Local pamidronate influences fracture healing in a rodent femur fracture model: an experimental study.

BACKGROUND: Bisphosphonates are a main component in the therapy of osteoporosis and other bone resorptive diseases. Previous studies have shown a positive effect of systemically applied bisphosphonates on fracture healing. Nevertheless high doses are related to side effects like osteonecrosis of the jaw, nephrotoxis and gastrointestinal symptoms. In this study we investigated the effect of locally applied pamidronate on fracture healing. METHODS: In a rodent model a simple femur fracture was set in female Wistar rats. We performed intramedullary fixation of the fracture and placed a collagen matrix around the fracture area. One group was treated with pamidronate, the other group with placebo via the matrix. To investigate the volume and quality of the callus we used micro-CT (µCT) and histology after 14 and 28 days. RESULTS: Our results show a positive influence of local applied pamidronate on callus volume. After 14 days an insignificant increase of callus volume in the treated animals was seen. 28 days after trauma the increase of callus volume in the treatment group was significantly higher in comparison to the control group. Osteonecrosis was not seen. CONCLUSIONS: Locally applied bisphosphonates increase the callus volume in fracture healing.

Increased infection rates of sinus floor elevations after the use of a bone filter.

OBJECTIVES: The collection of bone debris during the preparation of sinus floor augmentations is a commonly used technique for avoiding autologous bone transplants and thereby reducing donor site morbidity. However, the collected bone debris has a higher risk of bacterial contamination. The aim of this retrospective study was to analyse whether the use of a bone filter had an impact on the infection rates after sinus floor augmentation. MATERIALS AND METHODS: A retrospective analysis was conducted of 340 sinus floor elevations (136 using a bone filter) in 249 patients. The sinus floor elevations were performed with the lateral approach. RESULTS: Localised infection occurred in 7.0 % (24 of 340) of the sinus floor elevations. In 40.0 % of the cases, a bone filter was used, and in this group, the infection rate was 13.0 %. In the control group, the infection rate was 4.0 %. One hundred one patients received bone transplants from the iliac crest, and these patients had a lower infection rate of 2.0 %. Stepwise factor reduction, according to Akaike, showed the use of a bone filter to be the most relevant factor for postoperative infection. CONCLUSIONS: To reduce the amount of bacteria, full-mouth disinfection with chemical agents and a strict aspiration protocol should be used when a bone filter is applied. Antibiotic prophylaxis should be prescribed to reduce the risk of postoperative infections further. CLINICAL RELEVANCE: In use of a bone filter, there is the possibility of higher infection rates of sinus floor augmentations.

Three-dimensional image registration improves the long-term precision of in vivo micro-computed tomographic measurements in anabolic and catabolic mouse models.

Micro-computed tomography (micro-CT) is a widely used technique to track bone structural and mineral changes in small animals in vivo. Precise definition of volumes of interest (VOIs) in follow-up scans is required to accurately quantify these changes. To improve precision, VOIs can be transferred from baseline images onto follow-ups using image registration. We studied the performance of a registration procedure applied to in vivo data sets of anabolic and osteoporotic bone changes in mice. Micro-CT image data from two separate CD1 mouse data sets were studied. The first included a group treated with parathyroid hormone (PTH) and control and the second, an ovariectomy (OVX) group and control. Micro-CT was performed once per week for 4 weeks at the proximal tibia starting at treatment onset (PTH data set) or after surgery (OVX data set). A series consisting entirely of user-defined VOIs and a registered series where VOIs defined at baseline were transferred to follow-ups were created. Standard bone structural and mineral measurements were calculated. Image registration resulted in a 13-56 % reduction in precision error. Significant effects of registration to detect PTH-induced changes in BV/TV and trabecular BMD were observed. When changes were very pronounced or small, the qualitative improvement observed for the registered data set did not reach statistical significance. This study documents an increase in long-term precision of micro-CT measurements with image registration. Sensitivity to detect changes was improved but not uniform for all parameters. Future study of this technique on images with a smaller voxel size (<19 µm) may capture the effect in greater detail, in particular for trabecular thickness, where changes may be too small to be observed with the voxel size used here. Our results document the value of registration and indicate that the magnitude of improvement depends on the model and treatment chosen.

Comparison of platelet rich fibrin and collagen as osteoblast-seeded scaffolds for bone tissue engineering applications.

OBJECTIVES: The loss of jaw bone caused by different kinds of pathologies leads to dysfunction and reduced quality of life in affected patients. Thus, the pivotal goal in bone tissue engineering is to reconstruct these defects. The essential precondition for new tissue generation is an extracellular matrix which acts as a scaffold so that cells can migrate, differentiate, and proliferate. Fibrin, a biopolymer responsible for blood clot formation, has been shown to be suitable for tissue engineering applications. The aim of the present study is a comparison of platelet rich fibrin (PRF) with the commonly used collagen membrane BioGide(®) as a scaffold for human osteoblast cell seeding for bone tissue engineering. MATERIAL AND METHODS: Human osteoblasts were cultured with eluates from PRF (n = 7) and BioGide(®) (n = 8) membranes incubated in serum-free cell culture medium. Vitality of these cells was assessed by fluorescein diacetate and propidium iodide staining, biocompatibility with the lactate dehydrogenase test and proliferation levels with the MTT ([3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide]), and BrdU (5-bromo-2-deoxyuridine) tests. In addition, human osteoblasts were seeded on both membrane systems and cell growth was compared by the water soluble tetrazolium (WST-1) (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) test and scanning electron microscopy (SEM). Osteoblastic differentiation was assessed by alkaline phosphatase activity measured by ELISA in the supernatant of osteoblasts cultivated on PRF membranes (n = 10), PRF clots (n = 10), and BioGide(®) membranes (n = 10). RESULTS: Lactate dehydrogenase test values were higher for PRF compared to BioGide(®) . The BrdU test showed superior cell growth after cultivation in eluate from PRF than in eluate from BioGide(®) . The WST-1 assay demonstrated superior cell proliferation on PRF than on BioGide(®) . SEM revealed osteoblast colonization of both membranes. Cultivation of osteoblasts on PRF membranes and PRF clots showed significantly higher alkaline phosphatase activity than on BioGide(®) membranes. CONCLUSION: Metabolic activity and proliferation of human osteoblast cells in vitro were supported to a significant higher extent by eluates from PRF membranes. Both membranes are suitable as scaffolds for cultivation of human osteoblast cells in vitro; proliferation was significant higher on PRF membranes and on PRF clot than on BioGide(®) membranes.

Lipid-Iron Nanoparticle with a Cell Stress Release Mechanism Combined with a Local Alternating Magnetic Field Enables Site-Activated Drug Release.

Most available cancer chemotherapies are based on systemically administered small organic molecules, and only a tiny fraction of the drug reaches the disease site. The approach causes significant side effects and limits the outcome of the therapy. Targeted drug delivery provides an alternative to improve the situation. However, due to the poor release characteristics of the delivery systems, limitations remain. This report presents a new approach to address the challenges using two fundamentally different mechanisms to trigger the release from the liposomal carrier. We use an endogenous disease marker, an enzyme, combined with an externally applied magnetic field, to open the delivery system at the correct time only in the disease site. This site-activated release system is a novel two-switch nanomachine that can be regulated by a cell stress-induced enzyme at the cellular level and be remotely controlled using an applied magnetic field. We tested the concept using sphingomyelin-containing liposomes encapsulated with indocyanine green, fluorescent marker, or the anticancer drug cisplatin. We engineered the liposomes by adding paramagnetic beads to act as a receiver of outside magnetic energy. The developed multifunctional liposomes were characterized in vitro in leakage studies and cell internalization studies. The release system was further studied in vivo in imaging and therapy trials using a squamous cell carcinoma tumor in the mouse as a disease model. In vitro studies showed an increased release of loaded material when stress-related enzyme and magnetic field was applied to the carrier liposomes. The theranostic liposomes were found in tumors, and the improved therapeutic effect was shown in the survival studies.

Microdialysis in postoperative monitoring of microvascular free flaps: Experiences with a decision algorithm.

BACKGROUND: Reconstruction with free flaps has become a usual practice in maxillofacial surgery. Clinical monitoring is still the standard approach for postoperative follow-up, but can be difficult or impossible with intraorally situated or buried flaps. Microdialysis is a sampling technique that offers the possibility to monitor the metabolism of flaps continuously. It is a reliable method for early diagnosis of ischemia. MATERIALS AND METHODS: 48 microvascular free flaps applied following oral cancer resection were monitored with a microdialysis (MD) catheter, placed in the flap. Glucose, lactate, and lactate/pyruvate ratio were monitored using a bedside analyser for 5 days. 48 free flaps served as controls and were assessed (refill, flap temperature, and color) by clinical monitoring (CM). RESULTS: 12 flaps monitored by MD showed abnormal metabolism and underwent revision. Eight flaps were saved and four were lost within the first 5 days postoperatively. In addition, two flaps were lost at days 15 and 30 postoperatively, without previous complications. Four flaps assessed by CM developed complications, underwent revision, and were saved. In addition, five flaps were lost between the 8th and 23rd days postoperatively, without revision, due to missing previous clinical signs. CONCLUSION: Postoperative monitoring of free flaps using a microdialysis decision algorithm allows early diagnosis of anastomotic complications. It is a clinically feasible and sensitive monitoring method for microvascular flaps, allowing surgical revision to be undertaken before clinical alteration takes place.

An experimental study on antitumoral effects of KI-21-3, a synthetic fragment of antimicrobial peptide LL-37, on oral squamous cell carcinoma.

PURPOSE: The aim of this study was to investigate the oncolytic properties of KI-21-3, a shortened fragment of LL-37, against oral squamous cell carcinoma (OSCC) in an animal model. MATERIALS AND METHODS: Twelve athymic nude mice were divided into a therapy and a control group of six animals each. In both groups, SCC-4 cells were administered extraorally into the floor of the mouth in order to create an OSCC model. In the study group, KI-21-3 was applied intravenously during the 8th and 9th weeks. The subjects in the control group were injected with phosphate buffered saline solution in the same manner. During an examination period of 12 weeks, weight control was performed twice a week. Tumor growth was further controlled volumetrically via ultrasonography once a week with regular intervals. Following sacrifice, ablated tumoral tissues were immunohistochemically evaluated in order to determine the proliferation and apoptotic properties. RESULTS: The mean tumor weight in the AMP group was 0.0236 ± 0.023 g, which was 30% lower than the control group with the mean value of 0.01651 ± 0.012 g. In the control group, the approximate number of the proliferating cells per visualized field was fourfold higher compared to the therapy group. Moreover, in the control group, the number of apoptotic cells per visualized field was significantly lower compared to the therapy group. CONCLUSION: KI-21-3 showed considerable oncolytic properties on SCC-4 carcinoma cells via antiproliferative and caspase-3 apoptotic pathway. Further investigations are necessary to clarify the dose-dependent effects of this agent.

Increased survival rate by local release of diclofenac in a murine model of recurrent oral carcinoma.

Despite aggressive treatment with radiation and combination chemotherapy following tumor resection, the 5-year survival rate for patients with head and neck cancer is at best only 50%. In this study, we examined the therapeutic potential of localized release of diclofenac from electrospun nanofibers generated from poly(D,L-lactide-co-glycolide) polymer. Diclofenac was chosen since anti-inflammatory agents that inhibit cyclooxygenase have shown great potential in their ability to directly inhibit tumor growth as well as suppress inflammation-mediated tumor growth. A mouse resection model of oral carcinoma was developed by establishing tumor growth in the oral cavity by ultrasound-guided injection of 1 million SCC-9 cells in the floor of the mouth. Following resection, mice were allocated into four groups with the following treatment: 1) no treatment, 2) implanted scaffolds without diclofenac, 3) implanted scaffolds loaded with diclofenac, and 4) diclofenac given orally. Small animal ultrasound and magnetic resonance imaging were utilized for longitudinal determination of tumor recurrence. At the end of 7 weeks following tumor resection, 33% of mice with diclofenac-loaded scaffolds had a recurrent tumor, in comparison to 90%-100% of the mice in the other three groups. At this time point, mice with diclofenac-releasing scaffolds showed 89% survival rate, while the other groups showed survival rates of 10%-25%. Immunohistochemical staining of recurrent tumors revealed a near 10-fold decrease in the proliferation marker Ki-67 in the tumors derived from mice with diclofenac-releasing scaffolds. In summary, the local application of diclofenac in an orthotopic mouse tumor resection model of oral cancer reduced tumor recurrence with significant improvement in survival over a 7-week study period following tumor resection. Local drug release of anti-inflammatory agents should be investigated as a therapeutic option in the prevention of tumor recurrence in oral squamous carcinoma.

Quality of life in treatment of mandibular fractures using closed reduction and maxillomandibular fixation in comparison with open reduction and internal fixation--a randomized prospective study.

Treatment of mandibular fractures by open reduction and internal fixation (ORIF) is often assumed to be superior to treatment by close reduction and maxillomandibular fixation (MMF) because patients managed by ORIF seem to be rehabilitated earlier according to functional and social aspects. This assumption is often from surgeon's perspective, not taking into account patient's view point. This study highlights a comparative assessment between ORIF and MMF from the patients' perspective. Fifty six patients with mandibular fractures within the tooth bearing areas of the mandible were prospectively studied in a randomized controlled pattern for postoperative Quality of Life (QoL) after ORIF versus MMF. Both groups were analyzed preoperatively, at 1 day, 6 and 8 weeks regarding their QoL using the General Oral Health Assessment Index questionnaire (GOHAI). No significant statistical difference was found between the groups regarding overall QoL. Patients managed by MMF were more affected by psychosocial and physical domains whereas patients managed by ORIF were more affected by the pain domain. The results demonstrate that the treatment affects the psychosocial, physical and pain domain differentially. When both treatments are possible the patient's should be enlightened on the advantages and disadvantages of both treatment modalities to guide their choice of treatment.

Comparison of two different absorbable membranes for the coverage of lateral osteotomy sites in maxillary sinus augmentation: a preliminary study.

INTRODUCTION: Barrier membranes, both absorbable and non-absorbable, have been used in sinus augmentation for many years. Some years ago, a new autologous blood substrate called Platelet-Rich-Fibrin (PRF) was introduced, and to date, the supporting effect on bone regeneration has been controversial. This study aimed to evaluate the effect of PRF on bone regeneration when used as a barrier membrane at the lateral osteotomy site in sinus augmentation. MATERIAL AND METHODS: Twelve sinuses from six patients requiring bilateral sinus floor augmentation were treated with a two-stage surgical technique using sinus augmentation and implant placement after 5 months. The sinuses were grafted with autologous bone and bone-substitute material (Bio-Oss(®)) mixed in a 1:1 ratio and were covered in a randomized split-mouth design with a PRF or a conventional collagen membrane (Bio-Gide(®)), respectively. Five months later threaded titanium dental implants were inserted and bone specimens harvested with a trephine burr were evaluated histomorphometrically. RESULTS: Bone quality seemed to be equal at both sites of the grafted sinuses. Mean vital bone formation after 5 months was 17.0% and 17.2%, for the PRF and collagen sites, respectively. The mean of residual bone-substitute was 15.9% and 17.3% for PRF and collagen, respectively. No local complications, such as dehiscences or membrane exposures, were detected at either site in any of the treated patients. After 12 months all implants reached primary stability in the augmented maxillary sinus floor without any peri-implant tissue inflammation. CONCLUSIONS: Within the limits of the study the coverage of the lateral sinus window with two different absorbable membranes has been shown to result in a similar amount of vital bone formation and residual bone-substitute.

Magnesium-enhanced enzymatically mineralized platelet-rich fibrin for bone regeneration applications.

Membranes of the autologous blood-derived biomaterial platelet-rich fibrin (PRF) were mineralized enzymatically with calcium phosphate (CaP) by the incorporation of alkaline phosphatase (ALP) followed by incubation for 3 days in solutions of either 0.1 M calcium glycerophosphate (CaGP) or a combination of CaGP and magnesium glycerophosphate (CaGP:MgGP; both 0.05 M), resulting in the formation of two different PRF-mineral composites. Fourier transform infrared spectroscopy, transmission electron microscopy and selected area electron diffraction examinations showed that the CaP formed was amorphous. Inductively coupled plasma optical emission spectroscopy analysis revealed similar amounts of Ca and P in both composite types, while a smaller amount of Mg (Ca:Mg molar ratio = 10) was detected in the composites formed in the CaGP:MgGP solution, which was supported by the results of energy-dispersive x-ray spectroscopy-based elemental mapping. Scanning electron microscopy (SEM) imaging showed that the mineral deposits in PRF incubated in the CaGP:MgGP solution were markedly smaller. The mass percentage attributable to the mineral phase was similar in both composite types. MTT and WST tests with SAOS-2 cells revealed that incubation in the CaGP:MgGP solution had no negative effect on cytocompatibility and cell proliferation compared to the CaGP solution. Cells on all samples displayed a well-spread morphology as revealed by SEM imaging. In conclusion, the incorporation of Mg reduces mineral deposit dimensions and promotes cell proliferation.

Enzymatically induced mineralization of platelet-rich fibrin.

Membranes of the autologous blood-derived biomaterial platelet-rich fibrin (PRF) were functionalized by incorporation of alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, and subsequently incubated in calcium glycerophosphate (CaGP) solution to induce PRFs mineralization with calcium phosphate (CaP) to improve PRFs suitability as a material for bone replacement. Incorporated ALP retained its bioactivity and induced formation of CaP material within PRF membranes, as confirmed by SEM, EDS, FTIR, and von Kossa staining. The mass percentage attributable to CaP was quantified by lyophilization and measurement of the remaining mass fraction as well as by TGA. Cytocompatibility tests (LDH, MTT, and WST) with SAOS-2 cells showed that mineralized PRF did not release substances detrimental to cell vitality. Live/dead staining and SEM showed that mineralized PRF was colonized by cells. The results show that hydrogel biomaterials such as PRF can be mineralized through functionalization with ALP.