Biofabrication of Prevascularized Spheroids for Bone Tissue Engineering by Fusion of Microvascular Fragments with Osteoblasts

Overview

Journal Front Bioeng Biotechnol

Date 2024 Sep 25

PMID 39318668

Authors

Selina Wrublewsky

Jessica Schultz

Tekoshin Ammo

Caroline Bickelmann

Wolfgang Metzger

Thomas Spater

Tim Pohlemann

Michael D Menger

Matthias W Laschke

Affiliations

Soon will be listed here.

Abstract

Introduction: Spheroids are promising building blocks for scaffold-free bone tissue engineering. Their rapid vascularization is of major importance to guarantee their survival after transplantation. To achieve this, we herein introduce the biofabrication of prevascularized spheroids by fusion of adipose tissue-derived microvascular fragments (MVF) with osteoblasts (OB).

Methods: For this purpose, 200 MVF from donor mice and 5,000, 10,000 or 20,000 murine OB (MC3T3-E1) were co-cultured in a liquid overlay system for 3 days to generate OB + MVF spheroids. OB mono-culture spheroids served as controls.

Results And Discussion: During the generation process, the diameters of all spheroids progressively decreased, resulting in compact, viable spheroids of homogeneous sizes. MVF promoted the maturation of spheroids containing 5,000 OB, as shown by an accelerated decline of cell proliferation due to contact inhibition. Moreover, MVF most effectively reassembled into new microvascular networks within these small spheroids when compared to the other spheroid types, indicating the most beneficial MVF to OB ratio. Accordingly, these spheroids also showed a high angiogenic sprouting activity . In contrast to OB spheroids, they further rapidly vascularized after transplantation into dorsal skinfold chambers. This was caused by the interconnection of incorporated MVF with surrounding blood vessels. These findings indicate that OB + MVF spheroids may be suitable for bone tissue engineering, which should be next tested in appropriate bone defect models.

References

Wang X, Yang P . In vitro differentiation of mouse embryonic stem (mES) cells using the hanging drop method. J Vis Exp. 2008; (17). PMC: 3253613. DOI: 10.3791/825. View

Orth M, Altmeyer M, Scheuer C, Braun B, Holstein J, Eglin D . Effects of locally applied adipose tissue-derived microvascular fragments by thermoresponsive hydrogel on bone healing. Acta Biomater. 2018; 77:201-211. DOI: 10.1016/j.actbio.2018.07.029. View

Ruehle M, Krishnan L, Vantucci C, Wang Y, Stevens H, Roy K . Effects of BMP-2 dose and delivery of microvascular fragments on healing of bone defects with concomitant volumetric muscle loss. J Orthop Res. 2019; 37(3):553-561. DOI: 10.1002/jor.24225. View

Ampofo E, Rudzitis-Auth J, Dahmke I, Rossler O, Thiel G, Montenarh M . Inhibition of protein kinase CK2 suppresses tumor necrosis factor (TNF)-α-induced leukocyte-endothelial cell interaction. Biochim Biophys Acta. 2015; 1852(10 Pt A):2123-36. DOI: 10.1016/j.bbadis.2015.07.013. View

Ivascu A, Kubbies M . Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen. 2006; 11(8):922-32. DOI: 10.1177/1087057106292763. View

Sytkowski A, Davis K . Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the NASA rotating wall vessel bioreactor. In Vitro Cell Dev Biol Anim. 2001; 37(2):79-83. DOI: 10.1290/1071-2690(2001)037<0079:ECGADI>2.0.CO;2. View

Laschke M, Spater T, Menger M . Microvascular Fragments: More Than Just Natural Vascularization Units. Trends Biotechnol. 2020; 39(1):24-33. DOI: 10.1016/j.tibtech.2020.06.001. View

McClatchey A, Yap A . Contact inhibition (of proliferation) redux. Curr Opin Cell Biol. 2012; 24(5):685-94. DOI: 10.1016/j.ceb.2012.06.009. View

Metzger W, Sossong D, Bachle A, Putz N, Wennemuth G, Pohlemann T . The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. Cytotherapy. 2011; 13(8):1000-12. DOI: 10.3109/14653249.2011.583233. View

10.

Spater T, Frueh F, Nickels R, Menger M, Laschke M . Prevascularization of collagen-glycosaminoglycan scaffolds: stromal vascular fraction versus adipose tissue-derived microvascular fragments. J Biol Eng. 2018; 12:24. PMC: 6234670. DOI: 10.1186/s13036-018-0118-3. View

11.

Laschke M, Menger M . The dorsal skinfold chamber: A versatile tool for preclinical research in tissue engineering and regenerative medicine. Eur Cell Mater. 2016; 32:202-15. DOI: 10.22203/eCM.v032a13. View

12.

Meng R, Xu H, Di S, Shi D, Qian A, Wang J . Human mesenchymal stem cells are sensitive to abnormal gravity and exhibit classic apoptotic features. Acta Biochim Biophys Sin (Shanghai). 2011; 43(2):133-42. DOI: 10.1093/abbs/gmq121. View

13.

Hewett P . Isolation and Culture of Human Endothelial Cells from Micro- and Macro-vessels. Methods Mol Biol. 2016; 1430:61-76. DOI: 10.1007/978-1-4939-3628-1_4. View

14.

Simunovic F, Finkenzeller G . Vascularization Strategies in Bone Tissue Engineering. Cells. 2021; 10(7). PMC: 8306064. DOI: 10.3390/cells10071749. View

15.

Garcia P, Holstein J, Maier S, Schaumloffel H, Al-Marrawi F, Hannig M . Development of a reliable non-union model in mice. J Surg Res. 2007; 147(1):84-91. DOI: 10.1016/j.jss.2007.09.013. View

16.

Bialkowska K, Komorowski P, Bryszewska M, Milowska K . Spheroids as a Type of Three-Dimensional Cell Cultures-Examples of Methods of Preparation and the Most Important Application. Int J Mol Sci. 2020; 21(17). PMC: 7503223. DOI: 10.3390/ijms21176225. View

17.

Whatley B, Li X, Zhang N, Wen X . Magnetic-directed patterning of cell spheroids. J Biomed Mater Res A. 2013; 102(5):1537-47. DOI: 10.1002/jbm.a.34797. View

18.

Menger M, Laschke M, Nussler A, Menger M, Histing T . The vascularization paradox of non-union formation. Angiogenesis. 2022; 25(3):279-290. PMC: 9249698. DOI: 10.1007/s10456-022-09832-x. View

19.

Wang W, Yeung K . Bone grafts and biomaterials substitutes for bone defect repair: A review. Bioact Mater. 2018; 2(4):224-247. PMC: 5935655. DOI: 10.1016/j.bioactmat.2017.05.007. View

20.

Laschke M, Schank T, Scheuer C, Kleer S, Shadmanov T, Eglin D . In vitro osteogenic differentiation of adipose-derived mesenchymal stem cell spheroids impairs their in vivo vascularization capacity inside implanted porous polyurethane scaffolds. Acta Biomater. 2014; 10(10):4226-35. DOI: 10.1016/j.actbio.2014.06.035. View