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Medicina

Anvendelse af en tre-dimensionel Uniaxial Mekanisk Stimulation Bioreactor System til at fremkalde tenogen differentiering af senen-afledte stamceller

Published: August 01, 2020
doi:
10.3791/61278
, , , , , , ,
1Centre of Orthopaedic Translational Research, Medical School,University of Western Australia

Summary

Et tredimensionelt uniaxial mekanisk stimulationsbioreaktorsystem er et ideelt bioreaktor til tenogen-specifik differentiering af seneafledte stamceller og neosendannelse.

Abstract

Tendinopati er en almindelig kronisk senesygdom i forbindelse med inflammation og degeneration i et ortopædisk område. Med høj sygelighed, begrænset selvreparerende kapacitet og, vigtigst af alt, ingen endelige behandlinger, tendinopati stadig påvirker patienternes livskvalitet negativt. Sene-afledte stamceller (TDSCs), som primære prækursor celler af senenceller, spiller en afgørende rolle i både udviklingen af tendinopati, og funktionelle og strukturelle restaurering efter tendinopati. Således, en metode, der kan in vitro efterligne in vivo differentiering af TDSCs i senen celler ville være nyttig. Her beskriver denne protokol en metode baseret på et tredimensionelt (3D) uniaxial stretching system til at stimulere TDSCs at differentiere i sene-lignende væv. Der er syv faser af denne protokol: isolering af mus TDSCs, kultur og udvidelse af mus TDSCs, forberedelse af stimulation kultur medium for celleark dannelse, celle ark dannelse ved liggende i stimulation medium, forberedelse af 3D senen stamceller konstruere, samling af uniaxial-stretching mekanisk stimulation kompleks, og evaluering af den mekaniske stimuleret in vitro senen-lignende væv. Virkningen blev påvist ved histologi. Hele proceduren tager mindre end 3 uger. For at fremme ekstracellulær matrix aflejring, 4.4 mg/ml ascorbinsyre blev brugt i stimulation kultur medium. Et adskilt kammer med en lineær motor giver nøjagtig mekanisk belastning og er bærbar og let justeres, som anvendes til bioreaktoren. Lastningsordningen i denne protokol var 6% stamme, 0,25 Hz, 8 timer, efterfulgt af 16 h hvile i 6 dage. Denne protokol kunne efterligne celle differentiering i senen, hvilket er nyttigt for undersøgelsen af den patologiske proces med tendinopati. Desuden er senen-lignende væv potentielt bruges til at fremme senen healing i senen skade som en manipuleret autolog graft. Sammenfattende er denne protokol enkel, økonomisk, reproducerbar og gyldig.

Introduction

Tendinopati er en af de almindelige sportsskader. Det er hovedsageligt manifesteret ved smerte, lokale hævelse, nedsat muskelspændinger i det berørte område, og dysfunktion. Forekomsten af tendinopati er høj. Tilstedeværelsen af Achilles tendinopati er mest almindelig for mellem- og langdistanceløbere (op til 29%), mens tilstedeværelsen af patellar tendinopati er også høj i atleter af volleyball (45%), basketball (32%), atletik (23%), håndbold (15%), og fodbold (13%)1,2,,3,4,5. Men på grund af den begrænsede selvhelbredende evne af senen, og manglen på effektive behandlinger, tendinopati stadig påvirker patienternes liv negativt6,7. Desuden er patogenesen af tendinopati fortsat uklart. Der har været mange undersøgelser om dens patogenese, primært herunder “inflammation teori”, “degeneration teori”, “overforbrug teori”, og så videre8. På nuværende tidspunkt, mange forskere mente, at tendinopati skyldtes den mislykkede selvreparation til mikro-skader forårsaget af overdreven mekanisk lastning senen oplevelser9,10.

Senebaserede stamceller (TDSC’er) spiller som primære prækursorceller i senceller en afgørende rolle i både udviklingen af tendinopati og funktionel og strukturel genopretning eftertendinopati 11,12,13. Det blev rapporteret, at mekanisk stressstimulation kunne forårsage spredning og differentiering af osteocytter, osteoblaster, glatte muskelceller, fibroblaster, mesenkymale stamceller og andre kraftfølsommeceller 14,15,16,17,18. TDSC’er, som en af de mechanosensitive og multipotente celler, kan derfor ligeledes stimuleres til at differentiere ved mekanisk belastning19,20.

Forskellige mekaniske belastningsparametre (belastningsstyrke, belastningsfrekvens, lastetype og indlæsningsperiode) kan imidlertid få FDSC’erne til at differentiere sig i forskelligeceller 21. En effektiv og gyldig mekanisk belastningsordning er således meget vigtig for tenogenese. Desuden findes der forskellige typer bioreaktorer som stimuleringssystemer, der i øjeblikket anvendes til at levere mekanisk belastning til TDSC’er. Principperne for hver type bioreaktor er forskellige, så de mekaniske belastningsparametre, der svarer til forskellige bioreaktorer, er også forskellige. Derfor er der en enkel, økonomisk og reproducerbar stimuleringsprotokol i efterspørgslen, herunder typen af bioreaktor, det tilsvarende stimuleringsmedium og den mekaniske belastningsregime.

Denne artikel beskriver en metode baseret på en tre-dimensionel (3D) uniaxial stretching system til at stimulere TDSCs at differentiere i senen-lignende væv. Der er syv faser af protokollen: isolering af mus TDSCs, kultur og udvidelse af mus TDSCs, forberedelse af stimulation kultur medium for celle ark dannelse, celle ark dannelse ved liggende i stimulation medium, forberedelse af 3D senen stamcellekonstruktion, samling af uniaxial-stretching mekanisk stimulation kompleks, og evaluering af den mekaniske stimuleret in vitro senen-lignende væv. Hele proceduren tager mindre end 3 uger at opnå 3D-celle konstruktion, som er langt mindre end nogle eksisterende metoder22,23. Denne protokol har vist sig at være i stand til at få TDSCs til at differentiere sig til senevæv, og den er mere pålidelig end det nuværende almindeligt anvendte todimensionale (2D) stretchingsystem21. Virkningen blev påvist ved histologi. Kort sagt er denne protokol enkel, økonomisk, reproducerbar og gyldig.

Protocol

De beskrevne metoder blev godkendt og udført i overensstemmelse med retningslinjerne og forskrifterne fra University of Western Australia Animal Ethics Committee. 1. Isolering af mus TDSCs Aflive de 6-8-uger gamle C57BL/6 mus ved livmoderhalskræft dislokation. Høst patellar sener24 og Achilles sener25. Fordøje sener fra en med 6 ml type I kollagen (3 mg/ml) for 3 timer.BEMÆRK: Da senens størrelse i musen er…

Representative Results

Før mekanisk stimulation blev TDSC’erne dyrket til 100% sammenløb i komplet medium og viste en uorganiseret ultrastruken morfologi (Figur 2A). Efter 6 dages uniaxial stretching mekanisk belastning, ekstracellulære matrix (ECM) og celle justeringer var godt orienteret (Figur 2B). Cellerne var godt befolket og godt indhyllet i ECM efter mekanisk belastning. Cellemorfologi blev præsenteret for at være aflange og var mere ligner normal senecelle sammenlignet med den uden at strække (<s…

Discussion

Senen er en mechanosensitive fibrøst bindevæv. Ifølge tidligere forskning, overskydende mekanisk belastning kan føre til osteogene differentiering af senen stamceller, mens utilstrækkelig belastning ville føre til uordnede kollagen fiber struktur under senen differentiering21.

En fælles opfattelse er, at nøglen til en ideel bioreaktor er evnen til at simulere in vitro cellulære mikromiljø, at cellerne in vivo gennemgå. Derfor efterligner in vivo normale stres…

Declarações

The authors have nothing to disclose.

Acknowledgements

Forskningen blev udført, mens forfatteren var i modtagelsen af “en University of Western Australia International Fee Scholarship og en University Postgraduate Award på The University of Western Australia”. Dette arbejde blev støttet af National Natural Science Foundation of China (81802214).

Materials

Ascorbic acid Sigma-aldrich PHR1008-2G
Fetal bovine serum (FBS) Gibcoä by Life Technologies 1908361
Histology processor Leica TP 1020
Minimal Essential Medium (Alpha-MEM) Gibcoä by Life Technologies 2003802
Mouse Tendon Derived Stem Cell Isolated from Achilles tendons of 6- to 8-wk-old C57BL/6 mice. Then digested with type I collagenase (3 mg/ml; MilliporeSigma, Burlington, MA, USA) for 3 h and passed through a 70 mmcell strainer to yield single-cell suspensions.
Paraformaldehyde Sigma-aldrich 441244
Streptomycin and penicillin mixture Gibcoä by Life Technologies 15140122
Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System Centre of Orthopaedic Translational Research, Medical School, University of Western Australia Available from the corresponding author upon request. Or make it according to our design* *Wang T, Lin Z, Day RE, et al. Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system. Biotechnol Bioeng. 2013;110(5):1495–1507. doi:10.1002/bit.24809
Trypsin Gibcoä by Life Technologies 1858331
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Tags

Tendon-derived Stem CellsMechanical StimulationBioreactor SystemTenogenic DifferentiationTendinopathyCell Culture
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Chen, Z., Chen, P., Ruan, R., Chen, L., Yuan, J., Wood, D., Wang, T., Zheng, M. H. Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells. J. Vis. Exp. (162), e61278, doi:10.3791/61278 (2020).
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