Kara L. Spiller

Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds

In normal tissue repair, macrophages exhibit a pro-inflammatory phenotype (M1) at early stages and a pro-healing phenotype (M2) at later stages. We have previously shown that M1 macrophages initiate angiogenesis while M2 macrophages promote vessel maturation. Therefore, we reasoned that scaffolds that promote sequential M1 and M2 polarization of infiltrating macrophages should result in enhanced angiogenesis and healing. To this end, we first analyzed the in vitro kinetics of macrophage phenotype switch using flow cytometry, gene expression, and cytokine secretion analysis. Then, we designed scaffolds for bone regeneration based on modifications of decellularized bone for a short release of interferon-gamma (IFNg) to promote the M1 phenotype, followed by a more sustained release of interleukin-4 (IL4) to promote the M2 phenotype. To achieve this sequential release profile, IFNg was physically adsorbed onto the scaffolds, while IL4 was attached via biotin-streptavidin binding. Interestingly, despite the strong interactions between biotin and streptavidin, release studies showed that biotinylated IL4 was released over 6 days. These scaffolds promoted sequential M1 and M2 polarization of primary human macrophages as measured by gene expression of ten M1 and M2 markers and secretion of four cytokines, although the overlapping phases of IFNg and IL4 release tempered polarization to some extent. Murine subcutaneous implantation model showed increased vascularization in scaffolds releasing IFNg compared to controls. This study demonstrates that scaffolds for tissue engineering can be designed to harness the angiogenic behavior of host macrophages towards scaffold vascularization.

A novel method for the direct fabrication of growth factor-loaded microspheres within porous nondegradable hydrogels: Controlled release for cartilage tissue engineering

Because of similar mechanical properties to native cartilage, synthetic hydrogels based on poly(vinyl alcohol) (PVA) have been proposed for replacement of damaged articular cartilage, but they suffer from a complete lack of integration with surrounding tissue. In this study, insulin-like growth factor-1 (IGF-1), an important growth factor in cartilage regeneration, was encapsulated in degradable poly(lactic-co-glycolic acid) (PLGA) microparticles embedded in the PVA hydrogels in a single step based on a double emulsion. The release of IGF-1 from these hydrogels was sustained over 6 weeks in vitro. Poly(glycolic acid) (PGA) fiber scaffolds were wrapped around the hydrogels, seeded with chondrocytes, and implanted subcutaneously in athymic mice. The release of IGF-1 enhanced cartilage formation in the layers surrounding the hydrogels, in terms of the content of extracellular matrix components and mechanical properties, and increased integration between the cartilage layers and the hydrogels, according to gross observation of the cross-sections and histology. The compressive modulus of the cartilage-hydrogel constructs without IGF-1 was 0.07±0.02MPa, compared to 0.17-0.2MPa for hydrogels that contained IGF-1. The biochemical and mechanical markers of cartilage formation were not different between the low and high concentrations of IGF-1, despite an order of magnitude difference in concentration. This study shows that the sustained release of IGF-1 can enhance tissue formation and points to a possible strategy for effecting integration with surrounding tissue.

Time-Dependent Processes in Stem Cell-Based Tissue Engineering of Articular Cartilage

Articular cartilage (AC), situated in diarthrodial joints at the end of the long bones, is composed of a single cell type (chondrocytes) embedded in dense extracellular matrix comprised of collagens and proteoglycans. AC is avascular and alymphatic and is not innervated. At first glance, such a seemingly simple tissue appears to be an easy target for the rapidly developing field of tissue engineering. However, cartilage engineering has proven to be very challenging. We focus on time-dependent processes associated with the development of native cartilage starting from stem cells, and the modalities for utilizing these processes for tissue engineering of articular cartilage.

Relative Expression of Proinflammatory and Antiinflammatory Genes Reveals Differences between Healing and Nonhealing Human Chronic Diabetic Foot Ulcers

driven neoplasms (Reifenberger et al., 2004; Palomba et al., 2012). Our identification of somatic RAS mutations in vascular tumors has clinical relevance. Current therapies against these lesions are limited to steroids and β-blockers, which achieve mixed results, often limited to tumor size reduction without resolution (Wine Lee et al., 2014). Some infantile vascular tumors, such as VASC101, are unresponsive to such interventions (Wine Lee et al., 2014). These cases may harbor RAS mutations, and might respond to farnesyl transferase inhibitors or Raf/Mek/Erk inhibitors, which block signaling upstream or downstream of RAS. The finding that RAS mutation drives vascular tumors provides potential opportunities to develop targeted therapies for current drug-resistant lesions.

Design of semi-degradable hydrogels based on poly(vinyl alcohol) and poly(lactic-co-glycolic acid) for cartilage tissue engineering.

Articular cartilage damage is a persistent challenge in biomaterials and tissue engineering. Poly(vinyl alcohol) (PVA) hydrogels have shown promise as implants, but their lack of integration with surrounding cartilage prevents their utility. We sought to combine the advantages of PVA hydrogels with poly(lactic‐co‐glycolic acid) (PLGA) scaffolds, which have been successful in facilitating the integration of neocartilage with surrounding tissue. Through a novel double‐emulsion technique, PLGA microparticles and a high level of porosity were simultaneously incorporated into PVA hydrogels. The porosity, average pore size and swelling properties of the hydrogels were controlled by varying initial processing parameters, such as the relative amounts of PLGA and solvent. Average pore sizes were in the ranged 50–100 µm. The PLGA microparticles degraded within the hydrogels over time in aqueous conditions, resulting in increases in porosity and pore size. After 4 weeks in cell culture, immature cartilage tissue filled many of the pores of the hydrogels that initially contained PLGA, and proteoglycan production was proportional to the amount of PLGA. In contrast, there was little cell attachment and no proteoglycan production in control hydrogels without PLGA. The compressive moduli of the hydrogels were similar to that of healthy cartilage and increased over time from 0.05–0.1 to 0.3–0.7 MPa. The generation of a hybrid cartilage–hydrogel construct using this technique may finally allow the integration of PVA hydrogels with surrounding cartilage. Copyright

Analysis of the in vitro swelling behavior of poly(vinyl alcohol) hydrogels in osmotic pressure solution for soft tissue replacement

An osmotic solution was used to evaluate poly(vinyl alcohol) (PVA) hydrogels as potential non-degradable soft tissue replacements in vitro. Osmotic solutions are necessary in order to mimic the swelling pressure observed in vivo for soft tissues present in load-bearing joints. In vitro studies indicated that PVA hydrogels experience minimal changes in swelling with a polymer concentration of 20 wt.% PVA in phosphate-buffered saline solution (0 atm) and between 30 and 35 wt.% PVA in osmotic solution with a pressure of 0.95 atm. Swelling in osmotic pressure solutions caused decreases in the equilibrium hydrogel hydration. An investigation of hydrogel compressive modulus indicated that PVA hydrogels are within the range of articular cartilage, meniscal tissue, and the temporomandibular joint disk. Furthermore, it is possible to tailor PVA hydrogels through careful modification of the polymer concentration and freeze-thaw cycles during hydrogel preparation to match both a desired swelling ratio and a desired compressive modulus or porosity. The microstructure of the PVA hydrogels was also evaluated as a function of freeze-thaw cycles and polymer concentration to give an insight into the processes occurring during synthesis and swelling in osmotic solutions.

Differential gene expression in human, murine, and cell line-derived macrophages upon polarization.

The mechanisms by which macrophages control the inflammatory response, wound healing, biomaterial-interactions, and tissue regeneration appear to be related to their activation/differentiation states. Studies of macrophage behavior in vitro can be useful for elucidating their mechanisms of action, but it is not clear to what extent the source of macrophages affects their apparent behavior, potentially affecting interpretation of results. Although comparative studies of macrophage behavior with respect to cell source have been conducted, there has been no direct comparison of the three most commonly used cell sources: murine bone marrow, human monocytes from peripheral blood (PB), and the human leukemic monocytic cell line THP-1, across multiple macrophage phenotypes. In this study, we used multivariate discriminant analysis to compare the in vitro expression of genes commonly chosen to assess macrophage phenotype across all three sources of macrophages, as well as those derived from induced pluripotent stem cells (iPSCs), that were polarized towards four distinct phenotypes using the same differentiation protocols: M(LPS,IFN) (aka M1), M(IL4,IL13) (aka M2a), M(IL10) (aka M2c), and M(-) (aka M0) used as control. Several differences in gene expression trends were found among the sources of macrophages, especially between murine bone marrow-derived and human blood-derived M(LPS,IFN) and M(IL4,IL13) macrophages with respect to commonly used phenotype markers like CCR7 and genes associated with angiogenesis and tissue regeneration like FGF2 and MMP9. We found that the genes with the most similar patterns of expression among all sources were CXCL-10 and CXCL-11 for M(LPS,IFN) and CCL17 and CCL22 for M(IL4,IL13). Human PB-derived macrophages and human iPSC-derived macrophages showed similar gene expression patterns among the groups and genes studied here, suggesting that iPSC-derived monocytes have the potential to be used as a reliable cell source of human macrophages for in vitro studies. These findings could help select appropriate markers when testing macrophage behavior in vitro and highlight those markers that may confuse interpretation of results from experiments employing macrophages from different sources.

Controlled release of cytokines using silk-biomaterials for macrophage polarization

Polarization of macrophages into an inflammatory (M1) or anti-inflammatory (M2) phenotype is important for clearing pathogens and wound repair, however chronic activation of either type of macrophage has been implicated in several diseases. Methods to locally control the polarization of macrophages is of great interest for biomedical implants and tissue engineering. To that end, silk protein was used to form biopolymer films that release either IFN-γ or IL-4 to control the polarization of macrophages. Modulation of the solubility of the silk films through regulation of β-sheet (crystalline) content enabled a short-term release (4-8 h) of either cytokine, with smaller amounts released out to 24 h. Altering the solubility of the films was accomplished by varying the time that the films were exposed to water vapor. The released IFN-γ or IL-4 induced polarization of THP-1 derived macrophages into the M1 or M2 phenotypes, respectively. The silk biomaterials were able to release enough IFN-γ or IL-4 to repolarize the macrophage from M1 to M2 and vice versa, demonstrating the well-established plasticity of macrophages. High β-sheet content films that are not soluble and do not release the trapped cytokines were also able to polarize macrophages that adhered to the surface through degradation of the silk protein. Chemically conjugating IFN-γ to silk films through disulfide bonds allowed for longer-term release to 10 days. The release of covalently attached IFN-γ from the films was also able to polarize M1 macrophages in vitro. Thus, the strategy described here offers new approaches to utilizing biomaterials for directing the polarization of macrophages.

Kinetics and mechanics of clot contraction are governed by the molecular and cellular composition of the blood.

Platelet-driven blood clot contraction (retraction) is thought to promote wound closure and secure hemostasis while preventing vascular occlusion. Notwithstanding its importance, clot contraction remains a poorly understood process, partially because of the lack of methodology to quantify its dynamics and requirements. We used a novel automated optical analyzer to continuously track in vitro changes in the size of contracting clots in whole blood and in variously reconstituted samples. Kinetics of contraction was complemented with dynamic rheometry to characterize the viscoelasticity of contracting clots. This combined approach enabled investigation of the coordinated mechanistic impact of platelets, including nonmuscle myosin II, red blood cells (RBCs), fibrin(ogen), factor XIIIa (FXIIIa), and thrombin on the kinetics and mechanics of the contraction process. Clot contraction is composed of 3 sequential phases, each characterized by a distinct rate constant. Thrombin, Ca(2+), the integrin αIIbβ3, myosin IIa, FXIIIa cross-linking, and platelet count all promote 1 or more phases of the clot contraction process. In contrast, RBCs impair contraction and reduce elasticity, while increasing the overall contractile stress generated by the platelet-fibrin meshwork. A better understanding of the mechanisms by which blood cells, fibrin(ogen), and platelet-fibrin interactions modulate clot contraction may generate novel approaches to reveal and to manage thrombosis and hemostatic disorders.

Macrophage-based therapeutic strategies in regenerative medicine

Abstract Mounting evidence suggests that therapeutic cell and drug delivery strategies designed to actively harness the regenerative potential of the inflammatory response have great potential in regenerative medicine. In particular, macrophages have emerged as a primary target because of their critical roles in regulating multiple phases of tissue repair through their unique ability to rapidly shift phenotypes. Herein, we review macrophage‐based therapies, focusing on the translational potential for cell delivery of ex vivo‐activated macrophages and delivery of molecules and biomaterials to modulate accumulation and phenotype of endogenous macrophages. We also review current obstacles to progress in translating basic findings to therapeutic applications, including the need for improved understanding of context‐dependent macrophage functions and the myriad factors that regulate macrophage phenotype; potential species‐specific differences (e.g. humans versus mice); quality control issues; and the lack of standardized procedures and nomenclature for characterizing macrophages. Looking forward, the inherent plasticity of macrophages represents a daunting challenge for harnessing these cells in regenerative medicine therapies but also great opportunity for improving patient outcomes in a variety of pathological conditions. Graphical abstract Figure. No Caption available.