- Email: [email protected]
Mesenchymal stem cells cultured on a collagen scaffold: In vitro osteogenic differentiation
archives of oral biology 52 (2007) 64–73
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/arob
Mesenchymal stem cells cultured on a collagen scaffold: In vitro osteogenic differentiation E. Donzelli a,*, A. Salvade` a, P. Mimo b, M. Vigano` a, M. Morrone c, R. Papagna a,d, F. Carini a,d, A. Zaopo c, M. Miloso a, M. Baldoni a,d, G. Tredici a a
Dipartimento di Neuroscienze e Tecnologie Biomediche, Universita` degli Studi di Milano-Bicocca, Via Cadore 48, 20052 Monza, MI, Italy CORIMAV, Viale Sarca 222, 20126 Milano, Italy c Pirelli Labs S.p.A., Viale Sarca 222, 20126 Milano, Italy d Clinica Odontoiatrica, Ospedale San Gerardo, Monza, Italy b
article info
abstract
Article history:
Objective: Management of periodontal defects has always been a challenge in clinical
Accepted 18 July 2006
periodontics. Recently mesenchymal stem cells (MSC) have been proposed for tissue regeneration in periodontal disease and repair of large bone defects. Bone regeneration
Keywords:
has to be supported by a scaffold which has to be biocompatible, biodegradable, and able to
Mesenchymal stem cells
support cell growth and differentiation. The aim of this study was to evaluate osteogenic
Osteogenic differentiation
differentiation of MSC seeded on a collagen scaffold.
Collagen scaffold
Design: MSC were obtained from adult rat bone marrow, expanded and cultured in plastic
Periodontal disease
dishes or seeded in a collagen scaffold (Gingistat1). MSC were induced towards osteogenic differentiation using osteogenic supplements. Cell differentiation and calcium deposits were evaluated by immunoblotting, immunohistochemistry, histochemical techniques, enzymatic activity assay, and SEM–EDX analysis. Biomaterial in vitro degradation was evaluated by measuring mass reduction after incubation in culture medium. Results: Rat MSC osteogenic differentiation was demonstrated by osteopontin and osteocalcin expression and an increase in alkaline phosphatase activity. MSC were distributed homogeneously in the collagen scaffold. Nodular aggregates and alizarin red stained calcium deposits were observed in MSC induced towards osteogenic differentiation cultured in dishes or seeded in the collagen scaffold. SEM–EDX analysis demonstrated that calcium co-localized with phosphorous. The biomaterial in vitro degraded in 4–5 weeks. Conclusions: MSC from bone marrow differentiate towards osteogenic lineage, representing a suitable cell source for bone formation in periodontal regeneration. Gingistat1 collagen scaffold supports MSC distribution and differentiation, but its short degradation time may be a limitation for a future application in bone tissue regeneration. # 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Autogenous bone grafts are the classic therapeutic approaches to severe periodontal disease with atrophic alveolar crests.1 These surgical procedures are invasive requiring bone with-
drawal from non-oral sites and results are not always satisfactory and may be associated with donor site morbidity.2–5 Rarely is a bone graft able to regenerate all the components of the periodontium and, moreover, it often results in excessive apical migration of the epithelium.6–9 To
* Corresponding author. Tel.: +39 0264488119; fax: +39 0264488253. E-mail address: [email protected] (E. Donzelli). 0003–9969/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2006.07.007
archives of oral biology 52 (2007) 64–73
reduce the problems of autogenous bone grafts, a tissue engineering strategy for periodontal regeneration has been proposed.9,10 This approach requires the use of a biomaterial containing appropriately selected or primed cells, but at present both the cells and biomaterial to be used have yet to be defined. Mesenchymal stem cells (MSC) derived from adult bone marrow are a possible cell source for tissue engineering, in particular for applications in skeletal and hard tissue repair and also in periodontal tissue regeneration.11,12 Bone marrow percutaneous harvest from the patient supplies autogenous MSC in a simple and safe way. Due to their multilineage potential and plasticity, the MSC may be committed to forming nonhematopoietic tissues including bone, cartilage, tendons, and ligaments.13–16 MSC isolated from bone marrow differentiate into an osteogenic lineage when cultured in the presence of dexamethasone, ascorbic acid, and b-glycerolphosphate (osteogenic supplements),11,17 but also other trophic factors or drugs have been proposed for use either in vitro or in animal models.18,19 Bone formation from MSC requires a three-dimensional scaffold to drive cellular growth and differentiation.19–21 An ongoing increasing number of biomaterials have been proposed as scaffolds for tissue regeneration, with the aim of reproducing the milieu where the complex interaction between cells and their matrix occurs.22–27 A prerequisite for the use of an appropriate scaffold to support bone formation is an understanding of its biological and structural characteristics. To repair oral bone defects the use of scaffolds shaped to the bone defect has been proposed.28,29 The ideal scaffold must have certain characteristics: (a) it must be easy to shape it onto the bone defect, (b) it must have a good capacity for hosting, growing and maturating MSC, (c) it must form a barrier to neighboring tissues and (d) it must have a reabsorption time compatible with the time required for bone formation but not so long that it interferes with the bone substitution of the scaffold. In this study, we report our experience with adult rat MSC in forming bone in vitro and on a commercial collagen biomaterial which has already been used as a scaffold to support bone regeneration in severe cases of atrophic periodontitis. The aim of this study was to evaluate the structural characteristics, the degradation time and the capacity to support cell distribution and differentiation of a commercial collagen sponge.
2.
Materials and methods
2.1.
Materials
65
The materials used in the present study are listed in Table 1.
2.2.
Cell preparation and culture methods
Mesenchymal stem cells (MSC) were obtained from bone marrow of 10-week-old female Sprague–Dawley rats. Animal procedures were conducted in accordance with the European Communities Council Directive 86/609/EEC. Both femora and tibias were removed and soft tissues were detached. Metaphysis from both ends were resected and bone marrow cells were collected by flushing the diaphysis with 2 ml/bone of Eagle’s alpha minimum essential medium (aMEM) containing 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 250 mg/ml fungizone. A suspension of bone marrow cells was obtained by repeated aspiration of the cell preparation through a 25 gauge needle. Red cells were depleted by 5 min incubation in NH4Cl 0.84%. Cells were resuspended in 15 ml a-MEM medium plus 20% ES cell screened fetal bovine serum, plated in a polystyrene 75 cm2 tissue culture flask and cultured in a humidified atmosphere of 95% air with 5% CO2 at 37 8C. After 2 days, the culture medium was removed and new medium was added. The medium was changed two or three times a week and the floating cells were removed. After 14 days of culture, cells were passaged by trypsinization (0.05% trypsin/EDTA solution). As the culture reached almost complete confluence, cells were subcultured or plated for subsequent experiments.
2.3.
Osteogenic differentiation
MSC (passage 4–6) were seeded at approximately 3500 cells/cm2 on culture dishes in a culture medium composed of a-MEM medium plus 10% defined FBS and cultured until subconfluence occurred. After this period, cells were grown in the culture medium alone or in osteogenic medium (OS medium) consisting of the same culture medium with the addition of osteogenic supplements, 100 nM dexamethasone, 10 mM b-glycerolphosphate, and 0.05 mM 2-phosphate-ascorbic acid.
2.4.
Cell seeding and differentiation on collagen scaffolds
Gingistat1 was used as a scaffold for this study. Gingistat1 is a sponge made of lyophilized collagen (type I). Collagen
Table 1 – Materials used in the present study Materials
Purchased from
a-MEM, glutamine, penicillin/streptomicyn, fungizone, trypsin/EDTA ES cell screened FBS, defined FBS Dexamethasone, b-glycerol-phosphate, alizarin red Gingistat1 Primary antibodies anti-osteopontin, anti-actin and HRP-conjugated anti-goat secondary antibody Primary antibody anti-osteocalcin HRP-conjugated anti-mouse secondary antibody FITC-conjugated anti-mouse secondary antibody Other chemicals
BioWhittaker, Bergamo, Italy Hyclone, Logan, UT, USA Applichem GmbH, Darmstadt, Germany Vebas, Milano, Italy Santa Cruz Biotechnology, Santa Cruz, CA, USA Abcam, Cambridge, UK Chemicon, Temecula, CA, USA Rockland, Gilbertsville, PA, USA Sigma Chemicals Co., St. Louis, MO, USA
66
archives of oral biology 52 (2007) 64–73
sponges were cut, under sterile conditions, into 3 mm 3 mm 3 mm cubes. MSC were suspended at a concentration of 5 106 cells/ml and 106 cells were poured onto each scaffold through a 25 gauge needle. After 4 h at 37 8C the medium was replaced with fresh medium and the scaffolds were maintained in a humidified atmosphere at 37 8C with 5% CO2 in air. After 4 days, the cells were treated with OS medium or with culture medium alone. The medium was changed every 3–4 days. For each time point, an unseeded scaffold incubated with culture medium alone was used as a control. Histological analyses were performed at days 7, 14, 21, and 28 of culture. Collagen sponges were washed twice with PBS, fixed with 4% paraformaldehyde for 1 h at RT, embedded in paraffin with standard methods, cut into 7 mm sections and processed as subsequently described.
2.5.
Alizarin red staining
Cells were plated and treated for osteogenic differentiation as described. Paraformaldehyde (4%) fixed cells were incubated for 30 min at room temperature (RT) in a solution containing 1% alizarin red and 1% ammonium hydroxide. Cells were rinsed twice with distilled water and allowed to dry completely. MSC seeded or unseeded collagen scaffold sections were deparaffinized with xylene, hydrated to 70% alcohol and stained with alizarin red solution for 30 s. Excess dye was shaken off and stained sections were fixed with acetone (20 dips), aceton–xylene (20 dips), cleared with xylene and mounted in permanent medium.
2.6.
Alkaline phosphatase activity assay
Cells were plated and treated for osteogenic differentiation as described. Alkaline phosphatase activity was evaluated using the p-nitrophenol method30 in cells cultured in culture medium alone or in OS medium. Cells were washed twice with ice-cold phosphate buffered saline (PBS), and protein extracts were prepared in 10 mM Tris–HCl (pH 7.6) and 0.1% Triton X-100. Total protein extracts were prepared at days 0, 7, 14, 21 and 28 after OS medium induction. Alkaline phosphatase enzyme activity was calculated after measuring the absorbance of p-nitrophenol product formed at 405 nm on a microplate reader (Bio-Rad, Hercules, CA).
2.7.
Cell lysates and immunoblotting analysis
Cells were plated and treated for osteogenic differentiation as described Cells lysates were prepared as previously reported31 at days 0, 7, 14, 21 and 28 after OS medium induction. Protein concentration was estimated with a modified Bradford assay and aliquots (60 mg) were separated by 15% SDS-polyacrylamide gels. Immunoblotting analysis was performed according to the manufacturer’s instructions with primary antibody anti-osteopontin (1:1000), anti-osteocalcin (1:500) or anti-actin (1:500) and an appropriate HRP-conjugated secondary antibody. Antigens were revealed by a chemiluminescence detection kit.
2.8.
Immunofluorescence
Cells cultured on collagen coated glass slides were treated as described for osteogenic differentiation. Selected cultures were fixed with 4% paraformaldehyde for 10 min and treated for 10 min with 0.1 M glycine (in PBS). Slides were incubated for 1 h at RT with blocking solution (5% BSA, bovine serum albumine, 0.5% Triton X-100 in PBS) and subsequently for 30 min at 37 8C with 1 mg/ml RNAse in blocking solution. Incubation with primary antibody anti-osteopontin (1:200) was performed overnight at 4 8C. The following day, cells were rinsed with PBS and incubated at RT for 1 h in the dark with an appropriate FITC-conjugated secondary antibody (1:100) and 2.5 mg/ml propidium iodide (PI). Non-specific binding of secondary antibody was controlled by omitting the primary antibody. Slides were washed and mounted with glycerol. Microscopy analysis was performed with laser confocal microscopy (Radiance 2100; Biorad Laboratories, Hercules, CA, USA).
2.9.
Hematoxylin and eosin staining
MSC seeded or unseeded collagen scaffold sections were deparaffinized with xylene, hydrated to 70% alcohol and stained according to standard hematoxylin/eosin staining protocols and mounted in permanent medium.
2.10.
SEM–EDX analysis of MSC seeded collagen scaffolds
Unseeded or MSC seeded collagen scaffolds were treated with culture medium or with OS medium as previously described for up to 28 days. At days 7, 14, 21, and 28 collagen scaffolds were embedded in paraffin and scanning electron microscopy (SEM)–energy dispersive X-ray spectrometry (EDX) micro-analysis was performed on 7 mm sections. Each section was fixed to a sample holder with a conductive carbon ribbon. SEM observations were carried out with a Hitachi S-2700 model scanning electron microscope equipped with EDX (Oxford Instruments, model ISIS300). EDX micro-analysis was employed to detect the presence of calcium and phosphorous on the samples, and maps of the relative distribution of these elements were acquired operating at 12 kV with an acquisition time of 10 min. The quantity of Ca and P in each sample was measured and the Ca/P ratio was calculated.
2.11. In vitro degradation study of GingistatW collagen sponges The in vitro degradation study of Gingistat1 sponges was carried out by incubating each of 15 samples in 50 ml of aMEM medium supplemented with 20% ES-FBS in an aircirculation oven at 37 8C. At different time points, samples were removed from the culture medium, washed with plenty of distilled water, and then dried under reduced pressure in a vacuum oven at 50 8C for 2 h before being weighed. The masses of the collagen sponges before and after incubation in the culture medium were weighed on a Sartorius electronic balance with a resolution of 0.01 mg. The mass retained percentage of each sponge was
archives of oral biology 52 (2007) 64–73
67
calculated by comparing the dry weight (Mt) after the established incubation time (t) with the initial weight (Mi) according to the following formula:
mass retained ¼
Mt 100% Mi
3.
Results
3.1.
Establishment of primary cultures
It is generally accepted that mesenchymal stem cells (MSC) can be recognized as the adherent cells derived from bone marrow capable of extensive proliferation, with a fibroblastic profile and with the ability to differentiate into mesenchymal lineages. Cells obtained from bone marrow were cultured as described in Material and Methods and, after 15 days of culture, an almost homogeneous population of fibroblasticlike cells was observed throughout the flask with little evidence of round or floating cells. These cells were induced to differentiate into various lineages and were able to proliferate in vitro for a long time. The capacity to proliferate is maintained until the 30th passage, although the capacity to differentiate is reduced as the passage number increases. Because of their proliferative and differentiative abilities we refer to these cells as MSC. For the described experiments we used cells at the fourth to sixth passage.
Fig. 2 – MSC stained with alizarin red. MSC were treated with culture medium alone (a–d) or OS medium (e–h) and stained with alizarin red at day 7 (a and e), day 14 (b and f), day 21 (c and g) or day 28 (d and h) after induction.
Fig. 3 – ALP activity assay. ALP activity was evaluated in cells treated with OS medium with respect to cells treated with culture medium alone (mean W S.D. of three independent experiments).
3.2.
Fig. 1 – MSC after 7 days of induction. (a) CTRL culture treated with culture medium alone. (b) OS culture treated with OS medium. Bars = 50 mm.
Osteogenic differentiation
MSC were plated and treated for osteogenic differentiation as described for up to 28 days. Osteogenic differentiation was evaluated using morphological, biochemical and immunological techniques both in cells grown in OS medium (OS cultures) and in cells cultured in culture medium alone (CTRL cultures) which represented control cells. Proliferation and differentiation of CTRL and OS cultures were compared using phase contrast microscopy. In both CTRL and OS cultures, cells proliferated and reached almost complete confluence at day 7. In OS cultures, nodular aggregates of cells became evident at day 7 of culture (Fig. 1) and increased up to 28 days. These aggregates were characterized by deposits of amorphous material. In CTRL cultures similar cellular aggregates were observed, but they were smaller, were evident only after 21 days and lacked deposits. The nodular aggregates in OS cultures stained with alizarin red, demonstrating that the amorphous deposits observed at the microscope were calcium deposits. Alizarin red positive nodular aggregates were already present at day 14. At day 28,
68
archives of oral biology 52 (2007) 64–73
Osteopontin expression was detectable by immunoblotting analysis at day 7 of induction and increased afterwards. Osteocalcin expression was detectable by immunoblotting analysis at day 14 of induction and increased afterwards. (Fig. 4). Osteopontin expression was evaluated also by immunofluorescence. High magnification observation revealed an abundant cytoplasmic expression of the protein in OS cultures after 14 and 28 days of induction. The nodular cellular aggregates were particularly rich of these immunopositive cells. In CTRL cultures the signal was very weak (Fig. 5). Fig. 4 – Osteopontin and osteocalcin immunoblotting analysis. Cell lysates (60 mg/lane) were analyzed by immunoblotting using an antibody anti-osteopontin or anti-osteocalcin, as indicated. Reprobing was done with actin by immunoblotting as loading control.
positive alizarin red aggregates were larger and stained more intensively, indicating that a more extensive calcium deposition had occurred. CTRL cultures showed only minimal background staining (Fig. 2). Alkaline phosphatase (ALP) activity was elevated in OS cultures with respect to CTRL ones from 14 days of induction onwards and reached its maximum at 21 days, then decreasing to values close to those of CTRL cultures (Fig. 3).
3.3. Collagen scaffold: structure and MSC osteogenic differentiation Gingistat1 collagen sponge structure was observed at SEM, revealing the presence of interconnected pores with an average size of 300 mm. Hematoxylin–eosin staining on serial sections of MSC seeded onto the Gingistat1 collagen scaffold sponge demonstrated that the cells were distributed homogeneously throughout the whole sponge (Fig. 6). Osteogenic differentiation of MSC grown on collagen scaffolds was firstly evaluated with alizarin red staining on unseeded and MSC seeded scaffolds cultured in culture medium alone or in OS medium. Unseeded scaffolds showed a low basal staining that remained unchanged during the
Fig. 5 – Osteopontin immunofluorescence. Cells were grown on collagen coated glass slides and treated for 14 days with culture medium alone (a) or with OS medium (b). Osteopontin: FITC, green; nuclei: PI, red. Bars = 10 mm.
Fig. 6 – Hematoxylin/eosin staining of unseeded (a) or MSC seeded (b) collagen scaffolds cultured for 14 days in culture medium alone. Bars = 100 mm.
archives of oral biology 52 (2007) 64–73
69
cultured on polystyrene dishes were also present on the scaffolds (Fig. 7). In order to obtain also a quantitative evaluation of calcium deposition, we performed an EDX micro-analysis on sections of MSC seeded collagen scaffolds treated with culture medium alone or with OS medium. A scaffold without cells was processed as a negative control to take into account the possible presence of calcium or phosphorous in the untreated material. Calcium and phosphorous were detected on the samples incubated in OS medium (Fig. 8) and the chemical distribution maps thus collected indicate that these elements co-localize in the same areas of the sections (Fig. 9). A comparison of the calcium and phosphorous maps of the OS medium incubated samples clearly showed an increase in the presence of both chemical elements, with very low levels at day 7 up to very high levels at day 28. The distribution of calcium and phosphorous was homogeneous in all scaffold sections. The mean value of Ca/P ratio in the samples incubated with OS medium was about 1.2. Neither MSC seeded scaffolds treated with culture medium alone nor the unseeded scaffold showed any relevant presence of calcium or phosphorous, confirming that these chemical elements accumulated only onto scaffolds seeded with cells which had been exposed to the OS medium. The MSC seeded scaffold cultured for 28 days in culture medium alone showed a very low level of calcium and phosphorous. The study of the degradation of the collagen scaffold demonstrated that Gingistat1 discs degraded in about 4–5 weeks with a mostly linear trend. There was an approximately 20% mass loss at each week of incubation which was also reflected in a size decrease of the discs (Fig. 10).
4.
Fig. 7 – Alizarin red staining of unseeded (a) or MSC seeded (b and c) collagen scaffolds treated for 21 days with culture medium alone (a and b) or with OS medium (c). Bars = 50 mm.
whole observation period. MSC seeded collagen scaffolds cultured in culture medium alone stained weakly even after 28 days of culture. On the contrary, MSC seeded collagen scaffolds cultured in OS medium stained strongly with alizarin red starting at day 14 of culture. Alizarin red stained deposits similar to those observed in osteogenic differentiation of MSC
Discussion
The present study shows that the adult rat bone marrow is a suitable and feasible source of a great number of MSC and that the adult bone marrow derived MSC can be easily induced to differentiate into an osteogenic lineage. Furthermore, we have demonstrated that a commercial collagen spongy scaffold is biocompatible with the MSC and we propose some methods for evaluating those characteristics of the scaffold necessary for possible clinical use in periodontal diseases. In our experience, and according to many experimental studies, cells obtained from the bone marrow in toto, without selection of any particular population, constitute an optimal source of MSC which can then be differentiated towards an osteoblastic phenotype.11,32–34 The mesenchymal stem cell nature of the cells derived from the bone marrow of adult rats is demonstrated by their capacity of extensive proliferation and to differentiate towards several different lineages.13–16 In this context, the capacity of the bone marrow derived MSC to differentiate towards an osteogenic lineage and to deposit calcium has been demonstrated by several methods based on immunohistochemical and histochemical techniques, enzymatic activity assay, and SEM–EDX analysis. MSC committment to osteogenic differentiation was demonstrated by the expression of osteopontin, a phosphoprotein that possesses several calcium binding domains and is associated with cell attachment, proliferation, and
70
archives of oral biology 52 (2007) 64–73
Fig. 8 – SEM–EDX analysis of MSC seeded collagen scaffold sections. MSC seeded collagen scaffolds were cultured in culture medium alone (a, c, e and g) or OS medium (b, d, f and h) and analysed with SEM–EDX at 7 days (a and b), 14 days (c and d), 21 days (e and f) and 28 days (g and h) after induction. Calcium and phosphorous peaks are indicated by arrows (!).
Fig. 9 – Calcium and phosphorous maps of MSC seeded collagen scaffold sections. MSC seeded collagen scaffolds were cultured in OS medium for 14 days (a–c), 21 days (d–f) or 28 days (g–i) and analysed with SEM–EDX. Micrographs (a, d and g) and maps of the relative distribution of calcium (b, e and h) and phosphorous (c, f and i) were acquired. Bar = 100 mm.
archives of oral biology 52 (2007) 64–73
Fig. 10 – Collagen sponges in vitro degradation. Plot of collagen sponge mass retained percentage vs. time (mean W S.D. of three independent experiments).
mineralization of extra cellular matrix into bone, synthesized by bone forming cells35,36 and osteocalcin, a protein essential for the binding of calcium.36 These proteins are considered as lineage specific markers of osteoblastic differentiation.36 Also the increase in the levels of the alkaline phosphatase activity, an intracellular enzyme necessary for mineralization37 and considered to be an early marker of cells oriented towards osteogenic production,36 demonstrates that adult MSC exposed to OS medium differentiate towards osteoblastic lineage. According to other experimental studies, in OS medium treated MSC, ALP activity increases up to a peak and decreases to control level.17 The increase in ALP activity is a marker of the commitment towards osteoblastic lineage, while the subsequent decrease correlates with advanced matrix mineralization and a more mature phenotype.17,36 Calcium deposits in the matrix were demonstrated by alizarin red staining. This histological staining is based on the capacity of alizarin red to specifically stain matrix containing calcium and its positive appearance is considered an expression of bone matrix deposition.38 The co-localization of alizarin red stain with the deposit of amorphous material close to the nodular cell aggregates observed in OS cultures demonstrates that these amorphous deposits contain calcium and suggests that nodular cell aggregates are made up of cells committed to the osteoblastic lineage, confirmed by the high level of osteopontin expression in these structures. Several trophic factors have been proposed for inducing MSC osteogenic differentiation.39 In the present study, this target was reached in a simple and safe way using a cocktail of three different drugs, of which the key member was dexamethasone. Dexamethasone, as is the case with other glucocorticoids, has both a stimulatory and an inhibitory effect on osteogenic differentiation depending upon the dose. Dexamethasone is necessary for in vitro bone nodule formation and mineralization in marrow stroma-derived cell cultures,17,40 but it is also involved in adipogenesis of these cells in a timeand dose-related manner.15,41 The commercial lyophilized collagen sponge Gingistat1 is widely used as a scaffold for spontaneous bone regeneration
71
in periodontal diseases. Its wide and long-standing use reflects the good histo-compatibility of this kind of material. Due to the structural characteristics of the collagen sponge with large interconnected hollows, MSC are easily adsorbed and distributed to the entire scaffold. MSC seeded in the scaffold do not lose their capacity to be induced to differentiate. Thus, calcium deposits formed by MSC seeded on scaffolds and treated with OS medium are widely diffused. This has been demonstrated both with alizarin red staining and by EDX microanalysis. EDX enables qualitative and quantitative analyses of chemical elements within samples to be carried out using a SEM fitted with EDX equipment. By means of this method, it is possible to demonstrate that calcium and phosphorus co-localize, suggesting that calcium deposits in the scaffold are made by of hydroxyapatite. The Ca/P molar ratio of animal bones ranges from below to above 1.67 (the stoichiometric value for pure hydroxyapatite), depending on the species, age, and type of bone.42 When analysing the Ca/P ratio in MSC seeded scaffold treated for osteogenic differentiation our findings were in a fairly good agreement with the expected value. It has however to be noted that our results have been obtained in vitro, in an environment that present substantial differences from the natural bone. This fact could be responsible for the little differences observed. Altogether, the methods used to evaluate the cell distribution and the calcium deposits indicate that the Gingistat1 scaffold is suitable for supporting MSC and their commitment to form bone tissue. On the other hand, in vitro evaluation of the degradation time of the collagen sponge suggests that its use in in vivo experiments may be hindered by the scaffold’s complete dissolution prior to when bone formation is completed. This may be particularly true in the clinical use of this kind of scaffold since human adult MSC take more time with respect to those of rat to be committed towards an osteogenic phenotype (personal observations). Even if many similarities exist between MSC of rat and human origin, there are species differences that need to be considered in evaluating any kind of scaffold for clinical use in humans. Our results are to be confirmed using human MSC to evaluate if and at what extent the collagen scaffold is able to support human MSC osteogenic differentiation. Preliminary results using human MSC have confirmed the ability of these cells to growth and deposit calcium in the collagen scaffold. In conclusion, our results support the use of MSC for a tissue engineering approach to bone regeneration in periodontal diseases. The simple way in which MSC can be obtained from bone marrow, and the capacity of the entire population of bone derived cells to proliferate and differentiate, make this source of MSC particularly feasible for clinical application in restorative surgery. The possibility to greatly expand the number of MSC in vitro is a particularly important aspect since it suggests that in humans a limited bone marrow aspirate can supply an adequate number of cells for clinical applications. Further studies are required to evaluate alternative biomaterials with the same characteristics of biocompatibility as the Gingistat1 collagen scaffold, but which are able to provide the mechanical support for the time needed to allow tissue regeneration.
72
archives of oral biology 52 (2007) 64–73
Acknowledgments This work was supported by grants from Italian Minister for University and Scientific Research (MIUR), project FIRB 2001, protocol RBNE017HYL_001. We are grateful to Dr. L. Genton for language assistance.
references
1. Reynolds MA, Aichelmann-Reidy ME, Branch-Mays GL, Gunsolley JC. The efficacy of bone replacement grafts in the treatment of periodontal osseous defects. A systematic review. Ann Periodontol 2003;8:227–65. 2. Kenley RA, Yim K, Abrams J, Ron E, Turek T, Marden LJ, et al. Biotechnology and bone graft substitutes. Pharm Res 1993;10:1393–401. 3. Laurell L, Gottlow J, Zybutz M, Persson R. Treatment of intrabony defects by different surgical procedures. A literature review. J Periodontol 1998;69:303–13. 4. Seiler III JG, Johnson J. Iliac crest autogenous bone grafting: donor site complications. J South Orthop Assoc 2000;9:91–7. 5. Khan SN, Cammisa Jr FP, Sandhu HS, Diwan AD, Girardi FP, Lane JM. The biology of bone grafting. J Am Acad Orthop Surg 2005;13:77–86. 6. Schallhorn RG. Postoperative problems associated with iliac transplants. J Periodontol 1972;43:3–9. 7. Dragoo MR, Sullivan HC. A clinical and histological evaluation of autogenous iliac bone grafts in humans. I. Wound healing 2 to 8 months. J Periodontol 1973;44:599–613. 8. Moskow BS, Karsh F, Stein SD. Histological assessment of autogenous bone graft. A case report and critical evaluation. J Periodontol 1979;50:291–300. 9. Bartold PM, McCulloch CAG, Narayanan AS, Pitaru S. Tissue engineering: a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontol 2000;24:253– 69. 10. Betz RR. Limitations of autograft and allograft: new synthetic solutions. Orthopedics 2002;25(suppl):561S–70S. 11. Fibbe WE. Mesenchymal stem cells. A potential source for skeletal repair. Ann Rheum Dis 2002;61(suppl II):ii29–31. 12. Kawaguchi H, Hirachi A, Hasegawa N, Iwata T, Hamaguchi H, Shiba H, et al. Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cells. J Periodontol 2004;75:1281–7. 13. Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 1974;17:331–40. 14. Owen ME, Cave J, Joyner CJ. Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F. J Cell Sci 1987;87:731–8. 15. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–7. 16. Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med 2001;226:507–20. 17. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295–312. 18. Long MW. Osteogenesis and bone-marrow-derived cells. Blood Cells Mol Dis 2001;27:677–90. 19. Otto WR, Rao J. Tomorrow’s skeleton staff: mesenchymal repair of bone and cartilage. Cell Prolif 2004;37:97–110.
20. Yang S, Leong K-F, Du Z, Chua C-K. The design of scaffolds for use in tissue engineering. Part I. Traditional factors. Tissue Eng 2001;7:679–89. 21. Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000;21:2529–43. 22. Radice M, Brun P, Cortivo R, Scapinelli R, Battaliard C, Abatangelo G. Hyaluronan-based biopolymers as delivery vehicles for bone-marrow-derived mesenchymal progenitors. J Biomed Mater Res 2000;50:101–9. 23. Yamada Y, Boo JS, Ozawa R, Nagasaka T, Okazaki Y, Hata K, et al. Bone regeneration following injection of mesenchymal stem cells and fibrin glue with a biodegradable scaffold. J Craniomaxillofac Surg 2003;31:27–33. 24. El-Amin SF, Lu HH, Burems J, Mitchell J, Tuan RS, Laurencin CT. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering. Biomaterials 2003;4:1213–21. 25. Gunatillake PA, Adhikari R. Biodegradable synthetic polymers for tissue engineering. Eur Cell Mater 2003;5:1–16. 26. Kim H, Kim HW, Suh H. Sustained release of ascorbate-2phosphate and dexamethasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem cells. Biomaterials 2003;24:4671–9. 27. Meinel L, Karageorgiou V, Fajardo R, Snyder B, Shinde-Patil V, Zichner L, et al. Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Ann Biomed Eng 2004;32:112–22. 28. Garrett S. Periodontal regeneration around natal teeth. Ann Periodontol 1996;1:621–66. 29. Brekke JH, Toth JM. Principles of tissue engineering applied to progammable osteogenesis. J Biomed Mater Res 1998;43:380–98. 30. Mizutani K, Ikeda K, Kawai Y, Yamori Y. Resveratrol stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells. Biochem Biophys Res Commun 1998;253:859–63. 31. Donzelli E, Carfı` M, Miloso M, Strada A, Galbiati S, Bayssas M, et al. Neurotoxicity of platinum compounds: comparison of the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. J Neurooncol 2004;67:65–73. 32. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–4. 33. Bianco P, Riminucci M, Gronthos S, Gehron RP. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001;19:180–92. 34. Kotobuki N, Hirose M, Takakura Y, Ohgushi H. Cultured autologous human cells for hard tissue regeneration: preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs 2004;28:33–9. 35. Butler WT. The nature and significance of osteopontin. Connect Tissue Res 1989;23:123–36. 36. Aubin JE, Liu F, Malaval L, Gupta AK. Osteoblast and chondroblast differentiation. Bone 1995;17(suppl): 77S–83S. 37. Sugawara Y, Suzuki K, Koshikawa M, Ando M, Iida J. Necessity of enzymatic activity of alkaline phosphatase for mineralization of osteoblastic cells. Jpn J Pharmacol 2002;88:262–9. 38. Gregory CA, Gunn WG, Peister A, Prockop DJ. An alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 2004;329: 77–84. 39. Marie P, Debias F, Cohen-Solal M, de Vernejoul MC. New factors controlling bone remodelling. Joint Bone Spine 2000;67:150–6.
archives of oral biology 52 (2007) 64–73
40. Maniatopoulos C, Sodek J, Melcher AH. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res 1988;254:317–30. 41. Beresford JN, Bennett JH, Devlin C, LeBoy PS, Owen M. Evidence for an inverse relationship between the
differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci 1992;102:341–51. 42. LeGeros RZ. Properties of osteoconductive biomaterials: calcium phosphates. Clin Orthop Relat Res 2002;395: 81–98.
73
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/arob
Mesenchymal stem cells cultured on a collagen scaffold: In vitro osteogenic differentiation E. Donzelli a,*, A. Salvade` a, P. Mimo b, M. Vigano` a, M. Morrone c, R. Papagna a,d, F. Carini a,d, A. Zaopo c, M. Miloso a, M. Baldoni a,d, G. Tredici a a
Dipartimento di Neuroscienze e Tecnologie Biomediche, Universita` degli Studi di Milano-Bicocca, Via Cadore 48, 20052 Monza, MI, Italy CORIMAV, Viale Sarca 222, 20126 Milano, Italy c Pirelli Labs S.p.A., Viale Sarca 222, 20126 Milano, Italy d Clinica Odontoiatrica, Ospedale San Gerardo, Monza, Italy b
article info
abstract
Article history:
Objective: Management of periodontal defects has always been a challenge in clinical
Accepted 18 July 2006
periodontics. Recently mesenchymal stem cells (MSC) have been proposed for tissue regeneration in periodontal disease and repair of large bone defects. Bone regeneration
Keywords:
has to be supported by a scaffold which has to be biocompatible, biodegradable, and able to
Mesenchymal stem cells
support cell growth and differentiation. The aim of this study was to evaluate osteogenic
Osteogenic differentiation
differentiation of MSC seeded on a collagen scaffold.
Collagen scaffold
Design: MSC were obtained from adult rat bone marrow, expanded and cultured in plastic
Periodontal disease
dishes or seeded in a collagen scaffold (Gingistat1). MSC were induced towards osteogenic differentiation using osteogenic supplements. Cell differentiation and calcium deposits were evaluated by immunoblotting, immunohistochemistry, histochemical techniques, enzymatic activity assay, and SEM–EDX analysis. Biomaterial in vitro degradation was evaluated by measuring mass reduction after incubation in culture medium. Results: Rat MSC osteogenic differentiation was demonstrated by osteopontin and osteocalcin expression and an increase in alkaline phosphatase activity. MSC were distributed homogeneously in the collagen scaffold. Nodular aggregates and alizarin red stained calcium deposits were observed in MSC induced towards osteogenic differentiation cultured in dishes or seeded in the collagen scaffold. SEM–EDX analysis demonstrated that calcium co-localized with phosphorous. The biomaterial in vitro degraded in 4–5 weeks. Conclusions: MSC from bone marrow differentiate towards osteogenic lineage, representing a suitable cell source for bone formation in periodontal regeneration. Gingistat1 collagen scaffold supports MSC distribution and differentiation, but its short degradation time may be a limitation for a future application in bone tissue regeneration. # 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Autogenous bone grafts are the classic therapeutic approaches to severe periodontal disease with atrophic alveolar crests.1 These surgical procedures are invasive requiring bone with-
drawal from non-oral sites and results are not always satisfactory and may be associated with donor site morbidity.2–5 Rarely is a bone graft able to regenerate all the components of the periodontium and, moreover, it often results in excessive apical migration of the epithelium.6–9 To
* Corresponding author. Tel.: +39 0264488119; fax: +39 0264488253. E-mail address: [email protected] (E. Donzelli). 0003–9969/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2006.07.007
archives of oral biology 52 (2007) 64–73
reduce the problems of autogenous bone grafts, a tissue engineering strategy for periodontal regeneration has been proposed.9,10 This approach requires the use of a biomaterial containing appropriately selected or primed cells, but at present both the cells and biomaterial to be used have yet to be defined. Mesenchymal stem cells (MSC) derived from adult bone marrow are a possible cell source for tissue engineering, in particular for applications in skeletal and hard tissue repair and also in periodontal tissue regeneration.11,12 Bone marrow percutaneous harvest from the patient supplies autogenous MSC in a simple and safe way. Due to their multilineage potential and plasticity, the MSC may be committed to forming nonhematopoietic tissues including bone, cartilage, tendons, and ligaments.13–16 MSC isolated from bone marrow differentiate into an osteogenic lineage when cultured in the presence of dexamethasone, ascorbic acid, and b-glycerolphosphate (osteogenic supplements),11,17 but also other trophic factors or drugs have been proposed for use either in vitro or in animal models.18,19 Bone formation from MSC requires a three-dimensional scaffold to drive cellular growth and differentiation.19–21 An ongoing increasing number of biomaterials have been proposed as scaffolds for tissue regeneration, with the aim of reproducing the milieu where the complex interaction between cells and their matrix occurs.22–27 A prerequisite for the use of an appropriate scaffold to support bone formation is an understanding of its biological and structural characteristics. To repair oral bone defects the use of scaffolds shaped to the bone defect has been proposed.28,29 The ideal scaffold must have certain characteristics: (a) it must be easy to shape it onto the bone defect, (b) it must have a good capacity for hosting, growing and maturating MSC, (c) it must form a barrier to neighboring tissues and (d) it must have a reabsorption time compatible with the time required for bone formation but not so long that it interferes with the bone substitution of the scaffold. In this study, we report our experience with adult rat MSC in forming bone in vitro and on a commercial collagen biomaterial which has already been used as a scaffold to support bone regeneration in severe cases of atrophic periodontitis. The aim of this study was to evaluate the structural characteristics, the degradation time and the capacity to support cell distribution and differentiation of a commercial collagen sponge.
2.
Materials and methods
2.1.
Materials
65
The materials used in the present study are listed in Table 1.
2.2.
Cell preparation and culture methods
Mesenchymal stem cells (MSC) were obtained from bone marrow of 10-week-old female Sprague–Dawley rats. Animal procedures were conducted in accordance with the European Communities Council Directive 86/609/EEC. Both femora and tibias were removed and soft tissues were detached. Metaphysis from both ends were resected and bone marrow cells were collected by flushing the diaphysis with 2 ml/bone of Eagle’s alpha minimum essential medium (aMEM) containing 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 250 mg/ml fungizone. A suspension of bone marrow cells was obtained by repeated aspiration of the cell preparation through a 25 gauge needle. Red cells were depleted by 5 min incubation in NH4Cl 0.84%. Cells were resuspended in 15 ml a-MEM medium plus 20% ES cell screened fetal bovine serum, plated in a polystyrene 75 cm2 tissue culture flask and cultured in a humidified atmosphere of 95% air with 5% CO2 at 37 8C. After 2 days, the culture medium was removed and new medium was added. The medium was changed two or three times a week and the floating cells were removed. After 14 days of culture, cells were passaged by trypsinization (0.05% trypsin/EDTA solution). As the culture reached almost complete confluence, cells were subcultured or plated for subsequent experiments.
2.3.
Osteogenic differentiation
MSC (passage 4–6) were seeded at approximately 3500 cells/cm2 on culture dishes in a culture medium composed of a-MEM medium plus 10% defined FBS and cultured until subconfluence occurred. After this period, cells were grown in the culture medium alone or in osteogenic medium (OS medium) consisting of the same culture medium with the addition of osteogenic supplements, 100 nM dexamethasone, 10 mM b-glycerolphosphate, and 0.05 mM 2-phosphate-ascorbic acid.
2.4.
Cell seeding and differentiation on collagen scaffolds
Gingistat1 was used as a scaffold for this study. Gingistat1 is a sponge made of lyophilized collagen (type I). Collagen
Table 1 – Materials used in the present study Materials
Purchased from
a-MEM, glutamine, penicillin/streptomicyn, fungizone, trypsin/EDTA ES cell screened FBS, defined FBS Dexamethasone, b-glycerol-phosphate, alizarin red Gingistat1 Primary antibodies anti-osteopontin, anti-actin and HRP-conjugated anti-goat secondary antibody Primary antibody anti-osteocalcin HRP-conjugated anti-mouse secondary antibody FITC-conjugated anti-mouse secondary antibody Other chemicals
BioWhittaker, Bergamo, Italy Hyclone, Logan, UT, USA Applichem GmbH, Darmstadt, Germany Vebas, Milano, Italy Santa Cruz Biotechnology, Santa Cruz, CA, USA Abcam, Cambridge, UK Chemicon, Temecula, CA, USA Rockland, Gilbertsville, PA, USA Sigma Chemicals Co., St. Louis, MO, USA
66
archives of oral biology 52 (2007) 64–73
sponges were cut, under sterile conditions, into 3 mm 3 mm 3 mm cubes. MSC were suspended at a concentration of 5 106 cells/ml and 106 cells were poured onto each scaffold through a 25 gauge needle. After 4 h at 37 8C the medium was replaced with fresh medium and the scaffolds were maintained in a humidified atmosphere at 37 8C with 5% CO2 in air. After 4 days, the cells were treated with OS medium or with culture medium alone. The medium was changed every 3–4 days. For each time point, an unseeded scaffold incubated with culture medium alone was used as a control. Histological analyses were performed at days 7, 14, 21, and 28 of culture. Collagen sponges were washed twice with PBS, fixed with 4% paraformaldehyde for 1 h at RT, embedded in paraffin with standard methods, cut into 7 mm sections and processed as subsequently described.
2.5.
Alizarin red staining
Cells were plated and treated for osteogenic differentiation as described. Paraformaldehyde (4%) fixed cells were incubated for 30 min at room temperature (RT) in a solution containing 1% alizarin red and 1% ammonium hydroxide. Cells were rinsed twice with distilled water and allowed to dry completely. MSC seeded or unseeded collagen scaffold sections were deparaffinized with xylene, hydrated to 70% alcohol and stained with alizarin red solution for 30 s. Excess dye was shaken off and stained sections were fixed with acetone (20 dips), aceton–xylene (20 dips), cleared with xylene and mounted in permanent medium.
2.6.
Alkaline phosphatase activity assay
Cells were plated and treated for osteogenic differentiation as described. Alkaline phosphatase activity was evaluated using the p-nitrophenol method30 in cells cultured in culture medium alone or in OS medium. Cells were washed twice with ice-cold phosphate buffered saline (PBS), and protein extracts were prepared in 10 mM Tris–HCl (pH 7.6) and 0.1% Triton X-100. Total protein extracts were prepared at days 0, 7, 14, 21 and 28 after OS medium induction. Alkaline phosphatase enzyme activity was calculated after measuring the absorbance of p-nitrophenol product formed at 405 nm on a microplate reader (Bio-Rad, Hercules, CA).
2.7.
Cell lysates and immunoblotting analysis
Cells were plated and treated for osteogenic differentiation as described Cells lysates were prepared as previously reported31 at days 0, 7, 14, 21 and 28 after OS medium induction. Protein concentration was estimated with a modified Bradford assay and aliquots (60 mg) were separated by 15% SDS-polyacrylamide gels. Immunoblotting analysis was performed according to the manufacturer’s instructions with primary antibody anti-osteopontin (1:1000), anti-osteocalcin (1:500) or anti-actin (1:500) and an appropriate HRP-conjugated secondary antibody. Antigens were revealed by a chemiluminescence detection kit.
2.8.
Immunofluorescence
Cells cultured on collagen coated glass slides were treated as described for osteogenic differentiation. Selected cultures were fixed with 4% paraformaldehyde for 10 min and treated for 10 min with 0.1 M glycine (in PBS). Slides were incubated for 1 h at RT with blocking solution (5% BSA, bovine serum albumine, 0.5% Triton X-100 in PBS) and subsequently for 30 min at 37 8C with 1 mg/ml RNAse in blocking solution. Incubation with primary antibody anti-osteopontin (1:200) was performed overnight at 4 8C. The following day, cells were rinsed with PBS and incubated at RT for 1 h in the dark with an appropriate FITC-conjugated secondary antibody (1:100) and 2.5 mg/ml propidium iodide (PI). Non-specific binding of secondary antibody was controlled by omitting the primary antibody. Slides were washed and mounted with glycerol. Microscopy analysis was performed with laser confocal microscopy (Radiance 2100; Biorad Laboratories, Hercules, CA, USA).
2.9.
Hematoxylin and eosin staining
MSC seeded or unseeded collagen scaffold sections were deparaffinized with xylene, hydrated to 70% alcohol and stained according to standard hematoxylin/eosin staining protocols and mounted in permanent medium.
2.10.
SEM–EDX analysis of MSC seeded collagen scaffolds
Unseeded or MSC seeded collagen scaffolds were treated with culture medium or with OS medium as previously described for up to 28 days. At days 7, 14, 21, and 28 collagen scaffolds were embedded in paraffin and scanning electron microscopy (SEM)–energy dispersive X-ray spectrometry (EDX) micro-analysis was performed on 7 mm sections. Each section was fixed to a sample holder with a conductive carbon ribbon. SEM observations were carried out with a Hitachi S-2700 model scanning electron microscope equipped with EDX (Oxford Instruments, model ISIS300). EDX micro-analysis was employed to detect the presence of calcium and phosphorous on the samples, and maps of the relative distribution of these elements were acquired operating at 12 kV with an acquisition time of 10 min. The quantity of Ca and P in each sample was measured and the Ca/P ratio was calculated.
2.11. In vitro degradation study of GingistatW collagen sponges The in vitro degradation study of Gingistat1 sponges was carried out by incubating each of 15 samples in 50 ml of aMEM medium supplemented with 20% ES-FBS in an aircirculation oven at 37 8C. At different time points, samples were removed from the culture medium, washed with plenty of distilled water, and then dried under reduced pressure in a vacuum oven at 50 8C for 2 h before being weighed. The masses of the collagen sponges before and after incubation in the culture medium were weighed on a Sartorius electronic balance with a resolution of 0.01 mg. The mass retained percentage of each sponge was
archives of oral biology 52 (2007) 64–73
67
calculated by comparing the dry weight (Mt) after the established incubation time (t) with the initial weight (Mi) according to the following formula:
mass retained ¼
Mt 100% Mi
3.
Results
3.1.
Establishment of primary cultures
It is generally accepted that mesenchymal stem cells (MSC) can be recognized as the adherent cells derived from bone marrow capable of extensive proliferation, with a fibroblastic profile and with the ability to differentiate into mesenchymal lineages. Cells obtained from bone marrow were cultured as described in Material and Methods and, after 15 days of culture, an almost homogeneous population of fibroblasticlike cells was observed throughout the flask with little evidence of round or floating cells. These cells were induced to differentiate into various lineages and were able to proliferate in vitro for a long time. The capacity to proliferate is maintained until the 30th passage, although the capacity to differentiate is reduced as the passage number increases. Because of their proliferative and differentiative abilities we refer to these cells as MSC. For the described experiments we used cells at the fourth to sixth passage.
Fig. 2 – MSC stained with alizarin red. MSC were treated with culture medium alone (a–d) or OS medium (e–h) and stained with alizarin red at day 7 (a and e), day 14 (b and f), day 21 (c and g) or day 28 (d and h) after induction.
Fig. 3 – ALP activity assay. ALP activity was evaluated in cells treated with OS medium with respect to cells treated with culture medium alone (mean W S.D. of three independent experiments).
3.2.
Fig. 1 – MSC after 7 days of induction. (a) CTRL culture treated with culture medium alone. (b) OS culture treated with OS medium. Bars = 50 mm.
Osteogenic differentiation
MSC were plated and treated for osteogenic differentiation as described for up to 28 days. Osteogenic differentiation was evaluated using morphological, biochemical and immunological techniques both in cells grown in OS medium (OS cultures) and in cells cultured in culture medium alone (CTRL cultures) which represented control cells. Proliferation and differentiation of CTRL and OS cultures were compared using phase contrast microscopy. In both CTRL and OS cultures, cells proliferated and reached almost complete confluence at day 7. In OS cultures, nodular aggregates of cells became evident at day 7 of culture (Fig. 1) and increased up to 28 days. These aggregates were characterized by deposits of amorphous material. In CTRL cultures similar cellular aggregates were observed, but they were smaller, were evident only after 21 days and lacked deposits. The nodular aggregates in OS cultures stained with alizarin red, demonstrating that the amorphous deposits observed at the microscope were calcium deposits. Alizarin red positive nodular aggregates were already present at day 14. At day 28,
68
archives of oral biology 52 (2007) 64–73
Osteopontin expression was detectable by immunoblotting analysis at day 7 of induction and increased afterwards. Osteocalcin expression was detectable by immunoblotting analysis at day 14 of induction and increased afterwards. (Fig. 4). Osteopontin expression was evaluated also by immunofluorescence. High magnification observation revealed an abundant cytoplasmic expression of the protein in OS cultures after 14 and 28 days of induction. The nodular cellular aggregates were particularly rich of these immunopositive cells. In CTRL cultures the signal was very weak (Fig. 5). Fig. 4 – Osteopontin and osteocalcin immunoblotting analysis. Cell lysates (60 mg/lane) were analyzed by immunoblotting using an antibody anti-osteopontin or anti-osteocalcin, as indicated. Reprobing was done with actin by immunoblotting as loading control.
positive alizarin red aggregates were larger and stained more intensively, indicating that a more extensive calcium deposition had occurred. CTRL cultures showed only minimal background staining (Fig. 2). Alkaline phosphatase (ALP) activity was elevated in OS cultures with respect to CTRL ones from 14 days of induction onwards and reached its maximum at 21 days, then decreasing to values close to those of CTRL cultures (Fig. 3).
3.3. Collagen scaffold: structure and MSC osteogenic differentiation Gingistat1 collagen sponge structure was observed at SEM, revealing the presence of interconnected pores with an average size of 300 mm. Hematoxylin–eosin staining on serial sections of MSC seeded onto the Gingistat1 collagen scaffold sponge demonstrated that the cells were distributed homogeneously throughout the whole sponge (Fig. 6). Osteogenic differentiation of MSC grown on collagen scaffolds was firstly evaluated with alizarin red staining on unseeded and MSC seeded scaffolds cultured in culture medium alone or in OS medium. Unseeded scaffolds showed a low basal staining that remained unchanged during the
Fig. 5 – Osteopontin immunofluorescence. Cells were grown on collagen coated glass slides and treated for 14 days with culture medium alone (a) or with OS medium (b). Osteopontin: FITC, green; nuclei: PI, red. Bars = 10 mm.
Fig. 6 – Hematoxylin/eosin staining of unseeded (a) or MSC seeded (b) collagen scaffolds cultured for 14 days in culture medium alone. Bars = 100 mm.
archives of oral biology 52 (2007) 64–73
69
cultured on polystyrene dishes were also present on the scaffolds (Fig. 7). In order to obtain also a quantitative evaluation of calcium deposition, we performed an EDX micro-analysis on sections of MSC seeded collagen scaffolds treated with culture medium alone or with OS medium. A scaffold without cells was processed as a negative control to take into account the possible presence of calcium or phosphorous in the untreated material. Calcium and phosphorous were detected on the samples incubated in OS medium (Fig. 8) and the chemical distribution maps thus collected indicate that these elements co-localize in the same areas of the sections (Fig. 9). A comparison of the calcium and phosphorous maps of the OS medium incubated samples clearly showed an increase in the presence of both chemical elements, with very low levels at day 7 up to very high levels at day 28. The distribution of calcium and phosphorous was homogeneous in all scaffold sections. The mean value of Ca/P ratio in the samples incubated with OS medium was about 1.2. Neither MSC seeded scaffolds treated with culture medium alone nor the unseeded scaffold showed any relevant presence of calcium or phosphorous, confirming that these chemical elements accumulated only onto scaffolds seeded with cells which had been exposed to the OS medium. The MSC seeded scaffold cultured for 28 days in culture medium alone showed a very low level of calcium and phosphorous. The study of the degradation of the collagen scaffold demonstrated that Gingistat1 discs degraded in about 4–5 weeks with a mostly linear trend. There was an approximately 20% mass loss at each week of incubation which was also reflected in a size decrease of the discs (Fig. 10).
4.
Fig. 7 – Alizarin red staining of unseeded (a) or MSC seeded (b and c) collagen scaffolds treated for 21 days with culture medium alone (a and b) or with OS medium (c). Bars = 50 mm.
whole observation period. MSC seeded collagen scaffolds cultured in culture medium alone stained weakly even after 28 days of culture. On the contrary, MSC seeded collagen scaffolds cultured in OS medium stained strongly with alizarin red starting at day 14 of culture. Alizarin red stained deposits similar to those observed in osteogenic differentiation of MSC
Discussion
The present study shows that the adult rat bone marrow is a suitable and feasible source of a great number of MSC and that the adult bone marrow derived MSC can be easily induced to differentiate into an osteogenic lineage. Furthermore, we have demonstrated that a commercial collagen spongy scaffold is biocompatible with the MSC and we propose some methods for evaluating those characteristics of the scaffold necessary for possible clinical use in periodontal diseases. In our experience, and according to many experimental studies, cells obtained from the bone marrow in toto, without selection of any particular population, constitute an optimal source of MSC which can then be differentiated towards an osteoblastic phenotype.11,32–34 The mesenchymal stem cell nature of the cells derived from the bone marrow of adult rats is demonstrated by their capacity of extensive proliferation and to differentiate towards several different lineages.13–16 In this context, the capacity of the bone marrow derived MSC to differentiate towards an osteogenic lineage and to deposit calcium has been demonstrated by several methods based on immunohistochemical and histochemical techniques, enzymatic activity assay, and SEM–EDX analysis. MSC committment to osteogenic differentiation was demonstrated by the expression of osteopontin, a phosphoprotein that possesses several calcium binding domains and is associated with cell attachment, proliferation, and
70
archives of oral biology 52 (2007) 64–73
Fig. 8 – SEM–EDX analysis of MSC seeded collagen scaffold sections. MSC seeded collagen scaffolds were cultured in culture medium alone (a, c, e and g) or OS medium (b, d, f and h) and analysed with SEM–EDX at 7 days (a and b), 14 days (c and d), 21 days (e and f) and 28 days (g and h) after induction. Calcium and phosphorous peaks are indicated by arrows (!).
Fig. 9 – Calcium and phosphorous maps of MSC seeded collagen scaffold sections. MSC seeded collagen scaffolds were cultured in OS medium for 14 days (a–c), 21 days (d–f) or 28 days (g–i) and analysed with SEM–EDX. Micrographs (a, d and g) and maps of the relative distribution of calcium (b, e and h) and phosphorous (c, f and i) were acquired. Bar = 100 mm.
archives of oral biology 52 (2007) 64–73
Fig. 10 – Collagen sponges in vitro degradation. Plot of collagen sponge mass retained percentage vs. time (mean W S.D. of three independent experiments).
mineralization of extra cellular matrix into bone, synthesized by bone forming cells35,36 and osteocalcin, a protein essential for the binding of calcium.36 These proteins are considered as lineage specific markers of osteoblastic differentiation.36 Also the increase in the levels of the alkaline phosphatase activity, an intracellular enzyme necessary for mineralization37 and considered to be an early marker of cells oriented towards osteogenic production,36 demonstrates that adult MSC exposed to OS medium differentiate towards osteoblastic lineage. According to other experimental studies, in OS medium treated MSC, ALP activity increases up to a peak and decreases to control level.17 The increase in ALP activity is a marker of the commitment towards osteoblastic lineage, while the subsequent decrease correlates with advanced matrix mineralization and a more mature phenotype.17,36 Calcium deposits in the matrix were demonstrated by alizarin red staining. This histological staining is based on the capacity of alizarin red to specifically stain matrix containing calcium and its positive appearance is considered an expression of bone matrix deposition.38 The co-localization of alizarin red stain with the deposit of amorphous material close to the nodular cell aggregates observed in OS cultures demonstrates that these amorphous deposits contain calcium and suggests that nodular cell aggregates are made up of cells committed to the osteoblastic lineage, confirmed by the high level of osteopontin expression in these structures. Several trophic factors have been proposed for inducing MSC osteogenic differentiation.39 In the present study, this target was reached in a simple and safe way using a cocktail of three different drugs, of which the key member was dexamethasone. Dexamethasone, as is the case with other glucocorticoids, has both a stimulatory and an inhibitory effect on osteogenic differentiation depending upon the dose. Dexamethasone is necessary for in vitro bone nodule formation and mineralization in marrow stroma-derived cell cultures,17,40 but it is also involved in adipogenesis of these cells in a timeand dose-related manner.15,41 The commercial lyophilized collagen sponge Gingistat1 is widely used as a scaffold for spontaneous bone regeneration
71
in periodontal diseases. Its wide and long-standing use reflects the good histo-compatibility of this kind of material. Due to the structural characteristics of the collagen sponge with large interconnected hollows, MSC are easily adsorbed and distributed to the entire scaffold. MSC seeded in the scaffold do not lose their capacity to be induced to differentiate. Thus, calcium deposits formed by MSC seeded on scaffolds and treated with OS medium are widely diffused. This has been demonstrated both with alizarin red staining and by EDX microanalysis. EDX enables qualitative and quantitative analyses of chemical elements within samples to be carried out using a SEM fitted with EDX equipment. By means of this method, it is possible to demonstrate that calcium and phosphorus co-localize, suggesting that calcium deposits in the scaffold are made by of hydroxyapatite. The Ca/P molar ratio of animal bones ranges from below to above 1.67 (the stoichiometric value for pure hydroxyapatite), depending on the species, age, and type of bone.42 When analysing the Ca/P ratio in MSC seeded scaffold treated for osteogenic differentiation our findings were in a fairly good agreement with the expected value. It has however to be noted that our results have been obtained in vitro, in an environment that present substantial differences from the natural bone. This fact could be responsible for the little differences observed. Altogether, the methods used to evaluate the cell distribution and the calcium deposits indicate that the Gingistat1 scaffold is suitable for supporting MSC and their commitment to form bone tissue. On the other hand, in vitro evaluation of the degradation time of the collagen sponge suggests that its use in in vivo experiments may be hindered by the scaffold’s complete dissolution prior to when bone formation is completed. This may be particularly true in the clinical use of this kind of scaffold since human adult MSC take more time with respect to those of rat to be committed towards an osteogenic phenotype (personal observations). Even if many similarities exist between MSC of rat and human origin, there are species differences that need to be considered in evaluating any kind of scaffold for clinical use in humans. Our results are to be confirmed using human MSC to evaluate if and at what extent the collagen scaffold is able to support human MSC osteogenic differentiation. Preliminary results using human MSC have confirmed the ability of these cells to growth and deposit calcium in the collagen scaffold. In conclusion, our results support the use of MSC for a tissue engineering approach to bone regeneration in periodontal diseases. The simple way in which MSC can be obtained from bone marrow, and the capacity of the entire population of bone derived cells to proliferate and differentiate, make this source of MSC particularly feasible for clinical application in restorative surgery. The possibility to greatly expand the number of MSC in vitro is a particularly important aspect since it suggests that in humans a limited bone marrow aspirate can supply an adequate number of cells for clinical applications. Further studies are required to evaluate alternative biomaterials with the same characteristics of biocompatibility as the Gingistat1 collagen scaffold, but which are able to provide the mechanical support for the time needed to allow tissue regeneration.
72
archives of oral biology 52 (2007) 64–73
Acknowledgments This work was supported by grants from Italian Minister for University and Scientific Research (MIUR), project FIRB 2001, protocol RBNE017HYL_001. We are grateful to Dr. L. Genton for language assistance.
references
1. Reynolds MA, Aichelmann-Reidy ME, Branch-Mays GL, Gunsolley JC. The efficacy of bone replacement grafts in the treatment of periodontal osseous defects. A systematic review. Ann Periodontol 2003;8:227–65. 2. Kenley RA, Yim K, Abrams J, Ron E, Turek T, Marden LJ, et al. Biotechnology and bone graft substitutes. Pharm Res 1993;10:1393–401. 3. Laurell L, Gottlow J, Zybutz M, Persson R. Treatment of intrabony defects by different surgical procedures. A literature review. J Periodontol 1998;69:303–13. 4. Seiler III JG, Johnson J. Iliac crest autogenous bone grafting: donor site complications. J South Orthop Assoc 2000;9:91–7. 5. Khan SN, Cammisa Jr FP, Sandhu HS, Diwan AD, Girardi FP, Lane JM. The biology of bone grafting. J Am Acad Orthop Surg 2005;13:77–86. 6. Schallhorn RG. Postoperative problems associated with iliac transplants. J Periodontol 1972;43:3–9. 7. Dragoo MR, Sullivan HC. A clinical and histological evaluation of autogenous iliac bone grafts in humans. I. Wound healing 2 to 8 months. J Periodontol 1973;44:599–613. 8. Moskow BS, Karsh F, Stein SD. Histological assessment of autogenous bone graft. A case report and critical evaluation. J Periodontol 1979;50:291–300. 9. Bartold PM, McCulloch CAG, Narayanan AS, Pitaru S. Tissue engineering: a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontol 2000;24:253– 69. 10. Betz RR. Limitations of autograft and allograft: new synthetic solutions. Orthopedics 2002;25(suppl):561S–70S. 11. Fibbe WE. Mesenchymal stem cells. A potential source for skeletal repair. Ann Rheum Dis 2002;61(suppl II):ii29–31. 12. Kawaguchi H, Hirachi A, Hasegawa N, Iwata T, Hamaguchi H, Shiba H, et al. Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cells. J Periodontol 2004;75:1281–7. 13. Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 1974;17:331–40. 14. Owen ME, Cave J, Joyner CJ. Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F. J Cell Sci 1987;87:731–8. 15. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–7. 16. Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med 2001;226:507–20. 17. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295–312. 18. Long MW. Osteogenesis and bone-marrow-derived cells. Blood Cells Mol Dis 2001;27:677–90. 19. Otto WR, Rao J. Tomorrow’s skeleton staff: mesenchymal repair of bone and cartilage. Cell Prolif 2004;37:97–110.
20. Yang S, Leong K-F, Du Z, Chua C-K. The design of scaffolds for use in tissue engineering. Part I. Traditional factors. Tissue Eng 2001;7:679–89. 21. Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000;21:2529–43. 22. Radice M, Brun P, Cortivo R, Scapinelli R, Battaliard C, Abatangelo G. Hyaluronan-based biopolymers as delivery vehicles for bone-marrow-derived mesenchymal progenitors. J Biomed Mater Res 2000;50:101–9. 23. Yamada Y, Boo JS, Ozawa R, Nagasaka T, Okazaki Y, Hata K, et al. Bone regeneration following injection of mesenchymal stem cells and fibrin glue with a biodegradable scaffold. J Craniomaxillofac Surg 2003;31:27–33. 24. El-Amin SF, Lu HH, Burems J, Mitchell J, Tuan RS, Laurencin CT. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering. Biomaterials 2003;4:1213–21. 25. Gunatillake PA, Adhikari R. Biodegradable synthetic polymers for tissue engineering. Eur Cell Mater 2003;5:1–16. 26. Kim H, Kim HW, Suh H. Sustained release of ascorbate-2phosphate and dexamethasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem cells. Biomaterials 2003;24:4671–9. 27. Meinel L, Karageorgiou V, Fajardo R, Snyder B, Shinde-Patil V, Zichner L, et al. Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Ann Biomed Eng 2004;32:112–22. 28. Garrett S. Periodontal regeneration around natal teeth. Ann Periodontol 1996;1:621–66. 29. Brekke JH, Toth JM. Principles of tissue engineering applied to progammable osteogenesis. J Biomed Mater Res 1998;43:380–98. 30. Mizutani K, Ikeda K, Kawai Y, Yamori Y. Resveratrol stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells. Biochem Biophys Res Commun 1998;253:859–63. 31. Donzelli E, Carfı` M, Miloso M, Strada A, Galbiati S, Bayssas M, et al. Neurotoxicity of platinum compounds: comparison of the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. J Neurooncol 2004;67:65–73. 32. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–4. 33. Bianco P, Riminucci M, Gronthos S, Gehron RP. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001;19:180–92. 34. Kotobuki N, Hirose M, Takakura Y, Ohgushi H. Cultured autologous human cells for hard tissue regeneration: preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs 2004;28:33–9. 35. Butler WT. The nature and significance of osteopontin. Connect Tissue Res 1989;23:123–36. 36. Aubin JE, Liu F, Malaval L, Gupta AK. Osteoblast and chondroblast differentiation. Bone 1995;17(suppl): 77S–83S. 37. Sugawara Y, Suzuki K, Koshikawa M, Ando M, Iida J. Necessity of enzymatic activity of alkaline phosphatase for mineralization of osteoblastic cells. Jpn J Pharmacol 2002;88:262–9. 38. Gregory CA, Gunn WG, Peister A, Prockop DJ. An alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 2004;329: 77–84. 39. Marie P, Debias F, Cohen-Solal M, de Vernejoul MC. New factors controlling bone remodelling. Joint Bone Spine 2000;67:150–6.
archives of oral biology 52 (2007) 64–73
40. Maniatopoulos C, Sodek J, Melcher AH. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res 1988;254:317–30. 41. Beresford JN, Bennett JH, Devlin C, LeBoy PS, Owen M. Evidence for an inverse relationship between the
differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci 1992;102:341–51. 42. LeGeros RZ. Properties of osteoconductive biomaterials: calcium phosphates. Clin Orthop Relat Res 2002;395: 81–98.
73