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      Bone Progenitors Produced by Direct Osteogenic Differentiation of the Unprocessed Bone Marrow Demonstrate High Osteogenic Potential In Vitro and In Vivo

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          Abstract

          Tissue-engineered bone grafts seeded with mesenchymal stem cells (MSCs) have been sought as a replacement for bone grafts currently used for bone repair. For production of osteogenic constructs, MSCs are isolated from bone marrow (BM) or other tissues, expanded in culture, then trypsinized, and seeded on a scaffold. Predifferentiation of seeded cells is often desired. We describe here bone progenitor cells (BPCs) obtained by direct osteogenic differentiation of unprocessed BM bypassing isolation of MSCs. Human BM aspirates were incubated for 2 weeks with a commonly used osteogenic medium (OM), except no fetal calf serum, serum substitutes, or growth factors were added, because responding stem and/or progenitor cells were present in the BM milieu. The adherent cells remaining after the culture medium and residual BM were washed out, expressed high levels of bone-specific alkaline phosphatase (ALP) on their surface, demonstrated high ALP activity, were capable of mineralization of the intercellular space, and expressed genes associated with osteogenesis. These parameters in BPCs were similar and even at higher levels compared to MSCs subjected to osteogenic differentiation for 2 weeks. The yield of BPCs per 1 mL BM was 0.71±0.39×10 6. In comparison, the yield of MSCs produced by adhesion of mononuclear cells derived from the same amount of BM and cultured in a commercial growth medium for 2 weeks was 0.3±0.17×10 6. When a scaffold was added to the BM-OM mixture, and the mixture was cultured in a simple rotational bioreactor; the resulting BPCs were obtained already seeded on the scaffold. BPCs seeded on scaffolds were capable of proliferation for at least 6 weeks, keeping high levels of ALP activity, expressing osteogenic genes, and mineralizing the scaffolds. Autologous rat BPCs seeded on various scaffolds were transplanted into critical-size calvarial defects. Six weeks after transplantation of polylactic acid/polyglycolic acid scaffolds, 76.1%±18.3% of the defects were filled with a new bone, compared to 37.9%±28.4% in the contralateral defects transplanted with the scaffolds without cells.

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          Aging of mesenchymal stem cell in vitro

          Background A hot new topic in medical treatment is the use of mesenchymal stem cells (MSC) in therapy. The low frequency of this subpopulation of stem cells in bone marrow (BM) necessitates their in vitro expansion prior to clinical use. We evaluated the effect of long term culture on the senescence of these cells. Results The mean long term culture was 118 days and the mean passage number was 9. The average number of PD decreased from 7.7 to 1.2 in the 10th passage. The mean telomere length decreased from 9.19 Kbp to 8.7 kbp in the 9th passage. Differentiation potential dropped from the 6th passage on. The culture's morphological abnormalities were typical of the Hayflick model of cellular aging. Conclusion We believe that MSC enter senescence almost undetectably from the moment of in vitro culturing. Simultaneously these cells are losing their stem cell characteristics. Therefore, it is much better to consider them for cell and gene therapy early on.
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            Bone-grafting and bone-graft substitutes.

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              Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat calvarial bone defects.

              Tissue engineering has recently become available as a treatment procedure for bone augmentation. However, this procedure has several problems, such as high capital investment and expensive cell culture, complicated safety and quality management issues regarding cell handling, and patient problems with the invasive procedure of cell collection. Moreover, it was reported that stem cells secrete many growth factors and chemokines during their cultivation, which could affect cellular characteristics and behavior. This study investigated the effect of stem-cell-cultured conditioned media on bone regeneration. Cultured conditioned media from human bone marrow-derived mesenchymal stem cells (MSC-CM) enhanced the migration, proliferation, and expression of osteogenic marker genes, such as osteocalcin and Runx2, of rat MSCs (rMSCs) in vitro. MSC-CM includes cytokines such as insulin-like growth factor-1 and vascular endothelial growth factor. In vivo, a prepared bone defect of a rat calvarial model was implanted in five different rat groups using one of the following graft materials: human MSCs/agarose (MSCs), MSC-CM/agarose (MSC-CM), Dulbecco's modified Eagle's medium without serum [DMEM(-)]/agarose [DMEM(-)], PBS/agarose (PBS), and defect only (Defect). After 4 and 8 weeks, implant sections were evaluated using microcomputed tomography (micro-CT) and histological analysis. Micro-CT analysis indicated that the MSC-CM group had a greater area of newly regenerated bone compared with the other groups (p<0.05) and histological analysis at 8 weeks indicated that the newly regenerated bone bridge almost covered the defect. Interestingly, the effects of MSC-CM were stronger than those of the MSC group. In vivo imaging and immunohistochemical staining of transgenic rats expressing green fluorescent protein also showed that migration of rMSCs to the bone defect in the MSC-CM group was greater than in the other groups. These results demonstrated that MSC-CM can regenerate bone through mobilization of endogenous stem cells. The use of stem-cell-cultured conditioned media for bone regeneration is a unique concept that utilizes paracrine factors of stem cells without cell transplantation.
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                Author and article information

                Journal
                Biores Open Access
                Biores Open Access
                biores
                BioResearch Open Access
                Mary Ann Liebert, Inc. (140 Huguenot Street, 3rd FloorNew Rochelle, NY 10801USA )
                2164-7844
                2164-7860
                April 2012
                April 2012
                : 1
                : 2
                : 69-78
                Affiliations
                [ 1 ]Teva Pharmaceutical Industries LTD , Petach Tikva, Israel.
                [ 2 ]School of Dental Medicine, Tel Aviv University , Tel Aviv, Israel.
                Author notes
                Address correspondence to: Irene Ginis, MD, PhD, Teva Pharmaceutical Industries LTD, 5 Basel Street, Petach Tikva 49131, Israel. E-mail: irene@ 123456macrocure.com ; iginis1@ 123456gmail.com
                [*]

                Present address: Macrocure, LTD, Petach Tikva, Israel.

                [†]

                Present address: BrainStorm Cell Therapeutics LTD, Petach Tikva, Israel.

                Article
                10.1089/biores.2012.9904
                10.1089/biores.2012.9904
                3559218
                23514783
                a46d20eb-d400-4b21-b09f-278d76603b89
                Copyright 2012, Mary Ann Liebert, Inc.
                History
                Page count
                Figures: 6, Tables: 1, References: 35, Pages: 10
                Categories
                Original Research Articles

                bioengineering,bone progenitors,calvaria,mesenchymal stem cells,scaffold

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