Encapsulation of Human Mesenchymal Stem Cells in Phosphate Mineralized Alginate Beads

Alginate scaffolds show good promise for bone tissue engineering using stem cells. This is due to the fact that alginate is biocompatible, non-immunogenic, and in addition may direct differentiation of stem cells into a given phenotype. Finally, due to their ability to gel at physiological conditions, living and functional tissue are easily encapsulated into alginate beads. Alginate beads can be modified in a range of ways, not only to enhance the matrix stiffness and stability, but also to promote cell adhesion and direct differentiation towards a given phenotype. A method currently used to encourage bone growth is to mineralize the alginate beads, thus mimicking the structure of bone in vivo. Recently, Xie et al. (2011) demonstrated that enzymatic mineralization of alginate beads serves as a potent method for mineralizing beads as lower concentrations of CaCl2 is needed, which is beneficial for cell viability. In addition, the enzymatic method produces alginate beads with a uniformly distribution of calcium phosphate (CP) and stiffer mechanical properties. Mesenchymal Stem cells (MSCs) have the potential to differentiate into a variety of tissues, including cartilage, adipose, muscle and bone. MSCs extracted from bone marrow seem to possess the greatest potential for bone tissue engineering, as they are more easily differentiated into osteogenic phenotypes when compared with MSCs from e.g. adipose tissue. The main objective in the present study was to encapsulate MSCs into alginate beads mineralized with alkaline phosphatase (ALP) and study cell survival, as well as their potential to differentiate into mature osteoblasts inside the beads. To mineralize beads (ALP) was added to the alginate solution, whilst the precursors were added to the growth medium. The mineralization medium was changed every 3 hours (12 hours over night) for the first 2 days post encapsulation.Cell viability was surveyed by live/dead assay and imaging by Confocal Laser Scanning Microscopy (CLSM), and metabolic activity by Alamar Blue (AB) and colorimetric techniques. Examination of cell morphology was accomplished by phalloidin/DRAQ5 staining. Moreover, to investigate cell differentiation, PCR analysis on selected RNA candidates was performed. Quantification of mineral content was accomplished by running Alizarin Red-S (ARS-S) assay. ALP activity was determined using ALP assay. Finally, further investigation of cell- and alginate matrix structure was accomplished using scanning electron microscopy (SEM). To study capsule properties and cell survival, osteosarcoma cells were utilized as model cells. The main objective was to study bead stability, and how the beads and the cells within were affected by the enzymatic mineralization method. Alginate beads proved to be unstable and needed addition of CaCl2 for stabilization purposes. Furthermore, in order to recover cells, citrate was added to the cell/bead suspension. Initially, results were unsatisfactory, as little, or no, RNA was recovered. After optimizing the citrate treatment it was discovered that a sequential method needed to be utilized. Results demonstrated a successful recovery of cells, and RNA of excellent quality.Enzymatic mineralization of alginate beads was found to be a cell friendly way of mineralizing alginate beads. Mineralization of beads was observed in the light microscope, by visual inspection, as well as by SEM. Cell viability remained high when using a concentration of 0.25mg/ml ALP. The osteosarcoma cells proved to be good for optimizing the enzymatic mineralization method, but behaved differently compared with mesenchymal stem cells in terms of viability, and mineralization activity. Furthermore, mineralization of beads by addition of 0.25 mg/mL ALP compared with 0.5mg/mL appeared to be beneficial as image acquisition on CLSM was facilitated and slightly higher viability of cells was observed.In the second part of the present study, human MSCs were encapsulated into alginate beads. After initial experiments the optimal condition for cell survival, and bead stability was determined. Consequently, in the final experiment 16 million MSCs were encapsulated in beads containing 0.25mg/mL ALP, together with a sample without addition of ALP. At day 2 post encapsulation both samples were divided into two batches, one cultured in regular medium, and one in differentiation medium. All samples were stabilized with 7.5 mM of CaCl2.Observations in both light microscope and CLSM revealed that only beads given ALP were mineralized before reaching day 21. At day 21 the sample receiving no ALP, cultured in differentiation medium also appeared mineralized.Mesenchymal stem cells receiving differentiation medium were observed to differentiate into mature osteoblasts in the beads. This was verified by gene expression analysis, cell morphology studies, the presence of collagen in beads seen by SEM and analysis of ALP activity. Metabolic activity measurements confirmed little cell proliferation, nor cell death. However, an increased metabolic activity was observed for encapsulated cells cultured in regular medium relative to cells in differentiation medium. Cell morphology in differentiated samples was recognized by showing elongated actin filaments, compared with the ones cultured in regular medium, which appeared round in shape. The elongated filaments suggest that the cells are able to interact with the alginate matrix and/or minerals. The occurrence of collagen fibers in SEM images further confirmed presence of mature osteoblasts. Samples cultured in regular medium with or without added ALP both showed an increase in osterix expression until day 21 when the study was ended. This was surprising, as it inferred that the alginate matrix itself might influence differentiation of MSCs into osteoblasts, and that the minerals have little effect on differentiation. Runx2 expression was detected in all samples, including unencapsulated hMSCs. The expression of runx2 was at its maximum on day 21, when the study was ended.Encapsulating mesenchymal stem cells into alginate beads mineralized by the enzymatic method is cell friendly, and allows the cells to differentiate into mature osteoblasts when cultured in differentiation medium. Alginate without minerals seems to influence differentiation to a certain extent, suggesting that minerals are not needed for differentiation to occur. The minerals do, nonetheless, speed up the continuing mineralization process

Smac-mimetics reduce numbers and viability of human osteoclasts

Elevated activity of bone-degrading osteoclasts (OC) contributes to pathological bone degradation in diseases such as multiple myeloma. Several proinflammatory cytokines, including TNF, contribute to osteoclastogenesis. The receptor-interacting protein kinase 1 (RIPK1) regulates inflammation and cell death. It is recruited to the TNF-receptor complex, where it is ubiquitinated, and activates transcription factor NF-κB and mitogen-activated protein kinases (MAPK). Smac-mimetics (SM) is a group of drugs that block RIPK1 ubiquitination and shifts RIPK1 to activation of apoptosis or necroptosis. In this manuscript, we show that the two SM birinapant and LCL-161 reduced the number and viability of primary human OC, and induced TNF-dependent cell death in OC precursors (pre-OC). Birinapant was more cytotoxic than LCL-161 and induced predominantly apoptosis and to some degree necroptosis. Both inhibitors restrained osteoclastogenesis induced by myeloma patient bone-marrow aspirates. SM has gained attention as novel treatment strategies both for cancer and chronic inflammatory pathologies, but limited information has been available on interactions with primary human immune cells. As LCL-161 is in phase 2 clinical studies for multiple myeloma, we propose that SM might possess additional benefits in reducing bone degradation in myeloma patients. Taken together, we show that SM reduces human osteoclastogenesis, and that these compounds may represent promising drug candidates for pathological bone degradation

Chemerin is elevated in multiple myeloma patients and is expressed by stromal cells and pre-adipocytes

Abstract Chemerin is a recently discovered adipokine shown to be involved in both inflammatory and metabolic processes. Here, we demonstrate that chemerin serum levels are elevated in patients with multiple myeloma and that it increases with disease progression. We found that chemerin is expressed by stromal cells and preadipocytes, whereas its receptor CCRL2 is expressed by primary myeloma cells, suggesting a paracrine signaling loop between bone marrow stromal cells/adipocytes and myeloma cells. This is the first study exploring chemerin and its receptors in multiple myeloma

Revealing the influence of electron beam melted Ti-6Al-4V scaffolds on osteogenesis of human bone marrow-derived mesenchymal stromal cells

Porous Titanium-6Aluminum-4Vanadium scaffolds made by electron beam-based additive manufacturing (AM) have emerged as state-of-the-art implant devices. However, there is still limited knowledge on how they influence the osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs). In this study, BMSCs are cultured on such porous scaffolds to determine how the scaffolds influence the osteogenic differentiation of the cells. The scaffolds are biocompatible, as revealed by the increasing cell viability. Cells are evenly distributed on the scaffolds after 3 days of culturing followed by an increase in bone matrix development after 21 days of culturing. qPCR analysis provides insight into the cells' osteogenic differentiation, where RUNX2 expression indicate the onset of differentiation towards osteoblasts. The COL1A1 expression suggests that the differentiated osteoblasts can produce the osteoid. Alkaline phosphatase staining indicates an onset of mineralization at day 7 in OM. The even deposits of calcium at day 21 further supports a successful bone mineralization. This work shines light on the interplay between AM Ti64 scaffolds and bone growth, which may ultimately lead to a new way of creating long lasting bone implants with fast recovery times

Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

<div><p>Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (<i>RUNX2</i>, <i>COL1AI</i>, <i>SP7</i>, <i>BGLAP</i>) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (<i>SOST</i>) and dental matrix protein-1 (<i>DMP1</i>), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.</p></div

SEM micrographs of collagen fibrils in beads cultured in osteogenic medium.

<p>(A, C) low and high magnification of the space close to a cell producing collagen in ALP modified beads (ALP+). Mineral crystals similar to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120374#pone.0120374.g002" target="_blank">Fig. 2</a> are indicated by *; (B, D) low and high magnification micrographs of the space close to a cell producing collagen in unmineralized beads. Images were collected at 21 days post encapsulation.</p

SEM micrographs of the alginate network in ALP-modified (A, C, E) or unmodified (B, D, F) beads taken at day 21 post encapsulation.

<p>Cells were cultured in either growth medium (A, B) or osteogenic medium (C-F). Mineral particles with spherical morphology are clearly visible for heavily mineralized samples shown at high and low magnification in panel C and E.</p

Alkaline phosphatase activity in cells cultured in ALP-modified or unmodified beads for 9 days as indicated.

<p>p values indicate statistical significant differences by Sidak’s multiple comparison test.</p

Relative mRNA expression of osteoblast/ osteocyte markers.

<p>MSCs were cultured in unmodified (no ALP), ALP-modified (ALP) alginate beads or on traditional culture plates (2D). Samples were cultured in either growth medium (GM) or osteogenic medium (OM) for 21 days post encapsulation. RUNX2 (A), Osterix (SP7) (B), COL1A1 (C), and sclerostin (SOST) (E) mRNA expression are relative to mRNA expression in cells cultured on traditional culture plates in osteogenic medium for 7 days. Osteocalcin (BGLAP) (D) and DMP1 (F) mRNA expressions are relative to mRNA expression in cells in unmodified alginate beads cultured in growth medium for 7 days post encapsulation. ND = not detected.</p

Mineralization of alginate beads.

<p>Light microscopy of unmodified (A) or ALP-modified (B) beads taken at day 2 and day 21 post encapsulation. Cells were cultured in either growth medium (GM) or osteogenic medium (OM). Mineralized beads appear dark whereas unmineralized beads appear transparent. Scale bar 500μm.</p