Whole stromal fibroblast signature is linked to specific chemokine and immune infiltration patterns and to improved survival in NSCLC.

Cancer associated fibroblasts (CAF) are known to orchestrate multiple components of the tumor microenvironment, whereas the influence of the whole stromal-fibroblast compartment is less understood. Here, an extended stromal fibroblast signature was investigated to define its impact on immune cell infiltration. The lung cancer adenocarcinoma (LUAD) data set of the cancer genome atlas (TCGA) was used to test whole stroma signatures and cancer-associated fibroblast signatures for their impact on prognosis. 3D cell cultures of the NSCLC cancer cell line A549 together with the fibroblast cell line SV80 were used in combination with infiltrating peripheral blood mononuclear cells (PBMC) for in-vitro investigations. Immune cell infiltration was assessed via flow cytometry, chemokines were analyzed by immunoassays and RNA microarrays. Results were confirmed in specimens from NSCLC patients by flow cytometry or immunohistochemistry as well as in the TCGA data set. The TCGA analyses correlated the whole stromal-fibroblast signature with an improved outcome, whereas no effect was found for the CAF signatures. In 3D microtumors, the presence of fibroblasts induced infiltration of B cells and CD69+CD4+ T cells, which was linked to an increased expression of CCL13 and CXCL16. The stroma/lymphocyte interaction was confirmed in NSCLC patients, as stroma-rich tumors displayed an elevated B cell count and survival in the local cohort and the TCGA data set. A whole stromal fibroblast signature was associated with an improved clinical outcome in lung adenocarcinoma and in vitro and in vivo experiments suggest that this signature increases B and T cell recruitment via induction of chemokines.

The circadian transcription factor ARNTL2 is regulated by weight-loss interventions in human white adipose tissue and inhibits adipogenesis.

Misalignment of physiological circadian rhythms promotes obesity which is characterized by white adipose tissue (WAT) expansion. Differentiation of Adipose stem/progenitor cells (ASCs) contributes to WAT increase but the importance of the cellular clock in this process is incompletely understood. In the present study, we reveal the role of the circadian transcription factor Aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) in human ASCs, isolated from subcutaneous (s)WAT samples of patients undergoing routine elective plastic abdominal surgery. We show that circadian synchronization by serum-shock or stimulation with adipogenic stimuli leads to a different expression pattern of ARNTL2 relative to its well-studied paralogue ARNTL1. We demonstrate that ARNTL2 mRNA is downregulated in ASCs upon weight-loss (WL) whereas ARNTL2 protein is rapidly induced in the course of adipogenic differentiation and highly abundant in adipocytes. ARNTL2 protein is maintained in ASCs cooperatively by mechanistic Target of Rapamycin (mTOR) and Mitogen-activated Protein Kinase (MAPK) signalling pathways while ARNTL2 functions as an inhibitor on both circuits, leading to a feedback mechanism. Consistently, ectopic overexpression of ARNTL2 repressed adipogenesis by facilitating the degradation of ARNTL1, inhibition of Kruppel-Like Factor 15 (KLF15) gene expression and down-regulation of the MAPK-CCAAT/enhancer-binding protein ß (C/EBPß) axis. Western blot analysis of sWAT samples from normal-weight, obese and WL donors revealed that ARNTL2 protein was solely elevated by WL compared to ARNTL1 which underscores unique functions of both transcription factors. In conclusion, our study reveals ARNTL2 to be a WL-regulated inhibitor of adipogenesis which might provide opportunities to develop strategies to ameliorate obesity.

An organoid model derived from human adipose stem/progenitor cells to study adipose tissue physiology.

We established a functional adipose organoid model system for human adipose stem/progenitor cells (ASCs) isolated from white adipose tissue (WAT). ASCs were forced to self-aggregate by a hanging-drop technique. Afterwards, spheroids were transferred into agar-coated cell culture dishes to avoid plastic-adherence and dis-aggregation. Adipocyte differentiation was induced by an adipogenic hormone cocktail. Morphometric analysis revealed a significant increase in organoid size in the course of adipogenesis until d 18. Whole mount staining of organoids using specific lipophilic dyes showed large multi- and unilocular fat deposits in differentiated cells indicating highly efficient differentiation of ASCs into mature adipocytes. Moreover, we found a strong induction of the expression of key adipogenesis and adipocyte markers (CCAAT/enhancer-binding protein (C/EBP) ß, peroxisome proliferator-activated receptor (PPAR) γ, fatty acid-binding protein 4 (FABP4), adiponectin) during adipose organoid formation. Secreted adiponectin was detected in the cell culture supernatant, underscoring the physiological relevance of mature adipocytes in the organoid model. Moreover, colony formation assays of collagenase-digested organoids revealed the maintenance of a significant fraction of ASCs within newly formed organoids. In conclusion, we provide a reliable and highly efficient WAT organoid model, which enables accurate analysis of cellular and molecular markers of adipogenic differentiation and adipocyte physiology.

The Great Auricular Nerve Trigger Site: Anatomy, Compression Point Topography, and Treatment Options for Headache Pain.

BACKGROUND: Peripheral nerve decompression surgery can effectively address headache pain caused by compression of peripheral nerves of the head and neck. Despite decompression of known trigger sites, there are a subset of patients with trigger sites centered over the postauricular area coursing. The authors hypothesize that these patients experience primary or residual pain caused by compression of the great auricular nerve. METHODS: Anatomical dissections were carried out on 16 formalin-fixed cadaveric heads. Possible points of compression along fascia, muscle, and parotid gland were identified. Ultrasound technology was used to confirm these anatomical findings in a living volunteer. RESULTS: The authors' findings demonstrate that the possible points of compression for the great auricular nerve are at Erb's point (point 1), at the anterior border of the sternocleidomastoid muscle in the dense connective tissue before entry into the parotid gland (point 2), and within its intraparotid course (point 3). The mean topographic measurements were as follows: Erb's point to the mastoid process at 7.32 cm/7.35 (right/left), Erb's point to the angle of the mandible at 6.04 cm/5.89 cm (right/left), and the posterior aspect of the sternocleidomastoid muscle to the mastoid process at 3.88 cm/4.43 cm (right/left). All three possible points of compression could be identified using ultrasound. CONCLUSIONS: This study identified three possible points of compression of the great auricular nerve that could be decompressed with peripheral nerve decompression surgery: Erb's point (point 1), at the anterior border of the sternocleidomastoid muscle (point 2), and within its intraparotid course (point 3).

Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1-/CD34+/CD24+ Adipose Stem/Progenitor Cells.

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34-, DLK1+/CD34dim and DLK1-/CD34+ cells. We demonstrate that DLK1-/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPß are highly expressed. Further sorting of ASCs into DLK1-/CD34+/CD24- and DLK1-/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1-/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1-/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1-/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPß but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1-/CD34+/CD24+ relative to DLK1-/CD34+/CD24- subpopulations.

Anatomical basis of a safe mini-invasive technique for lengthening of the anterior gastrocnemius aponeurosis.

BACKGROUND: The surgical procedure itself of lengthening the gastrocnemius muscle aponeurosis is performed to treat multiple musculoskeletal, neurological and metabolical pathologies related to a gastro-soleus unit contracture such as plantar fasciitis, Achilles tendinopathy, metatarsalgia, cerebral palsy, or diabetic foot ulcerations. Therefore, the aim of our research was to prove the effectiveness and safety of a new ultrasound-guided surgery-technique for the lengthening of the anterior gastrocnemius muscle aponeurosis, the "GIAR"- technique: the gastrocnemius-intramuscular aponeurosis release. METHODS AND RESULTS: An ultrasound-guided surgical GIAR on ten fresh-frozen specimens (10 donors, 8 male, 2 females, 5 left and 5 right) was performed. Exclusion criteria of the donated bodies to science were BMI above 35 (impaired ultrasound echogenicity), signs of traumas in the ankle and crural region, a history of ankle or foot ischemic vascular disorder, surgery or space-occupying mass lesions. The surgical procedures were performed by two podiatric surgeons with more than 6 years of experience in ultrasound-guided procedures. The anterior gastrocnemius muscle aponeurosis was entirely transected in 10 over 10 specimens, with a mean portal length of 2 mm (± 1 mm). The mean gain at the ankle joint ROM after the GIAR was 7.9° (± 1.1°). No damages of important anatomical structures could be found. CONCLUSION: Results of this study indicate that our novel ultrasound-guided surgery for the lengthening of the anterior gastrocnemius muscle aponeurosis (GIAR) might be an effective and safe procedure.

CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (SPRY1) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: Insertion/Deletion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition.

Sprouty1 Prevents Cellular Senescence Maintaining Proliferation and Differentiation Capacity of Human Adipose Stem/Progenitor Cells.

The role of Ras-Mitogen-activated protein kinase (MAPK) signaling in cellular aging is not precisely understood. Recently, we identified Sprouty1 (SPRY1) as a weight-loss target gene in human adipose stem/progenitor cells (ASCs) and showed that Sprouty1 is important for proper regulation of adipogenesis. In the present study, we show that loss-of-function of Sprouty1 by CRISPR/Cas9-mediated genome editing in human ASCs leads to hyper-activation of MAPK signaling and a senescence phenotype. Sprouty1 knockout ASCs undergo an irreversible cell cycle arrest, become enlarged and stain positive for senescence-associated ß-galactosidase. Sprouty1 down-regulation leads to DNA double strand breaks, a considerably increased number of senescence-associated heterochromatin foci and induction of p53 and p21Cip1. In addition, we detect an increase of hypo-phosphorylated Retinoblastoma (Rb) protein in SPRY1 knockout ASCs. p16Ink4A is not induced. Moreover, we show that Sprouty1 knockout leads to induction of a senescence-associated secretory phenotype as indicated by the activation of the transcription factors NFκB and C/EBPß and a significant increase in mRNA expression and secretion of interleukin-8 (IL-8) and CXCL1/GROα. Finally, we demonstrate that adipogenesis is abrogated in senescent SPRY1 knockout ASCs. In conclusion, this study reveals a novel mechanism showing the importance of Sprouty1 for the prevention of senescence and the maintenance of the proliferation and differentiation capacity of human ASCs.

Postoperative Hypoparathyroidism in Thyroid Surgery: Anatomic-Surgical Mapping of the Parathyroids and Implications for Thyroid Surgery.

Hypoparathyroidism remains one of the most common complications in thyroid surgery. This study aims for an improved understanding of the complexity of the blood supply and the localisation of the parathyroids compared to the two most important intraoperative landmarks: the inferior laryngeal nerve (ILN) and Zuckerkandl's tubercle (ZT). We examined 103 laryngeal compounds to classify the blood supply and the localisation of the parathyroids. For intraoperative localisation we defined a Cartesian coordinate system with the ZT plane as x-axis and the course of the inferior laryngeal nerve as y-axis. The inferior thyroid artery (ITA) mainly supplies the parathyroids, whereas the superior thyroid artery provides a backup supply. It must be pointed out that 8.2% of parathyroids receive their blood directly from the thyroid gland. 73.5% of all parathyroids lie within 1 cm of the ILN and 1 cm cranial and 2.5 cm caudal to the ZT plane. Our described perimeters mark the most crucial areas during surgery and provide the surgeon with an anatomic mapping showing areas of special carefulness needed. One should keep bearing in mind all possible blood supply types of the parathyroids and therefore all branches should be handled with care.

Sprouty1 is a weight-loss target gene in human adipose stem/progenitor cells that is mandatory for the initiation of adipogenesis.

The differentiation of adipose stem/progenitor cells (ASCs) into adipocytes contributes to adipose tissue expansion in obesity. This process is regulated by numerous signalling pathways including MAPK signalling. In the present study, we show that weight loss (WL) interventions induce upregulation of Sprouty1 (SPRY1), a negative regulator of MAPK signalling, in human ASCs and elucidate the role of the Sprouty1/MAPK interaction for adipogenic differentiation. We found that the Sprouty1 protein levels are low in proliferating ASCs, increasing in density arrested ASCs at the onset of adipogenic differentiation and decreasing in the course of adipogenesis. Knock-down (KD) of Sprouty1 by RNA interference led to elevated MAPK activity and reduced expression of the early adipogenic transcription factor CCAAT/enhancer-binding protein ß (C/EBP ß), concomitant with an abrogation of adipogenesis. Intriguingly, co-treatment of Sprouty1 KD ASCs with differentiation medium and the pharmacological MEK inhibitor U0126 blunted ERK phosphorylation; however, failed to rescue adipogenic differentiation. Thus, the effects of the Sprouty1 KD are not reversed by inhibiting MAPK signalling although the inhibition of MAPK signalling by U0126 did not prevent adipogenic differentiation in wild type ASCs. In conclusion, we show that Sprouty1 is induced after WL in ASCs of formerly obese people acting as a negative regulator of MAPK signalling, which is necessary to properly trigger adipogenesis at early stages by a C/EBP ß dependent mechanism.

Peripheral Nerve Anatomy Revisited: Modern Requirements for Neuroimaging and Microsurgery.

Detailed anatomic investigation of peripheral nerve topography underlies the correct application of intraoperative neuromonitoring (IONM) and ultrasonography, both well-established methods to prevent nerve palsy during surgical operations and to elucidate pathomechanisms in disease. In this study, we analyzed the anatomy of selected peripheral nerves in the head and neck regions to improve the outcome of endocrine and migraine surgeries. Anatomic dissections of 204 hemilarynges were performed to study the topography of the inferior laryngeal nerve (ILN). Measurements were taken from the lower rim of the cricoid and from the Zuckerkandl tubercle to the beginning of the furcation of the ILN. For the analysis of peripheral nerves contributing to migraine pathogenesis, 22 hemifaces were investigated by dissection and ultrasonography. The supratrochlear and supraorbital nerves and their relationship to the corrugator supercilii muscle are described. For identification of the ILN, the cricoid offers a suitable intraoperative landmark. A single branch existed in 5% of specimens on the left side and in 3% on the right side. Bifurcation was present in 72.5% and 62% and trifurcation in 18% and 29% of cases, respectively. IONM signals from the vagus nerve were positive if derived proximal to and negative if derived distal to the branching off of a nonrecurrent ILN (nrILN). By ultrasonographic identification of a brachiocephalic trunk, an nrILN could be excluded. For migraine surgery, possible compression points of the supratrochlear and supraorbital nerves were identified, and a workflow algorithm for ultrasound visualization of these nerves is provided. Anat Rec, 302:1325-1332, 2019. © 2019 Wiley Periodicals, Inc.

Fibroblast feeder layer supports adipogenic differentiation of human adipose stromal/progenitor cells.

Adipose stromal/progenitor cells (ASCs) can differentiate into adipocytes in the course of adipogenesis. This process is governed by systemic factors and signals of the adipose stem cell niche. ASCs isolated from fat tissues and amplified in vitro provide an essential and reliable model system to study adipogenesis. However, current cell culture models routinely grow ASCs on plastic surfaces largely missing niche parameters. In the present communication, we employed human foreskin fibroblasts (HFFs) monolayers as feeder cells for ASCs, which were isolated from human subcutaneous white adipose tissue and amplified in vitro. We found that PPARγ2 and several adipocyte markers were significantly higher expressed in differentiated ASCs growing on feeder layers relative to plastic dishes. Moreover, a significant higher number of adipocytes was generated from ASCs cultured on feeder layer and these adipocytes contained larger fat droplets. Insulin strongly stimulated glucose uptake into adipocytes produced on feeder layer suggesting that these cells show characteristic metabolic features of fat cells.  Finally, we show that the HFF feeder layer allows adipogenic differentiation of low-density-seeded ASCs. In conclusion, we demonstrate that the HFF feeder layer increases adipocyte differentiation of ASCs and allows differentiation of low density seeded progenitor cells  into functional adipocytes.

Ultrasound-guided decompression surgery of the distal tarsal tunnel: a novel technique for the distal tarsal tunnel syndrome-part III.

BACKGROUND: The aim of this study was to provide a safe ultrasound-guided minimally invasive surgical approach for a distal tarsal tunnel release concerning nerve entrapments. METHODS AND RESULTS: The study was carried out on ten fresh-frozen feet. All of them have been examined by high-resolution ultrasound at the distal tarsal tunnel. The surgical approach has been marked throughout the course of the medial intermuscular septum (MIS, the lateral fascia of the abductor hallucis muscle). After the previous steps, nerve decompression was carried out through a MIS release through a 2.5 mm (± 0.5 mm) surgical portal. As a result, an effective release of the MIS has been obtained in all fresh-frozen feet. CONCLUSION: The results of our anatomic study indicate that this novel ultrasound-guided minimally invasive surgical approach for the release of the MIS might be an effective, safe and quick decompression technique treating selected patients with a distal tarsal tunnel syndrome.

Ultrasound-guided decompression surgery of the tarsal tunnel: a novel technique for the proximal tarsal tunnel syndrome-Part II.

BACKGROUND: The aim of this study is to provide a safe ultrasound-guided minimally invasive surgical approach for a proximal tarsal tunnel release concerning nerve entrapments. METHODS AND RESULTS: The study was carried out on ten fresh-frozen feet. All of them were examined by high resolution ultrasound at the medial ankle region. The surgical approach was marked throughout the course of the flexor retinaculum (laciniate ligament). Once the previous steps were done, the flexor retinaculum release technique was carried out with a 2-mm entry only. As a result, an effective and safe release of the flexor retinaculum was obtained in all fresh-frozen feet. CONCLUSION: The results of our anatomic study indicate that our novel ultrasound-guided minimally invasive surgical approach for the release of the flexor retinaculum might be an effective, safe and quick decompression technique treating selected patients with a proximal tarsal tunnel syndrome.

Clinical-anatomic mapping of the tarsal tunnel with regard to Baxter's neuropathy in recalcitrant heel pain syndrome: part I.

PURPOSE: Neuropathy of the Baxter nerve (BN) seems to be the first cause of the heel pain syndrome (HPS) of neurological origin. METHODS: 41 alcohol-glycerol embalmed feet were dissected. We documented the pattern of the branches of the tibial nerve (TN) and describe all relevant osteofibrous structures. Measurements for the TN branches were related to the Dellon-McKinnon malleolar-calcaneal line also called DM line (DML) for the proximal TT and the Heimkes Triangle for the distal TT. Additionally, we performed an ultrasound-guided injection procedure of the BN and provide an algorithm for clinical usage. RESULTS: The division of the TN was 16.4 mm proximal to the DML. The BN branches off 20 mm above the DML center or 30 mm distally to it. In most of the cases, the medial calcaneal branch (MCB) originated from the TN proximal to the bifurcation. Possible entrapment spots for the medial and lateral plantar nerve (MPN, LPN), the BN and the MCB are found within a circle of 5 mm radius with a probability of 80%, 83%, and 84%, respectively. In ten out of ten feet, the US-guided injection was precisely allocated around the BN. CONCLUSIONS: Our detailed mapping of the TN branches and their osteofibrous tubes at the TT might be of importance for foot and ankle surgeons during minimally invasive procedures in HPS such as ultrasound-guided ankle and foot decompression surgery (UGAFDS).

The influence of stromal cells and tumor-microenvironment-derived cytokines and chemokines on CD3+CD8+ tumor infiltrating lymphocyte subpopulations.

The tumor microenvironment has been identified as a major mediator of immunological processes in solid tumors. In particular, tumor-associated fibroblasts are known to interact with tumor infiltrating immune cells. We describe the influence of fibroblasts and tumor-microenvironment-derived cytokines on the infiltration capacity of CD3+CD8+ cytotoxic T lymphocyte subpopulations using a multicellular 3D co-culture system. 3D tumor microtissues were cultivated using a hanging drop system. Human A549 and Calu-6 cancer cell lines were incubated alone or together with the human fibroblast cell line SV80 for 10 d to form microtissues. On day 10, peripheral blood mononuclear cells (PBMC) were added with or without cytokine stimulation for 24 h. Infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of the microtissues and the infiltration of the PBMCs were analyzed by immunohistochemistry, and endogenous cytokine and chemokine expression was analyzed with a multi-cytokine immunoassay. Secretion of chemokines is increased in microtissues consisting of cancer cells and fibroblasts. PBMC infiltrate the whole spheroid in cancer cell monocultures, whereas in co-cultures of cancer cells and fibroblasts, PBMCs are rather localized at the margin. Activated CD69+ and CD49d+ T lymphocytes show an increased microtissue infiltration in the presence of fibroblasts. We demonstrate that the stromal component of cancer microtissues significantly influences immune cell infiltration. The presence of fibroblasts in cancer microtissues induces a shift of T lymphocyte infiltration toward activated T lymphocytes.

Development of a 3D angiogenesis model to study tumour - endothelial cell interactions and the effects of anti-angiogenic drugs.

The tumour microenvironment and tumour angiogenesis play a critical role in the development and therapy of many cancers, but in vitro models reflecting these circumstances are rare. In this study, we describe the development of a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines (A549 and Colo699) in combination with a fibroblast cell line (SV 80) and two different endothelial cell lines in a hanging drop technology. Endothelial cells aggregated either in small colonies in Colo699 containing microtissues or in tube like structures mainly in the stromal compartment of microtissues containing A549. An up-regulation of hypoxia and vimentin, ASMA and a downregulation of E-cadherin were observed in co- and tri-cultures compared to monocultures. Furthermore, a morphological alteration of A549 tumour cells resembling "signet ring cells" was observed in tri-cultures. The secretion of proangiogenic growth factors like vascular endothelial growth factor (VEGF) was measured in supernatants. Inhibition of these proangiogenic factors by using antiangiogenic drugs (bevacizumab and nindetanib) led to a significant decrease in migration of endothelial cells into microtissues. We demonstrate that our method is a promising tool for the generation of multicellular tumour microtissues and reflects in vivo conditions closer than 2D cell culture.

Increased internal femoral torsion can be regarded as a risk factor for patellar instability - A biomechanical study.

BACKGROUND: Increased internal femoral torsion is regarded as a risk factor for patellar instability. Biomechanical investigations confirming this hypothesis are missing. METHODS: Eight fresh-frozen cadaver knees were tested on a specially designed simulator. Patellar motion and patellofemoral pressure were evaluated for 0°, 10°, and 20° of increased internal and external femoral torsion with native and with transected medial patellofemoral ligaments used to simulate patellar instability. A regression analysis was used for statistical analysis. FINDINGS: In native medial patellofemoral ligaments, there were no significant changes in mean or peak pressures for any torsional states (P≥0.07). At 20° increased internal femoral torsion, there was a significant center of force shift towards the lateral side (P=0.01). Patellar shift was directed laterally at low knee flexion angles up to 30°. Lateral patellar tilt increased significantly at 10° and 20° of increased internal femoral torsion (P≤0.004). In transected medial patellofemoral ligaments, mean pressure (P≤0.005) and peak pressure (P≤0.02) decreased significantly for all torsional states. There was a significantly greater lateral center of force shift with increased internal femoral torsion (P≤0.04). Lateral patellar tilt increased significantly (P<0.001). Patellar shift did not change significantly with increased internal femoral torsion (P≥0.30). INTERPRETATION: In a native medial patellofemoral ligament, 20° of increased internal femoral torsion can be regarded as a significant risk factor for patellar instability. With an insufficient medial patellofemoral ligament, 10° of increased internal femoral torsion already represents a significant risk factor.

CD146 (MCAM) in human cs-DLK1-/cs-CD34+ adipose stromal/progenitor cells.

To precisely characterize CD146 in adipose stromal/progenitor cells (ASCs) we sorted the stromal vascular faction (SVF) of human abdominal subcutaneous white adipose tissue (sWAT) according to cell surface (cs) expression of CD146, DLK1 and CD34. This test identified three main SVF cell populations: ~50% cs-DLK1-/cs-CD34+/cs-CD146- ASCs, ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146+ and ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146- cells. All cells contained intracellular CD146. Whole mount fluorescent IHC staining of small vessels detected CD146+ endothelial cells (CD31+/CD34+/CD146+) and pericytes (CD31-/CD34-/CD146+ ASCs). The cells in the outer adventitial layer showed the typical ASC morphology, were strongly CD34+ and contained low amounts of intracellular CD146 protein (CD31-/CD34+/CD146+). Additionally, we detected wavy CD34-/CD146+ and CD34dim/CD146+ cells. CD34dim/CD146+ cells were slightly more bulky than CD34-/CD146+ cells. Both CD34-/CD146+ and CD34dim/CD146+ cells were detached from the inner pericyte layer and protruded into the outer adventitial layer. Cultured early passage ASCs contained low levels of CD146 mRNA, which was expressed in two different splicing variants, at a relatively high amount of the CD146-long form and at a relatively low amount of the CD146-short form. ASCs contained low levels of CD146 protein, which consisted predominantly long form and a small amount of short form. The CD146 protein was highly stable, and the majority of the protein was localized in the Golgi apparatus. In conclusion, the present study contributes to a better understanding of the spatial localization of CD34+/CD146+ and CD34-/CD146+ cells in the adipose niche of sWAT and identifies CD146 as intracellular protein in cs-DLK1-/cs-CD34+/cs-CD146- ASCs.