modules are referred to as (3)FnI, (5)FnI, (7)FnI and (9)FnI, respectively. We have previously reported that mutation of IGD motifs in modules (7)FnI and (9)FnI of MSF is sufficient to completely abolish the motogenic response of target adult skin fibroblasts. We now report that the IGD sequences in (3)FnI and (5)FnI are also capable of exhibiting motogenic activity when present within fragments of MSF. When present within (1-5)FnI, these sequences require the presence of serum or vitronectin for their motogenic activity to be manifest, whereas the IGD sequences in (7)FnI and (9)FnI are bioactive in the absence of serum factors. All MSF and IGD-containing peptides stimulated the phosphorylation of the integrin binding protein focal adhesion kinase (FAK) but did not necessarily affect migration. These results suggest that steric hindrance determines the motogenic activity of MSF and Fn, and that both molecules contain cryptic bioactive fragments.

Motogenic sites in human fibronectin are masked by long range interactions.

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains (4-5)FnI and (3)FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes (3)FnIII and disrupts the observed (4-5)FnI-(3)FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.

Differential involvement of TGF-beta1 in mediating the motogenic effects of TSP-1 on endothelial cells, fibroblasts and oral tumour cells.

The role of TSP-1 in tumour growth and angiogenesis remains controversial, with both stimulatory and inhibitory roles proposed. The effects of TSP-1 on the migration of endothelial cells, fibroblast and oral tumour cell lines were examined using the transmembrane assay. TSP-1 induced a bi-phasic effect on human and bovine endothelial cells: stimulation at low concentrations (0.1-10 microg/ml) and inhibition at high concentrations (25-100 microg/ml). FGF-2-stimulated endothelial cell migration was either further stimulated or inhibited by TSP-1, following the same bi-phasic dose response as in the absence of FGF-2. In contrast, TSP-1 stimulated the migration of human fibroblast and oral tumour cells in a dose dependent manner; a plateau was reached with 5-25 microg/ml and no inhibitory effect was observed. These effects were partly neutralised by antibodies to alphavbeta3 integrin. TGF-beta1 (0.1-200 ng/ml tested) mimicked the effects of TSP-1 on cell migration. Function-neutralising antibodies to TGF-beta1 completely abolished both the stimulatory and inhibitory effects of TSP-1 on endothelial migration, but had no effect on TSP-1-stimulated migration of fibroblast and oral tumour cells. The effects of TGF-beta1 were not affected by antibodies to TSP-1. These results indicate that the effects of TSP-1 on endothelial cell migration are mediated by TGF-beta1, whereas the effects on fibroblast and tumour cell migration are TGF-beta1-independent.

Individually addressable microelectrode array for monitoring oxygen and nitric oxide release.

We have fabricated a six individual addressable gold working electrode microarray. The device is wirebonded to an eight-pin DIL package that can be easily interconnected to an external multi-channel potentiostat. A polyion complex film coating on the electrode surface provides a suitable coating for the growth of cells. The responses of oxygen and nitric oxide were assessed on uncoated and coated devices using electroanalytical methods. The film coating reduced the diffusion current by approximately 20% in both cases. No changes in the electrochemical mechanism were observed. Simultaneous recordings were obtained for 2 h in the presence of the cells, thus the device is stable for the duration of the bioanalytical measurements. Measurements were conducted to study the simultaneous changes in oxygen and nitric oxide levels in cultured fibroblast cells in the presence of growth hormones that cause cell proliferation. Increases in oxygen consumption of the cells were coupled with increases in nitric oxide levels when in the presence of the growth hormones. Use of a biological detergent to cause an oxidative burst resulted in a large increase in the current for potentials set to detect nitric oxide and oxygen.

Co-expression by keratinocytes of migration stimulating factor (MSF) and a functional inhibitor of its bioactivity (MSFI).

Migration stimulating factor (MSF) is a potent autocrine and paracrine factor expressed by fibroblasts and epithelial cells in foetal skin, tumours and healing wounds. In tissue culture, MSF bioactivity is present in the conditioned medium of foetal and tumour derived fibroblasts, but not in normal adult fibroblasts or keratinocytes. The conditioned medium of early passage keratinocytes or a keratinocyte line (HaCaT) effectively inhibited the motogenic activity of rhMSF. Fractionation of keratinocyte conditioned medium by size-exclusion chromatography revealed the presence of bioactive MSF as well as a functional inhibitor of MSF (MSFI) in fractions corresponding to approximately 70 kDa and 25 kDa, respectively. MSFI was purified and identified as neutrophil gelatinase-associated lipocalin (NGAL or lipocalin-2). Immunostaining confirmed that keratinocytes expressed both MSF and NGAL, whereas normal adult fibroblasts did not express either. Recombinant and cell-produced NGAL neutralised the motogenic activity of rhMSF. NGAL is known to bind MMP-9 and promote the activity of this protease. In contrast, there was no evidence of NGAL-MSF binding in keratinocyte conditioned medium. MSF displays a number of bioactivities of relevance to cancer progression and wound healing. Our findings indicate a novel function of NGAL and a possible mechanism for regulating MSF activity in tissues.

The role of the fibronectin IGD motif in stimulating fibroblast migration.

The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 10(6) times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN.

EGF AND TGF-alpha motogenic activities are mediated by the EGF receptor via distinct matrix-dependent mechanisms.

EGF and TGF-alpha induce an equipotent stimulation of fibroblast migration and proliferation. In spite of their homologous structure and ligation by the same receptor (EGFR), we report that their respective motogenic activities are mediated by different signal transduction intermediates, with p70(S6K) participating in EGF signalling and phospholipase Cgamma in TGF-alpha signalling. We additionally demonstrate that EGF and TGF-alpha motogenic activities may be resolved into two stages: (a) cell "activation" by a transient exposure to either cytokine, and (b) the subsequent "manifestation" of an enhanced migratory phenotype in the absence of cytokine. The cell activation and manifestation stages for each cytokine are mediated by distinct matrix-dependent mechanisms: motogenetic activation by EGF requires the concomitant functionality of EGFR and the hyaluronan receptor CD44, whereas activation by TGF-alpha requires EGFR and integrin alphavbeta3. Manifestation of elevated migration no longer requires the continued presence of exogenous cytokine and functional EGFR but does require the above mentioned matrix receptors, as well as their respective ligands, i.e., hyaluronan in the case of EGF, and vitronectin in the case of TGF-alpha. In contrast, the mitogenic activities of EGF and TGF-alpha are independent of CD44 and alphavbeta3 functionality. These results demonstrate clear qualitative differences between EGF and TGF-alpha pathways and highlight the importance of the extracellular matrix in regulating cytokine bioactivity.

The expression of migration stimulating factor, a potent oncofetal cytokine, is uniquely controlled by 3'-untranslated region-dependent nuclear sequestration of its precursor messenger RNA.

Migration stimulating factor (MSF) is a truncated oncofetal fibronectin isoform expressed by fetal and tumor-associated cells. MSF mRNA is distinguished from other fibronectin isoforms by its size (2.1 kb) and the inclusion of a specific intronic sequence at its 3' end. Initial Northern blot analysis with a MSF-specific probe indicated the presence of this 2.1-kb transcript and an additional unexpected 5.9-kb RNA present in both MSF-secreting (fetal) and nonsecreting (adult) fibroblasts. Our investigations into the nature of these transcripts and their relationship to MSF protein secretion revealed that the 5.9-kb mRNA is a second MSF-encoding transcript. Both these mRNAs have identical coding sequence and differ only in the length of their intron-derived 3'-untranslated region (UTR). The 5.9-kb MSF mRNA is retained in the nucleus whereas the 2.1-kb mRNA is not. MSF-secreting fetal fibroblasts have significantly lower nuclear levels of the 5.9-kb mRNA and correspondingly higher cytoplasmic levels of the 2.1-kb transcript than their nonsecreting adult counterparts. Adult fibroblasts induced to secrete MSF by treatment with transforming growth factor-beta1 displayed similar changes in their respective levels of MSF mRNA, but not those of a control gene. When cloned downstream of a reporter gene, only the longer 3'-UTR retained coding sequence within the nucleus. We conclude that expression of MSF protein is regulated by 3'-UTR truncation of the 5.9-kb nuclear-sequestered "precursor" MSF mRNA and nuclear export of mature 2.1-kb message. Inducible 3'-UTR processing represents a novel regulatory mechanism involved in cancer pathogenesis that may open new avenues for therapeutic gene delivery.

Synthesis of an IGD peptidomimetic with motogenic activity.

Rational design and synthesis of an IGD peptidomimetic substrate with significant motogenic activity.

Migration-stimulating factor: a genetically truncated onco-fetal fibronectin isoform expressed by carcinoma and tumor-associated stromal cells.

Migration-stimulating factor (MSF) is a 70-kDa motogenic protein previously reported to be expressed by fetal and cancer patient fibroblasts cultured in vitro and present in the serum of breast cancer patients. A 2.2-kb full-length MSF cDNA has been cloned and shown to be a truncated isoform of fibronectin generated from its primary gene transcript by a hitherto unrecognized intron read-through mechanism. MSF cDNA is identical to the 5' end of fibronectin cDNA, up to and including exon III-1a, and terminates in a novel 195-nucleotide 3' sequence. This MSF unique sequence is derived from the intron immediately downstream of exon III-1a in the fibronectin gene and is not found in any previously identified "full-length" fibronectin cDNA. MSF mRNA is 1000-fold less abundant than full-length fibronectin message in fetal fibroblasts and exhibits rapid biphasic decay kinetics previously associated with oncogenes and stress response molecules. MSF recombinant protein exhibits a potent and substratum-dependent motogenic activity, with half-maximal response manifest at 0.1-1.0 pg/ml. This activity is (a) mediated by the IGD amino acid motif; and (b) not expressed by (i.e., cryptic within) full-length fibronectin. In situ hybridization and immunohistochemistry confirm that MSF is expressed by tumor-associated fibroblasts and additionally indicate that it is also expressed by carcinoma cells and tumor-associated vascular endothelial cells. MSF, as a consequence of its potent bioactivities and expression by both stromal and carcinoma cell populations, is well placed to function as an epigenetic effector promoting cancer development.

Evidence of a bi-phasic effect of thrombospondin-1 on angiogenesis.

Contradictory results have been reported regarding the association between vascularity (used as an index of angiogenesis) and thrombospondin-1 (TSP-1) in human tumours. In previous studies, the reported association was based on the estimated average TSP-1 value per tumour, with a sufficient number of specimens collectively analysed per tumour type. Given the extent of intra-tumour heterogeneity, we determined the association between TSP-1 and vascularity within individual specimens, based on the average values of TSP-1 and vascularity in 10-20 pre-selected areas per tumour. Cells expressing TSP-1 mRNA were visualised by in situ hybridisation and quantified by point counting. Vascularity was quantified by point counting and vessel density of von Willebrand Factor-positive vessels. In 10 ductal breast carcinomas, a direct correlation between TSP-1 and vascularity was found in 4 tumours, no correlation in 3 and an inverse correlation in 3. The effect of TSP-1 on endothelial cell migration in vitro was assessed in the Boyden chamber assay. TSP-1 stimulated cell migration at low concentrations (0.1-10 microg/ml) and was inhibitory at high concentrations (25-100 microg/ml). These results suggest that TSP-1 may elicit a concentration-dependent, bi-phasic, effect on angiogenesis.