Cells In Osteogenic Medium Research Articles

Induced pluripotent stem cell-derived smooth muscle cells to study cardiovascular calcification.

Cardiovascular calcification is the lead predictor of cardiovascular events and the top cause of morbidity and mortality worldwide. To date, only invasive surgical options are available to treat cardiovascular calcification despite the growing understanding of underlying pathological mechanisms. Key players in vascular calcification are vascular smooth muscle cells (SMCs), which transform into calcifying SMCs and secrete mineralizing extracellular vesicles that form microcalcifications, subsequently increasing plaque instability and consequential plaque rupture. There is an increasing, practical need for a large scale and inexhaustible source of functional SMCs. Here we describe an induced pluripotent stem cell (iPSC)-derived model of SMCs by differentiating iPSCs toward SMCs to study the pathogenesis of vascular calcification. Specifically, we characterize the proteome during iPSC differentiation to better understand the cellular dynamics during this process. First, we differentiated human iPSCs toward an induced-SMC (iSMC) phenotype in a 10-day protocol. The success of iSMC differentiation was demonstrated through morphological analysis, immunofluorescent staining, flow cytometry, and proteomics characterization. Proteomics was performed throughout the entire differentiation time course to provide a robust, well-defined starting and ending cell population. Proteomics data verified iPSC differentiation to iSMCs, and functional enrichment of proteins on different days showed the key pathways changing during iSMC development. Proteomics comparison with primary human SMCs showed a high correlation with iSMCs. After iSMC differentiation, we initiated calcification in the iSMCs by culturing the cells in osteogenic media for 17 days. Calcification was verified using Alizarin Red S staining and proteomics data analysis. This study presents an inexhaustible source of functional vascular SMCs and calcifying vascular SMCs to create an in vitro model of vascular calcification in osteogenic conditions, with high potential for future applications in cardiovascular calcification research.

Angio-osteogenic capacity of octacalcium phosphate co-precipitated with copper gluconate in rat calvaria critical-sized defect

ABSTRACT The objective of this study is to investigate the effects of octacalcium phosphate (OCP)-induced bone regeneration on angiogenesis regulated by the inclusion of copper ions in OCP in vitro and in vivo. Calcium (Ca)-deficient Cu-OCPs, containing 0.01 wt% Cu (low-Cu-OCP) and 0.12 wt% Cu (high-Cu-OCP), were synthesized with co7pper gluconate salt. The lattice parameters of Cu-OCPs tended to decrease slightly with Cu inclusion, as estimated by Rietveld analysis. Cu ions were released in OCP when the materials were incubated in the medium for human umbilical vein endothelial cells (HUVECs). The solubility of Cu-OCPs, estimated by the degree of supersaturation, was slightly higher than that of the original OCP. Cu-OCP tended to hydrolyze to an apatite structure while maintaining the crystal plate-like morphology when incubated with mesenchymal stem D1 cells in osteogenic media for 14 days. The specimens were characterized by selected area electron diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. Low-Cu-OCP significantly enhanced the HUVEC capillary cross-linking density. D1 cell differentiation was inhibited with the inclusion of Cu, even at low concentrations. The composite of low-Cu-OCP with a gelatin sponge (low-Cu-OCP/Gel) significantly enhanced angiogenesis coupled with bone regeneration when implanted in a rat calvarial critical-sized defect for 4 weeks, compared with the corresponding amount of Cu-containing Gel (Cu/Gel) or OCP/Gel materials through angiography and tissue histomorphometry. These results support the proposition that angiogenesis stimulated by low-Cu-OCP is closely related with enhanced bone regeneration.

Synthesis of N-methylpyridine-chlorofuranformamide analogs as novel OPG up-regulators and inhibitors of RANKL-induced osteoclastogenesis

The OPG/RANKL/RANK pathway is a promising target for the design of therapeutic agents used in the treatment of osteoporosis. E09241 with an N-methylpyridine-chlorofuranformamide structural skeleton was previously identified to decrease bone loss and thus protect against osteoporosis in ovariectomized rats through increasing osteoprotegerin (OPG) expression. In this study, 36 derivatives of E09241 (3a) were prepared. The synthesis, up-regulation of OPG activities, SAR (structure–activity relationship), and cytotoxicity of these compounds are presented. Compounds with good up-regulating OPG activities could inhibit RANKL (the receptor activator of nuclear factor-kappa B ligand)-induced osteoclastogenesis in RAW264.7 cells. Particularly, compounds 3c and 3i1 significantly reduced NFATc1 and MMP-9 protein expression through inhibition of the NF-κB and MAPK pathways in RANKL induced RAW264.7 cells. In addition, compounds 3c and 3v significantly promoted osteoblast differentiation in MC3T3-E1 cells in osteogenic medium, and compounds 3c, 3v, and 3i1 obviously increased OPG protein expression and secretion in MC3T3-E1 cells. Furthermore, the pharmacokinetic profiles, acute toxicity, and hERG K+ channel effects of compounds 3a, 3c, 3e, 3v, and 3i1 were investigated. Taken together, these results indicate that N-methylpyridine-chlorofuranformamide analog 3i1 could serve as a promising lead for the development of new agents for treating osteoporosis.

Aldo-keto reductase family 1 member B induces aortic valve calcification by activating hippo signaling in valvular interstitial cells

AimsCalcific aortic valve disease (CAVD) is a primary cause of cardiovascular mortality; however, its mechanisms are unknown. Currently, no effective pharmacotherapy is available for CAVD. Aldo-keto reductase family 1 member B (Akr1B1) has been identified as a potential therapeutic target for valve interstitial cell calcification. Herein, we hypothesized that inhibition of Akr1B1 can attenuate aortic valve calcification. Methods and resultsNormal and degenerative tricuspid calcific valves from human samples were analyzed by immunoblotting and immunohistochemistry. The results showed significant upregulation of Akr1B1 in CAVD leaflets. Akr1B1 inhibition attenuated calcification of aortic valve interstitial cells in osteogenic medium. In contrast, overexpression of Akr1B1 aggravated calcification in osteogenic medium. Mechanistically, using RNA sequencing (RNAseq), we revealed that Hippo-YAP signaling functions downstream of Akr1B1. Furthermore, we established that the protein level of the Hippo-YAP signaling effector active-YAP had a positive correlation with Akr1B1. Suppression of YAP reversed Akr1B1 overexpression-induced Runx2 upregulation. Moreover, YAP activated the Runx2 promoter through TEAD1 in a manner mediated by ChIP and luciferase reporter systems. Animal experiments showed that the Akr1B1 inhibitor epalrestat attenuated aortic valve calcification induced by a Western diet in LDLR−/− mice. ConclusionThis study demonstrates that inhibition of Akr1B1 can attenuate the degree of calcification both in vitro and in vivo. The Akr1B1 inhibitor epalrestat may be a potential treatment option for CAVD.

Multipotent Mesenchymal Stem Cells Derived from Sheep Bone Marrow: Isolation and Cryopreservation

Sheep are often used as preclinical large animal models for testing the therapeutic potential of multipotent mesenchymal stem cells (MMSCs). A cell culture with characteristics typical for MMSCs was obtained due to optimization of the method of cell isolation from sheep bone marrow (BM) in a Ficoll density gradient. The advantage of using SepMate-15 tube compared to the isolation by the classical method has been demonstrated. The obtained cells demonstrated a strong adhesion to the culture plastic, fibroblast-like morphology, and, being induced in vitro, the ability to differentiate into cells of adipose, bone, and cartilage tissues. Adipogenic differentiation was confirmed on day 14, when adipocytes with lipid vesicles positive for Oil Red O specific staining had been formed. On day 14, specific alkaline phosphatase activity appeared in cells in osteogenic medium. Von Kossa staining revealed the presence of insoluble calcium salts in the intercellular space. Chondrogenic differentiation appeared on day 14 and was accompanied with the formation of multilayer structures with an abandon matrix, in which the isogenic groups that resemble hyaline cartilage lacunae were visualized. On day 21, the cells formed tight microgranules with glycosaminoglycan matrixes. Cells were cryopreserved in three different media at the zero passage. A comparative analysis of cell viability was then performed, which revealed a relatively good cell state (more than 70% of live cells). The MMSCs culture obtained from sheep BM was deposited at two to three passages in the specialized Collection of Somatic Cell Cultures of Agricultural and Industrial Animals at the All-Russian Research Institute of Experimental Veterinary Medicine of Russian Academy of Science and could be used in preclinical studies.

99mTc-Methyl-Diphosphonate Binding to Mineral Deposits in Cultures of Marrow-Derived Mesenchymal Stem Cells in Osteogenic Medium.

The work is notable for describing a highly sensitive, quantitative, and nondestructive method for evaluating the in vitro amount of mineral accompanying different types of osteogenic differentiation of mesenchymal stem cells in a monolayer cell culture. What is so unique and useful about the method is that it has the potential to be used to define the kinetics of the differentiation process, reflected in the mineralization, without destroying the monolayer. Therefore, it remains intact for further experiments.

Glycan Profiling Shows Unvaried N-Glycomes in MSC Clones with Distinct Differentiation Potentials.

Different cell types have different N-glycomes in mammals. This means that cellular differentiation is accompanied by changes in the N-glycan profile. Yet when the N-glycomes of cell types with differing fates diverge is unclear. We have investigated the N-glycan profiles of two different clonal populations of mesenchymal stromal cells (MSCs). One clone (Y101), when differentiated into osteoblasts, showed a marked shift in the glycan profile toward a higher abundance of complex N-glycans and more core fucosylation. Yet chemical inhibition of complex glycan formation during osteogenic differentiation did not prevent the formation of functional osteoblasts. However, the N-glycan profile of another MSC clone (Y202), which cannot differentiate into osteoblasts, was not significantly different from that of the clone that can. Interestingly, incubation of Y202 cells in osteogenic medium caused a similar reduction of oligomannose glycan content in this non-differentiating cell line. Our analysis implies that the N-glycome changes seen upon differentiation do not have direct functional links to the differentiation process. Thus N-glycans may instead be important for self-renewal rather than for cell fate determination.

Incorporation of Fucoidan in β-Tricalcium phosphate-Chitosan scaffold prompts the differentiation of human bone marrow stromal cells into osteogenic lineage.

In our previous study, we reported the fabrication and characterization of a novel tricalcium phosphate-fucoidan-chitosan (TCP-Fu-Ch) biocomposite scaffold. However, the previous report did not show whether the biocomposite scaffold can exhibit osteogenic differentiation of human bone marrow stromal cells in osteogenic media and normal media supplemented with platelet-derived growth factor (PDGF-BB). On day 15, the release of osteocalcin, was significant in the TCP-Fu-Ch scaffold, when compared with that in the TCP-Ch scaffold, and the level of release was approximately 8 and 6 ng/ml in osteogenic and normal media supplemented with PDGF-BB, respectively. Scanning electron microscopy of the TCP-Fu-Ch scaffold demonstrated mineralization and apatite layer formation on day 14, while the addition of PDGF-BB also improved the osteogenic differentiation of the scaffold. An array of gene expression analysis demonstrated that TCP-Fu-Ch scaffold cultured in osteogenic and normal media supplemented with PDGF-BB showed significant improvement in the expression of collagen 1, Runt-related transcription factor 2, osteonectin, bone gamma-carboxyglutamate protein, alkaline phosphatase, and PPA2, but a decline in the expression of integrin. Altogether, the present study demonstrated that fucoidan-incorporated TCP-Ch scaffold could be used in the differentiation of bone marrow stromal cells and can be a potential candidate for the treatment of bone-related ailments through tissue engineering technology.

In-vitro differentiation of adult human bone marrow-derived mesenchymal stem cells into bone-forming cells

Background Bone tissue constitutes one of the most important units of the locomotive system. However, its regenerative capacity remains limited and bone diseases are still major socioeconomic issues. Stem cells have been the subject of much research over the recent years and comprise a promising tool for cellular therapy. Osteoblastic differentiation from human bone marrow-derived mesenchymal stem cells (hBM-MSCs) shows the best regenerative potential; thus, they are an ideal candidate as a source for osteogenic cells. Aim The aim of this study was to evaluate hBM-MSCs histologically to highlight the morphological changes occurring during their osteogenic differentiation. Materials and methods Cells from passage 9 were seeded in complete culture medium. When cells became confluent the osteogenic medium was applied. Osteogenic differentiation was detected histologically on days 7, 14, and 21 using light and electron microscopes. Results Cells in osteogenic medium became cuboidal in shape with extracellular deposits, which stain positively red with Alizarin red stain. Ultrastructurally, they revealed the picture of active secreting cells. Their cytoplasm showed euchromatic nucleus, multiple mitochondria, profiles of rough endoplasmic reticulum, well-developed Golgi complex, and lysosomes. Conclusion The hBM-MSCs represented a source of osteogenic differentiating cells. Moreover, passage 9 secured cells for further studies while maintaining their osteoblastic differentiation. In addition, the study shed light on the ultrastructure of these cells during differentiation, which revealed the characteristics of active secreting cells.

Effect of low-level diode laser on proliferation and osteogenic differentiation of dental pulp stem cells

This study assessed the effect of low-level laser irradiation (LLLI) on the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs). DPSCs were exposed to 810 nm laser light (0.1, 0.2, or 0.3 J cm−2) for 7 d (60 s daily). The negative control group (cells in regular medium) and positive control group (cells in osteogenic medium (OM)) were not lased. One group of cells in OM was irradiated with laser operated at 0.2 J cm−2. Cell viability was evaluated at 24 h and one week after the last day of laser irradiation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Osteogenic differentiation was assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and alizarin Red S staining. Cell proliferation was not affected by laser irradiation at 24 h except in one group (cells in OM exposed to laser at 0.2 J cm−2). However, one week after the last day of laser irradiation, it was significantly increased in groups exposed to laser at 0.1 or 0.2 J cm−2 and decreased in groups containing OM (P < 0.05). Osteoblast marker expression was observed in groups containing OM. LLLI at 0.2 J cm−2 dramatically enhanced cell differentiation. Laser at 0.3 J cm−2 increased bone sialoprotein (BSP) and decreased alkaline phosphatase (ALP). Mineralized nodules were only observed in groups containing OM. Considering these findings, LLLI may be used as a novel approach for preconditioning of DPSCs in vitro prior to bone tissue engineering.

Cobalt chloride supplementation induces stem-cell marker expression and inhibits osteoblastic differentiation in human periodontal ligament cells

ObjectiveLow oxygen tension is one of the crucial factors of the stem-cell niche. However, the long-term hypoxic culture of stem cells is difficult and requires special equipment. In this study, we investigated whether mimicking hypoxia using cobalt chloride (CoCl2) could maintain human periodontal ligament (HPDL) cell stemness. MethodsHPDL cells were treated with either 50 or 100μM CoCl2. Cell proliferation was determined by an MTT assay. The mRNA expression of stem-cell marker and osteogenic associated genes were analyzed by RT-PCR and Real-time PCR. Osteogenic differentiation was determined by assaying alkaline phosphatase activity and in vitro mineralization. ResultsThe results showed that the CoCl2 supplementation had no effect on cell proliferation. CoCl2 treatment increased the mRNA expression of the embryonic stem-cell markers REX1 and OCT4. Culturing HDPL cells in osteogenic medium containing CoCl2 resulted in a decrease in alkaline phosphatase activity, down-regulation of osteogenic associated gene expression, and suppression of mineralization. The use of Apigenin, an HIF-1α inhibitor, indicated that CoCl2 might inhibit osteogenic differentiation through an HIF-1α- dependent mechanism. ConclusionThis study shows that CoCl2 treatment can induce stem-cell marker expression and inhibit the osteoblastic differentiation of HPDL cells. These findings suggest the potential application of CoCl2 for maintaining the stem-cell state in the laboratory.

Enhanced Osteogenic Differentiation with 3D Electrospun Nanofibrous Scaffolds

Developing 3D scaffolds mimicking the nanoscale structure of the native extracellular matrix is important in tissue regeneration. In this study, we aimed to demonstrate the novelty of 3D nanofibrous scaffolds and compare their efficiency with 2D nanofibrous scaffolds. The 2D poly(L-lactic acid)/collagen nanofibrous scaffolds were 2D meshes fabricated by the conventional electrospinning technique, whereas the 3D poly(L-lactic acid)/collagen nanofibrous scaffolds were fabricated by a modified electrospinning technique using a dynamic liquid support system. The morphology, proliferation and differentiation abilities of human mesenchymal stem cells in osteogenic medium on both scaffolds were investigated. Compared with the 2D scaffolds, the 3D scaffolds significantly increased the expression of osteoblastic genes of the stem cells as well as the formation of bone minerals. In addition, the scanning electron microscopic and micro-computed tomographic images showed the dense deposition of bone minerals aligned along the nanofibers of the 3D scaffolds after 14 and 28 days cultured with the mesenchymal stem cells. As such, the 3D electrospun poly(L-lactic acid)/collagen nanofibrous scaffold is a novel bone graft substitute for bone tissue regeneration.

Culture human mesenchymal stem cells with calcium phosphate cement scaffolds for bone repair

Because of its moldability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for craniofacial and orthopedic applications. The objectives of this study were to investigate the response of human mesenchymal stem cells (hMSCs) to a high-strength CPC-chitosan scaffold and to examine cell proliferation and osteogenic differentiation. hMSCs were seeded onto CPC-chitosan composite, CPC control, and tissue culture polystyrene (TCPS). Alkaline phosphatase activity (ALP) and mineralization of hMSCs were measured. CPC-chitosan had a flexural strength (mean + or - SD; n = 5) of (19.5 + or - 1.4) MPa, higher than (8.0 + or - 1.4) MPa of CPC control (p < 0.05). The percentage of live hMSCs on CPC-chitosan was (90.5 + or - 1.3)% at 8 days, matching (90.7 + or - 3.8)% of CPC control (p > 0.1). The CPC-chitosan surface area covered by the attached hMSCs increased from (51 + or - 11)% at 1 day to (90 + or - 4)% at 8 days (p < 0.05), matching those of CPC control (p > 0.1). Hence, the CPC strength was significantly increased via chitosan without compromising the hMSC response. At 8 days, there was a significant increase in ALP of cells in osteogenic media (10.99 + or - 0.93) [(mM pNpp/min)/(microg DNA)] versus control media (3.62 + or - 0.40) (p < 0.05). hMSCs in osteogenic media exhibited greater mineralization area of (47.5 + or - 19.7)% compared with (6.1 + or - 2.3)% in control medium on TCPS (p < 0.05). In conclusion, hMSCs showed excellent attachment and viability on the strong and tough CPC-chitosan scaffold, matching the hMSC response on CPC control. hMSCs were successfully differentiated down the osteogenic lineage. Hence, the strong, in situ hardening CPC-chitosan scaffold may be useful as a moderate load-bearing vehicle to deliver hMSCs for maxillofacial and orthopedic bone tissue engineering.

Bone Morphogenetic Protein Receptor in the Osteogenic Differentiation of Rat Bone Marrow Stromal Cells

PurposeSeveral signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of bone marrow stromal cells (BMSCs). Bone morphogenetic protein (BMP) signaling has important effects on the process of skeletogenesis. In the present study, we tested the role of bone morphogenetic protein receptor (BMPR) in the osteogenic differentiation of rat bone marrow stromal cells in osteogenic medium (OM) with or without BMP-2.Materials and MethodsBMSCs were harvested from rats and cultured in OM containing dexamethasone, β-glycerophosphate, and ascorbic acid, with or without BMP-2 in order to induce osteogenic differentiation. The alkaline phosphatase (ALP) activity assay and von kossa staining were used to assess the osteogenic differentiation of the BMSCs. BMPR mRNA expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR).ResultsThe BMSCs that underwent osteogenic differentiation in OM showed a higher level of ALP activity and matrix mineralization. BMP-2 alone induced a low level of ALP activity and matrix mineralization in BMSCs, but enhanced the osteogenic differentiation of BMSCs when combined with OM. The OM significantly induced the expression of type IA receptor of BMPR (BMPRIA) and type II receptor of BMPR (BMPRII) in BMSCs after three days of stimulation, while BMP-2 significantly induced BMPRIA and BMPRII in BMSCs after nine or six days of stimulation, respectively.ConclusionBMSCs commit to osteoblastic differentiation in OM, which is enhanced by BMP-2. In addition, BMP signaling through BMPRIA and BMPRII regulates the osteogenic differentiation of rat BMSCs in OM with or without BMP-2.

A Functional RNAi Screen for Runx2-Regulated Genes Associated With Ectopic Bone Formation in Human Spinal Ligaments

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic ossification in the spinal ligaments, which enlarges with time and compresses the spinal cord, resulting in serious neurological symptoms. We previously reported that Runx2 expression was enhanced in spinal ligament cells from OPLL patients (OPLL cells). To clarify genes regulated by Runx2, Runx2 expression was first enhanced by culturing primary OPLL cells in osteogenic medium (OS induction) and then inhibited by siRNAs targeted to Runx2. DNA microarray demonstrated that in addition to chondrogenic factors such as connective tissue growth factor and cartilage oligomeric matrix protein, angiopoietin-1 was also significantly increased by OS induction and decreased by siRNAs for Runx2 in OPLL cells, suggesting that these genes are regulated by Runx2. However, these changes were not observed in non-OPLL control cells (from cervical spondylotic myelopathy patients). Furthermore, Runx2 was not decreased by siRNAs for angiopoietin-1. OS induction and RNAi inhibition of angiopoietin-1 expression was also observed in osteoblasts. Both siRNAs for Runx2 and angiopoietin-1 completely inhibited aggrecan-1 expression. These results suggest that angiopoietin-1 is downstream of Runx2 in both OPLL primary cells and osteoblasts. Angiopoietin-1 may play an important role in ectopic ossification.

Cotton wool‐like nanocomposite biomaterials prepared by electrospinning: In vitro bioactivity and osteogenic differentiation of human mesenchymal stem cells

The present study evaluates the in vitro biomedical performance of an electrospun, flexible, and cotton wool-like poly(lactide-co-glycolide) (PLGA)/amorphous tricalcium phosphate (ATCP) nanocomposite. Experiments on in vitro biomineralization, applicability in model defects and a cell culture study with human mesenchymal stem cells (hMSC) allowed assessing the application of the material for potential use as a bone graft. Scaffolds with different flame made ATCP nanoparticle loadings were prepared by electrospinning of a PLGA-based composite. Immersion in simulated body fluid showed significant deposition of a hydroxyapatite layer only on the surface of ATCP doped PLGA (up to 175% mass gain within 15 days for PLGA/ATCP 60:40). Proliferation and osteogenic differentiation of hMSC on different nanocomposites were assessed by incubating cells in osteogenic medium for 4 weeks. Proper adhesion and an unaffected morphology of the cells were observed by confocal laser scanning microscopy for all samples. Fluorometric quantification of dsDNA and analysis of ALP activity revealed no significant difference between the tested scaffolds and excluded any acute cytotoxic effects of the nanoparticles. The osteocalcin content for all scaffolds was 0.12-0.19 ng/ng DNA confirming osteogenic differentiation of human mesenchymal stem cells on these flexible bone implants.

Pre‐culture period of mesenchymal stem cells in osteogenic media influences their in vivo bone forming potential

The objective of this study was to investigate if the in vitro pre-culture period in osteogenic media of rat mesenchymal stem cells (MSCs), influences their ability to regenerate bone when implanted in a critical size cranial defect. MSCs were harvested from the bone marrow of 6-8 weeks old male Fisher rats and expanded in vitro in osteogenic media for different time periods (4, 10, and 16 days) in tissue culture plates (TCP), seeded on sintered titanium fiber meshes without the extracellular matrix (ECM) generated in vitro, and implanted in the rat cranium after 12 h. Thirty two adult Fisher rats received the implants, divided in four groups. Three groups were implanted with cells cultured for 4, 10, or 16 days in osteogenic media and at that time their alkaline phosphatase activity and mineral deposition denoted that they were at different stages of their osteoblastic maturation (undifferentiated MSC, committed, and mature Osteoblasts, respectively). MSCs cultured without osteogenic media for 6 days were used as controls. The constructs were retrieved 4 weeks later and processed for histomorphometric analysis. Implants seeded with cells that have been cultured with osteogenic media for only 4 days revealed the highest bone formation. The lowest bone formation was obtained with the implants seeded with MSCs cultured for 16 days in the presence of osteogenic media. The results of this study suggested that the in vitro pre-culture period of MSCs is a critical factor for their ability to regenerate bone when implanted to an orthotopic site.

Evidence That Osteogenic Progenitor Cells in the Human Tunica Albuginea May Originate from Stem Cells: Implications for Peyronie Disease1

Tissue ossification in Peyronie disease (commonly known as Peyronie's disease [PD]), a localized fibrotic lesion within the tunica albuginea (TA) of the penis, may result from osteogenic differentiation of fibroblasts, myofibroblasts, and/or adult stem cells in the TA, and may be triggered by chronic inflammation, oxidative stress, and profibrotic factors like transforming growth factor beta 1 (TGFB1). In this study, we have investigated whether cultures of cells from normal TA and PD plaques undergo osteogenesis, express markers for stem cells, and originate other cell lineages via processes modulated by TGFB1. We found that TA and PD cells in osteogenic medium (OM) expressed osteogenic markers, alkaline phosphatase, and osteopontin and underwent calcification. PD cells, but not TA cells, formed foci in soft agar that were positive for alkaline phosphatase and calcification and expressed the mRNAs for osteoblast-specific factors pleiotrophin and periostin and bone morphogenic protein 2. Both cultures expressed stem cell marker CD34 antigen but not protein tyrosine phosphatase, receptor type c. TA and PD cells expressed smooth-muscle cell markers smoothelin and transgelin. None of the cultures underwent adipogenesis in adipogenic medium. Incubation with TGFB1 increased osteogenesis and myofibroblast differentiation and reduced CD34 antigen expression in both cultures. TA and PD cells modulated the differentiation of the multipotent C3H 10T(1/2) cells in dual cultures, into osteoblasts and myofibroblasts. In conclusion, both TA and PD cultures contain cells, presumably stem cells, that undergo osteogenic and myofibroblast differentiation, and may induce these processes by paracrine interactions. This may explain progression of fibrosis in the PD plaque and its eventual calcification.

Influence of Emdogain&lt;sup&gt;®&lt;/sup&gt; Treatment onto K&lt;sub&gt;2&lt;/sub&gt;O Incorporated Calcium Metaphosphate on Osteogenic Activation

As a part of the efforts to develop a suitable scaffold optimizing bone regeneration that has similar physical properties to bone, we modified calcium metaphosphate (CMP) ceramics with K2O and evaluated their efficiency as a scaffold for tissue engineered bone tissue regeneration. Macroporous CMP ceramics modified by incorporation of 5% K2O to improve biodegradability were prepared to have 250 and 450 µm average pore sizes, respectively. The modified CMP ceramics were cultured with mouse primary calvarial osteoblastic cells in osteogenic media for 2 weeks and these cell-CMP ceramic constructs with or without Emdogain treatment were implanted in the SCID mice subcutaneous pouches. After 1, 2, and 3 weeks, the degree of ectopic bone formation was evaluated. The modified macroporous CMP ceramic-cell constructs treated with Emdogain induced ectopic bone formation, whereas the modified CMP ceramic-cell constructs without Emdogain treatment induced no ectopic bone formation. This result suggests that the Emdogain treatment on cell-scaffold constructs for tissue engineered bone regeneration may be effective for osteogenic activation of attached cells.

Bone induction by BMP-2 transduced stem cells derived from human fat

Purpose: We have isolated pluripotent mesenchymal progenitor cells in large numbers from liposuction aspirates (processed lipoaspirate cells or PLAs). This study examines the osteogenic potential of PLAs and bone marrow aspirate cells (BMAs), when exposed to either recombinant human bone morphogenetic protein (BMP)-2 (rh- BMP-2) or adenovirus containing BMP-2 cDNA (Ad- BMP-2). Methods: Liposuction aspirates underwent proteolytic digestion to obtain PLAs. After exposure to exogenous rh- BMP-2 or Ad- BMP-2 for four or seven days, PLAs and BMAs were assessed by histochemistry, spectrophotometry and RT-PCR. Western blotting and ELISA confirmed BMP gene transduction. Results were compared to osteoblasts and cells in osteogenic media only. PLA-Ad- BMP-2 cells were seeded on matrices and implanted in the hind limbs of SCID mice. Results: Analysis of quantified bone precursor assays including extracellular ALP histomorphometry, intracellular ALP spectrophotometry, and calcified extracellular matrix (von Kossa) histomorphometry revealed that PLAs treated with exogenous rh- BMP-2 or transduced with a BMP-2 containing adenovirus (PLA-Ad- BMP-2) produced more bone precursors than osteoblasts ( p=0.001). PLAs treated with exogenous rh- BMP-2 or PLA-Ad- BMP-2 also produced more bone precursors than BMAs ( p=0.001), except for day 7 ALP histomorphometry ( p=0.343). ELISA confirmed successful BMP-2 production by both progenitor cell groups transduced with Ad- BMP-2. H&E sections from collagen I matrices seeded with PLA-Ad- BMP-2 cells confirmed bone formation at six weeks. Conclusions: Liposuction aspirates contain PLAs that can be transfected with the BMP-2 gene, with rapid induction into the osteoblast phenotype at a rate comparable to rh- BMP-2 and osteoblast groups. Transduced PLAs produce more bone precursors with faster onset of calcified extracellular matrix than transduced BMAs. PLAs may be an ideal source of mesenchyme-lineage stem cells for gene therapy and tissue engineering.