We are grateful to the Biotechnology and Biological Sciences Research Council (BBSRC) for funding DH (BB/J01446X/1), CF &amp; VEM (Institute Strategic Programme Grant Funding BB/J004316/1). We also acknowledge Arthritis Research UK for funding to KAS (20413). We thank Miss Elaine Seawright for her assistance with the experiments detailed, Dr. Neil Mackenzie for his assistance with the lentiviral techniques, and Mr Darren Smith and Mr Alex Robertson (Roslin Institute BRF) for their assistance with the animal studies.

Procedures involving animal subjects were conducted in compliance with the U.K. Animals (Scientific Procedures) Act 1986 and animals were maintained in accordance with the Home Office published Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes.
NOTE: Experiments carried out using wild-type C57Bl/6J mice are well established and described below. Embryonic metatarsals from different mouse strains may similarly be cultured in vitro, although their growth and mineralization rates may differ to those detailed herein.
1. Dissection Conditions and Preparation of Instruments
<ol>
	<li>Perform all media preparation, tissues dissections and culture work in a laminar flow hood to ensure the sterility of media and embryonic rudiments.</li>
	<li>Allow culture and dissection media to equilibrate for at least 1 hr in a 37 &#176;C, 5% CO2 incubator prior to use.</li>
	<li>Sterilize dissection scissors, Dumont #5 and Dumont #4 tweezers alongside a pair of superFine Vannas micro scissors (8 cm straight, 3 mm blades) by autoclaving. After autoclaving, immerse the dissection equipment in 70% ethanol prior to use.</li>
</ol>
2. Preparation of Dissection and Culture Media
<ol>
	<li>Prepare dissection medium
	<ol>
		<li>Dilute &#945;-Minimum Essential Media (without nucleosides, &#945;MEM) in phosphate buffered saline (PBS) (1:13) and resuspend bovine serum albumin (BSA) at 2 mg/ml before filter sterilizing through a 0.22 &#181;m, 33 mm diameter syringe filter. 
		NOTE: Dissection medium can be made, filter sterilized and stored in aliquots at -20 &#176;C indefinitely.</li>
	</ol>
	</li>
	<li>Prepare culture medium
	<ol>
		<li>Culture embryonic metatarsals in 300 &#181;l of culture medium (in each well of a 24-well culture plate). Prepare the culture medium by adding 0.2% w/v BSA; 5 &#181;g/ml L-ascorbic acid phosphate; 1 mM &#946;-glycerophosphate (&#946;GP; optional &#8211; see results section); 0.05 mg/ml gentamicin and 1.25 &#181;g Amphotericin B to &#945;MEM and filter sterilize through a 0.22 &#181;m, 33 mm diameter syringe filter.</li>
	</ol>
	</li>
</ol>
3. Murine Embryonic Metatarsal Dissection and Culture
<ol>
	<li>Cull the pregnant mouse, harboring 15-day-old embryos, by cervical dislocation in accordance with home office guidelines in the U.K.</li>
	<li>Place the animal supine and disinfect skin by spraying with 70% ethanol. Using dissection scissors make a large incision in the midline, penetrating both the skin and the peritoneum to expose the abdominal cavity.</li>
	<li>Locate the two uterine horns of the animal in the dorsal region of the body cavity. Remove the uterine horns by firstly freeing each uterine from the mesometrium by careful incisions with dissection scissors and then finally cutting the uterine horns free at their base. Place uterine horns into dissection medium.</li>
	<li>Separate each embryo by cutting between implantation sites along the uterine horn and remove the embryos from their individual sacs using forceps. Cull embryos by decapitation in accordance with home office guidelines in the U.K.</li>
	<li>After disinfection of the skin by spraying with 70% ethanol, use Vannas micro scissors to remove the hind limbs from the embryos by incising around the proximal femur. This ensures as much of the hind limb as possible is removed. Place the hind limbs into a petri dish submerged in dissection medium prior to metatarsal dissection.</li>
	<li>Under a dissecting microscope, begin to remove the skin surrounding the embryonic foot, by pinching and pulling it off using the #5 tweezers whilst holding the hind limb steady using the #4 tweezers. This is most effectively performed by grasping the skin at around the level of the tibia and pulling towards the phalanges. The use of scissors at this stage to cut the very thin skin is not required.</li>
	<li>Carefully hold the tarsals of the embryonic foot with #4 tweezers and remove the first and fifth metatarsals by pinching off near the tarsals using #5 tweezers and discard.</li>
	<li>With the foot held in the same position, use the #5 tweezers to disrupt the connective tissues between the three remaining phalanges and metatarsals. Insert the tip of #5 tweezers at the joint between the phalanges and metatarsals to remove the phalanges from the metatarsals.</li>
	<li>Finally, use #5 tweezers to disrupt the connective tissues between the tarsals and metatarsals. Again place the tip of #5 tweezers in the joint space between the tarsals and metatarsals and gently free the metatarsals from the tarsals.</li>
	<li>Gently pick up the now free metatarsals with #4 tweezers and place in fresh dissection medium. 
	NOTE: Careful dissection should provide 6 metatarsals from each embryo.</li>
	<li>When all required metatarsals have been dissected, carefully place metatarsals individually into the pre-warmed 24-well plates containing 300 &#181;l of culture media. Culture metatarsals at 37 &#176;C in a 5 % CO2 incubator for up to 14 days. 
	NOTE: Do not change the media of E15 cultures until at least day 5 of culture as this affects their mineralization capability 26. The reasons for this are unclear but linear growth and matrix mineralization may be dependent upon stimulation by growth factors released from the metatarsals.</li>
</ol>
4. Analysis and Manipulation of Metatarsal Cultures
<ol>
	<li>On the day of dissection (day 0) make initial longitudinal measurements using a microscope with a digital camera attached and image analysis software. Measure the length of the central mineralization zone when it appears after a few days of culture.</li>
	<li>Periodically, as required, make further measurements (usually every 2nd day) until the final day of culture at which point metatarsal bones are processed dependent upon required outcomes (discussed in introduction).</li>
	<li>Supplement medium of metatarsal organ cultures with various exogenous factors e.g. growth factors, hormones and pharmacological agents from day 0 of culture 22,24. In all studies of this kind, control untreated metatarsals are required. 
	NOTE: Although it is advisable that the control bones are the equivalent rudiment from the contralateral leg, there have not been any differences in linear growth or mineralization potential of cultured E15 2nd, 3rd and 4th metatarsals (see below, Figure 2).</li>
</ol>

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The authors have nothing to disclose.

The culture of murine embryonic metatarsals provides a highly physiological model of endochondral growth and mineralization. Early studies have validated that E15 mouse metatarsals undergo a normal pattern of skeletal growth and differentiation. The cartilage (chondrocytes) and bone (osteoblasts and osteoclasts) cells and their respective collagenous matrices are indistinguishable from those observed in vivo. However, there are some recognized limitations of this model. The speed of osteoclast differentiation is impaired as is bone growth (but not cartilage differentiation). Bone growth is approximately 50% slower than that observed in vivo and may be due to deficiencies in systemic factors which influence the production of local factors (e.g., IGF-1, BMPs, etc.) 31. Also, it is well recognized that bone is sensitive to its mechanical environment and this is also the case for cultured metatarsals, which respond to changes in mechanical loading 32,33. Therefore, in unloaded situations, as described in this protocol, mineralization and mineral resorption may be compromised. Nevertheless, the metatarsal model offers the ability to directly measure linear bone growth which is not possible via 2D and 3D in vitro culture systems.
We have provided a comprehensive description of the protocol involved in the dissection and culture of E15 murine metatarsals and indeed, for the first time, confirm the validity of pooling the 2nd, 3rd and 4th metatarsals for experiments.
Metatarsals from E17/E18 are commonly used to investigate the mechanisms of bone growth owing to their extraordinary potential to grow ex vivo for long periods of time (i.e. beyond 14 days in culture). They are a well-established model to investigate the roles of growth factors on embryonic longitudinal bone growth. Additionally, the dissection and culture of postnatal metatarsals is commonly employed to delineate the mechanisms surrounding postnatal bone growth as it is understood that postnatal bone growth and fetal bone growth are regulated differently 21. The bone growth observed in cultured postnatal metatarsals is however significantly less than that observed in embryonic rudiments 25,34, limiting their potential in long term studies and highlighting the more systemic influence on bone growth at this postnatal stage. Indeed, 3 day old postnatal metatarsals from mice are typically the latest stage that can be used to obtain measurable growth 34.
Here we describe our protocol for the isolation of embryonic metatarsal bones at E15. The absence of hydroxyapaptite mineral in E15 metatarsal bones means that they provide an unrivaled model to investigate not only the mechanisms surrounding longitudinal bone growth, but also the initiation of skeletal mineralization. The mineralized matrix of the terminal hypertrophic chondrocyte zone provides a scaffold for invading osteoblasts to lay down a bone specific matrix (osteoid) which is subsequently mineralized, and as such this initial mineralization is vital for successful and functional bone formation 35. Indeed a number of recent reports utilizing E15 metatarsals have confirmed their unique ability for the investigation of the mineralization process 28,36,37. In addition, researchers have described the use of E15 metatarsals as a model of angiogenesis 38, highlighting the potential of embryonic metatarsals as model outside the bone growth/mineralization setting.
The protocol we describe requires only standard laboratory equipment including a laminar flow hood, a dissecting microscope for performing the dissection, and a CO2 incubator for the culture of excised rudiments. Furthermore, a very basic culture medium containing &#945;MEM, BSA, antibiotics, antimycotics and L-ascorbic acid (a co-factor in the synthesis of hydroxyproline and hydroxylysine, two essential amino acids for the production of collagen 39) avoids the use of undefined additives, such as animal sera. Traditionally, investigators have added &#946;GP to media used to culture embryonic metatarsals but as revealed in our results, the use of an additional phosphate source is not required for successful mineralization in this culture system. This is an important finding in our pursuit of understanding the mechanisms underpinning ECM mineralization.
Metatarsals have previously been infected with adenoviruses containing dominant-negative forms of Smad2 to explore the role of transforming growth factor &#946; in the regulation of long bone development 40. Here we also reveal the successful transfection of wild-type E15 metatarsal bones with GFP virus particles, indicating that lentiviral techniques could easily be adopted to manipulate gene expression in metatarsal cultures. Similarly, the metatarsal organ culture system is an excellent model in which to compare the growth of bones from genetically altered mice to better understand the role of a particular gene in the bone growth process. Our previous studies have utilized the metatarsal organ culture system to determine the role of suppressor of cytokine signalling-2 (SOCS2) in endochondral bone growth. Using metatarsal bones from mice deficient in SOCS2, we have shown that growth hormone is able to simulate their longitudinal growth independent of insulin like growth factor (IGF-1), unlike wild-type metatarsal bones which do not respond to growth hormone treatment 34,41. This highlights the extent to which metatarsal cultures can be manipulated to examine the effects of different genes and/or exogenous factors on endochondral bone growth.
In summary, we have detailed a method for the successful extraction and culture of embryonic metatarsal bones which may be manipulated and examined using a variety of analyses. This ex vivo model maintains cell-cell and cell-matrix interactions, as well as contains chondrocytes in different phases of chondrogenesis, therefore providing a more physiological model than cells in monolayer or 3D culture. Metatarsal organ cultures are therefore a unique model for investigating the molecular mechanisms responsible for endochondral ossification, and are essential to further our understanding of both bone physiology and pathophysiology

The skeleton is a highly intricate, dynamic and complex organ that has a range of functions spanning from locomotion, to ion homeostasis. Comprised of bone and cartilage, the skeleton consists of functionally distinct cell populations which operate to maintain it&#39;s structural, biochemical and mechanical integrity 1. One of the primary functions of the skeleton is its role in development and growth. These processes require the orchestration of multiple cellular populations which are regulated through tight endocrine and genetic control.
With the exception of the flat bones e.g. scapula, cranium and sternum, the mammalian skeleton is formed through the process known as endochondral ossification. This process, regulated both molecularly and spatially, begins with the condensation of mesenchymal cells derived primarily from the mesoderm 2. Under the influence of the transcription factor SOX9, cells in the interior of the condensations differentiate into chondrocytes, expressing and secreting cartilage specific extracellular matrix (ECM) components such as type II collagen and aggrecan. Cells at the margin of the condensation, express type I collagen and form the perichondrium, delineating the prospective bone 3. Later, the osteoprogenitors of the perichondrium differentiate into osteoblasts, directed by the expression of the transcription factors, Runx2 and osterix 4. This leads to a predominance of osteoblasts and this layer is renamed the periosteum which forms a mineralized bony collar around the mid-section of the bone. Vascular penetration of the periosteum and invasion of the cartilaginous scaffold occurs bringing with it, haematopoetically derived bone resorbing cells, osteoclasts, which resorb the chondrocyte remnants and much of the cartilaginous matrix 5. The spaces formed are filled by osteoprogenitors giving rise to the primary ossification center which progressively occupies the core of the diaphysis and merges with the periosteum. Other cells give rise to the bone marrow. Around birth, blood vessels and osteoprogenitors penetrate the cartilage rich matrix at either side (epiphysis) of the developing diaphysis to form the secondary ossification center. Situated between the epiphysis and diaphysis at either end of the long bones is a band of cartilage - the epiphyseal growth plate - which is responsible for coordinating linear growth of all long bones 6.
Chondrocytes within the growth plate initially proliferate and subsequently progress through morphologically distinct zones, coordinated by sequential expression of transcription and growth factors. The culmination of this sequence of events is the exit from the cell cycle and hypertrophy of chondrocytes at the center of this cartilage precursor. Hypertrophic chondrocytes strongly express type X collagen and matrix metalloproteinase-13 (MMP13) and their considerable volume enlargement in the direction of longitudinal growth provides the greatest contribution to bone growth in mammals 7,8. Furthermore, hypertrophic chondrocytes mineralize their surrounding ECM through the release of matrix vesicles, membrane bounded structures specialized for the production of amorphous calcium phosphate through their inclusion of phosphatases (tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1) and calcium channeling proteins (annexins) 9-11. This calcified cartilage is invaded by blood vessels from the underlying marrow resulting in osteoclast recruitment and matrix resorption. This is followed by osteoblast migration and alignment to the surface of the calcified cartilage remnants where they lay down a layer of osteoid which is rapidly mineralized. The woven bone of the primary spongiosa is later remodeled to lamellar bone of the secondary spongiosa 12. Epiphyseal fusion results in the cessation of endochondral ossification 13.
Endochondral ossification is under tight regulation by many endocrine and paracrine hormones and growth factors so as to prevent disturbed development and/or longitudinal bone growth 14. A better understanding of the molecular and cellular mechanisms underpinning these processes is therefore imperative in our pursuit of clinical advances towards human benefit. Previous research into the similarities and differences between growth plates of different ages, locations and species have greatly improved our understanding of the physiological mechanisms surrounding growth plate dynamics 15,16. However, the study of isolated chondrocytes is difficult, as removal of chondrocytes from their environment can lead to dedifferentiation, accompanied by loss of expression of key markers such as Col2a117.
The mouse metatarsal organ explant culture, pioneered by Prof. Elisabeth Burger and colleagues in the Netherlands, is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that seen in vivo 18. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in culture 19-21. Moreover, this model allows for direct examination of linear bone growth which is not possible in primary chondrocyte cultures. It also allows for the separation of systemic and local factors therefore permitting the specific analysis of the local effects on the growth plate.
The culture of metatarsals allows for the investigation of numerous parameters. Daily measurements of total length and of the mineralized regions of the rudiments can be performed with standard phase contrast microscopy and image analysis software. Histological, in situ hybridization and immunohistological staining may be readily performed on metatarsal sections, which allows for the spatial resolution of both cellular and molecular entities, a major advantage over traditional cell culture methods. The immunohistochemical approach includes the detection and quantification of 5-bromo-2&#39;-deoxyuridine (BrdU) uptake by proliferating chondrocytes 22. Additionally, further processing of metatarsal tissues may be performed to allow for downstream analysis of mRNA expression (RT-qPCR), protein and intracellular signaling analysis (Western blotting) or lipid composition (mass spectrometry). Most studies to date use cultured embryonic metatarsals rather than metatarsals from post-natal animals. Whilst both models can be informative, the study of embryonic bones has several advantages: they grow faster and can be exploited to study the initiation and progression of the mineralization process 23-25.
This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of embryonic day 15 (E15) murine embryos. Furthermore, the growth and mineralization of anatomically distinct metatarsals is shown.

<table><tbody><tr><td>Dissection scissors</td><td>World Precision Instruments</td><td>14393</td><td>Autoclave sterilise before use</td></tr><tr><td>Dumont #5 tweezers</td><td>World Precision Instruments</td><td>500342</td><td>Autoclave sterilise before use</td></tr><tr><td>Dumont #4 tweezers</td><td>World Precision Instruments</td><td>500339</td><td>Autoclave sterilise before use</td></tr><tr><td>SuperFine Vannas scissors</td><td>World Precision Instruments</td><td>501778</td><td></td></tr><tr><td>Absolute ethanol</td><td>Fisher Scientific</td><td>E/0650DF/17</td><td>Dilute to 70% v/v in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>&amp;alpha;-MEM without nucleosides</td><td>Thermo Fischer Scientific&amp;nbsp;</td><td>22561021</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A3059</td><td></td></tr><tr><td>Syringe filters (0.22 &amp;mu;m) 33 mm diameter</td><td>Merck Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>Phosphocholine chloride calcium salt tetrahydrate</td><td>Sigma Aldrich</td><td>P0378</td><td>Add directly to the media prior to experimentation</td></tr><tr><td>L-ascorbic acid-2-phosphate</td><td>Sigma Aldrich</td><td>A8960</td><td>Make up stock soution at 5mg/ml and aliquot at -20 &amp;deg;C</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C5080</td><td>Make up stock soution at 150mM and aliquot at -20 &amp;deg;C</td></tr><tr><td>&amp;beta;-glycerophosphate disodium salt hydrate</td><td>Sigma Aldrich</td><td>G9422</td><td>Make up stock soution at 1mM and aliquot at -20 &amp;deg;C</td></tr><tr><td>Gentamicin</td><td>Thermo Fischer Scientific&amp;nbsp;</td><td>15710049</td><td></td></tr><tr><td>Amphotericin B</td><td>Thermo Fischer Scientific&amp;nbsp;</td><td>15290018</td><td></td></tr><tr><td>Nunc cell-culture treated 24-well plates</td><td>Thermo Fischer Scientific&amp;nbsp;</td><td>142475</td><td></td></tr><tr><td>Polybrene</td><td>Sigma Aldrich</td><td>H9268</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fischer Scientific&amp;nbsp;</td><td>D3571</td><td></td></tr></tbody></table>

culture of murine embryonic metatarsals: a physiological model of endochondral ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

The aim of this method was to isolate embryonic metatarsals and culture them for examination of longitudinal bone growth and ECM mineralization. Using our protocol described herein, embryonic metatarsal bones were successfully dissected, as visualized by light microscopy. Measurements of metatarsal bones, conducted as indicated in Figure 1, showed their capacity to grow in longitudinal length and mineralize their ECM (Figures 2 and 3). Mineralization of the matrix is first noted in the mid-diaphysis of the bone rudiment and is easily observed by light microscopy. No staining is required although the uptake of calcein may offer enhanced visualization of newly formed mineral.
Studies utilizing embryonic metatarsals to date 22,27,28 have pooled the dissected 2nd, 3rd and 4th (central three) metatarsals, assuming in vitro growth and mineralization to be comparable. In vivo developmental of murine metatarsals, however, reveals that the 3rd and 4th digits have evidence of mineralization prior to all other metatarsals and phalanges 29. Assessment of the growth and mineralization potential of individually isolated and cultured E15 2nd, 3rd and 4th metatarsals revealed no significant differences in the appearance or extent of mineralization after 7 days of culture (Figure 2). No difference in the percentage increase from baseline was found during the culture period. As no differences in mineralization potential was noted the standard protocol of pooling the 2nd 3rd and 4th metatarsals appears valid for future studies.
Traditionally, investigators have added &#946;GP to media used to culture embryonic metatarsals to promote ECM mineralization. However, in cell culture studies, it has been shown that if TNAP enzyme is added to osteogenic media containing &#946;GP, ectopic hydroxyapatite mineral deposition still occurs even in the absence of cells 30. To examine the effects of various mineralization substrates and agonists on metatarsal mineralization capability, we cultured E15 metatarsals in culture media containing a range of supplements, and images and length measurements were recorded daily. Results indicated that E15 metatarsal bones still grow and mineralize their ECM in the absence of &#946;GP. E15 metatarsal bones cultured in ascorbic acid only media showed comparable increases in length and mineralization as metatarsals cultured in the presence of &#946;GP (Figure 3).
We have also shown that lentivirus may be used to transfect exogenous DNA into cultured metatarsals. Here we successfully transfected metatarsal bones on day 0 of culture (i.e. day of dissection) with 2 x 106 green fluorescent protein (GFP) virus particles per metatarsal. To enable virus transduction, polybrene was simultaneously added to cultures at 1:500. Cultures were left for 7 days, with no media changes as required for E15 metatarsal mineralization to occur, before being incubated with 4&#39;,6-diamidino-2-phenylindole (DAPI) stain for 5 min and then directly visualized using a confocal microscope (Figure 4). Our proof of concept experiments clearly show effective transduction. However it would be prudent to examine sections throughout the entirety of the metatarsal bone to assess their transfection and effective gene manipulation.
<img alt="Figure 1" src="/files/ftp_upload/54978/54978fig1.jpg" /> 
Figure 1: Standard Methodology used to Measure the Length and Mineralization Zone of Embryonic Metatarsals. (A) Total length measurements of E15 metatarsals on day 0 of culture (day of dissection) taken through the center of the metatarsal. (B) Measurements of total longitudinal length of a metatarsal after seven days in culture. Note the slight curvature in the metatarsal. (C) Measurement of the mineralization zone length after seven days in culture. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54978/54978fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54978/54978fig2.jpg" /> 
Figure 2: Growth and Mineralization Capacity of Anatomically Distinct E15 Metatarsals. Imaging and measurement of the 2nd, 3rd and 4th metatarsals from the hind limb of E15 mice was performed. (A) Serial images of the 2nd, 3rd and 4th metatarsals throughout the culture period. (B) Percentage increase in length from day 0 at each day of culture. (C) Mineral length of the 2nd, 3rd and 4th metatarsals over the culture period expressed as a percentage of the total metatarsal length. Data in are represented as mean &#177; standard error of the mean (S.E.M), (n = 6). (D) C57Bl/6J metatarsal lengths at each day of culture. Data in are represented as mean &#177; standard deviation (n = 6). Black bar = 1 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54978/54978fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54978/54978fig3.jpg" /> 
Figure 3: Growth and Mineralization Capacity of Wild-type E15 Metatarsals Cultured in various Osteogenic Media. (A) Serial images of metatarsals cultured in ascorbic acid only, 1 mM &#946;GP, 3 mM phosphocholine, 1.5 mM calcium chloride or 3 mM calcium chloride containing media. (B) Percentage increase in length from day 0 of metatarsals cultured with the various supplements. (C) Mineral length of metatarsals cultured in various media expressed as a percentage of the total metatarsal length. Data are represented as mean &#177; standard error of the mean (S.E.M), (n = 6). Black bar = 1 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54978/54978fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54978/54978fig4.jpg" /> 
Figure 4: Transfection of E15 Metatarsals with GFP Virus Particles. (A) E15 metatarsal bones were transfected with GFP virus particles for proof of concept. (B) Bones were stained with DAPI for DNA visualization. (C) Dual images indicated successful transfection of GFP virus throughout the entirety of the metatarsal bones. White bars = 0.5 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54978/54978fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification at JoVE.com

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

culture-murine-embryonic-metatarsals-physiological-model-endochondral

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.5K Views.  The University of Edinburgh. We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development. 

Video: Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Culturing and Measuring Fetal and Newborn Murine Long Bones

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.

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.

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Medicine

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Precise Growth Plate Injury Study Using Trilineage Fluorescent Mice

Tricolor Transgenic Murine Model for Studying Growth Plate Injury

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones are overloaded.&#160;Better treatments for severely fractured growth plates are needed because the response to injury is a bony bridge that prematurely fuses the growth plate, leading to stunted and/or crooked limbs. Murine models of growth plate injury are advantageous for mechanistic studies, but&#160;are challenging because it is difficult to visualize and precisely injure the small growth plates in young mice.&#160;We describe here an improved growth plate injury model using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X.
These mice show native fluorescence associated with the three primary substrata of the growth plate. A growth plate injury similar to a Salter-Harris Type II injury is created reproducibly with a bur using the hypertrophic section of the growth plate as a reference during live imaging under fluorescence stereo microscopy guidance. Frozen histology analysis of the native fluorescence simplifies assessing the cellular response to injury. This methodology represents a substantial leap in growth plate injury research, providing a detailed and reproducible method for investigating pathology and evaluating new therapeutic strategies.

This protocol describes an improved mouse model for adolescent bone growth plate injuries. Using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X, the primary matrices associated with three different substrata of the growth plate, injury placement is guided by native fluorescence under the microscope.

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

Biology

Artificial Cartilage Implants for Bone Regeneration via Endochondral Ossification

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

Author Spotlight: Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration 

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it ...

Improved Bone Marrow Injury Model for Intramembranous Bone Repair Studies

Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage template, the process of intramembranous ossification involves the direct conversion of skeletal stem and progenitor cells (SSPCs) into bone-forming osteoblasts. There are limited surgical methods to model this process in experimental mice. Here, we have improved upon a bone marrow injury model in mice to facilitate the study of bone repair via intramembranous ossification and to assess postnatal regulators of osteogenesis. This method is highly reproducible and user-friendly, and it allows temporal assessment of new bone formation in a short period (3-7 days post-injury) using micro-computed tomography (&#181;CT) and frozen section histology. Furthermore, the contributions of SSPCs and mature osteoblasts can be readily assessed using a combination of fluorescent reporter mice and this intramembranous bone marrow injury model. In clinical contexts, intramembranous bone formation is relevant for healing critical size defects, stabilized fractures, cortical defects, trauma from tumor resections, and joint replacements.

The murine bone marrow injury model is a useful tool for studying postnatal osteogenesis through intramembranous ossification. This simplified surgical protocol describes the bone marrow ablation procedure for downstream assessment of new bone formation and cellular responses following injury.

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

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