Mannose-6-phosphate facilitates early peripheral nerve regeneration in thy-1-YFP-H mice.

The formation of scar tissue following nerve injury has been shown to adversely affect nerve regeneration and evidence suggests that mannose-6-phosphate (M6P), a potential scar reducing agent that affects transforming growth factor (TGF)-ß activation, may enhance nerve regeneration. In this study we utilized thy-1-YFP-H mice - a transgenic strain expressing yellow fluorescent protein (YFP) within a subset of axons - to enable visual analysis of axons regenerating through a nerve graft. Using this strain of mouse we have developed analysis techniques to visualize and quantify regeneration of individual axons across the injury site following the application of either M6P or vehicle to the site of nerve injury. No significant differences were found in the proportion of axons regenerating through the graft between M6P- and vehicle-treated grafts at any point along the graft length. Maximal sprouting occurred at 1.0mm from the proximal graft ending in both groups. The maximum change in sprouting levels for both treatment groups occurred between the graft start and 0.5-mm interval for both treatment groups. The difference between repair groups was significant at this point with a greater increase seen in the vehicle group than the M6P group. The average length of axons regenerating across the initial graft entry was significantly shorter in M6P- than in vehicle-treated grafts, indicating that they encountered less impedance. Application of M6P appears to reduce the disruption of regenerating axons and may therefore facilitate quicker recovery; this is likely to result from altered scar tissue formation in M6P grafts in the early stages of recovery. This study also establishes the usefulness of our methods of analysis using the thy-1-YFP-H mouse strain to visualize and quantify regeneration at the level of the individual axon.

Randomized phase II clinical trial of avotermin versus placebo for scar improvement.

BACKGROUND: Scarring is a major problem following skin injury. In early clinical trials, transforming growth factor ß3 (avotermin) improved scar appearance. The aim of this study was to determine whether an injection of avotermin at the time of wound closure is effective in improving scar appearance. METHODS: Study RN1001-0042, a double-blind, randomized, within-patient, placebo-controlled trial, investigated the efficacy and safety of four doses of avotermin given once. Patients undergoing bilateral surgery to remove varicose leg veins by saphenofemoral ligation and long saphenous vein stripping were enrolled at 20 European centres. A total of 156 patients were randomized to receive one of four doses of avotermin (5, 50, 200 or 500 ng per 100 µl, at 100 µl per linear cm of wound margin), administered by intradermal injection to the groin and distal wound margins of one leg; placebo was administered to the other leg. Scar appearance was evaluated by an independent panel of lay people (lay panel), investigators and patients. The primary efficacy variable was lay panel Total Scar Score (ToScar), derived from visual analogue scale scores for groin scars between 6 weeks and 7 months. RESULTS: Avotermin 500 ng significantly improved groin scar appearance compared with placebo (mean lay panel ToScar difference 16·49 mm; P = 0·036). CONCLUSION: Avotermin 500 ng per 100 µl per linear cm of wound margin given once is well tolerated and significantly improves scar appearance.

The cell biology of suturing tendons.

Trauma by suturing tendon form areas devoid of cells termed "acellular zones" in the matrix. This study aimed to characterise the cellular insult of suturing and acellular zone formation in mouse tendon. Acellular zone formation was evaluated using single grasping sutures placed using flexor tendons with time lapse cell viability imaging for a period of 12h. Both tension and injury were required to induce cell death and cell movement in the formation of the acellular zone. DNA fragmentation studies and transmission electron microscopy indicated that cells necrosed. Parallel in vivo studies showed that cell-to-cell contacts were disrupted following grasping by the suture in tensioned tendon. Without tension, cell death was lessened and cell-to-cell contacts remained intact. Quantitative immunohistochemistry and 3D cellular profile mapping of wound healing markers over a one year time course showed that acellular zones arise rapidly and showed no evidence of healing whilst the wound healing response occurred in the surrounding tissues. The acellular zones were also evident in a standard modified "Kessler" clinical repair. In conclusion, the suture repair of injured tendons produces acellular zones, which may potentially cause early tendon failure.

TGF-beta3 and cancer: a review.

With the development of growth factors and growth factor modulators as therapeutics for a range of disorders, it is prudent to consider whether modulating the growth factor profile in a tissue can influence tumour initiation or progression. As recombinant human TGF-beta3 (avotermin) is being developed for the improvement of scarring in the skin it is important to understand the role, if any, of this cytokine in tumour progression. Elevated levels of TGF-beta3 expression detected in late-stage tumours have linked this cytokine with tumourigenesis, although functional data to support a causative role are lacking. While it has proved tempting for researchers to interpret a 'correlation' as a 'cause' of disease, what has often been overlooked is the normal biological role of TGF-beta3 in processes that are often subverted in tumourigenesis. Clarifying the role of this cytokine is complicated by inappropriate extrapolation of the data relating to TGF-beta1 in tumourigenesis, despite marked differences in biology between the TGF-beta isoforms. Indeed, published studies have indicated that TGF-beta3 may actually play a protective role against tumourigenesis in a range of tissues including the skin, breast, oral and gastric mucosa. Based on currently available data it is reasonable to hypothesize that administration of acute low doses of exogenous TGF-beta3 is unlikely to influence tumour initiation or progression.

Current scales for assessing human scarring: a review.

Patients can have wide-ranging problems related to scars, in terms of cosmesis, function, symptoms, psychological problems and overall quality of life issues. A range of treatments have been recommended for problematic scarring, however it has been acknowledged that the evidence base for most of the recommendations for scar therapy is limited, with few studies using validated measures of scar assessment in generating data. This review critically evaluates the subjective scar assessment scales developed to date and provides an insight into developments required in this area for the future. The principles of psychometric theory are discussed as a means of developing reliable and valid outcome measures and these are also applicable for measuring outcomes in other fields of plastic surgery research.

Skin stem and progenitor cells: using regeneration as a tissue-engineering strategy.

Cell plasticity and mesenchymal-epithelial interactions are regarded as a hallmark of embryonic development and are not believed to occur extensively in the adult. Recently, adult mesenchymal stem cells were reported to differentiate in culture into a variety of mature cell types, including epithelial cells. Progress in stem and progenitor cell biology and recognition of the unique properties of such cells may enable intelligent bioengineering design of replacement skin which allows regeneration to occur in vivo. Ideally, a scaffold-free environment which stimulates skin stem cells in situ to initiate cell signals that result in regeneration rather than scar formation is required. Various skin progenitor cell types are considered along with the signalling cascades that they affect. We also discuss a mammalian model of scar-free regeneration. Many of these mechanisms, if fully understood, could be harnessed after injury to perfectly restore the skin.

Perivascular cells in a skin graft are rapidly repopulated by host cells.

Survival of grafted tissues is dependent upon revascularisation. This study investigated revascularisation in a murine skin graft model, using two methods. The first involved 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) labelling of the wound bed, prior to replacing the skin graft, to allow tracking of host cells into the grafts. At time points between day 3 and day 14 post-surgery, DiI-labelled cells which had tracked into the grafts, were found to co-localise with CD31 positive endothelial cells and patent perfused vessels (fluorescein isothiocyanate (FITC)-dextran perfusion), to show possible association with the vasculature. To further differentiate between graft and host-derived cells, C57BL/6 wild-type grafts were placed on enhanced-green fluorescent protein (e-GFP) transgenic mouse hosts, and at set times post-grafting examined using confocal microscopy. Patent vessels were found at all depths of the graft by day 3. Host (DiI- or GFP-positive) cells were predominantly co-localised with graft vessels in grafts from day 3 onwards, with a similar morphology to control skin. Significantly more GFP labelled host cells were visualised in the superficial dermis at day 5 compared to day 3. Initial restoration of circulation appears to be due to linkage between existing graft and bed vessels, followed by an influx of host cells with a definite perivascular distribution. These findings have implications for skin autografts and tissue engineered skin substitutes.

The reinnervation pattern of wounds and scars may explain their sensory symptoms.

Anaesthesia, pruritus and pain are common in cutaneous scars. The reinnervation pattern of healing wounds and scars might help to explain these symptoms, as sensory neurotransmitters are known to be mediators of inflammation and healing. We quantified the regeneration patterns of blood vessels and nerves in excisional skin wounds as they matured into scars. Mice underwent 1cm(2) full thickness skin excisions. Wounds were harvested between five and 84 days. Sections underwent immunohistochemical staining for protein gene product 9.5 (PGP9.5) a pan-neuronal marker, and the sensory neuropeptides calcitonin gene related peptide (CGRP) and substance P (SP). The endothelial marker von Willebrand factor (VWF) was used to allow co-localisation and quantification of blood vessels. Nerve fibre density was quantified at multiple sites within wounds. There was no difference in the reinnervation/revascularisation pattern between peripheral and central sites. The density of PGP9.5, CGRP, SP and VWF peaked between 14 and 42 days, and levels of PGP9.5, CGRP and VWF all decreased to approximately those found in unwounded skin by 84 days (mature scar). SP levels, however, remained elevated at approximately twice the density found in unwounded skin. Increased densities of SP and CGRP in healing wounds could explain the unpleasant sensory symptoms of healing wounds.

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.

Spatial distribution of mast cells in chronic venous leg ulcers.

Chronic venous leg ulcers (CVUs) show chronic inflammation but different pathological changes occur in different parts of the ulcer. There is a lack of re-epithelialisation and defective matrix deposition in the ulcer base but epidermal hyperproliferation and increased matrix deposition in the surrounding skin. The role of mast cells in wound healing, inflammation, fibrosis and epidermal hyperproliferation has been extensively studied but less is known about their role in CVUs. In the present study, we investigated the distribution of mast cells in CVUs with specific consideration of the differences between the ulcer base and the skin surrounding the ulcer. Both histochemical and immunohistological methods were used to detect the mast cell marker tryptase in frozen sections of CVU biopsies. Mast cells were counted in the dermis of normal skin, in the ulcer base and in the skin surrounding the ulcer. Double immunofluorescence staining was used to study the location of mast cells in relation to blood vessels. In normal skin few mast cells were seen in the dermis but none in the epidermis. However in CVUs there was a significant increase in intact and degranulated mast cells in the surrounding skin and ulcer edge (184 per field, p<0.003) of CVUs and a significant reduction in the ulcer base (20.5 per field p<0.05) in comparison to normal skin (61 per field). In CVUs mast cells showed a characteristic location near the epithelial basement membrane whilst mast cell granules and phantom cells (mast cells devoid of granules) were predominantly seen in the epidermis. In the dermis, mast cells were seen associated with blood vessels. The marked increase in mast cells in the surrounding skin of CVUs and depletion of mast cells in the ulcer base could implicate mast cell mediators in the pathological changes in CVUs particularly in the epidermal and vascular changes occurring in the surrounding skin.

Genetic susceptibility to keloid disease: mutation screening of the TGFbeta3 gene.

Keloid disease (KD) is a fibroproliferative dermal tumour of unknown aetiology. The increased familial clustering in KD, its increased prevalence in certain races and its presence in identical twins suggest a strong genetic predisposition to keloid formation. Transforming growth factor beta isoforms (TGFbeta) play a central role in wound healing and fibrosis and have been implicated in KD pathogenesis. Recent data has suggested that TGFbeta(3) has an important role in scar formation. There is little known about the genetic variation present within the TGFbeta(3) gene, which contains seven exons and six introns spanning 43,000 base pairs of the human genome. Exons one to seven and the promoter region (1000 bp upstream from exon 1 in the 5'-flanking regions) were screened in 95 Caucasian KD cases and 95 Caucasian controls for the presence of novel mutations using a high throughput DHPLC mutation detection technology. There were no mutations identified in any of the exonic regions, however, multiple nondisease associated mutations were found in the promoter region of the TGFbeta(3) gene. These data demonstrate that there is no association between the exonic and promoter regions of TGFbeta(3) gene and keloid scarring in our cohort of Caucasian patients.

Mitochondrial mutation detection using enhanced multiplex denaturing high-performance liquid chromatography.

In this study, we investigated the presence of mutations within the mitochondrial genome in 40 Caucasian subjects using an enhanced multiplex denaturing high-performance liquid chromatography (DHPLC) approach. The enhanced DHPLC approach has increased sensitivity and throughput, and reduced analysis time per individual sample compared to conventional methods. This technique involved amplifying the mitochondrial genome in 18 fragments ranging in size from 300 to 2000 bp using a novel proofreading polymerase (Optimase, Transgenomic Inc., Omaha, NE) with a low misincorporation rate. Fourteen of these fragments underwent subsequent restriction digestion using a combination of five restriction enzymes to enable multiplex DHPLC analysis; the remaining four underwent conventional DHPLC. Using this complete mitochondrial genome-screening approach, we confirmed a number of previously reported mutations and additionally identified a large number of novel mutations using an enhanced DHPLC technique.

Harnessing wound healing and regeneration for tissue engineering.

Biomedical science has made major advances in understanding how cells grow into functioning tissue and the signalling mechanisms used to achieve this are slowly being dissected. Tissue engineering is the application of that knowledge to the building or repairing of organs, including skin, the largest organ in the body. Generally, engineered tissue is a combination of living cells and a supporting matrix. Besides serving as burn coverings, engineered skin substitutes can help patients with diabetic foot ulcers. Today, most of these ulcers are treated with an approach that includes antibiotics, glucose control, special shoes and frequent cleaning and bandaging. The results of such treatments are often disappointing and ineffectual, and scarring remains a major problem, mechanically, cosmetically and psychologically. Within our group we are attempting to address this by investigating novel approaches to skin tissue engineering. We are identifying novel therapeutic manipulations to improve the degree of integration between a tissue engineered dermal construct and the host by both molecular manipulation of growth factors but also by understanding and harnessing mechanisms of regenerative biology. For the purpose of this summary, we will concentrate primarily on the latter of these two approaches in that we have identified a novel mouse mutant that completely and perfectly regenerates skin and cartilaginous components following ear injury. This experimental animal will allow us to characterize not only novel genes involved in the regeneration process but also to utilize cells from such animals in artificial skin equivalents to assess their behaviour compared with normal cells. This approach should allow us to create a tissue-engineered substitute, which more closely resembles the normal regional microanatomy and physiology of the skin, allowing better integration to the host with minimal or no scarring.

Keloid disease: clinical relevance of single versus multiple site scars.

Much of our current understanding of keloid disease (KD) is based on anecdote rather than objective observation and statistical analysis. To elucidate further the aetiology of KD, we compared the profiles of patients with single versus multiple anatomical site keloid scars. We studied the clinical characteristics of 211 cases of keloid scarring, 137 (65%) females and 74 (35%) males. There were 122 cases with scars in single anatomical site and 89 cases with 369 scars in multiple anatomical sites entered into the study. Patients were of Afrocaribbean origin that presented to the department of Plastic and Reconstructive Surgery at the University of West Indies in Kingston, Jamaica. A total number of 491 keloid scars (single and multiple sites) were evaluated in the study. Data were collected on multiple parameters. The association of age of onset, anatomical area, cause of scarring, sex of the patient, presence or absence of family and medical history in patients with single as opposed to multiple site keloid scars were examined in detail and statistically evaluated. The formation of keloid scars in multiple anatomical sites was found to be statistically significant in that it was more common in younger age groups (p < 0.001) and in females (p < 0.001). Previous medical history, including other fibrotic disorders, was not statistically associated with development of keloid scars in multiple anatomical sites (p > 0.05). More than 50% (111) of all keloid cases had a positive family history of keloid scarring, and family history was strongly associated with the formation of keloid scars in multiple sites as opposed to a single anatomical site (p < 0.002). We conclude that in this particular study group female sex, younger age at presentation and the presence of a positive family history were associated with the development of keloid scars in multiple anatomical sites in Afrocaribbean individuals. This knowledge further emphasizes the need for genetic studies in KD, which may lead to better diagnostic and therapeutic regimes.

Description of site-specific morphology of keloid phenotypes in an Afrocaribbean population.

By examining the keloid scars of 211 Afrocaribbean patients presenting to the Plastic Surgery unit in Kingston, Jamaica, we have described site-specific morphologies of scarring; keloid disease is not a homogenous biological entity. All cases conformed to clinical criteria for diagnosis of keloid scarring: 369 keloid scars were present in 137 females (2-83 years; mean 29.6 years; SD+/-14.9 years) and 74 males (5-90 years, mean 29.5 years; SD+/-15.0 years). Morphologies were specific to each anatomical site: trunk scars (n=45,12.1%) were geometrically shaped with clear margins or irregular in outline, surface and margin; back single scars were well-demarcated botryoid but multiple scars were butterfly-shaped, spheroidal and irregular; chest scars (n=72,20.1%) were butterfly or nonbutterfly shaped found most commonly in the midsternal line; upper limb scars (n=57,15.3%) mostly in the deltoid region (propeller shaped) or elsewhere nodular, linear to irregular; ear (n=85,23%) commonest site being the lobe, having reniform to bulbous shape; face and neck (n=60,16.2%) scars were firm nodular to hard; posterior auricular scars were either horizontal and oblong-shaped or vertical and reniform in outline; scalp scars (n=11,2.8%) were commonest in the occipital area varying from small papules to large plaques; lower limb scars (n=39,10.5%) varied from propeller, butterfly, petalloid to dum-bell-shaped. Three plantar and eight pubic keloids were rare findings. Recognition of different morphological phenotypes is necessary in understanding genotypic predisposition and aiding diagnosis, treatment and prognosis of keloid scars.

Genetic susceptibility to keloid disease: transforming growth factor beta receptor gene polymorphisms are not associated with keloid disease.

Keloid disease (KD) is an abnormal form of scarring with a familial predisposition. Genetic studies have yet to identify the genes involved in KD. Transforming growth factor beta (TGF-beta) has multiple cellular activities including cellular proliferation, differentiation and extracellular matrix production. TGF-beta family members such as TGF-beta(1) and TGF-beta(2) are known to be involved in KD formation. However, we previously demonstrated a lack of association between common TGF-beta(1) and TGF-beta(2) polymorphisms and KD. Other studies have implicated TGF-beta receptors in KD pathogenesis. TGF-beta receptors were therefore selected as candidate-susceptibility genes for this condition. Single-nucleotide polymorphisms (SNPs) in TGF-beta receptors I, II and III (TGF-betaRI, TGF-betaRII and TGF-betaRIII) were identified and investigated for association with the risk of developing KD. A polymerase chain reaction-restriction fragment length polymorphism method was used for genotyping novel and known TGF-beta receptor polymorphisms. DNA samples from 92 KD cases and 181 controls were examined. There were no statistically significant differences in genotype or allele frequency distributions between cases and controls for the TGF-beta receptor SNPs. Therefore, these TGF-beta receptor polymorphisms are unlikely to be associated with keloid scarring. It is possible that other SNPs in other TGF-beta family members are associated with KD. To our knowledge, this is the first report of a case-control association study with KD and TGF-beta receptor gene polymorphisms.

Rapid denaturing high-performance liquid chromatography (DHPLC) for mutation scanning of the transforming growth factor beta3 gene using a novel proof-reading polymerase.

We have utilized a novel variation on the conventional denaturing high-performance liquid chromatography (DHPLC) technology, which we term rapid DHPLC, combining changes in instrumentation, cartridge technology and analysis conditions to enable significant increases in throughput to be achieved. In addition, the use of a novel proof-reading polymerase for sample amplification with a low misincorporation rate enables simplification of the DHPLC patterns and hence enhanced mutation detection recognition. This scheme for increasing DHPLC throughput has been tested by scanning the transforming growth factor (TGF) beta3 gene for the presence of mutations for which there is limited published or on-line data available regarding the presence of gene polymorphisms. TGFbeta isoforms have multiple roles in cell division, growth, proliferation, transformation and differentiation. TGFbeta3 is a TGFbeta cytokine isoform, and has an important role in embryogenesis, cell differentiation and wound healing. The TGFbeta3 gene consists of seven exons and six introns spanning 43 000 bp of the human genome on chromosome 14q23-24. The rapid DHPLC approach enabled scanning of all seven exons and part of the promoter region (1000 bp upstream from exon 1 in the 5'-flanking regions) of the TGFbeta3 gene in 95 Caucasian individuals in only 8 days, in comparison to the 17 days it would have previously taken. Mutations were clearly identified in the promoter region of the TGFbeta3 gene but were absent from the exonic regions. Understanding the genetic variations affecting the TGFbeta3 gene is important as this molecule has multiple regulatory functions on a variety of cell types.

Genetic susceptibility to Dupuytren's disease: transforming growth factor beta receptor (TGFbetaR) gene polymorphisms and Dupuytren's disease.

Dupuytren's disease (DD) is a benign fibroproliferative disease of unknown cause. It is a familial condition that commonly affects Caucasians. Genetic studies have yet to identify the genes involved in DD. Transforming growth factor beta (TGFbeta) family members are multifunctional; some play a central role in wound healing and fibrosis. Previous studies have implicated TGFbeta cytokines and receptors in DD. In the light of this evidence, TGFbeta receptors represent candidate susceptibility genes for this condition. In this study, we investigated the association of single nucleotide polymorphisms (SNPs) in TGFbeta receptors one, two and three (TGFbetaRI, RII and RIII) with the risk of DD formation. A polymerase chain reaction-restriction fragment length polymorphism method was used for genotyping novel and known TGFbeta receptor polymorphisms. DNA samples from 183 DD patients and 181 controls were examined. There was a statistically significant difference (p<0.05) in genotype frequency distributions between cases and controls for TGFbetaRI polymorphisms in the recessive model. However, there were no significant difference in genotype or allele frequency distributions between cases and controls for the TGFbetaRII and TGFbetaRIII SNPs.

Novel single nucleotide polymorphisms in the 3'-UTR of the TGFbetaRI and TGFbetaRIII genes.

Transforming growth factor beta (TGFbeta) family members are multifunctional cytokines that play a key role in cellular growth, proliferation and differentiation. Transmembrane signalling by TGFbeta occurs via a complex of the serine/threonine kinases TGFbeta type 1 (TGFbetaRI), type 2 (TGFbetaRII), and type 3 (TGFbetaRIII) receptors. Previous studies have implicated TGFbeta receptors (TGFbetaR) in a variety of important hereditary clinical disorders. Mutations of the TGFbetaR genes have been observed in several human cancers. The aim of this study was to identify and confirm novel single nucleotide polymorphisms (SNPs) in TGFbetaRI and RIII and to determine the relative allele and genotype frequencies of these SNPs. SNPs were identified from the examination of sequence alignments held in databases and were confirmed by DNA sequencing. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was devised for genotyping TGFbeta receptor polymorphisms. DNA samples from 91 controls were examined. The observed heterozygosities of TGFbetaRI and TGFbetaRIII gene polymorphisms in the control population were 43 and 33%, respectively, suggesting these SNPs could be useful markers in disease association studies.