Osteogenic Direction Research Articles

Induced differentiation of mesencymal precursor cells in hyaluronan hydrogel with covalently linked bisphosphonates

Event Abstract Back to Event Induced differentiation of mesencymal precursor cells in hyaluronan hydrogel with covalently linked bisphosphonates Dmitri Ossipov1*, Sujit Kootala1*, Yu Zhang1*, Vladimir Tolmachev2* and Jöns Hilborn1* 1 Uppsala University, Chemistry-Ångström Laboratory, Sweden 2 Uppsala University, Unit of Biomedical Radiation Sciences, Sweden Introduction: Repair or replacement of damaged tissues is often reliant on the ability of a biomaterial to control the delivery of growth factors[1]. Specific affinity interactions, which are engineered between the proteins and different matrices[2], exhibited capability to tune the rate of release while maintaining consistent levels of bioactivity. In this work we present an in situ cross-linkable hydrogel system that permits manipulation of entrapped cells in 3-D with the help of bisphosphonate (BP) ligands attached to the hyaluronic acid (HA) matrix and hence providing effective retention of bone morphogenetic protein-2 (BMP-2) in the matrix. Specifically, we demonstrate that (1) linking of predetermined amounts of BP ligands to HA matrix provide tailored degrees of BMP-2 release and that (2) BMP-2 sequestration caused by BPs provide a stable microenvironment for 3D entrapment of C2C12 myogenic progenitor cells in the hydrogel and promote their osteogenic differentiation (Fig. 1). Fig. 1. In situ encapsulation of myotube forming cells and BMP-2 in hyaluronic acid hydrogel to induce 3D differentiation of the cells through the controlled retention and release of the growth factor by the matrix-linked bisphosphonate ligands. Materials and Methods: HA-BP-SH derivative was synthesized by thiolation of the previously described HA-BP derivative[3] with 3,3'-dithiobis(propionic hydrazide). Results and Discussion: Three HA derivatives carrying 2-dithiopyridyl (HA-SSPy), thiol (HA-SH), and bisphosphonate and thiol groups (HA-BP-SH) were prepared to form two types of hydrogels by thiol-disulfide exchange cross-linking reaction[3]. The hydrogels were in situ formed by mixing of either HA-SSPy and HA-SH (giving control HA network) or HA-SSPy and HA-BP-SH (giving bisphosphonated HABP network). In both hydrogels, HA chains were cross-linked by disulfide bonds and equal cross-linking density was ensured by the same degree of substitution with the functional groups in all three derivatives. Loading of BMP-2 into the hydrogels was achieved in situ by pre-dissolving the growth factor in HA-SSPy solution. We observed that the release of BMP-2 from HA hydrogel was faster than from the HABP matrix. Moreover, the rate of the growth factor release could be tuned by changing the amount of BP groups in the matrix which was easily achieved by combination of HA-SH and HA-BP-SH components at different mass ratios. Next, C2C12 cells were encapsulated in the hydrogels together with the equal amount of BMP-2 and the obtained constructs were cultured for 5 days. Expression of alkaline phosphatase (ALP), a major late-stage osteogenic differentiation marker, was then measured. A 5-fold increase in ALP expression was detected for the cells cultured 3-D in HABP hydrogels as compared to HA hydrogels (Fig. 2). Fig. 2. ALP production by C2C12 cells cultured for 5 days in different hydrogels. Basing on the release results, we propose that the cells were exposed to BMP-2 for longer time and at higher concentrations in HABP hydrogel than in HA hydrogel. The retention of BMP-2 in HABP hydrogel was attributed to BP groups that bind and protect the growth factor. Conclusion: We present a novel hydrogel delivery system enabling sequestration of BMP-2 basing on use of in situ cross-linkable HA-BP conjugate. Application of this conjugate in combination with BMP-2 to cells in 3-D allows the cells to differentiate in osteogenic direction.

Comparative analysis of subpopulations of mesenchymal stromal cells of the bone marrow and fetal liver differing in sensitivity to 5-fluorouracil

It is shown that the effect of 5-fluorouracil (5-FU) on mesenchymal stromal cells (MSCs) of the bone marrow and fetal liver of the rat increases the population of cells with a reduced proliferative potential. It is shown that, in the course of passaging, the 5-FU-resistant MSCs of the bone marrow and fetal liver lose their ability to differentiate in the osteogenic direction more and less rapidly, respectively, than the 5-FU-sensitive MSCs.

Cultivation and differentiation change nuclear localization of chromosome centromeres in human mesenchymal stem cells.

Chromosome arrangement in the interphase nucleus is not accidental. Strong evidences support that nuclear localization is an important mechanism of epigenetic regulation of gene expression. The purpose of this research was to identify differences in the localization of centromeres of chromosomes 6, 12, 18 and X in human mesenchymal stem cells depending on differentiation and cultivating time. We analyzed centromere positions in more than 4000 nuclei in 19 mesenchymal stem cell cultures before and after prolonged cultivation and after differentiation into osteogenic and adipogenic directions. We found a centromere reposition of HSAX at late passages and after differentiation in osteogenic direction as well as of HSA12 and HSA18 after adipogenic differentiation. The observed changes of the nuclear structure are new nuclear characteristics of the studied cells which may reflect regulatory changes of gene expression during the studied processes.

Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices.

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 (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), 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.

Phenol Red Inhibits Chondrogenic Differentiation and Affects Osteogenic Differentiation of Human Mesenchymal Stem Cells in Vitro

The purpose with this study was to investigate the effect of phenol red (PR) on chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). hMSCs were differentiated into chondrogenic and osteogenic directions in DMEM with and without PR for 2, 7, 14, 21, and 28days. Gene expression of chondrogenic and osteogenic markers were analyzed by RT-qPCR. The presence of proteoglycans was visualized histologically. Osteogenic matrix deposition and mineralization were examined measuring the alkaline phophatase activity and calcium deposition. During chondrogenic differentiation PR decreased sox9, collagen type 2, aggrecan on day 14 and 21 (P < 0.05), and proteoglycan synthesis on day 21 and 28. Collagen type 10 was decreased on day 21 (P < 0.05). During osteogenic differentiation PR increased alkaline phosphatase on day 7 while decreased on day 21 (P < 0.05). PR increased collagen type 1 on day 7, 14, and day 21 (P < 0.05). The alkaline phosphatase activity was increased after 2, 7, and 14days (P < 0.05). The deposition of calcium was decreased on day 21 (P < 0.05). Our results indicate that PR should be removed from the culture media when differentiating hMSCs into chondrogenic and osteogenic directions due to the effects on these differentiation pathways.

Comparative characteristics of new lines of mesenchymal stem cells derived from human embryonic stem cells, bone marrow, and foreskin

New human nonimmortalized fibroblast-like cell lines were derived from various sources: from embryonic stem cells (ESCs) (the SC5-MSC and SC3a-MSC lines), from bone marrow of a 5- to 6-week-old fetus (the FetMSC line), and from foreskin of a 3-year-old child (the FRSN line). All the lines are successfully used as feeders during cultivation of human ESCs. The mean doubling time of the cell populations fluctuates depending on the line from 25.5 h in the SC5-MSC line to 38.8 h in the SC3a-MSC line. The growth curves indicate active cell proliferation of all lines. Numerical and structural karyotypical analysis has shown these lines to have a normal karyotype: 46, XX (SC5-MSC and SC3a-MSC) and 46, XY (FetMSC and FRSN). To determine the status of these lines, comparative analysis of surface markers was performed with the aid of flow cytofluorimetry, and expression of the c antigens characteristic of human mesenchymal stem cells (MSCs) was revealed: CD44, CD73, CD90, CD105, and HLA-ABC and the absence of expression of CD34 and HLA-DR. Interlinear differences in the expression level of the marker CD117 (c-kit) were revealed. Immunofluorescent and cytofluorimetric analysis of expression of surface markers and transcription factor Oct-4 that are characteristic of human ESCs has shown that, in all four lines, expression of TRA-1-60 and Oct-4 is absent, whereas in expression of SSEA-4 there are observed the interlinear differences not depending on the origin of cells. At present it is not yet clear whether the revealed interlinear differences affect essentially the functional status of mesenchymal stem cells. Immunofluorescent analysis in cells of all lines showed expression of markers of early differentiation into derivatives of three germ layers characterizing ESCs, which might possibly provide wide MSC possibilities during reparation of different tissue damages, depending on the corresponding microenvironment. The capability of cells of all lines for directed differentiation into the adipogenic and osteogenic directions was revealed.

Phenotypical properties and ability to multilineage differentiation of adipose tissue stromal cells during subculturing

Morphological and immunophenotypical properties of human adult adipose tissue stromal cells (ATSC) at cultivation passage 0 and 4 as well as their ability to induced in vitro differentiation into adipogenic and osteogenic directions were studied in this work. It was shown that primary cultures of ATSC were characterized by the presence of the lower number of cells expressing mesenchymal markers (CD73, CD105) than the cells of the 4th passage, but contained endothelial progenitor cells expressing CD34 and capable to form capillary-like structures within extracellular matrix. Both cell populations could equally differentiate into adipogenic and osteogenic lineages.

Overexpression of GDF5 through an Adenovirus Vector Stimulates Osteogenesis of Human Mesenchymal Stem Cells in vitro and in vivo

The use of stem cells combined with gene therapy could be an important way to facilitate bone regeneration. In this study, the aim was to investigate the potential of growth and differentiation factor-5 (GDF5) to genetically manipulate human mesenchymal stem cells (hMSCs) for bone regeneration. Recombinant adenovirus Ad-GDF5 and Ad-GFP were constructed and identified, and the titer of both were determined. Third-passage hMSCs were infected with adenovirus, and the expression of GDF5 was confirmed by detection of GFP-positive cells, GDF5 mRNA levels, Western blotting, and enzyme-linked immunosorbent assay (ELISA). hMSCs at passage 3 were divided into four groups: (1) an experimental group infected with Ad-GDF5, (2) a positive control group cultured with osteogenic differentiation medium, (3) a control group infected with Ad-GFP cultured with standard medium, and (4) a blank control group cultured with standard medium. Evaluation of cell morphology and proliferation, analysis of the expression of genes related to osteogenic differentiation, von Kossa staining, and immunofluorescent staining of collagen I were used to investigate the osteogenesis of cells among the groups. After culturing the cells for 2 days under each corresponding condition, the cells were detached and subcutaneously injected into the backs of nude mice to evaluate bone formation. Samples were collected for histological staining, protein Western blotting, and micro-computer tomography. When infected with Ad-GDF5, hMSCs could overexpress GDF5 for a prolonged period in vitro and reach a concentration of 160 ng/ml. Cells infected with Ad-GDF5 or cultured in osteogenic medium displayed osteogenic differentiation based on their histological and cellular properties and on their gene and protein expression patterns. Furthermore, Ad-GDF5 showed a better ability to upregulate the expression of collagen I, alkaline phosphatase, and osteocalcin mRNA than the osteogenic medium. Furthermore, Ad-GDF5 expression was associated with enhanced bone formation in vivo. Our findings suggest that hMSCs infected with Ad-GDF5 can differentiate in an osteogenic direction and may be a promising cell source for bone regeneration.

Stability of bone marrow stromal cells to low temperatures depending on degree of differentiation

Based on a previous study of the stability of a heterogenous population of keratinocytes against cold depending on their degree of differentiation, we studied in vitro the stability of rat bone marrow stem cells (BMSCs) against cold before and after their differentiation in the adipogenic or osteogenic direction. It was shown that, after the induction of differentiation, BMSCs were least stable against the action of low temperatures than the undifferentiated cells. The obtained data can serve as a basis for the further study of processes and mechanisms that affect the stability of BMSCs against cold depending on their degree of differentiation.

Regeneration of Skull Bones in Adult Rabbits after Implantation of Commercial Osteoinductive Materials and Transplantation of a Tissue-Engineering Construct

We performed a comparative study of reparative osteogenesis in rabbits with experimental critical defects of the parietal bones after implantation of commercial osteoinductive materials "Biomatrix", "Osteomatrix", "BioOss" in combination with platelet-rich plasma and transplantation of a tissue-engineering construct on the basis of autogenic multipotent stromal cells from the adipose tissue predifferentiated in osteogenic direction. It was found that experimental reparative osteogenesis is insufficiently stimulated by implantation materials and full-thickness trepanation holes were not completely closed. After transplantation of the studied tissue-engineering construct, the defect was filled with full-length bone regenerate (in the center of the regenerate and from the maternal bone) in contrast to control and reference groups, where the bone tissue was formed only on the side of the maternal bone. On day 120 after transplantation of the tissue-engineering construct, the percent of newly-formed bone tissue in the regenerate was 24% (the total percent of bone tissue in the regenerate was 39%), which attested to active incomplete regenerative process in contrast to control and reference groups. Thus, the study demonstrated effective regeneration of the critical defects of the parietal bones in rabbits 120 days after transplantation of the tissue-engineering construct in contrast to commercial osteoplastic materials for directed bone regeneration.

Dermal papilla cells induce keratinocyte tubulogenesis in culture

The ability of dermal papilla (DP) cells to induce hair growth was reported in many studies. However, early stages of hair follicle development and signals that govern this process are poorly understood. Therefore, an in vitro model may be a convenient system to study epithelial-mesenchymal interactions and early stages of epidermal morphogenesis, especially in humans. To investigate the role of DP cells in epidermal morphogenesis we modified the method of isolation of DP cells from hair follicle of human scalp and developed the three-dimensional model of epidermal morphogenesis. Isolated DP cells were able to differentiate in adipogenic and osteogenic directions and retained activity of alkaline phosphatase (AP) for seven passages in culture. DP cells were able to induce tubule-like structures in three-dimensional model in vitro and to reorganize collagen matrix. Prolonged cultivation of DP cells has been a big problem because of the loss of hair follicle-inducing ability and growth activity after several passages. To solve this problem we immortalized DP cells by the transfection of the human telomerase reverse transcriptase cDNA (hTERT). Immortalized DP-hTERT cells retained AP activity and demonstrated low ability to osteogenic differentiation. The conditioned medium collected from actively proliferated cells as well as DP-hTERT cells themselves were capable to induce tubulogenesis after prolonged keratinocyte cultivation.

Evaluation of the osteogenic potential and vascularization of 3D poly(3)hydroxybutyrate scaffolds subcutaneously implanted in nude rats

The aim of this study was to evaluate the osteogenic potential and the vascularization of embroidered, tissue engineered, and cell-seeded 3D poly(3)hydroxybutyrate (PHB) scaffolds in nude rats. Collagen I (coll I)- and collagen I/chondroitin sulfate (coll I/CS)-coated PHB scaffolds were seeded with human mesenchymal stem cells (hMSCs). Proliferation and differentiation were characterized by different biochemical assays in vitro. For animal experiments, the cells were cultivated on coll I- or coll I/CS-coated scaffolds and either expanded or osteogenically differentiated. Scaffolds were piled up to create a 3D scaffold pad and implanted subcutaneously into nude rats. In vitro hMSC showed proliferation and differentiation on PHB scaffolds. Alkaline phosphatase (ALP) and calcium increased in the differentiation medium and in the presence of coll I/CS. In vivo blood vessels were found in the scaffold-stack. Histological/immunohistological analyses of explanted scaffolds showed osteogenic markers such as osteopontin, osteonectin, and coll I around the PHB fibers. Coll I/CS-coated scaffolds with expanded hMSC showed higher values of ALP and calcium than the other combinations. Embroidered PHB scaffolds, coated with extracellular matrix components, provided an adequate environment and, therefore, a template for hMSC which could be differentiated in osteogenic direction.

Collagens and growth factors in heterotopic ossification

Heterotopic Ossification (HO) occurs as a consequence of several diseases and of various forms of trauma. HO is particularly frequent in paraplegic patients with spinal cord lesions. It is obvious that extraskeletal cells are able to differentiate into an osteogenic direction. However, the mechanisms of the induction process of HO and the stimulating agents are not precisely known. A novel tool for studying the ossification process at the level of transcription is the technique of non-radioactive in situ hybridisation. Using digoxigenin labeled cDNA probes we investigated the distribution patterns of types I, II and III collagen mRNAs and the mRNA of Transforming Growth Factor beta 1 (TGF-beta 1) in heterotopic ossification of pressure sores of paraplegic patients. The three collagen mRNAs as well as the TGF-beta 1 mRNA exhibited substantially divergent distribution patterns. Type I (alpha 1) collagen mRNA was predominantly detectable in preosteoblasts, chondroblasts and chondrocytes of the ossification zone. Type II (alpha 1) collagen mRNA was nearly exclusively found in cells of the chondrogenic lineage. Type III (alpha 1) collagen mRNA was detectable at low levels in soft tissue, but was strongly expressed by chondroblasts and chondrocytes of heterotopic cartilage. In contrast expression of TGF-beta 1 mRNA was found in a spatial different distribution pattern in areas of proliferation of mesenchymal tissue and in different stages of ectopic bone formation. As in the case of collagen Type I (alpha 1) and III (alpha 1) mRNAs the maximum of localization of TGF-beta 1 was detected in chondroblastic areas of heterotopic ossification. Taken together our in situ hybridization experiments provide evidence that chondrogenic cells play a central role in the process of HO with a phenotypic alteration in collagen type expression and a strong expression of TGF-beta 1 mRNA. These findings support individual in vivo function for TGF-beta 1 in local cellular regulation of ectopic bone formation.

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3-dimensional collagen sponges.

Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro, but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 x 10(6) cells are loaded, mineralization as measured by 85Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10(7) cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phosphatase positive cells. However; synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.

Osteogenic Stem Cells and the Stromal System of Bone and Marrow

According to current hypothesis, cells of the osteogenic lineage, which includes both osteoblasts and chondroblasts, are derived from a stromal stem cell in the postnatal organism. That there exist osteogenic precursors in association with the soft, fibrous tissue of the marrow stroma is well established. An osteogenic tissue comprised of cartilage and bone is formed when marrow or marrow cell suspensions are cultured in vivo within diffusion chambers. Bone with a functional marrow organ is formed when marrow or marrow cell suspensions are transplanted heterotopically, e.g., under the renal capsule. Cultures of marrow stromal fibroblasts are readily established in vitro from single-cell bone marrow suspensions. Such cultures do not demonstrate overt differentiation in an osteogenic direction in vitro. When transplanted in vivo, however, they differentiate to form cartilage and bone in diffusion chambers and bone with a functional marrow organ when transplanted heterotopically. Single-cell bone marrow suspensions can be cultured in vitro under conditions that facilitate the formation of stromal fibroblast colonies. Circumstantial evidence supports the conclusion that each colony is derived from a single initiating cell termed a colony-forming unit-fibroblastic (CFU-F). A proportion of CFU-F demonstrates extensive proliferative potential both in vitro and in vivo. In vitro the extensive proliferative potential of a subset of CFU-F has been shown to be associated with a capacity for extensive self-renewal. On transplantation in vivo, the progeny of a proportion of CFU-F has been shown to be capable of proliferating and differentiating into all the stromal cell lines necessary for the formation of bone and reconstitution of the hematopoietic inductive microenvironment. These findings provide strong circumstantial evidence to support the hypothesis that there are stem cells present within the marrow stroma that are capable of giving rise to cells of a number of different lineages, including those of the osteogenic lineage (chondroblasts and osteoblasts).

Mineralization in in Vitro Cultures of Rabbit Marrow Stromal Cells

Localized regions of mineralization were found in confluent cultures of rabbit marrow fibroblastic cells. The mineralized tissue developed within clusters of giant fat cells in the spaces between the cells. Investigations with light and electron microscopy demonstrated that in these sites there was some differentiation of the fibroblastic cells in an osteogenic direction, shown by changes to more polygonal shapes, and the synthesis of well-banded collagen similar to that found in bone tissue. Differentiation may be due, in part, to increased cell density in a confined space. Growth of the mineralized tissue was observed in the living cultures with a fluorescence microscope. Electron probe microanalysis confirmed that the mineral formed was hydroxyapatite. Initiating sites of mineralization included membranous vesicular bodies, lipid, and products of cellular degeneration. Once initiated, mineralization appeared to spread rapidly into adjacent collagenous and other structures, suggesting the appearance of a mixture of skeletal-type and dystrophic mineralization.