Tissue engineering. Part C, Methods最新文献

Isolated individual myofibers are valuable experimental models that can be used in various conditions to understand skeletal muscle physiology and pathophysiology at the tissue and cellular level. This report details a time- and cost-effective method for isolation of single myofibers from the flexor digitorum brevis (FDB) muscle in both young and aged mice. The FDB muscle was chosen for its documented history in single myofiber experiments. By modifying published methods for FDB myofiber isolation, we have optimized the protocol by first separating FDB muscle into individual bundles before the digestion, followed by optimizing the subsequent digestion medium conditions to ensure reproducibility. Morphological and functional assessments demonstrate a high yield of isolated FDB myofibers with sarcolemma integrity achieved in a shorter time frame than previous published procedures. This method could be also adapted to other types of skeletal muscle. Additionally, this highly reproducible method can greatly reduce the number of animals needed to yield adequate numbers of myofibers for experiments. Thus, this advanced method for myofiber isolation has the potential to accelerate research in skeletal muscle physiology and screening potential therapeutics "ex vivo" for muscle diseases and regeneration.

The heart contains diverse endothelial cell types. We sought to characterize the endocardial endothelial cells (EECs), which line the chambers of the heart. EECs are relatively understudied, yet their dysregulation can lead to various cardiac pathologies. Due to the lack of commercial availability of these cells, we reported our protocol for isolating EECs from porcine hearts and for establishing an EEC population through cell sorting. In addition, we compared the EEC phenotype and fundamental behaviors to a well-studied endothelial cell line, human umbilical vein endothelial cells (HUVECs). The EECs stained positively for classic phenotypic markers such as CD31, von Willebrand Factor, and vascular endothelial (VE) cadherin. The EECs proliferated more quickly than HUVECs at 48 h (1310 ± 251 cells vs. 597 ± 130 cells, p = 0.0361) and at 96 h (2873 ± 257 cells vs. 1714 ± 342 cells, p = 0.0002). Yet EECs migrated more slowly than HUVECs to cover a scratch wound at 4 h (5% ± 1% wound closure vs. 25% ± 3% wound closure, p < 0.0001), 8 h (15% ± 4% wound closure vs. 51% ± 12% wound closure, p < 0.0001), and 24 h (70% ± 11% wound closure vs. 90% ± 3% wound closure, p < 0.0001). Finally, the EECs maintained their endothelial phenotype by positive expression of CD31 through more than a dozen passages (three populations of EECs showing 97% ± 1% CD31⁺ cells in over 14 passages). In contrast, the HUVECs showed significantly reduced CD31 expression over high passages (80% ± 11% CD31⁺ cells over 14 passages). These important phenotypic differences between EECs and HUVECs highlight the need for researchers to utilize the most relevant cell types when studying or modeling diseases of interest.

Corneal nerves originate from the ophthalmic branch of the trigeminal nerve, which enters the cornea at the limbus radially from all directions toward the central cornea. The cell bodies of the sensory neurons of trigeminal nerve are located in the trigeminal ganglion (TG), while the axons are extended into the three divisions, including ophthalmic branch that supplies corneal nerves. Study of primary neuronal cultures established from the TG fibers can therefore provide a knowledge basis for corneal nerve biology and potentially be developed as an in vitro platform for drug testing. However, setting up primary neuron cultures from animal TG has been dubious with inconsistency among laboratories due to a lack of efficient isolation protocol, resulting in low yield and heterogenous cultures. In this study, we used a combined enzymatic digestion with collagenase and TrypLE to dissociate mouse TG while preserving nerve cell viability. A subsequent discontinuous Percoll density gradient followed by mitotic inhibitor treatment effectively diminished the contamination of non-neuronal cells. Using this method, we reproducibly generated high yield and homogenous primary TG neuron cultures. Similar efficiency of nerve cell isolation and culture was further obtained for TG tissue cryopreserved for short (1 week) and long duration (3 months), compared to freshly isolated tissues. In conclusion, this optimized protocol shows a promising potential to standardize TG nerve culture and generate a high-quality corneal nerve model for drug testing and neurotoxicity studies.

Mesenchymal stem cell and 3D printing-based bone tissue engineering present a promising technique to repair large-volume bone defects. Its success is highly dependent on cell attachment, spreading, osteogenic differentiation, and in vivo survival of stem cells on 3D-printed scaffolds. In this study, we applied human salivary histatin-1 (Hst1) to enhance the interactions of human adipose-derived stem cells (hASCs) on 3D-printed β-tricalcium phosphate (β-TCP) bioceramic scaffolds. Fluorescent images showed that Hst1 significantly enhanced the adhesion of hASCs to both bioinert glass and 3D-printed β-TCP scaffold. In addition, Hst1 was associated with significantly higher proliferation and osteogenic differentiation of hASCs on 3D-printed β-TCP scaffolds. Moreover, coating 3D-printed β-TCP scaffolds with histatin significantly promotes the survival of hASCs in vivo. The ERK and p38 but not JNK signaling was found to be involved in the superior adhesion of hASCs to β-TCP scaffolds with the aid of Hst1. In conclusion, Hst1 could significantly promote the adhesion, spreading, osteogenic differentiation, and in vivo survival of hASCs on 3D-printed β-TCP scaffolds, bearing a promising application in stem cell/3D printing-based constructs for bone tissue engineering.

This cross-sectional pilot study explored extracellular vesicle (EV)-derived gene expression of markers for bone turnover and pro-inflammatory cytokines in periodontal disease. Whole unstimulated saliva was collected from 52 participants (18 healthy, 13 gingivitis, and 21 stages III/IV periodontitis), from which salivary small extracellular vesicles (sEVs) were enriched using the size-exclusion chromatography method, and characterized by morphology, EV-protein, and size distribution, using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), and Nanoparticle Tracking Analysis (NTA), respectively. Bone turnover markers and pro-inflammatory cytokines in salivary sEVs were evaluated using reverse transcription PCR. Salivary sEVs morphology, mode, size distribution, and particle concentration were comparable between healthy, gingivitis, and periodontitis patients. The CD9+ subpopulation was significantly higher in periodontitis-derived salivary sEVs compared with healthy. The detection of sEVs mRNA for osterix and tumor necrosis factor-alpha was significantly decreased and increased, respectively, in periodontitis compared with healthy controls, with good discriminatory power for periodontitis diagnosis (area under the curve >0.72). This pilot study demonstrated that salivary sEVs mRNAs may serve as a potential noninvasive biomarker source for periodontitis diagnosis.

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.

The aim of this study was to test the suitability of calcium phosphate cement mixed with poly(lactic-co-glycolic acid) (CPC-PLGA) microparticles into a ring-shaped polymeric space-maintaining device as bone graft material for lateral bone augmentation. Therefore, the bone chambers were installed on the lateral portion of the anterior region of the mandibular body of mini-pigs. Chambers were filled with either CPC-PLGA or BioOss^® particles for comparison and left for 4 and 12 weeks. Histology and histomorphometry were used to obtain temporal insight in material degradation and bone formation. Results indicated that between 4 and 12 weeks of implantation, a significant degradation of the CPC-PLGA (from 75.1% to 23.1%), as well as BioOss material, occurred (from 40.6% to 14.4%). Degradation of both materials was associated with the presence of macrophage-like and osteoclast-like cells. Furthermore, a significant increase in bone formation occurred between 4 and 12 weeks for the CPC-PLGA (from 0.1% to 7.2%), as well as BioOss material (from 8.3% to 23.3%). Statistical analysis showed that bone formation had progressed significantly better using BioOss compared to CPC-PLGA (p < 0.05). In conclusion, this mini-pig study showed that CPC-PLGA does not stimulate lateral bone augmentation using a bone chamber device. Both treatments failed to achieve "clinically" meaningful alveolar ridge augmentation.