Influence of vitamin D receptor signaling and vitamin D on colonic epithelial cell fate decisions in ulcerative colitis.

BACKGROUND AND AIMS: Epidemiological studies have shown that subnormal levels of vitamin D (25(OH)D) are associated with a more aggravated clinical course of ulcerative colitis (UC). Despite an increased focus on the therapeutic importance of vitamin D and vitamin D receptor (VDR) signaling, the mechanisms underlying the effects of the vitamin D-VDR axis on UC remain elusive. Therefore, we aimed to investigate whether exposure to active vitamin D (1,25(OH)2D3)/VDR signaling in human organoids could influence the maintenance of the colonic epithelium. METHODS: Intestinal VDR expression was studied by immunohistochemistry, RNA expression arrays, and single-cell RNA sequencing of colonic biopsy specimens obtained from patients with UC and healthy individuals. To characterize the functional and transcriptional effects of 1,25(OH)2D3, we used patient-derived colonic organoids. The dependency of VDR was assessed by knocking out the receptor with CRISPR/Cas9. RESULTS: Our results suggest that 1,25(OH)2D3/VDR stimulation supports differentiation of the colonic epithelium and that impaired 1,25(OH)2D3/VDR signaling thereby may compromise the structure of the intestinal epithelial barrier, leading to flares of UC. Furthermore, a transcriptional response to VDR activity was observed primarily in fully differentiated cells at the top of the colonic crypt, and this response was reduced during flares of UC. CONCLUSIONS: We identified an important role of vitamin D signaling in supporting differentiated cell states in the human colonic epithelium, and thereby maintenance of the intestinal barrier integrity. This makes the vitamin D-VDR signaling axis an interesting target for therapeutic efforts to achieve and maintain remission in patients with UC.

Detecting host responses to microbial stimulation using primary epithelial organoids.

The intestinal epithelium is constantly exposed to microbes residing in the lumen. Traditionally, the response to microbial interactions has been studied in cell lines derived from cancerous tissues, e.g. Caco-2. It is, however, unclear how the responses in these cancer cell lines reflect the responses of a normal epithelium and whether there might be microbial strain-specific effects. To address these questions, we derived organoids from the small intestine from a cohort of healthy individuals. Culturing intestinal epithelium on a flat laminin matrix induced their differentiation, facilitating analysis of microbial responses via the apical membrane normally exposed to the luminal content. Here, it was evident that the healthy epithelium across multiple individuals (n = 9) demonstrates robust acute both common and strain-specific responses to a range of probiotic bacterial strains (BB-12Ⓡ, LGGⓇ, DSM33361, and Bif195). Importantly, parallel experiments using the Caco-2 cell line provide no acute response. Collectively, we demonstrate that primary epithelial cells maintained as organoids represent a valuable resource for assessing interactions between the epithelium and luminal microbes across individuals, and that these models are likely to contribute to a better understanding of host microbe interactions.

Differences in joint line level and posterior condylar offset during total knee replacement with use of gap-balancing and measured resection techniques-matched cohort study.

BACKGROUND: Total knee replacement (TKR) is considered one of the most common elective orthopaedic procedures. The main focus of TKR is to offer patient's symptomatic relief from persistent knee pain. To achieve this it is crucial to restore joint biomechanics by performing proper bone cuts. Some surgeons favor the measured resection technique, others prefer gap balancing technique. The researchers of the presented study performed TKR using these two techniques. The aim of this study was to compare the postoperative change in joint line and posterior condylar offset after TKR with use of anatomic knee design implants between gap balancing and measured resection techniques. METHODS: Two hundred twenty-five X-rays of patients who underwent TKR performed by a single surgeon between 2020 and 2021 were analyzed. The first group of patients (101) was operated with the use of gap balancing technique and the second group (124) was operated with the use of measured resection technique. Patients included in the study were > 50 years of age, had confirmed primary knee osteoarthritis, underwent primary TKR with a PS (posterior stabilized) knee implants without patella resurfacing and had at least 15 degree flexion contracture. T-student test and U Mann-Whitney test were used in statistical analysis of results, according to the normality of distribution examined with the Shapiro-Wilk test. Post-hoc analysis was performed using the Dwass-Steel-Crichtlow-Fligner test (DSCF). RESULTS: The postoperative analysis showed a significantly elevated joint line level in the gap balancing group (-2.6 ± 4.1 vs -0.7 ± 4.8, p < 0.0005). In the gap balancing group significantly more patients had joint lines elevated > 2 mm comparing to measured resection technique. The difference between pre- and postoperative PCO (posterior condylar offset) and PCOR (posterior condylar offset ratio) results had no significant differences (100.8 ± 11.8 vs 101 ± 12.5, p > 0.05) between the groups. CONCLUSIONS: The results of the study suggest that when it comes to restoring joint line level measured resection technique seems to be superior in comparison to the gap balancing technique. What is more, results indicate measured resection is equal in terms of restoring posterior condylar offset to the gap balancing technique. TRIAL REGISTRATION: NCT04164147, date of registration: November 14, 2019.

An organoid-based CRISPR-Cas9 screen for regulators of intestinal epithelial maturation and cell fate.

Generation of functionally mature organs requires exquisite control of transcriptional programs governing cell state transitions during development. Despite advances in understanding the behavior of adult intestinal stem cells and their progeny, the transcriptional regulators that control the emergence of the mature intestinal phenotype remain largely unknown. Using mouse fetal and adult small intestinal organoids, we uncover transcriptional differences between the fetal and adult state and identify rare adult-like cells present in fetal organoids. This suggests that fetal organoids have an inherent potential to mature, which is locked by a regulatory program. By implementing a CRISPR-Cas9 screen targeting transcriptional regulators expressed in fetal organoids, we establish Smarca4 and Smarcc1 as important factors safeguarding the immature progenitor state. Our approach demonstrates the utility of organoid models in the identification of factors regulating cell fate and state transitions during tissue maturation and reveals that SMARCA4 and SMARCC1 prevent precocious differentiation during intestinal development.

Transcriptional and epigenomic profiling identifies YAP signaling as a key regulator of intestinal epithelium maturation.

During intestinal organogenesis, equipotent epithelial progenitors mature into phenotypically distinct stem cells that are responsible for lifelong maintenance of the tissue. While the morphological changes associated with the transition are well characterized, the molecular mechanisms underpinning the maturation process are not fully understood. Here, we leverage intestinal organoid cultures to profile transcriptional, chromatin accessibility, DNA methylation, and three-dimensional (3D) chromatin conformation landscapes in fetal and adult epithelial cells. We observed prominent differences in gene expression and enhancer activity, which are accompanied by local changes in 3D organization, DNA accessibility, and methylation between the two cellular states. Using integrative analyses, we identified sustained Yes-Associated Protein (YAP) transcriptional activity as a major gatekeeper of the immature fetal state. We found the YAP-associated transcriptional network to be regulated at various levels of chromatin organization and likely to be coordinated by changes in extracellular matrix composition. Together, our work highlights the value of unbiased profiling of regulatory landscapes for the identification of key mechanisms underlying tissue maturation.

Comparison of Femoral Component Rotation between Robotic-Assisted vs. Soft-Tissue Tensor Total Knee Arthroplasty with Anatomic Implants.

Background and Objectives: Total knee arthroplasty (TKA) is the most effective treatment method for end-stage osteoarthritis. One of the most important aspects of this surgery is adequate implant positioning, as it guarantees the desired outcome of restoring limb biomechanics. Surgical technique is being continuously improved along with hardware development. There are two novel devices designed to help establish proper femoral component rotation: soft-tissue tensor and robotic-assisted TKA (RATKA). This study compared the femoral component rotation achieved with the use of three methods: RATKA, soft tissue tensioner and the conventional measured-resection technique, all of them utilizing anatomical design prosthesis components. Materials and Methods: A total of 139 patients diagnosed with end-stage osteoarthritis underwent total knee arthroplasty between December 2020 and June 2021. After the surgery, they were divided into three groups depending on procedure technique and implant type: Persona (Zimmer Biomet) + Fuzion Balancer, RATKA + Journey II BCS or conventional TKA + Persona/Journey. Postoperatively, a computed tomography examination was performed in order to measure femoral component rotation. All three groups were compared independently during statistical analysis. Fisher's exact, Kruskal-Wallis and Dwass-Steel-Crichtlow-Fligner tests were used for particular calculations. Results: Statistically significant differences in femoral component rotation between groups were noticed. However, in terms of values other than 0° in external rotation, no significant variance was revealed. Conclusions: Additional total knee arthroplasty instruments seem to improve the outcomes of the surgery, providing better component positioning than in the conventional measured-resection technique based only on bone landmarks.

Curve-on-curve technique does not improve tibial coverage in total knee arthroplasty in comparison to tibial tuberosity technique with use of anatomical implants: randomized controlled trial.

INTRODUCTION: During the last years, main attention while performing total knee replacement was paid to femoral component alignment; however, there is still lack of studies concerning tibial baseplate rotational alignment, especially in terms of anatomical designs of knee prosthesis. Some recent studies proved that tibial baseplate malrotation might be a cause of knee pain and patients' dissatisfaction. The aim of this study was to compare tibial component rotation and its coverage on the tibial plateau achieved with curve-on-curve and tibial tuberosity techniques (t-t technique) with use of anatomic knee designs with asymmetric tibial baseplate. MATERIALS AND METHODS: A total of 88 patients were randomly assigned in a 1:1 ratio to undergo total knee arthroplasty with use of the PERSONA PS (Zimmer Biomet) knee design with an asymmetric baseplate. The rotation of the tibial component was assessed and performed with two different techniques: curve-on-curve technique and tibial tuberosity technique. Tibial component rotation was measured on computed tomography (CT) scans using the method suggested by Benazzo et al. and designed for asymmetrical implants. For the measurement of the tibial bone coverage, the component surface area was outlined and measured on a proper CT section, then the tibial cut surface area was outlined and measured on a section just below the cement level. Pre- and post-operative range of motion was measured by another independent researcher 12 months post-operatively during follow-up visit. RESULTS: There was a statistically significant difference between both groups in median value of tibial rotation angle: 7° (interquartile range (IQR) = 0-12) in curve-on-curve technique group vs 2° (IQR-1-7) in tibial tuberosity technique group, probability value (p) = 0.0041, with values above 0 meaning external rotation of the component. There was no statistically significant difference between both groups in terms of range of motion (ROM) with average values of 124.3° ± 13.0° for curve-on-curve technique and 125.6° ± 12.8° for t-t technique with p = 0.45. There was a statistically insignificant difference between both groups in terms of coverage percentage in slight favor for curve-on-curve technique (85.9 ± 4.2 vs 84.5 ± 4.8, p = 0.17). CONCLUSION: In this study, no difference between the groups in terms of tibial bone coverage and range of motion was proved, even though both techniques differed significantly with values of tibial rotation. Future studies should be focused on influence of specific values of tibial rotation on patient-reported outcomes and survivorship of anatomic knee implants.

Hip hemiprosthesis due to femoral neck fracture in the elderly population - are we doing it right?

Introduction: Femoral neck fracture is one of the most common orthopaedic traumas affecting the elderly population. The standard treatment method is hip hemiarthroplasty and total hip arthroplasty. In hip hemiprostheses surgeons mainly have to reconstruct the femoral offset and limbs' length to obtain the correct gait biomechanics and a satisfactory surgical outcome.The aim of this study is to examine the radiological results of patients after hip hemiarthroplasty for femoral neck fracture and to evaluate the reconstruction of the femoral offset using standard neck angle stems. Material and methods: A consecutive series of 97 patients diagnosed with femoral neck fracture treated with a hip hemiprosthesis between 2017 and 2021 was identified and met the inclusion criteria. On preoperative images, the neck-shaft angle and the femoral offset on the healthy limb were measured. The femoral offset of the operated limb was measured on the postoperative X-rays. Results: There was a significant positive moderate correlation between neck-shaft angle and femoral offset change (r = 0.568, p < 0.0001). There was a statistically significant difference between femoral offset change and neck-shaft angle (24:52 vs. 14:7, p = 0.005). This means that in patients with coxa vara the change in femoral offset was more often < -5 mm. Less than half of operated patients had the femoral offset restored within a safe range (between -5 and 5 mm). Conclusions: Our study proved that it is sometimes hard to achieve femoral offset within a safe range while performing hip hemiarthroplasty in patients with coxa vara. The topic of using high offset stems in partial hip arthroplasty has not been thoroughly researched worldwide. However, taking into account the results of our study, during a hip hemiarthroplasty the usage of high offset stems for varus hips should be considered in order to improve the clinical outcome and improve patients' quality of life and functioning.

Comparison of Biomechanical Gait Parameters and Patient-Reported Outcome in Patients After Total Knee Arthroplasty With the Use of Fixed-Bearing Medial Pivot and Multi-radius Design Implants-Retrospective Matched-Cohort Study.

BACKGROUND: Total knee arthroplasty (TKA) is considered to be highly successful in treatment of end-stage osteoarthritis. There are multiple implant designs available on the market, and it is difficult to point which one is the best. The aim of this study was to compare the clinical and functional outcomes and gait pattern after TKA with the use of fixed-bearing medial pivot (K-Mod) vs multi-radius design (NexGen) implants and to compare them to norms for healthy patients with no osteoarthritis or arthroplasty procedure in anamnesis. METHODS: A group of 30 patients who received the medial pivot (MP) TKA and 33 patients who received the posterior-stabilized (PS) TKA between May and August of 2018 were included. All surgeries were performed in the level III academic hospital by a single surgeon. Every patient was asked to fulfill the The Western Ontario and McMaster Universities Arthritis Index (WOMAC) questionnaire preoperatively and 2 years postoperatively. Standard X-ray, biomechanical gait analysis using a motion capture system, and statistical analysis were performed at 2 years postoperatively. RESULTS: A total of 28 patients from either MP cohort (93%) or PS (85%) matched-control cohort completed the whole assessment at the final follow-up. There were statistically significant differences in a few gait parameters such as shorter mean step length both in operated and healthy limb, lower mean gait velocity, and lower mean walking cadence than the norm in both MP and PS groups. As to the WOMAC score, there was statistically significant improvement in both groups comparing preoperative and postoperative outcomes. Nevertheless, in the MP group, there was a significantly higher score, indicating worse outcomes, in the stiffness part of the WOMAC score than in the PS group. No significant differences were found between groups during radiological evaluation. CONCLUSIONS: There were satisfying and promising clinical, radiographic, and patient-reported outcomes in both MP and PS groups with very little difference in relation to norm values. However, both implants failed in fully restoring gait patterns similar to the healthy limb of the same patient.

Analysis of biomechanical gait parameters in patients after total hip replacement operated via anterolateral approach depending on size of the femoral head implant: retrospective matched-cohort study.

INTRODUCTION: Total hip replacement (THR) is considered one of the most effective medical procedures in treatment of osteoarthritis. Since its introduction, there has been a worldwide debate over proper implant selection in terms of size, bearing type and shape. Following study was designed to assess the importance of femoral head size in long-term follow-up. MATERIALS AND METHODS: A cohort of 30 patients with primary end stage osteoarthritis who underwent total hip replacement was analysed retrospectively. A homogenous group was chosen with no major differences in BMI. Patients' gait parameters were measured in a biomechanics laboratory using the 3D BTS Smart system. WOMAC and VAS questionnaires were used to assess patient reported outcome. RESULTS: The subgroup with larger implant head size had several outcomes significantly superior to the subgroup with standard head size and non-inferior to healthy hips. Following variables were measured during this study: time of support phase, time of swing phase, double support time, walking hip extension angle. CONCLUSIONS: Use of larger sized femoral heads during THR gives better results in terms of gait pattern. Since restoring the gait pattern is one of the aspects of rehabilitation and returning to daily activities it seems to be an important observation.

Does the anatomic design of total knee prosthesis allow for a better component fit than its nonanatomic predecessor? A matched cohort Study.

BACKGROUND: Total knee arthroplasty (TKA) is considered the most efficient treatment of end-stage osteoarthritis. There is an ongoing debate about proper implant designs and articulation types. One of the considered causes of unsatisfactory outcome and patients' dissatisfaction is femoral or tibial component overhanging, which can lead to chronic knee pain and restricted motion. The aim of this study is to compare radiological outcomes of TKA using an anatomic PERSONA Posterior-Stabilized (PS) knee design with its nonanatomic predecessor, the NexGen LPS. METHODS: A group of 39 patients who received the PERSONA PS system and 33 patients who received the NexGen LPS was included. PERSONA patients were matched to NexGen patients using a 0.1 propensity score threshold with priority given to exact matches. Anteroposterior, lateral, and long-leg radiographs were taken preoperatively and at 6 weeks postoperatively to perform radiological and statistical analysis. RESULTS: The PERSONA subgroup had statistically higher posterior condylar offset. There is no statistically significant difference in posterior condylar offset ratio. There were less cases of femoral notching, femoral overhang, and placing tibial baseplate in the medial overhang in the PERSONA PS subgroup than in the NexGen subgroup (each statistically significant). Occurrence of tibial underhang was not statistically significant. CONCLUSION: Radiological assessment in short-term follow-up showed excellent results for PERSONA knee design with better fit to native femur and tibia. In comparison to its predecessor, it also spares more bone tissue. As the aforementioned parameters are risk factors of lesser clinical outcomes, the PERSONA design gives high hopes for improvement of TKA satisfaction rate.

Machine learning workflows identify a microRNA signature of insulin transcription in human tissues.

Dicer knockout mouse models demonstrated a key role for microRNAs in pancreatic ß-cell function. Studies to identify specific microRNA(s) associated with human (pro-)endocrine gene expression are needed. We profiled microRNAs and key pancreatic genes in 353 human tissue samples. Machine learning workflows identified microRNAs associated with (pro-)insulin transcripts in a discovery set of islets (n = 30) and insulin-negative tissues (n = 62). This microRNA signature was validated in remaining 261 tissues that include nine islet samples from individuals with type 2 diabetes. Top eight microRNAs (miR-183-5p, -375-3p, 216b-5p, 183-3p, -7-5p, -217-5p, -7-2-3p, and -429-3p) were confirmed to be associated with and predictive of (pro-)insulin transcript levels. Use of doxycycline-inducible microRNA-overexpressing human pancreatic duct cell lines confirmed the regulatory roles of these microRNAs in (pro-)endocrine gene expression. Knockdown of these microRNAs in human islet cells reduced (pro-)insulin transcript abundance. Our data provide specific microRNAs to further study microRNA-mRNA interactions in regulating insulin transcription.

Hyperandrogenism and Metabolic Syndrome Are Associated With Changes in Serum-Derived microRNAs in Women With Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) remains one of the most common endocrine disorder in premenopausal women with an unfavorable metabolic risk profile. Here, we investigate whether biochemical hyperandrogenism, represented by elevated serum free testosterone, resulted in an aberrant circulating microRNA (miRNAs) expression profile and whether miRNAs can identify those PCOS women with metabolic syndrome (MetS). Accordingly, we measured serum levels of miRNAs as well as biochemical markers related to MetS in a case-control study of 42 PCOS patients and 20 Controls. Patients were diagnosed based on the Rotterdam consensus criteria and stratified based on serum free testosterone levels (≥0.034 nmol/l) into either a normoandrogenic (n = 23) or hyperandrogenic (n = 19) PCOS group. Overall, hyperandrogenic PCOS women were more insulin resistant compared to normoandrogenic PCOS women and had a higher prevalence of MetS. A total of 750 different miRNAs were analyzed using TaqMan Low-Density Arrays. Altered levels of seven miRNAs (miR-485-3p, -1290, -21-3p, -139-3p, -361-5p, -572, and -143-3p) were observed in PCOS patients when compared with healthy Controls. Stratification of PCOS women revealed that 20 miRNAs were differentially expressed between the three groups. Elevated serum free testosterone levels, adjusted for age and BMI, were significantly associated with five miRNAs (miR-1290, -20a-5p, -139-3p, -433-3p, and -361-5p). Using binary logistic regression and receiver operating characteristic curves (ROC), a combination panel of three miRNAs (miR-361-5p, -1225-3p, and -34-3p) could correctly identify all of the MetS cases within the PCOS group. This study is the first to report comprehensive miRNA profiling in different subgroups of PCOS women with respect to MetS and suggests that circulating miRNAs might be useful as diagnostic biomarkers of MetS for a different subset of PCOS.