Pub Date : 2024-05-02DOI: 10.1089/ten.TEA.2024.0037
Simon Andreas Mayer, Benjamin Thomas, Miriam Heuer, Jan C Brune, Volker Eras, Kilian Schuster, Leonard Knoedler, Rebecca Luisa Schaefer, Wilko Thiele, Jonathan P Sleeman, Arno Dimmler, Patrick Heimel, Ulrich Kneser, Amir K Bigdeli, Florian Falkner
The arteriovenous loop (AVL) model allows the in vivo engineering of axially vascularized flaps, the so-called AVL flaps. Although AVL flaps can be transplanted microsurgically to cover tissue defects, they lack an epithelial layer on the surface. Therefore, the objective of this study was to engineer axially vascularized AVL flaps with an accompanying epithelial layer for local defect reconstruction. In this study, AVLs were established in 20 male Lewis rats. Minimally invasive injection of keratinocytes onto the surface of the AVL flaps was performed on postoperative day (POD) 21. AVL flaps were explanted from 12 rats on POD 24 or POD 30, then the epithelium formed by the keratinocytes on the surface of the flaps was evaluated using immunofluorescence staining. In six other rats, the AVL flap was locally transposed to cover a critical defect in the rats' leg on POD 30 and explanted for analysis on POD 40. In two control rats, sodium chloride was applied instead of keratinocytes. These control flaps were also transplanted on POD 30 and explanted on POD 40. Our results revealed that 3 days after keratinocyte application, a loose single-layered epithelium was observed histologically on the AVL flaps surface, whereas after 9 days, a multilayered and structured epithelium had grown. The epithelium on the transplanted AVL flaps showed its physiological differentiation when being exposed to an air-liquid interface. Histologically, a layered epithelium identical to the rats' regular skin was formed. In the sodium chloride control group, no epithelium had been grown. This study clearly demonstrates that axially vascularized AVL flaps can be processed in the subcutaneous chamber by minimally invasive injection of keratinocytes. Thus, AVL flaps with an intact epithelial layer were engineered and could be successfully transplanted for local defect coverage in a small animal model.
动静脉环(AVL)模型可以在体内制作轴向血管化皮瓣,即所谓的动静脉环皮瓣。虽然动静脉瓣可通过显微手术移植覆盖组织缺损,但其表面缺乏上皮层。因此,本研究的目的是设计带有上皮层的轴向血管化 AVL 皮瓣,用于局部缺损的重建。在这项研究中,20 只雄性 Lewis 大鼠建立了 AVL。在术后第 21 天(POD)将角质细胞以微创方式注射到动静脉瓣表面。12 只大鼠的动静脉瓣在术后第 24 天或第 30 天被剥离,然后使用免疫荧光染色法评估角质细胞在瓣表面形成的上皮。在另外六只大鼠中,于 POD 30 将 AVL 皮瓣局部移位以覆盖大鼠腿部的严重缺损,并于 POD 40 取出进行分析。在两只对照组大鼠中,使用氯化钠代替角质细胞。这些对照组皮瓣也是在 POD 30 日移植,在 POD 40 日切除。我们的结果表明,应用角质形成细胞 3 天后,从组织学角度观察到 AVL 皮瓣表面有疏松的单层上皮,而 9 天后,多层和结构化的上皮已经生长出来。移植的动静脉瓣上皮在暴露于空气-液体界面时出现了生理性分化。在组织学上,形成了与大鼠正常皮肤相同的分层上皮。而氯化钠对照组则没有上皮生长。这项研究清楚地表明,轴向血管化的 AVL 皮瓣可通过微创注射角质形成细胞在皮下腔进行处理。因此,在小动物模型中,具有完整上皮层的反车辆皮瓣被设计出来,并可成功移植用于局部缺损覆盖。
{"title":"<i>In Vivo</i> Engineering and Transplantation of Axially Vascularized and Epithelialized Flaps in Rats.","authors":"Simon Andreas Mayer, Benjamin Thomas, Miriam Heuer, Jan C Brune, Volker Eras, Kilian Schuster, Leonard Knoedler, Rebecca Luisa Schaefer, Wilko Thiele, Jonathan P Sleeman, Arno Dimmler, Patrick Heimel, Ulrich Kneser, Amir K Bigdeli, Florian Falkner","doi":"10.1089/ten.TEA.2024.0037","DOIUrl":"https://doi.org/10.1089/ten.TEA.2024.0037","url":null,"abstract":"<p><p>The arteriovenous loop (AVL) model allows the <i>in vivo</i> engineering of axially vascularized flaps, the so-called AVL flaps. Although AVL flaps can be transplanted microsurgically to cover tissue defects, they lack an epithelial layer on the surface. Therefore, the objective of this study was to engineer axially vascularized AVL flaps with an accompanying epithelial layer for local defect reconstruction. In this study, AVLs were established in 20 male Lewis rats. Minimally invasive injection of keratinocytes onto the surface of the AVL flaps was performed on postoperative day (POD) 21. AVL flaps were explanted from 12 rats on POD 24 or POD 30, then the epithelium formed by the keratinocytes on the surface of the flaps was evaluated using immunofluorescence staining. In six other rats, the AVL flap was locally transposed to cover a critical defect in the rats' leg on POD 30 and explanted for analysis on POD 40. In two control rats, sodium chloride was applied instead of keratinocytes. These control flaps were also transplanted on POD 30 and explanted on POD 40. Our results revealed that 3 days after keratinocyte application, a loose single-layered epithelium was observed histologically on the AVL flaps surface, whereas after 9 days, a multilayered and structured epithelium had grown. The epithelium on the transplanted AVL flaps showed its physiological differentiation when being exposed to an air-liquid interface. Histologically, a layered epithelium identical to the rats' regular skin was formed. In the sodium chloride control group, no epithelium had been grown. This study clearly demonstrates that axially vascularized AVL flaps can be processed in the subcutaneous chamber by minimally invasive injection of keratinocytes. Thus, AVL flaps with an intact epithelial layer were engineered and could be successfully transplanted for local defect coverage in a small animal model.</p>","PeriodicalId":56375,"journal":{"name":"Tissue Engineering Part A","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140874050","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.
生物医学植入物、伤口愈合和病变组织的免疫反应通常涉及成纤维细胞和其他基质细胞的胶原沉积。胶原沉积失调可导致生物材料纤维化、心脏纤维化、脱钙、肝纤维化和肺纤维化等并发症,最终导致器官功能丧失或生物医学植入物失效。目前诱导胶原沉积的体外方法包括在大分子拥挤条件下或在涂有纤维连接蛋白的表面上培养细胞。然而,这些方法大多是通过单个细胞系进行验证的,培养条件和培养后处理对胶原沉积的综合影响尚未得到详细探讨。在这项工作中,我们使用 WI-38 人肺成纤细胞系评估了大分子拥挤与纤维连接蛋白涂层、甲醇固定与多聚甲醛固定、使用塑料基底与玻璃基底的影响。结果发现,在大分子拥挤条件下,纤连蛋白涂层可增强胶原沉积,而与大分子拥挤相比,更高的电镀密度可改善胶原 I 的沉积。胶原沉积在塑料基底上比玻璃基底上更明显。使用 WI-38 细胞、原代人类肺成纤维细胞、原代人类真皮成纤维细胞、原代小鼠肺成纤维细胞、原代小鼠真皮成纤维细胞和 L929 小鼠成纤维细胞系评估了原代细胞与细胞系、小鼠细胞与人类细胞的影响。与原代细胞相比,细胞系表现出更强的胶原蛋白 I 沉积能力。此外,还利用皮色红染色法对胶原沉积进行了量化,并通过对脱细胞细胞外基质进行皮色红染色,展示了基于平板的药物筛选方法。这项研究的结果提供了体外诱导多种类型细胞胶原沉积的详细条件,其应用包括材料开发、潜在抗纤维化疗法的开发以及疾病通路的机理研究。
{"title":"Factors Affecting the Evaluation of Collagen Deposition and Fibrosis <i>In Vitro</i>.","authors":"Parinaz Fathi, Vanathi Sundaresan, Andrea Lucia Alfonso, Anagha Rama Varma, Kaitlyn Sadtler","doi":"10.1089/ten.TEA.2023.0284","DOIUrl":"10.1089/ten.TEA.2023.0284","url":null,"abstract":"<p><p>Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current <i>in vitro</i> methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced <i>in vitro</i> in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on <i>in vitro</i> collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct <i>in vitro</i> experiments on collagen deposition.</p>","PeriodicalId":56375,"journal":{"name":"Tissue Engineering Part A","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11250831/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140177986","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-04-17DOI: 10.1089/ten.TEA.2023.0170
Adrienne K Scott, Katie M Gallagher, Stephanie E Schneider, Abhijit Kurse, Corey P Neu
Expansion of chondrocytes presents a major obstacle in the cartilage regeneration procedure, such as matrix-induced autologous chondrocyte implantation. Dedifferentiation of chondrocytes during the expansion process leads to the emergence of a fibrotic (chondrofibrotic) phenotype that decreases the chondrogenic potential of the implanted cells. We aim to (1) determine the extent that chromatin architecture of H3K27me3 and H3K9me3 remodels during dedifferentiation and persists after the transfer to a three-dimensional (3D) culture; and (2) to prevent this persistent remodeling to enhance the chondrogenic potential of expanded bovine chondrocytes, used as a model system. Chromatin architecture remodeling of H3K27me3 and H3K9me3 was observed at 0 population doublings, 8 population doublings, and 16 population doublings (PD16) in a two-dimensional (2D) culture and after encapsulation of the expanded chondrocytes in a 3D hydrogel culture. Chondrocytes were treated with inhibitors of epigenetic modifiers (epigenetic priming) for PD16 and then encapsulated in 3D hydrogels. Chromatin architecture of chondrocytes and gene expression were evaluated before and after encapsulation. We observed a change in chromatin architecture of epigenetic modifications H3K27me3 and H3K9me3 during chondrocyte dedifferentiation. Although inhibiting enzymes that modify H3K27me3 and H3K9me3 did not alter the dedifferentiation process in 2D culture, applying these treatments during the 2D expansion did increase the expression of select chondrogenic genes and protein deposition of type II collagen when transferred to a 3D environment. Overall, we found that epigenetic priming of expanded bovine chondrocytes alters the cell fate when chondrocytes are later encapsulated into a 3D environment, providing a potential method to enhance the success of cartilage regeneration procedures.
{"title":"Epigenetic Priming Enhances Chondrogenic Potential of Expanded Chondrocytes.","authors":"Adrienne K Scott, Katie M Gallagher, Stephanie E Schneider, Abhijit Kurse, Corey P Neu","doi":"10.1089/ten.TEA.2023.0170","DOIUrl":"10.1089/ten.TEA.2023.0170","url":null,"abstract":"<p><p>Expansion of chondrocytes presents a major obstacle in the cartilage regeneration procedure, such as matrix-induced autologous chondrocyte implantation. Dedifferentiation of chondrocytes during the expansion process leads to the emergence of a fibrotic (chondrofibrotic) phenotype that decreases the chondrogenic potential of the implanted cells. We aim to (1) determine the extent that chromatin architecture of H3K27me3 and H3K9me3 remodels during dedifferentiation and persists after the transfer to a three-dimensional (3D) culture; and (2) to prevent this persistent remodeling to enhance the chondrogenic potential of expanded bovine chondrocytes, used as a model system. Chromatin architecture remodeling of H3K27me3 and H3K9me3 was observed at 0 population doublings, 8 population doublings, and 16 population doublings (PD16) in a two-dimensional (2D) culture and after encapsulation of the expanded chondrocytes in a 3D hydrogel culture. Chondrocytes were treated with inhibitors of epigenetic modifiers (epigenetic priming) for PD16 and then encapsulated in 3D hydrogels. Chromatin architecture of chondrocytes and gene expression were evaluated before and after encapsulation. We observed a change in chromatin architecture of epigenetic modifications H3K27me3 and H3K9me3 during chondrocyte dedifferentiation. Although inhibiting enzymes that modify H3K27me3 and H3K9me3 did not alter the dedifferentiation process in 2D culture, applying these treatments during the 2D expansion did increase the expression of select chondrogenic genes and protein deposition of type II collagen when transferred to a 3D environment. Overall, we found that epigenetic priming of expanded bovine chondrocytes alters the cell fate when chondrocytes are later encapsulated into a 3D environment, providing a potential method to enhance the success of cartilage regeneration procedures.</p>","PeriodicalId":56375,"journal":{"name":"Tissue Engineering Part A","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139699002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-02-26DOI: 10.1089/ten.TEA.2023.0171
Chao Qi, Lu Cheng, Chuanqi Huang
Millions of people suffer from tracheal defect worldwide each year, while autograft and allograft cannot meet existing treatment needs. Tissue-engineered trachea substitutes represent a promising treatment for tracheal defect, while lack of precisely personalized treatment abilities. Therefore, development of an artificial trachea that can be used for personalized transplantation is highly desired. In this study, we report the design and fabrication of an artificial trachea based on sericin microsphere (SM) by microtissue engineering technology and three-dimensional (3D) printing for personalized repair of tracheal defect. The SM possessed natural cell adhesion and promoting cell proliferation ability. Then, the microtissue was fabricated by coincubation of SM with chondrocytes and tracheal epithelial cells. This microtissue displayed good cytocompatibility and could support seed cell adhesion and proliferation. After that, this microtissue was individually assembled to form an artificial trachea by 3D printing. Notably, artificial trachea had an encouraging complete cartilaginous and epithelial structure after transplantation. Furthermore, the artificial trachea molecularly resembled native trachea as evidenced by similar expression of trachea-critical genes. Altogether, the work demonstrates the effectiveness of microtissue engineering and 3D printing for individual construction of artificial trachea, providing a promising approach for personalized treatment of tracheal defect.
{"title":"Artificial Trachea from Microtissue Engineering and Three-Dimensional Printing for Tracheal Personalized Repair.","authors":"Chao Qi, Lu Cheng, Chuanqi Huang","doi":"10.1089/ten.TEA.2023.0171","DOIUrl":"10.1089/ten.TEA.2023.0171","url":null,"abstract":"<p><p>Millions of people suffer from tracheal defect worldwide each year, while autograft and allograft cannot meet existing treatment needs. Tissue-engineered trachea substitutes represent a promising treatment for tracheal defect, while lack of precisely personalized treatment abilities. Therefore, development of an artificial trachea that can be used for personalized transplantation is highly desired. In this study, we report the design and fabrication of an artificial trachea based on sericin microsphere (SM) by microtissue engineering technology and three-dimensional (3D) printing for personalized repair of tracheal defect. The SM possessed natural cell adhesion and promoting cell proliferation ability. Then, the microtissue was fabricated by coincubation of SM with chondrocytes and tracheal epithelial cells. This microtissue displayed good cytocompatibility and could support seed cell adhesion and proliferation. After that, this microtissue was individually assembled to form an artificial trachea by 3D printing. Notably, artificial trachea had an encouraging complete cartilaginous and epithelial structure after transplantation. Furthermore, the artificial trachea molecularly resembled native trachea as evidenced by similar expression of trachea-critical genes. Altogether, the work demonstrates the effectiveness of microtissue engineering and 3D printing for individual construction of artificial trachea, providing a promising approach for personalized treatment of tracheal defect.</p>","PeriodicalId":56375,"journal":{"name":"Tissue Engineering Part A","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139543588","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-01-31DOI: 10.1089/ten.TEA.2023.0240
Eileen Y Su, Christopher S Kennedy, Emmanuel E Vega-Soto, Brooke D Pallas, Samantha N Lukpat, Derek H Hwang, David W Bosek, Celeste E Forester, Claudia Loebel, Lisa M Larkin
Volumetric muscle loss (VML) is the loss of skeletal muscle that exceeds the muscle's self-repair mechanism and leads to permanent functional deficits. In a previous study, we demonstrated the ability of our scaffold-free, multiphasic, tissue-engineered skeletal muscle units (SMUs) to restore muscle mass and force production. However, it was observed that the full recovery of muscle structure was inhibited due to increased fibrosis in the repair site. As such, novel biomaterials such as hydrogels (HGs) may have significant potential for decreasing the acute inflammation and subsequent fibrosis, as well as enhancing skeletal muscle regeneration following VML injury and repair. The goal of the current study was to assess the biocompatibility of commercially available poly(ethylene glycol), methacrylated gelatin, and hyaluronic acid (HA) HGs in combination with our SMUs to treat VML in a clinically relevant large animal model. An acute 30% VML injury created in the sheep peroneus tertius (PT) muscle was repaired with or without HGs and assessed for acute inflammation (incision swelling) and white blood cell counts in blood for 7 days. At the 7-day time point, HA was selected as the HG to use for the combined HG/SMU repair, as it exhibited a reduced inflammation response compared to the other HGs. Six weeks after implantation, all groups were assessed for gross and histological structural recovery. The results showed that the groups repaired with an SMU (SMU-Only and SMU+HA) restored muscle mass to greater degree than the groups with only HG and that the SMU groups had PT muscle masses that were statistically indistinguishable from its uninjured contralateral PT muscle. Furthermore, the HA HG, SMU-Only, and SMU+HA groups displayed notable efficacy in diminishing pro-inflammatory markers and showed an increased number of regenerating muscle fibers in the repair site. Taken together, the data demonstrates the efficacy of HA HG in decreasing acute inflammation and fibrotic response. The combination of HA and our SMUs also holds promise to decrease acute inflammation and fibrosis and increase muscle regeneration, advancing this combination therapy toward clinically relevant interventions for VML injuries in humans.
{"title":"Repairing Volumetric Muscle Loss with Commercially Available Hydrogels in an Ovine Model.","authors":"Eileen Y Su, Christopher S Kennedy, Emmanuel E Vega-Soto, Brooke D Pallas, Samantha N Lukpat, Derek H Hwang, David W Bosek, Celeste E Forester, Claudia Loebel, Lisa M Larkin","doi":"10.1089/ten.TEA.2023.0240","DOIUrl":"10.1089/ten.TEA.2023.0240","url":null,"abstract":"<p><p>Volumetric muscle loss (VML) is the loss of skeletal muscle that exceeds the muscle's self-repair mechanism and leads to permanent functional deficits. In a previous study, we demonstrated the ability of our scaffold-free, multiphasic, tissue-engineered skeletal muscle units (SMUs) to restore muscle mass and force production. However, it was observed that the full recovery of muscle structure was inhibited due to increased fibrosis in the repair site. As such, novel biomaterials such as hydrogels (HGs) may have significant potential for decreasing the acute inflammation and subsequent fibrosis, as well as enhancing skeletal muscle regeneration following VML injury and repair. The goal of the current study was to assess the biocompatibility of commercially available poly(ethylene glycol), methacrylated gelatin, and hyaluronic acid (HA) HGs in combination with our SMUs to treat VML in a clinically relevant large animal model. An acute 30% VML injury created in the sheep peroneus tertius (PT) muscle was repaired with or without HGs and assessed for acute inflammation (incision swelling) and white blood cell counts in blood for 7 days. At the 7-day time point, HA was selected as the HG to use for the combined HG/SMU repair, as it exhibited a reduced inflammation response compared to the other HGs. Six weeks after implantation, all groups were assessed for gross and histological structural recovery. The results showed that the groups repaired with an SMU (SMU-Only and SMU+HA) restored muscle mass to greater degree than the groups with only HG and that the SMU groups had PT muscle masses that were statistically indistinguishable from its uninjured contralateral PT muscle. Furthermore, the HA HG, SMU-Only, and SMU+HA groups displayed notable efficacy in diminishing pro-inflammatory markers and showed an increased number of regenerating muscle fibers in the repair site. Taken together, the data demonstrates the efficacy of HA HG in decreasing acute inflammation and fibrotic response. The combination of HA and our SMUs also holds promise to decrease acute inflammation and fibrosis and increase muscle regeneration, advancing this combination therapy toward clinically relevant interventions for VML injuries in humans.</p>","PeriodicalId":56375,"journal":{"name":"Tissue Engineering Part A","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138814086","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-01Epub Date: 2024-02-01DOI: 10.1089/ten.TEA.2023.0219
Tia C Calabrese, Kristi Rothermund, Claire M Gabe, Elia Beniash, Lance A Davidson, Fatima N Syed-Picard
Challenges remain in simultaneously regenerating the multiple diverse tissues of the tooth root in a spatially organized manner. Previously, our research group has established that scaffold-free tissue engineering approaches enable dental pulp stem/progenitor cells (DPSCs) and periodontal ligament (PDL) stem/progenitor cells (PDLSCs) to self-assemble into dentin-pulp and PDL-cementum organoids, respectively. In this study, we leveraged the innate self-organizing capacity of DPSCs and PDLSCs to now engineer organoids that resemble the full tooth root. Scaffold-free engineered tissues were generated using a heterogeneous mixture of human DPSCs and PDLSCs. Within 2 days of construct formation, PDLSCs and DPSCs became spatially restricted to the periphery and center of the constructs, respectively, emulating their anatomical positions in the tooth root. Histological and microcomputed tomography analyses showed that organoids exhibited a striated mineral pattern with a central unmineralized core, surrounded by a mineralized tissue structure, enclosed within a second peripheral unmineralized tissue, similar to the natural tooth root. Interestingly, DPSCs gave rise to the central unmineralized tissue and the inner portion of the mineralized tissue, and PDLSCs generated the outer portion of the mineralized tissue and the peripheral soft tissue. Quantitative image analysis of immunofluorescent staining revealed increased dentin sialophosphoprotein expression in the region of mineralized tissue associated with DPSCs and increased cementum protein-1 expression in the portion formed by PDLSCs, demonstrating that tooth root organoids comprise two biochemically distinct mineralized tissues characteristic of dentin-like and cementum-like structures, respectively. In addition, PDL-associated protein-1 expression was localized to the peripheral soft tissue, suggesting the formation of a rudimentary PDL-like structure. This study demonstrates that DPSCs and PDLSCs have an inherent ability to orchestrate the formation of a full tooth root-like structure. These organoids present a biomimetic model system to study cellular dynamics driving dental tissue repair or could be utilized therapeutically as biological dental implants.
{"title":"Self-Assembly of Tooth Root Organoid from Postnatal Human Dental Stem Cells.","authors":"Tia C Calabrese, Kristi Rothermund, Claire M Gabe, Elia Beniash, Lance A Davidson, Fatima N Syed-Picard","doi":"10.1089/ten.TEA.2023.0219","DOIUrl":"10.1089/ten.TEA.2023.0219","url":null,"abstract":"<p><p>Challenges remain in simultaneously regenerating the multiple diverse tissues of the tooth root in a spatially organized manner. Previously, our research group has established that scaffold-free tissue engineering approaches enable dental pulp stem/progenitor cells (DPSCs) and periodontal ligament (PDL) stem/progenitor cells (PDLSCs) to self-assemble into dentin-pulp and PDL-cementum organoids, respectively. In this study, we leveraged the innate self-organizing capacity of DPSCs and PDLSCs to now engineer organoids that resemble the full tooth root. Scaffold-free engineered tissues were generated using a heterogeneous mixture of human DPSCs and PDLSCs. Within 2 days of construct formation, PDLSCs and DPSCs became spatially restricted to the periphery and center of the constructs, respectively, emulating their anatomical positions in the tooth root. Histological and microcomputed tomography analyses showed that organoids exhibited a striated mineral pattern with a central unmineralized core, surrounded by a mineralized tissue structure, enclosed within a second peripheral unmineralized tissue, similar to the natural tooth root. Interestingly, DPSCs gave rise to the central unmineralized tissue and the inner portion of the mineralized tissue, and PDLSCs generated the outer portion of the mineralized tissue and the peripheral soft tissue. Quantitative image analysis of immunofluorescent staining revealed increased dentin sialophosphoprotein expression in the region of mineralized tissue associated with DPSCs and increased cementum protein-1 expression in the portion formed by PDLSCs, demonstrating that tooth root organoids comprise two biochemically distinct mineralized tissues characteristic of dentin-like and cementum-like structures, respectively. In addition, PDL-associated protein-1 expression was localized to the peripheral soft tissue, suggesting the formation of a rudimentary PDL-like structure. This study demonstrates that DPSCs and PDLSCs have an inherent ability to orchestrate the formation of a full tooth root-like structure. These organoids present a biomimetic model system to study cellular dynamics driving dental tissue repair or could be utilized therapeutically as biological dental implants.</p>","PeriodicalId":56375,"journal":{"name":"Tissue Engineering Part A","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138833218","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Biomaterial scaffolds have been used successfully to promote the regenerative repair of small endometrial lesions in small rodents, providing partial restoration of gestational function. The use of rabbits in this study allowed us to investigate a larger endometrial tissue defect and myometrial injury model. A gelatin/polycaprolactone (GT/PCL) gradient-layer biofilm was sutured at the defect to guide the reconstruction of the original tissue structure. Twenty-eight days postimplantation, the uterine cavity had been restored to its original morphology, endometrial growth was accompanied by the formation of glands and blood vessels, and the fragmented myofibers of the uterine smooth muscle had begun to resemble the normal structure of the lagomorph uterine cavity, arranging in a circular luminal pattern and a longitudinal serosal pattern. In addition, the repair site supported both embryonic implantation into the placenta and normal embryonic development. Four-dimensional label-free proteomic analysis identified the cell adhesion molecules, phagosome, ferroptosis, rap1 signaling pathways, hematopoietic cell lineage, complement and coagulation cascades, tricarboxylic acid cycle, carbon metabolism, and hypoxia inducible factor (HIF)-1 signaling pathways as important in the endogenous repair process of uterine tissue injury, and acetylation of protein modification sites upregulated these signaling pathways.
{"title":"Restoration of Pregnancy Function Using a GT/PCL Biofilm in a Rabbit Model of Uterine Injury.","authors":"Di Huang, Jing Liu, Jie Yang, Junhui Liang, Jing Zhang, Qinyu Han, Jianlong Yu, Tingting Yang, Qi Meng, Thorsten Steinberg, Changzhong Li, Zhongle Chang","doi":"10.1089/ten.TEA.2023.0366","DOIUrl":"10.1089/ten.TEA.2023.0366","url":null,"abstract":"<p><p>Biomaterial scaffolds have been used successfully to promote the regenerative repair of small endometrial lesions in small rodents, providing partial restoration of gestational function. The use of rabbits in this study allowed us to investigate a larger endometrial tissue defect and myometrial injury model. A gelatin/polycaprolactone (GT/PCL) gradient-layer biofilm was sutured at the defect to guide the reconstruction of the original tissue structure. Twenty-eight days postimplantation, the uterine cavity had been restored to its original morphology, endometrial growth was accompanied by the formation of glands and blood vessels, and the fragmented myofibers of the uterine smooth muscle had begun to resemble the normal structure of the lagomorph uterine cavity, arranging in a circular luminal pattern and a longitudinal serosal pattern. In addition, the repair site supported both embryonic implantation into the placenta and normal embryonic development. Four-dimensional label-free proteomic analysis identified the cell adhesion molecules, phagosome, ferroptosis, rap1 signaling pathways, hematopoietic cell lineage, complement and coagulation cascades, tricarboxylic acid cycle, carbon metabolism, and hypoxia inducible factor (HIF)-1 signaling pathways as important in the endogenous repair process of uterine tissue injury, and acetylation of protein modification sites upregulated these signaling pathways.</p>","PeriodicalId":56375,"journal":{"name":"Tissue Engineering Part A","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140208371","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-30DOI: 10.1089/ten.TEA.2024.0030
Sophia Dalfino, Elena Olaret, Marco Piazzoni, Paolo Savadori, Izabela Stancu, Gianluca Tartaglia, Claudia Dolci, Lorenzo Moroni
Critical-sized mandibular bone defects, arising from, for example, resections after tumor surgeries, are currently treated with autogenous bone grafts. This treatment is considered very invasive and is associated with limitations such as morbidity and graft resorption. Tissue engineering approaches propose to use 3D scaffolds that combine structural features, biomaterial properties, cells, and biomolecules to create biomimetic constructs. However, mimicking the complex anatomy and composition of the mandible poses a challenge in scaffold design. In our study, we evaluated the dual effect of complex pore geometry and material composition on the osteogenic potential of 3D printed scaffolds. The scaffolds were made of polycaprolactone (PCL) alone (TCP0), or with a high concentration of β-tricalcium phosphate (β-TCP) up to 40% w/w (TCP40), with two complex pore geometries, namely a star- (S) and a diamond-like (D) shape. Scanning electron microscopy and microcomputed tomography images confirmed high fidelity during the printing process. The D-scaffolds displayed higher compressive moduli than the corresponding S-scaffolds. TCP40 scaffolds in simulated body fluid showed deposition of minerals on the surface after 28 days. Subsequently, we assessed the differentiation of seeded bone marrow-derived human mesenchymal stromal cells (hMSCs) over 28 days. The early expression of RUNX2 in the cell nuclei confirmed the commitment toward an osteogenic phenotype. Moreover, alkaline phosphatase (ALP) activity and collagen deposition displayed an increasing trend in the D-scaffolds. Collagen type I was mainly present in the deposited extracellular matrix (ECM), confirming deposition of bone matrix. Finally, Alizarin Red staining showed successful mineralization on all the TCP40 samples, with higher values for the S-shaped scaffolds. Taken together, our study demonstrated that the complex pore architectures of scaffolds comprised TCP40 stimulated osteogenic differentiation and mineralization of hMSCs in vitro. Future research will aim to validate these findings in vivo.
{"title":"Polycaprolactone/β-Tricalcium Phosphate Composite Scaffolds with Advanced Pore Geometries Promote Human Mesenchymal Stromal Cells' Osteogenic Differentiation.","authors":"Sophia Dalfino, Elena Olaret, Marco Piazzoni, Paolo Savadori, Izabela Stancu, Gianluca Tartaglia, Claudia Dolci, Lorenzo Moroni","doi":"10.1089/ten.TEA.2024.0030","DOIUrl":"https://doi.org/10.1089/ten.TEA.2024.0030","url":null,"abstract":"<p><p>Critical-sized mandibular bone defects, arising from, for example, resections after tumor surgeries, are currently treated with autogenous bone grafts. This treatment is considered very invasive and is associated with limitations such as morbidity and graft resorption. Tissue engineering approaches propose to use 3D scaffolds that combine structural features, biomaterial properties, cells, and biomolecules to create biomimetic constructs. However, mimicking the complex anatomy and composition of the mandible poses a challenge in scaffold design. In our study, we evaluated the dual effect of complex pore geometry and material composition on the osteogenic potential of 3D printed scaffolds. The scaffolds were made of polycaprolactone (PCL) alone (TCP0), or with a high concentration of β-tricalcium phosphate (β-TCP) up to 40% <i>w/w</i> (TCP40), with two complex pore geometries, namely a star- (S) and a diamond-like (D) shape. Scanning electron microscopy and microcomputed tomography images confirmed high fidelity during the printing process. The D-scaffolds displayed higher compressive moduli than the corresponding S-scaffolds. TCP40 scaffolds in simulated body fluid showed deposition of minerals on the surface after 28 days. Subsequently, we assessed the differentiation of seeded bone marrow-derived human mesenchymal stromal cells (hMSCs) over 28 days. The early expression of <i>RUNX2</i> in the cell nuclei confirmed the commitment toward an osteogenic phenotype. Moreover, alkaline phosphatase (ALP) activity and collagen deposition displayed an increasing trend in the D-scaffolds. Collagen type I was mainly present in the deposited extracellular matrix (ECM), confirming deposition of bone matrix. Finally, Alizarin Red staining showed successful mineralization on all the TCP40 samples, with higher values for the S-shaped scaffolds. Taken together, our study demonstrated that the complex pore architectures of scaffolds comprised TCP40 stimulated osteogenic differentiation and mineralization of hMSCs <i>in vitro</i>. Future research will aim to validate these findings <i>in vivo</i>.</p>","PeriodicalId":56375,"journal":{"name":"Tissue Engineering Part A","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140860844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-22DOI: 10.1089/ten.TEA.2023.0307
Martino Guiotto, Alison Clayton, Ryan Morgan, Wassim Raffoul, Andrew Hart, Mathis Riehle, Pietro di Summa
Adipose-derived stem cells (ADSC) are nowadays one of the most exploited cells in regenerative medicine. They are fast growing, capable of enhancing axonal elongation, support and locally stimulate Schwann cells (SCs), and protect de-innervated muscles from atrophy after a peripheral nerve injury. With the aim of developing a bio-safe, clinically translatable cell-therapy, we assessed the effect of ADSC pre-expanded with human platelet lysate in an in vivo rat model, delivering the cells into a 15 mm critical-size sciatic nerve defect embedded within a laminin-peptide-functionalized hydrogel (Biogelx-IKVAV) wrapped by a poly-ɛ-caprolactone (PCL) nerve conduit. ADSC retained their stemness, their immunophenotype and proliferative activity when tested in vitro. At 6 weeks post-implantation, robust regeneration was observed across the critical-size gap as evaluated by both the axonal elongation (anti-NF 200) and SC proliferation (anti-S100) within the human ADSC-IKVAV filled PCL conduit. All the other experimental groups manifested significantly lower levels of growth cone elongation. The histological gastrocnemius muscle analysis was comparable with no quantitative significant differences among the experimental groups. Taken together, these results suggest that ADSC encapsulated in Biogelx-IKVAV are a potential path to improve the efficacy of nerve regeneration. New perspectives can be pursued for the development of a fully synthetic bioengineered nerve graft for the treatment of peripheral nerve injury.
{"title":"Biogelx-IKVAV Is An Innovative Human Platelet Lysate-Adipose-Derived Stem Cells Delivery Strategy to Improve Peripheral Nerve Repair.","authors":"Martino Guiotto, Alison Clayton, Ryan Morgan, Wassim Raffoul, Andrew Hart, Mathis Riehle, Pietro di Summa","doi":"10.1089/ten.TEA.2023.0307","DOIUrl":"10.1089/ten.TEA.2023.0307","url":null,"abstract":"<p><p>Adipose-derived stem cells (ADSC) are nowadays one of the most exploited cells in regenerative medicine. They are fast growing, capable of enhancing axonal elongation, support and locally stimulate Schwann cells (SCs), and protect de-innervated muscles from atrophy after a peripheral nerve injury. With the aim of developing a bio-safe, clinically translatable cell-therapy, we assessed the effect of ADSC pre-expanded with human platelet lysate in an <i>in vivo</i> rat model, delivering the cells into a 15 mm critical-size sciatic nerve defect embedded within a laminin-peptide-functionalized hydrogel (Biogelx-IKVAV) wrapped by a poly-ɛ-caprolactone (PCL) nerve conduit. ADSC retained their stemness, their immunophenotype and proliferative activity when tested <i>in vitro</i>. At 6 weeks post-implantation, robust regeneration was observed across the critical-size gap as evaluated by both the axonal elongation (anti-NF 200) and SC proliferation (anti-S100) within the human ADSC-IKVAV filled PCL conduit. All the other experimental groups manifested significantly lower levels of growth cone elongation. The histological gastrocnemius muscle analysis was comparable with no quantitative significant differences among the experimental groups. Taken together, these results suggest that ADSC encapsulated in Biogelx-IKVAV are a potential path to improve the efficacy of nerve regeneration. New perspectives can be pursued for the development of a fully synthetic bioengineered nerve graft for the treatment of peripheral nerve injury.</p>","PeriodicalId":56375,"journal":{"name":"Tissue Engineering Part A","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140121495","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Extensively researched tissue engineering strategies involve incorporating cells into suitable biomaterials, offering promising alternatives to boost tissue repair. In this study, a hybrid scaffold, Gel-DCM, which integrates a photoreactive gelatin-hyaluronic acid hydrogel (Gel) with an oriented porous decellularized cartilage matrix (DCM), was designed to facilitate chondrogenic differentiation and cartilage repair. The Gel-DCM exhibited excellent biocompatibility in vitro, promoting favorable survival and growth of human adipose-derived stem cells (hADSCs) and articular chondrocytes (hACs). Gene expression analysis indicated that the hACs expanded within the Gel-DCM exhibited enhanced chondrogenic phenotype. In addition, Gel-DCM promoted chondrogenesis of hADSCs without the supplementation of exogenous growth factors. Following this, in vivo experiments were conducted where empty Gel-DCM or Gel-DCM loaded with hACs/hADSCs were used and implanted to repair osteochondral defects in a rat model. In the control group, no implants were delivered to the injury site. Interestingly, macroscopic, histological, and microcomputed tomography scanning results revealed superior cartilage restoration and subchondral bone reconstruction in the empty Gel-DCM group compared with the control group. Moreover, both hACs-loaded and hADSCs-loaded Gel-DCM implants exhibited superior repair of hyaline cartilage and successful reconstruction of subchondral bone, whereas defects in the control groups were predominantly filled with fibrous tissue. These observations suggest that the Gel-DCM can provide an appropriate three-dimensional chondrogenic microenvironment, and its combination with reparative cell sources, ACs or ADSCs, holds great potential for facilitating cartilage regeneration.
{"title":"A Hybrid Scaffold Induces Chondrogenic Differentiation and Enhances <i>In Vivo</i> Cartilage Regeneration.","authors":"Jiaming Zhao, Zexing Yan, Yufei Ding, Yike Dai, Ziyang Feng, Zhiyao Li, Lifeng Ma, Naicheng Diao, Ai Guo, Heyong Yin","doi":"10.1089/ten.TEA.2023.0344","DOIUrl":"10.1089/ten.TEA.2023.0344","url":null,"abstract":"<p><p>Extensively researched tissue engineering strategies involve incorporating cells into suitable biomaterials, offering promising alternatives to boost tissue repair. In this study, a hybrid scaffold, Gel-DCM, which integrates a photoreactive gelatin-hyaluronic acid hydrogel (Gel) with an oriented porous decellularized cartilage matrix (DCM), was designed to facilitate chondrogenic differentiation and cartilage repair. The Gel-DCM exhibited excellent biocompatibility <i>in vitro</i>, promoting favorable survival and growth of human adipose-derived stem cells (hADSCs) and articular chondrocytes (hACs). Gene expression analysis indicated that the hACs expanded within the Gel-DCM exhibited enhanced chondrogenic phenotype. In addition, Gel-DCM promoted chondrogenesis of hADSCs without the supplementation of exogenous growth factors. Following this, <i>in vivo</i> experiments were conducted where empty Gel-DCM or Gel-DCM loaded with hACs/hADSCs were used and implanted to repair osteochondral defects in a rat model. In the control group, no implants were delivered to the injury site. Interestingly, macroscopic, histological, and microcomputed tomography scanning results revealed superior cartilage restoration and subchondral bone reconstruction in the empty Gel-DCM group compared with the control group. Moreover, both hACs-loaded and hADSCs-loaded Gel-DCM implants exhibited superior repair of hyaline cartilage and successful reconstruction of subchondral bone, whereas defects in the control groups were predominantly filled with fibrous tissue. These observations suggest that the Gel-DCM can provide an appropriate three-dimensional chondrogenic microenvironment, and its combination with reparative cell sources, ACs or ADSCs, holds great potential for facilitating cartilage regeneration.</p>","PeriodicalId":56375,"journal":{"name":"Tissue Engineering Part A","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140337849","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}