Abstract
IntroductionTo stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs).MethodsIn the present study, we induced the overexpression of the growth factors bone morphogenetic protein 2 (BMP-2) and/or Indian hedgehog (IHH) in human MSCs by adenoviral transduction to increase their osteogenic potential. GFP and nontransduced MSCs served as controls. The influence of the respective genetic modification on cell metabolic activity, proliferation, alkaline phosphatase (ALP) activity, mineralization in cell culture, and osteogenic marker gene expression was investigated.ResultsTransduction had no negative influence on cell metabolic activity or proliferation. ALP activity showed a typical rise-and-fall pattern with a maximal activity at day 14 and 21 after osteogenic induction. Enzyme activity was significantly higher in groups cultured with osteogenic media. The overexpression of BMP-2 and especially IHH + BMP-2 resulted in a significantly higher mineralization after 28 days. This was in line with obtained quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses, which showed a significant increase in osteopontin and osteocalcin expression for osteogenically induced BMP-2 and IHH + BMP-2 transduced cells when compared with the other groups. Moreover, an increase in runx2 expression was observed in all osteogenic groups toward day 21. It was again more pronounced for BMP-2 and IHH + BMP-2 transduced cells cultured in osteogenic media.ConclusionsIn summary, viral transduction did not negatively influence cell metabolic activity and proliferation. The overexpression of BMP-2 in combination with or without IHH resulted in an increased deposition of mineralized extracellular matrix, and expression of osteogenic marker genes. Viral transduction therefore represents a promising means to increase the osteogenic potential of MSCs and the combination of different transgenes may result in synergistic effects.
Highlights
To stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs)
The augmentation of large bone defects usually requires the application of autologous bone graft (ABG)
Freshly reamed trabecular bone from the acetabulum of patients undergoing total hip arthroplasty was transferred to 50 ml conical tubes (Greiner Bio-One, Frickenhausen, Germany) containing Dulbecco’s Modified Eagle Medium (DMEM; PAA Laboratories, Linz, Austria) supplemented with 10% fetal bovine serum (FBS; PAA) and antibiotics (50 IU penicillin/ml and 50 μg streptomycin/ml, PAA)
Summary
To stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs). Treatment outcomes have improved because of advances regarding surgical techniques and implant design. Different factors, such as biomechanical instability and extensive soft tissue trauma, can promote the formation of nonunions and defects characterized by a restricted regenerative potential [1]. Such bone defects are associated with considerable surgical challenges and have a high socioeconomic impact, greatly curtailing affected patients’ quality of life [2,3]. Limiting associated factors include graft availability, comorbidity, and insufficient bony integration
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