Wound Neovascularization with Dermal Substitutes

April 14, 2012

This is the second of 2 articles detailing wound healing studies done in nude mice implanted with different biological materials.  Full thickness skin wounds were created on the dorsum and then different dermal replacements were implanted.  Healing was followed for 28 days and very detailed histological and immunostaining evaluations of the wounds were performed to quantify the healing process.  Most studies of wound healing use simple or highly subjective measures of healing, but one of the main goals of these studies was to use objective criteria to quantify healing and to do so in fine detail.

Below is the full text of this article and also a PDF link of higher quality.


Wound Neovascularization and Dermal Substitutes in Nude Mice

Neovascularization of Wounds Treated with Dermal Substitutes in Nude Mice

by

Michele M. Loor, MD, Anh-Tuan Truong, MD, Barbara A. Latenser, MD ^1,2,3 ,

Dorion E. Wiley, MD ^1,2, and Robert J. Walter, PhD ^1,2

¹ Sumner L. Koch Burn Center, Department of Trauma, John Stroger Jr. Hospital of Cook County  &

² Department of General Surgery, Rush University Medical Center, Chicago, Illinois

³ Current address:  Director, Burn Unit, University of Iowa, Iowa City, IA

Address Correspondence to:

Robert J. Walter, PhD

Department of Trauma, Suite 1300

John Stroger Jr. Hospital of Cook County

1900 West Polk Street

Chicago, IL 60612

Phone:  312.864.0578

Email:  rwalter@rush.edu

ABSTRACT

Background:  Dermal substitutes implanted into full-thickness skin wounds reduce wound contraction, improve cosmesis, and improve function.  These effects depend upon the development of optimal vascularization of the dermal substitute.

Study Design:  Full-thickness skin wounds were created on the dorsum of nude mice.  A dermal matrix (Integra®, AlloDerm®, acellular dermal matrix, Dermalogen®, or Dermagraft®) was implanted and covered by a mix of fibrin glue (FG) and human keratinocytes.  Wound healing was observed for 4 weeks.  Biopsies were immunostained for laminin followed by blood vessel quantitation using image processing.  Vessels were quantified in the superficial and deep dermis from the wound center, wound margin, and from peripheral unwounded dermis.

Results:  Extensive vascularity was seen at day 28 in implanted Dermagraft® and Dermalogen®.  AlloDerm®, ADM, and Integra® showed slower vessel ingrowth from the wound base and margins and, by day 28, showed diminished wound contraction. The average vessel size in wounds treated with Integra was greater than normal at both days 14 and 28.

Conclusions:  Dermagraft® and Dermalogen® underwent extensive granulation whereas AlloDerm®, Integra®, and ADM showed a more controlled, progressive vessel ingrowth.  For AlloDerm® and ADM, this pattern was associated with reduced wound contraction and increased epithelialization.

Keywords: neovascularization, dermal substitutes, wound healing, nude mice, fibrin glue

Abbreviations:  ADM: acellular dermal matrix, FG: fibrin glue, KC: keratinocytes, ACIS: automated cellular imaging system, WC: wound center, WM: wound margin, NP: normal peripheral tissue

INTRODUCTION

The treatment of full-thickness skin wounds poses a significant clinical challenge.  An open wound not only provides a portal of entry for microorganisms, but it also allows vital fluid and electrolytes to escape. The primary objective in treating an open wound is, therefore, early coverage.  Secondarily, healed wounds must be optimized for  function and appearance.  Full-thickness wounds lack a dermis and dermis regenerates poorly or not at all.  As a result, these wounds heal slowly with significant scarring and contracture.  Various synthetic collagen-based dermal matrices are now available for use in wounds.^1-11  Dermal replacements have space-filling properties and are intended to provide rapid wound coverage. They must also permit host cell infiltration and controlled neovascularization so that the dermal substitute may be quickly incorporated into the wound and subsuquently remodelled to form dermis that is as similar to unwounded dermis as possible.  However, the efficacy of the available dermal substitutes materials in the treatment of full-thickness wounds has, with the exception of our recent studies^12,13, seldom been carefully compared.

In most cases, placement of a dermal matrix is later followed by split-thickness skin grafting to provide definitive wound closure. The use of cultured autologous keratinocytes (KCs) has been studied extensively as an alternative to autografting, particularly in patients with greater than 40% total body surface area wounds who have limited donor site availability.^14-21 Several methods of KC delivery to wounds are available including combination with a fibrin sealant ^22-26 and application via a spray apparatus.^27-32  We have developed a spray method for applying KCs suspended in fibrin sealant to the wound surface.  In vitro and in vivo tests show that the viability and proliferative potential of the sprayed KCs remains very high.

Neovascularization is a key step in wound healing.  It is a complex process that is dependent upon appropriate interactions between the extracellular matrix, the migrating endothelial cells, and a number of growth factors.  Dermal substitutes composed of native (undenatured), allogenic extracellular matrix such as acellular dermal matrix (ADM) and AlloDerm® become readily vascularized, complement healing, and reduce contractive scarring in a variety of wound types.  We hypothesized that the mode and rate of neovascularization during the process of wound healing in the presence of ADM or AlloDerm® may be important determinants in final wound resolution.  The process of wound vascularization may be significantly altered with materials such as Integra®, Dermalogen®, and Dermagraft® which contain synthetic, highly modified, or denatured substances that may negatively affect wound resolution.

To evaluate this, dermal substitutes in conjunction with FG and KCs were introduced into full-thickness wounds on the dorsum of nude mice.  Paraffin-embedded sections from weekly biopsies were immunostained for laminin, an antigen found in the endothelial basement membrane, to assess and compare vascularization in these healing wounds.  The progress of wound healing and particularly dermal neovascularization was evaluated quantitatively over a period of four weeks based on digital imaging and analysis software. 

METHODS AND MATERIALS

      The following materials were used:

§         Tisseel® (Baxter Health, Deerfield, IL) is a two component system in which fibrinogen, calcium, thrombin, and a protease inhibitor are combined and dispensed onto a wound or other surface to form a fibrin clot.

§         Integra® (Ethicon, Somerville, NJ) is a bilayer artificial skin composed of a “dermal” layer of bovine collagen gel cross-linked with shark chondroitin-6-sulfate and an “epidermal” layer of polysiloxane polymer (which was removed for this study).  Integra® is indicated for partial-thickness wounds, but is being used increasingly for full-thickness wound treatment.

§         AlloDerm® (Lifecell Corp., Branchburg, NJ) is an undenatured collagen matrix derived from human skin that is treated to remove most of the cellular components.

§          Acellular Dermal Matrix (ADM) is a native dermal collagen matrix derived from human skin that is treated to remove all cellular components.  The preparation and characterization of this matrix material has been described previously ^11,33. Briefly, human cadaver skin was treated with Dispase to remove epithelial cells and then Triton-X detergent to remove all residual cells and cellular debris. 

§         Dermalogen® (Collagenesis Corp, Beverly, MA) is a powdered human dermal collagen matrix that has been treated to remove some cellular components and is used primarily for aesthetic surgery.

§         Dermagraft® (Smith & Nephew, Largo, FL) is comprised of a woven bioabsorbable polymer on and in which human dermal fibroblasts are grown and then devitalized.  This material is indicated for treating full-thickness wounds. 

Animals and Surgery

NIH homozygous male nude mice, 4 weeks of age (Taconic, Germantown, NY) were used. All surgical interventions were performed in the John H. Stroger, Jr. Hospital of Cook County Animal Facility using protocols approved by the IACUC.  Preoperative Kanamycin (25U/kg IM) was administered to the animals and ketamine/ xylazine was used for anesthesia.  Under aseptic conditions, 2 cm x 2 cm full-thickness wounds were excised down to the muscle fascia and then implanted with: 1) Integra®, 2) AlloDerm®, 3) ADM, 4) Dermalogen®, 5) Dermagraft®, 6) KCs + FG only, or 7) FG only.  Each group was comprised of at least 6 mice, three of which underwent weekly biopsies.  In the groups of mice that received dermal substitutes, the substitute was cut to size and sutured into the wound using 4-0 nylon sutures.  In the Dermalogen® group, 0.75 cc of the viscous suspension was placed into the wound.  Animals in groups 1-6 received human KCs sprayed onto the dermal substitutes or wound surface in combination with FG.  KCs (4 X 10⁵/ cc) were suspended in the thrombin component of Tisseel® and were sprayed such that the final number of KCs applied to each wound was 2 X 10⁵ in 1.0 cc of FG.  All defects were covered with a semi-permeable adhesive film (Op-Site, Smith & Nephew, Largo, FL), Xeroform (Sherwood Medical, St. Louis, MO), dry cotton gauze (Adaptic, Johnson & Johnson, New Brunswick, NJ), and finally with a fine stainless steel mesh fixed to the animals’ back with skin sutures.  This last was used to prevent the wounds from being disturbed by chewing or scratching.  The dressings were inspected daily.  Biopsies were performed on designated animals at 7, 14, 21, and 28 days post-surgery. The harvested biopsies were fixed in 10% buffered formalin, paraffin embedded, and sections stained with H&E or immunostained.³⁴ 

Human KC Culture and Preparation for Spraying

Human KCs, Epilife culture medium, supplements, and transfer solutions were obtained from Cascade Biologics (Portland, OR).  Cells were grown in 75 cm² flasks from expanded frozen stocks stored after the second passage.  After 7-10 days of proliferation and growth, flasks containing 50-80% confluent KCs were washed, trypsinized briefly to release cells from the substrate, trypsin was neutralized, and the suspension centrifuged at 20xg for 5 min and 4ºC.  The supernatant was discarded and the cells resuspended in fresh Epilife medium. Cells were mixed with the reconstituted thrombin/calcium component of the fibrin sealant kit at a 1:1 dilution.  The fibrinogen component was also reconstituted and diluted 1:1 with Epilife.  This components was stored at 4ºC until being sprayed onto wounds in conjunction with the fibrinogen component using a tuberculin syringe fitted with a spray head as indicated above.³⁴  

Immunostaining

Tissue sections were deparaffinized and antigen retrieval was performed by incubating specimens in pepsin (1mg/ ml 0.01N HCl) at 37ºC for 2 hours.  Following this, nonspecific binding was blocked using 1% bovine serum albumin and sections were then incubated in rabbit anti-mouse laminin IgG (Sigma, St. Louis, MO) at a 1:10 dilution followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (Cappel, Irvine, CA) at a dilution of 1:100.  Reaction product was generated using a Vector DAB/peroxide Developing Kit (Vector, Burlingame, VT) according to the manufacturer’s specifications with a 10 min developing time and specimens were counterstained with hematoxylin QS (Vector, Burlingame, VT).  Normal skin from previously unwounded mice was also immunostained for laminin to serve as a control.  Laminin staining marked the basement membrane of epidermis, nerve, muscle, and vessels.  Within the dermis, immunostained structures showing an open lumen were counted as blood vessels.  These were readily distinguished from the other positively stained structures by their location and morphology. 

Data Analysis

Wound characteristics were measured grossly and histologically.  Gross observations were made at days 7, 14, 21, and 28 post-surgery.  Wound contraction and degree of epithelialization were measured using UTHSCSA ImageTool software in conjunction with digital photographs of wounds  Vessel quantification was performed using the ChromaVision (San Juan Capistrano, CA) Automated Cellular Imaging System (ACIS) in conjunction with microvascular density (MVD) software.  Vessels were counted in the superficial (papillary) dermis, i.e., the region of dermis directly beneath the epidermis, and in the deep (reticular) dermis, i.e., immediately above the hypodermis.  Three different zones of each wound biopsy were evaluated in this way: the wound center (WC), wound margin (WM) and unwounded normal dermis (NP) peripheral to the wound margin.  Zone selection was standardized by defining: WC as the region equidistant from each wound margin; WM as the area directly adjacent to unwounded tissue; and NP as the area with normal skin structures and appendages at least 500 um away from the wound margin. Data obtained by image analysis included the microvascular density (number of vessels/ mm²) and the vessel area (µm²/ vessel) based on laminin immunostaining.   Results were analyzed by one-way ANOVA with Tukey’s post tests.

RESULTS

Gross wound observations

Each of the dermal substitutes except Dermalogen® reduced the amount of wound contraction as compared to wounds that received only sprayed KCs + FG (figure 1).  Thirty-five to 45% wound contraction was observed in wounds implanted with AlloDerm®, Dermagraft®, Integra®, or ADM at 28 days post-operatively.  Greater contraction (60%) was observed in the Dermalogen® and KCs + FG groups (figure 2).  Dermagraft®-implanted wounds showed poor epithelialization and poor incorporation into the wound with contraction limited only as long as the implant was retained.  Often Dermagraft® implants underwent partial or total spontaneous dehiscence and were rejected from the wound.

Laminin Immunostaining and Vessel Counts

Vessel counts were performed on immunostained sections from biopsies taken on days 14 and 28 (figures 2 and 3).  Six different regions were scored for each section: wound center, superficial and deep; wound margin, superficial and deep; normal peripheral dermis, superficial and deep.  For some parts of the analysis superficial and deep counts were combined to yield total counts representative of each region.  The primary control in this study was skin from previously unwounded animals (CTL) for which vessel counts in the superficial and deep regions were performed.  For each of the groups, additional internal controls were included by analyzing the number of vessels in the normal peripheral tissue flanking the wound.  For most of the experimental groups, this tissue exhibited vascularity similar to that of CTL skin. 

During the 28 day study period, the number of vessels seen in the superficial dermis rose from low levels at day 14 to supernormal levels at day 28 for Dermagraft® (p<0.05 vs CTL, one-way ANOVA,Tukey’s post tests) and Dermalogen® (figure 4).  Hypervascularity was seen in the superficial dermis for the KC + FG group beginning at day 14 with little change in vascularity between days 14 and 28.  AlloDerm®, ADM, and Integra® all showed minimal vascularity in the superficial dermis at day 14 (p<0.01 for ADM and AlloDerm vs CTL, p<0.05 for Integra vs CTL).  By day 28, the number of vessels in each of these groups approached the normal level but still remained somewhat hypovascular.

Similar patterns were seen for the number of vessels in the deep dermis over time (figure 5).  For AlloDerm®, there was limited vessel ingrowth in the deep dermis at day 14 (p<0.05 vs CTL)  and low levels at day 28.  The Integra® and ADM groups demonstrated an initial rise in vascularity at day 14 followed by a decrease in vessel number on day 28, such that both groups were ultimately hypovascular.  In contrast, the Dermagraft®, Dermalogen®, and KC + FG groupswere hypervascular  at day 28.  With regard to total vascularity (superficial + deep) at day 28, some of the dermal substitute groups were clearly hypervascular (Dermagraft®, p<0.001, and Dermalogen®, p<0.05) while others were hypovascular (Integra®, AlloDerm®, and ADM) compared to normal skin (CTL) (table 1). 

Vessel Area

The average area per blood vessel was also determined using the ACIS system for vessels present in the superficial and deep regions of the wound center.  The average vessel area in control normal mouse dermis (CTL) was used as the standard to which the values for implanted skin substitutes were compared. In the superficial dermis, larger than normal vessels were seen at day 14 for Integra (p<0.001 vs CTL), KC + FG, and AlloDerm.  In contrast, Dermagraft, Dermalogen, and ADM had smaller than normal vessels at day 14 (p> 0.05).  By day 28, vessel caliber for each of the groups approached normal with no significant differences from CTL, with the exception of Integra, which had persistently large vessels in the superficial region (p<0.01) (figure 6).  This same feature of large caliber vessels was also seen in the deep dermis of wounds treated with Integra at days 14 (p<0.01) and 28 (p<0.05) (figure 7).  In all other groups, vessel size was similar to CTL at both time points.

Degree of Epithelialization

            Digital imaging and analysis were used to determine the percent of the original wound area which was reepithelialized at days 7, 14, 21, and 28 (figure 8).  Wounds treated with AlloDerm,  ADM, and Integra underwent approximately 40% epithelialization by day 28.  In contrast, wounds treated  with Dermalogen or Dermagraft were less epithelialized (20% of original wound) at the conclusion of the study. Wounds treated with KC + FG alone were 35% epithelialized at day 28. 

DISCUSSION

Angiogenesis is integral to effective wound healing.  Blood vessels deliver oxygen, nutrients and inflammatory cells into the wound and provide conduits for the removal of metabolic by-products and debris from damaged tissue.  Appropriate angiogenesis is a complex process that involves endothelial cell division, selective degradation of vascular basement membrane and of surrounding extracellular matrix, and endothelial cell migration.³⁵  The level of organization of the extracellular matrix plays a key role in the regulation of neovascularization in that it provides support for migrating endothelial cells and acts as a reservoir for endothelial cell growth factors derived from the plasma or serum and from infiltrating fibroblasts, leukocytes, and migrating KCs.³⁶  It is thought that full-thickness wounds should undergo an initial phase of vigorous angiogenesis that is later followed by vessel regression, such that the final pattern of vascularization is similar to that of normal skin.³⁷

            However, no studies have been published directly comparing wound healing seen with different commercially-available dermal substitutes.  Thus, it is difficult to objectively determine which dermal substitutes are most useful in the treatment of full-thickness wounds.  Within this context, there are few studies which have examined the process of neovascularization in wounds implanted with dermal substitutes.  We hypothesized that wounds implanted with dermal substitutes composed of undenatured, allogenic extracellular matrix such as ADM and AlloDerm®, wound be readily incorporated and develop vascularization more similar to normal skin than dermal substitutes composed of synthetic, non-native, or denatured substances such as Integra®, Dermalogen®, and Dermagraft®.  To a large extent, this hypothesis was confirmed by the data presented here.

Grossly, wounds implanted with ADM, AlloDerm®, or Integra® demonstrated less wound contraction losing about 40% of their original size by day 28 and better cosmetic results than did Dermagraft®, Dermalogen®, or KC + FG only.  Our findings indicate that, of the dermal substitutes with native compositions, ADM and AlloDerm® underwent a gradual and limited pattern of neovascularization and were ultimately somewhat hypovascular at day 28.  In terms of vessel size, wounds treated with ADM and AlloDerm contained vessels of a caliber similar to CTL at both day 14 and 28.  For the highly modified or synthetic dermal substitutes, Dermagraft® and Dermalogen® (and KCs + FG alone), vascularization proceeded rapidly resulting in hypervascularity by day 14 or day 28. The size of the vessels in these wounds was also similar to CTL on days 14 and 28.

The results for Integra® were noteworthy in that despite its denatured, highly modified composition relative to normal dermis, it underwent a controlled pattern of vessel ingrowth that appeared to be conducive to improved wound healing.  This seems to confirm the previous claims that the collagen-GAG matrix of Integra® has been specifically designed to have pore sizes that ensure adequate microvascularization of the forming neodermis.^38-40 Notably, wounds in the Integra group contained vessels of  significantly larger areas than CTL at both days 14 and 28.  Upon further review of these specimens, it appears that they contain an unusually large number of tortuous vessels that are oriented perpendicularly to the wound surface.  These features may cause the vessel area calculations performed here to be skewed toward large vessel areas since more vessels were cut in longitudinal or tangential section due to their orientation in the tissue. This feature of Integra is likely related to the design of the Integra matrix, with the orientation of the pores allowing vessel growth in this configuration.  This result may be related to the improved wound healing observed with the use of Integra and clinical observations showing its ability to support overlying skin grafts. 

 Wounds in the Dermagraft® group demonstrated extensive granulation which may, to some extent, be attributable to its content of non-viable fibroblasts that may act as a source of vascular endothelial growth factor (VEGF).  Similarly, when viable fibroblasts are added to de-epidermized dermis, enhanced wound vascularization has been shown.⁴²  However, the present study illustrates that too much neovascularization can also result in impaired wound healing.  In the Dermalogen® and Dermagraft® groups hypervascularity at day 28 correlated with increased wound contraction and poor wound cosmesis.  Overall, there seems to be an optimal rate and final level of vascularization in healing wounds, where either increased or decreased rates or levels are associated with suboptimal healing. 

These results with Integra® and Dermagraft® also point up one important aspect of wound healing could not be tested in this model.  The model is insensitive to the xenogenic (in this case, non-mouse) nature of some of the components of these dermal substitutes because of the T cell immunodeficiency that characterizes nude mice.  Of course, it is this immunodeficiency that makes a study such as this possible.  Nonetheless, aspects of inflammation that might be triggered in humans by the presence of allogenic or xenogenic materials such as shark chondroitin sulfate, bovine collagen, or human fibroblasts are not seen in this model.  Interestingly, the dermal substitutes that contain such materials do not seem to evoke a strong immune response in humans.

Clinical experience tells us that certain minimum amounts of wound vascularization must be present and certain maximum amounts of granulation tissue may be tolerated for implanted skin grafts to survive.  The present study shows that the composition of implanted dermal substitutes can affect angiogenesis.  This will undoubtedly determine the level of oxygenation and the growth factor milieu (EGF, FGF, PDGF, VEGF, etc.) in the dermis.  Dermagraft® and Dermalogen® underwent extensive granulation, whereas AlloDerm®, Integra®, and ADM underwent limited vessel ingrowth that seemed to be conducive to the development of normal dermal structure, reepithelialization, and minimal wound contraction.  These data indicate that the rate and final extent of vascularization are important determinants in the efficacy of dermal substitution for the treatment of full-thickness wounds.  Further efforts to achieve one-step full-thickness wound closure will depend upon the use of dermal substitutes that can vascularize rapidly enough to support overlying KCs or an ultra-thin split-thickness graft, but will not induce overly abundant granulation tissue formation.  This is a realistic goal using currently available biomaterials but further refinements are needed to achieve optimal healing.

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FIGURES

 Figure 1. Percent of wound contraction over the 28-day study period for each of the dermal substitutes. Error bars represent SEM. Data was analyzed by one-way ANOVA with Tukey’s post tests.

Figure 2.  Laminin-stained cross-sections from the wound center following treatment with KC + FG only, Dermagraft, and Dermalogen on post-operative day 28 (above) and the corresponding gross appearance of these wounds at day 28 (below). Extensive vascularization is seen in both the superficial and deep dermis in all three of these wounds.Sutures mark the corners of the original wound.  Note the extensive contraction of wounds treated with KC + FG only and Dermalogen®.  Dermagraft®-implanted wounds showed poor epithelialization and poor incorporation into the wound with contraction limited only as long as the implant was retained.  Often part of the Dermagraft® implant underwent spontaneous dehiscence and was rejected from the wound. Magnification bar = 100 μm.

Figure 3.  Laminin-stained cross-sections from the wound center following treatment with AlloDerm, ADM, and Integra on post-operative day 28 (above) and the corresponding gross appearance of these wounds at day 28 (below). Controlled vessel ingrowth is seen in the AlloDerm and ADM groups. Note the numerous large and small cavities in the Integra® implant.  In life, these cavities held the collagen-chondroitin sulfate colloid that comprises Integra® and this colloid is still present at this time point. Several large vessels and vessels cut in tangential or longitudinal section are seen in this specimen.  The vessels appear to be growing around the cavities formed in the Integra implant.  In the gross photographs, sutures mark the corners of the original wound.  Note the limited contraction and nearly complete epithelialization of these wounds. Magnification bar = 100 μm.

  Figure 4.  Graph depicting changes in vessel number (mean±SEM) in the superficial dermis at the center of the wound over the study period of 28 days.  The dashed horizontal line represents the vascular counts in normal mouse skin (CTL). Note the hypovascularity of the Integra, AlloDerm, ADM, and Dermalogen groups at day 14 in comparison to CTL, and the hypervascularity seen with implanted Dermagraft at day 28. Data were analyzed using one-way ANOVA with Tukey’s post tests.

Figure 5.  Graph depicting changes in vessel number (mean±SEM) in the deep dermis at the center of the wound over the study period of 28 days.  The horizontal dashed line represents the vascular counts in normal mouse skin (CTL). Data were analyzed using one-way ANOVA with Tukey’s post tests.

Figure 6.  Graph depicting changes in vessel areas (mean±SEM) in the superficial dermis at the center of the wound over the study period of 28 days.  The dashed horizontal line represents the vessel size in normal mouse skin (CTL). Note that the vessels in wounds treated with Integra are larger than CTL at day 14 (p<0.001) and day 28 (p<0.01).  Data were analyzed using one-way ANOVA with Tukey’s post tests.

   Figure 7.  Graph depicting changes in vessel areas (mean±SEM) in the deep dermis at the center of the wound over the study period of 28 days.  The dashed horizontal line represents the vessel size in normal mouse skin (CTL). Note that the vessels in wounds treated with Integra are larger than CTL at day 14 (p<0.001) and day 28 (p<0.01).  Data were analyzed using one-way ANOVA with Tukey’s post tests.

Figure 8.  Graph depicting percentage of original wound area epithelialized over the study period of 28 days.  Error bars represent SEM.  At day 28, wounds implanted with AlloDerm, Integra, or ADM were approximately 40% reepithelialized, whereas wounds implanted with Dermagraft or Dermalogen were only 10-20% reepithelialized. 

Table 1.  Table of vessel numbers and sizes (mean±SEM) in the total dermis (superficial + deep) for each of the groups at day 28.

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