Dataset Information

In Situ Gene Expression in Native Cryofixed Bone Tissue.

ABSTRACT: Bone is a very complex tissue that is constantly changing throughout the lifespan. The precise mechanism of bone regeneration remains poorly understood. Large bone defects can be caused by gunshot injury, trauma, accidents, congenital anomalies and tissue resection due to cancer. Therefore, understanding bone homeostasis and regeneration has considerable clinical and scientific importance in the development of bone therapy. Macrophages are well known innate immune cells secreting different combinations of cytokines and their role in bone regeneration during bone healing is essential. Here, we present a method to identify mRNA transcripts in cryosections of non-decalcified rat bone using in situ hybridization and hybridization chain reaction to explore gene expression in situ for better understanding the gene expression of the bone tissues.

SUBMITTER: Nikovics K

PROVIDER: S-EPMC8962289 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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In Situ Gene Expression in Native Cryofixed Bone Tissue.

Nikovics Krisztina K Castellarin Cédric C Holy Xavier X Durand Marjorie M Morin Halima H Bendahmane Abdelhafid A Favier Anne-Laure AL

Biomedicines 20220218 2

Bone is a very complex tissue that is constantly changing throughout the lifespan. The precise mechanism of bone regeneration remains poorly understood. Large bone defects can be caused by gunshot injury, trauma, accidents, congenital anomalies and tissue resection due to cancer. Therefore, understanding bone homeostasis and regeneration has considerable clinical and scientific importance in the development of bone therapy. Macrophages are well known innate immune cells secreting different combi ...[more]

PMID: 35203694

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Project description:Emerging studies show the potential application of synthetic biomaterials that are intrinsically osteoconductive and osteoinductive as bone grafts to treat critical bone defects. Here, the biomaterial not only assists recruitment of endogenous cells, but also supports cellular activities relevant to bone tissue formation and function. While such biomaterial-mediated in situ tissue engineering is highly attractive, success of such an approach relies largely on the regenerative potential of the recruited cells, which is anticipated to vary with age. In this study, we investigated the effect of the age of the host on mineralized biomaterial-mediated bone tissue repair using critical-sized cranial defects as a model system. Mice of varying ages, 1-month-old (juvenile), 2-month-old (young-adult), 6-month-old (middle-aged), and 14-month-old (elderly), were used as recipients. Our results show that the bio-mineralized scaffolds support bone tissue formation by recruiting endogenous cells for all groups albeit with differences in an age-related manner. Analyses of bone tissue formation after 2 and 8 weeks post-treatment show low mineral deposition and reduced number of osteocalcin and tartrate-resistant acid phosphatase (TRAP)-expressing cells in elderly mice.Statement of significanceTissue engineering strategies that promote tissue repair through recruitment of endogenous cells will have a significant impact in regenerative medicine. Previous studies from our group have shown that biomineralized materials containing calcium phosphate minerals can contribute to neo-bone tissue through recruitment and activation of endogenous cells. In this study, we investigated the effect of age of the recipient on biomaterial-mediated bone tissue repair. Our results show that the age of the recipient mouse had a significant impact on the quality and quantity of the engineered neo-bone tissues, in which delayed/compromised bone tissue formation was observed in older mice. These findings are in agreement with the clinical observations that age of patients is a key factor in bone repair.

Project description:BackgroundAn important step in characterising the function of a gene is identifying the cells in which it is expressed. Traditional methods to determine this include in situ hybridisation, gene promoter-reporter fusions or cell isolation/purification techniques followed by quantitative PCR. These methods, although frequently used, can have limitations including their time-consuming nature, limited specificity, reliance upon well-annotated promoters, high cost, and the need for specialized equipment. In situ PCR is a relatively simple and rapid method that involves the amplification of specific mRNA directly within plant tissue whilst incorporating labelled nucleotides that are subsequently detected by immunohistochemistry. Another notable advantage of this technique is that it can be used on plants that are not easily genetically transformed.ResultsAn optimised workflow for in-tube and on-slide in situ PCR is presented that has been evaluated using multiple plant species and tissue types. The protocol includes optimised methods for: (i) fixing, embedding, and sectioning of plant tissue; (ii) DNase treatment; (iii) in situ RT-PCR with the incorporation of DIG-labelled nucleotides; (iv) signal detection using colourimetric alkaline phosphatase substrates; and (v) mounting and microscopy. We also provide advice on troubleshooting and the limitations of using fluorescence as an alternative detection method. Using our protocol, reliable results can be obtained within two days from harvesting plant material. This method requires limited specialized equipment and can be adopted by any laboratory with a vibratome (vibrating blade microtome), a standard thermocycler, and a microscope. We show that the technique can be used to localise gene expression with cell-specific resolution.ConclusionsThe in situ PCR method presented here is highly sensitive and specific. It reliably identifies the cellular expression pattern of even highly homologous and low abundance transcripts within target tissues, and can be completed within two days of harvesting tissue. As such, it has considerable advantages over other methods, especially in terms of time and cost. We recommend its adoption as the standard laboratory technique of choice for demonstrating the cellular expression pattern of a gene of interest.

Dataset Information

In Situ Gene Expression in Native Cryofixed Bone Tissue.

Publications

In Situ Gene Expression in Native Cryofixed Bone Tissue.

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