Microchannel Scaffolds Research Articles

A Biological 3D Printer for the Preparation of Tissue Engineering Micro-Channel Scaffold

The clinical applications of tissue engineering are still limited by the lack of a functional vascular supply in tissue-engineered constructs. In order to improve the pre-vascularization of tissue-engineered scaffold during in vitro culture, in this study, based on three-dimensional (3D) printing technology, using the crosslinking effect of coaxial fluids (sodium alginate and CaCl2) to prepare vessel-like hollow gel fibers, then layer by layer overlapping into 3D scaffold. The biological 3D printing platform was successfully developed and a coaxial nozzle module was introduced to generate a CaCl2-in-Alginate coaxial microfluidic. The inner core diameters of the prepared hollow gel fibers were 220~380 micrometers. In addition, the influence of materials concentration and dispensing rates on hollow fiber dimension were investigated, the cell-encapsulated in the printed hollow fibers was realized and the viability of endothelial cells (ECs) was studied with Laser scanning confocal microscopy (LSCM) and Live-Dead cell staining. The 3D scaffold built by hollow fibers could improve the phenomenon of diffusion constrain and enhance the survival rate of those ECs growing at a greater depth in the construct. This study provides a new theoretical basis for the vascularization of bone scaffold.

Design and fabrication of biomimetic multiphased scaffolds for ligament-to-bone fixation

Conventional ligament grafts with single material composition cannot effectively integrate with the host bones due to mismatched properties and eventually affect their long-term function in vivo. Here we presented a multi-material strategy to design and fabricate composite scaffolds including ligament, interface and bone multiphased regions. The interface region consists of triphasic layers with varying material composition and porous structure to mimic native ligament-to-bone interface while the bone region contains polycaprolactone (PCL) anchor and microchanneled ceramic scaffolds to potentially provide combined mechanical and biological implant-bone fixation. Finite element analysis (FEA) demonstrated that the multiphased scaffolds with interference value smaller than 0.5 mm could avoid the fracture of ceramic scaffold during the implantation process, which was validated by in-vitro implanting the multiphased scaffolds into porcine joint bones. Pull-out experiment showed that the initial fixation between the multiphased scaffolds with 0.47 mm interference and the host bones could withstand the maximum force of 360.31±97.51 N, which can be improved by reinforcing the ceramic scaffolds with biopolymers. It is envisioned that the multiphased scaffold could potentially induce the regeneration of a new bone as well as interfacial tissue with the gradual degradation of the scaffold and subsequently realize long-term biological fixation of the implant with the host bone.

Handcrafted Microwire Regenerative Peripheral Nerve Interfaces with Wireless Neural Recording and Stimulation Capabilities

A scalable microwire peripheral nerve interface was developed, which interacted with regenerated peripheral nerves in microchannel scaffolds. Neural interface technologies are envisioned to facilitate direct connections between the nervous system and external technologies such as limb prosthetics or data acquisition systems for further processing. Presented here is an animal study using a handcrafted microwire regenerative peripheral nerve interface, a novel neural interface device for communicating with peripheral nerves. The neural interface studies using animal models are crucial in the evaluation of efficacy and safety of implantable medical devices before their use in clinical studies. 16-electrode microwire microchannel scaffolds were developed for both peripheral nerve regeneration and peripheral nerve interfacing. The microchannels were used for nerve regeneration pathways as a scaffolding material and the embedded microwires were used as a recording electrode to capture neural signals from the regenerated peripheral nerves. Wireless stimulation and recording capabilities were also incorporated to the developed peripheral nerve interface which gave the freedom of the complex experimental setting of wired data acquisition systems and minimized the potential infection of the animals from the wire connections. A commercially available wireless recording system was efficiently adopted to the peripheral nerve interface. The 32-channel wireless recording system covered 16-electrode microwires in the peripheral nerve interface, two cuff electrodes, and two electromyography electrodes. The 2-channel wireless stimulation system was connected to a cuff electrode on the sciatic nerve branch and was used to make evoked signals which went through the regenerated peripheral nerves and were captured by the wireless recording system at a different location. The successful wireless communication was demonstrated in the result section and the future goals of a wireless neural interface for chronic implants and clinical trials were discussed together.

Microchannel-based regenerative scaffold for chronic peripheral nerve interfacing in amputees

Neurally controlled prosthetics that cosmetically and functionally mimic amputated limbs remain a clinical need because state of the art neural prosthetics only provide a fraction of a natural limb's functionality. Here, we report on the fabrication and capability of polydimethylsiloxane (PDMS) and epoxy-based SU-8 photoresist microchannel scaffolds to serve as viable constructs for peripheral nerve interfacing through in vitro and in vivo studies in a sciatic nerve amputee model where the nerve lacks distal reinnervation targets. These studies showed microchannels with 100 μm × 100 μm cross-sectional areas support and direct the regeneration/migration of axons, Schwann cells, and fibroblasts through the microchannels with space available for future maturation of the axons. Investigation of the nerve in the distal segment, past the scaffold, showed a high degree of organization, adoption of the microchannel architecture forming ‘microchannel fascicles’, reformation of endoneurial tubes and axon myelination, and a lack of aberrant and unorganized growth that might be characteristic of neuroma formation. Separate chronic terminal in vivo electrophysiology studies utilizing the microchannel scaffolds with permanently integrated microwire electrodes were conducted to evaluate interfacing capabilities. In all devices a variety of spontaneous, sensory evoked and electrically evoked single and multi-unit action potentials were recorded after five months of implantation. Together, these findings suggest that microchannel scaffolds are well suited for chronic implantation and peripheral nerve interfacing to promote organized nerve regeneration that lends itself well to stable interfaces. Thus this study establishes the basis for the advanced fabrication of large-electrode count, wireless microchannel devices that are an important step towards highly functional, bi-directional peripheral nerve interfaces.

A microchannel neural interface with embedded microwires targeting the peripheral nervous system

The essential purpose of neural interfaces is to record and stimulate neural activity. Careful analysis of neural signals allows them to be used to control technologies such as prosthetics and muscle stimulators that restore lost functionality caused by injury. Designing interfaces advanced enough to restore full functionality, however, is a challenge. Presented here is an animal study using the Texas regenerative peripheral nerve interface (TxNI), a novel neural interface device for communicating with peripheral nerves. Small extracellular potentials and concentration of current at the nodes of Ranvier increases the complexity of recording from axons. The TxNI is designed to obtain weak neural signals which are difficult to acquire using other techniques. The TxNI combines a PDMS (polydimethylsiloxane) microchannel scaffold with microwires (used as recording electrodes) embedded within the microchannels. Axonal regeneration is confined to the microchannels and electrodes are long enough to record from the nodes of Ranvier. Although several types of electrodes for peripheral nerve interfaces have been reported, they typically record unwanted signals from surrounding musculature and are subject to crosstalk. To address this, neural regeneration is directed through the TxNI in sealed microfluidic channels. The TxNI was successfully implanted in the rat sciatic nerve in the animal facility at UTPA. The details regarding its fabrication and implantation are described here. The present result is to be used to evaluate the effectiveness of the TxNI for use in advanced prosthetics. In particular, it aims to be used in a noninvasive brain-machine interface for bidirectional prosthetic arms.

Capillary action: enrichment of retention and habitation of cells via micro-channeled scaffolds for massive bone defect regeneration.

The development of a biomaterial substitute that can promote bone regeneration in massive defects has remained as a significant clinical challenge even using bone marrow cells or growth factors. Without an active, thriving cell population present throughout and stable anchored to the construct, exceptional bone regeneration does not occur. An engineered micro-channel structures scaffold within each trabecular has been designed to overcome some current limitations involving the cultivation and habitation of cells in large, volumetric scaffolds to repair massive skeletal defect. We created a scaffold with a superior fluid retention capacity that also may absorb bone marrow cells and provide growth factor-containing body fluids such as blood clots and/or serum under physiological conditions. The scaffold is composed of 3 basic structures (1) porous trabecular network (300-400μm) similar to that of human trabecular bones, (2) micro-size channels (25-70μm) within each trabecular septum which mimic intra-osseous channels such as Haversian canals and Volkmann's canals with body fluid access, diffusion, nutritional supply and gas exchange, and (3) nano-size pores (100-400nm) on the surface of each septum that allow immobilized cells to anchor. Combinatorial effects of these internal structures result in a host-adapting construct that enhances cell retention and habitation throughout the 3cm-height and 4cm-length bridge-shaped scaffold.

Effect of capillary action on bone regeneration in micro-channeled ceramic scaffolds

A new scaffold design was introduced with macro-pores and micro-channels, which greatly assisted in the initial bone marrow absorption and uniform cell distribution. Unfortunately, the underlying scientific reasons for the new scaffold׳s efficiency are currently unknown. Hence, we approached using a mathematical and experimental method to elucidate the new scaffold׳s efficiency. The mathematical formula describe rising fluid height in a narrow cylindrical vessel due to capillary action. Through the mathematical simulation, the maximum fluid heights at equilibrium for scaffold tubes of diameters 50, 150, 350, and 750µm were 156.6, 52.7, 22.6, and 10.5mm, respectively. The fluid would theoretically reach 90% of the maximum height at 900, 30, 3, and 0.3s, respectively. In the experiment, the fluid heights were observed from 30 to 600s. All the scaffolds had 50µm micro-channels with different macro-pore sizes of 150, 350, and 750µm. The media rose through macro-pores of the three scaffolds until 40, 15, and 10mm, respectively. The fluid heights were observed at about 2s and 0.5s after being immersed for the 350µm and 750µm macro-pore scaffolds. In the case of the 150µm sample, the fluid height was 30mm at about 30s and 40mm at about 75s. Since all samples had 50µm micro-channels, the fluid reached to the top of the scaffolds, eventually. The results showed that capillary action was highly dependent on the size of the tubes within the scaffold. They also confirmed the simulated data in both equilibrium height and the time trajectory. The data from both the experiment and the mathematical simulation proved our hypothesis that capillary action was the cause for the improvement in cell immigration in the new scaffold since the data matched each other in both equilibrium height and the time trajectory.

In vivo testing of a 3D bifurcating microchannel scaffold inducing separation of regenerating axon bundles in peripheral nerves

Artificial nerve guidance channels enhance the regenerative effectiveness in an injured peripheral nerve but the existing design so far has been limited to basic straight tubes simply guiding the growth to bridge the gap. Hence, one of the goals in development of more effective neuroprostheses is to create bidirectional highly selective neuro–electronic interface between a prosthetic device and the severed nerve. A step towards improving selectivity for both recording and stimulation have been made with some recent in vitro studies which showed that three-dimensional (3D) bifurcating microchannels can separate neurites growing on a planar surface and bring them into contact with individual electrodes. Since the growing axons in vivo have the innate tendency to group in bundles surrounded by connective tissue, one of the big challenges in neuro–prosthetic interface design is how to overcome it. Therefore, we performed experiments with 3D bifurcating guidance scaffolds implanted in the sciatic nerve of rats to test if this new channel architecture could trigger separation pattern of ingrowth also in vivo. Our results showed that this new method enabled the re-growth of neurites into channels with gradually diminished width (80, 40 and 20 µm) and facilitated the separation of the axonal bundles with 91% success. It seems that the 3D bifurcating scaffold might contribute towards conveying detailed neural control and sensory feedback to users of prosthetic devices, and thus could improve the quality of their daily life.

Mechanical stimulation of fibroblasts in micro‐channeled bacterial cellulose scaffolds enhances production of oriented collagen fibers

Cellulose perforated by micro-channels (Ø ~500 μm) has been investigated as a potential future scaffold material for meniscus implants. Scaffolds seeded with 3T6 fibroblasts were cultivated with mechanical stimulation in a compression bioreactor for enhanced collagen production. Constructs under dynamic compression at a frequency of 0.1 Hz and compression strain of 5% were compared to static cultures used as controls. The three-dimensional distributions of collagen fibers and fibroblasts in the cellulose scaffolds were studied under native, soft-matter conditions by combined second harmonic generation and coherent antiStokes Raman scattering microscopy, requiring no artificial sample preparation. Results showed that the micro-channels facilitated the alignment of cells and collagen fibers and that collagen production was enhanced by mechanical stimulation. Thus, cell-seeded, micro-channeled cellulose scaffolds provided guided tissue growth required to obtain an ultrastructure mimicking that of the meniscus.

Guiding the morphogenesis of dissociated newborn mouse retinal cells and hES cell-derived retinal cells by soft lithography-patterned microchannel PLGA scaffolds

Embryonic stem (ES) cell-derived photoreceptors are a promising cell source for enhanced in vitro models of retinal degenerative diseases, but the more differentiated characteristics of retinal cells do not typically develop in dissociated cell cultures. Therefore, we have reconstructed organized retinal tissue by seeding dissociated cells into an array of aligned units that more faithfully mimics the retina. We solvent-processed poly(lactic-co-glycolic acid) (PLGA) into a microchannel scaffold format to achieve this geometric constraint. We compared the effect of PLGA concentration on channel morphology and, along with other culture conditions, on the infiltration of dissociated newborn mouse retinal cells into the channels. Culturing scaffolds at the gas–liquid interface with low serum media increased infiltrated rod photoreceptor viability 18-fold over submerged, high serum cultures when evaluated after seven days. Rod photoreceptors and Müller glia aligned processes parallel to the microchannel walls. Otx2+ and Pax6+ subpopulations recapitulated lamination behavior. Further, we constructed scaffold/retinal pigment epithelium (RPE) co-cultures and observed rods extending rhodopsin-positive processes toward RPE cells, mimicking normal rod polarization and morphology. Finally, human embryonic stem cell-derived photoreceptors exhibited infiltration and morphological characteristics similar to mouse retinal cells inside the scaffolds. These findings constitute an important advance in generating tissue-level retinal models from dissociated cells for use as drug screening platforms and in regenerative medicine.

A new generation of sodium chloride porogen for tissue engineering

Porogen leaching is a widely used and simple technique for the creation of porous scaffolds in tissue engineering. Sodium chloride (NaCl) is the most commonly used porogen, but the current grinding and sieving methods generate salt particles with huge size variations and cannot generate porogens in the submicron size range. We have developed a facile method based on the principles of crystallization to precisely control salt crystal sizes down to a few microns within a narrow size distribution. The resulting NaCl crystal size could be controlled through the solution concentration, crystallization temperature, and crystallization time. A reduction in solution temperature, longer crystallization times, and an increase in salt concentration resulted in an increase in NaCl crystal sizes due to the lowered solubility of the salt solution. The nucleation and crystallization technique provides superior control over the resulting NaCl size distribution (13.78 ± 1.18 μm), whereas the traditional grinding and sieving methods produced NaCl porogens 13.89 ± 12.49 μm in size. The resulting NaCl porogens were used to fabricate scaffolds with increased interconnectivity, porous microchanneled scaffolds, and multiphasic vascular grafts. This new generation of salt porogen provides great freedom in designing versatile scaffolds for various tissue-engineering applications.

Precision microchannel scaffolds for central and peripheral nervous system repair

In previous studies, we demonstrated the ability to linearly guide axonal regeneration using scaffolds comprised of precision microchannels 2 mm in length. In this work, we report our efforts to augment the manufacturing process to achieve clinically relevant scaffold dimensions in the centimeter-scale range. By selective etching of multi-component fiber bundles, agarose hydrogel scaffolds with highly ordered, close-packed arrays of microchannels, ranging from 172 to 320 μm, were fabricated with overall dimensions approaching clinically relevant length scales. Cross-sectional analyses determined that the maximum microchannel volume per unit volume of scaffold approached 80%, which is nearly twice that compared to our previously reported study. Statistical analyses at various points along the length of the microchannels also show a significant degree of linearity along the entire length of the scaffold. Two types of multi-component fiber bundle templates were evaluated; polystyrene and poly(methyl methacrylate). The scaffolds consisting of 2 cm long microchannels were fabricated with the poly(methyl methacrylate) fiber-cores exhibited a higher degree of linearity compared to those fabricated using polystyrene fibers. It is believed that the materials process developed in this study is useful for fabricating high aspect ratio microchannels in biocompatible materials with a wide range of geometries for guiding nerve regeneration.

Regulating orientation and phenotype of primary vascular smooth muscle cells by biodegradable films patterned with arrays of microchannels and discontinuous microwalls

Vascular smooth muscle cells (vSMCs) cultured in vitro are known to exhibit phenotype hyperplasticity. This plasticity is potentially very useful in tissue engineering of blood vessels. The synthetic phenotype is necessary for cell proliferation on the tissue scaffold but the cells must ultimately assume a quiescent, contractile phenotype for normal vascular function. In vitro control of vSMC phenotype has been challenging. This study shows that microchannel scaffolds with discontinuous walls can support primary vSMC proliferation and, when the cells reach confluence inside the channels, transform the cell phenotype towards greater contractility and promote cell alignment. A thorough time-resolved study was undertaken to characterize the expression of the contractile proteins alpha-actin, calponin, myosin heavy chain (MHC) and smoothelin as a function of time and initial cell density on microchannel scaffolds. The results consistently indicate that primary vSMCs cultured on the microchannel substrate substantially align parallel to the microwalls, become more elongated and significantly increase their expression of contractile proteins only when the cells reach confluence. MHC immunostaining was visible in the micropatterned cells after confluence but not in flat substrate cells or non-confluent micropatterned cells, which further verifies the increased contractility of the confluent channel-constrained vSMCs. The higher total amount of deposited elastin and collagen in confluent flat cultures than in confluent micropatterned cultures also provides confirmation of the higher contractility of the channel-constrained cells. These results establish that our microchanneled film can trigger the switch of primary vSMCs from a proliferative state to a more contractile phenotype at confluence.

Bifurcating microchannels as a scaffold to induce separation of regenerating neurites

Many neural interfacing strategies, such as the sieve electrode and the cultured probe, rely on neurite growth to establish neural contact. But this growth is subject to natural fasciculation, compromising the effectiveness of these interfacing strategies by reducing potential selectivity. This in vitro study shows that the fasciculation mechanism can be manipulated by providing an appropriate microchannel scaffold to guide and influence growth, thereby achieving a high degree of selectivity. The microchannels employed have a bifurcation from a primary channel into two secondary channels. This bifurcating microstructure was able to support and promote fasciculated growth over 70% of the time for microchannels widths of 2.5, 5, 10 and 20 µm. Fasciculation is shown to be a strong force during ingrowth, with the initiation of neurite separation related to random spatial exploration. Narrower microchannels initiate separated growth better. Once separated growth starts fasciculation results in an even distribution of neurite growth across the bifurcation. The reduction from 20 µm to 10 µm wide channels also resulted in a 3-fold decrease in ingrowing neurites performing 180° turns to exit the microchannel via the entrance. No neurite turning was observed for both the 5 and 2.5 µm wide channels.

Synthesis and characterization of a biodegradable elastomer featuring a dual crosslinking mechanism.

The need for advanced materials in emerging technologies such as tissue engineering has prompted increased research to produce novel biodegradable polymers elastic in nature and mechanically compliant with the host tissue. We have developed a soft biodegradable elastomeric platform biomaterial created from citric acid, maleic anhydride, and 1,8-octanediol, poly(octamethylene maleate (anhydride) citrate) (POMaC), which is able to closely mimic the mechanical properties of a wide range of soft biological tissues. POMaC features a dual crosslinking mechanism, which allows for the option of the crosslinking POMaC using UV irradiation and/or polycondensation to fit the needs of the intended application. The material properties, degradation profiles, and functionalities of POMaC thermoset networks can all be tuned through the monomer ratios and the dual crosslinking mechanism. POMaC polymers displayed an initial modulus between 0.03 and 1.54 MPa, and elongation at break between 48% and 534% strain. In vitro and in vivo evaluation using cell culture and subcutaneous implantation, respectively, confirmed cell and tissue biocompatibility. POMaC biodegradable polymers can also be combined with MEMS technology to fabricate soft and elastic 3D microchanneled scaffolds for tissue engineering applications. The introduction of POMaC will expand the choices of available biodegradable polymeric elastomers. The dual crosslinking mechanism for biodegradable elastomer design should contribute to biomaterials science.

Chitosan Microchannel Scaffolds for Tendon Tissue Engineering Characterized Using Optical Coherence Tomography

Tendon tissue engineering requires the generation of a uniaxially orientated collagen type I matrix with several organization scales that confer mechanical functionality upon the tendon. A combination of factors in a dose- and time-dependent manner, such as growth factors and mechanical environment, may be the key to an in vitro-engineered tendon. To define the progress of tissue development within a scaffold, on-line systems need to be applied to monitor the newly generated matrix. To address this challenge, we designed a new porous chitosan scaffold with microchannels (diameter: 250 microm), which allows primary porcine tenocytes to proliferate in a bundle-like structure. The cell proliferation and extracellular matrix (ECM) production within the microchannels were successfully assessed under sterile conditions using optical coherence tomography (OCT). A semi-quantitative method that calculated the microchannel occupation ratio (the degree of cell proliferation and tissue turnover based on the total backscattered intensity in the microchannels) was developed. We further investigated the effect of different culture conditions on tendon cell matrix formation. Using a perfusion bioreactor, we demonstrated how fluid flow can increase (p < 1e(3)) ECM production within the microchannels significantly more than static culture. Our study illustrates how using a guiding scaffold in combination with the fast and non-destructive assessment of the microstructure using OCT allows discrimination between the parameters affecting the production and the organization of the ECM.

Chitosan Micro-Channel Scaffolds for Tendon Tissue Engineering Characterized Using Optical Coherence Tomography

Chitosan Micro-Channel Scaffolds for Tendon Tissue Engineering Characterized Using Optical Coherence Tomography

Influence of channel width on alignment of smooth muscle cells by high‐aspect‐ratio microfabricated elastomeric cell culture scaffolds

Engineered smooth muscle tissue requires ordered configurations of cells to reproduce native function, and microtechnology offers possibilities for physically and chemically controlling cell organization with high spatial resolution. In this work, poly(dimethylsiloxane) microchannel scaffolds, modified by layer-by-layer self-assembly of polyelectrolytes to promote cell adhesion, were evaluated for use as substrates for the culture of aligned smooth muscle cells. The hypothesis that narrower channels would result in better alignment was tested using channel width dimensions of 20, 30, 40, 50, and 60 microm, in addition to flat (control) surfaces. Alignment of cells was assessed by two different methods, each sensitive to a different aspect of cell alignment from fluorescence micrographs. Two-dimensional fast Fourier transform analysis was performed to analyze the orientation distribution of actin filaments in cells. This was complemented by connectivity analysis of stained nuclei to obtain nuclear orientation distributions. Both methods produced consistent data that support the hypothesis that narrow microchannels promote a highly aligned culture of smooth muscle cells, and the degree of alignment is dependent on the microchannel width. Precise replication of in vivo cell alignment in engineered tissue, with the ability to tailor specific surface chemistries of the scaffold to the desired application, will potentially allow the production of artificial tissue that more closely duplicates the structure and function of native tissue.