Safety and feasibility of cell-based therapy of autologous bone marrow-derived mononuclear cells in plate-stabilized proximal humeral fractures in humans.

BACKGROUND: Local implantation of ex vivo concentrated, washed and filtrated human bone marrow-derived mononuclear cells (BMC) seeded onto ß-tricalciumphosphate (TCP) significantly enhanced bone healing in a preclinical segmental defect model. Based on these results, we evaluated in a first clinical phase-I trial safety and feasibility of augmentation with preoperatively isolated autologous BMC seeded onto ß-TCP in combination with angle stable plate fixation for the therapy of proximal humeral fractures as a potential alternative to autologous bone graft from the iliac crest. METHODS: 10 patients were enrolled to assess whether cell therapy with 1.3 × 106 autologous BMC/ml/ml ß-TCP, collected on the day preceding the definitive surgery, is safe and feasible when seeded onto ß-TCP in patients with a proximal humeral fracture. 5 follow-up visits for clinical and radiological controls up to 12 weeks were performed. RESULTS: ß-tricalciumphosphate fortification with BMC was feasible and safe; specifically, neither morbidity at the harvest site nor at the surgical wound site were observed. Neither local nor systemic inflammation was noted. All fractures healed within the observation time without secondary dislocation. Three adverse events were reported: one case each of abdominal wall shingles, tendon loosening and initial screw perforation, none of which presumed related to the IND. CONCLUSIONS: Cell therapy with autologous BMC for bone regeneration appeared to be safe and feasible with no drug-related adverse reactions being described to date. The impression of efficacy was given, although the study was not powered nor controlled to detect such. A clinical trial phase-II will be forthcoming in order to formally test the clinical benefit of BMC-laden ß-TCP for PHF patients. Trial registration The study was registered in the European Clinical Trial Register as EudraCT No. 2012-004037-17. Date of registration 30th of August 2012. Informed consent was signed from all patients enrolled.

Cell-Based Therapy by Autologous Bone Marrow-Derived Mononuclear Cells for Bone Augmentation of Plate-Stabilized Proximal Humeral Fractures: A Multicentric, Randomized, Open Phase IIa study.

Proximal humerus fractures are common in an aging population. The standard operative treatment is open reduction internal fixation (ORIF) using an angular stable plate. However, this procedure has complications such as a relatively high rate of secondary dislocation, humeral head necrosis or nonunion caused by delayed bony consolidation. Autologous bone marrow mononuclear cells (BMC) combined with a ß-TCP scaffold could support bone healing and is considered clinically safe. This multicentric, randomized, open phase IIa clinical trial (Clinical Trials. Gov Identifier: NCT02803177, Eudra CT No: 2015-001820-51) evaluated whether autologous BMC with ß-TCP in addition to ORIF reduces the incidence of secondary dislocations in patients with proximal humerus fracture. Ninty-four patients equally divided between verum group (BMC+ß-TCP) and control group (ß-TCP only) were targeted and calculated. At the time of planned interim evaluation, ie, enrolment of 56 patients, no statistical difference in secondary dislocations or complications was demonstrated in either group after an observation period of 12 weeks. Radiographic bone healing and DASH score to determine shoulder function were comparable between both groups. Bone marrow harvest and BMC transplantation did not result in any severe adverse events. Therefore, the study was terminated after the interim analysis, as no other result could be expected. From the study results, it can be concluded that the application of autologous BMC is well tolerated, and bone healing can be achieved. Augmentation of bone defects with ß-TCP could be shown to be feasible and might be considered in other clinical situations.

Human endothelial-like differentiated precursor cells maintain their endothelial characteristics when cocultured with mesenchymal stem cell and seeded onto human cancellous bone.

INTRODUCTION: Cancellous bone is frequently used for filling bone defects in a clinical setting. It provides favourable conditions for regenerative cells such as MSC and early EPC. The combination of MSC and EPC results in superior bone healing in experimental bone healing models. MATERIALS AND METHODS: We investigated the influence of osteogenic culture conditions on the endothelial properties of early EPC and the osteogenic properties of MSC when cocultured on cancellous bone. Additionally, cell adhesion, metabolic activity, and differentiation were assessed 2, 6, and 10 days after seeding. RESULTS: The number of adhering EPC and MSC decreased over time; however the cells remained metabolically active over the 10-day measurement period. In spite of a decline of lineage specific markers, cells maintained their differentiation to a reduced level. Osteogenic stimulation of EPC caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression. Osteogenic stimulation of MSC significantly increased their metabolic activity whereas collagen-1α and alkaline phosphatase gene expressions declined. When cocultured with EPC, MSC's collagen-1α gene expression increased significantly. CONCLUSION: EPC and MSC can be cocultured in vitro on cancellous bone under osteogenic conditions, and coculturing EPC with MSC stabilizes the latter's collagen-1α gene expression.

Do antiosteoporotic drugs improve bone regeneration in vivo?

PURPOSE: Treatment of complex fractures in the elderly is a challenge for operative reconstruction due to degraded bone structure. Early peri-operative bone anabolic treatment could improve new bone formation, avoid implant loosening and accelerate fracture healing. METHODS: To compare the osteoanabolic potential of different drugs after distraction osteogenesis, 168 female Sprague-Dawley rats underwent lengthening of the right femur using a monolateral external fixator. Animals were randomly divided into six groups: vehicle-injected group, PTH(1-34), raloxifen, strontium ranelate, alendronate and simvastatin. Histomorphometry, CT-scanning, DEXA- and biomechanical analysis were performed to evaluate new bone formation, callus volume, mineralisation and biomechanical strength. Expression of bone metabolic mediators and differentiation indicators of distracted and intact bone were examined by RT-PCR and western blot. RESULTS: Histological analysis showed significant increase of the bone mass after treatment with PTH(1-34), raloxifen and strontium ranelate (p = 0.02). Raloxifen increased bone mineral content (BMC) of the whole distracted femur significantly (p = 0.007). Callus volume was significantly larger in the PTH(1-34), raloxifen and simvastatin groups (p = 0.001) compared to control. Ultimate load of distracted new formed bone was increased in PTH(1-34) and raloxifen groups. It seems that PTH(1-34) and raloxifen have a stronger effect on bone where a repair response is activated. Strontium ranelate demonstrates similar effects to PTH regarding new bone formation but shows low values for mineralisation and biomechanical strength. CONCLUSION: This study suggests that peri-operative treatment of complex and/or osteoporotic fractures with PTH(1-34) and raloxifen might be useful as a stimulator of bone formation and mineralisation to shorten the consolidation time in humans.

Improvement of Bone Healing by Neutralization of microRNA-335-5p, but not by Neutralization of microRNA-92A in Bone Marrow Mononuclear Cells Transplanted into a Large Femur Defect of the Rat.

Transplanted bone marrow mononuclear cells (BMC) support the healing of large bone defects. Neutralization of microRNA (MiR) that negatively affects key processes of the reparative response in BMC might help to further improve the beneficial effect of transplanted BMC in bone healing. Hence, the aim of this study was to evaluate if the neutralization of MiR-92A (vascularization) and MiR-335-5p (osteogenic differentiation) in BMC using specific antiMiRs leads to a further improvement of the BMC-supported therapy of large bone defects. BMC transiently transfected with antiMiR- 92A, antiMiR-335, antiMiR-92A, and antiMiR-355 or control antiMiR were seeded on ß-TCP (beta-tricalcium phosphate) and placed in a femoral large bone defect (5 mm) in Sprague-Dawley rats. Ultimate load as well as osseous integration of the ß-TCP-scaffolds were significantly improved in the antiMiR-335 group compared to the control group after 8 weeks, whereas neutralization of antiMiR-92A lead to an improvement of early vascularization after 1 week, but not to enhanced bone healing after 8 weeks. We demonstrated that the targeted inhibition of MiRs in transplanted BMC is a new approach that enhances BMC-supported bone healing.

Cultivation of EPC and co-cultivation with MSC on ß-TCP granules in vitro is feasible without fibronectin coating but influenced by scaffolds' design.

INTRODUCTION: Meanwhile, the osteoconductive properties of frequently used synthetic bone grafts can be improved by the use of osteoinductive cells and growth factors. Nevertheless, the cultivation of endothelial progenitor cells (EPC) seems to be difficult and requires a pre-conditioning of the scaffolds with fibronectin. Additionally, the influence of the scaffolds' design on cell cultivation is not fully elucidated. METHODS: As scaffold, a commercially available ß-tricalcium phosphate was used. 5 × 105 EPC, or 5 × 105 MSC or a combination of each 2.5 × 105 cells was seeded onto the granules. We investigated seeding efficiency, cell morphology, cell metabolism, adherence, apoptosis and gene expression of EPC and MSC in this in vitro study on days 2, 6 and 10. RESULTS: Total number of adherent cells was higher on the ß-TCP without fibronectin coating. The number of cells in all approaches significantly declined when a solid ß-TCP was used. Metabolic activity of MSC was comparable throughout the scaffolds and increased until day 10. Additionally, the amount of supernatants VEGF was higher for MSC than for EPC. DISCUSSION: Our results demonstrate that a coating of the scaffold for successful cultivation of EPC in vitro is not necessary. Furthermore, our study showed that structural differences of the scaffolds significantly influenced cell adherence and metabolic activity. Thereby, the influence on EPC seems to be higher than on MSC.

Influence of the induced membrane filled with syngeneic bone and regenerative cells on bone healing in a critical size defect model of the rat's femur.

INTRODUCTION: The induced membrane 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 rat's 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. Since little effort has been made to replace the need for autologous bone this study was performed to elucidate the influence of different stem cells and the membrane itself on bone healing in a critical size femur defect model in rats. Especially the question should be addressed whether the use of stem cells seeded on a ß-TCP scaffold is equivalent to syngeneic bone as defect filling in combination with the induced membrane technique. MATERIALS AND METHODS: A total of 96 male Sprague-Dawley (SD) rats received a 10 mm critical size defect of the femur, which was stabilized by a plate osteosynthesis and filled with PMMA cement. In a second step the spacer was extracted and the defects were filled with syngeneic bone, ß-TCP with MSC + EPC or BM-MNC. In order to elucidate the influence of the induced membrane on bone defect healing the induced membrane was removed in half of the operated femurs. The defect area was analysed 8 weeks later for bone formation (osteocalcin staining), bone mineral density (BMD) and bone strength (3-point bending test). RESULTS: New bone formation, bone mineral density and bone stiffness increased significantly, if the membrane was kept. The transplantation of biologically active material (syngeneic bone, stem cells on b-TCP) into the bone defect mostly led to a further increase of bone healing. Syngeneic bone had the greatest impact on bone healing however defects treated with stem cells were oftentimes comparable. CONCLUSION: For the first time we demonstrated the effect of the induced membrane itself and different stem cells on critical size defect healing. This could be a promising approach to reduce the need for autologous bone transplantation with its' limited availability and donor site morbidity.

Comparison of three different types of scaffolds preseeded with human bone marrow mononuclear cells on the bone healing in a femoral critical size defect model of the athymic rat.

Large bone defects often pose major difficulties in orthopaedic surgery. The application of long-term cultured stem cells combined with a scaffold lead to a significant improvement of bone healing in recent experiments but is strongly restricted by European Union law. Bone marrow mononuclear cells (BMC), however, can be isolated and transplanted within a few hours and have been proven effective in experimental models of bone healing. The effectivity of the BMC-supported therapy might be influenced by the type of scaffold. Hence, we compared three different scaffolds serving as a carrier for BMC in a rat femoral critical size defect with regard to the osteogenic activity in the defect zone. Human demineralized bone matrix (DBM), bovine cancellous bone hydroxyapatite ceramic (BS), or ß-tricalcium phosphate (ß-TCP) were seeded with human BMC and hereafter implanted into critically sized bone defects of male athymic nude rats. Autologous bone served as a control. Gene activity was measured after 1 week, and bone formation was analysed histologically and radiologically after 8 weeks. Generally, regenerative gene expression (BMP2, RUNX2, VEGF, SDF-1, and RANKL) as well as bony bridging and callus formation was observed to be most pronounced in defects filled with autologous bone, followed in descending order by DBM, ß-TCP, and BS. Although DBM was superior in most aspects of bone regeneration analysed in comparison to ß-TCP and BS, the level of autologous bone could not be attained.

Tissue engineered vascularized periosteal flap enriched with MSC/EPCs for the treatment of large bone defects in rats.

Vascularized periosteal flaps are used for complex cases if the reconstruction of large bone defects is necessary in modern trauma and orthopedic surgery. In this study, we combined this surgical procedure with ß­TCP scaffold and mesenchymal stem cells (MSCs) + endothelial progenitor cells (EPCs) as a tissue engineering approach to obtain optimum conditions for bone healing in rats. A critical size femoral defect was created in 80 rats allocated into 4 groups. Defects were treated according to the following protocol: i) vascularized periosteal flap alone; ⅱ) vascularized periosteal flap + ß­TCP scaffold; ⅲ) vascularized periosteal flap + ß­TCP scaffold + ligated vascular pedicle; and ⅳ) vascularized periosteal flap + ß­TCP scaffold + MSCs/EPCs. After 8 weeks, femur bones were extracted and analyzed for new bone formation, vascularization, proliferation and inflammatory processes and strength. Bone mineral density (BMD) and biomechanical stability at week 8 were highest in group 4 (flap + ß­TCP scaffold + MSCs/EPCs) compared to all the other groups. Stability was significantly higher in group 4 (flap + ß­TCP scaffold + MSCs/EPCs) in comparison to group 3 (ligated flap + ß­TCP scaffold). BMD was found to be significantly lower in group 3 (ligated flap + ß­TCP scaffold) compared to group 1 (flap) and group 4 (flap + ß­TCP scaffold + MSCs/EPCs). The highest density of blood vessels was observed in group 4 (flap + ß­TCP + MSCs/EPCs) and the values were significantly increased in comparison to group 3 (ligated flap), but not to group 1 (flap) and group 2 (flap + ß­TCP). The highest amounts of proliferating cells were observed in group 4 (flap + ß­TCP scaffold + MSC/EPCs). The percentage of proliferating cells was significantly higher in group 4 (flap + ß­TCP scaffold + MSCs/EPCs) in comparison to all the other groups after 8 weeks. Our data thus indicate that critical size defect healing could be improved if MSCs/EPCs are added to ß-TCP scaffold in combination with a periosteal flap. Even after 8 weeks, the amount of proliferating cells was increased. The flap blood supply is essential for bone healing and the reduction of inflammatory processes.

Effect of the harvest procedure and tissue site on the osteogenic function of and gene expression in human mesenchymal stem cells.

Evidence has indicated that mesenchymal stem cells (MSCs) harvested with the Reamer/Irrigator/Aspirator (RIA) procedure exhibited an improved osteogenic differentiation capability compared with MSCs obtained by bone marrow aspiration from the iliac crest. In the present study, we hypothesized that the harvest procedure indeed influences the osteogenic activity of human MSCs more than the tissue site itself. Concentration [by colony forming unit-fibroblast (CFU-F) assay], calcification (by von Kossa staining), collagen deposition, gene expression and the gene methylation of the bone morphogenetic protein (BMP)-2 pathway [BMP2, SMAD5 and runt-related transcription factor 2 (RUNX2)], the Wnt pathway [WNT3, dickkopf-1 (DKK1), low-density lipoprotein receptor­related protein 5 (LRP5) and ß-catenin] and osteogenic genes [alkaline phosphatase (ALP), collagen, type I, alpha 1 (COL1A) and osteocalcin] were analyzed in the MSCs isolated intraoperatively from the iliac crest with a spoon (n=14), from the femur with a spoon (n=7), from the femur with the RIA procedure (n=13) and from the iliac crest by fine-needle aspiration (n=8, controls). A Bonferroni-Holm corrected p-value <0.05 indicated a statistically significant difference. The concentration of CFU-F in the MSCs was increased in the RIA debris in comparison with that in the iliac crest aspirates (trend) and the femur (spoon, significant). Calcium deposition was highest in the femur-derived MSCs (by RIA) and was significantly increased in comparison with that in the iliac crest-derived MSCs (spoon, aspirate). The gene expression of BMP2, SMAD5, RUNX2, osteocalcin, and COL1A was significantly increased in the femur-derived MSCs (spoon) and the iliac crest aspirate derived-MSCs in comparison with that in the femur-derived MSCs (by RIA). There was no significant diversity between the samples obtained using a spoon (from the femur or iliac crest). Calcium deposition and osteogenic gene expression decreased significantly with the increasing passage number in all the samples. The methylation of genes did not correlate with their respective gene expression and inconsistent differences were observed between the groups. Herein, we provide evidence that the harvest procedure is a critical factor in the osteogenesis of MSCs in vitro. The MSCs isolated from the femur and iliac crest using a spoon exhibit no significant differences. The altered gene expression and function of the femur-derived MSCs (by RIA) may be due to the harsh isolation procedure. The variable differentiation ability of the MSCs, which depends on the harvest site and the harvest technique, as well as the rapid loss of the osteogenic differentiation capacity with the increasing culture duration should be taken into consideration when using MSCs as a potential therapeutic application for bone tissue engineering.

Treatment of Large Bone Defects with a Vascularized Periosteal Flap in Combination with Biodegradable Scaffold Seeded with Bone Marrow-Derived Mononuclear Cells: An Experimental Study in Rats.

INTRODUCTION: The surgical treatment of large bone defects continues to pose a major challenge in modern trauma and orthopedic surgery. In this study we test the effectiveness of a tissue engineering approach, using three-dimensional (3D) ß-tricalcium phosphate (ß-TCP) scaffolding plus bone marrow-derived mononuclear cells (BM-MNCs), combined with a vascularized periosteal flap, in a rat femur critical size defect model. METHODS: Eighty rats were randomly allocated into four equal groups. Under general anesthesia, critical size defects were created on their femurs and were treated with (1) Vascularized periosteal flap alone, (2) Vascularized periosteal flap+ß-TCP scaffolding, (3) Vascularized periosteal flap+ß-TCP scaffolding+ligated vascular pedicle, and (4) Vascularized periosteal flap+ß-TCP scaffolding+BM-MNCs. After 4 and 8 weeks animals were euthanized and the bone defects were harvested for analysis of new bone formation, vascularization, and strength using histology, immunohistology, micro-CT, and biomechanical testing, respectively. RESULTS: Group 1: (P. flap) Increase in new bone formation and vascularization. Group 2: (P. flap+scaffold) Increase in new bone formation and vascularization. Group 3: (P. flap+scaffold+ligated vascular pedicle) No new bone formation and no vascularization. Group 4: (P. flap+scaffold+BM-MNCs) A significant (p < 0.05) increase was seen in new bone formation, vascularization, and strength in bones treated with flaps, scaffold, and BM-MNCs, when compared with the other treatment groups. CONCLUSION: Combining a vascularized periosteal flap with tissue engineering approach (ß-TCP scaffolding and BM-MNC) results in significantly improved bone healing in our rat femur critical size bone defect model.

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 rat's 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.

Cell-based therapy by implanted human bone marrow-derived mononuclear cells improved bone healing of large bone defects in rats.

UNLABELLED: QUESTION/AIM: Cell-based therapy by cultivated stem cells (mesenchymal stem cells [MSC] and endothelial progenitor cells [EPC]) in a large-sized bone defect has already shown improved vascularization and new bone formation. However, these methods are clinically afflicted with disadvantages. Another heterogeneous bone marrow cell population, the so-called human bone marrow-derived mononuclear cells (BMC), has nevertheless been used clinically and showed improved vascularization in ischemic limbs or in the myocardium. For clinical use, a certified process has been established; thus, BMC were isolated from bone marrow aspirate by density gradient centrifugation, washed, cleaned, and given back to patients within several hours. This investigation tested the ability of human BMC seeded on beta-tricalcium phosphate (ß-TCP) and placed into a large bone defect in rats to improve the bone healing process in vivo. METHODS: Human EPC were isolated from buffy coat, and MSC or BMC, respectively, were isolated from bone marrow aspirate by density gradient centrifugation. 1.0×10(6) cells were loaded onto 750 µL ß-TCP (0.7-1.4 mm). Large femoral defects (6 mm) in athymic rats were created surgically and stabilized with an internal fixateur. The remaining defects were filled with ß-TCP granules alone (group 1), ß-TCP+EPC/MSC (group 2), or ß-TCP+BMC (group 3). After 8 weeks, histomorphometric analysis (new bone formation), radiological microcomputer tomography analysis (bony bridging), and biomechanical testing (three-point bending) were achieved. Moreover, a tumorigenicity study was performed to evaluate the safety of BMC implantation after 26 weeks. For statistical analysis, the Kruskal-Wallis test was used. RESULTS: Eight weeks after implantation of EPC/MSC or BMC, respectively, we detected a more significant new bone formation compared to control. In group 2 and 3, bony bridging of the defect was seen. In the control group, more chondrocytes and osteoid were detected. In the BMC and EPC/MSC group, respectively, less chondrocytes and a significantly more advanced bone formation were observed. The biomechanical stability of the bone regenerate was significantly enhanced if BMC and EPC/MSC, respectively, were implanted compared to control. Moreover, no tumor formation was detected either macroscopically or histologically after 26 weeks of BMC implantation. DISCUSSION: Implanted BMC suggest that a heterogeneous cell population may provide a powerful cellular therapeutic strategy for bone healing in a large bone defect in humans.

Characterization of bone marrow mononuclear cells on biomaterials for bone tissue engineering in vitro.

Bone marrow mononuclear cells (BMCs) are suitable for bone tissue engineering. Comparative data regarding the needs of BMC for the adhesion on biomaterials and biocompatibility to various biomaterials are lacking to a large extent. Therefore, we evaluated whether a surface coating would enhance BMC adhesion and analyze the biocompatibility of three different kinds of biomaterials. BMCs were purified from human bone marrow aspirate samples. Beta tricalcium phosphate (ß-TCP, without coating or coated with fibronectin or human plasma), demineralized bone matrix (DBM), and bovine cancellous bone (BS) were assessed. Seeding efficacy on ß-TCP was 95% regardless of the surface coating. BMC demonstrated a significantly increased initial adhesion on DBM and ß-TCP compared to BS. On day 14, metabolic activity was significantly increased in BMC seeded on DBM in comparison to BMC seeded on BS. Likewise increased VEGF-synthesis was observed on day 2 in BMC seeded on DBM when compared to BMC seeded on BS. The seeding efficacy of BMC on uncoated biomaterials is generally high although there are differences between these biomaterials. Beta-TCP and DBM were similar and both superior to BS, suggesting either as suitable materials for spatial restriction of BMC used for regenerative medicine purposes in vivo.

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.

Effect of sonic hedgehog/ß-TCP composites on bone healing within the critical-sized rat femoral defect.

The creation of entirely synthetically derived bone substitute materials which are as effective as autologous bone grafts is desirable. Osteogenesis involves the concerted action of several proteins within a signaling cascade. Hedgehog proteins act upstream of this cascade, inducing the expression of various bone morphogenetic proteins (BMPs) and promoting physiological bone healing. Therefore, the hypothesis that hedgehog signaling in bone defects improves bone healing more than BMP signaling alone was tested. Recombinant N-terminal sonic hedgehog protein (N-SHh), BMP-2 or a combination of the two was added to ß-tricalcium phosphate (ß-TCP) and 5-mm femoral midshaft defects in nude rats were filled with these composites. The defects were stabilized with mini-plates. After eight weeks, the animals were sacrificed and the femora were explanted. The radiological evaluation was followed by a three-point bending test and histological examination. BMP-2/ß-TCP composites showed a trend of increased stiffness compared with the controls (ß-TCP without protein). N-SHh/ß-TCP composites had lower stiffness compared with the control group and the N-SHh/BMP-2/ß-TCP composites also had lower average stiffness compared with the controls (all not significant). Histomorphometry, however, revealed abundant cartilage and bone core formation in the N-SHh-composite groups. The sum of the new cartilage and bone was highest in the combination group N-SHh/BMP-2 (not significant). The addition of N-SHh to bone substitute materials appears to delay bone healing at the applied concentration and observation time but also showed a trend for higher amounts of ossifying cartilage.

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 ß-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 5×10(5) rat EPCs. Vascularization after 1 week was significantly improved when EPCs were seeded onto BG40, compared to implanting BG40 alone. This indicates that Ca(2+) release improves EPC differentiation and is useful for enhanced early vascularization in critical size bone defects.

Effect of the Hedgehog-inhibitor cyclopamine on mice with osteosarcoma pulmonary metastases.

Chemoresistant metastases of osteosarcoma in humans limit survival in approximately one third of patients. Furthermore, aggressive chemotherapy can lead to side effects and occurrence of secondary malignancies in long time survivors. Therefore, supplemental medical strategies are worthwhile. The well-directed manipulation of cancer-signaling-cascades is an appealing approach. Targeting of the Hedgehog-pathway in cancer has led to promising results in vitro as well as in vivo in a number of different tumor types. Recently, the impact of cyclopamine, which inhibits Hedgehog signaling by binding to the receptor Smoothened, was shown in different human osteosarcoma cell lines in vitro and in vivo. In the present study we examined the influence of cyclopamine on early pulmonary metastases in vivo. Murine osteosarcoma cells, OS-50, were injected into the lateral tail vein of young BALB/c mice. Treatment with subcutaneous cyclopamine injections began after three days. Two weeks later, the animals were sacrificed and the number of pulmonary metastases was counted. We could observe a trend towards decreased metastases in the cyclopamine group (~20%). On the other hand, remarkable side effects were caused by the cyclopamine/ethanol/triolein preparation (mainly skin ulcerations).

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 neutrophil's 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.