ECM Composition Research Articles

Molecular analysis of muscle progenitor cells on extracellular matrix coatings and hydrogels

Development of an ex vivo culture system to expand satellite cells, the resident muscle stem cell population, will be necessary for the development of their use as therapeutics. The loss of the niche environment is often cited as the reason that culture results in both the loss of myogenic potential and low re-engraftment rates of these cells. Studies have shown that culture of satellite cells on more elastic substrates maintained their quiescence and potential and increased re-engraftment, but there was limited proliferation. We examined whether substrates composed of extracellular matrix proteins, as either coatings or hydrogels, could support expansion of this population whilst maintaining the potency of these cells. The collagen based hydrogels were very pliant and our studies demonstrate that stiffer substrates are necessary for in vitro proliferation and differentiation of satellite cells, and the ECM composition was not significantly important. Our data further indicates that culture on highly elastic substrates allowed satellite cells to down-regulate myogenic specific transcription factors, resulting in an expression profile similar to a Galert state. These satellite cells could be subsequently cultured on Matrigel and induced to differentiate. Proliferation and gene expression data further indicated that C2C12 cells are not a good proxy for studies of satellite cell proliferation and differentiation on alternative substrates. Statement of SignificanceAlthough biomaterials and muscle stem cell-based therapeutics for muscle loss due to trauma and disease have been studied intensely for the last decade, significant challenges remain; satellite cells lose their myogenic potential after in vitro culture, and do not re-engraft efficiently when delivered to skeletal muscle. We cultured adult mouse derived satellite cells and C2C12 cells on biomimetic, extracellular matrix based, hydrogels and coated plates and carried out a detailed analysis of proliferation, differentiation, and gene expression. These studies were designed to allow for the examination of the roles of both matrix composition and elasticity. The data demonstrated that satellite cells were most affected by elasticity. Examining the expression of lineage and cell cycle specific genes provided important insight into the behavior of both cell types, and point to fundamental differences that affect the interpretation of studies aimed at understanding the in vitro requirements of muscle progenitor cells. Understanding how satellite cells respond to various biochemical and biophysical cues at the molecular level will inform future efforts to design therapeutics for muscle regeneration.

Hybrid nanocomposite as a chest wall graft with improved integration by adipose-derived stem cells

Surgery of the chest wall is potentially required to cover large defects after removal of malignant tumours. Usually, inert and non-degradable Gore-Tex serves to replace the missing tissue. However, novel biodegradable materials combined with stem cells are available that stimulate the healing. Based on poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) and pure PLGA, a dual layer biodegradable hybrid nanocomposite was generated. Mouse adipose-derived stem cells were cultered on electrospun disks (ASCs of C57BL/6), and biomechanical tests were performed. The cell-seeded scaffolds were engrafted in C57BL/LY5.1 mice to serve as a chest wall substitute. Cell invasion into the bi-layered material, extent of CD45+ cells, inflammatory response, neo-vascularization and ECM composition were determined at 1 and 2 months post-surgery, respectively. The bi-layered hybrid nanocomposite was stable after a 2-week in vitro culture, in contrast to PLGA/aCaP without a PLGA layer. There was a complete biointegration and good vascularization in vivo. The presence of ASCs attracted more CD45+ cells (hematopoietic origin) compared to cell-free scaffolds. Inflammatory reaction was similar for both groups (±ASCs) at 8 weeks. A bi-layered hybrid nanocomposite fabricated of electrospun PLGA/aCaP and a reinforcing layer of pristine PLGA is an ideal scaffold for chest wall reconstruction. It is stable and allows a proper host tissue integration. If ASCs are seeded, they attract more CD45+ cells, supporting the regeneration process.

Transcriptomics of a KDELR1 knockout cell line reveals modulated cell adhesion properties

KDEL receptors (KDELRs) represent transmembrane proteins of the secretory pathway which regulate the retention of soluble ER-residents as well as retrograde and anterograde vesicle trafficking. In addition, KDELRs are involved in the regulation of cellular stress response and ECM degradation. For a deeper insight into KDELR1 specific functions, we characterised a KDELR1-KO cell line (HAP1) through whole transcriptome analysis by comparing KDELR1-KO cells with its respective HAP1 wild-type. Our data indicate more than 300 significantly and differentially expressed genes whose gene products are mainly involved in developmental processes such as cell adhesion and ECM composition, pointing out to severe cellular disorders due to a loss of KDELR1. Impaired adhesion capacity of KDELR1-KO cells was further demonstrated through in vitro adhesion assays, while collagen- and/or laminin-coating nearly doubled the adhesion property of KDELR1-KO cells compared to wild-type, confirming a transcriptional adaptation to improve or restore the cellular adhesion capability. Perturbations within the secretory pathway were verified by an increased secretion of ER-resident PDI and decreased cell viability under ER stress conditions, suggesting KDELR1-KO cells to be severely impaired in maintaining cellular homeostasis.

1794-P: Perineuronal Net Destabilization in Neural Network Dysfunction Common to Both T2D and Alzheimer’s Disease

The risk of Alzheimer’s disease (AD) and other forms of cognitive impairment is increased in patients with T2D. Both conditions are characterized by brain insulin resistance and regional brain hypometabolism, and the brain of T2D animal models also exhibit classical AD pathologies (e.g., P-tau, Aβ plaques). Here, we investigated whether pathological changes in brain ECM composition contribute to these shared defects. AD pathology in human hippocampus includes loss of perineuronal nets (PNNs), which are chondroitin sulfate (CS)-containing matrices that regulate key neurocircuit interactions critical to cognitive function. We report that as in humans, rat PNNs (identified by Wisteria floribunda agglutinin (WFA) labeling of CS) are comprised mainly of the CS-proteoglycan aggrecan (ACAN). Furthermore, we report a significant loss of stable WFA+/ACAN+ PNN structures in both the hypothalamus and hippocampus of ZDF rats, an established model of T2D that is also known to develop AD neuropathology, compared to aged-matched WT controls (Hypo: WFA 51.8±8.2% and ACAN 64.9±2.4% decrease; Hippo: WFA 43.9±5.2% and ACAN 25.9±3.8% decrease). PNN matrix stability is strongly influenced by CS composition, with an increase in 6S-CS promoting matrix destabilization. Using a novel mass spectrometry-based approach to characterize the CS composition isolated from brain tissue, we show that the sustained normalization of glycemia by fibroblast growth factor 1 (FGF-1) in T2D-ZDF rats is accompanied by both a reduction in hypothalamic 6S-CS abundance and restoration of normal WFA+/ACAN+ PNN structures (WFA: 2.0±0.2 and ACAN: 2.2±0.2-fold increase). Studies to determine if this is also true in hippocampus are ongoing. These results suggest that destabilized PNN structures cause neurocircuit dysfunction that affects glycemic control and impairs memory. FGF-1-induced normalization of PNNs underscores the potential for neurocircuit remodeling as a therapy for T2DM and AD. Disclosure K.M. Alonge: Research Support; Self; Novo Nordisk Inc. M.J. Reed: Stock/Shareholder; Spouse/Partner; Amgen Inc. M.A. Bentsen: None. C. Keene: None. M. Guttman: None. M.W. Schwartz: Consultant; Self; Novo Nordisk A/S. Research Support; Self; Novo Nordisk A/S. Funding National Institutes of Health; Novo Nordisk

The matrix: building more bioactive extracellular matrices via tuning of substrate stiffness

Background & Aim Mesenchymal stromal cells (MSCs) have long been at the forefront of cellular therapy and regenerative medicine, and they are being explored in more than 700 NIH-funded trials for their therapeutic potential. The low frequency of MSCs in the human body, poor yield of MSCs from tissue biopsies, and high number of MSCs required for clinical applications demand that tissue-isolated MSCs undergo a lengthy expansion ex vivo, during which they exhibit senescence and become unsuitable for therapeutic applications. Decellularized extracellular matrices (dECM) present a novel solution to this issue, as MSCs grown on dECM maintain greater viability as compared to those grown on conventional tissue culture substrates. The effect of environmental parameters on extracellular matrix bioactivity requires further elucidation. Particularly, we will investigate the influence of substrate stiffness on the composition of MSC-derived ECM, to produce customized dECM which enhances cellular growth and functionality. This is achieved by culturing MSCs on hydrogels of varying stiffnesses, and assessing the bioactivity of the ECM produced during culture. Methods, Results & Conclusion Mechanical properties of tunable stiffness polyacrylamide gels were verified using an Instron Microtester. Cells used were isolated from the decidua basalis, and ECM was sourced from decidua basalis hTERT-transfected MSCs. ECM deposition was induced by addition of ascorbic acid. Instron measurements confirm the elastic moduli of fully-hydrated polyacrylamide gels to be 4 kPa, 10 kPa, and 40 kPa, tailored to mimic the stiffness of adipose tissue, skeletal muscle tissue, and collagenous bone tissue, respectively. MSCs exhibit a spindle-shaped morphology when cultured on the hydrogels. Confocal microscopy reveals microarchitectural differences between ECM deposited on softer vs. stiffer hydrogels. We have shown that MSCs can be grown on hydrogels of varying stiffnesses, and can be induced to laydown extracellular matrix. The functionalization of the gels with collagen I to aid cell adhesion has been confirmed with immunofluorescence. The deposition of ECM has also been confirmed with confocal microscopy, and the alignment of ECM fibrils has been shown to depend on substrate stiffness. Further work will involve proteomic characterization of the dECMs, and culturing MSCs on the dECMs to assess bioactivity. We expect that the substrate stiffness will modulate the ECM bioactivity, allowing customization of tailored dECM for MSC expansion.

Cardiac tissue-derived extracellular matrix scaffolds for myocardial repair: advantages and challenges.

Decellularized extracellular matrix (dECM) derived from myocardium has been widely explored as a nature scaffold for cardiac tissue engineering applications. Cardiac dECM offers many unique advantages such as preservation of organ-specific ECM microstructure and composition, demonstration of tissue-mimetic mechanical properties and retention of biochemical cues in favor of subsequent recellularization. However, current processes of dECM decellularization and recellularization still face many challenges including the need for balance between cell removal and extracellular matrix preservation, efficient recellularization of dECM for obtaining homogenous cell distribution, tailoring material properties of dECM for enhancing bioactivity and prevascularization of thick dECM. This review summarizes the recent progresses of using dECM scaffold for cardiac repair and discusses its major advantages and challenges for producing biomimetic cardiac patch.

Designing a bioactive scaffold from coassembled collagen-laminin short peptide hydrogels for controlling cell behaviour.

Synthetic bioactive hydrogels have been widely recognized as key elements of emerging strategies for tissue engineering. The complex hierarchical structure and chemical composition of the natural ECM inspires us to design multi-component ECM mimics that have potential applications in biomedicine. Taking inspiration from natural proteins, we hypothesized that designing a multi-component synthetic matrix based on short ECM derived peptides will be highly beneficial for providing a functional scaffold, which still remains a challenge in the field of biomaterials. For the proof of concept, we designed a composite hydrogel scaffold inspired by the two essential components of native ECM, i.e. collagen and laminin, which play diverse roles in supporting cell growth. To the best of our knowledge, we have designed the shortest collagen inspired peptide sequence i.e. Nap-FFGSO, which has propensity to self-assemble in the presence of short laminin mimetic peptides. Interestingly, only 10% w/w of laminin peptides was sufficient to provide nucleation and growth of the nanostructures in the collagen inspired peptide and induces further self-assembly to create higher order structures. Such a nucleation and growth mechanism can trigger gelation in the collagen inspired peptides, which otherwise failed to form a gel under physiological conditions. We could achieve similar complexity in the designed matrix through utilization of simple non-covalent interactions, rather than covalent synthetic methodologies to create a dual functional matrix from the non-gelator collagen inspired peptide. The nanofibrous morphology generated through co-assembly can essentially mimic the structure and function of natural ECM, which enables the scaffold to communicate with cells through biochemical signals and promote cell growth, adhesion, proliferation and migration. We envisage that this structural mimicry of a native collagen fibrillar network using such a short peptide sequence can lead to new opportunities for developing next generation functional materials. The strategy of supramolecular assembly using multiple components could develop a plethora of viable biomaterials under physiological conditions.

The Expressional Pattern of Invasion-Related Extracellular Matrix Molecules in CNS Tumors

abstractAim of the study: Astrocytomas are primary CNS malignancies which infiltrate the peritumoral tissue, even when they are low-grade. Schwannomas are also primary CNS tumors, however, they do not show peritumoral infiltration similarly to brain metastases which almost never invade the neighboring parts of brain. Extracellular matrix is altered in composition in various cancer types and is proposed to play an important role in the development of invasiveness of astrocytic tumors. This study aims to identify differences in the ECM composition of CNS tumors with different invasiveness.Materials and methods: The mRNA and protein levels of ECM components were measured by QRT-PCR and mass-spectrometry, respectively, in grade II astrocytoma, NSCLC brain metastasis, schwannomas, and non-tumor brain control samples. Expressional data was analyzed statistically with ANOVA and nearest neighbor search.Results: There is a significant difference in the expressional pattern of invasion-related ECM components among various CNS tumors, especially among those of different embryonic origin. Non-invasive tumors show only slight differences in the expressional pattern of ECM molecules. Tumor samples can be separated based on their expressional pattern using statistical classifiers, therefore the ECM composition seems to be typical of various cancer types.Conclusions: Differences in the expressional pattern of the ECM could be responsible for the different invasiveness of various CNS tumors.

Tendon developmental plasticity and functional regeneration

Tendons enable movement by transferring force from muscle to bone. They are comprised of a highly organized collagen‐rich extracellular matrix. Disruption to the development and maintenance of tendons can lead to birth defects and result in injury in adults. We currently have an incomplete understanding of how tendons interact with their neighboring tissues to regulate the development and morphogenesis of the musculoskeletal system. In addition, once mature mammalian tendon tissue is formed, it has limited regenerative potential after injury. Central to this challenge is to uncover new mechanisms underlying tendon cell regulation during musculoskeletal development and regeneration. To accomplish this, we have turned to the zebrafish system because it is highly regenerative and amenable to genetic manipulations and live imaging. We have previously demonstrated that zebrafish tendons have molecular, developmental, morphological and biomechanical similarities to mammalian tendons, thereby making them an excellent vertebrate model to study tendon biology. However, no study has investigated their capacity to regenerate their tendons. Using the zebrafish, we developed methods to perform genetic tendon cell ablation, lineage tracing, live imaging, and mechanical testing to determine if developing and adult zebrafish can regenerate their tendon tissues. Following tendon cell ablation in developing zebrafish embryos, we observe defects in tendon gene expression and ECM composition. Cartilage and muscle tissue morphology was also disrupted following tendon cell loss. Tendon cells and the collagen organization returned after 1–2 weeks and 1–2 months, respectively. Interestingly, the pattern of the attachment sites was also restored, indicating the presence of regulatory programs that recruit and/or promote tendon cell proliferation and differentiation at the previous attachment sites. We are currently focused on identifying the developmental origins of the regenerating tendon cells and the pathways restoring the attachment site pattern. In addition to embryonic tendon regeneration, we also found that adult zebrafish appear to regenerate their collagen organization and mechanical properties after an acute physical injury. Together, our work demonstrates robust tendon regeneration at embryonic and adult stages in the zebrafish. These studies also provide a system to examine the morphogenetic and molecular events underlying tendon regeneration and musculoskeletal tissue‐tissue interactions, which has significance for regenerative biology approaches to musculoskeletal injuries.Support or Funding InformationNICHD HD06953, NIDCR DE024771, Charles H. Hood Foundation, HSCI Seed GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SAT-183 - Aspecific ECM composition regulates Smad dependent – TGFbeta1-induced EMT response in HepG2 cells engineered in cirrhotic and healthyliver 3D scaffolds

SAT-183 - Aspecific ECM composition regulates Smad dependent – TGFbeta1-induced EMT response in HepG2 cells engineered in cirrhotic and healthyliver 3D scaffolds

3D cell printing of in vitro stabilized skin model and in vivo pre-vascularized skin patch using tissue-specific extracellular matrix bioink: A step towards advanced skin tissue engineering

3D cell-printing technique has been under spotlight as an appealing biofabrication platform due to its ability to precisely pattern living cells in pre-defined spatial locations. In skin tissue engineering, a major remaining challenge is to seek for a suitable source of bioink capable of supporting and stimulating printed cells for tissue development. However, current bioinks for skin printing rely on homogeneous biomaterials, which has several shortcomings such as insufficient mechanical properties and recapitulation of microenvironment. In this study, we investigated the capability of skin-derived extracellular matrix (S-dECM) bioink for 3D cell printing-based skin tissue engineering. S-dECM was for the first time formulated as a printable material and retained the major ECM compositions of skin as well as favorable growth factors and cytokines. This bioink was used to print a full thickness 3D human skin model. The matured 3D cell-printed skin tissue using S-dECM bioink was stabilized with minimal shrinkage, whereas the collagen-based skin tissue was significantly contracted during in vitro tissue culture. This physical stabilization and the tissue-specific microenvironment from our bioink improved epidermal organization, dermal ECM secretion, and barrier function. We further used this bioink to print 3D pre-vascularized skin patch able to promote in vivo wound healing. In vivo results revealed that endothelial progenitor cells (EPCs)-laden 3D-printed skin patch together with adipose-derived stem cells (ASCs) accelerates wound closure, re-epithelization, and neovascularization as well as blood flow. We envision that the results of this paper can provide an insightful step towards the next generation source for bioink manufacturing.

Extracellular matrix directs phenotypic heterogeneity of activated fibroblasts

Activated fibroblasts are key players in the injury response, tumorigenesis, fibrosis, and inflammation. Dichotomous outcomes in response to varied stroma-targeted therapies in cancer emphasize the need to disentangle the roles of heterogeneous fibroblast subsets in physiological and pathophysiological settings. In wound healing, fibrosis, and myriad tumor types, fibroblast activation protein (FAP) and alpha-smooth muscle actin (αSMA) identify distinct, yet overlapping, activated fibroblast subsets. Prior studies established that FAPHi reactive fibroblasts and αSMAHi myofibroblasts can exert opposing influences in tumorigenesis. However, the factors that drive this phenotypic heterogeneity and the unique functional roles of these subsets have not been defined. We demonstrate that a convergence of ECM composition, elasticity, and transforming growth factor beta (TGF-β) signaling governs activated fibroblast phenotypic heterogeneity. Furthermore, FAPHi reactive fibroblasts and αSMAHi myofibroblasts exhibited distinct gene expression signatures and functionality in vitro, illuminating potentially unique roles of activated fibroblast subsets in tissue remodeling. These insights into activated fibroblast heterogeneity will inform the rational design of stroma-targeted therapies for cancer and fibrosis.

Extracellular matrix type modulates cell migration on mechanical gradients

Extracellular matrix composition and stiffness are known to be critical determinants of cell behavior, modulating processes including differentiation, traction generation, and migration. Recent studies have demonstrated that the ECM composition can modulate how cells migrate in response to gradients in environmental stiffness, altering a cell's ability to undergo durotaxis. These observations were limited to single varieties of extracellular matrix, but typically cells are exposed to environments containing complex mixtures of extracellular matrix proteins. Here, we investigate migration of NIH 3T3 fibroblasts on mechanical gradients coated with one or more type of extracellular matrix protein. Our results show that NIH 3T3 fibroblasts exhibit durotaxis on fibronectin-coated mechanical gradients but not on those coated with laminin, demonstrating that extracellular matrix type can act as a regulator of cell response to mechanical gradients. Interestingly, NIH 3T3 fibroblasts were also observed to migrate randomly on gradients coated with a mixture of both fibronectin and laminin, suggesting that there may be a complex interplay in the cellular response to mechanical gradients in the presence of multiple extracellular matrix signals. These findings indicate that specific composition of available adhesion ligands is a critical determinant of a cell's migratory response to mechanical gradients.

Paxillin genes and actomyosin contractility regulate myotome morphogenesis in zebrafish

Paxillin (Pxn) is a key adapter protein and signaling regulator at sites of cell-extracellular matrix (ECM) adhesion. Here, we investigated the role of Pxn during vertebrate development using the zebrafish embryo as a model system. We have characterized two Pxn genes, pxna and pxnb, in zebrafish that are maternally supplied and expressed in multiple tissues. Gene editing and antisense gene knockdown approaches were used to uncover Pxn functions during zebrafish development. While mutation of either pxna or pxnb alone did not cause gross embryonic phenotypes, double mutants lacking maternally supplied pxna or pxnb displayed defects in cardiovascular, axial, and skeletal muscle development. Transient knockdown of Pxn proteins resulted in similar defects. Irregular myotome shape and ECM composition were observed, suggesting an “inside-out” signaling role for Paxillin genes in the development of myotendinous junctions. Inhibiting non-muscle Myosin-II during somitogenesis altered the subcellular localization of Pxn protein and phenocopied pxn gene loss-of-function. This indicates that Paxillin genes are effectors of actomyosin contractility-driven morphogenesis of trunk musculature in zebrafish. Together, these results reveal new functions for Pxn during muscle development and provide novel genetic models to elucidate Pxn functions.

Abstract A40: A novel, biosynthetic 3D hydrogel system for breast cancer mechanobiology studies

Abstract Breast tumor cell-extracellular matrix (ECM) interactions drive malignant transformation, invasion, and metastasis of breast cancer cells and contribute to treatment resistance. The tumor microenvironment, composed of multiple ECM components, activate mechanochemical pathways associated with breast cancer progression and signaling of runt-related transcription factor 2 (Runx2). There is an urgent need to model these interactions in physiologically relevant three-dimensional (3D) models in vitro. We recently demonstrated that we can create 3D PEG hydrogel systems, containing type I collagen, Arg-Gly-Asp (RGD), and hyaluronic acid, whose stiffness can be tuned independent of the number of ECM components incorporated. In this study, we demonstrate that these hydrogels can be utilized to mimic the in vivo tumor microenvironment. We examined the behavior of breast cancer cell lines MDA-MB-231 (invasive) and MCF7 (non-invasive) as well as MCF10A (non-tumorigenic mammary epithelial cells) in our novel, biosynthetic hydrogel systems. We showed that these hydrogels supported growth for each cell line and formation of acinar structures over a 23-day period in vitro. In future studies, we will assess the migratory behaviors of these cell lines in vitro using microfluidic devices as well as Runx2 expression and activity to determine the influence of matrix physiochemical properties on the invasive potential of breast tumor cells. This novel, biosynthetic system represents a reproducible framework where tumor cell behavior may be quantified as a function of a complex system in which combined microenvironmental parameters, including matrix stiffness, ECM composition, and their interactions, are systematically varied. Citation Format: Amy Elissa Blatt, Bita Carrion, Rhima M. Coleman. A novel, biosynthetic 3D hydrogel system for breast cancer mechanobiology studies. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A40.

Oxymatrine inhibits the migration of human colorectal carcinoma RKO cells via inhibition of PAI-1 and the TGF-β1/Smad signaling pathway.

Transforming growth factor-β1 (TGF-β1) signaling has been shown to play a critical role in the development of epithelial-mesenchymal transition (EMT). PAI-1 is one of the most important target genes in the TGF-β/Smad signaling pathway, which can hinder the degradation of ECM composition and may promote cell invasion and migration. Oxymatrine (OM) is an alkaloid extracted from the Chinese herb Sophora flavescens Ait and has been demonstrated to inhibit the growth of various types of cancer cells including colorectal cancer. However, the anticancer effect of OM in colorectal cancer remains unclear. In the present study, we detected the expression of E-cadherin, α-SMA, FN, TGF-β1, PAI-1, Smad4, pP38 and pSmad2 in FHC, RKO and OM-treated RKO cells. We also detected pSmad2 and PAI-1 in RKO cells following the addition of SB203580 (a p38 MAPK inhibitor). The results showed that E-cadherin expression in RKO cells was significantly decreased, while PAI-1, TGF-β1, α-SMA, FN, Smad4, pSmad2 and pP38 expression levels were significantly increased in the RKO cells compared to levels in the FHC cells, which was almost completely reversed by OM. OM alleviated EMT induced in colorectal cancer via inhibition of TGF-β1/Smad signaling pathway activation by reducing P38-dependent increased expression of PAI-1. Hence, OM could be a novel therapeutic agent for colorectal cancer.

The effects of different doses of IGF-1 on cartilage and subchondral bone during the repair of full-thickness articular cartilage defects in rabbits

To investigate the effects of different doses of insulin-like growth factor 1 (IGF-1) on the cartilage layer and subchondral bone (SB) during repair of full-thickness articular cartilage (AC) defects. IGF-1-loaded collagen membrane was implanted into full-thickness AC defects in rabbits. The effects of two different doses of IGF-1 on cartilage layer and SB adjacent to the defect, the cartilage structure, formation and integration, and the new SB formation were evaluated at the 1st, 4th and 8th week postoperation. Meanwhile, after 1 week treatment, the relative mRNA expressions in tissues adjacent to the defect, including cartilage and SB were determined by quantitative real-time RT-PCR (qRT-PCR), respectively. Different doses of IGF-1 induced different gene expression profiles in tissues adjacent to the defect and resulted in different repair outcomes. Particularly, at high dose IGF-1 aided cell survival, regulated the gene expressions in cartilage layer adjacent defect and altered ECM composition more effectively, improved the formation and integrity of neo-cartilage. While, at low dose IGF-1 regulated the gene expressions in SB more efficaciously and subsequently promoted the SB remodeling and reconstruction. Different doses of IGF-1 induced different responses of cartilage or SB during the repair of full-thickness AC defects. Particularly, high dose of IGF-1 was more beneficial to the neo-cartilage formation and integration, while low dose of it was more effective for the SB formation.

Vascular smooth muscle cell durotaxis depends on extracellular matrix composition

Mechanical compliance has been demonstrated to be a key determinant of cell behavior, directing processes such as spreading, migration, and differentiation. Durotaxis, directional migration from softer to more stiff regions of a substrate, has been observed for a variety of cell types. Recent stiffness mapping experiments have shown that local changes in tissue stiffness in disease are often accompanied by an altered ECM composition in vivo. However, the importance of ECM composition in durotaxis has not yet been explored. To address this question, we have developed and characterized a polyacrylamide hydrogel culture platform featuring highly tunable gradients in mechanical stiffness. This feature, together with the ability to control ECM composition, allows us to isolate the effects of mechanical and biological signals on cell migratory behavior. Using this system, we have tracked vascular smooth muscle cell migration in vitro and quantitatively analyzed differences in cell migration as a function of ECM composition. Our results show that vascular smooth muscle cells undergo durotaxis on mechanical gradients coated with fibronectin but not on those coated with laminin. These findings indicate that the composition of the adhesion ligand is a critical determinant of a cell's migratory response to mechanical gradients.

Release of transgenic progranulin from a living hyaline cartilage graft model: An in vitro evaluation on anti-inflammation.

Osteoarthritis (OA) is a prevalent condition that compromises and even jeopardizes the life quality of millions of people. Common symptoms in OA includes joint stiffness and soreness, and they are often associated with inflammations to various extend. Due to the avascular and aneural nature of articular hyaline cartilage, it has limited self-repair capabilities; especially under inflammatory conditions, damages inflicted on cartilage are often irreversible. Hence, treatment approaches focus on anti-inflammation or articular cartilage replacement. In this study, an engineered, dual-functional living hyaline cartilage graft (LhCG), capable of releasing transgenic anti-inflammatory cytokine-progranulin (PGRN) is developed and envisioned to simultaneously fulfil both requirements. The therapeutic functionality of PGRN releasing LhCG is evaluated by co-culturing the constructs with tumor necrosis factor-alpha (TNFα) secreting THP-1 cells to simulate the inflammatory condition in arthritis. Non-transgenic LhCG constructs and non-coculture sample groups were set up as controls. Gene expression and ECM composition changes across samples were assessed to understand the effects of PGRN as well as inflammatory environment on the cartilage graft. Collectively, the results in this study suggest that in situ release of transgenic recombinant PGRN protects LhCG from induced inflammation in vitro; contrastively, in the absence of PGRN, cartilage grafts are at risk of being degraded and mineralized under exposure to TNFα signaling. This shows that cartilage graft itself can be at risk of degradation or calcification when implanted in arthritic microenvironment. Hence, the inflammatory microenvironment has to be considered in cartilage replacement therapy to increase chances of successful joint mobility restoration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2968-2977, 2016.

Engineering complex tissue-like microgel arrays for evaluating stem cell differentiation

Development of tissue engineering scaffolds with native-like biology and microarchitectures is a prerequisite for stem cell mediated generation of off-the-shelf-tissues. So far, the field of tissue engineering has not full-filled its grand potential of engineering such combinatorial scaffolds for engineering functional tissues. This is primarily due to the many challenges associated with finding the right microarchitectures and ECM compositions for optimal tissue regeneration. Here, we have developed a new microgel array to address this grand challenge through robotic printing of complex stem cell-laden microgel arrays. The developed microgel array platform consisted of various microgel environments that where composed of native-like cellular microarchitectures resembling vascularized and bone marrow tissue architectures. The feasibility of our array system was demonstrated through localized cell spreading and osteogenic differentiation of human mesenchymal stem cells (hMSCs) into complex tissue-like structures. In summary, we have developed a tissue-like microgel array for evaluating stem cell differentiation within complex and heterogeneous cell microenvironments. We anticipate that the developed platform will be used for high-throughput identification of combinatorial and native-like scaffolds for tissue engineering of functional organs.