细胞复合β-磷酸三钙生物陶瓷修复软骨缺损的实验研究

[目的]通过将骨髓间充质干细胞（MCSc）诱导的具有软骨细胞、成骨细胞表型的细胞接种到三维多孔β-磷酸三钙（β-TCP）生物陶瓷支架材料上，体外构建骨软骨复合体，探讨以β-TCP为载体建造组织工程化软骨修复骨软骨缺损的可行性。[方法]将β-TCP多孔陶瓷加工成圆柱状，并将其作为构建人工软骨的细胞支架。在支架材料上分别接种从犬骨髓干细胞培养成的具有软骨细胞、成骨细胞表型的细胞，将细胞-支架复合体共同培养1周后，移植到犬关节软骨缺损处。植入后12、16周末取材，进行大体观察、组织学及组织化学等观察。[结果]复合体体内移植后，在犬关节软骨缺损处有新生软骨形成，形成的软骨基本保持了支架材料原有形态。[结论]β-TCP多孔陶瓷可作为支架材料，复合细胞后具有修复软骨缺损的作用。     

组织工程骨软骨复合物的构建与形态学观察

目的探讨采用组织工程技术构建骨软骨复合物的可行性。方法将骨髓基质细胞(BMSCs)成诱导软骨后接种于快速成形的三维支架材料聚乳酸／聚羟乙酸共聚物(PLGA)构建组织工程软骨，经成骨诱导的BMSCs接种于聚乳酸／聚羟乙酸共聚物／磷酸三钙(PLGA／TCP)构建组织工程骨，在体外分别培养2周后，将两种工程化组织及两者以无损伤线缝合形成的组织工程骨软复合体分别植入自体股部肌袋，术后8周取材，行组织学观察。结果术后组织学观察表明。组织工程软骨在体内可形成软骨组织组织工程骨在体内可形成骨组织，两者的复合体在体内可形成骨软骨复合物。结论以骨髓基质细胞为种子细胞、以快速成形的生物降解材料为支架体外构建的组织工程骨软骨复合物，可在体内形成骨软骨组织，有望用于骨软骨缺损的修复。     

体内构建骨软骨复合体修复骨软骨缺损的实验研究

[目的]探讨软骨组织工程方法修复骨软骨缺损理想的种子细胞和支架材料。[方法]体外诱导骨髓基质干细胞向软骨细胞定向分化，分别与PLGA支架复合培养，并模仿马赛克骨软骨移植术在犬关节骨软骨缺损深层置入MSC-支架复合体，浅层置入诱导培养后的MSC-支架复合体，紧密压配，体内构建骨软骨复合体，观察其修复的情况。[结果]16周实验组缺损区完全由透明软骨修复，与周围正常软骨完全融合，组织观察显示以软骨细胞修复为主，软骨下骨形成，潮线基本恢复；阴性对照组缺损区主要由纤维组织修复，部分由透明软骨修复，组织观察显示以纤维细胞修复为主，混有部分软骨细胞；空白对照组完全由纤维组织修复。[结论]MSCs经向软骨细胞定向分化后复合PLGA支架材料，通过紧密压配方式与MSCs-PLGA支架在体内构建骨软骨复合体，可以有效修复骨软骨缺损。     

纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞修复兔膝关节软骨缺损的实验研究

目的：探讨纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞作为软骨组织工程支架的可行性及有效性,并为后续研究可注射性材料做基础。方法：体外分离培养软骨细胞后接种到纤维蛋白凝胶和脱钙骨基质支架材料体外培养4周,然后植入兔膝关节软骨缺损区继续培养4、8、12周后取材,分别行大体、组织学、Ⅱ型胶原免疫组织化学观察。并进行Wakitani评分,观察其体内修复关节缺损效果。结果：大体观察4周后,实验组软骨缺损区可有乳白色组织修复,12周可修复完全,并无明显凹凸感。光镜下8周可见大量软骨细胞修复,并在TB染色下见Ⅱ型胶原比4周时明显增多。12周时软骨陷窝结构形成,细胞形态排列及Ⅱ型胶原与正常软骨组织相近。结论：纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞可以作为软骨组织工程支架材料,能够用于再生修复软骨的缺损。并为构建可注射性修复材料提供途径。     

模拟微重力环境下层状修复体与软骨细胞复合培养的实验研究

[目的]探讨模拟微重力作为软骨组织工程培养方法的作用和胶原/壳聚糖/β-磷酸三钙(trical ciumphosphate,TCP)层状梯度修复体作为关节软骨组织工程支架的可行性.[方法]体外培养新西兰大白兔关节软骨细胞并扩增,吸附于多孔胶原/壳聚糖/β-磷酸三钙层状梯度修复体上,模拟微重力和普通环境下三维立体分别培养3周,通过生长曲线、倒置相差显微镜、组织学、扫描电镜及免疫组织化学检测微重力对软骨细胞培养的影响和支架在三维立体培养对软骨细胞的表型、增殖及功能的影响.[结果]软骨细胞/修复体体外培养3周,软骨细胞模拟微重力培养组明显比普通培养组在层状修复体上分布均匀,修复体中心软骨细胞数量明显较多,并分泌细胞基质,包裹在软骨细胞周围,Ⅱ型胶原免疫组织化学染色阳性.[结论]模拟微重力环境有利于软骨细胞在三维支架上的均匀增殖,有望成为软骨组织工程中的一种重要培养方法;胶原/壳聚糖/β-磷酸三钙层状梯度修复体,细胞相容性良好,有望成为一种比较理想的关节软骨组织工程支架材料.     

胶原/羟基磷灰石支架负载软骨细胞修复兔膝关节骨软骨缺损

目的 探讨胶原复合梯度羟基磷灰石(Col/HA)双相支架负载软骨细胞修复兔膝关节骨软骨缺损的可行性及疗效.方法 构建Col/HA双相支架,将软骨细胞种植于支架培养1周,再将软骨细胞-支架复合体移植修复兔膝关节股骨髁的骨软骨缺损,并对骨软骨缺损的修复进行检测.结果 光镜及扫描电镜观察显示软骨细胞在Col/HA支架中贴附良好,表型维持稳定,分泌胞外基质.大体观察和组织学检测显示,植入体内16周后实验组软骨层呈透明软骨样修复,软骨下骨缺损有新骨构建;对照组骨软骨缺损修复不良,组织学检测以纤维性组织或纤维软骨组织形成.Wakitani评分显示实验组修复组织优于对照组,差异有统计学意义(P＜0.05).结论 双相Col/HA复合支架可作为骨软骨组织工程支架,负载软骨细胞可修复兔膝关节骨软骨缺损,重建关节软骨的结构和功能.     

骨组织工程研究中的美学考量

目的：分别以珊瑚羟基磷灰石(CHA)、藻酸钙为成骨细胞载体，观察裸鼠体内的构建特定形态和可注射组织工程骨的可行性。方法：取兔髂骨，获取骨髓基质干细胞，经体外扩增培养诱导后，获得骨髓基质成骨细胞，分别与CHA和藻酸盐复合，植入和注射于裸鼠背部皮下，6、8周后进行大体观察、组织学和X线检查，观察软骨和骨的形成。以单纯CHA和藻酸盐植入作为对照。结果：植入CHA／成骨细胞组和注射藻酸盐/成骨细胞复合物组均有骨样组织形成，具备骨髓腔样结构，而对照组无骨形成。结论：CHA和藻酸盐均是较好的骨组织工程支架材料，复合成骨细胞后可构建特定形态和可注射组织工程骨。     

以β-磷酸三钙多孔陶瓷为载体建造组织工程化人工软骨

目的 通过将软骨细胞接种到三维多孔β－磷酸三钙（β－TCP）陶瓷支架材料上,探讨以β－TCP为载体建造组织工程化软骨的可行性。方法 将β－TPC多孔陶瓷加工成圆片状,并将其作为构建人工软骨的细胞支架。在支架材料上接种从兔关节软骨分离的软骨细胞,将细胞－陶瓷复合体置于旋转细胞培养系统（RCCS）内,培养1周后,将其移植到裸鼠背部皮下。植入术后4,8,16周取材,进行大体观察、组织学及组织化学等观察。结果 复合体体内移植后,在裸鼠皮下有新生软骨形成,形成的软骨基本保持了支架材料原有形态。结论 β－TPC多孔陶瓷是软骨细胞较适宜的贴附基质,以其为支架能够在体内建造出具有精确解剖形状的人工软骨。     

离心管内组织工程软骨培养的初步研究

目的　探讨几种材料与软骨细胞的生物相容性 ,离心管内培养形成组织工程软骨。方法　自 3周龄兔分离关节软骨细胞 ,按 3× 10 6细胞 /管于离心管内接种软骨细胞 ,离心 (10 0 0r/min) 5min后连续培养 2周。分别将脱脂脱蛋白骨、羟基磷灰石、糖蛋白微孔膜、胶原海绵和明胶海绵置于离心管底 ,按 3× 10 6细胞 /管加入软骨细胞 ,离心 (2 0 0r/min) 5min后连续培养 2周。含钙组织首先经脱钙处理后 ,与其它组织工程软骨行组织学检查。结果　软骨细胞无支架培养形成软骨 ,具有一定的骺软骨组织学特征。支架材料与软骨细胞有良好的生物相容性 ,均能形成软骨样组织。组织工程软骨具有不同的组织学结构 ,细胞及其细胞外基质形态差异明显。结论　支架材料与软骨细胞具有良好的生物相容性 ,软骨细胞于离心管内培养可形成组织工程软骨     

胶原凝胶包埋软骨细胞复合聚磷酸钙纤维/左旋聚乳酸支架异体移植修复兔关节软骨缺损

目的探讨胶原凝胶包埋软骨细胞复合聚磷酸钙纤维／左旋聚乳酸(CPPf／PLLA)支架异体移植修复兔关节软骨缺损的有效性和可行性。方法将胶原凝胶包埋的软骨细胞接种CPPf/PLLA支架构建的复合物体外培养3周，行倒置显微镜和扫描电镜观察，并将复合物异体移植入兔关节软骨缺损，术后4、8、12周取材，从大体、组织学和Ⅱ型胶原免疫组织化学对再生软骨组织进行评价。结果复合物体外培养3周，细胞被大量基质包裹，在支架内分布均匀；新形成的组织为透明软骨样组织、表面光滑且与周围组织整合良好、基质内有Ⅱ型胶原分布。结论胶原凝胶包埋软骨细胞接种CPPf／PLLA支架的方法能提高细胞一支架复合物构建质量，胶原凝胶复合CPPf／PLLA支架可作为软骨细胞载体修复关节软骨缺损。     

Reduction of Plasma Fibrinogen, Immunoglobulin G, and Immunoglobulin M Concentrations by Immunoadsorption Therapy with Tryptophan and Phenylalanine Adsorbents

Abstract Immunoadsorption (1A) therapy with tryptophan (TR-350) or phenylalanine (PH-350) adsorbents has been used to reduce the concentration of serum antibodies in human lymphocyte antigen (HLA)-immunized patients. Other forms of plasma purification have been reported to reduce the level of fibrinogen, which affects the blood properties. In this study we investigated the effects of IA therapy using both adsorbents on plasma fibrinogen and immunoglobulins G and M in 13 patients (8 patients were treated with TR-350, and 5 patients were treated with PH-350). During each session 1 plasma volume (2.8 ± 0.4 L of plasma) was processed through the immunocolumn and then returned to the patient together with the blood cells. Compared with the pretreatment values, the plasma fibrinogen, IgG, and IgM concentrations were significantly reduced after IA therapy (p < 0.01 for TR-350; p < 0.04 for PH-350). There was a positive correlation between the degree of reduction of plasma proteins and the number of IA treatments given. A nonpara-metric test (Wilcoxon's signed-rank test or the Mann-Whitney test) was used for statistical analysis. We conclude from our study that IA therapy effectively lowers the plasma levels of fibrinogen, IgG, and IgM and thus can be considered a valuable alternative to other blood purification methods.     

Is the recovery profile of mivacurium independent of the rate of decay of its plasma concentration in patients with normal plasma cholinesterase activity?

Background: The duration of action of muscle relaxants is poorly correlated to the rate of decay of their plasma concentration. The plasma concentration of mivacurium may rapidly decrease below its active concentration because of the extensive hydrolysis of mivacurium. By inflating a tourniquet on one upper limb for 3 min after the administration of atracurium, mivacurium or vecuronium, we studied the influence of the initial decline of their plasma concentration on their effect. Methods: In 50 patients anaesthetised with thiopental, isoflurane and fentanyl, the effect of bolus doses of 0.15 or 0.25 mg . kg^?1 mivacurium (MIV 15, MIV 25), 0.3 or 0.5 mg . kg^?1 atracurium (ATR 30, ATR 50) and 0.06 or 0.1 mg . kg^?1 vecuronium (VEC 06, VEC 10) were measured on both arms (evoked response of the adductor pollicis to train-of-four stimulation every 12 s), a tourniquet being applied on one arm just before and during 3 min after the muscle relaxant bolus. Results: Tourniquet inflation of 3 min almost abolished the neuromuscular effect of mivacurium. In the vecuronium groups and in the ATR 50 group, tourniquet inflation did not modify the maximum degree of depression of the twitch response. Also, the duration of action of vecuronium was unaffected by the tourniquet. In the ATR 30 group, times to return of the twitch response to 25% (duration 25%) and 75% (duration 75%) of control response were significantly shorter in the cuffed arm, 23 min vs 27 min, and 41 min vs 45 min, respectively. In the ATR 50 group, only duration 25% was significantly shorter in the cuffed arm (41 min vs 45 min). Conclusion: The results suggest that the rate of decline of the plasma concentration of mivacurium is so rapid, that a very low and almost clinically ineffective concentration is present as soon as 3 min after its administration. The results also indicate that the recovery from a mivacurium-induced neuromuscular blockade is not influenced by the rate of decay of its plasma concentration in patients with genotypically normal plasma cholinesterase.     

Membranes in Artificial Organs

Abstract: Membrane processes play a pivotal and enabling role in modern replacement therapy for acute and chronic organ failure and in the management of immunologic diseases. In fact, virtually all contemporary extracorporeal blood purification methods employ membrane devices, and the next generation of artificial organs and tissue engineering therapies are almost certain to be similarly grounded in membrane technology. In this short essay, we comment on the similarities and differences among synthetic membranes and their natural counterparts and also provide a critical overview of the demographics and technology of hemodialysis, hemofiltration, apheresis, oxygenation, and emerging membrane technologies and applications.     

The multiply injured child

Blunt trauma is the principal cause of childhood death in many developed countries. This review outlines the differences between adults and children with respect to resuscitation and treatment of orthopaedic injuries in a child with polytrauma. Recent advances in techniques of fracture stabilization are reported.     

Dilemma of Membrane Biocompatibility and Reuse

Abstract: Numerous articles have been published on the multiple use of dialyzers and on the effect of different reprocessing chemicals and techniques on the dialyzer biocompatibility and performance. The results often appear contradictory, especially those comparing standard biocompatibility parameters. Despite this confusion, a discerning review of the published works allows certain limited conclusions to be drawn. Reprocessing of used hemodialyzers changes the biocompatibility profile of a dialyzer as defined by the parameters complement activation. leukopenia, and cytokine release. The effect of reprocessing depends on the chemicals and reprocessing technique applied and also on the type of membrane polymer being subjected to the reprocessing procedure. Reports of pyrogenic reactions indicate that the flux of the membrane also influences how suitable it is for safe reuse. An increased risk of allergic and pyrogenic reactions appears to be associated with dialyzer reuse. Furthermore, there has been a lack of investigations into the immunologic effect of the layer of adsorbed and chemically altered proteins that remains on the inner surface of reprocessed dialyzers. We conclude that the clinical benefit of dialyzer reuse cannot be generally accepted from a biocompatibility point of view.     

The analgesic effect of racemic ketamine in patients with chronic ischemic pain due to lower extremity arteriosclerosis obliterans

Background : Ketamine in sub-dissociative doses has been shown to have analgesic and phantom-Limb pain, where conventional treatment has often failed. Chronic ischemic pain due to lower extremity arteriosclerosis obliterans often responds poorly to analgesics, and the pain-generating mechanisms are not well understood.
Methods : Eight patients with rest pain in the lower extremity due to arteriosclerosis obliterans were given sub-dissociative doses of 0.15, 0.30, or 0.45 mg/kg racemic ketamine and morphine 10 mg as a 5-min infusion on four separate days in a cross-over, double-blind, randomised protocol. Plasma levels of (S)- and (R)-ketamine and their nor-metabolites were analysed with an enantioselective high-performance liquid chromatography (HPLC) method. Pain levels were evaluated with a visual analogue scale (VAS).
Results : Individual pain levels were highly variable during and after all the infusions but the pooled pain levels showed a dose-dependent analgesic effect of ketamine with a transient but complete pain relief in all patients at the highest dose (0.45 mg/ kg). Side-effects, mainly disturbed cognition and perception, were pronounced and dose-dependent. Morphine 10 mg had an analgesic peak at 20 min and 5/8 patients had complete pain relief. The remaining 3 patients also had high baseline pain scores, indicating a higher analgesic potency for the 0.30 and 0.45 mg/ kg ketamine doses than for morphine 10 mg.
Conclusion : We have demonstrated a potent dose-dependent analgesic effect of racemic ketamine in clinical ischemic pain. Due to a narrow therapeutic window, this analgesic effect is probably best utilised in combination with other analgesics.     

Intravenous endotoxin does not increase tissue extravasation of albumin in rats

Background : It is unclear whether activation of the inducible nitric oxide synthase (iNOS) increases or decreases the extravasation of plasma.
Methods : Chloralose anaesthetised male Wistar rats received E. coli lipopolysacharide (LPS), 3 mg kg^-1 i.v., or the corresponding volume of saline, 3 or 5 h before the end of the experiment. Mean arterial pressure (MAP) and heart rate (HR) were recorded. Tissue clearance of radio-labelled albumin, during the last 2 h of each experiment, was determined by a double-isotope method. In separate animals, the serum concentration of nitrite and nitrate was determined, 5 h after LPS or the solvent.
Main Results : LPS initially decreased MAP and lastingly increased HR. In the 3-h LPS animals (n=8), tissue plasma clearance was lower in the heart and calf muscle and increased only in diaphragm, compared to corresponding control animals (n=8). In the 5-h LPS rats, clearance was lowered (n=8) in the entire gastrointestinal tract and in testes, compared to controls (n=8). The serum nitrite/nitrate concentration was higher in animals given LPS (n=6) than in controls (n=6).
Conclusion : After LPS, tissue clearance of albumin was not increased in any major tissue, in spite of increased serum levels of NO end products. Apparently, after activation of iNOS, the augmented release of NO is not necessarily associated with increased albumin extravasation.     

Adrenaline markedly improves thoracic epidural analgesia produced by a low-dose infusion of bupivacaine, fentanyl and adrenaline after major surgery: A randomised, double-blind, cross-over study with and without adrenaline

Background: Basic pharmacological research indicates that there are synergistic antinociceptive effects at the spinal cord level between adrenaline, fentanyl and bupivacaine. Our clinical experience with such a mixture in a thoracic epidural infusion after major surgery confirms this. The objectives of the present study were to evaluate the effects on postoperative pain intensity, pain relief and side effects when removing adrenaline from this triple epidural mixture. Methods: A prospective, randomised, double-blind, cross-over study was carried out in 24 patients after major thoracic or abdominal surgery. Patients with only mild pain when coughing during a titrated thoracic epidural infusion of about 10 ml · h^?1 of bupivacaine 1 mg · ml^?1, fentanyl 2 μg · ml^?1, and adrenaline 2 μg · ml^?1 were included. On the 1^st and 2^nd postoperative days each patient was given a double-blind epidural infusion, at the same rate, with or without adrenaline. The effect was observed for 4 h or until pain when coughing became unacceptable in spite of a rescue analgesic procedure. Rescue analgesia consisted of up to two epidural bolus injections per hour and i.v. morphine if necessary. All patients received rectal paracetamol 1 g, every 8 h. Fentanyl serum concentrations were measured with a radioimmunoassay technique at the start and end of each study period. Main outcome measures were extent of sensory blockade and pain intensity at rest and when coughing, evaluated by a visual analogue scale, a verbal categorical rating scale, the Prince Henry Hospital pain score, and an overall quality of pain relief score. Results: The number of hypaesthetic dermatomal segments decreased (P <0.001) and pain intensity at rest and when coughing increased (P <0.001) when adrenaline was omitted from the triple epidural mixture. This change started within the first hour after removing adrenaline. After 3 h pain intensity when coughing had increased to unacceptable levels in spite of rescue analgesia (epidural bolus injections and i.v. morphine). Within 15–20 min after restarting the triple epidural mixture with adrenaline, pain intensity was again reduced to mild pain when coughing. Serum concentration of fentanyl doubled from 0.22 to 0.45 ng · ml^?1 (P <0.01), and there was more sedation during the period without adrenaline. Conclusions: Adrenaline increases sensory block and improves the pain-relieving effect of a mixture of bupivacaine and fentanyl infused epidurally at a thoracic level after major thoracic or abdominal surgery. Serum fentanyl concentrations doubled and sedation increased when adrenaline was removed from the epidural infusion, indicating more rapid vascular absorption and systemic effects of fentanyl.