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BMSCs, also known as bone marrow-derived mesenchymal stem cells, provide an excellent source of progenitor cells for regenerative therapy. To assess whether osteoarthritis (OA) affects the regenerative potential of BMSCs we compared the proliferation and differentiation potential as well as the surface marker expression profile of OA- versus control BMSCs. BMSCs were isolated from bone marrow aspirates of n=14 patients with advanced-stage idiopathic hip OA (67±6years) and n=15 healthy individuals (61±4years). Proliferation was quantified by total DNA content and colony-forming-units of fibroblastsmax (CFU-F) assay. Differentiation assays included immunohistology, cell-specific alkaline phosphatase (ALP) activity, and osteogenic, chondrogenic as well as adipogenic marker gene qRT-PCR. Expression of BMSC-associated surface markers was analyzed using flow cytometry. No significant intergroup differences were observed concerning the proliferation potential, cell-specific ALP activity as well as adipogenic and osteogenic differentiation marker gene expressions. Interestingly, SOX9 gene expression levels were significantly increased in OA-BMSCs after 14days of chondrogenic stimulation (p<0.01). The surface markers CD73, CD90 and STRO-1 were elevated in relation to CD14, CD34 and CD45 in both groups (p<0.0001). Notably, OA-BMSCs showed significantly increased CD90 (p<0.01) and decreased CD166 (p<0.001) levels. Overall, the in vitro characteristics of BMSCs are not markedly influenced by OA. However, increased SOX9 and CD90 as well as reduced CD166 expression levels in OA-BMSCs warrant further investigation. These data will help to further understand the role of BMSC in OA and facilitate the application of autologous cell-based strategies for musculoskeletal tissue regeneration in OA patients., (Copyright © 2016 Roslin Cells Ltd. Published by Elsevier B.V. All rights reserved.)

Background: An increasing number of patients scheduled for total hip arthroplasty (THA) are obese and exhibit a different risk profile from that of patients of normal weight., Objectives: To provide an overview of the impact of obesity on the outcome of primary THA., Materials and Methods: Literature review and discussion of own epidemiological data., Results: Obese patients can expect as much functional improvement as non-obese patients after THA. However, peri- and postoperative complication (e.g., periprosthetic infection and dislocation) rates are reported to be increased in obese THA patients., Conclusions: The knowledge of obesity-associated risks is the prerequiste for successful THA in obese patients.

Background: Because of anatomical variations total hip arthroplasty (THA) can be demanding in patients with osteoarthritis secondary to hip dysplasia., Objectives: Depending on the degree of bony deformation, hip dislocation and soft tissue alteration numerous treatment strategies are available. This review describes current approaches that address frequent deformities., Materials and Methods: Review of relevant clinical studies, meta-analyses, and presentation of our own approach., Results: Pre-operative planning (based on a thorough clinical and radiographic examination) is essential. Acetabular reconstruction close to the primary acetabulum should always be intended. Roof augmentation and/or cup medialization can support stable bony implant fixation. Subtrochanteric shortening osteotomy of the femur is a demanding but reliable technique that avoids nerve damage in cases where inappropriate lengthening would be necessary (i.e., high riding dislocation)., Conclusions: Although the post-operative complication rate is elevated after THA for dysplastic hips compared with primary osteoarthritis, the overall functional results and implant survival are comparable.

Reduced tissue levels of endothelial progenitor cells (EPCs) and functional impairment of endothelium are frequently observed in patients with diabetes and cardiovascular disease. The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. The aim of this study was to investigate whether Icariin, a natural bioactive component known from traditional Chinese Medicine, can induce angiogenic differentiation and inhibit oxidative stress-induced cell dysfunction in bone marrow-derived EPCs (BM-EPCs), and, if so, through what mechanisms. We observed that treatment of BM-EPCs with Icariin significantly promoted cell migration and capillary tube formation, substantially abrogated hydrogen peroxide (H2 O2 )-induced apoptotic and autophagic programmed cell death that was linked to the reduced intracellular reactive oxygen species levels and restored mitochondrial membrane potential. Icariin downregulated endothelial nitric oxide synthase 3, as well as nicotinamide-adenine dinucleotide phosphate-oxidase expression upon H2 O2 induction. These antiapoptotic and antiautophagic effects of Icariin are possibly mediated by restoring the loss of mammalian target of rapamycin /p70S6K/4EBP1 phosphorylation as well as attenuation of ATF2 and ERK1/2 protein levels after H2 O2 treatment. In summary, favorable modulation of the angiogenesis and redox states in BM-EPCs make Icariin a promising proangiogenic agent both enhancing vasculogenesis and protecting against endothelial dysfunction., (© 2015 AlphaMed Press.)

Background: The clinical outcome of hip resurfacing (HR) as a demanding surgical technique associated with a substantial learning curve depends on the position of the femoral component. The aim of the study was to investigate the effects of the level of surgical experience on computer-assisted imageless navigation concerning precision of femoral component positioning, notching, and oversizing rate, as well as operative time., Methods: Three surgeons with different levels of experience in both HR and computer-assisted surgery (CAS) prepared the femoral heads of 54 synthetic femurs using the Durom(TM) Hip Resurfacing (Zimmer, Warsaw, IN, USA) system. Each surgeon prepared a total of 18 proximal femurs using the Navitrack® system (ORTHOsoft Inc., Montreal, Canada) or the conventional free-hand Durom(TM) K-wire positioning jig. The differences between planned and postoperative stem shaft angle (SSA) and anteversion angle in standardized x-rays were measured and the operative time, not including the time for calibrating the CAS-system, was documented. Notching was evaluated by the three surgeons in a randomized manner. Oversizing was determined by the difference of the preoperative determined cap and the cap size advised by the CAS-system., Results: CAS significantly reduced the overall mean deviation between planned and postoperative SSA in comparison with the conventional procedure (mean ± SD, 1 ± 1.7° vs. 7.4 ± 4.4°, P < 0.01) regardless of the surgeon's level of experience. The incidence of either varus or valgus SSA deviations exceeding 5° were 1/27 for CAS and 15/27 for the conventional method, respectively (P < 0.001), corresponding to a reduction by 97%. Using CAS, the rate of notching was reduced by 100%., Conclusions: The accuracy of femoral HR component orientation is significantly increased by use of CAS regardless of the surgeon's level of experience in our preclinical study. Thus, imageless computer-assisted navigation can be a valuable tool to improve implant positioning in HR for surgeons at any stage of their learning curve.

The principles of tissue engineering (TE) are widely used for bone regeneration concepts. Three-dimensional (3D) cultivation of autologous human mesenchymal stromal cells (MSCs) on porous scaffolds is the basic prerequisite to generate newly formed bone tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive analytical tool for the measurement of mRNA-levels in cells or tissues. For an accurate quantification of gene expression levels, stably expressed reference genes (RGs) are essential to obtain reliable results. Since the 3D environment can affect a cell's morphology, proliferation, and gene expression profile compared with two-dimensional (2D) cultivation, there is a need to identify robust RGs for the quantification of gene expression. So far, this issue has not been adequately investigated. The aim of this study was to identify the most stably expressed RGs for gene expression analysis of 3D-cultivated human bone marrow-derived MSCs (BM-MSCs). For this, we analyzed the gene expression levels of n=31 RGs in 3D-cultivated human BM-MSCs from six different donors compared with conventional 2D cultivation using qRT-PCR. MSCs isolated from bone marrow aspirates were cultivated on human cancellous bone cube scaffolds for 14 days. Osteogenic differentiation was assessed by cell-specific alkaline phosphatase (ALP) activity and expression of osteogenic marker genes. Expression levels of potential reference and target genes were quantified using commercially available TaqMan(®) assays. mRNA expression stability of RGs was determined by calculating the coefficient of variation (CV) and using the algorithms of geNorm and NormFinder. Using both algorithms, we identified TATA box binding protein (TBP), transferrin receptor (p90, CD71) (TFRC), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the most stably expressed RGs in 3D-cultivated BM-MSCs. Notably, genes that are routinely used as RGs, for example, beta actin (ACTB) and ribosomal protein L37a (RPL37A), were among the least stable genes. We recommend the combined use of TBP, TFRC, and HPRT1 for the accurate and robust normalization of qRT-PCR data of 3D-cultivated human BM-MSCs.

We investigated whether breast tumor cells can modulate the function of mesenchymal stromal cells (MSCs) with a special emphasis on their chemoattractive activity towards hematopoietic stem and progenitor cells (HSPCs). Primary MSCs as well as a MSC line (SCP-1) were cocultured with primary breast cancer cells, MCF-7, MDA-MB231 breast carcinoma or MCF-10A non-malignant breast epithelial cells or their conditioned medium. In addition, the frequency of circulating clonogenic hematopoietic progenitors was determined in 78 patients with breast cancer and compared with healthy controls. Gene expression analysis of SCP-1 cells cultured with MCF-7 medium revealed CXCL12 (SDF-1) as one of the most significantly downregulated genes. Supernatant from both MCF-7 and MDA-MB231 reduced the CXCL12 promoter activity in SCP-1 cells to 77% and 47%, respectively. Moreover, the CXCL12 mRNA and protein levels were significantly reduced. As functional consequence of lower CXCL12 levels, we detected a decreased trans-well migration of HSPCs towards MSC/tumor cell cocultures or conditioned medium. The specificity of this effect was confirmed by blocking studies with the CXCR4 antagonist AMD3100. Downregulation of SP1 and increased miR-23a levels in MSCs after contact with tumor cell medium as well as enhanced TGFβ1 expression were identified as potential molecular regulators of CXCL12 activity in MSCs. Moreover, we observed a significantly higher frequency of circulating colony-forming hematopoietic progenitors in patients with breast cancer compared with healthy controls. Our in vitro results propose a potential new mechanism by which disseminated tumor cells in the bone marrow may interfere with hematopoiesis by modulating CXCL12 in protected niches., (© 2014 UICC.)

Unlabelled: The treatment of loosened total hip replacement (THR) acetabular components may require the management of severe bone defects. Although being applied for decades, there is only limited scientific data about the osteointegration of cancellous bone allografts (CBA) and other void fillers. Monitoring of periprosthetic bone regeneration could possibly help to optimize this process thereby reducing late failure rates. The aim of this study was to show osteometabolic changes in periprosthetic CBA after THR revision with the use of sodium-[18F]-fluoride (NaF) and positron emission tomography (PET)., Patients, Methods: Twelve patients undergoing THR revision with the use of CBA were prospectively enrolled in the study. Nine patients completed all necessary examinations and were included in the evaluation. The temporal pattern of osteointegration was assessed via NaF-PET at one (PET1) and six weeks (PET2) after surgery. CBA, tantalum implants, supraacetabular regions ipsilateral and contralateral, and parasymphyseal pubic bones were delineated as volumes of interest (VOI) in postop CT scans, which were then merged with the PET data., Results: In comparison to the contralateral supraacetabular reference bone, a significant 1.5-fold increase of osteometabolic activity from PET1 to PET2 was seen in the CBA region. Also, the ipsilateral supraacetabular host bone showed a higher NaF-influx in week 6, compared to the first postoperative week. The supraacetabular site exhibited a significantly 1.8- to 2-fold higher influx and uptake than bone regions in non-operated sites. Tantalum implants had a low NaF influx at both time points investigated., Conclusion: Using NaF-PET osteometabolic changes of CBA and implant-bone-interfaces can be monitored. Applying this method we demonstrated early periprosthetic temporal bone regeneration patterns in THR cup revision patients.

The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Protection against reactive oxygen species (ROS)-mediated oxidative damage via antioxidant mechanisms is essential for tissue maintenance and shows therapeutic potential for patients suffering from cardiovascular and metabolic disorders. Salvianolic acid B (SalB), a natural bioactive component known from Traditional Chinese Medicine, has been reported to exert cellular protection in various types of cells. However, the underlying mechanisms involved are not fully understood. Here, we showed that SalB significantly promoted the migratory and tube formation abilities of human bone marrow derived-endothelial progenitor cells (BM-EPCs) in vitro, and substantially abrogated hydrogen peroxide (H2O2)-induced cell damage. SalB down-regulated Nox4 and eNOS, as well as nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase expression upon H2O2 induction that in turn prevents oxidative-induced endothelial dysfunction. Moreover, SalB suppressed the Bax/Bcl-xL ratio and caspase-3 activation after H2O2 induction. Furthermore, our results provide mechanistic evidence that activation of the mTOR/p70S6K/4EBP1 pathways is required for both SalB-mediated angiogenic and protective effects against oxidative stress-induced cell injury in BM-EPCs. Suppression of MKK3/6-p38 MAPK-ATF2 and ERK1/2 signaling pathways by SalB significantly protected BM-EPCs against cell injury caused by oxidative stress via reduction of intracellular ROS levels and apoptosis. Taken together, by providing a mechanistic insight into the modulation of redox states in BM-EPCs by SalB, we suggest that SalB has a strong potential of being a new proangiogenic and cytoprotective therapeutic agent with applications in the field of endothelial injury-mediated vascular diseases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Low back pain (LBP) is a common symptom in the populations of western countries, and adolescent athletes seem to be prone to LBP. The main objective of this study was to analyze the point (LBP within the last 48 h), 1-year (LBP within the last 12 months) and lifetime (LBP within the entire life) prevalence rates of LBP in adolescent athletes participating in various sports. We also assessed the characteristics of LBP and its association with potential risk factors. To this end, 272 competitive adolescent athletes involved in 31 different sports (158 males, 113 females, 15.4 ± 2.0 years, body mass index [BMI] 20.3 ± 2.4 kg/m(2)) were enrolled in a 10-month prospective clinical trial that included a questionnaire and physical examination. We found a point prevalence of 14%, a 1-year prevalence of 57%, and a lifetime prevalence of 66% for LBP. The mean age of first appearance of LBP was 13.1 ± 2.0 years. The lifetime prevalence was significantly higher in volleyball than in biathletes (74.3 vs. 45.7%, p = 0.015). Our findings confirm that LBP is a common symptom in adolescent athletes; LBP prevalence correlates with sports participation and individual competitive level. Adolescent athletes with LBP should receive a thorough diagnostic work-up and adapt training and technique correspondingly when indicated., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background and Purpose: With the increase of age, increased susceptibility to apoptosis and senescence may contribute to proliferative and functional impairment of endothelial progenitor cells (EPCs). The aim of this study was to investigate whether salidroside (SAL) can induce angiogenic differentiation and inhibit oxidative stress-induced apoptosis in bone marrow-derived EPCs (BM-EPCs), and if so, through what mechanism., Experimental Approach: BM-EPCs were isolated and treated with different concentrations of SAL for up to 4 days. Cell proliferation, migration and tube formation ability were detected by DNA content quantification, transwell assay and Matrigel-based angiogenesis assay. Gene and protein expression were assessed by qRT-PCR and Western blot respectively., Key Results: Treatment with SAL promoted cellular proliferation and angiogenic differentiation of BM-EPCs, and increased VEGF and NO secretion, which in turn mediated the enhanced angiogenic differentiation of BM-EPCs. Furthermore, SAL significantly attenuated hydrogen peroxide (H₂O₂)-induced cell apoptosis, reduced the intracellular level of reactive oxygen species and restored the mitochondrial membrane potential of BM-EPCs. Moreover, SAL stimulated the phosphorylation of Akt, mammalian target of rapamycin and p70 S6 kinase, as well as ERK1/2, which is associated with cell migration and capillary tube formation. Additionally, SAL reversed the phosphorylation of JNK and p38 MAPK induced by H₂O₂ and suppressed the changes in the Bax/Bcl-xL ratio observed after stimulation with H₂O₂., Conclusions and Implications: These findings identify novel mechanisms that regulate EPC function and suggest that SAL has therapeutic potential as a new agent to enhance vasculogenesis as well as protect against oxidative endothelial injury., (© 2014 The British Pharmacological Society.)

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.

Treatment of critical size bone defects is challenging. Recent studies showed that the cytokine stromal cell-derived factor 1 alpha (SDF-1α) has potential to improve the bone regenerative effect of low bone morphogenetic protein 2 (BMP-2) concentrations. The goal of this study was to demonstrate the combined effect of SDF-1α and BMP-2 on bone regeneration and stem cell recruitment using a critical size femoral bone defect model. A total of 72 mice were randomized to six groups. External fixators were implanted onto the right femur of each mouse and 3 mm defects were created. Depending on the group affiliation, adenovirally activated fat tissue grafts expressing SDF-1α or/and BMP-2 were implanted at the defect site. One day after operation, 1×10⁶ murine mesenchymal stromal cells (MSCs), lentivirally transduced to express the gene enhanced green fluorescent protein (eGFP), firefly luciferase, and CXCR4 were injected systemically in selected groups. Migration of the injected MSCs was observed by bioluminescence imaging on days 0, 2, 4, 6, 8, 10, 12, 14, 21, 28, and 42. After 6 weeks, animals were euthanized and 80 μm CT-scans were performed. For histological investigations, hematoxylin and eosin-, tartrate-resistant acid phosphatase-, alkaline phosphatase-, and anti-eGFP-stained sections were prepared. BMP-2 and SDF-1α combined at the defect site increased bone volume (BV) (2.72 mm³; 95% CI 1.95-3.49 mm³) compared with the negative control group (1.80 mm³; 95% CI 1.56-2.04 mm³; p<0.05). In addition, histological analysis confirmed a higher degree of bone healing in the BMP-2 and SDF-1α combined group compared with the negative control group. Bioluminescence imaging demonstrated higher numbers of migrated MSCs toward the defect site in the presence of both BMP-2 and SDF-1α at the defect site. Furthermore, eGFP-labeled migrated MSCs were found in all defect areas, when cells were injected. The ratio of osteoblasts to osteoclasts, assessed by immunohistological staining, was higher and thus showed a trend toward more bone formation for the combined use of BMP-2 and SDF-1α compared with all other groups. This study demonstrated that SDF-1α enhanced BMP-2 mediated bone healing in a critical size segmental bone defect model. Notably, both proteins alone also provided a cumulative effect on MSC attraction toward the site of injury.

Associated with the trend towards increased health consciousness and fitness, triathlon has established itself as a sport for masses. The goals of this study were to evaluate injury risk factors of non-professional triathletes and to compare prospective and retrospective evaluation methods. Using an online survey, 212 triathletes retrospectively answered a questionnaire about their training habits and injuries during the past 12 months. Forty-nine of these triathletes participated in a 12-month prospective trial. Injuries were classified with regard to the anatomical location, type of injury, incidence and associated risk factors. Most injuries occurred during running (50%) followed by cycling (43%) and swimming (7%). Fifty-four per cent (retrospective) and 22% (prospective) of the injuries were contusions and abrasions, 38% (retrospective) and 46% (prospective) were ligament and capsular injuries, 7% (retrospective) and 32% (prospective) were muscle and tendon injuries and 1% (retrospective) and 0% (prospective) were fractures. The incidence of an injury per 1000 training hours was 0.69 (retrospective) and 1.39 (prospective) during training and 9.24 (retrospective) and 18.45 (prospective) during competition. The main risk factor for injury in non-professional triathlon is participation in a competitive triathlon event. A retrospective design may underestimate the rate of overuse injuries.

Bone transplantation is frequently used for the treatment of large osseous defects. The availability of autologous bone grafts as the current biological gold standard is limited and there is a risk of donor site morbidity. Allogenic bone grafts are an appealing alternative, but disinfection should be considered to reduce transmission of infection disorders. Peracetic acid-ethanol (PE) treatment has been proven reliable and effective for disinfection of human bone allografts. The purpose of this study was to evaluate the effects of PE treatment on the biomechanical properties and microstructure of cancellous bone grafts (CBG). Forty-eight human CBG cylinders were either treated by PE or frozen at -20 °C and subjected to compression testing and histological and scanning electron microscopy (SEM) analysis. The levels of compressive strength, stiffness (Young's modulus), and fracture energy were significantly decreased upon PE treatment by 54%, 59%, and 36%, respectively. Furthermore, PE-treated CBG demonstrated a 42% increase in ultimate strain. SEM revealed a modified microstructure of CBG with an exposed collagen fiber network after PE treatment. We conclude that the observed reduced compressive strength and reduced stiffness may be beneficial during tissue remodeling thereby explaining the excellent clinical performance of PE-treated CBG.

Background: Metal-on-metal (MoM) tribological pairing results in less volumetric abrasion than pairing with the conventionally used polyethylene and is associated with a lower risk of material failure compared to other hard-hard pairings. An increased frequency of problem cases in recent years has led to a great increase in uncertainty. Against this background in this article the current aspects of epidemiology, etiology, diagnostics and treatment of complications in MoM hip joint endoprostheses will be discussed., Epidemiology and Etiology: Based on the results from national endoprosthesis registers and selected clinical studies an evaluation of the rate of local complications from MoM tribological pairings was undertaken. A differentiation was made between MoM pairings in pedicled small head prostheses (≤ 32 mm), large head (> 32 mm) and surface replacement (OFE) endoprostheses. Each year MoM endoprostheses release on average 10(12)-10(14) cobalt (Co) and chromium (Cr) nanoparticles per patient. This release of metal ions and particles can lead to a variety of tissue reactions., Diagnostics: A differentiation must be made between regular routine diagnostics within the framework of implant follow-up screening and specific investigations due to the occurrence of complaints. The diagnostics for patients treated with MoM hip endoprostheses consists of a standardized step-wise approach considering possible differential diagnoses and the utilization of modern laboratory chemical and radiological methods. When problems occur, a differentiation should preferentially be made between complaints not caused by metal and mechanical problems (e.g. prosthesis loosening and impingement) and symptoms due to periprosthetic infections., Therapy of Complications: The normal standards for hip endoprosthetics are also valid for periprosthetic infections, fractures and other general complications. Specific measures are, however, necessary for complications due to metal-specific risks.

Bone marrow stromal cells (BMSCs) are key cellular components for musculoskeletal tissue engineering strategies. Furthermore, recent data suggest that BMSCs are involved in the development of Osteoarthritis (OA) being a frequently occurring degenerative joint disease. Reliable reference genes for the molecular evaluation of BMSCs derived from donors exhibiting OA as a primary co-morbidity have not been reported on yet. Hence, the aim of the study was to identify reference genes suitable for comparative gene expression analyses using OA-BMSCs. Passage 1 bone marrow derived BMSCs were isolated from n=13 patients with advanced stage idiopathic hip osteoarthritis and n=15 age-matched healthy donors. The expression of 31 putative reference genes was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using a commercially available TaqMan(®) assay. Calculating the coefficient of variation (CV), mRNA expression stability was determined and afterwards validated using geNorm and NormFinder algorithms. Importin 8 (IPO8), TATA box binding protein (TBP), and cancer susceptibility candidate 3 (CASC3) were identified as the most stable reference genes. Notably, commonly used reference genes, e.g. beta-actin (ACTB) and beta-2-microglobulin (B2M) were among the most unstable genes. For normalization of gene expression data of OA-BMSCs the combined use of IPO8, TBP, and CASC3 gene is recommended., (© 2013.)

The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients' cells culture supernatants were almost 2-fold lower (P<0.01) than those in controls and this was paralleled by a reduced induction of migration of CD34(+) hematopoietic cells. Co-cultures of mesenchymal stromal cells from patients with CD34(+) cells from healthy donors resulted in reduced numbers of cobblestone area-forming cells and fewer colony-forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of stromal derived factor 1-alpha secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from patients with low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.

Mesenchymal stromal cells (MSCs) are one of the most frequently used cell sources for tissue engineering strategies. Cultivation of osteogenic MSCs is a prerequisite for cell-based concepts that aim at bone regeneration. Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) analysis is a commonly used method for the examination of mRNA expression levels. However, data on suitable reference genes for osteogenically cultivated MSCs is scarce. Hence, the aim of the study was to compare the regulation of different potential reference genes in osteogenically stimulated MSCs. Human MSCs were isolated from bone marrow aspirates of N = 6 hematologically healthy individuals, expanded by polystyrene-adherence, and maintained with and without osteogenic supplements for 14 days. Cellular proliferation and osteogenic differentiation were assessed by total DNA quantification, cell-specific alkaline phosphatase (ALP) activity and by qualitative staining for ALP and alizarin red, respectively. mRNA expression levels of N = 32 potential reference genes were quantified using the human Endogenous Control TaqMan® assays. mRNA expression stability was calculated using geNorm. The combined use of the most stable reference genes and DNA-damage-inducible alpha, Pumilio homolog 1, and large ribosomal protein P0 significantly improved gene expression accuracy as compared to the use of the commonly used reference genes beta actin and glyceraldehyde-3-phosphate dehydrogenase during qRT-PCR-based target gene expression analysis of osteogenically stimulated MSCs., (© 2013 American Institute of Chemical Engineers.)

Mesenchymal stromal cell (MSCs) are key cellular components for site-specific tissue regeneration. The chemokine stromal derived factor 1 alpha (SDF-1α) is known to attract stem cells via the C-X-C chemokine receptor-4 (CXCR4) receptor. The aim of the study was to develop a model for stem cell attraction using SDF-1α overexpressing fat tissue grafts. Murine MSCs were lentiviral transduced to express the genes for enhanced green fluorescent protein, firefly luciferace, and human CXCR4 (hCXCR4). Murine fat tissue was adenoviral transduced to express SDF-1α and red fluorescent protein transgenes. MSCs were cultured on transwells with SDF-1α containing supernatants from transduced fat tissue. The numbers of migrated MSCs in four groups (with hCXCR4 positive (+) or hCXCR4 negative (-) MSCs with or without SDF-1α containing supernatant) were investigated. After 36 h of culture, 9025 ± 925 cells migrated through the membrane of the transwells in group 1 (CXCR4+/SDF-1α+), 4817 ± 940 cells in group 2 (CXCR4-/SDF-1α+), 2050 ± 766 cells in group 3 (CXCR4+/SDF-1α-), and 2108 ± 426 cells in group 4 (CXCR4-/SDF-1α-). Both, the presence of SDF-1α and the expression of hCXCR4 significantly increased the migration rates (p < 0.0001). MSCs overexpressing the CXCR4 receptor by lentiviral transduction are highly attracted by medium from SDF-1α expressing fat tissue in vitro. Thus, SDF-1α activated tissue grafts may be a strategy to enhance site-specific musculoskeletal tissue regeneration., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: An increasing clinical pressure forces academic surgeons in Germany to decide between private life and research activities. How do those people decide and how do they develop their individual career plan?, Material and Methods: In an e-mail survey German orthopedic and trauma surgeons were interviewed on their way of reconciliation of private life, clinical duties and research. The survey included the same questions as the previous survey and a follow-up of 66 % was achieved., Results: The number of consultants in the questioned cohort increased from 44 % to 66 %. More than 80 % reported that the workload had increased which was accompanied by a more clinical orientation of research activities. When asked about personal priorities and wishes leisure time was ranked first, surgical skills, research and income followed in that order. The majority were content with the current situation and career path., Conclusions: This is the first study on occupational conditions in orthopedic and trauma surgeons in a time-line based manner. It became evident that a more clinical orientation of research is needed to match the interests of clinically engaged surgeons in orthopedics and traumatology.

Increasing data are available describing risk factors for the development of local and systemic adverse events following operations using metal-on-metal (MoM) hip implants. The prevalence and clinical relevance of metal-associated problems are, however, still under debate. They can be influenced by type and position of implant as well as patient-specific factors. Patients with small MoM heads (maximum diameter 32 mm) and subgroups of resurfacing arthroplasty can achieve good long-term survival. The use of large head MoM implants (diameters greater than 36 mm), however, is currently not advised due to the unsatisfactory results.

Background: The development of innovative therapies for bone regeneration requires the use of advanced site-specific bone defect small-animal models. The achievement of proper fixation with a murine model is challenging due to the small dimensions of the murine femur. The aim of this investigation was to find the optimal defect size for a murine critical-size bone defect model using external fixation method., Methods: An external fixation device was attached to the right femur of 30 mice. Femoral bone defects of 1 mm (n = 10), 2 mm (n = 10), and 3 mm (n = 10) were created. Wounds were closed without any additional treatment. To investigate bone healing during the 12-wk observation period, x-ray analysis, histomorphology, immunohistochemistry, and μCT scans were performed., Results: MicroCT analyses after 12 wk showed that 3/8 1-mm defects, 5/8 2-mm defects, and 8/8 3-mm defects remained as nonunions. The defect volumes were 0.36 ± 0.42 mm³ (1-mm group), 1.40 ± 0.88 mm³ (2-mm group), and 2.88 ± 0.28 mm³ (3-mm group; P < 0.001, between all groups)., Conclusion: Using external fixation, a defect size of 3 mm is necessary to reliably create a persisting femoral bone defect in nude mice., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: The aim of the study was to evaluate the effects of imageless computer-assisted surgery (CAS) on the accuracy of positioning of the femoral component and on the short-term clinical outcome in hip resurfacing (HR) using a randomised prospective design., Methods: A total of 75 consecutive patients undergoing HR were randomly allocated to CAS and conventional implantation, respectively. Preoperatively and six months post-operatively standardised pelvic anteroposterior X-ray images, the total Western Ontario and McMaster Universities Osteoarthritis Index, the Harris Hip Score and the EQ-5D utility index were evaluated in a blinded manner. The primary end point of the study was a post-operative femoral component malpositioning in five degrees or more either varus or valgus absolute deviation from the planned stem shaft angle., Results: Patient demographics and algofunctional scores did not differ between the CAS and conventional implantation samples. Using CAS fewer femoral components were positioned in five or more degrees absolute deviation (4/37 vs 12/38, Fisher's exact p = 0.047; 95 % confidence interval for the primary end point's incidence difference: +3 %; +39 %); the respective incidences of five or more degrees of varus deviation were 0/37 vs 5/38. One conversion to a stemmed prosthesis (CAS group) was performed for periprosthetic femoral neck fracture. Radiological signs of superolateral femoral neck/implant impingement were observed in two cases (one CAS-based and one conventional implantation)., Conclusions: The accuracy of femoral HR component positioning was significantly improved using CAS. However, one major complication necessitated early revision in the CAS group at six months of observation. Apart from that adverse event no inter-group differences were observed for the short-term clinical outcome. Future studies need to address the clinical long-term relevance of CAS in HR.

Reconstruction of extensive bone defects remains technically challenging and has considerable medical and financial impact on our society. Surgical procedures often require a bone/substitute graft to enhance and accelerate bone repair. Bone autografts are associated with morbidity related to bone harvesting and are limited in quantity. Alternatively, bone allografts expose the patient to the risk of transmission of infectious disease. Synthetic bone graft substitutes, such as calcium sulfates, hydroxyapatite, tricalcium phosphate, and combinations, circumvent some of the disadvantages of auto- and allografts, but have limited indications. Biomedical research has made possible the stimulation of the body's own healing mechanisms, either by delivering exogenous growth factors locally, or by stimulating their local production by gene transfer. Among all known factors having osteoinductive properties, only two bone morphogenetic proteins (for specific indications) and demineralized bone matrix have been approved for clinical use. In addition, ongoing research is exploring the efficacy of cell therapy and tissue engineering. The present report examines the composition, biological properties, indications, clinical experience and regulations of several of the biotherapeutics employed for bone reconstruction.

We tested the hypothesis that erythropoietin (EPO) enhances bone formation after posterolateral spinal fusion (PLF) in a rabbit model. Thirty-four adult rabbits underwent posterolateral intertransverse arthrodesis at the L5-L6 level using 2.0 g autograft per side. The animals were randomly divided into two groups receiving subcutaneous daily injections of either EPO or saline for 20 days. Treatment commenced 2 days preoperatively. Hemoglobin was monitored at baseline and 2, 4, and 6 weeks after fusion surgery. After euthanasia 6 weeks postoperatively, manual palpation, radiographic, and histomorphometric examinations were performed. Bone volume of the fusion mass was estimated by CT after 6 weeks. EPO increased bone fusion volume to 3.85 ccm (3.66-4.05) compared with 3.26 ccm (2.97-3.55) in the control group (p<0.01). EPO treatment improved vascularization of the fusion mass and increased hemoglobin levels (p<0.01). Fusion rate tended to be higher in the EPO group based on manual palpation, CT, and radiographic examinations. For the first time EPO has shown to augment bone formation after autograft PLF in a rabbit model. Increased vascularization provides a partial explanation for the efficacy of EPO as a bone autograft enhancer., (Copyright © 2011 Orthopaedic Research Society.)

Bone graft material is often required for the treatment of osseous defects. However, due to limitations and risks associated with autologous as well as allogenic bone grafting procedures, alternative strategies are needed. In this context, ex vivo tissue engineering (TE) strategies for de novo generation of bone tissue include the combined use of autologous bone-forming cells and three-dimensional (3D) porous scaffold materials serving as structural support for the cells. Three-dimensional cultivation of osteoprogenitor cells presents several challenges, for example, insufficient nutrient and oxygen transport to and removal of waste products from the cells at the interior of the scaffold. By providing physical stimulation of tissue-engineered constructs and resolving mass transport limitations bioreactor systems denote key components for bone TE strategies. A variety of dynamic 3D bioreactor concepts mimicking the native microenvironment in bone tissue, for example, spinner flasks, rotating wall vessel constructs, perfusion bioreactors, and systems based on mechanical or electromagnetic stimulation of cell/scaffold composites, have been developed. These techniques differ considerably with respect to ease of use, cost-effectiveness, and degree of additional osteogenic stimuli, as well as monitoring and manipulation options. This review provides an overview of the concepts, advantages, challenges, and potential future applications associated with current bioreactor systems for bone TE.

Mesenchymal stromal cells (MSCs) can be isolated from various tissues such as bone marrow aspirates, fat or umbilical cord blood. These cells have the ability to proliferate in vitro and differentiate into a series of mesoderm-type lineages, including osteoblasts, chondrocytes, adipocytes, myocytes and vascular cells. Due to this ability, MSCs provide an appealing source of progenitor cells which may be used in the field of tissue regeneration for both research and clinical purposes. The key factors for successful MSC proliferation and differentiation in vitro are the culture conditions. Hence, we here summarize the culture media and their compositions currently available for the differentiation of MSCs towards osteogenic, chondrogenic, adipogenic, endothelial and vascular smooth muscle phenotypes. However, optimal combination of growth factors, cytokines and serum supplements and their concentration within the media is essential for the in vitro culture and differentiation of MSCs and thereby for their application in advanced tissue engineering., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Objective: The use of mesenchymal stem cells (MSCs) for cartilage regeneration is hampered by lack of knowledge about the underlying molecular differences between chondrogenically stimulated chondrocytes and MSCs. The aim of this study was to evaluate differences in phenotype and gene expression between primary human chondrocytes and MSCs during chondrogenic differentiation in three-dimensional (3D) pellet culture (PC)., Materials and Methods: Chondrocytes isolated from cartilage samples obtained during total knee alloarthroplastic procedure (N=8) and MSCs, purified from bone marrow aspirates of healthy donors (N=8), were cultivated in PC under chondrogenic conditions. Immunohistology and quantitative reverse transcribing PCR (RT-PCR) were performed for chondrogenic-specific markers (i.e., Sox9, Collagen II). Global gene expression of the so-cultivated chondrocytes and MSCs was assessed by a novel approach of microarray-based pathway analysis. Refinement of data was done by hypothesis-driven gene expression omnibus (GEO) dataset comparison. Validation was performed with separate samples in transforming growth factor (TGF)β+ or TGFβ- conditions by use of quantitative real-time RT-PCR., Results/conclusions: Chondrogenic commitment of both cell types was observed. Interestingly, chondrocytes demonstrated an upregulated fatty acid/cholesterol metabolism which may give hints for future optimization of culture conditions. The novel microarray-based pathway analysis applied in this study seems suitable for the evaluation of whole-genome based array datasets in case when hypotheses can be backed with already existing GEO datasets. Future experiments should further explore the different metabolic behaviour of chondrocytes and MSC., (Copyright © 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background: Modern hip resurfacing as an alternative for stemmed total hip replacement therapy is associated with a specific risk profile. The aim of this study was therefore to assess the short- to midterm clinical and radiological outcome after introduction of the Durom™ Hip Resurfacing prosthesis in a consecutive series., Materials and Methods: A total of 132 hips (119 patients, 34 female, mean age 48±8,3 years) were evaluated functionally (Harris Hip Score, UCLA and Tegner activity scores) and radiologically with a mean follow-up period of 29 (6-60) months. Furthermore, preoperative ASA- and Charnley-scores, perioperative parameters as well as complications were registered., Results: During the observation period the Harris Hip Score improved by a mean of 36.6 points to 92.5±11.6 points (p<0.01). ULCA and Tegner scores improved by a mean of 3.1 and 1.6 to 7.1 and 4.0 points, respectively. Three patients (2.3%) needed revision surgery due to periprosthetic fracture, prosthesis infection, and aseptic loosening of the femoral component. In four patients (3.1%) an initial migration of the acetabular component not requiring surgical revision was observed radiologically., Conclusion: The Durom™ Hip Resurfacing prosthesis demonstrated a low revision rate and a good mid-term functional and radiological outcome. Due to acetabular cup migrations in a small number of patients we now use an implant with modified surface design.

Background Aims: Combining autologous bone precursor cells with cancellous bone allograft (CBA) offers an appealing strategy for skeletal regeneration. In this context, multipotent mesenchymal stromal cells (MSC) provide an excellent cell source because they are readily harvested from donors, expanded and differentiated in vitro. The aim of this study was to evaluate the proliferation, morphology, osteogenic differentiation and stem cell-related gene expression during static long-term ex vivo cultivation using human MSC and CBA under good manufacturing practice (GMP)-conforming conditions., Methods: MSC were isolated from healthy donors (n = 5) and cultivated on peracetic acid-sterilized CBA in the presence of 10% human platelet-rich plasma without osteogenic supplements. Total protein content, cell-specific alkaline phosphatase (ALP) activity and osteogenic marker gene expression levels were assessed. Stem cell-related gene expression was compared with MSC monolayer cultivation using microarray analysis. Furthermore, cellular distribution and morphology within the porous CBA were visualized by histology and scanning electron microscopy., Results: Effective adhesion, spreading, proliferation and intercellular contact of human MSC within the pores of CBA were observed during the study (< or = 42 days). Cell-specific ALP activity peaked after 3 weeks of cultivation. Gene expression of early, intermediate and late osteogenic marker genes was detectable during long-term cultivation. Microarray-based annotation and biologic interaction network data analysis indicated that expression levels of genes encoding crucial differentiation-regulating proteins and extracellular matrix components involved in the process of osteogenesis were induced in CBA-cultivated MSC., Conclusions: MSC-vitalized CBA offers an attractive GMP-grade bone-filling material. Further research is warranted to evaluate its bone-healing potential in vivo.

Aim: Academic careers often place the burden of research on top of clinical work. This conflict denotes a demanding task especially in surgical disciplines, such as orthopaedics and traumatology. Driven by changes in the social system, concerning, e.g., leisure and parentship, the future organisation of the academic workplace needs to address these individual issues. With the aim to evaluate individual motivation towards combining research, clinical work and private life and to receive suggestions for improvement we conducted a survey addressing residents employed in the orthopaedic and traumatological departments of German university hospitals., Method: With the support of the "Junges Forum der Deutschen Gesellschaft für Orthopädie und Unfallchirurgie (DGOU)", a survey, containing 21 questions about occupational and private issues, was sent out to the orthopaedic and traumatologic departments of 25 university hospitals in Germany. The focus of the questions was set on motivation, priorities and personal resources. It was possible to answer the questions by e-mail; later an anonymous online version of the survey was set in action as well., Results: Completely answered forms were received from n = 105 physicians, of which 88 % were judged as active researchers. Over 50 % of the participants were satisfied with their clinical career and with their research results. 96 % of the survey participants felt sure that most of the research could only be managed during spare time. A majority of the participants supported the idea of receiving leave of absence from clinical duties for research in order to serialise the scientific and clinical careers. Interestingly, when focusing on priority settings, salary aspects were ranked as low as research interests, falling behind the wish for a more intensive surgical training and more leisure time., Conclusions: Individual solutions seem to be necessary to achieve a scheduled efficiency of clinical and research careers. This includes structural developments (like a professionally headed lab) and the establishment of dynamic clinical structures. It seems possible that the clinical organisation of physicians in teams might offer solutions to cope with the demands of surgical training and clinical care on the one hand and research on the other hand., ((c) Georg Thieme Verlag KG Stuttgart . New York.)

Three-dimensional (3D) bone substitute material should not only serve as scaffold in large bone defects but also attract mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs) that are able to form new bone tissue. An additional crucial step is to attract BMSCs from the surface into deeper structures of 3D porous bone substitute scaffolds. Here we show that transient overexpression of CXCR4 in human BMSCs induced by mRNA transfection enhances stromal cell-derived factor-1alpha (SDF-1alpha)-directed chemotactic capacity to invade internal compartments of porous 3D bone substitute scaffolds in vitro and in vivo. In vitro native BMCSs invaded up to 500 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSCs invaded up to 800 mum within 5 days. In addition, 60% downregulation of endogenous SDF-1 transcription in BMSCs by endoribonuclease-prepared siRNA before CXCR4 mRNA transfection enhanced SDF-1alpha-directed migration of human BMSCs by 50%. Implantation of SDF-1alpha-releasing scaffolds seeded with transiently CXCR4-overexpressing BMSCs resulted in an increase of invasion into internal compartments of the scaffolds in a mouse model. In vivo native BMCS invaded up to 250 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSC invaded up to 500 mum within 5 days. Thus, the SDF-1alpha/CXCR4 chemoattraction system can be used to efficiently recruit BMSCs into SDF-1alpha-releasing 3D scaffolds in vitro and in vivo.

Treatment of severe bone defects remains a challenge in orthopaedic surgery and traumatology. Surgical techniques should provide primary stability to reach osseous integration and secondary remodeling of bone grafts and substitute materials. None of the currently available substitute materials provides osteoconduction and osteogenesis comparable to those of human allografts and autografts. To enhance osteoinductive and osteogenetic properties of these implants mesenchymal stem cells are used successfully in bone tissue engineering approaches. The aim of this report is to summarize the currently available data on bone tissue engineering and preliminary experience with a tissue engineered graft in acetabular revision surgery after loosening of a hip replacement.

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., (Copyright 2008 Wiley Periodicals, Inc.)

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.

Early results of contemporary hip resurfacing are encouraging and consequently an increasing number of this procedure has been performed worldwide. A theoretical advantage of hip resurfacing is that failed components can be revised safely and successfully revised to a conventional total hip arthroplasty. As the number of systematically analyzed failures is still limited, however, current data from the literature cannot substantially support this assumption. Our personal results indicate that the conversion of a failed femoral cup (i.e., due to neck fracture or aseptic loosening) to a conventional stem is a relatively simple and safe procedure. If and how potential wear of a firmly integrated acetabular component might have any impact on this type of revision, warrants further investigations. The conversion of acetabular components is influenced by the quality of the remaining pelvic bone stock and can therefore be compared to conventional revision surgery. However, as most providers of hip resurfacings systems only offer one-piece acetabular shells, the possibility of an isolated modular insert exchange is rare. In conclusion, the argument of easy revision surgery after hip resurfacing should be used with care.

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.

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.

The purpose of this study was to evaluate predictors of fracture-induced humeral head ischemia. Between February 1998 and December 2001, 100 intracapsular fractures of the proximal humerus, treated by open surgery, were included in a prospective surgical evaluation protocol (mean age, 60 years; minimum, 21 years; maximum, 88 years; 45 men; 57 right shoulders). Fracture morphology was assessed following a structured questionnaire and based on radiographic and intraoperative findings. Perfusion was assessed intraoperatively by observation of backflow after a borehole was drilled into the central part of the head in all shoulders and by intraosseous laser Doppler flowmetry in 46. Good predictors of ischemia were the length of the metaphyseal head extension (accuracy, 0.84 for calcar segments <8 mm), the integrity of the medial hinge (accuracy, 0.79 for disrupted hinge), and the basic fracture pattern (accuracy, 0.7 for combined types 2, 9, 10, 11, and 12). Moderate and poor predictors of ischemia were fractures consisting of four fragments (accuracy, 0.67), angular displacement of the head (accuracy, 0.62 for angulations over 45 degrees ), the amount of displacement of the tuberosities (accuracy, 0.61 for displacement over 10 mm), glenohumeral dislocation (accuracy, 0.49), head-split components (accuracy, 0.49), and fractures consisting of three fragments (accuracy, 0.38). When the above criteria (anatomic neck, short calcar, disrupted hinge) were combined, positive predictive values of up to 97% could be obtained. The most relevant predictors of ischemia were the length of the dorsomedial metaphyseal extension, the integrity of the medial hinge, and the basic fracture type determined with the binary description system.

The high sensitivity of pentagastrin stimulation in detecting primary or metastatic medullary thyroid cancer (MTC) suggests widespread expression of the corresponding receptor type on human MTC. Indeed, autoradiographic studies demonstrated cholecystokinin (CCK)-B/gastrin receptors not only in >90% of MTCs but in a high percentage of small cell lung cancers and potentially a variety of gastrointestinal adenocarcinomas. In a pilot study, we have demonstrated the feasibility of radiolabeled gastrin-I to target CCK-B receptor-expressing tissues in vivo in animals and patients (T. M. Behr et al., Eur. J. Nucl. Med., 25: 424-430, 1998). The aim of the present study was to systematically optimize, in a preclinical model, suitable radioligands for targeting CCK-B receptors in vivo. For this purpose, a variety of CCK/gastrin-related peptides, all having in common the COOH-terminal CCK-receptor binding tetrapeptide sequence Trp-Met-Asp-PheNH2 or derivatives thereof, were studied. They were radioiodinated by the Iodogen or Bolton-Hunter procedures. The peptides tested were members of the gastrin- or cholecystokinin families or possessed characteristics of both, which differ by the intramolecular position of a tyrosyl moiety (occurring in native or sulfated form). Their stability and affinity were studied in vitro and in vivo; their biodistribution and therapeutic efficacy were tested in nude mice bearing s.c. human MTC xenografts. Diethylene-triamine-pentaacetate derivatives of suitable peptides were synthesized, evaluated, and labeled with (111)In. All members of the CCK or gastrin family were stable in serum (with t(1/2)s of several hours at 37 degrees C); nevertheless, the stability of those peptides was highest that bore the NH2-terminal pGlu residues (e.g., big gastrin, gastrin-I, caerulein, and others) or D-amino acids. In accordance to their comparably low affinity, nonsulfated members of the CCK family showed fairly low uptake in the tumor and other CCK-B receptor-expressing tissues (e.g., the stomach). Sulfated CCK derivatives performed significantly better but additionally displayed a high uptake in normal, CCK-A receptor-expressing tissues (such as the liver/gallbladder, pancreas, and bowel). Best tumor uptake and tumor:nontumor ratios were obtained with members of the gastrin family, probably because of their selectivity and affinity for the CCK-B receptor subtype. Pilot therapy experiments in MTC bearing animals showed significant antitumor efficacy as compared with untreated controls. (111)In-Labeled diethylene-triamine-pentaacetate derivatives of minigastrin showed excellent targeting of CCK-B receptor-expressing tissues in animals and a normal human volunteer. These data suggest that CCK/gastrin analogues may be a useful new class of receptor binding peptides for diagnosis and therapy of CCK-B receptor-expressing tumors, such as MTC or small cell lung cancer. Nonsulfated gastrin derivatives may be preferable because of their CCK-B receptor selectivity, and hence, lower accretion in normal CCK-A receptor-expressing organs. Further preclinical as well as clinical studies are ongoing.

In Saxon prisons the risk of infection by HIV or hepatitis viruses differs from those in the Western German federal states in certain aspects. Particularly the intravenous use of drugs plays a minor role. Nevertheless there are other risks of transmission, and the general consumption of (non-injectable) drugs is estimated to be very high. Therefore a need exists for adequate and suitable measures of prevention, taking into account the specific situation in Saxon prisons. Supposedly the chances to structurally counteract the development of the same intravenous drug use habits as in Western Germany are low.