A Study on Symptoms of Pelvic Floor Dysfunction in Assigned Female at Birth Patients Diagnosed with Gender Dysphoria Undergoing Vaginectomy.

Background: A person diagnosed with gender dysphoria who was assigned female at birth (AFAB) may request a vaginectomy as part of gender-affirming treatment. The aim of this study was to investigate the impact of vaginectomy on symptoms of pelvic floor dysfunction (PFD). Methods: This is a cohort study on patient-reported symptoms of PFD in patients who were AFAB, diagnosed with gender dysphoria, and undergoing vaginectomy in a single surgical center. Patients responded to a questionnaire preoperatively and 1 year postoperatively. The questionnaire consisted of 33 questions, including a modified short-form version of the Pelvic Floor Distress Inventory (PFDI-20). Results: Twenty-three consecutive patients were included in the study and 20 patients (87%) completed the 1-year follow-up. The preoperative median PFDI-20 score was 24 (0-114) compared with 32 (0-168) at the 1-year follow-up (P = 0.07). Patients who had previously undergone neophallus construction with a metoidioplasty (n = 15) had no significant change between the preoperative and the 1-year postoperative PFDI-20 score [median 17.5 (0-114) and 27.5 (0-145) (P = 0.65), respectively]; whereas those with a groin flap phalloplasty (n = 5) had a significant increase in reported symptoms [median 37 (10-95) and 124 (45-168), respectively (P = 0.04)]. Conclusions: Overall, vaginectomy could be performed without any major impact on symptoms of PFD. However, this seemed to be true mainly for patients with previous metoidioplasty, whereas patients with previous groin flap phalloplasty reported worsening of symptoms.

Effects of amniotic fluid on human keratinocyte gene expression: Implications for wound healing.

Cutaneous wounds can lead to huge suffering for patients. Early fetal wounds have the capacity to regenerate without scar formation. Amniotic fluid (AF), containing hyaluronic acid (HA), may contribute to this regenerative environment. We aimed to analyse changes in gene expression when human keratinocytes are exposed to AF or HA. Human keratinocytes were cultured to subconfluence, starved for 12 h and then randomised to be maintained in (1) Dulbecco's modified Eagle's medium (DMEM), (2) DMEM with 50% AF, or (3) DMEM with 50% fetal calf serum (FCS). Transcriptional changes were analysed using microarray and enriched with WebGestalt and Enrichr. Additionally, eight diagnostic genes were analysed using semiquantitative real-time PCR to investigate epidermal differentiation and cellular stress after HA exposure as an alternative for AF exposure. The AF and FCS treatments resulted in enrichment of genes relating to varied aspects of epidermal and keratinocyte biology. In particular, p63-, AP1- and NFE2L2- (Nrf2) associated genes were found significantly regulated in both treatments. More genes regulated by FCS treatment were associated with inflammatory signalling, whilst AF treatment was dominantly associated with molecular establishment of epidermis and lipid metabolic activity. HA exposure mostly resulted in gene regulation that was congruent with the AF microarray group, with increased expression of ITGA6 and LOR. We conclude that AF exposure enhances keratinocyte differentiation in vitro, which suggests that AF constituents can be beneficial for wound-healing applications.

Transplantation of autologous cells and porous gelatin microcarriers to promote wound healing.

Definitive treatment to achieve wound healing in major burns frequently include skin transplantation, where split-thickness skin grafts is considered gold standard. This method is associated with several drawbacks. To overcome these hurdles, efforts have been made to develop tissue engineered skin substitutes, often comprised of a combination of cells and biomaterials. In the present study, we aimed to investigate transplantation of autologous keratinocytes and fibroblasts seeded on porous gelatin microcarriers using a porcine wound model. Pre-seeded microcarriers were transplanted to a total of 168 surgical full-thickness wounds (2cm diameter) on eight adult female pigs and covered with occlusive dressings. The experimental groups included wounds transplanted with microcarriers seeded with the combination of keratinocytes and fibroblasts, microcarriers seeded with each cell type individually, microcarriers without cells, each cell type in suspension, and NaCl control. Wounds were allowed to heal for one, two, four or eight weeks before being excised and fixated for subsequent histological and immunohistochemical analysis. In vitro, we confirmed that viable cells populate the surface and the pores of the microcarriers. In vivo, the microcarriers were to a large extent degraded after two weeks. After one week, all treatment groups, with the exception of microcarriers alone, displayed significantly thicker neo-epidermis compared to controls. After two weeks, wounds transplanted with microcarriers seeded with cells displayed significantly thicker neo-epidermis compared to controls. After four weeks there was no difference in the thickness of neo-epidermis. In conclusion, the experiments performed illustrate that autologous cells seeded on porous gelatin microcarriers stimulates the re-epithelialization of wounds. This method could be a promising candidate for skin transplantation. Future studies will focus on additional outcome parameters to evaluate long-term quality of healing following transplantation.

Tracking keratinocytes and melanocytes using carboxyfluorescein hydroxysuccinimidyl ester staining.

INTRODUCTION: The treatment of burn wounds and hypopigmentation conditions often require autologous transplantation of keratinocytes and melanocytes. Tracking transplanted cells to ascertain their contribution to tissue recapitulation presents a challenge. This study demonstrates a methodology based on passive staining with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) that enables localization of cells in tissue sections to investigate the fate of transplanted cells in wound re-epithelialisation. METHODS: Viability and migration of CFSE-stained keratinocytes and melanocytes were investigated using viability staining and scratch assays, while proliferation of cells was measured using flow cytometry. In addition, CFSE-stained keratinocytes and melanocytes were transplanted to a human ex vivo wound model, either in suspension, or with the aid of macroporous gelatine microcarriers. Wounds were analysed seven, 14 and 21 days post transplantation using cryosectioning and fluorescence microscopy. Sections from wounds with transplanted co-cultured keratinocytes and melanocytes were stained for pancytokeratin to distinguish keratinocytes. RESULTS: CFSE-staining of keratinocytes and melanocytes did not affect the viability, migration or proliferation of the cells. Transplanted cells were tracked in ex vivo wounds for 21 days, illustrating that the staining had no effect on wound re-epithelialisation. In conclusion, this study presents a novel application of CFSE-staining for tacking transplanted primary human keratinocytes and melanocytes.

Hyaluronic Acid Accelerates Re-epithelialization and Alters Protein Expression in a Human Wound Model.

BACKGROUND: Hyaluronic acid (HA), a large glycosaminoglycan involved in proliferation, migration, and tissue repair, is suggested to be an important factor for keratinocyte activation and re-epithelialization. The experimental hypothesis of this study was that HA accelerates re-epithelialization, and we aimed to investigate the effect of exogenous intradermal HA during deep dermal, incisional wound healing in vivo in humans, the primary endpoint being re-epithelialization. METHODS: A total of 8 standardized deep dermal incisional wounds (depth 1.6 mm, width 1.8 mm) per subject were induced in 10 healthy volunteers. Two of the wound sites per subject were pretreated with injections of HA and 2 with saline solution. At 2 time points (24 hours and 14 days), 2 biopsies for each treatment group (one for histology and one for proteomics) were taken. Skin erythema was measured at 24-hour intervals for 14 days as a surrogate measurement of inflammation. RESULTS: At 24 hours, 8 of 9 wounds pretreated with HA showed complete re-epithelization, whereas none of the wounds pretreated with saline had re-epithelized. Wounds pretreated with HA also showed a 10-fold regulation of 8 identified proteins involved in wound healing compared to wounds treated with saline solution. No difference in inflammation, as measured as erythema, could be seen between any of the groups. CONCLUSIONS: We conclude that HA accelerates re-epithelialization and stimulates an altered protein expression in vivo in human deep dermal incisional skin wounds, but has no effect on the inflammation process as measured by erythema.

Non-occlusive topical exposure of human skin in vitro as model for cytotoxicity testing of irritant compounds.

Testing of irritant compounds has traditionally been performed on animals and human volunteers. Animal testing should always be restricted and for skin irritancy mice and rabbits hold poor predictive value for irritant potential in humans. Irritant testing on human volunteers is restricted by the duration subjects can be exposed, and by the subjectivity of interpreting the visual signs of skin irritation. We propose an irritant testing system using viable human full thickness skin with the loss of cell viability in the exposed skin area as end point measurement. Skin was exposed to sodium dodecyl sulfate (SDS) at 20% concentration by non-occluded topical exposure to establish a positive control response and subsequent test compounds were statistically compared with the 20% SDS response. Cell viability and metabolism were measured with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The model presents correlation between increased concentration of SDS and decreased viability of cells in the exposed skin area (R(2) = 0.76). We propose the model to be used for cytotoxicity testing of irritant compounds. With fully intact barrier function, the model comprises all cells present in the skin with quantifiable end point measurement.

The expression pattern of PFKFB3 enzyme distinguishes between induced-pluripotent stem cells and cancer stem cells.

Induced pluripotent stem cells (iPS) have become crucial in medicine and biology. Several studies indicate their phenotypic similarities with cancer stem cells (CSCs) and a propensity to form tumors. Thus it is desirable to identify a trait which differentiates iPS populations and CSCs. Searching for such a feature, in this work we compare the restriction (R) point-governed regulation of cell cycle progression in different cell types (iPS, cancer, CSC and normal cells) based on the expression profile of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3) and phosphofructokinase (PFK1). Our study reveals that PFKFB3 and PFK1 expression allows discrimination between iPS and CSCs. Moreover, cancer and iPS cells, when cultured under hypoxic conditions, alter their expression level of PFKFB3 and PFK1 to resemble those in CSCs. We also observed cell type-related differences in response to inhibition of PFKFB3. This possibility to distinguish CSC from iPS cells or non-stem cancer cells by PFKB3 and PFK1 expression improves the outlook for clinical application of stem cell-based therapies and for more precise detection of CSCs.

Biodegradable Gelatin Microcarriers Facilitate Re-Epithelialization of Human Cutaneous Wounds - An In Vitro Study in Human Skin.

The possibility to use a suspended tridimensional matrix as scaffolding for re-epithelialization of in vitro cutaneous wounds was investigated with the aid of a human in vitro wound healing model based on viable full thickness skin. Macroporous gelatin microcarriers, CultiSpher-S, were applied to in vitro wounds and cultured for 21 days. Tissue sections showed incorporation of wound edge keratinocytes into the microcarriers and thicker neoepidermis in wounds treated with microcarriers. Thickness of the neoepidermis was measured digitally, using immunohistochemical staining of keratins as epithelial demarcation. Air-lifting of wounds enhanced stratification in control wounds as well as wounds with CultiSpher-S. Immunohistochemical staining revealed expression of keratin 5, keratin 10, and laminin 5 in the neoepidermal component. We conclude that the CultiSpher-S microcarriers can function as tissue guiding scaffold for re-epithelialization of cutaneous wounds.

Influence of acidic pH on keratinocyte function and re-epithelialisation of human in vitro wounds.

BACKGROUND: Chronic wounds are one of the greatest challenges for the healthcare system. Today, a plethora of dressings are used in the treatment of these wounds, each with specific influence on the wound environment. Due to differences in the permeability of the dressings the use will result in differences in the pH balance in the wound bed. However, little is known about how changes in the pH in the wound environment affect the different phases of the healing process. AIM: The aim of the present study was to investigate the effects of acidic pH on the regeneration phase by studying keratinocyte function in vitro and re-epithelialisation in an in vitro model of human skin. RESULTS: In vitro assays showed reduced viability and migration rates in human keratinocytes when pH was lowered. Real time PCR revealed differential expression of genes related to wound healing and environmental impairment. Tissue culture showed no re-epithelialisation of wounds subjected to pH 5.0 and moderate re-epithelialisation at pH 6.0, compared to controls at pH 7.4. CONCLUSION: The results indicate that lowering pH down to pH 5.0 in wounds is counterproductive in aspect of keratinocyte function which is crucial for successful wound healing.

Human melanocytes mitigate keratinocyte-dependent contraction in an in vitro collagen contraction assay.

Scarring is an extensive problem in burn care, and treatment can be especially complicated in cases of hypertrophic scarring. Contraction is an important factor in scarring but the contribution of different cell types remains unclear. We have investigated the contractile behavior of keratinocytes, melanocytes and fibroblasts by using an in vitro collagen gel assay aimed at identifying a modulating role of melanocytes in keratinocyte-mediated contraction. Cells were seeded on a collagen type I gel substrate and the change in gel dimensions were measured over time. Hematoxylin & Eosin-staining and immunohistochemistry against pan-cytokeratin and microphthalmia-associated transcription factor showed that melanocytes integrated between keratinocytes and remained there throughout the experiments. Keratinocyte- and fibroblast-seeded gels contracted significantly over time, whereas melanocyte-seeded gels did not. Co-culture assays showed that melanocytes mitigate the keratinocyte-dependent contraction (significantly slower and 18-32% less). Fibroblasts augmented the contraction in most assays (approximately 6% more). Non-contact co-cultures showed some influence on the keratinocyte-dependent contraction. Results show that mechanisms attributable to melanocytes, but not fibroblasts, can mitigate keratinocyte contractile behavior. Contact-dependent mechanisms are stronger modulators than non-contact dependent mechanisms, but both modes carry significance to the contraction modulation of keratinocytes. Further investigations are required to determine the mechanisms involved and to determine the utility of melanocytes beyond hypopigmentation in improved clinical regimes of burn wounds and wound healing.

Salinomycin induces activation of autophagy, mitophagy and affects mitochondrial polarity: differences between primary and cancer cells.

The molecular mechanism of Salinomycin's toxicity is not fully understood. Various studies reported that Ca(2+), cytochrome c, and caspase activation play a role in Salinomycin-induced cytotoxicity. Furthermore, Salinomycin may target Wnt/ß-catenin signaling pathway to promote differentiation and thus elimination of cancer stem cells. In this study, we show a massive autophagic response to Salinomycin (substantially stronger than to commonly used autophagic inducer Rapamycin) in prostrate-, breast cancer cells, and to lesser degree in human normal dermal fibroblasts. Interestingly, autophagy induced by Salinomycin is a cell protective mechanism in all tested cancer cell lines. Furthermore, Salinomycin induces mitophagy, mitoptosis and increased mitochondrial membrane potential (∆Ψ) in a subpopulation of cells. Salinomycin strongly, and in time-dependent manner decreases cellular ATP level. Contrastingly, human normal dermal fibroblasts treated with Salinomycin show some initial decrease in mitochondrial mass, however they are largely resistant to Salinomycin-triggered ATP-depletion. Our data provide new insight into the molecular mechanism of preferential toxicity of Salinomycin towards cancer cells, and suggest possible clinical application of Salinomycin in combination with autophagy inhibitors (i.e. clinically-used Chloroquine). Furthermore, we discuss preferential Salinomycins toxicity in the context of Warburg effect.

Differentiation of human dermal fibroblasts towards endothelial cells.

The ultimate goal of vascular tissue engineering is the production of functional grafts for clinical use. Difficulties acquiring autologous endothelial cells have motivated the search for alternative cell sources. Differentiation of dermal fibroblasts towards several mesenchymal lineages as well as endothelial cells has been proposed. The aim of the present study was to investigate the endothelial differentiation capacity of human dermal fibroblasts on a gene expression, protein expression and functional physiological level. Endothelial differentiation of fibroblasts was induced by culturing cells in 30% human serum, but not in fetal calf serum. Expression of proteins and genes relevant for endothelial function and differentiation was increased after induction. Furthermore, fibroblasts exposed to 30% human serum displayed increased uptake of low-density lipoprotein and formation of capillary-like networks. The results of this study may have an impact on cell sourcing for vascular tissue engineering, and the development of methods for vascularization of autologous tissue engineered constructs.

Hyaluronic acid, an important factor in the wound healing properties of amniotic fluid: in vitro studies of re-epithelialisation in human skin wounds.

Foetal wounds are unique in their ability to heal rapidly without forming scars. The amniotic fluid, rich in nutrients, growth factors, and hyaluronic acid, surrounds the foetus and is essential to foetal wound healing. The wound healing properties of foetal wounds may be the result of high concentrations of hyaluronic acid. This study aimed to verify that amniotic fluid induces re-epithelialisation in human skin wounds in vitro and to study whether this ability is dependent on hyaluronic acid. Standard deep dermal wounds were produced in vitro in human skin. The skin samples, with a central wound, were incubated in different culture media. Varying concentrations of amniotic fluid and amniotic fluid with added hyaluronidase were tested, and re-epithelialisation was assessed at 3, 7, and 12 days using light microscopy, after staining with haematoxylin and eosin. Amniotic fluid 50% resulted in a significantly higher (p < 0.05) grade of re-epithelialisation than Dulbecco's modified Eagle's medium and 10% amniotic fluid at all time points. When 50% amniotic fluid was compared with 10% foetal calf serum, no significant difference was found in grades of re-epithelialisation on days 3 and 12 and significantly higher grades of re-epithelialisation on day 7 (p < 0.05). Degradation of hyaluronic acid in the medium that contained 50% amniotic fluid gave significantly impaired re-epithelialisation (p < 0.05) on culture days 3 and 7. In conclusion, amniotic fluid promotes accelerated re-epithelialisation and hyaluronic acid is an important ingredient.

Osteogenically-induced human dermal fibroblasts as a tool to regenerate bone.

Engineering of bone tissue could help to overcome the need for extensive reconstruction and associated donor site morbidity, and it has been proposed that osteogenic biomaterials, which are scaffolds that contain osteocompetent cells, could be used to fill large bone defects. This study investigated the potential of osteogenically-induced human dermal fibroblasts cultured on gelatin microcarriers combined with platelet-rich plasma in a model of a femoral defect in athymic rats. Defects were transplanted with one of the following six combinations: 1 = sodium chloride, 2 = platelet-rich plasma, 3 = microcarriers + platelet-rich plasma, 4 = human dermal fibroblasts on microcarriers + platelet-rich plasma, 5 = human osteoblasts on microcarriers + platelet-rich plasma, and 6 = osteogenically induced human dermal fibroblasts on microcarriers + platelet-rich plasma. The femoral defects were assessed 4 weeks postoperatively with computed tomography (CT), routine histological staining, fluorescence in situ hybridisation, and polyclonal antibodies directed towards osteocalcin and osteonectin. Radiographs of all groups taken 4 weeks postoperatively showed unhealed defects. Femoral defects transplanted with osteogenically-induced human dermal fibroblasts on microcarriers (group 6) contained dense clusters of cells with large quantities of extracellular matrix. These clusters were exclusive to this group and stained strongly for osteocalcin and osteonectin. Fluorescence in situ hybridisation showed viable human cells in femoral defects that had been transplanted with microcarriers seeded with cells, which confirmed the survival of implanted cells. In conclusion, osteogenically-induced human dermal fibroblasts survived in this new niche, and bone-like structures were apparent in the defects.

Interpreted gene expression of human dermal fibroblasts after adipo-, chondro- and osteogenic phenotype shifts.

Autologous cell-based therapies promise important developments for reconstructive surgery. In vitro expansion as well as differentiation strategies could provide a substantial benefit to cellular therapies. Human dermal fibroblasts, considered ubiquitous connective tissue cells, can be coaxed towards different cellular fates, are readily available and may altogether be a suitable cell source for tissue engineering strategies. Global gene expression analysis was performed to investigate the changes of the fibroblast phenotype after four-week inductions toward adipocytic, osteoblastic and chondrocytic lineages. Differential gene regulation, interpreted through Gene Set Enrichment Analysis, highlight important similarities and differences of induced fibroblasts compared to control cultures of human fibroblasts, adipocytes, osteoblasts and articular chondrocytes. Fibroblasts show an inherent degree of phenotype plasticity that can be controlled to obtain cells supportive of multiple tissue types.

Cell expansion of human articular chondrocytes on macroporous gelatine scaffolds-impact of microcarrier selection on cell proliferation.

This study investigates human chondrocyte expansion on four macroporous gelatine microcarriers (CultiSpher) differing with respect to two manufacturing processes-the amount of emulsifier used during initial preparation and the gelatine cross-linking medium. Monolayer-expanded articular chondrocytes from three donors were seeded onto the microcarriers and cultured in spinner flask systems for a total of 15 days. Samples were extracted every other day to monitor cell viability and establish cell counts, which were analysed using analysis of variance and piecewise linear regression. Chondrocyte densities increased according to a linear pattern for all microcarriers, indicating an ongoing, though limited, cell proliferation. A strong chondrocyte donor effect was seen during the initial expansion phase. The final cell yield differed significantly between the microcarriers and our results indicate that manufacturing differences affected chondrocyte densities at this point. Remaining cells stained positive for chondrogenic markers SOX-9 and S-100 but extracellular matrix formation was modest to undetectable. In conclusion, the four gelatine microcarriers supported chondrocyte adhesion and proliferation over a two week period. The best yield was observed for microcarriers produced with low emulsifier content and cross-linked in water and acetone. These results add to the identification of optimal biomaterial parameters for specific cellular processes and populations.

Transplantation of acellular dermis and keratinocytes cultured on porous biodegradable microcarriers into full-thickness skin injuries on athymic rats.

In search of an optimal transplantation regime for sufficient dermal and epidermal regeneration after a full-thickness skin injury, wounds on athymic rats were grafted with split-thickness skin grafts or acellular human dermis followed by transplantation with human keratinocytes either in single-cell suspension or cultured on porous biodegradable microcarriers. After 2 weeks, all wounds grafted with acellular human dermis showed a well organised and vascularised dermal component and reepithelialisation on the grafted dermal matrix was complete 21 days after transplantation with human keratinocytes. Wounds grafted with human keratinocytes seeded on biodegradable microcarriers or split-thickness skin grafts displayed over time (i.e. 16-21 days post-transplantation) a significantly thicker epithelial cell layer in comparison to wounds grafted with keratinocytes in single-cell suspensions or microcarriers not seeded with cells. Furthermore, measurements of dermal thickness in the closed wounds 21 days after grafting showed a significantly thicker and well organised neodermal component in wounds transplanted with keratinocytes seeded on microcarriers or split-thickness skin grafts compared to all other wounds. Positive immunostaining towards von Willebrand factor revealed the plausible proangiogenic effects of transplantation with keratinocytes seeded on microcarriers. Analysis of representative tissue sections after fluorescence in situ hybridisation visualised that grafted human keratinocytes were present in the epidermal layers covering the wounds 16 and 21 days after transplantation, strongly indicating preservation of cell viability. These results shows that the use of biodegradable microcarriers in the culture of autologous keratinocytes for treatment of full-thickness wounds not only facilitate the cultivation, transportation and transplantation processes but also enhances the dermal regeneration induced by a dermal scaffold which results in a clinical result that is significantly superior to the one obtained when keratinocytes are transplanted in a single-cell suspension.

[Sex reassignment surgery in transsexuals]. / Kjønnskorrigerende kirurgi ved transseksualisme.

BACKGROUND: Rikshospitalet (The National Hospital) treats all transsexual patients in Norway. 50-70 patients are referred annually for assessment and about 20 of these are diagnosed as transsexual. Approximately 15 confirmed transsexuals, who have received hormone treatment, are referred to sex reassignment surgery per year. This article describes diagnosis and treatment of transsexualism with an emphasis on surgery. MATERIAL AND METHODS: The article is based on a non-systematic review of the literature and our own clinical experience. RESULTS: Patients are evaluated for sex reassignment surgery after at least one year of psychiatric evaluation and an additional year of endocrine treatment. Male-to-female patients are evaluated for breast augmentation if hormone therapy has given unsatisfactory results. Thereafter, genital surgery can be performed, i.e. the testis and corpus cavernosum are removed and a neovagina and neoclitoris are constructed. Female-to-male patients have the possibility to undergo breast reduction, hysterectomy, salpingoophorectomy and construction of a neopenis. If the patient responds well to male hormones, the clitoris can be straightened and reconstructed as a small neopenis with an erectile function (metaidoioplasty). Alternatively, phalloplasty is performed using the local groin flap or a microvascular free flap. INTERPRETATION: Sex reassignment surgery mainly consists of operations on breasts and genitalia.

Adipogenic, chondrogenic and osteogenic differentiation of clonally derived human dermal fibroblasts.

The apparent need of an autologous cell source for tissue engineering applications has led researchers to explore the presence of cells with stem cell plasticity in several human tissues. Dermal fibroblasts (FBs) are easy to harvest, expand in vitro and store, rendering them plausible candidates for cell-based therapies. The aim of the present study was to observe the effects of adipogenic, chondrogenic and osteogenic induction media on the phenotype of human FBs. Human preadipocytes obtained from fat tissue have been proposed as an adult stem cell source with suitable characteristics, and were used as control cells in regard to their differentiation potential. Routine staining, immunohistochemical analysis and alkaline phosphatase assay were employed, in order to study the phenotypic shift. FBs were shown to possess multilineage potential, giving rise to fat-, cartilage- and bone-like cells. To exclude contaminant progenitor cells or cell fusion giving rise to tissue with adipocyte-, chondrocyte- and osteoblast-like cells, single-cell cloning was performed. Single-cell-cloned FBs (sccFBs) displayed a similar differentiation potential as primary-culture FBs. The presence of 'stem-cell-specific' surface antigens was analyzed using flow cytometry. The results reveal that sccFBs have several of the markers associated with cells exhibiting stem cell plasticity. The findings presented here are corroborated by the findings of other groups, and suggest the use of human dermal FBs in cell-based therapies for the reconstruction of fat, cartilage and bone.

Engineering three-dimensional cartilage- and bone-like tissues using human dermal fibroblasts and macroporous gelatine microcarriers.

The creation of tissue-engineered cartilage and bone, using cells from an easily available source seeded on a suitable biomaterial, may have a vast impact on regenerative medicine. While various types of adult stem cells have shown promising results, their use is accompanied by difficulties associated with harvest and culture. The proposed inherent plasticity of dermally derived human fibroblasts may render them useful in tissue-engineering applications. In the present study, human dermal fibroblasts cultured on macroporous gelatine microcarriers encapsulated in platelet-rich plasma into three-dimensional constructs were differentiated towards chondrogenic and osteogenic phenotypes using specific induction media. The effect of flow-induced shear stress on osteogenic differentiation of fibroblasts was also evaluated. The generated tissue constructs were analysed after 4, 8 and 12 weeks using routine and immunohistochemical stainings as well as an enzyme activity assay. The chondrogenic-induced tissue constructs were composed of glycosaminoglycan-rich extracellular matrix, which stained positive for aggrecan. The osteogenic-induced tissue constructs were composed of mineralised extracellular matrix containing osteocalcin and osteonectin, with cells showing an increased alkaline phosphatase activity. Increased osteogenic differentiation was seen when applying flow-induced shear stress to the culture. Un-induced fibroblast controls did not form cartilage- or bone-like tissues. Our findings suggest that primary human dermal fibroblasts can be used to form cartilage- and bone-like tissues in vitro when cultured in specific induction media.