Predicting and Promoting Human Bone Marrow MSC Chondrogenesis by Way of TGFß Receptor Profiles: Toward Personalized Medicine.

The use of human mesenchymal stromal cells (hMSCs) for cartilage regeneration has been hampered by the inherent donor variation of primary monolayer expanded cells. Although CD markers are typically used to characterize cell populations, there is no correlation between CD marker profile and functional outcomes. Therefore, we aimed to discover novel predictive MSC chondrogenesis markers. The chondrogenic potential of primary human bone marrow MSCs (hBMSCs) over multiple passages was assessed by standard pellet culture. We confirmed that the ratio of TGFß-RI/TGFß-RII at the time of cell recovery from the tissue culture plastic reliably predicted chondrogenic potential. Furthermore, it is possible to prospectively characterize any human BMSC cell population as responders or non-responders with respect to chondrogenic differentiation potential. Transient increase of the ratio with siRNA knockdown of TGFß-RII reproducibly recovered the chondrogenic differentiation ability of non-responsive MSCs. Together this offers an opportunity to produce a more functionally characterized cell population for use in autologous cartilage repair therapies.

Correction to: Treatment of stage II medication-related osteonecrosis of the jaw with necrosectomy and autologous bone marrow mesenchymal stem cells.

In the Original publication of the article, the co-author has been misspelled as Fabian Duttenhöfer in the article "Treatment of stage II medication-related osteonecrosis of the jaw with necrosectomy and autologous bone marrow mesenchymal stem cells" published in October 2017, Volume 105, Issue 4 of Odontology. The correct name is "Fabian Duttenhoefer".

Clinical analysis of MatrixMANDIBLE Preformed Reconstruction Plate design.

MatrixMANDIBLE Preformed Reconstruction Plates (MMPRPs) were developed to overcome laborious bending procedures of conventional reconstruction plates. The design comprises three sizes with a nonbendable centerpiece and two bendable sections (proximal and distal). According to the surgical protocol unnecessary parts are trimmed after the last used screw hole. In the present retrospective study postoperative radiographs from 130 patients (average age 63 years) that received treatment with MMPRPs were assessed. There was no statistical correlation between plate-size, location (left/right) or age. 82.98% of the small and 91.80% of the medium MMPRPs were trimmed by at least the terminal screw hole of the ramus part. In all patients receiving a large MMPRP, the terminal screw hole of the ramus was unused accordingly all inserted large MMPRPs were trimmed by at least the terminal screw hole. The majority of the bridged defects were located within the area of the body indicating a feasible plate design. With the emergence of solid free form fabrication of Ti-alloys and economic need to reduce the waste of resources this study may help to further improve the MMPRP design and prevent the loss of medical-grade titanium.

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.

Pilot investigation of the molecular discrimination of human osteoblasts from different bone entities.

In oral and maxillofacial surgery, autologous grafts from the iliac crest remain the 'gold standard' for alveolar ridge reconstruction, whereas intraoral bone grafts are considered in smaller defects. To date, a comparison of the osteogenic potential of osteoblasts with regard to their tissue origin is missing. Primary osteoblasts have proven useful for the investigation of the tissue-specific osteogenic properties. The present study compares primary human alveolar (aHOBs) and iliac osteoblasts (iHOBs) derived from three female patients undergoing routine intraoral bone grafting. Proliferation potential of the osteoblasts was evaluated using real-time impedance monitoring. Relative gene expression of bone specific biomarkers was analyzed and quantified using quantitative polymerase chain reactions (qPCR). Immunohistochemistry and phase contrast microscopy were performed, as well as alkaline phosphatase assay and alizarin red staining to visualize morphology and mineralization capacity. A twofold faster proliferation rate of aHOBs compared with iHOBs (130 h vs. 80 h) was observed. Alkaline phosphatase activity and alizarin red staining in both HOBs indicated similar mineralization capacity. Gene expression of seven genes (BMP1, CSF-1, TGFBR1, ICAM1, VCAM1, SPP1 and DLX5) was significantly higher in iHOB than in aHOB samples. These data suggest a higher osteogenic potential of osteoblasts derived from the iliac crest compared with primary osteoblasts from the alveolar bone and may lead to a better understanding of the molecular impact of bone cells from different bone entities on bone regeneration in alveolar ridge reconstructions.

The validity of surgical clips as radiographic markers for the tumour resection cavity in head and neck cancer treatment.

BACKGROUND: A prerequisite of irradiation after advanced head and neck tumour resection is the accurate localization of the tumour resection margin. The purpose of the following study is to evaluate the use of surgical clips placed in the tumour resection margins for use as radiographic markers to facilitate focussed adjuvant radiation therapy. MATERIALS: To evaluate whether the clips remain predictive for the resection margin, we analysed the deviation of each clip in two postoperative CT scans on different days. Bone registration points were used to fuse the two CT scans in the region of the primary tumour and the distances between corresponding clips were measured. RESULTS: The tumour resection margins were labelled with an average of 18 titanium clips. In total 282 clips were evaluated. Metric analysis of clip deviation between the two postoperative CT scans found a mean distance of 4.5 mm ± 2.5 mm with a range of 0.5-11.8 mm. No significant statistical relationship of the clip differences as a function of time, the method of reconstruction or administered radiotherapy could be demonstrated. CONCLUSION: Placement of surgical clips in the cavity walls after complete tumour resection provides an easy and inexpensive approach for defining resection margins and allows for increased accuracy of adjuvant treatment. Clinical trial number DRKS00007534.

Injectable Hyaluronan Hydrogels with Peptide-Binding Dendrimers Modulate the Controlled Release of BMP-2 and TGF-ß1.

BMP-2 and TGF-ß1 released from injectable thermoresponsive hydrogels are studied in the presence and absence of branched macromolecules bearing BMP-2 or TGF-ß1 affinity binding peptides. The synthesized branched macromolecules and the gelling compositions before and after loading with either BMP-2 or TGF-ß1 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.

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.

Marking of tumor resection borders for improved radiation planning facilitates reduction of radiation dose to free flap reconstruction in head and neck cancer surgery.

Accurate localization of tumor resection borders is crucial for adjuvant radiotherapy. An improvement to adjuvant radiotherapy with the reduction of radiation doses to free flap reconstruction by virtual navigation procedures and titanium clips was evaluated. Thirty-three patients with oral cancer were prospectively included in the study. Following complete local excision of the primary tumor, resection borders were marked virtually using a navigation pointer and with titanium ligature clips. Postoperative delineation of tumor resection borders was examined. In five patients with microvascular free flap reconstruction a reduction of the radiation dose to the free flap reconstruction was achieved. The tumor resection borders in 30 patients were marked with titanium ligature clips. Surgical clip insertion was successful in 91%. We demonstrate a significant relationship between the reconstruction volume and the part of the target volume which will receive a reduced radiation dose. A cumulative dose of 60 Gy was administered to the target volume and a significant reduction of the administered radiation dose to the center of the flap could be demonstrated. We demonstrate an accurate delineation of the tumor resection margins. These improvements in tumor resection margin delineation allow for increased accuracy in adjuvant treatment and a reduction of radiation dose to the vascular free flap reconstruction.

Effect of Short-Term Stimulation with Interleukin-1ß and Differentiation Medium on Human Mesenchymal Stromal Cell Paracrine Activity in Coculture with Osteoblasts.

INTRODUCTION: Human mesenchymal stromal cells (hMSCs) exhibit the potential to accelerate bone healing by enhanced osteogenic differentiation. Interleukin-1ß is highly expressed during fracture healing and has been demonstrated to exert a significant impact on the differentiation behaviour of hMSCs. Here, we investigate the effect of 2-hour IL-1ß stimulation on the differentiation and paracrine activity of hMSCs in coculture with osteosarcoma cells in vitro. METHODS: hMSCs from 3 donors were incubated for 2 hours with 10 ng/mL IL-1ß and subsequently cocultured with MG63-GFP cells either in control or in differentiation medium in a transwell system for 28 days. Genetic and functional effects were investigated. RESULTS: hMSCs cultured in control medium exhibited a regulatory effect on cocultured MG63-GFP cells, resulting in upregulation of osteogenic gene expression in combination with increased ALP activity. However, while stimulated hMSCs cultured under differentiation conditions exhibit signs of osteogenic differentiation, osteogenic differentiation also caused an impaired regulatory effect on the cocultured MG63-GFP cells. CONCLUSION: Short stimulation of hMSCs has the potential to modify their long-term behaviour. In addition, undifferentiated hMSCs are able to regulate osteoblast differentiation; however, this regulatory function is lost upon osteogenic differentiation in vitro. This offers a novel approach for clinical cell therapy protocols.

Survival analysis of dental implants and implant-retained prostheses in oral cancer patients up to 20 years.

OBJECTIVES: The purpose of this study was to evaluate the long-term survival rate and potential influencing factors of dental implants and implant-retained prostheses in oral cancer patients who had undergone surgical tumor resection. MATERIAL AND METHODS: In the present study, 157 patients (95 females and 62 males with a mean age of 53.7 years) with 830 implants were included. All patients were diagnosed with a malignant tumor in the oral cavity and had undergone ablative surgery. In 55 patients (292 implants), the surgical procedure was followed by an additional radiochemotherapy (RCT) before implant placement. Nicotine users who received RCT were excluded from this study. Patients were clinically examined every 6 or 12 months according to a standard procedure. RESULTS: Of the 830 examined implants, 450 were placed in the maxilla and 380 in the mandible. A total of 65 implants were lost, 36 in the maxilla and 29 in the mandible; of these, 42 implants (65%) were documented as lost due to the patient's death. The mean observation period was 121 months. The cumulative survival rate was 94.9% at 3 years and 92.5% at 7 years. With an observation period up to 20 years, the cumulative survival rate remained constant after 11 years with 90.8%. Age, gender, and localization (maxilla/mandible) of implants did not show any influence on the survival of the implants. However, radiochemotherapy was determined as a significant factor influencing the survival rate. CONCLUSIONS: The results of this study demonstrate that the survival rate of implants was significantly lower in oral cancer patients who had been treated by ablative surgery and additional radiochemotherapy than in patients without RCT. Since there is no significant difference in the mortality rate of patients with additional RCT compared to patients who underwent sole ablative surgery, the higher loss ratio is due to a late failure of osseointegration. CLINICAL RELEVANCE: Dental implants in oral cancer patients who had been treated by ablative surgery show a high and steady cumulative survival rate after 11 years. Implant survival of patients with additional RCT is significantly lower. Non-smoking-irradiated patients seem to have a better implant survival.

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.

Follow-up of implant survival comparing ficoll and bone marrow aspirate concentrate methods for hard tissue regeneration with mesenchymal stem cells in humans.

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.

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.

Direct cell-cell contact between mesenchymal stem cells and endothelial progenitor cells induces a pericyte-like phenotype in vitro.

Tissue engineering techniques for the regeneration of large bone defects require sufficient vascularisation of the applied constructs to ensure a sufficient supply of oxygen and nutrients. In our previous work, prevascularised 3D scaffolds have been successfully established by coculture of bone marrow derived stem cells (MSCs) and endothelial progenitor cells (EPCs). We identified stabilising pericytes (PCs) as part of newly formed capillary-like structures. In the present study, we report preliminary data on the interactions between MSCs and EPCs, leading to the differentiation of pericyte-like cells. MSCs and EPCs were seeded in transwell cultures, direct cocultures, and single cultures. Cells were cultured for 10 days in IMDM 10% FCS or IMDM 5% FCS 5% platelet lysate medium. Gene expression of PC markers, CD146, NG2, αSMA, and PDGFR-ß, was analysed using RT-PCR at days 0, 3, 7, and 10. The upregulation of CD146, NG2, and αSMA in MSCs in direct coculture with EPCs advocates the MSCs' differentiation towards a pericyte-like phenotype in vitro. These results suggest that pericyte-like cells derive from MSCs and that cell-cell contact with EPCs is an important factor for this differentiation process. These findings emphasise the concept of coculture strategies to promote angiogenesis for cell-based tissue engineered bone grafts.

The effect of gabapentin-lactam hydroxamic acid derivatives on ovine osteoblast proliferation and phenotype: perspectives for tissue engineering application.

PURPOSE: Modern bone tissue engineering associated with mesenchymal stem cells (MSCs) provides promising treatment alternatives for the loss of bone, one of the foremost challenges in oral and craniofacial surgery today. The effect of gabapentin-lactam (GBP-L) and its analogs on osteogenic differentiated MSCs has not yet been deciphered. Consequently, this study investigates the effect of novel trans-8-tertbutylgabapentin-lactam (trans-8-TB-GBP-L) hydroxamic acid derivatives on metabolism, proliferation, and physiologic mineralization characteristics of ovine osteoblast cells. MATERIALS AND METHODS: Osteoblasts were extracted and prepared from sheep femoral heads and cultured in medium enriched with hydroxamic acid derivatives of trans-8-TB-GBP-L. The cell proliferation rate, cell metabolism, cell viability, and basic osteoblastic function were assessed. RESULTS: After 3 and 5 days of incubation, no significant increase in DNA content was detected in any of 12 test groups versus the control group. However, after 8 days of incubation, a significant increase of DNA contents in the test groups containing nanomolar concentrations of trans-8-TB-GBP-L hydroxamic acid derivatives was found. No significant aberration in metabolic activity was detected when any of the test substances were applied. ALP displayed similar activity rates among the test groups and the control at all time points. Calcification of osteoblastic cells occurred solely when nanomolar concentrations were used. CONCLUSION: Trans-8-TB-GBP-L hydroxamic acid derivatives do not interfere with physiologic function and phenotype of ovine osteoblasts. However, when applied at nanomolar concentrations, the assessed GBP-L derivatives significantly increased the cell proliferation rate after 8 days of incubation, indicating a dose-response curve with the maximum peak at nanomolar concentration and a retarded drug response between 5 and 8 days.

Effect of gabapentin-lactam and gamma-aminobutyric acid/lactam analogs on proliferation and phenotype of ovine mesenchymal stem cells.

PURPOSE: Classic tissue engineering consists of three components: scaffold, cells, and growth or differentiation factors. Currently, expensive bone morphogenetic proteins are the most common substance used for hard tissue regeneration. An alternative could be gamma-aminobutyric acid/lactam (GABA-lactam) analogs. MATERIALS AND METHODS: The effects of gabapentin-lactam, cis- and trans-8-tertbutyl-GABA-pentinlactam (trans-TB-GBP-L), and phenyl-GABA-lactam were tested in this study on ovine mesenchymal stem cell (MSC) proliferation. MSCs were selected from bone marrow aspirate concentrate by plastic adherence and amplified. Aliquots of the cells were incubated in medium, with four different concentrations of the GABA-lactam analogs dissolved in dimethyl sulfoxide. Cells in medium with and without dimethyl sulfoxide served as controls. Cell proliferation was tested with a nonradioactive assay. Before and after GABA-lactam analog influence, the MSC character was evaluated by the ability of the cells to differentiate into osteoblasts, chondrocytes, and adipocytes. RESULTS: Proliferation was significantly increased under the influence of the analogs, depending on their concentration. MSCs cultured in 1 nmol/L trans-TB-GBP-L showed the highest proliferation rate. The MSC character was not altered. CONCLUSIONS: GABA-lactam analogs could be suited to stimulate MSC proliferation for tissue engineering applications. Further in vivo studies are necessary to evaluate the possible clinical potential of GABA-lactam analogs for hard tissue regeneration.

Evaluation of bone substitute materials: comparison of flat-panel based volume CT to conventional multidetector CT.

Over the last decade tissue engineering has emerged as a key factor in bone regeneration within the field of cranio-maxillofacial surgery. Despite this in vivo analysis of tissue-engineered-constructs to monitor bone rehabilitation are difficult to conduct. Novel high-resolving flat-panel based volume CTs (fp-VCT) are increasingly used for imaging bone structures. This study compares the potential value of novel fp-VCT with conventional multidetector CT (MDCT) based on a sheep sinus floor elevation model. Calcium-hydroxyapatite reinforced collagen scaffolds were populated with autologous osteoblasts and implanted into sheep maxillary sinus. After 8, 16 and 24 weeks MDCT and fp-VCT scans were performed to investigate the volume of the augmented area; densities of cancellous and compact bone were assessed as comparative values. fp-VCT imaging resulted in higher spatial resolution, which was advantageous when separating closely related anatomical structures (i.e. trabecular and compact bone, biomaterials). Fp-VCT facilitated imaging of alterations occurring in test specimens over time. fp-VCTs therefore displayed high volume coverage, dynamic imaging potential and superior performance when investigating superfine bone structures and bone remodelling of biomaterials. Thus, fp-VCTs may be a suitable instrument for intraoperative imaging and future in vivo tissue-engineering studies.