The Effect of Intermittent Radiant Warming on the Contraction of Collagen Lattices Populated with Human Dermal Fibroblasts and o

Disclosure: This work was sponsored by an educational grant from Augustine Medical, Eden Prairie, Minnesota, USA, who also provided funding in order to present this work at scientific meetings. Introduction In recent years, a novel practical method of providing therapeutic radiant heat to clinical wounds has become available through the development of a warming device, the Warm-Up™ dressing (Augustine Medical Inc. Eden Prairie, Minnesota, USA). This device applies safe, precisely controlled heat to the wound while maintaining a moist environment. Several clinical studies using the warming system on various kinds of chronic wounds have shown improved healing.[1–5] A further benefit to the improved or complete healing with this treatment regimen was that patients frequently remarked on a decrease in the pain associated with their ulcers.[1,2] In the warming system, the infrared warming card is inserted into the plastic pocket attached to the top of the foam collar, which is sealed to the periwound skin by an adhesive (Figure 1). The dressing protects the wound without disrupting healing tissue. Any exudate is absorbed by the foam and a moist but not a wet environment is maintained. The temperature of the card is set to 38 degrees C and regulated by a temperature control unit powered by the battery or electrical power. The system is based on the fundamental physiological premise that most cellular physiological functions, enzymatic reactions, and biochemical reactions in the human body are optimized at 37 degrees C. Baseline skin and wound temperatures, however, are lower than core temperature. In our clinic, measurements of wound surface temperature, made with an infrared thermometer (ETI Ltd., Worthing, Sussex, United Kingdom) in 16 leg ulcer patients indicated an average ulcer surface temperature of 32.3 degrees C (range 28.8 degrees C–35.1 degrees C).[6] Application of the radiant warming raises the temperature of the periwound skin by about 2.4 degrees C in one hour.[4] Application of noncontact radiant heat has been shown to improve tissue oxygen in normal volunteers,[7] and this has been suggested as a possible mechanism contributing to improved healing. In patients with venous ulcers, raised oxygen tension and improved blood supply to the wound edge and surrounding skin have been demonstrated.[8] Another possible contributing mechanism to its efficacy is the stimulation of cell proliferation, which is important in several stages of wound healing. In-vitro studies have shown that intermittent radiant warming for seven days, following a protocol similar to that used in the clinic, stimulates the proliferation of human fibroblasts,[6] keratinocytes [unpublished], and dermal microvascular endothelial cells.[9] All these in-vitro studies used cells grown in monolayer, but in-vivo fibroblasts are in a three-dimensional matrix. The contraction of a wound, part of the normal healing process for an open wound, is the result of dynamic cell-matrix interactions principally between fibroblasts and collagen but also involving fibronectin and other proteins. These interactions can be influenced by a number of factors including the matrix substrate, cell-surface receptors, and the presence of cytokines.[10,11] Investigation of these interactions, which are involved in wound contraction in vivo, has been facilitated by the development of fibroblast-populated collagen lattices (FPCLs)[12] in which cells are suspended in a polymerized three-dimensional collagen matrix. When cast in the lattice, the fibroblasts, which initially are spherical after trypsinization, become elongated. As the fibroblast moves through the collagen matrix, it becomes intimately linked with the fibrils and pulls on them, resulting in contraction.[13] The ability of the lattices to contract can be affected by several factors, such as the concentration of collagen, the density of fibroblasts,14 and also the source of the fibroblasts.[11,15,16] To our knowledge, no research has focused primarily on the effects of temperature on the contraction of FPCLs, although Nishiyama, et al.,[17] included temperature in their extensive analysis of a number of factors influencing gel contraction. The study reported here aimed to investigate whether the intermittent use of the warming device, following a protocol similar to that used in the clinic, could have a direct effect on the contraction of collagen lattices and on the proliferation of fibroblasts within a three-dimensional matrix. Materials and Methods Fibroblast culture. Two strains of adult, human, normal, dermal fibroblasts from a 54-year-old subject and an 87-year-old subject, both from leg skin obtained from surgical discard tissue and one strain of leg-ulcer fibroblasts from the 87-year-old subject were used for this study. Cells were thawed from frozen stock and cultured in DMEM (Dulbecco’s Modified Eagle’s Medium; Gibco, Paisley, United Kingdom) containing 10 percent FCS (fetal calf serum), 50IU/mL penicillin, 50µg/mL streptomycin, 0.25µg/mL fungizone (all from GIBCO), in 75cm2 plastic flasks at 37 degrees C in a humidified atmosphere of five-percent CO2, 95-percent air. Cells from passages 5 to 7 were used for the experiments. Preparation of hydrated collagen lattices. Collagen lattices were prepared from type I collagen extracted from rat tail tendons as described by Bell,[12] and a working solution of 1.0mg/mL was prepared. Cells were trypsinized, resuspended in medium, and counted using a hemocytometer. The volume of suspension was adjusted to give a cell density of 2x105 cells/mL and placed on ice at 0 degrees C while the collagen polymerizing solution was made. The method of preparing collagen lattices was adapted from that of Montesano and Orci.[18] Reagents were dispensed into a 50mL centrifuge tube in the sequence: 4mL of 11.76mg/mL sodium bicarbonate solution; 2mL of 10x minimum essential medium (MEM), 10mL of collagen solution. The MEM was used because DMEM at 10 times concentration was no longer available from the manufacturing company due to precipitation problems. Sodium hydroxide 1.0M or 0.4M was added dropwise until the color changed to orange or orange/pink, indicating a pH of about 7.2. Then 4mL of the cell suspension was added, the solution mixed by gentle swirling, and 2mL of collagen cell suspension were dispensed into 32mm diameter petri dishes. This gave a density of 4x104 cells/mL (8x104 fibroblasts per lattice) and a collagen concentration of 0.5mg/mL in the final lattices. The lattices were incubated for 10 minutes at 37?C, then 1mL of DMEM/10-percent FCS medium was added, and the lattices were released from the sides and bottom of the petri dish with a spatula. This was considered as zero time. The lattices were observed under the microscope to ascertain that cells were uniformly distributed throughout the different planes of the collagen lattice. Warming protocol. For these experiments, the incubator temperature was adjusted to 32.5 degrees C±0.5 degrees C similar to average measured skin and wound temperatures.[6,19] Each plate was placed on a Microban plastic board and covered with the warming device, which was sealed to the board with a small tube inserted to allow equilibration with the five-percent CO2 atmosphere of the incubator. The plates were placed in the incubator, and warming cards were inserted in the plastic pockets of the dressings of the test plates (Figure 1). The warming protocol was started immediately by switching on the temperature control units linked to the warming cards, which have designated temperatures of 38 degrees C and 42 degrees C. The test lattices were treated by daily warming for three cycles of one hour with 1.5 hour intervals for one week while the control lattices were maintained at the incubator temperature. On certain days, the dressing was removed from the plates at the start of the day and contraction of the collagen lattices was assessed by placing the plates on transparent graph paper and optimizing visibility of the gel edges with light from a fiber optic lamp. Two diameters at right angles were measured using a hand magnifier, and the area was calculated from the average diameter and expressed as a percentage of initial area. For each lattice at a given time point, the percentage of initial area (% A0) was calculated as % A0 = 100 x At/A0 where A0 is initial lattice area At is lattice area at time “t.” A fresh dressing was then placed over each plate, and the warming regime for that day was begun. Cell proliferation in monolayer culture. For the ulcer fibroblasts from the 87-year-old subject, we carried out a limited monolayer experiment, because the low number of cells obtained after trypsinization was insufficient to prepare collagen lattices. Ulcer fibroblasts were seeded into three wells of two six-well plates at 3.8x104 cells/well. One plate was kept at 33 degrees C, and one was treated with the warming regime under a 38 degrees C dressing. Cells were counted by hemocytometer on Day 7. Temperature in the medium. In an incubator set to 32 to 33 degrees C, 2mL of culture medium were added to three wells in six-well plates placed on Microban plastic supports. A waterproof, stainless steel temperature sensor, accuracy ±0.1 degrees C (Merck, Lutterworth, United Kingdom), attached to a digital monitor (LIBRA Medical, Ascot, United Kingdom) was placed in the medium in one well per plate. The plates were then sealed with the warming device covers, and warming cards set to 38 degrees C or 42 degrees C were inserted. The plates were placed in an incubator for 4 to 5 hours to equilibrate and the warming cards switched on when the temperatures had stabilized. Temperatures were monitored for one hour with the warming cards switched on and for the following one and a half hours when the units were switched off. As only one temperature probe was available, separate runs were made for each warming card. The temperature of the incubator was monitored from the digital display on the incubator itself. Cell recovery from lattices. Bacterial collagenase Type IV (Sigma, Poole, United Kingdom) was dissolved at 4mg/mL (w/v) in DMEM serum-free medium, sterilized by filtration through a 0.2µ membrane (Acrodisc®, Gelman, Northampton, United Kingdom), and stored at 20 degrees C for later use. Fibroblasts were recovered from the FPCLs by enzymatic degradation of the collagen. Briefly, lattices were solubilized by adding 1.5mL of collagenase (4mg/mL) and incubating them at 37 degrees C for 120 minutes, followed by addition of 1.0mL of 0.25-percent (w/v) trypsin/0.02-percent (w/v) EDTA (ethylenediamine-tetraacetic acid) and further incubation for 20 minutes. The solution was then added to 4mL of DMEM/10-percent FCS in a centrifuge tube and centrifuged for eight minutes at 1000rpm. The cells were resuspended in 2mL of DMEM/10-percent FCS, and cell numbers were counted in a modified Fuchs-Rosenthal hemocytometer. Three lattices were solubilized immediately after preparation of the gels in order to ascertain initial cell numbers in the same conditions that were to be used for subsequent cell counts. At other time points, two lattices were solubilized for each temperature. Warming was continued for seven days (from Day 0 to Day 6 inclusive), and the final measurements were made on Day 7. Statistics. Four collagen lattices were used to measure contraction for each set of treatments and for the control set. Two extra collagen lattices were used at each time point to determine cell numbers. For the data at each time point, ANOVA analyses were carried out using the Statview 4.5 package. Fisher’s test was used to compare the mean percentage of initial area under different warming sets to identify significant differences at the 95-percent level. Results Temperature in the culture medium. The warming cards with control units with designated temperatures of 38 degrees C and 42 degrees C raised the temperature in the medium in an incubator at 33.2 degrees C to maxima of 36.2 degrees C and 38.1 degrees C, respectively (Figure 2). When the warming units were switched off, there was a rapid drop in the temperature of the medium in the first 30 minutes, followed by a slow decrease to the initial temperature by one hour under the 38 degrees C dressing and 1.5 hours under the 42 degrees C dressing. The difference of 0.4 degrees C between the recorded initial and final temperatures under the dressings and that of the incubator is probably due to the necessary use of a different probe and monitor for the incubator. Collagen lattice contraction. For these studies, the conditions of collagen concentration and cell density were selected so that lattice contraction would be slow enough to investigate the effect of the warming regime over several days. In preliminary experiments using this batch of collagen at 0.5mg/mL and 4x104 cells/mL, we found that at 33 degrees C, there was no contraction at 24 hours and about 10- to 20-percent contraction at 48 hours. For the warming experiments, we, therefore, decided to leave the collagen lattices in the dressings for 48 hours before removing them to make the first measurement. The fibroblasts seeded at the above density into the FPCL all eventually reorganized their surrounding collagenous environment and produced a decrease in FPCL dimensions. In all experiments, there was always the same trend of temperature dependence, with the intermittent warming treatment in the 42 degrees C dressing increasing contraction to a greater degree than the 38?C dressing, which induced greater contraction than controls (Figure 3). In a typical experiment using fibroblasts, passage 6, from a 54-year-old donor (Figure 4), the 42 degrees C dressing significantly enhanced lattice contraction by Day 3 to 45.6 percent of the initial area compared to 58.6 percent under the 38 degrees C dressing (pCell proliferation. By Day 3, there was a decrease in the number of cells recovered from the lattices at 33 degrees C. Intermittent warming in the 38 degrees C dressing either diminished this decrease or abolished it. However, in lattices in the 42 degrees C dressing, cell numbers had always increased. In a typical experiment with fibroblasts from the 54-year-old donor, cell numbers recovered from the lattices on Day 3 were 10.5±0.02x104 under the 42 degrees C dressing, 9.0±0.6x104 under the 38 degrees C dressing, and 7.3±0.3x104 in control lattices kept at 33 degrees C (Figure 6). The increase in cell numbers under the 42 degrees C dressing relative to controls was significant (p