Universidade Federal de Santa Catarina-UFSCDisciplina: BiomateriaisProfessores: Prof. Dr. – Ing. Márcio Celso Fredel Prof. Dra. Ana Paula Marzagão Casadei

Introdução Com o aumento do uso de materais artificiais

em medicina, compreender qual estímulo promove a reação celular desejada no tecido adjacente para melhorar a performance do implante, ainda é um desafio.

Dois aspectos importantes, interferem com a resposta celular:A química de superfícieA topografia da superfície

A reação celular frente à topografia da superfície Dirigir a migração celular é essencial para o

desenvolvimento do tecido. O movimento da célula de uma parte para outra deve estar sob controle.

A interação célula-célula tem um papel chave no controle da migação.

A possibilidade da química da matriz extra celular ou de gradientes quimiotáticos inteferirem no proceso, devem ser considerados.

Microfabricação e a definição de ceracterísticas de supefície Utilizando-se métodos aproveitados da tecnologia

de fabricação microeletrônica, é possível construir superfícies com estrutura perfeitamente definida, permitindo estudar o comportamento celular frente à diferentes superfícies.

Materiais sintéticos como silício, vidros e metais como titânio e suas ligas, têm permitido construir superfícies com características topográficas precisas, como cristas e sulcos.

Dependendo do espaço, as células podem se acumular sobre as cristas, extendendo-se ao longo delas, em um fenômeno chamado “ridges walking”.

Processo de fotolitografia

Fig. 1 Schematic representation of the photolithography process. The target surface (1) is cleaned and resist applied to the target material (2). A photomask is then created and developed onto the substrate (3), through the mask (4). The substrate is then etched (5), and the resist is removed using acetone (6), leaving the desired topography etched onto the substrate.

Tipos de topografia de superfície Fibras Sulcos repetidos Covas ou buracos Túneis ou tubos Substratos descontínuos Superfícies rugosas

Fibras Podem ser produzidas usando-se materiais

como titânio, PGA, PLA, Polipropileno, Quitosana, vidros, colágeno, etc.

Avanços na fabricação de fibras permite:Criar fibras de comprimento e diâmetros

controlados, formar malhas,Criar estruturas porosas sem perder as

propriedades mecânicas do material capazes de promover a migração e o crescimento celular

Túneis e tubos Vários tipos de células vivem ou formam

estruturas tubulares.

É necessário estudar o comportamento celular em superfícies concavas e convexas.

Substratos descontínuos Tradicionalmente tem-se utilizado a estrutura

de bordas contínuas para guiar a migração celular.

Atualmente tem-se estudado o desenvolvimento de superfícies com bordas descontínuas.

Investigações utilizando-se fibroblastos e células epiteliais têm demonstrado que a descontinuidade entre bordas, parece favorecer a migração e o crescimento celular.

Superfícies rugosas São definidas como sendo de topografia

produzidas aleatoriamente, a fim de aumentar a área de contato entre o tecido e o implante.

Medida por um índice de rugosidade – Ra.

TécnicasJateamentoPlasma spraying de titânioAtaque ácido (Ác. Nitrico e ac. Fluorídrio)

Nanotopografia e microtopografia Caracterizada pela presença de pilares

ou buracos em escala semelhante ao tamanho das moléculas como colágeno e outras matrizes extra celulares.

O objetivo é um bom ancorameto mecânico entre o tecido e o implante.

Representative images of surface topographies

(A) Epithelial cells growing on fibers, (B) actin staining of mesenchymal

stem cells growing on repeating grooves,

(C) P388D1 macrophages growing in cell traps,

(D), rat calvarial osteoblasts growing in ‘‘pit’’-type topographies,

(E) fibroblasts growing on a convex topography,

(F) ovine chondrocytes cultured in 240 mm-diameter quartz tube,

(G) microtubule staining of human gingival fibroblasts growing on a discontinuous edged topography,

(H) human gingival fibroblasts cultured on SLA roughened titanium.

Cell Types Sensitive to Topography

Comportamento celular e a topografia da superfície Adesão Alinhamento e orientação Guia por contato Guia por gap

Adesão Via de regra, a topografia da superfície

influencia a adesão celular, porém a natureza e a extensão dessa influência depende do tipo de célula envolvido e das dimensões da topografia.

Superfícies rugosas favorecem a adesão de algumas células, como osteoblastos e dificultam a adesão de outras, como fibroblastos, que preferem superfícies lisas.

As diferentes respostas celulares, permitem usar diferentes superfícies para selecionar o tipo de célula que se deseja favorecer em determinados locais.

Alinhamento e orientação No interior de alguns tecidos, a arquitetura e o

arranjo celular é um fator importante na determinação da função.

Alguns tecidos contam com a localização específica da célula para produção da MEC, e em alguns casos as células obedecem um padrão de alinhamento e orientação espacial, como em tendões, ligamentos e músculos.

Fibras e substratos com sulcos repetidos têm mostrado bons resultados no alinhamento de células e tecidos.

As células também variam em relação ao tempo de resposta frente ao substrato. Em sulcos, macrófagos respondem em 5 minutos, fibroblastos em 2 horas e condrócitos em mais de 6 horas.

Guia por contato Refere-se à tendência da célula de ser

guiada na sua locomoção por superfícies contínuas, como bordas físicas, sulcos, fibras ou outro tipo de estrutura topográfica.

Algumas células mostram um padrão de migração por contato com a superfície do substrato, enquanto outras como condócitos não mostram padrão de alinhamento paralelo ao eixo de sulcos.

Gap guidance Pesquias tem sido feitas para identificar

as possíveis respostas celulares frente a substratos com superfícies descontínuas.

Algumas células como fibroblastos gengivais e células epiteliais de ligamento periodontal tem apresentado respostas favoráveis frente a superfícies descontínuas.

Expressão genética de proteínas Enquanto o comportamento celular

frente a diferentes topografias de superfície tem sido bem estudados, as alterações na fisiologia celular em nível molecular permanecem obscuras.

Decifrar a expressão genética frente a diferentes superfícies, requer a seleção e análise de diferentes genes.

Introduction With the increased use of artificial

materials in medicine, understanding which stimuli promote desirable reactions from cells in surrounding tissues has become critical to improve implant performance.

two key aspects have been identified that influence cellular response, the chemistry the topography of the material.

SIGNIFICANCE OFTOPOGRAPHICAL REACTION Directed cell migration is essential during

development, where the movement of cells from one part to another must be under precise control.

cell–cell interactions and topography have been identified as playing key roles in this patterned migration.

The possibility that extracellular matrix chemistry or chemotactic gradients could be influential, can not be excluded.

IN VIVO APPLICATIONS OFCELL—TOPOGRAPHY INTERACTIONS

Fibers Since this time, fibers have been produced using materials

such as titanium, poly-glycolic acid, poly-lactic acid, polypropylene, glass, chitosan, ceramics, as well as natural materials such as fibronectin and collagen.

The advantages of fiber-based systems

be able to extrude the fibers to any length required as well as being able to knit the fibers into meshes.

the ability to create a porous structure without any loss of mechanical strength of the material, thus producing an implant that is able to withstand physiological biomechanical stimuli as well as promote cellular ingrowth.

Tunnels/Tubes

Several cell types within the body, such as endothelial cells in blood vessels, live within tube-like structures.

There has, however, been only scant attention paid to the response of cells to concave surfaces, in contrast to numerous reports of cell response to convex surfaces.

Discontinuous Substratum Traditionally, the theory of contact guidance centers

on the presence of a continuous edge, guiding a cell in a specified direction.

The design of the discontinuous edged topographies (DES) is such that essentially the topographies comprised repeated open square boxes, with gaps at each corner.

Investigations using fibroblasts and epithelial cells have shown that DES topographies have great potential to be used not only in vitro to investigate cell guidance, but also in vivo to organize tissue around percutaneous implants.

Roughened Surfaces Surface roughness was defined as a randomly produced topography.The rationale

for using rough surfaces is that the contact area between the tissue and the implant surface s increased, thus facilitating interaction between the tissues and the device.

The most widely accepted characterization is the average vertical amplitude or RA value, which is calculated from an average of the feature sizes. In general, the higher the RA value,the rougher is the surface.

The simplest way of producing a rough surface is to rub the material with abrasive particles.

Other techniques to produce rough surfaces include titanium plasma spraying, which involves the coating of melted droplets of titanium onto the material surface, which then cool to form protruding structures (RA value of ~5.85 mm).

Acid etching, commonly accomplished using an aqueous mixture of nitric acid and hydrofluoric acid at a ratio of 10 : 1, results in a surface that has an RA value of ~0.59 mm.

TYPES OF SURFACE TOPOGRAPHY

Microfabrication and Defined Surface Features.

With the advent of the use of techniques harnessed from the microelectronic industry, it has become possible to produce structures with precisely defined surfaces in cell biological studies, thus allowing the reaction of cells to defined topographic features to be observed.

Synthetic materials such as silicon and glass, cellulose acetate, epoxy, araldite, polystyrene and metals such as titanium and titanium alloys have all been employed to produce precisely characterize topographies.

Using such methods of fabrication, fibers of glass can be drawn out to precise dimensions, and structures such as ridges and grooves can be made using photolithography and etching techniques in silicon.

Depending on spacing, cells responded by accumulating on the ridges and extending along them. In some instances, the cells were guided in their migration by the ridges, a phenomenon termed “ridge walking”.

Microfabrication and Defined Surface Features

Nanotopography Vs. Microtopography One area on which many research laboratories are currently

focused is the use of nanometric sized surface features, such as pillars and holes.

The logic of using such small features is that they are in the same size range as molecules such as collagen and other extracellular matrix molecules that cells will encounter within the body.

One desired reaction at the implant surface is good mechanical interlocking, especially in bone contacting regions, because a major cause of implant failure is micromotion and friction at the tissue–implant interface.

CELL BEHAVIOR INFLUENCED BYSUBSTRATUM TOPOGRAPHY

Adhesion It has been long known that roughe surfaces generally promote

adhesion of some cell types such as osteoblasts, whereas cells such as fibroblasts prefer smooth surfaces.

The general rule to follow is that surface topography influences cell adhesion, although the nature and extent of the influences depends on the cell type involved, as well as the dimensions of the substratum topography.

The differential response of cell types to topography opens the possibility of using surfaces to select certain populations of cells based on their adhesive properties.

CELL BEHAVIOR INFLUENCED BYSUBSTRATUM TOPOGRAPHY Alignment and Orientation

Within many tissues, the architecture or spatial arrangement is an important determinant of function.

Many tissues such as bone, ligaments, tendons, and muscle rely on the specific position of the cells with regard to the surrounding matrix, and in many cases the cells have a determined alignment and orientation within the tissues.

Fibers, and repeating grooved substrates, in particular, have been shown to have a pronounced effect on cell and tissue alignment.

Different cell types also vary in their reaction time to grooved substrates, with macrophages responding within 5 min, fibroblasts within 2 hr, and chondrocytes up to 6 hr.

Contact Guidance Contact, or topographic guidance refers to the tendency of

cells to be guided in their direction of locomotion by a continuous, physical edge, whether this is a groove, fiber, or other type of topographical structure.

Many different cell types have been observed to show contact guidance, although cells such as chondrocytes have been observed to show guided migration without aligning parallel to the groove long axis.

Focal adhesions, have been put forward as drivers of contact guidance.

Gap Guidance It has been investigated the response of cells

to discontinuous topographies, with an emphasis on understanding the conditions for the oriented guidance of cells.

Results with gingival fibroblasts and periodontal ligament epithelial cells have shown that a continuous edge may not be a prerequisite to guiding a cell in a specific direction.

Analysis of cytoskeletal components suggests that a primary driver of gap guidance is focal adhesion stability.

Contact Guidance: Gene andProtein Expression Although the general behavior of cells on

topographical cues has been well described, the alterations in cels physiology at the molecular level remain elusive.

Initial attempts to decipher altered gene expression on topographical cues required gene selection and analysis.