Information

38.2: Bone - Biology


Skills to Develop

  • Classify the different types of bones in the skeleton
  • Explain the role of the different cell types in bone
  • Explain how bone forms during development

Bone, or osseous tissue, is a connective tissue that constitutes the endoskeleton. It contains specialized cells and a matrix of mineral salts and collagen fibers.

The mineral salts primarily include hydroxyapatite, a mineral formed from calcium phosphate. Calcification is the process of deposition of mineral salts on the collagen fiber matrix that crystallizes and hardens the tissue. The process of calcification only occurs in the presence of collagen fibers.

The bones of the human skeleton are classified by their shape: long bones, short bones, flat bones, sutural bones, sesamoid bones, and irregular bones (Figure (PageIndex{1})).

Long bones are longer than they are wide and have a shaft and two ends. The diaphysis, or central shaft, contains bone marrow in a marrow cavity. The rounded ends, the epiphyses, are covered with articular cartilage and are filled with red bone marrow, which produces blood cells (Figure (PageIndex{2})). Most of the limb bones are long bones—for example, the femur, tibia, ulna, and radius. Exceptions to this include the patella and the bones of the wrist and ankle.

Short bones, or cuboidal bones, are bones that are the same width and length, giving them a cube-like shape. For example, the bones of the wrist (carpals) and ankle (tarsals) are short bones (Figure (PageIndex{1})).

Flat bones are thin and relatively broad bones that are found where extensive protection of organs is required or where broad surfaces of muscle attachment are required. Examples of flat bones are the sternum (breast bone), ribs, scapulae (shoulder blades), and the roof of the skull (Figure (PageIndex{1})).

Irregular bones are bones with complex shapes. These bones may have short, flat, notched, or ridged surfaces. Examples of irregular bones are the vertebrae, hip bones, and several skull bones.

Sesamoid bones are small, flat bones and are shaped similarly to a sesame seed. The patellae are sesamoid bones (Figure (PageIndex{3})). Sesamoid bones develop inside tendons and may be found near joints at the knees, hands, and feet.

Sutural bones are small, flat, irregularly shaped bones. They may be found between the flat bones of the skull. They vary in number, shape, size, and position.

Bone Tissue

Bones are considered organs because they contain various types of tissue, such as blood, connective tissue, nerves, and bone tissue. Osteocytes, the living cells of bone tissue, form the mineral matrix of bones. There are two types of bone tissue: compact and spongy.

Compact Bone Tissue

Compact bone (or cortical bone) forms the hard external layer of all bones and surrounds the medullary cavity, or bone marrow. It provides protection and strength to bones. Compact bone tissue consists of units called osteons or Haversian systems. Osteons are cylindrical structures that contain a mineral matrix and living osteocytes connected by canaliculi, which transport blood. They are aligned parallel to the long axis of the bone. Each osteon consists of lamellae, which are layers of compact matrix that surround a central canal called the Haversian canal. The Haversian canal (osteonic canal) contains the bone’s blood vessels and nerve fibers (Figure (PageIndex{4})). Osteons in compact bone tissue are aligned in the same direction along lines of stress and help the bone resist bending or fracturing. Therefore, compact bone tissue is prominent in areas of bone at which stresses are applied in only a few directions.

Art Connection

Which of the following statements about bone tissue is false?

  1. Compact bone tissue is made of cylindrical osteons that are aligned such that they travel the length of the bone.
  2. Haversian canals contain blood vessels only.
  3. Haversian canals contain blood vessels and nerve fibers.
  4. Spongy tissue is found on the interior of the bone, and compact bone tissue is found on the exterior.

Spongy Bone Tissue

Whereas compact bone tissue forms the outer layer of all bones, spongy bone or cancellous bone forms the inner layer of all bones. Spongy bone tissue does not contain osteons that constitute compact bone tissue. Instead, it consists of trabeculae, which are lamellae that are arranged as rods or plates. Red bone marrow is found between the trabuculae. Blood vessels within this tissue deliver nutrients to osteocytes and remove waste. The red bone marrow of the femur and the interior of other large bones, such as the ileum, forms blood cells.

Spongy bone reduces the density of bone and allows the ends of long bones to compress as the result of stresses applied to the bone. Spongy bone is prominent in areas of bones that are not heavily stressed or where stresses arrive from many directions. The epiphyses of bones, such as the neck of the femur, are subject to stress from many directions. Imagine laying a heavy framed picture flat on the floor. You could hold up one side of the picture with a toothpick if the toothpick was perpendicular to the floor and the picture. Now drill a hole and stick the toothpick into the wall to hang up the picture. In this case, the function of the toothpick is to transmit the downward pressure of the picture to the wall. The force on the picture is straight down to the floor, but the force on the toothpick is both the picture wire pulling down and the bottom of the hole in the wall pushing up. The toothpick will break off right at the wall.

The neck of the femur is horizontal like the toothpick in the wall. The weight of the body pushes it down near the joint, but the vertical diaphysis of the femur pushes it up at the other end. The neck of the femur must be strong enough to transfer the downward force of the body weight horizontally to the vertical shaft of the femur (Figure (PageIndex{5})).

Link to Learning

View micrographs of musculoskeletal tissues as you review the anatomy.

Cell Types in Bones

Bone consists of four types of cells: osteoblasts, osteoclasts, osteocytes, and osteoprogenitor cells. Osteoblasts are bone cells that are responsible for bone formation. Osteoblasts synthesize and secrete the organic part and inorganic part of the extracellular matrix of bone tissue, and collagen fibers. Osteoblasts become trapped in these secretions and differentiate into less active osteocytes. Osteoclasts are large bone cells with up to 50 nuclei. They remove bone structure by releasing lysosomal enzymes and acids that dissolve the bony matrix. These minerals, released from bones into the blood, help regulate calcium concentrations in body fluids. Bone may also be resorbed for remodeling, if the applied stresses have changed. Osteocytes are mature bone cells and are the main cells in bony connective tissue; these cells cannot divide. Osteocytes maintain normal bone structure by recycling the mineral salts in the bony matrix. Osteoprogenitor cells are squamous stem cells that divide to produce daughter cells that differentiate into osteoblasts. Osteoprogenitor cells are important in the repair of fractures.

Development of Bone

Ossification, or osteogenesis, is the process of bone formation by osteoblasts. Ossification is distinct from the process of calcification; whereas calcification takes place during the ossification of bones, it can also occur in other tissues. Ossification begins approximately six weeks after fertilization in an embryo. Before this time, the embryonic skeleton consists entirely of fibrous membranes and hyaline cartilage. The development of bone from fibrous membranes is called intramembranous ossification; development from hyaline cartilage is called endochondral ossification. Bone growth continues until approximately age 25. Bones can grow in thickness throughout life, but after age 25, ossification functions primarily in bone remodeling and repair.

Intramembranous Ossification

Intramembranous ossification is the process of bone development from fibrous membranes. It is involved in the formation of the flat bones of the skull, the mandible, and the clavicles. Ossification begins as mesenchymal cells form a template of the future bone. They then differentiate into osteoblasts at the ossification center. Osteoblasts secrete the extracellular matrix and deposit calcium, which hardens the matrix. The non-mineralized portion of the bone or osteoid continues to form around blood vessels, forming spongy bone. Connective tissue in the matrix differentiates into red bone marrow in the fetus. The spongy bone is remodeled into a thin layer of compact bone on the surface of the spongy bone.

Endochondral Ossification

Endochondral ossification is the process of bone development from hyaline cartilage. All of the bones of the body, except for the flat bones of the skull, mandible, and clavicles, are formed through endochondral ossification.

In long bones, chondrocytes form a template of the hyaline cartilage diaphysis. Responding to complex developmental signals, the matrix begins to calcify. This calcification prevents diffusion of nutrients into the matrix, resulting in chondrocytes dying and the opening up of cavities in the diaphysis cartilage. Blood vessels invade the cavities, and osteoblasts and osteoclasts modify the calcified cartilage matrix into spongy bone. Osteoclasts then break down some of the spongy bone to create a marrow, or medullary, cavity in the center of the diaphysis. Dense, irregular connective tissue forms a sheath (periosteum) around the bones. The periosteum assists in attaching the bone to surrounding tissues, tendons, and ligaments. The bone continues to grow and elongate as the cartilage cells at the epiphyses divide.

In the last stage of prenatal bone development, the centers of the epiphyses begin to calcify. Secondary ossification centers form in the epiphyses as blood vessels and osteoblasts enter these areas and convert hyaline cartilage into spongy bone. Until adolescence, hyaline cartilage persists at the epiphyseal plate (growth plate), which is the region between the diaphysis and epiphysis that is responsible for the lengthwise growth of long bones (Figure (PageIndex{6})).

Growth of Bone

Long bones continue to lengthen, potentially until adolescence, through the addition of bone tissue at the epiphyseal plate. They also increase in width through appositional growth.

Lengthening of Long Bones

Chondrocytes on the epiphyseal side of the epiphyseal plate divide; one cell remains undifferentiated near the epiphysis, and one cell moves toward the diaphysis. The cells, which are pushed from the epiphysis, mature and are destroyed by calcification. This process replaces cartilage with bone on the diaphyseal side of the plate, resulting in a lengthening of the bone.

Long bones stop growing at around the age of 18 in females and the age of 21 in males in a process called epiphyseal plate closure. During this process, cartilage cells stop dividing and all of the cartilage is replaced by bone. The epiphyseal plate fades, leaving a structure called the epiphyseal line or epiphyseal remnant, and the epiphysis and diaphysis fuse.

Thickening of Long Bones

Appositional growth is the increase in the diameter of bones by the addition of bony tissue at the surface of bones. Osteoblasts at the bone surface secrete bone matrix, and osteoclasts on the inner surface break down bone. The osteoblasts differentiate into osteocytes. A balance between these two processes allows the bone to thicken without becoming too heavy.

Bone Remodeling and Repair

Bone renewal continues after birth into adulthood. Bone remodeling is the replacement of old bone tissue by new bone tissue. It involves the processes of bone deposition by osteoblasts and bone resorption by osteoclasts. Normal bone growth requires vitamins D, C, and A, plus minerals such as calcium, phosphorous, and magnesium. Hormones such as parathyroid hormone, growth hormone, and calcitonin are also required for proper bone growth and maintenance.

Bone turnover rates are quite high, with five to seven percent of bone mass being recycled every week. Differences in turnover rate exist in different areas of the skeleton and in different areas of a bone. For example, the bone in the head of the femur may be fully replaced every six months, whereas the bone along the shaft is altered much more slowly.

Bone remodeling allows bones to adapt to stresses by becoming thicker and stronger when subjected to stress. Bones that are not subject to normal stress, for example when a limb is in a cast, will begin to lose mass. A fractured or broken bone undergoes repair through four stages:

  1. Blood vessels in the broken bone tear and hemorrhage, resulting in the formation of clotted blood, or a hematoma, at the site of the break. The severed blood vessels at the broken ends of the bone are sealed by the clotting process, and bone cells that are deprived of nutrients begin to die.
  2. Within days of the fracture, capillaries grow into the hematoma, and phagocytic cells begin to clear away the dead cells. Though fragments of the blood clot may remain, fibroblasts and osteoblasts enter the area and begin to reform bone. Fibroblasts produce collagen fibers that connect the broken bone ends, and osteoblasts start to form spongy bone. The repair tissue between the broken bone ends is called the fibrocartilaginous callus, as it is composed of both hyaline and fibrocartilage (Figure (PageIndex{7})). Some bone spicules may also appear at this point.
  3. The fibrocartilaginous callus is converted into a bony callus of spongy bone. It takes about two months for the broken bone ends to be firmly joined together after the fracture. This is similar to the endochondral formation of bone, as cartilage becomes ossified; osteoblasts, osteoclasts, and bone matrix are present.
  4. The bony callus is then remodelled by osteoclasts and osteoblasts, with excess material on the exterior of the bone and within the medullary cavity being removed. Compact bone is added to create bone tissue that is similar to the original, unbroken bone. This remodeling can take many months, and the bone may remain uneven for years.

Scientific Method Connection

Decalcification of Bones Question: What effect does the removal of calcium and collagen have on bone structure?

Background: Conduct a literature search on the role of calcium and collagen in maintaining bone structure. Conduct a literature search on diseases in which bone structure is compromised.

Hypothesis: Develop a hypothesis that states predictions of the flexibility, strength, and mass of bones that have had the calcium and collagen components removed. Develop a hypothesis regarding the attempt to add calcium back to decalcified bones.

Test the hypothesis: Test the prediction by removing calcium from chicken bones by placing them in a jar of vinegar for seven days. Test the hypothesis regarding adding calcium back to decalcified bone by placing the decalcified chicken bones into a jar of water with calcium supplements added. Test the prediction by denaturing the collagen from the bones by baking them at 250°C for three hours.

Analyze the data: Create a table showing the changes in bone flexibility, strength, and mass in the three different environments.

Report the results: Under which conditions was the bone most flexible? Under which conditions was the bone the strongest?

Draw a conclusion: Did the results support or refute the hypothesis? How do the results observed in this experiment correspond to diseases that destroy bone tissue?

Summary

Bone, or osseous tissue, is connective tissue that includes specialized cells, mineral salts, and collagen fibers. The human skeleton can be divided into long bones, short bones, flat bones, and irregular bones. Compact bone tissue is composed of osteons and forms the external layer of all bones. Spongy bone tissue is composed of trabeculae and forms the inner part of all bones. Four types of cells compose bony tissue: osteocytes, osteoclasts, osteoprogenitor cells, and osteoblasts. Ossification is the process of bone formation by osteoblasts. Intramembranous ossification is the process of bone development from fibrous membranes. Endochondral ossification is the process of bone development from hyaline cartilage. Long bones lengthen as chondrocytes divide and secrete hyaline cartilage. Osteoblasts replace cartilage with bone. Appositional growth is the increase in the diameter of bones by the addition of bone tissue at the surface of bones. Bone remodeling involves the processes of bone deposition by osteoblasts and bone resorption by osteoclasts. Bone repair occurs in four stages and can take several months.

Art Exercise

[link]Which of the following statements about bone tissue is false?

  1. Compact bone tissue is made of cylindrical osteons that are aligned such that they travel the length of the bone.
  2. Haversian canals contain blood vessels only.
  3. Haversian canals contain blood vessels and nerve fibers.
  4. Spongy tissue is found on the interior of the bone, and compact bone tissue is found on the exterior.

[link]B

Review Questions

The Haversian canal:

  1. is arranged as rods or plates
  2. contains the bone’s blood vessels and nerve fibers
  3. is responsible for the lengthwise growth of long bones
  4. synthesizes and secretes matrix

B

The epiphyseal plate:

  1. is arranged as rods or plates
  2. contains the bone’s blood vessels and nerve fibers
  3. is responsible for the lengthwise growth of long bones
  4. synthesizes and secretes bone matrix

C

The cells responsible for bone resorption are ________.

  1. osteoclasts
  2. osteoblasts
  3. fibroblasts
  4. osteocytes

A

Compact bone is composed of ________.

  1. trabeculae
  2. compacted collagen
  3. osteons
  4. calcium phosphate only

C

Free Response

What are the major differences between spongy bone and compact bone?

Compact bone tissue forms the hard external layer of all bones and consists of osteons. Compact bone tissue is prominent in areas of bone at which stresses are applied in only a few directions. Spongy bone tissue forms the inner layer of all bones and consists of trabeculae. Spongy bone is prominent in areas of bones that are not heavily stressed or at which stresses arrive from many directions.

What are the roles of osteoblasts, osteocytes, and osteoclasts?

Osteocytes function in the exchange of nutrients and wastes with the blood. They also maintain normal bone structure by recycling the mineral salts in the bony matrix. Osteoclasts remove bone tissue by releasing lysosomal enzymes and acids that dissolve the bony matrix. Osteoblasts are bone cells that are responsible for bone formation.

Glossary

appositional growth
increase in the diameter of bones by the addition of bone tissue at the surface of bones
bone
(also, osseous tissue) connective tissue that constitutes the endoskeleton
bone remodeling
replacement of old bone tissue by new bone tissue
calcification
process of deposition of mineral salts in the collagen fiber matrix that crystallizes and hardens the tissue
compact bone
forms the hard external layer of all bones
diaphysis
central shaft of bone, contains bone marrow in a marrow cavity
endochondral ossification
process of bone development from hyaline cartilage
epiphyseal plate
region between the diaphysis and epiphysis that is responsible for the lengthwise growth of long bones
epiphysis
rounded end of bone, covered with articular cartilage and filled with red bone marrow, which produces blood cells
flat bone
thin and relatively broad bone found where extensive protection of organs is required or where broad surfaces of muscle attachment are required
Haversian canal
contains the bone’s blood vessels and nerve fibers
intramembranous ossification
process of bone development from fibrous membranes
irregular bone
bone with complex shapes; examples include vertebrae and hip bones
lamella
layer of compact tissue that surrounds a central canal called the Haversian canal
long bone
bone that is longer than wide, and has a shaft and two ends
osteoblast
bone cell responsible for bone formation
osteoclast
large bone cells with up to 50 nuclei, responsible for bone remodeling
osteocyte
mature bone cells and the main cell in bone tissue
osseous tissue
connective tissue that constitutes the endoskeleton
ossification
(also, osteogenesis) process of bone formation by osteoblasts
osteon
cylindrical structure aligned parallel to the long axis of the bone
resorption
process by which osteoclasts release minerals stored in bones
sesamoid bone
small, flat bone shaped like a sesame seed; develops inside tendons
short bone
bone that has the same width and length, giving it a cube-like shape
spongy bone tissue
forms the inner layer of all bones
suture bone
small, flat, irregularly shaped bone that forms between the flat bones of the cranium
trabeculae
lamellae that are arranged as rods or plates

38.2 Bone

Bone , or osseous tissue , is a connective tissue that constitutes the endoskeleton. It contains specialized cells and a matrix of mineral salts and collagen fibers.

The mineral salts primarily include hydroxyapatite, a mineral formed from calcium phosphate. Calcification is the process of deposition of mineral salts on the collagen fiber matrix that crystallizes and hardens the tissue. The process of calcification only occurs in the presence of collagen fibers.

The bones of the human skeleton are classified by their shape: long bones, short bones, flat bones, sutural bones, sesamoid bones, and irregular bones ( [link] ).

Shown are different types of bones: flat, irregular, long, short, and sesamoid.

Long bones are longer than they are wide and have a shaft and two ends. The diaphysis , or central shaft, contains bone marrow in a marrow cavity. The rounded ends, the epiphyses , are covered with articular cartilage and are filled with red bone marrow, which produces blood cells ( [link] ). Most of the limb bones are long bones&mdashfor example, the femur, tibia, ulna, and radius. Exceptions to this include the patella and the bones of the wrist and ankle.

The long bone is covered by articular cartilage at either end and contains bone marrow (shown in yellow in this illustration) in the marrow cavity.

Short bones , or cuboidal bones, are bones that are the same width and length, giving them a cube-like shape. For example, the bones of the wrist (carpals) and ankle (tarsals) are short bones ( [link] ).

Flat bones are thin and relatively broad bones that are found where extensive protection of organs is required or where broad surfaces of muscle attachment are required. Examples of flat bones are the sternum (breast bone), ribs, scapulae (shoulder blades), and the roof of the skull ( [link] ).

Irregular bones are bones with complex shapes. These bones may have short, flat, notched, or ridged surfaces. Examples of irregular bones are the vertebrae, hip bones, and several skull bones.

Sesamoid bones are small, flat bones and are shaped similarly to a sesame seed. The patellae are sesamoid bones ( [link] ). Sesamoid bones develop inside tendons and may be found near joints at the knees, hands, and feet.

The patella of the knee is an example of a sesamoid bone.

Sutural bones are small, flat, irregularly shaped bones. They may be found between the flat bones of the skull. They vary in number, shape, size, and position.


Integrative and perturbation-based analysis of the transcriptional dynamics of TGFβ/BMP system components in transition from embryonic stem cells to neural progenitors

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type β (TGFβ) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFβ system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions.

Keywords: TGFβ/BMP cell state transitions embryonic stem cells esiRNA genetic interaction neural differentiation signaling pathway.

©2019 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2019.


The effect of micro-macroporous biphasic calcium phosphate incorporated with polyphosphate on exophytic bone regeneration

In this study, the effect of micro-macroporous biphasic calcium phosphate(MBCP) incorporated with inorganic polyphosphate for bone regeneration in the calvaria of rabbit was evaluated.

The procedure of guided bone regeneration was performed with titanium reinforced expanded polytetrafluoroethylene(TR-ePTFE) membrane. Four animal groups were compared : 1) TR-ePTFE membrane for negative control group, 2) TR-ePTFE membrane filled with MBCP for positive control group, 3) TR-ePTFE membrane filled with MBCP soaked in 4% inorganic polyphosphate for experimental group I, and 4) TR-ePTFE membrane filled with MBCP soaked in 8% inorganic polyphosphate for experimental group II.

1. Negative control group showed the highest new bone formation at 16 weeks.

2. Positive control group showed the smallest new bone formation compared to other groups.

3. 8% inorganic polyphosphate induced more volume of bone formation, otherwise experimental group II did not show significant difference compared to negative control group.

These results suggest that inorganic polyphosphate has a promoting effect on bone regeneration, possibly by enhancing osteoconductivity of the carrier and by increasing osteoinductivity of the defected alveolar bone tissue.


MS4A4A: a novel cell surface marker for M2 macrophages and plasma cells

Current address: Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Current address: National Research Council, University of Prince Edward Island, Prince Edward Island, Canada.

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Current address: Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Alberta, Canada.

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Departments of Medicine and Oncology, University of Calgary, Calgary, Alberta, Canada

Calgary Laboratory Services, Foothills Medical Centre, Calgary, Alberta, Canada

Calgary Laboratory Services, Foothills Medical Centre, Calgary, Alberta, Canada

Calgary Laboratory Services, Foothills Medical Centre, Calgary, Alberta, Canada

Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, Alberta, Canada

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Dr JP Deans, Department of Biochemistry and Molecular Biology, Health Research Innovation Centre, Cumming School of Medicine, University of Calgary, 3280 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail: [email protected] Search for more papers by this author

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Current address: Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Current address: National Research Council, University of Prince Edward Island, Prince Edward Island, Canada.

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Current address: Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Alberta, Canada.

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Departments of Medicine and Oncology, University of Calgary, Calgary, Alberta, Canada

Calgary Laboratory Services, Foothills Medical Centre, Calgary, Alberta, Canada

Calgary Laboratory Services, Foothills Medical Centre, Calgary, Alberta, Canada

Calgary Laboratory Services, Foothills Medical Centre, Calgary, Alberta, Canada

Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, Alberta, Canada

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

Dr JP Deans, Department of Biochemistry and Molecular Biology, Health Research Innovation Centre, Cumming School of Medicine, University of Calgary, 3280 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail: [email protected] Search for more papers by this author

Abstract

MS4A4A is a member of the membrane-spanning, four domain family, subfamily A (MS4A) that includes CD20 (MS4A1), FcRβ (MS4A2) and Htm4 (MS4A3). Like the first three members of this family, transcription of MS4A4A appears to be limited to hematopoietic cells. To evaluate expression of the MS4A4A protein in hematopoietic cell lineages and subsets we generated monoclonal antibodies against extracellular epitopes for use in flow cytometry. In human peripheral blood we found that MS4A4A is expressed at the plasma membrane in monocytes but not in granulocytes or lymphocytes. In vitro differentiation of monocytes demonstrated that MS4A4A is expressed in immature but not activated dendritic cells, and in macrophages generated in the presence of interleukin-4 (‘alternatively activated’ or M2 macrophages) but not by interferon-γ and lipopolysaccharide (‘classically’ activated or M1 macrophages). MS4A4A was expressed in the U937 monocytic cell line only after differentiation. In normal bone marrow, MS4A4A was expressed in mature monocytes but was undetected, or detected at only a low level, in myeloid/monocytic precursors, as well as their malignant counterparts in patients with various subtypes of myeloid leukemia. Although MS4A4A was not expressed in healthy B lymphocytes, it was highly expressed in normal plasma cells, CD138+ cells from multiple myeloma patients, and bone marrow B cells from a patient with mantle cell lymphoma. These findings suggest immunotherapeutic potential for MS4A4A antibodies in targeting alternatively activated macrophages such as tumor-associated macrophages, and in the treatment of multiple myeloma and mantle cell lymphoma.

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Contents

Kenneth Lacovara [2] discovered the remains in the Cerro Fortaleza Formation in Santa Cruz Province, Patagonia, Argentina in 2005. Due to the large size of the bones and the remote location where they were found, it took his team four austral summers to fully excavate the remains. Mules, ropes and many team members were needed to finally get the field-jacketed bones to a truck.

In 2009, the fossils were transported to Philadelphia via an ocean freighter for preparation and study. Fossil preparation and analysis occurred at Drexel University, the Academy of Natural Sciences of Drexel University and the Carnegie Museum of Natural History. Dreadnoughtus schrani fossils will be returned to their permanent repository at the Museo Padre Molina in Rio Gallegos, Argentina. when?

The bones of both Dreadnoughtus specimens were scanned with a NextEngine 3D laser scanner. [3] Using the software Autodesk Maya, the scans of each bone were positioned in 3D space to create a digital articulated skeleton, which was then converted into 3D PDF files using the software GeoMagic. The high fidelity of these scans allowed Lacovara et al. (2014) to study the heavy fossils of Dreadnoughtus schrani in a way that was safe for the fossils and enhanced virtual and long-distance collaboration.

The holotype specimen, MPM-PV 1156, consists of a partial skeleton, somewhat preserved in its original layout, that comprises: a maxilla (jaw) fragment a tooth a posterior cervical vertebra cervical ribs multiple dorsal vertebrae and dorsal ribs the sacrum 32 caudal vertebrae and 18 haemal arches (bones from the tail) that include a sequence of 17 anterior and middle caudal vertebrae and their corresponding haemal arches found in their original layout the left pectoral girdle and forelimb minus the front foot both sternal plates all pelvic elements the left hind limb lacking a hind foot and right tibia metatarsals I and II and one claw from digit I.

The paratype, MPM-PV 3546, consists of a partially articulated postcranial skeleton of a slightly smaller individual whose remains were discovered in the same location as the holotype. It includes a partial anterior cervical vertebra, multiple dorsal vertebrae and ribs, the sacrum, seven caudal vertebrae and five haemal arches, a nearly complete pelvis, and the left femur. [3]

According to the research team that discovered the taxon, the genus name Dreadnoughtus "alludes to the gigantic body size of the taxon (which presumably rendered healthy adult individuals nearly impervious to attack)" and to the two Argentine dreadnoughts that served in the first half of the twentieth century, Rivadavia and Moreno. Thus, the genus name also honors the country in which Dreadnoughtus schrani was discovered. The name of the type species, schrani, was given in recognition of the American entrepreneur Adam Schran for his financial support of the project. [3]

Controversy over the mass/weight Edit

The researchers who described Dreadnoughtus schrani estimated its weight using Equation 1 of Campione and Evans (2012), [4] which allows the body mass of a quadrupedal animal to be estimated based only on the circumference of the humerus and femur. Using this scaling equation, they concluded that the Dreadnoughtus type specimen weighed about 59.3 tonnes (58.4 long tons 65.4 short tons). [3] By comparison, this would mean D. schrani weighed more than eight and a half times as much as a male African elephant and even exceeded the Boeing 737-900 airliner by several tons. [5] This very large mass estimate was quickly criticized. On SV-POW web blog, sauropod researcher Matt Wedel used volumetric models, based on the published figures, that yielded estimates between 36–40 tonnes (35–39 long tons 40–44 short tons), [6] or even as low as approximately 30 tonnes (30 long tons 33 short tons), based on a 20% shorter torso. [7] Researcher Gregory S Paul posted a response to Lacovara et al., pointing out that the error margins using equations based on limb bones are large using the same equation the Dreadnoughtus type specimen could have been anywhere between 44–74 tonnes (43–73 long tons 49–82 short tons). Using volumetric techniques based on a more accurate skeletal restoration, Paul estimated as low as 26 tonnes (26 long tons 29 short tons). [8]

A formal re-evaluation of the animal's weight was published in June 2015. In it, a research team led by Karl T. Bates compared the simple scaling equation results with results found using a volume-based digital model with various amounts of soft tissue and "empty space" for the respiratory system. They found that any model using the scale-based weight estimate would have meant the animal had an impossible amount of bulk (fat, skin, muscle, etc.) layered onto its skeleton. They compared their D. schrani volumetric model to those of other sauropods with more complete skeletons and better understood mass estimates to conclude that the D. schrani type specimen must have weighed in the range of 22.1–38.2 tonnes (21.8–37.6 long tons 24.4–42.1 short tons). [9] Lacovara disputes the methods used by Bates et al., arguing that the new study treats Dreadnoughtus as an exception to well-established mass estimate methods proven on living animals, and that the limb bones would be unnecessarily large if the new mass estimates were correct. [10] [11] In 2019 Gregory S. Paul estimated the weight of Dreadnoughtus type specimen at 31 tonnes (34 short tons) , slightly higher than his previous estimation. [12] In 2020 Dreadnoughtus was estimated at weighing 49,000 kilograms, 108,027 pounds [13]

The discovery of Dreadnoughtus schrani provides insight into the size and anatomy of giant titanosaurian sauropods, especially of the limbs and the shoulder and hip girdles. The majority of D. schrani bones are very well preserved. There is minimal deformation, especially in the limb bones. Fine features, such as locations of muscle attachment, are frequently clearly visible. Dreadnoughtus also has an unusually long neck for its body size, making up almost half of the animal's length.

Size Edit

Dreadnoughtus dimensions [3] [9]
Dimension Metric Imperial
Maximum mass ≈49,000 kilograms (49 t) 108,027lb [14] Total length
Total length ≈26 metres ≈85 feet
Head and neck length ≈12.2 m ≈40 ft
Neck-only length ≈11.3 m ≈37 ft
Torso and hip length ≈5.1 m ≈17 ft
Tail length ≈8.7 m ≈29 ft
Shoulder height ≈6 m ≈20 ft

Estimates based on measurements of the known parts of the skeleton suggest that the only known individual of Dreadnoughtus schrani was approximately 26 metres (85 ft) long and stood about 2 stories tall. [3] At 1.74 m, its scapula is longer than any other known titanosaur shoulder blade. [3] Its ilium, the top bone of the pelvis, is also larger than any other, measuring 1.31 m in length. [3] The forearm is longer than any previously known from a titanosaur, and it is only shorter than the long forearms of brachiosaurids, which had a more inclined body posture. [3] Only Paralititan [15] preserves a longer humerus (upper arm bone). Although each species likely had slightly different body proportions, these measurements demonstrate the massive nature of Dreadnoughtus schrani. [3] The current estimate for the mass of the type specimen is 49,000 kilograms, 108,027 pounds. [16]

Completeness Edit

Completeness may be assessed in different ways. Sauropod dinosaur skeletons are often recovered with little to no skull material, so completeness is often looked at in terms of postcranial completeness (i.e., the completeness of the skeleton excluding the skull). Completeness may also be assessed in terms of the numbers of bones versus the types of bones. The most important metric for understanding the anatomy of a fossil animal is the types of bones. The completeness statistics for Dreadnoughtus schrani are as follows:

The completeness of D. schrani compared with other extremely massive (over 40 metric tons) sauropods is as follows: [17]

Sauropod Skeletal Completeness Total Mirrored Postcranial Completeness
(i.e. types of bones)
Dreadnoughtus schrani 45.5% 70.4%
Turiasaurus riodevensis 44.1% 45.8%
Futalognkosaurus dukei 15.2% 26.8%
Paralititan stromeri 7.8% 12.7%
Argentinosaurus huinculensis 5.1% 9.2%
Antarctosaurus giganteus 2.3% 3.5%
Puertasaurus reuili 1.6% 2.8%

Thus, the skeleton of D. schrani is substantially more complete than those of all other extremely massive (>40 metric tons) dinosaurs. [3]

Posture Edit

All titanosaurs had what is called wide-gauge posture, a relative term to describe a stance in which the feet fell apart from the body midline. More derived titanosaurs had a greater degree of wide-gauge posture, [18] [19] with their limbs held more widely than their ancestors and contemporaneous counterparts. The stance of Dreadnoughtus schrani was clearly wide-gauge, but not to the degree of saltasaurids because the femoral condyles are perpendicular to its shaft rather than beveled. [3] This and the fact that the head of the femur was not turned in towards the body as in saltasaurids [18] support the phylogenetic conclusion that Dreadnoughtus was not a saltasaurid. The animal's broad sternal bones also demonstrate a wide pectoral girdle, giving it a broad-shouldered, broad-chested appearance. Paleontologist Kenneth Lacovara compared the animal's gait to an Imperial Walker. [20]

Although the forelimbs of D. schrani are longer than in any other previously known titanosaur, they are not significantly longer than the hind limbs. [3] Therefore, Lacovara et al. (2014) reconstructed its neck to have been held more horizontally, rather than anteriorly inclined in the manner of Brachiosaurus. [21]

Distinctive features Edit

The tail of Dreadnoughtus schrani has several characteristic features included in the diagnosis of the species. The first vertebra of the tail has a ridge on its ventral surface called a keel. In the first third of the tail, the bases of the neural spines are extensively subdivided into cavities caused by contact with air sacs (part of the dinosaur's respiratory system). In addition, the anterior and posterior boundaries of these neural spines have distinct ridges (pre- and postspinal laminae) connecting them to the pre- and postzygapophyses (the articulation points of the neural arches). In the middle of its tail, the vertebrae have a triangular process that extends over the centrum towards each preceding vertebra. [3]

Just like modern archosaurs with tails (crocodilians, for example), [22] D. schrani had bones below the vertebrae called chevrons or haemel arches. These bones connect with the ventral surface of the vertebrae and are “Y” shaped when viewed anteriorly. In Dreadnoughtus schrani the bottom stem of the “Y” is broadly expanded, likely for the attachment of muscles. [3]

The shoulder girdle and forelimb of D. schrani also exhibit unique features. An oblique ridge crosses the interior face of the scapular blade, extending from the top side near the far end of the blade to the bottom side near the base of the scapular blade. Finally, each end of the radius exhibits a unique form: the top, or proximal end, has a distinct concave embayment on its posterior face while the bottom, or distal end, is nearly square in shape instead of broadly expanded. [3]

Based on a cladistic analysis, Dreadnoughtus schrani appears to be a "derived" basal titanosaur that is not quite a lithostrotian. [3] Lacovara et al. (2014) note that because of the wide array of relatively "advanced" and "primitive" features in the skeleton of Dreadnoughtus schrani and the current instability of titanosaurian interrelationships, future analyses may find widely differing positions for it within Titanosauria.


Contents

The digit length is typically measured on the palmar (ventral) hand, from the midpoint of the bottom crease (where the finger joins the hand) to the tip of the finger. [1] Measurement of the digits on the dorsal hand, from the tip of the finger to the proximal phalange-bone protrusion (which occurs when digits are bent at 90 degrees to the palm), has recently also gained acceptance. [2] [3] A study has shown that, compared to the palmar digit ratio, the dorsal digit ratio is a better indicator of bone digit ratio. [3] Moreover, differential placing of flexion creases is a factor in the palmar digit ratio. [4]

Other digit ratios are also similarly calculated in the same hand.

The ratio of two digits in particular – the palmar 2nd (index finger) and 4th (ring finger) – is affected by fetal exposure to hormones, in particular to testosterone, and other androgens this 2D:4D ratio can be considered a crude measure for prenatal androgen exposure, with lower 2D:4D ratios pointing to higher prenatal androgen exposure. [5] [6] [7] [8] [9] [10] [11] [12] There are also studies that suggest that the palmar 2D:4D ratio is influenced by prenatal estrogen exposure, and that it thus correlates negatively not with prenatal testosterone alone, but rather with the prenatal testosterone-to-estrogen ratio (T:O). [13] [1] [14] [15]

In keeping with these hormonal differences, the digit ratios are sexually dimorphic, being lower in men than in women. In palmar digit ratios, strong sexual dimorphism occurs in those of digit 2. [16] [17] [18] In dorsal digit ratios, in contrast, strong sexual dimorphism occurs in those of digit 5, with women having shorter fifth digits on average. [2] Overall, the report of sexual dimorphism is much stronger in dorsal digit ratios [2] than in palmar digit ratios, especially as compared to the classic, palmar 2D:4D ratio. [19] Moreover, compared to palmar digit ratio, dorsal digit ratio is a better indicator of bone digit ratio. [3] Thus, while most of the earlier research has focused on palmar 2D:4D ratio, study of other digit ratios is also promising.

Experimental studies have shown prenatal testosterone injection produces male-typical changes in dermatoglyphics [20] and in palmar digit length, but not in bone digit length. [21] Moreover, this effect occurs in digit 2 but not in digit 4. [21] [22] Additionally, human epidermal tissues have only androgen receptors and no estrogen receptors-α. [23] Thus, it is likely that dermatoglyphic tissues in fingers may be more sensitive to prenatal testosterone effect, whereas, as reported above, bone digit ratios may be sensitive to testosterone-to-estrogen ratio. Hence, the palmar 2D:4D ratio reflects a combination of two different hormonal sensitivities. In support of this, a 2019 study has shown that differential placing of flexion creases contributes to sex differences in the palmar 2D:4D ratio. [4]

That a greater proportion of men have shorter index fingers than ring fingers than do women was noted in the scientific literature several times through the late 1800s, [24] with the statistically significant sex difference in a sample of 201 men and 109 women established by 1930, [25] after which time the sex difference appears to have been largely forgotten or ignored. In 1983, Glenn Wilson of King's College London published a study examining the correlation between assertiveness in women and their digit ratio, which found that women with a lower 2D:4D ratio reported greater assertiveness. [26] This was the first study to examine the correlation between digit ratio and a psychological trait within members of the same sex. [27] Wilson proposed that skeletal structure and personality were simultaneously affected by sex hormone levels in utero. [26] In 1998, John T. Manning and colleagues reported the sex difference in digit ratios was present in two-year-old children [28] and further developed the idea that the index was a marker of prenatal sex hormones. Since then, research on the topic has burgeoned around the world.

A 2009 study in Biology Letters argues: "Sexual differences in 2D:4D are mainly caused by the shift along the common allometric line with non-zero intercept, which means 2D:4D necessarily decreases with increasing finger length, and the fact that men have longer fingers than women", [29] which may be the basis for the sex difference in digit ratios and/or any putative hormonal influence on the ratios.

A 2011 paper by Zhengui Zheng and Martin J. Cohn reports "the 2D:4D ratio in mice is controlled by the balance of androgen to estrogen signaling during a narrow window of digit development". [5] The formation of the digits in humans, in utero, is thought to occur by 13 weeks, and the bone-to-bone ratio is consistent from this point into an individual's adulthood. [30] During this period if the fetus is exposed to androgens, the exact level of which is thought to be sexually dimorphic, the growth rate of the 4th digit is increased, as can be seen by analyzing the 2D:4D ratio of opposite sex dizygotic twins, where the female twin is exposed to excess androgens from her brother in utero, and thus has a significantly lower 2D:4D ratio. [31]

Importantly, there has been no correlation between the sex hormone levels of an adult and the individual's 2D:4D, [6] which implies that it is strictly the exposure in utero that causes this phenomenon.

A major problem with the research on this topic comes from the contradiction in the literature as to whether the testosterone level in adults can be predicted by the 2D:4D ratio. [6]

From a study of 136 males and 137 females at the University of Alberta: [32]

Assuming a normal distribution, the above lead to 95% prediction intervals for 2D:4D ratio of 0.889–1.005 for males and 0.913–1.017 for females.

From a 2018 study on a final sample of 249 graduate and undergraduate students from Warwick University, [33] proportionally balanced by gender:

The sex difference in 2D:4D is present before birth in humans. [14] [34] The ratio of testosterone to estradiol measured in 33 amniocentesis samples correlated with the child's subsequent 2D:4D ratio. [13] The conclusion of this research supported to an association between low 2D:4D and high levels of testosterone compared with estrogen, and high 2D:4D with low testosterone relative to estrogen.

The level of estrogen in the amniotic fluid is not correlated with higher 2D:4D, as researchers found no difference in estrogen levels between males and females. [13]

Several studies present evidence that digit ratios are heritable. [35] [36]

In a non-clinical sample of women, digit ratio correlated with anogenital distance in the expected direction. In other words, women with a greater anogenital distance, indicating greater prenatal androgen exposure, had a smaller digit ratio. [37]

Disorders of sex development Edit

Women with congenital adrenal hyperplasia (CAH), which results in elevated androgen levels before birth, have lower, more masculinized 2D:4D on average. [7] [8] [38] Other possible physiological effects include an enlarged clitoris and shallow vagina. [39] Males with CAH have more smaller (more masculine) digit ratios than control males, [7] [8] suggesting that prenatal androgens affect digit ratios. Amniocentesis samples show that prenatal levels of testosterone are in the high-normal range in males with CAH, while levels of the weaker androgen androstenedione are several fold higher than in control males. [40] [41] [42] These measures indicate that males with CAH are exposed to greater prenatal concentrations of total androgens than are control males.

A greater digit ratio occurs for men with Klinefelter's syndrome, who have reduced testosterone secretion throughout life compared to control males, than in their fathers or control males. [10]

Digit ratio in men may correlate with genetic variation in the androgen receptor gene. [43] Men with genes that produce androgen receptors that are less sensitive to testosterone (because they have more CAG repeats) have greater, i.e. more feminine, digit ratios. There are reports of a failure to replicate this finding. [44] However, men carrying an androgen receptor with more CAG repeats compensate for the less sensitive receptor by secreting more testosterone, [45] probably as a result of reduced negative feedback on gonadotropins. Thus, it is not clear that 2D:4D would be expected to correlate with CAG repeats, even if it accurately reflects prenatal androgen.

XY individuals with androgen insensitivity syndrome (AIS) due to a dysfunctional gene for the androgen receptor present as women and have feminine digit ratios on average, as would be predicted if androgenic hormones affect digit ratios. This finding also demonstrates that the sex difference in digit ratios is unrelated to the Y chromosome per se. [46]

Other animals Edit

In pheasants, the ratio of the 2nd to 4th digit of the foot has been shown to be influenced by manipulations of testosterone in the egg. [47]

Studies in mice indicate that prenatal androgen acts primarily by promoting growth of the fourth digit. [5]

It is not clear why digit ratio is influenced by prenatal hormones. There is evidence of other similar traits, e.g. otoacoustic emissions and arm-to-trunk length ratio, which show similar effects. Hox genes responsible for both digit and penis development [48] have been implicated in affecting these multiple traits (pleiotropy). Direct effects of sex hormones on bone growth might be responsible, either by regulation of Hox genes in digit development or independently of such genes. Likewise, it is unclear why digit ratio on the right hand should be more responsive than that on the left hand, as is indicated by the greater sex difference on the right than the left. [49] However, because no right–left difference has been found in sexual dimorphism of bone digit ratios (2D:4D [50] [51] [52] ) and dorsal digit ratios [53] and because differential placing of flexion creases contributes to sex differences in palmar digit ratio, [4] right–left differences in the placing of flexion creases may be determining right–left difference in palmar 2D:4D ratio.

One study on mice from 2011 suggests that the 2D:4D ratio correlates with prenatal sex hormone levels because the androgen receptor and estrogen receptor activity is higher in digit 4 than in digit 2. Inactivation of AR decreases growth of digit 4, which causes a higher 2D:4D ratio, whereas inactivation of estrogen receptor alpha (ER-α) increases growth of digit 4, which leads to a lower 2D:4D ratio. [5]

Manning and colleagues have shown that 2D:4D ratios vary greatly between different ethnic groups. In a study with Han, Berber, Uygur and Jamaican children as subjects, Manning et al. found that Han children had the highest mean values of 2D:4D (0.954±−0.032), they were followed by the Berbers (0.950±0.033), then the Uygurs (0.946±0.037), and the Jamaican children had the lowest mean 2D:4D (0.935±0.035). [54] [55] This variation is far larger than the differences between sexes in Manning's words, "There's more difference between a Pole and a Finn, than a man and a woman." [56]

The standard deviations associated with each given 2D:4D mean are considerable. For example, the ratio for Han children (0.954±−0.032) allows for a ratio as low as 0.922, while the ratio for Jamaican children (0.935±0.035) allows for a ratio as high as 0.970. Thus, some ethnic groups' confidence intervals overlap.

A 2008 study by Lu et al. found that the mean values of 2D:4D of the Hui and the Han in Ningxia were lower than those in European countries like Britain. [57]

In 2007 Manning et al. also found that mean 2D:4D varied across ethnic groups with higher ratios for Whites, Non-Chinese Asians, and Mid-Easterners and lower ratios in Chinese and Black samples. [58]

Two studies explored the question of whether geographical differences in 2D:4D ratios were caused by gene pool differences or whether some environmental variable associated with latitude might be involved (e.g., exposure to sunlight or different day-length patterns). The conclusions were that geographical differences in 2D:4D ratio were caused by genetic pool differences, not by geographical latitude. [59] [60]

Consanguinous parentage (inbreeding) has been found to lower the 2D:4D ratio in offspring, [61] which may account for some of the geographical and ethnic variation in 2D:4D ratios, as consanguinity rates depend on, among others, religion, culture, and geography. [62]

Some authors suggest that digit ratio correlates with health, behavior, and even sexuality in later life. Below is a non-exhaustive list of some traits that have been either demonstrated or suggested to correlate with either high or low digit ratio.

  • Increased risk of prostate cancer and prostate diseases in males. [63][64]
  • Slower utero fetal development in both sexes. [63]
  • Increased reproductive success in males. [54]
  • Longer penis in males. [65]
  • Left handedness or left hand preference [66]
  • Increased risk of breast cancer in females. [67]
  • Lowered sperm counts [28]
  • Increased risk for heart disease in males [68][69]
  • Increased risk of obesity and metabolic syndrome in males [70]
  • Reduced risk for prostate cancer[71][72]
  • Reduced birth size in males [73][74]
  • Increased reproductive success in females. [54]
  • Increased rate of ADHD in males [75][76][77][78]
  • Increased rate of Asperger syndrome and other autism spectrum disorders (when comparing digit ratio to general population) [79][80]
  • Increased risk in females for anorexia nervosa. [81]
  • Increased psychopathy in men with low digit ratio and high adult testosterone levels. [82]
  • Increased rate of alcohol dependency, [83] more frequent and more severe binge drinking [84]
  • Increased risk for depression in males [85]
  • Increased rate of schizophrenia[86]
  • Increased rate of borderline personality disorder. [87]
  • Increased rate of psychopathy in females and increased rate of callous affect (subscale of psychopathy) in males [88]
  • Reduced risk of alcohol dependency[83]
  • Reduced risk of video game addiction[89]
  • Increased anxiety in males [90]
  • Increased risk in females for bulimia. [81]
  • Reduced performance in sports [92]
  • Reduced financial trading ability [93]
  • Right handedness skills [94] (inconclusive) [95]
    in females [26] in females [96] in males [32][97][98][99][100][101] in females [102]
  • Hyperactivity and poor social cognitive function in girls [103]
  • Masculinized handwriting in females [104]
  • Perceived 'dominance' and masculinity of man's face [105][106]
  • In an orchestral context, rank and musical ability in males [107]
  • Right hand low digit ratio predicts academic performance [108]
  • Inverted U-shape relation between digit ratio and mathematical ability (participants with both high and low digit ratios earn lower grades in mathematics, while participants with intermediate digit ratios achieve the highest grades) [109]
  • Decreased empathy in men, in response to adult testosterone levels [82][110]
  • Higher propensity to attack without being provoked [111]
  • Increased risk-taking behavior in men [112]
  • Normative degrees of cooperation and sharing, as opposed to excessive altruism or egoism, which were both correlated with higher digit ratio [113]
  • Mean 2D:4D ratio among artists is lower than among controls [114]
  • Higher numeracy (compared to literacy) in children [115]
  • Higher criminal offending rates after puberty[116][117]
  • Attenuated socio-affective skills [118]
  • Conduct disorder in boys [119]
    traits correlated with digit ratio, higher being more feminized [120][121][122]
  • Greater openness personality factor [123] and superstitious beliefs among people with a higher digit ratio [124]
  • Higher exam scores among male students [47][115]
  • Higher neuroticism in both sexes with higher right hand digit ratio [125] and on left hand in females [96]
  • Higher left hand digit ratio in response to high adult testosterone levels predicts musical orchestra rank in females. [126]
  • Higher verbal fluency in both sexes. [63]
  • Higher visual recall in females. [127]
  • Higher literacy (compared to numeracy) in children [115]
  • Lesbians have a lower digit ratio, on average, than heterosexual women [128][129][56][130][131][132][133][134][135][136][137][138][139]
  • Bisexual men have a lower digit ratio than exclusively homosexual men and community volunteers recruited regardless of sexual orientation. [140]
  • Tendency toward polygamy [141]
  • Sexual preference for more masculine men among women [129] and gay men [142] with high digit ratio.
  • Lesbians are more likely to be femme and less likely to be butch with a high digit ratio. [130][143] Identical female twins discordant for sexual orientation still show the difference (lesbian less than straight, on average) in digit ratio. [132][139][128]
  • Homosexuality for men, according to some studies. [131][139][144] Other studies have disputed this some have shown that the digit ratio in homosexual men is similar to, [56][136][137][138][145] or lower than, [133][128][135][140] that of heterosexual men. One study concluded that differences are dependent on geographical variation, with gay men having lower or similar ratios to straight men in Europe, but higher or similar in the United States. [146] But this finding has been questioned in a meta-analysis including 18 studies, which suggested that ethnicity, rather than geography, explained the differences previously found in men of different sexual orientations. The meta-analysis concluded that no significant sexual orientation differences in digit ratio exist in men. [138]
  • Tendency toward monogamy [141]

Male-to-female transgender women Edit

A study in Germany has found a correlation between digit ratio and male-to-female transgender women. Trans women were found to have a higher digit ratio than males. This was not true for trans men, however, who were within the average range for females. [147]

Digit ratio and development Edit

There is some evidence that 2D:4D ratio may also be indicative for human development and growth. Ronalds et al. (2002) showed that men who had an above average placental weight and a shorter neonatal crown-heel length had higher 2D:4D ratios in adult life. [148] Kumar et al. [66] have reported that a similar effect of hand preference on digit lengths and digit ratios occurs oppositely among children and adults. Moreover, studies about 2D:4D correlations with face shape suggest that testosterone exposure early in life may set some constraints for subsequent development. Prenatal sex steroid ratios (in terms of 2D:4D) and actual chromosomal sex dimorphism were found to operate differently on human faces, but affect male and female face shape by similar patterns. [149] Fink et al. (2004) found that men with low (indicating high testosterone) and women with high (indicating high estrogen) 2D:4D ratios express greater levels of facial symmetry. [150]

2D:4D is being used alongside other methods to help understand Palaeolithic hand stencils found in prehistoric European and Indonesian cave painting. [151] [152] [153]


Acknowledgements

The authors acknowledge technical support from Wyatt Technology and especially J. Champagne. The authors also acknowledge the Genomics Resource Core facility (WCM) for their high-quality service. The authors thank C. Ghajar and J. Weiss for feedback on the manuscript and members of the Lyden laboratory for discussions. Our study was supported by the National Cancer Institute (U01-CA169538 to D.L.), the National Institutes of Health (NIH R01-CA169416 to D.L. and H.P. R01-CA218513 to D.L. and H.Z.), the US Department of Defense (W81XWH-13-10249 to D.L.), W81XWH-13-1-0425 (to D.L., J.Br.), the Sohn Conference Foundation (D.L., I.M., H.P. and H.Z.), the Children’s Cancer and Blood Foundation (D.L.), The Manning Foundation (A.H. and D.L.), The Hartwell Foundation (D.L.), The Nancy C. and Daniel P. Paduano Foundation (D.L.), The Starr Cancer Consortium (H.P. and D.L. D.L. and H.Z.), the Pediatric Oncology Experimental Therapeutic Investigator Consortium (POETIC D.L.), the James Paduano Foundation (D.L. and H.P.), the NIH/WCM CTSC (NIH/NCATS: UL1TR00457 to H.M. and H.Z. UL1TR002384 to D.L., H.M. and H.Z.), the Malcolm Hewitt Wiener Foundation (D.L.), the Champalimaud Foundation (D.L.), the Thompson Family Foundation (D.L., R.S.), U01-CA210240 (D.L.), the Beth Tortolani Foundation (J.Br.), the Charles and Marjorie Holloway Foundation (J.Br.), the Sussman Family Fund (J.Br.), the Lerner Foundation (J.Br.), the Breast Cancer Alliance (J.Br.), the Manhasset Women’s Coalition Against Breast Cancer (J.Br.), the National Institute on Minority Health and Health Disparities (NIMHD) of the NIH (MD007599 to H.M.), NIH/NCATS (UL1TR00457 to H.M.). C.R., A.M., D.F., A.F., A.S. and H.O. acknowledge FEDER (Fundo Europeu de Desenvolvimento Regional funds through COMPETE 2020) POCI, Portugal 2020 (NORTE-01-0145-FEDER-000029) and FCT – Fundação para a Ciência e a Tecnologia in the framework of the project ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274) and the FCT project POCI-01-0145-FEDER-016585 (PTDC/BBB-EBI/0567/2014). The authors acknowledge FCT for grants to A.M. (SFRH/BPD/75871/2011) and A.F. (SFRH/BPD/111048/2015). D.F. acknowledges FCT (SFRH/BD/110636/2015), the BiotechHealth PhD Programme (PD/0016/2012) and the American Portuguese Biomedical Research Fund.


At a nearby construction zone, workers have made a startling discovery. They uncovered several bones that look like they were buried some time ago. You are part of a team of forensic anthropologists who have been called in to analyze these bones. Unfortunately, the bones were heavily damaged by the construction equipment. The bones have all been mixed up, and several have been crushed. However, you think you can use the bones that are left to determine the number of bodies and the height of each individual.

When a body is discovered, it is important to learn as much as possible from the remains. Forensic anthropologists use mathematical formulas to estimate someone&rsquos height from the lengths of certain bones in their body.

1. Using a ruler or tape measure, measure the length of your femur in centimeters. This is the large bone that runs from your hip socket to your knee cap. The bone that sticks out near your hip is part of the femur and is called the greater trochanter . Record this information in the table below for you and your lab partners. Gather data from at least three people in your class.

*Use the femur length and the chart to calculate your height and compare that to your actual height. If your race isn't listed, you can find more formulas online.

2. Next, measure the length of your tibia. Start at the the tibial tuberosity (bump on your shin) to the medial malleolus , the bump on your ankle. Use the chart to calculate your height based on the tibia.

3. Finally, measure your ulna length by bending your arm and measuring from the proximal end of the ulna (elbow bump) to the distal end, the styloid process of the ulna . The styloid process is visible as a bump near your wrist. Use the chart to calculate your height based on the ulna.

4. Complete the table for at least 3 members in your group or class.

Name:
Actual Height (cm)
Femur Length (cm)
Calculated Height (cm)
Tibia Length (cm)
Calculated Height (cm)
Ulna Length (cm)
Calculated Height (cm)

Construction Site

The following bones were recovered from the construction site. A fellow forensic anthropologist has already classified the bones by sex and race.

Using the mathematical formulas, calculate the approximate height of each individual.

Bone # Bone Type Length (cm) Race Sex Calculated Height (cm)
1 Humerus 38.2 Caucasian Male
2 Femur 44.0 African-American Female
3 Ulna 25.4 Caucasian Male
4 Femur 52.4 Caucasian Male
5 Femur 43.9 African-American Female
6 Tibia 45.7 Caucasian Male

Discussion Questions:

1. Is it possible that any of the bones came from the same person? Which bones do you think might be the same person and provide an explanation for WHY you think so.

2. What is the minimum number of bodies buried at this site? What is the maximum number of bodies buried at this site? Explain your reasoning.

3. Consider a case where two females have the same femur lenght. Would you expect those famels to be the exact same height? Why or why not?

4. On the formula table, there is a symbol shown as ± . What does this symbol mean?

5. Consider your calculated heights and your actual height. Are they within the range that was expected. Suggest a reason for why a person's calculated height might not be accurate.


Materials and methods

Subject recruitment and generation of osteoclast-like cells

Details of the recruitment process and cell culture procedures used in this study have been described previously [3]. Briefly, the osteoclast eQTL study cohort comprises 158 women aged 30–70 years with self-reported European ancestry who attended the Bone Density Unit at Sir Charles Gairdner Hospital in Western Australia for a dual-energy X-ray absorptiometry BMD scan in 2016 (Hologic, Bedford, MA, USA). Exclusion criteria used during recruitment included presence of medical conditions or use of medications that are likely to influence osteoclastic bone resorption or the process of osteoclastogenesis. Osteoclast-like cells were generated using the conventional procedure peripheral blood mononuclear cells were first isolated from blood samples obtained from each individual by density gradient centrifugation using protocols well established in our laboratory [3, 49]. These cells were cultured in triplicate for 2 days in α-MEM supplemented with 25 ng/ml macrophage colony stimulating factor (M-CSF), then for a further 12 days in α-MEM supplemented with 25 ng/ml M-CSF and 100 ng/ml RANKL while osteoclastogenesis occurred. The osteoclastic character of the cultures was verified by staining for tartrate-resistant acid phosphatase (TRAP) as described previously [3] and by gene expression profiling (Additional file 1: Fig. S6).

Nucleic acid extraction

Genomic DNA for each participant was extracted from whole blood using the QIAamp DNA Blood Mini Kit (QIAGEN) according to the manufacturer’s instructions. At day 14 of culture, RNA and DNA were harvested from each set of triplicate osteoclast-like cell cultures using the AllPrep DNA/RNA Mini Kit (QIAGEN) according to the manufacturer’s instructions, with on-column DNase digestion for the RNA fraction. High-quality RNA was obtained, with all samples recording RNA integrity numbers (RINs) ≥ 9.7.

Genotyping and imputation

Genome-wide array genotyping was performed on the genomic DNA samples using the Illumina Infinium OmniExpress-24 BeadChip array. QC criteria applied to the genotype data included removal of individuals with a call rate < 90%, variants that were monomorphic, unmapped, had a MAF < 5%, Hardy-Weinberg equilibrium P < 5 × 10 −8 or call rate < 90%, leaving 572,898 variants for imputation. Genotype imputation was then performed by the Sanger Imputation Service using the Haplotype Reference Consortium (HRC) release 1.1 reference panel [50]. Any variants with an IMPUTE2 info score < 0.4 were removed from the imputed dataset. Relatedness testing and principal components analysis was performed on the genotype dataset using Plink v1.9 [51], with 10 principal components generated for use as covariates in the eQTL analysis to correct for population stratification.

Generation and processing of gene expression data

Transcriptome-wide quantitation of gene expression was performed on the osteoclast RNA samples using 50 bp single-end RNA-Seq on an Illumina HiSeq 2500. Raw read counts were generated for each gene (GENCODE v25), while those displaying a read count < 1 per million or expressed in < 10 individuals were removed. Trimmed mean of M value (TMM) normalisation and correction for total read count by conversion to counts per million (CPM) was performed on the gene expression data using the edgeR package [52]. Reads per kilobase per million (RPKM) values were also calculated for each gene using edgeR [52] to enable comparison of expression levels between different genes.

EQTL association analysis

The top genetic variant at each of the 1103 recently identified independent association signals for eBMD [19] was investigated for cis- (local) eQTL effects in the osteoclast samples. The eQTL analysis was performed on the TMM-normalised CPM gene expression values using the FastQTL software [53], which performs linear regressions between genotypes and gene expression values. Quantile normalisation was implemented using FastQTL (based on the rntransform function of the GenABEL package [54]) to ensure the gene expression values were normally distributed with a mean of 0 and standard deviation of 1. Only variants with a MAF ≥ 5% were included in the analysis, which was adjusted for the covariates patient age, RNA-Seq batch and 10 genomic principal components. Each variant was tested for association with the expression of any gene with a TSS located within a 1 Mb window on either side of the variant, consistent with the methods used by the GTEx project. Correction for multiple testing was performed using the Benjamini-Hochberg procedure [55], utilising a FDR of 5%.

Co-localisation analysis

To assess the probability that eBMD GWAS and osteoclast eQTL association signals residing in the same locus share the same causal variant, we performed a co-localisation analysis of the two datasets using the coloc package in R (default settings) [21]. This software uses a Bayesian framework to calculate posterior probabilities for 5 different scenarios (hypotheses) regarding the presence and sharing of causal variants between two genetic association datasets using summary statistics. A posterior probability of > 50% for hypothesis H4 for a specific locus (association with trait 1 and trait 2, one shared variant) indicates that co-localisation of the association signals in the two datasets due to a shared causal variant is the most likely of the 5 scenarios. We also performed co-localisation analysis using an updated version of the coloc software, named coloc2 [22]. Coloc2 performs alignment of the GWAS and eQTL summary results for each cis-eQTL region as a pre-processing step and includes optional changes implemented in the gwas-pw algorithm [56].

Summary-data-based Mendelian Randomisation analysis

To further characterise functionally relevant genes for the eBMD GWAS associations and to identify potential pleiotropic effects on gene expression and eBMD, we performed integration of the complete set of eBMD GWAS summary results with the osteoclast eQTL dataset (± 1 Mb) using the SMR software [23]. This package applies the principles of Mendelian randomisation [57] to test for association between gene expression and a trait due to a shared variant at a genetic locus. The software performs an SMR test, which detects dual association signals in the GWAS and eQTL datasets by testing for association between gene expression and the trait of interest at the top associated eQTL for each gene. The software also performs a heterogeneity in dependent instruments (HEIDI) test, which compares the association signals for nearby co-inherited markers in the GWAS and eQTL datasets. A significant HEIDI test indicates heterogeneity in the association profiles of the two datasets, thereby suggesting that the association signals seen in each dataset are less likely to be driven by the same causal variant. The osteoclast eQTL cohort genotype dataset was used as the reference panel in these analyses for estimation of LD, and only genes with at least 1 cis-eQTL association at P < 5 × 10 −8 were included (N = 1070). A Bonferroni multiple-testing corrected significance threshold of P < 4.7 × 10 −5 was used for the SMR test (PSMR), while a conservative significance threshold of P < 0.05 was set for the HEIDI test (PHEIDI) as an indicator of heterogeneity.

Osteoclast eQTL enrichment analysis

The GARFIELD software [24] was used to test for enrichment of osteoporosis risk variants among high-confidence osteoclast eQTL. GARFIELD performs LD pruning (r 2 > 0.1) of GWAS association results to generate an independent set of variants and then integrates this with annotations containing regulatory or functional information. Each variant is annotated to a functional category or regulatory feature if the variant or an LD proxy (r 2 > 0.8) is part of that annotation group. Fold enrichment is calculated using odds ratios at specified GWAS P value thresholds with significance assessed using a generalised linear model while accounting for MAF, distance to nearest TSS and number of LD proxies. A custom annotation category was generated containing all significant osteoclast eQTL associations identified at FDR 5%. Enrichment of eBMD GWAS variants in this annotation was assessed at four GWAS significance thresholds: P < 1 × 10 −5 , 1 × 10 −6 , 1 × 10 −7 and 1 × 10 −8 . The UK10K variant set was used as the reference population for these analyses, with correction for multiple testing performed using the Bonferroni method.

X-ray microcomputed tomography analysis of mouse bones

Ripk3-deficient mice were generated and phenotyped as described previously [58]. Femora were cleaned of soft tissue and fixed in 10% neutral buffered formalin for 48 h, followed by 24 h immersion in phosphate-buffered saline (PBS). Five pairs of 15-week-old male Ripk3 −/− mice and WT controls were used for the micro-CT analysis. Fixed femora were immersed in PBS and immobilised in a 2-ml tube prior to scanning of the distal femur using a Skyscan 1176 micro-CT instrument (Bruker, Kontich, Belgium). Scan parameters were as follows: 50 kV, 500 μA, 0.5 mm Al filter, exposure time 1 s, 0.4° rotation step, frame averaging of 2 and pixel resolution 8.89 μm. Scans were reconstructed using NRecon software (Bruker, Kontich, Belgium) using a constant threshold value and then analysed using the CTAn software (Bruker, Kontich Belgium). A trabecular region of interest was defined 0.5 mm below the base of the growth plate and 1 mm in height. A cortical region of interest was defined 3 mm below the growth plate and 1 mm in height. Trabecular extent was defined as the distance from the base of the growth plate to the furthest definable continuous trabecular element. Reported trabecular values were bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), trabecular extension (Tb.Ex) and BMD reported cortical values were cortical thickness (Ct.Th), cortical bone volume (Ct.BV), marrow volume (Ma.V), endocortical perimeter (Ec.Pm), periosteal perimeter (Ps.Pm) and tissue mineral density (TMD). Differences between WT and Ripk3 −/− samples were assessed using an unpaired t test.

Histomorphometric analysis of mouse bones

Mouse femur samples were formalin-fixed, decalcified and embedded in paraffin before being subjected to staining with H&E and a chromogenic TRAP substrate to characterise in vivo osteoblast and osteoclast parameters. Stained bone sections were scanned using a Scanscope XT machine (Aperio) at × 20 objective, with histomorphometric analysis performed using the BioQuant Osteo software (BioQuant). To characterise the trabecular bone, a region of interest located approximately 500 μm below the growth plate at the distal femur and 1 mm in height was defined. For the cortical bone analysis, we used a region located approximately 4 mm below the growth plate with a height of 1 mm. Two sections were analysed for each femur, with the mean of the two sets of measurements used for statistical analysis. Differences between the WT and Ripk3 −/− groups were assessed using an unpaired t test.


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