Eidesstattliche Erklärung / Affirmation - Ernst-Moritz-Arndt ...

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

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

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 

6

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 

7

— 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 

TGF-β family 

BMPs 

VEGF 

PDGF 

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 

Stimulate the production and deposition of ECM proteins 

Potent inducers of committed bone cell replication and osteoblast 

matrix production 

Autocrine and paracrine action mediated by their kinase receptors 

Induce early precursor bone cell replication and osteoblast 

commitment 

Regulates vascularization of developing bone and osteoclast 

activity, involved in bone repair 

Key component of a chondrocyte survival pathway, controls 

osteoblastic activity 

Potent mitogen and chemoattractant for target cells such as diploid 

fibroblasts and osteoblasts 

(Tang et al. 

2006; Bikle 

2008) 

(Ahdjoudj et al. 

2002; Ito and 

Miyazono 2003) 

(Duncan 1995; 

Zhao et al. 2000) 

(Zelzer and 

Olsen 2005; 

Papadopoulou et 

al. 2007) 

(Kim et al. 2007) 

8

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 

9

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). 

10

1.3. Bone surrogates 

Bone Formation 

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

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 

11

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). 

absorbable 

synthesized 

materials 

nonabsorbable 

Biomaterials 

cellular / acellular 

naturally derived 

materials 

absorbable nonabsorbable 

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 

12

epair 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 

13

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 

14

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). 

15

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 longtime 

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 

16

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. 

. 

17

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. 

18

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. 

19

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. 

21

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. 

22

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 singlestranded 

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. 

23

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, 7 th 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 

24

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

40x 

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). 

RT-PCR RT-PCR with SYBR ® -Green Dye 

1. Double-stranded 

DNA denaturation at 

2. Primer annealing at 

60°C 

3. DNA elongation at 

72°C 

New target sequences; 

Replication (step 1 to 3). 

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 -ΔΔC T 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, 

Thus, 

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. 

XT = X0 × (1 + Ex) C T,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) C T,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) C T,X - C T,R = K 

or XN × (1 + E) ΔC T = 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) -ΔC T 

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) -ΔC T,q / K × (1 + E) -ΔC T, 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 Mg 2+ 

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 

2.7. Statistical analysis 

amount of target = 2 -ΔΔC T . 

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). 

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. 

28 S 

18 S 

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- 

marker 

125 bp 

75 bp 

β-actin IGF1 IGF2 VEGF GDF8 

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

IGF1/beta-actin 

30 

25 

20 

15 

10 

5 

0 

IGF1 

* 

1 

control 6 w eeks 12 w eeks 

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 

(p0.05). The IGF2 mRNA amount 

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

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

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

expression (p

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 posttraumatic 

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

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. Ageassociated 

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

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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-3hydroxybutyrate 

(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

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

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

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. 

107,8g Tris 

55,0g boric acid 

5,8g EDTA 

pH 8,0; add 1000 ml aqua dest. 

TBE buffer Loading buffer for agarose gels 

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

PERSÖNLICHE ANGABEN 

AUSBILDUNG 

DR. TOMASZ JAKUB GREDES 

Familienstand: ledig 

Staatsangehörigkeit: Polen 

Nationalität: polnisch 

Geburtsdatum: 20. August 1975 

Geburtsort: Dzierżoniów (Polen) 

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 Kiefergelenkknorpel 

des Schweins (Sus scrofa 

domesticus) nach Vorverlagerung des 

Unterkiefers 

Seit 2007 Ausbildung zum Fachzahnarzt in der 

Kieferorthopädie- EMAU Greifswald 

WALTHER-RATHENAU-STR.47, 17489 GREIFSWALD• 

TELEFON 03834/867543 (dienstlich) 

E-MAIL: thomasgredes@yahoo.de 

60

BERUFSERFAHRUNG 


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 

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. 




61

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


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 




63


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) 


64




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-MAIL: THOMASGREDES@YAHOO.DE 

65

Eidesstattliche Erklärung / Affirmation - Ernst-Moritz-Arndt ...

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