Characterization of a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features and their potential for in vivo cartilage regeneration strategies

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Abstract

Background: Progenitor cells display interesting features for tissue repair and reconstruction. In the last years, such cells have been identified in different cartilage types. In this study, we isolated a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features by outgrowth from human nasal septum cartilage. These putative progenitor cells were comparatively characterized with mesenchymal stem cells (MSC) and human nasal septum chondrocytes with respect to their cellular characteristics as well as surface marker profile using flow cytometric analyses. Differentiation capacity was evaluated on protein and gene expression levels. Results: The migrative subpopulation differentiated into osteogenic and chondrogenic lineages with distinct differences to chondrocytes and MSC. Cells of the migrative subpopulation showed an intermediate surface marker profile positioned between MSC and chondrocytes. Significant differences were found for CD9, CD29, CD44, CD90, CD105 and CD106. The cells possessed a high migratory ability in a Boyden chamber assay and responded to chemotactic stimulation. To evaluate their potential use in tissue engineering applications, a decellularized septal cartilage matrix was either seeded with cells from the migrative subpopulation or chondrocytes. Matrix production was demonstrated immunohistochemically and verified on gene expression level. Along with secretion of matrix metalloproteinases, cells of the migrative subpopulation migrated faster into the collagen matrix than chondrocytes, while synthesis of cartilage specific matrix was comparable. Conclusions: Cells of the migrative subpopulation, due to their migratory characteristics, are a potential cell source for in vivo regeneration of nasal cartilage. The in vivo mobilization of nasal cartilage progenitor cells is envisioned to be the basis for in situ tissue engineering procedures, aiming at the use of unseeded biomaterials which are able to recruit local progenitor cells for cartilage regeneration.

Figures

  • Fig. 1 Expansion characteristics after passage 1 (a–c) and passage 5 (d–f). MnCPC proliferated up to 20 passages (g). Colony formation of mnCPC (h) and hCh (i) in 35 mm wells stained with crystal violet after 8 days. Representative images were chosen
  • Fig. 2 FACS analysis of surface marker expression. The expression of surface markers is given as median fluorescence intensity normalised to the respective isotype control. MSC (black n = 9), mnCPC (black and white stripes n = 10) and hCh (dark grey n = 9). *p ≤ 0.05. For determination of CD166, only 5–9 independent samples of each cell type were available
  • Fig. 3 The differentiation potential of mnCPC was compared to hCh and MSC and induced by growth factor supplemented differentiation media over a period of 21 (adipogenic and osteogenic differentiation) and 28 days (chondrogenic differentiation). Only MSC, cultured in adipogenic dif‑ ferentiation medium, exhibited fatty vacuoles positive with Oil red O staining (a). Strong calcium deposition—stained with von Kossa stain—was shown for MSC and mnCPC, only small deposits were found in hCh (b). In the pellet culture for chondrogenic differentiation, collagen type II was detected in all samples (c). All control groups, cultured in basal medium, were negative (d). The respective gene expression analysis (e–g) supported the findings visualised by histological and immunohistochemical staining. Graphs show individual values with median, n = 4–8 donors for each cell type, *p ≤ 0.05. Representative images were chosen
  • Fig. 4 Cell migration and response to chemotactic factors. MnCPC showed a significantly higher basal migration in a Boyden chamber assay com‑ pared to MSC and hCh (a). All three cell types responded comparably to chemotactic factors (b), stimulation with PDGF‑BB was always significant compared to basal migration (#). *p ≤ 0.05, n = 3 donors for each cell type
  • Fig. 5 GAG synthesis of mnCPC (a–c) and hCh (d–f) on DECM evaluated by alcian blue staining and quantified with DMMB assay (g). GAG synthesis was detectable on day 14 for mnCPC (a) and hCh (d). The GAG synthesis increased clearly from day 14 until day 42 (c, f). The DECM without cells contained no stainable GAG (h). Compared to mnCPC, hCh demonstrated a slightly higher synthesis and accumulation (g) of GAG during culture for up to 42 days. GAG content was normalised in respect to cell number and dry weight of unseeded DECM. The values represent the mean ± SD with n = 12 replicates
  • Fig. 6 Aggrecan synthesis of mnCPC (a–c) and hCh (d–f) on DECM evaluated on protein (a–f) and gene expression level (g). Aggrecan synthesis was detectable on day 14 for mnCPC (a) and hCh (d). The aggrecan synthesis increased clearly from day 14 until day 42 (c, f). Compared to mnCPC, hCh demonstrated a significantly higher expression of aggrecan (g). The values represent the mean ± SD with n = 8 donors for each cell type, *p ≤ 0.05
  • Fig. 7 Collagen type I production (a–f) as well as gene expression (g) was detected for mnCPC (a–c) and hCh (d–f). On gene expression level (g) mnCPC demonstrated a significantly higher collagen type I expression than hCh. The gene expression analysis correlated with the detection of col‑ lagen type I on protein level by immunohistochemical staining. The values represent the mean ± SD with n = 8 donors for each cell type, *p ≤ 0.05
  • Fig. 8 Immunohistochemical staining of collagen type II (a–f) as well as gene expression analysis (g) reflected the significantly higher collagen type II synthesis of hCh compared to mnCPC. On day 14, hCh demonstrated a slight collagen type II synthesis in the superficial cell layer on the construct surface (d) while mnCPC exhibited no collagen type II production. During the 3D culture for up to 42 days, collagen type II synthesis of hCh increased (d–f). This increase was additionally detected on gene expression level. mnCPC (a–c) demonstrated a slight collagen type II synthesis starting from day 28 (b, g) and increasing until day 42 (c, g). The scaffold matrix consists of collagen type II and reacted strongly with the collagen type II antibody (Fig. 6h). The values represent the mean ± SD with n = 8 donors for each cell type, *p ≤ 0.05

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Elsaesser, A. F., Schwarz, S., Joos, H., Koerber, L., Brenner, R. E., & Rotter, N. (2016). Characterization of a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features and their potential for in vivo cartilage regeneration strategies. Cell and Bioscience, 6(1). https://doi.org/10.1186/S13578-016-0078-6

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