Immunofluorescent localization of collagen types I,III and IV,fibronectin, laminin,and basement membrane proteoglycan in developing mouse skin

Summary The distribution of various extracellular matrix components was studied in frozen sections of embryonic (14–18 days) and early postnatal (birth and 4 days post parturn) dorsal mouse skin using monospecific antibodies and indirect immunofluorescence. Basement membrane zone components — type IV collagen, laminin and heparan sulphate proteoglycan — were found to be uniformly and unchangingly distributed along the dermal-epidermal junction. In contrast, the distribution of interstitial matrix components — types I and III collagen, and fibronectin — was heterogeneous and varied with the stages of hair development. Collagens became sparse and were eventually completely removed from the prospective dermal papilla and from a one-cell-thick sheath of dermal cells around hair buds. They remained absent from the dermal papilla throughout hair organogenesis. Fibronectin was always present around dermal papilla cells and was particularly abundant along the dermal-epidermal junction of hair rudiments, as well as underneath hair buds. In contrast, in interfollicular skin, collagens accumulated in increasing density, while fibronectin became progressively sparser. It thus appears that interstitial collagens and fibronectin are distributed in a manner which is related to hair morphogenesis. In morphogenetically active regions, collagen density is low, while that of fibronectin is high. Conversely, in histologically stabilized zones, collagen is abundant and fibronectin is sparse. This microheterogeneous distribution of interstitial collagens and of fibronectin might thus constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the development of skin and of cutaneous appendages.     

Cell culture from lizard skin: a tool for the study of epidermal differentiation

An in vitro system of isolated skin cells has been developed in order to address the understanding on the factors that control the shedding cycle and differentiation of lizard epidermis. The skin from the regenerating lizard tail has been separated in epidermis and dermis, cells have been dissociated, cultivated in vitro, and studied ultrastructurally after 1–30 days of culture condition. Dissociated keratinocytes after 12 days in culture show numerous cell elongations and contain bundles of keratin or sparse keratin filaments. These cells often contain one to three 0.5–3 μm large and dense “keratinaceous bodies”, an organelle representing tonofilament disassembling. Most keratinocytes have sparse tonofilaments in the cytoplasm and form shorter bundles of keratin in the cell periphery. The dissociated dermis mainly consists of mesenchymal cells containing sparse bundles of intermediate filaments. These cells proliferate and form multi-stratified layers and a dermal pellicle in about 2–3 weeks in vitro in our basic medium. Conversely, cultures of keratinocytes do not expand but eventually reduce to few viable cells within 2–3 weeks of in vitro condition. It is suggested that dermal cells sustain themselves through the production of growth factors but that epidermal cells requires specific growth factors still to be identified before setting-up an in vitro system that allows analyzing the control of the shedding cycle in lizards.     

Influence of dermal equivalent maturation on the development of a cultured skin equivalent.

Histologic and immunofluorescence methods were used to analyse the presence of fibronectin, chondroitin-4-sulphate and chondroitin-6-sulphate, type III and IV collagens, laminin, and keratins to assess the maturation level of cultured dermal and skin equivalents. In a first phase, fibroblasts in monolayer culture were compared with dermal equivalents in which fibroblasts are embedded in a type I collagen gel. Different fluorescent patterns were observed depending on the culture system used. A sequential appearance of macromolecules was noticed in dermal equivalents. Fibronectin was first detected after 4 days of culture, whereas chondroitin-4-sulphate and chondroitin-6-sulphate and type III collagen were present after 7 days. In contrast, all three macromolecules were detected at 24 h of culture in fibroblastic monolayer cultures. In a second phase, the quality of our skin equivalents was evaluated according to the seeding time of epidermal cells upon dermal equivalents (1, 4, or 7 days). A satisfactory stratification was obtained when keratinocytes were seeded after 4 and 7 days of dermal equivalent culture. Laminin and fibronectin were detected at the dermo-epidermal junction, but type IV collagen was absent. Various keratins, as detected by the AE1, AE2, and AE3 antibodies, were present in the epidermal layer. Following keratinocyte confluence, a change in the organization pattern of type III collagen in the dermal fraction of the skin equivalent was also noticed. Our comparative results show that seeding of epidermal cells on a more mature dermal equivalent leads to improved differentiation status of the epidermal layer.     

Effect of hydrocortisone on skin development in the chick embryo: ultrastructural, immunohistological, and biochemical analysis

The effect of hydrocortisone on the development of dorsal skin was analyzed in the chick embryo by (1) transmission electron microscopy, (2) indirect immunofluorescence histology of extracellular matrix components (collagen types I, III, and IV; fibronectin; and laminin), and (3) quantitative determination of collagen content and proline incorporation, between administration of the drug at 6 or 6.5 days and final retrieval of skin pieces at 11 days of incubation. Treatment caused the formation of featherless skin areas which exhibited an early maturation of the epidermis, a uniform distribution of interstitial collagen and rarefaction of fibronectin in the dermal extracellular matrix, and a significant increase of collagen content and proline incorporation in collagen noncollagen proteins, characterized by an increased hydroxyproline-to-proline ratio. The distribution of type IV collagen and of laminin was unchanged. The absence of feather formation in hydrocortisone-induced apteria is interpreted as resulting primarily from an early extinction of epidermal morphogenetic competence, and secondarily from modifications in the amount and distribution of extracellular matrix components in the dermis.     

Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermoepidermal junction in organotypic co-culture

Besides medical application as composite skin grafts, in vitro constructed skin equivalents (SEs) or organotypic co-cultures represent valuable tools for cutaneous biology. Major drawbacks of conventional models, employing collagen hydrogels as dermal equivalents (DEs), are a rather poor stability and limited life span, restricting studies to early phases of skin regeneration. Here we present an improved stabilised in vitro model actually providing the basis for skin-like homeostasis. Keratinocytes were grown on dermal equivalents (DEs) reinforced by modified hyaluronic acid fibres (Hyalograft-3D) and colonised with skin fibroblasts, producing genuine dermis-type matrix. These SEs developed a superior epidermal architecture with regular differentiation and ultrastructure, which occurred also faster than in SEs based on collagen-DEs. Critical aspects of differentiation, still unbalanced in early stages, were perfectly re-normalised, most strikingly the co-expression of keratins K1/K10 and downregulation of regeneration-associated keratins such as K16. The restriction of integrin and K15 distribution as well as keratinocyte proliferation to the basal layer underlined the restored tissue polarity, while the drop of growth rates towards physiological levels implied finally accomplishment of homeostasis. This correlated to faster basement membrane (BM) formation and ultrastructurally defined dermo-epidermal junction including abundant anchoring fibrils for strong tissue connection. Whereas the fibroblasts in the scaffold initially secreted a typical provisional regenerative matrix (fibronectin, tenascin), with time collagens of mature dermis (type I and III) were accumulating giving rise to an in vivo-like matrix with regularly organised bundles of striated collagen fibrils. In contrast to the more catabolic state in conventional DEs, the de novo reconstruction of genuine dermal tissue seemed to be a key element for maintaining prolonged normal keratinocyte proliferation (followed up to 8 wks), fulfilling the criteria of tissue-homeostasis, and possibly providing a stem cell niche.     

Development of sensory innervation in chick skin: comparison of nerve fibre and chondroitin sulphate distributions in vivo and in vitro

In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using themonoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.     

Changes in the extracellular matrix of the normal human breast during the menstrual cycle

Summary The normal human mammary gland undergoes a well defined sequence of histological changes in both epithelial and stromal compartments during the menstrual cycle. Studies in vitro have suggested that the extracellular matrix surrounding the individual cells plays a central role in modulating a wide variety of cellular events, including proliferation, differentiation and gene expression. We therefore investigated the distribution of a number of extracellular matrix molecules in the normal breast during the menstrual cycle. By use of indirect immunofluorescence, with specific antibodies, we demonstrated that laminin, heparan sulphate proteoglycan, type IV collagen, type V collagen, chondroitin sulphate and fibronectin undergo changes in distribution during the menstrual cycle, whereas collagen types I, III, VI and VII remain unchanged. These changes were most marked in the basement membrane, sub-basement membrane zone and delimiting layer of fibroblasts surrounding the ductules where basement membrane markers such as laminin, heparan sulphate proteoglycan, and type IV and V collagens appear greatly reduced during the mid-cycle period (days 8 to 22). These results suggest that some extracellular matrix molecules may act as medittors in the hormonal control of the mammary gland, whereas others may have a predominantly structural role.     

Interactions between chondroitin sulfate proteoglycan, fibronectin, and collagen

A proteoglycan isolated from a rat yolk sac tumor and characterized as a chondroitin sulfate proteoglycan with a smaller amount of dermatan sulfate was studied with respect to complex formation with collagen and fibronectin. The proteoglycan co-precipitated with native collagen from neutral salt solutions at 6 degrees C and 37 degrees C. Addition of fibronectin in such precipitation mixtures resulted in incorporation of fibronectin to the precipitate. Treatment of the proteoglycan with alkali to separate the glycosaminoglycan chains from the protein part and digestion of the protein part with papain greatly reduced the capacity of the proteoglycan to precipitate collagen and fibronectin. A defined extracellular matrix as represented by the complexes of collagen, proteoglycan, and fibronectin constructed here may be useful for studies on the biological effects of extracellular matrices. The multiple interactions of matrix macromolecules exemplified by these results may play a role in the formation of extracellular matrices and in the maintenance of their integrity.     

Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns.

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.     

Reconstitution of human epidermis in vitro is accompanied by transient activation of basal keratinocyte spreading

Frozen human cadaver skin obtained from the skin bank was thawed and incubated in serum-free medium for 1–2 days, after which the original epidermis could be removed mechanically. Transmission electron microscopic observations showed that the dermal matrix remaining behind contained intact bundles of collagen fibrils but no live cells and that a continuous lamina densa persisted in the basement membrane region. Indirect immunofluorescence analyses demonstrated linear staining of the basement membrane region by antibodies against laminin and type IV collagen and discontinuous staining with antibodies against fibronectin. Scanning electron microscopic observations revealed a normal topographical arrangement of dermal matrix papilla and interspersed crypts on the surface of the matrix. Epidermal cells placed on the dermal matrix attached in 1–2 h and spread by 24 h. After 1 week of culture the epidermis was reconstituted, at which time approximately 30% of the epidermal cells were basal keratinocytes and the remainder were more differentiated keratinocytes. A high degree of differentiation of the reconstituted epidermis was shown by the formation of hemidesmosomes along the basement membrane, the formation of desmosomes characterized by intercellular dense lines, and the presence of a cell layer containing keratohyalin granules. At various times during epidermal reconstitution, cells were harvested and tested in short-term assays for adhesion to fibronectin substrata. During the first several days there was a transient activation of basal keratinocyte spreading analogous to the modulation of keratinocyte spreading that we have observed during epidermal reconstitution in vivo.     

Analysis of heterogeneity of human keratinocyte populations by their adhesion to substrate and the keratin 19 to actin ratio

Keratin 19 is reported to be a marker for skin stem cells and that assumes an independent subpopulation of these cells in keratinocyte population. In addition, keratinocytes have natural affinity to extracellular matrix proteins. The aim of this work was to reveal subpopulations ofkeratinocytes cultivated on the substrates of collagen I type, laminin-2/4 and fibronectin, and distinguished by keratin 19/actin ratio (G/R). The area of cell projection on a substrate, perimeter, the spreading coefficient and G/R were measured. Both on fibronectin and laminin-2/4, keratinocyte populations were morphologically homogeneous with large cells comprising 12 and 20 percents of the population, respectively. On fibronectin, correlation between morphological parameters of the cells and the keratin 19/actin ratio was not found. The cells growth on collagen behaved as a heterogeneous population, with the large cells compressing more than 50 percents of the population. An average, the size of the cells growing on collagen was twice as large as than of the cells cultivated on fibronectin. On the other hand, correlation between the cell size and G/R was revealed in the cells growth both on collagen and laminin-2/4. The cells with lower G/R value display a larger size and extent of spreading. We assume that this correlation may be determined by alphal and alpha2, integrins characteristic of these cells. Our results cast doubts on whether stem cells are present in the culture of human skin keratinocytes.     

Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo

The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.     

Structure and binding properties of collagen type XIV isolated from human placenta

Collagen XIV was isolated from neutral salt extracts of human placenta and purified by several chromatographic steps including affinity binding to heparin. The same procedures also led to the purification of a tissue form of fibronectin. Collagen XIV was demonstrated by partial sequence analysis of its Col1 and Col2 domains and by electron microscopy to be a disulphide-linked molecule with a characteristic cross-shape. The individual chains had a size of approximately 210 kD, which was reduced to approximately 180 kD (domain NC3) after treatment with bacterial collagenase. Specific antibodies mainly to NC3 epitopes were obtained by affinity chromatography and used in tissue and cell analyses by immunoblotting and radioimmunoassays. Two sequences from NC3 were identified on fragments obtained after trypsin cleavage. They were identical to cDNA-derived sequences of undulin, a noncollagenous extracellular matrix protein. This suggests that collagen XIV and undulin may be different splice variants from the same gene. Heparin binding was confirmed in ligand assays with a large basement membrane heparan sulphate proteoglycan. This binding could be inhibited by heparin and heparan sulphate but not by chondroitin sulphate. In addition, collagen XIV bound to the triple helical domain of collagen VI. The interactions with heparin sulphate proteoglycan and collagen VI were not shared by the NC3 domain, or by reduced and alkylated collagen XIV. No or only low binding was observed for collagens I-V, pN- collagens I and III, and several noncollagenous matrix proteins, including laminin, recombinant nidogen, BM-40/osteonectin, plasma and tissue fibronectin, vitronectin, and von Willebrand factor. Insignificant activity was also shown in cell attachment assays with nine established cell lines.     

Tenascin-R and axon growth-promoting molecules are up-regulated in the regenerating visual pathway of the lizard (Gallotia galloti)

It is currently unclear whether retinal ganglion cell (RGC) axon regeneration depends on down-regulation of axon growth-inhibitory proteins, and to what extent outgrowth-promoting substrates contribute to RGC axon regeneration in reptiles. We performed an immunohistochemical study of the regulation of the axon growth-inhibiting extracellular matrix molecules tenascin-R and chondroitin sulphate proteoglycan (CSPG), the axon outgrowth-promoting extracellular matrix proteins fibronectin and laminin, and the axonal tenascin-R receptor protein F3/contactin during RGC axon regeneration in the lizard, Gallotia galloti. Tenascin-R and CSPG were expressed in an extracellular matrix-, oligodendrocyte/myelin- and neuron-associated pattern and up-regulated in the regenerating optic pathway. The expression pattern of tenascin-R was not indicative of a role in channeling or restriction of re-growing RGC axons. Up-regulation of fibronectin, laminin, and F3/contactin occurred in spatiotemporal patterns corresponding to tenascin-R expression. Moreover, we analyzed the influence of substrates containing tenascin-R, fibronectin, and laminin on outgrowth of regenerating lizard RGC axons. In vitro regeneration of RGC axons was not inhibited by tenascin-R, and further improved on mixed substrates containing tenascin-R together with fibronectin or laminin. These results indicate that RGC axon regeneration in Gallotia galloti does not require down-regulation of tenascin-R or CSPG. Presence of tenascin-R is insufficient to prevent RGC axon growth, and concomitant up-regulation of axon growth-promoting molecules like fibronectin and laminin may override the effects of neurite growth inhibitors on RGC axon regeneration. Up-regulation of contactin in RGCs suggests that tenascin-R may have an instructive function during axon regeneration in the lizard optic pathway.     

Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin

Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced by transgenic epidermal activation of β-catenin (EF skin). We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of β-catenin signalling. By contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal β-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis.     

Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.     

Matricellular hevin regulates decorin production and collagen assembly

Matricellular proteins such as SPARC, thrombospondin 1 and 2, and tenascin C and X subserve important functions in extracellular matrix synthesis and cellular adhesion to extracellular matrix. By virtue of its reported interaction with collagen I and deadhesive activity on cells, we hypothesized that hevin, a member of the SPARC gene family, regulates dermal extracellular matrix and collagen fibril formation. We present evidence for an altered collagen matrix and levels of the proteoglycan decorin in the normal dermis and dermal wound bed of hevin-null mice. The dermal elastic modulus was also enhanced in hevin-null animals. The levels of decorin protein secreted by hevin-null dermal fibroblasts were increased by exogenous hevin in vitro, data indicating that hevin might regulate both decorin and collagen fibrillogenesis. We also report a decorin-independent function for hevin in collagen fibrillogenesis. In vitro fibrillogenesis assays indicated that hevin enhanced fibril formation kinetics. Furthermore, cell adhesion assays indicated that cells adhered differently to collagen fibrils formed in the presence of hevin. Our observations support the capacity of hevin to modulate the structure of dermal extracellular matrix, specifically by its regulation of decorin levels and collagen fibril assembly.     

Collagen degradation in the rabbit skin during short-term tissue culture

Full thickness rabbit skin explants were cultured on plastic dish for 1 week and the sequential morphological changes were examined daily by light and electron microscopy. During the cultured period, bundles of dermal collagen fibres gradually loosened and were removed from the upper dermis and from the cut margin of the explant, which was covered by a sheet of migrating epidermal cells. In these areas, cells containing phagocytosed collagen fibrils were observed from the 3rd day to the end of the culture period. These cells containing phagocytosed collagen fibrils included dermal fibroblasts and macrophages, epidermal keratinocytes and endothelial cells lining blood vessels. The presence of acid phosphatase activity in vacuoles containing the collagen fibrils suggested that intracellular degradation of collagen was occurring. In addition, extracellular collagen degradation was recognized around fibroblasts and beneath the migrating epidermis by the high collagenolytic activity at these sites. These findings suggest that both intra- and extracellular collagen degradation may participate in collagen removal from dermal connective tissue in cultured skin explants.