Preparation and characterization of a hydrogel containing anti-VEGF (bevacizumab) liposomes to prevent bone tissue repair at injured growth plates.

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Table of contents

ACKNOWLEDGEMENTi

DECLARATIONii

List of figuresvi

Abbreviationsvi

Summary1

CHAPTER I INTRODUCTION3

1.1 Bone Growth and Function and Arrangement of the Growth Plate3

1.1.1The Resting zone4

1.1.2The Differentiation and Proliferative zone4

1.1.3The Hypertrophic and Provisional Calcification Zone5

1.2Growth Plate Injuries, Injury Responses and Repair Mechanism5

1.2.1Growth Plate Injuries, Their Classifications and Effects on Bone Growth5

1.2.2Injury Responses after a Growth Plate Fracture8

1.2.2.1Inflammatory Phase9

1.2.2.2Fibrogenic Phase10

1.2.2.3Osteogenic and Maturation Phases11

1.3Mechanisms of Bony Repair of Injured Cartilage of Growth Plate12

1.4Current Surgical Treatments for Injured Growth Plate and Its Limitation13

1.5Angiogenesis in Fracture Healing15

1.6Vascular Endothelial Growth Factor Neutralizing Antibody17

1.7In situ-Forming Hydrogels18

1.7.1Chitosan/ß-Glycerophosphate Thermosensitive Hydrogel19

1.7.1.1Effects of Chitosan on Cartilage Tissue Repair20

1.7.2Bevacizumab Encapsulated Liposome dispersed in Chitosan / ß-Glycerophosphate Thermosensitive Hydrogel21

chapter 2. AIMS AND HYPOTHESES22

Chapter 3 MATERIALS AND METHODS 23

3.1Chitosan Based Thermosensitive Hydrogel23

3.1.1Thermosensitive Solutions Preparation24

3.1.2Determination of Gelation Time:24

3.2Rheology25

3.3Preparation of Bevacizumab-Encapsulated Liposomes25

3.3.1Determination of Encapsulation Efficiency26

3.4Incorporation of Bevacizumab-Encapsulated-Liposomes into a Hydrogel (liposomal gel) and In vitro Release Analysis:27

3.5Enzyme-Linked Immunosorbent Assay27

3.6In Rats a Drill-Hole Growth Plate Injury Model28

3.6.1Animal Trial Design28

3.6.2Surgical Procedure29

3.6.3Specimen Collection29

3.6.3.1Blood Collection29

3.6.3.2Tissue Collection29

3.7Bone Histology30

3.7.1Bone Preparation30

3.7.2Micro-Computed Tomography Scans (µ-CT) and Bone Parameters Analysis31

3.7.3Histomorphological and Immunohistochemcial Analysis of Tibial:31

3.8Total RNA Isolation and Real-Time Quantitative Polymerase Chain Reaction:32

3.8.1RNA Extraction and cDNA Synthesis33

3.8.2Analysis of the expression of genes by Quantitative Real Time Polymerase Chain Reaction (RT-PCR)33

3.9Statistical Analysis34

Chatper 4 RESULTS35

4.1Characteristics of Chitosan/ß-Gp Hydrogels35

4.2Characteristics of Liposome37

References40

List of figures

Figure 1: Structure and function of different histologic zones in the growth plate9

Figure 2: Salter-Harris Classification System11

Figure 4: Picture33

Figure 5: Characteristics of Chitosan/ß-Gp Hydrogels (n=3)34

Figure 6: Viscosities of chitosan/ß-Gp hydrogels (chitosan: 6% (w/v), ß-Gp Salt: 10% (w/v), and chitosan: 10% (w/v), ß-Gp salt: 10% (w/v)) as a function of time at 37°C.35

Abbreviations

ANOVA

one-way analysis of variance

SEM

Standard error of mean

MSCs

Mesenchymal stem cells

OCN

Osteocalcin

Runx2

Runt-related transcription factor-2

TNF-a

Tumour necrosis factor-alpha

Summary

For the bone healing, bone development, and establishment, and growth of bone tumours, Angiogenesis is considered to be an enormous supplier. Growth plates are mainly situated at the end of all long bones, responsible for the lengthening of a child's skeleton via a tightly regulated process called endochondral ossification. The bone growth and function of the growth plate comprises of three zones, the resting zone, the differentiation, and proliferative zone and provisional and hypertrophic calcification zone. The bone tumours, impaired fracture healing therapy and skeletal defects ...