Dissertations / Theses on the topic 'Bone substitute; Biomaterials'

Bone graft substitute materials are becoming more common as alternative therapy strategies when bone defects in patients need to be treated. The interaction between bone cells and biomaterials occur at the surface of the materials. A great deal is known about the importance of surface topography and physic-chemical properties of biomaterials. It is also known that cells require proteins in order to interact with biomaterials. Less is known about how material properties and proteins interact forming the biological interface that cells will be exposed to, and that might determine if new bone is formed or not in the patient. The overall aim of the present thesis was to systematically investigate bone graft substitute material surface reactions and the interface in order to better understand how biomaterials may promote bone formation. Bio-Oss (BO) is a commonly used bone graft substitute material in reconstruction of periodontal and dentoalveolar bone defects. BO is mainly considered to be “osteoconductive”, but we could show that it does interact with a biological fluid (α-MEM cell culture medium) through dissolution/precipitation reactions. A significant reduction of calcium and phosphate levels in the medium was obtained even with low concentrations of BO. A release of silicon from the material was also demonstrated. An osteogenic response was seen in close contact to the BO particles when cultured with different types of pre-osteoblastic cells (Paper I). X-Ray Photoelectron Spectroscopy (XPS) with fast-frozen sample technique was used to further characterize the surface of BO, Frios Algipore (AP) and 45S5 Bioglass (BG). These three bone graft substitute materials are used as “model systems”, because they have all demonstrated newly formed bone on the surface after implantation in patients. From the XPS analysis it can be concluded that AP and BG acquired a positively charged surface while BO gained a negatively charged surface. Only AP and BG adsorb organic components (amino acids) from the medium (Paper II). Next we investigated initial surface reactions and the formation of a biological interface in the presence of proteins (serum) for the three biomaterials. The major findings were that in the presence of proteins BO underwent a surface charge reversal, all three biomaterials adsorbed proteins on the surface and all three biomaterials altered the chemical composition of the cell culture medium (Paper III-IV). Silicon (Si), which was released from BO as well as from BG, is interesting in relation to bone health. Positive effects of BG Si dissolution products on osteoblasts have been reported earlier. In the present study inhibitory interactions of Si on the RANK/RANKL/OPG signaling pathway as well as with gap junction intercellular communication in vitro are reported. These new findings implicate that Si could potentially be beneficial for patients with imbalance in bone remodeling (osteoporosis) and treatments of bone defects (Paper V). In conclusion, biomaterials of different origins interact with a solution resembling the extracellular tissue fluid. The dissolution-precipitation reactions are influenced by the material concentration used and should be taken into consideration when designing experiments and when biomaterials are used clinically. The presence of proteins will influence surface reactions, the formation of the biological interface and have implications on cellular responses. Possible dissolution products from the biomaterials should be investigated.  Si, a dissolution product, is shown to have an inhibitory effect on osteoclastogenesis and bone resorption in vitro. Potential clinical value of Si in treatment of patients with bone defects should be further investigated.

Large infected bone defects (IBD) are very complicated to treat due to their high variability; they often require multiple procedures. Bone autografts are the gold standard for treatment but have several drawbacks, such as a need for a second surgery site, limited grafting material, and donor site morbidity. The objective of this research was to develop a moldable synthetic bone grafting material capable of releasing both antimicrobial and osteogenic drugs over a clinically relevant time course for the treatment of IBDs. Current treatment methods for large IBDs require two separate procedures to treat the bone defect and the infection. This research sought to combine these two procedures into one implantable composite bone graft substitute for the treatment IBDs. To begin, the degradation and mechanical properties of the calcium sulfate (CS) based composite material were evaluated for different compositions. Next, the controlled drug release profiles from the composite was achieved by using a shell and core system incorporating poly(lactic-co-glycolic acid) microspheres (PLGAms). The release of vancomycin from the shell began immediately and continued over the course of 6 weeks, while the release of simvastatin from the core was delayed before being released over 4 weeks. Next, an infected, critically-sized rat femoral defect model was used to test different treatment methods with and without the composite bone graft substitute. Animals treated with locally released antibiotics had survivorship rates 24% higher than those treated with systemic antibiotics, and animals that received both antibiotics and an osteogenic drug had an increased amount of bone formation at 12 weeks compared to controls. Finally, several different anti-biofilm agents were evaluated for their ability to inhibit and/or disrupt the growth of Staphylococcus aureus (S. aureus) biofilms in vitro. Lysostaphin was the only drug investigated that was able to both inhibit and disrupt S. aureus biofilms. Furthermore, lysostaphin encapsulated into PLGAms maintained its bioactivity and may be useful for future incorporation into biofilm-combating materials. The bone grafting material developed here can be used to locally deliver drugs in a temporally controlled manner to reduce the number of procedures necessary for the treatment of complex IBDs.

O uso crônico de etanol prejudica diretamente o processo biológico de reparo ósseo, principalmente pela inibição das células osteoblásticas, podendo causar retardo de consolidação ou pseudoartrose. Vários biomateriais naturais ou sintéticos, têm sido utilizados como uma alternativa aos enxertos ósseos. Os biomateriais sintéticos, como o Beta Fosfato Tricálcico (-TCP) são biocompatíveis, biodegradáveis e osteocondutíveis, por orientar as células osteoblásticas, promovendo a neoformação óssea. Sendo assim, objetivou-se avaliar a influência da ingestão crônica de etanol no processo de reparo ósseo de defeitos cranianos tratados com -TCP® comparativamente aos preenchidos com coágulo sanguíneo e a sua interferência sobre a massa corporal. Foram utilizados 40 ratos machos (Rattus norvegicus), distribuídos aleatoriamente em 2 grupos: GAG - receberam água como dieta liquida, e GAL - receberam etanol a 25%. O GAL foi submetido inicialmente à adaptação gradativa ao álcool e depois permaneceu a 25% por 90 dias. Após o término desse período, todos os animais foram submetidos à cirurgia experimental. Nos ossos parietais de cada animal, foram confeccionadas duas cavidades 5,0 mm de diâmetro ao lado da sutura sagital, de forma que o parietal direito foi preenchido com -TCP® e o esquerdo com coágulo. Dessa forma, os grupos GAG e GAL foram divididos de acordo com o preenchimento dos defeitos: GC-AG (Grupo Coágulo Água), GC-AL (Grupo Coágulo Álcool), GB-AG (Grupo Biomaterial Água) e GB-AL (Grupo Biomaterial Álcool). O reparo ósseo foi observado de acordo com o período de eutanásia: 10, 20, 40 e 60 dias após a cirurgia. Após inclusão histológica, as peças foram submetidas à análises histomorfológica e histomorfométrica. Na análise da variação da massa corporal, observou-se que as massas iniciais do GAG foram maiores que GAL e as massas finais de GAG aumentou significativamente apenas em 60 dias e no GAL em todos os períodos. Na análise histomorfológica, observou-se nos períodos iniciais de todos os grupos a presença de tecido conjuntivo fibroso preenchendo toda área central do defeito. Os grupos com os defeitos preenchidos com -TCP apresentaram tecido reacional com as partículas envoltas por células inflamatórias. No período final de análise, observou-se em todos os grupos pequena neoformação óssea com a área central preenchida por tecido conjuntivo fibroso. Nos grupos GC-AL, GB-AG e GB-AL evidência de reação inflamatória menos intensa. Na análise histomorfométrica em relação à influência do tempo na neoformação óssea, os grupos GC-AG e GC-AL apresentaram o mesmo perfil gráfico em todos os períodos exceto 60 dias. O grupo GB-AG houve aumento substancial de neoformação em todo experimento e GB-AL pequeno aumento, mas não foi significante. Em relação à interferência da dieta, observou-se maior percentual de formação óssea nos grupos que consumiram água nos períodos 40 e 60 dias. E em relação ao tipo de preenchimento, observou-se no grupo GC-AG maior formação nos períodos de 20 e 40 dias. Já nos GB-AG e GB-AL a formação foi similar durante todo experimento. Conclui-se que o -TCP® não foi capaz de contribuir para melhor regeneração óssea, apresentando desempenho inferior nos animais que consumiram álcool.
Chronic use of ethanol directly affects the biological process of bone repair, primarily by inhibition of osteoblastic cells. This may cause delayed consolidation or nonunion. Various natural and synthetic biomaterials have been used as alternatives to bone grafting. Synthetic biomaterials such as beta tricalcium phosphate (-TCP) are biocompatible, biodegradable, and osteoconductive, guiding osteoblastic cells and promoting bone formation. Thus, the objective was to evaluate the influence of chronic ethanol intake on the bone healing process of cranial defects treated with - TCP® compared to those filled with blood clot. Forty male rats were used (Rattus norvegicus), randomly divided into two groups: GAG received water as a liquid diet, and GAL received 25% ethanol. The GAL group was initially subjected to gradual adaptation to the alcohol and then maintained at 25% for 90 days. At the end of this period, all animals were submitted to experimental surgery. In the parietal bone of each animal, two cavities were prepared, 5.0 mm in diameter on the side of the sagittal suture, and the right parietal was filled with -TCP® and left to clot. Thus, the GAG and GAL groups were divided according to the filling of defects: GC-AG (Clot Water Group); GC-AL (Clot Alcohol Group); GB-AG (Biomaterial Water Group); and GB-AL (biomaterial Alcohol Group). The bone repair was observed according to the euthanasia period: 10; 20; 40; and 60 days after surgery. After a histological inclusion, the pieces were submitted to histomorphological and histomorphometric analysis. The histomorphological analysis was observed in the initial periods of all groups fibrous connective tissue filling the entire central area of the defect. The groups of defects filled with -TCP showed tissue reaction to particles surrounded by inflammatory cells. At the end of the period of analysis, it was observed that in all groups there was little bone formation, with the central area filled with fibrous connective tissue. In the GC-AL, GB-AG, and GB-AL groups there was evidence of less intense inflammatory reaction. In histomorphometric analysis regarding the influence of time in osteogenesis, the GC-AG and GC-AL groups had the same graphic profile in all periods except 60 days. In the GB-AG group there was a substantial increase of formation in every experiment, and in the GB-AL a small increase, but it was not significant. Regarding the influence of diet, there was a higher percentage of bone formation in the group that consumed water in the 40- and 60-day periods, and in the type of filling, increased formation in the periods of 20 and 40 days was observed in the GC-AG group. In the GB-AG and GB-AL groups, the formation was similar throughout the experiment. It is concluded that the -TCP® was not able to contribute to better bone regeneration, with underperformance in animals that consumed alcohol.

One of the greatest obstacles to clinical translation of bone tissue engineering is the inability to effectively and efficiently vascularise scaffolds. This limits the size of defects that can be repaired, as blood perfusion is necessary to provide nutrient and waste exchange to tissue at the core of scaffolds. The goal of this work was to systematically explore whether architecture, at a scale of hundreds of microns, can be used to direct the growth of microvessels into the core of scaffolds. A pipeline was developed for the production of hydroxyapatite surfaces with controlled architecture. Three batches of hydroxyapatite were used with two different particle morphologies and size distributions. On sintering, one batch remained phase pure and the other two batches were biphasic mixtures of α-tricalcium phosphate (α-TCP) and hydroxyapatite. Sample production methods based on slip casting of a hydroxyapatite-gelatin slurry were explored. The most successful of these involved the use of curable silicone to produce moulds of high-resolution, three dimensional (3D) printed parts with the desired design. Parts were dried and sintered to produce patterned surfaces with higher resolution than obtainable through conventional 3D printing techniques. Given the difficulties associated with the structural reproducibility of concave pores architectures in 3D reported in the literature, in this work, a 2.5D model has been developed that varies architectural parameters in a controlled manner. Six contrasting architectures consisting of semi-circular ridges and grooves were produced. Grooves and ridges were designed to have widths of 330 μm and 660 μm, with periodicities, respectively, of 1240 μm and 630 μm. Groove depth was varied between 150 μm and 585 μm. Co-cultures of endothelial cells and osteoblasts were optimised and used to grow microcapillary-like structures (referred to as "microvessels") on substrates. Literature shows that these precursors to microcapillaries contain lumina and can produce functional vasculature, demonstrating their clinical promise. The effects of the composition and surface texture of grooved samples on microvessel formation were studied. It was found that surface microtopography and phase purity (α-TCP content) did not affect microvessel formation. However, hydroxyapatite architecture was found to significantly affect microvessel location and orientation. Microvessels were found to form predominantly in grooves or between convexities. Two metrics - the degree of alignment (DOA) and the degree of containment (DOC) - were developed to measure the alignment of endothelial cell structures and their localisation in grooves. For all patterned samples, the CD31 (an endothelial cell marker) signal was at least 2.5 times higher along grooves versus perpendicular to grooves. In addition, the average signal was at least two times higher within grooves than outside grooves for all samples. Small deep grooves had the highest DOA and DOC (6.13 and 4.05 respectively), and individual, highly aligned microvessels were formed. An image analysis method that compares sample X-ray microtomography sections to original designs to quantify architectural distortion was developed. This method will serve as a useful tool for improvements to architectural control for future studies. This body of work shows the crucial influence of architecture on microvessel self-assembly at the hundreds of micron scale. It also highlights that microvessel formation has a relatively low sensitivity to phase composition and microtopography. These findings have important implications for the design of porous scaffolds and the refinement of fabrication technologies. While important results were shown for six preliminary architectures, this work represents a toolkit that can be applied to screen any 2.5D architecture for its angiogenic potential. This work has laid the foundations that will allow elucidating the precise correspondence between architecture and microvessel organisation, ultimately enabling the "engineering" of microvasculature by tuning local scaffold design to achieve desirable microvessel properties.

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Tratamentos de superfície de materiais implantáveis no corpo humano são analisados incansavelmente dentro das áreas da saúde. É comum ser observado em artigos científicos o emprego de um recente e desconhecido biomaterial sendo analisado para este fim. O objetivo deste estudo foi avaliar o emprego do alendronato sobre a caracterização topográfica de implantes e discos de titânio in vitro, e sobre a superfície de implantes de titânio inseridos em coelhos após levantamento de seio maxilar, correlacionando as respostas biológicas e físicas do tratamento comparadas a protocolos bem estabelecidos da implantodontia. Utilizou-se seis discos e vinte e seis implantes dentários ambos de titânio comercialmente puro. Para as análises topográficas, discos e implantes se dividiram em 2 grupos de 3 amostras: Grupo CTL, submetidos a subtração ácida e grupo ALD, submetidos a tratamento biomimético com alendronato. Para os testes em animais, 5 implantes foram distribuídos por grupo, CTL e ALD, com intervalos de eutanásia de 7 e 40 dias. Após microscopia eletrônica de varredura, análise de espectroscopia por energia dispersiva, análise do ângulo de contato com a água, de energia livre de superfície e da presença de sítios doares de elétrons (base de Lewis), os grupos ALD apresentaram resposta satisfatória frente aos processos aceitos em saúde, sendo superiores em todas as análises. A resposta histométrica também foi mensurada resultando em maior tecido ósseo nas análises ELCOI e AON nos grupos ALD 40 dias. A discreta presença do TRAP aos 40 dias se refere a baixa reabsorção óssea no momento testado, fator relacionado ao uso de um bisfosfonato à nível local. A osteocalcina em 7 dias sugere uma atividade celular propícia à neoformação. Os tratamentos de biomiméticos são válidos uma vez que, frente aos testes realizados, melhoram a interação dos implantes a nível celular. O alendronato pode ser empregado para os fins propostos resultando em melhora do processo de reparo ósseo.
Surface treatments of implantable materials in the human body are analyzed tirelessly within the health areas. It is common to observe in scientific articles the use of a recent and unknown biomaterial being analyzed for this purpose. The objective of this study was to evaluate the use of alendronate on the t opographic c haracterization of implants and titanium discs in vitro , and over the surface of titanium implants inserted into rabbits after maxillary sinus lift, correlating the biological and physical responses of the treatment compared to well established implantology protocols. It was used six disks and twenty - six commercially pure titanium dental implants. For the topographic analyzes, disks and implants were divided into 2 groups of 3 samples: CTL group, submitted to acid subtraction and ALD group, submi tted to biomimetic treatment with alendronate. For the animal tests, 5 implants were distributed per group, CTL and ALD, with euthanasia intervals of 7 and 40 days. After scanning electron microscopy, analysis of energy dispersive spectroscopy, analysis of the water contact angle, free surface energy and the presence of electron ́s donor sites (Lewis base), the ALD groups showed a satisfactory response to the accepted processes in health, being superior in all the analyzes. The histometric response was also measured resulting in increased bone tissue in the ELCOI and AON analyzes in the ALD 40 days’ group. The discrete presence of TRAP at 40 days refers to low bone resorption at the time tested, a factor related to the use o f a bisphosphonate . Osteocalcin in 7 days suggests a cellular activity conducive to neoformation. Biomimetic treatments are valid since, given the tests performed, they improve the interaction at the cellular level. Alendronate can be used for the proposed applications resulting in a better bone repair process.

The number of clinical procedures performed in the USA using bone graft substitutes was estimated at 1.1 million in 2010 and is projected to reach 1.3 million in 2015. This increasing demand for bone graft substitutes is a result of an ever-ageing population coupled with recent reports in the clinical literature of concerns regarding the safety of allograft and recombinant bone morphogenetic proteins such as rh- BMP-2 and the supply of autograft, which has led to an increased clinical interest in synthetic alternatives to allograft; autograft; and recombinant growth factors. One such synthetic material is silicate-substituted hydroxyapatite (SiCaP). Mechanical testing revealed SiCaP to have similar mechanical behaviour to morcellised cancellous bone. In computated spinal and hip models the simulated stresses in SiCaP were determined to be low when in situ, indicating a stressshielding effect from the implanted metalwork and surrounding bone. We also found an inverse relationship between porosity and Young's Modulus. Our results indicated that the strut-porosity of a material substrate should be increased to maximise the potential for formation of a precursor to bone-like apatite after implantation in osseous defects and further confirmed previous reports that betatricalcium phosphate is less bioactive than hydroxyapatite. We demonstrated a direct link between the amount of strut-porosity and the osteoinductivity of SiCaP. We learned that adding a resorbable carrier phase did not impair the osteoinductive potential of SiCaP, suggesting that osteoinductivity is not necessarily determined in the first 24-48 hours post implantation. Most notably from our studies we determined that the osteoinductivity of SiCaP correlated with its performance in orthotopic defects. Our research confirmed our hypothesis that modifying the micron-scale physical structure of a hierarchical porous SiCaP based biomaterial influences its functional performance in vitro and such modifications can be applied to improve its performance outcomes in ectopic and orthotopic treatment sites in vivo.

An understanding of the interactions between orthopaedic and dental implant surfaces with the surrounding host tissue is critical in the design of next generation implants to improve osseointegration and clinical success rates. Critical to the process of osseointegration is the rapid establishment of a patent neovasculature in the peri-implant space to allow for the delivery of oxygen, nutrients, and progenitor cells. The central aim of this thesis is to understand how biomaterials regulate cellular and host tissue response to elicit a pro-angiogenic microenvironment at the implant/tissue interface. To address this question, the studies performed in this thesis aim to 1) determine whether biomaterial surface properties can modulate the production and secretion of pro-angiogenic growth factors by cells, 2) determine the role of integrin and VEGF-A signaling in the angiogenic response of cells to implant surface features, and 3) to determine whether neovascularization in response to an implanted biomaterial can be modulated in vivo. The results demonstrate that biomaterial surface microtopography and surface energy can increase the production of pro-angiogenic growth factors by osteoblasts and that these growth factors stimulate the differentiation of endothelial cells in a paracrine manner and the results suggest that signaling through specific integrin receptors affects the production of angiogenic growth factors by osteoblast-like cells. Further, using a novel in vivo model, the results demonstrate that a combination of a rough surface microtopography and high surface energy can improve bone-to-implant contact and neovascularization. The results of these studies also suggest that VEGF-A produced by osteoblast-like cells has both an autocrine and paracrine effect. VEGF-A silenced cells exhibited reduced production of both pro-angiogenic and osteogenic growth factors in response to surface microtopgraphy and surface energy, and conditioned media from VEGF-A silenced osteoblast-like cell cultures failed to stimulate endothelial cell differentiation in an in vitro model. Finally, the results show that by combining angiogenic and osteogenic biomaterials, new bone formation and neovascularization can be enhanced. Taken together, this research helps to provide a better understanding of the role of material properties in cell and host tissue response and will aid in the improvement of the design of new implants.

Pathological processes in the alveolar and facial bones can lead to bone loss that may not heal with complete regeneration. Biomaterials can be used to facilitate the healing process and/or as a bone substitute, but the mechanisms are not fully understood. Persistent leakage of bacteria/bacterial toxins, after root canal treatment, may lead to a residual bone defect. The healing is dependent on a placed dental biomaterial providing a tight seal. The composition of the filling material may also influence the healing process. The general aim of this study is to investigate surface properties and biological interactions of biomaterials used in dento-alveolar surgery. A dental biomaterial, a bonded compomer (DAP) containing a corroding glass filler, was used as a root end filling material, promoting a new operation technique. The healing (assessed according to Molven´s x-ray criteria) demonstrates a significant improvement in healing results for the compomer group, compared to a commonly used technique. The surface properties and biological interactions of DAP were analyzed. ICP-OES of DAP cell culture medium extract demonstrated a significant release of Sr, Si and F from the dental biomaterial. Human periodontal ligament (PDL) cells grew on and around DAP specimens without any sign of toxic reactions. DAP extract stimulated proliferation of PDL cells, but caused an inhibition of osteoblastic gene expression in mouse bone marrow cells. The surface properties of the glass containing compomer may contribute to improved healing of the periapical lesions. A bovine inorganic bone graft substitute (BO) is commonly used as a treatment option in dento-alveolar surgery with new bone formation in immediate close contact with BO material. ICP-OES dissolution analysis of cell culture media, after incubation with BO particles, demonstrated a dosedependent release of Si and a decrease of Ca and P. An uptake of Ca from the medium to the BO particle was demonstrated with calcium-45 labeling. The Si dissolution varied between different batches, possibly reflecting a variation in food intake in the animals. Stimulated osteogenic response was seen in close contact to the BO particles in cell cultures. Furthermore, it was clearly demonstrated that the study design is a critical factor for correctly understanding biomaterials’ biological interactions. The surface properties of three bone graft substitutes reported to have good results in dento-alveolar surgery were investigated, in order to establish whether or not dissolution-precipitation reactions could contribute to the bone healing. Dissolution-precipitation extracts of BO, bioactive glass 45S5 (BG) and a marine algae hydroxyl apatite (AP) in cell culture media were analyzed. Dissolution of Si at significant levels was detected for BO and 45S5 over time. Significant uptake levels of Ca and P from the culture were seen for both 45S5, BO and AP but at different times. Surface analysis of the biomaterials with SEM/EDAX, before and after immersion in cell culture media, revealed a smoothing of the surface morphology for 45S5 over time. No obvious alterations for BO and AP were detected. Ca/P ratio decreased significantly for 45S5, but no major changes were detected by XPS for BO or AP. XPS further demonstrated a surface charge for BO, changing from negatively to positively charged when exposed to serum. 45S5 and AP had positive surface charges, both in the absence and the presence of serum. These demonstrated surface changes in biomaterials could contribute to adherence of cells and subsequently affect bone healing. Conclusion: Biomaterials used in dento-alveolar surgery interact with biological surroundings through surface and dissolution-precipitation reactions which may have implications for bone healing.

This PhD thesis is a compendium of three publications, which sets out to broaden our knowledge and understanding of the topical application of bisphosphonates alone or mixed with a bone graft in alveolar bone defects, to evaluate the potential capacity of them to preserve/enhance alveolar new bone formation. In recent years, research has focused on improving bone substitutes to achieve faster and better regeneration by morphologic and biochemical modification. Bisphosphonates are a group of drugs that reduce bone resorption by inhibiting the formation, recruitment activity of mature osteoclasts; and promoting their apoptosis. In addition, some bisphosphonates enhance osteoblast differentiation and activity. Thence, it has been demonstrated that topical application of a bisphosphonates can minimize the bone resorption following muco-periostial flap surgery or in peri- implantitis; improve the osteoconductive and regenerative capacity of a biomaterial; prevent the surface resorption of onlay bone grafts; or reduce post-extraction dimensional changes. Mandibular second premolars (P2) and first molars (M1) were extracted from six Fox-Hound dogs. P2 were categorized as small defects (SD) and M1 as large defects (LD). Four random groups were created: SC (small control defects with MP3®), ST (small test defects MP3® + pamidronate), LC (large control defects with MP3®), and LT (large test defects MP3® + pamidronate). At four and eight weeks of healing the percentages of new bone formation (NB), residual grafts (RG) and connective tissue (CT) were analysed by histology and histomorphometric analysis. To complement the information already obtained from histological analysis, the samples were evaluated through scanning electron microscopy (SEM), and Energy dispersive X-ray spectroscopy (EDX), to identify the chemical elements present into the biomaterial and surrounding tissues, for understanding the biomaterial’s degradation process. The study was complemented with a systemic literature review of the articles published between January 2000 and December 2016, that evaluated in vivo the effects of the topical application of bisphosphonates on bone regeneration/preservation in alveolar defects. A total of 154 abstracts were identified, of which 18 potentially relevant articles were selected; a final total of nine papers were included for analysis. Histomorphometric and histologic analysis of the present pilot study demonstrated that after 4 and 8 weeks of healing, higher new bone formation for test groups (ST and LT) treated topically with pamidronate, compared with SC and LC respectively; residual graft was significantly higher in both control groups (SC and LC) compared to test (ST and LT) groups; and connective tissue percentage was higher in large defects (LC and LT) compared to small defects (SC and ST). SEM analysis revealed more mineralized bone in test groups (ST and LT) compared with control groups, demonstrated by higher percentages of Ca obtained from EDX spectroscopy. Within the limitations of this experimental study, the findings suggest that porcine xenografts (MP3®) modified with pamidronate favours the new bone formation and increased the porcine xenograft substitution/replacement after 4 and 8 weeks of healing. These results are in accordance with the conclusions obtained from the systematic review. Despite the comparison of the findings of the selected studies was made difficult by the heterogeneity of the articles, the topical application of bisphosphonate solution would appear to favour new bone formation in alveolar defects, and boosts the regenerative capacities of biomaterials resulting in increased bone density.

Procedimentos de levantamento do seio maxilar têm sido realizados para aumentar o volume ósseo e promover a estabilidade do implante, na região posterior de maxilas severamente atrofiadas. Ao longo dos anos, resultados de vários estudos demonstraram que alguns substitutos ósseos podem suportar implantes em função, após o levantamento de seio maxilar, igual ou melhor quando utilizado o osso autógeno. Neste estudo, foi avaliado o comportamento de um substituto ósseo completamente biodegradável (OsteoScaf™) no modelo experimental de levantamento de seio maxilar em coelhos. Além disso, ele foi comparado com o osso autógeno e outros dois substitutos ósseos, não totalmente biodegradáveis, disponíveis comercialmente (Bio-Oss® e BoneCeramic®). Avaliação clínica, tomografia computadorizada por feixe cônico, microtomografia computadorizada, análises microscópicas e análise molecular, através da técnica de PCR, foram realizadas após 2, 4 e 8 semanas de cirurgia. O levantamento de seio maxilar utilizando o osso autógeno demonstrou maior reabsorção, ao longo do tempo, comparado aos substitutos ósseos, os quais revelaram maior neoformação óssea após 8, 4 e 2 semanas, respectivamente. O grupo Bio-Oss® apresentou maior neoformação óssea, ao longo do tempo, quando comparado aos grupos Osteoscaf™ e BoneCeramic®, os quais foram qualitativamente emelhantes. O grupo BoneCeramic® mostrou uma resposta celular de células gigantes até 8 semanas. Concluiu-se que os substitutos ósseos, neste estudo, obtiveram melhor desempenho do que o osso autógeno, e o OsteoScaf™ demonstrou maior reabsorção do que os outros grupos, em todos períodos.
Maxillary sinus augmentation procedures have been applied to increase bone volume and to promote stability of implants in the severely atrophied posterior maxilla. Over the years, the outcomes of several studies have demonstrated that some bone substitutes can support implants in function after sinus augmentation as well as, or better than those with autogenous bone. Our experimental model evaluated the behavior of a fully biodegradable bone substitute (OsteoScaf™) in a rabbit sinus lift procedure. We compared this with autogenous bone and other two available non-biodegradable bone substitutes (Bio-Oss® and BoneCeramic®). Clinical evaluation, Cone Beam Computed Tomography, Microcomputed Tomography, microscopic and molecular evaluation were used for data analysis at 2, 4 and 8 weeks after sinus augmentation. Autogenous bone was more resorbed over time than the other materials. All bone substitutes showed more bone formation at 8, 4 and 2 weeks, respectively. Bio-Oss® showed more bone formation/timepoint than Osteoscaf™ and Boneceramic®, which were similar. Boneceramic® showed a florid giant cell response up to 8 weeks. We concluded that the bone graft materials all performed better than autogenous bone and OsteoScaf™ showed comparative bone growth yet greater degradation than the other two materials.

Bone regeneration or augmentation is often required prior to or concomitant with implant placement. With the limitations of many existing technologies, a biologically compatible synthetic bone grafting substitute that is osteogenic, bioerodible, and provides spacing-making functionality while acting as a drug delivery vehicle for bioactive molecules could provide an alternative to ‘gold standard’ techniques. In the first part of this work, calcium sulfate (CS) space-making synthetic bone grafts with uniformly embedded poly(β-amino ester) (PBAE) biodegradable hydrogel particles was developed to allow controlled release of bioactive agents. The embedded gel particles’ influence on the physical and chemical characteristics of CS was tested. Namely, the compressive strength and modulus, dissolution, and morphology, were studied. All CS samples dissolved via zero-order surface erosion consistent to one another. Compression testing concluded that the amount, but not size, of embedded gel particles significantly decreased (up to 75%) the overall mechanical strength of the composite. Release studies were conducted to explore this system’s ability to deliver a broad range of drug types and sizes. Lysozyme (model protein for larger growth factors like bone morphogenic protein [BMP]) was loaded into PBAE particles embedded in CS matrix. The release of simvastatin, a small molecule drug capable of up regulating BMP production, was also examined. The release of both lysozyme and simvastatin was governed by dissolution of CS. The second part of this work proposed a bilayered CS implant. The physical and chemical properties were characterized similarly to the CS composites above. Release kinetics of directly loaded simvastatin in either the shell, core, or both were investigated. A sequential release of simvastatin was witnessed giving foresight of the composite’s tunability. The sequential release of an antibacterial, metronidazole, loaded into poly(lactic-co-glycolic acid) (PLGA) particles embedded into the shell along with directly loaded simvastatin either in the shell, core, or both layers was also observed. Through controlled release of bioactive agents, as well as a tunable layered geometry, CS-based implants have the potential to be optimized in order to help streamline the steps required for the healing and regeneration of compromised bone tissue.

Injury to bone is one of the most prevalent and costly medical conditions. Clinical treatment of volumetric bone loss or hard-to-heal bony lesions often requires the use of proper bone grafting materials, with or without adjuvant anabolic therapeutics. Despite significant problems associated with autografting (donor site morbidity, limited supplies) and allografting (disease transmissions, high graft failure rates) procedures, synthetic bone grafts remain the least utilized clinically. Existing synthetic orthopaedic biomaterials rarely possess a combination of bone-like structural and biochemical properties required for robust osteointegration, scalable and user-friendly characteristics indispensable for successful clinical translations. This thesis tests the hypothesis that by recapitulating key structural elements and biochemical components of bone in 3- and 2-dimensional biomaterials, scalable synthetic bone grafts can be designed to enable expedited healing of hard-to-heal volumetric bone loss. Specifically, FlexBone, a 3-dimensional hydrogel scaffold encapsulating 50 wt% of structurally well integrated nanocrylstalline hydroxyapatite, the main inorganic component of bone, was developed. The large surface area of nanocrystalline hydroxyapatite combined with its intrinsic affinity to proteins and its excellent structural integration with the hydrogel matrix enabled FlexBone to both sequester endogenous protein signals upon press-fitting into an area of skeletal defect and to deliver exogenous protein therapeutics in a localized and sustained manner. We demonstrated that FlexBone enabled the functional healing of critical-size long bone defects in rats in 8 – 12 weeks with the addition of a very low dose of osteogenic growth factor BMP-2/7. This promising synthetic bone graft is now being explored for the delivery of multiple growth factors to expedite the healing of diabetic bony lesions. In addition, a 2-dimensional electrospun cellulose fibrous mesh was chemically modified with sulfate residues to mimic sulfated polysaccharide ECM components of skeletal tissues to enabled progenitor cell attachment and differentiation as well as controlled retention and localized/sustained delivery of protein therapeutics. This sulfated fibrous mesh is currently explored as synthetic periosteum to augment the osteointegration of devitalized structural allografts. Finally, a rat subcutaneous implantation model developed to examine the biocompatibility of newly developed biodegradable shape memory polymer bone substitutes is also presented.

Natural composites, in particular nacre, often combine high strength and toughness thanks to highly ordered architectures and controlled geometries of the reinforcement components. However, combining strength, toughness and resorbability in synthetic materials remains a challenge in particular in the field of bone graft substitutes. In the present study, calcium phosphate-(CaP-)based materials with designed architectures inspired from natural composite materials were achieved. CaP platelets obtained by precipitation in organic medium were first aligned in chitosan matrices by solvent casting in ambient conditions. Efficient strengthening was obtained with 15 vol-% ceramic, reaching cortical bone strength (150 MPa) and preserving good ductility (5 % deformation). In a weak magnetic field, high spatial arrangement without percolation was maintained up to 20 vol-%. With directional freezing, good alignment of the platelets could be pushed up to 50 vol-%. In parallel, in situ recrystallization of CaP blocks in hydrothermal conditions led to hierarchical structures. The strength and the work-of-fracture were enhanced (300%) thanks to a change of failure mode.

Introdução: os fenômenos envolvidos durante o processo de reparo após elevação do assoalho do seio maxilar, relacionados ao uso de enxertos e biomateriais, não são completamente conhecidos. Objetivos: avaliar clínica, radiológica e histomorfometricamente os resultados de neoformação óssea e desempenho de implantes inseridos em sítios previamente reconstruídos por meio de cirurgia de elevação do assoalho do seio maxilar utilizando osso bovino inorgânico, verificando se há correlação entre os mesmos. Metodologia: a amostra consistiu em 20 implantes inseridos após 7 a 11 meses, em 20 sítios de 10 pacientes adultos, consecutivamente, submetidos à cirurgia de levantamento do assoalho do seio maxilar e enxerto de osso bovino inorgânico. No momento da inserção do implante foi coletada uma amostra de tecido ósseo no sitio da perfuração, para a realização da histomorfometria. O sucesso dos implantes foi avaliado por critérios clínicos decorridos quatro meses de sua inserção. A densidade radiográfica das radiografias periapicais prévias à instalação de 13 dos implantes foi medida e correlacionada aos dados histomorfométricos e de sucesso. Resultados: um implante foi perdido. A porcentagem media de osso vital foi de 50.06%, a de não vital de 40.17%, e de tecido mole de 9,44%. Houve correlação positiva entre a densidade radiográfica e a quantidade de osso vital, e negativa entre esta e a quantidade de tecido mole. Não houve correlação entre o sucesso dos implantes e os demais achados. Conclusões: a cirurgia de elevação do seio maxilar mostrou-se segura a eficaz, permitindo inserção de implantes nos períodos citados. A radiografia periapical digitalizada apresentou potencial para estimar o percentual de osso vital após este tipo de procedimento.
Introduction: healing process after sinus floor elevation using biomaterials is not fully understood. Objectives: clinical, radiological and histomorphometric evaluation of healing, as well as implant results on previously sinus lift implant sites using inorganic bovine graft, testing for correlation between variables. Methods: 20 implants inserted after 7 to11 months, on 20 sitesof 10 adult consecutive patients,indicated for sinus floor elevation using inorganic bovine graft composed the study sample. On implant insertion a bone sample was trefined from implant site, for histomorphometry. Implant success was evaluated by clinical criteria 4 months after insertion. Radiographic density was measured before implant insertion on 13 sites and correlated to success and histomorphometric data. Results: one implant was lost. Vital bone was present on 50.06% of the sample areas, non vital on 40.17% and soft tissue one 9,44%. There was positive correlation between radiographic density and vital bone area, as weel as negative correlation with soft tissue area. There was not correlation between implant success and the other data. Conclusions: sinus lift presented as a safe and efficient treatmenta alternative for implant placement, on the healing periods described. Periapical radiography showed potential to estimate the vital bone and soft tissue formation after sinus lift using this kind of graft.

Wenn erworbene oder angeborene Knochendefekte aufgrund überkritischer Größe oder krankhafter Störungen nicht durch natürliche Regenerationsprozesse geheilt werden können, ist der Einsatz von Knochenersatzmaterialien notwendig. In der vorliegenden Arbeit ist es gelungen ein neuartiges Knochenersatzmaterial zu entwickeln und eingehend zu charakterisieren. Dazu wurden die Phasen Silikat und Kollagen in einem biomimetisch inspirierten Prozess zu einem Anorganik/Organik-Komposit verbunden. Calciumphosphatphasen konnten darüber hinaus als dritte Komponente hinzugefügt werden. Dafür wurden Herstellungsstrategien entwickelt, die Silikat in Form von Kieselsäure, Kollagen als hochkonzentrierte Suspension und gegebenenfalls Calciumphosphat als Pulver zu homogenen Mischungen vereinten. Als Zwischenprodukte wurden Komposithydrogele erhalten, deren Überführung in Xerogele in der Literatur als kritischer Schritt gilt, weil die dabei auftretenden Kapillarspannungen die Gelstruktur in der Regel irreversibel zerstören, wodurch das Material als Pulver oder Fragmente erhalten wird. Im vorliegenden Fall aber konnte die Gelfestigkeit in einem definierten Zusammensetzungsbereich durch die Kompositbildung und die kontrollierte Trocknung der Hydrogele so gesteigert werden, dass monolithische Proben von bis zu mehreren Kubikzentimetern Größe erhalten wurden. Diese konnten ohne weitere Verarbeitungsschritte einer Reihe von Untersuchungen zu mechanischen Eigenschaften, Bioaktivität, Degradabilität und Biokompatibilität unterzogen werden.

Combler une perte osseuse est une nécessité fréquente en chirurgie. L’usage d’implants biocéramiques est limité, conduisant au delà de quelques cm3, inaccessibles à l’ostéogénèse, à une hétérogénéité intrinsèquement fragile et une vascularisation interrompue. Les biomatériaux hybrides sont une solution. Ensemencée, la céramique est mise en culture en bioréacteur, un fluide y circulant afin d'alimenter les cellules et d’évacuer leurs déchets.L’étude porte sur une céramique en phosphate de calcium dont l’architecture interne consiste en pores sphériques interconnectés de quelques centaines de microns de diamètre. Le choix du débit et de l'écoulement à travers ce matériau reste jusqu’ici empirique. Pour optimiser la culture, il s'avère cependant nécessaire de savoir les déterminer en tout point afin de pouvoir les optimiser et les reproduire et, à terme, d'optimiser le choix de la biocéramique.L'étude numérique s'impose car les grandeurs physiques finales recherchées (vitesses et pressions locales) ne sont pas mesurables sur la structure microscopique. Or, ces grandeurs optimisées permettent de définir les grandeurs d'entrée optimales. Le protocole débute par une modélisation de la microstructure et le calcul de la taille du VER afin d'identifier la perméabilité intrinsèque du milieu. Une analyse statistique de la répartition des contraintes tangentielles en fonction des grandeurs moyennes homogénéisées permet ensuite d'identifier les paramètres d'entrée optimaux dans le VER. Finalement, une optimisation globale de la résolution des équations de Navier-Stokes sur la structure homogénéisée permet de définir le paramètre final, à savoir le débit de contrôle du bioréacteur
Repairing a bone loss is a frequent need in orthopaedic surgery. Though now much developed the use of bioceramic implants is limited: a bulk greater than a few cm3 does not allow a complete osteogenesis. So an intrinsic brittle heterogeneity remains with an interrupted vascularization. Hybrid biomaterials are a solution. Seeded ceramics are cultured in a bioreactor, a fluid flowing through the material to feed the cells and to clear their waste out.This study focuses on a calcium phosphate ceramic whose internal architecture consists of spherical interconnected pores of a few hundred microns in diameter. The choice of the flow rate or other flow parameters through the material remains far empiric. To optimize the cell culture, it is however necessary to know how to determine them at any point in order to optimize and reproduce them and eventually to optimize the choice of the bioceramic.A numerical study is necessary because the final desired physical parameters (local velocities and pressures) are not measurable on a microscopic structure. However, these quantities are used to define the optimal input values. The protocol begins in modelling the microstructure and determining the size of the representative elementary volume (REV) in order to identify intrinsic permeability of the material. A statistical analysis of the distribution of shear stresses based on homogenized average values then leads to identify optimal input parameters in the REV. Finally, a global optimization of solving of the Navier-Stokes equations on the homogenized structure leads to define the final parameter, namely the flow of control of the bioreactor

Für das Tissue Engineering von Knochen werden poröse dreidimensionale Substrate (Scaffolds) als Zellträger benötigt, die in der vorliegenden Arbeit über keramische Technologie hergestellt wurden. Neben dem strukturierten und getrockneten Verbundwerkstoff (Grünkörper) und der Sinterkeramik wurde auch der Zwischenzustand nach Ausheizen der organischen Phase (Braunkörper) evaluiert. Bei der Herstellung blieb die Architektur der parallel orientierten Kanalporen, die über den Sol-Gel-Prozess der gerichteten ionotropen Gelbildung des Alginates erzeugt wurde, in allen Materialzuständen erhalten. Die Herstellungstechnologie wurde derart optimiert, dass die neuartigen anisotropen Scaffolds allen prinzipiell gestellten Forderungen für das Tissue Engineering entsprachen – sie waren porös mit weithin einstellbarer Porengröße, sterilisierbar, gut handhabbar unter Zellkulturbedingungen, biokompatibel und degradabel. Der unerwartete Favorit der Biomaterialentwicklung, der Braunkörper – eine nanokristalline, poröse Hydroxylapatit-Biokeramik – lag in einer ersten in vivo-Studie nach 4 Wochen integriert im Knochen vor. Die beobachtete Knochenneubildung deutete auf eine osteokonduktive Wirkung des Materials hin. Die in der vorliegenden Arbeit untersuchten Technologien und Biomaterialien bieten eine Basis für weitere Forschung und motivieren zur Weiterentwicklung und Nutzung als Scaffold für das Tissue Engineering oder Knochenersatzmaterial unter Verwendung der interessanten Architektur.

Thanks to recent advances in science and engineering, the field of biomaterials stands poised to increase the effectiveness and longevity of established devices as well as to provide new options to biomedical engineers who work at designing future products. From its beginning, the field of bioengineering has focused on providing the best artificial devices - hearing aids, artificial limbs and other prostheses - to replace body parts that are missing, broken, or dysfunctional. Regeneration of body parts requires a biomaterial with a structure, components and chemical signals that allow the body ’s tissue cells to recognize, respond to, and remodel the material without rejecting it as foreign. Bone, cartilage and the major load bearing joints of the body all suffer degenerative changes with age and trauma. This area of research focus will seek solutions to the problems of osteoporosis, the fixation of implants in bone and the replacement of damaged bone and cartilage. This will be achieved through the development of non-metallic biomaterials and resorbable biomaterials that provide appropriate load bearing characteristics and the potential to interact suitably with the biology. The non-metallic materials for bone substitutes serve as scaffolds and may have modified surfaces to encourage natural tissue growth or the ability to be seeded with the hosts own cells before implantation. This will have applications for both bone and cartilage substitute materials. These bone substitutes biomaterials are the second-most implanted of all materials. Resorbable biomaterials, on the other hand, gradually disappear from the body as a result of hydrolysis, because are made from molecules similar to those in the human body, which resorb while the tissue is healing. This eliminates the need for a second surgery. The goal of this thesis is to explain the usefulness of these biomaterials in medical applications and especially in orthopaedics, focusing on the latest acquisitions. The first chapter makes an introduction in biomaterials, with emphasis on orthopaedic biomaterials. The second chapter contains information about: (1) the bone characteristics (anatomy and mechanics), in order to understand the basis for tissue engineered therapies and how damaged bones heal, (2) the non-metallic biomaterials (polymers, biodegradable polymers, ceramics and composites) for bone substitutes, giving examples of modern biomaterials used today and (3) the principles involved in the Modern Cementing Technique. The third chapter is a review of the chemistry of the polymers used in bioresorbable biomaterials, including synthesis and degradation, describe how properties can be controlled by proper synthetic controls such as copolymer composition, highlight special requirements for processing and handling, and presents in detail some of the commercial resorbable biomaterials.
Αυτή η διατριβή εξηγεί τη χρησιμότητα των μη μεταλλικών βιο-υλικών για τα υποκατάστατα οστών και των βιο-απορροφημένων βιο-υλικών στις ιατρικές εφαρμογές και ειδικά στις ορθοπεδικές, που εστιάζουν στις πιό πρόσφατες αποκτήσεις.

Natural composites, in particular nacre, often combine high strength and toughness thanks to highly ordered architectures and controlled geometries of the reinforcement components. However, combining strength, toughness and resorbability in synthetic materials remains a challenge in particular in the field of bone graft substitutes. In the present study, calcium phosphate-(CaP-)based materials with designed architectures inspired from natural composite materials were achieved. CaP platelets obtained by precipitation in organic medium were first aligned in chitosan matrices by solvent casting in ambient conditions. Efficient strengthening was obtained with 15 vol-% ceramic, reaching cortical bone strength (150 MPa) and preserving good ductility (5 % deformation). In a weak magnetic field, high spatial arrangement without percolation was maintained up to 20 vol-%. With directional freezing, good alignment of the platelets could be pushed up to 50 vol-%. In parallel, in situ recrystallization of CaP blocks in hydrothermal conditions led to hierarchical structures. The strength and the work-of-fracture were enhanced (300%) thanks to a change of failure mode.:Acknowledgements v Summary vii Background vii Thesis outline viii Part I: “Brick-and-mortar” structures with discrete reinforcement components ix Part II: “Textured” structures with continuous reinforcement components x Zusammenfassung xi Hintergrund xi Doktorarbeit Gliederung xii Teil I: “Ziegelmauer-Architektur” mit diskreten Verstärkungskomponenten xiii Teil II: “ Texturierte” Strukturen mit kontinuierlichen Verstärkungskomponenten xiv Chapter 1: General introduction 1 Bone grafting 1 CaP ceramics 1 How to improve toughness of CaP ceramics? 2 Importance of structure design: bio-inspiration 2 What mechanical properties should be reached? 5 Specific aims 5 Two general approaches to reach the goal 6 Nacre-inspired “brick-and mortar” structures (Part I) 6 Textured ceramic monoliths (Part II) 6 References 7 Chapter 2: Theoretical calculations 11 Introduction 12 Theoretical tensile strength of β-TCP platelets and critical size for flaw tolerance 13 Optimal aspect ratio 15 Composite strength and stiffness 17 Limitations 19 References 19 PART I: NACRE-INSPIRED “BRICK-AND-MORTAR” STRUCTURES Chapter 3: Synthesis of sub-micrometer calcium phosphate platelets 23 Introduction 24 ii Materials and Methods 25 Precipitation method 25 Reaction parameters 25 Characterization 26 Statistical analysis of results 28 Results 28 Reproducibility of standard experiments 28 Increase of the reagent volume to increase the productivity 30 Increase of the precursors concentration to increase the productivity and modify the particles 30 Increase of titration rate to simplify the process 32 Influence of temperature on the particles 35 Effect of the pH value on the particles 37 Effect of a longer reaction time on the particle stability 40 Study of the influence of variations of the Ca/P molar ratio 42 Discussion 43 Reproducibility 43 Productivity 44 CaP crystal shape 45 Crystal purity 47 Aspect ratio 48 Critical thickness 49 Uniformity of primary particles 50 Non agglomerated 51 General points 52 Conclusions 52 References 53 Chapter 4: Kinetics study of the calcium phosphate platelets growth 57 Introduction 58 Theory 58 Materials and methods 60 Materials and sample preparation 60 Characterization methods 61 Results 62 Visual observations during manipulations 62 SEM observations 62 XRD results 66 Size measurements 68 Kinetics calculations 70 Discussion 74 Nucleation and assembly mechanism 74 Reaction kinetics 76 Control of size and aspect ratio 76 Conclusions 77 References 78 Chapter 5: Structural design of bio-inspired composites by solvent casting 81 Foreword 82 Introduction 82 Experimental section 84 iii Synthesis of resorbable ceramic platelets 84 Solvent casting to prove the reinforcement efficiency of DCP platelets 84 Magnetization of the platelets 85 Maintaining the orientation during drying of an hydrogel matrix 86 Results 87 Synthesis of resorbable ceramic platelets 87 Solvent casting to prove the reinforcement efficiency of CaP platelets 87 Magnetization of the platelets 91 Maintaining the orientation during drying of an hydrogel matrix 93 Discussion 95 Detrimental effect of β-TCP platelets in chitosan 95 Efficient reinforcement with DCP platelets up to a given volume fraction 96 Threshold value for strength improvement 97 Fitting the experimental results with theoretical equations 98 Conclusions 101 References 101 Chapter 6: Biodegradable, strong and tough nacre-inspired structures obtained by freezecasting 105 Introduction 106 Experimental section 108 Synthesis of resorbable ceramic platelets 108 Preliminary freeze-casting tests with β-TCP-based slurries 108 Determination of adequate freeze-casting parameters for hydrogels-CaP slurries 108 Integration of CaP platelets and local planar alignment 109 Attempts to globally align porosity in two directions 109 Densification and consolidation 110 Tensile testing 110 Results 111 Preliminary freeze-casting tests with β-TCP-based slurries 111 Determination of adequate freeze-casting parameters for hydrogels-CaP slurries 112 Integration of CaP platelets and local planar alignment 113 Attempts to globally align porosity in two directions 119 Densification and consolidation 121 Tensile testing 121 Discussion 122 Conclusions 124 References 125 PART II: TEXTURED CERAMIC MONOLITHS Chapter 7: Micro-texturing by recrystallization of calcium phosphate blocks in hydrothermal conditions 127 Introduction 128 Materials and Methods 130 Samples characterization 132 Results 133 Macroscopic observations 133 Microstructural changes (SEM) 133 Crystalline phase conversion (XRD) 139 iv Mechanical properties 142 Fractured surfaces 142 Discussion 145 Conclusions 150 References 150 Chapter 8: Toughening of textured calcium phosphate blocks by polymer impregnation 155 Foreword 156 Introduction 156 Materials and Methods 157 Samples preparation 157 Characterization 158 Results 158 Porosity and microstructure 158 Composition 161 Mechanical properties 161 Discussion 162 Conclusions 164 References 164 Chapter 9: Synthesis and outlook 167 Curriculum Vitae 171

The issue of prosthetic infection leading to implant failure due to the formation of bacterial biofilms on biomaterial surfaces has been widely recognized as a major issue, often leading to revision surgery. The growing number of patients requiring synthetic biomaterials as implants is on the rise and so is the risk of infection arising from pre/peri-/post-operative surgical procedures. Traditional antibiotic treatment has led to the emergence of bacterial drug resistance. Therefore, the development of novel bactericidal methods to combat drug resistant microbial pathogens is the need of the hour. The first part of the thesis is an attempt to address prosthetic infection by the development of novel ultrasmall gold nanoparticles (AuNPs) which are cytocompatible and present a therapeutic dosage window for eliciting antimicrobial property. Towards this end, ultrasmall AuNPs with 0.8 nm and 1.4 nm gold core sizes, stabilized by monosulphonated triphenylphosphine ligand shells were synthesized. Such intricately designed AuNPs with ultrasmall gold cores and phosphine-based ligand chemistry were demonstrated to be highly potent bactericidal agents against staphylococci, the most common human pathogen causing biomaterial associated infection. The antibacterial efficacy of these AuNPs was significant even in mature staphylococcal biofilms. In another study, the application of high strength pulse magnetic fields (1-4 Tesla) was examined for bacterial growth inactivation in vitro. A magnetic field strength dependent decrease in bacterial viability with a concomitant increase in the production of reactive oxygen species (ROS) and longer doubling times were recorded. The mechanism of action was explained through an analytical model which involves ion-transport interference of essential ions like Ca2+ and Mg2+ and disruption of FeS clusters leading to inactivation of bacterial redox enzymes. On the contrary, such high magnetic fields did not pose any detrimental effects to eukaryotic cells under similar exposure. Additionally, the potency of low intensity direct current electric field (DC EF: 1V/cm) against biofilm formation by methicillin resistant Staphylococcus aureus (MRSA) was explored on antimicrobial surfaces of hydroxyapatite and Zinc oxide (HA-xZnO; x = 0, 5, 7.5 and 10 wt%). An EF exposure time dependent decline in the viability and stability of MRSA biofilms were noted. Further, EF treatment resulted in bacterial membrane depolarization and reduced biofilm formation on HA-ZnO composites, independent of the substrate composition. In summary, the above three studies were cases of the developmental methods to address prothetic infection. The second part of the thesis is focused on the development of magneto-responsive biomaterials as implants for orthopaedic applications. Under this category, the sintering/ hot pressing of hydroxyapatite-magnetite (HA-xFe3O4; x = 0, 5, 10, 20 and 40 wt%) powders in oxidizing and inert atmospheres was carried out and the resulting phases and microstructure were characterized. A detailed analysis of the phase assemblage by Rietveld refinement of the X-ray diffraction (XRD) data and Mössbauer spectroscopy revealed the major retention of Fe3O4 along with wustite (FeO) formation under reducing conditions while hematite (α-Fe2O3) was the oxidized product of conventional sintering in ambient atmosphere. A good correlation between the unit cell volume increases in HA observed from Rietveld refinements and Fe incorporation into the apatite lattice from Mössbauer spectral parameters was evident. Further, the Mössbauer data analysis indicated a preferential occupancy of Fe at the Ca(1) site under oxidizing conditions and Ca(2) site in inert atmosphere. The above phase analyses were further confirmed by X-ray photoelectron spectroscopy (XPS), Infrared spectroscopy (FT-IR) and CHN analysis. The microstructure of the hot-pressed samples observed under transmission electron microscope (TEM) divulged similar phases as deduced from XRD as well as the formation of translational Moire fringe patterns due to inference of overlapping crystal planes of HA and Fe3O4 in the HA-40 wt% composite. Such translational Moire fringes suggest a preferred arrangement and orientation of the crystallites resulting from hot-pressing, which correlated well with the room temperature magnetic measurements made with the help of a vibrating sample magnetometer (VSM). The compositional similarity of Fe doping in HA to that of the tooth enamel and bone presents these HA-Fe3O4 composites as potent dental/ orthopaedic implant materials. In the conclusive study, the hot-pressed HA-xFe3O4 composites were tested for their efficacy in supporting the osteogenesis of human mesenchymal stem cells (hMSCs) assisted by intermittent static magnetic field exposure. The magneto-responsive substrates were applied as platforms for the culture of hMSCs and the effect of static magnetic field (SMF) exposure on the viability, proliferation and differentiation of hMSCs were elucidated. With a mild compromise in viability, SMF triggered the osteogenic differentiation of hMSCs mediated by proliferative arrest in the G0/G1 phase and elevated intracellular calcium levels. The early bone marker genes - Runx2, Col IA and ALP were significantly up regulated upon SMF exposure on pure HA and HA-Fe3O4 composites. Further, the late osteogenic markers – OCN and OPN were detected exclusively in the HA-xFe3O4 (x = 10 and 40 wt%) composites. Matrix mineralization was enhanced and CaP nodules were detected on similar SMF treated HA-Fe3O4 composites. A substrate magnetization and time dependent modulation of gene expression was recorded which corroborated well with the temporal trending of osteogenic genes during bone development. In conclusion, substrate magnetization can be applied as a tool to modulate the behavior of stem cells and direct them towards osteogenic lineage. Such a pertinent combination of substrate magnetization and external magnetic field stimulation can be applied synergistically for stem cell based bone tissue engineering applications.

Wenn erworbene oder angeborene Knochendefekte aufgrund überkritischer Größe oder krankhafter Störungen nicht durch natürliche Regenerationsprozesse geheilt werden können, ist der Einsatz von Knochenersatzmaterialien notwendig. In der vorliegenden Arbeit ist es gelungen ein neuartiges Knochenersatzmaterial zu entwickeln und eingehend zu charakterisieren. Dazu wurden die Phasen Silikat und Kollagen in einem biomimetisch inspirierten Prozess zu einem Anorganik/Organik-Komposit verbunden. Calciumphosphatphasen konnten darüber hinaus als dritte Komponente hinzugefügt werden. Dafür wurden Herstellungsstrategien entwickelt, die Silikat in Form von Kieselsäure, Kollagen als hochkonzentrierte Suspension und gegebenenfalls Calciumphosphat als Pulver zu homogenen Mischungen vereinten. Als Zwischenprodukte wurden Komposithydrogele erhalten, deren Überführung in Xerogele in der Literatur als kritischer Schritt gilt, weil die dabei auftretenden Kapillarspannungen die Gelstruktur in der Regel irreversibel zerstören, wodurch das Material als Pulver oder Fragmente erhalten wird. Im vorliegenden Fall aber konnte die Gelfestigkeit in einem definierten Zusammensetzungsbereich durch die Kompositbildung und die kontrollierte Trocknung der Hydrogele so gesteigert werden, dass monolithische Proben von bis zu mehreren Kubikzentimetern Größe erhalten wurden. Diese konnten ohne weitere Verarbeitungsschritte einer Reihe von Untersuchungen zu mechanischen Eigenschaften, Bioaktivität, Degradabilität und Biokompatibilität unterzogen werden.

Für das Tissue Engineering von Knochen werden poröse dreidimensionale Substrate (Scaffolds) als Zellträger benötigt, die in der vorliegenden Arbeit über keramische Technologie hergestellt wurden. Neben dem strukturierten und getrockneten Verbundwerkstoff (Grünkörper) und der Sinterkeramik wurde auch der Zwischenzustand nach Ausheizen der organischen Phase (Braunkörper) evaluiert. Bei der Herstellung blieb die Architektur der parallel orientierten Kanalporen, die über den Sol-Gel-Prozess der gerichteten ionotropen Gelbildung des Alginates erzeugt wurde, in allen Materialzuständen erhalten. Die Herstellungstechnologie wurde derart optimiert, dass die neuartigen anisotropen Scaffolds allen prinzipiell gestellten Forderungen für das Tissue Engineering entsprachen – sie waren porös mit weithin einstellbarer Porengröße, sterilisierbar, gut handhabbar unter Zellkulturbedingungen, biokompatibel und degradabel. Der unerwartete Favorit der Biomaterialentwicklung, der Braunkörper – eine nanokristalline, poröse Hydroxylapatit-Biokeramik – lag in einer ersten in vivo-Studie nach 4 Wochen integriert im Knochen vor. Die beobachtete Knochenneubildung deutete auf eine osteokonduktive Wirkung des Materials hin. Die in der vorliegenden Arbeit untersuchten Technologien und Biomaterialien bieten eine Basis für weitere Forschung und motivieren zur Weiterentwicklung und Nutzung als Scaffold für das Tissue Engineering oder Knochenersatzmaterial unter Verwendung der interessanten Architektur.