Articular cartilage reconstruction using xenogeneic epiphyses slices

Reconstruction of articular cartilage defects using adult osteochondral allografts is an established clinical procedure, whose principal drawback is lack of lateral integration of the grafts to the surrounding tissue. Autologous chondrocytes transplantation is a sophisticated technique requiring cell culture and a staged operation. Its main draw back is the lack of mechanical strength early on. This study was conducted in order to evaluate the possibility of using embryonal epiphyses as a cartilage reconstruction tissue. A xenogeneic human to rabbit sub-acute osteochondral defect model was designed to evaluate the possibility of allogeneic implantation in humans. The following procedures were perfomed (n = 5): transplantation of 1. live epiphyses 2. live epiphyses with autogeneic periosteum 3. de-vitalized epiphyses and 4. devitalized epiphyses with autogeneic articular chondrocytes. A fifth control group did not receive any implant. Animals in groups 1 and 2 had a viable reconstruction of the articular surface with little evidence of rejection and without pannus formation. Animals in groups 3 and 4 became severely arthritic and the graft was resorbed. Nitric oxide synthase accumulation was reduced in group 1 and 2 as compared to groups 3, 4, and 5, indicating a joint preserving function of the epiphyseal grafts. Epiphyseal grafts appear to be a feasible procedure for reconstruction of articular cartilage defects even in a xenogeneic model. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antigen removal for the production of biomechanically functional,xenogeneic tissue grafts

Xenogeneic tissues are derived from other animal species and provide a source of material for engineering mechanically functional tissue grafts, such as heart valves, tendons, ligaments, and cartilage. Xenogeneic tissues, however, contain molecules, known as antigens, which invoke an immune reaction following implantation into a patient. Therefore, it is necessary to remove the antigens from a xenogeneic tissue to prevent immune rejection of the graft. Antigen removal can be accomplished by treating a tissue with solutions and/or physical processes that disrupt cells and solubilize, degrade, or mask antigens. However, processes used for cell and antigen removal from tissues often have deleterious effects on the extracellular matrix (ECM) of the tissue, rendering the tissue unsuitable for implantation due to poor mechanical properties. Thus, the goal of an antigen removal process should be to reduce the antigen content of a xenogeneic tissue while preserving its mechanical functionality. To expand the clinical use of antigen-removed xenogeneic tissues as biomechanically functional grafts, it is essential that researchers examine tissue antigen content, ECM composition and architecture, and mechanical properties as new antigen removal processes are developed.     

生殖细胞及性腺移植

生殖细胞和性腺移植研究近年来已取得了突破性进展。这两项技术对于农业、医学及动物繁殖学的研究具有深远的意义和很大的应用价值。本文从同源移植、异源移植、移植技术及其它移植相关问题等方面对生殖细胞和性腺移植进行了简要介绍,并阐述了近年来在这方面所取得的进展。     

Injection of xenogeneic cells into teleost fish elicits systemic and local cellular innate immune responses

The early innate immune response of the teleost gilthead seabream (Sparus aurata L.) against xenogeneic cells was studied. Fish received a single intraperitoneal injection of xenogeneic cells (tumour cell line), following which leucocyte mobilization, degranulation, peroxidase content, respiratory burst and phagocytic and cytotoxic activities were determined in both peritoneal exudate leucocytes (PELs) and head-kidney leucocytes (HKLs). The total number of PELs increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis of PEL and HKL suspensions revealed variations in the proportion of cell types. The percentage of HK acidophilic granulocytes significantly increased after 72 h, whereas PE acidophils increased after 4 h. Moreover, numbers of PE lymphocytes and monocyte-macrophages significantly increased during the experiment. The peroxidase content of the leucocytes was unaffected, although PEL degranulation was largely enhanced. This liberation of peroxidases correlated well with the enhancement of the oxidative respiratory burst activity in PELs, reflecting leucocyte activation. However, phagocytosis only increased in PELs 4 h after intraperitoneal injection, whereas the cytotoxic activity of HKLs increased 1 and 2 days post-injection but, in general, decreased in the PELs. Our data thus demonstrate that the appearance of xenogeneic cells involves leucocyte mobilization and innate immune-response activation at the site of invasion and in the head-kidney. Involvement of the various leucocyte types and potential modes of activation are discussed.This work was partially funded by the European Commission (QLRT-2001-00722). A. Cuesta and I. Salinas are fellows of Fundación CajaMurcia and Fundación Séneca, respectively.     

Effect of hybrid complement regulatory proteins on xenogeneic cells

To suppress C3 fragment deposition in the classical pathway complement activation on xenogeneic membranes, decay accelerating factor (DAF) was the most effective molecule among the complement regulatory proteins (CRPs) used in the present study. C3 fragment deposition was closely related to subsequent xenogeneic cell lysis. However, other molecules were also very effective in different ways and include phosphatidylinositol (PI)-anchored short consensus repeat (SCR) 2-4 of membrane cofactor protein (MCP-PI), PI-anchored C1 esterase inhibitor (C1-INH-PI), and PI-anchored SCR8-11 of complement receptor type 1 (CR1-PI). On the other hand, regarding a strategy for downregulating C4 fragment deposition, the use of only C1-INH-PI and PI-anchored SCR1-3 of the C4b-binding protein (C4bp-PI) was found to be effective.     

Porcine CD58: cDNA cloning and molecular dissection of the porcine CD58-human CD2 interface

The porcine ligands of human CD2 remain unknown in xenotransplantation despite being an important pathway of T cell costimulation. Of the two main candidates, i.e., CD48 and CD58, the cDNA of the most likely ligand poCD58 was cloned from CD48-negative endothelial cells costimulating human CD4(+) T cells through the CD2 pathway. The deduced protein sequence is 244 residues long and is 43% homologous to the human sequence. Based on similarity between porcine and human CD58 external V-set Ig-type domains, a structural model of poCD58-huCD2 interaction was built. Most of the charged residues located at the interface with huCD2 are highly conserved. Six putative hydrogen bonds between poCD58 and huCD2 were identified; five involve the same residues as in the syngeneic combination while the sixth is formed between an additional tyrosine in poCD58 and Arg48 in huCD2, increasing the complementarity between the two molecules. These structural data will help us to develop poCD58 blocking agents for xenotransplantation.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

Recovery and functionality of cryopreserved peripheral blood mononuclear cells using five different xeno-free cryoprotective solutions

In this study, we compared three commercially available and two widely used CPAs for their ability of cryopreserving PBMCs. Similar survival (81.0%) and recovery rate (73.7%) were observed among cells using these five CPAs. However, all the cryopreserved PBMCs exhibited a significantly lower survival rate when compared with the fresh samples (94.3%). We further evaluated effector cell subpopulation and tumoricidal activity of PBMC-derived cytokine-induced killing (CIK) cells and natural killing (NK) cells. Similar and high survival (CIK: 88.6%; NK: 87.5%) and recovery (CIK: 99.5%; NK: 99.7%) rates were detected in CIK and NK cells prepared from cryopreserved PBMCs using the five CPAs. The CD3⁺CD56⁺ effector percentage (27.3%) of cryopreserved PBMC-derived CIK cells using the five different CPAs and their tumoricidal activities on melanoma CHL-1 cells (45.7%) and bladder cancer cell line T-24 (44.7%) were similar but significantly lower than those of the fresh PBMC-derived controls (effector: 30.7%; CHL-1: 84.2%; T-24: 82.2%). Cryopreserved PBMC-derived NK cells also exhibited similar tumoricidal activities (CHL-1: 73.8%; T-24: 71.9%) but was significantly lower than that of the fresh control group. We were not able to identify a specific CPA that performed superior than others in PBMC cryopreservation.     

重组合异种骨治疗股骨非感染性骨不连的长期疗效观察

目的:探讨重组合异种骨治疗股骨非感染性骨不连的长期临床疗效。方法:对2000年1月至2006年9月间我院应用重组合异种骨(RBX)治疗的37例股骨非感染性骨不连患者进行回顾性分析,其中男26例,女11例;年龄4～70岁,平均31.6岁。骨折部位:股骨近端4例,中段30例,远端3例。骨不连类型:肥大型9例,营养不良型6例,萎缩型22例。固定方式:加压钢板24例,髓内钉11例,外固定架2例。结果:37例患者获得51-131个月的随访,平均90.2个月,骨不连一次手术愈合率:94.6%,4例股骨近端骨不连患者采用Harris评分系统评定疗效,优3例,良1例,差0例,优良率100%。3例股骨远端骨不连患者采用美国膝关节协会评分系统(KSS)评定疗效,优1例,例良1例,差1例,优良率66.7%。30例股骨干骨不连患者采用Harris评分系统和KSS评定疗效,优21例,良8例,差1例,优良率96.7。总优良率94.6%。长期观察均无免疫排斥反应表现。结论:RBX用于治疗股骨非感染性骨不连具有材料充足、骨折愈合率高、组织兼容性好长期应用无免疫排斥反应等优点,是一种良好的自体骨替代材料。