Effect of calcium phosphate ceramic substrate geometry on mesenchymal stromal cell organization and osteogenic differentiation.

The composition of calcium phosphate (CaP) ceramics in combination with surface features have been shown to influence biological performance, and micro- and nano-scale topography is known to stimulate osteogenic differentiation of mesenchymal stromal cells (MSCs). In view of this, adipose tissue derived MSCs were cultured on CaP disks featuring hemispherical concavities of various sizes (440, 800 or 1800 µm diameter). It was hypothesized that (i) surface concavities would promote cell proliferation, cellular organization within the concavities, and osteogenic differentiation, as a result of a more pronounced 3D micro-environment and CaP nucleation in concavities, and (ii) MSC proliferation and osteogenic differentiation would increase with smaller concavity size due to more rapidly occurring 3D cell-cell interactions. We found that concavities indeed affect cell proliferation, with 440 µm concavities increasing cell proliferation to a larger extent compared to 800 and 1800 µm concavities as well as planar surfaces. Additionally, concavity size influenced 3D cellular organization within the concavity volume. Interestingly, concavity size promoted osteogenic differentiation of cells, as evidenced by increased osteocalcin gene expression in 440 µm concavities, and osteocalcin staining predominantly for 440 and 800 µm concavities, but not for 1800 µm concavities and only slightly for planar surface controls.

Controlled Release of Chemotherapeutic Platinum-Bisphosphonate Complexes from Injectable Calcium Phosphate Cements.

Herein, we present a method to release chemotherapeutic platinum-bisphosphonate (Pt-BP) complexes from apatitic calcium phosphate cements (CPCs). Pt-BP-loaded hydroxyapatite nanoparticles (HA NPs) were added at different ratios to the powder phase of the cements, which contained poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres as porogens to accelerate their degradation. In vitro release kinetics of Pt-BP complexes revealed that the release rate of Pt species can be tuned by varying the amount of drug-loaded HA NPs as well as modifying the chemical structure of the Pt-BP complex to tailor its affinity with HA NPs. In addition, the incorporation of PLGA microspheres into the CPCs increased the degradation rate of the materials without affecting the release rate of Pt species. Finally, the antiproliferative activity of the free Pt-BP complexes and Pt-BP-loaded CPCs was evaluated using both human osteosarcoma cancer cells (MG-63) and human bone marrow-derived mesenchymal stromal cells (h-BMMSCs). This study demonstrated that both free Pt-BP complexes and the releasates from the CPCs were antiproliferative in a dose-dependent manner. Moreover, their antiproliferative activity was higher on MG-63 cells compared to h-BMMSC primary cells. In summary, it was shown that injectable CPCs can be rendered chemotherapeutically active by incorporation of HA NPs loaded with HA-binding Pt-BP complexes.

Effect of Nano-HA/Collagen Composite Hydrogels on Osteogenic Behavior of Mesenchymal Stromal Cells.

This study aimed to comparatively evaluate the in vitro effect of nanosized hydroxyapatite and collagen (nHA/COL) based composite hydrogels (with different ratios of nHA and COL) on the behavior of human mesenchymal stromal cells (MSCs), isolated from either adipose tissue (AT-MSCs) or bone marrow (BM-MSCs). We hypothesized that (i) nHA/COL composite hydrogels would promote the osteogenic differentiation of MSCs in an nHA concentration dependent manner, and that (ii) AT-MSCs would show higher osteogenic potential compared to BM-MSCs, due to their earlier observed higher proliferation and osteogenic differentiation potential in 2D in vitro cultures [1]. The obtained results indicated that AT-MSCs show indeed high proliferation, differentiation and mineralization capacities in nHA/COL constructs compared to BM-MSCs, but this effect was irrespective of nHA concentration. Based on the results of alkaline phosphatase (ALP) activity and osteocalcin (OCN) protein level, the osteogenic differentiation of BM-MSCs started in the beginning of the culture period and for AT-MSCs at the end of the culture period. At a molecular level, both cell types showed high expression of osteogenic markers (bone morphogenic protein 2 [BMP2], runt-related transcription factor 2 [RUNX2], OCN or COL1) in both an nHA concentration and time dependent manner. In conclusion, AT-MSCs demonstrated higher osteogenic potential in nHA/COL based 3D micro-environments compared to BM-MSCs, in which proliferation and osteogenic differentiation were highly promoted in a time dependent manner, irrespective of nHA amount in the constructs. The fact that AT-MSCs showed high proliferation and mineralization potential is appealing for their application in future pre-clinical research as an alternative cell source for BM-MSCs.

Coculture effects on the osteogenic differentiation of human mesenchymal stromal cells.

Cell-based bone regeneration is generally pursued based on single cell type approaches, for which human adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used, owing to their easy accessibility and relatively large yield. In view of multiple cell types involved in physiological bone regeneration, this study aimed to evaluate the osteogenic differentiation of AT-MSCs upon co-culture with endothelial cells or macrophages in a direct or indirect in vitro co-culture set-up. Our hypotheses were that 1) endothelial cells and macrophages stimulate AT-MSCs proliferation and osteogenic differentiation and that 2) these two cell types will more profoundly affect osteogenic differentiation of AT-MSCs in a direct compared to an indirect co-culture set-up, because of the possibility for both cell-cell interactions and effects of secreted soluble factors in the former. Osteogenic differentiation of AT-MSCs was stimulated by endothelial cells, particularly in direct co-cultures. Although initial numbers of AT-MSCs in co-culture with endothelial cells were 50% compared to monoculture controls, equal levels of mineralization were achieved. Macrophages showed a variable effect on AT-MSCs behavior for indirect co-cultures and a negative effect on osteogenic differentiation of AT-MSCs in direct co-cultures, the latter likely due to species differences of the cell types used. The results of this study demonstrate potential for cell combination strategies in bone regenerative therapies.

Signaling pathways involved in osteogenesis and their application for bone regenerative medicine.

Bone regeneration is a well organized but complex physiological process, in which different cell types and their activated signaling pathways are involved. In bone regeneration and remodeling processes, mesenchymal stem cells (MSCs) have a crucial role, and their differentiation during these processes is regulated by specific signaling molecules (growth factors/cytokines and hormones) and their activated intracellular networks. Especially the utilization of the molecular machinery seems crucial to consider prior to developing bone implants, bone-substitute materials, and cell-based constructs for bone regeneration. The aim of this review is to provide an overview of the signaling mechanisms involved in bone regeneration and remodeling and the osteogenic potential of MSCs to become a key cellular resource for such regeneration and remodeling processes. Additionally, an overview of possibilities to beneficially exploit cell signaling processes to optimize bone regeneration is provided.

Different unequal cross-over events between NCF1 and its pseudogenes in autosomal p47(phox)-deficient chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O2(-)) and other microbicidal oxidants due to mutations in one of the five components of the O2(-)-generating NADPH oxidase complex. The most common autosomal subtype of CGD is caused by mutations in NCF1, encoding the NADPH subunit p47(phox). Usually, these mutations are the result of unequal exchange of chromatid between NCF1 and one of its two pseudogenes. We have now investigated in detail the breakpoints within or between these (pseudo) NCF1 genes in 43 families with p47(phox)-deficient CGD by means of multiplex ligase-dependent probe amplification (MLPA). In 24 families the patients totally lacked NCF1 sequences, indicating that in these families the cross-over points are located between NCF1 and its pseudogenes. Six other families were compound heterozygous for a total NCF1 deletion and another mutation in NCF1 on the other allele. In 8 families, the patients lacked NCF1 exons 1-4 but had retained NCF1 exons 6-10, indicating that a cross-over point is located within NCF1 between exons 4 and 6. Similarly, in 4 families a cross-over point was located within NCF1 between exons 2 and 4. Similar cross-overs, in heterozygous form, were observed in family members of the patients. Several patients were compound heterozygous for total and partial NCF1 deletions. Thus, at least three different cross-over points exist within the NCF1 gene cluster, indicating that autosomal p47(phox)-deficient CGD is genetically heterogeneous but can be dissected in detail by MLPA.