Regenerative Engineering of a Biphasic Patient-Fitted Temporomandibular Joint Condylar Prosthesis.

Regenerative medicine approaches to restore the mandibular condyle of the temporomandibular joint (TMJ) may fill an unmet patient need. In this study, a method to implant an acellular regenerative TMJ prosthesis was developed for orthotopic implantation in a pilot goat study. The scaffold incorporated a porous, polycaprolactone-hydroxyapatite (PCL-HAp, 20wt% HAp) 3D printed condyle with a cartilage-matrix-containing hydrogel. A series of material characterizations was used to determine the structure, fluid transport, and mechanical properties of 3D printed PCL-HAp. To promote marrow uptake for cell seeding, a scaffold pore size of 152 ± 68 µm resulted in a whole blood transport initial velocity of 3.7 ± 1.2 mm·s-1 transported to the full 1 cm height. The Young's modulus of PCL was increased by 67% with the addition of HAp, resulting in a stiffness of 269 ± 20 MPa for etched PCL-HAp. In addition, the bending modulus increased by 2.06-fold with the addition of HAp to 470 MPa for PCL-HAp. The prosthesis design with an integrated hydrogel was compared with unoperated contralateral control and no-hydrogel group in a goat model for 6 months. A guide was used to make the condylectomy cut, and the TMJ disc was preserved. MicroCT assessment of bone suggested variable tissue responses with some regions of bone growth and loss, although more loss may have been exhibited by the hydrogel group than the no-hydrogel group. A benchtop load transmission test suggested that the prosthesis was not shielding load to the underlying bone. Although variable, signs of neocartilage formation were exhibited by Alcian blue and collagen II staining on the anterior, functional surface of the condyle. Overall, this study demonstrated signs of functional TMJ restoration with an acellular prosthesis. There were apparent limitations to continuous, reproducible bone formation, and stratified zonal cartilage regeneration. Future work may refine the prosthesis design for a regenerative TMJ prosthesis amenable to clinical translation.

Biodegradable electrospun patch containing cell adhesion or antimicrobial compounds for trachea repair in vivo.

Difficulty breathing due to tracheal stenosis (i.e. narrowed airway) diminishes the quality of life and can potentially be life-threatening. Tracheal stenosis can be caused by congenital anomalies, external trauma, infection, intubation-related injury, and tumors. Common treatment methods for tracheal stenosis requiring surgical intervention include end-to-end anastomosis, slide tracheoplasty and/or laryngotracheal reconstruction. Although the current methods have demonstrated promise for treatment of tracheal stenosis, a clear need exists for the development of new biomaterials that can hold the trachea open after the stenosed region has been surgically opened, and that can support healing without the need to harvest autologous tissue from the patient. The current study therefore evaluated the use of electrospun nanofiber scaffolds encapsulating 3D-printed PCL rings to patch induced defects in rabbit tracheas. The nanofibers were a blend of polycaprolactone (PCL) and polylactide-co-caprolactone (PLCL), and encapsulated either the cell adhesion peptide, RGD, or antimicrobial compound, ceragenin-131 (CSA). Blank PCL/PLCL and PCL were employed as control groups. Electrospun patches were evaluated in a rabbit tracheal defect model for 12 weeks, which demonstrated re-epithelialization of the luminal side of the defect. No significant difference in lumen volume was observed for the PCL/PLCL patches compared to the uninjured positive control. Only the RGD group did not lead to a significant decrease in the minimum cross-sectional area compared to the uninjured positive control. CSA reduced bacteria growth in vitro, but did not add clear value in vivo. Adequate tissue in-growth into the patches and minimal tissue overgrowth was observed inside the patch material. Areas of future investigation include tuning the material degradation time to balance cell adhesion and structural integrity.

Switching the intracellular pathway and enhancing the therapeutic efficacy of small interfering RNA by auroliposome.

Gene silencing using small-interfering RNA (siRNA) is a viable therapeutic approach; however, the lack of effective delivery systems limits its clinical translation. Herein, we doped conventional siRNA-liposomal formulations with gold nanoparticles to create "auroliposomes," which significantly enhanced gene silencing. We targeted MICU1, a novel glycolytic switch in ovarian cancer, and delivered MICU1-siRNA using three delivery systems-commercial transfection agents, conventional liposomes, and auroliposomes. Low-dose siRNA via transfection or conventional liposomes was ineffective for MICU1 silencing; however, in auroliposomes, the same dose gave >85% gene silencing. Efficacy was evident from both in vitro growth assays of ovarian cancer cells and in vivo tumor growth in human ovarian cell line-and patient-derived xenograft models. Incorporation of gold nanoparticles shifted intracellular uptake pathways such that liposomes avoided degradation within lysosomes. Auroliposomes were nontoxic to vital organs. Therefore, auroliposomes represent a novel siRNA delivery system with superior efficacy for multiple therapeutic applications.

The incorporation of poly(lactic-co-glycolic) acid nanoparticles into porcine small intestinal submucosa biomaterials.

Small intestinal submucosa (SIS) derived from porcine small intestine has been intensively studied for its capacity in repairing and regenerating wounded and dysfunctional tissues. However, SIS suffers from a large spectrum of heterogeneity in microarchitecture leading to inconsistent results. In this study, we introduced nanoparticles (NPs) to SIS with an intention of decreasing the heterogeneity and improving the consistency of this biomaterial. As determined by scanning electron microscopy and urea permeability, the optimum NP size was estimated to be between 200 nm and 500 nm using commercial monodisperse latex spheres. The concentration of NPs that is required to alter pore sizes of SIS as determined by urea permeability was estimated to be 1 mg/ml 260 nm poly(lactic-co-glycolic) acid (PLGA) NPs. The 1mg/ml PLGA NPs loaded in the SIS did not change the tensile properties of the unmodified SIS or even alter pH values in a cell culture environment. More importantly, PLGA NP modified SIS did not affect human mammary endothelial cells (HMEC-1) morphology or adhesion, but actually enhanced HEMC-1 cell growth.

Reinforced Electrospun Polycaprolactone Nanofibers for Tracheal Repair in an In Vivo Ovine Model.

Tracheal stenosis caused by congenital anomalies, tumors, trauma, or intubation-related damage can cause severe breathing issues, diminishing the quality of life, and potentially becoming fatal. Current treatment methods include laryngotracheal reconstruction or slide tracheoplasty. Laryngotracheal reconstruction utilizes rib cartilage harvested from the patient, requiring a second surgical site. Slide tracheoplasty involves a complex surgical procedure to splay open the trachea and reconnect both segments to widen the lumen. A clear need exists for new and innovative approaches that can be easily adopted by surgeons, and to avoid harvesting autologous tissue from the patient. This study evaluated the use of an electrospun patch, consisting of randomly layered polycaprolactone (PCL) nanofibers enveloping 3D-printed PCL rings, to create a mechanically robust, suturable, air-tight, and bioresorbable graft for the treatment of tracheal defects. The study design incorporated two distinct uses of PCL: electrospun fibers to promote tissue integration, while remaining air-tight when wet, and 3D-printed rings to hold the airway open and provide external support and protection during the healing process. Electrospun, reinforced tracheal patches were evaluated in an ovine model, in which all sheep survived for 10 weeks, although an overgrowth of fibrous tissue surrounding the patch was observed to significantly narrow the airway. Minimal tissue integration of the surrounding tissue and the electrospun fibers suggested the need for further improvement. Potential areas for further improvement include a faster degradation rate, agents to increase cellular adhesion, and/or an antibacterial coating to reduce the initial bacterial load.

Reduced urothelial regeneration in rat bladders augmented with permeable porcine small intestinal submucosa assessed by magnetic resonance imaging.

Augmentation enterocystoplasty remains the gold standard surgical bladder reconstruction procedure to increase the capacity and compliance of dysfunctional bladders. Since the use of the patient's intestine has severe risks of complications, alternative biodegradable matrices have been explored. Porcine small intestinal submucosa (SIS) has gained immense interests in bladder reconstruction due to its favorable properties. However, trials have shown inconsistent regeneration with SIS, attributed to the heterogeneity in microstructures and mechanical properties. We hypothesize that uneven SIS permeability to urine is a factor responsible for the inconsistency. We measured permeability to urine in situ using a contrast enhanced-magnetic resonance imaging (MRI), and evaluated urothelium regeneration using immunohistochemical staining of urothelial cell markers in SIS-augmented rat bladders. Results showed significant differences in permeability among SIS-augmented rat bladders. Commercial SIS scaffolds were then categorized into nonleaky and leaky groups based on MRI results. Hematoxylin and eosin staining showed higher numbers of inflammatory cells in leaky SIS on day 14 relative to nonleaky SIS. In addition, trichrome staining showed major changes in the distribution of collagen on day 28 between SIS-augmented bladder groups. Furthermore, expressions of urothelium-associated markers (cytokeratins AE1/AE3, claudin 4, and uroplakin III) were completed in bladders augmented with nonleaky SIS, whereas limited urothelial differentiation was noticed in leaky SIS-augmented bladders at post-augmentative day 14. These results show that scaffold permeability to urine may be responsible for variations in regenerative capacity of porcine SIS. Applications of MRI technique will be helpful to understand a relationship between biomaterial property and regenerative capacity. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1778-1787, 2018.

Biomatrices for bladder reconstruction.

There is a demand for tissue engineering of the bladder needed by patients who experience a neurogenic bladder or idiopathic detrusor overactivity. To avoid complications from augmentation cystoplasty, the field of tissue engineering seeks optimal scaffolds for bladder reconstruction. Naturally derived biomaterials as well as synthetic and natural polymers have been explored as bladder substitutes. To improve regenerative properties, these biomaterials have been conjugated with functional molecules, combined with nanotechology, or seeded with exogenous cells. Although most studies reported complete and functional bladder regeneration in small-animal models, results from large-animal models and human clinical trials varied. For functional bladder regeneration, procedures for biomaterial fabrication, incorporation of biologically active agents, introduction of nanotechnology, and application of stem-cell technology need to be standardized. Advanced molecular and medical technologies such as next generation sequencing and magnetic resonance imaging can be introduced for mechanistic understanding and non-invasive monitoring of regeneration processes, respectively.

Understanding roles of porcine small intestinal submucosa in urinary bladder regeneration: identification of variable regenerative characteristics of small intestinal submucosa.

Neuropathic bladders are the result from damages to the central or peripheral nervous system, and ultimately may require surgical reconstruction to increase bladder volumes and to reduce the risk of damages to the kidneys. Surgical reconstruction through bladder augmentation has traditionally been practiced using a segment of the ileum, colon, or stomach from the patient through enterocystoplasty. However, the use of gastrointestinal segments can lead to serious adverse consequences. Porcine small intestinal submucosa (SIS), a xenogeneic, acellular, biocompatable, biodegradable, and collagen-based bioscaffold is best known to encourage bladder regeneration without ex vivo cell seeding before implantation in various experimental and preclinical animal models. Although it has been demonstrated that SIS supports bladder cell growth in vitro, and SIS-regenerated bladders are histologically and functionally indistinguishable from normal functional tissues, clinical utilization of SIS for bladder augmentation has been hampered by inconsistent preclinical results. Several variables in SIS, such as the age of pigs, the region of the small intestine, and method of sterilization, can have different physical properties, biochemical characteristics, inflammatory cell infiltration, and regenerative capacity due to cellular responses in vitro and in vivo. These parameters are particularly important for bladder regeneration due to its specific biological function in urine storage. Clinical application of SIS for surgical bladder reconstruction may require graft materials to be prepared from a specific region of the small intestine, or to be further formulated or processed to provide uniform physical and biochemical properties for consistent, complete, and functional bladder regeneration.

Mammary analog secretory carcinoma of salivary gland with high-grade histology arising in hard palate, report of a case and review of literature.

Mammary gland analog secretary carcinoma (MASC) of salivary gland is typically a tumor of low histologic grade and behaves as a low-grade malignancy with relatively benign course. This tumor shares histologic features, immunohistochemical profile, and a highly specific genetic translocation, ETV6-NTRK3, with secretory carcinoma of breast. Histologically, it is often mistaken as acinic cell carcinoma, adenocarcinoma not otherwise specified, and other primary salivary gland tumors. Here we report a case of MASC with high-grade transformation and cervical lymph node metastases confirmed with ETV6-NTRK3 translocation arising in the hard palate of a 41 year-old adult. Interestingly, the metastatic carcinoma has lower grade than the original tumor which strongly support malignant transformation of the original tumor. Most commonly, MASC arises from the parotid gland and less often in minor salivary glands. Metastasis is relatively uncommon and high-grade histology has only been reported in four cases with three of them arising from the parotid gland and the location of the fourth one has not been reported. This is the first case with high grade histology that arise from minor salivary gland and it emphasizes the importance of molecular screening of salivary gland tumor with high-grade histology for ETV6-NTRK3 translocation. In our literature of 115 cases that includes the current case, MASC occurred predominantly in adult with only a few cases under 18 years of age and a male to female ratio of 1.2:1. Parotid gland is more commonly affected but there is also significant occurrence in minor salivary glands. Except for the cases with high grade histology, the overall prognosis is good.

Enhanced angiogenesis of modified porcine small intestinal submucosa with hyaluronic acid-poly(lactide-co-glycolide) nanoparticles: from fabrication to preclinical validation.

Hyaluronic acid-poly(de-co-glycolide) nanoparticles (HA-PLGA NPs) were synthesized to stabilize the porous structure of porcine small intestinal submucosa (SIS), to improve surface biocompatibility and to enhance performance in tissue regeneration. HA-PLGA NPs were characterized for size, zeta potential, surface morphology, and HA loading. Human microvascular endothelial cells responded to HA-PLGA NPs and HA-PLGA modified SIS (HA-PLGA-SIS) with elevated cell proliferation. HA-PLGA-SIS significantly enhanced neo-vascularization in an in ovo chorioallantoic membrane angiogenesis model. The angiogenic capability of the newly fabricated HA-PLGA-SIS was tested in a canine bladder augmentation model. Urinary bladder augmentation was performed in beagle dogs following hemi-cystectomy using HA-PLGA-SIS. The regenerated bladder was harvested at 10 weeks post augmentation and vascularization was evaluated using CD31 immunohistochemical staining. Bladder regenerated with HA-PLGA-SIS had significantly higher vascular ingrowth compared to unmodified SIS. This study shows that HA-PLGA NPs may represent a new approach for modifying naturally derived SIS biomaterials in regenerative medicine.