Platelet-rich plasma/osteoblasts complex induces bone formation via osteoblastic differentiation following subcutaneous transplantation.

BACKGROUND AND OBJECTIVE: Platelet-rich plasma (PRP) has been shown to enhance the maturation of bone grafts following local application and to have biological effects on osteoblasts in vitro. However, PRP is not applied by itself clinically as a result of its poor benefits in large bone defects. The present study was undertaken to develop a clinical alternative to autologous bone, by investigating the application of PRP in combination with osteoblastic cells and evaluating its effects after transplantation. MATERIAL AND METHODS: PRP and platelet-poor plasma (PPP) were prepared from blood, obtained from ddY mice, by two centrifugation steps. MC3T3-E1 cells were labeled with fluorescent carbocyanine just before transplantation. The combination of labeled cells and PRP gel was subcutaneously transplanted into the back of severe combined immunodeficient (SCID) mice, and the transplants were evaluated radiographically and immunohistologically after 4 wk. The effects of PRP were assessed by alkaline phosphatase (ALP) staining and von Kossa staining, and the expression of bone-related markers was analyzed by reverse transcription-polymerase chain reaction before transplantation. RESULTS: Before transplantation, PRP enhanced the expression of Osterix and bone sialoprotein mRNAs compared with PPP. Furthermore, PRP elevated ALP activity and induced the formation of mineralized nodules. After transplantation, the combination of labeled cells and PRP gel formed mineralized tissue, and the transplanted cells visualized in the tissue using fluorescence microscopy expressed osteocalcin and type I collagen. CONCLUSION: These results suggest that the application of a PRP/osteoblasts complex has beneficial effects for transplanting engineered cells into bone defects through the promotion of osteoblastic differentiation.

HLA class-I-restricted and colon-specific cytotoxic T cells from lamina propria lymphocytes of patients with ulcerative colitis.

We established cytotoxic T-lymphocyte (CTL) lines against colonic epithelial cell line from LPLs of patients with ulcerative colitis by continuous stimulation with human lymphocyte antigen A (HLA-A) matched colonic epithelial cell lines and clones from LPLs using polyclonal stimulation with phytohemagglutinin. The established CTL lines and clones showed cytotoxicity against HLA-A-matched colonic epithelial cell line but not against HLA-mismatched colonic epithelial cell lines, and HLA-A-matched lung and esophagus cell lines. The CTL response was HLA class I-restricted and mediated by CD8-positive T-lymphocytes. Moreover, the CTL line showed cytotoxicity against autologous B-LCLs pulsed with peptides extracted from HLA-A-matched colonic epithelial cell line but not against other organ-derived peptides pulsed and unpulsed autologous B-LCLs. CTL lines and clones established from LPLs of patients with ulcerative colitis showed colon-specific and HLA class I-restricted killing of human colonic epithelial cell line, suggesting that these CTLs may play a role in colonic epithelial cell damage in some patients with ulcerative colitis.

Generation of MHC class I-restricted cytotoxic T cell lines and clones against colonic epithelial cells from ulcerative colitis.

We established CTL lines and clones against colonic epithelial cells from PBLs of patients with ulcerative colitis by continuous stimulation with HLA-A locus-matched colonic epithelial cell lines. We developed a nonradioactive europium release cytotoxicity assay to detect CTLs. PBLs from 3 of 12 patients but not from any of 14 normal controls who shared at least one haplotype of HLA-A locus with two colonic epithelial cell lines, CW2 and ACM, showed increased cytotoxicity against these lines. Three CTL lines established from the PBLs of patients showed increased cytotoxicity against HLA-A locus-matched CW2 or ACM but not against matched lung or esophagus cell lines. The phenotypes of CTL lines were alpha beta-TCR+ CD3+ CD8+ CD16-. The CTL line MS showed increased cytotoxicity against freshly isolated colonic epithelial cells but not against cells with a different HLA-A locus. Two CTL clones were generated from MS and clone 3-2, expressing CD3+ CD8+ CD4- CD56-, showed high MHC class I-restricted cytotoxicity against the colonic epithelial cells. These results indicated that CTLs against colonic epithelial cells may contribute to epithelial cell damage in ulcerative colitis.

Mac1 discriminates unusual CD4-CD8- double-negative T cells bearing alpha beta antigen receptor from conventional ones with either CD4 or CD8 in murine lung.

Pulmonary intraparenchymal leukocytes were purified from normal mice. By flow cytometry, 20-30% of the lymphocytes were positive for the expression of Mac1, a cell-surface antigen largely restricted to macrophages, neutrophils and natural killer (NK) cells. Sorted Mac1+ lung lymphocytes were large and had abundant cytoplasm with few azurophilic granules. Because Mac1+ lymphocytes did not contain any asiallo GM1+ cells, they are not likely to be NK cells. By a two-color flow cytometric analysis, Mac1+ lymphocytes were demonstrated to be TCR-alpha beta intermediate+, TCR-gamma delta-, CD3intermediate+, CD4-, CD8-, Thy1-, CD5-, and B220-. These Mac1+ alpha beta T cells were not found in other organs such as spleen, thymus, liver, bone marrow and intestine of mice uninfected and infected with Mycobacterium bovis BCG. There was a considerable population of this unusual subset of alpha beta T cells in the lungs of congenitally athymic nude mice. In the Mac1+ alpha beta T-cell population, the proportions of V beta 8+ T cells and of forbidden T-cell clones expressing V beta 6 TCR were not much different from that in the conventional T-cell population. These results indicated that extrathymically developed alpha beta T cells reside in considerable proportions in the lung and that Mac1 clearly discriminates these cells from conventional ones. Interestingly, the proportion of these cells increased in the lungs of mice infected with M. bovis BCG, which raises a possibility that these cells may play some role in the host defense against mycobacterial infection.