A comparative study of skin cell activities in collagen and fibrin constructs

Collagen and fibrin are widely used in tissue engineering due to their excellent biocompatibility and bioactivities that support in vivo tissue formation. These two hydrogels naturally present in different wound healing stages with different regulatory effects on cells, and both of them are mechanically weak in the reconstructed hydrogels. We conducted a comparative study by the growth of rat dermal fibroblasts or dermal fibroblasts and epidermal keratinocytes together in collagen and fibrin constructs respectively with and without the reinforcement of electrospun poly(lactic acid) nanofiber mesh. Cell proliferation, gel contraction and elastic modulus of the constructs were measured on the same gels at multiple time points during the 22 day culturing period using multiple non-destructive techniques. The results demonstrated considerably different cellular activities within the two types of constructs. Co-culturing keratinocytes with fibroblasts in the collagen constructs reduced the fibroblast proliferation, collagen contraction and mechanical strength at late culture point regardless of the presence of nanofibers. Co-culturing keratinocytes with fibroblasts in the fibrin constructs promoted fibroblast proliferation but exerted no influence on fibrin contraction and mechanical strength. The presence of nanofibers in the collagen and fibrin constructs played a favorable role on the fibroblast proliferation when keratinocytes were absent. Thus, this study exhibited new evidence of the strong cross-talk between keratinocytes and fibroblasts, which can be used to control fibroblast proliferation and construct contraction. This cross-talk activity is extracellular matrix-dependent in terms of the fibrous network morphology, density and strength.     

Expression of CD13/aminopeptidase N and CD10/neutral endopeptidase on cultured human keratinocytes

Keratinocytes actively participate in immune response and inflammation by secreting cytokines and chemokines. Membrane-bound peptidases serve as negative loop in controlling concentration of peptide signalling molecules. Recently, they have also been proposed as additional mechanism of cell-to-cell interaction and as signalling molecules. In this study, we examined expression of two membrane-bound peptidases: aminopeptidase N (APN; EC 3.4.11.2; CD13) and neutral endopeptidase (NEP; EC 3.4.24.11; CD10) on nonstimulated cultured human keratinocytes obtained from healthy skin. Membrane expression of CD13 and CD10 was analysed by FACS and fluorescent microscope. Functional properties of CD13 and CD10 were examined by testing their enzymatic activity towards selective substrates. The data were compared to those obtained on cultured nonstimulated human skin fibroblasts expressing both CD13/APN and CD10/NEP. Approximately one-third (i.e. 31.7+/-2.8%; n=3) of cultured keratinocyte express CD13 as compared to fibroblasts which are 100% CD13(+) (n=3). Density of CD13 on keratinocytes is several times lower than on fibroblasts. Membrane CD13 expression on keratinocytes was associated with significant enzyme activity, which on the basis of substrate (L-Ala-betaNA) and inhibitor (bestatin, actinonin) selectivity could be ascribed to aminopeptidase N. Kinetic parameter V(max) revealed lower APN activity expressed on keratinocytes than on fibroblasts (V(max)=1.49+/-0.08 microM/60 min/5 x 10(4) cells for keratinocytes, n=3 versus V(max)=4.09+/-0.76 microM/60 min/5 x 10(4) cells for fibroblasts, n=3). Likewise, K(m) value of APN on keratinocytes was lower as compared to fibroblasts (K(m)=0.307+/-0.090 mM for keratinocytes, n=3 versus K(m)=0.766+/-0.065 mM for fibroblasts, n=3). CD13 demonstrated on cultured keratinocytes, is at least partly due to its constitutive expression since it was also found on freshly prepared epidermal skin cells. Inhibitors of APN, actinonin, bestatin and substance-P, as well as the APN blocking antibody WM-15, decreased keratinocytes growth. In contrast to membrane CD13 associated with APN enzyme activity, neither membrane CD10, nor its enzyme (NEP) activity could be found on the same keratinocyte samples. In conclusion, functional CD13, associated with APN activity, was found on about one third of cultured, non-stimulated keratinocytes, whereas no CD10/NEP was found on the same keratinocyte samples. Role of APN in regulation of keratinocyte growth is suggested, as its inhibition resulted in decreased keratinocyte growth.     

Response of human skin equivalents to Sarcoptes scabiei

Studies have shown that molecules in an extract made from bodies of the ectoparasitic mite, Sarcoptes scabiei De Geer, modulate cytokine secretion from cultured human keratinocytes and fibroblasts. In vivo, in the parasitized skin, these cells interact with each other by contact and cytokine mediators and with the matrix in which they reside. Therefore, these cell types may function differently together than they do separately. In this study, we used a human skin equivalent (HSE) model to investigate the influence of cellular interactions between keratinocytes and fibroblasts when the cells were exposed to active/burrowing scabies mites, mite products, and mite extracts. The HSE consisted of an epidermis of stratified stratum corneum, living keratinocytes, and basal cells above a dermis of fibroblasts in a collagen matrix. HSEs were inoculated on the surface or in the culture medium, and their cytokine secretions on the skin surface and into the culture medium were determined by enzyme-linked immunosorbent assay. Active mites on the surface of the HSE induced secretion of cutaneous T cell-attracting chemokine, thymic stromal lymphopoietin, interleukin (IL)-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1ra), IL-6, IL-8, monocyte chemoattractant protein-1, granulocyte/macrophage colony-stimulating factor, and macrophage colony-stimulating factor. The main difference between HSEs and monocultured cells was that the HSEs produced the proinflammatory cytokines IL-1alpha and IL-1beta and their competitive inhibitor IL-1ra, whereas very little of these mediators was previously found for cultured keratinocytes and fibroblasts. It is not clear how the balance between these cytokines influences the overall host response. However, IL-1ra may contribute to the depression of an early cutaneous inflammatory response to scabies in humans. These contrasting results illustrate that cell interactions are important in the host's response to burrowing scabies mites.     

Growth of herpes simplex virus in epidermal keratinocytes determines cutaneous pathogenicity in mice

Herpes simplex viruses (HSV)-1 and -2 isolated from genital lesions were examined for cutaneous pathogenicity and its correlation with cellular tropism. HSV-1 caused vesiculation, erosion/ulcer, and zosteriform lesions successively, but skin lesions of HSV-2 developed without vesiculation in some mice, and with statistically significantly less frequent vesiculation than HSV-1. Thus, the virological type of HSV was correlated with its cutaneous pathogenicity. The growth characteristics of HSV-1 and -2 were compared in cultured human embryonic lung (HEL) fibroblasts, human lung cancer A549 cells, human neonatal epidermal keratinocytes, human neonatal dermal fibroblasts, HeLa cells, and Vero cells. HSV-2 produced plaques that were 72% times the size of HSV-1 plaques in epidermal keratinocytes but 230%-500% the size in the other cells. The difference between HSV-1 and -2 in the ratio of plaque size to virus yield in epidermal keratinocytes was much larger (502 times) than the ratio of the other cells (5.57-28.8 times). Keratinocytes are the major constituent of the epidermal layer of the skin and the cells in which vesiculation and erosion/ulceration occur histologically. Therefore, the smaller spread of HSV-2 in keratinocytes of the epidermal layer and the greater spread in other cells of the dermal layer might reflect its lesser invasiveness in the epidermal layer despite larger invasiveness in the dermal layer, which is reflected in the low incidence of erosion/ulcer of the skin compared to HSV-1. Thus, the growth of HSV in epidermal keratinocytes appeared to correlate with the cutaneous pathogenicity causing vesiculation in the skin.     

Human skin cell cultures onto PLA50 (PDLLA) bioresorbable polymers: influence of chemical and morphological surface modifications

Poly(alpha-hydroxy acid)s derived from lactic and glycolic acid are bioresorbable polymers which can cover a large range of thermal, physical, mechanical, and biological properties. Human keratinocytes have been shown as able to grow on a poly(DL-lactic acid) film. However the keratinocyte growth was delayed with respect to culture on standard tissue culture polystyrene, even though the same plateau level was observed after 2 weeks. In order to improve the performance of poly(DL-lactic acid) films as skin culture support, their surface was modified by creating tiny cavities using a method based on the leaching out of poly(ethylene oxide) from poly(lactic acid)-poly(ethylene oxide) heterogeneous blends. The surface of the films was also chemically modified by alkaline attack with sodium hydroxide and by type-I collagen coating. Murine fibroblast cell line and primary cultures of human fibroblasts and of two types of keratinocytes were allowed to adhere and to grow comparatively on the different films. The presence of cavities affected neither the adhesion of dermal fibroblasts nor that of keratinocytes. Only keratinocyte proliferation was significantly reduced by the presence of cavities. Collagen coating improved skin cell adhesion and proliferation as well, except in the case of murine fibroblasts. In the case of the NaOH treatments, similar trends were observed but their extent depended on the treatment time. In the case of chemical modifications, fluorescence microscopy bore out adhesion and proliferation tendencies deduced from MTT tests.     

Use of human fibroblasts in the development of a xenobiotic-free culture and delivery system for human keratinocytes

Previous work has shown that keratinocytes can be cultured serum-free on an acid-functionalized, plasma-polymerized surface (for subsequent delivery to patients' wound beds) by inclusion of a fibroblast feeder layer. This study seeks to extend this work by substituting human for murine feeder cells in serum-free culture and examining the performance of keratinocytes expanded in this way to transfer to an in vitro human dermal wound bed model. We compared murine and human fibroblasts (both short-term dermal fibroblasts and a fetal lung fibroblast cell line MRC-5, which has a long history in human vaccine production), alternative methods for growth-arresting fibroblasts, establishing culture of cells serum-free, and the impact of culture with fibroblasts on the differentiation of the keratinocytes. Irradiated human and murine fibroblasts were equally effective in supporting initial keratinocyte expansion, both in the presence and absence of serum. Keratinocytes were significantly less differentiated, as assessed by measuring involucrin expression relative to DNA when grown serum-free with fibroblasts than when grown with serum. Initial cultures of fibroblasts and keratinocytes could be initiated serum-free but were much slower to establish than if serum were used. Transfer of keratinocytes from keratinocyte/fibroblast co-cultures cultured on a plasma polymer surface to a human dermal wound bed model was as successful as from monocultures in both serum and serum-free cultures. In summary, we have revisited a well-accepted methodology for expanding human keratinocytes for clinical use and avoided the use of bovine serum and a mouse fibroblast feeder layer by introducing an irradiated human fibroblast feeder layer.     

Biocompatibility and Growth of Human Keratinocytes and Fibroblasts on Biosynthesized Cellulose–Chitosan Film

Bacterial cellulose (BC)–chitosan (BCC) films made via bio-co-polymerization by Acetobacter xylinum were developed and characterized for physical and biological properties. With the incorporation of chitosan MW 3 × 10⁴ and 8 × 10⁴ into bacterial cellulose, the modified films (BCC-MW 30 000 and BCC-MW 80 000, respectively) became denser, with a smaller average pore size of 13.1–15.3 nm in dry form. The BCC films have no toxicity against L929 mouse fibroblast cells. Tissue compatibility was then evaluated by growth and spreading of human skin keratinocytes and fibroblasts. The results revealed that the growth of human skin keratinocytes and fibroblasts on the BCC films was comparable to that on the BC film; however, improvement of cell adhesion and spreading on the BCC films was observed in human skin keratinocytes. The results of the biological response experiments showed no significant difference between BCC-MW 30 000 and BCC-MW 80 000.     

Recessive epidermolysis bullosa simplex phenotype reproduced in vitro: ablation of keratin 14 is partially compensated by keratin 17

Recessive epidermolysis bullosa simplex (REBS) is characterized by generalized cutaneous blistering in response to mechanical trauma. This results from fragility of the basal keratinocytes that lack keratin tonofilaments because of homozygote null mutation in the keratin 14 gene. REBS patients display in addition focal dyskeratotic skin lesions with histology of epidermolytic hyperkeratosis (EHK) and tonofilament clumping in the suprabasal layers of the epidermis. In this study we examined whether it is possible to mimic in vitro the bullous and dyskeratotic cellular phenotype. For this purpose, fibroblasts from nondyskeratotic (K14-/-) and dyskeratotic (K14-/-) skin of a REBS patient and fibroblasts from a healthy donor (K14+/+) were isolated and incorporated into collagen matrices. Subsequently, fresh biopsies originating from the nondyskeratotic and dyskeratotic skin of the patient and from a healthy donor were placed onto the collagen matrices and cultured at the air-liquid interface. Epidermal morphogenesis was evaluated on the basis of tissue morphology and the expression of a series of keratins. The results of the present study indicate that basal cell vacuolization in REBS can be mimicked in vitro but not the EHK. Fibroblasts seem to play an important regulatory role in establishing the REBS phenotype. These findings suggest that wild-type fibroblasts may enhance the stability of K14-/- keratinocytes in vitro.     

Dermo-epidermal interactions and skin pharmacology]

Reconstruction of a living skin equivalent provides an in vitro model for the study of skin biology and pharmacology in a tissue organization whose cells differentiate similarly to skin cells in the body. This simplified skin equivalent, composed of normal or abnormal cells, is obtained in two steps: formation of a dermal equivalent is achieved first and this dermis is then covered with an epidermal equivalent. Each of these tissues, as well as the interactions between them, can then be studied. Using this model, we have demonstrated that normal fibroblasts promote epidermal growth, that psoriatic fibroblasts induce increased proliferation of normal keratinocytes, and that the effects of pharmacological agents (such as retinoids) on keratinocyte growth are modulated by fibroblasts.     

Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5 days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether with or without stimulation. The results demonstrate that the secretion of KGF and HGF in both unstimulated fibroblasts and in fibroblasts co-cultured with keratinocytes is dependent on the type of fibroblasts. In general, the periodontal fibroblasts had the highest level of cytokine production. This high level of growth factor production may influence the proliferation and the migration of junctional epithelium and thereby influence the development of periodontal disease.     

Expression of Thrombomodulin and Consequences of Thrombomodulin Deficiency during Healing of Cutaneous Wounds

Thrombomodulin is a cell surface anticoagulant that is expressed by endothelial cells and epidermal keratinocytes. Using immunohistochemistry, we examined thrombomodulin expression during healing of partial-thickness wounds in human skin and full-thickness wounds in mouse skin. We also examined thrombomodulin expression and wound healing in heterozygous thrombomodulin-deficient mice, compound heterozygous mice that have <1% of normal thrombomodulin anticoagulant activity, and chimeric mice derived from homozygous thrombomodulin-deficient embryonic stem cells. In both human and murine wounds, thrombomodulin was absent in keratinocytes at the leading edge of the neoepidermis, but it was expressed strongly by stratifying keratinocytes within the neoepidermis. No differences in rate or extent of reepithelialization were observed between wild-type and thrombomodulin-deficient mice. In chimeric mice, both thrombomodulin-positive and thrombomodulin-negative keratinocytes were detected within the neoepidermis. Compared with wild-type mice, heterozygous and compound heterozygous thrombomodulin-deficient mice exhibited foci of increased collagen deposition in the wound matrix. These findings demonstrate that expression of thrombomodulin in keratinocytes is regulated during cutaneous wound healing. Severe deficiency of thrombomodulin anticoagulant activity does not appear to alter reepithelialization but may influence collagen production by fibroblasts in the wound matrix.     

Conditioned medium from the three-dimensional culture of human umbilical cord perivascular cells accelerate the migration and proliferation of human keratinocyte and fibroblast

Abstract

Previous studies have reported that the conditioned medium (CM) of bone marrow-mesenchymal stem cells (BM-MSCs) stimulate the migration and proliferation of cell types involved in the wound healing process. However, these studies only show MSC-CM effects that were obtained using a two-dimensional (2D) culture. Recently, a three-dimensional (3D) culture has been considered to be a more physiologically appropriate system than the 2D culture. In addition, it has been shown that the procurement of BM-MSC is invasive, and other sources of MSC are thus being explored. Recently, perivascular cells (PVCs) have been considered as an alternative source of cells for dermal wound healing. Therefore, in this study, a PVC-conditioned medium (CM) was collected from a 3D culture (PVC-CM-3D) using highly porous polystyrene-based membranes and compared with PVC-CM from a 2D culture (PVC-CM-2D) to investigate the effects on the migration and proliferation of human keratinocytes and fibroblasts. Moreover, the PVC-CM components from the 2D and 3D cultures were identified using 2D gel electrophoresis. The migrations of the keratinocytes cells and fibroblasts were significantly higher with PVC-CM-3D than with the 2D culture; similarly, the proliferation of keratinocytes was also highly stimulated by PVC-CM-3D. Proteomic analyses of the PVC-CM revealed that type I collagen was highly expressed in the 3D-culture system. Microtubule-actin cross-linked factor 1 (KIAA0465), nebulin-related anchoring protein, and thioredoxin were specifically expressed only in PVC-CM-3D. In addition, more EVs could be isolated from the PVC-CM-3D, and EVs were found to stimulate keratinocyte migration. Taken together, 3D-culture using a polystyrene scaffold is demonstrated to be a better system for providing better physiological conditions; therefore, PVC-CM-3D could be a promising option for skin-wound healing.     

Study of novel chitosan-gelatin artificial skin in vitro

A novel absorbable scaffold composed of chitosan and gelatin was fabricated by freezing and lyophilizing methods, resulting in an asymmetric structure. This bilaminar texture is suitable for preparing a bilayer skin substitute. The methods employed to confirm the applicability of this chitosan-gelatin scaffold as an ideal skin substitute were a water uptake ability test, in vitro fibroblast proliferation, and scaffold tests in which fibroblasts were co-cultured with keratinocytes. The chitosan-gelatin scaffolds were more wettable and adsorbed more water than did chitosan alone. In static cell culture the thinner scaffold is better than the thicker one, and because of diffusion limitations in the scaffold, culture time must be within 3 weeks before transplantation to living tissues. Keratinocytes were co-cultured with fibroblasts in chitosan-gelatin scaffolds to construct an artificial bilayer skin in vitro. The artificial skin obtained was flexible and had good mechanical properties. Moreover, there was no contraction observed in the in vitro cell culture tests. The data from this study suggest that chitosan-gelatin scaffolds are suitable for skin tissue engineering goals.     

Human keratinocyte induction of rapid effector function in antigen-specific memory CD4+ and CD8+ T cells

The ability of human keratinocytes to present antigen to T cells is controversial and, indeed, it has been suggested that keratinocytes may promote T cell hyporesponsiveness. Furthermore, it is unclear whether keratinocytes can process antigen prior to MHC class I and class II presentation. We tested the ability of keratinocytes to induce functional responses in epitope-specific CD4+ and CD8+ memory T cells using peptides, protein and recombinant expression vectors as sources of antigen. Keratinocytes were able to efficiently process and present protein antigen to CD4+ T cells, resulting in cytokine secretion (Th1 and Th2). This interaction was dependent on keratinocyte expression of HLA class II and ICAM-1, which could be induced by IFN-gamma. In addition, keratinocytes could present virally encoded or exogenous peptide to CD8+ T cells, resulting in T cell cytokine production and target cell lysis. Finally, T cell lines grown using keratinocytes as stimulators showed no loss of function. These findings demonstrate that keratinocytes are able to efficiently process and present antigen to CD4+ and CD8+ memory T cells and induce functional responses. The findings have broad implications for the pathogenesis of cutaneous disease and for transcutaneous drug or vaccine delivery.     

Delayed healing of chronic leg ulcers can result from impaired trafficking of bone marrow-derived precursors of keratinocytes to the skin

In this paper, it is hypothesized that in chronic wounds the process of homing of bone marrow-derived precursors of keratinocytes is disturbed, and that the interaction between cutaneous T-cell attracting chemokine (CTACK/CCL27) and soluble P-selectin glycoprotein ligand-1 (PSGL-1) can be the cause of this impairment. Several studies have revealed that bone marrow-derived cells (BMDC) trans-differentiate into various cellular lineages, and probably they participate also in healing of wounded skin. Recent studies have demonstrated that BMDC can engraft into the epidermis, and probably they do not engraft into epidermis as keratinocyte stem cells, but rather as transient amplifying cells. So, bone marrow-derived keratinocytes build provisional epidermal layer, and later they are replaced by keratinocytes migrating from surrounding skin. Probably after injury BMDC are recruited by pro-inflammatory cytokines, like granulocyte-macrophage colony stimulating factor. Further homing to the skin is mediated by CTACK/CCL27. This chemokine is exclusively secreted by keratinocytes. In chronic wounds the recruitment of BMDC seems to be impaired. Inhibition of CTACK/CCL27 by as yet not determined factor could be the cause of delayed healing. PSGL-1 appears to be a good candidate for such inhibiting factor. PSGL-1 is expressed by several populations of leukocytes, and can be released from surface of activated neutrophils. It was demonstrated that soluble PSGL-1 binds CTACK/CCL27, and inhibits chemotaxis mediated by this chemokine. Because there are many activated neutrophils in the wound, it should be expected that wound exudate contains large amount of soluble PSGL-1. Thus, CTACK/CCL27 in the wound would be inhibited, and homing of bone marrow-derived precursors of keratinocytes would be disturbed. If this interaction were found to be the main cause of wound chronicity, above-mentioned molecules could be the potential targets for pharmaceutical agents.     

Structured drug-loaded bioresorbable films for support structures

Bioresorbable films can serve simultaneously as anatomic support structures and as drug delivery platforms. In the present study, bioresorbable PLLA films containing dexamethasone were developed through solution processing. The effect of processing parameters on the film morphology and the resulting mechanical properties was studied. A model describing the structuring of these films is suggested. Generally, the solvent evaporation rate determines the kinetics of drug and polymer crystallization and thus, both the mode of drug dispersion in the polymer and the resulting mechanical properties. Two types of structured films were studied: (1) a polymer film with drug located on its surface, obtained due to drug skin formation accompanied by a later polymer core formation; and (2) a polymer film with small drug particles and crystals distributed within the bulk, obtained by parallel solidification of the two components. A prototypical application of these films is an expandable biodegradable support structure (stent), which we have developed. This stent demonstrated good initial mechanical properties. The film structure has only a minor effect on the stent radial compression strength, but more significantly affects the tensile mechanical properties.     

Structured drug-loaded bioresorbable films for support structures.

Bioresorbable films can serve simultaneously as anatomic support structures and as drug delivery platforms. In the present study, bioresorbable PLLA films containing dexamethasone were developed through solution processing. The effect of processing parameters on the film morphology and the resulting mechanical properties was studied. A model describing the structuring of these films is suggested. Generally, the solvent evaporation rate determines the kinetics of drug and polymer crystallization and thus, both the mode of drug dispersion in the polymer and the resulting mechanical properties. Two types of structured films were studied: (1) a polymer film with drug located on its surface, obtained due to drug skin formation accompanied by a later polymer core formation; and (2) a polymer film with small drug particles and crystals distributed within the bulk, obtained by parallel solidification of the two components. A prototypical application of these films is an expandable biodegradable support structure (stent). which we have developed. This stent demonstrated good initial mechanical properties. The film structure has only a minor effect on the stent radial compression strength, but more significantly affects the tensile mechanical properties.     

Replication of varicella zoster virus in primary human keratinocytes.

The ability of varicella zoster virus (VZV) to infect and replicate in human keratinocytes in culture was examined. Primary human keratinocytes derived from the abdomen, breast, and foreskin were plated as monolayers and infected by co-cultivation with VZV infected fibroblasts (MRC-5 cells). Replication and spread of the virus was assayed by plaque assay and immunofluorescence of infected cells using a VZV specific monoclonal antibody. Although all three types of keratinocytes tested were capable of supporting productive VZV infection, the keratinocytes showed a 1.5 to 2 log reduction in virus yield as compared to infection of monolayer cultures of MRC-5 cells. Results from immunofluorescence studies and plaque assays indicate a slower rate of cell-to-cell spread of the virus. Testing of an anti-VZV compound in this novel assay system demonstrated an interesting sensitivity compared to that observed in conventional assay systems.