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1.
Biomaterials ; 28(6): 1036-47, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17081601

RESUMEN

Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 microm for expansion and differentiation of hMSCs. We cultivated the hMSC statically or in spinner flasks for 1, 7, 14 and 21 days and found that the 200-microm pore scaffolds exhibited a faster rate of osteogenic differentiation than did the 500-microm pore scaffolds as shown by an alkaline phosphatase activity assay and real-time reverse transcriptase polymerase chain reaction for 10 osteogenic markers. The 500-microm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by DNA content, scanning electron microscopy and fluorescence microscopy). Thus the porosity of a 3D microporous biomaterial may be used to steer hMSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small to intermediate size 3D scaffolds.


Asunto(s)
Cerámica/química , Hidroxiapatitas/química , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Osteoblastos/citología , Osteoblastos/fisiología , Osteogénesis/fisiología , Ingeniería de Tejidos/métodos , Materiales Biocompatibles/química , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Línea Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Humanos , Ensayo de Materiales , Oseointegración/fisiología
2.
J Biomed Mater Res A ; 79(3): 552-6, 2006 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-16788968

RESUMEN

Since its osteoinductive capacity has been established, demineralized bone matrix is considered a suitable alternative to bone autograft in the healing of osseous defects. The mechanisms of bone formation induction are still not fully understood. In this study we assessed the effects of a dispersion of bovine bone extract COLLOSS (BPE) with regard to proliferation and differentiation of a human mesenchymal stem cell line overexpressing human telomerase reverse transcriptase (hMSC-TERT). Proliferation rate was determined by (3)H-thymidine incorporation. The differentiation of hMSC-TERT cells to osteoblastic cells was assessed by means of measuring alkaline phosphatase activity and collagen synthesis in vitro. Both undifferentiated and osteoblast-differentiated hMSC-TERT cells were investigated for response to BPE. The metabolic responses to BPE were compared to unstimulated cells and cells stimulated with bovine collagen (COL). Undifferentiated hMSC-TERT cells responded to BPE with increased proliferation and decreased alkaline phosphatase activity. Osteoblastic differentiated hMSC-TERT cells had a diminished proliferative response and an increased alkaline phosphatase activity and collagen synthesis. Our study demonstrated significant metabolic effects of BPE on hMSC-TERT cells, which were highly dependant on the differentiated state of the cells.


Asunto(s)
Huesos/metabolismo , Diferenciación Celular/efectos de los fármacos , Extractos Celulares/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/biosíntesis , Humanos , Células Madre Mesenquimatosas/metabolismo
3.
Adv Exp Med Biol ; 585: 31-48, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17120775

RESUMEN

INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors and cytokines can induce and maintain lineage-specific differentiation. Due to anatomical and physiological similarities to humans, porcine research models have been proven valuable for the preclinical testing of tissue engineering protocols in large animals. The aim of this study was to evaluate optimized viral and non-viral ex vivo gene delivery systems with respect to gene transfer efficiency, maintenance of transgene expression, and safety issues using primary porcine MSCs as target cells. MATERIALS AND METHODS: MSCs were purified from bone marrow aspirates from the proximal tibiae of four 3-month-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno-associated virus (rAAV)-mediated and retroviral gene delivery. Each method for gene delivery was optimized. Gene transfer efficiency was compared on the basis of eGFP expression as assessed by fluorescence microscopy and fluorescence-activated flow cytometry. BMP-2 gene expression and osteogenic differentiation were evaluated by realtime quantitative RT-PCR and histochemical detection of alkaline phosphatase activity, respectively. RESULTS: Non-viral gene delivery methods resulted in transient eGFP expression by less than 2% of the cells. Using high titer rAAV-based vector up to 90% of the cells were transiently transduced. The efficiency of rAAV-mediated gene delivery was proportional to the rAAV vector titer applied. Retroviral gene delivery resulted in long-term transgene expression of porcine MSCs. A 26-fold increase in percentage of eGFP expressing cells (1.7%+/-0.2% versus 44.1% +/-5.0%, mean +/-SD) and a 68-fold increase in mean fluorescence intensity (327.4+/-56.6 versus 4.8+/-1.3) was observed by centrifugation of retroviral particles onto the target cell layer. Porcine MSCs that were BMP-2 transduced by optimized retroviral gene delivery demonstrated a significant increase in BMP-2 gene expression and showed increased osteogenic differentiation. Retrovirally transduced porcine MSCs were furthermore tested free of replication-competent viruses. DISCUSSION: The non-viral gene transfer methods applied were significantly less efficient compared to the viral methods tested. However, due to advantages with respect to safety issues and ease of handling, improvement of non-viral gene delivery to primary MSCs deserves further attention. The high efficiency of rAAV-mediated gene delivery observed at high titers can be explained by the ability of rAAV vector to transduce nondividing cells and by its tropism towards porcine MSCs. rAAV-mediated gene delivery resulted in transient transgene expression due to lack of stable AAV genome integration. MLV-mediated retroviral gene delivery can be considered a safe method for long-term transgene expression by porcine MSCs, and is therefore particularly attractive for advanced tissue engineering strategies requiring extended transgene expression.


Asunto(s)
Técnicas de Transferencia de Gen , Células Madre Mesenquimatosas/citología , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Virus de la Leucemia Murina/metabolismo , Ratones , Células 3T3 NIH , Retroviridae/metabolismo , Porcinos , Tibia/patología , Ingeniería de Tejidos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Transgenes
4.
Acta Biomater ; 18: 21-9, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25686557

RESUMEN

Bone metastasis is one of the leading causes of death in breast cancer patients. The current treatment is performed as a palliative therapy and the adverse side effects can compromise the patients' quality of life. In order to both effectively treat bone metastasis and avoid the limitation of current strategies, we have invented a drug eluting scaffold with clay matrix release doxorubicin (DESCLAYMR_DOX) to mechanically support the structure after resecting the metastatic tissue while also releasing the anticancer drug doxorubicin which supplements growth inhibition and elimination of the remaining tumor cells. We have previously demonstrated that this device has the capacity to regenerate the bone and provide sustained release of the anticancer drug in vitro. In this study, we focus on the ability of the device to inhibit cancer cell growth in vitro as well as in vivo. Drug-release kinetics was investigated and the cell viability test showed that the tumor inhibitory effect is sustained for up to 4weeks in vitro. Subcutaneous implantation of DESCLAYMR_DOX in athymic mice resulted in significant growth inhibition of human tumor xenografts of breast origin and decelerated multi-organ metastasis formation. Fluorescence images, visualizing doxorubicin, showed a sustained drug release from the DESCLAYMR device in vivo. Furthermore, local use of DESCLAYMR_DOX implantation reduced the incidence of doxorubicin's cardio-toxicity. These results suggest that DESCLAYMR_DOX can be used in reconstructive surgery to support the structure after bone tumor resection and facilitate a sustained release of anticancer drugs in order to prevent tumor recurrence.


Asunto(s)
Neoplasias/patología , Ingeniería de Tejidos/instrumentación , Animales , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxorrubicina/farmacología , Femenino , Fibrosis , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Soluciones , Carga Tumoral/efectos de los fármacos
5.
Biomaterials ; 25(23): 5375-85, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15130722

RESUMEN

In the interest of optimizing osteogenesis in in vitro, the present study sought to determine how porcine bone marrow stromal cell (BMSc) would respond to different concentrations of hyaluronan (HY) and its different combinations with dexamethasone (Dex) and recombinant human bone morphogenic protein-2 (rhBMP-2). Cellular proliferation was determined by 3H-thymidine incorporation into DNA at both Days 2 and 7 when BMSc was cultivated with HY at concentrations of 0, 0.5, 1.0, 2.0 and 4.0 mg/ml. HY accelerated cellular proliferation when compared with cultures in the absence of HY at both Days 2 and 7. BMSc proliferation under the high HY concentration of 4 mg/ml was significantly higher than under the other, lower HY concentrations of 0.5, 1.0 and 2.0 mg/ml. When BMSc were cultivated under HY at concentrations of 0, 1.0 and 4.0 mg/ml and its 12 combinations with rhBMP-2 at concentrations of 0 and 10 ng/ml and Dex (+, -) at both Days 2 and 7, cellular responses were examined by 3H-thymidine incorporation into DNA, cellular alkaline phosphatase (ALP) activity, and pro-collagen type I C-terminal propeptide production. HY accelerated cellular proliferation irrespective of the presence of Dex and rhBMP-2. HY increased expression of ALP activity at Day 7, whereas had inhibitory effect at Day 2. HY and Dex showed an interaction on expression of ALP acitivity irrespective of the HY dose by Day 7. Collagen synthesis was inhibited by HY irrespective of the presence of other factors at both Days 2 and 7. When BMSc were cultivated with HY of 4.0 mg/ml alone, its combinations with Dex (+) and 10 ng/ml rhBMP-2, and with DMEM/FBS alone, expression of bone-related marker genes was evaluated by real-time reverse transcription-polymerase chain reaction (Real-time RT-PCR) analysis. Osteocalcin was up-regulated under both rhBMP-2 and HY-Dex-rhBMP-2 at Day 2, as also under 4 mg/ml HY, Dex, HY-Dex, Dex-rhBMP-2, and HY-Dex-rhBMP-2 by Day 7. Type 1alpha1 collagen was induced by rhBMP-2 on Day 2, and by Dex-rhBMP-2 on Day 7. Osteonectin and type X collagen was only marginally induced by HY at Day 2. Type 1alpha1 collagen and type X collagen were down-regulated in the presence of 4 mg/ml HY by Day 7. These results suggest that HY stimulates BMSc proliferation, osteocalcin gene expression, and a secretion of enzymes such as that of ALP activity in vitro. More importantly, HY can interact with Dex and rhBMP-2 to generate direct and specific cellular effects, which could be of major importance in bone tissue engineering.


Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Proteínas Morfogenéticas Óseas/farmacología , Dexametasona/farmacología , Glicoproteínas/efectos de los fármacos , Ácido Hialurónico/farmacología , Ingeniería de Tejidos/métodos , Factor de Crecimiento Transformador beta/farmacología , Animales , Células de la Médula Ósea/citología , Proteína Morfogenética Ósea 2 , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/fisiología , Porcinos
6.
Biores Open Access ; 3(6): 278-85, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25469313

RESUMEN

Clinical trials using bone morphogenetic protein-2 (BMP2) for bone reconstruction have shown promising results. However, the relatively high concentration needed to be effective raises concerns for efficacy and safety. The aim of this study was to investigate the osteogenic effect of an alternative treatment strategy in which human bone marrow-derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with 10 and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured in osteogenic medium. The effects of preconditioning were analyzed with regard to proliferation and expression of osteogenic markers at both gene and protein level. The results were compared to those from cultures with continuous BMP2 stimulation. A significant increase in proliferation was seen with both precondition and continuous stimulation with BMP2, with no difference between the treatments. Preconditioning with BMP2 significantly increased gene expression of RUNX2, COLI, ALP, and OC, and protein levels of COLI and ALP. This was not found with continuous stimulation. The role of preconditioning with BMP2 in osteogenesis was validated by findings of increased gene expression of SMAD1 and an increase in dual phosphorylation of ser 463 and ser 465 in the SMAD 1/5/8 pathway. We concluded that preconditioning hMSCs with BMP2 stimulates osteogenesis: proliferation with matrix secretion and matrix maturation of hMSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could be avoided.

7.
Stem Cell Rev Rep ; 10(1): 69-78, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24052411

RESUMEN

Erythropoietin (EPO) is a pleiotropic growth factor. Of interest for skeletal tissue engineering, the non-hematopoietic capabilities of EPO include its osteogenic and angiogenic potencies. The main aim of this study was to investigate the dose-response relationship and determine the lowest effective dose of EPO that reliably increases the osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Additional aims were to elucidate the surface receptors and to investigate the role of the intracellular signaling pathways by blocking the mammalian target of rapamycin (mTOR), Jak-2 protein tyrosine kinase (JAK2), and phosphoinositide 3-kinases (PI3K). The primary outcome measures were two mineralization assays, Arsenazo III and alizarin red, applied after 10, 14, and 21 days. Moreover, alkaline phosphatase activity, cell number, and cell viability were determined after 2 and 7 days. A proportional dose-response relationship was observed. In vivo, the lowest effective dose of 20 IU/ml should be used for further research to accommodate safety concerns about adverse effects. Ex vivo, the most effective dose of 100 IU/ml could facilitate vascularization and bone ingrowth in cell-based scaffolds. The expression of non-hematopoietic receptors EPOR and CD131 was documented, and EPO triggered all three examined intracellular pathways. Future studies of the efficacy of EPO in cell-based tissue engineering can benefit from our findings.


Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/metabolismo , Eritropoyetina/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/efectos de los fármacos , Receptores de Eritropoyetina/metabolismo , Transducción de Señal/efectos de los fármacos , Androstadienos/farmacología , Células Cultivadas , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Células Madre Mesenquimatosas/citología , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Sirolimus/farmacología , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Tirfostinos/farmacología , Wortmanina
8.
Stem Cell Rev Rep ; 9(2): 132-9, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23135703

RESUMEN

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 28 days. 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 14 days (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.


Asunto(s)
Condrocitos/efectos de los fármacos , Medios de Cultivo/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteocitos/efectos de los fármacos , Fenolsulfonftaleína/farmacología , Agrecanos/antagonistas & inhibidores , Agrecanos/genética , Agrecanos/metabolismo , Fosfatasa Alcalina/metabolismo , Calcio/metabolismo , Diferenciación Celular , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Colágeno Tipo II/antagonistas & inhibidores , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo X/antagonistas & inhibidores , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Medios de Cultivo/química , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteocitos/citología , Osteocitos/metabolismo , Proteoglicanos/biosíntesis , Factor de Transcripción SOX9/antagonistas & inhibidores , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Adulto Joven
9.
J Biomed Mater Res A ; 97(3): 251-63, 2011 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-21442726

RESUMEN

Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 µm or 500 µm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 µm constructs as compared with 200 µm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 µm pores compared with 500 µm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific requirements of the scaffolds and cells, especially when natural biomaterials with varying morphology are used.


Asunto(s)
Cerámica/química , Hidroxiapatitas/química , Células Madre Mesenquimatosas/citología , Osteogénesis , Perfusión/instrumentación , Ingeniería de Tejidos/instrumentación , Andamios del Tejido/química , Adulto , Fosfatasa Alcalina/metabolismo , Células de la Médula Ósea/citología , Supervivencia Celular , Células Cultivadas , Expresión Génica , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Porosidad
10.
Acta Biomater ; 7(5): 2244-55, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21195810

RESUMEN

It is of high clinical relevance in bone tissue engineering that scaffolds promote a high seeding efficiency of cells capable of osteogenic differentiation, such as human bone marrow-derived mesenchymal stem cells (hMSCs). We evaluated the effects of a novel polycaprolactone (PCL) scaffold on hMSC seeding efficiency, proliferation, distribution and differentiation. Porous PCL meshes prepared by fused deposition modeling (FDM) were embedded in matrix of hyaluronic acid, methylated collagen and terpolymer via polyelectrolyte complex coacervation. Scaffolds were cultured statically and dynamically in osteogenic stimulation medium for up to 28 days. Compared to naked PCL scaffolds, embedded scaffolds provided a higher cell seeding efficiency (t-test, P<0.05), a more homogeneous cell distribution and more osteogenically differentiated cells, verified by a more pronounced gene expression of the bone markers alkaline phosphatase, osteocalcin, bone sialoprotein I and bone sialoprotein II. Dynamic culture resulted in higher amounts of DNA (day 14 and day 21) and calcium (day 21 and day 28), compared to static culture. Dynamic culture and the embedding synergistically enhanced the calcium deposition of hMSC on day 21 and day 28. This in vitro study provides evidence that hybrid scaffolds made from natural and synthetic polymers improve cellular seeding efficiency, proliferation, distribution and osteogenic differentiation.


Asunto(s)
Huesos/fisiología , Matriz Extracelular/metabolismo , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Fosfatasa Alcalina/metabolismo , Biomarcadores/metabolismo , Huesos/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Calcio/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Condrogénesis/genética , ADN/metabolismo , Matriz Extracelular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Microscopía Confocal , Microscopía Electrónica de Rastreo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Poliésteres/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coloración y Etiquetado , Telomerasa/metabolismo
11.
J Biomed Mater Res A ; 89(1): 96-107, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18431785

RESUMEN

Ex vivo engineering of autologous bone tissue as an alternative to bone grafting is a major clinical need. In the present study, we evaluated the effect of 3-D dynamic spinner flask culture on the proliferation, distribution, and differentiation of human mesenchymal stem cells (MSCs). Immortalized human MSCs were cultured on porous 75:25 PLGA scaffolds for up to 3 weeks. Dynamically cultured cell/scaffold constructs demonstrated a 20% increase in DNA content (21 days), enhanced ALP specific activity (7 days and 21 days), a more than tenfold higher Ca2+ content (21 days), and significantly increased transcript levels of early osteogenesis markers (e.g., COL1A1, BMP2, RUNX-2) as compared with static culture. Despite the formation of a dense superficial cell layer, markedly increased cell ingrowth was observed by fluorescence microscopy on day 21. Furthermore, increased extracellular matrix deposition was visualized by scanning electron microscopy after 1 and 3 weeks of dynamic culture. The observed increased ingrowth and osteogenic differentiation of 3-D dynamically cultured human MSCs can be explained by generation of fluid shear stress and enhanced mass transport to the interior of the scaffold mimicking the native microenvironment of bone cells. This study provides evidence for the effectiveness of dynamic culture of human MSCs during the initial phase of ex vivo osteogenesis.


Asunto(s)
Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Proliferación Celular , Células Madre Mesenquimatosas , Osteogénesis/fisiología , Fosfatasa Alcalina/metabolismo , Biomarcadores/metabolismo , Regeneración Ósea/fisiología , Calcio/metabolismo , Expresión Génica , Humanos , Ácido Láctico/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Ácido Poliglicólico/metabolismo , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Andamios del Tejido
12.
J Ethnopharmacol ; 125(1): 75-82, 2009 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-19549563

RESUMEN

AIM OF THE STUDY: To investigate the effects of a traditional Chinese medicine (TCM) formula extract, named as ZD-I, on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro. MATERIALS AND METHODS: When hMSCs cultivated in the basal medium with ZD-I, cell viability was assessed by MTT assay and cellular proliferation was assessed by SYBR green I assay. The effects of ZD-I on osteogenic differentiation of hMSCs were assessed by alkaline phosphatase (ALP) activity, mineralization assay and real-time RT-PCR. RESULTS: ZD-I (0.78-100 microg/ml) was non-cytotoxic. The 50% inhibitory concentration (IC50) of hMSCs was 200 microg/ml. ZD-I (0.78-50 microg/ml) stimulated the proliferation of hMSCs. ZD-I did not change ALP activity of hMSCs cultivated in osteogenic medium in the early stage (4 and 7 days), but ZD-I inhibited the mineralization of hMSCs through down-regulation of several osteogenic markers (e.g. osteocalcin, bone morphogenetic protein 2 and osteopontin) in the late stage. CONCLUSIONS: ZD-I stimulate cellular proliferation and decrease the bone mineral deposition of hMSCs. These results suggest ZD-I may play an important therapeutic role in osteoarthritic patients by improving proliferative capacity of hMSCs and inhibiting the mineralization of hMSCs.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medicina Tradicional China , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Extractos Vegetales/farmacología , Fosfatasa Alcalina/metabolismo , Medios de Cultivo , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Osteoblastos/citología , Osteoblastos/enzimología
13.
J Biomed Mater Res A ; 86(2): 448-58, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17975813

RESUMEN

Metallic implants are widely used in orthopedic surgery and dentistry. Durable osseous fixation of an implant requires that osteoprogenitor cells attach and adhere to the implant, proliferate, differentiate into osteoblasts, and produce mineralized matrix. In the present study, we investigated the interactions between human mesenchymal stem cells (MSCs) and smooth surfaces of titanium (Ti), tantalum (Ta), and chromium (Cr). Mean cellular area was quantified using fluorescence microscopy (4 h). Cellular proliferation was assessed by (3)H-thymidine incorporation and methylene blue cell counting assays (4 days). Osteogenic differentiation response was quantified by cell-specific alkaline phosphatase activity (ALP) assay (4 days), expression analysis of bone-related genes (4 days), and mineralization assay (21 days). Undifferentiated and osteogenically stimulated MSCs cultured on the different surfaces showed the same tendencies for proliferation and differentiation. MSCs exposed to Ti surfaces demonstrated enhanced proliferation compared with Ta and Cr surfaces. Cultivation of MSCs on Ta surfaces resulted in significantly increased mean cellular area and cell-specific ALP activity compared with the other surfaces tested. Cells cultured on Cr demonstrated reduced spreading and proliferation. In conclusion, Ta metal, as an alternative for Ti, can be considered as a promising biocompatible material, whereas further studies are needed to fully understand the role of Cr and its alloys in bone implants.


Asunto(s)
Materiales Biocompatibles/química , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Forma de la Célula , Cromo , Implantes Dentales/normas , Humanos , Prótesis Articulares/normas , Osteogénesis , Tantalio , Titanio
14.
Acta Orthop ; 76(1): 115-21, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15788319

RESUMEN

BACKGROUND: Cytokines released from intervertebral discs cultured in vitro have not been profiled, and the effect of these cytokines on human bone marrow stem cells is yet to be studied. MATERIALS AND METHODS: Intervertebral discs from 14 patients who had undergone spinal fusion surgery were cultured separately in vitro. Conditioned media were collected after 48 and 96 h of culture in serum-free Minimum Essential Medium (MEM). Profiling of the cytokines was conducted using pooled media. Conditioned medium from each patient was also tested in human bone marrow stem cell culture, and incorporation of alkaline phosphatase and 3H-thymidine incorporation was evaluated. RESULTS: Of the 18 cytokines screened, 12 were found to be positive, but only eotaxin, IP-10, Rantes IL-6 and IL-8 seemed to be present at high levels. There was a close correlation between IL-6 and IL-8 levels in the medium (R = 0.90, p < 0.001). When the conditioned media were added to human bone marrow stem cell cultures, cellular proliferation was stimulated (p = 0.02), but alkaline phosphatase activity remained unchanged. Cellular proliferation correlated negatively with IL-6 levels (R = -0.44, p = 0.04). INTERPRETATION: Intervertebral discs secrete certain cytokines into the medium when cultured in vitro, and conditioned media from cultured intervertebral discs stimulate proliferation of bone marrow stem cells.


Asunto(s)
Citocinas/metabolismo , Disco Intervertebral/citología , Adolescente , Adulto , Fosfatasa Alcalina/metabolismo , Células de la Médula Ósea/citología , División Celular , Células Cultivadas , Medios de Cultivo/análisis , Citocinas/análisis , Femenino , Humanos , Interleucinas/análisis , Interleucinas/metabolismo , Masculino , Células Madre/citología
15.
APMIS Suppl ; (109): 127-32, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-12874964

RESUMEN

The aim of this study was to investigate capability of cell attachment and ectopic bone formation in pigs after either ex vivo transplantation and expansion of bone marrow stem cells (BMSc) into three-dimensional porous tantalum, or porous tantalum supplemented with BMSc. After 24 hours incubation, cells adhering to the porous tantalum discs were quantified by means of scintillation counting of 3H-thymidine-labeled cells. After 7 days of incubation, the cell-loaded porous tantalum discs were harvested for histological analysis or implanted in the infrasternal muscle; an empty disc and disc implanted immediately after cell loading served as controls. All implants were taken out after 8 weeks of implantation and histological examination was performed. The results of in vitro cell attachment to the porous tantalum discs were not improved significantly with gelatin, collagen or fibronectin coatings. Histological analysis of cell loaded discs in vitro demonstrated viable BMSc within the 3-D tantalum structure. In vivo bone induction was demonstrated when the porous tantalum discs were cultured with BMSc. Our findings indicated that porous tantalum was suitable for cell attachment, and ectopic bone formation in pigs was achieved by means of BMSc cultured with porous tantalum. The present study suggests that cell-mediated hard bone tissue repair technology makes it possible to prefabricate autologous BMSc into three-dimensional trabecular metal in order to engineer bone tissue.


Asunto(s)
Células de la Médula Ósea , Huesos , Células Madre , Ingeniería de Tejidos , Animales , Células de la Médula Ósea/fisiología , Adhesión Celular/fisiología , Células Madre/fisiología , Porcinos , Tantalio
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