457 lines
117 KiB
XML
457 lines
117 KiB
XML
<document ID-DOI="10.1038/srep23099" ID-GBIF-Dataset="63d35c77-1116-4dc9-a30c-073ae11fde42" ID-PMC="PMC4791554" ID-PubMed="26975806" ID-Zenodo-Dep="3897580" checkinTime="1592226901665" checkinUser="jeremy" docAuthor="Mary Higby Schweitzer, Wenxia Zheng, Lindsay Zanno, Sarah Werning & Toshie Sugiyama" docDate="2016" docId="03D087F0FF83FFECFE41FCF3461FFB35" docLanguage="en" docName="Schweitzeretal2016.pdf.imf" docOrigin="Scientific Reports 6" docStyle="DocumentStyle{}" docTitle="Tyrannosaurus rex Osborn 1905" docType="treatment" docVersion="7" lastPageNumber="8" masterDocId="FFE9FF88FF80FFE4FFDEFFCC4306FFF1" masterDocTitle="Chemistry supports the identification of gender-specific reproductive tissue in Tyrannosaurus rex" masterLastPageNumber="23099" masterPageNumber="23099" pageNumber="3" updateTime="1668144149824" updateUser="ExternalLinkService">
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<mods:mods xmlns:mods="http://www.loc.gov/mods/v3">
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<mods:titleInfo>
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<mods:title>Chemistry supports the identification of gender-specific reproductive tissue in Tyrannosaurus rex</mods:title>
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</mods:titleInfo>
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<mods:name type="personal">
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<mods:role>
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<mods:roleTerm>Author</mods:roleTerm>
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<mods:namePart>Mary Higby Schweitzer</mods:namePart>
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<mods:affiliation>Department of Biological Sciences, North Carolina State University, Raleigh NC 27695, USA. North Carolina Museum of Natural Sciences, Raleigh NC 27601, USA</mods:affiliation>
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<mods:nameIdentifier type="email">schweitzer@ncsu.edu</mods:nameIdentifier>
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<mods:roleTerm>Author</mods:roleTerm>
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<mods:namePart>Wenxia Zheng</mods:namePart>
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<mods:affiliation>Department of Biological Sciences, North Carolina State University, Raleigh NC 27695, USA.</mods:affiliation>
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</mods:name>
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<mods:name type="personal">
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<mods:roleTerm>Author</mods:roleTerm>
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<mods:namePart>Lindsay Zanno</mods:namePart>
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<mods:affiliation>Department of Biological Sciences, North Carolina State University, Raleigh NC 27695, USA. North Carolina Museum of Natural Sciences, Raleigh NC 27601, USA</mods:affiliation>
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<mods:roleTerm>Author</mods:roleTerm>
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<mods:namePart>Sarah Werning</mods:namePart>
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<mods:affiliation>Department of Anatomy, Des Moines University, Des Moines IA 50312, USA.</mods:affiliation>
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</mods:name>
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<mods:name type="personal">
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<mods:role>
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<mods:roleTerm>Author</mods:roleTerm>
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</mods:role>
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<mods:namePart>Toshie Sugiyama</mods:namePart>
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<mods:affiliation>Department of Agrobiology, Niigata University, Niigata 9502181, Japan.</mods:affiliation>
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<mods:title>Scientific Reports</mods:title>
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<mods:part>
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<mods:date>2016</mods:date>
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<mods:number>2016-03-15</mods:number>
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<mods:detail type="volume">
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<mods:number>6</mods:number>
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<mods:start>23099</mods:start>
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<mods:identifier type="DOI">10.1038/srep23099</mods:identifier>
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<treatment ID-DOI="http://doi.org/10.5281/zenodo.5205205" ID-GBIF-Taxon="187974771" ID-Zenodo-Dep="5205205" LSID="urn:lsid:plazi:treatment:03D087F0FF83FFECFE41FCF3461FFB35" httpUri="http://treatment.plazi.org/id/03D087F0FF83FFECFE41FCF3461FFB35" lastPageId="8" lastPageNumber="8" pageId="3" pageNumber="3">
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<subSubSection pageId="3" pageNumber="3" type="nomenclature">
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<paragraph blockId="3.[415,1480,799,1972]" pageId="3" pageNumber="3">
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MB is clearly visible in both extant and extinct dinosaur bone (
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<figureCitation box="[1009,1062,830,852]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1</figureCitation>
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), and is morphologically and texturally distinct from overlying CB and/or TB in hand sample. MB is fibrous, vascular and randomly organized, non-lamellar woven bone, consistent with its rapid deposition (e.g.
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<bibRefCitation author="de Margerie, E. & Cubo, J. & Castanet, J." box="[913,928,880,894]" journalOrPublisher="C. R. Biologies" pageId="3" pageNumber="3" pagination="221 - 230" part="325" refId="ref6995" refString="40. de Margerie, E., Cubo, J. & Castanet, J. Bone typology and growth rate: testing and quantifying ' Amprino's rule' in the mallard (Anas platyrhynchos). C. R. Biologies 325, 221 - 230 (2002)." title="Bone typology and growth rate: testing and quantifying ' Amprino's rule' in the mallard (Anas platyrhynchos)" type="journal article" year="2002">
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<superScript attach="none" box="[913,928,880,894]" fontSize="6" pageId="3" pageNumber="3">40</superScript>
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</bibRefCitation>
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). In chicken (
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<figureCitation box="[1057,1146,884,906]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1A,B</figureCitation>
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) MB is distinct in color and texture from overlying CB (
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<figureCitation box="[604,675,910,932]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1A</figureCitation>
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) and TB (
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<figureCitation box="[770,841,910,932]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1B</figureCitation>
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). MB in ostrich femur (
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<figureCitation box="[1064,1158,910,932]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1C,D</figureCitation>
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) likewise shows visible differentiation in color and organization, but in hand sample, MB seems to grade from CB, becoming less dense and more spiculated as it extends toward the medullary cavity. Large erosion rooms (ER) are visible in the loosely organized MB (
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<figureCitation box="[463,534,990,1012]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1C</figureCitation>
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) and at the boundary between CB and forming MB, and white crystalline MB occasionally fills the erosion rooms (
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<figureCitation box="[568,640,1017,1039]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1C</figureCitation>
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, *). At higher magnification in ground section (
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<figureCitation box="[1102,1177,1017,1039]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1D</figureCitation>
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), a distinct separation between ostrich CB and MB can be seen that is not obvious in hand sample. Similarly, MB is attached to, but distinct from overlying
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<taxonomicName authorityName="Osborn" authorityYear="1905" box="[510,562,1071,1092]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="3" pageNumber="3" phylum="Chordata" rank="species" species="rex">
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<emphasis box="[510,562,1071,1092]" italics="true" pageId="3" pageNumber="3">T. rex</emphasis>
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</taxonomicName>
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CB in texture, color, vascularity and organization (
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<figureCitation box="[1048,1137,1070,1092]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1E,F</figureCitation>
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). The proximal femur shaft of
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<materialsCitation ID-GBIF-Occurrence="2636228303" collectionCode="MOR" pageId="3" pageNumber="3" specimenCode="MOR 1125">MOR 1125</materialsCitation>
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shows no expansion or distortion consistent with osteopetrosis in gross transverse section (
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<figureCitation box="[1354,1429,1097,1119]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1G</figureCitation>
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), but cortical bone (CB) is clearly distinct from hypothesized MB (arrows), which completely fills the medullary cavity. A red line marks the distinction between CB and MB, separated by erosion rooms. These areas of porous bone can be seen to extend from the medullary cavity almost to the periosteal surface in some regions (
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||
<figureCitation box="[1355,1429,1177,1199]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1G</figureCitation>
|
||
,*). A petrographic ground section is shown in
|
||
<figureCitation box="[817,892,1204,1226]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1H</figureCitation>
|
||
. Black arrows show a distinct line of separation between CB with large erosion rooms and adjacent MB, which is randomly oriented and appears fragmented. An apparent trabecular fragment (T) is seen interspersed with the MB (
|
||
<figureCitation box="[982,1058,1257,1279]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Fig. 1H</figureCitation>
|
||
).
|
||
<figureCitation box="[1076,1167,1257,1279]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="3" pageNumber="3">Figure S1</figureCitation>
|
||
shows an expanded view of this region, with more of the inner cortical bone visible. In this microscopic section, the contrast between dense CB with secondary osteons, the region of intense resorption, and the origin of non-lamellar MB is easily visualized.
|
||
</paragraph>
|
||
</subSubSection>
|
||
<subSubSection lastPageId="8" lastPageNumber="8" pageId="3" pageNumber="3" type="description">
|
||
<paragraph blockId="3.[415,1480,799,1972]" pageId="3" pageNumber="3">
|
||
Ground sections of
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[635,687,1338,1359]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="3" pageNumber="3" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[635,687,1338,1359]" italics="true" pageId="3" pageNumber="3">T. rex</emphasis>
|
||
</taxonomicName>
|
||
and ostrich CB is birefringent and anisotropic when observed using polarized light (
|
||
<figureCitation box="[422,529,1364,1386]" captionStart="Figure 2" captionStartId="4.[415,480,1167,1189]" captionTargetBox="[420,1360,129,1127]" captionTargetId="figure@4.[415,1375,124,1132]" captionTargetPageId="4" captionText="Figure 2. Computed tomographic imaging of MOR 1125 femur bone fragment showing morphological differentiation between MB and CB. (A–D) volumetric renderings and (E–G) cross sections. (A,B) High density cortical bone rendered transparent to visually isolate lower density medullary bone. (E) Density shown as spectrum from high (black) to low (white). Fragment is (C,D,G) color and (F) heat mapped.Color mapping key: (C,D,G) medullary bone (orange/red) and cortical bone (beige/yellow); heat mapping key (F): highest density (red) lowest density (blue). Sample shown in (A,C,E–G) cross sectional and (B,D) medial views." figureDoi="http://doi.org/10.5281/zenodo.3897586" httpUri="https://zenodo.org/record/3897586/files/figure.png" pageId="3" pageNumber="3">Fig. S2A,C</figureCitation>
|
||
), demonstrating the lamellar nature of this bone type. MB from ostrich and
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[1251,1304,1364,1385]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="3" pageNumber="3" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[1251,1304,1364,1385]" italics="true" pageId="3" pageNumber="3">T. rex</emphasis>
|
||
</taxonomicName>
|
||
(
|
||
<figureCitation box="[1316,1420,1364,1386]" captionStart="Figure 2" captionStartId="4.[415,480,1167,1189]" captionTargetBox="[420,1360,129,1127]" captionTargetId="figure@4.[415,1375,124,1132]" captionTargetPageId="4" captionText="Figure 2. Computed tomographic imaging of MOR 1125 femur bone fragment showing morphological differentiation between MB and CB. (A–D) volumetric renderings and (E–G) cross sections. (A,B) High density cortical bone rendered transparent to visually isolate lower density medullary bone. (E) Density shown as spectrum from high (black) to low (white). Fragment is (C,D,G) color and (F) heat mapped.Color mapping key: (C,D,G) medullary bone (orange/red) and cortical bone (beige/yellow); heat mapping key (F): highest density (red) lowest density (blue). Sample shown in (A,C,E–G) cross sectional and (B,D) medial views." figureDoi="http://doi.org/10.5281/zenodo.3897586" httpUri="https://zenodo.org/record/3897586/files/figure.png" pageId="3" pageNumber="3">Fig. S2B,D</figureCitation>
|
||
) lacks birefringence, supporting the randomly oriented, non-lamellar nature of MB tissues, consistent with rapidly deposited woven bone.
|
||
</paragraph>
|
||
<paragraph blockId="3.[415,1480,799,1972]" pageId="3" pageNumber="3">
|
||
To confirm density and organizational differences between MB and CB in this specimen of
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" higherTaxonomySource="GBIF" kingdom="Animalia" order="Dinosauria" pageId="3" pageNumber="3" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis italics="true" pageId="3" pageNumber="3">Tyrannosaurus rex</emphasis>
|
||
</taxonomicName>
|
||
, we employed high-resolution computed tomography (CT) (see Materials and Methods). MB in
|
||
<materialsCitation ID-GBIF-Occurrence="2636228301" box="[1373,1479,1470,1492]" collectionCode="MOR" pageId="3" pageNumber="3" specimenCode="MOR 1125">MOR 1125</materialsCitation>
|
||
is structurally unique and shows substantial density disparity with overlying cortical bone both in volumetric renderings (
|
||
<figureCitation box="[529,630,1523,1546]" captionStart="Figure 2" captionStartId="4.[415,480,1167,1189]" captionTargetBox="[420,1360,129,1127]" captionTargetId="figure@4.[415,1375,124,1132]" captionTargetPageId="4" captionText="Figure 2. Computed tomographic imaging of MOR 1125 femur bone fragment showing morphological differentiation between MB and CB. (A–D) volumetric renderings and (E–G) cross sections. (A,B) High density cortical bone rendered transparent to visually isolate lower density medullary bone. (E) Density shown as spectrum from high (black) to low (white). Fragment is (C,D,G) color and (F) heat mapped.Color mapping key: (C,D,G) medullary bone (orange/red) and cortical bone (beige/yellow); heat mapping key (F): highest density (red) lowest density (blue). Sample shown in (A,C,E–G) cross sectional and (B,D) medial views." figureDoi="http://doi.org/10.5281/zenodo.3897586" httpUri="https://zenodo.org/record/3897586/files/figure.png" pageId="3" pageNumber="3">Fig. 2A–D</figureCitation>
|
||
) and in two-dimensional transverse section (
|
||
<figureCitation box="[1053,1154,1523,1546]" captionStart="Figure 2" captionStartId="4.[415,480,1167,1189]" captionTargetBox="[420,1360,129,1127]" captionTargetId="figure@4.[415,1375,124,1132]" captionTargetPageId="4" captionText="Figure 2. Computed tomographic imaging of MOR 1125 femur bone fragment showing morphological differentiation between MB and CB. (A–D) volumetric renderings and (E–G) cross sections. (A,B) High density cortical bone rendered transparent to visually isolate lower density medullary bone. (E) Density shown as spectrum from high (black) to low (white). Fragment is (C,D,G) color and (F) heat mapped.Color mapping key: (C,D,G) medullary bone (orange/red) and cortical bone (beige/yellow); heat mapping key (F): highest density (red) lowest density (blue). Sample shown in (A,C,E–G) cross sectional and (B,D) medial views." figureDoi="http://doi.org/10.5281/zenodo.3897586" httpUri="https://zenodo.org/record/3897586/files/figure.png" pageId="3" pageNumber="3">Fig. 2E–G</figureCitation>
|
||
), independently substantiating the diagnosis of this tissue.
|
||
</paragraph>
|
||
<paragraph blockId="3.[415,1480,799,1972]" pageId="3" pageNumber="3">
|
||
We used Alcian blue histochemical stain (
|
||
<figureCitation box="[854,910,1577,1599]" captionStart="Figure 3" captionStartId="5.[415,480,1532,1554]" captionTargetBox="[421,1319,130,1488]" captionTargetId="figure@5.[415,1327,124,1492]" captionTargetPageId="5" captionText="Figure 3. Alcian blue histochemical stain capitalizes on the differential presence of sulfated glycosaminoglycans found in MB vs CB. Low (A) and high (B) magnification of demineralized, sectioned bone from a laying hen femur show MB (black arrows), forming along the borders of CB. Alcian blue stains MB intensely, but only lightly stains CB and TB, reflecting the differences in matrix composition.MB is shown to form around ovate vacancies that may represent vessels (red arrowheads).MB is sometimes found as islands within spicules of TB or CB (yellow arrows), possibly representing centripetal infilling of pre-existing vessel channels or erosion rooms with forming MB. Ostrich (C), shown at a lower magnification to encompass internal-most cortical bone and developing spicules of MB. The developmental pattern differs from that of chicken, reflecting macroscopic differences seen in hand sample, where MB and CB are not distinct, but more gradational in nature.Forming MB (black arrows) can be seen lining secondary osteons (leftmost black arrow), and as pockets of bone within pre-existing cortical bone (yellow arrows).Ovate open spaces within completely formed MB spicules are also seen (red arrowheads). Low (D) and high (E) magnifications of demineralized and sectioned T.rex CB show fibrous matrix that is lightly stained. Dinosaur MB in low (F), and high (G) magnifications show much more intense staining (black arrows).Matrix is fibrous, but is penetrated by ovate forms within deeply staining bone (F, red arrows).White spaces in F are sectioning artifact, where tissue and embedding material have pulled away from bone. Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897588" httpUri="https://zenodo.org/record/3897588/files/figure.png" pageId="3" pageNumber="3">Fig. 3</figureCitation>
|
||
) to capitalize on the chemical differences between CB and MB. This stain reacts with the acidic, sulfated glycosaminoglycan keratan sulfate
|
||
<bibRefCitation author="Yamamoto, T." box="[1174,1189,1600,1614]" journalOrPublisher="J. Electron Microsc. (Tokyo)" pageId="3" pageNumber="3" pagination="29 - 34" part="54" refId="ref6745" refString="35. Yamamoto, T. et al. Ultrastructrual and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans. J. Electron Microsc. (Tokyo) 54, 29 - 34, doi: 10.1093 / jmicro / dffi 097 (2005)." title="Ultrastructrual and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans" type="journal article" year="2005">
|
||
<superScript attach="left" box="[1174,1189,1600,1614]" fontSize="6" pageId="3" pageNumber="3">35</superScript>
|
||
</bibRefCitation>
|
||
, which is not found in cortical bone
|
||
<superScript attach="left" box="[463,497,1627,1641]" fontSize="6" pageId="3" pageNumber="3">36,37</superScript>
|
||
. Although Alcian blue lightly stains CB in both extant (chicken, ostrich) and extinct (
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[1326,1380,1631,1652]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="3" pageNumber="3" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[1326,1380,1631,1652]" italics="true" pageId="3" pageNumber="3">T. rex</emphasis>
|
||
</taxonomicName>
|
||
) dinosaur bone, staining of MB is much more intense in all cases, allowing microstructural and compositional differentiation of bone types.
|
||
<figureCitation box="[609,731,1684,1706]" captionStart="Figure 3" captionStartId="5.[415,480,1532,1554]" captionTargetBox="[421,1319,130,1488]" captionTargetId="figure@5.[415,1327,124,1492]" captionTargetPageId="5" captionText="Figure 3. Alcian blue histochemical stain capitalizes on the differential presence of sulfated glycosaminoglycans found in MB vs CB. Low (A) and high (B) magnification of demineralized, sectioned bone from a laying hen femur show MB (black arrows), forming along the borders of CB. Alcian blue stains MB intensely, but only lightly stains CB and TB, reflecting the differences in matrix composition.MB is shown to form around ovate vacancies that may represent vessels (red arrowheads).MB is sometimes found as islands within spicules of TB or CB (yellow arrows), possibly representing centripetal infilling of pre-existing vessel channels or erosion rooms with forming MB. Ostrich (C), shown at a lower magnification to encompass internal-most cortical bone and developing spicules of MB. The developmental pattern differs from that of chicken, reflecting macroscopic differences seen in hand sample, where MB and CB are not distinct, but more gradational in nature.Forming MB (black arrows) can be seen lining secondary osteons (leftmost black arrow), and as pockets of bone within pre-existing cortical bone (yellow arrows).Ovate open spaces within completely formed MB spicules are also seen (red arrowheads). Low (D) and high (E) magnifications of demineralized and sectioned T.rex CB show fibrous matrix that is lightly stained. Dinosaur MB in low (F), and high (G) magnifications show much more intense staining (black arrows).Matrix is fibrous, but is penetrated by ovate forms within deeply staining bone (F, red arrows).White spaces in F are sectioning artifact, where tissue and embedding material have pulled away from bone. Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897588" httpUri="https://zenodo.org/record/3897588/files/figure.png" pageId="3" pageNumber="3">Figure 3 A,B</figureCitation>
|
||
shows low (A) and high (B) magnification images of demineralized sectioned bone from a hen in lay. Trabeculae (T) are lightly stained, supporting compositional similarity with cortical bone, but MB stains a dark blue (black arrows), and shows a pattern of MB deposition on existing trabeculae. MB arises from and lines the trabeculae and internal layers of cortical bone, but also forms as pockets within CB and TB (
|
||
<figureCitation box="[422,493,1790,1812]" captionStart="Figure 3" captionStartId="5.[415,480,1532,1554]" captionTargetBox="[421,1319,130,1488]" captionTargetId="figure@5.[415,1327,124,1492]" captionTargetPageId="5" captionText="Figure 3. Alcian blue histochemical stain capitalizes on the differential presence of sulfated glycosaminoglycans found in MB vs CB. Low (A) and high (B) magnification of demineralized, sectioned bone from a laying hen femur show MB (black arrows), forming along the borders of CB. Alcian blue stains MB intensely, but only lightly stains CB and TB, reflecting the differences in matrix composition.MB is shown to form around ovate vacancies that may represent vessels (red arrowheads).MB is sometimes found as islands within spicules of TB or CB (yellow arrows), possibly representing centripetal infilling of pre-existing vessel channels or erosion rooms with forming MB. Ostrich (C), shown at a lower magnification to encompass internal-most cortical bone and developing spicules of MB. The developmental pattern differs from that of chicken, reflecting macroscopic differences seen in hand sample, where MB and CB are not distinct, but more gradational in nature.Forming MB (black arrows) can be seen lining secondary osteons (leftmost black arrow), and as pockets of bone within pre-existing cortical bone (yellow arrows).Ovate open spaces within completely formed MB spicules are also seen (red arrowheads). Low (D) and high (E) magnifications of demineralized and sectioned T.rex CB show fibrous matrix that is lightly stained. Dinosaur MB in low (F), and high (G) magnifications show much more intense staining (black arrows).Matrix is fibrous, but is penetrated by ovate forms within deeply staining bone (F, red arrows).White spaces in F are sectioning artifact, where tissue and embedding material have pulled away from bone. Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897588" httpUri="https://zenodo.org/record/3897588/files/figure.png" pageId="3" pageNumber="3">Fig. 3A</figureCitation>
|
||
, yellow arrows). This may be due to centripetal deposition and infilling of osteons or vessel channels with forming MB. Spicules of forming MB are occasionally penetrated by ovate “holes” that may represent vascular channels surrounded by MB matrix (red arrowheads). At higher magnifications, CB and MB are separated by large openings which may be vascular structures or, alternatively, erosion rooms that existed prior to MB deposition (
|
||
<figureCitation box="[481,550,1897,1919]" captionStart="Figure 3" captionStartId="5.[415,480,1532,1554]" captionTargetBox="[421,1319,130,1488]" captionTargetId="figure@5.[415,1327,124,1492]" captionTargetPageId="5" captionText="Figure 3. Alcian blue histochemical stain capitalizes on the differential presence of sulfated glycosaminoglycans found in MB vs CB. Low (A) and high (B) magnification of demineralized, sectioned bone from a laying hen femur show MB (black arrows), forming along the borders of CB. Alcian blue stains MB intensely, but only lightly stains CB and TB, reflecting the differences in matrix composition.MB is shown to form around ovate vacancies that may represent vessels (red arrowheads).MB is sometimes found as islands within spicules of TB or CB (yellow arrows), possibly representing centripetal infilling of pre-existing vessel channels or erosion rooms with forming MB. Ostrich (C), shown at a lower magnification to encompass internal-most cortical bone and developing spicules of MB. The developmental pattern differs from that of chicken, reflecting macroscopic differences seen in hand sample, where MB and CB are not distinct, but more gradational in nature.Forming MB (black arrows) can be seen lining secondary osteons (leftmost black arrow), and as pockets of bone within pre-existing cortical bone (yellow arrows).Ovate open spaces within completely formed MB spicules are also seen (red arrowheads). Low (D) and high (E) magnifications of demineralized and sectioned T.rex CB show fibrous matrix that is lightly stained. Dinosaur MB in low (F), and high (G) magnifications show much more intense staining (black arrows).Matrix is fibrous, but is penetrated by ovate forms within deeply staining bone (F, red arrows).White spaces in F are sectioning artifact, where tissue and embedding material have pulled away from bone. Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897588" httpUri="https://zenodo.org/record/3897588/files/figure.png" pageId="3" pageNumber="3">Fig. 3B</figureCitation>
|
||
, black arrow).
|
||
</paragraph>
|
||
<paragraph blockId="3.[415,1480,799,1972]" lastBlockId="4.[415,1480,1396,1952]" lastPageId="4" lastPageNumber="4" pageId="3" pageNumber="3">
|
||
Similarly, ostrich MB bone (
|
||
<figureCitation box="[714,785,1924,1946]" captionStart="Figure 3" captionStartId="5.[415,480,1532,1554]" captionTargetBox="[421,1319,130,1488]" captionTargetId="figure@5.[415,1327,124,1492]" captionTargetPageId="5" captionText="Figure 3. Alcian blue histochemical stain capitalizes on the differential presence of sulfated glycosaminoglycans found in MB vs CB. Low (A) and high (B) magnification of demineralized, sectioned bone from a laying hen femur show MB (black arrows), forming along the borders of CB. Alcian blue stains MB intensely, but only lightly stains CB and TB, reflecting the differences in matrix composition.MB is shown to form around ovate vacancies that may represent vessels (red arrowheads).MB is sometimes found as islands within spicules of TB or CB (yellow arrows), possibly representing centripetal infilling of pre-existing vessel channels or erosion rooms with forming MB. Ostrich (C), shown at a lower magnification to encompass internal-most cortical bone and developing spicules of MB. The developmental pattern differs from that of chicken, reflecting macroscopic differences seen in hand sample, where MB and CB are not distinct, but more gradational in nature.Forming MB (black arrows) can be seen lining secondary osteons (leftmost black arrow), and as pockets of bone within pre-existing cortical bone (yellow arrows).Ovate open spaces within completely formed MB spicules are also seen (red arrowheads). Low (D) and high (E) magnifications of demineralized and sectioned T.rex CB show fibrous matrix that is lightly stained. Dinosaur MB in low (F), and high (G) magnifications show much more intense staining (black arrows).Matrix is fibrous, but is penetrated by ovate forms within deeply staining bone (F, red arrows).White spaces in F are sectioning artifact, where tissue and embedding material have pulled away from bone. Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897588" httpUri="https://zenodo.org/record/3897588/files/figure.png" pageId="3" pageNumber="3">Fig. 3C</figureCitation>
|
||
) reacts more intensely to the stain, supporting its distinct chemical composition, but the pattern of deposition in this ratite differs from that of laying hen. Although CB is lightly stained, similar to the chicken (
|
||
<figureCitation box="[631,702,1396,1418]" captionStart="Figure 3" captionStartId="5.[415,480,1532,1554]" captionTargetBox="[421,1319,130,1488]" captionTargetId="figure@5.[415,1327,124,1492]" captionTargetPageId="5" captionText="Figure 3. Alcian blue histochemical stain capitalizes on the differential presence of sulfated glycosaminoglycans found in MB vs CB. Low (A) and high (B) magnification of demineralized, sectioned bone from a laying hen femur show MB (black arrows), forming along the borders of CB. Alcian blue stains MB intensely, but only lightly stains CB and TB, reflecting the differences in matrix composition.MB is shown to form around ovate vacancies that may represent vessels (red arrowheads).MB is sometimes found as islands within spicules of TB or CB (yellow arrows), possibly representing centripetal infilling of pre-existing vessel channels or erosion rooms with forming MB. Ostrich (C), shown at a lower magnification to encompass internal-most cortical bone and developing spicules of MB. The developmental pattern differs from that of chicken, reflecting macroscopic differences seen in hand sample, where MB and CB are not distinct, but more gradational in nature.Forming MB (black arrows) can be seen lining secondary osteons (leftmost black arrow), and as pockets of bone within pre-existing cortical bone (yellow arrows).Ovate open spaces within completely formed MB spicules are also seen (red arrowheads). Low (D) and high (E) magnifications of demineralized and sectioned T.rex CB show fibrous matrix that is lightly stained. Dinosaur MB in low (F), and high (G) magnifications show much more intense staining (black arrows).Matrix is fibrous, but is penetrated by ovate forms within deeply staining bone (F, red arrows).White spaces in F are sectioning artifact, where tissue and embedding material have pulled away from bone. Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897588" httpUri="https://zenodo.org/record/3897588/files/figure.png" pageId="4" pageNumber="4">Fig. 3C</figureCitation>
|
||
, left), staining intensifies with increasing depth toward the medullary cavity (black arrows), reflecting compositional differences from CB, and allowing differentiation of MB by chemistry when it is not as obvious histologically. As in the chicken, MB can be seen to form “pockets” within pre-existing CB/TB (yellow arrows) and also is penetrated by empty ovate structures (red arrowheads), supporting the possibility that MB will deposit around vascular tissues as well as pre-existing bone. Alternatively, it may be that this represents MB from a previous lay cycle that was not fully resorbed. CB from
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[1035,1086,1531,1552]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="4" pageNumber="4" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[1035,1086,1531,1552]" italics="true" pageId="4" pageNumber="4">T. rex</emphasis>
|
||
</taxonomicName>
|
||
(
|
||
<figureCitation box="[1098,1187,1530,1552]" captionStart="Figure 3" captionStartId="5.[415,480,1532,1554]" captionTargetBox="[421,1319,130,1488]" captionTargetId="figure@5.[415,1327,124,1492]" captionTargetPageId="5" captionText="Figure 3. Alcian blue histochemical stain capitalizes on the differential presence of sulfated glycosaminoglycans found in MB vs CB. Low (A) and high (B) magnification of demineralized, sectioned bone from a laying hen femur show MB (black arrows), forming along the borders of CB. Alcian blue stains MB intensely, but only lightly stains CB and TB, reflecting the differences in matrix composition.MB is shown to form around ovate vacancies that may represent vessels (red arrowheads).MB is sometimes found as islands within spicules of TB or CB (yellow arrows), possibly representing centripetal infilling of pre-existing vessel channels or erosion rooms with forming MB. Ostrich (C), shown at a lower magnification to encompass internal-most cortical bone and developing spicules of MB. The developmental pattern differs from that of chicken, reflecting macroscopic differences seen in hand sample, where MB and CB are not distinct, but more gradational in nature.Forming MB (black arrows) can be seen lining secondary osteons (leftmost black arrow), and as pockets of bone within pre-existing cortical bone (yellow arrows).Ovate open spaces within completely formed MB spicules are also seen (red arrowheads). Low (D) and high (E) magnifications of demineralized and sectioned T.rex CB show fibrous matrix that is lightly stained. Dinosaur MB in low (F), and high (G) magnifications show much more intense staining (black arrows).Matrix is fibrous, but is penetrated by ovate forms within deeply staining bone (F, red arrows).White spaces in F are sectioning artifact, where tissue and embedding material have pulled away from bone. Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897588" httpUri="https://zenodo.org/record/3897588/files/figure.png" pageId="4" pageNumber="4">Fig. 3D,E</figureCitation>
|
||
) was physically separated from underlying MB and analyzed separately (see Materials and Methods). The demineralized CB matrix is highly fibrous, and fibers show varying orientation. The matrix is only lightly stained, consistent with extant cortical bone samples, and neither the ovate structures or pockets of differentially stained regions were observed. In contrast, isolated fragments of demineralized
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[850,902,1637,1658]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="4" pageNumber="4" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[850,902,1637,1658]" italics="true" pageId="4" pageNumber="4">T. rex</emphasis>
|
||
</taxonomicName>
|
||
MB (
|
||
<figureCitation box="[954,1043,1636,1659]" captionStart="Figure 3" captionStartId="5.[415,480,1532,1554]" captionTargetBox="[421,1319,130,1488]" captionTargetId="figure@5.[415,1327,124,1492]" captionTargetPageId="5" captionText="Figure 3. Alcian blue histochemical stain capitalizes on the differential presence of sulfated glycosaminoglycans found in MB vs CB. Low (A) and high (B) magnification of demineralized, sectioned bone from a laying hen femur show MB (black arrows), forming along the borders of CB. Alcian blue stains MB intensely, but only lightly stains CB and TB, reflecting the differences in matrix composition.MB is shown to form around ovate vacancies that may represent vessels (red arrowheads).MB is sometimes found as islands within spicules of TB or CB (yellow arrows), possibly representing centripetal infilling of pre-existing vessel channels or erosion rooms with forming MB. Ostrich (C), shown at a lower magnification to encompass internal-most cortical bone and developing spicules of MB. The developmental pattern differs from that of chicken, reflecting macroscopic differences seen in hand sample, where MB and CB are not distinct, but more gradational in nature.Forming MB (black arrows) can be seen lining secondary osteons (leftmost black arrow), and as pockets of bone within pre-existing cortical bone (yellow arrows).Ovate open spaces within completely formed MB spicules are also seen (red arrowheads). Low (D) and high (E) magnifications of demineralized and sectioned T.rex CB show fibrous matrix that is lightly stained. Dinosaur MB in low (F), and high (G) magnifications show much more intense staining (black arrows).Matrix is fibrous, but is penetrated by ovate forms within deeply staining bone (F, red arrows).White spaces in F are sectioning artifact, where tissue and embedding material have pulled away from bone. Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897588" httpUri="https://zenodo.org/record/3897588/files/figure.png" pageId="4" pageNumber="4">Fig. 3F,G</figureCitation>
|
||
) are deeply stained relative to that seen in CB, and contain regions of even more intense staining (black arrows). The MB is deposited on, or retains, empty ovate “holes”, as seen in the other MB samples (
|
||
<figureCitation box="[799,863,1690,1712]" captionStart="Figure 3" captionStartId="5.[415,480,1532,1554]" captionTargetBox="[421,1319,130,1488]" captionTargetId="figure@5.[415,1327,124,1492]" captionTargetPageId="5" captionText="Figure 3. Alcian blue histochemical stain capitalizes on the differential presence of sulfated glycosaminoglycans found in MB vs CB. Low (A) and high (B) magnification of demineralized, sectioned bone from a laying hen femur show MB (black arrows), forming along the borders of CB. Alcian blue stains MB intensely, but only lightly stains CB and TB, reflecting the differences in matrix composition.MB is shown to form around ovate vacancies that may represent vessels (red arrowheads).MB is sometimes found as islands within spicules of TB or CB (yellow arrows), possibly representing centripetal infilling of pre-existing vessel channels or erosion rooms with forming MB. Ostrich (C), shown at a lower magnification to encompass internal-most cortical bone and developing spicules of MB. The developmental pattern differs from that of chicken, reflecting macroscopic differences seen in hand sample, where MB and CB are not distinct, but more gradational in nature.Forming MB (black arrows) can be seen lining secondary osteons (leftmost black arrow), and as pockets of bone within pre-existing cortical bone (yellow arrows).Ovate open spaces within completely formed MB spicules are also seen (red arrowheads). Low (D) and high (E) magnifications of demineralized and sectioned T.rex CB show fibrous matrix that is lightly stained. Dinosaur MB in low (F), and high (G) magnifications show much more intense staining (black arrows).Matrix is fibrous, but is penetrated by ovate forms within deeply staining bone (F, red arrows).White spaces in F are sectioning artifact, where tissue and embedding material have pulled away from bone. Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897588" httpUri="https://zenodo.org/record/3897588/files/figure.png" pageId="4" pageNumber="4">Fig. 3F</figureCitation>
|
||
, red arrowheads). The significance of these is not known, but they may represent pre-existing vascular channels on which MB is deposited.
|
||
</paragraph>
|
||
<caption ID-DOI="http://doi.org/10.5281/zenodo.3897586" ID-Zenodo-Dep="3897586" httpUri="https://zenodo.org/record/3897586/files/figure.png" pageId="4" pageNumber="4" startId="4.[415,480,1167,1189]" targetBox="[420,1360,129,1127]" targetPageId="4">
|
||
<paragraph blockId="4.[415,1464,1167,1323]" pageId="4" pageNumber="4">
|
||
<emphasis bold="true" pageId="4" pageNumber="4">
|
||
Figure 2. Computed tomographic imaging of
|
||
<materialsCitation ID-GBIF-Occurrence="2636228302" box="[873,981,1167,1189]" collectionCode="MOR" pageId="4" pageNumber="4" specimenCode="MOR 1125">MOR 1125</materialsCitation>
|
||
femur bone fragment showing morphological differentiation between MB and CB.
|
||
</emphasis>
|
||
(
|
||
<emphasis bold="true" box="[787,833,1194,1215]" pageId="4" pageNumber="4">A–D</emphasis>
|
||
) volumetric renderings and (
|
||
<emphasis bold="true" box="[1113,1157,1194,1216]" pageId="4" pageNumber="4">E–G</emphasis>
|
||
) cross sections. (
|
||
<emphasis bold="true" box="[1319,1358,1194,1215]" pageId="4" pageNumber="4">A,B</emphasis>
|
||
) High density cortical bone rendered transparent to visually isolate lower density medullary bone. (
|
||
<emphasis bold="true" box="[1294,1308,1221,1242]" pageId="4" pageNumber="4">E</emphasis>
|
||
) Density shown as spectrum from high (black) to low (white). Fragment is (
|
||
<emphasis bold="true" box="[978,1018,1247,1269]" pageId="4" pageNumber="4">C,D</emphasis>
|
||
,
|
||
<emphasis bold="true" box="[1024,1041,1247,1269]" pageId="4" pageNumber="4">G</emphasis>
|
||
) color and (
|
||
<emphasis bold="true" box="[1158,1171,1248,1269]" pageId="4" pageNumber="4">F</emphasis>
|
||
) heat mapped. Color mapping key: (
|
||
<emphasis bold="true" box="[468,531,1274,1296]" pageId="4" pageNumber="4">C,D,G</emphasis>
|
||
) medullary bone (orange/red) and cortical bone (beige/yellow); heat mapping key (
|
||
<emphasis bold="true" box="[1325,1338,1274,1295]" pageId="4" pageNumber="4">F</emphasis>
|
||
): highest density (red) lowest density (blue). Sample shown in (
|
||
<emphasis bold="true" box="[924,1013,1301,1323]" pageId="4" pageNumber="4">A,C,E–G</emphasis>
|
||
) cross sectional and (
|
||
<emphasis bold="true" box="[1219,1258,1301,1322]" pageId="4" pageNumber="4">B,D</emphasis>
|
||
) medial views.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph blockId="4.[415,1480,1396,1952]" lastBlockId="6.[415,1479,1844,1946]" lastPageId="6" lastPageNumber="6" pageId="4" pageNumber="4">
|
||
We exposed all bone types to high iron diamine (HID), a stain that reacts specifically to sulfated glycosaminoglycans
|
||
<bibRefCitation author="Yamamoto, T." box="[486,501,1766,1780]" journalOrPublisher="J. Electron Microsc. (Tokyo)" pageId="4" pageNumber="4" pagination="29 - 34" part="54" refId="ref6745" refString="35. Yamamoto, T. et al. Ultrastructrual and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans. J. Electron Microsc. (Tokyo) 54, 29 - 34, doi: 10.1093 / jmicro / dffi 097 (2005)." title="Ultrastructrual and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans" type="journal article" year="2005">
|
||
<superScript attach="right" box="[486,501,1766,1780]" fontSize="6" pageId="4" pageNumber="4">35</superScript>
|
||
</bibRefCitation>
|
||
. All are lightly stained, but MB in extant (
|
||
<figureCitation box="[899,952,1770,1792]" captionStart="Figure 4" captionStartId="6.[415,480,1597,1619]" captionTargetBox="[419,1371,130,1555]" captionTargetId="figure@6.[415,1375,124,1562]" captionTargetPageId="6" captionText="Figure 4. High iron diamine (HID) staining of demineralized CB and MB. (A) Low, and (B) high magnification of chicken femur showing deposition of darkly staining MB on pre-existing CB. (C) low, and (D) high magnification of ostrich femoral MB. Similar to the pattern seen using Alcian blue (Fig. 3), the distribution of MB is less distinct but can be chemically differentiated from pre-existing CB in a more mixed fashion. CB from T.rex femur in low (E) and high (F) magnification shows slight staining, as seen in modern samples, but staining is much more pronounced in T.rex MB in low (G) and higher (H) magnifications.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897590" httpUri="https://zenodo.org/record/3897590/files/figure.png" pageId="4" pageNumber="4">Fig. 4</figureCitation>
|
||
A–D, yellow arrowheads) and extinct dinosaur samples (
|
||
<figureCitation box="[422,521,1796,1818]" captionStart="Figure 4" captionStartId="6.[415,480,1597,1619]" captionTargetBox="[419,1371,130,1555]" captionTargetId="figure@6.[415,1375,124,1562]" captionTargetPageId="6" captionText="Figure 4. High iron diamine (HID) staining of demineralized CB and MB. (A) Low, and (B) high magnification of chicken femur showing deposition of darkly staining MB on pre-existing CB. (C) low, and (D) high magnification of ostrich femoral MB. Similar to the pattern seen using Alcian blue (Fig. 3), the distribution of MB is less distinct but can be chemically differentiated from pre-existing CB in a more mixed fashion. CB from T.rex femur in low (E) and high (F) magnification shows slight staining, as seen in modern samples, but staining is much more pronounced in T.rex MB in low (G) and higher (H) magnifications.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897590" httpUri="https://zenodo.org/record/3897590/files/figure.png" pageId="4" pageNumber="4">Fig. 4G,H</figureCitation>
|
||
, yellow arrowheads) reacts much more intensely to this stain. Chicken CB and MB (
|
||
<figureCitation box="[1342,1436,1796,1819]" captionStart="Figure 4" captionStartId="6.[415,480,1597,1619]" captionTargetBox="[419,1371,130,1555]" captionTargetId="figure@6.[415,1375,124,1562]" captionTargetPageId="6" captionText="Figure 4. High iron diamine (HID) staining of demineralized CB and MB. (A) Low, and (B) high magnification of chicken femur showing deposition of darkly staining MB on pre-existing CB. (C) low, and (D) high magnification of ostrich femoral MB. Similar to the pattern seen using Alcian blue (Fig. 3), the distribution of MB is less distinct but can be chemically differentiated from pre-existing CB in a more mixed fashion. CB from T.rex femur in low (E) and high (F) magnification shows slight staining, as seen in modern samples, but staining is much more pronounced in T.rex MB in low (G) and higher (H) magnifications.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897590" httpUri="https://zenodo.org/record/3897590/files/figure.png" pageId="4" pageNumber="4">Fig. 4A,B</figureCitation>
|
||
) are differentiated by intensity of staining, which reveals spicules of new bone growth along surfaces of the more lightly stained CB. Ostrich (
|
||
<figureCitation box="[683,779,1850,1872]" captionStart="Figure 4" captionStartId="6.[415,480,1597,1619]" captionTargetBox="[419,1371,130,1555]" captionTargetId="figure@6.[415,1375,124,1562]" captionTargetPageId="6" captionText="Figure 4. High iron diamine (HID) staining of demineralized CB and MB. (A) Low, and (B) high magnification of chicken femur showing deposition of darkly staining MB on pre-existing CB. (C) low, and (D) high magnification of ostrich femoral MB. Similar to the pattern seen using Alcian blue (Fig. 3), the distribution of MB is less distinct but can be chemically differentiated from pre-existing CB in a more mixed fashion. CB from T.rex femur in low (E) and high (F) magnification shows slight staining, as seen in modern samples, but staining is much more pronounced in T.rex MB in low (G) and higher (H) magnifications.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897590" httpUri="https://zenodo.org/record/3897590/files/figure.png" pageId="4" pageNumber="4">Fig. 4C,D</figureCitation>
|
||
) shows a similar pattern. Histological distinction between CB and MB is not as clear as in chicken, with stain intensity increasing with depth of tissue into the medullary cavity; however, like Alcian blue, HID stain is capable of differentiating bone types and follows the same pattern as the Alcian blue stain.
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[517,570,1931,1952]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="4" pageNumber="4" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[517,570,1931,1952]" italics="true" pageId="4" pageNumber="4">T. rex</emphasis>
|
||
</taxonomicName>
|
||
CB (
|
||
<figureCitation box="[617,704,1930,1952]" captionStart="Figure 4" captionStartId="6.[415,480,1597,1619]" captionTargetBox="[419,1371,130,1555]" captionTargetId="figure@6.[415,1375,124,1562]" captionTargetPageId="6" captionText="Figure 4. High iron diamine (HID) staining of demineralized CB and MB. (A) Low, and (B) high magnification of chicken femur showing deposition of darkly staining MB on pre-existing CB. (C) low, and (D) high magnification of ostrich femoral MB. Similar to the pattern seen using Alcian blue (Fig. 3), the distribution of MB is less distinct but can be chemically differentiated from pre-existing CB in a more mixed fashion. CB from T.rex femur in low (E) and high (F) magnification shows slight staining, as seen in modern samples, but staining is much more pronounced in T.rex MB in low (G) and higher (H) magnifications.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897590" httpUri="https://zenodo.org/record/3897590/files/figure.png" pageId="4" pageNumber="4">Fig. 4E,F</figureCitation>
|
||
) is minimally stained with HID, but within the matrix, blood vessels (BV, arrows) can be seen interspersed within the fibrous matrix. The much more intense reactivity of
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[1265,1319,1845,1866]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="6" pageNumber="6" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[1265,1319,1845,1866]" italics="true" pageId="6" pageNumber="6">T. rex</emphasis>
|
||
</taxonomicName>
|
||
MB to this stain (
|
||
<figureCitation box="[422,519,1871,1893]" captionStart="Figure 4" captionStartId="6.[415,480,1597,1619]" captionTargetBox="[419,1371,130,1555]" captionTargetId="figure@6.[415,1375,124,1562]" captionTargetPageId="6" captionText="Figure 4. High iron diamine (HID) staining of demineralized CB and MB. (A) Low, and (B) high magnification of chicken femur showing deposition of darkly staining MB on pre-existing CB. (C) low, and (D) high magnification of ostrich femoral MB. Similar to the pattern seen using Alcian blue (Fig. 3), the distribution of MB is less distinct but can be chemically differentiated from pre-existing CB in a more mixed fashion. CB from T.rex femur in low (E) and high (F) magnification shows slight staining, as seen in modern samples, but staining is much more pronounced in T.rex MB in low (G) and higher (H) magnifications.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897590" httpUri="https://zenodo.org/record/3897590/files/figure.png" pageId="6" pageNumber="6">Fig. 4G,H</figureCitation>
|
||
) relative to CB independently supports the presence of original compounds in these dinosaur materials. Data collection parameters were identical between modern and fossil bone samples for all histochemical stains.
|
||
<httpUri box="[480,570,1924,1946]" httpUri="https://static-content.springer.com/esm/art%3A10.1038%2Fsrep23099/MediaObjects/41598_2016_BFsrep23099_MOESM1_ESM.pdf" pageId="6" pageNumber="6">Figure S3</httpUri>
|
||
shows extant and dinosaur samples, demineralized and sectioned but unstained, as controls.
|
||
</paragraph>
|
||
<caption ID-DOI="http://doi.org/10.5281/zenodo.3897588" ID-Zenodo-Dep="3897588" httpUri="https://zenodo.org/record/3897588/files/figure.png" pageId="5" pageNumber="5" startId="5.[415,480,1532,1554]" targetBox="[421,1319,130,1488]" targetPageId="5">
|
||
<paragraph blockId="5.[415,1477,1532,1954]" pageId="5" pageNumber="5">
|
||
<emphasis bold="true" pageId="5" pageNumber="5">Figure 3. Alcian blue histochemical stain capitalizes on the differential presence of sulfated glycosaminoglycans found in MB vs CB.</emphasis>
|
||
Low (
|
||
<emphasis bold="true" box="[873,890,1558,1579]" pageId="5" pageNumber="5">A</emphasis>
|
||
) and high (
|
||
<emphasis bold="true" box="[1001,1016,1559,1580]" pageId="5" pageNumber="5">B</emphasis>
|
||
) magnification of demineralized, sectioned bone from a laying hen femur show MB (black arrows), forming along the borders of CB. Alcian blue stains MB intensely, but only lightly stains CB and TB, reflecting the differences in matrix composition. MB is shown to form around ovate vacancies that may represent vessels (red arrowheads). MB is sometimes found as islands within spicules of TB or CB (yellow arrows), possibly representing centripetal infilling of pre-existing vessel channels or erosion rooms with forming MB. Ostrich (
|
||
<emphasis bold="true" box="[934,950,1692,1714]" pageId="5" pageNumber="5">C</emphasis>
|
||
), shown at a lower magnification to encompass internal-most cortical bone and developing spicules of MB. The developmental pattern differs from that of chicken, reflecting macroscopic differences seen in hand sample, where MB and CB are not distinct, but more gradational in nature. Forming MB (black arrows) can be seen lining secondary osteons (leftmost black arrow), and as pockets of bone within pre-existing cortical bone (yellow arrows). Ovate open spaces within completely formed MB spicules are also seen (red arrowheads). Low (
|
||
<emphasis bold="true" box="[876,894,1825,1846]" pageId="5" pageNumber="5">D</emphasis>
|
||
) and high (
|
||
<emphasis bold="true" box="[1001,1015,1825,1846]" pageId="5" pageNumber="5">E</emphasis>
|
||
) magnifications of demineralized and sectioned
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[415,465,1852,1873]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="5" pageNumber="5" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[415,465,1852,1873]" italics="true" pageId="5" pageNumber="5">T. rex</emphasis>
|
||
</taxonomicName>
|
||
CB show fibrous matrix that is lightly stained. Dinosaur MB in low (
|
||
<emphasis bold="true" box="[1095,1108,1852,1873]" pageId="5" pageNumber="5">F</emphasis>
|
||
), and high (
|
||
<emphasis bold="true" box="[1221,1238,1852,1874]" pageId="5" pageNumber="5">G</emphasis>
|
||
) magnifications show much more intense staining (black arrows). Matrix is fibrous, but is penetrated by ovate forms within deeply staining bone (
|
||
<emphasis bold="true" box="[553,566,1905,1926]" pageId="5" pageNumber="5">F</emphasis>
|
||
, red arrows). White spaces in F are sectioning artifact, where tissue and embedding material have pulled away from bone. Scale bars as indicated.
|
||
</paragraph>
|
||
</caption>
|
||
<caption ID-DOI="http://doi.org/10.5281/zenodo.3897590" ID-Zenodo-Dep="3897590" httpUri="https://zenodo.org/record/3897590/files/figure.png" pageId="6" pageNumber="6" startId="6.[415,480,1597,1619]" targetBox="[419,1371,130,1555]" targetPageId="6">
|
||
<paragraph blockId="6.[415,1450,1597,1779]" pageId="6" pageNumber="6">
|
||
<emphasis bold="true" box="[415,1154,1597,1619]" pageId="6" pageNumber="6">Figure 4. High iron diamine (HID) staining of demineralized CB and MB.</emphasis>
|
||
(
|
||
<emphasis bold="true" box="[1167,1184,1597,1618]" pageId="6" pageNumber="6">A</emphasis>
|
||
) Low, and (
|
||
<emphasis bold="true" box="[1297,1312,1597,1618]" pageId="6" pageNumber="6">B</emphasis>
|
||
) high magnification of chicken femur showing deposition of darkly staining MB on pre-existing CB. (
|
||
<emphasis bold="true" box="[1320,1336,1624,1646]" pageId="6" pageNumber="6">C</emphasis>
|
||
) low, and (
|
||
<emphasis bold="true" box="[423,441,1651,1672]" pageId="6" pageNumber="6">D</emphasis>
|
||
) high magnification of ostrich femoral MB. Similar to the pattern seen using Alcian blue (Fig. 3), the distribution of MB is less distinct but can be chemically differentiated from pre-existing CB in a more mixed fashion. CB from
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[583,635,1704,1725]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="6" pageNumber="6" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[583,635,1704,1725]" italics="true" pageId="6" pageNumber="6">T. rex</emphasis>
|
||
</taxonomicName>
|
||
femur in low (
|
||
<emphasis bold="true" box="[775,789,1704,1725]" pageId="6" pageNumber="6">E</emphasis>
|
||
) and high (
|
||
<emphasis bold="true" box="[900,913,1704,1725]" pageId="6" pageNumber="6">F</emphasis>
|
||
) magnification shows slight staining, as seen in modern samples, but staining is much more pronounced in
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[901,952,1731,1752]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="6" pageNumber="6" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[901,952,1731,1752]" italics="true" pageId="6" pageNumber="6">T. rex</emphasis>
|
||
</taxonomicName>
|
||
MB in low (
|
||
<emphasis bold="true" box="[1070,1087,1730,1752]" pageId="6" pageNumber="6">G</emphasis>
|
||
) and higher (
|
||
<emphasis bold="true" box="[1217,1236,1731,1752]" pageId="6" pageNumber="6">H</emphasis>
|
||
) magnifications. Scale bars as indicated.
|
||
</paragraph>
|
||
</caption>
|
||
<caption ID-DOI="http://doi.org/10.5281/zenodo.3897592" ID-Zenodo-Dep="3897592" httpUri="https://zenodo.org/record/3897592/files/figure.png" pageId="7" pageNumber="7" startId="7.[415,480,960,982]" targetBox="[419,1467,130,919]" targetPageId="7">
|
||
<paragraph blockId="7.[415,1467,960,1222]" pageId="7" pageNumber="7">
|
||
<emphasis bold="true" pageId="7" pageNumber="7">Figure 5. Immunochemical staining of bone using monoclonal antibodies raised against the sulfated glycosaminoglycan keratan sulfate.</emphasis>
|
||
(
|
||
<emphasis bold="true" box="[777,950,987,1009]" pageId="7" pageNumber="7">A,C,E,G,I,K,M,O</emphasis>
|
||
) are overlay images showing tissue and localized binding; (
|
||
<emphasis bold="true" box="[508,666,1014,1036]" pageId="7" pageNumber="7">B,D,F,H,},L,N,P</emphasis>
|
||
) are fluorescent images using FITC label. Chicken CB (
|
||
<emphasis bold="true" box="[1192,1231,1014,1035]" pageId="7" pageNumber="7">A,B</emphasis>
|
||
) shows no binding; chicken MB (
|
||
<emphasis bold="true" box="[543,583,1040,1062]" pageId="7" pageNumber="7">C,D</emphasis>
|
||
) shows positive staining, with green fluorescent signal representing antibody-antigen complexes, arranged in globular clusters. Similarly, ostrich femoral CB (
|
||
<emphasis bold="true" box="[1096,1130,1068,1089]" pageId="7" pageNumber="7">E,F</emphasis>
|
||
) does not bind antibodies, but ostrich MB (
|
||
<emphasis bold="true" box="[535,578,1094,1116]" pageId="7" pageNumber="7">G,H</emphasis>
|
||
) is positive for binding using the same data collection parameters.
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[1211,1263,1095,1116]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="7" pageNumber="7" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[1211,1263,1095,1116]" italics="true" pageId="7" pageNumber="7">T. rex</emphasis>
|
||
</taxonomicName>
|
||
CB (
|
||
<emphasis bold="true" box="[1311,1335,1094,1116]" pageId="7" pageNumber="7">I,}</emphasis>
|
||
), does not show evidence of localized antibody binding, but sections of isolated MB (
|
||
<emphasis bold="true" box="[1118,1155,1121,1142]" pageId="7" pageNumber="7">K,L</emphasis>
|
||
) show localized specific binding to antibodies in a globular pattern, as seen in the chicken. (
|
||
<emphasis bold="true" box="[973,1017,1147,1168]" pageId="7" pageNumber="7">M,N</emphasis>
|
||
) cortical region of tarsometatarsus and (
|
||
<emphasis bold="true" box="[1404,1442,1147,1169]" pageId="7" pageNumber="7">O,P</emphasis>
|
||
) internal (medullary) region of chicken genetically diagnosed with avian osteopetrosis (Materials and Methods) exposed to anti-keratan sulfate antibodies. No binding is seen, using same data collection parameters.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph blockId="7.[415,1481,1312,1921]" pageId="7" pageNumber="7">
|
||
This pattern is repeated when Alcian blue is combined with HID stain, a common procedure to identify MB in modern birds
|
||
<bibRefCitation author="Yamamoto, T." box="[544,559,1336,1350]" journalOrPublisher="J. Electron Microsc. (Tokyo)" pageId="7" pageNumber="7" pagination="29 - 34" part="54" refId="ref6745" refString="35. Yamamoto, T. et al. Ultrastructrual and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans. J. Electron Microsc. (Tokyo) 54, 29 - 34, doi: 10.1093 / jmicro / dffi 097 (2005)." title="Ultrastructrual and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans" type="journal article" year="2005">
|
||
<superScript attach="left" box="[544,559,1336,1350]" fontSize="6" pageId="7" pageNumber="7">35</superScript>
|
||
</bibRefCitation>
|
||
. The dual stains (
|
||
<httpUri box="[721,786,1339,1361]" httpUri="https://static-content.springer.com/esm/art%3A10.1038%2Fsrep23099/MediaObjects/41598_2016_BFsrep23099_MOESM1_ESM.pdf" pageId="7" pageNumber="7">Fig. S4</httpUri>
|
||
) differentiate MB and CB in both extant and non-avian dinosaur samples. Differential distribution of sulfated glycosaminoglycans in forming bone is clearly visualized in both chicken (
|
||
<httpUri box="[422,525,1392,1415]" httpUri="https://static-content.springer.com/esm/art%3A10.1038%2Fsrep23099/MediaObjects/41598_2016_BFsrep23099_MOESM1_ESM.pdf" pageId="7" pageNumber="7">Fig. S4A,B</httpUri>
|
||
) and ostrich (
|
||
<httpUri box="[656,762,1392,1415]" httpUri="https://static-content.springer.com/esm/art%3A10.1038%2Fsrep23099/MediaObjects/41598_2016_BFsrep23099_MOESM1_ESM.pdf" pageId="7" pageNumber="7">Fig. S4C,D</httpUri>
|
||
) by staining intensity; similarly,
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[1070,1122,1393,1414]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="7" pageNumber="7" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[1070,1122,1393,1414]" italics="true" pageId="7" pageNumber="7">T. rex</emphasis>
|
||
</taxonomicName>
|
||
CB (
|
||
<httpUri box="[1170,1269,1392,1415]" httpUri="https://static-content.springer.com/esm/art%3A10.1038%2Fsrep23099/MediaObjects/41598_2016_BFsrep23099_MOESM1_ESM.pdf" pageId="7" pageNumber="7">Fig. S4E,F</httpUri>
|
||
) shows minimal reactivity, but isolated demineralized MB sections react intensely to the dual stains (
|
||
<httpUri box="[1168,1278,1419,1441]" httpUri="https://static-content.springer.com/esm/art%3A10.1038%2Fsrep23099/MediaObjects/41598_2016_BFsrep23099_MOESM1_ESM.pdf" pageId="7" pageNumber="7">Fig. S4G,H</httpUri>
|
||
).
|
||
</paragraph>
|
||
<paragraph blockId="7.[415,1481,1312,1921]" pageId="7" pageNumber="7">
|
||
Demineralized MB bone matrix also reacts to monoclonal antibodies specific to keratan sulfate (
|
||
<figureCitation box="[1349,1404,1446,1468]" captionStart="Figure 5" captionStartId="7.[415,480,960,982]" captionTargetBox="[419,1467,130,919]" captionTargetId="figure@7.[415,1471,124,925]" captionTargetPageId="7" captionText="Figure 5. Immunochemical staining of bone using monoclonal antibodies raised against the sulfated glycosaminoglycan keratan sulfate. (A,C,E,G,I,K,M,O) are overlay images showing tissue and localized binding; (B,D,F,H,},L,N,P) are fluorescent images using FITC label. Chicken CB (A,B) shows no binding; chicken MB (C,D) shows positive staining, with green fluorescent signal representing antibody-antigen complexes, arranged in globular clusters. Similarly, ostrich femoral CB (E,F) does not bind antibodies, but ostrich MB (G,H) is positive for binding using the same data collection parameters. T. rex CB (I,}), does not show evidence of localized antibody binding, but sections of isolated MB (K,L) show localized specific binding to antibodies in a globular pattern, as seen in the chicken.(M,N) cortical region of tarsometatarsus and (O,P) internal (medullary) region of chicken genetically diagnosed with avian osteopetrosis (Materials and Methods) exposed to anti-keratan sulfate antibodies. No binding is seen, using same data collection parameters." figureDoi="http://doi.org/10.5281/zenodo.3897592" httpUri="https://zenodo.org/record/3897592/files/figure.png" pageId="7" pageNumber="7">Fig. 5</figureCitation>
|
||
), allowing immunological differentiation of MB from CB or TB. Antibody-antigen complexes localize to globular structures within the MB matrix of Japanese quail
|
||
<bibRefCitation author="Yamamoto, T." box="[841,856,1496,1510]" journalOrPublisher="J. Electron Microsc. (Tokyo)" pageId="7" pageNumber="7" pagination="29 - 34" part="54" refId="ref6745" refString="35. Yamamoto, T. et al. Ultrastructrual and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans. J. Electron Microsc. (Tokyo) 54, 29 - 34, doi: 10.1093 / jmicro / dffi 097 (2005)." title="Ultrastructrual and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans" type="journal article" year="2005">
|
||
<superScript attach="left" box="[841,856,1496,1510]" fontSize="6" pageId="7" pageNumber="7">35</superScript>
|
||
</bibRefCitation>
|
||
, a pattern also seen in the present study for all samples tested. No binding is visualized when antibodies are exposed to chicken cortical bone (
|
||
<figureCitation box="[1136,1227,1526,1548]" captionStart="Figure 5" captionStartId="7.[415,480,960,982]" captionTargetBox="[419,1467,130,919]" captionTargetId="figure@7.[415,1471,124,925]" captionTargetPageId="7" captionText="Figure 5. Immunochemical staining of bone using monoclonal antibodies raised against the sulfated glycosaminoglycan keratan sulfate. (A,C,E,G,I,K,M,O) are overlay images showing tissue and localized binding; (B,D,F,H,},L,N,P) are fluorescent images using FITC label. Chicken CB (A,B) shows no binding; chicken MB (C,D) shows positive staining, with green fluorescent signal representing antibody-antigen complexes, arranged in globular clusters. Similarly, ostrich femoral CB (E,F) does not bind antibodies, but ostrich MB (G,H) is positive for binding using the same data collection parameters. T. rex CB (I,}), does not show evidence of localized antibody binding, but sections of isolated MB (K,L) show localized specific binding to antibodies in a globular pattern, as seen in the chicken.(M,N) cortical region of tarsometatarsus and (O,P) internal (medullary) region of chicken genetically diagnosed with avian osteopetrosis (Materials and Methods) exposed to anti-keratan sulfate antibodies. No binding is seen, using same data collection parameters." figureDoi="http://doi.org/10.5281/zenodo.3897592" httpUri="https://zenodo.org/record/3897592/files/figure.png" pageId="7" pageNumber="7">Fig. 5A,B</figureCitation>
|
||
) but these antibodies bind MB, in a regular, globular pattern in the laying hen (
|
||
<figureCitation box="[923,1022,1552,1574]" captionStart="Figure 5" captionStartId="7.[415,480,960,982]" captionTargetBox="[419,1467,130,919]" captionTargetId="figure@7.[415,1471,124,925]" captionTargetPageId="7" captionText="Figure 5. Immunochemical staining of bone using monoclonal antibodies raised against the sulfated glycosaminoglycan keratan sulfate. (A,C,E,G,I,K,M,O) are overlay images showing tissue and localized binding; (B,D,F,H,},L,N,P) are fluorescent images using FITC label. Chicken CB (A,B) shows no binding; chicken MB (C,D) shows positive staining, with green fluorescent signal representing antibody-antigen complexes, arranged in globular clusters. Similarly, ostrich femoral CB (E,F) does not bind antibodies, but ostrich MB (G,H) is positive for binding using the same data collection parameters. T. rex CB (I,}), does not show evidence of localized antibody binding, but sections of isolated MB (K,L) show localized specific binding to antibodies in a globular pattern, as seen in the chicken.(M,N) cortical region of tarsometatarsus and (O,P) internal (medullary) region of chicken genetically diagnosed with avian osteopetrosis (Materials and Methods) exposed to anti-keratan sulfate antibodies. No binding is seen, using same data collection parameters." figureDoi="http://doi.org/10.5281/zenodo.3897592" httpUri="https://zenodo.org/record/3897592/files/figure.png" pageId="7" pageNumber="7">Fig. 5C,D</figureCitation>
|
||
). Similarly, ostrich cortical bone is negative for binding (
|
||
<figureCitation box="[501,590,1579,1601]" captionStart="Figure 5" captionStartId="7.[415,480,960,982]" captionTargetBox="[419,1467,130,919]" captionTargetId="figure@7.[415,1471,124,925]" captionTargetPageId="7" captionText="Figure 5. Immunochemical staining of bone using monoclonal antibodies raised against the sulfated glycosaminoglycan keratan sulfate. (A,C,E,G,I,K,M,O) are overlay images showing tissue and localized binding; (B,D,F,H,},L,N,P) are fluorescent images using FITC label. Chicken CB (A,B) shows no binding; chicken MB (C,D) shows positive staining, with green fluorescent signal representing antibody-antigen complexes, arranged in globular clusters. Similarly, ostrich femoral CB (E,F) does not bind antibodies, but ostrich MB (G,H) is positive for binding using the same data collection parameters. T. rex CB (I,}), does not show evidence of localized antibody binding, but sections of isolated MB (K,L) show localized specific binding to antibodies in a globular pattern, as seen in the chicken.(M,N) cortical region of tarsometatarsus and (O,P) internal (medullary) region of chicken genetically diagnosed with avian osteopetrosis (Materials and Methods) exposed to anti-keratan sulfate antibodies. No binding is seen, using same data collection parameters." figureDoi="http://doi.org/10.5281/zenodo.3897592" httpUri="https://zenodo.org/record/3897592/files/figure.png" pageId="7" pageNumber="7">Fig. 5E,F</figureCitation>
|
||
), but MB shows a punctate pattern of antibody binding (
|
||
<figureCitation box="[1122,1218,1579,1601]" captionStart="Figure 5" captionStartId="7.[415,480,960,982]" captionTargetBox="[419,1467,130,919]" captionTargetId="figure@7.[415,1471,124,925]" captionTargetPageId="7" captionText="Figure 5. Immunochemical staining of bone using monoclonal antibodies raised against the sulfated glycosaminoglycan keratan sulfate. (A,C,E,G,I,K,M,O) are overlay images showing tissue and localized binding; (B,D,F,H,},L,N,P) are fluorescent images using FITC label. Chicken CB (A,B) shows no binding; chicken MB (C,D) shows positive staining, with green fluorescent signal representing antibody-antigen complexes, arranged in globular clusters. Similarly, ostrich femoral CB (E,F) does not bind antibodies, but ostrich MB (G,H) is positive for binding using the same data collection parameters. T. rex CB (I,}), does not show evidence of localized antibody binding, but sections of isolated MB (K,L) show localized specific binding to antibodies in a globular pattern, as seen in the chicken.(M,N) cortical region of tarsometatarsus and (O,P) internal (medullary) region of chicken genetically diagnosed with avian osteopetrosis (Materials and Methods) exposed to anti-keratan sulfate antibodies. No binding is seen, using same data collection parameters." figureDoi="http://doi.org/10.5281/zenodo.3897592" httpUri="https://zenodo.org/record/3897592/files/figure.png" pageId="7" pageNumber="7">Fig. 5G,H</figureCitation>
|
||
) at identical data collection parameters.
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[535,590,1607,1628]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="7" pageNumber="7" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[535,590,1607,1628]" italics="true" pageId="7" pageNumber="7">T. rex</emphasis>
|
||
</taxonomicName>
|
||
cortical bone is negative for antibody binding (
|
||
<figureCitation box="[1052,1132,1606,1628]" captionStart="Figure 5" captionStartId="7.[415,480,960,982]" captionTargetBox="[419,1467,130,919]" captionTargetId="figure@7.[415,1471,124,925]" captionTargetPageId="7" captionText="Figure 5. Immunochemical staining of bone using monoclonal antibodies raised against the sulfated glycosaminoglycan keratan sulfate. (A,C,E,G,I,K,M,O) are overlay images showing tissue and localized binding; (B,D,F,H,},L,N,P) are fluorescent images using FITC label. Chicken CB (A,B) shows no binding; chicken MB (C,D) shows positive staining, with green fluorescent signal representing antibody-antigen complexes, arranged in globular clusters. Similarly, ostrich femoral CB (E,F) does not bind antibodies, but ostrich MB (G,H) is positive for binding using the same data collection parameters. T. rex CB (I,}), does not show evidence of localized antibody binding, but sections of isolated MB (K,L) show localized specific binding to antibodies in a globular pattern, as seen in the chicken.(M,N) cortical region of tarsometatarsus and (O,P) internal (medullary) region of chicken genetically diagnosed with avian osteopetrosis (Materials and Methods) exposed to anti-keratan sulfate antibodies. No binding is seen, using same data collection parameters." figureDoi="http://doi.org/10.5281/zenodo.3897592" httpUri="https://zenodo.org/record/3897592/files/figure.png" pageId="7" pageNumber="7">Fig. 5I,J</figureCitation>
|
||
), but a globular pattern of antibody binding is visualized in sections of demineralized dinosaur MB (
|
||
<figureCitation box="[1025,1117,1632,1654]" captionStart="Figure 5" captionStartId="7.[415,480,960,982]" captionTargetBox="[419,1467,130,919]" captionTargetId="figure@7.[415,1471,124,925]" captionTargetPageId="7" captionText="Figure 5. Immunochemical staining of bone using monoclonal antibodies raised against the sulfated glycosaminoglycan keratan sulfate. (A,C,E,G,I,K,M,O) are overlay images showing tissue and localized binding; (B,D,F,H,},L,N,P) are fluorescent images using FITC label. Chicken CB (A,B) shows no binding; chicken MB (C,D) shows positive staining, with green fluorescent signal representing antibody-antigen complexes, arranged in globular clusters. Similarly, ostrich femoral CB (E,F) does not bind antibodies, but ostrich MB (G,H) is positive for binding using the same data collection parameters. T. rex CB (I,}), does not show evidence of localized antibody binding, but sections of isolated MB (K,L) show localized specific binding to antibodies in a globular pattern, as seen in the chicken.(M,N) cortical region of tarsometatarsus and (O,P) internal (medullary) region of chicken genetically diagnosed with avian osteopetrosis (Materials and Methods) exposed to anti-keratan sulfate antibodies. No binding is seen, using same data collection parameters." figureDoi="http://doi.org/10.5281/zenodo.3897592" httpUri="https://zenodo.org/record/3897592/files/figure.png" pageId="7" pageNumber="7">Fig. 5K,L</figureCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph blockId="7.[415,1481,1312,1921]" lastBlockId="8.[415,1479,136,185]" lastPageId="8" lastPageNumber="8" pageId="7" pageNumber="7">
|
||
Avian osteopetrosis is a viral-induced pathology which results in bone deposition on the endosteal and periosteal surfaces of affected long bones, and is usually accompanied by massive expansion in bone diameter. Because this bone is rapidly deposited and endosteally derived, it may superficially resemble MB in histological section. However, bone deposition is usually bilateral
|
||
<superScript attach="left" box="[960,994,1736,1750]" fontSize="6" pageId="7" pageNumber="7">39,41</superScript>
|
||
and results in massive increases in bone diameter, most often accompanied by periosteal reaction and abnormality
|
||
<bibRefCitation author="Barbosa, T. & Ramirez, M. & Hafner, S. & Cheng, S. & Zavala, G." box="[1037,1052,1762,1776]" journalOrPublisher="Avian Dis." pageId="7" pageNumber="7" pagination="981 - 989" part="54" refId="ref7046" refString="41. Barbosa, T., Ramirez, M., Hafner, S., Cheng, S. & Zavala, G. Forensic investigation of a 1986 outbreak of osteopetrosis in commercial brown layers reveals a novel avian leukosis virus-related genome. Avian Dis. 54, 981 - 989 (2010)." title="Forensic investigation of a 1986 outbreak of osteopetrosis in commercial brown layers reveals a novel avian leukosis virus-related genome" type="journal article" year="2010">
|
||
<superScript attach="left" box="[1037,1052,1762,1776]" fontSize="6" pageId="7" pageNumber="7">41</superScript>
|
||
</bibRefCitation>
|
||
. None of these features were seen grossly in our
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[454,508,1793,1814]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" kingdom="Animalia" order="Dinosauria" pageId="7" pageNumber="7" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[454,508,1793,1814]" italics="true" pageId="7" pageNumber="7">T. rex</emphasis>
|
||
</taxonomicName>
|
||
samples (
|
||
<figureCitation box="[602,675,1792,1815]" captionStart="Figure 1" captionStartId="2.[415,479,1530,1552]" captionTargetBox="[419,1332,130,1490]" captionTargetId="figure@2.[415,1337,124,1494]" captionTargetPageId="2" captionText="Figure 1. Morphological differentiation between MB and CB. (A) Mid shaft section from reproductively active laying hen femur shows textural differences between CB and MB. (B) more proximal region of hen femur shows that trabecular bone (T) can be differentiated from MB in hand sample, and that MB is deposited between trabeculae, infilling trabecular spaces. (C) MB in hand sample of ostrich femur appears to grade from CB, but can be differentiated by color and spiculation, as well as the presence of large erosion rooms (ER, arrows) at the boundary between layers. Infilling of erosion rooms with crystalline MB is also seen (*). (D) Ground section of ostrich at higher magnification shows clear separation of MB and CB. Bone fragment of MOR 1125 femur in (E) cross section and (F) medial, or medullary face orientation shows both textural and color differences between CB and MB, as well as the distinct separation between bone types.(G) Transverse section of MOR 1125 whole femur, showing almost complete infilling of the medullary cavity with MB. No gross deformation (corresponding to fracture callus) or bony expansion (corresponding to osteopetrosis) can be seen. Red line marks boundary between dense cortical bone and endosteal lamellar bone penetrated by multiple erosion rooms. Erosion rooms can be seen extending deep into the cortex in one region of the bone (*). (H) Petrographic ground section of deep CB layer and adjacent, internal MB of MOR 1125, showing change in texture and vascularity.Black arrows show distinct separation between innermost endosteal bone with erosion rooms, and region of MB deposition. Trabeculae (T) of laminar bone can be seen surrounded by MB.Scale bars as indicated." figureDoi="http://doi.org/10.5281/zenodo.3897582" httpUri="https://zenodo.org/record/3897582/files/figure.png" pageId="7" pageNumber="7">Fig. 1G</figureCitation>
|
||
), but we applied these antibodies to sections of bone from a chicken in which DNA analyses confirmed the presence of an avian leukosis virus, the group of retroviruses that induces avian osteopetrosis
|
||
<bibRefCitation author="Barbosa, T. & Ramirez, M. & Hafner, S. & Cheng, S. & Zavala, G." box="[492,507,1842,1856]" journalOrPublisher="Avian Dis." pageId="7" pageNumber="7" pagination="981 - 989" part="54" refId="ref7046" refString="41. Barbosa, T., Ramirez, M., Hafner, S., Cheng, S. & Zavala, G. Forensic investigation of a 1986 outbreak of osteopetrosis in commercial brown layers reveals a novel avian leukosis virus-related genome. Avian Dis. 54, 981 - 989 (2010)." title="Forensic investigation of a 1986 outbreak of osteopetrosis in commercial brown layers reveals a novel avian leukosis virus-related genome" type="journal article" year="2010">
|
||
<superScript attach="left" box="[492,507,1842,1856]" fontSize="6" pageId="7" pageNumber="7">41</superScript>
|
||
</bibRefCitation>
|
||
. Positive binding would indicate molecular similarities between the matrix of MB and osteopetrotic bone.
|
||
<figureCitation box="[474,598,1872,1894]" captionStart="Figure 5" captionStartId="7.[415,480,960,982]" captionTargetBox="[419,1467,130,919]" captionTargetId="figure@7.[415,1471,124,925]" captionTargetPageId="7" captionText="Figure 5. Immunochemical staining of bone using monoclonal antibodies raised against the sulfated glycosaminoglycan keratan sulfate. (A,C,E,G,I,K,M,O) are overlay images showing tissue and localized binding; (B,D,F,H,},L,N,P) are fluorescent images using FITC label. Chicken CB (A,B) shows no binding; chicken MB (C,D) shows positive staining, with green fluorescent signal representing antibody-antigen complexes, arranged in globular clusters. Similarly, ostrich femoral CB (E,F) does not bind antibodies, but ostrich MB (G,H) is positive for binding using the same data collection parameters. T. rex CB (I,}), does not show evidence of localized antibody binding, but sections of isolated MB (K,L) show localized specific binding to antibodies in a globular pattern, as seen in the chicken.(M,N) cortical region of tarsometatarsus and (O,P) internal (medullary) region of chicken genetically diagnosed with avian osteopetrosis (Materials and Methods) exposed to anti-keratan sulfate antibodies. No binding is seen, using same data collection parameters." figureDoi="http://doi.org/10.5281/zenodo.3897592" httpUri="https://zenodo.org/record/3897592/files/figure.png" pageId="7" pageNumber="7">Figure 5M,N</figureCitation>
|
||
is the cortical region of petrotic bone (Materials and Methods) and
|
||
<figureCitation box="[1251,1341,1872,1894]" captionStart="Figure 5" captionStartId="7.[415,480,960,982]" captionTargetBox="[419,1467,130,919]" captionTargetId="figure@7.[415,1471,124,925]" captionTargetPageId="7" captionText="Figure 5. Immunochemical staining of bone using monoclonal antibodies raised against the sulfated glycosaminoglycan keratan sulfate. (A,C,E,G,I,K,M,O) are overlay images showing tissue and localized binding; (B,D,F,H,},L,N,P) are fluorescent images using FITC label. Chicken CB (A,B) shows no binding; chicken MB (C,D) shows positive staining, with green fluorescent signal representing antibody-antigen complexes, arranged in globular clusters. Similarly, ostrich femoral CB (E,F) does not bind antibodies, but ostrich MB (G,H) is positive for binding using the same data collection parameters. T. rex CB (I,}), does not show evidence of localized antibody binding, but sections of isolated MB (K,L) show localized specific binding to antibodies in a globular pattern, as seen in the chicken.(M,N) cortical region of tarsometatarsus and (O,P) internal (medullary) region of chicken genetically diagnosed with avian osteopetrosis (Materials and Methods) exposed to anti-keratan sulfate antibodies. No binding is seen, using same data collection parameters." figureDoi="http://doi.org/10.5281/zenodo.3897592" httpUri="https://zenodo.org/record/3897592/files/figure.png" pageId="7" pageNumber="7">Fig. 5O,P</figureCitation>
|
||
is the internal (medullary) regions of the same bone. These controls are negative for all samples. In other control experiments, we omitted primary antibodies, but kept all other steps identical to test conditions, to control for spurious or non-specific binding of secondary antibodies or fluorescent label to the tissues (
|
||
<httpUri box="[1169,1237,163,185]" httpUri="https://static-content.springer.com/esm/art%3A10.1038%2Fsrep23099/MediaObjects/41598_2016_BFsrep23099_MOESM1_ESM.pdf" pageId="8" pageNumber="8">Fig. S5</httpUri>
|
||
).
|
||
</paragraph>
|
||
</subSubSection>
|
||
<subSubSection pageId="8" pageNumber="8" type="discussion">
|
||
<paragraph blockId="8.[415,1480,207,1220]" box="[415,744,207,233]" pageId="8" pageNumber="8">
|
||
<heading bold="true" box="[415,744,207,233]" fontSize="11" level="2" pageId="8" pageNumber="8" reason="0">
|
||
<emphasis bold="true" box="[415,744,207,233]" pageId="8" pageNumber="8">Discussion and Conclusion</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph blockId="8.[415,1480,207,1220]" pageId="8" pageNumber="8">
|
||
Medullary bone in extant birds is estrogen-dependent and linked to reproductive status and gender. Chemical differentiation of MB tissues in
|
||
<taxonomicName authorityName="Osborn" authorityYear="1905" box="[712,888,266,287]" class="Reptilia" family="Tyrannosauridae" genus="Tyrannosaurus" higherTaxonomySource="GBIF" kingdom="Animalia" order="Dinosauria" pageId="8" pageNumber="8" phylum="Chordata" rank="species" species="rex">
|
||
<emphasis box="[712,888,266,287]" italics="true" pageId="8" pageNumber="8">Tyrannosaurus rex</emphasis>
|
||
</taxonomicName>
|
||
implies that these factors can be extended deep into the theropod lineage. Homology can be inferred because of phylogenetic proximity, regional location within the skeleton, conserved histological features of endosteal derivation, high vascularity and isotropic arrangement of collagen fibers consistent with rapid deposition, as well as ephemeral nature (only 4 non-avian dinosaurs have been proposed to have this tissue), postcranial location, and now, molecular homologies. Identical tissues in multiple skeletal elements indicates a systemic process, consistent with MB deposition in extant birds, and the lack of periosteal reactive bone or abnormal bony enlargement in any elements negates an alternative hypothesis of avian osteopetrosis
|
||
<superScript attach="left" box="[541,575,448,462]" fontSize="6" pageId="8" pageNumber="8">39,42</superScript>
|
||
.
|
||
</paragraph>
|
||
<paragraph blockId="8.[415,1480,207,1220]" pageId="8" pageNumber="8">The fleeting nature of MB contributes to its rarity in the fossil record, but it may be possible, through careful study, to link MB unambiguously to other, less ephemeral traits, holding potential for rigorous examination of population structure, acquisition of reproductive novelties, and ontogenetic development in non-avian dinosaurs.</paragraph>
|
||
<paragraph blockId="8.[415,1480,207,1220]" pageId="8" pageNumber="8">
|
||
Original organic components are assumed to be completely destroyed during burial and fossilization processes over millions of years. However, we have shown that tissues
|
||
<superScript attach="left" box="[1056,1095,582,596]" fontSize="6" pageId="8" pageNumber="8">
|
||
<bibRefCitation author="Schweitzer, M. H." box="[1056,1071,582,596]" journalOrPublisher="Annu. Rev. Earth Planet. Sci." pageId="8" pageNumber="8" pagination="187 - 216" part="39" refId="ref7149" refString="43. Schweitzer, M. H. Soft tissue preservation in terrestrial Mesozoic vertebrates. Annu. Rev. Earth Planet. Sci. 39, 187 - 216, doi: 10.1146 / annurev-earth- 040610 - 133502 (2011)." title="Soft tissue preservation in terrestrial Mesozoic vertebrates" type="journal article" year="2011">43</bibRefCitation>
|
||
–
|
||
<bibRefCitation author="Avci, R." box="[1080,1095,582,596]" journalOrPublisher="Langmuir" pageId="8" pageNumber="8" pagination="3584 - 3590" part="21" refId="ref7226" refString="45. Avci, R. et al. Preservation of bone collagen from the late cretaceous period studied by immunological techniques and atomic force microscopy. Langmuir 21, 3584 - 3590 (2005)." title="Preservation of bone collagen from the late cretaceous period studied by immunological techniques and atomic force microscopy" type="journal article" year="2005">45</bibRefCitation>
|
||
</superScript>
|
||
, cells
|
||
<bibRefCitation author="Schweitzer, M. H. & Zheng, W. & Cleland, T. P. & Bern, M." box="[1148,1163,582,596]" journalOrPublisher="Bone" pageId="8" pageNumber="8" pagination="414 - 423" part="52" refId="ref7263" refString="46. Schweitzer, M. H., Zheng, W., Cleland, T. P. & Bern, M. Molecular analyses of dinosaur osteocytes support the presence of endogenous molecules. Bone 52, 414 - 423 (2013)." title="Molecular analyses of dinosaur osteocytes support the presence of endogenous molecules" type="journal article" year="2013">
|
||
<superScript attach="left" box="[1148,1163,582,596]" fontSize="6" pageId="8" pageNumber="8">46</superScript>
|
||
</bibRefCitation>
|
||
and fragments of original molecules
|
||
<superScript attach="left" box="[463,520,608,622]" fontSize="6" pageId="8" pageNumber="8">44,46–51</superScript>
|
||
can persist across geological time. Here we show the value of applying molecular methods to dinosaur bone to address important biological questions, using what is known of bone chemistry in homologous extant tissues.
|
||
</paragraph>
|
||
<paragraph blockId="8.[415,1480,207,1220]" pageId="8" pageNumber="8">
|
||
Recently, MB was suggested to exist in pterosaurs, based upon purported histological similarity. Prondvai and Stein
|
||
<bibRefCitation author="Prondvai, E. & Stein, K. H. W." box="[464,479,715,729]" journalOrPublisher="Sci. Rep." pageId="8" pageNumber="8" part="4" refId="ref7524" refString="52. Prondvai, E. & Stein, K. H. W. Medullary bone-like tissue in the mandibular symphyses of a pterosaur suggests non-reproductive significance. Sci. Rep. 4, doi: 10.1038 / srep 06253 (2014)." title="Medullary bone-like tissue in the mandibular symphyses of a pterosaur suggests non-reproductive significance" type="journal volume" year="2014">
|
||
<superScript attach="left" box="[464,479,715,729]" fontSize="6" pageId="8" pageNumber="8">52</superScript>
|
||
</bibRefCitation>
|
||
claimed to have identified the tissue in mandibulae of both juvenile and adult specimens, and proposed a redefinition of MB to include endosteal tissues that did not play a role in reproduction. These authors then concluded that the presence of MB could not be used for gender identification. However, the assignment of this pterosaur tissue to MB is dubious, in part because it was described
|
||
<emphasis box="[1059,1099,798,819]" italics="true" pageId="8" pageNumber="8">only</emphasis>
|
||
in mandibular symphyses, and was not identified in any postcrania examined
|
||
<bibRefCitation author="Prondvai, E. & Stein, K. H. W." box="[774,789,822,836]" journalOrPublisher="Sci. Rep." pageId="8" pageNumber="8" part="4" refId="ref7524" refString="52. Prondvai, E. & Stein, K. H. W. Medullary bone-like tissue in the mandibular symphyses of a pterosaur suggests non-reproductive significance. Sci. Rep. 4, doi: 10.1038 / srep 06253 (2014)." title="Medullary bone-like tissue in the mandibular symphyses of a pterosaur suggests non-reproductive significance" type="journal volume" year="2014">
|
||
<superScript attach="left" box="[774,789,822,836]" fontSize="6" pageId="8" pageNumber="8">52</superScript>
|
||
</bibRefCitation>
|
||
. In contrast, although MB occurs in some cranial material in a few birds, it is predominantly noted in the postcrania, specifically long bones (e.g.
|
||
<superScript attach="left" box="[1121,1178,848,862]" fontSize="6" pageId="8" pageNumber="8">3,9,10,19</superScript>
|
||
and references therein). It has never been reported in mandibulae of living birds, suggesting the tissue in pterosaurs is not homologous to MB. Furthermore, although MB can be induced to form in male birds with the administration of estrogen, it does not occur naturally in either males or juveniles, as was reported for pterosaurs. Because MB in living animals is estrogen dependent
|
||
<superScript attach="left" box="[556,590,955,969]" fontSize="6" pageId="8" pageNumber="8">14,34</superScript>
|
||
it cannot occur without the influence of these hormones. Thus the pterosaur tissues described by Prondvai and Stein
|
||
<bibRefCitation author="Prondvai, E. & Stein, K. H. W." box="[630,645,982,996]" journalOrPublisher="Sci. Rep." pageId="8" pageNumber="8" part="4" refId="ref7524" refString="52. Prondvai, E. & Stein, K. H. W. Medullary bone-like tissue in the mandibular symphyses of a pterosaur suggests non-reproductive significance. Sci. Rep. 4, doi: 10.1038 / srep 06253 (2014)." title="Medullary bone-like tissue in the mandibular symphyses of a pterosaur suggests non-reproductive significance" type="journal volume" year="2014">
|
||
<superScript attach="left" box="[630,645,982,996]" fontSize="6" pageId="8" pageNumber="8">52</superScript>
|
||
</bibRefCitation>
|
||
do not meet criteria for homology with MB in extant birds or non-avian dinosaurs as described.
|
||
</paragraph>
|
||
<paragraph blockId="8.[415,1480,207,1220]" pageId="8" pageNumber="8">Here, we demonstrate that the unique chemical composition of MB in birds is retained and can be identified in a non-avian theropod dinosaur, thereby supporting the homology of these tissues with MB in living birds. We show that it is possible to remove ambiguity associated with the assignment of MB in extinct taxa, using histochemical and immunological signatures, in cases where bone tissues retain original chemical components. The application of multiple and varied molecular techniques to fossils, as well as innovations in high resolution, nano-detection instrumentation will permit the exploration of sex-linked traits in theropod dinosaurs, offering a novel approach to investigate the paleobiology of birds and their extinct dinosaurian relatives.</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |