牛磺酸抑制新生大鼠心肌重塑的体外研究

目的 通过牛磺酸对新生大鼠心肌成纤维细胞增殖和胶原合成及对I／Ⅲ型胶原比值的影响,从细胞水平探讨其在体外抑制心肌重塑的作用。方法 在培养的心肌成纤维细胞中加入不同浓度的牛磺酸,用羟脯氨酸法测定胶原量,MTT比色法检测细胞增殖情况,SDS-PAGE电泳测胶原I／Ⅲ型比值。结果 100mmol／L、200mmol／L牛磺酸用药24h和48h及50mmol／L用药72h后能明显抑制心肌成纤维细胞的增殖及胶原的合成,I／Ⅲ型胶原比值明显下降。结论 牛磺酸能通过抑制心肌成纤维细胞增殖和胶原合成,降低I／Ⅲ型胶原比值起到抗心肌重塑的作用。     

超高效液相色谱-三重四级杆质谱法用于阿胶糕类食品中阿胶的鉴别及马、牛、羊、猪皮源成分的检测

目的:建立以特征肽为检测指标的阿胶糕中阿胶的鉴别方法与马皮、牛皮、羊皮、猪皮源成分的检测方法。方法:采用三氯乙酸沉淀法提取阿胶糕中的蛋白质,胰蛋白酶进行酶解处理,利用超高效液相色谱-三重四级杆质谱法(UHPLCQQQ MS),电喷雾离子源(ESI),正离子模式下扫描,采用多重反应监测模式,对特征肽目标离子进行检测。结果:经验证,鉴别方法和检查方法均均有良好的专属性,灵敏度高。市售样品测定结果显示,部分样品中检出阿胶成分,有的样品中检出牛皮源成分,有的样品中未检出任何皮源性成分。结论:所建立方法操作简便,专属性强,可用于阿胶糕中阿胶的鉴别和掺假杂皮胶的检测。     

The effect of a hydrolyzed collagen-based supplement on wound healing in patients with burn: A randomized double-blind pilot clinical trial

IntroductionBurn is among the most severe forms of critical illness, associated with extensive and prolonged physical, metabolic and mental disorders. The aim of this study was to assess the effect of an oral, low-cost, and accessible collagen-based supplement on wound healing in patients with burn.MethodsIn this randomized double-blind controlled pilot clinical trial, 31 men, 18–60 years, with 20–30% total body surface area burn were studied. Patients were randomly assigned to receive either a collagen-based supplement (1000 kcal) or an isocaloric placebo, for 4 weeks. Serum pre-albumin, rate of wound healing, length of hospital stay, and anthropometries were assessed at baseline, and the end of week 2 and 4.ResultsSerum pre-albumin was significantly higher at week 2 (29.7 ± 13.6 vs. 17.8 ± 7.5 mg/dL, P = 0.006) and week 4 (35.1 ± 7.6 vs. 28.3 ± 8.2 mg/dL, P = 0.023) in collagen than control group. Changes in pre-albumin concentration were also significantly higher in collagen group at week 2 (13.9 ± 9.8 vs. −1.9 ± 10.3 mg/dL, P < 0.001) and week 4 (19.2 ± 7.5 vs. 8.5 ± 10.1 mg/dL, P = 0.002). The Hazard ratio of wound healing was 3.7 times in collagen compared to control group (95% CI: 1.434–9.519, P = 0.007). Hospital stay was clinically, but not statistically, lower in collagen than control group (9.4 ± 4.6 vs. 13.5 ± 7 days, P = 0.063). There were no significant differences in weight, body mass index, dietary energy and protein intakes between the two groups.ConclusionThe findings showed that a hydrolyzed collagen-based supplement could significantly improve wound healing and circulating pre-albumin, and clinically reduce hospital stay in patients with 20–30% burn.     

Tendon regeneration induced by umbilical cord graft in a rabbit tendon defect model

Whether tendon regeneration can be induced using the umbilical cord as a whole‐graft structure is unknown. In this study, we explored the potential for tendon regeneration induction using an umbilical cord graft in a rabbit model of patella tendon defects. In 52 of 54 New Zealand White rabbits, the central third of the patella tendons of both hind legs was removed to create tendon defects. The rabbits were randomly divided into four groups, nonfilling (empty defect), refilling (defect refilled with resected tendon portion), Wharton's jelly (WJ) outside (WJO; defect filled with umbilical cord graft, WJ side facing outward), and WJ inside (WJI; same as WJO with WJ side facing inward) groups. Four rabbits from WJO and WJI groups were sacrificed for human CD 105 evaluation 1 month after surgery. Further histological, biomechanical, and gene expression analyses were performed at 3 and 6 months after surgery. The untreated patella tendons in the remaining two rabbits were harvested as normal biomechanical controls. Histological evaluation showed that the formed tissue structure fibers in the tendon defect area were much denser and more mature in the WJI group than in all other groups. Biomechanical testing showed that the failure load of the final tissue structure was the highest in the WJI group. Real‐time polymerase chain reaction indicated that the expression of most tendon‐related genes was upregulated in the WJI group at 6 months after surgery. We concluded that umbilical cord grafting induces effective tendon regeneration, particularly when the WJ side faces inward.     

Collagen from Turkey (Meleagris gallopavo) tendon: A promising sustainable biomaterial for pharmaceutical use

Collagen is the major fibrillar component and protein in both human and animal connective tissue. It is applied in medical preparations, e.g. wound dressings and tissue engineering. Meat and poultry production industries result in large amounts of organic waste, rich in collagen. Our aim was to isolate and characterize pepsin soluble collagen from turkey tendon. Structural analysis indicated the presence of α-chains from both collagen type I and III, β-dimers and γ-trimers, consistent with the estimated molecular weight of 477.3 kDa. Circular dichroism spectroscopy confirmed an intact triple helix. The collagen demonstrated excellent thermal stability, with denaturation temperatures (T_max) at 38.5 °C and 44.5 °C and partial refolding after extensive heating. Biocompatibility was confirmed through cell viability tests. The collagen was investigated for its potential drug carrier ability. Freeze dried collagen scaffolds containing prilocaine hydrochloride and riboflavin were prepared in the presence or absence of photo-crosslinking. Photochemical crosslinking was confirmed by SEM and enhanced mechanical properties were observed. Scaffolds had a significant slower in vitro release of the active ingredient than a reference solution. Altogether, our study suggests collagen from turkey tendon as a promising sustainable biomaterial for pharmaceutical use.     

Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo

We demonstrate that intrafibrillar, homogenous collagen biomineralization can be achieved by controlling self-assembly under mildly alkaline conditions. Using dense collagen (DC) gels as an osteoid model, we modulated their fibrillogenesis environment to evaluate the effects of fibrillogenesis pH on the protein charge distribution and ultimately on biomineralization. Cationic and anionic dye staining and electron cryomicroscopy analyses established that fibrillogenesis under mildly alkaline conditions promotes the formation of electronegative charges within the protein (anionic DC gels). These charges are stable upon titration of the gel pH to physiological values. Subsequent exposure of anionic DC gels to simulated body fluid induced the intrafibrillar biomineralization of the gels, promoting a rapid, extensive formation of carbonated hydroxyapatite, and strongly impacting gel mechanical properties. The generality and significance of this approach has been addressed by implanting freshly made anionic DC gels in vivo, in a rat subcutaneous model. Subcutaneous implants showed an extensive, homogenous biomineralization as early as at day 7, indicating that anionic collagen gels rapidly self-mineralize upon contact with body fluids in a non-osseous implantation site. The control of collagen fibrillogenesis pH provides not only new interpretations to what has been called the collagen mineralization enigma by demonstrating that neat collagen can intrafibrilarly self-mineralize, but it will also set a new starting point for the use of DC gels in bone regenerative medicine, in addition as potential applications as mineralized tissue model or as slow-release delivery carriers.     

Engineering multi-layered skeletal muscle tissue by using 3D microgrooved collagen scaffolds

Preparation of three-dimensional (3D) micropatterned porous scaffolds remains a great challenge for engineering of highly organized tissues such as skeletal muscle tissue and cardiac tissue. Two-dimensional (2D) micropatterned surfaces with periodic features (several nanometers to less than 100 μm) are commonly used to guide the alignment of muscle myoblasts and myotubes and lead to formation of pre-patterned cell sheets. However, cell sheets from 2D patterned surfaces have limited thickness, and harvesting the cell sheets for implantation is inconvenient and can lead to less alignment of myotubes. 3D micropatterned scaffolds can promote cell alignment and muscle tissue formation. In this study, we developed a novel type of 3D porous collagen scaffolds with concave microgrooves that mimic muscle basement membrane to engineer skeletal muscle tissue. Highly aligned and multi-layered muscle bundle tissues were engineered by controlling the size of microgrooves and cell seeding concentration. Myoblasts in the engineered muscle tissue were well-aligned and had high expression of myosin heavy chain and synthesis of muscle extracellular matrix. The microgrooved collagen scaffolds could be used to engineer organized multi-layered muscle tissue for implantation to repair/restore the function of diseased tissues or be used to investigate the cell–cell interaction in 3D microscale topography.     

Construction of fibroblast–collagen gels with orientated fibrils induced by static or dynamic stress: toward the fabrication of small tendon grafts

As a step toward the fabrication of small tendon grafts, fibroblast–collagen gels were constructed with orientated fibrils induced by static or dynamic loading. Three groups of gel samples, each consisting of 1.0 × 10⁶ fibroblasts and 2 mg type I collagen, were fabricated: freely contracted gels formed the control group; contraction-directed gels made up the static group (the gel contraction was directed perpendicular to an axis made by two anchors buried in the gels so that the constraint stress exerted by the two anchors was imposed on the gel); and for the dynamic group, a specific loading pattern (free contraction followed by cyclic stretching using a tensile bioreactor) was employed. Mechanical properties were evaluated by means of the uniaxial tension test. The gels of the static group had an ultimate stress of 350 ± 43.6 kPa and a material modulus of 548.8 ± 61.6 kPa, which were almost 5.2 times and 15.6 times, respectively, greater than those of the controls. The dynamic gels had an ultimate stress of 256.8 ± 80.7 kPa and a material modulus of 118.6 ± 23.5 kPa. These results show that the ultimate stress and material modulus of the static samples are much greater than those of the dynamic samples, which is the opposite of our expectations. Therefore, studies under other dynamic loading patterns and long-term culture are needed to clarify whether dynamic loading is superior to static loading.     

苦味酸-天狼星红偏振光法在早期肝纤维化诊断中的应用

目的　探讨早期肝纤维化诊断的特异性方法。方法　应用苦味酸天狼星红偏振光法对早期纤维化肝组织中胶原的性质及分布特点进行观察。结果　偏振光显微镜下可见随肝纤维化程度的不同清晰地显示嗜酸性胶原蛋白纤维束 ,其中Ⅰ型胶原纤维 (colⅠ )为粗大明亮的黄或红色纤维 ,显示很强的双折光性 ;Ⅲ型胶原纤维 (colⅢ )为绿色细纤维 ,呈疏网状 ,显示弱的双折光。经图像分析 ,其中S1期肝组织中colⅠ占 (10 5 8± 2 33) % ,colⅢ占(45 13± 2 6 7) % ;S2期肝组织中colⅠ占 (5 0 12± 2 5 8) % ,colⅢ占 (48 5 5± 1 6 6 ) %。结论　苦味酸天狼星红染色和偏振光法可确定肝纤维化组织胶原类型、分布、排列与含量 ,是明确早期肝纤维化分期较理想方法。