C-DOCTOR Publications

Below are some featured publications from C-DOCTOR ITP teams and leadership. Please email VyVy Nguyen to have your publication included here.

As part of the C-DOCTOR ITP Team Award Terms & Conditions, all publications supported in whole or part by C-DOCTOR funds must include the following acknowledgments: “Research reported in this publication was supported by the National Institute Of Dental & Craniofacial Research of the National Institutes of Health under award number U24 DE029463. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”


Allele-specific expression reveals genetic drivers of tissue regeneration in mice

Mack, K.L., Talbott, H.E., Griffin, M.F., Parker, J.B.L., Guardino, N.J., Spielman, A.F., Davitt, M.F., Mascharak, S., Downer, M., Morgan, A., Valencia, C., Akras, D., Berger, M.J., Wan, D.C., Fraser, H.B., Longaker, M.T.

View Abstract
In adult mammals, skin wounds typically heal by scarring rather than through regeneration. In contrast, "super-healer" Murphy Roths Large (MRL) mice have the unusual ability to regenerate ear punch wounds; however, the molecular basis for this regeneration remains elusive. Here, in hybrid crosses between MRL and non-regenerating mice, we used allele-specific gene expression to identify cis-regulatory variation associated with ear regeneration. Analyzing three major cell populations (immune, fibroblast, and endothelial), we found that genes with cis-regulatory differences specifically in fibroblasts were associated with wound-healing pathways and also co-localized with quantitative trait loci for ear wound-healing. Ectopic treatment with one of these proteins, complement factor H (CFH), accelerated wound repair and induced regeneration in typically fibrotic wounds. Through single-cell RNA sequencing (RNA-seq), we observed that CFH treatment dramatically reduced immune cell recruitment to wounds, suggesting a potential mechanism for CFH's effect. Overall, our results provide insights into the molecular drivers of regeneration with potential clinical implications.

Inhibition of a signaling modality within the gp130 receptor enhances tissue regeneration and mitigates osteoarthritis

Shkhyan, R., Flynn, C., Lamoure, E., Sarkar, A., Van Handel, B., Li, J., York, J., Banks, N., Van der Horst, R., Liu, N.Q., Lee, S., Bajaj, P., Vadivel, K., Harn, H.I.-C., Tassey, J., Lozito, T., Lieberman, J.R., Chuong, C.-M., Hurtig, M.S., Evseenko, D.

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Adult mammals are incapable of multitissue regeneration, and augmentation of this potential may shift current therapeutic paradigms. We found that a common co-receptor of interleukin 6 (IL-6) cytokines, glycoprotein 130 (gp130), serves as a major nexus integrating various context-specific signaling inputs to either promote regenerative outcomes or aggravate disease progression. Via genetic and pharmacological experiments in vitro and in vivo, we demonstrated that a signaling tyrosine 814 (Y814) within gp130 serves as a major cellular stress sensor. Mice with constitutively inactivated Y814 (F814) were resistant to surgically induced osteoarthritis as reflected by reduced loss of proteoglycans, reduced synovitis, and synovial fibrosis. The F814 mice also exhibited enhanced regenerative, not reparative, responses after wounding in the skin. In addition, pharmacological modulation of gp130 Y814 upstream of the SRC and MAPK circuit by a small molecule, R805, elicited a protective effect on tissues after injury. Topical administration of R805 on mouse skin wounds resulted in enhanced hair follicle neogenesis and dermal regeneration. Intra-articular administration of R805 to rats after medial meniscal tear and to canines after arthroscopic meniscal release markedly mitigated the appearance of osteoarthritis. Single-cell sequencing data demonstrated that genetic and pharmacological modulation of Y814 resulted in attenuation of inflammatory gene signature as visualized by the anti-inflammatory macrophage and nonpathological fibroblast subpopulations in the skin and joint tissue after injury. Together, our study characterized a molecular mechanism that, if manipulated, enhances the intrinsic regenerative capacity of tissues through suppression of a proinflammatory milieu and prevents pathological outcomes in injury and disease.

Purification and functional characterization of novel human skeletal stem cell lineages

Hoover, M.Y., Ambrosi, T.H., Steininger, H.M., Koepke, L.S., Wang, Y., Zhao, L., Murphy, M.P., Alam, A.A., Arouge, E.J., Butler, M.G.K., Takematsu, E., Stavitsky, S.P., Hu, S., Sahoo, D., Sinha, R., Morri, M., Neff, N., Bishop, J., Gardner, M., Goodman, S., Longaker, M., Chan, C.K.F.

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Human skeletal stem cells (hSSCs) hold tremendous therapeutic potential for developing new clinical strategies to effectively combat congenital and age-related musculoskeletal disorders. Unfortunately, refined methodologies for the proper isolation of bona fide hSSCs and the development of functional assays that accurately recapitulate their physiology within the skeleton have been lacking. Bone marrow-derived mesenchymal stromal cells (BMSCs), commonly used to describe the source of precursors for osteoblasts, chondrocytes, adipocytes and stroma, have held great promise as the basis of various approaches for cell therapy. However, the reproducibility and clinical efficacy of these attempts have been obscured by the heterogeneous nature of BMSCs due to their isolation by plastic adherence techniques. To address these limitations, our group has refined the purity of individual progenitor populations that are encompassed by BMSCs by identifying defined populations of bona fide hSSCs and their downstream progenitors that strictly give rise to skeletally restricted cell lineages. Here, we describe an advanced flow cytometric approach that utilizes an extensive panel of eight cell surface markers to define hSSCs; bone, cartilage and stromal progenitors; and more differentiated unipotent subtypes, including an osteogenic subset and three chondroprogenitors. We provide detailed instructions for the FACS-based isolation of hSSCs from various tissue sources, in vitro and in vivo skeletogenic functional assays, human xenograft mouse models and single-cell RNA sequencing analysis. This application of hSSC isolation can be performed by any researcher with basic skills in biology and flow cytometry within 1-2 days. The downstream functional assays can be performed within a range of 1-2 months.

Exploring the Overlooked Roles and Mechanisms of Fibroblasts in the Foreign Body Response

Parker, Jennifer B; Griffin, Michelle F; Spielman, Amanda F; Wan, Derrick C

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Significance: Foreign body response (FBR), wherein a fibrotic capsule forms around an implanted structure, is a common surgical complication that often leads to pain, discomfort, and eventual revision surgeries. Although believed to have some mechanistic overlap with normal wound healing, much remains to be discovered about the specific mechanism by which this occurs. Recent Advances: Current understanding of FBR has focused on the roles of the immune system and the biomaterial, both major contributors to FBR. However, another key player, the fibroblast, is often overlooked. This review summarizes key contributors of FBR, focusing on the roles of fibroblasts. As much remains to be discovered about fibroblasts' specific roles in FBR, we draw on current knowledge of fibroblast subpopulations and functions during wound healing. We also provide an overview on candidate biomaterials and signaling pathways involved in FBR. Critical Issues and Future Directions: While the global implantable medical devices market is considerable and continues to appreciate in value, FBR remains one of the most common surgical implant complications. In parallel with the continued development of candidate biomaterials, further exploration of potential fibroblast subpopulations at a transcriptional level would provide key insights into further understanding the underlying mechanisms by which fibrous encapsulation occurs, and unveil novel directions for antifibrotic and regenerative therapies in the future.

Topical vanadate improves tensile strength and alters collagen organisation of excisional wounds in a mouse model

Lintel, Hendrik; Abbas, Darren B; Mackay, Duncan J; Griffin, Michelle; Lavin, Christopher V; Berry, Charlotte E; Guardino, Nicholas J; Guo, Jason L; Momeni, Arash; Mackay, Donald R; Longaker, Michael T; Wan, Derrick C

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Wound dehiscence, oftentimes a result of the poor tensile strength of early healing wounds, is a significant threat to the post-operative patient, potentially causing life-threatening complications. Vanadate, a protein tyrosine phosphatase inhibitor, has been shown to alter the organisation of deposited collagen in healing wounds and significantly improve the tensile strength of incisional wounds in rats. In this study, we sought to explore the effects of locally administered vanadate on tensile strength and collagen organisation in both the early and remodelling phases of excisional wound healing in a murine model. Wild-type mice underwent stented excisional wounding on their dorsal skin and were divided equally into three treatment conditions: vanadate injection, saline injection control and an untreated control. Tensile strength testing, in vivo suction Cutometer analysis, gross wound measurements and histologic analysis were performed during healing, immediately upon wound closure, and after 4 weeks of remodelling. We found that vanadate treatment significantly increased the tensile strength of wounds and their stiffness relative to control wounds, both immediately upon healing and into the remodelling phase. Histologic analysis revealed that these biomechanical changes were likely the result of increased collagen deposition and an altered collagen organisation composed of thicker and distinctly organised collagen bundles. Given the risk that dehiscence poses to all operative patients, vanadate presents an interesting therapeutic avenue to improve the strength of post-operative wounds and unstable chronic wounds to reduce the risk of dehiscence.


Long-term functional regeneration of radiation-damaged salivary glands through delivery of a neurogenic hydrogel

Li, Jianlong; Sudiwala, Sonia; Berthoin, Lionel; Mohabbat, Seayer; Gaylord, Eliza A; Sinada, Hanan; Pacheco, Noel Cruz; Chiang, Jiun Chiun; Jeon, Oju; Lombaert, Isabelle M A; May, Alison J; Alsberg, Eben; Bahney, Chelsea S; Knox, Sarah M

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Salivary gland acinar cells are severely depleted after radiotherapy for head and neck cancer, leading to loss of saliva and extensive oro-digestive complications. With no regenerative therapies available, organ dysfunction is irreversible. Here, using the adult murine system, we demonstrate that radiation-damaged salivary glands can be functionally regenerated via sustained delivery of the neurogenic muscarinic receptor agonist cevimeline. We show that endogenous gland repair coincides with increased nerve activity and acinar cell division that is limited to the first week after radiation, with extensive acinar cell degeneration, dysfunction, and cholinergic denervation occurring thereafter. However, we found that mimicking cholinergic muscarinic input via sustained local delivery of a cevimeline-alginate hydrogel was sufficient to regenerate innervated acini and retain physiological saliva secretion at nonirradiated levels over the long term (>3 months). Thus, we reveal a previously unknown regenerative approach for restoring epithelial organ structure and function that has extensive implications for human patients.

Decellularized Adipose Matrices Can Alleviate Radiation-Induced Skin Fibrosis

Adem, Sandeep; Abbas, Darren B; Lavin, Christopher V; Fahy, Evan J; Griffin, Michelle; Diaz Deleon, Nestor M; Borrelli, Mimi R; Mascharak, Shamik; Shen, Abra H; Patel, Ronak A; Longaker, Michael T; Nazerali, Rahim S; Wan, Derrick C

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Objective: Radiation therapy is commonplace for cancer treatment but often results in fibrosis and atrophy of surrounding soft tissue. Decellularized adipose matrices (DAMs) have been reported to improve these soft tissue defects through the promotion of adipogenesis. These matrices are decellularized by a combination of physical, chemical, and enzymatic methods to minimize their immunologic effects while promoting their regenerative effects. In this study, we aimed at exploring the regenerative ability of a DAM (renuva®; MTF biologics, Edison, NJ) in radiation-induced soft tissue injury. Approach: Fresh human lipoaspirate or DAM was injected into the irradiated scalp of CD-1 nude mice, and volume retention was monitored radiographically over 8 weeks. Explanted grafts were histologically assessed, and overlying skin was examined histologically and biomechanically. Irradiated human skin was also evaluated from patients after fat grafting or DAM injection. However, integrating data between murine and human skin in all cohorts is limited given the genetic variability between the two species. Results: Volume retention was found to be greater with fat grafts, though DAM retention was, nonetheless, appreciated at irradiated sites. Improvement in both mouse and human irradiated skin overlying fat and DAM grafts was observed in terms of biomechanical stiffness, dermal thickness, collagen density, collagen fiber networks, and skin vascularity. Innovation: This is the first demonstration of the use of DAMs for augmenting the regenerative potential of irradiated mouse and human skin. Conclusions: These findings support the use of DAMs to address soft tissue atrophy after radiation therapy. Morphological characteristics of the irradiated skin can also be improved with DAM grafting.

Deferoxamine to Minimize Fibrosis During Radiation Therapy

Tevlin, Ruth; Longaker, Michael T; Wan, Derrick C

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Significance: By 2030, there will be >4 million radiation-treated cancer survivors living in the United States. Irradiation triggers inflammation, fibroblast activation, and extracellular matrix deposition in addition to reactive oxygen species generation, leading to a chronic inflammatory response. Radiation-induced fibrosis (RIF) is a progressive pathology resulting in skin pigmentation, reduced elasticity, ulceration and dermal thickening, cosmetic deformity, pain, and the need for reconstructive surgery. Recent Advances: Deferoxamine (DFO) is a U.S. Food and Drug Administration (FDA)-approved iron chelator for blood dyscrasia management, which has been found to be proangiogenic, to decrease free radical formation, and reduce cell death. DFO has shown great promise in the treatment and prophylaxis of RIF in preclinical studies. Critical Issues: Systemic DFO has a short half-life and is cumbersome to deliver to patients intravenously. Transdermal DFO delivery is complicated by its high atomic mass and hydrophilicity, preventing stratum corneum penetration. A transdermal drug delivery system was developed to address these challenges, in addition to a strategy for topical administration. Future Directions: DFO has great potential to translate from bench to bedside. An important step in translation of DFO for RIF prophylaxis is to ensure that DFO treatment does not affect the efficacy of radiation therapy. Furthermore, after an initial plethora of studies reporting DFO treatment by intravenous and subcutaneous routes, a significant advantage of recent studies is the success of transdermal and topical delivery. Given the strong foundation of basic scientific research supporting the use of DFO treatment on RIF, clinicians will be closely following the results of the ongoing human studies.

Plasmid encoding miRNA-200c delivered by CaCO3-based nanoparticles enhances rat alveolar bone formation

Remy, Matthew T; Ding, Qiong; Krongbaramee, Tadkamol; Hu, Jue; Mora Mata, Andrés V; Haes, Amanda J; Amendt, Brad A; Sun, Hongli; Buchakjian, Marisa R; Hong, Liu

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Aim: miRNAs have been shown to improve the restoration of craniofacial bone defects. This work aimed to enhance transfection efficiency and miR-200c-induced bone formation in alveolar bone defects via plasmid DNA encoding miR-200c delivery from CaCO3 nanoparticles. Materials & methods: The CaCO3/miR-200c delivery system was evaluated in vitro (microscopy, transfection efficiency, biocompatibility) and miR-200c-induced in vivo alveolar bone formation was assessed via micro-computed tomography and histology. Results: CaCO3 nanoparticles significantly enhanced the transfection of plasmid DNA encoding miR-200c without inflammatory effects and sustained miR-200c expression. CaCO3/miR-200c treatment in vivo significantly increased bone formation in rat alveolar bone defects. Conclusion: CaCO3 nanoparticles enhance miR-200cdelivery to accelerate alveolar bone formation, thereby demonstrating the application of CaCO3/miR-200c to craniofacial bone defects.

Wound healing, fibroblast heterogeneity, and fibrosis

Talbott, Heather E; Mascharak, Shamik; Griffin, Michelle; Wan, Derrick C; Longaker, Michael T

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Fibroblasts are highly dynamic cells that play a central role in tissue repair and fibrosis. However, the mechanisms by which they contribute to both physiologic and pathologic states of extracellular matrix deposition and remodeling are just starting to be understood. In this review article, we discuss the current state of knowledge in fibroblast biology and heterogeneity, with a primary focus on the role of fibroblasts in skin wound repair. We also consider emerging techniques in the field, which enable an increasingly nuanced and contextualized understanding of these complex systems, and evaluate limitations of existing methodologies and knowledge. Collectively, this review spotlights a diverse body of research examining an often-overlooked cell type-the fibroblast-and its critical functions in wound repair and beyond.

Transdermal deferoxamine administration improves excisional wound healing in chronically irradiated murine skin

Lintel, Hendrik; Abbas, Darren B; Lavin, Christopher V; Griffin, Michelle; Guo, Jason L; Guardino, Nicholas; Churukian, Andrew; Gurtner, Geoffrey C; Momeni, Arash; Longaker, Michael T; Wan, Derrick C

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Background: Radiation-induced skin injury is a well-known risk factor for impaired wound healing. Over time, the deleterious effects of radiation on skin produce a fibrotic, hypovascular dermis poorly suited to wound healing. Despite increasing understanding of the underlying pathophysiology, therapeutic options remain elusive. Deferoxamine (DFO), an iron-chelating drug, has been shown in prior murine studies to ameliorate radiation-induced skin injury as well as improve wound healing outcomes in various pathologic conditions when administered transdermally. In this preclinical study, we evaluated the effects of deferoxamine on wound healing outcomes in chronically irradiated murine skin. Methods: Wild-type mice received 30 Gy of irradiation to their dorsal skin and were left to develop chronic fibrosis. Stented excisional wounds were created on their dorsal skin. Wound healing outcomes were compared across 4 experimental conditions: DFO patch treatment, vehicle-only patch treatment, untreated irradiated wound, and untreated nonirradiated wounds. Gross closure rate, wound perfusion, scar elasticity, histology, and nitric oxide assays were compared across the conditions. Results: Relative to vehicle and untreated irradiated wounds, DFO accelerated wound closure and reduced the frequency of healing failure in irradiated wounds. DFO augmented wound perfusion throughout healing and upregulated angiogenesis to levels observed in nonirradiated wounds. Histology revealed DFO increased wound thickness, collagen density, and improved collagen fiber organization to more closely resemble nonirradiated wounds, likely contributing to the observed improved scar elasticity. Lastly, DFO upregulated inducible nitric oxide synthase and increased nitric oxide production in early healing wounds. Conclusion: Deferoxamine treatment presents a potential therapeutic avenue through which to target impaired wound healing in patients following radiotherapy.

Disrupting mechanotransduction decreases fibrosis and contracture in split-thickness skin grafting

Chen, Kellen; Henn, Dominic; Januszyk, Michael; Barrera, Janos A; Noishiki, Chikage; Bonham, Clark A; Griffin, Michelle; Tevlin, Ruth; Carlomagno, Theresa; Shannon, Tara; Fehlmann, Tobias; Trotsyuk, Artem A; Padmanabhan, Jagannath; Sivaraj, Dharshan; Perrault, David P; Zamaleeva, Alsu I; Mays, Chyna J; Greco, Autumn H; Kwon, Sun Hyung; Leeolou, Melissa C; Huskins, Savana L; Steele, Sydney R; Fischer, Katharina S; Kussie, Hudson C; Mittal, Smiti; Mermin-Bunnell, Alana M; Diaz Deleon, Nestor M; Lavin, Christopher; Keller, Andreas; Longaker, Michael T; Gurtner, Geoffrey C

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Burns and other traumatic injuries represent a substantial biomedical burden. The current standard of care for deep injuries is autologous split-thickness skin grafting (STSG), which frequently results in contractures, abnormal pigmentation, and loss of biomechanical function. Currently, there are no effective therapies that can prevent fibrosis and contracture after STSG. Here, we have developed a clinically relevant porcine model of STSG and comprehensively characterized porcine cell populations involved in healing with single-cell resolution. We identified an up-regulation of proinflammatory and mechanotransduction signaling pathways in standard STSGs. Blocking mechanotransduction with a small-molecule focal adhesion kinase (FAK) inhibitor promoted healing, reduced contracture, mitigated scar formation, restored collagen architecture, and ultimately improved graft biomechanical properties. Acute mechanotransduction blockade up-regulated myeloid CXCL10-mediated anti-inflammation with decreased CXCL14-mediated myeloid and fibroblast recruitment. At later time points, mechanical signaling shifted fibroblasts toward profibrotic differentiation fates, and disruption of mechanotransduction modulated mesenchymal fibroblast differentiation states to block those responses, instead driving fibroblasts toward proregenerative, adipogenic states similar to unwounded skin. We then confirmed these two diverging fibroblast transcriptional trajectories in human skin, human scar, and a three-dimensional organotypic model of human skin. Together, pharmacological blockade of mechanotransduction markedly improved large animal healing after STSG by promoting both early, anti-inflammatory and late, regenerative transcriptional programs, resulting in healed tissue similar to unwounded skin. FAK inhibition could therefore supplement the current standard of care for traumatic and burn injuries.

Harnessing a Feasible and Versatile ex vivo Calvarial Suture 2-D Culture System to Study Suture Biology

Quarto, Natalina; Menon, Siddharth; Griffin, Michelle; Huber, Julika; Longaker, Michael T

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As a basic science, craniofacial research embraces multiple facets spanning from molecular regulation of craniofacial development, cell biology/signaling and ultimately translational craniofacial biology. Calvarial sutures coordinate development of the skull, and the premature fusion of one or more, leads to craniosynostosis. Animal models provide significant contributions toward craniofacial biology and clinical/surgical treatments of patients with craniofacial disorders. Studies employing mouse models are costly and time consuming for housing/breeding. Herein, we present the establishment of a calvarial suture explant 2-D culture method that has been proven to be a reliable system showing fidelity with the in vivo harvesting procedure to isolate high yields of skeletal stem/progenitor cells from small number of mice. Moreover, this method allows the opportunity to phenocopying models of craniosynostosis and in vitro tamoxifen-induction of ActincreERT2;R26Rainbow suture explants to trace clonal expansion. This versatile method tackles needs of large number of mice to perform calvarial suture research.

Multi-omic analysis reveals divergent molecular events in scarring and regenerative wound healing

Mascharak, Shamik; Talbott, Heather E; Januszyk, Michael; Griffin, Michelle; Chen, Kellen; Davitt, Michael F; Demeter, Janos; Henn, Dominic; Bonham, Clark A; Foster, Deshka S; Mooney, Nancie; Cheng, Ran; Jackson, Peter K; Wan, Derrick C; Gurtner, Geoffrey C; Longaker, Michael T

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Regeneration is the holy grail of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward scarring or regenerative fates. We profile scarring versus YAP-inhibition-induced wound regeneration at the transcriptional (single-cell RNA sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell-surface barcoding, we integrate these data to reveal fibrotic and regenerative "molecular trajectories" of healing. We show that disrupting YAP mechanotransduction yields regenerative repair by fibroblasts with activated Trps1 and Wnt signaling. Finally, via in vivo gene knockdown and overexpression in wounds, we identify Trps1 as a key regulatory gene that is necessary and partially sufficient for wound regeneration. Our findings serve as a multi-omic map of wound regeneration and could have therapeutic implications for pathologic fibroses.

Immunomodulatory Microneedle Patch for Periodontal Tissue Regeneration

Zhang, Xuexiang; Hasani-Sadrabadi, Mohammad Mahdi; Zarubova, Jana; Dashtimighadam, Erfan; Haghniaz, Reihaneh; Khademhosseini, Ali; Butte, Manish J; Moshaverinia, Alireza; Aghaloo, Tara; Li, Song

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Periodontal diseases are caused by microbial infection and the recruitment of destructive immune cells. Current therapies mainly deal with bacteria elimination, but the regeneration of periodontal tissues remains a challenge. Here we developed a modular microneedle (MN) patch that delivered both antibiotic and cytokines into the local gingival tissue to achieve immunomodulation and tissue regeneration. This MN patch included a quickly dissolvable gelatin membrane for an immediate release of tetracycline and biodegradable GelMA MNs that contained tetracycline-loaded poly(lactic-co-glycolic acid) nanoparticles and cytokine-loaded silica microparticles for a sustained release. Antibiotic release completely inhibited bacteria growth, and the release of IL-4 and TGF-β induced the repolarization of anti-inflammatory macrophages and the formation of regulatory T cells in vitro. In vivo delivery of MN patch into periodontal tissues suppressed proinflammatory factors and promoted pro-regenerative signals and tissue healing, which demonstrated the therapeutic potential of local immunomodulation for tissue regeneration.


A comparative analysis of deferoxamine treatment modalities for dermal radiation-induced fibrosis

Lavin, Christopher V; Abbas, Darren B; Fahy, Evan J; Lee, Daniel K; Griffin, Michelle; Diaz Deleon, Nestor M; Mascharak, Shamik; Chen, Kellen; Momeni, Arash; Gurtner, Geoffrey C; Longaker, Michael T; Wan, Derrick C

View Abstract
The iron chelator, deferoxamine (DFO), has been shown to potentially improve dermal radiation-induced fibrosis (RIF) in mice through increased angiogenesis and reduced oxidative damage. This preclinical study evaluated the efficacy of two DFO administration modalities, transdermal delivery and direct injection, as well as temporal treatment strategies in relation to radiation therapy to address collateral soft tissue fibrosis. The dorsum of CD-1 nude mice received 30 Gy radiation, and DFO (3 mg) was administered daily via patch or injection. Treatment regimens were prophylactic, during acute recovery, post-recovery, or continuously throughout the experiment (n = 5 per condition). Measures included ROS-detection, histology, biomechanics and vascularity changes. Compared with irradiated control skin, DFO treatment decreased oxidative damage, dermal thickness and collagen content, and increased skin elasticity and vascularity. Metrics of improvement in irradiated skin were most pronounced with continuous transdermal delivery of DFO. In summary, DFO administration reduces dermal fibrosis induced by radiation. Although both treatment modalities were efficacious, the transdermal delivery showed greater effect than injection for each temporal treatment strategy. Interestingly, the continuous patch group was more similar to normal skin than to irradiated control skin by most measures, highlighting a promising approach to address detrimental collateral soft tissue injury following radiation therapy.

Disrupting biological sensors of force promotes tissue regeneration in large organisms

Chen, Kellen; Kwon, Sun Hyung; Henn, Dominic; Kuehlmann, Britta A; Tevlin, Ruth; Bonham, Clark A; Griffin, Michelle; Trotsyuk, Artem A; Borrelli, Mimi R; Noishiki, Chikage; Padmanabhan, Jagannath; Barrera, Janos A; Maan, Zeshaan N; Dohi, Teruyuki; Mays, Chyna J; Greco, Autumn H; Sivaraj, Dharshan; Lin, John Q ; Fehlmann, Tobias; Mermin-Bunnell, Alana M; Mittal, Smiti; Hu, Michael S; Zamaleeva, Alsu I; Keller, Andreas; Rajadas, Jayakumar; Longaker, Michael T; Januszyk, Michael; Gurtner, Geoffrey C

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Tissue repair and healing remain among the most complicated processes that occur during postnatal life. Humans and other large organisms heal by forming fibrotic scar tissue with diminished function, while smaller organisms respond with scarless tissue regeneration and functional restoration. Well-established scaling principles reveal that organism size exponentially correlates with peak tissue forces during movement, and evolutionary responses have compensated by strengthening organ-level mechanical properties. How these adaptations may affect tissue injury has not been previously examined in large animals and humans. Here, we show that blocking mechanotransduction signaling through the focal adhesion kinase pathway in large animals significantly accelerates wound healing and enhances regeneration of skin with secondary structures such as hair follicles. In human cells, we demonstrate that mechanical forces shift fibroblasts toward pro-fibrotic phenotypes driven by ERK-YAP activation, leading to myofibroblast differentiation and excessive collagen production. Disruption of mechanical signaling specifically abrogates these responses and instead promotes regenerative fibroblast clusters characterized by AKT-EGR1.

Aged skeletal stem cells generate an inflammatory degenerative niche

Ambrosi, Thomas H; Marecic, Owen; McArdle, Adrian; Sinha, Rahul; Gulati, Gunsagar S; Tong, Xinming; Wang, Yuting; Steininger, Holly M; Hoover, Malachia Y; Koepke, Lauren S; Murphy, Matthew P; Sokol, Jan; Seo, Eun Young; Tevlin, Ruth; Lopez, Michael; Brewer, Rachel E; Mascharak, Shamik; Lu, Laura; Ajanaku, Oyinkansola; Conley, Stephanie D; Seita, Jun; Morri, Maurizio; Neff, Norma F; Sahoo, Debashis; Yang, Fan; Weissman, Irving L; Longaker, Michael T; Chan, Charles K F

View Abstract
Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts1. Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system.

Mesenchymal stem cells and three-dimensional-osteoconductive scaffold regenerate calvarial bone in critical size defects in swine

Johnson, Zoe M; Yuan, Yuan; Li, Xiangjia; Jashashvili, Tea; Jamieson, Michael; Urata, Mark; Chen, Yong; Chai, Yang

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Craniofacial bones protect vital organs, perform important physiological functions, and shape facial identity. Critical-size defects (CSDs) in calvarial bones, which will not heal spontaneously, are caused by trauma, congenital defects, or tumor resections. They pose a great challenge for patients and physicians, and significantly compromise quality of life. Currently, calvarial CSDs are treated either by allogenic or autologous grafts, metal or other synthetic plates that are associated with considerable complications. While previous studies have explored tissue regeneration for calvarial defects, most have been done in small animal models with limited translational value. Here we define a swine calvarial CSD model and show a novel approach to regenerate high-quality bone in these defects by combining mesenchymal stem cells (MSCs) with a three-dimensional (3D)-printed osteoconductive HA/TCP scaffold. Specifically, we have compared the performance of dental pulp neural crest MSCs (DPNCCs) to bone marrow aspirate (BMA) combined with a 3D-printed HA/TCP scaffold to regenerate bone in a calvarial CSD (>7.0 cm2). Both DPNCCs and BMA loaded onto the 3D-printed osteoconductive scaffold support the regeneration of calvarial bone with density, compression strength, and trabecular structures similar to native bone. Our study demonstrates a novel application of an original scaffold design combined with DPNCCs or BMA to support regeneration of high-quality bone in a newly defined and clinically relevant swine calvarial CSD model. This discovery may have important impact on bone regeneration beyond the craniofacial region and will ultimately benefit patients who suffer from debilitating CSDs.

Skeletal stem and progenitor cells maintain cranial suture patency and prevent craniosynostosis

Menon, Siddharth; Salhotra, Ankit; Shailendra, Siny; Tevlin, Ruth; Ransom, Ryan C; Januszyk, Michael; Chan, Charles K F; Behr, Björn; Wan, Derrick C; Longaker, Michael T; Quarto, Natalina

View Abstract
Cranial sutures are major growth centers for the calvarial vault, and their premature fusion leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1+/- mouse model of coronal craniosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co-transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1+/- mice. Our study reveals that decrease and/or imbalance of skeletal stem/progenitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis.

Distinct skeletal stem cell types orchestrate long bone skeletogenesis

Ambrosi, Thomas H; Sinha, Rahul; Steininger, Holly M; Hoover, Malachia Y; Murphy, Matthew P; Koepke, Lauren S; Wang, Yuting; Lu, Wan-Jin; Morri, Maurizio; Neff, Norma F; Weissman, Irving L; Longaker, Michael T; Chan, Charles K F

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Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.

Polymer-Induced Liquid Precursor (PILP) remineralization of artificial and natural dentin carious lesions evaluated by nanoindentation and microcomputed tomography

Babaie, Elham; Bacino, Margôt; White, Joel; Nurrohman, Hamid; Marshall, Grayson W; Saeki, Kuniko; Habelitz, Stefan

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Objectives: The study evaluates the efficacy to remineralize artificial and natural dentin lesions through restorative dental procedures that include the Polymer-Induced Liquid Precursor (PILP) method comprising polyaspartic acid (pAsp). Methods: Novel ionomeric cement compositions based on bioglass 45S5 and pAsp mixtures, as well as conditioning solutions (conditioner) containing 5 mg/mL pAsp, were developed and tested on demineralized dentin blocks (3-4 mm thick) on shallow and deep lesions with the thickness of 140 μm ± 50 and 700 μm ± 50, respectively. In the first treatment group, 20 μL of conditioner was applied to demineralized shallow (n = 3) and deep (n = 3) lesion specimens for 20 s before restoration with glass ionomer cement (RMGIC). For the PILP cement treatment group, cement was applied onto the wet surface of the demineralized specimen for both shallow (n = 3) and deep (n = 3) artificial lesions after the application of the conditioner and before the final restoration. Sample groups were compared to RMGIC restoration, for both shallow and deep lesions (n = 3 each) and treatments in PILP-solution (n = 3 for deep lesions) without restoration for 4 weeks. All of the restored specimens were immersed in simulated body fluid (SBF) solution for 2 weeks and 4 weeks for shallow and deep lesions respectively to allow for remineralization. The artificial lesion specimens were evaluated for changes in the nanomechanical profile (E-modulus and hardness) using nanoindentation. Shallow lesions were analyzed by SEM under vacuum for changes in morphology caused by PILP treatments. Also, a pilot study on human third molars with moderate lesions in dentin (n = 3) was initiated to test the efficacy of treatments in natural lesions based on mineral densities using microcomputed tomography (μCT) at 0, 1, and 3 months. Results: This study showed that functional remineralization of artificial lesions using PILP-releasing restoratives occurred, indicated by an increase of the elastic modulus in shallow lesions and in the middle zone of deep artificial lesions. The mechanical improvement was significant when compared to RMGIC restoration without pAsp (P < 0.05). Nonetheless, recovery across artificial lesions was most significant when specimens were immersed into PILP-solution with restorative (P < 0.01). Furthermore, natural lesions increased in mineral volume content to a higher degree when the restorative treatment included the PILP-method (P < 0.05). However, none of the natural lesions recovered to full mineral degree regardless of the treatments. Clinical significance/conclusion: These findings indicate the benefit of PILP applications in the functional repair of dentin caries and illustrate the challenge to integrate the PILP-method into a restorative approach in minimally invasive dental procedures.

Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring

Mascharak, Shamik; desJardins-Park, Heather E; Davitt, Michael F; Griffin, Michelle; Borrelli, Mimi R; Moore, Alessandra L; Chen, Kellen; Duoto, Bryan; Chinta, Malini; Foster, Deshka S; Shen, Abra H; Januszyk, Michael; Kwon, Sun Hyung; Wernig, Gerlinde; Wan, Derrick C; Lorenz, H Peter; Gurtner, Geoffrey C; Longaker, Michael T

View Abstract
Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).

Cranial Suture Regeneration Mitigates Skull and Neurocognitive Defects in Craniosynostosis

Yu, Mengfei; Ma, Li; Yuan, Yuan; Ye, Xin; Montagne, Axel; He, Jinzhi; Ho, Thach-Vu; Wu, Yingxi; Zhao, Zhen; Sta Maria, Naomi; Jacobs, Russell; Urata, Mark; Wang, Huiming; Zlokovic, Berislav V; Chen, Jian-Fu; Chai, Yang

View Abstract
Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering development of urgently needed innovative therapies. Here, we show that Twist1+/- mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure, and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.


3D printing of hydroxyapatite/tricalcium phosphate scaffold with hierarchical porous structure for bone regeneration

Li, Xiangjia; Yuan, Yuan; Liu, Luyang; Leung, Yuen-Shan; Chen, Yiyu; Guo, Yuxing; Chai, Yang; Chen, Yong

View Abstract
Three-dimensional (3D)-printed scaffolds have attracted considerable attention in recent years as they provide a suitable environment for bone cell tissue regeneration and can be customized in shape. Among many other challenges, the material composition and geometric structure have major impacts on the performance of scaffolds. Hydroxyapatite and tricalcium phosphate (HA/TCP), as the major constituents of natural bone and teeth, possess attractive biological properties and are widely used in bone scaffold fabrication. Many fabrication methods have been investigated in attempts to achieve HA/TCP scaffolds with microporous structure enabling cell growth and nutrient transport. However, current 3D printing methods can only achieve the fabrication of HA/TCP scaffolds with certain range of microporous structure. To overcome this challenge, we developed a slurry-based microscale mask image projection stereolithography, allowing us to form a HA/TCP-based photocurable suspension with complex geometry including biomimetic features and hierarchical porosity. Here, the curing performance and physical properties of the HA/TCP suspension were investigated, and a circular movement process for the fabrication of highly viscous HA/TCP suspension was developed. Based on these investigations, the scaffold composition was optimized. We determined that a 30 wt% HA/TCP scaffold with biomimetic hierarchical structure exhibited superior mechanical properties and porosity. Cell proliferation was investigated in vitro, and the surgery was conducted in a nude mouse in vivo model of long bone with cranial neural crest cells and bone marrow mesenchymal stem cells. The results showed our 3D-printed HA/TCP scaffold with biomimetic hierarchical structure is biocompatible and has sufficient mechanical strength for surgery.

Creating Structured Hydrogel Microenvironments for Regulating Stem Cell Differentiation

Mills, David K; Luo, Yangyang; Elumalai, Anusha; Esteve, Savannah; Karnik, Sonali; Yao, Shaomian

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The development of distinct biomimetic microenvironments for regulating stem cell behavior and bioengineering human tissues and disease models requires a solid understanding of cell-substrate interactions, adhesion, and its role in directing cell behavior, and other physico-chemical cues that drive cell behavior. In the past decade, innovative developments in chemistry, materials science, microfabrication, and associated technologies have given us the ability to manipulate the stem cell microenvironment with greater precision and, further, to monitor effector impacts on stem cells, both spatially and temporally. The influence of biomaterials and the 3D microenvironment's physical and biochemical properties on mesenchymal stem cell proliferation, differentiation, and matrix production are the focus of this review chapter. Mechanisms and materials, principally hydrogel and hydrogel composites for bone and cartilage repair that create "cell-supportive" and "instructive" biomaterials, are emphasized. We begin by providing an overview of stem cells, their unique properties, and their challenges in regenerative medicine. An overview of current fabrication strategies for creating instructive substrates is then reviewed with a focused discussion of selected fabrication methods with an emphasis on bioprinting as a critical tool in creating novel stem cell-based biomaterials. We conclude with a critical assessment of the current state of the field and offer our view on the promises and potential pitfalls of the approaches discussed.

A new anabolic compound, LLP2A-Ale, reserves periodontal bone loss in mice through augmentation of bone formation

Jiang, Min; Liu, Lixian; Liu, Ruiwu; Lam, Kit S; Lane, Nancy E; Yao, Wei

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Background: Currently, there are no effective medications to reverse periodontal disease (PD)-induced bone loss. The objective of this study was to test a new anabolic compound, LLP2A-Ale, or with the combination treatment of mesenchymal stromal cell (MSC), in the treatment of bone loss secondary to PD. Methods: PD was induced in mice by placing a ligature around the second right molar. At one week after disease induction, the mice were treated with placebo, LLP2A-Ale, MSCs, or combination of LLP2A-Ale + MSCs, and euthanized at week 4. Results: We found that PD induced alveolar bone loss that was associated with reduced bone formation. LLP2A-Ale alone or in combination with MSCs sustained alveolar bone formation and reversed alveolar bone loss. Additionally, PD alone caused systemic inflammation and increased the circulating levels of G-CSF, IP-10, MIP-1a, and MIP2, which were suppressed by LLP2A-Ale +/- MSCs. LLP2A-Ale +/- MSCs increased bone formation at the peripheral skeletal site (distal femur), which was otherwise suppressed by PD. Conclusion: Our findings indicated that LLP2A-Ale treatment rescued alveolar bone loss caused by PD, primarily by increasing bone formation. LLP2A-Ale also attenuated the circulating levels of a series of inflammatory cytokines and reversed the PD-induced suppression of systemic bone formation.

Articular cartilage regeneration by activated skeletal stem cells

Murphy, Matthew P; Koepke, Lauren S; Lopez, Michael T; Tong, Xinming; Ambrosi, Thomas H; Gulati, Gunsagar S; Marecic, Owen; Wang, Yuting; Ransom, Ryan C; Hoover, Malachia Y; Steininger, Holly; Zhao, Liming; Walkiewicz, Marcin P; Quarto, Natalina; Levi, Benjamin; Wan, Derrick C; Weissman, Irving L; Goodman, Stuart B; Yang, Fan; Longaker, Michael T; Chan, Charles K F

View Abstract
Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage1. The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation2-4. Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis5-7. We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.

Differential antimicrobial and cellular response of electrolytically metalized halloysite nanotubes having different amounts of surface metallization

Humayun, Ahmed; Luo, Yangyang; Elumalai, Anusha; Mills, David K

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We demonstrate an electrolytic method to metalize the outer surface of halloysite nanotubes (HNTs). Different metal HNT (mHNT) combinations (copper, silver, zinc) were produced with metal content in the 5-30 wt% range. mHNTs were characterized using a Scanning Electron Microscope (SEM), energy-dispersive spectroscopy (EDS), X-ray fluorescence (XRF), Fourier-transform infrared spectroscopy (FTIR) and X-ray powder diffraction (XRD). Different amounts of surface/lumen metal content of a system can confer differing antimicrobial/cellular response; hence, it is essential to assess the antimicrobial/cellular response as a function of metal content. Cellular response after exposure to mHNTs was studied in Staphylococcus aureus and pre-osteoblasts, respectively. Coated mHNTs could easily be identified using the characterization methods, and contrasting bacterial and cellular responses were obtained, which we propose was due to the extent of metallization. These findings demonstrate the potential of this method for creating metal-coated HNTs and suggest they have potential as an implant coating solution.

Prophylactic treatment with transdermal deferoxamine mitigates radiation-induced skin fibrosis

Shen, Abra H; Borrelli, Mimi R; Adem, Sandeep; Diaz Deleon, Nestor M; Patel, Ronak A; Mascharak, Shamik; Yen, Sara J; Sun, Blake Y; Taylor 4th, Walter L; Januszyk, Michael; Nguyen, Dung H; Momeni, Arash; Gurtner, Geoffrey C; Longaker, Michael T; Wan, Derrick C

View Abstract
Radiation therapy can result in pathological fibrosis of healthy soft tissue. The iron chelator deferoxamine (DFO) has been shown to improve skin vascularization when injected into radiated tissue prior to fat grafting. Here, we evaluated whether topical DFO administration using a transdermal drug delivery system prior to and immediately following irradiation (IR) can mitigate the chronic effects of radiation damage to the skin. CD-1 nude immunodeficient mice were split into four experimental groups: (1) IR alone (IR only), (2) DFO treatment for two weeks after recovery from IR (DFO post-IR), (3) DFO prophylaxis with treatment through and post-IR (DFO ppx), or (4) no irradiation or DFO (No IR). Immediately following IR, reactive oxygen species and apoptotic markers were significantly decreased and laser doppler analysis revealed significantly improved skin perfusion in mice receiving prophylactic DFO. Six weeks following IR, mice in the DFO post-IR and DFO ppx groups had improved skin perfusion and increased vascularization. DFO-treated groups also had evidence of reduced dermal thickness and collagen fiber network organization akin to non-irradiated skin. Thus, transdermal delivery of DFO improves tissue perfusion and mitigates chronic radiation-induced skin fibrosis, highlighting a potential role for DFO in the treatment of oncological patients.


A Revised Perspective of Skeletal Stem Cell Biology

Ambrosi, Thomas H; Longaker, Michael T; Chan, Charles K F

View Abstract
Bone-related maladies are a major health burden on modern society. Loss of skeletal integrity and regeneration capacity through aging, obesity, and disease follows from a detrimental shift in bone formation and resorption dynamics. Targeting tissue-resident adult stem cells offers a potentially innovative paradigm in the development of therapeutic strategies against organ dysfunction. While the essential role of skeletal stem cells (SSCs) for development, growth, and maintenance of the skeleton has been generally established, a common consensus on the exact identity and definition of a pure bona fide SSC population remains elusive. The controversies stem from conflicting results between different approaches and criteria for isolation, detection, and functional evaluation; along with the interchangeable usage of the terms SSC and "mesenchymal stromal/stem cell (MSC)". A great number of prospective bone-forming stem cell populations have been reported with various characteristic markers, often describing overlapping cell populations with widely unexplored heterogeneity, species specificity, and distribution at distinct skeletal sites, bone regions, and microenvironments, thereby creating confusion that may complicate future advances in the field. In this review, we examine the state-of-the-art knowledge of SSC biology and try to establish a common ground for the definition and terminology of specific bone-resident stem cells. We also discuss recent advances in the identification of highly purified SSCs, which will allow detailed interrogation of SSC diversity and regulation at the single-cell level.

Skeletal Stem Cell-Schwann Cell Circuitry in Mandibular Repair

Jones, R Ellen; Salhotra, Ankit; Robertson, Kiana S; Ransom, Ryan C; Foster, Deshka S; Shah, Harsh N; Quarto, Natalina; Wan, Derrick C; Longaker, Michael T

View Abstract
Regenerative paradigms exhibit nerve dependency, including regeneration of the mouse digit tip and salamander limb. Denervation impairs regeneration and produces morphological aberrancy in these contexts, but the direct effect of innervation on the stem and progenitor cells enacting these processes is unknown. We devised a model to examine nerve dependency of the mouse skeletal stem cell (mSSC), the progenitor responsible for skeletal development and repair. We show that after inferior alveolar denervation, mandibular bone repair is compromised because of functional defects in mSSCs. We present mSSC reliance on paracrine factors secreted by Schwann cells as the underlying mechanism, with partial rescue of the denervated phenotype by Schwann cell transplantation and by Schwann-derived growth factors. This work sheds light on the nerve dependency of mSSCs and has implications for clinical treatment of mandibular defects.

Regulatory mechanisms of jaw bone and tooth development

Yuan, Yuan; Chai, Yang

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Jaw bones and teeth originate from the first pharyngeal arch and develop in closely related ways. Reciprocal epithelial-mesenchymal interactions are required for the early patterning and morphogenesis of both tissues. Here we review the cellular contribution during the development of the jaw bones and teeth. We also highlight signaling networks as well as transcription factors mediating tissue-tissue interactions that are essential for jaw bone and tooth development. Finally, we discuss the potential for stem cell mediated regenerative therapies to mitigate disorders and injuries that affect these organs.

Integrating the PILP-mineralization process into a restorative dental treatment

Bacino, Margot; Girn, Vishavjeet; Nurrohman, Hamid; Saeki, Kuniko; Marshall, Sally J; Gower, Laurie; Saeed, Ella; Stewart, Ray; Le, Thuan; Marshall, Grayson W; Habelitz, Stefan

View Abstract
The addition of charged polymers, like poly-aspartic acid (pAsp), to mineralizing solutions allows for transport of calcium and phosphate ions into the lumen of collagen fibrils and subsequent crystallization of oriented apatite crystals by the so-called Polymer-Induced Liquid Precursor (PILP) mineralization process, leading to the functional recovery of artificial dentin lesions by intrafibrillar mineralization of collagen. Objective: To evaluate the feasibility of applying the PILP method as part of a restorative treatment and test for effectiveness to functionally remineralize artificial lesions in dentin. Materials and methods: Two methods of providing pAsp to standardized artificial lesions during a restorative procedure were applied: (A) pAsp was mixed into commercial RMGI (resin modified glass ionomer) cement formulations and (B) pAsp was added at high concentration (25mg/ml) in solution to rehydrate lesions before restoring with a RMGI cement. All specimens were immersed in simulated body fluid for two weeks to allow for remineralization and then analyzed for dehydration shrinkage, integrity of cement-dentin interface, degree of mineralization, and changes in the nanomechanical profile (E-modulus) across the lesion. Results: After the remineralization treatment, lesion shrinkage was significantly reduced for all treatment groups compared to demineralized samples. Pores developed in RMGI when pAsp was added. A thin layer at the dentin-cement interface, rich in polymer formed possibly from a reaction between pAsp and the RMGI. When analyzed by SEM under vacuum, most lesions delaminated from the cement interface. EDS-analysis showed some but not full recovery of calcium and phosphorous levels for treatment groups that involved pAsp. Nanoindentations placed across the interface indicated improvement for RMGI containing 40% pAsp, and were significantly elevated when lesions were rehydrated with pAsp before being restored with RMGI. In particular the most demineralized outer zone recovered substantially in the elastic modulus, suggesting that functional remineralization has been initiated by pAsp delivery upon rehydration of air-dried demineralized dentin. In contrast, the effectiveness of the RMGI on functional remineralization of dentin was minimal when pAsp was absent. Significance: Incorporation of pAsp into restorative treatments using RMGIs promises to be a feasible way to induce the PILP-mineralization process in a clinical setting and to repair the structure and properties of dentin damaged by the caries process.


Mechanoresponsive stem cells acquire neural crest fate in jaw regeneration

Ransom, Ryan C; Carter, Ava C; Salhotra, Ankit; Leavitt, Tripp; Marecic, Owen; Murphy, Matthew P; Lopez, Michael L; Wei, Yuning; Marshall, Clement D; Shen, Ethan Z; Jones, Ruth Ellen; Sharir, Amnon; Klein, Ophir D; Chan, Charles K F; Wan, Derrick C; Chang, Howard Y; Longaker, Michael T

View Abstract
During both embryonic development and adult tissue regeneration, changes in chromatin structure driven by master transcription factors lead to stimulus-responsive transcriptional programs. A thorough understanding of how stem cells in the skeleton interpret mechanical stimuli and enact regeneration would shed light on how forces are transduced to the nucleus in regenerative processes. Here we develop a genetically dissectible mouse model of mandibular distraction osteogenesis-which is a process that is used in humans to correct an undersized lower jaw that involves surgically separating the jaw bone, which elicits new bone growth in the gap. We use this model to show that regions of newly formed bone are clonally derived from stem cells that reside in the skeleton. Using chromatin and transcriptional profiling, we show that these stem-cell populations gain activity within the focal adhesion kinase (FAK) signalling pathway, and that inhibiting FAK abolishes new bone formation. Mechanotransduction via FAK in skeletal stem cells during distraction activates a gene-regulatory program and retrotransposons that are normally active in primitive neural crest cells, from which skeletal stem cells arise during development. This reversion to a developmental state underlies the robust tissue growth that facilitates stem-cell-based regeneration of adult skeletal tissue.

Bioactive wound Closure Devices are highly Demanded

Ha, Pin; Golnazarian, Nicolette K; Soo, Chia; Zheng, Zhong

View Abstract
No abstract available

Identification of the Human Skeletal Stem Cell

Chan, Charles K F; Gulati, Gunsagar S; Sinha, Rahul; Tompkins, Justin Vincent; Lopez, Michael; Carter, Ava C; Ransom, Ryan C; Reinisch, Andreas; Wearda, Taylor; Murphy, Matthew; Brewer, Rachel E; Koepke, Lauren S; Marecic, Owen; Manjunath, Anoop; Seo, Eun Young; Leavitt, Tripp; Lu, Wan-Jin; Nguyen, Allison; Conley, Stephanie D; Salhotra, Ankit; Ambrosi, Thomas H; Borrelli, Mimi R; Siebel, Taylor; Chan, Karen; Schallmoser, Katharina; Seita, Jun; Sahoo, Debashis; Goodnough, Henry; Bishop, Julius; Gardner, Michael; Majeti, Ravindra; Wan, Derrick C; Goodman, Stuart; Weissman, Irving L; Chang, Howard Y; Longaker, Michael T

View Abstract
Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis.

Secretion of Shh by a Neurovascular Bundle Niche Supports Mesenchymal Stem Cell Homeostasis in the Adult Mouse Incisor

Zhao, Hu; Feng, Jifan; Seidel, Kerstin; Shi, Songtao; Klein, Ophir; Sharpe, Paul; Chai, Yang

View Abstract
No abstract available

Dental, Oral, and Craniofacial Regenerative Medicine: Transforming Biotechnologies for Innovating Patient Care

Giannobile, William V; Chai, Yang; Chen, Yong; Healy, Kevin E; Klein, Ophir; Lane, Nancy E; Longaker, Michael T; Lotz, Jeffrey C; Mooney, David J; Sfeir, Charles S; Urata, Mark; Wagner, William R; Wu, Benjamin M; Kohn, David H

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No abstract available

Deferoxamine Preconditioning of Irradiated Tissue Improves Perfusion and Fat Graft Retention

Flacco, John; Chung, Natalie; Blackshear, Charles P; Irizarry, Dre; Momeni, Arash; Lee, Gordon K; Nguyen, Dung; Gurtner, Geoffrey C; Longaker, Michael T; Wan, Derrick C

View Abstract
Background: Radiation therapy is a mainstay in the treatment of many malignancies, but collateral damage to surrounding tissue, with resultant hypovascularity, fibrosis, and atrophy, can be difficult to reconstruct. Fat grafting has been shown to improve the quality of irradiated skin, but volume retention of the graft is significantly decreased. Deferoxamine is a U.S. Food and Drug Administration-approved iron-chelating medication for acute iron intoxication and chronic iron overload that has also been shown to increase angiogenesis. The present study evaluates the effects of deferoxamine treatment on irradiated skin and subsequent fat graft volume retention. Methods: Mice underwent irradiation to the scalp followed by treatment with deferoxamine or saline and perfusion and were analyzed using laser Doppler analysis. Human fat grafts were then placed beneath the scalp and retention was also followed up to 8 weeks radiographically. Finally, histologic evaluation of overlying skin was performed to evaluate the effects of deferoxamine preconditioning. Results: Treatment with deferoxamine resulted in significantly increased perfusion, as demonstrated by laser Doppler analysis and CD31 immunofluorescent staining (p < 0.05). Increased dermal thickness and collagen content secondary to irradiation, however, were not affected by deferoxamine (p > 0.05). Importantly, fat graft volume retention was significantly increased when the irradiated recipient site was preconditioned with deferoxamine (p < 0.05). Conclusions: The authors' results demonstrated increased perfusion with deferoxamine treatment, which was also associated with improved fat graft volume retention. Preconditioning with deferoxamine may thus enhance fat graft outcomes for soft-tissue reconstruction following radiation therapy.


* Calvarial Defects: Cell-Based Reconstructive Strategies in the Murine Model

Murphy, Matthew P; Quarto, Natalina; Longaker, Michael T; Wan, Derrick C

View Abstract
Calvarial defects pose a continued clinical dilemma for reconstruction. Advancements within the fields of stem cell biology and tissue engineering have enabled researchers to develop reconstructive strategies using animal models. We review the utility of various animal models and focus on the mouse, which has aided investigators in understanding cranial development and calvarial bone healing. The murine model has also been used to study regenerative approaches to critical-sized calvarial defects, and we discuss the application of stem cells such as bone marrow-derived mesenchymal stromal cells, adipose-derived stromal cells, muscle-derived stem cells, and pluripotent stem cells to address deficient bone in this animal. Finally, we highlight strategies to manipulate stem cells using various growth factors and inhibitors and ultimately how these factors may prove crucial in future advancements within calvarial reconstruction using native skeletal stem cells.


Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal

Sun, Zhao; Yu, Wenjie; Sanz Navarro, Maria; Sweat, Mason; Eliason, Steven; Sharp, Thad; Liu, Huan; Seidel, Kerstin; Zhang, Li; Moreno, Myriam; Lynch, Thomas; Holton, Nathan E; Rogers, Laura; Neff, Traci; Goodheart, Michael J; Michon, Frederic; Klein, Ophir D; Chai, Yang; Dupuy, Adam; Engelhardt, John F; Chen, Zhi; Amendt, Brad A

View Abstract
Sox2 marks dental epithelial stem cells (DESCs) in both mammals and reptiles, and in this article we demonstrate several Sox2 transcriptional mechanisms that regulate dental stem cell fate and incisor growth. Conditional Sox2 deletion in the oral and dental epithelium results in severe craniofacial defects, including impaired dental stem cell proliferation, arrested incisor development and abnormal molar development. The murine incisor develops initially but is absorbed independently of apoptosis owing to a lack of progenitor cell proliferation and differentiation. Tamoxifen-induced inactivation of Sox2 demonstrates the requirement of Sox2 for maintenance of the DESCs in adult mice. Conditional overexpression of Lef-1 in mice increases DESC proliferation and creates a new labial cervical loop stem cell compartment, which produces rapidly growing long tusk-like incisors, and Lef-1 epithelial overexpression partially rescues the tooth arrest in Sox2 conditional knockout mice. Mechanistically, Pitx2 and Sox2 interact physically and regulate Lef-1, Pitx2 and Sox2 expression during development. Thus, we have uncovered a Pitx2-Sox2-Lef-1 transcriptional mechanism that regulates DESC homeostasis and dental development.


Modulation of noncanonical TGF-β signaling prevents cleft palate in Tgfbr2 mutant mice

Iwata, Jun-ichi; Hacia, Joseph G; Suzuki, Akiko; Sanchez-Lara, Pedro A; Urata, Mark; Chai, Yang

View Abstract
Patients with mutations in either TGF-β receptor type I (TGFBR1) or TGF-β receptor type II (TGFBR2), such as those with Loeys-Dietz syndrome, have craniofacial defects and signs of elevated TGF-β signaling. Similarly, mutations in TGF-β receptor gene family members cause craniofacial deformities, such as cleft palate, in mice. However, it is unknown whether TGF-β ligands are able to elicit signals in Tgfbr2 mutant mice. Here, we show that loss of Tgfbr2 in mouse cranial neural crest cells results in elevated expression of TGF-β2 and TGF-β receptor type III (TβRIII); activation of a TβRI/TβRIII-mediated, SMAD-independent, TRAF6/TAK1/p38 signaling pathway; and defective cell proliferation in the palatal mesenchyme. Strikingly, Tgfb2, Tgfbr1 (also known as Alk5), or Tak1 haploinsufficiency disrupted TβRI/TβRIII-mediated signaling and rescued craniofacial deformities in Tgfbr2 mutant mice, indicating that activation of this noncanonical TGF-β signaling pathway was responsible for craniofacial malformations in Tgfbr2 mutant mice. Thus, modulation of TGF-β signaling may be beneficial for the prevention of congenital craniofacial birth defects.


Epithelial fibroblast growth factor receptor 1 regulates enamel formation

Takamori, K; Hosokawa, R; Xu, X; Deng, X; Bringas Jr, P; Chai, Yang

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The interaction between epithelial and mesenchymal tissues plays a critical role in the development of organs such as teeth, lungs, and hair. During tooth development, fibroblast growth factor (FGF) signaling is critical for regulating reciprocal epithelial and mesenchymal interactions. FGF signaling requires FGF ligands and their receptors (FGFRs). In this study, we investigated the role of epithelial FGF signaling in tooth development, using the Cre-loxp system to create tissue-specific inactivation of Fgfr1 in mice. In K14-Cre;Fgfr1(fl/fl) mice, the apical sides of enamel-secreting ameloblasts failed to adhere properly to each other, although ameloblast differentiation was unaffected at early stages. Prior to eruption, enamel structure was compromised in the K14-Cre;Fgfr1(fl/fl) mice and displayed severe enamel defects that mimic amelogenesis imperfecta (AI), with a rough, irregular enamel surface. These results suggest that there is a cell-autonomous requirement for FGF signaling in the dental epithelium during enamel formation. Loss of Fgfr1 affects ameloblast organization at the enamel-secretory stage and, hence, the formation of enamel.


TGF-beta signaling and aplasia cutis congenita: proposed animal model

Zehnaly, Armen; Hosokawa, Ryoichi; Urata, Mark; Chai, Yang

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TGF-beta plays a role in cell migration, proliferation, and differentiation during embryonic development. This study investigated the effect of neural crest- or mesodermspecific loss of TGF-beta type II receptor in mice. These conditional knockout mice both exhibit skin defects of the skull associated with an underlying bone defect, a phenotype consistent with the human disorder aplasia cutis congenita. The authors suggest that TGF-3 type II receptor gene is a candidate gene for aplasia cutis congenita.