富血小板血浆联合同种异体骨治疗非感染性骨不连的临床应用

目的:评价富血小板血浆联合同种异体骨治疗非感染性骨不连的临床疗效。方法:回顾性分析我院创伤骨科2010年2月-2015年1月的非感染性骨不连患者,比较同种异体骨加富血小板血浆混合物与自体髂骨植骨治疗非感染性骨不连的疗效。结果:两组共随访非感染性骨不连患者69例,全部进行了术中断端植骨,其中富血小板血浆联合同种异体骨植骨组(PRP组)21例,其中愈合19例,未愈合2例,临床愈合率90.5%。自体髂骨植骨组(自体骨组)48例,愈合44例,4例未愈合,临床愈合率91.6%。PRP组平均手术持续时间为(108.73±13.91),自体骨组为(120.54±13.87)min,两组间有统计学意义(P0.05)。术后3月,PRP组患者X线骨痂评价标准平均评分值为(2.54±0.43)分,自体骨组为(2.62±0.45)分,两组间差异无统计学意义(P0.05)。结论:富血小板血浆混合同种异体骨植骨治疗骨不连较传统自体髂骨植骨可缩短手术时间,避免供骨区并发症,修复效果良好,为骨不连的治疗提供了一个新的方法。     

Platelet-Rich Plasma and Skeletal Muscle Healing: A Molecular Analysis of the Early Phases of the Regeneration Process in an Experimental Animal Model

Platelet-rich plasma (PRP) has received increasing interest in applied medicine, being widely used in clinical practice with the aim of stimulating tissue healing. Despite the reported clinical success, there is still a lack of knowledge when considering the biological mechanisms at the base of the activity of PRP during the process of muscle healing. The aim of the present study was to verify whether the local delivery of PRP modulates specific molecular events involved in the early stages of the muscle regeneration process. The right flexor sublimis muscle of anesthetized Wistar rats was mechanically injured and either treated with PRP or received no treatment. At day 2 and 5 after surgery, the animals were sacrificed and the muscle samples evaluated at molecular levels. PRP treatment increased significantly the mRNA level of the pro-inflammatory cytokines IL-1β, and TGF-β1. This phenomenon induced an increased expression at mRNA and/or protein levels of several myogenic regulatory factors such as MyoD1, Myf5 and Pax7, as well as the muscular isoform of insulin-like growth factor1 (IGF-1Eb). No effect was detected with respect to VEGF-A expression. In addition, PRP application modulated the expression of miR-133a together with its known target serum response factor (SRF); increased the phosphorylation of αB-cristallin, with a significant improvement in several apoptotic parameters (NF-κB-p65 and caspase 3), indexes of augmented cell survival. The results of the present study indicates that the effect of PRP in skeletal muscle injury repair is due both to the modulation of the molecular mediators of the inflammatory and myogenic pathways, and to the control of secondary pathways such as those regulated by myomiRNAs and heat shock proteins, which contribute to proper and effective tissue regeneration.     

Platelet-Rich Plasma and the Elimination of Neuropathic Pain

Peripheral neuropathic pain typically results from trauma-induced nociceptive neuron hyperexcitability and their spontaneous ectopic activity. This pain persists until the trauma-induced cascade of events runs its full course, which results in complete tissue repair, including the nociceptive neurons recovering their normal biophysical properties, ceasing to be hyperexcitable, and stopping having spontaneous electrical activity. However, if a wound undergoes no, insufficient, or too much inflammation, or if a wound becomes stuck in an inflammatory state, chronic neuropathic pain persists. Although various drugs and techniques provide temporary relief from chronic neuropathic pain, many have serious side effects, are not effective, none promotes the completion of the wound healing process, and none provides permanent pain relief. This paper examines the hypothesis that chronic neuropathic pain can be permanently eliminated by applying platelet-rich plasma to the site at which the pain originates, thereby triggering the complete cascade of events involved in normal wound repair. Many published papers claim that the clinical application of platelet-rich plasma to painful sites, such as muscle injuries and joints, or to the ends of nerves evoking chronic neuropathic pain, a process often referred to as prolotherapy, eliminates pain initiated at such sites. However, there is no published explanation of a possible mechanism/s by which platelet-rich plasma may accomplish this effect. This paper discusses the normal physiological cascade of trauma-induced events that lead to chronic neuropathic pain and its eventual elimination, techniques being studied to reduce or eliminate neuropathic pain, and how the application of platelet-rich plasma may lead to the permanent elimination of neuropathic pain. It concludes that platelet-rich plasma eliminates neuropathic pain primarily by platelet- and stem cell-released factors initiating the complex cascade of wound healing events, starting with the induction of enhanced inflammation and its complete resolution, followed by all the subsequent steps of tissue remodeling, wound repair and axon regeneration that result in the elimination of neuropathic pain, and also by some of these same factors acting directly on neurons to promote axon regeneration thereby eliminating neuropathic pain.     

Neural Differentiation of Human-Induced Pluripotent Stem Cells (hiPSc) on Surface-Modified Nanofibrous Scaffolds Coated with Platelet-Rich Plasma

The field of tissue engineering exploits living cells in a variety of ways to restore, maintain, or enhance tissues and organs. Between stem cells, human induced pluripotent stem cells (hiPSCs), are very important due to their wide abilities. Growth factors can support proliferation, differentiation, and migration of hiPSCs. Platelet-rich plasma (PRP) could be used as the source of growth factors for hiPSCs. In the present study, proliferation and neural differentiation of hiPSCs on surface-modified nanofibrous Poly-l-lactic acid (PLLA) coated with platelet-rich plasma was investigated. The results of in vitro analysis showed that on the surface, which was modified nanofibrous scaffolds coated with platelet-rich plasma, significantly enhanced hiPSCs proliferation and neural differentiation were observed. Whereas the MTT ([3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide]) results showed biocompatibility of surface-modified nanofibrous scaffolds coated with platelet-rich plasma and the usage of these modified nanoscaffolds in neural tissue engineering in vivo is promising for the future.

     

Platelet-Rich Plasma and Adipose-Derived Mesenchymal Stem Cells for Regenerative Medicine-Associated Treatments in Bottlenose Dolphins (Tursiops truncatus)

Dolphins exhibit an extraordinary capacity to heal deep soft tissue injuries. Nevertheless, accelerated wound healing in wild or captive dolphins would minimize infection and other side effects associated with open wounds in marine animals. Here, we propose the use of a biological-based therapy for wound healing in dolphins by the application of platelet-rich plasma (PRP). Blood samples were collected from 9 different dolphins and a specific and simple protocol which concentrates platelets greater than two times that of whole blood was developed. As opposed to a commonly employed human protocol for PRP preparation, a single centrifugation for 3 minutes at 900 rpm resulted in the best condition for the concentration of dolphin platelets. By FACS analysis, dolphin platelets showed reactivity to platelet cell-surface marker CD41. Analysis by electron microscopy revealed that dolphin platelets were larger in size than human platelets. These findings may explain the need to reduce the duration and speed of centrifugation of whole blood from dolphins to obtain a 2-fold increase and maintain proper morphology of the platelets. For the first time, levels of several growth factors from activated dolphin platelets were quantified. Compared to humans, concentrations of PDGF-BB were not different, while TGFβ and VEGF-A were significantly lower in dolphins. Additionally, adipose tissue was obtained from cadaveric dolphins found along the Spanish Mediterranean coast, and adipose-derived mesenchymal stem cells (ASCs) were successfully isolated, amplified, and characterized. When dolphin ASCs were treated with 2.5 or 5% dolphin PRP they exhibited significant increased proliferation and improved phagocytotic activity, indicating that in culture, PRP may improve the regenerative capacity of ASCs. Taken together, we show an effective and well-defined protocol for efficient PRP isolation. This protocol alone or in combination with ASCs, may constitute the basis of a biological treatment for wound-healing and tissue regeneration in dolphins.     

低智儿童与正常儿童中氨基酸的比较研究

我们对 30例低智儿童血清中TAU ,SER ,GLU ,GLY ,ALA ,VAL ,CYS ,MET ,ILE ,LEU ,TYR ,PHE ,TRP ,HIS ,ORN ,LYS ,ARG ,PRO ,18种游离氨基酸进行了测定。研究结果显示 :患儿血清中 11种游离氨基酸降低分别为 :TAU ,SER ,VAL ,MET ,ILE ,LEU ,TRY ,PHE ,ORN ,LYS ,TRP .氨基酸的失衡 ,儿童的蛋白质合成就将会受到严重的影响 ,因此将导致大脑的分化 ,发育受阻 ,引起智力低下。     

The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves: III. A Comparison between Some C(3) and C(4) Plants

The chlorophyll-based specific activity of cytochrome oxidase and three exclusively mitochondrial enzymes of the tricarboxylic acid cycle showed little variation between leaves of C₃ and C₄ plants or between mesophyll and bundle sheath cells of Atriplex spongiosa and Sorghum bicolor. However, a large, light-dependent transfer of label from intermediates of the tricarboxylic acid cycle to photosynthetic products was a feature of leaves of C₄ plants. This light-dependent transfer of label was barely detectable in leaves of C₃ plants and in leaves of F₁ and F₃ hybrids of Atriplex rosea (C₄) and Atriplex patula spp hastata (C₃). The light-dependent transfer of label to photosynthetic products in leaves of C₄ plants was inhibited by the tricarboxylic acid cycle inhibitors malonate and fluoroacetate. The requirement for continued tricarboxylic acid cycle activity was also indicated in experiments with specifically labeled succinate-¹⁴C. These experiments, together with the distribution of ¹⁴C in glucose prepared from sucrose-¹⁴C formed during the metabolism of succinate-2,3-¹⁴C, confirmed that the photosynthetic metabolism of malate and aspartate derived from the tricarboxylic acid cycle, and not the refixation of respiratory CO₂, was the main path of carbon from the cycle to photosynthesis.     

Platelet derived growth factor (PDGF) contained in Platelet Rich Plasma (PRP) stimulates migration of osteoblasts by reorganizing actin cytoskeleton

Platelet-rich plasma (PRP) is a platelet concentrate in a small volume of plasma. It is highly enriched in growth factors able to stimulate the migration and growth of bone-forming cells. PRP is often used in clinical applications, as dental surgery and fracture healing. Platelet derived growth factor (PDGF), is highly concentrated in PRP and it was shown in our previous studies to provide the chemotactic stimulus to SaOS-2 osteoblasts to move in a microchemotaxis assay. Aim of the present studies is to analyze the effects of a PRP pretreatment (short time course: 30–150 min) of SaOS-2 cells with PRP on the organization of actin cytoskeleton, the main effector of cell mobility. The results indicate that a pretreatment with PRP increases chemokinesis and chemotaxis and concomitantly induces the organization of actin microfilaments, visualized by immunocytochemistry, in a directionally elongated phenotype, which is characteristic of the cells able to move. PRP also produces a transient increase in the expression of PGDF α receptor. This reorganization is blocked by the immunoneutralization of PDGF demonstrating the responsibility of this growth factor in triggering the mechanisms responsible for cellular movements.     

Platelet derived growth factor (PDGF) contained in Platelet Rich Plasma (PRP) stimulates migration of osteoblasts by reorganizing actin cytoskeleton

Platelet-rich plasma (PRP) is a platelet concentrate in a small volume of plasma. It is highly enriched in growth factors able to stimulate the migration and growth of bone-forming cells. PRP is often used in clinical applications, as dental surgery and fracture healing. Platelet derived growth factor (PDGF), is highly concentrated in PRP and it was shown in our previous studies to provide the chemotactic stimulus to SaOS-2 osteoblasts to move in a microchemotaxis assay. Aim of the present studies is to analyze the effects of a PRP pretreatment (short time course: 30–150 min) of SaOS-2 cells with PRP on the organization of actin cytoskeleton, the main effector of cell mobility. The results indicate that a pretreatment with PRP increases chemokinesis and chemotaxis and concomitantly induces the organization of actin microfilaments, visualized by immunocytochemistry, in a directionally elongated phenotype, which is characteristic of the cells able to move. PRP also produces a transient increase in the expression of PGDF α receptor. This reorganization is blocked by the immunoneutralization of PDGF demonstrating the responsibility of this growth factor in triggering the mechanisms responsible for cellular movements.     

A Kinetic Study on the Interaction between Tazobactam (A Penicillanic Acid Sulphone Derivative) and Active-Site Serine beta-Lactamases

The interaction between tazobactam and several chromosome- and plasmid-encoded (TEM, SHV, PSE types) class A and C beta-lactamases was studied by spectrophotometry. Tazobactam behaved as a competitive inhibitor or inactivator able to restore in several cases the efficiency of piperacillin as a partner beta-lactam. A detailed kinetic analysis permitted measurement of the acylation efficiency for some cephalosporinases and broad-spectrum beta-lactamases; the presence of a turn-over of acyl-enzyme complex was also evaluated.     

A Comparison of Dark Respiration between C(3) and C(4) Plants

Lower respiratory costs were hypothesized as providing an additional benefit in C₄ plants compared to C₃ plants due to less investment in proteins in C₄ leaves. Therefore, photosynthesis and dark respiration of mature leaves were compared between a number of C₄ and C₃ species. Although photosynthetic rates were generally greater in C₄ when compared to C₃ species, no differences were found in dark respiration rates of individual leaves at either the beginning or after 16 h of the dark period. The effects of nitrogen on photosynthesis and respiration of individual leaves and whole plants were also investigated in two species that occupy similar habitats, Amaranthus retroflexus (C₄) and Chenopodium album (C₃). For mature leaves of both species, there was no relationship between leaf nitrogen and leaf respiration, with leaves of both species exhibiting a similar rate of decline after 16 h of darkness. In contrast, leaf photosynthesis increased with increasing leaf nitrogen in both species, with the C₄ species displaying a greater photosynthetic response to leaf nitrogen. For whole plants of both species grown at different nitrogen levels, there was a clear linear relationship between net CO₂ uptake and CO₂ efflux in the dark. The dependence of nightly CO₂ efflux on CO₂ uptake was similar for both species, although the response of CO₂ uptake to leaf nitrogen was much steeper in the C₄ species, Amaranthus retroflexus. Rates of growth and maintenance respiration by whole plants of both species were similar, with both species displaying higher rates at higher leaf nitrogen. There were no significant differences in leaf or whole plant maintenance respiration between species at any temperature between 18 and 42°C. The data suggest no obvious differences in respiratory costs in C₄ and C₃ plants.     

上海交通大学医学院附属新华医院崇明分院检验科

目的:探讨富血小板血浆(platelet-rich plasma,PRP)结合组织工程皮肤对裸鼠巨大创面修复的影响。方法:首先,利用密度梯度离心法制备富含生长因子的浓缩血小板的血浆,并测其所含生长因子的量;其次,在裸鼠的背侧部分构建大面积皮肤创面,分别用人工真皮,组织工程皮肤,碱性成纤维细胞生长因子组织工程皮肤,表皮生长因子组织工程皮肤和PRP结合组织工程皮肤修复裸鼠巨大创面;最后,手术后不同时间间隔收集组织标本,采用HE染色,PAS染色和免疫组化等方法评估创面愈合情况。结果:PRP结合组织工程皮肤组创面修复愈合情况最好。     

Elastoviscous Transitions of Articular Cartilage Reveal a Mechanism of Synergy between Lubricin and Hyaluronic Acid

When lubricated by synovial fluid, articular cartilage provides some of the lowest friction coefficients found in nature. While it is known that macromolecular constituents of synovial fluid provide it with its lubricating ability, it is not fully understood how two of the main molecules, lubricin and hyaluronic acid, lubricate and interact with one another. Here, we develop a novel framework for cartilage lubrication based on the elastoviscous transition to show that lubricin and hyaluronic acid lubricate by distinct mechanisms. Such analysis revealed nonspecific interactions between these molecules in which lubricin acts to concentrate hyaluronic acid near the tissue surface and promotes a transition to a low friction regime consistent with the theory of viscous boundary lubrication. Understanding the mechanics of synovial fluid not only provides insight into the progression of diseases such as arthritis, but also may be applicable to the development of new biomimetic lubricants.     

Cold Atmospheric Plasma (CAP) Changes Gene Expression of Key Molecules of the Wound Healing Machinery and Improves Wound Healing In Vitro and In Vivo

Cold atmospheric plasma (CAP) has the potential to interact with tissue or cells leading to fast, painless and efficient disinfection and furthermore has positive effects on wound healing and tissue regeneration. For clinical implementation it is necessary to examine how CAP improves wound healing and which molecular changes occur after the CAP treatment. In the present study we used the second generation MicroPlaSter ß® in analogy to the current clinical standard (2 min treatment time) in order to determine molecular changes induced by CAP using in vitro cell culture studies with human fibroblasts and an in vivo mouse skin wound healing model. Our in vitro analysis revealed that the CAP treatment induces the expression of important key genes crucial for the wound healing response like IL-6, IL-8, MCP-1, TGF-ß1, TGF-ß2, and promotes the production of collagen type I and alpha-SMA. Scratch wound healing assays showed improved cell migration, whereas cell proliferation analyzed by XTT method, and the apoptotic machinery analyzed by protein array technology, was not altered by CAP in dermal fibroblasts. An in vivo wound healing model confirmed that the CAP treatment affects above mentioned genes involved in wound healing, tissue injury and repair. Additionally, we observed that the CAP treatment improves wound healing in mice, no relevant side effects were detected. We suggest that improved wound healing might be due to the activation of a specified panel of cytokines and growth factors by CAP. In summary, our in vitro human and in vivo animal data suggest that the 2 min treatment with the MicroPlaSter ß® is an effective technique for activating wound healing relevant molecules in dermal fibroblasts leading to improved wound healing, whereas the mechanisms which contribute to these observed effects have to be further investigated.     

Platelet-Rich Plasma Promotes the Proliferation of Human Muscle Derived Progenitor Cells and Maintains Their Stemness

Human muscle-derived progenitor cells (hMDPCs) offer great promise for muscle cell-based regenerative medicine; however, prolonged ex-vivo expansion using animal sera is necessary to acquire sufficient cells for transplantation. Due to the risks associated with the use of animal sera, the development of a strategy for the ex vivo expansion of hMDPCs is required. The purpose of this study was to investigate the efficacy of using platelet-rich plasma (PRP) for the ex-vivo expansion of hMDPCs. Pre-plated MDPCs, myoendothelial cells, and pericytes are three populations of hMDPCs that we isolated by the modified pre-plate technique and Fluorescence Activated Cell Sorting (FACS), respectively. Pooled allogeneic human PRP was obtained from a local blood bank, and the effect that thrombin-activated PRP-releasate supplemented media had on the ex-vivo expansion of the hMDPCs was tested against FBS supplemented media, both in vitro and in vivo. PRP significantly enhanced short and long-term cell proliferation, with or without FBS supplementation. Antibody-neutralization of PDGF significantly blocked the mitogenic/proliferative effects that PRP had on the hMDPCs. A more stable and sustained expression of markers associated with stemness, and a decreased expression of lineage specific markers was observed in the PRP-expanded cells when compared with the FBS-expanded cells. The in vitro osteogenic, chondrogenic, and myogenic differentiation capacities of the hMDPCs were not altered when expanded in media supplemented with PRP. All populations of hMDPCs that were expanded in PRP supplemented media retained their ability to regenerate myofibers in vivo. Our data demonstrated that PRP promoted the proliferation and maintained the multi-differentiation capacities of the hMDPCs during ex-vivo expansion by maintaining the cells in an undifferentiated state. Moreover, PDGF appears to be a key contributing factor to the beneficial effect that PRP has on the proliferation of hMDPCs.     

Plasma Components and Platelet Activation Are Essential for the Antimicrobial Properties of Autologous Platelet-Rich Plasma: An In Vitro Study

Autologous platelet concentrates are successfully adopted in a variety of medical fields to stimulate bone and soft tissue regeneration. The rationale for their use consists in the delivery of a wide range of platelet-derived bioactive molecules that promotes wound healing. In addition, antimicrobial properties of platelet concentrates have been pointed out. In this study, the effect of the platelet concentration, of the activation step and of the presence of plasmatic components on the antimicrobial activity of pure platelet-rich plasma was investigated against gram positive bacteria isolated from oral cavity. The antibacterial activity, evaluated as the minimum inhibitory concentration, was determined through the microdilution two-fold serial method. Results seem to suggest that the antimicrobial activity of platelet-rich plasma against Enterococcus faecalis, Streptococcus agalactiae, Streptococcus oralis and Staphylococcus aureus is sustained by a co-operation between plasma components and platelet-derived factors and that the activation of coagulation is a fundamental step. The findings of this study may have practical implications in the modality of application of platelet concentrates.     

Molecular cloning of the cDNA coding for proline-rich protein (PRP): identity of PRP as C4b-binding protein

Proline-rich protein (PRP) is a plasma protein with a high proportion of proline residues and possessing lipid-binding properties. In order to clarify its structure, a human liver cDNA library was screened using anti-PRP antiserum. Several overlapping phage cDNA clones were isolated and the total nucleotide sequence of the cDNA, 2178 bp in length, was analyzed. The amino acid composition of PRP deduced from the cDNA was essentially the same as that reported for PRP. In a homology search, the cDNA sequence was almost completely the same as the previously reported cDNA sequence of C4b-binding protein. Furthermore, the reported molecular weights of the two proteins under both reduced and unreduced conditions were quite alike. These findings indicate that PRP is identical with C4bp.     

透明质酸在间充质干细胞向软骨细胞分化中的应用

随着组织工程学的发展,利用间充质干细胞(mesenchymal stem cells,MSCs)定向分化为软骨细胞,用于治疗骨性关节炎、关节创伤等因素造成的软骨缺损的研究方兴未艾。透明质酸(hyaluronic acid,HA) 是一种酸性多糖类生物大分子,亦是软骨基质的主要成分之一。由于其优良的生物相容性、可降解等特性,HA已成为优良的天然生物材料,其作为支架材料应用于软骨缺损修复已有一段历史。近年来又发现,HA除作为载体支架材料外,还可作为调节因子应用于MSCs向软骨细胞分化。以下将对近年来利用HA应用于MSCs向软骨细胞分化的研究进行总结,旨在为以MSCs为基础的组织工程化软骨的临床应用奠定基础。     

The Effect of Systemic Heparinisation and Haemodialysis on Plasma Octadeca-9,11-Dienoic Acid (9,11-La')

Systemic heparinisation induces a sharp rise not only in plasma total free fatty acids but also in 9.11-LA′ concentration and in the 9,11 -LA′/9,12-LA molar ratio. This “heparin effect” is enhanced by haemodialysis with cuprophan membranes but not with polycarbonate membranes.     

The Effect of Systemic Heparinisation and Haemodialysis on Plasma Octadeca-9,11-Dienoic Acid (9,11-La')

Systemic heparinisation induces a sharp rise not only in plasma total free fatty acids but also in 9.11-LA' concentration and in the 9,11 -LA'/9,12-LA molar ratio. This “heparin effect” is enhanced by haemodialysis with cuprophan membranes but not with polycarbonate membranes.