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Genes and Tissue Culture Technology - Presentation

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  • @Christine Wong Yee Theng Hi Christine, to answer your question. There is no guaranteeing the prevention of ectopic bone formation if mesenchymal stem cells are obtained from the bone marrow, however we can supplement the cells in vitro with cytokines/growth factors that are specific for tenocyte differentiation to induce the proliferation of tenocytes in vitro. An alternative would be to harvest the stem cells from non-bone origins, e.g. adipose tissue, which can reduce the risk of in vivo ectopic bone formation and ossification at the site of implantation. Hope that answers you question, thanks!
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  • For this approach, although it leads to a better healing of tendon defects but i have found out that the ectopic formation of bone has been reported and this might due to the MSC differentiation. So is there any specific requirements for the culture medium to control the MSC differentiation or any method to improve this ectopic formation?
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  • @RenHau Hi, I would say that there is no exact answer to your question as it all depends on the nature of the cells being cultured. While some may have shown results that suggest a method is more significant compared to the others, but really I would not take that as a result that applies to all cell types. Not to forget, external factors affect cell growth too. :) Thank you for your question.
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  • @LauSuetLing Cell-derived extracellular matrix (ECM) scaffolds have received considerable interest for tissue engineering applications nowadays. In this study, ECM scaffolds derived from mesenchymal stem cell (MSC), chondrocyte, and fibroblast were prepared by culturing cells in a selectively removable poly(lactic-co-glycolic acid) (PLGA) template. However, to be more specific, fibroblast is used to lay down the collagen matrix in tendon engineering and MSC is used to create autologous tenocytes that act as the seeding cells. Hope this answers your question! :)
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  • Hello :) Is there a specific way on how the scaffold of CDM for tendons is prepared? Thanks!
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Genes and Tissue Culture Technology - Presentation

  1. 1. Cell-derived Extracellular Matrices in Tissue Engineering for Tendon Injuries By Bryan Yap (0329882), Gregary Chan (0329443), Benjamin Lee (0324669), Ong Chon Phin (0329284) & Zayd Khairil (0323394) SCT60103 Genes and Tissue Culture Technology March 2018 Semester Dr Yap Wei Hsum
  2. 2. Basic Concepts: Tissue Engineering • Building substitutes with biological function in vitro to repair defects and replace the loss of function or failure of tissue/organ (Zhang et al. 2016) • Engineered scaffolds provide a 3D structure for growth of cells and cytokines, histocompatibility, and cell signalling (Zhang et al. 2016; Castells-Sala et al. 2013) • According to Longo et al. (2012), tissue engineering is based on founded on three main principles: a. Nonimmunogenic multipotent cells, b. Scaffold for mechanical support c. Growth factors/cytokines for cell differentiation and proliferation
  3. 3. Basic Concepts: Extracellular Matrices • Composition varies among different tissues; mainly organic compounds e.g. collagen, hyaluronic acid (Zhang et al. 2016) • Involved in regulation of cell proliferation, migration, adhesion, differentiation, homeostasis, and regeneration (Zhang et al. 2016) • Decellularized tissues retain in vivo structures but present problems of autologous scarcity, host responses and pathogen transfer (Lu et al. 2011) • Cell-derived matrices closely resemble native microenvironments, can be produced in vitro and readily customised using different cell types (Zhang et al. 2016) Composition of the ECM surrounding muscle, tendon and myotendinous junctions. (Subramanian & Schilling 2015)
  4. 4. Applications: Tendon Engineering ● Tendons are highly specialized tissues that join muscles to bones; Provide stability and facilitate movement (Ramos et al. 2015). ● Due to the general acellularity of tendons, there is limited regenerative capability; Injury often leads to scar formation and decreased mechanical function (Ramos et al. 2015). ● Biosynthetic materials e.g. CDM can be implicated in tendon repair strategies. ● The ideal scaffold for tendon engineering would possess the basic structure of the tendon, native extracellular matrix, and capability of cell seeding (Longo et al. 2012). Schematic of in vitro tendon tissue engineering using autologous tenocytes. (Bagnaninchi et al. 2007)
  5. 5. Applications ➔ Using adipose-derived mesenchymal stem cells, the Achilles tendon of an injured rabbit model showed ameliorated tendon restoration, exhibiting more organised ECM deposition (Schneider et al. 2017). ➔ Oriented multi-lamellar collagen I membrane grafted into the central region of the patellar tendon (PT) of New Zealand white rabbit species to assess the tendon regenerative properties. ➔ Good graft integration without adverse side effects display that collagen I membrane as an effective tool on repair of defective tendons (Dong & Lu 2016).
  6. 6. Applications ➔ Rat Achilles tendon injury model shows higher density of collagen fibers and Col III/Col I ratio reduced when using adipose- derived MSC ➔ Rat Achilles tendon defect model, use of bone-marrow mesenchymal stem cells increase overall tendon healing due to increased production of collagens (Schneider et al. 2017) ➔ Race horses that suffer superficial flexor digitorum longus tendon (SFDLT) lesions, the use of adipose-derived mesenchymal stem cells (ADMSC) enhances healing by inducing shorter duration of lameness and improved organisation of collagen fibers (Schneider et al. 2017)
  7. 7. Applications • Grafted specimens induced with IGF-1 showed lower collagen orientation in the midsubstance and tendon-bone interface, which enhanced biomechanical properties regenerated tendons (Dines, Grande, & Dines, 2007). • Immunohistochemical analysis staining are more emminent for type III than type I collagen in male Sprague Dawley rats (Ide & Tokunaga, 2018). • At 12 months postoperatively, the ultimate load to failure was significantly lower in the graft group. (Shearn et al. 2013)
  8. 8. Applications • A cell-derived collagen I matrix seeded with mesenchymal stem cells contracted by ~50%, resulting in a more compact and surgically manageable tissue for implantation (Awad et al. 1999). • The contraction traps cells and enhances delivery to repair site • Contractions of the ECM induce cytoskeletal and morphological changes that stimulate further production of new matrices Confocal micrograph of mesenchymal stem cells with red/green fluorescent staining (ANOVA IRM Stem Cell Centre 2018)
  9. 9. Current Developments • Using Mesenchymal Cells to develop into tenocytes instead of using tenocytes themselves (Chaudhury 2012) • Mesenchymal cells are abundant in amount compared to the little number of tenocytes that may be obtained from the patient • 3-Dimensional Culture Technique (McKee and Chaudhury 2017) • Static • Spheroid Culture • Biomaterials • Dynamic • Microcarriers • Microencapsulation • Microfluidics • Temperature-responsive Culture dishes • Decreasing the temperature to 20℃ will allow cells to maintain their cell to cell junction allowing an intact cell sheet to be extracted (Tang and Okano, 2014)
  10. 10. Schematic diagrams of the traditional two-dimensional (2D) monolayer cell culture (A) and three typical three-dimensional (3D) cell culture systems: cell spheroids/aggregates grown on matrix (B), cells embedded within matrix (C), or scaffold-free cell spheroids in suspension (D) (Edmonson, Broglie, Adcock and Yang 2014).
  11. 11. Challenges • The knowledge regarding the response of the tenocyte towards the dynamic microenvironment within the tendon is poor (Grier, Iyoha, and Harley 2016). • Expanding, purifying, and comparing populations of tenocytes proves difficult (Docheva et al., 2015). • Primary cell tenocytes differentiate rapidly when secreted from the body (Grier, Iyoha, and Harley 2016). • Tendons may face a wide range of non-linear mechanical deformation due to the organised collagen structure surrounded by proteoglycans (Bagnaninchi et al. 2007). • To use techniques developed in animal models and apply them in daily practice and in the operating theatre (Bagnaninchi et al. 2007).
  12. 12. Conclusion ● Tendon disorders are frequent and cause significant morbidity however the etiology of tendinopathies is largely unknown ● Scaffolds can provide an alternative for tendon augmentation and have enormous therapeutic potential ● Cell-derived matrices are being explored for tendon regeneration as they are more customisable, less immunogenic and reduces risk of graft rejection by the recipient as compared to tissue-derived or decellularised matrices ● Current research includes inducing MSC to tenocytes as primary tenocytes are difficult to obtain and differentiate too rapidly. Various techniques are being explored e.g. 3D cultures.
  13. 13. References • Awad, H.A., Butler, D.L., Boivin, G.P., Smith, F.N.L., Malaviya, P., Huibregtse, B., & Caplan, A.I., 1999, ‘Autologous Mesenchymal Stem Cell Mediated Repair of Tendon’, Tissue Engineering, vol. 5, no. 3, pp. 267-277 • Bagnaninchi, P.O., Yang, Y., El Haj, A.J., & Maffulli, N., 2007, ‘Tissue engineering for tendon repair’, British Journal of Sports Medicine, vol. 41, no. 8, pp. 1-5 DOI: 10.1136/bjsm.2006.030643 • Castells-Sala, C., Alemany-Ribes, M., Fernandez-Muiños, T., Recha- Sancho, L., Lopez-Chicon, P., Aloy-Reverte, C., Caballero-Camino, J., Marquez-Gil, A., Semino, C.E., 2013, ‘Current Applications of Tissue Engineering in Biomedicine’, Journal of Bioengineering and Bioelectronics. DOI:10.4172/2153-0777.S2-004
  14. 14. References • Dines, J.S., Grande, D.A., and Dines D.M., 2007. Tissue engineering and rotator dcuff tendon healing, Journal of Shoulder and Elbow Surgery, vol. 16, issue 5, pp S204-S207 • Dong C.J., & Lu Y.G., 2016, ‘Application of Collagen Scaffold in Tissue Engineering: Recent Advances and New Perspectives’, Polymers, vol 8(2), p. 42. DOI:10.3390/polym8020042 • Edmonson, R, Broglie, JJ, Adcock, AF, and Yang, L, 2014. ‘Three- Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors’, Assay Drug Dev Technol, vol 12, issue 4, pp. 204 – 218. DOI: 10.1089/adt.2014.573 • Fitzpatrick, LE, and Mc Devitt, TC, 2014. Cell-derived matrices for tissue engineering and regenerative medicine applications, Biomaterial Science, 2015 (1), 2, 12-24.
  15. 15. References • Grier, W.K., Iyoha, E.M., & Harley, B.A.C., 2016, ‘The influence of pore size and stiffness on tenocyte bioactivity and transcriptomic stability in collagen-GAG scaffolds’, Journal of the Mechanical Behavior of Biomedical Materials, vol. 65, pp. 295-305 DOI: http://dx.doi.org/10.1016/j.jmbbm.2016.08.034 • Ide, J., Tokunaga, T., 2018. Rotator cuff tendon-to-bone healing at 12 months after patch grafting of acellular dermal matrix in an animal model, Journal of Orthopaedic Science, vol. 23, issue2, pp 207-212 • Longo, U.G., Lamberti, A., Petrillo, S., Maffulli, N., & Denaro, V., 2012, ‘Scaffolds in Tendon Tissue Engineering’, Stem Cells International, vol. 2012. DOI:10.1155/2012/517165
  16. 16. References • Lu, H., Hoshiba, T., Kawazoe, N., Koda, I., Song, M., Chen, G., 2011, ‘Cultured cell-derived extracellular matrix scaffolds for tissue engineering’, Biomaterials, vol. 32, pp. 9658-9666. DOI:10.1016/j.biomaterials.2011.08.091 • Ramos, D., Peach, M.S., Mazzaocca, A.D., Yu, X., Kumbar, S.G., 2015, ‘Tendon tissue engineering’, Regenerative Engineering of Musculoskeletal Tissues and Interfaces, pp. 195-217 DOI:10.1016/B978-1-78242-301-0.00008-2 • Schneider, M., Angele, P., Jarvinen, T.A.H., & Docheva, D., 2017, ‘Rescue plan for Achilles: Therapeutics steering the fate and functions of stem cells in tendon wound healing’, Advanced Drug Delivery Reviews, pp. 1-24. DOI:https://doi.org/10.1016/j.addr.2017.12.016
  17. 17. References • Shearn, J.T., Kinneberg, K.R.C., Dyment, N.A., Galloway, M.T., Kenter, K, Wylie, C, and Butler, DL, 2013. Tendon Tissue Engineering: Progress, Challenges, and Translation to the Clinic, Journal Musculoskeletal and Neuronal Interactions, 11(2), pp 163- 173. • Subramanian, A., & Schilling, T.F., 2015, ‘Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix’, The Company of Biologists, vol. 142, pp. 4191-4204 DOI:10.1242/dev.114777 • Zhang, W., Zhu, Y., Guo, Q., Peng, J., Liu, S., Yang, J., Wang,. Y., 2016, ‘Cell-Derived Extracellular Matrix: Basic Characteristics and Current Applications in Orthopedic Tissue Engineering,’ Tissue Engineering: Part B, vol. 22(3), pp. 193-207. DOI: 10.1089/ten.teb.2015.0290

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