We describe the isolation of a nonhematopoietic (CD45 -, CD34 -, SH2 + , SH3 + , Thy-1 + , CD44 + ) human umbilical cord perivascular (HUCPV) cell population. Each HUCPV cell harvest (2-5 × 10 6 , depending on the length of cord available) gave rise to a morphologically homogeneous fibroblastic cell population, which expressed α-actin, desmin, vimentin, and 3G5 (a pericyte marker) in culture. We determined the colony-forming unit-fibroblast (CFU-F) frequency of primary HUCPV cells to be 1:333 and the doubling time, which was 60 hours at passage 0 (P0), decreased to 20 hours at P2. This resulted in a significant cell expansion, producing over 10 10 HUCPV cells within 30 days of culture. Furthermore, HUCPV cells cultured in nonosteogenic conditions contained a subpopulation that exhibited a functional osteogenic phenotype and elaborated bone nodules. The frequency of this CFU-osteogenic subpopulation at P1 was 2.6/10 5 CFU-F, which increased to 7.5/10 5 CFU-F at P2. Addition of osteogenic supplements to the culture medium resulted in these frequencies increasing to 1.2/10 4 and 1.3/10 4 CFU-F, respectively, for P1 and P2. CFU-O were not seen at P0 in either osteogenic or non-osteogenic culture conditions, but P0 HUCPV cells did contain a 20% subpopulation that presented neither class I nor class II cell-surface major histocompatibility complexes (MHC -/-). This population increased to 95% following passage and cryopreservation (P5). We conclude that, due to their rapid doubling time, high frequencies of CFU-F and CFU-O, and high MHC -/-phenotype, HUCPV cells represent a significant source of cells for allogeneic mesenchymal cell-based therapies. Stem Cells 2005;23:220-229

If dental implantology is an increasingly successful treatment modality, why should we still need to understand the mechanisms of peri-implant bone healing? Are there differences in cortical and trabecular healing? What does "poor quality" bone mean? What stages of healing are most important? How do calcium phosphate-coated implants accelerate healing? What is the mechanism of bone bonding? While there are still many aspects of peri-implant healing that need to be elucidated, it is now possible to deconvolute this biological reaction cascade, both phenomenologically and experimentally, into three distinct phases that mirror the evolution of bone into an exquisite tissue capable of regeneration. The first and most important healing phase, osteoconduction, relies on the recruitment and migration of osteogenic cells to the implant surface, through the residue of the peri-implant blood clot. Among the most important aspects of osteoconduction are the knock-on effects generated at the implant surface, by the initiation of platelet activation, which result in directed osteogenic cell migration. The second healing phase, de novo bone formation, results in a mineralized interfacial matrix equivalent to that seen in the cement line in natural bone tissue. These two healing phases, osteoconduction and de novo bone formation, result in contact osteogenesis and, given an appropriate implant surface, bone bonding. The third healing phase, bone remodeling, relies on slower processes and is not considered here. This discussion paper argues that it is the very success of dental implants that is driving their increased use in ever more challenging clinical situations and that many of the most important steps in the peri-implant healing cascade are profoundly influenced by implant surface microtopography. By understanding what is important in peri-implant bone healing, we are now able to answer all the questions listed above.

New three-dimensional (3D) scaffolds for bone tissue engineering have been developed throughout which bone cells grow, differentiate, and produce mineralized matrix. In this study, the percentage of cells anchoring to our polymer scaffolds as a function of initial cell seeding density was established; we then investigated bone tissue formation throughout our scaffolds as a function of initial cell seeding density and time in culture. Initial cell seeding densities ranging from 0.5 to 10 x 10(6) cells/cm(3) were seeded onto 3D scaffolds. After 1 h in culture, we determined that 25% of initial seeded cells had adhered to the scaffolds in static culture conditions. The cell-seeded scaffolds remained in culture for 3 and 6 weeks, to investigate the effect of initial cell seeding density on bone tissue formation in vitro. Further cultures using 1 x 10(6) cells/cm(3) were maintained for 1 h and 1, 2, 4, and 6 weeks to study bone tissue formation as a function of culture period. After 3 and 6 weeks in culture, scaffolds seeded with 1 x 10(6) cells/cm(3) showed similar tissue formation as those seeded with higher initial cell seeding densities. When initial cell seeding densities of 1 x 10(6) cells/cm(3) were used, osteocalcin immunolabeling indicative of osteoblast differentiation was seen throughout the scaffolds after only 2 weeks of culture. Von Kossa and tetracycline labeling, indicative of mineralization, occurred after 3 weeks. These results demonstrated that differentiated bone tissue was formed throughout 3D scaffolds after 2 weeks in culture using an optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks. Furthermore, after 6 weeks in culture, newly formed bone tissue had replaced degrading polymer.

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A phase I study was performed to determine the safety and tolerability of injecting autologous CD34؉ cells into five patients with liver insufficiency. The study was based on the hypothesis that the CD34 ؉ cell population in granulocyte colony-stimulating factor (G-CSF)-mobilized blood contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated a candidate CD34؉ stem cell population from the majority of the CD34 ؉ cells (99%) by adherence to tissue culture plastic. The adherent and nonadherent CD34؉ cells were distinct in morphology, immunophenotype, and gene expression profile. Reverse transcription-polymerase chain reactionbased gene expression analysis indicated that the adherent CD34؉ cells had the potential to express determinants consistent with liver, pancreas, heart, muscle, and nerve cell differentiation as well as hematopoiesis. Overall, the characteristics of the adherent CD34 ؉ cells identify them as a separate putative stem/progenitor cell population. In culture, they produced a population of cells exhibiting diverse morphologies and expressing genes corresponding to multiple tissue types. Encouraged by this evidence that the CD34 ؉ cell population contains cells with the potential to form hepatocyte-like cells, we gave G-CSF to five patients with liver insufficiency to mobilize their stem cells for collection by leukapheresis. Between 1 ؋ 10 6 and 2 ؋ 10 8 CD34 ؉ cells were injected into the portal vein (three patients) or hepatic artery (two patients). No complications or specific side effects related to the procedure were observed. Three of the five patients showed improvement in serum bilirubin and four of five in serum albumin. These observations warrant further clinical trials.

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Given the established anti-inflammatory properties of mesenchymal stromal cells (MSCs), we investigated their effect on inflammatory cell infiltration of ischemic cardiac tissue and cardiac function. We employed two types of MSCs, human bone marrow-derived (BM) MSCs and human umbilical cord perivascular cells in an experimental acute myocardial infarction (MI) model with the immune-deficient NOD/SCID gamma null mouse. Cells were infused 48 h after induction of MI and mice assessed 24 h later (72 h after MI) for bone marrow (BM), circulating and cardiac tissue-infiltrating monocytes/macrophages. We showed that in the presence of either MSC type, overall macrophage/monocyte levels were reduced, including pro-inflammatory M1-type macrophages, while the proportion of alternatively activated M2-type macrophages was significantly increased in the circulation and heart but not the BM. Moreover, we found decreased expression of IL-1β and IL-6, increased IL-10 expression and fewer apoptotic cardiomyocytes without changes in angiogenesis in the infarct area. Fractional shortening was enhanced 2 weeks after cell infusion but was similar to medium controls 16 weeks after MI. In vitro studies showed that BM MSCs increased the frequency of alternatively activated monocytes/macrophages, in part by MSC-mediated secretion of IL-10. Our data suggest a new mechanism for MSC-mediated enhancement of cardiac function, possibly via an IL-10 mediated switch from infiltration of pro-inflammatory to anti-inflammatory macrophages at the infarct site. Additional studies are warranted confirming the role of IL-10 and augmenting the anti-inflammatory effects of MSCs in cardiac regeneration.