RESUMO
Distraction osteogenesis (DO) is used for skeletal defects; however, up to 50% of cases exhibit complications. Previous mouse models of long bone DO have been anecdotally hampered by postoperative complications, expense, and availability. To improve clinical techniques, cost-effective, reliable animal models are needed. Our focus was to develop a new mouse tibial distractor, hypothesized to result in successful, complication-free DO. Methods: A lightweight tibial distractor was developed using CAD and 3D printing. The device was fixed to the tibia of C57Bl/6J mice prior to osteotomy. Postoperatively, mice underwent 5 days latency, 10 days distraction (0.15 mm every 12 hours), and 28 days consolidation. Bone regeneration was examined on postoperative day 43 using micro-computed tomography (µCT) and Movat's modified pentachrome staining on histology (mineralized volume fraction and pixels, respectively). Costs were recorded. We compared cohorts of 11 mice undergoing sham, DO, or acute lengthening (distractor acutely lengthened 3.0 mm). Results: The histological bone regenerate was significantly increased in DO (1,879,257 ± 155,415 pixels) compared to acute lengthening (32847 ± 1589 pixels) (P < 0.0001). The mineralized volume fraction (bone/total tissue volume) of the regenerate was significantly increased in DO (0.9 ± 0.1) compared to acute lengthening (0.7 ± 0.1) (P < 0.001). There was no significant difference in bone regenerate between DO and sham. The distractor was relatively low cost ($11), with no complications. Conclusions: Histology and µCT analysis confirmed that the proposed tibial DO model resulted in successful bone formation. Our model is cost-effective and reproducible, enabling implementation in genetically dissectible transgenic mice.
RESUMO
Cancer-associated fibroblasts (CAFs) are integral to the solid tumor microenvironment. CAFs were once thought to be a relatively uniform population of matrix-producing cells, but single-cell RNA sequencing has revealed diverse CAF phenotypes. Here, we further probed CAF heterogeneity with a comprehensive multiomics approach. Using paired, same-cell chromatin accessibility and transcriptome analysis, we provided an integrated analysis of CAF subpopulations over a complex spatial transcriptomic and proteomic landscape to identify three superclusters: steady state-like (SSL), mechanoresponsive (MR), and immunomodulatory (IM) CAFs. These superclusters are recapitulated across multiple tissue types and species. Selective disruption of underlying mechanical force or immune checkpoint inhibition therapy results in shifts in CAF subpopulation distributions and affected tumor growth. As such, the balance among CAF superclusters may have considerable translational implications. Collectively, this research expands our understanding of CAF biology, identifying regulatory pathways in CAF differentiation and elucidating therapeutic targets in a species- and tumor-agnostic manner.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias , Humanos , Fibroblastos Associados a Câncer/patologia , Proteômica , Microambiente Tumoral/genética , Fenótipo , Neoplasias/genética , Neoplasias/patologiaRESUMO
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.
Assuntos
Suturas Cranianas/metabolismo , Craniossinostoses/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Células-Tronco/metabolismo , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Suturas Cranianas/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Sistema Musculoesquelético/citologia , Sistema Musculoesquelético/metabolismo , Células-Tronco/citologia , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Via de Sinalização Wnt/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismoRESUMO
BACKGROUND: Bone retains regenerative potential into adulthood, and surgeons harness this plasticity during distraction osteogenesis. The underlying biology governing bone development, repair, and regeneration is divergent between the craniofacial and appendicular skeleton. Each type of bone formation is characterized by unique molecular signaling and cellular behavior. Recent discoveries have elucidated the cellular and genetic processes underlying skeletal development and regeneration, providing an opportunity to couple biological and clinical knowledge to improve patient care. METHODS: A comprehensive literature review of basic and clinical literature regarding craniofacial and long bone development, regeneration, and distraction osteogenesis was performed. RESULTS: The current understanding in craniofacial and long bone development and regeneration is discussed, and clinical considerations for the respective distraction osteogenesis procedures are presented. CONCLUSIONS: Distraction osteogenesis is a powerful tool to regenerate bone and thus address a number of craniofacial and appendicular skeletal deficiencies. The molecular mechanisms underlying bone regeneration, however, remain elusive. Recent work has determined that embryologic morphogen gradients constitute important signals during regeneration. In addition, striking discoveries have illuminated the cellular processes underlying mandibular regeneration during distraction osteogenesis, showing that skeletal stem cells reactivate embryologic neural crest transcriptomic processes to carry out bone formation during regeneration. Furthermore, innovative adjuvant therapies to complement distraction osteogenesis use biological processes active in embryogenesis and regeneration. Additional research is needed to further characterize the underlying cellular mechanisms responsible for improved bone formation through adjuvant therapies and the role skeletal stem cells play during regeneration.
Assuntos
Doenças Ósseas/cirurgia , Regeneração Óssea , Osteogênese por Distração , Osteogênese , Animais , Doenças Ósseas/fisiopatologia , Ossos Faciais/anormalidades , Ossos Faciais/fisiologia , Ossos Faciais/cirurgia , Humanos , Modelos Animais , Esqueleto/fisiologia , Esqueleto/cirurgia , Crânio/fisiologia , Crânio/cirurgiaRESUMO
Fibroblast heterogeneity has been shown within the unwounded mouse dorsal dermis, with fibroblast subpopulations being identified according to anatomical location and embryonic lineage. Using lineage tracing, we demonstrate that paired related homeobox 1 (Prrx1)-expressing fibroblasts are responsible for acute and chronic fibroses in the ventral dermis. Single-cell transcriptomics further corroborated the inherent fibrotic characteristics of Prrx1 fibroblasts during wound repair. In summary, we identify and characterize a fibroblast subpopulation in the mouse ventral dermis with intrinsic scar-forming potential.
Assuntos
Derme/metabolismo , Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Animais , Humanos , CamundongosRESUMO
Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.
Assuntos
Aderências Teciduais/metabolismo , Aderências Teciduais/patologia , Animais , Benzofenonas/farmacologia , Sistemas CRISPR-Cas , Células Cultivadas , Doxiciclina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Imunofluorescência , Gastroenteropatias/metabolismo , Gastroenteropatias/patologia , Humanos , Imuno-Histoquímica , Isoxazóis/farmacologia , Lipossomos/metabolismo , Camundongos , Células NIH 3T3 , Parabiose , RNA Mensageiro/metabolismo , Tamoxifeno/farmacologiaRESUMO
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.
Assuntos
Regeneração Óssea/fisiologia , Mandíbula/citologia , Mandíbula/metabolismo , Traumatismos Mandibulares/metabolismo , Neurônios/metabolismo , Células de Schwann/metabolismo , Células-Tronco/metabolismo , Animais , Regeneração Óssea/efeitos dos fármacos , Denervação , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Mandíbula/crescimento & desenvolvimento , Mandíbula/patologia , Traumatismos Mandibulares/tratamento farmacológico , Nervo Mandibular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Comunicação Parácrina/fisiologia , Traumatismos dos Nervos Periféricos/metabolismo , Fator de Crescimento Derivado de Plaquetas/uso terapêutico , Células de Schwann/citologia , Cicatrização/fisiologiaRESUMO
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. VIDEO ABSTRACT.
Assuntos
Desenvolvimento Ósseo/fisiologia , Osso e Ossos/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Osso e Ossos/metabolismo , Cartilagem/citologia , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Análise de Célula Única/métodos , Células-Tronco/citologia , Células Estromais/citologia , Transcriptoma/genéticaRESUMO
Targeted genetic dissection of tissues to identify precise cell populations has vast biological and therapeutic applications. Here we develop an approach, through the packaging and delivery of 4-hydroxytamoxifen liposomes (LiTMX), that enables localized induction of CreERT2 recombinase in mice. Our method permits precise, in vivo, tissue-specific clonal analysis with both spatial and temporal control. This technology is effective using mice with both specific and ubiquitous Cre drivers in a variety of tissue types, under conditions of homeostasis and post-injury repair, and is highly efficient for lineage tracing and genetic analysis. This methodology is directly and immediately applicable to the developmental biology, stem cell biology and regenerative medicine, and cancer biology fields.
Assuntos
Linhagem da Célula , Lipossomos/química , Tamoxifeno/análogos & derivados , Tecido Adiposo/metabolismo , Animais , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Modelos Animais de Doenças , Homeostase , Injeções Intraperitoneais , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Recombinases , Medicina Regenerativa , Pele/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Tamoxifeno/química , CicatrizaçãoRESUMO
There are limited methods available to study skeletal stem, progenitor, and progeny cell activity in normal and diseased contexts. Most protocols for skeletal stem cell isolation are based on the extent to which cells adhere to plastic or whether they express a limited repertoire of surface markers. Here, we describe a flow cytometry-based approach that does not require in vitro selection and that uses eight surface markers to distinguish and isolate mouse skeletal stem cells (mSSCs); bone, cartilage, and stromal progenitors (mBCSPs); and five downstream differentiated subtypes, including chondroprogenitors, two types of osteoprogenitors, and two types of hematopoiesis-supportive stroma. We provide instructions for the optimal mechanical and chemical digestion of bone and bone marrow, as well as the subsequent flow-cytometry-activated cell sorting (FACS) gating schemes required to maximally yield viable skeletal-lineage cells. We also describe a methodology for renal subcapsular transplantation and in vitro colony-formation assays on the isolated mSSCs. The isolation of mSSCs can be completed in 9 h, with at least 1 h more required for transplantation. Experience with flow cytometry and mouse surgical procedures is recommended before attempting the protocol. Our system has wide applications and has already been used to study skeletal response to fracture, diabetes, and osteoarthritis, as well as hematopoietic stem cell-niche interactions in the bone marrow.