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Epithelial and Stromal Developmental Patterns in a Novel Substitute of the Human Skin Generated with Fibrin-Agarose Biomaterials
Abstract
Development of human skin substitutes by tissue engineering may offer new therapeutic alternatives to the use of autologous tissue grafts. For that reason, it is necessary to investigate and develop new biocompatible biomaterials that support the generation of a proper human skin construct. In this study, we generated a novel model of bioengineered human skin substitute using human cells obtained from skin biopsies and fibrin-agarose biomaterials and we evaluated this model both at the ex vivo and the in vivo levels. Once the dermal fibroblasts and the epithelial keratinocytes were isolated and expanded in culture, we used fibrin-agarose scaffolds for the development of a full-thickness human skin construct, which was evaluated after 1, 2, 3 and 4 weeks of development ex vivo. The skin substitutes were then grafted onto immune-deficient nude mice and analyzed at days 10, 20, 30 and 40 postimplantation using transmission electron microscopy, histochemistry and immunofluorescence. The results demonstrated that the fibrin-agarose artificial skin had adequate biocompatibility and proper biomechanical properties. A proper development of both the bioengineered dermis and epidermis was found after 30 days in vivo, although the tissues kept ex vivo and those implanted in the animal model for 10 or 20 days showed lower levels of differentiation. In summary, our model of fibrin-agarose skin equivalent was able to reproduce the structure and histological architecture of the native human skin, especially after long-term in vivo implantation, suggesting that these tissues could reproduce the native skin.
... In this study, we present a hydrogel-based NI cancer model that considers the neural microenvironment and cancer cells in a reproducible in vitro system which consists of a three-dimensional model of NI squamous cell carcinoma combining epidermoid cells with a complete peripheral nerve segment immersed in a fibrine-agarose hydrogel. Fibrin-agarose advanced hydrogel has obtained successful results in a preclinical and clinical state [15,16] and also supported peripheral nerve regenerative function in vivo [17]. Now, we have used that hydrogel to build a unique model of NI that combines a highly biocompatible and natural biomaterial with the highly complex structure of the peripheral nerve to study the underlaying biological mechanism of NI. ...
... Previous studies showed that the combination of these two biomaterials resulted in a significant improvement in both biological and biomechanical properties of the hydrogel and allowed the generation of a wide variety of artificial substitutes [16][17][18]. In fact, this model has already shown promising preclinical results in multiple scenarios such as skin [15], cornea [19] and oral mucosa [18] grafting and as a nerve repair strategy [17], which allowed its successful translation to clinical practice [16]. However, this is the first time this hydrogel has been used to recreate complex pathological processes such as NI. ...
... In vitro models typically require more time for cells to express and assemble ECM components, mimicking the natural process that occurs in vivo. The results shown in our NI models are in agreement with previously published studies, which demonstrated that more in vitro culture time is needed for ECM molecule production [15]. ...
Neural Invasion (NI) is a key pathological feature of cancer in the colonization of distant tissues, and its underlying biological mechanisms are still scarcely known. The complex interactions between nerve and tumor cells, along with the stroma, make it difficult to reproduce this pathology in effective study models, which in turn has limited the understanding of NI pathogenesis. In this study, we have designed a three-dimensional model of NI squamous cell carcinoma combining human epidermoid carcinoma cells (hECCs) with a complete peripheral nerve segment encapsulated in a fibrine-agarose hydrogel. We recreated two vital processes of NI: a pre-invasive NI model in which hECCs were seeded on the top of the nerve-enriched stroma, and an invasive NI model in which cancer cells were immersed with the nerve in the hydrogel. Histological, histochemical and immunohistochemical analyses were performed to validate the model. Results showed that the integration of fibrin-agarose advanced hydrogel with a complete nerve structure and hECCs successfully generated an environment in which tumor cells and nerve components coexisted. Moreover, this model correctly preserved components of the neural extracellular matrix as well as allowing the proliferation and migration of cells embedded in hydrogel. All these results suggest the suitability of the model for the study of the mechanisms underlaying NI.
... NFAHs are artificial tissues that are used for diverse clinical applications depending on the encapsulated cell type. For the present study, the NFAH contained FBs and was generated as previously described [11]. Briefly, 4.16 ml of human plasma obtained from healthy blood donors was added to 7.5 × 10 5 FBs resuspended in Dulbecco's Modified Eagle´s Medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with gentamicin (20 µg/ ml) (Normon, Madrid, Spain) and 83 µl of tranexamic acid (Amchafibrin ® 500 mg; Rottapharm, Milan, Italy), as an anti-fibrinolytic agent. ...
... Fibrin agarose hydrogels were kept at 37 °C for 12 days. We then applied the nanostructuring technique, as described [11] (Fig. 1A). Briefly, we prepared an extrathick western blotting filter paper (ThermoFisher Scientific, Waltham, MA) with a 10-μm nylon net filter on top (Merck Millipore, Burlington, MA) to prevent adherence, and the hydrogel was placed over the filter. ...
... NFAH can be used for different clinical applications depending on the embedded cell type [11,[19][20][21][22]. The NFAH used in the present study contained FBs, thus acting as an artificial dermis. ...
Background
There remains much interest in improving cryopreservation techniques for advanced therapy medicinal products (ATMPs). Recently, human platelet lysate (hPL) has emerged as a promising candidate to replace fetal bovine serum (FBS) as a xeno-free culture supplement for the expansion of human cell therapy products. Whether hPL can also substitute for FBS in cryopreservation procedures remains poorly studied. Here, we evaluated several cryoprotective formulations based on a proprietary hPL for the cryopreservation of bioengineered tissues and cell therapy products.
Methods
We tested different xenogeneic-free, pathogen-inactivated hPL (ihPL)- and non-inactivated-based formulations for cryopreserving bioengineered tissue (cellularized nanostructured fibrin agarose hydrogels (NFAHs)) and common cell therapy products including bone marrow-derived mesenchymal stromal cells (BM-MSCs), human dermal fibroblasts (FBs) and neural stem cells (NSCs). To assess the tissue and cellular properties post-thaw of NFAHs, we analyzed their cell viability, identity and structural and biomechanical properties. Also, we evaluated cell viability, recovery and identity post-thaw in cryopreserved cells. Further properties like immunomodulation, apoptosis and cell proliferation were assessed in certain cell types. Additionally, we examined the stability of the formulated solutions. The formulations are under a bidding process with MD Bioproducts (Zurich, Switzerland) and are proprietary.
Results
Amongst the tissue-specific solutions, Ti5 (low-DMSO and ihPL-based) preserved the viability and the phenotype of embedded cells in NFAHs and preserved the matrix integrity and biomechanical properties similar to those of the standard cryopreservation solution (70% DMEM + 20% FBS + 10% DMSO). All solutions were stable at − 20 °C for at least 3 months. Regarding cell-specific solutions, CeA maintained the viability of all cell types > 80%, preserved the immunomodulatory properties of BM-MSCs and promoted good recovery post-thaw. Besides, both tested solutions were stable at − 20 °C for 18 months. Finally, we established that there is a 3-h window in which thawed NFAHs and FBs maintain optimum viability immersed in the formulated solutions and at least 2 h for BM-MSCs.
Conclusions
Our results show that pathogen-inactivated solutions Ti5 allocated for bioengineered tissues and CeA allocated for cells are efficient and safe candidates to cryopreserve ATMPs and offer a xenogeneic-free and low-DMSO alternative to commercially available cryoprotective solutions.
... NFAHs are arti cial tissues that are used for diverse clinical applications depending on the encapsulated cell type. For the present study, the NFAH contained FBs and was generated as previously described [11]. ...
... We then applied the nanostructuring technique, as described [11] (Fig. 1A). Brie y, we prepared an extrathick western blotting lter paper (ThermoFisher Scienti c, Waltham, MA) with a 10-µm nylon net lter on top (Merck Millipore, Burlington, MA) to prevent adherence, and the hydrogel was placed over the lter. ...
... Formulated hPL-based cryoprotective solutions preserve cell viability and functionality and sustain matrix integrity NFAH can be used for different clinical applications depending on the embedded cell type [11,[18][19][20][21]. The NFAH used in the present study contained FBs, thus acting as an arti cial dermis. ...
Background
There remains much interest in improving cryopreservation techniques for advanced therapy medicinal products (ATMPs). Recently, human platelet lysate (hPL) has emerged as a promising candidate to replace fetal bovine serum (FBS) as a xeno-free culture supplement for the expansion of human cell therapy products. Whether hPL can also substitute for FBS in cryopreservation procedures remains poorly studied. Here, we evaluated several cryoprotective formulations based on a proprietary hPL for the cryopreservation of bioengineered tissues and cell therapy products.
Methods
We tested different xenogeneic-free, pathogen-inactivated hPL (ihPL)- and non-inactivated-based formulations for cryopreserving bioengineered tissue (cellularized nanostructured fibrin agarose hydrogels (NFAHs)) and common cell therapy products including bone marrow-derived mesenchymal stromal cells (BM-MSCs), human dermal fibroblasts (FBs) and neural stem cells (NSCs). To assess the tissue and cellular properties post-thaw of NFAHs, we analyzed their cell viability, identity and structural and biomechanical properties. Also, we evaluated cell viability, recovery and identity post-thaw in cryopreserved cells. Further properties like immunomodulation, apoptosis and cell proliferation were assessed in certain cell types. Additionally, we examined the stability of the formulated solutions. The formulations are under a bidding process with MD Bioproducts (Zurich, Switzerland) and are proprietary.
Results
Amongst the tissue specific solutions, Ti5 (low-DMSO and ihPL-based) preserved the viability and the phenotype of embedded cells in NFAHs and preserved the matrix integrity and biomechanical properties similar to those of the standard cryopreservation solution (70% DMEM + 20% FBS + 10% DMSO). All solutions were stable at -20ºC for at least 3 months. Regarding cell specific solutions, CeA, maintained the viability of all cell types > 80%, preserved the immunomodulatory properties of BM-MSCs and promoted good recovery post-thaw. Besides, both tested solutions were stable at -20ºC for 18 months. Finally, we established that there is a 3-hour window in which thawed NFAHs and FBs, maintain optimum viability immersed in the formulated solutions; and, at least 2 h for BM-MSCs.
Conclusions
Our results show that pathogen-inactivated solutions Ti5 -allocated for bioengineered tissues- and CeA -allocated for cells- are efficient and safe candidates to cryopreserve ATMPs, and offer a xenogeneic-free and low-DMSO alternative to commercially available cryoprotective solutions.
... Among the different skin substitutes showing results, a model of bioengineered human skin originally generated at the University of Granada [20] called UGRSKIN demonstrated to be potentially useful at the preclinical level [21][22][23]. UGRSKIN consists of a fibrinagarose dermal substitute containing human fibroblasts and a stratified epithelium on top generated with autologous cultured keratinocytes. ...
... Despite the positive preliminary results, further research is needed in order to better understand the properties of these bioartificial tissues and thus improve their clinical efficiency. The UGRSKIN model consists of a biocompatible fibrin-agarose biomaterial containing human dermal fibroblasts within the biomaterial, and an epithelial layer on top generated with cultured human keratinocytes [20]. This skin substitute was approved by the Spanish Medicines Agency for autologous clinical use and is currently being applied for the treatment of severely burned patients [25]. ...
... Once primary cell cultures of human skin keratinocytes and fibroblasts were obtained, human skin substitutes were developed using fibrin-agarose based biomaterials as previously described [20,21]. In brief, a bioartificial dermal skin layer substitute was first generated by mixing 760 µL of human plasma as a fibrin source, 75 µL of DMEM containing 140,000 cultured human fibroblasts, 15 µL of tranexamic acid-as an antifibrinolytic agent-(Amchafibrin, Fides-Ecofarma, Valencia, Spain), 50 µL of a 2% solution of type VII agarose (Merck, Darmstadt, Germany) melted in PBS, and 100 µL of 1% CaCl2 solution (Merck, Darmstadt, Germany) per ml of mixture. ...
The most recent generation of bioengineered human skin allows for the efficient treatment of patients with severe skin defects. Despite UV sunlight can seriously affect human skin, the optical behavior in the UV range of skin models is still unexplored. In the present study, absorbance and transmittance of the UGRSKIN bioartificial skin substitute generated with human skin cells combined with fibrin-agarose biomaterials were evaluated for: UV-C (200–280 nm), -B (280–315 nm), and -A (315–400 nm) spectral range after 7, 14, 21 and 28 days of ex vivo development. The epidermis of the bioartificial skin substitute was able to mature and differentiate in a time-dependent manner, expressing relevant molecules able to absorb most of the incoming UV radiation. Absorbance spectral behavior of the skin substitutes showed similar patterns to control native skin (VAF > 99.4%), with values 0.85–0.90 times lower than control values at 7 and 14- days and 1.05–1.10 times the control values at 21- and 28-days. UV absorbance increased, and UV transmission decreased with culture time, and comparable results to the control were found at 21 and 28 days. These findings support the use of samples corresponding to 21 or 28 days of development for clinical purposes due to their higher histological similarities with native skin, but also because of their absorbance of UV radiation.
... However, the biocompatibility and regenerative potential of most currently available biomaterials is limited, and novel types of grafts capable of inducing effective bone regeneration are needed. One of the possible alternatives is the use of organic biomaterials based on natural components-such as human fibrin-which are known to have high biocompatibility, and have been successfully used in multiple regenerative applications, such as for the human cornea [16,17], oral mucosa [18], nerves [19], and skin [20,21]. Another possibility is the use of cells immersed in the biomaterial, since previous reports have demonstrated that the combination of biocompatible biomaterials with living cells is associated with a significant improvement in results in terms of the formation of bone tissue [10]-especially when stem cells are used [22]. ...
... Induction of efficient and successful bone regeneration is a challenge in maxillofacial surgery. In the present work, we used a combination of biocompatible fibrin-agarose biomaterials with potential utility in tissue engineering [16][17][18][19][20], and pre-differentiated AD-SCs that were previously demonstrated to have significant osteogenic potential [23]. Application of nanostructuration methods allowed us to generate three-dimensional Figure 7. Quantitative analysis of ECM components as determined by histochemistry (alizarin red, picrosirius red, alcian blue, and toluidine blue) and immunohistochemistry (osteocalcin and versican). ...
... Induction of efficient and successful bone regeneration is a challenge in maxillofacial surgery. In the present work, we used a combination of biocompatible fibrin-agarose biomaterials with potential utility in tissue engineering [16][17][18][19][20], and pre-differentiated ADSCs that were previously demonstrated to have significant osteogenic potential [23]. Application of nanostructuration methods allowed us to generate three-dimensional cylinder-type structures that were used to repair the mandible defects generated in an animal model. ...
Critical defects of the mandibular bone are very difficult to manage with currently available materials and technology. In the present work, we generated acellular and cellular substitutes for human bone by tissue engineering using nanostructured fibrin–agarose biomaterials, with and without adipose-tissue-derived mesenchymal stem cells differentiated to the osteogenic lineage using inductive media. Then, these substitutes were evaluated in an immunodeficient animal model of severely critical mandibular bone damage in order to assess the potential of the bioartificial tissues to enable bone regeneration. The results showed that the use of a cellular bone substitute was associated with a morpho-functional improvement of maxillofacial structures as compared to negative controls. Analysis of the defect site showed that none of the study groups fully succeeded in generating dense bone tissue at the regeneration area. However, the use of a cellular substitute was able to improve the density of the regenerated tissue (as determined via CT radiodensity) and form isolated islands of bone and cartilage. Histologically, the regenerated bone islands were comparable to control bone for alizarin red and versican staining, and superior to control bone for toluidine blue and osteocalcin in animals grafted with the cellular substitute. Although these results are preliminary, cellular fibrin–agarose bone substitutes show preliminary signs of usefulness in this animal model of severely critical mandibular bone defect.
... NFAHs are artificial tissues that are used for diverse clinical applications depending on the encapsulated cell type. For the present study, the NFAH contained FBs and was generated as previously described [11]. Briefly, 4.16 ml of human plasma obtained from healthy blood donors was added to 7.5 × 10 5 FBs resuspended in Dulbecco's Modified Eagle´s Medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with gentamicin (20 µg/ ml) (Normon, Madrid, Spain) and 83 µl of tranexamic acid (Amchafibrin ® 500 mg; Rottapharm, Milan, Italy), as an anti-fibrinolytic agent. ...
... Fibrin agarose hydrogels were kept at 37 °C for 12 days. We then applied the nanostructuring technique, as described [11] (Fig. 1A). Briefly, we prepared an extrathick western blotting filter paper (ThermoFisher Scientific, Waltham, MA) with a 10-μm nylon net filter on top (Merck Millipore, Burlington, MA) to prevent adherence, and the hydrogel was placed over the filter. ...
... NFAH can be used for different clinical applications depending on the embedded cell type [11,[19][20][21][22]. The NFAH used in the present study contained FBs, thus acting as an artificial dermis. ...
... In addition, agarose has been combined with other biomaterials, allowing the generation of biological scaffolds with high versatility towards specific soft tissue types. Among others, agarose biomaterials have been combined with fibrin to generate bioartificial human skin, cornea and oral mucosa [18][19][20], with platelet-rich plasma in cartilage tissue engineering [21], with collagen in the design of vascular endothelial networks [22], and with other biomaterials such as chitosan, graphene oxide and hydroxyapatite for the development of hard tissues [23,24]. Despite the positive results obtained with the use of agarose scaffolds in TE, the biological behavior of these hydrogels still needs further clarification. ...
... In fact, the clear reduction in metabolic activity found at the highest agarose concentrations and follow-up times may imply that highly concentrated agaroses should preferably be used as acellular scaffolds, as suggested for the human cartilage [36]. However, bioengineered tissues requiring an abundant cell population such as the human skin and cornea [3,19,37], should be used at the agarose concentrations showing the highest metabolic activity such as D2LE, LM and D5 agaroses at the concentration of 0.5%. Future experiments should determine the most proper conditions for the generation of cellular agarose-based biomaterials by tissue engineering. ...
... Again, the in vivo results support our hypothesis that the use of highly concentrated agaroses could be indicated in cases requiring long-term stability and low cellularity, such as the human intervertebral disk [41]. However, other tissues and organs with an abundant cell population requiring a progressive remodeling and biodegradation, such as the human skin and cornea [3,19,37], should use low concentrations of agarose. Although further studies are in need in this regard, this would be applicable not only to orthotypical cells obtained from the tissue to be reproduced in laboratory, but also to alternative cell sources like human mesenchymal stem cells (MSC) and other cell types, as previously suggested [42,43]. ...
Five agarose types (D1LE, D2LE, LM, MS8 and D5) were evaluated in tissue engineering and compared for the first time using an array of analysis methods. Acellular and cellular constructs were generated from 0.3–3%, and their biomechanical properties, in vivo biocompatibility (as determined by LIVE/DEAD, WST-1 and DNA release, with n = 6 per sample) and in vivo biocompatibility (by hematological and biochemical analyses and histology, with n = 4 animals per agarose type) were analyzed. Results revealed that the biomechanical properties of each hydrogel were related to the agarose concentration (p < 0.001). Regarding the agarose type, the highest (p < 0.001) Young modulus, stress at fracture and break load were D1LE, D2LE and D5, whereas the strain at fracture was higher in D5 and MS8 at 3% (p < 0.05). All agaroses showed high biocompatibility on human skin cells, especially in indirect contact, with a correlation with agarose concentration (p = 0.0074 for LIVE/DEAD and p = 0.0014 for WST-1) and type, although cell function tended to decrease in direct contact with highly concentrated agaroses. All agaroses were safe in vivo, with no systemic effects as determined by hematological and biochemical analysis and histology of major organs. Locally, implants were partially encapsulated and a pro-regenerative response with abundant M2-type macrophages was found. In summary, we may state that all these agarose types can be safely used in tissue engineering and that the biomechanical properties and biocompatibility were strongly associated to the agarose concentration in the hydrogel and partially associated to the agarose type. These results open the door to the generation of specific agarose-based hydrogels for definite clinical applications such as the human skin, cornea or oral mucosa.
... Among the different biomaterials that showed preclinical and clinical usefulness for the generation of human tissue substitutes, fibrin-agarose hydrogels show excellent biocompatibility and biomechanical properties and its porous fibrillar pattern allows diffusion and interchange of oxygen and nutrients [2,3,5,6]. Fibrin-agarose was previously used to generate bioartificial substitutes of the human cornea [7], sclera [8], oral mucosa [9], palate [10], nerve [11,12], cartilage [13] and skin [14,15]. In fact, a bioartificial model of tissue-engineered human skin (UGRSKIN), consisting of a dermal skin substitute based on fibrin-agarose biomaterials and dermal fibroblasts and an overlying epithelial layer, has been successfully translated to the clinical setting [16]. ...
... Primary cell cultures of dermal fibroblast cells were obtained from skin biopsies taken from healthy donors. Biopsies were carefully rinsed in phosphate-buffered saline and enzymatically digested in a 2 mg/ml Clostridium histolyticum collagenase I (Gibco-Thermo Fisher Scientific, Waltham, MA) solution at 37 °C for 6 h to obtain primary cell cultures of skin fibroblasts following previously described protocols [14,15]. Isolated human dermal fibroblasts were then cultured in Dulbecco's modified Eagle's medium -DMEM-(Merck Life Science, St. Louis, MO) supplemented with 10% fetal bovine serum (Merck Life Science) and 1% antibiotics/antimycotics (100 U/mL penicillin G, 100 mg/ mL streptomycin and 0.25 mg/mL amphotericin B; Merck Life Science) under standard cell culture conditions. ...
... Isolated human dermal fibroblasts were then cultured in Dulbecco's modified Eagle's medium -DMEM-(Merck Life Science, St. Louis, MO) supplemented with 10% fetal bovine serum (Merck Life Science) and 1% antibiotics/antimycotics (100 U/mL penicillin G, 100 mg/ mL streptomycin and 0.25 mg/mL amphotericin B; Merck Life Science) under standard cell culture conditions. Bioengineered human dermal skin substitutes (SS) were generated using fibrin-agarose biomaterials as previously described [7,9,11,14,15,28]. In brief, the following components were mixed per each ml of dermal skin substitute: 760 µl of human plasma -as a fibrin source-, 75 μl of DMEM containing 140,000 cultured human fibroblasts, 15 µl of tranexamic acid -as antifibrinolytic agent-(Amchafibrin, Fides-Ecofarma, Valencia, Spain), 50 μl of a 2% solution of type VII agarose (Merck Life Science) melted in PBS, and 100 μl of 1% CaCl 2 solution Drug loading = Initial Drug Amount mg Total Batch Weight(mg) ...
Background
Treatment of patients affected by severe burns is challenging, especially due to the high risk of Pseudomonas infection. In the present work, we have generated a novel model of bioartificial human dermis substitute by tissue engineering to treat infected wounds using fibrin-agarose biomaterials functionalized with nanostructured lipid carriers (NLCs) loaded with two anti-Pseudomonas antibiotics: sodium colistimethate (SCM) and amikacin (AMK).
Results
Results show that the novel tissue-like substitutes have strong antibacterial effect on Pseudomonas cultures, directly proportional to the NLC concentration. Free DNA quantification, WST-1 and Caspase 7 immunohistochemical assays in the functionalized dermis substitute demonstrated that neither cell viability nor cell proliferation were affected by functionalization in most study groups. Furthermore, immunohistochemistry for PCNA and KI67 and histochemistry for collagen and proteoglycans revealed that cells proliferated and were metabolically active in the functionalized tissue with no differences with controls. When functionalized tissues were biomechanically characterized, we found that NLCs were able to improve some of the major biomechanical properties of these artificial tissues, although this strongly depended on the type and concentration of NLCs.
Conclusions
These results suggest that functionalization of fibrin-agarose human dermal substitutes with antibiotic-loaded NLCs is able to improve the antibacterial and biomechanical properties of these substitutes with no detectable side effects. This opens the door to future clinical use of functionalized tissues.
... Among the different biomaterials that showed preclinical and clinical usefulness for the generation of human tissue substitutes, brin-agarose hydrogels show excellent biocompatibility and biomechanical properties and its porous brillar pattern allows diffusion and interchange of oxygen and nutrients (2,3,5,6). Fibrin-agarose was previously used to generate bioarti cial substitutes of the human cornea (7), sclera (8), oral mucosa (9), palate(10), nerve (11,12), cartilage (13) and skin (14,15). In fact, a bioarti cial model of tissue-engineered human skin (UGRSKIN), consisting of a dermal skin substitute based on brin-agarose biomaterials and dermal broblasts and an overlying epithelial layer, has been successfully translated to the clinical setting (16). ...
... Generation of non-functionalized human dermal skin substitutes (SS) by tissue engineering Primary cell cultures of dermal broblast cells were obtained from skin biopsies taken from healthy donors. Biopsies were carefully rinsed in phosphate-buffered saline and enzymatically digested in a 2 mg∕ml Clostridium histolyticum collagenase I (Gibco -Thermo Fisher Scienti c, Waltham, MA) solution at 37 °C for 6 h to obtain primary cell cultures of skin broblasts following previously described protocols (14,15). Isolated human dermal broblasts were then cultured in Dulbecco's modi ed Eagle's medium -DMEM-(Merck Life Science, St. Louis, MO) supplemented with 10% fetal bovine serum (Merck Life Science) and 1% antibiotics/antimycotics (100 U/mL penicillin G, 100 mg/ mL streptomycin and 0.25 mg/mL amphotericin B; Merck Life Science) under standard cell culture conditions. ...
... Isolated human dermal broblasts were then cultured in Dulbecco's modi ed Eagle's medium -DMEM-(Merck Life Science, St. Louis, MO) supplemented with 10% fetal bovine serum (Merck Life Science) and 1% antibiotics/antimycotics (100 U/mL penicillin G, 100 mg/ mL streptomycin and 0.25 mg/mL amphotericin B; Merck Life Science) under standard cell culture conditions. Bioengineered human dermal skin substitutes (SS) were generated using brin-agarose biomaterials as previously described (7,9,11,14,15,28). In brief, the following components were mixed per each ml of dermal skin substitute: 760 µl of human plasma -as a brin source-, 75 µl of DMEM containing 140,000 cultured human broblasts, 15 µl of tranexamic acid -as anti brinolytic agent-(Amcha brin, Fides-Ecofarma, Valencia, Spain), 50 µl of a 2% solution of type VII agarose (Merck Life Science) melted in PBS, and 100 µl of 1% CaCl 2 solution (Merck Life Science) -to promote brin polymerization-. ...
Background: Treatment of patients affected by severe burns is challenging, especially due to the high risk of Pseudomonas infection. In the present work, we have generated a novel model of bioartificial human dermis substitute by tissue engineering to treat infected wounds using fibrin-agarose biomaterials functionalized with nanostructured lipid carriers (NLCs) loaded with two anti-Pseudomonas antibiotics: sodium colistimethate (SCM) and amikacin (AMK).
Results: Results show that the novel tissue-like substitutes have strong antibacterial effect on Pseudomonas cultures, directly proportional to the NLC concentration. Free DNA quantification, WST-1 and Caspase 7 immunohistochemical assays in the functionalized dermis substitute demonstrated that neither cell viability nor cell proliferation were affected by functionalization in most study groups. Furthermore, immunohistochemistry for PCNA and KI67 and histochemistry for collagen and proteoglycans revealed that cells proliferated and were metabolically active in the functionalized tissue with no differences with controls. When functionalized tissues were biomechanically characterized, we found that NLCs were able to improve some of the major biomechanical properties of these artificial tissues, although this strongly depended on the type and concentration of NLCs.
Conclusions: These results suggest that functionalization of fibrin-agarose human dermal substitutes with antibiotic-loaded NLCs is able to improve the antibacterial and biomechanical properties of these substitutes with no detectable side effects. This opens the door to future clinical use of functionalized tissues.
... Our previous work evaluated the e cacy of a novel hemostatic agent made from a nanostructured brin and type VII agarose hydrogel (NFAH) [9]. NFAH has previously been used as a scaffold biomaterial in several preclinica [10], [11], [12] and clinical [13], [14]. This brin and agarose matrix is mainly derived from human plasma, has high exibility, elasticity and mechanical strength [15], [16] and has been proven to be biodegradable and biocompatible [11], [17]. ...
... NFAH has previously been used as a scaffold biomaterial in several preclinica [10], [11], [12] and clinical [13], [14]. This brin and agarose matrix is mainly derived from human plasma, has high exibility, elasticity and mechanical strength [15], [16] and has been proven to be biodegradable and biocompatible [11], [17]. However, in order to extend the half-life of this hemostatic agent, it is necessary to investigate a preservation method that maintains its structure and ensures long-term biological and therapeutic suitability. ...
Uncontrolled bleeding during surgery is associated with high mortality and prolonged hospital stay, necessitating the use of hemostatic agents. Fibrin sealant patches offer an efficient solution to achieve hemostasis and improve patient outcomes in liver resection surgery. We have previously demonstrated the efficacy of a nanostructured fibrin-agarose hydrogel (NFAH). However, for the widespread distribution and commercialization of the product, it is necessary to develop an optimal preservation method that allows for prolonged stability and facilitates storage and distribution. We investigated cryopreservation as a potential method for preserving NFAH using trehalose. Structural changes in cryopreserved NFAH (Cryo-NFAH) were investigated and comparative in vitro and in vivo efficacy and safety studies were performed with freshly prepared NFAH. We also examined the long-term safety of Cryo-NFAH versus TachoSil® in a rat partial hepatectomy model, including time to hemostasis, intra-abdominal adhesion, hepatic hematoma, inflammatory factors, histopathological variables, temperature and body weight, hemocompatibility and cytotoxicity. Structural analyses demonstrated that Cryo-NFAH retained most of its macro- and microscopic properties after cryopreservation. Likewise, hemostatic efficacy assays showed no significant differences with fresh NFAH. Safety evaluations indicated that Cryo-NFAH had a similar overall profile to TachoSil® up to 40 days post-surgery in rats. In addition, Cryo-NFAH demonstrated superior hemostatic efficacy compared with TachoSil® while also demonstrating lower levels of erythrolysis and cytotoxicity than both TachoSil® and other commercially available hemostatic agents. These results indicate that Cryo-NFAH is highly effective hemostatic patch with a favorable safety and tolerability profile, supporting its potential for clinical use.
... Fibrin-agarose (FA) biomaterials were first described by our group for the generation of cornea substitutes with promising results [27]. Subsequently, FA scaffolds were applied to the generation of numerous tissues and organs, such as the skin, nerve, tendon, sclera and oral mucosa [28][29][30][31][32]. Previous studies showed that the combination of these two materials resulted in a significant improvement of the biomaterials' biomechanical properties as compared to fibrin hydrogels, especially when nanostructuration is applied [33,34]. ...
... Development of an ideal biomaterial for clinical use is one of the main objectives of current research in tissue engineering. Although FA biomaterial showed promising results in tissue engineering of the cornea, skin, nerve, tendon, sclera and oral mucosa, the biocompatibility and biomechanical properties of this biomaterial still need to be improved [27][28][29][30][31][32]. In this regard, we recently demonstrated that different types of marine agaroses could have specific biological and biomechanical properties, suggesting that not all currently available marine agaroses polysaccharides show the same behavior both in vitro and in vivo [23]. ...
Development of an ideal biomaterial for clinical use is one of the main objectives of current research in tissue engineering. Marine-origin polysaccharides, in particular agaroses, have been widely explored as scaffolds for tissue engineering. We previously developed a biomaterial based on a combination of agarose with fibrin, that was successfully translated to clinical practice. However, in search of novel biomaterials with improved physical and biological properties, we have now generated new fibrin-agarose (FA) biomaterials using 5 different types of agaroses at 4 different concentrations. First, we evaluated the cytotoxic effects and the biomechanical properties of these biomaterials. Then, each bioartificial tissue was grafted in vivo and histological, histochemical and immunohistochemical analyses were performed after 30 days. Ex vivo evaluation showed high biocompatibility and differences in their biomechanical properties. In vivo, FA tissues were biocompatible at the systemic and local levels, and histological analyses showed that biointegration was associated to a pro-regenerative process with M2-type CD206-positive macrophages. These results confirm the biocompatibility of FA biomaterials and support their clinical use for the generation of human tissues by tissue engineering, with the possibility of selecting specific agarose types and concentrations for applications requiring precise biomechanical properties and in vivo reabsorption times.
... One of the ATMP approved for clinical use in Spain is the UGRSKIN bioartificial skin model generated in the laboratory for patients affected by large burns [2]. Like most full-thickness biological substitutes of human skin [3], UGRSKIN consists of an epidermal layer cultured on the surface of a dermal substitute, and requires the establishment of stromal and epithelial cell cultures [4]. Several methods have been described for the efficient generation of skin keratinocyte cell cultures [5]. ...
... Since publication of the first description of a culture technique to grow human keratinocytes [29], several methods have been described to efficiently culture human epithelial cells to produce ATMP. Application of these methods currently makes it possible to establish primary cell cultures from a small skin biopsy sample, which, in turn, allows bioartificial substitutes for human skin to be generated by tissue engineering in 3-4 weeks [2,4,30]. However, this timeframe may be too long when treating patients with severe burns. ...
Human skin keratinocyte primary cultures can be established from skin biopsies with culture media containing epithelial growth factor (EGF). Although current methods are efficient, optimization is required to accelerate the procedure and obtain these cultures in less time. In the present study, we evaluated the effect of novel formulations based on EGF-loaded nanostructured lipid carriers (NLC). First, biosafety of NLC containing recombinant human EGF (NLC-rhEGF) was verified in immortalized skin keratinocytes and cornea epithelial cells, and in two epithelial cancer cell lines, by quantifying free DNA released to the culture medium. Then we established primary cell cultures of human skin keratinocytes with basal culture media (BM) and BM supplemented with NLC-rhEGF, liquid EGF (L-rhEGF), or NLC alone (NLC-blank). The results showed that cells isolated by enzymatic digestion and cultured with or without a feeder layer had a similar growth rate regardless of the medium used. However, the explant technique showed higher efficiency when NLC-rhEGF culture medium was used, compared to BM, L-rhEGF, or NLC-blank. Gene expression analysis showed that NLC-rhEGF was able to increase EGFR gene expression, along with that of other genes related to cytokeratins, cell–cell junctions, and keratinocyte maturation and differentiation. In summary, these results support the use of NLC-rhEGF to improve the efficiency of explant-based methods in the efficient generation of human keratinocyte primary cell cultures for tissue engineering use.
... Para su desarrollo se proporciona al alumno, a través de la plataforma de recursos de la Universidad de Granada, acceso a una documentación escrita y una documentación audiovisual. La primera consiste en un texto con información básica sobre IT, un artículo científico en el que se describe la generación de un modelo de piel artificial [9] y un texto que resume el proceso de control sanitario que exige la trasferencia a la clínica de un medicamento tisular. El material docente e investigador aportado fue elaborado por el profesorado del departamento. ...
... El contenido está, sin embargo, estrechamente relacionado con ella, ya que constituye su soporte y fundamento conceptual y procedimental [2,8]. La implementación del aula se desarrolla, en nuestro modelo, tras la impartición previa de los dos temas básicos -tejido epitelial y tejido conjuntivo-a partir de los cuales los alumnos pueden acceder por sí mismos al nuevo conocimiento que se propone, al ser, en la mayoría de los casos, los dos elementos esenciales, objeto de sustitución biomimética, en los tejidos artificiales que se generan por IT [9,[20][21][22]. ...
... Engineered skins showed better cell differentiation and skin structure regeneration after 30 days of implantation in the in vivo environment compared to samples implanted for 10 or 20 days. Finally, the researchers confirmed that the fibrin-agarose-engineered skins can regenerate the histological structure of native human skin after long-term implantation in vivo (Carriel et al., 2012). Martin-Piedra et al. investigated the effects of a grafted fibrin-agarose skin substitute model (UGRSKIN) on severe burn patients for 3 months. ...
Skin, the largest biological organ, consists of three main parts: the epidermis, dermis, and subcutaneous tissue. Wounds are abnormal wounds in various forms, such as lacerations, burns, chronic wounds, diabetic wounds, acute wounds, and fractures. The wound healing process is dynamic, complex, and lengthy in four stages involving cells, macrophages, and growth factors. Wound dressing refers to a substance that covers the surface of a wound to prevent infection and secondary damage. Biomaterials applied in wound management have advanced significantly. Natural biomaterials are increasingly used due to their advantages including biomimicry of ECM, convenient accessibility, and involvement in native wound healing. However, there are still limitations such as low mechanical properties and expensive extraction methods. Therefore, their combination with synthetic biomaterials and/or adding bioactive agents has become an option for researchers in this field. In the present study, the stages of natural wound healing and the effect of biomaterials on its direction, type, and level will be investigated. Then, different types of polysaccharides and proteins were selected as desirable natural biomaterials, polymers as synthetic biomaterials with variable and suitable properties, and bioactive agents as effective additives. In the following, the structure of selected biomaterials, their extraction and production methods, their participation in wound healing, and quality control techniques of biomaterials-based wound dressings will be discussed.
... En grandes defectos cutáneos, los apósitos no bastan, y los sustitutos cutáneos autólogos desarrollados mediante ingeniería tisular se presentan como una alternativa terapéutica prometedora frente al homoinjerto cutáneo; de especial interés aquellos obtenidos a partir de células madre mesenquimales y células madre derivadas del tejido adiposo (11,12). También mediante la utilización de biomateriales de fibrina y agarosa se ha logrado generar piel humana artificial estructural y ultraestructuralmente análoga a la piel humana nativa (13). La vascularización de estos injertos supone el tema principal de numerosos estudios aún en fase experimental. ...
The aim of this work is to review the current state of the use of tissue engineering in the field of pediatric surgery. We conducted a search of articles in PubMed under the terms of “Pediatric Surgery AND Tissue Engineering”. All those articles that included in the name of the journal and / or of the article any of the following terms were selected: “bioengineered”, “engineering”, “pediatric”, “children” “pediatric surgery”; In addition to those articles resulting from this same search, which, in our opinion, should be included in our review because they also deal with issues related to tissue engineering applied to disciplines that are part of the daily healthcare task of a pediatric surgeon. 178 articles were obtained. After carrying out a first review, we eliminated those referring to the applications of tissue engineering in cardiovascular surgery, traumatology, dentistry, and otorhinolaryngology, as they are specialties not included within pediatric surgery in our setting. Finally, 117 articles distributed as follows were reviewed: 36 on the area of general surgery (6 bibliographic reviews, 30 experimental), 26 plastic surgery (3 clinical trials, 15 experimental, 8 bibliographic reviews), 33 urology (26 experimental, 7 bibliographic reviews), 22 cleft palate (1 clinical trial, 13 experimental, 8 bibliographic reviews). There are many works related to tissue engineering and its application to pediatric surgery and, although in the theoretical panorama tissue engineering promises to be a hopeful therapeutic alternative in many pathologies included within this specialty, and although there are multiple experimental studies that suggest a hopeful application in the clinic, there are few works that deal with its real application in pediatric patients.
... This is one of the factors that could explain the high SASS engraftment (98%) on severely burned patients [1]. However, the basement membrane could be formed after grafting in vivo as shown for biomaterial (collagen or human plasma)-based skin substitutes [39][40][41] and high engraftment has also been observed at discharge for this model (80-85%). Therefore, in vivo trials will be necessary to compare their regenerative potential. ...
Background
The aim of this in vitro study was to compare side-by-side two models of human bilayered tissue-engineered skin substitutes (hbTESSs) designed for the treatment of severely burned patients. These are the scaffold-free self-assembled skin substitute (SASS) and the human plasma-based skin substitute (HPSS).
Methods
Fibroblasts and keratinocytes from three humans were extracted from skin biopsies (N = 3) and cells from the same donor were used to produce both hbTESS models. For SASS manufacture, keratinocytes were seeded over three self-assembled dermal sheets comprising fibroblasts and the extracellular matrix they produced (n = 12), while for HPSS production, keratinocytes were cultured over hydrogels composed of fibroblasts embedded in either plasma as unique biomaterial (Fibrin), plasma combined with hyaluronic acid (Fibrin-HA) or plasma combined with collagen (Fibrin-Col) (n/biomaterial = 9). The production time was 46–55 days for SASSs and 32–39 days for HPSSs. Substitutes were characterized by histology, mechanical testing, PrestoBlue™-assay, immunofluorescence (Ki67, Keratin (K) 10, K15, K19, Loricrin, type IV collagen) and Western blot (type I and IV collagens).
Results
The SASSs were more resistant to tensile forces (p-value < 0.01) but less elastic (p-value < 0.001) compared to HPSSs. A higher number of proliferative Ki67+ cells were found in SASSs although their metabolic activity was lower. After epidermal differentiation, no significant difference was observed in the expression of K10, K15, K19 and Loricrin. Overall, the production of type I and type IV collagens and the adhesive strength of the dermal-epidermal junction was higher in SASSs.
Conclusions
This study demonstrates, for the first time, that both hbTESS models present similar in vitro biological characteristics. However, mechanical properties differ and future in vivo experiments will aim to compare their wound healing potential.
... The purpose of this study was to isolate and expand dermal fibroblasts and epithelial keratinocytes in fibrinagarose scaffolds to create a full-thickness human skin construct, which was then grafted onto immunodeficient nude mice to study its functional characteristics. 204 According to these results, artificial skin from fibrin-agarose was biocompatible and had appropriate biomechanical properties, suggesting that these tissues might be able to recreate native skin. The addition of agarose resulted in a significant improvement in biomechanical properties compared to fibrin hydrogels. ...
Fibrin is a promising natural polymer that is widely used for diverse applications, such as hemostatic glue, carrier for drug and cell delivery, and matrix for tissue engineering. Despite the significant advances in the use of fibrin for bioengineering and biomedical applications, some of its characteristics must be improved for suitability for general use. For example, fibrin hydrogels tend to shrink and degrade quickly after polymerization, particularly when they contain embedded cells. In addition, their poor mechanical properties and batch-to-batch variability affect their handling, long-term stability, standardization, and reliability. One of the most widely used approaches to improve their properties has been modification of the structure and composition of fibrin hydrogels. In this review, recent advances in composite fibrin scaffolds, chemically modified fibrin hydrogels, interpenetrated polymer network (IPN) hydrogels composed of fibrin and other synthetic or natural polymers are critically reviewed, focusing on their use for tissue engineering.
... www.nature.com/scientificreports/ treatment, tissue regeneration including heart/wound/skin, and attachment to brain and cornea [28][29][30][31] . Finally, its macromolecular networks caused by high cross-linking and the self-assembly of hydrogen bonds allow sufficient diffusion and transport to take place in the active layer of the memristive device. ...
Natural, organic, materials-based artificial synaptic devices have been in the spotlight for wearable/flexible devices due to their lightweight, biocompatibility, and scalability. In this study, an electronic memristive device based on agarose extracted from plants in the Rhodophyceae class was fabricated, and its memory characteristics and analog data processing capabilities were evaluated. The Al/agarose@gold nanoparticle (AuNP) film/indium-tin-oxide (ITO)-structured memristive device exhibited reliable resistive switching characteristics with excellent retention with a large Ron/Roff ratio of 10⁴. Also, analog conductance changes in our device were achieved with power consumption at the pJ level. This notable behavior could be maintained under mechanical deformations from a flat to a 4-mm bent state. In the recognition simulation based on the device's performance, an 91% accuracy and clear digit classification were achieved.
... Therefore, the differences observed between UC and cell cultures could be explained due to the lack of biological and physical factors in the 2D and 3D culture conditions. Actually, none of these cell culture methods were able to recreate the complex processes involved during UC development as expected, these results being supported by the partial differentiation observed within other models such as bioengineered skin [44,45] or cartilage substitutes [33,46,47]. ...
Wharton’s jelly stem cells (WJSC) from the human umbilical cord (UC) are one of the most promising mesenchymal stem cells (MSC) in tissue engineering (TE) and advanced therapies. The cell niche is a key element for both, MSC and fully differentiated tissues, to preserve their unique features. The basement membrane (BM) is an essential structure during embryonic development and in adult tissues. Epithelial BMs are well-known, but similar structures are present in other histological structures, such as in peripheral nerve fibers, myocytes or chondrocytes. Previous studies suggest the expression of some BM molecules within the Wharton’s Jelly (WJ) of UC, but the distribution pattern and full expression profile of these molecules have not been yet elucidated. In this sense, the aim of this histological study was to evaluate the expression of main BM molecules within the WJ, cultured WJSC and during WJSC microtissue (WJSC-MT) formation process. Results confirmed the presence of a pericellular matrix composed by the main BM molecules—collagens (IV, VII), HSPG2, agrin, laminin and nidogen—around the WJSC within UC. Additionally, ex vivo studies demonstrated the synthesis of these BM molecules, except agrin, especially during WJSC-MT formation process. The WJSC capability to synthesize main BM molecules could offer new alternatives for the generation of biomimetic-engineered substitutes where these molecules are particularly needed.
... They have been seen to provide better results as shown by a study using keratinocyte seeded hyaluronic acid membrane grafts on full-thickness wounds in nude mice wound models (Horch et al. 2019) In the recent times, newer combinations of biomaterials are being tested for better skin regeneration capabilities to increase their efficiency. Keratinocytes seeded on a fibrin-agarose scaffold were tested in vivo on nude mice models, which showed promising results (Carriel et al. 2012). ...
The vital role of structurally and functionally stable vasculature in engineered tissues is well-established in regenerative medicine. Large-volume, natural, and synthetic tissue constructs require a high degree of perfusion and nutrient diffusion to meet the physiological demands of the encapsulated cells. Additionally, cancer tissue models fabricated using various scaffolds and matrices also need to incorporate tumor-mimetic abnormal vasculature to study the influence of angiogenesis on tumor growth, progression, and anti-cancer drug delivery. In this chapter, prominent hydrogel-based matrices that have been developed for vascular tissue engineering as well as modeling of the tumor vasculature and angiogenesis are discussed. Various microenvironmental considerations (including biophysical and biochemical characteristics of the matrix) required for emulating vascular regeneration as well as tumor angiogenesis are described. A wide range of hydrogel-based models (including natural, synthetic and hybrid materials) and associated biofabrication strategies (spanning molecular design to macroscale materials processing) for creating vascularized scaffolds are elaborated. Overall, this chapter provides an overview to the reader on creation of engineered scaffolds for implementation in tissue vascularization and repair and in disease models for future applications in drug testing.
... That could be explained because as is shown in figures 4(a) and (b), the formed tissue in the scaffolds represents immature tissue, probably because of the short co-culture period of 14 d. Moreover, to obtain mature tissue it is necessary to have a culture time of approximately 4 weeks [28,29]. However, our objective was not to obtain artificial skin, but rather a skin substitute, checking if the incorporation of cells influences the healing process. ...
Hybrid scaffolds from natural and synthetic polymers have been widely used due to the complementary nature of their physical and biological properties. The aim of the present study, therefore, has been to analyze in vivo a bilayer scaffold of poly(lactide-co-glycolide) (PLGA)/fibrin electrospun membrane and fibrin hydrogel layer on a rat skin model. Fibroblasts were cultivated in the fibrin hydrogel layer and keratinocytes on the electrospun membrane to generate a skin substitute. The scaffolds without and with cells were tested in a full-thickness wound model in Wistar Kyoto rats. The histological results demonstrated that the scaffolds induced granulation tissue growth, collagen deposition and epithelial tissue remodeling. The wound-healing markers showed no difference in scaffolds when compared with the positive control. Activities of antioxidant enzymes were decreased concerning the positive and negative control. The findings suggest that the scaffolds contributed to the granulation tissue formation and the early collagen deposition, maintaining an anti-inflammatory microenvironment.
... Fibrin can be combined with agarose in the fabrication of bioartificial tissues, since this combination of fibrin and agarose demonstrated to provide the tissue with adequate biomechanical properties, including the Young modulus, stress at fracture and traction deformation, and permits its use as scaffolds (Ionescu et al., 2020). Several preclinical studies have successfully demonstrated the application of fibrin-agarose hydrogels in the repair of damaged human organs such as the cornea (Alaminos et al., 2006), skin (Carriel et al., 2012), nerves (Chato-Astrain et al., 2018), oral mucosa (Fernández-Valadés-Gámez et al., 2016) and cartilage (García-Martínez et al., 2017). These positive results have paved the way for the clinical translation of two bioartificial tissues, which are currently used for severe skin burns (Egea-Guerrero et al., 2019) and corneal ulcers (Rico-Sánchez et al., 2019). ...
Fibrin is widely used for tissue engineering applications. The use of blood derivatives, however, carries a high risk of transmission of infectious agents, necessitating the application of pathogen reduction technology (PRT). The impact of this process on the structural and biomechanical properties of the final products is unknown. We used normal plasma (PLc) and plasma inactivated by riboflavin and ultraviolet light exposure (PLi) to manufacture nanostructured cellularized fibrin-agarose hydrogels (NFAHs), and then compared their structural and biomechanical properties. We also measured functional protein C, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and coagulation factors [fibrinogen, Factor (F) V, FVIII, FX, FXI, FXIII] in plasma samples before and after inactivation. The use of PLi to manufacture cellularized NFAHs increased the interfibrillar spacing and modified their biomechanical properties as compared with cellularized NFAH manufactured with PLc. PLi was also associated with a significant reduction in functional protein C, FV, FX, and FXI, and an increase in the international normalized ratio (derived from the PT), APTT, and TT. Our findings demonstrate that the use of PRT for fibrin-agarose bioartificial tissue manufacturing does not adequately preserve the structural and biomechanical properties of the product. Further investigations into PRT-induced changes are warranted to determine the applications of NFAH manufactured with inactivated plasma as a medicinal product.
... Similar to unadulterated GAGs, fibrin-based hydrogels are mechanically weak and often exhibit shrinkage when used independently. Mixing fibrin hydrogels with agarose [177] or modified HA [ 129 , 178 , 179 ] allows a balance of biocompatibility and structural stability. Notably, Stark and co-workers found that polymerizing fibrin hydrogels within fibrous, esterified HA scaffold formed a stable FTSE capable of long-term culture with improved epidermal homeostasis [ 129 , 178 ]. ...
In vitro three-dimensional (3D) skin tissue models are critical tools in advancing our understanding of ba- sic skin physiology and function as well as in specific applications such as toxicity testing of dermatolog- ical compounds. However, the utilization of such skin models is often limited by the structural instability of the construct, lack of physiologically relevant features and weak barrier function. In this review, we highlight the current research effort s in hydrogel biomaterial selection and scaffold design that allow for maturation of engineered skin in vitro , with special emphasis on matured full-thickness (including epi- dermal and dermal compartments) skin. The different types of scaffold biomaterials, broadly categorized as natural, synthetic, or composite will also be discussed. At the same time, we will outline strategies for next-generation biomimetic skin templates incorporating skin appendages or perfusion systems that can more closely reflect the native skin environment.
... In previous models of bioengineered skin based on clotted plasma, coagulation is obtained by recalcification which usually takes between 15-30 min, is temperature dependent (it does not occur at temperatures <37 • C) [13,17,29,30] and constitutes solely the dermal layer. In a secondary step, keratinocytes need to be cultured over the fibroblasts-containing fibrin dermal equivalent, covered with culture medium and cultured for several days prior to its use [13,17]. ...
The skin is the largest organ in the human body, comprising the main barrier against the environment. When the skin loses its integrity, it is critical to replace it to prevent water loss and the proliferation of opportunistic infections. For more than 40 years, tissue-engineered skin grafts have been based on the in vitro culture of keratinocytes over different scaffolds, requiring between 3 to 4 weeks of tissue culture before being used clinically. In this study, we describe the development of a polymerizable skin hydrogel consisting of keratinocytes and fibroblast entrapped within a fibrin scaffold. We histologically characterized the construct and evaluated its use on an in vivo wound healing model of skin damage. Our results indicate that the proposed methodology can be used to effectively regenerate skin wounds, avoiding the secondary in vitro culture steps and thus, shortening the time needed until transplantation in comparison with other bilayer skin models. This is achievable due to the instant polymerization of the keratinocytes and fibroblast combination that allows a direct application on the wound. We suggest that the polymerizable skin hydrogel is an inexpensive, easy and rapid treatment that could be transferred into clinical practice in order to improve the treatment of skin wounds.
... The time spent at the air-liquid interface during our study is similar to the time needed for the epidermis to completely form in NHS, a duration that can also be found in other studies using different types of dermal compartments [33,34]. Some models can be found in the literature, however, where culture conditions were adjusted for a shorter duration at the air-liquid interface of 14 to 17 days [35][36][37][38]. ...
Healthy skin moLEdels produced by tissue-engineering often present a suboptimal skin barrier function as compared with normal human skin. Moreover, skin substitutes reconstructed according to the self-assembly method were found to be deficient in polyunsaturated fatty acids (PUFAs). Therefore, in this study, we investigated the effects of a supplementation of the culture media with docosahexaenoic acid (DHA) on the barrier function of skin substitutes. To this end, 10 μM DHA-supplemented skin substitutes were produced (n = 3), analyzed, and compared with controls (substitutes without supplementation). A Franz cell diffusion system, followed by ultra-performance liquid chromatography, was used to perform a skin permeability to testosterone assay. We then used gas chromatography to quantify the PUFAs found in the epidermal phospholipid fraction of the skin substitutes, which showed successful DHA incorporation. The permeability to testosterone was decreased following DHA supplementation and the lipid profile was improved. Differences in the expression of the tight junction (TJ) proteins claudin-1, claudin-4, occludin, and TJ protein-1 were observed, principally a significant increase in claudin-1 expression, which was furthermore confirmed by Western blot analyses. In conclusion, these results confirm that the DHA supplementation of cell culture media modulates different aspects of skin barrier function in vitro and reflects the importance of n-3 PUFAs regarding the lipid metabolism in keratinocytes.
... Features as biocompatibility, thermo-reversible gelation, and hydrophilicity make agarose ideal for cellular studies and gel electrophoresis. It has been broadly considered in TE, namely as cartilage and bone regeneration, wound healing, artificial pancreas, angiogenesis, neurogenesis, and spermatogenesis (Zarrintaj et al., 2018, Carriel et al., 2012, Carriel et al., 2013. Agarose is composed by (1→3)-β-D-galactopyranose-(1→4)-3,6-anhydro-β-L-galactopyranose unit, with molecular mass of ~120 kDa. ...
Recent advances in tissue engineering (TE) have shown that combining biomaterials, cells, and bioactive molecules are important to promote the regeneration of damaged tissues or as therapeutic systems. Porous three-dimensional structures with interconnected pore network are capable to guide the development of functional engineered tissues and afford the temporary mechanical support during in vivo implantation. Natural polymeric biomaterials have been used for 3D scaffolds fabrication, owing to its ability of mimicking the extracellular matrix, biocompatibility, biodegradability, and no immunological reactions, during tissue regeneration or wound healing. This chapter provides the most promising natural biopolymers—proteins, polysaccharides, and marine origin polymers—their properties and considerations specifically for scaffolding TE purposes. The recent works related to micro- and nano-particles, 3D porous scaffolds and hydrogel-based scaffolds as biomimetic strategies for TE and regeneration are also presented and discussed herein
... Several strategies have been proposed to overcome this limitation such as modifying in vitro culture conditions or combining polymers to engineer scaffolds with better mechanical properties. [26][27][28] For more details, we suggest the reader to see Refs. 9 and 29. ...
[This corrects the article DOI: 10.1063/5.0046376.].
... In general, most of the TESSs have demonstrated the establishment of a relatively well-stratified epithelium composed by viable and functional keratinocytes layers and a new-formed basal membrane. Some of these models achieve a closely similar histological pattern than a healthy human epidermis [94,97,136,137]. In order to specifically determine the expression of epithelial, dermal or ECM molecules in TESSs, immunohistochemical procedures are used. ...
Reconstruction of skin defects is often a challenging effort due to the currently limited reconstructive options. In this sense, tissue engineering has emerged as a possible alternative to replace or repair diseased or damaged tissues from the patient’s own cells. A substantial number of tissue-engineered skin substitutes (TESSs) have been conceived and evaluated in vitro and in vivo showing promising results in the preclinical stage. However, only a few constructs have been used in the clinic. The lack of standardization in evaluation methods employed may in part be responsible for this discrepancy. This review covers the most well-known and up-to-date methods for evaluating the optimization of new TESSs and orientative guidelines for the evaluation of TESSs are proposed.
... After in vitro characterization, we determined the vascularization potential of each cell type in a three-dimensional model of bioartificial oral mucosa stroma grafted in vivo on athymic mice. This model was previously used by our group for the in vivo evaluation of different models of human oral mucosa due to the incapability of nude mice to reject human xenografts.22,41,16 Results confirmed that HOM was fully biocompatible in vivo, and animals grafted with the different types of HOM were free from any significant complications or side effects. ...
Objective
The aim of this study was to generate novel models of bioartificial human oral mucosa with increased vascularization potential for future use as an advanced therapies medicinal product, by using different vascular and mesenchymal stem cell sources.
Background
Oral mucosa substitutes could contribute to the clinical treatment of complex diseases affecting the oral cavity. Although several models of artificial oral mucosa have been described, biointegration is a major issue that could be favored by the generation of novel substitutes with increased vascularization potential once grafted in vivo.
Methods
Three types of mesenchymal stem cells (MSCs) were obtained from adipose tissue, bone marrow, and dental pulp, and their in vitro potential was evaluated by inducing differentiation to the endothelial lineage using conditioning media. Then, 3D models of human artificial oral mucosa were generated using biocompatible fibrin-agarose biomaterials combined with human oral mucosa fibroblasts and each type of MSC before and after induction to the endothelial lineage, using human umbilical vein endothelial cells (HUVEC) as controls. The vascularization potential of each oral mucosa substitute was assessed in vitro and in vivo in nude mice.
Results
In vitro induction of MSCs kept in culture was able to increase the expression of VEGF, CD31, and vWF endothelial markers, especially in bone marrow and dental pulp-MSCs, and numerous proteins with a role in vasculogenesis become overexpressed. Then, in vivo grafting resulted in a significant increase in blood vessels formation at the interface area between the graft and the host tissues, with significantly positive expression of VEGF, CD31, vWF, and CD34 as compared to negative controls, especially when pre-differentiated MSCs derived from bone marrow and dental pulp were used. In addition, a significantly higher number of cells committed to the endothelial lineage expressing the same endothelial markers were found within the bioartificial tissue.
Conclusion
Our results suggest that the use of pre-differentiated MSCs could contribute to a rapid generation of a vascular network that may favor in vivo biointegration of bioengineered human oral mucosa substitutes.
... Another strategy is the PEGylation of the fibrinogen to prepare more consistent and stable fibrinbased hydrogels to prevent contraction, among other issues [39][40][41][42][43]. Alternatively, the preparation of composite materials consisting of a hyaluronic acid (HA)-derived solid scaffold filled with commercial fibrin hydrogel or the combination of a plasma-derived fibrin network with other natural polymer networks, such as agarose, has been utilized to prevent contraction, improve the mechanical properties and prepare in vitro skin models for long-term culture [44,45]. ...
Human plasma-derived bilayered skin substitutes have been successfully used by our group in different skin tissue engineering applications. However, several issues associated with their poor mechanical properties were observed, and they often resulted in rapid contraction and degradation. In this sense, hydrogels composed of plasma-derived fibrin and thiolated-hyaluronic acid (HA-SH, 0.05–0.2% w/v) crosslinked with poly(ethylene glycol) diacrylate (PEGDA, 2:1, 6:1, 10:1 and 14:1 mol of thiol to moles of acrylate) were developed to reduce the shrinking rates and enhance the mechanical properties of the plasma-derived matrices. Plasma/HA-SH-PEGDA hydrogels showed a decrease in the contraction behaviour ranging from 5% to 25% and an increase in Young's modulus. Furthermore, the results showed that a minimal amount of the added HA-SH was able to escape the plasma/HA-SH-PEGDA hydrogels after incubation in PBS. The results showed that the increase in rigidity of the matrices as well as the absence of adhesion cellular moieties in the second network of HA-SH/PEGDA, resulted in a decrease in contraction in the presence of the encapsulated primary human fibroblasts (hFBs), which may have been related to an overall decrease in proliferation of hFBs found for all hydrogels after 7 days with respect to the plasma control. The metabolic activity of hFB returned to the control levels at 14 days except for the 2:1 PEGDA crosslinking ratio. The metabolic activity of primary human keratinocytes (hKCs) seeded on the hydrogels showed a decrease when high amounts of HA-SH and PEGDA crosslinker were incorporated. Organotypic skins formed in vitro after 21 days with plasma/HA-SH-PEGDA hydrogels with an HA content of 0.05% w/v and a 2:1 crosslinking ratio were up to three times thicker than the plasma controls, evidencing a reduction in contraction, while they also showed better and more homogeneous keratin 10 (K10) expression in the supra-basal layer of the epidermis. Furthermore, filaggrin expression showed the formation of an enhanced stratum corneum for the constructs containing HA. These promising results indicate the potential of using these biomimetic hydrogels as in vitro skin models for pharmaceutical products and cosmetics and future work will elucidate their potential functionality for clinical treatment.
... However, current drawbacks of these types of hydrogel lies not only in their moderate tensile strength, but they also suffer a rapid degradation and contraction over time, limiting their reproducibility and lifespan [18][19][20]. PEGylation of the fibrinogen contained in human plasma or the combination with natural polymeric networks, such as alginate or agarose [21][22][23][24][25][26], have been utilized to overcome the aforementioned limitations. Another promising strategy could be the preparation of interpenetrating polymer network (IPN) hydrogels based on plasma-derived fibrin and extracellular matrix (ECM) components. ...
Dermo-epidermal equivalents based on plasma-derived fibrin hydrogels have been extensively studied for skin engineering. However, they showed rapid degradation and contraction over time and low mechanical properties which limit their reproducibility and lifespan. In order to achieve better mechanical properties, elasticity and biological properties, we incorporated a elastin-like recombinamer (ELR) network, based on two types of ELR, one modified with azide (SKS-N3) and other with cyclooctyne (SKS-Cyclo) chemical groups at molar ratio 1:1 at three different SKS (serine-lysine-serine sequence) concentrations (1, 3, and 5 wt.%), into plasma-derived fibrin hydrogels. Our results showed a decrease in gelation time and contraction, both in the absence and presence of the encapsulated human primary fibroblasts (hFBs), higher mechanical properties and increase in elasticity when SKSs content is equal or higher than 3%. However, hFBs proliferation showed an improvement when the lowest SKS content (1 wt.%) was used but started decreasing when increasing SKS concentration at day 14 with respect to the plasma control. Proliferation of human primary keratinocytes (hKCs) seeded on top of the hybrid-plasma hydrogels containing 1 and 3% of SKS showed no differences to plasma control and an increase in hKCs proliferation was observed for hybrid-plasma hydrogels containing 5 wt.% of SKS. These promising results showed the need to achieve a balance between the reduced contraction, the better mechanical properties and biological properties and indicate the potential of using this type of hydrogel as a testing platform for pharmaceutical products and cosmetics, and future work will elucidate their potential.
... However, we found several persisting issues associated with the low final concentration of fibrin (1.2 mg/mL) used in the plasma derived-fibrin hydrogels when they are placed in transwell inserts for in vitro applications: (1) their height is reduced by 30% during the first 24 h and by 70% after 21 days in culture [17]; and (2) they suffer rapid degradation due to the skin cells present in the culture, limiting their lifespan (usually to 17 days) [15,44]. Several strategies have been proposed to overcome the limitations of plasma-derived fibrin-based hydrogels in skin tissue engineering, for example, combining fibrin (blood plasma-derived) with other molecules such as PEG or agarose polymers [40][41][42]50,51] or the use of highly concentrated commercial fibrinogen. In our experience, the use of commercial fibrinogen in organotypic skin cultures produced worse keratinocyte proliferative and differentiation behavior in comparison with PPP-hydrogel cultures. ...
Human plasma-derived bilayered skin substitutes were successfully used by our group to produce human-based in vitro skin models for toxicity, cosmetic, and pharmaceutical testing. However, mechanical weakness, which causes the plasma-derived fibrin matrices to contract significantly, led us to attempt to improve their stability. In this work, we studied whether an increase in fibrin concentration from 1.2 to 2.4 mg/mL (which is the useful fibrinogen concentration range that can be obtained from plasma) improves the matrix and, hence, the performance of the in vitro skin cultures. The results show that this increase in fibrin concentration indeed affected the mechanical properties by doubling the elastic moduli and the maximum load. A structural analysis indicated a decreased porosity for the 2.4 mg/mL hydrogels, which can help explain this mechanical behavior. The contraction was clearly reduced for the 2.4 mg/mL matrices, which also allowed for the growth and proliferation of primary fibroblasts and keratinocytes, although at a somewhat reduced rate compared to the 1.2 mg/mL gels. Finally, both concentrations of fibrin gave rise to organotypic skin cultures with a fully differentiated epidermis, although their lifespans were longer (25–35%) in cultures with more concentrated matrices, which improves their usefulness. These systems will allow the generation of much better in vitro skin models for the testing of drugs, cosmetics and chemicals, or even to “personalized” skin for the diagnosis or determination of the most effective treatment possible.
... Several strategies have been proposed to overcome this limitation such as modifying in vitro culture conditions or combining polymers to engineer scaffolds with better mechanical properties. [26][27][28] For more details, we suggest the reader to see Refs. 9 and 29. ...
Over the last few years, several advances have been made toward the development and production of in vitro human skin models for the analysis and testing of cosmetic and pharmaceutical products. However, these skin models are cultured under static conditions that make them unable to accurately represent normal human physiology. Recent interest has focused on the generation of in vitro 3D vascularized skin models with dynamic perfusion and microfluidic devices known as skin-on-a-chip. These platforms have been widely described in the literature as good candidates for tissue modeling, as they enable a more physiological transport of nutrients and permit a high-throughput and less expensive evaluation of drug candidates in terms of toxicity, efficacy, and delivery. In this Perspective, recent advances in these novel platforms for the generation of human skin models under dynamic conditions for in vitro testing are reported. Advances in vascularized human skin equivalents (HSEs), transferred skin-on-a-chip (introduction of a skin biopsy or a HSE in the chip), and in situ skin-on-a-chip (generation of the skin model directly in the chip) are critically reviewed, and currently used methods for the introduction of skin cells in the microfluidic chips are discussed. An outlook on current applications and future directions in this field of research are also presented.
... As seen in the chronological review, a combination of dermal and epidermal components is needed to make an effective skin substitute [32][33][34]. Cuono and his colleagues demonstrated the fact that it is essential to have a dermal layer as a bed to put on the epidermal layer [15]. ...
The skin plays an important role in the maintenance of the human's body physiological homeostasis. It acts as a coverage that protects against infective microorganism or biomechanical impacts. Skin is also implied in thermal regulation and fluid balance. However, skin can suffer several damages that impede normal wound-healing responses and lead to chronic wounds. Since the use of autografts, allografts, and xenografts present source limitations and intense rejection associated problems, bioengineered artificial skin substitutes (BASS) have emerged as a promising solution to address these problems. Despite this, currently available skin substitutes have many drawbacks, and an ideal skin substitute has not been developed yet. The advances that have been produced on tissue engineering techniques have enabled improving and developing new arising skin substitutes. The aim of this review is to outline these advances, including commercially available skin substitutes, to finally focus on future tissue engineering perspectives leading to the creation of autologous prevascularized skin equivalents with a hypodermal-like layer to achieve an exemplary skin substitute that fulfills all the biological characteristics of native skin and contributes to wound healing.
... This biomaterial has been successfully used in several biomedical applications (such as skin, cornea, cartilage, sclera, and nerve tissue engineering) which confirmed its high biocompatibility and proregenerative properties ex vivo and in vivo. 13,14,16,22,34 In our study, tendon-derived fibroblasts showed active cell adhesion, high metabolic activity and low DNA-release in all the analyzed experimental conditions which were compared with the control groups. These new results are in line with our previous study in which NFAH and GP-NFAH supported fibroblast function and proliferation ex vivo. ...
Aims:
Achilles tendon injuries are a frequent problem in orthopaedic surgery due to their limited healing capacity and the controversy surrounding surgical treatment. In recent years, tissue engineering research has focused on the development of biomaterials to improve this healing process. The aim of this study was to analyze the effect of tendon augmentation with a nanostructured fibrin-agarose hydrogel (NFAH) or genipin cross-linked nanostructured fibrin-agarose hydrogel (GP-NFAH), on the healing process of the Achilles tendon in rats.
Methods:
NFAH, GP-NFAH, and MatriDerm (control) scaffolds were generated (five in each group). A biomechanical and cell-biomaterial-interaction characterization of these biomaterials was then performed: Live/Dead Cell Viability Assay, water-soluble tetrazolium salt-1 (WST-1) assay, and DNA-released after 48 hours. Additionally, a complete section of the left Achilles tendon was made in 24 Wistar rats. Animals were separated into four treatment groups (six in each group): direct repair (Control), tendon repair with MatriDerm, or NFAH, or GP-NFAH. Animals were euthanized for further histological analyses after four or eight weeks post-surgery. The Achilles tendons were harvested and a histopathological analysis was performed.
Results:
Tensile test revealed that NFAH and GP-NFAH had significantly higher overall biomechanical properties compared with MatriDerm. Moreover, biological studies confirmed a high cell viability in all biomaterials, especially in NFAH. In addition, in vivo evaluation of repaired tendons using biomaterials (NFAH, GP-NFAH, and MatriDerm) resulted in better organization of the collagen fibres and cell alignment without clinical complications than direct repair, with a better histological score in GP-NFAH.
Conclusion:
In this animal model we demonstrated that NFAH and GP-NFAH had the potential to improve tendon healing following a surgical repair. However, future studies are needed to determine the clinical usefulness of these engineered strategies. Cite this article: Bone Joint J 2020;102-B(8):1095-1106.
In developing three‐dimensional (3D) human skin equivalents (HSEs), preventing dermis and epidermis layer distortion due to the contraction of hydrogels by fibroblasts is a challenging issue. Previously, a fabrication method of HSEs was tested using a modified solid scaffold or a hydrogel matrix in combination with the natural polymer coated onto the tissue culture surface, but the obtained HSEs exhibited skin layer contraction and loss of the skin integrity and barrier functions. In this study, we investigated the method of HSE fabrication that enhances the stability of the skin model by using surface plasma treatment. The results showed that plasma treatment of the tissue culture surface prevented dermal layer shrinkage of HSEs, in contrast to the HSE fabrication using fibronectin coating. The HSEs from plasma‐treated surface showed significantly higher transepithelial electrical resistance compared to the fibronectin‐coated model. They also expressed markers of epidermal differentiation (keratin 10, keratin 14 and loricrin), epidermal tight junctions (claudin 1 and zonula occludens‐1), and extracellular matrix proteins (collagen IV), and exhibited morphological characteristics of the primary human skins. Taken together, the use of plasma surface treatment significantly improves the stability of 3D HSEs with well‐defined dermis and epidermis layers and enhanced skin integrity and the barrier functions.
A previously developed fibrin‐agarose skin model—UGRSKIN—showed promising clinical results in severely burnt patients. To determine the histological parameters associated to the biocompatibility and therapeutic effects of this model, we carried out a comprehensive structural and ultrastructural study of UGRSKIN grafted in severely burnt patients after 3 months of follow‐up. The grafted epidermis was analogue to native human skin from day 30th onward, revealing well‐structured strata with well‐differentiated keratinocytes expressing CK5, CK8, CK10, claudin, plakoglobin, filaggrin, and involucrin in a similar way to controls, suggesting that the epidermis was able to mature and differentiate very early. Melanocytes and Langerhans cells were found from day 30th onward, together with a basement membrane, abundant hemidesmosomes and lack of rete ridges. At the dermal layer, we found an interface between the grafted skin and the host tissue at day 30th, which tended to disappear with time. The grafted superficial dermis showed a progressive increase in properly‐oriented collagen fibers, elastic fibers and proteoglycans, including decorin, similarly to control dermis at day 60‐90th of in vivo follow‐up. Blood vessels determined by CD31 and SMA expression were more abundant in grafted skin than controls, whereas lymphatic vessels were more abundant at day 90th. These results contribute to shed light on the histological parameters associated to biocompatibility and therapeutic effect of the UGRSKIN model grafted in patients and demonstrate that the bioengineered skin grafted in patients is able to mature and differentiate very early at the epithelial level and after 60–90 days at the dermal level.
In this work we employ molecular dynamics simulations to investigate the first-order-reversal-curve distribution and switching-field distribution of magnetic elastomers. We model magnetic elastomers in a bead-spring approximation with permanently magnetized spherical particles of two different sizes. We find that a different fractional composition of particles affects the magnetic properties of elastomers obtained as a result. We prove that the hysteresis of the elastomer can be attributed to the broad energy landscape with multiple shallow minima and caused by dipolar interactions.
The dynamic nature of the wound healing is a well-studied process. With the rapid evolution of healthcare science, efforts are being made to design newer therapies, along with advancement in the available technologies. In this regard, a detailed knowledge about the molecular mechanisms underlying the healing process is necessary to develop a more effective and targeted therapeutic method. In addition to improve the therapeutic techniques, various wound healing models also need to be designed and studied for understanding the associated molecular intricacies of each phase of the healing process. Novel technologies are being combined with the long-practiced traditional therapeutic methods with the object of achieving a synergistic effect for faster healing with minimal scarring. With technological development, non-invasive wound assessment methods are being implemented by utilizing various imaging techniques and electromagnetic radiations, to minimize painful invasive wound analysis. Smart devices are also been developed with an aim to monitor the healing process in real time, which provides the ability to modulate treatment procedures according to the rate of healing. This chapter aims to give a brief overview on the emerging therapeutic methods which are being developed to aid in wound management strategies. An overview of the different invasive and non-invasive wound assessment and monitoring methods along with mathematically designed wound assessment models is also discussed in a nutshell. This would provide an idea about the possible areas for development on the currently available strategies for improving the healthcare and well-being of individuals.
A single biomaterial is disadvantageous for constructing skin in vitro, so a mixed biomaterial is more conducive to skin research. In this study, agarose‐chitosan scaffolds with a final concentration of 4% were constructed by freeze‐drying, in which the concentration ratios of agarose to chitosan were 1:3, 2:2, and 3:1. The scaffolds were coated with a 3 mg/ml collagen solution and the mechanical properties were evaluated by studying density, porosity, swelling rate, and degradation rate. The results demonstrated that the agarose‐chitosan scaffolds were porous, with porosity reaching 93%. Their densities ranged from 0.1 to 0.16 g/cm3. Analysis of Young's modulus showed that the mechanical properties of the agarose‐chitosan scaffolds were significantly enhanced when the agarose content in the agarose‐chitosan scaffolds was increased. Moreover, the density and Young's modulus of the agarose‐chitosan scaffolds of different concentration ratios were significantly different (p < 0.01). These scaffolds can withstand a certain amount of external pressure, such as that of human skin, making them more suitable for further skin replacement research. In addition, the results of MTT and immunofluorescence staining showed that the collagen‐coated agarose‐chitosan scaffolds were conducive to keratinocyte proliferation and differentiation. The MTT results revealed significant differences between the agarose‐chitosan scaffolds coated with collagen and the agarose‐chitosan scaffolds without collagen (p < 0.05). This study provides the potential for in vitro skin research and applications. This article is protected by copyright. All rights reserved
In vitro three-dimensional (3D) skin tissue models are critical tools in advancing our understanding of basic skin physiology and function as well as in specific applications such as toxicity testing of dermatological compounds. However, the utilization of such skin models is often limited by the structural instability of the construct, lack of physiologically relevant features and weak barrier function. In this review, we highlight the current research efforts in hydrogel biomaterial selection and scaffold design that allow for maturation of engineered skin in vitro, with special emphasis on matured full-thickness (including epidermal and dermal compartments) skin. The different types of scaffold biomaterials, broadly categorized as natural, synthetic, or composite will also be discussed. At the same time, we will outline strategies for next-generation biomimetic skin templates incorporating skin appendages or perfusion systems that can more closely reflect the native skin environment.
Statement of Significance
In vitro 3D human skin models are critical tools in advancing our understanding of skin physiology and function. Many of the existing reconstructed models are limited in terms of structure and complexity, thus failing to recapitulate native human skin. In order to address this, hydrogels have been identified as useful scaffold materials for fabricating the dermal equivalent of 3D skin models, allowing for greater flexibility and control in scaffold properties and cellular incorporation. This review aims to provide a critical discussion of the biomaterial selection and design strategies in the construction of hydrogel-based full-thickness skin equivalents. At the same time, we will offer insights into the future developments and technological advances which can accelerate the progress in this field.
The histological structure of human epithelial tissue is complex, but all epithelia share three major features: cohesion, polarity and attachment. These functions are mainly achieved by the presence of specialized structures such as intercellular junctions, polarity protein complexes and basement membranes. In the present review, we have analyzed the presence of each of these structures in several groups of animals that are considered to be at the base of the animal evolution tree. Interestingly, these characters seem to have evolved independently, and a careful histological and structural analysis of the phylogenetic tree shows different groups of animals in which epithelia are absent and groups in which cells show only some of the specialized structures found in differentiated epithelia. These findings could contribute to understand how epithelial tissues evolved and determine their current protective functions.
Advances in skin tissue engineering have promoted the development of artificial skin substitutes to treat large burns and other major skin loss conditions. However, one of the main drawbacks to bioengineered skin is the need to obtain a large amount of viable epithelial cells in short periods of time, making the skin biofabrication process challenging and slow. Enhancing skin epithelial cell cultures by using mesenchymal stem cells secretome can favor the scalability of manufacturing processes for bioengineered skin. The effects of three different types of secretome derived from human mesenchymal stem cells, e.g. hADSC-s (adipose cells), hDPSC-s (dental pulp) and hWJSC-s (umbilical cord), were evaluated on cultured skin epithelial cells during 24, 48, 72 and 120 h to determine the potential of this product to enhance cell proliferation and improve biofabrication strategies for tissue engineering. Then, secretomes were applied in vivo in preliminary analyses carried out on Wistar rats. Results showed that the use of secretomes derived from mesenchymal stem cells enhanced currently available cell culture protocols. Secretome was associated with increased viability, proliferation and migration of human skin epithelial cells, with hDPSC-s and hWJSC-s yielding greater inductive effects than hADSC-s. Animals treated with hWJSC-s and especially, hDPSC-s tended to show enhanced wound healing in vivo with no detectable side effects. Mesenchymal stem cells derived secretomes could be considered as a promising approach to cell-free therapy able to improve skin wound healing and regeneration.
In the present work, we evaluated the potential of maslinic acid (MA) to improve currently available keratinocyte culture methods for use in skin tissue engineering. Results showed that MA can increase cell proliferation and WST-1 activity of human keratinocytes after 24, 48, and 72 h, especially at the concentration of 5 μg/ml, without affecting cell viability. This effect was associated to a significant increase of KI-67 protein expression and upregulation of several genes associated to cell proliferation (PCNA) and differentiation (cytokeratins, intercellular junctions and basement membrane related genes). When human keratinocytes were isolated from skin biopsies, we found that MA at the concentration of 5 μg/ml significantly increased the efficiency of the explant and the cell dissociation methods. These results revealed the positive effects of MA to optimize human keratinocyte culture protocols for use in skin tissue engineering.
Polyunsaturated fatty acids (PUFAs) play an important role in the establishment and the maintenance of the skin barrier function. However, the impact of their derived lipid mediators remains unclear. Skin substitutes were engineered according to the self-assembly method with a culture medium supplemented with 10 μM of both α-linolenic acid (ALA) and linoleic acid (LA). We show that supplementation with ALA and LA decreased testosterone absorption through a tissue-engineered reconstructed skin model, thus indicating an improved skin barrier function following supplementation. The exogenously provided fatty acids were incorporated into the phospholipid and triglyceride fractions of the skin substitutes. Indeed, the dual supplementation increased the levels of eicosapentaenoic acid (15-fold), docosapentaenoic acid (DPA) (3-fold), and LA (1.5-fold) in the epidermal phospholipids while it increased the levels of ALA (>20-fold), DPA (3-fold) and LA (1.5-fold) in the epidermal triglycerides. The bioactive lipid mediator profile of the skin substitutes, including prostaglandins, hydroxy-fatty acids, N-acylethanolamines and monoacylglycerols, was next analyzed using liquid chromatography-tandem mass spectrometry. The lipid supplementation further modulated bioactive lipid mediator levels of the reconstructed skin substitutes, leading to a lipid mediator profile more representative of the one found in normal human skin. These findings show that an optimized supply of PUFAs via culture media is essential for the establishment of improved barrier function in vitro.
Statement of significance
: Supplementation of the culture medium with 10 μM of both α-linolenic acid (ALA) and linoleic acid (LA) improved the skin barrier function of a tissue-engineered skin model. The exogenously provided fatty acids were incorporated into the phospholipid and triglyceride fractions of the skin substitutes and further modulated bioactive lipid mediator levels, including prostaglandins, hydroxy-fatty acids, N-acylethanolamines and monoacylglycerols. These findings highlight the important role of ALA and LA in skin homeostasis and show that an optimized supply of polyunsaturated fatty acids via culture media is essential for the establishment of improved barrier function in vitro.
In addition to proteins and nucleic acids, polysaccharides are an important type of biomacromolecule widely distributed in plants, animals, and microorganisms. Polysaccharides are considered promising materials due to their significant bioactivities, natural abundance, immunoactivity and chemical modifiability. Due to the similarities of the biochemical properties of polysaccharides and the extracellular matrix of human bodies, polysaccharides are increasingly recognized and accepted. Furthermore, the degradation behavior of these macromolecules is generally nontoxic. Certain delicate properties, such as remarkable mechanical properties and tunable tissue response, can be obtained by modifying the functional groups on the surface of polysaccharide molecules. The applications of polysaccharide-based biomaterials in the tissue engineering field have been growing intensively in recent decades, e.g., bone/cartilage regeneration, cardiac regeneration, neural regeneration, and skin regeneration. This review summarizes the main essential properties of polysaccharides, including their chemical properties, crosslinking mechanisms, and biological properties, and focuses on the association between their structures and properties. The recent progress in polysaccharide-based biomaterials in various tissue engineering applications is reviewed, and the prospects for future studies are addressed as well. We intend this review to offer a comprehensive understanding of and inspiration for the research and development of polysaccharide-based materials in tissue engineering.
This review aims to be an update of Bioengineered Artificial Skin Substitutes (BASS) applications. At the first moment, they were created as an attempt to replace native skin grafts transplantation. Nowadays, these in vitro models have been increasing and widening their application areas, becoming important tools for research. This study is focus on the ability to design in vitro BASS which have been demonstrated to be appropriate to develop new products in the cosmetic and pharmacology industry. Allowing to go deeper into the skin disease research, and to analyze the effects provoked by environmental stressful agents. The importance of BASS to replace animal experimentation is also highlighted. Furthermore, the BASS validation parameters approved by the OECD (Organisation for Economic Cooperation and Development) are also analyzed. This report presents an overview of the skin models applicable to skin research along with their design methods. Finally, the potential and limitations of the currently available BASS to supply the demands for disease modeling and pharmaceutical screening are discussed.
Novel artificial tissues with potential usefulness in local-based therapies have been generated by tissue engineering using magnetic-responsive nanoparticles (MNPs). In this study, we performed a comprehensive in vivo characterization of bioengineered magnetic fibrin-agarose tissue-like biomaterials. First, in vitro analyses were performed and the cytocompatibility of MNPs was demonstrated. Then, bioartificial tissues were generated and subcutaneously implanted in Wistar rats and their biodistribution, biocompatibility and functionality were analysed at the morphological, histological, haematological and biochemical levels as compared to injected MNPs. Magnetic Resonance Image (MRI), histology and magnetometry confirmed the presence of MNPs restricted to the grafting area after 12 weeks. Histologically, we found a local initial inflammatory response that decreased with time. Structural, ultrastructural, haematological and biochemical analyses of vital organs showed absence of damage or failure. This study demonstrated that the novel magnetic tissue-like biomaterials with improved biomechanical properties fulfil the biosafety and biocompatibility requirements for future clinical use and support the use of these biomaterials as an alternative delivery route for magnetic nanoparticles.
Over the last quarter of a century there has been an emergence of a tissue engineering industry, one that has now evolved into the broader area of regenerative medicine. There have been 'ups and downs' in this industry; however, it now appears to be on a track that may be described as 'back to the future'. The latest data indicate that for 2007 the private sector activity in the world for this industry is approaching $2.5 billion, with 167 companies/business units and more than 6000 employee full time equivalents. Although small compared with the medical device and also the pharmaceutical industries, these numbers are not insignificant. Thus, there is the indication that this industry, and the related technology, may still achieve its potential and address the needs of millions of patients worldwide, in particular those with needs that currently are unmet.
Situations where normal autografts cannot be used to replace damaged skin often lead to a greater risk of mortality, prolonged hospital stay and increased expenditure for the National Health Service. There is a substantial need for tissue-engineered skin bioconstructs and research is active in this field. Significant progress has been made over the years in the development and clinical use of bioengineered components of the various skin layers. Off-the-shelf availability of such constructs, or production of sufficient quantities of biological materials to aid rapid wound closure, are often the only means to help patients with major skin loss. The aim of this review is to describe those materials already commercially available for clinical use as well as to give a short insight to those under development. It seeks to provide skin scientists/tissue engineers with the information required to not only develop in vitro models of skin, but to move closer to achieving the ultimate goal of an off-the-shelf, complete full-thickness skin replacement.
Keratins 1 (K1) and 10 (K10) are the predominant cytoskeletal intermediate filaments of epidermal cells during transition from the proliferative to the terminal differentiation stage. In situ, formation of the K1/K10 intermediate filament network occurs in the cytoplasm of cells with a preexisting cytoskeleton composed of keratins 5 and 14. To define cytoskeletal interactions permissive for formation of the K1/K10 filamentous network, active copies of mouse K1 and K10 genes were introduced into fibroblasts (NIH 3T3) which do not normally express these proteins. Transient and stable transfectants, as well as heterokaryons produced by fusions with epithelial cells, were evaluated for expression of K1 and K10 proteins and filament formation using specific antibodies. In contrast to keratin pairs K5/K14 and K8/K18, the K1/K10 pair failed to form an extensive keratin filament network on its own, although small isolated dense K1/K10 filament bundles were observed throughout the cytoplasm by EM. K1 and K10 filaments integrated only into the preexisting K5/K14 network upon fusion of the NIH 3T3 (K1/K10) cells with epithelial cells expressing endogenous K5/K14 or with NIH 3T3 cells which were transfected with active copies of the K5 and K14 genes. When combinations of active recombinant gene constructs for keratins 1, 5, 10, and 14 were tested in transient NIH 3T3 transfections, the most intact cytokeratin network observed by immunofluorescence was formed by the K5/K14 pair. The K1/K14 pair was capable of forming a cytoskeletal network, but the network was poorly developed, and usually perinuclear. Transfection of K10 in combination with K5 or K1 resulted in cytoplasmic agglomerates, but not a cytoskeleton. These results suggest that the formation of the suprabasal cytoskeleton in epidermis is dependent on the preexisting basal cell intermediate filament network. Furthermore, restrictions on filament formation appear to be more stringent for K10 than for K1.
Tissue engineering has created several original and new avenues in the biomedical sciences. There is ongoing progress, but the tissue-engineering field is currently at a crossroads in its evolution; the validity of this technique is well established. Thus, new clinical applications must appear rapidly, within a few years, so that it will have a true impact on patient care. The self-assembly approach of the Laboratoire d’Organogénèse Expérimentale (LOEX) should be at the forefront.
An artificial complete skin (dermis and epidermis) model has been developed in the Tissue engineering unit of the Centro Comunitario de Sangre y Tejidos del Principado de Asturias (CCST) and CIEMAT. This engineered skin has been employed for the treatment of severe epithelial injuries. In this paper, the clinical results obtained with this engineered skin during the last 18 months were evaluated.
(a) Culture: Cells (fibroblasts and keratinocytes) were obtained from biopsies by a double enzymatic digestion. After an expansion period, fibroblasts were seeded in an artificial dermis based on human plasma. Keratinocytes were seeded over this dermal surface. (b) Patients: 20 skin biopsies were processed (13 burned patients, 5 giant nevus, 1 GVHD, 1 neurofibromatosis), which came from different hospitals across the country. About 97,525 cm(2) of engineered skin were cultured.
The engineered skin took in all patients. The take percentage depended on previous pathology (burned patients 10-90%; non-critical patients 70-90%). The epithelization obtained was permanent in all cases. During the follow-up period, epithelial loss, blistering injuries or skin retractions were not observed. The aesthetic and functional results were acceptable.
This artificial skin has demonstrated to be useful for the definitive treatment of patients with severe skin injuries. This work shows that it is possible to produce this prototype in an hospitalarian laboratory and distribute it to different hospitals across the country.
Tissue-engineered skin is now a reality. For patients with extensive full-thickness burns, laboratory expansion of skin cells to achieve barrier function can make the difference between life and death, and it was this acute need that drove the initiation of tissue engineering in the 1980s. A much larger group of patients have ulcers resistant to conventional healing, and treatments using cultured skin cells have been devised to restart the wound-healing process. In the laboratory, the use of tissue-engineered skin provides insight into the behaviour of skin cells in healthy skin and in diseases such as vitiligo, melanoma, psoriasis and blistering disorders.
Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal oral mucosa using enzymatic treatments. Then we determined the viability of the cultured cells by electron probe quantitative X-ray microanalysis, and we demonstrated that most of the cells in the primary cultures were alive and had high K/Na ratios. Once cell viability was determined, we used the cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarose extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and the oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.
Skin graft contraction leading to loss of joint mobility and cosmetic deformity remains a major clinical problem. In this study we used a tissue-engineered model of human skin, based on sterilized human adult dermis seeded with keratinocytes and fibroblasts, which contracts by up to 60% over 28 days in vitro, as a model to investigate the mechanism of skin contraction. Pharmacologic agents modifying collagen synthesis, degradation, and cross-linking were examined for their effect on contraction. Collagen synthesis and degradation were determined using immunoassay techniques. The results show that skin contraction was not dependent on inhibition of collagen synthesis or stimulation of collagen degradation, but was related to collagen remodelling. Thus, reducing dermal pliability with glutaraldehyde inhibited the ability of cells to contract the dermis. So did inhibition of matrix metalloproteinases and inhibition of lysyl oxidase-mediated collagen cross-linking, but not transglutaminase-mediated cross-linking. In summary, this in vitro model of human skin has allowed us to identify specific cross-linking pathways as possible pharmacologic targets for prevention of graft contracture in vivo.
Keratins 1 (K1) and 10 (K10) are the predominant cytoskeletal intermediate filaments of epidermal cells during transition from the proliferative to the terminal differentiation stage. In situ, formation of the K1/K10 intermediate filament network occurs in the cytoplasm of cells with a preexisting cytoskeleton composed of keratins 5 and 14. To define cytoskeletal interactions permissive for formation of the K1/K10 filamentous network, active copies of mouse K1 and K10 genes were introduced into fibroblasts (NIH 3T3) which do not normally express these proteins. Transient and stable transfectants, as well as heterokaryons produced by fusions with epithelial cells, were evaluated for expression of K1 and K10 proteins and filament formation using specific antibodies. In contrast to keratin pairs K5/K14 and K8/K18, the K1/K10 pair failed to form an extensive keratin filament network on its own, although small isolated dense K1/K10 filament bundles were observed throughout the cytoplasm by EM. K1 and K10 filaments integrated only into the preexisting K5/K14 network upon fusion of the NIH 3T3 (K1/K10) cells with epithelial cells expressing endogenous K5/K14 or with NIH 3T3 cells which were transfected with active copies of the K5 and K14 genes. When combinations of active recombinant gene constructs for keratins 1, 5, 10, and 14 were tested in transient NIH 3T3 transfections, the most intact cytokeratin network observed by immunofluorescence was formed by the K5/K14 pair. The K1/K14 pair was capable of forming a cytoskeletal network, but the network was poorly developed, and usually perinuclear. Transfection of K10 in combination with K5 or K1 resulted in cytoplasmic agglomerates, but not a cytoskeleton. These results suggest that the formation of the suprabasal cytoskeleton in epidermis is dependent on the preexisting basal cell intermediate filament network. Furthermore, restrictions on filament formation appear to be more stringent for K10 than for K1.
Cytokeratins are fibrous intermediate-filament protein polymers present in almost all animal cells. Their function is related to epithelium structural maintenance, protection from mechanical trauma, and possibly communication between adjacent cells or cytoplasm components. Today there are 20 known cytokeratins, classified according to their molecular weight and pH as type I or acidic (cytokeratins 9–20) and type II or neutral-basic (cytokeratins 1–8). Cytokeratins are always expressed in specific pairs for each type of tissue, composed of one unit of type I and one unit of type II. Primary structural defects of cytokeratins are associated with various keratinization impairments. Two of the better characterized defects are bullous epidermolysis and epidermolytic hyperkeratosis. Anti-cytokeratin monoclonal antibodies are being used for diagnostic purposes to characterize the origin of poorly differentiated tumors and metastatic solid tumors.
Over the past two decades, the field of wound healing and tissue repair has witnessed tremendous advances resulting from the biological sciences, biomedical and tissue engineering, and greater clinical understanding of wounds and their pathophysiology. In large part because of these advances, clinicians are now able to offer and deliver more sophisticated and effective treatments to patients with acute wounds, chronic wounds, burns, and other types of injuries.
This report relies on published information focused on bioengineered skin and the authors' perspectives on the application of this technology in wound healing. In some cases, off-label applications of certain bioengineered skin constructs have been used to illustrate the spectrum of usefulness of these constructs.
Bioengineered skin (including acellular and cellular products; living and nonliving constructs; and epidermal, dermal, and bilayered therapeutic adjuncts) has resulted in very substantial and demonstrable improvements in wound care. Some of the constructs are U.S. Food and Drug Administration approved for treatment of burns and for impaired healing situations, including venous and diabetic foot ulcers.
The advances that have occurred in testing and proving the efficacy of bioengineered skin hold great promise for further improvements in the way this technology is used in the surgical field and in wound care. Advances in therapeutic agents have also led to greater understanding of pathophysiology. Thus, wound bed preparation as a concept and as an approach is in fact the result of the need to maximize the benefits of advanced therapies.
To create a scaffold that is suitable for the construction of tissue-engineered skin, a novel asymmetric porous scaffold with different pore sizes on either side was prepared by combining a collagen-chitosan porous membrane with fibrin glue. Tissue-engineered skin was fabricated using this asymmetric scaffold, fibroblasts, and a human keratinocyte line (HaCaT). Epidermal cells could be seen growing easily and achieved confluence on the fibrin glue on the upper surface of the scaffold. Scanning electron microscopy showed typical shuttle-like fibroblasts adhering to the wall of the scaffold and fluorescence microscopy showed them growing in the dermal layer of the scaffold. The constructed composite skin substitute had a histological structure similar to that of normal skin tissue after three weeks of culture. The results of our study suggest that the asymmetric scaffold is a promising biologically functional material for skin tissue engineering, with prospects for clinical applications.
Despite the rapid development of engineered skin, such skin still lacks skin appendages. Sweat glands, one of the skin appendages, play key roles in the maintenance of homeostasis and temperature regulation. In this study, we tested whether sweat glands could be integrated into engineered skin constructs to improve the quality of tissue regeneration. Using gelatin microspheres(containing epidermal growth factor [EGF]) as multifunctional vehicles, we cultured sweat gland cells (SGCs) on them and delivered SGCs-microspheres complex (SMC) into the engineered skin construct, which was created in vitro by culturing human keratinocytes on top of a fibroblast-embedded collagen-based matrix in an organotypic co-culture model. This engineered skin construct was then transplanted onto full-thickness cutaneous wounds in an athymic murine model. EGF-loaded microspheres displayed more cellular growth-promoting efficiency, and thus SMC was an available means for SGCs delivery. Constitution of the engineered skin constructs formed a skin-like pattern in vitro. Remarkably, SMC could differentiate toward a sweat gland-like structure in vitro within the hybrid matrix. Furthermore, the degree of wound healing in mice with this skin construct implantation was better than that with controls. This engineered skin construct could be used as a promising tool for regeneration of sweat glands in skin repair and a valuable engineered strategy for constitution of appendage-containing engineered skin models.
In this work we performed a study of cytokeratin (CK) expression profiling on human artificial oral mucosa developed in vitro by tissue engineering at different stages of maturation (from immature to well-developed stages) at the protein and mRNA levels. Human artificial oral mucosa was generated in the laboratory using fibrin-agarose biomaterials. As controls, we used human native normal oral mucosa and embryonic oral tissues. Our results demonstrated that human embryonic oral tissues tended to express CK8 and CK19. In contrast, monolayered bioengineered oral mucosa did not show any CK expression by immunohistochemistry, whereas bilayered and multilayered artificial oral mucosa showed several markers of stratified epithelia, but did not express CK10. These results suggest that the CK expression pattern is strongly dependent on the maturation state of the artificial tissues and that the CK expression profile of our model of artificial oral mucosa was partially similar to that of the non-keratinized human adult oral mucosa. However, the expression of CK8 by the artificial oral mucosa suggests that these samples correspond to an early stage of development while kept in vitro.
Development of human oral mucosa substitutes by tissue engineering may provide new therapeutic tools for the management of periodontal diseases. In this study we evaluated a fibrin-agarose human oral mucosa substitute both in vitro and in vivo.
In vitro bioengineered oral mucosa substitutes were developed from irrelevant biopsy samples of human oral gingiva. In vivo evaluation of the constructed tissues was performed by implantation into athymic nude mice. The expression of several epithelial markers was assessed by microarray analysis and immunohistochemistry.
Bioengineered oral mucosa samples kept in vitro developed a multilayered epithelium that expressed several cytokeratins, including some markers of simple epithelia (cytokeratins 7, 8 and 18), along with markers of stratified epithelia (cytokeratins 5 and 13) and of cell proliferation (proliferating cell nuclear antigen). Bioengineered tissues grafted in vivo onto nude mice exhibited very good biointegration with the host, showing a cytokeratin expression pattern that was very similar to that of normal native oral mucosa controls. Histological analysis of the artificial tissues demonstrated that oral mucosa substitutes evaluated in vivo were structurally mature, showing some typical structures of human native oral mucosa such as rete ridges and chorial papillae, along with numerous blood vessels at the fibrin-agarose stromal substitute. These structures were absent in samples evaluated in vitro.
The results indicate that this model of human oral mucosa, constructed using fibrin-agarose scaffolds, shows similarities to native oral mucosa controls and imply that bioengineered oral mucosa substitutes could eventually be used clinically.
Historically, the term 'keratin' stood for all of the proteins extracted from skin modifications, such as horns, claws and hooves. Subsequently, it was realized that this keratin is actually a mixture of keratins, keratin filament-associated proteins and other proteins, such as enzymes. Keratins were then defined as certain filament-forming proteins with specific physicochemical properties and extracted from the cornified layer of the epidermis, whereas those filament-forming proteins that were extracted from the living layers of the epidermis were grouped as 'prekeratins' or 'cytokeratins'. Currently, the term 'keratin' covers all intermediate filament-forming proteins with specific physicochemical properties and produced in any vertebrate epithelia. Similarly, the nomenclature of epithelia as cornified, keratinized or non-keratinized is based historically on the notion that only the epidermis of skin modifications such as horns, claws and hooves is cornified, that the non-modified epidermis is a keratinized stratified epithelium, and that all other stratified and non-stratified epithelia are non-keratinized epithelia. At this point in time, the concepts of keratins and of keratinized or cornified epithelia need clarification and revision concerning the structure and function of keratin and keratin filaments in various epithelia of different species, as well as of keratin genes and their modifications, in view of recent research, such as the sequencing of keratin proteins and their genes, cell culture, transfection of epithelial cells, immunohistochemistry and immunoblotting. Recently, new functions of keratins and keratin filaments in cell signaling and intracellular vesicle transport have been discovered. It is currently understood that all stratified epithelia are keratinized and that some of these keratinized stratified epithelia cornify by forming a Stratum corneum. The processes of keratinization and cornification in skin modifications are different especially with respect to the keratins that are produced. Future research in keratins will provide a better understanding of the processes of keratinization and cornification of stratified epithelia, including those of skin modifications, of the adaptability of epithelia in general, of skin diseases, and of the changes in structure and function of epithelia in the course of evolution. This review focuses on keratins and keratin filaments in mammalian tissue but keratins in the tissues of some other vertebrates are also considered.
We have carried out a sequential study of intercellular junction formation and differentiation on human corneal substitutes consisting of an artificial corneal stroma and a corneal epithelium, developed by tissue engineering. To generate these artificial human corneas, we developed a corneal stroma substitute, using fibrin and agarose scaffolds with human keratocytes immersed within, then cultured the human corneal epithelium on top. Electron microscopy and immunofluorescence analyses revealed that artificial corneas with one or two epithelial cell layers did not show any formation of intercellular junctions. In contrast, several types of cell-cell junction, especially desmosomes, were found in multilayered mature corneal substitutes. Concomitantly, the expression of genes encoding for plakoglobin 3 (PKG3), desmoglein 3 (DSG3) and desmoplakin (DSP), zonula occludens 1 (ZO-1) and 2 (ZO-2) and connexin 37 (Cx37) was higher in multilayered artificial corneas than in immature artificial corneas, as shown by both microarray and immunofluorescence. Although expression of ZO-1, ZO-2 and Cx37 proteins was homogeneous, PKG3, DSG3 and DSP expression was restricted to the most apical cell layers in artificial corneas submerged in culture medium at all times, whereas expression was higher in intermediate cell layers, similar to normal human control corneas, when corneal substitutes are submitted to air-liquid culture techniques. These results suggest that cultured corneal substitutes submitted to air-liquid culture technique tend to form a well-developed epithelium that is very similar to the epithelium of human native corneas, suggesting that these artificial corneas could eventually be used for clinical or in vitro purposes.
Progress in tissue engineering has led to the development of technologies allowing the reconstruction of autologous tissues from the patient's own cells. Thus, tissue-engineered epithelial substitutes produced from cultured skin epithelial cells undergo long-term regeneration after grafting, indicating that functional stem cells were preserved during culture and following grafting. However, these cultured epithelial sheets reconstruct only the upper layer of the skin and lack the mechanical properties associated to the connective tissue of the dermis. We have designed a reconstructed skin entirely made from human cutaneous cells comprising both the dermis and the epidermis, as well as a well-organized basement membrane by a method named the self-assembly approach. In this chapter, protocols to generate reconstructed skin and corneal epithelium suitable for grafting are described in details. The methods include extraction and culture of human skin keratinocytes, human skin fibroblasts as well as rabbit and human corneal epithelial cells, and a complete description of the skin reconstructed by the self-assembly approach and of corneal epithelium reconstructed over a fibrin gel.
Please cite this paper as: The skin: an indispensable barrier. Experimental Dermatology 2008.Abstract: The skin forms an effective barrier between the organism and the environment preventing invasion of pathogens and fending off chemical and physical assaults, as well as the unregulated loss of water and solutes. In this review we provide an overview of several components of the physical barrier, explaining how barrier function is regulated and altered in dermatoses. The physical barrier is mainly localized in the stratum corneum (SC) and consists of protein-enriched cells (corneocytes with cornified envelope and cytoskeletal elements, as well as corneodesmosomes) and lipid-enriched intercellular domains. The nucleated epidermis also contributes to the barrier through tight, gap and adherens junctions, as well as through desmosomes and cytoskeletal elements. During epidermal differentiation lipids are synthesized in the keratinocytes and extruded into the extracellular domains, where they form extracellular lipid-enriched layers. The cornified cell envelope, a tough protein/lipid polymer structure, resides below the cytoplasmic membrane on the exterior of the corneocytes. Ceramides A and B are covalently bound to cornified envelope proteins and form the backbone for the subsequent addition of free ceramides, free fatty acids and cholesterol in the SC. Filaggrin is cross-linked to the cornified envelope and aggregates keratin filaments into macrofibrils. Formation and maintenance of barrier function is influenced by cytokines, 3',5'-cyclic adenosine monophosphate and calcium. Changes in epidermal differentiation and lipid composition lead to a disturbed skin barrier, which allows the entry of environmental allergens, immunological reaction and inflammation in atopic dermatitis. A disturbed skin barrier is important for the pathogenesis of contact dermatitis, ichthyosis, psoriasis and atopic dermatitis.
Involucrin is a precursor of the insoluble protein envelope that is assembled in the outermost layers of the epidermis. The coding sequence of the protein contains a number of short tandem repeats that have been greatly altered during mammalian evolution. We have characterised eight mouse monoclonal antibodies raised against human involucrin, all of which bind to the protein in immunoprecipitation, immunoblot and immunohistochemical preparations. Each antibody was screened for cross-reactivity with gorilla, owl monkey, dog and pig involucrin and with a fragment of the human protein, expressed in lambda gt 11, that includes the entire early region of the modern segment of repeats. Three antibodies recognised involucrin in all of these assays. Four antibodies recognised primate involucrins and the lambda gt 11 fragment. One antibody, which showed cross-reactivity with lower molecular weight proteins, only recognised primate involucrins and therefore bound outside the early region of the modern segment. Since the antibodies can be used to detect involucrin both biochemically and histologically, in a range of species, they will have applications in further studies of the expression, function and evolution of the protein.
Closure of large skin wounds (i.e., burns, congenital giant nevus, reconstruction of traumatic injury) with split-thickness skin grafts requires extensive harvesting of autologous skin. Composite grafts consisting of collagen-glycosaminoglycan (GAG) substrates populated with cultured dermal fibroblasts and epidermal keratinocytes were tested in a pilot study on full-thickness burn wounds of three patients as an alternative to split-thickness skin. Light microscopy and transmission electron microscopy showed regeneration of epidermal and dermal tissue by 2 weeks, with degradation of the collagen-GAG implant associated with low numbers of leukocytes, and deposition of new collagen by fibroblasts. Complete basement membrane, including anchoring fibrils and anchoring plaques, is formed by 2 weeks, is mature by 3 months, and accounts for the absence of blistering of healed epidermis. All skin antigens tested (involucrin, filaggrin, laminin, collagens IV and VII, fibronectin, and chondroitin-sulfate) were expressed by 16 days after grafting. This cultured skin analogue provides an experimental alternative to split-thickness skin graft that develops histiotypic markers of skin anatomy and antigen expression after wound closure.
This study evaluates the use of composite grafts of cultured human keratinocytes and de-epidermalized, acellular human dermis to close full-thickness wounds in athymic mice. Grafts were transplanted onto athymic mice and studied up to 8 wk. Graft take was excellent, with no instances of infection or graft loss. By 1 wk, the human keratinocytes had formed a stratified epidermis that was fused with mouse epithelium, and by 8 wk the grafts resembled human skin and could be freely moved over the mouse dorsum. Immunostaining for keratins 10 and 16 and for involucrin revealed an initial pattern of epithelial immaturity, which by 8 wk had normalized to that of mature unwounded epithelium. Mouse fibroblasts began to infiltrate the acellular dermis as early as 1 wk. By 8 wk fibroblasts had completely repopulated the dermis, and blood vessels were evident in the most superficial papillary projections. Dermal elements, such as rete ridges and elastin fibers, which were present in the starting dermis, persisted for the duration of the experiment. Grafts using keratinocytes from dark-skinned donors as opposed to light-skin donors had foci of pigmentation as early as 1 wk that progressed to homogenous pigmentation of the graft by 6 wk. These results indicate that melanocytes that persist in vitro are able to resume normal function in vivo. Our study demonstrates that composite grafts of cultured keratinocytes combined with acellular dermis are a useful approach for the closure of full-thickness wounds.
The aim of this study was to develop a new keratinocyte culture system on a dermal equivalent suitable for skin wound closure. Our dermal matrix is based on a fibrin gel from plasma cryoprecipitate containing live human fibroblast (from human foreskin). Keratinocytes obtained from primary culture according to the Rheinwald and Green method, were seeded on the gel at different seeding ratios. In all cases, the keratinocytes plated on the dermal equivalent grew to confluence and stratified epithelium was obtained within 10-15 days in culture. Early expression of basal membrane proteins was detected by immunostaining with laminin and type IV collagen antibodies. Cell proliferation was detected both in the epidermal layer and in the fibroblast embedded in the gel as assessed by BrdU incorporation. Detachment of composite cultures from dishes or flasks is a simple and quick procedure without the need for dispase treatment. Grafting of composite cultures to nude mice gave rise to an orderly stratified, orthokeratinized epithelium resembling human epidermis. A number of advantages including a large expansion factor without the need of 3T3 feeder layer, the availability of fibrin/plasma cryoprecipitate from blood banks and the versatile manipulation of composite cultures suggest that this system could be suitable for the definitive coverage of severely burned patients.
In cases of severe burns, it seems necessary to excise burnt tissues as soon as possible and to cover the excised area immediately with a skin substitute, when few autografts are available. We report here the first clinical uses of a dermal substrate made of collagen--GAG--chitosan grafted immediately after early excision, then epidermalized either with autologous meshed autograft or with autologous cultured epidermis. The dermal substrate replaces the excised dermis by adhering to the underlying tissue, promoting fibrovascular ingrowth. Then after 15 days it can be epidermalized. The quality of the underlying dermis obtained permitted 100% take after epidermalization with large-meshed autograft, and tended to avoid the usual typical diamond aspect of the meshed skin. After epidermalization with autologous cultured autograft, the quality of the underlying dermis permits a good take. The best aspect is obtained by combining dermal substrate and autologous cultured epidermis. Even if it still does not replace the high quality of a homograft, this dermal substrate is a promising solution for replacement of dermis. It is always available, can be stored and is exempt from micro-organism transmission.
Extensive third degree burn wounds can be permanently covered by the transplantation of autologous cultured keratinocytes. Many modifications to Green and colleagues' original technique have been suggested, including the use of a fibrin matrix. However, the properties of the cultured cells must be assessed using suitable criteria before a modified method of culture for therapeutic purposes is transferred to clinical use, because changes in culture conditions may reduce keratinocyte lifespan and result in the loss of the transplanted epithelium.
To evaluate the performances of human keratinocytes grown on a fibrin matrix, we assay for their colony-forming ability, their growth potential and their ability to generate an epidermis when grafted onto athymic mice. The results of these experiments allowed us to compare side by side the performance for third degree burn treatment of autologous cultured epithelium grafts grown according to Rheinwald and Green on fibrin matrices with that of grafts grown directly on plastic surfaces.
We found that human keratinocytes cultured on a fibrin matrix had the same growth capacity and transplantability as those cultured on plastic surfaces and that the presence of a fibrin matrix greatly facilitated the preparation, handling, and surgical transplantation of the grafts, which did not need to be detached enzymatically. The rate of take of grafts grown on fibrin matrices was high, and was similar to that of conventionally cultured grafts. The grafted autologous cells are capable of generating a normal epidermis for many years and favor the regeneration of a superficial dermis.
We have demonstrated that: 1) fibrin matrices have considerable advantages over plastic for the culture of skin cells for grafting and that it is now possible to generate and transplant enough cultured epithelium from a small skin biopsy to restore completely the epidermis of an adult human in 16 days; and 2) the generated epidermis self-renews itself for years. The use of fibrin matrices thus significantly improves the transplantation of cultured epithelium grafts for extensive burns as recently demonstrated in a follow-up work.
Porous scaffolds composed of gelatin and beta-glucan were prepared using the freeze-drying method. The scaffold had an inter-connected pore structure with average pore size of 90-150 microm. Results for the contact angle and cell attachment revealed that a high gelatin content was suitable for cellular attachment and distribution in two- or three-dimensional fibroblast cultures, because the gelatin had acidic residues, and arginine-glycine-aspartic acid groups. To prepare a stratified wound dressing to mimic the normal human skin, fibroblasts and keratinocyte cells were isolated from a child's foreskin, and were co-cultured in gelatin/beta-glucan scaffolds were cross-linked using 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride. An in vivo study showed that after 1 week, the artificial dermis containing the fibroblasts enhanced the re-epithelialization of a full-thickness skin defect rather than the acellular scaffold.
The concept that mammalian epidermis is structurally organized into functional epidermal units has been proposed on the basis of stratum corneum (SC) architecture, proliferation kinetics, melanocyte:keratinocyte ratios (1:36), and, more recently, Langerhans cell: epidermal cell ratios (1:53). This article examines the concept of functional epidermal units in human skin in which the maintenance of phi (1.618034) proportionality provides a central organizing principle. The following empirical measurements were used: 75,346 nucleated epidermal cells per mm2, 1394 Langerhans cells per mm2, 1999 melanocytes per mm2, 16 (SC) layers, 900-microm2 corneocyte surface area, 17,778 corneocytes per mm2, 14-d (SC) turnover time, and 93,124 per mm2 total epidermal cells. Given these empirical data: (1) the number of corneocytes is a mean proportional between the sum of the Langerhans cell + melanocyte populations and the number of epidermal cells, 3393/17,778-17,778/93,124; (2) the ratio of nucleated epidermal cells over corneocytes is phi proportional, 75,346/17,778 approximately phi3; (3) assuming similar 14-d turnover times for the (SC) and Malpighian epidermis, the number of corneocytes results from subtraction of a cellular fraction equal to approximately 2/phi2 x the number of living cells, 75,436 - (2/phi2 x 75,346) approximately 17,778; and (4) if total epidermal turnover time equals (SC) turnover time x the ratio of living/dead cells, then compartmental turnover times are unequal (14 d for (SC) to 45.3 d for nucleated epidermis approximately 1/2phi) and cellular replacement rates are 52.9 corneocytes/69.3 keratinocytes per mm2 per h approximately 2/phi2. These empirically derived equivalences provide logicomathematical support for the presence of functional epidermal units in human skin. Validation of a phi proportional unit architecture in human epidermis will be important for tissue engineering of skin and the design of instruments for skin measurement.
Cytokeratins are fibrous intermediate-filament protein polymers present in almost all animal cells. Their function is related to epithelium structural maintenance, protection from mechanical trauma, and possibly communication between adjacent cells or cytoplasm components. Today there are 20 known cytokeratins, classified according to their molecular weight and pH as type I or acidic (cytokeratins 9-20) and type II or neutral-basic (cytokeratins 1-8). Cytokeratins are always expressed in specific pairs for each type of tissue, composed of one unit of type I and one unit of type II. Primary structural defects of cytokeratins are associated with various keratinization impairments. Two of the better characterized defects are bullous epidermolysis and epidermolytic hyperkeratosis. Anti-cytokeratin monoclonal antibodies are being used for diagnostic purposes to characterize the origin of poorly differentiated tumors and metastatic solid tumors.
Chronic wounds represent a major problem to our society. Therefore, advanced wound-healing strategies for the treatment of these wounds are expanding into the field of tissue engineering.
To develop a novel tissue-engineered, autologous, full-thickness skin substitute of entirely human origin and to determine its ability to heal chronic wounds.
Skin substitutes (fully differentiated epidermis on fibroblast-populated human dermis) were constructed from 3-mm punch biopsies isolated from patients to be treated. Acellular allodermis was used as a dermal matrix. After a prior 5-day vacuum-assisted closure therapy to prepare the wound bed, skin substitutes were applied in a simple one-step surgical procedure to 19 long-standing recalcitrant leg ulcers (14 patients; ulcer duration 0.5-50 years).
The success rate in culturing biopsies was 97%. The skin substitute visibly resembled an autograft. Eleven of the 19 ulcers (size 1-10 cm2) healed within 8 weeks after a single application of the skin substitute. The other eight larger (60-150 cm2) and/or complicated ulcers healed completely (n = 5) or continued to decrease substantially in size (n = 3) after the 8-week follow-up period. Wound healing occurred by direct take of the skin substitute (n = 12) and/or stimulation of granulation tissue/epithelialization (n = 7). Skin substitutes were very well tolerated and pain relief was immediate after application.
Application of this novel skin substitute provides a promising new therapy for healing chronic wounds resistant to conventional therapies.
A bilayered bioengineered living skin construct (LSC) consisting of viable human neonatal keratinocytes over a collagenous dermis seeded with dermal fibroblasts has been used extensively in difficult-to heal human wounds. Its biological properties include production of several mediators, cytokines, and growth factors and the ability to heal itself upon injury. In this study, we investigated the process of keratinocyte migration in LSC. At baseline, 6-mm punch biopsies of the construct were placed in serum-free medium (AIM-V) or Dulbecco's modified Eagle medium. At varying time points, the LSC samples were processed and analyzed using histology and immunohistochemistry. By 72 h, in a time-dependent manner, the overlying epidermis had migrated over and enveloped the entire underlying dermis, a process known as epiboly. Increasing concentrations of neutralizing antibodies to epidermal growth factor or interleukin-1 alpha down-regulated the extent of epiboly, as measured using computerized planimetry, but antibodies to transforming growth factor-beta 1 did not affect it. The consistent expression of laminin V, alpha3beta1 integrin, and vitronectin (epibolin) and its integrin receptor (alphavbeta5) characterized the tongue of migrating epidermis. Increasing concentrations of antibodies to vitronectin blocked the process of epiboly, as did antibodies to the alphavbeta5 integrin receptor, which mediates vitronectin-driven keratinocyte locomotion. This process of epiboly provides novel mechanisms of action for bioengineered skin constructs.
Before we can explain why so many closely related intermediate filament genes have evolved in vertebrates, while maintaining such dramatically tissue specific expression, we need to understand their function. The best evidence for intermediate filament function comes from observing the consequences of mutation and mis-expression, primarily in human tissues. Mostly these observations suggest that intermediate filaments are important in allowing individual cells, the tissues and whole organs to cope with various types of stress, in health and disease. Exactly how they do this is unclear and many aspects of cell dysfunction have been associated with intermediate filaments to date. In particular, it is still not clear whether the non-mechanical functions now being attributed to intermediate filaments are primary functions of these structural proteins, or secondary consequences of their function to respond to mechanical stress. We discuss selected situations in which responses to stress are clearly influenced by intermediate filaments.
The lack of sufficient oral mucosa available for intra-oral grafting is a major surgical problem, and new sources of oral tissues for clinical use are needed. In this regard, some models of engineered oral mucosa have been reported to date, but little is known about the structural and genetic mechanisms that occur during the process of development and maturation of these tissue substitutes. We have carried out a time-course study of the genes and morphological patterns of cell and tissue differentiation that develop in oral mucosa constructs after 3, 7, 11 and 21 days of development. Our electron microscopy and microarray analyses demonstrated that the oral mucosa constructs generated by tissue engineering undergo a progressive process of cell differentiation with the sequential formation and maturation of several layers of epithelium (with expression of stratifin, sciellin, involucrin, trichohyalin and kallikrein 7), intercellular junctions (with expression of plakophilin, desmocollin, desmoglein and cadherins), cytokeratins, a basement membrane (laminins, collagen IV) and the extracellular matrix (biglycan, matrix metalloproteinases). In conclusion, although the level and type of keratinization developed in vitro could be different, the oral mucosa substitutes were very similar to the native tissues.
Tissue-engineered skin is a significant advance in the field of wound healing. It has mainly been developed because of limitations associated with the use of autografts and allografts where the donor site suffers from pain, infection, and scarring. Recently, tissue-engineered skin replacements have been finding widespread application, especially in the case of burns, where the major limiting factor is the availability of autologous skin. The development of a bioartificial skin facilitates the treatment of patients with deep burns and various skin-related disorders. The present review gives a comprehensive overview of the developments and future prospects of scaffolds as skin substitutes for tissue repair and regeneration.
Our objective is to develop a synthetic biodegradable replacement dermal substitute for tissue engineering of skin and oral mucosa. Our in vivo criteria were that candidate scaffolds should allow surrounding cells to migrate fully into the scaffolds, enabling vasculogenesis and remodelling without invoking a chronic inflammatory response. We examined a total of six experimental electrospun polymer scaffolds: (1) poly-l-lactide (PLLA); (2) PLLA+10% oligolactide; (3) PLLA+rhodamine and (4-6) three poly(d,l)-lactide-co-glycolide (PLGA) random multiblock copolymers, with decreasing lactide/glycolide mole fractions (85:15, 75:25 and 50:50). These were evaluated for degradation in vitro up to 108 days and in vivo in adult male Wistar rats from 4 weeks to 12 months. In vivo, all scaffolds permitted good cellular penetration, with no adverse inflammatory response outside the scaffold margin and with no capsule formation around the periphery. The breakdown rate for each scaffold in vitro versus in vivo was similar, and an increase in the ratio of polyglycolide to polylactide correlated with an increase in breakdown rate, as expected. Scaffolds of PLLA were stable in vivo even after 12 months whereas scaffolds fabricated from PLGA 85:15 and 75:25 revealed a 50% loss of mass after 4 and 3 months, respectively. In vitro PLGA 85:15 and 75:25 scaffolds were able to support keratinocyte, fibroblast and endothelial cell growth and extracellular matrix production, with evidence of new collagen production after 7 days. In conclusion, the data supports the development of PLGA 85:15 and 75:25 electrospun polymer scaffolds as potential degradable biomaterials for dermal replacement.
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