Vet Dermatol 2014; 25: 475–e77
DOI: 10.1111/vde.12134
Development and characterization of an equine skin-equivalent model -Lluch*, Pilar Brazıs*, Rosa Maria Rabanal†, Dolors Fondevila† and Santiago Cerrato*, Laura Ramio Anna Puigdemont‡ s, Barcelona, Spain *UNIVET, Edifici Astrolabio, Avinguda Cerdanyola 92, 08172, Sant Cugat del Valle Departments of †Animal Medicine and Surgery and ‡ Pharmacology, Therapeutics and Toxicology, Facultat de Veterin aria, Edifici V, Universitat noma de Barcelona, 08913, Bellaterra, Barcelona, Spain Auto Correspondence: Anna Puigdemont, Department of Pharmacology, Therapeutics and Toxicology, Facultat de Veterin aria, Edifici V, Universitat noma de Barcelona, 08913 Bellaterra, Barcelona, Spain. E-mail: [email protected] Auto
Background – There is increasing interest in the biological and pathological study of equine skin owing to the high prevalence of cutaneous diseases in horses. However, knowledge of equine skin cell biology and cultures is limited by the low number of in vitro studies in the literature. Hypothesis/Objectives – The objective of the study was to develop and characterize an in vitro equine skin equivalent. Methods – Cultures of pure equine keratinocytes and dermal fibroblasts were obtained by enzymatic digestion of skin biopsies. Fibroblasts were embedded into type I collagen matrices to obtain dermal scaffolds, the surface of which was seeded with keratinocytes. The three-dimensional cultures were exposed to the air–liquid interface to enable epidermal stratification. Results – After 14 days in air-exposed conditions, histological analysis showed that keratinocytes underwent differentiation into a multilayered epidermis. Immunohistochemical studies revealed the expression of epidermal cytokeratin in keratinocytes, whereas vimentin was expressed in dermal fibroblasts, as expected in equine skin. Immunostaining of Ki67 showed proliferative keratinocytes in the stratum basale. A continuous basement membrane at the dermo-epidermal junction was also detected immunohistochemically through the expression of its major components (type IV collagen and laminin 5). Ultrastructural analysis by electron microscopy showed desmosomes located among keratinocytes in all layers and hemidesmosomes among the basal keratinocytes and lamina densa. Conclusions and clinical importance – This study reports, for the first time, the development of an in vitro equine skin-equivalent model that resembles equine skin morphologically, immunohistochemically and ultrastructurally.
Introduction The skin is the largest organ of the body in vertebrates and plays many important roles, such as barrier protection from chemical or physical insults, sensory function and regulation of homeostasis. The loss of skin integrity may result in an imbalance that can lead to several pathological complications, including infection, fluid loss or even death. Horses are often affected by self-inflected trauma when they respond to ‘fight or flight’ situations, predisposing them to massive skin wounds, especially on the limbs.1 Such traumatic skin lesions frequently heal slowly by second intention and may be associated with the formation of exuberant granulation tissue, infection or scarring.2 Some studies describe the use of skin autografts for the treatment of equine wounds.1,3–7 However, redundant donor skin is limited, and this treatment is useful only for small wounds.
Accepted 18 February 2014 Sources of Funding: This study was self-funded. Conflict of Interest: No conflicts of interest have been declared. © 2014 ESVD and ACVD, Veterinary Dermatology, 25, 475–e77.
An interesting alternative is tissue engineering of skin, a technique that has emerged in recent decades to regenerate injured or diseased tissue. The most frequent tissue-engineered skin techniques reported in human,8–10 canine11–13 and murine species14–16 are developed by expanding skin cells in the laboratory and incorporating them into a biological matrix to obtain a three-dimensional skin structure. Keratinocytes and dermal fibroblasts, major cell components of the epidermis and dermis, respectively, may be expanded in vitro at a much faster rate than in the patient. Thus, a large amount of autologous tissue-engineered skin can be obtained from a small skin biopsy, as reported in humans.17 To date, the development of equine skin equivalent (ESE) has been impeded by the difficulty of maintaining equine primary keratinocytes in culture long term. Two studies described media, techniques and procedures in the culturing and isolation of primary equine keratinocytes, but cells proliferated at low rates or were not passaged more than four times.18,19 Two recent studies, however, reported an equine keratinocyte culture grown for at least six passages20 and a three-dimensional culture system that enabled keratinocyte differentiation into an 475
Cerrato et al.
epidermis-like structure.21 Thus, efficient isolation and culture techniques have the potential to provide long-term cultures of equine keratinocytes that may be used to develop ESE, incorporating both dermal and epidermal compartments. The objective of this study was the development of an equine skin model that could provide a useful tool to investigate the behaviour of equine skin cells in physiological and pathological conditions and could be used to establish tissue-engineered skin therapy in horses.
Materials and methods Equine skin biopsy samples Equine skin biopsy samples (6 mm punch) from inguinal areas were obtained from horses that were euthanized for reasons not related to the present study. Samples were placed in sterile tubes containing Dulbecco’s modified Eagle’s medium (DMEM) comprised of 5% fetal bovine serum (FBS), penicillin (100 IU/mL), streptomycin (100 lg/mL) and amphotericin B (2.5 lg/mL; all reagents from Invitrogen, Carlsbad, CA, USA). Samples were preserved at 4°C.
Isolation and culture of equine skin cells Skin samples were cleaned with povidone-iodine and 70% ethanol. Fat tissue and blood vessels were removed from the skin. Samples were cut into small fragments and enzymatically dispersed based on previously described methods.17 Briefly, in this study samples were first digested with a type I collagenase solution (2 mg/mL; SigmaAldrich, St Louis, MO, USA) in DMEM and antibiotics for 4–6 h or until the dermis was completely disaggregated. Collagenase supernatant was washed, filtered with a cell strainer (100 lm) and centrifuged at 300g for 5 min to recover dermal fibroblasts. Dermal cells were seeded (2 9 105 cells/cm2) and grown in a humidified atmosphere at 37°C with 5% CO2 using DMEM supplemented with 10% FBS and antibiotics. The medium was changed twice a week, and cells were serially subcultured until used in the skin equivalent at the second and fifth passages. After complete removal of the collagenase solution, the remaining epidermal fragments were enzymatically digested with 0.05% trypsin–0.02% EDTA at 37°C to establish equine keratinocyte cultures based on previously described methods.17,22 The enzymatic solution was substituted by a fresh mixture every 30 min until no further cells were recovered. Trypsin supernatants were filtered using first a 100 lm cell strainer followed by a 40 lm cell strainer and centrifuged at 300g for 5 min to recover keratinocytes. Primary keratinocytes (1 9 105 cells/cm2) were then plated onto collagen-coated flasks and grown in a humidified atmosphere at 37°C with 5% CO2. The culture medium consisted of DMEM containing 5% FBS, 0.01 lg/mL epidermal growth factor (Austral Biologicals, San Ramon, CA, USA) and b-mercaptoethanol (0.1 mmol/L; Invitrogen), as previously described.21 The medium was changed every 2–3 days and subcultured when 90% confluence was reached.
Development of equine skin equivalent To obtain a three-dimensional ESE, 2.4 9 105 dermal fibroblasts were embedded in a 3 mL rat tail type I collagen solution (1.5 mg/ mL; Sigma-Aldrich). The collagen biomatrix was placed into a transwell chamber measuring 24 mm in diameter (Corning, Tewksbury, MA, USA) and cultured for 48 h prior to seeding 1 9 106 keratinocytes onto the biomatrix surface. After 24 h, ESEs were lifted to the air–liquid interface. The ESEs were cultured for 2 weeks, and the medium was changed three times per week.
Histological, immunohistochemical and ultrastructural studies of equine skin equivalent After 14 days at the air–liquid interface, samples were fixed in 10% formalin or 2% (w/v) paraformaldehyde (EM grade; Merck, Darms-
476
tadt, Germany) and 2.5% (v/v) glutaraldehyde solution (EM grade; TAAB Laboratories, Aldermaston, Reading, UK) in a 0.1 mol/L phosphate buffer (pH 7.4; Sigma-Aldrich). Formalin-fixed samples were dehydrated and embedded in paraffin wax. Sections (4 lm thick) were cut and stained with haematoxylin and eosin for routine histological evaluation and immunohistochemical study of pan-cytokeratin (K5, K6, K8 and K17), keratin 10, type IV collagen, laminin 5, vimentin and Ki67. Primary antibodies, used with the corresponding pretreatment for antigen retrieval and dilution conditions, consisted of mouse anti-human pan-cytokeratin (pretreatment of 0.01% trypsin for 20 min at room temperature, 1:100 dilution, Clone MNF116; Dako, Copenhagen, Denmark), citokeratin10 (pretreatment of citrate buffer for 20 min at 98°C plus 30 min at room temperature, 1:80 dilution, Clone DE-K10; Dako), goat anti-type IV collagen (pretreatment of type XIV protease for 8 min at 37°C, 1:100 dilution; Southern Biotechnology, Birmingham, AL, USA), rabbit antilaminin 5 (pretreatment of 0.1% proteinase K for 8 min at 37°C, 1:800 dilution; Dako), mouse anti-vimentin (pretreatment of citrate buffer for 20 min at 98°C plus 30 min at room temperature, 1:200 dilution, Clone V9; Dako) and mouse anti-Ki67 (pretreatment of citrate buffer for 20 min at 98°C plus 30 min at room temperature, 1:100 dilution, Clone MIB1; Dako). The detection system used for pan-cytokeratin, keratin 10, vimentin, Ki67 and laminin 5 was polymer–horseradish peroxidase anti-mouse or anti-rabbit (EnVision; Dako) for 30 min at room temperature. For type IV collagen, the secondary antibody used was a biotin-labelled rabbit anti-goat (Dako) at a 1:200 dilution in Trisbuffered saline for 1 h at room temperature. Diaminobenzidine was used as the detection system, and samples were counterstained with haematoxylin. For ultrastructural studies, samples fixed in paraformaldehyde– glutaraldehyde solution were rinsed four times with 100 mmol/L phosphate-buffered saline. Samples were then postfixed in 1% (w/v) osmium tetroxide (TAAB Laboratories) containing 0.8% (w/v) potassium hexacyanoferrate (III; Sigma-Aldrich) for 2 h and washed with deionized water four times, followed by dehydration in acetone. All procedures were performed at 4°C. Samples were dehydrated through a graded acetone series, embedded in Eponate 12 resin (Ted Pella Inc., Redding, CA, USA) and polymerized for 48 h at 60°C. Ultrathin sections (70 nm thick) were cut with a diamond knife (45°; Diatome, Biel, Switzerland), mounted on copper grids (200 mesh) and contrasted with conventional uranyl acetate (30 min) and Reynolds lead citrate solutions (5 min). Sections were observed with a Jeol 1400 transmission electron microscope (Jeol Ltd, Tokyo, Japan) equipped with a Gatan Ultrascan ES1000 CCD Camera (Gatan, Oxford, UK).
Results Isolation and culture of equine fibroblasts and keratinocytes Isolation of equine dermal fibroblasts was achieved by digesting the skin sample dermis in 2% collagenase solution. Primary cultures presented viability ratios of up to 95% and a high rate of proliferation, reaching confluence in
DOI: 10.1111/vde.12134
Development and characterization of an equine skin-equivalent model -Lluch*, Pilar Brazıs*, Rosa Maria Rabanal†, Dolors Fondevila† and Santiago Cerrato*, Laura Ramio Anna Puigdemont‡ s, Barcelona, Spain *UNIVET, Edifici Astrolabio, Avinguda Cerdanyola 92, 08172, Sant Cugat del Valle Departments of †Animal Medicine and Surgery and ‡ Pharmacology, Therapeutics and Toxicology, Facultat de Veterin aria, Edifici V, Universitat noma de Barcelona, 08913, Bellaterra, Barcelona, Spain Auto Correspondence: Anna Puigdemont, Department of Pharmacology, Therapeutics and Toxicology, Facultat de Veterin aria, Edifici V, Universitat noma de Barcelona, 08913 Bellaterra, Barcelona, Spain. E-mail: [email protected] Auto
Background – There is increasing interest in the biological and pathological study of equine skin owing to the high prevalence of cutaneous diseases in horses. However, knowledge of equine skin cell biology and cultures is limited by the low number of in vitro studies in the literature. Hypothesis/Objectives – The objective of the study was to develop and characterize an in vitro equine skin equivalent. Methods – Cultures of pure equine keratinocytes and dermal fibroblasts were obtained by enzymatic digestion of skin biopsies. Fibroblasts were embedded into type I collagen matrices to obtain dermal scaffolds, the surface of which was seeded with keratinocytes. The three-dimensional cultures were exposed to the air–liquid interface to enable epidermal stratification. Results – After 14 days in air-exposed conditions, histological analysis showed that keratinocytes underwent differentiation into a multilayered epidermis. Immunohistochemical studies revealed the expression of epidermal cytokeratin in keratinocytes, whereas vimentin was expressed in dermal fibroblasts, as expected in equine skin. Immunostaining of Ki67 showed proliferative keratinocytes in the stratum basale. A continuous basement membrane at the dermo-epidermal junction was also detected immunohistochemically through the expression of its major components (type IV collagen and laminin 5). Ultrastructural analysis by electron microscopy showed desmosomes located among keratinocytes in all layers and hemidesmosomes among the basal keratinocytes and lamina densa. Conclusions and clinical importance – This study reports, for the first time, the development of an in vitro equine skin-equivalent model that resembles equine skin morphologically, immunohistochemically and ultrastructurally.
Introduction The skin is the largest organ of the body in vertebrates and plays many important roles, such as barrier protection from chemical or physical insults, sensory function and regulation of homeostasis. The loss of skin integrity may result in an imbalance that can lead to several pathological complications, including infection, fluid loss or even death. Horses are often affected by self-inflected trauma when they respond to ‘fight or flight’ situations, predisposing them to massive skin wounds, especially on the limbs.1 Such traumatic skin lesions frequently heal slowly by second intention and may be associated with the formation of exuberant granulation tissue, infection or scarring.2 Some studies describe the use of skin autografts for the treatment of equine wounds.1,3–7 However, redundant donor skin is limited, and this treatment is useful only for small wounds.
Accepted 18 February 2014 Sources of Funding: This study was self-funded. Conflict of Interest: No conflicts of interest have been declared. © 2014 ESVD and ACVD, Veterinary Dermatology, 25, 475–e77.
An interesting alternative is tissue engineering of skin, a technique that has emerged in recent decades to regenerate injured or diseased tissue. The most frequent tissue-engineered skin techniques reported in human,8–10 canine11–13 and murine species14–16 are developed by expanding skin cells in the laboratory and incorporating them into a biological matrix to obtain a three-dimensional skin structure. Keratinocytes and dermal fibroblasts, major cell components of the epidermis and dermis, respectively, may be expanded in vitro at a much faster rate than in the patient. Thus, a large amount of autologous tissue-engineered skin can be obtained from a small skin biopsy, as reported in humans.17 To date, the development of equine skin equivalent (ESE) has been impeded by the difficulty of maintaining equine primary keratinocytes in culture long term. Two studies described media, techniques and procedures in the culturing and isolation of primary equine keratinocytes, but cells proliferated at low rates or were not passaged more than four times.18,19 Two recent studies, however, reported an equine keratinocyte culture grown for at least six passages20 and a three-dimensional culture system that enabled keratinocyte differentiation into an 475
Cerrato et al.
epidermis-like structure.21 Thus, efficient isolation and culture techniques have the potential to provide long-term cultures of equine keratinocytes that may be used to develop ESE, incorporating both dermal and epidermal compartments. The objective of this study was the development of an equine skin model that could provide a useful tool to investigate the behaviour of equine skin cells in physiological and pathological conditions and could be used to establish tissue-engineered skin therapy in horses.
Materials and methods Equine skin biopsy samples Equine skin biopsy samples (6 mm punch) from inguinal areas were obtained from horses that were euthanized for reasons not related to the present study. Samples were placed in sterile tubes containing Dulbecco’s modified Eagle’s medium (DMEM) comprised of 5% fetal bovine serum (FBS), penicillin (100 IU/mL), streptomycin (100 lg/mL) and amphotericin B (2.5 lg/mL; all reagents from Invitrogen, Carlsbad, CA, USA). Samples were preserved at 4°C.
Isolation and culture of equine skin cells Skin samples were cleaned with povidone-iodine and 70% ethanol. Fat tissue and blood vessels were removed from the skin. Samples were cut into small fragments and enzymatically dispersed based on previously described methods.17 Briefly, in this study samples were first digested with a type I collagenase solution (2 mg/mL; SigmaAldrich, St Louis, MO, USA) in DMEM and antibiotics for 4–6 h or until the dermis was completely disaggregated. Collagenase supernatant was washed, filtered with a cell strainer (100 lm) and centrifuged at 300g for 5 min to recover dermal fibroblasts. Dermal cells were seeded (2 9 105 cells/cm2) and grown in a humidified atmosphere at 37°C with 5% CO2 using DMEM supplemented with 10% FBS and antibiotics. The medium was changed twice a week, and cells were serially subcultured until used in the skin equivalent at the second and fifth passages. After complete removal of the collagenase solution, the remaining epidermal fragments were enzymatically digested with 0.05% trypsin–0.02% EDTA at 37°C to establish equine keratinocyte cultures based on previously described methods.17,22 The enzymatic solution was substituted by a fresh mixture every 30 min until no further cells were recovered. Trypsin supernatants were filtered using first a 100 lm cell strainer followed by a 40 lm cell strainer and centrifuged at 300g for 5 min to recover keratinocytes. Primary keratinocytes (1 9 105 cells/cm2) were then plated onto collagen-coated flasks and grown in a humidified atmosphere at 37°C with 5% CO2. The culture medium consisted of DMEM containing 5% FBS, 0.01 lg/mL epidermal growth factor (Austral Biologicals, San Ramon, CA, USA) and b-mercaptoethanol (0.1 mmol/L; Invitrogen), as previously described.21 The medium was changed every 2–3 days and subcultured when 90% confluence was reached.
Development of equine skin equivalent To obtain a three-dimensional ESE, 2.4 9 105 dermal fibroblasts were embedded in a 3 mL rat tail type I collagen solution (1.5 mg/ mL; Sigma-Aldrich). The collagen biomatrix was placed into a transwell chamber measuring 24 mm in diameter (Corning, Tewksbury, MA, USA) and cultured for 48 h prior to seeding 1 9 106 keratinocytes onto the biomatrix surface. After 24 h, ESEs were lifted to the air–liquid interface. The ESEs were cultured for 2 weeks, and the medium was changed three times per week.
Histological, immunohistochemical and ultrastructural studies of equine skin equivalent After 14 days at the air–liquid interface, samples were fixed in 10% formalin or 2% (w/v) paraformaldehyde (EM grade; Merck, Darms-
476
tadt, Germany) and 2.5% (v/v) glutaraldehyde solution (EM grade; TAAB Laboratories, Aldermaston, Reading, UK) in a 0.1 mol/L phosphate buffer (pH 7.4; Sigma-Aldrich). Formalin-fixed samples were dehydrated and embedded in paraffin wax. Sections (4 lm thick) were cut and stained with haematoxylin and eosin for routine histological evaluation and immunohistochemical study of pan-cytokeratin (K5, K6, K8 and K17), keratin 10, type IV collagen, laminin 5, vimentin and Ki67. Primary antibodies, used with the corresponding pretreatment for antigen retrieval and dilution conditions, consisted of mouse anti-human pan-cytokeratin (pretreatment of 0.01% trypsin for 20 min at room temperature, 1:100 dilution, Clone MNF116; Dako, Copenhagen, Denmark), citokeratin10 (pretreatment of citrate buffer for 20 min at 98°C plus 30 min at room temperature, 1:80 dilution, Clone DE-K10; Dako), goat anti-type IV collagen (pretreatment of type XIV protease for 8 min at 37°C, 1:100 dilution; Southern Biotechnology, Birmingham, AL, USA), rabbit antilaminin 5 (pretreatment of 0.1% proteinase K for 8 min at 37°C, 1:800 dilution; Dako), mouse anti-vimentin (pretreatment of citrate buffer for 20 min at 98°C plus 30 min at room temperature, 1:200 dilution, Clone V9; Dako) and mouse anti-Ki67 (pretreatment of citrate buffer for 20 min at 98°C plus 30 min at room temperature, 1:100 dilution, Clone MIB1; Dako). The detection system used for pan-cytokeratin, keratin 10, vimentin, Ki67 and laminin 5 was polymer–horseradish peroxidase anti-mouse or anti-rabbit (EnVision; Dako) for 30 min at room temperature. For type IV collagen, the secondary antibody used was a biotin-labelled rabbit anti-goat (Dako) at a 1:200 dilution in Trisbuffered saline for 1 h at room temperature. Diaminobenzidine was used as the detection system, and samples were counterstained with haematoxylin. For ultrastructural studies, samples fixed in paraformaldehyde– glutaraldehyde solution were rinsed four times with 100 mmol/L phosphate-buffered saline. Samples were then postfixed in 1% (w/v) osmium tetroxide (TAAB Laboratories) containing 0.8% (w/v) potassium hexacyanoferrate (III; Sigma-Aldrich) for 2 h and washed with deionized water four times, followed by dehydration in acetone. All procedures were performed at 4°C. Samples were dehydrated through a graded acetone series, embedded in Eponate 12 resin (Ted Pella Inc., Redding, CA, USA) and polymerized for 48 h at 60°C. Ultrathin sections (70 nm thick) were cut with a diamond knife (45°; Diatome, Biel, Switzerland), mounted on copper grids (200 mesh) and contrasted with conventional uranyl acetate (30 min) and Reynolds lead citrate solutions (5 min). Sections were observed with a Jeol 1400 transmission electron microscope (Jeol Ltd, Tokyo, Japan) equipped with a Gatan Ultrascan ES1000 CCD Camera (Gatan, Oxford, UK).
Results Isolation and culture of equine fibroblasts and keratinocytes Isolation of equine dermal fibroblasts was achieved by digesting the skin sample dermis in 2% collagenase solution. Primary cultures presented viability ratios of up to 95% and a high rate of proliferation, reaching confluence in
