Continuous stimulation with differentiation factors is necessary to enhance osteogenic differentiation of human mesenchymal stem cells in-vitro

Abstract

Bone defect treatment belongs to the most challenging fields in orthopedic surgery and requires the well-coordinated application of mesenchymal stem cells (MSC) and differentiation factors. MSC isolated from reaming material (RMSC) and iliac crest (BMSC) in combination with bone morphogenetic protein-7 (BMP-7) and insulin-like growth factor-1 (IGF-1) have been used. The short half-life of both factors limit their applications: a burst release of the factor can probably not induce sustainable differentiation. We stimulated MSC in osteogenic differentiation medium with three different concentrations of BMP-7 or IGF-1: Group A was stimulated continuously, group B for 24 h and group C remained without any stimulation. Osteogenic differentiation was measured after seven and 14 days by alizarin red staining and alkaline phosphatase (ALP) activity. Continuous stimulation led to higher levels of osteogenic differentiation than short-term stimulation. This could lead to a reconsideration of established application forms for differentiation factors, aiming to provide a more sustained release.

Human Platelet Lysate Can Replace Fetal Calf Serum as a Protein Source to Promote Expansion and Osteogenic Differentiation of Human Bone-Marrow-Derived Mesenchymal Stromal Cells

“…However, our study is the first to evaluate the osteogenic parameters' evolution under the influence of hPL over the differentiation period in two different concentrations, making it possible to track the influence of hPL on commonly used time points during osteogenic differentiation. This culture setting has been used regularly and covers the major steps of osteogenic differentiation by using the specified evaluation time points between one and 21 days of culture [36][37][38]49]: as described previously, the early phases in the differentiation of BMSC towards osteoblasts takes place from day five to 14 in culture and is characterized by ALP expression [48,50]. The calcification of the extracellular matrix produced during osteoblastic differentiation occurs from day 14 to 28 in culture [48,51,52].…”

“…In the ODM containing hPL, 2 IU/mL heparin (PL BioScience, Aachen, Germany) were added according to the manufacturer's instructions to avoid gel formation of the medium. The cells were incubated for up to 21 days at 37 • C, 5% CO 2 following well-established protocols [36][37][38]. Media were changed twice per week.…”

“…Media were changed twice per week. ALP correlates with osteogenic differentiation of BMSC since it is produced during differentiation towards osteoblasts [37,38]. The cells were washed with 1x PBS (Thermo Fisher Scientific, Dreieich, Germany), then lysed in 1% Triton buffer (Sigma Aldrich, Steinheim, Germany).…”

“…Alizarin red stains the calcium deposits in the extracellular matrix formed by the osteoblastic (precursor) cells [38]. The cells were washed with 1x PBS and then fixed in 70% ethanol (Carl Roth, Karlsruhe, Germany).…”

Gelatin‐coating increases in‐vivo bone formation capacity of three‐dimensional 45S5‐bioactive glass‐based crystalline scaffolds

“…Isolation, cultivation, expansion, and characterization of hBMSC from bone marrow aspirate were performed using standardized protocols as published previously (Dominici et al, ; Hoellig et al, ; Reible, Schmidmaier, Prokscha, Moghaddam, & Westhauser, ). In brief, hBMSC were separated from bone marrow aspirate by gradient centrifugation using Ficoll‐Paque PLUS (GE Healthcare, Munich, Germany).…”