Antioxidant therapies for wound healing: a clinical guide to currently commercially available products.

Many facets of wound healing under redox control require a delicate balance between oxidative stress and antioxidants. While the normal physiology of wound healing depends on low levels of reactive oxygen species and oxidative stress, an overexposure to oxidative stress leads to impaired wound healing. Antioxidants are postulated to help control wound oxidative stress and thereby accelerate wound healing. Many antioxidants are available over the counter or by prescription, but only one, Medihoney®, has been specifically FDA approved for wound healing. Here we review the existing evidence for the use of antioxidants for wound healing, with a review of the pertinent animal and clinical studies. Natural products and naturally derived antioxidants are becoming more popular, and we specifically review the evidence for the use of naturally derived antioxidants in wound healing. Antioxidant therapy for wound healing is promising, but only few animal studies and even fewer clinical studies are available. Because only few products have undergone FDA approval, the consumer is advised to scrutinize them for purity and contaminants prior to use, and this may require direct contact with the companies that sell them. As a field of science, the use of antioxidants for wound healing is in its infancy, and future studies will better elucidate the role of antioxidants in wound healing.

Adrenergic signaling in human oral keratinocytes and wound repair.

Catecholamines are present in saliva, but their influence on oral epithelium is not understood. Because psychological stress increases salivary catecholamines and impairs oral mucosal wound healing, we sought to determine if epithelial adrenergic signaling could link these two findings. We found that cultured human oral keratinocytes (HOK) express the α(2B)- and ß(2)-adrenergic receptors (ARs). Exposure of HOK to either epinephrine or the ß-AR agonist, isoproterenol, reduced migratory speed and decreased in vitro scratch wound healing. Incubation with the ß-AR antagonist timolol reversed the catecholamine-induced effects, indicating that the observed response is mediated by ß-AR. Epinephrine treatment decreased phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38; these decreases were also reversed with timolol. Cultured HOK express enzymes of the epinephrine synthetic pathway, and generate epinephrine. These findings demonstrate that stress-induced elevations of salivary catecholamines signal through MAPK pathways, and result in impaired oral keratinocyte migration required for healing.

Urothelial progenitor cells: regional differences in the rat bladder.

OBJECTIVES: Because the trigone is a unique region in the caudal bladder with a higher risk of neoplasia, we hypothesized that this area would have a high proportion of progenitor cells. As yet there is no marker nor methodology to specifically isolate urothelial stem cells, and thus demonstrate multi-potential differentiation and self-renewal. Here, our goal was to evaluate the distribution of progenitor cells that carry two general major attributes of stem cells: clonogenicity and proliferative capacity. MATERIALS AND METHODS: The bladders of Fisher rats were divided into caudal and cephalic segments and primary cultures were established from the harvested urothelial cells. RESULTS: We found that colony-forming efficiency was almost 2-fold higher for cells from the caudal bladder compared to the cephalic bladder. Doubling time was significantly faster for cells harvested from the caudal bladder at initial plating. This suggested that the caudal bladder harbours a higher density of urothelial progenitor cells. With passage to p4, the differences between the upper and lower bladder were lost, suggesting selection of proliferative cells with serial passage. Based on Ki-67 staining, there was no geographical difference in cell proliferation under normal homeostatic in vivo conditions. CONCLUSIONS: These results demonstrate geographical sequestration of urothelial progenitor cells to the area of the bladder that encompasses the bladder neck and trigone, which may be a factor in pathological disparities between the trigone and remaining bladder.

Economical LED based, real-time, in vivo imaging of murine corneal wound healing.

An optimal system for monitoring in vivo corneal wound healing is inexpensive, has utility for wounding and imaging, and is able to provide previews before photography. We outline such an imaging system that takes advantage of a consumer digital camera and an LED-based light source for fluorescein excitation. Using FVB/NJ mice, 2mm diameter, circular, axial corneal epithelial defects were created using a crescent blade. The corneal wounds were imaged every four hours until healed using a Nikon Coolpix 5400 camera attached to a Nikon SMZ-10A stereomicroscope, using the illumination from a 16 LED 464nm flashlight. The wound area was calculated, and the linear regressions of the linear phase of wound healing were compared using the F-test. The slopes of the linear regressions for the 6 trials of 4 mice/trial had an average of -52.95microm/h (SEM=0.55microm/h) and were statistically equivalent (p>0.05). The mean of the R(2) values for the linear regressions was 0.9546 (SEM=0.0121). The equivalent linear regressions and R(2)>0.90 suggest that the imaging system could precisely monitor the wound healing of multiple trials and of animals within each trial, respectively. Using a consumer digital camera and LED-based illumination, we have established a system that is economical, is used in both wounding and imaging, is operated by a single person, and is able to provide real-time previews to monitor corneal wound healing precisely.

Microneedle array for measuring wound generated electric fields.

A microneedle array has been fabricated and applied to the measurement of transdermal skin potentials in human subjects. Potential changes were recorded in the vicinity of superficial wounds, confirming the generation of a lateral electric field in human skin. The measured electric field decays with distance from the wound edge, and is directed towards the wound. The measurement of endogenous fields in skin is a prelude to the study of the therapeutic efficacy of applied electric fields to chronic non-healing wounds.

Cyclic AMP-dependent protein kinase A plays a role in the directed migration of human keratinocytes in a DC electric field.

Skin wound healing requires epithelial cell migration for re-epithelialization, wound closure, and re-establishment of normal function. We believe that one of the earliest signals to initiate wound healing is the lateral electric field generated by the wound current. Normal human epidermal keratinocytes migrate towards the negative pole, representing the center of the wound, in direct currents of a physiological strength, 100 mV/mm. Virtually nothing is known about the signal transduction mechanisms used by these cells to sense the endogenous electric field. To elucidate possible protein kinase (PK) involvement in the process, PK inhibitors were utilized. Two important findings have been described. Firstly, addition of 50 nM KT5720, an inhibitor of PKA, resulted in a 53% percent reduction in the directional response of keratinocytes in the electric field, while not significantly affecting general cell motility. The reduction was dose-dependent, there was a gradual decrease in the directional response from 5 to 50 nM. Secondly, addition of 1 microM ML-7, a myosin light chain kinase inhibitor, resulted in an approximate 31% decrease in the distance the cells migrated without affecting directional migration. The PKC inhibitors GF109203X at 4 microM and H-7 at 20 microM and W-7, a CaM kinase inhibitor, did not significantly alter either directed migration or cell migration, although they all resulted in a slight reduction in directional migration. D-erythro-sphingosine at 15 microM, a PKC inhibitor, had virtually no effect on either migration distance or directed migration. These findings demonstrate that divergent kinase signaling pathways regulate general cell motility and sustained directional migration and highlight the complexity of the signal transduction mechanisms involved. The inhibitor studies described in this paper implicate a role for PKA in the regulation of the directional migratory response to applied electric fields, galvanotaxis.

Wound dressings alter the colony-forming efficiency of keratinocytes in cultured sheet grafts.

Cultured keratinocyte grafts transplanted for skin wound repair are often affixed to a wound dressing to facilitate handling. In this study, the ability of five different types of wound dressings to support cell viability and maintain stem cell populations in the cultured grafts was determined. Postconfluent keratinocyte (NHK) sheets were attached to wound dressings for 24 h and then released by trypsinization. Cell viability was determined and NHKs were assessed for clonogenic capacity by colony-forming efficiency (CFE) assays. CFEs for NHKs exposed to a collagen-bonded, bilaminate membrane and a polyurethane film were significantly less than control. On the other hand, CFEs for NHKs exposed to a collagen/alginate dressing and to petrolatum-impregnated gauze were significantly greater than control. The choice of a wound dressing carrier has implications for maintaining long-term viability of the transplanted sheet of epithelium.

Divergent responses of ras-transfected and non-ras-transfected human keratinocytes to extracellular calcium.

Raising extracellular calcium (Ca(o)) induces terminal differentiation in cultured epidermal keratinocytes. The introduction of the ras oncogene into keratinocytes results in resistance to Ca(o)-mediated differentiation. To understand the signaling mechanism involved, we examined the Ca(o)-induced formation of inositol triphosphate (IP3) and changes in intracellular Ca2+ (Ca(i)) concentration in non-ras-transfected and ras-transfected HaCaT lines of human keratinocytes. When switched from 0.05- to 1.5-mM Ca(o) medium, the non-ras HaCaT line showed a rapid twofold increase in IP3 formation, whereas the IP3 level in the ras-transfected I-7 line was slightly affected. G-protein-coupled activation of phospholipase was intact in both lines, as evidenced by the generation of similar amounts of IP3 in response to addition of bradykinin or guanosine 5'-[gamma-thio]-triphosphate. Addition of 1.0 mM Ca(o) evoked similar Ca(i) responses in both non-ras- and ras-transfected cells: a transient elevation, followed by a sustained lower plateau. However, the two lines differed in their later responses: after being maintained in 1.0 mM Ca2+ for 24 h, the Ca(i) level was significantly lower in ras-transfected cells than in non-ras-transfected HaCaT cells. The Ca(o)-induced increase in Ca(i) in both lines was inhibited by the Ca2+ entry blocker SK&F 96365 or depolarization in high K+ bathing solution, demonstrating its dependence of calcium influx. The results suggest fundamental differences in the early signal that are generated in response to an increase in Ca(o) in ras-transfected keratinocytes, with the absence of a Ca(o)-induced rise in IP3--a signaling pathway defect that may play a role in the differentiation block the cells exhibit. In addition, the inability of ras-transfected cells to sustain a prolonged Ca(i) plateau may also contribute to their inability to differentiate in response to the Ca(o) signal.

Ultraviolet B-mediated phosphorylation of the small heat shock protein HSP27 in human keratinocytes.

Exposure of human keratinocytes to environmental stress is known to induce changes in the expression, phosphorylation, and subcellular relocalization of the 27 kDa heat shock protein. This study demonstrates that ultraviolet B (280-320 nM) irradiation with physiologic doses induces a dose-dependent phosphorylation of 27 kDa heat shock protein, generating the more acidic 27 kDa heat shock protein B, C, and D isoforms. Ultraviolet B also induces perinuclear cytoplasmic relocation and nuclear translocation of 27 kDa heat shock protein and caused aggregation of cytoplasmic actin filaments into a broad perinuclear distribution. The ultraviolet B-induced phosphorylation is reversible, returning to baseline levels 4 h after exposure, and this coincides with the reversal of ultraviolet B-induced actin reorganization. The ultraviolet B-induced phosphorylation is not affected by the protein kinase C inhibitor, GF 109203X, is partially inhibited by epidermal growth factor receptor tyrosine kinase inhibitor, PD 153035, and is substantially inhibited by the specific p38 mitogen-activated protein kinase inhibitor, SB 203580. In addition, pretreatment of cells with the anti-oxidant N-acetyl cysteine partially inhibits ultraviolet B-and oxidant-induced 27 kDa heat shock protein phosphorylation. The p38 mitogen-activated protein kinase cascade is thus the major transduction pathway for ultraviolet B-induced 27 kDa heat shock protein phosphorylation, and reactive oxygen species generated in response to ultraviolet B also contribute to this phosphorylation. As 27 kDa heat shock protein phosphorylation and relocalization has been associated with increased cell survival after environmental insult, our data suggest that ultraviolet B, in addition to initiating recognized cytotoxic events in keratinocytes, also initiates a signaling pathway that may provide cellular protection against this ubiquitous environmental insult.

Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease.

PURPOSE: To bioengineer a corneal surface replacement using ex vivo expanded, cultured corneal epithelial stem cells seeded on a matrix derived from amniotic membrane and use this bioengineered graft to manage difficult ocular surface disease. METHODS: Fourteen patients with ocular surface disease unresponsive to standard medical and surgical treatments, including seven patients with presumed limbal stem cell deficiency were chosen for transplantation of a bioengineered composite corneal surface in eye each. Presumed corneal stem cells were harvested from either the patient's or related donor's limbus, expanded ex vivo, and cultivated on a carrier of modified human amniotic membrane. The resulting composite cultured tissue was transplanted to the ocular surface of the diseased eye, from which the abnormal tissue had been surgically removed. Ten patients received autologous grafts, and four received allogeneic grafts. RESULTS: A successful outcome, defined as restoration or improvement of vision, along with maintenance of corneal re-epithelialization and absence or recurrence of surface disease was obtained in 6 of the 10 patients with autologous procedures and in all 4 allogeneic transplants. Follow-up ranged 6-19 months with a mean of 13 months. CONCLUSIONS: This novel technique documents that presumed corneal epithelial stem cells can be harvested safely from the limbus, expanded successfully in vitro, and grown on denuded amniotic membrane. The resultant composite cultured tissue can be transplanted and appears to successfully manage eyes with difficult ocular surface disease, including those with stem cell deficiency. This technique minimizes the threat of damage or depletion to the contralateral or donor limbus.

DC electric fields induce rapid directional migration in cultured human corneal epithelial cells.

After an epithelium is wounded, multiple soluble and extracellular matrix-associated signals induce a repair response. An often-overlooked signal is the endogenous electrical field established in the vicinity of the wound immediately upon disruption of epithelial integrity. Previous studies have detected lateral electric fields of approximately 42 mV mm-1 near bovine corneal wounds. In addition, electric fields on the order of 100-200 mV mm-1 have been measured lateral to wounds in mammalian epidermis. Here we report the migratory response of human corneal epithelial cells to DC electric fields of similar, physiologic magnitude. Our findings demonstrate that in a 100 mV mm-1 DC field, corneal epithelial cells demonstrate directed migration towards the cathode. The migratory speed and distances traversed by cultured human corneal epithelial cells is remarkably similar to those of cultured skin-derived keratinocytes under similar conditions; however, corneal epithelial cells demonstrate a more rapid directional response to the field than keratinocytes. These findings suggest that endogenous, wound-induced electric fields present in the cornea play an important role in human corneal wound healing, by orienting the directional response of migratory cells so that they efficiently re-epithelialize the wounded area.

Involucrin-positive keratinocytes demonstrate decreased migration speed but sustained directional migration in a DC electric field.

When skin is wounded, keratinocytes from the cut edges of the epidermis migrate over the wounded area to re-epithelialize the wound. It is not clear which cells of the epidermis have the capacity to migrate and contribute to this re-epithelialization: the less differentiated cells of the basal layer, or the more differentiated, involucrin-positive suprabasilar cells. Here we demonstrate that both involucrin-negative and involucrin-positive cells are able to respond to a directional cue for migration with sustained directional migration. When cultured keratinocytes are exposed to a physiologic DC electric field of 100 mV per mm as a cue to guide migration (galvanotaxis) they migrate toward the cathode with equivalent directionality. The involucrin-positive cells, however, display mean migration speeds approximately one half (23.6 microm per h) of the mean rate achieved by involucrin-negative cells (46.5 microm per h). Despite their decreased migration rates, involucrin-positive cells appear to possess an intact mechanism for sensing a directional signal, transducing that signal, and responding with sustained directional migration. Because electric fields are endogenous in skin wounds, it is likely that both the basal, involucrin-negative cells and the involucrin-positive suprabasilar cells respond to this cue with directional migration. The new observation that involucrin-positive cells can indeed migrate suggests that these cells may also contribute to wound re-epithelialization in vivo.

Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields.

Human keratinocytes migrate towards the negative pole in DC electric fields of physiological strength. This directional migration is promoted by epidermal growth factor (EGF). To investigate how EGF and its receptor (EGFR) regulate this directionality, we first examined the effect of protein tyrosine kinase inhibitors, including PD158780, a specific inhibitor for EGFR, on this response. At low concentrations, PD158780 inhibited keratinocyte migration directionality, but not the rate of migration; at higher concentrations, it reduced the migration rate as well. The less specific inhibitors, genistein, lavendustin A and tyrphostin B46, reduced the migration rate, but did not affect migration directionality. These data suggest that inhibition of EGFR kinase activity alone reduces directed motility, and inhibition of multiple tyrosine kinases, including EGFR, reduces the cell migration rate. EGFR redistribution also correlates with directional migration. EGFR concentrated on the cathodal face of the cell as early as 5 minutes after exposure to electric fields. PD158780 abolished EGFR localization to the cathodal face. These data suggest that EGFR kinase activity and redistribution in the plasma membrane are required for the directional migration of keratinocytes in DC electric fields. This study provides the first insights into the mechanisms of directed cell migration in electric fields.

Migration of human keratinocytes in electric fields requires growth factors and extracellular calcium.

Currents that leak out of wounds generate electric fields lateral to the wound. These fields induce directional locomotion of human keratinocytes in vitro and may promote wound healing in vivo. We have examined the effects of growth factors and calcium, normally present in culture medium and the wound fluid, on the directional migration of human keratinocytes in culture. In electric fields of physiologic strength (100 mV per mm), keratinocytes migrated directionally towards the cathode at a rate of about 1 microm per min. This directional migration requires several growth factors. In the absence of these growth factors, the cell migration rate decreased but directionality was maintained. Epidermal growth factor alone restored cell migration rates at concentrations as low as 0.2 ng per ml. Insulin at 5-100 microg per ml or bovine pituitary extract at 0.2%-2% vol/vol also stimulated keratinocyte motility but was not sufficient to fully restore the migration rate. Keratinocyte migration in electric fields requires extracellular calcium. Changes in calcium concentrations from 3 microM to 3.3 mM did not significantly change keratinocyte migration rate nor directionality in electric fields; however, addition of the chelator ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to migration medium reduced, and eventually abolished, keratinocyte motility. Our results show that (i) growth factors and extracellular calcium are required for electric field-induced directional migration of human keratinocytes, and (ii) keratinocytes migrate equally well in low and high calcium media.

Intracellular calcium oscillations in cell populations of ras-transfected I-7 subline of human HaCaT keratinocytes.

We have observed oscillations of intracellular Ca2+ (Ca[i]) concentration in populations of ras-transfected HaCaT keratinocytes of I-7 subline. In postconfluent monolayers of I-7 keratinocytes, an increase in extracellular Ca2+ (Ca[o]) concentration to 0.25-0.5 mM induced sinusoidal Ca(i) oscillations, which persisted longer than 1 h with amplitudes of 50-150 nM and periods of 5-10 min. Thapsigargin, which depletes internal Ca2+ stores, did not prevent Ca(o)-induced Ca(i) oscillations, and it also induced Ca(i) oscillations in the ras-transfected I-7 line. Removal of extracellular Ca2+ or addition of Ca2+-entry blocker La3+ or SK&F 96365 inhibited Ca(i) oscillations, suggesting that Ca(i) oscillations in ras-transfected HaCaT keratinocytes were dependent on Ca2+ influx across the plasma membrane. Because the Ca(o)-induced Ca(i) oscillations have been observed only in ras-transfected I-7 subline and not in its nontransfected parental HaCaT line, this may provide a partial explanation for the divergent responses of ras-transfected and nontransfected keratinocytes to Ca(o) signal for control of growth and differentiation.