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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 12, 2005 as doi:10.1096/fj.04-2978fje. |
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,1
* Department of Pathology, Vanderbilt University, Nashville, Tennessee, USA; and
Department of Veterans Affairs Medical Center, Nashville, Tennessee, USA
1 Correspondence: Department of Pathology, Vanderbilt University, C3321 Medical Center North, Nashville, Tennessee 37232-2562, USA. E-mail: jeff.davidson{at}vanderbilt.edu
SPECIFIC AIM
Normal wound repair is a dynamic process that is dependent on the appropriate responses of fibroblasts that appear predominantly in the wound after the inflammatory phase, proliferate to develop well-defined granulation tissue, and subsequently contribute significantly to the accumulation of connective tissue (primarily type I collagen) forming the scar. Historically, wound fibroblasts have been thought to derive primarily from the local recruitment and proliferation of resident fibroblasts. Recently, the identification and characterization of novel adult stem cell populations housed within the bone marrow (BM) compartment with developmental potentials beyond that of the pluripotent hematopoietic stem cell, has launched a flurry of investigations focused on examining the plasticity of adult, BM-derived progenitors. Therefore, the overall aim of this study was to examine the potential of adult, bone marrow-derived cells (BMDCs) to participate in wound repair as fibroblasts.
PRINCIPAL FINDINGS
1. BMDCs contribute to experimental granulation tissue beyond inflammation
The advent and widespread use of transgenic animal technologies has opened the door for investigations into the ontogeny of cell lineages once thought derived solely from the local proliferation of organ-specific cell populations, including the wound fibroblast. However, the majority of cell-tracing studies utilize transgenic donors that constitutively express a reporter gene, and therefore rely primarily upon phenotype of marked cells to determine the differentiation capacity of transplanted cells. To investigate the potential of BMDCs to migrate to sites of wound repair and participate in tissue remodeling as fibroblasts, we performed BM transplantation from donor transgenic animals to wild-type recipients. Our model is unique in that the resultant, chimeric animals express the versatile and well-characterized reporter genes ß-galactosidase (ß-gal) and luciferase (LUC) under control of the fibroblast-specific, collagen I 
2 chain (COL1A2) promoter-enhancer. Collagen I is the predominant collagen of the dermis and is highly up-regulated during wound repair. The promoter and enhancer elements for this fibrillar collagen, COL1A2, have been rigorously examined, and the construct used to generate the donor transgenic mouse line has been shown to direct expression to fibroblasts including those of the fascia and dermis. To elicit a naive granulation tissue response, polyvinyl alcohol sponges (PVA) were implanted into stable chimeras and subsequently analyzed for donor-derived nucleic acids and proteins. The PVA sponge implantation model was used due to its fixed dimensions and superiority in assessing quantitative biochemical and kinetic parameters of wound healing. Cells that participate in the PVA-induced granulation tissue response must migrate into the sponge from the circulation or from adjacent adipose, dermal, fascia, or muscle tissue. Sponges can be easily manipulated and examined at defined time points and thus facilitate the kinetic tracing of BMDCs to these naive wounds and their subsequent differentiation potential within the developing granulation tissue. The kinetics of BMDC infiltration were examined by real-time, quantitative PCR for the LUC transgene. Coincident with a decline in the inflammatory phase of wound repair, a statistically significant, 4-fold decline in BMDC numbers was observed between days 7 and 17 (P<0.02). Significant, 4-fold increases in BMDC numbers were observed between days 17 and 22 (P<0.02), suggesting an increase in the local recruitment and/or proliferation of BMDCs beyond inflammation to time points characterized by tissue remodeling.
2. BMDCs contribute significantly to COL1A2 promoter activation during tissue remodeling
To define further the phenotypic and physiologic contribution of BMDCs to the wound environment, quantitative real-time RT-PCR analysis was performed to examine COL1A2 specific promoter activation. Activation of the COL1A2 promoter by BMDCs was distinguishable, yet low, during the inflammatory phase of granulation tissue formation. As inflammation subsided, BM-derived COL1A2 promoter activation increased 4- and 6-fold at days 12 and 17, respectively. At the peak of tissue remodeling, day 22, a > 2-log increase in BMDC COL1A2 promoter activation was observed from day 7, 15-fold higher than at day 17. At day 27, there was a 2-fold logarithmic decline in BM-derived COL1A2 activation from day 22. These transcript level data strongly suggest that BMDCs contribute significantly to extracellular matrix deposition beyond inflammation. Moreover, up to a 2-fold logarithmic increase in the activation of the COL1A2 promoter per BMDC over the time course of these experiments indicates that the increase in BM-derived COL1A2 promoter activity was not simply due to an increase in cell number. Comparisons to total endogenous and transgenic COL1A2 promoter activity demonstrated that during inflammation (days 7–17) BMDCs contributed <6% of the total COL1A2 transcripts at any given time point. However, during tissue remodeling (day 22), BM-derived COL1A2 was responsible for 
40% of the COL1A2 levels (P <0.002). These data infer that as inflammation subsides and tissue remodeling ensues, a phenotypic switch takes place wherein BMDC populations shift to the expression of higher levels of COL1A2 synthesis, reflective of fibroblast differentiation. BM-derived, COL1A2 promoter activity decreased significantly at day 27. This may be due in part to regression of fibroplasia that occurs at later time points in the sponge model, whereby apoptosis results in decreased cellularity and protein synthesis. However, the observed selective decline in BM-derived COL1A2 expression suggests that the fibroblasts derived from the BM play a significant, though transient, role in the deposition of collagen I that characterizes the repair process.
3. BMDCs exhibit phenotypic characteristics of granulation tissue fibroblasts
To evaluate the phenotypic contribution of BMDCs to the wound environment, sponge material was analyzed by classic histochemical methodologies. Routine ß-gal staining demonstrated those BMDCs that activated the COL1A2 promoter exhibited a classic fibroblast morphology (Fig. 1
A) and localized within sites of collagen deposition (trichrome, Fig. 1B
). To further characterize these ß-gal-positive cells as fibroblasts, subsequent staining was performed with antisera generated to fibroblast-specific protein (FSP)-1. FSP-1 expression is confined to the cytoplasm of both resting and activated fibroblasts. FSP-1-positive fibroblasts were the predominant cell type within experimental granulation tissue at day 22 (Fig. 1D
), a subset of which coexpressed ß-gal, indicative of COL1A2 promoter activation (Fig. 1C
). Their contribution to wound repair is not trivial, in that a general surveillance of sections taken from a day 22 sponge demonstrated that up to 4% of cells within the granulation tissue at this time point coexpressed ß-gal and FSP-1. This particular animal’s percent chimerism was 10%, suggesting that BMDCs may contribute up to 40% of COL1A2 expressing cells and thus fibroblasts to developing granulation tissue.
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CONCLUSIONS AND SIGNIFICANCE
Collectively, these data further expand our knowledge regarding the plasticity of adult BMDCs to include a cell population that is competent to migrate to distant sites in response to injury, and participate in granulation tissue development and wound repair beyond inflammation as COL1A2/FSP-1-positive fibroblasts (Fig. 2
). Moreover, these data contradict our long-standing beliefs that wound fibroblasts are primarily derived from the local recruitment and proliferation of resident fibroblasts.
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Previous studies describe the contribution of BMDCs to dermal wound repair within hair follicles, sebaceous glands, vascular structures, and epidermal components including keratinocytes and dendritic cells. Bone marrow-derived myofibroblasts have been identified in early wounds 4 and 7 days postinjury, and cells with fibroblast morphology after 21 and 28 days. However, no in situ evidence of fibroblast differentiation by BMDCs, beyond phenotypic classification, has been reported nor their specific contribution to the deposition of extracellular matrix within the wound examined. Our results expand these investigations, follow BMDCs throughout the wound repair process, and are the first to demonstrate the kinetics of BMDC infiltration to the wound site, their phenotypic conversion to COL1A2/FSP-1-positive fibroblasts, and their significant contribution to the deposition of collagen during granulation tissue development and remodeling.
Several questions remain unresolved. First, whole, unfractionated BM was used in these experiments, and therefore it was not possible to assess the clonal origin of the described, BM-derived fibroblast population. Recent studies have separated putative mesenchymal and hematopoietic progenitors of the BM and examined their contribution to wound repair following transplantation. These investigations demonstrate that those cells that exhibit a fibroblast phenotype are derived from the mesenchymal, CD45 negative fraction and are therefore not seemingly HSC-derived. Future studies will include cellular fractionation of the BM to elucidate further the ontogeny of the BMDC population responsible for fibroblast differentiation during wound repair. Second, historically the origin of the wound fibroblast has been attributed to the local recruitment and proliferation of resident fibroblasts. Therefore, the influx and/or expansion of BMDCs that express high levels of COL1A2, coexpress FSP-1, exhibit a fibroblast phenotype, and localize to sites of collagen deposition within the developing granulation tissue was not intuitive based on our current understanding of the wound repair process. These data suggest that an as yet undefined cell population derived from the BM compartment contributes significantly to wound repair beyond inflammation, to later phases of granulation tissue maturation and remodeling as wound fibroblasts. It remains to be determined whether the observed secondary increase in BMDC number was due to further recruitment, local proliferation or both phenomena. More classic excisional wounds where COL1A2-expressing BMDCs may contribute to a greater or lesser degree to the fibroblast component of repair within the complex microenvironment of the skin must be evaluated. Third, it is well accepted that cells are summoned to sites of injury by local factors and that recruited cells undergo alterations in gene expression that result in changes in morphology, phenotype and functional characteristics. Therefore, the mechanism(s) by which BM-derived fibroblast progenitors are mobilized and recruited to sites of tissue injury, and what local cues elicit their differentiation within the wound site remain to be elucidated. Fourth, physiologic states associated with impaired or delayed wound healing, such as diabetes and aging, may affect the mobilization, recruitment, and/or differentiation of BM-derived fibroblast progenitors in response to tissue damage. Therefore, topical and/or systemic agents will be examined for their ability to function as therapeutic agents to potentiate wound repair not only by increasing the mobilization of fibroblast progenitors from the BM, but also their recruitment to and differentiation within the wound site.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2978fje;
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