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Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering.

ABSTRACT: Autogenous tissue grafting remains the gold standard in the treatment of critical sized bone and certain cartilage defects, while the translation of tissue engineered osteogenesis or chondrogenesis from the lab bench into clinical practice, utilizing natural or synthetic biomimetic devices, remains challenging. One of the crucial underestimated reasons for non-translatability could be the imprecision and inconsistency of generated gene expression profiles, utilizing improperly optimized and standardized quantitative gene assays. Utilizing GeNorm for downstream qRT-PCR applications, the stability of reference genes in relation to optimal cDNA amounts was assessed on human bone marrow-derived mesenchymal and adipose-derived stem cells neat and made to differentiate into chondrocytes including normal human derived chondrocytes and muscle tissue from rats. Results showed that reference genes can vary substantially across separately and/or combined cell lines and/or tissue types including treatment parameters. The recommendations to all bone and cartilage tissue engineers utilizing qRT-PCR is not to assume that reference gene stability and quantity remain conserved across cell lines or tissue types but to always determine, for each new experiment, the stability and normalization quantity of reference genes anew.

SUBMITTER: He T

PROVIDER: S-EPMC6173755 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering.

He Tao T Huang Yijiang Y Chak Juy Chi JC Klar Roland Manfred RM

Scientific reports 20181005 1

Autogenous tissue grafting remains the gold standard in the treatment of critical sized bone and certain cartilage defects, while the translation of tissue engineered osteogenesis or chondrogenesis from the lab bench into clinical practice, utilizing natural or synthetic biomimetic devices, remains challenging. One of the crucial underestimated reasons for non-translatability could be the imprecision and inconsistency of generated gene expression profiles, utilizing improperly optimized and stan ...[more]

PMID: 30291289

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Project description:Bone tissue engineering (TE) has the potential to transform the treatment of challenging musculoskeletal pathologies. To date, clinical translation of many traditional TE strategies has been impaired by poor vascularisation of the implant. Addressing such challenges has motivated research into developmentally inspired TE strategies, whereby implants mimicking earlier stages of a tissue's development are engineered in vitro and then implanted in vivo to fully mature into the adult tissue. The goal of this study was to engineer in vitro tissues mimicking the immediate developmental precursor to long bones, specifically a vascularised hypertrophic cartilage template, and to then assess the capacity of such a construct to support endochondral bone formation in vivo. To this end, we first developed a method for the generation of large numbers of hypertrophic cartilage microtissues using a microwell system, and encapsulated these microtissues into a fibrin-based hydrogel capable of supporting vasculogenesis by human umbilical vein endothelial cells (HUVECs). The microwells supported the formation of bone marrow derived stem/stromal cell (BMSC) aggregates and their differentiation toward a hypertrophic cartilage phenotype over 5 weeks of cultivation, as evident by the development of a matrix rich in sulphated glycosaminoglycan (sGAG), collagen types I, II, and X, and calcium. Prevascularisation of these microtissues, undertaken in vitro 1 week prior to implantation, enhanced their capacity to mineralise, with significantly higher levels of mineralised tissue observed within such implants after 4 weeks in vivo within an ectopic murine model for bone formation. It is also possible to integrate such microtissues into 3D bioprinting systems, thereby enabling the bioprinting of scaled-up, patient-specific prevascularised implants. Taken together, these results demonstrate the development of an effective strategy for prevascularising a tissue engineered construct comprised of multiple individual microtissue "building blocks," which could potentially be used in the treatment of challenging bone defects.

Dataset Information

Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering.

Publications

Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering.

Similar Datasets

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