CHANGES IN INSULIN LIKE GROWTH FACTORS, MYOSTATIN …

Aus der Poliklinik für Kieferorthopädie, Präventive Zahnmedizin und Kinderheilkunde

(Direktor: Prof. Dr. med. dent. T. Gedrange) Im Zentrum für Zahn-, Mund-, und Kieferheilkunde

(Geschäftsführender Direktor: Prof. Dr. med. dent. Dr. h.c.G. Meyer) der Medizinischen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald

CHANGES IN INSULIN LIKE GROWTH FACTORS, MYOSTATIN

AND VASCULAR ENDOTHELIAL GROWTH FACTOR

IN RAT MUSCULUS LATISSIMUS DORSI

BY POLY-3-HYDROXYBUTYRATE IMPLANTS

Inaugural-Dissertation

zur

Erlangung des akademischen Grades

Doktor der Zahnmedizin

(Dr. med. dent.)

der

Medizinischen Fakultät

der

Ernst-Moritz-Arndt-Universität

Greifswald

2009

vorgelegt von Dr.Tomasz Gredes

geboren am 20.08.1975

in Dzierżoniów (Polen)

Dekan: Univ.- Prof. Dr. rer. nat. H. K. Kroemer

Erster Gutachter: Prof. Dr. T. Gedrange

Zweiter Gutachter: PD Dr. A. Lupp

Ort, Raum: Greifswald, Hörsaal der Neuen Zahnklinik

Tag der Disputation: 17.02.2010

2

Moim Rodzicom, Babci i Siostrze

3

Contents List of abbreviations p. 5

1. Introduction p. 7

1.1. Regulation of bone remodelling p. 7

1.2. Bone formation p. 10

1.3. Bone surrogates p. 11

1.4. Poly-3-hydroxybutyrate – PHB p. 14

1.5. Aim of this study p. 16

2. Materials and Methods p. 18

2.1. Poly-3-hydroxybutyrate (PHB) p. 18

2.2. Experimental Design and Surgical Procedure p. 18

2.3. RNA-Isolation p. 20

2.4. Agarose gel electrophoresis p. 22

2.5. Reverse Transcriptase — Real-Time RT-PCR p. 23

2.6. Quantification of the gene expression - 2(-ΔΔCT) method p. 26

2.6.1. Derivation of the 2-ΔΔCT method p. 26

2.7. Statistical analysis p. 28

3. Results p. 29

3.1. Isolation of the total RNA from muscle samples p. 29

3.2. Examination of the primer specificity using standard RT-PCR p. 31

3.3. Quantification of the VEGF, IGF1, IGF2, and GDF8 mRNA expression p. 31

4. Discussion p. 35

5. References p. 39

6. Summary p. 48

7. Supplement p. 49

Legends of tables p. 56

Legends of figures p. 57

Affirmation p. 58

Acknowledgment p. 59

Curriculum vitae p. 60

Publication

4

http://dict.leo.org/ende?lp=ende&p=5tY9AA&search=list

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List of abbreviations

bp base pairs

BMP bone morphogenetic protein

BMSC bone marrow stromal cell

BMU basic multicellular unit

cDNA complementary desoxyribonucleic acid

CSF colony stimulating factor

e.g. exempli gratia

etc. et cetera

CT threshold cycle

DNA desoxyribonucleic acid

FGF fibroblast growth factor

GDF8 growth differentiation factor 8

HB D,L-β-hydroxybutyrate

IGF insulin-like growth factor

IL interleukin

M. musculus

MMSC multipotent mesenchymal stem cell

mRNA messenger ribonucleic acid

MSC mesenchymal stem cell

MyHC myosin heavy chain

PCR polymerase chain reaction

PDGF platelet derived growth factor

PHB poly (3-hydroxybutyrate)

5

http://dict.leo.org/ende?lp=ende&p=5tY9AA&search=list

http://dict.leo.org/ende?lp=ende&p=5tY9AA&search=of

http://dict.leo.org/ende?lp=ende&p=5tY9AA&search=abbreviations

PMN polymorphonuclear neutrophil

RNA ribonucleic acid

rRNA ribosomal ribonucleic acid

RT reverse transcription

RT-PCR real-time polymerase chain reaction

Runx2 runt-related transcription factor 2

S.E.M standard error of the mean

SSC skeletal stem cell

TGF transforming growth factor

TNF tumour necrosis factor

VEGF vascular endothelial growth factor

vis. videlicet

vs. versus

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1. Introduction

1.1. Regulation of bone remodelling

Bone is a dynamic tissue that constantly undergoes remodelling. The signal that initiates bone

remodelling has not been identified yet, but there is evidence that mechanical stress can alter

local bone architecture. This can be followed at osteocytes secreting paracrine factors such as

insulin-like growth factor (IGF-1) in response to mechanical forces (Lean et al. 1996).

This complex process requires interaction between different cell phenotypes regulated by

various biochemical and mechanical factors. This is a balance between the amount of bone

resorbed by osteoclasts and the amount of bone formed by osteoblast (Frost 1964). Bone

remodelling occurs in small packets of cells called basic multicellular units (BMU), which

turn over bone in multiple bone surfaces (Frost 1991). BMU consist of osteoblasts, other

bone-forming cells such as osteocytes and bone-lining cells, bone-resorbing cells osteoclasts,

the precursor cells of both, and their associated cells like endothelial and nerve cells

(Papachroni et al. 2009).

Osteoblasts are key components of the bone multi-cellular unit and play a seminal role in

bone remodelling, which is an essential function for maintenance of the structural integrity

and metabolic capacity of the skeleton. Osteoblasts originate from the non-hematopoietic part

of bone marrow which contains a group of fibroblast-like stem cells with osteogenic

differentiation potential, known as mesenchymal stem cells (MSCs) and also referred as

skeletal stem cells (SSCs), bone marrow stromal cells (BMSCs) or multipotent mesenchymal

stromal cells (MMSCs) (Abdallah and Kassem 2008; Heino and Hentunen 2008). MSCs are

capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts,

adipocytes, and chondrocytes (Dezawa et al. 2004; Luk et al. 2005).

Osteoblast growth and differentiation is determined by a complex array of growth factors and

signalling pathways. The following three families of growth factors influence the main

aspects of osteoblast activity and induce, mediate or modulate the effects of other bone

growth regulators:

— the transforming growth factor-β (TGF-β) family that promotes osteoblast

differentiation

— the insulin-like growth factors (IGFs), which induce osteoblastogenesis via activation

of Osterix gene expression

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— bone morphogenetic proteins (BMPs), the autocrine and paracrine anabolic action

mediated by their specific receptors (Zhou et al. 1993; Mundy 1994; Bikle 2008).

Furthermore, other growth factors, such as the vascular endothelial growth factor (VEGF) as

well as platelet derived growth factor (PDGF) are involved in osteoblast differentiation and

summarized in table 1.

Table 1. Main growth factors and its function in osteoblast differentiation.

Growth factor Effects on osteoblasts References

IGFs

IGF-1 triggers osteoblast proliferation, increases bone collagen synthesis and decreases collagen degradation

IGF-I and -II promote Osterix (Osx) expression in osteoblastic cells, trigger osteoblast induction in vitro and a transient increase in bone mass in vivo

(Tang et al. 2006; Bikle 2008)

TGF-β family

Stimulate the production and deposition of ECM proteins

Potent inducers of committed bone cell replication and osteoblast matrix production

(Ahdjoudj et al. 2002; Ito and Miyazono 2003)

BMPs

Autocrine and paracrine action mediated by their kinase receptors

Induce early precursor bone cell replication and osteoblast commitment

(Duncan 1995; Zhao et al. 2000)

VEGF

Regulates vascularization of developing bone and osteoclast activity, involved in bone repair

Key component of a chondrocyte survival pathway, controls osteoblastic activity

(Zelzer and Olsen 2005; Papadopoulou et al. 2007)

PDGF

Potent mitogen and chemoattractant for target cells such as diploid fibroblasts and osteoblasts

(Kim et al. 2007)

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Mechanisms that promote skeletal tissue specificity are necessary, because none of these

growth factors are specific for cells in the osteoblastic lineage. They involve interactions with

other circulating hormones (including glucocorticoids, sex steroids, parathyroid hormone or

prostaglandin E2) in addition to the action of specific intracellular mediators on bone-specific

transcription factors. It is certain that bone remodelling is regulated by systemic hormones

and by local factors (table 2 and 3) (Canalis 1983), which affect cells of both the osteoclast

and osteoblast lineages and exert their effects on the replication of undifferentiated cells, the

recruitment of cells, and the differentiated function of cells (Hill 1998).

Table 2. Local factors that regulate bone remodelling.

Growth factors that regulate bone remodelling

Fibroblast growth factors (FGF) Selected cytokines of the interleukin (IL)

Tumour necrosis factor (TNF) Colony-stimulating factor (CSF)

Table 3. Summary of bone remodelling regulating hormones.

Hormones that regulate bone remodelling

Polypeptide hormones

Parathyroid hormone Calcitonin

Insulin Growth hormone

Steroid hormones

1,25-Dihydroxyvitanin D3 Glucocorticoids

Sex steroids

Thyroid hormones

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Local factors have effects on cells of the same class (autocrine factors) or on cells of another

class within the tissue (paracrine factors). The presence of local factors is not unique to the

skeletal system, because non-skeletal tissues also synthesize, and respond to autocrine and

paracrine factors. Growth factors are also present in the circulation and may act as systemic

regulators of skeletal metabolism, but the locally produced factors have more direct and

important functions in cell growth (Hill et al. 1997).

Besides growth factors and regulating hormones, expression of transcription factors is

necessary and sufficient for mesenchymal cell differentiation. Runx2 is an essential bone-

specific transcription factor. It was recently shown, that the complete Runx2 gene inactivation

in transgenic mice leads to complete lack of intramembraneous and endochondral ossification

owing to lack of mature osteoblasts (Komori et al. 1997). In addition, heterozygous Runx2

mice demonstrate specific skeletal abnormalities that are also characteristic for the human

heritable skeletal disorder cleidocranial dysplasia (Otto et al. 1997).

1.2. Bone formation Bone formations results from a complex cascade of events involving proliferation of primitive

mesenchymal cells (osteoinduction), differentiation into osteoblast precursor cells, maturation

of osteoblasts, formation of matrix, and finally mineralization figure 1). Osteoblasts converge

at the bottom of the resorption cavity and form osteoid which begins to mineralize after 13

days. The osteoblasts continue to form and mineralize osteoid until the cavity is filled. At the

bottom of the cavity osteoblasts are plump and vigorous, they have tall nuclei, and they make

a thick layer of osteoid. The cells gradually flatten and become quiescent lining cells. Some of

the osteoblasts differentiate into osteocytes and become embedded in the matrix. The initial

event must be chemotactic attraction of osteoblasts or their precursors to sites of the

resorption defect. This is likely to be mediated by local factors produced during the resorption

process. The second event involved in the formation phase of the coupling phenomenon is

proliferation of osteoblast precursors. This is likely to be mediated by osteoblast-derived

growth factors and those growth factors released from bone during the resorption process (see

tables 1-2). The third event in the formation phase is the differentiation of the osteoblast

precursor into the mature cell. Several of the bone-derived growth factors can cause the

appearance of markers of the differentiated osteoblast phenotype, including expression of

alkaline phosphatase activity, type I collagen, and osteocalcin (Hill et al. 1997).

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Bone Formation

Figure 1. Regulation of bone formation (Roodman 2004).

1.3. Bone surrogates

One of the difficult clinic problems is a bone defect. These defects can be caused by

inflammation, congenital malformation, trauma or oncological surgery. The bone defects can

be a limiting factor in achievement of optimal orthodontic treatment. The conventional

biological methods of bone-defect management include autografting and allografting

cancellous bone, applying vascularised grafts of the fibula and iliac crest. The standard

treatments were continually improved and additionally new methods were searched (Burg et

al. 2000). The autografts often require longer operating time with the probability of infection,

pain, and hematoma. The allografting introduces the risk of disease and/or infection. It may

cause a lessening or complete loss of the bone inductive factors. Furthermore, the vascularised

grafts require a major elaborate microsurgical operative procedure (Bostrom and Mikos

1997).

Nowadays diverse bone substitutes were used for the creation of new bone in the patient. For

bone regeneration four components are essential: a morphogenetic signal, responsive host

cells response to the signal, a suitable carrier of this signal that can deliver it to specific sites

serving as scaffolding for the growth of the responsive host cells, and a viable, well

vascularised host bed (Harakas 1984; Croteau et al. 1999). The changes in material

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technology provide to development of bone tissue engineering. Materials used as bone tissue-

engineered scaffolds may be injectable or rigid, the latter requiring an operative implantation

procedure.

Bone tissue-engineered scaffolds are divided into acellular and cellular with drug delivery

overlapping in both areas (Burg et al. 2000). Materials on or in which no additional cellular

component is cultured will be classified as acellular. The cellular materials are classified as

scaffolds to which a cellular component is added prior to implantation. The materials

commonly used in all three approaches are ceramics, polymers or composites (Burg et al.

2000). The ceramics and polymers are either absorbable or non-absorbable, and the polymers

can be naturally derived or synthesized materials (figure 2).

Biomaterials cellular / acellular

synthesized materials

naturally derived materials

absorbable nonabsorbable

nonabsorbable

absorbable

Figure 2. The classification of biomaterials used for bone tissue engineering modified

according to Burg (Burg et al. 2000).

Bone tissue-engineering systems include demineralized bone matrix, collagen composites,

fibrin, calcium phosphate, polylactide, poly(lactide-co-glycolide), polylactide-polyethylene

glycol, hydroxyapatite, dental plaster, and titanium (Tsuruga et al. 1997; DeGroot et al. 2004).

The mechanisms by which bone can be repaired or regenerated using bone surrogates are

osteoinduction, osteoconduction, and osteointegration. Osteoinduction is defined as the ability

to stimulate the proliferation and differentiation of pluripotent cells. The stem cells directly

differentiate into osteoblasts, which form bone through direct mechanisms. In endochondral

bone formation, stem cells differentiate into chondroblasts and chondrocytes, subsequently

lay down a cartilaginous extracellular matrix, which then calcifies and remodels into lamellar

bone. Osteoinduction is routinely stimulated by osteogenic growth factors. Osteoinduction is a

basic biological mechanism that occurs regularly, e.g. in fracture healing but also in implant

incorporation (Albrektsson and Johansson 2001). An osteoinductive material allows the bone

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repair in a location that would normally not heal if left untreated (Ishaug et al. 1997; Helm

and Gazit 2005). Osteoconduction means that bone grows on a surface and is defined as the

ability to stimulate the attachment, migration, and distribution of vascular and osteogenic cells

within the graft material (Albrektsson and Johansson 2001). Several physical characteristics

can affect the graft osteoconductivity, including porosity, pore size, and three-dimensional

architecture. In addition, direct interactions between matrix proteins and their appropriate cell

surface receptors play a major role in the host response to the graft material. An

osteoconductive material guides repair in a location where normal healing will occur if left

untreated (Kulkarni et al. 1971; Helm and Gazit 2005). Osteointegration is defined as a direct

contact between living bone and implant, which means the formation of bony tissue around

the implant without the growth of fibrous tissue at the bone-implant interface. The

osteointegration is not an isolated phenomenon, but depends on previous osteoinduction and

osteoconduction (Albrektsson and Johansson 2001).

The ability of a graft material to produce bone independently is termed its direct osteogenic

potential. The main critical considerations in bone tissue-engineering scaffold design are

summarized in table 4.

Table 4. Selected critical consideration in bone tissue-engineering scaffold design (Peter et al. 1998; Burg et al. 2000)

Desirable qualities of a bone tissue-engineering scaffold

Available to surgeon on short notice Absorbs in predictable manner in concert with bone growth Adaptable to irregular wound site, malleable Maximal bone growth through osteoinduction and/or osteoconduction Correct mechanical and physical properties for application Good bony apposition

Promotes bone in-growth Does not induce soft tissue growth at bone/implant interface Average pore sizes approximately 200-400 μm No detrimental effects to surrounding tissue due to processing Sterilizable without loss of properties Absorbable with biocompatible components

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To have direct osteogenic activity the graft must contain cellular components that induce bone

formation directly. Polymers have been shown to be an excellent substrate for cellular or

bioactive molecule delivery. They can differ in their molecular weight, polydispersity,

crystallinity, and thermal transitions, allowing different absorption rates. Their relative

hydrophobicity and percent crystallinity can affect cellular phenotype (Hollinger and Schmitz

1997). Various types of biomaterials (minerals and non-mineral based materials as well as

natural and artificial polymers) with different characteristics have been used for studying

ossification and bone formation. For example, calcium phosphate ceramics include a variety

of ceramics such as hydroxyapatite, tricalcium phosphate, calcium phosphate cement, etc.

Local tissue responses to polymers in vivo depend on the biocompatibility of the polymer as

well as its degradative by-products (Hollinger and Battistone 1986). The mentioned ceramics

have excellent biocompatibility and bone bonding or bone regeneration properties. They have

been widely used in no or low load-bearing applications (Milosevski et al. 1999).

Furthermore, natural polymers like collagen have been used for bone tissue engineering

purposes (Hutmacher 2001; Lauer et al. 2001). Recently non-biodegradable and degradable

membranes have been tested for their appliance in bone defects (Zhao et al. 2000). Scores of

artificial polymers of diverse character are already in use for bone supply. One of them,

poly(3)hydroxybutyrate (PHB) with little inflammatory response after implantation due to its

form stability, may serve as a scaffold for tissue engineering (Gogolewski et al. 1993;

Schmack et al. 2000).

1.4. Poly-3-hydroxybutyrate — PHB

The lipidic polymer, poly-3-hydroxybutyrate (PHB) is found in the plasma membrane of

Escherichia coli in complex with calcium polyphosphorate (Reusch and Sadoff 1983).

Different types of microorganisms produce PHB from renewable sources from sugar and

molasses as intracellular storage materials. PHB is polyester (figure 3) with optical activity

and very good barrier properties. It has a high melting temperature (175°C), glass transition

temperature (15°C), and a high degree of crystallinity and is low permeable for O2, CO2, and

H2O (Holmes 1987; Miguel et al. 1997). This polymer is perfectly isotactic viz. the monomers

have all branch groups on the same side of the polymeric chain and are oriented in the same

way. This linear flexible molecule bearing electron-donating carbonyl ester oxygens at

intervals allows multiple bonding between the polymer chain and cation (Armand 1987; Gray

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1997). PHB does not contain any residues of catalysts like other synthetic polymers (Holmes

1987; Miguel et al. 1997).

a.

b.

–{ O – CH ( CH3 ) – ( C = O ) }n –

Figure 3. The polyester linkage creates a molecule which has 3-carbon segments separated by

oxygen atoms. The remainder of the monomer (a) becomes a side chain of the main

backbone of the polymer (b). In PHB the monomer unit is hydroxybutyric acid and

the side chain is a methyl group. PHB, with its short methyl side chain, is a very

crystalline and very brittle polymer. Industrially, it is difficult to use because the

temperature at which it melts is very close to the temperature at which it begins to

decompose. Its high degree of crystallinity causes it to crack easily.

PHB stiffness and brittleness depends on the degree of crystallinity, microstructure, and glass

temperature. The longer it is stored at room temperature, the more it becomes brittle. Because

of these findings its application is limited. Further application limitations are the poor process

ability and the low degradation (Mack et al. 2008).

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However, PHB will be used for the following applications:

1. In pharmacology as a material for cell and tablet packaging or microcapsules during

therapy.

2. In medicine and dentistry as surgical implant because of its compatibility with the

tissues of mammals and an undisturbed metabolism in the human blood.

3. In packaging industries as a biodegradable plastic for solving environmental pollution,

hygiene and textile.

Because of its unique combination of biodegradability and biocompatibility it is of great

interest for medical applications (Vogel et al. 2006). PHB completely degrades releasing a

normal component of blood and tissue, D, L-β-hydroxybutyrate (HB) and it is an ideal

biomaterial characterized by stability, lack of toxicity, compatibility in contact with tissue and

low inflammatory response after implantation (Gogolewski et al. 1993; Sevastianov et al.

2003; Mai et al. 2006; Suwantong et al. 2007; Shishatskaya et al. 2008).

The biocompatibility of PHB has been confirmed in vitro in cultures of cells of various

origins (Saad et al. 1999; Shishatskaya et al. 2004; Volova et al. 2004) and surrounding

muscle tissues (Mack et al. 2008).

1.5. Aim of this study

The amount of free microvascular bone grafts is limited due to lack of donor sites with

sufficient bone volume of adequate quality. A bone substitute creation ectopically in an

anatomical area having good vascular supply and an adequate vascular pedicle could reduce

this limitation (Warnke et al. 2004; Mai et al. 2006). Ectopic bone formation has been long-

time tested in several animal studies (Kusumoto et al. 1996; Buma et al. 2004; Meyer et al.

2004; Wiesmann et al. 2004; Kroese-Deutman et al. 2005). A bone surrogate human

application created from an osteoconductive biomaterial of bovine origin in combination with

growth factors and additional autogenous spongiose bone has also been described (Warnke et

al. 2004).

Muscles are known to have a considerable potential of adaptation. The extracellular matrix of

the muscle tissue surrounding the implant can integrate changes of the mechanical load of the

muscle and hereupon induce signalling cascades with a following adaptation of protein

synthesis and arrangement of the cytoskeleton (Kjaer 2004). Recently was shown, that

latissimus dorsi muscles are used for the growth and preparation of bone grafts for subsequent

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transplantation (Warnke et al. 2004). It was demonstrated that heterotopic bone induction and

custom vascularization is possible to form a bone replacement inside the latissimus dorsi

muscle in a human.

The aim of this study was to examine the effects of PHB implants on factors that regulate

vascularization or interaction with the extracellular matrix of the surrounding muscle tissue.

For this the mRNA expression of VEGF, IGF1, IGF2 as well as GDF8 should be analyzed

using quantitative RT-PCR in muscle tissue specimens from the Musculus latissimus dorsi of

rats. The muscle specimens were collected after subcutaneous implantation of PHB scaffolds

for six and twelve weeks.

.

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2. Materials and Methods

All used materials, chemicals, buffer, and equipment are summarized in table a-d in the

section “supplement”.

2.1. Poly-3-hydroxybutyrate

Fully biodegradable biotechnologically produced polyester PHB was used in powder form as

raw material. The powder with a molecular weight of 540 000 g/mol was granulated using a

twin screw extruder equipment. The PHB multifilaments were produced using a high-speed

melt spinning and spin drawing process (Mai et al., 2006; Schmack et al., 2000). Round

embroidery patches with a thickness of 1.2 mm and a diameter of 12 mm were generated

using an embroidery automat followed by coating with calf skin collagen type I. The average

macro mesh pore size of the embroidery was 200 µm and the total weight of the implant was

approximately 12 mg. A total of 24 implants were prepared. All implants were ultrasonically

cleaned in 70 % ethanol for 15 minutes and sterilised by ethylene oxide before the surgical

procedure.

2.2. Experimental Design and Surgical Procedure

The experiments were performed on twelve six-weeks-old adult male Wistar-King rats, with

approximately 200 g body mass. All surgical and experimental procedures were approved by

the Animal Welfare Committee of the State Government (no. 24-9168.11-1-2004-2).

For surgery, each rat was anesthetized with an intraperitoneal injection of pentobarbital at an

approximate dosage of 75 mg/kg. For the subcutaneous implants a 3 cm sagittal incision was

made in the skin in the midline of the back. A blunt dissection away from the midline

cranially and caudally to the left and the right of the spine was used to form subcutaneous

pockets on the surface of the Musculus latissimus dorsi (figure 4a.). In one of these pockets a

PHB embroidery was inserted (Figure 4b). The subcutaneous pocket was then closed around

the implant with a resorbable suture and the skin closed with a continuous suture. Eight rats

without any intervention served as control group.

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a. b.

Figure 4. Pictures of the surgical procedure.

a) The subcutaneous muscle pocket on the surface of the Musculus latissimus dorsi.

b) The PHB scaffold in a subcutaneous muscle pocket.

At the end of each time period (6 and 12 weeks after implantation) six rats were euthanized

with carbon dioxide. After euthanasia the implants with the surrounding tissues were retrieved

and prepared for molecular genetic evaluation (figure 5). Following a blunt dissection the

PHB surrounding tissue was carefully removed and frozen in liquid nitrogen.

skin

fascia

PHB scaffold

removed muscle tissue

M. latissimus dorsi

Figure 5. Schematic illustration of the PHB location and the removed muscle tissue.

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2.3. RNA-Isolation The total RNA was isolated using guanidinium-isothiocyanate (RNeasy Fibrous Tissue Mini

Kit, Qiagen, Valencia, CA, USA) according to the manufacturer’s instruction.

To reduce the viscosity of the lysates produced by disruption in Buffer RLT the fresh frozen

muscle tissue was homogenized. Homogenization shears high-molecular-weight genomic

DNA and other high-molecular-weight cellular components to create a homogeneous lysate.

Incomplete homogenization results in inefficient binding of RNA to the RNeasy spin column

membrane and therefore significantly reduced RNA yields.

For disruption the muscle tissue was immediately frozen in liquid nitrogen and ground to a

fine powder under liquid nitrogen using a mortar and pestle. The lysis buffer was added and

the homogenization was continued as quickly as possible in QIAshredder columns

(Qiashredder, Qiagen, Valencia, CA, USA). Total RNA purification from fibrous tissues,

such as skeletal muscle, can be difficult due to the abundance of contractile proteins,

connective tissue, and collagen. RNeasy Fibrous TissueMini Kits contain proteinase K, which

removes these proteins. 10 μl proteinase K were added to the homogenized lysate and the

solution was incubated at 55°C for 10 min. After centrifugation a small pellet of tissue debris

was formed. Thereafter ethanol was added to the supernatant to create conditions that promote

selective binding of RNA to the RNeasy membrane. The samples were then applied to the

RNeasy Mini spin column. Total RNA bound to the membrane, contaminants were efficiently

washed away, and high-quality RNA was eluted in RNase-free water (figure 6).

20

Figure 6. Schematic illustration of the RNeasy Fibrous Tissue Mini Kit Procedure (RNeasy Fibrous Tissue Handbook 11706, Qiagen).

The quality and yield of the RNA was determined by spectrophotometry at 260 nm and the

integrity examined by agarose gel electrophoresis with ethidium-bromide staining. The

quantification of the total RNA was performed using a NanoDrop®ND-1000 UV-Vis

Spectrophotometer (NanoDrop Technologies) which minimized the loss of RNA material

during the measurement procedure. Because of this only 2 μl RNA solution were used per

sample. After the RNA isolation 1µg of the total RNA was reverse transcribed in cDNA. The

cDNA was stored at -20°C.

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2.4. Agarose gel electrophoresis

The integrity and size distribution of the purified total RNA was checked by electrophoreses

on a denaturing at agarose gel. Both can be analysed by the survey of the respective ribosomal

RNAs (18S and 28S ribosomal RNA).

To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired

concentration, and then heated in a microwave oven until completely melted. Ethidium

bromide was added to the gel (final concentration 0.5 µg/ml) at this point to facilitate

visualization of DNA after electrophoresis. After cooling of the solution to about 60°C it was

poured into a casting tray containing a sample comb and allowed to solidify at room

temperature. After the gel has solidified the comb was removed and the gel was covered with

buffer, and samples containing RNA mixed with loading buffer were then filled into the

sample wells, the lid and power leads were placed on the apparatus, and a current was applied

(figure 7). The RNA was migrated towards the positive electrode. To visualize the RNA, the

gel was placed on an ultraviolet transilluminator.

Figure 7. Illustration of the pouring and loading of a horizontal agarose gel.

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2.5. Reverse Transcriptase-polymerase chain reaction — Real-Time RT-PCR

Changes in the mRNA amount could be measured by Real-Time RT-PCR using gene specific

primers according to the schema on figure 8. The concentrations of the primers were adjusted

as described in a manual for the Power Sybr-Green Master Mix (Applied Biosystems, Foster

City, CA, USA).

RNA isolation and analysis

First-strand cDNA synthesis

Real-Time PCR amplification

Continuous fluorescent measurement of PCR product during each cycle of PCR

Analysis of data

Figure 8. The procedure of the mRNA quantification using Real-time RT-PCR.

Because of the instability of RNAs and its digestion by ubiquitous RNases, the total-RNA

was transcribed into a more resistant form viz. complementary DNA (cDNA) (figure 9). The

High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA)

was used for this procedure. Reverse Transcription (RT reaction) is a process in which single-

stranded RNA is reverse transcribed into cDNA by using total cellular RNA or poly(A) RNA,

a reverse transcriptase enzyme, specific primers, dNTPs and an RNase inhibitor. The resulting

cDNA can be used in RT-PCR reaction.

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The High Capacity cDNA Reverse Transcription Kit delivers extremely high quantitative

single-stranded cDNA from total RNA. Although designed for short or long term archival of

cDNA, it yields very quantitative reverse transcription from 0.02 to 2 μg of total RNA for 20

μl.

Figure 9. Reverse transcription (The science of biology, 7th Edition, fig.16.8- schematic).

To quantify the expression of rat IGF1, IGF2, GDF8, VEGF and β-actin genes we applied the

Sybr-Green PCR Core Reagents (Applied Biosystems). Gene-specific PCR primers for the

genes were purchased from Qiagen (table 5).

Table 5. Information about RT-PCR primers. The sets of primers used for RT-PCR were obtained from Qiagen.

Gene Accession number Amplicon length

β-actin NM_031144 145 bp

IGF1 NM_001082479 135 bp

IGF2 XM_001064965 96 bp

VEGF NM_001110335 68 bp

GDF8 NM_019151 102 bp

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Parallel PCR assays for each gene target with cDNA samples and a “no-template control”

with water were performed parallel in all experiments on each 96-well plate. Reaction

mixtures contained in each well 12.5 µl of the Master Mix (Sybr-Green PCR Core Reagents)

and 300 nM of each primer. 2 µl of 1:50 diluted cDNA of each sample served as a template.

The specifity of the reaction was examined by creating a dissociation curve for each sample

and finally by checking the PCR products by agarose gel-electrophoresis. Quantitative real-

time polymerase chain reaction (RT-PCR) using the Applied Biosystems 7500 Real-Time

PCR System provided an accurate method for determination of levels of specific DNA

sequences in tissue samples. It is based on the detection of a fluorescent signal produced

proportionally during amplification of a PCR product (figure 10).

1. Double-stranded DNA denaturation at

2. Primer annealing at 60°C

New target sequences;

Replication (step 1 to 3).

40x

3. DNA elongation at 72°C

RT-PCR RT-PCR with SYBR® -Green Dye

Denaturation — when the DNA is denatured,

the SYBR®Green I Dye is released and fluorescence is

drastically reduced.

Polymerization — during extension, primers anneal and a PCR product is generated.

The SYBR®Green I Dye

fluoresces when bound to double-stranded DNA, fluorescence detected by the instrument. Figure 10. Standard PCR reaction and Sybr-Green Real-Time PCR; schematic illustration (van der Velden et al., 2003).

25

2.6. Quantification of the gene expression — 2(-ΔΔCT) method

To determine the quantity of the target-gene specific transcripts present in treated cells in

relation to untreated ones, their respective CT values were first normalized by subtracting the

CT value obtained from the β-actin control. The concentration of the gene-specific mRNA in

treated cells relative to untreated cells was calculated by subtracting the normalized CT values

obtained for untreated cells from those obtained from treated samples and the relative

concentration was determined (Livak and Schmittgen, 2001).

2.6.1. Derivation of the 2-ΔΔCT method

The equation that describes the exponential amplification of PCR is

Xn = X0 × (1 + Ex)n

where Xn - the number of target molecules at cycle n of the reaction,

X0 - the initial number of target molecules. EX is the efficiency of target

amplification,

n - the number of cycles. The threshold cycle (CT) indicates the fractional cycle

number at which the amount of amplified target reaches a fixed threshold.

Thus,

XT = X0 × (1 + Ex)CT,X = Kx

where XT - the threshold number of target molecules,

CT,X - the threshold cycle for target amplification,

Kx - a constant.

A similar equation for the endogenous reference (internal control gene) reaction is

RT = R0 × (1 + ER)CT,R = KR

26

where RT - the threshold number of reference molecules,

R0 - the initial number of reference molecules,

ER - the efficiency of reference amplification,

CT,R - the threshold cycle for reference amplification,

KR - a constant.

Dividing XT by RT gives the expression:

XT / RT = X0 × (1 + Ex)CT,X / R0 × (1 + ER)CT,R = Kx / KR = K

For real-time amplification using TaqMan probes, the exact values of XT and RT depend on a

number of factors including the reporter dye used in the probe, the sequence context effects

on the fluorescence properties of the probe, the efficiency of probe cleavage, purity of the

probe, and setting of the fluorescence threshold. Therefore, the constant K does not have to be

equal to one. Assuming efficiencies of the target and the reference are the same,

EX = ER = E

(X0 / R0) × (1 + E)CT,X - CT,R = K

or XN × (1 + E)ΔCT = K

where XN - equal to the normalized amount of target (X0 / R0) and

ΔCT - equal to the difference in threshold cycles for target and reference (CT,X- CT,R)

Rearranging gives the expression

XN = K × (1 + E)-ΔCT

The final step is to divide the XN for any sample q by the XN for the calibrator (cb):

XN,q / XN,cb = K × (1 + E)-ΔCT,q / K × (1 + E)-ΔCT,cb = (1 + E)-ΔΔCT

Here : ΔΔCT = - (ΔCT,q - ΔCT,cb )

27

For amplicons designed to be less than 150 bp and for which the primer and Mg2+

concentrations have been properly optimized, the efficiency is close to one. Therefore, the

amount of target normalized to an endogenous reference and relative to a calibrator, is given

by

amount of target = 2-ΔΔCT.

2.7. Statistical analysis

Statistical analysis was performed using the SigmaPlot Software (Systat Software, Inc.1735,

Technology Drive, Sn Jose, CA 95110, USA). The obtained values for the groups were

compared using Student’s unpaired t-test. Data are given as means ± SEM. p < 0.05 was

considered statistically significant.

28

3. Results

In this study 20 age-matched adult male Wistar rats were used. Eight animals served as

controls without PHB scaffolds on the surface of the M. latissimus dorsi. The remaining 12

animals received subcutaneous PHB scaffolds. All animals recovered from the operation and

healed uneventfully until the end of the experiments.

A good biocompatibility of the PHB implants could be observed macroscopically, because

one of the early responses to the PHB implants was capillary generation in the muscle tissue

surrounding the implants (figure 11).

Figure 11. PHB scaffold after 6 weeks of subcutaneous implantation with newly built capillaries.

3.1. Isolation of the total RNA from muscle samples

For the quantification of the different gene specific mRNAs four different muscle tissue

samples surrounding the PHB scaffold were prepared from six animals 6 and 12 weeks after

implantation, respectively. The total RNA was isolated from about 30 mg muscle tissue

according to the manufacturer’s instructions. The quality and yield of the total RNA was

determined by spectrophotometry at 260 nm using the NanoDrop® ND-1000 UV-Vis

spectrophotometer. Figure 12 shows exemplary the electropherogram of one muscle tissue

RNA probe.

29

Figure 12. The electropherogram at 260 nm using the NanoDrop® ND-1000 UV-Vis spectrophotometer of one muscle tissue RNA probe isolated from Musculus latissimus dorsi.

The integrity of the isolated total-RNA was examined by agarose gel electrophoresis with

ethidiumbromide staining. The electrophoresis of two different RNA probes shows two clear

bands of the respective size of the 18S and 28S rRNA. Furthermore, no degradation products

were found (figure 13).

28 S 18 S

Figure 13. Examples of RNA isolated from muscle run on an agarose gel and strained with ethidium bromide. 28S and 18S ribosomal RNA bands are indicated.

30

3.2. Examination of the primer specificity using standard RT-PCR

Using standard RT-PCR we detected signals for all gene transcripts mentioned above in the

M. latissimus dorsi. Standard RT-PCR yielded PCR products of the expected size. All

products were exemplary sequenced and revealed the expected DNA sequence. Figure 14

shows the RT-PCR results for VEGF, IGF1, IGF2,GDF8, and β-actin, respectively.

β-

β-actin IGF1 IGF2 VEGF GDF8

marker 125 bp 75 bp

Figure 14. Agarose gel electrophoresis of all used genes samples after RT-PCR for the verification of the purity and length of the amplicons.

3.3. Quantification of the VEGF, IGF1, IGF2, and GDF8 mRNA expression

Gene-specific RT-PCR was performed to quantify the expression of the VEGF, IGF1, IGF2

and GDF8 genes in M. latissimus dorsi specimens with and without PHB scaffold

implantation.

The relative expression pattern of the four tested genes in muscle tissue specimens

surrounding PHB scaffold after 6 and 12 weeks is shown in figures 15-18. Copy numbers of

the gene transcripts are given in relation to those of β-actin. A “no-template control” with

water was performed parallel in all experiments. Each series of experiments was performed

twice.

In untreated muscle tissue the relative copy number of VEGF was 3.9 ± 1.0. After six weeks

of implantation a significant increase in the expression level of VEGF was observed (treated

31

vs. non-treated: 7.9 ± 2.0 vs. 3.9 ± 1.0, p<0.05). In muscle tissue samples surrounding PHB

scaffolds a 2 fold increase of the VEGF mRNA amount was found compared to non-treated

muscle samples (Figure 15). Furthermore, a 1.6 fold up-regulation of the VEGF mRNA

expression was found after 12 weeks of the implantation of the PHB scaffolds (treated vs.

non-treated: 6.2 ± 1.6 vs. 3.9 ± 1.0, p<0.05). It is to note, that the level of the VEGF mRNA

expression was higher at 6 than at 12 weeks after treatment, but the mRNA amount was not

significantly decreased after 12 weeks compared to that after six weeks of implantation.

VEGF

0

2

4

6

8

10

12

1 control 6 weeks 12 weeks

VEG

F/be

ta-a

ctin

*

*

Figure 15. Relative expression of the VEGF mRNA in the M. latissimus dorsi of control rats and in the M. latissimus dorsi 6 and 12 weeks after PHB scaffold implantation. Mean ± S.E.M., Student’s t-test, * p< 0.05

The expression of the IGF1 gene was also significantly increased as compared to the controls

over the observed time period. In muscle tissue samples surrounding PHB scaffolds a 1.5 fold

increase of the IGF1 mRNA amount was found compared to non-treated muscle samples,

respectively (6 weeks, treated vs. non-treated: 29.0 ± 7.0 vs. 14.0 ± 3.0, p<0.05; 12 weeks,

treated vs. non-treated: 19.2 ± 3.0 vs. 14.2 ± 3.0, p<0.05, Figure 16). Like VEGF, the IGF1

mRNA expression decreased 12 weeks after implantation compared to 6 weeks after

treatment.

32

IGF1

0

5

10

15

20

25

30


IGF1

/bet

a-ac

tin

*

*

Figure 16. Relative expression of the IGF1 mRNA in the M. latissimus dorsi of control rats and in the M. latissimus dorsi 6 and 12 weeks after PHB scaffold implantation. Mean ± S.E.M., Student’s t-test, * p< 0.05

In case of the IGF2 mRNA, the expression was significantly up-regulated after 6 weeks

(p<0.05), but not significantly increased after 12 weeks (p>0.05). The IGF2 mRNA amount

was 1.5 times increased in M. latissimus dorsi surrounding PHB scaffolds compared to un-

treated muscle tissue specimens (treated vs. non-treated: 8.2 ± 1.8 vs. 5.5 ± 1.8; p<0.05; figure

17).

IGF2

0

2

4

6

8

10

12


IGF2

/bet

a-ac

tin

*

Figure 17. Relative expression of the IGF2 mRNA in the M. latissimus dorsi of control rats and in the M. latissimus dorsi 6 and 12 weeks after PHB scaffold implantation. Mean ± S.E.M.,Student’s t-test, * p< 0.05

33

In contrast to all other tested genes, we observed a significantly decreased GDF8 gene

expression (p<0.05) after 6 and 12 weeks, respectively. Moreover, the GDF8 mRNA levels

were identically after 6 and 12 weeks (6 weeks, treated vs. untreated: 2.9 ± 1.0 vs. 4.8 ± 1.6,

p<0.05; 12 weeks, treated vs. non-treated: 3.3 ± 0.8 vs. 4.8 ± 1.6; p<0.05).

GDF8

0

1

2

3

4

5

6

7


GD

F8/b

eta-

actin

* *

Figure 18. Relative expression of the GDF8 mRNA in the M. latissimus dorsi of control rats and in the M. latissimus dorsi 6 and 12 weeks after PHB scaffold implantation. Mean ± S.E.M., Student’s t-test, * p< 0.05

34

4. Discussion

In this study we have demonstrated for the first time that the natural polyester

polyhydroxybutyrate (PHB) has an influence on the expression of growth factors such as

vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF) as well as

myostatin also known as growth differentiation factor 8 (GDF8). We have found, that

subcutaneous implantation of PHB scaffolds increased the VEGF, IGF1, and IGF2 mRNA

expression in the Musculus latissimus dorsi of rats, whereas the myostatin mRNA was

significantly decreased. These reactions could be caused by the immunological adaptation of

the implant or by muscle regeneration / adaptation.

Wound healing, or wound repair, is an intricate process in which an organ or tissue repairs

itself after injury (Nguyen et al. 2009). The classic model of wound healing is divided into

three or four sequential, yet overlapping, phases: (1) hemostasis, (2) inflammation, (3)

proliferative and (4) remodeling phase. Within one hour after wounding, polymorphonuclear

neutrophils (PMNs) arrive at the wound site and become the predominant cells for the first

two days after injury. About two days after injury macrophages replace PMNs as the

predominant cells in the wound. It is well known that PHB shows an excellent

biocompatibility as evidenced by lack of toxicity, compatibility in contact with tissue and

blood (Saito et al. 1991; Clarotti et al. 1992; Gogolewski et al. 1993). The polymer was

implanted subcutaneously in different species and no abscess formation, or tissue necrosis

were observed (Gogolewski et al. 1993). Furthermore, PHB scaffolds expressed post-

traumatic inflammation with mononuclear macrophages, proliferating fibroblasts, and mature

vascularized fibrous capsules as typical tissue response (Gogolewski et al. 1993; Shishatskaya

et al. 2004; Qu et al. 2006; Shishatskaya et al. 2008).

Macrophages and other leukocytes such as helper T cells secrete cytokines resulting in

enhanced T cell division and increase of inflammation. It is known that pro-inflammatory

cytokines stimulate the expression of the nerve growth factor (Abe et al. 2007). Furthermore it

was shown that the cytokines transforming growth factor (TGF)-beta1, and interleukin (IL)-

1beta stimulate the production of VEGF from cultured conjunctival fibroblasts (Asano-Kato

et al. 2005). In our study an increased VEGF mRNA expression was detected in muscles

surrounding the PHB scaffold. It is to speculate that the increased VEGF mRNA expression

after implantation of the PHB scaffolds could be stimulated by cytokines released from

macrophages.

35

http://en.wikipedia.org/wiki/Granulocyte

http://en.wikipedia.org/wiki/Granulocyte

http://en.wikipedia.org/wiki/Helper_T_cell

http://en.wikipedia.org/wiki/Cytokines

The up-regulation of VEGF in the implant surrounding muscle tissue is necessary for wound

healing. One characteristic event of the proliferative phase of wound healing is angiogenesis,

the process of development and growth of new capillary blood vessels from pre-existing

vessels and the interaction with the extracellular matrix. Proliferation of capillary endothelial

cells is stimulated by VEGF (Klagsbrun and D'Amore 1996). The detection of the VEGF

mRNA in rat skeletal muscle is in agreement with previous studies. VEGF was identified to

be involved in the regulation of angiogenesis in skeletal muscles (Skorjanc et al. 1998;

Milkiewicz et al. 2001). Other angiogenesis factors, which are responsible for the maturation

of vascular networks in many tissues, have not been found in skeletal muscles so far.

However, the VEGF mRNA expression decreased over the entire investigation period and

resulted in delayed neo-vascularization of the implant. This time-dependent decrease of the

VEGF mRNA expression was also found after implantation of allogenous, acellular dermal

matrices on the gracilis muscle from rats (Mueller et al. 2009). Furthermore, recent studies

suggest that newly formed vessels are deleted by natural elimination (Peirce et al. 2004). The

VEGF induced growth of vessels is not accompanied by increased metabolic demand,

therefore durable presence is not essential for the function of the tissue.

Beside wound healing and immunological adaptation on the implant it is also possible that the

surrounding muscle tissue adapts and regenerates, because skeletal muscle has a remarkable

capacity to regenerate following exercise or injury. Muscle regeneration is a complex process

requiring the coordinated interaction between the myogenic satellite cells, growth factors,

cytokines, inflammatory components, vascular components and the extracellular matrix.

Differentiation, maturation, maintenance, and repair of skeletal muscle require ongoing

cooperation and coordination between an intrinsic regulatory programme controlled by

myogenic transcription factors and signalling pathways activated by hormones and growth

factors (Lassar and Munsterberg 1994; Naya and Olson 1999).

Important growth factors for muscle regeneration and differentiation are IGF1 for positive

adjustment of cell proliferation and myostatin as a negative growth factor. Besides, IGF2 is

also involved in muscle development and though IGF2 plays a crucial role in muscle

development (DeChiara et al. 1990; Marsh et al. 1997), its role in the repair / regeneration

process is less central and more secondary to other growth factors, for example IGF1. Age-

associated decrements in muscle repair process have also been shown to be associated with

the level of IGF gene expression. IGF1 might play a predominant role in protecting cells from

death mediated by myostatin (Yang et al. 2007). Thus, in the presence of IGF1, cells would be

arrested at G1/S (mitosis) and do not undergo apoptosis in response to myostatin. Myostatin

36

and IGF1 may regulate each other with a negative feedback mechanism to maintain

physiological homeostasis between cell growth and cell death during normal development.

This means that augmented IGF1 growth signal may require more myostatin-inhibitory

function to reach the balance between cell growth and cell death and vice versa. According to

other studies we could confirm that IGFs are expressed in muscles (Beck et al. 1987; Han et

al. 1987) and show that muscle fibres express increased IGF mRNA amounts after

implantation of PHB scaffolds. It is well established that endogenously produced IGF1 and

IGF2 can exert a strong positive effect on skeletal muscle differentiation (Florini et al. 1991;

Montarras et al. 1996). In contrast to these findings and the observation that elevated levels of

IGFs are sufficient to promote interstitial cell proliferation in otherwise untreated adult

skeletal muscle (Caroni and Grandes 1990), other findings support the hypothesis that the

early production of IGF1 by the inactive muscle fibre is involved in the initiation of the

proliferation reaction of muscle (Caroni et al. 1994). IGF1 was identified as a possible

initiator of restorative reactions in injured muscles (Caroni et al. 1994).

Recent results suggest that myostatin (GDF8) is a potent regulator of cell-cycle progression

and functions by regulating both the proliferation and differentiation steps of myogenesis

(Thomas et al. 2000; Langley et al. 2002; McCroskery et al. 2005). The role of myostatin has

been demonstrated in several studies not only during embryonic myogenesis, but also in

postnatal muscle growth. It is not known whether myostatin influences only muscle formation

or has also a function in the regulation of muscle metabolism (Ji et al. 1998). Lack of

myostatin results in accelerated regeneration and reduced fibrosis (McCroskery et al. 2005).

We observed 6 as well as 12 weeks after implantation a significantly decreased myostatin

expression. The decrease was about the same at both times. Several studies indicate that

myostatin might function as an inhibitor of satellite cell proliferation, suggesting a role of

myostatin in postnatal muscle growth and repair (Carlson et al. 1999; Wehling et al. 2000).

Consistent with this hypothesis, recent results from McCroskery et al. (McCroskery et al.

2003) indicate that myostatin is indeed expressed in satellite cells. Myostatin is known to

block hematogenesis and enhance chondrogenesis as well as epithelial cell differentiation

(Cieslak et al. 2003).

Moreover, muscles adapt to stress by change of fibre types and respective mRNA content.

Recently, Mack et al. (Mack et al. 2008) have shown that PHB implants can have an influence

on the myosin heavy chain (MyHC) isoform composition of the surrounding muscles. It was

found that MyHC isoform I increased significantly after the implantation of a PHB scaffold,

whereas the expression of the fast MyHC isoforms remains to be unchanged or decreased

37

(Mack et al. 2008). Accordingly, the basically fast rat Musculus latissimus dorsi adapted to a

slower phenotype with a more efficient energy utilisation and greater potential for

regeneration (Gedrange et al. 2003). Furthermore, in the course of adaptation and muscle

regeneration processes skeletal muscle are capable to change their anatomical characteristics.

These changes are often associated with changes in the intensity, duration, and frequency of

muscle activation by the central nervous system. It is known that the growth factors IGF1 and

IGF2 are candidates for muscle-derived nerve sprouting activity. These factors have been

shown to promote neurite outgrowth from sympathetic and sensory neurons in vitro (Recio-

Pinto et al. 1986) as well as innervated adult skeletal muscle (Caroni and Grandes 1990).

Muscle denervation or botulinum toxin-induced paralysis of adult rat skeletal muscle rapidly

led to elevated levels of IGF1 and IGF2 mRNA in the treated muscles (Ishii 1989). The

increase in the mRNA expression of both genes in our study could also cause nerve sprouting

and changes of the muscle activation.

Our results show that PHB implants in rat Musculus latissimus dorsi interact with the

surrounding muscle tissue and have influence on factors that regulate vascularization and

muscle adaptation. These changes in the mRNA expression of VEGF, GDF8, IGF1, and IGF2

are time-dependent. All changes occurred within six weeks and persisted up to twelve weeks

after implantation. These findings indicate on one hand a normal wound healing and on the

other hand improved muscle regeneration, stating that there is a synergistic effect between

PHB scaffolds and the surrounding muscle tissue. This synergistic effect should be further

elucidated.

38

5. References

Abdallah BM, Kassem M (2008) Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther 15:109-16

Abe Y, Akeda K, An HS, Aoki Y, Pichika R, Muehleman C, Kimura T, Masuda K (2007) Proinflammatory cytokines stimulate the expression of nerve growth factor by human intervertebral disc cells. Spine (Phila Pa 1976) 32:635-42

Ahdjoudj S, Lasmoles F, Holy X, Zerath E, Marie PJ (2002) Transforming growth factor beta2 inhibits adipocyte differentiation induced by skeletal unloading in rat bone marrow stroma. J Bone Miner Res 17:668-77

Albrektsson T, Johansson C (2001) Osteoinduction, osteoconduction and osseointegration. Eur Spine J 10 Suppl 2:S96-101

Armand M (1987). Current state of PEO-based electrolyte. London, 1. Elsevier Applied Science.

Asano-Kato N, Fukagawa K, Okada N, Kawakita T, Takano Y, Dogru M, Tsubota K, Fujishima H (2005) TGF-beta1, IL-1beta, and Th2 cytokines stimulate vascular endothelial growth factor production from conjunctival fibroblasts. Exp Eye Res 80:555-60

Beck F, Samani NJ, Penschow JD, Thorley B, Tregear GW, Coghlan JP (1987) Histochemical localization of IGF-I and -II mRNA in the developing rat embryo. Development 101:175-84

Bikle DD (2008) Integrins, insulin like growth factors, and the skeletal response to load. Osteoporos Int 19:1237-46

Bostrom RD, Mikos AG (1997). Tissue engineering of bone. Boston, Birkhäuser.

Buma P, Schreurs W, Verdonschot N (2004) Skeletal tissue engineering-from in vitro studies to large animal models. Biomaterials 25:1487-95

Burg KJ, Porter S, Kellam JF (2000) Biomaterial developments for bone tissue engineering. Biomaterials 21:2347-59

Canalis E (1983) The hormonal and local regulation of bone formation. Endocr Rev 4:62-77

39

Carlson CJ, Booth FW, Gordon SE (1999) Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading. Am J Physiol 277:R601-6

Caroni P, Grandes P (1990) Nerve sprouting in innervated adult skeletal muscle induced by exposure to elevated levels of insulin-like growth factors. J Cell Biol 110:1307-17

Caroni P, Schneider C, Kiefer MC, Zapf J (1994) Role of muscle insulin-like growth factors in nerve sprouting: suppression of terminal sprouting in paralyzed muscle by IGF-binding protein 4. J Cell Biol 125:893-902

Cieslak D, Blicharski T, Kapelanski W, Pierzchala M (2003) Investigation of polymorphisms in the porcine myostatin (GDF8, MSTN) gene. Czech J Anim Sci 48:69-75

Clarotti G, Schue F, Sledz J, Ait Ben Aoumar A, Geckeler KE, Orsetti A, Paleirac G (1992) Modification of the biocompatible and haemocompatible properties of polymer substrates by plasma-deposited fluorocarbon coatings. Biomaterials 13:832-40

Croteau S, Rauch F, Silvestri A, Hamdy RC (1999) Bone morphogenetic proteins in orthopedics: from basic science to clinical practice. Orthopedics 22:686-95; quiz 696-7

DeChiara TM, Efstratiadis A, Robertson EJ (1990) A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 345:78-80

DeGroot H, Donati D, Di Liddo M, Gozzi E, Mercuri M (2004) The use of cement in osteoarticular allografts for proximal humeral bone tumors. Clin Orthop Relat Res:190-7

Dezawa M, Kanno H, Hoshino M, Cho H, Matsumoto N, Itokazu Y, Tajima N, Yamada H, Sawada H, Ishikawa H, Mimura T, Kitada M, Suzuki Y, Ide C (2004) Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest 113:1701-10

Duncan RL (1995) Transduction of mechanical strain in bone. ASGSB Bull 8:49-62

Florini JR, Magri KA, Ewton DZ, James PL, Grindstaff K, Rotwein PS (1991) "Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II. J Biol Chem 266:15917-23

Frost H (1964). Dynamics of bone remodelling. Boston, Little Brown.

40

Frost H (1991) A new direction for osteoporosis research: a review and proposal. Bone Biodynamics 12:429-437

Gedrange T, Walter B, Tetzlaff I, Kasper M, Schubert H, Harzer W, Bauer R (2003) Regional alterations in fiber type distribution, capillary density, and blood flow after lower jaw sagittal advancement in pig masticatory muscles. J Dent Res 82:570-4

Gogolewski S, Jovanovic M, Perren SM, Dillon JG, Hughes MK (1993) Tissue response and in vivo degradation of selected polyhydroxyacids: polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/VA). J Biomed Mater Res 27:1135-48

Gray FM (1997). Polymer electrolytes. Cambridge.

Han VK, D'Ercole AJ, Lund PK (1987) Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus. Science 236:193-7

Harakas NK (1984) Demineralized bone-matrix-induced osteogenesis. Clin Orthop Relat Res:239-51

Heino TJ, Hentunen TA (2008) Differentiation of osteoblasts and osteocytes from mesenchymal stem cells. Curr Stem Cell Res Ther 3:131-45

Helm GA, Gazit Z (2005) Future uses of mesenchymal stem cells in spine surgery. Neurosurg Focus 19:E13

Hill PA (1998) Bone remodelling. Br J Orthod 25:101-7

Hill PA, Tumber A, Meikle MC (1997) Multiple extracellular signals promote osteoblast survival and apoptosis. Endocrinology 138:3849-58

Hollinger JO, Battistone GC (1986) Biodegradable bone repair materials. Synthetic polymers and ceramics. Clin Orthop Relat Res:290-305

Hollinger JO, Schmitz JP (1997) Macrophysiologic roles of a delivery system for vulnerary factors needed for bone regeneration. Ann N Y Acad Sci 831:427-37

Holmes PA (1987) Developents in crystalline polymers. Vol. 2. edited by Bassett D.C. Ed.

Hutmacher DW (2001) Scaffold design and fabrication technologies for engineering tissues--state of the art and future perspectives. J Biomater Sci Polym Ed 12:107-24

41

Ishaug SL, Crane GM, Miller MJ, Yasko AW, Yaszemski MJ, Mikos AG (1997) Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds. J Biomed Mater Res 36:17-28

Ishii DN (1989) Relationship of insulin-like growth factor II gene expression in muscle to synaptogenesis. Proc Natl Acad Sci U S A 86:2898-902

Ito Y, Miyazono K (2003) RUNX transcription factors as key targets of TGF-beta superfamily signaling. Curr Opin Genet Dev 13:43-7

Ji S, Losinski RL, Cornelius SG, Frank GR, Willis GM, Gerrard DE, Depreux FF, Spurlock ME (1998) Myostatin expression in porcine tissues: tissue specificity and developmental and postnatal regulation. Am J Physiol 275:R1265-73

Kim SJ, Kim SY, Kwon CH, Kim YK (2007) Differential effect of FGF and PDGF on cell proliferation and migration in osteoblastic cells. Growth Factors 25:77-86

Kjaer M (2004) Role of extracellular matrix in adaptation of tendon and skeletal muscle to mechanical loading. Physiol Rev 84:649-98

Klagsbrun M, D'Amore PA (1996) Vascular endothelial growth factor and its receptors. Cytokine Growth Factor Rev 7:259-70

Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T (1997) Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755-64

Kroese-Deutman HC, Ruhe PQ, Spauwen PH, Jansen JA (2005) Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants inserted at an ectopic site in rabbits. Biomaterials 26:1131-8

Kulkarni RK, Moore EG, Hegyeli AF, Leonard F (1971) Biodegradable poly(lactic acid) polymers. J Biomed Mater Res 5:169-81

Kusumoto K, Bessho K, Fujimura K, Konishi Y, Ogawa Y, Iizuka T (1996) Self-regenerating bone implant: ectopic osteoinduction following intramuscular implantation of a combination of rhBMP-2, atelopeptide type I collagen and porous hydroxyapatite. J Craniomaxillofac Surg 24:360-5

Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R (2002) Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. J Biol Chem 277:49831-40

42

Lassar A, Munsterberg A (1994) Wiring diagrams: regulatory circuits and the control of skeletal myogenesis. Curr Opin Cell Biol 6:432-42

Lauer G, Wiedmann-Al-Ahmad M, Otten JE, Hubner U, Schmelzeisen R, Schilli W (2001) The titanium surface texture effects adherence and growth of human gingival keratinocytes and human maxillar osteoblast-like cells in vitro. Biomaterials 22:2799-809

Lean JM, Mackay AG, Chow JW, Chambers TJ (1996) Osteocytic expression of mRNA for c-fos and IGF-I: an immediate early gene response to an osteogenic stimulus. Am J Physiol 270:E937-45

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-8

Luk JM, Wang PP, Lee CK, Wang JH, Fan ST (2005) Hepatic potential of bone marrow stromal cells: development of in vitro co-culture and intra-portal transplantation models. J Immunol Methods 305:39-47

Mack HB, Mai R, Lauer G, Mack F, Gedrange T, Franke R, Gredes T (2008) Adaptation of myosin heavy chain mRNA expression after implantation of poly(3)hydroxybutyrate scaffolds in rat m. latissimus dorsi. J Physiol Pharmacol 59 Suppl 5:95-103

Mai R, Hagedorn MG, Gelinsky M, Werner C, Turhani D, Spath H, Gedrange T, Lauer G (2006) Ectopic bone formation in nude rats using human osteoblasts seeded poly(3)hydroxybutyrate embroidery and hydroxyapatite-collagen tapes constructs. J Craniomaxillofac Surg 34 Suppl 2:101-9

Marsh DR, Criswell DS, Hamilton MT, Booth FW (1997) Association of insulin-like growth factor mRNA expressions with muscle regeneration in young, adult, and old rats. Am J Physiol 273:R353-8

McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R (2003) Myostatin negatively regulates satellite cell activation and self-renewal. J Cell Biol 162:1135-47

McCroskery S, Thomas M, Platt L, Hennebry A, Nishimura T, McLeay L, Sharma M, Kambadur R (2005) Improved muscle healing through enhanced regeneration and reduced fibrosis in myostatin-null mice. J Cell Sci 118:3531-41

Meyer U, Joos U, Wiesmann HP (2004) Biological and biophysical principles in extracorporal bone tissue engineering. Part I. Int J Oral Maxillofac Surg 33:325-32

43

Miguel O, Fernandez-Beridi MJ, Iruin JJj (1997) Survey on traansport properties of liquids, vapors and gases in biodegradable poly(3-hydroxybutyrate) (PHB). App Poly Sci 64:1849-1859

Milkiewicz M, Brown MD, Egginton S, Hudlicka O (2001) Association between shear stress, angiogenesis, and VEGF in skeletal muscles in vivo. Microcirculation 8:229-41

Milosevski M, Bossert J, Milosevski D, Gruevska A (1999) Preparation and properties of dense and porous calcium phosphate. Ceramics International 25:693-696

Montarras D, Aurade F, Johnson T, J II, Gros F, Pinset C (1996) Autonomous differentiation in the mouse myogenic cell line, C2, involves a mutual positive control between insulin-like growth factor II and MyoD, operating as early as at the myoblast stage. J Cell Sci 109 (Pt 3):551-60

Mueller CK, Lee SY, Schultze-Mosgau S (2009) Characterization of interfacial reactions between connective tissue and allogenous implants used for subdermal soft tissue augmentation. Int J Oral Maxillofac Surg

Mundy GR (1994) Peptides and growth regulatory factors in bone. Rheum Dis Clin North Am 20:577-88

Naya FJ, Olson E (1999) MEF2: a transcriptional target for signaling pathways controlling skeletal muscle growth and differentiation. Curr Opin Cell Biol 11:683-8

Nguyen DT, Orgill DP, Murphy GF (2009) Chapter 4: The pathophysiologic basis for wound healing and cutaneous regeneration. Biomaterials For Treating Skin Loss.:25-57

Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ (1997) Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89:765-71

Papachroni KK, Karatzas DN, Papavassiliou KA, Basdra EK, Papavassiliou AG (2009) Mechanotransduction in osteoblast regulation and bone disease. Trends Mol Med 15:208-16

Papadopoulou AK, Papachristou DJ, Chatzopoulos SA, Pirttiniemi P, Papavassiliou AG, Basdra EK (2007) Load application induces changes in the expression levels of Sox-9, FGFR-3 and VEGF in condylar chondrocytes. FEBS Lett 581:2041-6

44

Peirce SM, Price RJ, Skalak TC (2004) Spatial and temporal control of angiogenesis and arterialization using focal applications of VEGF164 and Ang-1. Am J Physiol Heart Circ Physiol 286:H918-25

Peter SJ, Miller MJ, Yasko AW, Yaszemski MJ, Mikos AG (1998) Polymer concepts in tissue engineering. J Biomed Mater Res 43:422-7

Qu XH, Wu Q, Zhang KY, Chen GQ (2006) In vivo studies of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) based polymers: biodegradation and tissue reactions. Biomaterials 27:3540-8

Recio-Pinto E, Rechler MM, Ishii DN (1986) Effects of insulin, insulin-like growth factor-II, and nerve growth factor on neurite formation and survival in cultured sympathetic and sensory neurons. J Neurosci 6:1211-9

Reusch RN, Sadoff HL (1983) D-(-)-poly-beta-hydroxybutyrate in membranes of genetically competent bacteria. J Bacteriol 156:778-88

Roodman GD (2004) Mechanisms of bone metastasis. N Engl J Med 350:1655-64

Saad B, Neuenschwander P, Uhlschmid GK, Suter UW (1999) New versatile, elastomeric, degradable polymeric materials for medicine. Int J Biol Macromol 25:293-301

Saito T, Tomita K, Juni K, Ooba K (1991) In vivo and in vitro degradation of poly(3-hydroxybutyrate) in rat. Biomaterials 12:309-12

Schmack G, Jehnichen D, Vogel R, Tandler B (2000) Biodegradable fibres of Poly (3-hydroxybutyrate) produced by high-speed melt spinning and spindrawing. Polymer Physics 38:2841-2850

Sevastianov VI, Perova NV, Shishatskaya EI, Kalacheva GS, Volova TG (2003) Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood. J Biomater Sci Polym Ed 14:1029-42

Shishatskaya EI, Voinova ON, Goreva AV, Mogilnaya OA, Volova TG (2008) Biocompatibility of polyhydroxybutyrate microspheres: in vitro and in vivo evaluation. J Mater Sci Mater Med 19:2493-502

Shishatskaya EI, Volova TG, Puzyr AP, Mogilnaya OA, Efremov SN (2004) Tissue response to the implantation of biodegradable polyhydroxyalkanoate sutures. J Mater Sci Mater Med 15:719-28

45

Skorjanc D, Jaschinski F, Heine G, Pette D (1998) Sequential increases in capillarization and mitochondrial enzymes in low-frequency-stimulated rabbit muscle. Am J Physiol 274:C810-8

Suwantong O, Waleetorncheepsawat S, Sanchavanakit N, Pavasant P, Cheepsunthorn P, Bunaprasert T, Supaphol P (2007) In vitro biocompatibility of electrospun poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fiber mats. Int J Biol Macromol 40:217-23

Tang LL, Xian CY, Wang YL (2006) The MGF expression of osteoblasts in response to mechanical overload. Arch Oral Biol 51:1080-5

Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J, Kambadur R (2000) Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem 275:40235-43

Tsuruga E, Takita H, Itoh H, Wakisaka Y, Kuboki Y (1997) Pore size of porous hydroxyapatite as the cell-substratum controls BMP-induced osteogenesis. J Biochem 121:317-24

van der Velden VH, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, van Dongen JJ (2003) Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. Leukemia 17:1013-34

Vogel R, Tandler B, Haussler L, Jehnichen D, Brunig H (2006) Melt spinning of poly(3-hydroxybutyrate) fibers for tissue engineering using alpha-cyclodextrin/polymer inclusion complexes as the nucleation agent. Macromol Biosci 6:730-6

Volova TG, Gladyshev MI, Trusova MY, Zhila NO, Kartushinskaya MV (2004) Degradation of bioplastics in natural environment. Dokl Biol Sci 397:330-2

Warnke PH, Springer IN, Wiltfang J, Acil Y, Eufinger H, Wehmoller M, Russo PA, Bolte H, Sherry E, Behrens E, Terheyden H (2004) Growth and transplantation of a custom vascularised bone graft in a man. Lancet 364:766-70

Wehling M, Cai B, Tidball JG (2000) Modulation of myostatin expression during modified muscle use. Faseb J 14:103-10

Wiesmann HP, Joos U, Meyer U (2004) Biological and biophysical principles in extracorporal bone tissue engineering. Part II. Int J Oral Maxillofac Surg 33:523-30

46

Yang W, Zhang Y, Li Y, Wu Z, Zhu D (2007) Myostatin induces cyclin D1 degradation to cause cell cycle arrest through a phosphatidylinositol 3-kinase/AKT/GSK-3 beta pathway and is antagonized by insulin-like growth factor 1. J Biol Chem 282:3799-808

Zelzer E, Olsen BR (2005) Multiple roles of vascular endothelial growth factor (VEGF) in skeletal development, growth, and repair. Curr Top Dev Biol 65:169-87

Zhao G, Monier-Faugere MC, Langub MC, Geng Z, Nakayama T, Pike JW, Chernausek SD, Rosen CJ, Donahue LR, Malluche HH, Fagin JA, Clemens TL (2000) Targeted overexpression of insulin-like growth factor I to osteoblasts of transgenic mice: increased trabecular bone volume without increased osteoblast proliferation. Endocrinology 141:2674-82

Zhao S, Pinholt EM, Madsen JE, Donath K (2000) Histological evaluation of different biodegradable and non-biodegradable membranes implanted subcutaneously in rats. J Craniomaxillofac Surg 28:116-22

Zhou H, Hammonds RG, Jr., Findlay DM, Martin TJ, Ng KW (1993) Differential effects of transforming growth factor-beta 1 and bone morphogenetic protein 4 on gene expression and differentiated function of preosteoblasts. J Cell Physiol 155:112-9

47

6. Summary

Bone defects can be a limiting factor in achievement of optimal orthodontic treatment.

Diverse bone substitutes were already used for the creation of new bone in the patient, such as

collagen composites, calcium phosphate, and titanium. Muscles are known to have a

considerable potential of adaptation. The latissimus dorsi muscle was used for the growth and

preparation of bone grafts for subsequent transplantation.

The aim of the present study was to identify the synergistic effect between the poly-3-

hydroxybutyrate (PHB) bone substitute and surrounding muscle tissue. To describe this effect,

changes of insulin like growth factors (IGF1, IGF2), myostatin (GDF8) and vascular

endothelial growth factor (VEGF) mRNA content were examined in the Musculus latissimus

dorsi of 12 Wistar-King rats after 6 and 12 weeks of PHB scaffold implantation. At each time

point six rats were killed and implants and surrounding tissues prepared for genetic

evaluation. Eight rats without any implants served as controls. RNA was extracted from

homogenized muscle tissue and reverse transcribed. Changes in the mRNA content were

measured by Real-Time RT-PCR using specific primers for IGF1, IGF2, GDF8, and VEGF.

The VEGF mRNA level was significantly increased (p<0.05) in muscle tissue specimens after

6 and 12 weeks of implantation compared to the controls. The expression of the IGF1 gene

was also significantly increased as compared to the controls over the observed time period

(p<0.05). Furthermore, the VEGF and IGF1 mRNA levels were higher after 6 than after 12

weeks of treatment. In the case of the IGF2 gene, the expression was significantly elevated

after 6 weeks (p<0.05), but not significantly increased after 12 weeks. In contrast to all other

tested genes we observed a significantly decreased GDF8 gene expression (p<0.05) both after

retrieval of implants after 6 as well as after 12 weeks. Moreover, the mRNA levels of GDF8

after 6 and 12 weeks were comparable the same.

Our results show that PHB implants in rat Musculus latissimus dorsi interact with the

surrounding muscle tissue and have influence on factors that regulate vascularization and

muscle adaptation. These findings indicate on one hand a normal wound healing and on the

other hand improved muscle regeneration, stating that there is a synergistic effect between

PHB scaffolds and the surrounding muscle tissue.

48

7. Supplement

Beta-actin

Rn_Actb_1_SG QuantiTect Primer Assay (200) (QT00193473)

Official gene name actin, beta

Official gene symbol Actb

Species Rat (Rattus norvegicus)

Entrez Gene ID 81822

Detected transcript NM_031144

Ensembl Transcript ID ENSRNOT00000001480

Length of detected transcript 1296 bp

Amplified exons*: 2/3

Amplicon length: 145 bp

Dye label / detection SYBR Green

Assay type QuantiTect Primer Assay

49

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=81822

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=&db=Nucleotide&cmd=search&term=NM_031144

http://www.ensembl.org/Rattus_norvegicus/transview?db=core&transcript=ENSRNOT00000001480

https://www1.qiagen.com/Products/Pcr/QuantiTect/PrimerAssays.aspx

VEGF

Rn_RGD:619991_1_SG QuantiTect Primer Assay (200) (QT00198954)

Official gene name vascular endothelial growth factor A

Official gene symbol Vegfa










50





IGF2

Rn_Igf2_1_SG QuantiTect Primer Assay (200) (QT00195594)

Official gene name insulin-like growth factor 2

Official gene symbol Igf2



Detected transcript XM_001064965







51


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=&db=Nucleotide&cmd=search&term=XM_001064965



GDF8

Rn_Mstn_1_SG QuantiTect Primer Assay (200) (QT00189406)

Official gene name myostatin

Official gene symbol Mstn










52





IGF1

Rn_Igf1_2_SG QuantiTect Primer Assay (200) (QT01745373)

Official gene name insulin-like growth factor 1

Official gene symbol Igf1










53





Material

Kits and biomaterials

Table a. Summary of all used kits and biomaterials.

material Manufacturer

High Capacity cDNA Archive Kit Applied Biosystems

AmpliTaq Gold DNA Polymerase Applied Biosystems

GeneAmp 10×PCR Gold Puffer Applied Biosystems

GeneAmp dNTP Mix Applied Biosystems

Power SYBR Green PCR Master Mix Applied Biosystems

25bp ladder Invitrogen

100bp ladder Invitrogen

RNeasy Fibrous Tissue Mini Kit Qiagen

Qiashredder Qiagen

Equipment Table b. Summary of all used equipments.

equipment manufacturer

7500 Real Time PCR System Applied Biosystems

Thermocycler Gene Amp PCR system 2400 Applied Biosystems

Geldetektionssystem Gel Doc 2000 Biorad

Centrifuge 5417 R Eppendorf

Photometer Eppendorf

Netzgerät EV 231 Consort

Biofuge Heraeus

Waage Portable Sartorius

Vortexer Vortex Genie 2.0 Scientific Industries

Microwelle Siemens

Pipettboy acu Tecnomaro

Gelkammern, Modell AGT-2-1 VWR International

54

Chemicals Table c. Summary of all used chemicals.

chemical manufacturer

Aqua ad injectabila B.Braun

Borsäure Roth

Bromphenolblau Roth

EDTA Merck

Ethanol absolut Roth

Ethidium bromide Roth

Liquid nitrogen

Glycerin Roth

2-Mercaptoethanol Roth

Natrium chloride Merck

peqGold Universal Agarose PeqLab

TRIS ICN Biomedicals

Table d. Buffer composition.

TBE buffer Loading buffer for agarose gels

107,8g Tris

55,0g boric acid

5,8g EDTA

pH 8,0; add 1000 ml aqua dest.

50mM EDTA

30% glycerin

Small amount of bromophenol blue

55

Legends of tables

Table 1. Main growth factors and its function in osteoblast differentiation.

Table 2. Local factors that regulate bone remodelling.

Table 3. Summary of bone remodelling regulating hormones.

Table 4. Selected critical consideration in bone tissue-engineering scaffold design.

Table 5. Information about RT-PCR primers.

Table a. Summary of all used kits and biomaterials.

Table b. Summary of all used equipments.

Table c. Summary of all used chemicals.

Table d. Buffer composition.

56

Legends of figures

Figure 1. Regulation of bone formation.

Figure 2. The classification of biomaterials used for bone tissue engineering.

Figure 3. The structure of the PHB monomer and polymer.

Figure 4. Pictures of the surgical procedure.

a) The subcutaneous muscle pocket on the surface of the Musculus latissimus dorsi.

b) The PHB scaffold in a subcutaneous muscle pocket.

Figure 5. Schematic illustration of the PHB location and the removed muscle tissue.

Figure 6. Schematic illustration of the RNeasy Fibrous Tissue Mini Kit Procedure.

Figure 7. Illustration of the pouring and loading of a horizontal agarose gel.

Figure 8. The procedure of the mRNA quantification using Real-time RT-PCR.

Figure 10. Standard PCR reaction and Sybr-Green Real-Time PCR.

Figure 11. PHB scaffold after 6 weeks of subcutaneous implantation with newly built

capillaries.

Figure 12. The electropherogram at 260 nm using the NanoDrop® ND-1000 UV-Vis

spectrophotometer of one muscle tissue RNA probe isolated from Musculus

latissimus dorsi.

Figure 13. Example of RNA isolated from muscle run on an agarose gel and strained with

ethidium bromide. 28S and 18S ribosomal RNA bands are indicated.

Figure 14. Agarose gel electrophoresis of all used genes samples after RT-PCR for the

verification of the purity and length of the amplicons.

Figure 15. Relative expression of the VEGF mRNA.

Figure 16. Relative expression of the IGF1 mRNA.

Figure 17. Relative expression of the IGF2 mRNA.

Figure 18. Relative expression of the GDF8 mRNA.

57

Eidesstattliche Erklärung / Affirmation Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig verfasst und keine

anderen als die angegebenen Hilfsmittel benutzt habe.

Die Dissertation ist bisher keiner anderen Fakultät vorgelegt worden.

Ich erkläre, dass ich bisher kein Promotionsverfahren erfolglos beendet habe und dass eine

Aberkennung eines bereits erworbenen Doktorgrades nicht vorliegt.

Datum Unterschrift

58

Danksagung / Acknowledgment

An vorrangiger Stelle möchte ich mich bei meinem Doktorvater Herrn Prof. Dr. Tomasz

Gedrange für die interessante Aufgabenstellung, die Anleitung und das ständige Interesse am

Fortgang der Arbeit sehr herzlich danken.

Ein ganz besonderer Dank geht an Frau Dr. Christiane Kunert-Keil für die großartige

Betreuung während meiner Doktorarbeit und ihre konstruktive Kritik, die in einem sehr

großen Maße zum Gelingen dieser Arbeit beitrugen. Sie hatte stets ein offenes Ohr für Fragen

und Probleme und stand mir jederzeit mit fachlichem Rat zur Seite.

Bei Frau Ingrid Pieper möchte ich mich ganz herzlich bedanken. Sie hat mich im Labor nicht

nur mit Hinweisen und Ideen unterstützt, sondern auch dafür gesorgt, dass der Spaß an der

Arbeit nicht verloren ging. Ich danke ihr ebenfalls für die Gespräche nicht nur über berufliche

Belange.

Meinem Kollegen, Herrn Dr. Phillip Eigenwillig danke ich für die grafische Unterstützung

dieser Arbeit und ein nettes und kollegiales Arbeitsklima in unserem Behandlungszimmer.

Den Mitgliedern der Abteilung für Kieferorthopädie der ZMKK danke ich für ihre

Hilfsbereitschaft, Freundlichkeit und die überaus angenehme Arbeitsatmosphäre.

Bei allen Mitarbeitern der Klinik mit Poliklinik für Innere Medizin C, Hämatologie,

Onkologie und Transplantationszentrum des Greifswalder Klinikums bedanke ich mich für

das gute Arbeitsklima und die Bereitstellung des Real-Time PCR Gerätes in der

Forschungsgruppe von Prof. Dr. Gottfried Dölken und Prof. Dr. Christian Schmidt.

Bei Herrn Prof. Dr. Alexander Wöll und Frau Dr. Silke Lucke möchte ich mich bedanken für

die freundliche Bereitschaft zum Korrekturlesen und ermunternde Worte.

Ein ausdrücklicher Dank gilt meinen Freunden: Herrn Dr. Ingo Merkl, Frau Dr. Margarita

Ruiz-Bamberg, Kantor Martin Rost, Herrn Dr. Thomas Klinke, Herrn André Wendel und

Familie Junge, für die moralische, verständnisvolle und auch anderweitig währende

Unterstützung.

Meiner Schwester Dr. Marzena Gredes danke ich für ihr Verständnis, ihre Unterstützung und

Ermutigungen.

Insbesondere möchte ich auf diesem offiziellen Weg meinen Eltern und meiner Oma, denen

diese Doktorarbeit gewidmet ist, danken. Sie haben mir jederzeit in jeder Hinsicht zur Seite

gestanden und mich nicht zuletzt fortlaufend ermuntert, diese Dissertation fertig zu stellen.

Prace ta dedykuj mojej Rodzinie, która zawsze we mnie wierzy i wspiera. Wam Kochani,

dedykuje ta prace.

59

D R . T O M A S Z J A K U B G R E D E S PERSÖNLICHE ANGABEN

Familienstand: ledig

Staatsangehörigkeit: Polen

Nationalität: polnisch

Geburtsdatum: 20. August 1975

Geburtsort: Dzierżoniów (Polen) AUSBILDUNG

09/1982 – 06/1990 Grundschule in Dzierżoniów

09/1990 – 06/1994 Lyzeum Nr.2 in Dzierżoniów, Abitur

10/1995 – 07/2000 Studium an der Wrocławer Universität, Fachrichtung Chemie (Diplom-Chemiker) 10/2000 – 07/2005 Studium an der Medizinischen Piastów-Śląskich-Universität in Wrocław, Fakultät für Zahnmedizin (Staatsexamen - Approbation als Zahnarzt)

12/2007 Promotion zum Dr.rer.med. an der EMAU Greifswald, Poliklinik für Kieferorthopädie Thema: Untersuchung der Genaktivität im Kiefer-gelenkknorpel des Schweins (Sus scrofa domesticus) nach Vorverlagerung des Unterkiefers

Seit 2007 Ausbildung zum Fachzahnarzt in der Kieferorthopädie- EMAU Greifswald

W A L T H E R - R A T H E N A U - S T R . 4 7 , 1 7 4 8 9 G R E I F S W A L D •

T E L E F O N 0 3 8 3 4 / 8 6 7 5 4 3 ( d i e n s t l i c h ) E - M A I L : t h o m a s g r e d e s @ y a h o o . d e

60

D R . T O M A S Z J A K U B G R E D E S

11/2009 Abgabe und Annahme der Promotion zur Erlangung des akademischen Grades “Dr.med.dent.“ mit dem Titel “Changes in insulin like growth factors, myostatin and vascular endothelial growth factor in rat musculus latissimus dorsi by poly-3-hydroxybutyrate implants” durch das Dekanat der Medizinischen Fakultät der EMAU Greifswald Rigorosum voraussichtlich im Februar 2010

BERUFSERFAHRUNG 10/2005 – 09/2006 Niederschlesiesches Zentrum für Pädiatrie (Dolnośląskie Centrum Pediatryczne im. J. Korczaka, Wrocław) - Zahnmedizinisches Assistenzjahr Seit 2006 wissenschaftlicher Mitarbeiter an der Poliklinik für Kieferorthopädie, Kinderzahnheilkunde und Präventive Zahnheilkunde, EMAU Greifswald Seit 2007 Weiterbildungsassistent an der Poliklinik für Kieferorthopädie,

Kinderzahnheilkunde und Präventive Zahnheilkunde, EMAU Greifswald

FORSCHUNGSPROJEKTE ITI Foundation 2007-2008

Early loading of palatal implants (ortho-implant type II): a prospective multicenter

randomized controlled clinical trial. (Mainz/Dresden/Greifswald)

DOT 2008-2009

Untersuchung der Anwendung von Knochenersatzpaste bei ausgeprägten Knochendefekten.

W A L T H E R - R A T H E N A U - S T R . 4 7 , 1 7 4 8 9 G R E I F S W A L D • T E L E F O N 0 3 8 3 4 / 8 6 7 5 4 3 ( d i e n s t l i c h )

E - M A I L : t h o m a s g r e d e s @ y a h o o . d e

61

D R . T O M A S Z J A K U B G R E D E S KONGRESSTEILNAHMEN

- International Annual Meeting of the Anatomische Gesellschaft; 30.03.-2.04.2007,

Giessen

- I. Internationales Fachsymposium zur Problematik der „Schlafatemstörungen“;

Greifswald, 07.07.2007

- Bone substitutes and endoprosthetic materials for structure conservation; Brunico 11-

14.03.2008, Italien

- II. Internationales Fachsymposium zur Problematik der „Schlafatemstörungen“;

Greifswald, 12.07.2008

- 84th Congress of the European Orthodontic Society- EOS; Lissabon, 10.-14.06.2008,

Portugal

- Internationale Zahnmedizinische Konferenz in der Periodontologie, 18.-20.09.2008,

Białystok, Polen

- XIV Zahnmedizinische Konferenz Expo-Andre in Toruń, 17-18.10.2008, Polen

- Knochenersatzmaterialen in der Kieferorthopädie; Brunico 16-19.03.2009, Italien

- 109th American Association of Orthodontists Annual Session in Boston,

Massachusetts, 1.-5.05.09, USA

- 85th Congress of the European Orthodontic Society- EOS Helsinki, 10.-14.06.2009,

Finnland

- Polish-German Life Science Forum, Szczecin 24.-25.09.2009, Polen

- XV Zahnmedizinische Konferenz Expo-Andre in Toruń, 15.-17.10.2009, Polen

PUBLIKATIONEN UND KONGRESSBEITRÄGE Veröffentlichungen:

1. A novel post-and-core restoration system--results of three years of clinical application of the "Wuerzburg Post". Rottner K, Boldt J, Proff P, Spassov A, Gredes T, Mack F, Richter EJ, J Physiol Pharmacol. 2008 9; 5:105-15.



62

http://www.ncbi.nlm.nih.gov/pubmed/19075331?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum



2. Evaluation of shape and size changes of bone and remodelled bone substitute after different fixation methods. Gedrange T, Mai R, Mack F, Zietek M, Borsos G, Vegh A, Spassov A, Gredes T., J Physiol Pharmacol. 2008 59; 5:87-94.

3. Adaptation of myosin heavy chain mRNA expression after implantation of

poly(3)hydroxybutyrate scaffolds in rat m. latissimus dorsi. Mack HB, Mai R, Lauer G, Mack F, Gedrange T, Franke R, Gredes T., J Physiol Pharmacol. 2008; 9; 5:95-103.

4. Physiological functions of the human finger.

Dumont C, Burfeind H, Kubein-Meesenburg D, Hosten N, Fanghanel J, Gredes T, Nagerl H., J Physiol Pharmacol. 2008 Nov;59 Suppl 5:69-74.

5. Different bone sesitivity to malformations induced by procarbazine in fetal rats.

Weingartner J, Proff P, Fanghanel J, Kundt G, Gedrange T, Kubein-Meesenburg D, Gredes T., J Physiol Pharmacol. 2008 Nov;59 Suppl 5:17-25.

6. A new design for post and core restorations implementing positive locking.

Richter EJ, Boldt J, Groth S, Proff P, Gredes T, Rottner K., Biomed Tech (Berl). 2008;53(5):234-41.

7. The influence of the root cross-section on the stress distribution in teeth restored with a positive-locking post and core design: a finite element study. Schilling KU, Rottner K, Boldt J, Proff P, Gredes T, Richter EJ, Reicheneder C., Biomed Tech (Berl). 2008;53(5):255-8.

8. Increased oxidative stress in mdx mice masticatory muscles

Spassov A., Gredes T., Gedrange T., Pavlovic D., Lupp A., Kunert-Keil C.; Am J Orthod and Dent Orth, submitted

9. Histological changes in masticatory muscles of mdx mice

Spassov A., Gredes T., Gedrange T., Lucke S., Pavlovic D., Kunert-Keil C., Arch Oral Biol 2009, accepted

10. Differential expression of MyHC isoforms in masticatory muscles of mdx mice

Spassov A., Gredes T., Gedrange T., Lucke S.,Morgenstern S., Pavlovic D., Kunert-Keil C., Europ J Orthod, submitted

11. Caveolin-1, caveolin-3 and VEGF expression in masticatory muscles of mdx mice

Kunert-Keil C., Gredes T., Lucke S., Morgenstern S., Pavlovic D., Gedrange T., Spassov A., Histochem Cell Biol, submitted



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http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Mack%20HB%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

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12. Expression of muscle specific growth factors in mdx mice masticatory muscles Spassov A., Gredes T., Gedrange T., Pavlovic D., Lucke S., Kunert-Keil C., Europ J Orthod submitted

13. Histological changes and changes in the myosin mRNA content of the porcine

masticatory muscles after masseter treatment with botulinum toxin A. Gedrange T., Gredes T., Mai R., Kuhn U.D., Kunert-Keil C., Fanghänel J., Spassov A., Clinical Oral Invest, submitted

14. Comparison of reference points in different methods of temporomandibular joint

imaging Gedrange T., Spassov A., Hietschold V., Gredes T., Fanghänel J., Laniado M., J Visualization, submitted

15. Orthodontic tooth movement into jaw regions treated with synthetic bone substitute

Gedrange T., Mai R., Weingaertner J., Fanghänel J., Allegrini S., Spassov A., Gredes T., Rottner K.,. Proff P., Adv Med Sci, submitted

16. Morphological evaluation of bone defect regeneration after treatment with two

different bone substitution materials on the basis of BONITmatrix® Kunert-Keil C., Gredrange T., Mai R., Spassov A., Franke R., Lucke S., Klinke T., Habersack K., Gredes T., J Physiol Pharmacol., submitted

Vorträge:

1. „Kształtowanie dziąsła przy implantach NobelBiocare“; Internationale Zahnmedizinische Konferenz in der Periodontologie, 18.-20.09.2008, Białystok, Polen

2. „Zastosowanie medycyny regeneracyjnej w stomatologii”; XIV Zahnmedizinische Konferenz Expo-Andre in Toruń, 17-18.10.2008, Polen

3. „Regenerative Zahnmedizin“; Knochenersatzmaterialen in der Kieferorthopädie; Brunico 16-19.03.2009, Italien

4. “Gene expression investigation of the pigs condylar cartilage after anterior mandibular displacement”; 109th American Association of Orthodontists Annual Session, Boston, Massachusetts, 1.-5.05.09, USA

Zitierbare Abstrakts und Kurzbeiträge:

1. Eur J Orthod 2008 30: 1-199 “CEPHALOMETRIC CHARACTERIZATION OF AN

ORTHODONTIC PATIENT SAMPLE” EUROPEAN ORTHODONTIC SOCIETY, 84th Congress 2008, 10–14 June, Lisbon (Portugal)

W A L T H E R - R A T H E N A U - S T R . 4 7 , 1 7 4 8 9 G R E I F S W A L D •

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T E L E F O N 0 3 8 3 4 / 8 6 7 5 4 3 ( d i e n s t l i c h ) E - M A I L : t h o m a s g r e d e s @ y a h o o . d e


2. Eur J Orthod 2008 30: 1-199 “SOFT TISSUE INTEGRATION IN THE NECK AREA

OF IMPLANTS” EUROPEAN ORTHODONTIC SOCIETY, 84th Congress 2008, 10–14 June, Lisbon (Portugal)

3. Eur J Orthod 2009 31; e21; “FIBRE TYPE DISTRIBUTION AND OXIDATIVE

STATE IN MASTICATORY MUSCLES OF MDX-MICE.” 85th EOS Tagung; 10-14.06.2009 Helsinki (Finland)

4. Eur J Orthod 2009 31; e61; “ORTHODONTIC TOOTH MOVEMENT INTO THE

MANDIBULAR ALVEOLUS AFTER TOOTH EXTRACTION.” 85th EOS Tagung; 10-14.06.2009 Helsinki (Finland)

5. Eur J Orthod 2009 31; e63; “CHANGES IN GENE MATERIAL OF THE PIG

MANDIBULAR CONDYLAR CARTILAGE IN RESPONSE TO MANDIBULAR PROTRUSION.” 85th EOS Tagung; 10-14.06.2009 Helsinki (Finland)

6. Eur J Orthod 2009 31; e63; “INFLUENCE OF BONE SUBSTITUTE ON RAT

MUSCLE.” 85th EOS Tagung; 10-14.06.2009 Helsinki (Finland) 7. Eur J Orthod 2009 31; e91; “CAVEOLIN-1, CAVEOLIN-3 AND VASCULAR

ENDOTHELIAL GROWTH FACTOR EXPRESSION IN MASTICATORY MUSCLES OF MDX MICE.” 85th EOS Tagung; 10-14.06.2009 Helsinki (Finland)


E - M A I L : T H O M A S G R E D E S @ Y A H O O . D E

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CHANGES IN INSULIN LIKE GROWTH FACTORS, MYOSTATIN …

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