Explant Technique Research Articles

Intraocular lens extraction using the cartridge pull-through technique.

Current intraocular lens (IOL) explantation techniques are limited to cutting the optic and removing the pieces through a small incision or folding single-piece acrylic IOLs using a 2-handed technique. Poor execution of IOL explantation can result in injury to intraocular structures, including the corneal endothelium and iris. The minimally invasive cartridge pull-through technique uses a cartridge for IOL implantation and new forceps optimized for secure grasping of the IOL for removal. This method involved less manipulation in the anterior chamber, thereby reducing the risks for complications such as corneal and iris injuries. A dropped IOL lying on the retinal surface can be extracted directly without lifting it onto the iris first. The cartridge pull-through technique offers a more streamlined and potentially safer approach for IOL explantation.

Easy surgical explantation technique for self-expanding transcatheter aortic valve: 'lasso technique'.

A prosthesis with a self-expanding nitinol frame firmly incorporates into the aortic wall and thus is difficult to explant. We introduce a novel technique that simplifies explantation.

Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers.

Human skin keratinocyte primary cultures can be established from skin biopsies with culture media containing epithelial growth factor (EGF). Although current methods are efficient, optimization is required to accelerate the procedure and obtain these cultures in less time. In the present study, we evaluated the effect of novel formulations based on EGF-loaded nanostructured lipid carriers (NLC). First, biosafety of NLC containing recombinant human EGF (NLC-rhEGF) was verified in immortalized skin keratinocytes and cornea epithelial cells, and in two epithelial cancer cell lines, by quantifying free DNA released to the culture medium. Then we established primary cell cultures of human skin keratinocytes with basal culture media (BM) and BM supplemented with NLC-rhEGF, liquid EGF (L-rhEGF), or NLC alone (NLC-blank). The results showed that cells isolated by enzymatic digestion and cultured with or without a feeder layer had a similar growth rate regardless of the medium used. However, the explant technique showed higher efficiency when NLC-rhEGF culture medium was used, compared to BM, L-rhEGF, or NLC-blank. Gene expression analysis showed that NLC-rhEGF was able to increase EGFR gene expression, along with that of other genes related to cytokeratins, cell–cell junctions, and keratinocyte maturation and differentiation. In summary, these results support the use of NLC-rhEGF to improve the efficiency of explant-based methods in the efficient generation of human keratinocyte primary cell cultures for tissue engineering use.

Molecular and genomic characterisation of a panel of human anal cancer cell lines

Anal cancer is a rare disease that has doubled in incidence over the last four decades. Current treatment and survival of patients with this disease has not changed substantially over this period of time, due, in part, to a paucity of preclinical models to assess new therapeutic options. To address this hiatus, we set-out to establish, validate and characterise a panel of human anal squamous cell carcinoma (ASCC) cell lines by employing an explant technique using fresh human ASCC tumour tissue. The panel of five human ASCC cell lines were validated to confirm their origin, squamous features and tumourigenicity, followed by molecular and genomic (whole-exome sequencing) characterisation. This panel recapitulates the genetic and molecular characteristics previously described in ASCC including phosphoinositide-3-kinase (PI3K) mutations in three of the human papillomavirus (HPV) positive lines and TP53 mutations in the HPV negative line. The cell lines demonstrate the ability to form tumouroids and retain their tumourigenic potential upon xenotransplantation, with varied inducible expression of major histocompatibility complex class I (MHC class I) and Programmed cell death ligand 1 (PD-L1). We observed differential responses to standard chemotherapy, radiotherapy and a PI3K specific molecular targeted agent in vitro, which correlated with the clinical response of the patient tumours from which they were derived. We anticipate this novel panel of human ASCC cell lines will form a valuable resource for future studies into the biology and therapeutics of this rare disease.

Comparison of the effects of co-transplantation of bone marrow hematopoietic stem cells and thymic multipotent stromal cells on the immune system of mice depending on methods

Physical interaction of multipotent stromal cells (MSCs) and hematopoietic stem cells (HSCs) is a modern approach to effective and focused changes in the properties of HSCs. Resulting of those contact interaction is significant activation of cells with following immune system restoration.
 The purpose of the study is to investigate the effect of co-transplantation of bone marrow hematopoietic stem cells (HSCs) and thymic multipotent stromal cells (MSCs) separately and as a union of cells on regeneration of the murine immune system, damaged by cyclophosphamide.
 MSCs were obtained from thymuses of C57BL mice using explant technique. Bone marrow cells (BMCs) were obtained by flushing out the femur with a nutrient medium. BMCs were cocultivated for 2 hours on the monolayer of thymus-derived MSCs. The immune deficiency of mice was modelled by the treatment with cyclophosphamide (CP). After that, the cells were co-transplanted in two methods (separately into different the retroorbital sinus and as a union after co-cultivation) and the parameters of the immune system were evaluated. It was shown, that separate co-transplantation of BMCs and thymus-derived MSCs is associated with the restoration of the number of bone marrow cells, thymus, spleen and lymph nodes with an increase in the proliferation index of lymph node cells by 1.4 times compared to control. It normalized the previous reduced concentration of hemoglobin and hematocrit in the blood. Co-transplantation had a suppressive effect on the blast transformation reaction, induced by phytohemagglutinin, by 4.3 times, but showed a stimulating effect on DTHR response by 1.6 times compared to control.
 Co-transplantation of the union of BMCs and MSCs is associated with the restoration of the number of bone marrow cells, spleen and lymph nodes. The level of spontaneous apoptosis of lymph node cells significantly increased by 3.3 times compared to control. It had not effect on hematological parameters, but is activated to impact the immune system. Thus, as a result of cells union administration showed normalization of the bactericidal activity of peritoneal macrophages, unlike the separate co-transplantation. This cells graft had a suppressive effect on the number of antibody-producing cells in the spleen by 4.2 times compared to control.
 Previous co-cultivation and contact interaction of cells change the properties of cell graft. The effect of co-transplantation of BMCs and thymic MSCs is not a simple additive effect of cells. It is acquiring the features typical to certain cell types, and the expression of new characteristics. We assume this phenomenon as a result development of complex cells cooperative processes in vivo and in vitro

Preliminary Evaluation of Proliferation, Wound Healing Properties, Osteogenic and Chondrogenic Potential of Dental Pulp Stem Cells Obtained from Healthy and Periodontitis Affected Teeth

Background: Dental pulp tissue within the central cavity of the tooth is composed of dental pulp stem cells (DPSC). These mesenchymal stem cells have good proliferative as well as differentiation potential. DPSC has been isolated even from teeth with inflamed pulps and is found to retain their proliferative and differentiation potential. Little research is available about the viability and differentiation potential of DPSC obtained from teeth with periodontitis. In the present study, the aim was to compare the morphological features, stem cell marker (MSC) expression, proliferation rate, migratory and wound healing properties, osteogenic and chondrogenic differentiation potential of DPSCs obtained from periodontally healthy teeth (hDPSCs) and periodontitis affected teeth (pDPSCs). Methods: Dental pulp tissue was obtained from periodontally healthy volunteers (n = 3) and patients with periodontitis undergoing extraction of mobile teeth (n = 3). DPSC were isolated using the explant technique and cultured. All the experiments were performed at early passage (Passage 2), late passage (Passage 6) and after cryopreservation. Morphological features of the hDPSCs and pDPSCs were ascertained using microscopy. The expression of cell surface stem cell markers was assessed by the flow cytometry method. The proliferation and growth rate of the cells were assayed by plotting a growth curve from 0–13 days of culture. The migratory characteristics were assessed by wound scratch assay. Osteogenic and chondrogenic differentiation of the cells was assessed using standard protocols with and without induction. Results: DPSCs were successfully obtained from periodontally healthy teeth (hDPSC) and periodontitis-affected teeth (pDPSCs). The data suggests that there were no morphological differences observed in early passage cells between the two cohorts. Cryopreservation did change the morphology of pDSPCs. There was no significant difference in the positive expression of mesenchymal markers CD73, CD90 and CD105 in early passage cells. However, serial passaging and cryopreservation affected the marker expression in pDPSCs. A faint expression of hematopoietic stem cell markers CD34, CD45 and MHC class II antigen HLA-DR was observed in both the cell types. The expression of HLA-DR is upregulated in pDPSCs compared to hDPSC. A significantly slower growth rate and slower wound healing properties was observed in pDPSCs compared to hDPSC. In late passage and after cryopreservation, the migratory ability of pDPSCs was found to be increased drastically. There was no significant difference in osteogenic potential between the two cell types. However, the chondrogenic potential of pDPSCs was significantly lower compared to hDPSc. Yet, pDPSCs showed enhanced osteogenesis and chondrogenesis at late passage as well as after cryopreservation. Conclusion: The results of this novel study shed light on the isolation of viable DPSC from periodontitis-affected teeth. These cells exhibit a slower growth rate and migratory characteristics compared to their healthy counterparts. There was no difference in osteogenic potential but a reduction in chondrogenic potential was seen in pDPSCs compared to hDPSC. The findings reveal that DPSC from periodontitis-affected teeth presents an easy and viable option for regenerative medicine application. Some additional nutritive factors and protocols may be required to attain better regenerative benefits while using pDPSCs.

Cryopreservation of Iranian Markhoz goat fibroblast cells as an endangered national genetic resource.

BackgroundThe continuous accessibility of local animals for sustainable use is being eroded annually. Thus, a strategic vision for the conservation of biodiversity is of far-reaching emphasis to deal with unprecedented challenges in the local population extension facing in the future. This study aimed to establish and cryopreserve endangered Markhoz goat (Capra hircus) fibroblast cell lines in vitro.Methods and resultsThese primary fibroblast cells were isolated from 58 Iranian Markhoz goats and individually cultured by explant technique in DMEM medium supplemented with 10% FBS and 2 mM L-Glutamine, in the presence of Penicillin (200 U/ml)—Streptomycin (200 mg/ml) during the first passage number. The extracted cell lines were confirmed morphologically as fibroblast cells. The population doubling time for DMEM-cultured cells was 23 ± 0.5 h. Chromosomal analysis indicated a total chromosome number of 2n = 60 with > 95% frequency. The cultured cells were checked for bacteria, fungi, yeast, and mycoplasma contaminations and the results were reported negative. The efficiencies of the fluorescent protein encoded by VSV-G (pMDG) and lentiviral pCSGW vectors reported in a range of 65% value. According to the species identification analysis, the goat cell lines were banked and confirmed without any miss- and cross-contamination.ConclusionsThe significant issue in this paper can be concluded about the first report of the establishment of endangered Markhoz goat cell banking inside the country. This study demonstrated the successful establishment of a genetically stable fibroblast bank as a valuable genetic resource for the endangered Iranian Markhoz goat breed.

Procedural outcome of lead explant and countertraction-assisted femoral lead extraction in Thai patients with cardiac implantable electronic device infection.

BackgroundCardiac implantable electronic device (CIED) implantation rate has been increasing worldwide. Despite proper surgical technique and preincisional intravenous antibiotics, the incidence of infected CIED remains high and leads to serious complications. When encountered with CIED infection, complete CIED system removal is indicated. Several lead extraction approaches have shown a high success rate. However, the facilities are limited in Thailand. In our current practice, we perform lead extraction using the Dotter basket snare femoral approach as our primary method. There are no prior data on this countertraction‐assisted transfemoral technique. Therefore, we aim to study the procedural outcome of countertraction‐assisted transfemoral lead removal technique of CIED infection in Thai patients.MethodsPatients diagnosed with CIED infection and with a history of device infection were retrospectively included. Simple manual removal was performed. In case of failure, we proceeded with the modified countertraction‐assisted transfemoral technique.ResultsThere were 35 patients in the study. The success rate was 94.3%. Most of the leads, 62.8%, were removed by simple manual traction. In the 37.1% who required further femoral approach lead extractions, procedural failure was observed in 5.7% and procedure‐related adverse events in 5.6%. CIED infection‐related death accounted for 5.7% and nosocomial infection‐related death, 2.8%.ConclusionThe success rate of CIED infection lead explant and countertraction‐assisted transfemoral lead extraction technique was high with small complications and can be performed without advanced facilities. However, the procedure required a main center with a cardiovascular thoracic surgery support team.

A pilot retrospective CT angio study of the internal pudendal arteries in male bodies, for the purpose of penis transplantation to trans men

Literature reports four successful cases where penile transplantation has been performed for cis men. To date, no penis transplantation has ever been attempted for trans men. Modern surgical techniques for penis reconstruction for (trans) men with gender incongruence present multiple drawbacks, and sometimes fail to meet patients' expectations. Penis transplantation could represent an alternative solution. With the aim of planning penis transplantation to trans men, a previous study from our same group suggested a surgical technique for explantation of penis and associated vessels, nerves and urethra en-bloc from a cadaver: further radiographic imaging of vascular anatomy was recommended. To measure length and diameter of the internal pudendal arteries at three set points in biological males, in order to confirm its viability for transplantation. A retrospective examination of existing CT images visualizing the internal pudendal arteries in 12 bodies, assigned male at birth, was performed. Diameters were measured in three points: 1. the take off of the artery; 2. distal to its rectal branch; and, 3. at the bifurcation of the two terminal branches of the artery, i.e. the bulbourethral artery and the dorsal artery of the penis. Mean diameters of the selected points were 2.4 mm, 1.9 mm, and 1.5 mm on both sides. Mean lengths of internal pudendal arteries were 111.2 mm on the left, and 120.1 mm on the right side. Diameters of the internal pudendal artery is sufficient for arterial anastomosis at all measured points. Lengths are sufficient to reach recipient vessels.

Proliferation rate and expression of stem cells markers during expansion in primary culture of pulp cells.

The aim of the present study was to evaluate the proliferation rate and the expression of stem cells markers during expansion in primary culture of dental pulp stem cells (DPSCs), comparing different techniques (explant and enzymatic digestion), subject ages (up to 40 and over 40) and cell passages (#2, #5 and #8). DPSCs were isolated using either the enzymatic digestion (ED) or explant (EX) technique. The number of days needed for the cells to reach confluence was determined. Immunophenotyping was performed by immunofluorescence and flow cytometry analysis using antibodies specific for nestin, vimentin, CD44, CD146, Oct3/4 and CD34. Data were subjected to three-way analysis of variance (n = 6/group). The ANOVA tests were complemented by Tukey's or t-tests (p < 0.05). The variables "donor age" and "technique" were analyzed to define the optimal desirability value using a response optimization. DPSCs presented a high proliferation rate from passages 2 to 5 while cells from passage 8 proliferated at a slower rate. For all markers, no significant difference was observed among passages, irrespective of the technique used or the donor's age. The mean fraction of specific antibodies was 73.7% (± 11.5), 49.0% (± 18.7), 80.1% (± 8.0), 45.2% (± 13.7), 64.7% (± 5.3) and 2.0% (± 1.5) for CD44, OCT, vimentin, nestin, CD146 and CD34, respectively. The highest optimal desirability value was obtained using the ED technique and cells from younger patients (d = 0.92). However, it was concluded that neither the isolation technique nor the donor age or cell passage significantly interfered with the stem cell phenotype and proliferation rate during cell expansion.

Feeder-Free Cell Culture of Labial Oral Mucosal Epithelium for Tissue-Engineering and Regenerative Medicine

Background.The cultured cheek mucosa epithelium (buccal epithelium, BE) is used for autologous transplants generation and tissue engineering. An alternative source of cells for these purposes may be the lip mucosa, covered, like BE, with non-keratinized stratified squamous epithelium, but with some histological distinctions.Aims to characterize the human lip mucosa as a promising source of epithelial cells for autologous transplantation and tissue engineering.Methods.Scrapings of the lip, cheek, and gum mucosa from five healthy volunteers were analyzed by cytofluorimetry to determine the level of desquamation and cytokeratin (CK) 10 and 13 expression. The lip mucosa of two patients was characterized using routine histological staining and fluorescence immunohistochemistry for CK 3, 4, 10, 13, and p63 marker. 35 samples of full-thickness strips of the patients lip mucosa were used to set the explant (n=18) and enzymatic (n=17) techniques for expansion epithelium. Culture systems with 1.05 and 0.06mM Calcium contained 5% fetal bovine serum, 5 g/ml human insulin, 5 g/ml hydrocortisone, 10 ng/ml human epidermal growth factor. Stable cultures were stained for p63, vimentin, zonula occludens-1 (ZO-1), and CK10. Software tools determined levels of their expression.Results.The number of cells in the lip and gum samples was significantly lower than from the cheek. The median number of CK13 positive cells was significantly different for the gum (6.4%) and cheek (64.8%, p=0.0089). Significant differences for CK10 positive cells were not observed. The epithelium of the lip mucosa was 72.13.6 m thick, relatively flat, and without keratinization sites. Samples were positively stained for CK 4 and 13, in the absence of expression of CK 3 and 10. The primary culture of epithelial cells obtained by explant technique was significantly more effective (p=0.001) in comparison with the enzymatic method. Stable cultures had a cobble-stone morphology in both culture systems. The levels of vimentin and p63 expression in both culture systems was not significantly differ. ZO-1 expression was 3.6-fold higher for 1.05-mMCa++medium (p=0.0006).Conclusions.Epithelium cell culture from the lip mucosa can be obtained by culturing explants without a feeder layer. Quality control steps have been developed for cultured cells and biopsy site.

A comparison of techniques for the explantation of osseointegrated dental implants

Data sources A search of electronic databases (Embase and PubMed) was carried out along with manual and grey searches of published and unpublished journals. Publication year was from first available until 23 August 2018.Study selection Titles and abstracts from the original search were reviewed by two authors. Studies were chosen for full-text analysis and data extraction after inter-reviewer agreement. Disagreement was resolved by discussion and Cohen's kappa was used to measure inter-reviewer agreement. An initial search gave 2,197 articles and, following screening, 18 publications were included in the study. Five articles were case series and ten were case reports describing one to nine cases. Three publications reported on comparatively large sample sizes, one prospectively and two retrospectively. None of the studies had control groups or blinding. The QUADAS-2 tool was used for quality assessment. Studies were deemed to have high, low or unclear levels of bias by two examiners. All were considered high risk of bias. Publications included fulfilled the following criteria: English language, human studies, endosseous osseointegrated dental implants, explantation technique described and reason for explantation clearly reported.Data extraction and synthesis Data extraction followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline process. Studies chosen for analysis were examined and the following data parameters were included: study design, number of patients, number of implants removed, implant system, reason for explantation, explantation technique and its success or failure, complications, flap access, socket grafting and immediate implant placement.Results The following five techniques for explantation of dental implants were identified: reverse torque, trephines, piezosurgery, burs and laser-assisted explantation. Reverse torque was the most commonly described technique (284 implants) with 87.7% success. Burs were used to remove 49 implants with 100% success, while trephines were used for explantation of 35 implants with 94% success. Piezosurgery and Er, Cr: YSGG laser removed 11 implants and one implant, respectively, with 100% success. One study reported perforation of the maxillary sinus floor following the use of a trephine technique, while another reported the fracture of three implants using reverse torque. The quality of the studies and lack of available data prevented further analysis. Results were presented in a narrative format.Conclusion The authors recommend reverse torque as the first choice for explantation. Despite its inferior success rate, it is the most conservative technique in terms of bone removal and flap access, meaning there is a greater opportunity for immediate implant placement.

Snaring technique for explantation of transcatheter aortic valve bioprosthesis

Snaring technique for explantation of transcatheter aortic valve bioprosthesis

A detailed explantation assessment protocol for patients with left ventricular assist devices with myocardial recovery.

Left ventricular assist device (LVAD) implantation for end-stage heart failure patients has been on the rise, providing a reliable long-term option. For some LVAD patients, longer term LV unloading leads to recovery; hence, the need for evaluating potential myocardial recovery and weaning eligibility has emerged. All patients who underwent contemporary LVAD explantation at our institution between 2009 and 2020 were included in the study. Patients in New York Heart Association I, left ventricular ejection fraction >40%, a cardiac index >2.4 l/min and a peak oxygen intake >50% predicted underwent a 4-phase weaning assessment. A minimally invasive approach using a titanium plug was the surgery of choice in the most recent explants. Kaplan-Meier curves were used to estimate the survival at 1 and 5 years. Twenty-six patients (17 HeartMate II, 9 HeartWare) underwent LVAD explantation after a median 317 days of support [IQ (212-518)], range 131-1437. Mean age at explant was 35.8 ± 12.7 years and 85% were males. Idiopathic dilated cardiomyopathy was the underlying diagnosis in 70% of cases. Thirteen (48%) patients were on short-term mechanical circulatory support and 60% required intensive care unit admission prior to the LVAD implantation. At 1 year, Kaplan-Meier estimated survival was 88%, whereas at 6 years, it was 77%. The average left ventricular ejection fraction at 1 year post-explant was 44.25% ± 8.44. The use of a standardized weaning protocol (echocardiographic and invasive) and a minimally invasive LVAD explant technique minimizes periprocedural complications and leads to good long-term device-free survival rates.

Novel Porcine Retina Cultivation Techniques Provide Improved Photoreceptor Preservation.

Age-related macular degeneration (AMD) is the leading cause of blindness in industrialized countries among people over 60 years. It has multiple triggers and risk factors, but despite intense research efforts, its pathomechanisms are currently not completely understood. AMD pathogenesis is characterized by soft drusen in Bruch’s membrane and involves the retinal pigment epithelium–Bruch’s membrane-choroid complex and adjacent structures, like photoreceptors. This study explores the potential of novel cultivation techniques to preserve photoreceptors in retinal explants to gain better insights in AMD pathology. The porcine retina explants were cultured for 4 and 8 days using three different explantation techniques, namely, control (photoreceptors facing down, touching the filter), filter (photoreceptors facing up, turned sample using a filter), and tweezers (photoreceptors facing up, turned sample using tweezers). Optical coherence tomography revealed that the tweezers method had the best capacity to limit thinning of the retinal explants. Both novel methods displayed advantages in maintaining outer segment thickness. Additionally, immunofluorescence evaluation revealed a better preservation of opsin+ cells and rhodopsin signal intensity in both novel methods, especially the tweezers method. Furthermore, RT-qPCR analysis demonstrated an upregulation of OPSIN and RHODOPSIN mRNA expression in tweezers samples at 8 days. Amacrine and bipolar cell numbers were not altered at day 4 of cultivation, while cultivation until 8 days led to reduced bipolar cell numbers. At 4 days, CALRETININ mRNA was upregulated in filter samples, but protein kinase C alpha expression was downregulated. Retinal ganglion cells were diminished in both novel techniques due to a direct physical contact with the insert. Remarkably, no difference in TUBB3 mRNA expression was detected among the techniques. Nevertheless, both novel methods exhibited an improved retention of photoreceptor cells. In conclusion, the tweezers technique was the most promising one. Due to the high homology of the porcine to the human retina, it provides a reasonable alternative to in vivo rodent models. Consequently, an adapted coculture system based on the current findings may serve as an ex vivo model suitable to analyze AMD pathomechanisms and novel therapeutic approaches.

TCT CONNECT-294 Outcomes Associated With Various Explant Techniques for Impella CP Removal

TCT CONNECT-294 Outcomes Associated With Various Explant Techniques for Impella CP Removal

Establishment and characterization of a cell line derived from fin of the endangered Yangtze sturgeon (Acipenser dabryanus).

Yangtze sturgeon (Acipenser dabryanus) is an endangered endemic freshwater fish of China. Cell-line is a potential means used for long-term preservation of germplasm resources and an ideal in vitro model in place of living organisms for biological studies. Here, culture condition and characterization of fin-derived cell in Yangtze sturgeon were carried out. Tissue explant techniques have been efficiently used in the Yangtze sturgeon caudal fin (YSCF) culture. The YSCF cell line showed a fibroblast-like morphology and stable growth in minimum essential medium eagle's (MEME) supplemented with 10-20% fetal bovine serum at 25°C. Cells were cryopreserved with preservative DMSO in liquid nitrogen and grew normally after recovery. No bacterial, fungal, or mycoplasma contamination was detected in the YSCF cells. Karyotype analysis of the YSCF cells showed that the chromosome numbers of the YSCF ranged from 242 to 273, and the modal chromosome number was identified as 264 at passage 9. The YSCF cells were confirmed from A. dabryanus by assay of 16S rRNA and COI. Furthermore, GFP reporter gene was successfully transferred into YSCF cells and expressed. The established YSCF cell lines will contribute to the preservation of germplasm resources and provide a useful vitro tool for further biological studies in sturgeon species.

Muscle Microbiopsy to Delineate Stem Cell Involvement in Young Patients: A Novel Approach for Children With Cerebral Palsy.

Cerebral palsy (CP), the single largest cause of childhood physical disability, is characterized firstly by a lesion in the immature brain, and secondly by musculoskeletal problems that progress with age. Previous research reported altered muscle properties, such as reduced volume and satellite cell (SC) numbers and hypertrophic extracellular matrix compared to typically developing (TD) children (>10 years). Unfortunately, data on younger CP patients are scarce and studies on SCs and other muscle stem cells in CP are insufficient or lacking. Therefore, it remains difficult to understand the early onset and trajectory of altered muscle properties in growing CP children. Because muscle stem cells are responsible for postnatal growth, repair and remodeling, multiple adult stem cell populations from young CP children could play a role in altered muscle development. To this end, new methods for studying muscle samples of young children, valid to delineate the features and to elucidate the regenerative potential of muscle tissue, are necessary. Using minimal invasive muscle microbiopsy, which was applied in young subjects under general anaesthesia for the first time, we aimed to isolate and characterize muscle stem cell-derived progenitors of TD children and patients with CP. Data of 15 CP patients, 3–9 years old, and 5 aged-matched TD children were reported. The muscle microbiopsy technique was tolerated well in all participants. Through the explant technique, we provided muscle stem cell-derived progenitors from the Medial Gastrocnemius. Via fluorescent activated cell sorting, using surface markers CD56, ALP, and PDGFRa, we obtained SC-derived progenitors, mesoangioblasts and fibro-adipogenic progenitors, respectively. Adipogenic, skeletal, and smooth muscle differentiation assays confirmed the cell identity and ability to give rise to different cell types after appropriate stimuli. Myogenic differentiation in CP SC-derived progenitors showed enhanced fusion index and altered myotube formation based on MYOSIN HEAVY CHAIN expression, as well as disorganization of nuclear spreading, which were not observed in TD myotubes. In conclusion, the microbiopsy technique allows more focused muscle research in young CP patients. Current results show altered differentiation abilities of muscle stem cell-derived progenitors and support the hypothesis of their involvement in CP-altered muscle growth.

What is the response profile of deciduous pulp fibroblasts stimulated with E. coli LPS and E. faecalis LTA?

BackgroundOral fibroblast immunological responses to bacterial stimuli are well known. However, there are few studies about pulp fibroblasts from deciduous teeth (HDPF) responses, which are important for the treatment of pulp infections in children. The aim of this study was to evaluate expression and production of inflammatory cytokines and chemokines by HDPF when challenged with bacterial antigens normally present in advanced caries lesions.MethodsTriplicate HDPF from 4 children (n = 4; 2 boys and 2 girls) were cultured by explant technique and challenged or not with Escherichia coli lipopolysaccharide/1 μg/mL (EcLPS) or Enterococcus faecalis lipoteichoic acid/1 μg/mL (EfLTA) for 6 and 24 h. Most of published studies employed immortalized cells, i.e., without checking possible gender and genetic variables. mRNA expression and protein production were evaluated by RT-qPCR and ELISA MILLIPLEX®, respectively, for Interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, Chemokine C-C motif ligand 2/monocyte chemoattractant protein 1 (CCL2/MCP-1), Chemokine C-C motif ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP1-α), Chemokine C-C motif ligand 5/ regulated on activation, normal T cell expressed and secreted (CCL5/RANTES), C-X-C motif chemokine 12/ stromal cell-derived factor 1 (CXCL12/SDF-1), Tumor Necrosis Factor-alpha (TNF-α), Interferon-gamma (IFN γ), Vascular Endothelial Growth Factor (VEGF), Colony stimulating factor 1 (CSF-1) and Macrophage colony-stimulating factor (M-CSF).ResultsEcLPS increased IL-1α, IL-1β, IL-8, CCL2, CCL5, TNF-α and CSF-1 mRNA and protein levels while EfLTA was only able to positively regulate gene expression and protein production of IL-8.ConclusionThe results of the present study confirmed our hypothesis, since pulp fibroblasts from deciduous teeth are capable of increasing gene expression and protein production after being stimulated with EcLPS and EfLTA.

Explantation of Cloudy Oculentis Intraocular Lens With Primary Pars Plana Vitrectomy and Deflooring of the Capsular Bag.

To report a novel technique for explantation of cloudy Oculentis intraocular lens (IOL). A retrospective review of patient's electronic records was performed. The technique includes pars plana vitrectomy, deflooring (large posterior capsulectomy) of the capsular bag, IOL explantation with preserving the anterior capsule, and a secondary IOL placement. Twenty eyes of 19 symptomatic patients were studied. Preoperative Snellen visual acuity ranged from 6/60 to 6/6; 15 eyes (75%) had visual acuity of 6/12 or better. Postoperative visual acuity ranged from 6/60 to 6/6; 18 eyes (90%) had visual acuity of 6/12 or better, and 2 eyes (10%) had visual acuity worse than 6/12. Among these eyes, 13 eyes (65%) had sulcus-fixated IOL, 5 eyes (25%) had sutureless scleral-fixated IOL, and 2 eyes (10%) had retropupillary iris clip IOL. Perioperative surgical complications included localized choroidal detachment in one eye and retinal detachment in another eye. In addition, most patients reported significant improvement in their postoperative vision satisfaction. Our new technique for explantation of cloudy Oculentis IOL provides a standard surgery allowing a safe predictable and repeatable removal of these lenses comparing with the anterior segment approach. This technique could also be used for removal of other IOLs.