The authors value the support of Liuying Chen for her administrative assistance and Long Zhao for his excellent technical assistance during the construction of bone extracellular matrix scaffolds. This study is supported by grants from the National Natural Science Foundation of China (31871413).

Animal care and use are conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication NO.80-23, revised in 1996) after approval from the Animal Ethics Committee of Sun Yat-sen University.
1. Bone preparation
<ol>
	<li>Obtain 4 to 6-week-old BALB/c mice (without sex-specific requirement). Euthanize a mouse aseptically by cervical dislocation and cut off fresh fibula, tibia and femur from a hindlimb with sterile surgical scissors. Pe...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+of+origin+in+osteosarcoma:+mesenchymal+stem+cells+or+osteoblast+committed+cells.">Cells of origin in osteosarcoma: mesenchymal stem cells or osteoblast committed cells.</a> Bone. 62, 56-63 (2014).</li><li>Sato, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+tumors+can+derive+from+Ng2/Cspg4-Expressing+pericytes+with+&#946;-Catenin+modulating+the+neoplastic+phenotype.">Mesenchymal tumors can derive from Ng2/Cspg4-Expressing pericytes with &#946;-Catenin modulating the neoplastic phenotype.</a> Cell Reports. 16, 917-927 (2016).</li><li>Patane, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MET+overexpression+turns+human+primary+osteoblasts+into+osteosarcomas.">MET overexpression turns human primary osteoblasts into osteosarcomas.</a> Cancer Research. 66, 4750-4757 (2006).</li><li>Poos, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+heterogeneity+of+osteosarcoma+-+shift+from+single+candidates+to+functional+modules.">Genomic heterogeneity of osteosarcoma - shift from single candidates to functional modules.</a> PLoS One. 10, 123082 (2015).</li><li>Martin, J. W., Squire, J. A., Zielenska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+osteosarcoma.">The genetics of osteosarcoma.</a> Sarcoma. 2012, 1-11 (2012).</li><li>Alfranca, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+microenvironment+signals+in+osteosarcoma+development.">Bone microenvironment signals in osteosarcoma development.</a> Cellular and Molecular Life Sciences. 72, 3097-3113 (2015).</li><li>Alford, A. I., Kozloff, K. M., Hankenson, K. 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Generally, OS can be classified as osteoblastic, chondroblastic, and &#64257;broblastic subtypes depending on its dominant histologic component. Its prognosis is dependent not only on histologic parameters but also on its anatomic site. It may occur inside the bones (in the intramedullary or intracortical compartment), on the surfaces of bones, and in extraosseous sites19. The emergence and heterogeneity of OS can be elucidated as a conjugation of oncogenic events and an adequate microenvironmenta...

Osteosarcoma (OS) usually occurs in actively growing areas, the metaphysis of long bones, during adolescence. More than 80% of the OS-affected sites have preference for the metaphysis of proximal tibia and proximal humerus as well as both distal and proximal femur, corresponding to the location of the growth plate1. OS comprises multiple cell subtypes with mesenchymal properties and considerable diversity in histologic features and grade. Evidences support mesenchymal stem cells (MSCs), osteoblasts committed precursors and pericytes as the cells of origin2,3,4,5. These cells can accumulate&#160;genetic or epigenetic alterations and give rise to OS under the influence of certain bone microenvironmental signals. Both intrinsic and extrinsic mechanisms result in the genomic instability and heterogeneity of OS, with multiple morphological and clinical phenotypes6,7. For individualized therapies or screening of new drugs, novel models need to be generated to against heterogeneity or other clinical disorders.
OS is an intra-osseous malignant solid tumor. The complexity and activity of surrounding microenvironment elements confer phenotypic and functional differences upon OS cells in different locations of a tumor. Bone extracellular matrix (BEM) provides a structural and biochemical scaffold for mineral deposition and bone remodeling. The organic portion of extracellular matrix (ECM) mainly consists of type I collagen secreted by osteoblastic lineage cells, while its mineralized portion is composed of calcium phosphate in the form of hydroxyapatite8. The dynamic role of ECM networks is to regulate cell adhesion, differentiation, cross-talk and tissue function maintenance9.
Demineralized BEM and ECM hydrogels have been successfully used in cell culture and can enhance cell proliferation10,11. Synthesized bone-like ECM can regulate the pool size, fate decisions and lineage progression of MSCs12,13,14. Moreover, results evidence its clinical significance to provide osteogenic activity by stimulating cellular processes during bone formation and regeneration15,16,17.
In this article, our group establishes a modified model and favorable alternative for three-dimensional long-term culture. OS cells injected into the tissue-derived BEM present a heterogeneously mesenchymal phenotype readily as compared to plastic two-dimensional cultures. BEM derived from site-specific homologous tissue show its dramatic advantage as being a native niche for OS cells in vitro and has great potential in OS theoretical and clinical research. This characterized BEM platform is simple but efficient for in vitro research and may be extended in modeling multiple cancers.

<table border="0" cellpadding="0" cellspacing="0" style="width:1049px;" width="1049">
	<tbody>
		<tr height="21">
			<td height="21">15 mL centrifuge tube</td>
			<td>Greiner</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">50 mL centrifuge tube</td>
			<td>Greiner</td>
			<td>227270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">6 cm cell culture dish</td>
			<td>Greiner</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">6-well plate</td>
			<td>Greiner</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>A9393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42">C57-BL/6J mouse</td>
			<td>Sun Yat-sen University Laboratory Animal Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25">CO2 incubator</td>
			<td>SHEL LAB</td>
			<td>SCO5A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42">Dibasic sodium phosphate</td>
			<td>Guangzhou Chemical Reagent Factory</td>
			<td>BE14-GR-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">DMEM/F12</td>
			<td>Sigma-Aldrich</td>
			<td>D0547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Hemocytometer</td>
			<td>BLAU</td>
			<td>717805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1487</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42">MG-63</td>
			<td>Chinese Academy of Science, Shanghai Cell Bank</td>
			<td></td>
			<td>Human osteosarcoma cell line</td>
		</tr>
		<tr height="42">
			<td height="42">MNNG/HOS</td>
			<td>Chinese Academy of Science, Shanghai Cell Bank</td>
			<td></td>
			<td>Human osteosarcoma cell line</td>
		</tr>
		<tr height="42">
			<td height="42">Phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>P4633</td>
			<td>A solution of phenol red is used as a pH indicator: its color exhibits a gradual transition from yellow to red over the pH range 6.6 to 8.0.</td>
		</tr>
		<tr height="21">
			<td height="21">Potassium chloride</td>
			<td>Sangon Biotech</td>
			<td>A100395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Potassium Phosphate Monobasic</td>
			<td>Sangon Biotech</td>
			<td>A501211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Sodium chloride</td>
			<td>Sangon Biotech</td>
			<td>A501218</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional bone extracellular matrix model for osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

After demineralization and decellularization, BEM appears to be translucent with stronger resilience and tenacity compared to native mouse bone. A little muscle residue and the space of medullary cavity can be clearly observed (Figure 1A, B). To determine the effective decellularization of BEM, BEM is embedded in paraffin after fixation, and then sliced into 3&#8211;5 &#956;m sections for hematoxylin-eosin (H&#38;E) staining. The thorough removal of cell nuclei is shown by b...

Watch this Scientific Journal Video about Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma at JoVE.com

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

This method provides an efficient strategy for preparing functional scaffolds for bone tumor research. Decellularized the bone extracelullar matrix, showed variable serocompatibility for the survival and activities of osteosarcoma cells. The main advantage of this technique is that osteosarcoma cells culture in bone-derived matrix show highly heterogenous morphology similar to a clinical osteosarcoma histopathology.The method provides ideal model for investigating the development, progression of drug sensitivities of bone tumors, such as osteosarcoma, Ewing sarcoma, and the other malignant tumors metastasis to bone. To begin, obtain four to six-week old BALB/c mice, after euthanizing a mouse using sterile surgical scissors, cut off fresh fibula, tibia, and femur from a hind limb. With the help of tweezers, peel off the epithelial tissue, and then remove as much of the soft tissue as possible.Rinse the leg bones with sterile 10 mM PBS solution twice to remove blood in a six-centimeter dish. Immerse the bones in a dish with 75%ethanol for three minutes then rinse with PBS twice. Store the clean bones in a sterile 50 milliliter centrifuge tube with sterile PBS tube at 80 degrees Celsius.Thaw the frozen bones at room temperature and then freeze again at 80 degrees Celsius for one hour. Subject the bones to more than two freeze-thaw cycles for cell lysis and tissue breakdown. Then, place the bones in a sterile 50 milliliter centrifuge tube filled with 0.5 normal HCl and incubate overnight at room temperature on an orbital shaker with gentle shaking to ensure complete and even coverage of the bones.After decalcification, decant the hydrochloric acid solution completely and rinse the bones under running water for one hour. Then, use distilled water to wash the bones twice for 15 minutes per wash on an orbital shaker. Completely decant the solution.To extract the lipids in a demineralized bones, place the bones in a 50 milliliter centrifuge tube with a 1:1 mixture of methanol and chloroform. Wrap the tube with tin foil to avoid light for prevention of chloroform decomposition. And put the tube on an orbital shaker for one hour.Then, use tweezers to transfer the bones into another tube of methanol with tin foil for 30 minutes. Remove the methanol completely, and wash with distilled water twice for 15 minutes on an orbital shaker. Decant final wash water and proceed under sterile condition.Rinse the bones in a six centimeter dish with sterile PBS for three minutes. Add 40 milliliters of sterile 0.05%TE solution into a 50 milliliter centrifuge tube and incubate bones for 23 hours in a carbon dioxide incubator at 37 degrees Celsius. Discard the TE solution, and rinse twice with sterile PBS supplemented with 90 g/mL ampicillin and 90 g/mL kanamycin for 15 minutes each on the orbital shaker.After decanting the final wash completely, replenish again with 40 milliliters of sterile PBS with antibiotics. Wash thoroughly for 24 hours at room temperature with gentle shaking to achieve effective sterilization of porous spaces. Then, transfer the bones into a 50 milliliter centrifuge tube filled with sterile PBS with antibiotics.The prepared Bone Extracellular Matrix can be stored at four degrees Celsius for two months. Immerse BEM in 75%ethanol in a dish and gently shake the dish by hand for 30 seconds. Then rinse with PBS for 30 seconds, twice.Transfer the BEM onto a clean six-well cell culture plate. Add two milliliters of complete culture medium to each well. Incubate the BEM overnight in a carbon dioxide incubator at 37 degrees Celsius.Obtain human OS cell lines in 100 microliters of pre-warmed PBS containing indicator phenol red. Use a pipette to suspend OS cells with the approximate concentration of 1 times 10 to the 5th. After the BEM is fully soaked in the medium, from proximal or distal epiphyses, pierce the needle to the medullary cavity of BEM and inject OS cells into BEM.Incubate the OS-BEM model for a minimum of two hours in a humidified 5%carbon dioxide atmosphere at 37 degrees Celsius to ensure the injected cells firmly adhere to BEM. Then, take out the plate from the incubator. Add one milliliter culture medium onto the plate, and keep it in the incubator overnight to completely coat the surface of the BEM culture.Gently transfer the OS-BEM model into a new well of a six-well plate with a sterile tweezer, and refeed one milliliter fresh culture medium. Culture the model for 14 days in the incubator. During the 14-day incubation, keep monitoring medium color.If the medium turns into orange, or even yellow, immediately refresh the medium by discarding half of the old medium and adding in new medium to maintain a healthy environment for OS cells. Keep monitoring cell status under the inverted fluorescence microscope. When OS cells expand to plate, gently transfer the OS-BEM model to another new well with sterile tweezers.After 14-day culture, gently rinse the OS-BEM model with PBS to remove the culture medium. Then transfer into a 15 milliliter centrifuge tube and add 10%buffered formalin to fix for histological identification. After demineralization and decellularization, BEM appears to be translucent with stronger resilience and tenacity compared to native mouse bone.A little muscle residue in the space of medullary cavity can be clearly observed. Bright-field imaging of the native bone and the decellularized BEM, shows the thorough removal of cell nuclei. The natural porous structure in collagen network arrangement is well-maintained in decellularized BEM.Additionally, immunohistochemical staining for Collagen I and Collagen IV shows the main components of extracellular matrix are preserved in mouse tibia after decellularization. During the 14-day culture, both periosteum and endosteum are infiltrated by the expansion of OS cells. OS cells on the decellularized BEM show highly heterogeneous morphology similar to the cytopathologic features of an OS section.Immunohistochemical analysis after culturing in BEM model for 14 days shows great advantages in long term cultures. Also, OS cells and BEM culture, highly express bone matrix glycoprotein, which is specific for osteoid matrix. This in vitro's three-dimensional model has been used to demonstrate a phenotypic heterogeneity and a regulatory mechanism of osteosarcoma dedifferentiation with success.

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Research

JoVE Journal

Cancer Research

7.1K visualizações.  Sun Yat-sen University. Este método fornece uma estratégia eficiente para o preparo de andaimes funcionais para a pesquisa de tumores ósseos. Descelularizou a matriz óssea extracelular, mostrou sorocompatibilidade variável para a sobrevivência e atividades das células osteossarcoma. A principal vantagem dessa técnica é que a cultura das células osteossarcoma na matriz derivada do osso mostra morfologia altamente heterogrógena semelhante a uma histopatologia osteossarcoma clínica.O método fornece ...