Culturing and Measuring Fetal and Newborn Murine Long Bones

Summary

Here, we present a method for ex vivo culture of long murine bones at both fetal and newborn stages, suitable for analyzing bone and cartilage development and homeostasis in controlled conditions while recapitulating the in vivo process.

Abstract

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy human bones makes particularly valuable the use of mammalian models, such as mouse and rat, to look into different aspects of bone growth and homeostasis. Additionally, the development of sophisticated genetic tools in mice allows more complex studies of long bone growth and asks for an expansion of techniques used to study bone growth. Here, we present a detailed protocol for ex vivo murine bone culture, which allows the study of bone and cartilage in a tightly controlled manner while recapitulating most of the in vivo process. The method described allows the culture of a range of bones, including tibia, femur, and metatarsal bones, but we have focused mainly on tibial culture here. Moreover, it can be used in combination with other techniques, such as time-lapse live imaging or drug treatment.

Introduction

Organ growth has to be tightly tuned to prevent the appearance of growth disorders, and involves the regulation of multiple cell types, molecular pathways and crosstalk among different parts of the body. Imaging techniques are essential to address the changes occurring over time in a growing embryo, both in normal conditions, as well as after a perturbation is induced in the system. Embryos with intrauterine development, such as the widely used rodent models, present an additional challenge for live imaging and drug treatment, which can be partially overcome by using ex vivo culture techniques. To successfully recapitulate the in vivo processes and obtain meaningful results, it becomes crucial to find the right culturing conditions for each organ or tissue.

Most bones of the mammalian skeleton grow through endochondral ossification, where the embryonic cartilage (composed of cells called chondrocytes) drives longitudinal growth and is gradually replaced by bone. This process happens at the growth plates, located at the end of the long bones, where three zones can be distinguished: resting, proliferative, and hypertrophic^1,2. First, the round progenitor chondrocytes in the resting zone transition into the cycling columnar chondrocytes in the proliferative zone. During the next stage of differentiation, these chondrocytes become hypertrophic and start secreting type X collagen. Hypertrophic chondrocytes orchestrate the subsequent steps of ossification: they secrete key signaling molecules, such as connective tissue growth factor, bone morphogenetic proteins and Indian hedgehog, and direct the mineralization of the matrix, recruit blood vessels to the central part of the bone, and, upon apoptosis, allow osteoblasts (bone-forming cells) to invade the matrix to form the primary ossification center^3,4. The mineralized matrix facilitates the penetration of blood vessels through which osteoblasts migrate to replace this degraded cartilage with a bone matrix⁵. Most osteoblasts invade the cartilage matrix from the perichondrium, a fibrous layer that wraps the cartilage⁶. Alternatively, a proportion of hypertrophic chondrocytes are able to survive and transdifferentiate to osteoblasts^7,8,9. The final length of the bone is due to the accumulated growth of the transient cartilage, whose growth rate in turn depends on the number and size of the hypertrophic chondrocytes, and their matrix production¹⁰. Additionally, it was recently shown that the duration of the last hypertrophy phase correlates with the final length of the bone¹¹. Therefore, tight regulation of the proliferation and differentiation of these cells is required to ensure proper bone size.

Despite of the substantial knowledge acquired over the years on the organization and development of the growth plates, most of these conclusions are based on the observation of fixed histological sections. Tissue sectioning provides valuable information about this process, but can be ridden with technical artifacts, so it cannot be always reliably used to estimate morphological or size changes between different stages. Additionally, as bone growth is a dynamic process, the static two-dimensional (2D) images offer a limited insight into the movement of the cells in the growth plate, while time-lapse imaging on live tissue could offer valuable information on the behavior of the chondrocytes in the growth plate.

All these limitations can be potentially resolved using ex vivo bone cultures. While bone culture protocols have been developed some time ago, they were limitedly applied to murine long bones. Most of the studies use chick bones due to the technical advantages offered by the chick model^12,13. Organotypic cultures (air/liquid interface) were applied to chick embryonic femurs, which were maintained in culture for 10 days¹⁴. The sophisticated genetic tools available in mouse make this model very appealing to be used in ex vivo bone culture. The studies that used mice to look into bone growth worked mostly with metatarsal bones¹⁵, probably due to their small size and greater numbers obtained per embryo¹⁶. Although traditionally considered long bones, metatarsi enter senescence (characterized by reduced proliferation and involution of the growth plate¹⁷) earlier than other long bones in vivo, and therefore their continuous growth ex vivo does not really recapitulate the in vivo process. For the purposes of this article, we will use the term long bones for bones from the proximal and intermediate limb regions. Several previous studies used long murine bones, such as tibia, in ex vivo cultures and observed a substantial growth of the cartilage but little ossification¹⁸. We also used tibial cultures recently, mainly to study chondrocyte dynamics¹⁹. Other studies used femoral heads from young mice²⁰ or only the distal part of the femur for culture²¹. Some more recent works successfully combine the ex vivo culture of full bones with time-lapse imaging to acquire three-dimensional (3D) movies of chondrocytes in living mouse tissue^22,23. The authors managed to observe previously unnoticed events in the rearrangement of chondrocytes to the proliferative zone²³ in a good example of the potential application of bone ex vivo culture. The alternative, i.e., analyzing static images, requires indirect and complex techniques. This was exemplified by a recent study assessing the importance of transversally-oriented clones for cartilage growth, where genetic tracing with multicolor reporter mouse strains coupled with mathematical modeling were used²⁴. Therefore, ex vivo culture might help gain insight into dynamic processes in a faster and more straightforward way.

Here, we present a method for murine long bones culture, which can be combined with different molecular treatments and/or with time-lapse live imaging. This protocol adapts the methods used in previous reports^15,18,25, but addresses some additional issues and focuses on long bones such as the tibia, rather than metatarsal bones. Finally, it explores the potential of using statistically powerful paired comparisons by culturing left and right bones separately in the presence of different substances.

Protocol

All the experiments should be carried out following the local governmental and institutional guidelines of ethical handling of laboratory animals.

1. Preparations Prior to the Day of Bone Culture

1. Set up timed mouse matings to obtain fetuses and pups from embryonic day 14.5 (E14.5) and onward.
   NOTE: The culture of long bones can be successfully applied to different mouse strains; in the present protocol, outbred Swiss Webster wild-type mice are used.
2. Prepare dissection medium (adapted from Houston et al.¹⁵): dilute α-minimum essential medium (α-MEM) or Dulbecco’s modified Eagle’s medium (DMEM) 1/13 in phosphate-buffered saline (PBS) and 2 mg/mL bovine serum albumin (BSA) and filter sterilize through a 0.22 µm, 33 mm diameter syringe filter. Store aliquots at -20 °C.
3. The day before extraction of the fetuses, prepare the serum-free bone culture medium composed of DMEM containing 0.2% BSA, 0.5 mM L-glutamine, 40 U/mL penicillin/streptomycin, 0.05 mg/mL ascorbic acid, and 1 mM betaglycerophosphate. Filter sterilize through a 0.22 µm, 33 mm diameter syringe filter and store at 4 °C for up to 1 month.
4. On the day of the culture and prior to mouse culling, prepare 24-well plates and 60 mm dishes with dissection medium and keep them ice cold. Prewarm the bone culture medium in a 37 °C water bath. Spray 80% (v/v) ethanol on the tools (tweezers and small scissors) to be used for fetus handling. Transfer a binocular scope to a Class II biosafety cabinet.

2. Culture of Fetus and Newborn Tibia and Femur

1. Cull the pregnant mouse through cervical dislocation at the desired gestational stage (ranging from E14.5 to E18.5). If newborn pups are used, remove them from the mother one by one and cull by decapitation.
2. Place the mouse on her back and sterilize the abdominal region by spraying 80% (v/v) ethanol on its surface.
3. Cut the skin and the abdominal muscle with small scissors to access the uterine horns.
4. Extract the uterus from the abdominal cavity with the help of tweezers and small scissors, removing the mesometrium and cutting the base of the horns. Place the uterus in a 60 mm Petri dish with ice-cold dissection medium and keep the culture dish on ice during the whole procedure.
5. Transfer the Petri dish with the uterus to the biosafety cabinet and work there from now on.
6. Separate individual fetuses with scissors by cutting between the sacs.
7. Transfer individual sac under a dissection stereomicroscope in a clean 60 mm dish with dissection medium and open them up with tweezers to separate the fetuses from the placentas and clean them from membranes.
   NOTE: Work with one embryo at a time, while keeping the rest on ice.
8. Decapitate the fetuses and transfer the body to a clean new 60 mm dish with a 1 mL cut sterile pipette.
9. Remove the skin of the fetuses or pups with tweezers starting from the back and peeling it out till the toes.
10. Separate the hindlimbs from the body by cutting with the tweezers close to the spine and transfer them to a clean dish with ice-cold dissection medium.
11. Separate the tibia from the femur with tweezers by carefully introducing them between the surface cartilage of distal femur and proximal tibia.
12. Remove the hip bones from the proximal femur and the calcaneus bone and the fibula from the tibia.
13. Carefully remove the soft tissue from the femur and the tibia by nipping and pulling it off.
    NOTE: It is important to remove as much soft tissue as possible, taking special care in removing the tissue that connects the two cartilage poles, but avoiding damaging the cartilage, the perichondrium, and the bones.
14. Place the four bones (left and right tibias and femurs) in the first well of the 24-well plate with a plastic 1 mL sterile pipette. Alternatively, to compare the effect of different treatments on left and right limbs, place contralateral limbs in different wells.
    NOTE: Extra care should be taken when the bones are transferred to the wells, as they can easily stick to the pipette.
15. Proceed the same way with as many fetuses as necessary.
    NOTE: Change the dish with the dissection medium as soon as it gets too clouded, as it is important to see clearly the dissected bones.
16. When all the desired bones are transferred to the wells, remove the dissection medium with a plastic 1 mL sterile pipette and take extra care not to aspirate the bones.
    1. Depending on the purpose of experiment, pictures can be taken of the bones before removing the dissection medium, as timepoint zero of the experiment. Take pictures with a microscope attached to a digital camera and annotate the exposure and scale used. To ensure easy and reliable measurements, take pictures with good contrast to distinguish the mineralized part.
17. Add 1 mL of culture medium to each well. If any treatment is intended on the bones (doxycycline, tamoxifen, growth factors, etc.), it should be added now.
18. To observe the effect of growth inhibition in the culture conditions, treat the left tibias with retinoic acid (RA, 500 nM), while incubating the right tibias with an equivalent volume of vehicle (dimethyl sulfoxide [DMSO], final concentration 0.1%) as a control.
19. Leave the bones to grow for two days or more in a cell culture incubator under standard cell culture conditions (at 37 °C in a 5% CO₂ incubator).
20. To assess proliferation, add 5-ethynyl-2’-deoxyuridine (EdU) or 5-bromo-2’-deoxyuridine (BrdU) to the medium at a final concentration of 10 µM 1−2 h before fixation.
    NOTE: The stock concentration of EdU is 20 mM.
    CAUTION: EdU and BrdU are thymidine analogues and can be toxic and mutagenic.
21. Thaw 4% paraformaldehyde (PFA) and fix the bones by immersion in PFA in individual 2 mL tubes.
    CAUTION: PFA is toxic and designated as a probable human carcinogen. Avoid breathing paraformaldehyde powder and vapors. EdU and BrdU are thymidine analogues and can be toxic and mutagenic.
22. After a brief 10 min fixation in PFA at room temperature, transfer bones to PBS for picture acquisition at final timepoint. Then place bones back into PFA for overnight fixing at 4 °C.
23. After fixation bones can be processed for desired downstream applications.

3. Measurement and Analysis of the Full Length of the Bone and of the Mineralized Region

1. Use an image editing software to measure the length of the bones, taking into account the scale of the image. Measure both total length of the bone and the mineralized region. Start the measurements from the first dark cells at one end until the last ones at the other end.
   NOTE: The mineralized region is characterized by the darker color and is easily distinguished from the cartilage.
2. To calculate the growth rate, defined as the average increase in length per day, divide the difference between the final length of the bone and the initial one by the number of days in culture.

Results

Bone culture can be performed starting from different stages. In Figure 1A-D, a comparison between cultured tibia and freshly extracted ones at equivalent stages is shown. The first observation is that up to two days of culture the size achieved is comparable to the in vivo bone growth for both cartilage and mineralized bone (Figure 1A,B,D). Longer culture periods lead to bigger differences betwe...

Discussion

Bone ex vivo culture methods have been used for some time to assess the biology of bone growth²⁸, but have been seldom applied to murine long bones. With the development of imaging techniques, ex vivo bone culture offers an attractive way to study bone growth in real time in a setting closely resembling the in vivo conditions. In this scenario, it is important to define the conditions in which the growth of long bones is comparable to their growth in vivo.

In the presen...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-Ethynyl-2'-deoxyuridine	Santa Cruz	CAS 61135-33-9
5-Bromo-2′-deoxyuridine	Sigma	B5002
50 mL Conical Centrifuge Tubes	Falcon	352070
60 mm TC-treated Center Well Organ Culture Dish, 20/Pack, 500/Case, Sterile	Falcon	353037
Adobe Photoshop	Adobe	CS4
Ascorbic acid	Sigma	A92902
Base unit for the scope	Zeiss	435425-9100-000
Betaglycerophosphate	Sigma	G9422
Binocular scope	Zeiss	STEMI-2000
Bovine Serum Albumin (BSA) fraction v	Roche/Sigma	10735086001
DigiRetina 500 camera	Aunet
Dissection kit	Cumper Robbins	PFS00034
DMEM	Gibco	11960044
DMSO	Sigma	D8418
Eppendorf 2 mL tubes	Eppendorf	0030120094
Ethanol 96%	Merk	159010
Forceps Dumont#5 Inox08	Fine Science Tools	T05811
Heracell 150 CO2 incubator	Thermo Fisher	51026282
Minimum Essential Medium Eagle	Sigma	M2279
Multiwell 24 well	Falcon	353047
Paraformaldehyde	Sigma	158127
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140-122
Plastic pipettes 1 mL Sterile Individually wrapped	Thermo	273
Syringe filter 0.2 um	Life Sciences	PN4612
Terumo syringe 20 mL	Terumo	DVR-5174
Tretinoin (retinoic acid)	Sigma	PHR1187-3X
Trinocular scope	Aunet	AZS400T