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Falls among the elderly are a major source of injury, often leading to serious fractures, hospitalization, and death. Osteoporosis (OP) is a global problem intimately related with these fractures, characterized by reduced bone mass, increased bone fragility. There exists a high failure rate in the translation of treatments to osteoporotic populations. Mesenchymal stromal cell (MSC) transplantation as a therapeutic strategy for OP has not yet been examined in clinical trials. This may be attributed to the mixed findings of pre-clinical studies aimed at determining the efficacy of MSC therapy towards bone regeneration in OP. The most common animal model of OP is ovariectomy (OVX) that simulates post-menopausal estrogen loss. A plethora of bone regeneration studies have used OVX models with 12-16 weeks post-OVX periods and have generally reported positive results from a variety of treatment modalities, including MSC therapy. However, the use of the minimum post-OVX period may not be appropriate to reflect the global changes in regenerative potential of OP patients. In our research group's previous study, MSC were isolated from a minimum 60 week post-OVX rat model, representing a severe case of OP. The MSC isolated from these animals are a unique cell population that we expect may better represent the outcomes of autologous cell therapies for the older patient population in the clinic. In the present study, adipose and bone marrow derived MSC from OVX and age-matched animals were evaluated for their osteogenic and adipogenic differentiation potentials in culture through passage 10. Results from this study suggest that bone marrow derived-MSC maintain their phenotype and functionality more effectively than adipose derived-MSC in OP. Further investigations used regenerative medicine approaches for cell expansion on keratin protein coated microcarriers in static culture. Hair-derived keratin biomaterials have demonstrated their utility as carriers of biologics and drugs for tissue engineering. An optimal microcarrier was selected that demonstrated superior retention of the protein coating through electrostatic interactions and high cell viability. Finally, the integration of cell-microcarriers into a perfusion bioreactor system was explored. Preliminary results demonstrated the feasibility of MSC growth and differentiation on microcarrier based packed beds. Moreover, AD-MSC from OP rats were unresponsive to both inductive media and shear stress related osteogenic cues. These results highlight the complexity and challenges associated with the MSC regenerative strategy. / Doctor of Philosophy / Osteoporosis is a skeletal disease that results in reduced bone mass, increased bone fragility and fracture risk. Osteoporotic patients who experience falls suffer serious fractures, hospitalization, and poor bone healing. Several different therapies have been developed for the treatment of osteoporosis, though many are unable to translate from the bench to the clinical population. A popular treatment being investigated is the application of mesenchymal stromal cells (MSC) for fracture repair and the reversal of osteoporotic bone losses. However, cells isolated from aged and osteoporotic patients have been shown to have deficient bone forming properties. Nevertheless, animal models of osteoporosis applying this treatment report amelioration of bone loss. This work seeks to examine a more clinically relevant rat model of osteoporosis. Typical osteoporosis models use an ovariectomy procedure to simulate post-menopausal bone loss on relatively young animals and conduct short-term studies. These studies may not accurately reflect the global regenerative changes in osteoporosis patients or the impaired MSC properties. Adipose and bone marrow derived MSC from a long term ovariectomy model were investigated for their regenerative potentials. MSC growth and bone forming potential was evaluated on keratin protein coated microcarriers in both static and perfusion cultures. Results from this study suggest that bone marrow derived MSC maintain their phenotype and functionality more effectively than adipose derived MSC in osteoporosis. Further preliminary results demonstrated the feasibility of MSC growth and differentiation on microcarrier based packed beds. These results highlight the complexity and challenges associated with the MSC regenerative strategy.

No description available.

A presença da hipertensão arterial pode comprometer a qualidade da reparação óssea, pois a doença é caracterizada por alterações fisiopatológicas vasculares e do metabolismo mineral. Com o objetivo de avaliar a neoformação e o remodelamento ósseo, este trabalho investigou o processo da reparação óssea em ratos geneticamente hipertensos (SHR) e de seus controles Wistar-Kyoto (WKY). Defeitos bicorticais de 2mm de diâmetro no lado direito e de 5mm no lado esquerdo, foram realizados com trefinas na região do ângulo de mandíbula. Os animais, divididos em grupos de cinco indivíduos cada, foram sacrificados após 2, 3, 5, 10, 15, 30, 60 e 90 dias pós-operatórios; as mandíbulas foram removidas, fixadas em formol a 10%, descalcificadas em ácido fórmico a 20%, incluídas em parafina e as secções histológicas, de 7m de espessura, coradas com hematoxilina e eosina. As imagens foram capturadas com aumento de 40x e a área do defeito mensurada pelo programa de histometria digital Image J versão 1.4. A análise estatística revelou que não houve diferença significante na comparação entre as linhagens WKY e SHR (p = 0,884), independente dos períodos ou lados avaliados; entre períodos, nas linhagens WKY (p = 0,101) e SHR (p = 0,479), independente dos lados avaliados; entre períodos por linhagens no lado direito; e entre linhagens por lados, esquerdo com p = 0,466 e direito com p = 0,689, independente do fator período. Houve diferença estatisticamente significante entre o lado esquerdo e o direito (p < 0,001), independente das linhagens e períodos avaliados; entre os lados por linhagens, WKY e SHR, ambas com p < 0,001; entre períodos por linhagens no lado esquerdo, no qual o grupo WKY de 15 dias apresentou área menor que o grupo WKY de 60 dias e o grupo SHR de 10 dias, e o grupo WKY de 60 dias apresentou área maior que o grupo SHR de 30 dias e SHR de 60 dias. Apesar das alterações encontradas no lado esquerdo, que podem ser atribuídas à remodelação funcional do osso da mandíbula, não houve diferenças significantes na reparação do defeito de 5mm e de 2mm entre ratos espontaneamente hipertensos e de seus controles Wistar-Kyoto. / Arterial hypertension may affect the quality of bone repair because this disease is characterized by physiopathological vascular and bone metabolism changes. With the objective of evaluating the bone neoformation and remodeling, this study investigated the process of bone repair in spontaneously hypertensive rats (SHR) and their match controls Wistar-Kyoto (WKY). Through-in-through defects were done with trephine burs in the mandibular angle area, of 2mm diameter on the right side and 5mm diameter on the left side. The animals were divided into groups of five individuals each one and killed after 2, 3, 5, 10, 15, 30, 60 and 90 postoperative days; the mandibles were removed, fixed in 10% formalin solution, decalcified with 20% formic acid, and embedded in paraffin; the histological sections of 7m thickness were stained with hematoxylin and eosin. The images were captured with 40x magnification and the defect area was measured by the image processing program Image J version 1.4. The statistical analysis showed that there is no significant difference in the comparison of WKY and SHR strains (p = 0,884), independent of periods or sides; among periods, in the WKY strain (p = 0,101) and SHR one (p = 0,479), independent of sides; among periods by strains on the right side; and among strains by sides, left side with p = 0,466 and right side with p = 0,689, independent of periods. There is a significant difference between left and right side (p < 0,001), independent of strains and periods; between sides by strains, WKY and SHR, both with p < 0,001; among periods by strains on the left side, which WKY 15 days group showed an area smaller than WKY 60 days and SHR 10 days groups, and WKY 60 days group showed an area bigger than SHR 30 days and SHR 60 days groups. Despite the changes founded on the left side that could be attributed to the functional remodeling of the mandibular bone, there were no differences in the bone repair of 5mm and 2mm diameter defects between spontaneously hypertensive rats and their match controls Wistar-Kyoto.

A presença da hipertensão arterial pode comprometer a qualidade da reparação óssea, pois a doença é caracterizada por alterações fisiopatológicas vasculares e do metabolismo mineral. Com o objetivo de avaliar a neoformação e o remodelamento ósseo, este trabalho investigou o processo da reparação óssea em ratos geneticamente hipertensos (SHR) e de seus controles Wistar-Kyoto (WKY). Defeitos bicorticais de 2mm de diâmetro no lado direito e de 5mm no lado esquerdo, foram realizados com trefinas na região do ângulo de mandíbula. Os animais, divididos em grupos de cinco indivíduos cada, foram sacrificados após 2, 3, 5, 10, 15, 30, 60 e 90 dias pós-operatórios; as mandíbulas foram removidas, fixadas em formol a 10%, descalcificadas em ácido fórmico a 20%, incluídas em parafina e as secções histológicas, de 7m de espessura, coradas com hematoxilina e eosina. As imagens foram capturadas com aumento de 40x e a área do defeito mensurada pelo programa de histometria digital Image J versão 1.4. A análise estatística revelou que não houve diferença significante na comparação entre as linhagens WKY e SHR (p = 0,884), independente dos períodos ou lados avaliados; entre períodos, nas linhagens WKY (p = 0,101) e SHR (p = 0,479), independente dos lados avaliados; entre períodos por linhagens no lado direito; e entre linhagens por lados, esquerdo com p = 0,466 e direito com p = 0,689, independente do fator período. Houve diferença estatisticamente significante entre o lado esquerdo e o direito (p < 0,001), independente das linhagens e períodos avaliados; entre os lados por linhagens, WKY e SHR, ambas com p < 0,001; entre períodos por linhagens no lado esquerdo, no qual o grupo WKY de 15 dias apresentou área menor que o grupo WKY de 60 dias e o grupo SHR de 10 dias, e o grupo WKY de 60 dias apresentou área maior que o grupo SHR de 30 dias e SHR de 60 dias. Apesar das alterações encontradas no lado esquerdo, que podem ser atribuídas à remodelação funcional do osso da mandíbula, não houve diferenças significantes na reparação do defeito de 5mm e de 2mm entre ratos espontaneamente hipertensos e de seus controles Wistar-Kyoto. / Arterial hypertension may affect the quality of bone repair because this disease is characterized by physiopathological vascular and bone metabolism changes. With the objective of evaluating the bone neoformation and remodeling, this study investigated the process of bone repair in spontaneously hypertensive rats (SHR) and their match controls Wistar-Kyoto (WKY). Through-in-through defects were done with trephine burs in the mandibular angle area, of 2mm diameter on the right side and 5mm diameter on the left side. The animals were divided into groups of five individuals each one and killed after 2, 3, 5, 10, 15, 30, 60 and 90 postoperative days; the mandibles were removed, fixed in 10% formalin solution, decalcified with 20% formic acid, and embedded in paraffin; the histological sections of 7m thickness were stained with hematoxylin and eosin. The images were captured with 40x magnification and the defect area was measured by the image processing program Image J version 1.4. The statistical analysis showed that there is no significant difference in the comparison of WKY and SHR strains (p = 0,884), independent of periods or sides; among periods, in the WKY strain (p = 0,101) and SHR one (p = 0,479), independent of sides; among periods by strains on the right side; and among strains by sides, left side with p = 0,466 and right side with p = 0,689, independent of periods. There is a significant difference between left and right side (p < 0,001), independent of strains and periods; between sides by strains, WKY and SHR, both with p < 0,001; among periods by strains on the left side, which WKY 15 days group showed an area smaller than WKY 60 days and SHR 10 days groups, and WKY 60 days group showed an area bigger than SHR 30 days and SHR 60 days groups. Despite the changes founded on the left side that could be attributed to the functional remodeling of the mandibular bone, there were no differences in the bone repair of 5mm and 2mm diameter defects between spontaneously hypertensive rats and their match controls Wistar-Kyoto.

Indiana University-Purdue University Indianapolis (IUPUI) / The objective of this study was to evaluate the in vivo performance of the novel press-fit dental implant fabricated via electron beam melting (EBM, Southern Methodist Univ.) and compare it to a commercially-available porous-coated press-fit dental implant (Endopore, Innova Corp.). Twelve cylindrical shaped implants 3 mm in diameter x 5 mm long were made by EBM (Southern Methodist Univ.) using Ti6Al4V ELI alloy. Twelve commercial implants (Endopore, Innova Corp.) of the same geometry were used as controls. Samples were implanted in rabbit tibia and retrieved six weeks postoperatively. Six specimens from each implant type were embedded undecalcified, sectioned, and stained with toluidine blue (Sigma) for histomorphometry analysis. Bone-to-implant contact (BIC) was measured. On the six remaining samples from each implant type, the mechanical properties were evaluated by pushout test on a material testing machine. The samples were loaded at a loading rate of 1 mm/min. The pushout strength was measured and the apparent shear stiffness was calculated. The results were analyzed with a paired-t test. The histology shows osteointegration of surrounding bone with both implant types. Bone was found to grow into the porous space between the beads. Both the Endopore (Innova Corp.) and the EBM (Southern Methodist Univ.) showed similar BIC. The mean BIC for the Endopore (Innova Corp.) and EBM (Southern Methodist Univ.) implant were 35 ± 6% and 32 ± 9%, respectively. It failed to reach statistical significance (p > 0.05). The peak pushout force for Endopore (Innova Corp.) and EBM (Southern Methodist Univ.) implants were 198.80 ± 61.29 N and 243.21 ± 69.75 N, respectively. The apparent shear stiffness between bone and implant for the Endopore (Innova Corp.) and EBM (Southern Methodist Univ.) implants were 577.36 ± 129.99 N/mm; and 584.48 ± 146.63 N/mm, respectively. Neither the peak pushout force nor the apparent shear stiffness of the implants was statistically different between the two groups (p > 0.05). The results suggest that the implants manufactured by EBM (Southern Methodist Univ.) perform equally well as the commercial implant Endopore (Innova Corp.) in this current animal model.

No description available.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Insulin is a mitogen for many cells and commonly signals through the classical, mitogenic Raf- MEK-ERK or metabolic PB-kinase pathways. Insulin deficiency or type I diabetes causes severe osteopenia. Obese patients with type II diabetes or insulin resistance, a disease associated with defective insulin signaling pathways and high levels of circulating insulin, have increased or normal bone mineral density. The question of whether hyperinsul inemia preserves bone mass is frequently raised. However, there is still a lot of controversy on the role of insulin as an osteoanabolic agent and this question still remains unanswered. A critical role for insulin signaling in bone building osteoblasts has recently been demonstrated with IRS-l knock-out mice. These mice developed low-turnover osteopenia due to impaired proliferation and differentiation, stressing the importance of osteoblastic IRS-l for maintaining normal bone formation. In the present study it was found that insulin does function in vitro as an osteoblast mitogen. This was illustrated in three relatively immature osteoblast (MBA-15.4, -15.6 mouse and MG- 63 human) cell lines, which responded to insulin with significant increases in proliferation. In the MBA -15.4 preosteoblasts insulin stimulation of proliferation was comparable to the welldescribed mitogen, TPA. The UMR-I06 cell line expresses markers of differentiated osteoblasts, and was much less responsive to insulin treatment. The difference in proliferative potential may be due to differences between spontaneously transformed cell lines, or the stage of cell differentiation. UOI26, a MEKI/2 inhibitor and wortmannin, a PB-kinase inhibitor, were used to investigate the pathway used by insulin to signal and activate ERK and osteoblast proliferation. In MBA-15.4 mouse preosteoblasts, GF-containing FCS was completely dependent on MEK for DNA synthesis. In contrast, in both MBA-15.4 and more mature MBA-15.6 osteoblasts, insulininduced proliferation was resistant to the inhibitors alone or in combination. Higher MEKinhibitor concentrations had no effect, and proliferation was also increased by the inhibitors in several experiments. This indicated that the classical, insulin mitogenic pathway was not involved in MBA-15.4 proliferation. Wortmannin had no effect on either insulin- or 20% FCSstimulated proliferation, but inhibited activation of Akt/PKB, the metabolic downstream target of PI3-kinase. Insul in signal ing to ERK was both MEK-and PI3-kinase- dependent, but this had no effect on proliferation. In contrast, FCS-stimulated ERK activation and proliferation was almost completely dependent on MEK-ERK activation. Proliferative signaling in the MG-63 human osteoblastic cell line in response to insulin was partially dependent on MEK and partially dependent on PB-kinase. In contrast, signaling in response to the phorbol ester, TPA, was partially dependent on PI3K but totally dependent on MEK-ERK. This indicates that the signal converges on ERK, suggesting the involvement of a PB-kinase upstream of a dominant MEK-ERK pathway. The differences found here between mouse and human insulin mitogenic signaling pathways indicate that there may be species differences between osteoblast signaling pathways, with mouse cells being independent and human cells being dependent on MEK for DNA synthesis in response to insulin. The effects of glucocorticoids on insulin mitogenic signaling in osteoblasts were also investigated, because chronic long-term steroid use results in excessive bone loss. The PTP inhibitor, sodium orthovanadate, reversed GC-impaired TPA- and FCS- induced proliferation in MBA-1SA and MG-63 preosteoblasts. PTPs, such as SHP-l and PTP-IB, dephosphorylate and inactivate phosphorylated kinases. Both SHP-l and PTPlB associated with kinases in the mitogenic signaling cascade of MBA-lS.4 preosteoblasts growing rapidly in 10% FCS. Further, SHP-I co-irnmunoprecipitated with active, tyrosine phosphorylated ERK, which may indicate that it can dephosphorylate and inactivate ERK. However, since the MEK-ERK or PB-kinase pathways are not important in insulin-induced proliferation in mouse osteoblasts, the PTPs are unlikely to be role players in the negative regulation of this signaling pathway. This was confirmed by the finding that vanadate was unable to reverse GC-induced decreases in insulinstimulated DNA synthesis. This suggests that vanadate-sensitive PTPs may not be important in the negative regulation of insulin-induced mouse osteoblast proliferation, and provides further evidence of a novel insulin mitogenic pathway in the MBA-lSA but not MG-63 osteoblastic cell line. / AFRIKAANSE OPSOMMING: Insulien is 'n mitogeen vir baie selle en gelei na binding aan die insulien reseptor, intrasellulêre seine via die klassieke, mitogeniese Raf-MEK-ERK of die metaboliese PB-kinase seintransduksie pad. 'n Insulien gebrek of tipe I diabetes veroorsaak osteopenie. Vetsugtige pasiënte met insulien weestandigheid of tipe II diabetes, 'n siekte wat geassosieer word met foutiewe insulien seintransduksie en hoë vlakke van sirkuierende insulien, het verhoogde of normale been mineraal digtheid (BMD). Die vraag of hiper insulin ernie 'n verlies aan beenmassa teëwerk word dikwels gevra. Teenstrydigheid oor die rol van insulien as 'n osteo-anaboliese stof bestaan egter steeds en hierdie vraag bly dus onbeantwoord. Dat insulien seintransduksie wel 'n kritiese rol speel in beenvormende osteoblaste is onlangs bevestig in studies met muise waarvan die geen vir IRS-l uitgeslaan is. Hierdie muise ontwikkel 'n lae omset osteopenie weens verswakte proliferasie en differensiasie. fn hierdie studie is gevind dat insulien wel in vitro as 'n osteoblast mitogeen kan funksioneer. Dit is in drie relatief onvolwasse (MBA-15.4, -15.6 muis en MG-63 mens) sellyne geillistreer, deur betekenisvolle verhogings in insulien-geaktiveerde proliferasie. In MBA-15.4 preosteoblaste is die persentasie verhoging in insulien-gestimuleerde proliferasie vergelykbaar met dié van die bekende mitogeniese forbolester, TPA. Die UMR-I06 sellyn het kenmerke van gedifferensieerde osteoblaste, en was baie minder responsief op insulien behandeling. Die verskil in die proliferasie vermoë van die verskillende sellyne kan die gevolg wees van verskille wat bestaan tussen spontaan getransformeerde sellyne of die stadium van sel differensiasie. 'n MEK 1/2 inhibitor, UO126 en 'n PB-kinase inhibitor, wortmannin, is gebruik om die insulien seintransduksie pad noodsaaklik vir die aktivering van ERK en osteoblast proliferasie te bepaal. In MBA-1S.4 muis pre-osteoblaste, was fetale kalf SenlTI1(FKS)-geinduseerde DNA sintese totaal afhanklik van MEK. Beide die MBA-15.4 en die meer volwasse MBA-15.6 muis osteoblaste was weerstandig teen die inhibitors op hulle eie, of in kombinasie. Verhoogde MEK-inhibitor konsentrasies het geen verdere effek gehad nie en in verskeie eksperimente is 'n verhoging in preliferasie selfs waargeneem met MEK-inhibisie. Hierdie resultate dui aan dat die klassieke insulien mitogeniese pad nie betrokke is in MBA-I5.4 gestimuleerde selproliferasie nie. Wortmannin het geen effek gehad op insulien- of20% FKS-gestimuleerde DNA sintese nie, maar het wel die aktivering van PB-kinase se metaboliese teiken, AktJPKB geinhibeer. Insulien seintransduksie aktiveer dus ERK deur beide MEK en PB-kinase, maar het geen effek op proliferasie gehad nie. FKS-gestimuleerde ERK aktivering en proliferasie was totaal afhanlik van MEK-ERK aktivering. Insulien-geaktiveerde DNA sintese in die mens MG-63 osteoblaste was gedeeltelik afhanklik van beide MEK en PB-kinase. Alhoewel IPA ook PB-kinase kon aktiveer, was dit totaal afhanklik van MEK vir DNA sintese. Dit dui aan dat daar 'n PB-kinase stroom-op van 'n dominante MEK-ERK seintransduksie pad voorkom. Die verskille wat ons dus waargeneem het in insulien mitogeniese seintransduksie tussen muis en mens, kan aandui dat insuliengestimuleerde seintranduksie paaie kan verskil van spesie tot spesie. Dit is bevestig met die muisselle wat onafhanklik is en mens selle wat afhanklik is van MEK aktivering vir insuliengeaktiveerde DNA sintese. Kroniese, langtermyn steroied behandeling kan beenverlies veroorsaak en die effek van glukokortikoide (GK) op die insulien mitogeniese pad in osteoblaste is dus ook ondersoek. Natrium-ortovanadaat, 'n proteien tirosien fosfatase (PIP) inhibitor het GK-verlaagde proliferasie in repons tot beide IPA- en FKS behandeling herstel in MBA-lSA en MG-63 preosteoblaste. PIPs soos SHP-l en PIP-l B funksioneer deur gefosforileerde kinases te defosforileer en dus te inaktiveer. Beide SHP-l and PIP-lB kon assosieer met kinases in die mitogeniese insulien seintransduksie pad van vinnig groeiende MBA-IS A preosteoblaste in 10% FKS. Verder het SHP-I ook geko-immunopresipiteer met aktiewe, tirosien-gefosforileerde ERK, wat aandui dat SHP-I met ERK assosieer om dit te defosforileer en inaktiveer. Die MEKERK of PB-kinase paaie is nie belangrik vir insulien-geaktiveerde seintransduksie in muis osteoblaste nie. Dit is dus onwaarskynlik dat die PIPs 'n rol sal speel in die negatiewe regulering van hierdie seintransduksie paaie. Die ontdekking dat vanadaat nie glukokortikoiedverlaagde insulien-geaktiveerde DNA sintese kan herstel nie, toon dat vanadaat-sensitiewe PIPs nie 'n rol speel in insulien-geaktiveerde proliferasie in muisselle nie. Hierdie bevinding het verder bevestig dat 'n nuwe insulien mitogeniese pad in die MBA-ISA, maar nie die MG-63 selle moontlik bestaan.