The purpose of this study was to compare the effects of two types of stem/progenitor cells on the healing of critical sized bone defects in a rat model. Endothelial progenitor cells (EPCs), a novel cell type with previously demonstrated effects on both osteogenesis and angiogenesis, were compared to both a control group (no cells), and a treatment group of mesenchymal stem cells (MSCs). The hypothesis was that EPCs would demonstrate both superior bone healing and angiogenesis, when compared to MSCs and controls. EPCs, MSCs, or a control carrier were placed in surgically stabilized bone defects in a rat femur and both bone formation and angiogenesis were assessed. EPC treated defects demonstrated significantly more bone formation and angiogenesis at the bone defect site than MSC or control treated defects. These results strongly suggest that EPCs are more effective than MSCs for therapeutic osteogenesis and angiogenesis in a bone defect model.

No description available.

Bones have a large intrinsic capacity for repair and regeneration following an injury, however, an estimated 5-10% of nearly 8 million fractures that occur every year in the United States lead to nonunions. The process of bone regeneration is a complex trait that brings together different complements of molecular and cellular interactions to carry out its necessary mechanical functions. These interactions may be attributable to the effects of genetic variations that contribute to differences in bone morphology and fracture healing. This study was undertaken to determine how genetic variability that controls phenotypic qualities of bone affect rates and patterns of fracture healing. Three inbred strains of mice (A/J (AJ), C57BL/6J (B6), and C3H/HeJ (C3)) with known structural and biomechanical differences resulting from fetal bone development were examined. Transverse fractures were generated in the femur and healing traits were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), micro-computed tomography (μCT), biomechanical torsional testing, and cartilage contrast-enhanced micro-computed tomography (CECT). The temporal analysis of gene expression revealed that B6 had the longest duration of chondrocyte maturation and the greatest relative expression of osteogenic genes relative to either C3 or AJ. While AJ and C3 exhibited similar patterns of chondrogenesis, AJ initiated osteogenesis earlier than C3. These results suggest that compared to either AJ or B6, the C3 strain exhibited the least temporal coordination between the chondrogenic and osteogenic stages. Consistent with the relative patterns of RNA expression, μCT evaluations at day 21 post fracture showed that B6 had higher callus mineralization than AJ and C3. μCT, cartilage CECT, and biomechanical testing revealed less tissue mineralization and more cartilage near the fracture gap, which indicated a less developed bony bridge in C3 calluses at day 21 post fracture. The lack of large amounts of cartilage in calluses of all strains by day 21 indicated that all strains had initiated osteogenesis by this time. Taken together, these results showed that mice with different genetic backgrounds express different patterns of mobilization and renewal of skeletal stem cells with differing temporal progressions of chondrogenic and osteogenic differentiation. These data further show that these variations affect the phenotypic properties of fracture calluses during the process of fracture healing.

Indiana University-Purdue University Indianapolis (IUPUI) / Fracture repair is costly and difficult to treat. One of the main causations of nonunion is a lack of essential blood supply. The needed blood is supplied by the growth of new blood vessels, a process known as angiogenesis, that invade the damaged tissue early in the healing process. We proposed using bone tissue engineering as an effective therapy. This therapy uses stem cells to aid in tissue regeneration. Endothelial progenitor cells (EPCs) were selected due to their ability to form tube-like networks in vitro. EPCs were isolated from murine bone marrow and lung tissue. We tested EPC’s tube forming, proliferative, and wound migration ability in vitro. To test their ability in vivo we created a femoral fracture in young and old mice. EPCs were seeded to the fracture site upon a collagen scaffold. The in vitro studies displayed that the bone marrow and lung-derived endothelial cells presented EPC traits. In the mouse fracture model bone marrow, endothelial cells did not significantly improve the healing process. In the future, we want to improve our cell extraction and purification method, as well as test a new stem cell delivery biomaterial. We also want to select and use a growth factor (GF) that can help to promote bone regeneration in tandem with the EPCs.

Atrophic non-union is a major complication following fracture of a bone. It represents a biological failure of the fracture healing process and occurs in 5-10% of cases. A number of factors predispose to atrophic non-union including high energy injuries, open fractures, diabetes, and smoking. Atrophic non-unions cause immense patient morbidity and consume large amount of health care resources. Bone grafts taken from the iliac crest contain biologic components required for fracture healing and are considered as the gold standard treatment of aseptic atrophic non-union. However, harvesting bone grafts from the iliac crest is associated with significant patient morbidity which can reduce quality of life. Mesenchymal stem cells (MSCs) have the ability to proliferate and undergo multilineage differentiation. The emergence of MSC therapy provides an alternative strategy for treating impaired fracture healing. MSCs contribute to normal fracture healing both directly as bone progenitor cells and indirectly as mediator secreting cells. Although a number of studies have shown that MSCs can promote bone regeneration in small animal fresh critical size defects, this is not analogous to most clinical aseptic atrophic non-unions which do not have a significant bone gap. There remains therefore a clinical need for an appropriate strategy for using stem cells in atrophic non-unions. Thus, the aim of this study aim was to develop a clinically relevant strategy to promote fracture healing in an atrophic non-union model using the percutaneous injection of MSCs as a minimally invasive technique. An atrophic non-union model was established and validated. A small (1 mm) non-critical size defect was created at the mid shaft tibia and the fracture site was stabilised using an external fixator. Atrophic non-union was induced by stripping the periosteum for one bone diameter either side of the osteotomy site and curettage of the intramedullary canal over the same distance. The procedure reliably created an atrophic non-union. Fracture healing was evaluated using (1) serial radiography, (2) micro-computed tomography, (3) histomorphology and (5) biomechanical testing. Fracture scoring systems including the radiographic union scale in tibia (RUST) and the Lane & Sandhu score were validated in a preclinical model. A simple sample preparation technique for evaluating bone mechanical properties was developed and used to assess the stiffness and strength of the fracture repair. Percutaneous injection of MSCs locally into the fracture site in the early ‘post-injury’ period at three weeks after induction of atrophic non-union was found to improve the fracture healing process significantly (83% of cases), while MSCs implantation in the late ‘post-injury’ period at eight weeks after induction of atrophic non-union showed no significant improvement of fracture healing (20% of cases). Percutaneous local implantation of MSCs rescued the fracture healing process in cases destined to progress to atrophic non-union. In clinical practice, there may be an advantage using MSCs from a universal donor as the processes of MSC isolation and preparation are expensive and time consuming. To investigate the feasibility of using non-autologous cells, the atrophic non-union was used to determine the bone regenerative potential of using xenogeneic donor hMSCs in an atrophic non-union. The results demonstrated that the therapeutic effect of using hMSCs in a xenogeneic manner to promote fracture healing in the rat atrophic non-union model was comparable with rMSCs (88% of cases in both hMSCs and rMSCs) and there were neither significant clinical adverse effects nor adverse immune responses with the xenogeneic transplantation. However, MSCs did not persist at the fracture following injection. Perivascular stem cells (PSCs) taken from adipose tissue, which is an expendable source, have advantages over conventional MSCs as they are a defined and homogenous population and can be used without culture expansion. The administration of PSC using percutaneous injection improved the fracture healing process in atrophic non-union (60% of cases). This suggested that PSCs may present an appropriate choice for use in cell therapies to promote fracture healing in atrophic non-union. The results from this thesis can be applied to the development of a clinically relevant strategy using MSCs as a minimally invasive technique to promote fracture healing in atrophic non-union, in particular (1) the effectiveness of a cell therapy is likely to be highly dependent of the timing of injection relative to the stage of fracture healing, (2) hMSCs were as effective as rMSCs in promoting fracture healing, suggesting that it may be feasible to use an allogeneic strategy in humans, (3) the injected MSCs were not detectable even in case of successful repair, suggesting that they may act through a paracrine effect and (4) PSCs isolated from adipose tissue contributed to fracture healing in the atrophic non-union model, suggesting that adipose tissues can be used as an alternative cell sources for bone repair.

Η διαδικασία πώρωσης καταγμάτων των οστών συμπεριλαμβάνει την ενεργοποίηση και αλληλεπίδραση διαφόρων κυττάρων, που ρυθμίζονται από βιοχημικά και μηχανικά σήματα. Στην παρούσα διπλωματική εργασία μελετάται το μαθηματικό μοντέλο πώρωσης καταγμάτων οστών, συμπεριλαμβανομένου μόνο των βιοχημικών ερεθισμάτων. Το μοντέλο αυτό, που αναπτύχθηκε αρχικά από τους Geris L. et al. (2008), περιλαμβάνει μία σειρά μερικών μη γραμμικών διαφορικών εξισώσεων που περιγράφουν την χωροχρονική εξέλιξη των συγκεντρώσεων και των πυκνοτήτων των κυτταρικών τύπων, των τύπων εξωκυττάριας θεμέλιας ουσίας και των αυξητικών παραγόντων που συμμετέχουν στη διαδικασία πώρωσης. Η προσομοίωση του μαθηματικού μοντέλου πώρωσης οστών έγινε μέσω υπολογιστικού κώδικα πεπερασμένων όγκων στο Matlab. Ιδιαίτερη έμφαση δίνεται στην διαδικασία της αγγειογένεσης που λαμβάνει χώρα κατά την πώρωση των καταγμάτων και αποτελεί σημαντικό παράγοντα για την αποκατάσταση των οστών και την πλήρη επαναφορά τους στην αρχική κατάσταση. Για την καλύτερη κατανόηση των διαδικασιών αγγειογένεσης, εκτός από την μελέτη του μαθηματικού μοντέλου της πώρωσης των οστών, πραγματοποιήθηκε μελέτη των βιολογικών διαδικασιών επούλωσης δερματικής πληγής και προσομοίωση με υπολογιστικό κώδικα στο Matlab του απλοποιημένου μαθηματικού μοντέλου της αγγειογένεσης στην επούλωση δερματικών πληγών. / The process of fracture healing involves the action and interaction of many cells, regulated by biochemical and mechanical signals. This postgraduate dissertation studies a mathematical bone fracture healing model for the case of normal fracture healing including only the biochemical factors (a bioregulatory model). The mathematical model, which was originally established by Geris L. et al. (2008), consists of a system of nonlinear partial differential equations describing the spatiotemporal evolution of concentrations and densities of the cell types, extracellular matrix types and growth factors indispensable to the healing process. The simulation of mathematical model was held by a computational finite volume code in Matlab. Particular emphasis is given to the process of angiogenesis, which occurs during fracture healing and is a key factor for bone repair and restore of the original state. For a better understanding of angiogenesis processes, a study in biological processes for dermal wound healing was held and a simulation of a simplified mathematical model of angiogenesis in healing dermal wounds by a computational code in Matlab.

by Yeung Hiu Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 158-171). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT (English & Chinese) --- p.iii / TABLE OF CONTENT --- p.viii / INDEX FOR FIGURES --- p.xii / INDEX FOR TABLES --- p.xvi / Chapter 1. --- INTRODUCTION --- p.2 / Chapter 1.1. --- History of Distraction Osteogenesis --- p.3 / Chapter 1.2. --- Clinical Application of Distraction Osteogenesis --- p.5 / Chapter 1.2.1. --- Limb-Lengthening --- p.5 / Chapter 1.2.2. --- Correction of Deformities and Non-Unions --- p.5 / Chapter 1.2.3. --- Bone Transport --- p.6 / Chapter 1.2.4. --- Reconstruction of the mandible --- p.7 / Chapter 1.3. --- Bone-specific Alkaline Phosphatase (BALP) --- p.8 / Chapter 1.4. --- Osteocalcin --- p.9 / Chapter 1.5. --- Bone Growth Factors --- p.11 / Chapter 1.6. --- Fibroblast Growth Factors (FGFs) --- p.12 / Chapter 1.6.1. --- Acidic Fibroblast Growth Factor (aFGF) --- p.13 / Chapter 1.6.2. --- Basic Fibroblast Growth Factor (bFGF) --- p.14 / Chapter 1.7. --- Transforming Growth Factor-pi (TGF-β1) --- p.16 / Chapter 1.8. --- Fracture Healing --- p.18 / Chapter 1.8.1. --- Histology --- p.18 / Chapter 1.8.2. --- Growth Factor Expression --- p.18 / Chapter 1.9. --- Distraction Osteogenesis --- p.19 / Chapter 1.9.1. --- Histology --- p.19 / Chapter 1.9.2. --- Growth Factor Expression --- p.20 / Chapter 1.10. --- Aim of the Study --- p.21 / Chapter 2. --- METHODOLOGY --- p.23 / Chapter 2.1. --- Animal Model --- p.23 / Chapter 2.1.1. --- Source of Animal --- p.23 / Chapter 2.1.2. --- Animal Operation --- p.23 / Chapter 2.1.3. --- Fracture Healing Model --- p.24 / Chapter 2.1.4. --- Distraction Osteogenesis Model --- p.24 / Chapter 2.2. --- Sample Collection --- p.25 / Chapter 2.2.1. --- Tissue Sample Collection and Preparation --- p.25 / Chapter 2.2.1.1. --- Test for the Complete Decalcification of the Calluses --- p.26 / Chapter 2.2.2. --- Blood Sample Collection and Storage --- p.26 / Chapter 2.3. --- Bone Mineral Density Measurement of the Distracted Callus and the Fracture Callus --- p.27 / Chapter 2.3.1. --- Fracture Healing Group --- p.27 / Chapter 2.3.2. --- Distraction Osteogenesis Group --- p.28 / Chapter 2.4. --- Serum Bone Specific Alkaline Phosphatase (BALP) Activity --- p.28 / Chapter 2.4.1. --- Wheat Germ Lectin (WGL) Precipitation of BALP --- p.28 / Chapter 2.4.1.1. --- Reagent --- p.28 / Chapter 2.4.1.2. --- Preparation and Measurement of Samples --- p.29 / Chapter 2.4.1.3. --- Auto-analyzer Setup --- p.30 / Chapter 2.5. --- Quantification of the Osteocalcin in Serum --- p.30 / Chapter 2.5.1. --- Reagent and Sample Preparation --- p.31 / Chapter 2.5.2. --- Detection Procedures --- p.31 / Chapter 2.6. --- Localization of the Growth Factors in Distraction Osteogenesis and Fracture Healing --- p.32 / Chapter 2.6.1. --- Immunohistochemistry of the Growth Factors --- p.33 / Chapter 2.6.1.1. --- Reagents and Solution Preparation --- p.33 / Chapter 2.6.1.2. --- Experimental Procedure --- p.36 / Chapter 2.6.1.3. --- Evaluation of Immunohistochmical Staining Results --- p.37 / Chapter 2.6.2. --- Verification of the Primary Antibody Used in the Study --- p.37 / Chapter 2.6.2.1. --- Tissue Preparation --- p.37 / Chapter 2.6.2.2. --- Antibody to Acidic Fibroblast Growth Factor (aFGF) --- p.38 / Chapter 2.6.2.2.1. --- Immunohistochemistry of Goat Brain and Growth Plate --- p.38 / Chapter 2.6.2.2.2. --- Dot Blot --- p.38 / Chapter 2.6.2.2.2.1. --- Materials and Reagents --- p.38 / Chapter 2.6.2.2.2.2. --- Procedures --- p.39 / Chapter 2.6.2.2.3. --- Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.41 / Chapter 2.6.2.2.3.1. --- Materials and Reagents --- p.41 / Chapter 2.6.2.2.3.2. --- Procedures --- p.42 / Chapter 2.6.2.2.4. --- Western Blotting --- p.43 / Chapter 2.6.2.2.4.1. --- Materials and Reagents --- p.43 / Chapter 2.6.2.2.4.2. --- Procedures --- p.44 / Chapter 2.6.2.3. --- Antibody to Basic Fibroblast Growth Factor --- p.45 / Chapter 2.6.2.4. --- Antibody to Transforming Growth Factor-β1 --- p.45 / Chapter 3. --- RESULTS --- p.53 / Chapter 3.1. --- Animal Model --- p.53 / Chapter 3.1.1. --- Fracture Healing Animal Model --- p.53 / Chapter 3.1.1.1. --- Radiography of Fracture Healing --- p.53 / Chapter 3.1.2. --- Distraction Osteogenesis Animal Model --- p.54 / Chapter 3.1.2.1. --- Gross Morphology of Distraction Osteogenesis --- p.54 / Chapter 3.1.2.2. --- Radiography of Distraction Osteogenesis --- p.55 / Chapter 3.2. --- Bone Mineral Density (BMD) Measurement --- p.56 / Chapter 3.2.1. --- In Fracture Healing --- p.56 / Chapter 3.2.2. --- Distraction Osteogenesis --- p.57 / Chapter 3.3. --- Bone-specific Alkaline Phosphatase Activity in Goat Serum --- p.59 / Chapter 3.3.1 --- ", Fracture Healing" --- p.59 / Chapter 3.3.2. --- Distraction Osteogenesis --- p.59 / Chapter 3.4. --- Serum Osteocalcin Measurement --- p.60 / Chapter 3.4.1. --- Fracture Healing --- p.60 / Chapter 3.4.2. --- Distraction Osteogenesis --- p.60 / Chapter 3.5. --- Histology --- p.61 / Chapter 3.5.1. --- Fracture Healing --- p.61 / Chapter 3.5.2. --- Distraction Osteogenesis --- p.64 / Chapter 3.6. --- Verification of Primary Antibody Used in the Study --- p.67 / Chapter 3.6.1. --- Antibody to Acidic Fibroblast Growth Factor --- p.67 / Chapter 3.6.1.1. --- Dot Blot --- p.67 / Chapter 3.6.1.2. --- Western Blotting --- p.68 / Chapter 3.6.1.3. --- Immunohistochemistry of Goat Brain and Growth Plate --- p.68 / Chapter 3.6.2. --- Antibody to Basic Fibroblast Growth Factor --- p.69 / Chapter 3.6.2.1. --- Dot Blot --- p.69 / Chapter 3.6.2.2. --- Immunohistochemistry of Goat Brain and Growth Plate --- p.69 / Chapter 3.6.3. --- Antibody to Transforming Growth Factor-β1 --- p.70 / Chapter 3.6.3.1. --- Western Blotting --- p.70 / Chapter 3.6.3.2. --- Immunohistochemistry of Growth Plate --- p.70 / Chapter 3.7. --- Localization of Growth Factors in Fracture Healing and Distraction Osteogenesis --- p.70 / Chapter 3.7.1. --- Acidic Fibroblast Growth Factor --- p.71 / Chapter 3.7.1.1. --- Fracture Healing --- p.71 / Chapter 3.7.1.2. --- Distraction Osteogenesis --- p.72 / Chapter 3.7.2. --- Basic Fibroblast Growth Factor --- p.73 / Chapter 3.7.2.1. --- Fracture Healing --- p.73 / Chapter 3.7.2.2. --- Distraction Osteogenesis --- p.74 / Chapter 3.7.3. --- Transforming Growth Factor-β1 --- p.75 / Chapter 3.7.3.1. --- Fracture Healing --- p.75 / Chapter 3.7.3.2. --- Distraction Osteogenesis --- p.76 / Chapter 4. --- DISCUSSION --- p.142 / Chapter 4.1. --- The Biochemical Events in Fracture Healing --- p.142 / Chapter 4.2. --- The Biochemical Events in Distraction Osteogenesis --- p.147 / Chapter 4.3. --- Limitations of the present study --- p.153 / Chapter 4.4. --- Future Study --- p.154 / Chapter 5. --- CONCLUSION --- p.156 / BIBLIOGRAPHY --- p.158

Understanding the recruitment of multipotent skeletal progenitor cells and the factors that influence their differentiation would be helpful in providing a means for harnessing the regenerative capacity of skeletal progenitor cells in bone tissue engineering. In order to track the recruitment of skeletal stem cells in fracture healing, transgenic mice containing a Tamoxifen-inducible Cre recombinase that had been placed under the control of a 2.4 kb Prx1 promotor were used to induce conditional expression in periosteal skeletal stem cells that express the Prx1 gene. In order to initially see the cells expressing Prx1, a green fluorescent protein gene (GFP) had also been put downstream to the Prx1 promotor. We then crossed these Prx1CreER-GFP transgenic mice with a second strain containing the Beta-galactosidase gene that becomes constitutively expressed after recombination by the Cre recombinase. The enzymatic activity of Beta-galactosidase was then used to generate a colormetric staining reaction that was used to visualize the cells in which recombination had occurred based on a blue staining product. The recombination activity should only be present in Prx1 expressing cells and their progeny. The goal of the present study was to assess several different approaches to optimize the Beta-galactosidase enzymatic staining protocol and to visualize the Prx1-expressing cells during fracture healing. These studies further examined those populations of cells in the fracture calluses that became labeled and arose from the stem cell populations that had expressed Prx1 at post-operative day 7 and 14. The optimization of a staining method for histology will allow this study to track Prx1 cell fates in a fracture model both in response to specific drug treatments, mechanical loading of the fracture during healing and under pathological conditions that effect healing.

The objective of this project was to evaluate the effects of local endothelial progenitor cell (EPC) therapy on bone regeneration in a segmental defect in the rat femur. Animals from the EPC-treated (N=28) and control (N=28) groups were sacrificed at 1, 2, 3, and 10 weeks post-operatively. Bone healing was evaluated with radiographic, histological, and micro computed tomography (micro-CT) scans. Radiographically; mean scores of the EPC group at 1, 2, and 3 weeks were significantly higher compared to control group. At 10 weeks, all the animals in the EPC group had complete union (7/7), but in the control group none achieved union (0/7). Histologically, specimens from EPC-treated animals had abundant new bone formation compared to controls. Micro-CT assessment showed significantly improved parameters of bone healing for the EPC group compared to control group. In conclusion, local EPC therapy significantly enhanced bone regeneration in a segmental bone defect in rat femur.

The objective of this project was to evaluate the effects of local endothelial progenitor cell (EPC) therapy on bone regeneration in a segmental defect in the rat femur. Animals from the EPC-treated (N=28) and control (N=28) groups were sacrificed at 1, 2, 3, and 10 weeks post-operatively. Bone healing was evaluated with radiographic, histological, and micro computed tomography (micro-CT) scans. Radiographically; mean scores of the EPC group at 1, 2, and 3 weeks were significantly higher compared to control group. At 10 weeks, all the animals in the EPC group had complete union (7/7), but in the control group none achieved union (0/7). Histologically, specimens from EPC-treated animals had abundant new bone formation compared to controls. Micro-CT assessment showed significantly improved parameters of bone healing for the EPC group compared to control group. In conclusion, local EPC therapy significantly enhanced bone regeneration in a segmental bone defect in rat femur.