WOUND HEALING WITH NASA LED

 

 

Experiments using an ischemia animal model system provide pre-clinical data relevant to human healing problems, chronic non-healing wounds.

 

LED-Wound Healing in Rats

 

An ischemic wound is a wound in which there is a lack of oxygen to the wound bed due to an obstruction of arterial blood flow. Tissue ischemia is a significant cause of impaired wound healing which renders the wound more susceptible to infection, leading to chronic, non-healing wounds. Despite progress in wound healing research, we still have very little understanding of what constitutes a chronic wound, particularly at the molecular level, and have minimal scientific rationale for treatment.

 

In order to study the effects of NASA LED technology and hyperbaric oxygen therapy (HBO), we developed a model of an ischemic wound in normal Sprague Dawley rats. Two parallel 11-cm incisions were made 2.5 cm apart on the dorsum of the rats leaving the cranial and caudal ends intact. The skin was elevated along the length of the flap and two punch biopsies created the wounds in the center of the flap. A sheet of silicone was placed between the skin and the underlying muscle to act as a barrier to vascular growth, thus increasing the ischemic insult to the wounds. The four groups, each consisting of 15 rats, in this study include: the control (no LED or HBO), HBO only, LED (880nm) only, and LED and HBO in combination. The HBO was supplied at 2.4 atm for 90 minutes, and the LED was delivered at a fluence of 4J/cm2 for fourteen consecutive days. A future study will incorporate the combination of three wavelengths (670nm, 728nm, and 880nm) in the treatment groups.

 

The wounds were traced manually on days 4, 7, 10, and 14. These tracings were subsequently scanned into a computer and the size of the wounds was tracked using SigmaScan Pro software. Figure ! depicts the change in wound size over the course of the 14-day experiment. The combination of HBO and LED (880nm) proves to have the greatest effect in wound healing in terms of this qualitative assessment of wound area. At day 7, wounds of the HBO and LED (880nm) group are 36% smaller than those of the control group. That size discrepancy remains even by day 10. The LED (880nm) alone also showed to speed  wound closure. On day 7, the LED (880 nm) treated wounds are 20% smaller than the control wounds. By day 10, the difference between these two groups will be closed.

Hence, the early differences are the most important in terms of determining the optimal effects of a given treatment. This can be seen in Figure 1 at day 14 when the points are converging due to the fact that the wounds are healing.

 

Analysis of the biochemical makeup of the wounds at days 4, 7 and 14 is currently underway. The day 0 time point was determined by evaluating the punch biopsy samples from the original surgery. The levels of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were determined using ELISA (enzyme linked immunosorbent assay). The changes in the VFGF concentration throughout the 14-day experiment can be seen in Figure 2. The LED (880nm) group experiences a VEGF peak at day 4 much like the control group. In contrast, the hyperoxic effect of the HBO and LED (880nm) can be seen at day 4. The VEGF level for the group receiving both treatments is markedly higher at day 4 than the HBO treatment. Hence, there is a more uniform rise and fall to the VEGF level in the combined treatment group as opposed to the sudden increases seen in the control, LED only, and HBO only groups. By day 14, the HBO treated groups have dropped closer to the normal level than the LED (880nm) only or control groups.

 

The synergistic effects of HBO and LED (880nm) can be seen easily in Figure 3. The pattern of the changes in basic fibroblast growth factor (FGF-2) concentration is similar to that of the VEGF data. It is clear that the LED (880nm) day 4 peak is higher than the day 4 peak of the control group. These peaks can be attributed to the hypoxic effect of the tissue ischemia created in the surgery. The hyperoxia of the HBO therapy has a greater effect on suppressing the FGF-2 concentration at day 4 than the VEGF concentration at the same time point. The synergy of the two treatments is evident when looking at the HBO and LED (880nm) treated group. The concentration of FGF-2 at day 4 is significantly enhanced by the LED (880nm) treatment. Whereas, the level would normally drop off by day 7 for a LED-only treated wound, the HBO effect seizes control causing the concentration of FGF-2 to plateau. Hence, an elevated FGF-2 concentration is achieved throughout the greater part of the 14 day treatment with both HBO and LED (880) therapies. Further analysis of the excised wounds will include matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9) determination by ELISA, histological examination, and RNA extraction.

 

FIGURE 1. Change in wound size(%) in rat ischemic wound model Time (Days).

 

LED-Wound Healing in Human Subjects

 

Preclinical and clinical LED-Wound Healing studies were reported previously (Whelan et al., 1999, 2000); and additional human trials are summarized below:

Submarine atmospheres are low in oxygen and high in carbon dioxide, which compounds the absence of crew exposure to sunlight, making wound healing slower than on the surface. An LED array with 3 wavelengths combined in a single unit (670, 720, 880 nm) was delivered to Naval Special Warfare Group-2 in Norfolk and d data collection system has been implemented for musculoskeletal training injuries treated with NASA LEDs. Data collection instruments now include injury diagnosis, day from injury, range of motion measured with goniometer, pain intensity scales reported on scale 1-10, girth –circumferential measurements in cm, percent changes over time in all of the aforementioned parameters, and number of LED-treatments required for the subject to be fit –for-full-duty(FFD). Data have also been received from Naval-LEDs and results indicate>40% improvement in musculoskeletal training injuries. Data has also been received form the USS Salt Lake City(submarine SSN 716 on Pacific deployment) reporting 50% faster(7 day) healing of lacerations in crew members compared to untreated control healing (approximately 14days)

FIGURE 2. Change in vascular endothelial growth factor (VEGF) concentration (µg/mg Protein) vs. Time (Day) in rat ischemic wound model.

 

FIGURE 3. Change in basic fibroblast growth factor (FGF-2) concentration (µg/mg Protein) vs. Time (Day) in rat ischemic wound model.

 

In addition to ischemic and chronic wound healing, we have recently begun using NASA LEDs to promote healing of acute oral lesions in pediatric leukemia patients. As a final life-saving effort, leukemia patients are given healthy bone marrow from an HLA-matched donor. Prior to the transplant, the patient is given a lethal does of chemo and radiation therapy in order to destroy their own, cancerous, bone marrow. Because many chemotherapeutic drugs as well as radiation therapy kill all rapidly dividing cells indiscriminately, the mucosal linings of the mouth and gastrointestinal tract are often damaged during the treatment. As a result of these GI effects, patients often develop ulcers in their mouths (oral mucositis), and suffer from nausea and diarrhea. Oral mucositis is a significant risk for this population as it can impair the ability to eat and drink, and poses a risk for infection in this immunocompromized patient. Because lasers have been shown to speed healing of oral mucositis (Barasch, et al,1995), we have recently expanded the wound-healing abilities of NASA LEDs to include these oral lesions. Beginning on the day after the last dose of chemotherapy, we treat one side of the mouth with a 688nm LED at 4J/cm2 daily until the lesions are healed. Dental clinicians monitor the rate of healing by using an Oral Mucositis Index (Schubert, et al, 1992) and a Visual Analog Scale to assess mouth pain. Although many BMT patients must receive intravenous feeding due to their oral mucositis, all of the patients we have treated with LEDs have been able to eat, drink, and talk. All have had nausea, diarrhea, and sore throats, indicating mucositis elsewhere in their GI tract but their oral cavities have been markedly less affected by mucosal ulcers. This study has only included 10% of our target subject number (3/30), and the data so far is preliminary (figurel), but reports by the attending oncologists reveal that these patients have developed significantly less oral mucositis than was expected, especially Patient 2 who receive Melphalan, which is notorious for causing severe mucositis. All patients have had Patient Controlled Analgesia (PCA) with morphine sulfate, but all have reported that it was not their mouths that caused them to activate it.

 

Patlent #1

16 year old male with recurrent Hodgkin’s Disease

Received radiation therapy and Cytoxan, ARA-Cprior to Bone Marrow Transplant

Began LED treatment for Mucositis of June 22,2000

LED treatment ended on July 10,2000 when patient was discharged fom the hospital.

Tolerated oral feeds and reported infrequent use of analgesics for mouth pain.

Patient #2

16year old male with AML (recurrent)

Received radiation therapy and Melphalan, Methotrexate, and Cyclosporin prior to BMT

Began LED treatments for Mucositis on June 22, 2000

Completed LED treatments on July 21, 2000

Tolerated oral feeds and reported infrequent use of analgesics for mouth pain.

Patient #3

6 year old male with relapsed T-Cell ALL

Radiation therapy and ARA-C w/o Aaparaginase, Daunomycin, Doxorubicin, Idarubicin.

This is this child’s second Bone Marrow Transplant. He developed oral mucositis severe enough to require admission to the ICU and a trach with vent assisted respirations with his first BMT in another facility.

LED treatments began July 12, 2000, and were completed August 11, 2000.

He has minimal mouth sores and is able to tolerate some oral feeds. Mucositis continues in his throat and lower GI system as evidenced by nausea and diarrhea.

 

Further In Vitro LED Cell Growth Studies

In order to better understand the effects of LEDs on cell growth and proliferation, we have measured radiolabeled thymidine incorporation in virto in several cell lines treated with LEDs at various wavelengths and energy levels.

As previously reported (Whelan, 2000), 3T3 fibroblasts (mouse derived skin cells) responded extremely well to LED exposure. Cell growth increased 150-200% over untreated controls. Additionally, we have treated osteoblasts(rat derived bone cells), and  L6 rat skeletal muscle cells with LEDs  and have found that both fibroblasts and particularly osteoblasts demonstrated a growth-phase specificity to LED treatment, responding only when cells are in the growth phase. In these experiments, fibroblasts and osteoblasts at a concentration of 110 cells/well were seeded in 24 well plates with a well diameter of 2 square centimeters. DNA synthesis was determined on the second, third and fourth days in culture for both fibroblasts (figure 1) and osteoblasts (figure 2). Exposure to LED irradiation accelerated the growth rate of fibroblasts and osteoblasts in culture for 2 to 3 days (growing phase), but showed no significant change in growth rate for cells in culture at 4 days (stationary phase). These data are important demonstrations of cell-cell contact inhibition, which occurs in vitro once cell cultures approach confluence. This is analogous in vivo to a healthy organism, which will regenerate healing tissue, but stop further rowth when healing is complete. It is important to demonstrate that LED treatment accelerates this normal healing

Patient 1- Pain intensity scale vs.              Patient 1 – Degree of ulcerative

Observation number reported during         mucositis changes vs. observation

BMT. Light was applied to the left           number during BMT. (Scale: 0=

Cheek. Even though the left cheek           no change; 1=mild; 2=moderate;

experienced the greatest degree             3=severe change). The light was

of ulcerative mucositis, the patient            applied to the left mucosa.

Reported the least pain in this area.

Patient 2 – Pain intensity scale              Patient 2 – Degree of ulcerative 

(100mm) vs. observation number           mucositis changes vs. observation

 reported during BMT.                     number during BMT.

Patient 3 – Pain intensity scale            Patient 3 – Degree of ulcerative

(1-6) vs. observation number              mucositis changes vs. observation

Reported during BMT.                     Number during BMT.

 

FIGURE 4. LED-treatment results in three patients with mucositis.

 

 and tissue regeneration without producing overgrowth or neoplastic transformation. Similar data is also currently under study using skeletal muscle cells A human gingival fibroblast cell line and a human epithelial cell line have been acquired for further study and both cell lines will be cultured and treated with LED light at 688nm and doses of 4 and 8J/cm2.

 

FIGURE 5. 3T3 Fibroblast DNA Synthesis – LED – response, 4& 8J energy/cm2 using combined wavelengths of 670nm, 728nm & 880nm showing growth phase specificity (% change from Control vs. # of days in culture).

 

 

FIGURE 6. Osteoblast murine MC3T3 – E1  cells DNA Synthesis- LED-response at 4J energy / cm2 using individual wavelengths of 670nm, 728nm, 880nm showing growth phase specificity (% change from Control vs. # of days in culture).

We have begun collaborating with Dr. Neal Pellis and Dr. Dennis Morrison at Johnson Space Center in Houston. Their experiments consist of: (1) Using LEDs for Photodynamic Therapy (PDT) with the PDT drugs “microencapsulated” using microgravity technology, which will further improve the selectivity of PDT because “microcapsules” lodge selectively in cancer-tumor capillaries.

(2) Using LEDs to stimulate cell healing mechanisms in “simulated” microgravity, using the Johnson Space Center “Rotating Bioreactor” and then proceed to Bioreactor experiments with the LEDs treating cells in the actual microgravity environment of space flight. This addresses the need for “countermeasures” under the new NASA Bioastronautics Initiative. The effects of simulated microgravity on cellular damage and repair mechanisms using bioreactors and using near –IR light ( 630-880nm) to stimulate wound repair at the cellular level as a possible countermeasure for wound healing and cellular repair during space the related research background that could be used for an Earth-based analogue to determine whether or not the near-IR light affects key mechanisms in microgravity. This is an important topic that we should study under the new Bioastronautics Initiative.

 

ACKNOWLEDGMENTS

We wish to thank Karen Zeqiri for assistance in manuscript preparation. The LED arrays were provided by Quantum Devices, Inc., Barneveld, WI. We also gratefully acknowledge the Department of Defense, Air Force Material Command , Armstrong Laboratories, Davis Hyperbaric Laboratory, Brooks Air Force Base, TX for providing the hyperbaric chamber used in this research. The hyperbaric oxygen treatments of our human subjects were performed by Estelle Woodard, C.R.T., C.H.T. This work was supported by the National Aeronautics and Space Administration, Marshall Space Flight Center SBIR grants: NAS8-99015, Children’s Hospital Foundation, the MACC Fund and Quantum Devices, Inc.

 

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