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1.
Front Genome Ed ; 6: 1401163, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38903529

RESUMO

Recent advancements in genome editing techniques, notably CRISPR-Cas9 and TALENs, have marked a transformative era in biomedical research, significantly enhancing our understanding of disease mechanisms and helping develop novel therapies. These technologies have been instrumental in creating precise animal models for use in stem cell research and regenerative medicine. For instance, we have developed a transgenic pig model to enable the investigation of LGR5-expressing cells. The model was designed to induce the expression of H2B-GFP under the regulatory control of the LGR5 promoter via CRISPR/Cas9-mediated gene knock-in. Notably, advancements in stem cell research have identified distinct subpopulations of LGR5-expressing cells within adult human, mouse, and pig tissues. LGR5, a leucine-rich repeat-containing G protein-coupled receptor, enhances WNT signaling and these LGR5+ subpopulations demonstrate varied roles and anatomical distributions, underscoring the necessity for suitable translational models. This transgenic pig model facilitates the tracking of LGR5-expressing cells and has provided valuable insights into the roles of these cells across different tissues and species. For instance, in pulmonary tissue, Lgr5+ cells in mice are predominantly located in alveolar compartments, driving alveolar differentiation of epithelial progenitors via Wnt pathway activation. In contrast, in pigs and humans, these cells are situated in a unique sub-basal position adjacent to the airway epithelium. In fetal stages a pattern of LGR5 expression during lung bud tip formation is evident in humans and pigs but is lacking in mice. Species differences with respect to LGR5 expression have also been observed in the skin, intestines, and cochlea further reinforcing the need for careful selection of appropriate translational animal models. This paper discusses the potential utility of the LGR5+ pig model in exploring the role of LGR5+ cells in tissue development and regeneration with the goal of translating these findings into human and animal clinical applications.

2.
Nat Protoc ; 18(11): 3413-3459, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37735235

RESUMO

Over the past 5 years, our laboratory has systematically developed a structure-guided library approach to evolve new adeno-associated virus (AAV) capsids with altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity. Here, we provide a detailed protocol describing two distinct evolution strategies using structurally divergent AAV serotypes as templates, exemplified by improving CNS gene transfer efficiency in vivo. We outline four major components of our strategy: (i) structure-guided design of AAV capsid libraries, (ii) AAV library production, (iii) library cycling in single versus multiple animal models, followed by (iv) evaluation of lead AAV vector candidates in vivo. The protocol spans ~95 d, excluding gene expression analysis in vivo, and can vary depending on user experience, resources and experimental design. A distinguishing attribute of the current protocol is the focus on providing biomedical researchers with 3D structural information to guide evolution of precise 'hotspots' on AAV capsids. Furthermore, the protocol outlines two distinct methods for AAV library evolution consisting of adenovirus-enabled infectious cycling in a single species and noninfectious cycling in a cross-species manner. Notably, our workflow can be seamlessly merged with other RNA transcript-based library strategies and tailored for tissue-specific capsid selection. Overall, the procedures outlined herein can be adapted to expand the AAV vector toolkit for genetic manipulation of animal models and development of human gene therapies.


Assuntos
Capsídeo , Dependovirus , Animais , Humanos , Capsídeo/química , Dependovirus/genética , Terapia Genética/métodos , Técnicas de Transferência de Genes , Proteínas do Capsídeo/genética , Vetores Genéticos , Transdução Genética
4.
NPJ Regen Med ; 8(1): 40, 2023 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-37528116

RESUMO

A network of co-hepato/pancreatic stem/progenitors exists in pigs and humans in Brunner's Glands in the submucosa of the duodenum, in peribiliary glands (PBGs) of intrahepatic and extrahepatic biliary trees, and in pancreatic duct glands (PDGs) of intrapancreatic biliary trees, collectively supporting hepatic and pancreatic regeneration postnatally. The network is found in humans postnatally throughout life and, so far, has been demonstrated in pigs postnatally at least through to young adulthood. These stem/progenitors in vivo in pigs are in highest numbers in Brunner's Glands and in PDGs nearest the duodenum, and in humans are in Brunner's Glands and in PBGs in the hepato/pancreatic common duct, a duct missing postnatally in pigs. Elsewhere in PDGs in pigs and in all PDGs in humans are only committed unipotent or bipotent progenitors. Stem/progenitors have genetic signatures in liver/pancreas-related RNA-seq data based on correlation, hierarchical clustering, differential gene expression and principal component analyses (PCA). Gene expression includes representative traits of pluripotency genes (SOX2, OCT4), endodermal transcription factors (e.g. SOX9, SOX17, PDX1), other stem cell traits (e.g. NCAM, CD44, sodium iodide symporter or NIS), and proliferation biomarkers (Ki67). Hepato/pancreatic multipotentiality was demonstrated by the stem/progenitors' responses under distinct ex vivo conditions or in vivo when patch grafted as organoids onto the liver versus the pancreas. Therefore, pigs are logical hosts for translational/preclinical studies for cell therapies with these stem/progenitors for hepatic and pancreatic dysfunctions.

5.
FASEB J ; 37(6): e22975, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37159340

RESUMO

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/ß-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.


Assuntos
Intestinos , Humanos , Suínos , Animais , Camundongos , Hibridização in Situ Fluorescente , Células-Tronco , Íleo , Colo , Proteínas de Fluorescência Verde/genética , Receptores Acoplados a Proteínas G/genética
6.
Amino Acids ; 55(6): 743-756, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37036518

RESUMO

Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr2) isolated from Day-12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via activation of mechanistic target of rapamycin (MTOR) cell signaling pathway in pTr2 cells. Porcine ADM at 10-7 M stimulated (P < 0.05) pTr2 cell proliferation, migration and adhesion by 1.4-, 1.5- and 1.2-folds, respectively. These ADM-induced effects were abrogated (P < 0.05) by siRNA-mediated knockdown of ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL), as well as by rapamycin, the inhibitor of MTOR. Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph acts on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses.


Assuntos
Adrenomedulina , Proteínas Proto-Oncogênicas c-akt , Gravidez , Feminino , Suínos , Animais , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adrenomedulina/farmacologia , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proliferação de Células , Sirolimo/farmacologia
7.
Nat Commun ; 13(1): 5947, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36210364

RESUMO

Recombinant adeno-associated viral (AAV) vectors are a promising gene delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Effective clinical translation is confounded, at least in part, by differences in AAV biology across animal species. Here, we tackle this challenge by sequentially evolving AAV capsid libraries in mice, pigs and macaques. We discover a highly potent, cross-species compatible variant (AAV.cc47) that shows improved attributes benchmarked against AAV serotype 9 as evidenced by robust reporter and therapeutic gene expression, Cre recombination and CRISPR genome editing in normal and diseased mouse models. Enhanced transduction efficiency of AAV.cc47 vectors is further corroborated in macaques and pigs, providing a strong rationale for potential clinical translation into human gene therapies. We envision that ccAAV vectors may not only improve predictive modeling in preclinical studies, but also clinical translatability by broadening the therapeutic window of AAV based gene therapies.


Assuntos
Dependovirus , Edição de Genes , Animais , Dependovirus/metabolismo , Terapia Genética , Vetores Genéticos/genética , Humanos , Macaca/genética , Camundongos , Suínos , Transdução Genética
8.
Front Vet Sci ; 9: 965316, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36311661

RESUMO

Mice with severe combined immunodeficiency are commonly used as hosts of human cells. Size, longevity, and physiology, however, limit the extent to which immunodeficient mice can model human systems. To address these limitations, we generated RAG2 -/- IL2RG y/- immunodeficient pigs and demonstrate successful engraftment of SLA mismatched allogeneic D42 fetal liver cells, tagged with pH2B-eGFP, and human CD34+ hematopoietic stem cells after in utero cell transplantation. Following intrauterine injection at day 42-45 of gestation, fetuses were allowed to gestate to term and analyzed postnatally for the presence of pig (allogeneic) and human (xenogeneic) B cells, T-cells and NK cells in peripheral blood and other lymphoid tissues. Engraftment of allogeneic hematopoietic cells was detected based on co-expression of pH2B-eGFP and various markers of differentiation. Analysis of spleen revealed robust generation and engraftment of pH2B-eGFP mature B cells (and IgH recombination) and mature T-cells (and TCR-ß recombination), T helper (CD3+CD4+) and T cytotoxic (CD3+CD8+) cells. The thymus revealed engraftment of pH2B-eGFP double negative precursors (CD4-CD8-) as well as double positive (CD4+, CD8+) precursors and single positive T-cells. After intrauterine administration of human CD34+ hematopoietic stem cells, analysis of peripheral blood and lymphoid tissues revealed the presence of human T-cells (CD3+CD4+ and CD3+CD8+) but no detectable B cells or NK cells. The frequency of human CD45+ cells in the circulation decreased rapidly and were undetectable within 2 weeks of age. The frequency of human CD45+ cells in the spleen also decreased rapidly, becoming undetectable at 3 weeks. In contrast, human CD45+CD3+ T-cells comprised >70% of cells in the pig thymus at birth and persisted at the same frequency at 3 weeks. Most human CD3+ cells in the pig's thymus expressed CD4 or CD8, but few cells were double positive (CD4+ CD8+). In addition, human CD3+ cells in the pig thymus contained human T-cell excision circles (TREC), suggesting de novo development. Our data shows that the pig thymus provides a microenvironment conducive to engraftment, survival and development of human T-cells and provide evidence that the developing T-cell compartment can be populated to a significant extent by human cells in large animals.

9.
Tissue Eng Part A ; 28(19-20): 833-844, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35925753

RESUMO

Current cellular hydrogel-based skin grafts composed of human dermal fibroblasts and a hydrogel scaffold tend to minimize contraction of full-thickness skin wounds and support skin regeneration. However, there has been no comparison between the sources of the dermal fibroblast used. Products using human adult or neonatal foreskin dermal fibroblasts are often expanded in vitro and used after multiple passages without a clear understanding of the effects of this initial production step on the quality and reproducibility of the cellular behavior. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may influence cellular properties and contractile behavior in a fibroblast-populated collagen matrix. Using porcine skin as a model based on its similarity to human skin in structure and wound healing properties, we isolated porcine dermal fibroblasts of three different donor ages for use in a 2D proliferation assay and in a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1 in vitro. However, after five passages in culture, there was no difference in contractility between ages. These results show how cellular responses in a hydrogel scaffold differ based on donor age and time in culture in vitro, and suggest that consistency in the cellular component of bioengineered skin products could be beneficial in the biomanufacturing of consistent, reliable skin grafts and graft in vivo models. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding. Impact statement Little is known about the impact of donor cell age and time in culture on the contraction of cellular, hydrogel-based skin grafts. These results show how cellular phenotypes of porcine fibroblasts differ based on donor age and time in culture. This information is beneficial when addressing important inconsistencies in biomanufacturing of bioengineered skin grafts and in vitro models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. The information from this study can be extrapolated to future in vivo studies using human dermal fibroblasts in an in vivo model to help determine the best donor age and time in culture for optimal wound healing outcomes or more reproducible in vitro testing constructs.


Assuntos
Hidrogéis , Cicatrização , Adulto , Recém-Nascido , Humanos , Suínos , Animais , Hidrogéis/farmacologia , Reprodutibilidade dos Testes , Colágeno/química , Fibroblastos
10.
iScience ; 25(8): 104695, 2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-35865132

RESUMO

Over 11% of the world's population experience hearing loss. Although there are promising studies to restore hearing in rodent models, the size, ontogeny, genetics, and frequency range of hearing of most rodents' cochlea do not match that of humans. The porcine cochlea can bridge this gap as it shares many anatomical, physiological, and genetic similarities with its human counterpart. Here, we provide a detailed methodology to process and image the porcine cochlea in 3D using tissue clearing and light-sheet microscopy. The resulting 3D images can be employed to compare cochleae across different ages and conditions, investigate the ontogeny of cochlear cytoarchitecture, and produce quantitative expression maps of LGR5, a marker of cochlear progenitors in mice. These data reveal that hair cell organization, inner ear morphology, cellular cartography in the organ of Corti, and spatiotemporal expression of LGR5 are dynamic over developmental stages in a pattern not previously documented.

11.
Sci Rep ; 12(1): 9104, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35650234

RESUMO

Hair follicle stem cells are key for driving growth and homeostasis of the hair follicle niche, have remarkable regenerative capacity throughout hair cycling, and display fate plasticity during cutaneous wound healing. Due to the need for a transgenic reporter, essentially all observations related to LGR5-expressing hair follicle stem cells have been generated using transgenic mice, which have significant differences in anatomy and physiology from the human. Using a transgenic pig model, a widely accepted model for human skin and human skin repair, we demonstrate that LGR5 is a marker of hair follicle stem cells across species in homeostasis and development. We also report the strong similarities and important differences in expression patterns, gene expression profiles, and developmental processes between species. This information is important for understanding the fundamental differences and similarities across species, and ultimately improving human hair follicle regeneration, cutaneous wound healing, and skin cancer treatment.


Assuntos
Folículo Piloso , Células-Tronco , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Folículo Piloso/metabolismo , Humanos , Morfogênese , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Pele , Células-Tronco/metabolismo , Suínos
12.
J Athl Train ; 57(9-10): 978-989, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34964874

RESUMO

CONTEXT: Pediatric anterior cruciate ligament (ACL) injury rates are increasing and are highest in female adolescents. Complete ACL tears are typically surgically reconstructed, but few guidelines and very limited data exist regarding the need for surgical reconstruction or rehabilitation for partial ACL tears in skeletally immature patients. OBJECTIVE: To evaluate the effects of partial (anteromedial bundle) and complete ACL transection on joint laxity and tissue forces under anterior and rotational loads in male and female stifle joints throughout skeletal growth in the porcine model. DESIGN: Descriptive laboratory study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: We studied 60 male and female Yorkshire crossbreed pigs aged 1.5, 3, 4.5, 6, and 18 months (n = 6 pigs per age per sex). MAIN OUTCOME MEASURE(S): Joint laxity was measured in intact, anteromedial bundle-transected, and ACL-transected joints under applied anterior-posterior drawer and varus-valgus torque using a robotic testing system. Loading of the soft tissues in the stifle joint was measured under each condition. RESULTS: Anterior-posterior joint laxity increased by 13% to 50% (P < .05) after anteromedial bundle transection and 75% to 178% (P < .05) after ACL transection. Destabilization after anteromedial bundle transection increased with age (P < .05) and was greater in late female than late male adolescents (P < .05). In anteromedial bundle-transected joints, the posterolateral bundle resisted the anterior load. In ACL-transected joints, the medial collateral ligament (MCL) contribution was largest, followed by the medial meniscus. The MCL contribution was larger and the medial meniscus contribution was smaller in male versus female specimens. CONCLUSIONS: Partial ACL transection resulted in moderate increases in joint laxity, with the remaining bundle performing the primary ACL function. Destabilization due to partial ACL transection (anteromedial bundle) was largest in late adolescent joints, indicating that operative treatment should be considered in active, late-adolescent patients with this injury. Increased forces on the MCL and medial meniscus after ACL transection suggested that rehabilitation protocols may need to focus on protecting these tissues.


Assuntos
Lesões do Ligamento Cruzado Anterior , Instabilidade Articular , Masculino , Feminino , Humanos , Animais , Suínos , Lesões do Ligamento Cruzado Anterior/cirurgia , Ligamento Cruzado Anterior/cirurgia , Ligamento Cruzado Anterior/fisiologia , Fenômenos Biomecânicos , Articulação do Joelho , Cadáver
13.
J Orthop Res ; 40(8): 1853-1864, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-34751996

RESUMO

Pediatric anterior cruciate ligament (ACL) injuries are on the rise, and females experience higher ACL injury risk than males during adolescence. Studies in skeletally immature patients indicate differences in ACL size and joint laxity between males and females after the onset of adolescence. However, functional data regarding the ACL and its anteromedial and posterolateral bundles in the pediatric population remain rare. Therefore, this study uses a porcine model to investigate the sex-specific morphology and biomechanics of the ACL and its bundles throughout skeletal growth. Hind limbs from male and female Yorkshire pigs aged early youth to late adolescence were imaged using magnetic resonance imaging to measure the size and orientation of the ACL and its bundles, then biomechanically tested under anterior-posterior drawer using a robotic testing system. Joint laxity decreased (p < 0.001) while joint stiffness increased (p < 0.001) throughout skeletal growth in both sexes. The ACL was the primary stabilizer against anterior tibial loading, while the functional role of the anteromedial bundle increased with age (p < 0.001), with an earlier increase in males. ACL and posterolateral bundle cross-sectional area and ACL and anteromedial bundle length were larger in males than females during adolescence (p < 0.01 for all), while ACL and bundle sagittal angle remained similar between sexes. Additionally, in situ ACL stiffness versus cross-sectional area regressions were significant across skeletal growth (r2 = 0.75, p < 0.001 in males and r2 = 0.64, p < 0.001 in females), but not within age groups. This study has implications for age and sex-specific surgical intervention strategies and suggests the need for human studies.


Assuntos
Lesões do Ligamento Cruzado Anterior , Instabilidade Articular , Adolescente , Idoso , Animais , Ligamento Cruzado Anterior , Fenômenos Biomecânicos , Criança , Feminino , Humanos , Articulação do Joelho , Masculino , Suínos , Tíbia
14.
ACS Biomater Sci Eng ; 7(11): 5175-5188, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34597013

RESUMO

Gelatin methacryloyl (GelMA) hydrogels have emerged as promising and versatile biomaterial matrices with applications spanning drug delivery, disease modeling, and tissue engineering and regenerative medicine. GelMA exhibits reversible thermal cross-linking at temperatures below 37 °C due to the entanglement of constitutive polymeric chains, and subsequent ultraviolet (UV) photo-cross-linking can covalently bind neighboring chains to create irreversibly cross-linked hydrogels. However, how these cross-linking modalities interact and can be modulated during biofabrication to control the structural and functional characteristics of this versatile biomaterial is not well explored yet. Accordingly, this work characterizes the effects of synergistic thermal and photo-cross-linking as a function of GelMA solution temperature and UV photo-cross-linking duration during biofabrication on the hydrogels' stiffness, microstructure, proteolytic degradation, and responses of NIH 3T3 and human adipose-derived stem cells (hASC). Smaller pore size, lower degradation rate, and increased stiffness are reported in hydrogels processed at lower temperature or prolonged UV exposure. In hydrogels with low stiffness, the cells were found to shear the matrix and cluster into microspheroids, while poor cell attachment was noted in high stiffness hydrogels. In hydrogels with moderate stiffness, ones processed at lower temperature demonstrated better shape fidelity and cell proliferation over time. Analysis of gene expression of hASC encapsulated within the hydrogels showed that, while the GelMA matrix assisted in maintenance of stem cell phenotype (CD44), a higher matrix stiffness resulted in higher pro-inflammatory marker (ICAM1) and markers for cell-matrix interaction (ITGA1 and ITGA10). Analysis of constructs with ultrasonically patterned hASC showed that hydrogels processed at higher temperature possessed lower structural fidelity but resulted in more cell elongation and greater anisotropy over time. These findings demonstrate the significant impact of GelMA material formulation and processing conditions on the structural and functional properties of the hydrogels. The understanding of these material-process-structure-function interactions is critical toward optimizing the functional properties of GelMA hydrogels for different targeted applications.


Assuntos
Gelatina , Hidrogéis , Materiais Biocompatíveis , Humanos , Metacrilatos , Engenharia Tecidual
15.
CRISPR J ; 3(5): 409-418, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33095051

RESUMO

The use of CRISPR-Cas and RNA-guided endonucleases has drastically changed research strategies for understanding and exploiting gene function, particularly for the generation of gene-edited animal models. This has resulted in an explosion in the number of gene-edited species, including highly biomedically relevant pig models. However, even with error-free DNA insertion or deletion, edited genes are occasionally not expressed and/or translated as expected. Therefore, there is a need to validate the expression outcomes gene modifications in vitro before investing in the costly generation of a gene-edited animal. Unfortunately, many gene targets are tissue specific and/or not expressed in cultured primary cells, making validation difficult without generating an animal. In this study, using pigs as a proof of concept, we show that CRISPR-dCas9 transcriptional activators can be used to validate functional transgene insertion in nonexpressing easily cultured cells such as fibroblasts. This is a tool that can be used across disciplines and animal species to save time and resources by verifying expected outcomes of gene edits before generating live animals.


Assuntos
Animais Geneticamente Modificados/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , RNA Guia de Cinetoplastídeos/metabolismo , Transativadores/metabolismo , Transgenes , Animais , Proteína 9 Associada à CRISPR/genética , Células Cultivadas , Expressão Gênica , Técnicas de Transferência Nuclear , RNA Guia de Cinetoplastídeos/genética , Suínos , Transativadores/genética
17.
J Biomech ; 101: 109636, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32005549

RESUMO

Anterior cruciate ligament (ACL) injuries are increasingly common in the skeletally immature population. As such there is a need to increase our understanding of the biomechanical function of the joint following partial and complete ACL injury during skeletal growth. In this work, we aimed to assess changes in knee kinematics and loading of the remaining soft tissues following both partial and complete ACL injury in a porcine model. To do so, we applied anterior-posterior tibial loads and varus-valgus moments to stifle joints of female pigs ranging from early juvenile to late adolescent ages and assessed both kinematics and in-situ loads carried in the bundles of the ACL and other soft tissues including the collateral ligaments and the menisci. Partial ACL injury led to increased anterior tibial translation only in late adolescence and small increases in varus-valgus rotation at all ages. Complete ACL injury led to substantial increases in translation and rotation at all ages. At all ages, the medial collateral ligament and the medial meniscus combined to resist the majority of applied anterior tibial load following complete ACL transection. Across all ages and flexion angles, the contribution of the MCL ranged from 45 to 90% of the anterior load and the contribution of the medial meniscus ranged from 14 to 35% of the anterior load. These findings add to our current understanding of age-specific functional properties of both healthy and injured knees during skeletal growth.


Assuntos
Lesões do Ligamento Cruzado Anterior/complicações , Instabilidade Articular/complicações , Instabilidade Articular/fisiopatologia , Joelho de Quadrúpedes/crescimento & desenvolvimento , Joelho de Quadrúpedes/fisiopatologia , Animais , Fenômenos Biomecânicos , Feminino , Rotação , Suínos
18.
PLoS One ; 14(10): e0219637, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31644571

RESUMO

Prior studies have analyzed growth of musculoskeletal tissues between species or across body segments; however, little research has assessed the differences in similar tissues within a single joint. Here we studied changes in the length and cross-sectional area of four ligaments and tendons, (anterior cruciate ligament, patellar tendon, medial collateral ligament, lateral collateral ligament) in the tibiofemoral joint of female Yorkshire pigs through high-field magnetic resonance imaging throughout growth. Tissue lengths increased by 4- to 5-fold from birth to late adolescence across the tissues while tissue cross-sectional area increased by 10-20-fold. The anterior cruciate ligament and lateral collateral ligament showed allometric growth favoring change in length over change in cross-sectional area while the patellar tendon and medial collateral ligament grow in an isometric manner. Additionally, changes in the length and cross-sectional area of the anterior cruciate ligament did not increase as much as in the other ligaments and tendon of interest. Overall, these findings suggest that musculoskeletal soft tissue morphometry can vary within tissues of similar structure and within a single joint during post-natal growth.


Assuntos
Ligamento Cruzado Anterior , Articulação do Joelho , Imageamento por Ressonância Magnética , Ligamento Patelar , Animais , Ligamento Cruzado Anterior/diagnóstico por imagem , Ligamento Cruzado Anterior/crescimento & desenvolvimento , Feminino , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/crescimento & desenvolvimento , Especificidade de Órgãos , Ligamento Patelar/diagnóstico por imagem , Ligamento Patelar/crescimento & desenvolvimento , Suínos
19.
J Biomech Eng ; 141(12)2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31513698

RESUMO

Partial and complete anterior cruciate ligament (ACL) injuries occur in both pediatric and adult populations and can result in loss of joint stability and function. The sigmoidal shape of knee joint function (load-translation curve) under applied loads includes a low-load region (described by slack length) followed by a high-load region (described by stiffness). However, the impact of age and injury on these parameters is not fully understood. The current objective was to measure the effects of age and injury on the shape of joint function in a porcine model. In response to an applied anterior-posterior tibial load, in situ slack did not change (p > 0.05), despite sevenfold increases in joint size with increasing age. Joint stiffness increased from an average of 10 N/mm in early youth to 47 N/mm in late adolescence (p < 0.05). In situ ACL stiffness increased similarly, and changes in in situ joint stiffness and ACL stiffness were highly correlated across ages. With complete ACL injury, in situ slack length increased by twofold to fourfold and in situ stiffness decreased threefold to fourfold across ages (p < 0.05). Partial ACL injury resulted in less dramatic, but statistically significant, increases in joint slack and significant decreases in in situ joint stiffness in the adolescent age groups (p < 0.05). This work furthers our understanding of the interaction between joint biomechanics and ACL function throughout growth and the impact of ACL injury in the skeletally immature joint.

20.
Clin Orthop Relat Res ; 477(9): 2161-2174, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31373947

RESUMO

BACKGROUND: ACL injuries are becoming increasingly common in children and adolescents, but little is known regarding age-specific ACL function in these patients. To improve our understanding of changes in musculoskeletal tissues during growth and given the limited availability of pediatric human cadaveric specimens, tissue structure and function can be assessed in large animal models, such as the pig. QUESTIONS/PURPOSES: Using cadaveric porcine specimens ranging throughout skeletal growth, we aimed to assess age-dependent changes in (1) joint kinematics under applied AP loads and varus-valgus moments, (2) biomechanical function of the ACL under the same loads, (3) the relative biomechanical function of the anteromedial and posterolateral bundles of the ACL; and (4) size and orientation of the anteromedial and posterolateral bundles. METHODS: Stifle joints (analogous to the human knee) were collected from female Yorkshire crossbreed pigs at five ages ranging from early youth to late adolescence (1.5, 3, 4.5, 6, and 18 months; n = 6 pigs per age group, 30 total), and MRIs were performed. A robotic testing system was used to determine joint kinematics (AP tibial translation and varus-valgus rotation) and in situ forces in the ACL and its bundles in response to applied anterior tibial loads and varus-valgus moments. To see if morphological changes to the ACL compared with biomechanical changes, ACL and bundle cross-sectional area, length, and orientation were calculated from MR images. RESULTS: Joint kinematics decreased with increasing age. Normalized AP tibial translation decreased by 44% from 1.5 months (0.34 ± 0.08) to 18 months (0.19 ± 0.02) at 60° of flexion (p < 0.001) and varus-valgus rotation decreased from 25° ± 2° at 1.5 months to 6° ± 2° at 18 months (p < 0.001). The ACL provided the majority of the resistance to anterior tibial loading at all age groups (75% to 111% of the applied anterior force; p = 0.630 between ages). Anteromedial and posterolateral bundle function in response to anterior loading and varus torque were similar in pigs of young ages. During adolescence (4.5 to 18 months), the in situ force carried by the anteromedial bundle increased relative to that carried by the posterolateral bundle, shifting from 59% ± 22% at 4.5 months to 92% ± 12% at 18 months (data for 60° of flexion, p < 0.001 between 4.5 and 18 months). The cross-sectional area of the anteromedial bundle increased by 30 mm throughout growth from 1.5 months (5 ± 2 mm) through 18 months (35 ± 8 mm; p < 0.001 between 1.5 and 18 months), while the cross-sectional area of the posterolateral bundle increased by 12 mm from 1.5 months (7 ± 2 mm) to 4.5 months (19 ± 5 mm; p = 0.004 between 1.5 and 4.5 months), with no further growth (17 ± 7 mm at 18 months; p = 0.999 between 4.5 and 18 months). However, changes in length and orientation were similar between the bundles. CONCLUSION: We showed that the stifle joint (knee equivalent) in the pig has greater translational and rotational laxity in early youth (1.5 to 3 months) compared with adolescence (4.5 to 18 months), that the ACL functions as a primary stabilizer throughout growth, and that the relative biomechanical function and size of the anteromedial and posterolateral bundles change differently with growth. CLINICAL RELEVANCE: Given the large effects observed here, the age- and bundle-specific function, size, and orientation of the ACL may need to be considered regarding surgical timing, graft selection, and graft placement. In addition, the findings of this study will be used to motivate pre-clinical studies on the impact of partial and complete ACL injuries during skeletal growth.


Assuntos
Envelhecimento/fisiologia , Ligamento Cruzado Anterior/fisiologia , Desenvolvimento Musculoesquelético/fisiologia , Amplitude de Movimento Articular/fisiologia , Animais , Fenômenos Biomecânicos , Cadáver , Modelos Animais , Rotação , Suínos , Tíbia/fisiologia , Torque
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