RESUMO
INTRODUCTION: The goal is to propose a material scientific hypothesis for the atomic arrangement of calcium phosphates during the mineralization of bones. MATERIALS AND METHODS: It was reached by the analysis of bones of healthy and osteoporotic rats using analytical transmission electron microscopic methods. RESULTS: Electron diffraction patterns show hydroxyapatite (HAP) as dominant phase within the mineralized areas. In the electron energy loss spectrum, a double peak of the phosphorous L-edge seems to be a characteristic feature of the phosphorous binding in biological HAP. The hypothesis bases on periodic features on the collagen surface which agree with distances between oxygen atoms in the (200) plane of octacalcium phosphate (OCP). Bridge pillars for the HAP network consist of OCP coupled with a half unit cell on collagen by oxygen-hydrogen bridges. Possibly, the metastable OCP bridges are only a transient step, while the mineralization is starting. OCP and HAP couple by similar distances of calcium atoms in an interface close to the (100) planes of the OCP and the HAP network. To reach the perfect overlap of the equidistant Ca atoms, the HAP network has to be rotated by 22.5° around the a-axis, 11.5° around the c-axis of HAP, and 10.1° around an axis perpendicular to a and c. CONCLUSIONS: A supercell based on this idea is able to explain the dominance of HAP in the electron diffraction patterns, the arrangement of the (002) lattice planes perpendicular to the collagen fiber axis, and sections of high-resolution TEM images.
Assuntos
Biomineralização/fisiologia , Osso e Ossos/fisiologia , Animais , Osso e Ossos/ultraestrutura , Fosfatos de Cálcio/química , Durapatita/química , Feminino , Minerais/química , Ratos Sprague-Dawley , Difração de Raios XRESUMO
The degradation mechanism of human trabecular bone harvested from the central part of the femoral head of a patient with a fragility fracture of the femoral neck under conditions of senile osteoporosis was investigated by high-resolution electron microscopy. As evidenced by light microscopy, there is a disturbance of bone metabolism leading to severe and irreparable damages to the bone structure. These defects are evoked by osteoclasts and thus podosome activity. Podosomes create typical pit marks and holes of about 300-400 nm in diameter on the bone surface. Detailed analysis of the stress field caused by the podosomes in the extracellular bone matrix was performed. The calculations yielded maximum stress in the range of few megapascals resulting in formation of microcracks around the podosomes. Disintegration of hydroxyapatite and free lying collagen fibrils were observed at the edges of the plywood structure of the bone lamella. At the ultimate state, the disintegration of the mineralized collagen fibrils to a gelatinous matrix comes along with a delamination of the apatite nanoplatelets resulting in a brittle, porous bone structure. The nanoplatelets aggregate to big hydroxyapatite plates with a size of up to 10 x 20 µm2. The enhanced plate growth can be explained by the interaction of two mechanisms in the ruffled border zone: the accumulation of delaminated hydroxyapatite nanoplatelets near clusters of podosomes and the accelerated nucleation and random growth of HAP nanoplatelets due to a nonsufficient concentration of process-directing carboxylated osteocalcin cOC.
Assuntos
Osteoporose , Podossomos , Apatitas , Osso e Ossos/diagnóstico por imagem , Humanos , OsteoclastosRESUMO
Human mesenchymal stem cells (hMSCs) from bone marrow are considered a promising cell source for bone tissue engineering applications because of their ability to differentiate into cells of the osteoblastic lineage. Mechanical stimulation is able to promote osteogenic differentiation of hMSC; however, the use of hydrostatic pressure (HP) has not been well studied. Artificial extracellular matrices containing collagen and chondroitin sulfate (CS) have promoted the expression of an osteoblastic phenotype by hMSCs. However, there has been little research into the combined effects of biochemical stimulation by matrices and simultaneous mechanical stimulation. In this study, artificial extracellular matrices generated from collagen and/or CS were coated onto polycaprolactone-co-lactide substrates, seeded with hMSCs and subjected to cyclic HP at various time points during 21 days after cell seeding to investigate the effects of biochemical, mechanical, and combined biochemical and mechanical stimulations. Cell differentiation was assessed by analyzing the expression of alkaline phosphatase (ALP) at the protein- and mRNA levels, as well as for calcium accumulation. The timing of HP stimulation affected hMSC proliferation and expression of ALP activity. HP stimulation after 6 days was most effective at promoting ALP activity. CS-containing matrices promoted the osteogenic differentiation of hMSCs. A combination of both CS-containing matrices and cyclic HP yields optimal effects on osteogenic differentiation of hMSCs on scaffolds compared with individual responses.
Assuntos
Colágeno/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Fosfatase Alcalina/metabolismo , Osso e Ossos/metabolismo , Diferenciação Celular/fisiologia , Sulfatos de Condroitina/metabolismo , Durapatita/metabolismo , Matriz Extracelular/metabolismo , Humanos , Pressão Hidrostática , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Poliésteres , Engenharia Tecidual/métodosRESUMO
Glucuronic acid (GlcA) and phosphoserine (pS) carrying acidic functional groups were used as model molecules for glycosaminoglycans and phosphoproteins, respectively to mimic effects of native biomolecules and influence the mineralization behaviour of collagen I. Collagen substrates modified with GlcA showed a stable interaction between GlcA and collagen fibrils. Substrates were mineralized using the electrochemically assisted deposition (ECAD) in a Ca(2+)/H( x )PO (4) ((3-x)) electrolyte at physiological pH and temperature. During mineralization of collagen-GlcA matrices, crystalline hydroxyapatite (HA) formed earlier with increasing GlcA content of the collagen matrix, while the addition of pS to the electrolyte succeeded in inhibiting the transformation of preformed amorphous calcium phosphate (ACP) to HA. The lower density of the resulting mineralization and the coalesced aggregates formed at a certain pS concentration suggest an interaction between calcium and the phosphate groups of pS involving the formation of complexes. Combining GlcA-modified collagen and pS-modified electrolyte showed dose-dependent cooperative effects.
Assuntos
Materiais Biomiméticos/química , Líquidos Corporais/química , Substitutos Ósseos/química , Colágeno Tipo I/química , Ácido Glucurônico/química , Minerais/química , Fosfosserina/química , Cristalização/métodos , Teste de MateriaisRESUMO
A new concept for modular biosurface engineering of titanium implants based on the self-assembly of complementary oligonucleotides was biochemically investigated and optimized. This study describes the synthesis and characterization (RP-HPLC and Sakaguchi assay) of oligodeoxyribonucleotide (ODN) conjugates of the hexapeptide GRGDSP containing the RGD sequence as the recognition motif for cellular adhesion receptors (integrins). The peptide was chosen exemplarily as a model molecule, because it is a simple but potent bioactive molecule and relatively well investigated. The conjugation products must fulfill two main requirements: (I) the ability to hybridize and (II) the preservation of biological activity of the RGD peptide for the enhancement of osteoblast adhesion. In the following text, the term "hybridization" is generally used for Watson-Crick base pairing. The ability of the conjugates to hybridize to surface-immobilized complementary ODN was verified by competitive hybridization with radiolabeled ((32)P) complementary strands and by hybridization experiments using a quartz crystal microbalance (QCM). Surface hybridization was further characterized using different adsorption isotherms (e.g., Freundlich and Frumkin types), since the type of isotherm and the derived thermodynamic parameters may reveal characteristic differences between ODN and conjugates thereof. Biological activity of the conjugates was examined in vitro with osteoblasts. The cells were either cultured directly on the ODN-GRGDSP modified titanium implants or used for competition adhesion studies with dissolved ODN-GRGDSP conjugates. All results support the successful establishment of the new surface modification system. Hybridization of RGD peptide-modified nucleic acids to ODN-modified titanium implant materials is thus a promising method for osteoblast attachment in a modular and self-organizing system on implant surfaces.
Assuntos
Oligonucleotídeos/química , Oligopeptídeos/química , Osteoblastos/citologia , Próteses e Implantes , Titânio/química , Titânio/metabolismo , Adsorção , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Adesão Celular , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Humanos , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Oligopeptídeos/metabolismo , Osteoblastos/metabolismo , Quartzo/química , Propriedades de SuperfícieRESUMO
In this study, we have demonstrated that the modification of hyaluronan (hyaluronic acid; Hya) with sulfate groups led to different binding affinities for recombinant human bone morphogenetic protein-4 (rhBMP-4). The high-sulfated sHya2.8 (average degree of sulfation (D.S.) 2.8) exhibited the tightest interaction with rhBMP-4, followed by the low-sulfated sHya1.0, as determined with surface plasmon resonance (SPR), ELISA, and competition ELISA. Unmodified Hya, chondroitin-sulfate (CS), and heparan sulfate (HS) showed significantly less binding affinity. SPR data could be fitted to an A + B = AB Langmuir model and binding constants were evaluated ranging from 13 pM to 5.45 microM. The interaction characteristics of the differentially sulfated Hyas are promising for the incorporation of these modified polysaccharides in bioengineered coatings of biomaterials for medical applications.
Assuntos
Proteína Morfogenética Óssea 4/química , Ácido Hialurônico/química , Proteínas Imobilizadas/química , Ésteres do Ácido Sulfúrico/química , Técnicas Biossensoriais , Proteína Morfogenética Óssea 4/genética , Sulfatos de Condroitina/química , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Imobilizadas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ressonância de Plasmônio de SuperfícieRESUMO
Textile chitosan fiber scaffolds were developed and tested in terms of biocompatibility for human bone marrow stromal cells (hBMSCs). A part of the scaffolds was further modified by coating with fibrillar collagen type I in order to biologize the surface. hBMSCs of two donors were used for cell culture experiments in vitro. Confocal laser scanning microscopy (CLSM) as well as scanning electron microscopy (SEM) revealed fast attachment and morphological adaptation of the cells on both the raw chitosan fibers and the collagen-coated scaffolds. Cells were osteogenically induced after 3 days and cultivated for up to 28 days on the scaffolds. Activity of lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) was analyzed to evaluate proliferation as well as osteogenic differentiation. We found a 3.5-6-fold increase in the cell number, whereas the collagen coating did not noticeably influence these factors. Osteogenic differentiation was confirmed by the course of ALP activity and immunostaining of osteocalcin. The feature of the collagen-coated as well as the raw chitosan fiber scaffolds to support attachment, proliferation, and differentiation of hBMSCs suggests a potential application of chitosan fibers and textile chitosan scaffolds for the tissue engineering of bone.
Assuntos
Materiais Biocompatíveis/química , Células da Medula Óssea/citologia , Quitosana/química , Células Estromais/citologia , Têxteis , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Fosfatase Alcalina/metabolismo , Contagem de Células , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , L-Lactato Desidrogenase/metabolismo , Teste de Materiais , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Developing new biopolymer-based materials with bio-identical properties is a significant challenge in modern science. One interesting route to this goal involves the biomineralization of collagen, a pre-structured and widely available protein, into a material with interesting properties. A prerequisite for biomineralization is the ability of cations (e.g., calcium) to bind to the protein and to result in concert with appropriate anions (e.g., phosphate) in composite material with e.g., bone-like properties. In order to increase the number of binding sites it is necessary to modify the protein prior to mineralization. For this glucuronic acid (GA) was used due to its carbonyl and carboxyl groups to derivatize proteinogenic amino groups transferring them into negatively charged carboxyl groups. Our experiments showed for the first time, that Nepsilon-carboxymethyllysine is the major product of in vitro non-enzymatic glycosylation of collagen by glucuronic acid. For an unequivocal determination of the reaction products, the lysine residues of collagen and of the model peptide were carboxymethylated through a reductive alkylation with glyoxalic acid and compared to the glucuronic acid derivatives. Beside their identical mass spectra the common structure elements could be confirmed with FTIR. Thus, in the context of matrix engineering, by producing Nepsilon-carboxymethyllysine, glucuronic acid offers a convenient way of introducing additional stable acidic groups into protein matrices.
Assuntos
Biopolímeros/química , Colágeno/química , Ácido Glucurônico/química , Lisina/análogos & derivados , Aminoácidos/análise , Biomimética/métodos , Lisina/síntese química , Espectrometria de Massas , Estrutura Molecular , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Two novel scaffold models made of chitosan fibers were designed, fabricated, and investigated. Raw chitosan fibers were either tightened between plastic rings or were processed into stand-alone scaffolds. Chitosan fiber scaffolds were further modified by coating with a thin layer of fibrillar collagen type I to biologize the surface. Cell culture experiments were carried out using murine osteoblast-like cells (7F2). Confocal laser scanning microscopy (cLSM) as well as scanning electron microscopy (SEM) revealed fast attachment and morphological adaptation of the cells on both the raw chitosan fibers and the collagen-coated scaffolds. Cells were cultivated for up to 4 weeks on the materials and proliferation as well as osteogenic differentiation was quantitatively analyzed in terms of lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity. We found a 14-16-fold increase of cell number and the typical pattern of ALP activity, whereas the collagen coating does not remarkably influence these parameters. The maintenance of osteogenic phenotype on the novel materials was furthermore confirmed by immunostaining of osteocalcin and study of matrix mineralization. The feature of the collagen-coated but also the raw chitosan fiber scaffolds to support the attachment, proliferation, and differentiation of osteoblast-like cells suggest a potential application of chitosan fibers and textile chitosan scaffolds for the tissue engineering of bone.
Assuntos
Materiais Biocompatíveis/química , Quitosana/química , Osteoblastos/metabolismo , Têxteis , Fosfatase Alcalina/metabolismo , Animais , Cálcio/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colorimetria/métodos , L-Lactato Desidrogenase/metabolismo , Camundongos , Microscopia Confocal , Microscopia Eletrônica de VarreduraRESUMO
Tibia trabeculae and vertebrae of rats as well as human femur were investigated by high-resolution TEM at the atomic scale in order to reveal snapshots of the morphogenetic processes of local bone ultrastructure formation. By taking into account reflections of hydroxyapatite for Fourier filtering the appearance of individual alpha-chains within the triple-helix clearly shows that bone bears the feature of an intergrowth composite structure extending from the atomic to the nanoscale, thus representing a molecular composite of collagen and apatite. Careful Fourier analysis reveals that the non-collagenous protein osteocalcin is present directly combined with octacalcium phosphate. Besides single spherical specimen of about 2 nm in diameter, osteocalcin is spread between and over collagen fibrils and is often observed as pearl necklace strings. In high-resolution TEM, the three binding sites of the γ-carboxylated glutamic acid groups of the mineralized osteocalcin were successfully imaged, which provide the chemical binding to octacalcium phosphate. Osteocalcin is attached to the collagen structure and interacts with the Ca-sites on the (100) dominated hydroxyapatite platelets with Ca-Ca distances of about 9.5 Å. Thus, osteocalcin takes on the functions of Ca-ion transport and suppression of hydroxyapatite expansion.
Assuntos
Calcificação Fisiológica/fisiologia , Fosfatos de Cálcio/metabolismo , Colágeno/metabolismo , Fêmur/metabolismo , Osteocalcina/metabolismo , Tíbia/metabolismo , Animais , Apatitas/metabolismo , Sítios de Ligação/fisiologia , Plaquetas/metabolismo , Cálcio/metabolismo , Durapatita/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Controlling the differentiation of human mesenchymal stem cells (hMSC) and providing tissue functions in engineered constructs before implantation are major challenges. Beside the additives in culture media, the artificial niches inside a scaffold can serve this purpose. To prepare niches favoring the osteoblastic differentiation of hMSCs, components of the extracellular matrix of bone were immobilized on fabrics of poly(3-hydroxybutyrate). Aqueous gels of fibrillar bovine collagen I, with or without addition of chondroitin sulphate (CS), were immobilized on the textile scaffold, sub-structured in a freeze-drying process, and cross-linked. hMSCs of four donors were isolated from bone marrow. After expansion, the cells were seeded dynamically onto the scaffolds. From thereon, the culture was transferred into perfused vessels and partly submitted to dexamethasone to promote osteogenic differentiation. During their 4 weeks of culture, the cells' distribution and morphology throughout the scaffolds were characterized by laser scanning microscopy (LSM) and scanning electron microscopy (SEM). Photospectrometrically the cells' viability (MTT) and alkaline phosphatase (ALP) production were assessed. The transcription of osteoblast-specific markers was elucidated with polymerase chain reaction (PCR) tests. Cells on CS-containing scaffolds in the presence of dexamethasone showed the highest ALP production. PCR monitored an increase of osteoblastic markers. All scaffolds showed higher calcium deposition than cell-free controls. These results lead to the conclusion that a niche containing CS renders the differentiation of hMSCs toward osteoblastic cells more specific.
Assuntos
Diferenciação Celular , Matriz Extracelular/química , Hidroxibutiratos/química , Células-Tronco Mesenquimais/fisiologia , Polímeros/química , Adulto , Fosfatase Alcalina/biossíntese , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Sulfatos de Condroitina/química , Colágeno Tipo I/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Dexametasona/farmacologia , Marcadores Genéticos , Humanos , Hidrogéis , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Confocal , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Osteogênese/efeitos dos fármacos , Reação em Cadeia da Polimerase , Fatores de Tempo , Transcrição GênicaRESUMO
Besides osteoconductive and osteoinductive signals, angiogenesis plays a crucial role in bone development and regeneration and consequently in the integration of implants. Therefore we investigated in this study the binding and release behaviour of vascular endothelial growth factor (VEGF) from Ti6Al4V surfaces coated with 3-dimensional collageneous matrices, some additionally modified with heparin. The heparin was incorporated using different methods: a) adsorptive immobilization b) crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) or c) incorporating during self-assembly of fibrils followed by cross-linking. For unmodified samples, maximum VEGF adsorption was reached with 85 ng VEGF/cm(2). On all 3d-collagen coated surfaces studied (with or without heparin), no saturation could be observed in the range of 0-256 ng VEGF/cm(2).Improved release kinetics were observed for the modified coatings. The initial burst of VEGF within the first 24 h was diminished. From the third day of delivery heparinized matrices showed a higher release of VEGF than the pure collagen matrix and the unmodified reference surface, respectively. In vitro, the proliferation of human dermal microvascular endothelial cells was increased with released VEGF from all investigated samples compared to a VEGF-free control. After 7 days highest increases in cell numbers were observed with solutions from heparinized matrices. It is concluded that functionalization of Ti6Al4V surfaces with heparinized collageneous matrices and VEGF leads to advantageous properties concerning the impact on angiogenesis and thus may improve bone regeneration in the microenvironment of implants.
Assuntos
Materiais Revestidos Biocompatíveis , Células Endoteliais/citologia , Matriz Extracelular , Teste de Materiais , Titânio , Fator A de Crescimento do Endotélio Vascular , Ligas , Regeneração Óssea , Técnicas de Cultura de Células , Colágeno/química , Preparações de Ação Retardada , Matriz Extracelular/química , Heparina/química , Humanos , Teste de Materiais/métodos , Neovascularização Fisiológica , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/químicaRESUMO
Studies in developmental and cell biology have established the fact that responses of cells are influenced to a large degree by morphology and composition of the extracellular matrix. Goal of this work is to use this basic principle to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM), utilizing the natural self-assembly potential of collagen in combination with further ECM components in close analogy to the situation in vivo. Aiming at load-bearing applications in bone contact, collagen type I in combination with the proteoglycan decorin and the glycosaminoglycan chondroitin sulfate (CS) was used; fibrillogenesis, fibril morphology, and adsorption of differently composed fibrils onto titanium were assessed. Both decorin and CS could be integrated into the fibrils during fibrillogenesis, the amount bound respectively desorbed depending on the ionic strength of fibrillogenesis buffer. Including decorin always resulted in a significant decrease of fibril diameter, CS in only a slight decrease or even increase, depending on the collagen preparation used. No significant changes in adsorption to titanium could be detected. Osteoblastic cells showed different reactions for cytoskeletal arrangement and osteopontin expression depending on the composition of the ECM, with CS enhancing the osteoblast phenotype.
Assuntos
Sulfatos de Condroitina , Materiais Revestidos Biocompatíveis , Proteínas da Matriz Extracelular , Osteoblastos , Próteses e Implantes , Proteoglicanas , Titânio , Animais , Bovinos , Colágeno/ultraestrutura , Decorina , RatosRESUMO
In this work the osteoconductive potential of coatings for titanium implants using different extracellular matrix components was evaluated. Cylindrical implants with two defined cavities A and B were coated with collagen type I, type III, or RGD peptide, and placed in the femur of goats together with an uncoated reference state. Bone contact and volume were determined after 5 and 12 weeks implantation, using both histomorphometry and synchrotron radiation micro computed tomography (SR muCT) as the methods complement each other: SR muCT allows for a high precision of bone detection due to the large number of analysed slices per sample, while histology offers a better lateral resolution and the possibility of additionally determining bone contact. Both methods revealed similar tendencies in bone formation for the differently bio-functionalized implants, with the SR muCT data resulting in significant differences. After 5 and 12 weeks, all three coatings showed a significant increase in bone volume over the uncoated reference, with the highest results for the collagen coatings. The coating consisting of just the RGD-sequence to improve cell adhesion showed only a slight improvement compared with the reference material. For uncoated titanium, RGD, and especially collagen type I, the response in cavity A, situated in denser bone, was stronger than in cavity B. Collagen type III, on the other hand, appeared to be the more effective coating in areas of lesser bone density as represented by cavity B. These results indicate that matrix molecules (or combinations thereof) are capable of generating the appropriate signals for the specific microenvironment around implants and can thus accelerate the bone formation process and increase the stability of implants.
Assuntos
Materiais Biocompatíveis/farmacologia , Regeneração Óssea , Substitutos Ósseos , Materiais Revestidos Biocompatíveis/química , Osseointegração , Titânio/farmacologia , Animais , Desenvolvimento Ósseo , Osso e Ossos/metabolismo , Bovinos , Colágeno Tipo I/química , Colágeno Tipo III/química , Feminino , Fêmur , Cabras , Microscopia Eletrônica de Varredura , Oligopeptídeos/química , Próteses e Implantes , Propriedades de Superfície , Síncrotrons , Fatores de Tempo , Titânio/química , Tomografia Computadorizada por Raios XRESUMO
The molecular structure of collagen type 1 can be understood as the result of evolutionary selection in the process of formation of calcium phosphate based biocomposites acting as load bearing components in living organisms. The evolutionary selection fulfills the principle of 'survival of the fittest' in a particular biological environment. Disk-like post-nucleation complexes of Ca2(HPO4)3 2- organized in ribbon-like assemblies in the metastable octacalcium phosphate (OCP) phase, and Ca3 triangles in the stable HAP phase had formed the crystallographic motifs in this selection process. The rotational as well as the translational symmetry of the major tropocollagen (TC) helix agree nearly perfectly with the corresponding symmetries of the OCP structure. The sequence of (Gly-X-Y) motifs of the three α chains constituting the TC molecule enables an optimized structural fit for the nucleation of Ca3 triangles, the directed growth of nanostructured OCP, and the subsequent formation of hydroxyapatite (HAP) in collagen macrofibrils by a topotaxial transition. The known connection between genetic defects of collagen type 1 and Osteogenesis imperfecta should motivate the search for similar dependences of other bone diseases on a disturbed molecular structure of collagen on the genetic scale.
RESUMO
Collagen type I as a robust fibre protein and main component of the extracellular matrix of most tissues is increasingly utilized for surface engineering of biomaterials using different immobilization methods. In the present work we studied the mineralization behaviour of fibrillar collagen type I in simulated body fluid as a measure for conformational changes caused by adsorptive immobilization or immobilization by partial incorporation into the anodic oxide layer on c.p.-titanium using microscopic and vibration spectroscopic methods. Adsorptive immobilization on highly oriented pyrolytic graphite (HOPG) and c.p.-titanium without collagen were used as references. In the initial phase (1-24 h) the kinetics of formation and the morphology of calcium phosphate phases (CPP) are strongly influenced both by the substrate and the immobilization method. Compared to HOPG both types of immobilization on titanium increasingly inhibit the formation of CPP. For longer times (30 d) these initial differences disappear-mineralization product on titanium, irrespective of the presence of collagen, is a mixture of amorphous calcium phosphate and octacalcium phosphate. Contrary to this the mineralization of HOPG substrates results in hydroxy apatite. This is discussed with respect to the conditions during the immobilization as well as the resulting interactions between substrate and immobilized collagen. It is shown that the mineralization process exhibits a high sensitivity with respect to conformational changes caused by these interactions. Possible cell biological relevance of these conformational changes is discussed.
Assuntos
Líquidos Corporais/química , Materiais Revestidos Biocompatíveis/química , Colágeno Tipo I/química , Colágeno Tipo I/ultraestrutura , Minerais/síntese química , Adsorção , Colágenos Fibrilares/química , Colágenos Fibrilares/ultraestrutura , Grafite/química , Teste de Materiais , Conformação Proteica , Relação Estrutura-Atividade , Propriedades de Superfície , Titânio/químicaRESUMO
PURPOSE: Type-I collagen, the major structural protein in bone, has beneficial properties regarding bone regeneration. Little is known about the potential effects of collagen coating on orthopedic implants. METHODS: 3 to 6 microg/cm2 of lyophilized type-I collagen was absorbed on titanium rods. Six coated and uncoated pins of 0.9 mm diameter were inserted into the tibia of adult male Wistar rats for 1, 2, 4, 7, 14, and 28 days. Specimens were embedded in methacrylate-based Technovit 9100N resin. From one portion cutting and grinding sections were obtained. The implant was removed from the other half that was depolymerized, sectioned, and mounted for immunohistochemistry. RESULTS: At day 4, the interface around the collagen-coated implants displayed a granulation tissue with higher numbers of cathepsin D-positive mononucleated cells compared to the uncoated implants (p<0.05). Active osteoblasts, reactive for osteopontin, were increased around the collagen-coated pins at day 4 and 7 (p<0.01). After 28 days of implantation, direct bone contact averaged 74.9% around the collagen-coated implants and 62.1% around uncoated implants (NS). The amount of newly formed bone averaged 76.3% around the collagen-coated pins and 67.8% around the uncoated pins (NS). The histomorphometric findings were confirmed by SRmicroCT in two specimens. CONCLUSIONS: The earlier observation of mononuclear phagocytozing cells and the earlier and higher expression of bone-specific matrix proteins suggest an increased early bone remodeling around titanium pins through collagen coating. A tendency towards increased bone formation was observed around the coated implants.
Assuntos
Colágeno Tipo I , Implantes Experimentais , Tíbia/cirurgia , Titânio , Animais , Catepsina D/análise , Imuno-Histoquímica , Masculino , Ratos , Ratos WistarRESUMO
Studies in developmental and cell biology have established the fact that responses of cells are influenced to a large degree by morphology and composition of the extracellular matrix. Goal of this work is to use this basic principle to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM). Aiming at load-bearing applications in bone contact, in this study the modification of titanium surfaces with the collagen types I and III in combination with fibronectin was undertaken; fibrillogenesis, fibril morphology and adsorption of type I, III and I/III-cofibrils onto titanium were assessed. Increasing the collagen type III amount resulted in a decrease of fibril diameter, while no significant changes in adsorption could be detected. The amount of fibronectin bound to the heterotypic fibrils depended on fibrillogenesis parameters such as ionic strength or concentration of phosphate, and varied with the percentage of integrated type III collagen.
Assuntos
Ligas/química , Colágeno Tipo III/química , Colágeno Tipo I/química , Fibronectinas/química , Alumínio/química , Estrutura Quaternária de Proteína , Titânio/químicaRESUMO
Responses of osteoblastic cells are influenced by morphology and composition of the extracellular matrix, and this fact has been used to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM). In this study, the effect of the collagen types I and III on adhesion, proliferation, and differentiation was studied, using primary osteoblastic cells from rat calvariae. Differences in alkaline phosphatase activity (ALP) and collagen synthesis were observed between differently composed collagen coatings. An increase in collagen type III resulted in an increase in collagen synthesis and a concomitant decrease in ALP activity and Ca deposition. Initial adhesion mechanism of the cells depended on the substrate (titanium, collagen, fibronectin).
Assuntos
Ligas/química , Colágeno Tipo III/química , Colágeno Tipo I/química , Fibronectinas/química , Osteoblastos/fisiologia , Alumínio/química , Animais , Estrutura Quaternária de Proteína , Ratos , Crânio/fisiologia , Fatores de Tempo , Titânio/químicaRESUMO
In order to investigate the effects of different degrees of bioactivity of xerogels on connexin 43 (cx43) signaling of osteoclasts a cell culture approach was developed. Cells isolated from peripheral blood mononuclear cells were cultured in combination with the xerogels and were harvested for further investigations on day 1, day 5, and day 10. By means of quantitative PCR increased cx43 mRNA levels and coincident decreasing mRNA levels of the calcium sensing receptor, TRAP, and Cathepsin K were detected with increasing bioactivity of the xerogel samples. Additionally, osteoclasts cultured on tissue culture plates were used to perform principle investigations on cell differentiation by means of transmission electron microscopy, life cell imaging, and immunofluorescence, and the results demonstrated that cx43-signaling could be attributed to migration and fusion of osteoclast precursors. Therefore, the positive correlation of cx43 expression with high xerogel bioactivity was caused by proceeding differentiation of the osteoclasts. Finally, the presently observed pattern of cx43 signaling refers to strong effects regarding bioactivity on cx43-associated cell differentiation of osteoclasts influenced by extracellular calcium ions.