Camilo Mora-Navarro

Decellularized extracellular matrix (dECM) scaffolds are critical for tissue engineering and regenerative medicine, yet standardized methods for real-time monitoring of decellularization remain lacking. Current protocols typically use fixed treatment durations to match endpoint control quality attributes (CQAs), such as final dsDNA content in the scaffold, without a mechanistic understanding of DNA removal kinetics. This knowledge gap leads to either incomplete decellularization (CQA rejections) or unnecessary processing time, compromising dECM scaffold quality and process efficiency. In this paper, we develop a model-based mathematical monitoring framework for rapid assessment of DNase-treated porcine lung tissue that integrates dual-optical sensing of supernatant DNA release via ultraviolet–visible (UV–vis) absorbance and fluorescence. Decellularization supernatant was collected every 10 min over a 100-min treatment window in each sample group. Signals were well described by a first-order accumulation model C(t) = C0 + (Cmax – C0) (1 – e–kt), yielding an interpretable kinetic descriptor k and a model-based progress metric. A linear relationship was observed between the initial tissue mass and rate constant (R2 = 0.9998), enabling mass-aware prediction of release kinetics. When explored in dimensionless coordinates, normalized trajectories collapsed onto the universal curve f(τ) = 1 – e–τ that supports shared kinetics across sensing modalities. Model-derived in-process metrics were associated with endpoint residual tissue dsDNA quantified by QuantiFluor tissue digests and revealed an accessibility-limited gap between supernatant signal completion and tissue-level DNA removal within the treatment window. Together, these results provide the foundation of a practical process analytical technology style framework for quantitative decellularization monitoring and quality control, supporting mass-aware forecasting of process progress and adaptive protocol development.

Controlling bacterial presence and inflammation in the larynx remains a significant challenge, especially in contexts where infection and immune responses can affect tissue health, prompting growing interest in developing bioactive materials that can be delivered locally and noninvasively to modulate microbial load and inflammatory responses. In this study, we developed and characterized aerosolizable formulations derived from decellularized extracellular matrix (dECMx) of porcine vocal fold lamina propria (VFLPx) and thyroid cartilage (Cx). These formulations were evaluated for their antibacterial and immunomodulatory effects. The dECMx formulations exhibited heterogeneous microstructures and aggregate sizes smaller than 5 μm, which is consistent with requirements for aerosol delivery. Antibacterial activity against Staphylococcus aureus was assessed through colony-forming unit (CFU) quantification and bacterial growth kinetics, comparing the liquid and aerosol forms. In liquid form, VFLPx reduced CFU counts by ∼66% and Cx by ∼16%, both showing statistically significant reductions compared to controls. In aerosolized form, both formulations demonstrated comparable antibacterial efficacy, with approximately ∼47% reductions in CFU for VFLPx and Cx. Immunomodulatory potential was evaluated using a THP-1 ascGFP reporter cell line, which was stimulated with LPS to activate NF-κB signaling. Exposure to both dECMx formulations significantly attenuated the LPS-induced GFP fluorescence, indicating a suppression of NF-κB-mediated activation. These in vitro results demonstrate that liquid dECM-derived formulations maintain significant antibacterial activity after processing and aerosolization while also inhibiting inflammatory pathways in human macrophages. This highlights their potential as localized treatments to effectively control bacterial infections without causing inflammation.

In this study, we developed a biosensor using a gold screen-printed electrode (Au-SPE) functionalized with mercaptoundecanoic acid (MUA) and an antibody for detecting the vascular-endothelial cadherin (CD144) as a endothelial biomarker protein on extracellular vesicles (EVs) isolated from saliva. The MUA functionalization provides a stable platform for immobilizing the CD144 antibody, ensuring the detection of the target protein. This biosensor combines Au-SPE technology with an immunoassay, offering a rapid, sensitive, and non-invasive method for detection of CD144 carried by EVs. Characterization of saliva-derived EVs using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) confirmed their morphology and size, which fell within the expected range of 80–180 nm. NTA indicated a lower concentration of particles in saliva-EVs than in serum-EVs (controls), highlighting the need for sensitive detection of EV cargos in this type of EV. Immunodetection confirmed the presence of CD144 in both saliva and serum-derived EVs, with higher concentrations in serum. Functionalization of Au-SPEs with MUA and CD144 antibodies was confirmed by significant resistance changes, and atomic force microscopy (AFM) was used to verify the preservation of EV morphology and their capturing post-immune adsorption. A calibration curve demonstrated the high sensitivity of the biosensor prototype for detecting CD144-positive EVs, with a limit of detection (LOD) of 0.111 ng mL⁻¹ and a limit of quantification (LOQ) of 0.37 ng mL⁻¹, requiring only 3 μL of EV-sample. This biosensor shows potential as a novel method for detecting and studying endothelial biomarkers associated with cardiovascular disease in EVs isolated from saliva, a capability not currently available with existing tools. Furthermore, it provides a key platform for expanding research to other biomarkers and diseases by monitoring protein cargos in the EVs, enhancing its utility across diverse clinical applications.

Head and neck cancer encompasses a diverse group of malignant neoplasms originating in regions such as the oral cavity, oropharynx, hypopharynx, larynx, sinonasal cavities, and salivary glands. HNC represents a significant public health challenge, and recent reports indicate an increment in the incidence of HNC in young adults. In 2020, approximately 377,700 new HNC cases and 177,800 HNC-related deaths were reported globally. Major risk factors include tobacco smoking, alcohol consumption, and human papillomavirus (HPV) infections. HNC impacts vital functions such as breathing, swallowing, and speech. Treatments for this type of cancer within this complex anatomy include surgery, radiotherapy, and chemotherapy combinations. Radiotherapy is often an essential component of both curative and palliative HNC treatment, balancing tumor control with the preservation of function and appearance. However, its use can damage adjacent normal tissues, causing acute or chronic toxicity. One complication of HNC irradiation is VF fibrosis, which leads to severe voice impairments, significantly affecting patients’ quality of life. Fibrosis involves excessive and aberrant deposition of extracellular matrix, driven by factors such as TGF-β1 and inflammatory cytokines, which ultimately impair the flexibility and function of VF. Current radiation-induced fibrosis treatments primarily focus on symptom management and include systemic therapies like corticosteroids, anti-inflammatory drugs, and antioxidants. However, these treatments have limited efficacy. Experimental approaches targeting molecular pathways involved in fibrosis are being explored. Given the limitations of these treatments, advancing research is crucial to develop more effective therapeutic strategies that can significantly improve the quality of life for HNC patients, especially those vulnerable to VF fibrosis.

Macrophages are critical to the immune response, serving multiple essential roles in maintaining tissue homeostasis and providing immune protection. These cells also interact with and influence the extracellular matrix (ECM) by sensing and responding to its components. Such interactions between macrophages and the ECM are mediated through the secretion and uptake of various biomacromolecules, such as cytokines and the extracellular vesicles, including exosomes and microvesicles. These vesicles are pivotal in regulating cellular behaviors that affect the organism’s overall function. Moreover, macrophages are integral to the repair mechanisms that alter tissue structure and functionality during tissue remodeling. This chapter will delineate how macrophages interact with the ECM and discuss potential therapeutic strategies leveraging these interactions. It will conclude with a discussion of the challenges ahead, highlighting the importance of understanding macrophage-ECM dynamics for advancing basic biology and clinical applications.

The goal of tissue decellularization is to efficiently remove unwanted cellular components, such as DNA and cellular debris, while retaining the complex structural and molecular milieu within the extracellular matrix (ECM). Decellularization protocols to date are centered on customized tissue-specific and lab-specific protocols that involve consecutive manual steps which results in variable and protocol-specific ECM material. The differences that result from the inconsistent protocols between decellularized ECMs affect consistency across batches, limit comparisons between results obtained from different laboratories, and could limit the transferability of the material for consistent laboratory or clinical use. The present study is the first proof-of-concept towards the development of a standardized protocol that can be used to derive multiple ECM biomaterials (powders and hydrogels) via a previously established automated system. The automated decellularization method developed by our group was used due to its short decellularization time (4 hours) and its ability to reduce batch-to-batch variability. The ECM obtained using this first iteration of a unified protocol was able to produce ECM hydrogels from skin, lung, muscle, tendons, cartilage, and laryngeal tissues. All hydrogels formed in this study were cytocompatible and showed gelation and rheological properties consistent with previous ECM hydrogels. The ECMs also showed unique proteomic composition. The present study represents the first step towards developing standardized protocols that can be used on multiple tissues in a fast, scalable, and reproducible manner.

Extracellular matrix (ECM) is a complex structure composed of bioactive molecules representative of the local tissue microenvironment. Decellularized ECM biomaterials harness these biomolecules for regenerative medicine applications. One potential therapeutic application is the use of vocal fold (VF) specific ECM to restore the VFs after injury. ECM scaffolds are derived through a process of decellularization, which aims to remove unwanted immunogenic biomolecules (e.g. DNA) while preserving the composition of the ECM. The effectiveness of the decellularization is typically assessed at the end by quantifying ECM attributes such as final dsDNA content. However, batch-to-batch variability in ECM manufacturing remains a significant challenge for the standardization, cost-effectiveness, and scale-up process. The limited number of tools available for in-process control heavily restricts the uncovering of the correlations between decellularization process parameters and ECM attributes. In this study, we developed a technique applicable to both the classical batch method and semi-continuous decellularization systems to trace the decellularization of two laryngeal tissues in real-time. We hypothesize that monitoring the bioreactor’s effluent absorbance at 260 nm as a function of time will provide a representative DNA release profile from the tissue and thus allow for process optimization. The DNA release profiles were obtained for laryngeal tissues and were successfully used to optimize the derivation of VF lamina propria-ECM (auVF-ECM) hydrogels. This hydrogel had comparable rheological properties to commonly used biomaterials to treat VF injuries. Also, the auVF-ECM hydrogel promoted the down-regulation of CCR7 by THP-1 macrophages upon lipopolysaccharide stimulation in vitro suggesting some anti-inflammatory properties. The results show that absorbance profiles are a good representation of DNA removal during the decellularization process thus providing an important tool to optimize future protocols.

Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may aide in understanding how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue resulting in junction specific features. ECM materials were derived from the achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue specific pre-hydrogel digests. C2C12 myoblasts and tendon fibroblasts were cultured in tissue-specific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. ECM conditioned media had only a minor effect on upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. Using tissue specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscle-tendon junctions to study the expression of MTJ specific proteins. Impact Statement The muscle-tendon junction is an important feature of muscle-tendon units; however, despite cross-talk between the two tissue types, it is overlooked in current research. Deconstructing the cell-matrix interactions will allow the opportunity to study significant junction specific features and markers that should be included in tissue models of the muscle-tendon unit, while gaining a deeper understanding of the natural junction. This research aims to inform future methods to engineer a more relevant multi-tissue platform to study the muscle-tendon unit.