Academic literature on the topic 'Osteogenesis in rat'

MicroRNAs are small noncoding RNAs involved in numerous biological processes. Emerging pieces of evidence suggest that microRNAs play important roles in osteogenesis and skeletal homeostasis. Recent studies indicated the significant regulation function of mir-21 in osteogenesisin vitro, but little information is known about its veritable functionsin vivo. In the present study, we aimed to investigate the effect of mir-21 intervention on osteogenic differentiation of rats bone marrow derived mesenchymal stem cells (rBMSCs) and repair capacity in rats closed femur fracture model with internal fixation. The results showed that the upregulation of mir-21 not only increased the expression of osteopontin and alkaline phosphatase in rBMSCs but also promoted mineralization in the condition of osteogenic induction. Furthermore, the bone healing properties were also improved in fracture healing model according to the results of micro-CT, mechanical test, and histological analysis. The current study confirms that the overexpression of mir-21 could promote osteogenesis and accelerate bone fracture healing, which may contribute to a new therapeutic way for fracture repair.

The objective of this work was to investigate the osteogenetic ability of the human dental pulp stem cells (hDPSCs) derived from human third molars. We induced alkaline phosphatase (ALP) activity and bone morphogenetic protein 2 (BMP-2) mRNA expression, the markers for bone formation, in hDPSCs by using osteoinductive factors. The implantation of hDPSCs with collagen sponge promoted osteogenesis and fracture healing in the femur of an immunocompromised rat, which was a bone defect model for pseudoarthrosis. Histological analyses revealed that after implantation of the hDPSCs, the size and number of osteoblasts and the rates of osteoid production and mineralization increased to an appreciable extent, whereas the rate of bone resorption decreased. We believe that hDPSC implantation is a simple and safe procedure that can be beneficial in bone regeneration therapy in clinical practice.

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.

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.

This chapter discusses the anatomy and physiology of the bone, including mineralization, and outlines techniques in bone remodelling. It describes formation and resorption hormonal markers that are part of the bone remodelling cycle, such as procollagens and serums. It describes how diagnostic measures in these formation markers are increased for focal bone disorders like Paget’s disease, fibrous dysplasia, osteomalacia, bone metastases, myeloma, primary hyperparathyroidism, thyrotoxicosis, and acromegaly. The chapter also discusses osteoporosis, including causes, symptoms, and treatment options. Clinical suggestions for bone diagnoses and diseases are provided, based on dual-energy X-ray absorptiometry (commonly abbreviated as DXA), plain radiography, and bone biopsy. The chapter also defines osteogenesis imperfecta and describes its epidemiology and management. In addition, it outlines sclerosing bone disorders such as osteopetrosis, pycnodysostosis, and hyperostosis type Worth, as well as fibrodysplasia ossificans progressive.

Bone marrow is a reservoir of mesenchymal stem cells and osteoprogenitors. It was previously shown [1, 2] and we have recently verified that in vitro cultures of bone marrow undergo osteogenesis without addition of osteoinductive stimulants. We hypothesized that the in vitro ossifying bone marrow organ culture system can be used as a model to investigate the dynamics of the osteogenesis process and its mechanoresponsiveness in terms of expression of key osteoinductive factors. The outcomes of these studies can be used to develop more effective tissue engineered substitutes for bone regeneration by the utilization of multiple osteogenic factors with complex/sequential delivery mechanisms.

Pharmaceutical bone growth stimulation holds promise for prevention and treatment bone disorders, and the enhancement of fracture healing. Bone growth hormones have begun to have limited clinical use, but can illicit adverse side effects. Recent studies have shown that short peptides (less than 15 amino acids) derived from the protein sequence of Vitamin D Binding Protein (DBP), can enhance bone formation (osteogenesis). These peptides may have potential as controllable bone growth stimulators without the adverse side effects and cost of bone growth hormones. Rats, injected every other day for two weeks with DBP-based peptide fragments ranging from 3 to 13 amino acids in length, were euthanized and the tibias and femurs were scanned by peripheral quantitative computerized tomography (pQCT) to determine bone density and cross-sectional geometric properties. The bones were then tested in three-point bending to determine strength and bending modulus. Injection of DBP-based peptides over only a 2-week period resulted in significant (p<0.05) increases in bone density and material properties in the experimental rat bones in comparison to controls injected with saline. The short length of these effective peptides suggests their use not only in systemic injections but also as clinically convenient pills taken orally for pharmaceutically induced bone growth stimulation.

Osteoporosis is a debilitating disease characterized as decreased bone mass and structural deterioration of bone tissue. Osteoporotic bone tissue turns itself into altered structure, which leads to weaker bones that are more susceptible for fractures. While often happening in elderly, long-term bed-rest patients, e.g. spinal cord injury, and astronauts who participate in long-duration spaceflights, osteoporosis has been considered as a major public health thread and causes great medical cost impacts to the society. Mechanobiology and novel stimulation on regulating bone health have long been recognized. Loading induced bone fluid flow, as a critical mechanotransductive promoter, has been demonstrated to regulate cellular signaling, osteogenesis, and bone adaptation [4]. As one of the factors that mediate bone fluid flow, intromedullary pressure (ImP) creates a pressure gradient that further influence the magnitude of mechanotransductory signals [5]. As for a potential translational development of ImP, our group has recently introduced a novel, non-invasive dynamic hydraulic stimulation (DHS) on bone structural enhancement. Its promising effects on inhibition of disuse bone loss has been shown with 2 Hz loading through a 4-week hindlimb suspension rat study followed by microCT analysis. At the cellular level, mesenchymal stem cells (MSCs) are defined by their self-renewal ability and that to potentially differentiate into the cells that form tissues such as bone [1]. To further elucidate the cellular effects of DHS and its potential mechanism on bone quality enhancement, the objective of this study was to measure MSC quantification in response to the in vivo mechanical signals driven by DHS.

Mandibular distraction osteogenesis is a medical procedure for lengthening the mandible bone by stimulating natural bone-healing mechanisms via a mechanical device that exerts a force on the mandible in one or more directions. Many mandibular distraction devices must be placed externally and most rely on the patient to manually actuate the device each day. This project focuses on the design of an automatically actuated, single degree-of-freedom, implantable distraction device that would be minimally visible after installation. Such a device could reduce errors from patient compliance and would be an important first step toward increasing the capability of future devices. A simple motor and leadscrew system was used with a custom designed impact transmission and controller. A test was conducted on a prototype to determine the feasibility of the design and measure the overall system efficiency. The device was able to move the required 70 N load at a rate of about 1 mm per minute. Compared to an equivalent device utilizing a planetary gearhead to amplify the torque, the impact coupling is significantly less efficient. However, the necessary increase in battery size has only a small impact on the total device length. For a system with the same motor and force output, the impact coupling system is shorter than the gearhead-based system due to a 50% reduction in transmission length.

Lower limb length discrepancy (LLD), defined by unequal length of paired lower limbs, contributes to lower back pain, osteoarthritis of the hip, and stress fractures [1–3]. The Center for Disease Control and Prevention estimated that there were approximately 700 children born with LLD each year in US [4]. Patients may receive distraction osteogenesis treatment, in which an osteotomy is performed on the shorter limb, and mechanical force is applied to gradually distract the two halves of the bone during the healing process. This stretches the bone callus during healing to achieve desired limb length upon callus consolidation [5]. The current correction devices are external fixators that leave unsightly scars and are prone to infection [6]. While recently developed intramedullary devices address many of the persistent issues with external lengthening devices, size limitations and potential damage to the bone growth plates make them impractical for use in children [7, 8]. The proposed research addresses an unmet need by developing a novel implantable extramedullary device for LLD correction that is targeted for pediatric use. The device will be implantable, submuscular, and fixed to the outside surface of the bone (extramedullary), thus allowing for use in children without concern for injury to the growth plates. The device’s function will be similar to an external fixator; however, it will not require exposed hardware, which increases risk of infection, or muscle penetration from the pins, which causes pain. Additionally, the device incorporates real-time control of the distraction rate, reducing the risk of complications arising from fixed rate distraction such as premature consolidation and non-union of the callus. [9–11]. The investigators of this study have previously designed and constructed a distraction mechanism prototype and test frame [10]. The current study aims to validate the real-time controller of the prototype.