An Injectable Reverse Thermal Gel for Minimally Invasive Coverage of Mouse Myelomeningocele

Abstract

Background: Myelomeningocele (MMC) results in lifelong neurologic and functional deficits. Currently, prenatal repair of MMC closes the defect, resulting in a 50% reduction in postnatal ventriculoperitoneal shunting. However, this invasive fetal surgery is associated with significant morbidities to mother and baby. We have pioneered a novel reverse thermal gel (RTG) to cover MMC defects in a minimally invasive manner. Here, we test in-vitro RTG long-term stability in amniotic fluid and in vivo application in the Grainy head-like 3 (Grhl3) mouse MMC model. Materials and methods: RTG stability in amniotic fluid (in-vitro) was monitored for 6 mo and measured using gel permeation chromatography and solutionegel transition temperature (lower critical solution temperature). E16.5 Grhl3 mouse fetuses were injected with the RTG or saline and harvested on E19.5. Tissue was assessed for RTG coverage of the gross defect and inflammatory response by immunohistochemistry for macrophages. Results: Polymer backbone molecular weight and lower critical solution temperature remain stable in amniotic fluid after 6 mo. Needle injection over the MMC of Grhl3 fetuses successfully forms a stable gel that covers the entire defect. On harvest, some animals demonstrate >50% RTG coverage. RTG injection is not associated with inflammation. Conclusions: Our results demonstrate that the RTG is a promising candidate for a minimally invasive approach to patch MMC. We are now poised to test our RTG patch in the large preclinical ovine model used to evaluate prenatal repair of MMC.

Evaluation of scaffolding, inflammatory response, and wound healing support of a reverse thermal gel for myelomeningocele patching

“…8,12,14,21 We demonstrated that the RTG can be applied with simple small gauge needle and act as an MMC patch. 8 In addition, the incorporation of biomaterial patches for prenatal MMC closure aims to not only provide a protective barrier but also to help facilitate tissue growth and allow complete skin closure over the defect area. In order to support skin tissue growth over the defect, the RTG should possess scaffolding properties to encourage skin cell growth and proliferation.…”

“…PSHU-PNIPAAm (RTG) was synthesized as described. 8 Briefly, poly (serinol hexamethylene urea) (PSHU) was synthesized by mixing urea (0.360 g), N-BOC-serinol (1.149 g) 8 precipitated in warm water (45 C), followed by purification in dialysis tubing with molecular weight cut off (MWCO) 12,000-14,000 Daltons (Da) against 9 L of ultrapure water with daily water changes. The purified PNIPAAm-COOH was lyophilized at −45 C for 72 h and stored at RT.…”

“…The other half of tissue was embedded for frozen processing using a previously described protocol. 8 Primary antibodies for frozen sections included CD68 (1:250) and F4/80 (1:250) diluted in blocking buffer in a humidified chamber overnight at 4 C. Secondary antibodies included: goat anti-rabbit Alexa 594 for CD68 (1:250), goat anti-rat Alexa 488 for F4/80 (1:250). IHC slides were imaged with a Nikon Eclipse Ti confocal microscope and cells were counted using ImageJ software (National Institutes of Health: http://rsbweb.nih.…”

“…The development of a reverse thermal gel (RTG) is a unique solution to deliver an early, minimally invasive patch to cover MMC defects using the technical advances of fetoscopic surgery. 8 The RTG exists as a solution at room temperature that will instantly form a solid gel upon injection into physiologically temperature substances such as tissues. The injectable, in situ gel forming properties with a lower critical solution temperature (LCST) of 34 C make a RTG ideal for a minimally invasive approach for MMC patching.…”

“…Our lab has previously shown the RTG has rapid in situ gelling properties in amniotic fluid, stability after longterm exposure to amniotic fluids, minimal inflammatory induction, and can be localized to the MMC defect in mouse embryos through simple injection. 8,9 However, these in utero mouse studies were 2-3 days in length due to short murine gestation, thus limiting the evaluation of the RTG's ability to support cellular growth and longterm inflammatory response. Surgical closure of skin is the ideal closure for a complete patch and seal of the MMC defect to prevent further CSF leakage and resolve hindbrain herniation.…”