Introduction: MSCs were demonstrated to exist within peripheral blood (PB) of several mammalian species including human, guinea pig, mice, rat, and rabbit. We have found increased numbers of circulating MSCs in human peripheral blood after fracture and in patients with cancers. We have also compared the difference between circulating MSCs and bone marrow MSCs and evaluated their potential clinical applications in tissue engineering and cell therapy.

Methods and findings: Using culture conditions similar to those defined for bone marrow derived mesenchymal stromal cells (BMMSCs), we have isolated and expanded multi-colony and single colony derived PBMSCs strains from the GFP transgenic rats. Aspects of molecular, cellular and developmental properties of this poorly characterized peripheral blood subpopulation were examined. PBMSCs share some common phenotypic characteristics with BMMSCs, but are distinguishable in gene expression profile by cDNA microarray analysis, with 84 up-regulated and 83 down-regulated genes (> 2 fold, E-B/B-E> 100, P< 0.05). Most of these genes are related to cell proliferation, differentiation, cyto-skeleton, and calcium/iron homeostasis. Differentially expressed genes with fold change ≥10 were further confirmed with quantitative real time RT-PCR, and these genes are: retinol-binding protein 1 (CRBP1), cadherin 2, bone morphogenetic protein 6 (BMP6), SRY-box containing gene 11 (Sox11), the aquaporin 1 (AQP1), and so on, and they can be potential targets for further investigations. We have demonstrated that single colony derived PBMSCs strains possess extensive proliferation and multipotent differentiation potentials into osteoblasts, adipocytes, chondrocytes, endothelial cells and neuronal cells. In terms of potential clinical implications of PBMSCs, we have demonstrated that allogenic PB-MSCs enhance bone regeneration in rabbit ulna critical-sized bone defect model. We also demonstrated that BM-MSCs can be recruited towards to the sites of bone fracture and participate fracture healing. We are now working on using MSCs as a gene delivery vehicle for management of would healing or cancer therapy, and ways of enhancing the homing and recruitment MSCs towards to specific sites after their systemic delivery.

Conclusion: Taken the above data together, PB-MSCs may be a new cell source for cell therapy, tissue engineering and gene therapy strategies.

The thrombin-related peptide, TP508, a synthetic 23 amino acid peptide, has been shown to promote soft tissue, cartilage and fracture repair. We have previously demonstrated that two injections of TP508 have signifi-cantly enhanced bone consolidation in a rabbit model of distraction osteogenesis. This study was to test if a single injection of TP508 in a slow-releasing preparation will have the similar effects.

Unilateral tibial osteoectomies were stabilized with M100 Orthofix lengtheners in 17 male adult NZW rabbits. After 7 days, lengthening was initiated at a rate of 1.4 mm/day for 6 days. The following treatments were given: Group 1: TP508 in saline (300ug/300ul, n=6) was injected into the osteotomy gap at day of surgery and into the lengthening gap at end of lengthening. Group 2 (Control): Dextran gel (300ul, n=6) and Group 3: 300ul Dextran gel mixed with microspheres containing 300ug TP508 (n=5), was injected into the lengthening gap at end of lengthening. All animals were terminated 2 weeks after lengthening. Bone formation was assessed by weekly radiography and the specimens were subject to pQCT, microCT and histology examinations.

On radiographies there was more bone formation in the TP508 treated groups than that of the control group at 1st week post-lengthening and complete union was seen in 50% rabbits in Group 1, 33% in Group 2, and 60% in Group 3 at termination. The mean BMD of the regenerates was significantly higher in the TP508 treated groups than that of the control group (p< 0.05). MicroCT analysis demonstrated advanced bone formation in the TP508 treated animals. For histology, the regenerates were mainly consisted of woven bone of neocortilization and callus remodelling in Groups 1 and 3, whereas in Group 2, focal defects with cartilaginous tissues were frequently seen.

In conclusion we have demonstrated that a single injection of TP508 in the form of slow releasing micro-spheres has enhanced bone consolidation during distraction osteogenesis. TP508 may therefore be applied in the slow-releasing preparation for augmenting bone formation at reduced doses, costs and risks of infections through repeated injections.

Introduction: The existence of circulating skeletal stem cells in the peripheral blood from different species including adult mouse and human has been found and documented. The circulating skeletal stem cells may provide a new source of stem cells that may be used for bone regeneration and tissue engineering applications. The aim of this study was to investigate the existence of circulating osteogenic stem cells in the rat peripheral blood, and to compare their osteogenic potentials with bone marrow mesenchymal stem cells (BMMSCs).

Methods: Whole blood from twelve female 3-month old SD rats was harvested by cardiac puncture and bone marrows were also collected. Mononuclear cells from both bone marrow and peripheral blood (PBMNCs) were isolated by Lymphoprep density gradient centrifugation method, and plated at a density of 300000 to 400000/cm2 in flasks with á-MEM medium and 15% FCS. The colony forming efficiency (CFE) was calculated after 10–14 days culture. The osteogenic, adipogenic, and chondrogenic differentiation potential of both BMMSCs and peripheral blood mononuclear cell subset were examined and compared under different specific culture conditions. In addition, both BMMSCs and peripheral blood mononuclear cell subset were seeded into absorbable porous calcium phosphate substitute and implanted subcutaneously into SCID mice for 12 weeks, and the implants were examined histologically.

Results: After 10–14 days in culture, the adherent fibroblast-like colonies were formed in the PBMNCs, with CFE ranging from 1.3 to 3.5 per 10000000 cells. Under osteogenic conditions, both BMMSCs and PBMNCs subset were positive for bone markers such as ALP, type I collagen and osteocalcin; bone nodules were formed in BMMSCs and PBMNCs subset long-term culture with positive Von Kossa and Alizarin Red S staining. Under adipogenic conditions, PBMNCs subset and BMMSCs were positive for Oil Red O and C/EBP á immunostaining. For chondrogenic differentiation studies, PBMNCs subset and BMMSCs were positive for type II collagen and they had Alcian blue positive nodules formation. After implantation with calcium phosphate substitutes in SCID mice for 12 weeks, osteoid and bony tissues were evident in the implants both loaded with PBMNCs subset and BMSCSs.

Conclusions: A subset of mononuclear cells that have multi-differentiation potential similar to BMMSCs exists in the rat peripheral blood. Our present study has shown that these circulating stem cells possess osteogenic potential in vitro and in vivo. Further work is ongoing to investigate the roles of PBMNCs subset in fracture healing and their recruiting and homing mechanisms.

Introduction: The existence of peripheral blood (PB) derived mesenchynal stem cells (PBMSCs) have been documented in several species including human. The circulating skeletal stem cells may provide a new source of stem cells that may be used for skeletal and other tissue engineering applications. The objective of this study is to further investigate and compare the biological characteristics of the PBMSCs with bone marrow derived MSCs in the GFP rats.

Methods: The peripheral blood (PB) from the GFP rats was harvested by cardiac puncture using syringes containing sodium heparin. Mononuclear cells were isolated by density gradient centrifugation method and plated at a density of 1–3~105/cm2 in flasks with D-MEM medium containing 15% FCS. The bone marrow (BM) was also collected for obtaining BMMSCs, the bone chips for osteoblastic cells, and the skin for skin fibroblasts. The phenotypes of the cells were characterized by immunocytochemistry (ICC), and flow cytometry methods. Gene expression profiles of 3-paired PBMSCs and BMMSCs cDNA samples were examined by Affymetrix gene chips microarray analysis. The multipotent differentiation potentials of PBMSCs into osteoblasts, chondrocytes, and adipocytes were examined under specific inductive conditions and checked with lineage specific markers. Finally, the osteogenic potential of the PBMSCs was examined by an in vivo implantation model in which the PBMSCs were seeded with HA-TCP powder complexes, and implanted subcutaneously in the severe compromised immunodeficiency (SCID) mice for 12 weeks, whereas the bone-derived osteoblasts and skin fibroblasts were used as controls.

Results: Compared with the BMMSCs, the PBMSCs shared some but not all common surface markers as demonstrated by (ICC) and flow cytometry examinations. The osteogenic differentiation of PBMSCs was defined with positive staining of type I collagen and osteocalcin; positive staining for alkaline phosphatase and Von Kossa staining for mineralized bone nodules. Adipogenic differentiation was evidenced by positive Oil red-O staining for accumulated lipids, and chondrogenic differentiation by positive type II collagen and Saferinin O positive staining. For gene expression profiles, in the Affymetrix chip general analysis, 83 genes were up regulated and 84 genes down regulated in the PBMSCs (vs BMMSCs, > 2 fold, E-B/B-E> 100, p< 0.05). Most of which genes are related to cell proliferation, differentiation, cytoskeleton, and calcium/iron homeostasis. After 12 weeks implantation in SCID mice, newly formed lamellar bone was clearly evident in the groups with PBMSCs implants, so as in the groups with osteoblasts implants, but only fibrous tissue was found in the group implanted with skin fibroblasts.

Discussion: This study demonstrated that the multi-potent PBMSCs in the GFP rats resemble BMMSCs in many aspects, but they are distinguishable from the BMMSCs in some biological characteristics and gene profiles. Our study has confirmed that these PBMSCs possess osteogenic potential in vitro and in vivo, suggesting that these circulating stem cells could serve as an alternative source as bone marrow derived MSCs for tissue engineering purposes.

During bone development and repair, angiogenesis, osteogenesis and bone remodeling (resorption) are closely associated processes with some common mediators involved. BMPs, VEGF and other cytokines are released from bone during bone resorption. Recent study showed that VEGF caused a dose- and time-dependent increase in bone resorption in vitro and in vivo, and BMP-2 markedly enhanced osteoclast differentiation induced by sRANKL and M-CSF in mouse osteoclast culture system. The aim of this study was to further examine the effects of VEGF and BMP-2 on osteoclastogenesis using in vitro human osteoclast culture system. Mononuclear cells were isolated by Lympo-Prep density gradient centrifugation from bone marrow washouts in bone samples from patients undergone total hip replacement. Mononuclear cells were plated at a density of 1 x 106/cm2 in a T-75 flask with aMEM and 15% FCS. The first medium change was made at day 7, when the floating cells were collected from the withdrawn media by centrifugation, and plated in a separate flask. The non-adherent cells in the 2nd flask were harvested again 24 hours later in a similar fashion. The non-adherent cells were then cultured in 24-well plates or calcium phosphate (Ca-P) coated plates, with osteoclast-inducing media (OC media) containing sRANKL 30 ng/ml and M-CSF 30 ng/ml, media were changed every 4 days. After 4 days culture in OC media, rhBMP-2 (3, 30, 300 ng/ml) and VEGF (25 ng/ml) were added respectively or in combination to the cell culture, and the culture was kept for total 16 days. The number of TRAP positive multinuclear cells in each well and the resorptive pit areas on the Ca-P coated plates were calculated and compared. Osteoclastic cell phenotype was defined by expressing tartrate resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and resorptive pit assay. By day 12–14, osteoclastic cells were found in all the experimental groups, they were positive for TRAP and VNR. The number of TRAP+ multinuclear cells were significantly reduced (p< 0.05, t-test) when rhBMP-2 (30 and 300 ng/ml) were present, and this was further reduced (p< 0.01) when rhVEGF was added together with rhBMP-2, comparing to the culture with OC media alone. Extensive lacunar resorption pits in the Ca-P coated plates were found in the culture treated with OC media and OC media with rhVEGF (25 ng/ml). The resorption pit areas were, however, significantly reduced when rhBMP-2 was added at 30 and 300 ng/ml with or without rhVEGF (25 ng/ml, p< 0.05, t-test). The presence of low concentration of rhBMP-2 (3 ng/ml) with VEGF had no effect on osteoclast number or the areas of resorption pit formation. In contrary to previous findings in the mouse osteoclast culture system, the present study had shown that the presence of rhBMP-2 at 30 and 300 ng/ml had strongly inhibited osteoclast differentiation and bone resorptive capability in the human osteoclast culture system, and the inhibition was further enhanced by the presence of rhVEGF. This study implies that VEGF and BMP-2 may be important, yet to be defined regulators, for osteoclastogenesis.

Introduction: Peripheral blood derived mesenchymal stem cells (PBMSCs) are multipotent cells capable of forming bone, cartilage, fat, and other connective tissues. Bone marrow derived mesenchymal stem cells (BMMSCs) have promoted repair a critical-sized bone defect in several animal models including mouse, rat, rabbit, and dog. The aim of this study was to investigate whether or not the use of allogenic BMMSCs and PBMSCs could regenerate a critical-sized bone defect in rabbit ulnae.

Methods: Rabbit peripheral blood mononuclear cells (PBMNCs) were isolated by density gradient centrifugation method and cultured at a density of 100,000/ cm2 in flasks with DMEM 15% FCS. Colony forming efficiency (CFE) was calculated and their multipotential differentiations into bone, cartilage, and fat were examined under different induction conditions. Specific differentiation markers were examined using cytochemistry and immunocytochemistry methods in the PBMSCs. Critical-sized ulna bone defects, 20 mm in length, were created in the mid-diaphysis of both ulnae in twelve 6 month old NZW rabbits. The ulnar defects were treated as the following 5 groups: empty control (n=4), PBMSCs/Skelite (multi-phase porous calcium phosphate resorbable substitute, EBI Company, USA) (n=5), BMMSCs/Skelite (n=4), PBMNCs/Skelite (n=5), and Skelite alone (n=5). All animals were sacrificed 12 weeks after treatment. The bone regeneration was evaluated by regular radiography, and all samples were subject to peripheral quantitative computed tomography (pQCT) and histological examination at the end point.

Results: The CFE of PBMSCs ranged from 1.2 to 13 per million mononuclear cells. Spindle and polygonal shaped cells were found in the primary PBMSCs colony, showing similar differentiation potential with BMMSCs. Mineralized bone nodules formed under osteogenic media were positive for Alizarin Red S staining in the PBMSCs. Chondrogenic differentiation was identified in serum free media containing TGF-¦Â1 (10 ng/ml), with type II collagen expression and Alcian blue positive nodule formation. Adipocytic differentiation was tested with or without adipogenic media, with positive Oil Red O staining for lipid accumulation and CEBP¦Á expression in the PBMSCs. After twelve weeks implantation, the ulnar defects were not healed in the empty control group; the total bone density in PBMSCs/Skelite and BMMSCs/Skelite treated defects were greater than that of PBMNCs/Skelite and Skelite alone treated groups (p< 0.05), with higher score of X-ray evaluation (p< 0.05). Histologically, there were a greater amount of new bone present in both the PBMSCs/Skelite and BMMSCs/Skelite treated groups compared to the PBMNCs/Skelite and Skelite alone treated groups.

Conclusions: This study demonstrated that PBMSCs were multipotent cells; allogenic PBMSCs loaded onto porous calcium phosphate resorbable substitute had enhanced bone regeneration of a critical-sized segmental defect in the rabbit ulna. PBMSCs may be a new source of osteogenic stem cells for bone regeneration and tissue engineering, and further investigations are undergoing to clarify their functions.

Introduction: Sufficient quantity of osteogenic cells is an essential aspect for a successful cell therapy in the treatment of difficult bone fractures and defects. At present, this was achieved by culturing bone marrow and bone-derived cells in a relatively long duration. A large number of the non-adherent mesenchymal stem cells were discarded during medium change. We hypothesize that collecting the non-adherent cells and re-plating them may result in more osteogenic cells in the same duration of cell culture. The aim of this study was to investigate the possibility of enhancing number of osteogenic cells by collecting non-adherent cells in the pull-off media and to examine their osteognic potentials.

Methods: Mononuclear cells were isolated by density gradient centrifugation method from bone marrow washouts in the bone samples obtained from 5 patients undergone total hip replacement. Mononuclear cells were plated at a density of 1 x 10⁶/cm² in a T-75 flask with αMEM medium and 15% FCS. The first medium change was made at day 7 and every 3 days thereafter. For the first three times of medium change, the removed media were centrifuged at 250 g for 10 minutes and plated in a separate T-75 (first time change) and T-25 flask (for the 2nd and 3rd times change). The non-adherent cells from the second and the third puff-off flasks were also collected and plated in separate T-25 flasks. Thus, 1xT-75 flask and 4xT-25 flasks of non-adherent cells resulted from the original T-75 flask. The cells in all flasks were harvested at 21 days from the day when the original flask was set up. The total number of cells in all pull off flasks were counted and compared with that of the original T-75 flask. Rate of cell proliferation with or without osteogenic growth medium were also examined by MTT method for passage 1 of both cells types. Osteogenic differentiation was defined with immunocytochemistry of bone markers: ALP, type I collagen, Osteocalcin and cbfa1. It is planed that cells of passage 2 will be mixed with HA powders and to be implanted into the SCID mice to examine the in-vivo osteogenic potential of these cells.

Results: Mesenchymal stem cells (MSCs) derived from the non-adherent population of human bone marrow culture have demonstrated having similar cell proliferation and differentiation potential in vitro, when compared to the MSCs derived from the adherent cell population. These cells expressed bone markers such as: ALP, type I collagen, osteocalcin and cbfa1. When the non-adherent cells were collected and cultured accumulatively, the total number of MSCs was increased to an average of 39.7% (36.6%–42.9%), compared to the number of cells obtained from the original T-75 flask.

Conclusions: Collecting the non-adherent cell population in the bone marrow culture appeared to result in more MSCs. This harvesting method may be used as a non-invasive way for enhancing MSC numbers in a given period of time. Further in vitro and in vivo studies of these MSCs of non-adherent origins may provide information for optimizing cell culture protocols for rapid expanding the osteogenic cells in vitro. This will facilitate the clinical applications of human osteogenic cell therapy.

Introduction: Recently, co-transplantion of mesenchy-mal stem cells (MSCs) with hematopoietic stem cells (HSCs) has been shown to alleviate complications such as GVHD and speeding recovery of HSCs. This in vivo finding suggests that coculture of MSCs and HSCs may enhance their growth potentials in vitro. As the large-scale expansion of HSCs has been achieved by NASA’s suspension culture system, we further examined the effects of this suspension culture system (rotary bio-reactor) on MSCs’ proliferation and differentiation potentials in vitro.

Methods: Mononuclear cell fractions (MNCs) of human bone marrow aspirates (n=6, ages 46–81) were collected by density gradient centrifugation. The cells were inoculated into bioreactor (RCCS, Synthecon Inc., Texas, USA) at the concentration 1x10⁶ cells/ml, in Myelocult^TM medium supplemented with 50ng/ml SCF, 20ng/ ml rhIL-3 and rhIL-6 (10ng/ml SCF, 2ng/ml IL-3 and IL-6 after the first feeding) and 10-6 M hydrocortisone for 8 days. The medium was fully exchanged after 3 days and 20% daily thereafter. Total cell numbers in the bioreactor were counted daily using hemacytometer. Cells from day 1, 4, and 8 cultures were subjected to tri-color flow cytometry examination using CD34, CD44, and Stro-1 antibodies. By the end of 8 day culture, the output cells were resuspended in DMEM medium with 10% FBS and cultured in T75 flasks at 1x10⁵ cells/cm2 for further 3 weeks. Upon harvest, half of the attached MSCs were prepared for western blotting assay using various antibodies. The other half was further cultured for 13–28 days in osteogenic, chondrogenic, and adipogenic induction medium respectively. Cell differentiation results were examined by histology staining, immunohistochemistry (ICC) and transmission electron microscope (TEM) examinations.

Results: After 8-day culture in bioreactor, flow-cytometric analysis confirmed that two cell populations, CD34+CD44+ (HSCs) and Stro-1+CD44+ (MSCs), increased 8-fold and 29-fold respectively, when compared to the values of the MNCs prior to bioreactor treatment. Cell counting revealed that the total cell expansion over 8 days was 9-fold above the number of the input MNCs. Western blotting data confirmed that bioreactor-expanded MSCs population remained in their early-stage with the expression of primitive MSCs markers such as CD105 (endoglin, SH-2) and Vimentin, whereas no expression of differentiation markers including osteocalcin (osteogenesis), Type II collegen (chondrogenesis) and C/EBPα (adipogenesis). Upon differentiation induction, the bioreactor-expanded MSCs were capable of differentiating into osteocytes, chondrocytes, and adipocytes as evidenced by histology staining, ICC and TEM examinations.

Discussion: Our study has shown that the percentage of MSCs (Stro-1+CD44+) increased 29 folds in the bone marrow derived MNCs after they have been cultured with Myelocult¢â medium in bioreactor for 8 days. The suspension culture system did not affect the subsequent in vitro proliferation and differentiation potentials of MSCs. Current study indicates that rotary bioreactor may be used to rapidly expand the numbers of traditionally attachment-dependent MSCs from bone marrow-derived MNCs, which may be very useful in clinical tissue engineering applications.

Introduction: The existence of peripheral blood (PB) derived mesenchymal stem cells (PB-MSCs) have been documented in different mammalian species including young and adult human. However, the number of PB-MSCs is low in normal adult human blood. We have demonstrated previously that there was an increase in the number of PB-MSCs following long bone fracture and in the patients suffering from fracture non-union. The present study was to compare the biological characteristics of the PB-MSCs from fracture non-union patients, with human bone marrow derived MSCs (BM-MSCs).

Methods: 200 mls PB was collected from 9 patients suffering from fracture non-union. The mononuclear cells (MNCs) were isolated by density gradients centrifugation and cultured in á-MEM containing 15% FBS. The PB-MNCs from normal donors (n=8) and BM-MSCs from patients underwent total hip replacement were used as controls. The colony forming efficiency (CFE) of the PB-MSCs was calculated, and the phenotypes of PB-MSCs and BM-MSCs were compared using immunocytochemistry and flow cytometry methods. Their multipotent differentiation potentials into osteoblasts, chondrocytes, adipocytes, neurogenic and angiogenic cells were examined under specific inductive culture media. The in vivo osteogenic potential of PB-MSCs was examined by implanting the HA-TCP blocks seeded with PB-MSCs into the SCID mice for 12 weeks.

Results: After 28 days in culture, fibroblastic colonies were formed in the PB-MNCs cultures in 5 of 9 fracture non-union patients, with CFE ranging from 2.08–2.86 per 10^8 MNCs. No fibroblastic colony was seen in PB-MNCs cultures of the 8 normal donors. Under flow cytometry examination, PB-MSCs and BM-MSCs were CD34 (low) and CD105+, but PB-MSCs were CD29-, CD44-, and ALP (low), whereas BM-MSCs were CD29+, CD44+, and ALP (high). Under specific differentiation inductions, the PB-MSCs differentiated into osteoblastic cells (ALP+, type I collagen+, osteocalcin+ and Alizarin red+; chondrocytes (type II collagen+ and Alcian Blue nodules formation); adipocytes (Oil red-O positive lipid accumulation). Neurogenic differentiation was confirmed by positive neuro-filament staining, and differentiation into endothelial cells was evident with tube formation in 2D culture, and positive staining for VW factor and CD31. After implantation in the SCID mice for 12 weeks, newly formed woven bones were found in the biomaterials seeded with PB-MSCs, and they were positive for human osteocalcin immunostaining.

Discussion: This study indicated that there were more PB-MSCs in the peripheral circulation of the fracture non-union patients than that in the normal subjects. This may be due to a continous systemic response for recruiting MSCs from remote bone marrow sites, with attempt to repair the fracture(s). The PB-MSCs were clearly multi-potential cells, which had shared some common phenotypic markers with BM-MSCs, as well as many distinguishable makers from the BM-MSCs. The recruitment of the PB-MSCs through circulation might be a general phenomenon of systemic responses in many pathological conditions, such as fracture or wound healing and other systemic diseases. Further understanding the roles of PB-MSCs in diseases and repair may lead to novel therapeutic strategies.