Musculoskeletal Tissue Research Articles

Development of tropoelastin-functionalized anisotropic PCL scaffolds for musculoskeletal tissue engineering.

The highly organized extracellular matrix (ECM) of musculoskeletal tissues, encompassing tendons, ligaments and muscles, is structurally anisotropic, hierarchical and multi-compartmental. These features collectively contribute to their unique function. Previous studies have investigated the effect of tissue-engineered scaffold anisotropy on cell morphology and organization for musculoskeletal tissue repair and regeneration, but the hierarchical arrangement of ECM and compartmentalization are not typically replicated. Here, we present a method for multi-compartmental scaffold design that allows for physical mimicry of the spatial architecture of musculoskeletal tissue in regenerative medicine. This design is based on an ECM-inspired macromolecule scaffold. Polycaprolactone (PCL) scaffolds were fabricated with aligned fibers by electrospinning and mechanical stretching, and then surface-functionalized with the cell-supporting ECM protein molecule, tropoelastin (TE). TE was attached using two alternative methods that allowed for either physisorption or covalent attachment, where the latter was achieved by plasma ion immersion implantation (PIII). Aligned fibers stimulated cell elongation and improved cell alignment, in contrast to randomly oriented fibers. TE coatings bound by physisorption or covalently following 200 s PIII treatment promoted fibroblast proliferation. This represents the first cytocompatibility assessment of novel PIII-treated TE-coated PCL scaffolds. To demonstrate their versatility, these 2D anisotropic PCL scaffolds were assembled into 3D hierarchical constructs with an internally compartmentalized structure to mimic the structure of musculoskeletal tissue.

Recent Advances in the Application of Natural and Synthetic Polymer-Based Scaffolds in Musculoskeletal Regeneration.

The musculoskeletal system plays a critical role in providing the physical scaffold and movement to the mammalian body. Musculoskeletal disorders severely affect mobility and quality of life and pose a heavy burden to society. This new field of musculoskeletal tissue engineering has great potential as an alternative approach to treating large musculoskeletal defects. Natural and synthetic polymers are widely used in musculoskeletal tissue engineering owing to their good biocompatibility and biodegradability. Even more promising is the use of natural and synthetic polymer composites, as well as the combination of polymers and inorganic materials, to repair musculoskeletal tissue. Therefore, this review summarizes the progress of polymer-based scaffolds for applications of musculoskeletal tissue engineering and briefly discusses the challenges and future perspectives.

Differences in Medial and Lateral Gastrocnemius Stiffness after Exercise-Induced Muscle Fatigue.

Muscles are affected at the cellular level by exercised-induced fatigue, inducing changes in their stiffness. Examining muscle stiffness can improve the knowledge of various pathologic conditions, such as pain and injury. The objective of this study was to examine the stiffness of the medial gastrocnemius (MG) muscle and the lateral gastrocnemius (LG) muscle to determine the changes in stiffness, and to assess the differences in the stiffness between the MG and the LG, as affected by muscle fatigue measured using shear wave elastography (SWE) and a MyotonPRO after inducing muscle fatigue. A total of 35 healthy young adults participated in the study. The stiffness of the MG and the LG were assessed before and after a muscle fatigue protocol (MFP), which included three sets of 50 eccentric contractions of the calf muscles of the dominant leg, at rest, and at maximum voluntary contraction (MVC). The measurements were taken with SWE and the MyotonPRO simultaneously. Compared to baseline, the resting stiffness of the MG and the LG significantly increased immediately, 24 h, and 48 h after muscle fatigue (p < 0.05); however, during MVC, the stiffness of the MG decreased (p < 0.05) and that of the LG showed no change (p > 0.05). When the stiffness of the MG and the LG were compared before and after the MFP, changes in the stiffness of the MG were significantly greater than those in the LG (p < 0.05). This signifies that the MG was more affected by the exercise-induced muscle fatigue than was the LG. The assessment of musculoskeletal tissue and its characteristics, before and after eccentric exercise, is crucial in the prevention of overuse injuries associated with repeated exposure to both low and high levels of force.

Multiple congenital malformations arise from somatic mosaicism for constitutively active Pik3ca signaling.

Recurrent missense mutations of the PIK3CA oncogene are among the most frequent drivers of human cancers. These often lead to constitutive activation of its product p110α, a phosphatidylinositol 3-kinase (PI3K) catalytic subunit. In addition to causing a broad range of cancers, the H1047R mutation is also found in affected tissues of a distinct set of congenital tumors and malformations. Collectively termed PIK3CA-related disorders (PRDs), these lead to overgrowth of brain, adipose, connective and musculoskeletal tissues and/or blood and lymphatic vessel components. Vascular malformations are frequently observed in PRD, due to cell-autonomous activation of PI3K signaling within endothelial cells. These, like most muscle, connective tissue and bone, are derived from the embryonic mesoderm. However, important organ systems affected in PRDs are neuroectodermal derivatives. To further examine their development, we drove the most common post-zygotic activating mutation of Pik3ca in neural crest and related embryonic lineages. Outcomes included macrocephaly, cleft secondary palate and more subtle skull anomalies. Surprisingly, Pik3ca-mutant subpopulations of neural crest origin were also associated with widespread cephalic vascular anomalies. Mesectodermal neural crest is a major source of non-endothelial connective tissue in the head, but not the body. To examine the response of vascular connective tissues of the body to constitutive Pik3ca activity during development, we expressed the mutation by way of an Egr2 (Krox20) Cre driver. Lineage tracing led us to observe new lineages that had normally once expressed Krox20 and that may be co-opted in pathogenesis, including vascular pericytes and perimysial fibroblasts. Finally, Schwann cell precursors having transcribed either Krox20 or Sox10 and induced to express constitutively active PI3K were associated with vascular and other tumors. These murine phenotypes may aid discovery of new candidate human PRDs affecting craniofacial and vascular smooth muscle development as well as the reciprocal paracrine signaling mechanisms leading to tissue overgrowth.

Major economic burden of musculoskeletal and connective tissue diseases in Slovenia

Abstract Background Musculoskeletal and connective tissue diseases represent a growing public health problem, a great burden on health systems and place a heavy burden on society as a whole. Patients with musculoskeletal and connective tissue diseases often retire early, and any early retirement that could be prevented represents a loss of human capital, which means great social and economic damage to society. The present study was conducted in order to calculate for the first time in Slovenia the costs of the six most common diseases of the musculoskeletal and connective tissue diseases for the period from 2016-2018. Methods The calculated burden of musculoskeletal and connective tissue diseases was based on the calculation of various direct and indirect costs cross-sectionally at the level of one year. The methodology of National Transfer Accounts (NTA) was used for the calculation of indirect costs. Six major diagnoses based on the International Classification of Diseases and Related Health Problems for statistical purposes were selected for calculation. Data were obtained from different national routine databases. Results Calculations of the economic burden of musculoskeletal and connective tissue diseases in Slovenia showed that the economic costs of six selected diagnoses in the period 2016-2018 averaged about 5% of total health expenditure or 0.4% of gross domestic product in this period. The highest direct costs were hospitalizations, followed by costs for medicines, first curative visits at the primary level and visits to the outpatient clinic at the secondary level. Conclusions The results of the first calculation of the burden of musculoskeletal and connective tissue diseases in Slovenia showed a high economic burden of these diseases in the period from 2016 to 2018. The economic burden is underestimated and would be significantly higher considering all diagnoses from this group of diseases. Key messages

A Review of Recent Advances in Natural Polymer-Based Scaffolds for Musculoskeletal Tissue Engineering

A Review of Recent Advances in Natural Polymer-Based Scaffolds for Musculoskeletal Tissue Engineering

Can Radiomics and Machine Learning Predict Diagnosis in Musculoskeletal Myxoid Soft Tissue Tumors?

Introduction: Differentiating benign and malignant musculoskeletal myxoid soft tissue tumors is challenging due to their shared clinical, imaging, and histologic features. We assess if radiomics and machine learning can assist in differentiating benign and malignant musculoskeletal myxoid tumors. Methods: Forty patients with a pre-treatment MRI and a histologically confirmed myxoid soft tissue tumor (20 myxomas and 20 myxofibrosarcomas) on final resection pathology were reviewed. Baseline clinical features consisted of patient age, sex, tumor size, tumor depth, tumor location, pain, and tumor as an incidental finding. Manual image segmentation of the tumor was performed on T1 and T2 images by attending clinicians. A LASSO model was used for MRI feature reduction. The performance of five machine learning models were implemented using cross validation and compared with classify benign and malignant myxoid soft tissue tumors. Results: The five models using T1 and T2 radiomics features + clinical features achieved mean area under the ROC curves (AUC) of 0.729 to 0.780 and accuracy of 0.696 to 0.757. The highest AUC achieved was 0.780 using a support vector machine model. All combined radiomics + clinical feature models outperformed the classification models using clinical features alone. Conclusion: Classification models using T1 and T2 radiomics features + clinical features perform better in differentiating benign and malignant myxoid soft tissue tumors than models using clinical features alone. Future studies include validation of these preliminary findings in a larger and separate data set, and comparison of the diagnostic performance of radiomics to manual review of images by clinicians.

The Significance of the Nucleic Acid Test (NAT) to Prevent Viral Contamination in Musculoskeletal Tissue Transplantation.

Objective The present study aims to highlight the significance of the nucleic acid test (NAT) for musculoskeletal tissue donation and to compare the sensitivity of this test on the different available platforms. Method The present study is a retrospective survey in a human tissue bank database and an integrative literature review encompassing the last 10 years. The PubMed portal and the SCOPUS, CINAHL, and Web of Science databases were queried for articles. Results We found no specific studies on the use and sensitivity of NAT in braindead tissue donors. The information presented in the present study consists of specific contents intended for the Brazilian Blood Transfusion Network (Hemorrede Transfusional Nacional, in Portuguese) and internal retrospective data from a tissue bank located at a city in the state of São Paulo, Brazil. Conclusions The NAT is effective in blood samples from living patients. However, since biochemical reactions in braindead patients can be different, specific research, platforms, or both are crucial to tissue banks.

Translating musculoskeletal bioengineering into tissue regeneration therapies.

Musculoskeletal injuries and disorders are the leading cause of physical disability worldwide and a considerable socioeconomic burden. The lack of effective therapies has driven the development of novel bioengineering approaches that have recently started to gain clinical approvals. In this review, we first discuss the self-repair capacity of the musculoskeletal tissues and describe causes of musculoskeletal dysfunction. We then review the development of novel biomaterial, immunomodulatory, cellular, and gene therapies to treat musculoskeletal disorders. Last, we consider the recent regulatory changes and future areas of technological progress that can accelerate translation of these therapies to clinical practice.

Stable human cartilage progenitor cell line stimulates healing of meniscal tears and attenuates post-traumatic osteoarthritis.

Meniscal tearing in the knee increases the risk of post-traumatic osteoarthritis (OA) in patients. The therapeutic application of tissue-specific mesenchymal progenitor cells is currently being investigated as an emerging biologic strategy to help improve healing of musculoskeletal tissues like meniscal fibrocartilage and articular hyaline cartilage. However, many of these approaches involve isolating cells from healthy tissues, and the low yield of rare progenitor populations (< 1% of total cells residing in tissues) can make finding a readily available cell source for therapeutic use a significant logistical challenge. In the present study, we investigated the therapeutic efficacy of using expanded cartilage-derived and bone marrow-derived progenitor cell lines, which were stabilized using retroviral SV40, for repair of meniscus injury in a rodent model. Our findings indicate that these cell lines express the same cell surface marker phenotype of primary cells (CD54+, CD90+, CD105+, CD166+), and that they exhibit improved proliferative capacity that is suitable for extensive expansion. Skeletally mature male athymic rats treated with 3.2 million cartilage-derived progenitor cell line exhibited approximately 79% greater meniscal tear reintegration/healing, compared to injured animals that left untreated, and 76% greater compared to animals treated with the same number of marrow-derived stromal cells. Histological analysis of articular surfaces also showed that cartilage-derived progenitor cell line treated animals exhibited reduced post-traumatic OA associated articular cartilage degeneration. Stable cell line treatment did not cause tumor formation or off-target engraftment in animals. Taken together, we present a proof-of-concept study demonstrating, for the first time, that intra-articular injection of a stable human cartilage-derived progenitor cell line stimulates meniscus tear healing and provide chondroprotection in an animal model. These outcomes suggest that the use of stable cell lines may help overcome cell source limitations for cell-based medicine.

Enhancing cartilage repair with optimized supramolecular hydrogel-based scaffold and pulsed electromagnetic field.

Functional tissue engineering strategies provide innovative approach for the repair and regeneration of damaged cartilage. Hydrogel is widely used because it could provide rapid defect filling and proper structure support, and is biocompatible for cell aggregation and matrix deposition. Efforts have been made to seek suitable scaffolds for cartilage tissue engineering. Here Alg-DA/Ac-β-CD/gelatin hydrogel was designed with the features of physical and chemical multiple crosslinking and self-healing properties. Gelation time, swelling ratio, biodegradability and biocompatibility of the hydrogels were systematically characterized, and the injectable self-healing adhesive hydrogel were demonstrated to exhibit ideal properties for cartilage repair. Furthermore, the new hydrogel design introduces a pre-gel state before photo-crosslinking, where increased viscosity and decreased fluidity allow the gel to remain in a semi-solid condition. This granted multiple administration routes to the hydrogels, which brings hydrogels the ability to adapt to complex clinical situations. Pulsed electromagnetic fields (PEMF) have been recognized as a promising solution to various health problems owing to their noninvasive properties and therapeutic potentials. PEMF treatment offers a better clinical outcome with fewer, if any, side effects, and wildly used in musculoskeletal tissue repair. Thereby we propose PEMF as an effective biophysical stimulation to be 4th key element in cartilage tissue engineering. In this study, the as-prepared Alg-DA/Ac-β-CD/gelatin hydrogels were utilized in the rat osteochondral defect model, and the potential application of PEMF in cartilage tissue engineering were investigated. PEMF treatment were proven to enhance the quality of engineered chondrogenic constructs in vitro, and facilitate chondrogenesis and cartilage repair in vivo. All of the results suggested that with the injectable self-healing adhesive hydrogel and PEMF treatment, this newly proposed tissue engineering strategy revealed superior clinical potential for cartilage defect treatment.

Mechanoresponsive regulation of fibroblast-to-myofibroblast transition in three-dimensional tissue analogues: mechanical strain amplitude dependency of fibrosis

The spatiotemporal interaction and constant iterative feedback between fibroblasts, extracellular matrix, and environmental cues are central for investigating the fibroblast-induced musculoskeletal tissue regeneration and fibroblast-to-myofibroblast transition (FMT). In this study, we created a fibroblast-laden 3D tissue analogue to study (1) how mechanical loading exerted on three-dimensional (3D) tissues affected the residing fibroblast phenotype and (2) to identify the ideal mechanical strain amplitude for promoting tissue regeneration without initiating myofibroblast differentiation. We applied uniaxial tensile strain (0, 4, 8, and 12%) to the cell-laden 3D tissue analogues to understand the interrelation between the degree of applied mechanical loading amplitudes and FMT. Our data demonstrated that 4% mechanical strain created an anabolic effect toward tissue regeneration, but higher strain amplitudes over-stimulated the cells and initiated fibrotic tissue formation. Under increased mechanical strain amplitudes, fibroblasts were activated from a homeostatic state to a proto-myofibroblast state which resulted in increased cellularity accompanied by increased expressions of extracellular matrix (ECM) components, activation stressors (TGF-β1 and TGF-βR1), and profibrotic markers. This further transformed fibroblasts into α-smooth muscle actin expressing myofibroblasts. Understanding the interplay between the applied degree of mechanical loading exerted on 3D tissues and residing fibroblast phenotypic response is important to identify specific mechanomodulatory approaches for tissue regeneration and the informed mechanotherapy-guided tissue healing strategies.

Imaging features and biopsy yield of soft tissue metastatic lesions: 10-year single tertiary center experience.

To evaluate imaging features of soft tissue metastases, technical factors associated with diagnostic yield of image-guided biopsy, and clinical impact of biopsy results on patient outcomes. A total of 1605 image-guided soft tissue biopsies were retrospectively identified from December 2010 to December 2020. Included lesions were histologically proven musculoskeletal soft tissue metastases. Lesions were excluded if intraabdominal, intrathoracic, retroperitoneal, associated with osseous lesions or surgical scar implants or arising from skin or lymph nodes. Image guidance modality, needle size, number of cores, and lesion location, size, and depth from skin were recorded. Patient demographics, malignancy history, biopsy-driven changes in management, and survival rate after biopsy were collected. Forty-six patients met the inclusion criteria with a biopsy diagnostic yield of 44/46 (95.7%). Metastases were most commonly located truncal (82.6%, p < 0.001) and intramuscular (78.3%, p < 0.001). A total of 37/46 (80.4%) biopsies were US-guided. And 9/46 (19.6%) were CT-guided. There was no significant difference in the number of cores or mean needle gauge between diagnostic and nondiagnostic biopsies. At time of review, 23 (50%) patients were deceased, with a mean survival of 13.5months after biopsy. The majority (71.7%) of patients had a known primary malignancy at time of biopsy, most commonly lung (24.2%) and breast (24.2%). Overall survival showed no association with anatomic location (p > 0.83) or tissue type (p > 0.34). The most common biopsy-driven outcome was initiation of chemotherapy, immunotherapy, and/or radiotherapy (52.2%). Image-guided biopsy for soft tissue metastases has high diagnostic yield and commonly influences clinical management. Metastases were most commonly intramuscular in the trunk and are associated with poor prognosis.

Advances in The Application and Research of Magnetic Resonance Diffusion Kurtosis Imaging in The Musculoskeletal System.

Magnetic resonance diffusion kurtosis imaging (DKI) is an emerging magnetic resonance imaging (MRI) technique that can reflect microstructural changes in tissue through non-Gaussian diffusion of water molecules. Compared to traditional diffusion weighted imaging (DWI), the DKI model has shown greater sensitivity for diagnosis of musculoskeletal diseases and can help formulate more reasonable treatment plans. Moreover, DKI is an important auxiliary examination for evaluation of the motor function of the musculoskeletal system. This article briefly introduces the basic principles of DKI and reviews the application and research of DKI in the evaluation of disorders of the musculoskeletal system (including bone tumors, soft tissue tumors, spinal lesions, chronic musculoskeletal diseases, musculoskeletal trauma, and developmental disorders) as well as the normal musculoskeletal tissues. EVIDENCE LEVEL: 5 TECHNICAL EFFICACY: 1.

Digital volume correlation for the characterization of musculoskeletal tissues: Current challenges and future developments.

Biological tissues are complex hierarchical materials, difficult to characterise due to the challenges associated to the separation of scale and heterogeneity of the mechanical properties at different dimensional levels.The Digital Volume Correlation approach is the only image-based experimental approach that can accurately measure internal strain field within biological tissues under complex loading scenarios. In this minireview examples of DVC applications to study the deformation of musculoskeletal tissues at different dimensional scales are reported, highlighting the potential and challenges of this relatively new technique.The manuscript aims at reporting the wide breath of DVC applications in the past 2 decades and discuss future perspective for this unique technique, including fast analysis, applications on soft tissues, high precision approaches, and clinical applications.

State-of-the-art techniques for imaging the vascular microenvironment in craniofacial bone tissue engineering applications.

Vascularization is a crucial step during musculoskeletal tissue regeneration via bioengineered constructs or grafts. Functional vasculature provides oxygen and nutrients to the graft microenvironment, facilitates wound healing, enhances graft integration with host tissue, and ensures the long-term survival of regenerating tissue. Therefore, imaging de novo vascularization (i.e., angiogenesis), changes in microvascular morphology, and the establishment and maintenance of perfusion within the graft site (i.e., vascular microenvironment or VME) can provide essential insights into engraftment, wound healing, as well as inform the design of tissue engineering (TE) constructs. In this review, we focus on state-of-the-art imaging approaches for monitoring the VME in craniofacial TE applications, as well as future advances in this field. We describe how cutting-edge in vivo and ex vivo imaging methods can yield invaluable information regarding VME parameters that can help characterize the effectiveness of different TE constructs and iteratively inform their design for enhanced craniofacial bone regeneration. Finally, we explicate how the integration of novel TE constructs, preclinical model systems, imaging techniques, and systems biology approaches could usher in an era of "image-based tissue engineering."

Effects of Gabapentin and Pregabalin on Calcium Homeostasis: Implications for Physical Rehabilitation of Musculoskeletal Tissues.

In this review, we discuss the mechanism of action of gabapentinoids and the potential consequences of long-term treatment with these drugs on the musculoskeletal system. Gabapentinoids, such as gabapentin (GBP) and pregabalin (PGB) were designed as antiepileptic reagents and are now commonly used as first-line treatment for neuropathic pain and increasingly prescribed off-label for other pain disorders such as migraines and back pain. GBP and PGB exert their analgesic actions by selectively binding the α2δ1 auxiliary subunit of voltage-sensitive calcium channels, thereby inhibiting channel function. Numerous tissues express the α2δ1 subunit where GBP and PGB can alter calcium-mediated signaling events. In tissues such as bone, muscle, and cartilage, α2δ1 has important roles in skeletal formation, mechanosensation, and normal tissue function/repair that may be affected by chronic use of gabapentinoids. Long-term use of gabapentinoids is associated with detrimental musculoskeletal outcomes, including increased fracture risk. Therefore, understanding potential complications is essential for clinicians to guide appropriate treatments.

Multicolor Histochemical Staining for Identification of Mineralized and Non-Mineralized Musculoskeletal Tissue: Immunohistochemical and Radiological Validation in Decalcified Bone Samples.

Histochemical staining of paraffin-embedded decalcified bone samples is commonly used in preclinical research of musculoskeletal diseases, enabling the visualization of multiple tissue components by the application of chromogens. The purpose of this study was to introduce a novel multicolor staining protocol involving optimized chemical reagents and procedure, allowing the identification of high-mineralized bone, low-mineralized fracture callus, cartilage and skeletal muscle fibers simultaneously. Fractured femur and healthy tail vertebra samples from adult male Sprague–Dawley rats were decalcified with EDTA and formic acid, respectively, followed by paraffin embedding, tissue sectioning and multicolor staining. Conventional Movat’s pentachrome and safranin O / fast green staining were conducted in parallel for comparison. Immunohistochemical staining of collagen type-X and micro-CT analysis were included to further validate the efficacy of the staining method. The multicolor staining allowed visualization of major musculoskeletal tissue components in both types of decalcified samples, providing quality outcomes with fewer chemical reagents and simplified procedures. Immunohistochemical staining demonstrated its capacity for identification of the endochondral ossification process during fracture healing. Micro-CT imaging validated the staining outcome for high-mineralized skeletal tissue. The application of the multicolor staining may facilitate future preclinical research involving decalcified paraffin-embedded samples.

Whole Body Vibration Improves Brain and Musculoskeletal Health by Modulating the Expression of Tissue-Specific Markers: FNDC5 as a Key Regulator of Vibration Adaptations.

Whole body vibration (WBV) is well known to exert beneficial effects on multiple tissues, improving synaptic transmission, muscle mass, bone quality, and reducing anxiety and depressive behavior. However, the underlying molecular mechanisms are not yet fully understood, and organs and tissues may respond differently to the vibratory stimulus depending on multiple factors. Therefore, we investigated the WBV effects on the brain and musculoskeletal tissue of 4-month-old young mice, evaluating synaptic plasticity by electrophysiological recordings and tissue organization by histology and histomorphometric analysis. Specifically, WBV protocols were characterized by the same vibration frequency (45 Hz), but different in vibration exposure time (five series of 3 min for the B protocol and three series of 2 min and 30 s for the C protocol) and recovery time between two vibration sessions (1 min for the B protocol and 2 min and 30 s for the C protocol). In addition, immunohistochemistry was conducted to evaluate the expression of fibronectin type III domain-containing protein 5 (FNDC5), as well as that of tissue-specific markers, such as brain-derived neurotrophic factor (BDNF) in brain, myostatin in muscle and collagen I (COL-1) in bone. Our results suggest that the WBV effects depend closely on the type of protocol used and support the hypothesis that different organs or tissues have different susceptibility to vibration. Further studies will be needed to deepen our knowledge of physiological adaptations to vibration and develop customized WBV protocols to improve and preserve cognitive and motor functions.

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment.

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (<40 µm in thickness) of COL1 fibers that extend over distances less than 500 µm and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 µm in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.