Comparison of two decellularized dermal equivalents

Immunologically inert allogeneic acellular dermal scaffolds provide a matrix with molecular architecture close to native tissues, which synthetic scaffolds cannot. Not all nature‐derived scaffolds possess the same biological and physical properties. The different properties of scaffolds supporting cellular growth used for manufacturing tissue engineered grafts could lead to different implantation results. The scaffold properties should be carefully considered in order to meet the expected outcomes of tissue engineered grafts. In this report, we evaluated the cellular growth on AlloDerm® and Allopatch, 2 acellular scaffolds derived from human cadaver skin, using a fabricated 3D organotypic culture with primary human oral keratinocytes to produce an ex vivo produced oral mucosa equivalent (EVPOME). A well stratified epithelium could be constructed on both scaffolds. AlloDerm® and Allopatch EVPOMEs were also implanted into severe combined immunodeficiency mice to compare the ingrowth of blood vessels into the dermal component of the two EVPOMEs. Blood vessel counts were 3.3 times higher (p = .01) within Allopatch EVPOMEs than within AlloDerm® EVPOMEs. An oral and skin keratinocyte co‐culture, separated by a physical barrier to create a cell‐free zone, was used to evaluate cell migration on AlloDerm® and Allopatch. Slower cell migration was observed on Allopatch than on AlloDerm®.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143690/1/term2530.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143690/2/term2530_am.pd

The potential of label-free nonlinear optical molecular microscopy to non-invasively characterize the viability of engineered human tissue constructs

AbstractNonlinear optical molecular imaging and quantitative analytic methods were developed to non-invasively assess the viability of tissue-engineered constructs manufactured from primary human cells. Label-free optical measures of local tissue structure and biochemistry characterized morphologic and functional differences between controls and stressed constructs. Rigorous statistical analysis accounted for variability between human patients. Fluorescence intensity-based spatial assessment and metabolic sensing differentiated controls from thermally-stressed and from metabolically-stressed constructs. Fluorescence lifetime-based sensing differentiated controls from thermally-stressed constructs. Unlike traditional histological (found to be generally reliable, but destructive) and biochemical (non-invasive, but found to be unreliable) tissue analyses, label-free optical assessments had the advantages of being both non-invasive and reliable. Thus, such optical measures could serve as reliable manufacturing release criteria for cell-based tissue-engineered constructs prior to human implantation, thereby addressing a critical regulatory need in regenerative medicine

Characterizing Morphology and Nonlinear Elastic Properties of Normal and Thermally Stressed Engineered Oral Mucosal Tissues Using Scanning Acoustic Microscopy

This study examines the use of high-resolution ultrasound to monitor changes in the morphology and nonlinear elastic properties of engineered oral mucosal tissues under normal and thermally stressed culture conditions. Nonlinear elastic properties were determined by first developing strain maps from acoustic ultrasound, followed by fitting of nonlinear stress?strain data to a 1-term Ogden model. Testing examined a clinically developed ex vivo produced oral mucosa equivalent (EVPOME). As seeded cells proliferate on an EVPOME surface, they produce a keratinized protective upper layer that fills in and smoothens out surface irregularities. These transformations can also alter the nonlinear stress/strain parameters as EVPOME cells differentiate. This EVPOME behavior is similar to those of natural oral mucosal tissues and in contrast to an unseeded scaffold. If ultrasonic monitoring could be developed, then tissue cultivation could be adjusted in-process to account for biological variations in their development of the stratified cellular layer. In addition to ultrasonic testing, an in-house-built compression system capable of accurate measurements on small (?1.0?1.5?cm2) tissue samples is presented. Results showed a near 2.5-fold difference in the stiffness properties between the unstressed EVPOME and the noncell-seeded acellular scaffold (AlloDerm?). There were also 4?greater differences in root mean square values of the thickness in the unseeded AlloDerm compared to the mature unstressed EVPOME; this is a strong indicator for quantifying surface roughness.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140241/1/ten.tec.2012.0467.pd

Human Oral Mucosa Tissue-Engineered Constructs Monitored by Raman Fiber-Optic Probe

In maxillofacial and oral surgery, there is a need for the development of tissue-engineered constructs. They are used for reconstructions due to trauma, dental implants, congenital defects, or oral cancer. A noninvasive monitoring of the fabrication of tissue-engineered constructs at the production and implantation stages done in real time is extremely important for predicting the success of tissue-engineered grafts. We demonstrated a Raman spectroscopic probe system, its design and application, for real-time ex vivo produced oral mucosa equivalent (EVPOME) constructs noninvasive monitoring. We performed in vivo studies to find Raman spectroscopic indicators for postimplanted EVPOME failure and determined that Raman spectra of EVPOMEs preexposed to thermal stress during manufacturing procedures displayed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, giving a Raman metric to distinguish between healthy and compromised postimplanted constructs. This study is the step toward our ultimate goal to develop a stand-alone system, to be used in a clinical setting, where the data collection and analysis are conducted on the basis of these spectroscopic indicators with minimal user intervention.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140248/1/ten.tec.2013.0622.pd

Tissue-Engineered Constructs of Human Oral Mucosa Examined by Raman Spectroscopy

A noninvasive quality monitoring of tissue-engineered constructs is a required component of any successful tissue-engineering technique. During a 2-week production period, ex vivo produced oral mucosa-equivalent constructs (EVPOMEs) may encounter adverse culturing conditions that might compromise their quality and render them ineffective. We demonstrate the application of near-infrared Raman spectroscopy to in vitro monitoring of EVPOMEs during their manufacturing process, with the ultimate goal of applying this technology in situ to monitor the grafted EVPOMEs. We identify Raman spectroscopic failure indicators for less-than optimal EVPOMEs that are stressed by higher temperature and exposure to higher than normal concentration of calcium ions. Raman spectra of EVPOMEs exposed to thermal and calcium stress showed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs. We compared these results to histology and glucose consumption measurements, demonstrating that Raman spectroscopy is more sensitive and specific to changes in proteins' secondary structure not visible by HandE histology. We also exposed the EVPOMEs to rapamycin, a cell growth inhibitor and cell proliferation capacity preserver, and distinguished between EVPOMEs pretreated with 2?nM rapamycin and controls, using the ratio of the Amide III envelope to the phenylalanine band as an indicator.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140240/1/ten.tec.2012.0287.pd

Tissue-Engineered Constructs of Human Oral Mucosa Examined by Raman Spectroscopy

A noninvasive quality monitoring of tissue-engineered constructs is a required component of any successful tissue-engineering technique. During a 2-week production period, ex vivo produced oral mucosa-equivalent constructs (EVPOMEs) may encounter adverse culturing conditions that might compromise their quality and render them ineffective. We demonstrate the application of near-infrared Raman spectroscopy to in vitro monitoring of EVPOMEs during their manufacturing process, with the ultimate goal of applying this technology in situ to monitor the grafted EVPOMEs. We identify Raman spectroscopic failure indicators for less-than optimal EVPOMEs that are stressed by higher temperature and exposure to higher than normal concentration of calcium ions. Raman spectra of EVPOMEs exposed to thermal and calcium stress showed correlation of the band height ratio of CH(2) deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs. We compared these results to histology and glucose consumption measurements, demonstrating that Raman spectroscopy is more sensitive and specific to changes in proteins' secondary structure not visible by H&E histology. We also exposed the EVPOMEs to rapamycin, a cell growth inhibitor and cell proliferation capacity preserver, and distinguished between EVPOMEs pretreated with 2 nM rapamycin and controls, using the ratio of the Amide III envelope to the phenylalanine band as an indicator

Tuft cells are required for a rhinovirus-induced asthma phenotype in immature mice.

Infection of immature mice with rhinovirus (RV) induces an asthma-like phenotype consisting of type 2 inflammation, mucous metaplasia, eosinophilic inflammation and airways hyperresponsiveness which is dependent on IL-25 and type 2 innate lymphoid cells (ILC2s). Doublecortin-like kinase (DCLK)-1+ tuft cells are a major source of IL-25. We sought to determine the requirement of tuft cells for the RV-induced asthma phenotype in wild-type mice and mice deficient in Pou2f3, a transcription factor required for tuft cell development. C57Bl/6 mice infected with RV-A1B on day 6 of life and RV-A2 on day 13 of life showed increased DCLK1+ positive tuft cells in the large airways. Compared to wild-type mice, RV-infected Pou2f3-/- mice showed reductions in IL-25 mRNA and protein expression, ILC2 expansion, type 2 cytokine expression, mucous metaplasia, lung eosinophils and airway methacholine responsiveness. We conclude that airway tuft cells are required for the asthma phenotype observed in immature mice undergoing repeated RV infections. Furthermore, RV-induced tuft cell development provides a mechanism by which early life viral infections could potentiate type 2 inflammatory responses to future infections.http://deepblue.lib.umich.edu/bitstream/2027.42/192075/2/166136.1-20231207162808-covered-e0fd13ba177f913fd3156f593ead4cfd.pdfPublished onlineDescription of 166136.1-20231207162808-covered-e0fd13ba177f913fd3156f593ead4cfd.pdf : Accepted versio