This document summarizes research on using cell-derived extracellular matrices for tendon tissue engineering and repair. It discusses how cell-derived matrices more closely resemble native tissue microenvironments compared to decellularized tissues. Studies show cell-derived matrices seeded with mesenchymal stem cells can improve tendon healing in animal models by enhancing extracellular matrix deposition and collagen organization. Current research focuses on developing three-dimensional culture techniques to better understand tendon cell behavior and generate tissues for implantation. Challenges include a lack of knowledge about the tendon microenvironment and difficulties expanding and comparing tendon cell populations.

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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