Cell-mediated Degradation Research Articles

-RAMP3 promotes hepatocellular carcinoma tumor cell-mediated CCL2 degradation by supporting membrane distribution of ACKR2

This study aimed to explore the potential bind of Receptor Activity-Modifying Protein 3 (RAMP3) with atypical chemokine receptor 2 (ACKR2), and their cooperative regulation on the degradation of the immunosuppressive chemokine CCL2 in the tumor microenvironment of HCC. Bioinformatic analysis was conducted using available bulk-tissue RNA-seq, single-cell RNA-seq, and protein–protein interaction datasets. Human HCC cell line Huh7 and HepG2 and mouse HCC cell line Hepa1-6 were utilized for experiments. Results showed that RAMP3 binds with ACKR2 in HCC tumor cells and promotes the membrane distribution of ACKR2 through RAB4-positive vesicles. RAMP3 promotes CCL2 scavenging through ACKR2 in HCC cells. Mouse RAMP3 inhibited the proliferation of mouse liver cancer cell line (Hepa1-6)-derived syngeneic tumors through ACKR2, reduced the intratumoral concentration of CCL2 in the tumor, and inhibited the phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) and protein kinase B (AKT). In addition, mouse RAMP3 inhibited CD11b+/Gr-1 + myeloid cell infiltration and neovascularization in the tumors through ACKR2. In TCGA-LIHC, RAMP3low/ACKR2low group had the worst progression-free interval (PFI), while the RAMP3high/ACKR2high group had the best overall survival (OS). In summary, restoring RAMP3 expression in HCC cells may generate synergistic support for the anticancer effect of ACKR2.

Amivantamab efficacy in wild-type EGFR NSCLC tumors correlates with levels of ligand expression

Amivantamab is an FDA-approved bispecific antibody targeting EGF and Met receptors, with clinical activity against EGFR mutant non-small cell lung cancer (NSCLC). Amivantamab efficacy has been demonstrated to be linked to three mechanisms of action (MOA): immune cell-mediated killing, receptor internalization and degradation, and inhibition of ligand binding to both EGFR and Met receptors. Among the EGFR ligands, we demonstrated that amphiregulin (AREG) is highly expressed in wild-type (WT) EGFR (EGFRWT) NSCLC primary tumors, with significantly higher circulating protein levels in NSCLC patients than in healthy volunteers. Treatment of AREG-stimulated EGFRWT cells/tumors with amivantamab or with an AREG-targeting antibody inhibited ligand-induced signaling and cell/tumor proliferation/growth. Across 11 EGFRWT NSCLC patient-derived xenograft models, amivantamab efficacy correlated with AREG RNA levels. Interestingly, in these models, amivantamab anti-tumor activity was independent of Fc engagement with immune cells, suggesting that, in this context, the ligand-blocking function is sufficient for amivantamab maximal efficacy. Finally, we demonstrated that in lung adenocarcinoma patients, high expression of AREG and EGFR mutations were mutually exclusive. In conclusion, these data 1) highlight EGFR ligand AREG as a driver of tumor growth in some EGFRWT NSCLC models, 2) illustrate the preclinical efficacy of amivantamab in ligand-driven EGFRWT NSCLC, and 3) identify AREG as a potential predictive biomarker for amivantamab activity in EGFRWT NSCLC.

Quantifying and Controlling the Proteolytic Degradation of Cell Adhesion Peptides.

Peptides are widely used within biomaterials to improve cell adhesion, incorporate bioactive ligands, and enable cell-mediated degradation of the matrix. While many of the peptides incorporated into biomaterials are intended to be present throughout the life of the material, their stability is not typically quantified during culture. In this work, we designed a series of peptide libraries containing four different N-terminal peptide functionalizations and three C-terminal functionalizations to better understand how simple modifications can be used to reduce the nonspecific degradation of peptides. We tested these libraries with three cell types commonly used in biomaterials research, including mesenchymal stem/stromal cells (hMSCs), endothelial cells, and macrophages, and quantified how these cell types nonspecifically degraded peptides as a function of terminal amino acid and chemistry. We found that peptides in solution which contained N-terminal amines were almost entirely degraded by 48 h, irrespective of the terminal amino acid, and that degradation occurred even at high peptide concentrations. Peptides with C-terminal carboxylic acids also had significant degradation when cultured with the cells. We found that simple modifications to the termini could significantly reduce or completely abolish nonspecific degradation when soluble peptides were added to cells cultured on tissue culture plastic or within hydrogel matrices, and that functionalizations which mimicked peptide conjugations to hydrogel matrices significantly slowed nonspecific degradation. We also found that there were minimal differences in peptide degradation across cell donors and that sequences mimicking different peptides commonly used to functionalize biomaterials all had significant nonspecific degradation. Finally, we saw that there was a positive trend between RGD stability and hMSC spreading within hydrogels, indicating that improving the stability of peptides within biomaterial matrices may improve the performance of engineered matrices.

A Rheological Study on the Effect of Tethering Pro- and Anti-Inflammatory Cytokines into Hydrogels on Human Mesenchymal Stem Cell Migration, Degradation, and Morphology.

Polymer-peptide hydrogels are being designed as implantable materials that deliver human mesenchymal stem cells (hMSCs) to treat wounds. Most wounds can progress through the healing process without intervention. During the normal healing process, cytokines are released from the wound to create a concentration gradient, which causes directed cell migration from the native niche to the wound site. Our work takes inspiration from this process and uniformly tethers cytokines into the scaffold to measure changes in cell-mediated degradation and motility. This is the first step in designing cytokine concentration gradients into the material to direct cell migration. We measure changes in rheological properties, encapsulated cell-mediated pericellular degradation and migration in a hydrogel scaffold with covalently tethered cytokines, either tumor necrosis factor-α (TNF-α) or transforming growth factor-β (TGF-β). TNF-α is expressed in early stages of wound healing causing an inflammatory response. TGF-β is released in later stages of wound healing causing an anti-inflammatory response in the surrounding tissue. Both cytokines cause directed cell migration. We measure no statistically significant difference in modulus or the critical relaxation exponent when tethering either cytokine in the polymeric network without encapsulated hMSCs. This indicates that the scaffold structure and rheology is unchanged by the addition of tethered cytokines. Increases in hMSC motility, morphology and cell-mediated degradation are measured using a combination of multiple particle tracking microrheology (MPT) and live-cell imaging in hydrogels with tethered cytokines. We measure that tethering TNF-α into the hydrogel increases cellular remodeling on earlier days postencapsulation and tethering TGF-β into the scaffold increases cellular remodeling on later days. We measure tethering either TGF-β or TNF-α enhances cell stretching and, subsequently, migration. This work provides rheological characterization that can be used to design new materials that present chemical cues in the pericellular region to direct cell migration.

Osteogenic effects of covalently tethered rhBMP-2 and rhBMP-9 in an MMP-sensitive PEG hydrogel nanocomposite

While bone morphogenic protein-2 (BMP-2) is one of the most widely studied BMPs in bone tissue engineering, BMP-9 has been purported to be a highly osteogenic BMP. This work investigates the individual osteogenic effects of recombinant human (rh) BMP-2 and rhBMP-9, when tethered into a hydrogel, on encapsulated human mesenchymal stem cells (MSCs). A matrix-metalloproteinase (MMP)-sensitive hydrogel nanocomposite, comprised of poly(ethylene glycol) crosslinked with MMP-sensitive peptides, tethered RGD, and entrapped hydroxyapatite nanoparticles was used. The rhBMPs were functionalized with free thiols and then covalently tethered into the hydrogel by a thiol-norbornene photoclick reaction. rhBMP-2 retained its full bioactivity post-thiolation, while the bioactivity of rhBMP-9 was partially reduced. Nonetheless, both rhBMPs were highly effective at enhancing osteogenesis over 12-weeks in a chemically-defined medium. Expression of ID1 and osterix, early markers of osteogenesis; collagen type I, a main component of the bone extracellular matrix (ECM); and osteopontin, bone sialoprotein II and dentin matrix protein I, mature osteoblast markers, increased with increasing concentrations of tethered rhBMP-2 or rhBMP-9. When comparing the two BMPs, rhBMP-9 led to more rapid collagen deposition and greater mineralization long-term. In summary, rhBMP-2 retained its bioactivity post-thiolation while rhBMP-9 is more susceptible to thiolation. Despite this shortcoming with rhBMP-9, both rhBMPs when tethered into this hydrogel, enhanced osteogenesis of MSCs, leading to a mature osteoblast phenotype surrounded by a mineralized ECM. Statement of significanceOsteoinductive hydrogels are a promising vehicle to deliver mesenchymal stem cells (MSCs) for bone regeneration. This study examines the in vitro osteoinductive capabilities when tethered bone morphogenic proteins (BMPs) are incorporated into a degradable biomimetic hydrogel with cell adhesive ligands, matrix metalloproteinase sensitive crosslinks for cell-mediated degradation, and hydroxyapatite nanoparticles. This study demonstrates that BMP-2 is readily thiolated and tethered without loss of bioactivity while bioactivity of BMP-9 is more susceptible to immobilization. Nonetheless, when either BMP2 or BMP9 are tethered into this hydrogel, osteogenesis of human MSCs is enhanced, bone extracellular matrix is deposited, and a mature osteoblast phenotype is achieved. This bone-biomimetic hydrogel is a promising design for stem cell-mediated bone regeneration.

Enzyme-Mediated Nerve Growth Factor Release from Nanofibers Using Gelatin Microspheres.

Spinal cord injury is a complex environment, with many conflicting growth factors present at different times throughout the injury timeline. Delivery of multiple growth factors has received mixed results, highlighting a need to consider the timing of delivery for possibly antagonistic growth factors. Cell-mediated degradation of delivery vehicles for delayed release of growth factors offers an attractive way to exploit the highly active immune response in the spinal cord injury environment. In this study, growth factor-loaded gelatin microspheres (GMS) combined with methacrylated hyaluronic acid (MeHA) were electrospun to create GMS fibers (GMSF) for delayed release of growth factors (GFs). GMS were successfully combined with MeHA while electrospinning, with an average fiber diameter of 365 ± 10 nm and 44% ± 8% fiber alignment. GMSF with nerve growth factor (NGF) was tested on dissociated chick dorsal root ganglia cells. We further tested the effect of M1 macrophage-conditioned media (M1CM) to simulate macrophage invasion after spinal cord injury for cell-mediated degradation. We hypothesized that neurons grown on GMSF with loaded NGF would exhibit longer neurites in M1CM, showing a release of functional NGF, as compared with controls. GMSF in M1CM was significantly different from MeHA in serum-free media (SFM) and M0-conditioned media (M0CM), as well as GMSF in M0CM (p < 0.05). Moreover, GMSF + NGF in all media conditions were significantly different from MeHA in SFM and M0CM (p < 0.05). The goal of this study was to develop a biomaterial system where drug delivery is triggered by immune response, allowing for more control and longer exposure to encapsulated drugs. The spinal cord injury microenvironment is known to have a robust immune response, making this immune-medicated drug release system particularly significant for directed repair.

One-Pot Synthesis of Double-Network PEG/Collagen Hydrogel for Enhanced Adipogenic Differentiation and Retrieval of Adipose-Derived Stem Cells.

Hydrogels are widely used in stem cell therapy due to their extensive tunability and resemblance to the extracellular matrix (ECM), which has a three-dimensional (3D) structure. These features enable various applications that enhance stem cell maintenance and function. However, fast and simple hydrogel fabrication methods are desirable for stem cells for efficient encapsulation and to reduce adverse effects on the cells. In this study, we present a one-pot double-crosslinked hydrogel consisting of polyethylene glycol (PEG) and collagen, which can be prepared without the multi-step sequential synthesis of each network, by using bio-orthogonal chemistry. To enhance the adipogenic differentiation efficiency of adipose-derived stem cells (ADSCs), we added degradable components within the hydrogel to regulate matrix stiffness through cell-mediated degradation. Bio-orthogonal reactions used for hydrogel gelation allow rapid gel formation for efficient cell encapsulation without toxic by-products. Furthermore, the hybrid network of synthetic (PEG) and natural (collagen) components demonstrated adequate mechanical strength and higher cell adhesiveness. Therefore, ADSCs grown within this hybrid hydrogel proliferated and functioned better than those grown in the single-crosslinked hydrogel. The degradable elements further improved adipogenesis in ADSCs with dynamic changes in modulus during culture and enabled the retrieval of differentiated cells for potential future applications.

Injury-related cell death and proteoglycan loss in articular cartilage: Numerical model combining necrosis, reactive oxygen species, and inflammatory cytokines.

Osteoarthritis (OA) is a common musculoskeletal disease that leads to deterioration of articular cartilage, joint pain, and decreased quality of life. When OA develops after a joint injury, it is designated as post-traumatic OA (PTOA). The etiology of PTOA remains poorly understood, but it is known that proteoglycan (PG) loss, cell dysfunction, and cell death in cartilage are among the first signs of the disease. These processes, influenced by biomechanical and inflammatory stimuli, disturb the normal cell-regulated balance between tissue synthesis and degeneration. Previous computational mechanobiological models have not explicitly incorporated the cell-mediated degradation mechanisms triggered by an injury that eventually can lead to tissue-level compositional changes. Here, we developed a 2-D mechanobiological finite element model to predict necrosis, apoptosis following excessive production of reactive oxygen species (ROS), and inflammatory cytokine (interleukin-1)-driven apoptosis in cartilage explant. The resulting PG loss over 30 days was simulated. Biomechanically triggered PG degeneration, associated with cell necrosis, excessive ROS production, and cell apoptosis, was predicted to be localized near a lesion, while interleukin-1 diffusion-driven PG degeneration was manifested more globally. Interestingly, the model also showed proteolytic activity and PG biosynthesis closer to the levels of healthy tissue when pro-inflammatory cytokines were rapidly inhibited or cleared from the culture medium, leading to partial recovery of PG content. The numerical predictions of cell death and PG loss were supported by previous experimental findings. Furthermore, the simulated ROS and inflammation mechanisms had longer-lasting effects (over 3 days) on the PG content than localized necrosis. The mechanobiological model presented here may serve as a numerical tool for assessing early cartilage degeneration mechanisms and the efficacy of interventions to mitigate PTOA progression.

Scar-Degrading Endothelial Cells as a Treatment for Advanced Liver Fibrosis.

Deposition of extracellular matrix (ECM) in the liver is an important feature of liver cirrhosis. Recovery from liver cirrhosis is physiologically challenging, partially due to the ECM in scar tissue showing resistance to cell-mediated degradation by secreted matrix metalloproteinases (MMPs). Here, a cell-mediated ECM-degradation screening system (CEDSS) in vitro is constructed for high-throughput searching for cells with tremendous degradation ability. ECM-degrading liver sinusoidal endothelial cells (dLSECs) are screened using CEDSS, which exhibit 17 times the ability to degrade collagen when compared to other cells. The degradation ability of dLSECs is mediated by the upregulation of MMP9. In particular, mRNA expression of MMP9 shows an 833-fold increase in dLSECs compared to normal endothelial cells (nLSECs), and MMP9 is regulated by transcription factor c-Fos. In vivo, single intrasplenic injection of dLSECs alleviates advanced liver fibrosis in mice, while intraperitoneal administration of liver-targeting peptide-modified dLSECs shows enhanced fibrosis-targeting effects. Degradative human umbilical vein endothelial cells (dHUVECs) prove their enhanced potential of clinical translation. Together, these results highlight the potential of ECM-degrading endothelial cells in alleviating advanced liver fibrosis, thus providing remarkable insights in the development of ECM-targeting therapeutics.

Biodegradable Interpolycomplexes for Anti-Erosion Stabilization of Soil and Sand.

A linear anionic polysaccharide, sodium alginate, electrostatically interacts with a cationic polysaccharide, quaternized hydroxyethyl cellulose ethoxylate, in aqueous solution, thus giving an interpolyelectrolyte complex. Aqueous solutions of the initial polysaccharides and polycomplexes with an excess of the cationic or anionic polymers were used for the stabilization of soil and sand against water erosion. Physicochemical, mechanical and biological properties of the polymers and coatings were characterized by gravimetric analysis, viscosimetry, mechanical strength assessment, cell viability, and cell-mediated degradation with the following main conclusions. (a) Non-stoichiometric polycomplexes with an excess of cationic or anionic units ("cationic" and "anionic" polycomplexes, respectively) form transparent solutions or stable-in-time dispersions. (b) The complexation results in a decrease in the viscosity of polymer solutions. (c) A complete dissociation of polycomplexes to the initial components is achieved in a 0.2 M NaCl solution. (d) Soil/sand treatment with 1 wt% aqueous solutions of polymers or polycomplexes and further drying lead to the formation of strong composite coatings from polymer(s) and soil/sand particles. (e) Cationic polycomplexes form stronger coatings in comparison with anionic polycomplexes. (f) The polymer-soil coatings are stable towards re-watering, while the polymer-sand coatings show a much lower resistance to water. (g) The individual polysaccharides demonstrate a negligible toxicity to Gram-negative and Gram-positive bacteria and yeast. (h) The addition of Bacillus subtilis culture initiates the degradation of the polysaccharides and polycomplexes. (i) Films from polysaccharides and polycomplexes decompose down to small fragments after being in soil for 6 weeks. The results of the work are of importance for constructing water-resistant, low toxicity and biodegradable protective coatings for soil and sand.

In Vivo Biocompatibility Investigation of an Injectable Calcium Carbonate (Vaterite) as a Bone Substitute including Compositional Analysis via SEM-EDX Technology.

Injectable bone substitutes (IBS) are increasingly being used in the fields of orthopedics and maxillofacial/oral surgery. The rheological properties of IBS allow for proper and less invasive filling of bony defects. Vaterite is the most unstable crystalline polymorph of calcium carbonate and is known to be able to transform into hydroxyapatite upon contact with an organic fluid (e.g., interstitial body fluid). Two different concentrations of hydrogels based on poly(ethylene glycol)-acetal-dimethacrylat (PEG-a-DMA), i.e., 8% (w/v) (VH-A) or 10% (w/v) (VH-B), were combined with vaterite nanoparticles and implanted in subcutaneous pockets of BALB/c mice for 15 and 30 days. Explants were prepared for histochemical staining and immunohistochemical detection methods to determine macrophage polarization, and energy-dispersive X-ray analysis (EDX) to analyze elemental composition was used for the analysis. The histopathological analysis revealed a comparable moderate tissue reaction to the hydrogels mainly involving macrophages. Moreover, the hydrogels underwent a slow cellular infiltration, revealing a different degradation behavior compared to other IBS. The immunohistochemical detection showed that M1 macrophages were mainly found at the material surfaces being involved in the cell-mediated degradation and tissue integration, while M2 macrophages were predominantly found within the reactive connective tissue. Furthermore, the histomorphometrical analysis revealed balanced numbers of pro- and anti-inflammatory macrophages, demonstrating that both hydrogels are favorable materials for bone tissue regeneration. Finally, the EDX analysis showed a stepwise transformation of the vaterite particle into hydroxyapatite. Overall, the results of the present study demonstrate that hydrogels including nano-vaterite particles are biocompatible and suitable for bone tissue regeneration applications.

Computational Modeling Intervertebral Disc Pathophysiology: A Review

Lower back pain is a medical condition of epidemic proportion, and the degeneration of the intervertebral disc has been identified as a major contributor. The etiology of intervertebral disc (IVD) degeneration is multifactorial, depending on age, cell-mediated molecular degradation processes and genetics, which is accelerated by traumatic or gradual mechanical factors. The complexity of such intertwined biochemical and mechanical processes leading to degeneration makes it difficult to quantitatively identify cause–effect relationships through experiments. Computational modeling of the IVD is a powerful investigative tool since it offers the opportunity to vary, observe and isolate the effects of a wide range of phenomena involved in the degenerative process of discs. This review aims at discussing the main findings of finite element models of IVD pathophysiology with a special focus on the different factors contributing to physical changes typical of degenerative phenomena. Models presented are subdivided into those addressing role of nutritional supply, progressive biochemical alterations stemming from an imbalance between anabolic and catabolic processes, aging and those considering mechanical factors as the primary source that induces morphological change within the disc. Limitations of the current models, as well as opportunities for future computational modeling work are also discussed.

Supramolecular Click Product Interactions Induce Dynamic Stiffening of Extracellular Matrix-Mimetic Hydrogels.

Progressive stiffening of the extracellular matrix (ECM) is observed in tissue development as well as in pathologies such as cancer, cardiovascular disease, and fibrotic disease. However, methods to recapitulate this phenomenon in vitro face critical limitations. Here, we present a poly(ethylene glycol)-based peptide-functionalized ECM-mimetic hydrogel platform capable of facile, user-controlled dynamic stiffening. This platform leverages supramolecular interactions between inverse-electron demand Diels-Alder tetrazine-norbornene click products (TNCP) to create pendant moieties that undergo non-covalent crosslinking, stiffening a pre-existing network formed via thiol-ene click chemistry over the course of 6 h. Pendant TNCP moieties have a concentration-dependent effect on gel stiffness while still being cytocompatible and permissive of cell-mediated gel degradation. The robustness of this approach as well as its simplicity and ease of translation give it broad potential utility.

FLASH: Fluorescently LAbelled Sensitive Hydrogel to monitor bioscaffolds degradation during neocartilage generation

Regenerative therapies based on photocrosslinkable hydrogels and stem cells are of growing interest in the field of cartilage repair. Cell-mediated degradation is critical for the successful clinical translation of implanted hydrogels. However, characterising cell-mediated degradation, while simultaneously monitoring the deposition of a distinct new matrix, remains a major challenge.In this study we generated a Fluorescently LAbelled Sensitive Hydrogel (FLASH) to correlate the degradation of a hydrogel bioscaffold with neocartilage formation. Gelatine Methacryloyl (GelMA) was covalently bound to the FITC fluorophore to generate FLASH and bioscaffolds were produced by casting different concentrations of FLASH GelMA, with and without human adipose-derived stem cells (hADSCs) undergoing chondrogenesis. The loss of fluorescence from FLASH bioscaffolds was correlated with changes in mechanical properties, expression of chondrogenic markers and accumulation of a cartilaginous extracellular matrix. The ability of the system to be used as a sensor to monitor bioscaffold degradability during chondrogenesis was evaluated in vitro, in a human ex vivo model of cartilage repair and in a full chondral defect in vivo rabbit model. This study represents a step towards the generation of a high throughput monitoring system to evaluate de novo cartilage formation in tissue engineering therapies.

A comparative study of brain tumor cells from different age and anatomical locations using 3D biomimetic hydrogels

Brain tumors exhibit vast genotypic and phenotypic diversity depending on patient age and anatomical location. Hydrogels hold great promise as 3D in vitro models for studying brain tumor biology and drug screening, yet previous studies were limited to adult glioblastoma cells, and most studies used immortalized cell lines. Here we report a hydrogel platform that supports the proliferation and invasion of patient-derived brain tumor cell cultures (PDCs) isolated from different patient age groups and anatomical locations. Hydrogel stiffness was tuned by varying poly(ethylene-glycol) concentration. Cell adhesive peptide (CGRDS), hyaluronic acid, and MMP-cleavable crosslinkers were incorporated to facilitate cell adhesion and cell-mediated degradation. Three PDC lines were compared including adult glioblastoma cells (aGBM), pediatric glioblastoma cells (pGBM), and diffuse pontine intrinsic glioma (DIPG). A commonly used immortalized adult glioblastoma cell line U87 was included as a control. PDCs displayed stiffness-dependent behavior, with 40 Pa hydrogel promoting faster tumor proliferation and invasion. Adult GBM cells exhibited faster proliferation than pediatric GBM, and DIPG showed slowest proliferation. These results suggest both patient age and tumor location affects brain tumor behaviors. Adult GBM PDCs also exhibited very different cell proliferation and morphology from U87. The hydrogel reported here can provide a useful tool for future studies to better understand how age and anatomical locations impacts brain tumor progression using 3D in vitro models.

Determining How Human Mesenchymal Stem Cells Change Their Degradation Strategy in Response to Microenvironmental Stiffness.

During the wound healing process, human mesenchymal stem cells (hMSCs) are recruited to the injury where they regulate inflammation and initiate healing and tissue regeneration. To aid in healing, synthetic cell-laden hydrogel scaffolds are being designed to deliver additional hMSCs to wounds to enhance or restart the healing process. These scaffolds are being designed to mimic native tissue environments, which include physical cues, such as scaffold stiffness. In this work, we focus on how the initial scaffold stiffness hMSCs are encapsulated in changes cell-mediated remodeling and degradation and motility. To do this, we encapsulate hMSCs in a well-defined synthetic hydrogel scaffold that recapitulates aspects of the native extracellular matrix (ECM). We then characterize cell-mediated degradation in the pericellular region as a function of initial microenvironmental stiffness. Our hydrogel consists of a 4-arm poly(ethylene glycol) (PEG) end-functionalized with norbornene which is chemically cross-linked with a matrix metalloproteinase (MMP) degradable peptide sequence. This peptide sequence is cleaved by hMSC-secreted MMPs. The hydrogel elastic modulus is varied from 80 to 2400 Pa by changing the concentration of the peptide cross-linker. We use multiple particle tracking microrheology (MPT) to characterize the spatiotemporal cell-mediated degradation in the pericellular region. In MPT, fluorescently labeled particles are embedded in the material, and their Brownian motion is measured. We measure an increase in cell-mediated degradation and remodeling as the post-encapsulation time increases. MPT also measures changes in the degradation profile in the pericellular region as hydrogel stiffness is increased. We hypothesize that the change in the degradation profile is due to a change in the amount and type of molecules secreted by hMSCs. We also measure a significant decrease in cell speed as hydrogel stiffness increases due to the increased physical barrier that needs to be degraded to enable motility. These measurements increase our understanding of the rheological changes in the pericellular region in different physical microenvironments which could lead to better design of implantable biomaterials for cell delivery to wounded areas.

Age-dependent regulation of cell-mediated collagen turnover.

Although aging represents the most important epidemiologic risk factor for fibrotic disease, the reasons for this are incompletely understood. Excess collagen deposition in tissues is the sine qua non of tissue fibrosis and can be viewed as an imbalance between collagen production and collagen degradation. Yet we still lack a detailed understanding of the changes that take place during development, maturation, and aging in extracellular matrix (ECM) dynamics. Resolution of fibrosis is impaired in aging, and this impairment may explain why age is the most important risk factor for fibrotic diseases, such as idiopathic pulmonary fibrosis. However, ECM dynamics and impaired resolution of fibrosis in aging remain understudied. Here we show that cell-mediated collagen uptake and degradation are diminished in aged animals and this finding correlates with downregulation of the collagen endocytic receptor mannose receptor, C-type 2 (Mrc2). We identify myeloid zinc finger-1 as a potentially novel transcriptional regulator of Mrc2, and both this transcription factor and Mrc2 are downregulated in multiple tissues and organisms in an age-dependent manner. Thus, cell-mediated degradation of collagen is an essential process that promotes resolution of fibrosis, and impairment in this process contributes to age-related fibrosis.

Tuning the immune reaction to manipulate the cell-mediated degradation of a collagen barrier membrane

In order to elicit a desired barrier function in guided bone regeneration (GBR) or guided tissue regeneration (GTR), a barrier membrane has to maintain its integrity for a certain period of time to guarantee the regeneration of target tissue. Due to the complexity and variety of clinical conditions, the healing time required for tissue regeneration varies from one case to another, which implies the need for tailoring the barrier membranes to diverse conditions via manipulating their degradation property. As a “non-self” biomaterial, a barrier membrane will inevitably trigger host-membrane immune response after implantation, which entails the activation of phagocytic cells. In the degradation process of a barrier membrane, the cell-mediated degradation may play a more vital role than enzymatic and physicochemical dissolution; however, limited studies have been carried out on this topic. In this context, we investigated the cell-mediated degradation and illustrated the possible key cells and mediators for immunomodulation via in vivo and in vitro studies. We discovered that IL-13, a key cytokine mainly released by T helper 2 cells (Th2), induced the formation of foreign body giant cells (FBGCs), thus resulting in membrane degradation. Neutralizing IL-13 could suppress membrane degradation and formation of FBGC. The contributions of this study are (1) unveiling the immune mechanisms underlying the cell-mediated collagen membrane degradation; (2) allowing the formation of an “immunodegradation” strategy to develop an “immune-smart” barrier membrane to manipulate its degradation; (3) providing the key regulatory immune cells and cytokines for the immunomodulation target in collagen membrane degradation. Statement of SignificanceThe significance of this research includes:(1)Unveiling the immune mechanisms underlying the cell-mediated collagen membrane degradation;(2)Allowing the formation of an “immunodegradation” strategy to develop an “immune-smart” barrier membrane to manipulate its degradation;(3)Providing the key regulatory immune cells and cytokines for the immunomodulation target in collagen membrane degradation, which may allow accurate and customized modulation during tissue/bone regenerative process.

Progress on cell-mediated degradation of bone materials

Biomaterials have been widely used as bone grafts for bone tissue repair. The application of biomaterials needs to consider various aspects of material properties such as biocompatibility, mechanical strength and plasticity. It is also necessary for bone repair to consider the degradability of materials. Previous studies have shown that biomaterials can be degraded by physical, chemical and biological ways. Cell-mediated degradation is an important part of the biodegradation process of , mainly carried out by the biological behavior of macrophages and osteoclasts and reactive oxygen species, enzymes and acidic metabolites secreted by them. Illustration of cell-mediated degradation of biological materials helps us understand the biological behavior of cells better, to accurately design and manufacture more effective bone repair materials, which is conducive to initial stability during material implantation, in line with the consistence of material degradation and new bone formation, promoting bone regeneration and bone repair.

IDG-SW3 Osteocyte Differentiation and Bone Extracellular Matrix Deposition Are Enhanced in a 3D Matrix Metalloproteinase-Sensitive Hydrogel

Osteocytes reside within a heavily mineralized matrix making them difficult to study in vivo and to extract for studies in vitro. IDG-SW3 cells are capable of producing mineralized collagen matrix and transitioning from osteoblasts to mature osteocytes, thus offering an alternative to study osteoblast to late osteocyte differentiation in vitro. The goal for this work was to develop a 3D degradable hydrogel to support IDG-SW3 differentiation and deposition of bone ECM. In 2D, the genes Mmp2 and Mmp13 increased during IDG-SW3 differentiation and were used as targets to create a MMP-sensitive poly(ethylene glycol) hydrogel containing the peptide crosslink GCGPLG-LWARCG and RGD to promote cell attachment. IDG-SW3 differentiation in the MMP-sensitive hydrogels improved over non-degradable hydrogels and standard 2D culture. Alkaline phosphatase activity at day 14 was higher, Dmp1 and Phex were 8.1-fold and 3.8-fold higher, respectively, and DMP1 protein expression was more pronounced in the MMP-sensitive hydrogels compared to non-degradable hydrogels. Cell-encapsulation density (cells/ml precursor) influenced formation of dendrite-like cellular process and mineral and collagen deposition with 80×106 performing better than 2×106 or 20×106, while connexin 43 was not affected by cell density. The cell density effects were more pronounced in the MMP-sensitive hydrogels over non-degradable hydrogels. This study identified that high cell encapsulation density and a hydrogel susceptible to cell-mediated degradation enhanced mineralized collagen matrix and osteocyte differentiation. Overall, a promising hydrogel is presented that supports IDG-SW3 cell maturation from osteoblasts to osteocytes in 3D.