Mathematical modelling of soft callus formation in early murine bone repair

Fracture healing is a complicated coupling of many processes. Yet despite the apparent complexity, fracture repair is usually effective. There is, however, no comprehensive mathematical model addressing the multiple interactions of cells, cytokines and oxygen that includes extra-cellular matrix production and that results in the formation of the early stage soft callus. This thesis develops a one dimensional continuum transport model in the context of early fracture healing. Although fracture healing is a complex interplay of many local factors, critical components are identified and used to construct an hypothesis about regulation of the evolution of early callus formation. Multiple cell lines, cellular differentiation, oxygen levels and cytokine concentrations are examined as factors affecting this model of early bone repair. The model presumes diffusive and chemotactic cell migration mechanisms. It is proposed that the initial signalling regime and oxygen availability arising as consequences of bone fracture, are sufficient to determine the quantity and quality of early soft callus formation. Readily available software and purpose written algorithms have been used to obtain numerical solutions representative of various initial conditions. These numerical distributions of cellular populations reflect available histology obtained from murine osteotomies. The behaviour of the numerical system in response to differing initial conditions can be described by alternative in vivo healing pathways. An experimental basis, as illustrated in murine fracture histology, has been utilised to validate the mathematical model outcomes. The model developed in this thesis has potential for future extension, to incorporate processes leading to woven bone deposition, while maintaining the characteristics that regulate early callus formation.

Effects of scaffold architecture on mechanical characteristics and osteoblast response to static and perfusion bioreactor cultures

Tissue engineering focuses on the repair and regeneration of tissues through the use of biodegradable scaffold systems that structurally support regions of injury whilst recruiting and/or stimulating cell populations to rebuild the target tissue. Within bone tissue engineering, the effects of scaffold architecture on cellular response have not been conclusively characterized in a controlled-density environment. We present a theoretical and practical assessment of the effects of polycaprolactone (PCL) scaffold architectural modifications on mechanical and flow characteristics as well as MC3T3-E1 preosteoblast cellular response in an in vitro static plate and custom-designed perfusion bioreactor model. Four scaffold architectures were contrasted, which varied in inter-layer lay-down angle and offset between layers, whilst maintaining a structural porosity of 60 ± 5%. We established that as layer angle was decreased (90° vs. 60°) and offset was introduced (0 vs. 0.5 between layers), structural stiffness, yield stress, strength, pore size and permeability decreased, whilst computational fluid dynamics-modeled wall shear stress was increased. Most significant effects were noted with layer offset. Seeding efficiencies in static culture were also dramatically increased due to offset (~45% to ~86%), with static culture exhibiting a much higher seeding efficiency than perfusion culture. Scaffold architecture had minimal effect on cell response in static culture. However, architecture influenced osteogenic differentiation in perfusion culture, likely by modifying the microfluidic environment.

Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity

Introduction The clinically known importance of patient sex as a major risk factor for compromised bone healing is poorly reflected in animal models. Consequently, the underlying cellular mechanisms remain elusive. Because mesenchymal stem cells (MSCs) are postulated to regulate tissue regeneration and give rise to essential differentiated cell types, they may contribute to sex-specific differences in bone healing outcomes. Methods We investigated sex-specific variations in bone healing and associated differences in MSC populations. A 1.5 mm osteotomy gap in the femora of 8 male and 8 female 12-month-old Sprague-Dawley rats was stabilized by an external fixator. Healing was analyzed in terms of biomechanical testing, bridging and callus size over time (radiography at 2, 4, and 6 weeks after surgery), and callus volume and geometry by μCT at final follow-up. MSCs were obtained from bone marrow samples of an age-matched group of 12 animals (6 per gender) and analyzed for numbers of colony-forming units (CFUs) and their capacity to differentiate and proliferate. The proportion of senescent cells was determined by β-galactosidase staining. Results Sex-specific differences were indicated by a compromised mechanical competence of the callus in females compared with males (maximum torque at failure, p = 0.028). Throughout the follow-up, the cross-sectional area of callus relative to bone was reduced in females (p ≤ 0.01), and the bridging of callus was delayed (p 2weeks = 0.041). μCT revealed a reduced callus size (p = 0.003), mineralization (p = 0.003) and polar moment of inertia (p = 0.003) in female animals. The female bone marrow contained significantly fewer MSCs, represented by low CFU numbers in both femora and tibiae (p femur = 0.017, p tibia = 0.010). Functional characteristics of male and female MSCs were similar. Conclusion Biomechanically compromised and radiographically delayed bone formation were distinctive in female rats. These differences were concomitant with a reduced number of MSCs, which may be causative for the suboptimal bone healing.

LCC15-MB : a vimentin-positive human breast cancer cell line from a femoral bone metastasis

The LCC15-MB cell line was established from a femoral bone metastasis that arose in a 29-year-old woman initially diagnosed with an infiltrating ductal mammary adenocarcinoma. The tumor had a relatively high (8%) S-phase fraction and 1/23 positive lymph nodes (LN). Both the primary tumor and LN metastasis were positive for estrogen receptor (ER) and progesterone receptor (PgR), but lacked erbB2 expression. Approximately one year later, the patient presented with a 0.8 cm comedo-type intraductal mammary adenocarcinoma in the left breast that was negative for ER and PgR, but positive for erbB2. Thirty-five months after the initial diagnosis she was treated for acute skeletal metastasis, and stabilized with a hip replacement. At this time, tumor cells were removed from surplus involved bone, inoculated into cell culture, and developed into the LCC15-MB cell line. The bone metastasis was a poorly differentiated adenocarcinoma lacking ER, PgR, and erbB2, characteristics shared by the LCC15-MB cells, although ER can be re-expressed by treatment of the LCC15-MB cells for 5 days with 75 μM 5-aza-2'-deoxycytidine. The LCC15-MB cell line is tumorigenic when implanted subcutaneously in NCr nu/nu mice and produces long-bone metastases after intracardiac injection. Although the bone metastasis from which the LCC15-MB cell line was derived lacked vimentin (VIM) expression, the original primary tumor and lymph node metastasis were strongly VIM positive, as are LCC15-MB cells in vitro and in nude mice. The karyotype and isozyme profiles of LCC15-MB cells are consistent with its origin from a human female, with most chromosome counts in the hypertriploid range. Thirty-two marker chromosomes are present. These cells provide an in vitro/in vivo model in which to study the inter-relationships between ER, VIM, and bone metastasis in human breast cancer.

Calculation of the speed of walking of individuals with transfemoral amputation fitted with bone-anchored prosthesis

Typically, the walking ability of individuals with a transfemoral amputation (TFA) can be represented by the speed of walking (SofW) obtained in experimental settings. Recent developments in portable kinetic systems allow assessing the level of activity of TFA during actual daily living outside the confined space of a gait lab. Unfortunately, only minimal spatio-temporal characteristics could be extracted from the kinetic data including the cadence and the duration on gait cycles. Therefore, there is a need for a way to use some of these characteristics to assess the instantaneous speed of walking during daily living. The purpose of the study was to compare several methods to determine SofW using minimal spatial gait characteristics.

Spatio-temporal characteristics of locomotion and functional outcomes of individuals with transfemoral amputation fitted with OPRA fixation

The conventional method of attachment of prosthesis involves a socket. A new method relying on osseointegrated fixation has emerged in the last decades. It has significant prosthetic benefits. Only a few studies demonstrated the biomechanical benefits. The ultimate aim of this study was to characterise the functional outcome of individuals with lower limb amputation fitted with osseointegrated fixation, which can be assess through temporal and spatial gait characteristics. The specific objective of this study was to present the key temporal and spatial gait characteristics of individuals with transfemoral amputation (TFA).

Temporal and spatial gait characteristics of transfemoral amputees fitted with osseointegrated fixation: preliminary data

The conventional method of attachment of prosthesis involves on a socket. A new method relying on osseointegrated fixation is emerging. It has significant prosthetic benefits. Only a few studies demonstrated the biomechanical benefits. The ultimate aim of this study was to characterise the functional outcome of transfemoral amputees fitted with osseointegrated fixation, which can be assess through temporal and spatial gait characteristics. The specific objective of this preliminary study was to present the key temporal and spatial gait characteristics.

Load applied on a bone-anchored transfemoral prosthesis: characterisation of a prosthesis : a pilot study

The objectives of this study were (A) to record the inner prosthesis loading during activities of daily living (ADL), (B) to present a set of variables comparing loading data, and (C) to provide an example of characterisation of two prostheses. The load was measured at 200 Hz using a multi-axial transducer mounted between the residuum and the knee of an individual with unilateral transfemoral amputation fitted with a bone-anchored prosthesis. The load was measured while using two different prostheses including a mechanically (PRO1) and a microprocessor controlled (PRO2) knee during six ADL. The characterisation of prosthesis was achieved using a set of variables split into four categories, including temporal characteristics, maximum loading, loading slopes and impulse. Approximately 360 gait cycles were analysed for each prosthesis. PRO1 showed a cadence improved by 19% and 7%, a maximum force on the long axis reduced by 11% and 19%, as well as an impulse reduced by 32% and 15% during descent of incline and stairs compared to PRO2, respectively. This work confirmed that the proposed apparatus and characterisation can reveal how changes of prosthetic components are translated into inner loading.

A humanized tissue-engineered in vivo model to dissect interactions between human prostate cancer cells and human bone

Currently used xenograft models for prostate cancer bone metastasis lack the adequate tissue composition necessary to study the interactions between human prostate cancer cells and the human bone microenvironment. We introduce a tissue engineering approach to explore the interactions between human tumor cells and a humanized bone microenvironment. Scaffolds, seeded with human primary osteoblasts in conjunction with BMP7, were implanted into immunodeficient mice to form humanized tissue engineered bone constructs (hTEBCs) which consequently resulted in the generation of highly vascularized and viable humanized bone. At 12 weeks, PC3 and LNCaP cells were injected into the hTEBCs. Seven weeks later the mice were euthanized. Micro-CT, histology, TRAP, PTHrP and osteocalcin staining results reflected the different characteristics of the two cell lines regarding their phenotypic growth pattern within bone. Microvessel density, as assessed by vWF staining, showed that tumor vessel density was significantly higher in LNCaP injected hTEBC implants than in those injected with PC3 cells (p\0.001). Interestingly, PC3 cells showed morphological features of epithelial and mesenchymal phenotypes suggesting a cellular plasticity within this microenvironment. Taken together, a highly reproducible humanized model was established which is successful in generating LNCaP and PC3 tumors within a complex humanized bone microenvironment. This model simulates the conditions seen clinically more closely than any other model described in the literature to date and hence represents a powerful experimental platform that can be used in future work to investigate specific biological questions relevant to bone metastasis.

Site and gender specificity of inheritance of bone mineral density

Differences in genetic control of BMD by skeletal sites and genders were examined by complex segregation analysis in 816 members of 147 families with probands with extreme low BMD. Spine BMD correlated more strongly in male-male comparisons and hip BMD in female-female comparisons, consistent with gender- and site-specificity of BMD heritability. Introduction: Evidence from studies in animals and humans suggests that the genetic control of bone mineral density (BMD) may differ at different skeletal sites and between genders. This question has important implications for the design and interpretation of genetic studies of osteoporosis. Methods: We examined the genetic profile of 147 families with 816 individuals recruited through probands with extreme low BMD (T-score < −2.5, Z-score < −2.0). Complex segregation analysis was performed using the Pedigree Analysis Package. BMD was measured by DXA at both lumbar spine (L1-L4) and femoral neck. Results: Complex segregation analysis excluded purely monogenic and environmental models of segregation of lumbar spine and femoral neck BMD in these families. Pure polygenic models were excluded at the lumbar spine when menopausal status was considered as a covariate, but not at the femoral neck. Mendelian models with a residual polygenic component were not excluded. These models were consistent with the presence of a rare Mendelian genotype of prevalence 3–19 %, causing high BMD at the hip and spine in these families, with additional polygenic effects. Total heritability range at the lumbar spine was 61–67 % and at the femoral neck was 44–67 %. Significant differences in correlation of femoral neck and lumbar spine BMD were observed between male and female relative pairs, with male-male comparisons exhibiting stronger lumbar spine BMD correlation than femoral neck, and female-female comparisons having greater femoral neck BMD correlation than lumbar spine. These findings remained true for parent-offspring correlations when menopausal status was taken into account. The recurrence risk ratio for siblings of probands of a Z-score < −2.0 was 5.4 at the lumbar spine and 5.9 at the femoral neck. Conclusions: These findings support gender- and site-specificity of the inheritance of BMD. These results should be considered in the design and interpretation of genetic studies of osteoporosis.

Genetic variation in the LDL receptor-related protein 5 (LRP5) gene : Association with bone health and metabolic parameters

Bone mass accrual and maintenance are regulated by a complex interplay between genetic and environmental factors. Recent studies have revealed an important role for the low-density lipoprotein receptor-related protein 5 (LRP5) in this process. The aim of this thesis study was to identify novel variants in the LRP5 gene and to further elucidate the association of LRP5 and its variants with various bone health related clinical characteristics. The results of our studies show that loss-of-function mutations in LRP5 cause severe osteoporosis not only in homozygous subjects but also in the carriers of these mutations, who have significantly reduced bone mineral density (BMD) and increased susceptibility to fractures. In addition, we demonstrated for the first time that a common polymorphic LRP5 variant (p.A1330V) was associated with reduced peak bone mass, an important determinant of BMD and osteoporosis in later life. The results from these two studies are concordant with results seen in other studies on LRP5 mutations and in association studies linking genetic variation in LRP5 with BMD and osteoporosis. Several rare LRP5 variants were identified in children with recurrent fractures. Sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses revealed no disease-causing mutations or whole-exon deletions. Our findings from clinical assessments and family-based genotype-phenotype studies suggested that the rare LRP5 variants identified are not the definite cause of fractures in these children. Clinical assessments of our study subjects with LPR5 mutations revealed an unexpectedly high prevalence of impaired glucose tolerance and dyslipidaemia. Moreover, in subsequent studies we discovered that common polymorphic LRP5 variants are associated with unfavorable metabolic characteristics. Changes in lipid profile were already apparent in pre-pubertal children. These results, together with the findings from other studies, suggest an important role for LRP5 also in glucose and lipid metabolism. Our results underscore the important role of LRP5 not only in bone mass accrual and maintenance of skeletal health but also in glucose and lipid metabolism. The role of LRP5 in bone metabolism has long been studied, but further studies with larger study cohorts are still needed to evaluate the specific role of LRP5 variants as metabolic risk factors.

Scaffolds for bone tissue engineering: role of surface patterning on osteoblast response

The fabrication of tissue engineering scaffolds necessitates amalgamation of a multitude of attributes including a desirable porosity to encourage vascular invasion, desired surface chemistry for controlled deposition of calcium phosphate-based mineral as well as ability to support attachment, proliferation, and differentiation of lineage specific progenitor cells. Scaffold fabrication often includes additional surface treatments to bring about desired changes in the surface chemistry. In this perspective, this review documents the important natural and synthetic scaffolds fabricated for bone tissue engineering applications in tandem with the surface treatment techniques to maneuver the biocompatibility of engineered scaffolds. This review begins with a discussion on the fundamental concepts related to biocompatibility as well as the characteristics of the biological micro-environment. The primary focus is to discuss the effects of surface micro/nano patterning on the modulation of bone cell response. Apart from reviewing a host of experimental studies reporting the functionality of osteoblast-like bone cells and stem cells on surface modified or textured bioceramic/biopolymer scaffolds, theoretical insights to predict cell behavior on a scaffold with different topographical features are also briefly analyzed.

Effect of temperature and mixing conditions on quality and consistency of poly(methyl methacrylate) bone cement

The mixing of poly(methyl methacrylate) (PMMA) bone cement has been studied to develop methods for preparing a consistently high quality cement. A novel droplet test experimental procedure was developed that characterised the wetting characteristics involved in bone cement mixing. Using this technique it was established that increased wetting occurred by mixing bone cement at a lower temperature (-28 degreesC) than normal mixing at room temperature.

Cyclosporin A and short-term methotrexate versus cyclosporin A as graft versus host disease prophylaxis in patients with severe aplastic anemia given allogeneic bone marrow transplantation from an HLA-identical sibling:results of a GITMO/EBMT randomized trial

A randomized trial was carried out comparing cyclosporin A (CsA) and short-term methotrexate (MTX) versus CsA alone for graft versus host disease (GVHD) prophylaxis in patients with severe aplastic anemia (SAA) undergoing allogeneic bone marrow transplantation (BMT) from a compatible sibling. Seventy-one patients (median age, 19 years; range, 4-46 years) were randomized to receive either CsA and MTX or CsA alone for the first 3 weeks after BMT. Subsequently, both groups received CsA orally, with gradual drug reduction until discontinuation 8 to 12 months after BMT. Patients randomized in both arms had comparable characteristics and received the same preparative regimen (ie, cyclophosphamide 200 mg/kg over 4 days). The median time for neutrophil engraftment was 17 days (range, 11-31 days) and 12 days (range, 4-45 days) for patients in the CsA/MTX group and the CsA alone group, respectively (P =.01). No significant difference was observed in the probability of either grade 2, grade 3, or grade 4 acute GVHD or chronic GVHD developing in the 2 groups. The Kaplan-Meier estimates of 1-year transplantation-related mortality rates for patients given either CsA/MTX or CsA alone were 3% and 15%, respectively (P =.07). With a median follow-up of 48 months from BMT, the 5-year survival probability is 94% for patients in the CsA/MTX group and 78% for those in the CsA alone group (P =. 05). These data indicate that the use of CsA with MTX is associated with improved survival in patients with SAA who receive transplants from compatible siblings. (Blood. 2000;96:1690-1697)

Bone properties and skeletal maturity in adolescent males, as assessed by quantitative ultrasound

ABSTRACT Background: Previous studies have implied that weight-bearing, intense and prolonged physical activities optimize bone accretion during the grow^ing years. The majority of past inquiries have used dual-energy X-ray absorptiometry (DXA) to examine bone strength and hand-wrist radiography to determine skeletal maturity in children. Recently, quantitative ultrasound (QUS) technologies have been developed to examine bone properties and skeletal maturity in a safe, noninvasive and cost-effective manner. Objective: The purpose of this study was to compare bone properties and skeletal maturity in competitive male child and adolescent athletes with minimallyactive, age-matched controls, using QUS technology. >. Methods: In total, 224 males were included in the study. The 115 pre-pubertal boys aged 10-12 years consisted of control, minimally-active children (n=34), soccer players (n=26), gymnasts (n=25) and hockey players (n=30). In addition, the 109 late-pubertal boys aged 14-16 years consisted of control, minimally-active adolescents (n=31), soccer players (n=30), gymnasts (n=17) and hockey players (n=31). The athletic groups were elite level players that predominantly trained year-round. Physical activity, nutrition and sports participation were assessed with various questionnaires. Anthropometries, such as height, weight and relative body fat percentage (BF%) were assessed using standard measures. Skeletal strength and age were evaluated using bone QUS. Lastly, salivary testosterone (sT) concentration was measured using Radioimmunoassay (RIA). Results: Within each age group, there were no significant differences between the activity groups in age and pubertal stage. An age effect was apparent in all variables, as expected. A sport effect was noted in all physical characteristics: the child and adolescent gymnasts were shorter and lighter than other sports groups. Adiposity was greater in the controls and in the hockey players. All child subjects were pubertal stage (fanner) I or II, while adolescent subjects were pubertal stage IV or V. There were no differences in daily energy and mineral intakes between sports groups. In both age groups, gymnasts had a higher training volume than other athletic groups. Bone speed of sound (50s) was higher in adolescents compared with the children. Gymnasts had signifieantly higher radial 50S than controls, hockey and soccer players in both age cohorts. Hockey athletes also had higher radial 50S than controls and soccer players in the child and adolescent groups, respectiyely. Child gymnasts and soccer players had greater tibial 50S compared with the hockey players and control groups. Likewise, adolescent gymnasts and soccer players had higher tibial SoS compared with the control group. No interaction was apparent between age and type of activity in any of the bone measures. » Lastly, maturity as assessed by sT and secondary sex characteristics (Tanner stage) was not different between sports group within each age group. Despite the similarity in chronological age, androgen levels and sexual maturity, differences between activity groups were noted in skeletal maturity. In the younger group, hockey players had the highest bone age while the soccer players had the lowest bone age. In the adolescent group, gymnasts and hockey players were characterized by higher skeletal maturity compared with controls. An interaction between the age and sport type effects was apparent in skeletal maturity, reflecting the fact that among the children, the soccer players were significantly less mature than the rest of the groups, while in the adolescents, the controls were the least skeletally mature. Summary and Conclusions: In summary, radial and tibial SOS are enhanced by the unique loading pattern in each sport (i.e, upper and lower extremities in gymnastics, lower extremities in soccer), with no cumulative effect between childhood and adolescence. That is, the effect of sport participation on bone SOS was apparent already among the young athletes. Enhanced bone properties among athletes of specific sports suggest that participation in these sports can improve bone strength and potential bone health.