Type V collagen: heterotypic type I/V collagen interactions in the regulation of fibril assembly

Type V collagen is a quantitatively minor fibrillar collagen with a broad tissue distribution. The most common type V collagen isoform is alpha1(V)(2) alpha2(V) found in cornea. However, other isoforms exist, including an [alpha1(V)alpha2(V)alpha3(V)] form, an alpha1(V)(3) homotrimer and hybrid type V/XI forms. The functional role and fibrillar organization of these isoforms is not understood. In the cornea, type V collagen has a key role in the regulation of initial fibril assembly. Type I and type V collagen co-assemble into heterotypic fibrils. The entire triple-helical domain of the type V collagen molecules is buried within the fibril and type I collagen molecules are present along the fibril surface. The retained NH(2)-terminal domains of the type V collagen are exposed at the surface, extending outward through the gap zones. The molecular model of the NH(2)-terminal domain indicates that the short alpha helical region is a flexible hinge-like region allowing the peptide to project away from the major axis of the molecule; the short triple-helical regions serve as an extension through the hole zone, placing the tyrosine-rich domain at the surface. The assembly of early, immature fibril intermediates (segments) is regulated by the NH(2)-terminal domain of type V collagen. These NH(2)-terminal domains alter accretion of collagen molecules onto fibrils and therefore lateral growth. A critical density would favor the initiation of new fibrils rather than the continued growth of existing fibrils. Other type V collagen isoforms are likely to have an important role in non-cornea tissues. This role may be mediated by supramolecular aggregates different from those in the corneal stroma or by an alteration of the interactions mediated by tissue-specific type V collagen domains generated by different isoforms or aggregate structures. Presumably, the aggregate structure or specific domains are involved in the regionalization of fibril-associated macromolecules necessary for the tissue-specific regulation of later fibril growth and matrix assembly stages.     

胶原调制磷酸钙矿化成核位点的红外光谱研究

对胶原、胶原 /CaCl2 、胶原 /羟基磷灰石等几种体系的红外光谱进行了比较研究。发现胶原矿化后 ,红外光谱中胶原的酰胺Ⅰ ,Ⅱ ,Ⅲ带的吸收强度显著降低 ,Ⅱ ,Ⅲ带接近消失 ,Ⅰ带发生红移现象。提出胶原中的羧基 (COOH )、羰基 (CO )为胶原矿化的两类成核位点 ,钙盐在成核位点上结晶。脱钙胶原(胶原 /羟基磷灰石经盐酸处理 )的红外光谱与纯胶原相似 ,酰胺Ⅰ ,Ⅱ ,Ⅲ带的吸收强度恢复正常 ,表明 :钙盐对胶原中酰胺基的影响是由于钙盐在该成核位点上结晶而导致的“包裹”作用。根据酰胺 3个吸收带的不同振动方式 ,用“包裹”作用解释了胶原 /钙盐复合物中 ,酰胺Ⅰ与Ⅱ ,Ⅲ带的降低程度不同的原因。分析了胶原 /磷酸钙生物矿化的作用机制。     

Transcutaneous application of carbon dioxide improves contractures after immobilization of rat knee joint

Objective: Joint contractures are a major complication following joint immobilization. However, no fully effective treatment has yet been found. Recently, carbon dioxide (CO₂) therapy was developed and verified this therapeutic application in various disorders. We aimed to verify the efficacy of transcutaneous CO₂ therapy for immobilization-induced joint contracture. Method: Twenty-two Wistar rats were randomly assigned to three groups: caged control, those untreated after joint immobilization, and those treated after joint immobilization. The rats were treated with CO₂ for 20 min once a daily either during immobilization, (prevention) or during remobilization after immobilization (treatment). Knee extension motion was measured with a goniometer, and the muscular and articular factors responsible for contractures were calculated. We evaluated muscle fibrosis, fibrosis-related genes (collagen Type 1α1 and TGF-β1) in muscles, synovial intima''s length, and fibrosis-related proteins (Type I collagen and TGF-β1) in the joint capsules. Results: CO₂ therapy for prevention and treatment improved the knee extension motion. Muscular and articular factors decreased in rats of the treatment group. The muscular fibrosis of treated rats decreased in the treatment group. Although CO₂ therapy did not repress the increased expression of collagen Type 1α1, the therapy decreased the expression of TGF-β1 in the treatment group. CO₂ therapy for treatment improved the shortening of the synovial membrane after immobilization and decreased the immunolabeling of TGF-β1 in the joint capsules. Conclusions: CO₂ therapy may prevent and treat contractures after joint immobilization, and appears to be more effective as a treatment strategy for the deterioration of contractures during remobilization.     

Effects of Combined Stretching and Clenbuterol on Disuse Atrophy in Rat Soleus Muscle

Background and Purpose: Clinically, disuse muscle atrophy is often seen among patients who are severely debilited and are on prolonged bed rest. Common physical therapy interventions are not successful in preventing disuse muscle atrophy early in the medical treatment of critically ill patients. In situations such as this, the use of a β₂-adrenergic agonist such as clenbuterol (Cb) may be of benefit in preventing atrophy. Also, recent studies have suggested that stretching is possible in preventing disuse muscle atrophy and the decline in muscle strength. The objective of this study was to evaluate the effects of Cb medication combined with stretching (ST) on rat soleus muscle (SOL) during the progression of disuse muscle atrophy. Subjects: Thirty-five male Wistar rats were used in this study. Methods: The rats were divided into five groups: control (CON), hindlimb-unweighting (HU) only, HU+ST, HU+Cb medication, and HU+ST+Cb groups. The right SOL in stretching groups was maintained a stretched position for one hour daily by passively dorsiflexing the ankle joint under non-anesthesia. The experimental period was 2 weeks. Results: In the ST group, peak twitch tension per cross-sectional area in soleus muscle was significantly larger than in the Cb group, while there was no significant difference between the CON and ST groups. The conversion of type I to type II fibers that was observed in the Cb group was not recognized in the combined ST and Cb group. Discussion and Conclusion: Distinct effect of combined stretching and Cb medication was not recognized statistically. The results indicate that Cb affects muscle morphological characteristics while stretching affects contractile properties. These data suggest that a combined ST and Cb intervention considered the type-specificity of muscle fiber may be need more consideration for preventing disuse muscle atrophy and the decline in muscle strength.     

Effects of aging and maternal protein restriction on the muscle fibers morphology and neuromuscular junctions of rats after nutritional recovery

Changes in the nutritional status of mothers may predispose their offspring to neuromuscular disorders in the long term. This study evaluated the effects of maternal protein restriction during pregnancy and lactation on the muscle fibers and neuromuscular junctions (NMJs) of the soleus muscle in the offspring of rats at 365 days of age that had undergone nutritional recovery. Wistar rats were divided into two groups: control (CG) – the offspring of mothers fed a normal protein diet (17%) and restricted (RG) – offspring of mothers fed a low protein diet (6%). After lactation, the male pups received standard chow ad libitum. At 365 days, samples of soleus muscle were collected for muscle fiber analysis (HE staining, NADH-TR reaction and ultrastructure), intramuscular collagen quantification (picrosirius red staining) and NMJs analysis (non-specific esterase technique). The cross-sectional area of type I fibers was reduced by 20% and type IIa fibers by 5% while type IIb fibers increased by 5% in the RG compared to the CG. The percentage of intramuscular collagen was 19% lower in the RG. Disorganization of the myofibrils and Z line was observed, with the presence of clusters of mitochondria in both groups. Regarding the NMJs, in the RG there was a reduction of 10% in the area and 17% in the small diameter and an increase of 7% in the large diameter. The results indicate that the effects of maternal protein restriction on muscle fibers and NMJs seem to be long-lasting and irreversible.     

胶原纤维网络和癌细胞的力学微环境

文章以第一类胶原纤维网络为例, 着重分析了癌细胞三维微环境的多尺度结构及力学特征. 对于细胞与细胞外介质结合的蛋白集团、单个细胞以及细胞群体, 分别由单个纤维(或亚纤维)、纤维集束以及纤维网络整体来决定相应的力学环境. 文章同时也讨论了胶原纤维(及其类似材料) 的局限性.     

Skin aging and natural photoprotection

Aging of skin is a continuous process that may be enhanced by sun exposure. Photoaging may provoke changes different from aging. Epidermal changes involve thinning of stratum spinosum and flattening of the dermo-epidermal junction. The senescent keratinocytes becomes resistant to apoptosis and may survive for a long time giving time for DNA and protein damage to accumulate with possible implication for carcinogenesis. The numbers of melanocytes decrease with age with dysregulation of melanocyte density resulting in freckles, guttate hypo-melanosis, lentigines and nevi. The number of dendritic Langerhans cells also decreases with age and the cells get less dendrites and have reduced antigen-trapping capacity. Aging involves dermal changes such as damage to elastic and collagen fibers giving thickened, tangled, and degraded non-functional fibers. Collagen intermolecular cross-links are stable and essential for stability and tensile strength. Cross-links increase with age converting divalent cross-links into mature trivalent cross-links of, e.g. histidinohydroxylysinonorleucine. Two mechanisms are involved; an enzyme-controlled process of maturation and a non-enzymatic glycosylation, the Maillard reaction leading to cross-links in proteins such as in collagen between arginine and lysine. Such may be seen with age and in diabetes mellitus. However, autofluorescence studies have shown that UVR reduces collagen cross-links. Natural photoprotection involves thickening of stratum corneum by sunlight and increased pigmentation. This leads to a factor 2 increase in photoprotection from spring until after-summer. The constitutive pigmentation is independent of age and thickness of stratum corneum is likewise independent of age. The minimal erythema dose is thus the same through life, when corrected for pigmentation or measured in areas with constitutive pigmentation.     

温度对鱼鳞胶原蛋白二级结构的影响

从草鱼鱼鳞中提取酶溶性胶原蛋白(PSC),通过SDS-PAGE电泳分析为典型Ⅰ型胶原蛋白且达到电泳纯。在此基础上利用傅里叶变换红外光谱(FTIR)、拉曼光谱(Raman)和圆二色谱(CD)研究了温度对鱼鳞胶原蛋白二级结构的影响。FTIR分析表明：鱼鳞胶原蛋白具有典型的胶原蛋白特征吸收带,酰胺Ⅰ、酰胺Ⅱ和酰胺Ⅲ带的特征吸收频率分别出现在1658,1552和1238cm-1处。随温度升高,酰胺A和酰胺B峰位向低波数移动,1658cm-1处吸收峰裂解成多个吸收峰;1552cm-1处的吸收峰在35℃微略红移,随后发生明显蓝移;1238cm-1处吸收峰随温度升高向低波数移动。在拉曼光谱中,胶原蛋白的酰胺Ⅰ、酰胺Ⅱ和酰胺Ⅲ带的特征吸收频率分别出现在1669,1557和1245cm-1处,都较红外光谱的波数高;此外,921和855cm-1处脯氨酸的特征谱峰在拉曼光谱中体现出来。圆二色谱分析表明,胶原蛋白溶液在221.6和204.4nm分别有一正、负峰,具有典型胶原蛋白三螺旋结构的特征圆二色谱峰型。胶原蛋白冻干品的FTIR光谱和Raman谱线大都在35～60℃时发生波数和强度改变,而胶原蛋白乙酸溶液的CD谱线在20～35℃之间发生剧烈改变。由此可以判断胶原蛋白在固态和溶液状态下,变性温度存在一定差异,胶原蛋白冻干品比其乙酸溶液更稳定。     

Collagen fibers evaluation after rhBMP-2 insertion in critical-sized defects

The objective of this investigation was to assess the quantity of collagen fibers with different dosages of recombinant human bone morphogenetic protein, type 2 (rhBMP-2) associated with two different carriers, monoolein and poloxamer gels, in critical bone defects created in the calvaria of Wistar rats. Forty male adult Wistar rats were divided into eight groups of 5 animals each—group I: critical bone defect with application of 1 μg of rhBMP-2 combined with monoolein gel; group II: 3 μg of rhBMP-2 combined with monoolein gel; group III: 7 μg of rhBMP-2 combined with monoolein gel; group IV: 1 μg of rhBMP-2 combined with poloxamer gel; group V: 3 μg of rhBMP-2 combined with poloxamer gel; group VI: 7 μg of rhBMP-2 combined with poloxamer gel; group VII: monoolein gel only and group VIII: poloxamer gel only. A critical-sized defect of 6 mm diameter was produced in the left parietal bone using a surgical round bur and a high-speed micromotor. The bone defects were filled according to the group that animals belonged and after two weeks the rats were perfused and their calvarial bones were removed for histological processing, and collagen fibers quantification. Differences among the eight groups were statistically analyzed by Anova and Bonferroni test (p < 0.05). The results did not show statistical difference between the groups, in exception, between the comparisons II and III. According to the experimental methodology used in this research, it was observed, in a general way, a qualitative inverse relationship between collagen fibers presence and rhBMP-2 quantity inserted in the critical bone defect, associated or not to a material carrier.     

Atomic force microscopy investigation of chemically stabilized pericardium tissue

Native and chemically stabilized porcine pericardium tissue was imaged by the contact mode atomic force microscopy (AFM), in air. Chemically stabilized pericardium is used as a tissue-derived biomaterial in various fields of the reconstructive and replacement surgery. Collagen type I is the main component of the fibrous layer of the pericardium tissue. In this study, the surface topography of collagen fibrils in their native state in tissue and after chemical stabilization with different cross-linking reagents: glutaraldehyde (GA), dimethyl suberimidate (DMS) and tannic acid (TA) was investigated. It has been found that chemical stabilization causes considerable changes in the surface topography of collagen fibrils as well as in the spatial organization of the fibrils within the tissue. The observed changes in the D-spacing pattern of the collagen fibril correspond to the formation of intrafibrilar cross-links, whereas formation of interfibrilar cross-links is mainly responsible for the observed tangled spatial arrangement of fibrils and crimp structure of the tissue surface. The crimp structure was distinctly seen for the GA cross-linked tissue. Surface heterogeneity of the cross-linking process was observed for the DMS-stabilized tissue. SDS-PAGE electrophoresis was performed in order to evaluate the stabilization effect of the tissues treated with the cross-linking reagents. It has been found that stabilization with DMS, GA or TA enhances significantly the tissue resistance to SDS/NaCl extraction. The relation between the tissue stability and changes in the topography of the tissue surface was interpreted in terms of different nature of cross-links formed by DMS, GA and TA with collagen.     

Nuclear microscopy of normal and necrotic skeletal muscle fibers: an intracellular elemental microanalysis

The in situ total elemental composition and elemental concentrations present in mouse soleus (type I) and gastrocnemius (type IIA) muscle fibers were analyzed by using nuclear microscopy (NM). Elemental changes in necrotic fibers, induced by intramuscluar injection with snake venom (Pseudechis australis), were also studied 3 h post-injection. Nuclear microscopy is a new method based on nuclear technology that utilizes the interaction between a million-electron-volt nuclear particle beam and the muscle sample (in the case of the present study). Elemental analysis was done at the parts per million (ppm) level of sensitivity on unfixed, rapidly frozen and unstained single fast- and slow-twitch muscle fibers with imaging capabilities of μm spatial resolution and in multi-elemental mode. In total, 12 different intracellular elements were mapped, co-localized and analyzed in single normal and necrotic skeletal muscle fibers from mice. Elements such as potassium, sulfur, phosphorus, chlorine and sodium were found in concentrations from 1000 to 18 000 ppm. Unlike conventional electron-probe X-ray microanalysis, NM also detected and analyzed the trace elements such as magnesium, calcium, iron and zinc that were found in concentrations of 50 to 1000 ppm. Other elements — copper, manganese and rubidium — were also detected in concentrations of less than 50 ppm. The trace elements calcium, iron and zinc were more abundant in the soleus than the gastrocnemius (the level of iron was statistically significant). Calcium, sodium and chlorine were significantly elevated in venom-induced necrotic soleus muscle fibers.     

Joint Function and Arthropathy Severity in Patients with Hemophilia

Background: The Arnold-Hilgartner classification is one of the most popular evaluation systems for the progression hemophilic arthropathy. A previous study reported an association between arthropathy severity and arc range of motion (ROM). However, associations between arthropathy severity and angular ROM and muscle strength remain unclear. AIM: The purpose of this study was to clarify the association between joint function and arthropathy severity in hemophilia. Methods: We studied the knee, ankle, and elbow joints of 31 patients with hemophilia (PWH). The condition of the affected joints was evaluated on the basis of the interview data, joint function measurements, and roentgenography of the affected joints. In assessment of joint function, we evaluated knee strength (flexor, extensor) and grip strength as well as the passive ROM of the elbow, knee, and ankle. During the interview, all patients were asked about the history of intra-articular bleeding over the past year and pain. Results: As arthropathy severity worsened, knee flexor strength, knee extensor strength, grip strength, and ROM (elbow flexion, elbow extension, knee flexion, knee extension, and ankle extension) significantly decreased. Even patients with mild arthropathies experienced knee extensor weakness and extension limitation. In addition, joint function of severe ankle arthropathy was significantly related to the history of intra-articular bleeding and pain. Conclusion: Our results suggest that physical therapy is necessary to improve joint function in PWH and mild or no arthropathy. Pain control and prophylactic hematological management are necessary for patients with severe arthropathy because intra-articular bleeding and pain significantly decrease joint function.     

Unexpected ultrastructral changes in bone osteiod collagens in osteogenesis imperfecta

Osteogenesis Imperfecta (OI) is a heterogeneous, inherited bone disorder usually resulting from a defect in collagen synthesis or function. The Sillence classification recognises four OI subtypes of which type III is the severe, progressively deforming form. Here, we report distinctive ultrastructural abnormalities of bone osteoid collagen fibrils from three patients with OI type III and compared with normal controls. Collagen biochemistry of these patients showed normal alpha1(I) and alpha2(I) chains, despite the structurally abnormal collagen fibrils.

The expected lamellar organisation of normal osteoid was absent in the bone biopsies of these patients. In addition their collagen fibrils had frayed edges and no periodicity was observed in most of these fibrils. These collagen fibrils were also flower like, twisted, spiralled and sparsely distributed throughout a very thick osteoid with patchy mineralisation.

These structurally abnormal collagens may not be able to provide the nucleating and scaffolding sites for normal mineralisation and may lead to the bone fragility observed in OI.     

The fibril structure of type V collagen triple-helical domain

Although the triple-helical structure of fibrillar collagen is regarded in general as being quite similar, each type of collagen molecule has inherent characteristics in the triple-helical domain. Few studies have ever been performed in terms of the aggregate structure of the triple-helical domain of fibrillar collagen. Reconstituted aggregates from the purified triple-helical domain of each type of fibrillar collagen might amplify the subtle differences in the structural characteristics of each type of collagen molecule. In this study, the reconstituted aggregate structure of pepsin-treated type V collagen (type Vp collagen), that is, virtually its triple-helical domain was characterized by transmission electron microscopy. Pepsin-treated type I (type Ip) and type II (type IIp) collagen were compared with type Vp collagen. Unique features of the aggregate structure of the triple-helical domain of the type V collagen can be summarized as follows:These results suggested that the lateral packing of the triple-helical domain of type V collagen is determined by its molecular structure. The characteristics of type Vp collagen fibrils might be explained by their characteristic amino acid composition. A significant feature of the triple-helical domain of type V collagen is the high content of glycosylated hydroxylysine residues. Molecular model building of the collagenous structure suggests that a change in surface roughness is conspicuous by incorporating the glycosylated hydroxylysine residues. More than a ten-fold content of bulky glycosylated hydroxylysine residues in type V collagen compared to that of type I might have a significant influence on both the intermolecular and interfibrillar interactions of the triple-helical domain of type V collagen molecule.     

Differences in Cartilage Repair between Loading and Unloading Environments in the Rat Knee

We investigated the histopathological and immunohistochemical effects of loading on cartilage repair in rat full-thickness articular cartilage defects. A total of 40 male 9-week-old Wistar rats were studied. Full-thickness articular cartilage defects were created over the capsule at the loading portion in the medial condyle of the femur. Twenty rats were randomly allocated into each of the 2 groups: a loading group and a unloading group. Twenty rats from these 2 groups were later randomly allocated to each of the 2 groups for evaluation at 1 and 2 weeks after surgery. At the end of each period, knee joints were examined histopathologically and immunohistochemically. In both groups at 1 and 2 weeks, the defects were filled with a mixture of granulation tissue and some remnants of hyaline cartilage. The repair tissue was not stained with toluidine blue in both groups. Strong staining of type I collagen was observed in the repair tissue of both groups. The area stained with type I collagen was smaller in the unloading group than in the loading groups, and the stained area was smaller at 2 weeks than at 1 week. In the staining for type II collagen, apparent staining of type II collagen was observed in the repair tissue of both groups at 1 week. At 2 weeks, there was a tendency toward a higher degree of apparent staining in the loading group than in the unloading group. Accordingly, these results indicated that loading and unloading in the early phase of cartilage repair have both merits and demerits.     

Disuse atrophy alterations in normal and low temperature environments during hindlimb unloading in Syrian hamsters

This study examined whether a hypothermic environment reduces experimentally-induced atrophy of skeletal muscle, as judged by histochemical findings. The hind limbs of hamsters in a hypothermic group were suspended and flexed into plantar positions at the ankle joint, and housed for one week at 8 to 12 degree celsius in a temperature-controlled room, while the normothermic group was housed at 23 to 25 degree celsius. Hypothermia did not significantly alter the average caloric intake, and the animals from the hypothermic group lost a significant amount of body weight when compared with the normothermic group. The hypothermic group retained more muscle wet-weight and myofibers cross-sectional area in the soleus and gastrocnemius muscles compared with the normothermic group. Our results indicate that a hypothermic environment inhibits short-term muscle atrophy. This inhibition may be caused by the increased caloric intake combined with a state similar to hibernation in low-temperature environments.     

Antibacterial and Miscibility Properties of Chitosan/Collagen Blends

The miscibility, bioactivity, and antibacterial properties of chitosan/collagen specimens were systematically studied. The specimens were prepared by blending collagen and chitosan with varying deacetylation degrees in solutions; the collagen molecules had been extracted from pigskins using the acid swelling-pepsin digestion method. To understand the miscibility properties of collagen and chitosan molecules, the intrinsic viscosity and differential scanning calorimetry analysis of collagen, chitosan, and collagen/chitosan specimens were performed. The instrinsic viscosity measurements suggested that chitosan and collagen molecules with varying deacetylation degrees were miscible at molecular level for all compositions and degrees of deacetylation of chitosan/collagen mixture solutions prepared in this study. Fourier transform infrared analyses suggested that the percentage of preserved triple helix structures present in collagen molecules in collagen/chitosan specimens decreased with increasing chitosan contents, since the ratios of peak absorbance at 1239 cm^?1 of amide III and 1455 cm^?1 of C?H bending of collagen/chitosan specimens decreased significantly with increase in their chitosan contents. Abnormally high denaturation temperatures (T_d) were observed as the chitosan contents of collagen/chitosan specimens reached 40 wt%, at which T_d of collagen molecules was even higher than that of the corresponding pure chitosan molecules with varying deacetylation degrees. The antibacterial activity of collagen/chitosan blends increased consistently with increasing deacetylation degrees and concentrations of chitosan molecules in collagen/chitosan solutions. Possible explanations for these interesting thermal denaturation, antibacterial, and miscibility properties of chitosan/collagen specimens are reported.     

Effects and mechanisms of cold atmospheric plasma on skin wound healing of rats

Cold atmospheric plasma (CAP) has been reported as a novel potential way to promote skin wound healing. The aim of this study was to explore the effects of an atmospheric‐pressure plasma generated in helium gas through dielectric barrier discharge on skin wound healing in rats and its mechanism. A total of 44 adult male Sprague–Dawley rats were divided into the CAP group and the control group after a full‐thickness skin wound model was established. The animals in the CAP group received CAP treatment (driving frequency 17 kHz, root‐mean‐square [rms] values of the discharge voltage 1.33 kV, rms values of the discharge current 10.7 mA, helium flow rate 11 slpm [standard litres per minute]) for 1 min. After that, the size of the healed area of the wound, morphological changes in the wound, wound neovascularization, collagen deposition, and the level of inflammatory factors were recorded. The results showed that compared to that of the control group, the size of the wound area in the CAP group significantly decreased, the contraction and re‐epithelialization of the wounds were obviously speeded up, and the protein level of smooth muscle actin markedly increased. However, the levels of collagen type I A2, collagen type III, and the deposition of collagen bundles did not change. In addition, it was found that the level of inflammatory factors (such as tumour necrosis factor and interleukin‐1β) significantly increased at 24 hr after the creation of the wound. These results suggest that CAP treatment in this condition could promote skin wound healing by accelerating the contraction, re‐epithelialization, neovascularization, and inflammatory response.