Ligament and meniscus regeneration using cells in peripheral blood

1.富血小板血浆本研究的目的是检验富血小板血浆（PRP）在体外和体内增强乳腺组织再生的假设。在体外研究中，制备单层胰岛细胞培养物，并进行3-（4，5-二甲基噻唑-2基）-5-（3-羧基甲氧基苯基）-2-（4-磺基苯基）-2H-四唑内盐测定和5-溴-20-脱氧尿苷测定以评估在PRP存在下的增殖行为。进行阿新蓝测定以评估细胞外基质（ECM）合成。实时荧光定量聚合酶链反应检测纤维软骨相关信使核糖核酸（mRNA）的表达。在体内研究中，在兔半月板的无血管区域中创建1.5 mm直径的全层缺损。明胶水凝胶（GH）被用作PRP生长因子的药物递送系统。A组：GH + PRP ; B组：GH+贫血小板血浆; C组：单纯GH。每组W ...更多信息 在术后4、8和12周进行组织学评价。PRP刺激脱氧核糖核酸合成和ECM合成（p < 0.05）。与PRP培养的半月板细胞相比，双糖链蛋白聚糖和核心蛋白聚糖的mRNA表达增加（p < 0.05）。组织学发现显示明胶水凝胶的残余物在4周时存在，表明水凝胶可以控制释放约4周。12周时缺损部位的组织学评分显示，接受PRP + GH的动物的尿道修复显著优于其他两组。这些发现表明PRP促进了骨缺损的愈合。外周血干细胞（人循环CD 34+细胞）新血管生成是韧带愈合初始阶段的关键过程。成人循环CD 34+细胞是一种内皮/造血祖细胞富集的细胞群，据报道有助于新生血管生成，但CD 34+细胞用于韧带愈合的治疗潜力仍不清楚。因此，我们进行了一系列的实验，以测试我们的假设，韧带愈合是通过血管生成的CD 34+细胞的支持。在免疫缺陷大鼠内侧副韧带损伤后，局部移植粒细胞刺激因子动员的外周血CD 34+细胞和去端胶原（CD 34+组），或仅移植去端胶原（对照组）。逆转录-聚合酶链反应（RT-PCR）和免疫组化染色在损伤部位表现出的分子和组织学表达的人特异性标志物的内皮细胞在CD 34+组在第1周。以毛细血管密度和血管内皮生长因子mRNA表达为指标，观察CD 34+细胞移植的内源性效应。真实的时间RT'PCR检测结果显示，CD 34+组韧带特异性标志物基因表达明显高于对照组。通过宏观和组织学检查评估的韧带愈合通过CD 34+细胞移植增强。我们的数据表明，循环的人CD 34+细胞可能在韧带愈合过程中发挥重要作用，通过在受损组织中的新血管形成促进有利的环境，从而导致适当的韧带愈合。少

1. Platelet-Rich PlasmaThe objective of the study was to test the hypothesis that platelet-rich plasma (PRP) enhances meniscaltissue regeneration in vitro and in vivo. In the in vitro study, monolayer meniscal cell cultures were prepared, and 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay and 5-bromo-20-deoxyuridine assay were performed to assess proliferative behavior in the presence of PRP. Alcian blue assay was performed to assess extracellular matrix (ECM) synthesis. To detect the fibrocartilage-related messenger ribonucleic acid (mRNA) expressions, real-time polymerase chain reaction was performed. In the in vivo study, 1.5-mm-diameter full-thickness defects were created in the avascular region of rabbit meniscus. Gelatin hydrogel (GH) was used as the drug delivery system for PRP growth factors. The defects were filled as follows : Group A, GH with PRP ; Group B, GH with platelet-poor plasma ; Group C, GH only. Each group w … More as evaluated histologically at 4, 8, and 12 weeks after surgery. PRP stimulated deoxyribonucleic acid synthesis and ECM synthesis ( p < 0.05). Meniscal cells cultured with PRP showed greater mRNA expression of biglycan and decorin ( p < 0.05). Histological findings showed that remnants of gelatin hydrogels existed at 4 weeks, indicating that the hydrogels could control release for approximately 4 weeks. Histological scoring of the defect sites at 12 weeks revealed significantly better meniscal repair in animals that received PRP with GH than in the other two groups. These findings suggest that PRP enhances the healing of meniscal defects.2. Peripheral Blood Stem Cells (Human Circulating CD34+ Cells)Neoangiogenesis is a key process in the initial phase of ligament healing. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to contribute to neoangiogenesis, however the therapeutic potential of CD34+ cells for ligament healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that ligament healing is supported by CD34+ cells via vasculogenesis. Granulocyte-stimulating factor mobilized peripheral blood CD34+ cells with atelocollagen (CD34+ group), or only atelocollagen (control group) were locally transplanted after creating medial collateral ligament injury in immunodeficient rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining at the injury site demonstrated molecular and histological expression of human-specific markers for endothelial cells in the CD34+ group at week 1. Endogeneous effect assessed by capillary density and mRNA expression of vascular endothelial growth factor was found by CD34+ cell transplantation. Gene expression of ligament-specific marker in the CD34+ group assessed by real time RT'PCR was significant higher than in the control group. Ligament healing assessed by macroscopic and histological examinations was enhanced by CD34+ cell transplantation. Our data suggest circulating human CD34+ cells may play an essential role in the ligament healing process by promoting a favorable environment through neovascularization in the damaged tissue, resulting in appropriate ligament healing. Less