RICERCA ITALIANA     

EXTRACELLULAR MATRIX: REGULATORY PHENOMENA, INTERMOLECULAR INTERACTIONS AND FUNCTIONAL MODIFICATIONS

Università degli Studi di Bologna

Abstract

The extracellular matrix (ECM) is a substance underlying all epithelia and endothelia and surrounding all the cells of connective tissues, thus giving tissues and organs mechanical support and physical resistance. In addition to its merely mechanical functions, the ECM strongly influences cell behaviour (adhesion, diffusion, migration) and the patterns of gene expression in adjacent cells.
The ECM comprises structural molecules such as fibrillar and non fibrillar collagens, proteoglycans (PGs) and, in elastic connective tissues, elastin fibers. Changes in the composition of these elements and their interactions give rise to different tissues with a different functional role and response.
The goal of this project is to analyze "in vitro" the interactions that small leucine- rich proteoglycans (SLRPs) have with fibrillar collagens and with syntetic peptides or obtained from specific fragmentation ( CNBr peptides); to analyze the behavior and the modifications received "in vivo" from ECM when it is found to be in functional conditions of particular clinical interest, such as muscle-tendon stretching; to analyze the interaction of titanium implants with host bone.
We expect to find modifications of the ECM under functional stimuli and therefore to evaluate the possible consequences at a physiological and functional level.
Unit 3 aims to examine the type of binding that SLRPs such as decorin (DCN), fibromodulin (FM) and biglycan (BGN) can contract with fibrillar collagens (I,II,III,V), with peptides deriving from collagen specific defragmentation (CNBr peptides) and synthetic peptides. These observations will influence the analysis of SRLPs extracted from tendons subjected, or not, to mechanical forces (stretching).
Unit 2 aims to reproduce "in vivo" (rats) clinical conditions specific to the practice of stretching, in order to look for chemical and structural modifications of tendon fibrillar collagen and DCN connected fibrils. The research is based on the microscopical analysis of the collagen fibrils (crimps, diameter, banding), on the possible neo-fibrillogenesis (labelled prolyn analysis) and on the study of SLRPs extracted from tendons after stretching.
It also aims to examine with LM, TEM, SEM the "in vivo" role that ECM has in the adhesion of implant surfaces to host bone (sheep). The response of the host bone to implants with different surfaces (smooth and rough) is also examined "in vitro" by LM, SEM, and TEM analysis. Adhesion, proliferation, ALP-ase activity, type I collagen, osteocalcin and osteoprotegerin production are also analyzed on osteoblasts, stem cells and endotelial cells plated on titanium disks with various surfaces.
Unit 1 is involved in the study of proteoglican-collagen interactions in stretched tendons using ultrastructural morphological techniques (Tapping mode Atomic Force Microscopy and High resolution SEM) that allow single molecules observation and recognition of reaction products of matrix macromolecules. <<<

Principal Investigator

Alessandro RUGGERI Università degli Studi di BOLOGNA

Research Objectives

Among many functions of the extracellular matrix (ECM) the role of physical support and resistance for tissues and organs has been widely studied by the project research units. In particular, we have focused on the structural and functional aspects of two ECM components: collagen fibrils and proteoglycans. Recent analysis of these components has moved to an in-depth study of their composition and reciprocal interactions under physiological conditions and to their possible adaptations when they are influenced by functional conditions different from the phisyological ones.
The project's objective is to investigate the limitations beyond which ECM components subjected to external stimuli undergo qualitative and quantitative changes and to analyze which of these components changes are compatible with ongoing physiological organization of the ECM.
The project will address possible changes in the constitution and structure of collagen fibrils in fibrous tissues (tendons) subjected to functional stimuli bordering on the limit of physiological conditions; the influence of metal surfaces of varying geometry and micromorphology on the osteogenic activity of bone tissue located at the interface; the influence of interactions of small proteoglycans belonging to fibrous tissues with fibrillar collagens when these collagens undergo treatments affecting their size or composition (using peptides from natural or synthetic sources) and chemical features (following derivatizations undertaken under mild conditions).

The analysis of tendon functional responses will be undertaken by stretching the tendon muscle complex of experimental animals (rats). Possible qualitative and quantitative changes will be analyzed in fibrilar collagens and proteoglycans under routine physiological conditions and after stretching for short and long periods.
Making reference to literature reports on changes in the fibrils and proteoglycans of tendons subjected to prolonged physical exercise and starting from the observation that prolonged stretching lengthens the muscle-tendon unit, it is envisaged that stretching also entails structural modifications. This analysis aims to disclose possible negative effects of uncontrolled stretching during training.
The analysis of metal surfaces with different potential effects on osteogenesis aims to enhance osteointegration at the bone-implant interface, and therefore to shorten clinical intervention times with an improvement in the cost-benefit ratio. Also on the basis of previous personal experience, rough implant surfaces have a positive effect on osteointegration and among these sand-blasting roughened surfaces yield the best results.
The current project aims to disclose the mechanisms underlying this enhanced osteointegration process by analyzing both the "in vitro" response of cells involved in the osteogenic line (osteoblasts, stem cells and endothelial cells) and possible changes in the ECM fostering cell activation. "In vitro" tests will be flanked by "in vivo" experiments (sheep) in which morphological and immunohistochemical findings will shed light on the influence exerted by the geometry and micromorphology of the implant surface.
The analysis of interactions between fibrillar collagens and two small matrix proteoglycans (decorin and fibromodulin) will be based on previous research undertaken by Unit 3. The tested approach entails changing the molecular size of two fibrillar collagens - type I and type II - by specific chemical cleavage yielding natural sequence peptides whose features have already been determined. In addition, treatments will be done to change the chemical characteristics of the collagens and their peptides by chemical modifications under mild conditions. This procedure will produce derivatives which have already been well characterised. These two approaches will detail some of the aspects of the interactions between the small proteoglycans and collagens, as described in form B submitted by Unit 3.
Using these methods, the main objectives of the current project are to extend studies on biglycan, another small matrix proteoglycan structurally and functionally correlated to the two already studied, and to involve all three small proteoglycans in binding studies with specially designed collagen sequence synthetic peptides to add more detail to our knowledge of the collagen portions binding the small proteoglycans studied and their biological and functional features.
The project also aims to conclude the study of the interactions of small proteoglycans with fibrillar collagens studying tendons under physiological conditions and tendons subjected to stretching.
The study will be extended to the tendon sheaths, with share with tendons the same anatomical site but have to withstand different functional loads, and it will take advantage of the techniques and the experience the Unit 1 has gained in the 3D visualization of macromolecular complexes. The comparison among tissues respectively subjected to extreme, high and no functional load should allow us to develop a three-dimensional, structural model of the intermolecular relationships that determine the biomechanical specifications of a given tissue. This also includes such structural entities as the collagen VI microfibrils, whose presence has been assesses but whose functional significance is still open to speculations. <<<

First Results

By a study of the interactions between fibrillar collagens and SLRPs, we expect to characterize the relationship between analysed SLRPs (DCN, FM, BGN) and fibrillar collagens and their derivatives. The final purpose is to objectify and justify the variations of the interaction among these components in different metabolic and/or functional conditions.
By a morpho-functional study of the muscle-tendon unit subjected tostretching we expect to identify after a long-term treatment qualitative and quantitative variations of the ECM's components such as collagens and proteoglycans. In fact it is demonstrated that prolonged stretching leads to elongation of the muscle-tendon unit and it is likely that a qualitative as well as quantitative renewal of the components themselves would take place.
By a study of the relationship between bone matrix and implant surfaces with different roughness, we expect to find qualitative modifications of the ECM at the bone/implant interface.
These modifications may be detected "in vitro" by the analysis of matrix proteins selective adsorption and by the different behavior that osteoblasts, mesenchymal and endothelial cells may assume for their relationship with the surface. <<<

Timescale

24 months

National and international background

The extracellular matrix (ECM) is a substance underlying any epithelium and endothelium and surrounding all connective tissue cells, thus giving tissues and organs mechanical support and physical resistance. In addition to its merely mechanical functions, the ECM strongly influences cell behaviour (adherence, diffusion, migration) and the patterns of gene expression in adjacent cells [1].
The ECM comprises structural molecules such as collagen fibrils, proteoglycans (PGs) and, in elastic connective tissues, elastin fibers. These constituents are associated with equally important glycoproteins and other protein molecules and form a three-dimensional network enveloping the cells and influencing their metabolic activity.
Changes in the composition of these elements and their interactions give rise to different tissues with a different functional role. Likewise, a complex system of cell to cell, cell to matrix and matrix to matrix interactions, as well as the production of enzymes, enzyme activators/inhibitors, growth factors, etc. underlies ECM remodelling.
Fibrillar collagens in particular (types I, II, III, V and XI) and proteoglycans are ubiquitous components and are present in all connective tissues. The structural and functional role of fibrillar collagens has been the subject of extensive research and the Laboratory of Electron Microscopy of the Institute of Human Anatomy in Bologna, in particular, has a long track of direct involvement in morphological and ultrastructural research on the structural components of the ECM. It has dealt with the cross-banding and the microfibrillar arrangement of genetically distinct collagens [2-9], the histochemical and ultrastructural features of PGs and the mutual interactions between collagen and PGs [10-16] as well as the structure and the spatial layout of elastic fibres [17-19].
There is now a substantial consensus on the fact that collagen types II and XI coform heteropolymeric fibrils typical of cartilages, while types I, III and V also coform mixed fibrils, distinct from the former ones and diffuse in the remaining connective tissues. In skeletal tissues, such as bone and tendon, fibrils are mostly made of type I, show a multi-modal diameter distribution and are tightly packed in thick bundles. By contrast, in the connectival stroma of parenchymatous organs, but also in nerve sheaths, in skin, in the cornea and in the blood vessels, collagen fibrils contain greater amounts of types III and V and form an homogeneous population of slender fibrils, gathered in flexuous bundles with a strikingly unimodal diameter distribution [20]. Their inner structure differs too, as has been repeatedly confirmed by freeze-etching [21], cross-linking analysis [22], atomic force microscopy [23,24] and electron microtomography [25].
Proteoglycans - in particular the small, leucine-rich proteoglycans (SLRPs [26,27]) - are usually entirely bound to the collagen fibril surface by means of their protein core and project their side chains in the interfibrillar space, forming a three-dimensional lattice extended across the whole ECM.
Several studies have indicated that different SLRPs bind different locations on the collagen fibril [28-30]. It has been shown that various molecular fragments of type I and II collagens interact with decorin; this interaction is abolished by N-acetylation of Lys/Hyl residues but is modulated by N-methylation of Lys/Hyl, thus indicating that these residues are directly involved in the interaction [31].
The interaction of the core protein with the collagen fibril surface is known to be one of the factors which limit the lateral growth during fibrillogenesis, and is now established (both in the clinical practice and in knock-out animal models) that any alteration of this fraction has evident effects on the three-dimensional layout of the ECM. The proteoglycan fraction, moreover, changes with time in a distinctive pattern, in both quality and quantity, during histogenetic processes. It has been also shown that decorin binds TGF-beta [32] and cell receptors, such as a low-affinity protein-protein interaction between decorin and ECF receptors.
The molecular relations among the glycosaminoglycan part of PGs are generally less known. At variance with the prevailing model [28] other, multiple interaction patterns have been described [33-35]. Although these interactions are only partially understood, they appear to have a paramount importance in the functional behaviour of connective tissues [35,36].
Among the many cases of dynamic modification of the matrix or of the ECM reaction to different functional or biological stimulations, the behaviour of tendons and ligaments subjected to prolonged tension or stretching bears a particular interest. It has been shown that stretching endows some components of the ECM such as the PGs with a greater ability to transmit tensile stresses in tendons and/or withstand the applied load [37]. PGs therefore represent also an elastic, deformable structure able to redistribute the functional load among adjoining fibrils [38]. The succession of qualitatively and quantitatively different proteoglycans occurring in tissue maturation suggests that a similar mechanism may also be at work in the remodelling processes brought about by variations in mechanical load, and that have so far been paid scarce attention.
Another example of structural and functional adaptation of the ECM in response to external stimuli is represented, in dentistry and in orthopaedics, by the different interactions of bone with metallic surfaces (usually titanium), which may differ in geometry and micro morphology.
Most recent research was intended to assess how the matrix proteins interact with the implant surface either by inhibiting or promoting the response of the cells involved in neo-osteogenetic processes [39]. In vivo it has been shown that the implant roughness positively influences the osteointegration process, as evidenced morphologically with a progressive reduction of the residual gap at the bone-implant interface in early time. This process takes place through a gradual substitution of the initial blood clot in the granulation tissue with osteoid tissue, that successively turns into primary bone [40,41]. Previous studies of our group have shown that implants with different roughness (macro- and micro-roughness) induce a different response in the cells that interact with them [42].
As a consequence of their clinical relevance, endosseous implants as a whole have been the subject of several studies. Most of the effort has been however, directed towards the chemical aspects of the bone-implant interaction, and toward the assessment of such parameters as osteoconductivity, integrability, biocompatibility or, on the contrary, the cytotoxicity or the long term physical-chemical stability. Far lesser attention has been paid to the mechanical aspect of the bone-implant interface, even if all integration and degradation processes obviously occur only on the surfaces of mutual contact. <<<