Biological Compatibility Profile on Biomaterials for Bone Regeneration

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 remodeling^1,2. However, bone regeneration potential is limited to the size of the bone fracture^1,3. 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....

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 .......

Materials

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