Isolation and Culture of Hair Follicle Dermal Sheath Mesenchymal Stromal Cells.

To date, little is published on the characterization and therapeutic potential of human mesenchymal stromal cells (MSCs) derived from hair follicle dermal sheath (DS). We present protocols for the isolation and culture of human DS-MSCs starting with the use of a dissecting microscope to separate out dermal sheaths from hair follicles for trypsin digestion. We also present the protocols for the adipogenic, osteogenic, and chondrogenic differentiation of these DS-MSCs as we seek to harness these cells for potential applications in stem cell therapy and tissue engineering.

Asian Cleft Rhinoplasty: The Open Structural Approach.

BACKGROUND: Asian cleft rhinoplasty is a unique procedure with specific challenges. OBJECTIVES: This paper presents our experience with the use of an open structural technique for Asian cleft rhinoplasty utilizing a complete autologous approach. METHODS: An open approach that reconstructs the malpositioned nasal cartilage was utilized. Centrally, the septocolumella graft fixed securely in the midline with extender spreader grafts was utilized to project and lengthen the nose. The dislocated lateral crura on the cleft side was completely detached from the accessory cartilages and mobilized off the vestibular lining. The deficient medial crura was lengthened with the lateral crural steal procedure. The resultant shortened lateral crura was then reconstructed with the lateral crural strut graft (LCSG). This gave the versatility needed to bring the tip cartilages into a more anatomic and symmetrical position. Tip suturing and grafting was performed and dorsal augmentation achieved through diced cartilage wrapped in deep temporal fascia. At closure, when indicated, a modified Tajima reverse-U excision of the vestibular lining was performed to correct the alar hooding on the cleft side. Alar base modifications were done as indicated. RESULTS: From January 2010 to December 2015, 35 Asian patients underwent open cleft rhinoplasty. There were 18 female patients and 17 male patients. Twenty-nine patients were unilateral cleft and 6 were bilateral cleft. The mean follow up was 23 months. All patients were highly satisfied with the functional and aesthetic improvement of the procedure. The complications and revision rates were low. CONCLUSIONS: The autologous open structural approach can predictably and consistently give excellent results for Asian cleft rhinoplasty. The result attainable is superior to results attainable before adopting this approach for our patients. LEVEL OF EVIDENCE: 4.

Probing the Role of Integrins in Keratinocyte Migration Using Bioengineered Extracellular Matrix Mimics.

Bioengineered extracellular matrix (ECM) mimetic materials have tunable properties and can be engineered to elicit desirable cellular responses for wound repair and tissue regeneration. By incorporating relevant cell-instructive domains, bioengineered ECM mimics can be designed to provide well-defined ECM-specific cues to influence cell motility and differentiation. More importantly, bioengineered ECM surfaces are ideal platforms for studying cell-material interactions without the need to genetically alter the cells. Here, we showed that bioengineered ECM mimics can be employed to clarify the role of integrins in keratinocyte migration. Particularly, the roles of α5ß1 and α3ß1 in keratinocytes were examined, given their known importance in keratinocyte motility. Two recombinant proteins were constructed; each protein contains a functional domain taken from fibronectin (FN-mimic) and laminin-332 (LN-mimic), designed to bind α5ß1 and α3ß1, respectively. We examined how patient-derived primary human keratinocytes migrate when sparsely seeded as well as when allowed to move collectively. We found, consistently, that FN-mimic promoted cell migration while the LN-mimic did not support cell motility. We showed that, when keratinocytes utilize α5ß1 integrins on FN-mimics, they were able to form stable focal adhesion plaques and stabilized lamellipodia. On the other hand, keratinocytes on LN-mimic utilized primarily α3ß1 integrins for migration and, strikingly, cells were unable to activate Rac1 and form stable focal adhesion plaques. Taken together, employment of our bioengineered mimics has allowed us to clarify the roles of α5ß1 and α3ß1 integrins in keratinocyte migration, as well as further provided a mechanistic explanation for their differences.

A Novel Interferon Regulatory Factor 6 Mutation in an Asian Family With Van der Woude Syndrome.

Van der Woude syndrome (VWS) is a rare autosomal dominant genetic disorder characterized by orofacial clefting and lip pits. Mutations in the transcription factor interferon regulatory factor 6 gene (IRF6) have been identified in individuals with VWS. We performed direct sequencing of the gene for molecular investigation of a proband with Bangladeshi-Malay ancestry. A novel transition mutation (c.113T>C), which resulted in an amino acid substitution (p.Ile38Thr) in the deoxyribonucleic acid-binding domain was detected. Testing of family members showed that the mutation segregated with the VWS phenotype for members of her immediate family. Although there is some phenotypic variability, all of the affected members are of the female gender.

In vitro characterization of human hair follicle dermal sheath mesenchymal stromal cells and their potential in enhancing diabetic wound healing.

BACKGROUND AIMS: Little is published on the characterization and therapeutic potential of human mesenchymal cells derived from hair follicle (HF) dermal sheath (DS). In this study, we isolated and characterized HF DS-mesenchymal stromal cells (DS-MSCs) with respect to the bone marrow mesenchymal stromal cells (BM-MSCs). We further tested if DS-MSC-conditioned medium (CM), like what was previously reported for BM-MSC CM, has superior wound-healing properties, in both in vitro and in vivo wound models compared with skin fibroblast CM. METHODS: DS-MSCs were isolated from HF and cultured in vitro to assess long-term growth potential, colony-forming efficiency (CFE), expression of CD surface markers and differentiation potential. The cytokine expression of DS-MSC CM was determined through an antibody-based protein array analysis. The wound-healing effects of the CM were tested in vitro with the use of human cell cultures and in vivo with the use of a diabetic mouse wound model. RESULTS: In vitro results revealed that DS-MSCs have high growth capacity and CFE while displaying some phenotypes similar to BM-MSCs. DS-MSCs strongly expressed many surface markers expressed in BM-MSCs and could also differentiate into osteoblasts, chondrocytes and adipocytes. DS-MSCs secreted significantly higher proportions of paracrine factors such as interleukin-6 (IL-6), IL-8 and growth-related oncogene. DS-MSC-CM demonstrated enhanced wound-healing effects on human skin keratinocytes, fibroblasts and endothelial cells in vitro, and the wound-healing time in diabetic mice was found to be shorter, compared with vehicle controls. CONCLUSIONS: Human HF DS stromal cells demonstrated MSC-like properties and might be an alternative source for therapeutic use in wound healing.

Breast cancer resistance protein identifies clonogenic keratinocytes in human interfollicular epidermis.

INTRODUCTION: There is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs. METHODS: We investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract. RESULTS: Immunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, ß1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models. CONCLUSIONS: These data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis.

Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.

De novo 2.3 Mb microdeletion of 1q32.2 involving the Van der Woude Syndrome locus.

BACKGROUND: Van der Woude syndrome is the most common among syndromes which include cleft lip and/or cleft palate as one of the presentations. It is usually caused by mutations in the interferon regulatory factor 6 (IRF6) gene. CASE PRESENTATION: We previously reported on a patient with suspected deletion of the IRF6 gene. Using the Affymetrix Human SNP 6.0 Array, the interstitial deletion has been confirmed and found to be approximately 2.327-2.334 Mb within the 1q32.2 region. Although several known genes were deleted, the patient has no other phenotype apart from the orofacial presentations typical of VWS. The same deletion was not present in either parent and his two siblings were also phenotypically normal. CONCLUSIONS: Other than IRF6, the genes which are deleted in this patient appear to be insensitive to copy number and haploinsufficiency. We compared the deletion in this patient with another case which was also mapped by high resolution array but had additional phenotypic features.

CASE REPORT Journey of a Noma Face.

OBJECTIVE: Noma, or cancrum oris, is rare in developed countries. Surgeons are likely to encounter this disease only in the context of a medical mission. While it is tempting to approach noma sequelae as an oncologic resection, an understanding of the disease process will reveal that the challenge is quite different. In addition, unlike the oncologic patient who desires rapid return to an aesthetically normal facies, the adult noma patient with chronic history of noma sequelae may be more accepting of a functional but less aesthetic outcome. METHODS: We describe a noma patient with soft-tissue losses involving right cheek, nasal ala, upper lip and oral commissure, and severe trismus who underwent staged reconstructive surgery. RESULTS: The objectives of temporomandibular joint release, facial defect coverage, correction of occlusal cant, and restoration of lower facial symmetry were met. The final planned stage of reconstruction was declined as the patient had regained sufficient self-confidence to participate in social activities. CONCLUSIONS: Surgeons from developed countries rarely encounter adult patients with noma sequelae. While reconstructive principles remain the same, noma reconstruction must be approached differently from oncologic resection and a staged approach is often necessary. Although complete correction may be planned to restore function and aesthetics, the noma patient may eventually be satisfied with a functional but less aesthetic outcome.

Identification of IRF6 gene variants in three families with Van der Woude syndrome.

Van der Woude syndrome is the most common cause of syndromic orofacial clefting. It is characterised by the presence of lip pits, cleft lip and/or cleft palate. It is transmitted in an autosomal dominant manner, with high penetrance and variable expressivity. Several mutations in the interferon regulatory factor 6 (IRF6) gene have been found in VWS families, suggesting that this gene is the primary locus. We screened for mutations in this gene in three families in our population. There was a recurrent nonsense mutation within exon 9 of the gene for a Malay family consisting of five affected members with different presentations. We also found a co-segregating rare polymorphism which would result in a non-synonymous change 23 bases downstream of the nonsense mutation. This polymorphism was present in <1% of the Malay subjects screened, but was not found among the Chinese and Indians in our population. For another family, a 396C-->T mutation (R45W in the DNA-binding domain) was found in the proband, although the possibility of a genetic defect elsewhere could not be excluded because his mother and twin sister (both unaffected) also had this variant. In the third case with complete absence of family history, a de novo deletion spanning the whole IRF6 gene was detected in the child with VWS. This case of haploinsufficiency caused disruption of orofacial development but not other organ systems as the child has no other medical or developmental abnormalities despite the deletion of at least five other genes.

A modified technique of percutaneous subclavian venous catheterization in the oedematous burned patient.

Infraclavicular subclavian venepuncture in the oedematous burned patient is often difficult because of increased depth of the vein. In addition, proper patient positioning is not easily achieved because of extensive burns, generalised oedema and bulky dressings. To overcome these difficulties, a modified technique of infraclavicular subclavian venepuncture has been developed. The introducer needle is bent to create a mild curvature. It is inserted at a point 1-2 cm inferior to the palpable lower border of the clavicle along the junction of the middle and medial thirds of the bone, advanced along the deep surface of the clavicle and directed at the superior border of the suprasternal notch. This medial point of insertion shortens the distance of access to the subclavian vein. The curve allows the tip to be kept close to the undersurface of the clavicle as the needle is advanced, thereby reducing the risk of injury to deep structures. The advantages of the modified technique are demonstrated in anatomical dissections. This technique is a viable alternative when conventional techniques fail.

Effect of argon-plasma treatment on proliferation of human-skin-derived fibroblast on chitosan membrane in vitro.

Chitosan is not only a nontoxic, biocompatible, and biodegradable polymer, but has also a chemical structure similar to glycosaminoglycans (GAGs), which promote scarless wound healing of skin. In this study, chitosan membranes were treated with argon plasma to improve their surface hydrophilicity. The results showed that the water contact angles of these surface-treated membranes were significantly reduced from 60.76 to 11.57 degrees . The total surface energy was increased from 41.06 to 67.31 mJ/m(2), with 60-86.95% improvement in the gamma-negative component and a 20% difference in the nonpolar component. Argon-plasma-treated chitosan membranes exhibited excellent attachment, migration, and proliferation of the human-skin-derived fibroblasts (hSFs) compared to the untreated ones. It was found that the duration of argon-plasma treatment influenced the cell proliferation, and the optical densities in MTT assay were enhanced. Argon-plasma treatment improved the surface hydrophilicity of chitosan membranes and promoted the attachment and proliferation of hSFs.

Suppression of transforming growth factor beta/smad signaling in keloid-derived fibroblasts by quercetin: implications for the treatment of excessive scars.

BACKGROUND: Keloids are characterized by abnormal proliferation and overproduction of extracellular matrix. Quercetin, a dietary compound, has strong antioxidant and anticancer properties. Previous studies by the authors have shown that quercetin inhibits fibroblast proliferation, collagen production, and contraction of keloid and hypertrophic scar-derived fibroblasts. Quercetin also blocks the signal transduction of insulin-like growth factor-1 in keloid fibroblasts. This study assessed the effects of quercetin on the transforming growth factor (TGF)-beta/Smad-signaling pathway in keloid-derived fibroblasts, which may be an important biologic mechanism of this proliferative scarring. METHODS: Keloid fibroblasts were isolated from keloid tissue specimens. Cells were treated with quercetin at different concentrations, then harvested, and subjected to immunoblotting analysis. RESULTS: Quercetin significantly inhibited the expression of TGF-beta receptors 1 and 2 in keloid fibroblasts at three concentrations (low, medium, and high). Quercetin also strongly suppressed the basal expression of Smad2, Smad3, and Smad4 as well as the phosphorylation of Smad2 and Smad3 and the formation of the Smad2-Smad3-Smad4 complex. CONCLUSIONS: Taken together, these data suggest that quercetin effectively blocks the TGF-beta/Smad-signaling pathway in keloid fibroblasts. These data indicate that quercetin-based therapies for keloids should be investigated further.

Synchronous activation of ERK and phosphatidylinositol 3-kinase pathways is required for collagen and extracellular matrix production in keloids.

Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2-5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I-III, laminin beta2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I-III production and significantly decreased laminin beta2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I-III and laminin beta2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.

Dietary compounds inhibit proliferation and contraction of keloid and hypertrophic scar-derived fibroblasts in vitro: therapeutic implication for excessive scarring.

BACKGROUND: Keloid and hypertrophic scars commonly occur after injuries. Overproliferation of fibroblasts, overproduction of collagen, and contraction characterize these pathologic scars. Current treatment of excessive scars with intralesional corticosteroid injections used individually or in combination with other methods often have unsatisfactory outcome, frustrating both the patient and the clinician. The phytochemical compounds are well known as potential anticancer agents. We have investigated the inhibitory effects of compounds on keloid fibroblasts (KF) and hypertrophic scar-derived fibroblasts (HSF). METHODS: Fibroblasts were cultured from nontreated earlobe keloids and burn hypertrophic scars. Ten compounds (three hydroxybenzoic and four hydroxycinnamic acid derivatives, two flavonols [quercetin and kaempferol], and turmeric curcumin) were tested with fibroblasts. The inhibitory effects of compounds on fibroblasts was assessed by proliferation assays, fibroblast-populated collagen lattice (FPCL) contraction, and electron microscopy. RESULTS: The phytochemicals significantly inhibited KF and HSF proliferation in a dose- and time-dependent manner. In the hydroxybenzoic and flavonol groups, increasing inhibitory effects seemed to depend on increasing numbers of hydroxyl groups in their chemical structures. This phenomenon was not observed in the hydroxycinnamic acid group. The phytochemicals inhibited fibroblast proliferation by inducing cell growth arrest but not apoptosis. The reversibility of growth inhibition occurred when the compounds were removed from the culture and fresh media was replaced. Slower reversibility of growth inhibition was observed in the groups treated with quercetin, chlorogenic acid, or curcumin. The compounds quercetin, gallic acid, protocatechuic acid, and chlorogenic acid were the strongest inhibitors of FPLC contraction by HTFs. When the compounds were washed out of the lattices and replaced by fresh medium, the FPCL contraction was resumed. The resumption of FPCL contraction was slowest in the quercetin-treated group, indicating again the strong inhibitory effect of quercetin. CONCLUSION: From this in vitro study, quercetin seemed to have good potent effects to inhibit proliferation and contraction of excessive scar-derived fibroblasts.

Role of IGF system of mitogens in the induction of fibroblast proliferation by keloid-derived keratinocytes in vitro.

Keloids are proliferative dermal growths representing a pathological wound-healing response. We report high proliferation rates in normal (NF) and keloid-derived fibroblasts (KF) cocultured with keloid-derived keratinocytes (KK). IGF binding protein (IGFBP)-3 mRNA and secreted IGFBP-3 in conditioned media were increased in NF cocultured with KK compared with NF but markedly reduced in KF cocultured with KK or normal keratinocytes (NK). IGFBP-2 and IGFBP-4 mRNA levels were elevated, whereas IGFBP-5 mRNA was decreased in KF cocultured with KK or NK. Significant increases in IGFBP-2 and -4 mRNA in KF cocultured with KK did not correlate with protein secretion. Downstream IGF signaling cascade components, phospho-Raf, phospho-MEK1/2, phospho-MAPK, PI-3 kinase, phospho-Akt, and phospho-Elk-1, were elevated in KF cocultured with KK. Addition of recombinant human IGFBP-3 or antibodies against IGF-I or IGF-IR significantly inhibited proliferation of KF. The bioavailability of IGF-I may be related to the levels of IGFBP-3 produced, which in turn influences KF proliferation, suggesting that modulation of IGF-I, IGF-IR, and IGFBP-3, individually or in combination, may represent novel approaches to the treatment of keloids.

Fibroblasts cocultured with keloid keratinocytes: normal fibroblasts secrete collagen in a keloidlike manner.

Keloid scars represent a pathological response to cutaneous injury, reflecting a new set point between synthesis and degradation biased toward extracellular matrix (ECM) collagen accumulation. Using a serum-free two-chamber coculture model, we recently demonstrated a significant increase in normal fibroblast proliferation when cocultured with keloid-derived keratinocytes. We hypothesized that similar keratinocyte-fibroblast interactions might influence fibroblast collagen production and examined conditioned media and cell lysate from coculture for collagen I and III production by Western blot, allied with Northern analysis for procollagen I and III mRNA. Normal fibroblasts cocultured with keloid keratinocytes produced increased soluble collagen I and III with a corresponding increase in procollagen I and III mRNA transcript levels. This was associated with decreased insoluble collagen from cell lysate. When keloid fibroblasts were cocultured with keloid keratinocytes, both soluble and insoluble collagen were increased with associated procollagen III mRNA upregulation. Transmission electron microscopy of normal fibroblasts cocultured with keloid keratinocytes showed an ECM appearance similar to in vivo keloid tissue, an appearance not seen when normal fibroblasts were cocultured with normal keratinocytes.

Differences in collagen production between normal and keloid-derived fibroblasts in serum-media co-culture with keloid-derived keratinocytes.

Keloids are characterized by the deposition of excessive extracellular-matrix collagen by abnormal fibroblasts in response to cutaneous injury. Studies to date have largely concentrated on the role of the keloid fibroblast in the pathogenesis of this lesion. Recent studies have highlighted the important concept of epithelial-mesenchymal interactions in normal skin biology. Extrapolating this to keloids in two recent serum-free in vitro studies, we demonstrated increased growth and proliferation, as well as induction of keloid-like collagen secretory characteristics in normal fibroblasts co-cultured with keloid-derived keratinocytes. Most fibroblast culture work to date has been performed in nutrient and growth factor-rich serum media. To investigate how a serum co-culture system might influence epithelial-mesenchymal interactions, [3H] proline incorporation was examined in normal and keloid fibroblasts co-cultured in serum with keratinocytes derived either from normal skin or keloid tissue. Results showed increased [3H] proline incorporation when normal fibroblasts were co-cultured with keloid keratinocytes, which was significantly increased when keloid fibroblasts were co-cultured with keloid keratinocytes. Taken with previous results, this study demonstrates a good correlation between both serum and serum-free co-culture systems, and supports the significance of epithelial-mesenchymal interactions in keloid pathogenesis.