Intelligent Computing in Medical Ultrasonic System

兵庫県立大学大学院201

High calcium bioglass enhances differentiation and survival of endothelial progenitor cells, inducing early vascularization in critical size bone defects

Early vascularization is a prerequisite for successful bone healing and endothelial progenitor cells (EPC), seeded on appropriate biomaterials, can improve vascularization. The type of biomaterial influences EPC function with bioglass evoking a vascularizing response. In this study the influence of a composite biomaterial based on polylactic acid (PLA) and either 20 or 40% bioglass, BG20 and BG40, respectively, on the differentiation and survival of EPCs in vitro was investigated. Subsequently, the effect of the composite material on early vascularization in a rat calvarial critical size defect model with or without EPCs was evaluated. Human EPCs were cultured with β-TCP, PLA, BG20 or BG40, and seeding efficacy, cell viability, cell morphology and apoptosis were analysed in vitro. BG40 released the most calcium, and improved endothelial differentiation and vitality best. This effect was mimicked by adding an equivalent amount of calcium to the medium and was diminished in the presence of the calcium chelator, EGTA. To analyze the effect of BG40 and EPCs in vivo, a 6-mm diameter critical size calvarial defect was created in rats (n = 12). Controls (n = 6) received BG40 and the treatment group (n = 6) received BG40 seeded with 5×105 rat EPCs. Vascularization after 1 week was significantly improved when EPCs were seeded onto BG40, compared to implanting BG40 alone. This indicates that Ca2+ release improves EPC differentiation and is useful for enhanced early vascularization in critical size bone defects

Injectable Cell-based Tissue Engineered Bone Formulations

PhDCurrent golden standard therapy for bone repair and regeneration involves the use of auto grafts. Nevertheless, there are many drawbacks associated with auto grafts including donor site morbidity, requirement for an invasive surgery, post-operative pain and infection. The use of injectable tissue engineered bone is an attractive alternative, providing a minimally invasive approach to regenerate bone. It offers faster healing, less pain and exact conformation to irregular defects. The present work is designed to achieve injectable formulations of tissue engineered bone that fulfil the requirements needed. It involves investigation of potential polymeric binders that are biocompatible, biodegradable and allow bone formation when combined with cells. Chitosan binders were tested for biocompatibility, biodegradability, gelation, angiogenic potential and osteogenic differentiation and bone formation when mixed with goat and human bone marrow derived mesenchymal stem cells (gMSCs, hMSCs). An in vivo bone formation study was performed to investigate the bone formation ability of gMSCs in contact with chitosan binder. Chick chorioallantoic membrane assay was carried out to examine the angiogenic potential of the chitosan binder combined with/without hMSCs. Furthermore, MC3T3-El cells were employed to assess the osteogenic potential of cells exposed to chitosan polymeric systems. Chitosan binder was proved to be an attractive polymer to carry cell-scaffold combination. hMSCs were able to survive and differentiate along the osteogenic lineage when encapsulated with 1.5% (w/v) chitosan-15% (w/v) glycerol phosphate (GP)-0.18% (w/v) hydroxyethyl cellulose (HEC) in a 14-day study. Furthermore, chitosan-GP-HEC solutions demonstrated fast gelation at 37°C. Chitosan was biodegradable following 42 days in the presence/absence of lysozyme. Moreover, gMSCs combined with chitosan binder produced 24.6 ± 13.7% bone comparable to the control group after a 6-week implantation in mice. Chitosan was shown to be nonangiogenic unlike hMSCs which showed angiogenic potential. Also, chitosan was found to be osteogenic at 2 and 0.05 mg/ml concentrations

Dual Phase Engineered Tissue for Enhanced Bone Formation.

Large bone defects are a significant clinical problem in the United States and worldwide. “Non-unions” are fractures that fail to heal due to a lack of blood supply to the defect site. In our approach to bone regeneration, we create modular engineered tissues (“microbeads”) designed to form bone, and combine them with a surrounding vascularizing tissue to generate a dual-phase injectable matrix for enhanced bone formation. In the first Aim, human bone marrow mesenchymal stem cells (bmMSC) or human adipose stem cells (AdSC) were embedded in collagen/fibrin (COL/FIB) or collagen/fibrin/hydroxyapatite (COL/FIB/HA) microbeads. Both cell types mineralized microbeads, indicating differentiation towards the osteogenic lineage. The second Aim used a co-culture model of bmMSC and human umbilical vein endothelial cells in COL/FIB composite hydrogels to create a vasculogenic matrix. Cell ratio and matrix composition were varied in a systematic manner. Vascular network formation increased in vitro with increasing fibrin content in composite materials, although the 40/60 COL/FIB and pure fibrin materials exhibited similar responses. Hydroxyapatite (HA) was found to recover endothelial network formation in unconstrained hydrogels. Over 7 days of dorsal subcutaneous implantation in nude mice, these matrices exhibited increasing neovascularization, though there was no significant effect of HA. The final Aim combined osteogenic microbeads with a surrounding vasculogenic matrix to evaluate the effect of this dual-phase tissue in vivo. Both vasculogenesis and osteogenesis were examined in a subcutaneous bone formation model in the mouse at 4 and 8 weeks. Blood flow measured by Doppler imaging was not significantly different between any conditions at any time point, except at 8 weeks where the vasculogenic matrix alone was lower than all other groups. Micro-computed tomography of ectopic bone demonstrated significantly higher bone volume in the osteogenic microbead condition at 4 weeks and both the blank and osteogenic microbead conditions at 8 weeks, compared to the dual osteogenic/vasculogenic condition. These data suggest an inhibitory effect of the vasculogenic component on bone formation in the non-ischemic model. Dual-phase implants may be more effective in ischemic orthotopic bone regeneration models, and these results demonstrate that such constructs can be designed, fabricated, and delivered for therapeutic use.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99786/1/ramrao_1.pd

The use of an induced muscle flap to reconstruct mandibular defects

The treatment of challenging large osseous defects presents a formidable problem for orthopaedic and maxillofacial surgeons. Autogenous bone grafting is the present method of choice to replace the lost tissue, but supplies of autologous bone are limited and harvesting of the graft is associated with donor site morbidity. Artificial biomaterials hold much promise, but do not, by themselves, supply the osteoprogenitor cells needed for bone formation. Moreover, there are often issues with resorption of the scaffold used in the biomaterial, as well as limited vascularity. This study investigates the novel application of a composite bone mineral (Cerament TM Spine Support) as an injectable bone cement loaded with cytokines and seeded with induced mesenchymal stromal cells, for maxillofacial reconstruction in rabbits. This study aims to test the feasibility of converting a pedicled muscle flap into bone to reconstruct a critical-size defect in the mandible as the above three components would theoretically have the combined effect of osteoconduction, osteoinduction and osteogenesis. The study included a comprehensive assessment of bone regeneration using plain radiography, Cone Beam computerized Tomography (CBCT), Micro-Computerized Tomography (micro-CT) and histology. Results at 3 months following surgery showed areas of bone formation and remnants of residual cement throughout the muscle and connective tissue in the surgical defect. Data analysis showed that complete bone integration or incorporation for the reconstruction of the surgical defect was not achieved. However, the regenerated bone displayed a high degree of remodeling with an intricate network of woven bone trabeculae within the cement. The bone was thicker in the bucco-lingual direction and exhibited more red and fatty marrow spaces compared to the contralateral (non-operated) side. The study confirmed that bone formation within a muscle flap in the maxillofacial region is possible. However, a wide range of variation in the patterns of bone formation was seen among the samples. The findings demonstrated the remarkable potential for the use of autologous muscle flaps as bioreactors for injectable scaffoldings, BMP, rMSCs to facilitate the reconstruction of maxillofacial bony defects

Microspheres for bone regeneration and localised delivery of enzymes

Tese de mestrado. Faculdade de Engenharia. Universidade do Porto. 199

The Synergistic Effect of Bone Graft Substitute Architecture and Mechanical Environment on hMSCs Responses in vitro

PhD thesisPorous silicate substituted hydroxyapatite (SiHA) as synthetic bone graft substitute (BGS) shows excellent bone repair in vivo. It is accepted that the mechanical environment to which cells are exposed regulates cellular differentiation. One mechanism by which BGS architecture may regulate bone formation could be through influencing the shear stress distribution of interstitial fluid. The aim of this study was to investigate the combined influence of BGS architecture and fluid shear environment on human mesenchymal stem cells (hMSCs) responses. hMSCs were cultured on SiHA BGS with defined porosity. A 3D in-house perfusion bioreactor system was established, and two shear stress profiles were applied in this study: 1) continuous basal perfusion rate (BPR) at 0.07 ml/min; 2) BPR with a period of high perfusion rate (pHPR) every day at 2.5 ml/min. The cytoskeleton of hMSCs was reorganized under perfusion conditions compared with under static condition. Shear stress induced both ERK1/2 and pEKR1/2 translocation from the cytoplasm to nucleus. hMSCs cultured in BPR profile differentiated towards osteogenic lineage, while pHPR induced hMSCs to differentiate towards chondrogenic lineage. Gene expression of osx, sox9, runx2 and col ii was not dependent on BGS micro-porosity under static condition. However, the expression of osteogenic transcription factor osx increased significantly with increasing BGS micro-porosity under BPR condition, whereas the expression of chondrogenic markers like sox9, runx2 and col ii decreased with increasing BGS micro-porosity under pHPR condition after 3 days. Nifedipine was used to block L-type voltage-sensitive Ca2+ channel (VSCC) activity. The translocation of ERK1/2 and pEKR1/2 from the cytoplasm to nucleus was found to be dependent on L-type VSCCs. Both BPR induced osteogenic differentiation and pHPR induced chondrogenic differentiation were found to be modulated by L-type VSCCs. The findings of this PhD thesis demonstrate that the future evaluation of porous BGS bioactivity should be conducted under carefully selected perfusion conditions, and the results of this thesis suggest that chondrogenic markers should also be used as one of the indicators for BGS performance in addition to conventional osteogenic markers, as early chondrogenic activity may denote the onset of osteochondral bone formation. This would also argue for longer term culture to further monitor cell fate and the development of any extracellular matrix (ECM) produced

Toward a novel tissue engineering method for repairing critically sized craniofacial bone defects

Non-healing craniofacial bone defects are a major clinical problem. Tissue engineering has the potential to provide a next-generation solution, but specific focus on clinical translatable technologies is needed. A translatable approach combining using enhanced bone marrow and decellularized trabecular bone scaffolds is first investigated. The drawbacks of this approach, including difficulty of scaffold production and invasive cell harvesting, prompted the development of a new approach in which 3D printing of scaffolds is combined with adipose-derived stem cells signaled with platelet-derived growth factor BB. 3D printing was successfully used to create porous, anatomically shaped scaffolds with polycaprolactone. The lack of osteoinductive properties of polycaprolactone was addressed by development of a hybrid material consisting of bone extracellular matrix particles embedded in polycaprolactone and this material was demonstrated to be both printable and bioactive. Platelet-derived growth factor BB was examined as a suitable biomolecule for bone engineering by investigation of its osteoinductive effects on both marrow-derived and adipose-derived mesenchymal stem cells. Platelet-derived growth factor BB was found to be osteoinductive to adipose-derived but not to marrow-derived mesenchymal stem cells with both correlative and loss-of-function evidence, the latter of which made use of a reducible delivery vehicle developed specifically for siRNA delivery. Finally, the fate of transplanted cells is addressed by investigation of chemical exchange saturation transfer magnetic resonance imaging, found to be suitable for non-invasive and longitudinal in vivo monitoring

Implementation of the 3R principle in musculoskeletal research – Refinement measures and in vitro replacement methods

Musculoskeletal disorders are a challenging clinical problem. Each year, millions of patients worldwide experience bone fractures and 10–15% of these fractures suffer from impaired healing. The global prevalence for osteoarthritis is higher than ever before due to an increased life expectancy and rise in associated risk factors such as physical inactivity and obesity. Sophisticated complex treatment plans with novels biologics allowed to effectively achieve remission in patients with rheumatoid arthritis, however, about 25% of the patients still suffers from moderate or even high disease activity. Thus, further fundamental, and translational preclinical research is imperative to tackle the unmet medical needs for musculoskeletal conditions and ensure health throughout the life course. The current goldstandard in preclinical research is the use of animal models, i.e. mainly rodents (mouse, rat). However, during recent years, we have witnessed the failure of promising therapeutics in clinical testing albeit being based on strong evidence from animal experiments. Therefore, it can be speculated that trans-species differences might be responsible for the limited transferability of findings to the human patient. The 3R principle (replace, reduce, refine) published by Russell and Burch in 1959 can be used as a framework for the humane use of animals in research. Moreover, it can enhance and ensure scientific quality and integrity in studies using animals, thereby accelerating the translational process. To enhance the current knowledge on refinement measures in fundamental research studies and to provide evidence-based data on pain management protocols in laboratory animals, we evaluated two analgesics, tramadol and buprenorphine in the drinking water, for their efficiency and side effects on experimental readout in the mouse-osteotomy model. Furthermore, we developed novel in vitro approaches to evade cross-species differences and to replace lab animal usage with a specific focus on fracture healing and joint pathologies. In detail, to recapitulate the initial phase of fracture healing, we specifically focused on integrating the interaction between immune cells and mesenchymal stromal cells/bone-related cells, exemplified by artificial fracture hematoma models containing mesenchymal stromal cells and the combination with three-dimensional scaffold-free bone-like constructs (fracture gap model). This tissue-engineered macroscale approach was used in parallel to mimic cartilage degradation during the onset of osteoarthritis in vitro, which was later extended towards an osteochondral unit model by integrating a tricalcium phosphate-based bone equivalent to recapitulate key features of rheumatoid arthritis. Together, within this thesis, I provide an overview of the variety of approaches towards the active implementation of the 3R principle in musculoskeletal-related preclinical research. Thereby, I specifically underline the importance of equivalently prioritizing all 3Rs to effectively rethink traditional research approaches in biomedicine for continuous improvement in animal welfare and successful human patient-driven translation.Erkrankungen des muskuloskelettalen Systems sind ein herausforderndes klinisches Problem. Jedes Jahr erleiden Millionen von Patienten weltweit Knochenbrüche und bei 10-15 % dieser Frakturen kommt es zu Heilungsstörungen. Die weltweite Prävalenz von Osteoarthrose ist aufgrund der gestiegenen Lebenserwartung und der Zunahme der damit verbundenen Risikofaktoren, wie Bewegungsmangel und Übergewicht, höher als je zuvor. Dank ausgeklügelter komplexer Behandlungspläne mit neuartigen Biologika konnte bei Patienten mit rheumatoider Arthritis eine wirksame Remission erreicht werden, allerdings leiden etwa 25 % der Patienten immer noch unter einer mäßigen oder sogar hohen Krankheitsaktivität. Daher ist weiterführende Forschung unerlässlich, um den verbleibenden medizinischen Bedarf im Bereich der muskuloskelettalen Erkrankungen zu decken. Der derzeitige Goldstandard in der präklinischen Forschung ist die Verwendung von Tiermodellen, insbesondere Nagetieren (Maus, Ratte). Dennoch sind in den letzten Jahren immer wieder neue Therapeutika in der klinischen Testung gescheitert, trotz vielversprechender Daten aus dem Tierversuch. Speziesübergreifende Unterschiede werden für die begrenzte Übertragbarkeit der Ergebnisse auf den menschlichen Patienten verantwortlich gemacht. Das von Russell und Burch 1959 veröffentlichte 3R-Prinzip (Replace, Reduce, Refine) kann als Rahmen für den humanen Einsatz von Tieren in der Forschung dienen sowie die Qualität und Integrität von Tierversuchen sicherstellen und so den Translationsprozess beschleunigen. Um das derzeitige Wissen über Refinement-Maßnahmen zu erweitern und evidenzbasierte Daten zu Schmerzbehandlungsprotokollen bei Labortieren bereitzustellen, haben wir zwei Analgetika, Tramadol und Buprenorphin im Trinkwasser, auf ihre Wirksamkeit und ihre Nebenwirkungen im Maus-Osteotomie-Modell untersucht. Darüber hinaus haben wir neue in vitro Ansätze entwickelt mit speziellem Fokus auf die Frakturheilung und Gelenkpathologien. Um die Anfangsphase der Frakturheilung zu rekapitulieren, konzentrierten wir uns insbe-sondere auf die Interaktion zwischen Immunzellen und mesenchymalen Stromazellen/Knochenzellen, z. B. durch die Kombination von Frakturhämatom-Modellen mit dreidimensionalen trägerfreien knochenähnlichen Konstrukten (Frakturspaltmodell). Ein vergleichbarer Ansatz wurde verwendet, um den Knorpelabbau während der beginnenden Osteoarthrose in vitro zu imitieren. Später wurde dieser Ansatz auf ein Modell der osteochondralen Einheit ausgeweitet, um die Hauptmerkmale der rheumatoiden Arthritis zu rekapitulieren. In dieser Arbeit gebe ich einen Überblick über die Vielfalt der Ansätze zur aktiven Implementierung des 3R-Prinzips in der präklinischen muskuloskelettalen Forschung. Dabei unterstreiche ich insbesondere die gleichwertige Priorisierung aller 3R, um eine kontinuierliche Verbesserung des Tierschutzes und eine erfolgreiche, auf den menschlichen Patienten ausgerichtete Translation zu gewährleisten