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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Published: November 16th, 2018

DOI:

10.3791/58077

1Department of Pharmacology and Toxicology, University of Vienna, 2Laboratory of Experimental Allergy, Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna

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 ....

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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

  1. OB isolation from neonatal mouse calvaria using enzymatic digestion
    1. Euthanize 1–2 day old neonatal pups (60 in total) by decapitation and place.......

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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.......

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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 .......

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This research project has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 263363.

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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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