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1.
Clin Genet ; 97(3): 396-406, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31794058

RESUMO

The 2017 classification of Ehlers-Danlos syndromes (EDS) identifies three types associated with causative variants in COL1A1/COL1A2 and distinct from osteogenesis imperfecta (OI). Previously, patients have been described with variable features of both disorders, and causative variants in COL1A1/COL1A2; but this phenotype has not been included in the current classification. Here, we expand and re-define this OI/EDS overlap as a missing EDS type. Twenty-one individuals from 13 families were reported, in whom COL1A1/COL1A2 variants were found after a suspicion of EDS. None of them could be classified as affected by OI or by any of the three recognized EDS variants associated with COL1A1/COL1A2. This phenotype is dominated by EDS-related features. OI-related features were limited to mildly reduced bone mass, occasional fractures and short stature. Eight COL1A1/COL1A2 variants were novel and five recurrent with a predominance of glycine substitutions affecting residues within the procollagen N-proteinase cleavage site of α1(I) and α2(I) procollagens. Selected variants were investigated by biochemical, ultrastructural and immunofluorescence studies. The pattern of observed changes in the dermis and in vitro for selected variants was more typical of EDS rather than OI. Our findings indicate the existence of a wider recognizable spectrum associated with COL1A1/COL1A2.


Assuntos
Colágeno Tipo I/genética , Doenças do Tecido Conjuntivo/classificação , Síndrome de Ehlers-Danlos/classificação , Variação Genética , Osteogênese Imperfeita/classificação , Adolescente , Adulto , Criança , Pré-Escolar , Colágeno Tipo I/ultraestrutura , Tecido Conjuntivo/ultraestrutura , Doenças do Tecido Conjuntivo/genética , Demografia , Síndrome de Ehlers-Danlos/genética , Feminino , Heterozigoto , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Osteogênese Imperfeita/genética , Fenótipo , Adulto Jovem
2.
Med Mol Morphol ; 53(1): 21-27, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31144043

RESUMO

The acetabular labrum is frequently damaged with advancing age. As collagen fibers are the main sources of strength, knowledge of their ultrastructure is important to determine the cause of age-induced changes. We aimed to investigate the ultrastructure of collagen fibers constituting the acetabular labrum using scanning electron microscopy (SEM). Acetabular labrum samples obtained during total hip arthroplasty were studied. The samples were specially prepared to observe the steric construction of collagen fibrils constituting the acetabular labrum under light microscopy followed by SEM. The acetabular labrum was mostly composed of cartilage tissue, consisting of chondrocytes and collagen type II, with a layer of collagen type I. In adults, chondrocytes with a rich cytoplasm were surrounded by a dense network of fine type II collagen fibrils, and small bundles of type I collagen fibrils were interposed in the cartilage layer. In elderly individuals, the chondrocytes atrophied and both type I and II collagen fibrils were sparse. We suggest that cartilage has three to five layers, consisting of type I and type II collagen fibrils with a solid cartilage substrate. In elderly individuals, the density of chondrocytes decreases and the cellular shape and architecture of collagen fibrils also changes.


Assuntos
Acetábulo/ultraestrutura , Envelhecimento/patologia , Cartilagem Articular/ultraestrutura , Condrócitos/ultraestrutura , Articulação do Quadril/ultraestrutura , Acetábulo/patologia , Acetábulo/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/fisiologia , Artroplastia de Quadril/métodos , Cartilagem Articular/patologia , Cartilagem Articular/cirurgia , Colágeno Tipo I/ultraestrutura , Colágeno Tipo II/ultraestrutura , Feminino , Articulação do Quadril/patologia , Articulação do Quadril/cirurgia , Humanos , Imageamento Tridimensional , Masculino , Microscopia Eletrônica de Varredura , Necrose/patologia , Necrose/cirurgia
3.
Nat Commun ; 10(1): 4886, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653854

RESUMO

Unraveling the mechanisms that govern the formation and function of invadopodia is essential towards the prevention of cancer spread. Here, we characterize the ultrastructural organization, dynamics and mechanical properties of collagenotytic invadopodia forming at the interface between breast cancer cells and a physiologic fibrillary type I collagen matrix. Our study highlights an uncovered role for MT1-MMP in directing invadopodia assembly independent of its proteolytic activity. Electron microscopy analysis reveals a polymerized Arp2/3 actin network at the concave side of the curved invadopodia in association with the collagen fibers. Actin polymerization is shown to produce pushing forces that repel the confining matrix fibers, and requires MT1-MMP matrix-degradative activity to widen the matrix pores and generate the invasive pathway. A theoretical model is proposed whereby pushing forces result from actin assembly and frictional forces in the actin meshwork due to the curved geometry of the matrix fibers that counterbalance resisting forces by the collagen fibers.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/ultraestrutura , Actinas/ultraestrutura , Neoplasias da Mama/patologia , Colágeno Tipo I/ultraestrutura , Metaloproteinase 14 da Matriz/metabolismo , Podossomos/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Matriz Extracelular , Humanos , Microscopia Eletrônica , Modelos Teóricos , Invasividade Neoplásica , Podossomos/metabolismo , Polimerização , Proteólise
4.
Zhonghua Kou Qiang Yi Xue Za Zhi ; 54(7): 475-480, 2019 Jul 09.
Artigo em Chinês | MEDLINE | ID: mdl-31288328

RESUMO

Objective: To observe the effect of adenosine triphosphate (ATP) phosphorylation on type Ⅰ collagen mineralization and explore the role of small molecule compound ATP in biomimetic mineralization. Methods: Fourier transform infrared spectroscopy (FT-IR) was used to analyze the phosphorylation of collagen molecules by different concentrations (0, 25, 50, 100 mmol/L) of ATP. The concentration of 50 mmol/L ATP was chosen to construct the phosphorylated collagen mineralization model. Transmission electron microscopy (TEM) was used to observed the ultrastructure of mineralized collagen and the collagen mineralization rate was further calculated by ImageJ software. The surface morphology of the collagen gel ATP group and the control group was observed by scanning electron microscopy (SEM) and the elemental analysis was performed by using an X-ray energy spectrometer. The artificial demineralized dentin samples were mineralized for 2 days and 4 days to compare the effect of ATP on dentin remineralization by SEM. Results: FT-IR analysis showed that the formation of new peaks at wavenumbers of 642, 818, and 902 cm(-1) indicated that ATP can phosphorylate type Ⅰ collagen. Through TEM and SEM observation, the mineralization degree of type Ⅰ collagen and demineralized dentin pretreated with 50 mmol/L ATP were significantly higher than that of the control group. Compared with the control group [(31.65±1.62)%], the mineralization rate of collagen in the ATP group [(100±0)%] was significantly increased after 2 days of mineralization (P<0.05). Conclusions: ATP phosphorylation can effectively promote the mineralization process of type Ⅰ collagen.


Assuntos
Trifosfato de Adenosina , Materiais Biomiméticos , Colágeno Tipo I , Trifosfato de Adenosina/farmacologia , Materiais Biomiméticos/química , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Colágeno Tipo I/ultraestrutura , Dentina/química , Dentina/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fosforilação , Espectroscopia de Infravermelho com Transformada de Fourier
5.
Sci Rep ; 9(1): 7280, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086263

RESUMO

Type I Collagen is one of the most abundant proteins of the extracellular matrix of the most organs. During chronological aging or in diseases, type I collagen undergoes biochemical and structural changes which can impact biomechanical and physiological properties of organs. In this study, we have investigated the age-related changes in the molecular organization of type I collagen in rat tails tendon using polarized Raman spectroscopy. Our results show that Amide I, amide III as well as the bands related to proline and hydroxyproline are highly sensitive to polarization and age-related. On the other hand, 1453 and 1270 cm-1 do not show any preferential orientation. Depolarization and anisotropic ratios were used to provide information about the changes in orientation of collagen fibers with aging. The anisotropy degree of Raman bands increase from adult to old collagen, indicating a higher collagen fibers alignment to the fascicle backbone axis in old tendons, and consequently a higher straightness of collagen fibers. These data were correlated to those obtained using polarized second harmonic generation technique. Polarized Raman mapping showed a more homogeneous spatial distribution of collagen fibers alignment to the fascicle axis in old tendon. This confirms a higher straightness of collagen fiber with aging.


Assuntos
Envelhecimento/patologia , Colágeno Tipo I/ultraestrutura , Microscopia de Geração do Segundo Harmônico/métodos , Análise Espectral Raman/métodos , Tendões/ultraestrutura , Animais , Ratos , Ratos Wistar , Tendões/patologia
6.
Mar Drugs ; 17(3)2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30813606

RESUMO

The aim of this study is to investigate the physicochemical properties, biosafety, and biocompatibility of the collagen extract from the skin of Nile tilapia, and evaluate its use as a potential material for biomedical applications. Two extraction methods were used to obtain acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from tilapia skin. Amino acid composition, FTIR, and SDS-PAGE results showed that ASC and PSC were type I collagen. The molecular form of ASC and PSC is (α1)2α2. The FTIR spectra of ASC and PSC were similar, and the characteristic peaks corresponding to amide A, amide B, amide I, amide II, and amide III were 3323 cm-1, 2931 cm-1, 1677 cm-1, 1546 cm-1, and 1242 cm-1, respectively. Denaturation temperatures (Td) were 36.1 °C and 34.4 °C, respectively. SEM images showed the loose and porous structure of collagen, indicting its physical foundation for use in applications of biomedical materials. Negative results were obtained in an endotoxin test. Proliferation rates of osteoblastic (MC3T3E1) cells and fibroblast (L929) cells from mouse and human umbilical vein endothelial cells (HUVEC) were increased in the collagen-treated group compared with the controls. Furthermore, the acute systemic toxicity test showed no acute systemic toxicity of the ASC and PSC collagen sponges. These findings indicated that the collagen from Nile tilapia skin is highly biocompatible in nature and could be used as a suitable biomedical material.


Assuntos
Materiais Biocompatíveis/química , Ciclídeos , Colágeno Tipo I/química , Proteínas de Peixes/química , Animais , Materiais Biocompatíveis/isolamento & purificação , Linhagem Celular , Colágeno Tipo I/isolamento & purificação , Colágeno Tipo I/ultraestrutura , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/ultraestrutura , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Microscopia Eletrônica de Varredura , Pele/química , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier
7.
Nanomedicine ; 17: 319-328, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30771503

RESUMO

Bone regeneration is a highly orchestrated process crucial for endogenous healing procedures after accidents, infections or tumor therapy. Changes in surface nanotopography are known to directly affect the formation of osteogenic cell types, although no direct linkage to the endogenous nanotopography of bone was described so far. Here we show the presence of pores of 31.93 ±â€¯0.97 nm diameter on the surface of collagen type I fibers, the organic component of bone, and demonstrate these pores to be sufficient to induce osteogenic differentiation of adult human stem cells. We further applied SiO2 nanoparticles thermally cross-linked to a nanocomposite to artificially biomimic 31.93 ±â€¯0.97 nm pores, which likewise led to in vitro production of bone mineral by adult human stem cells. Our findings show an endogenous mechanism of directing osteogenic differentiation of adult stem cells by nanotopological cues and provide a direct application using SiO2 nanocomposites with surface nanotopography biomimicking native bone architecture.


Assuntos
Células-Tronco Adultas/citologia , Colágeno Tipo I/ultraestrutura , Nanoporos/ultraestrutura , Osteogênese , Adulto , Materiais Biocompatíveis/química , Regeneração Óssea , Células Cultivadas , Colágeno Tipo I/química , Humanos , Nanocompostos/química , Nanocompostos/ultraestrutura , Porosidade , Dióxido de Silício/química , Tecidos Suporte/química
8.
Acta Biomater ; 86: 171-184, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30616076

RESUMO

Although several biomaterials for bone regeneration have been developed in the last decades, clinical application of bone morphogenetic protein 2 is clinically only approved when applied on an absorbable bovine collagen I scaffold (ACS) (Helistat; ACS-H). In research, another ACS, namely Lyostypt (ACS-L) is frequently used as a scaffold in bone-linked studies. Nevertheless, until today, the influence of ACS alone on bone healing remains unknown. Unexpectedly, in vitro studies using ASC-H revealed a suppression of osteogenic differentiation and a significant reduction of cell vitality when compared to ASC-L. In mice, we observed a significant delay in bone healing when applying ACS-L in the fracture gap during femoral osteotomy. The results of our study show for the first time a negative influence of both ACS-H and ACS-L on bone formation demonstrating a substantial need for more sophisticated delivery systems for local stimulation of bone healing in both clinical application and research. STATEMENT OF SIGNIFICANCE: Our study provides evidence-based justification to promote the development and approval of more suitable and sophisticated delivery systems in bone healing research. Additionally, we stimulate researchers of the field to consider that the application of those scaffolds as a delivery system for new substances represents a delayed healing approach rather than a normal bone healing which could greatly impact the outcome of those studies and play a pivotal role in the translation to the clinics. Moreover, we provide impulses on underlying mechanism involving the roles of small-leucine rich proteoglycans (SLRP) for further detailed investigations.


Assuntos
Colágeno Tipo I/farmacologia , Consolidação da Fratura/efeitos dos fármacos , Osteotomia , Tecidos Suporte/química , Animais , Regeneração Óssea/efeitos dos fármacos , Calo Ósseo/patologia , Calcificação Fisiológica/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/ultraestrutura , Modelos Animais de Doenças , Endotélio/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fator de Necrose Tumoral alfa/metabolismo , Microtomografia por Raio-X
9.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(6): 638-643, 2019 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-31955538

RESUMO

OBJECTIVE: To investigate the effects of bio-crosslinker genipin pretreatment on type Ⅰ collagen mineralization. METHODS: Type Ⅰ collagen gels were prepared and pretreated with 0.5wt%genipin (experimental group) and deionized water (control group) for 2 h, respectively. The pretreated products were subjected to Fourier transform infrared spectroscopy (FT-IR). Reconstituted collagen fibrils were pretreated with genipin or deionized water for 2 h and were mineralized for 4 h. The collagen density and mineralization degree were examined with transmission electron microscopy (TEM) and analyzed with ImageJ software. Then scanning electron microscopy (SEM) and TEM were used to observe the mineralization of cross-linked demineralized dentin collagen. RESULTS: FT-IR spectrum showed that the genipin was crosslinked with collagen. TEM observation and ImageJ results showed that after 4 h mineralization, the mineralization effect of 0.5wt% genipin group was significantly better than that of the control group[(73.3±5.3)%vs.(7.4±3.5)%,P<0.01]. TEM and SEM observation showed that the mineralization rate of type Ⅰ collagen and demineralized dentin pretreated with genipin were significantly faster than that of the control group. CONCLUSIONS: The study demonstrates that 0.5 wt% concentration of genipin can significantly promote the mineralization of type Ⅰ collagen.


Assuntos
Colágeno Tipo I , Iridoides , Colágeno Tipo I/química , Colágeno Tipo I/ultraestrutura , Iridoides/química , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Espectroscopia de Infravermelho com Transformada de Fourier
10.
J Biomed Mater Res A ; 107(2): 312-318, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29896910

RESUMO

For efficient manufacturing of fibrous collagen-based materials by electrospinning, the search on optimal rheological parameters is of the great importance. Rheological characteristics and denaturation of collagen in aqueous dispersions were studied as a function of shear rate and acetic acid concentration in the range of 3-9% w/w at temperature from 20 to 40°C. It was shown that an increase in temperature, acetic acid concentration of the collagen dispersion leads to a significant decrease in its viscosity. It was found that helical conformation of the collagen macromolecules is preserved up to 31°C. An increase in acetic acid concentration leads to a reduction of denaturation temperature. The complex viscosity of collagen dispersions exhibits a sharp drop, followed by a rapid growth of damping factor in the temperature range from 22 to 35°C. Both storage (G') and loss (G″) moduli increase with frequency and collagen concentration. It was revealed that optimal parameters for electrospinning of highly concentrated collagen dispersions can be achieved by adjusting of the concentration of acetic acid, temperature, and stirring speed. As a result, collagen nonwoven materials with diameter from 100 to 700 nm were obtained. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 312-318, 2019.


Assuntos
Colágeno Tipo I/química , Nanofibras/química , Animais , Bovinos , Colágeno Tipo I/ultraestrutura , Nanofibras/ultraestrutura , Conformação Proteica em alfa-Hélice , Desnaturação Proteica , Reologia , Temperatura , Viscosidade
11.
Mar Drugs ; 16(10)2018 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-30257422

RESUMO

Collagen from a marine resource is believed to have more potential activity in bone tissue engineering and their bioactivity depends on biochemical and structural properties. Considering the above concept, pepsin soluble collagen (PSC) and acid soluble collagen (ASC) from blue shark (Prionace glauca) skin were extracted and its biochemical and osteogenic properties were investigated. The hydroxyproline content was higher in PSC than ASC and the purified collagens contained three distinct bands α1, α2, and ß dimer. The purity of collagen was confirmed by the RP-HPLC profile and the thermogravimetric data showed a two-step thermal degradation pattern. ASC had a sharp decline in viscosity at 20⁻30 °C. Scanning electron microscope (SEM) images revealed the fibrillar network structure of collagens. Proliferation rates of the differentiated mouse bone marrow-mesenchymal stem (dMBMS) and differentiated osteoblastic (dMC3T3E1) cells were increased in collagen treated groups rather than the controls and the effect was dose-dependent, which was further supported by higher osteogenic protein and mRNA expression in collagen treated bone cells. Among two collagens, PSC had significantly increased dMBMS cell proliferation and this was materialized through increasing RUNX2 and collagen-I expression in bone cells. Accordingly, the collagens from blue shark skin with excellent biochemical and osteogenic properties could be a suitable biomaterial for therapeutic application.


Assuntos
Osso e Ossos/metabolismo , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Tubarões , Engenharia Tecidual/métodos , Animais , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Colágeno Tipo I/química , Colágeno Tipo I/isolamento & purificação , Colágeno Tipo I/ultraestrutura , Células-Tronco Mesenquimais , Camundongos , Microscopia Eletrônica de Varredura , Osteoblastos , Osteogênese/efeitos dos fármacos , Pepsina A/química , Pele/química , Solubilidade , Viscosidade
12.
Am J Physiol Endocrinol Metab ; 315(4): E650-E661, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29894201

RESUMO

Widespread use of pancreatic islet transplantation for treatment of type 1 diabetes (T1D) is currently limited by requirements for long-term immunosuppression, limited donor supply, and poor long-term engraftment and function. Upon isolation from their native microenvironment, islets undergo rapid apoptosis, which is further exacerbated by poor oxygen and nutrient supply following infusion into the portal vein. Identifying alternative strategies to restore critical microenvironmental cues, while maximizing islet health and function, is needed to advance this cellular therapy. We hypothesized that biophysical properties provided through type I oligomeric collagen macroencapsulation are important considerations when designing strategies to improve islet survival, phenotype, and function. Mouse islets were encapsulated at various Oligomer concentrations (0.5 -3.0 mg/ml) or suspended in media and cultured for 14 days, after which viability, protein expression, and function were assessed. Oligomer-encapsulated islets showed a density-dependent improvement in in vitro viability, cytoarchitecture, and insulin secretion, with 3 mg/ml yielding values comparable to freshly isolated islets. For transplantation into streptozotocin-induced diabetic mice, 500 islets were mixed in Oligomer and injected subcutaneously, where rapid in situ macroencapsulation occurred, or injected with saline. Mice treated with Oligomer-encapsulated islets exhibited rapid (within 24 h) diabetes reversal and maintenance of normoglycemia for 14 (immunocompromised), 90 (syngeneic), and 40 days (allogeneic). Histological analysis showed Oligomer-islet engraftment with maintenance of islet cytoarchitecture, revascularization, and no foreign body response. Oligomer-islet macroencapsulation may provide a useful strategy for prolonging the health and function of cultured islets and has potential as a subcutaneous injectable islet transplantation strategy for treatment of T1D.


Assuntos
Colágeno Tipo I/uso terapêutico , Diabetes Mellitus Experimental/cirurgia , Diabetes Mellitus Tipo 1/cirurgia , Sobrevivência de Enxerto , Secreção de Insulina , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/metabolismo , Sobrevivência de Tecidos , Animais , Colágeno Tipo I/ultraestrutura , Técnicas de Cultura , Derme/química , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Colágenos Fibrilares/uso terapêutico , Técnicas In Vitro , Ilhotas Pancreáticas/anatomia & histologia , Camundongos , Microscopia Confocal , Polimerização , Suínos
13.
J Struct Biol ; 203(3): 255-262, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29758270

RESUMO

Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa)n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502GFPGER507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI.


Assuntos
Colágeno Tipo I/química , Osteogênese Imperfeita/genética , Conformação Proteica , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Dicroísmo Circular , Colágeno Tipo I/genética , Colágeno Tipo I/ultraestrutura , Glicina/genética , Humanos , Ligação de Hidrogênio , Mutação de Sentido Incorreto , Osteogênese Imperfeita/patologia , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína
14.
Spine J ; 18(6): 1081-1087, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29477753

RESUMO

BACKGROUND CONTEXT: Studies over the past 20 years have revealed that there are fibrous connective tissues between the suboccipital muscles, nuchal ligament, and cervical spinal dura mater (SDM). This fibrous connection with the SDM is through the posterior atlanto-occipital or atlantoaxial interspaces and is called the myodural bridge (MDB). Researchers have inferred that the MDB might have important functions. It was speculated that the function of MDB might be related to proprioception transmission, keeping the subarachnoid space and the cerebellomedullary cistern unobstructed, and affecting the dynamic circulation of the cerebrospinal fluid. In addition, clinicians have found that the pathologic change of the MDB might cause cervicogenic or chronic tension-type headache. Previous gross anatomical and histologic studies only confirmed the existence of the MDB but did not reveal the fiber properties of the MDB. This is important to further mechanical and functional research on the MDB. PURPOSE: Multiple histologic staining methods were used in the present study to reveal the various origin and fiber properties of the MDB. Muscles and ligaments participating in forming the MDB at the posterior atlanto-occipital or atlantoaxial interspaces were observed, and the fiber properties of the MDB were confirmed. The present study provides a basis for speculating the tensile force values of the MDB on the SDM and a morphologic foundational work for exploring the physiological functions and clinical significances of the MDB. STUDY DESIGN: Anatomical and histologic analyses of suboccipital structures that communicate with the SDM at the posterior atlanto-occipital or atlantoaxial interspaces were carried out. METHODS: Multiple histologic staining methods were used to evaluate the histologic properties and composition of the MDB at the posterior atlanto-occipital or atlantoaxial interspaces in five formalin-fixed head-neck human specimens. RESULTS: The results show that the MDB traversing the atlanto-occipital interspace originated from the rectus capitis posterior minor (RCPmi). The MDB traversing the atlantoaxial interspace originated mainly from the RCPmi, rectus capitis posterior major, and obliquus capitis inferior. These fibers form the vertebral dural ligament in the atlantoaxial interspace and connect with SDM. The MDB is mainly formed by parallel running type I collagen fibers; thus, suboccipital muscle could pull SDM strongly through the effective force propagated by the MDB during head movement. CONCLUSIONS: Myodural bridge is mainly formed by parallel running type I collagen fibers; thus, it can transmit the strong pull from the diverse suboccipital muscles or ligaments during head movement. The results of the present study will serve as a basis for further biomechanical and functional MDB research.


Assuntos
Vértebras Cervicais/anatomia & histologia , Dura-Máter/anatomia & histologia , Ligamentos/anatomia & histologia , Músculos do Pescoço/anatomia & histologia , Pescoço/anatomia & histologia , Colágeno Tipo I/ultraestrutura , Humanos , Orientação Espacial
15.
J Bone Miner Res ; 33(6): 1166-1182, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29461659

RESUMO

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen. © 2018 American Society for Bone and Mineral Research.


Assuntos
Colágeno Tipo I/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Imagem Molecular/métodos , Osteoblastos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Linhagem Celular , Colágeno Tipo I/ultraestrutura , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Camundongos , Osteoblastos/ultraestrutura , Imagem com Lapso de Tempo
16.
Methods Cell Biol ; 143: 79-95, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29310793

RESUMO

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders.


Assuntos
Colágeno Tipo I/ultraestrutura , Matriz Extracelular/ultraestrutura , Imagem Molecular/métodos , Proteólise , Coloração e Rotulagem/métodos , Animais , Colágeno Tipo I/química , Doenças do Tecido Conjuntivo/etiologia , Doenças do Tecido Conjuntivo/patologia , Matriz Extracelular/química , Humanos , Imuno-Histoquímica/métodos , Microscopia Eletrônica de Varredura/instrumentação , Microscopia Eletrônica de Varredura/métodos , Imagem Molecular/instrumentação , Coloração e Rotulagem/instrumentação
17.
Int J Biol Macromol ; 107(Pt A): 549-559, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28911806

RESUMO

The present study isolated and characterized the barramundi (Lates calcarifer) skin collagen (BC) and tilapia (Oreochromis niloticus) skin collagen (TC). The yields of BC and TC by enzymatic extraction were 47.3±3.7% and 52.6±6.1% respectively, dry weight. The SDS-PAGE profile indicated both collagens were mainly type I with two different α chains. FTIR spectra and X-ray diffraction confirmed that the triple helical structure of both collagens was not affected by pepsin digestion. The denaturation (Td) and melting temperature (Tm) were 36.8 and 109.6°C for BC, 37.6 and 113.7°C for TC, measured by rheometer and DSC. This high thermal stability could be attributed to the high imino acid content (205.9 and 210.9 residues/1000 residues) of BC and TC. Fibril-forming studies indicated BC exhibited higher ability than (p<0.05) that of TC, especially under the effect of NaCl. One major characteristic of this result showed that NaCl had the markedly effects of promoting collagen forming fibrils, and electron microscopic observation corroborated this phenomenon. In general, barramundi and tilapia skin collagen with high thermal stability and good fibril-forming ability may be suitable for use as an alternative to mammalian-derived collagen in biomaterials, pharmaceuticals and cosmetics.


Assuntos
Colágeno Tipo I/química , Proteínas de Peixes/química , Percas/metabolismo , Pele/química , Tilápia/metabolismo , Animais , Colágeno Tipo I/isolamento & purificação , Colágeno Tipo I/ultraestrutura , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/ultraestrutura , Pepsina A/química , Conformação Proteica em alfa-Hélice , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/ultraestrutura , Estabilidade Proteica , Proteólise
18.
Int J Biol Macromol ; 106: 516-522, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28801096

RESUMO

Collagens were extracted from the scales and skin of Ctenopharyngodon idella (C. idella) as raw materials using an acid-enzyme hybrid method. The structural properties of the extracted collagens were compared using ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and differential scanning calorimetry. Additionally, the in vitro self-aggregation behaviors of the two types of collagens (fish skin- and scale-derived collagens) were compared using turbidimetric assays, aggregation assays, and scanning electron microscopy (SEM). The results showed that both types of extracted collagen were typical type I collagen with two α chains and intact triple-helical structures. The denaturation temperatures of the collagens from fish scales and skin were 34.99°C and 39.75°C, respectively. Both types of collagens were capable of self-aggregation in neutral salt solution at 30°C, with aggregation degrees of 28% and 27.33% for the scale and skin collagens, respectively. SEM analysis revealed that both types of collagens could self-aggregate into interwoven fibers, and the fish scale-derived collagen had a more pronounced reticular fiber structure with a striped periodic D-band pattern of collagen fibrils, whereas the collagen fibers from the self-aggregation of fish skin-derived collagen had a certain degree of disruption without any D-band pattern.


Assuntos
Escamas de Animais/química , Colágeno Tipo I/química , Proteínas de Peixes/química , Agregados Proteicos , Pele/química , Animais , Carpas/metabolismo , Colágeno Tipo I/isolamento & purificação , Colágeno Tipo I/ultraestrutura , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/ultraestrutura , Extração Líquido-Líquido/métodos , Especificidade de Órgãos , Conformação Proteica em alfa-Hélice , Desnaturação Proteica , Solubilidade , Temperatura
19.
Cells Tissues Organs ; 204(5-6): 261-269, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29055948

RESUMO

As a result of restrictions on animal experimentation, improved skin equivalents (SEs) are needed as alternative test models. This work investigated the effects of avian collagen on the construction of SEs, and to the best of our knowledge is the first study to do so. Hematoxylin and eosin and immunohistochemical staining were used to analyze the SEs. In models containing avian collagen as a dermal equivalent (DE) ingredient, fibroblast proliferation increased by about 60% relative to the control model. Immunohistochemical staining showed that the expression of proliferating cell nuclear antigen (PCNA) and p63 increased in the avian collagen models, while the expression of involucrin, integrin α6, and integrin ß1 remained unchanged. Next, DEs were cryopreserved to allow the easier creation of SEs. Keratinocytes were seeded on thawed DEs, and SEs were constructed. Avian collagen increased the viability of DEs relative to the control. Furthermore, avian collagen increased the expression of PCNA and p63 in keratinocytes on thawed DEs. The results indicate that DEs containing avian collagen can be thawed as needed after cryopreservation. Avian collagen can improve the construction of SEs and be used as part of a dermal kit for SE construction.


Assuntos
Proteínas Aviárias/química , Materiais Biocompatíveis/química , Colágeno Tipo I/química , Fibroblastos/citologia , Pele Artificial , Animais , Aves , Linhagem Celular , Proliferação de Células , Colágeno Tipo I/ultraestrutura , Criopreservação , Humanos , Ratos
20.
J Proteome Res ; 16(8): 2914-2923, 2017 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-28696707

RESUMO

Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.


Assuntos
Colágeno Tipo I , Ciclofilinas/deficiência , Dentina/ultraestrutura , Animais , Colágeno Tipo I/ultraestrutura , Ciclofilinas/fisiologia , Dentina/patologia , Matriz Extracelular/patologia , Matriz Extracelular/ultraestrutura , Glicosilação , Hidroxilação , Lisina/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Knockout , Osteogênese Imperfeita , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Processamento de Proteína Pós-Traducional
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