Dissertations / Theses on the topic 'Osteogenesis in rat'

The objective of the present study was to synthesize porous, biodegradable poly (D, l- lactide-co-glycolide) PLGA-B-HA (Bovine hydroxyapatite) composite and evaluate the effect of ceramic content on bone marrow cell differentiation in vitro. A macroporous biodegradable PLGA-B-HA composite with the pore size varying from 0.1 to 1000?? and a highly interconnected structure was fabricated using the freeze-drying/lyophilization technique. A pilot study was done to determine the effects of B-HA on to the osteoblast function. The main study was done to determine the effect of the increase in B-HA concentration on to the mesenchymal stem cell differentiation. Morphological characteristics of the composites were analyzed using FTIR and SEM/EDX analysis. The composites were seeded with neonatal rat calvarial osteoblasts (NRCO). The polymer: ceramic ratio in this study was 35%:65%. For comparison parallel experiments involving pure HA-200 discs were performed. SEM results indicated a higher proliferation and mineralization on PLGA-B-HA composites than pure HA discs. In addition, we evaluated the in vitro characteristics of PLGA-B-HA composites with varying ratios, i.e., 1:1, 1:2 and 1:3, seeded with rat marrow cells. FTIR indicated an increase in the area under the ceramic peak as ceramic concentration was increased. In addition, the average roughness values increased in the order of 1:3 andgt; 1:2 andgt; 1:1. Both compressive strength and modulus of 1:1 were significantly higher than 1:2 and 1:3 PLGA-B-HA composites. No significant difference in compressive modulli and strengths could be observed for 1:2 and 1:3 PLGA-B-HA composites. Cellular activity was determined by measuring AP activity, total protein analysis and osteocalcin concentration. Evaluation of alkaline phosphatase activity showed bone cells attached to 1:3 (PLGA-B-HA) expressed significantly higher alkaline phosphatase as compared to 1:1 and 1:2 PLGA-B-HA composites. In addition, cells seeded on to 1:3 composites secreted significantly higher osteocalcin and at a relatively short time period as compared to the other samples. Corrosion studies (ICP) and pH values indicate minimal difference in the concentration of Ca and P and pH in tissue culture media for all the samples at the end of all time periods. Hence we conclude that an increase in the ceramic concentration stimulated mesenchymal stem cell differentiation thereby promoting osteogenesis.

Les variations de contraintes mécaniques appliquées sur l'os entrainent des modifications du métabolisme osseux. La diminution des contraintes mécaniques telle qu'elle existe dans le modèle du rat suspendu, induit une perte osseuse rapide dès le début de la suspension qui se stabilise par la suite. L'augmentation des contraintes par un exercice de type dynamique (la course sur tapis roulant) induit un gain osseux. Les mécanismes cellulaires responsables de ces modifications sont encore mal connus. Grâce à la mise au point d'une méthode d'isolement des cellules ostéoformatrices à partir du tibia de rat, nous avons pu isoler les cellules des facteurs environnementaux et les maintenir en culture primaire. Les études in vitro sur les différents modèles animaux précités, ont permis de montrer que la diminution de contrainte entraine une inhibition de la maturation des ostéoblastes conduisant à une perte osseuse, qui cesse dès que les contraintes sur l'os se retrouvent au niveau initial. Inversement, l'exercice stimule la différenciation des ostéoblastes en ostéoblastes matures aboutissant à un gain osseux qui diminue les contraintes mécaniques de l'os. Nous avons pu étudier l'effet direct de la microgravité sur les cellules ostéoblastiques grâce au développement d'un module de culture et de techniques d'investigations adaptées aux conditions expérimentales du vol spatial biocosmos x. Les résultats de cette expérience ont montré que la cellule ostéoblastique est capable de s'adapter à son nouvel environnement physique en modifiant son activité cellulaire et l'organisation de son cytosquelette. Nous pensons que les contraintes mécaniques pourraient avoir une influence sur le cytosquelette des ostéoblastes qui modifierait l'activité cellulaire, permettant à l'os de s'adapter à son nouvel environnement

Bone is a remarkable multifunctional tissue with the ability to regenerate and remodel without generating any scar tissue. However, bone loss due to injury or diseases can be a great challenge and affect the patient significantly. Transplanting bone graft from one site in the patient to the site of fracture or bone void, i.e. autologous bone grafting is commonly used throughout the world. The transplanted bone not only fills voids, but is also bone inductive, housing the particular cells that are needed for bone regeneration. Nevertheless, a regenerative complement to autograft is of great interest and importance because the benefits from an off-the-shelf product with as good of healing capacity as autograft will circumvent most of the drawbacks with autograft. With a regenerative-medicine approach, the use of biomaterials loaded with bioactive molecules can avoid donor site morbidity and the problem of limited volume of material. Two such regenerative products that utilize bone morphogenetic protein 7 and 2 have been used for more than a decade in the clinic. However, some severe side effects have been reported, such as severe swelling due to inflammation and ectopic bone formation. Additionally, the products require open surgery, use of supra physiological doses of the BMPs due to poor localization and retention of the growth factors. The purpose of this thesis was to harness the strong inductive capability of the BMP-2 by optimizing the carrier of this bioactive protein, thereby minimizing the side effects that are associated with the clinical products and facilitating safe and localized bone regeneration at the desired site. We focused on an injectable hyaluronan-based carrier. The strategy was to use the body’s own regenerative pathway to stimulate and enhance bone healing in a manner similar to the natural bone-healing process. The hyaluronan-based carrier has a similar composition to the natural extracellular matrix and is degraded by resident hyaluronidase enzymes. Earlier studies have shown a more controlled release and improved mechanical properties when adding a weight of 25 percent of hydroxyapatite, a calcium phosphate that constitutes the inorganic part of the bone matrix. In Paper I, the aim was to improve the carrier by adding other forms of calcium phosphate. The results indicated that the bone formation was enhanced when using nano-sized hydroxyapatite. We wished to further develop the carrier system but were lacking an animal model with high output and easy access. We also wanted to provide paired data and were committed to the 3 Rs of refinement, reduction and replacement. To meet these challenges, we developed and refined an animal model, and this is described in Paper II. In Paper III, we characterized and optimized the handling properties of the carrier. In Paper IV, we discovered the importance of crushing the material, thus enhancing permeability and enlarging the surface area. In Paper V, we sought to further optimize biomaterial properties of the hydrogel through covalently bonding of bisphosphonates to the hyaluronan hydrogel. The results demonstrated exceptional retention of the growth factor BMP-2. In Paper VI, the in vivo response related to the release of the growth factor was examined by combining a SPECT/PET/µCT imaging method to visualize both the retention of the drug, and the in-vivo response in terms of mineralization.

Bone is a remarkable multifunctional tissue with the ability to regenerate and remodel without generating any scar tissue. However, bone loss due to injury or diseases can be a great challenge and affect the patient significantly. Autologous bone grafting is commonly used throughout the world. Autograft both fills the void and is bone inductive, housing the particular cells that are needed for bone regeneration. However, a regenerative complement to autograft is of great interest as the use of biomaterials loaded with bioactive molecules can avoid donor site morbidity and the problem of a limited volume of material. Two such regenerative products that utilise bone morphogenetic protein (BMP)-7 and -2 have been used for more than a decade clinically. Unfortunately, several side effects have been reported, such as severe swelling due to inflammation and ectopic bone formation. Additionally, the products require open surgery and use of supra physiological doses of the BMPs due to poor localisation and retention of the growth factor. The purpose of this thesis was to harness the strong inductive capacity of the BMP-2 by optimising the carrier of this bioactive protein, thereby minimising the side effects that are associated with the clinical products and facilitating safe and localised bone regeneration. We focused on an injectable hyaluronan-based carrier developed through polymer chemistry at the University of Uppsala. The strategy was to use the body’s own regenerative pathway to stimulate and enhance bone healing in a manner similar to the natural bone-healing process. The hyaluronan-based carrier has a similar composition to the natural extracellular matrix and is degraded by resident enzymes. Earlier studies have shown improved properties when adding hydroxyapatite, a calcium phosphate that constitutes the inorganic part of the bone matrix. In Paper I, the aim was to improve the carrier by adding other forms of calcium phosphate. The results indicated that bone formation was enhanced when using nano-sized hydroxyapatite. In Paper II, we discovered the importance of crushing the material, thus enhancing permeability and enlarging the surface area. We wished to further develop the carrier system, but were lacking an animal model with relatively high throughput, facilitated access, paired data, and we were also committed to the 3Rs of refinement, reduction, and replacement. To meet these challenges, we developed and refined an animal model, and this is described in Paper III. In Paper IV, we sought to further optimise the biomaterial properties of the hydrogel through covalent bonding of bisphosphonates to the hyaluronan hydrogel. This resulted in exceptional retention of the growth factor BMP-2. In Paper V, SPECT/PET/µCT was combined as a tri-modal imaging method to allow visualisation of the biomaterial’s in situ action, in terms of drug retention, osteoblast activity and mineralisation. Finally, in Paper VI the correlation between existing in vitro results with in vivo outcomes was observed for an array of biomaterials. The study identified a surprisingly poor correlation between in vitro and in vivo assessment of biomaterials for osteogenesis.

In comparison to their ecophysiological and behavioral aspects, the skeletal system of African mole-rats (Bathyergidae) has been relatively understudied. Only a few studies have assessed their skeletal system, but these have mostly focused on their cranial and dental systems, with little attention on their postcranial skeleton. This PhD thesis provides a considerable amount of information about the functional anatomy, morphological diversity and postnatal bone morphogenesis of the appendicular system of these subterranean mammals. African mole-rats are small mammals highly adapted to the hypogeous niche that feed on underground roots and tubers. They forage, mate, breed and to some extent even disperse underground. For this, they build extensive burrow systems primarily with their chisel-like teeth, but also using their forelimbs for scratch-digging. One of the most exceptional features of bathyergids is their wide spectrum of social organization, which is unique among mammals and ranges from solitary, social and eusocial. Here, the eusocial naked mole-rat, Heterocephalus glaber, has been the most studied species. The physiology of African mole-rats is also exceptional among rodents and other mammals, showing low metabolic rates and body temperatures, as well as slow somatic growth rates. They also show enhanced fitness and prolonged longevity, features that have been associated to a life protected from both climatic extremes and predation, as well as to intergenerational transfer of information, communal care of young and shared foraging endeavors in social species. For these reasons, bathyergids represent a unique animal model to explore their skeletal adaptations to fossoriality and life underground. The aim of this research was to assess the patterns of bone growth and development to understand how adults attain their final phenotype. A comprehensive sample (N = 506) of all six bathyergid genera including seven species and comprising individuals of both sexes and of different ontogenetic stages was studied. Stylopodial (humerus and femur) and zeugopodial (ulna and tibia-fibula) bones (n = 1133) were analyzed using multiple quantitative analyses of variance (ANOVA, MANOVA), ordination (PCA, DA) and regression (RMA, OLS, equality of slopes), as well as bone labeling techniques and detailed qualitative descriptions of their midshaft bone histology. Chapter 3 shows that the specialized phenotype of the only scratch-digger bathyergid Bathyergus suillus underwent considerable morphological changes during ontogeny, e.g. juveniles showed externally more robust bones with thin cortical walls, whereas adults presented slender bones with significantly thicker cross-sections. Such changes are probably related to the increased digging demands and agonistic behaviors of the developing young. However, other aspects of their anatomy expressed perinatally, such as greater external epicondylar robustness, well-developed olecranon, teres major and deltoid processes, suggest a major role of genetic factors in their development. This chapter applied for first time the conceptualization of developmental modules to long bones, and showed that the periosteal module had higher variability and tended to grow faster than the endochondral module. Chapter 4 analyzed the morphological diversity within Bathyergidae using comparative anatomy and morpho-functional indices and showed that most species shared a highly specialized fossorial morphology and that only the naked mole-rats were morphologically divergent (having a simplified phenotype), resembling the condition of non-fossorial closest relatives of the Bathyergidae. Nevertheless, the novel inclusion of three ecomorphological categories (solitary scratch-diggers, solitary chisel-tooth diggers and social chisel-tooth diggers) in this study, showed significant differences among the groups. In general, social species appeared to have a phenotype more specialized to increase digging ability and locomotor performance, whereas solitary species showed a relatively less specialized fossorial phenotype, and a diminished locomotor ability. This may contribute to foraging strategies in social species which are known to have more complex and relatively longer burrow systems as compared to solitary species. Chapter 5 assesses the ossification patterns of the endochondral and periosteal modules, and shows that in general most bathyergids have relatively similar endochondral growth rates, irrespective of social behavior or digging strategy, although the periosteal module showed relatively higher growth rates and a higher degree of variation as compared to the endochondral module, thus appearing to be considerably less dependent on body size and genetic factors. Naked mole-rats showed the lowest growth rates among bathyergids. Considering the basal phylogenetic position of H. glaber within the family, a neotenic condition is suggested for this species, and suggests accelerated bone growth rates for the evolution of the other bathyergids. Chapter 6 provides a comprehensive description of the pattern of bone modelling in bathyergids and includes an assessment of their bone dynamics using fluorochrome labeling. All bathyergids analyzed showed increased cortical bone thickening during ontogeny, as well as low rates of endosteal bone resorption. Also, all species showed high histodiversity, limited remodeling (i.e. development of secondary osteons) and they do not ever develop Haversian bone tissues. This thesis concludes that the combination of social strategy and type of excavation had an impact on the evolution of the bathyergid appendicular system. On one hand, it was evidenced that the development of fore- and hindlimbs are not constrained by intrinsic factors (as suggested for other mammals), and that the limbs develop at similar growth rates, resulting in relatively symmetrical limb proportions. This is suggested to improve locomotion within burrows and represents an adaptation to the subterranean lifestyle, which is also observed in other fossorial mammals. This thesis further discusses how environmental factors and specific behaviors and locomotor modes, may represent strong selective pressures on limb adaptation and evolution. Similarly, a proximo-distal pattern of variation was observed, where zeugopodial elements were more variable than stylopodial elements, probably because they are in direct interaction with the substrate, so they can evolve morphological adaptations for particular habitats and locomotor behaviors. Importantly, these adaptations are most likely mediated by heterochronic modifications of their ossification modules, especially intramembranous ossification, which is known to be more responsive to environmental factors, whilst the endochondral modules would be more conservative, perhaps because a stronger genetic regulation in postnatal life. Further research on long bone modules is necessary to understand the specificity of such changes. Despite the comparatively simplified phenotype of H. glaber, they showed a larger morphospace as compared to other bathyergids, indicating a wider intraspecific variability. This agrees with previous observations suggesting skeletal plasticity for this species. It is suggested that living in large colonies results in diminished selective pressures for limb specialization but has an impact on increasing trait variability within members of the colony. This study showed that the integration of multiscale techniques and multivariate analysis of combined skeletal phenotypes (i.e. forelimb + hindlimb) offer a better understanding of adaptations to the hypogeous environment. The findings of this study also highlight the importance of considering developmental modularity of long bones for assessment of bone adaptations, particularly for understanding the differential effects of intrinsic and extrinsic factors regulating endochondral and intramembranous ossification.

The effects of the rate of distraction and of local infusion of IGF-1 upon bone deposition during mandibular distraction osteogenesis was studied in a rabbit model. Five groups of rabbits were studied. All rabbits, except sham operated controls, underwent distraction to 15 mm. The variables studied were the rate of distraction (0.5 mm twice a day versus 1.5 mm twice a day) and the effects of local IGF-1 infusion via osmotic infusion pumps. Analysis by DEXA scanning and three point bending 28 days after the end of distraction demonstrated no difference in density or strength of bone between the experimental groups. Histological examination demonstrated non-union across the distraction gap to be more common in rapidly distracted rabbits. Histomorphometric analysis demonstrated higher mineral apposition rates and less un-mineralised osteoid with slow as opposed to rapid distraction (p = 0.0001). Infusion of exogenous IGF-1 also resulted in a small increase in mineral apposition rate which was significant at slow but not a rapid distraction. Bone densitometry and three point bending results did not reveal any effect of distraction rate or IGF-1 infusion other than greater stiffness associated with IGF-1 infusion during rapid distraction (p = 0.01). It seems probable that the overwhelming stimulus to new bone formation produced by distraction renders the anabolic effects of IGF-1 less significant making it detectable by only the more sensitive analysis. This may be due to maximal stimulation of IGF-1 production by slow distraction thus rendering the administration of exogenous IGF-1 relatively superfluous. Rapid distraction may produce less stimulus to growth factor synthesis or the level of production maybe insufficient to cope with a higher requirement. The complete union of rapidly distracted rabbits who received exogenous IGF-1 may be as a result of levels being restored to optimal.

INTRODUÇÃO: A Osteogênese Imperfeita (OI) é uma doença genética do tecido conjuntivo caracterizada por fragilidade óssea e grande suscetibilidade de fraturas aos mínimos traumas. OBJETIVO: Avaliar e descrever o tratamento com pamidronato de sódio cíclico nas formas moderada e grave de Osteogenesis Imperfecta (OI) em um Centro Referência de Tratamento para OI no Sul do Brasil. METODOLOGIA: Foi realizado um estudo de coorte retrospectivo com crianças e adolescentes, segundo os critérios da Organização Mundial de Saúde (OMS), de ambos os gêneros, com diagnóstico de OI nas formas moderada e grave que receberam tratamento cíclico de pamidronato de sódio no CROI – HCPA no período de 2002 a 2012. Os parâmetros clínicos foram obtidos durantes as consultas médicas para acompanhamento dos pacientes com OI e internações para tratamento com pamidronato de sódio. Os dados bioquímicos foram coletados durante a internação dos pacientes para infusão cíclica de pamidronato de sódio. Cálcio, Fósforo e Fosfatase Alcalina foram coletados sistematicamente. A densidade mineral óssea foi mensurada através do DXA (dual energy x-ray absoptometry) em coluna lombar (L1-L4) e corpo total. Para a análise dos dados foi utilizado Statistical Package for the Social Sciences (SPSS) Version 18. Foram considerados valores de significativos p < 0,05. RESULTADOS: Foram revisados dados de prontuário de 48 pacientes com OI, sendo 3 excluídos da amostra por apresentarem dados incompletos. A mediana da taxa de fraturas/mês reduziu significativamente após o primeiro ano de tratamento para todos os tipos de OI (p<0,01). Também para os tipos III e IV houve redução significativa da taxa de fraturas antes e após 1 ano de tratamento. Houve redução de 71,4% no número de fraturas após o tratamento na amostra geral. Esta redução foi maior na OI tipo III (86%) e tipo IV (78,6%) seguido do tipo I (60%). A mobilidade dos pacientes apresentou melhora significativa ao final do tratamento (p=0,004). Houve aumento significativo na DMO do corpo total do 1° ano para 6° em diante (p<0,001). Em relação à coluna lombar (L1-L4) o aumento foi observado a partir do 4° ano (p<0,001). Vinte e quatro pacientes (54,5%) tiveram alguma intercorrência durante o tratamento, sendo a maioria destas observadas no primeiro ciclo de tratamento. Quanto à adesão ao tratamento, a média do percentual foi de 92,3% (± 10,7). Houve associação positiva e significativa entre adesão ao tratamento e o número de fraturas por ano (rs=0,319; p=0,033), ou seja, maiores percentuais de adesão são obtidos em indivíduos com maior número de fraturas por ano. CONCLUSÃO: Nossos dados mostraram a variabilidade clínica da OI e a sua melhora ao longo do tratamento com pamidronato. Os resultados sugerem um incremento da DMO dos pacientes ao longo do tratamento e principalmente a redução das taxas de fratura ao longo do tratamento. O uso de pamidronato foi bem tolerado, com eventos adversos leves.
BACKGROUND: Osteogenesis Imperfecta (OI) is a genetic connective tissue disorder characterized by bone fragility and susceptibility to fractures to minimal trauma. OBJECTIVE: To evaluate and describe the treatment of cyclic sodium pamidronate in moderate and severe forms of Osteogenesis imperfecta (OI) at a Reference Center for OI Treatment in Southern Brazil. METHODS: A retrospective cohort study was conducted with children and adolescents diagnosed with OI in moderate and severe forms receiving cyclical sodium pamidronate from 2002 to 2012. The clinical data were obtained at hospitalization for treatment with sodium pamidronate and at follow-up visits. Biochemical data as calcium, phosphorus and alkaline phosphatase were systematically collected. Bone mineral density was measured using DXA (Dual Energy X-ray Absoptometry).For data analysis SPSS V. 18 was used. We considered significant p < 0.05. RESULTS: Medical charts were reviewed from 48 patients with OI and three were excluded due to incomplete data. The median fracture per month rate decreased significantly after the first year of treatment for all types of OI (p <0.01). Also for the types III and IV there was a significant reduction in the rate of fractures before and after 1 year of treatment. We observed a reduction of 71.4% in the number of fractures after treatment in the general sample. This reduction was higher in Type III (86%) and type IV (78.6%) followed by type I (60%). The median fracture/month rate decreased significantly after the first year of treatment for all types of OI (p <0.01). Also for the types III and IV there was a significant reduction in the rate of fractures before and after 1 year of treatment. In relation to the mobility of patients improved significantly after the end of treatment (p = 0.004). Was it is observed that regardless of the OI, a significant increase in BMD of the total body of 1 year to 6 onwards (p <0.001). In relation the spine (L1-L4) is increased from the 4 th year (P <0.001). Twenty-four patients (54.5%) had some problems during treatment, most of these observed in the first treatment cycle. As for adherence to treatment, the mean percentage was 92.3% (± 10.7). Of the total sample, 26 patients (57.8%) fully completed the full treatment. There were significant positive association between adherence to treatment and the number of fractures per year (rs = 0.319, p = 0.033), that is, higher adhesion percentages are obtained in individuals with more fractures per year. CONCLUSION: Our data showed improvement of BMD and mobility and decreasing of fracture rate with cyclic pamidronate treatment. The treatment was well tolerated with mild adverse events.

Orientador: Jose Ricardo de Albergaria-Barbosa
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-07-21T20:50:26Z (GMT). No. of bitstreams: 1 Silva_ClaudiaJordao_M.pdf: 4090867 bytes, checksum: bfd1ff3ed0e4830528fe82ad6f674596 (MD5) Previous issue date: 1996
Resumo: O propósito deste trabalho foi avaliar histologicamente o processo de reparo de defeitos ósseos, criados cirurgicamente em calvária de coelho, com ou sem enxerto ósseo e submetidos ou não a eletroestimulação. Foram utilizados 18 coelhos, sendo que cada um deles recebeu duas osteotomias na calvária. Na osteotomia do osso parietal direito o fragmento removido foi imediatamente reposicionado, sendo que no parietal esquerdo o fragmento foi desprezado. Os animais dos grupos tratados receberam a eletroestimulação por uma hora, durante 21 dias. Já os animais do grupo controle foram submetidos as mesmas condições de stress, porém com o aparelho desligado. Os períodos de sacrifício foram as quatro, oito e quinze semanas, compreendendo seis animais sacrificados em cada período. A análise dos espécimes sob a microscopia óptica revelou neoformação óssea em todos os animais. Nos animais com enxerto ósseo houve uma reparação mais acentuada quando comparados com o grupo sem enxerto. A eletroestimulação parece ter alterado o processo de reparo ósseo de maneira discreta
Abstract: The purpose of this research was to histologicalIy evaluate the healing process in bone defects surgically created in calvaria of rabbits, with or without bone grafts under the action of eletrotherapy or not. It were utilized 18 rabbits with 2 perfurations in each calvaria. In the right parietal bone perforation the fragment removed was immediately grafted in its place. lu the left parietal bone the fragment was thrown away. The animais in the treated group received 1 hour of eletrotherapy dayly, during 21 days. The animals in the control group were submitted to the same stress conditions, but the stimulator was turned off. The sacrifice periods were 4, 7 and 15 weeks, with 6 sacrificed animais in each group. The analysis of the specimens under optical microscopy revealed bone formation in all animals. In the animals with the bone grafted there was a more noticeable healing when compared to the animais in the control group. With regard the electrotherapy it seems to affect the bone healing mildly
Mestrado
Cirurgia e Traumatologia Buco-Maxilo-Faciais
Mestre em Clínica Odontológica

RESUMO Introdução: Osteogênese imperfeita (OI) é uma enfermidade que leva à fragilidade e redução da massa óssea, não secundária a qualquer outra condição conhecida. Devido à importância do estado nutricional para a saúde óssea, há necessidade de melhor conhecimento sobre o estado nutricional, a composição corporal e a ingestão alimentar de indivíduos com OI. Objetivo: Avaliar o estado nutricional e o consumo alimentar de adolescentes e adultos com OI. Métodos: Estudo caso-controle, com adolescentes e adultos de ambos os sexos. Todos os indivíduos foram submetidos a avaliações do estado nutricional (IMC, comprimento supino e envergadura), da composição corporal e densidade mineral óssea (DMO) pelo Dual energy X-ray absorptiometry DXA, ingestão alimentar (diário alimentar de 3 dias), avaliação bioquímica [cálcio total, fósforo, creatinina, albumina, CTX, PTH e vitamina D], avaliação da atividade física, determinação da Taxa de Metabolismo Basal (TMB). Para análise dos resultados foram utilizados One Way ANOVA, teste T-Student e testes de correlação de Pearson e considerados os valores de p<0,05. Resultados: Participaram do estudo 26 indivíduos com OI (50% OI tipo I/ 50% OI tipo III) e 8 indivíduos saudáveis pareados por sexo e idade. O nº de fraturas foi maior nos indivíduos com OI tipo III, em média 60 fraturas comparado a 19 no OI tipo I. A DMO em coluna lombar L1-L4 foi significativamente maior no grupo controle comparado aos tipos de OI I e III [-0,4(0,5) vs. -2,7(1,0) e - 2,7(0,6) g/cm2, p<0,05], respectivamente. Segundo o IMC, 100% dos indivíduos do grupo controle encontram-se eutróficos, 46% dos indivíduos com OI tipo III obesos e 31% dos indivíduos OI tipo I com sobrepeso. Quanto ao percentual de gordura corporal, encontrou-se 14% de indivíduos com excesso de gordura corporal no grupo controle, 83% nos indivíduos com OI tipo III e 42% nos indivíduos com OI tipo I. No grupo caso houve correlação negativa entre número de fraturas e massa magra e correlações positivas entre número de fraturas e porcentagem de gordura corporal e IMC. Não houve diferença na ingestão de cálcio e de fósforo entre os grupos. Entretanto, a vitamina D foi significativamente menor no grupo controle. Somando-se a ingestão e a suplementação de nutrientes, 58% e 12% dos indivíduos do grupo caso não alcançaram os valores recomendados de ingestão de cálcio e vitamina D, respectivamente. Os indivíduos com OI tipo III apresentaram concentrações séricas de fósforo significativamente maiores quando comparados aos com OI tipo I [4,3 (0,8) vs. 3,3 (0,4)mg/dl, p<0,05], respectivamente. Foi observada 71%, 77% e 69% de indivíduos com insuficiência de vitamina D no grupo controle, OI tipos I e III, respectivamente. Quanto à TMB, observou-se diferença significativa na TMB em kcal/dia entre os gêneros dos indivíduos adultos com OI tipo III, sendo significativamente maior no feminino. Conclusão: Observou-se elevada porcentagem de indivíduos com OI com excesso de peso e excesso de gordura corporal, principalmente entre os indivíduos com OI tipo III. A ingestão de cálcio e vitamina D foi inferior a recomendação na maioria dos indivíduos com OI e também no grupo controle. Estes resultados apontam a necessidade de uma intervenção nutricional direcionada a estes pacientes, uma vez que o estado nutricional e alimentar adequado podem contribuir para a saúde óssea.
Background: Osteogenesis imperfecta (OI) is a disease that leads to fragility and reduced bone mass, not secondary to another known condition. Due to the importance of nutritional status for bone health, there is a need for better knowledge on the nutritional status, body composition and dietary intake of individuals with OI. Objective: To evaluate the nutritional status and nutrient intakes of adolescents and adults with OI. Methods: A case-control study with adolescents and adults of both genders. Nutritional status (BMI, supine length and armspan), body composition and bone mineral density (BMD) by Dual Energy X-Ray Absorptiometry - DXA, dietary intake (3 days Dietary Records), biochemical measurements (total calcium, phosphorus, albumin, creatinina, PTH, CTX and vitamin D), physical activity assessment and determination of basal metabolic rate (BMR) were evaluated. Statistical analyses comprised One Way ANOVA and Student-T test to calculated differences between groups and Pearson\'s correlation test. Significance was considered when p<0.05. Results: There were 26 subjects with OI (50% OI type I / type III OI 50%) and 8 healthy subjects matched by sex and age in the control group. The number of fractures was higher in subjects with OI type III, an average of 60 fractures compared to 19 in the OI type I. The BMD in the lumbar spine L1-L4 was significantly higher in the control group compared to the types of OI I and III [-0.4 (0.5) vs -2.7 (1.0) and -2.7 (0.6) g/cm2, p<0.05], respectively. According to BMI, 100% of subjects in the control group were considered normal weight, 46% of individuals with OI type III are obese and 31% of individuals type I OI, overweight. Regarding percentage of body fat, 14% of individuals with in the control group, 83% in OI type III and 42% in individuals with type I presented excess of body fat. In the case group, there was negative association between number of fractures and fat-free body mass and positive association between number of fractures and body fat% and BMI. There was no difference of calcium and phosphorus intakes between the groups. However, vitamin D was significantly lower in the control group. In addition considering foods and supplementation, 58% and 12% of individuals in the case group did not reach the recommended levels of calcium and vitamin D, respectively. Individuals with type III showed serum phosphorus significantly higher when compared with OI type I [4.3 (0.8) vs. 3.3 (0.4) mg/dl, p<0.05], respectively. Was observed 71%, 77% and 69% of insufficiency vitamin D in individuals of control group, OI types I and III, respectively. A significant difference between BMR in kcal/day between genders of adults with OI type III was observed being significantly higher in females. Conclusion: There was a high percentage of individuals with OI with higher body weight and body fat, mainly among OI type III. The calcium and vitamin D intakes was under recommended levels in most individuals with OI and in the control group. These results demonstrates that a nutritional intervention program is necessary for these patients, such an adequate nutritional status and dietary pattern could contribute to bone health.

O hormônio da paratireóide (PTH) tem sido utilizado como um agente anabólico ósseo para o tratamento de condições de osteopenia / osteoporose, prevenção e consolidação de fraturas. O papel do fator de crescimento semelhante à insulina I (IGF-I), como um potencial mediador dos efeitos anabólicos do PTH, é controverso. O rato dwarf pode ser adequado para o estudo dessas interações in vivo, uma vez que a os níveis séricos de hormônio do crescimento (GH) encontram-se reduzidos a cerca de 6% dos valores normais em fêmeas e os níveis séricos de IGF-I, a cerca de 10% dos valores normais, mas estes animais são saudáveis e sem malformações esqueléticas. Os objetivos deste estudo foram: 1 - Avaliar o rato dwarf (dw-/dw-) como um modelo animal para o estudo dos efeitos da deficiência do GH e do IGF-I sobre o esqueleto e o metabolismo ósseo; 2 - Comparar os efeitos do tratamento com PTH sobre o esqueleto e formação óssea em ratos dwarf e em ratos Lewis, sua linhagem de origem. A partir de 9 semanas de idade, ratas Lewis e dwarf receberam injeções por via subcutânea, diariamente, por duas semanas, com medicamento placebo ou fragmento de hormônio paratireóideo humano, hPTH 1-34, na dose de 50 g / kg de peso corpóreo (N = 7-13/grupo). Foram realizadas avaliações do peso corpóreo semanalmente e, por ocasião da eutanásia, na 11ª semana, foram coletadas amostras de sangue para realização de dosagens séricas de IGF-I (ELISA). As vértebras lombares e as metáfises proximais das tíbias foram avaliadas por meio de histomorfometria óssea. Os fêmures direitos foram mensurados e analisados por tomografia quantitativa periférica computadorizada (pQCT). Os níveis séricos de IGF-I mostraram-se três vezes menores nas ratas dwarf quando comparados aos observados nas ratas Lewis, a despeito do tratamento com PTH, que não provocou aumento de IGF-I em nenhum dos dois grupos. No entanto, o PTH aumentou significativamente o volume ósseo trabecular em ambos os grupos, dwarf (p<0.003) e Lewis (p < 0.0001) comparados aos seus respectivos grupos controle, efeito associado ao aumento da espessura e da distância trabeculares. As ratas dwarf tratadas com PTH também exibiram aumentos de 7 a 13 vezes na superfície de mineralização e na taxa de formação óssea respectivamente, quando comparadas às ratas dwarf tratadas com placebo, enquanto as ratas Lewis tratadas com PTH mostraram aumentos de 3 e 4 vezes quando comparadas as ratas Lewis tratadas com placebo. A taxa de aposição mineral, indicativa de atividade osteoblástica, estava aumentada nas ratas dwarf e Lewis tratadas com PTH (p<0.0001) comparadas aos seus respectivos grupos controle. As análises pela pQCT das metáfises femorais distais revelaram que todos os parâmetros estruturais do osso trabecular (BMC total, BMD total, BMC trabecular e BMD trabecular) também apresentaram valores significativamente aumentados nas ratas, Lewis e dwarf, tratadas com PTH, quando comparadas às ratas tratadas com placebo (p<0.0001). Ao se considerar os parâmetros para o osso cortical, praticamente todos os valores obtidos nas diáfises femorais (BMC total, BMD total, BMC cortical, BMD cortical, área cortical, espessura cortical, circunferência periosteal e endosteal) não mostraram qualquer efeito do tratamento com PTH nos dois grupos. Em conclusão, o PTH induziu efeitos anabólicos altamente significativos no tecido ósseo trabecular das tíbias e vértebras lombares, a despeito dos baixos níveis circulantes de IGF-I em animais da linhagem dwarf. A resposta positiva ao tratamento com PTH confirma a sua utilização terapêutica como potente agente anabólico ósseo mesmo em face à deficiência no eixo GH/IGF-I
The parathyroid hormone (PTH) has been used as a bone anabolic agent to treat osteopenic/osteoporotic conditions, prevention and healing of fractures. The role of insulin-like growth factor I (IGF-I) as a potential mediator for the bone anabolic effects of PTH is controversial. The dwarf rat (dw-/dw-) may be suitable to study these interactions in vivo, since GH synthesis is selectively reduced to about 6% of normal in females, and serum IGF-I levels are about 10% of normal, but these animals are healthy without skeletal malformations. The objectives of this study were: 1- Evaluate the dwarf rat (dw-/dw-) as an animal model for studies of the effects of GH and IGF-I deficiency on the skeleton and bone metabolism; 2- Compare the skeletal effects of PTH treatment in dwarf rats and their background strain, Lewis rats. At 9 weeks of age, female Lewis and dwarf rats were injected SC daily for 2 weeks with vehicle or human parathyroid hormone fragment, hPTH 1-34, at a dose of 50 g/kg body weight (N=7- 13/group). The body weight was evaluated weekly and at the time of euthanasia, at 11 weeks, blood samples were collected. Serum IGF-I was measured by ELISA, and cancellous bone histomorphometry was performed in the lumbar vertebral body and tibial proximal metaphysis. The right femurs were measured, scanned and analyzed by peripheral quantitative computed tomography (pQCT). Serum levels of IGF-I were nearly 3-fold lower in dwarf rats compared with Lewis rats regardless of treatment, but PTH treatment did not increase serum IGF-I in either Lewis or dwarf rats. However, PTH significantly increased cancellous bone volume in both dwarf (P<0.003) and Lewis rats (P<0.0001) when compared to vehicle-treated rats, which was associated with increased trabecular width and decreased trabecular separation. PTH-treated dwarf rats also exhibited 7- and 13-fold increases in mineralizing surface and bone formation rate respectively, compared to vehicle-treated dwarf rats, while PTH-treated Lewis rats showed 3- and 4-fold increases when compared to vehicle-treated Lewis rats. Mineral apposition rate, an index of osteoblast activity, was increased in PTH-treated dwarf rats (P<0.0001) and in Lewis rats (P<0.0001) compared to their respective control groups. The pQCT analyses of the distal femoral metaphysis revealed that cancellous bone structural parameters (total BMC, total BMD, trabecular BMC, and trabecular BMD) also presented significantly higher values in PTH-treated dwarf and Lewis rats, when compared to vehicle treated rats (P<0.0001). When considering cortical bone parameters, almost all the values obtained at the femoral shafts (total BMC, total BMD, cortical BMC, cortical area, cortical thickness, periosteal and endocortical circumferences) did not show any PTH treatment effect in either groups. In conclusion, PTH induced highly significant anabolic effects in vertebral and tibial cancellous bone despite low circulating levels of IGF-I in dwarf rats. The positive response to PTH treatment confirms its therapeutic use as a potent bone anabolic agent, even in the face of GH/IGF-I deficiency

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.

我们的研究是基于发现镁金属的成骨现象。在我们组之前的工作中，我们发现大鼠股骨骨髓腔内植入镁棒后，在很短时间内（一周后）就会在股骨骨膜下部位形成新骨。这种镁导致的成骨现象是怎样发生的，以及我们能否利用镁金属的这种特性去促进骨质疏松骨折的愈合？对这两个问题的解答便构成了本篇论文的主要内容。
因为镁诱导的新生骨产生在骨膜下方，并且植入镁棒产生的成骨现象在骨膜剥除的部位消失，所以我们认为骨膜是镁成骨的关键点。骨膜是富含感觉神经纤维和干细胞的组织，而且动物骨的感觉神经分布主要集中在骨膜（约占总的神经数量的99%以上）。骨膜神经末端不仅仅感知痛觉触觉和温度觉，而且在外界刺激下释放感觉神经递质。神经递质包裹于处于神经末梢的囊泡当中，CGRP 是经典的也是分布最广的感觉神经递质。在动物体内，血液中CGRP 的含量随年龄的增加而减少，同时骨内的镁含量也随之流失。这也是老年龄动物骨折愈合较慢的原因之一。所以我们提出本课题的研究假设：镁金属降解产生的镁离子作用于骨膜部位的感觉神经末梢，刺激神经递质CGRP 的释放。骨膜内增多的CGRP 作用于骨膜内的干细胞进行成骨分化，最后形成新骨。我们进一步检测镁的这种成骨作用能否促进骨质疏松鼠骨折的愈合。
首先我们用过量的辣椒素破坏大鼠股骨的感觉神经末端之后，镁的成骨显著减少，这说明镁的成骨作用相当程度上依赖于通过骨膜的神经组织。我们通过免疫组化染色及蛋白定量测定发现，植入镁后的骨组织内CGRP（降钙素基因相关肽，一种感觉神经末端分泌的主要神经递质）含量增加了一倍多。我们用CGRP 受体拮抗剂同样发现可以部分抑制镁的成骨作用。我们推测镁降解过程中产生的镁离子在骨膜部位增加了感觉神经递质的释放，骨膜部位增多的神经递质作用于骨膜源性间充质干细胞以及骨髓源性间充质干细胞想成骨方向分化成骨。体外试验结果表明，CGRP 在高浓度下显著促进骨膜及骨髓源性干细胞的成骨分化。我们从大鼠的脊髓腰段L3-5 背根神经节分离出背根神经节神经元，在体外用荧光对神经元内的突触小泡进行染色，发现当培养液中的镁离子浓度升高时（1-2mM），这些富含神经递质CGRP 的突触小泡不但数目增加，而且从胞体中心向轴突末梢迁移。在这个过程中，我们同时记录到显著的镁离子内流。实验结果表明，镁离子可以促进神经元的复极化以及神经递质向轴突末端迁移聚集，从而在下一次刺激中释放出更多的神经递质。同时体外干细胞分化实验结果表明高浓度的镁离子（5-10mM）显著促进干细胞的成骨分化。对干细胞和神经元的胞内镁离子内流检测发现，在胞外镁离子浓度升高的情况下，胞内镁离子内流主要通过一种膜通道MagT1。至此，关于镁成骨的机制可以归纳为：镁金属在降解过程中产生的镁离子作用于骨膜感觉神经末梢，使之释放出更多的神经递质，增加释放的神经递质和镁离子共同促进分布在骨膜和骨髓的干细胞进行成骨分化，从而增加成骨。
镁的成骨效应使之有很大的潜力用于骨质疏松骨折的修复。由于镁金属强度不足以直接用来固定大鼠骨折，所以我们设计了一种中空的不锈钢针管作为髓内骨折固定针。针管中部与骨折线对应的部位开出一些小孔，细的镁棒可以插入针管，在体内镁降解产生的镁离子可以从中部的小孔释放出去发挥其成骨效应，进而促进骨折的愈合。我们用卵巢切除大鼠进行闭合性骨折造模，然后用我们设计的髓内针固定。X 射线结果表明，手术后第二、四周镁治疗组骨折愈合组织的面积和宽度显著大于对照组。Micro-CT 扫描结果也同样表明，镁治疗组骨折部位愈合组织的总体积和骨组织体积在术后第四周显著大于对照组。组织学染色表明，在术后第二周，镁治疗组的骨折部位的骨膜内成骨大量增加，并且有大量间充质细胞充塞与骨折部位。第术后第四周，更多的软骨组织形成于镁治疗组的骨折部位。荧光双染色结果也表明，镁治疗组的骨折部位在第四周有更多的新生骨形成。第八周和十二周的偏振光图像表明，镁治疗组的骨折愈合部位形成的胶原纤维比对照组更规则且更多更亮。这说明在骨折愈合后期软骨内成骨以及编制骨向层状骨转化的过程中，镁治疗组的骨重建更加规则。最后在第十二周的力学实验结果证明，镁治疗组的骨干所能承受的最大压力显著高于对照组（大约增强了27%）。这部分体内试验证明镁金属可以加速并优化大鼠骨质疏松骨折的愈合，而且我们设计的中空含镁髓内针可以作为将来临床新型骨折髓内固定针的原型。
结论：我们对镁成骨的作用和其机制进行了比较深入全面的研究，并初步证实镁金属可以用于动物骨质疏松骨折的修复。我们的研究结果为将来镁金属在临床尤其是骨科领域的实际应用提供了一些基本的理论依据。
In the rodent femur, almost 99% of all sensory nerves are distributed densely in the periosteum. Neuropeptides encapsulated in the synaptic vesicles are located at the axon terminals and released through exocytosis after being stimulated at the sensory nerve endings. The neuropeptides released from nerve endings have an osteo-anabolic effect on osteoblasts. Among the many kinds of neuropeptides, which include α-calcitonin gene-related peptide (CGRP), substance P, and other amino molecules, CGRP is the classical and dominantly distributed peptide in sensory nerve endings. In aged animals, decreased serum CGRP and loss of bone Mg content may be the factors inhibiting fracture healing.
In this study, Mg was found to significantly promote new bone formation in the subperiosteal cortical region after it was intramedullarily implanted in the rat femur canal. Histomorphological analysis revealed that the newly formed bone grew from periosteum, a fibrous membrane constituted of blood vessels, sensory serves, and mesenchymal stem cells, and did not form any cartilage-like tissue, the latter of which is a feature of intramembranous ossification. Observation that Mg-induced new bone formation disappeared at the periosteum-stripped region revealed the existence of an interaction between the periosteum and Mg ions.
Based on previous findings, this study examined the following hypotheses: (1) Mg ions from Mg implanted in the rat femur canal act on sensory nerve endings in the periosteum and promote neuropeptide CGRP release, (2) mass CGRP release in the periosteum promotes periosteum-derived stem cells osteoblastogenesis and leads to new bone formation. Mg ions affect synaptic replasticity in dorsal root ganglia neurons, and (3) pure Mg metal affects fracture healing in ovariectomized (OVX) rats.
Neuropeptide CGRP plays a pivotal role in Mg-induced new bone formation. This hypothesis was supported by femur bone analysis showing that CGRP content significantly increased in Mg-implanted femur bone compared to control femur bone. When rat sensory nerves were destroyed by administration of high-dose capsaicin, induction of new bone formation by Mg implantation significantly decreased, proving that sensory nerves play an important role in Mg-induced osteogenesis. Because neuropeptide CGRP from sensory nerve endings may play a pivotal role in Mg’s osteogenic process, the effective CGRP antagonist BIBN4096bs was administered to Mg-implanted rats. Administration of the CGRP antagonist significantly reduced newly formed bone volume after Mg implantation. To examine whether this phenomenon is dependent on the interaction between neuropeptides and MSCs, which are richly distributed in the periosteum, periosteum-derived stem cells (PDSCs) and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from the periosteum and bone marrow, respectively. It was observed that high concentrations of CGRP significantly promoted osteogenic differentiation in both PDSCs and BMSCs while high concentrations of CGRP had an obvious chemotaxis effect on BMSCs.
Mg increases CGRP release by affecting DRG neurons. The results of immunochemical staining and ELISA CGRP quantification analysis of femur samples showed that femur CGRP content in Mg-implanted samples was almost twice that of controls. Previous studies reported that Mg ions could promote neural synaptic replasticity in hippocampus neurons in vitro. This study examined the hypothesis that Mg ions could promote synaptic replasticity in DRG neurons. The neural synaptic vesicles, which contain neuropeptides of DRG neurons, including CGRP, derived from the L3-5 dorsal root ganglion were stained in vitro. The synaptic vesicles were found to significantly increase in number when their medium was changed from Mg-free medium to Mg-rich medium of 1 mM and 2 mM and to migrate from the neuron body to its axon terminals. These results proved that Mg could facilitate neuron replasticity and prompt synaptic vesicle aggregation at axon terminals, indicating that much neuropeptide release occurs after stimulation. Real-time recording of the intracellular Mg signal revealed that DRG neuron Mg influx significantly increased after Mg medium had been added and that Mg influx into neurons was mainly through the membrane Mg ion channel MagT1. Implantation of Mg ions (MgCl₂) of high concentration was found to promote stem cell (PDSCs and BMSCs) osteogenic differentiation. Although the mechanism of Mg’s osteogenic effect on stem cells was not thoroughly studied, cellular Mg influx was found to increase in high-Mg medium through the membrane ion channel MagT1.
Mg accelerated bone fracture in ovariectomized rats. Mg metal is too soft to repair bone fracture in animal models. To overcome this challenge, we designed a novel intramedullary nail containing Mg to accelerate osteoporotic bone fracture healing in ovariectomized (OVX) rats. The novel nail is a hollow stainless steel needle with several interlacing arranged holes drilled midway through the needle. The Mg pin is inserted into the needle canal and Mg ions released through the holes on the needle reach the fracture line during degradation in vivo. Our findings indicate that use of this Mg-containing intramedullary nail could accelerate bone fracture healing in OVX rats. Review of post-surgery X-ray results showed that the fracture callus of the Mg-treated group was significantly larger than that of the control group at weeks 2 and 4. Review of micro-computed tomography (micro-CT) scanning images indicated that both the total volume and area of callus bone in the Mg-treated group exceeded those of the control group at week 4. However, no significant difference was found between the two groups regarding callus area and volume at week 12.
Histomorphological analysis showed a wider intramembranous ossification area and woven bone area in the Mg-treated group at weeks 2 and 4 and more cartilage tissue at the callus site in the Mg-treated group at week 4. Double fluorescence labeling staining revealed more densely stained newly formed bone in the Mg-treated group than the control group at week 4, indicating accelerated callus bone formation in the Mg-treated group. The callus was observed to be undergoing endochondral ossification and woven bone remodeling at weeks 8 and 12. Review of polarized light images showed brighter and more regularly arranged collagen fibers in the Mg-treated group compared to the control group. Biomechanical testing at week 12 revealed that the ultimate load of shaft bone in the Mg-treated group had increased 30% more than that of the control group. These results indicate that the novel Mg-containing intramedullary nail designed in this study could significantly accelerate and optimize osteoporotic fracture healing in OVX rat model.
Significance: The results of this study contribute to a thorough understanding of the osteogenic effect of Mg by explicating its bioeffect on neurons and stem cells. The novel Mg-containing intramedullary nail designed in this study appears promising in osteoporotic fracture healing and to have many potential clinical applications.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Zhang, Yifeng.
Thesis (Ph.D.) Chinese University of Hong Kong, 2015.
Includes bibliographical references (leaves 170-180).
Abstracts also in Chinese.