Biologic scaffolds are extensively used in various clinical applications such as musculotendinous reconstruction, hernia repair or wound healing. Biologic scaffolds used in these applications vary in species, breed and tissue of origin, and other variables that affect their properties. Decellularization and sterilization processes also determine the characteristics of these scaffolds. The goal of the present study is to compare the composition and mechanical properties of decellularized porcine placental scaffolds from three different porcine breeds: Landrace, York and Duroc. Placental extracellular matrix (ECM) scaffolds from the three porcine breeds preserved the amnion/chorion ECM structure and the basement membrane markers laminin and collagen type IV. ECM placental scaffolds showed similar contents of collagen, elastin and lipids, and minimal differences in glycosaminoglycans content. Mechanical properties from the three breeds ECM placental scaffolds were also similar and stable for 24 months. While this study serves as preliminary characterization of porcine ECM scaffolds, future studies will determine their compatibility and suitability for tissue engineering applications.

Suture materials are the most common bioimplants in surgical and clinical practice, playing a crucial role in wound healing and tendon and ligament repair. Despite the assortment available on the market, sutures are still affected by significant disadvantages, including failure in mimicking the mechanical properties of the tissue, excessive fibrosis, and inflammation. This study introduces a mandrel-less electrodeposition apparatus to fabricate continuous microfiber wires of indefinite length. The mandrel-less biofabrication produces wires, potentially used as medical fibers, with different microfiber bundles, that imitate the hierarchical organization of native tissues, and tailored mechanical properties. Microfiber wire morphology and mechanical properties are characterized by scanning electron microscopy, digital image processing, and uniaxial tensile test. Wires are tested in vitro on monocyte/macrophage stimulation and in vivo on a rat surgical wound model. The wires produced by mandrel-less deposition show an increased M2 macrophage phenotype in vitro. The in vivo assessment demonstrates that microfiber wires, compared to the medical fibers currently used, reduce pro-inflammatory macrophage response and preserve their mechanical properties after 30 days of use. These results make this microfiber wire an ideal candidate as a suture material for soft tissue surgery, suggesting a crucial role of microarchitecture in more favorable host response.

BACKGROUND CONTEXT Poly-ether-ether-ketone (PEEK) has been the most commonly used biomaterial for spinal fusion for many years, primarily because it meets the necessary mechanical requirements to support the pressures to which the spinal column is subjected. However, the biologic response to PEEK has serious limitations including a persistent foreign body response, very modest osseointegration which can result in micromotion, pain, or worse, delamination requiring surgical reintervention. Recent advances in our understanding of immunobiology show that PEEK elicits a proinflammatory phenotype among cells of the innate immune system, including macrophages. Numerous studies have shown that macrophage phenotype can be predictive of clinical outcome. The host immune response to biomaterials used in spine surgery has been largely ignored in deference to mechanical and biomaterial properties such as peak load bearing, porosity and surface characteristics. Pro-inflammatory (M1-like) macrophages have been shown to release several mediators of cell behavior such as IL-6, IL-1b, and TNF-a, that are osteoclastogenic, while pro-healing (M2-like) macrophages are associated with the release of osteoblastogenic signaling molecules. The emergence of "immunomodulatory biomaterials" is directed toward the biologic consequences associated with permanent implants such as spinal fusion materials. Surface modification of such materials represents one approach for developing more host friendly, biocompatible materials while retaining the required mechanical properties. PURPOSE The objective of the present study was to characterize and compare the macrophage phenotype elicited by the ZFuze polymer, PEEK, titanium-based alloys, or iterations of modified ZFuze polymer. The ability of these materials to promote differentiation of osteoblast-like cells in vitro was also determined. STUDY DESIGN/SETTING Macrophage activation study: Primary bone marrow-derived derived macrophages were isolated from C57bl6/j mice and plated on one of the following test articles: ZFuzeTM, PEEK, rough surface titanium alloy, or iterations of modified ZFuze polymer. Gene and protein expression were determined through qPCR and immunolabeling, respectively. Osteoblast differentiation assay: Saos-2 cells were purchased from ATCC. 1*104 cells/cm2 were plated into 6 well tissue culture plates containing test articles composed of: ZFuzeTM, PEEK, rough surface titanium alloy, or iterations of modified ZFuze polymer. Gene and protein expression were determined through qPCR and ELISA, respectively. OUTCOME MEASURES qPCR, immunolabeling, and ELISA. METHODS Culture of bone marrow derived macrophages: Bone marrow was isolated from the femurs and tibias of C57bl/6 mice and subsequently cultured in complete growth media, including Dulbecco's modified Eagles medium, 10% fetal bovine serum (FBS), 10% L929 supernatant, 0.1% beta-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM nonessential amino acids, and 10 mM HEPES buffer, for 7 days with complete media changes every 48 hours until mature bone marrow-derived macrophages were obtained. Macrophage activation: Mature macrophages were exposed to the following treatments for 24 hours: complete media (M0 control), 20 ng/ml IFN-γ and 100 ng/mL lipopolysaccharide LPS (M1 control), 20 ng/mL interleukin IL-4 (M2 control), PEEK, ZFuze, rough-surface titanium alloy, or ZFuze surface modification iterations. For the cytokine challenge study, cells were exposed for 6 hours to 20 ng/ml IFN-γ and 100 ng/mL LPS, washed and then placed in 10% FBS 1% P/S DMEM for 24 hours. Macrophage immunolabeling: Fixed cells were washed with 1X PBS followed by incubation in a blocking solution composed of PBS, 0.1% Triton-X, 0.1% Tween-20, 4% goat serum, and 2% bovine serum albumin for 1 hour at room temperature. Cells were then incubated in a solution of one of the following primary antibodies: anti-F4/80 at 1:100 dilution as a pan-macrophage marker, anti-inducible nitric oxide synthase (iNOS) at 1:100 dilution as an M1-like marker, and anti-Fizz1 and anti-Arginase1 at 1:200 dilution, each as M2-like markers. Nuclei were counterstained with DAPI. Saos-2 culture: Saos-2 cells were purchased from ATCC. Cells were grown in culture medium comprised of EMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. Medium was changed every 48 hours. 1*104 cells/cm2 were plated into 6 well tissue culture plates containing test articles composed of either ZFuze, PEEK, rough surface titanium, or iterations of modified ZFuze polymer. Upon reaching confluence, fresh medium was added for 24 hours, and subsequently collected for protein expression determination by ELISA, and then the cells were trypsinized for cell counting and subsequently lysed with TRIzol reagent for RNA isolation. Isolated RNA was then converted to cDNA and gene expression was assessed by qPCR: GAPDH, BMP2, and BMP4. Collected medium was assessed for relative levels of: osteocalcin, BMP2, BMP4, and BMP7. Relative gene and protein expression levels were normalized to cell number and quantified to make relative assessments of differentiation status as a function of test article. RESULTS Macrophage gene and protein expression: Across three biological replicates, results of qPCR analyses show that ZFuze promoted an M2-like phenotype with expression of Fizz1, and decreased expression of IL-1b and TNF-a compared to PEEK and titanium, which elicited a more "M1-like" macrophage phenotype. Further, zeolite-loaded ZFuze promoted significantly greater expression of Fizz1 by both gene and protein expression than unloaded ZFuze or other test article comparators. Saos-2 osteoblast-like cell gene and protein expression: Aggregated biological replicates show that BMP4 gene expression was upregulated in a statistically significant manner in Saos-2 cells exposed to ZFuze, but not PEEK or rough surface titanium alloy. The expression of BMP2 was also significantly elevated at the translational level in ZFuze, but not PEEK or rough surface titanium, exposed Saos-2 cells. CONCLUSIONS ZFuze and its iterations elicit a favorable and more biocompatible in vitro immune profile than PEEK or titanium. ZFuze also supports increased expression of osteoblastic differentiation markers' differentiation of progenitor cells in vitro. FDA DEVICE/DRUG STATUS ZFuze (Approved for this indication). Poly-ether-ether-ketone (PEEK) has been the most commonly used biomaterial for spinal fusion for many years, primarily because it meets the necessary mechanical requirements to support the pressures to which the spinal column is subjected. However, the biologic response to PEEK has serious limitations including a persistent foreign body response, very modest osseointegration which can result in micromotion, pain, or worse, delamination requiring surgical reintervention. Recent advances in our understanding of immunobiology show that PEEK elicits a proinflammatory phenotype among cells of the innate immune system, including macrophages. Numerous studies have shown that macrophage phenotype can be predictive of clinical outcome. The host immune response to biomaterials used in spine surgery has been largely ignored in deference to mechanical and biomaterial properties such as peak load bearing, porosity and surface characteristics. Pro-inflammatory (M1-like) macrophages have been shown to release several mediators of cell behavior such as IL-6, IL-1b, and TNF-a, that are osteoclastogenic, while pro-healing (M2-like) macrophages are associated with the release of osteoblastogenic signaling molecules. The emergence of "immunomodulatory biomaterials" is directed toward the biologic consequences associated with permanent implants such as spinal fusion materials. Surface modification of such materials represents one approach for developing more host friendly, biocompatible materials while retaining the required mechanical properties. The objective of the present study was to characterize and compare the macrophage phenotype elicited by the ZFuze polymer, PEEK, titanium-based alloys, or iterations of modified ZFuze polymer. The ability of these materials to promote differentiation of osteoblast-like cells in vitro was also determined. Macrophage activation study: Primary bone marrow-derived derived macrophages were isolated from C57bl6/j mice and plated on one of the following test articles: ZFuzeTM, PEEK, rough surface titanium alloy, or iterations of modified ZFuze polymer. Gene and protein expression were determined through qPCR and immunolabeling, respectively. Osteoblast differentiation assay: Saos-2 cells were purchased from ATCC. 1*104 cells/cm2 were plated into 6 well tissue culture plates containing test articles composed of: ZFuzeTM, PEEK, rough surface titanium alloy, or iterations of modified ZFuze polymer. Gene and protein expression were determined through qPCR and ELISA, respectively. qPCR, immunolabeling, and ELISA. Culture of bone marrow derived macrophages: Bone marrow was isolated from the femurs and tibias of C57bl/6 mice and subsequently cultured in complete growth media, including Dulbecco's modified Eagles medium, 10% fetal bovine serum (FBS), 10% L929 supernatant, 0.1% beta-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM nonessential amino acids, and 10 mM HEPES buffer, for 7 days with complete media changes every 48 hours until mature bone marrow-derived macrophages were obtained. Macrophage activation: Mature macrophages were exposed to the following treatments for 24 hours: complete media (M0 control), 20 ng/ml IFN-γ and 100 ng/mL lipopolysaccharide LPS (M1 control), 20 ng/mL interleukin IL-4 (M2 control), PEEK, ZFuze, rough-surface titanium alloy, or ZFuze surface modification iterations. For the cytokine challenge study, cells were exposed for 6 hours to 20 ng/ml IFN-γ and 100 ng/mL LPS, washed and then placed in 10% FBS 1% P/S DMEM for 24 hours. Macrophage immunolabeling: Fixed cells were washed with 1X PBS followed by incubation in a blocking solution composed of PBS, 0.1% Triton-X, 0.1% Tween-20, 4% goat serum, and 2% bovine serum albumin for 1 hour at room temperature. Cells were then incubated in a solution of one of the following primary antibodies: anti-F4/80 at 1:100 dilution as a pan-macrophage marker, anti-inducible nitric oxide synthase (iNOS) at 1:100 dilution as an M1-like marker, and anti-Fizz1 and anti-Arginase1 at 1:200 dilution, each as M2-like markers. Nuclei were counterstained with DAPI. Saos-2 culture: Saos-2 cells were purchased from ATCC. Cells were grown in culture medium comprised of EMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. Medium was changed every 48 hours. 1*104 cells/cm2 were plated into 6 well tissue culture plates containing test articles composed of either ZFuze, PEEK, rough surface titanium, or iterations of modified ZFuze polymer. Upon reaching confluence, fresh medium was added for 24 hours, and subsequently collected for protein expression determination by ELISA, and then the cells were trypsinized for cell counting and subsequently lysed with TRIzol reagent for RNA isolation. Isolated RNA was then converted to cDNA and gene expression was assessed by qPCR: GAPDH, BMP2, and BMP4. Collected medium was assessed for relative levels of: osteocalcin, BMP2, BMP4, and BMP7. Relative gene and protein expression levels were normalized to cell number and quantified to make relative assessments of differentiation status as a function of test article. Macrophage gene and protein expression: Across three biological replicates, results of qPCR analyses show that ZFuze promoted an M2-like phenotype with expression of Fizz1, and decreased expression of IL-1b and TNF-a compared to PEEK and titanium, which elicited a more "M1-like" macrophage phenotype. Further, zeolite-loaded ZFuze promoted significantly greater expression of Fizz1 by both gene and protein expression than unloaded ZFuze or other test article comparators. Saos-2 osteoblast-like cell gene and protein expression: Aggregated biological replicates show that BMP4 gene expression was upregulated in a statistically significant manner in Saos-2 cells exposed to ZFuze, but not PEEK or rough surface titanium alloy. The expression of BMP2 was also significantly elevated at the translational level in ZFuze, but not PEEK or rough surface titanium, exposed Saos-2 cells. ZFuze and its iterations elicit a favorable and more biocompatible in vitro immune profile than PEEK or titanium. ZFuze also supports increased expression of osteoblastic differentiation markers' differentiation of progenitor cells in vitro.

Tissue engineering materials play a key role in how closely the complex architectural and functional characteristics of native healthy tissue can be replicated. Traditional natural and synthetic materials are superseded by bespoke materials that cross the boundary between these two categories. Here we present hydrogels that are derived from decellularised extracellular matrix and those that are synthesised from de novo alpha-helical peptides. We assess in vitro activation of murine macrophages to our hydrogels and whether these gels induce an M1-like or M2-like phenotype. This was followed by the in vivo immune macrophage response to hydrogels injected into rat partial-thickness abdominal wall defects. Over 28 days we observe an increase in mononuclear cell infiltration at the hydrogel-tissue interface without promoting a foreign body reaction and see no evidence of hydrogel encapsulation or formation of multinucleate giant cells. We also note an upregulation of myogenic differentiation markers and the expression of anti-inflammatory markers Arginasel, IL-10, and CD206, indicating pro-remodelling for all injected hydrogels. Furthermore, all hydrogels promote an anti-inflammatory environment after an initial spike in the pro-inflammatory phenotype. No difference between the injected site and the healthy tissue is observed after 28 days, indicating full integration. These materials offer great potential for future applications in regenerative medicine and towards unmet clinical needs. Statement of Significance Materials play a key role in how closely the complex architectural and functional characteristics of native healthy tissue can be replicated in tissue engineering. Here we present injectable hydrogels derived from decellularised extracellular matrix and de novo designed alpha-helical peptides. Over 28 days in the rat abdominal wall we observe an increase in mononuclear cell infiltration at the hydrogel-tissue interface with no foreign body reaction, no evidence of hydrogel encapsulation and no multinucleate giant cells. Our data indicate pro-remodelling and the promotion of an anti-inflammatory environment for all injected hydrogels with evidence of full integration with healthy tissue after 28 days. These unique materials offer great potential for future applications in regenerative medicine and towards designing materials for unmet clinical needs. (C) 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) raises questions regarding their biologic functions and their potential theranostic application. Unlike liquid-phase extracellular vesicles (e.g., exosomes), MBV are tightly bound to the ECM, which makes their isolation and harvesting more challenging. The indiscriminate use of different methods to harvest MBV can alter or disrupt their structural and/or functional integrity. The objective of the present study was to compare the effect of various MBV harvesting methods upon yield, purity, and biologic activity. Combinations of four methods to solubilize the ECM (collagenase [COL], liberase [LIB], or proteinase K [PK] and nonenzymatic elution with potassium chloride) and four isolation methods (ultracentrifugation, ultrafiltration [UF], density barrier, and size exclusion chromatography [SEC]) were used to isolate MBV from urinary bladder-derived ECM. All combinations of solubilization and isolation methods allowed for the harvesting of MBV, however, distinct differences were noted. The highest yield, purity, cellular uptake, and biologic activity were seen with MBV isolated by a combination of liberase or collagenase followed by SEC. The combination of proteinase K and UF was shown to have detrimental effects on bioactivity. The results show the importance of selecting appropriate MBV harvesting methods for the characterization and evaluation of MBV and for analysis of their potential theranostic application. Impact statement Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) has raised questions regarding their biologic functions and their potential theranostic application. This study demonstrates that the harvesting methods used can result in samples with physical and biochemical properties that are unique to the isolation and solubilization methods used. Consequently, developing harvesting methods that minimize sample contamination with ECM remnants and/or solubilization agents will be essential in determining the theranostic potential of MBV in future studies.

The host innate immune response to a surgical mesh is arguably the most important determinant of tissue remodeling and functional outcome. Macrophage phenotype and the associated secretion of pro-inflammatory or anti-inflammatory cytokines during the first 10–14 days following implantation has been strongly associated with downstream events such as chronic inflammation vs. functional tissue remodeling, respectively, and the associated clinical consequences. A persistent, pro-inflammatory (M1-like) macrophage phenotype is typically associated with fibrosis and scarring. In contrast, an early transition to a regulatory, pro-remodeling (M2-like) macrophage phenotype is predictive of organized, site-appropriate connective tissue deposition. The ratio of M2-like to M1-like macrophages in the early post-implantation period defines the microenvironmental milieu and the associated tissue response. The present study evaluated the early macrophage response to a synthetic non-resorbable (Bard® Mesh), synthetic resorbable (TIGR® Matrix Surgical Mesh and GORE® BIO-A® Tissue Reinforcement), synthetic mesh composed of the naturally occurring molecule 4-hydroxybutyrate, (Phasix™ Mesh), and a biologic surgical mesh composed of dermal extracellular matrix (Strattice™ Reconstructive Tissue Matrix); all of which are used in ventral hernia repair. The spatiotemporal distribution of pro-inflammatory (CD68+CD86+TNF-α+) and pro-remodeling (CD68+CD206+) macrophages, and the remodeling response in terms of vascularization, total number of infiltrating cells, presence of multinucleate giant cells (MNGC), and cell layer thickness around the implanted materials was evaluated at 3, 7, 14, 21, and 35 days post implantation. Results showed an association of the synthetic non-resorbable and resorbable meshes with a robust, pro-inflammatory response within 3 days of implantation, and an increased presence of MNGC around the mesh fibers at longer time points. Phasix™ Mesh was associated with an increased presence of M2-like macrophages immediately adjacent to the mesh fibers at earlier time points, and a favorable tissue remodeling outcome at 35 days. Results of the present study are consistent with the premise that an early shift of M1-like to M2-like macrophages is associated with favorable outcomes, including reduced fibrosis, at later time points.

The regenerative healing response of injured skeletal muscle is dependent upon an appropriately timed switch from a local type-I to a type-II immune response. Biologic scaffolds derived from extracellular matrix (ECM) have been shown to facilitate a macrophage phenotype transition that leads to downstream site-appropriate functional tissue deposition and myogenesis. However, the mechanisms by which ECM directs the switching of immune cell phenotype are only partially understood. Herein, we provide the first evidence that matrix bound nanovesicles (MBV) embedded within ECM-scaffolds are a rich and stable source of interleukin-33 (IL-33), an alarmin/cytokine with emerging reparative properties. We show that IL-33 encapsulated within MBV bypass the classical IL33/ST2 receptor signaling pathway to direct macrophage differentiation into the reparative, pro-remodeling M2 phenotype, which in turn facilitates myogenesis of skeletal muscle progenitor cells. Our results suggest the potential of IL-33+ MBV as a clinical therapy to augment the restorative efficacy of existing ECM-based and non-ECM based approaches.

The ability of skeletal muscle to regenerate after acute injury is compromised in the case of disease or massive traumatic injury (i.e., volumetric muscle loss). An understanding of the mechanisms by which skeletal muscle can acutely regenerate has informed the development of tissue engineering and regenerative medicine–based approaches to treat muscle injuries traditionally considered irreparable using both cell-based and acellular approaches. This chapter discusses the use of stem cells and diverse scaffold materials, synthetic, biosynthetic, and biologic in nature, to promote functional myogenesis. The preclinical and clinical progress of each of these methods and barriers to their further utility as a clinical therapy are discussed.

The early macrophage response to biomaterials has been shown to be a critical and predictive determinant of downstream outcomes. When properly prepared, bioscaffolds composed of mammalian extracellular matrix (ECM) have been shown to promote a transition in macrophage behavior from a proinflammatory to a reg-ulatory/anti-inflammatory phenotype, which in turn has been associated with constructive and functional tissue repair. The mechanism by which ECM bioscaffolds promote this phenotypic transition, however, is poorly understood. The present study shows that matrix-bound nanovesicles (MBV), a component of ECM bioscaffolds, are capable of recapitulating the macrophage activation effects of the ECM bioscaffold from which they are derived. MBV isolated from two different source tissues, porcine urinary bladder and small intestinal submucosa, were found to be enriched in miRNA125b-5p, 143-3p, and 145-5p. Inhibition of these miRNAs within macrophages was associated with a gene and protein expression profile more consistent with a proinflammatory rather than an anti-inflammatory/regulatory phenotype. MBV and their associated miRNA cargo appear to play a significant role in mediating the effects of ECM bioscaffolds on macrophage phenotype. miRNA inhibition shows opposite protein expression in BMDM

Macrophage presence and phenotype are critical determinants of the healing response following injury. Downregulation of the pro-inflammatory macrophage phenotype has been associated with the therapeutic use of bioscaffolds composed of extracellular matrix (ECM), but phenotypic characterization of macrophages has typically been limited to small number of non-specific cell surface markers or expressed proteins. The present study determined the response of both primary murine bone marrow derived macrophages (BMDM) and a transformed human mononuclear cell line (THP-1 cells) to degradation products of two different, commonly used ECM bioscaffolds; urinary bladder matrix (UBM-ECM) and small intestinal submucosa (SIS-ECM). Quantified cell responses included gene expression, protein expression, commonly used cell surface markers, and functional assays. Results showed that the phenotype elicited by ECM exposure (MECM) is distinct from both the classically activated IFNγ+LPS phenotype and the alternatively activated IL-4 phenotype. Furthermore, the BMDM and THP-1 macrophages responded differently to identical stimuli, and UBM-ECM and SIS-ECM bioscaffolds induced similar, yet distinct phenotypic profiles. The results of this study not only characterized an MECM phenotype that has anti-inflammatory traits but also showed the risks and challenges of making conclusions about the role of macrophage mediated events without consideration of the source of macrophages and the limitations of individual cell markers.

The early macrophage response to biomaterials has been shown to be a critical and predictive determinant of downstream outcomes. When properly prepared, bioscaffolds composed of mammalian extracellular matrix (ECM) have been shown to promote a transition in macrophage behavior from a proinflammatory to a regulatory/anti-inflammatory phenotype, which in turn has been associated with constructive and functional tissue repair. The mechanism by which ECM bioscaffolds promote this phenotypic transition, however, is poorly understood. The present study shows that matrix-bound nanovesicles (MBV), a component of ECM bioscaffolds, are capable of recapitulating the macrophage activation effects of the ECM bioscaffold from which they are derived. MBV isolated from two different source tissues, porcine urinary bladder and small intestinal submucosa, were found to be enriched in miRNA125b-5p, 143-3p, and 145-5p. Inhibition of these miRNAs within macrophages was associated with a gene and protein expression profile more consistent with a proinflammatory rather than an anti-inflammatory/regulatory phenotype. MBV and their associated miRNA cargo appear to play a significant role in mediating the effects of ECM bioscaffolds on macrophage phenotype.