scholarly journals A Single-Cell Culture System for Dissecting Microenvironmental Signaling in Development and Disease of Cartilage Tissue

2021 ◽  
Vol 9 ◽  
Author(s):  
Jade Tassey ◽  
Arijita Sarkar ◽  
Ben Van Handel ◽  
Jinxiu Lu ◽  
Siyoung Lee ◽  
...  
Keyword(s):  
Single Cell ◽  
Articular Chondrocytes ◽  
3D Culture ◽  
Cartilage Regeneration ◽  
Culture Method ◽  
Cartilage Tissue ◽  
Surrounding Matrix ◽  
Clonogenic Potential

Cartilage tissue is comprised of extracellular matrix and chondrocytes, a cell type with very low cellular turnover in adults, providing limited capacity for regeneration. However, in development a significant number of chondrocytes actively proliferate and remodel the surrounding matrix. Uncoupling the microenvironmental influences that determine the balance between clonogenic potential and terminal differentiation of these cells is essential for the development of novel approaches for cartilage regeneration. Unfortunately, most of the existing methods are not applicable for the analysis of functional properties of chondrocytes at a single cell resolution. Here we demonstrate that a novel 3D culture method provides a long-term and permissive in vitro niche that selects for highly clonogenic, colony-forming chondrocytes which maintain cartilage-specific matrix production, thus recapitulating the in vivo niche. As a proof of concept, clonogenicity of Sox9IRES–EGFP mouse chondrocytes is almost exclusively found in the highest GFP+ fraction known to be enriched for chondrocyte progenitor cells. Although clonogenic chondrocytes are very rare in adult cartilage, we have optimized this system to support large, single cell-derived chondrogenic organoids with complex zonal architecture and robust chondrogenic phenotype from adult pig and human articular chondrocytes. Moreover, we have demonstrated that growth trajectory and matrix biosynthesis in these organoids respond to a pro-inflammatory environment. This culture method offers a robust, defined and controllable system that can be further used to interrogate the effects of various microenvironmental signals on chondrocytes, providing a high throughput platform to assess genetic and environmental factors in development and disease.

scholarly journals 3D Hepatic Organoid-Based Advancements in LIVER Tissue Engineering

2021 ◽  
Vol 8 (11) ◽  
pp. 185
Author(s):  
Amit Panwar ◽  
Prativa Das ◽  
Lay Poh Tan
Keyword(s):  
Tissue Engineering ◽  
Self Assembly ◽  
Liver Tissue ◽  
3D Culture ◽  
Culture Method ◽  
Culture Conditions ◽  
Phenotypic Properties ◽  
Liver Tissue Engineering

Liver-associated diseases and tissue engineering approaches based on in vitro culture of functional Primary human hepatocytes (PHH) had been restricted by the rapid de-differentiation in 2D culture conditions which restricted their usability. It was proven that cells growing in 3D format can better mimic the in vivo microenvironment, and thus help in maintaining metabolic activity, phenotypic properties, and longevity of the in vitro cultures. Again, the culture method and type of cell population are also recognized as important parameters for functional maintenance of primary hepatocytes. Hepatic organoids formed by self-assembly of hepatic cells are microtissues, and were able to show long-term in vitro maintenance of hepato-specific characteristics. Thus, hepatic organoids were recognized as an effective tool for screening potential cures and modeling liver diseases effectively. The current review summarizes the importance of 3D hepatic organoid culture over other conventional 2D and 3D culture models and its applicability in Liver tissue engineering.

scholarly journals Chondrogenic Potential of Dental-Derived Mesenchymal Stromal Cells

Osteology ◽  
2021 ◽  
Vol 1 (3) ◽  
pp. 149-174
Author(s):  
Naveen Jeyaraman ◽  
Gollahalli Shivashankar Prajwal ◽  
Madhan Jeyaraman ◽  
Sathish Muthu ◽  
Manish Khanna
Keyword(s):  
Tissue Engineering ◽  
Mesenchymal Stromal Cells ◽  
Tissue Regeneration ◽  
Stromal Cells ◽  
Cartilage Tissue Engineering ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Dentin Matrix

The field of tissue engineering has revolutionized the world in organ and tissue regeneration. With the robust research among regenerative medicine experts and researchers, the plausibility of regenerating cartilage has come into the limelight. For cartilage tissue engineering, orthopedic surgeons and orthobiologists use the mesenchymal stromal cells (MSCs) of various origins along with the cytokines, growth factors, and scaffolds. The least utilized MSCs are of dental origin, which are the richest sources of stromal and progenitor cells. There is a paradigm shift towards the utilization of dental source MSCs in chondrogenesis and cartilage regeneration. Dental-derived MSCs possess similar phenotypes and genotypes like other sources of MSCs along with specific markers such as dentin matrix acidic phosphoprotein (DMP) -1, dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and STRO-1. Concerning chondrogenicity, there is literature with marginal use of dental-derived MSCs. Various studies provide evidence for in-vitro and in-vivo chondrogenesis by dental-derived MSCs. With such evidence, clinical trials must be taken up to support or refute the evidence for regenerating cartilage tissues by dental-derived MSCs. This article highlights the significance of dental-derived MSCs for cartilage tissue regeneration.

scholarly journals Spatiotemporal regulation of endogenous MSCs using a functional injectable hydrogel system for cartilage regeneration

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Yunsheng Dong ◽  
Yufei Liu ◽  
Yuehua Chen ◽  
Xun Sun ◽  
Lin Zhang ◽  
...  
Keyword(s):  
Cartilage Tissue Engineering ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Cartilage Defects ◽  
In Vivo Experiments ◽  
Spatiotemporal Regulation ◽  
Stromal Cell Derived Factor ◽  
Hydrogel System

AbstractHydrogels have been extensively favored as drug and cell carriers for the repair of knee cartilage defects. Recruiting mesenchymal stem cells (MSCs) in situ to the defect region could reduce the risk of contamination during cell delivery, which is a highly promising strategy to enhance cartilage repair. Here, a cell-free cartilage tissue engineering (TE) system was developed by applying an injectable chitosan/silk fibroin hydrogel. The hydrogel system could release first stromal cell-derived factor-1 (SDF-1) and then kartogenin (KGN) in a unique sequential drug release mode, which could spatiotemporally promote the recruitment and chondrogenic differentiation of MSCs. This system showed good performance when formulated with SDF-1 (200 ng/mL) and PLGA microspheres loaded with KGN (10 μΜ). The results showed that the hydrogel had good injectability and a reticular porous structure. The microspheres were distributed uniformly in the hydrogel and permitted the sequential release of SDF-1 and KGN. The results of in vitro experiments showed that the hydrogel system had good cytocompatibility and promoted the migration and differentiation of MSCs into chondrocytes. In vivo experiments on articular cartilage defects in rabbits showed that the cell-free hydrogel system was beneficial for cartilage regeneration. Therefore, the composite hydrogel system shows potential for application in cell-free cartilage TE.

Influence of Chondrocyte Zone on Co-Cultures With Mesenchymal Stem Cells in HA Hydrogels for Cartilage Tissue Engineering

2012 ◽  
Author(s):  
Minwook Kim ◽  
Jason A. Burdick ◽  
Robert L. Mauck
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Hyaluronic Acid ◽  
Articular Chondrocytes ◽  
Cartilage Tissue Engineering ◽  
3D Culture ◽  
Cartilage Tissue ◽  
Cell Type

Mesenchymal stem cells (MSCs) are an attractive cell type for cartilage tissue engineering in that they can undergo chondrogenesis in a variety of 3D contexts [1]. Focused efforts in MSC-based cartilage tissue engineering have recently culminated in the formation of biologic materials possessing biochemical and functional mechanical properties that match that of the native tissue [2]. These approaches generally involve the continuous or intermittent application of pro-chondrogenic growth factors during in vitro culture. For example, in one recent study, we showed robust construct maturation in MSC-seeded hyaluronic acid (HA) hydrogels transiently exposed to high levels of TGF-β3 [3]. Despite the promise of this approach, MSCs are a multipotent cell type and retain a predilection towards hypertrophic phenotypic conversion (i.e., bone formation) when removed from a pro-chondrogenic environment (e.g., in vivo implantation). Indeed, even in a chondrogenic environment, many MSC-based cultures express pre-hypertrophic markers, including type X collagen, MMP13, and alkaline phosphatase [4]. To address this issue, recent studies have investigated co-culture of human articular chondrocytes and MSCs in both pellet and hydrogel environments. Chondrocytes appear to enhance the initial efficiency of MSC chondrogenic conversion, as well as limit hypertrophic changes in some instances (potentially via secretion of PTHrP and/or other factors) [5–7]. While these findings are intriguing, articular cartilage has a unique depth-dependent morphology including zonal differences in chondrocyte identity. Ng et al. showed that zonal chondrocytes seeded in a bi-layered agarose hydrogel construct can recreate depth-dependent cellular and mechanical heterogeneity, suggesting that these identities are retained with transfer to 3D culture systems [8]. Further, Cheng et al. showed that differences in matrix accumulation and hypertrophy in zonal chondrocytes was controlled by bone morphogenic protein [9]. To determine whether differences in zonal chondrocyte identity influences MSC fate decisions, we evaluated functional properties and phenotypic stability in photocrosslinked hyaluronic acid (HA) hydrogels using distinct, zonal chondrocyte cell fractions co-cultured with bone marrow derived MSCs.

scholarly journals Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications

Materials ◽  
2019 ◽  
Vol 12 (4) ◽  
pp. 640 ◽  
Author(s):  
Serena Duchi ◽  
Stephanie Doyle ◽  
Timon Eekel ◽  
Cathal D. O’Connell ◽  
Cheryl Augustine ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Cartilage Regeneration ◽  
Light Sheet ◽  
Cartilage Tissue ◽  
Cartilage Defects ◽  
Label Free ◽  
Microscopy Imaging ◽  
Microscopy Technique

Cartilage defects and diseases remain major clinical issues in orthopaedics. Biomanufacturing is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. However, several limitations of in vitro and experimental animal models pose serious challenges to the translation of preclinical findings into clinical practice. Ex vivo models are of great value for translating in vitro tissue engineered approaches into clinically relevant conditions. Our aim is to obtain a viable human osteochondral (OC) model to test hydrogel-based materials for cartilage repair. Here we describe a detailed step-by-step framework for the generation of human OC plugs, their culture in a perfusion device and the processing procedures for histological and advanced microscopy imaging. Our ex vivo OC model fulfils the following requirements: the model is metabolically stable for a relevant culture period of 4 weeks in a perfusion bioreactor, the processing procedures allowed for the analysis of 3 different tissues or materials (cartilage, bone and hydrogel) without compromising their integrity. We determined a protocol and the settings for a non-linear microscopy technique on label free sections. Furthermore, we established a clearing protocol to perform light sheet-based observations on the cartilage layer without the need for tedious and destructive histological procedures. Finally, we showed that our OC system is a clinically relevant in terms of cartilage regeneration potential. In conclusion, this OC model represents a valuable preclinical ex vivo tool for studying cartilage therapies, such as hydrogel-based bioscaffolds, and we envision it will reduce the number of animals needed for in vivo testing.

scholarly journals Effect of different aged cartilage ECM on chondrogenesis of BMSCs in vitro and in vivo

2020 ◽  
Vol 7 (6) ◽  
pp. 583-595
Author(s):  
Xiuyu Wang ◽  
Yan Lu ◽  
Wan Wang ◽  
Qiguang Wang ◽  
Jie Liang ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Current Knowledge ◽  
Cartilage Tissue Engineering ◽  
Cartilage Regeneration ◽  
Biological Properties ◽  
Cartilage Tissue ◽  
Before And After ◽  
Marrow Mesenchymal Stem Cells

Abstract Extracellular matrix (ECM)-based biomaterials are promising candidates in cartilage tissue engineering by simulating the native microenvironment to regulate the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) without exogenous growth factors. The biological properties of ECM scaffolds are primarily depended on the original source, which would directly influence the chondrogenic effects of the ECM materials. Despite the expanding investigations on ECM scaffolds in recent years, the selection of optimized ECM materials in cartilage regeneration was less reported. In this study, we harvested and compared the articular cartilage ECM from newborn, juvenile and adult rabbits. The results demonstrated the significant differences in the mechanical strength, sulphated glycosaminoglycan and collagen contents of the different aged ECM, before and after decellularization. Consequently, different compositional and mechanical properties were shown in the three ECM-based collagen hydrogels, which exerted age-dependent chondrogenic inducibility. In general, both in vitro and in vivo results suggested that the newborn ECM promoted the most chondrogenesis of BMSCs but led to severe matrix calcification. In contrast, BMSCs synthesized the lowest amount of cartilaginous matrix with minimal calcification with adult ECM. The juvenile ECM achieved the best overall results in promoting chondrogenesis of BMSCs and preventing matrix calcification. Together, this study provides important information to our current knowledge in the design of future ECM-based biomaterials towards a successful repair of articular cartilage.

scholarly journals Metabolomic Profiling and Mechanotransduction of Single Chondrocytes Encapsulated in Alginate Microgels

2020 ◽  
Author(s):  
Jacob P. Fredrikson ◽  
Priyanka Brahmachary ◽  
Ebru Erdoğan ◽  
Zach Archambault ◽  
Ronald K. June ◽  
...  
Keyword(s):  
Single Cell ◽  
Dynamic Compression ◽  
Cartilage Tissue Engineering ◽  
Cell Encapsulation ◽  
Cartilage Tissue ◽  
Human Articular Cartilage ◽  
Mechanical Loads ◽  
Single Cell Encapsulation

AbstractHuman articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened resulting in increased chondrocyte stress. As chondrocytes are responsible for cartilage synthesis and maintenance, it is important to understand how mechanical loads affect cellular responses of chondrocytes. Many studies have examined the chondrocyte response to in vitro mechanical loading by embedding in stiff agarose. However, these experiments are mostly performed in the absence of PCM which may obscure important responses to mechanotransduction. Here, we demonstrate that drop-based microfluidics allows culture of single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes form PCM over 10 days in these single cell microenvironments. Single cell microgels and monolayer controls were encapsulated in high stiffness agarose to mimic the cartilage PCM. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.

scholarly journals A novel construct with biomechanical flexibility for articular cartilage regeneration

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Baixiang Cheng ◽  
Teng Tu ◽  
Xiao Shi ◽  
Yanzheng Liu ◽  
Ying Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Hydrostatic Pressure ◽  
In Vitro Study ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Temporomandibular Joints ◽  
Marrow Mesenchymal Stem Cells

Abstract Background Although tissue-engineered cartilage has been broadly studied, complete integration of regenerated cartilage with residual cartilage is still difficult for the inferior mechanical and biochemical feature of neocartilage. Chondrogenesis of mesenchymal stem cells can be induced by biophysical and biochemical factors. Methods In this study, autologous platelet-rich fibrin (PRF) membrane was used as a growth factor-rich scaffold that may facilitate differentiation of the transplanted bone marrow mesenchymal stem cells (BMSCs). At the same time, hydrostatic pressure was adopted for pre-adjustment of the seed cells before transplantation that may promote the mechanical flexibility of neocartilage. Results An in vitro study showed that the feasible hydrostatic pressure stimulation substantially promoted the chondrogenic potential of in vitro-cultured BMSC/PRF construct. In vivo results revealed that at every time point, the newborn tissues were the most favorable in the pressure-pretreated BMSC/PRF transplant group. Besides, the transplantation of feasible hydrostatic pressure-pretreated construct by BMSC sheet fragments and PRF granules could obviously improve the integration between the regenerated cartilage and host cartilage milieu, and thereby achieve boundaryless repair between the neocartilage and residual host cartilage tissue in rabbit temporomandibular joints. It could be concluded that feasible hydrostatic pressure may effectively promote the proliferation and chondrogenic differentiation of BMSCs in a BMSC/PRF construct. Conclusion This newly formed construct with biomechanical flexibility showed a superior capacity for cartilage regeneration by promoting the mechanical properties and integration of neocartilage.

scholarly journals Emulsion-free chitosan–genipin microgels for growth plate cartilage regeneration

2021 ◽  
pp. 088532822199989
Author(s):  
Christopher Erickson ◽  
Michael Stager ◽  
Michael Riederer ◽  
Karin A Payne ◽  
Melissa Krebs
Keyword(s):  
Growth Plate ◽  
Bone Growth ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Growth Plate Cartilage ◽  
Rat Growth ◽  
Growth Plate Injury ◽  
Syringe Filter

The growth plate is a cartilage tissue near the ends of children’s long bones and is responsible for bone growth. Injury to the growth plate can result in the formation of a ‘bony bar’ which can span the growth plate and result in bone growth abnormalities in children. Biomaterials such as chitosan microgels could be a potential treatment for growth plate injuries due to their chondrogenic properties, which can be enhanced through loading with biologics. They are commonly fabricated via an emulsion method, which involves solvent rinses that are cytotoxic. Here, we present a high throughput, non-cytotoxic, non-emulsion-based method to fabricate chitosan–genipin microgels. Chitosan was crosslinked with genipin to form a hydrogel network, and then pressed through a syringe filter using mesh with various pore sizes to produce a range of microgel particle sizes. The microgels were then loaded with chemokines and growth factors and their release was studied in vitro. To assess the applicability of the microgels for growth plate cartilage regeneration, they were injected into a rat growth plate injury. They led to increased cartilage repair tissue and were fully degraded by 28 days in vivo. This work demonstrates that chitosan microgels can be fabricated without solvent rinses and demonstrates their potential for the treatment of growth plate injuries.

scholarly journals Cartilage Tissue Engineering Approaches Need to Assess Fibrocartilage When Hydrogel Constructs Are Mechanically Loaded

2022 ◽  
Vol 9 ◽  
Author(s):  
Hamed Alizadeh Sardroud ◽  
Tasker Wanlin ◽  
Xiongbiao Chen ◽  
B. Frank Eames
Keyword(s):  
Tissue Engineering ◽  
Collagen Type ◽  
Hyaline Cartilage ◽  
Imaging Techniques ◽  
Cartilage Tissue Engineering ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Type I

Chondrocytes that are impregnated within hydrogel constructs sense applied mechanical force and can respond by expressing collagens, which are deposited into the extracellular matrix (ECM). The intention of most cartilage tissue engineering is to form hyaline cartilage, but if mechanical stimulation pushes the ratio of collagen type I (Col1) to collagen type II (Col2) in the ECM too high, then fibrocartilage can form instead. With a focus on Col1 and Col2 expression, the first part of this article reviews the latest studies on hyaline cartilage regeneration within hydrogel constructs that are subjected to compression forces (one of the major types of the forces within joints) in vitro. Since the mechanical loading conditions involving compression and other forces in joints are difficult to reproduce in vitro, implantation of hydrogel constructs in vivo is also reviewed, again with a focus on Col1 and Col2 production within the newly formed cartilage. Furthermore, mechanotransduction pathways that may be related to the expression of Col1 and Col2 within chondrocytes are reviewed and examined. Also, two recently-emerged, novel approaches of load-shielding and synchrotron radiation (SR)–based imaging techniques are discussed and highlighted for future applications to the regeneration of hyaline cartilage. Going forward, all cartilage tissue engineering experiments should assess thoroughly whether fibrocartilage or hyaline cartilage is formed.

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