Journal of Biomedical Materials Research Part A

BackgroundHydrogel-based 3D culture systems are emerging as a valuable tool for preclinical screening of cell-based immunotherapies against solid and hematological malignancies, such as chimeric antigen receptor T (CAR-T) cells. Hydrogels can influence T cell function in a non-desired manner due to their mechanical properties and chemical composition, potentially skewing results in preclinical testing of novel immunotherapeutic compounds. MethodsIn this study, we assess CD4+ T and CAR-T cell activation and proliferation in chemically-undefined matrices (Matrigel and basement membrane extract, BME) and compare them to a synthetic nanofibrillar cellulose (NFC) hydrogel. ResultsRheometric analyses show that NFC is more rigid than Matrigel and BME. Murine CD4+ T cells acquire a regulatory T cell (Treg) phenotype in Matrigel and BME, while this is not observed in NFC. Proliferation and activation of human T cells are higher in NFC than in Matrigel or BME. Similarly, we show that CAR-T cell activation and proliferation is significantly impaired in Matrigel and BME, in contrast to NFC. ConclusionsOur findings highlight the impact of hydrogel choice on (CAR-)T cell behavior, with direct implications for preclinical immunotherapy testing. In contrast to Matrigel and BME, NFC offers a chemically-defined 3D environment where T cell function is preserved. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSIn 3D (preclinical) tumor-killing assays for evaluating engineered T cell cytotoxicity, the surrounding matrix can influence immune cell phenotype and function, potentially skewing T cell activity. Basement membrane hydrogels such as Matrigel and basement membrane extract (BME), widely used as scaffolds for 3D culture, are inherently heterogeneous and contain extracellular matrix components that can influence lymphocyte function. What this study addsHere, we show that (CAR-)T cell function is significantly reduced in Matrigel and BME as compared to standard (2D) culture conditions. In contrast, (CAR-)T cell activity is preserved in synthetic nanofibrillar cellulose (NFC) gels. Importantly, murine T cells spontaneously acquire a Treg phenotype in Matrigel and BME. T cell proliferation and cytokine secretion are >10-fold lower in Matrigel than in NFC. Similarly, CAR-T cell survival and expansion are 10-fold higher in NFC than in Matrigel or BME. How this study might affect research, practice or policyWe report that the intrinsic cytotoxic and proliferative potential of (CAR-)T cells can be underestimated when performing assays in 3D cultures based on Matrigel or BME. As an alternative, we suggest the use of chemically defined synthetic gels, and we show that nanofibrillar cellulose hydrogels are suitable 3D matrices for preserving T cell phenotype and activation.

Volumetric muscle loss (VML) is an irreversible muscle injury that results in chronic functional impairment. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) can facilitate tissue repair through immunomodulatory, angiogenic, and anti-fibrotic effects. However, their low yield and poor on-site retention limit their therapeutic efficacy. Hypoxia can boost MSC metabolism, proliferation, and EV production. Hypoxic (3% O2) preconditioning of MSCs increased the yield of EVs (30-300 nm) by 1.5-fold but decreased the expression of characteristic EV markers (i.e., CD81, ICAM, and FLOT1). Fibrin hydrogels promote skeletal muscle regeneration and can sequester EVs via integrins or electrostatic interactions. We hypothesized that encapsulating EVs in fibrin hydrogels would further enhance regeneration and prolong the retention of EVs at the VML injury site. VML was created by removing [~]20% of the gastrocnemius-soleus muscles mass in mice using a 3 mm biopsy punch. EVs (4.48x1010 particles/mL) derived from MSCs cultured under hypoxic (Hypo-EV) or normoxic (Norm-EV) conditions were encapsulated within fibrin hydrogels and implanted at the VML injury site. Fibrin hydrogels containing PBS (PFG) were used as controls. On day 14 post-injury, Norm-EV treatment resulted in increased muscle mass, angiogenesis, and myofiber regeneration relative to the Hypo-EV group. Both the Norm-EV and Hypo-EV treatment groups reduced macrophage infiltration at the injury site compared to the PFG. These findings highlight that while both Norm-EV and Hypo-EV exhibit immunomodulatory effects, they differ in their regenerative potential. We speculate that hypoxic conditions could have caused MSCs to prioritize survival over repair-promoting activities, thereby producing EVs with less pro-regenerative signals. The increased quantity of EVs in response to hypoxia doesnt compensate for their diminished regenerative potential, highlighting the importance of quality over quantity when considering EVs for therapeutic applications. Graphical AbstractJain et al., Comparative Effects of Hypoxic vs. Normoxic Mesenchymal Stem Cell-Derived Extracellular Vesicles on Tissue Repair Following Volumetric Muscle Loss (VML) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/697216v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@79a1feorg.highwire.dtl.DTLVardef@17a5f41org.highwire.dtl.DTLVardef@103c2e8org.highwire.dtl.DTLVardef@1f13e13_HPS_FORMAT_FIGEXP M_FIG C_FIG

Severe traumatic brain injury (TBI) is a life-threatening condition characterized by internal brain swelling and commonly treated using a two-stage surgical approach. The interval between surgeries, generally spaced weeks to months, is associated with secondary neurologic complications from leaving the brain unprotected. Hydrogels may reshape severe TBI treatment by enabling a single-stage surgical intervention, capable of being implanted at the initial surgery, remaining flexible to accommodate brain swelling, and calibrated to regenerate bone after brain swelling has subsided. The current study evaluated the use of a pentenoate-modified hyaluronic acid (PHA) polymer with thiolated devitalized tendon (TDVT) for calvarial bone regeneration in a rat TBI model. Additionally, PHA-TDVT hydrogels encapsulating microspheres containing bone morphogenetic protein-2 (BMP-2) were investigated to enhance bone regeneration. All hydrogel precursor formulations exhibited sufficient yield stress for surgical placement. The addition of TDVT to the crosslinked hydrogels increased the average compressive modulus. In vitro cell studies revealed that the PHA-TDVT hydrogel with the highest concentration of BMP-2 microspheres (i.e., PHA-TDVT+{micro}100) significantly improved calcium deposition and osteogenic gene expression. Minimal in vivo bone regeneration was observed for all hydrogel groups; however, BMP-2 microsphere addition fortuitously reduced motor skill impairment and brain atrophy. The PHA-TDVT+{micro}100 group had 2.8 times greater reach index and 2.3 times lower brain atrophy values compared to the negative control (p<0.05). Overall, hydrogels with controlled release of BMP-2 may provide neuroprotective benefits in TBI treatment. Future studies should explore BMP-2 delivery strategies to enhance both bone and brain recovery in rat TBI studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/649206v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@136d4d4org.highwire.dtl.DTLVardef@cee564org.highwire.dtl.DTLVardef@13612e8org.highwire.dtl.DTLVardef@1134b4c_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceSevere traumatic brain injury (TBI) is a life-threatening condition characterized by internal brain swelling and is commonly treated using a two-stage surgical approach. Complications associated with the two-stage treatment paradigm include secondary neurologic impairment, termed syndrome of the trephined (SOT). SOT is often reversible once the second surgery is performed, whereas a single-stage TBI treatment paradigm may avoid the occurrence of SOT altogether. Utilizing hydrogels comprised of pentenoate-modified hyaluronic acid and thiolated devitalized tendon encapsulating microspheres containing bone morphogenetic protein-2 (BMP-2), the current study demonstrated improvements in motor skill function and reductions in brain atrophy in a rat TBI model. The introduction of hydrogels with controlled release of BMP-2 as a neuroprotective strategy for TBI application offers a promising approach for single-stage TBI treatment.

Skeletal muscle cannot regenerate after volumetric muscle loss (VML), a traumatic injury defined as the loss of > 20% of a muscles mass. VML directly reduces the number of myofibers and causes axonal degeneration of nerves, resulting in reduced muscle function and impaired neuromuscular junctions (NMJs). Biosponge (BSG) scaffolds, composed of gelatin, collagen, and laminin-111, have been shown to improve muscle mass, cross-sectional area, and myofiber number following VML. However, improvements in NMJ quantity were not observed. Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that enhances motor unit survival and neurite outgrowth. In this work, BSG scaffolds were electrostatically coupled with GDNF via gelatin nanoparticles (GNPs) to support myofiber regeneration and preserve NMJs post-VML in a rodent model. In vitro determination of release kinetics revealed an initial burst release of surface bound GDNF with almost an equivalent amount of electrostatically bound GDNF retained within the BSG post 1 week of incubation at 37{degrees}C in phosphate buffered saline (PBS). To create the VML injury in male Lewis rats (10-12 weeks old), [~]20% of the muscle mass was removed from the tibialis anterior (TA) muscle of both hindlimbs. Relative to BSG+GNP alone, treatment with BSG+GNP+GDNF showed a significant increase ([~]25%) in peak isometric torque at 6 weeks post-injury. Qualitative and quantitative histological analysis of NMJs revealed an enhanced overlap between pre- and post-synaptic structures in the BSG+GNP+GDNF group. Additionally, the incorporation of GDNF slowed BSG remodeling and degradation. Overall, these results suggest that the BSG-mediated delivery of GDNF is an effective strategy for mitigating NMJ loss and enhancing muscle recovery following VML. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/693478v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1719d84org.highwire.dtl.DTLVardef@1c6bceorg.highwire.dtl.DTLVardef@1e9a9a5org.highwire.dtl.DTLVardef@180ad57_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Abstract Tadiwala et al., 2025 Biosponges embedded with GDNF promote neuromuscular recovery following volumetric muscle loss.

Decellularized extracellular matrix (ECM) hydrogels present a novel, clinical intervention for a myriad of regenerative medicine applications. The source of ECM is typically the same tissue to which the treatment is applied; however, the need for tissue specific ECM sources has not been rigorously studied. We hypothesized that tissue specific ECM would improve regeneration through preferentially stimulating physiologically relevant processes (e.g. progenitor cell proliferation and differentiation). One of two decellularized hydrogels (tissue specific skeletal muscle or non mesoderm-derived lung) or saline were injected intramuscularly two days after notexin injection in mice (n=7 per time point) and muscle was harvested at days 5 and 14 for histological and gene expression analysis. Both injectable hydrogels were decellularized using the same detergent and were controlled for donor characteristics (i.e. species, age). At day 5, the skeletal muscle ECM hydrogel significantly increased the density of Pax7+ satellite cells in the muscle. Gene expression analysis at day 5 showed that skeletal muscle ECM hydrogels increased expression of genes implicated in muscle contractility. By day 14, skeletal muscle ECM hydrogels improved muscle regeneration over saline and lung ECM hydrogels as shown through a shift in fiber cross sectional area distribution towards larger fibers. This data indicates a potential role for muscle-specific regenerative capacity of decellularized, injectable muscle hydrogels. Further transcriptomic analysis of whole muscle mRNA indicates the mechanism of tissue specific ECM-mediated tissue repair may be immune and metabolism pathway-driven. Taken together, this suggests there is benefit in using tissue specific ECM for regenerative medicine applications.Competing Interest StatementKLC is co-founder, board member, consultant, receives income, and has equity interest in Ventrix, Inc.View Full Text

BACKGROUNDCartilage damage affects 25 million people globally each year. Tissue engineering strategies such as microfracture and matrix induced autologous chondrocyte implantation (MACI) are currently being used in the clinic; however, they are accompanied by their own limitations such as donor site morbidity, rapid clearance from the injury site, and extensive cost. To overcome these limitations, the tissue engineering field has shown increasing interest in the use of decellularized extracellular matrix (dECM) biomaterials due to their heightened integration with native tissue and regeneration rates. METHODSThe Gottardi Lab has developed a new dECM material sourced from porcine meniscus decellularization (MEND), in which elastin fibers are removed via enzymatic digestion, resulting in channels that can be easily recellularized. RESULTSIn this work we demonstrate that MEND can be seeded with bone-marrow derived mesenchymal stem cells (MSCs), achieving a uniform distribution of cell nuclei throughout the cross section of the scaffold. We also show that MEND retains its native structure in the presence of MSCs and can support chondrogenesis comparably to other commonly used tissue engineering materials such as methacrylated type I collagen and gelatin/hyaluronic acid hydrogels. CONCLUSIONOverall, MEND is a promising new dECM biomaterial for cartilage regeneration.

Human mesenchymal stem cells (MSCs) have demonstrated promise when delivered to damaged tissue or tissue defects for their cytokine secretion and inflammation modulation behaviors that can promote repair. Insulin-like growth factor 1 (IGF-1) has been shown to augment MSCs viability and survival and promote their secretion of cytokines that signal to endogenous cells, in the treatment of myocardial infarction, wound healing, and age-related diseases. Biomaterial cell carriers can be functionalized with growth factor-mimetic peptides (i.e. IGF-1 mimicking peptides) to enhance MSC function while promoting cell retention and minimizing off-target effects seen with direct administration of soluble growth factors. Here, we functionalized alginate hydrogels with three distinct IGF-1 peptide mimetics and the integrin-binding peptide, cyclic RGD. One IGF-1 peptide mimetic (IGM-3) in combination with integrin ligand was found to activate Akt and ERK1/2 signaling and support survival of serum-deprived MSCs. MSCs encapsulated in alginate hydrogels that presented both IGM-3 and cRGD showed a significant reduction in pro-inflammatory cytokine secretion when challenged with interleukin-1{beta}. Finally, MSCs cultured within the cRGD/IGM-3 hydrogels were able to blunt pro-inflammatory gene expression of human primary cells from degenerated intervertebral discs. These studies indicate the potential to leverage cell adhesive and IGF-1 growth factor peptide mimetics together to control therapeutic secretory behavior of MSCs. Significance StatementInsulin-like growth factor 1 (IGF-1) plays a multifaceted role in stem cell biology and may promote proliferation, survival, migration, and immunomodulation for MSCs. In this study, we functionalized alginate hydrogels with integrin-binding and IGF-1 peptide mimetics to investigate their impact on MSC function. Encapsulating MSCs in the dual peptide (cRGD/IGM-3) hydrogels enhanced their ability to reduce inflammatory cytokine production and promote anti-inflammatory gene expression in cells from degenerative human intervertebral discs exposed to proteins secreted by the MSC. This approach suggests a new way to retain and augment MSC functionality using IGF-1 peptide mimetics, offering an alternative to co-delivery of cells and high dose soluble growth factors for tissue repair and immune-system modulation.

Regenerating functional skin without the formation of scar tissue remains an important goal for Tissue Engineering. Current hydrogel-based grafts minimize contraction of full-thickness skin wounds and support skin regeneration using adult or neonatal foreskin dermal fibroblasts, which are often expanded in vitro and used after multiple passages. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may also influence the functionality of 3D skin constructs by affecting fibroblast-mediated graft contraction. To validate these predicted differences in fibroblast phenotype and resulting 3D graft model contraction, we isolated porcine dermal fibroblasts of varying donor age for use in a 2D proliferation assay and a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1. However, after 5 passages in culture, there was no difference in contractility between groups. These results show how cellular responses differ based on donor age and time in culture, which could account for important differences in biomanufacturing of 3D hydrogel-based skin grafts. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding. IMPACT STATEMENTLittle is known about the impact of donor age and time in culture on the contraction of the 3D hydrogel-based graft. These results show how cellular phenotypes differ based on donor age and time in culture, which could account for important inconsistencies in biomanufacturing of skin grafts and in vitro models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. Future in vivo studies could help determine the best donor age and time in culture for improved wound healing outcomes or more reproducible in vitro testing constructs.

The immune system is a vital regulator of tissue repair after trauma and the response to implantable scaffolds for regenerative medicine. Decellularized extracellular matrix (ECM) scaffolds promote tissue integration and remodeling following traumatic injury in part due initiating a pro-reparative Type-2 immune response. However, exogenous soluble inflammatory immune signals can be introduced during scaffold implantation, including microbial products in contaminated surgical fields or during immunotherapy to treat autoimmunity and cancer. It remains largely unknown how such immune mediators modulate the ECM scaffold immune environment and subsequent scaffold remodeling. In the present study, we co-delivered 3 distinct inflammatory immune adjuvants (cyclic di-AMP [CDA], monophosphoryl lipid A [MPLA], and granulocyte colony stimulating factor [GM-CSF]) with small intestinal submucosa (SIS) ECM in a murine volumetric muscle loss injury model, evaluating acute (1 week) and long-term (8-week) immune environments and scaffold remodeling. High parameter spectral cytometry, histologic analysis, and PCR revealed differential potentiation of the ECM scaffold microenvironment. Type 2 immune programs including IL-4, eosinophils, CD4 T cells, and CD206/CD86 macrophage ratios were induced in all ECM groups but attenuated by varying amounts with the CDA and MPLA co-delivery. By 8-weeks, inflammation had largely subsided, and histologically ECM with GM-CSF or MPLA showed the greatest degradation and remodeling into adipose tissue. These findings suggest that early pro-inflammatory compound delivery does not abrogate the ECM immune environment but does attenuate some programs while inducing others. These have long-term effects on scaffold remodeling and should be a consideration for surgical reconstruction in patients receiving immune stimulatory therapies.

Tissue decellularization has demonstrated widespread applications across numerous organ systems for tissue engineering and regenerative medicine applications. Decellularized tissues are expected to retain structural and/or compositional features of the natural extracellular matrix (ECM), enabling investigation of biochemical factors and cell-ECM interactions that drive tissue homeostasis, healing, and disease. However, the dense collagenous tendon matrix has limited the efficacy of traditional decellularization strategies without the aid of harsh chemical detergents and/or physical agitation that disrupt tissue integrity and denature proteins involved in regulating cell behavior. Here, we adapted and established the advantages of a detergent-free decellularization method that relies on Latrunculin B actin destabilization, alternating hypertonic-hypotonic salt and water incubations, nuclease-assisted elimination of cellular material, and protease inhibitor supplementation under aseptic conditions. Compared with previous tendon decellularization studies, our method minimized collagen denaturation while adequately removing cells and preserving bulk tissue alignment and mechanical properties. Furthermore, we demonstrated that decellularized tendon ECM-derived coatings isolated from different mouse strains, injury states (i.e., naive and acutely injured/provisional), and anatomical sites harness distinct biochemical cues and robustly maintain tendon cell viability in vitro. Together, our work provides a simple and scalable decellularization method to facilitate mechanistic studies that will expand our fundamental understanding of tendon ECM and cell biology. Impact StatementIn this study, we present a decellularization method for tendon that does not rely on any detergents or physical processing techniques. We assessed the impact of detergent-free decellularization using tissue, cellular, and molecular level analyses and validated the preservation of tendon structural organization, collagen molecular integrity, and ECM-associated biological cues that are essential for studying physiological cell-ECM interactions. Lastly, we demonstrated the success of this method on healthy and injured tendon environments, across mouse strains, and for different types of tendons, illustrating the utility of this approach for isolating the contributions of biochemical cues within unique tendon ECM microenvironments.

Multiple groups have reported on the impact of hydrogel stiffness on vascular network formation in vitro, with overall findings indicating that less stiff hydrogels better support vasculogenesis. However, the majority of this research utilized hydrogels with static stiffness, even though vasculogenesis occurs in tandem with changes in extracellular matrix material properties. To that end, we hypothesize that dynamic modulation of hydrogel stiffness during the process of vasculogenesis, recapitulating changes observed during embryonic development, would improve vascular network formation. Using our Collagen I/Norbornene-modified hyaluronic acid hydrogel system, we swelled in additional crosslinker and photoinitiator and exposed the hydrogel to UV light, enabling hydrogel stiffening at user-defined time points with no significant effect on cell viability. We observed that in situ stiffening at early time points, prior to the onset of significant cell migration, resulted in more robust vascular network formation relative to unstiffened controls, while stiffening at later time points disrupted existing vascular networks. These trends continued in in vivo experiments in nude mice, with cell-laden hydrogels stiffened at early time points resulting in improved blood flow, while those stiffened at later time points had the opposite effect. We hypothesized that this was due to differential impacts of focal adhesion kinase (FAK) activation following in situ stiffening, as supplementation with a Rho kinase inhibitor, downstream of FAK, partially reversed the effects of in situ stiffening. Taken together, this research demonstrates the benefits of incorporating dynamic cues into hydrogel design to create more physiologically relevant vasculature.

Interleukin 2 (IL-2) is a promising therapy for autoimmune type 1 diabetes (T1D), but the short half-life in vivo (less than 6 minutes) limits effective tissue exposure to IL-2. Tissue exposure is required for the tolerogenic effects of IL-2. We have developed an injectable hydrogel that incorporates heparin polymers to enable the sustained release of IL-2. This platform uses clinical grade and commercially available materials, including collagen, hyaluronan, and heparin, to deliver IL-2 by slowly degrading and releasing IL-2 over a two-week period in vivo. We find that heparin potentiates the activity of IL-2 and IL-2-mediated expansion of Foxp3+ regulatory T cell (Treg). Hydrogel-mediated IL-2 release showed a reduction of CD4+ and CD8+ T cells and an increase of FoxP3+ Treg in the lymph nodes of injected mice. Moreover, in the Non-Obese Diabetic (NOD) mouse model of T1D once-weekly administration of IL-2 hydrogels prevented diabetes onset as efficiently as 3x weekly repeated injections of soluble IL-2. Together these data suggest that heparin-containing hydrogels may have benefit in delivering low-dose IL-2 and promoting immune tolerance in autoimmune diabetes.

There are limited biomaterials for skeletal muscle regeneration. This study aimed to apply a decellularization protocol in a muscle flap model and investigate its patency. Twenty-six gracilis-muscle (GM) flaps were harvested from 13 rats. GMs were divided into groups of either 1) normal (control), 2) perfusion with 1% sodium dodecyl sulfate or SDS for 48h, followed by Triton X-100 or TX100, or lastly, 3) perfusion with SDS for 72h, followed by TX100. The morphology, microcirculatory network patency, and residual DNA content (DNAC) were evaluated. Decellularized muscle (DM) for 72h was more translucent than DM-48h. Despite longer decellularization, the DM-72h microcirculatory network maintained its integrity, except when the dye infiltrated from the muscle edges. Compared to normal, all DM had significantly lower DNAC (normal of 1.44 g/mg vs. DM-48h of 0.37 g/mg vs. DM-72h of 0.089 g/mg; P < 0.001). The DNAC of the DM-72h group was significantly lower than DM-48h (P< 0.001). We report successful GM flap decellularization. Longer decellularization led to lower DNAC, which did not compromise circulation. Our protocol may be applicable as a free-flap scaffold model for transplantation in the future. Statement of clinical significanceThe impact of our work involves a reproducible skeletal muscle decellularization protocol to later apply in translational research.

IntroductionPrimary breast cancer surgery can compromise aesthetics and quality-of-life for breast cancer patients. While breast reconstruction improves these outcomes, current methods are limited by suboptimal aesthetic outcomes and potential complication risks. There is an urgent clinical need for improved approaches to post surgical reconstruction for breast cancer patients. Adipose-derived stromal cells (ADSCs) with biological scaffolds are being widely evaluated for tissue engineering applications in the field of reconstruction. AimsThis study aimed to assess the biomechanical properties, biocompatibility, adipogenic potential of ADSCs encapsulated in modified hyaluronic acid derivatives in vitro; and efficacy and tissue integration of this construct in vivo in a murine breast cancer and reconstruction model. MethodsADSCs were obtained, with informed consent, from female breast cancer patients undergoing autologous breast reconstruction or cosmetic procedures (n=8) aged 47{+/-}12 years. Modified hyaluronic acid solution was combined with 1x106 ADSCs/mL and crosslinked using hydrogen peroxide and horseradish peroxidase. Youngs modulus, cell viability and adipogenic potential of the cell-loaded hydrogels were assessed in vitro. In vivo, hydrogels combined with murine ADSCs were grafted into a murine breast cancer model and tissues were harvested for immunohistochemistry after 4 weeks. ResultsADSCs were characterised via morphology, Colony forming unit-fibroblast (CFU-F) assay, flow cytometry and multilineage differentiation. The cell-loaded hydrogels had a compressive Youngs modulus of 7.35{+/-}0.96 kPa after 21 days in culture, similar to human breast adipose tissue ([~]10 kPa). High ADSC viability was observed after 21 days in culture, and ADSCs differentiated into mature adipocytes. After 4 weeks in vivo, hydrogels exhibited adipocytes, vascular endothelium, and pericyte-like cells. ConclusionThis study demonstrates the potential suitability of modified hyaluronic acid hydrogels encapsulating ADSCs for adipose tissue engineering for post breast cancer reconstruction.

Immune regulation plays a crucial role during the regeneration process, and it determines the fate of inflammation after tissue injury or infection. Neutrophils serve as the primary immune cells recruited to the site of inflammation, initiating and directing the subsequent inflammatory cascade following implantation. This study investigated the effects of the in vitro standard foetal bovine serum (FBS), either in the culture medium or as a surface coating, as well as type I collagen coating on responses of neutrophils isolated from human peripheral blood using 3D-printed polycaprolactone (PCL) scaffolds. Neutrophil activity was evaluated by assessing metabolic activity and metabolomic profiles, reactive oxygen species (ROS) production, and inflammation-related markers via high throughput proximity extension assay. Type I collagen coating modified the metabolomic profile of neutrophils and MMP-9 release but had minimal effect on ROS generation. In contrast, the presence of FBS in the culture medium significantly influenced neutrophil behavior, leading to significant changes in metabolic activity, cytotoxicity, and the secretion of inflammation-associated molecules, even at concentrations as low as 1% (v/v). These findings highlight the importance of assessing the use of FBS in neutrophil culture models, particularly those isolated from humans, and emphasize the development of alternative platforms for investigating neutrophil-cell interactions in a more physiologically relevant manner. HighlightsO_LINeutrophil response to FBS(1-10%) and collagen coatings on PCL scaffolds was tested. C_LIO_LIFBS impacts neutrophil activation and alters metabolite composition of the medium. C_LIO_LIFBS increased the release of inflammation-related molecules such as NE, IL-8 and VEGFA. C_LIO_LICollagen changed neutrophil metabolites and decreased MMP-9 release. C_LIO_LIResults addressed the FBS bias and the need for physiologically relevant culture models. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/687474v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@37ba15org.highwire.dtl.DTLVardef@99ed20org.highwire.dtl.DTLVardef@19efb44org.highwire.dtl.DTLVardef@825111_HPS_FORMAT_FIGEXP M_FIG C_FIG

Expansion of chondrocytes presents a major obstacle in the cartilage regeneration procedure matrix-induced autologous chondrocyte implantation (MACI). Dedifferentiation of chondrocytes during the expansion process leads to the emergence of a fibrotic (chondrofibrotic) phenotype that decreases the chondrogenic potential of the implanted cells. We aim to 1) determine the extent that chromatin architecture of H3K27me3 and H3K9me3 remodels during dedifferentiation and persists when expanded chondrocytes are transferred to a 3D culture; and 2) to prevent this persistent remodeling to enhance the chondrogenic potential of expanded chondrocytes. Chromatin architecture remodeling of H3K27me3 and H3K9me3 was observed at 0, 8 and 16 population doublings in a two-dimensional (2D) culture and after encapsulation of the expanded chondrocytes in a three-dimensional (3D) hydrogel culture. Chondrocytes were treated with inhibitors of epigenetic modifiers (epigenetic priming) for 16 population doublings and then encapsulated in 3D hydrogels. Chromatin architecture of chondrocytes and gene expression were evaluated before and after encapsulation. We observed a change in chromatin architecture of epigenetic modifications H3K27me3 and H3K9me3 during chondrocyte dedifferentiation. Although inhibiting enzymes that modify H3K27me3 and H3K9me3 did not alter the dedifferentiation process in 2D culture, applying these treatments during the 2D expansion did increase the expression of select chondrogenic genes and protein deposition of type II collagen when transferred to a 3D environment. Overall, we found that epigenetic priming of expanded chondrocytes alters the cell fate when chondrocytes are later encapsulated into a 3D environment, providing a potential method to enhance the success of cartilage regeneration procedures.

Extracellular vesicles (EVs) are versatile transporters of genetic cargo with enormous potential in the therapeutic setting. Scalable production of EVs, and routes to overcome rapid clearance are required. Biocompatible hydrogels may support precise, localized delivery of EVs to target sites. This study aimed to establish sustained production of EVs in a scalable 3D dynamic bioreactor and to fabricate hydrogels using tyramine-modified hyaluronic acid (HA-TA) to study EV integration and release patterns. MDA-MB-231 cells transduced with lentiviral GFP fused with CD63, were cultured in a 20kD dynamic hollow fiber bioreactor and GFP-EVs harvested over five weeks. GFP-EVs were characterized by Nanoparticle Tracking Analysis(NTA), Western Blot(WB) and Transmission Electron Microscopy(TEM). Tyramine modified hyaluronic acid(HA-TA) hydrogels were formulated via enzymatic crosslinking using hydrogen peroxide and horseradish peroxidase, to investigate EV release patterns in static and dynamic conditions. Hydrogel swelling was recorded at 1-72 hrs and hydrogels were loaded with GFP-EVs to assess distribution and release by Scanning Electron Microscopy(SEM) and NTA respectively. GFP-EV uptake was assessed by confocal microscopy. Longitudinal GFP expression was demonstrated in transduced cells and released EVs throughout bioreactor culture. TEM and NTA demonstrated successful isolation of EVs of 30-200 nm in size with intact lipid bilayers (average 4x109 EVs/harvest). Initial harvests exhibited subpopulations of larger EVs, which disappeared upon serum withdrawal. WB verified the presence of EV markers CD63, TSG101, and CD81. HA-TA hydrogels were successfully formed and swelling assays revealed the requirement for higher concentrations of HA-TA and crosslinkers for scaffold stability and continued swelling. GFP-EVs were successfully incorporated into the hydrogels with variable release patterns observed over time, depending on EV concentration and hydrogel formulation. EV clusters in hydrogels were visualized by SEM. Investigation of GFP-EV release patterns under static and dynamic conditions highlighted a significant increase in release under fluid flow conditions. Efficient transfer of released EVs to recipient cells was also demonstrated in vitro. The data demonstrate the potential for scalable production of engineered EVs in serum free conditions and subsequent incorporation into HA-TA hydrogels for sustained release. These biocompatible hydrogels hold promise for tuneable delivery of therapeutic EVs in a variety of disease settings.

Systemic delivery of immunotherapy is dose-limited and often causes serious immune-related adverse events. Intratumoral injections can reduce systemic immunotoxicities and increase immunotherapy concentrations within a tumor. However, high pressures associated with direct tumor injection limits injectate retention, as low viscosity, saline-based solutions rapidly leak out of tumors. Viscoelastic solids, such as hydrogels, can improve local retention of co-formulated immunotherapies and provide sustained delivery. Prior work demonstrated that a chitosan-based hydrogel, XCSgel, was shear-thinning, self-healing, injectable, biocompatible, and clinically imageable. Here, we investigated XCSgel as a localized intratumoral delivery platform in the context of murine models of orthotopic triple-negative breast cancer. The intratumoral retention of immunotherapeutics co-formulated in XCSgel was characterized both ex vivo and in vivo via fluorescence imaging. Histopathological responses to intratumoral injections of XCSgel alone were scored by a veterinary pathologist. Initial antitumor studies evaluated a range of antitumor cytokines co-formulated with XCSgel. Subsequent antitumor and rechallenge studies evaluated the efficacy of a single intratumoral injection of interleukin-12 (IL-12) co-formulated in XCSgel (XCSgel-IL12) to control the growth of primary and abscopal tumors while inducing protective immunity. Pharmacokinetics studies quantified the systemic dissemination of IL-12 and consequent production of interferon-gamma following intratumoral injection with XCSgel co-formulation. Spectral flow cytometry was used to document changes in the tumor-immune microenvironment (TIME). XCSgel resisted tumor leakage and slowly released three model cytokines. XCSgel could be tuned for faster or slower release of embedded therapeutics. XCSgel-IL12 outperformed XCSgel formulations with other commonly used antitumor cytokines. A single injection of XCSgel-IL12 eliminated 86% E0771 and 20% mWnt orthotopic primary TNBC tumors. Mice rendered tumor-free resisted a live tumor challenge. XCSgel-IL12 also eliminated 67% untreated abscopal E0771 tumors. XCSgel-IL12 induced profound changes to the TIME, including a 3-fold reduction in the frequency of exhausted CD8+ T cells and a 3.2-fold increase in activated, proliferating CD8+ T cells. XCSgel is a promising localized delivery platform well-suited to enhance the retention and antitumor activity of potent immunotherapeutics. A single injection of XCSgel-IL12 can eliminate both primary and abscopal solid tumors, indicating that systemic immunotherapy may not be required for systemic control of cancer. SynopsisA novel injectable hydrogel, XCSgel, can localize and slowly release immunotherapies to eliminate primary and abscopal murine triple negative breast cancer tumors with a single injection.

Mesenchymal stromal cells (MSCs) have shown promise as a treatment for osteoarthritis (OA); however, effective translation has been limited by numerous factors ranging from high variability and heterogeneity of hMSCs, to suboptimal delivery strategies, to poor understanding of critical quality and potency attributes. The objective of the current study was to assess the effects of biomaterial encapsulation in alginate microcapsules on human MSC (hMSC) secretion of immunomodulatory cytokines in an OA microenvironment and therapeutic efficacy in treating established OA. Lewis rats underwent Medial Meniscal Transection (MMT) surgery to induce OA. Three weeks post-surgery, after OA was established, rats received intra-articular injections of either encapsulated hMSCs or controls (saline, empty capsules, or non-encapsulated hMSCs). Six weeks post-surgery, microstructural changes in the knee joint were quantified using contrast enhanced microCT. Encapsulated hMSCs attenuated progression of OA including articular cartilage degeneration (swelling and cartilage loss) and subchondral bone remodeling (thickening and hardening). A multiplexed immunoassay panel (41 cytokines) was used to profile the in vitro secretome of encapsulated and non-encapsulated hMSCs in response to IL-1{square}, a key cytokine involved in OA. Non-encapsulated hMSCs showed an indiscriminate increase in all cytokines in response to IL-1{square} while encapsulated hMSCs showed a highly targeted secretory response with increased expression of some pro-inflammatory (IL-1{beta}, IL-6, IL-7, IL-8), anti-inflammatory (IL-1RA), and chemotactic (G-CSF, MDC, IP10) cytokines. These data show that biomaterial encapsulation using alginate microcapsules can modulate hMSC paracrine signaling in response to OA cytokines and enhance the therapeutic efficacy of the hMSCs in treating established OA.

Novel therapeutics have sought to stimulate the endogenous repair mechanisms in the mammalian myocardium as the native regenerative potential of the adult cardiac tissue is limited. In particular, a myocardial matrix derived injectable hydrogel has shown efficacy and safety in various animal myocardial infarction (MI) including evidence of increased myocardium. In this study, investigation on the properties of this myocardial matrix material demonstrated its native capability as an effective reactive oxygen species (ROS) scavenger that can protect against oxidative stress and maintain cardiomyocyte proliferation in vitro. In vivo assessment of of myocardial matrix hydrogel treatment post-MI demonstrated increased thymidine analog uptake in cardiomyocytes compared to saline controls along with co-staining with cell cycle progression marker, phospho-histone H3. Overall, this study provides further evidence that properties of the myocardial matrix hydrogel promote an environment supportive of cardiomyocytes undergoing cell cycle progression.