An abnormality in glucocorticoid receptor expression differentiates steroid responders from nonresponders in keloid disease.

BACKGROUND: Glucocorticoids (GCs) are first-line treatment for keloid disease (KD) but are limited by high incidence of resistance, recurrence and undesirable side-effects. Identifying patient responsiveness early could guide therapy. METHODS: Nineteen patients with KD were recruited at week 0 (before treatment) and received intralesional steroids. At weeks 0, 2 and 4, noninvasive imaging and biopsies were performed. Responsiveness was determined by clinical response and a significant reduction in vascular perfusion following steroid treatment, using full-field laser perfusion imaging (FLPI). Responsiveness was also evaluated using (i) spectrophotometric intracutaneous analysis to quantify changes in collagen and melanin and (ii) histology to identify changes in epidermal thickness and glycosaminoglycan (GAG) expression. Biopsies were used to quantify changes in glucocorticoid receptor (GR) expression using quantitative reverse transcriptase polymerase chain reaction, immunoblotting and immunohistochemistry. RESULTS: At week 2, the FLPI was used to separate patients into steroid responsive (n = 12) and nonresponsive groups (n = 7). All patients demonstrated a significant decrease in GAG at week 2 (P < 0.05). At week 4, responsive patients exhibited significant reduction in melanin, GAG, epidermal thickness (all P < 0.05) and a continued reduction in perfusion (P < 0.001) compared with nonresponders. Steroid-responsive patients had increased GR expression at baseline and showed autoregulation of GR compared with nonresponders, who showed no change in GR transcription or protein. CONCLUSIONS: This is the first demonstration that keloid response to steroids can be measured objectively using noninvasive imaging. FLPI is a potentially reliable tool to stratify KD responsiveness. Altered GR expression may be the mechanism gating therapeutic response.

Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy.

BACKGROUND: Overproduction of collagen and its abnormal assembly are hallmarks of keloid scars. Type I/III collagen ratios are altered in keloids compared with normal skin. Fibroblasts from different sites in keloid tissue, perilesional compared with intralesional and extralesional sites, show differential apoptosis and contraction. Additionally, early vs. later cell culture passages display differential collagen expression. We therefore hypothesize that keloid fibroblasts from the growing margin of the keloid express higher levels of collagen type I and III, and that collagen production is altered by extended cell culture passage. OBJECTIVES: (i) To measure collagen I and III at mRNA and protein levels quantitatively in keloid fibroblasts, growth media and tissue sections; and (ii) to perform tissue staining for collagen I and III expression in different lesional sites. METHODS: Keloid fibroblast cultures from intralesional, perilesional and extralesional sites (n = 8 separate keloid cases, yielding 64 biopsy samples) were established from passage 0 to passage 3. Collagen I and III mRNA was quantified using quantitative reverse transcription-polymerase chain reaction. We also measured the protein levels quantitatively by developing a highly specific and sensitive capture sandwich enzyme-linked immunosorbent assay. A novel in-cell Western blotting was carried out in addition to haematoxylin and eosin and Herovici staining on keloid tissue sections for collagen I and III. RESULTS: Collagen types I and III were significantly higher (P < 0·03) in fibroblasts from the growing margin (perilesional site) compared with extralesional and intralesional keloid biopsy sites. As the passage number increased, the amount of collagen I significantly (P < 0·05) decreased and the collagen I/III ratio also decreased. CONCLUSIONS: Our data show that cells from the growing margin of keloid scars have a higher production of collagen I and III compared with other lesional sites. Additionally, temporal extension of cell passage affects collagen production. Clinically these findings may influence selection and interpretation of extended cell passage and provide future direction for lesional site-specific therapy in keloid scars.

Characterization of hyaluronan and TSG-6 in skin scarring: differential distribution in keloid scars, normal scars and unscarred skin.

BACKGROUND: Hyaluronan (HA) is a major component of the extracellular matrix (ECM) with increased synthesis during tissue repair. Tumour necrosis factor-stimulated gene-6 (TSG-6) is known to catalyze the covalent transfer of heavy chains (HC1 and HC2) from inter-α-inhibitor (IαI) onto HA, and resultant HC•HA complexes have been implicated in physiological and pathological processes related to remodelling and inflammation. OBJECTIVE: The aims of this study were to determine the expression of HA, TSG-6 and the IαI polypeptides in unscarred skin, normal scars and keloid scars. METHODS: Formalin-fixed paraffin-embedded sections of unscarred skin, normal scars and keloid scars were prepared from patient samples collected during scar revision surgery. Haematoxylin and eosin, as well as immunofluorescent staining for HA, TSG-6 and the three polypeptide chains of IαI (i.e. HC1, HC2 and bikunin) were performed. RESULTS: All skin types stained positive for TSG-6, HC1, HC2 and bikunin, associated with keratinocytes, fibroblasts and skin appendages all in close proximity to HA. Keloid lesions showed altered HA organization patterns compared with unscarred skin and normal scars. TSG-6 staining was significantly more intense in the epidermis compared with the dermis of all sample types. There was a significant reduction in TSG-6 levels within keloid lesions compared with the dermis of unscarred skin (P=0.017). CONCLUSION: TSG-6 is expressed in unscarred skin, where its close association with HA and IαI could give rise to TSG-6-mediated HC•HA formation within this tissue. A reduction in the beneficial effects of TSG-6, caused by diminished protein levels in keloid lesions, could contribute to this abnormal scarring process.

Differential distribution of haematopoietic and nonhaematopoietic progenitor cells in intralesional and extralesional keloid: do keloid scars provide a niche for nonhaematopoietic mesenchymal stem cells?

BACKGROUND: Keloid disease is a benign, quasineoplastic disease with a high recurrence rate. Mesenchymal-like stem cells (MLSC) have previously been demonstrated in keloid scars and may be involved in keloid pathobiology. However, as these cells have only been examined by single colour fluorescence activated cell sorting (FACS) alone, they need to be more comprehensively characterized so that the key cellular contributors to keloid scars can be better understood. OBJECTIVES: To identify and characterize MLSC in intralesional and extralesional keloid, and to distinguish haematopoietic stem cells (HSC) from mesenchymal stem cells (MSC). METHODS AND PATIENTS: Punch biopsies from intralesional (top, middle and margin) and extralesional keloid scar sites were obtained from 17 patients with a keloid. Multicolour FACS analysis using antibodies specific for HSC markers CD34 and CD117 and MSC markers CD13, CD29, CD44 and CD90 was performed on freshly isolated keloid scar cells and on passage 0 and 1 cells. This was complemented by real-time quantitative polymerase chain reaction (PCR) and immunohistological in situ analyses. RESULTS: Keloid scars contain distinct subpopulations of MLSCs. Cells positive for CD13, CD29, CD44 and CD90 were found to be significantly (P<0·05) higher in the top and middle compartments of keloid scars compared with extralesional skin, where cells positive for CD34, CD90 and CD117 (representing HSCs) predominated. A unique population of CD34+ cells (cells positive for CD13, CD29, CD34, CD44 and CD90) were found in keloid scars and in extralesional skin. FACS and quantitative PCR analysis showed that many of the MSC markers were progressively downregulated and all HSC markers were lost during extended keloid fibroblast culture up to passage 1. CONCLUSION: We have found distinct subpopulations of haematopoietic and nonhaematopoietic MSC in keloid scars, whereby HSC accumulate extralesionally, while keloids seem to provide a niche environment for nonhaematopoietic MSC. Future therapy of keloids may have to target differentially both stem cell populations in order to deprive these tumours of their regenerative cell pools.

Treatment of symptomatic abnormal skin scars with electrical stimulation.

OBJECTIVE: To evaluate the effect of non-invasive biofeedback electrical stimulation on symptomatic abnormal skin scars. METHOD: Thirty patients with over 140 scars with long-term pain and itch were recruited into the study. Patients monitored the intensity of symptoms (pain and itching) on a numerical rating scale. In addition, a modified Manchester scar scale was used to objectively assess digital photographs of each scar in terms of colour, contour, distortion and texture, while a non-invasive spectrophotometric intracutaneous analysis was used to monitor the scars' physical characteristics. RESULTS: The electrical stimulation device resulted in a clinically and statistically significant (p < 0.05) reduction of symptoms and scar scores. Pain and itch scores were both reduced to a median score of 0 by 2 months, from a baseline of 7 and 6 respectively. Scar scores were reduced from a baseline of 14 to a median score of 11 by 2 months. CONCLUSION: These results give a preliminary indication of the potential role of non-invasive biofeedback electrical stimulation in the management of chronic scar pain and itch. However, further large scale controlled studies are warranted to elucidate its overall efficacy and mechanistic action. CONFLICT OF INTEREST: Funding was provided from Fenzian Ltd for this study.

Mobilization of endothelial progenitor cells into the circulation in burned patients.

BACKGROUND: Bone marrow-derived endothelial progenitor cells (EPCs) have been detected in the peripheral blood of patients following thermal injury. EPCs migrate to sites of active neovascularization in response to mediators released after trauma, contributing to wound healing. The aim was to characterize levels and kinetics of EPCs in burned patients, then relate these to key mobilizing factors, vascular endothelial growth factor (VEGF) and the chemokine (C-X-C motif) ligand 12 (CXCL 12), and compare them with those in healthy subjects. METHODS: The study included 19 adult patients with superficial or full-thickness burns and 50 blood donor volunteer controls. EPCs, identified by cell surface markers CD45(dim/-), CD133+, CD144+ and VEGF receptor 2, were quantified by four-colour flow cytometry. Plasma VEGF and CXCL12 were measured using enzyme-linked immunosorbent assay. RESULTS: Burned patients showed a rapid rise in EPC levels within 24 h, a ninefold increase compared with controls, returning to basal levels by 72 h. Body surface area burned correlated strongly with the degree of mobilization. EPC levels correlated significantly with rises in plasma VEGF and CXCL12. CONCLUSION: Thermal injury induced a rapid rise in EPCs that was proportional to the extent of the burn and significantly correlated with levels of angiogenic cytokines. Such cytokines may be used to stimulate EPCs as a future therapeutic target in burned patients.

What triggers in trigger finger? The flexor tendons at the flexor digitorum superficialis bifurcation.

To define the role of the flexor tendons in trigger finger, a high-resolution ultrasound examination was performed in 20 trigger fingers and 20 normal contralateral digits in three digital postures: full extension, mid-flexion and near-full flexion. Precise measurements of diameter and cross-sectional area of the combined tendon mass were recorded at five clearly defined locations: summit of the metacarpal head, proximal lip of the proximal phalanx (PP) and at 1/8, 1/4 and 1/2 length of the PP. In the normal tendons, there was an anatomical thickening, not previously appreciated at 1/4 length PP, in the region of the FDS bifurcation. This anatomical region moved proximally on finger flexion to the A1 pulley. In trigger fingers, the flexor tendons had greater diameter (sagittal view) and cross-sectional area than the normal side at all locations (p < 0.01, p < 0.001), with an even greater increase in diameter in the FDS bifurcation area (p < 0.001). Trigger fingers also had thicker A1 pulleys (p < 0.001). Triggering occurs on flexing the finger when the enlarged combined flexor tendon mass at the specific anatomical region of the FDS bifurcation impacts on the thickened A1 pulley, resisting its excursion.

The cellular effect of a single interrupted suture on tendon.

The effects on cell and matrix morphology of a single interrupted suture are described in rabbit (vascular) and mouse (avascular) digital flexor tendons. This model of tendon injury is reproducible and suitable for quantitative histological analysis. Tendons analysed at day 1, 3, 5, 7 and 14 after wounding demonstrated a well-demarcated "acellular zone" around the suture within 24 hours and persisting over 14 days. The placement of an untied suture in tendon did not produce this effect but tying and releasing the tied knot did. The rapidity of onset suggests that cells move from the zone of injury into less mechanically strained tissue. The acellular zone was apparent in rabbit hind paw flexor tendon which is vascularised and the corresponding tendon in mouse which has no intrinsic blood vessels. This phenomenon highlights biological events that must be considered in parallel with the current trend for multistrand locking flexor tendon suture repairs.

A culture force monitor for measurement of contraction forces generated in human dermal fibroblast cultures: evidence for cell-matrix mechanical signalling.

Non-contractile cells are able to exert 'tensional' forces on collagen substrate. Although such forces have been implicated in contraction during tissue repair their importance and mechanism of action are difficult to assess without quantitative data. Currently, the most widely used model is not a direct measure of force and has serious deficiencies as a model of wound contraction. A Culture Force Monitor, CFM, has been developed in this study, to measure directly the forces generated by fibroblasts and other cell types in culture. Under model conditions, a peak force of 1 x 10(-10) Newton/cell was generated (assuming participation of all cells) over a range of cell densities. Contraction of a collagen gel over 2 days was in 3 phases; initial contraction, linear increase and equilibrium. The final, maximum force was produced at the equilibrium phase (24 h) and balanced the restraining force of the CFM. Cell force responses during the initial and linear phases (corresponding to cell attachment) indicated that contraction of the cells responded in a rapid and subtle manner to changes in the mechanical properties of their substrate.

Enhanced early sensory outcome after nerve repair as a result of immediate post-operative re-learning: a randomized controlled trial.

We assessed the use of guided plasticity training to improve the outcome in the first 6 months after nerve repair. In a multicentre randomized controlled trial, 37 adults with median or ulnar nerve repair at the distal forearm were randomized to intervention, starting the first week after surgery with sensory and motor re-learning using mirror visual feedback and observation of touch, or to a control group with re-learning starting when reinnervation could be detected. The primary outcome at 3 and 6 months post-operatively was discriminative touch (shape texture identification test, part of the Rosen score). At 6 months, discriminative touch was significantly better in the early intervention group. Improvement of discriminative touch between 3 and 6 months was also significantly greater in that group. There were no significant differences in motor function, pain or in the total score. We conclude that early re-learning using guided plasticity may have a potential to improve the outcomes after nerve repair. LEVEL OF EVIDENCE II.