Recommendations on clinical proof of efficacy for potential scar prevention and reduction therapies.

Cutaneous scarring is an enormous medical problem with approximately 100 million patients acquiring scars each year. Scar prevention/reduction represents a significant, and largely unmet, clinical need. Research into the prophylactic modulation of scar outcome differs from research into other disease processes as the scar is not present at the start of the study; measurements of changes from baseline are impossible. Final scar morphology is influenced by many variables. A fundamental principle that should be observed in the prospective evaluation of scar prevention/reduction therapies is that, if left untreated, wounds in treatment and control groups should have healed with identical scars. Observation of this principle will allow the detection of true treatment effects. The many variables that influence scar morphology mean that the evaluation of potential pharmaceutical products for this indication favors the use of self-controlled designs in clinical trials. In this article, we review variables that affect scar morphology and recommend the self-controlled design for clinical trials aiming to establish proof of efficacy of scar prevention and reduction pharmaceuticals. With no pharmaceutical products currently licensed for this indication, this represents a new therapeutic area. The principles discussed will also have direct relevance to the wider fields of wound healing and regenerative medicine.

Transforming growth factor-beta1 stimulation enhances Dupuytren's fibroblast contraction in response to uniaxial mechanical load within a 3-dimensional collagen gel.

PURPOSE: A function of fibroblasts is the generation of cytomechanical force within their surrounding extracellular matrix. Abnormalities in force generation may be the cause of many pathologic conditions including scarring, and some fibroproliferative disorders such as Dupuytren's disease, which is the focus of this report. METHODS: This work investigated the cytomechanical responses of Dupuytren's-derived fibroblasts to externally applied mechanical force using a culture force monitor model, with and without stimulation with the fibrosis-linked cytokine, transforming growth factor-beta1 (TGF-beta1). We compared these responses with cytomechanical responses of fibroblasts derived from the transverse carpal ligament. RESULTS: Dupuytren's fibroblasts display a significantly greater ability to contract a collagen matrix compared with control fibroblasts, with a maximum generated force of 131 dynes (p < .001). These cells did not exhibit a characteristic plateau phase in the contraction, which indicates a delay in achieving tensional homeostasis from Dupuytren's-derived cells. After being subjected to uniaxial overload and underload, Dupuytren's fibroblasts responded by increased force generation, whereas control fibroblasts responded by a reduction in force in response to an overload, and contraction in response to an underload. These changes were exacerbated by the addition of the profibrotic factor TGF-beta1, with a significant increase in generated force for all cell types, in particular during the early phase of fibroblast attachment and contraction, and a positive contraction gradient in response to overloading forces. CONCLUSIONS: These data suggest that cells derived from this fibrotic disease display characteristic abnormalities in force generation profiles. Their default response to loading or underloading is contraction, or increased force generation. This work highlights the role of TGF-beta1 as a mechano-transduction cytokine, which has an influence on the early phase cell of force generation, as well as a role in mechanical responses of cells to external mechanical stimuli. This, in turn, may influence the progression of Dupuytren's disease and the high rates of recurrence seen postoperatively.

Purinergic receptors are part of a functional signaling system for proliferation and differentiation of human epidermal keratinocytes.

We investigated the expression of P2X5, P2X7, P2Y1 and P2Y2 receptor subtypes in normal human epidermis and in relation to markers of proliferation (PCNA and Ki-67), keratinocyte differentiation (cytokeratin K10 and involucrin) and markers of apoptosis (TUNEL and anticaspase-3). Using immunohistochemistry, we showed that each of the four receptors was expressed in a spatially distinct zone of the epidermis, suggesting different functional roles for these receptors. Functional studies were performed on primary cultures of human keratinocytes and on explanted rat skin, where different P2 receptor subtype agonists and antagonists were applied to cultured keratinocytes or injected subcutaneously into the skin, respectively. An increase in cell number was caused by low doses of the nonspecific P2 receptor agonist ATP, the P2Y2 receptor agonist UTP (p<0.001), and the P2Y1 receptor agonist 2MeSADP (p<0.05). There was a significant decrease in cell number as a result of treatment with the P2X5 receptor agonist ATPgammaS (p<0.001) and the P2X7 receptor agonist BzATP (p<0.001). Suramin caused a significant block in the effect of 100 microm ATP (p<0.01) and 1000 microm ATP (p<0.001) on cell number. These results imply that different purinergic receptors have different functional roles in the human epidermis with P2Y1 and P2Y2 receptors controlling proliferation, while P2X5 and P2X7 receptors control early differentiation, terminal differentiation and death of keratinocytes, respectively.

Expression of purinergic receptors in non-melanoma skin cancers and their functional roles in A431 cells.

We investigated the use of purinergic receptors as a new treatment modality for nonmelanoma skin cancers. Purinergic receptors, which bind adenosine 5'-tri-phosphate, are expressed on human cutaneous keratinocytes. Previous work in rat and human epidermis suggested functional roles for purinergic receptors in the regulation of proliferation, differentiation, and apoptosis. Immunohistochemical analysis of frozen sections in human basal cell carcinomas and squamous cell carcinomas for P2X5, P2X7, P2Y1, P2Y2, and P2Y4 receptors was performed, accompanied by detailed analysis of archive material of tumor subtypes in paraffin sections. Functional studies were performed using a human cutaneous squamous cell carcinoma cell line (A431), where purinergic receptor subtype agonists were applied to cells and changes in cell number were quantified via a colorimetric assay. Immunostaining in paraffin sections was essentially the same as that in frozen sections, although more detail of the subcellular composition was visible. P2X5 and P2Y2 receptors were heavily expressed in basal cell carcinomas and squamous cell carcinomas. P2X7 receptors were expressed in the necrotic center of nodular basal cell carcinomas and in apoptotic cells in superficial multifocal and infiltrative basal cell carcinomas, and squamous cell carcinomas. P2Y1 receptors were only expressed in the stroma surrounding tumors. P2Y4 receptors were found in basal cell carcinomas but not in squamous cell carcinomas. P2X5 receptors appear to be associated with differentiation. The P2X7 receptor agonist benzoylbenzoyl-adenosine 5'-triphosphate and high concentrations of adenosine 5'-triphosphate (1000-5000 microM) caused a significant reduction in A431 cell number (p<0.001), whereas the P2Y2 receptor agonist uridine 5'-triphosphate caused a significant amount of proliferation (p<0.001). We have demonstrated that non-melanoma skin cancers express functional purinergic receptors and that P2X7 receptor agonists significantly reduce cell numbers in vitro.

The contractile properties and responses to tensional loading of Dupuytren's disease--derived fibroblasts are altered: a cause of the contracture?

Dupuytren's disease causes disability because of the development of finger flexion deformities, with distinct nodule and cord formation. This results in physical shortening of the diseased fascial tissue through a combination of cell-mediated contraction and matrix remodeling. It is this fixed tissue fabric shortening that prevents finger extension. In this experimental study, the relative contractile properties of Dupuytren nodule- and cord-derived fibroblasts were quantified in a culture force monitor model, in comparison with normal carpal ligament fibroblasts. Nine nodule, 10 cord, and four carpal ligament fibroblast cell lines were studied; each cell line was derived from a separate patient. The contractile forces generated by nodule and cord fibroblasts were significantly greater than the force generated by carpal ligament fibroblasts. There were also significant differences between nodule- and cord-derived fibroblasts, with the nodule cells demonstrating the greatest contractile force generation. The contraction profiles of both cord and nodule Dupuytren fibroblasts demonstrated delays in the attainment of tensional homeostasis, with an absence of a plateau phase by 20 hours. After the contraction phase, cell-seeded constructs were subjected to a series of four uniaxial mechanical overloads and cellular responses were monitored during each subsequent 30-minute period. Dupuytren nodule and cord fibroblast responses were significantly altered, compared with carpal ligament fibroblasts, exhibiting an increased and opposite response. Dupuytren fibroblasts, particularly nodule fibroblasts, exhibited increased force generation and a delay in reaching tensional homeostasis. The data suggest that these cells have an inherently higher basal tension and contractile ability. This results in increased shortening of the matrix, and the delay in reaching tensional homeostasis might exacerbate this response. These results represent a theoretical framework regarding the fundamental processes involved in the pathogenesis and progression of clinical flexion deformities in Dupuytren disease.

Skin tension or skin compression? Small circular wounds are likely to shrink, not gape.

The final appearance of a scar may be influenced by tension or mechanical factors [Borges AF. Scar prognosis of wounds. Br J Plast Surg 1960;13:47-54; Arem AJ, Madden JW. Effects of stress on healing wounds. J Surg Res 1976;20:93-102; Burgess LP, Morin GV, Rand M, et al. Wound healing. Relationship of wound closing tension to scar width in rats. Arch Otolaryngol Head Neck Surg 1990;116:798-802; Meyer M, McGrouther DA. A study relating wound tension to scar morphology in the pre-sternal scar using Langer's technique. Br J Plast Surg 1991;44:291-4] Karl Langer suggested that information could be gained about the tension inherent in skin, in all directions, by observing the wound edge retraction that occurred after making circular skin incisions [Langer K. On the anatomy and physiology of the skin II. Skin tension. Br J Plast Surg 1978;31:93-106]. Circular wounds may be used to demonstrate the orientation of the dominant axis of 'tension' in the skin but is this always a tensile stress as opposed to a compressive stress? This is the second article in a series documenting the mechanical properties of circular punch biopsy wounds. The aim of this study was to make detailed observations of the dimensional distortions of circular wounds on the face and neck, from which deductions could be made with regard to mechanical stress. One hundred and seventy-five benign head and neck lesions were excised from 72 volunteers using circular dermal punch biopsies. The distortions of the resulting wounds were observed to be elliptical in most cases. Measurements were taken of the maximum and minimum diameters of the wound and expressed as ratios of the size of the punch biopsy used for excision. The change in area from the area of the punch biopsy to that of the wound was also calculated. The maximum diameter of the wound was smaller than the diameter of the punch biopsy in 40.6% of cases, the minimum diameter of the wound was smaller in 97.7% of cases and the area of the wound was smaller than that of the punch biopsy in 90.3%. These dimensional changes varied between sites (P=0.0005, P=0.0001 and P<0.0001, respectively). We conclude that the reported rhomboidal or lattice structure [Ridge MD, Wright V. The directional effects of skin. A bioengineering study of skin with particular reference to Langer's Lines. J Invest Dermatol 1966;46:341-6] of skin has individual components which are under tensional force due to elastic retraction. Wounds smaller than the rhomboidal unit will reduce in area, due to the intact tensional forces in the individual dermal components, giving an appearance of the skin overall being under compression. Larger wounds, disrupting more of the lattice structure, will gape.

Use of a non-contact 3D digitiser to measure the volume of keloid scars: a useful tool for scar assessment.

Keloid scars often fail to respond to treatment, so research into new therapeutic regimes is important. However, research is limited by a scarcity of reliable, objective scar assessment tools. The volume of a keloid scar should decrease with successful treatment. This study demonstrates the use of a non-contact 3D digitiser to measure digitally the volume of a keloid scar. The scanner was used to scan 62 keloid scars and one fine-line normal scar. The scan took approximately 9s to complete. The volume was measured using 3D reverse modelling software. A previously validated scar assessment scale was used to score the scars according to their physical parameters. A significant correlation was found between volume and the scar score (Pearson's r=0.627, p<0.001). Linear regression was also statistically significant (p<0.001, R(2)=0.44). Therefore it was possible to predict the scar score from the measured volume. This technique could allow monitoring of a patient on treatment, or comparison of treatments in a research setting. It overcomes previous problems with the measurement of scar volume as it is quantitatively objective and well-tolerated.

Scarring occurs at a critical depth of skin injury: precise measurement in a graduated dermal scratch in human volunteers.

BACKGROUND: The association between scarring and the depth of dermal injury or burn is clinically recognized but not quantified. The authors tested the hypothesis that there is a critical depth beyond which a fibrous scar develops. METHODS: A novel jig produced a wound that was deep dermal at one end and superficial dermal at the other. Pilot studies in cadaveric and ex vivo breast skin confirmed the depth of injury. Healthy volunteers had a standardized dermal wound made on the lateral aspect of the hip. Digital photography recorded the surface appearance of wound healing and scar development. High-frequency ultrasound demonstrated the depth of the healing wound and subsequent scar in vivo. RESULTS: One hundred thirteen human subjects participated in the clinical study. Mean length of follow up was 28.6 +/- 13.2 weeks. The deep dermal end of the wound healed with a visible scar and the superficial end had no visible residual mark after week 18. The initial length of injury was 51.3 +/- 0.6 mm, which reduced to a scar of 34.9 +/- 1.0 mm at 36 weeks (corresponding areas were 196.6 +/- 7.5 mm and 92.7 +/- 9.4 mm). High-frequency ultrasound analysis showed a gradual reduction in scar thickness at the deep end and no detectable scar at the shallow end. The transition point between scar and no scar marked the threshold depth for scarring. This was calculated as 0.56 +/- 0.03 mm, or 33.1 percent of normal hip skin thickness. CONCLUSIONS: The dermal scratch provides a well-tolerated, standardized, and reproducible wound model for investigating the healing response to dermal injury of different depths. There is a threshold depth of dermal injury at which scarring develops.

5-fluorouracil selectively inhibits collagen synthesis.

BACKGROUND: Fibroproliferative disorders, such as Dupuytren's contracture of the hand, are characterized by excessive production of collagen. 5-Fluorouracil has been used to treat fibroproliferative disorders of the eye and skin and is thought to inhibit thymidylate synthetase blocking DNA replication. 5-Fluorouracil has been shown to down-regulate fibroblast proliferation and differentiation in vitro. METHODS: This study investigated the dose-dependent effect of 5-fluorouracil on fibroblast extracellular matrix production. Fibroblasts were derived from tendon and primary Dupuytren's disease of the hand, a fibroproliferative disorder of the palmar aponeurosis (n = 4 patients). Total collagen synthesis was determined by means of the incorporation of radiolabeled proline. Fibroblast secretion of the profibrotic factor transforming growth factor-beta1 (TGF-beta1) was measured by a sandwich enzyme-linked immunosorbent assay. Gene expression of collagen types I and III and TGF-beta1 were quantified by means of reverse-transcriptase polymerase chain reaction assays. RESULTS: The authors found that 5-fluorouracil caused a dose-dependent, selective, and specific decrease in collagen production by Dupuytren's fibroblasts compared with noncollagenous protein synthesis. By contrast, procollagen types I and III mRNA were unaffected by 5-fluorouracil treatment. These changes did not appear to be mediated by alterations in the endogenous secretion of TGF-beta1 or its autocrine effect on collagen metabolism. CONCLUSIONS: The clinical implication is that 5-fluorouracil could possibly reduce extracellular matrix production and therefore reduce recurrence of Dupuytren's disease of the hand.

Purinergic receptor expression in the regeneration epidermis in a rat model of normal and delayed wound healing.

This study investigated changes in the protein expression of purinergic receptors in the regenerating rat epidermis during normal wound healing, in denervated wounds, and in denervated wounds treated with nerve growth factor (NGF), where wound healing rates are normalized. Excisional wounds were placed within denervated, pedicled, oblique, groin skin flaps, and in the contralateral abdomen to act as a control site. Six rats had NGF-treated wounds and six had untreated wounds. Tissue was harvested at day four after wounding. The re-epithelializing wound edges were analyzed immunohistochemically for P2X(5), P2X(7), P2Y(1) and P2Y(2) receptors, and immunostaining of keratinocytes was quantified using optical densitometry. In normal rat epidermis, P2Y(1) and P2Y(2) receptors were found in the basal layer where keratinocytes proliferate; P2X(5) receptors were associated with proliferating and differentiating epidermal keratinocytes in basal and suprabasal layers; P2X(7) receptors were associated with terminally differentiated keratinocytes in the stratum corneum. In the regenerating epidermis of denervated wounds, P2Y(1) receptor protein expression was significantly increased in keratinocytes (P<0.001) but P2Y(1) receptors (P<0.001) compared with untreated denervated wounds. In innervated wounds, NGF treatment enhanced expression in keratinocytes. P2X(5) (P>0.001) and P2Y(1) receptor protein (P<0.001) expression in keratinocytes. P2X(7) receptors were absent in all experimental wound healing preparations. P2X(5), P2X(7), P2Y(1) and P2Y(2) receptor protein expression in the regenerating epidermis was altered both during wound healing and also by NGF treatment. Possible roles for purinergic signalling and its relation to NGF in wound healing are discussed.

The early surface cell response to flexor tendon injury.

PURPOSE: The migratory response of surface fibroblasts to flexor tendon injury was studied by their selective labeling with a vital dye. METHOD: The surfaces of 30 rat deep flexor tendons were bathed in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI), a vital dye for 5 minutes. The residual dye was removed by thorough irrigation. A partial tenotomy was made in the stained section by cutting out a central window. Semiquantitative cell counts and position of stained fibroblasts were noted by examination under fluorescent light at 0, 1, 3, 5, and 7 days. RESULTS: The surface fibroblasts readily took up the vital dye at day 0. By day 1 dyed cells had moved into the cut. By day 3 they had migrated laterally into the core substance of the tendon. Core dyed cell counts at days 1, 3, 5, and 7 were significantly different compared with day 0 core dyed cell counts. CONCLUSIONS: This cell migration from the surface of the cut to the tendon core is likely to be vital in the early stages of tendon healing.