1. Induction of Bone Morphogenetic Protein-6 in Skin Wounds
Induction of Bone Morphogenetic Protein-6 in Skin Wounds. Delayed Reepitheliazation and Scar Formation in BMP-6 Overexpressing Transgenic Mice 
Sibylle Kaiser, Peter Schirmacher, Armin Philipp, Martina Protschka, Ingrid Moll, Karin Nicol, Manfred Blessing 
Journal of Investigative Dermatology 
Volume 111, Issue 6, Pages (December 1998)
DOI: /j x
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Distribution of BMP-6 in wounded skin on the protein level. Five micrometer cryostat sections from 1 (a), 3 (b, c), 5 (d), and 13 (e, f) d old wounds were stained with polyclonal BMP-6 specific anti-sera. BMP-6 was hardly detectable 1 d after incision (a,arrow denotes site of incision), but was strongly upregulated 3 d after wounding in the epidermis and in hair follicles (short arrows inb) close to the site of the incision (long arrow inb). Spindle-shaped cells like cells invading the wound space also showed BMP-6-specific staining (arrows inc,arrowheads denote dermal–epidermal border). BMP-6 expresssion was most prominent between days 5 and 7 after injury at the original site of incision (arrow ind) and in the newly formed epithelium that migrates beneath the wound scab (d). When reepitheliazation was completed elevated BMP-6 expression persisted in the epidermis covering the wound bed and at the wound periphery (e,arrows denote wound margins). BMP-6 was also detected at the merging site of epidermal wedges (f).Scale bars: (a, b, d, e) 100 μm; (c, f) 50 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Divergent onset of expression and distribution of BMP-6 RNA and protein in skin wounds. Five micrometer successive cryostat sections of full-thickness wounds were analyzed byin situ hybridization (a–d) and immunohistochemistry (e–h) at days 1 (a, e), 5 (b, f), 13 (c, g), and 25 (d, h) after incision. BMP-6 RNA was induced within 24 h after wounding throughout the hyperplastic epidermis at the wound edge including the basal epidermal layer (a), whereas no protein accumulation was detected at this time (e). Note restriction of BMP-6 RNA to suprabasal layers in normal neonatal skin (insert ina). Keratinocytes at the leading edge of the regenerating epithelium expressed BMP-6-specific RNA (b), but not the protein (f). Prominent BMP-6-specific RNA expression persisted until 13 d after incision throughout the thickened epidermis (c), whereas the protein always remained restricted to the suprabasal epidermal layer of differentiated epithelium (g). When epidermal thickness decreased, the levels of BMP-6-specific RNA and protein declined (d, h) and the RNA became restricted to suprabasal layers (d). The junction of epidermis and dermis is depicted byarrowheads. In all panels, the wound is located to the left-hand side.Scale bars: 50 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Localization of BMP-6 on RNA and protein levels in nonhealing human wounds (a–d) and normally healing human wounds (e). Five micrometer sections of a chronic skin ulcer were subjected toin situ hybridization with a BMP-6-specific anti-sense cRNA probe (a, b) or to immunohistochemistry using an antibody to BMP-6 (c, d). BMP-6-specific RNA was present throughout the hyperplastic epithelium covering the wound bed, including the basal keratinocytes at the leading edge of the migrating tongue (a, b). On the protein level, BMP-6 was also detected throughout the epidermal layer covering the wound bed (c) but was not expressed by basal keratinocytes at the leading edge of the migrating tongue (d). Immunohistochemistry for BMP-6 on a section from a 3 d old normally healing human skin wound (e). Note the absence of BMP-6 reactivity in keratinocytes migrating into the wound bed.Arrows in (a), (c), and (e) indicate the tip of the migrating epithelium, the dashed lines in (b) and (d) denote the border between epidermis and wound bed.Scale bars: (a, c) 500 μm; (b, d) 25 μm; (e) 100 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Comparison of the progress in wound healing in control (left) and BMP-6 transgenic mice (right) at various time points after incision. At day 2 post-wounding (2 d) control and transgenic wounds were covered by a dehydrated scab. At day 9 post-wounding (9 d) some control wounds had lost their scab, whereas the earliest time point transgenic wounds lost the scab was day 15 post-wounding (15 d). Twenty-one days after incision (21 d) all wounds had lost their scab and showed complete reepitheliazation.Scale bar: 1 cm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Re-epitheliazation and tissue remodeling. Hematoxylin and eosin stained skin sections from 7 d old (a, c) and 13 d old (b, d) full-thickness wounds from control mice (a, b) and transgenic mice (c, d). After 7 d the wounds of control mice were covered completely by migrating keratinocytes (a). Thirteen days after injury the original wound area of control mice was covered by a stratified epithelium (b). In some transgenic wounds the epidermal wedges were still close to the wound margins 7 d after incision (c). Thirteen days after incision migrating edges of transgenic keratinocytes still had not merged and the wound bed was still covered by an eschar (d). Boxed areas in (a)–(d) are shown at higher magnifications in (a′)-(d′).Arrows in (a)–(d) denote the site of original incision,arrowheads in (c) and (c′)-(d′) denote tips of epidermal wedges. Migrating keratinocytes of a 7 d old control wound formed a complete epithelium over the wound bed, denoted byarrowheads (a′). Accumulation of fibrous bundles at the site of the original wound of a 13 d old control mouse indicated maturation of granulation tissue (arrows inb′). Transgenic keratinocytes had just begun to migrate under the wound scab 7 d after injury (c′). Granulation tissue in a 13 d old transgenic wound still showed a much higher cellularity (d′).Scale bars: (a–d) 500 μm; (a′–d′) 25 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Dissolution of granulation tissue. Differential interference contrast photomicrographs taken from sections of wound margins in normal (a) and transgenic (b, c) mice 13 d (a, b) and 25 d (c) after incision. The corresponding epifluorescence photomicrographs are shown after staining with a Mac-1-specific antibody (a′,b′). Black and whitearrows denote the original sites of incision. Note the abundance of Mac-1 positive cells in 13 d old wounds from transgenics (b, b′) when compared with controls (a, a′).Scale bars: 50 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
Prolonged proliferation of keratinocyes at the wound margin of BMP-6 transgenic mice. Differential interference contrast (a, b) and corresponding epifluorescence photomicrographs (a′,b′) of 5 μm sections of 13 d old wounds from control (a) and transgenic mice (b), which were incubated with fluorescein labeled anti-BrdU antibody. Black and whitearrows denote the original site of incision. The wound is located to the right-hand side. Note that the number of S-phase keratinocytes distal to the wound is greatly reduced in interfollicular epidermis of normal mice (arrowheads ina′) as compared with transgenics (arrowheads inb′). Follicular proliferation is prominent in both transgenics and controls (a′,b′).Scale bars: 50 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions