Progress 01/01/24 to 12/31/24
Outputs
Target Audience:This project centers on exploring the efficacy of non-antibiotic biotherapeutics to control antibiotic-resistant bacteria. Antimicrobial resistance is an expanding public health concern, with methicillin-resistant S. aureus (MRSA) as a prominent example. It is proposed that domestic animals, including those of agricultural importance, can function as reservoirs and shedders of MRSA, and MRSA transmission between animals and humans in both ways has been reported. This projectserves the interests of diverse audiences invested in antimicrobial stewardship. We delivered science-based knowledge to these audiences by presenting our data at conferences, including the Conference of Research Workers in Animal Diseases (CRWAD) and the North American Veterinary Regenerative Medicine Association (NAVRMA) Conference. We have also delivered our findingsto a broader audience, including horse owners, veterinarians, and people from the equine industry, where and when applicable. Women who work on this project are the PDDr. Rebececca Harman, technician Kelly Oxford, post-doc Aarthi Rajesh, and Combined degree graduate student Megan Fahey. This research provides a platform for each team memberto gain skills and make connections in agricultural animal research and outreach. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Post-doctoral Associate Dr. Aarthi Rajesh: Dr. Rajesh's PhD work focused on wound healing in mouse models. Since joining ourlab in the spring of 2022, she has been doing in vitro experimental work with equine immune cells and in vivo mouse wound healing studies. She is currently optimizing ex vivo equine skin explant models. Dr. Rajesh was trained in various techniques, including flow cytometry, confocal microscopy, and image analysis. Working on thisproject, has required her tooptimize exisitingprotocols and establishnew protocols. She has learned new techniques and developed analytical skills to interpret data. She has implementedinnovative ideas and presenteddata to the scientific community. Graduate Student Megan Fahey: Megan Fahey is a combined DVM-PhD degree student in the lab. Megan was heavily involved in the in vivo preliminary study of the experimental skin wound infection model in horses and tookthe lead role in data analysis and presentation for this study. She has characterized mesenchymal stromal cells (MSCs) isolated from a novel tissue source and is currently conducting in vitro anti-bacterial experiments. Megan hasgained first-hand experiencesin developing, establishing, and executing experimental in vivo protocols. She isdeveloping a range of laboratory skills. She has also had the opportunity to improve her scientific writing and presentation skills. How have the results been disseminated to communities of interest?This reporting period,results from our workhave been presented at the national CRWAD Conference to an audience with interests in microbiology, immunology, and veterinary research as well as at a Univeristy work-in-progress seminar to an audience interested in stem cell therapies.Our results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. What do you plan to do during the next reporting period to accomplish the goals?During the next (final) reporting period, Dr.Aarthi Rajesh will finalize and submit another paper for publication, describing her in vitro work focused on theeffects of the equine mesenchymal stromal cell (MSC) secretome on immune cells. She will also finsh her experiments using ex vivo explant infection models and prepare that work for publication.
Impacts
What was accomplished under these goals?
Aim 1: We published a manuscript in a peer-reviewed journal describing the effects of the mesenchymal stromal cell (MSC) secretome on bacterial clearance, immuno modulationand wound healing in vivo using a mouse model. We also expanded our in vitro studies on the ability of the MSC secretome to controlMRSA growth using MSCs isolated from a novel tissue source (dental pulp) and MRSA field strains isolated from horse skin wounds. Aim 2: We are currently optimizing a 3D skin MRSA biofilm explant model, and refining methods to study the indirect anti-MRSA effects of the MSC secretome via stimulation of local immune cell populations. Aim 3: We publishes a manuscript in a peer-reviewed journal describing the results of our pilot study of the ability of the MSC secretome to control MRSA and promote wound closure in an in vivo horse model. We also presented this data at a national scientific meeting and a local work in progress seminar.
Publications
- Type:
Other
Status:
Published
Year Published:
2024
Citation:
Fahey MJ. Mesenchymal stromal cell (MSC) secreted products as a novel anti-MRSA treatment for equine skin wounds.
Stem Cell Program Work-in-Progress Seminar, Cornell University.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Aarthi Rajesh, Esther Da Eun Ju, Kelly A. Oxford, Rebecca M. Harman, Gerlinde R. Van de Walle,
The mesenchymal stromal cell secretome promotes tissue regeneration and increases macrophage infiltration in acute and methicillin-resistant Staphylococcus aureus-infected skin wounds in vivo,
Cytotherapy,
Volume 26, Issue 11,
2024,
Pages 1400-1410,
ISSN 1465-3249,
https://doi.org/10.1016/j.jcyt.2024.06.007.
(https://www.sciencedirect.com/science/article/pii/S1465324924007588)
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Fahey MJ, Harman RM, Thomas MA, et al. Preliminary in vivo investigation of the mesenchymal stromal cell secretome as a novel treatment for methicillin-resistant Staphylococcus aureus in equine skin wounds. Veterinary Surgery. 2024; 53(8): 1377-1389. doi:10.1111/vsu.14170
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Treatment of equine thorax wounds wtih the mesenchyaml stromal cell (MSC) secretome. CRWAD Conference, Chicago, IL.
|
Progress 01/01/23 to 12/31/23
Outputs
Target Audience:This project centers on exploring the efficacy of non-antibiotic biotherapeutics to control antibiotic-resistant bacteria. Antimicrobial resistance is an expanding public health concern, with methicillin-resistant S. aureus (MRSA) as a prominent example. It is proposed that domestic animals, including those of agricultural importance, can function as reservoirs and shedders of MRSA, and MRSA transmission between animals and humans in both ways has been reported. This project thus serves the interests of diverse audiences invested in antimicrobial stewardship. We delivered science-based knowledge to these audiences by presenting our data at conferences, including the Conference of Research Workers in Animal Diseases (CRWAD) and the North American Veterinary Regenerative Medicine Association (NAVRMA) Conference. Moreover, we have been delivering our findings via outreach to a broader audience, including horse owners, veterinarians, and people from the equine industry, where and when applicable. Also, women who have been working on this project are the PD, Dr. Gerlinde Van de Walle, her Research Support Specialist, Rebecca Harman, her post-doc Aarthi Rajesh, and her Combined degree graduate student Megan Fahey. This research provides a platform for them to gain skills and status in animal agriculture and outreach. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?* Post-doctoral Associate Dr. Aarthi Rajesh: Postdoctoral training under Dr. Van de Walle Dr. Rajesh has obtained her PhD on wound healing and since joining the lab in the spring of 2022, has been doing all in vitro experimental work with the equine immune cells. Dr. Rajesh was trained in various techniques, including flow cytometry, confocal microscopy, and image analysis. Working on the project, greatly strengthens her competences in optimizing protocols as well as her independence to establishing new protocols, realizing innovative ideas and presenting data to the scientific community. Further, the project allows her to develop new analytical skills to interpret data and she has been using her expertise in mouse models of wound healing. In addition to broadening her skills related to immunology and molecular bacteriology, she also is preparing two manuscripts for submission to peer reviewed journals. * Graduate Student Megan Fahey: Training under Dr. Van de Walle Megan Fahey is a combined DVM-PhD degree student In the Van de Walle lab. Megan has been involved in the in vivo preliminary study of the experimental skin wound infection model in horses and she has taken the lead role in analyzing the collected data and preparing a manuscript for submission to a peer reviewed journal. Working on this project helps Megan to develop important lab skills including bacteriology, histology and immunohistochemistry. Further, she gained first-hand experiences in developing, establishing, and executing experimental in vivo protocols. How have the results been disseminated to communities of interest?The results from our work thus far have been presented at the CRWAD conference to an audience with interests in microbiology, immunology, and veterinary research; at the North American Veterinary Regenerative Medicine Association (NAVRMA) Conference to an audience with interests in stem cell biology in veterinary species; and at the New York Immunology Conference to an audience with interest in immunology. Moreover, our results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. What do you plan to do during the next reporting period to accomplish the goals?The results from Aim 1 demonstrate that the equine MSC secretome has antibacterial, pro-healing and immunomodulatory properties. Experiments are currently underway to test the exact macromolecular subunit of the secretome responsible for these different functions, especially the immunomodulatory properties. Our upcoming experiments will include in vitro functional experiments to compare the responses of neutrophils and macrophages in the presence of either the extracellular vesicle (EV) fraction or the EV-depleted soluble fraction of the MSC secretome. From what we have learned from the pilot study in horses, we are returning to in vitro experiments to optimize the MSC secretome treatment scheme. Firstly, because we found an MSC tissue source-dependent effect of the secretome on immune cells, notably the best effect with bone marrow-derived MSCs, we want to explore whether MSC tissue source might also influence the antibacterial effects of the secretome. In addition to the adipose-, peripheral blood-, and bone marrow-derived MSCs that we have previously used and studied, we have successfully isolated MSCs from equine dental pulp. These tissue sources vary in their accessibility and invasiveness for collection which is another important factor to consider when developing novel biologics. Planktonic bacterial inhibition and biofilm assays are underway to compare the anti-MRSA effect of the MSC secretome from these different tissue sources. These experiments parallel our goals for Aim 2 to optimize the MSC delivery method by considering other factors. We also learned that the hydroxyethyl cellulose negatively impacted wound healing, which in part could be explained by reduced availability of bioactive secretome factors. Consequently, we will explore alternative hydrogels, such as polyethylene glycol (PEG) that is being proposed to improve wound healing when delivered as PEG hydrogel-cellulose nanocomposite, and plan to test the efficacy of these hydrogels both in vivo using a mouse model and ex vivo using a horse skin explant model we previously developed in our lab.
Impacts
What was accomplished under these goals?
This project centers on exploring the efficacy of non-antibiotic biotherapeutics to control antibiotic-resistant bacteria. Antimicrobial resistance is an expanding public health concern, with methicillin-resistant S. aureus (MRSA) as a prominent example. It is proposed that domestic animals, including those of agricultural importance, can function as reservoirs and shedders of MRSA, and MRSA transmission between animals and humans in both ways has been reported. Our laboratory studies the therapeutic potential of equine mesenchymal stromal cells (MSCs), a type of adult stem cells isolated from the blood of horses, in equine wound management. A common complication of equine wounds is infection with bacteria, such as MRSA, and we recently found that equine MSCs secrete factors that can inhibit the growth of MRSA. This project will work on further studying the potential of equine MSC secreted factors to control MRSA. The specific science objectives are to identify which specific MSC secreted factors exhibit the anti-MRSA activity and how they can be delivered effectively to the skin. Moreover, the efficacy of MSC secreted factors to control MRSA will be examined in vivo in horses with infected wounds. Once we understand how equine MSC exert their anti-MRSA effects, we can develop evidence-based strategies to (i) control MRSA infections in horses and reduce the negative impacts of these infections in horses, (ii) prevent transmission of MRSA from horses to humans, and (ii) decrease the use of antibiotics by using these MSC-derived secreted factors as a non-antibiotic biotherapeutic to control antibiotic-resistant bacteria, not only in veterinary medicine but potentially also in human medicine. Accomplishments for this second year of the project include: Aim 1: We examined the impact of equine bone marrow-derived mesenchymal stromal cell (BM-MSC) secretome, which showed the best immunomodulatory capabilities in vitro, on Methicillin-Resistant Staphylococcus Aureus (MRSA) infected wounds in vivo using a mouse model. Mice were subjected to two full-thickness punch biopsies (6 mm) and subsequently infected with 1x107 MRSA. Mice were separated into three groups (n = 4) and each group was treated with either (i) DMEM, (ii) BM-MSC secretome and (iii) antibiotic (muricin) groups. Daily photographs of the wound were taken, and the area of the wound was measured in a blinded manner. Tissue samples were collected on days 3 and 10 post-wounding for subsequent histological and immunofluorescence staining. Morphometrically, the wounds treated with the BM-MSC secretome exhibited similar closure rate as compared to the DMEM-treated wounds. Although the wounds treated with antibiotics demonstrated better closure than the other two groups, this difference did not reach statistical significance. On day 3, there was notable reduction in bacterial colony forming units in both the BM-MSC secretome- and antibiotic-treated wounds. Overall, these results suggest that the BM MSC-CM does play an antibacterial role in infected wounds, at levels comparable to antibiotics. Masson's trichrome staining indicated significant resolution of the granulation tissue formation in BM-MSC secretome- and antibiotic-treated groups at day 10 post-wounding. Immunofluorescence staining for neutrophils in the wounds did not reveal any changes in neutrophil counts at any of the time points tested. Meanwhile, there was a significant increase in the number of macrophages in the BM-MSC secretome-treated wounds when compared to both the DMEM- and antibiotic-treated groups. To further characterize these macrophages, we assessed whether they exhibited a predominantly M1- or M2-like phenotype. Tissues were double stained with iNOS (M1) and CD206 (M2) markers and we found that the infiltrating macrophages at day 10 were primarily CD206+ (M2) cells. M2 macrophages are associated with wound healing and tissue repair. Notably, there were also double positive stained macrophages present in the wounds. More recent evidence has shown that M1+M2+ hybrid macrophages play a part in the resolution of wound healing and scarring. Ongoing experiments are being conducted to elucidate the component(s) of the secretome that is (are) responsible for the observed immunomodulation. Various protocols for the isolation of extracellular vesicles, an important subfraction of the secretome, are currently underway. Working on aim 1 led to a change in knowledge. Using a skin wound mouse model, we showed that the BM-MSC secretome improved wound healing of acute wounds and displayed antimicrobial effects in chronic, MRSA-infected wounds together with an increased infiltration of pro-healing macrophages. These results corroborate our in vitro experiments that equine MSC secretome plays an antibacterial, pro-healing, and immunomodulatory role in vivo. A manuscript describing these results is in preparation and will be submitted later this month. Aim 2: We mixed the secretome with 5% hydroxyethyl cellulose (HEC), at a 1:1 ratio, to see whether we can deliver functional secretome in the form of a gel. Using 6 mm punch biopsies, we inflicted wounds in mice skin and compared the rate of wound closure among four groups: (i) DMEM only, (ii) BM-MSC secretome only, (iii) DMEM + HEC, and (iv) BM-MSC secretome + HEC. Mice treated with HEC (groups iii and iv) exhibited slower wound closure compared to the other groups, suggesting that HEC negatively impacts wound healing, both in itself and/or by limiting the availability of MSC secretome components in the wound. Aim 3: We completed the analyses of our pilot skin wound model in horses. Last year, two horses were enrolled in a pilot study of cutaneous wounds experimentally infected with MRSA or left uninfected. Muricin, a triple antibiotic ointment (positive control), culture medium (negative control), or the MSC secretome was applied topically for three days following wound creation. We've completed analysis of data collected from this study in the areas of (i) bacterial load, (ii) rate of healing and (iii) histological analysis. (i) Bacterial load within the wounds was significantly higher in MRSA inoculated wounds at day 1, indicating successful inoculation. (ii) Wound closure and healing were assessed from photographs taken at days 0, 1, 2, 3, 7, 14, 21, and 28. At day 28, non-inoculated wounds were smaller in wound area than MRSA-inoculated wounds, indicating slightly delayed healing because of MRSA inoculation. (iii) Histologic acute inflammation score (HAIS) and histologic repair score (HRS) were graded from biopsies taken at days 0, 7, and 28 by a board-certified pathologist and dermatologist. No significant differences were seen between inoculation status or with treatment. Although no significant improvement of wound healing parameters was seen with the MSC secretome treatment in this pilot study, these data showed no negative reactions, for example an inflammatory reaction, to MSC secretome treatment in horses. This pilot study and subsequent data analysis established a cutaneous wound healing model with MRSA infection. Further optimization of MSC secretome application to wounds in ongoing. The manuscript for this pilot study is in preparation and is expected to be submitted later this month.
Publications
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Fahey MJ. Mesenchymal stromal cell (MSC) secreted products as a novel anti-MRSA treatment for equine skin wounds. Biomedical and Biological Sciences Work-in-Progress Seminar, Cornell University.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Rajesh A, Ju EDE, Oxford K, Harman RM, Van de Walle GR, Effects of equine mesenchymal stromal cells on innate immunity in vitro and in vivo. Cornell Stem Cell Program Retreat, Cornell University.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Rajesh A, Ju EDE, Oxford K, Harman RM, Van de Walle GR, Effects of equine mesenchymal stromal cells on innate immunity in vitro and in vivo. New York Immunology Conference, Cooperstown, NY.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Rajesh A, Oxford K, Harman RM, Van de Walle GR, Evaluating the efficacy of equine mesenchymal stromal cell secretome on innate immune responses in vitro and in vivo. North American Veterinary Regenerative Medicine Association (NAVRMA), Kona, Hawaii.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Rajesh A, Ju EDE, Oxford K, Harman RM, Van de Walle GR, Evaluating the effects of equine bone marrow mesenchymal stromal cell secretome on MRSA-infected wounds in vivo. Annual Meeting of CRWAD, Chicago, IL.
|
Progress 01/01/22 to 12/31/22
Outputs
Target Audience:This project centers on exploring the efficacy of non-antibiotic biotherapeutics to control antibiotic-resistant bacteria. Antimicrobial resistance is an expanding public health concern, with methicillin-resistant S. aureus (MRSA) as a prominent example. It is proposed that domestic animals, including those of agricultural importance, can function as reservoirs and shedders of MRSA, and MRSA transmission between animals and humans in both ways has been reported. This project thus serves the interests of diverse audiences invested in antimicrobial stewardship. We delivered science-based knowledge to these audiences by presenting our data at conferences, including the Conference of Research Workers in Animal Diseases (CRWAD). Moreover, we have been delivering our findings via outreach to a broader audience, including horse owners, veterinarians, and people from the equine industry, where and when applicable. Also, women who have been working on this project are the PD, Dr. Gerlinde Van de Walle, her Research Support Specialist, Rebecca Harman, and her post-doc Aarthi Rajesh. The in vivo preliminary experiment of an equine MRSA wound healing model that was performed in year 1 of the project involved the board-certified equine surgeon Dr. Michelle Delco, her graduate student Megan Fahey, and 3 female veterinary students helping with horse care. This research provides a platform for them to gain skills and status in animal agriculture and outreach. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?* Post-doctoral Associate Dr. Aarthi Rajesh: Postdoctoral training under Dr. Van de Walle Dr. Rajesh has obtained her PhD on wound healing and since joining the lab in the spring of 2022, has been doing all in vitro experimental work with the equine immune cells. Dr. Rajesh was trained in various techniques, including flow cytometry, confocal microscopy, and image analysis. Working on the project, greatly strengthens her competences in optimizing protocols as well as her independence to establishing new protocols, realizing innovative ideas and presenting data to the scientific community. Further, the project allows her to develop new analytical skills to interpret data. The work broadens her skills related to immunology and molecular bacteriology. * Graduate Student Megan Fahey: Training under Dr. Delco Megan Fahey is a combined DVM-PhD degree student at Cornell university, working in the lab of her mentor Dr. Delco. Megan has been involved in the in vivo preliminary study of the experimental skin wound infection model in horses and she is taking an important lead in analyzing the collected data. This will help Megan to develop important lab skills including bacteriology, histology and immunohistochemistry. Further, she gained first-hand experiences in developing, establishing, and executing experimental in vivo protocols. How have the results been disseminated to communities of interest?The results from our work thus far have been presented at the CRWAD conference to an audience with interests in microbiology, immunology, and veterinary research; and internally at the Baker Institute to faculty and trainees. Moreover, our results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. What do you plan to do during the next reporting period to accomplish the goals?We will continue with our experiments to complete aim 1. Specifically, experiments will be repeated with MRSA-infected wounds in vivo in mice to assess the efficacy of MSC treatment during the different phases of MRSA infections. Bacterial load in wounds will be determined on days two, six and ten-post wounding, to compare the bacterial clearance in control, BM-MSC secretome-treated, and antibiotic-treated groups. Masson's trichrome staining will be used to determine the impact of BM-MSC secretome on different aspects of healing, including re-epithelialization, and granulation tissue formation. Immunofluorescence staining for different immune cell markers will be carried out to investigate the changes in immune cell populations at the different phases of healing in both acute and MRSA-infected wounds. This information will help us guide in what to specifically focus on for analysis of the equine biopsies. We will also identify the secretome sub-fractions responsible for the antimicrobial and immunomodulatory effects, i.e., the soluble fraction or extracellular vesicles. This information will be important when moving on to aim 2, where we will explore MSC secretome delivery systems using a 3D skin MRSA biofilm explant model ex vivo.
Impacts
What was accomplished under these goals?
This project centers on exploring the efficacy of non-antibiotic biotherapeutics to control antibiotic-resistant bacteria. Antimicrobial resistance is an expanding public health concern, with methicillin-resistant S. aureus (MRSA) as a prominent example. It is proposed that domestic animals, including those of agricultural importance, can function as reservoirs and shedders of MRSA, and MRSA transmission between animals and humans in both ways has been reported. Our laboratory studies the therapeutic potential of equine mesenchymal stromal cells (MSCs), a type of adult stem cells isolated from the blood of horses, in equine wound management. A common complication of equine wounds is infection with bacteria, such as MRSA, and we recently found that equine MSCs secrete factors that can inhibit the growth of MRSA. This project will work on further studying the potential of equine MSC secreted factors to control MRSA. The specific science objectives are to identify which specific MSC secreted factors exhibit the anti-MRSA activity and how they can be delivered effectively to the skin. Moreover, the efficacy of MSC secreted factors to control MRSA will be examined in vivo in horses with infected wounds. Once we understand how equine MSC exert their anti-MRSA effects, we can develop evidence-based strategies to (i) control MRSA infections in horses and reduce the negative impacts of these infections in horses, (ii) prevent transmission of MRSA from horses to humans, and (ii) decrease the use of antibiotics by using these MSC-derived secreted factors as a non-antibiotic biotherapeutic to control antibiotic-resistant bacteria, not only in veterinary medicine but potentially also in human medicine. Accomplishments for this first year of the project include: Aim 1: We evaluated the efficacy of the secretome from equine MSC from three different tissue sources, namely bone marrow (BM), fat (AD), and peripheral blood, (PB) on the functionality of equine innate immune cells. MRSA is known to exhibit immune evasion strategies by inhibiting the functionality of neutrophils and macrophages, thus stimulation of these immune cells is important for fighting MRSA infections. We used neutrophil migration assays to evaluate the impact of the MSC secretome on neutrophil chemotaxis. We found that the mean number of neutrophils doubled with AD- and almost tripled with BM- MSC secretome compared to control wells. There was no significant difference in chemotaxis when treated with PB-MSCs. We also evaluated whether the MSC secretome affected the proportions of neutrophil subsets during chemotaxis, based on a human study that found that immature or banded neutrophils played a more substantial role in controlling MRSA infections. The phenotype of equine neutrophils post migration was predominantly segmented (< 70%) in the control wells and those incubated with AD- and PB-MSC secretome. In contrast, there were equal numbers of segmented (50%) and banded (47%) neutrophils when treated with BM-MSC secretome, suggesting a potential higher anti-MRSA activity. When looking at the phagocytic capacities of neutrophils and macrophages, we found a decreased phagocytic capacity of neutrophils when incubated with MSC secretome, and no difference in phagocytotic ability of macrophages treated with secretome from either MSC source when compared to control cells. When evaluating the production of reactive oxygen species (ROS) by both neutrophils and macrophages, there was a clear trend towards increased ROS production when cells were incubated with MSC secretome, however, this did not reach significance due to the variations observed in the assays. Due to the strongest increase in neutrophil chemotaxis and highest number of banded neutrophils with the BM-MSC secretome, this source was used for in vivo wound healing experiments, using the mouse as a preliminary model. Mice were separated into control (DMEM) and treated groups (n = 4 per group). Two full-thickness (4 mm) wounds were created on each mouse and the wounds were treated with either DMEM or BM-MSC secretome every day for ten days. The wounds were photographed every day, and the size of the wounds was measured blinded. Tissues were harvested on day 10 (remodeling phase). Experiments were repeated to obtain tissues at day 2 (inflammatory phase) and day 6 (proliferative phase) post wounding for further histological analyses using Masson's trichrome staining. Wounds treated with BM-MSC secretome showed accelerated wound closure when compared to controls. Trichrome staining analyses of the tissues revealed that the accelerated wound closure in the treated group was characterized by enhanced granulation tissue formation with increased vasculature at days 2 and 6 post-wounding. At day 10, MSC secretome-treated wounds appeared more like normal skin with better resolution of granulation tissue, increased collagen deposition, and regeneration of hair follicles. Working on aim 1 led to a change in knowledge. For the first time, we showed that the MSC secrete factors that alter the functions of neutrophils and macrophages in vitro, important first line defense mechanisms to combat wound infections, suggesting that the equine MSC secretome might have the potential to restore impaired immune cell functions in infected wounds. Moreover, we found that the BM-MSC secretome improved wound healing in vivo. Aim 3: We performed a preliminary pilot study of an experimental skin wound MRSA infection model in horses. To this end, two horses were enrolled in the study, with each horse receiving 12 full thickness skin wounds on the torso, 6 per side. The wounds on one side of each horse were inoculated with MRSA, the wounds on the other side were not inoculated. On each side, following treatments were administered topically, in duplicate; (i) positive control (antibiotic ointment), (ii) negative control (base culture medium) and (iii) MSC secretome. The procedures of creating and inoculating the wounds went smoothly, and the administration of treatments was successful. Wound status was determined by (i) bacterial load in wounds, based on culturing swabs collected from wounds at intervals for 4 weeks (ii) rate of healing, based on scoring photographs as well as measuring wound area, at intervals for 6 weeks, and (iii) histological analysis of wound margin biopsies taken at 2 time points during the process of wound healing. The bacterial data from the first couple of days showed a trend towards the MSC secretome reducing bacterial wound loads when compared to the negative control DMEM-treated wounds. A consensus wound scoring table was established and is being used for blind scoring (ongoing). Wound margins have been calculated and data are being analyzed. Biopsies have been submitted for histology and will be evaluated blinded by a board-certified anatomic pathologist. A preliminary screening showed differences in granulation tissue formation, but we don't know yet if this is related to the treatments as the analysis will not be unblinded until after all analyses are finished. This successful preliminary study informed us that our general study design and protocols are appropriate for a larger experiment, and we intend to conduct a study with more horses in the future, with some modification. For example, we will decrease the size of each wound to 2.5 cm2, which will make measuring wound area more accurate and increase the ratio of wound margin to "center". With more wound margin, it will be easier to capture subtle differences in re-epithelialization and will give us a larger area to biopsy. We also will start the treatments 2 days post-wounding, to give bacterial infections a chance to become solidly established before treatment.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Rajesh A, Harman RM, Van de Walle GR. Impact of equine mesenchymal stromal cell secretome on innate immune response during wound healing. 103rd Annual Meeting of CRWAD
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Rajesh A. Impact of equine mesenchymal stromal cell secretome on innate immune response during wound healing. Baker Institute seminar series, Cornell University.
|