99mTc-HDP Labeling—A Non-Destructive Method for Real-Time Surveillance of the Osteogenic Differentiation Potential of hMSC during Ongoing Cell Cultures

Abstract

99-Metastabil Technetium (99mTc) is a radiopharmaceutical widely used in skeletal scintigraphy. Recent publications show it can also be used to determine the osteogenic potential of human mesenchymal stem cells (hMSCs) by binding to hydroxyapatite formed during bone tissue engineering. This field lacks non-destructive methods to track live osteogenic differentiation of hMSCs. However, no data about the uptake kinetics of 99mTc and its effect on osteogenesis of hMSCs have been published yet. We therefore evaluated the saturation time of 99mTc by incubating hMSC cultures for different periods, and the saturation concentration by using different amounts of 99mTc activity for incubation. The influence of 99mTc on osteogenic potential of hMSCs was then evaluated by labeling a continuous hMSC culture three times over the course of 3 weeks, and comparing the findings to cultures labeled once. Our findings show that 99mTc saturation time is less than 0.25 h, and saturation concentration is between 750 and 1000 MBq. Repeated exposure to γ-radiation emitted by 99mTc had no negative effects on hMSC cultures. These new insights can be used to make this highly promising method broadly available to support researchers in the field of bone tissue engineering using this method to track and evaluate, in real-time, the osteogenic differentiation of hMSC, without any negative influence on the cell viability, or their osteogenic differentiation potential.

Impact of Anti-Mycotic Drugs on the Osteogenic Response of Bone Marrow Mesenchymal Stem Cells In Vitro

“…This shows that PMMA does not affect the binding of 99m Tc-HDP to hydroxyapatite. This result is in line with prior studies, which showed that 99m Tc-HDP binds in a highly specific manner to hydroxyapatite [45][46][47]. Proof of this principle is fundamental for the understanding that 99m Tc-HDP uptake is directly proportional to hydroxyapatite concentrations in the probes, and consequently, is a reliable and sensitive method for accessing osteogenesis [47].…”

“…The radioactive tracer 99m Technetium ( 99m Tc) bound to Hydroxydiphosphonate (HDP) directly binds to newly formed hydroxyapatite and can be quantified by an activimeter [46]. This method is a non-destructive way to measure the osteogenic potential of cells, and consequently, cell cultures remain intact for further assessment [47]. To evaluate the effects of anti-mycotic drugs on cell amounts, we performed DAPI staining and automated digital cell counts.…”

Impact of Antibiotic-Loaded PMMA Spacers on the Osteogenic Potential of hMSCs

“…In summary, it can be stated that the formation of calcium complexes took place in the osteogenically differentiated groups. As earlier studies showed, these calcium complexes are equivalent in structure to hydroxyapatite [31]. Therefore, a successful osteogenesis took place.…”

“…Here, a radioactive tracer ( 99m Tc) bound to a polyphosphate (HDP) directly binds to newly formed hydroxyapatite while this uptake can be visualized with a gamma camera and precisely quantified using an activimeter [30]. Beyond that, this is a nondestructive way to measure the amount of hydroxyapatite formed so the cell cultures remain intact for further assessment [31].…”