In vitro skin models to study epithelial regeneration from the hair follicle.

The development of dermal equivalents (DEs) for the treatment of burns has contributed toward efficient wound closure. A collagen-glycosaminoglycan DE (C-GAG) grafted with hair follicles converted a full-thickness wound to partial-thickness resulting in complete wound closure and restored hair. In this study we compared the ability of both intact pilosebaceous units (PSU) or truncated hair follicles (THF) to regenerate a multilayered epidermis in vitro when implanted into de-epidermalized dermis (DED) or C-GAG with the epidermis generated in vivo using C-CAG. Keratinocytes explanted from the outer root sheath of PSU and THF in both DED and C-GAG but only formed a multilayered epidermis with PSU in DED. PSU were more effective at forming multilayered epidermis in DED than THF. Both DED and C-GAG skin expressed proliferation (PCNA), differentiation (K1, K10), hyperproliferation (K6, K16), basal (K14), putative stem cell (p63), extracellular matrix protein (Collagen IV), mesenchymal (vimentin) and adherens junction (ß-catenin) markers. These data suggest DEs supported initial maintenance of the implanted hair follicles, in particular PSU, and provide an excellent model with which to investigate the regulation of hair follicle progenitor epithelial cells during epidermal regeneration. Although neither PSU nor THF formed multilayered epidermis in C-CAG in vitro, hair follicles implanted into engrafted C-GAG on a burns patient resulted in epithelial regeneration and expression of proliferation and differentiation markers in a similar manner to that seen in vitro. However, the failure of C-GAG to support epidermal regeneration in vitro suggests in vivo factors are essential for full epidermal regeneration using C-GAG.

An in vitro skin model to study the effect of mesenchymal stem cells in wound healing and epidermal regeneration.

The development of new wound therapies, such as bioengineered skin equivalents, is an ongoing process. Multi-potent mesenchymal stem cells (MSCs) give rise to many tissue lineages and have been implicated in wound healing making them a potential candidate for cell-based bioengineered products for injured tissue. In this study, we investigated the mesenchymal/epithelial interactions of cultured MSCs in comparison to cultured fibroblasts on epidermal proliferation, differentiation, and extracellular matrix (ECM) protein expression using a de-epidermalized dermis (DED) skin model. We also studied whether MSCs can transdifferentiate to keratinocytes using the same model. Keratinocytes were cultured on unseeded DED or DED populated with fibroblasts or MSCs at an air-liquid interface to induce epidermal differentiation. Fibroblasts or MSCs were also seeded on the papillary surface of the DED alone or on the reticular surface. General histology and immunostaining was performed on the skin equivalents to examine the expression of pan keratin (K) (K1, K5, K6, and K18) and protein markers for epidermal differentiation (K10), hyperproliferation (K6), proliferation (PCNA), ECM component (collagen type IV), and mesenchymal marker (vimentin). Keratinocyte-fibroblast skin model and keratinocyte-MSC skin model both displayed an epidermal phenotype similar to epidermis in vivo. Positive expression of proliferation, differentiation and ECM protein markers was observed. MSCs failed to adopt an epithelial phenotype in the DED skin model. Our findings highlight the potential use of MSCs in bioengineered tissue for the treatment of wounds.

In vivo effects of tailored laminin-332 α3 conjugated scaffolds enhances wound healing: a histomorphometric analysis.

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides. In our previous work, we have shown the tethering of laminin-332 α3 chain to type I collagen scaffold using microbial transglutaminase (mTGase), promotes cell adhesion, migration, and proliferation. In this study, we evaluated the wound healing properties of tailored laminin-332 α3 chain (peptide A: PPFLMLLKGSTR) tethered to a type I collagen scaffold using mTGase by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide B: PPFLMLLKGSTREAQQIVM) or lysine (peptide C: PPFLMLLKGSTRKKKKG) in rat full-thickness wound model at two different time points (7 and 21 days). Histological evaluations were assessed for wound closure, epithelialization, angiogenesis, inflammatory, fibroblastic cellular infiltrations, and quantified using stereological methods (p < 0.05). Peptide A and B tethered to collagen scaffold using mTGase stimulated neovascularization, decreased the inflammatory cell infiltration and prominently enhanced the fibroblast proliferation which significantly accelerated the wound healing process. We conclude that surface modification by incorporating motif of laminin-332 α3 chain (peptide A: PPFLMLLK GSTR) domain and transglutaminase substrate to the laminin-332 α3 chain (peptide B: PPFLMLLKGSTREAQQIVM) using mTGase may be a potential candidate for tissue engineering applications and skin regeneration.

α6 Integrin and CD44 enrich for a primary keratinocyte population that displays resistance to UV-induced apoptosis.

Epidermal human keratinocytes are exposed to a wide range of environmental genotoxic insults, including the UV component of solar radiation. Epidermal homeostasis in response to cellular or tissue damage is maintained by a population of keratinocyte stem cells (KSC) that reside in the basal layer of the epithelium. Using cell sorting based on cell-surface markers, we have identified a novel α6 integrin(high+)/CD44(+) sub-population of basal keratinocytes. These α6 integrin(high+)/CD44(+) keratinocytes have both high proliferative potential, form colonies in culture that have characteristics of holoclones and have a unique pattern of resistance to apoptosis induced by UVB radiation or by agents that induce single- or double strand DNA breaks. Resistance to UVB induced apoptosis in the α6 integrin(high+)/CD44(+) cells involved increased expression of TAp63 and was overcome by PI-3 kinase inhibition. In marked contrast, the α6 integrin(high+)/CD44(+) cells were sensitive to apoptosis induced by the cross-linking agent cisplatin, and imatinib inhibition of c-Abl blocked the ability of cisplatin to kill α6 integrin(high+)/CD44(+) cells. Our findings reveal a population of basal keratinocytes with long-term proliferative properties that display specific patterns of apoptotic resistance that is dependent upon the genotoxic stimulus, and provide insights into how these cells can be targeted with chemotherapeutic agents.

Patient-related keloid scar assessment and outcome measures.

BACKGROUND: Keloid scars cause pain, itching, functional limitation, and disfigurement, leading to psychological distress. Progress in treatment regimens is hindered by the lack of a universally accepted outcome measure. The Patient and Observer Scar Assessment Scale is a tool for the assessment of scars, incorporating an assessment by both clinician and patient. This study evaluates its application to keloids and compares it to the widely used Vancouver Scar Scale, which is considered the standard mode of assessment for scars. METHODS: Three observers using the two scales assessed 34 patients with 41 keloid scars independently. Patients evaluated their own scars simultaneously using the patient component of the Patient and Observer Scar Assessment Scale. Internal consistency, interobserver reliability, and convergent validity were examined. RESULTS: Both components of the Patient and Observer Scar Assessment Scale had high internal consistency (0.82 and 0.86 for patient and observer components, respectively); those rates were higher than the rate for the Vancouver Scar Scale (0.65). Interobserver reliability was "substantial" for the Vancouver Scar Scale (0.65) and "almost perfect" for the observer component of the Patient and Observer Scar Assessment Scale (0.85). Convergent validity was very strong (0.83, p < 0.01), although the patient component did not correlate well with either of the observer scales. Patients rated their scars worse than the observer average for 83 percent of the scars, and were influenced by color, stiffness, thickness, and irregularity (p < 0.05). CONCLUSION: The findings support the use of the Patient and Observer Scar Assessment Scale as a reliable and valid method of assessing keloid scars in a clinical context. CLINICAL QUESTION/LEVEL OF EVIDENCE: Diagnostic, II.

Online marketing strategies of plastic surgeons and clinics: a comparative study of the United Kingdom and the United States.

The cosmetic surgery market is a rapidly growing sector of healthcare, and the use of marketing strategies is now an integral part of any cosmetic surgery practice. In this study, the authors review 50 Web sites from practitioners in London and New York to quantify the utilization of online marketing, comparing results between the United Kingdom and the United States.

Chemical chaperones protect epidermolysis bullosa simplex keratinocytes from heat stress-induced keratin aggregation: involvement of heat shock proteins and MAP kinases.

Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by mutations in keratin genes (KRT5 or KRT14), with no existing therapies. Aggregates of misfolded mutant keratins are seen in cultured keratinocytes from severe EBS patients. In other protein-folding disorders, involvement of molecular chaperones and the ubiquitin-proteasome system may modify disease severity. In this study, the effects of heat stress on keratin aggregation in immortalized cells from two patients with EBS (KRT5) and a healthy control were examined with and without addition of various test compounds. Heat-induced (43 °C, 30 minutes) aggregates were observed in all cell lines, the amount of which correlated with the donor phenotype. In EBS cells pre-exposed to proteasome inhibitor, MG132, and p38-mitogen-activated protein kinase (MAPK) inhibitor, SB203580, the proportion of aggregate-positive cells increased, suggesting a role of proteasomes and phosphorylation in removing mutated keratin. In contrast, aggregates were reduced by pretreatment with two chemical chaperones, trimethylamine N-oxide (TMAO) and 4-phenylbutyrate (4-PBA). TMAO also modulated stress-induced p38/c-jun N-terminal kinase (JNK) activation and expression of heat shock protein (HSPA1A), the latter of which colocalized with phosphorylated keratin 5 in EBS cells. Taken together, our findings suggest therapeutic targets for EBS and other keratinopathies.

The E2 protein of human papillomavirus type 8 increases the expression of matrix metalloproteinase-9 in human keratinocytes and organotypic skin cultures.

Non-melanoma skin cancer (NMSC) is the most frequent human cancer of Caucasian populations. Although the ultraviolet irradiation is a key contributor to the establishment of this keratinocyte malignancy, the infection by some types of human papillomavirus (HPV) has also been implicated in NMSC development. Cancers occur as a result of a complex series of interactions between the cancer cell and its surrounding matrix. The matrix metalloproteinases (MMPs) play a role in degrading the extracellular matrix. MMP9 is an important gelatinase involved in processes such as cell migration, invasion and metastasis. In this report, we demonstrated by EMSA experiments that the MMP9 promoter contains a binding site for the transcriptional regulator E2 of HPV8. Transient reporter gene assays showed that HPV8-E2 activated the MMP9 promoter in a dose-dependent manner in human epidermal keratinocytes. An E2 transactivation-defective mutant (I73L) as well as a DNA-binding deficient mutant (R433K) demonstrated no activation of the MMP9 promoter, suggesting that both an intact transactivation and DNA-binding domain are required for E2 activation of the MMP9-promoter. The functional role of the E2 binding site within the MMP9 promoter was also confirmed by mutating the E2 binding site. In organotypic cultures of human skin, an overexpression of MMP9 was observed in suprabasal layers of the HPV8 E2-expressing epidermis thus confirming the results of the monolayer cultures. These results demonstrate that the early gene E2 of HPV8 is able to increase the expression of MMP9 by direct activation of the MMP9-promoter.

Application of autologous derived-platelet rich plasma gel in the treatment of chronic wound ulcer: diabetic foot ulcer.

The treatment of chronic wounds remains problematic, despite new insight into the cellular and molecular basis of wound healing. Although the aetio-pathogenesis of chronic wounds is said to be multi-factorial, it is evident from literature that effective and adequate wound debridement has produced the most consistent effect in chronic wound treatment. There is a growing body of evidence that suggests that wound healing in chronic diabetic foot ulcers is growth factor dependent and that the therapeutic delivery of these growth factors to wounds topically, has the potential ability to accelerate wound healing in conjunction with conventional wound care. Autologous derived platelet concentrate is activated to release growth factors that are stored in the platelet granules. These secretory proteins include cytokines and growth factors such as transforming growth factor-beta, vascular endothelia growth factor, platelet derived growth factor, and so on. The enhancement of soft tissue healing by the application of autologous derived platelet rich plasma gel (APG) is supported by basic science and some clinical studies. This review article will attempt to provide a concise report of current concepts on the use of APG in treating chronic ulcers.

Human papillomavirus 5 and 8 E6 downregulate interleukin-8 secretion in primary human keratinocytes.

Human papillomaviruses (HPVs) of the genus Betapapillomavirus appear to be involved in the early stages of skin cancer development, since both the prevalence and viral load are higher in precancerous actinic keratoses than in skin cancers. Interleukin-8 (IL-8) is an inflammatory cytokine that serves to alert the surrounding tissue after UV-induced damage. We examined the effects of the E2, E6 and E7 proteins of HPV8 and the E6 proteins of various HPV genotypes on IL-8 secretion from primary keratinocytes. HPV5 and HPV8 E6 showed the highest downregulation of basal IL-8 secretion. HPV8 E6 also negatively modulated IL-8 mRNA expression and protein secretion upon UVB irradiation. The downregulation of IL-8 in actinic keratoses may weaken the response to UV-induced damage and thus favour the accumulation of UVB-induced mutations.

Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion.

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth.

Characterization of immortalized human epidermolysis bullosa simplex (KRT5) cell lines: trimethylamine N-oxide protects the keratin cytoskeleton against disruptive stress condition.

BACKGROUND: Epidermolysis bullosa simplex (EBS) is an autosomal inherited mechano-bullous disease, characterized by intraepidermal blistering and skin fragility caused by mutations in the keratin (KRT) 5 or 14 genes. Despite a vast knowledge about the intermediate filament pathology in this disease, the progress in therapy has been slow. Animal models and well-characterized continuous cell culture models of EBS are needed prior to clinical testing. OBJECTIVES: Our aim was to generate immortalized cell lines as an in vitro model for the study of EBS and test a chemical chaperone, trimethylamine N-oxide (TMAO), as a putative novel therapy. METHODS: We generated four immortalized cell lines, two each from an EBS patient with a KRT5-mutation (V186L) and a healthy control, using human papillomavirus 16 (HPV16) E6E7 as transducer. Cell lines were established in serum-free and serum-containing medium and assessed for growth characteristics, keratin expression profiles, ability to differentiate in organotypic cultures, and response to heat stress with and without the presence of TMAO. RESULTS: All cell lines have been expanded >160 population doublings and their cellular characteristics are similar. However, the formation of cytoplasmic keratin filament aggregates in response to heat-shock treatment differed between EBS and normal cell lines. Notably, serum-free established EBS-cell line was most vulnerable to heat shock but both cell lines exhibited significant reduction in the number of keratin aggregates containing cells by TMAO. CONCLUSION: The immortalized cell lines represent a suitable model for studying novel therapies for EBS. TMAO is a promising new agent for future development as a novel EBS therapy.

Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin-blistering disorder caused by mutations in the COL7A1 gene that lead to reduced type-VII collagen and defective anchoring fibrils at the dermal-epidermal junction (DEJ). Presently there are no effective treatments for this disorder. Recent mouse studies have shown that intradermal injections of normal human fibroblasts can generate new human type-VII collagen and anchoring fibrils at the DEJ. To assess potential clinical benefits in humans, we gave single intradermal injections of allogeneic fibroblasts to five subjects with RDEB. We noted increased type-VII collagen at the DEJ at 2 weeks and at 3 months following injection and increased anchoring fibrils, although none of these had normal morphology. No adverse effects, clinical or immunopathologic, were noted. We believe the major effect of allogeneic fibroblasts is to increase the recipients' own COL7A1 mRNA levels with greater deposition of mutant type-VII collagen at the DEJ and formation of additional rudimentary anchoring fibrils. Nevertheless, this mutant protein may be partially functional and capable of increasing adhesion at the DEJ. This is the first study demonstrating that intradermal injections of allogeneic fibroblasts have therapeutic potential in human subjects with RDEB.

Epidermal repair results from activation of follicular and epidermal progenitor keratinocytes mediated by a growth factor cascade.

Reepithelialization of human suction blister wounds was examined in five normal human volunteers over a period of 14 days postwounding to understand the control of keratinocyte migration, proliferation, and differentiation in acute wound healing in a controlled model. The hypothesis that morphological changes and progenitor activation result from altered cytokines and growth factor expression [in particular interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), transforming growth factor alpha (TGF-alpha), TGF-beta 1, and keratinocyte growth factor] was tested using semiquantitative immunohistochemistry combined with reverse transcriptase-polymerase chain reaction of samples from the blister roof, edge, and base. Parallel changes in keratin expression were examined using a wide range of well-established antibodies to multiple keratins and in situ hybridization for keratin 16 (K16), a marker of the hyperproliferative (mucoregenerative) phenotype. Longitudinal morphological, semiquantitative cytokine and growth factor expression, and histometric histone and cytokeratin profiles suggest three phases to reepithelialization: phase 1, or the acute activation phase, early in the first 24 hours postwounding is characterized by epidermal expression of IL-1beta and IL-6, and dermal expression of TGF-beta1, as basal, upper outer root sheath, and putative interfollicular transit amplifying keratinocytes become committed to mitosis; phase 2, or the early activation phase, late in the second 24 hours postwounding, characterized by epidermal expression of TGF-alpha and IL-6 with concurrent suprabasal K16 expression and migration with continued proliferation, and dermal expression of keratinocyte growth factor and IL-6; and phase 3 or restitution over the following 2 weeks, characterized by the return of normal homeostasis, including bulge activation as evidenced by K19 expression.

Hyalomatrix: a temporary epidermal barrier, hyaluronan delivery, and neodermis induction system for keratinocyte stem cell therapy.

Keratinocyte stem cell technology provides at least an adjuvant therapy to clinically close large cutaneous wounds (e.g., burn wounds). Here, the performance of keratinocyte cultures depends primarily on the quality of the bed to which they are applied. Clinical take rates for cultured keratinocyte grafts are optimal when applied to a vascularized dermal bed with minimal bacterial colonization. In the absence of autologous dermis, staged reconstruction with a dermal equivalent or dermal regeneration template is required. A novel product, Hyalomatrix, is a bilayer of an esterified hyaluronan scaffold beneath a silicone membrane. The scaffold delivers hyaluronan to the wound bed, and the silicone membrane acts as a temporary epidermal barrier. The product has been investigated in a controlled, porcine, acute full-thickness excisional wound model. Cultured autologous keratinocytes (CAKs) were delivered on Laserskin to acute full-thickness wounds treated with Hyalomatrix within chambers, and graft take rates were assessed longitudinally using image analysis. In the absence of chambers, wound contraction was assessed. Clinical CAK take rates fall sequentially with delay in application post-Hyalomatrix pre-treatment, but repeated pre-treatment removed this, with maximal take of 57.2% at 5 weeks post-wounding. In the absence of chambers, more-complete wound closure resulted from edge re-epithelialization and contraction, by a factor of 5 at 1 month, and was achieved at least 2 weeks sooner in the gold standard controls of split-thickness autograft to an acute or pre-treated wound bed. Wound contraction and late neodermal morphology (1 year) were similar in pre-treated CAKs and split-thickness autograft wounds. In this model, the Hyalomatrix wound bed pre-treatment increase in CAK take appeared to be dose dependent. The product appeared to act as a hyaluronan delivery system rather than a dermal regeneration template. The silicone membrane may limit wound bed colonization, and the combination of this temporary barrier with hyaluronan delivery and neodermis induction has been termed a barrier-delivery-induction system. The development of similar systems for serial application offers an alternative to a dermal regeneration template when CAKs are engrafted in the hostile, colonized environment of large burn wounds.

A deafness-associated mutant human connexin 26 improves the epithelial barrier in vitro.

A large proportion of recessive nonsyndromic hearing loss is due to mutations in the GJB2 gene encoding connexin 26 (Cx26), a component of a gap junction. Within different ethnic groups there are specific common recessive mutations, each with a relatively high carrier frequency, suggesting the possibility of heterozygous advantage. Carriers of the R143W GJB2 allele, the most prevalent in the African population, present with a thicker epidermis than noncarriers. In this study, we show that (R143W)Cx26-expressing keratinocytes form a significantly thicker epidermis in an organotypic coculture skin model. In addition, we show increased migration of cells expressing (R143W)Cx26 compared to (WT)Cx26-overexpressing cells. We also demonstrate that cells expressing (R143W)Cx26 are significantly less susceptible to cellular invasion by the enteric pathogen Shigella flexneri than (WT)Cx26-expressing cells. These in vitro studies suggest an advantageous effect of (R143W)Cx26 in epithelial cells.

Hyaluronic acid: the scientific and clinical evidence.

Hyaluronic acid is a naturally occurring biopolymer whose molecular structure is highly conserved between mammalian species. First described in 1934, it has since been used across a wide variety of medical fields as diverse as neurosurgery and cutaneous wound healing. Presently it has reached prominence in cosmetic practice where it is now the injectable dermal filler of choice for most surgeons. We used our experience of this technology with searches in the English language literature for the purpose of a systematic review. We present an overview, including the scientific evidence for its use in wound healing and, briefly, in other fields. We summarise the evidence for and against hyaluronic acid and provide a resumé of the current technologies available in fields such as skin regeneration and wound healing, in addition to cosmetic surgery. This overview is not intended to teach the reader about the various formulations currently on the market or how to use these materials clinically - rather to provide a solid scientific background enabling the reader to understand the attributes (and otherwise) of the material. We hope to allow clinicians to assess the evidence for a material now in common use in order that they may be fully aware of its properties.

Culture of human keratinocytes on polypyrrole-based conducting polymers.

Variously loaded polypyrrole films, including those containing proteins and polysaccharides, were prepared on gold-coated polycarbonate coverslips. The characteristics of human keratinocytes were studied on these films by microscopy, biochemical assays, and immunocytochemistry. We found keratinocyte viability to be load dependent. For chloride, polyvinyl sulphate, dermatan sulphate, and collagen-loaded polypyrrole films, keratinocyte viability as assessed by the AlamarBlue assay was respectively 47.22, 60.43, 87.71, and 22.65% of tissue culture polystyrene controls after 5 days. This was found to require a previously unreported polymer washing step prior to cell seeding due to the observed toxicity of untreated films. In the case of bare polycarbonate and gold substrates, viability was respectively 75.44 and 61.04% of tissue culture polystyrene controls after 5 days. Keratinocytes stained positive for PCNA (proliferation), K10 (suprabasal differentiation), and K16 (hyperproliferation) markers although cell morphology was poor for organotypical cultures on dermatan- loaded polypyrrole compared with de-epidermalized dermis. From our studies, we concluded that optimized polypyrrole films adequately support keratinocyte growth in submerged cultures with some improvements needed for organotypical cultures. Polypyrrole composites are attractive candidates for tissue-engineering applications since they may incorporate biomolecules and are electrically addressable with the potential to both direct and report on cell activity.