The authors thank Mario Nomura, M.D. and Rodolfo D&#237;az for their help with the histology, and Pierrick Fournier, Ph.D. for his valuable comments to improve the quality of the paper.

All mice used in these assays were obtained from BALB/c mice strains, using male and female mice indiscriminately. Previous culture experiments have also been performed using other strains, such as FVB, Swiss mice, CD-1, and CsA mice11,12,14. All mice were housed according to National Institutes of Health (NIH) guidelines, Appendix Q. Procedures involving animal subjects have been approved by the Institutional Animals Care and Use Committee (IACUC) at the Center for Scientific Research and Higher Education at Ensenada (CICESE).
1. Calvarial dissection
<ol>
	<li>Sterilize dissection instruments (e.g., 1 x 2 teeth tissue forceps, straight surgical scissors, dissecting scissors, fine-tipped tweezers, fine curved tip dissecting forceps, dissecting forceps, scalpel). Sterile distilled water can be used to clean the surgical tools between each dissection, and sterile 1x PBS can be used for cleaning each hemi-calvaria.</li>
	<li>Place the sterile dissection instruments, water, 1x PBS, and required materials to carry out the procedure (e.g., micropipettes, precipitate glasses, sterile Petri dishes) in a laminar flow hood. 
	NOTE: Carry out the entire procedure under sterile conditions.</li>
	<li>Add 12 mL of sterile 1x PBS into two 10 cm Petri dishes.</li>
	<li>Select the pups and place them near the hood. 
	NOTE: Dissect the calvarias from 5-7 day old pups.</li>
	<li>Pick and hold the mouse carefully using dissecting forceps.</li>
	<li>Decapitate the mouse using dissecting scissors and place the head in a Petri dish with PBS. 
	NOTE: Decapitation is not suitable for older mice. If the experiment must be done with older mice, the method of euthanasia should be modified according to the animal experimentation rules.</li>
	<li>Hold the head firmly by the nose area with 1 x 2 teeth tissue forceps and remove the skin over the skull until the calvaria is visible.</li>
	<li>Identify the sutures (i.e., sagittal, coronal, and lambdoid) of the skull on the exposed calvaria.</li>
	<li>Penetrate with the tip of the microscissors approximately 2 mm behind the lambdoid suture and make a straight cut alongside it.</li>
	<li>Insert the tip of the microscissors on the rear side of the skull. Make a straight cut along the distal side of the lambdoid suture towards the coronal suture. Proceed similarly with the other lambdoid suture.</li>
	<li>Make another cut (at a 45&#176; angle) to connect the end of the cut made with the cut of the anterior fontanel.</li>
	<li>Repeat steps 1.10 and 1.11 with the other lambdoid suture. 
	NOTE: It is important to identify the sutures to make appropriate cuts and to be able to perform adequate embedding and data analysis.</li>
	<li>Use fine-tip forceps to remove the calvaria and place it in a Petri dish. With a scalpel, make a straight cut from the posterior fontanel along the sagittal suture through the coronal suture and ending in the anterior fontanel to obtain two hemi-calvarias.</li>
	<li>Pick up each of the hemi-calvarias with the fine-tipped tweezers and place them in a new Petri dish containing PBS.</li>
</ol>
2. Calvaria culture
<ol>
	<li>Add 1 mL of high-glucose DMEM medium supplemented with 0.1% bovine serum albumin (BSA) and 1% antibiotic/antimycotic (i.e., penicillin and streptomycin) in the wells of a 24 well plate. With fine-tip forceps, pick up each hemi-calvaria and place it in a well. 
	NOTE: Put the hemi-calvarias concave side down.</li>
	<li>Incubate the hemi-calvarias for 24 h at 37 &#176;C with 5% CO2.</li>
	<li>Remove the culture medium from each well and add 1 mL of fresh media containing the compound to test or conditioned media from other cells. 
	NOTE: Use at least three hemi-calvarias for each treatment group and use negative and positive controls.</li>
	<li>Incubate the hemi-calvarias for 7 days, changing the media every 2-3 days.</li>
</ol>
3. Culture with cancer cells
<ol>
	<li>Select a Petri dish with cancer cells at 80-90% confluence and trypsinize them. To trypsinize, wash the cancer cells 1x using PBS (5 mL per 75 cm2 flask) followed by incubation in an HBSS solution containing 0.05% trypsin and 0.53 mM EDTA (2 mL per 75 cm2 flask).</li>
	<li>Transfer the cell suspension into a 15 mL conical tube and centrifuge at 800 x g for 5 min at room temperature (RT).</li>
	<li>Remove and discard the supernatant.</li>
	<li>Resuspend the pellet in 2 mL of DMEM containing 2% fetal bovine serum (FBS) and 1% antibiotic/antimycotic. Count live cells.</li>
	<li>Assign hemi-calvarias to each study group (i.e., the negative control and coculture group for RNA or histology analysis).</li>
	<li>Remove the culture media from the hemi-calvarias and add 1 mL of DMEM containing 2% FBS and 1% antibiotic/antimycotic supplemented or not supplemented with cancer cells. 
	NOTE: The amount of cells to add can change depending on the cell line tested. With cancer cells like MDA-MB-231 or PC-3, 500,000 cells per well work appropriately.</li>
	<li>Incubate for 24 h at 37 &#176;C with 5% CO2. Pass each hemi-calvaria to a new well to retain only cancer cells that adhered to the bone tissue.</li>
	<li>Incubate for 7 days at 37 &#176;C with 5% CO2 and change the media every 2-3 days.</li>
</ol>
4. Fixation
<ol>
	<li>Cut squares of tissue paper to wrap the hemi-calvarias. 
	NOTE: Biopsy foam pads or steel capsules for small biopsies can also be used.</li>
	<li>Use straight fine-tip forceps to pick up each hemi-calvaria from the sagittal suture, place on a tissue paper, and wrap.</li>
	<li>Place the wrapped hemi-calvaria inside a labeled embedding cassette.</li>
	<li>Place the cassette into a container with 10% phosphate-buffered formalin.</li>
	<li>Fix for 24 h at 4 &#176;C.</li>
</ol>
5. Decalcification
<ol>
	<li>Remove the formalin, add 1x PBS, and agitate the tissues for 30 min to rinse the hemi-calvarias.</li>
	<li>Remove the PBS and add 10% EDTA (pH = 8, 0.34 M). 
	NOTE: Verify that the solution covers the tissues completely.</li>
	<li>Decalcify the tissues for 48 h at 4 &#176;C.</li>
	<li>Discard the EDTA solution and add 1x PBS to rinse the tissue.</li>
	<li>Store the hemi-calvarias in 70% ethanol until the histology processing.</li>
</ol>
6. Tissue processing
<ol>
	<li>Dehydrate the tissues with rounds of 96% ethanol, 60 min (3x), followed by 100% ethanol, 60 min (3x).</li>
	<li>Replace the ethanol by 100% xylene, 60 min (3x).</li>
	<li>Incubate the cassettes in paraffin wax, 60 min (2x).</li>
</ol>
7. Embedding
<ol>
	<li>Open the cassettes, carefully remove the tissue paper, and unwrap the calvaria.</li>
	<li>Pick up each calvaria carefully and stack all the hemi-calvarias from the same group in the same orientation.</li>
	<li>Put some paraffin in a mold.</li>
	<li>Pick up all hemi-calvarias with the forceps and place them inside the mold with the sagittal suture down towards the base of the mold. 
	NOTE: Orientation of the hemi-calvarias in the mold during inclusion is crucial to obtain consistent histological sections and reproducible results because this orientation will allow posterior identification of the front and parietal bones from the calvarias and the coronal suture facilitating the quantitative assessment.</li>
	<li>Release the forceps and verify that the hemi-calvarias stay in place.</li>
	<li>Place the labeled cassette on top of the mold and fill it with more paraffin to cover the hemi-calvarias.</li>
	<li>Move the molds to the cold surface.</li>
</ol>
8. Sectioning
<ol>
	<li>Trim 500-600 &#181;m of the sagittal suture with the microtome.</li>
	<li>Cut 4 &#181;m thick sections, and collect the sections needed.</li>
	<li>Trim another 300 &#181;m.</li>
	<li>Cut and collect another six 4 &#181;m thick sections.</li>
	<li>Trim the block 300 &#181;m further and collect more sections.</li>
	<li>Mount the sections onto glass microscope slides.</li>
	<li>Dry the slides at RT.</li>
</ol>
9. Staining
<ol>
	<li>Immerse the sections in 100% xylene for 3 min.</li>
	<li>Submerge in 100% ethanol for 1 min.</li>
	<li>Merge in 96% ethanol for 1 min.</li>
	<li>Immerse in 80% ethanol for 1 min.</li>
	<li>Submerge in 70% ethanol for 1 min.</li>
	<li>Rinse in water for 3 min.</li>
	<li>Submerge in hematoxylin for 3 min.</li>
	<li>Rinse in water until the section is clear.</li>
	<li>Submerge in a saturated lithium carbonate solution for 10 sec.</li>
	<li>Rinse in water for 3 min.</li>
	<li>Submerge in 96% ethanol for 1 min.</li>
	<li>Submerge in eosin for 3 min.</li>
	<li>Immerse in 96% ethanol for 1 min.</li>
	<li>Submerge in 100% ethanol for 1 min.</li>
	<li>Submerge in 100% xylene for 3 min.</li>
	<li>Add some per mount-mounting medium to the slide and protect it with a coverslip. 
	NOTE: You can complement the hematoxyline and eosin (H&#38;E) staining with tartrate-resistant acid phosphatase (TRAP) staining to evaluate osteoclast and osteolysis.</li>
</ol>
10. Quantitative assessment: defining area for analysis
<ol>
	<li>Examine the sections under low power (i.e., 4x) to identify the orientation and the sutures.</li>
	<li>Define the coronal suture and identify the long bone surface on one side and the short surface on the other.</li>
	<li>Identify the coronal suture under 40x magnification, and move two or three optical fields away from the suture along the long surface. Capture images of this area for analysis.</li>
	<li>Define the old bone area and the new bone area. The eosin Y with orange G stains the old bone darker and the new bone lighter.</li>
	<li>Measure the total bone area of the old and new bone with Image J software using the color threshold tool. Express the results as &#181;m2.</li>
</ol>
11. Data analysis
<ol>
	<li>Analyze the results using statistical software. Significant differences between groups can be determined using appropriate tests (e.g., nonparametric Mann-Whitney U test as is done herel).</li>
</ol>

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	<li>Boyce, B., Coleman, R. E., Abrahamsson, P. A., Hadji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+biology+and+pathology.">Bone biology and pathology.</a> Handbook of Cancer-Related Bone Disease. , 3-21 (2012).</li><li>Clark, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy and Physiology: Understanding the Human Body. 474, (2005).</li><li>Fournier, P. G. J., Ju&#225;rez, P., Guise, T. A., Heymann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-bone+interactions:+there+is+no+place+like+bone.">Tumor-bone interactions: there is no place like bone.</a> Bone Cancer: Primary Bone Cancers and Bone Metastases. , 13-28 (2014).</li><li>Ribatti, D., Mangialardi, G., Vacca, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stephen+Paget+and+the+"seed+and+soil"+theory+of+metastatic+dissemination.">Stephen Paget and the "seed and soil" theory of metastatic dissemination.</a> Clinical and Experimental Medicine. 6 (4), 145-149 (2006).</li><li>Guise, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vicious+cycle+of+bone+metastases.">The vicious cycle of bone metastases.</a> Journal of Musculoskeletal &amp; Neuronal Interactions. 2 (6), 570-572 (2002).</li><li>Mundy, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+bone+metastasis.">Mechanisms of bone metastasis.</a> Cancer. 80 (8), 1546-1556 (1997).</li><li>Mundy, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+to+bone:+causes,+consequences+and+therapeutic+opportunities.">Metastasis to bone: causes, consequences and therapeutic opportunities.</a> Nature Reviews Cancer. 2 (8), 584-593 (2002).</li><li>Chong, S. K. 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The authors declare no competing interests.

Here, we describe the protocol for a calvarial ex vivo model to evaluate bone formation or resorption and to study the interactions of cancer cells with calvarial mouse bone. The critical steps of this technique are the dissection, culture, embedding, and histomorphometrical analysis of the calvarias. During the dissection of the calvarias, it is crucial to cut the hemi-calvarias into a trapezoid, as it will strongly facilitate the orientation during the paraffin inclusion. When studying cancer cell interactions...

Bone is a dynamic connective tissue that has several functions, including supporting the muscles, protecting the internal organs and bone marrow, and storing and releasing calcium and growth factors1,2. To maintain its integrity and proper function, bone tissue is continuously under the process of remodeling. In general terms, a cycle of bone remodeling can be divided into bone resorption and bone formation1. An imbalance between these two phases of bone remodeling can lead to the development of bone pathologies. Also, diseases such as breast cancer often affect bone integrity; approximately more than 70% of patients in advanced stages have or will have bone metastases. When breast cancer cells enter the bones, they affect bone metabolism, resulting in excessive resorption (osteoclastic lesions) and/or formation (osteoblastic lesions)3.
To understand the biology of bone diseases and develop new treatments, it is necessary to understand the mechanisms involved in bone remodeling. In cancer research, it is essential to investigate the bone metastasis process and its relation to the metastatic microenvironment. In 1889, Stephen Paget hypothesized that metastases occur when there is compatibility between the tumor cells and the target tissue, and suggested that the metastatic site depends on the affinity of the tumor for the microenvironment4. In 1997, Mundy and Guise introduced the concept of the &#34;vicious cycle of bone metastases&#34; to explain how tumor cells modify the bone microenvironment to achieve their survival and growth, and how the bone microenvironment promotes their growth by providing calcium and growth factors5,6,7.
To characterize the mechanisms involved in bone remodeling and bone metastasis and to evaluate molecules with possible therapeutic potential, it has been necessary to develop in vitro and in vivo models. However, these models currently present many limitations, such as the simplified representation of the bone microenvironment, and their cost8,9. The culture of bone explants ex vivo has the advantage of maintaining the three-dimensional organization as well as the diversity of bone cells. In addition, experimental conditions can be controlled. The explant models include the culture of metatarsal bones, femoral heads, calvarias, and mandibular or trabecular cores10. The advantages of the ex vivo models have been demonstrated in diverse studies. In 2009, Nordstrand and collaborators reported the establishment of a coculture model based on the interactions between bone and prostate cancer cells11. Also, in 2012, Curtin and collaborators reported the development of a three-dimensional model using ex vivo cocultures12. The purpose of such ex vivo models is to recreate the conditions of the bone microenvironment as accurately as possible to be able to characterize the mechanisms involved in normal or pathological bone remodeling and evaluate the efficacy of new therapeutic agents.
The present protocol is based on the procedures published by Garrett13 and Mohammad et al.14. Neonatal mouse calvaria cultures have been used as an experimental model, as they retain the three-dimensional architecture of the bone under development and bone cells, including cells at all stages of differentiation (i.e., osteoblasts, osteoclasts, osteocytes, stromal cells) that lead to mature osteoclasts and osteoblasts, as well as the mineralized matrix14. The ex vivo model does not represent the pathological process of bone diseases totally. However, effects on bone remodeling or cancer-induced bone osteolysis can be accurately measured.
Briefly, this protocol consists of the following steps: the dissection of calvarias from 5-7 day old mice, calvaria preculture, calvaria culture applications (e.g., culture in the presence of insulin, cancer cells or conditioned medium, and even agents with therapeutic potential, according to the aim of the investigation), bone fixation and calvaria decalcification, tissue processing, histological analysis, and result interpretation.

<table><tbody><tr><td>24 well cell culture</td><td>Corning</td><td>CLS3524</td><td></td></tr><tr><td>24 well non tissue culture</td><td>Falcon</td><td>15705-060</td><td></td></tr><tr><td>2 mL cryovial</td><td>SSI</td><td>2341-S0S</td><td></td></tr><tr><td>Antibiotics-Antimycotic</td><td>Corning</td><td>30-004-CI</td><td></td></tr><tr><td>BSA</td><td>Biowest</td><td>P6154-100GR</td><td></td></tr><tr><td>Centrifugue</td><td>Eppendorf</td><td>22628188</td><td>Centrifuge 5810R</td></tr><tr><td>Coverslips</td><td>Corning</td><td>2935-24X50</td><td></td></tr><tr><td>Cytoseal resin</td><td>Richard Allen</td><td>8310-10</td><td></td></tr><tr><td>DMSO</td><td></td><td>D2650-100ML</td><td></td></tr><tr><td>Dulbecco&#039;s Modification of Eagles Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>Dulbecco&#039;s PBS (10X)</td><td>Corning</td><td>20-031-CV</td><td></td></tr><tr><td>Ebedding Cassettes</td><td>Sigma</td><td>Z672122-500EA</td><td></td></tr><tr><td>EDTA</td><td>Golden</td><td>26400</td><td></td></tr><tr><td>Embedding Workstation</td><td>Thermo Scientific</td><td>A81000001</td><td></td></tr><tr><td>Eosin</td><td>Golden</td><td>60600</td><td></td></tr><tr><td>Ethanol absolute</td><td>JALMEK</td><td>E5325-17P</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Biowest</td><td>BIO-S1650-500</td><td></td></tr><tr><td>Filters</td><td>Corning</td><td>CLS431229</td><td></td></tr><tr><td>Forceps and scissors</td><td>LANCETA HG</td><td>74165</td><td></td></tr><tr><td>Formalin buffered 10%</td><td>Sigma</td><td>HT501320</td><td></td></tr><tr><td>Glass slides 25 x 75 mm</td><td>Premiere</td><td>9105</td><td></td></tr><tr><td>Harris&#039;s Hematoxylin</td><td>Jalmek</td><td>SH025-13</td><td></td></tr><tr><td>High profile blades</td><td>Thermo Scientific</td><td>1001259</td><td></td></tr><tr><td>Histoquinet</td><td>Thermo Scientific</td><td>813150</td><td>STP 120</td></tr><tr><td>Insulin from bovine pancreas</td><td>Sigma</td><td>16634</td><td></td></tr><tr><td>Microscope</td><td>ZEISS</td><td>Axio Scope.A1</td><td></td></tr><tr><td>Microtome</td><td>Thermo Scientific</td><td>905200</td><td>MICROM HM 355S</td></tr><tr><td>Mouse food, 18% prot, 2018S</td><td>Harlan</td><td>T.2018S.15</td><td></td></tr><tr><td>Neubauer</td><td>VWR</td><td>631-0696</td><td></td></tr><tr><td>Orange G</td><td>Biobasic</td><td>OB0674-25G</td><td></td></tr><tr><td>Paraffin</td><td>Paraplast</td><td>39601006</td><td></td></tr><tr><td>Paraffin Section Flotation Bath</td><td>Electrothermal</td><td>MH8517X1</td><td></td></tr><tr><td>Petri dish</td><td>Corning</td><td>CLS430167</td><td></td></tr><tr><td>Phloxin B</td><td>Probiotek</td><td>166-02072</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Corning</td><td>25-051-CI</td><td></td></tr><tr><td>Wax dispenser</td><td>Electrothermal</td><td>MH8523BX1</td><td></td></tr><tr><td>Xylene</td><td>Golden</td><td>534056-500ML</td><td></td></tr></tbody></table>

measuring bone remodeling and recreating the tumor-bone microenvironment using calvaria co-culture and histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

To evaluate bone formation in the calvarial model, we cultivated the hemi-calvarias in media with or without 50 &#181;g/mL of insulin. Tissue sections were prepared and stained with H&#38;E. In these conditions, the histology showed that the structural integrity of the calvarial bone was maintained, allowing the identification of its different components (Figure 1). The calvarias treated with insulin presented an increase in the amount of bone tissue compared...

Watch this Scientific Journal Video about Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry at JoVE.com

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

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Cancer Research

6.8K Views.  Center for Scientific Research and Higher Education at Ensenada. Het doel van deze procedure is om een ex vivo model te hebben om bot remodelleren te evalueren en de tumor-bone micro-omgeving te recreëren met behulp van calvaria cultuur of co-cultuur met kankercellen. De meeste calvaria techniek is een orgaancultuur systeem met behulp van calvaria botten van neonatale muizen om bot remodelleren te evalueren. U het therapeutisch potentieel van diverse moleculen testen, of zelfs de bot-tumor micro-omgeving nabootsen wanneer u coculturen met kankercellen geb...

Video: Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Developmental Biology

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Medicine

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Bioengineering

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 ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long follow-ups necessary for detection and sporadic occurrence, it is challenging to assess the pathogenesis ofparticle-induced osteolysis in clinical cases. Hence, optimal animal models are required for further studies. The murine model of calvarial osteolysis established by exposure to CoCrMo particles is an effective and valid tool for assessing the interactions between particles and various cells in aseptic loosening. In this model, CoCrMo particles were first obtained by high-vacuum three-electrode direct current and resuspended in phosphate-buffered saline at a concentration of 50 mg/mL. Then, 50 &#181;L of the resulting suspension was applied to the middle of the murine calvaria after separation of the cranial periosteum by sharp dissection. After two weeks, the mice were sacrificed, and calvaria specimens were harvested; qualitative and quantitative evaluations were performed by hematoxylin and eosin staining and micro computed tomography. The strengths of this model include procedure simplicity, quantitative evaluation of bone loss, rapidity of osteolysis development, potential use transgenic or knockout models, and a relatively low cost. However, this model cannot to be used to assess the mechanical force and chronic effects of particles in aseptic loosening. Murine calvarial osteolysis model generated by exposure to CoCrMo particles is an ideal tool for assessing the interactions between wear particles and various cells, e.g., macrophages, fibroblasts, osteoblasts and osteoclasts, in aseptic loosening.

This manuscript describes a murine calvarial osteolysis model by exposure to CoCrMo particles, which constitutes an ideal animal model for assessing the interactions between wear particles and various cells in aseptic loosening.

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

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