Osteogenic predifferentiation of human bone marrow-derived stem cells by short-term mechanical stimulation.

It is commonly accepted that bone marrow-derived stem cells (BMSCs) have to be expanded in vitro, but a prolonged time in culture decreases their multilineage potential. Mechanical and biological stimuli have been used to improve their osteogenic potential. While long-term stimulation has been shown to improve osteogenic differentiation, it remains to be seen whether short-term stimulation is also sufficient.We investigated the influence of 24 hours' cyclic loading (0.05Hz, 4kPa) on gene expression of human BMSCs in three-dimensional fibrin-DMEM constructs (n=7) in a compression bioreactor using DNA-array technology. Expression of the following genes showed a significant increase after mechanical stimulation: 2.6-fold osteopontin (OPN) and integrin-ß1 (ITGB1), 2.2-fold transforming growth factor-ß-receptor 1 (TGF-ß-R1) and 2.4-fold SMAD5 expression, compared to controls without mechanical stimulation (p<0.05 each). Platelet-derived growth factor-α (PDGF-α ) and annexin-V were also significantly overexpressed, the mechanical stimulation resulting in a 1.8-fold and 1.6-fold expression (p<0.05).Cells were identified as osteoblast precursors with a high proliferative capacity. Given the identical in-vitro environment for both groups, the increase in gene expression has been interpreted as a direct influence of cyclic mechanical stimulation on osteogenic differentiation. It may be postulated that short-term mechanical stimulation results in an improved osseous integration of tissue engineered grafts in bone defect healing.

Matrix metalloprotease activity is an essential link between mechanical stimulus and mesenchymal stem cell behavior.

Progenitor cells are involved in the regeneration of the musculoskeletal system, which is known to be influenced by mechanical boundary conditions. Furthermore, matrix metalloproteases (MMPs) and tissue-specific inhibitors of metalloproteases (TIMPs) are crucial for matrix remodelling processes that occur during regeneration of bone and other tissues. This study has therefore investigated whether MMP activity affects mesenchymal stem cell (MSC) behavior and how MMP activity is influenced by the mechanical stimulation of these cells. Broad spectrum inhibition of MMPs altered the migration, proliferation, and osteogenic differentiation of MSCs. Expression analysis detected MMP-2, -3, -10, -11, -13, and -14, as well as TIMP-2, in MSCs at the mRNA and protein levels. Mechanical stimulation of MSCs led to an upregulation of their extracellular gelatinolytic activity, which was consistent with the increased protein levels seen for MMP-2, -3, -13, and TIMP-2. However, mRNA expression levels of MMPs/TIMPs showed no changes in response to mechanical stimulation, indicating an involvement of post-transcriptional regulatory processes such as alterations in MMP secretion or activation. One potential regulatory molecule might be the furin protease. Specific inhibition of MMP-2, -3, and -13 showed MMP-13 to be involved in osteogenic differentiation. The results of this study suggest that MSC function is controlled by MMP activity, which in turn is regulated by mechanical stimulation of cells. Thus, MMP/TIMP balance seems to play an essential role in transferring mechanical signals into MSC function. Disclosure of potential conflicts of interest is found at the end of this article.

Mesenchymal stem cells regulate angiogenesis according to their mechanical environment.

In fracture and bone defect healing, MSCs largely drive tissue regeneration. MSCs have been shown to promote angiogenesis both in vivo and in vitro. Angiogenesis is a prerequisite to large tissue reconstitution. The present study investigated how mechanical loading of MSCs influences their proangiogenic capacity. The results show a significant enhancement of angiogenesis by conditioned media from mechanically stimulated compared with unstimulated MSCs in two-dimensional tube formation and three-dimensional spheroid sprouting assays. In particular, proliferation but not migration or adhesion of endothelial cells was elevated. Promotion of angiogenesis was dependent upon fibroblast growth factor receptor 1 (FGFR1) signaling. Moreover, stimulation of tube formation was inhibited by vascular endothelial growth factor receptor (VEGFR) tyrosine kinase blocking. Screening for the expression levels of different soluble regulators of angiogenesis revealed an enrichment of matrix metalloprotease 2, transforming growth factor beta1, and basic fibroblast growth factor but not of vascular endothelial growth factor in response to mechanical stimulation. In conclusion, mechanical loading of MSCs seems to result in a paracrine stimulation of angiogenesis, most likely by the regulation of a network of several angiogenic molecules. The underlying mechanism appears to be dependent on the FGFR and VEGFR signaling cascades and might be mediated by an additional cross-talk with other pathways.

VCAM-1-positive stromal cells from human bone marrow producing cytokines for B lineage progenitors and for plasma cells: SDF-1, flt3L, and BAFF.

The differentiation of B lymphocytes in the bone marrow is guided by the surrounding microenvironment determined by cytokines, adhesion molecules, and the extracellular matrix. These microenvironmental factors are mainly provided by stromal cells. In this paper, we report the identification of a VCAM-1-positive stromal cell population by flow cytometry. This population showed the expression of cell surface markers known to be present on stromal cells (CD10, CD13, CD90, CD105) and had a fibroblastoid phenotype in vitro. Single cell RT-PCR analysis of its cytokine expression pattern revealed transcripts for haematopoietic cytokines important for either the early B lymphopoiesis like flt3L or the survival of long-lived plasma cells like BAFF or both processes like SDF-1. Whereas SDF-1 transcripts were detectable in all VCAM-1-positive cells, flt3L and BAFF were only expressed by some cells suggesting the putative existence of different subpopulations with distinct functional properties. In summary, the VCAM-1-positive cell population seems to be a candidate stromal cell population supporting either developing B cells and/or long-lived plasma cells in human bone marrow.

Effects of tacrolimus, cyclosporin A and sirolimus on MG63 cells.

The reduction in bone mineral density after organ transplantation results in increased morbidity (post-transplantation bone disease) and remains an unsolved problem. A connection with the long-term application of nonglucocorticoidal immunosuppressants is the subject of controversial discussion. We hypothesized that such substances have an influence on the skeletal system on the cellular level by modulating osteoblast differentiation. Therefore, we investigated the effects of tacrolimus, cyclosporin A and sirolimus as representative substances of nonglucocorticoidal immunosuppressants on cell proliferation and expression of bone tissue-specific genes of human osteoblasts (MG63). None of the examined substances affected cell proliferation, but all influenced the gene expression pattern towards change in cell differentiation. In detail, collagen III and XII, matrix metalloproteinase 2, SMAD2, epithelial growth factor receptor, annexin V and osteonectin expression were increased by all of the examined substances. Tacrolimus, cyclosporin A and sirolimus influence intracellular signalling pathways, transmembranous receptors and bone-specific matrix synthesis. They do not have antiproliferative or toxic effects. We postulate that the shown changes of osteoblast differentiation cause post-transplantation disease.

The Zweymüller threaded cup: a choice in revision? Migration analysis and follow-up after 6 years.

Excellent long-term results are reported for threaded cups with an osteointegrable surface in primary interventions. So far, little data are available on the use of such implants in revision cases. We investigated 30 loosened Aesculap PM threaded cups (Tuttlingen, Germany) with a smooth surface, revised with a conical, corundum-blasted Zweymüller threaded cup (Zweymüller Alloclassic CSF cup, Zimmer, Warsaw, Ind). Aseptic radiological loosening was seen in 8% of cases, and a survival rate of 95% was achieved after 6.1 years. Average migration was 1.4 mm cranially and 0.7 mm medially. The Harris hip score improved from 42 points preoperatively to 75 points at follow-up. Good medium-term results are possible after revision using a threaded cup with an osteointegrable surface, but not if segmental acetabular margin defects are present.

Autologous bone marrow-derived cells enhance muscle strength following skeletal muscle crush injury in rats.

Insufficient post-traumatic skeletal muscle regeneration with consecutive functional deficiency continues to be a serious problem in orthopedic and trauma surgery. Transplantation of autologous muscle precursor cells has shown encouraging results in muscle trauma treatment but is associated with significant donor site morbidity. In contrast to this, bone marrow-derived (BMD) cells can be obtained without any functional deficit by puncture. The goal of this study was to examine whether regular muscle regeneration can be improved by local application of autologous BMD cells in a rat model of blunt skeletal muscle trauma. One week after standardized open blunt crush injury to the left soleus muscle, 10(6) autologous BMD cells were injected into the traumatized muscle of male Sprague Dawley rats. Rats of the control group received saline solution as treatment. Three weeks after application, the fast twitch and tetanic contraction capacity of the soleus muscles was measured bilaterally by stimulating the sciatic nerves. Contraction forces of injured soleus muscles in control animals recovered to 39 +/- 10% (tetanic) and 59 +/- 12% (fast twitch) of the contralateral noninjured soleus muscles (p < 0.001). In contrast, autologous BMD cell injection significantly restored contractile forces to 53 +/- 8% (tetanic) and 72 +/- 13% (fast twitch) compared to those observed in contralateral noninjured soleus muscles. Thus, muscle function was significantly increased by BMD cell treatment (tetanic, p = 0.014; fast twitch, p = 0.05). In conclusion, autologous BMD cell grafting leads to an increase in contraction force, 14% in tetanic and 13% in fast twitch stimulation, demonstrating its potential to improve functional outcome after skeletal muscle crush injury.

Simulation of cell differentiation in fracture healing: mechanically loaded composite scaffolds in a novel bioreactor system.

Cell differentiation during bone healing following a fracture is influenced by various biological and mechanical factors. We introduce a method for the examination of cell and tissue differentiation simulating a fracture gap in vitro. A closed bioreactor system allows the imitation of the biological, mechanical, and biochemical conditions in vitro. The initial hematoma formed in a fracture is simulated with a mixed construct composed of lyophilized cancellous bone and a fibrin matrix in a sandwich configuration. The construct may be loaded with osteoprogenitor cells. Exemplarily, constructs were loaded with rabbit periosteal cells and cultivated under mechanical loading with 7 kPa at 0.05 Hz for up to two weeks. During the observation period, cell morphology and correlating protein synthesis changed under mechanical stimulation. Cell differentiation differed between the various regions of the constructs. The periosteal cells were arranged perpendicularly to the mechanical loading and differentiated to osteoblastic forms with rising collagen type I synthesis, constant alkaline phosphatase activity, and initiation of the calcification of the extracellular matrix. The observed pattern of cell and tissue differentiation was similar to the one seen in the early phase of bone healing. In conclusion, the presented method allows simulation of cell and tissue differentiation during the early phase of fracture healing. It could serve as an in vitro model for the examination of mechanical and pharmacological influences during the early phase of bone healing on a cellular level.

Local application of a collagen type I/hyaluronate matrix and growth and differentiation factor 5 influences the closure of osteochondral defects in a minipig model by enchondral ossification.

This pilot study evaluated the effect of growth and differentiation factor-5 (rhGDF-5) combined with a collagen type I/hyaluronate matrix (c/h) on osteochondral defect repair in a minipig model. Defects created in both medial femoral condyles of 20 minipigs were treated with c/h (n = 10), c/h + rhGDF-5 (n = 10) or were left empty. After 3 and 12 months, five animals of each group were sacrificed. Evaluation included macroscopic and histological scoring and quantitative histomorphometry of synthesized bone. C/h and c/h + rhGDF-5 treatment increased trabecular bone formation in the upper third of the defect compared to empty controls, showing significance for c/h + rhGDF-5 (p = 0.05) but not between c/h and c/h + rhGDF-5 treatment. Cartilage regeneration and macroscopic outcome were not improved by c/h or c/h + rhGDF-5 treatment. Since c/h remnants were seen even one year postoperatively in the defect, possibly inhibiting further bone and cartilage healing, other matrices in combination with rhGDF-5 may provide further improvement in osteochondral defect treatment.