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Spatiotemporally controlled microchannels of periodontal mimic scaffolds.

ABSTRACT: Physiologic bioengineering of the oral, dental, and craniofacial complex requires optimized geometric organizations of fibrous connective tissues. A computer-designed, fiber-guiding scaffold has been developed to promote tooth-supporting periodontal tissue regeneration and functional restoration despite limited printing resolution for the manufacture of submicron-scaled features. Here, we demonstrate the use of directional freeze-casting techniques to control pore directional angulations and create mimicked topographies to alveolar crest, horizontal, oblique, and apical fibers of natural periodontal ligaments. For the differing anatomic positions, the gelatin displayed varying patterns of ice growth, determined via internal pore architectures. Regardless of the freezing coordinates, the longitudinal pore arrangements resulted in submicron-scaled diameters (~50 µm), along with corresponding high biomaterial porosity (~90%). Furthermore, the horizontal + coronal ([Formula: see text]) freezing orientation facilitated the creation of similar structures to major fibers in the periodontal ligament interface. This periodontal tissue-mimicking microenvironment is a potential tissue platform for the generation of naturally oriented ligamentous tissues consistent with periodontal ligament neogenesis.

SUBMITTER: Park CH

PROVIDER: S-EPMC4237636 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Spatiotemporally controlled microchannels of periodontal mimic scaffolds.

Park C H CH Kim K H KH Rios H F HF Lee Y M YM Giannobile W V WV Seol Y J YJ

Journal of dental research 20140911 12

Physiologic bioengineering of the oral, dental, and craniofacial complex requires optimized geometric organizations of fibrous connective tissues. A computer-designed, fiber-guiding scaffold has been developed to promote tooth-supporting periodontal tissue regeneration and functional restoration despite limited printing resolution for the manufacture of submicron-scaled features. Here, we demonstrate the use of directional freeze-casting techniques to control pore directional angulations and cre ...[more]

PMID: 25216511

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Project description:Successful periodontal repair and regeneration requires the coordinated responses from soft and hard tissues as well as the soft tissue-to-bone interfaces. Inspired by the hierarchical structure of native periodontal tissues, tissue engineering technology provides unique opportunities to coordinate multiple cell types into scaffolds that mimic the natural periodontal structure in vitro. In this study, we designed and fabricated highly ordered multicompartmental scaffolds by melt electrowriting, an advanced 3-dimensional (3D) printing technique. This strategy attempted to mimic the characteristic periodontal microenvironment through multicompartmental constructs comprising 3 tissue-specific regions: 1) a bone compartment with dense mesh structure, 2) a ligament compartment mimicking the highly aligned periodontal ligaments (PDLs), and 3) a transition region that bridges the bone and ligament, a critical feature that differentiates this system from mono- or bicompartmental alternatives. The multicompartmental constructs successfully achieved coordinated proliferation and differentiation of multiple cell types in vitro within short time, including both ligamentous- and bone-derived cells. Long-term 3D coculture of primary human osteoblasts and PDL fibroblasts led to a mineral gradient from calcified to uncalcified regions with PDL-like insertions within the transition region, an effect that is challenging to achieve with mono- or bicompartmental platforms. This process effectively recapitulates the key feature of interfacial tissues in periodontium. Collectively, this tissue-engineered approach offers a fundament for engineering periodontal tissue constructs with characteristic 3D microenvironments similar to native tissues. This multicompartmental 3D printing approach is also highly compatible with the design of next-generation scaffolds, with both highly adjustable compartmentalization properties and patient-specific shapes, for multitissue engineering in complex periodontal defects.

Project description:Rebuilding the tooth-supporting tissues (periodontium) destroyed by periodontitis remains a clinical challenge. Periodontal ligament cells (PDLCs), multipotent cells within the periodontal ligament (PDL), differentiate and form new PDL and mineralized tissues (cementum and bone) during native tissue repair in response to specific extracellular matrix (ECM) cues. Thus, harnessing ECM cues to control PDLC activity ex vivo, and ultimately, to design a PDLC delivery vehicle for tissue regeneration is an important goal. In this study, poly(ethylene glycol) hydrogels were used as a synthetic PDL ECM to interrogate the roles of cell-matrix interactions and cell-mediated matrix remodeling in controlling PDLC activity. Results showed that PDLCs within matrix metalloproteinase (MMP)-degradable hydrogels expressed key PDL matrix genes and showed a six to eightfold increase in alkaline phosphatase (ALP) activity compared with PDLCs in nondegradable hydrogel controls. The increase in ALP activity, commonly considered an early marker of cementogenic/osteogenic differentiation, occurred independent of the presentation of the cell-binding ligand RGD or soluble media cues and remained elevated when inhibiting PDLC-matrix binding and intracellular tension. ALP activity was further increased in softer hydrogels regardless of degradability and was accompanied by an increase in PDLC volume. However, scaffolds that fostered PDLC ALP activity did not necessarily promote hydrogel ECM mineralization. Rather, matrix mineralization was greatest in stiffer, MMP-degradable hydrogels and required the presence of soluble media cues. These divergent outcomes illustrate the complexity of the PDLC response to ECM cues and the limitations of current scaffold materials. Nevertheless, key biomaterial design principles for controlling PDLC activity were identified for incorporation into scaffolds for periodontal tissue regeneration. Impact statement Engineered scaffolds are an attractive approach for delivering periodontal ligament cells (PDLCs) to rebuild the tooth-supporting tissues. Replicating key extracellular matrix (ECM) cues within tissue engineered scaffolds may maximize PDLC potential. However, the identity of important ECM cues and how they can be harnessed to control PDLC activity is still unknown. In this study, matrix degradability, cell-matrix binding, and stiffness were varied using synthetic poly(ethylene glycol) hydrogels for three-dimensional PDLC culture. PDLCs exhibited dramatic and divergent responses to these cues, supporting further investigation of ECM-replicating scaffolds for control of PDLC behavior and periodontal tissue regeneration.

Project description:BackgroundMethods for the electrical inhibition of cardiac excitation have long been sought to control excitability and conduction, but to date remain largely impractical. High-amplitude alternating current (AC) stimulation has been known to extend cardiac action potentials (APs), and has been recently exploited to terminate reentrant arrhythmias by producing reversible conduction blocks. Yet, low-amplitude currents at similar frequencies have been shown to entrain cardiac tissues by generation of repetitive APs, leading in some cases to ventricular fibrillation and hemodynamic collapse in vivo. Therefore, an inhibition method that does not lead to entrainment - irrespective of the stimulation amplitude (bound to fluctuate in an in vivo setting) - is highly desirable.Methodology/principal findingsWe investigated the effects of broader amplitude and frequency ranges on the inhibitory effects of extracellular AC stimulation on HL-1 cardiomyocytes cultured on microelectrode arrays, using both sinusoidal and square waveforms. Our results indicate that, at sufficiently high frequencies, cardiac tissue exhibits a binary response to stimulus amplitude with either prolonged APs or no effect, thereby effectively avoiding the risks of entrainment by repetitive firing observed at lower frequencies. We further demonstrate the ability to precisely define reversible local conduction blocks in beating cultures without influencing the propagation activity in non-blocked areas. The conduction blocks were spatiotemporally controlled by electrode geometry and stimuli duration, respectively, and sustainable for long durations (300 s).Conclusion/significanceInhibition of cardiac excitation induced by high-frequency AC stimulation exhibits a binary response to amplitude above a threshold frequency, enabling the generation of reversible conduction blocks without the risks of entrainment. This inhibition method could yield novel approaches for arrhythmia modeling in vitro, as well as safer and more efficacious tools for in vivo cardiac mapping and radio-frequency ablation guidance applications.

Project description:Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.

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Spatiotemporally controlled microchannels of periodontal mimic scaffolds.

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Spatiotemporally controlled microchannels of periodontal mimic scaffolds.

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