Analysis of Metabolic Products by Response Surface Methodology for Production of Human-like Collagen II

Recombinant Escherichia coli BL21 is used to produce human-like collagen. The key constituents of media are optimized using response surface methodology (RSM). Before thermal induction, the highest biomass production and the lowest production of some hazardous by-products, especially acetic acid, were obtained in the media containing 0.085 mol·L-1 glucose and 0.019 mol·L-1 nitrogen (carbon-nitrogen ratio, 4.47:1). After thermal induction, when the concentrations of glucose and nitrogen in the media were 0.065 mol·L-1 and 0.017 mol·L-1 , respectively (carbon-nitrogen ratio, 3.82:1), the productivity of human-like collagen per cell was the highest while that of acetic acid was the lowest. The extended analysis showed that the production of lactic acid and propionic acid increased while that of some intermediate acids of the tricarboxylic acid cycle decreased if the dose of glucose increased.

Process Control for Production of Human-like Collagen in Fed-batch Culture of Escherichia coli BL 21

Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5min and 4min intervals, oxygen-enrichment methods and inducement strength on the cell yield and human-like collagen production were investigated. The studies showed that nitrogen source feeding in fast cycle could result in higher human-like collagen production than that in slow cycle; and the feedback regulation of glucose, increase of the pressure of fermentation bioreactor, and supply of oxygen-enriched air could all increase cell yield and human-like collagen production. The effects of inducement strength on protein expression were found important. When OD600 reached 90-100, the cultivation temperature was increased to 42℃ to begin induction for 2-3 h, and then shifted to 39℃ for 5-6h induction, the cell density and human-like collagen production could reach 96g·L-1 [DCW (dry cell mass)] and 19.8% (g·g-1 DCW) respectively.

Optimization of Fermentation Process for Human-like Collagen Production of Recombinant Escherichia coli Using Response Surface Methodology

In order to improve the production of human-like collagen III(HLC III)by fed-batch culture of recombinant Escherichia coli BL21,the Plackett-Burman and Box-Behnken design were applied to optimize the fermentation process parameters.Three variables(induction time,inoculum age and pH),which have significant effects on HLC III production,were selected from eight variables by Plackett-Burman design.With the regression coefficient analysis in the Box-Behnken design,a relationship between HLC III production and three significant factors was obtained,and the optimum levels of the three variables were as follows:induction time 3.2h,inoculum age 12.6 h and pH 6.7.The 3D response surface plots and 2D contour plots created by the Box-Behnken design showed that the interaction between induction time and pH and that between innoculum age and pH were significant.An average 9.68 g·L1HLC III production was attained in the validation experiment under optimized condition,which was 80%higher than the yield of 5.36 g·L1before optimization.

Kinetics of High Cell Density Fed-batch Culture of Recombinant Escherichia coli Producing Human-like Collagen

The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Yx/s were determined as 1.75h, 0.65h^-1 and 0.51g·g^-1, respectively. In the fed-batch culture, different specific growth rates were set at （0.15, 0.2, 0.25h^-1） by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L^-1 and 10.2g·L^-1 respectively. The modal predictions are in good agreement with the experimental data.

Progress of Domestic Recombinant Human-Like Collagen Research and Application

By intercepting the most effective fragment of human collagen’s gene,recombinant human-like collagen is produced by genetic engineering technology and biotechnology.The collagen has good biocompatibility and can promote cell formation.Recombinant human-like collagen has great potential application value in medical biological materials,cosmetics and foods.In recent years,many specialized enterprises which are engaged in human-like collagen raw materials production have emerged.In this paper,a brief summary of the current recombinant human-like collagen raw material’s production and its latest research and development have been made.

Mineralization of Hydroxyapatite Regulated by Recombinant Human-like Collagen

We reported recombinant human-like type I collagen inducing growth of hydroxyapatite crystals in vitro in the form of self-assembly of nano-fibrils of mineralized collagen resembling extracellular matrix, which obey the same rules, but is superior to the collagen derived from animal tissues because the latter may carry diseases of animals and cause immunological reactions. The mineralized collagen fibrils aligned parallel to each other to form mineralized collagen fibers. Hydroxyapatite nanocrystals grew on the surface of these collagen fibrils with the c-axis of nanocrystals of HA orienting along the longitudinal axis of the fibrils.

Human-Like Decision-Making of Autonomous Vehicles in Dynamic Traffic Scenarios

With the maturation of autonomous driving technology, the use of autonomous vehicles in a socially acceptable manner has become a growing demand of the public. Human-like autonomous driving is expected due to the impact of the differences between autonomous vehicles and human drivers on safety.Although human-like decision-making has become a research hotspot, a unified theory has not yet been formed, and there are significant differences in the implementation and performance of existing methods. This paper provides a comprehensive overview of human-like decision-making for autonomous vehicles. The following issues are discussed: 1) The intelligence level of most autonomous driving decision-making algorithms;2) The driving datasets and simulation platforms for testing and verifying human-like decision-making;3) The evaluation metrics of human-likeness;personalized driving;the application of decisionmaking in real traffic scenarios;and 4) The potential research direction of human-like driving. These research results are significant for creating interpretable human-like driving models and applying them in dynamic traffic scenarios. In the future, the combination of intuitive logical reasoning and hierarchical structure will be an important topic for further research. It is expected to meet the needs of human-like driving.

Calcium-chelating peptides from rabbit bone collagen:characterization,identification and mechanism elucidation

This study aimed to characterize and identify calcium-chelating peptides from rabbit bone collagen and explore the underlying chelating mechanism.Collagen peptides and calcium were extracted from rabbit bone by instant ejection steam explosion(ICSE)combined with enzymatic hydrolysis,followed by chelation reaction to prepare rabbit bone peptide-calcium chelate(RBCP-Ca).The chelating sites were further analyzed by liquid chromatography-tandem mass(LC-MS/MS)spectrometry while the chelating mechanism and binding modes were investigated.The structural characterization revealed that RBCP successfully chelated with calcium ions.Furthermore,LC-MS/MS analysis indicated that the binding sites included both acidic amino acids(Asp and Glu)and basic amino acids(Lys and Arg),Interestingly,three binding modes,namely Inter-Linking,Loop-Linking and Mono-Linking were for the first time found,while Inter-Linking mode accounted for the highest proportion(75.1%),suggesting that chelation of calcium ions frequently occurred between two peptides.Overall,this study provides a theoretical basis for the elucidation of chelation mechanism of calcium-chelating peptides.

Estimation of cancer cell migration in biomimetic random/oriented collagen fiber microenvironments

Increasing data indicate that cancer cell migration is regulated by extracellular matrixes and their surrounding biochemical microenvironment,playing a crucial role in pathological processes such as tumor invasion and metastasis.However,conventional two-dimensional cell culture and animal models have limitations in studying the influence of tumor microenvironment on cancer cell migration.Fortunately,the further development of microfluidic technology has provided solutions for the study of such questions.We utilize microfluidic chip to build a random collagen fiber microenvironment(RFM)model and an oriented collagen fiber microenvironment(OFM)model that resemble early stage and late stage breast cancer microenvironments,respectively.By combining cell culture,biochemical concentration gradient construction,and microscopic imaging techniques,we investigate the impact of different collagen fiber biochemical microenvironments on the migration of breast cancer MDA-MB-231-RFP cells.The results show that MDA-MB-231-RFP cells migrate further in the OFM model compared to the RFM model,with significant differences observed.Furthermore,we establish concentration gradients of the anticancer drug paclitaxel in both the RFM and OFM models and find that paclitaxel significantly inhibits the migration of MDA-MB-231-RFP cells in the RFM model,with stronger inhibition on the high concentration side compared to the low concentration side.However,the inhibitory effect of paclitaxel on the migration of MDA-MB-231-RFP cells in the OFM model is weak.These findings suggest that the oriented collagen fiber microenvironment resembling the late-stage tumor microenvironment is more favorable for cancer cell migration and that the effectiveness of anticancer drugs is diminished.The RFM and OFM models constructed in this study not only provide a platform for studying the mechanism of cancer development,but also serve as a tool for the initial measurement of drug screening.

TGF-β1和Collagen-Ⅲ在牦牛感染细粒棘球蚴肝纤维化中的表达

研究转化生长因子β1(TGF-β1)、Ⅲ型胶原(Collagen-Ⅲ)在牦牛感染细粒棘球蚴导致肝纤维化中的表达及其意义。收集西藏地区40例细粒棘球蚴感染的牦牛肝组织和血清样本,同时收集未感染的肝组织和血清进行对照。采用苏木素-伊红(HE)染色法和天狼猩红染色法观察牦牛肝脏病理变化和肝纤维化程度;酶联免疫吸附试验(ELISA)和免疫组化法(IHA)分别检测肝组织和血清中TGF-β1、Collagen-Ⅲ的表达水平及分布;RT-PCR检测肝细胞内TGF-β1和Collagen-Ⅲ基因mRNA的相对表达水平。HE染色和天狼猩红染色结果显示,与未感染细粒棘球蚴病牦牛肝组织相比,感染棘球蚴周围的肝组织肝界板细胞破坏,呈虫蚀状,感染肝组织外围纤维化,细粒棘球蚴寄生的牦牛肝脏组织引起组织增生,纤维化;ELISA结果显示感染细粒棘球蚴牦牛血清样中细胞因子TGF-β1、Collagen-Ⅲ的含量明显超过未感染组(P<0.05),且免疫组化的结果显示它们的表达主要存在于细胞基质中;RT-PCR显示细粒棘球蚴感染肝组织中TGF-β1、Collagen-Ⅲ的mRNA相对表达量超出未感染组(P<0.05)。结果表明,细粒棘球蚴感染期间TGF-β1、Collagen-Ⅲ在细粒棘球蚴感染引起的肝纤维化中起重要作用。

Preparation,characterization and antioxidant activity analysis of three Maillard glycosylated bone collagen hydrolysates from chicken,porcine and bovine

Bone collagen hydrolysates(peptides)derived from byproduct of animal product processing have been used to produce commercially valuable products due to their potential antioxidant activity.Maillard glycosylated reaction is considered as a promising method to enhance the antioxidant activity of peptides.Hence,this research aims at investigating the Maillard glycosylation activity and antioxidant activity of bone collagen hydrolysates from different sources.In this study,3 glycosylated bone collagen hydrolysates were prepared and characterized,and cytotoxicity and antioxidant activity were analyzed and evaluated.The free amino groups loss,browning intensity,and fluorescence intensity of G-Cbcp(glycosylated chicken bone collagen hydrolysates(peptides))were the heaviest,followed by G-Pbcp(glycosylated porcine bone collagen hydrolysates(peptides))and G-Bbcp(glycosylated bovine bone collagen hydrolysates(peptides)).The results of amino acid analysis showed that amino acid composition of different bone collagen hydrolysates was significantly different and the amino acid decreased to different degrees after Maillard glycosylated reaction,which may lead to differences in Maillard glycosylated reaction activity.Furthermore,the 3 glycosylated hydrolysates showed no significant cytotoxicity.The results showed that glycosylation process significantly increased the antioxidant activity of bone collagen hydrolysates,and G-Cbcp showed the strongest antioxidant activity,followed by G-Pbcp and G-Bbcp.Therefore,compared with the bone collagen hydrolysates,3 glycosylated hydrolysates showed significant characteristic and structural changes,and higher antioxidant activity.

Molecular mechanism of hypoxia and alpha-ketoglutaric acid on collagen expression in scleral fibroblasts

AIM:To investigate the molecular mechanisms underlying the influence of hypoxia and alpha-ketoglutaric acid(α-KG)on scleral collagen expression.METHODS:Meta-analysis and clinical statistics were used to prove the changes in choroidal thickness(ChT)during myopia.The establishment of a hypoxic myopia model(HYP)for rabbit scleral fibroblasts through hypoxic culture and the effects of hypoxia andα-KG on collagen expression were demonstrated by Sirius red staining.Transcriptome analysis was used to verify the genes and pathways that hypoxia andα-KG affect collagen expression.Finally,real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used for reverse verification.RESULTS:Meta-analysis results aligned with clinical statistics,revealing a thinning of ChT,leading to scleral hypoxia.Sirius red staining indicated lower collagen expression in the HYP group and higher collagen expression in the HYP+α-KG group,showed that hypoxia reduced collagen expression in scleral fibroblasts,whileα-KG can elevated collagen expression under HYP conditions.Transcriptome analysis unveiled the related genes and signaling pathways of hypoxia andα-KG affect scleral collagen expression and the results were verified by RT-qPCR.CONCLUSION:The potential molecular mechanisms through which hypoxia andα-KG influencing myopia is unraveled and three novel genes TLCD4,TBC1D4,and EPHX3 are identified.These findings provide a new perspective on the prevention and treatment of myopia via regulating collagen expression.

Anti-abrasion collagen fiber-based membrane functionalized by UiO-66-NH_(2)with ultra-high efficiency and stability for oil-in-water emulsions separation

Membrane separation strategies offer promising platform for the emulsion separation.However,the low mechanical strength of membrane separation layers and the trade-off between separation flux and efficiency present significant challenges.In this study,we report a CFM@UiO-66-NH_(2)membrane with high separation flux,efficiency and stability,through utilizing a robust anti-abrasion collagen fiber membrane(CFM)as the multifunctional support and UiO-66-NH_(2)by an in-situ growth as the separation layer.The high mechanical strength of the CFM compensated for the weakness of the separation layer,while the charge-breaking effect of UiO-66-NH_(2),along with the size sieving of its constituent separating layers and the capillary effect of the collagen fibers,contributed to the potential for efficient separation.Additionally,the CFM@UiO-66-NH_(2)membrane exhibited superhydrophilic properties,making it suitable for separating oil-in-water microemulsions and nanoemulsions stabilized by anionic surfactants.The membrane demonstrated remarkable separation efficiencies of up to 99.960%and a separation flux of370.05 L·m^(-2)·h^(-1).Moreover,it exhibits stability,durability,and abrasion resistance,maintaining excellent separation performance even when exposed to strong acids and alkalis without any damage to its structure and performance.After six cycles of reuse,it achieved a separation flux of 417.97 L·m^(-2)·h^(-1)and a separation efficiency of 99.747%.Furthermore,after undergoing 500 cycles of strong abrasion,the separation flux remained at 124.39 L·m^(-2)·h^(-1),with a separation efficiency of 99.992%.These properties make it suitable for the long-term use in harsh operating environments.We attribute these properties to the electrostatic effect resulting from the amino group on UiO-66-NH_(2)and its in-situ growth on the CFM,which forms a size-screening separation layer.Our work highlights the potential of the CFM@UiO-66-NH_(2)membrane as an environmentally friendly size-screening material for the efficient emulsion wastewater separation.

Acupotomy ameliorates knee osteoarthritis-related collagen deposition and fibrosis in rabbit skeletal muscle through the TGF-β/Smad pathway

Objective To investigate the effects of acupotomy on skeletal muscle fibrosis and collagen deposition in a rabbit knee osteoarthritis(KOA)model.Methods Rabbits(n=18)were randomly divided into control,KOA,and KOA+acupotomy(Apo)groups(n=6).The rabbits in the KOA and Apo groups were modeled using the modified Videman's method for 6 weeks.After modeling,the Apo group was subjected to acupotomy once a week for 3 weeks on the vastus medialis,vastus lateralis,rectus femoris,biceps femoris,and anserine bursa tendons around the knee.The behavior of all animals was recorded,rectus femoris tissue was obtained,and histomorphological changes were observed using Masson staining and transmission electron microscopy.The expression of transforming growth factor-β1(TGF-β1),Smad 3,Smad 7,fibrillar collagen types I(Col-I)and III(Col-III)was detected using Western blot and real-time polymerase chain reaction(RT-PCR).Results Histological analysis revealed that acupotomy improved the microstructure and reduced the collagen volume fraction of rectus femoris,compared with the KOA group(P=.034).Acupotomy inhibited abnormal collagen deposition by modulating the expression of fibrosis-related proteins and mRNA,thus preventing skeletal muscle fibrosis.Western blot and RT-PCR analysis revealed that in the Apo group,Col-I,and Col-III protein levels were significantly lower than those in the KOA group(both P<.01),same as Col-I and Col-III mRNA levels(P=.0031;P=.0046).Compared with the KOA group,the protein levels of TGF-β1 and Smad 3 were significantly reduced(both P<.01),as were the mRNA levels of TGF-β1 and Smad 3(P=.0007;P=.0011).Conversely,the levels of protein and mRNA of Smad 7 were significantly higher than that in the KOA group(P<.01;P=.0271).Conclusion Acupotomy could alleviate skeletal muscle fibrosis and delay KOA progress by inhibiting collagen deposition through the TGF-β/Smad pathway in the skeletal muscle of KOA rabbits.

Skin care efficacy study of recombinant humanized collagen based on in vitro level

Studying the skin care efficacy of recombinant humanized collagen based on in vitro level.The stability of the recombinant humanized collagen was first analyzed by treating at different temperatures,then its skincare efficacy based on in vitro level was evaluated by detecting the inhibition rate of elastase,the inhibition rate of collagenase,the protein content of type I collagen in human fibroblasts,the inhibition of reactive oxygen species(ROS)with human keratinocytes,and the effects of the recombinant humanized collagen on the expression of hyaluronic acid(HA),filaggrin(FLG)and transglutaminase 1(TGM1)in keratinocytes.The results showed that recombinant humanized collagen was able to maintain stability at temperatures below 70℃.With regard to its skincare efficacy,recombinant humanized collagen could inhibit elastase and collagenase activities and promote the increase of type I collagen content in human fibroblasts.It also showed good inhibition of ROS in keratinocytes in vitro and could increase the expression of HA,FLG,and TGM1 in keratinocytes.In short,the recombinant humanized collagen exhibited a favourable skin care effect in vitro level.This study proved that it has potential firming,anti-wrinkle,moisturizing,and repairing efficacy,and is a valuable cosmetic raw material.

Clinical efficacy of intradermal type Ⅰ collagen injections in treating skin photoaging in patients from high-altitude areas

BACKGROUND Photoaging,a result of chronic sun exposure,leads to skin damage and pigmentation changes.Traditional treatments may have limitations in high-altitude areas like Yunnan Province.Intradermal Col Ⅰ injections stimulate collagen production,potentially improving skin quality.This study aims to assess the efficacy and safety of this treatment for photoaging.AIM To evaluate the efficacy and safety of intradermal typeΙcollagen(ColΙ)injection for treating photoaging.METHODS This prospective,self-controlled study investigated the impact of intradermal injections of ColΙon skin photodamage in 20 patients from the Yunnan Province.Total six treatment sessions were conducted every 4 wk±3 d.Before and after each treatment,facial skin characteristics were quantified using a VISIA skin detector.Skin thickness data were assessed using the ultrasound probes of the Dermalab skin detector.The Face-Q scale was used for subjective evaluation of the treatment effect by the patients.RESULTS The skin thickness of the right cheek consistently increased after each treatment session compared with baseline.The skin thickness of the left cheek significantly increased after the third through sixth treatment sessions compared with baseline.The skin thickness of the right zygomatic region increased after the second to sixth treatment sessions,whereas that of the left zygomatic region showed a significant increase after the fourth through sixth treatment sessions.The skin thickness of both temporal regions significantly increased after the fifth and sixth treatment sessions compared with baseline(P<0.05).These findings were also supported by skin ultrasound images.The feature count for the red areas and wrinkle feature count decreased following the treatment(P<0.05).VISIA assessments also revealed a decrease in the red areas after treatment.The Face-QSatisfaction with Facial Appearance Overall and Face-Q-Satisfaction with Skin scores significantly increased after each treatment session.The overall appearance of the patients improved after treatment.CONCLUSION Intradermal ColΙinjection improves photoaging,with higher patient satisfaction and fewer adverse reactions,and could be an effective treatment method for populations residing in high-altitude areas.

Corneal collagen cross-linking in patients with keratoconus from the Dresden protocol to customized solutions:theoretical basis

Keratoconus is an ectatic condition characterized by gradual corneal thinning,corneal protrusion,progressive irregular astigmatism,corneal fibrosis,and visual impairment.The therapeutic options regarding improvement of visual function include glasses or soft contact lenses correction for initial stages,gas-permeable rigid contact lenses,scleral lenses,implantation of intrastromal corneal ring or corneal transplants for most advanced stages.In keratoconus cases showing disease progression corneal collagen crosslinking(CXL)has been proven to be an effective,minimally invasive and safe procedure.CXL consists of a photochemical reaction of corneal collagen by riboflavin stimulation with ultraviolet A radiation,resulting in stromal crosslinks formation.The aim of this review is to carry out an examination of CXL methods based on theoretical basis and mathematical models,from the original Dresden protocol to the most recent developments in the technique,reporting the changes proposed in the last 15y and examining the advantages and disadvantages of the various treatment protocols.Finally,the limits of non-standardized methods and the perspectives offered by a customization of the treatment are highlighted.

Topological Structure-Modulated Collagen Carbon as Two-in-One Energy Storage Configuration toward Ultrahigh Power and Energy Density

Efficient energy storage devices with suitable electrode materials,that integrate high power and high energy,are the crucial requisites of the renewable power source,which have unwrapped new possibilities in the sustainable development of energy and the environment.Herein,a facile collagen microstructure modulation strategy is proposed to construct a nitrogen/oxygen dual-doped hierarchically porous carbon fiber with ultrahigh specific surface area(2788 m^(2)g^(-1))and large pore volume(4.56 cm^(3)g^(-1))via local microfibrous breakage/disassembly of natural structured proteins.Combining operando spectroscopy and density functional theory unveil that the dual-heteroatom doping could effectively regulate the electronic structure of carbon atom framework with enhanced electric conductivity and electronegativity as well as decreased diffusion resistance in favor of rapid pseudocapacitive-dominated Li^(+)-storage(353 mAh g^(-1)at 10 A g^(-1)).Theoretical calculations reveal that the tailored micro-/mesoporous structures favor the rapid charge transfer and ion storage,synergistically realizing high capacity and superior rate performance for NPCF-H cathode(75.0 mAh g^(-1)at 30 A g^(-1)).The assembled device with NPCF-H as both anode and cathode achieves extremely high energy density(200 Wh kg^(-1))with maximum power density(42600 W kg^(-1))and ultralong lifespan(80%capacity retention over 10000 cycles).

Exosomes from umbilical cord mesenchymal stromal cells promote the collagen production of fibroblasts from pelvic organ prolapse

BACKGROUND Pelvic organ prolapse(POP)involves pelvic organ herniation into the vagina due to pelvic floor tissue laxity,and vaginal structure is an essential factor.In POP,the vaginal walls exhibit abnormal collagen distribution and decreased fibroblast levels and functions.The intricate etiology of POP and the prohibition of trans-vaginal meshes in pelvic reconstruction surgery present challenges in targeted therapy development.Human umbilical cord mesenchymal stromal cells(hucMSCs)present limitations,but their exosomes(hucMSC-Exo)are promising therapeutic tools for promoting fibroblast proliferation and extracellular matrix remodeling.suppressed inflammation in POP group fibroblasts,stimulated primary fibroblast growth,and elevated collagen I(Col1)production in vitro.High-throughput RNA-seq of fibroblasts treated with hucMSC-Exo and miRNA sequencing of hucMSC-Exo revealed that abundant exosomal miRNAs downregulated matrix metalloproteinase 11(MMP11)expression.CONCLUSION HucMSC-Exo normalized the growth and function of primary fibroblasts from patients with POP by promoting cell growth and Col1 expression in vitro.Abundant miRNAs in hucMSC-Exo targeted and downregulated MMP11 expression.HucMSC-Exo-based therapy may be ideal for safely and effectively treating POP.

Progress in the extraction and application of collagen peptides

Collagen peptide is the product of complete hydrolysis of collagen,which has a relatively small molecular weight and is more easily absorbed than proteins and amino acids.Collagen peptide not only has unique nutritional value,but also has certain physiological functions,which makes it has great potential value in various fields,so it has set off a wave of research on collagen peptide in the biological world.This paper describes the sources and extraction methods of collagen peptides,and describes the research progress and application of collagen peptides in the medical,food,material and skin care industries according to their physiological functions,which will provide new ideas for the future research of collagen peptides.