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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 the heads in a Petri dish with sterile 1x phosphate-buffered saline (PBS).
    2. Hold the head by the nape of the neck, and cut the skin away us....

Representative Results

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.......

Discussion

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 .......

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
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)Orthovita2102-1405
Mouse osteoblast culture
Alizarin Red SSigma-AldrichA5533
ALP assay buffer (2x) pH 10.4Dissolve 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 concentrateThermo ScientificC7027Dilute the concentrated cell lysis buffer stock 20-fold in ultrapure water. 
Cell viability reagent: Presto Blue cell viability reagentInvitrogen, Thermo Fisher ScientificA13261
Collagenase type IV from clostridium histolyticumSigma-AldrichC5138
DC protein assay kit IIBio Rad5000112DC 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-AldrichD8418Steril-filter DMSO before usage. 
Dispase II (neutral protease, grade II)Roche4942078001
EnzCheck Phosphatase Assay Kit for alkaline phosphatase activity Invitrogen, Thermo Fisher ScientificE12020EnzCheck 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-AldrichF7524
Formalin solution neutral buffered 10%Sigma-AldrichHT501128
GlycineSigma-AldrichG8898
Hexadecylpyrdinium (cetylpyridinium) chloride monohydrateSigma-AldrichC9002
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrateSigma-AldrichA8960
Magnesium chloride hexahydrateSigma-AldrichM2670
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-AldrichP4333
Phosphate-buffered saline 1x (PBS) pH 7.4Gibco, Thermo Fisher Scientific 10010023
Sigmafast BCIP/NBTSigma-AldrichB5655
Trypsin-EDTA (0.5%), no phenol red (10x)Gibco, Thermo Fisher Scientific 15400054Dilute the concentrated stock solution 10-fold in 1x PBS. 
Tween20Sigma-AldrichP1379
Ultrapure water: Cell culture water pyrogen freeVWRL0970-500
Wash bufferAdd  0.05% Tween20 to 1x PBS.
Zinc chlorideSigma-Aldrich1.08816
β-Glycerophosphate disodium salt hydrateSigma-AldrichG5422
24-well suspension culture plateGreiner bio-one662102
24-well tissue culture plateGreiner bio-one662160
50 mL conical tubeGreiner bio-one227261
96-well black plateGreiner bio-one655076
96-well transparent plateGreiner bio-one655001
Centrifuge: VWR Mega Star 600RVWR
CO2-Incubator: HeraCell 240Thermo Scientific
Cryovial 2 mLGreiner bio-one122263
Disposable filter unit FP30/0.2 CA-SGE Healthcare Life Sciences Whatman10462200For sterile filtration of enzyme solution.
Flatbed scanner: Epson Perfection 1200 PhotoEpson
Infinite M200 Pro microplate readerTecan
Microcentrifuge tube: Eppendorf tube safe-lock 0.5 mLVWR20901-505
Microcentrifuge tube: Eppendorf tube safe-lock 1.5 mLVWR21008-959
Shaker Swip (orbital and horizontal) Edmund Buehler 
Shaking incubator GFL3032GFL 3032
Single-use pipet: serum pipetGreiner bio-one612301
Sterile instruments (scissors, tweezer, curved forceps)
Tissue culture plate (10 cm)Greiner bio-one664960
Mouse osteoblast-osteoclast co-culture
1α,25-Dihydroxyvitamin D3Sigma-Aldrich179361000x concentrated stock (10 µM in ethanol 100%)
AcetoneVWR Chemicals22065.327
Bone growth medium (BGM)α-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin
Distilled waterCarl Roth3478.4
Ethanol (100% vol/vol)VWR Chemicals20821.365
Ethanol (70% vol/vol)VWR Chemicals93003.1006
Fast red violet LB saltSigma-AldrichF3381
Fetal bovine serum (FBS)Sigma-AldrichF7524
Formalin solution neutral buffered 10%Sigma-AldrichHT501128
MEM alpha Medium (α-MEM)Gibco Life Technologies 11900-073
N,N-DimethylformamideSigma-AldrichD4551
Naphthol AS-MX phosphateSigma-AldrichN4875
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-AldrichP4333
Phosphate-buffered saline 1x (PBS) pH 7.4Gibco, Thermo Fisher Scientific 10010023
Prostaglandin E2 (PGE2)Cayman Chemical Company90030161000x concentrated stock (1 mM in ethanol 100%)
Sodium acetate trihydrateSigma-AldrichS7670
Sodium tartrate dibasic dihydrateSigma-AldrichS8640
Sterile instruments (scissors, forceps, scalpel)
TRAP buffer pH 5.0Dissolve 40 mM sodium acetate, 10 mM sodium tartrate in ultrapure water and adjust to pH 5.
Ultrapure water: Cell culture water pyrogen freeVWRL0970-500
24-well suspension culture plateGreiner bio-one662102
24-well tissue culture plateGreiner bio-one662160
50 mL conical tubeGreiner bio-one227261
Centrifuge: VWR Mega Star 600RVWR
CO2-Incubator: HeraCell 240Thermo Scientific
Diamond wafering blade (10.2 cm x 0.3 cm)Buehler
Isomet low-speed sawBuehler
Petri dish (6 cm)Greiner bio-one628,161
Screw cap glass bottle 20 mLCarl RothEXY4.1
Single-use sterile syringe 1 mLHenry Schein Animal Health9003016
Single-use pipet: serum pipetGreiner bio-one612301
Sterile needle: Hypodermic needle RW 27 G x 3/4''Henry Schein Animal Health9003340
Mouse calvarial defect model
Analgesia: Buprenorphine (Bupaq)Richter Pharma AG
Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun)Richter Pharma AG/Bayer HealthCare
Cold sterilant: SafeSept MaxHenry Schein Animal Health9882765
Ethanol (70% vol/vol)VWR Chemicals93003.1006
Eye ointment: VitA POS 5 gUrsapharm
Normal saline solution (0.9% sodium chloride solution)Fresenius Kabi
Phosphate-buffered saline 1x (PBS) pH 7.4Gibco, Thermo Fisher Scientific 10010023
Povidone iodine solution: Braunol 1000 mLB. Braun3864154
Roti-Histofix 4.5% buffered formalinCarl Roth2213.6
Tissue adhesive: Histoacryl 5 x 0.5 mL B. Braun Surgical1050060
Personal protective equipment: surgical gloves, cap and mask, gownHenry Schein Animal Health1045073, 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 Health9003340, 9003630
Single-use sterile syringe 1 mLHenry Schein Animal Health9003016
Heating plate: PhysitempRothacher MedicalTCAT-2LV
Sterile gauze swabsHenry Schein Animal Health220192
Sterile disposable scalpel (Figur 20)Henry Schein Animal Health9008957
Surgical/dental drill: Implantmed SI-923W&H Dentalwerk Bürmoos GmbH16929000
Handpiece type S-IIW&H Dentalwerk Bürmoos GmbH30056000
Trephine, diameter 4 mmHager&Meisinger GmbH229040
Irrigation tubing set 2.2 mW&H Dentalwerk Bürmoos GmbH4363600
Periosteal elevator 2 mm / 3 mmHenry Schein Animal Health472683
Non-resorbable suture: Polyester green, DS19, met. 1.5, USP 4/0 75 cmHenry Schein Animal Health300715
In vitro immune responses
Anesthesia for euthanasia: Ketamine (Ketamidor), Xylazine (Rompun)Richter Pharma AG/Bayer HealthCare
Cell Proliferation ELISA, BrdU (colorimetric) Assay KitSigma-Aldrich11647229001The kit contains BrdU labeling solution, fixation solution (FixDenat), Anti-BrdU antibody solution, washing buffer and substrate solution. 
Concanavalin A (ConA)MP Biomedicals150710
Culture medium splenocytesRPMI 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 Chemicals93003.1006
Fetal bovine serum (FBS)Gibco, Thermo Fisher Scientific 10500064
GentamicinGibco, Thermo Fisher Scientific 15750037
Mouse IL-1β ELISA Ready-SET-Go!Invitrogen, Thermo Fisher Scientific88-7013-88
Mouse IL-2 ELISA MAX StandardBioLegend431001
Mouse IL-4 ELISA MAX StandardBioLegend431101
Mouse INF-γ ELISA MAX StandardBioLegend430801
Non-essential amino acids solutionGibco, Thermo Fisher Scientific 11140050
Penicillin-streptomycin Gibco, Thermo Fisher Scientific 15140122
Phosphate-buffered saline 1x (PBS) pH 7.4Gibco, Thermo Fisher Scientific 10010023
Red blood cell (RBC) lysis buffer: BD Pharm LyseBD Bioscience555899
RPMI 1640 medium, HEPESGibco, Thermo Fisher Scientific 52400025
β-MercaptoethanolGibco, Thermo Fisher Scientific 31350010
50 mL conical tubeGreiner bio-one227261
96-well tissue culture plateGreiner bio-one655180
Centrifuge: VWR Mega Star 600RVWR
Cell strainer 40 µmBD Bioscience352340
Infinite M200 Pro microplate readerTecan
Single-use sterile syringe 1 mLHenry Schein Animal Health9003016
Sterile surgical instruments (forceps, glass slides)
High throughput intraperitoneal model
Anesthesia: Ketamine (Ketamidor), Xylazine (Rompun)Richter Pharma AG/Bayer HealthCare
Cold sterilant: SafeSept MaxHenry Schein Animal Health9882765
Ethanol (70% vol/vol)VWR Chemicals93003.1006
Eye ointment: VitA POS 5 gUrsapharm
Mouse IL-1β ELISA Ready-SET-Go!Invitrogen, Thermo Fisher Scientific88-7013-88
Mouse IL-2 ELISA MAX StandardBioLegend431001
Mouse IL-4 ELISA MAX StandardBioLegend431101
Phosphate-buffered saline 1x (PBS) pH 7.4Gibco, Thermo Fisher Scientific 10010023
Povidone iodine solution: Braunol 1000 mLB. Braun3864154
Shandon Rapid-Chrome Kwik-Diff Staining KitThermo Scientific9990700For differential cell count.
Heating plate: PhysitempRothacher MedicalTCAT-2LV
Hemocytometer: Neubauer chamberCarl RothPC72.1
Non-resorbable suture: Ethibond Excel 4-0Ethicon6683H
Resorbable suture: PolysorbCovidienSL-5628
Shandon centrifuge for cytopsinThermo Scientific
Single-use sterile syringe 1 mLHenry Schein Animal Health9003016
Sterile gauze swabsHenry Schein Animal Health220192
Sterile needles: Hypodermic needles RW 27 G x 3/4'' and 25 GHenry Schein Animal Health9003340, 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 MaxHenry Schein Animal Health9882765
Ethanol (70% vol/vol)VWR Chemicals93003.1006
Eye ointment: VitA POS 5 gUrsapharm
Phosphate-buffered saline 1x (PBS) pH 7.4Gibco, Thermo Fisher Scientific 10010023
Povidone iodine solution: Braunol 1000 mLB. Braun3864154
Roti-Histofix 4.5% buffered formalinCarl Roth2213.6
Heating plate: PhysitempRothacher MedicalTCAT-2LV
Non-resorbable suture: Ethibond Excel 4-0Ethicon6683H
Personal protective equipment: surgical gloves, cap and mask, gownHenry Schein Animal Health1045073, 1026614, 9009062, 370406
Single-use sterile syringe 1 mLHenry Schein Animal Health9003016
Sterile gauze swabsHenry Schein Animal Health220192
Sterile instruments (scalpel, scissors, forceps, needle holder)
Sterile needle: Hypodermic needles RW 27 G x 3/4'' Henry Schein Animal Health9003340, 420939

References

  1. Einhorn, T. A., Gerstenfeld, L. C. Fracture healing: mechanisms and interventions. Nature Reviews Rheumatology. 11 (1), 45-54 (2015).
  2. Marsell, R., Einhorn, T. A. The biology of fracture healing. Injury. 42 (6), 551-555 (2011).<....

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