The direction of human mesenchymal stem cells into the chondrogenic lineage is influenced by the features of hydrogel carriers
Graphical abstract
Introduction
Low back pain is a major public health issue in the western world, which afflicts 70–85% of all people at some point in their life (Andersson, 1999, Freemont et al., 2002), and the prevalence of low back pain is reported to be as high as 84% (Airaksinen et al., 2006). In the Global Burden of Disease 2010 study it was concluded that low back pain causes the most disability compared with almost 300 other types of diseases or impairing medical conditions (Hoy et al., 2014). One of the main causes of low back pain has been proposed to be degeneration of the intervertebral discs (IVDs) (Freemont et al., 2002, Luoma et al., 2000). The IVD constitutes of two main types of tissues, the annulus fibrosus (AF) and the hydrogel-like nucleus pulposus (NP) (Humzah and Soames, 1988, Roughley, 2004). The chondrocyte-like cells produce the extracellular matrix (ECM) consisting mainly of proteoglycans and collagens. The expression of biomarkers in common for NP cells and chondrocytes in articular cartilage includes e.g. sex determining region box 9 (SOX9), collagen type II and aggrecan. SOX9 is a transcription factor necessary for the differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes/chondrocyte-like cells (Chimal-Monroy et al., 2003, Goldring et al., 2006). IVD degeneration includes increased cell death, presence of ECM degrading enzymes, lack of nutrient supply and loss of gel-like structure in the NP, leading to tissue dehydration, reduced IVD height and fissure formation (Urban and Roberts, 2003, Adams and Roughley, 2006). Current treatments for low back pain include physiotherapy and surgery, but these treatments are not fully dealing with the underlying factors and open surgery includes several risks e.g. postoperative infections (Fritzell et al., 2001, Vedicherla and Buckley, 2016). Minimal invasive treatments for halting or diminishing IVD degeneration are therefore desired and transplantation of cells, so called cell therapy, has been suggested (Gruber et al., 2002, Brisby et al., 2004, Henriksson et al., 2009, Hiyama et al., 2008, Feng et al., 2011). Stem cells have been considered for this purpose and hMSCs are of the most interest since these cells are easily obtained surgically, are able to differentiate into chondrocytes and to deposit ECM (Chamberlain et al., 2007, Pittenger et al., 1999, Svanvik et al., 2010). Different types of biomaterials such as collagen and hyaluronan hydrogels have been suggested as cell carriers to support cells transplanted into degenerated IVD (Hilborn, 2011, Sakai et al., 2003, Sawamura et al., 2009, Kisiday et al., 2002, Levett et al., 2014). Hydrogels, which are suitable for minimally invasive transplantations, would act as shock absorbers and may restore the IVD height (Schmidt et al., 2008, Silva-Correia et al., 2011). Hydrogels are further suggested to facilitate the distribution and differentiation of transplanted cells within the IVD (Chan and Gantenbein-Ritter, 2012, Henriksson et al., 2012, Fernandez-Muinos et al., 2015, Yamaoka et al., 2006). It is desired that the transplanted hMSCs attach to and migrate within the hydrogel. In situ, the hMSCs should further be able to differentiate into chondrocyte-like cells and produce ECM. Selected growth factors delivered by e.g. injections into the degenerated IVD are hypothesized to, either alone or in combination with cell therapy, increase cell proliferation, proteoglycan production and IVD height, thus regenerating the IVD by reversing the low grade IVD degeneration in situ (Revell et al., 2007, Masuda, 2008, Seelbach et al., 2015). Several in vitro approaches as well as in vivo studies including smaller and larger animal models have demonstrated the plausibility of this conjecture (Henriksson et al., 2009, Sakai et al., 2003, Henriksson et al., 2015, Masuda et al., 2004).
In this study, growth hormone (GH), Genotropin®, was applied, which is used clinically for patients with growth disorders including insufficient height and weight development (Albertsson-Wikland et al., 2008, Janssen et al., 1997).
The aim of the present study was to compare and examine cell survival and potential differentiation of hMSCs into chondrocyte-like cells, when cultured in two different types of hydrogel cell carriers. Further, the aim was to investigate if stimulation with growth hormone (Genotropin®) could improve chondrogenesis and ECM accumulation of hMSCs when encapsulated in these hydrogels.
Section snippets
Ethical permission
The study was approved by the local human ethics committee (ethical permission no: 532-04) and samples were collected from donors undergoing spine surgery with informed consent from all of the donors.
Isolation and culture of hMSCs
hMSCs were isolated from the donors; a female, age 31 years (donor 1), a male, age 43 years (donor 2) and a male, age 49 years (donor 3).
hMSCs were isolated from bone marrow aspirates (iliac crest) by centrifugation in Ficoll tubes (Becton Dickinson, Franklin Lakes, USA) and thereafter amplified,
Flow cytometry results of hMSCs
The flow cytometry analysis showed that about 38% of total events (passage 4) from each donor possess hMSCs properties. Of this cell population (P1) 99% cells showed double positive expression of the hMSCs markers CD105 and CD166 and negative expression (<1%) of CD45, a hematopoietic stem cell marker. (Fig. 1)
Cell viability in hydrogels
The Tunel assay analysis including cell counting demonstrated that the cells remained viable in the Hydromatrix® and Puramatrix® hydrogel cell cultures for at least 28 days (end point of
Discussion
The results from this in vitro study demonstrate good potential for cell survival and differentiation capacity of hMSCs in both of types of hydrogels investigated, especially in the Hydromatrix® hydrogel. The differences regarding chondrogenic differentiation capacity of the cells within the two gels during the study period suggests that relatively similar hydrogel carriers may influence the differentiation of hMSCs into chondrocyte-like cells differently.
The positive outcome of cell viability
Conclusions
In conclusion, the hMSCs were viable in both the investigated hydrogels for the full time of the experiment, 28 days, indicating a good potential for these types of hydrogels to be used as cell carriers when transplanting hMSCs into degenerated human IVDs. In this study, based on the in vitro results, Hydromatrix® hydrogel appeared the more promising carrier for hMSCs in terms of cell viability, ECM production and differentiation of hMSCs into chondrocyte-like cells when compared to the
Conflict of interest
No competing financial conflicts exist.
Acknowledgements
The study was supported with grants from The Swedish Research Council, ALF Västra Götaland, Inga-Britt and Arne Lundberg Foundation, Dr Felix Neubergh Foundation and from BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg Sweden. Electron microscopy images were taken at the Electron Microscopy Unit core facility at the Sahlgrenska Academy at the University of Gothenburg. Statistical data analysis was performed at the Bioinformatics Core Facility, at the Sahlgrenska
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The first two authors contributed equally.