Cell therapy for bone repair.

When natural bone repair mechanisms fail, autologous bone grafting is the current standard of care. The osteogenic cells and bone matrix in the graft provide the osteo-inductive and osteo-conductive properties required for successful bone repair. Bone marrow (BM) mesenchymal stem cells (MSCs) can differentiate into osteogenic cells. MSC-based cell therapy holds promise for promoting bone repair. The amount of MSCs available from iliac-crest aspirates is too small to be clinically useful, and either concentration or culture must therefore be used to expand the MSC population. MSCs can be administered alone via percutaneous injection or implanted during open surgery with a biomaterial, usually biphasic hydroxyapatite/ß-calcium-triphosphate granules. Encouraging preliminary results have been obtained in patients with delayed healing of long bone fractures or avascular necrosis of the femoral head. Bone tissue engineering involves in vitro MSC culturing on biomaterials to obtain colonisation of the biomaterial and differentiation of the cells. The biomaterial-cell construct is then implanted into the zone to be treated. Few published data are available on bone tissue engineering. Much work remains to be done before determining whether this method is suitable for the routine filling of bone tissue defects. Increasing cell survival and promoting implant vascularisation are major challenges. Improved expertise with culturing techniques, together with the incorporation of regulatory requirements, will open the way to high-quality clinical trials investigating the usefulness of cell therapy as a method for achieving bone repair. Cell therapy avoids the drawbacks of autologous bone grafting, preserving the bone stock and diminishing treatment invasiveness.

Human and rodent bone marrow mesenchymal stem cells that express primitive stem cell markers can be directly enriched by using the CD49a molecule.

Bone marrow (BM) from human and rodent species contains a population of multipotential cells referred to as mesenchymal stem cells (MSCs). Currently, MSCs are isolated indirectly by using a culture step and then the generation of fibroblast colony-forming units (CFU-fs). Unprocessed or native BM MSCs have not yet been fully characterised. We have previously developed a direct enrichment method for the isolation of MSCs from human BM by using the CD49a protein (alpha1-integrin subunit). As the CD49a gene is highly conserved in mammals, we have evaluated whether this direct enrichment can be employed for BM cells from rodent strains (rat and mouse). We have also studied the native phenotype by using both immunodetection and immunomagnetic methods and have compared MSCs from mouse, rat and human BM. As is the case for human BM, we have demonstrated that all rodent multipotential CFU-fs are contained within the CD49a-positive cell population. However, in the mouse, the number of CFU-fs is strain-dependent. Interestingly, all rat and mouse Sca-1-positive cells are concentrated within the CD49a-positive fraction and also contain all CFU-fs. In human, the colonies have been detected in the CD49a/CD133 double-positive population. Thus, the CD49a protein is a conserved marker that permits the direct enrichment of BM MSCs from various mammalian species; these cells have been phenotyped as true BM stem cells.

Induction of tissue factor expression on human umbilical vein endothelial cells by cell-specific HLA class I antibody: preliminary data.

Donor-specific antibodies may play an important role in the development of chronic allograft rejection process. However, the mechanisms leading to intimal vascular proliferation and fibrosis remain poorly understood. The aim of this study was to examine whether donor-specific HLA antibodies induce overexpression of tissue factor (TF) by endothelial cells. HLA typed human umbilical vein endothelial cells (HUVEC) were incubated for 1 to 12 hours with LPS (10 microg/mL), and increasing concentrations (1 to 500 microg/mL) of anti-HLA A1 antibody specific for an antigen expressed by HUVEC and of an anti-HLA A2 antibody for which A2 was not expressed by the HUVEC. Expression of TF mRNA transcripts was quantified using real time Q-RT PCR and TF activity was tested in cell lysates of cultured HUVEC using a chromogenic TF activity assay. HUVEC-specific anti-HLA A1 antibody at low concentrations (10 microg/mL) induced both a significant increase of TF mRNA transcripts after 1 hour of incubation and TF activity after 3 hours incubation compared to incubation with medium alone or with the nonspecific anti-HLA A2 antibody (n = 4 for all experiments, P < .05). These data show for the first time that specific anti-HLA antibody can induce overexpression of TF on endothelial cells. TF, a transmembrane glycoprotein involved not only in the onset of the coagulation cascade, but also in cell proliferation and anti-apoptotic processes, may play a role in the development of alloantibody-induced chronic rejection.

Critical analysis and efficacy of BMPs in long bones non-union.

Non-union of long bone fractures is considered as multifactorial. The management of this entity continues to be difficult often requiring multiple procedures with unpredictable results. Bone morphogenetic proteins (BMPs) are power agents being used in the clinical setting for a variety of pathological conditions where tissue regeneration is required. This article analyses the currently existing evidence of the efficacy of BMPs for the management of non-union of long bone fractures.

Adhesion of CD34+ marrow precursors to human stroma is related to alphaSM actin expression by human marrow myofibroblasts.

We have studied the adhesion of human marrow CD34(+) precursors to stromal layer of nontransformed human marrow myofibroblasts (normal stroma) and to different stromal cell lines immortalized by T (PU-34) or t and T (L88/5, L87/4, L2Ori-, KM-102) oncogenes from simian virus 40 and E6, E7 oncogenes from human papilloma virus 16 (HS-27A, HS-23). Flow cytometry and Western blotting studies showed that cells from all lines were stromal myofibroblasts similar to normal stroma. Using an original method of adhesion measurement, we found that adhesion of CD34(+) cells was significantly increased on PU-34 cell layer as compared to normal stroma (43% vs. 27%) whereas adhesion on HS-27A and HS-23 was significantly decreased (11% and 8.5%, respectively), and adhesion on L88/5, L87/4, KM-102 and L2Ori- was negligible to nil (<6%). Adhesion of CD34(+) cells to stromal layers paralleled the expression of alpha smooth muscle (alphaSM) actin within the microfilaments of the cells from the different lines and was inversely correlated to their anchorage-independent growth in semisolid agar. These data show that adhesion to the stromal layer of CD34(+) cells is related to the alphaSM actin microfilamentous network in marrow myofibroblasts and that transformation can negatively affect this microfilamentous network and therefore adhesion of hematopoietic precursors.

Human marrow stromal precursors are alpha 1 integrin subunit-positive.

In this work we studied the expression of adhesion molecules on primate human and non-human marrow stromal cells (primary cultures and lines) and on human CD34(+) hematopoietic normal and leukemic precursors. Differential expression of alpha1 integrin subunit was observed, since this molecule was intensely expressed by marrow stroma but not detected on CD34(+) cells. We used this difference to select, in fresh bone marrow samples, alpha 1-positive cells. We found that all stromal precursors giving rise to colony-forming units-fibroblasts (CFU-F) were present in the alpha 1-positive fraction. No colonies were detected in the alpha 1-negative fraction even after 2 weeks of culture. Phenotypic studies of stromal cells derived from alpha1-positive cells and grown in long-term marrow culture indicated that these cells were similar to stromal cells from primary cultures. We also observed early upregulation of alpha 4 and alpha 2 integrin subunits in cultures derived from alpha1-positive cells with maximal expression by day 10 (26 and 51%, respectively) preceding a gradual decline to low to nil values at day 30 (4.5 and 12%). These data indicate that alpha 1 integrin subunit is a marker for both mature stromal cells and stromal precursors, while alpha 2 and alpha 4 integrin subunits are expressed primarily by immature cells.

HCA, an immunoglobulin-like adhesion molecule present on the earliest human hematopoietic precursor cells, is also expressed by stromal cells in blood-forming tissues.

We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38(-/lo), Thy-1(+), rho123(lo), CD34(+) hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1(+) progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34(+) CD38(-) hematopoietic precursors, and CD3(+) CD6(+) peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34(+) rho123(med/lo) progenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.

Adhesion of Hematopoietic Precursors to Human Stroma: Studies Using Normal Marrow Stromal Myofibroblasts and a Stromal Cell Line Transformed by SV40.

Adhesion of hematopoietic precursors to the marrow microenvironment appears to be a prerequisite for proliferation and differentiation of hematopoietic cells. In this report, we have studied the adhesion of CFU-GM from marrow CD34+ precursors to human marrow myofibroblasts and to an human stromal cell line, L2Ori-, transformed by a vector comprising the whole of the SV40 viral sequence except for the origin of replication. This Stro-l(+) cell line presents characteristics similar to those of vascular smooth muscle cells, since (i) few cells were α-SM actin(+)while all cells were vimentin(+) but desmine(-) and a metavinculin band was consistently detected, (ii) all cells contained lysosomes filled with glycoproteins recognized by the monoclonal antibody 1B10, (iii) we detected EDa(+) EDb(-) pericellular fibronectin and intracellular ß1 and ß laminins and (iv) the cytokine expression pattern was similar to that of cells from colony-derived cell lines. Transformation was confirmed by abnormal and irregular growth (hallmarked by crises with rather slow growth between crises), and the presence of some very large cells with several nuclei. Although presenting an usual stromal phenotype, this cell line could not sustain hematopoiesis from marrow CD34+ cells in coculture due to a complete inability of adhesion of CD34+ cells (0% of adherent CFU-GM vs. 20% on normal stromal myofibroblasts). The lack of adhesion was explained by abnormal expression of adhesion molecules and molecules involved in the organization of extracellular matrix: (1) at the membrane level: the lack of VCAM-1 and significant differences in the distribution of CD44 and integrins α1, α3, α4 and ß as compared to normal stroma; (2) at the level of focal adhesions: the predominance of the 200 kD fragment of talin (as opposed to that of 230 kD in normal stroma), and a significantly decreased expression of vinculin and α-actinin; (3) at the level of microfilaments: the decrease in polymerized actin and a large decrease of α-SM actin synthesis; and (4) at the level of extracellular matrix: very few fibronectin fibres. These data show that transformation can strongly and negatively affect the function of hematopoiesis maintenance by disrupting intercellular and extracellular matrix adhesion mechanisms of hematopoietic cells to the stroma, in particular by affecting the fibronexus. Our data suggest the need for extreme caution when using SV40 transformed cell lines and instead, make the case for the use of other means of immortalization (such as thermosensitive T, other transforming sequences, introduction of inducible promoters).