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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 ....
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
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.......
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 .......
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.
....Name | Company | Catalog Number | Comments |
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 |
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