The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.
Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of β-tricalcium phosphate (β-TCP) disks. We also tested a β-TCP/Collagen (β-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.
Bone repair is a complex process that begins with hematoma formation, inflammation, callus formation and then remodeling1,2. However, bone regeneration potential is limited to the size of the bone fracture1,3. For instance, large bone fractures caused by trauma, cancer or osteoporosis may not heal and are termed non-union bone fractures. These bone lesions often require treatment to promote healthy physiological bone repair and regeneration. Currently, autograft and allograft bone transplantation is the approach of choice4 with 2.2 million bone replacement procedures annually5. Though these procedures have a high success rate, there may be complications, for example, limited availability of bone, infection, donor site morbidity, and rejection4. New alternatives for bone tissue engineering are being sought to address these challenges.
The design of biomaterials based on natural or synthetic polymers, bioceramics or metals in combination with cells and bioactive molecules is on the rise6. Our current understanding of physiological bone healing and healing in the context of biomaterials depends on multiple factors such as mechanical properties and multiple local and systemic factors including cells from the circulation and fracture site7,8,9. Biomaterials for bone regeneration aim to promote osteogenicity and osseointegration10 and are ideally biocompatible, biodegradable, and porous (promoting cell migration, oxygen, and nutrients). They also need to be sufficiently strong to support the fracture site to relieve pain. Additionally, inflammatory factors are required to initiate the healing process. However, if the biomaterial induces excessive inflammation and allergic responses, this might limit or inhibit bone healing11,12. Thus, an interdisciplinary approach is necessary to evaluate biomaterials developed for bone repair.
In this study, we present a pre-clinical evaluation of representative materials, 1) Orthovita Vitoss foam which is a commercially available cancellous bone graft substitute consisting of tricalcium phosphate composed of nanometer-sized pure β-tricalcium phosphate (β-TCP) particles and Type 1 bovine collagen (C) (β-TCP/C foam) and 2) β-TCP disks. Here, we illustrate biocompatibility testing of these biomaterials using primary osteoblast (OB) and osteoclast (OC) assays, an in vivo model of bone repair, an immunological assessment comprising in vitro T lymphocyte proliferation and cytokine production, and in vivo immunogenicity and allergenicity, as previously reported13.
The procedures were done with BALB/c mice following all guidelines for the Care and Use of Laboratory Animals of the Austrian Ministry of Education, Science and Research and were approved by the Committee on the Ethics of the Austrian Ministry of Education, Science and Research.
1. Primary Mouse OB Culture
2. Mouse OB-OC Co-culture to Derive Mature OCs
3. Critical-sized Mouse Calvarial Defect Model
4. In Vitro Immune Responses
5. High Throughput Intraperitoneal Model
6. Subchronic Subcutaneous Model
To assess β-TCP for its effectiveness as a biomaterial for bone repair, we used in vitro and in vivo screening methods. Firstly, we measured the OB responses to the β-TCP disks compared with baseline medium alone controls. Figure 2 demonstrates the OB viability in response to β-TCP disks at 7 and 14 days of culture. Cell viability measured from metabolically active cells in the culture wells was the same for OBs with medium in tissue culture plastic as well as with β-TCP disks indicating that this biomaterial is neither enhancing nor suppressing OB proliferation.
To further evaluate the OBs, we measured ALP activity as a marker of differentiation using qualitative and quantitative approaches. Figure 2 illustrates ALP enzyme activity in culture wells after 7 days of culture. OBs in the wells with the medium alone had baseline ALP activity, while in optimal mineralization medium (MM) OBs had intense ALP staining, reflecting a high level of OB differentiation. In contrast, the OBs plated on β-TCP disks differentiated less than the OBs incubated in MM. In a quantitative assay, ALP concentration was 77% higher for the wells containing MM compared with the baseline medium alone, whereas ALP concentration was 40% lower in the cells cultured on β-TCP disks compared to MM controls. Although these results demonstrate that the cells grown on β-TCP disks differentiated less than those with optimal conditions of plastic with MM, they differentiated sufficiently on the biomaterials.
Another critical feature of OBs is their capacity to induce mineralization, which is an essential step in bone healing. We stained cultured OB cells with ARS after 14 days and found that mineralization was higher for MM controls compared to OBs cultured in medium alone and on β-TCP disks in culture wells (Figure 2). When we measured the ARS concentration, we found that the MM controls were more than 45% higher than the β-TCP group. These data illustrate that OBs cultured on plastic in the presence of MM mature, differentiate and mineralize better than those with medium alone and on β-TCP disks.
To determine how OCs respond to β-TCP disks, we used a culturing technology in which OBs are co-cultured with bone marrow OC precursors followed by the examination of OC morphology. OB-OC co-cultures were observed at 5 days and differed substantially between the cells grown on plastic with OC DM and the cells grown on bone slices and β-TCP. On plastic, the OCs were large and widespread whereas the OCs on physiological substrates were smaller, less-spread out and irregularly-shaped (Figure 3). To quantitate the OCs, we enumerated TRAP+ OCs and found that there were higher numbers when incubated with β-TCP (1755 ± 21.41/cm2) compared to tissue culture controls (1140 ± 15.71/cm2) and bone slices (709 ± 59.69/cm2), suggesting enhanced OC differentiation on β-TCP disks (Figure 3).
To determine a commercially available β-TCP/C foam in vivo, we used a critical-sized calvarial defect model in mice. We show representative histological sections processed by an undecalcified histological technique with glycol methyl methacrylate embedding and Levai Laczko staining. MicroCT and histology may be used to evaluate new bone formation within the defect area. Here, we show an example with histological sections in Figure 4. When the surgically-induced bone defect was left empty (sham), we observed a thin layer covering the entire defect, but no significant bone formation was present at 12 weeks post operation confirming the critical size of the created bone fracture. In contrast, when the defect contained β-TCP/C foam, there were β-TCP/C foam remnants surrounded by dense fibrous tissue including some blood vessels and inflammatory cells bridging the defect area without evidence of bone formation.
To evaluate the foreign body response, we assessed immunological and allergic reactions to the biomaterials, using an in vitro assay. Figure 5 demonstrates that when naïve splenocytes were incubated with medium alone or with β-TCP/C foam, the naïve spleen cells did not respond by proliferating or producing IL-2, IL-4, and IFN-γ cytokines. In contrast, in ConA containing cultures, cell proliferation and cytokine production increased except for IL-1β. Cell responses were unaffected when co-cultured with ConA and β-TCP/C foam compared with ConA alone, indicating that β-TCP/C foam neither increased nor decreased in vitro responses.
To determine whether β-TCP/C foam induced an in vivo immune response, we implanted it 1) intraperitoneally and measured inflammatory cell counts and cytokine concentrations in the peritoneal lavage fluid and 2) subcutaneously and evaluated inflammation and fibrosis on histological sections of the implantation site. In Table 1, the cell differential in the peritoneal lavage fluid reveals that the total number of inflammatory cells was significantly higher in the β-TCP/C foam implanted mice compared with the sham controls. Furthermore, there were increased numbers of all cell types. In Figure 6A, there are higher concentrations of IL-1β, IL-2, and IL-4 cytokines in the β-TCP/C foam compared with the sham controls. In β-TCP/C foam s.c. implanted mice, we observed an inflammatory response with foreign body giant cells on H&E-stained sections (Figure 6B) and evidence of fibrosis on Masson's Trichrome stained sections (Figure 6C) at 8 weeks. In contrast, the implantation site of the sham controls had minimal inflammation and no fibrosis (Figure 6C).
Figure 1: Calvaria removal for primary OB cell isolation diagram. The diagram illustrates how to remove the calvarium with 4 cuts (red dashed line) using curved scissors. The first cut is perpendicular to right (R) eye socket from X1 to X2, and the second is perpendicular to left (L) eye socket from X3 to X4. The third cut is to separate the calvarium at the front from X4 to X2, and the fourth cut is to separate the back from X3 to X1. The calvarium is then free to be removed. Please click here to view a larger version of this figure.
Figure 2: β-TCP-induced in vitro OB differentiation and maturation. OB viability and proliferation on days 7 and 14 for the cells cultured in medium alone (bars) or β-TCP (open bars) (mean ± SEM; n=3). ALP activity quantification of cell lysates and normalization to the protein content (µM DIFMU/µg protein, mean ± SEM, n = 3) with representative images illustrating ALP-stained culture wells from day 7. Mineralization quantified from ARS-stained cultures by a cetylpyridinium chloride extraction method shown as the concentration of ARS (µM ARS, mean ± SEM, n = 3) with representative ARS-stained culture wells on day 14. Groups include bone growth medium alone (BGM); Mineralization medium (MM); β-TCP. Please click here to view a larger version of this figure.
Figure 3: β-TCP-induced in vitro OC differentiation. Representative photomicrographs show TRAP+ MNCs at day 5 after co-culturing mouse OBs and bone marrow OC precursors. Endpoint analysis of TRAP+ multinucleated cells (MNCs) demonstrates the absolute count of TRAP+ MNCs (≥3 nuclei) per cm2 (mean ± SEM, n = 3) ***p <0.001. Groups include OC differentiation medium alone (DM); Bone; β-TCP. Please click here to view a larger version of this figure.
Figure 4: In vivo evaluation of β-TCP/C foam bone grafts in a critical-sized calvarial defect model. Non-healing calvarial defects created in 8-week old female BALB/c (n = 3) mice using a 4 mm dental trephine. Treatment groups included sham control (empty defect) and defects treated with β-TCP/C foam. Representative histological sections prepared at 12 weeks post-implantation. Formalin-fixed tissue glycol methyl methacrylate-embedded sections (80–100 µm) stained with Levai Laczko dye. Photomicrographs shown at low (left) and high (right) magnification. Black triangles indicate the bone defect. Black * denotes bone tissue and white * refers to β-TCP/C foam. Please click here to view a larger version of this figure.
Figure 5: β-TCP/C foam-induced in vitro cell proliferation and cytokine production. Splenocytes from naïve BALB/c mice cultured in medium alone, with β-TCP/C foam or ConA. Supernatant cell proliferation (BrdU), and production of IL-1β, IL-2, IL-4, and IFN-γ (medium alone ●, ConA ○, β-TCP/C foam ■, β-TCP/C foam and ConA □). Proliferation results presented as the mean of triplicate samples (O.D. ± SEM) in the BrdU assay and the mean of duplicate samples (pg/mL ± SEM) for cytokine concentration from two independent experiments. *p <0.05 is considered significant for biomaterial vs. medium and biomaterial and ConA vs. ConA alone. Please click here to view a larger version of this figure.
Figure 6: In vivo immune response of β-TCP/C foam bone grafts in a rapid high throughput i.p. and subchronic mouse model. (A) Female BALB/c mice implanted i.p. with β-TCP/C foam or without added materials (sham). Seven days later, peritoneal lavage analyzed for cytokine concentrations (data presented as mean cytokine concentrations pg/mL ± SEM). These data are representative of two independent experiments (n = 5). *p <0.05 is considered significant compared to sham. (B-C) Female BALB/c mice (n = 5) implanted s.c. with β-TCP/C foam or without added materials (sham). At 8 weeks after implantation, skin from the implantation sites stained with H&E (B) and Masson's Trichrome (C) to evaluate inflammation and fibrosis, respectively. Please click here to view a larger version of this figure.
Sham | Vitoss | ||
Cell counts x 106 (% of total cells) | |||
Macrophages | 1.240 ± 0.051 (88.1) | 3.262 ± 0.380 (70.4) | p <0.001 |
Eosinophils | 0.120 ± 0.011 (8.5) | 1.181 ± 0.254 (25.5) | p <0.01 |
Neutrophils | 0.002 ± 0.001 (0.2) | 0.029 ± 0.011 (0.6) | ns |
Lymphocytes | 0.048 ± 0.020 (3.2) | 0.148 ± 0.041 (3.5) | ns |
Total cell count | 1.410 ± 0.066 | 4.620 ± 0.452 | p <0.001 |
Table 1: In vivo immune response of β-TCP/C foam bone grafts in a rapid high throughput i.p. mouse model. Female BALB/c mice were implanted i.p. with β-TCP/C foam or without materials (sham). Seven days later, peritoneal lavage was obtained and analyzed for inflammatory cell number and differential cell counts (data presented as mean cell counts ± SEM). These data are representative of two independent experiments (n = 5).
Here, we show a multidisciplinary approach for the preclinical assessment of biocompatibility for representative biomaterials developed for bone regeneration and repair. We tested the responses of OBs, OCs, and the in vivo healing response in a critical bone defect model in mice as well as in vitro and in vivo immune responses. We aimed to demonstrate how the assays work and summarize the data and conclusions derived from the examination of the biomaterials. We show that our strategy generates a valuable profile of bone biomaterial biocompatibility.
Primary cell assays were used to evaluate OB and OC function. OBs are responsible for bone formation and physiological repair. They must remain viable, differentiate, and induce mineralization. In this study, we show how to perform assays for cell viability, ALP, and ARS as markers of physiologically differentiated cells with the capacity to mineralize. The controls for the assays included medium alone, which provides a baseline and osteogenic mineralization medium (MM), which optimized differentiation and mineralization of OBs on plastic. The latter control group was a reference standard for the biomaterial. For OC evaluation, we co-cultured OBs and bone marrow-derived OC precursors, differentiated the precursors into multinucleated cells, stained them with TRAP, an osteoclastic enzyme widely used to identify OCs in vitro15 and then enumerated the cells using light microscopy. These assays are state-of-the-art and did not require modifications. However, we noted a limitation related to the quality of isolated primary OBs to mineralize. Preserved OBs are stored in liquid nitrogen and used within one year for optimal results.
OB attachment and activity were higher on tissue culture plastic than on the β-TCP disks tested. When we assessed OCs, we observed the expected differences between the response to plastic and bone, which is the physiologic substrate16. In comparison, bone and β-TCP induced similar morphological changes. The TRAP assay enumeration of OCs in response to the β-TCP disks showed that the numbers were significantly different between bone slices and β-TCP. β-TCP induced higher OC differentiation than on bone slices. For OC differentiation, TRAP is a well-established assay. There were no significant modifications necessary in this method. However, to obtain the best results, it is essential not to incubate the cells for too long, or all cells of monocytic origin will become TRAP-positive.
To address the in vivo response, we used β-TCP/C foam as an exemplary biomaterial because it contains β-TCP, which was used in the in vitro assays and collagen and promotes bone healing13. Although β-TCP/C foam is commercially available and used clinically for bone repair17,18, it is only one of many different types of materials that would be interesting to study, e.g., biphasic calcium phosphate (hydroxyapatite/β-TCP) as well as demineralized human bone in these assays to determine how biological responses differ between materials. For the in vivo response, we implanted β-TCP/C foam into a critical-sized calvarial bone defect in mice and 12 weeks later assessed histology and showed differences compared with sham controls. It is also possible to evaluate the responses with microCT which provides complimentary information19. The defect in the sham control mice had no significant bone formation, as expected, whereas β-TCP/C foam induced an inflammatory response, fibrosis, and angiogenesis, which is the evidence of the early phase of bone formation. This method has been demonstrated previously in JOVE20. However, our approach differed in that we used a modified "elevator" technique with a periosteal elevator to reduce the risk of injuring the dura mater by the trephine. We reasoned that the dura mater plays a significant role in the healing process of calvarial defects by producing osteogenic cells and osteoinductive factors3,21,22,23. Notably, the material implanted, the size of the defect, and method for creating the defect influences bone regeneration of calvarial defects. Another modification in the procedure involved the stabilization of the biomaterial in the calvarial defect with a biocompatible tissue glue that is routinely used clinically for wound closure. This modification guaranteed that the material in the defect area would not be displaced during healing.
Inflammation regulates the early phase of bone healing, but too much inflammation or allergic responses may reduce repair11. The ideal immune response to biomaterials is to initiate an inflammatory cascade that promotes bone formation. However, certain biomaterials might cause a foreign body response leading to an array of inflammatory signals that cause fibrosis or allergic sensitization. In our studies, we evaluated immune responses to β-TCP/C foam and found that it was not toxic to naïve spleen cells and did not interfere with T lymphocyte expansion or function (cytokine secretion) when added to cultures with ConA. In the intraperitoneal experiments, β-TCP/C foam induced inflammation, and there was some evidence of an increase in eosinophilia and macrophages with concomitant increases in Th1- and Th2-type cytokines. In the subcutaneous implantation experiments, β-TCP/C foam also induced inflammation, but there was no evidence for chronic, destructive inflammatory or allergic responses, which suggests that β-TCP/C foam is biocompatible. These models provide information on the immune response to biomaterials. Firstly, the in vitro model addresses the effect of the biomaterial on naïve immune cells in the presence of a mitogen to provide evidence that there is no suppression of the mitogenic response caused by the biomaterial. Secondly, the intraperitoneal model provides a fast 7 day readout of the type of immune response, e.g., allergic and inflammation as observed by the type of inflammatory cell infiltration and the cytokine profile. Thirdly, the subcutaneous, subchronic model illustrates the tissue response over a longer period, allows for the evaluation of chronic inflammation, fibrosis, antibody titers, and can be used to test repeated implantation for immunological memory responses. These models have been previously published and are shown here without any modifications13. It is crucial that there are appropriate negative and positive controls for these models. We suggest performing all three models to avoid the limitations of each method. While the models shown are well established in other areas of immunology, the approach for testing biomaterials is recent.
In summary, bone and immune assays provide a biological compatibility profile on a biomaterial. For bone, OB and OC responses to the biomaterial provide preliminary data necessary before performing complicated and expensive animal experiments and to adhere to the 3Rs principle. Immunological in vitro assays provide data on antigen cross-reactivity and cytotoxicity, which may also preclude further animal experiments. The rapid high throughput experiments offer results on inflammatory and cytokine response (e.g., type of T lymphocyte responses), while the subchronic model is useful because of data on the duration of inflammation and the potential for damaging fibrosis. This novel interdisciplinary approach which includes bone and immune responses to biomaterials offers an excellent pre-clinical assessment of biocompatibility for future applications in the materials field.
The authors have nothing to disclose.
This research project has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 263363.
Biomaterials | |||
β-tricalcium phosphate disks (β-TCP, 14 mm) | Disks were sintered in a muffle furnace at a temperature of 1150 °C. A detailed description of the production can be found in Zimmer et al (doi: 10.1002/jbm.a.34639). Samples were UV-irradiated (15 min for each side) before using in cell cultures. | ||
Orthovita Vitoss foam (β-TCP/C foam) | Orthovita | 2102-1405 | |
Mouse osteoblast culture | |||
Alizarin Red S | Sigma-Aldrich | A5533 | |
ALP assay buffer (2x) pH 10.4 | Dissolve 200 mM glycine, 2 mM magnesium chloride, 2 mM zinc chloride in ultrapure water and adjust to pH 10. | ||
Bone growth medium (BGM) | α-MEM + 10% heat-inactivated FBS + 1% penicillin/streptomycin | ||
Cell lysis buffer: CyQuant cell lysis buffer 20x concentrate | Thermo Scientific | C7027 | Dilute the concentrated cell lysis buffer stock 20-fold in ultrapure water. |
Cell viability reagent: Presto Blue cell viability reagent | Invitrogen, Thermo Fisher Scientific | A13261 | |
Collagenase type IV from clostridium histolyticum | Sigma-Aldrich | C5138 | |
DC protein assay kit II | Bio Rad | 5000112 | DC protein assay kit contains reagent A, reagent B, reagent S and BSA for the reference standard. For reagent A', add 20 µL reagent S to 1000 µL reagent A. |
Dimethylsulfoxide (DMSO) | Sigma-Aldrich | D8418 | Steril-filter DMSO before usage. |
Dispase II (neutral protease, grade II) | Roche | 4942078001 | |
EnzCheck Phosphatase Assay Kit for alkaline phosphatase activity | Invitrogen, Thermo Fisher Scientific | E12020 | EnzCheck Phosphatase Assay Kit contains 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), N,N-dimethylformamide and 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU). |
Fetal bovine serum (FBS) | Sigma-Aldrich | F7524 | |
Formalin solution neutral buffered 10% | Sigma-Aldrich | HT501128 | |
Glycine | Sigma-Aldrich | G8898 | |
Hexadecylpyrdinium (cetylpyridinium) chloride monohydrate | Sigma-Aldrich | C9002 | |
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate | Sigma-Aldrich | A8960 | |
Magnesium chloride hexahydrate | Sigma-Aldrich | M2670 | |
MEM alpha medium (α-MEM) | Gibco Life Technologies | 11900-073 | |
Osteogenic mineralization medium (MM) | α-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin + 50 µg/mL ascorbic acid + 5 mM β-glycerophosphate | ||
Penicillin-streptomycin | Sigma-Aldrich | P4333 | |
Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
Sigmafast BCIP/NBT | Sigma-Aldrich | B5655 | |
Trypsin-EDTA (0.5%), no phenol red (10x) | Gibco, Thermo Fisher Scientific | 15400054 | Dilute the concentrated stock solution 10-fold in 1x PBS. |
Tween20 | Sigma-Aldrich | P1379 | |
Ultrapure water: Cell culture water pyrogen free | VWR | L0970-500 | |
Wash buffer | Add 0.05% Tween20 to 1x PBS. | ||
Zinc chloride | Sigma-Aldrich | 1.08816 | |
β-Glycerophosphate disodium salt hydrate | Sigma-Aldrich | G5422 | |
24-well suspension culture plate | Greiner bio-one | 662102 | |
24-well tissue culture plate | Greiner bio-one | 662160 | |
50 mL conical tube | Greiner bio-one | 227261 | |
96-well black plate | Greiner bio-one | 655076 | |
96-well transparent plate | Greiner bio-one | 655001 | |
Centrifuge: VWR Mega Star 600R | VWR | ||
CO2-Incubator: HeraCell 240 | Thermo Scientific | ||
Cryovial 2 mL | Greiner bio-one | 122263 | |
Disposable filter unit FP30/0.2 CA-S | GE Healthcare Life Sciences Whatman | 10462200 | For sterile filtration of enzyme solution. |
Flatbed scanner: Epson Perfection 1200 Photo | Epson | ||
Infinite M200 Pro microplate reader | Tecan | ||
Microcentrifuge tube: Eppendorf tube safe-lock 0.5 mL | VWR | 20901-505 | |
Microcentrifuge tube: Eppendorf tube safe-lock 1.5 mL | VWR | 21008-959 | |
Shaker Swip (orbital and horizontal) | Edmund Buehler | ||
Shaking incubator GFL3032 | GFL | 3032 | |
Single-use pipet: serum pipet | Greiner bio-one | 612301 | |
Sterile instruments (scissors, tweezer, curved forceps) | |||
Tissue culture plate (10 cm) | Greiner bio-one | 664960 | |
Mouse osteoblast-osteoclast co-culture | |||
1α,25-Dihydroxyvitamin D3 | Sigma-Aldrich | 17936 | 1000x concentrated stock (10 µM in ethanol 100%) |
Acetone | VWR Chemicals | 22065.327 | |
Bone growth medium (BGM) | α-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin | ||
Distilled water | Carl Roth | 3478.4 | |
Ethanol (100% vol/vol) | VWR Chemicals | 20821.365 | |
Ethanol (70% vol/vol) | VWR Chemicals | 93003.1006 | |
Fast red violet LB salt | Sigma-Aldrich | F3381 | |
Fetal bovine serum (FBS) | Sigma-Aldrich | F7524 | |
Formalin solution neutral buffered 10% | Sigma-Aldrich | HT501128 | |
MEM alpha Medium (α-MEM) | Gibco Life Technologies | 11900-073 | |
N,N-Dimethylformamide | Sigma-Aldrich | D4551 | |
Naphthol AS-MX phosphate | Sigma-Aldrich | N4875 | |
Osteoclast differentiation medium (DM) | α-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin + 1 nM 1,25-(OH)2-vitamin D3 + 1 µM prostaglandin E2 | ||
Penicillin-streptomycin | Sigma-Aldrich | P4333 | |
Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
Prostaglandin E2 (PGE2) | Cayman Chemical Company | 9003016 | 1000x concentrated stock (1 mM in ethanol 100%) |
Sodium acetate trihydrate | Sigma-Aldrich | S7670 | |
Sodium tartrate dibasic dihydrate | Sigma-Aldrich | S8640 | |
Sterile instruments (scissors, forceps, scalpel) | |||
TRAP buffer pH 5.0 | Dissolve 40 mM sodium acetate, 10 mM sodium tartrate in ultrapure water and adjust to pH 5. | ||
Ultrapure water: Cell culture water pyrogen free | VWR | L0970-500 | |
24-well suspension culture plate | Greiner bio-one | 662102 | |
24-well tissue culture plate | Greiner bio-one | 662160 | |
50 mL conical tube | Greiner bio-one | 227261 | |
Centrifuge: VWR Mega Star 600R | VWR | ||
CO2-Incubator: HeraCell 240 | Thermo Scientific | ||
Diamond wafering blade (10.2 cm x 0.3 cm) | Buehler | ||
Isomet low-speed saw | Buehler | ||
Petri dish (6 cm) | Greiner bio-one | 628,161 | |
Screw cap glass bottle 20 mL | Carl Roth | EXY4.1 | |
Single-use sterile syringe 1 mL | Henry Schein Animal Health | 9003016 | |
Single-use pipet: serum pipet | Greiner bio-one | 612301 | |
Sterile needle: Hypodermic needle RW 27 G x 3/4'' | Henry Schein Animal Health | 9003340 | |
Mouse calvarial defect model | |||
Analgesia: Buprenorphine (Bupaq) | Richter Pharma AG | ||
Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun) | Richter Pharma AG/Bayer HealthCare | ||
Cold sterilant: SafeSept Max | Henry Schein Animal Health | 9882765 | |
Ethanol (70% vol/vol) | VWR Chemicals | 93003.1006 | |
Eye ointment: VitA POS 5 g | Ursapharm | ||
Normal saline solution (0.9% sodium chloride solution) | Fresenius Kabi | ||
Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
Povidone iodine solution: Braunol 1000 mL | B. Braun | 3864154 | |
Roti-Histofix 4.5% buffered formalin | Carl Roth | 2213.6 | |
Tissue adhesive: Histoacryl 5 x 0.5 mL | B. Braun Surgical | 1050060 | |
Personal protective equipment: surgical gloves, cap and mask, gown | Henry Schein Animal Health | 1045073, 1026614, 9009062, 370406 | |
Sterile instruments (scalpel, scissors, forceps, needle holder) | |||
Sterile needles: Hypodermic needles RW 27 G x 3/4'' and 30 G x 1/2'' | Henry Schein Animal Health | 9003340, 9003630 | |
Single-use sterile syringe 1 mL | Henry Schein Animal Health | 9003016 | |
Heating plate: Physitemp | Rothacher Medical | TCAT-2LV | |
Sterile gauze swabs | Henry Schein Animal Health | 220192 | |
Sterile disposable scalpel (Figur 20) | Henry Schein Animal Health | 9008957 | |
Surgical/dental drill: Implantmed SI-923 | W&H Dentalwerk Bürmoos GmbH | 16929000 | |
Handpiece type S-II | W&H Dentalwerk Bürmoos GmbH | 30056000 | |
Trephine, diameter 4 mm | Hager&Meisinger GmbH | 229040 | |
Irrigation tubing set 2.2 m | W&H Dentalwerk Bürmoos GmbH | 4363600 | |
Periosteal elevator 2 mm / 3 mm | Henry Schein Animal Health | 472683 | |
Non-resorbable suture: Polyester green, DS19, met. 1.5, USP 4/0 75 cm | Henry Schein Animal Health | 300715 | |
In vitro immune responses | |||
Anesthesia for euthanasia: Ketamine (Ketamidor), Xylazine (Rompun) | Richter Pharma AG/Bayer HealthCare | ||
Cell Proliferation ELISA, BrdU (colorimetric) Assay Kit | Sigma-Aldrich | 11647229001 | The kit contains BrdU labeling solution, fixation solution (FixDenat), Anti-BrdU antibody solution, washing buffer and substrate solution. |
Concanavalin A (ConA) | MP Biomedicals | 150710 | |
Culture medium splenocytes | RPMI medium + 10% heat-inactivated FBS + 1% penicillin-streptomycin solution + 0.1% gentamicin + 0.2% ß-mercaptoethanol + 1% non-essential amino acids | ||
Ethanol (70% vol/vol) | VWR Chemicals | 93003.1006 | |
Fetal bovine serum (FBS) | Gibco, Thermo Fisher Scientific | 10500064 | |
Gentamicin | Gibco, Thermo Fisher Scientific | 15750037 | |
Mouse IL-1β ELISA Ready-SET-Go! | Invitrogen, Thermo Fisher Scientific | 88-7013-88 | |
Mouse IL-2 ELISA MAX Standard | BioLegend | 431001 | |
Mouse IL-4 ELISA MAX Standard | BioLegend | 431101 | |
Mouse INF-γ ELISA MAX Standard | BioLegend | 430801 | |
Non-essential amino acids solution | Gibco, Thermo Fisher Scientific | 11140050 | |
Penicillin-streptomycin | Gibco, Thermo Fisher Scientific | 15140122 | |
Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
Red blood cell (RBC) lysis buffer: BD Pharm Lyse | BD Bioscience | 555899 | |
RPMI 1640 medium, HEPES | Gibco, Thermo Fisher Scientific | 52400025 | |
β-Mercaptoethanol | Gibco, Thermo Fisher Scientific | 31350010 | |
50 mL conical tube | Greiner bio-one | 227261 | |
96-well tissue culture plate | Greiner bio-one | 655180 | |
Centrifuge: VWR Mega Star 600R | VWR | ||
Cell strainer 40 µm | BD Bioscience | 352340 | |
Infinite M200 Pro microplate reader | Tecan | ||
Single-use sterile syringe 1 mL | Henry Schein Animal Health | 9003016 | |
Sterile surgical instruments (forceps, glass slides) | |||
High throughput intraperitoneal model | |||
Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun) | Richter Pharma AG/Bayer HealthCare | ||
Cold sterilant: SafeSept Max | Henry Schein Animal Health | 9882765 | |
Ethanol (70% vol/vol) | VWR Chemicals | 93003.1006 | |
Eye ointment: VitA POS 5 g | Ursapharm | ||
Mouse IL-1β ELISA Ready-SET-Go! | Invitrogen, Thermo Fisher Scientific | 88-7013-88 | |
Mouse IL-2 ELISA MAX Standard | BioLegend | 431001 | |
Mouse IL-4 ELISA MAX Standard | BioLegend | 431101 | |
Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
Povidone iodine solution: Braunol 1000 mL | B. Braun | 3864154 | |
Shandon Rapid-Chrome Kwik-Diff Staining Kit | Thermo Scientific | 9990700 | For differential cell count. |
Heating plate: Physitemp | Rothacher Medical | TCAT-2LV | |
Hemocytometer: Neubauer chamber | Carl Roth | PC72.1 | |
Non-resorbable suture: Ethibond Excel 4-0 | Ethicon | 6683H | |
Resorbable suture: Polysorb | Covidien | SL-5628 | |
Shandon centrifuge for cytopsin | Thermo Scientific | ||
Single-use sterile syringe 1 mL | Henry Schein Animal Health | 9003016 | |
Sterile gauze swabs | Henry Schein Animal Health | 220192 | |
Sterile needles: Hypodermic needles RW 27 G x 3/4'' and 25 G | Henry Schein Animal Health | 9003340, 420939 | |
Sterile surgical instruments (scalpel, scissors, forceps, needle holder) | |||
Trypan blue solution 0.4% | Gibco, Thermo Fisher Scientific | 15250061 | |
Subchronic subcutaneous model | |||
Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun) | Richter Pharma AG/Bayer HealthCare | ||
Cold sterilant: SafeSept Max | Henry Schein Animal Health | 9882765 | |
Ethanol (70% vol/vol) | VWR Chemicals | 93003.1006 | |
Eye ointment: VitA POS 5 g | Ursapharm | ||
Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
Povidone iodine solution: Braunol 1000 mL | B. Braun | 3864154 | |
Roti-Histofix 4.5% buffered formalin | Carl Roth | 2213.6 | |
Heating plate: Physitemp | Rothacher Medical | TCAT-2LV | |
Non-resorbable suture: Ethibond Excel 4-0 | Ethicon | 6683H | |
Personal protective equipment: surgical gloves, cap and mask, gown | Henry Schein Animal Health | 1045073, 1026614, 9009062, 370406 | |
Single-use sterile syringe 1 mL | Henry Schein Animal Health | 9003016 | |
Sterile gauze swabs | Henry Schein Animal Health | 220192 | |
Sterile instruments (scalpel, scissors, forceps, needle holder) | |||
Sterile needle: Hypodermic needles RW 27 G x 3/4'' | Henry Schein Animal Health | 9003340, 420939 |