The effect of type I collagen on osteochondrogenic differentiation in adipose-derived stromal cells in vivo, M. Alonso, S. Claros, J. Becerra and J.A. Andrades, Volume 10, issue 6, pages 597–610
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Abstract
Background
Recent studies have demonstrated that adipose-derived adult stromal cells (ADASCs) offer great promise for cell-based therapies due to their ability to differentiate towards bone, cartilage and fat. The objective of this study was to investigate whether type I collagen would elicit in vivo bone formation of passaged rat ADASCs placed extraskeletally.
Methods
After expansion to 1–4 passages (P1-P4), cells were incubated in osteogenic medium containing dexamethasone, ascorbic acid, and ß-glycerol phosphate for 2–4 weeks. Undifferentiated cells were maintained in Dulbecco′s modified Eagle′s medium with 10% fetal bovine serum. Osteogenic differentiation in vitro was confirmed by alkaline phosphatase and von Kossa staining as well as by gene expression of bone marker proteins including alkaline phosphatase, osteopontin, type I collagen, and osteocalcin. For in vivo osteogenesis, diffusion chambers were filled with 1 X 106 cells mixed with (or without) type I collagen gel and implanted subcutaneously into rats. Controls included chambers exposed to I) undifferentiated cells (with or without collagen), II) collagen without cells, and III) empty chambers (n=5 per group).
Results
Four weeks after implantation, in vivo bone and cartilage formation was demonstrated in implants containing 4-week osteoinduced p1 and p4 cells wrapped in the collagen gel, as confirmed by Goldner′s trichrome and Alcian blue staining, respectively. Newly formed bone stained positive for type I collagen. Control implants had no bone or cartilage and were primarily filled with fibrous tissue at that time interval.
Discussion
Although the influence of different matrices on the in vivo osteogenic capability of ADASCs is not fully understood, these findings suggest that type I collagen may support the survival and/or expression of osteogenic and chondrogenic phenotype in passaged rat ADASCs in vivo.
Introduction (p. 598)
Line 37: In combination with appropriate biomaterials and growth factors, autologous and syngeneic bone marrow-derived stem cells (BMSCs) have been shown to enhance bone repair significantly in animal fracture models [9,10] and differentiate into various human tissue types in vitro and in vivo [11,12].
Line 43: Although it remains to be determined whether adipose-derived adult stromal cells, termed ADASCs, meet the definition of stem cells, they are multipotential, available in large numbers and easily accessible and attach and proliferate rapidly in culture, making them an attractive cell source for tissue engineering [17–20].
Line 59: This study demonstrates that passaged rat ADASCs (rADASCs) mixed with type I collagen gel are capable of forming bone-like and cartilage tissue in vivo when implanted with diffusion chambers. These findings support further the potential application of these cells for tissue regeneration.
Methods
Cell isolation and culture (p. 598)
Line 43: 2 mM L-glutamine
Osteogenic differentiation (p. 598)
Line 51: After primary culture in the control medium (DMEM+10% FBS) and expansion to 1–4 passages, the rADASCs were trypsinized and replated onto 75-cm2 flasks at a density of 2 x 104/cm2.
RT-PCR determination
Line 89 (p. 599): (Real, Paterna, Valencia, Spain)
Line 91 (p. 599): (Roche, Branford, CT, USA)
Line 102 (p. 600): Triplicate PCR reactions were amplified using primers designed rat Aldolase A (ALD) as a control for assessing PCR efficiency.
Flow cytometry analysis (p. 600)
Line 110: (Gibco, West Palm Beach, FL, USA)
Line 116: (R&D Systems, Madrid, Spain); AbD Serotec (Madrid, Spain)
Line 130: (DakoCytomation, Glostrup, Denmark)
Immunofluorescence (p. 600)
Line 123: (Calbiochem-Novabiochem Co., San Diego, CA, USA)
Results
Expression of osteoblast-specific genes (p. 600)
Line 161: Although the osteogenic induction conditions used in this study were specific for the bone lineage and did not result in the expression of Col II, consistent with cartilage differentiation, expression of Sox9 (specific to chondrogenic differentiation) was noted at the 2- and 4-week time points in the differentiated ADAS cells. Expression levels of Runx2 and Osx, transcription factors essential for bone formation and osteoblastic differentiation, were observed in osteo-induced ADASC at days 14 and 28. Furthermore, for all three of these genes (Sox9, Runx2 and Osx), the expression was not restricted to osteogenic induction, as basal expression was seen in ADASCs maintained in control medium (Figure 2).
Line 175: Additional osteoblast-specific genes were investigated, including ALP, OP, BSP, ON, OC, Col I, Postn and the homeodomain proteins Msx1 and Msx2 (Figure 2).
Line 182: As shown in Figure 2, expression of the bone matrix proteins OP, ON and Col I was observed with consistent expression levels in both differentiated and control ADASCs.
Line 141: In addition to these matrix proteins, ADASCs also expressed TGF-β1 and Postn, both before and after induction.
Line 148: Finally, both osteo-induced and control ADASCs expressed Msx1, a gene involved in osteoblast differentiation, whereas Msx2 was not detected at any time point in either differentiated or control ADASCs.
Figure 1 and Figure 2: OM=Osteogenic medium
Figure 5: PSH=Picrosirius-hematoxylin
Discussion
Line 187 (p. 603): Expression of the key transcription factors Runx2, Osx and Msx1 that bind to the promoters of several osteogenic genes in rADASCs indicates their osteogenic commitment [33–37].
Line 223 (p. 607): In contrast to findings in previously reported studies, in which elevated levels of osteogenic markers were prominent in the less passaged rat BMSCs (P0 through P2 cells) [51,52], the expression of osteogenic markers in our DEX-treated ADASC cultures did not decrease with passing.
Line 228 (p. 607): According to Ter Brugge and Jansen [52], rBMSCs continuously cultured in the presence of DEX initially showed high ALP expression and abundant mineralization, but no ALP activity and calcification were found in cells passaged three times.
Line 244 (p. 607): Sugiura et al. [51] reported that the in vivo osteogenic potential of BMSCs tended to decrease with passages.
Line 289 (p. 608): It has been demonstrated that a large number of multipotent cells can easily be obtained by human lipoaspiration and induced to differentiate into osteoblasts [5,7,25,26].
Line 298 (p. 608): Moreover, the efficacy of various osteoinductive scaffolds such as type I collagen [7,58] and the osteoinductive capability of various growth factors can be studied by using these cells [4,7,59].
Table 1. Oligonucleotide primer sequences for RT–PCRs (p. 599)
References
- Sugiura F., Kitoh H., Ishiguro N. Osteogenic potential of rat mesenchymal stem cells after several passages. Biochem. Biophys. Res. Commun. 2004; 316: 233–239
- Ishikawa H, Kitoh H, Sugiura F, et al. The effect of recombinant human bone morphogenetic protein-2 on the osteogenic potential of rat mesenchymal stem cells after several passages. Acta Orthop 2007; 78: 285–292