Extracellular Matrix Coatings Research Articles

In Vitro Cell Surface Marker Expression on Mesenchymal Stem Cell Cultures does not Reflect Their Ex Vivo Phenotype.

Cell surface marker expression is one of the criteria for defining human mesenchymal stem or stromal cells (MSC) in vitro. However, it is unclear if expression of markers including CD73 and CD90 reflects the in vivo origin of cultured cells. We evaluated expression of 15 putative MSC markers in primary cultured cells from periosteum and cartilage to determine whether expression of these markers reflects either the differentiation state of cultured cells or the self-renewal of in vivo populations. Cultured cells had universal and consistent expression of various putative stem cell markers including > 95% expression CD73, CD90 and PDPN in both periosteal and cartilage cultures. Altering the culture surface with extracellular matrix coatings had minimal effect on cell surface marker expression. Osteogenic differentiation led to loss of CD106 and CD146 expression, however CD73 and CD90 were retained in > 90% of cells. We sorted freshly isolated periosteal populations capable of CFU-F formation on the basis of CD90 expression in combination with CD34, CD73 and CD26. All primary cultures universally expressed CD73 and CD90 and lacked CD34, irrespective of the expression of these markers ex vivo indicating phenotypic convergence in vitro. We conclude that markers including CD73 and CD90 are acquired in vitro in most 'mesenchymal' cells capable of expansion. Overall, we demonstrate that in vitro expression of many cell surface markers in plastic-adherent cultures is unrelated to their expression prior to culture.

Laminin as a Key Extracellular Matrix for Proliferation, Differentiation, and Maturation of Porcine Muscle Stem Cell Cultivation.

Extracellular matrix (ECM) proteins play a crucial role in culturing muscle stem cells (MuSCs). However, there is a lack of extensive research on how each of these proteins influences proliferation and differentiation of MuSCs from livestock animals. Therefore, we investigated the effects of various ECM coatings-collagen, fibronectin, gelatin, and laminin-on the proliferation, differentiation, and maturation of porcine MuSCs. Porcine MuSCs, isolated from 14-day-old Berkshire piglets, were cultured on ECM-coated plates, undergoing three days of proliferation followed by three days of differentiation. MuSCs on laminin showed higher proliferation rate than others (p<0.05). There was no significant difference in the mRNA expression levels of PAX7, MYF5, and MYOD among MuSCs on laminin, collagen, and fibronectin (p>0.05). During the differentiation period, MuSCs cultured on laminin exhibited a significantly higher differentiation rate, resulting in thicker myotubes compared to those on other ECMs (p<0.05). Also, MuSCs on laminin showed higher expression of mRNA related with maturated muscle fiber such as MYH1 and MYH4 corresponding to muscle fiber type IIx and muscle fiber type IIb, respectively, compared with MuSCs on other ECM coatings (p<0.05). In summary, our comparison of ECMs revealed that laminin significantly enhances MuSC proliferation and differentiation, outperforming other ECMs. Specifically, muscle fibers cultured on laminin exhibited a more mature phenotype. These findings underscore laminin's potential to advance in vitro muscle research and cultured meat production, highlighting its role in supporting rapid cell proliferation, higher differentiation rates, and the development of mature muscle fibers.

Bone morphogenetic protein signaling inhibitor improves differentiation and function of 3D muscle construct fabricated using C2C12

Functional tissue-engineered artificial skeletal muscle tissue has great potential for pharmacological and academic applications. This study demonstrates an in vitro tissue engineering system to construct functional artificial skeletal muscle tissues using self-organization and signal inhibitors. To induce efficient self-organization, we optimized the substrate stiffness and extracellular matrix (ECM) coatings. We modified the tissue morphology to be ring-shaped under optimized self-organization conditions. A bone morphogenetic protein (BMP) inhibitor was added to improve overall myogenic differentiation. This supplementation enhanced the myogenic differentiation ratio and myotube hypertrophy in two-dimensional cell cultures. Finally, we found that myotube hypertrophy was enhanced by a combination of self-organization with ring-shaped tissue and a BMP inhibitor. BMP inhibitor treatment significantly improved myogenic marker expression and contractile force generation in the self-organized tissue. These observations indicated that this procedure may provide a novel and functional artificial skeletal muscle for pharmacological studies.

A Protein-Adsorbent Hydrogel with Tunable Stiffness for Tissue Culture Demonstrates Matrix-Dependent Stiffness Responses.

Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an in vitro system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, we present a novel hydrophobic hydrogel system via chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, we show that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, we reveal that stiffness mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. Our material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function.

The Use of Small-Molecule Compounds for Cell Adhesion and Migration in Regenerative Medicine.

Cell adhesion is essential for cell survival, communication, and regulation, and it is of fundamental importance in the development and maintenance of tissues. Cell adhesion has been widely explored due to its many important roles in the fields of tissue regenerative engineering and cell biology. This is because the mechanical interactions between a cell and its extracellular matrix (ECM) can influence and control cell behavior and function. Currently, biomaterials for regenerative medicine have been heavily investigated as substrates for promoting a cells' adhesive properties and subsequent proliferation, tissue differentiation, and maturation. Specifically, the manipulation of biomaterial surfaces using ECM coatings such as fibronectin extracted from animal-derived ECM have contributed significantly to tissue regenerative engineering as well as basic cell biology research. Additionally, synthetic and natural bioadhesive agents with pronounced abilities to enhance adhesion in numerous biological components and molecules have also been assessed in the field of tissue regeneration. Research into the use of facilitative bioadhesives has aimed to further optimize the biocompatibility, biodegradability, toxicity levels, and crosslinking duration of bioadhesive materials for improved targeted delivery and tissue repair. However, the restrictive drawbacks of some of these bioadhesive and animal-derived materials include the potential risk of disease transmission, immunogenicity, poor reproducibility, impurities, and instability. Therefore, it is necessary for alternative strategies to be sought out to improve the quality of cell adhesion to biomaterials. One promising strategy involves the use of cell-adhesive small molecules. Small molecules are relatively inexpensive, stable, and low-molecular-weight (<1000 Da) compounds with great potential to serve as efficient alternatives to conventional bioadhesives, ECM proteins, and other derived peptides. Over the past few years, a number of cell adhesive small molecules with the potential for tissue regeneration have been reported. In this review, we discuss the current progress using cell adhesive small molecules to regulate tissue regeneration.

A cell retrievable strategy for harvesting extracellular matrix as active biointerface

Extracellular matrix (ECM) provides a variety of physical and chemical cues for cells. Here, a very simple and smart method is developed to glue living cells away for harvesting their ECMs. The obtained ECM coatings show less cell fragment residues comparing with those obtained by the traditional cell lysis. The glued cell sheets can even be re-cultured and reused after transferring to new environment. This moderate way well maintains the activity of the ECM proteins, which can promote cell adhesion and growth. Strikingly, the ECM coatings acquired from different functional cells can guide stem cell differentiation, which is attributed to the natural physical and biochemical cues on ECM coatings. Consequently, this method provides a substantial progress for preparing natural ECM coatings and shows promising potential in regenerative medicine and other related fields of biomedical engineering.

Differentiating Human Pluripotent Stem Cells to Cardiomyocytes Using Purified Extracellular Matrix Proteins.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into cardiomyocytes (hESC-CMs and iPSC-CMs, respectively), which hold great promise for cardiac regenerative medicine and disease modeling efforts. However, the most widely employed differentiation protocols require undefined substrates that are derived from xenogeneic (animal) products, contaminating resultant hESC- and iPSC-CM cultures with xenogeneic proteins and limiting their clinical applicability. Additionally, typical hESC- and iPSC-CM protocols produce CMs that are significantly contaminated by non-CMs and that are immature, requiring lengthy maturation procedures. In this review, we will summarize recent studies that have investigated the ability of purified extracellular matrix (ECM) proteins to support hESC- and iPSC-CM differentiation, with a focus on commercially available ECM proteins and coatings to make such protocols widely available to researchers. The most promising of the substrates reviewed here include laminin-521 with laminin-221 together or Synthemax (a synthetic vitronectin-based peptide coating), which both resulted in highly pure CM cultures. Future efforts are needed to determine whether combinations of specific purified ECM proteins or derived peptides could further improve CM maturation and culture times, and significantly improve hESC- and iPSC-CM differentiation protocols.

Modulation of human iPSC-derived hepatocyte phenotype via extracellular matrix microarrays

In vitro human liver models are essential for drug screening, disease modeling, and cell-based therapies. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHeps) mitigate sourcing limitations of primary human hepatocytes (PHHs) and enable precision medicine; however, current protocols yield iHeps with very low differentiated functions. The composition and stiffness of liver's extracellular matrix (ECM) cooperatively regulate hepatic phenotype in vivo, but such effects on iHeps remain unelucidated. Here, we utilized ECM microarrays and high content imaging to assess human iHep attachment and functions on ten major liver ECM proteins in single and two-way combinations robotically spotted onto polyacrylamide gels of liver-like stiffnesses; microarray findings were validated using hydrogel-conjugated multiwell plates. Collagen-IV supported higher iHep attachment than collagen-I over 2 weeks on 1 kPa, while laminin and its combinations with collagen-III, fibronectin, tenascin C, or hyaluronic acid led to both high iHep attachment and differentiated functions; laminin and its combination with tenascin or fibronectin led to similar albumin expression in iHeps and PHHs. Additionally, several collagen-IV-, laminin-, fibronectin-, and collagen-V-containing combinations on 1 kPa led to similar or higher CYP3A4 staining in iHeps than PHHs. Lastly, collagen-I or -III mixed with laminin, collagen-IV mixed with lumican, and collagen-V mixed with fibronectin led to high and stable functional output (albumin/urea secretions; CYP1A2/2C9/3A4 activities) in iHep cultures versus declining PHH numbers/functions for 3 weeks within multiwell plates containing 1 kPa hydrogels. Ultimately, these platforms can help elucidate ECM's role in liver diseases and serve as building blocks of engineered tissues for applications. Statement of significanceWe utilized high-throughput extracellular matrix (ECM) microarrays and high content imaging to assess the attachment and differentiated functions of iPSC-derived human hepatocyte-like cells (iHep) on major liver ECM protein combinations spotted onto polyacrylamide gels of liver-like stiffnesses. We observed that iHep responses are regulated in unexpected ways via the cooperation between ECM stiffness and protein composition. Using this approach, we induced mature functions in iHeps on substrates of physiological stiffness and select ECM coatings at higher levels over 3+ weeks than analogous primary human hepatocyte cultures, which is useful for building platforms for drug screening, disease modeling, and regenerative medicine.

Extracellular Matrix Coatings on Cardiovascular Materials—A Review

Vascular transplantation is an effective and common treatment for cardiovascular disease (CVD). However, the low biocompatibility of implants is a major problem that hinders its clinical application. Surface modification of implants with extracellular matrix (ECM) coatings is an effective approach to improve the biocompatibility of cardiovascular materials. The complete ECM seems to have better biocompatibility, which may give cardiovascular biomaterials a more functional surface. The use of one or several ECM proteins to construct a surface allows customization of coating composition and structure, possibly resulting in some unique functions. ECM is a complex three-dimensional structure composed of a variety of functional biological macromolecules, and changes in the composition will directly affect the function of the coating. Therefore, understanding the chemical composition of the ECM and its interaction with cells is beneficial to provide new approaches for coating surface modification. This article reviews novel ECM coatings, including coatings composed of intact ECM and biomimetic coatings tailored from several ECM proteins, and introduces new advances in coating fabrication. These ECM coatings are effective in improving the biocompatibility of vascular grafts.

3D porous scaffolds from wheat glutenin for cultured meat applications

Scaffolds suitable for use in food products are crucial components for the production of cultured meat. Here, wheat glutenin, an inexpensive and abundant plant-based protein, was used to develop 3D porous scaffolds for cultured meat applications. A physical cross-linking method based on water annealing was developed for the fabrication of porous glutenin sponges and fibrous aligned scaffolds. The pore sizes ranged from 50 to 250 μm, with compressive modulus ranges from 0.5 to 1.9 kPa, depending on the percentage of glutenin (2%–5%) used in the process. The sponges were stable in PBS with refrigeration for at least six months after water annealing. The glutenin scaffolds supported the proliferation and differentiation of C2C12 mouse skeletal myoblasts and bovine satellite cells (BSCs) without the need to add specific cell adhesive proteins or other coatings. The low cost and food safe production process avoided the use of toxic cross-linkers and animal-derived extracellular matrix (ECM) coatings, suggesting that this as approach is a promising system for scaffolds useful in cultivated meat applications.

Long-term adherence of human brain cells invitro is enhanced by charged amine-based plasma polymer coatings.

SummaryAdvances in cellular reprogramming have radically increased the use of patient-derived cells for neurological research in vitro. However, adherence of human neurons on tissue cultureware is unreliable over the extended periods required for electrophysiological maturation. Adherence issues are particularly prominent for transferable glass coverslips, hindering imaging and electrophysiological assays. Here, we assessed thin-film plasma polymer treatments, polymeric factors, and extracellular matrix coatings for extending the adherence of human neuronal cultures on glass. We find that positive-charged, amine-based plasma polymers improve the adherence of a range of human brain cells. Diaminopropane (DAP) treatment with laminin-based coating optimally supports long-term maturation of fundamental ion channel properties and synaptic activity of human neurons. As proof of concept, we demonstrated that DAP-treated glass is ideal for live imaging, patch-clamping, and optogenetics. A DAP-treated glass surface reduces the technical variability of human neuronal models and enhances electrophysiological maturation, allowing more reliable discoveries of treatments for neurological and psychiatric disorders.

Guided Self-Assembly of ES-Derived Lung Progenitors into Biomimetic Tube Structures That Impact Cell Differentiation

Chemically directed differentiation of pluripotent stem cells (PSCs) into defined cell types is a potent strategy for creating regenerative tissue models and cell therapies. In vitro observations suggest that physical cues can augment directed differentiation. We recently demonstrated that confining human PSC-derived lung progenitor cells in a tube with a diameter that mimics those observed during lung development results in the alteration of cell differentiation towards SOX2−SOX9+ lung cells. Here we set out to assess the robustness of this geometric confinement effect with respect to different culture parameters in order to explore the corresponding changes in cell morphometry and determine the feasibility of using such an approach to enhance directed differentiation protocols. Culture of progenitor cells in polydimethylsiloxane (PDMS) tubes reliably induced self-organization into tube structures and was insensitive to a variety of extracellular matrix coatings. Cellular morphology and differentiation status were found to be sensitive to the diameter of tube cells that were cultured within but not to seeding density. These data suggest that geometric cues impose constraints on cells, homogenize cellular morphology, and influence fate status.

Extracellular Matrix Optimization for Enhanced Physiological Relevance in Hepatic Tissue-Chips.

The cellular microenvironment is influenced explicitly by the extracellular matrix (ECM), the main tissue support biomaterial, as a decisive factor for tissue growth patterns. The recent emergence of hepatic microphysiological systems (MPS) provide the basic physiological emulation of the human liver for drug screening. However, engineering microfluidic devices with standardized surface coatings of ECM may improve MPS-based organ-specific emulation for improved drug screening. The influence of surface coatings of different ECM types on tissue development needs to be optimized. Additionally, an intensity-based image processing tool and transepithelial electrical resistance (TEER) sensor may assist in the analysis of tissue formation capacity under the influence of different ECM types. The current study highlights the role of ECM coatings for improved tissue formation, implying the additional role of image processing and TEER sensors. We studied hepatic tissue formation under the influence of multiple concentrations of Matrigel, collagen, fibronectin, and poly-L-lysine. Based on experimental data, a mathematical model was developed, and ECM concentrations were validated for better tissue development. TEER sensor and image processing data were used to evaluate the development of a hepatic MPS for human liver physiology modeling. Image analysis data for tissue formation was further strengthened by metabolic quantification of albumin, urea, and cytochrome P450. Standardized ECM type for MPS may improve clinical relevance for modeling hepatic tissue microenvironment, and image processing possibly enhance the tissue analysis of the MPS.

Modulation of Synthetic Tracheal Grafts with Extracellular Matrix Coatings.

Synthetic scaffolds for the repair of long-segment tracheal defects are hindered by insufficient biocompatibility and poor graft epithelialization. In this study, we determined if extracellular matrix (ECM) coatings improved the biocompatibility and epithelialization of synthetic tracheal grafts (syn-TG). Porcine and human ECM substrates (pECM and hECM) were created through the decellularization and lyophilization of lung tissue. Four concentrations of pECM and hECM coatings on syn-TG were characterized for their effects on scaffold morphologies and on in vitro cell viability and growth. Uncoated and ECM-coated syn-TG were subsequently evaluated in vivo through the orthotopic implantation of segmental grafts or patches. These studies demonstrated that ECM coatings were not cytotoxic and, enhanced the in vitro cell viability and growth on syn-TG in a dose-dependent manner. Mass spectrometry demonstrated that fibrillin, collagen, laminin, and nephronectin were the predominant ECM components transferred onto scaffolds. The in vivo results exhibited similar robust epithelialization of uncoated and coated syn-TG patches; however, the epithelialization remained poor with either uncoated or coated scaffolds in the segmental replacement models. Overall, these findings demonstrated that ECM coatings improve the seeded cell biocompatibility of synthetic scaffolds in vitro; however, they do not improve graft epithelialization in vivo.

In vitro generation of peri-islet basement membrane-like structures

The peri-islet extracellular matrix (ECM) is a key component of the microenvironmental niche surrounding pancreatic islets of Langerhans. The cell anchorage and signaling provided by the peri-islet ECM is critical for optimum beta cell glucose responsiveness, but islets lose this important native ECM when isolated for transplantation or in vitro studies. Here, we established a method to construct a peri-islet ECM on the surfaces of isolated rat and human islets by the co-assembly from solution of laminin, nidogen and collagen IV proteins. Successful deposition of contiguous peri-islet ECM networks was confirmed by immunofluorescence, western blot, and transmission electron microscopy. The ECM coatings were disrupted when assembly occurred in Ca2+/Mg2+-free conditions. As laminin network polymerization is divalent cation dependent, our data are consistent with receptor-driven ordered ECM network formation rather than passive protein adsorption. To further illustrate the utility of ECM coatings, we employed stem cell derived beta-like cell clusters (sBCs) as a renewable source of functional beta cells for cell replacement therapy. We observe that sBC pseudo-islets lack an endogenous peri-islet ECM, but successfully applied our approach to construct a de novo ECM coating on the surfaces of sBCs.

Human Platelet Lysate Acts Synergistically With Laminin to Improve the Neurotrophic Effect of Human Adipose-Derived Stem Cells on Primary Neurons in vitro.

BackgroundDespite the advancements in microsurgical techniques and noteworthy research in the last decade, peripheral nerve lesions have still weak functional outcomes in current clinical practice. However, cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit has shown promising results in animal studies. Human platelet lysate (hPL) has been adopted to avoid fetal bovine serum (FBS) in consideration of the biosafety concerns inherent with the use of animal-derived products in tissue processing and cell culture steps for translational purposes. In this work, we investigate how the interplay between hPL-expanded hADSC (hADSChPL) and extracellular matrix (ECM) proteins influences key elements of nerve regeneration.MethodshADSC were seeded on different ECM coatings (laminin, LN; fibronectin, FN) in hPL (or FBS)-supplemented medium and co-cultured with primary dorsal root ganglion (DRG) to establish the intrinsic effects of cell–ECM contact on neural outgrowth. Co-cultures were performed “direct,” where neural cells were seeded in contact with hADSC expanded on ECM-coated substrates (contact effect), or “indirect,” where DRG was treated with their conditioned medium (secretome effect). Brain-derived nerve factor (BDNF) levels were quantified. Tissue culture plastic (TCPS) was used as the control substrate in all the experiments.ResultshPL as supplement alone did not promote higher neurite elongation than FBS when combined with DRG on ECM substrates. However, in the presence of hADSC, hPL could dramatically enhance the stem cell effect with increased DRG neurite outgrowth when compared with FBS conditions, regardless of the ECM coating (in both indirect and direct co-cultures). The role of ECM substrates in influencing neurite outgrowth was less evident in the FBS conditions, while it was significantly amplified in the presence of hPL, showing better neural elongation in LN conditions when compared with FN and TCPS. Concerning hADSC growth factor secretion, ELISA showed significantly higher concentrations of BDNF when cells were expanded in hPL compared with FBS-added medium, without significant differences between cells cultured on the different ECM substrates.ConclusionThe data suggest how hADSC grown on LN and supplemented with hPL could be active and prone to support neuron–matrix interactions. hPL enhanced hADSC effects by increasing both proliferation and neurotrophic properties, including BDNF release.

Porous polyetheretherketone microcarriers fabricated via hydroxylation together with cell-derived mineralized extracellular matrix coatings promote cell expansion and bone regeneration.

Porous microcarriers have aroused increasing attention recently by facilitating oxygen and nutrient transfer, supporting cell attachment and growth with sufficient cell seeding density. In this study, porous polyetheretherketone (PEEK) microcarriers coated with mineralized extracellular matrix (mECM), known for their chemical, mechanical and biological superiority, were developed for orthopedic applications. Porous PEEK microcarriers were derived from smooth microcarriers using a simple wet-chemistry strategy involving the reduction of carbonyl groups. This treatment simultaneously modified surface topology and chemical composition. Furthermore, the microstructure, protein absorption, cytotoxicity and bioactivity of the obtained porous microcarriers were investigated. The deposition of mECM through repeated recellularization and decellularization on the surface of porous MCs further promoted cell proliferation and osteogenic activity. Additionally, the mECM coated porous microcarriers exhibited excellent bone regeneration in a rat calvarial defect repair model in vivo, suggesting huge potential applications in bone tissue engineering.

Interrogating cardiac muscle cell mechanobiology on stiffness gradient hydrogels.

Extracellular matrix (ECM) remodeling is a major facet of cardiac development and disease, yet our understanding of cardiomyocyte mechanotransduction remains limited. To enhance our understanding of cardiomyocyte mechanosensation, we studied stiffness-driven changes to cell morphology and mechanomarker expression in H9C2 cells and neonatal rat cardiomyocytes (NRCMs). Linear stiffness gradient polyacrylamide hydrogels (2-33 kPa) coated with ECM proteins including Collagen I (Col), Fibronectin (Fn) or Laminin (Ln) were used to represent necrotic, healthy, and infarcted cardiac tissue on a continuous stiffness gradient. Cell size, cell shape and nuclear size were found to be mechanosensitive in H9C2 cells, as was the expression or nuclear translocalization of the mechanomarkers Lamin-A, YAP, and MRTF-A. Minor differences were observed between the different ECM coatings, with the same overarching stiffness-dependent trends being observed across Col, Fn and Ln coated hydrogels. Inhibition of mechanotransduction in H9C2 cells using blebbistatin or Y27632 resulted in disruptions to cell shape, nuclear shape, and nuclear size, however, trends in cell size and mechanomarker expression were not significantly attenuated. Mechanosensation in NRCMs was much less marked, with no significant changes in cell morphology being detected, although YAP did become increasingly nuclear localized with increasing stiffness. In α-actinin positive cells, striations formed with regular structure and frequency at all stiffnesses for Col and Fn coated hydrogels, but not Ln coated gels. In this study, we used our stiffness gradient hydrogels to comprehensively map the relationship between ECM stiffness and cardiac cell phenotype and found that less mature H9C2 cardiac cells are more sensitive to ECM changes than the more developed neonatal cardiomyocytes.

Azide-Functional Extracellular Matrix Coatings as a Bioactive Platform for Bioconjugation.

In recent years, the development and application of decellularized extracellular matrices (ECMs) for use as biomaterials have grown rapidly. These cell-derived matrices (CDMs) represent highly bioactive and biocompatible materials consisting of a complex assembly of biomolecules. Even though CDMs mimic the natural microenvironment of cells in vivo very closely, they still lack specifically addressable functional groups, which are often required to tailor a biomaterial functionality by bioconjugation. To overcome this limitation, metabolic glycoengineering has emerged as a powerful tool to equip CDMs with chemical groups such as azides. These small chemical handles are known for their ability to undergo bioorthogonal click reactions, which represent a desirable reaction type for bioconjugation. However, ECM insolubility makes its processing very challenging. In this contribution, we isolated both the unmodified ECM and azide-modified clickECM by osmotic lysis. In a first step, these matrices were concentrated to remove excessive water from the decellularization step. Next, the hydrogel-like ECM and clickECM films were mechanically fragmentized, resulting in easy to pipette suspensions with fragment sizes ranging from 7.62 to 31.29 μm (as indicated by the mean d90 and d10 values). The biomolecular composition was not impaired as proven by immunohistochemistry. The suspensions were used for the reproducible generation of surface coatings, which proved to be homogeneous in terms of ECM fragment sizes and coating thicknesses (the mean coating thickness was found to be 33.2 ± 7.3 μm). Furthermore, they were stable against fluid-mechanical abrasion in a laminar flow cell. When primary human fibroblasts were cultured on the coated substrates, an increased bioactivity was observed. By conjugating the azides within the clickECM coatings with alkyne-coupled biotin molecules, a bioconjugation platform was obtained, where the biotin-streptavidin interaction could be used. Its applicability was demonstrated by equipping the bioactive clickECM coatings with horseradish peroxidase as a model enzyme.

Creation of a contractile biomaterial from a decellularized spinach leaf without ECM protein coating: An in vitro study.

Myocardial infarction (MI) results in the death of cardiac tissue, decreases regional contraction, and can lead to heart failure. Tissue engineered cardiac patches containing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) can restore contractile function. However, cells within thick patches require vasculature for blood flow. Recently, we demonstrated fibronectin coated decellularized leaves provide a suitable scaffold for hiPS-CMs. Yet, the necessity of this additional coating step is unclear. Therefore, we compared hiPS-CM behavior on decellularized leaves coated with collagen IV or fibronectin extracellular matrix (ECM) proteins to noncoated leaves for up to 21 days. Successful coating was verified by immunofluorescence. Similar numbers of hiPS-CMs adhered to coated and noncoated decellularized leaves for 21 days. At Day 14, collagen IV coated leaves contracted more than noncoated leaves (3.25 ± 0.39% vs. 1.54 ± 0.60%; p < .05). However, no differences in contraction were found between coated leaves, coated tissue culture plastic (TCP), noncoated leaves, or noncoated TCP at other time points. No significant differences were observed in hiPS-CM spreading or sarcomere lengths on leaves with or without coating. This study demonstrates that cardiac scaffolds can be created from decellularized leaves without ECM coatings. Noncoated decellularized leaf surfaces facilitate robust cell attachment for an engineered tissue patch.