Mechanical stimulation and mitogen-activated protein kinase signaling independently regulate osteogenic differentiation and mineralization by calcifying vascular cells.

Ectopic calcification of vascular tissue is associated with several cardiovascular pathologies and likely involves active regulation by vascular smooth muscle cells and osteoblast-like vascular cells. This process often occurs in sites with altered mechanical environments, suggesting a role for mechanical stimuli in calcification. In this study, we investigated the effect of mechanical stimulation on the proliferation, osteogenic differentiation, calcification, and mitogen-activated protein kinase (MAPK) signaling in calcifying vascular cells (CVCs), a subpopulation of aortic smooth muscle cells putatively involved in vascular calcification. Application of equibiaxial cyclic strain (7%, 0.25 Hz) to CVCs had no effect on cell proliferation, but accelerated alkaline phosphatase expression and significantly increased mineralization by 3.1-fold over unstrained cells. Fluid motion in the absence of strain also enhanced mineralization, but to a lesser degree. Because MAPK pathways mediate mechanically regulated osteoblast differentiation, we tested whether similar signaling was involved in mineralization by CVCs. In static cultures, pharmacological inhibition of the extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase pathways significantly attenuated mineral production by as much as -94%, compared with uninhibited CVCs. Strikingly, although mechanical stimulation activated each of the MAPK pathways, inhibition of these pathways had no effect on the mechanically induced enhancement of alkaline phosphatase activity or mineralization. These novel data indicate that mechanical signals regulate calcification by CVCs, and although MAPK signaling is critical to CVC osteogenic differentiation and mineralization, it is not involved directly in transduction of mechanical signals to regulate these processes under the conditions utilized in this study.

Shape retaining injectable hydrogels for minimally invasive bulking.

PURPOSE: Particle migration, poor shape definition and/or rapid resorption limit the success of current urethral bulking agents. We propose that shape defining porous scaffolds that allow cell infiltration and anchoring, and may be delivered in a minimally invasive manner may provide many advantageous features. MATERIALS AND METHODS: Alginate hydrogels were prepared with varying degrees of covalent cross-linking and different pore characteristics. Dehydrated scaffolds were compressed into smaller, temporary forms, introduced into the dorsal subcutaneous space of CD-1 mice by minimally invasive delivery through a 10 gauge angiocatheter and rehydrated in situ with a saline solution delivered through the same catheter. Ionically cross-linked calcium alginate gel served as a control. Specimens were harvested at 2, 6, 12 and 24 weeks to evaluate implant shape retention and volume, cell infiltration and calcification, and the presence of an inflammatory response. RESULTS: A total of 90 scaffolds were implanted and 95% were recovered at the site of injection. All of these scaffolds successfully rehydrated and 80% recovered and maintained their original 3-dimensional shape for 6 months. Scaffold volume and tissue infiltration varied depending on the degree of alginate cross-linking. Highly cross-linked materials (20% and 35%) demonstrated the best volume maintenance with the latter facilitating the most tissue infiltration. The inflammatory response was minimal except with the 80% cross-linked material. Calcification was not observed in covalently cross-linked scaffolds. In contrast, 98% of calcium alginate implants were calcified. CONCLUSIONS: Shape retaining porous hydrogels meet many of the requirements necessary for a successful injectable bulking agent and offer advantages over currently used agents.

Shape-defining scaffolds for minimally invasive tissue engineering.

BACKGROUND: Minimally invasive surgical procedures are increasingly important in medicine, but biomaterials consistent with this delivery approach that allow one to control the structure of the material after implantation are lacking. Biomaterials with shape-memorizing properties could permit minimally invasive delivery of cell transplantation constructs and enable the formation of new tissues or structures in vivo in desired shapes and sizes. METHODS: Macroporous alginate hydrogel scaffolds were prepared in a number of predefined geometries, compressed into significantly smaller, different "temporary" forms, and introduced into immunocompromised mice by means of minimally invasive surgical delivery through a small catheter. Scaffolds were rehydrated in situ with a suspension of cells (primary bovine articular chondrocytes) or cell-free medium and delivered through the same catheter. Specimens were harvested at 1 hr to evaluate the efficacy of cell delivery and the recovery of scaffold geometry, and at 8 and 24 weeks to evaluate neotissue formation. RESULTS: A high percentage (88%) of scaffolds that were introduced with a catheter and rehydrated with cells had recovered their original shape and size within 1 hr. This delivery procedure resulted in cartilage structures with the geometry of the original scaffold by 2 months and histologically mature appearing tissue at 6 months. CONCLUSIONS: Shaped hydrogels, formed by covalently cross-linking, can be structurally collapsed into smaller, temporary shapes that permit their minimally invasive delivery in vivo. The rapid recovery of scaffold properties facilitates efficient cell seeding in vivo and permits neotissue formation in desired geometries.

Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway.

Physical stimuli play critical roles in the development, regeneration, and pathology of many mesenchymal tissues, most notably bone. While mature bone cells, such as osteoblasts and osteocytes, are clearly involved in these processes, the role of their progenitors in mechanically mediated tissue responses is unknown. In this study, we investigated the effect of cyclic substrate deformation on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Application of equibiaxial cyclic strain (3%, 0.25Hz) to hMSCs cultured in osteogenic media inhibited proliferation and stimulated a 2.3-fold increase in matrix mineralization over unstrained cells. The strain stimulus activated the extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase pathways, but had no effect on c-Jun N-terminal kinase phosphorylation or activity. Strain-induced mineralization was largely mediated by ERK1/2 signaling, as inhibition of ERK1/2 attenuated calcium deposition by 55%. Inhibition of the p38 pathway resulted in a more mature osteogenic phenotype, suggesting an inhibitory role for p38 signaling in the modulation of strain-induced osteogenic differentiation. These results demonstrate that mechanical signals regulate hMSC function, suggesting a critical role for physical stimulation of this specific cell population in mesenchymal tissue formation.