Skin wounds comprise a broad spectrum of clinical manifestations, depending on their aetiology, clinical course, and anatomic location as well as individual pathophysiologic factors. Routine wound bed assessment is based on the clinical evaluation of wound characteristics such as size, location, the presence of cavities, sinuses, and fistulae. Other features may include the presence of necrosis, granulation, infection, or other signs of critical colonization.1 Besides assessment of the wound area itself, the condition of surrounding skin is of crucial importance. Wound margins may present oedematous, white, shiny, warm, red, dry, or scaling ultimately serving as prognostic factors for the individual healing capacity.2 While visual inspection is the established procedure in clinical dermatology, the method is inherently subjective, often yielding inaccurate descriptions of wound conditions, and do not permit an ultrastructural analysis of the wound tissue.3
In that regard, routine histology remains the gold standard for morphologic evaluation of cutaneous wound healing.4 However, while histology still plays an important role in skin research and the development of innovative wound dressings, there are substantial limitations. The invasive character of biopsies does not allow an assessment over time and the method may not be feasible for evaluation of large or recurrent wounds or patients with significant impairment of wound healing or high risk of infection. Lastly, tissue removal and histological processing may result in artifacts, further limiting its clinical applicability.
On the other hand, wound healing has been extensively studied in a variety of animal species including amphibians, rodents, and pigs. Animal models allow the infliction of skin wounds under standardized conditions and an investigation of the influence of pharmacological and physical treatments on tissue repair.5 For investigational purposes rodents can be genetically modified to over-express cytokines of interest thereby gaining new insights on their respective influence on cutaneous wound healing.67.–8 There are, however, significant differences in tissue repair between the species, such that regenerative capacities of amphibians cannot be compared to wound healing in mammals and rodents. Therefore, the model has to be carefully chosen and experiments in human skin still represent the gold standard for research in the field of tissue repair.
Considering these limitations, a number of noninvasive imaging techniques have been evaluated for their applicability for assessment of human skin wounds at different stages of wound healing.9 Among them, in vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical imaging tool for noninvasive evaluation of normal and diseased skin in vivo and in real time. Previous reports have shown its diagnostic suitability for inflammatory, neoplastic, and proliferative skin disorders7 and its applicability to study skin diseases over time.10 Altintas and co-workers have first employed CLSM for evaluation of burn wounds in human volunteers, whereby aspects of microcirculation, inflammation, and histomorphology have been described.11,12 Their findings supported the use of CLSM as an adjunctive tool for evaluation and management of burn wounds.
Based on these findings, the present study was designed to test the feasibility of in vivo reflectance CLSM for evaluation of skin wounds, including acute and chronic, superficial and deep dermal skin wounds in this noninvasive analysis. We used CLSM for assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. By means of analyzing features of cutaneous inflammation, vascularisation, and epithelialisation as biomorphological endpoints of therapy, the goal of this evaluation was to study the role of CLSM in modern wound management.
Materials and Methods
Patients and Study Procedures
The research protocol was approved by the Charité University Hospital Subcommittee on Human Studies, at the Institutional Review Board. A total of 15 patients (8 male, 7 female, SPT I-III, aged 29 to 82) were enrolled in this clinical evaluation and assigned to the individual study goals (Table 1). Written consent was obtained prior to enrollment and all clinical investigation was conducted according to GCP and the Declaration of Helsinki principles. All 15 subjects completed the study, and data of all subjects were included in the analysis.
Study groups and procedures. Procedures and evaluations timepoints in study group 1 (epidermal wounds), study group 2 (superficial dermal wounds), and study group 3 (deep ulcers).
|Study groups||Study participants||Study procedures||Evaluation site||Evaluation time point|
|Group I||Healthy individuals(4F/1 M)||Contact cryosurgery at −32 °C for 10 seconds on the volar forearm, Removal of blister roof Clinical exam, serial CLSM evaluation||Wound area and surrounding skin||Days 0, 1, 2, 7, 14, 28|
|Group II||Patients with skin cancer receiving split skin grafts(4F/1 M)||Routine tumor excision followed by split skin graftsClinical exam, serial CLSM evaluation of the skin graft donor site||Wound area, wound margin||Day 5|
|Group III||Patients with chronic leg ulcers(2F/3 M)||Routine wound management with serial follow-up; clinical exam, CLSM evaluation of wound margins||Wound margin||Serial evaluations during therapeutic follow-up|
Group I (superficial epidermal wounds): To study the events of cutaneous wound repair following cryosurgery a total of () healthy volunteers were included; each participant was treated with contact cryosurgery at for 10 s on the volar forearm, followed by removal of the blister roof on day 1 after cryosurgery. Clinical and CLSM evaluation were performed at baseline (day 0) and follow-up (at days 1, 2, 7, 14, and week days, respectively).
Group II (superficial dermal wounds): To evaluate the healing of deeper wounds involving epidermis and dermis, the respective donor sites of skin grafts from () patients undergoing skin transplantation were included. Patients were recruited from the skin surgery unit at the Department of Dermatology, Venereology, and Allergology at the Charité University Hospital (Berlin, Germany) and seen for their routine surgical care and follow-up. Clinical and CLSM evaluation were performed at baseline and follow-up at skin graft donor sites at day 5. Superficial dermal skin wounds were created by a split skin grafting procedure, using an electric dermatome (GA 630, Aesculap, Tuttlingen, Germany), whereby skin grafts of uniform thickness of approximately 0.3 mm were obtained for surgical reconstruction following the removal of skin cancer. This procedure was performed under local anesthesia.13
This model was chosen since the surgical use of the dermatome creates wounds at standardized depths involving the dermis stimulating the proliferation and migration of fibroblasts, myofibroblasts, and endothelial cells within the wound bed. Evaluations were not performed until day 5, since the wound bed is covered by a sterile dressing until that time point.
Group III (deep dermal wounds): To study the events of cutaneous wound repair in chronic skin ulcers a total of () patients were included for evaluation. Patients were recruited from the outpatient wound clinic at the Department of Dermatology, Venerology, and Allergology at the Charité University Hospital (Berlin, Germany) where they were seen for treatment of chronic leg ulcers due to chronic venous insufficiency (Widmer stage III). All patients were treated according to the guidelines of modern wound managements.14 The individual response to the conservative wound bed preparation was graded: poor (no response within 4 weeks), moderate (some visible signs of epithelialization at the wound margin), and good (epithelialization and decreasing wound size). Clinical and noninvasive evaluation by CLSM was performed at baseline and consecutive follow-ups up to 4 weeks following inclusion.
Confocal Laser Scanning Microscopy
CLSM evaluation was performed using a commercially available system (Vivascope 1500®, Mavig GmbH, Munich, Lucid-Tech Inc., Henrietta, NY).15 A detailed description of the technique and the device used has been published previously.16,17 Briefly, the system employs an 830 nm Diode laser, generating a power of less than 30 mW at tissue level. CLSM imaging is based on tissue illumination and detection of scattered and reflected light through a small aperture, thereby producing the high lateral resolution of CLSM images, which lies around 0.5 to 1 microns. Axial resolution (optical section thickness) is about 3 to 5 microns. Optical sectioning is performed from superficial to deeper layers, by progressively moving the focus of the objective down the -axis. Images are resolved in grey-scale and imaging is performed at video-rate (real-time) at 9 frames per second.
The size of individual images obtained in the horizontal plane ( direction) is microns; the microscope permits horizontal mapping (Vivablockﬁ function) of areas from to and vertical mapping (Vivastack® function) whereby the system allows software-assisted imaging using pre-set or individually programmed imaging steps and penetration depths. Imaging depth (-axis) was determined for study group 1 (superficial epidermal wounds) relative to the surface by zeroing the micrometer at the most superficial discernable skin layer. In study groups II and III imaging depth was restricted due to exudation and crust formation. In these study groups respective depths were assessed by the morphological appearance of the investigated tissue.
CLSM Evaluation Parameters
In each participant, one or more skin sites were selected for imaging following clinical examination; for each skin site, a systematic horizontal mapping was performed and four to six individual images were captured in axial sections beginning with the stratum corneum (SC), through the entire epidermis, and into the upper reticular dermis.
CLSM evaluation parameters included features of cutaneous wound repair on a cellular, morphological and architectural level, as well as the documentation of dynamic processes such as blood flow and inflammation, and the successive events of wound healing. CLSM evaluation parameters for respective study groups are listed in Table 2.
CLSM evaluation parameters. Morphological aspects of CLSM analysis based on established histological features in comparison to normal skin. The abscence/presence of respective CLSM features was recorded during in vivo examination and by retrospective image analysis. Semiquantitative scoring was performed for chronic skin wounds, whereby a score of 0= none, 1= mild, 2= moderate, and 3=severe was assigned to individual patients.
|CLSM parameter||Group I||Group II||Group III|
|Epidermis||Loss of SC integrity after blister removal Stage of re-epithelialization Restoration of SC integrity||Visualization of slough formation-Inflammatory infiltrate Impetiginization||Morphology/integrity of epidermal architecture|
|Architectural changes in granular/spinous/basal layer Dermoepidermal junction||Stage of re-epithelialization Presence of keratinocytes Presence of corneocytes||Stage of re-epithelialization-Presence of keratinocytes|
|Spongiosis, Exocytosis (presence/absence)||Morphology of adjacent epidermal keratinocytes||Spongiosis, exocytosis(semiquantitative scoring)|
|Dermis||Superficial dermal capillariesNumber/morphology (dilatation/elongation)||Superficial dermal capillaries Number/morphology (dilatation/elongation)||Superficial dermal capillaries Number/morphology (dilatation/elongation)|
|Inflammatory infiltrate||Inflammatory infiltrate||Inflammatory infiltrate(semiquantitative scoring)|
|Reactive changes in subjacent dermal tissue inflammatory infiltrate Morphology of collagen bundles||Morphology of collagen bundles Scar formation/presence of fibrosis||Morphology of collagen bundles|
All images underwent descriptive morphological analysis, including a second, retrospective analysis by an expert in the field. CLSM features of inflammation, vascularization and re-epithelialization were based on those previously published for CLSM,18 and/or in correlation with established histological features,19 and described in comparison to normal skin. Image analysis was performed by using a simple present/absent scheme. Semiquantitative image analysis was performed for the presence of inflammatory cells for chronic skin wounds; respective grading ranged from , , , and based on the number of detected inflammatory cells/per field of view. At least 4 to 6 images were analyzed per wound.18
CLSM Findings of Superficial Epidermal Wounds (Group I)
CLSM imaging of normal skin showed the characteristic features of SC appearing as a bright somewhat cohesive layer, arranged in typical fields, folds, and furrows. At the level of the dermoepidermal junction dermal papillae with dark appearance surrounded by a rim of somewhat bright basal cells were visualized. Within dermal papillae capillaries seen as bright canalicular structures are observed (Fig. 1, normal skin). Real-time imaging of normal skin showed regular blood flow within the blood vessels of the dermal papillae without significant leukocyte-endothelial interaction (Video 1, Quick time, 1.4 MB).
CLSM imaging of the test site 30 min after cryotherapy showed an increased distension of skin folds and furrows, while respecting the original architecture and tension lines. At the level of the spinous layer CLSM identified focal areas with thickened and blurred intercellular demarcations (Fig. 2) likely corresponding to early spongiosis. CLSM imaging at the level of the DEJ showed less well-defined dermal papillae and some blood vessels.
Real-time imaging showed increased blood flow in somewhat dilated vessels and presence of lymphocytes rolling along the blood vessel wall 30 min after cryo-induced damage (Video 2, Quick time 3.7 MB). The distinction of lymphocytes is based upon their larger diameter compared to that of erythrocytes in the surrounding blood flow (Fig. 1, 30 min) (Video 2, Quick time 3.7 MB). At the level of the superficial/papillary dermis mainly perivascular inflammatory infiltrate is observed.
Following blister removal on day 1, the superficial epidermal defect becomes visible on CLSM evaluation, by showing a central, well circumscribed dark area surrounded by the remaining SC with bright appearance. CLSM imaging at the level of the dermis showed ill-defined dermal papillae compared to normal skin, with noted increase of blood flow and elongation of superficial capillaries. The fibrous structures within the dermis showed increased brightness on CLSM evaluation, resulting in a more distinct appearance compared to normal skin (Fig. 1, day 1).
CLSM imaging on day 7 showed bright digitate protrusions from the wound margins corresponding to thin sheets of keratinocytes migrating towards the wound center. On individual images, these sheets correspond to loosely aggregated, somewhat elongated keratinocytes or fields of bright, polygonal corneocytes in CLSM evaluation (Fig. 3, day 7).
CLSM imaging on day 14 showed continued wound closure, whereby only a small, residual epidermal defect may be visualized. At the level of the dermoepidermal junction, the restoration of a regular honeycomb pattern may be observed. In addition, sparse inflammatory cells and occasional presence of dendritic shaped bright cells were visualized. While the latter most likely correspond to epidermal dendritic cells, no further distinction may be made by CLSM. Within dermal papillae, superficial dermal capillaries were visualized in regular density and distribution. Dermal fibrous structures showed no apparent structural alteration, resembling the delicate network seen in normal skin (Fig. 3, day 14).
CLSM imaging at week 4 revealed complete re-epithelialization with regular epidermal and dermal architecture, and no residual inflammation (data not shown).
CLSM Findings of Superficial Dermal Wounds (Group II)
Clinical evaluation of split skin donor sites at day 5 revealed serohemorrhagic crusting of variable thickness and the formation of a preliminary wound matrix within the wound bed. CLSM imaging at the level of the wound surface revealed crusting/slough formation seen as amorphous bright area [Fig. 4(a) to 4(c)]. One patient showed the presence of block-like formations and areas of clefting, which appear to evolve under dry wound conditions [Fig. 4(b)]. In addition, numerous bright, round to oval structures of approximately 6 to 10 μm were visualized within the wound surface likely corresponding to inflammatory cells and cell remnants. While inflammatory cells have been described as bright, round to oval cells of approximately 8 to 10 μm, macrophages have previously been described as bright, roundish-plump cells of 12 to 15 microns in diameter.22,23 Moreover, oblong or segmented bright cells, with 9 to 15 microns in diameter are seen on CLSM that might correspond to Polymorphonuclear cells [Fig. 4(c)].
CLSM evaluation within the wound matrix showed inflammatory cells in variable density in the absence of a regular epidermis [Fig. 4(d) to 4(e)]. The detailed visualization of superficial dermal vasculature within the wound bed was only possible in cases with mild inflammation, minimal crusting, or in close vicinity to the wound margin [Fig. 4(f)]. Vasculature seemed elongated and widened compared to normal skin, appearing as bright, canalicular structures due to their content of erythrocytes and visible blood flow upon in vivo CLSM evaluation. In addition, inflammatory cells may be visualized in perivascular and interstitial distribution [Fig. 4(f)].
CLSM imaging at skin sites adjacent to the dermal wound showed spongiosis [Fig. 4(g) to 4(h)] and partial alteration of the adjacent epidermal architecture with elongation of granular keratinocytes due to incipient wound contraction [Fig. 4(h)]. The presence of aggregates of polygonal corneocytes corresponds to initial re-epithelialization protruding towards to wound center [Fig. 4(i)].
CLSM Findings of Deep/Chronic Skin Wounds (Group III)
The evaluation of chronic skin wounds included different subsets of patients; two () patients with chronic skin ulcers with a history of poor response to topical therapy; two () patients with chronic skin ulcers and a history of good response to therapy, rendering only residual superficial erosions for CLSM evaluation; lastly, the evaluation included one () patient with a chronic skin ulcer following re-epithelialization under topical wound management.
In the subset studied during this evaluation, those patients with poor response to therapy showed a reduced or absent inflammatory infiltrate upon CLSM evaluation of wound margins within the epidermis and superficial dermis, while exhibiting marked spongiosis with granular and spinous layers (Fig. 5, Pat. 1). Those patients with moderate response to therapy featured spongiosis at the level of the granular and spinous layers, and presence of inflammatory cells in moderate density interspersed within keratinocytes and superficial dermis (Fig. 5, Pat. 2). One patient with early re-epithelialization showed marked inflammation with the presence of numerous round to oval bright cells interspersed between epidermal keratinocytes and superficial dermal collagen deposits (Fig. 5, Pat. 3). Spongiosis at the level of the spinous and granular layer was invariably present in all examined skin sites [Fig. 5(d) to 5(f)] while the level of extent varied between the studied subsets of patients.
CLSM images obtained at the level mid superficial dermal layer of all three patients show aggregates of fibrous structures seen as bright strands of variable thickness, arranged in a reticulated pattern and somewhat haphazard orientation. Patients with moderate and good wound healing show occasional round to oval bright inflammatory cells. The absence of well-defined dermal papillae indicates a loss of regular epidermal architecture and skin atrophy. Within the dermis increased vascularization may be seen by presence of elongated and/or dilated canalicular dermal blood vessels (Fig. 5).
Cutaneous wound healing is a dynamic, well-orchestrated process involving soluble mediators, blood cells, extracellular matrix, and parenchymal cells. The process can be divided in three overlapping phases—inflammation, tissue formation, and tissue remodeling—and has been well described by histological analyses.19
During the inflammatory phase a provisional matrix is formed and infiltrating neutrophils clean the tissue of foreign particles and bacteria. Blood vessels in the wound bed are dilating. The phase of tissue formation is characterized by the formation of granulation tissue within the wound bed, consisting of migrating fibroblasts and angiogenesis19 followed by the phase of tissue remodeling. Tissue remodeling is characterized by the controlled restitution of blood vessels in the granulation tissue as a result of apoptosis and increasing collagen synthesis and deposition within the wound matrix.
Using the model of cryosurgery induced epidermal wounds, the process of epidermal tissue repair was studied by serial CLSM evaluations of the wound bed and wound margin, beginning at superficial layers and reaching into the upper dermis. CLSM was able to visualize the consecutive events of wound formation and wound healing, from loss of SC integrity until complete re-epithelialization. At the early phase of skin response inflammatory cells were seen within the epidermis, while the presence of a honeycombed pattern corresponded to the restoration of normal epidermal architecture at day 14. Thirty minutes after the cryo-induced damage, focal areas with findings previously ascribed to spongiosis were observed, including thickened and blurred intercellular demarcations. Previous CLSM studies related these characteristics to focal minimal to mild spongiosis.18,20,21
Interestingly, although cryo-induced skin wounds are limited to the epidermis upon clinical inspection, CLSM was able to visualize transient alterations of dermal papillae and superficial dermal vasculature. Upon sequential CLSM imaging leukocyte-endothelial interactions were observed as rolling and tethering of lymphocytes (Video 2, Quick time 3.7 MB). The presence of these intravascular lymphocytes and the dermal inflammatory infiltrate [Fig. 1(k)] may represent reactive changes as part of the local inflammatory response as it has been described in inflammatory skin conditions.24
The resolution of CLSM images allowed a morphological description of the inflammatory phase (day 1) dominated by exocytosis. The tissue formation phase was characterized by CLSM showing reactive keratinocyte proliferation and their progressive migration into the wound bed from the surrounding wound margin. Thereby, CLSM was able to monitor the process of re-epithelialization over time. During the phase of tissue remodeling, CLSM allowed for the visualization of the progressive normalization of the dermoepidermal architecture with formation of a regular honeycombed pattern and the restoration of all epidermal layers between days 7 and 14. Occasional dendritic cells found 14 days after injury might correspond to epidermal dendritic cells repopulating the regenerating epidermis.25
Therefore, CLSM enables the investigator to observe the onset of inflammation, the dynamics of wound closure, and the time point of completed tissue repair. Our findings are confirmatory of preliminary investigations using CLSM in the evaluation of epidermal wounds and previous studies using fluorescence CLSM for the evaluation of wound repair in suction blisters. In these studies, morphologic features of the individual phases of tissue repair were visualized, thereby defining the respective stages of wound healing over time.26,27
Next, we set out to study the applicability of CLSM for the evaluation of wound healing in superficial dermal wounds. For that purpose we have chosen the split skin grafts donor site model as representative to investigate the classical evolution of wound healing in a clinical setting. The harvesting of the skin with a dermatome allows the creation of wounds, standardized in surface area and depth. Following the mechanical removal of the epidermis, the wound surface consists of slough and subjacent dermis. CLSM enabled the visualization of slough as an amorphous bright structure of moderate refractility containing numerous round to oval bright cells that, due to their segmented appearance, might correspond to polymorphonuclear cells and cell remnants.
While CLSM has already been established for investigations of the biofilm in vitro,3031.32.–33 it remains to be elucidated whether in vivo CLSM could be used for biofilm research of wounds and other surfaces of the body. In our studies, CLSM was suitable for the visualization of the inflammatory cells within the dermis and for the evaluation of incipient re-epithelialization along the wound margins. At day 5, aspects of neovascularization as well as re-epithelialization were found at the wound edge.
Based on these findings the inflammatory phase and the tissue formation phase seem to overlap in dermal wounds at day 5. This is supportive of previous findings from routine histology at this timepoint.19 For the purpose of this study, we did not evaluate the phase of tissue remodeling.Therefore, no conclusions may be drawn as to the influence of local inflammation on the structural and cosmetic outcome of individual test sites.
Another goal of this investigation was to test the feasibility of CLSM for the evaluation of deep dermal skin wounds, including patients with chronic venous leg ulcers. Not unexpectedly, CLSM was unable to evaluate the area of the wound bed due to significant slough formation, limiting the analysis to the respective wound margins. While the wound edge is the starting point for the migration of keratinocytes into the wound bed, it is also exposed to the aggressive and proteolytic environment of the adjacent wound bed.34,35 The latter may significantly reduce the regenerative potential of the wound margin, ultimately leading to a lack of re-epithelialization. Therefore, the evaluation of the wound edge and the surrounding skin gives valuable information to the clinician and allows for individual adaptation of the wound management protocol.
Considering the heterogeneity of evaluated patients, it was not surprising to find a broad variety of CLSM findings corresponding to the inflammation, re-epithelialization, and the remodeling at the wound edge.
In those wounds with good healing, polygonal granular keratinocytes and thin, more or less cohesive sheets of corneocytes were found which are consistent with incipient re-epithelialization. The wound margins also showed signs of tissue remodelling, such as increased vascularization and aspects of fibrosis. In addition, CLSM was able to show the loss of the dermal papillae, indicating the disruption of the regular architecture. This feature of tissue remodelling is also found in stasis dermatitis, as it occurs in patients suffering from chronic venous insufficiency.
From the small subset of patients evaluated, however, we did see a trend towards increased inflammation in wounds with good healing tendency, as opposed to nonhealing skin ulcers, showing almost no inflammatory cells and only mild spongiosis. Although there is not much evidence, it may be speculated, whether a certain amount and composition of inflammatory infiltrate might be necessary for the wound healing process as the inflammation phase is the earliest phase in tissue repair.
Overall, the findings of this investigation indicate that CLSM may serve as an adjunct noninvasive diagnostic tool for the evaluation of cutaneous wound healing. With a resolution that is comparable to routine histology, cellular, and morphological details of superficial and deep, acute and chronic skin wounds may be visualized, whereby aspects of inflammation, vascularization, and re-epithelialization may be documented on a cellular level. CLSM allows a semiquantitative analysis of the extent of edema and inflammatory infiltrate, thereby giving important information about the individual healing phase and stage of epithelialization. However, in contrast to immunohistochemical analysis in routine histology, CLSM cannot differentiate between the inflammatory cells in vivo. Moreover, the image analysis is inherently subjective. This limitation is addressed by a number of research groups investigating the use of computer-assisted analysis of CLSM images.3637.–38
With an imaging area of , CLSM is particularly suitable for the evaluation of small experimental wounds since the entire wound geography including the fine layers of emerging epithelialization can be evaluated over time. Based on the experience of this study, CLSM imaging may include an assessment of the wound closure rate. Moreover, functional parameters such as the measurement of the blood flow and the adherence of inflammatory cells to the blood vessel walls might be valuable in future studies.
Overall, superficial epidermal wounds are most suited for noninvasive evaluation. The CLSM evaluations of the wound bed of superficial and deep dermal wounds, however, were complicated by variable crust/slough formation and the presence of mild-to-moderate exudate following surgery. Thus, the placement and immobilization of the CLSM objective posed a significant challenge, ultimately interfering with an evaluation of underlying dermal structures.
In summary, CLSM may contribute to an understanding of the consecutive events of wound healing and assist in the morphological analysis of skin wounds on a microscopic level. Considering respective limitations, different techniques may be used complementary for comprehensive wound analysis and may ultimately play a role in the prognostic assessment and therapeutic management of acute and chronic skin wounds. In that regard, the role of CLSM with respect to wound healing may include pharmacological testing of topical drugs and wound dressings, including the observation of product related side-effects in vivo, thereby avoiding risks potentially associated with histomorphological evaluations.
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