Dissertations / Theses on the topic 'Bone regeneration'

Bone defects caused by trauma, tumor resection or disease present a significant clinical problem. Failures in 'high risk' fractures and large bone defects have been reported to be as high as 30-50%. The drawbacks associated with current bone grafting procedures have stimulated the search for improved techniques for bone repair. Tissue engineering/regenerative medicine approaches promote tissue repair by providing a combination of physical and biological cues through structural scaffolds and bioactive agents. Though they have demonstrated significant promise for bone regeneration, very little has been translated to clinical practice. The goal of this thesis was to investigate the potential of electrospun nanofiber mesh scaffolds for bone regeneration. Nanofiber meshes were utilized in a three-pronged approach. First, we validated their ability to robustly support osteogenic cell functions, including proliferation and matrix mineralization. We also demonstrated their efficacy as a cell delivery vehicle. Second, we investigated the effects of modulating nanofiber bioactivity and orientation on stem cell programming. Our results indicate that functionalization of nanofiber meshes with a collagen-mimetic peptide enhanced the migration, proliferation and osteogenic differentiation of cells. Fiber alignment improved cell migration along the direction of fiber orientation. Finally, a nanofiber mesh based hybrid system for growth factor delivery was developed for bone repair and tested in a challenging animal model. The delivery of bone morphogenetic protein (BMP) via this system resulted in the functional restoration of limb function, and in fact proved more efficacious than the current clinical standard for BMP delivery. The studies performed in this thesis have suggested novel techniques for improving the repair of clinically challenging bone defects. They indicate that the delivery of BMP via the hybrid system may reduce the dose and side effects of BMP, thereby broadening the use of BMP based bone augmentation procedures. Therefore, this nanofiber mesh based system has the potential to become the standard of care for clinically challenging bone defects, including large bone defects, open tibial fractures, and nonunions.

Master of Science in Dentistry
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The biomedical burden of large bone defects caused by trauma, infection, tumours or inherent genetic disorders remains a clinical challenge. Autologous bone or autograft continue to be the clinical “gold standard” for most effective bone regeneration, which is limited by bone supply and donor site morbidity. Thus, current synthetic substitutes need to be improved to match the performance of autografts. Bone tissue engineering is an attractive approach for regeneration of bone especially in relation to critical sized defects and a scaffold with osteoinductive properties and adequate mechanical properties is expected to enhance bone formation. The aim of the study is to enhance bone regeneration and the concept is based on adequate design of three dimensional scaffolds that mimic the structure of bone, which by virtue of its inherent properties have the ability to localise fluids rich in osteoinductive factors. The hydrogels and composites were all synthesized with a base polymer polyvinyl alcohol (PVA), which is both robust, biocompatible and FDA approved material. Facile methods of crosslinking such as air-drying and freeze-drying which introduces a level of porosity in the materials due to the lyophilisation process, were used to render the hydrogels insoluble, and conditions optimised to yield materials with properties suitable for soft bone tissue applications. PVA hydrogels were synthesized and characterised, results indicated that water uptake, glass transition and tensile strength were influenced by varying concentration of the polymer solution. A new type of dual network (DN) hydrogel composed of PVA and alginate was developed and optimised. Characterisations by spectral and thermal analysis, confirmed incorporation of alginate within the PVA network structure. Hydration dynamics and tensile properties, indicated that DN formed from a PVA base crosslinked by two freeze thaw cycles yielded tough hydrogels with controlled swelling, making them suitable for soft tissue applications as well as the diversity of being further incorporated with ceramic fillers for the development of bone composite substitutes. Incorporation of bioglass® 45S5 within the polymeric network structure of the DN led to enhancement of the mechanical properties such as tensile strength and fracture toughness as well as imparting bioactivity within the hydrogel composites, which was demonstrated by the development of hydroxy carbonated apatite on the surface and internal structure of the composites, this result was further corroborated by the increase in tensile strength and stiffness of the composites when placed in simulated body fluid over a period of 28 days. The second group of hydrogel composites was composed of a PVA fluid phase and calcium metaphosphate (CMP) ceramic phase, crosslinked by freeze drying. This hydrogel composite was developed to have a high mineral content with properties that closely resemble the properties of bone based on its inorganic/organic nanocomposite structure. Compression and water uptake behaviour of the composite could be modulated by varying concentration of PVA and the composite system properties were found to be suitable and lie within the range values for trabecular bone. In vitro cell culture tests were used to assess biocompatibility and the selected scaffolds were seeded with human osteoblast cells (HOB) and were evaluated by MTT, and live dead staining. All the systems were found to be biocompatible and cells were able to attach and proliferate within the scaffolds. Biofunctionality was assessed on the scaffolds which all showed a peak increase in alkaline phosphatase activity (ALP) at day 14, an important bone marker indicating osteoblast differentiation.

Mestrado em Ciência e Engenharia de Materiais
Tissue engineering research attempts to satisfy the needs of support, reinforcement and in some cases organization of the regenerating tissue with a controlled supply of bioactive substances that might positively influence the behaviour of incorporated or ingrowing cells. As demonstrated by the recent advances on biomaterials, the ideal scaffold for tissue regeneration should offer a 3D interconnected porous structure behaving as a template to promote cells adhesion and proliferation and vascularisation as well thus stimulating the new tissue ingrowth. A special interest has been focused on chitosan (CH - the partially deacetylated derivative of chitin) scaffolds for bone regeneration due to its biological and physical properties, in spite of some drawbacks regarding its lack of mechanical strength and bioactivity. The incorporation of bioactive calcium phosphates materials in the polymer matrix is expected to reinforce chitosan scaffolds improving their mechanical performance and osteoconductivity. In the present work, chitosan based scaffolds were produced by freeze-drying CH solutions containing calcium phosphate (CaP) particles, either as fibers of hydroxyapatite (HA), platelets of monetite or a mixture of both. CaP particles were prepared by a wet precipitation method. The calcium phosphate precipitation was monitored by taking a number of samples during 3-days. Evolution of the morphology and crystal phase composition of the precipitated particles were followed by scanning electron microscopy (SEM), N2 adsorption using the BET isotherm (BET), and X-ray diffraction (XRD). It was observed that the increase of refluxing temperature allowed a faster transformation of octacalcium phosphate fibers into HA fibers, hence shortening the precipitation time required for obtaining HA fibers, Chitosan based scaffolds suspensions at two different pH values were frozen at three different temperatures before freeze-drying (thermally induced phase separation-TIPS). SEM, XRD, microcomputed tomography (μ-CT) and Fourier transformed infrared spectroscopy (FTIR) were used to analyze the physical and chemical properties of the composite scaffolds. Compressive mechanical tests were also undertaken to characterize the materials. Bioactivity studies were performed in simulated body fluid (SBF) solutions by monitoring the Ca and P concentration variations of SBF solutions. Highly interconnected macroporous scaffolds with a pore size ranging from of 50 to 250μm, interconnectivity around 91-98.5%, and porosity higher than 80% were obtained. The freezing temperature and the pH of chitosan solution/suspension revealed to play a significant influence in the pore structure. The higher pH (pH=5) and the higher freezing temperature (T=0ºC) were found as the most favourable conditions for ice crystal growth which resulted in larger pores. It was also observed that CaP particles incorporation in the CH matrix increased the scaffold mechanical strength which was also conditioned by the pore size and by the reinforcing particle morphology. The bioactivity studies revealed the CaP contribution for the scaffold bioactivity. The composite scaffolds having brushite and HA (obtained at pH=2) exhibited enhanced bioactivity as compared to composite CH/HA scaffolds based. CH based scaffolds were also prepared by incorporating HA granules loaded with dexamethasone (DEX), a drug model, in CH solution. The granules were obtained by spray drying HA nanosized particles suspended in DEX solution. The drug release profiles of DEX were determined in phosphate-buffered solution (PBS) by DEX concentration evaluation in the releasing medium by Ultraviolet (UV) spectroscopy at the wavelength of 242 nm. Among the different DEX release patterns corresponding to the various DEX loading methodologies which were tested, an adequate release profile could be selected: it showed that the release of 80% of the DEX loaded amount could be ensured during ~30 days, thus enabling a prolonged and slowest DEX release as compared to literature reports. It is thus found that the CH scaffolds engineered with a calcium phosphate based drug delivery system (DDS) provides the desirable association of a bioactive and osteoconductive matrix with an in situ controlled release of a therapeutic agent. These results point out an additional potential of the composite CH/HA scaffolds for behaving as a controlled drug release system (DDS).
A investigação em engenharia de tecidos (ET) tem procurado soluções para as necessidades de reforço e de regeneração dos tecidos recorrendo por vezes a substâncias bioactivas que podem favorecer a proliferação celular. Os avanços recentes em ET têm beneficiado da utilização de matrizes tridimensionais porosas (scaffolds) que permitem a adesão, proliferação e regeneração das células bem como a vascularização, estimulando a formação de novo tecido. A obtenção de scaffolds de quitosano (CH) para a regeneração óssea tem merecido especial interesse devido às suas propriedades biológicas e físicas, apresentando no entanto o inconveniente da falta de resistência mecânica e de bioatividade. A obtenção de scaffolds compósitos por incorporação na matriz polimérica de materiais bioactivos de fosfato de cálcio, permite reforçar o scaffold, melhorando o seu desempenho mecânico e a sua osteocondutividade. No presente trabalho, produziram-se scaffolds compósitos de quitosano/hidroxiapatite por processos de congelamento e liofilização de suspensões de fosfatos de cálcio (CaP) em soluções de CH. Utilizaramse CaP sintetizados laboratorialmente, quer na forma de fibras de hidroxiapatite (HA), quer de lamelas de monetite, quer de mistura dos dois. Os CaP foram sintetizados por um método de precipitação em meio aquoso, tendo-se monitorizado a precipitação de fosfato de cálcio durante 3 dias. Avaliou-se a evolução das fases cristalinas e da morfologia das partículas precipitadas por microscopia eletrónica de varrimento (SEM), difracção de raios X (XRD) e por adsorção de N2 usando a isotérmica de BET. Os resultados evidenciaram que o aumento da temperatura de refluxo acelera a transformação das fibras de octacalcium fosfato em fibras de HÁ, permitindo reduzir o tempo de precipitação total para obtenção de fibras de HA As soluções de quitosano e as suspensões de HAP em solução de CH, a dois valores de pH (pH=2 e pH= 5), foram congeladas a três temperaturas diferentes antes de serem liofilizadas. Caracterizaram-se os scaffolds por SEM, DRX, microtomografia computorizada (μ-CT) e espectroscopia de infravermelhos com transformada de Fourier (FTIR), tendo-se ainda avaliado o seu comportamento mecânico em compressão. Obtiveram-se scaffolds compósitos macroporosos com porosidade superior a 80%, tamanho de poro na gama 50-250μm e porosidade interconectada com grau de interconexão de 91-98.5%. Verificou-se que o tamanho e morfologia de poro dos scaffolds é condicionado pelo pH das suspensões e pela temperatura de congelamento. O valor de pH mais elevado (pH=5) e a temperatura de congelamento mais elevada (T=0ºC) são as condições que mais favorecem o crescimento de cristais de gelo e por conseguinte a formação de poros de maior dimensão. Verificou-se também que a incorporação de partículas de CaP na matriz polimérica de CH aumenta a resistência mecânica do scaffold, que é também condicionada pelo tamanho de poro e pela morfologia da partícula de CaP. O estudo do comportamento bioactivo dos scaffolds compósitos em soluções simuladoras do plasma humano (SBF), monitorizando a variação das concentrações de Ca e P na solução de SBF, evidenciou o contributo das partículas de CaP para a bioactividade do scaffold. Os scaffolds compósitos em que coexistem brushite e HA (preparados a pH=2) evidenciaram bioactividade superior á dos scaffolds compósitos CH/HA. Preparam-se também scaffolds incorporando grânulos de hidroxiapatite carregados com um fármaco modelo, a dexametasona (DEX), na solução inicial de CH. Os grânulos obtiveram-se por atomização de suspensões de HA nanométrica em solução de DEX. Construíram-se os perfis de libertação da DEX em solução tampão fosfato (PBS) por determinação da concentração de DEX por espectroscopia de ultravioleta (UV) ao comprimento de onda de 242 nm. Entre as várias curvas de libertação de DEX decorrentes das diferentes metodologias testadas para carregamento do fármaco, evidenciou-se um perfil de libertação de DEX segundo o qual cerca de 80% da DEX é libertado ao longo de ~30 dias, assegurando-se assim uma libertação mais lenta e prolongada do que as referidas na literatura para a DEX As características dos scaffolds compósitos preparados no presente trabalho apontam os materiais produzidos como promissores para aplicação em engenharia de tecidos, apresentando como potencial adicional a capacidade de se comportarem como sistemas de libertação controlada de fármacos.

Thesis (Ph.D)--University of Tennessee Health Science Center, 2007.
Title from title page screen (viewed on October 8, 2007). Research advisor: Joo L. Ong, Ph.D. Document formatted into pages (xiii, 128 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 106-114).

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

Phosphate glass with additions of sodium, magnesium and/or calcium were investigated for their potential to be used as the reinforcing phase in a completely degradable long fibre composite. Glasses were prepared from phosphate salts as opposed to oxides and melted under air in platinum/gold crucibles. The effect of cation addition on the material properties and biocompatibility was investigated. Glasses were characterised using a number of complimentary techniques, including: XRD, XPS, DSC, IR and EDX. The findings from these techniques were used to explain the observed thermal and dissolution properties. The thermal and dissolution properties were found to be dependant on both the thermal history and composition of the phosphate glass. For a phosphate glass with low cation content, the temperature and length of time held at that temperature increased the Tg by 10 C for sodium phosphate glass and slightly improved the durability of sodium phosphate glass containing 10 mol.% MgHPO4, as phosphate chain growth was greater under those conditions. Addition of divalent cations increased the Tg of phosphate glasses from 295 C for sodium phosphate glass by up to 150 C with the addition of 50 mol.% MgHPO4. The dissolution rate was decreased exponentially with the addition of calcium phosphate or magnesium phosphate to sodium phosphate glass. Rates as low as 1x10-7 g/(cm2.h) were achieved with the addition of 50 mol.% divalent cation phosphates. The divalent cations inhibited phosphate chain growth but formed a new network based upon divalent cation/non bridging oxygen cross-links. These cross-links were found to exert greater influence over the material properties then the phosphate chain length. Cell culture assays were used to establish the biocompatibility of phosphate glasses with different compositions. Preliminary tests were conducted with craniofacial derived osteoblast like cells cultured on glass surfaces. Initial assays performed showed that the most durable glasses sustained the greatest amount of proliferation and differentiation over a seven day period. The most promising glass compositions, 40 Ca, 40 Mg, 30 Mg/20 Ca and 20 Mg/30 Ca, and were selected for longer term osteoblast culture and short term macrophage culture. Long term osteoblast culture showed that cells were able to attach, spread and proliferate throughout the 28 day duration of the study. Assays performed on the culture showed that cells were differentiating, producing specific osteoblast markers for each of the three differentiation phases of proliferation, matrix maturation and mineralisation. ECM production and mineralisation was confirmed on all surfaces tested via type I collagen staining and alizarin red staining respectively. Over the 28 day period, it was found that the composition did not have a significant effect on the production of the osteoblast markers, namely alkaline phosphatase, collagen, osteocalcin and mineral deposition. Immunological studies show that macrophages are not activated by the presence of phosphate glass. This demonstrated that phosphate glass has shown potential for use a biomaterial.

Currently available cements and granules for bone repair include devices based on glass ionomer cement (GICs) technology. These cements are based on the setting reaction between an aluminium containing fluorosilicate glass, poly (acrylic acid) (PAA) and a setting modifier. The glass powder is acid degradable, which crosslinks with the ionised acid, resulting in a matrix of polyacrylates salts with reacted glass particles. However, bone demineralisation, as well as neurotoxicity in craniofacial applications are drawbacks associated with aluminium. These disadvantages have created a scientific interest on developing aluminium free compositions with the potential to be used as cements and bone grafts. Therefore, new glass compositions have been researched to substitute the alumina (Al2O3) with oxides such as ZnO, GeO, MgO, and TiO. However, no previous detailed study has investigated variations of the classic Hench Bioglass® composition (45SiO2-24.5Na2O-24.5CaO-6P2O5 wt. %) for preparation of cements, with Mirvakily studying this system, but focusing solely on one cement composition (Mirvakily, 2009). In the present work, eight glasses were prepared and characterised by X-ray diffraction (XRD), differential thermal analysis (DTA), scanning electron microscopy (SEM), and Fourier transformed infrared spectroscopy (FTIR-ATR). These glasses were mixed with varying quantities of poly (acrylic acid), and a phosphoric acid solution (H3PO4 (sol)), to evaluate their cement forming properties and setting times. The most suitable glasses were chosen along with the optimised cement combination, this being a powder/liquid (P/L) ratio of two, 10.7 % PAA powder and 25% H3PO4 (sol), to further investigate their mass change, ion release and conductivity when immersed in distilled water, along with their setting chemistry. Results showed that glasses based on a SiO2-Na2O-CaO-SrO-P2O5 system could be used to produce setting pastes that were stable in distilled water, with net setting times varying between 34 min and 115 min. The setting mechanism was found to have similarities to GICs, by release of Ca2+, Sr2+, and Na+ after PAA ionisation and formation of the respective polyacrylate salts. The use of phosphoric acid was found to be essential to prevent the gelation in water of aged cements (set for one day, 37 °C), suggesting that this addition aided glass dissolution and precipitation of phosphate containing salts. Their mass loss in distilled water, reached its maximum after one day for the cements prepared with the 45S5 and 45S5Sr10 glasses (with lower SiO2 content), while this peak was observed after one week for the cements prepared with the 49P9 and 53P4 glasses (with higher SiO2 content). The cement dissolution was further confirmed by the release of Si, Ca, Na, and P; with the direct in vitro short-term test showing that the cement based on the 45S5 glass was not cytotoxic. The study carried out with the commercial cement Serenocem™, showed that its dissolution and ion release was very low, which suggested the limited solubility of the Al containing glass. Therefore, the findings of this research provide an alternative system for the preparation of aluminium free cements for craniofacial applications, showing that no additional ionic substitution was required to produce a setting cement with ion release comparable to that of bioactive glasses.

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, June 2005.
Includes bibliographical references (p. 38-39).
In this thesis, the chemical/mechanical properties and biocompatibility of gelatin were investigated to produce a gelatin scaffold for the release of bone morphogenetic proteins (BMPs) from composite particles. This delivery system, designed to regenerate bone, holds much promise as an alternative to bone grafts. The chemical properties of gelatin were examined through zeta potential measurements, swelling studies, optical microscopy, environmental scanning electron microscopy (ESEM), and collagenase degradation. Compressive tests and mercury porosimetry were performed to study the mechanical and structural properties of the scaffold. The biocompatibility of the scaffold was determined through cell optical imaging and DNA quantification studies. Based on findings of this research, the material choices were made and the synthesis method for the gelatin scaffold was developed. Gelatin A, 300B, derived from bovine collagen, with an isoelectric point of [approx.] 9, was selected. Crosslinking was accomplished by reacting 10 w/v% glutaraldehyde with 10 w/v% gelatin solution. The most effective crosslinking condition was found to be 5 hours at room temperature. Glycine rinses were conducted to cap any non- reacted (toxic) aldehyde groups, and the necessary length of time was found to be at least 48 hours at 37⁰C. Finally, based on pore size distribution and mechanical stability, an optimal lyophilization method was developed with initial freezing at -20⁰C for 1 day, followed by lyophilization of the scaffold for 1-2 days. In terms of mechanical properties of the gelatin and amount of protein delivered, the most effective loading of poly(lactic-co-glycolic acid)/apatite/protein composite particles was found to be 10% of the mass of the gelatin.
by Elizabeth A. Hager.
S.B.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 1998.
Includes bibliographical references (leaves 59-68).
Current methods of bone repair rely on autografts (bone from a donor site) and allografts (bone from human cadaver). However, these methods are plagued with disadvantages. There is a clear and urgent need to provide alternatives for regenerating and repairing bone. Bone is known to be one of the many connective tissues in the body that are responsive to exogenous electrical stimulation. Based on this principle, this thesis explores the potential of using an electrically conducting polymer, polypyrrole, as a substrate for bone regeneration. Optically transparent thin films of polypyrrole, with a polyanionic dopant, poly(styrenesulfonate), were synthesized electrochemically and characterized by X-Ray Photoelectron Spectroscopy, UV/VIS spectroscopy, Scanning Electron Microscopy and by electrical conductivity measurements. In this study, Bone Marrow Stromal Cells (BMSC), which are the progenitor cells to bone cells (osteoblasts), were used as the in vitro model system. Their viability, proliferation and differentiation capabilities were evaluated on polypyrrole, in the absence and presence of electrical stimulation. Results indicate that polypyrrole is ideally suited as a substratum for BMSC growth and differentiation. The application of an electrical stimulus through the polypyrrole substrate was found to induce the differentiation of BMSC towards an osteogenic lineage. Thus, polypyrrole, by virtue of its conductive properties, its in vitro biocompatibility and its flexibility in altering surface characteristics, has an exciting potential as a suitable interactive substrate for bone regeneration.
by Nahid Rahman.
S.M.

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

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 1998.
Includes bibliographical references (leaves 59-68).
Current methods of bone repair rely on autografts (bone from a donor site) and allografts (bone from human cadaver). However, these methods are plagued with disadvantages. There is a clear and urgent need to provide alternatives for regenerating and repairing bone. Bone is known to be one of the many connective tissues in the body that are responsive to exogenous electrical stimulation. Based on this principle, this thesis explores the potential of using an electrically conducting polymer, polypyrrole, as a substrate for bone regeneration. Optically transparent thin films of polypyrrole, with a polyanionic dopant, poly(styrenesulfonate), were synthesized electrochemically and characterized by X-Ray Photoelectron Spectroscopy, UV/VIS spectroscopy, Scanning Electron Microscopy and by electrical conductivity measurements. In this study, Bone Marrow Stromal Cells (BMSC), which are the progenitor cells to bone cells (osteoblasts), were used as the in vitro model system. Their viability, proliferation and differentiation capabilities were evaluated on polypyrrole, in the absence and presence of electrical stimulation. Results indicate that polypyrrole is ideally suited as a substratum for BMSC growth and differentiation. The application of an electrical stimulus through the polypyrrole substrate was found to induce the differentiation of BMSC towards an osteogenic lineage. Thus, polypyrrole, by virtue of its conductive properties, its in vitro biocompatibility and its flexibility in altering surface characteristics, has an exciting potential as a suitable interactive substrate for bone regeneration.
by Nahid Rahman.
S.M.

Mestrado em Materiais e Dispositivos Biomédicos
Um dos grandes desafios da investigação biomédica é ultrapassar as taxas de rejeição de próteses implantadas melhorando as suas propriedades como biomateriais, garantindo assim maior qualidade de vida aos pacientes. Grande parte destas próteses é constituída por componentes metálicas que, por serem inertes, surge uma necessidade de as melhorar. Uma das soluções reside no revestimento do metal por um polímero, de preferência com capacidade de induzir a regeneração óssea. Neste trabalho testou-se a adesão entre o aço 316L, material muito utilizado como biomaterial, e o ácido poli(L-láctico) (PLLA), um polímero, biocompatível, de biodegradação controlável, bioabsorvível, piezoeléctrico e aprovado pela Food and Drug Administration (FDA). O filme de PLLA foi depositado no aço por spin-coating e procedeu-se à investigação do efeito de diferentes variáveis na adesão, nomeadamente tratamento físico de superfície (por polimento), tratamento químico de superfície (por silanização), peso molecular do PLLA, cristalinidade do filme, espessura, e imersão numa solução tampão de fosfatos (PBS). A adesão entre os dois materiais foi estudada utilizando um teste qualitativo, o teste da fita-cola, seguindo a norma ASTM D3359. Observou-se que os filmes preparados da solução de PLLA de menor peso molecular apresentaram os melhores resultados no teste da fita-cola, principalmente quando depositada nas amostras de aço com maior rugosidade. O efeito da espessura do filme, foi testado com diferentes concentrações da solução de PLLA de menor peso molecular, concluindo-se que quanto menor a concentração da solução de polímero, menor a espessura do filme e melhor a sua adesão ao substrato. Por conseguinte, estas condições de polimento (P180 e P400) foram selecionadas para prosseguirem para caracterização adicional: cristalização e posterior ensaio de degradação em fluido sintético (PBS), com a duração de uma semana, um mês e dois meses. Os resultados apontam para uma significativa perda de adesão, uma vez que a adesão do filme ao substrato resultou enfraquecida após a imersão. Ensaios preliminares de silanização dos substratos de aço não revelaram melhorias significativas da adesão dos filmes ao substrato comparativamente aos obtidos por tratamento físico da superfície. Em conclusão, os resultados deste trabalho mostram que é possível produzir revestimentos de PLLA sobre aço 316L e controlar a adesão do filme de PLLA ao substrato de aço através de tratamentos de superfície e de variações nas características do filme. Assim a combinação destes dois materiais parece ser adequada para potenciais aplicações biomédicas.
One of the major challenges in biomedical research is to overcome the rejection rates of implanted prostheses improving their properties as biomaterials, thus ensuring greater quality of life for the patients. Many of this prosthesis include inert metallic components, hence the necessity of improvement. One of the solutions lies in the polymeric coating, preferably one with the ability to induce bone regeneration. In this study we tested the adhesion between the 316L stainless steel, a material widely used as a biomaterial, and poly (L-lactic acid) (PLLA), a polymer, biocompatible, with controlled biodegradation, bioabsorbable, piezoelectric and approved by the Food and Drug Administration (FDA). The PLLA film was deposited onto the stainless steel samples by spin-coating and proceeded to the investigation of the effect of different variables in the adhesion, namely substrate surface physical treatment (by grinding), substrate surface chemical treatment (by silanization), PLLA molecular weight, film crystallinity, film thickness and immersion into phosphate buffered saline (PBS) solution. The adhesion between both materials was studied using a qualitative test, the tape test, following a standard (ASTM D3359). It was observed that films prepared with the lower molecular weight PLLA solution presented the best results in the tape test, especially when deposited onto the substrates with higher roughness. The effect of film thickness was tested with different solution concentrations of the lower molecular weight PLLA solution, concluding that the lower the solution concentration, the thinner the film and the better the adhesion of the film to the substrate. Therefore, these polishing conditions (P180 and P400) were chosen for further characterization: crystallization and subsequent degradation assay in a synthetic fluid (PBS) for one week, one month and two months. These results point at a significant loss of adhesion, since the adhesion of the film to the substrate after immersion resulted weakened. Preliminary tests of silanization of steel substrates showed no significant improvements in the film adhesion to the substrate, when compared to the results already obtained only with a surface physical treatment. In conclusion, the results obtained during this work show that it is possible to produce PLLA coatings on 316L stainless steel substrates and to control the adhesion of PLLA films to substrate through surface treatments and variations in the film characteristics. Therefore, the combination of these materials appears to be potentially suitable for biomedical applications.

Doutoramento em Ciência e Engenharia dos Materiais
Bioactive glasses and glass-ceramics are a class of third generation biomaterials which elicit a special response on their surface when in contact with biological fluids, leading to strong bonding to living tissues. The purpose of the present study was to develop diopside based alkali-free bioactive glasses in order to achieve good sintering behaviour, high bioactivity, and a dissolution/ degradation rates compatible with the target applications in bone regeneration and tissue engineering. Another aim was to understand the structure-property relationships in the investigated bioactive glasses. In this quest, various glass compositions within the Diopside (CaMgSi2O6) – Fluorapatite (Ca5(PO4)3F) – Tricalcium phosphate (3CaO•P2O5) system have been investigated. All the glasses were prepared by melt-quenching technique and characterized by a wide array of complementary characterization techniques. The glass-ceramics were produced by sintering of glass powders compacts followed by a suitable heat treatment to promote the nucleation and crystallization phenomena. Furthermore, selected parent glass compositions were doped with several functional ions and an attempt to understand their effects on the glass structure, sintering ability and on the in vitro bio-degradation and biomineralization behaviours of the glasses was made. The effects of the same variables on the devitrification (nucleation and crystallization) behaviour of glasses to form bioactive glass-ceramics were also investigated. Some of the glasses exhibited high bio-mineralization rates, expressed by the formation of a surface hydroxyapatite layer within 1–12 h of immersion in a simulated body fluid (SBF) solution. All the glasses showed relatively lower degradation rates in comparison to that of 45S5 Bioglass®. Some of the glasses showed very good in vitro behaviour and the glasses co-doped with zinc and strontium showed an in vitro dose dependent behaviour. The as-designed bioactive glasses and glass–ceramic materials are excellent candidates for applications in bone regeneration and for the fabrication of scaffolds for tissue engineering.
Os vidros e vitrocerâmicos bioactivos fazem parte da chamada terceira geração de biomateriais, i.e., materiais que estimulam uma resposta especial quando em contacto com fluidos biológicos, capaz de conduzir ao estabelecimento de ligações fortes entre a sua superfície e os tecidos vivos. O presente estudo visou o estudo e desenvolvimento de vidros bioactivos à base de diópsido e isentos de metais alcalinos que apresentem um bom comportamento na sinterização, elevados índices de bioactividade, e taxas de dissolução / degradação compatíveis com as almejadas aplicações em regeneração óssea e em engenharia de tecidos. Procurou-se ainda entender as relações entre a estrutura e as propriedades dos vidros bioactivos estudados. De acordo com esta perspectiva, estudaram-se várias composições de vidros bioactivos pertencentes ao sistema Diópsido (CaMgSi2O6) – Fluorapatite (Ca5(PO4)3F) – Fosfato de tricálcico (3CaO•P2O5). Todas as composições vítreas foram preparados por fusão, seguida de fritagem em água fria, e caracterizados através de um conjunto de técnicas complementares de caracterização. Os vitrocerâmicos foram obtidos por sinterização das fritas de vidro moídas e compactadas, seguida de tratamento térmico adequado para promover os fenómenos de nucleação e cristalização. Além disso, algumas composições vítreas seleccionadas foram dopadas com vários iões funcionais e os seus efeitos na estrutura vítrea, na sua propensão para a sinterização, e nos comportamentos in vitro em termos de biodegradação e bio-mineralização foram avaliados. Os efeitos das mesmas variáveis no processo de devitrificação (nucleação e cristalização) dos vidros e formação de materiais vitrocerâmicos foram também investigados. Algumas composições de vítreas apresentaram taxas de bio-mineralização elevadas, expressas através da formação de camadas superficiais de hidroxiapatite após 1-12 h de imersão num fluido fisiológico simulado (SBF). Todas as composições vítreas apresentaram taxas de degradação mais baixas quando comparadas com a do 45S5 Bioglass®. Alguns vidros bioactivos revelaram comportamentos in vitro excelentes, sendo a taxa de biomineralização dos co-dopados com zinco e estrôncio dependente da dose incorporada de dopantes. Os materiais estudados demostraram boa aptidão para aplicações em regeneração óssea e para o fabrico de estruturas de suporte em engenharia de tecidos.

The repair of complex bone defects remains a surgical challenge and it is hoped that tissue-engineering may offer an unlimited source of bone tissue and circumvent many of the drawbacks associated with current clinical approaches. Currently, the majority of bone tissue-engineering research has been focused on the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Tissue-engineering via the endochondral ossification pathway to prepare a hypertrophic cartilage graft, however, may be a more advantageous approach. This tissue is able to survive the relatively low oxygen tensions found in defects and it may provide growth factors that promote angiogenesis and bone tissue regeneration. Surprisingly little research has been directed at hypertrophic cartilage engineering. The aim of this project was therefore to investigate the ability of nasal chondrocytes to form a hypertrophic cartilage graft capable of regenerating bone tissue in vivo. The methodology used in this study was based on the typical tissue-engineering approach and encompassed scaffold fabrication, tissue characterisation and in vivo studies. Rat nasal chondrocytes consistently differentiated to form hypertrophic cartilage grafts on both PGA and PLLA/calcium phosphate scaffold materials. These grafts expressed collagen type X and alkaline phosphatase, co-located with large chondrocytes, in a tissue that had morphological features in common with hypertrophic cartilage. Gene and protein expression studies demonstrated a decrease in hyaline cartilage markers and an increase in markers for hypertrophic chondrocytes as differentiation proceeded. Vital and decellularised grafts cultured on the PGA scaffold material significantly increased bone regeneration in vivo when compared to empty defects (4mm) in the rat cranium. Decellularised grafts that were reseeded with MSCs also promoted significant bone tissue regeneration after 12 weeks. This regeneration, however, occurred at a slower rate compared to the other grafts evaluated. Foetal calf serum (FCS) was shown to have a significant effect on the differentiation of the rat nasal chondrocytes. A defined, serum-free medium was therefore developed which was able to support the hypertrophic differentiation of the chondrocytes. Finally proof of concept studies demonstrated the ability of a Quasi-vivo® bioreactor to support hypertrophic differentiation of nasal chondrocytes under flow conditions. It was concluded that nasal chondrocytes could be seeded onto scaffold materials and cultured under defined conditions to prepare a hypertrophic cartilage graft capable of stimulating bone tissue regeneration in vivo. The use of a decellularised hypertrophic cartilage demonstrated substantial clinical potential for use as a new regenerative graft material.

Long bone defects can cause complex challenges to both doctor and patient. Current clinical strategies for treating patients with long bone segment defects are very poor. Sensate implantable biomimetic polybutylene terephthalate (PBT) scaffolds develop faster and more complete bone ingrowth than simple porous scaffolds with the same pore size and overall porosity. They have provided the opportunity to monitor healing and could be used to study regeneration of bone in defects. Previous studies have successfully created small sensate scaffolds for canine femoral condylar resurfacing in the stifle joint (the knee). The shape and size of these scaffolds could be modified to act as bone supporting scaffolds for larger segment bone regeneration. Sensors on these scaffolds could be used to determine loading and detect healing as bone ingrowth occurred. A biomimetic scaffold was wired and loaded to 250 N. The stiffness of this porous segmental replacement scaffold was found to be lower than the modulus of the material. A sheep bone was wired and loaded with compression to 250 N, and with cantilever bending to 50 N. The stiffness was found to be similar to previously reported stiffness for sheepfemora. Limitations to the study include the irregular size, shape, and composition of the sheep bone, as well as differences in loading compared to a human bone.

Knochen ist ein multifunktionales Organ und zugleich ein biologisches Material. In dieser Arbeit wird der Heilungsverlauf eines Knochenbruchs (als biologisches Material) näher untersucht mit Hilfe von Computermodellen. Im menschlichen Körper kommt es nach einem Bruch zu einer vollständigen Regeneration des Knochens, ohne dass eine Narbe nach der Heilung zurückbleibt. In grob 10% der Frakturen kommt es jedoch zu Komplikationen bis zu einem Nicht-Heilen des Bruches. Das Ziel von intensiver interdisziplinärer Forschung ist es daher, nicht nur die medikamentöse Behandlung solcher Komplikationen zu verbessern, sondern auch durch externe, biophysikalische Stimulation die Heilung anzuregen. Gewöhnlich heilt ein Knochenbruch nicht direkt (Primäre Knochenheilung), das heißt durch Bildung von neuem Knochen im Knochenspalt, sondern über Sekundäre Knochenheilung. Während der sekundären Heilung bildet sich vorübergehend zusätzliches Gewebe außerhalb des Frakturspaltes, der so genannte Kallus, der die Aufgabe hat, den Bruch zu stabilisieren. Im Kallus werden im Laufe der Heilung verschiedene Gewebearten gebildet (z.B. Bindegewebe, Knorpel und Knochen). Die Gewebe werden von spezialisierten biologischen Zellen gebildet. Die spezialisierten Zellen entwickeln sich aus mesenchymalen Stammzellen (d.h. sie differenzieren), die in den Kallus wandern. Hauptziel der Arbeit ist das bessere Verständnis der mechano-biologischen Regulation der Gewebeformation während der Heilung eines normalen Knochenbruches. Dazu wurden Computersimulationen durchgeführt und mit experimentellen Daten eines Schafmodels verglichen.
Bone is a multifunctional organ, a biological material and is able to fully restore bone fractures without leaving a scar. However, in about 10% of the bone fractures, healing does not lead to a successful reunion of the broken bone ends. Intensive interdisciplinary research therefore looks for new ways to promote healing not only by medication, but also by external biophysical stimulation. Usually, bone fractures do not heal by a direct bridging of the fracture gap with newly formed bone (primary bone healing). Instead, secondary bone healing proceeds indirectly via the formation of an external callus (additional tissue). Within the callus, intricate tissue type patterns are formed, which evolve during the healing progression. Stem cells differentiate into specialized cells, which lay down different tissues such as fibrous tissue, cartilage and bone. This cell differentiation can be biophysically stimulated, e.g. by mechanical deformation of the cytoskeleton. The main aim of this thesis was to connect the microscopic cell response to mechanical stimulation with the macroscopic healing progression. Simple rules for cell behaviour were implemented in a computer model, the progression of healing was simulated and the outcome of the simulations was compared to results from animal experiments. In comparison to existing simulations of bone healing, this study approached the problem from a more physical viewpoint and linked experimental in vivo data and computer modelling.

There exists a dire need for improved therapeutics to achieve predictable and effective bone regeneration. Non-viral gene therapy is a safe method that can efficiently transfect target cells, therefore is a promising approach to overcoming the drawbacks of protein delivery of growth factors. The goal of this study was to employ cost-effective biomaterials to deliver genetic materials (DNA or RNA) in a controlled manner in order to address the high cost issues, safety concerns, and lower transfection efficiencies that exist with protein and gene therapeutic approaches. To achieve our goal, we set several aims: 1) To assess the bone regeneration capacity of polyethylenimine (PEI)-chemically modified ribonucleic acid (cmRNA) (encoding bone morphogenetic protein-2 (BMP-2)) activated matrices, compared to PEI-plasmid DNA (BMP-2)-activated matrices. 2) To explore the osteogenic potential of cmRNA-encoding BMP-9, in comparison to cmRNA-encoding BMP-2. 3) To use collagen membranes as integral components of a guided bone regeneration protocol and to enhance the bioactivity of collagen membranes by incorporating plasmid DNA (pDNA) or cmRNA encoding bone morphogenetic protein-9 (BMP-9). 4) To test whether the delivery of pDNA encoding BMP-2 (pBMP-2) and fibroblast growth factor-2 (pFGF-2) together can synergistically promote bone repair in a leporine model of diabetes mellitus, a condition that is known to be detrimental to union. 5) To investigated whether there is a synergistic effect on bone regeneration following delivery of pBMP-2 and pFGF-2, insulin and/or vitamin D. These investigations together provided new insights regarding the appropriate treatment methods for patients with fractures. Here we develop and test a non-viral gene delivery system for bone regeneration in challenging animal models utilizing a scaffold carrying PEI-nucleic acid complexes. We utilized three kinds of pDNA encoding either BMP-2, BMP-9 or FGF-2 as well as two kinds of cmRNA encoding either BMP-2 or BMP-9 formulated into PEI complexes. The fabricated nanoplexes were assessed for their size, charge, in vitro cytotoxicity, and capacity to transfect human bone marrow stromal cells (BMSCs). The in vivo functional potency of different nanoplexes embedded in scaffolds was evaluated using a calvarial bone defect model in rats, diaphyseal long bone radial defects in a diabetic rabbit model and intramuscular implantation in a diabetic rat. The results indicate that our non-viral gene delivery system induced migration and differentiation of resident cells to enhance bone regeneration. Together these findings suggest that scaffolds loaded with non-viral vectors harboring cmRNA or pDNA encoding osteogenic proteins may be a powerful tool for stimulating bone regeneration with significant potential for clinical translation.

To synthesize a polyurethane (PU) foam-like scaffold which could perform both as a resorbable membrane and as a cavity filling material in oro-maxillary bone defects. MATERIALS & METHODS: A PU foam was synthesized via a onepot reaction starting from a pre-polymerized isocyanate and a biocompatible polyester diol, using water as a foaming agent. Different foaming conditions were examined, with the aim of creating a dense/porous functional graded material. The obtained PU was characterized in terms of morphological and mechanical properties. Biocompatibility assessment was performed in combination with bonemarrow- derived human mesenchymal stromal cells (hBMSC). RESULTS: PU showed a highly porous structure, consisting of interconnected round pores with a diameter larger than 200 μm. Degradation test showed a slow degradation (ca. 1% weight loss after 6 weeks). Mechanical properties were strongly dependent upon foaming conditions, and not significantly affected by in vitro degradation process. In vitro biocompatibility assessments combined with hBMSCs proved the materials non cytotoxic, with cell viability values higher than 95% after 24 hours. CONCLUSIONS: This work demonstrates the feasibility of fabricating biphasic dense/porous polyurethane foams by a confined foaming reaction. Results support the potential application of the synthesized materials in the treatment of oro-maxillary bone defects.

Background: Bone-grafting materials are required in orthopaedic surgery to treat bone defects. Bone formation assessment is required for the development of new strategies and approaches and for quality assurance and quality control of currently available materials. Approaches to the assessment of bone formation are yet to be systematically established, quantified and standardized. Aims: the overall aim of this study was to establish a set of comprehensive quantitative approaches for the assessment of bone formation and to evaluate the role of osteoblastic cells, growth factors, and scaffolds on this process. Materials & methods: both in vitro and in vivo parameters for osteoblast phenotype and bone formation were tested in osteosarcoma cell lines, Saos-2 and U2OS cells, mesenchymal cell line, C2C12 cells, primary adipose derived stromal cells (ADSCs), platelet rich plasma (PRP), and morselized bone grafts. The in vitro parameters used were measurement of alkaline phosphatase (ALP) activity, detection of bone nodules and biomineralization, and quantification of immunocytochemistry and conventional RT-PCR of osteoblast genotyping. In vivo parameters involved ectopic bone formation in nude mice and nude rats and a tibial defect model in nude rats. Histomorphometric and quantitative immunohistochemical analyses were also performed. Results: The in vitro characterization and ectopic bone formation capabiltity of Saos-2 and U2OS cells have been established. Saos-2 cell line, which presents many osteoblast genotype and phenotype, is a stable positive control for both in vitro and in vivo bone formation assessments. The measurement of ALP activity in both solid and liquid phases has been standardized. Both the genotype and phenotype of osteoblast lineage cells has been quantitatively assessed during the capability testing of ADSCs and PRP. Quantitative assessment of new bone formation and related protein markers in vivo has been successfully established through the testing of the biological properties of gamma irradiated morselized bone grafts. Conclusion: A comprehensive knowledge of the assessment of bone regeneration and formation in vitro and in vivo has been integrated and developed through years of study. A whole set of in vitro and in vivo approaches for the assessment of bone formation has been modified and standardized to best suit the different clinical applications. This thesis provides an outline of both in vitro and in vivo bone formation assessment and their clinical applications.

Thesis (M. S.)--Biology, Georgia Institute of Technology, 2009.
Committee Chair: Boyan Barbara; Committee Member: Guldberg Robert; Committee Member: Lovachev Kiril; Committee Member: Schwartz Zvi. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Long-term exposure to pharmacological doses of glucocorticoids has been associated with the development of osteopenia and avascular necrosis. Bone loss may be partially attributed to a steroid-induced decrease in the osteoblastic differentiation of multipotent progenitor cells found in the bone marrow. In order to determine if there is a change in the osteogenic potential of the bone marrow stroma following glucocorticoid treatment, Sprague-Dawley rats were administered methylprednisolone for up to six weeks, then sacrificed at 0, 2, 4, or 6 weeks during treatment or 4 weeks after cessation of treatment. Femurs were collected and analyzed for evidence of steroid-induced osteopenia and bone marrow adipogenesis. Although glucocorticoid treatment did inhibit bone growth, differences in ultimate shear stress and mineral content were not detected. The volume of marrow fat increased with increasing duration of treatment, but returned to near control levels after cessation of treatment. Marrow stromal cells were isolated from tibias, cultured in the presence of osteogenic supplements, and analyzed for their capacity to differentiate into osteoblast-like cells in vitro. Glucocorticoid treatment diminished the absolute number of isolated stromal cells, but did not inhibit the relative levels of bone-like mineral deposition or osteocalcin expression and secretion. Although pharmacological glucocorticoid levels induce bone loss in vivo, physiologically equivalent concentrations have been shown to enhance the formation of bone-like tissue in vitro. However, glucocorticoids have also been reported to inhibit proliferation and type I collagen synthesis in marrow stromal cell cultures. In order to assess the effects of intermittent dexamethasone treatment on the progression of osteogenesis in rat marrow stromal cell culture, this synthetic glucocorticoid was removed from the culture medium after a variable period of initial supplementation. Cell layers were analyzed for total cell number, collagen synthesis, phenotypic marker expression, and matrix mineralization. Prolonged supplementation with dexamethasone decreased proliferation, but did not significantly affect collagen synthesis. Furthermore, increased treatment duration was found to increase bone sialoprotein expression and mineral deposition. The duration of glucocorticoid treatment may be a key factor for controlling the extent of differentiation in vitro.
Master of Science

Bioactive glasses have the ability to bond to bone in vivo, but they are brittle and cannot be used in load bearing applications. In this thesis, a process was developed to toughen bioactive glasses by forming a hybrid material for bone tissue engineering using the sol-gel process. As a first step, in preparation for polymer incorporation into the sol-gel process, the pH of the sol-gel synthesis had to be raised to milder pH conditions to prevent acid chain scission hydrolysis of the polymer. Solgel glasses were synthesised under the modified conditions and no adverse effects were found due to raising the pH of synthesis from pH < 1 to 5.5. These mild pH conditions were then used to synthesise hybrids of silica and calcium salt poly(γ-glutamic acid) (γCaPGA). γCaPGA was used as the toughening agent and as a low temperature calcium source with 3-glycidoxypropyl trimethoxysilane (GPTMS) providing covalent coupling between the inorganic and organic components. Hybrids of 40 wt% γCaPGA of all molecular weights tested (120 to 30 kDa) had large strain to failure (> 26 %) which showed that γCaPGA hybrids successfully softened the brittle behaviour of sol-gel glasses. However, the polymer dissolved preferentially due to its hydrophilic nature. All γCaPGA hybrids were found to form hydroxycarbonate apatite (HCA) within one week in SBF, even though they contained a low calcium concentration of 5 wt% when compared with 17.5 wt% Ca in Bioglass®. Formation of HCA is the first step in bonding to bone in vivo which is a fundamental requirement of materials for bone tissue engineering. Calcium was not only important for bioactivity, but also for ionic crosslinking, which improved compressive strength and reduced strain to failure when compared with identical hybrids made without ionic crosslinking. Although hybrids synthesised with γCaPGA dissolved too quickly for bone applications, calcium chelating polymers have been shown to offer great promise for bone tissue engineering.

Thesis (M.S.)--University of Alabama at Birmingham, 2008.
Additional advisors: Susan L. Bellis, Renato P. Camata, Thomas L. Clemens, Timothy M. Wick. Description based on contents viewed Feb. 10, 2009; title from PDF t.p. Includes bibliographical references (p. 60-65).

Healing large bone defects remains a clinical challenge. While autografts are the gold standard treatment for large bone defects, they are limited by availability and donor site pain. Growth factor treatments such as BMP therapy provide a promising alternative but are expensive and present clinical safety concerns, primarily due to delivery of BMPs at supraphysiological doses. Integrins are ECM receptors which mediate crucial cell functions such as adhesion and differentiation. Therefore, understanding the role of integrins in bone formation and directing desired interactions may enable modulation of host cell functions for therapeutic applications. In this work, beta 1 integrins were deleted in osteolineage cells of transgenic mice at three different stages of differentiation to elucidate their role in bone development. We also engineered bioartificial PEG-based matrices which target the pro-osteogenic alpha 2 beta 1 integrin to promote bone healing. Conditional deletion of beta 1 integrins in osteochondroprogenitor cells under the Twist 2 promoter resulted in severe pre-natal skeletal mineralization defects and embryonic lethality. Targeted deletion of beta 1 integrins in osterix-expressing osteoprogenitors resulted in growth abnormalities, reduced calvarial mineralization, impaired femur development, and tooth defects. However, mice lacking beta 1 integrins in osteocalcin-expressing osteoblasts and osteocytes displayed only a mild skeletal phenotype, indicating that beta 1 integrins play an important role in early skeletal development, but are not required for mature osteoblast function. PEG hydrogels functionalized with the integrin-specific GFOGER ligand enhanced bone regeneration, induced defect bridging in combination with low doses of rhBMP-2 and stimulated improved bone healing compared collagen sponges, which are the clinical standard delivery vector for BMP-2 therapy. These results suggest that treatment with bioartificial integrin-specific PEG hydrogels may be a promising clinical strategy for bone regeneration in large bone defects.

Tese de doutoramento em Engenharia de Materiais
In the orthopedics field, one of the major hurdles that surgeons face on daily basis is the need for bone replacing materials to restore defects that lose capability for self repair. To date, autograft materials remain as the “gold standard” for bone repair; however donor-site morbidity and limitations on the amount of tissue that can be collected have led scientists to search for new materials capable to induce bone repair. The main objective of this thesis was the conception of membranes that comprise pivotal characteristics and properties to impart bone repair. The first part of this thesis addressed the production of membranes that have the capacity of osteointegration, i.e., that possess an adequate bone-bond ability. We hypothesized that, in some situations, the two sides of the membranes are in contact with distinct biological environments in which one side faces a region in which osteointegration should be ideally promoted. For the proof of concept, the first work describes the development and characterization of poly(D,L-lactic acid)/Bioglass® (PDLLA/BG) composite membranes with asymmetric bioactivity. Such asymmetry was obtained by an adjusted solvent casting method that promoted a nonuniform distribution of the inorganic component along the membrane thickness. The membranes presented a good integration between the polymeric and inorganic fractions. Moreover, only the inorganic-rich face promoted the deposition of bone-like apatite. Additionally, the composite membranes were found to be stiffer compared with the pure polymer. The results indicate that the proposed asymmetric PDLLA/BG membranes could have potential to be used in guided bone regeneration therapies. This work was validated by the use of synthetic PDLLA polymer, a material widely used in bone repair but biologically inert. The subsequent studies of this thesis make use of natural polymers once they are biologically active possessing similarities with the bone extracellular matrix. In this context, the bone-bonding ability was also demonstrated by the preparation of chitosan/Bioglass® (CHI/BG) composite membranes where their potential to induce a bone-like layer was demonstrated upon immersion in simulated body fluids (SBF). Although in literature the bioactivity (capacity to form a bonelike apatite layer under physiological-like conditions) of such chitosan-based systems has been reported, such studies never demonstrate what happens with the mechanical properties of the material during this process. For that, dynamic mechanical analysis (DMA) experiments were performed in real time while the samples were immersed in simulated body fluid at 37ºC, being subjected to different tension loads. It was possible to follow the formation of a bone-like apatite using microscopic and spectroscopic techniques, and, simultaneously, the variations of the mechanical/viscoelastic properties were also evaluated. Moreover, as in in-vivo conditions bone tissues are subjected to mechanical stimuli with distinct intensities, we demonstrated that the membranes can present different calcification kinetics by varying the dynamic strain amplitude. With the same reasoning, we also developed CHI/BG composite membranes with nano-sized BG particles (CHI/nBG). The results demonstrated that CHI/nBG membranes possess enhanced mechanical properties and higher bioactivity in comparison with the CHI/BG membranes that contained micron-sized BG particles. Although bone-bonding ability is an important characteristic, it is not the only requirement for inducing bone healing upon implanting a device for such purposes. The second part of this thesis focused on the preparation of membranes that hold osteoconductive and osteoinductive properties. To achieve this goal, we transpose the Layer-by-Layer (LbL) technology for the production of nanostructured bioactive free-standing (FS) polymeric membranes that could be directly used to cover bone defects and actively assist bone healing. To this end, an adequate protocol was used to detach the multilayer films from lowsurface energy substrates in mild conditions. The FS membranes, prepared using chitosan and alginate, were crosslinked at various extents and their biocompatibility was validated. Additionally, in order to enhance osteoinductive properties of the membranes, bone morphogenetic protein 2 (BMP-2) – an osteogenic growth factor already approved to be used in the clinic - was loaded in the membranes. The BMP-2 incorporation was proven successful, once improved osteogenesis was detected in cells when cultured in presence of membranes leaded with the growth factor. Taken altogether, distinct methods and strategies to tailor membranes with topographical, chemical and biological signals were obtained in this thesis. The developed systems present valuable properties for the enhancement of bone healing and have characteristics that make them suitable to be potentially used in the clinics.
No campo ortopédico, um dos principais obstáculos que os cirurgiões enfrentam diariamente é a necessidade de materiais de substituição óssea para restaurar defeitos que perdem a capacidade de auto-reparação. Até à data, enxertos autólogos são os materiais de referência para a reparação óssea. No entanto problemas relacionados com a morbilidade da zona dadora, limitações na quantidade de tecido que pode ser recolhido levaram cientistas em busca de novos materiais capazes de induzir a regeneração óssea. O principal objectivo desta tese foi a produção de membranas que possuem características e propriedades fundamentais que permitam acelerar a regeneração óssea. Na primeira parte desta tese foram desenvolvidas membranas com capacidade de osteointegração, ou seja, membranas com capacidade de promover a ligação com o osso. Uma das hipóteses colocada foi de que, em alguns casos, os dois lados da membrana estão em contacto com ambientes biológicos distintos em que um lado enfrenta uma região em que a osteointegração deve ser idealmente promovida, devendo por isso apresentar propriedades assimétricas. Para a prova de conceito, o primeiro trabalho descreve o desenvolvimento e a caracterização de membranas compósitas com bioactividade assimétrica à base de ácido poliláctico com partículas de biovidro (PDLLA/BG). Esta assimetria foi conseguida através de um método optimizado de evaporação de solvente que levou a uma distribuição não -uniforme do componente inorgânico ao longo da espessura da membrana. Os testes efectuados demontraram que as membranas apresentam uma boa integração entre as fracções polimérica e inorgânica. Além disso, apenas o lado mais rico em material inorgânico promoveu uma camada de apatite, o principal componente mineral presente no osso. As membranas compósitas desenvolvidas demonstraram serem mais rígidas quando comparadas com membranas de polímero puro. Os resultados indicam que as membranas assimétricas de PDLLA/BG propostas têm efectivamente potencial para serem utilizados em terapias de regeneração óssea guiada. Este trabalho foi validado pela utilização de PDLLA que é um polímero sintético e é largamente utilizado na reparação do osso, mas biologicamente inerte. Os estudos subsequentes desta tese fazem uso de polímeros naturais, uma vez que são estes podem ser biologicamente activos e possuem semelhanças com a matriz extracelular do osso. Neste contexto, a capacidade de ligação membrana-osso foi igualmente demonstrada através da preparação de membranas de compósito quitosano/biovidro (CHI/BG). Neste estudo, o potencial para induzir uma camada semelhante ao osso foi demonstrada após imersão numa solução que simula o plasma sanguíneo (SBF). Embora na literatura já tenha sido reportada a bioactividade de materais à base de quitosano, tais estudos não demonstram o que acontece com as propriedades mecânicas do material durante este processo. Para isso, ensaios dinâmico-mecânicos foram realizados em tempo real, com as amostras imersas em SBF a 37°C. Através desta análise foi possível seguir simultaneamente a formação de apatite e as variações das propriedades mecânicas e viscoelásticas das membranas. Esta análise também foi complementada através de técnicas microscópicas e espectroscópicas. Além disso, in-vivo, os tecidos ósseos são submetidos a estímulos mecânicos com diferentes intensidades. Para simular estes eventos as membranas foram submetidas a diferentes cargas de tensão e, foi demonstrado que mediante o estímulo aplicado os materiais apresentam diferentes cinéticas de calcificação. Seguindo o mesmo raciocínio, também se desenvolveram membranas compósitas com nanopartículas de biovidro. Os resultados demonstraram que estas membranas possuem propriedades mecânicas melhoradas e maior bioactividade em comparação com as outras que continham micropartículas de BG. Embora a capacidade de integração com o osso seja uma característica importante, este não é o único requisito para induzir a regeneração óssea. Assim, a segunda parte desta tese centrou-se na preparação de membranas que possuem propriedades de osteocondução e osteoindução. Para atingir este fim, a tecnologia Layer-by-Layer (LbL) foi utilizada para a produção de membranas poliméricas bioactivas nanoestruturadas - free-standing (FS). Estas podem ser usadas diretamente para cobrir defeitos ósseos e ajudar activamente na consolidação óssea. Um protocolo adequado foi usado para destacar as membranas dos seus de substratos por forma a não danificar as mesmas. As membranas FS, preparadas à base de alginato e quitosano foram reticuladas e sua biocompatibilidade foi validada. Neste estudo, a fim de melhorar as propriedades osteoindutoras das membranas, a proteína morfogenética óssea 2 (BMP-2)- um factor de crescimento osteogénico já aprovado para ser utilizado na prática clínica foi impregnado nas membranas. A BMP-2 foi incorporada com sucesso nas membranas uma vez que foi detectada osteogénese nas células quando cultivadas na presença destas membranas. No âmbito desta tese, foram desenvolvidos distintos métodos e estratégias para produzir membranas com propriedades topográficas, químicas e biológicas adequadas para regeneração óssea. Os sistemas desenvolvidos apresentam propriedades valiosas para a melhoria desta e têm características que os tornam adequados para serem potencialmente utilizados na prática clínica.

Piezoelectric materials have shown large potential on hard tissue applications due to their ability to stimulate osteogeneses and osseointegration. Barium titanate (BT) is a well-known piezoelectric ceramic. This work reports the consequences of calcium acceptance in BT lattice without compromising the formation of piezoelectric tetragonal phase under physiological conditions. Analytical reagents CaCO3, BaCO3 and TiO2 were used to prepare, via solid state reaction, Ba(1-x)CaxTiO3 (BCT), 0 ≤ x ≤ 0.3 mixtures. Materials were sintered from 1150°C to 1450°C under air and N2 atmospheres. Composites were prepared from Ba(1-x)CaxTiO3 (0 ≤ x ≤ 0.15) and hydroxyapatite (HP) at 10/90 and 20/80 (HP/BCT wt%) proportions. Reagents and produced ceramics were characterized by DTA-TG, granulometry, X-ray diffraction, FTIR, Raman and SEM/EDS. Samples were polarized by corona poling at 110°C, 1 hour, tip potential -15kV and -2kV grid potential for subsequent bioactivity essays. The polarization was analysed by thermally stimulated depolarization currents. Calcium substitution up to 15 mol% deformed the known BaTiO3 lattice without compromising the tetragonal phase stability, maintaining Curie point between 123 °C and 125 °C. All materials tested were non cytotoxic. Corona poling was successfully done to BCT samples. Hydroxyapatite reacts with BCT while sintered at 1350 °C forming different phases, but materials original structures are partially maintained. Early stage bioactivity studies made after both polarized and unpolarized samples were immersed in SBF (simulated body fluid) for 7 days. Results from ICP-AES supported by SEM/EDS point to the materials enhanced ability while polarized to deposit calcium and phosphor ions on its surface.