Fabian Duttenhoefer

In vivo comparison of hard tissue regeneration with ovine mesenchymal stem cells processed with either the FICOLL method or the BMAC method

INTRODUCTION The aim of this work was to analyse the suitability of mesenchymal stem cell isolation by FICOLL density centrifugation and the closed bone marrow aspirate concentrate (BMAC) system for sinus augmentation with bovine bone mineral (BBM) in the sheep model. METHODS 16 sheep underwent sinus augmentation with BBM and MSCs; they were divided between two groups with survival points of 8 and 16 weeks. For the FICOLL control arm three, and for the BMAC test arm, five augmentations were performed for each time point. The derived cell numbers were counted; a colony forming unit (CFU) assay was performed; the pluripotency of the MSCs was proved; histological and histomorphometrical analysis were performed. RESULTS The approach of using BBM and MSCs in combination with fibrin adhesive was sufficient for new bone formation as the FICOLL experiment indicated. However, due to significantly lower cell numbers isolated using the BMAC in sheep, less new bone was formed in the test arm. CONCLUSIONS The BMAC system is well suited for human MSC isolation but it needs to be optimized to fit sheep cell characteristics if it is to be used in this animal model.

Multivalent dendrimers presenting spatially controlled clusters of binding epitopes in thermoresponsive hyaluronan hydrogels

The controlled presentation of biofunctionality is of key importance for hydrogel applications in cell-based regenerative medicine. Here, a versatile approach was demonstrated to present clustered binding epitopes in an injectable, thermoresponsive hydrogel. Well-defined multivalent dendrimers bearing four integrin binding sequences and an azido moiety were covalently grafted to propargylamine-derived hyaluronic acid (Hyal-pa) using copper-catalyzed alkyne-azide cycloaddition (CuAAC), and then combined with pN-modified hyaluronan (Hyal-pN). The dendrimers were prepared by synthesizing a bifunctional diethylenetriamine pentaacetic acid core with azido and NHBoc oligo(ethylene glycol) aminoethyl branches, then further conjugated with solid-phase synthesized RGDS and DGRS peptides. Azido terminated pN was synthesized by reversible addition-fragmentation chain transfer polymerization and reacted to Hyal-pa via CuAAC. Nuclear magnetic resonance (NMR), high performance liquid chromatography, size exclusion chromatography and mass spectroscopy proved that the dendrimers had well-defined size and were disubstituted. NMR and atomic absorption analysis confirmed the hyaluronan was affixed with dendrimers or pN. Rheological measurements demonstrated that dendrimers do not influence the elastic or viscous moduli of thermoresponsive hyaluronan compositions at a relevant biological concentration. Finally, human mesenchymal stromal cells were encapsulated in the biomaterial and cultured for 21days, demonstrating the faculty of this dendrimer-modified hydrogel as a molecular toolbox for tailoring the biofunctionality of thermoresponsive hyaluronan carriers for biomedical applications.

Long-Term Survival of Dental Implants Placed in the Grafted Maxillary Sinus: Systematic Review and Meta-Analysis of Treatment Modalities

Background A prevalent modality to increase the amount of available bone prior to implantation is grafting of the maxillary sinus. Multiple factors such as the surgical technique, moment of implant placement as well as grafting materials and membranes are known to affect implant survival. However, the role of different factor combinations and associated reciprocal effects remain unclear. Conventional statistical methods do not consider inconsistency of study designs and do not take covariables into account. Hence, a systematic research and meta-analysis was conducted to investigate the influence of various treatment modalities on implant survival in the grafted maxillary sinus. Materials and Methods A meta-analysis was conducted according to the PRISMA guidelines. Articles published from 1980 through January 2013 were electronically and manually searched in MEDLINE (Ovid), the Cochrane Register of Controlled Trials, the Database of Abstracts of Effects, and the Cochrane Database of Systematic Reviews. Clinical reports on single intervention sinus augmentation with root-form implants, a minimum of 10 patients and 6 months of loading were eligible for inclusion if implant survival was stated or calculable. Results were calculated by non-parametric univariate Kaplan-Meier analysis and Bayesian multivariate interval-censored Cox regression. Results A total of 122 publications on 16268 endosseous implants placed in grafted maxillary sinus were included. The treatment parameters surgical approach, grafting material and implant type showed no selective preference. However, application of membranes showed a significantly reduced hazard-ratio, independent of other co-factors. Conclusions The use of membranes is the most significant factor to achieve long-term implant survival in sinus augmentation procedures. More data exceeding 3 years follow-up are needed to address prospective confounding and improve clinical evidence.

Follow-Up of Implant Survival Comparing Ficoll and Bone Marrow Aspirate Concentrate Methods for Hard Tissue Regeneration with Mesenchymal Stem Cells in Humans

Abstract Objective: Clinical follow-up of implant survival in 11 patients comparing two different methods for mesenchymal stem cell (MSC) isolation (Ficoll and bone marrow aspirate concentrate [BMAC]) applied in maxillary sinus augmentation. Methods: Mononuclear cells, including MSCs, were concentrated with either Ficoll (control group, n=6 sinus) or BMAC (test group, n=12 sinus) and transplanted in combination with bovine bone mineral. A total of 50 implants were placed in a second surgical intervention (17 Ficoll/33 BMAC) and loaded after 4 months. Overall implant survival was assessed with a Kaplan-Meier model using package survival under R. Results: Implant survival of the Ficoll group was 100% compared with the BMAC group, which had 93.4% survival (95% confidence interval, 0.849–1). The difference between the groups was not significant (p=0.381). Conclusion: The BMAC system is an effective and suitable “chair-side” method for clinical application in hard tissue regeneration.

Injectable Hyaluronan Hydrogels with Peptide-Binding Dendrimers Modulate the Controlled Release of BMP-2 and TGF-β1

BMP-2 and TGF-β1 released from injectable thermoresponsive hydrogels are studied in the presence and absence of branched macromolecules bearing BMP-2 or TGF-β1 affinity binding peptides. The synthesized branched macromolecules and the gelling compositions before and after loading with either BMP-2 or TGF-β1 are characterized physico-chemically and show a significantly lower amount of proteins released in the presence of the affinity binding peptide macromolecules. This study illustrates the potential of affinity binding peptide functionalized dendrimers to modulate the local delivery and availability of growth factors important for musculoskeletal regeneration therapies.

Magnetic resonance imaging in zirconia‐based dental implantology

OBJECTIVES X-ray-based planning and post-implantation assessment of titanium implants is the commonly accepted standard to date. However, new implant materials such as zirconia (ZrO2 ) have become available, and magnetic resonance imaging may be a valuable alternative with these implants. The present in vitro study investigated artifacts produced by titanium and zirconia implants in magnetic resonance imaging (MRI) and assessed the accuracy of pre-implant planning and post-implantation assessment comparing MRI to standard X-ray-based imaging modalities: Orthopantomogram (OPT), cone beam (CBCT), and computed tomography (CT). MATERIALS AND METHODS Twelve porcine mandibles were prepared and scanned (MRI, OPT, CBCT, μCT), and bone height above the nerve canal was measured. Specimens were implanted with either two titanium or zirconia implants and rescanned to investigate the influence of implant materials on post-implantation assessment. MRI and μCT artifacts were quantified with implants embedded in gelatin phantoms and porcine specimens. RESULTS Compared with CBCT set as standard, μCT, OPT, and MRI showed similar accuracy in pre-op bone height measurements. Post-implantation, while titanium implants induced a strong B0 -field distortion resulting in extensive signal voids, zirconia implants were clearly depictable with only minor distortions. CONCLUSIONS Excellent contrast, limited artifacts, radiation-free and accurate implant assessment may indicate that MRI is a valuable imaging alternative for zirconia-based implant dentistry.

Long-term peri-implant bone level changes of non-vascularized fibula bone grafted edentulous patients

Long-term results of reconstructions and prosthetic rehabilitation of patients presenting severely atrophied edentulous ridges remains a challenge for clinicians. Among the various available augmentation materials there is evidence that avascular fibula bone grafts possess a reliable resistance against resorption and may thus provide a valuable source to reduce the loss of vertical bone height after reconstruction of the severely atrophied mandible and maxilla. The purpose of the present study was to assess long-term crestal bone level stability in avascular fibula bone grafts. 8 edentulous female patients (average age 70.6 years) with Class-VI-atrophy and less than 5 mm residual bone volume received onlay-grafting with avascular fibula bone grafts and were monitored with a mean observation time of 133.7 months (121-186). A total of 39 implants were placed in the maxilla and mandible. Three patients received immediate and five patients delayed implant placement 3 months after grafting. All patients were provided with bar-retained dentures. Postoperative evaluation included clinical implant success (Buser) and radiographic examinations (orthopantomogram) to quantify crestal bone resorption. Grafting was successfully performed in all patients with no regrafting necessary. All implants but one, lost 2 years after abutment connection, remained successfully integrated and fulfilled the Buser criteria, rendering to a success rate of 97%. Mean bone resorption after 10 years was mesial 1.4 mm and distal 1.4 mm at each implant-site. Maximum bone resorption occurred between postoperative and first year, thereafter no significant resorption was measured in re-examinations up to 15 years. Avascular fibula grafts are a reliable bone graft for augmentation procedures in atrophied edentulous ridges. Dental implants that integrated in the autogenous fibular bone grafts showed a stable crestal peri-implant bone level up to 15 years after implant placement.

Endothelial Progenitor Cell Fraction Contained in Bone Marrow-Derived Mesenchymal Stem Cell Populations Impairs Osteogenic Differentiation.

In bone tissue engineering (TE) endothelial cell-osteoblast cocultures are known to induce synergies of cell differentiation and activity. Bone marrow mononucleated cells (BMCs) are a rich source of mesenchymal stem cells (MSCs) able to develop an osteogenic phenotype. Endothelial progenitor cells (EPCs) are also present within BMC. In this study we investigate the effect of EPCs present in the BMC population on MSCs osteogenic differentiation. Human BMCs were isolated and separated into two populations. The MSC population was selected through plastic adhesion capacity. EPCs (CD34+ and CD133+) were removed from the BMC population and the resulting population was named depleted MSCs. Both populations were cultured over 28 days in osteogenic medium (Dex+) or medium containing platelet lysate (PL). MSC population grew faster than depleted MSCs in both media, and PL containing medium accelerated the proliferation for both populations. Cell differentiation was much higher in Dex+ medium in both cases. Real-time RT-PCR revealed upregulation of osteogenic marker genes in depleted MSCs. Higher values of ALP activity and matrix mineralization analyses confirmed these results. Our study advocates that absence of EPCs in the MSC population enables higher osteogenic gene expression and matrix mineralization and therefore may lead to advanced bone neoformation necessary for TE constructs.

Can Mesenchymal Stem Cells and Novel Gabapentin-Lactam Enhance Maxillary Bone Formation?

PURPOSE Novel gabapentin-lactam (GBP-L) has shown its potency in enhancing new bone formation (NBF) in vitro. The objective of the present preclinical trial was to investigate the in vivo performance of GBP-L. MATERIALS AND METHODS Bilateral sinus floor augmentations in 10 adult sheep were conducted. Bovine bone mineral (BBM) and mesenchymal stem cells (MSCs) combined with novel GBP-L were placed into the test sinus of each sheep. The BBM and MSCs alone served as the control on the contralateral side. Simultaneously, 3 dental implants were inserted in each maxillary sinus. The animals were sacrificed after 8 and 16 weeks, and the amount of NBF was analyzed using histomorphometry. The osteogenic potency of the MSCs was demonstrated using the colony-forming unit and differentiation assay. Statistical evaluation was performed using the Wilcoxon signed rank test and 3-factorial nonparametric analysis of variance. RESULTS The histologic examination showed NBF in tight contact with the original bone in the control and test groups. The NBF was not significantly different between the test and control sites (P > .05). However, a highly significant difference in NBF between the apical and coronal sites in the specimens from the control and test groups was detected (P < .05). GBP-L did not alter the multipotency of the MSCs or impair NBF. CONCLUSIONS Bone formation is initiated from the residual alveolar crest and along the implant. The elected mode of GBP-L application did not induce faster NBF. Alternate forms of application (eg, slow release or systemic administration) might clarify the controversial in vitro findings.

Sp1-dependent regulation of PPARα in bone metabolism.

PURPOSE Successful repair and regeneration in bone tissue engineering vastly depends on proper interaction between the tissue-engineered construct and the recipients immune system. In clinical application, adverse responses to bioartificial implants may result in chronic inflammation and loss of the implant. It is known that prolonged inflammation linked to NF-κB inflammatory pathways inhibits bone-forming activity of osteoblast cells. Contributing to orchestrate inflammatory processes, the ligand-activated transcription factor peroxisome proliferator-activated receptor alpha (PPARα) holds inhibitory effects on NF-κB and CEBβ activity. Sp1, a widely expressed transcription factor, has been linked to PPAR pathways, cellular homeostasis, and responsiveness to environmental perturbation. Formerly not being characterized, the role of PPARα in inflammatory-mediated bone loss requires further investigation. The aim of the present study was to identify regulatory transcription factor binding sites (TFBS) on the PPAR alpha promoter and to assess the role of Sp1 and associated proteins in its regulation. MATERIALS AND METHODS In a first set of experiments, polymerase chain reaction assessed the presence of PPARα gene expression in isolated murine bone tissue. Deletion mutagenesis was performed on the human PPARα (hPPARα) promoter gene, and the deletion constructs were transiently transfected to murine osteoblasts to identify important TFBS. PPARα promoter-driven reporter gene expression was monitored in response to overexpression and repression of Sp1 to analyze functional transcription factor recruitment to the PPARα promoter. RESULTS This study could demonstrate that the full-length hPPARα promoter contains inhibiting promoter regions and that hPPARα basal expression can be significantly increased by deletion mutagensis. Sp1 TFBS proved functional in the regulation of PPARα promoter activity, and the first five Sp1 motifs on the PPARα promoter were sufficient to significantly increase PPARα expression. Additional transient co-transfection experiments could not detect any direct effect of NF-κB/IκB downstream pathway on the regulation of PPARα promoter activity. Taken together, we could demonstrate that Sp1 plays a key role in transcriptional regulation of PPARα promoter activity and gene expression. CONCLUSION This study provides further insight on Sp1-dependent PPARα regulatory mechanisms and suggests that Sp1-regulated PPARα expression plays a key role in inflammatory mediated bone loss.