Optimal Concentration of Thrombin to Activate Platelet for Wound Healing

PURPOSE: Platelet transplantation is a novel therapeutic strategy for acceleration of wound healing. When applying platelets, efficacy of adding thrombin to stimulate growth factor release from platelets has already been proven. However, no quantitative data of the thrombin treatment has been reported. The purpose of this study is to determine the optimal thrombin concentration to maximize growth factor release of platelets. In particular, this study was designed to quantify levels of platelet derived growth factor(PDGF)-BB, which is a major growth factor containing in the platelets, in vitro. METHODS: Fresh platelets were obtained from a blood bank. They were suspended in DMEM/F-12 and incubated with thrombin of various concentrations. The concentrations of thrombin tested were 0, 6.25, 12.5, 25, 50, 100, 200, and 400IU/mL. After 30 minutes, 1, 3, 5, and 7 days, the levels of PDGF-BB were measured using enzyme linked immunosorbent assay. Platelets from four donors were included in this study. Each sample was tested in triplicate and the mean value was used as a data for each sample. RESULTS: The addition of thrombin increased the level of PDGF-BB. Increases in storage time of platelets resulted in decreased levels of PDGF-BB. Higher levels of PDGF were detected in consort with increased thrombin concentrations. However, there was no significant difference between samples of 200 and 400IU/mL concentrations. CONCLUSION: The results indicate that adding thrombin accelerates the release of growth factors from platelets and the optimal thrombin concentration to maximize this function is 200IU/mL.

Effect of Lipoaspirate Cell Autograft on Proliferation and Collagen Synthesis of Diabetic Fibroblasts in Vitro

PURPOSE: Human lipoaspirate cells are relatively easy to obtain in large quantities without cell culture. The aim of this in vitro pilot study is to identify the effects of cell therapy using uncultured lipoaspirate cells on cell proliferation and collagen synthesis of diabetic fibroblasts, which are the major contributing factors in wound healing. METHODS: In order to get diabetic fibroblasts, dermis tissues were obtained from foot skin of diabetic patients who underwent debridements or toe amputations (n = 4). In order to isolate lipoaspirate cells, the same diabetic patients' abdominal adipose tissues were obtained by liposuction. The diabetic fibroblasts were co-cultured with or without autogenous lipoaspirate cells using porous culture plate insert. Initial numbers of the lipoaspirate cells and diabetic fibroblasts seeded were 15,000 cells/well, respectively. For cell proliferation assay, two treatment groups were included. In group I, diabetic fibroblasts were cultured with the insert having no cells, which serves as a control. In group II, the lipoaspirate cells were added in the culture plate insert. For collagen synthesis assay, one additional group (group III) was included for a reference, in which diabetic fibroblasts were not seeded in the well and only lipoaspirate cells inside the insert were incubated without diabetic fibroblasts. RESULTS: One hundred to one hundred sixty thousand lipoaspirate cells were isolated per ml of aspirated adipose tissue. After 3-day incubation, the mean cell numbers in group I and II were 17,294/well and 22,163/well. The mean collagen level in group I, II, and III were 29, 41, and 2 ng/mL, respectively. These results imply that both cell proliferation and collagen synthesis in the lipoaspirate cell treatment group were 28 and 44 percents higher than in the control group, respectively (p < 0.05). CONCLUSION: Uncultured lipoaspirate cell autografts may stimulate the wound healing activity of diabetic fibroblasts.

Effect of Oncostatin M on Wound Healing Activity of Diabetic Fibroblasts in vitro

PURPOSE: Oncostatin M(OSM) has been known as a role in fibrosis and anti-inflammatory effects of various organs and tissues. Although there have been a number of studies which are focused on the roles and mechanisms of OSM, there are few reports on its effects in chronic wound healing. The purpose of this study is to evaluate the effects of OSM in wound healing activities of dermal fibroblasts of chronic wound in vitro. In particular, this study is focused on cell proliferation and synthesis of collagen and glycosaminoglycan(GAG), which are the major components of the extracellular matrices, of diabetic fibroblasts. METHODS: Fibroblasts were isolated from excess skin that was obtained from diabetic foot ulcer patients who underwent debridement. The isolated fibroblasts were cultivated in presence of OSM(100ng/mL). Cell proliferation, collagen synthesis and GAG levels were compared. RESULTS: All the components tested in this study increased in OSM treatment group. In particular, collagen and GAG synthesis demonstrated statistically significant increases(p<0.05 in the Mann-Whitney U- test). CONCLUSION: These results indicate that OSM increases wound healing activities of dermal fibroblasts of chronic wound in vitro.

Effect of Oncostatin M on Proliferation and Matrix Synthesis of Dermal Fibroblasts

PURPOSE: Oncostatin M(OSM) is a multifunctional cytokine that belongs to the interleukin(IL)-6 family. Although there have been a number of studies that focused on the role and mechanism of OSM in various organs and tissues, there are few reports on its effect on wound healing. The final purpose of this project is to evaluate the effect of OSM on wound healing. This pilot study was designed to investigate the effect of OSM on proliferation and matrix synthesis of human dermal fibroblasts, which are the major components of the wound healing. METHODS: Excess skin that was obtained from patients who underwent skin grafts, was used for this study. From this material, fibroblasts were isolated and cultured. The cultured fibroblasts were treated with one of four concentrations of OSM. The OSM concentrations used were 0, 50, 100, and 200ng/ml, respectively. After the OSM treatment, cell proliferation was determined by the MTT assay, collagen synthesis by the C1CP method, GAG levels by the Blyscan Dye method. The parameter levels of each group were compared. RESULTS: OSM treatment increased all the components tested in the study. In particular, cell proliferation, GAG synthesis demonstrated statistically significant increases(p<0.05 in the Mann-Whitney U-test). The highest increase in all the components was obtained at a 100ng/ml concentration of OSM. CONCLUSION: The results of the present study indicate that OSM stimulates proliferation and matrix synthesis of human dermal fibroblast and the optimal concentration for wound healing is 100ng/mL.

Clinical Application of Adipose Derived Stromal Cell Autograft for Wound Coverage

PURPOSE: Skin and soft tissue defect is one of the major challenges faced by plastic surgeons. Adipose derived stromal cells, which can be harvested in large quantities with low morbidity, display multilineage mesodermal potential. Therefore, adipose derived stromal cells have been met with a great deal of excitement by the field of tissue engineering. Recently, Adipose derived stromal cells have been isolated and cultured to use soft tissue restoration. In order to apply cultured cells for clinical purpose, however, FDA approved facilities and techniques are required, which may be difficult for a clinician who cultures cells in a laboratory dedicated to research to utilize this treatment for patients. In addition, long culture period is needed. Fortunately, adipose derived stromal cells are easy to obtain in large quantities without cell culture. The purpose of this study is to present a possibility of using uncultured adipose derived stromal cells for wound coverage. METHODS: Seven patients who needed skin and soft tissue restoration were included. Five patients had diabetic foot ulcers, 1 patient got thumb amputation, and 1 patient had tissue defect caused by resection of squamous cell carcinoma. The patients' abdominal adipose tissues were obtained by liposuction. The samples were digested with type I collagenase and centrifuged to obtain adipose derived stromal cells. The isolated adipose derived stromal cells were applied over the wounds immediately after the wound debridement. Fibrin was used as adipose derived stromal cells carrier. Occlusive dressing was applied with films and foams and the wounds were kept moist until complete healing. RESULTS: One hundred to one hundred sixty thousand adipose derived stromal cells were isolated per ml aspirated adipose tissue. All patients' wounds were successfully covered with the grafted adipose derived stromal cells in a 17 to 27 day period. No adverse events related to this treatment occurred. CONCLUSION: The use of uncultured adipose derived stromal cells was found to be safe and effective treatment for wound coverage without donor site morbidity.

Comparison of Doppler and CT Angiography as a Predictor of Healing Diabetic Foot Ulcers

PURPOSE: Adequate tissue oxygenation is considered as an essential factor for wound healing. In the non- diabetic population, an uncompromised macrocirculation generally leads to adequate tissue oxygenation. On the contrary, the macrocirculation in diabetic patients may not correlate with tissue oxygenation because of structural changes in the capillary basement membrane. Nevertheless, many medical professionals in Korea rely on macrocirculation evaluation when predicting wound healing potential of the diabetic ulcers. The purpose of this study is to compare reliability of two common macrocirculation assessment methods, Doppler probing and CT angiography, on tissue oxygenation in diabetic foot patients. METHODS: Doppler and CT angiography scores were given according to the patency of the anterior and posterior tibial arteries. Tissue oxygenation was measured by transcutaneous partial oxygen tension(TcpO2). Doppler and CT angiography scores were statistically analyzed against TcpO2 values. Sixty-eight diabetic foot ulcer patients were included in this study. RESULTS: The test was carried out on Doppler score and TcpO2 variables displayed a p-value of 0.0202, and concluded that the two variables were statistically dependent. The test used to determine for linear trends between Doppler scores and TcpO2 variables displayed a p-value of 0.0149, displaying statistical linear trend between the two variables. On the contrary, the tests between CT angiography scores and TcpO2 variables showed p-values of 0.1242 and 0.6590, that means no correlation between CT angiography and TcpO2 scores. CONCLUSION: Doppler probing is more reliable than CT angiography in predicting tissue oxygenation of diabetic foot ulcers.

A Novel Method of Dermis Graft for Better Outcome

The two major concerns in skin grafting are poor color match in the recipient site and the donor site morbidity. A new skin graft(dermis graft; deepithelialized split thickness skin graft), was used to minimize these problems. The important aspects of this method involve immediate return of epidermis to the donor site and restoration of the recipient site's epidermis by inducing epithelialization from adjacent skin. From April of 2001 to March of 2004, dermis graft and a conventional split thickness skin graft(STSG) were performed in 53 and 33 patients, respectively. The healing time, the scar condition, and the patients' satisfaction were compared. Regarding the recipient sites, the wounds of the dermis graft(n=53) and STSG(n=33) had reepithelialized after 15.5+/-1.9 and 11.8+/-1.6 days, respectively. The scarring were less severe on the dermis graft in terms of pigmentation, height, and vascularity(p<0.05). No significant difference in pliability was detected. The patients' satisfaction with the dermis graft was also better. Concerning the donor sites, the wounds healed within 7.5+/-0.8 and 12.8+/-1.1 days, respectively. In terms of scar quality and patients' satisfaction, the dermis graft(n=26) showed better results. The dermis graft is superior to conventional STSG both aesthetically and functionally in both the recipient and donor sites.

A New Experimental Model for the Evaluation of Wound Healing: Porous Polyethylene Implantation

Tissue regeneration, such as neovascularization and collagen synthesis, is the most important element throughout the whole process of wound healing. However, this research lacks an appropriate experimental model for the quantitative analysis of the extent of tissue regeneration that takes place in the animal experiments. This lack motivated us to introduce our experimental model, using Medpor(R)(porous polyethylene implant). Our model is designed to analyze the degree of tissue regeneration quantitatively on the basis of the which concept of "the amount of tissue ingrown during a definite time period within a definite volume unit." After medpor discs were loaded with fibroblast, they were implanted at the back of 2 white rats. In control group the medpor discs were not load with cells. Six discs per group(total 12 discs) were used in this study. At three time intervals from 1 to 3 weeks, the implanted discs were harvested and processed for histologic study. The microvascular density method(MVD) was adopted to observe neovascularization, of which only obvious microvessels were counted under the light microscopy(x 100), while the focus of the microscope was fixed at the center of the sample unit segmented. Following the 1st, 2nd and 3rd week observation periods, the noticeable change was identified in the ingrowths of soft tissues of both control and fibroblast group. Regarding MVD, there was no significant difference among groups at 1 and 2 weeks. At 3 weeks, there was significant difference in MVD between the 2 groups; the MVD of fibroblast group(26 and 34) was markedly higher than that of the control group(14 and 16). It is expected that the our experimental model will have a wide application to the whole of research concerned with processes of wound healing involving angiogenesis, regeneration experimentation, etc.

Effect of Transplantation of Human Mesenchymal Stem Cells and Dermal Fibroblasts on the Angiogenesis in Rats

It has been established that the graft of cryopreserved dermal fibroblast is able to improve wound healing. Transplantation of mesenchymal stem cells has the ability to undergo site-specific differentiation and participates in wound healing process. The wound healing process requires angiogenesis and the formation of a vascular network throughout the newly formed tissue. In this study we examined the effect of xeno- transplantation of fresh human mesenchymal stem cells and dermal fibroblasts on the angiogenesis. Human mesenchymal stem cells and human fibroblasts were isolated from bone marrow and dermis of the same patients and were grown in culture respectively. We have developed a porous polyethylene disc as an experimental model system for angiogenesis. Single block of polyethylene was cut into discs which were 5 mm in diameter and 3 mm in thickness. Four discs were soaked in a solution containing 4 106 cells and 1 ml thrombin. After porous polyethylene discs were loaded with the cell-thrombin composite, they then were coated with fibrinogen. After the discs were cleaned from surrounding fibrin, they were implanted in the back of Sprague-Dawley rats. In group I, the discs were filled with fibrin alone without cells. In group II and III, the discs were loaded with fibroblasts and mesenchymal stem cells respectively. Eight rats and 16 discs per group(total 48 discs) were used in this study. After creating 6 pockets in the back of a rat, 6 discs(2 discs per group) were implanted. At three different time intervals from 1 to 3 weeks, the implanted discs were harvested and processed for histological study. A longitudinal section was cut with a thickness of 6 micrometers in the very middle of a disc. Histological study was carried out to examine the formation of microvessels in the implants. Microvascular density was measured by counting the number of microvessels in the very middle of a biopsy specimen under 100 magnification field. Only fibrinoid materials and inflammatory cells were detected in most of specimens in the first week under 100 magnification field of the very middle. There was no significant differences in microvascular density among the three groups. In the second week, extracellular matrices including microvessels were detected in all the 3 groups. The microvascular density of group III(17.75/ 100 magnification field) was significantly higher than that of group II(10.50/100 magnification field) and group I (10.25/100 magnification field). The third week specimens showed that most of the pores of the implants contained extracellular matrices. Significantly greater differences were seen in the microvascular density. The microvascular densities averaged 52.88, 26.12, and 17.50 per 100 magnification field for group III, II, and I respectively. The results indicate that transplantation of mesenchymal stem cells and dermal fibroblasts can significantly improve the angiogenesis in the wound healing process and mesenchymal stem cells are superior to dermal fibroblasts.