mesenchymal stem cell-based bone tissue engineering · 24 international dental journal of...

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INTERNATIONAL DENTAL JOURNAL OF STUDENT’S RESEARCH| Oct 2012-Jan 2013| Volume 1| Issue 3

REVIEW ARTICLE _______________________________________________________________________________________

Mesenchymal Stem Cell-based Bone Tissue Engineering

Mohammad A. Javaid1, Mari T. Kaartinen2

1MSc Student, Faculty of Dentistry, McGill

University, Montreal, Canada

2PhD, Associate Professor, Director of

Division of Biomedical Sciences, Faculty of

Dentistry, McGill University, Montreal,

Canada

Corresponding Author

M. Javaid, BDS

Faculty of Dentistry, McGill University,

3640 University Street, Montreal, QC,

Canada H3A 2B2

E-mail: [email protected]

Access this Article Online

Abstract Every year more than 2.2 million people

worldwide undergo bone grafting due to

critical sized bone defects arising from

trauma, disease, tumor resection and

accidents. These large bone defects that do

not heal by themselves are still a challenge

to reconstructive surgery and many

approaches are being used to replace the lost

bone. The newest methods involve the use of

patient’s own mesenchymal stem cells which

are incorporated into the biomaterials that

are shaped and modelled to the contours of

the defect. These grafts can incite and

promote natural healing and replacement of

bone without inducing immune responses

and rejection of the graft. Mesenchymal

stem cells have the potential to develop into

bone forming cells under appropriate

conditions and since they can be isolated

from many adult tissues, they can be ideal

for tissue regeneration. In this review we

discuss current materials and use of

mesenchymal stem cells for development of

tissue engineered bone constructs. Many

promising results have been obtained both,

in vitro and in vivo from animal experiments

as well as from clinical studies.

Assembly of this article was supported by

grant from Canadian Institutes of Health

Research (CIHR) to Mari T. Kaartinen.

Introduction Trauma, disease, tumor resection and

accidents frequently result in bone defects

and fractures that need reconstruction or

bone grafting. According to American

Academy of Orthopedic Surgeons more than

6.3 million people suffer from bone fractures

each year [1]. Many of these defects are

critical size bone defects that cannot heal

autonomously and hence require major

reconstruction work. Bone grafting is the

second most common transplantation

procedure after blood transplantation. In

2005 alone, more than 500,000 bone graft

procedures were performed in U.S. alone [1]

and every year more than 2.2 million people

undergo orthopedics, neurosurgery and

dentistry related bone grafting procedures

worldwide [2].

Critical size bone defects, which can be

defined as the smallest intraosseous defects

in a specific bone and species that do not

heal spontaneously in the lifetime of the

animal [3-6], are still a major challenge to

reconstructive surgery. Bone grafting

related materials, approaches and

procedures are rapidly incorporating

elements from novel research results and

significant progress has been made to

improve healing. Bone grafting can be

brought about by using autografts, allografts

and xenografts. Of these, autografts (bone

from the patient) and allografts (bone from

another human) have been used for decades

for this purpose, however, these approaches

have limitations. The disadvantage with

autografts is limitation of supply, risk of

donor site morbidity, pain, paraestheia,

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inflammation, hematoma and prolong

rehabilitation[7]. Using allografts exposes

patient to possible transmission of donor

pathogens to the recipient body and

triggering of host immune responses.

Xenografts (bone from other spieces) are also

used but are not ideal substitute as they

may lead to infection, toxicity,

immunogenicity, disease transmission and

rejection [7]. All these limitations have lead

the scientists to search for a better bone

substitutes with improved prognosis and

longevity in the host. Tissue engineering

approaches have been able to generate

synthetic materials that can replace patient

tissue, and materials that can stimulate

tissue synthesis. The use of biological

substitutes to critical size bone defects

completely eliminates the use of donors and

therefore transmission of diseases, immune

response and a possible rejection. However,

synthetic materials can also be rejected by

the patient and often results in

incapsulation of material by fibrotic tissue.

Large defects also require vascular growth

into the site to provide nutrients into area.

Lack of this can result in necrosis of the site.

Therefore, tissue engineering and

biomaterials scientists have aimed to

develop materials that have specific

qualities and can integrate well into the

existing tissue (osteointegration).

Furthermore, such qualities as to promote

bone tissue synthesis, i.e., osteogenesis have

also been vigorously sought. Material that is

capable of inducing bone formation via

stimulating cellular activity is called

osteoinductive (osteoinduction)[8-10].

Material whose surface allows bone growth

is called osteoconductive (osteoconduction)

[2, 8, 10-12]. Furthermore, an engineered

bone construct should be structurally and

morphologically similar to bone and

resorbable so that it can be eventually

replaced by new bone with all it’s tissue

components including vasculature [7]. It

should be also cost effective and easy to

handle [2]. This review discusses

characterstics of osteoinductive bone

substitutes which can carry cells into graft

side to accelerate tissue synthesis and to

improve osteointegration. We focus

particularly on mesenchymal stem cells

which have shown great promise in tissue

regeneration.

Tissue Engineered Bone The development of biological substitutes for

bone tissue engineering capabale of

osteoinduction has focused on using three

basic building blocks; scaffolds, cells and growth factors [7]. These all have the

potential to promote tissue regeneration

individually but when used in combination

their effects are greately enhanced. Many

biomaterials and several cell types and

number of growth factors have the potential

to enhance osteogenesis in an engineered

bone construct and have been studied

extensively over the last decade.

Scaffolds for bone constructs

The scaffold should fully integrate into

surrounding bone providing the initial three

dimensional structure which can allow the

cells to adhere, proliferate, differentiate and

lay down extra cellular matrix. The scaffold

should structural and morphological

characteristics close to bone. Bone is

primarily composed of inorganic

hydroxyapatite and organic matrix which is

mainly (about 95%) of collagen type I [13]. A

good scaffold is biocompatible, biodegradable

and porous for it to be used for construction

of a biological tissue. This means that even

the breakdown products of the scaffold

should be nontoxic to the body. It should

also be resorbable to allow the growing bone

to take its place gradually and it should

have porous three dimensional structures to

accommodate implanted cells. Ideal size of

pores is around 200-900 micrometers [7]. If

pores are too small they do not allow the

cells to migrate into the deeper layers of the

material and if pores are too large they

compromise the strength of the scaffold.

Bioceramics

Scaffolds made out of different materials

have been used in cell-based bone constructs

and have advantages and disadvantages.

Bioceramics can be divided into two main

types; natural which include coral-derived

calcium carbonate/hydroxyapatite and

synthetic which comprise of synthetic

hydroxyapatite and tricalcium phosphate.

These materials have been tested and are

currently used in wide array of clinical

domains including orthopedics, plastic

surgery, maxillofacial surgery and

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periodontal repair [7]. Hydroxyapatite

based calcium phosphate compounds [14-16]

and bioactive glass [17] are of special

interest for development of engineered bone

constructs due to their high mechanical

strength and osteoconductive nature [18].

According to some reports they also possess

osteoinductive characteristics [18] which

makes them excellent choice as a scaffolding

material and hence have been used in many

studies and case reports. Bioceramics also

have deficiencies. They tend to have low

biodegradability and bind to the growth

factors too tightly leading to a slow release

which may not be very suitable for extensive

clinical applications [7]. Also they tend to be

very brittle and have a low tensile strength

[19, 20].

Polymers

Polymer materials can be divided into two

categories; naturally occurring and

synthetically prepared. Natural polymers

include collagen, alginate and chitosan.

Polyglycolic acid (PGA), polylactic acid

(PLA), polylactic-co-glycolic acid (PLGA),

polycarprolacton (PCL) and polypropylene

fumarate (PPF) fall into the category of

synthetically prepared polymers [7]. Natural

polymers including collagen, alginate and

chitosan have been used in studies over the

years and have the advantage of being easily

soluble in physiological fluid but they incur

the risk of pathogen transmission and

immunogenicity. To overcome this

drawback, synthetic polymers have been

designed and used. They have the

advantages of having lower risk to induce

infection, reduced toxicity and being suitable

for affordable mass production. Also their

mechanical, physical and chemical

properties can be modified to resemble that

of natural bone. However, so far these

synthetic polymers have not been

osteoconductive and have decreased

strength which tends to deteriorate further

if pores are incorporated [7].

Composites

Composite materials have been developed to

improve the qualities that neither

bioceramics nor polymers posses. As the

name suggests, the idea behind a composite

is to combine the advantages of bioceramics

with qualities of polymers.

Hydroxyapatite/Poly-L-lactic acid

composites are an example of commercially

produced composites which are used in

tissue engineering. Bioceramics provide the

increased mechanical qualities and

incorporate osteoconductiveness whereas

polymers increase degradability and reduce

the brittleness of the ceramics[7].

Cells used in scaffolds

Integration of tissue forming cells into

grafting materials to improve

osteointegration has been attempted since

early 1950s [21]. Obvious choice has been

the use of osteoblasts which are the bone

forming cells and thus found in the bone

naturally. Osteoblasts can be acquired

autologously from the patient's bone through

biopsy and this eliminates the possibility of

a potential immune response. However, the

number of osteoblasts in each bone biopsy is

usually very low and the cell pool needs to

be expanded prior to integrating them into

the actual scaffold. The expansion

requirement presents numerous problems,

for example, studies have shown that their

proliferation is slow [7] and that they have a

limited life [22]. Also compared to other cells

for example stromal cells, osteoblasts are

less convenient source for bone tissue

engineering [23]. Therefore, they are not an

ideal candidate cells for tissue engineering

purposes.

Other types of cells, such as embryonic stem

cells have been considered. These cells have

great self-renewal potential; they are

totipotent and can differentiate into three

germ layers and thus also osteoblasts.

However, there are major ethical and legal

dilemmas related to using human embryonic

cells [24-26]. There is a possibility of

immunological incompatibility [27-29] and

teratoma formation [27, 30] which has

inhibited scientists to fully develop their use

in bone constructs [31].

Mesenchymal stem cells (MSCs) have

tremendous osteogenic differentiation

potential which is maintained for an

extended period of time allowing for

engineering of bone constructs with

autologous cells even for adult. Research in

MSC use in bone grafts was suggested in the

late 90’s [32, 33] and research on their use

has resulted in thousands of publications.

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Here we summarize the current knowledge

on MSC and bone tissue engineering.

Mesenchymal stem cells (MSCs)

Uses of cells that, a) can readily obtained in

high quantities from patient itself and b)

become osteoblasts in the scaffold, would be

ideal cell type for bone tissue engineering.

Osteoblasts arise and differentiate from

mesenchymal stem cells (MSCs) in vivo.

MSCs are nonhematopoietic stromal,

adherent cells that have the capacity to

differentiate into several other types of cells

including osteoblasts[34, 35], adipocytes[34,

36] and chondrocytes [34, 37, 38]. Their

differentiation into different lineage cells

results in formation of mesenchymal tissues

such as bone, muscle, cartilage, tendon,

ligament and adipose tissue [39]. In addition

to these, MSCs have been observed to

differentiate under appropriate conditions

into tenocytes[38], myocytes[38, 40-42],

neurons[42-47], and endothelial cells [39,

48]. Studies have even shown differentiation

of MSCs into astrocytes [38, 42], type 1

pnemocytes, lung fibroblasts, type 1 and 2

epithelial cells, myofibroblasts, retinal

pigment epithelial cells, skin epithelial cells

, sebaceous duct cells and tubular epithelial

cells in kidney [37].

Studies have also shown that MSCs play a

positive role in tissue repair [49-60] and it is

possible that this capacity of MSCs to induce

tissue repair is linked to the fact that MSCs

have also been shown to modulate immune

responses [31, 61]. MSCs are known to

inhibit T cell proliferation [62], B cell

proliferation and secretion of antibody [63]

and suppress differentiation, maturation

and activation of dendritic cells [64]. MSCs

have also been known to inhibit natural

killer cell proliferation [7, 65]. Therefore,

these immunomodulatory properties of

mesenchymal stem cells make them ideal

candidate for engineering of bone tissue

using techniques of tissue engineering [39].

Since mesenchymal stem cells are known to

be non-immunogenic their clinical

application may not require

immunosuppression and hence may also

reduce the incidence of opportunistic

infections [39].

Source of MSCs

MSCs are virtually present in all postnatal

tissues and for research purposes, they have

been isolated from other species including

mice, rat, cats, dogs, rabbits and pigs [66,

67]. In humans their presence has been

confirmed in many organs [37] and MSCs

have been isolated from skeletal muscle,

adipose tissue, umbilical cord, synovioum ,

circulatory system, dental pulp, amniotic

fluid, blood, liver, bone marrow, lung of fetus

[37] during first and second trimester of

pregnancy [66, 67]. In adults they can be

isolated from various tissues including bone

marrow, trabecular bone, dermis,

periosteum, pericyte, muscles, adipose

tissue, blood and synovial membrane [7, 68].

Cell isolation can be done for example from

bone marrow with gradient centrifugation

[69]. Isolation of MSCs from bone marrow is

considered to yield most MSCs and is

currently most commonly adopted method

for procurement of MSCs for various studies

[31, 68]. However, for actual clinical

purposes the use of bone marrow might not

be most desirable method due to its

invasiveness and thus alternate sources,

such as adipose tissue, are being tested [70-

73].

MSC differentiation

Since MSC can differentiate to large number

of different type of cells, their differentiation

must be carefully controlled if used in tissue

engineering approaches. Mesenchymal stem

cells maintain the self-renewal capacity for

long periods of time. Although mesenchymal

stem cell number in bone marrow aspirate is

quite low ranging from 0.001% to 0.01% [7],

these cells can be expanded quite easily with

standard culture techniques. MSCs when

-

glycerophosphate and dexamethasone for 2-

3 weeks differentiate into osteoblasts [12].

Furthermore dexamethasone has been

shown to stimulate osteoinduction and

subsequent bone formation by MSCs [74-77].

The mechanism how dexamethasone might

promote osteoblast differentiation from MSC

is not entirely known, however, it is well

known that ascorbic acid (vitamin C) which

is a cofactor for lysyl- and prolyl

hydroxylases (enzymes critical for collagen

biosynthesis) induces collagen type I and

fibronectin matrix deposition [78-84]. The

elaboration of this matrix is the first step of

bone formation and requirement for further

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differentiation of the MSC/preosteoblast into

-Glycerophophate is

generally used in cell cultures to induce

mineralization in vitro (hydroxyapatite

deposition) [85, 86] -Glycerophoshate is

cleaved into phosphate in the cultures via

alkaline phosphatase activity that is

dramatically increased when cells reach

their osteoblastic phenotype. Other

phosphate sources for mineralization also

can exist, amongst them are adenosine 5′-

triphosphate (ATP), pyridoxal-5′-phosphate

(PLP) and phosphoethanolamine (PEA) [87-

91].

Large number of growth factors also control

MSC differentiation into osteoblasts, they

include bone morphogenetic protein, insulin-

like growth factor, fibroblast growth factors

[1] and some studies have attempted to

integrate these into MSC containing

constructs [92]. For example, MSC

differentiation was shown improved in a

study where MSCs were engineered to

express bone morphogenetic protein-2. The

transfected cells had increased osteogenic

activity in vitro and in vivo in nude mouse

model which is immunocompromised and

thus suitable for transplantation studies. In

these mice, the engineered cells made more

new bone by the end of week 1 after

transplantation. Authors’ work

demonstrates that MSC can be transfected

and their differentiation can be controlled

and augmented in this manner. This has

obvious benefits to their use in bone tissue

engineering [75].

MSCs in bone tissue engineering

MSCs based therapies utilize three different

approaches: 1) incorporation of MSCs in

three dimentional scaffolds for replacement

of lost tissues, 2) therapies in which

defective host cells are replaced with normal

allogenic donor cells and 3) therapies in

which MSCs are used to produce cytokines

and growth factors to carry out reparative or

degenerative events [93]. Different MSCs

based models have been used for

regeneration of bone in animal and clinical

studies. In this section we will review some

of these studies.

MSC-based bone implants in animal studies

Kadiyala and co-workers carried out an in vivo study (published in 1997, [13]) using rat

model. Hydroxyapatite tricalcium phosphate

(HA/TCP) cylinders; 4mm in diameter and

8mm in length were prepared. The cylinders

contained a central canal about 1mm in

diameter. MSCs isolated from rat were

loaded in the HA/TCP cylinder and the

construct was placed in 8mm artificially

created bone defect in rat femora. Healing

was assessed 4 and 8 weeks after the

operation and results showed that without

MSCs there was no healing in defects

whereas MSCs loaded cylinders showed

increased bone formation by the end of week

4 and the defect showed complete healing by

the end of week 8 with excellent union at

host-implant junction. In 1998 Bruder and

co-workers [94] carried out a study on canine

model. They isolated MSCs from bone

marrow and loaded them onto HA/TCP

ceramic cylinders. These cylinders were

placed in 21mm bone defect artificially

created in femora of female canines and

healing was evaluated with radiographs

taken at 4 weeks interval. In the end of

week 16 the animals were sacrificed and

bone healing was assessed. Radiographic

evaluation revealed that in all the femora

that were left untreated atrophic non-union

was observed. In defects which had been

fitted with control cylinders without cells,

had numerous fractures which became more

pronounced with time. In experiments using

MSC-containing cylinders rapid bone

formation was observed with excellent union

at the host-implant interface - woven and

lamellar bone filled the pores in MSC loaded

implant. Furthermore, there was no callus

formation in cell free cylinder in contrast to

formation of a large collar of bone that fully

integrated and became contiguous with

callus in MSC loaded implant-host interface.

In 2003 Holy and co-workers [95] carried out

a study using MSC loaded onto Poly lactide-

co-glycolide (PLGA) to heal bone defects in

rabbits. MSC were isolated from femora and

iliac crest and expanded ex vivo. The cells

were then loaded onto PLGA scaffold which

were placed in bone defects in rabbit femur.

The controls received cell free implants or

were left untreated. Radiographs were taken

at 2 weeks interval and animals were

sacrificed at week 8 postoperatively.

Radiographs revealed that there was 77%

bone formation in cell loaded constructs

compared to 33% in cell free implants at

week 2 and 100% compared to 77% by the

end of week 4. At the end of week 6 cell

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loaded implants showed radio opacity

throughout the defect whereas there was

limited radio opacity in cell free implants

[95]. Diao and co-workers [96] used human

umbilical cord MSCs that were seeded onto

hydroxyapatite/collagen/poly lactic acid

composite cubes. The implants were placed

in the back of mice. The controls were fitted

with cell free cell free scaffold cylinders.

Animals were sacrificed at 4, 8 and 12 weeks

postoperatively. Analyses revealed that in

cell free implant no bone like tissue was

formed during the entire healing period. In

MSC loaded implants osteoid deposition was

evident by the end of week 4. The scaffold

material was continuously replaced by

newly laid down woven bone and significant

bone deposition in the MSC-containing

implants was observed [96].

MSC-based bone implants in clinical studies MSC have also been used in human studies.

Quarto and coworkers [97] used MSCs to

treat bone defects in human subjects. They

treated 7 cm long bone defect using MSCs

loaded onto hydroxyapatite cylinders. MSCs

were isolated from bone marrow of each

subject and expanded ex vivo. Cells were

loaded onto macroporous hydroxyapatite

scaffold whose shape and size was made to

conform to the individual bone defect in each

subject. Initially external fixation was

provided for mechanical support and

stability which was removed later. Two

months post implantation callus formation

was detected in the defect site followed by

integration of construct into host bone and

complete union at the host-implant

interface. All patients recovered full bone

function and there were no complications or

loss of function 15 months post implantation

for the last patient and 27 months for the

first patient. In another study done by

Marcacci and co-workers in 2007 [98] similar

outcome was observed. Their study was very

prominent as they had a long follow up

period of 6 to 7 years. In this study four

patients were treated using MSCs-based

constructs with large bone defects and

alternatives with poor prognosis. MSCs were

extracted from iliac crest marrow aspirates

of each patient. The scaffold material was

100% hydroxyapatite. Cylinders were made

out according to size and shape of individual

bone defect. Patients were evaluated every

month using radiography which was

followed by computed tomography scan (CT

scan) after 6-10 months post surgery. This

was followed by clinical, radiographic and

CT evaluation every year. No subject

reported any significant pain or discomfort

following the surgery nor was there any

swelling or infection at the implant site.

Callus formation was detected at the host

implant interface after 1 to 2 months post

surgery. Gradually the radiolucency at bone-

implant interface disappeared. Complete

integration of implant into the host bone

was achieved in 5 to 7 months. In this study

regenerated bone was evaluated over a long

period of time. Physiological bone formation

followed by progressive integration of

implant in the host bone was observed [98].

Growth factors and vascularization of the bone constructs

Relation between osteogenesis and

angiogenesis is well established and fracture

repair requires neovascularisation of the

new tissue as osteogenesis and maintenance

of normal bone remodelling requires

abundant oxygen and nutrient and cell

supply via vasculature [99]. Therefore,

vascularisation is a key element in wound

and fracture healing and recovery from bone

defect/fracture and engineered bone

constructs. Small defects reply on nutrient

diffusion to the healing site, however, large

defects require rapid neovascularization.

Defects in this vascularization process can

lead to tissue necrosis. The newly formed

blood vessels sustain the granulation tissue

and allow survival of cells by providing

essential nutrients and oxygen to the site

[100]. Tissue engineered bone constructs

face nutritional limitations due to very

limited inherent microvasculature. This

repair site is especially sensitive during first

days after implantation of the bone

construct. Although, hypoxia serves as a

signal to stimulate angiogenesis via

induction of hypoxia induced factor-1 (HIF-

1) and subsequent promotion of vascular

growth factor expression [101-103], repair of

large defects face the danger of cell death if

nutrients and oxygen are not provided to the

site [104-106]. To accelerate the

vascularization process (circumventing

hypoxia) and transition from oxygen-

dependent granulation tissue to

vascularized bone, many vascular growth

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factors have been incorporated directly into

the bone construct. Growth factors like

VEGF, BMP-2, PDGF-BB, IGF and TGF-

beta have been documented to increase

angiogenesis. Out of these growth factors

VEGF exerts its effect directly where as

others produce their effect by regulation of

VEGF [1]. Another approach which has

been taken up recently is the incorporation

of endothelial cells in the implants for

formation of microvasculature in the bone

construct. Various studies have shown that

endothelial cells can form a capillary like

network in the engineered construct which

when implanted in the defect site can

connect and merge (anastomoses or

inosculation) with the host’s microvessels.

This approach is particularly directed to

solve problems of decreased blood supply in

early days after implantation. It has been

demonstrated that endothelial cells can form

capillary like network within tissue

engineered constructs without need of

external growth factors [107] and since

endothelial cells can be derived

autologously, chances of immune rejection

are reduced. Studies have shown that

microvasculature formed by endothelial cells

can become functional through inosculation

as early as 96 hours post transplantation

[107]. It has also been observed that this

vasculature forming potential of endothelial

cells is enhanced by MSCs. In a study by

Wu, and co-workers [100] it was revealed

that MSC when injected to wound site

increased capillary density and

angiogenesis. MSCs modulate their effect by

inducing endothelial cell migration,

proliferation as well as formation of

capillary like tubular network [100].

Endothelial cells also have positive influence

on proliferation of mesenchymal stem cells

and osteoblasts. Studies have revealed that

endothelial cells when co-cultured with

MSCs and osteoblasts increase their

proliferation [99]. Lastly, microsurgery

techniques have also been used to establish

blood vessels in the bone construct. Most

commonly used techniques are flap

fabrication and arteriovenous loop creation

[1]. Although available and still used in

some cases, these procedures are laborious

and require second surgery and risk donor

site morbidity. With better understanding of

angiogenesis and advancement in the field of

tissue engineering these procedures may be

entirely replaced by promising MSC-

endothelial cell-based implants.

Conclusions Bone regeneration using tissue engineered

bone substitutes are slowly replacing

autografts, allograft and xenografts.

Numerous studies show that MSCs based

bone constructs have the potential to

regenerate critical size bone defects with

substantially lower risk of transmission of

disease, immune response or graft rejection.

Although MSC-based bone repair shows

tremendous potential, a few challenges

remain. Future years in bone tissue

engineering will focus on developing simple,

rapid and safe MSC isolation from patients

from tissue other than bone marrow -

adipose tissue being an interesting and

promising candidate [108-111]. The field will

also aim to generate smart biomaterials

capable of both osteoinduction and

stimulation of formation of microvascular

capillary like network [7]. This field will

benefit from understanding all aspects of

biomaterials science, cell-cell, cell-material

interactions and as well as basic

understanding of cell coupling, cellular

behavior and characteristics of MSCs.

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