Synthesis of scaffold-free, three dimensional, osteogenic constructs following culture of skeletal osteoprogenitor cells on glass surfaces

Latifa Alghfeli,Divyasree Parambath,Shaista Manzoor,Helmtrud I Roach,Richard O.C Oreffo,Ahmed T El-Serafi

doi:10.1016/j.bonr.2021.101143

Latifa Alghfeli, Divyasree Parambath + Show 4 more

Open Access

https://doi.org/10.1016/j.bonr.2021.101143

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Abstract

BackgroundEfficient differentiation of stem cells into three-dimensional (3D) osteogenic construct is still an unmet challenge. These constructs can be crucial for patients with bone defects due to congenital or traumatic reasons. The modulation of cell fate and function as a consequence of interaction with the physical and chemical properties of materials is well known. MethodsThe current study has examined the osteogenic differentiation potential of human skeletal populations following culture on glass surfaces, as a monolayer, or in glass tubes as a pellet culture. The 3D prosperities were assessed morphometrically and the differentiation was evaluated through molecular characterization as well as matrix formation. ResultsEarly temporal expression of alkaline phosphatase expression of skeletal populations was observed following culture on glass surfaces. Skeletal populations seeded on glass tubes, adhered as a monolayer to the tube base and subsequently formed 3D pellets at the air -media interface. The pellets cultured on glass displayed 4.9 ± 1.3 times the weight and 2.9 ± 0.1 the diameter of their counterpart cultured in plastic tubes and displayed enhanced production of osteogenic matrix proteins, such a collagen I and osteonectin. The size and weight of the pellets correlated with surface area in contrast to cell numbers seeded. Global DNA methylation level was decreased in pellets cultured on glass. In contrast, gene expression analysis confirmed upregulation extracellular matrix proteins and osteogenesis-related growth factors. ConclusionThis simple approach to the culture of skeletal cells on glass tubes provides a scaffold-free, 3D construct platform for generating pellets enabling analysis and evaluation of tissue development and integration of multiple constructs with implications for tissue repair and regenerative application on scale-up.

Highlights

Efficient differentiation of stem cells into three-dimensional (3D) osteogenic construct is still an unmet challenge
To attempt addressing these issues, tissue engi neering and regenerative medicine have emerged as approaches to Abbreviations: 3D, three-dimensional; A/S, Alcian blue/Sirius red/Weigert's haematoxylin; Alkaline phosphatase (ALP), Alkaline Phosphatase; BMP, bone morphogenic protein; BMSC, human bone marrow stromal; CSF, colony stimulating factor; Ct, Cycle threshold; EGF, epidermal growth factor; FC, Fetal bone cells; FCS, Fetal Calf Serum; FGF, fibroblast growth factor; FN1, fibronectin; gDNA, genomic DNA; GLI, GLI family zinc finger 1; HIPPIE, Human Integrated Protein Interaction Reference; iMSC, immortalized human bone marrow derived, mesenchymal stem cells; ITGA3, integrin A3; membrane-type metalloproteinase (MMP), matrix metalloprotease; P/S, penicillin and streptomycin; R, re ceptor; TGF, β transforming growth factor beta; TGFBR2 transforming growth factor beta receptor 2 VDR, vitamin D receptor; vWF, von Willebrand factor
MG63 cells cultured on glass slides express alkaline phosphatase earlier than cells maintained on tissue culture plastic

Summary

Introduction

Efficient differentiation of stem cells into three-dimensional (3D) osteogenic construct is still an unmet challenge. Current approaches to regenerate lost or damaged skeletal tissue hold a number of limitations including, but not limited to host-integration, scale, functionality and cost To attempt addressing these issues, tissue engi neering and regenerative medicine have emerged as approaches to Abbreviations: 3D, three-dimensional; A/S, Alcian blue/Sirius red/Weigert's haematoxylin; ALP, Alkaline Phosphatase; BMP, bone morphogenic protein; BMSC, human bone marrow stromal; CSF, colony stimulating factor; Ct, Cycle threshold; EGF, epidermal growth factor; FC, Fetal bone cells; FCS, Fetal Calf Serum; FGF, fibroblast growth factor; FN1, fibronectin; gDNA, genomic DNA; GLI, GLI family zinc finger 1; HIPPIE, Human Integrated Protein Interaction Reference; iMSC, immortalized human bone marrow derived, mesenchymal stem cells; ITGA3, integrin A3; MMP, matrix metalloprotease; P/S, penicillin and streptomycin; R, re ceptor; TGF, β transforming growth factor beta; TGFBR2 transforming growth factor beta receptor 2 VDR, vitamin D receptor; vWF, von Willebrand factor. The ability of these cells to differentiate into the osteogenic and chondrogenic lineages as well as their ability for selfassembly into an osteochondral construct that resemble, in part, the cross section of the fetal bone construct (El-Serafi et al, 2011a; Mir malek-Sani et al, 2006)

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