RESUMEN
The aim of this study was to elucidate the impact of autologous umbilical cord blood cells (USSC) on bone regeneration and biomechanical stability in an ovine tibial bone defect. Ovine USSC were harvested and characterized. After 12 months, full-size 2.0 cm mid-diaphyseal bone defects were created and stabilized by an external fixateur containing a rigidity measuring device. Defects were filled with (i) autologous USSC on hydroxyapatite (HA) scaffold (test group), (ii) HA scaffold without cells (HA group), or (iii) left empty (control group). Biomechanical measures, standardized X-rays, and systemic response controls were performed regularly. After six months, bone regeneration was evaluated histomorphometrically and labeled USSC were tracked. In all groups, the torsion distance decreased over time, and radiographies showed comparable bone regeneration. The area of newly formed bone was 82.5 ± 5.5% in the control compared to 59.2 ± 13.0% in the test and 48.6 ± 2.9% in the HA group. Labeled cells could be detected in lymph nodes, liver and pancreas without any signs of tumor formation. Although biomechanical stability was reached earliest in the test group with autologous USSC on HA scaffold, the density of newly formed bone was superior in the control group without any bovine HA.
Asunto(s)
Regeneración Ósea , Sangre Fetal/citología , Osteogénesis , Tibia/química , Animales , Fenómenos Biomecánicos , Movimiento Celular , Fracturas Óseas/diagnóstico , Fracturas Óseas/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/ultraestructura , Proyectos Piloto , Ovinos , Tibia/patología , Andamios del Tejido , Cicatrización de HeridasRESUMEN
Drugs may have a significant effect on postoperative bone healing by reducing the function of human mesenchymal stromal cells (hMSC) or mature osteoblasts. Although cefazolin is one of the most commonly used antibiotic drugs in arthroplasty to prevent infection worldwide, there is a lack of information regarding how cefazolin affects hMSC and therefore may have an effect on early bone healing. We studied the proliferation and migration capacity of primary hMSC during cefazolin treatment at various doses for up to 3 days, as well as the reversibility of the effects during the subsequent 3 days of culture without the drug. We found a time- and dose-dependent reduction of the proliferation rate and the migratory potential. Tests of whether these effects were reversible revealed that doses ≥ 250 µg/mL or treatments longer than 24 h irreversibly affected the cells. We are the first to show that application of cefazolin irreversibly inhibits the potential of hMSC for migration to the trauma site and local proliferation. Cefazolin should be administered only at the required dosage and time to prevent periprosthetic infection. If long-term administration is required and delayed bone healing is present, cefazolin application must be considered as a cause of delayed bone healing.
Asunto(s)
Cefazolina/toxicidad , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Adolescente , Adulto , Anciano , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Curación de Fractura/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Adulto JovenRESUMEN
BACKGROUND: Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost. MATERIAL/METHODS: Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of the iliac crest followed by density gradient centrifugation and flow cytometry with defined antigens (CD105+/73+/45-/14-). The cells were seeded and incubated as follows: without additives (Group 0; donor A/B/C), with 10(-7) M iloprost only (Group 0+ilo; A/B), with indomethacin only in concentrations of 10(-6) M (Group 1, A), 10(-5) M (Group 2, B), 10(-4) M (Group 3, A/B), and together with 10(-7) M iloprost (Groups 4-6, A/B/C). On Day 10 and 28, UV/Vis spectrometric and immunocytochemical assays (4 samples per group and donor) were performed to investigate cell proliferation (cell count measurement) and differentiation towards the osteoblastic lineage (CD34-, CD45-, CD105+, type 1 collagen (Col1), osteocalcin (OC), alkaline phosphatase (ALP), Runx2, Twist, specific ALP-activity). RESULTS: Indomethacin alone suppressed BMSC differentiation towards the osteoblastic lineage by downregulation of Runx2, Col1, and ALP. In combination with indomethacin, iloprost increased cell proliferation and differentiation and it completely suppressed Twist expression at Day 10 and 28. Iloprost alone did not promote cell proliferation, but moderately enhanced Runx2 and Twist expression. However, the proliferative effects and the specific ALP-activity varied donor-dependently. CONCLUSIONS: Iloprost partially antagonized the suppressing effects of indomethacin on BMSC differentiation towards the osteoblast lineage. It enhanced the expression of Runx2 and, only in the presence of indomethacin, it completely suppressed Twist. Thus, in the treatment of avascular osteonecrosis or painful bone marrow edema, the undesirable effects of indomethacin might be counterbalanced by iloprost.
Asunto(s)
Células de la Médula Ósea/citología , Epoprostenol/farmacología , Indometacina/farmacología , Células Madre Mesenquimatosas/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Adulto , Fosfatasa Alcalina/metabolismo , Antígenos CD/metabolismo , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Epoprostenol/administración & dosificación , Femenino , Humanos , Iloprost/farmacología , Inmunohistoquímica , Indometacina/administración & dosificación , L-Lactato Deshidrogenasa/metabolismo , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteocalcina/metabolismoRESUMEN
Dexamethasone/ascorbic acid/glycerolphosphate (DAG) and bone morphogenic protein (BMP)-2 are potent agents in cell proliferation and differentiation pathways. This study investigates the in vitro interactions between dexamethasone and BMP-2 for an osteoblastic differentiation of mesenchymal stem cells (MSCs). Bone marrow-derived human MSCs were cultured with DAG (group A), BMP-2 + DAG (group B), and DAG + BMP-2 combined with a porous collagen I/III scaffold (group C). RT-PCR, ELISA, immuncytochemical stainings and flow cytometry analysis served to evaluate the osteogenic-promoting potency of each of the above conditions in terms of cell morphology/viability, antigen presentation, and gene expression. DAG induced collagen I secretion from MSCs, which was further increased by the combination of DAG + BMP-2. In comparison, the collagen scaffold and the control samples showed no significant influence on collagen I secretion of MSCs. DAG stimulation of MSCs led also to a steady but not significant increase of BMP-2 level. A DAG and more, a DAG + BMP-2, stimulation increased the number of mesenchymal cells (CD105+/CD73+). All samples showed mRNA of ALP, osteopontin, Runx2, Twist 1 and 2, Notch-1/2, osteonectin, osteocalcin, BSP, and collagen-A1 after 28 days of in vitro culture. Culture media of all samples showed a decrease in Ca(2+) and PO(4) (2-) concentration, whereas a collagen-I-peak only occurred at day 28 in DAG- and DAG + BMP-2-stimulated bone marrow cells. In conclusion, BMP-2 enhances DAG-induced osteogenic differentiation in mesenchymal bone marrow cells. Both agents interact in various ways and can modify osteoblastic bone formation.
Asunto(s)
Antiinflamatorios/farmacología , Proteínas Morfogenéticas Óseas/farmacología , Dexametasona/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Biomarcadores/metabolismo , Células de la Médula Ósea , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Combinación de Medicamentos , Técnica del Anticuerpo Fluorescente Indirecta , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
We have examined the distribution of immunoreactivity for GAP-43 in the developing and adult brain of a diprotodontid metatherian, the tammar wallaby ( Macropus eugenii). The distribution of GAP-43 immunoreactivity in the neonatal wallaby brain was strikingly heterogeneous, in contrast to that reported for the newborn polyprotodontid opossum. Immunoreactivity for GAP-43 in the developing wallaby brain showed a caudal-to-rostral spatiotemporal gradient, with the brainstem well in advance of the telencephalon throughout the first 100 days of postnatal life. In many regions examined, GAP-43 immunoreactivity passed through the following phases: 1. intense immunoreactivity in developing fiber tracts and occasional somata; 2. diffuse homogeneous immunoreactivity; 3. selective loss of immunoreactivity in particular nuclei or cortical regions. In the isocortex, selective loss of GAP-43 immunoreactivity in the somatosensory and visual cortex (at postnatal day 115) coincided with the maturation of the laminar distribution of terminal thalamocortical axonal fields. Within adult cortical regions, GAP-43 immunoreactivity was highest in layer I of all regions, lower layers (V and VI) of primary somatosensory and visual cortices, layers II/III of motor and cingulate cortex, and layer IV of entorhinal cortex. Our findings suggest that, while patterning of GAP-43 immunoreactivity in the mature brain is similar across meta- and eutheria, there may be early developmental differences in the distribution of GAP-43 immunoreactivity between poly- and diprotodontid metatheria.