We recently reported that systemic administration of peripheral blood (PB) CD34+ cells, an endothelial progenitor cell (EPC)-enriched population, contributed to fracture healing via vasculogenesis/angiogenesis. However, pathophysiological role of EPCs in fracture healing process has not been fully clarified. Therefore, we investigated the hypothesis whether mobilization and incorporation of bone marrow (BM)-derived EPCs may play a pivotal role in appropriate fracture healing. Serial examinations of Laser doppler perfusion imaging and histological capillary density revealed that neovascularization activity at the fracture site peaked at day 7 post-fracture, the early phase of endochondral ossifification. Fluorescence-activated cell sorting (FACS) analysis demonstrated that the frequency of BM cKit+Sca1+Lineage- (Lin-) cells and PB Sca1+Lin- cells, which are EPC-enriched fractions, significantly increased post-fracture. The Sca1+ EPC-derived vasuculogenesis at the fracture site was confirmed by double immunohistochemistry for CD31 and Sca1. BM transplantation from transgenic donors expressing LacZ transcriptionally regulated by endothelial cell-specific Tie-2 promoter into wild type also provided direct evidence that EPCs contributing to enhanced neovascularization at the fracture site were specifically derived from BM. Animal model of systemic administration of PB Sca1+Lin- Green Fluorescent Protein (GFP)+ cells further confirmed incorporation of the mobilized EPCs into the fracture site for fracture healing. These findings indicate that fracture may induce mobilization of EPCs from BM to PB and recruitment of the mobilized EPCs into fracture sites, thereby augment neovascularization during the process of bone healing. EPCs may play an essential role in fracture healing by promoting a favorable environment through neovascularization in damaged skeletal tissue.

BackgroundCarbon dioxide (CO2) therapy refers to the transcutaneous administration of CO2 for therapeutic purposes. This effect has been explained by an increase in the pressure of O2 in tissues known as the Bohr effect. However, there have been no reports investigating the oxygen dissociation of haemoglobin (Hb) during transcutaneous application of CO2 in vivo. In this study, we investigate whether the Bohr effect is caused by transcutaneous application of CO2 in human living body.MethodsWe used a novel system for transcutaneous application of CO2 using pure CO2 gas, hydrogel, and a plastic adaptor. The validity of the CO2 hydrogel was confirmed in vitro using a measuring device for transcutaneous CO2 absorption using rat skin. Next, we measured the pH change in the human triceps surae muscle during transcutaneous application of CO2 using phosphorus-31 magnetic resonance spectroscopy (31P-MRS) in vivo. In addition, oxy- and deoxy-Hb concentrations were measured with near-infrared spectroscopy in the human arm with occulted blood flow to investigate O2 dissociation from Hb caused by transcutaneous application of CO2.ResultsThe rat skin experiment showed that CO2 hydrogel enhanced CO2 gas permeation through the rat skin. The intracellular pH of the triceps surae muscle decreased significantly 10 min. after transcutaneous application of CO2. The NIRS data show the oxy-Hb concentration decreased significantly 4 min. after CO2 application, and deoxy-Hb concentration increased significantly 2 min. after CO2 application in the CO2-applied group compared to the control group. Oxy-Hb concentration significantly decreased while deoxy-Hb concentration significantly increased after transcutaneous CO2 application.ConclusionsOur novel transcutaneous CO2 application facilitated an O2 dissociation from Hb in the human body, thus providing evidence of the Bohr effect in vivo.

Hypertrophic nonunion usually results from insufficient fracture stabilization. Therefore, most hypertrophic nonunions simply require the stabilization of the nonunion site. However, the reasons why union occurs without treating the nonunion site directly is not well understood biologically. In this study, we hypothesized that the intervening tissue at the hypertrophic nonunion site (nonunion tissue) could serve as a reservoir of mesenchymal progenitor cells and investigated whether the cells derived from nonunion tissue had the capacity for multilineage mesenchymal differentiation. After nonunion tissue was obtained, it was cut into strips and cultured. Homogenous fibroblastic adherent cells were obtained. Flow cytometry revealed that the adherent cells were consistently positive for mesenchymal stem cell related markers CD13, CD29, CD44, CD90, CD105, CD166, and negative for the hematopoietic markers CD14, CD34, CD45, and CD133, similar to control bone marrow stromal cells. In the presence of lineage-specific induction factors, the adherent cells differentiated in vitro into osteogenic, chondrogenic, and adipogenic cells. These results demonstrated for the first time that hypertrophic nonunion tissue contains multilineage mesenchymal progenitor cells. This suggests that hypertrophic nonunion tissue plays an important role during the healing process of hypertrophic nonunion by serving as a reservoir of mesenchymal cells that are capable of transforming into cartilage and bone forming cells. ß

BackgroundThe discovery of microRNA (miRNA) has revealed a novel type of regulatory control for gene expression. Increasing evidence suggests that miRNA regulates chondrocyte, osteoblast, and osteoclast differentiation and function, indicating miRNA as key regulators of bone formation, resorption, remodeling, and repair. We hypothesized that the functions of certain miRNAs and changes to their expression pattern may play crucial roles during the process of fracture healing.MethodsStandard healing fractures and unhealing fractures produced by periosteal cauterization at the fracture site were created in femurs of seventy rats, with half assigned to the standard healing fracture group and half assigned to the nonunion group. At post-fracture days 3, 7, 10, 14, 21, and 28, total RNA including miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture day 14. For further analysis, we selected highly up-regulated five miRNAs in the standard healing fracture group from the microarray data. Real-time PCR was performed with miRNA samples at each time point above mentioned to compare the expression levels of the selected miRNAs between standard healing fractures and unhealing fractures and investigate their time-course changes.ResultsMicroarray and real-time polymerase chain reaction (PCR) analyses on day 14 revealed that five miRNAs, miR-140-3p, miR-140-5p, miR-181a-5p, miR-181d-5p, and miR-451a, were significantly highly expressed in standard healing fractures compared with unhealing fractures. Real-time PCR analysis further revealed that in standard healing fractures, the expression of all five of these miRNAs peaked on day 14 and declined thereafter.ConclusionOur results suggest that the five miRNAs identified using microarray and real-time PCR analyses may play important roles during fracture healing. These findings provide valuable information to further understand the molecular mechanism of fracture healing and may lead to the development of miRNA-based tissue engineering strategies to promote fracture healing.

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Objective. Human embryonic stem cells (ESCs)provide an unlimited supply of pluripotent cells for articular cartilage tissue engineering and regenerative medicine applications. Articular cartilage is an avascular tissue with precise polarity and organization comprising 3 distinct functional zones: surface, middle, and deep. To date, attempts at differentiating human ESCs into articular chondrocytes have been unsuccessful. The majority of studies have focused on chondrogenic (but not specifically articular cartilage) differentiation. Furthermore, previous investigations of induction of chondrogenesis by human ESCs required embryoid body formation; however, embryoid body formation often results in heterogeneous differentiation. The present study was undertaken to determine the in vitro chondrogenic potential of bone morphogenetic protein 7 (BMP-7) and transforming growth factor ␤1 (TGF␤1)-induced human ESC differentiation toward the articular cartilage phenotype.Methods. Dissociated single human ESCs were cultured and passaged on a gelatin-coated flask. The human ESCs were cultured as an aggregate in a pellet culture system for 14 days in basal chondrogenic medium (CM), CM with TGF␤1, CM with BMP-7, or CM with both TGF␤1 and BMP-7.Results. The size and wet weight of the cartilage pellets and glycosaminoglycan levels increased, with the smallest, intermediate, and greatest increases, respectively, observed with CM plus TGF␤1 treatment, CM plus BMP-7 treatment, and CM plus TGF␤1 and BMP-7 treatment (compared with CM treatment alone). The largest size and highest weight of the pellet was in the group in which TGF␤1 and BMP-7 were added to the medium. However, expression of the genes for cartilage-specific aggrecan and type II collagen II, as assessed by determination of messenger RNA levels, was highest in the BMP-7-treated group. Superficial zone protein (SZP)/lubricin, a marker of the superficial zone articular chondrocyte, was not detectable under identical culture conditions.Conclusion. These results demonstrate an efficient and reproducible model system of human ESCinduced chondrogenesis, using a novel direct plating method in which intervening embryoid body formation does not occur. Further work is needed for optimization of conditions to obtain the articular cartilage phenotype that includes the superficial zone marker as demonstrated by SZP/lubricin synthesis.Articular cartilage is an avascular tissue and lacks regeneration capacity, possibly because the normal mechanisms of initiation of tissue repair by humoral factors and attendant recruitment of stem/progenitor cells to the injury site by vasculature do not occur. Osteoarthritis is a common joint disease affecting articular cartilage in older individuals, and current treatment options, other than total joint replacement, are limited. Smaller injuries can be treated with cell-based therapies, e.g., autologous chondrocyte or bone marrow-derived mesenchymal stem cell (MSC) transplantation (1,2). However, the self-renewal and proliferative capacity of chondroc...

Superficial-zone protein (SZP), also known as lubricin, is a key mediator of boundary lubrication and plays an important role in the functional integrity of the diarthrodial joint. The aim of this investigation was to examine the role of transforming growth factor beta (TGF-beta) and interleukin-1 beta (IL-1beta) on the expression of SZP in various compartments of the bovine knee joint: the superficial zone of articular cartilage, synovium, meniscus, and anterior and posterior cruciate ligaments. The effects of TGF-beta1 and IL-1beta on SZP expression were examined in explants and cells from the different tissue compartments. TGF-beta1 up-regulated the expression of SZP in cultured explants, but IL-1beta down-regulated it. Quantitative analysis of secreted proteins in the medium of the cells demonstrated significant stimulation by TGF-beta1 and inhibition by IL1-beta of the accumulation of SZP protein in all four tissues. Real-time polymerase chain reaction analysis revealed that TGF-beta1 significantly up-regulated SZP expression and that IL-1beta down-regulated it. These results revealed the modulation of SZP expression in various compartments of the knee joint by TGF-beta1 and IL-1beta. In addition, SZP was found to be immunolocalized at the surface layer of cells in histological sections of all four tissue compartments. Collectively, results of the current study on regulation of SZP expression by TGF-beta and IL-1 help provide new insights, into tissue engineering strategies to repair and regenerate the different tissue compartments in the articular joint with optimal lubrication.

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