| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,*
Departments of
* Microbiology and Immunology and
Surgery, Burn and Shock Trauma Institute, Loyola University Medical Center, Maywood, Illinois
Correspondence: Luisa A. DiPietro, Loyola University Medical Center, 2160 S. 1st Ave., BSTI, Building 110, Maywood, IL 60153. E-mail: ldipiet{at}lumc.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: healing • keratinocytes • inflammation • diabetic
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Our overall goal is to elucidate the role of neutrophils and their products in wound healing. In this study, we investigated healing of murine full-thickness dermal excisional and incisional wounds following the depletion of neutrophils.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Antisera and monoclonal antibody (mAb)
Rabbit anti-mouse neutrophil serum was purchased from Intercell Technologies (Hopewell, NJ). The rabbit preimmune serum was kindly provided by Dr. Katherine L. Knight. The hybridoma RB6-8C5, which was originally developed by Dr. Robert Coffman (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA), secretes a monoclonal rat anti-mouse Gr-1 antibody [4
,5
]. This clone was a kind gift from Dr. C. Brown (University of Michigan, Ann Arbor). The anti-Gr-1 antibody was purified by precipitation of the hybridoma culture supernatant with ammonium sulfate as described elsewhere [6]. Precipitated protein was subsequently dialyzed against phosphate-buffered saline (PBS) and was then subjected to affinity protein A chromatography (Protein G Sepharose 4 Fast Flow, Amersham Pharmacia Biotech, Piscataway, NJ). Concentrated antibody was eluted from the column, and the concentration was determined by spectrophotometry at 280 nm.
Fluorescence-activated cell sorter (FACS) analysis
A total of 3 ml murine blood was collected by cardiac puncture into heparinized tubes. Red blood cells were lysed by addition of Qiagen EL erythrocytes lysis buffer, according to the manufacturer’s instructions (Qiagen, Valencia, CA). White blood cells (5x105) were resuspended in 0.5 ml PBS + 1% bovine serum albumin (BSA) to block nonspecific binding. Cells were then incubated with 1 µg/ml monoclonal anti-Gr-1 antibody or rabbit anti-mouse neutrophil serum diluted 1:100 in 0.5 ml for 30 min on ice. Cells were washed in two changes of PBS/BSA and subsequently resuspended in 0.5 ml of the appropriate secondary antibody solution. For the RB6-8C5-stained cells, a fluorescein isothiocyanate (FITC)-conjugated goat anti-rat immunoglobulin G (IgG; Sigma Chemical Co., St. Louis, MO) was used (1:32, final concentration 31.3 µg/ml). For the rabbit anti-mouse neutrophil serum-labeled cells, a FITC-conjugated goat anti-rabbit antibody (Sigma Chemical Co.) was used (1:80, final concentration 12.5 µg/ml). After a 30-min incubation on ice, cells were washed in two changes of PBS/BSA and two changes of PBS. Analysis was performed on a FACScaliber (Becton Dickinson, San Jose, CA).
In vivo neutrophil depletion
To induce neutropenia, female BALB/c mice were injected intraperitoneally (i.p.) with 150 µl rabbit anti-mouse neutrophil serum or rabbit preimmune serum. Successful depletion of peripheral neutrophils was confirmed by differential counts of blood smears prepared from the injected animals. Blood was collected by removing 2 mm of the tail tip from anesthetized mice. To prepare blood smears, 2 µl murine blood was pipetted onto a 45° tilted glass slide and was then evenly distributed by dragging a second glass slide over the first one. Blood smears were air-dried for 30 min and stained with Giemsa Wright stain (Sigma Chemical Co.) for 1 min. All slides were then covered with an equal volume of distilled water and incubated for an additional 10 min. Slides were then rinsed with distilled water and air-dried. A total of 200 cells per slide was counted, and the percent neutrophils was determined.
Excisional injury model and tissue collection
Sixteen hours post-injection of rabbit anti-mouse neutrophil or control serum, neutropenic and control mice were anesthetized with Halothane (Halocarbon Laboratories, River Edge, NJ) and shaved on their dorsum. A dermal biopsy punch (Acu Punch, Acuderm, Ft. Lauderdale, FL) was used to produce six full-thickness dermal excisional wounds. Each wound measured 3 mm in diameter and extended through the panniculus carnosus on the dorsal skin of each mouse. At days 1, 2, 3, and 5 post-wounding, mice were killed, and the wounds were excised. None of the wounds displayed any signs of infection. Wound tissue was embedded in Tissue Freezing Medium (Triangle Biomedical Science, Durham, NC) and was frozen at -70°C for biochemical analysis. Some day-1 wounds were embedded in paraffin for preparation of 1 µm sections.
Analysis of wound myeloperoxidase (MPO)
The measurement of MPO levels in tissue has been shown to reflect neutrophil content. MPO content in wound tissue was determined using previously described methods [7
,8
]. Briefly, wounds were homogenized in 2 ml 20 mM phosphate buffer, pH 7.4. Homogenates were centrifuged at 12,000 g for 45 min, and the supernatant was decanted. The pellets were resuspended in 1 ml 50 mM phosphate buffer containing 10 mM EDTA and 0.5% hexadecyltrimethylammonium bromide (HTAB). After a freeze-thaw cycle, the samples were briefly sonicated and incubated at 60°C for 2 h. The samples were centrifuged at 500 g for 10 min, and the supernatant was transferred to 1.5 ml tubes and directly subjected to analysis or stored at -20°C. For analysis, a standard curve ranging from 0 to 3.0 units/ml MPO was generated. Aliquots of samples (50 µL) or standards were placed in 12 x 75 mm glass tubes with 500 µl assay buffer (0.1 M phosphate buffer, pH 5.4, 1% HTAB, 0.43 mg/ml 3,3',5,5'-tetramethylbenzidine). The reactions were started by the addition of 50 µl 15 mM H2O2, incubated at 37°C for 15 min, and stopped with 1.0 ml cold 0.2 M sodium acetate, pH 3.0. The absorbance of each sample and standard was read at 655 nm within 10 min of completion. All samples and standards were tested in duplicates.
Analysis of re-epithelialization
To analyze the degree of re-epithelialization, 10 µm sections were prepared from frozen, embedded wounds. Sections were then stained with hematoxylin and eosin (H&E; Sigma Chemical Co.) and visualized under 100x power using a light microscope. The wound width covered by epithelium as well as the total wound width were measured and used to calculate % re-epithelialization with the formula: % re-epithelialization = re-epithelialized wound width x 100/total wound width. The analysis of wound closure was conducted in a blinded manner.
Incisional injury model and wound disruption strength
Mice were injected with the rabbit anti-mouse neutrophil or control serum, as described above. Sixteen hours post-injection, the mice were anesthetized by i.p. injection of Ketamine (Abbott Laboratories, North Chicago, IL) and Xylaject (Phoenix Pharmaceutical, St. Joseph, MO). The fur of the ventral and dorsal skin was removed by shaving, and the skin was washed briefly with 70% isopropanol. A 2-cm incisional wound extending through the dermis and the panniculus carnosus was prepared in the paraspinal region and closed using sterile surgical clips. After 5 days, the mice were lightly anesthetized with Halothane to remove the surgical clips. At days 7 and 14 post-injury, wounds were harvested. From each wound, two strips perpendicular to the incision were excised and subjected to tensiometry. In this procedure, sequentially increased tension was applied to the excised wound strips. Wound disruption strength is defined as the load value of wound disruption. The tensiometer used was designed and built by the Department of Surgery and Instrument Models Facility at the University of Vermont (Burlington).
Collagen content
The collagen content of wounds was assessed by determining the amount of hydroxyproline present [9
]. Frozen wound tissue was hydrolized in 2.0 ml 6 N HCl for 3 h at 130°C or overnight at 110°C. The reaction was stopped with 2.5 N NaOH and diluted 40-fold with dH2O. A 0.05 mol/L N-chloro-p-toluene-sufonamide solution (1 ml) was added to 2 ml of the diluted hydrolysate and incubated for 20 min at room temperature. Perchloric acid (1 ml 3.15 mol/L) was added to the samples and incubated for an additional 5 min at room temperature. p-Dimethylaminobenzaldehyde (1 ml 20%) was then added followed by a 20-min incubation at 60°C. The samples were cooled with cold tap water, and the absorbance was measured at 557 nm. The hydroxyproline content was determined by comparison to a standard curve. All reagents in this assay were purchased from Sigma Chemical Co.
Macrophage staining
For analysis of macrophage content, sections from the central portion of each wound were stained with MOMA-2, a rat mAb specific for murine macrophages (Serotec, Raleigh, NC). Histologic sections were fixed in acetone for 30 min and pretreated with 0.3% H2O2 in methanol to block endogenous peroxidase. After three 5-min washes in PBS, sections were incubated with normal mouse serum (1:10; Harlan Sprague Dawley) for 30 min. Sections were incubated in primary antibody MOMA-2 (1:500) for 30 min, washed three times in PBS, and incubated in secondary antibody (biotinylated mouse anti-rat Ig, 13.0 µg/ml; The Jackson Laboratory) for another 30 min. Subsequently, sections were incubated with avidin-biotin-horseradish peroxidase complex (Vector Laboratories, Burlingame, CA) for 30 min and were again washed in PBS. Color development was performed with 3,3'-diaminobenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for 10 min, and sections were counterstained with Gills hematoxylin (Sigma Chemical Co.). To quantitate macrophages, MOMA-2-positive cells were counted in five random high-power fields (HPF) within the wound bed. The analysis was conducted in a blinded manner.
Statistical analysis
Data were analyzed using GraphPad Prism, version 2.01 (GraphPad Software, San Diego, CA). The means and SEM were calculated for each data set. A one-way or two-way ANOVA followed by an unpaired t-test was used for comparison of groups. Values of P< 0.05 were considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
|
|
To determine if neutrophil depletion affects the collagen content present in the wound, we compared the amount of collagen in wounds of neutropenic and control mice. The hydroxyproline content, indicative of collagen presence, was not significantly different between neutrophil-depleted and control mice (Fig. 5 ). These data suggest that neutrophils do not affect the collagen content in the wound bed.
|
To determine whether neutrophils also influence the regeneration of tissue strength, we studied the effects of neutropenia on the development of wound disruption strength in an incisional wound model. In this model of a clean incision, approximated by sterile surgical clips, the least amount of epithelial damage occurs. This wound closely resembles a surgical incision and allows for quantification of dermal healing. Measurements of wound disruption strength provide highly quantifiable estimates of the efficacy of subepidermal repair.
Quantifying wound disruption strength, we found no significant difference between the neutropenic and control groups (day 7: 100.3±5.9 g compared with 90.9±10.6 g, P>0.4, n=12; day 14: 281.1±14.6 g compared with 270.6±15.7 g, P>0.6, n=12; Fig. 6 ). These results suggest that neutrophils do not affect the regeneration of tissue strength during normal wound healing.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Despite growing interest in how inflammatory cells contribute to the pathology of nonhealing wounds and immune-mediated tissue damage, little is known about how neutrophils affect normal healing. In the present study, we used a neutropenic mouse model of normal wound healing to determine if neutrophils affect the repair process. We found that 1) wounds of neutrophil-depleted mice exhibited significantly accelerated re-epithelialization, 2) neutrophil depletion had no effect on wound disruption strength or collagen content, 3) neutrophil depletion did not influence the number of macrophages at the wound site, and 4) the healing impairment of re-epithelialization displayed in diabetic mice was alleviated by neutrophil depletion. These results suggest that in an uninfected wound, neutrophils retard wound closure by inhibiting re-epithelialization but not the overall dermal repair. Additionally, in case of poorly healing wounds with excess neutrophils at the wound site, neutrophils may contribute significantly to the impaired healing phenotype.
Apart from their role to control infection, neutrophils have previously been regarded as a cell type with minor influence on healing of uninfected wounds. This concept is largely derived from a study conducted by Simpson and Ross in 1972 [18 ], which investigated the effects of neutrophil depletion on wound healing in a guinea pig model. In this study, no discernable differences in fibrin content or distribution were observed in wounds of neutropenic and control guinea pigs. Further, no differences in percent wound volume occupied by macrophages or in macrophage phagocytosis were seen. Using a similar approach of neutrophil depletion to study the effects of neutropenia on wound healing in a murine model, we obtained results consistent with but also extending the study conducted by Simpson and Ross [18 ]. In the murine study described herein, we also did not find significant differences in collagen deposition, wound disruption strength, or macrophage infiltration in wounds of neutropenic mice. Apart from these parameters of healing that take place in the dermal layer of the skin, our study complements the study conducted by Simpson and Ross [18 ] by investigating epidermal healing in the absence of neutrophils. The fact that re-epithelialization, but not any of the processes in the dermis, was accelerated by neutrophil depletion strongly suggests a direct effect of neutrophil presence on keratinocyte function. The nature of a possible neutrophil-keratinocyte interaction remains to be characterized. Our results also suggest that epidermal and dermal healing are not necessarily linked, as epithelialization, but not the regeneration of tensile strength, was accelerated in the absence of neutrophils.
In the current study, we chose a murine model of cutaneous healing in which components of the repair process, in particular, infiltration of inflammatory cells, have been extensively studied. Although our data suggest an overall negative influence of neutrophils in normal wound healing, previous study of neutrophil function supports both a positive [19 ] and a negative [20 ] role for neutrophils in normal tissue repair. Neutrophils produce a variety of growth factors that could promote repair of injured tissue, among them interleukin-8 [21 ] and vascular endothelial growth factor [22 ]. On the contrary, neutrophils produce many bioactive substances that can oppose the repair process by inducing further tissue damage. For instance, within the wound, neutrophils secrete a battery of proteases that can convert wound cytokines to an active or inactive form and can induce substantial tissue damage. For example, the proteases neutrophil elastase and PR-3 are capable of cleaving elastin and a variety of ECM proteins, including fibronectin, laminin, vitronectin, and collagen IV. Apart from serving as a supporting scaffold, the ECM influences cellular function such as migration and proliferation. Briggaman et al. [23 ] demonstrated that incubation of skin with neutrophil elastase leads to separation of the dermal and epidermal layers. It is interesting that the addition of neutrophils to a monolayer of keratinocytes leads to a protease-dependent keratinocyte detachment [24 ].
An important caveat to our experimental approach is the use of a polyclonal rabbit anti-mouse neutrophil serum as a depleting agent. This antiserum appears to exhibit low reactivity to monocytes, which could lead to a decrease in monocyte numbers. However, we did not observe a difference in the numbers of macrophages at the wound site (Fig. 7) . In fact, although not statistically significant, there appears to be a trend to more macrophages infiltrating the wound bed of neutrophil-depleted mice when compared with control mice. As macrophages play a critical role in wound healing, additional macrophages would be expected to accelerate wound healing [11 ]. One interesting possibility is that an enhancement in macrophage function, favorable to healing, in tandem with the depletion of neutrophils, accounts for the accelerated re-epithelialization observed in neutrophil-depleted mice.
Taken together, these reports in the literature and our own findings lead to speculate that neutrophils at the wound site directly inhibit keratinocyte migration and possibly proliferation. In wounds, neutrophils localize to the subepidermal layer and are, hence, in close contact with keratinocytes. Thus, neutrophil-derived proteases could degrade ECM, fibrin clot components, and other proteins at the site of keratinocyte migration and proliferation. This proteolysis could lead to keratinocyte detachment. As keratinocytes absolutely require contact to the ECM components for migration and proliferation [25 ], a now, at least partly, unattached keratinocyte would not be able to mediate re-epithelialization until the cell regained attachment. The above-described observation that keratinocytes detach in the presence of neutrophils in vitro supports this possibility.
It is possible that the extent of degradation of ECM components by neutrophil-derived proteases at the site of injury may not actually lead to keratinocyte detachment but may still impede keratinocyte migration. Rather, a limited amount of proteolysis of provisional matrix components in the vicinity of keratinocytes could stall re-epithelialization, as keratinocytes lose their directionality (Fig. 9 ). Pilcher et al. [26 ] proposed a model in which interaction and then cleavage of collagen I is used by keratinocytes to determine their direction during re-epithelialization. The proteolysis of type I collagen, mediated by keratinocyte-derived collagenase-1, may serve as a "molecular compass" for migrating keratinocytes to move forward. Based on Pilcher’s model of keratinocyte migration [26 ], a degradation of provisional matrix components by neutrophil-derived proteases could stall re-epithelialization even if keratinocytes are not detached.
|
In summary, we have shown that neutropenia, induced by injection of a rabbit anti-mouse neutrophil serum, accelerates the rate of wound epithelial closure without altering the overall quality of the dermal healing process. This is also true for wounds of diabetic mice, in which excess inflammation appears to participate in healing impairment. Thus, although the inflammatory phase is a vital response to tissue injury, a subset of inflammatory cells may in fact retard wound closure. Further detailed studies on the mechanism of neutrophil-mediated delay in healing will be necessary. Ultimately, re-evaluating the necessity of complete neutrophil function in a setting of sterile surgical wound healing may be indicated.
|
ACKNOWLEDGEMENTS |
|---|
Received August 21, 2002; accepted December 5, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  K. A. Radek, L. A. Baer, J. Eckhardt, L. A. DiPietro, and C. E. Wade Mechanical unloading impairs keratinocyte migration and angiogenesis during cutaneous wound healing J Appl Physiol, May 1, 2008; 104(5): 1295 - 1303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Delp Unraveling the complex web of impaired wound healing with mechanical unloading and physical deconditioning J Appl Physiol, May 1, 2008; 104(5): 1262 - 1263. [Full Text] [PDF]
|
|
|
|
 |
|
 K. T. Keylock, V. J. Vieira, M. A. Wallig, L. A. DiPietro, M. Schrementi, and J. A. Woods Exercise accelerates cutaneous wound healing and decreases wound inflammation in aged mice Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2008; 294(1): R179 - R184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Tonks, E. Dudley, N. G. Porter, J. Parton, J. Brazier, E. L. Smith, and A. Tonks A 5.8-kDa component of manuka honey stimulates immune cells via TLR4 J. Leukoc. Biol., November 1, 2007; 82(5): 1147 - 1155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. W. J. van de Sande, A. Fahal, H. Verbrugh, and A. van Belkum Polymorphisms in Genes Involved in Innate Immunity Predispose Toward Mycetoma Susceptibility J. Immunol., September 1, 2007; 179(5): 3065 - 3074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Shaykhiev and R. Bals Interactions between epithelial cells and leukocytes in immunity and tissue homeostasis J. Leukoc. Biol., July 1, 2007; 82(1): 1 - 15. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Agren and M. Werthen The Extracellular Matrix in Wound Healing: A Closer Look at Therapeutics for Chronic Wounds International Journal of Lower Extremity Wounds, June 1, 2007; 6(2): 82 - 97. [Abstract] [PDF]
|
|
|
|
|
|
D. Marsolais, C. H. Cote, and J. Frenette Pifithrin-{alpha}, an inhibitor of p53 transactivation, alters the inflammatory process and delays tendon healing following acute injury Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R321 - R327. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Mori, K. T. Power, C. M. Wang, P. Martin, and D. L. Becker Acute downregulation of connexin43 at wound sites leads to a reduced inflammatory response, enhanced keratinocyte proliferation and wound fibroblast migration J. Cell Sci., December 15, 2006; 119(24): 5193 - 5203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Godbout, O. Ang, and J. Frenette Early voluntary exercise does not promote healing in a rat model of Achilles tendon injury J Appl Physiol, December 1, 2006; 101(6): 1720 - 1726. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. G. Engeland, J. A. Bosch, J. T. Cacioppo, and P. T. Marucha Mucosal Wound Healing: The Roles of Age and Sex Arch Surg, December 1, 2006; 141(12): 1193 - 1197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Qualls, A. M. Kaplan, N. van Rooijen, and D. A. Cohen Suppression of experimental colitis by intestinal mononuclear phagocytes J. Leukoc. Biol., October 1, 2006; 80(4): 802 - 815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ishida, T. Kondo, A. Kimura, K. Matsushima, and N. Mukaida Absence of IL-1 Receptor Antagonist Impaired Wound Healing along with Aberrant NF-{kappa}B Activation and a Reciprocal Suppression of TGF-beta Signal Pathway J. Immunol., May 1, 2006; 176(9): 5598 - 5606. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ueno, B. L. Lyons, L. M. Burzenski, B. Gott, D. J. Shaffer, D. C. Roopenian, and L. D. Shultz Accelerated Wound Healing of Alkali-Burned Corneas in MRL Mice Is Associated with a Reduced Inflammatory Signature Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4097 - 4106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Radek, A. M. Matthies, A. L. Burns, S. A. Heinrich, E. J. Kovacs, and L. A. DiPietro Acute ethanol exposure impairs angiogenesis and the proliferative phase of wound healing Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1084 - H1090. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Sosne, P. L. Christopherson, R. P. Barrett, and R. Fridman Thymosin-{beta}4 Modulates Corneal Matrix Metalloproteinase Levels and Polymorphonuclear Cell Infiltration after Alkali Injury Invest. Ophthalmol. Vis. Sci., July 1, 2005; 46(7): 2388 - 2395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Daley, J. S. Reichner, E. J. Mahoney, L. Manfield, W. L. Henry Jr., B. Mastrofrancesco, and J. E. Albina Modulation of Macrophage Phenotype by Soluble Product(s) Released from Neutrophils J. Immunol., February 15, 2005; 174(4): 2265 - 2272. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Stramer, W. Wood, M. J. Galko, M. J. Redd, A. Jacinto, S. M. Parkhurst, and P. Martin Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration J. Cell Biol., February 14, 2005; 168(4): 567 - 573. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. Wilgus, V. K. Bergdall, K. L. Tober, K. J. Hill, S. Mitra, N. A. Flavahan, and T. M. Oberyszyn The Impact of Cyclooxygenase-2 Mediated Inflammation on Scarless Fetal Wound Healing Am. J. Pathol., September 1, 2004; 165(3): 753 - 761. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Martin and S. M. Parkhurst Parallels between tissue repair and embryo morphogenesis Development, July 1, 2004; 131(13): 3021 - 3034. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Scott, K M Khan, C R Roberts, J L Cook, and V Duronio What do we mean by the term "inflammation"? A contemporary basic science update for sports medicine Br. J. Sports Med., June 1, 2004; 38(3): 372 - 380. [Abstract] [Full Text] [PDF]
|
|
