Evaluation of a Novel Thiol-Norbornene-Functionalized Gelatin Hydrogel for Bioprinting of Mesenchymal Stem Cells.

Introduction: Three-dimensional bioprinting can be considered as an advancement of the classical tissue engineering concept. For bioprinting, cells have to be dispersed in hydrogels. Recently, a novel semi-synthetic thiolene hydrogel system based on norbornene-functionalized gelatin (GelNB) and thiolated gelatin (GelS) was described that resulted in the photoclick hydrogel GelNB/GelS. In this study, we evaluated the printability and biocompatibility of this hydrogel system towards adipose-tissue-derived mesenchymal stem cells (ASCs). Methods: GelNB/GelS was synthesized with three different crosslinking densities (low, medium and high), resulting in different mechanical properties with moduli of elasticity between 206 Pa and 1383 Pa. These hydrogels were tested for their biocompatibility towards ASCs in terms of their viability, proliferation and differentiation. The extrusion-based bioprinting of ASCs in GelNB/GelS-high was performed to manufacture three-dimensional cubic constructs. Results: All three hydrogels supported the viability, proliferation and chondrogenic differentiation of ASCs to a similar extent. The adipogenic differentiation of ASCs was better supported by the softer hydrogel (GelNB/GelS-low), whereas the osteogenic differentiation was more pronounced in the harder hydrogel (GelNB/GelS-high), indicating that the differentiation fate of ASCs can be influenced via the adaption of the mechanical properties of the GelNB/GelS system. After the ex vivo chondrogenic differentiation and subcutaneous implantation of the bioprinted construct into immunocompromised mice, the production of negatively charged sulfated proteoglycans could be observed with only minimal inflammatory signs in the implanted material. Conclusions: Our results indicate that the GelNB/GelS hydrogels are very well suited for the bioprinting of ASCs and may represent attractive hydrogels for subsequent in vivo tissue engineering applications.

The Edmonton Obesity Staging System Predicts Postoperative Complications After Abdominoplasty.

BACKGROUND: This study investigates the relationship between Edmonton Obesity Staging System (EOSS) and the occurrence of postoperative complications after abdominoplasty in massive weight loss patients. METHODS: A single-institution retrospective review of patients undergoing abdominoplasty between 2009 and 2019 after massive weight loss. Demographic data, laboratory findings, known risk factors for postoperative complications, as well as data on major and minor complications were extracted from the patient charts. Logistic regression models were used to investigate the relationship between the variables. RESULTS: Four hundred and five patients were included in the study. The prevalence of EOSS stages was: 0 (no comorbidities, N = 151, 37%), 1 (mild conditions, N = 40, 10%), 2 (moderate conditions, N = 149, 36%) and 3 (severe conditions, N = 70, 17%). Regression analysis showed that, controlling for body mass index (BMI), BMI Δ (maximal BMI - BMI at presentation), bariatric surgery, volume of resected tissue, and duration of surgery, EOSS stage significantly associated with the occurrence of postoperative complications. Compared with EOSS stage 0, EOSS stages 2 and 3 patients were associated with significantly more minor and major complications, respectively. The volume of resected tissue, BMI Δ, and age were associated with the occurrence of major complications. A regression model of comorbidities comprising the EOSS revealed a significant association of variables diabetes mellitus and hypertension with the occurrence of postoperative complications. CONCLUSIONS: Edmonton Obesity Staging System is a robust predictor of postoperative complications in abdominoplasty.

In vivo evaluation of bioprinted prevascularized bone tissue.

Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three-dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose-derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion-based bioprinting and drop-on-demand (DoD) bioprinting, respectively. The computer-generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical-sized bone defects.

Calmodulin Regulated Spectrin Associated Protein 1 mRNA is Directly Regulated by miR-126 in Primary Human Osteoblasts.

Vascularization is essential for bone development, fracture healing, and bone tissue engineering. We have previously described that coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves differentiation of both cell types. Investigating the role of microRNAs (miRNAs) in this system, we found that miR-126 is highly upregulated in hOBs following coculturing with HUVECs. In this study we performed miR-126 gain-of-function and loss-of-function experiments in hOBs followed by microarray analysis in order to identify targets of miR-126. The transcript cluster IDs were sieved by applying cut-off criteria and by selecting transcripts which were upregulated following miR-126 downregulation and vice versa. The calmodulin regulated spectrin associated protein 1 (CAMSAP1) mRNA was confirmed to be differentially regulated by miR-126. Using the luciferase reporter assay it was demonstrated that CAMSAP1 is directly targeted by miR-126. In this study, we show that miR-126 and CAMSAP1 directly interact in hOBs. This finding has potential implications for tissue engineering applications. J. Cell. Biochem. 118: 1756-1763, 2017. © 2016 Wiley Periodicals, Inc.

Prospective 3D analysis of facial soft tissue augmentation with calcium hydroxylapatite.

BACKGROUND: Facial rejuvenation is an expanding field with an increasing number of treatment modalities. Several non-autologous filler materials are available for soft tissue augmentation. Calcium hydroxylapatite (CaOH) is aimed at increasing collagen neosynthesis and thereby producing long-term augmentation effects. Despite a multitude of observational reports, the field is suffering from lack of quantitative morphometric evaluation methods. OBJECTIVE: The objective of this proof-of-principle study was to investigate whether the effects of facial tissue augmentation with CaOH (RADIESSE™) can be quantified and followed up using 3D surface scanning. METHODS: 3 female subjects received augmentation of the mid and lower face with CaOH. The faces were recorded prior, directly after, and two weeks and six months after the injection using standardized photos and 3D scanning. Computational analysis allowed quantifying the change in volume and displacement of the facial surface. Additionally, a patient satisfaction questionnaire was administered. RESULTS: In all subjects, increase in facial volume could be quantified and was present after two weeks and six months. CONCLUSIONS: 3D surface scanning is an adequate tool for objective quantification of changes after facial augmentation with filler materials. Persistent volume augmentation after CaOH injections could be quantified after two weeks and six months. Evidence level: IV.

Transcriptomic Changes in Osteoblasts Following Endothelial Cell-Cocultivation Suggest a Role of Extracellular Matrix in Cellular Interaction.

Vascularization is important for bone development, fracture healing and engineering of artificial bone tissue. In the context of bone tissue engineering, it was shown that coimplantation of human primary umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) results in the formation of functional blood vessels and enhanced bone regeneration. Implanted endothelial cells do not only contribute to blood vessel formation, but also support proliferation, cell survival and osteogenic differentiation of coimplanted hOBs. These effects are partially mediated by direct heterotypic cell contacts. In a previous report we could show that cocultivated hOBs strongly increase the expression of genes involved in extracellular matrix (ECM) formation in HUVECs, suggesting that ECM may be involved in the intercellular communication between hOBs and HUVECs. The present study aimed at investigating whether comparable changes occur in hOBs. We therefore performed a microarray analysis of hOBs cultivated in direct contact with HUVECs, revealing 1,004 differentially expressed genes. The differentially expressed genes could be assigned to the functional clusters ECM, proliferation, apoptosis and osteogenic differentiation. The microarray data could be confirmed by performing quantitative real time RT-PCR on selected genes. Furthermore, we could show that the ECM produced by HUVECs increased the expression of the osteogenic differentiation marker alkaline phosphatase (ALP) in hOBs. In summary, our data demonstrate that HUVECs provoke complex changes in gene expression patterns in cocultivated hOBs and that ECM plays and important role in this interaction. J. Cell. Biochem. 117: 1869-1879, 2016. © 2016 Wiley Periodicals, Inc.

miR-126 modulates angiogenic growth parameters of peripheral blood endothelial progenitor cells.

Vascularization plays an important role in tissue engineering applications. It is known that implantation of differentiated endothelial cells or endothelial progenitor cells (EPCs) from cord blood (cbEPCs) gives rise to the formation of a complex functional neovasculature, whereas EPCs isolated from peripheral blood (pbEPCs) have a limited capability to form blood vessels upon implantation. MicroRNA-126 (miR-126) has been shown to have pro-angiogenic effects in vivo. In this study, we investigated whether modulation of miR-126 expression in pbEPCs may alter their angiogenic properties. Gain of function and loss of function experiments revealed that miR-126 has anti-angiogenic effects in pbEPCs. Overexpression of miR-126 resulted in decreased proliferation, migration, invasion and tube formation, while inhibition of miR-126 induced the opposite effects. However, modulation of miR-126 expression did not influence apoptotic susceptibility of pbEPCs. This study provides evidence that inhibition of miR-126 improves angiogenesis-related growth parameters in pbEPCs and may represent a therapeutic option to ameliorate the angiogenic and vasculogenic properties of pbEPCs.

Osteoblastic alkaline phosphatase mRNA is stabilized by binding to vimentin intermediary filaments.

Vascularization is essential in bone tissue engineering and recent research has focused on interactions between osteoblasts (hOBs) and endothelial cells (ECs). It was shown that cocultivation increases the stability of osteoblastic alkaline phosphatase (ALP) mRNA. We investigated the mechanisms behind this observation, focusing on mRNA binding proteins. Using a luciferase reporter assay, we found that the 3'-untranslated region (UTR) of ALP mRNA is necessary for human umbilical vein endothelial cells (HUVEC)-mediated stabilization of osteoblastic ALP mRNA. Using pulldown experiments and nanoflow-HPLC mass spectrometry, vimentin was identified to bind to the 3'-UTR of ALP mRNA. Validation was performed by Western blotting. Functional experiments inhibiting intermediate filaments with iminodipropionitrile and specific inhibition of vimentin by siRNA transfection showed reduced levels of ALP mRNA and protein. Therefore, ALP mRNA binds to and is stabilized by vimentin. This data add to the understanding of intracellular trafficking of ALP mRNA, its function, and have possible implications in tissue engineering applications.

miR-126 regulates platelet-derived growth factor receptor-α expression and migration of primary human osteoblasts.

Adequate vascularization is an essential requirement for bone development, fracture healing and bone tissue engineering. We have previously described the coculture of primary human osteoblasts (hOBs) and human endothelial cells (HUVECs), designed to investigate the interactions between these cells. In this system, we showed that cocultivation of these two cell types leads to a downregulation of platelet-derived growth factor receptor-α (PDGFR-α) in hOBs, which was a consequence of reduced mRNA stability. In the current study we investigated the possible involvement of microRNAs in this process. Firstly, we performed a microarray analysis of osteoblastic miRNAs following cocultivation with HUVECs, revealing an upregulation of miR-126. This result was confirmed by RT-qPCR, and we observed that the increase is dependent on direct cell-to-cell contacts. Gain-of-function and loss-of-function experiments showed that miR-126 is a negative regulator of PDGFR-α mRNA. Additionally, migration of hOBs was inhibited by miR-126 overexpression and stimulated by miR-126 inhibition. Addition of PDGFR-α blocking antibody to hOB culture also inhibited hOB migration. There was no effect of miR-126 modulation on osteoblast proliferation, apoptosis rate or differentiation. In conclusion, we report that the miR-126/PDGFR-α system regulates the migratory behavior of human osteoblasts, without exerting effects on cell survival and differentiation.

In Situ Deactivation of Interleukin-6 Enhances Early Peripheral Nerve Regeneration in a Murine Injury Model.

BACKGROUND: Systemic alteration of interleukin-6 (IL-6) influences peripheral nerve regeneration. We investigated the potential influences of in situ (at the coaptation site) IL-6 modulation in a peripheral-nerve-transection/sciatic-nerve-graft in vivo model. METHODS: We quantified the elongation of regenerating axons, the number of arborizing axons, and the number of branches per arborizing axon 7 days after the injury in mice expressing axonal fluorescent proteins (thy-1-YFP mice). Sciatic nerves from nonexpressing mice (C57Bl6 or IL-6(-/-) mice) were grafted into those expressing yellow fluorescent protein. We altered the in situ IL-6 concentration by loading a topical gelatin sponge with an inhibiting IL-6 receptor antibody or IL-6 combined with a soluble IL-6 receptor. Sciatic nerves from IL-6(-/-) mice were grafted into an additional group. The contralateral sham-operated side served as control in all the groups. RESULTS: Axonal elongation increased significantly with the in situ application of the IL-6 receptor antibody, while topical IL-6 significantly reduced the regeneration distance. The number of arborizing axons increased significantly in nerves grafted from IL-6(-/-) mice, whereas branches per arborizing axons remained stable. CONCLUSION: In situ IL-6 receptor inhibition and IL-6(-/-) nerve grafting enhance early peripheral nerve regeneration in an acute murine injury model.

Aesthetic surgery performed by plastic surgery residents: an analysis of safety and patient satisfaction.

BACKGROUND: Aesthetic surgery is an integral component of plastic surgery. Despite its importance, adequate training in aesthetic surgery is met with challenges. Although the educational benefit of resident clinics has been demonstrated, such clinics are rarely found outside the United States. The objective of the present study was to assess safety and patient satisfaction associated with aesthetic surgery procedures performed by plastic surgery residents at a German academic medical center. METHODS: The study had 2 components, namely, a retrospective chart review and an administration of a patient satisfaction survey. Only patients who underwent a surgical intervention by a plastic surgery resident between 2003 and 2011 were included in the study. Parameters of interest included age, sex, procedure performed, number of procedures, revenue (in &OV0556;), length of follow-up, revision rate, and postoperative complication rate. Patient satisfaction was assessed by the client satisfaction questionnaire-8. RESULTS: A total of 273 aesthetic procedures were performed in 206 patients with an increase in recent years. The median follow-up period was 49.5 months. The most frequently performed procedures were liposuction (n = 59), breast augmentation (n = 53), and upper eyelid blepharoplasty (n = 31). One hundred ninety-two (90.3%) patients had an uneventful postoperative course. The client satisfaction questionnaire-8 questionnaire was completed by 110 patients (response rate, 50.2%). The median value of 28 indicates a high degree of patient satisfaction. An association between occurrence of major complications and patient satisfaction was seen. CONCLUSIONS: Aesthetic surgery performed by plastic surgery residents under supervision by attending physicians is safe and provides for high levels of patient satisfaction postoperatively. Offering these services may be able to bridge the gap between providing high-quality aesthetic surgery training while yet recruiting an increasing number of patients who may appreciate the lower fees associated with these services.

Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells.

Genetic Parkinson disease (PD) has been associated with mutations in PINK1, a gene encoding a mitochondrial kinase implicated in the regulation of mitochondrial degradation. While the studies so far examined PINK1 function in non-neuronal systems or through PINK1 knockdown approaches, there is an imperative to examine the role of endogenous PINK1 in appropriate human-derived and biologically relevant cell models. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblasts taken from three PD patients with nonsense (c.1366C>T; p.Q456X) or missense (c.509T>G; p.V170G) mutations in the PINK1 gene. These cells were differentiated into dopaminergic neurons that upon mitochondrial depolarization showed impaired recruitment of lentivirally expressed Parkin to mitochondria, increased mitochondrial copy number, and upregulation of PGC-1α, an important regulator of mitochondrial biogenesis. Importantly, these alterations were corrected by lentiviral expression of wild-type PINK1 in mutant iPS cell-derived PINK1 neurons. In conclusion, our studies suggest that fibroblasts from genetic PD can be reprogrammed and differentiated into neurons. These neurons exhibit distinct phenotypes that should be amenable to further mechanistic studies in this relevant biological context.

Vascularization Strategies in Bone Tissue Engineering.

Bone is a highly vascularized tissue, and its development, maturation, remodeling, and regeneration are dependent on a tight regulation of blood vessel supply. This condition also has to be taken into consideration in the context of the development of artificial tissue substitutes. In classic tissue engineering, bone-forming cells such as primary osteoblasts or mesenchymal stem cells are introduced into suitable scaffolds and implanted in order to treat critical-size bone defects. However, such tissue substitutes are initially avascular. Because of the occurrence of hypoxic conditions, especially in larger tissue substitutes, this leads to the death of the implanted cells. Therefore, it is necessary to devise vascularization strategies aiming at fast and efficient vascularization of implanted artificial tissues. In this review article, we present and discuss the current vascularization strategies in bone tissue engineering. These are based on the use of angiogenic growth factors, the co-implantation of blood vessel forming cells, the ex vivo microfabrication of blood vessels by means of bioprinting, and surgical methods for creating surgically transferable composite tissues.

Efficacy and Cost-Benefit Analysis of Magnetic Resonance Imaging in the Follow-Up of Soft Tissue Sarcomas of the Extremities and Trunk.

There is no consensus regarding follow-up after soft tissue sarcoma (STS) treatment. This study examines the efficacy and the cost-benefit of MRI imaging for discovering recurrence. A retrospective analysis was performed, collecting data on patient demography, tumor characteristics, treatment, and follow-up. Imaging was correlated to the clinical course, and sensitivity, specificity, and predictive values were calculated. The number needed to screen and costs of finding recurrence are reported. Amongst 216 sarcomas, 73 (35%) exhibited local recurrence during a follow-up of 5.3 ± 3.5 years. 173 entities had complete MRI follow-up with 58 (34%) local recurrences. Thirty-three (57%) were discovered by MRI, 8 (14%) by clinical presentation, and 17 (29%) simultaneously. There was a sensitivity of 100.00%, a specificity of 89%, a positive predictive value of 32%, and a negative predictive value of 100% for detecting local recurrence with MRI. Our data confirm the modalities and intervals proposed by the German guidelines for sarcoma care. The recommended MRI intervals should not be extended. MRI is more cost-effective than clinical examination; still, both modalities should be performed together to discover the maximum number of recurrences.

Gene expression profiling of substantia nigra dopamine neurons: further insights into Parkinson's disease pathology.

Parkinson's disease is caused by a progressive loss of the midbrain dopamine (DA) neurons in the substantia nigra pars compacta. Although the main cause of Parkinson's disease remains unknown, there is increasing evidence that it is a complex disorder caused by a combination of genetic and environmental factors, which affect key signalling pathways in substantia nigra DA neurons. Insights into pathogenesis of Parkinson's disease stem from in vitro and in vivo models and from postmortem analyses. Recent technological developments have added a new dimension to this research by determining gene expression profiles using high throughput microarray assays. However, many of the studies reported to date were based on whole midbrain dissections, which included cells other than DA neurons. Here, we have used laser microdissection to isolate single DA neurons from the substantia nigra pars compacta of controls and subjects with idiopathic Parkinson's disease matched for age and postmortem interval followed by microarrays to analyse gene expression profiling. Our data confirm a dysregulation of several functional groups of genes involved in the Parkinson's disease pathogenesis. In particular, we found prominent down-regulation of members of the PARK gene family and dysregulation of multiple genes associated with programmed cell death and survival. In addition, genes for neurotransmitter and ion channel receptors were also deregulated, supporting the view that alterations in electrical activity might influence DA neuron function. Our data provide a 'molecular fingerprint identity' of late-stage Parkinson's disease DA neurons that will advance our understanding of the molecular pathology of this disease.

New paradigm in training of undergraduate clinical skills: the NEPTUNE-CS project at the Split University School of Medicine.

Clinical skills' training is arguably the weakest point in medical schools' curriculum. This study briefly describes how we at the Split University School of Medicine cope with this problem. We consider that, over the last decades, a considerable advancement in teaching methodologies, tools, and assessment of students has been made. However, there are many unresolved issues, most notably: (i) the institutional value system, impeding the motivation of the teaching staff; (ii) lack of a strong mentoring system; (iii) organization, timing, and placement of training in the curriculum; (iv) lack of publications pertinent to training; and (v) unwillingness of patients to participate in student training. To improve the existing training models we suggest increased institutional awareness of obstacles, as well as willingness to develop mechanisms for increasing the motivation of faculty. It is necessary to introduce changes in the structure and timing of training and to complement it with a catalog, practicum, and portfolio of clinical skills. At Split University School of Medicine, we developed a new paradigm aimed to improve the teaching of clinical skills called "Neptune-CSS," which stands for New Paradigm in Training of Undergraduate Clinical Skills in Split.

Improved accuracy of breast volume calculation from 3D surface imaging data using statistical shape models.

BACKGROUND: Three-dimensional (3D) scanning is an established method of breast volume estimation. However, this method can never be entirely precise, since the thoracic wall cannot be imaged by the surface scanner. Current methods rely on interpolation of the posterior breast border from the surrounding thoracic wall. Here, we present a novel method to calculate the posterior border and increase the accuracy of the measurement. METHODS: Using principal component analysis, computed tomography images were used to build a statistical shape model (SSM) of the thoracic wall. The model was fitted to 3D images and the missing thoracic wall curvature interpolated (indirect volumetry). The calculations were evaluated by ordinary least squares regression between the preoperative and postoperative volume differences and the resection weights in breast reduction surgery (N = 36). Also, an SSM of the breast was developed, allowing direct volumetry. Magnetic-resonance images (MRI) and 3D scans were acquired from 5 patients in order to validate the direct 3D volumetry. RESULTS: Volumetry based on a SSM exhibited a higher determination coefficient (R2 = 0,737) than the interpolation method (R2 = 0,404). The methods were not equivalent (p = 0.75), suggesting that the methods significantly differ. There was no influence of BMI on the correlation in either method. The MRI volumetry had a strong correlation with the 3D volumetry (R2 = 0,978). CONCLUSION: The SSM-based method of posterior breast border calculation is reliable and superior to the currently used method of interpolation. It should serve as a basis of software applications aiming at calculation of breast volume from 3D surface scanning data.

In vivo evaluation of an electrospun gelatin nonwoven mat for regeneration of epithelial tissues.

One major objective in epithelial tissue engineering is to identify a suitable biomaterial that supports epithelial tissue formation. Therefore, the purpose of this study is to elucidate a novel electrospun gelatin nonwoven mat (NWM) for epithelial tissue engineering purposes in vivo. This NWM was seeded with either human gingival keratinocytes (GK, in coculture with gingival fibroblast) or human skin epithelial keratinocytes (EK, in coculture with skin dermal fibroblasts). These constructs were ex vivo cultured for 4 days before subcutaneous implantation into athymic nude mice. After 7 days, the constructs were explanted and investigated by immunohistology. Our results show that GK form a stratified epithelium on the surface of the NWM, mostly independent of a fibroblastic counterpart. Like native mucosa, the regenerated epithelium showed expression of epidermal growth factor receptor, cytokeratin-14 and -1, and involucrin. Only the expression of the basement membrane constituent laminin 5 was more pronounced in cocultures. Comparing GK and skin EK, we found that skin EK form a less developed epithelial tissue. Furthermore, the NWM allows not only for epithelial tissue formation by GK, but also for infiltration of human fibroblasts and mouse immune cells, thus representing a biomaterial with potential regenerative capacity for oral mucosa tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1605-1614, 2019.

Is there a place for medical students in research laboratories? A student's perspective.

Based on personal experience gained in two medical schools, one from a developed and one from a developing European country, I present a short discourse on the introduction of undergraduate medical students to research. The potential benefit of research for professional profile and overall competency building is emphasized and the possibility of counterbalancing the present crisis in academic medicine through undergraduate research is discussed.