Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

Summary

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

Abstract

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Introduction

Bone defects, whether caused by trauma, tumor resection, congenital anomalies, or infection, represent a major challenge for regenerative medicine. These alterations compromise the structural integrity of the skeletal system, leading to discomfort, functional impairment, and a reduction in patients' quality of life.

To overcome these challenges, innovative bone repair strategies have emerged, with a focus on enhancing osteogenesis and bone tissue regeneration. These approaches include the use of implantable, injectable, or 3D-printable bone substitutes, which can be of natural origin (e.g., bio-sourced macromolecules, animal-derived hyd....

Protocol

This study has been approved by an ethics and animal welfare committee and by the French National Veterinary and Food Administration under number G44171.

1. Preparation and culture of osteochondral explants

1. Harvest sheep joint specimens from freshly euthanized animals in an aseptic environment. Position the sheep in a supine position and shave the left hind limb. Prepare by disinfecting with alcohol around the knee joint. Use lateral parapatellar arthrotomy to expo.......

Discussion

Repair of bone defects is a major challenge in regenerative medicine to restore mobility, reduce pain, and improve the quality of life of affected individuals. The use of explant models offers a number of advantages compared to in vivo studies for the investigation of bone defect repair. In addition to ethical considerations, this model allows for the rigorous control of experimental conditions and the reduction of biological variability, thereby facilitating the generation of more accurate and reproducible resu.......

Materials

Name	Company	Catalog Number	Comments
0.20 filters	VWR	28145-501
18 G needle (1,2x40 mm)	Sterican	4665120
3 mL syringe	HENKE-JECT	8300005762
37% hydrochloric acid	VWR	1.00317.1000
Acetic acid (glacial)	Sigma	A6283
Acetone	VWR	20063-365
Alcian Blue 8GX	VWR	361186
Ammonium hydroxide	VWR	318612
Apatitic tricalcium phosphate	Centre for Biomedical and Healthcare Engineering (Mines Saint Etienne, France)	TV26U
Azophloxine	Sigma	210633
Benzoyl peroxide	Sigma	8.01641.0250
BMP2	Medtronic	InductOs 1.5 mg/mL
Brillant crocein	Aldrich	2107507
CTVox	Bruker	-
DataViewer	Skyscan	-
Diamond blade	Struers	MOD13
Diamond saw	Struers	Accutom-50
DiaPro Mol B3 diamond solution	Struers	40600379
DiaPro Nap B1 diamond solution	Struers	40600373
Dibasic sodium phosphate (Na2HPO4)	Sigma	102404598
Dibutyl Phtalate	Chimie-Plus Laboratoires	28656
DragonFly software	ORS	2022.1.0.1231.
Dulbecco's Modified Eagle Medium (DMEM) high glucose, GlutaMAX(TM), pyruvate	ThermoFisher Scientific	31966-021
Eosine Y- Surgipath	Sigma	1002830105
Erythrosin B	Sigma	102141057
Ethanol absolute	VWR	20820362
Eukitt	Dutscher	6.00.01.0003.06.01.01
Falcon 50 mL	Sarstedt	62.547.254
Ferric chloride hexahydrate (FeCl3, 6H2O)	Merck	1.03943.0250
Fetal Bovine Serum (FBS)	Eurobio	CVFSVF00
Fuchsine acid	Merck	1.05231.0025
Hank's Balanced Salt Solution (HBSS)	Biosera	MS01NG100J
Hematoxylin	Sigma	86.118.9
Isostatic press	Nova Suisse	Pmax 1500 bars
Laser diffraction granulometry	Malvern	Mastersizer 3000
Light green	Prolabo	28947135
Lithium carbonate	Sigma	A13149
MD-Mol polishing cloth	Struers	40500077
Methylcyclohexane	VWR	8.06147.1000
Methylcyclohexane	VWR	8.06147.1000
Methylcyclohexane	VWR	8.06147.1000
Methylmethacrylate	Sigma	8.00590.2500
Micro-CT, micro-scanner	Bruker	Skyscan 1272
Monobasic sodium phosphate (NAH2PO4)	Sigma	71496
Mortar	Fritsch	Pulverisette 6
N,N, Dimethylanilin	Sigma	803060
Nanoindentation station	Anton Paar	NHT2
ND-Nap polishing cloth	Struers	40500080
OATS Osteochondral Autograft Transfer System Set, 4,75 mm	Arthrex	AR-1981-04S
OATS Osteochondral Autograft Transfer System Set, 8 mm	Arthrex	AR-1981-08S
Orange G	Ral	M15
Paraformaldehyde (PFA)	Sigma	P6148
Peel-a-way disposable embbedding moulds	Polysciences, Inc	18646C-1
Penicillin/Streptomycin (P/S)	ThermoFisher Scientific	15140122
Phosphate Buffered Saline (PBS)	ThermoFisher Scientific	10010023
Phosphomolybdic acid	Sigma	221856-100 g
Phosphotungstic acid	Aldrich	12863-5
Polishing machine	Sturers	Dap V
Poupinel	MEMMERT	TV26U
Raman microspectrometer	Renishaw	InVia Qontor
Safran du Gâtinais	Labonord	11507737
Scanning electron microscope	Carl Zeiss	Evo LS 10
SEM	Zeiss	Carl Zeiss Evo LS10
SiC foils/Grinding papers	Struers	40400008 (#320), 40400011 (#1000), 40400122 (#2000), 40400182 (#4000)
Silver paint	Electron microscopy sciences	12686-15
Standard stub with Faraday cup, carbon, aluminium and silicon standards	Micro-Analysis Consultants Ltd	8602
T25 flask	Corning	430639
Xylene	VWR	28975.325
Xylidine Ponceau	Aldrich	19.976-1