The Topical Application of Cryopreserved Keratinocytes Accelerates Steroid-Impaired Cutaneous Healing in Rats by Releasing Insul

Introduction Chronic wounds, such as diabetic, venous, or pressure ulcers, cause severe socioeconomic burden. Diabetic foot ulcers alone, for example, are responsible for more than $1 billion in medical treatment costs in the United States each year.[1] Another common cause for the development of recurrent ulcerations is long-term steroid use.[2] It has been shown that systemic steroid therapy decreases growth factor levels in both blood and wound fluid[3] and reduces the expression of adhesion molecules, cytokines, and chemokines.[4,5] However, growth factors play a key role in wound healing as regulators of extracellular matrix remodeling.[6,7] Transplantation of living cells to achieve wound closure and restore local growth factor deficiency and cellular dysfunction is attractive. The enhanced healing is attributed to the secretion of various growth factors and cytokines by the transplanted cells.[8] The major disadvantage is a time delay in growing confluent cell layers.[9] Furthermore, keratinocyte sheets tend to be thin and fragile because of their lacking dermal component. Fibrin glue has been established as a matrix to prevent blistering and guarantee a better adhesion to the wound bed after transplantatio.[10] Optimal supportive wound care is also crucial because survival of the transplanted cells is related to nutritional conditions in the wound bed.[11] Occlusive dressings create an improved environment for epidermal regeneration by providing a barrier against infection and controlling water loss.[12] Through cryopreservation of allogenic cells the problem of culturing autologous cells to confluent monolayers is addressed and cells can now be stored for later use.[13] To maintain healthy wound tissue, these cryopreserved cells are attached to various materials and function as biological dressings.[14] The aim of this study was to determine the following: 1) The in-vitro release of insulin-like growth factor I (IGF-I) out of cryopreserved rat keratinocytes; and 2) the in-vivo biological activity of the released IGF-I using a steroid wound model. Material and methods Animals. Forty male Sprague-Dawley rats (mean body weight 360±6g), were kept under controlled conditions with constant temperature and a 12-hour light/12-hour dark cycle. Water and standard rodent laboratory chow (Purina Ground Laboratory Chow, Purina, Atlanta, Georgia) were supplied ad libitum. Surgery. Animals were anesthetized with a intraperitoneal mixture of ketamine (100mg/kg; Ketanest®, Parke-Davis B.V., Germany) and xylazine (15 mg/kg; Rompun®, Bayer, Leverkusen, Germany). Prior to surgery animals received a single subcutaneous injection of 18mg/kg methylprednisolone-acetate (Depomedrate® , Pharmacia-Upjohn GmbH, Erlangen, Germany). The dorsum was shaved and disinfected with isopropyl alcohol. Full-thickness excisional wounds were created on the back of each rat by sterile biopsy punches (8mm in diameter), equidistant from the midline. A daily dressing change was performed for seven days. A special device was sutured around the wound to guarantee the adherence of the dressing to the ulcer surface and to allow easy dressing changes. On Day 7, rats were euthanized, wounds were measured photoplanimetrically, surgically removed, fixed in formalin, and embedded in paraffin. Wound size measurements were assigned using a blind design, in which the investigator had no knowledge of the type of treatment. Treatment groups. In a preliminary experiment, healthy (n=5) and steroid (n=10) animals were treated with a standard hydrogel dressing (Hydrosorb®, Hartmann, Germany). A second steroid group (n=12) received the dressing containing cryopreserved keratinocytes (KK dressing), and a third steroid group (n=13) received IGF-I gel (10µg IGF-I dissolved in 0.2-percent methylcellulose) once a day for seven days. Application of IGF-I. Stock IGF-I (Tebu®, Frankfurt, Germany) was stored at 4°C at a concentration of 100µg/mL. IGF-I gel contains a total amount of 10µg IGF-I, dissolved in 200µl 0.2-percent methylcellulose (Sigma®, Frankfurt, Germany). It was applied into the wound with a sterile pipet. The wound was additionally covered by an occlusive hydrogel dressing (Hydrosorb®, Hartmann, Germany). Cultured keratinocytes dressing (KK dressing). Rat foreskin (ageIGF-I measurement. Releasates from the KK dressing and IGF-I dissolved in methylcellulose gel were evaluated for their biological activity. KK dressing and IGF-I gel were incubated in 1mL 0.1-percent BSA phosphate-buffered saline solution. Samples were collected after 10, 20, 30, 60, 120, and 1,440 minutes. The IGF-I concentration was measured by ELISA (IBL Hamburg, Germany). Statistics. Results are documentated as mean ± SEM. Differences between the groups were calculated with the Wilcoxon rank test. Results In-vitro experiments. Approximately 3.3µg IGF-I/cm2 was released out of the keratinocyte dressing. Most IGF-I was released within the first 10 minutes after incubation. At later time points, there was no significant further release (Figure 1). In-vivo experiments. Compared to healthy rats, animals treated with a single subcutaneous injection of methyl-prednisolone acetate demonstrated significantly larger wounds after seven days treatment with control dressing (44.5±7.5 vs. 26.2±5mm2; p=0.001) (Figure 2). Both IGF-I gel (26±5.3 vs. 44.5±7.5mm2; p=0.0001) and KK dressing (26±4 vs. 44.5±7.5mm2; p=0.0001) significantly reduced wound size in steroid rats compared to treatment with standard hydrogel dressings (Figure 3). Discussion Cutaneous healing processes are dependent upon protein synthesis, matrix deposition, cellular migration, and replication. Extracellular matrix molecules and growth factors regulate the interactions between the aforementioned processes.[6,15] Prior investigations have shown that steroid treatment decreases growth factor levels in both blood and wound fluid.[3,16] Wound IGF-I levels of steroid-treated animals are significantly decreased.[17,18] Furthermore, steroids reduce collagen synthesis in vivo[19] and in human skin fibroblasts in vitro.[20] Therefore, local administration of dissolved growth factors has been utilized to accelerate impaired healing.[21,22] However, Puolakkainen, et al., pointed out that the vehicle of topical growth factor application plays a significant role for its potential benefit on healing.[23] It is also known that the application of a combination of growth factors is superior to treatment with a single growth factor,[21,24] and there is further evidence that platelet-derived wound healing factors (PDWHFs) have beneficial effects on healing.[25–27] In addition, growth factors applied to open wounds have a changing bioavailability. For example, dissolved IGF-I can be rapidly degraded by proteases in the wound bed[28] or inactivated through an acidic wound environment.[29] This emphasizes the importance of adequate additional wound treatment. Rats receiving a subcutaneous injection of steroids show significantly larger wounds compared to healthy animals after seven days of treatment with control dressing (p=0.001). The application of dissolved IGF-I in a concentration of 50µg/mL significantly accelerated repair (p=0.0001). However, the dressing containing cryopreserved keratinocytes demonstrated the same effect although the concentration of IGF-I is nearly 15-fold less. Keratinocytes are a well documented source of growth factors and cytokines in addition to IGF-I.[30] This new dressing does not primarily act through the biological activity of viable keratinocytes; instead, it is the releaseate of the cryopreserved cells that provides multiple growth promoting factors and, therefore, stimulates healing comparable to the effect of PDWHFs. In this study, we chose to focus on IGF-I as one of the growth promoting factors released. This might explain why the KK dressing was as effective as IGF gel in vivo. In vitro, the biological dressing released approximately 3.3µg/mL IGF-I. Most was delivered within the first 10 minutes, with no significant release at later timepoints. However, the in-vivo rate of release from the dressing has to be estimated as much slower because the whole dressing does not become hydrated on the wound surface at the same time. Furthermore, IGF-I receptors might be downregulated if a high amount of IGF-I is delivered to a wound in a short time period.[31,32] The surface of full-thickness wounds is mostly irregular. Growth factors applied topically in a liquid formulation might leak out of the wound site. This problem is overcome by the hydrogel component of the new dressing. The IGF-I released from the wound cover remains on the wound surface, because the hydrogel polymer working as an occlusive dressing creates a moist chamber, which prevents water loss and thus prevents the loss of the released growth factor. In the past, keratinocytes were mainly used as confluent cell layers and transplanted to achieve wound closure.[33,34] The major disadvantages were a time delay of three to four weeks until cells reached confluence and difficult surgical handling. Due to the absence of a dermis, this neoepidermis remains very fragile, and spontaneous blistering on minor trauma may occur, even years later.[9] Keratinocytes transplanted in cell layers are likely to develop infection, and the healing rate is dependent on the condition of the wound bed.[35] The approach taken in this study was not wound closure but wound coverage. The hydrogel polymer component of the dressing provides a physical barrier against the outside environment protecting the wound from injuries, preventing infection, removing excessive exudate, and maintaining thermal isolation.[36] The cellular component (keratinocytes) enhances healing by releasing growth factors. We measured IGF-I as one such growth factor. Using cryopreservation, the problem posed by the time delay in growing confluent cell layers is overcome. However, these results cannot automatically be anticipated in a clinical setting. Additionally, cryopreserved keratinocytes do not express HLA class I antigens, which might induce rejection reactions.[37]