Noninvasive Assessment of Progenitor Cell Persistence in Wound Beds of Immunocompetent Mice

Abstract: Synthetic grafts have become a clinical standard for the acute and chronic surgical care of wounds. While these grafts provide basic wound site coverage, the final outcomes of repair are often inadequate in terms of minimizing scar formation, restoring tissue functionality, and maintaining durability of the repaired site. Accordingly, there is a significant unmet need to develop a “next generation” graft matrices that can actively enhance tissue repair, remodeling and regeneration. To this end, the development of molecular tools to better understand the molecular physiology of wound repair is essential. In the experiments described here, we exploited advances in technologies that enable the noninvasive monitoring of endogenous gene expression, to evaluate the long-term persistence of grafted and engrafted progenitor cells in the wound bed. We show how a preclinical model using an actin promoter-driven bioluminescent reporter gene can provide a quantitative and non-invasive image assessment of bone marrow cell persistence in an Integra® (Integra LifeSciences, Plainsboro, NJ) graft for at least 2 weeks. This approach, along with other cell and tissue-specific promoters, can be used to develop wound healing models in which it is possible to better understand the pathophysiological relevance of tissue repair therapies. We suggest that the development of an increasing number of transgenic reporter mice that express bioluminescent genes under the regulation of specific promoters can be used as donors to characterize the molecular physiology of wound healing and evaluate the biological effectiveness of innovative engineered biomaterials. From the Department of Surgery, University of California San Diego Address correspondence to: Andrew Baird, PhD Division of Trauma, Surgical Critical Care, and Burns Department of Surgery University of California San Diego 212 Dickinson Street, MC 8236 San Diego, CA 92103 Phone: 619-543-2905 Email: abaird@ucsd.edu   Synthetic matrices have been widely deployed as grafts for acute and chronic wounds, providing physical protection and coverage of the wound bed while supporting wound healing and optimizing subsequent autogenous skin grafting.   While these matrices are currently imperfect with respect to the rate of wound closure, the decrease in final tensile strength, and the extent of scarring, the capacity for these matrices to provide a protected microenvironment for the recruitment and integration of progenitor cells has become an important objective of graft matrix development. For example, many studies have examined the capacity of dissociated keratinocytes, primary cultured fibroblasts and purified progenitor cells from blood and bone marrow, to be applied to the wound bed with the goal of accelerating the rate of wound healing.^1–4 Although these strategies have helped identify specific stem cell/stem cell-like populations within the dermis, advances in skin cell biology and tissue engineering have yet to yield a biologically, structurally, cosmetically, and economically adequate substitute for skin.   In the experiments described here, we examine and then discuss the potential of emerging bioluminescent technologies to evaluate the role of progenitor cells—specifically, their persistence in the process of repair and regeneration in the wound bed. We examined the persistence of donor bioluminescent syngeneic mouse bone marrow cells when injected in a murine model of cutaneous wound healing that uses an Integra® (Integra LifeSciences, Plainsboro, NJ) graft. The results largely corroborate findings obtained with transplanted bone marrow cells in a graft but gain particular relevance because of the immunocompetent mouse used.

Materials and Methods

  Surgery. Integra was sutured into a 1.5 cm full-thickness section of skin on the dorsum of FVB strain-matched mice. We have previously characterized the wound healing and vascular response to this specific graft and have chosen Integra because of its reproducibility, availability and the fact that it is widely used in the UCSD Burn unit.^5,6   Cell transplantation. Immediately following placement of the graft, whole bone marrow cells were aspirated from the long bone marrow of a transgenic Actin-Fluc transgenic donor mouse using standard collection techniques and after washing and counting, 1 x 106 cells were injected in a volume of 150 µL saline under the graft.   Quantification and imaging of cellular persistence. An IVIS Lumina CCD animal imaging system (Caliper Life Science, Hopkinton, MA) was used to detect bioluminescence and the grafts were non-invasively imaged after intraperitoneally injecting the substrate D-luciferin (Caliper Life Science, Hopkinton, MA) at a dose of 1.5 mg in 150 uL of normal saline. Following a five minute incubation to allow for steady-state delivery of the substrate, images were acquired at the following CCD camera settings: Field of View A, 5-minute duration of acquisition, medium binning, and F-stop 1. The dose of luciferin and the time of imaging after injection of luciferin were each optimized separately in preliminary experiments. Quantification of the presence of bone marrow cells was performed using Living Image Software as recommended by the manufacturer (Caliper Life Science). Regions of interest (ROI) were selected using a consistent template for quantification and data was obtained from at least four animals in each group. The photons of light emitted in the ROI of the area encompassing the graft were measured as well as an additional control area away from the graft over the right flank using the same template.   Luminescence was calculated by dividing number of photons of light emitted from the graft area over the control area. These data were analyzed by using the Kruskal-Wallis test for nonparametric data, where significance was determined when P < 0.05. Injections and data acquisitions were performed and optimized according to the manufacturer’s recommendations.

Results

  Over the course of the experiment, homogeneous healing through wound contraction lead to complete wound closure between 14–21 days. Quantitative data was acquired from the same ROI from the same animals over each different time. As shown in Figure 1A and 1B, exposure-matched images establish that luminescence was detectable in the graft area throughout the duration of the study but strongest on day 7 after grafting. The specific location of the persisting bone marrow-derived stem cells (BMSCs) in the wound bed could also be highlighted using contrast enhancement in the Living Image software (Figure 1C). This feature takes full advantage of the high sensitivity of light production and enables a qualitative localization of even the smallest quantities of bioluminescence generated by the luciferase transgene present in the transplanted cells. The collection of quantitative data enables an assessment the progenitor cell contribution to remodeling in the wound bed.

Discussion

  Several preclinical models have been deployed to study the ultimate destiny of transplanted BMSCs. Here, we hypothesized that the decrease in bioluminescence between days 7 and 14 post-grafting reflected a “culling” of the transplanted bone marrow cells by the host’s immune system, which would continue until cells are fully integrated into regenerated tissue. We specifically noted the remarkably long duration in which whole bone marrow cells persisted in the wound. This suggested that while they may have a functional role in the initial stages of wound healing, it does not presumably involve proliferation because the signal is decreased rather than propagated through the wound. Alternatively, the bioluminescent cells that were grafted directly into the wound bed may have migrated to other organ systems and locations. To evaluate this possibility, the grafted animals were sacrificed by cervical ligation and the organs imaged individually ex vivo. We did not observe any differences in the bioluminescence of the internal organs between control and transplanted animals (data not shown). Because the experimental approach measures the expression of luciferase activity as a surrogate for transplanted cells, it is possible that the transplanted BMSC lose their ability to express the transgene due to terminal differentiation or gene loss. Finally, cell fusion between host and transplanted cells may abrogate the luciferase signal. PCR and more invasive detection techniques like immunostaining can evaluate the fate and characteristics of the transplanted cells that persist, but they do not provide kinetic information. Together with quantitative noninvasive imaging, it may be possible to manipulate BMSCs to facilitate wound healing, and optimize their contribution to the kinetics of repair and regeneration.   Most previous research evaluating the fate of stem and progenitor cells transplanted into the wound bed was performed in model systems like chimeric mice, immunodeficient hosts, or undifferentiated stem cells that were specifically designed to minimize donor cell rejection.^2,4,7–11 This is the first study demonstrating a specific and quantifiable example of the persistence of progenitor cells in the wound bed in immunocompetent hosts. While we show that only a small portion of the BMSCs remain in the wound bed, they are indeed relatively long-lasting and point to the need for further assessments of their long term contribution to repair and regeneration in the wound bed. The specificity of the model can also be extended by deploying more cell-type specific promoters other than actin to monitor specific responses in the wound bed. Because the only significant bioluminescence signal detected was emitted from the transplanted cells, there is an excellent signal: noise ratio compared to non-transplantation models and noninvasive imaging enables detection, quantification and localization of discrete changes in the expression of the luciferase reporter.   In the case of chronic wounds, large acute wounds and burns, there is a significant unmet need to develop alternative strategies for synthetic grafts that incorporate growth factors, genes and progenitor cells so as to yield a clinically significant improvement in wound healing.^12–15 In the experiments here, we focused on the question of stem cell persistence and the deployment of its noninvasive quantitation in the wound bed. Several years ago, Falanga and Sabolinski¹⁶ demonstrated that the presence of fetal allogenic skin cells within a synthetic skin-substitute matrix could accelerate wound healing. Indeed, using traditional methods such as in situ hybridization or fluorescent cell labeling to track donor cells in skin wound healing, these models have shown that donor cells persist in the wound bed in some cases for up to 2.5 years.^1,3 Similarly, tagging of BMSC with a number of different reporters (ie, green fluorescent protein, lac Z, and firefly luciferase) can be used to monitor their incorporation and differentiation in the wound bed.^7,17–19 While these studies demonstrated that dermal fibroblasts and bone marrow-derived cells can be tracked as they incorporated into the wound bed, they all required ex vivo tissue analyses at multiple time-points over the duration of the study. These invasive approaches remain impractical to assess and quantify donor cell persistence over the extended kinetics of tissue repair.   Noninvasive imaging with bioluminescent reporter models. In contrast to invasive techniques that require biopsy and ex vivo tissue for analyses, the development of transgenic bioluminescent reporter models (ie, the detection of firefly luciferase reporter) enables the use of noninvasive methods to analyze the hosts’ molecular response to injury. These reporter systems take advantage of naturally occurring light producing enzymes that were originally identified from a variety of sources, including bacteria, marine crustaceans and insects.²⁰ These luciferase enzymes self-emit photons of light in the presence of a substrate, usually luciferin, and a source of energy, such as ATP.²⁰ When activated, light emission can be detected and quantified using a cooled coupled device (CCD) camera and the appropriate software.²¹ When the luciferase gene is incorporated into the genome of a transgenic animal and specifically placed under the control of an endogenous promoter, luciferase gene expression, and therefore the production of light, reports when that promoter is activated.^22,23 Because different promoters are activated at different times and in different locations during angiogenesis, fibrosis, inflammation, and wound consolidation, the detection of light reflects the spatiotemporal activation of endogenous and specific gene pathways. This transgenic bioluminescence reporter system has several distinct advantages over traditional imaging techniques: background luminescence is extremely low, no external excitation light is needed, rapid enzyme turnover rate enables real-time measurements, and there is a linear relationship between the concentration of luciferase enzyme and the peak height of emitted light. Furthermore, repeated administration of D-luciferin is well-tolerated by animals even in serial imaging where animals are injected at 3, 7, 14, and 21 days with no apparent effect on wound healing. Taken together, changes in light emitted can be detected over a range of five orders of magnitude, allowing for both sensitive and quantitative in-vivo imaging of reporter gene expression.²⁴   The most extensively studied of the luciferase enzymes is obtained from the North American firefly, Photinus pyralis, which emits light at a wavelength of 550–570 nm with a peak at 562 nm.^20,22 This firefly luciferase (Fluc) is extremely versatile and has been used in many different bioluminescence applications.^21,25–27 When transgenic mice expressing Fluc under the control of the constitutively expressed β-Actin promoter are used as cell, tissue, and organ donors to non-luciferase expressing mice, then luminescence can be measured noninvasively and quantitatively after bone marrow, pancreatic islet cell, and cardiac transplants.²⁸ For example, Cao et al²⁸ have shown that luminescence was directly correlated with engraftment and transplant survival, and inversely correlated with rejection.   Bioluminescence imaging has also found particular use in the study of tumorigenesis and cancer metastases. Bioluminescent transgenic mice using the promoter for Vascular Endothelial Growth Factor-A (VEGF-A), a well-described angiogenic factor, have been used to evaluate murine mammary tumor progression and reveal that luciferase activity can be detected, and quantified, before the presence of palpable tumors.²⁹ Similarly, when Prostate-Specific Antigen promoter controls luciferase, transgenic mice have been used to study prostate tumorigenesis and the results show that increased luminescence correlates with increased tumor load.³⁰ When the same mice were depleted of androgens the tumors regressed and the luminescence decreased.³⁰   Applying bioluminescent imaging to wound healing paradigms. While some quenching of bioluminescence occurs when overlying tissues are > 2 cm thick, bioluminescence imaging with Fluc is a highly quantifiable technique when detecting cells located in a skin graft or wound. Accordingly, this technology is particularly useful for studies of wound healing.^5,6,20,31 For example, the role of inflammation in host-biomaterial interactions can be studied in NF-κB-luciferase transgenic mice that have the luciferase gene under the control of the NF-κB binding element promoter.^32,33 In this model, increased luminescence of host tissues, both locally and at distant organs, is readily observed when a gelatin-based implant is placed under the skin.³³ When the inflammatory cascade is triggered by an administration of LPS, luminescence dramatically increases both locally and in distant organs.   Traditional hypotheses of wound healing have assumed that the cells involved in tissue repair and regeneration are fibroblasts, endothelial cells, and keratinocytes and that they originate from surrounding normal skin located adjacent to the site of injury.³⁴ However, recent models of tissue repair have highlighted important roles for BMSC in this process.³⁵ These cells, which originate from the non-hematopoietic stem cells of the bone marrow, are not restricted to a specific lineage, and have been shown to “home” to the area of injury, reduce inflammation, engraft into the wounded tissue, differentiate into tissue-specific cells, and assist in tissue repair.^36-40 This process is also observed in skin wounding. Tagged BMSC have been shown to home to injured skin from bone marrow, proliferate at wound edges and differentiate into skin-specific cell types.^{17–19,41–43} Taken together, these studies imply that BMSC facilitate wound healing by differentiating into cells that specialize in tissue repair and regeneration, thus increasing the amount of tissue-repairing cells in the wound and accelerating repair. Alternatively, BMSC may facilitate wound healing by acting in a paracrine fashion and secreting cytokines and growth factors into the local wound area, which in turn recruits and activates more cells from the surrounding tissue.^36,44   Bioluminescent imaging has the potential to play a significant role in the field of tissue engineering, where real-time and continuous measurements of tissue growth over time are critical. Logeart-Avramoglou et al⁹ recently reported on the validity of bioluminescent imaging in a tissue engineering model using extracellular matrix scaffolds seeded with bioluminescent stem cells. The authors demonstrated that light emitted from bioluminescent cells is linearly correlated with the number of cells present. Furthermore, they showed that the results from bioluminescent imaging are comparable to those obtained by traditional fluorescent imaging. Lastly, they established that the minimal number of labeled cells that are needed to produce a detectable signal is 15-fold less with bioluminescent imaging then with fluorescent imaging. This increased sensitivity in bioluminescent imaging is particularly evident in studies that have shown persistence of luminescent cells within the wound bed for extended periods of time, even up to 90 days after implantation.^2,4,9,10   The use of bioluminescent imaging techniques in the analysis of wound healing is an expanding area of research, where it is being used to establish the kinetics of cell homing and engraftment. It is interesting to note that applications of this technology will also have particular significance in defining the spatiotemporal changes in endogenous gene expression, depending on the promoter used, within the wound bed. Biotherapeutic drug development for wound healing is currently hampered by a need for multiple drugs, at different times, acting on different cells to elicit different responses, all likely dependent on whether the therapeutic target is present during angiogenesis, wound consolidation/closure or scarring phases. Using the kinetic information obtained by dissecting the molecular physiology of each endpoint of the wound healing response, it should be possible to engineer biosynthetic and biodynamic matrices that deliver drugs into the wound bed where they are needed, when they are needed, and for the duration that they are needed. The validation of such engineered therapeutics will require the development and testing of innovative models such as the bioluminescent reporter model we describe here in which to evaluate their efficacy, exploiting a better understanding of the molecular physiology of the wound bed.

References

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