Tissue-engineered oral epithelium (ΤΕΟΕ) was developed after comparing various culture conditions, including submerged (SUB) and air-liquid interface (ALI) human cell expansion options. Barrier formation was evaluated via transepithelial electrical resistance (TEER) and calcein permeation via spectrofluorometry. TEOE was further assessed for long-term viability via live/dead staining and development of intercellular connections via transmission electron microscopy. Tissue architecture was evaluated via histochemistry and the expression of pancytokeratin (pCK) via immunohistochemistry. The effect of two commonly used dental resinous monomers on TEOE was evaluated for alterations in cell viability and barrier permeability. ALI/keratinocyte growth factor-supplemented (ALI-KGS) culture conditions led to the formation of an 8-20-layer thick, intercellularly connected epithelial barrier. TEER values of ALI-KGS-developed TEOE decreased compared with all other tested conditions, and the established epithelium intensively expressed pCK. Exposure to dental monomers affected the integrity and architecture of TEOE and induced cellular vacuolation, implicating hydropic degeneration. Despite structural modifications, the permeability of TEOE was not substantially affected after exposure to the monomers. In conclusion, the biological properties of the TEOE mimicking the physiological functional conditions and its value as biocompatibility assessment tool for dental materials were characterized.
{"title":"Tissue-Engineered Oral Epithelium for Dental Material Testing: Toward <i>In Vitro</i> Biomimetic Models.","authors":"Foteini Machla, Paraskevi Kyriaki Monou, Chrysanthi Bekiari, Dimitrios Andreadis, Evangelia Kofidou, Emmanuel Panteris, Orestis L Katsamenis, Maria Kokoti, Petros Koidis, Imad About, Dimitrios Fatouros, Athina Bakopoulou","doi":"10.1089/ten.TEC.2024.0154","DOIUrl":"10.1089/ten.TEC.2024.0154","url":null,"abstract":"<p><p>Tissue-engineered oral epithelium (ΤΕΟΕ) was developed after comparing various culture conditions, including submerged (SUB) and air-liquid interface (ALI) human cell expansion options. Barrier formation was evaluated via transepithelial electrical resistance (TEER) and calcein permeation via spectrofluorometry. TEOE was further assessed for long-term viability via live/dead staining and development of intercellular connections via transmission electron microscopy. Tissue architecture was evaluated via histochemistry and the expression of pancytokeratin (pCK) via immunohistochemistry. The effect of two commonly used dental resinous monomers on TEOE was evaluated for alterations in cell viability and barrier permeability. ALI/keratinocyte growth factor-supplemented (ALI-KGS) culture conditions led to the formation of an 8-20-layer thick, intercellularly connected epithelial barrier. TEER values of ALI-KGS-developed TEOE decreased compared with all other tested conditions, and the established epithelium intensively expressed pCK. Exposure to dental monomers affected the integrity and architecture of TEOE and induced cellular vacuolation, implicating hydropic degeneration. Despite structural modifications, the permeability of TEOE was not substantially affected after exposure to the monomers. In conclusion, the biological properties of the TEOE mimicking the physiological functional conditions and its value as biocompatibility assessment tool for dental materials were characterized.</p>","PeriodicalId":23154,"journal":{"name":"Tissue engineering. Part C, Methods","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142296173","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1089/ten.TEC.2024.0190
Tianbai Wang, Sung Yeon Kim, Yifan Peng, Jane Zheng, Matthew D Layne, Joanne E Murphy-Ullrich, Michael B Albro
Transforming growth factor beta (TGF-β) is a potent growth factor that regulates the homeostasis of native cartilage and is administered as an anabolic supplement for engineered cartilage growth. The quantification of TGF-β activity in live tissues in situ remains a significant challenge, as conventional activity assessments (e.g., Western blotting of intracellular signaling molecules or reporter cell assays) are unable to measure absolute levels of TGF-β activity in three-dimensional tissues. In this study, we develop a quantification platform established on TGF-β's autoinduction response, whereby active TGF-β (aTGF-β) signaling in cells induces their biosynthesis and secretion of new TGF-β in its latent form (LTGF-β). As such, cell-secreted LTGF-β can serve as a robust, non-destructive, label-free biomarker for quantifying in situ activity of TGF-β in live cartilage tissues. Here, we detect LTGF-β1 secretion levels for bovine native tissue explants and engineered tissue constructs treated with varying doses of media-supplemented aTGF-β3 using an isoform-specific ELISA. We demonstrate that: 1) LTGF-β secretion levels increase proportionally to aTGF-β exposure, reaching 7.4- and 6.6-fold increases in native and engineered cartilage, respectively; 2) synthesized LTGF-β exhibits low retention in both native and engineered cartilage tissue; and 3) secreted LTGF-β is stable in conditioned media for 2 weeks, thus enabling a reliable biological standard curve between LTGF-β secretion and exposed TGF-β activity. Accordingly, we perform quantifications of TGF-β activity in bovine native cartilage, demonstrating up to 0.59 ng/mL in response to physiological dynamic loading. We further quantify the in situ TGF-β activity in aTGF-β-conjugated scaffolds for engineered tissue, which exhibits 1.81 ng/mL of TGF-β activity as a result of a nominal 3 μg/mL loading dose. Overall, cell-secreted LTGF-β can serve as a robust biomarker to quantify in situ activity of TGF-β in live cartilage tissue and can be potentially applied for a wide range of applications, including multiple tissue types and tissue engineering platforms with different cell populations and scaffolds.
{"title":"Autoinduction-Based Quantification of <i>In Situ</i> TGF-β Activity in Native and Engineered Cartilage.","authors":"Tianbai Wang, Sung Yeon Kim, Yifan Peng, Jane Zheng, Matthew D Layne, Joanne E Murphy-Ullrich, Michael B Albro","doi":"10.1089/ten.TEC.2024.0190","DOIUrl":"10.1089/ten.TEC.2024.0190","url":null,"abstract":"<p><p>Transforming growth factor beta (TGF-β) is a potent growth factor that regulates the homeostasis of native cartilage and is administered as an anabolic supplement for engineered cartilage growth. The quantification of TGF-β activity in live tissues <i>in situ</i> remains a significant challenge, as conventional activity assessments (e.g., Western blotting of intracellular signaling molecules or reporter cell assays) are unable to measure absolute levels of TGF-β activity in three-dimensional tissues. In this study, we develop a quantification platform established on TGF-β's autoinduction response, whereby active TGF-β (aTGF-β) signaling in cells induces their biosynthesis and secretion of new TGF-β in its latent form (LTGF-β). As such, cell-secreted LTGF-β can serve as a robust, non-destructive, label-free biomarker for quantifying <i>in situ</i> activity of TGF-β in live cartilage tissues. Here, we detect LTGF-β1 secretion levels for bovine native tissue explants and engineered tissue constructs treated with varying doses of media-supplemented aTGF-β3 using an isoform-specific ELISA. We demonstrate that: 1) LTGF-β secretion levels increase proportionally to aTGF-β exposure, reaching 7.4- and 6.6-fold increases in native and engineered cartilage, respectively; 2) synthesized LTGF-β exhibits low retention in both native and engineered cartilage tissue; and 3) secreted LTGF-β is stable in conditioned media for 2 weeks, thus enabling a reliable biological standard curve between LTGF-β secretion and exposed TGF-β activity. Accordingly, we perform quantifications of TGF-β activity in bovine native cartilage, demonstrating up to 0.59 ng/mL in response to physiological dynamic loading. We further quantify the <i>in situ</i> TGF-β activity in aTGF-β-conjugated scaffolds for engineered tissue, which exhibits 1.81 ng/mL of TGF-β activity as a result of a nominal 3 μg/mL loading dose. Overall, cell-secreted LTGF-β can serve as a robust biomarker to quantify <i>in situ</i> activity of TGF-β in live cartilage tissue and can be potentially applied for a wide range of applications, including multiple tissue types and tissue engineering platforms with different cell populations and scaffolds.</p>","PeriodicalId":23154,"journal":{"name":"Tissue engineering. Part C, Methods","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142296168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1089/ten.TEC.2024.0214
Eladio Muñoz, Ana Carolina Loyola, Leticia Pitol-Palin, Roberta Okamoto, Jamil Shibli, Michel Messora, Arthur Belém Novaes, Sergio Scombatti de Souza
This study evaluated the efficacy of synthetic bone blocks, composed of hydroxyapatite (HA) or β-tricalcium phosphate (B-TCP), which were produced by additive manufacturing and used for the repair of critical size bone defects (CSDs) in rat calvaria. Sixty rats were divided into five groups (n = 12): blood clot (CONTROL), 3D-printed HA (HA), 3D-printed β-TCP (B-TCP), 3D-printed HA + autologous micrograft (HA+RIG), and 3D-printed β-TCP + autologous micrograft (B-TCP+RIG). CSDs were surgically created in the parietal bone and treated with the respective biomaterials. The animals were euthanized at 30 and 60 days postsurgery for microcomputed tomography (micro-CT) histomorphometric, and immunohistochemical analysis to assess new bone formation. Micro-CT analysis showed that both biomaterials were incorporated into the animals' calvaria. The HA+RIG group, especially at 60 days, exhibited a significant increase in bone formation compared with the control. The use of 3D-printed bioceramics resulted in thinner trabeculae but a higher number of trabeculae compared with the control. Histomorphometric analysis showed bone islands in close contact with the B-TCP and HA blocks at 30 days. The HA blocks (HA and HA+RIG groups) showed statistically higher new bone formation values with further improvement when autologous micrografts were included. Immunohistochemical analysis showed the expression of bone repair proteins. At 30 days, the HA+RIG group had moderate Osteopontin (OPN) staining, indicating that the repair process had started, whereas other groups showed no staining. At 60 days, the HA+RIG group showed slight staining, similar to that of the control. Osteocalcin (OCN) staining, indicating osteoblastic activity, showed moderate expression in the HA and HA+RIG groups at 30 days, with slight expression in the B-TCP and B-TCP+RIG groups. The combination of HA blocks with autologous micrografts significantly enhanced bone repair, suggesting that the presence of progenitor cells and growth factors in the micrografts contributed to the improved outcomes. It was concluded that 3D-printed bone substitute blocks, associated with autologous micrografts, are highly effective in promoting bone repair in CSDs in rat calvaria.
{"title":"Synthetic Bone Blocks Produced by Additive Manufacturing in the Repair of Critical Bone Defects.","authors":"Eladio Muñoz, Ana Carolina Loyola, Leticia Pitol-Palin, Roberta Okamoto, Jamil Shibli, Michel Messora, Arthur Belém Novaes, Sergio Scombatti de Souza","doi":"10.1089/ten.TEC.2024.0214","DOIUrl":"10.1089/ten.TEC.2024.0214","url":null,"abstract":"<p><p>This study evaluated the efficacy of synthetic bone blocks, composed of hydroxyapatite (HA) or β-tricalcium phosphate (B-TCP), which were produced by additive manufacturing and used for the repair of critical size bone defects (CSDs) in rat calvaria. Sixty rats were divided into five groups (<i>n</i> = 12): blood clot (CONTROL), 3D-printed HA (HA), 3D-printed β-TCP (B-TCP), 3D-printed HA + autologous micrograft (HA+RIG), and 3D-printed β-TCP + autologous micrograft (B-TCP+RIG). CSDs were surgically created in the parietal bone and treated with the respective biomaterials. The animals were euthanized at 30 and 60 days postsurgery for microcomputed tomography (micro-CT) histomorphometric, and immunohistochemical analysis to assess new bone formation. Micro-CT analysis showed that both biomaterials were incorporated into the animals' calvaria. The HA+RIG group, especially at 60 days, exhibited a significant increase in bone formation compared with the control. The use of 3D-printed bioceramics resulted in thinner trabeculae but a higher number of trabeculae compared with the control. Histomorphometric analysis showed bone islands in close contact with the B-TCP and HA blocks at 30 days. The HA blocks (HA and HA+RIG groups) showed statistically higher new bone formation values with further improvement when autologous micrografts were included. Immunohistochemical analysis showed the expression of bone repair proteins. At 30 days, the HA+RIG group had moderate Osteopontin (OPN) staining, indicating that the repair process had started, whereas other groups showed no staining. At 60 days, the HA+RIG group showed slight staining, similar to that of the control. Osteocalcin (OCN) staining, indicating osteoblastic activity, showed moderate expression in the HA and HA+RIG groups at 30 days, with slight expression in the B-TCP and B-TCP+RIG groups. The combination of HA blocks with autologous micrografts significantly enhanced bone repair, suggesting that the presence of progenitor cells and growth factors in the micrografts contributed to the improved outcomes. It was concluded that 3D-printed bone substitute blocks, associated with autologous micrografts, are highly effective in promoting bone repair in CSDs in rat calvaria.</p>","PeriodicalId":23154,"journal":{"name":"Tissue engineering. Part C, Methods","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142296171","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1089/ten.TEC.2024.0229
Gustavo Henrique Doná Rodrigues Almeida, Mariana Sversut Gibin, Jaqueline de Carvalho Rinaldi, Victória Hellen de Souza Gonzaga, Camila Rodrigues Thom, Rebeca Piatniczka Iglesia, Raquel Souza da Silva, Iorrane Couto Fernandes, Rafael Oliveira Bergamo, Luan Stefani Lima, Beatriz Lopomo, Giovanna Vitória Consani Santos, Thais Naomi Gonçalves Nesiyama, Francielle Sato, Mauro Luciano Baesso, Luzmarina Hernandes, Flávio Vieira Meirelles, Ana Claudia Oliveira Carreira
Biomaterials derived from biological matrices have been widely investigated due to their great therapeutic potential in regenerative medicine, since they are able to induce cell proliferation, tissue remodeling, and angiogenesis in situ. In this context, highly vascularized and proliferative tissues, such as the uterine wall, present an interesting source to produce acellular matrices that can be used as bioactive materials to induce tissue regeneration. Therefore, this study aimed to establish an optimized protocol to generate decellularized uterine scaffolds (dUT), characterizing their structural, compositional, and biomechanical properties. In addition, in vitro performance and in vivo biocompatibility were also evaluated to verify their potential applications for tissue repair. Results showed that the protocol was efficient to promote cell removal, and dUT general structure and extracellular matrix composition remained preserved compared with native tissue. In addition, the scaffolds were cytocompatible, allowing cell growth and survival. In terms of biocompatibility, the matrices did not induce any signs of immune rejection in vivo in a model of subcutaneous implantation in immunocompetent rats, demonstrating an indication of tissue integration after 30 days of implantation. In summary, these findings suggest that dUT scaffolds could be explored as a biomaterial for regenerative purposes, which is beyond the studies in the reproductive field.
{"title":"Development and Biocompatibility Assessment of Decellularized Porcine Uterine Extracellular Matrix-Derived Grafts.","authors":"Gustavo Henrique Doná Rodrigues Almeida, Mariana Sversut Gibin, Jaqueline de Carvalho Rinaldi, Victória Hellen de Souza Gonzaga, Camila Rodrigues Thom, Rebeca Piatniczka Iglesia, Raquel Souza da Silva, Iorrane Couto Fernandes, Rafael Oliveira Bergamo, Luan Stefani Lima, Beatriz Lopomo, Giovanna Vitória Consani Santos, Thais Naomi Gonçalves Nesiyama, Francielle Sato, Mauro Luciano Baesso, Luzmarina Hernandes, Flávio Vieira Meirelles, Ana Claudia Oliveira Carreira","doi":"10.1089/ten.TEC.2024.0229","DOIUrl":"10.1089/ten.TEC.2024.0229","url":null,"abstract":"<p><p>Biomaterials derived from biological matrices have been widely investigated due to their great therapeutic potential in regenerative medicine, since they are able to induce cell proliferation, tissue remodeling, and angiogenesis <i>in situ</i>. In this context, highly vascularized and proliferative tissues, such as the uterine wall, present an interesting source to produce acellular matrices that can be used as bioactive materials to induce tissue regeneration. Therefore, this study aimed to establish an optimized protocol to generate decellularized uterine scaffolds (dUT), characterizing their structural, compositional, and biomechanical properties. In addition, <i>in vitro</i> performance and <i>in vivo</i> biocompatibility were also evaluated to verify their potential applications for tissue repair. Results showed that the protocol was efficient to promote cell removal, and dUT general structure and extracellular matrix composition remained preserved compared with native tissue. In addition, the scaffolds were cytocompatible, allowing cell growth and survival. In terms of biocompatibility, the matrices did not induce any signs of immune rejection <i>in vivo</i> in a model of subcutaneous implantation in immunocompetent rats, demonstrating an indication of tissue integration after 30 days of implantation. In summary, these findings suggest that dUT scaffolds could be explored as a biomaterial for regenerative purposes, which is beyond the studies in the reproductive field.</p>","PeriodicalId":23154,"journal":{"name":"Tissue engineering. Part C, Methods","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142296170","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1089/ten.TEC.2024.0271
Amir M Alsharabasy, Abhay Pandit
Although routine two-dimensional (2D) cell culture techniques have advanced basic cancer research owing to their simplicity, cost-effectiveness, and reproducibility, they have limitations that necessitate the development of advanced three-dimensional (3D) tumor models that better recapitulate the tumor microenvironment. Various biomaterials have been used to establish these 3D models, enabling the study of cancer cell behavior within different matrices. Hyaluronic acid (HA), a key component of the extracellular matrix (ECM) in tumor tissues, has been widely studied and employed in the development of multiple cancer models. This review first examines the role of HA in tumors, including its function as an ECM component and regulator of signaling pathways that affect tumor progression. It then explores HA-based models for various cancers, focusing on HA as a central component of the 3D matrix and its mobilization within the matrix for targeted studies of cell behavior and drug testing. The tumor models discussed included those for breast cancer, glioblastoma, fibrosarcoma, gastric cancer, hepatocellular carcinoma, and melanoma. The review concludes with a discussion of future prospects for developing more robust and high-throughput HA-based models to more accurately mimic the tumor microenvironment and improve drug testing. Impact Statement This review underscores the transformative potential of hyaluronic acid (HA)-based hydrogels in developing advanced tumor models. By exploring HA's dual role as a critical extracellular matrix component and a regulator of cancer cell dynamics, we highlight its unique contributions to replicating the tumor microenvironment. The recent advancements in HA-based models provide new opportunities for more accurate studies of cancer cell behavior and drug responses. Looking ahead, these innovations pave the way for high-throughput, biomimetic platforms that could revolutionize drug testing and accelerate the discovery of effective cancer therapies.
虽然常规的二维细胞培养技术因其简便、成本效益高和可重复性强而推动了基础癌症研究,但它们也有局限性,因此有必要开发先进的三维肿瘤模型,以更好地再现肿瘤微环境。各种生物材料已被用于建立这些三维模型,以便研究癌细胞在不同基质中的行为。透明质酸(HA)是肿瘤组织细胞外基质的关键成分,已被广泛研究并用于多种癌症模型的开发。本综述首先探讨了HA在肿瘤中的作用,包括其作为细胞外基质(ECM)成分和影响肿瘤进展的信号通路调节器的功能。然后探讨了基于 HA 的各种癌症模型,重点关注作为三维基质核心成分的 HA 及其在基质内的调动,以便对细胞行为和药物测试进行有针对性的研究。讨论的肿瘤模型包括乳腺癌、胶质母细胞瘤、纤维肉瘤、胃癌、肝细胞癌和黑色素瘤。综述最后讨论了开发更强大和高通量的基于 HA 的模型的未来前景,以更准确地模拟肿瘤微环境并改进药物测试。
{"title":"Hyaluronan-Based Hydrogels for 3D Modeling of Tumor Tissues.","authors":"Amir M Alsharabasy, Abhay Pandit","doi":"10.1089/ten.TEC.2024.0271","DOIUrl":"10.1089/ten.TEC.2024.0271","url":null,"abstract":"<p><p>Although routine two-dimensional (2D) cell culture techniques have advanced basic cancer research owing to their simplicity, cost-effectiveness, and reproducibility, they have limitations that necessitate the development of advanced three-dimensional (3D) tumor models that better recapitulate the tumor microenvironment. Various biomaterials have been used to establish these 3D models, enabling the study of cancer cell behavior within different matrices. Hyaluronic acid (HA), a key component of the extracellular matrix (ECM) in tumor tissues, has been widely studied and employed in the development of multiple cancer models. This review first examines the role of HA in tumors, including its function as an ECM component and regulator of signaling pathways that affect tumor progression. It then explores HA-based models for various cancers, focusing on HA as a central component of the 3D matrix and its mobilization within the matrix for targeted studies of cell behavior and drug testing. The tumor models discussed included those for breast cancer, glioblastoma, fibrosarcoma, gastric cancer, hepatocellular carcinoma, and melanoma. The review concludes with a discussion of future prospects for developing more robust and high-throughput HA-based models to more accurately mimic the tumor microenvironment and improve drug testing. Impact Statement This review underscores the transformative potential of hyaluronic acid (HA)-based hydrogels in developing advanced tumor models. By exploring HA's dual role as a critical extracellular matrix component and a regulator of cancer cell dynamics, we highlight its unique contributions to replicating the tumor microenvironment. The recent advancements in HA-based models provide new opportunities for more accurate studies of cancer cell behavior and drug responses. Looking ahead, these innovations pave the way for high-throughput, biomimetic platforms that could revolutionize drug testing and accelerate the discovery of effective cancer therapies.</p>","PeriodicalId":23154,"journal":{"name":"Tissue engineering. Part C, Methods","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142354422","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01Epub Date: 2024-10-07DOI: 10.1089/ten.TEC.2024.0230
Sik-Loo Tan, Chee-Ken Chan, T Sara Ahmad, Seow-Hui Teo, Wuey-Min Ng, Lakshmi Selvaratnam, Tunku Kamarul
<p><p>Mesenchymal stromal cells (MSCs) have immense potential for use in musculoskeletal tissue regeneration; however, there is still a paucity of evidence on the effect of tenogenic MSCs (TMSCs) in tendon healing <i>in vivo</i>. This study aimed to determine the effects of growth differentiation factor 5 (GDF5)-induced rabbit MSCs (rbMSCs) on infraspinatus tendon healing in a New Zealand white rabbit model. In this study, bone marrow-derived rbMSCs were isolated, and 100 ng/mL GDF5 was used to induce tenogenic differentiation in rbMSC. The effects of GDF5 on rbMSC <i>in vitro</i> were assessed by total collagen assay, gene expression analysis, and immunofluorescence staining of tenogenic markers; native tenocytes isolated from rabbit tendon were used as a positive control. In <i>in vivo</i>, a window defect was created on the infraspinatus tendons bilaterally. After 3 weeks, the rabbits (<i>n</i> = 18) were randomly divided into six groups and repaired with various interventions: (1) surgical suture; (2) fibrin glue (FG); (3) suture and FG; (4) suture, FG, and rabbit tenocytes (rbTenocyte); (5) suture, FG, and rbMSCs, and (6) suture, FG, and TMSC. All animals were euthanized at 6 weeks postoperatively. The <i>in vitro</i> GDF5-induced rbMSCs (or TMSC) showed increased total collagen expression, augmented scleraxis (<i>SCX</i>), and type-I collagen (<i>COL1A1</i>) mRNA gene expression levels. Immunofluorescence showed similar expression in GDF5-induced rbMSC to that of rbTenocyte. <i>In vivo</i> histological analysis showed progressive tendon healing in the TMSC-treated group; cells with elongated nuclei aligned parallel to the collagen fibers, and the collagen fibers were in a more organized orientation, along with macroscopic evidence of tendon callus formation. Significant differences were observed in the cell-treated groups compared with the non-cell-treated groups. Histological scoring showed a significantly enhanced tendon healing in the TMSC- and rbMSC-treated groups compared with the rbTenocyte group. The <i>SCX</i> mRNA expression levels, at 6 weeks following repair, were significantly upregulated in the TMSC group. Immunofluorescence showed COL-1 bundles aligned in parallel orientation; this was further confirmed in atomic force microscopy imaging. SCX, TNC, and TNMD were detected in the TMSC group. In conclusion, GDF5 induces tenogenic differentiation in rbMSCs, and TMSC enhances tendon healing <i>in vivo</i> compared with conventional suture repair. Impact Statement Tendon tears and degeneration are debilitating clinical conditions. To date, the suture method is the only gold standard for repairing tendons. Mesenchymal stromal cells (MSCs) have been suggested for many years for their potential in tissue regeneration, especially in tendon-degenerative conditions. Growth differentiation factor 5 (GDF5) has been reported to induce human MSC into a tenogenic lineage (or TMSC), hence a potential cell source for tendon regeneration. This st
{"title":"Growth Differentiation Factor 5-Induced Mesenchymal Stromal Cells Enhance Tendon Healing.","authors":"Sik-Loo Tan, Chee-Ken Chan, T Sara Ahmad, Seow-Hui Teo, Wuey-Min Ng, Lakshmi Selvaratnam, Tunku Kamarul","doi":"10.1089/ten.TEC.2024.0230","DOIUrl":"10.1089/ten.TEC.2024.0230","url":null,"abstract":"<p><p>Mesenchymal stromal cells (MSCs) have immense potential for use in musculoskeletal tissue regeneration; however, there is still a paucity of evidence on the effect of tenogenic MSCs (TMSCs) in tendon healing <i>in vivo</i>. This study aimed to determine the effects of growth differentiation factor 5 (GDF5)-induced rabbit MSCs (rbMSCs) on infraspinatus tendon healing in a New Zealand white rabbit model. In this study, bone marrow-derived rbMSCs were isolated, and 100 ng/mL GDF5 was used to induce tenogenic differentiation in rbMSC. The effects of GDF5 on rbMSC <i>in vitro</i> were assessed by total collagen assay, gene expression analysis, and immunofluorescence staining of tenogenic markers; native tenocytes isolated from rabbit tendon were used as a positive control. In <i>in vivo</i>, a window defect was created on the infraspinatus tendons bilaterally. After 3 weeks, the rabbits (<i>n</i> = 18) were randomly divided into six groups and repaired with various interventions: (1) surgical suture; (2) fibrin glue (FG); (3) suture and FG; (4) suture, FG, and rabbit tenocytes (rbTenocyte); (5) suture, FG, and rbMSCs, and (6) suture, FG, and TMSC. All animals were euthanized at 6 weeks postoperatively. The <i>in vitro</i> GDF5-induced rbMSCs (or TMSC) showed increased total collagen expression, augmented scleraxis (<i>SCX</i>), and type-I collagen (<i>COL1A1</i>) mRNA gene expression levels. Immunofluorescence showed similar expression in GDF5-induced rbMSC to that of rbTenocyte. <i>In vivo</i> histological analysis showed progressive tendon healing in the TMSC-treated group; cells with elongated nuclei aligned parallel to the collagen fibers, and the collagen fibers were in a more organized orientation, along with macroscopic evidence of tendon callus formation. Significant differences were observed in the cell-treated groups compared with the non-cell-treated groups. Histological scoring showed a significantly enhanced tendon healing in the TMSC- and rbMSC-treated groups compared with the rbTenocyte group. The <i>SCX</i> mRNA expression levels, at 6 weeks following repair, were significantly upregulated in the TMSC group. Immunofluorescence showed COL-1 bundles aligned in parallel orientation; this was further confirmed in atomic force microscopy imaging. SCX, TNC, and TNMD were detected in the TMSC group. In conclusion, GDF5 induces tenogenic differentiation in rbMSCs, and TMSC enhances tendon healing <i>in vivo</i> compared with conventional suture repair. Impact Statement Tendon tears and degeneration are debilitating clinical conditions. To date, the suture method is the only gold standard for repairing tendons. Mesenchymal stromal cells (MSCs) have been suggested for many years for their potential in tissue regeneration, especially in tendon-degenerative conditions. Growth differentiation factor 5 (GDF5) has been reported to induce human MSC into a tenogenic lineage (or TMSC), hence a potential cell source for tendon regeneration. This st","PeriodicalId":23154,"journal":{"name":"Tissue engineering. Part C, Methods","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142005382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1089/ten.tec.2024.0246
Leopold Klein, Dietmar W Hutmacher
Tissue engineering research fundamentally relies on experiments to advance knowledge, utilizing various models for research on both humans and animals. With scientific progress, experimental models have become increasingly complex over time. This complexity sometimes blurs the distinction between categories, making terminology less consistent. In biomedical research, three overarching terms are commonly used to characterize experimental environments: in vitro, ex vivo, and in vivo. While in vitro translates from Latin as "in glass," referring historically to experimental conditions in a test tube or petri dish, in vivo experiments occur within a living organism's natural environment. Conversely, ex vivo originates from living tissue outside its host environment while striving to maintain conditions as close to the host surroundings as possible. In the tissue engineering and regenerative medicine (TE&RM) community, there needs to be more clarity between in vitro and ex vivo terminology, with historical definitions sometimes disregarded and new terms often introduced without rigorous scientific justification. At this juncture, the question arises of when to refer to experiments as in vitro or ex vivo or whether the terms may be used synonymously in some instances. Moreover, what criteria must ex vivo experiments meet to be legitimately defined as such? This perspective is intended to address questions that would assist the TE&RM community in better understanding the differences between in vitro and ex vivo models. Impact Statement In the tissue engineering & regenerative medicine literature, the terms "in vitro" and "ex vivo" are often used interchangeably to describe experiments. This interchangeable usage can lead to a compromised interpretation of research results and, consequently, misleading scientific conclusions and teachings. This perspective aims to provide clarity on the various definitions of experimental designs. It also highlights the issue of using terms with inconsistent meanings that have origins dating back to the distant past. It's important to note that scientific definitions constantly evolve, and there is a scientifically rooted responsibility to evaluate and rethink the current state of affairs critically.
{"title":"Straddling the Line Between <i>In Vitro</i> and <i>Ex Vivo</i> Investigations.","authors":"Leopold Klein, Dietmar W Hutmacher","doi":"10.1089/ten.tec.2024.0246","DOIUrl":"https://doi.org/10.1089/ten.tec.2024.0246","url":null,"abstract":"<p><p>Tissue engineering research fundamentally relies on experiments to advance knowledge, utilizing various models for research on both humans and animals. With scientific progress, experimental models have become increasingly complex over time. This complexity sometimes blurs the distinction between categories, making terminology less consistent. In biomedical research, three overarching terms are commonly used to characterize experimental environments: <i>in vitro</i>, <i>ex vivo</i>, and <i>in vivo</i>. While <i>in vitro</i> translates from Latin as \"in glass,\" referring historically to experimental conditions in a test tube or petri dish, <i>in vivo</i> experiments occur within a living organism's natural environment. Conversely, <i>ex vivo</i> originates from living tissue outside its host environment while striving to maintain conditions as close to the host surroundings as possible. In the tissue engineering and regenerative medicine (TE&RM) community, there needs to be more clarity between <i>in vitro</i> and <i>ex vivo</i> terminology, with historical definitions sometimes disregarded and new terms often introduced without rigorous scientific justification. At this juncture, the question arises of when to refer to experiments as <i>in vitro</i> or <i>ex vivo</i> or whether the terms may be used synonymously in some instances. Moreover, what criteria must <i>ex vivo</i> experiments meet to be legitimately defined as such? This perspective is intended to address questions that would assist the TE&RM community in better understanding the differences between <i>in vitro</i> and <i>ex vivo</i> models. Impact Statement In the tissue engineering & regenerative medicine literature, the terms \"in vitro\" and \"ex vivo\" are often used interchangeably to describe experiments. This interchangeable usage can lead to a compromised interpretation of research results and, consequently, misleading scientific conclusions and teachings. This perspective aims to provide clarity on the various definitions of experimental designs. It also highlights the issue of using terms with inconsistent meanings that have origins dating back to the distant past. It's important to note that scientific definitions constantly evolve, and there is a scientifically rooted responsibility to evaluate and rethink the current state of affairs critically.</p>","PeriodicalId":23154,"journal":{"name":"Tissue engineering. Part C, Methods","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142475417","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01Epub Date: 2024-10-04DOI: 10.1089/ten.TEC.2024.0252
Simon Kwoon-Ho Chow, Qi Gao, Alexa Pius, Mayu Morita, Yasemin Ergul, Masatoshi Murayama, Issei Shinohara, Mehmet Sertac Cekuc, Chao Ma, Yosuke Susuki, Stuart B Goodman
<p><p>This review explores the regenerative potential of key progenitor cell types and therapeutic strategies to improve healing of complex fractures and bone defects. We define, summarize, and discuss the differentiation potential of totipotent, pluripotent, and multipotent stem cells, emphasizing the advantages and shortcomings of cell therapy for bone repair and regeneration. The fundamental role of mesenchymal stem cells is highlighted due to their multipotency to differentiate into the key lineage cells including osteoblasts, osteocytes, and chondrocytes, which are crucial for bone formation and remodeling. Hematopoietic stem cells (HSCs) also play a significant role; immune cells such as macrophages and T-cells modulate inflammation and tissue repair. Osteoclasts are multinucleated cells that are important to bone remodeling. Vascular progenitor (VP) cells are critical to oxygen and nutrient supply. The dynamic interplay among these lineages and their microenvironment is essential for effective bone restoration. Therapies involving cells that are more than "minimally manipulated" are controversial and include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs, derived from early-stage embryos, possess pluripotent capabilities and have shown promise in preclinical studies for bone healing. iPSCs, reprogrammed from somatic cells, offer personalized medicine applications and can differentiate into various tissue-specific cell lines. Minimally manipulative cell therapy approaches such as the use of bone marrow aspirate concentrate (BMAC), exosomes, and various biomaterials for local delivery are explored for their effectiveness in bone regeneration. BMAC, which contains mostly immune cells but few mesenchymal and VPs, probably improves bone healing by facilitating paracrine-mediated intercellular communication. Exosome isolation harnesses the biological signals and cellular by-products that are a primary source for cell crosstalk and activation. Safe, efficacious, and cost-effective strategies to enhance bone healing using novel cellular therapies are part of a changing paradigm to modulate the inflammatory, repair, and regenerative pathways to achieve earlier more robust tissue healing and improved physical function. Impact Statement Stem cell therapy holds immense potential for bone healing due to its ability to regenerate damaged tissue. Nonmanipulated bone marrow aspirate contains mesenchymal stem cells that promote bone repair and reduce healing time. Induced pluripotent stem cells offer the advantage of creating patient-specific cells that can differentiate into osteoblasts, aiding in bone regeneration. Other delivery methods, such as scaffold-based techniques, enhance stem cell integration and function. Collectively, these approaches can improve treatment outcomes, reduce recovery periods, and advance our understanding of bone healing mechanisms, making them pivotal in orthopedic research and regenerative medici
这篇综述探讨了主要祖细胞类型的再生潜力,以及改善复杂骨折和骨缺损愈合的治疗策略。我们定义、总结并讨论了全能干细胞、多能干细胞和多能干细胞的分化潜力,强调了细胞疗法在骨修复和再生方面的优势和不足。间充质干细胞(MSCs)具有多能性,可分化成骨母细胞、骨细胞和软骨细胞等关键系细胞,对骨的形成和重塑至关重要,因此强调了间充质干细胞的基本作用。造血干细胞(HSCs)也发挥着重要作用;巨噬细胞和 T 细胞等免疫细胞可调节炎症和组织修复。破骨细胞是多核细胞,对骨重塑非常重要。血管祖细胞对氧气和营养供应至关重要。这些细胞系及其微环境之间的动态相互作用对有效的骨修复至关重要。涉及 "微操作 "以上细胞的疗法存在争议,其中包括胚胎干细胞(ESC)和诱导多能干细胞(iPSC)。胚胎干细胞来源于早期胚胎,具有多能能力,在临床前研究中已显示出骨愈合的前景。目前正在探索微操作细胞治疗方法,如使用浓缩骨髓抽吸物(BMAC)、外泌体和各种生物材料进行局部给药,以提高其在骨再生中的有效性。BMAC 主要含有免疫细胞,但间质细胞和血管祖细胞很少,它可能通过促进旁分泌介导的细胞间交流来改善骨愈合。外泌体分离利用的生物信号和细胞副产物是细胞串联和激活的主要来源。利用新型细胞疗法促进骨愈合的策略安全、有效、成本效益高,是不断变化的模式的一部分,可调节炎症、修复和再生途径,使组织愈合更早、更强健,并改善身体功能。
{"title":"The Advantages and Shortcomings of Stem Cell Therapy for Enhanced Bone Healing.","authors":"Simon Kwoon-Ho Chow, Qi Gao, Alexa Pius, Mayu Morita, Yasemin Ergul, Masatoshi Murayama, Issei Shinohara, Mehmet Sertac Cekuc, Chao Ma, Yosuke Susuki, Stuart B Goodman","doi":"10.1089/ten.TEC.2024.0252","DOIUrl":"10.1089/ten.TEC.2024.0252","url":null,"abstract":"<p><p>This review explores the regenerative potential of key progenitor cell types and therapeutic strategies to improve healing of complex fractures and bone defects. We define, summarize, and discuss the differentiation potential of totipotent, pluripotent, and multipotent stem cells, emphasizing the advantages and shortcomings of cell therapy for bone repair and regeneration. The fundamental role of mesenchymal stem cells is highlighted due to their multipotency to differentiate into the key lineage cells including osteoblasts, osteocytes, and chondrocytes, which are crucial for bone formation and remodeling. Hematopoietic stem cells (HSCs) also play a significant role; immune cells such as macrophages and T-cells modulate inflammation and tissue repair. Osteoclasts are multinucleated cells that are important to bone remodeling. Vascular progenitor (VP) cells are critical to oxygen and nutrient supply. The dynamic interplay among these lineages and their microenvironment is essential for effective bone restoration. Therapies involving cells that are more than \"minimally manipulated\" are controversial and include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs, derived from early-stage embryos, possess pluripotent capabilities and have shown promise in preclinical studies for bone healing. iPSCs, reprogrammed from somatic cells, offer personalized medicine applications and can differentiate into various tissue-specific cell lines. Minimally manipulative cell therapy approaches such as the use of bone marrow aspirate concentrate (BMAC), exosomes, and various biomaterials for local delivery are explored for their effectiveness in bone regeneration. BMAC, which contains mostly immune cells but few mesenchymal and VPs, probably improves bone healing by facilitating paracrine-mediated intercellular communication. Exosome isolation harnesses the biological signals and cellular by-products that are a primary source for cell crosstalk and activation. Safe, efficacious, and cost-effective strategies to enhance bone healing using novel cellular therapies are part of a changing paradigm to modulate the inflammatory, repair, and regenerative pathways to achieve earlier more robust tissue healing and improved physical function. Impact Statement Stem cell therapy holds immense potential for bone healing due to its ability to regenerate damaged tissue. Nonmanipulated bone marrow aspirate contains mesenchymal stem cells that promote bone repair and reduce healing time. Induced pluripotent stem cells offer the advantage of creating patient-specific cells that can differentiate into osteoblasts, aiding in bone regeneration. Other delivery methods, such as scaffold-based techniques, enhance stem cell integration and function. Collectively, these approaches can improve treatment outcomes, reduce recovery periods, and advance our understanding of bone healing mechanisms, making them pivotal in orthopedic research and regenerative medici","PeriodicalId":23154,"journal":{"name":"Tissue engineering. Part C, Methods","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142296172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-23DOI: 10.1089/ten.TEC.2024.0141
Hui Che, Mischa Selig, Jasmin C Lauer, Melanie L Hart, Bernd Rolauffs
Micropatterns (MPs) are widely used as a powerful tool to control cell morphology and phenotype. However, methods for determining the effectiveness of how well cells are controlled by the shape of MPs have been inconsistently used and studies rarely report on this topic, indicating lack of standardization. We introduce an evaluation score that quantitatively assesses the MP fabrication quality and effectiveness, which can be broadly used in conjunction with all currently available MP design types. This score uses four simple and quick steps: (i) scoring MP and (ii) background fabrication quality, (iii) defining the type(s) of MP of interest, and (iv) assigning so-called efficiency descriptors describing cell behavior. These steps are based on visual inspection and quick categorization of various aspects of MP fabrication quality and cell behavior, presented in illustrations and microscopy image examples intended to serve as a reference "atlas." To illustrate the advantage of using this score, we determined differences in cell morphology and F-actin intensity between scored versus nonscored cells. These measurements, which could be different in other studies, were chosen because both are understood as markers of cell phenotype and function. We combined intensity-calibrated immunofluorescence microscopy and image-based single cell protein analysis. Most important, significant differences in cell morphology and cytoskeletal protein content between scored versus nonscored cells were noted: the unconditional inclusion of all experimental read-outs (i.e., all MP data regardless of MP quality and effectiveness) into the final results significantly misjudged the experimental readouts versus only including experimental read-outs of quality-controlled and effective MPs, identified by scoring. Specifically, nonscoring underestimated the F-actin intensity per cell and quantitative cellular morphometric descriptors circularity and solidity and overestimated aspect ratio. Scoring improved the precision of cellular readouts, advocating the use of a MP quality and efficiency score as a quantitative decision-supporting tool in deciding whether or not particular MPs should be used for experiments, saving time and money. This simple scoring methodology can be used for improving MP fabrication, comparing results across studies, benefiting basic science studies and potential future clinical use of MPs by introducing standardization.
{"title":"Simple Methodology to Score Micropattern Quality and Effectiveness.","authors":"Hui Che, Mischa Selig, Jasmin C Lauer, Melanie L Hart, Bernd Rolauffs","doi":"10.1089/ten.TEC.2024.0141","DOIUrl":"10.1089/ten.TEC.2024.0141","url":null,"abstract":"<p><p>Micropatterns (MPs) are widely used as a powerful tool to control cell morphology and phenotype. However, methods for determining the effectiveness of how well cells are controlled by the shape of MPs have been inconsistently used and studies rarely report on this topic, indicating lack of standardization. We introduce an evaluation score that quantitatively assesses the MP fabrication quality and effectiveness, which can be broadly used in conjunction with all currently available MP design types. This score uses four simple and quick steps: (i) scoring MP and (ii) background fabrication quality, (iii) defining the type(s) of MP of interest, and (iv) assigning so-called efficiency descriptors describing cell behavior. These steps are based on visual inspection and quick categorization of various aspects of MP fabrication quality and cell behavior, presented in illustrations and microscopy image examples intended to serve as a reference \"atlas.\" To illustrate the advantage of using this score, we determined differences in cell morphology and F-actin intensity between scored versus nonscored cells. These measurements, which could be different in other studies, were chosen because both are understood as markers of cell phenotype and function. We combined intensity-calibrated immunofluorescence microscopy and image-based single cell protein analysis. Most important, significant differences in cell morphology and cytoskeletal protein content between scored versus nonscored cells were noted: the unconditional inclusion of all experimental read-outs (i.e., all MP data regardless of MP quality and effectiveness) into the final results significantly misjudged the experimental readouts versus only including experimental read-outs of quality-controlled and effective MPs, identified by scoring. Specifically, nonscoring underestimated the F-actin intensity per cell and quantitative cellular morphometric descriptors circularity and solidity and overestimated aspect ratio. Scoring improved the precision of cellular readouts, advocating the use of a MP quality and efficiency score as a quantitative decision-supporting tool in deciding whether or not particular MPs should be used for experiments, saving time and money. This simple scoring methodology can be used for improving MP fabrication, comparing results across studies, benefiting basic science studies and potential future clinical use of MPs by introducing standardization.</p>","PeriodicalId":23154,"journal":{"name":"Tissue engineering. Part C, Methods","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142112244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-23DOI: 10.1089/ten.TEC.2024.0228
Étienne Savard, Brice Magne, Caroline Simard-Bisson, Christian Martel, Danielle Larouche, Robert Gauvin, Véronique J Moulin, Lucie Germain
Hypertrophic scarring is a common complication in severely burned patients who undergo autologous skin grafting. Meshed skin grafts tend to contract during wound healing, increasing the risk of pathological scarring. Although various technologies have been used to study cellular contraction, current methods for measuring contractile forces at the tissue level are limited and do not replicate the complexity of native tissues. Self-assembled skin substitutes (SASSs) were developed at the "Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX" (LOEX) and are used as permanent full-thickness skin grafts. The autologous skin substitutes are produced using the self-assembly method, allowing the cultured cells to produce their own extracellular matrix (ECM) leading to a tissue-engineered substitute resembling the native skin. The level of contraction of the SASSs during the fabrication process is patient-dependent. Thus, because of its architecture and composition, SASS is an interesting model to study skin contraction in vitro. Unfortunately, standard measurement methods are unsuited for SASS contraction assessment, mainly due to incompatibilities between the SASS manufacturing process and the current contraction force measurement methods. Here, we present an innovative contraction measurement method specifically designed to quantify the contractile behavior of tissue-engineered substitutes, without disrupting the protocol of production. The method uses C-shape anchoring frames that closes at different speed and magnitude according to the tissue contractile behavior. A finite element analysis model is then used to associate the frame deformation to a contractile force amplitude. This paper shows that the method can be used to measure the contraction force of tissues produced with cells displaying different contractile properties, such as primary skin fibroblasts and myofibroblasts. It can also be used to study the effects of cell culture conditions on tissue contraction, such as serum concentration. This protocol can be easily and affordably applied and tuned to many regenerative medicine applications or contraction-related pathological studies.
肥厚性瘢痕是接受自体皮肤移植的严重烧伤患者常见的并发症。网状植皮容易在伤口愈合过程中收缩,增加病理性瘢痕的风险。虽然研究细胞收缩的技术多种多样,但目前在组织层面测量收缩力的方法有限,无法复制原生组织的复杂性。自组装皮肤替代物(SASSs)由 "拉瓦尔大学有机实验研究中心"(Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX)开发,可用作永久性全厚皮肤移植。自体皮肤替代物是利用自组装方法制造的,培养细胞可产生自身的细胞外基质(ECM),从而形成与原生皮肤相似的组织工程替代物。在制造过程中,SASS 的收缩程度取决于患者。因此,由于其结构和组成,SASS 是研究体外皮肤收缩的有趣模型。遗憾的是,标准测量方法不适用于 SASS 收缩评估,这主要是由于 SASS 的制造过程与当前的收缩力测量方法不兼容。在此,我们介绍一种创新的收缩力测量方法,该方法专门用于量化组织工程代用品的收缩行为,且不会破坏生产程序。该方法使用 C 型锚定框架,根据组织收缩行为以不同的速度和幅度闭合。然后使用有限元分析模型将框架变形与收缩力振幅联系起来。本文表明,该方法可用于测量由具有不同收缩特性的细胞(如原生皮肤成纤维细胞和肌成纤维细胞)生成的组织的收缩力。它还可用于研究血清浓度等细胞培养条件对组织收缩力的影响。该方案可轻松、经济地应用于许多再生医学应用或与收缩相关的病理研究。
{"title":"Design of an innovative method for measuring the contractile behaviour of engineered tissues.","authors":"Étienne Savard, Brice Magne, Caroline Simard-Bisson, Christian Martel, Danielle Larouche, Robert Gauvin, Véronique J Moulin, Lucie Germain","doi":"10.1089/ten.TEC.2024.0228","DOIUrl":"https://doi.org/10.1089/ten.TEC.2024.0228","url":null,"abstract":"<p><p>Hypertrophic scarring is a common complication in severely burned patients who undergo autologous skin grafting. Meshed skin grafts tend to contract during wound healing, increasing the risk of pathological scarring. Although various technologies have been used to study cellular contraction, current methods for measuring contractile forces at the tissue level are limited and do not replicate the complexity of native tissues. Self-assembled skin substitutes (SASSs) were developed at the \"Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX\" (LOEX) and are used as permanent full-thickness skin grafts. The autologous skin substitutes are produced using the self-assembly method, allowing the cultured cells to produce their own extracellular matrix (ECM) leading to a tissue-engineered substitute resembling the native skin. The level of contraction of the SASSs during the fabrication process is patient-dependent. Thus, because of its architecture and composition, SASS is an interesting model to study skin contraction in vitro. Unfortunately, standard measurement methods are unsuited for SASS contraction assessment, mainly due to incompatibilities between the SASS manufacturing process and the current contraction force measurement methods. Here, we present an innovative contraction measurement method specifically designed to quantify the contractile behavior of tissue-engineered substitutes, without disrupting the protocol of production. The method uses C-shape anchoring frames that closes at different speed and magnitude according to the tissue contractile behavior. A finite element analysis model is then used to associate the frame deformation to a contractile force amplitude. This paper shows that the method can be used to measure the contraction force of tissues produced with cells displaying different contractile properties, such as primary skin fibroblasts and myofibroblasts. It can also be used to study the effects of cell culture conditions on tissue contraction, such as serum concentration. This protocol can be easily and affordably applied and tuned to many regenerative medicine applications or contraction-related pathological studies.</p>","PeriodicalId":23154,"journal":{"name":"Tissue engineering. Part C, Methods","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142296169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}