Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: factor; FGF-23; phosphatonin; microarray; expression; phosphate; time; gene; RT-PCR; growth; fibroblast. Includes bibliographical references (p. 127-136).

It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the DED in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.

Objetivo: Avaliar por meio de análises radiográfica e histológica os efeitos da associação do laser de diodo e do fator de crescimento de fibroblastos no tratamento de dentes de ratos reimplantados tardiamente. Método: Cinquenta ratos (Rattus norvegicus albinus, Wistar) tiveram seus incisivos superiores direitos extraídos, a papila dental removida, e por via retrógrada, o preparo e preenchimento dos canais radiculares com pasta de hidróxido de cálcio. Em seguida, os espécimes foram aleatoriamente divididos em cinco grupos: C+ (controle positivo)- reimplante imediato; C- (controle negativo)- reimplante tardio, mantidos em meio ambiente seco por sessenta minutos, sem tratamento adicional; LA (laser de alta potência)-reimplante tardio associado ao tratamento prévio da superfície radicular com irradiação por varredura com laser diodo de alta potência (810nm, modo contínuo, fibra óptica de 600&#956;m, 1,5W input, 30s); FGF (Fator de crescimento de fibroblastos)- reimplante tardio associado à aplicação do fator de crescimento de fibroblastos 50 &#956;g na concentração de 0,2% FGF-2 em gel de hidroxipropilmetilcelulose (HPMC) a 3% sobre a superfície radicular lingual e no interior do alvéolo dental de cada espécime; LA+FGF (Laser de alta potência e FGF)- reimplante tardio, com tratamento prévio da superfície radicular com laser diodo nos mesmos parâmetros do grupo LA, associado à aplicação do FGF-2 da mesma forma que no grupo FGF. A eutanásia dos animais ocorreu após sessenta dias. Os espécimes foram radiografados digitalmente e processados para análise descritiva dos eventos histológicos e análise da porcentagem de fibras colágenas do tipo I por meio do método Picrosirius Red. Resultados: Na análise radiográfica, o grupo LA+FGF apresentou o menor número e porcentagem de áreas reabsorvidas (p<0,05). Com a análise dos eventos histológicos por meio de escores, o grupo LA apresentou as menores médias de reabsorção substitutiva e inflamatória e anquilose, seguido pelo grupo LA+FGF e não diferindo estatisticamente do grupo controle positivo. O grupo LA+FGF apresentou o melhor reparo periodontal entre os grupos experimentais, com a maior quantidade de fibras colágenas espessas, (p<0,05). Conclusões: A irradiação da superfície radicular com laser de diodo de alta potência associada ou não ao FGF-2 reduziu a incidência de reabsorções radiculares externas e anquilose. A aplicação do FGF-2 favoreceu o reparo do ligamento periodontal, embora a reinserção das fibras não tenha ocorrido em todos os espécimes. / Aim: The aim of this study was to evaluate, by radiographic and histological analyses, the effects of high-power diode laser irradiation and fibroblast growth factor (FGF) in the treatment of delayed replanted rat teeth. Methods: Fifty rats ratos (Rattus norvegicus albinus, Wistar) had their maxillary right incisors extracted, the dental papilla removed, and by a retrograde way, the root canals were prepared and filled with calcium hydroxide paste. Subsequently, the specimens were randomly assigned to five groups: C+ (positive control)- immediate replantation; C- (negative control)- delayed replantation, left on the bench for sixty minutes, without additional treatment; LA (Diode laser high potency)- delayed replantation and root surface treatment scanning irradiation with a high-power diode laser (810nm, continuous mode, optic fiber 600&#956;m, 1.5W input, 30s); FGF (Fibroblast Growth Factor) - delayed replantation and topical application of 50&#956;g 0,2% FGF-2 gel in 3% hydroxylpropyl methylcellulose matrix (HPMC) over the lingual root surface and into dental alveolus of each specimen; LA+FGF (Diode laser high potency + Fibroblast Growth Factor)- delayed replantation and root surface treatment irradiation with a diode laser using the same parameters as those used for LA group, associated to topical application of FGF-2 in the same way as FGF group. The rats were euthanized after sixty days of replantation. The specimens were digitally radiographed and processed for descriptive histological events analysis and percentage analysis of type I collagen fibers by the Picrosírius red method. Results: By radiographic analysis, the minnor number and percentage of resorpted areas were seen in LA+FGF group (p<0.05). In the histological analysis through scores, the LA group showed lower means of replacement and inflammatory resorption and ankylosis and followed by LA+FGF group and they were not statistically different of positive control group. The LA+FGF group showed the best periodontal healing of all experimental groups, with the greater number of thick collagen fibers stained (p<0,05). Conclusions: The irradiation of root surface treatment with high-power diode laser associated or not to FGF-2 reduced the occurrence of external root resorption and ankylosis. The FGF-2 application favored the periodontal ligament healing, but the fibers reinsertion not occurred in all the specimens.