FACULDADE DE ENGENHARIA DA UNIVERSIDADE DO PORTO

Bonelike® Associated to Stem Cells for the Regeneration of the Bone

Tissue

Ana Cláudia da Costa Lopes

MEB 2008/2010 25 de Outubro de 2010

2

Resumo

A engenharia de tecidos é uma ciência em evolução com o objectivo de restaurar

danos em tecidos ou órgãos que não sejam capazes de uma regeneração rápida,

completa e bem sucedida. Os substitutos ósseos fazem parte deste objective,

porém por vezes apresentam dificuldades difíceis de ultrapassar como a rejeição

imunológica, a necessidade de mais que uma cirurgia, a morbilidade associada ao

local de colheita, a transferência de doenças infecciosas entre outras. Todas estas

desvantagens estão associadas às diferentes origens dos enxertos ósseos.

Os enxertos ósseos devem por isso eliminar a possibilidade das desvantagens

referidas se realizarem bem como possuir propriedades que aumentem o ritmo do

processo de regeneração integrando as células da forma mais semelhante

possível ao organismo in vivo.

São várias as linhagens celulares que estão envolvidas no processo de

regeneração in vivo, porém as mais importantes são sem dúvida as células

estaminais mesenquimais, presentes na medula óssea, que podem originar tecido

ósseo, cartilagíneo e adiposo. Estas células estaminais mesenquimais originam as

células que intervêm nos processos de remodelação óssea.

O objectivo deste trabalho é avaliar a resposta biológica das células presentes no

aspirado da medula óssea com diferentes formatos de Bonelike®, substituto ósseo

sintético. Tendo estes dois formatos, Bonelike® Pellets e Bonelike® Poro,

características diferentes com diferentes propósitos clínicos, irá ser avaliada a

aderência, crescimento e migração das células nestes materiais e verificar a

diferenciação das células estaminais mesenquimais em células da linhagem

osteoblástica.

Os resultados mostraram que ambos os formatos de Bonelike®, permitem o

crescimento, a proliferação e induzem a diferenciação das células mesenquimais

em direcção a uma linhagem osteoblástica.

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Abstract

Tissue engineering is a science still in evolution with the goal of repair damaged

tissues and organs which are not capable of a quick, complete and well succeed

regeneration. Bone substitutes are part of that goal, although sometimes they

present some hard difficulties to pass through like immunological rejection, the

need of an extra surgery, morbidity associated to the donor site, transmission of

infectious diseases, among others. All these disadvantages are associated to the

different origins of the bone grafts.

The perfect bone grafts should eliminate the possibility of some of those

disadvantage occur as well as possess special properties that improve the rhythm

of the regeneration process integrating the cells the most similar way as in the

organism in vivo.

Several cellular lineages are involved in the regeneration process in vivo, however

the most important group are the mesenchymal stem cells, presents in bone

marrow, that can originate osteogenic, chondrogenic and adipogenic cells. These

mesenchymal cells can differentiate into all the cells that interfere in the bone

remodeling process.

The aim of the present work was to evaluate the biological response of the cells

presents in bone marrow aspirate with different formulations of Bonelike®, a

synthetic bone graft. Bonelike® Pelletes and Bonelike® Poro have different

properties and so different clinical applications in which adherence, growth and

migration of cells were tested as well as the differentiation of mesenchymal stem

cells into a osteoblastic lineage.

Results showed that both formulations of Bonelike®, allow the growth, proliferation

and induce the differentiation of mesenchymal cells towards to an osteoblastic

commitment.

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Acknowledgements

I am grateful to my supervisor Professor José Domingos Santos and Professora

Maria Ascensão Lopes.

My specials thanks to Professora Helena Fernandes and to all the members of

Laboratório de Farmacologia da Faculdade de Medicina Dentária da Universidade

do Porto, specially Raquel Almeida.

I would like to thank my family for the encouragement and support given during

these two years.

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Contents

Resumo ................................................................................................................... 1

Abstract .................................................................................................................... 3

Acknowledgements .................................................................................................. 4

Chapter 1 ................................................................................................................. 9

General introduction ................................................................................................. 9

1. Bone Physiology .................................................................................................. 9

1.1 Morphology ..................................................................................................... 9

1.2 Bone Cells .................................................................................................... 11

1.2.1 Osteoblasts ............................................................................................ 11

1.2.2 Osteocysts ............................................................................................. 12

1.2.3 Osteoclasts ............................................................................................ 14

1.3 Bone remodeling .......................................................................................... 14

1.3.1 Healing process ..................................................................................... 15

2. Bone marrow...................................................................................................... 17

2.1 Stem cells ..................................................................................................... 17

2.1.1 Origin ..................................................................................................... 17

2.1.2 Differentiation ......................................................................................... 20

2.1.3 Lineage Cells ......................................................................................... 21

2.1.4 Identification ........................................................................................... 25

3. Bone grafts ........................................................................................................ 29

3.1 Autografts ..................................................................................................... 29

3.2 Allografts ...................................................................................................... 29

3.3 Xenografts .................................................................................................... 29

3.4 Synthetic....................................................................................................... 30

3.4.1 Bonelike® ............................................................................................... 31

3.4.1.1 Composition .................................................................................... 31

3.4.1.2 Structure and Properties ................................................................. 32

4. Tissue Engineering ............................................................................................ 34

Chapter 2 ............................................................................................................... 37

Experimental Procedures ....................................................................................... 37

Preparation of Bonelike® .................................................................................... 37

In vitro biological studies .................................................................................... 38

Results and Discussion .......................................................................................... 41

Bonelike® characterization.................................................................................. 41

In vitro biological studies .................................................................................... 44

Conclusion ............................................................................................................. 59

References ............................................................................................................ 60

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List of Figures and Tables

Figure 1 -Bone structure. In this squeme is represented the Haversian system and the osteocytes[6]. ........................................................................ 11

Figure 2 -Proliferation vs Differentiation in osteoblastic lineage [3]. ...................... 12

Figure 3 -Schematic representation of the influence of the osteocytes in the remodelating process. O –osteocyst; L –cellular lineage; B -osteoblast; C –osteoclast [3]. ............................................................. 13

Figure 4 -Plasticity of a Hematopoietic stem cell [22]. .......................................... 20

Figure 5 -Different lineages that a HPSC can originate [17]. ................................ 22

Figure 6 -Different lineages that a MSC can originate [3]. .................................... 24

Figure 7 -Process of identification of surface markers using fluorescent tags [17]. .................................................................................................... 26

Figure 8 -FACS method, separation of SC by their fluorescence [17]. ................. 27

Figure 9 -CD34 expression in the HPSC that will originate platelets [35]. ............ 28

Figure 10 -Bonelike® Pellets structure. SEM analysis. .......................................... 32

Figure 11 -Bonelike® Poro 2000-6000µm. SEM analysis. ..................................... 33

Figure 12 –Principal Intervenients in the tissue regeneration process in tissue engineering. ....................................................................................... 35

Figure 13 -Microstructures of bone substitutes. SEM analysis. ............................ 35

Figure 14 –Cell separator from the Kit with the three distinct parts. From the top, cell poor plasma, nucleated cell concentrate and on the bottom the red blood cells. ................................................................................... 38

Figure 15 –Bonelike® Pellets. a) pellets with some irregular details (arrows) in surface, b) microporosity present in the pellets surface (arrows). SEM analysis. .................................................................................... 42

Figure 16 –Bonelike® Poro samples. a) high level of porosity, pores with different sizes, b) irregular surface with microposity (arrow). SEM analysis. ............................................................................................. 43

Figure 17 -Bonelike® Pellets seeded with the bone marrow concentrate, at 1 day of culture, a),b) showing some erythrocytes (arrows). SEM analysis. ............................................................................................. 45

Figure 18 -Bonelike® Pellets colonized with bone marrow cells, at 4 to 23 days. a) presence of defects through the surface; b), c), d), f) cells take advantage of microposity to attache to surface material (arrows); e), g) cluster of cells on the material surface (arrows). SEM analysis. .... 47

Figure 19 -Bonelike® Pellets colonized with bone marrow cells. a) cluster of cells at day 23, b) evidence of mineralized deposits associated with the cell layer within the cell clusters (arrow). SEM analysis. .............. 48

Figure 20 -Bonelike® Pellets colonized with bone marrow cells, at 23 days. Representative spectrum of the mineralized deposits. X-ray spectrum. ........................................................................................... 48

Figure 21 -Bonelike® Pellets colonized with bone marrow cells on day 23. a) cluster of cells, and cells growing on defects present on the surface(see arrows); b) control culture; 20x.. ..................................... 49

Figure 22 -Cell growth rate over colonized Bonelike ® Pellets and control cultures. MTT assay. *significantly different from control. ................. 50

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Figure 23 -ALP activity measured over colonized Bonelike® Pellets and control cultures. * significantly different from control. ..................................... 51

Figure 24 -Bonelike® Poro, at 1 day of culture. a), b) erythrocytes on the porous surface (arrows). SEM analysis. ........................................................ 52

Figure 25 -Bonelike® Poro colonized with bone marrow cells, at 4 to 23 days. a), b) cells taking advantage of microporosity to attach to the material (arrows); c) cells colonizing the interior of pores; d), e), f) bridges made by cells through some irregularities of the material (arrows); g), h) several layers of cells adapted to the topological morphology of the material’s surface(arrows). SEM analysis. ............................... 53

Figure 26 -Bonelike® Poro colonized with bone marrow cells, at 23 days. a), b), c) and d) abundant mineralized deposits closely associated with the cell layer (see arrows). SEM analysis. ............................................... 55

Figure 27 -Bonelike® Poro colonized with bone marrow cells, at 23 days. X-ray spectrum of the mineralized deposits. ............................................... 56

Figure 28 -Bonelike® Poro colonized with bone marrow cells at day 23. a) exuberant proliferation through the entire scaffold; b) control culture; 20x. .................................................................................................... 56

Figure 29 –Cell growth rate over colonized Bonelike® Poro and control cultures. MTT assay. *significantly different from control. ............................... 57

Figure 30 -ALP activity measured over colonized Bonelike® Poro and control cultures. * significantly different from control. ..................................... 58

Table 1 -Embryonic germ layers from which differentiated tissues develop,

adapted from Stem Cells: Scientific Progress and Future Research Directions [17]. ................................................................................... 18

8

Chapter 1

General introduction

9

Chapter 1

General introduction

1. Bone Physiology

Bone is structurally complex, live and mineralized. It shows some rigidity but it

keeps a certain degree of elasticity that allows him to absorb energy and when

under stress and tension it can deform reversibly [1-3].

Bone plays important functions in our body, among the most important ones are:

mechanical support, connection to muscles which allows locomotion [4] , protection

of the vital organs and bone marrow (BM), they also act like ions reserve, calcium

and phosphorus are the principal, so this way bone became the primary

responsible for the internal homeostasis, and for the main place for production of

blood cells (hematopoiesis ) [2].

During the life period of human body, bone mass suffers constantly remodeling

process, that include formation of new bone and reabsorption, these mechanisms

are integrating part of the skeleton renewal, that include loss and gain of bone

mass (mineral homeostasis) [2, 4]. When bone is required its deposition is induced

on the other and as soon as it stops the lack of activity translates into reabsorption.

The majority of bone matrix is composed by collagen type I (90%), and all the

molecules are organized in fibril. Proteins and peptidoglycans are also a part of this

matrix [2]. The mineral phase is composed by ions such as calcium and

phosphate with a similar composition to hydroxyapatite (HA) [Ca10(PO 4)6(OH)2].

1.1 Morphology In our body exists three main types of bone: the long bones, like tibia and femur,

flat bones such as skull and jaw, and short bones like carpal and tarsal.

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It can even be found in bone structure two anatomically different zones: epiphysis

and diaphysis. These two parts are separated by a layer of cartilage, which is

composed by proliferative cells, it’s responsible for the longitudinal growth of the

bone. Diaphysis is the middle region that allows mechanical stability [5]. BM can be

found in this area.

Morphologically bone is divided in: cortical bone and spongy or trabecular bone.

The external part is made of a thin layer, 80-90% of that layer is mineralized. The

compact bone is denser that the spongy one, so it has two functions: mechanical

and protection [2]. For having BM spongy bone has functions mostly related to

metabolic process.

Cortical bone is composed by a thin layer of collagen fibers with parallel disposition

or concentrically around canal with vases – the Haversian channel. These systems

have in their central axis, the Havers channel, this channel have concentrically

lamellas with fibers around [5]. These channels communicate each other, and with

marrow cavity and the external surface of the bone trough transversal channels

called Volkman channels, this can be distinguished from the Havers because they

don’t show this concentrically lamellas.

The Haversian system (Figure 1) is a structural unit of the cortical bone, and they

can be found in the region of the diaphysis.

These systems also have a central channel with blood vessels (one arteriole and

one venule) and these vessels are also surrounded by lamellas. Between the

lamellas may occur many lacunas which communicate with the central channel. In

the lacunas several osteocyctes are presents, arranged in circles around the

Havers channel. Havers system or osteon are isolated and separated from the

others osteon with cement.

Spongy bone is formed by a thin layer of trabeculae, and in their gaps can found

the BM.

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Figure 1 -Bone structure. In this squeme is represented the Haversian system and the osteocytes[6].

1.2 Bone Cells The three main types of cells that are responsible for the formation, management

and the reabsorption of the bone are the osteoblasts, osteocysts and the

osteoclasts respectively. The progenitor cells are originated from the mesenchymal

ones, during the post-natal period they localized mostly in the periosteum and

endeosteum, which are the layers that cover the inner and outer surface of the

bone, respectively.

1.2.1 Osteoblasts These are the cells responsible for the bone formation, they have their origin in

mesenchymal stem cells (MSC), these stem cells (SC) are locally stimulated by

growth factors like fibroblast grow factor, bone morphogenic proteins and

transcription factors, such as proteins that bound themselves to DNA of the

eurcariotic cells to allow a connection between the RNA-polimerase enzim and the

DNA, to make possible transcription and translation, such as Runx2 and Osterix [3,

7].

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The MSC suffer a division, and then differentiate in a pre-osteoblast cell, as can be

seen in Figure 2. Along this process of differentiation there are a number of

markers that permit the identification of the stage of maturation the cells are [7].

Beside these morphological characteristics, the analysis of the expression of

certain molecules allows the osteoblast identification. The Cbfa-1 (core-binding

factor A1) is an osteoblastic transcription factor express in a differentiation stage

[8], the Osteopontin is especially detected during the proliferation stage of these

cells, and the alkaline phosphatase is an indicator of the level of the osteoblastic

differentiation, the bone sialoprotein is expressed is the stage of maturation of

osteoblasts [3, 7]. The recently formed bone matrix, together with the active

osteoblasts that are not yet calcified is called osteoid.

Figure 2 -Proliferation vs Differentiation in osteoblastic lineage [3].

Another substance is also sintethized by the osteoblasts, the osteoprotogerin is an

inhibitor of the differentiation of monocytes and macrophages into osteoclasts [3].

1.2.2 Osteocysts Osteoblasts may be arrested in the calcified matrix and then they differentiated in

osteocyctes. The osteocyctes function in a grid, every osteon is a major structure

that plays an important role in the management and metabolic activity in the bone

tissue (Figure 3) forming this way a three dimensional and sensitive structure like a

specialized communication system.

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This structure offers two advantages:

1) An enormous contact surface bone-cells, twice superior to the contact area that

osteoblasts have;

2) An extensive communication system, intra and extra cellular between the inner

places and the bone surface.

It is thought that osteocytes can be involved in the calcium diffusion process from

the inside to the outside of the bone and vice-versa.

Figure 3 -Schematic representation of the influence of the osteocytes in the remodelating process. O –osteocyst; L –cellular lineage; B -osteoblast; C –osteoclast [3].

In the initial stage (USE) the normal mechanical strength provides a regular flux of

interstitial fluid through the lacunas. This flux keeps the osteocysts viable and

promotes their activation, so the osteoblastic activity is suppressed as well as

osteoclastic attack. However in a situation where the amount of flux increased

(Overuse) the osteocysts are activated and start to produce signals for osteoblastic

recruitment. A subsequent bone formation reduces the activity of osteoblasts until

the initial and regular pressures can be achieved. When a decreased (Disuse) of

the flux provided by the mechanical strength happens, the osteocysts are

inactivated by lack of that same flux. This inactivation leads to a release of

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recruitment signal for the osteoclasts. The bone reabsorption caused by the

osteoclastic activity leads to a reestablishment of the normal bone mechanical use.

1.2.3 Osteoclasts The bone reabsorption is essential to skeleton maturation, including the remodeling

processes and bone growth [1], as for example the moving process of tooth.

Osteoclasts are large cells, originated thought the fusion of several cells from the

monocyto-macrophage system which have their origin in cells of BM [3, 4]. The BM

cells, when under the effect of growth factors synthesized by osteoblasts (like the

Macrophage Colony Stimulating Factor), promote the differentiation and

multiplication of monocytes and macrophages as well as their fusion into

osteoclasts [3, 4].

The regulation of osteoclastic activity is also dependent on the presence or

absence of some hormones. The paratiroide hormone acts directly on osteoblasts,

which secrete interleukin 11 that stimulate the osteoclasts. The calcitonin also has

a direct effect on osteoclasts, promote the decrease of bone reabsorption [3].

1.3 Bone remodeling To be such a dynamic tissue, after growing is completed, bone undergoes

constants remodeling processes in structure. These processes function as an

adaptation to the needs and effort developed by the organism [9]. For instance a

professional tennis player has different needs in his superior members, so he

probably will have different structure at bone level. The bone size, his shape and

bone mass distribution will be different in his superior members [3].

It has already been seen, that histologically, exist two types of bone, the cortical

and the spongy bone. The cortical bone is reshaped by the removal and the filling

of osteons (or Havers systems). The active osteoclasts reabsorb the damaged

bone forming this way a lacuna. So if a certain depth is reached in the lacuna,

starts a new stage and the osteoclasts reabsorbed all the osteon. In the next phase

osteoblasts start to produce and depositing bone. In the process of formation of a

15

new osteon, the osteoblasts can be trapped in the matrix, if this happens they

transform into osteocysts.

Due to the different structure (absence of Havers systems) that spongy bone has, it

does not follow the same path of cortical bone and the osteoblasts do not get

arrested in the matrix during the remodeling process. The osteoclasts can be

activated and start to reabsorb bone, if osteoclasts receive signals to stop their

activity, the osteoblasts can begin to do their function and restore the missing bone

[3].

The sequence of events in bone remodeling can be described as in the following

order:

1) Osteocysts connected between them;

2) Small fracture that leads to the osteocysts apoptosis;

3) The site where osteocysts undergoes apoptosis provides the information to

the activated osteoclasts about where the damage occurs;

4) Osteoclasts reabsorbed the damaged bone and remove all the dead cells by

phagocytosis;

5) Reversal process happens, formation of cement;

6) Osteoblasts start depositing the new bone;

7) And as a consequence some of them can be trapped in the matrix

transforming themselves into osteocysts, rebuilding that way the grid of

osteocysts.

1.3.1 Healing process The healing process involves the same cells that the bone remodeling process

needs [10].

The bone shows an incredible ability to regenerate most part of the damage

through the proliferation of the osteoprogenitors cells [1]. After a fracture occur the

following sequence of steps happens:

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i) The blood vessel broke so as consequence a hematoma is formed, which

leads to an afflux of inflammatory cells and the substances secreted by

them, like growth factors activate the osteoblastic progenitor cells;

ii) The hematoma is transformed in a fibrous tissue that some way provides

some support to the boundaries of the damaged site but does not have

enough toughness to support the surrounding tissues.

iii) The osteoblasts begin to deposit the new formed and still immature bone.

This type of primary bone tissue is characterized by an irregular disposition

of collagen;

iv) The MSC give origin to the hyaline cartilage that involves the entire fractured

site;

v) The new bone in the fractured site mineralize;

vi) In the end all the primary bone that has been deposited is remodel and

transformed into secondary bone with regular structure and lamellar shape

with layers of collagen [11, 12].

The age has an important role in the healing process. The rate of a fracture healing

is largely superior in children compared to adults and may be related to

vascularization as well as the periosteum cells that have a less effective cellular

response in relation with the growth factors in people with advantage age [9, 13].

The nutrition is especially important, the calcium and phosphorus have to be in

good levels to a good healing process [9].

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2. Bone marrow

BM is one of the places in the human body where is possible to find SC.

SC originate all the bone forming cells, those responsible for reabsorption as well

as all the cells in blood and immunological system. It is by definition the

hematopoietic organ. From BM also come progenitor cells of cartilage and

adipogenic tissue. The blood vessels surrounding the BM constitute by their own a

physical barrier to the immature blood cells stopping them to leave the BM before

their complete maturation.

2.1 Stem cells A SC is an undifferentiated cell capable of self-renewal for indefinites periods of

time [14-16]. SC undergoes an asymmetric division and the result is a cell equal to

the first and other cell called progenitor cell. So in the end of the division process

we will have one SC that originates two different cells. Every time a division

happens a SC is perpetuating his population. The progenitor cell will follow is

differentiation path and raise eventually cells more specialize and committed with a

specific function, forming this way a cell lineage.

In a regular embryo the cells cross many stages, in the beginning they are

undifferentiated, then committed and at last differentiated. The zygote for instance

although his totipotency is not considered a SC because when it divides it will not

generate cells equal to itself.

SC are now being study as possible carriers of genes to specific tissues in human

body, these investigations have been made mostly for therapy cancer [17]. It is

thought that in the future these cells will probably be able to correct cromossomic

anomalies in the first’s stages of development [17].

2.1.1 Origin Which make these cells so special is mostly:

1) Being undifferentiated cells and not specialize;

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2) Their capacity to self-renewing and division for long periods;

3) Being able to differentiate into distinct cell lineages.

A totipotent cell, the zygote, has potential to originate all kinds of cells and tissues.

The zygote passes through divisions and then differentiates in a developed

organism, so it has an unlimited potential of differentiation.

The pluripotent SC are capable of give rise cells from the three embrionary layers

(Table 1) mesoderm, endoderm and ectoderm. All together can originate any kind

of tissue in an adult organism except the placenta and support tissues of uterus.

The only sources of pluripotent SC are those who have been isolated and

cultivated from human embryos.

Embryonic Germ Layer Differentiated Tissue

Endoderm Thymus Thyroid, parathyroid glands

Larynx, trachea, lung Gastrointestinal (GI)

organs(liver,pancreas) Lining of the respiratory tract

Mesoderm Bone marrow (blood) Lymphatic tissue

Skeletal, smooth, and cardiac muscle Connective tissues(bone, cartilage)

Heart and blood vessels (vascular system)

Ectoderm Skin Neural tissue (neuroectoderm)

Adrenal medulla Connective tissue of the head and face

Eyes, ears

Table 1 -Embryonic germ layers from which differentiated tissues develop, adapted from Stem Cells: Scientific Progress and Future Research Directions [17].

The unipotent SC are usually adult SC meaning that they have the capacity of only

differentiates in one cell lineage. However if a tissue had suffered any damage and

some types of cells are needed the pluripotent SC are activated.

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The SC can have two possible origins: the embryo and the adult organism. There

are two major types of SC: adult and embrionary stem cells (ESC). ESC are

descendents from an internal cellular mass present in blastocyst (4-5 day) [14, 18].

Until 2001 the scientific community has discovered 30 cellular lineages of

pluripotent human cells which can differentiate from the blastocyst cells [17].

By now scientists already have identified adult SC in brain, BM, peripheral blood,

blood vessels, skin epithelium, digestive system, cornea, retina, dental pulp, liver,

pancreas, hair follicle and in oral mucosa. In another words it have been founded

adult SC in organs descendent from the three embrionary layers.

This kind of SC in an adult organism may proliferate for long periods (as definition

of adult SC for all the life of the organism) [14]. Adult SC are rare, difficult to isolate

and purify in laboratory which makes it very hard to get for research. Their

insufficient number is definitely a limiting factor with respect to using them to

repopulate a damaged area. However it’s possible to expand small populations

with a bioreactor, studies have been done in order to make this situation a reality

[19-21].

Besides their origin one of the main differences between ESC and adult SC are

their different capacity of differentiation. ESC are clearly pluripotents and adults SC

are only unipotent [16]. But could adult SC be removed from is natural environment

and being induced or manipulated so they can differentiate into types of cells that

initially they would not become? There are not any definitive answers yet.

ESC can be achieved in great numbers in vitro and they can stay in the

undifferentiation stage for many generations although their tendency to form

tumors are still a problem [14]. If the difficulties associated to the adult SC were

considered the ideal conditions in laboratory for these cells to proliferate without

differentiating has not yet been discovered. The problem is particularly more

severe with the isolated hematopoietic progenitor stem cells (HPSC) from the

blood or from the BM. Despite proliferation may occur in case of transplant to a

human or to an animal when in in vitro conditions they stop proliferating or do it in a

limited way.

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2.1.2 Differentiation Differentiation is the cellular process when a cell unspecialized became committed

to a certain cellular lineage. For differentiation to occur in a SC or in a progenitor

cells it is necessary that biological conditioning happens. The signals that modulate

this procedure in specialize cells are until now unknown.

When a SC suffers its first division gives rise to a cell exactly like the first one and

other called progenitor cell. The progenitor cell enters in a division process and

originates two cells, one is a progenitor cell and the other could be a cell with a

higher level of specialization. These cells are responsible for replacing all the

tissues in damaged areas and maintaining the organs functionality.

Some SC are gifted with plasticity. Plasticity is a recent termination that is applied

every time that an adult SC from a specific tissue as the ability to generate a

specialize cell from another kind of tissue (Figure 4). Unfortunately this process is

not well understood yet. A recent example of this termination is that under specific

experimental conditions the adult SC from de BM may originate cells that look like

neurons [17]. Evidences suggest that giving to an adult SC the proper environment

some will be capable of a genetic reprogramming which leads to cells with

characteristic from other kind of tissues [17].

Figure 4 -Plasticity of a Hematopoietic stem cell [22].

Some intrinsic, inherent to the cell, and extrinsic factors like inductors interfere in

the differentiation process of SC and progenitor cells. Niche is the termination that

includes the surrounding environment and these factors. The surrounding cells in

a niche are those who keeps the SC in a quiescent stage or in a slowly rate of

division. The environmental factors shape the surrounding cells which will induce

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the SC to divide. So we can assume that cells fate is controlled and defined by

environmental and inductors factors [17].

2.1.3 Lineage Cells The human body has as many cells lineages as types of tissue and specialize

cells. In this situation is interesting look deeper in two types of SC in the BM, the

HPSC and the MSC. In Figure 5 all lineages that one single HPSC may originate

are represented as well as their functions.

All blood cells including the cells from the immunological system have their origin in

a HPSC which are a pluripotent cell [23]. With the intervention of several

environmental factors like cytokines these progenitors cells highly undifferentiated

(derived from hematopoietic stem cell) may originate all cells existing in blood [24].

One of the lineages that a HPSC could give rise is a myeloid lineage. The myeloid

progenitors cells might differentiate itself into: megakaryocytes (multinucleated

cells which form blood platelets), erythroblasts (multiply and differentiate in

erythrocytes), myeloblasts (originate neutrophils, eosinophils and basophils),

monoblasts (monocytes precursors) and denditric cells. In response to mediators,

chemokines, leukocytes migrate from blood to the tissues where they repair the

injured tissue and remove bacteria, parasites and dead cells which can lead to a

serious infection. After their migration into some tissues the monocytes from the

blood differentiate into macrophages [18].

Cells with greater relevance in the immunological system are lymphocytes which

are originated from a progenitor cell from the BM. We can distinguish two types of

lymphocytes: the T lymphocytes responsible for the immune response and the B

lymphocytes that as a major task of producing the antibodies and the natural killer

cells which are also related to the T lymphocytes. In case of transplant is only

necessary that one single HPSC is present in the transplanted cells for a total

reposition of all cells of the immune system and the blood [25].

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Figure 5 -Different lineages that a HPSC can originate [17].

One of the difficulties scientists face is that HPSC behave like white blood cells in

culture which make it more difficult to identify simply by morphology. Still exists

many difficulties relating to culture and expansion of this type of SC without the

differentiation process occurs. If it was possible keep these cells in culture without

their differentiation scientist probably would have the chance to study deeper and

looking for more knowledge about their molecular activity, their regulators and

substances that intervene in the self-renewal process allowing higher rates of

success in transplants with HPSC.

The HPSC may be found in the BM, being the BM their most classical source, as

well as in peripheral blood, in the umbilical cord blood (UCB) and in the

hematopoietic system of the fetus. The SC existing in the BM can be coerced to

migrate into the blood stream through the injection of a cytokine, like the stimulate

factor of granulocytes. One day before the harvest this cytokine is injected in the

patient. To the harvesting a catheter is inserted intravenously, so the blood passes

through a filter system that separates the white blood cells and the CD34+, so that

the erythrocytes can be returned to the patient. This method has been chosen by

several doctors for being less invasive then a lumbar puncture.

23

About the MSC, another group of cells that come from the BM [26], their biologic

and clinic interest increase dramatically in the last decades evidence of that is the

huge number of investigation groups that devote their time to these group of cells.

The MSC has also the ability of self-renewing and they can differentiate at least in

three cell lineages. They support the HPSC expansion through the cytokines

expression and through the reconstruction of the hematopoietic environment [14,

24].

The different possibilities of in vitro differentiation are connected with

environmental factors, usually combinations of chemical inductors, growth factors

and cytokines. There are many regulators like Leptin (peptide hormone discovered

in 1994) which is an important factor of mesenchymal differentiation [15]. Has a

significant effect in promoting osteogenesis and in inhibition of adipogenesis in

MSC form the BM [15].

Characteristics of MSC do not generate consensus among the scientists, many

laboratories use different techniques to isolate and expand MSC.

In an attempt to solve this problem The Mesenchymal and Tissue Stem Cell

Committee of the International Society for Cellular Therapy proposed a set of

characteristics that cells must have in order to be classified as MSC [27, 28]:

• Adherence to plastic;

• Specific surface antigen expression;

• Multipotent differentiation potential.

First the MSC should be adherent to plastic culture surface [23, 29], second 95% of

MSC must express the protein molecules CD105, CD73 and CD90 being that

expression verified by flow cytometry [23, 28]. Additionally these cells mustn’t

express CD45, CD34 and CD14 [29] (classical hematopoietic markers) [28]. CD45

is a typically specific marker of the surface of the progenitor cells of white blood

cell. New markers that may be discovered in the future could change some of

these criteria. MSC have to finally be able to differentiate into osteoblasts,

adipocytes and chondroblasts under controlled in vitro conditions (Figure 6).

24

Figure 6 -Different lineages that a MSC can originate [3].

Cells that are plastic-adherent but do not differentiate into lineage cells originated

by MSC are considered to be fibroblasts, which have a similar morphology [26].

The best place to harvest MSC is in the BM, but the rate of proliferation and

differentiation of this organ decrease with age. Harvest MSC from the BM is an

invasive method that may have some risks to the patient. So in the last few years

some researchers have been trying to find another source of MSC [15, 30], the

umbilical cord is one of them and has the advantage of not causing any damage

neither to the mother neither to the child, cells from umbilical cord can be

cryopreserved.

The umbilical cord has two arteries and one vein, protected by a connective tissue

called Wharton’s jelly. Inside the abundant extracellular matrix of Wharton’s jelly it

can be found the recent described population of stem cells named Human

Umbilical Cord Perivascular Cells (HUCPVC) [31].

HUCPVC have showed high potential of proliferation and capacity to differentiate

into osteogenic phenotype [31]. They demonstrate even a higher potential of

proliferation than MSC from the BM and they are also capable of differentiation into

osteoblasts, adipocytes and chondrocytes [31].

It is also proved that differentiation in an osteoblastic lineage by the human

umbilical cord perivascular cells happened in a faster rhythm compared to the MSC

from the BM. In an attempt to isolate and characterize MSC from the BM and from

25

the UCB, in the same conditions, only those harvested from the BM could be

successfully isolated [32].

The UCB is also a source of HPSC, but they do not have mesenchymal

progenitors in it [33]. However the MSC circulate in the blood of premature fetus

and may be isolated with success. Romanov et al [33] tried to obtain MSC from the

subendothelial layer of human umbilical cord vein. They reached the conclusion

that a population with characteristics of the MSC showed a morphologic

resemblance with fibroblasts, expressing mesenchymal surface markers and are

capable of differentiation at least in an osteoblastic and adipogenic lineage.

They discovered that a lower percentage of MSC can be found in UCB and a

higher percentage in the matrix. The cells from the blood and from the matrix of the

umbilical cord multiply faster than the cells from the BM, showing less

differentiation [33].

2.1.4 Identification The most common way to identify any SC types is through molecules existing on

the surface of those cells (markers), specific for every cellular lineage. Some

markers are only expressed in some phases of their cellular cycle, with allows us to

know in which phase of the cycle that cell is in [27].

Flow cytometry allow the evaluation of cellular viability, that have an important role

in medical diagnosis in cases of diseases from immunological system, detection of

quantities and cellular types presents in blood.

26

Flow cytometry uses different light emissions (different wave lengths) that match to

different specific dyes for different molecules. Usually the fluorescent dyes are

connected to protein molecules.

For identification of every cellular lineage is necessary using a complementary

molecule in the cell surface (Figure 7), this molecule has aggregate a fluorescent

dye and his color will be detected by flow cytometry.

Figure 7 -Process of identification of surface markers using fluorescent tags [17].

Another method that besides giving us the identification of different molecules in a

heterogeneous population in suspension, can also allow their separation is named

Fluorescent Activated Cell Sorting (FACS). This method is showed in Figure 8.

Cells in suspension are crossed by a laser beam that detects their fluorescence.

Fluorescent cells stay negatively charged, while the non-fluorescent stays

positively charged. The charge differences permit the separation of SC from the

others cells present in suspension. Using this method is possible to have an entire

population of cells that express all the same markers and can be later expanded in

vitro.

27

Figure 8 -FACS method, separation of SC by their fluorescence [17].

The HPSC expresses mostly in their progenitor phase the CD34 molecule , and

further it advances in their cycle his expression decrease until is not present.

In the Figure 9 it can been confirmed that a HPSC expresses the CD34 molecule

before his maturation process, in the differentiation phase [34]. However this

protein is not by itself an exclusive indicator of the presence of HPSC [24]. The

myeloid lineage expresses mostly the CD15 and the CD33, while the lymphoid

lineage the CD3 and CD19 molecules.

28

Figure 9 -CD34 expression in the HPSC that will originate platelets [35].

.

The greater the information available about the different specific cell markers much

better work could be done and a higher quality of laboratory techniques could be

achieved. Ethical questions will have to be taken under concern and legislation

would have to be made in order to prevent future controversies related to the use

and origin of some cells, like cells from the fetus in name of the scientific progress.

29

3. Bone grafts

Bone grafts are materials used to substitute bone, their main goal is promote

osteoregeneration in injured areas.

3.1 Autografts Autograft is an autologous graft and still is until today the gold standard graft, is

basically the harvest of bone from one site to another site of transplantation in the

same patient [36]. The major limitations of this type of grafts are their amount of

availability and the nature of the harvest procedure, it is also usually associated to

these grafts high morbidity to donor site [36]. However they do not possess the risk

of infection and immunological rejection.

3.2 Allografts These are allogenic grafts and have their origin in post mortem human bone tissue.

With these grafts it is easier to solve some problems characteristic of the autografts

such as elimination of the second surgical procedure and quantity of tissue

available once it is possible use bone banks. On the other hand the risk of

infectious disease transmission to the patient is the biggest problem [36]. Among

these diseases some contaminations of HIV and Hepatitis B and C and bacterial

infection has already been reported [37]. Another major concern regarding this type

of grafts is that the processes used to their preparation, preservation and

sterilization could modify their physical-chemical properties affecting this way their

biological behavior.

3.3 Xenografts Xenografts are grafts with an animal origin, usually bovine. The advantages of

xenografts include their relative abundant supply, ease of use and potentially

favorable clinical performance. The major concern regarding to these procedures is

30

the risk of disease transmission such a bacterial, viral and prion transmission.

Immune response is also a problem related to the safety of this material [38].

The manufacturing process of some of these bone substitutes includes heating for

several hours at temperatures above 1000ºC transforming the material into

ceramics [39]. So products may lose their unique microporosity from native bone

and therefore their osteoconductive capacity could be reduced. The resorption rate

may also be affected because ceramics cannot be such well degraded as

nonsintered materials [39].

3.4 Synthetic For many years the synthetic bone grafts have had a great development. Ideally

these are supposed to promote the cellular adhesion, differentiation and

proliferation of cells. So is particularly important understand the behavior and

structure of the extracellular matrix. These materials may have similar chemical

properties and biological structure to human bone. From clinical point of view these

grafts eliminate the disease transmission risk as well as the need for a second

surgical procedure [2].

There are a huge number of factors that can influence the cellular growth which

can interfere with the necessary mineralization of the osteoblasts that have an

important role in the bone regeneration [40]. And there are some essential

properties that a synthetic graft should have like osteoinduction, osteoconductivity

and promote osteogenesis which is the cellular process of new bone formation.

Osteoinduction include recruitment, proliferation and differentiation of

osteoprogenitors cells into osteoblasts, and osteoconductivity is related to the

structural framework and environment requested to cellular migration, attachment

and growth [2].

The most commonly used materials are Hydroxyapatite (HA) Ca10(PO4)6(OH)2,

Tricalcium phosphatase Ca3(PO4)2 in a α and β forms and bioglasses [37]. Calcitite

from Calcitek, Cerasorb from Curasan AG, Bioglass® from Novabone and

31

Bonelike® from MedMat Innovation-Biosckin are examples of some of these

products available in market.

Biocompatibility of HA as material to bone replacement was tested by flow

cytometry, concluding that it was compatibility with cellular growth [41, 42].

3.4.1 Bonelike ® Bonelike® is a synthetic bone graft, developed by J. D. Santos and co-authors

(Patent WO0068164), based on glass-reinforced hydroxyapatite [2].

It has the ideal environment for bone cell adhesion, proliferation and differentiation.

Due to its chemical composition is reabsorbed in a slow and controlled way and

interfere in the bone’s natural process of remodeling [2]. For being a synthetic bone

graft is completely safe regarding to disease transmission.

Bonelike® can be used in different forms: granular form, dense blocks or in a 3D

macroporous structure depending on the clinical purpose.

Many clinic trials in orthopedic had already been made using Bonelike® granules

implanted in bone defects in tibia [43], macroporous cylinder blocks implanted in

patient’s tibia [44] and wedge in treatment of medial compartment osteoarthritis

[45]. In all of them it has been showed that Bonelike® promote osteoprogenitors cell

differentiation into osteoblasts and has a positive effect during the formation of a

mineralized matrix [2, 46-48]. Clinical tests in maxillofacial surgery have

demonstrated that granules of Bonelike® have been successfully used in the

treatment bone defects caused by cyst removal [49, 50], as well as used in coating

titanium dental implants in order to promote osteointegration [51].

3.4.1.1 Composition

Bonelike® is prepared with the incorporation of a CaO-P2O5 based glass in the HA

matrix by means of a liquid phase sintering process in order to increase the

mechanical properties of HA and to introduce ions commonly found in bone tissue.

The incorporation of glass instigate the decomposition of the HA part to a

secondary phase, β-TCP. At higher temperatures this β- phase changes to α-TCP.

32

Once these phases have higher solubility than hydroxyapatite, Bonelike® releases

to the medium ions such as calcium, phosphorous, sodium and fluoride which

induce osteoblastic differentiation leading to a strong connection between bone

and the bone graft [2].

3.4.1.2 Structure and Properties

This synthetic bone graft has two important characteristics. Firstly Bonelike®

enhance bioactivity by reproducing the inorganic phase of HA in bone which

contains several ionic substitution which modulates its biological behavior and

secondly improve mechanical properties by using CaO-P2O5 based glasses that

act as liquid phase sintering process of HA [2, 52].

Different forms of Bonelike® have different porosities, densities and in

consequence their superficial areas are different to. These properties are

fundamental so the proper type of Bonelike® could be chosen to the correct clinical

situation.

Pores of Bonelike® by allow diffusion of proteins and essential growth factors

present in the patient’s blood into Bonelike® interstitial spaces, promote

osteoconductivity enhancement.

Microstructure of Bonelike® Pellets (Figure 10) is positive for surface adsorption of

native proteins in patient’s blood allowing the scaffold to be recognized by the

organism.

Figure 10 -Bonelike® Pellets structure. SEM analysis.

33

Bonelike® Poro has higher interconnective porosity that facilitates the blood vessel

and osteoblastic cells migration, through the interior of the bone graft increasing

the rate of formation of new bone. Porous materials permit the growth of a capillary

vascular network and the establishment of cell-cell contact between the bone graft

and osteoprogenitors cells that are absolutely essential for growth and cellular

organization.

Figure 11 -Bonelike® Poro 2000-6000µm. SEM analysis.

Different types of porosity present in Bonelike® Poro, micro meso and

macroporosity (Figure 11) together with the degree of interconnectivity improve the

nutrients and gases diffusion in the physiological fluids as well as the removal of

metabolic waste.

34

4. Tissue Engineering

Tissue engineering is a term which includes concepts from life sciences, such as

biology and engineering, with surgical procedures to develop strategies for the

regeneration of tissue.

The procedures used in tissue engineering are now being used in a large number

to optimize medical treatments and develop new methods. The main goal to

achieve is speeding up the healing and damaged tissues. The application of

allografts and total joint replacement are the most successful options in bone

regeneration. On the other hand some problems are still happening due to their

mechanical and biological properties [53].

In tissue engineering these procedures are part of routine: cell harvesting,

signaling molecules and a support matrix (scaffold). A scaffold is a polymeric

support where the cells will be seeded together with growth factors for a posterior

implantation in human body to promote bone regeneration in the damaged area.

This technique seeks to create a faster growth of the cells as much as is possible,

so they can differentiate and proliferate like they were in is natural environment

replacing the injured tissues by functional ones.

Cells from adult organs can be used as sources for cultured cells [54]. Cells are

dissociated from tissue by the use of enzymes or mechanical forces to disrupt the

extracellular matrix. Cells in culture can be maintained as single cells, as small

explants of intact tissue, or as whole segments of organs.

Cells are the most important factor in any regenerative process [55]. Sometimes is

necessary to expand cell population so that an effective process of regeneration

may occur, that is accomplished with bioreactors [19-21].

For a successful regeneration of the injured tissues the cells, signals and the

scaffold has to establish a connection so they can interact and restore the

damaged area forming a complete regenerated and functional tissue (Figure 12).

Scaffolds can be formed by many materials like membranes, gels, ceramics, cells

also can have different origins, stem cells, adult organs, and signals have the task

of inducing cells to form an extracellular matrix.

35

Cells must react to their surrounding and be able to integrate the new environment

and synthesizing extracellular matrix [16, 56].

Figure 12 –Principal intervenients in the tissue regeneration process in tissue engineering.

Concerning the case of bone tissue is essential de capability of supporting

additional loads, so scaffolds must be able to do this while the cells reconstruct the

biomatrix (Figure 13) [57].

Figure 13 -Microstructures of bone substitutes. SEM analysis.

Knowledge at the molecular level of cell interaction with synthetic materials has

advanced rapidly in the last decade so the designs of new materials for tissue

regeneration tend to be designed more carefully.

36

Chapter 2

Experimental procedures

37

Chapter 2

Experimental Procedures

Preparation of Bonelike ® Bonelike ® Pellets

Pellets were fabricated according to the Patent WO0068164. Preparation of pellets

begins with the mixing of Hydroxyapatite and bioglass with microcrystalline

cellulose, followed by extrusion and spheronization. Pellets were then dried at

60ºC, after the drying the thermal treatment was made. The thermal treatment has

two steps, in the first one heating at a temperature of 600ºC causes the

combustion of the microcrystalline cellulose, followed by a temperature increase to

1300ºC for 1h, for sintering the pellets follow by natural cooling inside the furnace.

The obtained pellets show a particle size range of 1000-4000µm.

Bonelike ® Poro

Bonelike® Poro is the result of a suspension composed of hydroxyapatite, bioglass,

microcrystalline cellulose and polyvinyl alcohol (PVA) and subsequent combustion

of the microcrystalline cellulose and sintering at 1300ºC. PVA and microcrystalline

cellulose both act as agents of formation of porosity and PVA, being an organic

ligand, allows keeping all the solid components together in the suspension avoiding

their deposition. Bonelike® Poro show a particle size range of 2000-6000µm.

In both materials surface area was achieved by BET method and porosity was

determined by Mercury Porosimetry. Bonelike® Pellets and Poro were autoclaved

before used in the biological studies.

38

In vitro biological studies Bone Marrow Concentrate

Human bone marrow was collected during orthopedic surgical procedure, with

patient informed consent. The Biomet Kit MarrowStimTM Concentration System was

used to achieve a rapid preparation of autologous concentrate of nucleated cells

from a sample of bone marrow aspirate (Figure 14).

The needle was coated with heparin before the extraction of the bone marrow.

After the extraction of the bone marrow aspirate the cell separator was loaded with

the aspirate and centrifuged at 3200RPM for 15 min. After removing the plasma

from the cell separator, the portion of nucleated cell concentrate was extracted.

The concentrate of nucleated cells obtained was about 3mL.

Figure 14 –Cell separator from the Kit with the three distinct parts. From the top, cell poor plasma, nucleated cell concentrate and on the bottom the red blood cells.

Flow Cytometry

Flow cytometry showed that the bone marrow concentrate contained 6x107

nucleated cells and 2,5x105 hematopoietic cells. This concentrate was diluted with

minimum essential medium (α-MEM) and the resulting cell suspension was seeded

at 3x106 cell/cm2 over 0.10g/cm2 of Bonelike® (Pellets or Poro) placed in 24-well

plates, and cultured for 30 days. Cultures performed in standard tissue culture

plates (absence of materials) were used as control.

39

Cell Culture

Cultures – seeded Bonelike® and control - were performed in α-MEM containing

15% fetal bovine serum (FBS), 100µg/mL penicillin, 10 IU/mL streptomycin,

2.5µg/ml fungizone and supplemented with 50µg/mL ascorbic acid and 10mM of β-

glycerophosphate. Incubation was carried out in a humidified atmosphere of 95%

air and 5% CO2 at 37ºC. Culture medium was changed twice a week.

Previous studies showed that Bonelike® pre-incubation was not necessary to

enhance the performance of the material.

MTT

Cell viability/proliferation of the seeded Bonelike® samples and control was

determined by MTT assay, that relies on the ability of viable cells to oxidize MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble blue

formazan product. Cultures were incubated with 0.5mg/mL-1 of MTT for the last 4h

of the culture period tested. Then, the materials were transferred to a new plate,

the formed formazan salt was dissolved with dimethylsulphoxide, and the

absorbance was determined at 600nm. MTT assay was performed at days 1, 4, 9,

17, 23 and 30 of culture time. .

Total protein and ALP activity

Total protein (TP) content regarding seeded Bonelike® and control was determined

in 0.1M NaOH cell lysates according to the method of Lowry using bovine serum

albumin as standard.

ALP activity was assayed in cell lysates (obtained by the treatment of the seeded

material with 0.1% triton), by the hydrolyses of p-nitrophenol phosphate in alkaline

buffer solution, pH 10.3, for 30 min, and colorimetric determination of p-nitrophenol

at 405 nm. Both ALP and Total protein content assays were performed at days 9,

17, 23 and 30 of culture time. ALP activity was normalized by total protein content,

40

and was expressed in nanomoles of p-nitrophenol produced per min per µg of

protein (nmolmin-1/µgprotein).

SEM

For SEM observation, cells were fixed with 1.5% glutaraldehyde in 0.14M sodium

cacodylate. Samples were dehydrated in graded series of alcohols and further

dried with Hexamethyldisilazane. Samples were mounted onto aluminium supports,

super-coated with gold, and observed in a Joel JSM 35C scanning electron

microscope equipped with an X-ray energy dispersive spectroscopy voyager

XRMA System, Noran Instruments.

Statistical analysis

Three replicas were performed for each assay. Results of ALP activity and MTT

are presented as mean ± standard deviation (SD). Comparisons between seeded

Bonelike® samples and the control were performed by the Student’s t-test.

Differences were considered statistically significant at p< 0.05.

41

Results and Discussion

Bonelike ® characterization Bonelike® samples were prepared in the Laboratory of Medmat Innovation,

Biosckin SA, and were examined by SEM (Figure 15). Bonelike® is a mixture of

hydroxyapatite and bioglass, which result in a triphasic mixture of hydroxyapatite,

α- and β- tricalcium phosphate (α-TCP and β–TCP) [2, 47, 52], under the shape of

spherical granules named Bonelike® Pellets, and bigger scaffolds with a

heterogeneous shape, but with macroporous structure named Bonelike® Poro.

β-TCP is resorbed in the body at a very fast rhythm, however the α-TCP does not

have the same resorb rate as the β-TCP. α-TCP when in contact with human

plasma hydrolyzes itself. So, in vivo as the β-TCP is resorb from the beginning, the

scaffold will not just dissolve away, because the α–TCP which have a slower rate

of resorption maintain the structure of macroporous scaffold which will be later

resorbed by the osteoclasts cells [58-60] .

The α-TCP and β- TCP phases are much more soluble than the hydroxyapatite, so

as Bonelike® is implanted in the organism, releases calcium and phosphorous that

induce differentiation of osteoblastic cells [2, 47, 52]. Livingston et al showed that

hydroxyapatite combined with β-TCP stimulate the osteogenic differentiation of

mesenchymal stem cells [61, 62]. This material besides stimulating osteoblastic

differentiation without adding growth factors also enhance the osteoblast-specific

gene expression [63, 64].

Muller et al consider that surface chemistry has a bigger role in the differentiation

process than the topography [63], however Zhang et al showed that is the surface

structure of the material that interfere more in cells differentiation [64] and Kasten

et al even say that pore distribution and size as well the surface structure are the

essential features for osteogenic differentiation [65].

Bonelike® Pellets showed a particle size range of 1000-4000µm. Pellets have a

microporosity determined by mercury porosimetry of 25.3% and a surface area of

0.0171m2/g as determined by BET method. These pellets present microposity and

42

their surface have some rugosity that allow cells to attach and migrates through the

scaffold, as it may be seen in Figure 15.

Figure 15 –Bonelike® Pellets. a) pellets with some irregular details (arrows) in surface, b) microporosity present in the pellets surface (arrows). SEM analysis.

In terms of clinical applications, Bonelike® Pellets are suitable for fill small bone

defects and their size and spherical shape permit the injectability through a syringe

if it is necessary, so this material can been seen as an injectable bone graft

substitute.

Scaffolds of Bonelike® Poro showed a particle size range of 2000-6000µm, (see

Figure 16), with macro (pore diameter range 224.62 µm), meso (pore diameter

range 19.097 µm) and microporosity (pore diameter range <2.0 µm) which results

in an interconnective porosity presenting 70% of total porosity determined by

mercury porosimetry. This characteristic influence the type of topography of the

surface with 0.1152m²/g of surface area as determined by BET method, being

much more heterogeneous compared to that of the Bonelike® Pellets.

Concluding both materials have the same composition but differ in terms of shape,

micro and macroporosity distribution as well as total porosity.

Microposity allows the attachment of cells, meso and macroporosity permit the

migration of cells through the interior of scaffold with the possibility of colonization

43

of a much bigger area. Bonelike® Poro have interconnective porosity which

facilitate the growth of blood vessels, vascularization, through the interior of

scaffold allowing bone growth through the inside [66]. Formation of new blood

vessels and the establishment of a capillary network induce the interactions cell-

cell which are essential for cellular organization [17, 67, 68]. Studies have proved

the importance of cell-cell contact in stem cell differentiation [69, 70], and those

interaction can be controlled by a cell-material interaction with particular attention

to the design of the surface material [69]. Bonelike® Poro show a high percentage

of porosity, this particular structure is achieved with the use of microcrystalline

cellulose and polyvinyl alcohol [71-73].

Figure 16 –Bonelike® Poro samples. a) high level of porosity, pores with different sizes, b) irregular surface with microposity (arrow). SEM analysis.

From the clinical point of view, the different shape of Bonelike® Poro scaffolds

allows the surgeon to handle separately every scaffold with tweezers, which

sometimes is useful in surgery, their irregular shape can also permit a better

fixation at the bone defect decreasing this way the movement of the material in the

damaged area. Bonelike® Poro is intended to be used in large bone defects in

orthopaedic applications.

Topological structure of the surface material could influence cell behavior as well

as morphology, proliferation and growth activity [74], so for both formulation of

44

Bonelike® Pellets and Poro, differences in result are expected due to their different

shape, structure and porosity.

In vitro biological studies

A concentrate of mononuclear cells prepared from a human bone marrow aspirate

was seeded over Bonelike® Pellets and Bonelike® Poro, in order to evaluate the

cell response to the material regarding attachment, growth and osteoblastic

differentiation.

Flow cytometry (data not shown) revealed that prepared bone marrow concentrate

contained 6x107 nucleated cells and 2.5x105 hematopoietic stem cells. Some

studies show that the percentage of MSC in the nucleated cells harvested from the

BM is only 0.01 to 0.001% of the total nucleated cells [23, 75]. MSC present in the

nucleated component of the bone marrow concentrate, originate the osteoblastic

lineage cells, so they are the most important cells in terms of bone reconstitution

[23, 31]. Osteoclasts originated from MSC are also very important in the

regeneration process that involves calcium phosphate (Ca-P) scaffolds, they

contribute to the Ca-P degradation and also stimulate osteoblastic activity [64, 76,

77]. However, in vivo hematopoietic progenitor stem cells (HPSC) are also

important, as these cells are responsible for the new vascularization of the

damaged area, with constant supply of nutrients and essential growth factors, as

well as the immunological behavior [17, 24]. In vivo MSC and HPSC are both

essential to a successful bone regeneration process [24, 27].

Colonized materials were cultured for 30 days in experimental conditions known to

favor the development of the osteoblast phenotype, namely in the presence of

ascorbic acid and beta-glycerophosphate (bGP) [7]. Ascorbic acid plays an

important role in the production of the collagenous bone extracellular matrix, some

studies showed that the presence of various concentrations of this compound

results in a dose-dependent synthesis of collagen and suggested that the resulting

increase in the accumulation of the extracellular matrix is associated with a higher

45

alkaline phosphatase (ALP) activity and ability to form a mineralized matrix [78,

79]. bGP is routinely added to bone cell cultures to induce osteogenesis and

promote calcium phosphate deposition [80-82]. The mechanism by which bGP

induces mineralization is closely linked to the high ALP activity of bone cell

cultures, this compound is rapidly hydrolyzed by ALP to produce high levels of

local phosphate ions providing the chemical conditions for mineral deposition [80].

ALP is an enzyme expressed by MSC during the osteogenesis and is considered a

marker for their differentiation [63, 83, 84].

Bonelike ® Pellets

Bonelike® Pellets were cultured with a concentrate of nucleated cells prepared from

a bone marrow aspirate, as described in the section “Experimental procedures”.

Cultures were maintained for 30 days. SEM appearance of the colonized material

at early culture time (1 day) is presented in Figure 17. The concentrate of BM

contained some erythrocytes, which were removed during subsequent changes of

the culture medium (see arrows).

Figure 17 -Bonelike® Pellets seeded with the bone marrow concentrate, at 1 day of culture, a),b) showing some erythrocytes (arrows). SEM analysis.

46

SEM images regarding culture times from 4 to 23 days (see Figure 18) showed

that MSC were able to attach and spread over the surface of Bonelike®, taking

advantage of the surface microporosity and topological structure. At day 4, cells

showed an elongated fibroblastic morphology that changed progressively to a more

extended and polygonal appearance at days 9 and 17. Cells used the topographic

irregularities on the surface to adhere, which is clearly visible at days 9 and 17 (see

arrows). Cells proliferated with culture time, and by days 17 and 23, abundant cell

clusters were visible over the material surface. These clusters appeared to be

associated with particular surface features, namely small defects scattered over

the surface, where it is easier to cells to create niches. Within the cell

clusters/niches, cell layer appeared well organized with established cell-to-cell

communication (days 17 and 23) abundant cytoplasmic extensions and a perfect

adaptation to the underlying material microporosity and topography at days 9, 17

and 23 of culture. By day 23 of culture, the cell clusters present evidences of matrix

mineralization, Figure 19 (arrow). Mineralized deposits were identified in close

association with the cell layer.

Mesenchymal stem cells are able to differentiate in an osteogenic lineage [26, 31,

33]. Studies concerning the development of the osteoblast phenotype suggest a

temporal sequence of differentiation involving the adhesion of the mesenchymal

stem cells to a biological (pre-existing bone tissue) or artificial (biomaterial scaffold)

substratum, active cell proliferation accompanied by the production of a

collagenous extracellular matrix and expression of osteoblast-related markers, like

ALP (seen in Figure 23) and extracellular proteins, and, ultimately, matrix

mineralization (Figure 20) [3, 7, 47, 85].

Figure 20 shows a representative spectrum only of the mineralized deposits

signaled in Figure 19 b), displaying the presence of Calcium and Phosphorous

peaks.

47

Figure 18 -Bonelike® Pellets colonized with bone marrow cells, at 4 to 23 days. a) presence of

defects through the surface; b), c), d), f) cells take advantage of microposity to attache to surface

material (arrows); e), g) cluster of cells on the material surface (arrows). SEM analysis.

Day 4

Day 9

Day 17

Day 23

48

Figure 19 -Bonelike® Pellets colonized with bone marrow cells. a) cluster of cells at day 23, b) evidence of mineralized deposits associated with the cell layer within the cell clusters (arrow). SEM

analysis.

Figure 20 -Bonelike® Pellets colonized with bone marrow cells, at 23 days. Representative spectrum of the mineralized deposits. X-ray spectrum.

Figure 21 shows colonized Bonelike® Pellets, after being incubated with MTT, a

cell viability/proliferation assay. MTT assay provides a general view of the spatial

49

distribution of cells through the material. Bone marrow cells showed specific

preferential attachment locations, growing in niches, apparently associated with

small defects on the material surface. This type of behavior is reinforced by the

SEM analyses of the colonized Pellets.

Cells seeded over Bonelike® Pellets attached to preferential locations on the

material surface, like some defects characterized by higher irregularities on the

material surface as can be seen in Figure 21 a) (arrows), forming well-organized

cell clusters, perfectly adapted to the underlying surface. This is in line with

previous studies showing that cells can guide themselves according to

morphological patterns of the surface [86-88].

Figure 21 -Bonelike® Pellets colonized with bone marrow cells on day 23. a) cluster of cells, and cells growing on defects present on the surface(see arrows); b) control culture; 20x..

Figure 22 presents the cell growth rate observed on the colonized Bonelike®

Pellets, compared to that found on control cultures (performed on standard tissue

culture plates). Control cultures presented a lag phase until day 9, with a low

growth rate, but, afterwards, cell growth increased significantly, especially from

days 17 to 23, maximum values were achieved by day 23 and, after that, cell

proliferation decreased.

Colonized Bonelike® Pellets presented a similar behavior in the first days, with a

lag phase until day 9, but cell growth increased significantly from day 9 to 17.

50

Maximum values were attained on day 17, and after that cell growth begin to

decrease.

As seen in Figure 22 from the day 17 until day 30 big differences can be noted in

cell growth on Bonelike® Pellets and control cultures. Day 17 is where values

presented by both cultures most differ. Some hypothesis may be formulated to

explain this different behavior, such as cells seeded on Pellets have a much faster

rate of growth due to the material composition of Bonelike® that has the ability to

release ionic species like fluoride and sodium which improve the cellular growth

and promote the differentiation towards to an osteoblastic lineage [89], and also

Bonelike® topography, microporosity is an important factor to consider as already

mentioned before. Cells from control cultures were all exposed the same way to

the culture environment, such fact did not happen with cells on the Pellets that

could form niches in the irregularities mentioned above that could offer better

growing spots as can be seen in Figure 21 (arrows).

Figure 22 -Cell growth rate over colonized Bonelike ® Pellets and control cultures. MTT assay. *significantly different from control.

Results regarding ALP activity are presented in Figure 23. In control cultures, ALP

activity increased from days 9 to 23, where maximum values were achieved, after

that, ALP activity decreased. In the colonized Bonelike® Pellets, ALP activity

0

20

40

60

80

100

1 4 9 17 23 30

Cel

l Gro

wth

Rat

e%

Days

Bonelike® Pellets vs. control culture

Bonelike® Pellets

Control

*

**

51

increased significantly from days 9 to 17, decreasing after that. In the present

work, bone marrow cells cultured in standard tissue culture plates (control cultures)

presented a similar behavior to that mentioned above and established for the

development of the osteoblast phenotype. After a lag phase of few days, most

probably due to the adaptation to the culture conditions and the low number of

mesenchymal stem cells known to be present in the bone marrow, cells entered a

period of exponential cell growth, followed by a slow decrease of the growth rate.

Figure 23 -ALP activity measured over colonized Bonelike® Pellets and control cultures. * significantly different from control.

Being ALP a well known marker of osteoblastic differentiation as mentioned before

Figure 23 demonstrate that cells seeded on Bonelike® Pellets present higher levels

of ALP sooner that cells from control cultures , day 17, and with this is possible to

conclude that material (composition and structure) influence positively the

growth/differentiation of MSC into an osteoblastic lineage [63-65, 87]. The

maximum value for ALP/TP in control cultures was achieved by day 23, later than

the maximum value of Bonelike® Pellets, also reinforces the idea that is the

material that improve cell destiny direct to an osteoblastic commitment.

0

0,005

0,01

0,015

0,02

0,025

0,03

0,035

0,04

9 17 23 30

nmol

ALP

/ µ

g pr

otei

n

Days

Bonelike® Pellets ALP/TP

Bonelike® Pellets

Control

**

52

Bonelike ® Poro

A concentrate of nucleated cells prepared from a bone marrow aspirate was

seeded over Bonelike® Poro, and the seeded materials were cultured for 30 days.

This material compared to Bonelike® Pellets has different levels of porosity (macro,

meso and microporosity) with much higher surface area then Bonelike® Pellets and

therefore provides to cells the opportunity to adhere and attache to a huge quantity

of locations throughout the scaffold. The concentrate of bone marrow contained

some erythrocytes and, at early culture times (1 day), blood cells were observed on

the material surface, as shown by SEM image in Figure 24. Blood cells take also

advantage of the topography of the surface material, using the different levels of

porosity and the irregular morphology to establish themselves. These cells were

progressively removed during the subsequent culture medium changes.

In Figure 25, images of colonized Bonelike® Poro regarding culture times from days

4 to 23 are shown. Cells use the irregular surface of the material to attach taking

advantage of the microporosity, as observed in 4-day cultures. As the culture time

advances, cells proliferate through the material, days 9 and 17, and the

establishments of cellular bridges between surface defects are clearly seen.

Figure 24 -Bonelike® Poro, at 1 day of culture. a), b) erythrocytes on the porous surface (arrows). SEM analysis.

53

Figure 25 -Bonelike® Poro colonized with bone marrow cells, at 4 to 23 days. a), b) cells taking advantage of microporosity to attach to the material (arrows); c) cells colonizing the interior of

pores; d), e), f) bridges made by cells through some irregularities of the material (arrows); g), h) several layers of cells adapted to the topological morphology of the material’s surface(arrows). SEM

analysis.

Day 4

Day 9

Day 17

Day 23

54

By day 17, cells started covering the macropores of the material of thin layers with

abundant cell-to-cell contact. This behavior is more explicit by day 23, with dense

cell layers perfectly adapted to the highly heterogeneous porous surface. In a study

made by Bignon et al macroporosity and interconnective pores size influence the

penetration of cells through the scaffold, and that migration is performed by cells

with their cytoplasmic extensions that can hanged to the micropores [66, 90, 91].

On seeded Bonelike® Poro, attached cells were able to colonize the entire

heterogeneous scaffold forming a continuous cell layer perfectly adapted to the

porous surface. Bonelike® Poro is intended to find application in large bone defects

such as in orthopaedic applications.

The macroporosity will allow the growth of blood vessels in the interior of the

scaffold favoring the constant supply of nutrients and growth factors and the

removal of metabolic waste products. The microporosity will be suitable for cell

adhesion, proliferation and differentiation. The colonization of the entire porous

scaffold is expected to speed up the regeneration process of the bone defect.

For a cell and tissue growth, porosity is absolutely essential thus it affects the

biological activity of cells [64, 91, 92] as other study as proved the relation between

pore size and biological response to implantation [91-93], so porosity and pore size

can influence the protein production and also ALP activity in this type of TCP

materials [65].

SEM observation of the colonized Bonelike® Pellets (Figure 19) and Bonelike®

Poro (Figure 26) at later incubation times (day 23) showed well-organized cell

layers, with abundant cell-to-cell contact and associated calcium phosphate

mineralized deposits (arrows). These results suggest that mesenchymal stem cells

were able to attach, proliferate and completely differentiate into the osteoblastic

lineage.

55

Figure 26 -Bonelike® Poro colonized with bone marrow cells, at 23 days. a), b), c) and d) abundant mineralized deposits closely associated with the cell layer (see arrows). SEM analysis.

The x-ray spectrum of the mineralized deposits seen on colonized Bonelike® Poro

is represented in Figure 27, showing Calcium and Phosphorous peaks which

indicates the mineralization signalized by the arrows in Figure 26. In this spectrum

the Ca and P represented are only from the mineralized crystals on top of cell

layers and not belonging to the scaffold.

Colonized scaffolds of Bonelike® Poro incubated with MTT, Figure 28, showed an

exuberant cell proliferation through the entire scaffold, as observed in SEM

images.

56

Figure 27 -Bonelike® Poro colonized with bone marrow cells, at 23 days. X-ray spectrum of the mineralized deposits.

Figure 28 -Bonelike® Poro colonized with bone marrow cells at day 23. a) exuberant proliferation through the entire scaffold; b) control culture; 20x.

Results regarding the cell distribution over Bonelike® Pellets and Bonelike® Poro,

evaluated by the observation of the colonized material after the MTT assay (low

magnification images) and SEM observation (high magnification images), revealed

that the cells were sensitive to the surface morphology showing different patterns

in spatial distribution through the scaffold. In addition, significant differences

57

between the two formulations related to structure, porosity and surface topography

were evident which may have influenced cellular growth.

Control cultures have a slow initial growth until day 9, but cell growth rate

increased significantly from days 9 to 17, decreasing slowly after that. Cell growth

rate observed on colonized Bonelike® Poro is shown in Figure 29. Seeded

Bonelike® Poro samples show a slow growth rate until day 4, but cell proliferation

increased significantly from days 4 to 17. Maximum growth was achieved around

days 17-23, followed by a slow decrease. The significance in the difference of

values form control cultures and on seeded Bonelike® Poro are particular noticed

from day 17 to 23.

Figure 29 –Cell growth rate over colonized Bonelike® Poro and control cultures. MTT assay. *significantly different from control.

Results from Bonelike® Poro day 1 to day 17 are significantly different from those

presented in control culture, so the highest growth associated to cells seeded on

Bonelike® may be related to the higher surface area available to cells attach and

proliferate and composition of the material according to aforementioned studies

related to the release of ionic species like sodium and fluor that enhance

osteoblastic cellular growth. Cells have optimal conditions regarding porosity

(70%), topography of the surface and chemical composition, with plenty of spots to

0

10

20

30

40

50

60

70

80

90

100

1 4 9 17 23 30

Cel

l Gro

wth

Rat

e%

Days

Bonelike® Poro vs. control culture

Bonelike® Poro

Control

*

*

*

*

58

attache migrate and colonize through the entire scaffold (interconnective porosity)

being constantly stimulated to a commitment with the osteoblastic lineage by the

release of ions of calcium and phosphate, all of these leads to a faster rate of

growth/differentiation compared to cells from control culture which only received

medium supplemented with ascorbic acid and β-glycerophosphate.

On colonized Bonelike® Poro, ALP activity attained maximum values by days 17-

23, whereas in control cultures maximum values were found at day 23 (Figure 30)

suggesting that the growing cells were engaged with an osteoblastic differentiation

process, as it is well established that ALP is synthesized by osteoblastic-lineage

cells and is involved in the mineralization of the extracellular matrix [3, 7, 85]. After

that, ALP activity decreases in both conditions throughout the remaining culture

time.

Figure 30 -ALP activity measured over colonized Bonelike ® Poro and control cultures. *

significantly different from control.

As mentioned about cells seeded on Bonelike® Pellets, for those seeded on

Bonelike® Poro is also true, porosity affects positively ALP activity [65]. Zhang et al

proved that some osteogenic markers like ALP where highly expressed in porous

in Ca-P than in dense-smooth materials [64].

0

0,005

0,01

0,015

0,02

0,025

0,03

0,035

0,04

9 17 23 30

nmol

ALP

/ µ

g pr

otei

n

Days

Bonelike® Poro ALP/TP

Bonelike® Poro

Control

*

59

Conclusion

Bonelike® Pellets and Bonelike® Poro formulations to be used, respectively, in the

regeneration of small and large bone defects, allowed the attachment, proliferation

and osteoblastic differentiation of mesenchymal stem cells present in the

concentrate of mononuclear cells prepared from an aspirate of bone marrow. In

addition, both material formulations induced significantly the cell proliferation,

compared with a standard tissue culture surface.

Cells growing on Bonelike® Pellets in vitro show to prefer locations with some

irregularities on the material surface as starting point to colonize the rest of the

scaffold, On the other hand, cells seeded on Bonelike® Poro present an

homogeneous distribution through the entire scaffold, which is more suitable for a

faster cellular growth due to structure and topography. On both formulations of

Bonelike® cells present a higher and faster rate of growth compared to the control

cultures, which means that material has a positive influence on the cellular

behavior towards to a faster proliferation and differentiation of the osteoblastic

cells.

Despite being very difficult extrapolating from in vitro to in vivo conditions, results

suggest that using a mixture of Bonelike® Pellets or Bonelike® Poro formulations

with an autologous bone marrow concentrate to fill the bone defect will improve the

clinical outcome of the regenerative process.

It is possible infer that with the natural supply of nutrients and removal of metabolic

waste cells will rapidly colonize the entire scaffold, which is the best process to a

complete regeneration of the bone defect. Cells in their natural environment with all

the essentials factors will provide a faster process of healing, which is the main

goal for bone substitutes.

60

References

[1] Lieberman, J.R. and G.E. Friedlaender, Bone Regeneration and Repair - Biology and Clinical Applications. 2005: Humana Press.

[2] Santos, J.D., Bonelike Graft for Regenerative Bone Applications, in Surface Engineered Surgical Tools and Medical Devices, M.J. Jackson and W. Ahmed, Editors. 2006, Springer. p. 477-512.

[3] Bilezikion, J.P., L.G. Raisz, and T.J. Martin, Principles of Bone Biology. 3ª ed. Vol. 1. 2008: Academic Press.

[4] Felix Bronner, M.C.F.-C., Janet Rubin, Bone Resorption. Vol. 2. 2005: Springer.

[5] White, T.D. and P.A. Folkens, The Human Bone Manual. 2005: Elsevier Academic Press.

[6] Junqueira and Carneiro, Histologia Básica Texto e Atlas. 10ª ed. 2005: Guanabara Koogan.

[7] Marcus J., J.F., Wiebke D., Xinning L., David C., Akos C., Wolf P., Sabine L., Rudiger K., Dexamethasone Modulates BMP-2 Effects on Mesenchymal Stem Cells in Vitro. Orthopaedic research Society, 2008. 26: p. 1440-1448.

[8] Shinji OGAWA, H.H., Masanori FUJIWARA, Shuzo TAGASHIRA, Takashi KATSUMATA, and Hiroshi TAKADA*, Cbfal, an Essential Transcription Factor for Bone Formation, Is Expressed in Testis from the Same Promoter Used in Bone. DNA RESEARCH 2000. 7: p. 181-185.

[9] Petite, H. and R. Quarto, Engineered Bone. 2005: Eureka.com/ Landes Biosciences.

[10] Harry C. Blair, M.Z., paul H. Schlasinger, Mechanisms balancing skeletal matrix synthesis and degradation. Biochem. Journal, 2002. 364: p. 329-341.

[11] Dudek, R.W., High-Yield Histology. 2 ed. 2000: Lippincott Williams & Wilkins.

[12] Martin Braddock, P.H., Callum Campbel, Patrick Ashcroft, Born Again Bone: Tissue Enginering for Bone Repair. News Physiol. Sci., 2001. 16.

[13] KT Wright, W.E.M., A Osman, S Roberts, J Trivedi, BA Ashton, WEB Johnson The cell culture expansion of bone marrow stromal cells from humans with spinal cord injury: implications for future cell transplantation therapy. Spinal Cord, 2008: p. 1-7.

[14] Burt, R.K., Y. Loh, and W. Pearce, Clinical Applications of Blood-Derived and Marrow-Derived Stem Cells for Nonmalignant Diseases. JAMA, 2008. 299: p. 925-936.

[15] Chang, Y.-J., et al., Disparate Mesenchyme-Lineage in Mesenchymal Stem Cells form Human Bone Marrow and Umbilical Cord Blood. Stem Cells, 2006. 24: p. 679-685.

[16] Susan X. Hsiong, t.B., Nathaniel huebsch, David J. Mooney Cyclic Arginine-Glycine-Aspartate Peptides Enhance Three-Dimensional Stem Cell Osteogenic Differentiation. Tissue Engineering, 2009. 15.

[17] Stem Cells: Scientific Progress and Future Research Directions. 2001.

61

[18] Walter C. Low, C.M.V., Stem Cells and Regenerative Medicine. 2008: World Scientific.

[19] Pankaj Godara, C.D.M., Robert E Norton, Design of bioreactors for mesenchymal stem cell tissue engineering. Journal of Chemical Technology and Biotechnology, 2008. 83: p. 408-420.

[20] James A. King, W.M.M., Bioreactor Development for Stem Cell Expansion and Controlled Differentiation. Curr Opin Chem Biol, 2007: p. 394-398.

[21] Xi Chen, H.X., Chao Wan, Mervyn McCaigue, Gang Li, Bioreactor Expansion of Human Adult Bone Marrow-Derived Mesenchymal Stem Cells. Stem Cells, 2006. 24: p. 2052-2059.

[22] Burmester, G.-R. and A. Pezzutto, Color Atlas of Immunology. 2003: Thieme.

[23] D. McGonagle, A.E., E. A. Jones, The relevance of mesenchymal stem cells in vivo for future orthopaedic strategies aimed at fracture repair. Current Orthopaedics, 2007. 21: p. 262-267.

[24] Silva, C.L.d., et al., Modelling of ex vivo expansion/maintenance of hematopoietic stem cells. Bioprocess Biosyst Eng, 2003. 25: p. 365-369.

[25] Quesenberry, P.J., et al., Stem Cell Biology and Gene Therapy. 1998: Wiley-Liss, Inc.

[26] Sudo, K., et al., Mesenchymal Progenitors Able to Differentiate into Osteogenic, Chondrogenic, and/or Adipogenic Cells In Vitro Are Present in Most Primary Fibroblast-Like Cell Populations. Stem Cells, 2007. 25: p. 1610-1617.

[27] Burt, R.K., et al., Clinical Applications of Blood-Derived and Marrow-Derived Stem Cells for Nonmalignant Diseases. JAMA, 2008. 299: p. 925-936.

[28] Dominici, M., et al., Minimal criteria for defining multipotent msesnchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006. 8: p. 315-317.

[29] Yuko Segawa, T.M., hatsune Makino, Akimoto Nimura, tomoyuki Mochizuki, Young-Jin Ju, Yoichi Ezura, Akihiro Umezawa, Ichiro Sekiya, Mesenchymal Stem Cells Derived from Synovium, Meniscus, Anterior Cruciate Ligament, and Articular Chindrocytes Share Similar Gene Expression Profiles. Journal of Orthopaedic Research, 2009. 27: p. 435-441.

[30] Romanov, Y.A., et al., Searching for Alternative Sources of Postnatal Human Mesenchymal Stem Cells: Candidate MSC-Like Cells from Umbilical Cord. Stem Cells, 2003. 21: p. 105-110.

[31] Baksh, D., R. Yao, and R.S. Tuan, Comparison of Proliferative and Multilineage Differentiation Potencial of Human Mesenchymal Stem Cells Deriverd from Umbilical Cord and Bone MArrow. Stem Cells, 2007. 25: p. 1384-1392.

[32] Katia Marechi, E.b., Wanda Piacibello, Massimo aglietta, Enrico Madon, Franca Fagioli, Isolation of human mesenchymal stem cells: bone marrow versus umbilical cord blood. Haematologica, 2001. 86: p. 1099-1100.

62

[33] Romanov, Y.A., V.A. Svintsitska, and V.N. Smirnov, Searching for Alternative Sources of Postnatal Human Mesenchymal Stem Cells: Candidate MSC-Like Cells from Umbilical Cord. Stem Cells, 2003. 21: p. 105-110.

[34] Jung Park, V.S., Viktor Wixler, Holm Schneider Umbilicar Cord Blood Stem Cells: Induction of Differentiation into Mesenchymal Lineages By Cell-Cell Contacts with Various Mesenchymal Cells. Tissue Engineering, 2009. 15.

[35] Audet, J. and W.L. Stanford, Stem Cells in Regenerative Medicine: Methods and Protocols. 2009: Humana Press, Springer Protocols.

[36] Mandi J. Lopez, K.R.M., Nakia D. Spencer, Jade N. Borneman, Ronald Horswell, Paul Anderson, Gang Yu, Lorrie Gaschen, Jeffrey M. Gimble, Acceleration of Spinal Fusion Using Syngeneic and Allogeneic Adult Adipose Derived Stem Cells in a Rat Model Journal of Orthopaedic Research, 2008. 27: p. 366-373.

[37] D. tadic, M.E., A Thorough physicochemical characterization of 14 calcium phosphate-based bone substitution materials in comparison to natural bone. Biomaterials, 2004. 25: p. 987-994.

[38] Cato T. Laurencin, S.F.E.-A., Xenotransplantation in Orthopaedic Surgery. Journal of the American Academy of Orthopaedic Surgeons, 2008. 16: p. 4-8.

[39] Birgit Wenz, B.O., Martin Horst, Analysis of the risk of transmiting bovine spongiform encephalopathy through bone grafts derived from bovine bone. Biomaterials, 2001. 22: p. 1599-1606.

[40] L. Saldana, S.S.-S., I. Izquierdo-Barba, F. Bensiamar, L. Munuera, M. Vallet-Regi, N. Vilaboa, Calcium phosphate-based particles influence osteogenic maturation of human mesenchymal stem cells. Acta Biomaterialia, 2009. 5: p. 1294-1305.

[41] Ferraz, M.P., et al., Flow cytometry analysis of the effects of pre-immersion on the biocompatibility of glass-reinforced hydroxyapatite plasma-spayed coatings. Biomaterials, 2000. 21: p. 813-820.

[42] Lopes, M.A., et al., Flow cytometry for assessing biocompatibility. Journal Biomed Mater Res, 1998. 41(649-656).

[43] Gutierres, M., et al., Histological and scanning electron microscopy analyses of bone/implant interface using the novel Bonelike synthetic bone graft. J Orthop Res, 2006. 24(5): p. 953-8.

[44] Gutierres, M., et al., Bone ingrowth in macroporous Bonelike for orthopaedic applications. Acta Biomater, 2008. 4(2): p. 370-7.

[45] Gutierres, M., et al., Opening wedge high tibial osteotomy using 3D biomodelling Bonelike macroporous structures: case report. J Mater Sci Mater Med, 2007. 18(12): p. 2377-82.

[46] Ferraz, M.P., et al., In vitro Growth and differentiation of osteoblast-like human bone marrow cells on glass reinforced hydroxyapatite plasma sprayed-coatings. Journal of Materials Science: Materials in Medicine. 10: p. 567-576.

[47] M. A. Costa, M.G., L. Almeida, M. A. Lopes, J. D. Santos M. H. Fernandes, In Vitro Mineralization of Human Bone Marrow Cells Cultured on Bonelike. Key Engineering Materials, 2004. 254-256: p. 821-824.

63

[48] Gomes, P.S., J.D. Santos, and M.H. Fernandes, Cell-induced response by tetracyclines on human bone marrow colonized hydroxyapatite and Bonelike. Acta Biomater, 2008. 4(3): p. 630-7.

[49] Duarte, F., J.D. Santos, and A. Afonso, Medical Applications of Bonelike in Maxillofacial Surgery. Materials Science Forum, 2004. 455-456: p. 370-373.

[50] Sousa, R.C., et al., A clinical report of bone regeneration in maxillofacial surgery using Bonelike synthetic bone graft. J Biomater Appl, 2007. 22(4): p. 373-85.

[51] Lobato, J.V., et al., Titanium dental implants coated with Bonelike: Clinical case report. Elsevier, 2005.

[52] Oliveira, J.M., et al., Bonelike/PLGA hybrid materials for bone regeneration: preparation route and physicochemical characterisation. J Mater Sci Mater Med, 2005. 16(3): p. 253-9.

[53] Victor M. Golberg, A.I.C., Orthopedic Tissue Engineering. Basic Science and Practice. 2204: Marcel Dekker, Inc.

[54] Saltzman, W.M., Tissue Engineering: Engineering Principles for the Design of Replacement Organs and Tissues. 2204: Oxford University Press.

[55] Anup K. Kundu, C.B.K., Andrew J. Putnam, Extracellular Matrix Remodeling, Integrin Expression, and Downstream Signaling Pathways Influence the Osteogenic Differentiation of Mesenchymal Stem Cells on Poly(Lactide-Co-Glycolide) Substrates. Tissue Engineering, 2009. 15.

[56] Jason A. Burdick, G.V.-N., Engineered Microenvironments for Controlled Stem Cell Differentiation. Tissue Engineering, 2009. 15.

[57] Leenaporn Jongpaiboonkit, W.J.K., William L. Murphy, Screening for 3D Environments That Support Human Mesenchymal Stem Cell Viability Using Hydrogel Arrays. Tissue Engineering, 2009. 15.

[58] Tas, A.C., Preparatioonf Porous Bioceramics by a Simple PVA-Processing Route. Key Engineering Materials, 2004. 264-268: p. 2079-2082.

[59] J. Wiltfang, H.A.M., K. A. Schlegel, S. Schultze-Mosgau, F. R. Kloss, S. Rupprecht, P. Kessler, Degradation Characteristics of alpha and beta Tri-Calcium-Phosphate (TCP) in Minipigs. J Biomed Mater Res (Appl Biomater) 2002. 63: p. 115-121.

[60] Hans Albert Merten, J.r.W., Ulrike Grohmann,Johannes Franz Hoenig,, Intraindividual Comparative Animal Study of alpha- and beta-Tricalcium Phosphate Degradation in Conjunction with Simultaneous Insertion of Dental Implants. Journal of Craniofacial Surgery, 2001. 12

[61] T. Livingston Arinzeha, T.T., J. Mcalaryb, G. Daculsic, A comparative study of biphasic calcium phosphate ceramics for human mesenchymal stem-cell-induced bone formation. Biomaterials, 2005. 26: p. 3631-3638.

[62] Y. M. Lee, Y.J.S., Y. T. Lim, S Kim, S. B. Han, I. C. Rhyu, S. H. Baek, S. J. Heo, J. Y. Choi, P. R. Klokkevold and C. P. Chung, Tissue-engineered growth of bone by marrow cell transplantation using porous calcium metaphosphate matrices. J. Biomed. Mater. Res., 2001. 54: p. 216-223.

64

[63] Petra Müller, U.B., Annette Diener, Frank Lüthen, Marianne Teller, Ernst-Dieter Klinkenberg, Hans-Georg Neumann, Barbara Nebe, Andreas Liebold, Gustav Steinhoff, Joachim Rychly, Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells. J. Cell. Mol. Med., 2008 12: p. 281-291.

[64] Lingli Zhang, N.H., Megumi Maeda, Takashi Minowa, Toshiyuki Ikoma, Hongsong Fan and Xingdong Zhang, Porous hydroxyapatite and biphasic calcium phosphate ceramics promote ectopic osteoblast differentiation from mesenchymal stem cells. SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS, 2009. 10: p. 9.

[65] Philip Kasten, I.B., Philipp Niemeyer, Reto Luginbuhl, Marc Bohner, Wiltrud Richter, Porosity and pore size of b-tricalcium phosphate scaffold can influence protein production and osteogenic differentiation of human mesenchymal stem cells: An in vitro and in vivo study. Acta Biomaterialia 2008. 4: p. 1904-1915.

[66] A. Bignon, J.C., J. Chevalier, G. Fantozzi, J. P. Carret, P. Chavassieux, G. Boivin, M. Melin, D. Hartamn Effect of micro- and macroporosity of bone substitutes on their mechanical properties and cellular response. Journa of Materials Science: Materials in Medicine, 2003. 14: p. 1089-1097.

[67] James J. Moon , J.L.W., Vascularization of Engineered Tissues: Approaches to Promote Angiogenesis in Biomaterials. Current Topics in Medicinal Chemistry, 2008. 8: p. 300-310.

[68] Bischoff, J., Cell Adhesion in Vascular Biology. The American Society for Clinical Investigation, Inc., 1997. 99 p. 373-376.

[69] Jian Tang, R.P., Jiandong Ding, The regulation of stem cell differentiation by cell-cell contact on micropatterned material surfaces. Biomaterials, 2010. 31: p. 2470-2476.

[70] Bina Rai, J.L.L., Zophia X.H. Lim, Robert E. Guldberg, Dietmar W. Hutmacher, Simon M. Cool, Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCLeTCP scaffolds. Biomaterials 2010: p. 1-11.

[71] Tas, A.C., Preparation of Porous Bioceramics by a Simple PVA Processing Route. Key Engineering Materials, 2004. 264-268: p. 2079-2082.

[72] Qing JIE, K.L.A.J.Z., YIFENG SHI AND QIN LI, JIANG CHANG AND RUODING WANG, Preparation of Macroporous Sol-Gel Bioglass Using PVA Particles as Pore Former. Journal of Sol-Gel Science and Technology 2004. 30: p. 49-61.

[73] Chien-Wen Wang, M.Y., Hsien-Chang Chang, Shinn-Jyh Ding, Degradation Behavior of Porous Calcium Phosphates. Journal of Medical and Biological Engineering, 2003. 23: p. 159-164.

[74] Bo-Yi Yu, P.-H.C., Yi-Ming Suna, Yu-Tsang Lee, Tai-Horng Young, Topological micropatterned membranes and its effect on the morphology and growth of human mesenchymal stem cells (hMSCs). Journal of Membrane Science 2006. 273: p. 31-37.

[75] HUSEIN K. SALEM, C.T., Mesenchymal Stromal Cells: Current Understanding and Clinical Status. STEM CELLS, 2010. 28: p. 585-596.

65

[76] Masahiro Yamada, M.S., Yasuo Yamashita, Shohei Kasugai, Histological and Histomorphometrical Comparative Study of the Degradation and Osteoconductive Characteristics of a- and b-Tricalcium Phosphate in Block Grafts. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2006.

[77] Jianxi Lu, M.D., Jacques Dejou,Gilles Koubi, Pierre Hardouin, Jacques Lemaitre,Jean-Pierre Proust, The Biodegradation Mechanism of Calcium Phosphate Biomaterials in Bone. Journal of Biomed Mater, 2002. 63: p. 408-412.

[78] Stein GS, L.J., Molecular mechanisms mediated proliferation-diferentiation interrelationships during progressive development of the osteoblast phenotype: update. End Rev, 1995. 4: p. 290-7.

[79] RT., F., The role of ascorbic acid in mesenchymal diferentiation. Nutr Rev, 1992. 5: p. 50:60.

[80] Bellows CG, A.J., Heersche JNM. , Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone Min, 1991. 40: p. 14:27.

[81] M.J. Coelho, M.H.F., Human bone cell cultures in biocompatibility testing. Part II: e!ect of ascorbic acid, b-glycerophosphate and dexamethasone on osteoblastic di!erentiation. Biomaterials 2000. 21: p. 1095-1102.

[82] N. FRATZL-ZELMAN, P.F., H. HO¨ RANDNER, B. GRABNER, F. VARGA, A. ELLINGER, K. KLAUSHOFER, Matrix Mineralization in MC3T3-E1 Cell Cultures Initiated by b-Glycerophosphate Pulse. Bone 1998. 23: p. 511-520.

[83] Limin Wang, N.H.D., Lynda F. Bonewald and Michael S. Detamore, Osteogenic Differentiation of Human Umbilical Cord Mesenchymal Stromal Cells in Polyglycolic Acid Scaffolds. TISSUE ENGINEERING, 2010. 16.

[84] Xu, J.L.M.a.H.H.K., Mesenchymal stem cell proliferation and differentiation on an injectable calcium phosphate - chitosan composite scaffold. Biomaterials, 2009 30: p. 2675-2682.

[85] Limin Wang, N.H.D., Lynda F. Bonewald, and Michael S. Detamore, Osteogenic Differentiation of Human Umbilical Cord Mesenchymal Stromal Cells in Polyglycolic Acid Scaffolds. TISSUE ENGINEERING:, 2010. 16.

[86] Adalberto L. Rosaa, M.M.B., Richard van Noort, Osteoblastic differentiation of cultured rat bone marrow cells on hydroxyapatite with different surface topography. Dental Materials, 2003. 19: p. 768-772.

[87] Bo-Yi Yu, P.-H.C., Yi-Ming Suna, Yu-Tsang Lee, Tai-Horng Young, Topological micropatterned membranes and its effect on the morphology and growth of human mesenchymal stem cells (hMSCs). Journal of Membrane Science 2006. 273: p. 31-37.

[88] Buddy D. Ratner, A.S.H., Frederick J. Schoen, Jack E. Lemons, Biomaterials in science. An Introduction to Materials in Medicine, ed. n. edition. 2004: Elsevier Academic Press.

[89] Yongsheng Wang , S.Z., Xianting Zeng , Lwin Lwin Ma ,Wenjian Weng, Weiqi Yan , Min Qian, Osteoblastic cell response on fluoridated hydroxyapatite coatings. Acta Biomaterialia 2007. 3: p. 191-197.

66

[90] Yiwei Wanga, H.-I.C., David F. Wertheimb, Allan S. Jonesc, Chris Jacksond, Allan G.A. Coombes, Characterisation of the macroporosity of polycaprolactone-based biocomposites and release kinetics for drug delivery. Biomaterials 2007. 28: p. 4619-4627.

[91] PETER X. MA, J.-W.C., Biodegradable Polymer Scaffolds with Well-Defined Interconnected Spherical Pore Network. TISSUE ENGINEERING

Volume 2001. 7.

[92] F.J. O’Briena, B.A.H., I.V. Yannasc, L.J. Gibson, The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials 2005. 26: p. 433-441.

[93] von Doernberg MC, v.R.B., Bohner M, Grunenfelder S, van Lenthe GH, Muller R, et al., In vivo behavior of calcium phosphate scaffolds with four different pore sizes. Biomaterials, 2006. 27: p. 5186-98.