Article
Dynamic compression induces osteogenic differentiation of hMSC on 3D electrospun PCL scaffolds
Search Medline for
Authors
Published: | October 23, 2017 |
---|
Outline
Text
Objectives: Osteogenic and osteochondral defects such as osteoporosis, osteochondrosis dissecans and bone fractures still remain a challenge for orthopedic surgery because of lack of healthy bone substance. According to the current gold standard missing bone tissue is augmented with autologous cancellous bone grafts from the crista iliaca. This involves a second operation field with associated donor site morbidity. The use of allogeneic spongiosa is less favoured, because unlike autologous cancellous bone, allogenic bone is depleted of live cells due to the risk of graft versus host disease. Here, we developed a human mesenchymal stem cell (MSC)-based bone substitute using 3D electrospun poly-caprolactone (PCL) scaffolds within a mechanoreactor, which induces osteogenic differentiation by dynamic compression without addition of any other inductors.
Material: We used 3D electrospun PCL scaffolds that are biodegradable within 9 month rendering further operations and its risks unnecessary. PCL is very robust and already established for clinical use in human medicine. Furthermore we used a mechanoreactor performing dynamic compression under sterile cell culture conditions using femoral head BMMSC with the consent of patients which underwent artificial hip surgery.
Methods: hMSC were seeded on PCL scaffolds and on TCPS as a control for 21 days. Scaffolds treated in the bioreactor system undergo dynamic compression with 0.8N (3500 Pa) for 4h a day compared to scaffolds without mechanical stimulation. MSC on scaffolds and on TCPS were cultured either in osteogenic induction medium containing DMEM low glucose, FCS, LGPS, ascorbic acid, dexamethasone and ß-glycerophosphate or in stem cell expansion medium for both, dynamic and static conditions. Cytotoxicity of scaffolds and the bioreactor system for hMSC was analysed by live/dead staining. Osteogenic differentiation was analysed using real time RT-PCR, scanning electron microscopy (SEM) with EDX analysis, alkaline phosphatase assay, Alizarin red and van Kossa staining. Furthermore we used the MEK/ERK-Inhibitor U0126 to analyse the molecular pathways of the osteogenic induction via dynamic compression.
Results and Conclusion: PCL scaffolds were not cytotoxic towards hMSC. Cells underwent osteogenic differentiation during the 21-day-culture period in the mechanoreactor even without osteogenic induction media. Osteogenic differentiation was scored by real time RT-PCR, SEM and EDX analysis as well as alizarin red staining, van Kossa staining and alkaline phosphatase assay. U0126 had no influence on the osteogenic differentiation via dynamic compression.
To conclude, we established a bioreactor setting to produce allogeneic MSC-based PCL-grafts as bone substitutes, which has no immunogenic potency and can be individually modulated to the defect.
Next, we will focus on the molecular mechanism transducing mechanostimulation to osteogenic differentiation and investigate effects of the produced hMSC-based scaffolds on bone healing in animal models.