Abstract A new animal model was developed to evaluate the effect of bovine native bone morphogenetic protein (BMP) on the healing of segmental, critical-sized bone defects. Laboratory-bred adult beagle dogs were used in the study. A 2 cm corticoperiosteal defect was created using an oscillating saw in mid-ulna, and the defect was treated with bone grafts and implants fixed by an intramedullary Kirschner wire through predrilled holes in the middle of the implant. Plate and screw fixation was also used in some groups. Coral, hydroxyapatite and demineralized xenograft bone were placed in the defects with or without BMP. Autografts and allografts were used as controls. The BMP was extracted from bovine diaphyseal bone. The follow-up period was 36 weeks. Radiographs were taken at regular intervals during the follow-up period, and bone formation and bone union were evaluated. The radiographs were digitized, and callus was measured and CT scans obtained to define bone density. At the end of the study, the bones were harvested and tested mechanically in a torsion machine until failure. After mechanical testing, the bones were reconstructed and histological sections were made. With autograft and allograft bone grafts, healing was nearly complete. Hydroxyapatite and demineralized xenograft bone did not result in healing of the bone defect, while coral enhanced bone formation, but the healing was not comparable to autografts or allografts. Hydroxyapatite implants did not resorb during the 36 weeks of follow-up to enhance bone healing, and there was a fibrous capsule around the hydroxyapatite implants in histology. Xenograft bone was resorbed, and very little bone formation and extensive fibrosis were seen at the implant site. Coral was resorbed and gradually replaced by new bone, but did not heal the defect completely. With every implant, added BMP had a positive effect on healing as evaluated either radiographically, mechanically or histologically. Coral was the most optimal carrier material for BMP among the materials tested in this study. The animal model seems to be suitable for studying the healing of bone defects, as all the animals were physically active from the first postoperative day and did not seem to have problems with motion during the follow-up period. Intramedullary fixation lacks rotational stability, which may have a negative effect on healing. The bones fixed with a plate and screws showed better scores in radiographs and were mechanically stronger, although the study groups were too small to allow definitive conclusions. As a conclusion, none of the transplants or implants were equally efficient as cortical autograft in healing segmental ulnar defects. BMP did not enhance the poor capacity of hydroxyapatite and xenograft bone to heal the bone defect. According to the present findings, the composite implant consisting of coral and BMP seemed to be the best of the composite implants tested.

Please read the abstract in die section front of this document. / Dissertation (MChD)--University of Pretoria, 2009. / Oral Pathology and Oral Biology / unrestricted

Adipositas und Typ-2-Diabetes sind häufige Erkrankungen des Stoffwechsels. Zur Basistherapie der Adipositas und des Typ-2-Diabetes gehören eine gesunde Ernährungs- weise und die Erhöhung der körperlichen Aktivität unter anderem mit dem Ziel der Gewichtsreduktion. Vermehrte Bewegung führt neben der Verbesserung der körperlichen Leistungsfähigkeit zur Fettmassenreduktion, Verbesserungen der Hyperglykämie, Lipo- proteinstoffwechsels und des Adipokinprofils. Bone morphogenetic proteins (BMPs) werden im Fettgewebe produziert und spielen eine wichtige Rolle in der Adipogenese und Transdifferenzierung von Adipozyten. Während ein Zusammenhang zwischen der BMP-7-Serumkonzentration und Adipositas vor kurzem belegt wurde, ist bisher nicht bekannt, ob weitere BMPs wie BMP-2 und -4 mit Adipositas und Typ-2-Diabetes assoziiert sind. Ziel dieser Arbeit war es deshalb zu untersuchen, ob die BMP-2 und -4 Serumkonzentrationen im Zusammenhang mit Körpergewicht, Fett- verteilung und Parametern des Glukosestoffwechsels bei Patienten mit Adipositas und Typ-2-Diabetes (n=213) stehen. Im Rahmen von drei Interventionsstudien wurde der Einfluss einer hypokalorischen Ernährungsweise über sechs Monate (n=19), eines 45,3 ± 7,4 kg Gewichtsverlustes ein Jahr nach bariatrischer Chirurgie (n=32) sowie eines zwölf- wöchigen Trainingsprogramms (n=60) auf die BMP-2- und -4-Serumkonzentrationen untersucht. Zusätzlich wurde die BMP-2-und -4-mRNA-Expression in humanen omentalen und subkutanen Fettgewebsproben von 161 Patienten charakterisiert. Die BMP-2- und -4-Serumkonzentrationen und die BMP-2- und -4-mRNA-Expression im viszeralen Fettgewebe korrelieren signifikant mit dem BMI und dem Körperfettgehalt. Zirkulierende BMP-4-Spiegel sind geschlechtsabhängig und bei Patienten mit T2D signifikant niedriger als bei gesunden Kontrollpatienten. Sowohl eine moderate Gewichts- reduktion durch kalorienreduzierte Ernährung als auch ein Gewichtsverlust von 45,3 ± 7,4 kg nach bariatrischer Chirurgie führen zu einer signifikanten Reduktion der zirkulierenden BMP-2- und -4-Spiegel. Das zwölfwöchige Trainingsprogramm führte lediglich zu einer signifikanten Reduktion der BMP-2-Serumkonzentration und zu signifikanten Ver- besserung der Leistungsfähigkeit, von Parametern des Glukosestoffwechsels und der Serumkonzentrationen von Adiponektin und Interleukin-6. Zusammengefasst zeigen die Daten, dass erhöhte Serumkonzentrationen von BMP-2 und 4 mit Adipositas assoziiert sind und durch Gewichtsreduktion und Erhöhung der körperlichen Aktivität verringert werden können. Die BMP-2- und -4-mRNA-Expression im viszeralen Fettgewebe kann zu erhöhten Serumkonzentrationen dieser Adipokine bei viszeraler Fettverteilung beitragen.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18895号 / 医博第4006号 / 新制||医||1009(附属図書館) / 31846 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 武藤 学, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第20297号 / 人健博第45号 / 新制||人健||4(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 坪山 直生, 教授 山田 重人, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) coordinate tissue development and postnatal remodelling by regulating proliferation, differentiation and apoptosis. However, their role in wound healing remains unclear. To study this, transgenic mice overexpressing Smad1 (K14-caSmad1) or the BMP antagonist Noggin (K14-Noggin) were utilised, together with human and mouse ex vivo wound healing models and in vitro keratinocyte culture. In wild-type mice, transcripts for Bmpr-1A, Bmpr-II, Bmp ligands and Smad proteins were decreased following tissue injury, whilst Bmpr-1B expression was up-regulated. Furthermore, immunohistochemistry revealed a down-regulation of BMPR-1A in hair follicles adjacent to the wound in murine skin, whilst in murine and human wounds, BMPR-1B and phospho-Smad-1/5/8 expression was pronounced in the wound epithelial tongue. K14-caSmad1 mice displayed retarded wound healing, associated with reduced keratinocyte proliferation and increased apoptosis, whilst K14-Noggin mice exhibited accelerated wound healing. Furthermore, microarray analysis of K14-caSmad1 epidermis revealed decreased expression of distinct cytoskeletal and cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a versus controls. Human and mouse keratinocyte proliferation and migration were suppressed by BMP-4/7 both in vitro and ex vivo, whilst they were stimulated by Noggin. Additionally, K14-caSmad1 keratinocytes showed retarded migration compared to controls when studied in vitro. Furthermore, Bmpr-1B silencing accelerated migration and was associated with increased expression of Krt16, Krt17 and Myo5a versus controls. Thus, this study demonstrates that BMPs inhibit proliferation, migration and cytoskeletal re-organization in epidermal keratinocytes during wound healing, and raises a possibility that BMP antagonists may be used for the future management of chronic wounds.

No / Bone morphogenetic protein (BMP) signaling plays a key role in the control of skin development and postnatal remodeling by regulating keratinocyte proliferation, differentiation, and apoptosis. To study the role of BMPs in wound-induced epidermal repair, we used transgenic mice overexpressing the BMP downstream component Smad1 under the control of a K14 promoter as an in vivo model, as well as ex vivo and in vitro assays. K14-caSmad1 (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) mice exhibited retarded wound healing associated with significant inhibition of proliferation and increased apoptosis in healing wound epithelium. Furthermore, microarray and quantitative real-time reverse-transcriptase-PCR (qRT-PCR) analyses revealed decreased expression of a number of cytoskeletal/cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myosin VA (Myo5a), in the epidermis of K14-caSmad1 mice versus wild-type (WT) controls during wound healing. BMP treatment significantly inhibited keratinocyte migration ex vivo, and primary keratinocytes of K14-caSmad1 mice showed retarded migration compared with WT controls. Finally, small interfering RNA (siRNA)-mediated silencing of BMPR-1B in primary mouse keratinocytes accelerated cell migration and was associated with increased expression of Krt16, Krt17, and Myo5a compared with controls. Thus, this study demonstrates that BMPs inhibit keratinocyte proliferation, cytoskeletal organization, and migration in regenerating skin epithelium during wound healing, and raises a possibility for using BMP antagonists for the management of chronic wounds.

No / Bone morphogenetic protein (BMP) signaling is involved in the regulation of a large variety of developmental programs, including those controlling organ sizes. Here, we show that transgenic (TG) mice overexpressing the BMP antagonist noggin (promoter, K5) are characterized by a marked increase in size of anagen hair follicles (HFs) and by the replacement of zig-zag and auchen hairs by awl-like hairs, compared with the age-matched WT controls. Markedly enlarged anagen HFs of TG mice show increased proliferation in the matrix and an increased number of hair cortex and medulla cells compared with WT HFs. Microarray and real-time PCR analyses of the laser-captured hair matrix cells show a strong decrease in expression of Cdk inhibitor p27(Kip1) and increased expression of selected cyclins in TG vs. WT mice. Similar to TG mice, p27(Kip1) knockout mice also show an increased size of anagen HFs associated with increased cell proliferation in the hair bulb. Primary epidermal keratinocytes (KC) from TG mice exhibit significantly increased proliferation and decreased p27(Kip1) expression, compared with WT KC. Alternatively, activation of BMP signaling in HaCaT KC induces growth arrest, stimulates p27(Kip1) expression, and positively regulates p27(Kip1) promoter activity, thus further supporting a role of p27(Kip1) in mediating the effects of BMP signaling on HF size. These data suggest that BMP signaling plays an important role in regulating cell proliferation and controls the size of anagen HFs by modulating the expression of cell-cycle-associated genes in hair matrix KC.

Chronic patellar tendinopathy is a degenerative tendon disorder characterized by chronic activity-related, anterior knee pain associated with localized tenderness, swelling and impaired performance, which is a common clinical problem in athletes. The pathogenesis of patellar tendinopathy is still largely unknown, although tendon overuse is the most commonly suggested etiological factor, and treatment is usually symptomatic. / Histopathologically, the predominant feature of patellar tendinopathy is tendinosis, which is characterized by progressive tissue degeneration with a failed healing response and the absence of inflammatory cells. Hypercellularity with non-tenocyte phenotype cells and tissue metaplasia, including hyaline metaplasia, fibrocartilaginous metaplasia and bony metaplasia were observed in clinical patellar tendinopathy samples. The degeneration of patellar tendon in patellar tendinopathy is an active cell-mediated process rather than a passive degenerative process. Using a patellar tendinopathy animal model, we observed the presence of chondrocytic and osteoblastic phenotype / markers in patellar tendinopathy samples with or without ossification, which was consistent with the findings in clinical samples. Interestingly, chondrocyte makers were expressed by healing tendon cells at week 2 which became round prior to their expression in the chondrocyte-like cells at week 4. This leads us to speculate that erroneous differentiation of tendon-derived stem cells (TDSCs) identified recently in tendon tissues by our group, to chondrocyte / osteoblasts, due to alteration of mechanical and biological microenvironment during overuse, may lead to the ectopic chondro-ossification and failed healing in patellar tendinopathy. Osteo-chondrogenic BMPs, such as BMP-2, BMP-4 and BMP-7 might be possible factors regulating the osteo-chondrogenic differentiation of TDSCs in the pathogenesis of patellar tendinopathy. / In conclusion, our results have provided new insights about the pathological mechanisms of patellar tendinopathy involving the resident stem cells, osteo-chondrogenic BMPs and mechanical overloading. Erroneous differentiation of TDSCs to chondrocytes / osteoblasts due to ectopic osteo-chondrogenic BMP-2 expression, which were induced by repetitive tensile loading stimulation, might account for the chondro-ossification and failed healing in patellar tendinopathy. Re-directing of stem cells for tenogenic differentiation by blocking the ectopic expression of osteo-chondrogenic BMPs may help to promote tendon healing in patellar tendinoapthy. / In this study, we hypothesized that (1) TDSCs isolated from pathological patellar tendon of the CI model will exhibit higher osteogenic and chondrogenic differentiation potential but lower proliferative capacity compared to TDSCs isolated from healthy patellar tendon. Rat pathological tendon in our collagenase-induced failed healing animal model will harbor more TDSCs compared to healthy patellar tendon. (2) Osteo-chondrogenic BMPs, such as BMP-2, BMP-4, and BMP-7, will be expressed ectopically in both preclinical and clinical samples of patellar tendinopathy. (3) BMP-2 will promote osteo-chondrogenic differentiation and inhibit tenogenic differentiation of TDSCs in vitro. (4) Repetitive tensile loading will increase the expression of BMP-2 in TDSCs in vitro. / Our results showed that TDSCs isolated from the collagenase-induced tendinopathic patellar tendon of the animal model exhibited higher osteogenic/chondrogenic differentiation potential as well as lower proliferative capacity, supporting that there might be some defects in the TDSCs from the animal model, which might undergo osteo-chondrogenic differentiation and hence reduced the pool of TDSCs for tendon repair in the development of patellar tendinopathy. The higher clonogenicity and increased yield of TDSCs in tendinopathic patellar tendon might be caused by a compensation for the impaired differentiation potential and proliferative capacity of TDSCs. The histopathological features of our clinical patellar tendinopathy were characterized by tissue degeneration. Non-tenocyte phenotype cells and tissue metaplasia, such as chondrocyte-like cells and endochondral ossification were also observed. We observed the ectopic expression of osteo-chondrogenic BMP-2, BMP-4 and BMP-7 in both our animal model and clinical samples of patellar tendinopathy, which might trigger the erroneous differentiation of TDSCs to non-tenocytes. Indeed, we further showed that BMP-2 could promote the osteo-chondrogenic and inhibit tenogenic differentiation of TDSCs in vitro, which might provide a possible explanation for ectopic chondro-ossification and failed healing in patellar tendinopathy. In addition, our results also showed that in vitro repetitive cyclic tensile loading could increase the expression of BMP-2 in TDSCs, which might provide a possible explanation for the ectopic expression of BMP-2 in patellar tendinopathy. / This study aimed to compare the osteogenic / chondrogenic differentiation potential, proliferative capacity and yield of TDSCs isolated from rat healthy patellar tendon and pathological tendon in our collagenase-induced failed tendon healing animal model of patellar tendinopathy in vitro. The histopathological characteristics of our clinical patellar tendinopathy with or without ossification were examined. The ectopic expression of BMP-2, BMP-4, and BMP-7 in both human and rat samples of patellar tendinopathy was also examined. The effects of BMP-2 on the osteogenic, chondrogenic and tenogenic differentiation of TDSCs was further investigated in vitro. The effect of repetitive tensile loading on the expression of BMP-2 in TDSCs was studied in vitro. / Rui, Yunfeng. / Advisers: Kai Ming Chan; Po Yee Lui. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 172-193). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Les adipocytes (cellules spécialisées dans le stockage des graisses) sont formés à partir de cellules immatures appelées cellules progénitrices lors du processus d'adipogenèse. Chez l'homme, les différentes étapes de ce processus sont mal connues ainsi que les signaux qui le régulent. La première partie de mon travail de thèse a eu pour but de caractériser la cellule intermédiaire entre la cellule progénitrice et l'adipocyte : le préadipocyte. La deuxième partie a consisté à évaluer le rôle des protéines morphogénétiques de l'os (ou BMP), des inducteurs de l'adipogenèse décrits chez la souris, dans l'adipogenèse humaine. Nous avons montré que les BMP2, 4 et 7 sont produites dans le tissu gras humain et BMP7 est modulée par l'obésité. Les BMP2 et 4 induisent l'adipogenèse des cellules progénitrices humaines mais seule la BMP7 permet la production d'adipocytes particuliers " beiges " décrits pour consommer les lipides et produire de la chaleur. Ces travaux affinent nos connaissances sur les mécanismes impliqués dans l'expansion du tissu gras et permettront d'élaborer des stratégies pour lutter contre le développement des pathologies liées à l'obésité. / Adipocytes (cells specialized in fat storage) arise from immature cells, called progenitor cells, during the process of adipogenesis. In human, the different stages of adipogenesis are not well defined as well as the signals involved in adipogenic modulation. The first part of my thesis work aimed to characterize the intermediate cell state between progenitor cell and mature adipocyte: the preadipocyte. The second part aimed to evaluate the role of bone morphogenetic proteins (BMPs) in human adipogenesis. In mice, BMP2 and BMP4 induce classical adipogenesis whereas BMP7 leads to the production of "brite" adipocytes with the capacity to use lipids to produce heat. We have shown that BMP2, 4 and 7 are produced in human fat depots and BMP7 is modulated by obesity. BMP2 and 4 induce classical adipogenesis and BMP7 only induces brite adipogenesis from human progenitor cells. These works improve our knowledge about the mechanisms involved in the expansion of fat depot and may allow the identification of new strategies to fight against the development of obesity-associated pathologies.