Not scheduled
ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Polain, Claire (MERLN institute )


Introduction: The formation and outgrowth of nerve fibers during fracture healing is a highly important process. These nerve fibers are distributed throughout the entirety of skeletal tissue, being crucial for sensing and responding to stimuli such as mechanical load. Bone morphogenic proteins (BMPs) are commonly used for bone tissue regeneration, due to their osteogenic character. However, this growth factor family does not induce the formation of nerves. Thus, identifying nerve growth cues, intrinsically expressed during bone healing may be advantageous. The clinical use of recombinant protein poses several limitations. These include, but are not limited to, adverse side effects in patients due to the unavoidable use of supraphysiological amounts along with high costs. Gene- and transcript-therapies are proposed as alternatives to recombinant protein use. Transcript therapy is based on the application of protein-coding messenger RNA (mRNA) to induce in situ protein production by the local cells. As mRNA is unstable and immunogenic, chemical modifications to mRNA (cmRNA) increase its stability and biocompatibility. BMP-2 cmRNA has been proven to induce osteogenesis in vitro and in vivo1-3.
In this study, expression of semaphorin-3A (SEMA-3A), neurofilament 200 (NF-200), and b3-tubulin (B3T) in rat BMP2 cmRNA-treated bone specimens was evaluated. These molecules are crucial in neuron development.
Methodology: In rats, 5 mm femoral defects were treated with a collagen sponge containing 5-50µg BMP-2 cmRNA. As a comparison, 11µg of recombinant human (rh) BMP-2 was used. Bone tissue explants were harvested at 4- and 8- weeks after implantation. Expression of SEMA-3A, NF-200, and B3T was assayed by qPCR and immunohistochemistry (IHC). For qPCR, explants were homogenized in TRIzol using a TissueLyser (Qiagen, Germany; 3 min at 30 Hz). RNA extraction and subsequent cDNA synthesis were performed using standard protocols and kits3. Human β-tubulin was used as the housekeeping gene. The ∆∆Ct method was used to treat the data. For IHC, explants were fixated, decalcified, and embedded in paraffin. Staining was performed using DAPI and an anti-SEMA-3A, anti-NF-200, or anti-B3T antibody (Abcam, UK). Samples were imaged using a Slide Scanner microscope (Nikon, Japan) for fluorescence microscopy.
Results and conclusion: Healing doses of BMP-2 cmRNA (25µg and 50µg) showed the highest expression of SEMA-3A, NF-200 and B3T. SEMA-3A, NF-200 and B3T expression was particularly observed in callus tissue and de novo formed bone. The expression of SEMA-3A was less specific than that of NF-200 and B3T, as SEMA-3A has several functions other than neurogenesis. This expression was observed as early as 4 weeks after cmRNA treatment and persisted up to 8 weeks. Similar expression patterns were observed when using the rhBMP-2 counterpart. Conversely, untreated defects showed little to no expression of these genes.
Funding: This project was supported by NIH No. R01 AR074395 from NIAMS. C. Polain thanks the H2020 EU project cmRNAbone No. 874790 for additional support.
References: 1. E.R. Balmayor, et al. Biomaterials 87 (2016)131-146. 2. W. Zhang, et al. Tissue Engineering Part A (2019)131-144. 3. R.E. De La Vega, et al. Science Advances (2022) In Press.


Presentation materials

There are no materials yet.