In Vivo Evaluation of Novel PLA/PCL Polymeric Patch in Rats for Potential Spina Bifida Coverage.

BACKGROUND: Current therapeutic materials for spina bifida repair showed a limited number of options in the market, and none of them have all the requirements as the ideal patch. In fact, sometimes the surgical procedures pose substantial challenges using different patches to fully cover the spina bifida lesion. For this purpose, a tailored patch made of poly (L-lactic acid) and poly (ε-caprolactone) blend was designed and validated in vitro to accomplish all these requirements but was never tested in vivo. MATERIAL AND METHODS: In our present study, the designed patch was analyzed in terms of rejection from the animal when implanted subcutaneously and as a dural substitute in the spinal cord. Inflammatory reaction (Iba1), astrogliosis (GFAP), was analyzed and functional interaction with spinal cord tissue assessing the (%motor-evoked potentials /compound motor action potential) by electrophysiology. RESULTS: No evidence of adverse or inflammatory reactions was observed in both models of subcutaneous implantation, neither in the neural tissue as a dural substitute. No signs of astrogliosis in the neural tissue were observed, and no functional alteration with improvement of the motor-evoked potential's amplitude was detected after 4 wk of implantation as a dural substitute in the rat spinal cord. CONCLUSIONS: Designed patch used as a dural substitute will apparently not produce inflammation, scar formation, or tethering cord and not induce any adverse effect on regular functions of the spinal cord. Further studies are needed to evaluate potential improvements of this novel polymeric patch in the spinal cord regeneration using spina bifida models.

Single-Access Fetal Endoscopy (SAFE) for myelomeningocele in sheep model I: amniotic carbon dioxide gas approach.

BACKGROUND: This study aimed to assess the feasibility of single-access fetal endoscopy (SAFE) for the management of myelomeningocele (MMC) using intrauterine carbon dioxide as a distension medium in a sheep model. METHODS: This prospective experimental case-control study investigated 12 lamb fetuses that had a myelomeningocele-like defect surgically created on the 75th day of gestation. Four fetuses remained untreated (control group), and eight fetuses had MMC repair using two fetoscopic approaches with carbon dioxide used to distend the amniotic cavity. A collagen patch was placed over the defect and secured with surgical sealant. Four animals had a two-port fetoscopic procedure, and four animals had SAFE. Clinical and pathologic studies were performed after delivery. RESULTS: This study confirmed the validity of the animal MMC model. None of the control animals was able to stand or walk, and all had a significant defect in the lumbar area with continuous leakage of cerebrospinal fluid, ventriculomegaly, and a Chiari-II malformation. All the treated animals, independently of the number of ports used in the repair, were able to walk and had a closed defect with resolution of the Chiari malformation. CONCLUSIONS: The SAFE patch and glue coverage of surgically created fetal MMC is feasible and effective in restoring gross neurologic function in the fetal lamb model.

Biodegradation of poly(L-lactic acid) and poly(ε-caprolactone) patches by human amniotic fluid in an in-vitro simulated fetal environment.

Open spina bifida or myelomeningocele (MMC) is a devastating neurologic congenital defect characterized by primary failure of neural tube closure of the spinal column during the embryologic period. Cerebrospinal fluid leak caused by the MMC spinal defect in the developing fetus can result in a constellation of encephalic anomalies that include hindbrain herniation and hydrocephalus. The exposure of extruded spinal cord to amniotic fluid also poses a significant risk for inducing partial or complete paralysis of the body parts beneath the spinal aperture by progressive spinal cord damage in-utero. A randomized trial demonstrated that prenatal repair by fetal surgery, sometimes using patches, to cover the exposed spinal cord with a watertight barrier is effective in reducing the postnatal neurologic morbidity as evidenced by decreased incidence and severity of postnatal hydrocephalus and the reduced need for ventricular-peritoneal shunting. Currently, the use of inert or collagen-based patches are associated with high costs and inadequate structural properties. Specifically, the inert patches do not degrade after implantation, causing the need for a post-natal removal surgery associated with trauma for the newborn. Our present study is aimed towards in-vitro degradation studies of a newly designed patch, which potentially can serve as a superior alternative to existing patches for MMC repair. This novel patch was fabricated by blending poly(L-lactic acid) and poly(ε-caprolactone). The 16-week degradation study in amniotic fluid was focused on tracking changes in crystallinity and mechanical properties. An additional set of designed patches was exposed to phosphate-buffered saline (PBS), as a time-paired control. Crystallinity studies indicate the progress of hydrolytic degradation of the patch in both media, with a preference to bulk erosion in phosphate buffered saline and surface erosion in amniotic fluid. Mechanical testing results establish that patch integrity is not compromised up to 16 weeks of exposure either to body fluids analog (PBS) or to amniotic fluid.

Using poly(l-lactic acid) and poly(ɛ-caprolactone) blends to fabricate self-expanding, watertight and biodegradable surgical patches for potential fetoscopic myelomeningocele repair.

Our study focuses on the development and characterization of a self-expanding, watertight and biodegradable patch for fetoscopic myelomeningocele (MMC) prenatal repair. We fabricated poly(l-lactic acid) (PLA) and poly(ɛ-caprolactone) (PCL) blend films by solution casting. Formulation c with average glass transition temperature of 37.6 ± 1.2°C was chosen for temporospatial recovery. Favorable results from surface studies reflected homogeneous dispersion of polymers in the blend. The cytotoxicity was studied in human foreskin fibroblasts. The blend film was cytocompatible, evidenced by matching percentage of live cells in exposed and control solutions. Subsequently, liquid water permeability experiments confirmed watertight nature of films. Finally, in vitro degradation was investigated in phosphate buffered saline (PBS) and amniotic fluid (AF) separately for 16 weeks. Similar weight loss (n = 6, p = 0.912) and significantly different (n = 3, p = 0.025) surface roughness was observed in PBS and AF, respectively, at 16 weeks. Functional group analysis displayed increasing carbonyl and hydroxyl bonds in PBS and AF, respectively, over time, indicating progression of hydrolytic degradation. Favorable characterization results provide strong evidence to employ PLA-PCL blend films as surgical patches in fetoscopic MMC repair. Designed patch serves as standalone system to successfully tackle impending hurdles of MMC repair and proves to be a superior alternative compared to existing patches. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 295-305, 2019.

Cell necrosis, intrinsic apoptosis and senescence contribute to the progression of exencephaly to anencephaly in a mice model of congenital chranioschisis.

Exencephaly/anencephaly is one of the leading causes of neonatal mortality and the most extreme open neural tube defect with no current treatments and limited mechanistic understanding. We hypothesized that exencephaly leads to a local neurodegenerative process in the brain exposed to the amniotic fluid as well as diffuse degeneration in other encephalic areas and the spinal cord. To evaluate the consequences of in utero neural tissue exposure, brain and spinal cord samples from E17 exencephalic murine fetuses (maternal intraperitoneal administration of valproic acid at E8) were analyzed and compared to controls and saline-injected shams (n = 11/group). Expression of apoptosis and senescence genes (p53, p21, p16, Rbl2, Casp3, Casp9) was determined by qRT-PCR and protein expression analyzed by western blot. Apoptosis was measured by TUNEL assay and PI/AV flow cytometry. Valproic acid at E8 induced exencephaly in 22% of fetuses. At E17 the fetuses exhibited the characteristic absence of cranial bones. The brain structures from exencephalic fetuses demonstrated a loss of layers in cortical regions and a complete loss of structural organization in the olfactory bulb, hippocampus, dental gyrus and septal cortex. E17 fetuses had reduced expression of NeuN, GFAP and Oligodendrocytes in the brain with primed microglia. Intrinsic apoptotic activation (p53, Caspase9 and 3) was upregulated and active Caspase3 localized to the layer of brain exposed to the amniotic fluid. Senescence via p21-Rbl2 was increased in the brain and in the spinal cord at the lamina I-II of the somatosensory dorsal horn. The current study characterizes CNS alterations in murine exencephaly and demonstrates that degeneration due to intrinsic apoptosis and senescence occurs in the directly exposed brain but also remotely in the spinal cord.

Fetoscopic coverage of experimental myelomeningocele in sheep using a patch with surgical sealant.

OBJECTIVE: Assess the feasibility of a fetoscopic patch coverage method for myelomeningocele repair in a sheep model. STUDY DESIGN: Experimental study. A myelomeningocele-like defect was created in 15 fetal sheep on day 75 of gestation. Six remained untreated, whereas 9 underwent fetoscopic coverage of the defect on day 95 of gestation using an inert patch secured with surgical sealant. Clinical and histological examinations were performed after delivery. RESULTS: Four valid newborn lambs were obtained in each group. Mean fetoscopic surgical time was 26.9 (SD=7.4)min. All untreated animals had an open lumbar defect with cerebrospinal fluid leakage, paraplegia, urinary incontinence, and Chiari malformation. All treated animals had a closed defect and were able to walk; one had weak bladder control, and another mild Chiari malformation. CONCLUSION: In a chronic myelomeningocele model in fetal sheep, fetoscopic repair using a sealed patch results in simple, fast, satisfactory neural tube closure and averts neurological damage and Chiari malformation.

Inert patch with bioadhesive for gentle fetal surgery of myelomeningocele in a sheep model.

OBJECTIVE: Current techniques used in foetal myelomeningocele repair can require considerable manipulation of fragile foetal tissues to obtain tension-free closure. The aim of this study was to assess the feasibility of a simple foetal coverage method without foetal tissue manipulation to provide closure of the neural tube defect in myelomeningocele. STUDY DESIGN: This is an experimental study performed in 15 foetal sheep with lumbar myelomeningocele, surgically created on day 75 of gestation. Five foetuses remained untreated. Ten underwent coverage with inert sheeting (5 Silastic; 5 Silastic+Marlex) secured by surgical tissue adhesive without suturing on day 95; none of them underwent foetal muscle or skin manipulation. Clinical and subsequent histological examinations were performed at 48h after birth. The Chi-square, Fisher exact, and Mann-Whitney U tests, when appropriate, were used for the comparisons. RESULTS: The mean operating time for foetal coverage was 7.1 (SD=1.6)min. All untreated animals were unable to walk, had sphincter incontinence, showed an open defect, histological spinal cord damage, and a large Chiari malformation. All covered animals were able to walk, had sphincter continence, showed almost complete closure of the defect with regeneration of several soft tissue layers, and minimum Chiari malformation. CONCLUSION: In a surgical myelomeningocele model in sheep, a simple, fast and gentle coverage method using a sealed patch avoids foetal tissue manipulation and enables adequate closure of the neural tube defect, providing regeneration of several tissue layers that protect the spinal cord, and significantly reducing Chiari II malformation.