Self-assembled Collagen Fibrils Research Articles

Zero dimensional Graphene Quantum Dots self-assembled collagen 3D bio-matrices for soft tissue regeneration

The current study focuses on self-assembly of collagen fibrils with Graphene Quantum Dots (GQD) and its tissue regenerative applications. GQDs were synthesised by the liquid exfoliation method using ethyl acetoacetate (EAA) as the solvent. The photoluminescence property of the GQD was established by the UV-Vis absorption spectrum. Characteristic increase in thermal stability, enhanced tensile strength was found after the self-assembly of collagen with GQD. The GQDs self-assembled collagen bio-matrices showed 3D surface topology mimicking the extracellular matrix. The GQDs self-assembled collagen bio-matrices did not show toxicity in the structural cells namely fibroblast cells and exhibited increased biocompatibility as indicated by enhanced cell adhesion, proliferation and cell migration of fibroblast cells. Angio-proliferative effect as indicated by an enhanced blood vessel formation and intact unruptured blood vessels was observed in the GQD self-assembled collagen. The study opens a way forward for the use of zero-dimensional graphene based 3-dimensional self-assembled collagen biomatrices for soft tissue regenerative applications.

Self-assembly of collagen fibrils on graphene oxide and their hybrid nanocomposite films

In order to explore the distribution, conformation and interaction of collagen on GO nanosheet surfaces, the mechanism of self-assembly of collagen was investigated in the presence of GO nanosheets. Atomic force microscopy (AFM) was employed to observe the conformation of self-assembled collagen fibrils on the GO nanosheets surfaces. The collagen concentration and incubation time mainly affect the size of the collagen fibrils while the pH of the dispersion determines the self-assembly sites of collagen fibrils on the GO nanosheets surfaces. This pH-dependent adsorption is attributed to the interfacial interactions between the tunable ionization of the collagen molecules and the amphiphilic GO nanosheets. Vacuum-assisted self-assembly technology confirmed that GO nanosheets can direct the self-assembly of collagen molecules and form nacre-like nanocomposites. The GO/collagen nanocomposite films combine the remarkable properties of GO nanosheets and collagen to form functional nanocomposites with well-ordered hierarchical structures. Further, strong interfacial interactions between GO nanosheets with collagen fibrils result in enhanced mechanical properties and biocompatibility of nanocomposite films, which is conducive to enhance the neuronal differentiation of SH-SY5Y cells. Overall, this work provides fresh insight into the interactions between GO and collagen, which is essential for the design and manufacture of bioinspired nanocomposites with tailored mechanical properties.

Mechanically-reinforced and highly adhesive decellularized tissue-derived hydrogel for efficient tissue repair

Hydrogels derived from decellularized tissue (DT) have demonstrated huge potential in regenerative medicine. However, the hydrogel resulting from a thermally-induced crosslinking process via the self-assembly of collagen fibrils in DT exhibits weak mechanical properties. This restricts its application in tissue regeneration that requires strong mechanical properties and structural integrity of the hydrogel constructs. To overcome the aforementioned challenges of the present DT-derived hydrogels, we developed a DT hydrogel equipped with oxidative crosslinking chemistry by conjugating catechol moieties to the extracellular matrix in DT. The catechol-modified DT (DT-CA) constructed hydrogel instantly upon oxidation via catechol–catechol adducts, exhibiting 10 times stronger mechanical properties compared to that of the unmodified DT hydrogel. The oxidative crosslinking also increased the adhesiveness and physical integrity of the DT hydrogel, allowing for the shaping of scaffolds without using any supportive material, which could not be accomplished with conventional DT hydrogels. DT-CA hydrogel demonstrated an enhanced osteogenic differentiation of human stem cells and accelerated formation of new bones in a mouse model of the critical-sized calvarial defect. In addition, the patch type of DT-CA facilitated wound healing by mediating efficient topical delivery of growth factors. Similarly, other phenolic adhesive moiety (pyrogallol) could be introduced to improve DT hydrogel for promoting wound regeneration. Collectively, the results of this study support the applicability of mechanically-reinforced, adhesive DT hydrogel for effective tissue regeneration.

Dentin remineralization induced by nanobioactive glass in association with RGDS peptide

The aim of this study was to investigate the binding ability of a arginine-glycine-aspartate-serine (RGDS) peptide to dentin and its effect on the dentin remineralization, dentin cohesive strength and intrafibrillar mineralization induced by nanobioactive glass (nBG). The demineralized dentin discs were treated with RGDS and/or nBG and then incubated in stimulated body fluid for 2 weeks. The results of scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction and confocal laser scanning microscopy showed that RGDS could bind to dentin and promote the attachment of nBG. Compared to the nBG group, the RGDS + nBG group induced more apatite formation on the dentin surface and apatite rods inside the dentinal tubules. The results of transmission electron microscopy showed crystal lattice formation in the demineralized dentin matrix treated with nBG or RGDS + nBG, indicating the remineralization of dentin. The RGDS + nBG-treated dentin had the highest dentin cohesive strength in all groups. The self-assembled collagen fibrils exposed to RGDS + nBG exhibited intrafibrillar mineralization. In conclusion, RGDS promoted nBG-induced apatite formation and dentin collagen matrix remineralization, thus enhancing the dentin strength. The results lay a foundation for the wide application of RGDS and nBG in dentin erosion, dentin hypersensitivity, dentin bonding interface improvement, and regenerative dentistry.

Modeling Fibrillogenesis of Collagen-Mimetic Molecules

One of the most robust examples of self-assembly in living organisms is the formation of collagen architectures. Collagen type I molecules are a crucial component of the extracellular matrix, where they self-assemble into fibrils of well-defined axial striped patterns. This striped fibrillar pattern is preserved across the animal kingdom and is important for the determination of cell phenotype, cell adhesion, and tissue regulation and signaling. The understanding of the physical processes that determine such a robust morphology of self-assembled collagen fibrils is currently almost completely missing. Here, we develop a minimal coarse-grained computational model to identify the physical principles of the assembly of collagen-mimetic molecules. We find that screened electrostatic interactions can drive the formation of collagen-like filaments of well-defined striped morphologies. The fibril axial pattern is determined solely by the distribution of charges on the molecule and is robust to the changes in protein concentration, monomer rigidity, and environmental conditions. We show that the striped fibrillar pattern cannot be easily predicted from the interactions between two monomers but is an emergent result of multibody interactions. Our results can help address collagen remodeling in diseases and aging and guide the design of collagen scaffolds for biotechnological applications.

The Effect of Cholesterol on Membrane Binding and Self-Assembly of Collagen Fibrils.

Collagen is a skeleton of native extracellular matrix (ECM) that is known to provide mechanical and structural stability. In an attempt to develop a new connective cellular model with the surrounding ECM without further experimental complications, such as the reconstitution of ECM receptors, we designed the experiments and discovered that the fibrillogenesis of membrane-bound collagen is not spontaneous as it is in the form of free collagen in bulk solution. The confocal microscopic results suggest that cholesterol is a crucial component that facilitates the fibril formation on the membrane surface. In situ X-ray and neutron reflectivity on Langmuir monolayer and solid-supported lipid bilayer models, respectively, reveal two features of cholesterol effects on the collagen fibril formation. Mainly, cholesterol increases the lateral lipid headgroup separation on the membrane surface, which promotes the association degree of collagen monomers. It also enhances the elastic modulus of the membrane to impede membrane filtration by the collagen assemblies.

Self-assembled collagen fibrils from the swim bladder of Bester sturgeon enable alignment of MC3T3-E1 cells and enhance osteogenic differentiation.

Collagen is the most abundant protein in animals, and its polymer, collagen fibrils, regulate cellular proliferation, differentiation, and migration. Low antigenicity, biocompatibility, and biodegradability make collagen fibrils suitable functional scaffolds for tissue engineering. In a previous study, we found that the type I atelocollagen purified from the swim bladder of Bester sturgeon (swim bladder collagen, SBC) showed high fibril-forming ability, producing thicker fibrils faster than porcine collagen. In this study, we report a novel method to coat cell culture wells with highly aligned collagen fibrils using the SBC. Two types of fibrils with different thicknesses were prepared by changing the crosslinking treatment timing. The oriented, thick collagen fibrils induced pre-osteoblastic MC3T3-E1, pre-adipocytic 3T3-L1, pre-myocytic C2C12, and fibroblastic L929 cells to align in the same direction, whereas the oriented, fine fibrils made a cell network with their long pseudopods. Cellular proliferation was inhibited on both fibrils. Furthermore, both fibrils induced the early differentiation of MC3T3-E1 cells without differentiation stimuli. In contrast, the morphology of pre-chondrocytic ATDC5 cells on both fine and thick fibrils extended very short pseudopods and continued to maintain a spherical shape without stretching, suggesting a distinct effect by the fibrils. The newly developed fibril coatings are in the form of a thin film, thereby providing good visibility of the cell structure, including cell-cell and cell-ECM interactions, using a phase contrast microscope. The fibril coatings have high potential as a useful tool for tissue engineering research.

A Biomimetic Model for Mineralization of Type-I Collagen Fibrils.

The bone and dentin mainly consist of type-I collagen fibrils mineralized by hydroxyapatite (HAP) nanocrystals. In vitro biomimetic models based on self-assembled collagen fibrils have been widely used in studying the mineralization mechanism of type-I collagen. In this chapter, the protocol we used to build a biomimetic model for the mechanistic study of type-I collagen mineralization is described. Type-I collagen extracted from rat tail tendon or horse tendon is self-assembled into fibrils and mineralized by HAP in vitro. The mineralization process is monitored by cryoTEM in combination with two-dimensional (2D) and three-dimensional (3D) stochastic optical reconstruction microscopy (STORM), which enables in situ and high-resolution visualization of the process.

Collagen Structure-Function Relationships from Solid-State NMR Spectroscopy.

The extracellular matrix of a tissue is as important to life as the cells within it. Its detailed molecular structure defines the environment of a tissue's cells and thus their properties, including differentiation and metabolic status. Collagen proteins are the major component of extracellular matrices. Self-assembled collagen fibrils provide both specific mechanical properties to handle external stresses on tissues and, at the molecular level, well-defined protein binding sites to interact with cells. How the cell-matrix interactions are maintained against the stresses on the tissue is an important and as yet unanswered question. Similarly, how collagen molecular and fibrillar structures change in aging and disease is a crucial open question. Solid-state NMR spectroscopy offers insight into collagen molecular conformation in intact in vivo and in vitro tissues, and in this Account we review how NMR spectroscopy is beginning to provide answers to these questions. In vivo 13C,15N labeling of the extracellular matrix has given insight into collagen molecular dynamics and generated multidimensional NMR "fingerprints" of collagen molecular structure that allow comparison of local collagen conformation between tissues. NMR studies have shown that charged collagen residues (Lys, Arg) adopt extended-side-chain conformations in the fibrillar structure to facilitate charge-charge interactions between neighboring collagen molecules, while hydrophobic residues (Leu, Ile) fold along the collagen molecular axis to minimize the hydrophobic area exposed to surrounding water. Detailed NMR and molecular modeling work has shown that the abundant Gly-Pro-Hyp (Hyp = hydroxyproline) triplets in collagen triple helices confer well-defined flexibility because the proline is conformationally metastable, in contrast to the expectation that these triplets confer structural rigidity. The alignment of the Gly-Pro-Hyp triplets within the fibril structure means that the Gly-Pro-Hyp molecular flexibility generates fibril flexibility. The fibrillar bands of Gly-Pro-Hyp are highly correlated with collagen ligand binding sites, leading to the hypothesis that the fibril alignment of Gly-Pro-Hyp triplets is essential to protect collagen-ligand binding against external stresses on the tissue. Non-enzymatic chemistry between collagen side-chain amine groups (Lys, Arg) and reducing sugars-glycation-is an important source of matrix structural change in aging and disease. Glycation leads to stiffening of collagen fibrils, which is widely speculated to be the result of intermolecular cross-linking. The chemistry of non-enzymatic glycation has been extensively detailed through NMR studies and has been shown to lead to side-chain modifications as the majority reaction products, rather than intermolecular cross-links, with resultant molecular misalignment in the fibrils. Thus, a picture is beginning to emerge in which collagen glycation causes stiffening through misalignment of collagen molecular flexible regions rather than intermolecular cross-linking, meaning that new thinking is needed on how to alleviate collagen structural changes in aging and disease.

Insight into the collagen assembly in the presence of lysine and glutamic acid: An in vitro study

The aim of this study is to evaluate the effects of two different charged amino acids in collagen chains, lysine and glutamic acid, on the fibrillogenesis process of collagen molecules. The turbidity, zeta potential, and fiber diameter analysis suggest that introducing the positively charged lysine into collagen might improve the sizes or amounts of the self-assembled collagen fibrils significantly. Conversely, the negatively charged glutamic acid might restrict the self-assembly of collagen building blocks into a higher order structure. Meanwhile, the optimal fibrillogenesis condition is achieved when the concentration of lysine reaches to 1mM. Both scanning electron microscopy (SEM) and atomic force microscope (AFM) analysis indicates that compared to pure collagen fibrils, the reconstructed collagen-lysine co-fibrils exhibit a higher degree of inter-fiber entanglements with more straight and longer fibrils. Noted that the specific D-period patterns of the reconstructed collagen fibrils could be clearly discernible and the width of D-banding increases steadily after introducing lysine. Besides, the kinetic and thermodynamic collagen self-assembly analysis confirms that the rate constants of both the first and second assembly phase decrease after introducing lysine, and lysine could promote the process of collagen fibrillogenesis obeying the laws of thermodynamics.

Rapid biomimetic mineralization of collagen fibrils and combining with human umbilical cord mesenchymal stem cells for bone defects healing

Collagen biomineralization is regulated by complicated interactions between the collagen matrix and non-collagenous extracellular proteins. Here, the use of sodium tripolyphosphate to simulate the templating functional motif of the C-terminal fragment of non-collagenous proteins is reported, and a low molecular weight polyacrylic acid served as a sequestration agent to stabilize amorphous calcium phosphate into nanoprecursors. Self-assembled collagen fibrils served as a fixed template for achieving rapid biomimetic mineralization in vitro. Results demonstrated that, during the mineralization process, intrafibrillar and extrafibrillar hydroxyapatite mineral with collagen fibrils formed and did so via bottom-up nanoparticle assembly based on the non-classical crystallization approach in the presence of these dual biomimetic functional analogues. In vitro human umbilical cord mesenchymal stem cell (hUCMSC) culture found that the mineralized scaffolds have a better cytocompatibility in terms of cell viability, adhesion, proliferation, and differentiation into osteoblasts. A rabbit femoral condyle defect model was established to confirm the ability of the n-HA/collagen scaffolds to facilitate bone regeneration and repair. The images of gross anatomy, MRI, CT and histomorphology taken 6 and 12weeks after surgery showed that the biomimetic mineralized collagen scaffolds with hUCMSCs can promote the healing speed of bone defects in vivo, and both of the scaffolds groups performing better than the bone defect control group. As new bone tissue formed, the scaffolds degraded and were gradually absorbed. All these results demonstrated that both of the scaffolds and cells have better histocompatibility.

Biomimetic intrafibrillar silicification of collagen fibrils

Event Abstract Back to Event Biomimetic intrafibrillar silicification of collagen fibrils Changmin Hu1* and Mei Wei2* 1 University of Connecticut, Institute of Materials Science, United States 2 University of Connecticut, Materials Science & Engineering, Institute of Materials Science, United States Introduction: Silicon is the most abundant element on earth, and the study of the effect of silicon on biological system has attracted extensive attention. Si-containing biomaterials have been found to be osteoinductive as well as to be able to stimulate neovascularization, induce apatite deposition, and increase extracellular matrix (ECM) synthesis.[1] Since type I collagen is the most abundant component of ECM, it has been used extensively as the ideal component to produce silicified hybrid materials. The biomimetic silicification of collagen fibrils is templated by the self-assembly of triple helix collagen molecules, inspired by biomimetic silicification of collagen fibrils from diatom frustules. In this abstract, we report two approaches to fabricate biomimetic intrafibrillar silicification of collagen fibrils. In the first approach, a two-step process was used, where collagen fibrils were self-assembled and then silicification occurred. In the second approach, a single-step approach was employed, where the self-assembly and silicification of collagen fibrils occurred simultaneously. A combination of poly(allylamine) hydrochloride (PAH) and sodium tripolyphosphate (TPP) was used to act as a stabilizing agent and a templating analog, respectively. Methods: Type I collagen stock solution (4.5 mg/mL), 1.5% PAH stabilized silica precursors (PAH-SA), and collagen self-assembling (CSA) solution were prepared as reported previously.[2][3][4] In two-step approach, collagen molecules self-assembled in the CSA solution at 37 °C for 1 day, and then PAH-SA precursors were added (Col/SA weight ratio is 3:4) to the collagen fibrils and incubate at 37 °C for 1 day (T-Col-PAH-SA). In single-step approach, collagen stock solution was diluted using CSA, and then PAH-SA solution (Col/SA=3:4) was added dropwise to the collagen solution, then the mixture was incubated at 37 °C for 1 day to form S-Col-PAH-SA. In two-step approach, collagen molecules self-assembled in CSA solution at 37 °C for 1 day, TPP (1 wt%) was added, and then followed with PAH-SA precursors at a ratio of Col/SA=3:4 to silicify collagen fibrils at 37 °C for 1 day (T-Col-PAH-SA-TPP). In the later approach, collagen stock solution was diluted using CSA, and TPP was added at a concentration of 1 wt%. Then PAH-SA solution (Col/SA=3:4) was added dropwise to the collagen solution, and finally the mixture was incubated at 37 °C for 1 day (S-Col-PAH-SA-TPP). Results: Intrafibrillar silicified collagen fibrils were produced using either a two-step or a single-step approach (Fig. 1B1, 1B2). PAH was used to stabilize silica precursors to form fluidic nanoprecursors (Fig. 2), and TPP was used to template silica deposition within collagen fibrils (Fig. 1A1, 1B1). Fig. 1A1 and 1A2 show that the single-step process for S-Col-PAH-SA produces twisted collagen fibrils, indicating that the self-assembly of collagen fibrils and the silicification process occurred simultaneously. Both two-step and single-step processes for preparation of Col-PAH-SA-TPP produce silicified collagen fibrils with clear D-bandings (Fig. 1B1, 1B2), indicating the formation of intrafibrillar silicification. Fig. 1 TEM images of T-Col-PAH-SA (A1), S-Col-PAH-SA (A2), T-Col-PAH-SA-TPP (B1), and S-Col-PAH-SA-TPP (B2) fibrils. In B1 and B2, D-banding of the fibrils is clearly seen. Fig. 2 (A) Low and (B) high magnification images of the fluidic silica precursors infiltrated into the collagen fibrils during the collagen self-assembly process. White arrow in B: Vague D-banding. Conclusions: Biomimetic intrafibrillar silicification of collagen fibrils has been successfully achieved using either a single-step or a two-step approach at the presence of PAH for stabilizing silica to form fluidic nanoprecursors and TPP as a templating analog. The authors would like to thank the support from NSF grants (CBET-1133883 and CBET-1347130) and GEMS Fellowship.

A continuum model for hierarchical fibril assembly

Most of the biological polymers that make up our cells and tissues are hierarchically structured. For biopolymers ranging from collagen, to actin, to fibrin and amyloid fibrils this hierarchy provides vitally important versatility. The structural hierarchy must be encoded in the self-assembly process, from the earliest stages onward, in order to produce the appropriate substructures. In this letter, we explore the kinetics of multistage self-assembly processes in a model system which allows comparison to bulk probes such as light scattering. We apply our model to recent turbidimetry data on the self-assembly of collagen fibrils. Our analysis suggests a connection between diffusion-limited aggregation kinetics and fibril growth, supported by slow, power-law growth at very long time scales.

Effect of the self-assembly of collagen on crystallisation of calcium carbonate in aqueous solution

In order to investigate how the self-assembly of organic matrix influences crystallisation and growth of inorganic minerals, we selected collagen as the matrix and conducted three experiments of crystallisation of CaCO3 in different reaction systems: H2O system, as-assembled collagen fibrils system and self-assembling of collagen system. It is found that (i) the self-assembly process of organic matrix had a remarkable effect on the morphology of inorganic minerals: CaCO3 crystals formed in the as-assembled collagen fibrils system were global clusters and those formed in the self-assembling of collagen system appeared as interlaced networks and (ii) the organic matrix decided the polymorph of crystals: CaCO3 crystals were calcite in the H2O system and appeared vaterite in the collagen system. From this study, we can conclude that the self-assembly of collagen fibrils greatly affect the crystallisation and growth of CaCO3. Such results are significant in understanding the mechanism of biomineralisation in calcified tissues in general, and useful in the synthesis of biominerals. (a) CaCO3 formed in the as-assembled collagen fibrils system. (b) CaCO3 formed in the self-assembling of collagen monomer system.The TEM images of samples obtained in the as-assembled collagen fibrils and self-assembling of collagen monomer system, were observed, respectively. The result shows that crystals CaCO3 formed in the as-assembled collagen fibrils system were global clusters; crystals CaCO3 formed in the self-assembling of collagen monomer system appeared interlaced networks.

Expression of type I procollagen genes.

All of the type I collagen in connective tissue is the product of one structural gene for the pro alpha 1(I) chain and another for the pro alpha 2(I) chain of type I procollagen. An intriguing question therefore is how the expression of the two genes differs in mineralizing and non-mineralizing tissues. One approach that our laboratory has pursued to answer this and related questions is to develop a new system whereby one can examine the self-assembly of collagen fibrils de novo by controlled enzymic cleavage of procollagen to collagen under physiological conditions. The system has made it possible for the first time to define thermodynamic parameters for the self-assembly process. We are now using the system to define the normal kinetics for fibril formation. The results should make it possible to study the effects of other components of extracellular matrix on fibril assembly, including the effects of bone-specific components that initiate mineralization. A second approach has been to define mutations in type I procollagen genes that cause increased brittleness of bone. Over a dozen mutations in type I procollagen genes have been found in probands with osteogenesis imperfecta. One of the surprises has been that at least 25% of the probands with lethal variants of osteogenesis imperfecta have mutations in type I procollagen genes. Another surprise has been the observation that a number of the mutations are tissue specific in terms of their phenotypic manifestations even though the same abnormal pro alpha chains are being synthesized in a variety of tissues.

Self-assembly of mineralized collagen composites

Abstract This paper presents a review of the current understanding of the structure, self-assembly mechanisms, and properties of mineralized collagen fibril composites in connective tissues, such as in lamellar bones, woven bones, zebrafish skeletal bone, and ivory. Recent work involving biomimetic synthesis of new materials with the structure of mineralized collagen is described. The focus in the paper is mainly on materials containing type I collagen, with mineralization by Ca–P crystals although some other systems are also described. Investigation and simulation of naturally occurring fibril structures can offer some new ideas in the design and fabrication of new functional materials, for applications such as bone grafts or for use as scaffolds in tissue engineering and biomimetic engineering materials. The development of bone grafts based on the mineralization of self-assembled collagen fibrils in vivo and in vitro is an active area of research. This kind of bone graft composite has already shown great promise and success in clinical applications, on account of its compositional and structural similarity to autologous bone. It is suggested that future work in this should focus on both basic theoretical aspects as well as the development of applications. In particular issues including control of morphology, incorporation of foreign ions, interaction with biomolecules, and the assembly of organic and inorganic phases are all still not well understood. In the area of applications, the design of composite materials with a hierarchical structure closer to that of natural hard tissues, and the synthesis of bone grafts and tooth regenerative materials, as well as biomimetic functional materials, are areas currently being examined by many research groups.

Electrochemically-assisted deposition of biomimetic hydroxyapatite–collagen coatings on titanium plate

A biomimetic bone-like composite, made of self-assembled collagen fibrils and carbonate hydroxyapatite nanocrystals, has been performed by an electrochemically-assisted deposition on titanium plate. The electrolytic processes have been carried out using a single type I collagen molecules suspension in a diluted Ca(NO3)2 and NH4H2PO4 solution at room temperature and applying a constant current for different periods of time. Using the same electrochemical conditions, carbonate hydroxyapatite nanocrystals or reconstituted collagen fibrils coatings were obtained. The reconstituted collagen fibrils, hydroxyapatite nanocrystals and collagen fibrils/apatite nanocrystals coatings have been characterized chemically, structurally and morphologically, as well as for their ability to bind fibronectin (FN). Fourier Transform Infrared microscopy has been used to map the topographic distribution of the coating components at different times of electrochemical deposition, allowing to single out the individual deposition steps. Moreover, roughness of Ti plate has been found to affect appreciably the nucleation region of the inorganic nanocrystals. Laser scanning confocal microscopy has been used to characterize the FN adsorption pattern on a synthetic biomimetic apatitic phase, which exhibits a higher affinity when it is inter-grown with the collagen fibrils. The results offer auspicious applications in the preparation of medical devices such as biomimetic bone-like composite-coated metallic implants.

Early bone apposition in vivo on plasma-sprayed and electrochemically deposited hydroxyapatite coatings on titanium alloy

Three different implants, bare Ti–6Al–4V alloy, Ti–6Al–4V alloy coated with plasma-sprayed hydroxyapatite (PSHA), and Ti–6Al–4V alloy coated with electrochemically deposited hydroxyapatite (EDHA), were implanted into canine trabecular bone for 6h, 7, and 14 days, respectively. Environmental scanning electron microscopy study showed that PSHA coatings had higher bone apposition ratios than those exhibited by bare Ti–6Al–4V and EDHA coatings after 7 days; however, at 14 days after implantation, EDHA and PSHA coatings exhibited similar bone apposition ratios, much higher than that for bare Ti–6Al–4V. The ultrastructure of the bone/implant interface observed by transmission electron microscope showed that the earliest mineralization (6h—7 days) was in the form of nano-ribbon cluster mineral deposits with a Ca/P atomic ratio lower than that of hydroxyapatite. Later-stage mineralization (7–14 days) resulted in bone-like tissue with the characteristic templating of self-assembled collagen fibrils by HA platelets. Though adhesion of EDHA coatings to Ti–6Al–4V substrate proved problematical and clearly needs to be addressed through appropriate manipulation of electrodepositon parameters, the finely textured microstructure of EDHA coatings appears to provide significant advantage for the integration of mineralized bone tissue into the coatings.

Controlled Self-Assembly of Collagen Fibrils by an Automated Dialysis System

In vitro self-assembled collagen fibrils form a variety of different structures during dialysis. The self-assembly is dependent on several parameters, such as concentrations of collagen and alpha1-acid glycoprotein, temperature, dialysis time, and the acid concentration. For a detailed understanding of the assembly pathway and structural features like banding pattern or mechanical properties it is necessary to study single collagen fibrils. In this work we present a fully automated system to control the permeation of molecules through a membrane like a dialysis tubing. This allows us to ramp arbitrary diffusion rate profiles during the self-assembly process of macromolecules, such as collagen. The system combines a molecular sieving method with a computer assisted control system for measuring process variables. With the regulation of the diffusion rate it is possible to control and manipulate the collagen self-assembly process during the whole process time. Its performance is demonstrated by the preparation of various collagen type I fibrils and native collagen type II fibrils. The combination with the atomic force microscope (AFM) allows a high resolution characterization of the self-assembled fibrils. In principle, the represented system can be also applied for the production of other biomolecules, where a dialysis enhanced self-assembly process is used.

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants in vitro

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6). Penetration of cartilage by mAb was determined by immunofluorescence on frozen sections and correlated with changes to the extracellular matrix and chondrocytes by morphometric analysis of sections stained with toluidine blue. The effects of mAbs on matrix components were examined by Fourier transform infrared microspectroscopy (FTIRM). A possible role of Fc-binding was investigated using F(ab)2 from CIIC1. All three mAbs to CII penetrated the cartilage explants and CIIC1 and M2139, but not CIIF4, had adverse effects that included proteoglycan loss correlating with mAb penetration, the later development in cultures of an abnormal superficial cellular layer, and an increased proportion of empty chondrons. FTIRM showed depletion and denaturation of CII at the explant surface in the presence of CIIC1 or M2139, which paralleled proteoglycan loss. The effects of F(ab)2 were greater than those of intact CIIC1. Our results indicate that mAbs to CII can adversely affect preformed cartilage, and that the specific epitope on CII recognised by the mAb determines both arthritogenicity in vivo and adverse effects in vitro. We conclude that antibodies to CII can have pathogenic effects that are independent of inflammatory mediators or Fc-binding.