Resorbability of bone substitute biomaterials by human osteoclasts
Arndt F. Schilling , 1, Wolfgang Linhart 1, Sandra Filke 1, Matthias Gebauer , Thorsten Schinke , Johannes M. Rueger and Michael Amling ,
Department of Trauma, Hand, and Reconstructive Surgery, Center for Biomechanics, Hamburg University School of Medicine, Martinistrasse 52, Hamburg 20246, Germany
Received 24 July 2003; accepted 22 October 2003. Available online 6 December 2003.
Biomaterials
Volume 25, Issue 18 , August 2004, Pages 3963-3972
Abstract
Third generation biomaterials are being designed with the aim that once implanted they will help the body to heal itself. One desirable characteristic of these materials in bone is their ability to be remodeled, i.e. that osteoclasts resorb the material and it is subsequently replaced by newly formed bone through osteoblastic activity. So far the only way to test this biological property of bone substitutes are animal experiments with all their limitations like ethics, costs and limited transferability to man. The present study was designed, to develop a human in vitro assay, allowing to generate human osteoclasts directly on the biomaterial. The assay was validated using calcium phosphate cement and PMMA as biomaterials. Quantification was performed by raster electron microscopy and computer assisted image analysis. Dentin was used as internal standard. Our assay shows iso-bone resorbability of calcium phosphate cement in comparison to unresorbable PMMA cement. Both current clinical orthopedic practice and future skeletal engineering may profit from the availability and use of a test system for the assessment of resorption quality. The assay presented here allows to address this question of resorbability and to select the best materials for the use as bone substitutes in specific patients.
Author Keywords: Author Keywords: Bioresorption; Biomimetic material; Calcium phosphate cement; Cell culture; In vitro test; Osteoclast
Article Outline
1. Introduction
For almost a century autogenous bone grafting has been successfully used to augment repair of skeletal defects [1 and 2]. The distinct advantages of cancellous and cortical autografts namely being osteoinductive and osteoconductive are compromised by its disadvantages including donor site morbidity, increased duration of the surgical procedure and limited availability [3]. The virtually unlimited supply of allografts and xenografts on the other hand is offset by problems with the immune system and potential transmittal of infectious diseases. This was the basis for biomedical materials to enter the scene in bone replacement some 50 years ago. In the beginning the aim was to produce biologically inert implants causing a minimum of immune response within the body. Already 20 years ago this paradigm changed from bioinert toward bioactive materials with controlled action and reaction in the physiological environment.
Bone is a living organ that is constantly remodeled. It permanently replaces old worn tissue by newly formed bone, thereby evading impairment of the bone structure by wear and tear. It repairs damages and adjusts its structure to external stress [4]. This leads to a bone, which is perfectly adapted to the load it has to bear. This directional self-renewal is one of the main advantages of the living bone tissue over any inanimate biomaterial, which is by definition subject to wear and tear. Thus, an ideal, bioactive material for bone substitution should be able to be resorbed and replaced by new bone, before e.g. cyclic loading can compromise the stability of the biomaterial. While it might seem desirable for a "restitution ad integrum" to have a fast remodeling process that disposes the body of the biomaterial as soon as possible, stability is the most important factor for the patient. Thus the patient's ability to form new bone must be balanced with the speed of resorption of the biomaterial.
The only cell type that is capable of resorbing bone is the osteoclast [5 and 6]. Unfortunately, it is very difficult, to access osteoclasts. This is mainly due to two properties of these cells. First, under physiologic conditions, the osteoclast is a very scarce cell type, covering only 1% of the bony surface [7]. Second, calcified tissue is literally hard to handle, which makes it nearly impossible to purify satisfying numbers of authentic cells for in vitro studies.
However, since the discovery of RANKL, a molecule required for osteoclast differentiation [8, 9 and 10], it has become possible to generate osteoclasts from mononuclear precursors in vitro, without the support of osteoblastic stromal cells.
The objective of this study was to optimize the culture conditions for human osteoclasts, making it possible to let them grow directly on biomaterials. We were able to generate multinuclear cells that express the specific markers of terminally differentiated osteoclasts. These cells are functionally active and resorb dentin in the pit assay. They prosper on the tested biomaterials and were also capable of resorbing the surface of these materials. By cocultivation of biomaterials with dentin in the same culture, we were able to establish a system that allows quantification of resorption relative to bone. Use of this system might help biomedical engineers to optimize their compounds for the use in man and assist clinicians to choose the ideal biomaterial for the specific needs of their patients.
2. Material and methods
2.1. Chemicals and antibodies
Standard lab chemicals were from Merck (Darmstadt, Germany) and Sigma (Deisenhofen, Germany). Na-heparin was obtained from Roche (Mannheim, Germany). Ficoll-Paque came from Amersham Pharmacia Biotech (Uppsala, Sweden). Alpha-MEM, fetal bovine serum (FBS), penicillin-streptomycin-solution, recombinant human macrophage-colony-stimulating factor (M-CSF) and trypsin (10×) were purchased from Sigma (Deisenhofen, Germany). Phosphate buffered saline (PBS) was from GIBCO BRL, Life Technologies Inc. (Rockville, MD, USA). Soluble recombinant human receptor-activator for NfkappaB Ligand (RANKL) came from Peprotech Ltd. (London, UK). Calcitonin receptor antibody was purchased from Santa Cruz Biotechnology (Sata Cruz, USA) Rhodamine-phalloidine was obtained from Molecular Probes (Leiden, The Netherlands). Fluorescent anti-mouse secondary antibodies were purchased from Boehringer Mannheim (Mannheim, Germany). Biotinylated secondary antibody, DAB and HRP marked streptavidin antibody and Fluorsave came from DAKO Diagnostika (Hamburg, Germany).
2.2. Media, solutions, buffer
Cells were cultivated in alphaMEM containing 0.22% sodium bicarbonate, 10% FBS and 1% penicillin/streptomycin, 20 ng/ml M-CSF and 40 ng/ml RANKL. Fixation of cells was done in PBS containing 3.7% formaldehyde. For trypsination alphaMEM was used containing 0.22 g sodium carbonate, 10% Trypsin-EDTA (10×) and 1% penicillin/streptomycin. As immunochemistry-solution PBS was used containing 0.1% BSA and 0.05% saponin. For the blocking solution, 150
l of serum of the host of the secondary antibody were added to 2.85 ml immunochemistry-solution. TRAP buffer was a solution with 40 m
sodium-acetate and 10 m
sodium-tartrate. TRAP staining solution was 5 mg Naphtol AS-MX Phosphate, 500
l N-N-Dimethylformamide and 30 mg Fast Red Violett LB Salt in 50 ml TRAP-buffer.
2.3. Biomaterials and dentin
Calcium phosphate cement (Biobon) and polymethylmetacrylate (PMMA, Palamed G20) were obtained from Merck (Darmstadt, Germany). Dentin (ivory) was kindly provided by German customs in accordance with the international laws for the protection of species.
2.4. RT-PCR analysis
Total RNA was isolated from cultured human osteoclasts following the TRIzol-protocol (Invitrogen). cDNA synthesis from 1
g of total RNA was performed with the cDNA-CYCLE-Kit (Invitrogen) according to the manufacturer's instructions. The obtained cDNA was dissolved in a total volume of 100
l of sterile water. To normalize the cDNA amount in the samples, 3
l of the resulting cDNA of each probe was used for PCR analysis of the housekeeping gene beta-actin. PCR reactions were performed with 27 cycles. Products were detected in ethidium bromide (0.1%)-stained 1.0% agarose gels and cDNA volumes were adjusted for consecutive analyses by densitometry (Quantity One, Bio-Rad). Programs and primers for the measurement of steady-state levels of mRNA were as follows. A unique 4 min period for complete denaturation at 94°C in the beginning, followed by 27 primer-specific cycles consisting of 30 s denaturation at 94°C, 45 s annealing at a primer specific temperature (carbonic anhydrase type 2 (CA-2) 56.0°C; beta-actin 60.0°C) and 45 s primer extension at 72°C, with an additional 5 min at 72°C for the final extension in the end.
All PCR reactions were performed in an iCycler (Bio-Rad, Germany). Amplification of beta-actin cDNA served as an internal standard (housekeeping gene).
2.5. Primers and PCR-chemicals
Small DNA Low Melt Agarose came from Biozym Diagnostik GmbH (Oldendorf, Germany). Recombinant Taq DNA Polymerase and 10×PCR buffer were from Invitrogen (Karlsruhe, Germany). DNeasy Tissue Kit was obtained from Qiagen (Hilden, Germany). 100 bp DNA-ladder came from Biowhittaker Molecular Applications (Vallensbaek Strand, Denmark). 25 m
dNTP-solution was bought from Biozym Diagnostik GmbH (Oldendorf, Germany). Primer were bought from Invitrogen (Karlsruhe, Germany).
2.6. Isolation of hematopoietic stem cells
Bone marrow, peripheral human blood and buffy coats were obtained from healthy volunteers. To prevent clotting, bone marrow and peripheral blood were treated with 1% sodium-heparin and stored on ice before processing.
Bone marrow, peripheral blood and buffy coat were each subjected to the same protocol for isolation of mononuclear cells, which contain the cells, from which osteoclasts originate. This mononuclear cell fraction will, therefore, be called hematopoietic or osteoclastic precursors in the following text. Density gradient centrifugation was used to separate the mononuclear precursor cells from other formed elements in blood.
PBS and Ficoll-Paque were heated to 37°C. A volume of 50 ml cell-solution (bone marrow, peripheral blood or buffy coat, respectively) were diluted with PBS to 200 ml and transferred to 4×50 ml tubes. Tubes of 8×50 ml were filled with 25 ml Ficoll-Paque. The 200 ml with PBS diluted cell-solution was carefully layered on top of the Ficoll-Paque. The tubes were centrifuged with 350g at 20°C for 30 min. After this centrifugation step, the mononuclear hematopoietic precursors accumulate at the interface between PBS and Ficoll-Paque. These cells were transferred in fresh tubes resulting in 4 tubes with 30-40 ml with mononuclear cells. These tubes were filled to 50 ml with PBS and centrifuged with 350g at 20°C for 10 min. Pellets were washed with 50 ml PBS and resuspended in 1 ml PBS. Cell solution of 10
l were stained with 990
l Trypan-blue to assess viability and counted in a Neubauer counting chamber. To dispose the culture of contaminating lymphocytes, the cells were purified for adherence. Therefore, the non-adherent cells were washed off at day one of culture, so that only the adherent monocytes were used for cultivation. Cells were cultivated in alphaMEM containing 0.22% sodium bicarbonate, 10% FBS and 1% penicillin/streptomycin, 20 ng/ml M-CSF and 40 ng/ml RANKL for up to 28 days.
For the determination of variability of the amount of cells between samples, cells were counted on day 1 after the adherence purification step.
2.7. TRAP-staining
Cells were fixed for 5 min in 3.7% buffered formaldehyde, air-dried for 2 min. The cells were stained in TRAP-staining solution for 10 min. Cells with a positive staining for TRAP containing 3 or more nuclei were counted as osteoclasts.
2.8. Immunofluorescence and confocal laser-microscopy
For actin staining, cells on microscopic cover slips were dehydrated in −20°C acetone for 5 min and air dried on parafilm. They were then incubated in Rhodamine-phalloidine, diluted in PBS 1:40 for 20 min in the dark. For double labeling additional steps also were performed in the dark to prevent bleaching. Cover slips were rinsed in PBS and then incubated for 20 min in blocking solution to inhibit unspecific binding. The primary antibody (mouse-anti-vitronectin (23C6)) was diluted 1:40 in PBS and the cells were incubated in it for 120 min. Then the cover slips were washed 3×5 min in immunochemistry-solution. The secondary antibody (Alexa Fluor 488 anti-mouse) was diluted 1:100 and was used to incubate the cells for 60 min. After 3×5 min washing, the cells were mounted with Fluosave and stored overnight at 4°C. Analysis was done with an Olympus confocal laser microscope using a combined argon/krypton laser at 488 and 568 nm wavelengths.
2.9. Calcitonin receptor staining
For staining of the calcitonin receptor (CTR), cells were fixed for 10 min in 3.7% buffered formalin. Endogen peroxidase was blocked with 3% H2O2 in methanol for 10 min. Then cells were washed for 5 min in PBS. Cells were incubated for 1 h with CTR-antibody diluted 1:500. Cells were washed 5 min in PBS. Cells were incubated with secondary antibody (biotinylated anti-goat-IgG; dilution 1:200) for 60 min followed by 5 min washing in PBS. HRP-marked streptavidin antibody was diluted 1:200 and incubated with the cells for 30 min. After 5 min washing in PBS the staining was developed by DAB for 6 min. Cells were rinsed in aqua dest. For counterstaining of the nuclei, the cells were stained with hämalaun diluted 1:5 in aqua dest for 1 min. Cells were washed for 10 min in aqua dest, for 5 min in 100% EtOH. They were air-dried and mounted on microscopic slides for documentation.
2.10. Raster electron microscopy and pit assay on dentin, calcium phosphate and PMMA
Dentin was cut to a thickness of 1 mm. Calcium phosphate and PMMA were casted on glass to obtain a smooth surface and cut to 8×8 mm. For the assessment of resorbability, cells were cultivated in 6-well-dishes for up to 28 days on dentin alone, calcium phosphate with dentin and PMMA with dentin (n=5). At the end of the culture period, cells were washed off with PBS. The material was air-dried and sputtered with gold in a sputter coater 108 auto device (Cressington, Watford, UK). They were analyzed in a Leo 435vp raster electron microscope (Leo, Oberkochen, Germany) at 150 mA and 20 kV. Resorbed area was quantified using UTHSCSA Image Tool (UTHSCSA San Antonio, TX, USA).
2.11. Statistics
Statistical analysis was done using ANOVA. p<0.05 was accepted as statistically significant. Error bars represent standard deviation (SD).
3. Results
3.1. Source of human stem cells
Our first goal was to find the optimal source for human osteoclast precursors for our assay. We followed three approaches based on different stem cell sources: (i) Bone marrow, which has the highest concentration of hematopoietic stem cells and is used in mouse coculture experiments to generate osteoclasts. (ii) Peripheral human blood, which is known to contain peripheral stem cells. (iii) Buffy coat, a waste product from the manufacture of blood bottles, containing platelets and most of the white blood cells. We found that all three sources yielded reasonable numbers of monocytic precursor cells to design experiments (35−740×106 cells/50 ml). While bone marrow has as expected a higher percentage of hematopoietic stem cells, than peripheral blood, this can be compensated by using buffy coats, where monocytes from 500 ml are pooled, increasing the yield for 50 ml buffy coat approximately ten-fold (408×106 cells) versus 50 ml blood (48×106 cells). Based on that, we decided to perform all following experiments with precursors obtained from buffy coat.
3.2. Stem cell density
Our second goal was to find the ideal density of precursors for human osteoclast development. We used the mononuclear precursor cells isolated from buffy coat in concentrations from 104 to 108 cells/ml. After a culture period of 14 days, multinuclear cells positive for TRAP, a marker of differentiated osteoclasts, were counted. Osteoclast yield increased in a dose-dependent fashion from precursor densities of 104 cells/ml culture medium to 107 cells/ml culture medium (Fig. 1A). Increasing the density to 108 cells/ml culture medium does not result in osteoclasts any more, probably because the cell layer in the culture dish becomes too thick to be nourished. 107 precursors only yield 65% more osteoclasts than 106 precursors with 10 times more precursors. As osteoclasts are almost confluent at a precursor density of 106 cells/ml we decided to use 2×106 cells/ml to get confluent human osteoclast cultures.
Fig. 1. The cultivation of human ostoclasts is critically dependent on cell density of the precursors (A) and on pH value of the medium (B). Increasing the initial precursor density tenfold leads to a 1.5-2-fold increase of osteoclasts. Cell densities of 108 cells/ml culture medium do not result in osteoclastogenesis any more (A). An acidic environment favors the development of human osteoclasts. Between pH 6.9 and pH 7.1 a statistically significant better result can obtained compared to the physiological pH 7.3 (B).
3.3. Osteoclast culture conditions
Next we wanted to optimize the culture conditions for human osteoclast development. Therefore, we tested osteoclast development at physiologic pH (7.3), under basic conditions (pH 7.5) and in the acidic range (pH 7.1, 6.9 and 6.7). As it is known, that bone resorption is increased in chronic metabolic acidosis [11], we reasoned, that a more acid environment might favor osteoclast development. In fact, we found a more than four-fold increase of osteoclasts compared to physiologic pH in an acidic culture environment with an optimum between pH 6.9 and pH 7.1 (p<0.01) (Fig. 1B).
3.4. Human osteoclast characterization
To ensure, that the generated cells are indeed osteoclasts we tested them for important specific characteristics after 28 days of culture. First we analyzed the morphology of the resulting cells (multinuclearity, formation of actin rings with podosomes). A confluent layer of multinucleated cells was observed in all cultures at 28 days by light microscope (Fig. 2A). The multinucleated cells form actin rings and podosomes that are observed by confocal microscopy after rhodamine-phalloidine labeling of actin (Fig. 2B).
Fig. 2. The cultivated cells show all morphological characteristics of osteoclasts. In the confluent cultures, more than 90% of the surface is covered by multinucleated cells (A, Pappenheim staining, magnification 200×). These cells show the formation of actin rings (B, confocal microscopy, actin-staining, magnification 400×) and podosomes (B insert).
Next we analyzed the expression of osteoclast specific markers (TRAP, calcitonin receptor, vitronectin receptor and carbonic anhydrase type II). Starting from confluent precursors (Fig. 3A), the cells begin to express TRAP already after 4 days of culture (Fig. 3B). After 28 days high levels of TRAP expression were detected in all multinucleated cells after 28 days ( Fig. 3C), as was the expression of the osteoclast specific [12] calcitonin receptor ( Fig. 3D). The alphavbeta3 subunit of the vitronectin receptor (Fig. 3E), which is essential for osteoclastic function [13 and 14] was also highly expressed. The time course expression carbonic anhydrase type II (CA2) was studied by RT-PCR at 1 day, 4 days, 14 days and 28 days ( Fig. 3F), as it is known, that CA2 plays an important part in the resorption process. CA2 is necessary for the acidification of the resorption compartment. Thus, patients with an inactivating mutation of CA2 develop osteopetrosis [15]. This is cell autonomous, as the osteopetrosis can be cured by bone marrow transplantation [16]. We used CA2 as a marker for osteoclastic activity. The expression of carbonic anhydrase type II was specifically upregulated in our culture during osteoclast differentiation.
Fig. 3. The cultivated human cells (A day 1 of culture) start to fuse and express TRAP at day 4 of culture (B, red: TRAP, magnification 100×), at day 28 more than 90% of the multinucleated cells express TRAP (C, red: TRAP, magnification 100×). These cells also express the calcitonin receptor (D, brown: CTR, blue: nuclei (Hämalaun staining), magnification 200×) and the vitronectin receptor alphavbeta3 (C, green: alphavbeta3, red: actin, confocal microscopy, magnification 500×); molecular expression analysis shows a marked increase in carbonic anhydrase 2, documenting the accretive differentiation over 28 days (semi-quantitative RT-PCR, normalized to beta-actin).
For comparison of different culture samples, it is essential to ensure, that the initial cell densities do not vary. We evaluated this by counting the adherent mononuclear cells after 1 day of culture. The variance in cell count between samples was 4.6%.
Last, we analyzed the functional activity of the cells in the pit assay. The first resorption pits could be detected after 3 weeks of culture (Fig. 4A). After 4 weeks of culture, more than 80% of the surface were resorbed (Fig. 4B). Thus at 28 day of culture, the cells show all important characteristics of osteoclasts and are highly functionally active.
Fig. 4. Human osteoclasts in culture start resorbing the surface of dentin at 3 weeks (A). After 4 weeks more than 80% of the surface was covered with resorption pits (B). (Light microscopy, toluidine staining, magnification 100×).
3.5. Resorbability of biomaterials
Having established this culture system to a high level of reliability and reproducibility, we next wanted to test the resorbability of biomaterials. First we focused on two biomaterials that are already used clinically, calcium phosphate cement and PMMA. For standardization, dentin was cocultivated in every culture. Osteoclast precursors were seeded on the evenly sized materials and cultivated to differentiation in five independent experiments. As a control, five additional chips of calcium phosphate and PMMA were cultivated for the same period of time in medium containing no cells to monitor cell-independent hydrolytic degradation of the respective compounds. After 4 weeks of culture the PMMA, dentin and calcium phosphate cement were analyzed by raster electron microscopy (Fig. 5) and the resorption area of the surface was quantified using an image processing system.
Fig. 5. Surface of PMMA (A/B), dentin (C/D) and calcium phosphate (E/F) after 28 days of culture without cells (A/C/E) and with human osteoclasts (B/D/F). While the surface is unchanged after 4 weeks in medium alone, the cultured human osteoclasts are able to resorb calcium phosphate leaving only islets of the former surface (Raster electron microscopy, magnification 100×).
While there was no resorption detectable on PMMA in our cultures (Fig. 5B), 72.5±9.8% of the calcium phosphate surface was resorbed ( Fig. 5F), compared to 75.5±21.2% of the surface of dentin ( Fig. 5D). Controls treated for the same time with the same medium without the cells showed no sign of hydrolytic degradation ( Figs. 5A, C and E). To circumvent the problem of inter-culture differences in activity of the cells, we used the dentin as an internal control. Normalizing by calculating the ratio between resorption on the biomaterial and resorption of dentin in the same culture, makes it possible to compare different materials independent of these inter-culture changes. We propose to name this ratio the "relative resorbability coefficient" (RRC). Dentin has an RRC of 1, PMMA has a RRC of 0 while the calcium phosphate biomaterial used in this study has an RRC of 0.95.
4. Discussion
Biomaterials are increasingly used for the augmentation and replacement of harmed organs [17]. Better understanding of the bone structure and the advantages of a self repairing biologic system have led to a back-to-nature shift in concept from "as solid as possible" to bioactive materials [18]. The ideal bone substitution material should be osteoinductive, osteoconductive and only stay in the body as long as necessary to replace the defect by newly formed bone [19]. As every patient is different in bone structure, type of injury and illness, this goal can only be achieved, if the expert clinician has a variety of different biomaterials to tailor an individual treatment. Therefore, we aimed at developing a system that allows relative quantification of resorbability and resorption speed for biomaterials preclinically.
4.1. Preclinical testing of biomaterials
Cell culture and animal experiments have a long history in the preclinical evaluation of materials designed to be implanted into the human body [20, 21, 22 and 23]. Toxicity and biocompatibility can easily be studied in cell culture, making it possible to rule out some potential candidates at an early stage of development. Tests on resorbability, however, were almost completely restricted to animal experiments using, e.g. rats [24] or rabbits [25] with all their limitations, because authentic human osteoclasts were not available in sufficient amounts for sensible cell culture experiments. This is mainly due to the fact, that under physiologic conditions, osteoclasts only cover 1% of the bone surface [7]. Additionally, they endue a specialized cytoskeleton allowing a very strong attachment to the bone, making it difficult to isolate them.
The first approach to overcome these obstacles in the animal model was to feed laying hens a calcium depleted diet and letting them lay eggs [26]. To provide enough calcium for the egg-shell, the birds had to release the calcium from its reservoir in the bone. This leads to a massive increase in osteoclast number, making it possible to obtain reasonable amounts of osteoclasts for molecular and in vitro studies. However it turned out, that avian osteoclasts have some important differences to mammalian osteoclasts (e.g. resorption pits of chick osteoclasts are five times bigger than rat osteoclasts [27]) limiting the application of this model to study human diseases. Based on the fact, that osteoclasts arise from the hematopoietic system [28], a mammal coculture system was designed [29]. By using osteoblasts stimulated with vitamin D, cocultured hematopoietic stem cells differentiate and fuse into osteoclasts. This model in turn permitted the discovery of a cell-surface molecule on the osteoblast (RANKL) and its receptor on the osteoclast (RANK) [8, 30 and 31], that are essential for osteoclast differentiation. By using recombinant RANKL in combination with a stimulator of the hematopoietic precursors (M-CSF), it became possible to grow osteoclasts in vitro from hematopoietic cells without the need for osteoblasts. Also a immortalized mouse osteoclast precursor cell line was developed from mice transgenic for both bcl-X(L) and simian virus 40 large T antigen [32]. This permitted nearly unlimited access to osteoclast precursors in the mouse model, however, with the restrictions associated with cell lines in respect to understanding physiology.
The only human cell source for resorbing cells that could be used for such experiments was the giant cell tumor [33]. However, this approach has two major disadvantages. First, the giant cell tumor is a rare disease, only accessible at highly specialized orthopedic centers. Second and more important, the cells are highly aggressive and activated cells, which do not display the physiology of human osteoclasts. The latter fact almost precludes drawing any conclusion about the behavior of the biomaterial in a physiologic environment from such experiments. Thus, we set out to develop a cell culture system for the evaluation of biomaterial resorption using non-tumor human osteoclasts. To approximate physiology of human bone resorption in vitro, we wanted to differentiate primary human osteoclast precursors into active human osteoclasts directly on the surface of biomaterials in vitro.
4.2. Human osteoclast culture system
We evaluated three different sources for precursors and decided to use buffy coat, a waste product from the manufacturing of blood bottles that is easily accessible in great quantities. This helped us to design standardized experiments. However, the system reported here even works with 50 ml venous blood directly obtained from a patient, principally allowing individual testing of cell substrate-interaction in patients with specific disorders, e.g. cell-autonomous osteoclast defects. In the course of optimizing the culture conditions, we were able to show, that a pH value slightly below the physiologic value favors human osteoclast development, which resembles the findings in rabbits [34]. Also, we established the optimal precursor cell density for the generation of confluent human osteoclast cultures on biomaterials.
The morphology of the generated confluent cells is consistent with osteoclast morphology. The large multinucleated cells display the typical cytoskeletal features like the formation of actin rings and the appearance of podosomal attachments. Furthermore, they express the specific osteoclastic markers like TRAP, calcitonin receptor and vitronectin receptor. As osteoclasts are by definition the only cells capable of resorbing bone [35], we proved their osteoclastic nature in a dentin resorption assay. Time course experiments showed the first signs of osteoclastic differentiation already at day 4 of culture, while fully functional cells needed at least 3 weeks of differentiation in the culture environment presented here.
4.3. Human biomaterial resorption assay
While there are reports of other cells containing degradation products of biomaterials [36], the osteoclasts is by far the most important cell for the resorption of bone substitute materials. By cultivating precursor cells directly on the biomaterial, the physiological conditions leading to resorption are mimicked as closely as possible. Therefore, several questions can be answered in one experiment: Can human cells grow on the respective biomaterial? Is there any toxic effect on the cells? Is the biomaterial stable against hydrolytic destruction? Can it be resorbed by human osteoclasts? Thus, the cell culture system presented in this paper represents an excellent model for the evaluation of biomaterials for the use in bone. However admittedly, the system described here does not cover if the material is osteoconductive or osteoinductive. In vivo newly formed bone on the surface of the material might be protective against osteoclast mediated bone resorption. This question can only be answered in vivo, but our model allows the preselection of promising compounds. By cocultivation of dentin, with the studied biomaterials, it becomes possible to define a RRC. This coefficient allows comparison of different biomaterials in respect to resorption by osteoclasts and normalizes potential inter-individual donor effects. However, the great yield of osteoclast precursors from buffy coat even allowed in this study to perform the experiments for the evaluation of calcium phosphate cement, PMMA and dentin with cells from the same donor.
Using our system may facilitate the development of new compounds designed for implantation. Biomedical engineers can construct and test materials for different purposes limiting costs and the need of animal experiments. The generated results are instantly valid in respect to human osteoclastic resorption. If a material is desired to be remodeled like bone (e.g. bone substitution), the RRC should be around 1, if it should be removed quicker (e.g. delivery of drugs), the coefficient needs to be higher, and if it should not be resorbed at all (e.g. implant fixation), RRC should preferably be 0 like in PMMA. In this way, the model system presented here allows standardized classification of biomaterials based upon their resorbability. This would enable the orthopedic surgeon to choose the appropriate material for the needs of his specific patient.
Acknowledgements
The authors thank Dr. A.R. Zander (Department of Bone Marrow Transplantation, Hamburg University School of Medicine, Germany) for generously providing human bone marrow and Dr. P. Kühnl (Department for Transfusion Medicine, Hamburg University School of Medicine, Germany) for generously providing buffy coats.
Also the authors thank Dr. Michael Horton (Bone and Mineral Center, University College, London, UK) for generously providing the 23C6-antibody against the alphaVbeta3 subunit of the vitronectin-receptor.
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