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Researchers get stem cells to differentiate into NP cells with hydrogel carrier
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Investigators at the AO Research Institute, in Davos, Switzerland, showed that when bone marrow-derived mesenchymal stem cells were applied to an intervertebral disc the cell viability could be preserved and possibly enhanced by using a thermoreversible hyaluronan-based hydrogel carrier.
According to Mauro Alini, PhD, who recently presented these findings, the hydrogel carrier allowed the mesenchymal stem cells (MSC) to differentiate into nucleus pulposus (NP) or intervertebral disc (IVD) cells once the cultured MSCs were injected into IVDs.
“Human mesenchymal stem cell differentiation toward the disc phenotype can be achieved in a whole intervertebral disc model by using a hyaluronan-based thermoreversible hydrogel carrier,” Alini said. The process did not require any supplementation with growth factors, like the GDF-5 factor, he noted.
Alini and colleagues won a research grant from the North American Spine Society for this study.
Advantages of hyaluronan-based carrier
The researchers chose a hyaluronan-based thermoreversible hydrogel carrier functionalized with poly(N-isopropyl acrylamide) (pNIPAM) for its capacity to form a gel at 30°C to 32°C. The gel reliquified when it was cooled down to room temperature, which made it easy to retrieve the cells later, according to Alini.
Alini and colleagues hoped to learn what happens to human MSCs after they are injected into the IVD. Zhu and colleagues showed MSCs recognize hyaluronan, Alini said when he presented the results. “[They] induce metabolic pathways and growth factors,” according to Alini.
Image: Alini M
“We have a limited knowledge of the molecular characteristics of intervertebral discs and also the phenotype is not known,” Alini said.
For this study, he and colleagues identified IVD-specific molecules that indicate differentiation of human MSCs, such as cytokeratin-19 (KRT-19), forkhead box protein F1 (FOXF1) and carbonic anhydrase (CA12). They either cultured human bone marrow-derived MSCs in hyaluronan-pNIPAM for 7 days in chondrogenic medium and then applied them to a cavity they created in bovine caudal IVDs or suspended them in hyaluronan-pNIPAM, applying the gel immediately afterwards, according to the abstract.
“We can stimulate the cells in vitro before or directly inject them into the intervertebral disc, stimulate with different growth factors [and] manipulate by gene therapy in order to promote or induce differentiation,” Alini said.
The researchers approached the IVD from the endplate and used a punch approach to retrieve the MSCs. They cultured them for 1 week and performed gene expression analyses to identify their disc-like differentiation, including the KRT-19, FOXF1, and CA 12 genes, collagen type I and II (COL1 and COL2), aggrecan (ACAN), transcription factor SOX-9, the cluster of differentiation 24 (CD24), and hyaluronan synthase I and II (HAS1 and HAS2).
Gene expression
The results showed the viability of the MSCs was >90% in vitro and ex vivo within the IVD cavity. The SOX9, COL2 and KR-19 expression increased in vitro; while ex vivo COL2, CD24 and FOXF1 expression increased. The researchers concluded that when the MSCs expressed these genes, they also differentiated into NP or IVD cells, but not into chondrocytes.
There were no significant differences between in vitro and ex vivo cultures for the other genes, according to Alini.
“Cells were only injected in the disc with the hyaluronic acid, which also means that the milieu...where you inject the cells, can influence the mesenchymal stem cells to differentiate towards the tissue where you inject them,” Alini said.
In phase 2 of the study, the researchers hope to apply different loading regimes — physiological or detrimental — with low or high frequency to understand how the cells behave in this type of system, Alini said. – by Renee Blisard Buddle
- References:
- Alini M. Thermoreversible hyaluronan-based hydrogels support mesenchymal stem cells disc-like differentiation in vitro and ex-vivo. Presented at: North American Spine Society Meeting; Oct. 24-27, 2012; Dallas.
- Zhu H. Stem Cells. 2006;24(4);928-935.
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For more information:
- Mauro Alini, PhD, can be reached at AO Research Institute Davos, Clavadelerstrasse 8, Davos, Switzerland; email: mauro.alini@aofoundation.org.
Disclosure: Alini has no relevant financial disclosures.