In Vitro Preparation of Actively Maturing Bovine Articular Cartilage Explants for X-Ray Phase Contrast Imaging

Summary

This protocol describes the preparation of in vitro bovine articular cartilage for imaging in high resolution with X-rays. These explants actively undergo postnatal maturation. We describe here the necessary steps from the biopsy to data analysis of 3D X-ray phase contrast imaging, passing through explant culture, tissue fixation and synchrotron preparation.

Abstract

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of tissue engineering strategies and potentially repairing diseased or damaged cartilage. In general, postnatal maturation of the articular cartilage, which is a wholesale change in collagen structure and function of the tissue to accommodate growth of the organism, occurs over a timescale ranging from months to years. Conversely dissolution of the structural organization of the cartilage that also occurs over long timescales is the hallmark of tissue degeneration. Our ability to study these biological processes in detail have been enhanced by the findings that growth factors can induce precocious in vitro maturation of immature articular cartilage. The developmental and disease related changes that occur in the joint involve bone and cartilage and an ability to co-image these tissues would significantly increase our understanding of their intertwined roles.

The simultaneous visualization of soft tissue, cartilage and bone changes is nowadays a challenge to overcome for conventional preclinical imaging modalities used for the joint disease follow-up. Three-dimensional X-ray Phase-Contrast Imaging methods (PCI) have been under perpetual developments for 20 years due to high performance for imaging low density objects and their ability to provide additional information compared to conventional X-ray imaging.

In this protocol we detail the procedure used in our experiments from biopsy of the cartilage, generation of in vitro matured cartilage to data analysis of image collected using X-ray phase contrast imaging.

Introduction

Immature articular cartilage is an adequate support to initiate morphological, structural and biomolecular changes¹ in order to obtain an adult joint-specific function. The principal change is reorganization of collagen fibrils from one displaying a parallel orientation with respect to the surface in immature cartilage to one where fibrils deeper in the tissue are perpendicular in mature cartilage. Pseudo-stratification of adult cartilage is evident through the reorganization of resident chondrocytes along the direction of collagen fibril orientation with cells at the surface disc-like and parallel to the surface and in the deeper zones cells b....

Protocol

All methods described here have been approved by the Ethical Research Committee of Swansea University and biopsy materials were acquired under license from the Department for Environment, Food & Rural Affairs (DEFRA), UK. This protocol follows the animal care guidelines of our institutions.

1. Explant Cultures

1. Preparation of the explants from bovine legs scalp
   1. Prepare absorbent protector and a scalpel, standard surgical scalpel with #10 scalpel blade.
   2. Spr.......

Discussion

We presented a complete study from the sample preparation to the image visualization, including the data acquisition protocols, for the study of in vitro fast maturating articular cartilage. Results of a synchrotron imaging session showed the goodness of the model.

In the model presented here, some observations and limits must be mentioned. This “accelerated maturation” occurs within 21 days of culture. For longer culture periods, explant plugs begin to degrade with change.......

Materials

Name	Company	Catalog Number	Comments
Material : Biological products
DMEM/F-12 (1:1) (1X) + GlutaMAX Dulbecco's Modified Eagle Medium F-12 Nutriment Mixture (Ham) 500mL	Gibco by life technologies	31331-028
Gentamicin Reagent Solution, 50 mg/mL	Gibco by life technologies	15750-060
HEPES, special preparation, 1M, pH 7.5 filtered	Sigma	H-3375
ITS, Insulin-Transferrin-Selenium	Gibco by life technologies	51500-057
L-Ascorbic Acid-2-Phosphate, sesquimagnesium salf hydrate, 95%	Sigma	A8960-5G
Neutral Buffer Formalin (NBF)	Sigma Aldrich	HT501128
phosphate buffered saline (PBS) pH 7.4	Gibco by life technologies	10010023
Culture equipments:
Absordent Protector,Benchkote	WhatmanTM	Cat No. 2300731,	Polysterene Backed, 460cm*50m
Accurpette VWR
Autoclavable Disposal Bag			For disposal of contamined plastic laboratory ware neck should be left open to allow penetration of steam, Hazardous Waste, STERILIN (white bag)
biopsy punches	MILTEX by KAI	ref 33-36	4 & 6 mm diameter
Clinical waste for alternative treatment Medium Duty			(UN-approved weight 5kg, Un-closure methods, UN- SH4/Y5/S/II/GB/4/06 (orange bag)
Eppendorf tubes			0.5mL and 1.5 mL
Falcon tubes			15mL and 50 mL
free of detectable RNase, DNase, DNA&Pyrogens 1000ul Bevelled Graduated, filter tip	Starlab, TipOne (sterile)	S1122-1830
free of detectable RNase, DNase, DNA&Pyrogens 20µl Bevelled Graduated, filter tip	Starlab, TipOne (sterile)	S1120-1810
free of detectable RNase, DNase, DNA&Pyrogens 200ul Bevelled Graduated, filter tip	Starlab, TipOne (sterile)	S1110-1810
Incubator			Incubator at 37°C, humidified atmosphere with 5% CO2
Optical microscope
Pipette-boy			25mL-, 10mL-, and 5mL sterile plastic-pipettes
Pipettes (25-10-5 ml)	CellStar, Greiner Bio-one
Plastic tweezers	Oxford Instrument	AGT 5230
Scalpel
Tips			P1000, P200 and P10 with P1000, P200 and P10 tips (sterile)
Tissue culture hood
Vacuum pump
Water bath 37°C
well plates			12 & 24 well plates
Protection equipment:
face shield
gloves
lab coat
safety goggles
Data acquisition equipment:
Fiji software			open source Software
PyHST reconstruction toolkit			open source Software