layer-layer-collagen-deposition-microfluidic-devices-for-microtissue

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Massachusetts General Hospital, Harvard Medical School

Shriners Hospitals for Children-Boston

Osteoblasts respond to mechanical stimulation by changing morphology, gene expression and matrix mineralization. Introducing surface topography on biomaterials, independently of mechanical loading, has been reported to give similar effects. In the current study, using a nanotextured surface, and mechanical loading, we aimed to develop a multi-factorial model in which both parameters interact. Mechanical stimulation to osteoblast-like cells was applied by longitudinal stretch in parallel direction to the nanotexture (300 nm wide and 60 nm deep grooves), with frequency of 1 Hz and stretch magnitude varying from 1% to 8%. Scanning electron microscopy showed that osteoblast-like cells subjected to mechanical loading oriented perpendicularly to the stretch direction. When cultured on nanotextured surfaces, cells aligned parallel to the texture. However, the parallel cell direction to the nanotextured surface was lost and turned to perpendicular when parallel stretch to the nanotexture, greater than 3% was applied to the cells. This phenomenon could not be achieved when a texture with micro-sized dimensions was used. Moreover, a significant synergistic effect on upregulation of fibronectin and Cfba was observed when dual stimulation was used. These findings can lead to a development of new biomimetic materials that can guide morphogenesis in tissue repair and bone remodeling.

The interaction between nanoscale surface features and mechanical loading and its effect on osteoblast-like cells behavior.

The immune response to an implanted biomaterial is orchestrated by macrophages. In this study various nanogrooved patterns were created by using laser interference lithography and reactive ion etching. The created nanogrooves mimic the natural extracellular matrix environment. Macrophage cell culture demonstrated that interleukin 1β and TNF-α cytokine production were upregulated on nanogrooved substrates. In vivo subcutaneous implantation in a validated mouse cage model for 14 days demonstrated that nanogrooves enhanced and guided cell adhesion, and few multinucleated cells were formed. In agreement with the in vitro results, cytokine production was found to be nanogroove dependent, as interleukin 1β, TNF-α, TGF-β and osteopontin became upregulated. The results indicate that biomaterial surface texturing, especially at the nanometric scale, can be used to control macrophage activation to induce a wound healing response, rather than a profound inflammatory response. From the Clinical Editor: The authors investigate various nano-grooved patterns that mimic the natural extracellular matrix environment and demonstrate (both in macrophage cultures and in vivo) that interleukin 1β and TNF-α cytokine production is dependent upon surface texturing at the nanometric scale. They propose that modified surfaces may trigger macrophage activation to promote a wound healing response.

In vitro and in vivo evaluation of the inflammatory response to nanoscale grooved substrates.

Mimicking the structural nanomolecular extracellular matrix with synthetically designed nanosized materials is a relatively new approach, which can be applied in the field of bone tissue engineering. Likewise, bone tissue-engineered constructs can be aided in their development by the use of several types of mechanical stimuli. In this study, we wanted to combine nanotextured biomaterials and centrifugation in one multifactorial system. Mesenchymal stem cells were isolated from rat bone marrow, and cultured on a nanogrooved polystyrene substrate (200-nm-wide pitch with a depth of 50 nm). Constant centrifugation of 10 g was applied to cells up to 7 days. Results showed that on a nanogrooved substrate osteoblast-like cells align parallel to the groove direction. Centrifugation of 10 g also affected cell morphology on a smooth surface. Moreover, cell alignment was significantly reduced for cells grown on nanogrooved substrates, which were subsequently subjected to centrifugation. Independently, both stimuli increased the number of cells after 7 days of culture. However, when both stimuli were combined, an additive effect on cell number was observed, followed by an enhanced effect on osteocalcin mRNA expression and matrix mineralization. In conclusion, biomaterial surface modification as well as centrifugation are effective means to enhance bone cell behavior, moreover, readily available to many tissue engineers.

Substrate nanotexture and hypergravity through centrifugation enhance initial osteoblastogenesis.

Cells in situ are surrounded with defined structural elements formed by the nanomolecular extracellular matrix (ECM), and at the same time subjected to different mechanical stimuli arising from variety of physiological processes. In this study, using a nanotextured substrate mimicking the structural elements of the ECM and simulated microgravity, we wanted to develop a multifactorial model and understand better what guides cells in determining the morphological cell response. In our set-up, bone precursor cells from rat bone marrow were isolated and cultured on nanotextured polystyrene substrate (pitch 200 nm, depth 50 nm). Simulated microgravity was applied to the cells, using a random positioning machine (RPM). The results demonstrated that cells cultured on nanotextured substrate align parallel to the grooves and re-align significantly, but not completely, when subjected to simulated microgravity. The nanotextured substrate increased cell number and alkaline phosphatase (ALP) activity, whereas simulated microgravity decreased cells number and ALP activity. When the nanotextured substrate and simulated microgravity were combined together, the negative effect of the simulated microgravity ALP and cell number was reversed. In conclusion, absence of mechanical load in simulated microgravity has a negative effect on initial osteoblastogenesis, and nanotextured surfaces can partly reverse such a process.

Nanostructured substrate conformation can decrease osteoblast-like cell dysfunction in simulated microgravity conditions.

Periodontal ligament (PDL) cells play an important role in regulating osseous remodeling and ligament formation. Mechanical loading and the specific cellular environment are involved in these processes, regulating cell behavior. However, most in vitro experimental setups investigate mechanical loading or substrate texture separately and thus do not fully represent the PDL microenvironment. Therefore, the authors investigated the influence of combined mechano-topographical stimuli on PDL cell morphology, proliferation, and osteogenic and ligament differentiation.

Regulation of periodontal ligament cell behavior by cyclic mechanical loading and substrate nanotexture.

Laser profiling of titanium has been of considerable interest in the field of oral implantology. However, very few pre-clinical and clinical studies have been performed with laser-treated implants, especially focusing on isotropic roughness topography. The aim of the study was to compare the cortical bone response of Ti-implants discs treated with pico-sec pulsed laser (LAS) and conventional grit-blasted/acid-etched (GAE) method.

In vivo comparison between laser-treated and grit blasted/acid etched titanium.

Designing biomaterial surfaces to control the reaction of the surrounding tissue is still considered to be a primary issue, which needs to be addressed systematically. Although numerous in vitro studies have described different nano-metrically textured substrates capable to influence bone cellular response, in vivo studies validating this phenomenon have not been reported. In this study, nano-grooved silicon stamps were produced by laser interference lithography (LIL) and reactive ion etching (RIE) and were subsequently transferred onto the surface of 5 mm diameter Titanium (Ti) discs by nanoimprint lithography (NIL). Patterns with pitches of 1000 nm (500 nm ridge and groove, 150 nm depth), 300 nm (150 nm ridge and groove, 120 nm depth; as well as a 1:3 ratio of 75 nm ridge and 225 nm groove, 120 nm depth) and 150 nm (75 nm ridge and groove, 30 nm depth) were created. These samples were implanted in a rabbit tibia cortical bone. Histological evaluation and histomorphometric measurements were performed, comparing each sample to conventional grit-blasted/acid-etched (GAE) titanium controls. Results showed a significantly higher bone-to-implant contact at 4 weeks for the 300 nm (1:3) specimens, compared to GAE (p = 0.006). At 8 weeks, there was overall more bone contact compared to 4 weeks. However, no significant differences between the nano-textured samples and the GAE occurred. Further studies will need to address biomechanical testing and the use of trabecular bone models.

The effect of nanometric surface texture on bone contact to titanium implants in rabbit tibia.

The development of emerging in vitro tissue culture platforms can be useful for predicting human response to new compounds, which has been traditionally challenging in the field of drug discovery. Recently, several in vitro tissue-like microsystems, also known as 'organs-on-a-chip', have emerged to provide new tools for better evaluating the effects of various chemicals on human tissue.

Organs-on-a-chip: a new tool for drug discovery.

The development of long-term human organotypic liver-on-a-chip models for successful prediction of toxic response is one of the most important and urgent goals of the NIH/DARPA's initiative to replicate and replace chronic and acute drug testing in animals. For this purpose, we developed a microfluidic chip that consists of two microfluidic chambers separated by a porous membrane. The aim of this communication is to demonstrate the recapitulation of a liver sinusoid-on-a-chip, using human cells only for a period of 28 days. Using a step-by-step method for building a 3D microtissue on-a-chip, we demonstrate that an organotypic in vitro model that reassembles the liver sinusoid microarchitecture can be maintained successfully for a period of 28 days. In addition, higher albumin synthesis (synthetic) and urea excretion (detoxification) were observed under flow compared to static cultures. This human liver-on-a-chip should be further evaluated in drug-related studies. Biotechnol. Bioeng. 2015;9999: 1-6. © 2015 Wiley Periodicals, Inc.

Article	Year
The interaction between nanoscale surface features and mechanical loading and its effect on osteoblast-like cells behavior. Biomaterials\| PubMed ID: 20647152	2010
In vitro and in vivo evaluation of the inflammatory response to nanoscale grooved substrates. Nanomedicine : nanotechnology, biology, and medicine\| PubMed ID: 21704595	2012
Substrate nanotexture and hypergravity through centrifugation enhance initial osteoblastogenesis. Tissue engineering. Part A\| PubMed ID: 22871341	2013
Nanostructured substrate conformation can decrease osteoblast-like cell dysfunction in simulated microgravity conditions. Journal of tissue engineering and regenerative medicine\| PubMed ID: 22941788	2014
Regulation of periodontal ligament cell behavior by cyclic mechanical loading and substrate nanotexture. Journal of periodontology\| PubMed ID: 23215671	2013
In vivo comparison between laser-treated and grit blasted/acid etched titanium. Clinical oral implants research\| PubMed ID: 23346926	2014
The effect of nanometric surface texture on bone contact to titanium implants in rabbit tibia. Biomaterials\| PubMed ID: 23380354	2013
Organs-on-a-chip: a new tool for drug discovery. Expert opinion on drug discovery\| PubMed ID: 24620821	2014
Long-term maintenance of a microfluidic 3D human liver sinusoid. Biotechnology and bioengineering\| PubMed ID: 26152452	2015

Ljupcho Prodanov

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