The osteo-inductive activity of bone-marrow-derived mononuclear cells resides within the CD14+ population and is independent of the CD34+ population.

Bone marrow mononuclear cells (BMC) seeded on a scaffold of ß-tricalcium phosphate (ß-TCP) promote bone healing in a critical-size femur defect model. Being BMC a mixed population of predominantly mature haematopoietic cells, which cell type(s) is(are) instrumental for healing remains elusive. Although clinical therapies using BMC are often dubbed as stem cell therapies, whether stem cells are relevant for the therapeutic effects is unclear and, at least in the context of bone repair, seems dubious. Instead, in light of the critical contribution of monocytes and macrophages to tissue development, homeostasis and injury repair, in the current study it was hypothesised that BMC-mediated bone healing derived from the stem cell population. To test this hypothesis, bone remodelling studies were performed in an established athymic rats critical-size femoral defect model, with ß-TCP scaffolds augmented with complete BMC or BMC immunomagnetically depleted of stem cells (CD34+) or monocytes/macrophages (CD14+). Bone healing was assessed 8 weeks after transplantation. Compared to BMC-augmented controls, when CD14- BMC, but not CD34- BMC were transplanted into the bone defect, femora possessed dramatically decreased biomechanical stability and new bone formation was markedly reduced, as measured by histology. The degree of vascularisation did not differ between the two groups. It was concluded that the monocyte fraction within the BMC provided critical osteo-inductive cues during fracture healing. Which factors were responsible at the molecular levels remained elusive. However, this study marked a significant progress towards elucidating the mechanisms by which BMC elicit their therapeutic effects, at least in bone regeneration.

Autologous cell-based therapy for treatment of large bone defects: from bench to bedside.

OBJECTIVES: Reconstruction of long segmental bone defects is demanding for patients and surgeons, and associated with long-term treatment periods and substantial complication rates in addition to high costs. While defects up to 4-5 cm length might be filled up with autologous bone graft, heterologous bone from cadavers, or artificial bone graft substitutes, current options to reconstruct bone defects greater than 5 cm consist of either vascularized free bone transfers, the Masquelet technique or the Ilizarov distraction osteogenesis. Alternatively, autologous cell transplantation is an encouraging treatment option for large bone defects as it eliminates problems such as limited autologous bone availability, allogenic bone immunogenicity, and donor-site morbidity, and might be used for stabilizing loose alloplastic implants. METHODS: The authors show different cell therapies without expansion in culture, with ex vivo expansion and cell therapy in local bone defects, bone healing and osteonecrosis. Different kinds of cells and scaffolds investigated in our group as well as in vivo transfer studies and BMC used in clinical phase I and IIa clinical trials of our group are shown. RESULTS: Our research history demonstrated the great potential of various stem cell species to support bone defect healing. It was clearly shown that the combination of different cell types is superior to approaches using single cell types. We further demonstrate that it is feasible to translate preclinically developed protocols from in vitro to in vivo experiments and follow positive convincing results into a clinical setting to use autologous stem cells to support bone healing.

Establishment and characterization of the Masquelet induced membrane technique in a rat femur critical-sized defect model.

The Masquelet induced membrane technique for reconstructing large diaphyseal defects has been shown to be a promising clinical treatment, yet relatively little is known about the cellular, histological and biochemical make-up of these membranes and how they produce this positive clinical outcome. We compared cellular make-up, histological changes and growth factor expression in membranes induced around femur bone defects and in subcutaneous pockets at 2, 4 and 6 weeks after induction, and to the periosteum. We found that membranes formed around bone defects were similar to those formed in subcutaneous pockets; however, both were significantly different from periosteum with regard to structural characteristics, location of blood vessels and overall thickness. Membranes induced at the femur defect (at 2 weeks) and in periosteum contain mesenchymal stem cells (MSCs; STRO-1+ ) which were not found in membranes induced subcutaneously. BMP-2, TGFß and VEGF were significantly elevated in membranes induced around femur defects in comparison to subcutaneously induced membranes, whereas SDF-1 was not detectable in membranes induced at either site. We found that osteogenic and neovascular activity had mostly subsided by 6 weeks in membranes formed at both sites. It was conclude that cellular composition and growth factor content in induced membranes depends on the location where the membrane is induced and differs from periosteum. Osteogenic and neovascular activity in the membranes is maximal between 2 and 4 weeks and subsides after 6. Based on this, better and quicker bone healing might be achieved if the PMMA cement were replaced with a bone graft earlier in the Masquelet technique. Copyright © 2013 John Wiley & Sons, Ltd.

Decreased inflammatory responses of human lung epithelial cells after ethanol exposure are mimicked by ethyl pyruvate.

BACKGROUND AND PURPOSE: Leukocyte migration into alveolar space plays a critical role in pulmonary inflammation resulting in lung injury. Acute ethanol (EtOH) exposure exerts anti-inflammatory effects. The clinical use of EtOH is critical due to its side effects. Here, we compared effects of EtOH and ethyl pyruvate (EtP) on neutrophil adhesion and activation of cultured alveolar epithelial cells (A549). EXPERIMENTAL APPROACH: Time course and dose-dependent release of interleukin- (IL-) 6 and IL-8 from A549 were measured after pretreatment of A549 with EtP (2.5-10 mM), sodium pyruvate (NaP, 10 mM), or EtOH (85-170 mM), and subsequent lipopolysaccharide or IL-1beta stimulation. Neutrophil adhesion to pretreated and stimulated A549 monolayers and CD54 surface expression were determined. KEY RESULTS: Treating A549 with EtOH or EtP reduced substantially the cytokine-induced release of IL-8 and IL-6. EtOH and EtP (but not NaP) reduced the adhesion of neutrophils to monolayers in a dose- and time-dependent fashion. CD54 expression on A549 decreased after EtOH or EtP treatment before IL-1beta stimulation. CONCLUSIONS AND IMPLICATIONS: EtP reduces secretory and adhesive potential of lung epithelial cells under inflammatory conditions. These findings suggest EtP as a potential treatment alternative that mimics the anti-inflammatory effects of EtOH in early inflammatory response in lungs.

Endothelial progenitor cells improve directly and indirectly early vascularization of mesenchymal stem cell-driven bone regeneration in a critical bone defect in rats.

Early vascularization of a composite in a critical bone defect is a prerequisite for ingrowth of osteogenic reparative cells to regenerate bone, since lack of vessels does not ensure a sufficient nutritional support of the bone graft. The innovation of this study was to investigate the direct and indirect effects of endothelial progenitor cells (EPCs) and cotransplanted mesenchymal stem cells (MSCs) on the in vivo neovascularization activity in a critical size defect at the early phase of endochondral ossification. Cultivated human EPCs and MSCs were loaded onto ß-TCP in vitro. A critical-sized bone defect (5 mm) was created surgically in the femoral diaphysis of adult athymic rat and stabilized with an external fixateur. The bone defects were filled with ß-TCP, MSCs seeded on ß-TCP, EPCs seeded on ß-TCP, and coculture of MSCs and EPCs seeded on ß-TCP or autologous bone of rat. After 1 week, the rats were sacrificed. Using quantitative CD34 immunohistochemistry as well as qualitative analysis of vascularization (staining of MHC and VEGF) in decalcified serial sections were performed by means of an image analysis system. Fluorescence microscopy analyzed the direct effects and indirect effects of human implanted EPCs for vessel formation at bone regeneration site. Formation of a primitive vascular plexus was also detectable in the ß-TCP, MSC, or autologous bone group, but on a significantly higher level if EPCs alone or combined with MSCs were transplanted. Moreover, highest amount of vascularization were detected when EPCs and MSCs together were implanted. Early vascularization is improved by transplanted EPCs, which formed new vessels directly. Indeed the indirect effect of EPCs to vascularization is much higher. Transplanted EPC release chemotactic factors (VEGF) to recruit EPCs of the host and stimulate vascularization in the bone defect. Transplantation of human EPCs displays a promising approach to improve early vascularization of a scaffold in a critical bone defect. Moreover, coculture of EPCs and MSCs demonstrate also a synergistic effect on new vessel formation and seems to be a potential osteogenic construct for in vivo application.

Mesenchymal stem cell (MSC) and endothelial progenitor cell (EPC) growth and adhesion in six different bone graft substitutes.

INTRODUCTION: Several different synthetic and allograft bone graft substitutes are used clinically to treat large bone defects. In contrast to the "gold standard" of autologous bone grafts, these do not contain bone-forming (MSC) or vessel-forming (EPC) cells. In order to achieve the same level of success enjoyed by autologous bone grafts, they must be compatible with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC). In a previous study, we seeded MSC onto six different bone graft substitutes and then measured the cell adhesion, viability, differentiation, and morphology. In the present study, we seeded both MSC and EPC onto the same six bone graft substitutes and measured the same parameters. METHODS: In vitro, 125,000 MSC and 125,000 EPC were seeded onto Chronos(®), Vitoss(®), Actifuse(®), Biobase(®), Cerabone(®), and Tutoplast(®). Cell adhesion (fluorescence microscopy) and viability (MTT assay) were measured on days 2, 6, and 10. Osteogenic (cbfa-1, alkaline phosphatase [ALP], osteocalcin, collagen-1 alpha [Col1A]) and endothelial (von Willebrand factor [vWF], vascular endothelial growth factor [VEGF], kinase domain receptor [KDR]) gene expression were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Morphology was described by scanning electron microscopy (SEM) at day 2. RESULTS: MSC adhered significantly better to Tutoplast(®), Chronos(®), Actifuse(®), and Biobase(®). EPC adhered better to Actifuse(®), Chronos(®), Biobase(®), and Tutoplast(®). Viability increased over time when seeded on Tutoplast(®) and Chronos(®). Osteogenic and endothelial gene expression were detectable at day 10 in cells seeded on Chronos(®), Actifuse(®), and Tutoplast(®). The best morphology of MSC and EPC was found on Tutoplast(®), Chronos(®), Actifuse(®), and Biobase(®). CONCLUSION: When bone graft substitutes are used to help fill large defects, it is important that their interaction with these cells be supportive of bone healing.

Number and proliferative capacity of human mesenchymal stem cells are modulated positively in multiple trauma patients and negatively in atrophic nonunions.

Mesenchymal stem cells (MSCs) participate in regenerative osteogenesis by generating bone-forming cells. To examine the proliferative capacity of MSC populations from bone marrow and their relationship to trauma severity (multiple trauma, monofracture, atrophic nonunion), we quantified colony properties of human MSCs in vitro. Serum levels of mediators associated with bone formation were also assessed. Fifty-five individuals were enrolled in this study (13 multiple trauma patients, 15 patients with monofracture, 20 patients with atrophic nonunions, 7 healthy volunteers). The colony forming unit-fibroblast (CFU-F) assay was used to quantify total colony number, mean cell density per colony, and mean colony area. MSC phenotype was established using flow cytometry and osteogenic differentiation. MSCs obtained from multiple-trauma patients yielded the highest reservoir. Significant differences in colony numbers of MSCs in female subjects were found between multiple-trauma patients (mean +/- SD 48 +/- 21 CFU-F/culture) and healthy volunteers (18.7 +/- 3.3 CFU-F/culture, P < 0.05), patients with monotrauma (15 +/- 10 CFU-F/culture, P < 0.05), and patients with atrophic nonunions (6.3 +/- 4.1 CFU-F/culture, P < 0.05). In male participants, significant differences were found between patients with nonunions (14 +/- 14 CFU-F/culture) and healthy volunteers (54 +/- 17 CFU-F/culture, P < 0.05) as well as multiple-trauma patients (59 +/- 25 CFU-F/culture, P < 0.05). The highest proliferative capacity (cell density) was seen in multiple-trauma patients. These data suggest that trauma severity and gender affect the reservoir and proliferation capacity of bone marrow-derived MSCs.

[The influence of bisphosphonates on fracture healing]. / Einfluss der Bisphosphonate auf die Frakturheilung.

As the population ages and the prevalence of osteoporotic fractures increases, perioperative medical care of the elderly will continue to present challenges. Bisphosphonates, in combination with calcium and vitamin D have become the first-line therapy for patients with osteoporosis. Thus, one of the frequently asked questions concerning such patients is whether individuals who have recently sustained a fracture should take inhibitors of bone resorption. This discussion is relevant because many of the patients treated with bisphosphonates do have fractures, such as patients with osteoporosis, tumor bone disease, Paget's disease or osteogenesis imperfecta. A recent fracture should not preclude the initiation of therapy, because bisphosphonates have not been shown to interfere with overall fracture strength. Bisphosphonates appear to affect callus formation differently from either estrogen or raloxifene, but no significant difference in callus strength was seen 16 weeks after fracture. In addition, current studies demonstrate a significant reduction in periprosthetic bone loss after uncemented primary hip arthroplasty.

Intermittent parathyroid hormone (1-34) enhances mechanical strength and density of new bone after distraction osteogenesis in rats.

Distraction osteogenesis is used both for leg lengthening and for bone transportation in the treatment of fractures and nonunions. The main problem with this method is that the time until full recovery may be up to a year, partly because of the time needed for the new formed bone to consolidate and become strong enough for weight bearing. We have studied whether intermittent parathyroid hormone (PTH(1-34)) could accelerate the consolidation of new formed bone after distraction osteogenesis in rats. Forty-seven, 3-months-old male Sprague-Dawley rats underwent lengthening of the right femur using an external fixator. After a middiaphyseal osteotomy and a 7-day latency period, the callus was distracted during 10 days, with a distraction rate of 0.25 mm twice a day. The consolidation time was either 20 days or 40 days after distraction was completed. A dose of 60 microg of human PTH(1-34)/kg body weight/injection or vehicle was given every second day beginning 30 days before the rats were killed. Both femura of each rat were subjected to mechanical testing and dual-energy X-ray absorptiometry. Blinded histological examination was done for the distracted femura. In the 20 days consolidation experiment, PTH(1-34) increased ultimate load (56%), stiffness (117%), total regenerate callus volume (58%), callus BMC (24%) and histologic bone density (35%) compared to untreated distraction osteogenesis specimens. In the 40 days consolidation experiment, PTH(1-34) increased ultimate load (54%), stiffness (55%), callus BMC (33%) and histologic bone density (23%) compared to untreated distraction osteogenesis specimens. Total regenerate callus volume was unchanged. The contralateral femur also became stronger, stiffer and denser under PTH(1-34) treatment, but to a lesser degree. PTH(1-34) might become useful to shorten the consolidation time after distraction osteogenesis in humans.

Estudio citogenético de leucemias / Cytogenetic study in leukemias

La citogenética ha adquirido un valor indiscutible en el diagnóstico, pronóstico y seguimiento de las leucemias. Es un hecho conocido que las anomalías cromosómicas forman parte del fenotipo de la célula tumoral. Con las mejoras en las técnicas citogenéticas se ha podido establecer aberraciones cromosómicas específicas, no aleatorias en diferentes tipos de leucemias. El ejemplo clásico lo constituye la leucemia mieloide crónica (LMC), resultado de una transformación neoplásica monoclonal de la célula pluripotencial. Está caracterizada por presentar un 90 por ciento de los casos, un cromosoma Philadelphia (cromosoma 22 acortado), producto de una translocación recíproca entre un cromosoma 9 y 22. Esta aberración cromosómica, además de constituir un sello diagnóstico, permitió la localización del proto-oncogen c-abl (en el cromosoma 9) que al yuxtaponerse (mediante translocación al bcr del cromosoma 22) da como resultado la producción celular de una proteína kinasa anormal, que juega un rol muy importante en la cancerogénesis. El estudio cromosómico sirve de gran apoyo clínico, de ahí el interés de montar estas técnicas. Desde febrero de 1990 iniciamos el estudio citogenético de leucemias, realizando cultivos de médulña ósea y en ocasiones de blastos de sangre periférica. Se realizaron en forma sistémica cultivo directo de 24 y de 48 horas y un minucioso análisis. Nuestra experiencia muestra un 70 por ciento de éxito en la obtención de resultados. De un total de 30 pacientes, 21 arrojaron resultados positivos. De éstos, 8 (35 por ciento) no presentaron anomalías cromosómicas, 134 (65 por ciento) las presentaron. De éstos, 13.5 fueron POhiladelkphia positivo y 8 presentaron anomalías numéricas y/o estructurales, entre las cuales cuenta una translocación aberrante, t (9;11;22). La clínica y la utilidad prestada por el estudio citogenético son analizados individualmente en los casos con resultados positivos

Estudios cromosomicos en lactantes con desnutricion calorica proteica severa. / Chromosomal studies in infants with severe protein-calorie malnutrition

Existe controversia sobre el efecto de la desnutricion a nivel cromosomico. La falta de consenso podria deberse a heterogeneidad de los tipos clinicos de desnutricion y/o a factores medio ambientales adversos, con frecuencia presentes en ninos desnutridos. Se estudiaron los Intercambios de Cromatides Hermanas (ICH), alteraciones estructurales y polimorfismos cromosomicos utilizando diferentes tecnicas de bandeo, en 5 lactantes con desnutricion calorico-proteica severa primaria (DCP), sin antecedentes de prematurez, problemas perinatales, afecciones geneticas, metabolicas, neurologicas o malformaciones ni de infecciones, drogas o radiaciones en el ultimo mes. Como controles se estudiaron 5 lactantes eutroficos, de edad y condiciones semejantes. No hubo diferencias significativas en el numero de ICH; ni en la frecuencia de alteraciones estructurales entre los dos grupos. Nuestros resultados permiten concluir que la DCP severa "per se" no producira alteraciones cromosomica