UNIVERSITY GHENT

FACULTY PHARMACEUTICAL SCIENCE

Department of Pharmaceutics

Laboratory for Pharmaceutical Technology

Academy year 2009-2010

PREPARATION AND CHARACTERIZATION OF

HYDROXYAPATITE - ALENDRONATE COMPOSITES

Katrien COLSON

Eerste Master in de Farmaceutische Zorg

Promotor

Prof. dr. apr. J. P. Remon

Commissarissen

Prof. dr. apr. J. Demeester

Dr. apr. K. Raemdonck

COPYRIGHT

"The author and the promoters give the authorization to consult and to copy parts of this thesis

for personal use only. Any other use is limited by the laws of copyright, especially concerning

the obligation to refer to the source whenever results from this thesis are cited."

May 25, 2010

Promoter Author

Prof. dr. apr. J. P. Remon Katrien Colson

DANKWOORD

I would like to mention several people, that have helped

and supported me during the time I had to write my thesis.

First of all, I would like to thank Prof. dr. apr. J.P.

Remon, who gave me the opportunity to experience

Erasmus and write my thesis in Pavia.

Subsequently, a word of thanks to Prof. Conti, for the

assistance during my stay in Pavia. Also thanks to

Priscilla for all the help with my thesis and the daily aid

in the laboratory. I appreciate their good care while I was

sick.

I would like to thank the Italians girls at the laboratory,

Romain, Luca and Kaja for the great moments in Pavia.

Thanks to Julie for the support and friendship in Italy and

Belgium.

At last, I would like to thank my family, who supported me

and Van Eenaeme Christian for reading my thesis.

TABLE OF CONTENTS

1. INTRODUCTION ............................................................................................................... 1

1.1. BONE TISSUE REGENERATION ............................................................................... 1

1.1.1. Tissue regeneration ............................................................................................... 1

1.1.2. Biomineralization .................................................................................................. 2

1.1.2.1. Bone ................................................................................................................. 2

1.1.3. Bone diseases .......................................................................................................... 5

1.2 BISPHOSPHONATES .................................................................................................... 8

1.2.1. Structure – Activity interaction ........................................................................... 9

1.2.2. Effects of BPs on osteoclasts ............................................................................... 11

1.2.3. Classification ........................................................................................................ 12

1.2.4. BPs and implants ................................................................................................. 13

1.3. CALCIUM PHOSPHATES ......................................................................................... 14

1.3.1. Hydroxyapatite (HA) .......................................................................................... 14

2. OBJECTIVES .................................................................................................................... 16

3. MATERIALS ..................................................................................................................... 18

4. METHODS ........................................................................................................................ 19

4.1. NANOCOMPOSITES ................................................................................................. 19

4.1.1. Preparation of hydroxyapatite-alendronate sodium composites .................... 19

4.1.2. Characterization of HA – ALN composites ...................................................... 20

4.1.2.1. Transmission Electron Microscopy (TEM) .................................................... 20

4.1.2.2. Scanninig electron microscope (SEM) ........................................................... 20

4.1.2.3. Brunauer-Emmett-Teller (BET) ..................................................................... 21

4.1.2.4. Fourier transform spectroscopy ...................................................................... 22

4.1.2.5. Differential Scanning Calorimeter (DSC) ...................................................... 22

4.2. DETERMINATION OF ALENDRONATE BY NINHYDRIN ................................. 23

4.2.1. Construction of calibration curve ...................................................................... 24

4.2.2. Adsorption and desorption curve ...................................................................... 25

4.2.2.1. Determination of the amount of alendronate adsorbed to HA ....................... 25

4.2.2.1. ALN release from HA-ALN composites ....................................................... 26

5. RESULTS AND DISCUSSION ........................................................................................ 27

5.1. CHARACTERIZATION OF HA-ALN COMPOSITES ............................................. 28

5.1.1. TEM images ......................................................................................................... 28

5.1.2. SEM images ......................................................................................................... 30

5.1.3. BET analysis ........................................................................................................ 34

5.1.4. DSC analysis ........................................................................................................ 35

5.1.5. FTIR analysis ....................................................................................................... 38

5.2. DETERMINATION OF ALENDRONATE ................................................................ 40

5.2.1. Construction of ALN calibration curve by a ninhydrin assay ........................ 40

5.2.2. Adsorption curve ................................................................................................. 42

5.2.3. ALN release from HA-ALN composites (desorption curve) ........................... 44

6. CONCLUSION .................................................................................................................. 46

7. REFERENCES .................................................................................................................. 47

LIST OF ABBREVIATIONS

HA: Hydroxyapatite

NCPs: Noncollagenous proteins

BSP: Bone sialoprotein

ON : Osteonectin

OP : Osteopontin

OC : Osteocalcin

BPs: Bisphosphonates

FPP: farnesylpyrophosphate

GGPP: geranylgeranylpyrophosphate

DDS: Drug delivery system

ALN: Alendronate

TEM: Transmission electron microscopy

SEM: Scanning electron microscopy

FTIR: Fourier transform infrared microscopy

BET: Brunauer-Emmett-Teller method

DSC: Differential scanning colorimetry

1

1. INTRODUCTION

1.1. BONE TISSUE REGENERATION

1.1.1. Tissue regeneration

Nowadays bone regeneration is a rising discipline that has become essential

in our society. The prominent aim of this regenerative medicine is to regenerate bone

tissue. Although osseous tissue is able to repair internal tissues without any scars, there

are particular situations where bone regeneration by replacement is needed. In general

we can agree that bone tissue engineering is inevitable when the damage has reached a

critical limit, whereby non-healing and critical-size bone defects arise. Those critical

injuries may arise after fracture comminution, infections, diseases or simply an

inadequate repair management of the bone tissues. The causes of these injuries can be

acquired or congenital (Mourino & Boccaccini, 2009).

Briefly it can be assumed that bone regeneration demands four important

elements: a morphogenetic signal, host cells that will answer this specific signal, a

vehicle and a host bed, which is well vascularised. It is obvious that to insure this

medical therapy, research, understanding and collaboration of many fields of science is

demanded (Burg et al., 2000).

Before considering bone and biomineralization, it is useful to consider three

critical considerations in bone tissue engineering, specifically osteoconduction,

osteoinduction and osseointegration. Osteoinduction is the possibility of a graft or

material to generate bone formation by sending signals. It is an active process that starts

from nonosseous environments and it leads to pluripotential cells, chondrocytes and

osteoblasts. On the other hand when the graft or the material is the matrix, we use the

term osteoconduction. Here the materials aid the cells and capillaries to form bone.

There is also a certain demand for growth factors and vascularization. In contrast to

osteoinduction, osteoconduction is a passive process. The direct contact and interaction

between living bone and an implant material correspond to osseointegration (Burg et al.,

2000; Palmer et al., 2008).

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1.1.2. Biomineralization

Biomineralization is a natural process by which biominerals are produced in a

controlled way by living organisms. Biomineralized tissues have a hierarchical structure

constituted by two main phases: the organic and inorganic phase. The first plays a

crucial role in the mineralized tissues by holding the structure and is produced

biological. The latter phase is the other component of the matrix and contains nano- and

microscale amorphous or chrystalline minerals. We call these minerals biominerals.

Briefly we can say that biominerals are inorganic chemical substances, who are

normally crystalline. By several techniques we can distinguish them, based on

remarkable chemical compositions and distinctive physical and mechanical properties,

such as hardness, colour, melting temperature, etc. These biominerals are the structural

basis of bones in mammals (Palmer et al., 2008;

http://en.wikipedia.org/wiki/Biomineralization).

Biominerals in bones can be impaired by demineralization. Demineralization

consists of the removal of the inorganic parts or the biominerals from the matrix.

Osteoclasts may fulfill this process, specifically called bone resorption. Bone resorption

can be considered as a physiological and pathological demineralization conducted by

osteoclasts. It is at this point where bone tissue engineering plays a major role. We can

discover and learn how mineralized tissues were built and which functions they

accomplish. Once we understand this we can use our knowledge to develop therapeutic

approaches in repair and healing of human tissues by producing synthetic hybrid

materials (Palmer et al., 2008).

1.1.2.1. Bone

Bone is a complex, hierarchical, dynamic, well vascularized connective

tissue, with a three-dimensional internal structure. Bones are hard and strong tissues.

Bone tissues are the most highly organized hybrids in biology. The bone matrix is

composed of an organic (30%) and an inorganic (70%) fraction. The organic part of the

matrix includes collagen, glycoproteins, proteoglycans and sialoproteins. The minerals

form the inorganic phase. The most abundant mineral found in mammals, particularly in

bones, is carbonated hydroxyapatite (HA). Carbonated HA or Ca10(PO4,CO3)6(OH)2 is a

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calcium phosphate mineral. The term apatite in HA is based on the chemical structure

A4B6(MO4)6X2. The X is a hydroxide ion. The letters A and B coincide both with

calcium and MO4 is a phosphate group (Palmer et al., 2008; Hill et al., 1998;

http://www.technion.ac.il/~mdcourse/274203/lect5.html).

Bone is an unique tissue which undergoes non stop remodelling and has many

functions. The continuously regeneration protects the body from damage and it prevents

the body against extreme aging. The main mechanical functions of bone are:

- support of the body

- protect vital organs (lung, heart, brain)

- attachment for muscles

- movement

Bone is also a storage location for bone marrow, in which stem cells are produced. Stem

cells form blood cellular components in a process called haematopoiesis. This is the

synthetic function of bone. The last important function of bone is the metabolic activity.

Bone maintains a mineral reservoir. It is a homeostasis regulator, important for the

regulation, storage and release of calcium and phosphates. Bone releases these minerals

in function of the body’s physiological needs. In addition to the storage of minerals,

bone is also responsible for the storage of fat. At last bone operates as an acid-base

balance. It buffers blood against extreme pH changes by adsorbing or releasing alkaline

salts (Palmer et al., 2008; http://en.wikipedia.org/wiki/Osseous_tissue).

The structure of bone is complex, there are molecular and cellular

components. The hierarchical architecture is composed of seven different levels. The

first level contains water, HA, collagen and other proteins and is the basis and primary

building block for the other levels. The crystals of HA are mostly nanoscale crystals

with platelets as dominant morphology. HA confers rigidity to bone. Collagen is a

structural protein and one of the organic biomacromolecules of bone. Into bone we find

type I collagen, which is an extracellular matrix molecule, synthesized intracellularly as

tropocollagen. The tropocollagen units join to each other in a parallel way, producing

the “hole zone”. The “hole zones” are crucial for mineralization because they form the

site of mineral nucleation. Moreover, the size of this zone influences the mineral growth.

4

The noncollagenous proteins (NCPs) constitute 10% of the total proteins. NCPs include

organic components as bone sialoprotein (BSP), osteonectin (ON), osteopontin (OP) and

osteocalcin (OC) (Palmer et al., 2008).

The mineralized collagen fibrils are forming the second level. The HA

crystals are concentrated in the “hole zones” and their axis is nearly parallel to the long

axis of the collagen fibril (Palmer et al., 2008).

A collection of these mineralized collagen fibrils, associated with other

arrangements, constitute the third level. A pattern of this collection forms the fourth

level of hierarchy (Palmer et al., 2008).

The fifth level is made of cellular components. This level consists of the

osteoclasts and osteoblasts. The sixth level of bone is characterized by a compact

(cortical) and a spongy (trabecular or cancellous) osseous tissue. Cortical bone has a

minimum of gaps and holes and it has a low porosity of 5 – 10%. It consists of

cylindrical units, called osteons or Haversian systems. Cancellous bone has on the other

hand a huge porosity, which is needed to give space to marrow and blood vessels. The

actual difference between those two osseous tissues is that blood covers bone in

cancellous bone and that bone covers blood in the cortical bone. Furthermore,

cancellous bone has no osteons. The entire bone, on macroscopic scale, is the seventh

and last level of the hierarchy of the bone structure (Palmer et al., 2008).

The main cellular components of bone are osteoblasts and osteoclasts. The

osteoblasts are immature bone cells. They are active, mononuclear, bone forming cells.

They are located on the bone surface, which is not yet mineralized. They derive from

mesenchymal osteoprogenitor cells and they form an extracellular protein mixture,

called osteoid, by producing organic components, containing type I collagen. The

osteoid includes, next to collagen, NCPs and polysaccharides. Bone is formed when

osteoid is mineralized by binding of calcium salts. Osteoblasts are responsible for

osteoid secretion. In addition, they secrete an enzyme, called alkaline phosphatase, that

plays a major role in the mineralization of bone. Finally, osteoblasts are important for

calcium and phosphates concentrations to stimulate apatite mineralization. They

indirectly control the levels of bone resorption. After fulfilling their functions they

5

undergo apoptosis or they differentiate into osteocytes (Palmer et al., 2008;

http://www.technion.ac.il/~mdcourse/274203/lect5.html).

Osteocytes are mature bone cells. They originate from osteoblasts, that

migrate to and become trapped into the bone matrix. Osteocytes are star-shaped and

form the lacunae in the bone matrix. They are in contact and communicate with other

osteocytes and osteoblasts through dendritic processes. The main function of osteocytes

is to give signals in response to mechanical stress, in case of matrix resorption or

formation. The other functions of osteocytes are matrix maintenance, calcium

homeostasis and formation of bone (Palmer et al., 2008;

http://www.technion.ac.il/~mdcourse/274203/lect5.html).

Osteoclasts are mature, multi-nucleated secretory cells. The osteoclast

appears to be derived from a monocyte-granulocyte precursor cell. They are located on

the bone surface in shallow depressions, called the Howship’s lacunae. Their main

function is to remove or resorb bone (bone resorption). The osteoclast undergoes

changes in its structure to fulfill this resorption process. The cell membrane changes into

a “ruffled border”, the actin cytoskeleton of the osteoclast forms a “sealing zone”. This

is a ring that will accomplish the dissolution of the HA crystals. Lysosomal cysteine

proteases and matrix metalloproteases continue and complete together the breakdown of

the organic phase of the matrix after hydrolysis by the osteoclasts. Osteoclasts dissolve

both the inorganic and organic part of the matrix. Normally, bone resorption is followed

by new bone formation and is essential for growth, healing, remodelling and regulation

of calcium in the body (Palmer et al., 2008;

http://www.technion.ac.il/~mdcourse/274203/lect5.html).

1.1.3. Bone diseases

As we know bone tissue is able to heal and remodel itself with a minimum of

scars. However, the ability and capacity of regeneration is not infinite. When people

have severe injuries or a specific disease, bone loss occurs without healing. The loss of

bone or fragile bone tissue can be very dangerous, particularly in elderly patients. The

presence of weak bones can give many complications for human health and it may affect

life expectancy and quality of life (Palmer et al., 2008).

6

There are many pathologies related to mineralization of bone tissues and they

may impair the quality of life. Not only mineral density may be decreased, but

microarchitecture can be disrupted as well. Important examples of bone diseases are

osteoporosis, osteogenesis imperfecta, renal osteodystrophy, osteomalacia,

osteomyelitis, Paget’s disease, tumor induced osteolysis and hypercalcemia of

malignancy etc.

Osteoporosis may be a metabolic bone disease, by which the entire skeleton

can be affected. In general there is a reduction of peak bone mass due to an increased

bone porosity and an excessive bone resorption. However, bone formation is mostly

normal. The illness is most common in women after menopause (postmenopausal) or by

senile loss of bone. Bone loss occurs without specific symptoms, so osteoporosis is a

silent disease. Risk factors for osteoporosis are :

Elderly women

Medical problems (rheumatoid arthritis, chronic bronchitis,

hyperthyroidism and chronic liver disease can increase the

development of osteoporosis)

Genetic influence (having a family member with osteoporosis)

Medication (glucocorticoids)

Like many bone tissue related pathologies, there is an increased incidence of bone

fractures and the underlying mechanism is an imbalance between bone resorption and

bone formation. These fractures will normally not occur in healthy people and can be

fatal for patients, suffering from osteoporosis (http://www.osteoporosestichting.nl/wat-

is-osteoporose.html).

Similar to osteoporosis, there is a fulminate osteoclastic activity in Paget’s

disease and renal osteodystrophy. Paget’s disease is characterized by a mixture of

osteoblastic and osteoclastic phases, that leads to an unorganized bone formation, with

disordered, enlarged, brittle bone. It may be caused through a viral infection of the

paramyxovirus, or it can be transferred by genes. Paget’s disease is a chronic disease,

without particularly symptoms, but the illness can be associated with pain in bones, hair

loss and headaches. Early diagnosis and treatment is very important. Children of

7

affected people should perform tests every two years. In renal osteodystrophy, the

skeletal changes are due to a chronic renal disease. The kidneys are regulators of the

maintenance of calcium and phosphates in the blood. When the kidneys are affected and

have lost a part of their ability to maintain the ion levels in blood, renal osteodystrophy

may occur. Similar to Paget’s disease, there are no specific symptoms: in adults bone

fractures, bone deformities occur, while slow bone growth is common in children

(http://en.wikipedia.org/wiki/Paget's_disease_of_bone).

Osteogenesis imperfecta is one of the most common congenital disease of

connective tissue and it is a type of osteoporosis. More specifically, osteogenesis

imperfecta is a group of genetic diseases, of four different types. Type І and type Π are

best known. Type І of osteogenesis imperfecta is characterized by autosomal dominant

genes and is usually lethal. Type Π needs two copies of the mutant gene to cause the

illness and is not lethal. So they are both caused by a mutation in genes, whereby

abnormalities of type І collagen composition or type І collagen production originate.

Increased frequency of fractures is common (http://www.orpha.net/data/patho/GB/uk-

OI.pdf).

Osteomalacia is characterized by an accumulation of unmineralized bone

tissue. People suffering of osteomalacia have weak and soft bones, caused by

demineralization. Osteomalacia may be due to a poor diet or to a poor absorption of

minerals, such as calcium, from food. Depletion of calcium in bones may be the result.

The symptoms of this metabolic bone disease vary depending on the stage of

development of the disease and on the person himself. In general the symptoms are

weak bones, bone fractures, bone softening and bone pain.

Hypermalacia occurs often after advanced malignancies. Hypermalacia is

common in patients suffering from lung cancer, breast cancer or renal cell carcinoma.

Specific mediators (cytokines and prostaglandins) affect the matrix of bone, leading to

release and loss of calcium. The kidneys reabsorb more calcium in the renal tubules. The

result is an increased concentration of calcium ions in the blood. Without treatment,

patients suffering from hypermalacia will die, due to excessive dehydration. They have

to take their illness seriously. Dehydration is caused by nausea, constipation and cramps

(Mehrotra, 2009).

8

Hereafter bisphosphonates will be discussed. These are drugs used against

bone diseases. In particular bone diseases with an excessive osteoclast activity. This is

their most important clinical use. (Table 1.1)

TABLE 1.1.: MAJOR APPLICATIONS IN WICH BISPHOSPHONATES CAN BE

USED CURENTLY AND IN THE FUTURE. (Russell & Rogers, 1999)

1.2 BISPHOSPHONATES

Bisphosphonates (BPs) are a class of powerful drugs. Since many years, BPs

are a well known treatment for systemic metabolic bone diseases. A variety of specific

bone diseases, caused by an increased amount of osteoclasts or with an increased

activity of osteoclasts, are the main targets of these drugs. They perform their function

by acting as strong inhibitors of the osteoclast-mediated bone resorption. After their

pharmacological action, they are mostly excreted unmetabolized by the kidneys. The

BPs are used to treat Paget’s disease, tumor-induced osteolysis and hypercalcemia and

postmenopausal osteoporosis. Moreover, BPs would seem to promote fixation and

prevent migration of implants, such as nails and screws.

*Bone scanning agents

*Inhibition of calcification

Heterotopic bone formation

Dental calculus

*Reducing bone resorption

Paget’s disease

Hypercalcemia of malignancy

Multiple myeloma

Bone metastases, especially breast cancer

Osteoporosis

Glucocorticosteroid-induced bone loss

*Newer and potential clinical indications

Extended use in specific indications

Used in children in osteogenesis imperfecta

Use in cardiac or liver transplantation

Wider use to prevent glucocorticosteroid

induced bone loss in children and adult of both

genders and with a spectrum of underlying diseases

Extended use in cancers to optimize antitumor effect

and survival

Prevention of bone loss and erosions rheumatoid

arthritis

Possible applications in other joint diseases

Reduction of bone loss associated with periodontal Disease

Prevention of loosening of joint prosthesis

9

BPs are discovered during attempts to find stable analogous of

pyrophosphates. Pyrophosphates are natural compounds, present in body fluids such as

plasma and urine, which are able to inhibit calcification under physiological conditions.

Because pyrophosphates become inactive after oral administration, due to hydrolysis in

the gastrointestinal tract, more stable nonhydrolyzable analogous are demanded. BPs

seem to be stable, nonhydrolyzable analogous, because the labile P-O-P bond of

pyrophosphate is replaced by a stable P-C-P bond (Figure 1.1.). BPs have the same high

affinity for bone minerals as pyrophosphates. Like pyrophosphates, BPs are able to

inhibit calcification by binding to new formed HA crystals. Secondly BPs have the

possibility to inhibit the dissolution of HA crystals. Next to these physico-chemical

effects, BPs have cellular effects, resulting in inhibition of bone resorption (Russell et

al., 1999; Rogers et al., 1999).

FIGURE 1.1.: THE CHEMICAL STRUCTURE OF A GEMINAL

BISPHOSPHONATE AND AN INORGANICE PYROPHOSPHATE. (Russell &

Rogers, 1999)

1.2.1. Structure – Activity interaction

As said before, the difference between BPs and pyrophosphates is a single

carbon atom that bridges the two phosphates groups, rather than an oxygen atom. The P-

C-P bond is important to resist the acid and hydrolytic enzymes in the gastrointestinal

tract and the bond is responsible for the high affinity of BPs for carbonated HA crystals.

BPs contain next to the two important phosphate groups, two side chains (R1 and R2 ). In

the presence of a hydroxyl group at the R1 position (as in etidronate), the affinity and

binding of BPs to bone minerals increases (Figure 1.2.; 1.3.). After adsorption, this leads

10

to inhibiting both, the crystallization of calcium salts and dissolution of HA crystals

(Russell et al., 1999; Rogers et al., 1999).

FIGURE 1.2.: SCHEME OF THE FUNCTIONAL DOMAINS OF A GEMINAL

BONE-ACTIVE BISPHOSPHONATE. (Russell & Rogers, 1999)

The antiresorptive potency of BPs depend on the phosphate groups and both

side chains. The two phosphate groups and a hydroxyl group at R1 position are necessary

to target the bone mineral surfaces (enhance bone affinity). Once localized in and

adsorbed to bone, the structure of the R2 side chain becomes essential and is the major

determinant (Figure 1.2.). BPs containing a primary basic nitrogen atom in their alkyl R2

side chain (as in alendronate) have potent antiresorptive qualities. Substitution of the

primary amino nitrogen atom for a tertiary amino nitrogen (as in ibandronate) or for a

nitrogen atom within a heterocyclic ring (as in risedronate) enhances the ability of BPs

to interact with specific molecular targets, resulting in more potent inhibition of bone

resorption (Russell et al., 1999; Rogers et al., 1999).

FIGURE 1.3.: STRUCTURES OF DIFFERENT BISPHOSPHONATES. (Rogers et al.,

1999)

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1.2.2. Effects of BPs on osteoclasts

As previously discussed, BPs have an antiresorptive potency, which is

accomplished by affecting the osteoclasts directly. Osteoclasts are the cells in bone that

are most likely to come in contact with BPs. During bone resorption, bone minerals are

more exposed. This occurs through the dissolution of HA bone crystals performed by an

acidic pH of the environment. In this way the ability of BPs to adsorb to bone mineral,

makes them osteoclast-specific. Osteoclasts are endocytic and internalize the BPs

present in the resorption place. Once the BPs are present in the cells, they interfere with

essential pathways for normal cell function, structure and survival of the cells. Due to

inhibition or damage of metabolic pathways, the cell functions are affected. In this way,

BPs can induce apoptotic cell death in osteoclasts (Figure 1.4.) (Russell et al., 1999;

Rogers et al., 1999).

FIGURE 1.4.: “THE ROUTE BY WICH BPs AFFECT BONE-RESORBING

OSTEOCLASTS”. (Russell & Rogers, 1999)

12

1.2.3. Classification

There are two main classes of BPs. The classification is based on the

molecular mechanism by which they affect osteoclasts. The first class contains the less

potent BPs with short or little side chains, such as etidronate. These BPs are similar to

pyrophosphates, act as prodrugs and are transformed in the body into nonhydrolyzable

metabolites (Russell et al., 1999; Rogers et al., 1999; Boanini et al., 2007).

Aminoacyl-tRNA synthetases are enzymes, which are able to catalyze the

reaction by which nucleotide analogous are formed. Those specific enzymes of type Π

subclass form normally ATP molecules and have the possibility to replace the

pyrophosphate group by bisphosphonates, producing analogous of ATP (Figure 1.5.).

The ATP metabolites contain the P-O-P bond in stead of the β,γ-pyrophoshate group

(PPi). The structure of the catalytic site of the type Π subclass differs from that of type І

subclass and is able to bind with BPs with short chains. The more potent BPs,

containing nitrogen are to large for the catalytic site. Once the metabolites are formed,

they will accumulate in the cell cytoplasm and they will prevent the intracellular activity

of enzymes and pathways, leading to final negative effects on cell function and cell life

(Russell et al., 1999; Rogers et al., 1999).

1. ENZYME + AMINO ACID + ATP <=> AMINO-ACYL-AMP + PPi

Reaction 1 can be reversed using BPs in stead of PPi, resulting in a metabolic analogue of

ATP.

2. AMINO-ACYL-AMP + BPs <=> AMINO ACID + ATP ANALOGUE

FIGURE 1.5.: REACTION 2 SHOWS THE FORMATION OF THE NUCLEOTIDE

ANALOGOUS CONTAINING BISPHOSPHATES. (Russell et al., 1999; Rogers et al.,

1999)

The second class contains the larger, nitrogen-containing, more potent,

antiresorptive bisphosphonates, such as alendronate. Because of their bulkier structure,

these BPs can not be metabolized by the former enzymes (aminoacyl-tRNA

13

synthetases). These BPs interfere and inhibit the mevalonate pathway. By affecting the

mevalonate pathway this bisphosphonates will indirectly cause a lot of damage to the

osteoclast, including functions and structures. If mevalonate is no longer formed, a

decrease and stop of production of isoprenoid lipids occur. These lipids, particularly in

farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), are the

essential components in prenylation of small GTPases. Without prenylation, the small

GTPases loose their function of being important signalling proteins that regulate the cell

processes in the osteoclasts (Russell et al., 1999; Rogers et al., 1999).

1.2.4. BPs and implants

In orthopaedic surgery, the usage of screws and nails is omnipresent. These

implants are used to repair fractures. Their fixation and removal resistance have to be

optimal, in order to fulfill their function. The bone, which is inclosing the implants,

provides the strength of implants fixation. Due to the trauma or fracture, the bone acting

as a nut for the implants, can be resorbed. Loss of bone leads to failure of the implants

fixation and to microinstability. Microinstability may result in bone resorption (Figure

1.6.). That is where BPs come in. Systemic or local treatment with BPs may inhibit the

loss of bone around implants, enhancing their removal resistance, fixation and migration

(Aspenberg, 2009).

FIGURE 1.6.: MICROINSTABILITY OF A SCREW, LEADING TO BONE

RESORPTION. (Aspenberg, 2009)

14

1.3. CALCIUM PHOSPHATES

Calcium phosphates are bioactive, absorbable inorganic ceramics. Due to

their chemical and crystal similarities to bone minerals, calcium phosphates are good

candidates, as materials for bone tissue engineering. Bone exists for more than 50% of

HA. For this reason, it is logical that HA and related calcium phosphates (tetracalcium

phosphate (Ca4P2O9), amorphous calcium phosphate, α-TCP (Ca3(PO4)2) and β-TCP

(Ca3(PO4) 2)) have an excellent biocompatibility with bone tissues. The use of calcium

phosphates guarantees minimal immunological reactions, rejections and systemic

toxicity. Besides the good biocompatibility, calcium phosphates have more advantages,

such as an excellent osteoconductivity. The inorganic materials have certain

osteoconductive properties and can bind directly to bone (under particular conditions),

but they possess no osteoinductive ability. The attachment, differentiation and

proliferation into the specific cells is always ensured (Rezwan et al., 2006; Mourino et

al., 2010).

Calcium phosphates do have negative aspects. Their degradation occurs

slowly, especially the crystalline phase and they are brittle. The mechanical properties of

healthy normal bones, depend on the biological location in the body and on the

adjustments of that location, such as porosity, composition of bone and crystallinity.

Synthetic produced calcium phosphates with a high crystallinity, a low porosity and a

small grain size, give the best mechanical properties, such as a high stiffness, good

strength (compressive and tensile) and a great fracture toughness. The mechanical

properties in bones will always be better, due to the presence of tough and flexible

collagen fibers. Despite their good biocompatibility, calcium phosphates are not suitable

for load-bearing applications. This is due to their particular low mechanical strength

comparing to bone and to their slow degradation rate (Rezwan et al., 2006; Mourino et

al., 2010).

1.3.1. Hydroxyapatite (HA)

Hydroxyapatite is probably the most efficient calcium phosphate used in

tissue engineering. The usage of synthetic HA is increased. Because of the possibility to

control and lower the crystallinity of synthetic HA, the great biological features of HA,

15

such as nontoxicity, lack of inflammatory and immunitary responses can be enhanced.

Because of the unique well organized way in which apatite crystals are formed in bone,

there have to be differences between the biological and synthetic HA. The synthetic HA

has a higher surface area, because of the smaller crystal size. This feature of synthetic

HA allows more adsorption of molecules on the HA surface. The formation of synthetic

HA can be achieved using a variety of methods. Sol-gel synthesis and precipitation

reaction are often used, but to simulate the biological formed HA, direct growth of HA

is the key (Palmer et al., 2008; Palazzo et al., 2007).

16

2. OBJECTIVES

Nowadays, bone implants are the surgical strategy to repair bone fractures

caused by bone loss correlated to bone diseases. Essential in this regenerative medicine

is the fixation of bone implants and the integration between bone and implants. Implant

failure and revision surgery is often seen. Aseptic loosening of bone, which is

surrendering the implants is the main cause of implant failure. Loss of bone arises after

destructive processes, in particularly through production of stress shielding wear debris

and through micromotion at the bone-implant surface. There is need of a therapy that

could inhibit bone loss during initial remodelling. After many studies, it is assumed that

periprosthetic osteoclast activity is the main factor responsible for periprosthetic bone

resorption and bone loss. The therapy has to create more time and enhance the chance

that the bone implant lasts until the bone fracture is healed. In this way, bone-implant

interface longevity is improved.

Bisphosphonates (BPs) are drugs potentially used in the clinical treatment of

several systemic metabolic bone diseases. Recently it has become clear that BPs can be

used as an effective strategy in avoiding bone implant loosening. BPs accomplish this by

inhibition of osteoclast activity. The oral and intravenous administration of BPs doesn’t

permit to obtain high biodisponibility in the body. The poor biodisponibility of BPs is

caused by degradation of the drugs on gastric intestinal level or through a quick removal

from the damaged place in bone. An alternative route to administrate drug in high

concentration is a local treatment by drug delivery systems (DDS), such as microspheres

and scaffolds. The problem, correlated with the preparation of these systems with BPs,

is the tendency of BPs to flush away from the drug delivery systems. The reason why

BPs don’t prefer to stay in drug delivery systems is due to their hydrophilic properties,

resulting in a low encapsulation efficiency.

The aim of this research is to synthesize hydroxyapatite-alendronate (HA-

ALN) composites, by exploiting the affinity of BPs for calcium ions of hydroxyapatite.

With these HA-ALN composites higher encapsulation efficiencies could be obtained. In

particular, the research is focused on the study of parameters that affect the loading of

the drug on hydroxyapatite and on the characterization of the prepared HA-ALN

composites. The aim is to observe which parameters favour the adsorption of BPs to

17

HA, resulting in obtaining higher local concentrations of BPs in the body, to fulfill their

function. Adsorption and desorption curves will be obtained by ninhydrin reactions. The

characterization of the prepared HA-ALN composites is performed by several

techniques, such as transmission electron microscopy, scanning electron microscopy,

Fourier transform infrared microscopy, differential scanning colorimetry and the

Brunauer-Emmett-Teller method.

18

3. MATERIALS

HYDROXY APATITE NANOPOWDER

( HAP, < 200 nm, ≥ 97% synthetic, Sigma-Aldrich, Saint Louis, USA)

ALENDRONATE SODIUM

(ALN, Batch 70225, Discovery Fine Chemicals, Bournemouth, United Kingdom)

NINHYDRIN PURUM

(Crystalline, assay spec. ≥ 95,0% (UV), Sigma-Aldrich, Saint Louis, USA)

19

4. METHODS

4.1. NANOCOMPOSITES

4.1.1. Preparation of hydroxyapatite-alendronate sodium composites

A 30 mM alendronate solution (maximum solubility of ALN in water: 10

mg/ml) was prepared by dissolving the drug powder in filtered water (0,22 µm). The

hydroxyapatite nanopowder was suspended in 1 ml of the alendronate drug solution, in

Eppendorfs having a capacity of 2 ml, at 37 °C for 24 hours at 700 rpm, using a

thermomixer (Thermomixer comfort 5355, Eppendorf, Germany). This to load the

alendronate on hydroxyapatite nanocrystals, resulting in a chemical complex between

ALN and HA. Subsequently, the solid apatite-drug conjugates were washed with filtered

water (0,22 µm) at 11.000 rpm for 3 minutes at room temperature by an ultracentrifuge

(Centrifuge 5417 C/R, Eppendorf, Germany) to remove drug that was physically

adsorbed. Finally, after third washing, the supernatant was removed and the precipitate

was freezed. The freezed samples have to be in freeze dryer (Freeze dryer Lio-5P,

Cinquepascal, Italy) for 24 hours. The composites are stored at 4 °C until further

characterization (Seshima et al., 2005).

The composition of three different types of composites, prepared by the

method reported above, are listed in Table 4.1. The same samples were characterized by

several techniques.

TABLE 4.1.: THE COMPOSITION OF PREPARED SAMPLES

Batches HA-

ALN

ratio

(mg/mg)

HA

conc.

(mg)

Parameters

A 1:1 100 Incubation under

stirring at 700 rpm

for 24 hours at 37°C

B 5:1 50

C 10:1 10

20

4.1.2. Characterization of HA – ALN composites

HA–ALN composites were characterized by SEM, TEM and BET techniques

to determinate morphological properties and were characterized by FTIR and DSC to

determinate chemico-physical properties and the interaction between inorganic and

organic components.

4.1.2.1. Transmission Electron Microscopy (TEM)

Transmission electron microscopy is a valuable method in the research of

materials. TEM is a type of microscope, using electron beams for imaging solid samples

at atomic resolution. Because of the high resolution of the TEM, structural information

can be acquired. Electrons interact and pass through a thin sample. A two dimensional

projection of the sample is showed on a fluorescent screen, resulting in a shadow image.

Depending on the different densities of the sample, varied darkness occurs. Completely

dried samples are needed for maintaining a high vacuum throughout the whole

instrument (Mathews et al., 2000).

Sample preparation was made by suspending composites powder in distilled

water before fixing the suspension on a mesh grid. The TEM images were obtained by a

Zeiss EM 900 instrument (Jena, Germany), with objective lens of 30 µm, used at 80 kV.

4.1.2.2. Scanninig electron microscope (SEM)

The scanning electron microscope is an other type of microscope, critical for

characterization of solid materials. The images made by SEM give information about

the external morphology, topography and composition. Due to the main advantage of a

SEM, a great depth of field, a lot of information can be gathered about the surface

structure of a sample. The SEM uses beams of high-energy electrons to generate the

images. The incident electrons scan over the surface of the sample and interact with

electrons of the sample, producing secondary electrons. The emitted secondary electrons

are attracted by a detector and translated into signals that contain information about the

sample’s surface topography. The preparation of samples for SEM is a minimal, but

critical step. The solid samples must be fixed and dried to be stable in vacuum and have

21

to fit into the microscope chamber. By low vacuum sputter coating, the samples are

coated with a thin, electrically conductive layer of commonly gold. The resolution of the

SEM is not high enough to image individual atoms, but this method is excellent to

obtain clear views of sample surfaces and to image bulk materials (Mathews et al.,

2000;http://serc.carleton.edu/research_education/geochemsheets/techniques/SEM.htm).

The samples to be analysed were placed on a sample holder, dried overnight

and gold sputtered under vacuum. The analysis is performed by a SEM (Jeol,CX,

Temcan, Tokyo, Japan).

4.1.2.3. Brunauer-Emmett-Teller (BET)

The Brunauer-Emmett-Teller method is a physisorption analyse, to determine

the total surface area of solid materials. The total surface area is measured by the

adsorption of a certain amount of gas molecules. Gas molecules of known size (as

nitrogen) adsorb on the external and internal surfaces of the solid materials (adsorbent).

After covering the samples, the surface and irregularities can be characterized. Nitrogen

is often used as adsorbate, because of its advantaged properties. Nitrogen is a inert gas,

resulting in no interaction between the gas molecules and the surface of the samples.

Samples preparation is an essential and necessary step. Samples have to be clean and

“outgassed”. To outgas, samples are taken to an elevated temperature under vacuum. In

sample tubes under high vacuum, the “outgassed” samples are able to attract the

nitrogen molecules. Adsorption of gasses on solid materials are characterized by type Π

adsorption isotherms (sigmoid). Type Π isotherms prefer to be calculated by the BET

equation, which is an extension of the Langmuir theory. The BET equation (1)

represents the equilibrium between a sample and its surrounded gas and is used to

measure the total surface area.

ypp

p

=

ky`

1+

``

1

kpy

pk (1)

where: y: mass of gas adsorbed

y`: mass of gas adsorbed (for each unit mass of adsorbent) when

the adsorbent is covered with a monolayer of gas

22

p: equilibrium pressure (mm Hg) of the adsorbed gas

p`: saturation pressure of adsorbed gas (mm Hg)

k: constante

Sample preparation: about 400 mg of sample was outgassed under vacuum in

a suitable measurement burette at 300 °C for 8 hours. Then the N2 adsorption/desorption

isotherm was obtained at the temperature of liquid nitrogen and the equilibrium time is 3

min. The employed instrument is a Thermo Electron Sorptomatic of 1990.

4.1.2.4. Fourier transform spectroscopy

The Fourier transform infrared spectroscope is a method for defining the

identity of samples. FTIR detects functional groups, chemical bonds (for example

covalent bonds) and the molecular structure of organic and inorganic components.

Samples have characteristic vibration frequencies based on their specific structure. The

specific vibrations depend on the nature and number of atoms, bonds and functional

groups in a certain sample. The analyze is based on the absorption of electromagnetic

radiation with a wavelength between 2,5 µm and 25 µm (infrared area). The absorbed

infrared energy is typical for each sample and leads to a distinctive molecular

fingerprint. FTIR is a quicker and cheaper variant of the traditional infrared

spectroscope. In FTIR, infrared light is guided through an interfermeter, samples are

exposed to a single pulse of radiation, obtaining a Fourier transformation. A Fourier

transformation is a mathematical operation, whence a infrared spectrum can be

calculated. Identified materials of the FTIR library are necessary to analyze the FTIR

spectral pattern. The presence of characteristic peaks of ALN, HA and HA-ALN

composites was investigated into a 4000-800 cm-1

range by spectrophotometer, Perkin

Elmer 1600 series FTIR.

4.1.2.5. Differential Scanning Calorimeter (DSC)

Characterization of materials (in particularly polymers), is often performed by

a differential scanning calorimeter. DSC is a highly sensitive, fast and reliable thermo

analytical method for defining fingerprints of samples. The samples to be analyzed and

reference samples are subjected to a controlled temperature program. The two samples

23

are heated (or cooled), resulting in chemical or physical reactions. These specific

reactions are connected with production or consumption of heat. The aim of calorimetry

is to measure heat. During the chemical and physical reactions, exchange of heat occurs.

The exchanged heat causes a temperature change. DSC measures the amount of heat

required to maintain both, samples and reference samples at the same temperature. In

function of the temperature, samples and reference samples are scanned. The reference

sample is assumed to have a well-known heat capacity.

Thermograms were obtained by a STARe SW 8.10 instrument (New Castle,

UK). To perform the analysis, 8-10 mg of powder has to be weighted. This amount is

transferred into an aluminium pan; the pans were warmed from 0 °C to 300 °C at 10

°C/min. The data were obtained by Thermal Solution software (TA, Instrument, New

Castle, UK).

4.2. DETERMINATION OF ALENDRONATE BY NINHYDRIN

Ninhydrin or 2,2-dihydroxyindane-1,3-dione is a chemical reagent used for

the determination of amino acids, primary amines and ammonia. Alendronate has a

primary amine in his R2 side chain. The primary aliphatic amino group of ALN reacts

with the ninhydrin reagent. During the assay, sodium bicarbonate will be added. The

presence of this substance is needed for the reaction of ALN with ninhydrin through a

oxidative deamination of the primary amino group. Subsequently the formed product

will condensate with the reduced form of ninhydrin. The final product is a purple

colored reaction product, called Ruhemenn’s purple (Figure 4.1.) (Taha & Youssef,

2003).

Alendronate sodium has no detected chromophore and no adsorbance in the

UV/vis spectrum. Thus the drug can not be determined through common direct

spectrophotometric methods. Therefore the ninhydrin is used, with Ruhemenn”s purple

as final product, which has as maximum absorbance at 568 nm (Taha & Youssef, 2003).

24

FIGURE 4.1.: THE REACTION MECHANISME BETWEEN ALENDRONATE

SODIUM AND NINHYDRIN, RESULTING IN A COLORED FINAL PRODUCT

THAT WILL BE MEASURED BY A SPECTROFOTOMETRIC METHOD. (Taha &

Youssef, 2003)

4.2.1. Construction of calibration curve

Stock solution

Alendronate sodium solution (10 mg/ml) was prepared in filtered water (0.22 μm).

Reagent solutions

Ninhydrin 0.2% w/v in methanol

Sodium bicarbonate 0.1 M in distilled water

Sample preparations

Aliquots of stock solution, equivalent to 300 – 1200 μg of alendronate, were

transferred into glass tubes of 10 ml. For each sample, 0.5 ml sodium bicarbonate and

2.5 ml ninhydrin reagent were added and the mixture was heated in a water bath at 90 ±

5 °C for 20 minutes. The tubes were cooled at room temperature for 15 à 20 minutes,

subsequently the volume was made up to the mark with distilled water. If there is

coloration, the absorbance, measured after 30 minutes, was read at 568 nm with a UV-

25

vis spectrophotometer (UV Spectrophotometer DU- 7500 Beckman,Fullenton, CA,

USA). The actual calibration curve is shown in 5.2.1.

4.2.2. Adsorption and desorption curve

4.2.2.1. Determination of the amount of alendronate adsorbed to HA

In order to asses the amounts of alendronate adsorbed to HA, three different

batches of composites, at increasing HA content (1:1, 5:1, 10:1), were prepared (Table

4.2.). 1 ml of ALN solution (10 mg/ml) was added to HA powder and the mixture was

incubated at 37°C at 700 rpm. The adsorption curve was made by taking 100 µl of ALN

supernatant at scheduled time (6, 24, 48, 72 h). Alendronate was determined by a

colorimetric method using ninhydrin test as reported in 4.2. The alendronate content into

composite was determined as follows:

W1 – W2 = Wt

where: W1 = amount of alendronate initially added to the solution (mg)

W2 = amount of alendronate in the supernatant (mg)

Wt = amount adsorbed onto hydroxyapatite (mg)

After one singular uptake of ALN solution, 100 µl of fresh water was added to each

sample to respect sink conditions.

TABLE 4.2.: COMPOSITION OF BATCHES TO DETERMINE CONCENTRATION

OF ALN ADSORBED TO HYDROXYAPATITE.

Batches HA-ALN

ratio

(mg/mg)

Parameters Sample Times

A 1:1 Incubation under

stirring at 700

rpm

at 37°C

6, 24, 48, 72

hours

B 5:1

C 10:1

26

4.2.2.1. ALN release from HA-ALN composites

Prepare PBS buffer solution (pH = 7,4)

Weight 1,19 g Na2HPO4.12H20

Weight 0,095 g KH2PO4

Weight 4 g NaCl

Weight 0,05 g of 0,01% NaN3

Add and dissolve the powders into an Erlenmeyer of 500 ml

with filtrated water (0,22 µm)

Prepare desorption curve

A quantity (50 mg) of HA-ALN composites at three different ratio (1:1, 5:1,

10:1) were prepared by adding 1 ml of PBS solution at pH 7.4 (PBS preparation is

described above) to each sample. The samples (Table. 4.3.) were incubated at 37°C. The

ALN release was determined at scheduled times (6, 24, 48, 72 hours) by collecting 100

µl of PBS supernatant and determining the ALN concentration with a ninhydrin assay.

After one singular uptake of PBS, 100 µl of fresh buffer was added to each sample to

respect the sink conditions. Desorption curve is shown in 5.2.3.

TABLE 4.3.: COMPOSITION OF THE BATCHES TO DETERMINE ALN RELEASE

FROM COMPOSITES.

Batches HA-ALN ratio

(mg/mg)

ALN (mg) Parameters Sample Times

1 1:1 10

Stirrer at 700 rpm

at 37°C

6, 24, 48, 72 hours

2 5:1 10

3 10:1 10

27

5. RESULTS AND DISCUSSION

Nowadays a lot of articles describe the great affinity of BPs for bone mineral,

in particularly a great affinity for the hydroxyapatite crystals present in bone. The

articles indicate that this great affinity between the drug and the hydroxyapatite crystals,

is based on several specific interactions. These interactions depend on the structure of

BPs and occur with natural and synthetic HA. Studies shown in previous articles could

confirm the intermolecular interaction between a nitrogen-bisphosphonate and HA. The

intermolecular interaction is based on the binding between the phosphate groups of ALN

and the calcium atoms present in HA.

The dominant intermolecular interaction between ALN and HA, which

constitutes the ALN-HA composite, is an electrostatic interaction. The electrostatic

interaction originates in the relatively large partial negative charge that both elements of

ALN share and the positive charge of the calcium atoms of HA. The amino nitrogen

atom bears one electron pair, resulting in a partial negative charge. The oxygen atoms of

the phosphate groups of ALN have partial negative charges. The distance between the

two interacting charges defines the strength of the intermolecular interaction (Neves et

al.,2002).

The interaction can be shown on images of the surface of the HA crystals.

Previous studies indicate that on the surface of HA, six calcium atoms are needed to

surround one hydroxyl group of BPs. There are three calcium atoms in a plane and the

remaining three in adjacent plane (Figure 5.1.). “Hydroxyapatite crystal is constituted by

layers by layers of phosphate and hydroxide groups intercalated with layers of calcium

ions” (Neves et al.,2002).

The presence of the amino group in the end of the side chain in the isolated

molecules is very important. First of all, it ensures the pharmacological activity in situ.

The amino nitrogen forms a OH---N hydrogen bond. This is a intramolecular bond,

which is responsible for the most stable conformer of the molecule. When the amino

group forms a tridentate binding with calcium atoms, the intramolecular bond will not

remain. This is chemisorption of BPs to bone mineral (Neves et al.,2002).

28

FIGURE 5.1.: “THREE-DIMENSIONAL AND TOP VIEW OF HYDROXYAPATITE: +

ARE CA ATOMS; >-- ARE PHOSPHATE GROUPS.” (Neves et al.,2002)

5.1. CHARACTERIZATION OF HA-ALN COMPOSITES

Characterization of the HA-ALN composites has been carried out in order to

investigate the interaction between ALN and HA. A number of techniques have been

used in order to characterize the HA-.ALN composites.

5.1.1. TEM images

The images received from the TEM, show us the presence of a fine network,

due to a great resolution of about 30 nm. This network refers to the filaments of HA-

ALN complexation, is present in the three batches of HA-ALN-composites and is

independent of the HA-ALN ratio. The fine network is absent in the image of only HA

powder (Figure 5.2.a), because without a certain amount of BPs, no interaction can

occur. The presence of black nanospheres confirms that HA does not completely interact

with ALN. There are saturation limits, above ALN will not longer bind to HA.

The fibers that compose the network of HA-ALN composite 1:1 (Figure

5.2.d) are more interconnected and more fine, than those observed in HA-ALN

composite 10:1 (Figure 5.2.b). We think that the reason of this result originates from the

fact that with an increasing amount of HA, the drug (ALN) is more dispersed in the HA

powder. Consequential there is more interaction between ALN and HA in HA-ALN

composite 10:1. This seems logical as there is more mineral to bind. The HA-ALN

composite 10:1 has the largest interaction between HA and ALN and seems to be

preferred in the future for healing bone structures. There are no TEM images reported of

29

ALN powder alone, because ALN dissolves in water during sample preparation. So

ALN can not be seen by TEM resolution.

FIGURE 5.2.: TEM IMAGES OF HA-ALN SYSTEMS: a) HA POWDER; b) HA-ALN

COMPOSITE 10:1.

FIGURE 5.2.: TEM IMAGES OF HA-ALN SYSTEMS: c) HA-ALN COMPOSITE

5:1; d) HA-ALN COMPOSITE 1:1.

a b

c d

f

30

5.1.2. SEM images

The SEM micrographs show us the same network, which was revealed in the

TEM images. In image 5.3.a, HA powder appears as aggregates of porous nanoparticles.

HA powder underwent the same process as the HA-ALN composites (in water at 37 °C

at 700 rpm). The HA-ALN composites 1:1, 5:1, and 10:1 are shown in figure 5.3.b, 5.3.c

and 5.3.d. respectively. They have become aggregates with variable dimensions (from 2

µm to 100 µm) and with a highly porous structure. The highly porous structures are

foams, where nanoparticles and needle-shaped particles are present with micrometric

dimension. When taken a closer look, we can conclude that for HA-ALN composites

with a decreasing amount of HA, aggregates enriched of pores arise. The SEM image of

HA-ALN composite 10:1 shows aggregates with an irregular surface and superficial thin

filaments. In the HA-ALN composite 10:1, there is less porosity, but the maximum

amount of HA enhances the osteoconductivity and biocompatibility of systems.

FIGURE 5.3.a: SEM MICROGRAPH OF HA POWDER (SINGLE GRANULE).

31

FIGURE 5.3.b1: SEM MICROGRAPH OF HA-ALN COMPOSITE (1:1) (SINGLE

GRANULE).

FIGURE 5.3.b2: SEM MICROGRAPH OF HA-ALN COMPOSITE (1:1),

PARTICULAR OF THE SURFACE WITH THE PRESENCE OF

INTERCONNECTED FIBRES.

1

2

32

FIGURE 5.3.c1: SEM MICROGRAPH OF HA-ALN COMPOSITE (5:1): GROUPING

OF COMPOSITES.

FIGURE 5.3.c2: SEM MICROGRAPH OF HA-ALN COMPOSITE (5:1): SURFACE

OF A COMPOSITE GRANULE.

1

2

33

FIGURE 5.3.d1: SEM MICROGRAPH OF HA-ALN COMPOSITE (10:1) AT A

MAGNIFICATION OF 100 µm.

FIGURE 5.3.d2: SEM MICROGRAPH OF HA-ALN COMPOSITE (10:1) AT

MAGNIFICATION OF 10 µm.

2

1

34

5.1.3. BET analysis

Table 5.1. shows that the total surface area doesn’t significantly change, when

we compare HA powder and the two batch of composites. An other value, which have

been calculated by the same machine and obtained by the Barret-Joyner-Halenda

method and the Dollimore-Heal method, is the pore size of the materials. We see that

the size of the pores are in the range of 43- 48 nm in both cases. Therefore, the total

surface area and the pore size are independent from the HA-ALN ratio.

TABLE 5.1.: A SUMMARY OF THE RESULTS OBTAINED FROM BET

MEASUREMENTS.

BET MEASUREMENTS

Sample BET

m2/g

Spec.Volume

BET (cm3/g)

Pore size D.H.

nm

Pore size B.J.H.

nm

HA powder 21 0.030 2,5-3,5

3,0-4,5

HA-ALN

10:1 26 0.043 43-48 43-48

HA-ALN

5:1 26 0.041 43-48 43-48

35

5.1.4. DSC analysis

In figure 5.4.a, a DSC curve of ALN is shown. It indicates an endothermic

peak at 122,49 °C, which corresponds to loss of adsorbed water (ΔH = 482,9 J/g). At

268,04 °C, we notice an endo-exothermal effect, corresponding to the melting of

crystalline ALN, followed by decomposition. In figures 5.4.c and 5.4.d, the DSC curves

of respectively HA-ALN composites 10:1 and 5:1 are reported. The characteristic peak,

when ALN melts, is absent. This is due to the interaction between HA and ALN. Only a

weak hollow is observed in the temperature range 50-150 °C of the HA-ALN composite

5:1, probably due to the removal of water surface. The DSC curve of HA-ALN

composite 1:1 (Figure 5.5.e) has a peak, near to the melting point of pure ALN. This

indicates the presence of free ALN in the particular sample. DSC indicates the change of

crystalline free ALN powder into amorphous bonded ALN to HA.

FIGURE 5.4.a: DSC CURVE OF ALN POWDER AT 10 °C/min.

124.05°C

122.49°C482.9J/g

272.38°C

268.04°C97.15J/g

-60

-40

-20

0

20

40

He

atF

low

(m

W)

0 50 100 150 200 250 300

Temperature (°C)

36

-3

-2

-1

0

1

Heat

Flo

w (

mW

)

0 50 100 150 200 250 300

Temperature (°C)

-2.0

-1.5

-1.0

-0.5

0.0

0.5

He

at

Flo

w (

mW

)

0 50 100 150 200 250 300

Temperature (°C)

FIGURE 5.4.b: DSC CURVE OF HA POWDER AT 10°C/min.

FIGURE 5.4.c: DSC CURVE OF HA-ALN COMPOSITES (10:1) AT 10°C/min.

37

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

He

atF

low

(m

W)

0 50 100 150 200 250 300

Temperature (°C)

251.99°C

223.90°C15.61J/g

-4

-3

-2

-1

0

He

at

Flo

w (

mW

)

0 50 100 150 200 250 300

Temperature (°C)

FIGURE 5.4.d: DSC CURVE OF HA-ALN COMPOSITE (5:1) AT 10°C/min.

FIGURE 5.4.e: DSC CURVE OF HA-ALN COMPOSITE (1:1) AT 10°C/min.

38

5.1.5. FTIR analysis

In figure 5.5.a and 5.5.b, the spectrums of HA-ALN composite 1:1, show the

intensity of the characteristic peaks of ALN- N-H stretching and C-N respectively at

3500 cm-1

and at 1100-900 cm-1

. They are dimly visible, because the HA adsorptions

prevail. Looking closer to the spectrum of HA, we observe bands at 3572 cm-1

, assigned

to stretching of OH groups. Spectrums of HA also show a broad peak between 1150 cm-

1 and 850 cm

-1, due to vibrations of the PO4 groups. Considering the composites spectra

in the range of 3500-800 cm-1

(Figure 5.5.c; 5.5.d), the spectrums of HA and HA-ALN

composites are practical overlapping. The only difference that can be observed is a

broad band of the composite spectra between the range of 3400-2800 cm-1

. This broad

band is the same band present in the ALN spectra. Finally, the composites with a lower

amount of HA, show at 3700 cm-1

, an intensity peak, which correspond with the OH

stretching, who is reduced compared with the same intensity peak in the HA spectrum.

FIGURE 5.5.a: INFRARED SPECTRA (3800-2200 cm-1

) OF THE HA POWDER (a);

ALN POWDER (b); PHYSICAL MIXTURE OF HA AND ALN (1:1)(c).

(c)

92

94

96

%T

(b)

60

70

80

90

%T

(a)

90

92

94

96

98

%T

2200 2400 2600 2800 3000 3200 3400 3600 3800 Wavenumbers (cm-1)

39

FIGURE 5.5.b: INFRARED SPECTRA (2200-700 cm-1

) OF HA POWDER (a); ALN

POWDER (b); PHYSICAL MIXTURE OF HA AND ALN (1:1) (c).

FIGURE 5.5.c: INFRARED SPECTRA (3800-2200 cm-1

) OF HA-ALN COMPOSITE

(10:1) (a); HA-ALN COMPOSITE (5:1) (b); HA POWDER (c); ALN POWDER (d).

(c)

20

40

60

80

%T

(b)

20

40

60

80

%T

(a)

20

40

60

80

%T

800 1000 1200 1400 1600 1800 2000 Wavenumbers (cm-1)

(d)

60

70

80

90

%T

(c)

90 92 94

96 98

%T

(b)

85

90

95

%T

(a)

92

94

96

98

%T

2200 2400 2600 2800 3000 3200 3400 3600 3800

Wavenumbers (cm-1)

40

FIGURE 5.5.d: INFRARED SPECTRA (2200-700 cm-1

) OF HA-ALN COMPOSITE

(10:1) (a); HA-ALN COMPOSITE (5:1) (b); HA POWDER (c); ALN POWDER (d).

5.2. DETERMINATION OF ALENDRONATE

5.2.1. Construction of ALN calibration curve by a ninhydrin assay

A critical step of experiments was to establish an opportune and safe method

to determine and to calculate the amount of ALN bonded to HA. In literature, there are

many methods for determination of ALN, such as an analytical HPLC and a colorimetric

method, using an UV-visible spectrophotometer. After studies of the ALN structure, the

most desirable method could be chosen. ALN has an amino group, a distinctive target

for ALN, which can easily bind to ninhydrin reagent, resulting in the formation of

purple solution, which allows us to quantify the bounded ALN. Moreover, the amino

group of ALN is the only amino group present in the prepared solution, so the ninhydrin

can only bind to the drug, without emerging interference problems. The calibration

curve was built, following the described method in 4.2.1. In table 5.2. the concentration

of ALN and the corresponding absorbance (568 nm) are reported.

(d)

60 70 80 90

%T

(c)

20 40 60 80

%T

(b)

20 40

60

80 %T

(a)

20 40 60

80 %T

800 1000 1200 1400 1600 1800 2000 Wavenumbers (cm-1)

41

TABLE 5.2.: ABSORBANCE VALUES, AT 568 nm, WHICH CORRESPOND TO

THE SPECIFIC ALN CONCENTRATION.

FIGURE 5.6.: CALIBRATION CURVE OF ALN: A GOOD CORRELATION

COEFFICIENT (r) OF 0,9959; THE EQUATION WILL BE UTILIZED TO

DETERMINE THE ALN ADSORBED TO HA.

Samples ALN concentration (μg) ABS at 568 nm

1 300 0,13738

2 333,3 0,16677

3 416,7 0,25018

4 600 0,38087

5 700 0,54729

6 800 0,62132

7 1000 0,823123

8 1166,7 0,969187

ALN calibration curve

y = 0,001x - 0,1601 R 2 = 0,9959

0

0,2

0,4

0,6

0,8

1

1,2

0 200 400 600 800 1000 1200 1400

ALN concentration (µg)

Abs (568 nm)

42

5.2.2. Adsorption curve

Figures 5.7 and 5.8 show the percentage of alendronate adsorbed to

hydroxyapatite of three different batches. The type of interaction between drug and

mineral is been explained previously; in this paragraph parameters that can influence the

drug adsorption will be discussed. Those parameters are time, rate, temperature of

processing incubation and molar ratio between the two components. It is clear from

plots that the ALN adsorbed at 6h is very low for all batches (A, B, C), ~ 40%. After 6h

the three batches follow different trends. Batch A curve follows linear trend until 48h,

when it begins to increase reaching the largest ALN adsorbed percentage of 60% at 72h.

The curve of batch B and C instead, notably increase until 24h, where batch B reaches a

plateau with 70% of ALN adsorbed, while batch C reaches maximum ALN adsorbed

value of ~ 80%. Differently from other batches, batch C curve doesn’t assume linear

trend but it decreases from 24h to 48h until an adsorbed value of 60% ALN is reached.

The trend of these three different batches depends mainly from soaking time and molar

ratio between ALN and HA.

By evaluation of soaking time, it is observed that the uptake of ALN reaches

a plateau and high value at 24 hours for batches B and C. Figure 5.8. shows only a light

deflection, from 24h to 48h, for batches C due to fact that adsorption and releasing

reaction can simultaneously occur. By evaluation of molar ratio instead it is possible to

observe that with increasing molar ratio in favour to HA, ALN adsorption is more rapid

due to high area surface that enhances HA-ALN interaction. Batch A in fact, with 1:1

molar ratio, shows a low adsorption trend, only at 72h it reaches 60% of adsorption.

Another parameter fundamental for reaction is the shaking rate: with high

oscillations of thermomixer, for powders in solution, this augment the number of

possible interactions between two phases. A preliminary study of adsorption in static

conditions, confirmed a low percentage of adsorption yet to 24 hours. Figure 5.8. shows

the percentage ALN adsorbed at 24h for three batches A, B, C that is respectively 46.79

%, 66.45 % and 79.50 %.

43

TABLE 5.3.: SAMPLE COMPOSITION OF HA-ALN COMPOSITES FOR ALN

ADSORPTION STUDY.

FIGURE 5.7.: ADSORPTION CURVE OF BATCHES A,B,C.

Batches HA-ALN ratio

(mg/mg)

Parameters Sample Times

A 1:1 Incubation under

stirring at 700 rpm

at 37°C

6, 24, 48, 72

hours

B 5:1

C 10:1

Adsorption curves of ALN onto HA

0

2

4

6

8

10

0 10 20 30 40 50 60 70 80

Time (hours)

% ALN adsorbed to HA

Batch A Batch B Batch C

44

ALN adsorption onto HA

Batch A

Batch B

Batch C

0

10

20

30

40

50

60

70

80

90

100

Batches

AL

N a

dso

rbed

(%

)

FIGURE 5.8.: ALENDRONATE PERCENTAGE ADSORBED TO HA AT 24

HOURS.

5.2.3. ALN release from HA-ALN composites (desorption curve)

ALN release behaviour is important in order to the function of the drug to

fulfill its action on bone mineral. ALN carries on its pharmacologically action by the

presence of the amino group and phosphate groups, that are tight involved into HA-ALN

binding. Figure 5.9. shows ALN release profiles from composites at different HA-ALN

ratios. ALN is not released in the first 24h of incubation. The explanation is the

hydrophobic nature of hydroxyapatite and the complexity of composites agglomerates

observed by SEM that avoids the enter of water and consequently the release of drug in

the release medium. ALN begins to be released from HA in all batches after 24 h: it is

released faster with increasing HA-ALN molar ratio, corresponding to the lower

network complexity observed by TEM. In fact ALN is released from batch A slower due

to the high interconnectivity between components, in particular at 48h and 96h only ~

40% passes into buffer solution. ALN is released completely only in batch C where it

can produce a plateau at 96 h; while Batch B reaches at 96h a maximum value of ~70%.

45

ALN release

0

20

40

60

80

100

0 50 100 150

Time (hours)

AL

N c

on

cen

trati

on

(%

) Batch A Batch B Batch C

FIGURE 5.9.: ALN PERCENTAGE RELEASED FROM HA-ALN COMPOSITES OF

BATCHES A,B,C.

46

6. CONCLUSION

We have synthesized three batches of HA-ALN composites at three different

molar ratios (10:1, 5:1 and 1:1). Adsorption and desorption curves, obtained by

ninhydrin assays, confirm the presence of the interaction between ALN and HA due to

surface charge of the molecules. In particular, HA-ALN composites (10:1) show the

best adsorption and desorption profiles in terms of rate and loaded-released percentage.

The adsorption curve of HA-ALN composite (10:1) shows already after 24h an absorbed

percentage of 79,50 %. The desorption curve of the same HA-ALN composite shows a

high percentage of release (~ 90%) after 96h, due to the low network complexity.

Morphological and physico-chemical characterization of the three different

composites allow us to obtain useful structural information for clarifying the interactions

between ALN and HA. TEM images reproduce networks, corresponding to the present

interaction between HA and ALN. SEM micrographs reveal the porosity present in the

composites. BET analysis describe the same surface area for the different batches.

Finally, this approach for synthesizing HA-ALN composites offers a

relevant tool as possible application as biomaterials to repair bone tissues. The HA-ALN

composites enhance the encapsulation efficiency.

47

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