Abstract
Aim: To explore the feasibility of culturing mesenchymal stem cells in an hydroxyapatite-fibrin matrix held by a mesh scaffold and inducing osteogenic differentiation of these cells. The aim was to obtain bone-material in vitro in a desired form. Materials and Methods: Rat mesenchymal stem cells were mixed with fibrin and nanocrystalline hydroxyapatite in tubular scaffolds constructed from a poly(L-lactic acid) mesh, and cultured under standard and osteogenic differentiating conditions. Cell viability, cytotoxicity and alkaline phosphatase activity were followed for 3 weeks. Living cells and the expression of bone markers were visualized by fluorescence staining and immunofluorescence staining, respectively. Attachment of cells to the scaffold mesh surface was examined by scanning electron microscopy. Results: Cell viability decreased and cytotoxicity increased rapidly during the first day of culture but stabilized gradually afterwards, indicating fast adaptation of the cells in the matrix-scaffold environment. From day 17, cytotoxicity started to decrease, paralleled by an increase in alkaline phosphatase activity, indicating osteogenic differentiation. A large number of living cells were visible in the matrix and on the mesh scaffold. Expression of collagen type I, osteoponin, osteocalcin and core binding factor 1 were evident under osteogenic differentiation conditions. Conclusion: The three-dimensional construction of a fibrin-hydroxyapatite matrix in a biocompatible poly(L-lactic acid) as mesh-scaffold provides a promising carrier for producing bone-material in vitro in a desired form for applications in regenerative medicine.
Ostim® (Heraeus Kulzer, Hanau, Germany), a synthetic nanocrystalline hydroxyapatite gel, is an approved bone replacement material frequently used in dental applications (1-3). Several studies have reported that hydroxyapatite enhanced the proliferation of osteoblastic and mesenchymal stem cells (MSCs) under standard culturing conditions, and promoted osteogenic differentiation of these cells under differentiation conditions in the presence of dexamethasone, β-glycerolphosphate, ascorbic acid phosphate and fluvastatin (4-9). Furthermore, previous studies have shown good biocompatibility and osteoinductive effects of combined fibrin-hydroxylapatite matrices (10-13). However, these cell-containing matrices were cultured as two-dimensional sheets.
The aim of the present study was to evaluate the feasibility of an approach for culturing MSCs in a three-dimensional nanocrystalline hydroxyapatite-fibrin matrix that is structurally supported by a poly(L-lactic acid) (PLLA) mesh scaffold. This PLLA mesh has an inter-connecting porous structure and therefore enables medium exchange in vitro and nutrition supply and vascularization in vivo. Our goal was to develop a carrier for culturing and differentiating MSCs in vitro for obtaining bone substitutes in a desired form for applications in regenerative medicine.
Materials and Methods
Culturing cells in the carriers. MSCs were prepared from bone marrow of Sprague-Dawley-rats as described elsewhere (14). A total of 1×106 cells were resuspended in a matrix gel made of 2 ml fibrin and 0.6 g nanocrytalline hydroxyapatite (Ostim®; Heraeus Kulzer, Hanau, Germany), and packed into a 2.28 mm2 (10 mm in length and 6 mm in diameter) scaffold made of PLLA. The PLLA had a porosity of 92% and density of 164 g/m2. The scaffolds were sterilized by γ-radiation.
Each of the fibrin-hydroxyapatite-PLLA carriers with MSCs was placed in one well of a 12-well-plate and covered with 2 ml basic medium containing 10% fetal calf serum. For osteogenic differentiation, the basic medium was complemented with osteogenic differentiation-inducing additives dexamethasone phosphate (Sigma-Aldrich, Hamburg, Germany), β-glycerolphosphate (Sigma-Aldrich, Hamburg, Germany) and ascorbic acid phosphate (Sigma-Aldrich, Hamburg, Germany). The effect of fluvastatin (Sigma-Aldrich, Hamburg, Germany), a known pro-osteogenic supplement (15), was investigated by adding it to the basic medium and the osteogenic medium. Each condition was examined in four to eight replicates.
Assays. At different time intervals, 100 μl conditioned medium was taken from the wells containing the scaffold with cells for measuring cell proliferation, cytotoxicity and alkaline phosphatase activity using CellTiter-Blue (Promega, Mannheim, Germany), CytoTox-One (Promega, Mannheim, Germany) and p-nitrophenol (Sigma-Aldrich, Hamburg, Germany) based assays, respectively.
At the end of the experimental period of 21 days, vital cells were visualized by double staining with fluorescein diacetate/propidium iodide (Sigma-Aldrich, Hamburg, Germany). Detailed structures of cells in the matrix and on the PLLA scaffold surface were studied in frozen paraffin sections of 50 μm thickness by scanning electron microscopy.
The expression of bone-specific markers type I collagen, osteopontin, osteocalcin and core binding factor 1 (CBFA1) in cells in the scaffolds were determined by immunostaining with specific antibodies. For collagen type I, a goat polyclonal IgG antibody (clone N19; Merck Millipore, Darmstadt, Germany) was used at a dilution of 1:100. As the secondary antibody, Alexa Fluor® 546-coupled donkey anti-goat antibody (clone GK1.5; Life Technologies, Darmstadt, Germany) was used at a dilution of 1:500. For osteopontin and osteocalcin staining, a rabbit polyclonal IgG antibody (clone N45.1; Merck-Millipore, Darmstadt, Germany) was used at a dilution of 1:100. As the secondary antibody, Alexa Fluor® 466 (clone W6/32) goat anti-rabbit antibody at a dilution of 1:500 was used (Life Technologies). The diluent for the antibodies was 1% bovine serum albumin in phosphate buffered saline (PBS). Prior to immunostaining, all specimens were fixed using 4% paraformaldehyde in PBS. 2-(4-carbamimidoylphenyl) (Carl Roth GmbH, Karlsruhe, Germany) was used for nuclear counterstaining at a dilution of 1:10,000.
Results
The construction of the three-dimensional carrier-containing cells in the fibrin-hydroxyapatite matrix held by a mesh PLLA scaffold is illustrated in Figure 1.
Cell viability decreased rapidly in the first day but stabilized under standard culture conditions or slowed-down under osteogenic differentiation conditions (Figure 2A). Likewise, cytocoxicity also increased rapidly during the first day of culture but stabilized thereafter (Figure 2B). Alkaline phosphatase activity decreased more gradually and reached a plateau phase at day 5 (Figure 2C). Around day 18, alkaline phosphatase activity started to increase again, coupled with a decrease of cytotoxicity under both standard and osteogenic conditions. Alkaline phosphatase activity was generally higher under osteogenic culture conditions. This effect was more prominent when the activity was normalized against cell viability, representing the activity per cell (Figure 2D).
We also tested fluvastatin as an additive to both standard and osteogenic cultures. However, no effect was observed (data not shown). At the end of the experiment on day 21, a large number of living cells was visible in the matrix (Figure 3A). SEM images generated at the end of the experiment show cells attached to the textile scaffold (Figure 3B).
Cells cultured in scaffolds under differentiation-inducing conditions stained positively for antibodies directed against collagen type I, osteoponin, osteocalcin and CBFA1 (Figure 4).
Discussion
We successfully cultured MSCs in a three-dimensional carrier consisting of a fibrin-hydroxyapatite matrix and a mesh PLLA scaffold, and induced osteogenic differentiation. The initial decrease of cell viability paralleled by an increase of cytotocixity likely corresponds to death of some cells in the process of settling into the new environment. Our findings demonstrate that the porous PLLA mesh and the three-dimensional hydroxyapatite-fibrin matrix, with a diameter of 6-mm, provide an adequate supply of medium that supports in vitro culturing of vital cells.
Under osteogenic differentiation conditions, the decrease of cell viability slowed-down after the initial adaptation phase but persisted until the end of the experiment. This may be explained at least partially by the well-known association of proliferation arrest and osteogenic differentiation (16). This explanation is also supported by the generally higher alkaline phosphatase activity. However, the higher starting viability in the osteogenic culture, likely resulting from more viable cells at the beginning of the experiment, may also contribute to the difference in the viability dynamics between the two cultural conditions.
The increase of the alkaline phosphatase activity at the end of the culture period that was also observed in the culture without osteogenic differentiation additives may be explained by the osteogenic differentiation-inducing effect of the hydroxyapatite-fibrin matrix itself (4-8). However, expression of the bone markers osteoponin and osteocalcin was only detected in cells cultured with osteogenic additives.
A: Illustration of the three-dimensional hydroxyapatite-fibrin matrix held by the poly(L-lactic acid) (PLLA)-mesh scaffold. B: The PLLA mesh tube. C: The microporous structure of the PLLA mesh (100-fold magnification).
Conclusion
This three-dimensional construction with fibrin-hydroxyapatite as matrix and biocompatible PLLA as mesh scaffold provides a promising carrier for culturing MSCs and inducing osteogenic differentiation for producing bone-material in vitro in a desired form for regenerative medicine.
Viability (A), cytotoxicity (B), alkaline phosphatase activity (C) and alkaline phosphatase activity relative to viability (D) of cells cultured in the carriers as illustrated in Figure 1 under standard (control) and osteogenic differentiation (Osteo) conditions. Data are shown as means from 2-6 measurements of relative fluorescence units (rfu) (A, B), absorbance (C) and absorbance divided by rfu. Error bars represent the standard deviation.
Cells after 21 days' culture. A: Living cells stained with fluorescein diacetate (green) and dead cells stained with propidium iodide (red). B: Scanning electron microscopy revealing cells attached to the poly(L-lactic acid) mesh.
Immunohistochemical staining of bone differentiation markers, collagen type I (A), osteopontin (B), osteocalcin (C) and core binding factor 1 (CBFA1) (D) in cells cultured under osteogenic differentiation conditions.
Acknowledgements
The Authors thank Professor T. Gries, Institute for Textile Technology (ITA), University of Aachen, Aachen, Germany, for providing the PLLA tubes and the electron microscopy images; Heraeus Kulzer, Hanau, Germany, for providing Ostim®; and Dr. L. Kluwe for her valuable contribution in preparing the manuscript.
Footnotes
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↵* These Authors contributed equally to this study.
- Received February 14, 2014.
- Revision received March 3, 2014.
- Accepted March 4, 2014.
- Copyright © 2014 The Author(s). Published by the International Institute of Anticancer Research.
