No description available.

Mesenchymal stem cell (MSC) differentiation is known to be influenced by a range of environmental stimuli. MSC-based bone regeneration strategies would benefit from the identification of scaffold material properties which intrinsically promote osteoblast lineage progression. The aim of this work was to contribute to the understanding of elucidating cause-effect relationships between extracellular matrix (ECM) signaling and osteogenic MSC differentiation using poly(ethylene glycol) diacrylate (PEGDA) hydrogels as a material platform. First, the effect of several ECM proteins associated with bone morphogenesis or bone fracture healing on MSC osteogenesis was investigated. Second, collagen-mimetic proteins (Scl2) were modified in order to incorporate them in a 3D network, and cell adhesion and activation of cell signaling were evaluated, as well. Finally, the influence of integrin α1 and α2 binding on human MSC (hMSC) osteogenesis was investigated toward the goal of deconvoluting the impact of integrin-based interactions on associated cell behavior. In terms of the osteoinductivity of select ECM components, the results showed that both FG and LN enhanced the osteogenic response of encapsulated MSC cells. In addition, the integrin-based interactions supported by these ECM components indicated that integrin α2 and α6 appeared to play an important role in MSC osteogenesis. Regarding Scl2 protein studies, Scl2-1, Scl2-2, and Scl2-3 were functionalized with photocrosslinking sites to enable incorporation into a 3D hydrogel matrix. characterization studies confirmed that the functionalization of the Scl2 proteins did not disrupt triple helix conformation, integrin binding, or cell adhesion. Also, initial cell studies confirmed specific hMSC adhesion to Scl2 proteins and appropriate activation of different MAP kinase pathways. Finally, Scl2 proteins were conjugated into PEGDA hydrogels and their effect on hMSC osteogenesis was evaluated. The results indicated that both PEG-Scl2-2 and PEG-Scl2-3 were osteoinductive, but in different ways. Therefore, to gain insight into the origins of the observed osteogenic responses, the influence of p38 pathway in osteogenesis of hMSC was investigated in order to establish its potential causative relationship with Scl2 proteins. The results of the p38 inhibition studies suggested p38 pathway may regulate osteogenesis in hMSCs. Further research is needed for investigation of detailed mechanism of osteogenesis regulation.

Stuttgart, Univ., Diss., 2008.

Thesis (doctoral)--Ludwig Maximilians-Universität zu München, 1979.

Thesis (M.S.)--University of Alabama in Birmingham, School of Dentistry.

Zugl.: Diplomarbeit.

No description available.

In spite of vigorous research efforts to date the induction of bone formation by macroporous coral-derived constructs when implanted heterotopically in the rectus abdominis muscle of the non-human primate Chacma baboon Papio ursinus has not yet been resolved and needs to be assigned. More importantly, the apparent redundancy of molecular signals singly initiating the induction of bone formation in primate species and the heterotopic induction of endochondral bone formation by the mammalian recombinant human transforming growth factor –β3 (rhTGF-β3) isoform have not yet been assigned and need to be mechanistically resolved. Using the rectus abdominis muscle of Papio ursinus the study sought to molecularly determine how coral-derived macroporous constructs and doses of the hTGF-β3 isoform initiate the induction of bone formation. To elucidate the function of osteoclastogenesis and Ca2+, biomimetic coral-derived 7% hydroxyapatite/calcium carbonate (7% HA/CC) devices were supplemented either with 240 μg zoledronate bisphosphonate, an osteoclast binding antagonist, or 500 μg of the calcium channel blocker verapamil hydrochloride. Additionally but in separate coralderived bioreactors, 125 μg rhTGF-β3 and/or 125 μg hNoggin were added to answer the question of how TGF-β3 induces bone formation. All devices were then subsequently implanted within heterotopic sites of the rectus abdominis muscle of 6 Papio ursinus and left in vivo for 15, 60 and 90 days. Harvested specimens were subjected to histomorphometrical and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Collagen Type IV expression supported by extensive vascularisation was detected and observed respectively in all implants after 15 days in vivo. Importantly the zoledronate treated specimens possessed delayed tissue patterning and morphogenesis,

Several members of the bone morphogenetic protein/osteogenic protein (BMP/OP) and transforming growth factor beta (TGF-B) families are molecular regulators o f cartilage and bone regeneration. However, their precise mode o f signal transduction and combined interactions are poorly understood. The presence of several molecular forms o f these growth factors suggests multiple functions in vivo as well as synergistic interactions during both embryonic bone development and regeneration o f cartilage and bone in postnatal life. A functional bioassay for identification of osteogenic proteins within the bone matrix has been established. The heterotopic bioassay in rats, together with the improved purification methods, has led to the purification o f native BMPs. In rats, heterotopic and orthotopic implantation o f TGF- 13 singly fails to initiate new bone formation whereas implantation o f BMPs/OPs elicit the local differentiation o f new bone, at both sites. This study presents data which shows that co-administration of TGF-B1 with OP-1 delivered by a collagenous carrier, and implanted in the subcutaneous site o f rats results in synergistic induction o f bone formation. Changes in hist-logical, biochemical and molecular response o f the effects o f the morphogens were vu bed over 7, 12 and 21 days post implantation. Induced cartilage and bone were analyzed by alkaline phosphatase activity, calcium content, Northern blots and histological examination of subcutaneous implants in the rats. Single applications o f TGF-B1 (0.01pg, 0.03pg and O.lpg) on days 7, 12 and 21 gave rise to negligible alkaline phosphatase activity (0.03-0.09 U/ mg protein) and calcium content (0.05-0.6 pg/mg tissue). Histological examination showed that all TGF-B1 implants did not exhibit any sign o f bone formation. On day 7, OP-1 implants (O .lpg and 0.3 pg) elicited negligible alkaline phosphatase activity and calcium content. However, higher doses o f OP-1 (1 pg and 3 pg) elicited alkaline phosphatase activity o f 0.1 U/mg protein and 0.2 U/mg protein. Combination TGF-Bl (O.Olpg, 0.0.3pg and O.lpg) with OP-1 (O.lpg, 0.3pg, Ip g and 3pg) slightly increased the activity o f alkaline phosphatase activity (0.2 U/mg protein-0.4 U/mg protein). OP-1 singly gave rise to very low calcium levels o f 0.4 pg/mg tissue to 0.5 pg/mg tissue. Addition o f TGF-Bl to OP-1 resulted in calcium content rising from 0.2 to 1 pg/mg tissue. Histologically, the specimens o f single OP-1 applications did not show any sign o f bone formation. On addition o f TGF-Bl to OP-1 the specimens showed signs o f the beginning o f chondroblastic differentiation. On day 12 alkaline phosphatase activity elicited by single applications o f OP-1 ranged from 0.1 U/mg protein to 1 U/mg protein. Addition of l i , : 7- Bl increased the alkaline phosphatase from 0.8 U/mg protein to 7 U/mg protein. Calcium levels resulting from single applications of OP-1 ranged from 0.1 to 15 pg/mg tissue Auer addition of TGF-Bl to OP-1 calcium levels rose from 5 to 20 pg/mg tissue. Histological analysis showed formation o f cartilage in specimens both of OP-1 solo and OP-1 in combination with TGF-B1. On day 21 alkaline phosphatase activity was reduced to a range o f 0.1-0.5 U/mg protein upon single applications o f OP-1. Addition TGF-131 resulted in a further decrease in alkaline phosphatase activity. Calcium levels were 10-68 jag/mg tissue on single applications o f OP-1. Addition o f TGF-131 to OP-1 increased the calcium levels in the range o f 2-70 gg/mg tissue. Histological examination o f the 3 jig OP-1 solo specimens showed complete chondrolysis whereas the OP-1 (3|ig)/ TGF-B1 (0.03 and 0.1 gg) specimens showed the differentiation o f bone marrow. Tissues generated in the rat subcutaneous space at 7, 12 and 21 days post implantation elicited mKNA expression o f OP-1, BMP-3 and TGF-B1. These results indicate that at least in part, the matrix-induced endochondral bone formation involves the expression o f some members o f the TGF-B superfamily. Type II collagen (chondrogenesis marker) and type IV collagen (angiogenesis marker) mRNAs were also detected on days 12 and 21, respectively. The present data suggests that TGF-B up regulates the temporal activity o f OP-1 to induce bone formation. Co-administiation of TGF-B 1 to OP-1 caused an increase in the alkaline phosphatase activity and calcium content, markers o f bone formation, which implies that when TGF-B is mixed with OP-1, the cascade o f bone formation is accelerated. These results may have important therapeutic implications. The rapidity o f tissue morphogenesis with bone marrow formation is important for regeneration o f bone in older patients where repair phenomena are temporally delayed and the healing process is slower than in younger patients. The expression of multiple members o f the TGF-13 superfamily indicates that the cascade of bone formation incorporates some members o f the TGF-B superfamily and this may form the basis for synergistic molecular therapeutics for cartilage and bone regeneration in clinical contexts.

by Lee Kwong Man, Simon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 88-94. / ABSTRACT --- p.I / ACKNOWLEDGEMENTS --- p.IV / TABLE OF CONTENTS --- p.V / Chapter CHAPTER ONE - --- INTRODUCTION --- p.1 / Chapter CHAPTER TWO - --- STRUCTURE OF CARTILAGE / Chapter 2.1 --- Characteristics of Cartilage --- p.4 / Chapter 2.2 --- Types of Cartilage --- p.4 / Chapter 2.3 --- Matrix --- p.5 / Chapter 2.3.1 --- Collagen --- p.5 / Chapter 2.3.2 --- Proteoglycan --- p.7 / Chapter 2.4 --- Diffusion of Solultes in Cartilage --- p.9 / Chapter 2.5 --- Chondroytes --- p.10 / Chapter CHAPTER THREE - --- PHYSIOLOGICAL CHANGES WITHIN CARTILAGE / Chapter 3.1 --- Endochondral Ossification --- p.12 / Chapter 3.2 --- Hormone Responses on Cartilage --- p.16 / Chapter 3.3 --- Degradative enzymatic system --- p.17 / Chapter 3.3.1 --- Evidence for the Role of Enzymes in osteoarthritis --- p.18 / Chapter 3.3.2 --- Neutral Protease Acivity --- p.20 / Chapter 3.3.3 --- Neutal proteases in osteoarthritis --- p.21 / Chapter 3.3.4 --- Collagenase activity in articular cartilage --- p.22 / Chapter CHAPTER FOUR - --- METHODOLOGIES / Chapter 4.1 --- Isolation of Chick Growth Plate and Articular Chondrocytes --- p.24 / Chapter 4.2 --- Countercurrent Centrifugal Elutriation --- p.25 / Chapter 4.3 --- Size Determination of Chondrocytes --- p.26 / Chapter 4.4 --- Chondrocyte Cell Culture --- p.28 / Chapter 4.5 --- Flow Cytometry Cell Cycle Analysis of Elutriated Chondrocytes --- p.28 / Chapter 4.6 --- Thymidine Incorporation Assay on Elutriated Chondrocytes --- p.29 / Chapter 4.7 --- Sulfur Incorporation Assay on Elutriated Chondrocytes --- p.30 / Chapter 4.8 --- Hyalurondiase Assay on Elutriated Chondrocytes --- p.31 / Chapter 4.9 --- Alkaline Phosphatase Assay on Elutriated Chondrocytes --- p.32 / Chapter 4.10 --- Acid Phosphatase Assay --- p.33 / Chapter 4.10.1 --- Total Acid Phosphatase Assay on Elutriated Chondrocytes --- p.33 / Chapter 4.10.2 --- Optimal pH Determination of Phosphatase in Isolated Chondrocytes --- p.33 / Chapter 4.10.3 --- Enzyme Kinetics of Acid Phosphatase of Isolated Growth Plate and Articular Chondrocytes --- p.34 / Chapter 4.10.4 --- Tartrate Inhibition Effect on Acid Phosphatase of Growth Plate and Articular Chondrocytes --- p.35 / Chapter 4.10.5 --- Distribution of Acid Phosphatase Isoenzymes Among Chondrocytes of Different Size --- p.35 / Chapter 4.11 --- Hormonal Effects on Acid and Alkaline Phosphatase Activities in Growth Plate and Articular Chondrocytes --- p.36 / Chapter CHAPTER FIVE - --- RESULTS / Chapter 5.1 --- Morphology of the Isolated Chick Chondrocytes --- p.39 / Chapter 5.2 --- Countercurrent Centrifugal Elutriation VI --- p.39 / Chapter 5.3 --- Thymidine Incorporation Assay on Elutriated Chondrocytes --- p.44 / Chapter 5.4 --- Flow Cytometer Cell Cycle Analysis of Elutriated Chondrocytes --- p.44 / Chapter 5.5 --- Sulfate Incorporation Assay on Elutriated Chondrocytes --- p.48 / Chapter 5.6 --- Hyaluronidase Assay on Elutriated Chondrocytes on --- p.48 / Chapter 5.7 --- Alkaline Phosphatase Assay on Elutriaed Chondrocytes --- p.48 / Chapter 5.8 --- Acid Phosphatase Assay --- p.52 / Chapter 5.8.1 --- pH Curve of Phosphatase in Isolated Chondrocytes --- p.52 / Chapter 5.8.2 --- Enzyme Kinetics of Acid Phosphatase oflsolated Growth Plate and Articular Chondrocytes --- p.52 / Chapter 5.8.3 --- Tartrate Inhibition Effect on Acid Phosphatae of Growth Plate and Articular Chondrocytes --- p.55 / Chapter 5.8.4 --- Distribution of Acid Phosphatase Isoenzymes Among Chondrocytes in Different Size --- p.57 / Chapter 5.9 --- Hormonal Effects on Acid and Alkaline Phosphatase Activities in Growth Plate and Articular Chondrocytes --- p.59 / Chapter CHAPTER SIX - --- DISCUSSION / Chapter 6.1 --- Identification of Chondrocyte Subpopulations --- p.63 / Chapter 6.2 --- Characterization of Chondrocyte Subpopulations --- p.72 / Chapter 6.3 --- Characterization of Acid Phosphatase in Chick Chondrocytes --- p.74 / LIST OF FIGURES --- p.84 / LIST OF TABLES --- p.87 / REFERENCES --- p.88 / Chapter APPENDIX I --- Principle of Countercurrent Centrifugal Elutriation --- p.95 / Chapter APPENDIX II --- Principle of Flow Cytometry --- p.98 / Chapter APPENDIX III --- Reagents for Experiments --- p.103