Toll-like receptor 2/6-dependent stimulation of mesenchymal stem cells promotes angiogenesis by paracrine factors.

Reconstruction of critical size bone defects represents a major challenge in orthopaedic surgery. Insufficient angiogenesis is a limiting factor for engraftment of large-scale tissue transplants. Transplantation or stimulation of local mesenchymal stem cells (MSCs) represents a potential solution to enhance angiogenesis. We recently identified angiogenic properties for the Toll-like receptor (TLR) 2/6 agonist MALP-2 and now investigated if MALP-2 could be used to stimulate MSCs in order to promote angiogenesis in vitro and in vivo. Human MSCs from the bone marrow of healthy subjects were isolated, cultured and expanded in vitro and were shown to be positive for mesenchymal stem cells markers as well as for the MALP-2 receptors TLR2 and TLR6. MALP-2 directly enhanced migration but not proliferation of human MSCs. Conditioned medium from MALP-2 stimulated MSCs significantly increased proliferation, migration and tube formation of endothelial cells. Analysis of the conditioned medium from MSCs revealed that MALP-2 stimulation enhanced the secretion of several chemokines and growth factors including vascular endothelial growth factors (VEGF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Finally, we studied MALP-2 effects on MSCs in a sheep model of tissue engineering in vivo. Therefore, MSCs were isolated from the iliac crest of black head sheep and co-cultivated with MALP-2 ex vivo. Implantation of autologous MSCs within a scaffold cylinder into the M. latissimus dorsi significantly enhanced vessel density of these constructs after 6 months. We here present the first evidence that TLR2/6-dependent stimulation of MSCs promotes angiogenesis in vitro and in vivo offering a novel strategy for therapeutic angiogenesis, e.g., for tissue engineering of bone.

[Reconstruction of complex midfacial defects with individualized titanium implants]. / Rekonstruktion komplexer Mittelgesichtsdefekte durch individualisierte Titanimplantate.

BACKGROUND: Loss of hard and soft tissue structures of the midface due to resection or trauma is associated with substancial functional and aesthetic deficits. Besides reconstruction of bony contours for preservation of orbit position and facial symmetry, reconstruction often requires simultaneous transplantation of soft tissue flaps for separation of nasal and oral cavities and refilling of soft tissue volume deficits. PATIENTS AND METHODS: A well-established procedure of our institution will be demonstrated in 10 exemplary patients, in which titanium meshes are customized for individual defect situations using computer-assisted techniques in combination with soft tissue transfer if required. RESULTS: According to our experience, this procedure provides satisfactory results in functional as well as in aesthetic respects. Especially in patients with loss of bony structures of the orbit and preservation of orbital contents, this procedure forms optimal preconditions for prevention of enophthalmos and diplopia by preservation of the original orbital volume. CONCLUSION: Individualized titanium implants should be used more frequently in clinical routine for reconstruction of complex midfacial defects.

Comparison of two beta-tricalcium phosphate composite grafts used for reconstruction of mandibular critical size bone defects.

OBJECTIVE: The aim was to compare osseointegration of blood perfused beta-tricalcium phosphate cylinders (beta-TCPB) with similar composites that were additionally loaded with cancellous bone and bone marrow (beta-TCPB/BM/CB) for mandibular reconstruction. METHODS: Twelve German Black-Headed sheep with an average weight of 72.5 +/- 10 kg underwent segmental resection of the right hemi-mandible. The animals that were assigned to group A (n = 6) were reconstructed using beta-TCPB while the sheep that were assigned to group B received beta-TCPB/BM/CB grafts. Tissue quality was histologically assessed and bone-, scaffold-, cartilage- and fibrous-tissue area were estimated using semiautomated histomorphometrical software. RESULTS: Composite grafts that were loaded with bone marrow and cancellous bone (beta-TCPB/BM/CB) exhibited significant (p<0.01) higher amounts of bone formation than beta-TCPB. The patients that were assigned to group B achieved defect union and a high grade of bone maturation. Residual ceramic remnants were rare and disconnected. Bone maturity within group A was inferior and none of the specimens showed defect union. The defect centre was still occupied by a ceramic core. CLINICAL SIGNIFICANCE: Bone and bone marrow augmented beta-tricalcium phosphate composites may qualify as a promising alternative to autograft bone for mandibular reconstruction in human and veterinary medicine.

Heat-shock protein expression and topical treatment with tacrolimus in oral lichen planus: an immunohistochemical study.

Oral lichen planus (OLP) is a chronic inflammatory mucosal disease of unknown etiology. Usually asymptomatic, the disorder is occasionally complicated by extensive painful erosions. Topical corticosteroids are the mainstay of treatment, but a new topical therapy with tacrolimus has been described previously. The aim of the current study was to examine the expression of heat-shock protein 70 (HSP70) in biopsy specimens from 11 OLP lesions before and after topical treatment with tacrolimus. Immunostaining was performed with anti-HSP70 antibody as the primary layer. Clinically, there was a rapid improvement with topical tacrolimus treatment in 10 out of the 11 patients. The moderate increase in HSP70 expression after treatment with tacrolimus was not significant. It was concluded that topical tacrolimus has no effect on the expression of HSP70 in OLP.

In vivo tissue engineered bone versus autologous bone: stability and structure.

This pilot study investigated the biomechanical properties of prefabricated, vascularized bioartificial bone grafts, which may provide an alternative bone source for the restoration of segmental osseous defects. Vascularized bioartificial bone grafts comprise an artificial customized scaffold made of beta-tricalcium phosphate. Bone formation along the prefabricated scaffold is induced by autogenous cancellous bone. Vascularization of the bone graft is provided by the host's vascular system. Within 6 months, a mammalian bioreactor (sheep were used in the present study) creates heterotopic vascularized bioartificial bone grafts of a predetermined anatomical shape, which can be harvested for reconstructing osseous defects. The bioartificial bone grafts in this study contained up to 25% bone tissue, as shown by histomorphometric analysis and computed tomography. Moreover, unconfined compression tests revealed that the constructs had mechanical characteristics similar to those of ovine cancellous bone. Therefore, this method could be applied to generate vascularized prefabricated bone substitutes for critical-size defects.

[Prefabrication of vascularized facial bones]. / Präfabrikation vaskularisierter Gesichtsknochen.

Critical size defects in the craniomaxillofacial region often result from ablative tumor surgery, inflammation and posttraumatic deformities. To date, autologous bone grafts are still the gold standard for the reconstruction of these defects; however, they are frequently associated with severe donor site morbidity as well as functional and aesthetic compromises. In this context various resorbable and non-resorbable bone replacement materials have been developed and intensively investigated. Particularly in critical size defects these materials fail due to their lack of osteogenic potential and endogenous vascularization. The combination of alloplastic osteoconductive scaffolds, osteogenic cells, and axial prevascularization in bioartificial bone grafts might present an innovative approach for the microsurgical reconstruction of critical size defects.

[Prefabrication of bone transplants]. / Präfabrikation von Knochentransplantaten.

Prefabrication of bone transplants is a promising option for large defects of the long bones, especially if there is compromised vascularization of the defect. This is especially true for postinfection bone defects and other types of atrophic nonunion. The generation of a foreign body membrane (Masquelet's technique) has been investigated in order to ameliorate the response of the host tissue surrounding the defect. In an experimental animal study, a blood vessel within a bone construct could be used to generate customized, vascularized osteogenic constructs that can be used to treat large bone defects in the future.

En bloc prefabrication of vascularized bioartificial bone grafts in sheep and complete workflow for custom-made transplants.

The aim of this pilot study was to determine, in a new experimental model, whether complex bioartificial monoblocs of relevant size and stability can be prefabricated in a defined three-dimensional design, in which the latissimus dorsi muscle serves as a natural bioreactor and the thoracodorsal vessel tree is prepared for axial construct perfusion. Eighteen sheep were included in the study, with six animals in each of three experimental groups. Vitalization of the ß-tricalcium phosphate-based constructs was performed by direct application of unmodified osteogenic material from the iliac crest (group A), in vivo application of nucleated cell concentrate (NCC) from bone marrow aspirate (group B), and in vitro cultivation of bone marrow stromal cells (BMSC) in a perfusion bioreactor system (group C). The contours of the constructs were designed digitally and transferred onto the bioartificial bone grafts using a titanium cage, which was bent over a stereolithographic model of the defined subvolume intraoperatively. At the end of the prefabrication process, only the axial vascularized constructs of group A demonstrated vital bone formation with considerable stability. In groups B and C, the applied techniques were not able to induce ectopic bone formation. The presented computer-assisted workflow allows the prefabrication of custom-made bioartificial transplants.

Investigation of perfusion in osseous vessels in close vicinity to piezo-electric bone cutting.

Cutting bones by piezosurgery leads to failure of perfusion at the site of the osteotomy, the cause of which cannot be identified immediately. Among other things the formation of vascular thrombi by the transmission of oscillations from the piezoelectric unit to the bone may be responsible. We used three output levels of oscillation that were predefined by the system. The outer cortical bone of the calvaria of rats (n=24) was removed horizontally and the intraosseous vessels exposed at the surface of the osteotomy. The blood flow was then examined repeatedly using intravital fluorescence microscopy. To calculate the transmission of oscillations to the bone, the spatial oscillation frequency of each calvarium and the contact pressure during removal of bone in vitro (n=18) were also examined. After removal of the bone there was constant blood flow at all three levels of oscillation output. In no case did an individual vessel seem to be occluded. The excitation oscillation of the bone was established at 2000 Hz in all spatial directions, irrespective of the predefined oscillation output. The application of piezosurgery does not cause the formation of vascular thrombi in the bone. This probably results from the oscillation damping properties of bone.

Elongated coronoid process: CT-based quantitative analysis of the coronoid process and review of literature.

Impingement of the enlarged coronoid processes against the medial surfaces of the zygomatic arches and posterior surfaces of the body of the zygomatic bones results in mechanical restriction of the mouth opening. The authors introduce a helpful tool for easy assessment and estimatation of the length of the coronoid process, measured on the CT scans of 40 patients (20 adults, 20 adolescents) and report a case of a 13-year-old boy suffering from restricted mouth opening caused by bilateral hyperplasia of the coronoid process. The CT based analysis resulted in a mean length of the coronoid process of 13.02mm in adults and 12.43mm in adolescents. The 13-year-old boy had a length of nearly 2cm. For comparison, a coronoid/condyle ratio was developed. This ratio showed a value of 0.78 for all patients compared with a value of about 2.0 for the boy. The literature review revealed comparable results to the reported case. Most of the patients were adolescent, male and presented a median history of 2 years until correct diagnosis.

Comparison of different harvesting methods from the flat and long bones of rats.

Different harvesting methods have been developed for bony augmentation before implantation. The aim of the present study was to assess the viability of endochondral (femoral) and membranous (mandibular) bone cells harvested by different methods under standard conditions in an animal model, and to investigate the surface of the bone in the harvested area. Samples of mandibular and femoral bone were harvested using a drilling burr, a piezoelectrical device, or a Safescraper(®). Blocks of bone that had been harvested with cutting forceps were used as controls. The size of the samples was measured and they were examined by conventional microscopy and immunohistochemical analysis; osteoblast-like cells were also cultured. The surface of the harvested area was analysed with scanning and conventional microscopy. There was no significant difference between mandibular and femoral bone in the size of particles harvested, but bone chips were significantly smaller when a drilling device had been used in both harvesting areas. Viability of cells in these smaller particles was significantly less than among cells harvested with a piezoelectrical device or Safescraper(®). Scanning microscopy showed a smooth bony surface where a drilling burr or piezoelectrical device had been used, whereas small disruptions were observed after the Safescraper(®) had been used. Harvesting of particulate bone is feasible using a drilling burr, piezoelectrical device, or Safescraper(®) from mandibular and femoral bone. The piezoelectrical device and the Safescraper(®) gave comparable results concerning the viability of osteoblast-like cells, and so are preferred to a drilling burr.

Sandwich osteotomy for vertical and transversal augmentation of the posterior mandible.

The aim of this study is to describe the treatment outcome after alveolar ridge augmentation in the atrophic posterior mandible by segmental sandwich osteotomy combined with an interpositional autograft prior to placement of endosseous implants. Thirteen consecutive patients (five males, mean age 48 years, and eight females, mean age 61 years) were included in this study. The postoperative course was uneventful in six patients. Sensory disturbances in the mental nerve were found in five patients, all of them with hypoaesthesia. None of these patients complained of permanent sensory disturbances. Vertical gain ranged from 2.0 to 7.8mm (mean value 4.61mm). Horizontal gain ranged from 2.0 to 6.3 (mean value 3.42mm). A total of 41 implants were placed in 22 surgical sites, 12 weeks after bone reconstruction. In conclusion, segmental mandibular sandwich osteotomy is recommended to meet the dimensional requirements of preimplant bone augmentation in atrophic posterior mandible.

[Effectiveness of neck dissection in metastasizing mouth carcinoma. Uni- and multivariate analysis of factors of influence]. / Effektivität der Neck dissection beim metastasierenden Mundhöhlenkarzinom. Uni- und multivariate Analyse von Einflussfaktoren.

BACKGROUND: The purpose of this study was to evaluate the oncologic effectiveness of radical and different types of modified neck dissections with preservation of n. accessorius, v. jugularis interna, and m. sternocleidomastoideus and to identify prognostic factors for regional control and survival in univariate and multivariate analysis. METHODS: This retrospective study included 373 patients with squamous cell carcinoma of the oral cavity who underwent 401 neck dissections between January 1986 and December 1995 at the Department for Oral and Maxillofacial Surgery, Hanover Medical School. RESULTS: The 5-year regional control was estimated at 87%. Relapse occurred only within the first 2 years after neck dissection. The number of positive nodes, metastases without lymphatic tissue, preparation of metastases from the carotid artery and cranial base, and preoperative radiochemotherapy were analyzed as prognostic factors with significant influence. The grade of metastases, extracapsular spread, lymphangiosis carcinomatosa, and postoperative radiation showed no prognostic significance. DISCUSSION: The comparison of recurrent metastases after radical and modified neck dissection demonstrated that as the extent of neck disease increased there was a tendency toward improved regional control after radical neck dissection.

[Adenoid cystic carcinoma of the major and minor salivary glands. Retrospective analysis of 74 patients]. / Adenoidzystische Karzinome der grossen und kleinen Speicheldrüsen. Retrospektive Analyse von 74 Patienten.

PURPOSE: In this study we give a clinical review of our experience with adenoid cystic carcinoma (ACC) and compare our results with those reported in the literature. PATIENTS: Between 1981 and 2000, 74 patients with ACC of the head and neck were treated at the Department of Oral and Maxillofacial Surgery, Hannover Medical School. RESULTS: The average age at diagnosis was 58 years. There were 38 men and 36 women. The primary site was the parotid gland in 4 cases, the submandibular and sublingual gland in 21 cases, the lacrimal gland in 1 case, the minor salivary glands of the oral cavity and oropharynx in 44 cases, and the nasal cavity and maxillary sinus in 4 cases. There were 19 T1, 15 T2, 9 T3, and 31 T4 tumors with perineural invasion of 32 tumors. R0 resection was performed in 45 cases. Fourteen patients received postoperative radiation. There were only five N1 and two N2b necks. All patients were staged M0 at presentation. Local control rates at 5, 10, and 15 years were 64%, 56%, and 52% with a mean local control time of 11.1 years. Tumor size ( p< or =0.001), margin status ( p< or =0.001), and perineural invasion ( p

[Malignant epithelial salivary gland tumors. Clinical review of 2 decades]. / Maligne epitheliale Speicheldrüsentumoren. Klinischer Rückblick über zwei Jahrzehnte.

PURPOSE: In this retrospective study we give a clinical review of our experience with different tumor entities of salivary gland cancer. PATIENTS: Between January 1983 and December 2002, a total of 155 patients with carcinomas of the salivary glands received initial treatment at the Department of Oral and Maxillofacial Surgery, Hanover Medical School. RESULTS: There were 51.0% adenoid cystic carcinomas, 27.1% mucoepidermoid carcinomas, 7.7% adenocarcinomas, 6.5% polymorphous adenocarcinomas, and altogether 7.7% other less frequent tumor entities. Complete resection was achieved for 63.5% of patients with high-grade carcinomas and for 80.0% of patients with low-grade carcinomas. Postoperative radiation was administered to 26.2% of patients with high-grade carcinomas and 13.3% of patients with low-grade carcinomas. Overall local control rates at 5, 10, and 15 years were 77.5%, 66.2%, and 59.0%. Overall regional control rates at 5, 10, and 15 years were 93.5%, 87.6%, and 85,4%. Overall distant control rates at 5, 10, and 15 years were 85.9%, 77,2%, and 73,6%. Overall survival rates at 5, 10, and 15 years were 65,9%, 48,0%, and 39,8%. There was a significant difference between patients with low-grade and high-grade carcinomas. Polymorphous adenocarcinomas showed the best prognosis, followed by low-grade mucoepidermoid carcinomas, adenoid cystic carcinomas, adenocarcinomas, and high-grade mucoepidermoid carcinomas. The number of the remaining tumor entities was too small to be divided into independent subgroups for statistical analysis. Tumor entity, tumor stage, and margin status significantly influenced prognosis. Statistically, we were not able to demonstrate a positive effect for postoperative radiation. CONCLUSION: Grading is important, but should be considered in the context of stage. In the future, patients with salivary gland carcinomas should be randomized for prospective multicenter clinical trials, which could provide reliable information about adjuvant treatment modalities and their results even for rare subtypes of salivary gland cancer.