Clinical experience in cell-based therapeutics: disc chondrocyte transplantation A treatment for degenerated or damaged intervertebral disc

Disc herniation treated by discectomy results in a significant loss of nucleus material and disc height. Biological restoration through the use of autologous disc chondrocyte transplantation offers a potential to achieve functional integration of disc metabolism and mechanics. Chondrocytes that have been removed from damaged cartilaginous tissues maintain a capacity to proliferate, produce and secrete matrix components and respond to physical stimuli such as dynamic loading. Nucleus regeneration using autologous cultured disc-derived chondrocytes (ADCT) has been demonstrated in a canine model and in clinical pilot studies. In 2002 a prospective, controlled, randomised, multi-center study, EuroDISC, comparing safety and efficacy of autologous disc chondrocyte transplant, chondrotransplant DISC, plus discectomy (ADCT), with discectomy alone was initiated. A dog model was used to investigate the hypothesis that autologous disc chondrocytes can be used to repair damaged intervertebral disc. Disc chondrocytes were harvested and expanded in culture under controlled and defined conditions, returned to the same animals from which they had been sampled (autologous transplantation) via percutaneous delivery. The animals were analyzed at specific times after transplantation by several methods to examine whether disc chondrocytes integrated with the surrounding tissue, produced the appropriate intervertebral disc extracellular matrix, and might provide a formative solution to disc repair. The clinical goals of the EuroDISC study, were to provide long-term pain relief, maintain disc height and prevent adjacent segment disease. Interim analysis was performed after 2 years; Oswestry (low back pain/disability), Quebec Back-Pain Disability Scale, as well as Prolo and VAS score were used for the evaluation. Disc height was assessed by MRI. In the context of degenerative changes in an injury model: () autologous disc chondrocytes were expended in culture and returned to the disc by a minimally invasive procedure after 12 weeks; () disc chondrocytes remained viable after transplantation as shown by bromodeoxyuridine incorporation and maintained a capacity for proliferation after transplantation as depicted by histology; () transplanted disc chondrocytes produced an extracellular matrix that displayed composition similar to normal intervertebral disc tissue. Positive evidence of Proteoglycan content was supported by accepted histochemical staining techniques such as Safranin O-Fast Green; () both Type II and Type I collagens were demonstrated in the regenerated intervertebral disc matrix by immunohistochemistry after chondrocyte transplantation; and () when the disc heights were analyzed for variance according to treatment a statistically significant-correlation between transplanting cells and retention of disc height was achieved. A clinically significant reduction of low back pain in the ADCT-treated group was shown by all three pain score systems. The median total Oswestry score was 2 in the ADCT-treated group compared with 6 in the control group. Decreases in the disability index and VAS score in ADCT-treated patients correlated strongly with the reduction of low back pain. Decreases in disc height over time were only found in the control group, and of potential significance, intervertebral discs in adjacent segments appeared to retain hydration when compared to those adjacent to levels that had undergone discectomy without cell intervention. Autologous chondrocyte transplantation is technically feasible and biologically relevant to repairing disc damage and retarding disc degeneration.     

Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc

When a fragment of a Drosophila imaginal disc is cultured in growth permissive conditions, it either regenerates the missing structures or duplicates the pattern present in the fragment. This kind of pattern regulation is known to be epimorphic, i.e. the new pattern is generated by proliferation in a specialized tissue called the blastema. Pattern regulation is accompanied by the healing of the cut surfaces restoring the continuous epithelia. Wound healing has been considered to be the inductive signal to commence regenerative cell divisions. Although the general outlines of the proliferation dynamics in a regenerating imaginal disc blastema have been well studied, little is known about the mechanisms driving cells into the regenerative cell cycles. In this study, we have investigated the role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila wing imaginal disc. By utilizing in vivo and in vitro culturing of incised and fragmented discs, we have been able to visualize the dynamics in cellular architecture and gene expression involved in the healing and regeneration process. Our results directly show that homotypic wound healing is not a prerequisite for regenerative cell divisions. We also show that JNK signaling participates in imaginal disc wound healing and is regulated by the physical dynamics of the process, as well as in recruiting cells into the regenerative cell cycles. A model describing the determination of blastema size is discussed.     

经皮椎间孔镜与Mast Quadrant通道技术治疗腰椎间盘突出症临床疗效比较

目的:研究和比较经皮椎间孔镜(Percutaneous Transforaminal Endoscopic Discectomy,PTED)与Mast Quadrant通道技术治疗腰椎间盘突出症(Lumbar disc herniation,LDH)的临床疗效及安全性。方法:回顾性分析2008年至2015年在本院接受手术治疗的单节段LDH患者90例,其中接受经皮椎间孔镜椎间盘突出髓核摘除术(PTED)患者58例,接受Mast Quadrant通道系统下腰椎间盘切除术患者32例,按手术方式的不同分为PTED组与Quadrant组。结合随访资料,评价并比较两组病例手术时间、术中出血量、术后住院天数、并发症发生情况。手术效果按照视觉疼痛模拟评分(VAS)、0swestry功能障碍指数(ODI)和改良Mac Nab标准进行评定。结果:椎间孔镜组术中出血量、术后住院天数、并发症发生率、术后1天、7天腰痛VAS评分及ODI均优于Quadrant组(P0.05);椎间孔镜组手术时间长于Quadrant组(P0.05);两组术后腿痛VAS评分、手术优良率比较差异无统计学意义(P0.05)。结论:经皮椎间孔镜与Mast Quadrant通道技术均能有效治疗腰椎间盘突出症,在严格掌握手术适应证和禁忌症的前提下,经皮椎间孔镜手术能够明显减少出血及并发症,是一种治疗腰椎间盘突出症理想的微创手术方式,但长期疗效有待进一步临床研究。     

Chemonucleolysis for relief of sciatica due to a herniated intervertebral disc.

Chemonucleolysis is the nonoperative chemical removal of displaced lumbar disc material. The enzyme chymopapain, which has a wide margin of safety between its effective therapeutic and toxic doses, is effective in the management of sciatica due to a herniated intervertebral disc. The patient will have leg pain as the dominant symptom and a 50% reduction in straight-leg raising with or without bowstring discomfort and crossover pain. Neurologic symptoms and signs are usual, as are abnormal results of contrast studies, which will verify the level of involvement. In 220 randomly selected patients who met criteria for the diagnosis of sciatica due to a herniated intervertebral disc and did not have psychogenic or nonorganic spinal pain, a spinal stenosis or a history of a previous, unsuccessful operation to relieve the sciatica, chemonucleolysis had a success rate of 80%. The only complications were a severe anaphylactic reaction in two patients and lesser, delayed reactions in five others. All of the reactions were successfully treated. Of the 45 patients in whom chemonucleolysis was unsuccessful, 38 underwent a laminectomy. In 3 of the 38 the results of chemonucleolysis were initially good, but later the disc herniation recurred; thus, the long-term treatment failure rate was 1.4%.     

Regeneration following duplication in imaginal wing disc fragments of Drosophila melanogaster

Fragments from the imaginal wing disc of Drosophila melanogaster were cultured in vivo for periods up to 28 days. One type of edge fragment first duplicated and then ceased to grow, but others often continued to grow following initial duplication and regenerated structures characteristic of other areas of the disc. After 28 days of culture, about 50% of fragments from the presumptive ventral hinge region of the disc grew extensively and produced regenerated as well as original structures. The regenerated structures in some implants were produced at the line of mirror-image symmetry. Regeneration was associated with fragment growth and in many cases was accompanied by loss of duplicate structures. Fragments which were only duplicated after the culture period could in some cases be stimulated to grow by additional culture in fresh hosts, but the results of coculturing two fragments in each host show that culture conditions alone do not control growth and regulation in the fragments. The large, normally regenerating fragment, complementary to the ventral fragment, did not appear to grow following regeneration and only occasionally produced supernumerary structures during prolonged culture. Intact wing discs cultured under similar conditions never produced supernumerary structures. Our results suggest that a duplicated pattern is less stable than a complete, regenerated pattern, which in turn is less stable than an intact disc. We propose that the growth of duplicated disc fragments is stimulated by polarity reversals present at lines of mirror-image symmetry.     

笼养猕猴疝根治手术的探讨

目的对发生嵌闭性疝的猕猴,采用疝手术治疗法挽救其生命。方法各种猕猴疝经确诊,实施手术治疗。结果共实施各种猕猴疝手术7例,手术成功率100%,愈后恢复良好。结论根据疝的位置和临床症状,确诊疝的种类,采取相应的疝根治手术疗法,7例猕猴疝病例手术后经过治疗护理,没有手术并发症及手术死亡,手术治疗法是治疗猕猴疝最有效的方法,对相关的灵长类动物外科手术及保护珍贵猕猴动物资源,减少猕猴因各种疝引起的痛苦或死亡均有一定的参考价值。     

Regulative interaction of mature imaginal disc Fragments with embryonic and immature disc tissues inDrosophila

Summary These experiments examined whether inDrosophila immature imaginal disc tissue and tissues from embryonic stages can influence pattern regulation in a disc fragment in the same way as can mature imaginal discs. Immature imaginal discs, or the cells of whole embryos, were mixed with a test fragment (presumptive notum) from a mature wing disc. The immature tissues in each mixture were genetically marked and had been heavily irradiated (25 Kr gamma) prior to mixing to prevent growth and maturation during subsequent culture in vivo. Alteration of the regulative behavior of the test fragment (that is, regeneration of wing) thus provided an assay for the communication of positional information by the immature tissues. The results suggest that this capacity arises well before competence to metamorphose, as early as the 16th hour of embryonic development, whereas prior to 16 h, essentially no stimulation of regeneration occurred. It is suggested that the imaginal disc (or presumptive disc) cells of the embryo may have been responsible for this early stimulatory capacity.     

Homologies of positional information in thoracic imaginal discs ofDrosophila melanogaster

Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.     

Growth and maturation of roker Raja clavata L. in the Solway Firth

Two hundred and seventy-one Raja clavata taken by otter trawl from the Solway Firth were examined for size, sex and state of maturity. Size relationships for both sexes were similar. Total length and disc width were linearly related as were disc width with wing width. Males and females appear to mature at 42 and 45 cm disc width respectively. The Solway population is heavily exploited by an unrestricted commercial fishery and a considerable proportion (48·6%) of the presently retained catch is immature. It is suggested that fishing pressure has brought about a reduction in the size at which female fish mature.     

Compartments and distal outgrowth in theDrosophila imaginal wing disc

Summary Peripheral tissue of the imaginal wing disc gives rise to the proximal mesothoracic structures of the adult. Pieces of peripheral tissue, which have no regenerative capacity when cultured as intact fragments, are capable of distal outgrowth (regeneration) after dissociation and reaggregation. This ability depends on the region of the disc periphery from which the fragment is taken. Extensive distal outgrowth occurs in reaggreages of a fragment containing equal proportions of tissue from anterior and posterior developmental compartments. The extent of outgrowth decreases as the proportion of posterior tissue is reduced, so that a fragment containing only anterior tissue shows no regeneration after dissociation. Limited distal outgrowth occurs in reaggregates of a wholly posterior fragment, but the regenerative capacity is increased greatly when a small amount of anterior tissue is included. It is concluded that distal outgrowth in the wing disc requires an interaction between cells of the anterior and posterior compartments.     

A functional polymorphism in THBS2 that affects alternative splicing and MMP binding is associated with lumbar-disc herniation

Lumbar-disc herniation (LDH), one of the most common musculoskeletal diseases, has strong genetic determinants. Recently, several genes that encode extracellular matrix (ECM) proteins in the intervertebral disc have been reported to associate with LDH. Thrombospondins (THBSs) 1 and 2 are good candidates for the LDH susceptibility gene: They are intervertebral disc ECM proteins that regulate the effective levels of matrix metalloproteinases (MMPs) 2 and 9, which are key effectors of ECM remodeling. Here, we report that THBS2 is associated with LDH in Japanese populations. An intronic SNP in THBS2 (IVS10-8C --> T; rs9406328) showed significant association (p = 0.0000028) with LDH in two independent Japanese populations. This SNP, located in a polypyrimidine tract upstream of the 3' splice site of intron 10, exerts allelic differences on exon 11 skipping rates in vivo, with the susceptibility allele showing increased skipping. Skipping of exon 11 results in decreased THBS2 interaction with MMP2 and MMP9. Further, a missense SNP in MMP9 (Q279R; rs17576) is also strongly associated with LDH in the Japanese population (p = 0.00049) and shows a combinatorial effect with THBS2 (odds ratio 3.03, 95% confidence interval 1.58-5.77). Thus, a splicing-affecting SNP in THBS2 and a missense SNP in MMP9 are associated with susceptibility to LDH. Our data indicate that regulation of intervertebral disc ECM metabolism by the THBS2-MMP system plays an essential role in the etiology and pathogenesis of LDH.     

Red cell lactic acid dehydrogenase in the family Macropodidae

Lactic acid dehydrogenase isoenzyme patterns from red cells of members of the family Macropodidae are described as they appear in disc acrylamide gels. The reproducibility of patterns, which are determined by the relative intensities of LDH isoenzymes, is noted. Of the 473 animals tested, 471 had red cell LDH patterns belonging to one of five patterns. The distribution of these five patterns among the different genera and species is discussed, and it is suggested that investigation of LDH patterns by this method might assist in the classification of members of the family Macropodidae.     

A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.     

A fragment comprising the last third of bovine serum albumin which accounts for almost all the antigenic reactivity of the native protein.

The fragmentation of native bovine serum albumin by trypsin has been studied in aqueous solution under various conditions with regard to the yield and size of the fragments obtained. From a partial tryptic hydrolysate at pH 8.2 (40 degrees, 1 hour), a homogeneous fragment was isolated in high yield by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose. The molecular weight of the fragment by gel filtration on calibrated Sephadex G-100 columns and by sodium dodecyl sulfate electrophoresis was 22,500. After reduction of the disulfide bonds followed by alkylation of the resultant thiol groups with iodoacetamide, the fragment retained homogeneity by disc electrophoresis and its molecular weight remained unchanged, indicating that it was composed of a single polypeptide chain. From its amino acid composition, sequence of the first 20 residues, and actions of carboxypeptidases A or B, it was unequivocally assigned to positions 377-571 in albumin. The inhibitory activity of the fragment was 90 to 93% towards the immune reaction of the protein with the IgG fraction of the antisera. The IgGfraction accounted for 96% of the total antibody activity in the antisera. An immunoabsorbent of fragment 377-571 removed 89 to 95% of the antibody to albumin. A fluorescent derivative of the fragment, which retained full immunochemical activity, was found to bind 2 mol of antibody/mol of peptide. The disulfides in peptide 377-571 were essential for its immunochemical reaction because the latter was entirely abolished upon reduction and S-alkylation of the disulfides. Since this fragment comprised only a third of the albumin molecule, but accounted for 90 to 95% of its antigenic reactivity, the results indicated that native albumin carries identical repeating antigenic reactive sites.     

保守疗法治疗腰椎间盘突出症的短期临床疗效分析

目的:分析保守疗法治疗腰椎间盘突出症的短期临床疗效。方法:选取2013年1月~2013年12月来我科就诊并采取保守疗法治疗的69例腰椎间盘突出症患者,对其临床资料进行回顾性分析。其中,37例采用了电针、推拿、中频、牵引和功能锻练等综合疗法(综合疗法治疗组),32例仅采用了药物治疗(单纯药物对照组),比较两组治疗前后的JOA评分及VAS疼痛评分,并比较其临床疗效。结果:治疗后,两组患者的腰椎功能JOA评分均较治疗前明显改善(P0.05),且综合保守治疗组的JOA评分明显高于单纯药物对照组(p0.05);两组患者的VAS疼痛评分均较治疗前明显降低(P0.05),且综合保守治疗组患者的VAS疼痛评分降低程度明显高于单纯药物对照组(P0.05)。单纯药物对照组的总有效率为71.87%,而综合保守治疗组的总有效率为91.89%,较单纯药物对照组显著升高(P0.05)。结论:与单纯药物治疗相比,采用综合保守疗法治疗腰间盘突出症的短期临床疗效更好,可更有效改善患者的腰椎功能并缓解其疼痛。     

长沙地区两种常见蚯蚓及太平Ⅱ号蚓的EST、LDH同工酶研究

本文报道了长沙地区两种常见蚯蚓——日本杜拉蚓、舒脉环毛蚓以及人工养殖的太平Ⅱ号蚓为实验材料,应用聚丙烯酰胺凝胶圆盘电泳方法,分析ＥＳＴ、ＬＤＨ两种同工酶。实验结果表明ＥＳＴ同工酶酶谱复杂,ＬＤＨ同工酶具多态现象。ＥＳＴ同工酶能正确反映出种属间的差异。因此ＥＳＴ同工酶酶谱可成为寡毛类分类学上分子水平的一项生化指标     

Cloning and sequence analysis of the gene encoding L-lactate dehydrogenase from Lactococcus lactis: evolutionary relationships between 21 different LDH enzymes.

Lactate dehydrogenase (LDH; EC1.1.1.27) is a key enzyme in the fermentation of milk by lactic acid bacteria used in the dairy industry. An 800-bp DNA fragment containing part of the gene (ldh) encoding LDH was amplified from Lactococcus lactis in a polymerase chain reaction using primers designed from the partial amino acid sequence of a lactococcal LDH. This fragment was radioactively labelled and used to probe a phage lambda library of Lc. lactis genomic DNA. Fragments containing ldh were subcloned from lambda to pUC13 and pUC18 and a 1.2-kb region was sequenced. The deduced aa sequence reveals that the lactococcal LDH is highly homologous to the LDHs of other organisms. The active site and several other domains of unknown function are highly conserved between all LDH enzymes (prokaryotic and eukaryotic). An evolutionary study of LDH sequences clearly divides the prokaryotic from the eukaryotic enzymes except for the Bifidobacterium longum LDH which anomalously groups with the eukaryotic enzymes. The LDHs from Gram-positive bacteria form a separate group from the enzymes from the Gram-negative organisms. The lactococcal LDH is phylogenetically closest to the streptococcal LDH.     

Short aggrecan gene repetitive alleles associated with lumbar degenerative disc disease in Turkish patients

We investigated a possible association between aggrecan gene polymorphism and lumbar degenerative disc disease in Turkish patients. One hundred 20-30-year-old patients with or without low back pain were selected for the study. Lumbar magnetic resonance imaging was performed on all patients. The patient group had low back pain clinically and degenerative disc disease radiographically. The control group included patients with and without low back pain: all were negative radiographically for degenerative disc disease. Genomic DNA was extracted from all participants. A PCR assay were used to evaluate variable number of tandem repeat polymorphism of aggrecan gene alleles to determine if there was any correlation with degenerative disc disease. Significant associations were found between short repeated alleles of the aggrecan gene and severe disc degeneration. A significant association was also found between short repeated alleles of the aggrecan gene and multilevel disc herniation as well as extrusion and sequestration types of disc herniation. In Turkish population, short repeated alleles of the aggrecan gene are associated with increased disc degeneration and disc herniation.