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How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials
Biomaterials 23 (2002) 3319–3328
How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials a . M. Wiedmann-Al-Ahmada,*, R. Gutwalda, G. Lauerb, U. Hubner , R. Schmelzeisena a
Klinik und Poliklinik fur . Mund-, Kiefer- und Gesichtschirurgie, Albert-Ludwigs-Universitat . Freiburg, Hugstetterstraße 55, D-79106 Freiburg, Germany b Klinik und Poliklinik fur Carl G. Carus, Technische Universitat . Mund-, Kiefer- und Gesichtschirurgie, Universitatsklinikum . . Dresden, Fetscher Straße 74, D-01307, Dresden, Germany Received 5 April 2001; accepted 8 January 2002
Abstract The optimization of seeding and culturing of human osteoblast-like cells on three collagen-based biomaterials (bovine, equine and calf collagen membrane) was studied by cell proliferation and cell colonization (scanning electron microscopy) analysis. Osteoblasts of five patients were seeded onto the three biomaterials and two different parameters were varied: the time intervals between initial seeding and adding culture medium (2 h, 6 h, 12 h, 24 h) and the seeding concentration (1 105, 1 106, 2 106 cells/ml) of cells onto biomaterials. The results of the study demonstrated that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration effects the cell proliferation and the cell colonization. The best proliferation rate was achieved by adding the culture medium 2 h after initial seeding and with a seeding density of 1 105 cells/ml. Moreover, all three biomaterials resulted in different proliferation rates. The best proliferation rate resulted with the bovine collagen membrane. In conclusion, the examined parameters are very important for the development of the tissue engineering techniques and in a larger perspective also for reconstructive surgery. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Human osteoblast-like cells; Biomaterials; Seeding; Cultivation; Cell proliferation; Cell colonization
1. Introduction The application of tissue engineered bone grafts in the reconstruction of facial and maxillary sceletal defects requires cell carrier matrices which have no cytotoxic effect and allow a three-dimensional growth of human osteoblasts. Usually, the osteoblast-like cells are grown adherent to the floor of the culture dish very well but for grafting, the growth on support materials is necessary. Over the past few years, especially in the development of the tissue engineering techniques, new biomaterials have been introduced for the use in reconstructive surgery. These commercially available cell carrier matrices differ in composition, pore size and pore density, as well as in permeability and durability [1]. However, most of them are bioabsorbable which is an advantage for tissue
*Corresponding author. Tel.: +1-49-761-270-4989; fax: +1-49-761270-4817. E-mail address: [email protected] (M. Wiedmann-Al-Ahmad).
engineering and reconstructive surgery as the materials need not to be removed and consequently, a second surgical procedure is not required. Studies about the cultivation and growth of epithelial cells and human gingival keratinocytes on different biomaterials were published regarding their attachment and spreading including the protein adsorption behaviour onto various implant materials such as titanium, gold, hydroxylapatite, carbonate apatite and polystyrene substrates [2–7]. Especially, the influence of the surface topography of such implant materials on biomolecule or cell adherence, orientation and morphogenesis has been widely reported and plays an important role [8–10]. Our recent study [11] describes the cultivation of gingival keratinocytes on different permeable polycarbonate membranes with various pore sizes and on nylon. The results indicated that primary keratinocyte cultures, established according to the explant culture technique [12–14], formed a confluent cell layer on both support materials. Whereas, using secondary cultures, established according to the disperse culture technique [15], a confluent cell layer was only observed on the polycarbonate membranes.
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 0 1 9 - 4
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In contrast, only few data exist about the attachment and growth of human osteoblast-like cells on such cell carriers. Less data is also available about the requirements a biomaterial must meet such that the cells attach very well and about the healing of such tissue engineered constructs in humans. Different aspects of culturing osteoblasts were monitored such as their differentiation during culturing and their cell invasion into porous materials [16–20]. Studies about osteoblast-material interactions have shown that they depend on various factors such as topography, chemistry and surface energy [16,21]. For example, the protein adsorption is influenced by the pH, the temperature and the ionic composition as well as by the surface roughness [22–24]. Till now, in case of human osteoblast-like cells and bioabsorbable carrier materials, the search for the ideal material as well as the optimization for culture and seeding conditions on various biomaterials continues. In this paper, we focus on the seeding and cultivation procedure in order to optimize the attachment and growth of human osteoblasts on various cell carriers. A second aspect is the comparison of three different biomaterials with respect to promoting attachment and proliferation of human osteoblasts. The three different biomaterials examined were a native bovine collagen membrane, a native equine collagen membrane and a native calf collagen membrane. The aims of this study were to optimize the culture conditions of osteoblasts and to find a useful cytocompatible biomaterial for the surgical management of intraoral applications. This would be of interest to develop tissue engineering straightforward for creating autologous grafts, which can be applied soon in reconstructive surgery.
2. Materials and methods 2.1. Biomaterials Three different biomaterials were used for the cultivation of human osteoblast-like cells and for potential subsequent grafting: a native bovine collagen membrane (Tissue Vliess, Baxter, Heidelberg, Germany), a native equine collagen membrane (Resorbas, Nurnberg, . Germany) and a native calf collagen membrane (Osteovits, B. Braun-Dexon GmbH, Spangenberg, Germany). 2.2. Isolation and cultivation of human osteoblast-like cells For cultivation of osteoblasts, pieces of corticolamellar bone of the maxilla from five different patients of each sex in the age range of 23–65 yr were crumbled into explants (size 2 mm 2 mm) and seeded on culture flasks (25 cm2, Greiner, Frickenhausen, Germany) using
Opti-minimal essential medium (Opti-MEM, Gibco Laboratories Life Technologies, Inc, Grand Island, NY, USA) pH 7.2 with 10% foetal calf serum (FCS) and kept in a humidified atmosphere of 5% CO2 at 371C (Heraeus, Hanau, Germany). The osteoblast-like cells which migrated onto the floor of the culture dish form a confluent layer after 4–5 weeks (primary culture) and the first passage was used for the growth experiments on the various biomaterials. 2.3. Preparation of the cell culture plates Before cell seeding onto the biomaterials the 24-well culture plates (Costar, NY, USA) were humidified with culture medium. The three different plasma-sterilized biomaterials (1 cm2) were placed into the wells: one for scanning electron microscopic study, one for alkaline phosphatase assay and one for the type I collagen determination. For the proliferation test smaller samples of the three materials were placed in 96-well plates (Corning, NY, USA): one well for the medium blank, one well with the biomaterial only, and three wells with the biomaterial and the seeded cells. 2.4. Seeding the cells onto biomaterials The confluent osteoblast cultures (primary culture) were detached from the culture flask by incubation with 0.5% trypsin (Gibco, Paisley, Scotland) in phosphatebuffered saline (PBS) for 8 min at 371C. The bone cell solution was filtered through a 100 mm cell-strainer (Falcon, Heidelberg, Germany) in a 50 ml tube (Falcon, Heidelberg, Germany), centrifuged (Biofuge Strato, Heraeus, Hanau, Germany, 1120 g, 12 min, 301C) and resuspended in 1 ml culture medium. The cells were transferred into a 75 cm2 culture flask (Greiner, Frickenhausen, Germany), filled up with 25 ml culture medium. After 14 days, the cells of the first passage were detached again from the culture flask with 0.5% trypsin, centrifuged and resuspended in 1 ml culture medium. After staining with trypanblue (1:1; v/v), the cells still alive were counted in a chamber by light microscopy (Zeiss Axiovert 135, Jena, Germany). Then, the cells were seeded onto the biomaterials of constant size. Two different parameters were tested in this study (the materials were seeded with osteoblasts of five patients; for each parameter and each test as triplicate): *
Time interval between initial cell seeding and adding sufficient volume of culture medium to cover biomaterial cubes completely 100 ml of 106 cells/ml was seeded onto the three different biomaterials with a sterile syringe. The cells were incubated for 2, 6, 12 and 24 h, respectively, at 371C in 5% CO2 atmosphere before adding the
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culture medium to a total volume of 1 ml. After three days of incubation in total, scanning electron microscopic studies and proliferation assays followed. Seeding concentration To examine the optimal seeding concentration, three different cell concentrations (1 105, 1 106 and 2 106 cells/ml) were seeded by a sterile syringe onto the biomaterial cubes. The total volume was 100 ml. The plates were incubated for 2 h at 371C in 5% CO2 atmosphere. Then, the culture medium was added and a further incubation for 3 days followed.
Additionally, plates with 1 105 cells/ml were incubated for 1 week at 371C in 5% CO2 atmosphere for the detection of alkaline phophatase and collagen. Once in a week the medium was changed. Parallel to seeding the cells (second passage) onto the biomaterials an aliquot of the same passage was seeded in cell culture plates. This serves as control for the cell proliferation as well as for the detection of the cells as osteoblasts. 2.5. Assay for osteocalcin For the quantification of osteocalcin in the cell culture supernatant of human maxillar osteoblast-like cells, the osteocalcin ELISA (DAKO, Glostrup, Denmark) was performed according to the manufacturer’s instructions. In brief, the standards, the curve control and the cell culture supernatants were premixed with biotinylated osteocalcin, incubated in microwells precoated with anti-osteocalcin for 1 h, washed and incubated with peroxidase-conjugated streptavidin for 15 min, which binds strongly to the biotinylated osteocalcin. After a further washing step the chromogenic substrate was added and incubated for 30 min. The reaction was stopped by 2 m H2SO4 and the absorbance at 450 nm was measured. Osteocalcin is exclusively synthesized by osteoblasts and is believed to prevent premature mineralization of newly formed, but yet disorganized bone matrix [25]. 2.6. Alkaline phosphatase assay and morphometry For the staining of maxillar osteoblast-like cells an alkaline phosphatase assay kit (Sigma, Deisenhofen, Germany) was used. The culture dishes were air dried, fixed in a citrate-aceton-formaldehyde solution for 30 s and rinsed gently with Aqua dest. Incubation with alkaline phosphatase staining solution for 15 min protected from direct light and a washing step with Aqua dest followed. The citrate-aceton-formaldehyde solution as well as the alkaline phosphatase staining solution was prepared according to the manufacturer’s instructions. The culture dishes were counterstained
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with neutral red for 5 min, rinsed with Aqua dest. and dried mounted with cover slips. Positive staining for alkaline phosphatase (red–violet) was identified by light microscopy and evaluated by morphometry using the computer program Analysis 3.1 (Soft Imaging System, Munster, . Germany). 2.7. Detection of type I collagen For the quantification of type I collagen the cells were washed in PBS for 5 min, fixed with 70% ethanol for 1 h, washed in PBS for 5 min, allowed to air dry and washed again in PBS for 5 min. After an incubation of 0.3% H2O2 in methanol for 30 min, unspecific immune reactions were blocked with 1% bovine serum albumin for 10 min, before the anti-collagen I antibody (Sigma, Deisenhofen, Germany) was administered for 1 h. An incubation of the biotin-conjugated secondary antibody (Vectastain Elite Kit, Vector Laboratories, Burlingame USA) for 45 min and an incubation of avidin mixed with biotin-conjugated peroxidase (Vectastain Elite Kit, Vector Laboratories, Burlingame USA) for 30 min followed. Sections were rinsed between each incubation step three times with PBS for 5 min. The immunoreaction was developed by diaminobenzidine-solution (0.05 mg/l DAB/0.05 m Tris-HCl pH 7.3/0.01% H2O2) at room temperature. The sections were counterstained with hematoxylin (Merck, Darmstadt, Germany) for 10 s and mounted in 40% glycerin (Merck, Darmstadt, Germany) in PBS. The evaluation was done by light microscopy and the computer program Analysis 3.1 (Soft Imaging System, Munster, . Germany). 2.8. Cell proliferation analysis For cell proliferation analysis, the nonradioactive assay EZ4UFEASY FOR YOU (Biozol diagnostica GmbH; Eching, Germany) was used. This method is based on the finding that living cells are capable of reducing slightly yellow coloured tetrazolium salts to intense red coloured formazan derivatives by an intracellular reduction system, mostly located in the mitochondria [26]. These formazan derivatives are excreted into the culture medium and the absorbance can be measured with a microplate reader. The amount of coloured formazan derivatives correlates with the amount of living cells in the sample. The proliferation assay was carried out according to the manufacturers’ instructions. 2.9. Cell colonization analysis The cell colonization analysis was assessed by scanning electron microscopy. For scanning electron microscopy the samples were fixed in 4% paraformaldehyde for 2 h at room temperature and incubated in
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8% formaldehyde for 2 days at 41C. The samples were dehydrated in graded alcohol (30%, 50%, 70%, 80%, 90%, each one time and two times in 99.8%). After critical point drying (CPD 030 Baltec, Wallruf, Germany), according to standard procedure using liquid carbon dioxide, the samples were sputtered with gold palladium (Plano, Germany) in the SCD 040 (Balzers Union, Wallruf, Germany). The probes were examined via Zeiss Leo 32 scanning electron microscope (Zeiss, Kochern, Germany) at 10 kV. Images were digitized.
the seeding concentration as well as the time of adding the culture medium after seeding the osteoblast-like cells onto the biomaterials. The values of the standard deviation are between 0.03 and 0.1 (see Fig. 1 error bars). Osteoblast-like cells seeded on bovine collagen showed in each test significantly more vital cells than the other two biomaterials, equine collagen and calf collagen independent of which patient was used. The highest proliferation rate could be measured with a seeding concentration of 1 105 cells/ml and adding the culture medium 2 h after seeding the cells onto the biomaterials.
3. Results 3.2. Scanning electron microscopy 3.1. Cell proliferation and vitality The vitality and the proliferation capacity of the osteoblast-like cells from five different patients seeded each onto three different biomaterials was studied by the EZ4UFEASY FOR YOU test. The average of the measured absorbance is shown in Fig. 1 depending on
The surface of the three collagen biomaterials with respect to the osteoblast morphology, attachment and tissue invasion was studied by the scanning electron microscope. Fig. 2 shows the biomaterials without seeding cells as control. Whereas, the bovine and the equine collagen membrane show a similar structure with
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Fig. 1. Cell proliferation analysis of human osteoblast-like cells seeded onto native bovine, native equine and native calf collagen membrane. The time intervals between initial cell seeding and adding culture medium were varied: 2 h (A), 6 h (B), 12 h (C) and 24 h (D). In each test different seeding concentrations (1 105, 1 106, 2 106 cells/ml) were used. Error bars represent standard deviations.
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the biomaterials showed that there is the best cell density of intact osteoblasts when adding the culture medium after 2 h. These results were independent of the patient material used and independent of the biomaterial used (Figs. 3 and 4; calf collagen membrane not shown). In Figs. 5–7 different concentrations of osteoblast-like cells for seeding were used. It could be observed that the osteoblast-like cells of all five patients grow to form confluent sheets in the presence of bovine collagen, equine collagen and calf collagen. The surface of the biomaterials was completely covered with the osteoblast-like cells. The best result in covering the biomaterial surface was achieved with a seeding concentration of 1 105 cells/ml (Figs. 5A–7A). No differences were visible between the patient material (data not shown) and between the different collagen membranes. A higher seeding concentration showed much more not intact osteoblast-like cells, round cells were visible as well as incomplete cell sheets. The proliferation and vitality test (EZ4UFEASY FOR YOU) confirmed these morphological findings that using a seeding concentration of 1 105 cells/ml is optimal, and that the part of living cells, able to proliferate, is extremely reduced when applying higher seeding concentrations than 1 105 cells/ml (Fig. 1 and see section results, cell proliferation). 3.3. Osteocalcin determination, alkaline phosphatase assay and determination of type I collagen The characterization of the cells seeded on the three different biomaterials as osteoblasts was determined by the amount of osteocalcin, the relative amount of alkaline phosphatase activity and the relative amount of the presence of cells expressing type I collagen. Gingival keratinocytes were used as control. The amount of osteocalcin was 5.48 mg/l at a concentration of 1 105 cells/ml (standard deviation 0.94), 10.18 mg/l at a concentration of 1 106 cells/ml (standard deviation 0.88) and 12.66 mg/l at a concentration of 2 106 cells/ml (standard deviation 0.96). The alkaline staining of these cells typically resulted intensively positive (about 59%) whereas the human gingival keratinocytes did not. Immunocytochemistry of the fixed cells showed the presence of type I collagen in about 74% of the cells. Fig. 2. Scanning electron microscopic study of three native collagen membranes of different origins without osteoblasts. Bovine (A), equine (B) and calf (C) collagen membrane.
an extensive pore mesh (Fig. 2A and 2B), the calf collagen membrane shows a smooth surface with small pores (Fig. 2C). Comparing the different time intervals (2, 6, 12 and 24 h) of adding culture medium after seeding the cells on
4. Discussion The attachment and proliferation of human osteoblast-like cells on biomaterials is required for tissue engineering of bone autografts. Bone autografts are regularly necessary for skeletal reconstructions in cases involving large defects created by tumor resection, trauma and skeletal abnormalities and they have the
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Fig. 3. Scanning electron microscopic study of human osteoblast-like cells cultivated onto native bovine collagen membrane. Different time intervals between initial cell seeding and adding culture medium are shown: 2 h (A), 6 h (B), 12 h (C) and 24 h (D). Arrows showing the living cells ( ) and the cells which were not viable anymore ( ).
Fig. 4. Scanning electron microscopic study of human osteoblast-like cells cultivated onto native equine collagen membrane. Different time intervals between initial cell seeding and adding culture medium are shown: 2 h (A), 6 h (B), 12 h (C) and 24 h (D). Arrows showing the living cells ( ), the cells which were not viable anymore ( ) and the biomaterial itself ( ).
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Fig. 5. Scanning electron microscopic study of human osteoblast-like cells seeded onto native bovine collagen membrane with different seeding concentrations: 1 105 cells/ml (A), 1 106 cells/ml (B) and ), the cells 2 106 cells/ml (C). Arrows showing the living cells ( which were not viable anymore ( ) and the biomaterial itself ( ).
Fig. 6. Scanning electron microscopic study of human osteoblast-like cells seeded onto native equine collagen membrane with different seeding concentrations: 1 105 cells/ml (A), 1 106 cells/ml (B) and 2 106 cells/ml (C). Arrows showing the living cells ( ), the cells which were not viable anymore ( ) and the biomaterial itself ( ).
disadvantage of donor morbidity [27,28]. The use of biomaterials instead or in combination with bone autografts and even more tissue engineering has the
potential to overcome this disadvantage. So far bone cements, metals especially titanium and ceramics are in use but they are not biodegradable and do not enhance
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Fig. 7. Scanning electron microscopic study of human osteoblast-like cells seeded onto native calf collagen membrane with different seeding concentrations: 1 105 cells/ml (A), 1 106 cells/ml (B) and 2 106 cells/ml (C). Arrows showing the living cells ( ), the cells which were not viable anymore ( ) and the biomaterial itself ( ).
the bone ingrowth [29]. However, for tissue engineering of bone and eventually for proper bone reconstruction biomaterials which are bioresorbable, biocompatible,
not cytotoxic and which allow the bone ingrowth should be used. Collagen matrices provide these properties. To take from these collagen-based biomaterials a step further to tissue engineering, we addressed the problem of attachment and proliferation of human bone cells on such collagen based materials, which are registered for use on humans. Consequently, this study deals with the optimization of seeding and culturing of human osteoblast-like cells on three collagen membranes: bovine collagen, equine collagen and calf collagen. Two parameters were analyzed in this study: the first was the time interval between seeding of osteoblasts and adding of culture medium and the second was the seeding concentration. The aims were to clarify if the time interval between seeding and adding culture medium and how the seeding density of osteoblasts on the biomaterial affect cell adhesion and proliferation. Preliminary experiments showed that it is necessary to seed small volumes of cell solutions (about 100 ml) on the biomaterials. By applying bigger volumes not only the biomaterial is surrounded by the cell solution but the whole culture dish. Consequently, the cells drift on the ground of the culture plates and are not able to fix on the biomaterial. The results of our study showed that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration significantly affect the osteoblast proliferation, analyzed by the EZ4UFEASY FOR YOU test. Concerning the seeding concentration it was visible that using a seeding density of 105 cells/ml a good attachment of the cells to the biomaterial and the highest proliferation rate was observed, suggesting that at higher seeding concentrations only a part of the seeded osteoblast-like cells were able to attach to the biomaterial, the rest were not viable anymore. Similar results were reported by Ishaug [27] who studied the bone formation by culturing stromal osteoblasts in three dimensional, biodegradable poly(DL-lactic-co-glycolic acid) foams. Although the equine collagen membrane showed a similar structure as the bovine collagen membrane by scanning electron microscopy (Fig. 2A and 2B), the best proliferation rate was observed with the bovine collagen; the equine collagen and calf collagen followed. This suggests that bovine collagen meets all requirements for the cultivation of human osteoblasts and that the seeding density as well as the time interval between seeding and culturing of the cells are important parameters for tissue engineered constructs but the pore structures tested did not affect cell proliferation. Scanning electron microscopic studies to observe the cell morphology correlated with the proliferation data. A seeding density of 1 105 cells/ml showed the best result: the cells were well spread and flattened whereas a higher seeding density showed less spread and more rounded cells suggesting that a part of the cells were not viable
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anymore. Concerning the time interval between seeding osteoblasts and adding culture medium nearly the same results were observed: Bovine collagen showed always the highest proliferation rate compared with the other two biomaterials used in this study. The best proliferation rate was achieved by adding the culture medium 2 h after seeding the cells. Human osteoblast-like cells used for the seeding procedure in this study could be characterized as osteoblasts by the determination of osteocalcin, of the type I collagen level as well as by the enzymatic activity of alkaline phosphatase. Osteocalcin, an extracellular noncollagenous matrix protein, produced exclusively by osteoblasts, was found in all of our seeded cells. The positive alkaline phosphatase staining was reported in many studies as an indicator for osteoblasts, for the formation of new bone and as a marker for cell differentiation in cultures [30,31]. Type I collagen was expressed in approximately 74% of the cells. The levels of type I collagen may depend on the duration of the cultivation and the supplements because other publications report other levels [31]. In contrast to our study, Mailhot and Borke used the third passage of humanderived bone cells for type I collagen immunocytochemistry. In our study, this was performed with the first passage. In conclusion, the data in this study demonstrate that the point in time of adding the culture medium after initial cell seeding on the biomaterial and the seeding concentration effect the cell proliferation and cell colonization. Consequently, these are very important parameters for the development of tissue engineering and in a larger perspective also for the use in humans. Additionally, it could be shown that all three biomaterials used in this study resulted in different proliferation rates. Moreover, the best proliferation rate resulted with the bovine collagen membrane. Consequently, further studies with further biomaterials are necessary to compare the proliferation rate and cell colonization of osteoblasts with the aim to find the optimal material for skeletal reconstruction.
Acknowledgements Thanks to Dr. Dr. R. Schimming for his valuable proposals.
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How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials a . M. Wiedmann-Al-Ahmada,*, R. Gutwalda, G. Lauerb, U. Hubner , R. Schmelzeisena a
Klinik und Poliklinik fur . Mund-, Kiefer- und Gesichtschirurgie, Albert-Ludwigs-Universitat . Freiburg, Hugstetterstraße 55, D-79106 Freiburg, Germany b Klinik und Poliklinik fur Carl G. Carus, Technische Universitat . Mund-, Kiefer- und Gesichtschirurgie, Universitatsklinikum . . Dresden, Fetscher Straße 74, D-01307, Dresden, Germany Received 5 April 2001; accepted 8 January 2002
Abstract The optimization of seeding and culturing of human osteoblast-like cells on three collagen-based biomaterials (bovine, equine and calf collagen membrane) was studied by cell proliferation and cell colonization (scanning electron microscopy) analysis. Osteoblasts of five patients were seeded onto the three biomaterials and two different parameters were varied: the time intervals between initial seeding and adding culture medium (2 h, 6 h, 12 h, 24 h) and the seeding concentration (1 105, 1 106, 2 106 cells/ml) of cells onto biomaterials. The results of the study demonstrated that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration effects the cell proliferation and the cell colonization. The best proliferation rate was achieved by adding the culture medium 2 h after initial seeding and with a seeding density of 1 105 cells/ml. Moreover, all three biomaterials resulted in different proliferation rates. The best proliferation rate resulted with the bovine collagen membrane. In conclusion, the examined parameters are very important for the development of the tissue engineering techniques and in a larger perspective also for reconstructive surgery. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Human osteoblast-like cells; Biomaterials; Seeding; Cultivation; Cell proliferation; Cell colonization
1. Introduction The application of tissue engineered bone grafts in the reconstruction of facial and maxillary sceletal defects requires cell carrier matrices which have no cytotoxic effect and allow a three-dimensional growth of human osteoblasts. Usually, the osteoblast-like cells are grown adherent to the floor of the culture dish very well but for grafting, the growth on support materials is necessary. Over the past few years, especially in the development of the tissue engineering techniques, new biomaterials have been introduced for the use in reconstructive surgery. These commercially available cell carrier matrices differ in composition, pore size and pore density, as well as in permeability and durability [1]. However, most of them are bioabsorbable which is an advantage for tissue
*Corresponding author. Tel.: +1-49-761-270-4989; fax: +1-49-761270-4817. E-mail address: [email protected] (M. Wiedmann-Al-Ahmad).
engineering and reconstructive surgery as the materials need not to be removed and consequently, a second surgical procedure is not required. Studies about the cultivation and growth of epithelial cells and human gingival keratinocytes on different biomaterials were published regarding their attachment and spreading including the protein adsorption behaviour onto various implant materials such as titanium, gold, hydroxylapatite, carbonate apatite and polystyrene substrates [2–7]. Especially, the influence of the surface topography of such implant materials on biomolecule or cell adherence, orientation and morphogenesis has been widely reported and plays an important role [8–10]. Our recent study [11] describes the cultivation of gingival keratinocytes on different permeable polycarbonate membranes with various pore sizes and on nylon. The results indicated that primary keratinocyte cultures, established according to the explant culture technique [12–14], formed a confluent cell layer on both support materials. Whereas, using secondary cultures, established according to the disperse culture technique [15], a confluent cell layer was only observed on the polycarbonate membranes.
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 0 1 9 - 4
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In contrast, only few data exist about the attachment and growth of human osteoblast-like cells on such cell carriers. Less data is also available about the requirements a biomaterial must meet such that the cells attach very well and about the healing of such tissue engineered constructs in humans. Different aspects of culturing osteoblasts were monitored such as their differentiation during culturing and their cell invasion into porous materials [16–20]. Studies about osteoblast-material interactions have shown that they depend on various factors such as topography, chemistry and surface energy [16,21]. For example, the protein adsorption is influenced by the pH, the temperature and the ionic composition as well as by the surface roughness [22–24]. Till now, in case of human osteoblast-like cells and bioabsorbable carrier materials, the search for the ideal material as well as the optimization for culture and seeding conditions on various biomaterials continues. In this paper, we focus on the seeding and cultivation procedure in order to optimize the attachment and growth of human osteoblasts on various cell carriers. A second aspect is the comparison of three different biomaterials with respect to promoting attachment and proliferation of human osteoblasts. The three different biomaterials examined were a native bovine collagen membrane, a native equine collagen membrane and a native calf collagen membrane. The aims of this study were to optimize the culture conditions of osteoblasts and to find a useful cytocompatible biomaterial for the surgical management of intraoral applications. This would be of interest to develop tissue engineering straightforward for creating autologous grafts, which can be applied soon in reconstructive surgery.
2. Materials and methods 2.1. Biomaterials Three different biomaterials were used for the cultivation of human osteoblast-like cells and for potential subsequent grafting: a native bovine collagen membrane (Tissue Vliess, Baxter, Heidelberg, Germany), a native equine collagen membrane (Resorbas, Nurnberg, . Germany) and a native calf collagen membrane (Osteovits, B. Braun-Dexon GmbH, Spangenberg, Germany). 2.2. Isolation and cultivation of human osteoblast-like cells For cultivation of osteoblasts, pieces of corticolamellar bone of the maxilla from five different patients of each sex in the age range of 23–65 yr were crumbled into explants (size 2 mm 2 mm) and seeded on culture flasks (25 cm2, Greiner, Frickenhausen, Germany) using
Opti-minimal essential medium (Opti-MEM, Gibco Laboratories Life Technologies, Inc, Grand Island, NY, USA) pH 7.2 with 10% foetal calf serum (FCS) and kept in a humidified atmosphere of 5% CO2 at 371C (Heraeus, Hanau, Germany). The osteoblast-like cells which migrated onto the floor of the culture dish form a confluent layer after 4–5 weeks (primary culture) and the first passage was used for the growth experiments on the various biomaterials. 2.3. Preparation of the cell culture plates Before cell seeding onto the biomaterials the 24-well culture plates (Costar, NY, USA) were humidified with culture medium. The three different plasma-sterilized biomaterials (1 cm2) were placed into the wells: one for scanning electron microscopic study, one for alkaline phosphatase assay and one for the type I collagen determination. For the proliferation test smaller samples of the three materials were placed in 96-well plates (Corning, NY, USA): one well for the medium blank, one well with the biomaterial only, and three wells with the biomaterial and the seeded cells. 2.4. Seeding the cells onto biomaterials The confluent osteoblast cultures (primary culture) were detached from the culture flask by incubation with 0.5% trypsin (Gibco, Paisley, Scotland) in phosphatebuffered saline (PBS) for 8 min at 371C. The bone cell solution was filtered through a 100 mm cell-strainer (Falcon, Heidelberg, Germany) in a 50 ml tube (Falcon, Heidelberg, Germany), centrifuged (Biofuge Strato, Heraeus, Hanau, Germany, 1120 g, 12 min, 301C) and resuspended in 1 ml culture medium. The cells were transferred into a 75 cm2 culture flask (Greiner, Frickenhausen, Germany), filled up with 25 ml culture medium. After 14 days, the cells of the first passage were detached again from the culture flask with 0.5% trypsin, centrifuged and resuspended in 1 ml culture medium. After staining with trypanblue (1:1; v/v), the cells still alive were counted in a chamber by light microscopy (Zeiss Axiovert 135, Jena, Germany). Then, the cells were seeded onto the biomaterials of constant size. Two different parameters were tested in this study (the materials were seeded with osteoblasts of five patients; for each parameter and each test as triplicate): *
Time interval between initial cell seeding and adding sufficient volume of culture medium to cover biomaterial cubes completely 100 ml of 106 cells/ml was seeded onto the three different biomaterials with a sterile syringe. The cells were incubated for 2, 6, 12 and 24 h, respectively, at 371C in 5% CO2 atmosphere before adding the
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*
culture medium to a total volume of 1 ml. After three days of incubation in total, scanning electron microscopic studies and proliferation assays followed. Seeding concentration To examine the optimal seeding concentration, three different cell concentrations (1 105, 1 106 and 2 106 cells/ml) were seeded by a sterile syringe onto the biomaterial cubes. The total volume was 100 ml. The plates were incubated for 2 h at 371C in 5% CO2 atmosphere. Then, the culture medium was added and a further incubation for 3 days followed.
Additionally, plates with 1 105 cells/ml were incubated for 1 week at 371C in 5% CO2 atmosphere for the detection of alkaline phophatase and collagen. Once in a week the medium was changed. Parallel to seeding the cells (second passage) onto the biomaterials an aliquot of the same passage was seeded in cell culture plates. This serves as control for the cell proliferation as well as for the detection of the cells as osteoblasts. 2.5. Assay for osteocalcin For the quantification of osteocalcin in the cell culture supernatant of human maxillar osteoblast-like cells, the osteocalcin ELISA (DAKO, Glostrup, Denmark) was performed according to the manufacturer’s instructions. In brief, the standards, the curve control and the cell culture supernatants were premixed with biotinylated osteocalcin, incubated in microwells precoated with anti-osteocalcin for 1 h, washed and incubated with peroxidase-conjugated streptavidin for 15 min, which binds strongly to the biotinylated osteocalcin. After a further washing step the chromogenic substrate was added and incubated for 30 min. The reaction was stopped by 2 m H2SO4 and the absorbance at 450 nm was measured. Osteocalcin is exclusively synthesized by osteoblasts and is believed to prevent premature mineralization of newly formed, but yet disorganized bone matrix [25]. 2.6. Alkaline phosphatase assay and morphometry For the staining of maxillar osteoblast-like cells an alkaline phosphatase assay kit (Sigma, Deisenhofen, Germany) was used. The culture dishes were air dried, fixed in a citrate-aceton-formaldehyde solution for 30 s and rinsed gently with Aqua dest. Incubation with alkaline phosphatase staining solution for 15 min protected from direct light and a washing step with Aqua dest followed. The citrate-aceton-formaldehyde solution as well as the alkaline phosphatase staining solution was prepared according to the manufacturer’s instructions. The culture dishes were counterstained
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with neutral red for 5 min, rinsed with Aqua dest. and dried mounted with cover slips. Positive staining for alkaline phosphatase (red–violet) was identified by light microscopy and evaluated by morphometry using the computer program Analysis 3.1 (Soft Imaging System, Munster, . Germany). 2.7. Detection of type I collagen For the quantification of type I collagen the cells were washed in PBS for 5 min, fixed with 70% ethanol for 1 h, washed in PBS for 5 min, allowed to air dry and washed again in PBS for 5 min. After an incubation of 0.3% H2O2 in methanol for 30 min, unspecific immune reactions were blocked with 1% bovine serum albumin for 10 min, before the anti-collagen I antibody (Sigma, Deisenhofen, Germany) was administered for 1 h. An incubation of the biotin-conjugated secondary antibody (Vectastain Elite Kit, Vector Laboratories, Burlingame USA) for 45 min and an incubation of avidin mixed with biotin-conjugated peroxidase (Vectastain Elite Kit, Vector Laboratories, Burlingame USA) for 30 min followed. Sections were rinsed between each incubation step three times with PBS for 5 min. The immunoreaction was developed by diaminobenzidine-solution (0.05 mg/l DAB/0.05 m Tris-HCl pH 7.3/0.01% H2O2) at room temperature. The sections were counterstained with hematoxylin (Merck, Darmstadt, Germany) for 10 s and mounted in 40% glycerin (Merck, Darmstadt, Germany) in PBS. The evaluation was done by light microscopy and the computer program Analysis 3.1 (Soft Imaging System, Munster, . Germany). 2.8. Cell proliferation analysis For cell proliferation analysis, the nonradioactive assay EZ4UFEASY FOR YOU (Biozol diagnostica GmbH; Eching, Germany) was used. This method is based on the finding that living cells are capable of reducing slightly yellow coloured tetrazolium salts to intense red coloured formazan derivatives by an intracellular reduction system, mostly located in the mitochondria [26]. These formazan derivatives are excreted into the culture medium and the absorbance can be measured with a microplate reader. The amount of coloured formazan derivatives correlates with the amount of living cells in the sample. The proliferation assay was carried out according to the manufacturers’ instructions. 2.9. Cell colonization analysis The cell colonization analysis was assessed by scanning electron microscopy. For scanning electron microscopy the samples were fixed in 4% paraformaldehyde for 2 h at room temperature and incubated in
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8% formaldehyde for 2 days at 41C. The samples were dehydrated in graded alcohol (30%, 50%, 70%, 80%, 90%, each one time and two times in 99.8%). After critical point drying (CPD 030 Baltec, Wallruf, Germany), according to standard procedure using liquid carbon dioxide, the samples were sputtered with gold palladium (Plano, Germany) in the SCD 040 (Balzers Union, Wallruf, Germany). The probes were examined via Zeiss Leo 32 scanning electron microscope (Zeiss, Kochern, Germany) at 10 kV. Images were digitized.
the seeding concentration as well as the time of adding the culture medium after seeding the osteoblast-like cells onto the biomaterials. The values of the standard deviation are between 0.03 and 0.1 (see Fig. 1 error bars). Osteoblast-like cells seeded on bovine collagen showed in each test significantly more vital cells than the other two biomaterials, equine collagen and calf collagen independent of which patient was used. The highest proliferation rate could be measured with a seeding concentration of 1 105 cells/ml and adding the culture medium 2 h after seeding the cells onto the biomaterials.
3. Results 3.2. Scanning electron microscopy 3.1. Cell proliferation and vitality The vitality and the proliferation capacity of the osteoblast-like cells from five different patients seeded each onto three different biomaterials was studied by the EZ4UFEASY FOR YOU test. The average of the measured absorbance is shown in Fig. 1 depending on
The surface of the three collagen biomaterials with respect to the osteoblast morphology, attachment and tissue invasion was studied by the scanning electron microscope. Fig. 2 shows the biomaterials without seeding cells as control. Whereas, the bovine and the equine collagen membrane show a similar structure with
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Fig. 1. Cell proliferation analysis of human osteoblast-like cells seeded onto native bovine, native equine and native calf collagen membrane. The time intervals between initial cell seeding and adding culture medium were varied: 2 h (A), 6 h (B), 12 h (C) and 24 h (D). In each test different seeding concentrations (1 105, 1 106, 2 106 cells/ml) were used. Error bars represent standard deviations.
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the biomaterials showed that there is the best cell density of intact osteoblasts when adding the culture medium after 2 h. These results were independent of the patient material used and independent of the biomaterial used (Figs. 3 and 4; calf collagen membrane not shown). In Figs. 5–7 different concentrations of osteoblast-like cells for seeding were used. It could be observed that the osteoblast-like cells of all five patients grow to form confluent sheets in the presence of bovine collagen, equine collagen and calf collagen. The surface of the biomaterials was completely covered with the osteoblast-like cells. The best result in covering the biomaterial surface was achieved with a seeding concentration of 1 105 cells/ml (Figs. 5A–7A). No differences were visible between the patient material (data not shown) and between the different collagen membranes. A higher seeding concentration showed much more not intact osteoblast-like cells, round cells were visible as well as incomplete cell sheets. The proliferation and vitality test (EZ4UFEASY FOR YOU) confirmed these morphological findings that using a seeding concentration of 1 105 cells/ml is optimal, and that the part of living cells, able to proliferate, is extremely reduced when applying higher seeding concentrations than 1 105 cells/ml (Fig. 1 and see section results, cell proliferation). 3.3. Osteocalcin determination, alkaline phosphatase assay and determination of type I collagen The characterization of the cells seeded on the three different biomaterials as osteoblasts was determined by the amount of osteocalcin, the relative amount of alkaline phosphatase activity and the relative amount of the presence of cells expressing type I collagen. Gingival keratinocytes were used as control. The amount of osteocalcin was 5.48 mg/l at a concentration of 1 105 cells/ml (standard deviation 0.94), 10.18 mg/l at a concentration of 1 106 cells/ml (standard deviation 0.88) and 12.66 mg/l at a concentration of 2 106 cells/ml (standard deviation 0.96). The alkaline staining of these cells typically resulted intensively positive (about 59%) whereas the human gingival keratinocytes did not. Immunocytochemistry of the fixed cells showed the presence of type I collagen in about 74% of the cells. Fig. 2. Scanning electron microscopic study of three native collagen membranes of different origins without osteoblasts. Bovine (A), equine (B) and calf (C) collagen membrane.
an extensive pore mesh (Fig. 2A and 2B), the calf collagen membrane shows a smooth surface with small pores (Fig. 2C). Comparing the different time intervals (2, 6, 12 and 24 h) of adding culture medium after seeding the cells on
4. Discussion The attachment and proliferation of human osteoblast-like cells on biomaterials is required for tissue engineering of bone autografts. Bone autografts are regularly necessary for skeletal reconstructions in cases involving large defects created by tumor resection, trauma and skeletal abnormalities and they have the
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Fig. 3. Scanning electron microscopic study of human osteoblast-like cells cultivated onto native bovine collagen membrane. Different time intervals between initial cell seeding and adding culture medium are shown: 2 h (A), 6 h (B), 12 h (C) and 24 h (D). Arrows showing the living cells ( ) and the cells which were not viable anymore ( ).
Fig. 4. Scanning electron microscopic study of human osteoblast-like cells cultivated onto native equine collagen membrane. Different time intervals between initial cell seeding and adding culture medium are shown: 2 h (A), 6 h (B), 12 h (C) and 24 h (D). Arrows showing the living cells ( ), the cells which were not viable anymore ( ) and the biomaterial itself ( ).
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Fig. 5. Scanning electron microscopic study of human osteoblast-like cells seeded onto native bovine collagen membrane with different seeding concentrations: 1 105 cells/ml (A), 1 106 cells/ml (B) and ), the cells 2 106 cells/ml (C). Arrows showing the living cells ( which were not viable anymore ( ) and the biomaterial itself ( ).
Fig. 6. Scanning electron microscopic study of human osteoblast-like cells seeded onto native equine collagen membrane with different seeding concentrations: 1 105 cells/ml (A), 1 106 cells/ml (B) and 2 106 cells/ml (C). Arrows showing the living cells ( ), the cells which were not viable anymore ( ) and the biomaterial itself ( ).
disadvantage of donor morbidity [27,28]. The use of biomaterials instead or in combination with bone autografts and even more tissue engineering has the
potential to overcome this disadvantage. So far bone cements, metals especially titanium and ceramics are in use but they are not biodegradable and do not enhance
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Fig. 7. Scanning electron microscopic study of human osteoblast-like cells seeded onto native calf collagen membrane with different seeding concentrations: 1 105 cells/ml (A), 1 106 cells/ml (B) and 2 106 cells/ml (C). Arrows showing the living cells ( ), the cells which were not viable anymore ( ) and the biomaterial itself ( ).
the bone ingrowth [29]. However, for tissue engineering of bone and eventually for proper bone reconstruction biomaterials which are bioresorbable, biocompatible,
not cytotoxic and which allow the bone ingrowth should be used. Collagen matrices provide these properties. To take from these collagen-based biomaterials a step further to tissue engineering, we addressed the problem of attachment and proliferation of human bone cells on such collagen based materials, which are registered for use on humans. Consequently, this study deals with the optimization of seeding and culturing of human osteoblast-like cells on three collagen membranes: bovine collagen, equine collagen and calf collagen. Two parameters were analyzed in this study: the first was the time interval between seeding of osteoblasts and adding of culture medium and the second was the seeding concentration. The aims were to clarify if the time interval between seeding and adding culture medium and how the seeding density of osteoblasts on the biomaterial affect cell adhesion and proliferation. Preliminary experiments showed that it is necessary to seed small volumes of cell solutions (about 100 ml) on the biomaterials. By applying bigger volumes not only the biomaterial is surrounded by the cell solution but the whole culture dish. Consequently, the cells drift on the ground of the culture plates and are not able to fix on the biomaterial. The results of our study showed that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration significantly affect the osteoblast proliferation, analyzed by the EZ4UFEASY FOR YOU test. Concerning the seeding concentration it was visible that using a seeding density of 105 cells/ml a good attachment of the cells to the biomaterial and the highest proliferation rate was observed, suggesting that at higher seeding concentrations only a part of the seeded osteoblast-like cells were able to attach to the biomaterial, the rest were not viable anymore. Similar results were reported by Ishaug [27] who studied the bone formation by culturing stromal osteoblasts in three dimensional, biodegradable poly(DL-lactic-co-glycolic acid) foams. Although the equine collagen membrane showed a similar structure as the bovine collagen membrane by scanning electron microscopy (Fig. 2A and 2B), the best proliferation rate was observed with the bovine collagen; the equine collagen and calf collagen followed. This suggests that bovine collagen meets all requirements for the cultivation of human osteoblasts and that the seeding density as well as the time interval between seeding and culturing of the cells are important parameters for tissue engineered constructs but the pore structures tested did not affect cell proliferation. Scanning electron microscopic studies to observe the cell morphology correlated with the proliferation data. A seeding density of 1 105 cells/ml showed the best result: the cells were well spread and flattened whereas a higher seeding density showed less spread and more rounded cells suggesting that a part of the cells were not viable
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anymore. Concerning the time interval between seeding osteoblasts and adding culture medium nearly the same results were observed: Bovine collagen showed always the highest proliferation rate compared with the other two biomaterials used in this study. The best proliferation rate was achieved by adding the culture medium 2 h after seeding the cells. Human osteoblast-like cells used for the seeding procedure in this study could be characterized as osteoblasts by the determination of osteocalcin, of the type I collagen level as well as by the enzymatic activity of alkaline phosphatase. Osteocalcin, an extracellular noncollagenous matrix protein, produced exclusively by osteoblasts, was found in all of our seeded cells. The positive alkaline phosphatase staining was reported in many studies as an indicator for osteoblasts, for the formation of new bone and as a marker for cell differentiation in cultures [30,31]. Type I collagen was expressed in approximately 74% of the cells. The levels of type I collagen may depend on the duration of the cultivation and the supplements because other publications report other levels [31]. In contrast to our study, Mailhot and Borke used the third passage of humanderived bone cells for type I collagen immunocytochemistry. In our study, this was performed with the first passage. In conclusion, the data in this study demonstrate that the point in time of adding the culture medium after initial cell seeding on the biomaterial and the seeding concentration effect the cell proliferation and cell colonization. Consequently, these are very important parameters for the development of tissue engineering and in a larger perspective also for the use in humans. Additionally, it could be shown that all three biomaterials used in this study resulted in different proliferation rates. Moreover, the best proliferation rate resulted with the bovine collagen membrane. Consequently, further studies with further biomaterials are necessary to compare the proliferation rate and cell colonization of osteoblasts with the aim to find the optimal material for skeletal reconstruction.
Acknowledgements Thanks to Dr. Dr. R. Schimming for his valuable proposals.
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