- 作者：Marianna Peroglio ; David Eglin ; Lorin M. Benneker ; Mauro Alini ; Sibylle Grad
- 关键词：Human mesenchymal stem cells ; Intervertebral disc ; Thermoreversible hydrogel ; Hyaluronan ; Whole-organ culture
- 刊名：The Spine Journal
- 出版年：November, 2013
- 年：2013
- 卷：13
- 期：11
- 页码：1627-1639
- 全文大小：1096 K
Background context
The fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection.Purpose
To optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in聽vitro, and evaluate whether preconditioning contributes to a better hMSC response ex聽vivo.
Study design
In聽vitro and ex聽vivo whole-organ culture of hMSCs.
Methods
In聽vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor 尾1 or growth and differentiation factor 5 (GDF-5). Ex聽vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype.
Results
The HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in聽vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex聽vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs.
Conclusions
Hyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in聽vitro and ex聽vivo. Further in聽vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration.