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Biology

Integreret knogledannelse gennem in vivo endokondral ossifikation ved hjælp af mesenkymale stamceller

Published: July 14, 2023
doi:
10.3791/65573
, , ,
1Department of Molecular Craniofacial Embryology, Graduate School of Medical and Dental Sciences,Tokyo Medical and Dental University, 2Department of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences,Tokyo Medical and Dental University, 3Department of Regenerative and Reconstructive Dental Medicine, Graduate School of Medical and Dental Sciences,Tokyo Medical and Dental University

Summary

Knogleterapi via endokondral ossifikation ved implantering af kunstigt bruskvæv fremstillet af mesenkymale stamceller har potentialet til at omgå ulemperne ved konventionelle terapier. Hyaluronsyrehydrogeler er effektive til opskalering af ensartet differentierede brusktransplantater samt skabelse af integreret knogle med vaskularisering mellem smeltede transplantater in vivo.

Abstract

Konventionel knogleregenereringsterapi ved hjælp af mesenkymale stamceller (MSC’er) er vanskelig at anvende på knogledefekter, der er større end den kritiske størrelse, fordi den ikke har en mekanisme til at inducere angiogenese. Implantering af kunstigt bruskvæv fremstillet af MSC’er inducerer angiogenese og knogledannelse in vivo via endokondral ossifikation (ECO). Derfor kan denne ECO-medierede tilgang være en lovende knogleregenereringsterapi i fremtiden. Et vigtigt aspekt af den kliniske anvendelse af denne ECO-medierede tilgang er at etablere en protokol til forberedelse af nok brusk til at blive implanteret til at reparere knogledefekten. Det er især ikke praktisk at designe en enkelt masse podet brusk af en størrelse, der er i overensstemmelse med formen på den faktiske knogledefekt. Derfor skal brusk, der skal transplanteres, have egenskaben til at danne knogle integreret, når flere stykker implanteres. Hydrogeler kan være et attraktivt værktøj til opskalering af vævsmanipulerede transplantater til endokondral ossifikation for at opfylde kliniske krav. Selvom mange naturligt afledte hydrogeler understøtter MSC-bruskdannelse in vitro og ECO in vivo, er det optimale stilladsmateriale til at opfylde behovene ved kliniske anvendelser endnu ikke fastlagt. Hyaluronsyre (HA) er en afgørende bestanddel af bruskens ekstracellulære matrix og er et biologisk nedbrydeligt og biokompatibelt polysaccharid. Her viser vi, at HA-hydrogeler har fremragende egenskaber til at understøtte in vitro-differentiering af MSC-baseret bruskvæv og fremme endokondral knogledannelse in vivo.

Introduction

Autolog knogle er stadig guldstandarden til reparation af knogledefekter på grund af traumer, medfødte defekter og kirurgisk resektion. Autogen knogletransplantation har imidlertid betydelige begrænsninger, herunder donorsmerter, risiko for infektion og begrænset knoglevolumen, der kan isoleres fra patienterne 1,2,3,4. Talrige biomaterialer er blevet udviklet som knoglesubstitutter, der kombinerer naturlige eller syntetiske polymerer med mineraliserede materialer såsom calciumphosphat eller hydroxyapatit 5,6. Knogledannelse i disse konstruerede materialer opnås normalt ved anvendelse af det mineraliserede materiale som primingmateriale for at tillade stamceller at differentiere direkte til osteoblaster gennem den intramembrane ossifikation (IMO) proces7. Denne proces mangler det angiogene trin, hvilket resulterer i utilstrækkelig in vivo vaskularisering af transplantatet efter implantation 8,9,10, og derfor er tilgange, der anvender en sådan proces, muligvis ikke optimale til behandling af store knogledefekter 11.

Strategier, der anvendes til at rekapitulere den endokondrale ossifikationsproces (ECO), en medfødt mekanisme i skeletogenese under udvikling, har vist sig at overvinde betydelige problemer forbundet med traditionelle IMO-baserede tilgange. I ECO frigiver chondrocytter i bruskskabelonen vaskulær endotelvækstfaktor (VEGF), som fremmer vaskulær infiltration og ombygning af bruskskabelonen til knogle12. Den ECO-medierede tilgang til osteogenese via bruskremodellering og angiogenese, som også aktiveres under reparation af brud, bruger kunstigt skabt bruskvæv afledt af MSC’er som primingmateriale. Chondrocytter kan tolerere hypoxi i knogledefekter, fremkalde angiogenese og omdanne et vaskulært brusktransplantat til angiogent væv. Talrige undersøgelser har rapporteret, at MSC-baserede brusktransplantater genererer knogle in vivo ved at implementere et sådant ECO-program 13,14,15,16,17,18,19,20,21.

Et væsentligt krav til den kliniske anvendelse af denne ECO-medierede tilgang er, hvordan man forbereder den ønskede mængde brusktransplantat i kliniske omgivelser. Forberedelse af klinisk brusk af en størrelse, der passer til den faktiske knogledefekt, er ikke praktisk. Derfor skal graftbrusk danne knogle integreret, når flere fragmenter implanteres22. Hydrogeler kan være et attraktivt værktøj til opskalering af vævsmanipulerede transplantater til endokondral ossifikation. Mange naturligt afledte hydrogeler understøtter MSC-bruskdannelse in vitro og ECO in vivo 23,24,25,26,27,28,29,30,31,32; Det optimale støttemateriale til at opfylde de kliniske applikationskrav er dog forblevet ubestemt. Hyaluronsyre (HA) er et biologisk nedbrydeligt og biokompatibelt polysaccharid, der findes i bruskens ekstracellulære matrix33. HA interagerer med MSC’er via overfladereceptorer såsom CD44 for at understøtte kondrogen differentiering 25,26,28,30,31,32,34. Derudover fremmer HA-stilladser IMO-medieret osteogen differentiering af humane tandmassestamceller35, og stilladser kombineret med kollagen fremmer ECO-medieret osteogenese36,37.

Her præsenterer vi en metode til fremstilling af HA-hydrogeler ved hjælp af knoglemarvsafledte voksne humane MSC’er og deres anvendelse til hypertrofisk kondrogenese in vitro og efterfølgende endokondral ossifikation in vivo38. Vi sammenlignede egenskaberne ved HA med kollagen, et materiale, der i vid udstrækning anvendes inden for knoglevævsteknik med MSC’er og et nyttigt materiale til opskalering af kunstige transplantater til endokondral ossifikation17. I en immunkompromitteret musemodel blev HA- og kollagenkonstruktioner podet med humane MSC’er evalueret for in vivo ECO-potentiale ved subkutan implantation. Resultaterne viser, at HA-hydrogeler er fremragende som stillads for MSC’er til at skabe kunstige brusktransplantater, der tillader knogledannelse gennem ECO.

Protokollen er opdelt i to trin. For det første fremstilles konstruktioner af humane MSC’er podet på hyaluronanhydrogel og differentieres til hypertrofisk brusk in vitro. Dernæst implanteres de differentierede konstruktioner subkutant i en nøgen model for at inducere endokondral ossifikation in vivo (figur 1).

Protocol

Denne protokol bruger 4 uger gamle nøgne hanmus. Opstald fire mus i et bur under en 12 timers lys/mørk cyklus ved 22-24 °C og 50-70% relativ luftfugtighed. Alle dyreforsøg blev udført i overensstemmelse med retningslinjerne godkendt af Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (godkendelses-id: A2019-204C, A2020-116A og A2021-121A). 1. Fremstilling af buffere og reagenser Forbered mesenkymal stamcellevækstmedium (MSC-væ…

Representative Results

MSC-indkapslede HA-hydrogeler blev dyrket i kondrogent medium suppleret med TGFβ3, en inducer af kondrogenese41 (trin 4.1). Vi sammenlignede egenskaberne af HA med kollagen, som har vist sig at være effektive til at skabe MSC-baserede kunstige brusktransplantater til endokondral ossifikation, som beskrevet tidligere38. Udifferentierede MSC’er blev ikke inkluderet som negative kontroller i denne undersøgelse, fordi det er blevet påvist, at udifferentierede MSC’er kræver…

Discussion

Brug af passende stilladsmaterialer, der fremmer overgangen fra hypertrofisk brusk til knogle, er en lovende tilgang til at opskalere MSC-baserede konstruerede hypertrofiske brusktransplantater og behandle knogledefekter af klinisk signifikant størrelse. Her viser vi, at HA er et fremragende stilladsmateriale til at understøtte differentieringen af MSC-baseret hypertrofisk bruskvæv in vitro og til at fremme endokondral knogledannelse in vivo38. Desuden viste det sig in viv…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev støttet af et tilskud til videnskabelig forskning (KAKENHI) fra Japan Society for the Promotion of Science (JSPS) (bevilling nr. JP19K10259 og 22K10032 til MAI).

Materials

0.25w/v% Trypsin-1mmol/L EDTA.4Na Solution FUJIFILM Wako Pure Chemical  209-16941
Antisedan Nippon Zenyaku Kogyo
ascorbate-2-phosphate Nacalai Tesque 13571-14
Bambanker GC Lymphotec CS-02-001
basic fibroblastic growth factor Reprocell RCHEOT002 
bovine serum albumin FUJIFILM Wako Pure Chemical  012-23881 7.5 w/v%
Countess Automated Cell Counter with cell counting chamber slides and Trypan Blue stain 0.4% Invitrogen C10283
dexamethasone Merck D8893
Domitor Nippon Zenyaku Kogyo
Dormicum Astellas Pharma
Dulbecco's Modified Eagle Medium Merck D6429 high glucose
Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham Merck D6421
Fetal bovine serum Hyclone SH30396.03
Gentamicin sulfate FUJIFILM Wako Pure Chemical  1676045  10 mg/mL
Haccpper Generator TechnoMax CH-400-5QB 50 ppm hypochlorous acid water
Human Mesenchymal Stem Cells Lonza PT-2501
HyStem Cell Culture Scaffold Kit Merck HYS020
IL-1ß PeproTech AF-200-01B
ITS-G supplement FUJIFILM Wako Pure Chemical  090-06741 ×100
L-Alanyl-L-Glutamine FUJIFILM Wako Pure Chemical  016-21841 200mmol/L (×100)
L-proline Nacalai Tesque 29001-42
L-Thyroxine Merck T1775
MSCGM Mesenchymal Stem Cell Growth Medium
BulletKit		 Lonza PT-3001
paraffin FUJIFILM Wako Pure Chemical  165-13375
PBS / pH7.4 100ml Medicago 09-2051-100
TGF-β3  Proteintech HZ-1090
Vetorphale Meiji Seika Kaisha
Visiocare Ointment SAVAVET/SAVA Healthcare
β-glycerophosphate FUJIFILM Wako Pure Chemical  048-34332
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Tags

Mesenchymal Stem CellsBone FormationIn VivoEndochondral OssificationArtificial CartilageBone RegenerationAngiogenesisScaffoldCartilage DifferentiationCritical Size Bone DefectECO-mediated Approach
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Yamazaki, S., Lin, Y., Marukawa, E., Ikeda, M. Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells. J. Vis. Exp. (197), e65573, doi:10.3791/65573 (2023).
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