Background. Following a motorcycle accident, a 9-year-old boy experienced a complete right-sided ( dominant) arm and hand paralysis with total sensory loss, Horner syndrome and severe constant pain. This study assessed the long-term outcome of spinal cord surgery undertaken on the patient, focusing on the restored hand function and related cortical activity. The study follows on from previous reports on the same patient. Investigations. Clinical functional and electrophysiological examinations. Functional MRI of cortical activity. Diagnosis. Complete brachial plexus (C5-T1) avulsion from the spinal cord. Management. Spinal cord surgery to restore motor trajectories.

Treatment of necrotizing fasciitis (NF) includes radical surgical debridement often resulting in large wounds that need to be closed with methods including split-thickness skin grafts (STSG), local flaps, or guided tissue regeneration procedures. In this case report, a 45 year-old Caucasian male was surgically treated for a benign left groin hernia, developed NF, and was transferred to the authors burn unit. The wound was treated initially with wide debridement and with a brief delay before finally closing the wound. A collagen matrix such as Integra (R) Dermal Regeneration Template (Integra LifeSciences, Plainsboro, NJ) in combination with STSG and negative pressure wound treatment, can provide fast recovery resulting in pliable, functional skin.

The aim of this thesis was to develop and evaluate a new way of monitoring blood flow with microdialysis. A thin catheter consisting of a semipermeable membrane is implanted in the tissue being studied. The catheter is perfused by a solution that closely resembles interstitial fluid, and small water-soluble substances are allowed to diffuse passively through the pores of the membrane with the aim at reaching equilibrium with the surrounding tissue.  The minimally invasive character of microdialysis, and its ability to sample from the organ being studied, make microdialysis attractive in most research settings as well as for clinical surveillance. It has, however, become increasingly evident that microdialysis under conditions of non-equilibrium - for example, fluctuating regional blood flow, will alter the results gained. We have therefore aimed to explore the possibilities of developing a new marker of blood flow that will yield information about changes in blood flow that occur in the area of the microdialysis catheter itself.

We hypothesised that the changes in the diffusion of exogenous urea could be used as markers of changes in tissue blood flow. The theoretical basis for this approach is that the mass transfer of urea will increase across the dialysis membrane secondary to increased blood flow. As removal of urea from the vicinity of the dialysis membrane increases with increased blood flow, the concentration gradient of urea between the perfusate and tissue will also increase. This in turn will result in a greater loss of urea from the perfusate. The changes noted in retrieval of urea from dialysate by the system are therefore thought to be inversely related to changes in blood flow. We tested our hypothesis in two species of animal (rat and pig) and in man, and in three organ systems (muscle, liver, and skin), and present four papers that indicate that the urea clearance technique provides reliable and reproducible results. The technique was evaluated against conventional metabolic markers (lactate and glucose), the ethanol clearance technique (microdialysis), laser Doppler perfusion imaging (LDPI), and polarisation light spectroscopy (TiVi).

We present evidence that the urea clearance technique can be used to assess blood flow in the organs studied reliably and reproducibly with microdialysis. The microdialysis technique is minimally invasive and safe for the recipient, and catheters can easily be implanted during operation to monitor organs at risk. Urea is easily analysed as a standard assay among other “basic” metabolic markers (in a standard microdialysis kit) and has favourable characteristics with a standardised measurement system that is routinely used for monitoring metabolites in the clinic. The technique is also effective when used at lower perfusate flow rates (<1 μl/minute), which is advantageous as the recovery of metabolic markers increases at low perfusate flow rates.

Objective: The aim of this study was to evaluate the urea clearance technique for the measurement of drug-induced blood flow changes in human skin, and compare it with two non-invasive techniques: polarization light spectroscopy and laser Doppler perfusion imaging.

Methods: Fifteen microdialysis catheters were placed intracutaneously on the volar aspect of the forearms of healthy human subjects, and were perfused with nitroglycerine, noradrenaline, and again nitroglycerine, to induce local tissue hyperaemia, hypoperfusion, and hyperaemia, respectively.

Results: Urea clearance, but not the other techniques, detected the changes in blood flow during all three periods of altered flow.  The last hyperaemic response was detected by all three methods.

Conclusion: Urea clearance can be used as a relatively simple method to estimate blood flow changes during microdialysis of vasoactive substances, in particular when the tissue is preconditioned in order to enhance the contrast between baseline and the responses to the provocations. Our results support that, in the model described, urea clearance was superior to the optical methods as it detected both the increases and decrease in blood flow, and the returns to baseline between these periods.

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. © 2006 Journal of Pediatric Urology Company.

Spider silk is an interesting biomaterial for medical applications. Recently, a method for production of recombinant spider silk protein (4RepCT) that forms macroscopic fibres in physiological solution was developed. Herein, 4RepCT and Mersilk(TM) (control) fibres were implanted subcutaneously in rats for seven days, without any negative systemic or local reactions. The tissue response, characterised by infiltration of macrophages and multinucleated cells, was similar with both fibres, while only the 4RepCT-fibres supported ingrowth of fibroblasts and newly formed capillaries. This in vivo study indicates that 4RepCT-fibres are well tolerated and could be used for medical applications, e. g., tissue engineering.

Skin tags (acrochordons) are exceedingly common benign skin lesions. A novel medical device in the form of a flat adhesive patch applies pressure to the base of a skin tag, leading to its removal within 3-6 days. The device was used in a clinical trial to treat and remove skin tags of the neck, upper torso, and axillae in volunteers. In this study, a total of 177 skin tags were treated in 32 individuals. One hundred seventy-two lesions fulfilled intention to treat (ITT) criteria. A majority of ITT lesions (90%) reached final assessment. Successful outcome was highest (90%) for lesions up to 1 mm in base. For lesions up to 2 mm, the rate of successful outcome was 76 percent. The desired outcome was seen in 65 percent of all ITT lesions. The cosmetic outcome after removal was excellent. Discomfort was assessed as minimal during all stages of the procedure. Analysis of data on blood flow in the skin tags during the treatment showed that the outcome was influenced by whether a decrease in blood flow was achieved immediately after application and at 2-3 days, but that the degree of occlusion was not critical. The results of this study illustrate that the device presents a new option for the management of unmet needs in the treatment of skin tags.

Silver is commonly used in wound dressings and topical formulations to assist in the management of wounds that are infected or at risk of becoming infected. They provide potent broad-spectrum antimicrobial activity, but should not cause sustained staining of the skin, dermal or systemic accumulation of silver, or discomfort to the patient. However, clinicians and healthcare personnel have been concerned about topical staining of the skin and complaints of additional pain from patients treated with certain silver dressings. Some delay in re-epithelialization has also been noticed and reported. The reasons for this are not clear, and the authors believed further study regarding the possible effects of silver accumulation and silver dressings effect on re-epithelialization was required. The authors studied possible silver accumulation and re-epithelialization in normal human dermal skin. The results showed that most of the dressings or treatments discolored the wound surface and that there was a dermal accumulation of what were assumed to be silver particles. Varying grades of accumulation were found in deep dermal tissue, particularly around blood vessels, depending on the dressing used. The results also indicated that all of the tested products delayed re-epithelialization in this model.

Transplantation of autologous cultured keratinocytes in single cell suspension is useful in the treatment of burns. The reduced time needed for culture, and the fact that keratinocytes in suspension can be transported from the laboratory to the patient in small vials, thus reducing the costs involved and be stored (frozen) in the clinic for transplantation when the wound surfaces are ready, makes it appealing. We found few published data in the literature about actual cell survival after transplantation of keratinocytes in single cell suspension and so did a comparative in vitro study, considering commonly used application techniques. Human primary keratinocytes were transplanted in vitro in a standard manner using different techniques. Keratinocytes were counted before and after transplantation, were subsequently allowed to proliferate, and counted again on days 4, 8, and 14 by vital staining. Cell survival varied, ranging from 47% to >90%, depending on the technique. However, the proliferation assays showed that the differences in numbers diminished after 8 days of culture. Our findings indicate that a great number of cells die during transplantation but that this effect is diminished if cells are allowed to proliferate in an optimal milieu. A burned patient’s wounds cannot be regarded as the optimal milieu, and using less harsh methods of transplantation may increase the take rate and wound closing properties of autologous keratinocytes transplanted in a single cell suspension.

Sex steroid exposure constitutes a risk factor for breast cancer, but little is known about the effects of sex steroids on the normal breast, largely because of the lack of convenient models. We have developed a method of culturing normal breast tissue ex vivo. We have applied this method to investigate the effects of estradiol and progesterone on the key angiogenic mediator, vascular endothelial growth factor (VEGF), in the breast. Whole breast tissue was obtained from routine reduction mammoplasty. Tissue biopsies were cultured in vitro for 1–3 weeks, and the expression of luminal cytokeratin 18 was determined by immunohistochemistry. As an application, tissue biopsies were treated in vitro for 1 week with or without estradiol or estradiol and progesterone. Estrogen receptor, progesterone receptor, and Ki–67 were analyzed, and VEGF levels were examined by quantitative immunoassay and immunohistochemistry. Whole breast tissue was cultured ex vivo for 1 week with preserved morphology. Increased detachment of the luminal epithelium was observed after 2 weeks. Estradiol increased extracellular levels of VEGF in normal breast tissue biopsy medium. The addition of progesterone had neither stimulatory nor inhibitory effects on secreted VEGF. The method of whole breast tissue culturing thus provide a means by which to explore the biology of normal breast tissue. Our results suggest that estradiol exerts pro-angiogenic effects in normal breast by increasing levels of biologically active VEGF.

We report our observations of cross-modal interactions between sight and touch using functional magnetic resonance imaging (fMRI). Experiments were devised to show that sight and touch are linked in a cross-modal arrangement, and two separate experiments were done in an MRI scanner. In the first, the subject's right hand was stimulated with a brush; in the second, a video sequence was presented to the subject inside the scanner through video goggles in visual three-dimensional stereo, showing one brushstroke every second on a hand in the same manner as the subject had just previously experienced. The result was that both the primary and the secondary somatosensory cortexes were activated in the participants when the hands were touched, and when the subjects saw only a hand being touched in the same manner. The results indicated cross-modal links between sight and touch of the hand in humans.

Full thickness skin wounds in humans heal with scars, but without regeneration of the dermis. A degradable poly(urethane urea) scaffold (PUUR), Artelon® is already used to reinforce soft tissues in orthopaedics, and for treatment of osteoarthritis of the hand, wrist and foot. In this paper we have done in vitro experiments followed by in vivo studies to find out whether the PUUR is biocompatible and usable as a template for dermal regeneration. Human dermal fibroblasts were cultured on discs of PUUR, with different macrostructures (fibrous and porous). They adhered to and migrated into the scaffolds, and produced collagen. The porous scaffold was judged more suitable for clinical applications and 4 mm Ø, 2 mm-thick discs of porous scaffold (12% w/w or 9% w/w polymer solution) were inserted intradermally in four healthy human volunteers. The implants were well tolerated and increasing ingrowth of fibroblasts was seen over time in all subjects. The fibroblasts stained immunohistochemically for procollagen and von Willebrand factor, indicating neocollagenesis and angiogenesis within the scaffolds. The PUUR scaffold may be a suitable material to use as a template for dermal regeneration. ©2008 Landes Bioscience.

In the course of development of a new type of filler for the correction of small defects in soft tissues we studied macroporous gelatine spheres as culture substrate, transplantation vehicle, and biodegradable scaffold for guided regeneration of soft tissues in vivo. We injected intradermally in nude mice gelatine spheres that had either been preseeded with human fibroblasts or preadipocytes, or left unseeded. We compared the extent of regenerated tissue with that found after injections of saline or single-cell suspensions of human fibroblasts or preadipocytes. Routine histological examinations and immunohistochemical staining for von Willebrand factor (indicating neoangiogenesis) were made after 7, 21, and 56 days. Injected saline or single-cell suspensions had no effect. However, a quick and thorough tissue regeneration with developing neoangiogenesis was elicited by the gelatine spheres and the effect of spheres preseeded with preadipocytes surpassed the effect of spheres preseeded with fibroblasts, which in turn surpassed the effect of unseeded gelatine spheres. We suggest that minor soft tissue defects such as wrinkles or creases can be corrected by injection of naked macroporous gelatine spheres, whereas larger defects are best corrected by injection of macroporous gelatine spheres preseeded with fibroblasts, or preadipocytes, or both.

Correcting soft tissue defects by autologous fat grafting is a routine procedure in plastic surgery. Its efficacy and safety has been discussed extensively and several techniques of lipoinjection have been developed. However, one is bound to overcorrect by 30%-70% or need to repeat the procedure because of resorption of the transplant. The reasons are that many of the transplanted cells are already differentiated, and also that there is no nutritional support to the inner cell layers when they are transplanted as fragments. By culturing autologous adipocytes one can ensure that only non-differentiated, but committed, preadipocytes are transplanted and the procedure can be done in a way that ensures optimal nutritional support for the cells. In the present study we have compared our cell culture technique with two common clinical ways of processing liposuction material and found that (pre)adipocytes survive and proliferate significantly better in cell culture.

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.

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The apparent need of an autologous cell source for tissueengineering applications has led researchers to explore thepresence of cells with stem cell plasticity in several humantissues. Dermal fibroblasts (FBs) are easy to harvest, expandin vitro and store, rendering them plausible candidates forcell-based therapies. The aim of the present study was toobserve the effects of adipogenic, chondrogenic and osteogenicinduction media on the phenotype of human FBs.Human preadipocytes obtained from fat tissue have beenproposed as an adult stem cell source with suitable characteristics,and were used as control cells in regard to their differentiationpotential. Routine staining, immunohistochemicalanalysis and alkaline phosphatase assay were employed,in order to study the phenotypic shift. FBs were shown topossess multilineage potential, giving rise to fat-, cartilageandbone-like cells. To exclude contaminant progenitor cellsor 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 similardifferentiation potential as primary-culture FBs. The pres-ence of ‘stem-cell-specific’ surface antigens was analyzedusing flow cytometry. The results reveal that sccFBs haveseveral of the markers associated with cells exhibiting stemcell plasticity. The findings presented here are corroboratedby the findings of other groups, and suggest the use of humandermal FBs in cell-based therapies for the reconstructionof fat, cartilage and bone.

BACKGROUND: Thirst is a commonly experienced source of discomfort among patients receiving mechanical ventilation during intensive care. Dehydration, endotracheal tubes, electrolyte disturbances and the use of various medicines can contribute to the feeling of thirst. At present, the knowledge of nurses' perceptions of the feelings of thirst in mechanically ventilated patients are limited. PURPOSE: The purpose of this study was to describe registered and enrolled intensive care nurses' perceptions of the feelings of thirst in mechanically ventilated adult patients and to investigate the interventions to reduce thirst. METHOD: Ten registered intensive care nurses and 10 enrolled nurses, aged 32-59 years, were interviewed. They all had at least one year's experience in caring for adult, mechanically ventilated patients. The data were analysed using a phenomenographic methodology. FINDINGS: Four categories of perceptions among registered and enrolled nurses, regarding thirst in mechanically ventilated patients were identified: reasons for thirst, signs of thirst, preventing or reducing thirst and the perception that patients had no feelings of thirst. CONCLUSIONS: Registered and enrolled nurses wish to reduce sources of discomfort among their patients but they do not always realise that patients feel thirst and therefore relieving it has a low priority among staff. Relieving thirst is a human need that must be recognised in nursing education and on intensive care units.

The pathogenesis of trigger finger has generally been ascribed to primary changes in the pulley. Histological examination of the affected tendons has rarely been done. We studied biopsies from tendons of trigger fingers from 29 patients and compared these to biopsies from six intact tendons. We used a modified Movin score, which describes the tendinosis of the Achilles tendon. Trigger finger tendons had a high score (14.2; SD, 2.2) consistent with tendinosis, while the controls were almost normal (2.5; SD, 1.9). This suggests that the tendon is also affected, and that trigger finger is a form of tendinosis.

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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 andlt; 0.05) grade of re-epithelialisation than Dulbeccos modified Eagles 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 andlt; 0.05). Degradation of hyaluronic acid in the medium that contained 50% amniotic fluid gave significantly impaired re-epithelialisation (p andlt; 0.05) on culture days 3 and 7. In conclusion, amniotic fluid promotes accelerated re-epithelialisation and hyaluronic acid is an important ingredient.

Platelet rich plasma (PRP) has been investigated for its beneficial use in cartilage tissue engineering previously. Here, we address the effect of using PRP as encapsulating agent for gelatin-supported chondrocyte pellet culture in vitro. Furthermore, the concept of using dynamic centrifugation to stimulate extracellular matrix (ECM) formation of the chondrocytes is explored. Human articular chondrocytes were expanded on macroporous gelatin microcarriers in a spinner flask system. The cell-seeded microcarriers were allowed to form pellets with or without re-calcified citrated PRP, and subjected to dynamic centrifugation (f = 0.0125 Hz) for a total of 16 min every other day using a standard tabletop centrifuge. Three acceleration curves with differing top speeds (corresponding to 500 g, 1500 g and 3000 g respectively) were used for the experimental groups and unstimulated controls were set for comparison. Pellets were kept in culture for up to 12 weeks, paraffin embedded and sectioned for histological and immunohistochemical analysis. Results showed increasing numbers of cells and ECM with time, as well as a gradual degradation of the gelatin microcarriers, indicating ongoing cell proliferation and metabolism throughout the culture period. Cell densities and ECM formation were more pronounced in the PRP-containing groups after four weeks, although this difference diminished with time. At the last time point several cartilage markers were found in the produced ECM, however including the fibrocartilaginous marker collagen type I. Dynamic centrifugation did not visibly increase the ECM accumulation over the 12-week duration of this experiment, although non-conclusive indications of collagen fiber organization were seen in the two groups with the highest acceleration limits at the last time point.

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.

Biodegradable macroporous gelatin microcarriers fixed with blood-derived biodegradable glue are proposed as a delivery system for human autologous chondrocytes. Cell-seeded microcarriers were embedded in four biological glues - recalcified citrated whole blood, recalcified citrated plasma with or without platelets, and a commercially available fibrin glue - and cultured in an in vitro model under static conditions for 16 weeks. No differences could be verified between the commercial fibrin glue and the blood-derived alternatives. Five further experiments were conducted with recalcified citrated platelet-rich plasma alone as microcarrier sealant, using two different in vitro culture models and chondrocytes from three additional donors. The microcarriers supported chondrocyte adhesion and expansion as well as extracellular matrix (ECM) synthesis. Matrix formation occurred predominantly at sample surfaces under the static conditions. The presence of microcarriers proved essential for the glues to support the structural takeover of ECM proteins produced by the embedded chondrocytes, as exclusion of the microcarriers resulted in unstable structures that dissolved before matrix formation could occur. Immunohistochemical analysis revealed the presence of SOX-9- and S-100-positive chondrocytes as well as the production of aggrecan and collagen type I, but not of the cartilage-specific collagen type II. These results imply that blood-derived glues are indeed potentially applicable for encapsulation of chondrocyte-seeded microcarriers. However, the static in vitro models used in this study proved incapable of supporting cartilage formation throughout the engineered constructs.

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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.

During the past decades, several adult stem cell populations from a range of tissues have been characterized. Since principally all human cells contain the same genetic material, the specific gene expression profile determines the cell phenotype. The notion of terminally differentiated somatic cells being necessarily restricted to one phenotype has been challenged, and instead an inherent range of plasticity for any given cell type has been suggested. We have in previous work shown that normal human dermal fibroblasts have an inherent plasticity and can be induced to differentiate towards adipogenic, chondrogenic, endotheliogenic and osteogenic lineages when subjected to defined induction media. The aim of the present study was to further study the differentiation of human dermal fibroblasts on a gene expression level. This was achieved by employing genome wide expression analysis using microarray technology. Selected gene expression was also evaluated over time using real-time PCR. Several master regulatory genes important for lineage commitment, as well as phenotypically relevant genes, were found regulated in the respective induced cultures. The results obtained in this study strengthen previously published results showing an inherent ability for controllable phenotype alteration of human dermal fibroblasts in vitro. We conclude that adipogenic, chondrogenic, endotheliogenic and osteogenic induction results in novel phenotypes that show a genetic readiness for lineage-specific biological functionality.

Objective: To analyse the outcome of exercise testing and myocardial perfusion scintigraphy (MPS) in primary care patients with chest pain of new onset.

Design: Prospective, observational. Patients aged 20-79 years, consulting due to chest pain of new onset, were enrolled consecutively.

Setting: Three primary care health centres in south-eastern Sweden.

Patients: 191 patients where the possibility of stable ischaemic heart disease (IHD) could not be excluded by clinical examination alone.

Main outcome measures: Exercise test results, when equivocal completed by MPS.

Results: Exercise testing revealed IHD in 14 (7%) and no IHD in 134 (70%) of the cases. In 43 (23%) the exercise test results were equivocal. Thirty-nine of these patients underwent MPS, which showed IHD in 19 and no IHD in 20 cases. Among previously diagnosed cardiovascular disease and risk factors only atrial fibrillation in the male group showed a significant correlation to the outcome IHD.

Conclusion: Exercise testing and MPS are both useful when investigating chest pain patients in primary care.

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 micro-carriers. 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.

Tissue engineering applies principles of biology and engineering to the development of functional substitutes for damaged or lost tissues. Tools for the neo-generation of tissue in tissue engineering research include cells, biomaterials and soluble factors.

One main obstacle in tissue engineering is the limited availability of autologous tissue specific progenitor cells. This has led to interest into using autologous cells with stem cell plasticity. Bone marrow derived stem cells were the first adult stem cells shown to have multilineage potential. Since, several reports have been published indicating that cells from other tissues; fat, muscle, connective tissue e.g., possess potential to differentiate into lineages distinct from their tissue of origin.

The optimal cell type for use in tissue engineering applications should be easy to obtain, cultivate and store. The human dermal fibroblast is an easily accessible cell source, which after routine cell expansion gives a substantial cell yield from a small skin biopsy. Hence, the dermal fibroblast could be a suitable cell source for tissue engineering applications.The main aim of this thesis was to investigate the differentiation capacity of human dermal fibroblasts, and their possible applications in bone and cartilage tissue engineering applications.

Human dermal fibroblasts were shown to differentiate towards adipogenic, chondrogenic, and osteogenic phenotypes upon subjection to specific induction media. Differentiation was seen both in unrefined primary cultures and in clonal populations (paper I). Fibroblasts could be used to create three-dimensional cartilage- and bone like tissue when grown in vitro on gelatin microcarriers in combination with platelet rich plasma (paper II). 4 weeks after in vivo implantation of osteogenic induced fibroblasts into a fracture model in athymic rats, dense cell clusters and viable human cells were found in the gaps, but no visible healing of defects as determined by CT-scanning (paper III). After the induction towards adipogenic, chondrogenic, endotheliogenic and osteogenic lineages, gene expression analysis by microarray and quantitative real-time-PCR found several master regulatory genes important for lineage commitment, as well as phenotypically relevant genes regulated as compared to reference cultures (paper IV).

In conclusion, results obtained in this thesis suggest an inherent ability for controllable phenotype alteration of human dermal fibroblasts in vitro. We conclude that dermal fibroblasts could be induced towards adipogenic, chondrogenic, endotheliogenic or osteogenic novel phenotypes which suggest a genetic readiness of differentiated fibroblasts for lineage-specific biological functionality, indicating that human dermal fibroblasts might be a suitable cell source in tissue engineering applications.

Fracture healing is a complex event involving cells and growth factors. When healing is impaired it substantially affects quality of life and increases medical costs. To overcome difficulties with impaired bone healing several methods using biomaterials have been tested. Osteogenic biomaterials, which are scaffolds loaded with osteocompetent cells, have been proposed when the defect is large. In this study we wanted to investigate the potential of osteogenic induced human dermal fibroblasts grown on gelatin microcarriers combined with platelet rich plasma (PRP) in a femoral gap surgical model in athymic rats. The gaps were transplanted with one of the following six combinations: 1; NaCl, 2; PRP, 3; microcarriers + PRP, 4; human dermal fibroblasts on microcarriers + PRP, 5; human osteoblasts on microcarriers + PRP, 6; osteogenic induced human dermal fibroblasts on microcarriers + PRP. The gaps were analysed 4 weeks postoperatively with computer tomography, routine histological staining, fluorescence in situ hybridization (FISH) and polyclonal antibodies directed towards osteocalcin and osteonectin. Radiographs taken 4 weeks post surgery did not reveal callus in any of the groups. Gaps transplanted with osteogenic induced human dermal fibroblasts on microcarriers (group 6) contained dense cell clusters with large amounts of extracellular matrix. These cell clusters were not found in the other groups and stained highly positive for osteocalcin and osteonectin. FISH analysis revealed viable human cells in gaps filled with cell-seeded microcarriers confirming survival of transplanted cells. In conclusion osteogenic induced human dermal fibroblasts survive in this new niche and display bonelike structures in the gaps.

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.

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.

Organ dysfunction and failure are important for burned patients as they increase morbidity and mortality. Recent evidence has suggested that organ injuries are occurring earlier after burns, and are more common, than previously thought. In this study we have assessed the extent to which liver function, assessed by the plasma disappearance rate of indocyanine green (PDRICG), is affected in patients with severe burns. This is a prospective, descriptive exploratory study at a national burn centre. Consecutive adult patients with a total burned body surface area (TBSA%) of 20% or more, were examined prospectively by dynamic (PDRICG) and static liver function tests (plasma: bilirubin concentration, prothrombin complex, and alanine aminotransferase and alkaline phosphatase activities). Early liver dysfunction was common, as assessed by both dynamic (7 of 17) and static liver function tests (6-17 of 17). A regression model showed that changes in PDRICG were associated with age, TBSA%, plasma bilirubin concentration, plasma C-reactive protein concentration, and cardiac index. Persistent and advanced hepatic dysfunction was associated with mortality. The PDRICG seems to give a comprehensive assessment of liver function after major burns. Hepatic dysfunction seems to be as common as dysfunction in other organs. We interpret the recorded effects on liver function as part of a multiple organ dysfunction syndrome, primarily induced by the burn itself. However, this needs to be further investigated.

Background: Young women have been reported to be more likely to survive than men after severe trauma. Girls also have less inflammation and hypermetabolism after major burns. Yet burned women have been found to have a twofold greater risk of death than men. Our aim was to find out if there is a sex-related difference in mortality after thermal injury, particularly in the age group between 16 years and 49 years, when hormonal differences would be most influential. Methods: All patients admitted to the Linkoping University Hospital Burn Unit with thermal injuries during the years 1993-2008 were included and the variables percentage burned total body surface area (TBSA%), age, type of burn, mechanical ventilation, and year were included in a multiple regression (Poisson log) model. Results: Of 1,119 patients with thermal injury, 792 (71%) were men. Crude mortality was 5% among men, and 8% among women (p = 0.04). After adjustment for age and TBSA%, there was no correlation between mortality and sex, in any age group. Eight men and four women died in the group of young adults (16-49 years) in which TBSA% correlated with mortality (p andlt; 0.01) but age did not. Mortality was 14% (32 of 221) among the men and 23% (23 of 102) of women in the group of older adults (50 years and older), and both age and TBSA% correlated with mortality (p andlt; 0.001). Conclusions: There is no relevant sex-related difference in survival after thermal injury. The conclusion is, however, tempered by the few deaths, particularly among younger adults.

Conservative treatment of the cubital tunnel syndrome was evaluated in a randomised study of 70 patients with mild or moderate symptoms (Dellon, 1989). All patients were informed about the cause of symptoms and allocated to three groups: night splinting, nerve gliding and control. Evaluation consisted of Canadian Occupational Performance Measure, visual analogue pain scales, strength measurements and neurophysiological examination, before treatment and after six months. Fifty-seven patients were followed for six months. Fifty-one (89.5%) were improved at the follow-up. There were no significant differences between the groups in any of the recorded variables. Night splints and nerve gliding exercises did not add favourably. Routine neurophysiological examination seems unnecessary since 76% of the patients with typical symptoms had normal findings and 75% with pathological findings improved. Patients with mild or moderate symptoms have a good prognosis if they are informed of the causes of the condition and how to avoid provocation.

Disorders causing pain in the elbow region is a common problem and is one of the most frequent forms of work-related health problems. These conditions are thus of major importance from the public health point of view, as well as from that of the suffering individual. Both sensory and motor function may be impaired, particularly in cases where a nerve is involved, resulting in severely impaired hand function.

“Tennis elbow” (lateral epicondylalgia) has been found to be the second most frequently diagnosed musculoskeletal disorder of the upper extremity in the primary health-care setting. “Golfer’s elbow” (medial epicondylalgia) is not that commonly encountered. It has been stated that tennis or golfer’s elbow syndromes are self-limiting. Even so, clinical experience has shown that there are a few cases where symptoms have a painful and long-lasting course, resistant to many forms of therapy. The outcomes of frequently adopted management regimes for treatment of epicondylalgia or nerve entrapments around the elbow were examined in the following five studies:

I: A randomised, prospective study of 38 patients with tennis elbow (lateral epicondylalgia). Groups were assigned to eccentric exercises or stretching. Eccentric exercise gave somewhat better results.In a second part of the study, a 4-year follow-up of 127 patients who used eccentric exercises for tennis elbow was performed. Patients showed decreased pain and increased grip-strength after 3 months treatment. At the time of publication this study was the first to examine eccentric exercises for this condition.

II: A retrospective analysis of long-term results from 51 patients treated with surgical release of the common extensor origin because of “chronic tennis elbow”. Eighty-seven per cent of the patients rated themselves as completely recovered or improved.

III: A randomised, prospective study of 70 patients with ulnar neuropathy in the forearm (cubital tunnel syndrome). Groups assigned the commonly recommended elbow brace at night or nerve gliding exercises were compared with a control group (information only). The majority of patients improved considerably, both subjectively and objectively, after a 3 months period, regardless of group. The study thus showed the effectiveness of information and expectance, and that orthosis or nerve gliding exercises added nothing further to the result.

IV: A retrospective study of 205 patients treated with surgery for suspected nerve entrapment in the forearm. Followup, almost 4 years later, demonstrated a subjective improvement in two of three patients, but only 3% experienced complete relief of all symptoms.

V: A prospective long-term study on 20 patients with golfer’s elbow (medial epicondylalgia) treated with eccentric exercises over 3 months. The results showed decreased pain and increased gripstrength. This is the first study published on the management of this disorder with eccentric exercises.

Fifty-five elbows in 53 patients affected by lateral epicondylitis for more than a year were operated on with lateral extensor release. Fifty-one patients (53 elbows) were followed-up 90 months postoperatively by two independent observers using Verhaar's score and the subjective grading scheme described by Svernlöv and Adolfsson. According to Verhaar's score 26 (49%) were excellent or good and 27 (51%) fair or poor. Women had significantly worse results than men (p

Background: Eccentric exercises have been successfully used in the treatment of Achilles and patellar tendinopathies, as well as lateral epicondylalgia. No studies have explored the same treatment for medial epicondylalgia (golfer’s elbow).

Methods: Twenty consecutive adults with a clinical diagnosis of medial epicondylalgia were treated with eccentric exercise over 3 months in this prospective case series. The programme was based on home training. There were 11 women and nine men, with a mean age of 47 years, and a mean duration of symptoms of 19 months. At baseline, at 3 months and after a mean of 11 years, pain was assessed by three independent visual analogue scales (VAS) and grip strength using a Jamar dynamometer (Sammons Preston Inc., Jackson, MI, USA). At the 11-year follow-up, patients also reported their satisfaction on a 100-mm VAS and completed the Disabilities of Arm, Shoulder and Hand questionnaire.

Results: Pain decreased significantly in all measured variables at both assessment occasions (all p < 0.0001). Grip strength in the affected arm/hand increased significantly after 3 months (p = 0.009).

Discussion: Twelve weeks of eccentric training reduced pain and increased grip strength in patients with medial epicondylalgia. It is a safe, uncomplicated and cost-effective method, with only a minimum contribution from a physiotherapist.

The outcome of decompression for long-standing symptoms of nerve entrapments in the proximal forearm was investigated in a retrospective study of 205 patients using a self-assessment questionnaire, 45 months after the operation. The questionnaire consisted of visual analogue scale recordings of pre- and postoperative pain during rest and activity, questions about remaining symptoms and appreciation of the result and the Disabilities of Arm, Shoulder and Hand form (DASH). Altogether, 59% of the patients were satisfied, 58% considered themselves improved, and 3% as being entirely relieved of all symptoms. Pain decreased significantly (P = 0.001). There was a significant correlation between preoperative duration and patient perceived post-operative pain. Preoperative pain was a chief complaint, and pain reduction appears to be the principal gain of the operation. Although the majority of the patients benefited from the operation, a substantial proportion was not satisfied. There is apparently room for improvement of the diagnostic and surgical methods applied in this study.

Bakgrunn. Oslo universitetssykehus,Rikshospitalet har siden 1979 hattlandsfunksjon for behandling avpasienter med transseksualisme. Påårsbasis henvises 50–70 pasienter tilutredning, hvorav rundt 20 pasienterblir diagnostisert som transseksuelle.Årlig henvises omtrent 15 pasienter avdem som oppfyller kriterier for transseksualisme,og som er blitt endokrinologiskbehandlet, til kirurgisk intervensjon.I denne artikkelen beskrivesdiagnose og behandling av transseksualisme,med hovedvekt på kirurgi.Materiale og metode. Artikkelen erbasert på ikke-systematisk litteraturgjennomgangog egne kliniske og vitenskapeligeerfaringer.Resultater. Etter minimum ett årspsykiatrisk utredning og diagnostikk,og ett års påfølgende hormonellbehandling, vurderes pasientene i forholdtil kjønnskorrigerende kirurgi.Hos pasienter som konverteres framann til kvinne, vurderes brystforstørrendekirurgi hvis ikke hormonbehandlingalene har gitt tilfredsstillenderesultat. I tillegg er det aktuelt medgenital kirurgi hvor testikler og svamplegemenefjernes, og hvor neovaginaog neoklitoris konstrueres. For pasientersom konverteres fra kvinne tilmann, er det aktuelt å gjøre brystreduserendeinngrep, hysterektomi og salpingo-ooforektomi samt neopeniskonstruksjon.Ved god respons på hormonbehandlingkan klitoris rettes ut ogkonstrueres til en neopenis med ereksjonsmulighet(metoidioplastikk).Alternativt gjøres falloplastikk medbruk av lokalt vev fra lysken eller friemikrovaskulære lapper.Fortolkning. Kjønnskorrigerende kirurgived transseksualisme omfatterhovedsakelig konverterende operasjonerpå bryst og genitalia.