Metoidioplasty and groin flap phalloplasty as two surgical methods for the creation of a neophallus in female-to-male gender-confirming surgery: A retrospective study comprising 123 operated patients.

BACKGROUND: In gender-confirming surgery of the female-to-male gender dysphoric patient, there is currently no ideal method for the creation of a neophallus. Historically, in our clinic, groin flap phalloplasty (GFP) has been the dominating method, but during the last 20 years, it has gradually been replaced with metoidioplasty (MP). The aim of this study was to investigate whether this change of method has influenced factors such as the frequency of complications and the number of operations needed to complete the reconstruction of the neophallus. METHODS: This is a retrospective, single-centre, study comprising 123 consecutive female-to-male patients receiving a neophallus by GFP or MP between 2002 and 2015 at Linköping University Hospital, Sweden. RESULTS: One-hundred twenty-three patients underwent 126 primary surgical procedures (39 GFPs and 87 MPs) with the intention of reconstructing a neophallus. The mean number of procedures required in the GFP group was 5.2 ± 2.7 compared with that of 2.4 ± 1.7 in the MP group (p < 0.001). In the GFP group, 18/39 (46.2%) had a documented complication compared with 30/87 (34.5%) in the MP group; however, the difference was not statistically significant (p = 0.21). CONCLUSIONS: The present study shows that the shift in method from GFP to MP has resulted in a decreased number of complications as well as a decrease in total surgical occasions. Both methods were found to be associated with relatively high frequencies of complications, however, mostly minor.

Autologous in vitro cultured urothelium in hypospadias repair.

OBJECTIVE: To treat severe hypospadias with a transplant of autologous in vitro cultured urothelial cells on acellular dermis. PATIENTS AND METHODS: During 2000-2002 six patients aged 14-44 months with severe hypospadias were treated surgically with autologous urothelial cell transplants. All were born with scrotal or perineal hypospadias and pronounced chordee. All patients were subjected to a two-staged procedure starting with repair of the chordee. Urothelial cell harvesting via bladder lavage was performed during the first operation. The neourethra was constructed by using a transplant with cultured urothelium in an on-lay fashion. Patients have been followed 3-5.5 years. RESULTS: All six boys are voiding through their neourethra without straining and have no residual urine after micturition. Five patients are using a standing voiding position and present bell shaped, urinary flow curves. One developed a stricture treated conservatively with persisting good effect (after more than 5 years). Two developed a fistula requiring surgical correction that was uneventful. The last patient developed an obstruction in the proximal anastomosis that was treated with an internal urethrotomy. Cosmetic appearance is good in all cases with good parental satisfaction. Urethroscopy in all patients show a wide penile neourethra. Biopsies indicate a mucosal lining consisting of urothelial cells in three cases. CONCLUSION: This technique is feasible for treatment of a selected group of hypospadias where pronounced chordee and shortage of preputial and penile skin complicates the creation of a neourethra. It may have other clinical implications including disorders such as bladder exstrophy and cloacal malformations, as well as mutilating traumatic injuries or cancer therapy.

Tissue engineering--body parts from the Petri dish.

The development of methods to regenerate human tissues and organs by tissue engineering (TE), will have a dramatic influence on many medical specialities in the future. The essence of plastic surgery is to reconstruct disrupted and damaged tissues by the use of a plethora of techniques spanning from small local skin flaps to highly advanced microsurgery and free composite grafts. However, these methods only focus on moving tissue from one part of the patient to another without actual regeneration. To be able to take the next step in development of the speciality it is of necessity to address this issue. Hence it follows naturally that plastic surgeons lead and represent the driving force of the development within the research of tissue engineering. In this paper we would like to present active research and also give an overview of areas in tissue engineering that are of special interest to the plastic surgeon.

Mammary epithelial cell and adipocyte co-culture in a 3-D matrix: the first step towards tissue-engineered human breast tissue.

Reconstruction of the female breast after cancer surgery is a demanding task where the methods used today suffer from several disadvantages. In the present study we have investigated the possibility to use tissue engineering methods to regenerate human autologous breast tissue. Human mammary epithelial cells and preadipocytes were derived from breast tissue biopsies from healthy women undergoing reduction mammoplasty, and the two celltypes were co-cultured with conventional cell culture methods as well as in 3-D matrices. The study shows that it is possible to harvest both human mammary epithelial cells and preadipocytes in a single session, propagate several subcultures, and that the cells maintain a normal intercellular distribution and growth-pattern when co-cultured in a 3-D collagen gel. We propose that growth and formation of a tissue closely resembling normal human breast tissue be readily obtained in the described in vitro cell culture set-up using basic tissue engineering principles. This concept may be of great importance in the development of new methods for reconstruction of the human breast.

Tissue engineering in urology.

Techniques that are aimed at regeneration of human tissues and organs (tissue engineering) have recently entered into clinical practice. Tissue engineering is currently among the fastest growing areas in medicine, and involves the application of the principles of biology and engineering to the development of functional substitutes for damaged tissues. One of the main limitations of reconstructive surgery in the genitourinary tract is the lack of autologous tissue. This could be changed by the ability to cultivate the patient's own tissues in vitro, or by stimulating the cells in vivo into regeneration of new tissues. The present review discusses how tissue engineering can be used to regenerate some of the tissues of the genitourinary tract. Even though these methods have only recently been introduced clinically into genitourinary medicine, numerous scientific studies have been reported that indicate that these techniques may be of great importance in the near future.

Effects of continuous stretching on cell proliferation and collagen synthesis in human burn scars.

Hypertrophic and contracted scars are common complications of deep and partial thickness burns, and the usual way to prevent them is to stretch the burn are actively as well as passively. However, little has been written about the effects of stretching on burn scar tissue at a cellular and molecular level. The stretching usually results in an increased area of skin, and a central question is whether this is caused by stimulation of cell proliferation or decreased cell density, which could lead to impaired quality of the skin. In the present study a new in vitro model was developed and used to study the effects of stretching on the proliferative activity as well as on the synthesis of collagen in human burn scars. Proliferation was measured quantitatively by thymidine incorporation and spatially by immunohistochemistry. The net proliferation in the burn scar was decreased after one day, and significantly decreased after six days of continuous stretching (p = 0.02). However, immunohistochemistry showed increased proliferation in the basal layer of the epidermis while the proliferative activity in the dermal cells was inhibited. Collagen synthesis was decreased after six days of stretching whereas no effect was shown after one day. These findings indicate that static stretching of a human burn scar results in inhibition of proliferation in dermal cells leading to a low cell density in the dermis which, combined with increased collagen synthesis, could lead to reduced biomechanical strength.

A biodegradable bovine collagen membrane as a dermal template for human in vivo wound healing.

A bovine collagen membrane was used as a template for dermal regeneration in human full thickness wounds. Healing was allowed for 7, 21, or 42 days. The formation of neodermis, basement membrane, and terminal differentiation were assessed histologically and immunohistochemically. The collagen template was neovascularised within 7 days, and from day 21 small vessels were detected throughout the transplanted area. The procollagen content decreased whereas the number of fibroblasts increased with time. Collagen type IV was not detected after 7 days but was deposited with time from the wound edges and inwards over the transplanted area. Re-epithelialisation was complete at day 7 and terminal differentiation was similar to normal human skin from day 21. We have shown the time course of dermal and epidermal healing with the aid of a ready-to-use biodegradable collagen membrane. This material may be used as a true dermal template-because of the evidence of dermal regeneration and, in addition, its availability and ease of handling.

[Recreation of tissue--the plastic surgeon's spring-board in to the 21st century]. / Nyskapa vävnad--plastikkirurgins språngbräda in i 2000-talet.

During recent years, a new field has appeared, in which the principles of life sciences and engineering are applied to the development of methods of regenerating human tissue and organs. Since the emergence of this interdisciplinary field, plastic surgeons have been deeply involved in its development, both in the early stages and in introducing the methods into clinical practice. The article consists in discussion of the possibilities these methods offer and the impact they may have on the field of plastic and reconstructive surgery.

Specific binding of proinsulin C-peptide to human cell membranes.

Recent reports have demonstrated beneficial effects of proinsulin C-peptide in the diabetic state, including improvements of kidney and nerve function. To examine the background to these effects, C-peptide binding to cell membranes has been studied by using fluorescence correlation spectroscopy. Measurements of ligand-membrane interactions at single-molecule detection sensitivity in 0.2-fl confocal volume elements show specific binding of fluorescently labeled C-peptide to several human cell types. Full saturation of the C-peptide binding to the cell surface is obtained at low nanomolar concentrations. Scatchard analysis of binding to renal tubular cells indicates the existence of a high-affinity binding process with K(ass) > 3.3 x 10(9) M(-1). Addition of excess unlabeled C-peptide is accompanied by competitive displacement, yielding a dissociation rate constant of 4.5 x 10(-4) s(-1). The C-terminal pentapeptide also displaces C-peptide bound to cell membranes, indicating that the binding occurs at this segment of the ligand. Nonnative D-C-peptide and a randomly scrambled C-peptide do not compete for binding with the labeled C-peptide, nor were crossreactions observed with insulin, insulin-like growth factor (IGF)-I, IGF-II, or proinsulin. Pretreatment of cells with pertussis toxin, known to modify receptor-coupled G proteins, abolishes the binding. It is concluded that C-peptide binds to specific G protein-coupled receptors on human cell membranes, thus providing a molecular basis for its biological effects.

Cultured autologous keratinocytes on a cell-free dermis in the treatment of full-thickness wounds.

The purpose of this study was to establish a method for transplantation of autologous keratinocytes on an allogenic cell-free dermis. From four healthy volunteers two full thickness skin grafts, 1 x 1 cm, were taken. The epidermis was separated from the dermis enzymatically and the cells of the dermal part were removed by incubation in Triton X-100. Keratinocytes were seeded on a cell-free dermis and the combined graft transplanted back to one of the wounds of the donor of the keratinocytes. The other wound was covered with cell-free dermis without keratinocytes. After 2, 3, 4 and 6 weeks, respectively, the grafted wounds were removed from the subjects and investigated histologically and immunohistochemically regarding re-epithelialisation, fibroblast ingrowth and angiogenesis. The wounds covered with cell-free dermis and keratinocytes showed a complete epidermal coverage 2 weeks after transplantation, in contrast to the wounds covered with un-seeded dermis which only showed epidermal coverage at the wound edges. There was also a marked difference concerning fibroblast ingrowth and angiogenesis. In this study we have shown that autologous keratinocytes can be seeded on a cell-free dermis and transplanted as a kerato-dermal graft which stimulate re-epithelialisation as well as fibroblast ingrowth and angiogenesis in the wound.

Endogenously formed nitric oxide modulates cell growth in bladder cancer cell lines.

OBJECTIVES: Nitric oxide (NO) is formed in many mammalian tissues, and a growing body of evidence suggests that NO is involved in cell growth and cell differentiation. Low concentrations of NO can stimulate cell growth; high concentrations result in cytostatic/cytotoxic effects. It has previously been shown that intravesical treatment with bacille Calmette-Guérin (BCG) for bladder cancer increases NO production in the human urinary bladder and that NO inhibits bladder cancer cell growth in vitro. In this study, we investigated nitric oxide synthase (NOS) activity in different bladder cancer cells and the role of the NO precursor L-arginine in cell proliferation. METHODS: NOS activity was assessed by citrulline assay in cultured normal human urothelial cells and bladder cancer cell lines T24 and MBT-2 before and after treatment with cytokines. We also measured cell growth at various L-arginine concentrations and after addition of the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) in unstimulated and cytokine-stimulated cells. RESULTS: Normal urothelial cells, as well as T24 and MBT-2 cells, showed calcium-dependent NOS activity under basal conditions. The bladder cancer cell lines also showed calcium-independent NOS activity in contrast to the normal cells. After cytokine treatment, both the normal cells and the cancer cell lines showed a marked increase in calcium-independent NOS activity. There was a dose-dependent stimulation of cell growth in the cancer cell lines after L-arginine addition, and this effect could be antagonized by L-NNA. Cytokine treatment inhibited cell growth, and this inhibition was partly reversed by L-NNA. CONCLUSIONS: Normal urothelial cells and bladder cancer cell lines MBT-2 and T24 show NOS activity, and cytokine treatment induces calcium-independent NOS activity. Our results suggest that endogenous activity of the constitutively expressed form of NOS in unstimulated cells promotes cell proliferation, and NO production secondary to increased activity of the inducible form of NOS after cytokine treatment inhibits cell growth.

Immobilised heparin accelerates the healing of human wounds in vivo.

Locally produced growth factors are of great importance in wound healing in human skin. Wound fluid from chronic wounds contains low concentrations of growth factors possibly because of rapid degradation as a result of the high concentration of proteases. Many growth factors involved in wound healing bind to heparin and are thereby stabilised and activated. We have recently shown that heparin in combination with chitosan stimulates re-epithelialisation in an in vitro model of human wound healing. In the present study we investigated the effects of a chitosan-heparin membrane on wound healing in 10 split-thickness graft donor sites in human skin. The chitosan-heparin membrane stimulated healing of the donor sites both when judged macroscopically in a blinded fashion and when biopsy specimens from the treated and untreated parts of the wound were investigated microscopically. We hypothesise that the beneficial effects of the chitosan-heparin membrane result from slow release of heparin into the wound area which protects locally produced growth factors. The result is increased stabilisation and concentration of growth factors in the wound area, which stimulate healing. We believe that these results may be important in the treatment of wounds that are reluctant to heal.

Modeling of wound healing processes in human skin using tissue culture.

To facilitate the investigation of the complex process that leads to healing of a human skin wound we developed standardized and repeatable in vitro models for both incisional and burn wounds. Wounds with a standardized area and depth were created in normal human skin biopsies which were then incubated in vitro. It was shown, by cultivation, that both dermal and epidermal cells maintained their viability during a 14-day in vitro incubation if exposed to at least 2% fetal calf serum. By incubating in 10% serum, the skin samples were stimulated to completely re-epithelialize the wounded area. Because a large number of standardized wounds can be obtained from each donor, the re-epithelialization process can be studied histologically and immunohistochemically at several adjacent time points. The ability to keep the cells in the wound area viable without stimulating healing by incubating the wounds in suboptimal serum concentrations implies a way of studying the stimulatory effects of different agents, such as growth factors, on the wound healing process. There were some marked discrepancies in the healing process between the incisional and burn wounds which resemble the in vivo situation, indicating that the in vitro models could be used to more closely study differences between healing in different types of wounds. Our findings suggest that in vitro tissue culture can be of great value in attempting to better understand the complex process of wound healing in human skin.

Melanocytes in cultured epithelial grafts are depleted with serial subcultivation and cryopreservation: implications for clinical outcome.

Patchy hypopigmentation often occurs unpredictably in the skin regenerated from cultured epidermal autografts, especially when that skin is grown from frozen cells, serially passaged, or both. The impact of serial subcultivation and cryopreservation on melanocyte viability in the cultured epidermal autograft culture system was investigated. Serial subcultivation of human keratinocytes through as many as eight passages was performed, and melanocyte densities in confluent cultures at each passage were determined after specific labeling of melanocytes. The experimental cells were frozen before cultivation and between passages to determine the effect of standard cryopreservation on melanocyte survival. Freshly passaged cells that had not been frozen served as controls. Melanocytes were gradually depleted during fresh passage of epidermal cells but persisted through as many as seven passages. Freezing before or after the first passage or between subsequent passages resulted in a complete loss of melanocytes by the third or fourth passage. The findings suggest that cryopreservation should be avoided during cultured epidermal autograft production to optimize melanocyte survival and minimize pigmentation abnormalities that may occur after grafting.

Culture of human urothelial cells on a cell-free dermis for autotransplantation.

OBJECTIVE: Techniques for in vitro culturing and autotransplantation have been developed for a variety of human cells and are used today in several fields of medicine. In reconstructive surgery within the genitourinary tract, autologous urothelial cells cultured in vitro could be of considerable value but have not yet been used clinically. The aim of this study was to facilitate transplantation of cultured urothelium by establishing a reliable method for culturing urothel on an immunologically inert and biodegradable structure. METHODS: Normal human urothelial cells were cultured in vitro using a feeder-cell system. To achieve an optimal carrier structure, cells were removed enzymatically from a split thickness skin graft. Human urothelial cells were then seeded on the cell-free dermis and incubated in vitro. The seeded dermis samples were investigated histologically and with immunohistochemical methods at days 7, 14 and 21. RESULTS: The human urothelial cells incubated in vitro reached confluence after 7-10 days and the cells could be cultured through 9 passages with preserved proliferative potential. When the cells were seeded on a cell-free dermis they attached, formed colonies and became confluent and stratified up to three cell layers after 21 days of incubation. The urothelial origin of the cells was confirmed by immunohistochemical staining against cytokeratin. CONCLUSION: The advantages of culturing the urothelial cells on a cell-free dermis include a short time lag until grafts are available, probably facilitated transplantation procedure, transplantation of undifferentiated cells and the formation of a vascularised base under the new urothelium. The method described in this study may be of great value in providing autologous urothelium for reconstructive surgery in the genitourinary tract.

Fibroblasts derived from human chronic diabetic wounds have a decreased proliferation rate, which is recovered by the addition of heparin.

We have studied the growth kinetics of fibroblasts derived from uninjured skin and chronic wounds in non-diabetic and diabetic (IDDM) patients. DNA measurements during the first 24 h after cell starvation showed that fibroblasts derived from chronic wounds, both non-diabetic and diabetic, display a decreased adhesion and proliferation. When determining the rate of proliferation after another 48, 72 and 96 h, a significant decrease in the proliferation rate was found in the chronic wound fibroblasts compared to those from uninjured skin. Furthermore, we have investigated the effects of heparin, hyaluronic acid and other heparin-like substances on the proliferation of non-diabetic and diabetic fibroblasts. We found that these substances stimulated the proliferation of human fibroblasts derived from both normal skin and chronic wounds measured as DNA content. Stimulation with heparin normalized the proliferation of the diabetic chronic wound fibroblasts. This effect was independent of the presence of serum. The effect of heparin was dose-dependent and most pronounced during the first 24 h of stimulation. These results suggest that heparin may be of importance in the treatment of chronic diabetic wounds.

Characterization and partial purification of a keratinocyte-derived growth factor with wound-healing properties.

We have previously described the mitogenic and wound-healing properties of keratinocyte-conditioned medium (KCM). In this study we investigated the effect of KCM on the activation of second messenger systems and the expression of proto-oncogene in cultured human skin fibroblasts. We also present a partial purification of the factor responsible for the mitogenic and wound-healing effects of KCM. KCM was shown to increase the expression of the proto-oncogenes c-fos, c-myc and c-jun. The effect of KCM on three second messenger systems was investigated. The extracellular release of choline metabolites was increased by 40 per cent when cells were stimulated with KCM whereas the formation of cAMP and hydrolysis of phosphatidyl inositol (PI) was unaffected. KCM was purified by ion exchange chromatography and filtration. The biologically active fraction was eluted from an SP column and retained its activity after filtration through a 3-kDa filter. The fraction was inactivated by heat and acid, indicative of a peptide origin. Furthermore, the active fraction was shown to increase the extracellular release of choline metabolites and to stimulate re-epithelialization in wounds in human skin in vitro comparable to KCM. The study indicates that human keratinocytes produce a < 3 kDa peptide which may be partly responsible for the growth stimulatory and wound-healing properties of KCM.

Tissue expression of transforming growth factor-beta1 and transforming growth factor-alpha during wound healing in human skin explants.

In the dynamic and complex process of wound healing, locally produced growth factors are important mediators, although their actual roles have not been fully established. In the present study, the presence of transforming growth factor-beta1 and -alpha during the re-epithelialization of full-thickness wounds was investigated in an in vitro model of wound healing in human skin. The amounts of transforming growth factor-beta1 and -alpha secreted from the wound area were measured with enzyme immunoassays, and immunohistochemistry was used to study the localization of these two growth factors in the healing wound. The wounds were followed until they were completely re-epithelialized. The results showed a continuous increase in secreted transforming growth factor-beta1 throughout the re-epithelialization phase of healing followed by a decrease after its completion. The keratinocytes migrating out from the wound edges showed intense staining for transforming growth factor-beta1 which declined to the level of the surrounding epidermis after the wound was covered by a new epidermis. After the skin was wounded, a decrease both in secreted transforming growth factor-alpha and in immunostaining for this growth factor was apparent. Even though a minor increase in the immunoreactivity for transforming growth factor-alpha occurred after the completion of re-epithelialization, no increase in secreted transforming growth factor-alpha could be detected by enzyme immunoassay. These data suggest that keratinocytes modulate their expression of transforming growth factor-beta1 and -alpha during the wound healing process in human skin and that these changes may be controlled in part by autocrine pathways.

Heparin-chitosan complexes stimulate wound healing in human skin.

The effect of heparin ionically linked to chitosan on the stimulation of re-epithelialisation of full thickness wounds in human skin was investigated in an in vitro model. After seven days of incubation, heparin-chitosan gel stimulated 9/10 of the full thickness wounds to re-epithelialise compared with only 3/10 of the wounds that were covered with chitosan gel or membrane, and none of the wounds incubated without gel or membrane or with heparin solution alone. Both dermal and epidermal cells were viable after the incubation time. Furthermore, the stimulatory effect of the heparin-chitosan complexes depended on the concentration of heparin in the complex. We hypothesise that these effects are caused by stabilisation and activation of growth factors that bind to immobilised heparin.