Decreased osteogenesis, increased cell senescence and elevated Dickkopf-1 secretion in human fracture non union stromal cells

The delicately orchestrated process of bone fracture healing is not always successful and long term non union of fractured bone occurs in 5-20% of all cases. Atrophic fracture non unions have been described as the most difficult to treat and this is thought to arise through a cellular and local failure of osteogenesis. However, little is known about the presence and osteogenic proficiency of cells in the local area of non union tissue. We have examined the growth and differentiation potential of cells isolated from human non union tissues compared with normal human bone marrow mesenchymal stromal cells (BMSC). We report the isolation and culture expansion of a population of non union stromal cells (NUSC) which have a CD profile similar to that of BMSC, i.e. CD34-ve, CD45-ve and CD105+ve. The NUSC demonstrated multipotentiality and differentiated to some extent along chondrogenic, adipogenic and osteogenic lineages. However, and importantly, the NUSC showed significantly reduced osteogenic differentiation and mineralization in vitro compared to BMSC. We also found increased levels of cell senescence in NUSC compared to BMSC based on culture growth kinetics and cell positivity for senescence associated beta galactosidase (SA-beta-Gal) activity. The reduced capacity of NUSC to form osteoblasts was associated with significantly elevated secretion of Dickkopf-1 (Dkk-1) which is an important inhibitor of Wnt signalling during osteogenesis, compared to BMSC. Conversely, treating BMSC with levels of rhDkk-1 that were equivalent to those levels secreted by NUSC inhibited the capacity of BMSC to undergo osteogenesis. Treating BMSC with NUSC conditioned medium also inhibited the capacity of the BMSC to undergo osteogenic differentiation when compared to their treatment with BMSC conditioned medium. Our results suggest that the development of fracture non union is linked with a localised reduced capacity of cells to undergo osteogenesis, which in turn is associated with increased cell senescence and Dkk-1 secretion.

A prototype bone screw design for osteoporotic bone

Poster. Introduction: One in five menand one half of women over the age of 50 will experience a bone fracture, whichis frequently accompanied by poor bone health. This combination of poor bonehealth and fracture is a two edge sword, because not only does poor bone healthmake fractures more likely, it also reduces the efficacy of standard fracturetreatments. Currently available surgical fixation devices that were originallydeveloped for healthy bone, such as pins, plates and bone screws, are often noteffective for patients with osteoporosis, resulting in unsatisfactory outcomesor longer and more painful recovery times. One major issue is the design ofbone screws, which can loosen or pull-out from osteoporotic bone. Osteopenicscrews with larger outer thread diameters have been developed to try andaddress this problem. The larger diameter screws have been shown to be 60–70 %stronger in lab tests of individual screws but the larger diameter screwscannot be used with the standard spacing in fixation plates without the risk ofcausing fractures between the screws. In addition, many fractures occur nearjoints where there is not room to increase the spacing between screws.Therefore, new bone screws are needed for treatment of fractures in osteoporoticbone. Materials and Methods: Afterdeveloping a novel bone screw design, we fabricated screws using rapidprototyping methods. Screws were inserted into 10 pcf density sawbones polyurethanefoam as a model for osteoporotic bone. Pull-out tests were conducted using theprototype bone screw design and the standard screw design for comparison inaccordance with ASTM 543-13. Results and Discussion: Ourprototype screws have the same outer diameter as standard bone screws, but haveoptimised threads. For pull-out tests in 10 psf density sawbones poly-urethanefoam, the prototype screw design was 60 % stronger than the standard bone screwdesign (p<0.01). Conclusion: Our novel bonescrew design provides significant improvement in standard tests with syntheticbone material. Additional tests are needed to determine if the bone screwswould be suitable for human trials.

Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing

Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and β1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of β1 and β3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing. © 2014 Springer-Verlag.

Nitric oxide-dependent bone marrow progenitor mobilization by carbon monoxide enhances endothelial repair after vascular injury

Carbon monoxide (CO) has emerged as a vascular homeostatic molecule that prevents balloon angioplasty-induced stenosis via antiproliferative effects on vascular smooth muscle cells. The effects of CO on reendothelialization have not been evaluated.

An in vitro comparison of the incorporation, growth, and chondrogenic potential of human bone marrow versus adipose tissue mesenchymal stem cells in clinically relevant cell scaffolds used for cartilage repair

Aim. To compare the incorporation, growth, and chondrogenic potential of bone marrow (BM) and adipose tissue (AT) mesenchymal stem cells (MSCs) in scaffolds used for cartilage repair. Methods. Human BM and AT MSCs were isolated, culture expanded, and characterised using standard protocols, then seeded into 2 different scaffolds, Chondro-Gide or Alpha Chondro Shield. Cell adhesion, incorporation, and viable cell growth were assessed microscopically and following calcein AM/ethidium homodimer (Live/Dead) staining. Cell-seeded scaffolds were treated with chondrogenic inducers for 28 days. Extracellular matrix deposition and soluble glycosaminoglycan (GAG) release into the culture medium was measured at day 28 by histology/immunohistochemistry and dimethylmethylene blue assay, respectively. Results. A greater number of viable MSCs from either source adhered and incorporated into Chondro-Gide than into Alpha Chondro Shield. In both cell scaffolds, this incorporation represented less than 2% of the cells that were seeded. There was a marked proliferation of BM MSCs, but not AT MSCs, in Chondro-Gide. MSCs from both sources underwent chondrogenic differentiation following induction. However, cartilaginous extracellular matrix deposition was most marked in Chondro- Gide seeded with BM MSCs. Soluble GAG secretion increased in chondrogenic versus control conditions. There was no marked difference in GAG secretion by MSCs from either cell source. Conclusion. Chondro-Gide and Alpha Chondro Shield were permissive to the incorporation and chondrogenic differentiation of human BM and AT MSCs. Chondro-Gide seeded with BM MSCs demonstrated the greatest increase in MSC number and deposition of a cartilaginous tissue.