Extracellular Matrix Scaffold-Assisted Tumor Vaccines Induce Tumor Regression and Long-Term Immune Memory.

Injectable scaffold delivery is a strategy to enhance the efficacy of cancer vaccine immunotherapy. The choice of scaffold biomaterial is crucial, impacting both vaccine release kinetics and immune stimulation via the host response. Extracellular matrix (ECM) scaffolds prepared from decellularized tissues facilitate a pro-healing inflammatory response that promotes local cancer immune surveillance. Here, an ECM scaffold-assisted therapeutic cancer vaccine that maintains an immune microenvironment consistent with tissue reconstruction is engineered. Several immune-stimulating adjuvants are screened to develop a cancer vaccine formulated with decellularized small intestinal submucosa (SIS) ECM scaffold co-delivery. It is found that the STING pathway agonist cyclic di-AMP most effectively induces cytotoxic immunity in an ECM scaffold vaccine, without compromising key interleukin 4 (IL-4) mediated immune pathways associated with healing. ECM scaffold delivery enhances therapeutic vaccine efficacy, curing 50-75% of established E.G-7OVA lymphoma tumors in mice, while none are cured with soluble vaccine. SIS-ECM scaffold-assisted vaccination prolonged antigen exposure is dependent on CD8+ cytotoxic T cells and generates long-term antigen-specific immune memory for at least 10 months post-vaccination. This study shows that an ECM scaffold is a promising delivery vehicle to enhance cancer vaccine efficacy while being orthogonal to characteristics of pro-healing immune hallmarks.

Creation and preclinical evaluation of a novel mussel-inspired, biomimetic, bioactive bone graft scaffold: direct comparison with Infuse bone graft using a rat model of spinal fusion.

OBJECTIVE: Infuse bone graft is a widely used osteoinductive adjuvant; however, the simple collagen sponge scaffold used in the implant has minimal inherent osteoinductive properties and poorly controls the delivery of the adsorbed recombinant human bone morphogenetic protein-2 (rhBMP-2). In this study, the authors sought to create a novel bone graft substitute material that overcomes the limitations of Infuse and compare the ability of this material with that of Infuse to facilitate union following spine surgery in a clinically translatable rat model of spinal fusion. METHODS: The authors created a polydopamine (PDA)-infused, porous, homogeneously dispersed solid mixture of extracellular matrix and calcium phosphates (BioMim-PDA) and then compared the efficacy of this material directly with Infuse in the setting of different concentrations of rhBMP-2 using a rat model of spinal fusion. Sixty male Sprague Dawley rats were randomly assigned to each of six equal groups: 1) collagen + 0.2 µg rhBMP-2/side, 2) BioMim-PDA + 0.2 µg rhBMP-2/side, 3) collagen + 2.0 µg rhBMP-2/side, 4) BioMim-PDA + 2.0 µg rhBMP-2/side, 5) collagen + 20 µg rhBMP-2/side, and 6) BioMim-PDA + 20 µg rhBMP-2/side. All animals underwent posterolateral intertransverse process fusion at L4-5 using the assigned bone graft. Animals were euthanized 8 weeks postoperatively, and their lumbar spines were analyzed via microcomputed tomography (µCT) and histology. Spinal fusion was defined as continuous bridging bone bilaterally across the fusion site evaluated via µCT. RESULTS: The fusion rate was 100% in all groups except group 1 (70%) and group 4 (90%). Use of BioMim-PDA with 0.2 µg rhBMP-2 led to significantly greater results for bone volume (BV), percentage BV, and trabecular number, as well as significantly smaller trabecular separation, compared with the use of the collagen sponge with 2.0 µg rhBMP-2. The same results were observed when the use of BioMim-PDA with 2.0 µg rhBMP-2 was compared with the use of the collagen sponge with 20 µg rhBMP-2. CONCLUSIONS: Implantation of rhBMP-2-adsorbed BioMim-PDA scaffolds resulted in BV and bone quality superior to that afforded by treatment with rhBMP-2 concentrations 10-fold higher implanted on a conventional collagen sponge. Using BioMim-PDA (vs a collagen sponge) for rhBMP-2 delivery could significantly lower the amount of rhBMP-2 required for successful bone grafting clinically, improving device safety and decreasing costs.

Transfer learning in a biomaterial fibrosis model identifies in vivo senescence heterogeneity and contributions to vascularization and matrix production across species and diverse pathologies.

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells' (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo-derived senescence signature (SenSig) using a foreign body response-driven fibrosis model in a p16-CreERT2;Ai14 reporter mouse. We identified pericytes and "cartilage-like" fibroblasts as senescent and defined cell type-specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34-CSF1R-TGFßR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.

An immunologically active, adipose-derived extracellular matrix biomaterial for soft tissue reconstruction: concept to clinical trial.

Soft tissue reconstruction remains an intractable clinical challenge as current surgical options and synthetic implants may produce inadequate outcomes. Soft tissue deficits may be surgically reconstructed using autologous adipose tissue, but these procedures can lead to donor site morbidity, require multiple procedures, and have highly variable outcomes. To address this clinical need, we developed an "off-the-shelf" adipose extracellular matrix (ECM) biomaterial from allograft human tissue (Acellular Adipose Tissue, AAT). We applied physical and chemical processing methods to remove lipids and create an injectable matrix that mimicked the properties of lipoaspirate. Biological activity was assessed using cell migration and adipogenesis assays. Characterization of regenerative immune properties in a murine muscle injury model revealed that allograft and xenograft AAT induced pro-regenerative CD4+ T cells and macrophages with xenograft AAT additionally attracting eosinophils secreting interleukin 4 (Il4). In immunocompromised mice, AAT injections retained similar volumes as human fat grafts but lacked cysts and calcifications seen in the fat grafts. The combination of AAT with human adipose-derived stem cells (ASCs) resulted in lower implant volumes. However, tissue remodeling and adipogenesis increased significantly in combination with ASCs. Larger injected volumes of porcine-derived AAT demonstrated biocompatibility and greater retention when applied allogeneicly in Yorkshire cross pigs. AAT was implanted in healthy volunteers in abdominal tissue that was later removed by elective procedures. AAT implants were well tolerated in all human subjects. Implants removed between 1 and 18 weeks demonstrated increasing cellular infiltration and immune populations, suggesting continued tissue remodeling and the potential for long-term tissue replacement.

IL-17 and immunologically induced senescence regulate response to injury in osteoarthritis.

Senescent cells (SnCs) are implicated in the pathogenesis of age-related diseases including osteoarthritis (OA), in part via expression of a senescence-associated secretory phenotype (SASP) that includes immunologically relevant factors and cytokines. In a model of posttraumatic OA (PTOA), anterior cruciate ligament transection (ACLT) induced a type 17 immune response in the articular compartment and draining inguinal lymph nodes (LNs) that paralleled expression of the senescence marker p16INK4a (Cdkn2a) and p21 (Cdkn1a). Innate lymphoid cells, γδ+ T cells, and CD4+ T cells contributed to IL-17 expression. Intra-articular injection of IL-17-neutralizing antibody reduced joint degeneration and decreased expression of the senescence marker Cdkn1a. Local and systemic senolysis was required to attenuate tissue damage in aged animals and was associated with decreased IL-17 and increased IL-4 expression in the articular joint and draining LNs. In vitro, we found that Th17 cells induced senescence in fibroblasts and that SnCs skewed naive T cells toward Th17 or Th1, depending on the presence of TGF-ß. The SASP profile of the inflammation-induced SnCs included altered Wnt signaling, tissue remodeling, and cell-cycle pathways not previously implicated in senescence. These findings provide molecular targets and mechanisms for senescence induction and therapeutic strategies to support tissue healing in an aged environment.

Interleukin 17 and senescent cells regulate the foreign body response to synthetic material implants in mice and humans.

Medical devices and implants made of synthetic materials can induce an immune-mediated process when implanted in the body called the foreign body response, which results in formation of a fibrous capsule around the implant. To explore the immune and stromal connections underpinning the foreign body response, we analyzed fibrotic capsules surrounding surgically excised human breast implants from 12 individuals. We found increased numbers of interleukin 17 (IL17)-producing γδ+ T cells and CD4+ T helper 17 (TH17) cells as well as senescent stromal cells in the fibrotic capsules. Further analysis in a murine model demonstrated an early innate IL17 response to implanted synthetic material (polycaprolactone) particles that was mediated by innate lymphoid cells and γδ+ T cells. This was followed by a chronic adaptive CD4+ TH17 cell response that was antigen dependent. Synthetic materials with varying chemical and physical properties implanted either in injured muscle or subcutaneously induced similar IL17 responses in mice. Mice deficient in IL17 signaling established that IL17 was required for the fibrotic response to implanted synthetic materials and the development of p16INK4a senescent cells. IL6 produced by senescent cells was sufficient for the induction of IL17 expression in T cells. Treatment with a senolytic agent (navitoclax) that killed senescent cells reduced IL17 expression and fibrosis in the mouse implant model. Discovery of a feed-forward loop between the TH17 immune response and the senescence response to implanted synthetic materials introduces new targets for therapeutic intervention in the foreign body response.

The Canary in the Coal Mine: Biomaterial Implants to Monitor Cancer Recurrence.

Synthetic biomaterials implanted in the body induce a foreign body reaction characterized by chronic inflammation and fibrosis. In this issue of Cancer Research, Oakes and colleagues used biomaterial implants and their associated immunologic activity to develop a "metastasis sensor" for detection of tumor burden at distal sites. A scoring system was developed from computational analysis of gene expression patterns from implant biopsies that could predict the presence of tumor. This unexpected use of biomaterials for early detection of cancer provides a more accurate systemic sampling compared with blood or liquid biopsies and alleviates the need for inefficient imaging and biopsy sampling from potential metastatic target tissues.See related article by Oakes et al., p. 602.

Two-Year Follow-Up and Remodeling Kinetics of ChonDux Hydrogel for Full-Thickness Cartilage Defect Repair in the Knee.

OBJECTIVE: To determine performance and repair kinetics of the ChonDux hydrogel scaffold for treating focal articular cartilage defects in the knee over 24 months. DESIGN: This assessor-blinded trial evaluates ChonDux hydrogel scaffold implantation in combination with microfracture in 18 patients across 6 sites. Male and female patients 18 to 65 years of age with full-thickness femoral condyle defects 2 to 4 cm2 in area were enrolled. Eligible patients received ChonDux treatment followed by rehabilitation. Defect volume fill was evaluated after 3, 6 (primary outcome), 12, 18, and 24 months by assessor blinded magnetic resonance imaging (MRI) analysis. Secondary outcomes were T2-weighted MRI relaxation time and patient surveys via visual analogue scale (VAS) pain and International Knee Documentation Committee (IKDC) knee function scoring. RESULTS: ChonDux maintained durable tissue restoration over 24 months with final defect percent fill of 94.2% ± 16.3% and no significant loss of fill volume at any time points. Tissues treated with ChonDux maintained T2 relaxation times similar to uninjured cartilage between 12 and 24 months. VAS pain scoring decreased between 1 and 6 weeks, and IKDC knee function scores improved by approximately 30.1 with ChonDux over 24 months. CONCLUSION: ChonDux treatment is a safe adjunct to microfracture therapy and promotes stable restoration of full thickness articular cartilage defects for at least 24 months.

A biologic scaffold-associated type 2 immune microenvironment inhibits tumor formation and synergizes with checkpoint immunotherapy.

Biomaterials in regenerative medicine are designed to mimic and modulate tissue environments to promote repair. Biologic scaffolds (derived from decellularized tissue extracellular matrix) promote a wound-healing (proregenerative) immune phenotype and are used clinically to treat tissue loss, including in the context of tumor resection. It is unknown whether a biomaterial microenvironment that encourages tissue formation may also promote tumor development. We implanted a urinary bladder matrix (UBM) scaffold, which is used clinically for wound management, with syngeneic cancer cell lines in mice to study how wound-healing immune responses affect tumor formation and sensitivity to immune checkpoint blockade. The UBM scaffold created an immune microenvironment that inhibited B16-F10 melanoma tumor formation in a CD4+ T cell-dependent and macrophage-dependent manner. In-depth immune characterization revealed an activated type 2-like immune response that was distinct from the classical tumor microenvironment, including activated type 2 T helper T cells, a unique macrophage phenotype, eosinophil infiltration, angiogenic factors, and complement. Tumor growth inhibition by PD-1 and PD-L1 checkpoint blockade was potentiated in the UBM scaffold immune microenvironment. Engineering the local tumor microenvironment to promote a type 2 wound-healing immune signature may serve as a therapeutic target to improve immunotherapy efficacy.

Divergent immune responses to synthetic and biological scaffolds.

The immune system plays a critical role in wound healing and the response to biomaterials. Biomaterials-directed regenerative immunology is an immunoengineering strategy that targets the immune system to promote tissue repair. Biomaterial scaffolds employed in regenerative medicine can be broadly classified as biological (such as those derived from the tissue extracellular matrix) or synthetic. Here, we show in depth the divergent myeloid response to biological versus synthetic biomaterial scaffolds. While neutrophil depletion and changes in physical properties such as shape and mechanics can modulate the pro-inflammatory myeloid immune response to synthetic materials to a degree, the overall general divergent myeloid responses persist. Biologic scaffolds elicit a type-2-like immune response with upregulation of genes such as Il4, Cd163, Mrc1 and Chil3, as well as genes associated with damage-associated molecular patterns providing another possible mechanism by which ECM scaffolds promote wound healing via amplification of endogenous wound-associated signaling pathways. Synthetic materials recruit a high proportion of neutrophils which is compounded by material stiffness and by the presence of an injury. Understanding the complex immune response to biomaterial classes will help in the efficient design of immunoengineering strategies and optimizing regenerative and reducing foreign body fibrotic responses to scaffolds.

Biological scaffold-mediated delivery of myostatin inhibitor promotes a regenerative immune response in an animal model of Duchenne muscular dystrophy.

Recent studies have reported that the immune system significantly mediates skeletal muscle repair and regeneration. Additionally, biological scaffolds have been shown to play a role in polarizing the immune microenvironment toward pro-myogenic outcomes. Moreover, myostatin inhibitors are known to promote muscle regeneration and ameliorate fibrosis in animal models of Duchenne muscular dystrophy (DMD), a human disease characterized by chronic muscle degeneration. Biological scaffolds and myostatin inhibition can potentially influence immune-mediated regeneration in the dystrophic environment, but have not been evaluated together. Toward this end, here we created an injectable biological scaffold composed of hyaluronic acid and processed skeletal muscle extracellular matrix. This material formed a cytocompatible hydrogel at physiological temperatures in vitro When injected subfascially above the tibialis anterior muscles of both WT and dystrophic mdx-5Cv mice, a murine model of DMD, the hydrogel spreads across the entire muscle before completely degrading at 3 weeks in vivo We found that the hydrogel is associated with CD206+ pro-regenerative macrophage polarization and elevated anti-inflammatory cytokine expression in both WT and dystrophic mice. Co-injection of both hydrogel and myostatin inhibitor significantly increased FoxP3+ regulatory T cell modulation and Foxp3 gene expression in the scaffold immune microenvironment. Finally, delivery of myostatin inhibitor with the hydrogel increased its bioactivity in vivo, and transplantation of immortalized human myoblasts with the hydrogel promoted their survival in vivo This study identifies a key role for biological scaffolds and myostatin inhibitors in modulating a pro-regenerative immune microenvironment in dystrophic muscle.

Proteomic composition and immunomodulatory properties of urinary bladder matrix scaffolds in homeostasis and injury.

Urinary bladder matrix (UBM) is used clinically for management of wounds and reinforcement of surgical soft tissue repair, among other applications. UBM consists of the lamina propria and basal lamina of the porcine urinary bladder, and is decellularized as part of the process to manufacture the medical device. UBM is composed mainly of Collagen I, but also contains a wide variety of fibrillar and basement membrane collagens, glycoproteins, proteoglycans and ECM-associated factors. Upon application of the biomaterial in a traumatic or non-traumatic setting in a mouse model, there is a cascade of immune cells that respond to the damaged tissue and biomaterial. Here, through the use of multicolor flow cytometry, we describe the various cells that infiltrate the UBM scaffold in a subcutaneous and volumetric muscle injury model. A wide variety of immune cells are found in the UBM scaffold immune microenvironment (SIM) including F4/80+ macrophages, CD11c+ dendritic cells, CD3+ T cells and CD19+ B cells. A systemic IL-4 upregulation and a local M2-macrophage response were observed in the proximity of the implanted UBM. The recruitment and activation of these cells is dependent upon signals from the scaffold and communication between the different cell types present.

Intra-articular Injection of Urinary Bladder Matrix Reduces Osteoarthritis Development.

Micronized porcine urinary bladder matrix (UBM) is an extracellular matrix biomaterial that has immunomodulatory and pro-regenerative properties. The objective of this study was to assess the ability of UBM to alter disease progression in a mouse model of post-traumatic osteoarthritis (OA). Ten-week-old wild-type C57BL/6 male mice underwent anterior cruciate ligament transection (ACLT) to induce OA. Two weeks after ACLT, UBM (50 mg/mL) or saline was injected into the mouse joint. At 4 and 8 weeks post-ACLT, cartilage integrity was assessed using OARSI scoring of histology, pain was evaluated, and joints were harvested for quantitative RT-PCR analysis of cartilage-specific and inflammatory gene expression. UBM-treated animals showed improved cartilage integrity at 4 and 8 weeks and reduced pain at 4 weeks compared to saline-injected mice. Animals injected with UBM expressed higher levels of genes encoding structural cartilage proteins, such as collagen2α1 and aggrecan, as well as anti-inflammatory cytokines, including interleukins 10 and 4. UBM decreased cartilage degeneration in the murine ACLT model of OA, which may be due to reduced inflammation in the joint and maintenance of high expression levels of proteoglycans.

Bi-layered polyurethane - Extracellular matrix cardiac patch improves ischemic ventricular wall remodeling in a rat model.

As an intervention to abrogate ischemic cardiomyopathy, the concept of applying a temporary, local patch to the surface of the recently infarcted ventricle has been explored from a number of design perspectives. Two important features considered for such a cardiac patch include the provision of appropriate mechanical support and the capacity to influence the remodeling pathway by providing cellular or biomolecule delivery. The objective of this report was to focus on these two features by first evaluating the incorporation of a cardiac extracellular matrix (ECM) component, and second by evaluating the impact of patch anisotropy on the pathological remodeling process initiated by myocardial infarction. The functional outcomes of microfibrous, elastomeric, biodegradable cardiac patches have been evaluated in a rat chronic infarction model. Ten weeks after infarction and 8 wk after patch epicardial placement, echocardiographic function, tissue-level structural remodeling (e.g., biaxial mechanical response and microstructural analysis), and cellular level remodeling were assessed. The results showed that the incorporation of a cardiac ECM altered the progression of several keys aspects of maladaptive remodeling following myocardial infarction. This included decreasing LV global mechanical compliance, inhibiting echocardiographically-measured functional deterioration, mitigating scar formation and LV wall thinning, and promoting angiogenesis. In evaluating the impact of patch anisotropy, no effects from the altered patch mechanics were detected after 8 wk, possibly due to patch fibrous encapsulation. Overall, this study demonstrates the benefit of a cardiac patch design that combines both ventricle mechanical support, through a biodegradable, fibrillary elastomeric component, and the incorporation of ECM-based hydrogel components.

Immunomodulation and Mobilization of Progenitor Cells by Extracellular Matrix Bioscaffolds for Volumetric Muscle Loss Treatment.

Acellular bioscaffolds composed of extracellular matrix (ECM) have been effectively used to promote functional tissue remodeling in both preclinical and clinical studies of volumetric muscle loss, but the mechanisms that contribute to such outcomes are not fully understood. Thirty-two C57bl/6 mice were divided into eight groups of four animals each. A critical-sized defect was created in the quadriceps muscle and was repaired with a small intestinal submucosa ECM bioscaffold or left untreated. Animals were sacrificed at 3, 7, 14, or 56 days after surgery. The spatiotemporal cellular response in both treated and untreated groups was characterized by immunolabeling methods. Early time points showed a robust M2-like macrophage phenotype following ECM treatment in contrast to the predominant M1-like macrophage phenotype present in the untreated group. ECM implantation promoted perivascular stem cell mobilization, increased presence of neurogenic progenitor cells, and was associated with myotube formation. These cell types were present not only at the periphery of the defect near uninjured muscle, but also in the center of the ECM-filled defect. ECM bioscaffolds modify the default response to skeletal muscle injury, and provide a microenvironment conducive to a constructive healing response.

Developing a pro-regenerative biomaterial scaffold microenvironment requires T helper 2 cells.

Immune-mediated tissue regeneration driven by a biomaterial scaffold is emerging as an innovative regenerative strategy to repair damaged tissues. We investigated how biomaterial scaffolds shape the immune microenvironment in traumatic muscle wounds to improve tissue regeneration. The scaffolds induced a pro-regenerative response, characterized by an mTOR/Rictor-dependent T helper 2 pathway that guides interleukin-4-dependent macrophage polarization, which is critical for functional muscle recovery. Manipulating the adaptive immune system using biomaterials engineering may support the development of therapies that promote both systemic and local pro-regenerative immune responses, ultimately stimulating tissue repair.

Tissue matrix arrays for high-throughput screening and systems analysis of cell function.

Cell and protein arrays have demonstrated remarkable utility in the high-throughput evaluation of biological responses; however, they lack the complexity of native tissue and organs. Here we spotted tissue extracellular matrix (ECM) particles as two-dimensional (2D) arrays or incorporated them with cells to generate three-dimensional (3D) cell-matrix microtissue arrays. We then investigated the responses of human stem, cancer and immune cells to tissue ECM arrays originating from 11 different tissues. We validated the 2D and 3D arrays as representative of the in vivo microenvironment by means of quantitative analysis of tissue-specific cellular responses, including matrix production, adhesion and proliferation, and morphological changes after culture. The biological outputs correlated with tissue proteomics, and network analysis identified several proteins linked to cell function. Our methodology enables broad screening of ECMs to connect tissue-specific composition with biological activity, providing a new resource for biomaterials research and further understanding of regeneration and disease mechanisms.

Naturally derived and synthetic scaffolds for skeletal muscle reconstruction.

Skeletal muscle tissue has an inherent capacity for regeneration following injury. However, severe trauma, such as volumetric muscle loss, overwhelms these natural muscle repair mechanisms prompting the search for a tissue engineering/regenerative medicine approach to promote functional skeletal muscle restoration. A desirable approach involves a bioscaffold that simultaneously acts as an inductive microenvironment and as a cell/drug delivery vehicle to encourage muscle ingrowth. Both biologically active, naturally derived materials (such as extracellular matrix) and carefully engineered synthetic polymers have been developed to provide such a muscle regenerative environment. Next generation naturally derived/synthetic "hybrid materials" would combine the advantageous properties of these materials to create an optimal platform for cell/drug delivery and possess inherent bioactive properties. Advances in scaffolds using muscle tissue engineering are reviewed herein.

Predicting in vivo responses to biomaterials via combined in vitro and in silico analysis.

The host response to both synthetic and biologically derived biomaterials is a temporally regulated, complex process that involves multiple interacting cell types. This complexity has classically limited the efficacy of in vitro assays for predicting the in vivo outcome, necessitating the use of costly animal models for biomaterial development. The present study addressed these challenges by developing an in vitro assay that characterized the dynamic inflammatory response of human monocyte-derived-macrophages to biomaterials, coupled with quasi-mechanistic analysis in silico analysis: principal component analysis (PCA) and dynamic network analysis (DyNA). Synthetic and extracellular matrix (ECM)-derived materials were evaluated using this method, and were then associated with the in vivo remodeling and macrophage polarization response in a rodent skeletal muscle injury model. PCA and DyNA revealed a distinct in vitro macrophage response to ECM materials that corresponded to constructive remodeling and an increased M2 macrophage presence in vivo. In contrast, PCA and DyNA suggested a response to crosslinked ECM and synthetic materials characteristic of a foreign body reaction and dominant M1 macrophage response. These results suggest that in silico analysis of an in vitro macrophage assay may be useful as a predictor for determining the in vivo host response to implanted biomaterials.