Caroline Seebach

Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism and growth behaviour of human mesenchymal stem cells (MSC) in vitro

INTRODUCTION Various synthetic bone-graft substitutes are used commercially as osteoconductive scaffolds in the treatment of bone defects and fractures. The role of bone-graft substitutes is changing from osteoconductive conduits for growth to an delivery system for biologic fracture treatments. Achieving optimal bone regeneration requires biologics (e.g. MSC) and using the correct scaffold incorporated into a local environment for bone regeneration. The need for an unlimited supply with high quality bone-graft substitutes continue to find alternatives for bone replacement surgery. MATERIALS AND METHODS This in vitro study investigates cell seeding efficiency, metabolism, gene expression and growth behaviour of MSC sown on six commercially clinical available bone-graft substitutes in order to define their biological properties: synthetic silicate-substituted porous hydroxyapatite (Actifuse ABX), synthetic alpha-TCP (Biobase), synthetic beta-TCP (Vitoss), synthetic beta-TCP (Chronos), processed human cancellous allograft (Tutoplast) and processed bovines hydroxyapatite ceramic (Cerabone). 250,000 MSC derived from human bone marrow (n=4) were seeded onto the scaffolds, respectively. On days 2, 6 and 10 the adherence of MSC (fluorescence microscopy) and cellular activity (MTT assay) were analysed. Osteogenic gene expression (cbfa-1) was analysed by RT-PCR and scanning electron microscopy was performed. RESULTS The highest number of adhering cells was found on Tutoplast (e.g. day 6: 110.0+/-24.0 cells/microscopic field; p<0.05) followed by Chronos (47.5+/-19.5, p<0.05), Actifuse ABX (19.1+/-4.4), Biobase (15.7+/-9.9), Vitoss (8.8+/-8.7) and Cerabone (8.1+/-2.2). MSC seeded onto Tutoplast showed highest metabolic activity and gene expression of cbfa-1. These data are confirmed by scanning electron microscopy. The cell shapes varied from round-shaped cells to wide spread cells and cell clusters, depending on the bone-graft substitutes. Processed human cancellous allograft is a well-structured and biocompatible scaffold for ingrowing MSC in vitro. Of all other synthetical scaffolds, beta-tricalcium phosphate (Chronos) have shown the best growth behaviour for MSC. DISCUSSION Our results indicate that various bone-graft substitutes influence cell seeding efficiency, metabolic activity and growth behaviour of MSC in different manners. We detected a high variety of cellular integration of MSC in vitro, which may be important for bony integration in the clinical setting.

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.

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 β-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 β-TCP, MSCs seeded on β-TCP, EPCs seeded on β-TCP, and coculture of MSCs and EPCs seeded on β-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 β-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.

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, TGFβ 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

Use of Recombinant Activated Factor VII (Novoseven) in Trauma and Surgery: Analysis of Outcomes Reported to an International Registry

The objective was to evaluate the efficacy and safety of recombinant activated factor VII in patients with massive bleeding. Forty-five patients with severe massive hemorrhage requiring= 14 transfusion units of packed red blood cells received recombinant activated factor VII. Postdrug blood loss and transfusion requirements were assessed, and mortality was compared with predicted outcomes. Blood loss was markedly reduced in 40 of 43 (93.0%) patients, and transfusion requirements decreased after recombinant activated factor VII administration. Mortality rate in trauma patients who had massive hemorrhage was significantly reduced compared with predictions using scoring systems. This may be associated with the use of recombinant activated factor VII. This study failed to demonstrate an improvement in surgical patients. The absence of concurrent controls prevents definitive conclusions regarding actual safety or efficacy of recombinant activated factor VII.

Functional outcome and complications following PHILOS plate fixation in proximal humeral fractures

OBJECTIVES Proximal humeral fractures account for approximately 5% of all fractures. New plating techniques have been developed to improve stability. The purpose of this study was to evaluate functional outcome following plate fixation with the Proximal Humeral Internal Locking System (PHILOS) and to analyze potential implant-related complications. METHODS The PHILOS plate was used for internal fixation of displaced proximal humeral fractures in 28 patients (20 females, 8 males; mean age 60.7+/-12.9 years). Fractures were caused by low-energy trauma (fall from standing height) in 21 patients, and by an accident while skiing or cycling in seven patients. Involvement was on the right in 16 cases and on the left in 12 cases. According to the Neer classification, 8, 12, and 8 patients had displaced 2-, 3-, or 4-part fractures, respectively. All patients received a similar physical therapy program following internal fixation with the PHILOS plate. The patients were assessed clinically and radiographically after a mean follow-up of 25.2+/-11.8 months. Functional outcome was assessed using the Constant-Murley score adjusted for age and gender. Range of motion and shoulder abduction strength were measured. The patients were also evaluated with the Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire and a visual analog scale (VAS). Complications during the follow-up period were recorded. RESULTS Twenty fractures (71.4%) healed in good anatomical position. At the end of the follow-up period, the mean Constant-Murley score was 57.9+/-21.7, and the mean age- and gender-adjusted Constant-Murley score was 67.5+/-23.6. The results were excellent or good in 16 patients (57.1%), moderate in one patient (3.6%), and poor in 11 patients (39.3%). The mean DASH and VAS scores were 28.3+/-24.3 and 75.4+/-21.2, respectively. Eleven complications (39.3%) were seen during the follow-up period. Reoperation was required in eight patients (72.3%). Complications included avascular necrosis of the humeral head in two patients (7.2%), subacromial impingement in six patients (21.4%), loosening of a locking head screw in one patient (3.6%), and transiently decreased radial nerve sensation in two patients (7.2%). Subacromial impingement was mainly caused by the superior plate position. CONCLUSION Our results demonstrate that the PHILOS plate provides sufficient fracture stabilization in the treatment of proximal humeral fractures of elderly patients.

High calcium bioglass enhances differentiation and survival of endothelial progenitor cells, inducing early vascularization in critical size bone defects.

Early vascularization is a prerequisite for successful bone healing and endothelial progenitor cells (EPC), seeded on appropriate biomaterials, can improve vascularization. The type of biomaterial influences EPC function with bioglass evoking a vascularizing response. In this study the influence of a composite biomaterial based on polylactic acid (PLA) and either 20 or 40% bioglass, BG20 and BG40, respectively, on the differentiation and survival of EPCs in vitro was investigated. Subsequently, the effect of the composite material on early vascularization in a rat calvarial critical size defect model with or without EPCs was evaluated. Human EPCs were cultured with β-TCP, PLA, BG20 or BG40, and seeding efficacy, cell viability, cell morphology and apoptosis were analysed in vitro. BG40 released the most calcium, and improved endothelial differentiation and vitality best. This effect was mimicked by adding an equivalent amount of calcium to the medium and was diminished in the presence of the calcium chelator, EGTA. To analyze the effect of BG40 and EPCs in vivo, a 6-mm diameter critical size calvarial defect was created in rats (n = 12). Controls (n = 6) received BG40 and the treatment group (n = 6) received BG40 seeded with 5×105 rat EPCs. Vascularization after 1 week was significantly improved when EPCs were seeded onto BG40, compared to implanting BG40 alone. This indicates that Ca2+ release improves EPC differentiation and is useful for enhanced early vascularization in critical size bone defects.

Alteration of Masquelet's induced membrane characteristics by different kinds of antibiotic enriched bone cement in a critical size defect model in the rat's femur.

The Masquelet technique for the treatment of large bone defects consists of a 2-stage procedure. In the first stage, a polymethylmethacrylate (PMMA) cement spacer is inserted into the bony defect of a rats femur and over a period of 2-4 weeks a membrane forms that encapsulates the defect/spacer. In a second operation the membrane is opened, the PMMA spacer is removed and the resulting cavity is filled with autologous bone. Different kinds of bone cements are available, with or without supplemental antibiotics. Both might influence the development and the characteristics of the induced membrane which might affect the bone healing response. Hence, this comparative study was performed to elucidate the effect of different bone cements with or without supplemental antibiotics on the development of an induced membrane in a critical size femur defect model in rats. A total of 72 male SD rats received a 10mm critical size defect of the femur which was stabilised by a plate osteosynthesis and filled with either Palacos+Gentamycin, Copal Gentamycin+Vancomycin, Copal+Gentamycin+Clindamycin or Copal Spacem. The induced membranes were analysed after two, four and six weeks (wks) after insertion of the cement spacers (n=6/group). Paraffin embedded histological sections of the membrane were microscopically analysed for membrane thickness, elastic fibres, vascularisation and proliferation by an independent observer blinded to the group setup. The thickness of the induced membrane increased significantly from 2 wks (553 μm) to 6 wks (774 μm) in group Palacos+Gentamycin whereas membrane thickness decreased significantly in groups Copal+Gentamycin+Clindamycin (682-329 μm) and Copal Spacem (916 μm to 371 μm). The comparison between the groups revealed significantly increased membrane thickness in group Palacos+Gentamycin and Copal Gentamycin+Vancomycin in comparison to group Copal+Gentamycin+Clindamycin six weeks after induction. However, the fraction of elastic fibres was significantly increased in groups Copal+Gentamycin+Clindamycin (71%, 80%) and Copal Spacem (82%, 81%) after 2 and 4 weeks in comparison to the groups Palacos+Gentamycin (56%, 57%) and Copal Gentamycin+Vancomycin (63%, 69%). Those differences however were partly diminished after 6 wks. The ratio of immature (vWF+) to more mature (CD31+) blood vessels increased significantly in groups Palacos+Gentamycin and Copal Gentamycin+Vancomycin whereas no significant alterations were noted in groups Copal+Gentamycin+Clindamycin and Copal Spacem. For the first time we demonstrated that thickness and proportion of elastic fibres in induced membranes were influenced by the type of cement and the kind of supplemental antibiotics being used. Whether these alterations of the induced membrane have an effect on bone healing remains to be proven in future studies.

Safety evaluation of a bioglass–polylactic acid composite scaffold seeded with progenitor cells in a rat skull critical-size bone defect

Treating large bone defects represents a major challenge in traumatic and orthopedic surgery. Bone tissue engineering provides a promising therapeutic option to improve the local bone healing response. In the present study tissue biocompatibility, systemic toxicity and tumorigenicity of a newly developed composite material consisting of polylactic acid (PLA) and 20% or 40% bioglass (BG20 and BG40), respectively, were analyzed. These materials were seeded with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) and tested in a rat calvarial critical size defect model for 3 months and compared to a scaffold consisting only of PLA. Serum was analyzed for organ damage markers such as GOT and creatinine. Leukocyte count, temperature and free radical indicators were measured to determine the degree of systemic inflammation. Possible tumor occurrence was assessed macroscopically and histologically in slides of liver, kidney and spleen. Furthermore, the concentrations of serum malondialdehyde (MDA) and sodium oxide dismutase (SOD) were assessed as indicators of tumor progression. Qualitative tissue response towards the implants and new bone mass formation was histologically investigated. BG20 and BG40, with or without progenitor cells, did not cause organ damage, long-term systemic inflammatory reactions or tumor formation. BG20 and BG40 supported bone formation, which was further enhanced in the presence of EPCs and MSCs. This investigation reflects good biocompatibility of the biomaterials BG20 and BG40 and provides evidence that additionally seeding EPCs and MSCs onto the scaffold does not induce tumor formation.

Trauma-activated polymorphonucleated leukocytes damage endothelial progenitor cells: probable role of CD11b/CD18-CD54 interaction and release of reactive oxygen species.

Endothelial progenitor cells (EPCs) and polymorphonucleated leukocytes (PMNLs) migrate to and accumulate at the site of tissue injury where they express complementary sets of surface receptors (CD11b/CD18, CD54), suggesting a possible cellular interaction. Trauma-activated PMNLs release inflammatory mediators and reactive oxygen species (ROS) produced by the NADPH oxidase, which may negatively impact EPCs. To characterize the interactions between PMNLs and EPCs, we identified common surface receptors and measured the role played by NADPH oxidase and neutrophil elastase. Polymorphonucleated leukocytes were obtained from either healthy volunteers or multiple-trauma patients. After stimulation with either n-formyl-l-methionyl-l-leucyl-l-phenylalanine or phorbol 12-myristate 13-acetate, the PMNLs were incubated with DiL-prestained EPCs in a ratio of 20:1 for 3 h. Early EPCs were isolated from buffy coat. Endothelial progenitor cell killing was measured by flow cytometry, and necrotic EPCs were identified by measuring the uptake of 7-aminoactinomycin. We found that blocking CD11b, CD18, or CD54 on the EPC surface with monoclonal antibodies or blocking the intracellular production of ROS by neutralizing neutrophils NADPH oxidase with a diphenyliodonium chloride pretreatment protected EPCs, enhancing its survival, whereas inhibiting neutrophil elastase had no effect on survival. Furthermore, we observed that native PMNLs obtained from multiple-trauma patients damaged EPCs, whereas native PMNLs from healthy volunteers did not. Our results demonstrate that EPCs and PMNLs do interact via complementary receptors and that this interaction results in PMNL-derived ROS-induced EPC damage. The effect of neutrophil-derived elastase was found to be negligible. These findings suggest that EPC damage by activated PMNLs may contribute to impaired wound healing observed after severe trauma.