Endogenus chondrocytes immobilized by G-CSF in nanoporous gels enable repair of critical-size osteochondral defects.

Injured articular cartilage is a leading cause for osteoarthritis. We recently discovered that endogenous stem/progenitor cells not only reside in the superficial zone of mouse articular cartilage, but also regenerated heterotopic bone and cartilage in vivo. However, whether critical-size osteochondral defects can be repaired by pure induced chemotatic cell homing of these endogenous stem/progenitor cells remains elusive. Here, we first found that cells in the superficial zone of articular cartilage surrounding surgically created 3 × 1 mm defects in explant culture of adult goat and rabbit knee joints migrated into defect-filled fibrin/hylaro1nate gel, and this migration was significantly more robust upon delivery of exogenous granulocyte-colony stimulating factor (G-CSF). Remarkably, G-CSF-recruited chondrogenic progenitor cells (CPCs) showed significantly stronger migration ability than donor-matched chondrocytes and osteoblasts. G-CSF-recruited CPCs robustly differentiated into chondrocytes, modestly into osteoblasts, and barely into adipocytes. In vivo, critical-size osteochondral defects were repaired by G-CSF-recruited endogenous cells postoperatively at 6 and 12 weeks in comparison to poor healing by gel-only group or defect-only group. ICRS and O'Driscoll scores of articular cartilage were significantly higher for both 6- and 12-week G-CSF samples than corresponding gel-only and defect-only groups. Thus, endogenous stem/progenitor cells may be activated by G-CSF, a Food and Drug Administration (FDA)-cleared bone-marrow stimulating factor, to repair osteochondral defects.

Alkaline activation of endogenous latent TGFß1 by an injectable hydrogel directs cell homing for in situ complex tissue regeneration.

Utilization of the body's regenerative potential for tissue repair is known as in situ tissue regeneration. However, the use of exogenous growth factors requires delicate control of the dose and delivery strategies and may be accompanied by safety, efficacy and cost concerns. In this study, we developed, for the first time, a biomaterial-based strategy to activate endogenous transforming growth factor beta 1 (TGFß1) under alkaline conditions for effective in situ tissue regeneration. We demonstrated that alkaline-activated TGFß1 from blood serum, bone marrow fluids and soaking solutions of meniscus and tooth dentin was capable of increasing cell recruitment and early differentiation, implying its broad practicability. Furthermore, we engineered an injectable hydrogel (MS-Gel) consisting of gelatin microspheres for loading strong alkaline substances and a modified gelatin matrix for hydrogel click crosslinking. In vitro models showed that alkaline MS-Gel controllably and sustainably activated endogenous TGFß1 from tooth dentin for robust bone marrow stem cell migration. More importantly, infusion of in vivo porcine prepared root canals with alkaline MS-Gel promoted significant pulp-dentin regeneration with neurovascular stroma and mineralized tissue by endogenous proliferative cells. Therefore, this work offers a new bench-to-beside translation strategy using biomaterial-activated endogenous biomolecules to achieve in situ tissue regeneration without the need for cell or protein delivery.

mTOR deletion in neural crest cells disrupts cardiac outflow tract remodeling and causes a spectrum of cardiac defects through the mTORC1 pathway.

A critical cell type participating in cardiac outflow tract development is a subpopulation of the neural crest cells, the cardiac neural crest cells (NCCs), whose defect causes a spectrum of cardiovascular abnormalities. Accumulating evidence indicates that mTOR, which belongs to the PI3K-related kinase family and impacts multiple signaling pathways in a variety of contexts, plays a pivotal role for NCC development. Here, we investigated functional roles of mTOR for cardiac neural crest development using several lines of mouse genetic models. We found that disruption of mTOR caused NCC defects and failure of cardiac outflow tract separation, which resulted in a spectrum of cardiac defects including persistent truncus arteriosus, ventricular septal defect and ventricular wall defect. Specifically, mutant neural crest cells showed reduced migration into the cardiac OFT and prematurely exited the cell cycle. A number of critical factors and fundamental signaling pathways, which are important for neural crest and cardiomyocyte development, were impaired. Moreover, actin dynamics was disrupted by mTOR deletion. Finally, by phenotyping the neural crest Rptor and Rictor knockout mice respectively, we demonstrate that mTOR acts principally through the mTORC1 pathway for cardiac neural crest cells. Altogether, these data established essential roles of mTOR for cardiac NCC development and imply that dysregulation of mTOR in NCCs may underline a spectrum of cardiac defects.

Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo.

BACKGROUND: Periosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs. METHODS: We detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in ß-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16. RESULTS: Wnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/ß-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity. CONCLUSION: Together, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.

mTOR plays a pivotal role in multiple processes of enamel organ development principally through the mTORC1 pathway and in part via regulating cytoskeleton dynamics.

We herein report that deletion of mTOR in dental epithelia caused defective development of multiple cell layers of the enamel organ, which culminated in tooth malformation and cystogenesis. Specifically, cells of the stellate reticulum and stratum intermedium were poorly formed, resulting in cystic changes. The pre-ameloblasts failed to elongate along the apical-basal axis and persisted vigorous expression of Sox2 and P63, which are normally downregulated during cytodifferentiation. Expression of amelogenic markers was also attenuated in mutants. Cell proliferation and cell sizes in mutants were significantly reduced over time. Importantly, we found reduced amounts and aberrant aggregations of cytoskeletal components in mutants, along with attenuated expression of cytoskeleton regulator Cdc42, whose epithelial deletion causes a similar phenotype. Moreover, disruption of actin assembly in an organ culture system affected cell proliferation and cytodifferentiation of tooth germs, supporting a causative role of mTOR-regulated cytoskeleton dynamics for the observed phenotype of mTOR mutant mice. In further support of this view, we showed that mTOR overactivation caused increased cytoskeletal component synthesis and assembly, along with accelerated cytodifferentiation in the enamel organ. Finally, we demonstrated that mTOR regulated enamel organ development principally through the mTORC1 pathway.

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies - Secondary Publication.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:485-502, 2020.

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2019 American Society for Bone and Mineral Research.

Long-term Evaluation of Meniscal Tissue Formation in 3-dimensional-Printed Scaffolds With Sequential Release of Connective Tissue Growth Factor and TGF-ß3 in an Ovine Model.

BACKGROUND: Artificial meniscal scaffolds are being developed to prevent development of osteoarthritis after meniscectomy. Previously, it was reported that 3-dimensional (3D) anatomic scaffolds loaded with connective tissue growth factor (CTGF) and transforming growth factor ß3 (TGF-ß3) achieved meniscal regeneration in an ovine model. This was a relatively short-term study (3 months postoperative), and outcome analyses did not include magnetic resonance imaging (MRI). PURPOSE: To evaluate long-term outcome of meniscal replacement with growth factor-laden poly-ε-caprolactone (PCL) scaffolds. STUDY DESIGN: Controlled laboratory study. METHODS: Anatomically shaped ovine meniscal scaffolds were fabricated from PCL with a 3D printer based on MRI data. Skeletally mature sheep (N = 34) were randomly allocated to 3 groups: scaffold without growth factor (0-µg group), scaffold with CTGF microspheres (µS) (5 µg) + TGF-ß3 µS (5 µg) (5-µg group), and scaffold with CTGF µS (10 µg) + TGF-ß3 µS (10 µg) (10-µg group). Unilateral medial meniscal replacement was performed. Animals were euthanized at 6 or 12 months. Regenerated meniscus, articular cartilage status, and synovial reaction were evaluated quantitatively with gross inspection, histology, and MRI. Kruskal-Wallis and Dunn tests were used to compare the 3 groups. RESULTS: Remnants of the PCL scaffold were evident in the 6-month specimens and were decreased but still present at 12 months in most animals. There were no significant differences among groups in gross inspection, histology, or MRI for either meniscal regeneration or articular cartilage protection. All experimental groups exhibited articular cartilage degeneration as compared with control (nonoperated). In terms of synovitis, there were no clear differences among groups, suggesting that growth factors did not increase inflammation and fibrosis. MRI revealed that meniscal extrusion was observed in most animals (82.7%). CONCLUSION: Previously, the combination of CTGF and TGF-ß3 was shown to stimulate mesenchymal stem cells into a fibrochondrocyte lineage. CTGF and TGF-ß3 did not aggravate synovitis, suggesting no adverse response to the combination of 3D-printed PCL scaffold combined with CTGF and TGF-ß3. Further work will be required to improve scaffold fixation to avoid meniscal extrusion. CLINICAL RELEVANCE: A significant advantage of this technique is the ability to print custom-fit scaffolds from MRI-generated templates. In addition, average-size menisci could be printed and available for off-the-shelf applications. Based on the 1-year duration of the study, the approach appears to be promising for meniscal regeneration in humans.

Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix.

Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/ß-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.

Epigenetic and therapeutic implications of dnmt3b in temporomandibular joint osteoarthritis.

Temporomandibular joint (TMJ) arthritis causes severe debilitation and has few treatment options. Here, we found a small molecule, DNA methyltransferase 3B (Dnmt3b), as a putative therapeutic target, partially rescued osteoarthritic phenotype. Dnmt3b was detected differentially expressed in cell zones of mandibular condylar cartilage and the expression of Dnmt3b decreased in the progression of TMJ osteoarthritis. Dnmt3b deficiency using conditional knockout mice led to the onset of osteoarthritis-like conditions including cartilage clefts, cartilage matrix loss and premature chondrocyte hypertrophy, which suggested that Dnmt3b functioned as a osteoarthritis suppressor. Dnmt3b gain-of-function in TMJ stem/progenitor cells showed increases in collagen type II but decreases in collagen type X, whereas Dnmt3b knockdown had opposite effects with attenuated collagen type II but increased collagen type X. Dnmt3b acted via Wnt/ß-catenin signaling and Dnmt3b regulated TMJ stem/progenitor cells differentiation by inducing their premature progression towards hypertrophic chondrocytes through ß-catenin transnucleation and activation. Finally, local Dnmt3b delivery partially rescued cartilage degradation in experimentally induced osteoarthritis. Thus, novel molecules in articular cartilage, such as Dnmt3b, may have therapeutic effects for TMJ osteoarthritis.

A simple layer-stacking technique to generate biomolecular and mechanical gradients in photocrosslinkable hydrogels.

Physicochemical and biological gradients are desirable features for hydrogels to enhance their relevance to biological environments for three-dimensional (3D) cell culture. Therefore, simple and efficient techniques to generate chemical, physical and biological gradients within hydrogels are highly desirable. This work demonstrates a technique to generate biomolecular and mechanical gradients in photocrosslinkable hydrogels by stacking and crosslinking prehydrogel solution in a layer by layer manner. Partial crosslinking of the hydrogel allows mixing of prehydrogel solution with the previous hydrogel layer, which makes a smooth gradient profile, rather than discrete layers. This technique enables the generation of concentration gradients of bovine serum albumin in both gelatin methacryloyl (GelMA) and poly(ethylene glycol) diacrylate hydrogels, as well as mechanical gradients across a hydrogel containing varying gel concentrations. Fluorescence microscopy, mechanical testing, and scanning electron microscopy show that the gradient profiles can be controlled by changing both the volume and concentration of each layer as well as intensity of UV exposure. GelMA hydrogel gradients with different Young's moduli were successfully used to culture human fibroblasts. The fibroblasts migrated along the gradient axis and showed different morphologies. In general, the proposed technique provides a rapid and simple approach to design and fabricate 3D hydrogel gradients for in vitro biological studies and potentially for in vivo tissue engineering applications.

Optimizing Clinical Use of Biologics in Orthopaedic Surgery: Consensus Recommendations From the 2018 AAOS/NIH U-13 Conference.

Concern that misinformation from direct-to-consumer marketing of largely unproven "biologic" treatments such as platelet-rich plasma and cell-based therapies may erode the public trust and the responsible investment needed to bring legitimate biological therapies to patients have resulted in calls to action from professional organizations and governing bodies. In response to substantial patient demand for biologic treatment of orthopaedic conditions, the American Academy of Orthopaedic Surgeons convened a collaborative symposium and established a consensus framework for improving and accelerating the clinical evaluation, use, and optimization of biologic therapies for musculoskeletal diseases. The economic and disease burden of musculoskeletal conditions is high. Of the various conditions discussed, knee osteoarthritis was identified as a "serious condition" associated with substantial and progressive morbidity and emerged as the condition with the most urgent need for clinical trial development. It was also recognized that stem cells have unique characteristics that are not met by minimally manipulated mixed cell preparations. The work group recommended that minimally manipulated cell products be referred to as cell therapy and that the untested and uncharacterized nature of these treatments be clearly communicated within the profession, to patients, and to the public. Minimum standards for product characterization and clinical research should also be followed. A framework for developing clinical trials related to knee OA was agreed upon. In addition to recommendations for development of high-quality multicenter clinical trials, another important recommendation was that physicians and institutions offering biologic therapies commit to establishing high-quality patient registries and biorepository-linked registries that can be used for postmarket surveillance and quality assessments.

mTOR acts as a pivotal signaling hub for neural crest cells during craniofacial development.

mTOR is a highly conserved serine/threonine protein kinase that is critical for diverse cellular processes in both developmental and physiological settings. mTOR interacts with a set of molecules including Raptor and Rictor to form two distinct functional complexes, namely the mTORC1 and mTORC2. Here, we used novel genetic models to investigate functions of the mTOR pathway for cranial neural crest cells (NCCs), which are a temporary type of cells arising from the ectoderm layer and migrate to the pharyngeal arches participating craniofacial development. mTOR deletion elicited a proliferation deficit and excessive apoptosis of post-migratory NCCs, leading to growth arrest of the facial primordia along with midline orofacial clefts. Furthermore, NCC differentiation was impaired. Thus, NCC derivatives, such as skeletons, vasculatures and neural tissues were either rudimentary or malformed. We further demonstrate that disruption of mTOR caused P53 hyperactivity and cell cycle arrest in cranial NCCs, and lowering P53 activity by one copy reduction attenuated the severity of craniofacial phenotype in NCC-mTOR knockout mice. Remarkably, NCC-Rptor disruption caused a spectrum of defects mirroring that of the NCC-mTOR deletion, whereas NCC-Rictor disruption only caused a mild craniofacial phenotype compared to the mTOR and Rptor conditional knockout models. Altogether, our data demonstrate that mTOR functions mediated by mTORC1 are indispensable for multiple processes of NCC development including proliferation, survival, and differentiation during craniofacial morphogenesis and organogenesis, and P53 hyperactivity in part accounts for the defective craniofacial development in NCC-mTOR knockout mice.

Profiling of Stem/Progenitor Cell Regulatory Genes of the Synovial Joint by Genome-Wide RNA-Seq Analysis.

Synovial joints suffer from arthritis and trauma that may be severely debilitative. Despite robust investigations in the roles of individual genes in synovial joint development and arthritis, little is known about global profiles of genes that regulate stem/progenitor cells of a synovial joint. The temporomandibular joint is a poorly understood synovial arthrosis with few clinical treatment options. Here, we isolated the articular and mature zones of the mandibular condyle by laser capture microdissection, performed genome-wide profiling, and analyzed molecular signaling pathways relevant to stem/progenitor cell functions. A total of 804 genes were differentially expressed between the articular and mature zones. Pathway analyses revealed 29 enriched signaling pathways, including the PI3K-Akt, Wnt, and Toll-like receptor signaling pathways that may regulate stem/progenitor cell homeostasis and differentiation into the chondrocyte lineage. Upstream regulator analyses further predicted potential upstream key regulators such as Xbp1, Nupr1, and Hif1a, and associated underlying mechanism networks were described. Among the multiple candidates of growth and transcriptional factors that may regulate stem/progenitor cells, we immunolocalized Sox9, Ihh, Frzb, Dkk1, Lgr5, and TGFß3 in the articular and mature zones. These findings provide a comprehensive genetic mapping of growth and transcriptional genes in the articular and mature zones of a synovial joint condyle. Differentially expressed genes may play crucial roles in the regulation of stem/progenitor cells in development, homeostasis, and tissue regeneration.

BMAL1 Deficiency Contributes to Mandibular Dysplasia by Upregulating MMP3.

Skeletal mandibular hypoplasia (SMH), one of the common types of craniofacial deformities, seriously affects appearance, chewing, pronunciation, and breathing. Moreover, SMH is prone to inducing obstructive sleep apnea syndrome. We found that brain and muscle ARNT-like 1 (BMAL1), the core component of the molecular circadian oscillator, was significantly decreased in mandibles of juvenile SMH patients. Accordingly, SMH was observed in circadian-rhythm-disrupted or BMAL1-deficient mice. RNA sequencing and protein chip analyses suggested that matrix metallopeptidase 3 (MMP3) is the potential target of BMAL1. Interestingly, in juvenile SMH patients, we observed that MMP3 was obviously increased. Consistently, MMP3 was upregulated during the whole growth period of 3-10 weeks in Bmal1-/- mice. Given these findings, we set out to characterize the underlying mechanism and found BMAL1 deficiency enhanced Mmp3 transcription through activating p65 phosphorylation. Together, our results provide insight into the mechanism by which BMAL1 is implicated in the pathogenesis of SMH.

Treatment of Necrotic Teeth by Apical Revascularization: Meta-analysis.

Each year ~5.4 million children and adolescents in the United States suffer from dental infections, leading to pulp necrosis, arrested tooth-root development and tooth loss. Apical revascularization, adopted by the American Dental Association for its perceived ability to enable postoperative tooth-root growth, is being accepted worldwide. The objective of the present study is to perform a meta-analysis on apical revascularization. Literature search yielded 22 studies following PRISMA with pre-defined inclusion and exclusion criteria. Intraclass correlation coefficient was calculated to account for inter-examiner variation. Following apical revascularization with 6- to 66-month recalls, root apices remained open in 13.9% cases (types I), whereas apical calcification bridge formed in 47.2% (type II) and apical closure (type III) in 38.9% cases. Tooth-root lengths lacked significant postoperative gain among all subjects (p = 0.3472) or in subgroups. Root-dentin area showed significant increases in type III, but not in types I or II cases. Root apices narrowed significantly in types II and III, but not in type I patients. Thus, apical revascularization facilitates tooth-root development but lacks consistency in promoting root lengthening, widening or apical closure. Post-operative tooth-root development in immature permanent teeth represents a generalized challenge to regenerate diseased pediatric tissues that must grow to avoid organ defects.

Regenerative Endodontics for Adult Patients.

The goal of endodontics is to save teeth. Since inception, endodontic treatments are performed to obturate disinfected root canals with inert materials such as gutta-percha. Although teeth can be saved after successful endodontic treatments, they are devitalized and therefore susceptible to reinfections and fractures. The American Association of Endodontists (AAE) has made a tremendous effort to revitalize disinfected immature permanent teeth in children and adolescents with diagnoses including pulp necrosis or apical periodontitis. The American Dental Association (ADA) in 2011 issued several clinical codes for regenerative endodontic procedures or apical revascularization in necrotic immature permanent teeth in children and adolescents. These AAE and ADA initiatives have stimulated robust interest in devising a multitude of tissue engineering approaches for dental pulp and dentin regeneration. Can the concept of regenerative endodontics be extended to revitalize mature permanent teeth with diagnoses including irreversible pulpitis and/or pulp necrosis in adults? The present article was written not only to summarize emerging findings to revitalize mature permanent teeth in adult patients but also to identify challenges and strategies that focus on realizing the goal of regenerative endodontics in adults. We further present clinical cases and describe the biological basis of potential regenerative endodontic procedures in adults. This article explores the frequently asked question if regenerative endodontic therapies should be developed for dental pulp and/or dentin regeneration in adults, who consist of the great majority of endodontic patients.

Exosomes Mediate Epithelium-Mesenchyme Crosstalk in Organ Development.

Organ development requires complex signaling by cells in different tissues. Epithelium and mesenchyme interactions are crucial for the development of skin, hair follicles, kidney, lungs, prostate, major glands, and teeth. Despite myriad literature on cell-cell interactions and ligand-receptor binding, the roles of extracellular vesicles in epithelium-mesenchyme interactions during organogenesis are poorly understood. Here, we discovered that ∼100 nm exosomes were secreted by the epithelium and mesenchyme of a developing tooth organ and diffused through the basement membrane. Exosomes were entocytosed by epithelium or mesenchyme cells with preference by reciprocal cells rather than self-uptake. Exosomes reciprocally evoked cell differentiation and matrix synthesis: epithelium exosomes induce mesenchyme cells to produce dentin sialoprotein and undergo mineralization, whereas mesenchyme exosomes induce epithelium cells to produce basement membrane components, ameloblastin and amelogenenin. Attenuated exosomal secretion by Rab27a/b knockdown or GW4869 disrupted the basement membrane and reduced enamel and dentin production in organ culture and reduced matrix synthesis and the size of the cervical loop, which harbors epithelium stem cells, in Rab27aash/ash mutant mice. We then profiled exosomal constituents including miRNAs and peptides and further crossed all epithelium exosomal miRNAs with literature-known miRNA Wnt regulators. Epithelium exosome-derived miR135a activated Wnt/ß-catenin signaling and escalated mesenchymal production of dentin matrix proteins, partially reversible by Antago-miR135a attenuation. Our results suggest that exosomes may mediate epithelium-mesenchyme crosstalk in organ development, suggesting that these vesicles and/or the molecular contents they are transporting may be interventional targets for treatment of diseases or regeneration of tissues.

Pyrintegrin Induces Soft Tissue Formation by Transplanted or Endogenous Cells.

Focal adipose deficiency, such as lipoatrophy, lumpectomy or facial trauma, is a formidable challenge in reconstructive medicine, and yet scarcely investigated in experimental studies. Here, we report that Pyrintegrin (Ptn), a 2,4-disubstituted pyrimidine known to promote embryonic stem cells survival, is robustly adipogenic and induces postnatal adipose tissue formation in vivo of transplanted adipose stem/progenitor cells (ASCs) and recruited endogenous cells. In vitro, Ptn stimulated human adipose tissue derived ASCs to differentiate into lipid-laden adipocytes by upregulating peroxisome proliferator-activated receptor (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα), with differentiated cells increasingly secreting adiponectin, leptin, glycerol and total triglycerides. Ptn-primed human ASCs seeded in 3D-bioprinted biomaterial scaffolds yielded newly formed adipose tissue that expressed human PPARγ, when transplanted into the dorsum of athymic mice. Remarkably, Ptn-adsorbed 3D scaffolds implanted in the inguinal fat pad had enhanced adipose tissue formation, suggesting Ptn's ability to induce in situ adipogenesis of endogenous cells. Ptn promoted adipogenesis by upregulating PPARγ and C/EBPα not only in adipogenesis induction medium, but also in chemically defined medium specifically for osteogenesis, and concurrently attenuated Runx2 and Osx via BMP-mediated SMAD1/5 phosphorylation. These findings suggest Ptn's novel role as an adipogenesis inducer with a therapeutic potential in soft tissue reconstruction and augmentation.

Regenerative Endodontics by Cell Homing.

Apical revascularization (AR) and platelet-rich plasma have been used to restore dental pulp vitality in infected immature permanent teeth. Two regenerative therapies are cell transplantation and cell homing. This article updates and benchmarks these therapies with cell homing. A case report concluded that AR increased root length; however, quantitative and statistical assessments disproved this. Regenerative endodontic therapies require prospective clinical trials demonstrating safety and efficacy. These therapies are intrinsically susceptible to procedural and patient variations. Cell homing uses novel molecules that drive therapeutic efficacy, and may be less sensitive to procedural and patient variations.