In vitro studies of composite biomaterials and human adipose stem cells in bone and tendon tissue engineering applications

Kaisa Vuornos

Research output: Book/ReportDoctoral thesisCollection of Articles

Abstract

Critical size bone defects and other musculoskeletal injuries due to illness or trauma continue to contribute to increasing health care costs in connection to the growth of the aging population and obesity related complications in the industrialized countries. Tissue engineering with stem cells, biomaterials, and soluble factors offers feasible solutions to ease the growing need for suitable musculoskeletal implants with the promise of stem cell therapies to remedy failure or lack of tissue function. Adult stem cells, such as multipotent mesenchymal stem cells of adipose tissue, are easily available and abundant stem cells. Human adipose stem cells (hASCs) have the potential to differentiate towards besides adipogenic, also osteogenic, tenogenic, chondrogenic, and myogenic lineages in specific conditions. Critical size bone defects of maxillofacial area have already been treated by combining hASCs with biomaterials and growth factors. A tissue specific functional biomaterial supporting structure, a scaffold, offers physical support and guides stem cell growth, development, and differentiation in combination with the chemical induction provided by specific soluble factors of cell culture media. In particular, the composite biomaterial scaffolds offer an attractive alternative in order to harness the combined feasible properties of the different components. Moreover, the three-dimensional (3D) scaffold offers the differentiating stem cells a microenvironment mimicking the natural.

The aim of this thesis was to investigate novel in vitro musculoskeletal applications combining hASCs with suitable biomaterials and soluble factors for bone and tendon tissue engineering and vascularized bone construct applications. Firstly, tenogenic medium (TM) was optimized to support tenogenic differentiation of hASCs and used to induce formation of tendon-like matrix on mechanically suitable braided 3D polymer scaffolds. Secondly, 3D hydrogels supportive of hASC osteogenic differentiation were tested in combination with bioactive glass (BaG) extract based osteogenic medium (BaG OM) with support from evolving hydrogel mechanical properties. Thirdly, concomitant matrix mineralization and microvascularization formation were investigated in composite 3D hydrogels with hASC and human umbilical vein endothelial cell
(HUVEC) coculture by comparing BaG extract based endothelial-osteogenic medium (BaG EM-OM) and endothelial cell growth medium-2 (EGM-2) together with assessing the effect of osteogenic preconditioning of hASCs.

The results showed that the optimized TM with both growth and differentiation factor-5 and L-ascorbic acid together with the support of 3D braided poly(L/D) lactide (PLA) 96L/4D copolymer filament scaffolds significantly enhanced tendon-like matrix production of hASCs compared to other tested media groups or different 3D scaffold structure. The combined optimized chemical support of TM and 3D mechanical support of PLA 96/4 were shown essential for efficient and fast tendon-like matrix formation by hASCs.

In 3D hydrogels, robust hASC osteogenic differentiation and mineralization with BaG OM without any added growth factors was reported. Natural polymers collagen type I (COL) and gellan gum (GG) hydrogels were compared for hASC osteogenic induction for minimally invasive bone tissue engineering applications. In addition, ionic crosslinking of GG with the BaG extract was tested and was found to significantly increase mineralization even in the control condition of regular osteogenic medium. While both the hydrogels elicited strong hASC mineralization, hASCs embedded in COL with the BaG OM induction showed significantly higher gene expression of osteogenic marker genes, and also strong immunocytochemical staining of late osteogenic marker osteocalcin (OCN) was observed, thus confirming efficacious hASC osteogenic differentiation in in vitro 3D hydrogel culture mimicking physiological conditions.

In 3D GG-COL composite hydrogels in hASC-HUVEC coculture, both BaG EM-OM and EGM-2 media supported osteogenic as well as endothelial marker gene expression. Whereas hydroxyapatite mineralization and strong OCN staining were detected in the BaG EM-OM condition, in comparison, strong production of mature endothelial marker CD31 and elongated tube-like structures of hASCs and HUVECs with initial prevascular network formation were apparent in the EGM-2 condition. However, osteogenesis and vasculogenesis processes were deemed mutually inhibitory in these conditions in coculture in 3D GG-COL composite hydrogels.

In conclusion, these novel methods for in vitro 3D culture have potential for considerable future significance and impact on the field of musculoskeletal regenerative medicine applications.
Original languageEnglish
Place of PublicationTampere
PublisherTampere University
ISBN (Electronic)978-952-03-1643-3
ISBN (Print)978-952-03-1642-6
Publication statusPublished - 2020
Publication typeG5 Doctoral dissertation (article)

Publication series

NameTampere University Dissertations - Tampereen yliopiston väitöskirjat
Volume287
ISSN (Print)2489-9860
ISSN (Electronic)2490-0028

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