Erratum: A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro.

This corrects the article 10.3791/64016.

Preoperative Assessment of Medication-Related Osteonecrosis of the Jaw Using [18F]fluoride Positron Emission Tomography (PET)/CT and [18F]fluorodeoxyglucose PET/MRI in Correlation with Histomorphometry and Micro-CT-A Prospective Comparative Study.

OBJECTIVES: The purpose of this study was to investigate the imaging characteristics of medication-related osteonecrosis of the jaw (MRONJ) using [18F]fluoride positron emission tomography/computed tomography (PET/CT) and [18F]fluorodeoxyglucose (FDG) PET/magnetic resonance imaging (MRI) for preoperative assessment and to correlate them with microarchitectural and histomorphometric data with respect to clinical findings. METHODS: Twelve patients (five female; mean age 75 ± 7.6 yr) with symptomatic MRONJ underwent both scans on the same day, and imaging findings were used to plan surgical interventions for seven patients. Bone tracer uptake was classified as high, medium, or low, and surgical samples were evaluated using Micro-CT and histomorphometric analysis. RESULTS: CT showed medullary sclerosis in all patients, and MRI revealed gadolinium enhancement in four patients. PET imaging revealed remarkably elevated [18F]fluoride uptake and moderately increased [18F]FDG uptake in MRONJ compared to healthy jawbones, with both differences being statistically significant. [18F]fluoride uptake was associated with necrosis, bacteria, and inflammatory tissue. Micro-CT data did not show significant differences, but histomorphometric analysis revealed higher osteocyte and lacunae densities in the high [18F]fluoride uptake group, and more necrotic bone in the medium [18F]fluoride uptake group. Bacteria were observed in all areas. CONCLUSIONS: In summary, [18F]fluoride PET accurately identified MRONJ extent, revealing functional changes in jawbone remodeling not visible on CT. [18F]FDG PET showed differences in bone and soft tissue, though less pronounced. This method aids in evaluating disease activity and guiding treatment planning, requiring further research for optimal surgical approaches based on tracer uptake.

Three-Dimensionally Cultured Jaw Periosteal Cells Attenuate Macrophage Activation of CD4+ T Cells and Inhibit Osteoclastogenesis.

The implementation of a successful therapeutic approach that includes tissue-engineered grafts requires detailed analyses of graft-immune cell interactions in order to predict possible immune reactions after implantation. The phenotypic plasticity of macrophages plays a central role in immune cell chemotaxis, inflammatory regulation and bone regeneration. The present study addresses effects emanating from JPC-seeded ß-TCP constructs (3DJPCs) co-cultivated with THP-1 derived M1/M2 macrophages within a horizontal co-culture system. After five days of co-culture, macrophage phenotype and chemokine secretion were analyzed by flow cytometry, quantitative PCR and proteome arrays. The results showed that pro-inflammatory factors in M1 macrophages were inhibited by 3DJPCs, while anti-inflammatory factors were activated, possibly affected by the multiple chemokines secreted by 3D-cultured JPCs. In addition, osteoclast markers of polarized macrophages were inhibited by osteogenically induced 3DJPCs. Functional assays revealed a significantly lower percentage of proliferating CD4+ T cells in the groups treated with secretomes from M1/M2 macrophages previously co-cultured with 3DJPCs compared to controls without secretomes. Quantifications of pit area resorption assays showed evidence that supernatants from 3DJPCs co-cultured with M1/M2 macrophages were able to completely suppress osteoclast maturation, compared to the control group without secretomes. These findings demonstrate the ability of 3D cultured JPCs to modulate macrophage plasticity.

Mechanical and Functional Improvement of ß-TCP Scaffolds for Use in Bone Tissue Engineering.

Autologous bone transplantation is still considered as the gold standard therapeutic option for bone defect repair. The alternative tissue engineering approaches have to combine good hardiness of biomaterials whilst allowing good stem cell functionality. To become more useful for load-bearing applications, mechanical properties of calcium phosphate materials have to be improved. In the present study, we aimed to reduce the brittleness of ß-tricalcium phosphate (ß-TCP). For this purpose, we used three polymers (PDL-02, -02a, -04) for coatings and compared resulting mechanical and degradation properties as well as their impact on seeded periosteal stem cells. Mechanical properties of coated and uncoated ß-TCP scaffolds were analyzed. In addition, degradation kinetics analyses of the polymers employed and of the polymer-coated scaffolds were performed. For bioactivity assessment, the scaffolds were seeded with jaw periosteal cells (JPCs) and cultured under untreated and osteogenic conditions. JPC adhesion/proliferation, gene and protein expression by immunofluorescent staining of embedded scaffolds were analyzed. Raman spectroscopy measurements gave an insight into material properties and cell mineralization. PDL-coated ß-TCP scaffolds showed a significantly higher flexural strength in comparison to that of uncoated scaffolds. Degradation kinetics showed considerable differences in pH and electrical conductivity of the three different polymer types, while the core material ß-TCP was able to stabilize pH and conductivity. Material differences seemed to have an impact on JPC proliferation and differentiation potential, as reflected by the expression of osteogenic marker genes. A homogenous cell colonialization of coated and uncoated scaffolds was detected. Most interesting from a bone engineer's point of view, the PDL-04 coating enabled detection of cell matrix mineralization by Raman spectroscopy. This was not feasible with uncoated scaffolds, due to intercalating effects of the ß-TCP material and the JPC-formed calcium phosphate. In conclusion, the use of PDL-04 coating improved the mechanical properties of the ß-TCP scaffold and promoted cell adhesion and osteogenic differentiation, whilst allowing detection of cell mineralization within the ceramic core material.

Autologous Tooth Transplantation in Craniofacial Malformations.

OBJECTIVE: To evaluate the applicability of transplanted teeth in young patients with craniofacial anomalies. DESIGN: Observational study. SETTING: Comprehensive Centre for Cleft Palate and Craniofacial Malformations. PATIENTS/PARTICIPANTS: Patients with craniofacial anomalies who underwent tooth transplantation. Only children with complete clinical and radiological documentation and a follow-up period of at least 1.5 years were included. INTERVENTIONS: Tooth transplantation. MAIN OUTCOME MEASURE(S): Retrospective evaluation of clinical records, pre- and postoperative radiographs, and operative charts. Clinical characteristics of patients, preoperative parameters and postoperative outcome parameters were collected. RESULTS: A total of 17 patients with 23 tooth transplantations were included. The median follow-up period was 6.7 years. The pooled survival and success rates were 91%. Notably, one out of two teeth that were transplanted into the bone grafted alveolar cleft site had to be extracted, which might indicating a higher risk for this procedure. In total, two transplanted teeth had to be extracted during the follow-up period, one due to external resorption and the other one due to perio-endo lesion. One patient needed endodontic treatment due to pulp necrosis. CONCLUSION: We consider tooth transplantation to be a reliable and suitable procedure in the dental rehabilitation of young patients with craniofacial anomalies and fitting concomitant circumstances. We encourage craniofacial teams to reconsider this option more frequently in appropriate cases.

Prospective Evaluation of Children with Robin Sequence following Tübingen Palatal Plate Therapy.

BACKGROUND: To assess the long-term functional orthodontic outcome of the Tübingen palatal plate (TPP) in children with Robin sequence (RS) in comparison to age- and sex-matched healthy controls. METHODS: Between 09/2019 and 10/2020, we performed orthodontic assessments in 41 children at our Department of Orthodontics. Included were patients with RS (17 non-syndromic; four syndromic) and healthy controls (n = 22, average age in both groups 9.9 y). Facial analyses of 2D images, digital study casts and cephalometric measurements were made. RESULTS: The orthodontic examinations showed no statistically significant group differences regarding functional extraoral, intraoral and pharyngeal parameters, or in skeletal patterns. The relationship between the upper and lower incisors was significantly increased (overjet 4 (2-10) vs. 3 (0-9) mm; p = 0.01) with a significant deficit in the lower face proportions (Jaw Index 4.15 (1.9-9.6) vs. 2.98 (0-9); p = 0.02; Facial convexity angle 157 (149-173) vs. 159 (149-170); p = 0.01). CONCLUSION: Children with RS treated with the TPP showed normal long-term functional orthodontic outcomes, thanks to the functional adaption of the stomatognathic system. However, soft tissue growth did not completely match skeletal growth, resulting in a more convex facial profile.

Differences in analysis and treatment of upper airway obstruction in Robin sequence across different countries in Europe.

The goal of this study was to explore the availability of diagnostic and treatment options for managing upper airway obstruction (UAO) in infants with Robin Sequence (RS) in Europe. Countries were divided in lower- (LHECs, i.e., PPP per capita < $4000) and higher-health expenditure countries (HHECs, i.e., PPP per capita ≥ $4000). An online survey was sent to European healthcare professionals who treat RS. The survey was designed to determine the availability of diagnostic tools such as arterial blood gas analysis (ABG), pulse oximetry, CO2 analysis, polysomnography (PSG), and sleep questionnaires, as well as to identify the used treatment options in a specific center. Responses were received from professionals of 85 centers, originating from 31 different countries. It was equally challenging to provide care for infants with RS in both LHECs and HHECs (3.67/10 versus 2.65/10, p = 0.45). Furthermore, in the LHECs, there was less access to ABG (85% versus 98%, p = 0.03), CO2 analysis (45% versus 70%, p = 0.03), and PSG (54% versus 93%, p < 0.01). There were no significant differences in the accessibility concerning pulse oximetry, sleep questionnaires, home saturation monitoring, nasopharyngeal tubes, Tuebingen plates, and mandibular distraction.    Conclusion: This study demonstrates a large difference in available care for infants with RS throughout Europe. LHECs have less access to diagnostic tools in RS when compared to HHECs. There is, however, no difference in the availability of treatment modalities between LHECs and HHECs. What is Known: • Patients with Robin sequence (RS) require complex and multidisciplinary care. They can present with moderate to severe upper airway obstruction (UAO). There exists a large variety in the use of diagnostics for both UAO treatment indications and evaluations. In most cases, conservative management of UAO in RS is sufficient. Patients with UAO that persist despite conservative management ultimately need surgical intervention. To determine which intervention is best suitable for the individual RS patient, the level of UAO needs to be determined through diagnostic testing. • There is a substantial variation among institutions across Europe for both diagnostics and treatment options in UAO. A standardized, internationally accepted protocol for the assessment and management of UAO in RS could guide healthcare professionals in the timing of assessment and indications to prevent escalation of UAO. Creating such a protocol might be a challenge, as there are large financial differences between countries in Europe (e.g., health expenditure per capita in purchasing power parity in international dollars ranges from $600 to over $8500). What is New: • There is a substantial variation in the availability of objective diagnostic tools between European countries. Arterial blood gas analysis, CO2 analysis and polysomnography are not equally accessible for lower-healthcare expenditure countries (LHECs) compared to higher-healthcare expenditure countries (HHECs). These differences are not only limited to availability; there is also a difference in quality of these diagnostic tools. Surprisingly, there is no difference in access to treatment tools between LHECs and HHECs. • There is national heterogeneity in access to tools for diagnosis and treatment of RS, which suggests centralization of health care, showing that specialized care is only available in tertiary centers. By centralization of care for RS infants, diagnostics and treatment can be optimized in the best possible way to create a uniform European protocol and ultimately equal care across Europe. Learning what is necessary for adequate monitoring could lead to better allocation of resources, which is especially important in a low-resource setting.

How osteogenic is dexamethasone?-effect of the corticosteroid on the osteogenesis, extracellular matrix, and secretion of osteoclastogenic factors of jaw periosteum-derived mesenchymal stem/stromal cells.

Dexamethasone (dexa) is commonly used to stimulate osteogenic differentiation of mesenchymal stem/stromal cells (MSCs) in vitro. However, it is paradoxical that glucocorticoids (GCs) such as dexa lead to bone loss and increased fracture risk in patients undergoing glucocorticoid therapy, causing glucocorticoid-induced osteoporosis (GIOP). In a recent publication, we demonstrated that osteogenic differentiation of progenitor cells isolated from jaw periosteal tissue (JPCs) does not depend on dexa, if the medium is supplemented with human platelet lysate (hPL) instead of fetal bovine serum (FBS). This allows the in vitro conditions to be much closer to the natural situation in vivo and enables us to compare osteogenic differentiation with and without dexa. In the present study, we demonstrate that the absence of dexa did not reduce mineralization capacity, but instead slightly improved the osteogenic differentiation of jaw periosteal cells. On the other hand, we show that dexa supplementation strongly alters the gene expression, extracellular matrix (ECM), and cellular communication of jaw periosteal cells. The secretome of periosteal cells previously treated with an osteogenic medium with and without dexa was used to investigate the changes in paracrine secretion caused by dexa. Dexa altered the secretion of several cytokines by jaw periosteal cells and strongly induced osteoclast differentiation of peripheral blood mononuclear cells (PBMCs). This study demonstrates how dexa supplementation can influence the outcome of in vitro studies and highlights a possible role of periosteal cells in the pathogenesis of glucocorticoid-induced osteoporosis. The methods used here can serve as a model for studying bone formation, fracture healing, and various pathological conditions such as (glucocorticoid-induced) osteoporosis, osteoarthritis, bone cancer, and others, in which the interactions of osteoblasts with surrounding cells play a key role.

Accuracy of Three-Dimensional Soft-Tissue Prediction Considering the Facial Aesthetic Units Using a Virtual Planning System in Orthognathic Surgery.

Virtual surgical planning (VSP) is commonly used in orthognathic surgery. A precise soft-tissue predictability would be a helpful tool, for determining the correct displacement distances of the maxilla and mandible. This study aims to evaluate the soft-tissue predictability of the VSP software IPS CaseDesigner® (KLS Martin Group, Tuttlingen, Germany). Twenty patients were treated with bimaxillary surgery and were included in the study. The soft-tissue simulation, done by the VSP was exported as STL files in the engineering software Geomagic Control XTM (3D systems, RockHill, SC, USA). Four months after surgery, a 3D face scan of every patient was performed and compared to the preoperative simulation. The quality of the soft-tissue simulation was validated with the help of a distance map. This distance map was calculated using the inter-surface distance algorithm between the preoperative simulation of the soft-tissue and the actual scan of the postoperative soft-tissue surface. The prediction of the cranial parts of the face (upper cheek, nose, upper lip) was more precise than the prediction of the lower areas (lower cheek, lower lip, chin). The percentage of correctly predicted soft-tissue for the face in total reached values from 69.4% to 96.0%. The VSP system IPS CaseDesigner® (KLS Martin Group; Tuttlingen, Germany) predicts the patient's post-surgical soft-tissue accurately. Still, this simulation has to be seen as an approximation, especially for the lower part of the face, and continuous improvement of the underlying algorithm is needed for further development.

Plaster Casts vs. Intraoral Scans: Do Different Methods of Determining the Final Occlusion Affect the Simulated Outcome in Orthognathic Surgery?

A virtual occlusal adjustment in orthognathic surgery has many advantages; however, the haptic information offered by plaster casts is missing when using intraoral scans. Feeling the interferences may be helpful in defining the best possible occlusion. Whether the use of a virtual occlusal adjustment instead of the conventional approach has a significant effect on the postsurgical position of the jaws is a question that remains unanswered. This study compares a virtual method to the conventional method of defining the final occlusion. Twenty-five orthognathic patients were included. Bimaxillary and single-jaw orthognathic surgery (mandible only) was simulated. The two methods were compared regarding discrepancies in the simulated postsurgical position of the mandible, measured three-dimensionally using MeshLab (MeshLab 2020.12 3D). An analysis using SPSS revealed no significant differences between the tested methods (p-values: 0.580 to 0.713). The mean absolute discrepancies ranged from 0.14 mm to 0.72 mm, laying within the scope of the clinically acceptable inaccuracies of an osteosynthesis in orthognathic surgery. The lack of haptic information in virtual planning had no relevant influence on the definition of the final occlusion and the simulated postsurgical outcome. However, in individual cases, plaster models might still be helpful in finding the adequate occlusion, especially in the sagittal dimension and in cases of patients with an anterior open bite, but this remains to be tested.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro.

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

Impact of Fluid Dynamics on the Viability and Differentiation Capacity of 3D-Cultured Jaw Periosteal Cells.

Perfused bioreactor systems are considered to be a promising approach for the 3D culturing of stem cells by improving the quality of the tissue-engineered grafts in terms of better cell proliferation and deeper penetration of used scaffold materials. Our study aims to establish an optimal perfusion culture system for jaw periosteal cell (JPC)-seeded scaffolds. For this purpose, we used beta-tricalcium phosphate (ß-TCP) scaffolds as a three-dimensional structure for cell growth and osteogenic differentiation. Experimental set-ups of tangential and sigmoidal fluid configurations with medium flow rates of 100 and 200 µL/min were applied within the perfusion system. Cell metabolic activities of 3D-cultured JPCs under dynamic conditions with flow rates of 100 and 200 µL/min were increased in the tendency after 1, and 3 days of culture, and were significantly increased after 5 days. Significantly higher cell densities were detected under the four perfused conditions compared to the static condition at day 5. However, cell metabolic and proliferation activity under dynamic conditions showed flow rate independency in our study. In this study, dynamic conditions increased the expression of osteogenic markers (ALPL, COL1A1, RUNX2, and OCN) compared to static conditions and the tangential configuration showed a stronger osteogenic effect than the sigmoidal flow configuration.

Pre-Conditioning with IFN-γ and Hypoxia Enhances the Angiogenic Potential of iPSC-Derived MSC Secretome.

Induced pluripotent stem cell (iPSC) derived mesenchymal stem cells (iMSCs) represent a promising source of progenitor cells for approaches in the field of bone regeneration. Bone formation is a multi-step process in which osteogenesis and angiogenesis are both involved. Many reports show that the secretome of mesenchymal stromal stem cells (MSCs) influences the microenvironment upon injury, promoting cytoprotection, angiogenesis, and tissue repair of the damaged area. However, the effects of iPSC-derived MSCs secretome on angiogenesis have seldom been investigated. In the present study, the angiogenic properties of IFN-γ pre-conditioned iMSC secretomes were analyzed. We detected a higher expression of the pro-angiogenic genes and proteins of iMSCs and their secretome under IFN-γ and hypoxic stimulation (IFN-H). Tube formation and wound healing assays revealed a higher angiogenic potential of HUVECs in the presence of IFN-γ conditioned iMSC secretome. Sprouting assays demonstrated that within Coll/HA scaffolds, HUVECs spheroids formed significantly more and longer sprouts in the presence of IFN-γ conditioned iMSC secretome. Through gene expression analyses, pro-angiogenic genes (FLT-1, KDR, MET, TIMP-1, HIF-1α, IL-8, and VCAM-1) in HUVECs showed a significant up-regulation and down-regulation of two anti-angiogenic genes (TIMP-4 and IGFBP-1) compared to the data obtained in the other groups. Our results demonstrate that the iMSC secretome, pre-conditioned under inflammatory and hypoxic conditions, induced the highest angiogenic properties of HUVECs. We conclude that pre-activated iMSCs enhance their efficacy and represent a suitable cell source for collagen/hydroxyapatite with angiogenic properties.

Neurocognitive development in isolated Robin sequence treated with the Tuebingen palatal plate.

OBJECTIVES: We aimed to determine the neurocognitive development of cleft palate patients with and without Robin sequence (RS). MATERIALS AND METHODS: Children with isolated RS with cleft palate and children with cleft palate only (CPO) were contacted at the age of 5-6 years. All RS children had undergone initial polygraphic sleep study (PG) with a mixed-obstructive apnea index (MOAI) of ≥ 3/h and were consequently treated with the Tuebingen palatal plate. A standardized clinical examination as well as a neuropediatric and neuropsychological examination included the Wechsler Pre-school and Primary Scale of Intelligence (WPPSI-III), Kaufman Assessment Battery for Children (K-ABC), and an assessment of developmental milestones. RESULTS: In total, 44 children (22RS, 22CPO) were included. RS children were younger at study (70.5 ± 7.3 and 75.2 ± 7.5 months; P = .035). Both groups achieved the evaluated milestones within the normed time frame. WPPSI-III and K-ABC results showed no group differences. Mean values for Verbal IQ (101.8 ± 11.1 vs. 97.1 ± 15.7), Performance IQ (102.9 ± 12.1 vs. 99.6 ± 14.5), Processing Speed Quotient (98.9 ± 15.6 vs. 94.5 ± 15.7), Full-Scale IQ (103.2 ± 12.1 vs. 98.4 ± 15.3), and Sequential Processing Scale (102.1 ± 13.1 vs. 94.2 ± 17.3) were within the reference range (IQ 85-115) for RS and CPO children, respectively, indicating average performance of both groups. CONCLUSION: No neurocognitive, physical, or mental impairments were detected suggesting that RS children having upper airway obstruction (UAO) treated early and effectively may use their potential for an age-appropriate neurocognitive development. CLINICAL RELEVANCE: Tuebingen palatal plate treatment successfully releases UAO. Thus, isolated RS does not necessarily result in developmental delay or an impaired neurocognitive outcome. TRIAL REGISTRATION: Deutsches Register Klinischer Studien, DRKS00006831, https://www.drks.de/drks_web/.

Intraoral scanning of neonates and infants with craniofacial disorders: feasibility, scanning duration, and clinical experience.

OBJECTIVE: The aim of this study was to evaluate intraoral scanning (IOS) in infants, neonates, and small children with craniofacial anomalies for its feasibility, scanning duration, and success rate. Impression taking in vulnerable patients can be potentially life-threatening, with the risk of airway obstruction and aspiration of impression material. The advantage of increasingly digitalized dentistry is demonstrated. MATERIALS AND METHODS: IOS was captured with the Trios 3® (3Shape, Copenhagen, Denmark) intraoral scanner. The underlying disorders were divided into cleft lip and palate (CLP), Trisomy 21 (T21), Robin Sequence (RS), Treacher Collins syndrome (TC), and isolated mandibular retrognathia (MR). Scan data were analysed by scanning duration, number of images, possible correlations of these factors with the different craniofacial disorders, patient age, and relationship between first and subsequent scans. Clinical experiences with the repeated digital impressions are described. RESULTS: Patient data of 141 scans in 83 patients were analysed within an 11-month period. Patients had a median age of 137 days. Median scanning duration was 138 seconds, resulting in a median of 352 images. There was a statistically significant difference in scanning duration (P = 0.001) between infants and neonates. IOS took longest in patients with CLP (537 seconds) and shortest in T21 patients (21 seconds), although there was no statistically significant difference between aetiologies. There was no statistically significant difference between first and subsequent scans in scanning duration. In four cases the IOS had to be repeated, and one patient ultimately required conventional impression taking (all CLP patients; success rate 94%). No severe adverse events occurred. CONCLUSION: IOS is a fast, safe, and feasible procedure for neonates, small children, and infants with craniofacial malformations. One special challenge for both technician and user was identified in patients with CLP, though implementing this new approach of digital impression taking was otherwise found to be highly successful in everyday clinical routine.

Speech Development in Cleft Palate with and without Robin Sequence.

BACKGROUND: Robin sequence is defined as the triad of micrognathia, glossoptosis, and upper airway obstruction. In up to 85 percent, it is associated with cleft palate. Many studies have reported worse speech development in Robin sequence children after cleft palate repair. The authors investigated speech development in isolated Robin sequence with cleft palate versus children with cleft palate only at the age of 5 to 6 years. METHODS: All Robin sequence children were treated with the Tübingen palatal plate after birth. Data were collected using the German version of the Great Ormond Street Speech Assessment. Audio and video recordings were reviewed and analyzed separately by two blinded senior phoniatricians based on the German version of the Universal Reporting Parameters for Cleft Palate Speech, and scored to enable comparability of speech outcomes. RESULTS: Forty-four children (Robin sequence, n = 22; cleft palate only, n = 22) were included. Robin sequence children were significantly older at surgery (11.8 months versus 7.1 months; p < 0.001) but younger at study (70.5 months versus 75.2 months; p = 0.035). They also had more severe cleft of the palate (p = 0.006). All children studied showed good to very good speech development without serious impairment. None of the reported parameters on the German version of the Universal Reporting Parameters for Cleft Palate Speech showed significant group differences; the median total score in the Robin sequence group was 23 (interquartile range, 16.5 to 27.5) versus 19 (interquartile range, 17 to 23) in the cleft palate-only group. Statistical analysis revealed no significant effect of group (Z = -1.47; p = 0.14). CONCLUSIONS: No group differences in speech development were found at age 5 to 6 years. Isolated Robin sequence does not necessarily represent a risk for impaired speech development. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, II.

Secretomes derived from osteogenically differentiated jaw periosteal cells inhibit phenotypic and functional maturation of CD14+ monocyte-derived dendritic cells.

The jaw periosteal tissue is generally recognized as a suitable source for the isolation of mesenchymal stem cells (MSCs). In previous studies we showed evidence that two- and three-dimensionally cultured jaw periosteum-derived MSCs (JPCs) are able to induce a more immature phenotype of dendritic cells (DCs). To further expand our knowledge of JPCs' immunoregulative function, we investigated the effects of JPC secretomes derived from undifferentiated (CO) or osteogenically differentiated cells (treated with or without dexamethasone: OB+/-D) on CD14+ monocyte-derived DCs (MoDCs). We detected a remarkably reduced formation of MoDC homotypic clusters under the influence of secretomes from osteogenically induced JPCs. Further, significantly decreased numbers of CD83+ cells, up-regulated CD209 and down-regulated CD80, CD86 and CD197 expression levels were detected on the surface of MoDCs. Whereas secretomes from JPCs osteogenically stimulated with dexamethasone significantly enhanced FITC-dextran uptake capacity of MoDCs, the increase by secretomes of JPCs treated without dexamethasone did not reach significance. The analysis of mixed lymphocyte reactions revealed that OB+/-D secretomes were able to significantly reduce the numbers of proliferating CD14- peripheral blood mononuclear cells (PBMCs) and of proliferating CD4+ T cells. The OB-D secretome significantly promoted the expansion of regulatory CD25+ T cells. Regarding gene expression of MoDCs, remarkably up-regulated mRNA expression of CD209, HLA-DRA, CSF3, IL10 and IL8 was detected when DCs were cultured in the presence of OB+/-D secretomes. At the same time, secretomes seemed to have an impact in the down-regulation of IFNγ and IL12B gene expression. At protein level, OB+/-D secretomes significantly up-regulated IL-10 and IDO (indoleamine-pyrrole 2,3-dioxygenase) levels whereas IL-12/IL-23p40 levels were down-regulated in supernatants of MoDCs when cultured under the presence of OB+/-D secretomes. Taken together, while secretomes from untreated JPCs had only little effects on the process of maturation of MoDCs, secretomes derived from osteogenically induced JPCs were able to inhibit the phenotypic and functional maturation of MoDCs.

Analysis of the Influence of Jaw Periosteal Cells on Macrophages Phenotype Using an Innovative Horizontal Coculture System.

Jaw periosteum-derived mesenchymal stem cells (JPCs) represent a promising cell source for bone tissue engineering in oral and maxillofacial surgery due to their high osteogenic potential and good accessibility. Our previous work demonstrated that JPCs are able to regulate THP-1-derived macrophage polarization in a direct coculture model. In the present study, we used an innovative horizontal coculture system in order to understand the underlying paracrine effects of JPCs on macrophage phenotype polarization. Therefore, JPCs and THP-1-derived M1/M2 macrophages were cocultured in parallel chambers under the same conditions. After five days of horizontal coculture, flow cytometric, gene and protein expression analyses revealed inhibitory effects on costimulatory and proinflammatory molecules/factors as well as activating effects on anti-inflammatory factors in M1 macrophages, originating from multiple cytokines/chemokines released by untreated and osteogenically induced JPCs. A flow cytometric assessment of DNA synthesis reflected significantly decreased numbers of proliferating M1/M2 cells when cocultured with JPCs. In this study, we demonstrated that untreated and osteogenically induced JPCs are able to switch macrophage polarization from a classical M1 to an alternative M2-specific phenotype by paracrine secretion, and by inhibition of THP-1-derived M1/M2 macrophage proliferation.

Angiogenic Potential of VEGF Mimetic Peptides for the Biofunctionalization of Collagen/Hydroxyapatite Composites.

Currently, the focus on bioinspired concepts for the development of tissue engineering constructs is increasing. For this purpose, the combination of collagen (Coll) and hydroxyapatite (HA) comes closest to the natural composition of the bone. In order to confer angiogenic properties to the scaffold material, vascular endothelial growth factor (VEGF) is frequently used. In the present study, we used a VEGF mimetic peptide (QK) and a modified QK-peptide with a poly-glutamic acid tag (E7-QK) to enhance binding to HA, and analyzed in detail binding efficiency and angiogenic properties. We detected a significantly higher binding efficiency of E7-QK peptides to hydroxyapatite particles compared to the unmodified QK-peptide. Tube formation assays revealed similar angiogenic functions of E7-QK peptide (1µM) as induced by the entire VEGF protein. Analyses of gene expression of angiogenic factors and their receptors (FLT-1, KDR, HGF, MET, IL-8, HIF-1α, MMP-1, IGFBP-1, IGFBP-2, VCAM-1, and ANGPT-1) showed higher expression levels in HUVECs cultured in the presence of 1 µM E7-QK and VEGF compared to those detected in the negative control group without any angiogenic stimuli. In contrast, the expression of the anti-angiogenic gene TIMP-1 showed lower mRNA levels in HUVECs cultured with E7-QK and VEGF. Sprouting assays with HUVEC spheroids within Coll/HA/E7-QK scaffolds showed significantly longer sprouts compared to those induced within Coll/HA/QK or Coll/HA scaffolds. Our results demonstrate a significantly better functionality of the E7-QK peptide, electrostatically bound to hydroxyapatite particles compared to that of unmodified QK peptide. We conclude that the used E7-QK peptide represents an excellently suited biomolecule for the generation of collagen/hydroxyapatite composites with angiogenic properties.

Influence of Human Jaw Periosteal Cells Seeded ß-Tricalcium Phosphate Scaffolds on Blood Coagulation.

Tissue engineering offers auspicious opportunities in oral and maxillofacial surgery to heal bone defects. For this purpose, the combination of cells with stability-providing scaffolds is required. Jaw periosteal cells (JPCs) are well suited for regenerative therapies, as they are easily accessible and show strong osteogenic potential. In this study, we analyzed the influence of uncoated and polylactic-co-glycolic acid (PLGA)-coated ß-tricalcium phosphate (ß-TCP) scaffolds on JPC colonization and subsequent osteogenic differentiation. Furthermore, interaction with the human blood was investigated. This study demonstrated that PLGA-coated and uncoated ß-TCP scaffolds can be colonized with JPCs and further differentiated into osteogenic cells. On day 15, after cell seeding, JPCs with and without osteogenic differentiation were incubated with fresh human whole blood under dynamic conditions. The activation of coagulation, complement system, inflammation, and blood cells were analyzed using ELISA and scanning electron microscopy (SEM). JPC-seeded scaffolds showed a dense cell layer and osteogenic differentiation capacity on both PLGA-coated and uncoated ß-TCP scaffolds. SEM analyses showed no relevant blood cell attachment and ELISA results revealed no significant increase in most of the analyzed cell activation markers (ß-thromboglobulin, Sc5B-9, polymorphonuclear (PMN)-elastase). However, a notable increase in thrombin-antithrombin III (TAT) complex levels, as well as fibrin fiber accumulation on JPC-seeded ß-TCP scaffolds, was detected compared to the scaffolds without JPCs. Thus, this study demonstrated that besides the scaffold material the cells colonizing the scaffolds can also influence hemostasis, which can influence the regeneration of bone tissue.