Growth of Human Keratinocytes on ADM in Vitro

May 1, 2012

This study was an attempt to use the acellular dermal matrix material that we had developed earlier into a substitute for full-thickness skin.  ADM could be used as a dermal filler or substitute in situations where the dermis was damaged or absent but, by itself, ADM lacked an epithelial barrier to infection and fluid loss.  Here we used ADM as the substrate for growing human keratinocytes in culture. A patent layer of keratinocytes would provide an effective epithelial barrier over the ADM.

However, keratinoctes are fairly difficult to grow due to their stringent growth requirements.  They can be easily and irreversibly damaged by the tryptic enzymes used for cell transfers, require carefully controlled soluble calcium levels for optimal growth and differentiation, must be handled carefully during transfers to minimize damage, require a number of growth factors and hormones to achieve reasonable or good survival and growth. 

This sensitivity to growth conditions complicated this study considerably.  We added a range of different attachment factors, growth factors, or extracellular matrix components to the ADM in an effort to promote keratinocytes attachment and growth on the ADM substrate.  The following paper describes the results of these experiments.

The full paper and a link to the PDF are shown below.

Growth of Human Keratinocytes on ADM in Vitro

Growth of Human Keratinocytes on 

Acellular Dermal Matrix in Vitro

By

Lawrence J. Jennings, MD¹, Evangeline Z. DeSagun, MD²,

Marella Hanumadass, MD¹, and Robert J. Walter, PhD³ 

¹ Burn Center, Cook County Hospital, Chicago, IL 60612

² Department of Pathology, Cook County Hospital, Chicago, IL 60612

 ³ Department of Surgery, Cook County Hospital and Department of General Surgery,          Rush University Medical Center, Chicago, IL 60612

Running Title:    Keratinocyte growth on ADM

Keywords:  keratinocytes, acellular dermal matrix, matrigel, dermis, collagen, laminin, chondroitin sulfate, dermatan sulfate, hyaluronate, fibronectin, heparin, polylysine, fetal calf serum, artificial skin, wound healing, burn

Correspondence should be addressed to:

Robert J. Walter, PhD

Division of Surgical Research

Department of Surgery

Cook County Hospital

627 South Wood Street

Chicago, Illinois   60612 

Telephone (312) 633-7237;  FAX (312) 633-8347

E-mail: rwalter@rush.edu

ABSTRACT

            A composite skin substitute composed of acellular dermal matrix (ADM) covered by a layer of human keratinocytes (KCs) would be very useful in treating burns or other injuries.  To determine the factors necessary to support KC attachment and growth on human ADM, we treated ADMs with different extracellular matrix components and cell attachment factors, plated KCs onto the treated ADM, and then cultured the KCs for three or ten days.  At these times, ADMs were frozen, cryosectioned, and assessed for KC attachment and growth.  ADM prepared in the presence of azide was mildly toxic to KCs that came into direct contact with it even after it had been extensively washed.  ADM prepared with antibiotics provided a much better substrate for KC attachment and growth, but growth was still not optimal.  Despite pretreating the ADM with a range of basement membrane constituents (laminin, Matrigel, collagen IV), extracellular matrix components (collagen I, chondroitin or dermatan sulfate, hyaluronate, fibronectin), or other attachment factors (heparin, polylysine), keratinocyte growth was not significantly improved.  Pretreatment of the ADM with fetal calf serum (20% or 100%) or co-culture with 2%fetal calf serum, fibroblast conditioned medium, or 3T3 cells significantly promoted keratinocyte attachment to and growth on ADM such that confluent monolayers formed.

INTRODUCTION

In extensively burned patients, prompt replacement of damaged skin is essential to limit the morbidity and mortality associated with these injuries.  Since donor sites for autografts are limited and the donor sites themselves become a source of long-term morbidity, alternative sources of non-immunogenic skin and functional skin substitutes are being developed (1-6).  In this pursuit, cultured epithelial autografts derived from the growth of autogenic keratinocytes (KCs) in vitro (7,8) and several types of dermal substitutes have been used with varying degrees of success (9-14).  Dermal substitutes composed of gelled type I collagen (6,15), type I collagen mixed with glycosaminoglycan (16), or fibrin (17) is conducive to the attachment of human KCs, their subsequent proliferation, and the formation of differentiated multilayers.  In addition, many basement membrane and extracellular matrix components (ECM) including type IV collagen, laminin, fibronectin, and RGD tripeptide (arg-gly-asp) (18,19) have been found to improve human KC attachment and sometimes their proliferation on a variety of substrates used for tissue culture.

Using de-epidermized dermis, Krejci et al. (20) found that KCs from foreskin explants readily grew out onto the papillary, but not the reticular, dermal surface in vitro.  This suggested that one or more components of the basement membrane might be required for KC attachment and growth on native dermis.  However, pretreatment of the dermis with fibronectin or type IV collagen did not improve KC outgrowth onto the reticular surface of de-epidermized dermis.  Ralston et al. (21) have also shown the importance of basement membrane antigens in facilitating KC attachment and growth on de-epidermized dermis.  While de-epidermized dermis is a useful substrate for studying KC growth and differentiation, it seems to retain a variety of cell-associated antigens that may make it unsuitable for implantation into an immunocompetent host (22).  A more thoroughly decellularized material, acellular dermal matrix (ADM), is derived from human cadaver skin that has been processed to remove the epidermis and all cellular components leaving only the connective tissue dermal matrix.  It has been found to be very weakly- or non-antigenic and is an effective dermal substitute when used in conjunction with onlay grafts of ultrathin split-thickness skin (3,23,24).  However, ADM is a poor substrate for KC growth in vitro.

Rennekampff et al. (25) found that human KCs attached poorly to AlloDerm®, one type of ADM, in vitro and that added fibronectin did not improve this poor adherence.  Further, we have found that ADMs prepared by treating skin using either the NaCl/ sodium dodecyl sulfate method (like AlloDerm®) or the dispase/Triton X-100 method (3) serve as poor substrates for KC growth in serum-free, low-Ca⁺⁺ medium (26).  We have also reported that several ECMs including glycosaminoglycans (chondroitin, dermatan, and keratan sulfates, and hyaluronic acid), components of the basal lamina (collagen types IV and VII, laminin), and cell attachment factors (e.g., fibronectin) are partially depleted or absent from these ADMs (14).  Because many of these tissue components are known to promote epithelial attachment to underlying connective tissue and to support epithelial cell proliferation, we hypothesized that KC attachment and growth on ADM might be improved by replenishing one or more of these factors or by employing other agents to promote cell attachment.  Here we report the effects of a variety of crude and purified ECMs, growth factors, and cell attachment factors on KC attachment to and proliferation on dispase/ Triton human ADM prepared in the presence of either azide or antibiotics. 

ABBREVIATIONS USED

acellular dermal matrix, ADM; extracellular matrix components, ECMs; fetal calf serum, FCS; keratinocyte, KC; keratinocyte growth medium, KGM; phosphate buffered saline, PBS

MATERIALS AND METHODS

Preparation of ADM

Normal human skin, 0.012 inches thick, obtained from cadavers using a dermatome (Padgett Electro-Dermatome, Padgett Instruments, Inc., Kansas City, MO) was washed in RPMI-1640 containing 10% human serum, and then frozen at -80°C.  Tissue was obtained according the ethical guidelines established in the 1975 Declaration of Helsinki and the Institutional Review Board.  Cryopreserved skin was thawed rapidly at 37°C in saline and was then treated with 2.5 units/ml dispase II (Boehringer Mannheim, Indianapolis, IN) in phosphate buffered saline (PBS) containing 0.2 mM CaCl₂ at 4°C for 24 hours to remove the epidermis and other cellular components from the dermal matrix.  Subsequently, the dermal matrix was incubated in buffered 0.5% Triton X-100 (U. S. Biochemical Corp., Cleveland, OH) for 24 hours at room temperature with continuous shaking.  Dispase-Triton ADM was then extensively washed with PBS and stored at 4°C until use.  All solutions were filter-sterilized and all procedures were performed aseptically.  Either sodium azide (0.02% w/v) or a cocktail of antibiotics (300 U/ml penicillin, 0.3 mg/ml streptomycin, 0.75 μg/ml fungizone, 50 mg/ml gentamycin) was present at all times during both of these steps to deter microbial growth (3). 

ADM Treatment and Keratinocyte Culture

ADMs were washed extensively with sterile saline, cut into 10X10 mm pieces, treated with ECMs or attachment factors for 24 h at room temperature, rinsed thoroughly with PBS, attached to sterile stainless steel mesh, and placed into 24-well culture plates.  The following ECMs and attachment factors were used: laminin (12 μg/cm²), fibronectin (10 μg/cm²), Matrigel (60 μl/cm²), fibrin (0.5 mg/cm²), collagen type I (0.25 mg/cm²), collagen type IV (2.5 μg/cm²), heparin (5 mg/cm²), chondroitin sulfate (5 mg/cm²), dermatan sulfate (0.5 mg/cm²), hyaluronic acid (0.2 mg/cm²), fetal calf serum (FCS; 2%, 20%, 100%), and polylysine (0.1%, 0.001%).  Types I and IV collagen, Matrigel, and laminin were from Collaborative Biomedical, Bedford, MA; hyaluronic acid from Seikagaku, Tokyo, Japan; plasma fibronectin, polylysine, fibrinogen, thrombin, chondroitin sulfate, and dermatan sulfate from Sigma Chemicals, St. Louis, MO; heparin from US Biochemicals, Cleveland, OH; and FCS from Gibco BRL, Grand Island, NY.

Human KCs obtained from foreskin were maintained in KGM supplemented with bovine pituitary extract (Clonetics, Temecula, CA) and were trypsinized and plated into 24-well plates (100,000 cells/ well) containing treated ADMs.  Cultures were continued for 3 or 10 days in KGM alone, in KGM supplemented with 10% human fibroblast-conditioned medium or 2% FCS or in KGM with co-cultured 3T3 fibroblasts.

Tissue Preparation and Staining

ADMs were removed from culture vessels, embedded in Tissue Freezing Medium (Triangle Biomedical, Durham, NC), frozen, and cut in cross-section at a thickness of 8 mm using an IEC Minotome cryostat.  Sections were picked up on chrome-albumin subbed glass slides and allowed to dry at room temperature.  Dried cryosections were fixed in formalin and 95% ethanol (1:9), rinsed in water, and stained using H&E.  The nuclei of KCs attached to the ADM were counted in twenty 40X microscope fields (the field diameter was 0.45 mm) and this number was then averaged and converted to cells per linear mm of ADM cross-section (cells/ mm).

Statistical Analysis

Cell counts for each group were averaged and means ± SEM calculated.  Unpaired t-tests or ANOVA were used to compare groups and p values less than 0.05 were considered significant.

RESULTS

As seen in Figure 1, all of the ADMs prepared in the presence of azide showed relatively few attached KCs.  KC attachment (Figures 2 and 3) for the ADMs pretreated with 100% FCS, fibroblast-conditioned medium, or grown in the presence of 2% FCS showed improved KC attachment and growth after 3 days in culture (8.6, 10.7, and 10.5 cells per mm of cross-sectioned ADM, respectively) as compared to untreated ADM (5.4 cells per mm) but these differences were not significant.  Polylysine did not affect cell attachment but reduced cell viability.  Laminin, chondroitin sulfate, dermatan sulfate, heparan sulfate, and collagen IV did not significantly affect KC attachment and growth on ADM.  All groups tested showed fewer attached KCs after 10 days in culture than after 3 days (p<0.001, ANOVA).  On the tenth day of culture, the cell numbers in the 2% FCS group were significantly (p=0.03, t-test) greater than those of the controls.

Greatly improved cell attachment and growth (Figure 4) were noted in ADM processed in the presence of antibiotics (i.e., without azide) as compared to ADM processed in the presence of azide (p<0.001; ANOVA).  Nevertheless, fewer cells were present after 10 days in culture for most groups (Figures 5 and 6; e.g., 7.8 vs. 2.0 cells/mm for the control group on days 3 and 10, respectively).  However, for the KCs grown on ADM pretreated with FCS (20% or 100%), or cultured in the presence of human fibroblast-conditioned medium or 2% FCS (Figure 6), cell proliferation continued after the third day.  As a result, continuous KC monolayers were observed on the ADM by the tenth day (e.g., 10.9 cells/mm at 3 days and 23.5 cells/mm at 10 days for the 20% FCS group).  By the tenth day in culture, the cell numbers in the 2% FCS, 20% FCS, human fibroblast conditioned medium, and co-cultured 3T3 cell groups were significantly (p<0.003, p<0.001, p<0.003, p<0.04, respectively; t-tests) greater than those of the control.  ADMs treated with other attachment factors showed fewer attached KCs after 10 days in culture than after 3 days. 

DISCUSSION

Despite extensive washing, residual azide may have remained associated with ADMs prepared in the presence of azide, resulting in poor KC attachment and survival in the 3 and 10 day groups (see Figures 2 and 3).  Interestingly, the only KCs affected were those directly in contact with the ADM.  KCs that attached to the bottom of the ADM-containing culture wells proliferated normally during the ten day culture period.  Thus, the azide or other deleterious substance(s) was not released into the culture medium and affected only cells in direct contact with the ADM.  Cell numbers declined for most groups on azide-treated ADM between the third and tenth days in culture.  To some extent, this can be attributed to the aforementioned azide effect, but a similar decline was also seen for many of the groups in which KCs were grown on azide-free ADM (e.g., untreated, fibronectin, chondroitin sulfate, dermatan sulfate, etc.)(see Figure 5).  There are several possible reasons that such a varied assortment of basement membrane components, glycosaminoglycans, and other ECMs would fail to improve KC attachment to and growth on ADM.  These supplements may not have bound to the ADM initially or they may have bound but: 1) subsequently dissociated from the ADM, 2) the ADM may have exerted an overriding inhibitory effect on KC growth, or 3) do not benefit KC growth.

To assure initial binding of the supplements, they were applied at the concentrations and under conditions similar to those described elsewhere or as recommended by the manufacturers.  However, most other studies that use ECMs to enhance cell attachment or growth in culture permit them to air-dry onto plastic tissue culture substrates or dermal matrices (see e.g., (19,27,28)) or incorporate them into semi-synthetic matrices during fabrication (16,29).  The latter method was not feasible here and air-drying was deemed undesirable because of the attendant potential for denaturation or modification of ADM components due to surface tension forces.  While such alterations might not adversely affect the results of the present in vitro studies, denatured ADMs used for implantation would likely evince greater antigenicity and less stability, thereby adversely affecting wound healing.  Instead, in the present study, we sought to exploit the known binding affinities of the ECM materials for type I collagen and other components of the hydrated, native ADM in order to effect their attachment to the dermal matrix.  To assess the initial binding to and retention by ADM, we used fluorescein-conjugated anti-fibronectin to detect fibronectin on cryosectioned ADM using fluorescence microscopy.  This confirmed that supplemental fibronectin did bind to the ADM under the conditions used here for the fibronectin, FCS (2%, 20%, 100%) co-cultured 3T3 cells, and fibroblast conditioned medium groups and that this fibronectin remained bound for the entire ten day culture period (data not shown).  Based on this finding and the known binding affinities of the various supplements used, these supplements very likely bound initially to the ADM, but some may have subsequently dissociated from the ADMs or were degraded during extended cell culture thereby abrogating any potentially beneficial effects to KC proliferation.  Indeed, Hanthamrongwit et al. (30)noted that chondroitin sulfate dissociated rapidly after it had been applied to collagen sponges used as in vitro KC substrates. 

Components of the basement membrane including laminin-5 (17), type IV collagen (17,18), and Matrigel (a mixture of type IV collagen, laminin, heparan sulfate, and entactin) (18,28) have each been shown to promote KC attachment and proliferation on tissue culture plastic and on some types of dermal matrices (21).  Related connective tissue components such as type I collagen gels, plasma fibronectin, and RGD have shown variable effects on KC attachment ranging from inhibition to stimulation (18,19,28,29).  However in the present study, as in that of Krejci et al. (20) where type IV collagen and fibronectin were tested on second-cut dermis, each of these compounds had little effect on KC attachment and growth.  Glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, and hyaluronate are also known to improve in vitro KC attachment and proliferation (2,16,30-32) and fibrin glue has been found to be effective as a substrate for KC growth (17,33).  However, as with the present study, Shakespeare and Shakespeare (34) reported that fibrin appeared to inhibit KC attachment to dermal collagen.  The above-mentioned basement membrane components and ECMs may have been ineffective in the present system because most of them form gels at the concentrations employed for cell culture and these gels may infiltrate poorly into the ADM.  As a result, the gelled material may readily dissociate or detach from the ADM together with any attached KCs.  Our observations of cryostat sections tend to confirm this for type I collagen, Matrigel, hyaluronate, and chondroitin sulfate.  Further studies to quantify the extent of the initial binding to and dissociation from ADM of these supplements are needed and are in progress.

The effects of polylysine and heparin on KC growth on ADM were also evaluated.  These agents were studied because of their known ability to bind to surfaces by electrostatic interactions (polylysine) or through specific binding domains (heparin) on both cell surfaces and collagen.  KCs grown on ADMs pretreated with polylysine at high concentration (0.1%) were non-viable probably due to the continued release of residual polylysine from the ADM since other cells in the same wells, attached to the bottom of the culture plate, were also killed.  At lower concentrations, these substances were found to have no effect on KC binding and proliferation.

KCs grown on azide-free ADM cultured in the presence of human fibroblast-conditioned medium (which also contained FCS at a final concentration of 2%) developed into confluent monolayers on the ADM by the tenth day in culture.  Under similar conditions, we have also seen that multilayers developed when these monolayer cultures were moved to the air-liquid interface (unpublished data).  For the purposes of the present study, the formation of monolayers was used as the end-point.  It is not entirely surprising that fibroblast-conditioned medium and co-cultured 3T3 cells enhance KC growth on ADM since the latter is the classical method for stimulating KC growth in vitro (35) and this method has also succeeded in improving KC growth on dermal substitutes in other studies (18,20,21,28,36).  However, even pretreatment of the ADM with FCS (20% or 100%) was sufficient to permit extensive KC attachment, proliferation, and monolayer development by the tenth day in culture.  Because plasma fibronectin is one of the most abundant cell attachment factors found in serum, we tested the effect of purified fibronectin on KC attachment to and proliferation on ADM, but as described above, it had no effect.  Thus, we cannot determine precisely how FCS benefits KC attachment and proliferation in this system.  FCS may exert a non-specific blocking effect, coating the ADM with protein and masking sites or substances that would otherwise inhibit KC attachment and growth.  Alternatively, in experiments where 2% FCS was present during the entire ten day culture period, FCS may provide a continuous source of factors (e.g., plasma fibronectin, mitogens, nutritional factors) that enhance KC attachment and proliferation.  Because KC survival was reduced in all other treatment groups between the third and tenth days in culture, we speculate that either the KCs underwent apoptosis due to inadequate cell-substrate interaction or the nutritional requirements for KCs growing on ADM are altered such that KGM is not sufficient to maintain viability.

In conclusion, it seems that the presence of azide during ADM preparation is detrimental to the subsequent growth of KCs on ADM, but that KC attachment and proliferation are greatly improved on ADM prepared in the presence of antibiotics rather than azide.  Nonetheless, a variety of purified ECMs and other cell attachment factors known to be effective in promoting cell attachment or growth in other culture systems were ineffective in promoting KC growth on ADM.  Alternative methods for applying these supplements to the ADM such as chemical cross-linking, lyophilization, or continuous application throughout the culture period are currently under study.  The presence of FCS, co-cultured fibroblasts, fibroblast-conditioned medium, or pretreatment of the ADM with FCS resulted in good KC attachment and growth on ADM and resulted in growth of confluent KC monolayers on ADM.  These studies show that dispase/ Triton ADM can be a good substrate for KC growth and indicate that it has future potential for use in the production of KC-ADM composites for use in implantation.

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FIGURES

  Figure 1

Human KCs growing on the surface of human ADM after 3 days (left) or 10 days (right) in culture.  ADM was prepared in the presence of azide, pretreated with the substances shown for 24 hrs, washed, and then KCs were plated onto the ADM.  For some of the treatment groups (10% human fibroblast conditioned medium, 2% FCS, or co-cultured 3T3 cell groups), the KCs were maintained for the entire 3 or 10 day period in the presence of these additional factors.  After 3 or 10 days in culture, ADMs were cryosectioned and photographed. H&E stain.  Magnification bar = 400 μm

 

 Figure 2

Human azide-prepared ADM was pretreated for 24 hr as shown, washed in buffer, human keratinocytes were plated onto the treated ADM in 24-well plates (100,000/ well), and the cultures continued for 3 (open bars) or 10 (cross-hatched bars) days in KGM.  ADMs were cryosectioned and adherent cells counted in twenty 40X fields.  Data are expressed as means of cell counts ± SEM.  The number associated with each bar represents the number of experiments (n) performed using each compound; bars for 3 and 10 day groups are overlapping not stacked.

 Figure 3

Human azide-prepared ADM was pretreated for 24 hr as shown, washed in buffer, human keratinocytes were plated onto the ADM in 24-well plates (100,000/ well), and the cultures continued for 3 (open bars) or 10 (cross-hatched bars) days in KGM.  Alternatively, keratinocytes were maintained in the presence of 10% human fibroblast (hu Fb) conditioned medium (which contained 2% FCS), 2% FCS alone, or co-cultured 3T3 cells for the entire 3 or 10 day period.  ADMs were cryosectioned and adherent cells counted in twenty 40X fields.  Data are expressed as means of cell counts ± SEM.  The number associated with each bar represents the number of experiments (n) performed using each compound; bars for 3 and 10 day groups are overlapping not stacked.

Figure 4

Human KCs growing on the surface of human ADM after 3 days (left) or 10 days (right) in culture.  ADM was prepared in the absence of azide, pretreated with the substances shown for 24 hrs, washed, and then KCs were plated onto the ADM.  For some of the treatment groups (10% human fibroblast conditioned medium, 2% FCS, or co-cultured 3T3 cell groups), the KCs were maintained for the entire 3 or 10 day period in the presence of these additional factors.  After 3 or 10 days in culture, ADMs were cryosectioned and photographed. H&E stain.  Magnification bar = 400 μm

 

Figure 5

Human ADM prepared in antibiotics was pretreated for 24 hr as shown, washed in buffer, human keratinocytes were plated onto the ADM in 24-well plates (100,000/ well), and the cultures continued for 3 (open bars) or 10 (cross-hatched bars) days in KGM.  ADMs were cryosectioned and adherent cells counted in twenty 40X fields.  Data are expressed as means of cell counts ± SEM.  The number associated with each bar represents the number of experiments (n) performed using each compound; bars for 3 and 10 day groups are overlapping not stacked.

 Figure 6

Human ADM prepared in antibiotics was pretreated for 24 hr as shown, washed in buffer, human keratinocytes were plated onto the ADM in 24-well plates (100,000/ well), and the cultures continued for 3 (open bars) or 10 (cross-hatched bars) days in KGM.  Alternatively, keratinocytes were maintained in the presence of 10% human fibroblast (hu Fb) conditioned medium, 2% FCS, or co-cultured 3T3 cells for the entire 3 or 10 day period.  ADMs were cryosectioned and adherent cells counted in twenty 40X fields.  Data are expressed as means of cell counts ± SEM.  The number associated with each bar represents the number of experiments (n) performed using each compound; bars for 3 and 10 day groups are overlapping not stacked.

 TABLE 1

Keratinocyte Attachment and Growth on Azide-Sterilized ADM

Effect of Basement Membrane and Extracellular Matrix Components

Treatment	Human keratinocytes/ mm of ADM cross-section
ADM with azide	3 day cultures	n	10 day cultures	n
control, untreated	5.4 ± 1.7	5	2.7 ± 0.7	5
laminin	3.6 ± 0.5	3	0.8 ± 0.2	3
plasma fibronectin	2.1 ± 0.2	3	1.7 ± 0.3	3
Matrigel	1.0 ± 0.2	2	0.8 ± 0.3	2
collagen type IV	2.1 ± 0.5	2	0.5 ± 0.2	2
collagen type I	7.8 ± 3.3	3	1.4 ± 0.7	3
chondroitin sulfate	1.4 ± 0.5	2	0.4 ± 0.1	2
dermatan sulfate	4.6 ± 1.0	2	0.2 ± 0.1	2
hyaluronic acid	3.7 ± 0.8	2	1.2 ± 0.5	2

Human ADM was pretreated for 24 hr with the compounds shown above, washed in buffer, human keratinocytes were plated onto them in 24-well plates (100,000/ well), and the cultures continued for 3 or 10 days in KGM.  ADMs were cryosectioned and adherent cells counted in twenty 40X fields.  Data are expressed as means of cell counts from the indicated number (n) of  experiments.  Means ± SEM.

TABLE 2

Keratinocyte Attachment and Growth on Azide-Sterilized ADM

Effect of Attachment Agents

Treatment	Human keratinocytes/ mm of ADM cross-section
ADM with azide	3 day cultures	n	10 day cultures	n
control, untreated	5.4 ± 1.7	5	2.7 ± 0.7	5
FCS - 2%	10.5 ± 4.5	3	8.0 ± 1.9 (p=0.03)	3
FCS - 20%	4.4 ± 0.7	5	3.6 ± 0.5	5
FCS - 100%	8.6 ± 2.3	2	4.6 ± 1.2	2
polylysine - 0.1%	5.3 (non-viable)	4	5.0 (non-viable)	4
polylysine - 0.001%	6.8 ± 1.2	4	5.0 ± 1.3	4
heparin	0.9 ± 0.5	2	0.4 ± 0.5	2
hu Fb conditioned medium	10.7 ± 2.1	3	4.7 ± 1.6	3
co-cultured 3T3 cells	8.8 ± 2.8	5	4.8 ± 1.8	5

Human ADM was pretreated for 24 hr with the compounds shown above, washed in buffer, human keratinocytes were plated onto them in 24-well plates (100,000/ well), and the cultures continued for 3 or 10 days in KGM.  Alternatively, keratinocytes were maintained in the presence of 10% human fibroblast (hu Fb) conditioned medium, 2% FCS, or co-cultured 3T3 cells for the entire 3 or 10 day period.  ADMs were cryosectioned and adherent cells counted in twenty 40X fields.  Data are expressed as means of cell counts from the indicated number (n) of  experiments.  Means ± SEM.

TABLE 3

Keratinocyte Attachment and Growth on Antibiotic-Sterilized ADM

Effect of Basement Membrane and Extracellular Matrix Components

Treatment	Human keratinocytes/ mm of ADM cross-section
ADM without azide	3 day cultures	n	10 day cultures	n
control, untreated	7.8 ± 1.7	9	2.0 ± 0.7	9
laminin	6.4 ± 1.5	2	1.1 ± 0.2	2
plasma fibronectin	7.8 ± 2.7	9	2.0 ± 0.5	9
Matrigel	4.7 ± 1.6	2	3.3 ± 0.6	2
collagen type IV	6.1 ± 1.2	2	1.5 ± 0.6	2
collagen type I	6.7 ± 1.5	2	3.2 ± 1.5	2
chondroitin sulfate	13.6 ± 4.8	2	4.1 ± 1.4	2
dermatan sulfate	7.1 ± 2.6	2	5.0 ± 1.5	2
hyaluronic acid	11.8 ± 4.1	5	5.6 ± 1.1	5

Human ADM was pretreated for 24 hr with the compounds shown above, washed in buffer, human keratinocytes were plated onto them in 24-well plates (100,000/ well), and the cultures continued for 3 or 10 days in KGM.  ADMs were cryosectioned and adherent cells counted in twenty 40X fields.  Data are expressed as means of cell counts from the indicated number (n) of experiments.  Means ± SEM.

TABLE 4

Keratinocyte Attachment and Growth on Antibiotic-Sterilized ADM

Effect of Attachment Agents

Treatment	Human keratinocytes/ mm of ADM cross-section
ADM without azide	3 day cultures	n	10 day cultures	n
control, untreated	7.8 ± 1.7	9	2.0 ± 0.7	9
fibrin	2.6 ± 1.5	2	1.8 ± 0.5	2
FCS - 2%	16.2 ± 6.3	7	19.6 ± 5.1 (p=0.001)	7
FCS - 20%	10.9 ± 4.3	6	23.5 ± 5.9 (p=0.001)	6
FCS - 100%	10.2 ± 3.2	2	18.5 ± 4.8	2
polylysine - 0.001%	7.7 ± 1.6	5	8.9 ± 1.8	5
heparin	4.7 ± 2.5	2	4.0 ± 2.0	2
hu Fb conditioned medium	13.6 ± 5.4	7	18.7 ± 5.3 (p=0.003)	7
co-cultured 3T3 cells	14.1 ± 4.8	7	15.6 ± 5.7 (p=0.04)	7

Human ADM was pretreated for 24 hr with the compounds shown above, washed in buffer, human keratinocytes were plated onto them in 24-well plates (100,000/ well), and the cultures continued for 3 or 10 days in KGM.  Alternatively, keratinocytes were maintained in the presence of 10% human fibroblast (hu Fb) conditioned medium, 2% FCS, or co-cultured 3T3 cells for the entire 3 or 10 day period.  ADMs were cryosectioned and adherent cells counted in twenty 40X fields.  Data are expressed as means of cell counts from the indicated number (n) of experiments.  Means ± SEM.

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