Towards in vivo application of oxygen-releasing microspheres for enhancing bone
regeneration
Buizer, Arina
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Chapter 2
Static versus vacuum cell seeding
on high and low porosity ceramic
scaffolds
A.T. Buizer, A.G. Veldhuizen, S.K. Bulstra, R. Kuijer
Abstract
An adequate cell seeding technique is essential for effective bone regeneration on cell-seeded constructs of porous tricalcium phosphates. In previous studies, dynamic cell seeding, in which an external force is applied to seed cells on a biomaterial, resulted in more homogeneous cell seeding in low porosity scaffolds than static seeding. The optimal cell seeding technique for high porosity scaffolds has not been defined yet. Human mesenchymal stem cells were isolated from bone marrow and characterized. The cells were seeded on low porosity (45%) and high porosity (90%) tricalcium phosphate scaffolds using a static and a vacuum seeding technique. LIVE/DEAD® staining of the cell-scaffold complexes followed by confocal laser scanning microscopy was used to measure cell proliferation, cell distribution and cell viability at one, three and seven days after seeding. Cell proliferation was also quantified using a DNA quantification assay. Neither static nor vacuum seeding resulted in homogeneous cell seeding on both low and high porosity scaffolds. Cell density was lower on the inside than on the outside of the scaffolds. On low porosity scaffolds, the vacuum method yielded the highest numbers of cells compared to the static method. Low porosity scaffolds were seeded most homogeneously using the static seeding method. Seven days after seeding, the numbers of adherent cells were comparable for both scaffold types and independent of the cell seeding technique used. In conclusion, on high porosity scaffolds, static seeding results in more homogeneous cell seeding and it is easier to use than a vacuum seeding technique.
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Introduction
Regenerative medicine is a rapidly developing research field, which aims at the restoration or preservation of body functions by regenerating tissues or organs. In orthopedic surgery regenerative medicine can be of great use in bone replacement. At this moment, autologous bone grafts are the standard treatment for bone defects. Autologous grafts have osteoconductive, osteogenic and osteoinductive capacities, and excellent biomechanical properties1,2. Disadvantages of the use of autologous
bone grafts are the occurrence of donor site morbidity, the limited availability, the creation of a second operation site and the increased cost of harvest3,4.
Constructs of porous tricalcium phosphate (TCP) seeded with human mesenchymal ‘stem’ cells (hMSCs) seem a promising replacement for autologous bone grafts. TCP resembles the bone mineral composition. The material is osteoconductive, it is biodegradable and it is available in virtually unlimited amounts5,6. A disadvantage is
the lack of osteoinductive capacity in most types of material7,8. The biomechanical
properties of TCP are influenced by its porosity. Highly porous materials have lower mechanical stability than low porosity scaffold materials9. However, high porosity
scaffold materials show higher ingrowth of bone cells and tissue than low porosity materials10,11. Furthermore, a high pore interconnectivity allows for rapid ingrowth of
bone cells.
hMSCs show great potential as a cell component on different scaffold materials for bone regeneration. First, hMSCs present in bone marrow and periosteum take part in the bone growth and regeneration process by nature. Second, hMSCs are capable of differentiation into several cell types, amongst which osteogenic, chondrogenic and adipogenic lineages. Third, the cells are readily available in several types of tissues and can, for example, easily be isolated from bone marrow aspirates. Fourth, due to their trophic effect hMSCs support attraction, growth and development of host cells12–15.
Ideally, cells should be distributed homogeneously throughout the scaffold after seeding16–19. An optimal seeding technique should have a high cell seeding efficiency,
which means that a high percentage of cells in the seeding suspension should adhere to the scaffold16,18,19. Furthermore, cell damage due to the seeding procedure should
be minimal. In addition, a good cell seeding method is reproducible and easy to use. In case of point of care seeding, the technique should also be rapid16,19.
Cell seeding techniques can be divided into two groups: static and dynamic seeding. In static seeding, a cell seeding suspension is mixed with a scaffold without application of an external force. Examples of static seeding techniques are pipetting a cell suspension
on top of a scaffold or soaking scaffold granules in a suspension. Static seeding techniques are the most frequently used as they are easy to use and do not expose cells to potentially damaging forces16,18. However, these techniques lead to a lower cell
seeding efficiency and poorer homogeneous distribution of the cells throughout the scaffold than dynamic seeding techniques. In dynamic seeding cells are applied on a scaffold using an external force, such as in application of vacuum16, application of
low pressure19, centrifugation15 or oscillating perfusion18. These methods may yield
higher numbers of adherent cells throughout TCP scaffolds and cell seeding can be more efficient and homogeneous. The prolonged seeding time and the complexity of the seeding techniques are the most important disadvantages of dynamic cell seeding techniques. Besides these, each type of dynamic cell seeding technique has its specific drawbacks16.
In clinical settings, TCP materials with a broad range of porosities are used20–22. It is not
clear yet whether the propagated dynamic cell seeding techniques are suitable for scaffolds of all porosities. Therefore, we compared a static and a vacuum hMSC seeding technique on both highly porous TCP and on lower porosity TCP scaffolds, focusing on cell proliferation, cell distribution throughout the scaffolds and cell seeding efficiency.
Materials and methods
Materials
Cell culture medium, fetal bovine serum (FBS), a solution of 0,5 µg amphotericin/ml, 200 IU penicillin/ml and 200 IU streptomycin/ml (Antibiotic-Antimycotic), Insulin Transferrin Selenium (ITS+), RNAse, the CyQuant Cell Proliferation Assay Kit, the LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells and trypsin/ethylenediaminetetraacetic acid (EDTA) were all purchased from Invitrogen, Paisley, United Kingdom. All other chemicals were purchased from Sigma, Steinheim, Germany, unless stated otherwise.
Isolation and culture of MSCs
Bone marrow was acquired from three females, aged 58, 63, and 87 years, undergoing total hip surgery. The reaming debris from the femur was collected using a Jamshidi bone marrow aspiration needle (Care Fusion, McGaw Park, IL, USA). The study protocol was approved by the ethics committee of the University Medical Center Groningen. For each bone marrow sample four portions of 3 ml were taken. Each portion was diluted with 5 ml of phosphate-buffered saline (PBS) and subsequently processed by centrifugation over a density gradient (Histopaque 1077). Further processing took
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place according to the manufacturer’s instructions. After isolation, mononuclear cells were seeded in T75 tissue culture flasks (Greiner Bio-one, Alphen aan den Rijn, the Netherlands) and cultured in minimal essential medium-alpha (α-MEM) supplemented with 10% heat-inactivated FBS, 0,1 mM Ascorbic Acid-2-Phosphate (AA2P, Fluka, Steinheim, Germany) and 2% Antibiotic-Antimycotic. The cells were grown in a humidified atmosphere with 5% CO2 at 37°C. Medium was changed twice a week. At 70% confluence, the cells were harvested and frozen in liquid nitrogen in full culture medium containing 7,5% DMSO (passage 1). Cells were thawed and cultured under 2D-conditions until passage two before they were used in experiments.
Characterization of MSCs
hMSC antigen expression profile was assessed using a BD Stemflow Human MSC Analysis Kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions on a FACS machine (FACSCalibur, Beckton Dickinson, Franklin Lakes, NJ, USA). FACS data were analyzed using the Kaluza software package (Beckman Coulter, Brea, CA, USA).
Differentiation potential of the isolated cells was assessed by differentiation into osteogenic, adipogenic and chondrogenic cells. For osteogenic differentiation, 7500 cells per well were plated in a 24-well tissue culture plate (Greiner Bio-one). The cells were incubated with osteogenic medium (α-MEM, 2% Antibiotic-Antimycotic, 10% heat inactivated FBS, 100 nM dexamethasone, 0,05 mM β-glycerophosphate and 0,05 mM AA2P). After two weeks of culture, an alkaline phosphatase assay (Leukocyte alkaline phosphatase kit) was performed according to the manufacturer’s instructions. For adipogenic differentiation, 10.000 cells per well were plated in a 24-well tissue culture plate. The cells were incubated with adipogenic medium (α-MEM, 2% Antibiotic-Antimycotic, 10% heat inactivated FBS, 1 µM dexamethasone, 0,5 mM IBMX, 60 µM indomethacin and 10 µM human insulin) for three weeks. After this culture period, the cells were fixed in 3,7% paraformaldehyde (Boom, Meppel, the Netherlands). An 0,3 weight percentage Oil Red O stock solution in 99% 2-propanol was diluted 1,67 times in water and used for staining the cells. For chondrogenic differentiation, cell pellets of 250.000 cells were incubated with chondrogenic medium (α-MEM, 2% Antibiotic-Antimycotic, 0,1 mM AA2P, 0,1 µM dexamethasone, 1% ITS+, 40 µg/ml L-proline and 10 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN, USA)) for two weeks. After two weeks, the cell pellets were fixated in 3,7% paraformaldehyde and embedded in paraffin. Sections of 5 µm were deparaffinized, stained with an 1% Alcian blue (Boom) in 0,1 M HCl solution and counterstained with a 0,2 weight percentage Nuclear Fast Red (Merck, Darmstadt, Germany) staining solution.
Scaffolds
Two β-tricalcium phosphate scaffold materials were used. A 45% porosity scaffold (Bio-1TM sticks, Science & Bio Materials, Lourdes, France), with pore sizes ranging from 250
to 400 µm, was adjusted to approximately 4 x 4 x 4 mm blocks23,24. Of a 90% porosity
scaffold (VitossTM, Orthovita, Malvern, PA, USA), with 75% pores of 100-1000 µm and
25% pores of 1-100 µm, granules of approximately 4 x 4 x 4 mm in size were used. The pore interconnectivity of the latter material was 88-92%7.
Cell seeding methods
For cell seeding, cell suspensions of 0,5 x 106, 1,0 x 106 and 1,5 x 106 cells/ml were
used. The 0,5 x 106 cells/ml suspension was seeded on both scaffold types and using
the vacuum seeding method as well as the static seeding method. The 1,0 x 106 and
1,5 x 106 cells/ml suspensions were only seeded on high porosity scaffolds using the
static seeding method.
For the pipetting seeding method, scaffolds were placed in 48-well tissue culture plates (Greiner Bio-one), one scaffold per well. 50 µl of cell suspension were pipetted on top of each scaffold.
The vacuum seeding method was described before16. Briefly, a sterile luer lock syringe
(Omnifix luer lock, Braun, Melsungen, Germany) was filled with 150 µl of cell suspension and three scaffolds. The volume of these scaffolds and cell suspension was called one volume. Additionally, one milliliter of air was drawn into the syringe. The syringe was capped with a luer lock cap (Combi-Stopper, Braun). Vacuum was created by pulling back the plunger for the same amount as one volume. A five-second vacuum was created trice interrupted by 10 seconds pauses. One scaffold per well was placed in separate tissue culture wells of a 48-well plate.
After cell seeding, the culture plates with scaffolds were incubated in a humidified atmosphere with 5% CO2 at 37°C for one hour. After one hour the scaffolds were placed in different, sterile tissue culture wells, one scaffold per well. Fresh medium was added to the cell-seeded scaffolds.
Assessment of cell proliferation using DNA quantification
At one, three and seven days after seeding cell growth on scaffolds was determined. At each time point three cell-scaffold constructs per patient were washed in PBS at 37°C once and subsequently frozen at -80°C until final assays took place. For releasing the cells from the scaffold a modified technique described earlier by Piccinini et al25
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was used. Briefly, the cell-scaffold complexes were thawed and incubated in 100 µl of a RNAse solution containing 180 mM NaCl, 1 mM EDTA, 1 mg/ml RNAse and 1 x component B of CyQuant Cell Proliferation Assay Kit, dissolved in nuclease free water for 1 hour at room temperature. Then, 100 µl of a freshly prepared proteinase K solution (1 mM EDTA, 1 mM iodoacteamide (Merck), 50 mM TRIS, 800 mM HK2PO4 (Merck), 1 mg/ml proteinase K and 10 μg/ml pepstatin A in nuclease free water) was added and incubation was continued for 16 hours at 56°C. Finally, 200 µl of CyQuant dye solution (Component A 2x, component B 1x) was added to the lysate and mixed. 200 µl of the cell lysate-CyQuant mixture per scaffold was pipetted into a 96-well plate. Plates were read in a plate reader (Fluostar Optima, BMG labtech, Olfenburg, Germany) at excitation/emission wavelengths of 480/520 nm. A DNA quantification standard curve was created using standard quantities of lambda DNA. The mathematic formula based on this standard curve was used to calculate DNA quantities, which were converted in numbers of cells assuming that each human cell contains 7,5 pg of DNA.
Assessment of cell distribution in scaffolds
At one, three and seven days after seeding cell distribution throughout the scaffolds was determined. In preparation, a 2 µM calcein AM and 4 µM EthD-1 solution was made according to the manufacturer’s instructions of the LIVE/DEAD® Viability/ Cytotoxicity Kit for mammalian cells. At each time point three cell-scaffold complexes per patient were washed in PBS at 37°C once. Subsequently, 0,5 ml of LIVE/DEAD® staining solution was added to the scaffolds, followed by 30-45 minutes incubation in the dark at room temperature. The fluorescence at the outside of the scaffolds was investigated using a confocal laser scanning microscope (CLSM; TCS SP2, Leica microsystems, Heidelberg, Germany). Of one out of three scaffolds per patient an area of 375 x 375 µm wide and 100 µm thick was scanned, the others were only inspected. After examining the outside, the scaffolds were broken in half using a scalpel, and the insides of the scaffolds were scanned or inspected as well. The number of live and dead cells was counted on each scan.
Cell seeding efficiency
After cell seeding on the scaffolds, the cells were allowed to adhere to the scaffolds for one hour before the scaffolds were placed in fresh, sterile wells. For static seeded cells, the cells that remained in the original wells after 1 h incubation time were released by incubation with trypsin/EDTA for 3 min. Subsequently, the cells were suspended in standard medium and counted with an automatic cell counter (Scepter Cell Counter, Millipore, Billerica, MA, USA). For the vacuum seeded cells, after the seeding procedure the seeding syringe was flushed with 0,5 ml of trypsin/EDTA, and the remaining cells
were thus suspended. The cell suspension was placed in a sterile tissue culture well and trypsin was blocked by the addition of 0,5 ml standard medium. After one hour of incubation, medium was removed, the adjacent cells were washed with PBS once and released from the tissue culture plastic using trypsin/EDTA. Subsequently, the cells were counted using an automatic cell counter. Cell seeding efficiency was calculated using the following formula:
cell seeding efficiency =
(
number of seeded cells - number of leftover cells)
*100% number of seeded cellsStatistical analysis
Statistical analyses were done using SPSS 20. For normally distributed data, a t-test was used. For not normally distributed data a Mann-Whitney-U-test was used. A p value of 0,05 was considered to be significant.
Results
Characterization of human mesenchymal stem cells
The isolated cells were characterized according to the criteria as stated by the International Society for Cellular Therapy (ISCT)26. Firstly, we selected hMSCs from
the white cell fraction of bone marrow cells by their ability to adhere to tissue culture plastic. Secondly, the antigen expression profile of the cells was in concordance with the ISCT guidelines (Figure 1a-1d). Lastly, differentiation into osteogenic, adipogenic and chondrogenic lineages was accomplished (Figure 1e-1j).
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Figure 1 Characterization of hMSCs. Pictures 1a-1d are graphs representing flow cytometric analyses of
antigen expression profiles in hMSCs, and 1e-1j represent differentiation of hMSCs (a) Antigen expression profile positive for expression of CD73. (b) Antigen expression profile positive for expression of CD90. (c) Antigen expression profile positive for expression of CD105. (d) Antigen expression profile negative for expression of a cocktail of CD34, CD45, CD11b, CD19 and HLA-DR. All MSCs used in this study had similar profiles. (e) Alkaline Phosphatase staining results in marked blue staining of alkaline phosphatase produc-ing cells in osteogenic differentiation of hMSC. (f) In the blank measurements only slight stainproduc-ing is visible. (g) After Oil Red O staining red stained lipid vacuoles are visible in differentiated hMSC. (h) The blank hMSCs do not show any Oil Red O staining. (i) The blue staining of proteoglycan deposits by Alcian Blue staining and the pink staining of cell nuclei is clearly visible in chondrogenic differentiated hMSCs, as well as in staining of human chondrocytes as a positive control (j). (Scale bars in pictures 1e-1j indicate 50 µm.)
Cell distribution in scaffolds
When seeding a cell suspension of 0,5 x 106 cells/ml, both static and vacuum seeding
techniques resulted in significantly lower cell adherence to the inside than to the outside of the material on both types of scaffold material (Figure 2 and Table 1). However, relatively more cells were found on the inside of high porosity scaffolds than on the inside of low porosity scaffolds (difference not significant). Hence, there was a trend towards more homogenous cell distribution on high porosity scaffolds than on low porosity scaffolds, especially when the static seeding technique was used.
On high porosity scaffolds, the absolute number of live cells adherent to the scaffolds was highest if the static seeding technique was used. On low porosity scaffolds, the absolute number of live cells adherent to the scaffolds was higher when the vacuum seeding technique was used. These differences were not significant.
At seeding densities of 1,0 x 106 and 1,5 x 106 cells/ml, only the static seeding method
was tested and only on high porosity scaffold material. A higher number of adherent live and dead cells was found on the outside of the scaffold than on the inside of the scaffold in all three tested seeding densities. Cell distribution was thus not homogeneous throughout the scaffold material. The absolute numbers of live and dead cells increased as the seeding density increased, both on the outside and on the inside of the scaffold (figure 3). No plateau level was found. Only at the day three time point the number of live cells on the high porosity scaffold was the highest at a seeding density of 1,0 x 106 cells/ml. At the other time points, a seeding density of 1,5
x 106 cells/ml resulted in the highest cell attachment, while a seeding density of 0,5
x 106 cells/ml always resulted in the lowest number of live cells on the scaffold. The
differences between seeding densities per time point were not significant, except for day seven. Then, the 0,5 x 106 seeding density resulted in significantly lower numbers
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Figure 2 Example of CLSM scan of scaffold materials after LIVE/DEAD® staining. Live cells are shown asgreen stained cells, dead cells are shown as red stained cells. Scale bars indicate 50 µm.
Scaffold type Low porosity High porosity
Cell seeding technique Static Vacuum Static Vacuum
Outside/Inside
O
utside
Inside Outside Inside Outside Inside Outside Inside
Day 1 Average 12,7 0,7 4,5 3,0 11,0 9,7 10,0 3,0 SD 3,1 0,5 1,5 2,2 6,5 2,2 10,6 2,2 Day 3 Average 8,0 0,0 27,0 1,7 9,7 9,7 7,3 4,0 SD 3,3 0,0 22,0 1,2 5,2 3,7 9,0 3,6 Day 7 Average 5,0 2,3 10,7 2,0 3,0 5,0 4,3 5,0 SD 1,6 2,6 0,5 1,6 2,4 3,6 0,9 3,6
Table 1 Numbers of cells per scanned area of a scaffold, per scaffold type, seeding technique and on the
Figure 3 Average number of live cells of all patients on the outside and inside, of low porosity scaffolds at
three cell seeding densities, as assayed using LIVE/DEAD® staining. Error bars represent standard error of the mean.
Cell proliferation
Cell proliferation was assessed using both LIVE/DEAD® staining and DNA quantification. As was found using LIVE/DEAD® staining, the static seeding technique resulted in the highest numbers of adherent cells on both scaffold types one day after seeding (figure 4). On the high porosity scaffolds, the situation remained the same on day three, but on day seven static seeding performed worst. On low porosity scaffolds, the highest number of adherent cells to the scaffolds was accomplished using vacuum cell seeding on days three and seven. None of the differences in numbers of live cells between seeding techniques and scaffold types were significant.
The results of the CyQuant cell proliferation assay are depicted in figure 5. On high porosity scaffolds, vacuum seeding resulted in more adherent cells on the scaffolds than did static seeding on days one and three. After seven days, absolute cell numbers on high porosity scaffolds were similar using both cell seeding techniques. On low porosity scaffolds, initially vacuum cell seeding resulted in the highest numbers of cells adherent to the scaffold. At days three and seven, static seeding resulted in higher numbers of cells adherent to the scaffold than the vacuum technique did. None of the differences in absolute cell numbers between seeding techniques and scaffold types were significant.
Using both techniques and on both scaffold types, the absolute cell numbers decreased over time after seeding.
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Figure 4 Histogram representing average numbers of live cells per scanned area of a scaffold on the inside
and outside of scaffolds seeded with a cell suspension of 0,5 x 106 cells/ml, as measured using LIVE/DEAD®
staining. Averages are shown per seeding technique, per scaffold material and per time point. Error bars represent standard error of the mean.
Figure 5 Histogram representing numbers of cells per scaffold (averages of all patients) as measured using
the CyQuant cell proliferation assay. A calibration curve of 0-1000 ng of lambda DNA was used to calculate the numbers. Error bars represent standard error of the mean.
Cell viability
The percentage of dead cells on both scaffold types seemed higher after vacuum cell seeding than after static cell seeding (Figure 6). However, the difference in dead cell percentages between vacuum and static seeding was not significant.
Figure 6 Average percentage of live and dead cells per scaffold material and per seeding technique of all
patients as measured using LIVE/DEAD® staining. Averages were calculated for a seeding density of 0,5 x 106 cells/ml. Error bars represent standard error of the mean.
Cell seeding efficiency
Cell seeding efficiency was not dependent on the scaffold material (table 2). With the vacuum cell seeding technique a cell seeding efficiency of approximately 88-89% was found, while with the static seeding technique the efficiency was around 91-92% (not significant).
Cell seeding method and scaffold material Cell seeding efficiency (%)
Static-low porosity 91,4 ± 5,4
Static-high porosity 92,1 ± 3,4
Vacuum – low porosity 88,0 ± 4,5
Vacuum – high porosity 89,6 ± 4,6
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Discussion and conclusion
The aim of this study was to investigate which cell seeding method is most suitable for seeding hMSCs on low porosity and high porosity TCP scaffolds. Criteria for the optimal cell seeding method were homogeneous cell seeding, low cell damage rates, high cell seeding efficiency and ease of use of the cell seeding method.
The cells used in this study met the ISCT criteria for human MSCs. Both in low and high porosity scaffolds, less cells adhered to the insides than to the outsides of the scaffolds, in both tested seeding techniques. In porous scaffolds, air may be trapped inside the pores of the scaffold. This trapped air may prevent a cell suspension from penetrating into the center of the scaffold, so that cells are not seeded homogeneously throughout the scaffold27,28. In addition, trapping of cell plugs in the pores at the outside of the
scaffold may prevent the penetration of cells into the inside of the scaffold27,29.
When using a vacuum cell seeding method in low porosity β-TCP scaffolds, cells penetrated into the center of the scaffold better and cell viability was not adversely influenced by vacuum treatment. These results were in concordance with earlier findings in our lab16. Other authors investigating cell seeding techniques for a higher
porosity β-TCP than we tested, namely 70-75%, found dynamic seeding techniques to be superior to static cell seeding techniques in terms of cell seeding homogeneity18,19,30.
We observed that when scaffolds came in contact with a cell suspension, the cell suspension seemed to be aspirated by high porosity scaffolds. This aspiration effect was not observed in low porosity scaffolds. Perhaps the air trapped inside the pores of the scaffolds is expelled easier from high porosity scaffolds than from low porosity scaffolds. Therefore, there is more room for a cell suspension to settle in the core of a scaffold. The higher air expulsion in high porosity scaffolds could be an explanation why static cell seeding resulted in more homogeneous cell seeding than in low porosity scaffolds. To our knowledge, there are presently no studies available that investigated cell seeding techniques for high porosity β-TCP scaffolds. Solchaga et al27
compared static and vacuum seeding of hMSCs on > 80% porous sponges made out of a hyaluronic acid derivative. In their study, the most homogeneous cell seeding was accomplished with a vacuum cell seeding technique. However, the tested sponges were made out of a hydrophobic material, which rejects water-based solutions, such as cell culture medium. An aspiration effect, as the one observed in our experiments, seems less likely in these materials. Therefore, an external force may be needed to force cells into the center of a hyaluronic acid derivative scaffold.
In our study, three cell seeding densities were tested using a static cell seeding technique on a high porosity TCP scaffold. The cell seeding density that resulted in the highest number of adherent cells was 1,5 x 106 cells/ml. At the time points after
day one, the dispersion of the cell counts is large. It is expected that cell death occurs shortly after seeding. After a day, the cells that survived the seeding procedure start proliferating colony wise. Therefore, areas with low cell adherence can be found next to areas with cell colonies. This inhomogeneous cell growth may have led to a larger variation in cell counts after day one. Tan et al16 seeded MC3T3-E1 cells on
β-TCP at different seeding densities and measured equal cell retention rates at one hour after seeding, so the higher the initial seeding density, the higher the number of cells attached to the scaffold. They too did not find a plateau level. Holy et al31 seeded
rat bone marrow cells on poly (lactic-co-glycolic) acid (PLGA) scaffolds using a static seeding technique at different seeding densities. They concluded that bone formation on the scaffold was more influenced by culture time than by initial seeding density. Similar results were found when human alveolar osteoblasts were seeded on poly (ε-caprolactone)-tricalcium phosphate scaffolds using a static seeding technique by Zhou et al32.
Cell proliferation results as assayed using LIVE/DEAD® staining seem contradictory to the results as assayed using DNA quantification. Nucleic acids are negatively charged, while ceramics tend to be positively charged. Therefore, nucleic acids show a tendency to adhere to calcium containing scaffolds through electrochemical bonding25.
However, nucleic acids have a greater affinity for electrochemical bond formation with phosphate ions in a solution than with a ceramic material. Therefore, a phosphate buffer helps to release nucleic acids from ceramic materials and thus leads to more adequate nucleic acid quantification. For that, the modified technique as described by Piccinini et al25 was used in this study. However, we noticed that especially after
vacuum cell seeding, a high percentage of dead cells was visible on both scaffold types after LIVE/DEAD® staining. The nucleic acids that are released after cell deaths also adhere to the scaffold, and these nucleic acids are not washed after a PBS wash. That means that the nucleic acids originating from dead cells are also counted in the total amounts of extracted nucleic acids from the scaffolds. This may result in distorted DNA quantification and therefore distorted results.
There was a tendency towards higher cell death after vacuum seeding than after static cell seeding. To our knowledge, no other researchers considered cell death in their vacuum cell seeding experiments. We also investigated cell survival after vacuum treatment and cell survival without vacuum treatment (data not shown). In cells that were seeded using vacuum, cell growth was not significantly different from
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cells not treated with vacuum. These data corroborate with earlier findings of other authors16,27,30,33.
The cell seeding efficiency did not differ significantly between scaffold materials and cell seeding methods. We found cell seeding efficiencies of around 90%, similar as found by Zhou et al, who seeded human alveolar osteoblasts on a poly (ε-caprolactone)-tricalcium phosphate scaffolds with 45% porosity using a static seeding technique32. In other studies, cell seeding efficiencies of 80-90% were only
accomplished using dynamic seeding techniques, but not with static cell seeding18,27.
Tan et al16 seeded MC3T3-E1 cells on 45% porosity β-TCP scaffolds and found 60%
seeding efficiency using a vacuum seeding technique and 57% efficiency using a static seeding technique. However, they found initial retention rates of 80-95% at one hour after cell seeding. Roh et al15 found seeding efficiencies on a poly (glycolic acid) mesh
of 25% using a static seeding protocol and 35% using a centrifugal seeding technique. The differences between our findings and previous outcomes may be caused by a difference in scaffold material properties, different seeding techniques and different methodology for analysis of cell seeding efficiencies.
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