This project introduces some knowledge about the basics involved in finding the

constituents of bone. This Project deals with the principle of qualitative analysis of
cation and anion.

Skeletal system plays an integral part of most of the animals what is that it
makes it form an integral part? The solution of this can be understood more
clearly from this project.

INDEX

CONTENTS PAGE NO

Bone – Introdution 6

Functions 7

Characteristics 8

Cellular Structure 10

Molecular Structure 11

Types of Bones 13

Formation 14

Re-Modelling 16

Paracrine Cell Signalling 17

Experiment Analysis 18

Report of the project 19

Results 20
Disorders 21

Osteology 22

Terminology 23,24

Bibliography 25

Significance of project:

 Deals with analysis of bone ash

 Provides all basic ideas about concentration of various salts present

 Provides information about ion exchange reaction

This project indeed would be a revolution in the world where there is increasing
worry about problems of bone like osteoporosis and in this industrial age amount of
calcium content in bone is also reducing; this project would indeed be a very good
solution.

Bone

Bones are rigid organs that form part of the endoskeleton of vertebrates. They

function to move, support, and protect the various organs of the body, produce red
and white blood cells and store minerals. Bone tissue is a type of dense connective
tissue. Because bones come in a variety of shapes and have a complex internal and
external structure they are lightweight, yet strong and hard, in addition to fulfilling
their many other functions. One of the types of tissue that makes up bone is the
mineralized osseous tissue, also called bone tissue, that gives it rigidity and
a honeycomb-like three-dimensional internal structure. Other types of tissue found
in bones include marrow, endosteum and periosteum, nerves, blood vessels and
cartilage. There are 206 bones in the adult human body and 270 in an infant

Functions

Bones have eleven main functions:

Mechanical

 Protection — Bones can serve to protect internal organs, such as the skull

protecting the brain or the ribsheart and lungs.protecting the
 Shape — Bones provide a frame to keep the body supported.

 Movement — Bones, skeletal muscles, tendons, ligaments and joints function

together to generate and transfer forces so that individual body parts or the
whole body can be manipulated in three-dimensional space. The interaction
between bone and muscle is studied in biomechanics.

 Sound transduction — Bones are important in the mechanical aspect of

overshadowed hearing.

 Blood production — The marrow, located within the medullary cavity of long

bones and interstices of cancellous bone, produces blood cells in a process
called haematopoiesis.

Synthetic

Metabolic

 Mineral storage — Bones act as reserves of minerals important for the body,
most notably calciumphosphorus.and
 Growth factor storage — Mineralized bone matrix stores important growth
factors such as insulin-like growth factors, transforming growth factor, bone
morphogenetic proteins and others.

 Fat Storage — The yellow bone marrow acts as a storage reserve of fatty
acids.
  Acid-base balance — Bone buffers the blood against excessive pH changes by
absorbing or releasing alkaline salts.

 Detoxification — Bone tissues can also store heavy metals and other foreign
elements, removing them from the blood and reducing their effects on other
tissues. These can later be gradually released for excretion.

 Endocrine organ – Bone controls phosphate metabolism by

releasing fibroblast growth factor – 23 (FGF-23), which acts on kidneys to
reduce phosphate re absorption.

Characteristics

The primary tissue of bone, osseous tissue, is a relatively hard and

lightweight composite material, formed mostly of calcium phosphate in the chemical
arrangement termed calcium hydroxylapatite (this is the osseous tissue that gives
bones their rigidity). It has relatively high compressive strength but poor tensile
strength of 104-121 MPa, meaning it resists pushing forces well, but not pulling
forces. While bone is essentially brittle, it does have a significant degree
of elasticity, contributed chiefly by collagen. All bones consist of living and
dead cells embedded in the mineralized organic matrix that makes up the osseous
tissue.

Individual bone structure

Bone is not a uniformly solid material, but rather has some spaces between its hard
elements.

Compact bone or (Cortical bone)

The hard outer layer of bones is composed of compact bone tissue, so-called due to
its minimal gaps and spaces. This tissue gives bones their smooth, white, and solid
appearance, and accounts for 80% of the total bone mass of an adult skeleton.
Compact bone may also be referred to as dense bone.

Trabecular bone
Filling the interior of the bone is the trabecular bone tissue (an open
cell porousnetwork also called cancellous or spongy bone), which is composed of a
network of rod- and plate-like elements that make the overall organ lighter and
allowing room for blood vessels and marrow. Trabecular bone accounts for the
remaining 20% of total bone mass but has nearly ten times the surface area of
compact bone. If for any reason there is an alteration in the strain to which the
cancellous subjected there is a rearrangement of the trabeculae. Although adult
bone exists in both cancellous and compact forms, there is no microscopic
difference between the two.

Cellular structure

There are several types of cells constituting the bone;

 Osteoblasts are mononucleate bone-forming cells that descend

from osteoprogenitor cells. They are located on the surface of osteoid seams
and make a protein mixture known as osteoid, which mineralizes to become
bone. The osteiod seam is a narrow region of newly formed organic matrix,
not yet mineralized, located on the surface of a bone. Osteoid is primarily
composed of Type I collagen. Osteoblasts also manufacture hormones, such
as prostaglandins, to act on the bone itself. They robustly produce alkaline
phosphatase, anenzyme that has a role in the mineralisation of bone, as well
as many matrix proteins. Osteoblasts are the immature bone cells.
 Bone lining cells are essentially inactive osteoblasts. They cover all of the
available bone surface and function as a barrier for certain ions.

 Osteocytes originate from osteoblasts that have migrated into and become
trapped and surrounded by bone matrix that they themselves produce. The
spaces they occupy are known as lacunae. Osteocytes have many processes
that reach out to meet osteoblasts and other osteocytes probably for the
purposes of communication. Their functions include to varying degrees:
formation of bone, matrix maintenance and calcium homeostasis. They have
also been shown to act as mechano-sensory receptors — regulating the
bone’s response to stress and mechanical load. They are mature bone cells.
  Osteoclasts are the cells responsible for bone resorption (remodeling of bone
to reduce its volume). Osteoclasts are large, multinucleated cells located on
bone surfaces in what are called Howship’s lacunae or resorption pits. These
lacunae, or resorption pits, are left behind after the breakdown of the bone
surface. Because the osteoclasts are derived from a monocyte stem-cell
lineage, they are equipped withphagocytic-like mechanisms similar to
circulating macrophages. Osteoclasts mature and/or migrate to discrete bone
surfaces. Upon arrival, active enzymes, such as tartrate resistant acid
phosphatase, are secreted against the mineral substrate.

Molecular structure

Matrix

The majority of bone is made of the bone matrix. It has inorganic and organic
parts. Bone is formed by the hardening of this matrix entrapping the cells. When
these cells become entrapped from osteoblasts they become osteocytes.

Inorganic

The inorganic is mainly crystalline mineral salts and calcium, which is present in the
form of hydroxyapatite. The matrix is initially laid down as unmineralised osteoid
(manufactured by osteoblasts). Mineralisation involves osteoblasts
secreting vesicles containing alkaline phosphatase. This cleaves the phosphate
groups and acts as the foci for calcium and phosphate deposition. The vesicles then
rupture and act as a centre for crystals to grow on.

Organic

The organic part of matrix is mainly composed of Type I collagen. This is

synthesised intracellularly as tropocollagen and then exported, forming fibrils. The
organic part is also composed of various growth factors, the functions of which are
not fully known. Factors present
includeglycosaminoglycans, osteocalcin, osteonectin, bone sialo
protein, osteopontin and Cell Attachment Factor. One of the main things that
distinguishes the matrix of a bone from that of another cell is that the matrix in
bone is hard.

Woven or lamellar

Two types of bone can be identified microscopically according to the pattern of

collagen forming the osteoid (collagenous support tissue of type I collagen
embedded in glycosaminoglycan gel

1) woven bone characterised by haphazard organisation of collagen fibers and is

mechanically weak, and

2) lamellar bone which has a regular parallel alignment of collagen into sheets
(lamellae) and is mechanically strong.

Woven bone is produced when osteoblasts produce osteoid rapidly which occurs
initially in all fetalbones (but is later replaced by more resilient lamellar bone). In
adults woven bone is created afterfractures or in Paget’s disease. Woven bone is
weaker, with a smaller number of randomly oriented collagen fibers, but forms
quickly; it is for this appearance of the fibrous matrix that the bone is
termedwoven. It is soon replaced by lamellar bone, which is highly organized
in concentric sheets with a much lower proportion of osteocytes to surrounding
tissue. Lamellar bone, which makes its first appearance in the fetus during the third
trimester,[3] is stronger and filled with many collagen fibers parallel to other fibers
in the same layer (these parallel columns are called osteons). In cross-section, the
fibers run in opposite directions in alternating layers, much like in plywood,
assisting in the bone’s ability to resist torsion forces. After a fracture, woven bone
forms initially and is gradually replaced by lamellar bone during a process known as
“bony substitution.”

These terms are histologic, in that a microscope is necessary to differentiate

between the two.

Types
There are five types of bones in the human body: long, short, flat, irregular and
sesamoid.

 Long bones are characterized by a shaft, the diaphysis, that is much greater

in length than width. They are comprised mostly of compact bone and lesser
amounts of marrow, which is located within the medullary cavity, and spongy
bone. Most bones of the limbs, including those of the fingers and toes, are
long bones. The exceptions are those of the wrist, ankleand kneecap.
 Short bones are roughly cube-shaped, and have only a thin layer of compact
bone surrounding a spongy interior. The bones of the wrist and ankle are
short bones, as are thesesamoid bones.

 Flat bones are thin and generally curved, with two parallel layers of compact
bones sandwiching a layer of spongy bone. Most of the bones of the skull are
flat bones, as is thesternum.

 Irregular bones do not fit into the above categories. They consist of thin
layers of compact bone surrounding a spongy interior. As implied by the
name, their shapes are irregular and complicated. The bones of the spinehips
are irregular bones.and

 Sesamoid bones are bones embedded in tendons. Since they act to hold the
tendon further away from the joint, the angle of the tendon is increased and
thus the leverage of the muscle is increased. Examples of sesamoid bones
are the patella and the pisiform.Compared to woven bone , lamellar bone
formation takes place more slowly. The orderly deposition of collagen fibers
restricts the formation of osteoid to about 1 to 2 µm per day.

Lamellar bone requires a relatively flat surface to lay the collagen fibers in parallel
or concentric layers.

Formation

The formation of bone during the fetal stage of development occurs by two
processes: Intramembranous ossification and endochondral ossification.
Intramembranous ossification

Intramembranous ossification mainly occurs during formation of the flat bones of

the skull; the bone is formed from mesenchyme tissue. The steps in
intramembranous ossification are:

1. Development of ossification center

2. Calcification

3. Formation of trabeculae

4. Development of periosteum

Endochondral ossification

Endochondral ossification, on the other hand, occurs in long bones, such as limbs;
the bone is formed from cartilage. The steps in endochondral ossification are:

1. Development of cartilage model

2. Growth of cartilage model

3. Development of the primary ossification center

4. Development of the secondary ossification center

5. Formation of articular cartilage and epiphyseal plate

Endochondral ossification begins with points in the cartilage called “primary

ossification centers.” They mostly appear during fetal development, though a few
short bones begin their primary ossification after birth. They are responsible for the
formation of the diaphyses of long bones, short bones and certain parts of irregular
bones. Secondary ossification occurs after birth, and forms the epiphyses of long
bones and the extremities of irregular and flat bones. The diaphysis and both
epiphyses of a long bone are separated by a growing zone of cartilage
(the epiphyseal plate). When the child reaches skeletal maturity (18 to 25 years of
age), all of the cartilage is replaced by bone, fusing the diaphysis and both
epiphyses together (epiphyseal closure).
Bone marrow

Bone marrow can be found in almost any bone that holds cancellous tissue.
In newborns, all such bones are filled exclusively with red marrow, but as the child
ages it is mostly replaced by yellow, or fatty marrow. In adults, red marrow is
mostly found in the marrow bones of the femur, the ribs, the vertebrae and pelvic
bones.

Remodeling

Remodeling or bone turnover is the process of resorption followed by replacement

of bone with little change in shape and occurs throughout a person’s life.
Osteoblasts and osteoclasts, coupled together via paracrine cell signalling, are
referred to as bone remodeling units.

Purpose

The purpose of remodeling is to regulate calcium homeostasis, repair micro-

damaged bones (from everyday stress) but also to shape and sculpture the
skeleton during growth.

Calcium balance

The process of bone resorption by the osteoclasts releases stored calcium into the
systemic circulation and is an important process in regulating calcium balance. As
bone formation actively fixes circulating calcium in its mineral form, removing it
from the bloodstream, resorption actively unfixes it thereby increasing circulating
calcium levels. These processes occur in tandem at site-specific locations.

Repair

Repeated stress, such as weight-bearing exercise or bone healing, results in the

bone thickening at the points of maximum stress (Wolff’s law). It has been
hypothesized that this is a result of bone’s piezoelectric properties, which cause
bone to generate small electrical potentials under stress. [4]
Paracrine cell signalling

The action of osteoblasts and osteoclasts are controlled by a number of

chemical factors which either promote or inhibit the activity of the bone remodelling
cells, controlling the rate at which bone is made, destroyed or changed in shape.
The cells also use paracrine signalling to control the activity of each other.

Osteoblast stimulation

Osteoblasts can be stimulated to increase bone mass through increased secretion

of osteoid and by inhibiting the ability of osteoclasts to break down osseous tissue.

Bone building through increased secretion of osteoid is stimulated by the secretion

of growth hormone by the pituitary, thyroid hormone and the sex hormones
(estrogens and androgens). These hormones also promote increased secretion
of osteoprotegerin.[5] Osteoblasts can also be induced to secrete a number
of cytokines that promote reabsorbtion of bone by stimulating osteoclast activity
and differentiation from progenitor cells. Vitamin D, parathyroid hormone and
stimulation from osteocytes induce osteoblasts to increase secretion of RANK-ligand
and interleukin 6, which cytokines then stimulate increased reabsorbtion of bone by
osteoclasts. These same compounds also increase secretion ofmacrophage colony-
stimulating factor by osteoblasts, which promotes the differentiation of progenitor
cells into osteoclasts, and decrease secretion of osteoprotegerin.

Osteoclast inhibition

The rate at which osteoclasts resorb bone is inhibited by calcitonin and

osteoprotegerin. Calcitonin is produced by parafollicular cells in thethyroid gland,
and can bind to receptors on osteoclasts to directly inhibit osteoclast activity.
Osteoprotegerin is secreted by osteoblasts and is able to bind RANK-L, inhibiting
osteoclast stimulation.

Experimental Analysis

Materials Required
PARTICULARS QUANTITY

Rib Bone 2 Pieces

Beaker 150 ml

Test Tube 7 nos

Evaporating Dish 1 no

Ring Stand 1 no

Bunsen Burner 1 no

Test Tube Holder 2 nos

Filter Paper -

PH Paper -

Dil. Nitric Acid 200 ml

1% Ammonium Hydroxide 100 ml

1% Silver Nitrate 25 ml

1% Ammonium Chloride 50 ml

Acetic Acid 100 ml

1% Potassium Thiocyanate 25 ml

Distilled Water As Reqd

Report of Project

EXPERIMENT OBSERVATION
  A strip of bone was burnt in Yellowish white precipitate was
evaporating dish obtained
 2 gms of bone as was
weighed
 To it dilute nitric acid was On adding Nitric acid the ash
added sparingly dissolved
 It was diluted with water and
the ash was completely
dissolved
 The above solution was
filtered and the residue (left
on the filter paper) was
discarded
 Ammonium hydroxide was The pH was made to 8.6
added to the filtrate (left on Whitish brown precipitate of
the beaker) Magnesium ammonium phosphate
was obtained
 The solution was made basic.
The basicity was checked with
the help of pH paper
 The solution was filtered and
the residue was isolated
 The filtrate was separated White precipitate of Silver
into two test tubes chloride was obtained
 Silver nitrate was added to White residue of calcium
one of the test tubes
 To the other test tube Carbonate was obtained
ammonium chloride and
ammonium carbonate was
added simultaneously and
boiled
 To the solution left, dilute HCL Red colour solution marking the
was added followed by presence of Iron was obtained
Potassium thiocyanate

Result

Extrapolation from the above observations

Constituents of bone ash identified were:

1. I. Calcium
2. II. Phosphate

3. III. Chloride

4. IV. Magnesium

5. V. Iron

Apart from this Calcium and Phosphate which is found maximum in bone
was estimated from the precipitate got. This was done by weighing the
precipitate

 Weight of Calcium carbonate:-                                               1.7 g

 Weight of Mg (NH4) PO4 :-                                                     1.1 g

 Weight of Ca in 2g of sample:-                                            0.68 g

 Weight of Phosphorous:-                                                    0.24 g

 % of Ca:-                                                                                34%

 % of Phosphorous:-                                                             12%

Disorders

There are many disorders of the skeleton. One of the more prominent
is osteoporosis.

Osteoporosis

Osteoporosis is a disease of bone, leading to an increased risk of fracture. In

osteoporosis, the bone mineral density (BMD) is reduced, bone microarchitecture is
disrupted, and the amount and variety of non-collagenous proteins in bone is
altered. Osteoporosis is defined by the World Health Organization (WHO) in women
as a bone mineral density 2.5 standard deviations below peak bone mass (20-year-
old sex-matched healthy person average) as measured by DXA; the term
“established osteoporosis” includes the presence of a fragility fracture.[6]
Osteoporosis is most common in women after the menopause, when it is
called postmenopausal osteoporosis, but may develop in men and
premenopausal women in the presence of particular hormonal disorders and
other chronic diseases or as a result of smoking and medications,
specifically glucocorticoids, when the disease is called steroid- or glucocorticoid-
induced osteoporosis (SIOP or GIOP).

Osteoporosis can be prevented with lifestyle advice and medication, and preventing
falls in people with known or suspected osteoporosis is an established way to
prevent fractures. Osteoporosis can be treated with bisphosphonates and various
other medical treatments.

Other

Other disorders of bone include:

 Bone fracture
 Osteomyelitis

 Osteosarcoma

 Osteogenesis imperfecta

 Osteochondritis Dissecans

 Bone Metastases

 Neurofibromatosis type I

Osteology

The study of bones and teeth is referred to as osteology. It is frequently used

in anthropology, archeology and forensic science for a variety of tasks. This can
include determining the nutritional, health, age or injury status of the individual the
bones were taken from. Preparing fleshed bones for these types of studies can
involve maceration – boiling fleshed bones to remove large particles, then hand-
cleaning.
Typically anthropologists and archeologists study bone tools made by Homo sapiens
and Homo neanderthalensis. Bones can serve a number of uses such as projectile
points or artistic pigments, and can be made from endoskeletal or external bones
such as antler or tusk.

Alternatives to bony endoskeletons

There are several evolutionary alternatives to mammillary bone; though they have

some similar functions, they are not completely functionally analogous to bone.

 Exoskeletons offer support, protection and levers for movement similar to

endoskeletal bone. Different types of exoskeletons include shells,carapaces
(consisting of calcium compounds or silica) and chitinous exoskeletons.
 A true endoskeleton (that is, protective tissue derived from mesoderm) is
also present in Echinoderms. Poriferaspicules and a spongin fiber network.
(sponges) possess simple endoskeletons that consist of calcareous or
siliceous

Exposed bone

Bone penetrating the skin and being exposed to the outside can be both a natural
process in some animals, and due to injury:

 A deer’s antlers are composed of bone.

 Instead of teeth, the extinct predatory fish Dunkleosteus had sharp edges of
hard exposed bone along its jaws.

 A compound fracture occurs when the edges of a broken bone puncture the
skin.

 Though not strictly speaking exposed, a bird’s beak is primarily bone covered

in a layer of keratin over a vascular layer containing blood vessels and nerve
endings.

Terminology

Several terms are used to refer to features and components of bones throughout the body:
Bone feature Definition

articular A projection that contacts an adjacent bone.

process

articulation The region where adjacent bones contact each other — a joint.

canal A long, tunnel-like foramen, usually a passage for notable nerves

or blood vessels.

condyle A large, rounded articular process.

crest A prominent ridge.

eminence A relatively small projection or bump.

epicondyle A projection near to a condyle but not part of the joint.

facet A small, flattened articular surface.

foramen An opening through a bone.

fossa A broad, shallow depressed area.

fovea A small pit on the head of a bone.

Labyrinth A cavity within a bone.

line A long, thin projection, often with a rough surface. Also known as
a ridge.

malleolus One of two specific protuberances of bones in the ankle.

meatus A short canal.

process A relatively large projection or prominent bump.(gen.)

ramus An arm-like branch off the body of a bone.

sinus A cavity within a cranial bone.

spine A relatively long, thin projection or bump.

suture Articulation between cranial bones.

trochanter One of two specific tuberosities located on the femur.

tubercle A projection or bump with a roughened surface, generally smaller

than a tuberosity.

tuberosity A projection or bump with a roughened surface.

Bibliography

 Biology Investigations

-        Otto, Towle, Crider

 Concise Inorganic Chemistry

-        J.D.Lee

 Wikipedia

 NCERT Biology and chemistry Textbook

 The Journal of Biological Chemistry

-        Anon