Research Spotlight

Chang, B., Ma, C. and Liu, X. (2020). “Nanofibrous Tubular Three-Dimensional Platform for Single Dental Pulp Stem Cell Polarization.” ACS Appl Mater Interfaces Nov 30. [Epub ahead of print.].

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Dental pulp stem cells (DPSCs) are the primary stem cell source for regenerative endodontics. DPSCs need to undergo a polarization process and retain the permanent polarization status to perform the function of odontoblasts. However, the factors that control DPSC polarization and its underlying mechanism remain unknown. In this study, we established a unique nanofibrous tubular three-dimensional (3D) platform to explore DPSC polarization. The 3D platform has a “clean” background and confines one single DPSC in each microisland of the platform; therefore, it is capable of deciphering any signal that initiates or regulates DPSC polarization. Using the biomimetic platform, we identified that the nanofibrous tubular architecture is the crucial factor to initiate DPSC polarization. Dynamic morphological observation showed that the cellular process of the polarized DPSCs continuously extended and reached a plateau at 72 h. Meanwhile, Golgi apparatus, a cell polarization marker, continuously moved from a juxtanuclear region, passed the nucleus, and eventually settled down at a final position that was a few micrometers away from the nucleus. Inhibition of microfilament and microtubule polymerization demonstrated the indispensable role of cytoskeleton reorganization in modulating DPSC polarization. In addition, cell tension was involved in the regulation of DPSC polarization. The findings of this work expand the in-depth understanding of DPSC polarization, which helps design new bioinspired materials for regenerative endodontics.

Wang, J., Xie, X., Muench, N.A., Massoudi, D., Xu, C., Greenspan, D.S. and Feng, J.Q. (2020). “Proteinase bone morphogenetic protein 1, but not tolloid-like 1, plays a dominant role in maintaining periodontal homeostasis.” J Periodontol Nov 9. [Epub ahead of print.].

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BACKGROUND: Periodontitis is caused by multiple factors involving a bacterial challenge and a susceptible host, although there is no report on gene mutation directly linked to this common disease. Mutations in the proteinase bone morphogenetic protein 1 (BMP1) were identified in patients with osteogenesis imperfecta, who display some dentin defects and alveolar bone loss. We previously reported essential roles of BMP1 and tolloid-like 1 (TLL1), two closely related extracellular proteinases with overlapping functions, in mouse periodontium growth by simultaneous knockout (KO) of both genes, although the separate roles of BMP1 and TLL1 have remained unclear. Here, we have investigated whether and how BMP1 and TLL1 separately maintain periodontal homeostasis by comparing single Bmp1 KO and Tll1 KO with double KO (dKO) phenotypes. METHODS: Floxed Bmp1 and/or Tll1 alleles were deleted in transgenic mice via ubiquitously expressed Cre(ERT2) induced by tamoxifen treatment starting at 4-weeks of age (harvested at 18-weeks of age). Multiple approaches, including X-ray, micro-CT, calcein and alizarin red double-labeling, scanning electron microscopy, and histological and immunostaining assays, were used to analyze periodontal phenotypes and molecular mechanisms. RESULTS: Both Bmp1 KO and double KO mice exhibited severe periodontal defects, characterized by periodontal ligament (PDL) fiber loss and ectopic ossification in the expanded PDL area, and drastic reductions in alveolar bone and cementum volumes, whereas Tll1 KO mice displayed very mild phenotypes. Mechanistic studies revealed a sharp increase in the uncleaved precursor of type I collagen (procollagen I), leading to defective extracellular matrices. CONCLUSIONS: BMP1, but not TLL1, is essential for maintaining periodontal homeostasis. This occurs at least partly via biosynthetic processing of procollagen I, thereby maintaining appropriate levels of procollagen I and its activated products such as mature collagen I.

Lu, M., Bowlus, C.L., Lindor, K., Rodriguez-Watson, C.V., Romanelli, R.J., Haller, I.V., Anderson, H., VanWormer, J.J., Boscarino, J.A., Schmidt, M.A., Daida, Y.G., Sahota, A., Vincent, J., Li, J., Trudeau, S., Rupp, L.B. and Gordon, S.C. (2020). “Validity of an Automated Algorithm to Identify Cirrhosis Using Electronic Health Records in Patients with Primary Biliary Cholangitis.” Clin Epidemiol 12: 1261-1267.

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BACKGROUND: Biopsy remains the gold standard for determining fibrosis stage in patients with primary biliary cholangitis (PBC), but it is unavailable for most patients. We used data from the 11 US health systems in the FibrOtic Liver Disease Consortium to explore a combination of biochemical markers and electronic health record (EHR)-based diagnosis/procedure codes (DPCs) to identify the presence of cirrhosis in PBC patients. METHODS: Histological fibrosis staging data were obtained from liver biopsies. Variables considered for the model included demographics (age, gender, race, ethnicity), total bilirubin, alkaline phosphatase, albumin, aspartate aminotransferase (AST) to platelet ratio index (APRI), Fibrosis 4 (FIB4) index, AST to alanine aminotransferase (ALT) ratio, and >100 DPCs associated with cirrhosis/decompensated cirrhosis, categorized into ten clusters. Using least absolute shrinkage and selection operator regression (LASSO), we derived and validated cutoffs for identifying cirrhosis. RESULTS: Among 4328 PBC patients, 1350 (32%) had biopsy data; 121 (9%) were staged F4 (cirrhosis). DPC clusters (including codes related to cirrhosis and hepatocellular carcinoma diagnoses/procedures), Hispanic ethnicity, ALP, AST/ALT ratio, and total bilirubin were retained in the final model (AUROC=0.86 and 0.83 on learning and testing data, respectively); this model with two cutoffs divided patients into three categories (no cirrhosis, indeterminate, and cirrhosis) with specificities of 81.8% (for no cirrhosis) and 80.3% (for cirrhosis). A model excluding DPCs retained ALP, AST/ALT ratio, total bilirubin, Hispanic ethnicity, and gender (AUROC=0.81 and 0.78 on learning and testing data, respectively). CONCLUSION: An algorithm using laboratory results and DPCs can categorize a majority of PBC patients as cirrhotic or noncirrhotic with high accuracy (with a small remaining group of patients’ cirrhosis status indeterminate). In the absence of biopsy data, this EHR-based model can be used to identify cirrhosis in cohorts of PBC patients for research and/or clinical follow-up.

Tashkandi, M.M., Alsaqer, S.F., Alhousami, T., Ali, F., Wu, Y.C., Shin, J., Mehra, P., Wolford, L.M., Gerstenfeld, L.C., Goldring, M.B. and Bais, M.V. (2020). “LOXL2 promotes aggrecan and gender-specific anabolic differences to TMJ cartilage.” Sci Rep 10(1): 20179.

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In the United States, 5-12% of adults have at least one symptom of temporomandibular joint (TMJ) disorders, including TMJ osteoarthritis (TMJ-OA). However, there is no chondroprotective agent that is approved for clinical application. We showed that LOXL2 is elevated in the regenerative response during fracture healing in mice and has a critical role in chondrogenic differentiation. Indeed, LOXL2 is an anabolic effector that attenuates pro-inflammatory signaling in OA cartilage of the TMJ and knee joint, induces chondroprotective and regenerative responses, and attenuates NF-kB signaling. The specific goal of the study was to evaluate if adenoviral delivery of LOXL2 is anabolic to human and mouse TMJ condylar cartilage in vivo and evaluate the protective and anabolic effect on cartilage-specific factors. We employed two different models to assess TMJ-OA. In one model, clinical TMJ-OA cartilage from 5 different samples in TMJ-OA cartilage plugs were implanted subcutaneously in nude mice. Adenovirus LOXL2 -treated implants showed higher mRNA levels of LOXL2, ACAN, and other anabolic genes compared to the adenovirus-Empty-treated implants. Further characterization by RNA-seq analysis showed LOXL2 promotes proteoglycan networks and extracellular matrix in human TMJ-OA cartilage implants in vivo. In order to evaluate if LOXL2-induced functional and sex-linked differences, both male and female four-month-old chondrodysplasia (Cho/+) mice, which develop progressive TMJ-OA due to a point mutation in the Col11a1 gene, were subjected to intraperitoneal injection with Adv-RFP-LOXL2 every 2 weeks for 12 weeks. The data showed that adenovirus delivery of LOXL2 upregulated LOXL2 and aggrecan (Acan), whereas MMP13 expression was slightly downregulated. The fold change expression of Acan and Runx2 induced by Adv-RFP-LOXL2 was higher in females compared to males. Interestingly, Adv-RFP-LOXL2 injection significantly increased Rankl expression in male but there was no change in females, whereas VegfB gene expression was increased in females, but not in males, as compared to those injected with Adv-RFP-Empty in respective groups. Our findings indicate that LOXL2 can induce specifically the expression of Acan and other anabolic genes in two preclinical models in vivo. Further, LOXL2 has beneficial functions in human TMJ-OA cartilage implants and promotes gender-specific anabolic responses in Cho/+ mice with progressive TMJ-OA, suggesting its merit for further study as an anabolic therapy for TMJ-OA.

Tolia, V.N. and Clark, R.H. (2020). “The Denominator Matters! Lessons from Large Database Research in Neonatology.” Children (Basel) 7(11).

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Observational studies from large datasets are becoming more common in neonatology. In this review, we highlight the importance of the denominator in study design and interpretation including examples of bias from source data, weight-based categories, age-related bias, and diagnosis-based denominators.