22 research outputs found
Transient hypoxia improves matrix properties in tissue engineered cartilage
No full textAdult articular cartilage is a hypoxic tissue, with oxygen tension ranging from <10% at the cartilage surface to <1% in the deepest layers. In addition to spatial gradients, cartilage development is also associated with temporal changes in oxygen tension. However, a vast majority of cartilage tissue engineering protocols involves cultivation of chondrocytes or their progenitors under ambient oxygen concentration (21% O2), that is, significantly above physiological levels in either developing or adult cartilage. Our study was designed to test the hypothesis that transient hypoxia followed by normoxic conditions results in improved quality of engineered cartilaginous ECM. To this end, we systematically compared the effects of normoxia (21% O2 for 28 days), hypoxia (5% O2 for 28 days) and transient hypoxiareoxygenation (5% O2 for 7 days and 21% O2 for 21 days) on the matrix composition and expression of the chondrogenic genes in cartilage constructs engineered in vitro. We demonstrated that reoxygenation had the most effect on the expression of cartilaginous genes including COL2A1, ACAN, and SOX9 and increased tissue concentrations of amounts of glycosaminoglycans and type II collagen. The equilibrium Young's moduli of tissues grown under transient hypoxia (510.01 +/- 28.15kPa) and under normoxic conditions (417.60 +/- 68.46kPa) were significantly higher than those measured under hypoxic conditions (279.61 +/- 20.52kPa). These data suggest that the cultivation protocols utilizing transient hypoxia with reoxygenation have high potential for efficient cartilage tissue engineering, but need further optimization in order to achieve higher mechanical functionality of engineered constructs. (c) 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 544553, 201
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Preconditioning Methods in Cartilage Tissue Engineering: Influences of Silk Material Properties and Hypoxia on Chondrogenesis
Full text linkCartilage has limited intrinsic healing potential, due to the low cell density and the lack of blood supply. Current treatments for cartilage repair rarely restore full structure and function to the native state. Tissue engineering holds promise to create cartilage grafts capable to withstand the stresses present in joints. More than 90% of articular cartilage tissue is composed of extracellular matrix and is located in the loading environment under low oxygen tension in knee joints. To form engineered constructs with mechanical properties compatible to native tissue, scaffolds should provide structural support, maintain cell phenotype and subsequently promote tissue development. The focus of this dissertation is on utilizing the physiological conditions found in joints to regulate biological behavior of cells. The first factor that was studied was the extracellular matrix.
Two formats of silk fibroin-hydrogel and porous scaffolds - were examined for their potential as a supporting material for creating cartilage tissue constructs. The composite silk made from nano-fibers and hydrogel - a structure resembling the collagen network and proteoglycan in native cartilage - improved equilibrium and dynamic modulus of engineered tissue by 50% and 60%, respectively, in comparison to silk hydrogel without fibers. The second factor studied was the modulation of oxygen level, which is a major regulator during native cartilage development. Chondrogenic differentiation was induced in human embryonic stem cells under hypoxic conditions, in conjunction with biochemical cues from bovine chondrocytes.
As a result, SOX9, a key transcription factor of cartilaginous lineage, was upregulated in the induced cells. Subsequent cultivation under normoxic conditions resulted in robust formation of cartilage tissue. Taken together, studies conducted in my thesis work address two major challenges in cartilage tissue engineering: i) providing cells with structural and mechanical properties similar to native ECM for generating in vitro cartilaginous tissue and ii) preconditioning cells with physiological environment for directing chondrogenic differentiation
Using Vertebrate Stem and Progenitor Cells for Cellular Agriculture, State-of-the-Art, Challenges, and Future Perspectives
No full textGlobal food systems are under significant pressure to provide enough food, particularly protein-rich foods whose demand is on the rise in times of crisis and inflation, as presently existing due to post-COVID-19 pandemic effects and ongoing conflict in Ukraine and resulting in looming food insecurity, according to FAO. Cultivated meat (CM) and cultivated seafood (CS) are protein-rich alternatives for traditional meat and fish that are obtained via cellular agriculture (CA) i.e., tissue engineering for food applications. Stem and progenitor cells are the building blocks and starting point for any CA bioprocess. This review presents CA-relevant vertebrate cell types and procedures needed for their myogenic and adipogenic differentiation since muscle and fat tissue are the primary target tissues for CM/CS production. The review also describes existing challenges, such as a need for immortalized cell lines, or physical and biochemical parameters needed for enhanced meat/fat culture efficiency and ways to address them
Silk microfiber-reinforced silk hydrogel composites for functional cartilage tissue repair
No full textCartilage tissue lacks an intrinsic capacity for self-regeneration due to slow matrix turnover, a limited supply of mature chondrocytes and insufficient vasculature. Although cartilage tissue engineering has achieved some success using agarose as a scaffolding material, major challenges of agarose-based cartilage repair, including non-degradability, poor tissue-scaffold integration and limited processing capability, have prompted the search for an alternative biomaterial. In this study, silk fiber-hydrogel composites (SF-silk hydrogels) made from silk microfibers and silk hydrogels were investigated for their potential use as a support material for engineered cartilage. We demonstrated the use of 100% silk-based fiber-hydrogel composite scaffolds for the development of cartilage constructs with properties comparable to those made with agarose. Cartilage constructs with an equilibrium modulus in the native tissue range were fabricated by mimicking the collagen fiber and proteoglycan composite architecture of native cartilage using biocompatible, biodegradable silk fibroin from Bombyx mari. Excellent chondrocyte response was observed on SF-silk hydrogels, and fiber reinforcement resulted in the development of more mechanically robust constructs after 42 days in culture compared to silk hydrogels alone. Thus, we demonstrate the versatility of silk fibroin as a composite scaffolding material for use in cartilage tissue repair to create functional cartilage constructs that overcome the limitations of agarose biomaterials, and provide a much-needed alternative to the agarose standard. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved
Effect of Silver Sulfadiazine and Metallic Ions on Properties of Thai Silk Fibroin/Gelatin Films for Anti-Bacterial Applications
Full text linkThis study aimed to develop Thai silk fibroin/gelatin (SF/GA) films incorporating various concentrations of silver sulfadiazine (SSD) and to investigate the effects of SSD and metallic ions (Ag(I) and Cu(II)) on chemical conformation of the SF/GA films. We found that the incorporation of SSD in the films changed the conformation of silk fibroin protein by increasing β-sheet content. The same phenomena was also observed with the other 2 metallic ions, Ag(I) and Cu(II), incorporated in the SF/GA films. This effect of metallic ions on the SF conformation transition in the SF/GA films may be similar to the phenomenon occurred during natural spinning process of the Bombyx mori silkworm. The SF/GA films incorporating SSD had therefore more stability, less water-insoluble fraction and extended degradation rate than the SF/GA films without SSD due to the higher content of stable β-sheet conformation. When cultured with L929 mouse fibroblast cells according to ISO 10993 part 5 standard, the SF/GA films incorporating SSD at all concentrations showed no cytotoxicity. The SSD released from the films also showed obvious anti-bacterial activity against Staphylococcus aureus (Gram positive) and Escherichia coli (Gram negative) bacteria. We suggested that the SF/GA films incorporating SSD may be useful for some medical applications in which anti-bacterial effect was required
Using Vertebrate Stem and Progenitor Cells for Cellular Agriculture, State-of-the-Art, Challenges, and Future Perspectives
No full textAbstract
Global food systems are under significant pressure to provide enough food, particularly protein-rich foods whose demand is on the rise in times of crisis and inflation, as presently existing due to post-COVID-19 pandemic effects and ongoing conflict in Ukraine and resulting in looming food insecurity, according to FAO. Cultivated meat (CM) and cultivated seafood (CS) are protein-rich alternatives for traditional meat and fish that are obtained via cellular agriculture (CA) i.e., tissue engineering for food applications. Stem and progenitor cells are the building blocks and starting point for any CA bioprocess. This review presents CA-relevant vertebrate cell types and procedures needed for their myogenic and adipogenic differentiation since muscle and fat tissue are the primary target tissues for CM/CS production. The review also describes existing challenges, such as a need for immortalized cell lines, or physical and biochemical parameters needed for enhanced meat/fat culture efficiency and ways to address them
Human adipose-derived cells can serve as a single-cell source for the in vitro cultivation of vascularized bone grafts
No full textOrthopaedic surgery often requires bone grafts to correct large defects resulting from congenital defects, surgery or trauma. Great improvements have been made in the tissue engineering of bone grafts. However, these grafts lack the vascularized component that is critical for their survival and function. From a clinical perspective, it would be ideal to engineer vascularized bone grafts starting from one single-cell harvest obtained from the patient. To this end, we explored the potential of human adipose-derived mesenchymal stem cells (hASCs) as a single-cell source for osteogenic and endothelial differentiation and the assembly of bone and vascular compartments within the same scaffold. hASCs were encapsulated in fibrin hydrogel as an angioinductive material for vascular formation, combined with a porous silk fibroin sponge to support osteogenesis, and subjected to sequential application of growth factors. Three strategies were evaluated by changing spatiotemporal cues: (a) induction of osteogenesis prior to vasculogenesis; (b) induction of vasculogenesis prior to osteogenesis; or (c) simultaneous induction of osteogenesis and vasculogenesis. By 5 weeks of culture, bone-like tissue development was evidenced by the deposition of bone matrix proteins, alkaline phosphatase activity and calcium deposition, along with the formation of vascular networks, evidenced by endothelial cell surface markers, such as CD31 and von Willebrand factor, and morphometric analysis. Most robust development of the two tissue compartments was achieved by sequential induction of osteogenesis followed by the induction of vasculogenesis. Taken together, the collected data strongly support the utility of hASCs as a single-cell source for the formation of vascularized bone tissue.We gratefully acknowledge funding support of this work by the National Institutes of Health (NIH; Grant Nos DE161525 and EB02520, to GVN) and an FCT PhD Grant (No. SFRH/BD/42316/2007, to CC). The authors thank Darja Marolt and Supansa Yodmuang for their help with the experiments and David Kaplan for providing the silk scaffolds used in this study
A novel gelatin/chitooligosaccharide/demineralized bone matrix composite scaffold and periosteum-derived mesenchymal stem cells for bone tissue engineering
No full textAbstract Background A novel biodegradable scaffold including gelatin (G), chitooligosaccharide (COS), and demineralized bone matrix (DBM) could play a significant part in bone tissue engineering. The present study aimed to investigate the biological characteristics of composite scaffolds in combination of G, COS, and DBM for in vitro cell culture and in vivo animal bioassays. Methods Three-dimensional scaffolds from the mixture of G, COS, and DBM were fabricated into 3 groups, namely, G, GC, and GCD using a lyophilization technique. The scaffolds were cultured with mesenchymal stem cells (MSCs) for 4 weeks to determine biological responses such as cell attachment and cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, cell morphology, and cell surface elemental composition. For the in vivo bioassay, G, GC, and GCD, acellular scaffolds were implanted subcutaneously in 8-week-old male Wistar rats for 4 weeks and 8 weeks. The explants were assessed for new bone formation using hematoxylin and eosin (H&E) staining and von Kossa staining. Results The MSCs could attach and proliferate on all three groups of scaffolds. Interestingly, the ALP activity of MSCs reached the greatest value on day 7 after cultured on the scaffolds, whereas the calcium assay displayed the highest level of calcium in MSCs on day 28. Furthermore, weight percentages of calcium and phosphorus on the surface of MSCs after cultivation on the GCD scaffolds increased when compared to those on other scaffolds. The scanning electron microscopy images showed that MSCs attached and proliferated on the scaffold surface thoroughly over the cultivation time. Mineral crystal aggregation was evident in GC and greatly in GCD scaffolds. H&E staining illustrated that G, GC, and GCD scaffolds displayed osteoid after 4 weeks of implantation and von Kossa staining confirmed the mineralization at 8 weeks in G, GC, and GCD scaffolds. Conclusion The MSCs cultured in GCD scaffolds revealed greater osteogenic differentiation than those cultured in G and GC scaffolds. Additionally, the G, GC, and GCD scaffolds could promote in vivo ectopic bone formation in rat model. The GCD scaffolds exhibited maximum osteoinductive capability compared with others and may be potentially used for bone regeneration
