Noch immer gibt es trotz neuester medizinischer Operationstechniken Patienten, bei denen eine Fraktur zu einer verzögerten Heilung führt oder gar nicht heilt. Indizien häufen sich, dass das Immunsystem, seine Zellen und Zytokine eine wichtige Rolle im Prozess der Heilung einer Fraktur spielen könnten. In dieser Arbeit wurde der Einfluss des adaptiven Immunsystems auf die Fraktur-heilung untersucht. Dazu wurde der Heilungsverlauf in Wildtyp Mäusen mit RAG1-/- Mäusen, Mäusen ohne reife B- und T-Lymphozyten, verglichen. Histologische, immunhistologische sowie Mikroarray Analysen der verschiedenen Tierstämme lassen darauf schließen, dass T-Lymphozyten einerseits der Schlüssel für ein gutes Heilungsergebnis sind, andererseits ihr Fehlen maßgeblich zu einer Veränderung der Heilung führt. Diese resultierte darin, dass die Knorpelphase verkürzt war und eine ungeordnete Bildung von Geflechtknochen einsetzte. / Despite of advanced methods in trauma surgery some patients suffer from a delayed healing or a non-union one. There is more and more evidence that the immune system with its cells and mediators could play an important role in such healing situations. Hence this thesis investigated the role of the adaptive immune system in the process of fracture healing comparing healing in wildtype mice and RAG1-/- mice. RAG1-/- mice do not have mature B- and T-lymphocytes. Histological, immunohistological and microarry analyses of different mice strains reveal that T lymphocytes are the key cell for a normal healing outcome. Their absence leads to a different healing outcome with a diminished cartilage phase and an irregular distribution of woven bone in the whole callus region.

As fraturas são consideradas um dos maiores problemas de saúde pública pelo custo e morbidade a elas associados. O objetivo deste estudo foi investigar os efeitos do ultrassom de baixa intensidade (LIPUS) e do laser de baixa potência (LLLT) sobre o reparo ósseo de tíbia de ratos (Wistar) utilizando a microtomografia por raio-x (&#956CT) e a análise histomorfométrica. Uma osteotomia total no terço médio foi fixada com fio de Kirschner (0,89 mm) e utilizado um espaçador polimérico de 2 mm de espessura e 1 mm de diâmetro entre os fragmentos proximal e distal para induzir retardo de consolidação da fratura. Foram utilizados 24 animais separados aleatoriamente em 4 grupos experimentais: Grupo Controle: sem tratamento do retardo da consolidação (n = 5); Grupo Laser: tratamento da fratura com laser (n = 6); Grupo LIPUS: tratamento da fratura com LIPUS (n = 7) e; Grupo LIPUS + Laser: tratamento da fratura com LIPUS e Laser (n = 7). Os tratamentos com LIPUS e/ou Laser foram iniciados após 5 semanas da cirurgia e realizados durante 5 dias consecutivos de tratamento, seguidos por 2 dias sem serem tratados até totalizarem 12 sessões terapêuticas. Os parâmetros de irradiação laser foram: 808 nm; 100 mW; 2500 mW/cm² , 125 J/cm² durante 50 segundos. Os parâmetros do LIPUS foram: 1,5 MHz, ciclo de trabalho 1:4, intensidade de 30 mW/cm², 20 minutos por sessão. Quando as duas técnicas foram associadas, o laser foi aplicado perpendicularmente ao LIPUS no décimo minuto de aplicação do LIPUS. Radiografias da tíbia foram feitas para acompanhar a evolução da fratura após a cirurgia, antes dos tratamentos e antes da eutanásia. Após a eutanásia foram realizadas avaliações microtomográficas por raio-x e histomorfométricas para análise do reparo ósseo. Na análise microtomográfica do reparo ósseo o tecido neoformado de reparo (calo ósseo) e o tecido normal não foram isolados. Na análise estatística foi utilizada a ANOVA one-way com posthoc de Tukey. Não foi constatada diferença significativa para maioria das variáveis, exceto para o fator padrão trabecular (Tb.Pf) no grupo LIPUS+Laser comparado aos grupos LIPUS (p = 0.02), Laser (p = 0.02) e Controle (p = 0.01), para a conectividade (Conn) no grupo Laser comparada ao grupo Controle (p = 0.04) e para a densidade de conectividade (D.Conn) no grupo LIPUS+Laser comparado ao grupo Controle (p = 0.04). As imagens histológicas mostram maior quantidade de tecido esponjoso no grupo LIPUS+Laser. Parâmetros morfométricos e histomorfométricos não revelaram um avanço do reparo ósseo na comparação entre os grupos com tratamento em relação ao grupo controle provavelmente devido a não reabsorção do espaçador polimérico pelo organismo dos animais. A &#956CT é uma técnica de grande potencial que permite análises qualitativas e quantitativas e reconstruçoes 2D e 3D. / Bone fractures are important public health problems due to the morbidity and high cost related to them. The aim of this study was to evaluate the effects of low pulsed intensity ultrasound (LIPUS) and infrared low level laser therapy (LLLT) in the bone repair of a transverse rat tibia osteotomy with assessement by x-ray microtomography (&#956CT) and histomorphometry. An easily implemented transverse osteotomy with a Kirchner wire and 2 mm width polymeric spacer beads established a delayed union in the fracture. Twenty four rats were divided into four experimental groups: Control Group - untreated; Laser Group - treated with infrared LLLT; LIPUS Group treated with low intensity pulsed ultrasound; LIPUS+Laser Group - treated with low intensity pulsed ultrasound and infrared LLLT. The treatments with infrared LLLT (808 nm, 100 mW, 2500 mW/cm², 125 J/cm², 50 seconds) and LIPUS (1,5 MHz, 1:4 duty cicle, 30 mW/cm², 20 minutes) started 5 weeks after the surgery following a sequence of 5 days on and 2 days off until 12 sessions were reached. When LLLT and LIPUS therapies were used simultaneously there was a 90º angle between the laser and the ultrasound beams. The surgical technique and the bone repair were assessed by x-ray radiography. One-way analysis of variance (one-way ANOVA) and Tukey post-hoc were used for statistical analysis. The normal bone and the callus were kept together in the &#956CT analysis. There was no significant difference between treatments results except for the parameters trabecular pattern factor - Tb.Pf [US+Laser group x LIPUS group (p = 0.02); US+Laser group x Laser group (p = 0.02) and US+Laser group x Control group (p = 0.01)], connectivity - Conn [Laser group x Control group (p = 0.04)] and connectivity density D.Conn [US+Laser group x Control group (p = 0.04)]. The histology showed greater amount of spongeous bone in the LIPUS+Laser group. The morphometric and histomorphometric assessments didn´t show a faster bone repair when treated groups were compared against the control one. The non absorption of the polymeric spacer bead is probably the reason of these findings. The &#956CT is a powerful technique that allows 2D and 3D quantitative analysis and reconstructions.

No description available.

INTRODUÇÃO: O objetivo deste estudo foi de avaliar a eficácia de um centrifugado osteogênico de medula óssea para estimular a consolidação de osteotomias da fíbula em coelhos. MÉTODOS: Este estudo experimental envolveu a utilização de dez coelhos machos adultos da raça Nova Zelândia albino. Realizou-se uma osteotomia transversa médio-diafisária da fíbula direita, seguida da adição local de uma esponja de colágeno absorvível embebida em um centrifugado osteogênico, obtido pela centrifugação de aspirado de medula óssea do osso ilíaco ipsilateral. A fíbula esquerda foi utilizada como controle, sendo feita a mesma osteotomia, porém neste caso adicionando-se somente a esponja de colágeno absorvível. O centrifugado de medula óssea elaborado em laboratório foi submetido à contagem do número de células nucleadas e a teste de viabilidade celular antes de ser administrada no local das osteotomias. Após quatro semanas os animais foram sacrificados para estudo dos calos ósseos formados. Os critérios de avaliação foram a mensuração da densidade mineral utilizando-se a densitometria óssea com DEXA, do volume do calo com tomografia computadorizada multi-slice e dos tecidos formados por meio de histomorfometria. RESULTADOS: O método utilizado para a centrifugação dos aspirados de medula óssea resultou em uma concentração média de células nucleadas três vezes maior que o número destas células nos aspirados originais, sem destruição celular significativa. A utilização deste centrifugado osteogênico resultou em um aumento médio na densidade mineral óssea dos calos de 40,3% e da quantidade relativa de tecido ósseo de 9,4%, sem aumento significativo nas quantidades relativas de cartilagem ou fibrose. Não houve aumento significativo no volume dos calos ósseos. CONCLUSÃO: A administração de centrifugado osteogênico de medula óssea utilizado neste estudo favoreceu a consolidação óssea de osteotomias experimentais em coelhos, observando-se uma melhora qualitativa do calo ósseo. / INTRODUTION: The purpose of this study was to evaluate the efficacy of a centrifuged osteogenic bone-marrow aspirate to stimulate rabbit fibular osteotomies healing. METHOD: Ten white New Zeeland rabbits were used. A transverse middle-diaphysis fibular osteotomy was performed at the right fibula, where a collagen absorbable sponge embedded in the osteogenic centrifuged bone marrow aspirate was inserted. The left fibula was used as the control group, where the collagen absorbable sponge was inserted without the osteogenic centrifuged aspirate. The centrifuged bone-marrow aspirate was arranged at the laboratory and submitted to nuclear cell count and cell viability test. The rabbits were killed at four weeks after surgery to evaluate bone callus formation. The results analysis was performed with DEXA bone densitometry to evaluate callus mineral mass, multislice computer tomography to evaluate callus volume and histomorphometry to evaluate the relative rate of tissue formation. RESULTS: The bone-marrow centrifugation technique increased the number of nucleated cells by three compared with the number of that cells in the original bone-marrow aspirates, without significant nucleated cell dead. The apply of centrifuged osteogenic bone-marrow aspirate resulted in an increased callus bone mineral mass by 40,3%, and increased relative rate of bone tissue formation by 9,4%, without increase the relative rate of cartilage or fibrous tissue. There was not increased callus volume. CONCLUSION: This study shows that the centrifuged osteogenic bone-marrow aspirate was able to improve the healing of experimental fibular osteotomies in rabbits by qualitative improve of bone callus.

TP508 is a 23 amino acid peptide derived from human prothrombin. It helps wound healing in both soft tissues and bones. In our previous study, we have demonstrated that TP508 retains chondrocyte in a less mature differentiation state while expanding the cartilage mass, indicating it may partly help bone healing by expand the cartilage template in the endochondral bone formation stage. In our current study, we want to demonstrate that TP508 also blocks chondrocyte apoptosis. We used rat costochondral growth plate chondrocytes as our model. We first established chelerythrine as an apoptogen in chondrocytes. TP508 is able to block apoptosis caused by chelerythrine. Chelerythrine also causes an increase in NO production, which is known to cause both pathological and physiological apoptosis of chondrocyte, and blocking NO production can in turn block apoptosis caused by them. TP508 is also able to block NO production caused by chelerythrine. Therefore, TP508 may partially block chondrocyte apoptosis by blocking NO production. From all above, we conclude that besides decreasing chondrocyte differentiation, TP508 also blocks their apoptosis, so as to conserve the cartilage template in endochondral bone formation

No description available.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O objetivo do estudo foi verificar os efeitos do carbonato de lodenafil no processo de consolidaÃÃo Ãssea nos fÃmures de ratos reduzidos com fio de Kirshner. Os fatores vasculares sÃo extremamente importantes para a formaÃÃo do calo Ãsseo. O carbonato de lodenafil causa efeitos vasodilatadores, pertence ao grupo dos inibidores da fosfodiesterase seletiva para o tipo 5 (PDE5), enzima que hidrolisa monofosfato de guanosina cÃclico (GMPc). O GMPc induz o relaxamento da musculatura lisa, sendo utilizado no tratamento da disfunÃÃo erÃtil. Foram utilizados 36 ratos machos, adultos, da linhagem Wistar, com o peso mÃdio de 300g. Todos os animais foram submetidos a cirurgia em que era realizada a fratura da diÃfise femoral com uma guilhotina romba. Os animais foram divididos em 2 grupos: o grupo lodenafil, que recebeu, via oral, doses diÃrias de carbonato de lodenafil (10mg/kg) e o grupo controle. Os animais foram sacrificados com 7, 14 e 28 dias de pÃs-operatÃrio. Houve duas mortes, dois falsos trajetos do fio e em trÃs animais nÃo foi estabelecida a fratura. Foram realizados estudo radiogrÃfico com filme de mamÃgrafo para analisar a densidade Ãptica e Ãrea do calo Ãsseo, medida em mm e estudo histolÃgico, utilizando amostras coradas com picrosirius red sob a luz poralizada do microscÃpio, para quantificar a formaÃÃo de colÃgeno tipo I e tipo III na regiÃo cortical prÃxima a fratura e no calo Ãsseo. A densidade de colÃgeno tipo I na regiÃo cortical do fÃmur prÃximo à fratura verificada no grupo lodenafil foi significantemente menor que a observada no grupo controle com 28 dias de consolidaÃÃo Ãssea (**P = 0,0028). Os resultados encontrados nÃo sÃo consistentes o suficiente para afirmar que este fÃrmaco exerce algum efeito na consolidaÃÃo Ãssea. Verificou-se que o Carbonato de lodenafil nÃo influenciou no processo de consolidaÃÃo Ãssea nos fÃmures de ratos reduzidos com fio de Kirshner. / The study was to investigate the effects of Lodenafil Carbonate in the process of bone healing in femurs of rats with reduced KirshnerÂs wire. The vascular factors are extremely important for the formation of callus. Lodenafil Carbonate cause vasodilatory effects, belongs to the group of selective phosphodiesterase type 5(PDE5) inhibitors, that enzyme inhibits cyclic guanosine monophosphate (GMPc) and relaxation of smooth muscles and are used to treat erectile dysfunction. It used 36 male rats, adult male rats, with the average weight of 300g. All animals underwent surgery in which fractures to the femoral shaft with a blunt guillotine. The animals were divided into 2 groups: lodenafil group, wich received oral doses of carbonate lodenafil (10mg/kg) and the control group. The animals were sacrificed at 7, 14 and 28 days postoperatively. There were two deaths, two false paths of the wire and three animals was not established fracture. Were performed radiographic studies with film mammography to analyze the optical density and area of callus, measured in mm  and histological study, using samples stained with picrosirius red under microscope light polarization, to quantify formation of collagen type I and type III in the cortical region near the fracture and callus. The density of type I collagen in the cortical region of the femur near the fracture observed in group lodenafil was significantly lower than that observed in the control group at 28 days of bone (** P = 0.0028). The results are not consistent enough to say that this drug has an effect on bone healing. It was found that the carbonate lodenafil not influence the process of bone healing in femurs of rats with reduced wire Kirshner.

Abstract Various bone proteins and growth factors are needed during the bone healing cascade. If the body cannot produce sufficient quantities of these factors, bone trauma healing can be improved with an implant that contains the required growth factors. However, an added bone protein extract needs a suitable delivery system to protect the proteins from degradation and to release them gradually, promoting new bone formation. This study focused on evaluating and optimization of the bone forming capacity of various scaffold systems of reindeer bone protein extract formulations using different experimental models. The tested carrier systems for various reindeer bone protein extract doses were collagen sponge and bioactive glass granules in critical-size defect model of rat femur. Calcium salt compositions (beta-tricalcium phosphate (β-TCP), hydroxyapatite or calcium sulphate) in disc and compressed pellet forms were tested in the thigh muscle pouch model of mouse. Various β-TCP granules combined with polyethylene/glycerol and stearic acid gel were tested in a hole defect model of sheep femur and humerus. Control groups involved carrier materials with no protein extract or untreated defects. In the sheep study, reference materials also included autograft and demineralized bone matrix (DBM). New bone formation, bone healing, and carrier resorption were evaluated based on radiographs, peripheral computerized tomography (pQCT), mechanical tests, histological examination, and micro-CT. New bone formation and bone union were markedly better in groups receiving higher doses of the extract and with follow-ups of six or more weeks, compared to empty defect or carrier without extract. Resorptions of carrier materials in active groups were faster and more active than in the control groups. The greatest bone formation occurred in the groups that had the bone protein extract readily available, which indicated that bone forming factors are required in sufficient concentrations at an early stage. The micro-CT analysis showed that bone formation in the groups with the extract was comparable to autograft, while the least bone formation was observed in the DBM and untreated groups. The present study indicated that the tested reindeer bone protein extract can be used to improve bone formation with various carriers. The study suggests that an inorganic carrier material together with stearic acid is the one of most suitable carrier alternatives for this extract. The developed medical device in paste form can be an alternative for autograft use. / Tiivistelmä Luun paraneminen vaatii erilaisia proteiineja ja kasvutekijöitä. Jos elimistö ei pysty tuottamaan riittävää määrää näitä tekijöitä, luumurtuma ei parane luonnollisesti vaan vaatii hoitoa, jossa murtuma alueelle viedään tarvittavia kasvutekijöitä. Kasvutekijät kiinnitetään tarkoituksenmukaiseen kantaja-aineeseen, jonka avulla kasvutekijöiden vapautumista ja toimintaa voidaan säädellä. Tutkimuksen tavoitteena oli arvioida ja optimoida erilaisissa eläinmalleissa porosta eristetyn luuproteiiniuutteen kantaja-ainesysteemien toimivuutta. Testatut kantaja-ainemateriaalit olivat kollageenihuopa ja bioaktiivinen lasirae. Näitä testattiin useilla luuproteiiniannoksilla, ja mallina oli rotan reisiluun kriittisen koon murtuma. Hiiren reisilihasmallilla testattiin kalsiumsuolayhdistelmiä (beta trikalsiumfosfaatti (β-TCP), hydroksiapatiitti ja kalsiumsulfaatti) tablettina ja pellettinä. Lampaan reisi- ja olkaluiden reikämurtumamallilla testattiin erilaisia β-TCP-rakeita yhdistettynä polyetyleenistä, glyserolista ja steariinihaposta valmistettuun geeliin. Kontrollisryhmistä toinen sisälsi kantaja-aineen ilman luuproteiiniuutetta, toisessa ryhmässä murtuma jätettiin kokonaan hoitamatta. Lammaskokeessa verrattiin lisäksi omaluusiirteen ja demineralisoidun luumateriaalin (DBM) toimivuutta verrattuna poron luuproteiiniuutteeseen. Uudisluun muodostuminen, murtuman paraneminen ja kantaja-aineen hajoaminen arvioitiin natiiviröntgenillä, perifeerisellä tietokonetomografialla (pQCT), mekaanisin testein, histologisesti ja mikro-CT:llä. Luuproteiiniuutetta sisältäneillä ryhmillä oli luun paraneminen ja kantaja-aineen hajoaminen merkittävästi parempaa kuin uutetta sisältämättömillä ryhmillä. Uudisluun muodostuminen oli suurempaa korkeammilla annoksilla ja pitemmillä seuranta-ajoilla. Suurin uudisluun muodostus mitattiin ryhmillä, joissa luuproteiiniuute oli heti käytettävissä implantoinnin jälkeen. Tämä osoittaa, että varsinkin murtuman paranemisen alkuvaiheissa tarvitaan luuta muodostavia kasvutekijöitä riittävinä pitoisuuksina. Mikro-CT-analyysit osoittivat, että luuproteiiniuuttetta sisältäneet ryhmät olivat verrannollisia omaluusiirrehoidolle. Vähiten uudisluuta havaittiin DBM ja tyhjäreikäryhmissä. Tutkimus osoittaa, että poron luuproteiiniuutetta erilaisten kantajamateriaalien kanssa voidaan käyttää parantamaan luun muodostumista. Erityisesti epäorgaaninen kantajamateriaali steariinihapon kanssa on yksi soveltuvimmista vaihtoehdoista luu-uutteelle. Kehitelty pastamutoinen lääkinnällinen laite, joka sisälsi poron luuproteiiniuutteen ja kalsiumsuolakantaja-aineen, osoittautui vaihtoehdoksi omaluusiirrehoidolle.

As fraturas são consideradas um dos maiores problemas de saúde pública pelo custo e morbidade a elas associados. O objetivo deste estudo foi investigar os efeitos do ultrassom de baixa intensidade (LIPUS) e do laser de baixa potência (LLLT) sobre o reparo ósseo de tíbia de ratos (Wistar) utilizando a microtomografia por raio-x (&#956CT) e a análise histomorfométrica. Uma osteotomia total no terço médio foi fixada com fio de Kirschner (0,89 mm) e utilizado um espaçador polimérico de 2 mm de espessura e 1 mm de diâmetro entre os fragmentos proximal e distal para induzir retardo de consolidação da fratura. Foram utilizados 24 animais separados aleatoriamente em 4 grupos experimentais: Grupo Controle: sem tratamento do retardo da consolidação (n = 5); Grupo Laser: tratamento da fratura com laser (n = 6); Grupo LIPUS: tratamento da fratura com LIPUS (n = 7) e; Grupo LIPUS + Laser: tratamento da fratura com LIPUS e Laser (n = 7). Os tratamentos com LIPUS e/ou Laser foram iniciados após 5 semanas da cirurgia e realizados durante 5 dias consecutivos de tratamento, seguidos por 2 dias sem serem tratados até totalizarem 12 sessões terapêuticas. Os parâmetros de irradiação laser foram: 808 nm; 100 mW; 2500 mW/cm² , 125 J/cm² durante 50 segundos. Os parâmetros do LIPUS foram: 1,5 MHz, ciclo de trabalho 1:4, intensidade de 30 mW/cm², 20 minutos por sessão. Quando as duas técnicas foram associadas, o laser foi aplicado perpendicularmente ao LIPUS no décimo minuto de aplicação do LIPUS. Radiografias da tíbia foram feitas para acompanhar a evolução da fratura após a cirurgia, antes dos tratamentos e antes da eutanásia. Após a eutanásia foram realizadas avaliações microtomográficas por raio-x e histomorfométricas para análise do reparo ósseo. Na análise microtomográfica do reparo ósseo o tecido neoformado de reparo (calo ósseo) e o tecido normal não foram isolados. Na análise estatística foi utilizada a ANOVA one-way com posthoc de Tukey. Não foi constatada diferença significativa para maioria das variáveis, exceto para o fator padrão trabecular (Tb.Pf) no grupo LIPUS+Laser comparado aos grupos LIPUS (p = 0.02), Laser (p = 0.02) e Controle (p = 0.01), para a conectividade (Conn) no grupo Laser comparada ao grupo Controle (p = 0.04) e para a densidade de conectividade (D.Conn) no grupo LIPUS+Laser comparado ao grupo Controle (p = 0.04). As imagens histológicas mostram maior quantidade de tecido esponjoso no grupo LIPUS+Laser. Parâmetros morfométricos e histomorfométricos não revelaram um avanço do reparo ósseo na comparação entre os grupos com tratamento em relação ao grupo controle provavelmente devido a não reabsorção do espaçador polimérico pelo organismo dos animais. A &#956CT é uma técnica de grande potencial que permite análises qualitativas e quantitativas e reconstruçoes 2D e 3D. / Bone fractures are important public health problems due to the morbidity and high cost related to them. The aim of this study was to evaluate the effects of low pulsed intensity ultrasound (LIPUS) and infrared low level laser therapy (LLLT) in the bone repair of a transverse rat tibia osteotomy with assessement by x-ray microtomography (&#956CT) and histomorphometry. An easily implemented transverse osteotomy with a Kirchner wire and 2 mm width polymeric spacer beads established a delayed union in the fracture. Twenty four rats were divided into four experimental groups: Control Group - untreated; Laser Group - treated with infrared LLLT; LIPUS Group treated with low intensity pulsed ultrasound; LIPUS+Laser Group - treated with low intensity pulsed ultrasound and infrared LLLT. The treatments with infrared LLLT (808 nm, 100 mW, 2500 mW/cm², 125 J/cm², 50 seconds) and LIPUS (1,5 MHz, 1:4 duty cicle, 30 mW/cm², 20 minutes) started 5 weeks after the surgery following a sequence of 5 days on and 2 days off until 12 sessions were reached. When LLLT and LIPUS therapies were used simultaneously there was a 90º angle between the laser and the ultrasound beams. The surgical technique and the bone repair were assessed by x-ray radiography. One-way analysis of variance (one-way ANOVA) and Tukey post-hoc were used for statistical analysis. The normal bone and the callus were kept together in the &#956CT analysis. There was no significant difference between treatments results except for the parameters trabecular pattern factor - Tb.Pf [US+Laser group x LIPUS group (p = 0.02); US+Laser group x Laser group (p = 0.02) and US+Laser group x Control group (p = 0.01)], connectivity - Conn [Laser group x Control group (p = 0.04)] and connectivity density D.Conn [US+Laser group x Control group (p = 0.04)]. The histology showed greater amount of spongeous bone in the LIPUS+Laser group. The morphometric and histomorphometric assessments didn´t show a faster bone repair when treated groups were compared against the control one. The non absorption of the polymeric spacer bead is probably the reason of these findings. The &#956CT is a powerful technique that allows 2D and 3D quantitative analysis and reconstructions.

There are approximately 600,000 cases of delayed or aberrant fracture healing in people each year, with a small subset of these fractures experiencing disunion. Dietary phosphate deficiency has been shown to impair oxidative phosphorylation and decrease BMP-2 mediated chondrogenic differentiation during fracture healing. Prior studies using pre-committed chondro-progenitor ATDC5 cell line grown in phosphate deficient media showed that energy consumption was linked to protein production and collagen hydroxylation but inversely related to matrix mineralization. The goal of this study was to further define the relationship between energy consumption and BMP-2 mediated stem cell chondrogenic differentiation and further examine how dietary phosphate, and promotion of collagen hydroxylation via ascorbate availability effected these processes. C3H10T1/2 murine cells, a multi-potential cell line, were expanded in pre-differentiation growth medium (DMEM with 10% FBS and 1% Pen/Strep). Once cells reached 60% confluence (day 0), they were grown in differentiating media (α-MEM with 5% FBS and 1X insulin-transferrin-selenium) containing either 100% (1mM) or 25% (0.25mM) inorganic phosphate (Pi), ± 200ng/mL BMP-2(BMP), and ±0.2 mM L-ascorbic acid (AA). In total, there were 8 groups with varying combinations of these three substances. Intracellular lipid, total DNA, protein, and hydroxyproline (HP) content were examined. Chondrocyte gene expression (Col2a1, Acan, ColXa1) and adipocyte gene expression (Pparg, Plin1, Ucp1) were measured to check for cell lineage commitment and specific differentiation of the C3H10T1/2. All measurements were acquired at day 8. The +BMP differentiation media groups contained significantly less DNA content and more protein content than the –BMP differentiation media groups (both p<0.0001). There was also a significant interaction between phosphate and ascorbic acid treatment (p=0.0296), with 25% Pi +AA groups producing significantly more protein than 100% Pi +AA groups. Hydroxyproline production was not different in 100% Pi or 25% Pi conditions (p=0.2951). AA presence in culture media led to greater HP production than culture media lacking AA (p=0.0035) There was a trend of an interaction between phosphate content and AA availability (p=0.0744). 100% Pi ±AA groups produced significantly different amounts of HP while 25% Pi ±AA groups did not produce significantly different amount of HP. Col2a1, Acan, and ColXa1 expression were all increased in +BMP groups. Ascorbic acid treatment groups expressed significantly more Col2a1and Acan than –AA groups. 100% Pi media led to greater Acan expression over 25% Pi groups (p=0.0009), whereas 25% Pi media trended to lead to greater ColXa1 expression over 100% Pi groups (p=0.0734). Pparg and Plin1 expression were increased in the 25% Pi condition. There were no significant differences in expression of Ucp1. C3H10T1/2 cells were significantly affected by phosphate concentration, BMP-2 treatment, and ascorbic acid supplementation. Phosphate deficiency hindered maturation of early chondrocytes into proliferating chondrocytes while also promoting MSC differentiation into the adipocyte cell lineage. Hypertrophic chondrocyte expression was decreased in phosphate deficient media, which may coincide with increased protein production observed in low phosphate conditions. BMP-2 promoted chondrogenesis which resulted in increased protein production. Whereas, lack of ascorbic acid in cell culture media led to decreased hydroxyproline production.