Articular cartilage is a hydrated soft tissue with a fibrous solid matrix characterized by high porosity and low permeability. It is the bearing material of diarthrodial joints, permitting motion and transmitting loads with extraordinarily low friction. This function may be disrupted pathologically by osteoarthritis, a disease where cartilage becomes weakened and eroded. Osteoarthritis creates pain during normal activities like walking or grasping, thus diminishing quality of life. The disease affects nine percent of Americans and is one of the leading causes of disability worldwide. There is presently no cure or prevention for osteoarthritis, only palliative treatments designed to help patients manage pain and regain mobility. New such treatments are developed in part by advancing the science of cartilage mechanics, structure and function, and this dissertation presents novel contributions toward this effort: Chapters 2, 3, and 4 enhance our knowledge of the structure-function relationships critical to our understanding of cartilage friction and load support. Whereas most prior theoretical and experimental studies have focused on the analysis of small cylindrical explants, or idealized joint geometries such as cylindrical or spherical articular layers, these chapters describe novel investigations performed on whole articular layers of the shoulder and knee joints. Insights from these investigations have a direct impact on our formulation of design objectives in cartilage tissue engineering, whose purpose is to grow constructs that reproduce the functional properties of native cartilage. The studies presented in this chapter are critical to ongoing tissue engineering studies in our laboratory, which has pioneered the development of anatomically sized cartilage constructs. Finally, Chapter 5 describes the development of a novel clinical treatment for thumb osteoarthritis that uses bent osteochondral allografts (living bone and cartilage from human donors) to replace the eroded thumb trapezial articular layer with a healthy and thick articular layer from another joint such as the knee. This highly promising treatment strategy overcomes the limitation of size mismatch between donor and recipient which had relegated osteochondral allograft surgery to a niche treatment. Like other fibrous tissues, cartilage exhibits tension-compression nonlinearity, meaning it can be 100 times stiffer in tension than in compression. Tension-compression nonlinearity allows compressive physiologic joint loads to be supported by tensile stress within the collagen fibers and elevated fluid pressure, effectively shielding the solid matrix from compressive load. According to theory, fluid load support derives directly from tension-compression nonlinearity. Fluid load support is also a dominant mechanism of cartilage lubrication. Because cartilage is 80 to 90% water, most of the contact traction on the porous cartilage surface takes the form of hydrostatic fluid pressure. Friction forces only occur upon solid-on-solid contact, so cartilage friction is nearly negligible, even for joint contact forces that may routinely exceed three or four times the body’s total weight. The dependence of friction on fluid load support is demonstrated by experiments that simultaneously measure interstitial fluid pressure and friction - a transient rise in friction occurs as pressure subsides and fluid drains from the tissue. These structure-function relationships have been identified over decades of research, mostly through small cartilage explant studies, which have supported hypothesized mechanisms under non-physiologic conditions. Therefore, in situ studies utilizing intact, naturally-congruent articular surfaces under physiologic loading conditions would significantly extend and validate these principles. For example, friction may rise nearly 100-fold after only 1 hour in cartilage explant experiments, yet there is no evidence that normal daily activities spanning 16 hours or more lead to cartilage damage. Can fluid load support sustain low friction under these relatively harsh conditions? To date, no study has examined this question, so Chapter 2 of this work addresses the hypothesis that the friction coefficient of diarthrodial joints can remain low over a full day of loading at physiologic speeds and load magnitudes. Another question that may be uniquely addressed by an in situ analysis is: What is the complete state of stress within naturally-congruent cartilage layers? A primary hypothesis for the initiation and progression of osteoarthritis is that the state of stress within articular cartilage may exceed a threshold beyond which the tissue is unable to repair itself. Since the complete stress tensor within a material is immeasurable, techniques such as finite element analysis must be used to examine the state of stress. Additionally, a theoretical framework such as mixture theory may be used to examine the stresses in the fluid and solid constituents of the tissue separately, making it possible to test theories of solid matrix damage. Chapter 3 of this work uses this strategy to examine the hypothesis that physiologic solid matrix stresses within anatomically-shaped, biphasic, tension-compression nonlinear cartilage layers are primarily tensile, despite the fact that the articular layers are loaded in compression. The proteoglycan content of articular cartilage gives the tissue an osmotic swelling pressure that is resisted by tensile stresses in the collagen fibrils, even in the absence of external loads. This charge effect may be additionally incorporated into a mixture theory finite element analysis to examine the role of osmotic swelling on the solid matrix stresses in a physiologic, in situ analysis. This capability has only been developed recently and is explored for the first time in Chapter 4. The final part of this work translates basic cartilage science into a clinical therapy for thumb joint osteoarthritis, a common site for this disease. The current gold-standard treatment for thumb joint osteoarthritis replaces the trapezium bone with a soft-tissue tendon autograft, relieving pain but significantly weakening hand strength. Living osteochondral allograft transplantation may provide a relatively straightforward treatment alternative, though this procedure has not been used for the thumb due to the inadequate availability of suitable allografts. The ideal allograft would have a relatively thick articular layer to provide sufficient compliance for promoting joint congruence with the mating metacarpal surface, and surface curvatures that match the saddle-shaped anatomy of the distal trapezial articular surface to reproduce the normal joint motions. A potential solution that would provide suitable trapezium osteochondral allografts for patients involves precisely machining and bending allografts from a lower extremity joint with thicker cartilage, such as the distal femoral surface of the knee, to match the shape and curvature of the trapezium. Such bent osteochondral allografts would provide all the desired benefits of the ideal arthroplasty. Chapter 5 outlines the development of this novel technology, including proof of concept and feasibility demonstrations, business strategy and market analysis.

A Engenharia de Tecidos tem como objetivo desenvolver alternativas para o tratamento de doenças degenerativas e regeneração de tecidos lesionados. O princípio básico da Engenharia de Tecidos é promover o crescimento de células sobre um substrato. Esse substrato deve reproduzir a matriz extracelular, influenciando a diferenciação e as funções celulares. Para isso, neste trabalho, nanopartículas poliméricas de poli(3-hidroxibutirato-co- 3-hidroxivalerato) (PHBHV), com diferentes diâmetros hidrodinâmicos, foram sintetizadas pelo método de emulsão-evaporação do solvente. A concentração do surfactante, a velocidade de agitação durante a emulsificação e o tempo de agitação foram variados a fim de se analisar a influência desses parâmetros no diâmetro hidrodinâmico, índice de polidispersidade e estabilidade coloidal das emulsões. Os parâmetros que mais influenciaram no diâmetro hidrodinâmico e no índice de dispersidade foram a concentração de surfactante e a amplitude de sonicação da emulsão, respectivamente. Após a obtenção das dispersões com estabilidade coloidal, realizou-se a modificação química da superfície dessas partículas utilizando os aminosilanos, 3-aminopropiltrimetoxisilano (APTMS) e 3- aminopropiltrietoxisilano (APTES). Essa modificação química na superfície das nanopartículas teve como objetivo a mimetização da matriz extracelular, permitindo a adesão e proliferação das células. As partículas modificadas foram caracterizadas por espalhamento de luz dinâmico (DLS), microscopia de força atômica (AFM), calorimetria exploratória diferencial (DSC), Espectroscopia de Infravermelho por Transformada de Fourier (FTIR) e Ressonância Magnética Nuclear de Hidrogênio (RMN 1H). Os resultados de FTIR mostraram o aparecimento de pico referente ao grupo amino, que foi indicativo da modificação química da superfície das nanopartículas. Os resultados de RMN 1H mostraram o sinal dos grupos CH3 do silano APTMS no espectro das nanopartículas modificadas por APTMS, e nas partículas modificadas por APTES, foi possível identificar o sinal referente aos grupos CH2 do APTES. A partir desses resultados comprovou-se a modificação química da superfície das nanopartículas pelos aminosilanos. / Tissue engineering aims to develop alternatives to treat damaged tissues by promoting tissue regeneration. The basic principle of Tissue Engineering is to promote the growth of cells on a substrate. This substrate must reproduce the extracellular matrix, influencing particular cell functions and differentiation fate. For this purpose, at the present work, polymeric nanoparticles of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) with different hydrodynamic diameters were synthesized by emulsion-solvent evaporation technique. The concentration of the surfactant, stirring speed during emulsification and stirring time were varied in order to analyze the influence of these parameters on hydrodynamic diameter, polydispersity and colloidal stability. The parameters that most influenced the hydrodynamic diameter and polydispersity index were the variation in the surfactant concentration and the variation of emulsion sonication amplitude, respectively. After that, the nanoparticles had their surface modified by 3-aminopropyltrimethoxysilane (APTMS) and 3- aminopropyltriethoxysilane (APTES). The aim of this chemical modification was to mimic the extracellular matrix, allowing the adhesion and proliferation of cells. The modified particles were characterized by dynamic light scattering (DLS), atomic force microscopy (AFM), differential scanning calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR). The FTIR results showed a peak of the amino group, which was an indicative of the nanoparticles surface chemical modification. 1H NMR results showed the signal of the CH3 groups of the APTMS silane in the spectrum of the APTMS-modified nanoparticles and in the APTES-modified particles it was possible to identify signal relating to the CH2 groups of the APTES. From these results, it was verified the nanoparticles surface chemical modification by the aminosilanes.

Bone undergoes constant turnover throughout life and has the capacity to regenerate itself. However, the repair of critical size defects, caused by bone diseases such as osteoporosis, can be more problematic. Therefore, there is a clinical need for a bone graft substitute that can be used at sites of surgical intervention to enhance bone regeneration. Poly(vinylphosphonic acid-co-acrylic acid) (PVPA-co-AA) has recently been identified as a potential candidate for use in bone tissue scaffolds. It is hypothesised that PVPA-co-AA can mimic the action of bisphosphonates – a class of drugs used in the treatment of osteoporosis – by binding to calcium ions from bone mineral surfaces. In this way, bisphosphonates can affect bone turnover by increasing the activity of osteoblasts and reducing osteoclast activity. Although PVPA-co-AA has been shown to improve bone formation, the mechanism of action has so far not been fully elucidated. Therefore, this work aims to understand the effect of copolymer composition on the properties of PVPA-co-AA, and thus to determine its effect on osteoblast adhesion and proliferation. PVPA-co-AA copolymers have been synthesised with a range of monomer feed ratios. It was found that a VPA content of 30 mol % led to the greatest calcium binding affinity of the copolymer and is thus expected to lead to enhanced bone formation and mineralisation of the matrix produced by osteoblast cells. The release profile of PVPA-co-AA from electrospun PCL scaffolds was investigated. It was shown that all of the PVPA-co-AA was released into aqueous media within 8 h of immersion. It was also found that the calcium chelation from osteogenic differentiation media significantly increased within the first 8 h. Therefore, it was concluded that PVPA-co-AA is released from the scaffolds, where it can then bind to calcium ions from the bone mineral surface to promote mineralisation, thus acting as a mimic of non-collagenous proteins, which are present in the extracellular matrix (ECM) of bone. Hydrogels of PVPA-co-AA have been produced and the effect of monomer feed ratio (0-50 mol % VPA) on the properties of the gels was explored. It was found that an increase in VPA content led to greater hydrogel swelling and increased porosities. Hydrogels that contained 30 and 50 mol % VPA were shown to have similar morphologies to the native ECM of bone. Rheological testing showed that hydrogels with higher VPA contents were more flexible and could be deformed to a large extent without permanent deformation of their structure. An increase in osteoblast adhesion and proliferation was observed for hydrogels with 30 and 50 mol % VPA content as well as superior cell spreading. Osteoblast cell metabolic activity also increased as a function of VPA content in the hydrogels. This work indicates that hydrogels of PVPA-co-AA, with VPA contents of 30 or 50 mol %, are ideal for use as bone tissue scaffolds. Furthermore, the mechanical and cell adhesion properties of the gels can be tuned by altering the copolymer composition. Finally, composite hydrogels of PVPA-co-AA and hydroxyapatite (HA) have been produced and investigated for their ability to remove fluoride ions from groundwater. It was found that the fluoride uptake ability of PVPA-HA hydrogels was significantly enhanced when compared with HA powder alone. Furthermore, the fluoride uptake was dependent on many factors, including pH, contact time and the presence of competing ions. It was possible to regenerate the hydrogel to remove the fluoride ions, and thus it was shown that the material can be used a number of times with only a slight reduction in its fluoride uptake capacity.

<p>Validerat; 20130918 (global_studentproject_submitter)</p>

Pre-clinical studies are a necessary step in the process of material and drug testing. For this, high-throughput monolayer cell cultures are conducted followed by in vivo animal experiments. However, animal use is ethically questionable and in many cases yields misleading results. In vitro three dimensional (3D) tissue engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer cultures and are less ethically questionable than animal models. Therefore, an in vitro biomimetic musculoskeletal junction (MSKjct) is required as a more relevant pre-clinical testbed. This thesis describes the steps taken to co-culture 3D TE skeletal muscle and bone models as a material testbed and towards an in vitro MSKjct.

In the United Kingdom 1 in 6 - 8000 children are born missing one or both of their ears. The surgical technique most commonly used to reconstruct ears requires surgeons to remove ribs from the patient, and the cartilage from the ribs is used to carve a new ear. This procedure involves many risks including significant pain, punctured lung and chest deformity. Therefore the ability to ‘grow’ an ear would be a major advancement. Stem cells show huge promise in tissue engineering and regenerative medicine. Approved stem cell technology must be evaluated with regards to safety, purity, identity, potency and efficacy prior to biologic licensing and clinical use. Therefore, access to ethically sourced tissue for research is fundamental to the successful delivery of novel therapies. Adipose tissue provides an abundant and accessible source of stem cells for clinical translation. Within the first section of this thesis, the perceptions and attitudes of patients towards the donation and use of adipose tissue for research are sought. Based on this information, a tissue bank with all appropriate ethical approval to collect, process, store and distribute adipose tissue and adipose derived stem cells is established. The second part of this thesis demonstrates the specific identity, location and frequency of stem cells within adipose tissue; revealing them to reside in a perivascular niche. Using this data, protocols to rapidly purify stem cells from adipose tissue using Fluorescence Activated Cell Sorting are developed. The frequency of cells, and both the patient and procedure based variables that can affect this yield are also examined. The final section of this thesis uses a high-throughput microarray platform to screen thousands of polymers to identify potential substrates that can support the attachment, stable proliferation and subsequent differentiation of stem cells purified from adipose tissue. From the initial screen, 5 distinct polymers have been identified, characterised and their effects on the stem cells examined and quantified. Combined together, these elements provide significant advances in our understanding, and the basis for on going research to deliver a tissue engineered ear for use in human ear reconstruction.

No description available.

Osteoarthritis is a debilitating and irreversible disease afflicting the synovial joints. It is characterized by pain and hindered mobility. Given that osteoarthritis has no cure, current treatments focus on pain management. Ultimately, however, a patient's pain and immobility necessitates joint replacement surgery. An attractive alternative to this treatment paradigm, tissue engineering is a promising strategy for resurfacing the osteoarthritis-afflicted cartilage surface with a biochemically and biomechanically similar tissue to the healthy native cartilage tissue. The most successful cartilage tissue engineered systems to date can repeatably grow constructs ~4 mm in diameter with native proteoglycan and compressive mechanical properties. Unfortunately, as symptomatic cartilage typically presents only once lesions span large regions of the joint (~25 mm in diameter), these small construct are of limited use in clinical practice. Numerous attempts to simply grow a construct large enough to span the size of an osteoarthritic lesion have shown that the growth of large engineered tissues develop heterogeneous properties, emphasizing the need for culture protocols to enhance tissue homogeneity and robustness. In particular, as nutrient limitations drive heterogeneous growth in engineered cartilage, developing strategies to improve nutrition throughout the construct are critical for clinical translation of the technology. To this end, our lab has successfully supplemented nutrient channels within large engineered cartilage constructs to improve the functional properties of developing tissue. However, it is unknown what the optimal nutrient channel spacing is for growing large cartilage constructs of anatomical scale. Additionally, the fundamental factors and mechanisms which drive tissue heterogeneity have not been implicated, making the results of channel-spacing optimizations difficult to translate across different systems. Computational models of growth, faithful to the physics and biology of engineered tissue growth, may serve as an insightful and efficient tool for optimally designing culture protocols and construct geometries to ensure homogeneous matrix deposition. Such computational tools, however, are not presently available, owing to the unresolved mechanical and biological growth phenomena within developing engineered cartilage. This dissertation seeks to develop and implement computational models for predicting the biochemical and biomechanical growth of engineered tissues and apply these models to optimizing tissue culture strategies. These models are developed in two stages: 1) based on our recent characterization of the nutrient demands of engineered cartilage, models are developed to simulate the spatial biochemical deposition of matrix within tissue constructs and, subsequently, 2) based on models of biochemical matrix deposition we develop models for simulating the mechanical growth of tissue constructs. To accomplish these tasks, we first develop models simulating glucose availability within large tissue constructs using system-specific modeling based on our recent characterization of the glucose demands of engineered cartilage. These models led to early insight that we had to enhance the supply of glucose within large tissue constructs to ensure maximal matrix synthesis throughout culture. Experimental validations confirmed that increasing glucose supply enhanced matrix deposition and growth in large tissue constructs. However, even despite the increased glucose supply, increasing the size of constructs demonstrated that severe matrix heterogeneities were still present. Subsequent nutrient characterization led to the finding that TGF-ß transport was significantly hindered within large tissue constructs. Incorporating the influence of glucose and TGF-ß into the computational model growth kinetics. Using both nutrients, models recreated the heterogeneous matrix deposition evident in our earlier work and could account for the role of cell seeding density and construct geometry on tissue growth. The insights gathered from this modeling analysis led to important changes in our culture protocols: we could reduce the dose of TGF-ß from 10 ng/mL to 1 ng/mL for constructs cultured with channels, saving considerable expense while still maintaining a high level of matrix synthesis throughout the construct. In the presence of sufficient nutrition, we witnessed an unprecedented level of matrix deposition and physical growth of the constructs. In fact, by using developmentally physiologic cell seeding densities (120 million cells/mL) and providing adequate nutrition, constructs physically grew to 9-times their originally cast size. Despite such encouraging growth, tissue function properties plateaued at sub-physiologic levels. For insight into the connection between matrix deposition and tissue mechanics, we extended the computational growth models to consider the mechanisms underlying physical growth. Interestingly, we found that a large matrix synthesis mismatch between proteoglycans and collagen gave rise to the excessive tissue swelling. Computational models of this matrix synthesis mismatch predicted the high tissue swelling displayed experimentally only when a damage-able collagen fiber material was implemented. Together, the experimental and modeling evidence suggested a new mechanism of cartilage growth: the high proteoglycan deposition creates a swelling pressure within the nascent tissue which outcompetes the restraining force of newly deposited collagens; this rapid tissue swelling disrupts a functional collagen network from forming. Subsequent analysis suggested that the disruption of the collagen network prevented the formation of collagen crosslinks, stymieing the development of native functional properties. Based on this insight into the mechanisms of cartilage growth, we developed a culture systems to improve tissue functional properties. Modeling analysis indicated two novel routes for improving tissue mechanics: either through 1) reducing the swelling response (synthesis and deposition) of proteoglycans or 2) enhancing and reinforcing the newly synthesized collagen to prevent disruptions brought on by tissue swelling. We developed a cage culture system for resisting the swelling pressure of deposited proteoglycans and reenforcing the deposition of new collagens. Using this cage system, we grew tissue constructs with enhanced functional properties using two separate scaffold systems – agarose and a cartilage-derived matrix hydrogel – suggesting this mechanism of growth is fundamental to engineered cartilage development. This work has generated a novel paradigm towards engineering cartilage constructs using biomimetic strategies. Performing simulations with the validated, computational growth models allowed anatomically-sized cartilage constructs to grow into the largest, homogeneous cartilage constructs grown to date. Models presented a new level of insight into the nutrient demands of developing tissues, allowing for the first time the successful development of large tissue constructs grown with developmentally physiologic cell seeding densities. In this way, tissue constructs growth followed a biomimetic approach, based on the high cell densities and cartilage canals and vasculature present during fetal cartilage development. Adequate nutrition led to high levels of tissue growth not previously experienced in vitro, a result of adequately nourishing primary chondrocytes, a cell type which preferentially deposits proteoglycans over collagen. We therefore developed a cage-based growth system to resist the proteoglycan-induced tissue swelling in a manner similar to the fetal development of cartilage where the resident cells synthesize more collagens than proteoglycans. Together, the use of nutrient growth models, high cell seeding densities, and culture systems to strengthen the collagen-framework of de novo cartilage proved beneficial for engineering anatomically-sized cartilage constructs. The fundamental mechanisms identified here are likely to be universal across a number of engineered cartilage systems and will be adapted to more clinically-relevant cell sources in future our work.

Made available in DSpace on 2014-12-17T15:43:50Z (GMT). No. of bitstreams: 1 HaroldoAOJ_DISSERT.pdf: 2217261 bytes, checksum: 1a1221344f95ad964f692521743b5714 (MD5) Previous issue date: 2013-03-01 / The regeneration of bone defects with loss of substance remains as a therapeutic challenge in the medical field. There are basically four types of grafts: autologous, allogenic, xenogenic and isogenic. It is a consensus that autologous bone is the most suitable material for this purpose, but there are limitations to its use, especially the insufficient amount in the donor. Surveys show that the components of the extracellular matrix (ECM) are generally conserved between different species and are well tolerated even in xenogenic recipient. Thus, several studies have been conducted in the search for a replacement for autogenous bone scaffold using the technique of decellularization. To obtain these scaffolds, tissue must undergo a process of cell removal that causes minimal adverse effects on the composition, biological activity and mechanical integrity of the remaining extracellular matrix. There is not, however, a conformity among researchers about the best protocol for decellularization, since each of these treatments interfere differently in biochemical composition, ultrastructure and mechanical properties of the extracellular matrix, affecting the type of immune response to the material. Further down the arsenal of research involving decellularization bone tissue represents another obstacle to the arrival of a consensus protocol. The present study aimed to evaluate the influence of decellularization methods in the production of biological scaffolds from skeletal organs of mice, for their use for grafting. This was a laboratory study, sequenced in two distinct stages. In the first phase 12 mice hemi-calvariae were evaluated, divided into three groups (n = 4) and submitted to three different decellularization protocols (SDS [group I], trypsin [Group II], Triton X-100 [Group III]). We tried to identify the one that promotes most efficient cell removal, simultaneously to the best structural preservation of the bone extracellular matrix. Therefore, we performed quantitative analysis of the number of remaining cells and descriptive analysis of the scaffolds, made possible by microscopy. In the second stage, a study was conducted to evaluate the in vitro adhesion of mice bone marrow mesenchymal cells, cultured on these scaffolds, previously decellularized. Through manual counting of cells on scaffolds there was a complete cell removal in Group II, Group I showed a practically complete cell removal, and Group III displayed cell remains. The findings allowed us to observe a significant difference only between Groups II and III (p = 0.042). Better maintenance of the collagen structure was obtained with Triton X-100, whereas the decellularization with Trypsin was responsible for the major structural changes in the scaffolds. After culture, the adhesion of mesenchymal cells was only observed in specimens deccelularized with Trypsin. Due to the potential for total removal of cells and the ability to allow adherence of these, the protocol based on the use of Trypsin (Group II) was considered the most suitable for use in future experiments involving bone grafting decellularized scaffolds / A regenera??o de defeitos ?sseos com perda de subst?ncia permanece um desafio terap?utico na ?rea m?dica. ? consenso ser o osso aut?geno, o material mais adequado para esta finalidade, por?m h? limita??es at? para o seu uso, especialmente a quantidade insuficiente no pr?prio doador. Pesquisas de engenharia tecidual evidenciam que os componentes da matriz extracelular (MEC) s?o geralmente conservados entre as diferentes esp?cies sendo bem toleradas, mesmo em receptores xen?genos. Assim, diversos estudos t?m sido realizados na busca por um arcabou?o substituto do osso aut?geno atrav?s da t?cnica de descelulariza??o. Para a obten??o destes arcabou?os, os tecidos devem passar por um processo de remo??o celular, que cause m?nimos efeitos adversos na composi??o, atividade biol?gica e integridade mec?nica na matriz extracelular remanescente. Entretanto, h? controv?rsias acerca do melhor protocolo de descelulariza??o, j? que cada um desses tratamentos interfere de maneira diferente na composi??o bioqu?mica, ultraestrutura e comportamento mec?nico da matriz extracelular, afetando o tipo de resposta imunol?gica ao material. Ademais o baixo arsenal de pesquisas envolvendo a descelulariza??o de tecidos ?sseos representa mais um obst?culo ? chegada de um consenso protocolar. O presente estudo teve como objetivo avaliar a influ?ncia dos m?todos de descelulariza??o na produ??o de arcabou?os biol?gicos a partir de ?rg?os ?sseos de camundongos, visando sua utiliza??o para enxertia. Trata-se de um estudo laboratorial, sequenciado em duas etapas distintas. Na primeira fase foram avaliadas 12 hemi-calv?rias de camundongos, divididas em tr?s grupos (n=4) e submetidas a tr?s diferentes protocolos de descelulariza??o (SDS [Grupo I], Tripsina [Grupo II], Triton X-100 [Grupo III]). Buscou-se identificar aquele que promove a mais eficiente remo??o celular, simultaneamente a melhor preserva??o estrutural da MEC ?ssea. Para tanto, foi realizada an?lise quantitativa do n?mero de c?lulas remanescentes e an?lise descritiva dos arcabou?os, possibilitadas por microscopia. Na segunda etapa, foi realizado um estudo in vitro para avaliar a ades?o de c?lulas mesenquimais da medula ?ssea de camundongos, cultivadas sobre arcabou?os previamente descelularizados. Atrav?s da contagem manual de c?lulas nos arcabou?os, verificou-se total remo??o celular no Grupo II, remo??o praticamente completa no Grupo I, e perman?ncia de c?lulas e remanescentes no Grupo III. Os achados permitiram observar diferen?a significativa apenas entre os Grupos II e III (p=0,042). Melhor manuten??o da estrutura col?gena foi obtida com o Triton X-100, ao passo que a descelulariza??o com Tripsina foi respons?vel pelas maiores altera??es estruturais nos arcabou?os. Ap?s o cultivo, a ades?o de c?lulas mesenquimais s? foi observada nas calv?rias descelularizadas com Tripsina. Devido ao potencial de remo??o total das c?lulas e ? capacidade de permitir a ades?o destas, o protocolo baseado no uso da Tripsina (Grupo II) foi considerado o mais adequado para uso em experimentos futuros, que envolvam enxertia de arcabou?os ?sseos descelularizados

A odontologia moderna, mesmo usando as suas técnicas mais primorosas, na prática, ainda recupera a perda dental com implantes metálicos recobertos por coroas protéticas. Há um empenho coletivo dos cientistas em criar técnicas de desenvolvimento dental in vitro na busca por maneiras de recuperar, de maneira biológica, a ausência dental. Já é possível criar estruturas similares a dentes a partir de células-tronco de origem dental (polpa de dentes permanentes e decíduos) e não dental (células-tronco embrionárias, células-tronco da medula óssea e da crista neural) por meio de recombinação dos tecidos epiteliais e mesenquimais de germes dentais. As técnicas de reconstrução tecidual nunca estiveram tão perto do desenvolvimento da terceira dentição mas a ciência ainda tem muito a aprender no que concerne o estudo da biologia dental e engenharia de tecidos. Não basta saber como um dente se desenvolve; há de se entender como ele interage com o organismo do qual faz ou fará parte. É com esta preocupação que nos propomos a estudar se pode haver uma correlação entre o desenvolvimento do sistema nervoso e vascular de um germe dental transplantado com a posição que ele é implantado e/ou com a presença de tecido ósseo que no leito receptor. Ademais, buscamos saber se o estágio de desenvolvimento do germe dental a ser transplantado pode influenciar a formação de tecido nervoso e vascular na polpa dental ou não. Nossos resultados revelaram que o local do sítio do implante influencia diretamente o desenvolvimento dental e que isto é tempo dependente. A vascularização e a reinervação da polpa dental nos espécimes implantados nas tíbias é mais semelhante ao grupo controle que os implantados nos rins e isto independe da posição de implantação dental. Entretanto, a polpa dental dos germes implantados nos rins parece estar comumente mais sadia, conter mais odontoblastos viáveis e ser capaz de produzir tecidos mineralizados como a osteodentina. / Contemporary dentistry, even using modern techniques, still deal with missing teeth using metal implants coated by prosthetic crowns. However, there is a worldwide effort to develop a biotooth using in vitro techniques. In this way it is already possible to generate structures similar to teeth using recombination of odontogenic and non odontogenic cells in tissue engineering experiments. The transplant of the recombined cells into a host is a necessary and major step to obtain the biotooth. In this context, at the same time that the development of an appropriate sensorial and vascular system in the biotooh is required, there are many unclear questions about it. Therefore, herein we intend to analyze (I) whether may exist a correlation between the stage of development and vascular and nervous re-growth in the dental pulp after tooth transplantation; (II) if the absence or presence of bone could influence this processes or (III) if the position of implantation could change the vascular and/or nervous development in the transplanted tooth. Our results showed that the site of implantation directly alter tooth development modifying morphogenesis and expression of different vascular, perivascular and neural markers in a time dependant way. The re-growth of the vascular and neural tissue on samples transplanted to the tibia is more similar to the control group than the kidney ones and it is non dependant of the position of implantation. However, the pulp tissue of the samples transplanted under the kidney capsule seemed to be healthier as they were capable of producing mineralized tissue such as osteodentin and still had live odontoblasts.