Indiana University-Purdue University Indianapolis (IUPUI) / The effects of hydroxyapatite in a DAP-loaded MC carrier on dental pulp stem cells Introduction: Regenerative endodontic procedures (REP) require disinfection techniques to eliminate bacteria from the infected immature root canal system and promote new growth of the pulp-dentin complex. Double antibiotic paste (DAP), a mixture of ciprofloxacin and metronidazole, has shown efficacy in doing so while minimizing cytotoxicity on dental pulp stem cells (DPSC). Stem cells, scaffolding, and growth factors are necessary in the maturation, proliferation, and differentiation of mesenchymal stem cells into the root canal system. Nano-hydroxyapatite (n-HA) has a history of biocompatibility and, in addition, has shown promising effects as a tissue bioengineering material. Objective: The aim of this in vitro study was to investigate the proliferation and mineralization of DPSC in the presence of 1% DAP and methylcellulose (MC) with varying concentrations of nano-hydroxyapatite. Materials and Methods: DPSC were plated in 24-well plates containing culture media. The next day, semi-permeable 0.1 mm Transwell chambers were inserted into the wells to separate the reservoirs for medicaments. Treatment paste composed of methylcellulose containing 1% DAP with either 0.25%, 0.50%, or 1.0% nano-hydroxyapatite was added along with culture media. Methylcellulose alone and calcium hydroxide (Ultracal) were used as control groups. After 3 days, cells were evaluated for cytotoxic effects using an MTS proliferation assay (n = 10, in triplicate). DPSCs were also cultured with these medicaments for 7 days in osteogenic media and evaluated for alkaline phosphatase (ALP) activity and mineralization activity (n = 13, in triplicate). Comparisons between groups for differences in mineralization, BSA, and ALP activity were performed using analysis of variance (ANOVA), with different variances allowed for each group and a random effect included in the model to account for correlation within each of the three trials. A simulation-based model was used to adjust for multiple comparisons. Results: Addition of n-HA treatment groups increased mineralization significantly greater than calcium hydroxide, with MC alone and MC+DAP+0.5% HA providing the greatest effect. Regarding ALP, all HA concentrations performed significantly greater than MC and DAP concentrations. Proliferation demonstrated similar metabolic activity in all experimental groups with few comparisons significant. Conclusion: The challenge in REPs is to maintain survival, and preferably promote the proliferation and development of DPSCs into the pulp-dentin complex with a consistent treatment outcome. The combination of DAP with hydroxyapatite may allow for both disinfection and improved mineralization and cellular differentiation. This contribution has shown significant ability to increase stem cell differentiation into an osteogenic lineage as well as calcium deposition, indicating end goal results of regenerative procedures.

published_or_final_version / abstract / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

Active targeting with controlled delivery of therapeutic agents to bone is an ideal approach for treatment of several bone diseases. Since bisphosphonates (BPs) are known to have high affinity to bone mineral and are being widely used in treatment of osteoporosis, they are well-suited for drug targeting to bone. For this purpose, bifunctional hydrazine-bisphosphonates (HBPs) with spacers of various lengths and lipophilicity were synthesized and studied. Crystal growth inhibition assays demonstrated that the HBPs with shorter spacers bound more strongly to bone mineral, hydroxyapatite (HA), than did alendronate. HBPs were also demonstrated to be non-toxic to MC3T3-E1 pre-osteoblasts. The targeted delivery of the HBP-conjugated model drug, 4-nitrobenzaldehyde, was demonstrated through hydrolysis of the hydrazone linkage at the low pH of bone resorption and wound healing sites. In another series of experiments, a method to orient proteins on HA surfaces was developed to improve protein bioactivity. Enhanced green fluorescent protein (EGFP) and β-lactamase were used as model proteins. These proteins have a Ser or Thr at their N-terminus, which was oxidized to obtain a single aldehyde group that was subsequently used for bonding HBPs of various length and lipophilicity through formation of a hydrazone bond. The amount of protein immobilized through various HBPs was determined and found not to be exclusively dependent on the length of HBPs. The enzymatic activity of HBP-immobilized β-lactamase, measured with cefazolin as substrate, was found to be higher than β-lactamase that was simply adsorbed on HA. In a third set of studies, HBPs were evaluated for delivering parathyroid hormone (PTH) to bone mineral to enhance cell responses for bone formation. PTH was oxidized and conjugated to HBPs, followed by targeting to bone wafers. In vitro bioassays demonstrated that HBP-targeted PTH stimulated greater synthesis of cAMP in pre-osteoblasts compared to surfaces with simply adsorbed PTH. HBPs were also found to have similar pro-apoptotic activity to widely used alendronate. Overall, HBPs can be used for drug delivery to bone and oriented immobilization of proteins and peptides, with or without anti-osteoclastic action, for a variety of applications including bone tissue engineering.

Device associated infections are a growing problem in the field of orthopaedics and dentistry. Bacteria adhering to implant surfaces and subsequent biofilm formation are challenging to treat with systemic administered antibiotics. Functionalization of implant surfaces with therapeutic coatings that are capable of inhibiting bacterial adhesion are therefore considered as a straight forward strategy to treat and prevent implant related infections. In this thesis, the use of crystalline, arc deposited TiO2 and biomimetic hydroxyapatite (HA) coatings were evaluated with respect to their potential as antibacterial surface modifications for bone-anchored implants. UV light induced photocatalysis of anatase dominated TiO2 coated surfaces was shown to provide a bactericidal effect against S. epidermidis under clinically relevant illumination times and doses. Major parts of the drug release work carried out was based on biomimetic HA (HA-B) coated fixation pins. The analysis of the coating characteristics revealed that the nanoporous structure of HA-B coatings in addition to the chemical composition and surface charge are essential parameters that influence the drug carrier performance. Loading by adsorption was demonstrated to be a feasible approach to quickly incorporate antibiotics. The controlled release of antibiotics was shown to facilitate bactericidal effects against S. aureus over application-relevant time periods, even when exposed to biomechanical forces during insertion into bone model materials. Antibiotic incorporation during coating growth was shown to promote somewhat longer drug release time periods than those obtained using adsorption loading. In summary, functionalization of implant surfaces with bioactive and biocompatible coatings is a promising concept to impact the clinical success for bone-anchored applications. The additional feature of optional, on-demand antibacterial properties of these coatings through either on-site drug release or photocatalytic antibacterial treatment is advantageous for the prevention and effective treatment of devices-associated infections. Both strategies provide an immediate response to the implant contamination by bacteria and are believed to contribute towards minimizing the origin of post-surgical infections, while at the same time improving the interfacial stability between implant and bone.

Soil phosphorus (P) is often unavailable in SK soils due to immobilization by microbial biomass and complexation with cations. The prohibition of synthetic fertilizer use in organic systems means farmers rely on crop rotation or approved inputs to supply P for crops. Legumes in crop rotation add P to soil through decomposition and deposition, and approved fertilizers such as bone meal (BM), rock phosphate (RP), and composted manure add P to soil through dissolution. Arbuscular mycorrhizal fungi (AMF) improve crop access to soil P. The fungi colonize roots of host crops, allowing roots to reach immobile pockets of soil P. Colonization by AMF is usually decreased or delayed following partial fallow periods, non-host plants, and the addition of soluble P fertilizers. This thesis consists of two studies. For the first, the effects of crop rotation were tested on AMF colonization and soil P dynamics. Colonization by AMF of mycorrhizal crops was examined following a non-mycorrhizal crop, a partial fallow period, and mycorrhizal crops. All crops were colonized evenly (63-70%) at flowering despite non-mycorrhizal and partial fallow periods, and the sequence most depleted in soil N (wheat-barley) had the lowest colonization in August (36%). The second study evaluated soil P and plant N and P after applications of BM, hydroxyapatite (HAP), and sheep manure compost. Compost application increased plant P uptake compared to the control (1.26 vs. 0.71 mg pot-1), while applications of BM and HAP alone did not. Compost application did not affect AMF colonization of wheat (Triticum aestivum L.). Overall this research highlights the importance of legumes and composted manure use in organic systems. Legume use in crop rotation simultaneously increased soil P deposition and may have preserved AMF communities despite fallow periods and non-host crops in rotation. Conditions normally affecting AMF colonization in conventional systems did not apply. The use of composted manure in the greenhouse study resulted in the greatest P uptake and concentrations in wheat. Thus the use of legumes and composted manure may increase P availability to crops directly and indirectly: directly through soil P deposition and perhaps indirectly through the preservation of AMF communities.

The unsafe use of needles propagates cross infections with bloodborne pathogens and reduces the positive impact of vaccinations on global health. While a plethora of needle-free injection devices exist, the reformulation of protein-based vaccines is largely empirical and costly, which presents a barrier to their widespread clinical application. This thesis contributes to the identification of approaches that facilitate rapid vaccine reformulation and enhance the immunogenicity of needle-free dry-powder vaccines with the help of novel antigen delivery platforms. We hypothesised that the thermodynamic stabilisation of diphtheria toxin mutant 197 (CRM197), a glycoconjugate vaccine carrier protein, may enhance its structural preservation during spray-freeze-drying (SFD), and that its formulation in either soluble, surface-adsorbed, or nanoparticle form impacts the elicited immune response. Differential scanning fluorimetry was used to study the effect of excipients on the thermal stability of CRM197. Dry-powder formulation of CRM197 used i) encapsulation into a thermodynamically stabilising excipient matrix by SFD, ii) surface-immobilisation via physisorption onto a novel potassium-doped hydroxyapatite (kHA) carrier microparticle formed by molten salt synthesis, and iii) chemical conjugation and surface presentation on amphiphilic block copolymer nanoparticles that were incorporated into SFD-powders (SFD-NP). The structural integrity of CRM197 was assessed by size separation in addition to various spectral and thermal analysis methods. The immunogenicity of dry-powder CRM197 formulations was subsequently tested in vivo. The results suggest that the thermodynamic stability of CRM197 in solution does not ensure its structural stability during SFD. While needle-free dermal vaccination with kHA-adsorbed CRM197 induced comparable antibody titres to conventional IM injection of alum-adjuvanted CRM197, needle-free SFD and SFD-NP powders were less immunogenic. The highest mean IgG titre and most balanced Th1/Th2 response was achieved with nanoparticle-conjugated CRM197 by IM, which outperformed the current clinical standard. Therefore, future vaccine design should combine thermodynamic and kinetic stability screening, and place special emphasis on the delivery and structural presentation of the antigen to the immune system.

The field of tissue engineering aims to develop viable substitutes with the ability to repair and regenerate the functions of damaged tissue. Common practices to supplement bone regeneration in larger defects include bone graft biomaterials such as autografts, allografts, xenografts, and synthetic biomaterials. Autologous bone grafting is the current gold-standard procedure used to replace missing or damaged bone. However, these grafts have disadvantages such as donor site morbidity, limited availability, and the need for a secondary surgery. The focus of this study is to tissue engineer a lyophilized gelatin composite sponge composed of hydroxyapatite (HA), chitin whiskers (CW), and preparations rich in growth factors (PRGF) to provide sufficient structural support to the defect site while enhancing the body’s own reparative capacity, ultimately eliminating the need for autologous tissue harvesting or repeat operations. The present study investigates several in vitro evaluations on multiple compositions of modified gelatin sponge scaffolds for use in bone graft applications. Gelatin sponges were fabricated via freeze-drying, enhanced with PRGF, HA, and/or CW, and cross-linked with 50 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) either during or post gelation. Initial evaluation of all scaffold combinations indicated that scaffolds released contents up to 90 days, EDC cross-linking during gelation allowed for more protein release, and had the ability to swell. Since the incorporation of PRGF, HA, and CW increased cell infiltration, and production of cell-created mineral matrix over 90 days in culture, these scaffolds were further characterized. Freeze-dried gelatin sponges enhanced with PRGF, HA, and CW and cross-linked during gelation with EDC (PHCE) were mineralized (M) in 5x revised simulated body fluid (r-SBF) for 1 hour to create a bone-like mineral surface. Gelatin EDC scaffold controls (GE), GE-M, PHCE, and PHCE-M scaffolds were characterized for their ability to swell, mineralizing potential, surface morphology, growth factor incorporation and release, uniaxial compression properties, and cell attachment, proliferation, infiltration, and protein/cytokine secretion.. After mineralization, scanning electron microscopy showed sparse clusters of mineral deposition for GE-M scaffolds while PHCE-M scaffolds exhibited a more uniform mineral deposition. Both GE and PHCE scaffolds were porous structures that swelled up to 50% of their original volume upon hydration. Over 21 days incubation, PHCE-M scaffolds cumulatively released about 30% of their original protein content, significantly more than all other scaffolds. Multiplex Luminex assays confirmed the successful incorporation of PRGF growth factors within PRGF sponges. For acellular uniaxial compression testing, PHCE-M scaffolds reported lower Young’s modulus values (1.3 - 1.6 MPa) when compared to GE and GE-M scaffolds (1.6 – 3.2 MPa). These low modulus values were comparable to values of tissue found in early stages of bone healing. DAPI (4',6-diamidino-2-phenylindole) staining and imaging showed an increase in initial cell attachment and infiltration of PHCE and PHCE-M scaffolds on day 1. GE-M scaffolds also appeared to attach more cells than the GE control. MTS cell proliferation assay results indicated that on days 4 and 7, PHCE scaffolds increased cell proliferation (compared to GE controls). MTS also illustrated that the addition of a mineral coating increases and decreases cell proliferation on GE-M and PHCE-M scaffolds, respectively. Multiplexer analysis of MG-63 protein/cytokine secretion suggests that cells are responding in a bone regenerative fashion on all scaffolds, as evidence of osteocalcin secretion. Little to no secretion of osteopontin, IL-1β, and TNF-α demonstrates that scaffolds are not influencing cells to secrete factors associated with bone resorption. The compressive mechanical properties of cellularized scaffolds did not differ much from acellular scaffolds. The collective results indicated increased cellular attachment, infiltration, and bone regenerative protein/cytokine secretion by cells on GE-M scaffolds, which support the addition of a bone-like mineral surface on GE scaffolds. Cellularized PHCE and PHCE-M scaffolds report similar advantages as well as Young’s modulus values in the range of native tissues present in the early stages of bone healing. The results of this study propose that the developed PHCE and PHCE-M scaffolds exhibit good cellular responses and mechanical properties for use in early bone healing applications.

Estudos dosimétricos em hidroxiapatita (HAp) podem ser utilizados para a determinação da dose absorvida em tecidos duros em diversas situações, tais como acidentes radiológicos, controle de processos de esterilização e datação arqueológica. Essa tese apresenta estudos da resposta à dose de radiação tanto do sinal de Ressonância Paramagnética Eletrônica (RPE) quanto de termoluminescência (TL) para HAp. A fauna de mamíferos fósseis encontrada na Planície Costeira do Rio Grande do Sul é conhecida desde o final do século XIX, porém ainda pouco se sabe sobre seu contexto bio e cronoestratigráfico. Neste trabalho foram datadas, por RPE, onze amostras de dentes de mamíferos extintos, coletados no Arroio Chuí e ao longo da praia, proveniente do sul da costa do Rio Grande do Sul. As idades obtidas para essas amostras contribuem para um melhor entendimento da origem dos depósitos fossilíferos. Em um segundo momento do trabalho, é proposto um novo procedimento de decomposição de espectros complexos de RPE, voltados para a dosimetria e datação. O método consiste na utilização de funções do pacote de funções do software \"Easyspin\", que é gratuito, associadas a métodos de minimização de funções. Após a validação, o método foi aplicado na decomposição de espectro de duas amostras de dentes de Stego-mastodon waringi, provenientes do nordeste do Brasil. A decomposição visa verificar o efeito que tem as componentes sobrepostas ao sinal dosimétrico sobre o cálculo da dose acumulada e mostrou-se útil para melhorar a precisão na determinação da dose. Por fim, hidroxiapatita sintética carbonatada do tipo-A e hidroxiapatita natural extraída de dentes fósseis foram caracterizadas por TL. Os resultados obtidos por essa técnica mostrou que esses dois tipos de amostra são capazes de responder a radiação ionizante. Contudo, esses mesmos resultados também revelaram a impossibilidade de se fazer dosimetria por termoluminescência com fins à datação, para esse tipo de amostra. / Dosimetric studies on hydroxyapatite (HAp) can be used to determine the absorbed dose in hard tissues in several situation, such as radiologic accidents, control of sterilization process and archaeological dating. This PhD thesis presents studies about radiation dose response assessed by electron paramagnetic resonance (EPR) as well as thermoluminescence in HAp. The fossil mammalian fauna from the Coastal Plain of Rio Grande do Sul State has been known since the late XIX century; however, its biostratigraphic and chronostratigraphic context is still poorly known. The present works describes the results of electron spin resonance (ESR) dating on teeth of extinct mammals collected along the Chuí Creek and coastline. The ages obtained for these samples contribute to a better knowledge about the origin of these fossil. In a second stage of this work, we propose a new EPR spectrum deconvolution (or decomposition) procedure aimed to dosimetry and dating. The method uses the EasySpin (a freeware software) set of functions associated with minimization procedures. After validation, the method was applied in spectrum deconvolution of two Stegomastodon waringi enamel samples, originated from northeast of Brazil. The spectrum econvolution is aimed to verify the superposed components effects on the dosimetric signal over the accumulated dose calculation. The results have shown to be helpful in improving the dose calculation accuracy. In the last stage of this work, synthetic carbonated A-typ hydroxyapatite and natural one were investigated by Thermoluminescence. The obtained results shown that the two samples respond linearly to the ionizing radiation dose. However, the short thermoluminescent glow-peak lifetime suggest that it\'s inadequate for dating purpose.

Este trabalho propõe a síntese de vários materiais nanoestruturados pelo processo sol-gel. Foram preparados sóis a base de óxido de cério, hidroxiapatita e magnetita. Filmes a base de paládio metálico foram obtidos pela técnica de decomposição térmica e redução em atmosfera de nitrogênio. A caracterização estrutural dos materiais preparados foi realizada por DRX; a caracterização morfológica foi feita mediante SAXS, MET e MEV; a caracterização química por EDS; a caracterização térmica por DSC; a caracterização magnética por RPE e a caracterização relaxometrica por IRM. No caso da hidroxiapatita, foram realizadas medidas de FTIR. Nanopartículas a base de óxido de cério e Pdo com propriedades catalíticas foram depositadas na forma de filme sobre substratos de alumínio anodizado e na região da cabeça de pistões automotivos de motor a gasolina. Os catalisadores foram suportados nos poros que os anodizados apresentam. Os pistões foram submetidos a testes catalíticos e apresentaram redução nas emissões de CO, compostos orgânicos voláteis e NOx. Além disso, verificou-se a diminuição no consumo de combustível e aumento na eficiência do motor automotivo a gasolina. A síntese de nanopartículas a base de hidroxiapatita produz um material de morfologia nanométrica, apresentando um comportamento ferromagnético atribuído ao ferro como impureza nos precursores da síntese. Também foram obtidas sóis estáveis a base de nanopartículas de hidroxiapatita revestidas por ácido oléico. A síntese e caracterização do ferrofluido à base de nanopartículas de magnetita superparamagnéticas levaram a fabricação de um agente de contraste negativo, característica principal para aplicações na IRM. / This work proposes the synthesis of several nonostructured materials by the sol-gel process. Cerium oxide, hydroxyapatite and magnetite based sols were prepared. Metallic palladium based films were obtained by the thermal decomposition technique followed by reduction under nitrogen atmosphere. Structural characterizations of the prepared materials were accomplished by XRD; morphologic characterization were carried out by SAXS, TEM and SEM, chemical characterization by EDS, thermal characterization using DSC, magnetic characterization by EPR, and the relaxometry characterization by MRI. FTIR\'s measurements were also accomplished in hydroxyapatite samples. Cerium oxide and palladium nanoparticles presenting catalytic properties were deposited as a film on anodized aluminum substrates and on automotive piston heads for gasoline motors. The catalyzers were supported by pores present in anodized aluminum surfaces. Catalytic test of the pistons have shown emission reduction of CO, organic volatile compounds, and NOx. Moreover, decrease in the fuel consumption and increase in automotive motor efficiency were verified. The synthesis of nanoparticles based on hydroxyapatite gives rise to a material of nanometric morphology. It presents a ferromagnetic behavior due to the iron as impurity in the synthesis precursors. Stable sols of hydroxyapatite nanoparticles covered by oleic acid were also obtained. Synthesis and characterization of ferrofluid based on superparamagnetic nanoparticles of magnetite lead to the production of an agent for negative contrast. It is the main characteristic for applications in MRI.