Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation

Jörg Handschel,Christian Naujoks,Hans-Peter Wiesmann,Norbert Kübler,Rita Depprich,Ulrich Meyer,Lydia Lammers

doi:10.1186/1746-160x-7-12

Jörg Handschel, Christian Naujoks + Show 5 more

Open Access

https://doi.org/10.1186/1746-160x-7-12

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Abstract

Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs) in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG). After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin.

Highlights

Bony defects have various causes and often turn out to be a major therapeutic challenge
The mineralization was most prominent in the centre of the sphere, as demonstrated in histological sections stained with microscopy (TEM) confirmed the presence of scorching crystallites in the mineralized area, which appeared after 21 days of cultivation (Figure 3a)
Theses crystals were typically embedded in an extracellular matrix containing numerous collagenous fibrils (Figure 3b)

Summary

Introduction

Bony defects have various causes and often turn out to be a major therapeutic challenge. The reconstruction of bone using autologous grafts has been recognized as the gold standard because it provides biological active cells with osteoinductive properties and avoids any immunological reactions [1]. Artificial materials and extracorporeal tissue formation are alternative approaches for the reconstruction of bone defects, because they neither cause donor-site lesions nor is their availabilty restricted. Bone is a highly specialized tissue of the organism which is generated by mineralization of the extracellular matrix called osteoid. A key feature of bone tissue is the presence of biological active apatite crystals. These crystals were formatted by the mineralization of the extracellular matrix (osteoid) with calcium and phosphate ions. The process of mineralization can be monitored histologically by special stainings like alizarin red or ultrastructurally by transmission (TEM) and scanning electron microscopy (SEM)

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