Generation and characterization of two immortalized dermal fibroblast cell lines from the spiny mouse (Acomys).

The spiny mouse (Acomys) is gaining popularity as a research organism due to its phenomenal regenerative capabilities. Acomys recovers from injuries to several organs without fibrosis. For example, Acomys heals full thickness skin injuries with rapid re-epithelialization of the wound and regeneration of hair follicles, sebaceous glands, erector pili muscles, adipocytes, and dermis without scarring. Understanding mechanisms of Acomys regeneration may uncover potential therapeutics for wound healing in humans. However, access to Acomys colonies is limited and primary fibroblasts can only be maintained in culture for a limited time. To address these obstacles, we generated immortalized Acomys dermal fibroblast cell lines using two methods: transfection with the SV40 large T antigen and spontaneous immortalization. The two cell lines (AcoSV40 and AcoSI-1) maintained the morphological and functional characteristics of primary Acomys fibroblasts, including maintenance of key fibroblast markers and ECM deposition. The availability of these cells will lower the barrier to working with Acomys as a model research organism, increasing the pace at which new discoveries to promote regeneration in humans can be made.

Label-Free Quantification of Microscopic Alignment in Engineered Tissue Scaffolds by Polarized Raman Spectroscopy.

Monitoring of extracellular matrix (ECM) microstructure is essential in studying structure-associated cellular processes, improving cellular function, and for ensuring sufficient mechanical integrity in engineered tissues. This paper describes a novel method to study the microscale alignment of the matrix in engineered tissue scaffolds (ETS) that are usually composed of a variety of biomacromolecules derived by cells. First, a trained loading function was derived from Raman spectra of highly aligned native tissue via principal component analysis (PCA), where prominent changes associated with specific Raman bands (e.g., 1444, 1465, 1605, 1627-1660, and 1665-1689 cm-1) were detected with respect to the polarization angle. These changes were mainly caused by the aligned matrix of many compounds within the tissue relative to the laser polarization, including proteins, lipids, and carbohydrates. Hence this trained function was applied to quantify the alignment within ETS of various matrix components derived by cells. Furthermore, a simple metric called Amplitude Alignment Metric (AAM) was derived to correlate the orientation dependence of polarized Raman spectra of ETS to the degree of matrix alignment. It was found that the AAM was significantly higher in anisotropic ETS than isotropic ones. The PRS method revealed a lower p-value for distinguishing the alignment between these two types of ETS as compared to the microscopic method for detecting fluorescent-labeled protein matrices at a similar microscopic scale. These results indicate that the anisotropy of a complex matrix in engineered tissue can be assessed at the microscopic scale using a PRS-based simple metric, which is superior to the traditional microscopic method. This PRS-based method can serve as a complementary tool for the design and assessment of engineered tissues that mimic the native matrix organizational microstructures.

Spiny mice (Acomys) cells fail to engraft in NOD scid gamma.

Immune cells and stromal cells regulate wound healing and regeneration through complex activation patterns with spatiotemporal variation. The scarless regeneration of Spiny mice (Acomys species) is no exception; differential activation of immune and stromal cell populations seems to play a role in its remarkable regenerative capacity. To elucidate the role and interplay of Acomys immune cells in mammalian regeneration, we sought to create Acomys-Mus chimeras by transplanting bone marrow (BM) from Acomys into NOD Scid Gamma (NSG), a severely immunodeficient mouse line often used in creating humanized mice. Here, we report that Acomys BM cells fail to reconstitute and differentiate when transferred to irradiated NSG adults and neonates. In addition, we did not detect the presence of donor cells nor observe the onset of Graft versus Host Disease (GvHD)-like pathology, even after transplanting Acomys splenocytes in Acomys-Mus chimeras suggesting early graft failure. Overall, these results demonstrate the adoptive transfer of Acomys BM alone is not sufficient to establish Acomys hematopoietic system in NSG mouse.

Surgeon confidence in planning total shoulder arthroplasty improves after consulting a clinical decision support tool.

PURPOSE: Software algorithms are increasingly available as clinical decision support tools (CDSTs) to support shared decision-making. We sought to understand if patient-specific predictions from a CDST would impact orthopedic surgeons' preoperative planning decisions and corresponding confidence. METHODS: We performed a survey study of orthopedic surgeons with at minimum of 2 years of independent shoulder arthroplasty experience. We generated patient profiles for 18 faux cases presenting with glenohumeral osteoarthritis and emailed 93 surgeons requesting their recommendation for anatomic or reverse total shoulder arthroplasty for each case and their certainty in their recommendation on a 4-point Likert scale. The thirty respondents were later sent a second survey with the same cases that now included predicted patient-specific outcomes and complication rates generated by a CDST. RESULTS: Initial recommendations and changes in recommendation varied widely by surgeon and by case. After viewing the results of the CDST, surgeons switched from anatomic to reverse recommendations in 46 instances (12% of initial anatomic) and from reverse to anatomic in 22 instances (6% of initial reverse). Overall, surgeon change in confidence increased significantly across all responses (p = 0.0001), with certain cases and certain surgeons having significant changes. Change in confidence did not correlate with surgeon-specific factors, including years in practice. CONCLUSION: The addition of CDST reports to preoperative planning for anatomic and reverse total shoulder arthroplasty informed decision-making but did not direct recommendations uniformly. However, the CDST information provided did increase surgeon confidence regardless of implant selection and irrespective of surgeon experience.

Biomaterial Design Inspired by Regenerative Research Organisms.

The efficacy of implanted biomaterials is largely dependent on the response of the host's immune and stromal cells. Severe foreign body response (FBR) can impede the integration of the implant into the host tissue and compromise the intended mechanical and biochemical function. Many features of FBR, including late-stage fibrotic encapsulation of implants, parallel the formation of fibrotic scar tissue after tissue injury. Regenerative organisms like zebrafish and salamanders can avoid fibrosis after injury entirely, but FBR in these research organisms is rarely investigated because their immune competence is much lower than humans. The recent characterization of a regenerative mammal, the spiny mouse (Acomys), has inspired us to take a closer look at cellular regulation in regenerative organisms across the animal kingdom for insights into avoiding FBR in humans. Here, we highlight how major features of regeneration, such as blastema formation, macrophage polarization, and matrix composition, can be modulated across a range of regenerative research organisms to elucidate common features that may be harnessed to minimize FBR. Leveraging a deeper understanding of regenerative biology for biomaterial design may help to reduce FBR and improve device integration and performance.

Breathing patterns and CO2 production in adult spiny mice (Acomys cahirinus).

The spiny mouse (Acomys) is a precocial mammal with unique regenerative abilities. We used whole-body plethysmography to describe the breathing patterns and CO2 production (VCO2) of adult spiny mice (n = 10 male, 10 female) and C57BL/6 mice (n = 9 male, 11 female). During quiet breathing, female but not male spiny mice had lower tidal volumes and CO2 production vs. C57BL/6 mice. During extended hypoxia (30 min), male and female spiny mice decreased VCO2 and tidal volume to a greater degree than C57BL/6 mice. During an acute hypoxic-hypercapnic respiratory challenge (10% O2, 7% CO2), male and female spiny mice had blunted ventilatory responses as compared to C57BL/6 mice, primarily from a diminished increase in respiratory rate. These data establish a baseline for studies of respiratory physiology and neurobiology in spiny mice in the context of their remarkable regenerative capacity and their unique background of a desert dwelling species.

Matrix produced by diseased cardiac fibroblasts affects early myotube formation and function.

The extracellular matrix (ECM) provides both physical and chemical cues that dictate cell function and contribute to muscle maintenance. Muscle cells require efficient mitochondria to satisfy their high energy demand, however, the role the ECM plays in moderating mitochondrial function is not clear. We hypothesized that the ECM produced by stromal cells with mitochondrial dysfunction (Barth syndrome, BTHS) provides cues that contribute to metabolic dysfunction independent of muscle cell health. To test this, we harnessed the ECM production capabilities of human pluripotent stem-cell-derived cardiac fibroblasts (hPSC-CFs) from healthy and BTHS patients to fabricate cell-derived matrices (CDMs) with controlled topography, though we found that matrix composition from healthy versus diseased cells influenced myotube formation independent of alignment cues. To further investigate the effects of matrix composition, we then examined the influence of healthy- and BTHS-derived CDMs on myotube formation and metabolic function. We found that BTHS CDMs induced lower fusion index, lower ATP production, lower mitochondrial membrane potential, and higher ROS generation than the healthy CDMs. These findings imply that BTHS-derived ECM alone contributes to myocyte dysfunction in otherwise healthy cells. Finally, to investigate potential mechanisms, we defined the composition of CDMs produced by hPSC-CFs from healthy and BTHS patients using mass spectrometry and identified 15 ECM and related proteins that were differentially expressed in the BTHS-CDM compared to healthy CDM. Our results highlight that ECM composition affects skeletal muscle formation and metabolic efficiency in otherwise healthy cells, and our methods to generate patient-specific CDMs are a useful tool to investigate the influence of the ECM on disease progression and to investigate variability among diseased patients. STATEMENT OF SIGNIFICANCE: Muscle function requires both efficient metabolism to generate force and structured extracellular matrix (ECM) to transmit force, and we sought to examine the interactions between metabolism and ECM when metabolic disease is present. We fabricated patient-specific cell derived matrices (CDMs) with controlled topographic features to replicate the composition of healthy and mitochondrial-diseased (Barth syndrome) ECM. We found that disease-derived ECM negatively affects metabolic function of otherwise healthy myoblasts, and we identified several proteins in disease-derived ECM that may be mediating this dysfunction. We anticipate that our patient-specific CDM system could be fabricated with other topographies and cell types to study cell functions and diseases of interest beyond mitochondrial dysfunction and, eventually, be applied toward personalized medicine.

Experimental and Computational Models of Transport of Galectin-3 Through Glycosylated Matrix.

Altered extracellular matrix (ECM) production is a hallmark of many fibroproliferative diseases, including certain cancers. The high incidence of glycan-rich components within altered ECM makes the use of glycan-binding proteins such as Galectin-3 (G3) a promising therapeutic strategy. The complexity of ECM as a rich 3D network of proteins with varied glycosylation states makes it challenging to determine the retention of glycan-binding proteins in altered ECM environments. Computational models capable of predicting the transport of glycan-binding proteins in altered ECM can benefit the design and testing of such proteins and associated novel therapeutic strategies. However, such computational models require many kinetic parameters that cannot be estimated from traditional 2D pharmacokinetic assays. To validate transport properties of G3 in 3D ECM constructs, we developed a species transport model that includes diffusion and matrix-binding components to predict retention of G3 fusion proteins in glycan-rich ECM. By iteratively comparing our computational model to experimental results, we are able to determine a reasonable range of parameters for a robust computational model of G3 transport. We anticipate this overall approach to building a data-driven model is translatable to other ECM-targeting therapeutic strategies.

Label-free quantification of soft tissue alignment by polarized Raman spectroscopy.

The organization of proteins is an important determinant of functionality in soft tissues. However, such organization is difficult to monitor over time in soft tissue with complex compositions. Here, we establish a method to determine the alignment of proteins in soft tissues of varying composition by polarized Raman spectroscopy (PRS). Unlike most conventional microscopy methods, PRS leverages non-destructive, label-free sample preparation. PRS data from highly aligned muscle layers were utilized to derive a weighting function for aligned proteins via principal component analysis (PCA). This trained weighting function was used as a master loading function to calculate a principal component score (PC1 Score) as a function of polarized angle for tendon, dermis, hypodermis, and fabricated collagen gels. Since the PC1 Score calculated at arbitrary angles was insufficient to determine level of alignment, we developed an Amplitude Alignment Metric by fitting a sine function to PC1 Score with respect to polarized angle. We found that our PRS-based Amplitude Alignment Metric can be used as an indicator of level of protein alignment in soft tissues in a non-destructive manner with label-free preparation and has similar discriminatory capacity among isotropic and anisotropic samples compared to microscopy-based image processing method. This PRS method does not require a priori knowledge of sample orientation nor composition and appears insensitive to changes in protein composition among different tissues. The Amplitude Alignment Metric introduced here could enable convenient and adaptable evaluation of protein alignment in soft tissues of varying protein and cell composition. STATEMENT OF SIGNIFICANCE: Polarized Raman spectroscopy (PRS) has been used to characterize the of organization of soft tissues. However, most of the reported applications of PRS have been on collagen-rich tissues and reliant on intensities of collagen-related vibrations. This work describes a PRS method via a multivariate analysis to characterize alignment in soft tissues composed of varying proteins. Of note, the highly aligned muscle layer of mouse skin was used to train a master function then applied to other soft tissue samples, and the degree of anisotropy in the PRS response was evaluated to obtain the level of alignment in tissues. We have demonstrated that this method supports convenient and adaptable evaluation of protein alignment in soft tissues of varying protein and cell composition.

Spiny mouse (Acomys): an emerging research organism for regenerative medicine with applications beyond the skin.

The spiny mouse (Acomys species) has emerged as an exciting research organism due to its remarkable ability to undergo scarless regeneration of skin wounds and ear punches. Excitingly, Acomys species demonstrate scar-free healing in a wide-range of tissues beyond the skin. In this perspective article, we discuss published findings from a variety of tissues to highlight how this emerging research organism could shed light on numerous clinically relevant human diseases. We also discuss the challenges of working with this emerging research organism and suggest strategies for future Acomys-inspired research.

Quantifying cellular forces: Practical considerations of traction force microscopy for dermal fibroblasts.

Traction force microscopy (TFM) is a well-established technique traditionally used by biophysicists to quantify the forces adherent biological cells exert on their microenvironment. As image processing software becomes increasingly user-friendly, TFM is being adopted by broader audiences to quantify contractility of (myo)fibroblasts. While many technical reviews of TFM's computational mechanics are available, this review focuses on practical experimental considerations for dermatology researchers new to cell mechanics and TFM who may wish to implement a higher throughput and less expensive alternative to collagen compaction assays. Here, we describe implementation of experimental methods, analysis using open-source software and troubleshooting of common issues to enable researchers to leverage TFM for their investigations into skin fibroblasts.

Potential Applications of Microfluidics to Acute Kidney Injury Associated with Viral Infection.

The kidneys are susceptible to adverse effects from many diseases, including several that are not tissue-specific. Acute kidney injury is a common complication of systemic diseases such as diabetes, lupus, and certain infections including the novel coronavirus (SARS-CoV-2). Microfluidic devices are an attractive option for disease modeling, offering the opportunity to utilize human cells, control experimental and environmental conditions, and combine with other on-chip devices. For researchers with expertise in microfluidics, this brief perspective highlights potential applications of such devices to studying SARS-CoV-2-induced kidney injury.

Raman Spectroscopy Methods to Characterize the Mechanical Response of Soft Biomaterials.

Raman spectroscopy has been used extensively to characterize the influence of mechanical deformation on microstructure changes in biomaterials. While traditional piezo-spectroscopy has been successful in assessing internal stresses of hard biomaterials by tracking prominent peak shifts, peak shifts due to applied loads are near or below the resolution limit of the spectrometer for soft biomaterials with moduli in the kilo- to mega-Pascal range. In this Review, in addition to peak shifts, other spectral features (e.g., polarized intensity and intensity ratio) that provide quantitative assessments of microstructural orientation and secondary structure in soft biomaterials and their strain dependence are discussed. We provide specific examples for each method and classify sensitive Raman characteristic bands common across natural (e.g., soft tissue) and synthetic (e.g., polymeric scaffolds) soft biomaterials upon mechanical deformation. This Review can provide guidance for researchers aiming to analyze micromechanics of soft tissues and engineered tissue constructs by Raman spectroscopy.

Animal Models to Study Emerging Technologies Against SARS-CoV-2.

New technologies are being developed toward the novel coronavirus SARS-CoV-2 to understand its pathogenesis and transmission, to develop therapeutics and vaccines, and to formulate preventive strategies. Animal models are indispensable to understand these processes and develop and test emerging technologies; however, the mechanism of infection for SARS-CoV-2 requires certain similarities to humans that do not exist in common laboratory rodents. Here, we review important elements of viral infection, transmission, and clinical presentation reflected by various animal models readily available or being developed and studied for SARS-CoV-2 to help bioengineers evaluate appropriate preclinical models for their emerging technologies. Importantly, applications of traditional mice and rat models are limited for studying SARS-CoV-2 and development of COVID-19. Non-human primates, Syrian hamsters, ferrets, cats, and engineered chimeras mimic the human infection more closely and hold strong potential as animal models of SARS-CoV-2 infection and progression of resulting human disease.

Tunable methacrylated hyaluronic acid-based hydrogels as scaffolds for soft tissue engineering applications.

Hyaluronic acid (HA)-based biomaterials have been explored for a number of applications in biomedical engineering, particularly as tissue regeneration scaffolds. Crosslinked forms of HA are more robust and provide tunable mechanical properties and degradation rates that are critical in regenerative medicine; however, crosslinking modalities reported in the literature vary and there are few comparisons of different scaffold properties for various crosslinking approaches. In this study, we offer direct comparison of two methacrylation techniques for HA (glycidyl methacrylate HA [GMHA] or methacrylic anhydride HA [MAHA]). The two methods for methacrylating HA provide degrees of methacrylation ranging from 2.4 to 86%, reflecting a wider range of properties than is possible using only a single methacrylation technique. We have also characterized mechanical properties for nine different tissues isolated from rat (ranging from lung at the softest to muscle at the stiffest) using indentation techniques and show that we can match the full range of mechanical properties (0.35-6.13 kPa) using either GMHA or MAHA. To illustrate utility for neural tissue engineering applications, functional hydrogels with adhesive proteins (either GMHA or MAHA base hydrogels with collagen I and laminin) were designed with effective moduli mechanically matched to rat sciatic nerve (2.47 ± 0.31 kPa). We demonstrated ability of these hydrogels to support three-dimensional axonal elongation from dorsal root ganglia cultures. Overall, we have shown that methacrylated HA provides a tunable platform with a wide range of properties for use in soft tissue engineering.

Treatment of volumetric muscle loss in mice using nanofibrillar scaffolds enhances vascular organization and integration.

Traumatic skeletal muscle injuries cause irreversible tissue damage and impaired revascularization. Engineered muscle is promising for enhancing tissue revascularization and regeneration in injured muscle. Here we fabricated engineered skeletal muscle composed of myotubes interspersed with vascular endothelial cells using spatially patterned scaffolds that induce aligned cellular organization, and then assessed their therapeutic benefit for treatment of murine volumetric muscle loss. Murine skeletal myoblasts co-cultured with endothelial cells in aligned nanofibrillar scaffolds form endothelialized and aligned muscle with longer myotubes, more synchronized contractility, and more abundant secretion of angiogenic cytokines, compared to endothelialized engineered muscle formed from randomly-oriented scaffolds. Treatment of traumatically injured muscle with endothelialized and aligned skeletal muscle promotes the formation of highly organized myofibers and microvasculature, along with greater vascular perfusion, compared to treatment of muscle derived from randomly-oriented scaffolds. This work demonstrates the potential of endothelialized and aligned engineered skeletal muscle to promote vascular regeneration following transplantation.

Mechanical Characterization by Mesoscale Indentation: Advantages and Pitfalls for Tissue and Scaffolds.

Regenerative medicine and tissue engineering are hindered by the lack of consistent measurements and standards for the mechanical characterization of tissue and scaffolds. Indentation methods for soft matter are favored because of their compatibility with small, arbitrarily shaped samples, but contact mechanics models required to interpret data are often inappropriate for soft, viscous materials. In this study, we demonstrate indentation experiments on a variety of human biopsies, animal tissue, and engineered scaffolds, and we explore the complexities of fitting analytical models to these data. Although objections exist to using Hertz contact models for soft, viscoelastic biological materials since soft matter violates their original assumptions, we demonstrate the experimental conditions that enable consistency and comparability (regardless of arguable misappropriation). Appropriate experimental conditions involving sample hydration, the indentation depth, and the ratio of the probe size to sample thickness enable repeatable metrics that are valuable when comparing synthetic scaffolds and host tissue, and bounds on these parameters are carefully described and discussed. We have also identified a reliable quasistatic parameter that can be derived from indentation data to help researchers compare results across materials and experiments. Although Hertz contact mechanics and linear viscoelastic models may constitute oversimplification for biological materials, the reporting of such simple metrics alongside more complex models is expected to support researchers in tissue engineering and regenerative medicine by providing consistency across efforts to characterize soft matter. Impact Statement To engineer replacement tissue requires a deep understanding of its biomechanical properties. Mesoscale indentation (between micron and millimeter length scales) is well-suited to characterize tissue and engineered replacements as it accommodates small, oddly shaped samples. However, it is easy to run afoul of the assumptions for common contact models when working with biological materials. In this study, we describe experimental procedures and modeling approaches that allow researchers to take advantage of indentation for biomechanical characterization while minimizing its weaknesses.

Unique behavior of dermal cells from regenerative mammal, the African Spiny Mouse, in response to substrate stiffness.

The African Spiny Mouse (Acomys spp.) is a unique outbred mammal capable of full, scar-free skin regeneration. In vivo, we have observed rapid reepithelialization and deposition of normal dermis in Acomys after wounding. Acomys skin also has a lower modulus and lower elastic energy storage than normal lab mice, Mus musculus. To see if the different in vivo mechanical microenvironments retained an effect on dermal cells and contributed to regenerative behavior, we examined isolated keratinocytes in response to physical wounding and fibroblasts in response to varying substrate stiffness. Classic mechanobiology paradigms suggest stiffer substrates will promote myofibroblast activation, but we do not see this in Acomys dermal fibroblasts (DFs). Though Mus DFs increase organization of α-smooth muscle actin (αSMA)-positive stress fibers as substrate stiffness increases, Acomys DFs assemble very few αSMA-positive stress fibers upon changes in substrate stiffness. Acomys DFs generate lower traction forces than Mus DFs on pliable surfaces, and Acomys DFs produce and modify matrix proteins differently than Mus in 2D and 3D culture systems. In contrast to Acomys DFs "relaxed" behavior, we found that freshly isolated Acomys keratinocytes retain the ability to close wounds faster than Mus in an in vitro scratch assay. Taken together, these preliminary observations suggest that Acomys dermal cells retain unique biophysical properties in vitro that may reflect their altered in vivo mechanical microenvironment and may promote scar-free wound healing.

Quantitative assessment of intestinal stiffness and associations with fibrosis in human inflammatory bowel disease.

Inflammatory bowel disease (IBD) continues to increase in prevalence in industrialized countries. Major complications of IBD include formation of fibrotic strictures, fistulas, reduced absorptive function, cancer risk, and the need for surgery. In other chronic gastrointestinal disease models, stiffness has been shown to precede fibrosis; therefore, stiffness may be a reasonable indicator of progression toward stricture formation in IBD patients. Herein, we seek to quantify tissue stiffness and characterize fibrosis in patients with IBD and to compare mechanical properties of unaffected human tissue to common animal species used for IBD studies. Inflamed and unaffected tissue from IBD patients and unaffected tissue from mice, pigs, and cows were indented using a custom device to determine the effective stiffness. Histology was performed on matched tissues, and total RNA was isolated from IBD tissue samples and used for gene expression analysis of pro-fibrotic genes. We observed an increase in the effective stiffness (steady-state modulus, SSM) (p < 0.0001) and increased expression of the collagen type I gene (COL1A1, p = 0.01) in inflamed tissue compared to unaffected areas in our IBD patient cohort. We also found that increased staining of collagen fibers in submucosa positively correlated with SSM (p = 0.093). We determined that unaffected animal bowel stiffness is significantly greater than similar human tissues, suggesting additional limitations on animal models for translational investigations regarding stiffness-related hypotheses. Taken together, our data support development of tools for evaluation of bowel stiffness in IBD patients for prognostic applications that may enable more accurate prediction of those who will develop fibrosis and more precise prescription of aggressive therapies.