VU Research Portal

Cutaneous and oral wound closure in vitro; Role of salivary peptides and cytokines Boink, M.A.

2017

document version Publisher's PDF, also known as Version of record

Link to publication in VU Research Portal

citation for published version (APA) Boink, M. A. (2017). Cutaneous and oral wound closure in vitro; Role of salivary peptides and cytokines.

General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.

• Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ?

Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

E-mail address: [email protected]

Download date: 23. Sep. 2021 4. Different properties of dermis, adipose and gingiva mesenchymal stromal cells

Mireille Boink Lenie van den Broek Sanne Roffel Kamran Nazmi Jan Bolscher Amit Gefen Enno Veerman Susan Gibbs

Wound Repair and Regeneration, 2016;24:100-109 CHAPTER 4 WOUND HEALING POTENTIAL OF DERMIS, ADIPOSE AND GINGIVA MSC

Introduction Abstract Immediately after to the or oral mucosa, the body responds with a series of Oral wounds heal faster and with better quality than skin wounds. Deep skin overlapping phases to repair and regenerate the damaged tissue, i.e. the inflammatory wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic phase, proliferative phase and remodeling phase. Wound healing is initiated by immediate . Differences in wound healing and final scar quality might be related to differences contraction of the wound margins. In the inflammatory phase bacteria and debris are removed in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and factors are secreted to attract and activate cells that are involved in the proliferative and extrinsic signals, such as human salivary histatin, epidermal and phase. During the proliferative phase multiple processes take place, such as , transforming growth factor beta1. Dermis-, adipose- and gingiva derived MSC were cell migration, formation and re-epithelialization. Transforming growth compared for their regenerative potential with regards to proliferation, migration and factor β1 (TGFβ1) is involved in regulating angiogenesis and deposition of extracellular matrix contraction. Proliferation was assessed by cell counting and migration using a matrix (ECM), such as fibronectin and collagen and is also important in wound contraction scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed (1,2). Mesenchymal stromal cells (MSC) (also known as fibroblasts in dermis and gingiva) play in MSC populated collagen gels, and also in skin and gingival full thickness tissue an important role by depositing ECM. In the remodeling phase, ECM is further synthesized, engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared remodeled and realigned. The quality of the final scar is dependent on the interplay between to skin derived MSC, gingiva MSC showed greater proliferation and migration capacity, all above mentioned phases. Excessive or abnormal ECM deposited by MSC can lead to and less matrix contraction in full thickness tissue equivalents, which may partly abnormal scar formation (e.g. hypertrophic scar). explain the superior oral wound healing. Epidermal keratinocytes were required for Even though the general processes leading to wound healing described above are enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, comparable in skin and oral mucosa, clear clinical differences have been reported. Wounds and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin in the oral mucosa heal faster and with a far better scar quality than wounds in the skin (3,4). enhanced migration without influencing proliferation or matrix contraction in all three It is currently unknown why these differences exist. Most likely both intrinsic properties of the

MSC, indicating that salivary peptides may have a beneficial effect on wound closure cells within the oral mucosa as well as interactions with the surrounding tissue environment 4 Chapter in general. Transforming growth factor beta1 enhanced contraction and alpha smooth are involved, leading to the superior oral wound healing. For example, we have previously muscle actin expression in all three MSC types when incorporated into collagen gels. shown that using identical methods to culture organotypic tissue engineered skin and oral Understanding the mechanisms responsible for the superior oral wound healing will mucosa, the difference in epithelial keratin expression observed in these native tissues is due aid us to develop advanced strategies for optimal skin regeneration, wound healing and only to properties of the keratinocytes and/or fibroblasts. This is in contrast to the increased scar formation. proliferation observed in oral epithelium compared to skin, which is not due to keratinocyte- fibroblast interactions, and therefore must be regulated by other external factors such as cross-talk with additional neighboring cells or (5,6). The increased proliferation in native oral epithelium leads to a thicker (more cell layers) epithelium, as well as a higher baseline turnover than skin, both of which are not observed in in vitro tissue equivalent culture (5-7). The major extrinsic difference between skin and oral mucosa is the presence of saliva. In addition to being rich in , saliva creates a humid environment which has been shown to be beneficial for wound healing (7,8). Wound licking promotes cutaneous wound healing in mice (9) and in rats it was shown that desalivation delays oral wound healing (10). The proteins within human saliva include classical growth factors, as well as , cathelicidin and trefoil factors known for their positive effect on wound healing (11-13). (EGF) plays an important role in re-epithelialization, by increasing proliferation and migration (1); it also promotes granulation tissue formation (14). EGF is found in rodent saliva as well as human saliva, but one of the great differences between rodent and human saliva is the amount of EGF, which is found abundantly in rodent saliva,

60 61 CHAPTER 4 WOUND HEALING POTENTIAL OF DERMIS, ADIPOSE AND GINGIVA MSC

but only in trace amounts in human saliva (15). This suggests that other factors are present in Dermal MSC: After removal of all adipose tissue and large vessels, dermis (same size as human saliva which may stimulate wound closure. Importantly, we have shown that human gingiva tissue, see below) was washed in phosphate buffered saline (PBS) (B. Braun, saliva contains histatins, which are the predominant peptides responsible for enhanced Melsungen, Germany) and incubated overnight on dispase II solution (Roche, Mannheim, oral keratinocyte and fibroblast migration (16). This strongly implicates their role as an Germany) at 4˚C. The next day epithelial sheets were separated from the dermis and the extrinsic factor in oral wound healing. Histatins are a family of peptides which are specifically dermis was incubated in collagenase type II (Gibco, Life Technologies, Grand Island, USA) produced in salivary glands of higher primates and are encoded by two genes, HTN1 and / dispase II in Hanks balanced salt solution (HBSS)(Gibco) at 37˚C for 2-3 h. Enzymatic HTN3. The latter encodes for histatin 3 and derivatives which possess antimicrobial activities activity was neutralized by adding PBS containing 0.1% bovine serum albumin (BSA)(Sigma- (17,18). HTN1 encodes for histatin 1 and 2 that have been found to enhance keratinocyte and Aldrich, St. Louis, USA). Tissue debris was removed by filtration (filter chamber: NPI, Emmer- fibroblast migration in vitro (15,16,19,20) Compascuum, the Netherlands). MSC medium, consisting of Dulbecco’s modified Eagle In superficial wounds mostly dermal MSC are involved in wound healing. However, medium (DMEM) (Lonza, Verviers, Belgium) containing 1% ultroserG (UG) (Biosepra, Cergy- in deep cutaneous wounds the MSC within the exposed underlying adipose tissue may Saint-Christophe, France) and 1% penicillin-streptomycin (P/S) (Gibco) was added. Dermal also play a role in the healing process. Previously we have shown that both dermal and MSC were harvested by centrifugation (300 x g, 5 min) and suspended in MSC medium. The adipose derived MSC express typical surface markers, as well as show multi-lineage suspension was passed through a 100 μm and 40 μm cell strainer (Becton Dickinson Falcon, (adipogenic, chondrogenic and osteogenic) differentiation capacity (21). Others have shown Erembodegem, Belgium). that gingival MSC also have typical stem cell characteristics (22,23). In wound healing, dermal MSC are associated with normotrophic scar formation and adipose MSC with hypertrophic Adipose MSC: Adipose tissue was carefully dissected from skin and large vessels were scar formation, whereas gingival MSC are associated with superior scar quality (24-26). removed. Adipose tissue was washed several times with PBS, cut into small pieces and However, the underlying mechanism for the differences in quality of the final scar is still largely incubated overnight at 4˚C in HBSS. The next day it was incubated in collagenase type II/ unknown. Therefore, in this study we compared the intrinsic capacity of the three different dispase II in HBSS at 37˚C for 2 h and then treated the same as dermis (see above).

MSC populations to proliferate, migrate and contract a matrix, since these are major events 4 Chapter resulting in wound healing and tissue regeneration. Additionally, matrix contraction and the Gingival MSC: Gingiva biopsies (8-15 mm2; material of multiple donors was pooled if pieces expression of α smooth muscle actin (αSMA), which represent scar forming myofibroblasts, were too small) were washed with PBS + 1% P/S and incubated overnight on dispase II. was investigated in full thickness skin and gingiva equivalents which consisted of a fully The next day, epithelial sheets were separated from the lamina propria and incubated in differentiated epithelium on a MSC populated connective tissue matrix. Finally, the extrinsic collagenase type II / dispase II in HBSS at 37˚C for 2-3 h. After this, MSC medium was added effect of human salivary histatin on these MSC was investigated in comparison to EGF and and the solution was passed through a 100 μm and 40 μm cell strainer. TGFβ1. EGF is reported to influence migration and proliferation and TGFβ1 is reported as pro- fibrotic and increases matrix contraction (1). Furthermore, TGFβ1 expression is lower in fetal Single-cell suspensions derived from the three different MSC types were seeded at skin (which is associated with scarless healing) compared to postnatal skin. Understanding approximately 3.5 x 104 cells/cm2. Cells were cultured in MSC medium for the proliferation the mechanisms responsible for the superior oral wound healing will aid us to develop assay, the migration assay and prior to constructing the tissue equivalents or in DMEM advanced strategies for optimal skin wound healing and scar formation. containing 1% P/S and 5% fetal bovine serum (FBS) (Thermo Fischer Scientific, Waltham, USA) for the collagen contraction gels. Medium was changed twice a week and cultures were Methods passaged when 90% confluent using 0.5 mM EDTA/ 0.05% trypsin (Gibco). The cells were

Isolation and culture of mesenchymal stromal cells from human skin and gingiva maintained at 37˚C in a humidified atmosphere containing 5% CO2. First passages MSC were Human abdominal skin was obtained after informed consent from healthy adult donors stored in liquid nitrogen until further use, except for the proliferation assay. MSC were used in undergoing corrective abdominal plastic surgery; gingiva was obtained after informed all experiments at passage 3, again except for the proliferation assay. Dermis- and adipose- consent from healthy adult donors after molar tooth extraction or implant surgery. Tissue derived MSC were donor matched, gingiva was not donor matched with skin biopsies. Table was used in an anonymous fashion in accordance with the “Code for Proper Use of Human 1 shows the experimental set-up and the different donors used in the experiments. Tissues” as formulated by the Dutch Federation of Medical Scientific Organizations (www. fmwv.nl) and following procedures approved by the institutional review board of the VU University medical centre.

62 63 CHAPTER 4 WOUND HEALING POTENTIAL OF DERMIS, ADIPOSE AND GINGIVA MSC

Table 1: Experimental set-up and donors used in experiments passage keratinocytes were stored in liquid nitrogen until further use in constructing skin Experiment type Experiment Dermal MSC Adipose MSC Gingival MSC and gingiva equivalents. Proliferation 1 Donor 1 Donor 1 Donor 2 2 Donor 3 Donor 3 Donor 4 Peptide synthesis Histatin 2 (Hst2) (RKFHEKHHSHREFPFYGDYGSNYLYDN) was synthesized by solid-phase 3 Donor 5 Donor 5 Donor 6 peptides synthesis using Fmoc chemistry with a Syro II synthesizer (Biotage, Uppsala, Sweden). Purification was conducted by ultimate 3000 RP-HPLC (Thermo Scientific) and Migration 1 Donor 7 Donor 7 Donor 8 + 9 authenticity was confirmed by mass spectrometry (MALDI-TOF) (Bruker Daltonik GmbH 2 Donor 10 Donor 10 Donor 11 Germany) as previously described (27).

3 Donor 12 Donor 12 Donor 13 + 14 + 15 Proliferation assay Freshly isolated MSC were grown to max 90% confluency in DMEM containing 1% P/S and 1% 4 Donor 16 Donor 16 Donor 17 + 18 + 19 UG, counted in duplicate using an Accu Chip, (Digital Bio, Sedul, Korea) and seeded in 6-well plates (passage 1) with a cell density of 4 x 103 cells/cm2. The cells were left to attach for 4 h Contraction of 1 Donor 20 Donor 20 Donor 21 and were then exposed to different concentrations of Hst2 (0.05, 0.5, 5 and 50 μM), 10 ng/ml

collagen gels 2 Donor 22 Donor 22 Donor 23 rhEGF and 10 ng/ml rhTGFβ1 (R&D Systems, Minneapolis, USA). Vehicle (H2O) supplemented 3 Donor 24 Donor 24 Donor 25 medium was used as negative control. After 4 days of culture, cells were trypsinized, and reseeded at a dilution of 1:4 (passage 2). Culture medium was supplemented with Hst2, 4 Donor 26 Donor 26 Donor 27 + 28 rhEGF and rhTGFβ1 as described above. After an additional 4 days (8 days total), cells were

harvested and counted in duplicate dishes (max 90% confluency). This number was corrected 4 Chapter Contraction of 1 Donor 29 Donor 29 Donor 30 + 31 + 32 for the dilution factor 1:4 and divided by the starting number of cells, to give a fold increase connective- and 2 Donor 33 Donor 33 Donor 34 + 35 in proliferation after 8 days of culture. full thickness tissue equivalents 3 Donor 36 Donor 36 Donor 37 - 44 4 Donor 45 Donor 45 Migration assay The migration assay was performed exactly as described earlier (21). In brief, confluent MSC All donors were consenting adults (age 18 – 65) and biopsies were obtained from healthy abdominal skin or monolayers in a 48-well plate were incubated with serum free medium (DMEM containing 1% gingiva with no signs on inflammation. The donors used within the experiments are indicated. In some cases P/S and 0.1% BSA) for four days to ensure growth arrest. After four days a scratch was drawn with when gingiva biopsies were small, donors were pooled as indicated. a plastic disposable pipette tip. The cultures were washed twice with PBS to remove detached cells and then exposed to different concentrations of Hst2 (0.05, 0.5, 5 and 50 μM) in serum free Isolation and culture of keratinocytes from human skin and gingiva medium. Medium supplemented with 10 ng/ml rhEGF was used as a positive control, vehicle After overnight incubation on dispase II (see dermal and gingival MSC), epithelial sheets supplemented medium was used as negative control. Phase contrast micrographs were taken were separated from the dermis and lamina propria. The epithelial sheets were incubated in after drawing the scratch and after four days of exposure. Data were analyzed using an image 0.05% trypsin for 10 min, keratinocyte medium was added and cell suspension was passed processing algorithm which has been described in detail in our previous work (28-30). This through a 100 μm and 40 μm cell strainer. Keratinocytes were then seeded in tissue culture algorithm is designed to non-destructively and quantitatively determine the scratch area. dishes coated with 0.5 μg/cm2 human placental collagen IV (Sigma-Aldrich) in a density Specifically, micrographs were segmented into two regions: denuded areas and cell-populated of approximately 4 x 104 cells/cm2, cultured in keratinocyte medium and maintained at areas. The segmentation process automatically distinguishes between the two regions based

37˚C in a humidified atmosphere containing 7.5% CO2. Keratinocyte medium consisted of on local texture homogeneity measures, where any denuded areas are characterized by higher DMEM/ Ham’s F-12 (Gibco) (3:1, v/v), 1% UG, 1% P/S, 1 μM hydrocortisone (Sigma-Aldrich), local texture homogeneity with respect to cell-populated areas. Standard deviation (SD) of 1μM isoproterenol (Sigma-Aldrich), 0.1 μM insulin (Sigma-Aldrich) and 2 ng KGF for skin pixel intensities is used as the measure of texture homogeneity; lower SD values correspond to keratinocytes and 1 ng/ml rhEGF for gingival keratinocytes (both Sigma-Aldrich). First higher texture homogeneities, and vice versa.

64 65 CHAPTER 4 WOUND HEALING POTENTIAL OF DERMIS, ADIPOSE AND GINGIVA MSC

Contraction assay Histological and immunohistochemical analysis Contraction was measured using two different methods, the first method used only MSC in Paraffin embedded sections (5 μm) of the connective tissue equivalents, as well as the full collagen gels; the second method used human skin and gingiva equivalents. thickness equivalents were either stained with haematoxylin and eosin for morphological Collagen matrix: Collagen gels were prepared using 4 mg/ml collagen in 0.1% acetic acid analysis, or with an αSMA (clone 1A4; 1:200, Dako, Glostrup, Denmark), followed by isolated from rat tails. The collagen solution was mixed on ice with MSC (2 x 105 cells/gel). Envision (Dako) for immunohistochemical analysis, essentially as described by the supplier. Subsequently, 1 ml of collagen suspension was pipetted into each well of a 12-wells plate (Costar, Corning Incorporated, New York, USA). After one hour of incubation at 37˚C in a Statistical analysis humidified atmosphere containing 5% CO2 to allow gel polymerization, the gels were gently All data are presented as mean ± standard error mean. Differences were considered significant detached with the aid of a spatula. They were then exposed to different concentrations of when *P < 0.05, **P < 0.01, ***P <0.001. Statistics were calculated in GraphPad Prism (San Hst2 (0.05, 0.5, 5 and 50 μM) in DMEM containing 1% P/S and 5% FCS. Medium supplemented Diego, CA, USA) and method used is indicated in figure legends and text. with 10 ng/ml TGFβ1 was used as a positive control, vehicle supplemented medium was used as negative control. Medium was changed three times a week. Photographs were Results taken with the Canon Powershot G12 digital camera at the beginning, before each medium Adipose derived MSC show less proliferation than dermal and gingival MSC refreshment and at the end of the experiment. The diameter of the cultures was measured In order to close a wound, MSC need to proliferate to generate cells which can in turn migrate using NIS-Elements AR 3.2 software. All cultures were formalin-fixed and paraffin embedded into the wound bed. Therefore, we first determined whether the three types of MSC showed using standard methods. differences with regards to proliferation (Figure 1). Whereas no statistically significant difference was found between the fold increase in number of cells after an 8 day study period Skin and gingiva equivalents: Skin equivalents (containing dermal or adipose MSC) and for dermal and gingival MSC, adipose MSC clearly showed a lower proliferation compared to gingiva equivalents were created using the same method as previously described (31). In either dermal (significant) or gingival (trend) MSC. 5 short, mesenchymal cells (4 x 10 ) were seeded into 2.2 x 2.2 cm collagen-elastin-matrix Next, it was determined whether the wound closing factor histatin was able to 4 Chapter (Matriderm®; Dr. Suwelack Skin & Health Care, Billerbeck, Germany) and cultured submerged influence proliferation. Supplementation of culture medium with histatin did not influence in culture medium containing DMEM /Ham’s F-12 (3:1), 2% UG, 1% P/S, 5 μg/ml insulin, 50 the proliferation of any of the MSC subtypes (data not shown). μg/ml L-ascorbic acid (Sigma-Aldrich), and 5 ng/ml rhEGF in a 0.4 μm pore size transwell (Cat nr 3450, Costar) in a 37°C, 5% CO2 atmosphere. After three weeks of culturing the connective tissue equivalents were harvested. For the full thickness tissue equivalents, keratinocytes (5 x 105 cells/culture) were seeded onto the MSC-populated matrix. After three days of submerged culture, equivalents were further cultured at the air-liquid interface in DMEM/ Ham’s F-12 (3:1), 0.2% UG, 1% P/S, 1 μM hydrocortisone, 1 μM isoproterenol, 0.1 μM insulin, 10 μM L-carnitine (Sigma-Aldrich), 10 mM L-serine (Sigma-Aldrich), 1 μM dl-α-tocopherol acetate (Sigma-Aldrich), enriched with a lipid supplement, for another ten days until a total of five weeks culture had been reached. The lipid supplement contained 25 μM palmitic acid, 15 μM linoleic acid, 7 μM arachidonic acid and 24 μM BSA, all obtained from Sigma-Aldrich. The cultures received new culture medium twice a week. Photographs were taken with a Canon Figure 1: Intrinsic proliferation of dermis-, adipose and gingiva-derived MSC Powershot G12 digital camera at the first medium refreshment (four days after seeding MSC Fold increase in total cell count of MSC after 8 days of culture relative to the cell number at the start of the in the matrix), after three weeks of culture resulting in connective tissue MSC equivalents or experiment is shown. Cells were passaged (1:4 dilution) once after four days (see Materials and Methods). after five weeks of culture resulting in full thickness tissue equivalents. The surface area of the Symbols represent three different experiments. Filled symbols represent donor matched dermal and adipose cultures was measured using NIS-Elements AR 3.2 software. All cultures were formalin-fixed MSC, open symbols represent non donor matched gingiva. See table 1 for details. *P < 0.05 (ordinary one-way ANOVA with Tukey’s multiple comparisons test). and paraffin embedded using standard methods.

66 67 CHAPTER 4 WOUND HEALING POTENTIAL OF DERMIS, ADIPOSE AND GINGIVA MSC

Gingival MSC migrate faster than dermal and adipose MSC significantly more migration response to 5 μM Hst 2, and dermal MSC (but not adipose MSC) During wound healing, MSC migrate from the healthy tissue at the wound boundaries into showed significantly more migration to rhEGF compared to gingiva MSC (P < 0.05). However, the wound bed to form granulation tissue. To determine whether MSC derived from dermis, when taking into account that intrinsic migration of gingival MSC was twice that of dermal adipose tissue and gingiva showed different intrinsic migration rates, a scratch assay was and adipose MSC, Hst2 and rhEGF supplementation of gingival MSC cultures showed a performed (Figure 2A). All MSC types migrated as individual cells, rather than as a front in similar net coverage of the scratch area compared to that observed for dermis- and adipose- the 4 day study period. Under culture conditions where serum was absent to ensure that no derived MSC cultures. proliferation occurred, the intrinsic migration rate of both dermis- and adipose-derived MSC was similar during the four day study period, whereas gingival MSC migrated twice as fast as Dermal, adipose and gingival MSC contract collagen gels to a similar extent either dermal or adipose MSC (Figure 2B). Contraction of a wound is part of the healing process and results in a decreased wound area. To explore possible differences in the ability of dermis-, adipose- and gingiva-derived MSC to contract a connective tissue matrix, the MSC were seeded into a collagen gel (Figure 3A). Collagen gel contraction was measured as the reduction in diameter of the gels over a period of 13 days, expressed as percentage of the original gel diameter. All three MSC types induced comparable levels of contraction of the collagen gels: gingival MSC: 61% reduction; adipose MSC: 56% reduction; and dermal MSC: 52% reduction in collagen gel diameter (Figure 3B). Chapter 4 Chapter

Figure 2: Migration properties of dermis-, adipose- and gingiva-derived MSC A: Representative photographs showing intrinsic dermal MSC migration in a scratch assay directly after introducing the scratch at day 0 (left) and after four days of migration (right). B: Intrinsic MCS migration measured as extent of closure of the area of the scratch introduced into a confluent layer of MSC derived from dermal, adipose and gingiva tissue, after four days. Symbols represent four different experiments. Filled symbols represent donor matched dermal and adipose MSC, open symbols represent non donor matched gingiva. See table 1 for details. *P < 0.05 (ordinary one-way ANOVA with Tukey’s multiple comparisons test. C: Extrinsic MSC migration. At time of introduction of the scratch cultures were supplemented with Hst2, rhEGF (10ng/ ml: positive control), or vehicle (H2O, negative control). Four days later the extent of migration (area covered) was determined with an image processing algorithm (see Materials and Methods). *P < 0.05, **P < 0.01 (repeated measures one-way ANOVA with Dunnett’s multiple comparison test). Bars represent means ± SEM of three independent experiments in quadruplicate relative to vehicle exposed control cultures.

Next the influence of histatin on migration of the three MSC types was determined. Stimulation Figure 3: Contraction properties of dermis-, adipose- and gingiva-derived MSC in collagen gels of MSC derived from dermis, adipose tissue and gingiva with different concentrations of Hst2 A: Representative photographs showing dermal MSC-mediated contraction of collagen gels exposed to control or TGFβ1 treatment for 0, 8 and 13 days. (0.05, 0.5, 5 and 50μM) enhanced the migration of all three MSC types (Figure 2C). Using a B: Contraction of collagen gels populated with dermal, adipose and gingival derived MSC, exposed to control two-way ANOVA with Tukey’s multiple comparison test, adipose (but not dermal) MSC show medium and TGFβ1 for up to 13 days. *P < 0.05 (two-way ANOVA with Tukey’s multiple comparisons test). Bars represent means ± SEM of four independent experiments in duplicate. 68 69 CHAPTER 4 WOUND HEALING POTENTIAL OF DERMIS, ADIPOSE AND GINGIVA MSC

Addition of TGFβ1, known for its capacity to enhance dermal matrix contraction, resulted in Contraction of full thickness skin and gingiva equivalents a further 20% contraction of collagen gels, independent of the type of MSC they contained Because the margins of wounds are epithelialized during wound contraction, we next (Figure 3). In contrast histatin did not influence contraction of the MSC populated collagen determined whether differences in contraction occurred in full thickness skin and gingiva gels (Figure 4A). Furthermore histatin did not influence the TGFβ1 mediated enhanced equivalents (Figure 5). The full thickness tissue equivalents consisted of a fully differentiated contraction of MSC populated collagen gels in any way (Figure 4B). epithelium on a MSC populated matrix (Figure 5B) while the connective tissue equivalents consisted only of a MSC populated matrix (Figure 5A). Full thickness skin equivalents constructed with adipose derived MSC showed more contraction than skin equivalents constructed with dermal MSC (both constructed with epidermal keratinocytes). Full thickness gingiva equivalents (constructed with gingiva keratinocytes) showed a similar degree of contraction as full thickness skin equivalents containing dermis MSC (Figure 5C). In order to confirm that the differences observed in contraction between the full thickness tissue equivalent model and the collagen gel model described above were not due to differences in methodology, contraction of connective tissue equivalent matrices (in the absence of keratinocytes) was also determined. In line with collagen gel contraction experiments, no difference in contraction was observed between the three MSC types in the absence of keratinocytes, indicating that keratinocytes are required to enhance adipose MSC mediated contraction (Figure 5C).

Absence of αSMA expression in full thickness gingival equivalents As α SMA expressing myofibroblasts are involved in wound contraction, we determined whether

there was a correlation between the degree of matrix contraction and αSMA expression. 4 Chapter Immunohistochemical staining showed absence of αSMA expression in the connective tissue equivalents, independent of the type of MSC they contained (Figure 5A). This is in line with our observation that the three types of MSC caused a similar degree of matrix contraction (collagen gels and connective tissue equivalents) in the absence of keratinocytes. However, the full thickness skin equivalent, containing adipose MSC and a differentiated epidermal layer, showed strong αSMA expression (Figure 5B). The αSMA expressing myofibroblasts were located mainly underneath the epidermis. Less αSMA expression was observed in skin equivalents containing dermal derived MSC and very little αSMA expression was observed Figure 4: Effect of TGFβ1 and histatin on contraction of collagen gels populated with dermis-, adipose- and in gingival equivalents. Gingiva MSC were capable of expressing αSMA, since when collagen gingiva-derived MSC gels were exposed to TGFβ1, αSMA was strongly expressed in the tissue sections (data not A: Contraction of collagen gels populated with dermal, adipose and gingival MSC, supplemented with vehicle shown). Histatin had no influence on αSMA expression in this experiment in any MSC type.

(H2O, negative control) TGFβ1 (10 ng/ml; positive control) or Hst2 for up to 13 days. ***P < 0.005 (repeated measures one-way ANOVA with Dunnett’s multiple comparisons test). Bars represent means ± SEM of four independent experiments in duplicate.

B: Collagen gel contraction. Cultures were supplemented with vehicle (H2O, negative control), TGFβ1 (10 ng/ ml; positive control), Hst2 (50 μM), or a combination of Hst2 and TGFβ1, for up to 13 days. **P < 0.01 (repeated measures one-way ANOVA with Bonferroni’s multiple comparisons test). Bars represent means ± SEM of three independent experiments in duplicate.

70 71 CHAPTER 4 WOUND HEALING POTENTIAL OF DERMIS, ADIPOSE AND GINGIVA MSC

Discussion In this study we show that migration, proliferation and matrix contraction characteristics of dermal, adipose and gingival MSC differs, and may in part explain the differences observed between skin and oral wound healing. Gingiva MSC showed a greater proliferative and migration capacity than adipose derived MSC, and less matrix contraction in full thickness tissue equivalents. These properties could be related to the enhanced scar free healing observed in the oral cavity. Furthermore, epidermal keratinocytes are required for enhanced adipose MSC matrix contraction and αSMA expression, and may therefore contribute to adverse scar formation in deep cutaneous wounds. Histatin enhanced migration without influencing matrix contraction or proliferation in all three MSC, indicating that saliva peptides may have a beneficial effect on wound closure in general. Cell proliferation is an essential part of wound closure. It is well recognized that gingival epithelium is highly proliferative compared to skin epidermis (5), but studies directly comparing skin and gingiva MSC proliferation are scarce. In our study, the proliferation rate of dermal and gingival MSC was similar. These results are in line with Lee et al., who described that oral mucosal MSC proliferate slightly (not significantly) faster than dermal MSC (32). However, we observed a lower proliferation rate for adipose MSC compared to either gingival or dermal MSC, the latter of which has already been described in literature (33). This finding is in line with the clinical observation that a decreased rate of wound closure in deep cutaneous wounds (where

adipose tissue is exposed) correlates to a greater chance of developing a hypertrophic scar (34). 4 Chapter During wound healing, cell proliferation goes hand in hand with cell migration in order to close the wound. We found that gingival MSC had a two-fold higher intrinsic migration capability compared to either dermal or adipose MSC. This is in line with the clinical observation that oral wounds heal faster than skin wounds (3,25) and indicates not only enhanced re-epithelialization, but also enhanced MSC migration plays a role in the better oral wound closure. Surprisingly, the saliva derived histatin peptide had a greater effect on adipose MSC migration and rhEGF on dermal MSC migration than on gingival MSC migration. However Figure 5: Contraction properties and αSMA expression of connective- and full thickness tissue equivalents gingival MSC had a notably higher intrinsic migration, which is probably the reason why little with dermis-, adipose- and gingiva-derived MSC additional migration upon stimulation was observed with gingiva MSC. Our findings indicate A and B: Representative photographs of HE and αSMA staining on 5 μm sections of connective tissue equivalents that a high rate of MSC migration in the oral cavity is intrinsically regulated by the MSC and (A) and full thickness tissue equivalents (B) with dermal, adipose or gingiva MSC in a collagen-elastin matrix; 20x magnification therefore there is a smaller effect of environmental factors. Furthermore, our results indicate C: Contraction of connective- and full thickness tissue equivalent, composed of skin keratinocytes (with dermal that in particular an enhanced MSC migration as well as MSC proliferation may contribute to and adipose MSC), or gingival keratinocytes (with gingival MSC). Connective tissue equivalents were cultured enhanced oral wound closure compared to skin. 21 days, full thickness tissue equivalents were cultured 35 days. Symbols represent three different experiments. In addition to cell proliferation and migration, faster wound closure is associated with Filled symbols represent donor matched dermal and adipose MSC, open symbols represent non donor matched contraction from the wound margins in order to decrease the size of the wound. The final gingiva. See table 1 for details. * P <0.05 (ordinary one-way ANOVA with Tukey’s multiple comparison test). scar quality of the closed wound is determined in part by the extent of tissue contraction. We found that in the absence of keratinocytes, the extent of matrix contraction and number of αSMA expressing myofibroblasts in the presence or absence of Hst2 was virtually similar for all three MSC types. However, when MSC were cultured together with keratinocytes, for example in the full tissue equivalent, skin equivalents constructed with adipose derived MSC showed

72 73 CHAPTER 4 WOUND HEALING POTENTIAL OF DERMIS, ADIPOSE AND GINGIVA MSC significantly more contraction than gingiva equivalents or skin equivalents constructed with Reference List dermal MSC and surprisingly high amounts of αSMA expressing myofibroblasts underneath 1. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and the epidermis. Our results indicate that a very specific crosstalk occurs in our model between cytokines in wound healing. Wound Repair Regen 2008;16(5):585-601. epidermal keratinocytes and adipose MSC, which occurs to a lesser extent between dermal 2. Klass BR, Branford OA, Grobbelaar AO, Rolfe KJ. The effect of epigallocatechin-3-gallate, a MSC and is absent between gingiva keratinocytes and MSC, which may play a pivotal role in constituent of green tea, on transforming growth factor-beta1-stimulated wound contraction. hypertrophic scar formation. Further research is required to identify the soluble mediators Wound Repair Regen 2010;18(1):80-8. involved. Clearly the MSC respond differently to keratinocyte soluble mediators with only adipose 3. Engeland CG, Bosch JA, Cacioppo JT, Marucha PT. Mucosal wound healing: the roles of age MSC undergoing a strong phenotypic change to a αSMA expressing myofibroblasts which can and sex. Arch Surg 2006;141(12):1193-7. enhance matrix contraction. Our observations, although unexplainable at present, are in line 4. Larjava H, Wiebe C, Gallant-Behm C, Hart DA, Heino J, Hakkinen L. Exploring scarless healing with the clinical observation that a higher risk of hypertrophic scar formation exists after deep of oral soft tissues. J Can Dent Assoc 2011;77:b18. cutaneous wounding and that there is rarely a scar visible after wounding of oral mucosa (4,34). 5. Vriens AP, Waaijman T, van den Hoogenband HM, de Boer EM, Scheper RJ, Gibbs S. Comparison We do realize though that our model is relatively simple, including only keratinocytes and MSC, of autologous full-thickness gingiva and skin substitutes for wound healing. Cell Transplant and that other skin residential cells most likely influence these processes as well. 2008;17(10-11):1199-209. Our study showed no significant differences in contraction between the three MCS types 6. Gibbs S, Ponec M. Intrinsic regulation of differentiation markers in human epidermis, hard (in the absence of keratinocytes), although there was a trend towards more contraction of palate and buccal mucosa. Arch Oral Biol 2000;45(2):149-58. gingival MSC than dermal MSC in our free floating collagen gels. This is in line with other studies 7. Field FK, Kerstein MD. Overview of wound healing in a moist environment. Am J Surg with human tissue that show that oral mucosal MSC contract collagen gels more than dermal 1994;167(1A):2S-6S. MSC (35-38), while Lee et al. stated that dermis contracts more than oral mucosa (32). 8. Jones V, Grey JE, Harding KG. Wound dressings. BMJ 2006;332(7544):777-80. Interestingly, the contraction observed in all three types of MSC in either collagen gels 9. Hutson JM, Niall M, Evans D, Fowler R. Effect of salivary glands on wound contraction in mice.

or connective tissue equivalents (without keratinocytes) was independent of αSMA expressing Nature 1979;279(5716):793-5. 4 Chapter myofibroblasts since negligible αSMA expression was observed. Literature is conflicting on 10. Bodner L, Dayan D, Pinto Y, Hammel I. Characteristics of palatal wound healing in desalivated this subject. While some authors found αSMA expression higher in skin than in oral mucosa, rats. Arch Oral Biol 1993;38(1):17-21. in monolayers and free floating as well as attached-relaxed collagen gels (37,38), Lygoe et al. 11. Niyonsaba F, Ushio H, Nakano N, Ng W, Sayama K, Hashimoto K, Nagaoka I, Okumura showed greater basal αSMA expression in monolayers of oral mucosa MSC than in dermal MSC K, Ogawa H. Antimicrobial peptides human beta-defensins stimulate epidermal keratinocyte (36). In an in vivo study with red Ducoc pigs, in both the unwounded situation as well as at 14 migration, proliferation and production of proinflammatory cytokines and chemokines. J Invest and 60 days after wounding there was more αSMA expression found in the oral mucosa than Dermatol 2007;127(3):594-604. in the dermis (39). It was also shown that desalivated mice show less wound contraction (9) 12. Woo JS, Jeong JY, Hwang YJ, Chae SW, Hwang SJ, Lee HM. Expression of cathelicidin in human and there were less myofibroblasts found in desalivated rats (10). Whether saliva also enhances salivary glands. Arch Otolaryngol Head Neck Surg 2003;129(2):211-4. αSMA expression in the oral mucosa is currently unknown. Stephens et al. suggested that 13. Storesund T, Hayashi K, Kolltveit KM, Bryne M, Schenck K. Salivary trefoil factor 3 enhances contraction may be caused by increased movement of the oral fibroblasts through the collagen migration of oral keratinocytes. Eur J Oral Sci 2008;116(2):135-40. gel, coupled with intimate association with collagen fibrils (35). However, we did find that 14. Buckley A, Davidson JM, Kamerath CD, Woodward SC. Epidermal growth factor increases gingival MSC, similar to dermis and adipose MSC, were able to produce αSMA when stimulated granulation tissue formation dose dependently. J Surg Res 1987;43(4):322-8. with TGFβ1 (data not shown). 15. Oudhoff MJ, Kroeze KL, Nazmi K, van den Keijbus PA, van ‘t HW, Fernandez-Borja M, Hordijk Taken together our results indicate that intrinsic properties of MSC together with crosstalk PL, Gibbs S, Bolscher JG, Veerman EC. Structure-activity analysis of histatin, a potent wound with epithelial cells result in differential proliferative, migratory and matrix contraction healing peptide from human saliva: cyclization of histatin potentiates molar activity 1,000-fold. properties of dermal, adipose and gingival MSC which may in turn influence wound closure FASEB J 2009;23(11):3928-35. and the quality of the final scar. Since the saliva peptide Hst2 stimulates skin MSC migration to 16. Oudhoff MJ, Bolscher JG, Nazmi K, Kalay H, van ‘t HW, Amerongen AV, Veerman EC. Histatins an even greater extent than gingiva MSC migration, without influencing matrix contraction or are the major wound-closure stimulating factors in human saliva as identified in a cell culture αSMA expression, Hst2 may possibly be a novel skin wound healing therapeutic. assay. FASEB J 2008;22(11):3805-12.

74 75 CHAPTER 4 WOUND HEALING POTENTIAL OF DERMIS, ADIPOSE AND GINGIVA MSC

17. Oppenheim FG, Xu T, McMillian FM, Levitz SM, Diamond RD, Offner GD, Troxler RF. Histatins, 31. van den Broek LJ, Niessen FB, Scheper RJ, Gibbs S. Development, validation and testing of a a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, human tissue engineered hypertrophic scar model. ALTEX 2012;29(4):389-402. primary structure, and fungistatic effects on Candida albicans. J Biol Chem 1988;263(16):7472-7. 32. Lee HG, Eun HC. Differences between fibroblasts cultured from oral mucosa and normal skin: 18. Helmerhorst EJ, van’t Hof W, Veerman EC, Simoons-Smit I, Nieuw Amerongen AV. Synthetic implication to wound healing. J Dermatol Sci 1999;21(3):176-82. histatin analogues with broad-spectrum antimicrobial activity. Biochem J 1997;326 ( Pt 1):39-45. 33. Al-Nbaheen M, Vishnubalaji R, Ali D, Bouslimi A, Al-Jassir F, Megges M, Prigione A, Adjaye J, 19. Oudhoff MJ, Blaauboer ME, Nazmi K, Scheres N, Bolscher JG, Veerman EC. The role of salivary Kassem M, Aldahmash A. Human stromal (mesenchymal) stem cells from bone marrow, adipose histatin and the human cathelicidin LL-37 in wound healing and innate immunity. Biol Chem tissue and skin exhibit differences in molecular phenotype and differentiation potential. Stem Cell 2010;391(5):541-8. Rev 2013;9(1):32-43. 20. Oudhoff MJ, van den Keijbus PA, Kroeze KL, Nazmi K, Gibbs S, Bolscher JG, Veerman EC. 34. Deitch EA, Wheelahan TM, Rose MP, Clothier J, Cotter J. Hypertrophic burn scars: analysis of Histatins enhance wound closure with oral and non-oral cells. J Dent Res 2009;88(9):846-50. variables. J Trauma 1983;23(10):895-8. 21. Kroeze KL, Jurgens WJ, Doulabi BZ, van Milligen FJ, Scheper RJ, Gibbs S. Chemokine-mediated 35. Stephens P, Davies KJ, al-Khateeb T, Shepherd JP, Thomas DW. A comparison of the ability of migration of skin-derived stem cells: predominant role for CCL5/RANTES. J Invest Dermatol intra-oral and extra-oral fibroblasts to stimulate extracellular matrix reorganization in a model of 2009;129(6):1569-81. wound contraction. J Dent Res 1996;75(6):1358-64. 22. Mitrano TI, Grob MS, Carrion F, Nova-Lamperti E, Luz PA, Fierro FS, Quintero A, Chaparro A, 36. Lygoe KA, Wall I, Stephens P, Lewis MP. Role of vitronectin and fibronectin receptors in oral Sanz A. Culture and characterization of mesenchymal stem cells from human gingival tissue. J mucosal and dermal myofibroblast differentiation. Biol Cell 2007;99(11):601-14. Periodontol 2010;81(6):917-25. 37. Shannon DB, McKeown ST, Lundy FT, Irwin CR. Phenotypic differences between oral and 23. Zhang Q, Shi S, Liu Y, Uyanne J, Shi Y, Shi S, Le AD. Mesenchymal stem cells derived from skin fibroblasts in wound contraction and growth factor expression. Wound Repair Regen human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related 2006;14(2):172-8. tissue destruction in experimental colitis. J Immunol 2009;183(12):7787-98. 38. McKeown ST, Barnes JJ, Hyland PL, Lundy FT, Fray MJ, Irwin CR. Matrix metalloproteinase-3

24. van den Broek LJ, Limandjaja GC, Niessen FB, Gibbs S. Human hypertrophic and keloid scar differences in oral and skin fibroblasts. J Dent Res 2007;86(5):457-62. 4 Chapter models: principles, limitations and future challenges from a tissue engineering perspective. Exp 39. Mak K, Manji A, Gallant-Behm C, Wiebe C, Hart DA, Larjava H, Hakkinen L. Scarless healing of Dermatol 2014;23(6):382-6. oral mucosa is characterized by faster resolution of inflammation and control of myofibroblast 25. Glim JE, van EM, Niessen FB, Everts V, Beelen RH. Detrimental dermal wound healing: what action compared to skin wounds in the red Duroc pig model. J Dermatol Sci 2009;56(3):168-80. can we learn from the oral mucosa? Wound Repair Regen 2013;21(5):648-60. 26. van den Bogaerdt AJ, van der Veen VC, van Zuijlen PP, Reijnen L, Verkerk M, Bank RA, Middelkoop E, Ulrich MM. Collagen cross-linking by adipose-derived mesenchymal stromal cells and scar-derived mesenchymal cells: Are mesenchymal stromal cells involved in scar formation? Wound Repair Regen 2009;17(4):548-58. 27. Bolscher JG, Oudhoff MJ, Nazmi K, Antos JM, Guimaraes CP, Spooner E, Haney EF, Garcia Vallejo JJ, Vogel HJ, van’t Hof W, Ploegh HL, Veerman EC. Sortase A as a tool for high-yield histatin cyclization. FASEB J 2011;25(8):2650-8. 28. Topman G, Sharabani-Yosef O, Gefen A. A standardized objective method for continuously measuring the kinematics of cultures covering a mechanically damaged site. Med Eng Phys 2012;34(2):225-32. 29. Topman G, Lin FH, Gefen A. The influence of ischemic factors on the migration rates of cell types involved in cutaneous and subcutaneous pressure ulcers. Ann Biomed Eng 2012;40(9):1929- 39. 30. Topman G, Lin FH, Gefen A. The natural medications for wound healing - Curcumin, Aloe-Vera and Ginger - do not induce a significant effect on the migration kinematics of cultured fibroblasts. J Biomech 2013;46(1):170-4.

76 77