This research was presented as a poster at the 44th Annual Conference on Shock GOES Virtual Meeting in Portland, OR, USA (October 12-15, 2021).
Chronic wounds are a serious medical condition affecting over 6 million people in the United States. Biofilms, which alter the host immune response and establish a microenvironment that prevents wound healing, are intimately associated with the development of chronic wounds. Current treatment options do not specifically target the underlying molecular mechanisms of biofilm pathology. Paracrine factors secreted by mesenchymal stem cells (MSCs) have been demonstrated to play an important role in wound healing. The objective of this study was to examine the effects of the paracrine factors secreted from MSCs on reducing infection and accelerating wound closure in biofilm-infected wounds.
MSCs were cultured by seeding on an extracellular matrix (ECM). The supernatant from MSCs containing paracrine factors were applied to mature
The paracrine factors from MSCs grown on ECM were found to reduce
The results indicate that paracrine factors from reprogrammed MSCs accelerated wound healing and reduced the bacterial burden in biofilm-infected wounds. Future studies are needed to further characterize this phenomenon.
Chronic wounds are a major public health issue resulting in a significant financial burden on the healthcare system [
In chronic wounds, a constant state of inflammation due to complex interactions between microbes and the wound microenvironment exists [
Human MSCs were isolated from the iliac crest bone marrow of healthy donors after obtaining written informed consent. Institutional review board approval of the protocol was grant-ed by Tulane University. MSCs were harvested from donors using previously described techniques [
MSCs were seeded onto porcine small intestinal submucosa (SIS; Surgisis Biodesign, Cook Surgical, West Lafayette, IN, USA) as previously described [
Cell culture media was completely exchanged 24 hours prior to collection of paracrine factors for the static/ECM seeded MSCs. The collected media was fractionated using 3 kDa Am-icon Ultra-4 centrifugal filters (Millipore, Billerica, MA, USA) at 3,200 ×
All samples were analyzed using the Milliplex Multi Analyte Profiling Human Cytokine/Chemokine 14 analyte premixed kit (Millipore) following the manufacturer's instructions. Samples were assayed in duplicate wells (25 μL per well) and the mean was determined. The plates were read with a Bioplex analyzer (Bio-Rad, Hercules, CA, USA). Cytokine concentrations were calculated in pg/mL based upon an eight-point five-parameter logistic standard curve for each cytokine. The values were normalized at each time to the number of viable cells. The cytokines studied included: granulocyte macrophage colony stimulating factor (GM-CSF), interferon (IFN)-gam-ma, interleukin (IL)-1b, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-alpha.
Adult BALB/cJ male mice (8–12 weeks old) obtained from the Jackson Laboratory (Bar Harbor, ME, USA) were used for all experiments (n=17). All animals were housed in individual cages under constant temperature and humidity with a 12-hour light/dark cycle with access to chow and water ad libitum throughout the study. All animal procedures and care were reviewed and approved by the Tulane University Institutional Animal Care and Use Committee (IACUC No. #4261) and performed in accordance with all Association for Assessment and Accreditation of Laboratory Animal Care International and IACUC standards.
Mice were individually anesthetized with isoflurane. Hair was removed from the dorsal surface with a depilatory agent and rinsed with an alcohol wipe. The site was prepped with beta-dine and draped. Sterile technique was maintained throughout the entire procedure. A 5mm punch biopsy was used to outline a pattern for the wound on one side of the mouse's dorsum. One wound was created per mouse. Full thickness wounds were created using Iris scissors to extend through the panniculus carnosus. A 10-mm square-shaped splint fash-ioned from a 0.5mm thick silicone sheet (Grace Bio-Labora-tories, Bend, OR, USA) was positioned with the wound cen-tered within the splint. An immediate-bonding adhesive (Kra-zy Glue; Elmer's Inc., Columbus, OH, USA) fixed the splint to the skin, followed by interrupted 4-0 PDS sutures to stabilize its position. A 10-mm square antimicrobial dressing (Telfa; Covidien, Mansfield, MA, USA) with a 5mm hole in the cen-ter to prevent contamination of the wound with any surrounding skin bacteria was placed over the silicone splint and the wound was dressed with a sterile semi-occlusive transparent film dressing (Tegaderm; 3M Health Care, St. Paul, MN, USA). This dressing did not contain any antimicrobial agents. The mice were wrapped around their midline with a self-ad-herent wrap (Coban; 3M Health Care) to protect the wound. All mice received the same type of dressing.
All mice received 0.9% NaCl (50 cc/kg) and buprenorphine (0.01–0.03 mg/kg) subcutaneously each day following surgery. They were also given a liquid diet (DietGel; ClearH2 O, Portland, ME, USA) to avoid dehydration post-surgery. Mice were weighed every 2 days during dressing changes and were euthanized if they lost greater than 30% of their original body weight.
On post-surgery day 2, mice in the biofilm group (n=6) and the treatment group (n=6) were inoculated with 105 CFU
Outline of study groups for murine wound healing study. Day, post-surgery day.
Digital photographs of each wound were taken every 2 days. A camera was affixed onto a stand to allow for a standard distance to the mice. The area of each wound was measured in pixels using image analysis software (Image J, v1.44; NIH, Bethesda, MD, USA). Post-wounding measurements were normalized to the corresponding day 0 wound area and re-corded as a percentage of the original wound area. Validation of area measurements was performed as previously described [
Concentrated media (100 µL) from MSCs grown on SIS for 7 days was mixed with an equal amount of petroleum ointment (Aquaphor; Beiersdorf Inc., Wilton, CT, USA) and applied topically to each wound in the treatment group at post-surgery day 4. The same ointment without MSCs was applied to wounds of the other (control and biofilm) groups on the same day.
At post-surgery days 5, 7, and 9, mice were sacrificed from all three groups. A 5mm punch biopsy was used to excise the wound. All tissues were weighed and homogenized in sterile saline. The homogenate was serially diluted and plated on LB agar and incubated at 37°C overnight to quantitate CFU. Car-diac puncture was also performed at the time of sacrifice to obtain blood cultures and 0.1 mL was plated on LB agar and incubated at 37°C overnight.
Wounds were harvested using a 10-mm punch biopsy and bi-sected at their largest diameter for staining. They were pre-served in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). A pathologist blinded to the samples evaluated the epithelial gap size in each sample with light microscopy at a magnification of 200×.
Data are presented as the mean±standard error of the mean. The Shapiro-Wilk test was used to assess for normal distribution of the data (SPSS version 27; IBM Corp., Armonk, NY, USA). Significance of the treatment group compared to the biofilm group was determined using an unpaired two-tailed Student t-test. A P-value ≤0.05 was considered to be statistically significant.
Detectable levels of all 14 cytokines were found in the supernatant from MSCs grown on the ECM. Higher levels of the pro-inflammatory cytokines GM-CSF, IL-6, IL-8, and MCP-1 were detected after 7 days of growth. There was no change in levels of IFN-gamma, IL-1b, IL-2, IL-4, IL-7, IL-10, IL-12, IL-13, and TNF-alpha detected at 7 days.
In pilot studies, it was determined that inoculums of biofilm >105 CFU led to a significant weight loss and mortality (data not shown). Therefore, the inoculum of 105 CFU was chosen. No mice were found to have >102 CFU in the blood cultures at the time of sacrifice. At day 0, the BALB/cJ mice ranged in weight from 20.7–31.6 g. Average weight in the control group was 26.1±1.7, average weight of the biofilm group was 28.2± 0.5, and average weight of the treatment group was 26.2±0.6. No statistical significance was found (P>0.05).
To confirm the presence of delayed healing in infected wounds, H&E staining was performed to evaluate the samples for wound closure (
Histology of wounds from control and biofilm mice (H&E stain, ×200).
Morphological changes in the wounds for all three groups during the course of the experiment are shown in
Photographs of gross wounds from mice groups (control, biofilm, and treatment).
The control group had an average of 26.1%±6.5% of the original wound area by post-surgery day 9. In contrast, the biofilm group had an average of 81.4%± 0.3% of the original wound remaining on post-surgery day 9 (post-infection day 7). The treatment group had 39.8%±10.2% of the original wound area remaining at post-surgery day 9 (post-infection day 7 and post-treatment day 5).
The biofilm control, which represented wounds infected with biofilm and treated only with ointment not containing any paracrine factors had 91.4% of the original wound area remaining at post-surgery day 9. All data were determined to be nor-mally distributed (
Percentage of wound remaining in mice groups (control, biofilm, and treatment). a) P=0.03 vs. biofilm day 7; b) P<0.001 vs. biofilm day 9.
Average percentage of wound remaining open for control group, biofilm group, and biofilm group treated with paracrine factors from mesenchymal stem cells
% Open wound | Control (n=5) | Biofilm (n=6) | Treatment (n=6) | P-value | Degree of freedom | T statistic |
---|---|---|---|---|---|---|
Post-surgery day 5 | 76.6 (7.5) | 100 (0) | 83.6 (8.6) | 0.09 | 10 | 1.907 |
Post-surgery day 7 | 47.7 (5.8) | 98.2 (0.8) | 67.4 (11.8) | 0.03
|
10 | 2.604 |
Post-surgery day 9 | 26.1 (6.5) | 91.4 (0.3) | 39.8 (10.2) | <0.001
|
10 | 5.057 |
Values are presented as average (standard error of the mean).
Statistical significance, biofilm vs. biofilm+treatment.
The numbers of viable bacteria in wounds were quantified by plating tissue homogenates (
Quantitative bacteriology for wound biopsies at 5, 7, and 9 days after surgery
Group | Day 5 | Day 7 | Day 9 |
---|---|---|---|
Control (n=5) | <102 (0) | <102 (0) | <102 (0–50) |
Biofilm (n=6) | 1.9×106 (4.8×105–4.8×106) | 8.0×106 (2.7×104–1.7×107) | 8.9×106 (1.2×104–3.4×107) |
Treatment (n=6) | 6.2×106 (1.0×106–1.1×107) | 8.4×105 (8.0×105–8.7×105) | 6.8×105 (5.5×105–8.1×105) |
Results are mean counts in colony forming units per gram of body tissue. The sample range is also shown.
Chronic wounds are a significant source of morbidity and mortality in the healthcare field. This study used a well-established
Previous studies have demonstrated that stem cells have an effect on the wound healing process [
There are few well-studied biofilm models and even fewer have been used to evaluate the different treatment strategies of biofilms [
There are several limitations to this study. First, the number of mice for the treatment group was too low to reach statistical significance. In addition, mouse skin heals primarily by contraction in comparison to human skin which heals largely by epithelization. The healing process of the animal model was altered through using a silicon ring to prevent contraction, however, there are still some limitations with translating these results to human skin [
In conclusion, the results from this study demonstrated a novel treatment strategy for chronic wounds using the Paracrine factors secreted from reprogrammed MSCs to decrease bacteria counts and accelerate wound closure in an animal study. Wound healing is a dynamic process that involves many different types of cells. Existing strategies to heal chronic wounds that focus on one or two agents are too simplistic. MSC-based paracrine factor therapies offer the advantage of several cytokines, growth factors, and peptides being secreted at different stages, which may be critical for the development of novel strategies to heal wounds.
We would like to thank Dr. Lisa Morici from Tulane University in New Orleans, LA for providing the bacterial strain. We would also like to acknowledge Dr. Donald Phinney from Scripps Institute in Juniper, FL for providing the mesenchymal stem cells. We also thank Dr. Rodney Shackelford from Tulane University in New Orleans, LA for providing the histologic analysis.
No potential conflict of interest relevant to this article was reported.
Data on mice weights and experimental strategy
Supplemental data can be found at:
Results of Shapiro-Wilk normality test
Supplemental data can be found at: