Copper economy in Chlamydomonas: prioritized allocation and reallocation of copper to respiration vs. photosynthesis.

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.

Basic leucine zipper transcription activators - tools to improve production and quality of human erythropoietin in Nicotiana benthamiana.

Human erythropoietin (hEPO) is one of the most in-demand biopharmaceuticals, however, its production is challenging. When produced in a plant expression system, hEPO results in extensive plant tissue damage and low expression. It is demonstrated that the modulation of the plant protein synthesis machinery enhances hEPO production. Co-expression of basic leucine zipper transcription factors with hEPO prevents plant tissue damage, boosts expression, and increases hEPO solubility. bZIP28 co-expression up-regulates genes associated with the unfolded protein response, indicating that the plant tissue damage caused by hEPO expression is due to the native protein folding machinery being overwhelmed and that this can be overcome by co-expressing bZIP28.

Epimorphic regeneration approach to tissue replacement in adult mammals.

Urodeles and fetal mammals are capable of impressive epimorphic regeneration in a variety of tissues, whereas the typical default response to injury in adult mammals consists of inflammation and scar tissue formation. One component of epimorphic regeneration is the recruitment of resident progenitor and stem cells to a site of injury. Bioactive molecules resulting from degradation of extracellular matrix (ECM) have been shown to recruit a variety of progenitor and stem cells in vitro in adult mammals. The ability to recruit multipotential cells to the site of injury by in vivo administration of chemotactic ECM degradation products in a mammalian model of digit amputation was investigated in the present study. Adult, 6- to 8-week-old C57/BL6 mice were subjected to midsecond phalanx amputation of the third digit of the right hind foot and either treated with chemotactic ECM degradation products or left untreated. At 14 days after amputation, mice treated with ECM degradation products showed an accumulation of heterogeneous cells that expressed markers of multipotency, including Sox2, Sca1, and Rex1 (Zfp42). Cells isolated from the site of amputation were capable of differentiation along neuroectodermal and mesodermal lineages, whereas cells isolated from control mice were capable of differentiation along only mesodermal lineages. The present findings demonstrate the recruitment of endogenous stem cells to a site of injury, and/or their generation/proliferation therein, in response to ECM degradation products.

Engineering of a plant-produced virus-like particle to improve the display of the Plasmodium falciparum Pfs25 antigen and transmission-blocking activity of the vaccine candidate.

Malaria kills around 409,000 people a year, mostly children under the age of five. Malaria transmission-blocking vaccines work to reduce malaria prevalence in a community and have the potential to be part of a multifaceted approach required to eliminate the parasites causing the disease. Pfs25 is a leading malaria transmission-blocking antigen and has been successfully produced in a plant expression system as both a subunit vaccine and as a virus-like particle. This study demonstrates an improved version of the virus-like particle antigen display molecule by eliminating known protease sites from the prior A85 variant. This re-engineered molecule, termed B29, displays three times the number of Pfs25 antigens per virus-like particle compared to the original Pfs25 virus-like particle. An improved purification scheme was also developed, resulting in a substantially higher yield and improved purity. The molecule was evaluated in a mouse model and found to induce improved transmission-blocking activity at lower doses and longer durations than the original molecule.

Resistance to infection of five different materials in a rat body wall model.

BACKGROUND: Infection occurs after approximately 1% of hernia repair procedures. The resistance to infection of the repair materials is therefore an important consideration. We evaluated the infection resistance of five different materials in a rat model of body wall repair, two of which, urinary bladder matrix (UBM-ECM) and Revive, were not previously evaluated in a controlled model of infection. MATERIALS AND METHODS: An inoculum of 1 × 10(8) colony forming units of Staphylococcus aureus was delivered to the wound site following implantation of an autograft, UBM-ECM, Proceed, Prolene, or Revive. Infection was monitored by white blood cell counts, body temperature, bacterial culture, and histomorphologic analysis of the implant site. RESULTS: Infection was shown in all groups through increased white blood cell count and body temperature. Animals with UBM-ECM returned to pre-surgery body temperature before all other groups. Substantial bacterial clearance was found in the autograft, UBM-ECM, and Prolene. Histomorphologic analysis showed evidence for persistent bacterial infection in Prolene, Proceed, and Revive 28 d after implantation, whereas the autograft and UBM-ECM appeared free of infection. The autograft showed a pyogranulomatous inflammatory reaction at 28 d while UBM-ECM was similar to uninfected controls. CONCLUSIONS: Superior infection resistance was shown by UBM-ECM compared with the other materials, which were substantially equivalent. Histomorphologic analysis clearly showed an increased ability to resist persistent bacterial infection for UBM-ECM. Our results suggest UBM-ECM may be useful as a repair material in areas of high risk for infection.

Safety and immunogenicity of a plant-derived recombinant protective antigen (rPA)-based vaccine against Bacillus anthracis: A Phase 1 dose-escalation study in healthy adults.

BACKGROUND: The potential use of Bacillus anthracis as a bioterrorism weapon requires a safe and effective vaccine that can be immediately distributed for mass vaccination. Protective antigen (PA), a principal component of virulence factors edema toxin and lethal toxin of B. anthracis, has been the topic of extensive research. Previously, full-length PA (PA83) was manufactured using a transient plant-based expression system. Immunization with this PA83 antigen formulated with Alhydrogel® adjuvant elicited strong neutralizing immune responses in mice and rabbits and protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. This Phase 1 study evaluates this vaccine's safety and immunogenicity in healthy human volunteers. METHODS: This first-in-human, single-blind, Phase 1 study was performed at a single center to investigate the safety, reactogenicity, and immunogenicity of the plant-derived PA83-FhCMB vaccine at four escalating dose levels (12.5, 25, 50 or 100 µg) with Alhydrogel® in healthy adults 18-49 years of age (inclusive). Recipients received three doses of vaccine intramuscularly at 28-day intervals. Safety was evaluated on days 3, 7, and 14 following vaccination. Immunogenicity was assessed using an enzyme-linked immunosorbent assay (ELISA) and a toxin neutralizing antibody (TNA) assay on days 0, 14, 28, 56, 84, and 180. RESULTS: All four-dose ranges were safe and immunogenic, with no related serious adverse events observed. Peak ELISA Geometric Mean Concentration (GMC) and TNA ED50 Geometric Mean Titer (GMT) were noted at Day 84, 1 month after the final dose, with the most robust response detected in the highest dose group. Antibody responses decreased by Day 180 across all dose groups. Long-term immunogenicity data beyond six months was not collected. CONCLUSIONS: This is the first study demonstrating a plant-derived subunit anthrax vaccine's safety and immunogenicity in healthy adults. The results support further clinical investigation of the PA83-FhCMB vaccine. ClinicalTrials.gov identifier. NCT02239172.

Functional complementation and genetic deletion studies of KirBac channels: activatory mutations highlight gating-sensitive domains.

The superfamily of prokaryotic inwardly rectifying (KirBac) potassium channels is homologous to mammalian Kir channels. However, relatively little is known about their regulation or about their physiological role in vivo. In this study, we have used random mutagenesis and genetic complementation in K(+)-auxotrophic Escherichia coli and Saccharomyces cerevisiae to identify activatory mutations in a range of different KirBac channels. We also show that the KirBac6.1 gene (slr5078) is necessary for normal growth of the cyanobacterium Synechocystis PCC6803. Functional analysis and molecular dynamics simulations of selected activatory mutations identified regions within the slide helix, transmembrane helices, and C terminus that function as important regulators of KirBac channel activity, as well as a region close to the selectivity filter of KirBac3.1 that may have an effect on gating. In particular, the mutations identified in TM2 favor a model of KirBac channel gating in which opening of the pore at the helix-bundle crossing plays a far more important role than has recently been proposed.

NMR structural analysis of the soluble domain of ZiaA-ATPase and the basis of selective interactions with copper metallochaperone Atx1.

A Cu(I) metallochaperone, Atx1, interacts with the amino-terminal domain of a Cu(I)-transporting ATPase, PacS(N), but not with a domain of related Zn-transporting ATPase, ZiaA(N) in Synechocystis PCC 6803. This is thought to prevent ZiaA(N) from acquiring Cu(I), which it binds more tightly than Zn. Solution structures of Atx1, PacS(N), and the heterodimer were previously described. Here we report solution structural studies of the ZiaA(N) soluble domain. Apo-ZiaA(N) has a typical ferredoxin-like fold followed by an atypical 34 residues of unstructured polypeptide containing a His(7) motif. ZiaA(N) competes with the metallochromic indicator 4-(2-pyridylazo)resorcinol for 1 equiv of Zn, which can be displaced by thiol-modifying p-mercuriphenylsulfonic acid, establishing that a high-affinity site involves thiols of the CXXC motif within the ferredoxin-like fold. A single equivalent of Zn affects nuclear magnetic resonance signals arising from the CXXC motif as well as all seven His residues. The presence of NMR-line broadening in both sites implies that Zn(1)-ZiaA(N) undergoes exchange phenomena, consistent with CXXC-bound Zn coincidentally sampling various His ligands. These Zn-dependent dynamic changes could either aid metal transfer or alter intramolecular interactions. No formation of Atx1-Cu(I)-ZiaA(N) heterodimers was observed, and in the presence of equimolar ZiaA(N) and PacS(N), only Atx1-Cu(I)-PacS(N) complexes were detected. Residues flanking the CXXC motif of PacS(N) (R(13)-ASS(20)) differ in charge and bulk from those of ZiaA(N) (D(18)-KLK(25)) and make contacts in the Atx1-Cu(I)-PacS(N) complex. Crucially, swapping these residues flanking the CXXC motifs of ZiaA(N) and PacS(N) reciprocally swaps partner choice by Atx1. These few residues of the two ATPases have diverged during evolution to bias Atx1 interactions in favor of PacS(N) rather than ZiaA(N.).

Plant-Produced Subunit Vaccine Candidates against Yellow Fever Induce Virus Neutralizing Antibodies and Confer Protection against Viral Challenge in Animal Models.

Yellow fever (YF) is a viral disease transmitted by mosquitoes and endemic mostly in South America and Africa with 20-50% fatality. All current licensed YF vaccines, including YF-Vax® (Sanofi-Pasteur, Lyon, France) and 17DD-YFV (Bio-Manguinhos, Rio de Janeiro, Brazil), are based on live attenuated virus produced in hens' eggs and have been widely used. The YF vaccines are considered safe and highly effective. However, a recent increase in demand for YF vaccines and reports of rare cases of YF vaccine-associated fatal adverse events have provoked interest in developing a safer YF vaccine that can be easily scaled up to meet this increased global demand. To this point, we have engineered the YF virus envelope protein (YFE) and transiently expressed it in Nicotiana benthamiana as a stand-alone protein (YFE) or as fusion to the bacterial enzyme lichenase (YFE-LicKM). Immunogenicity and challenge studies in mice demonstrated that both YFE and YFE-LicKM elicited virus neutralizing (VN) antibodies and protected over 70% of mice from lethal challenge infection. Furthermore, these two YFE-based vaccine candidates induced VN antibody responses with high serum avidity in nonhuman primates and these VN antibody responses were further enhanced after challenge infection with the 17DD strain of YF virus. These results demonstrate partial protective efficacy in mice of YFE-based subunit vaccines expressed in N. benthamiana. However, their efficacy is inferior to that of the live attenuated 17DD vaccine, indicating that formulation development, such as incorporating a more suitable adjuvant, may be required for product development.

Between a rock and a hard place: trace element nutrition in Chlamydomonas.

Photosynthetic organisms are among the earliest life forms on earth and their biochemistry is strictly dependent on a wide range of inorganic nutrients owing to the use of metal cofactor-dependent enzymes in photosynthesis, respiration, inorganic nitrogen and sulfur assimilation. Chlamydomonas reinhardtii is a photosynthetic eukaryotic model organism for the study of trace metal homeostasis. Chlamydomonas spp. are widely distributed and can be found in soil, glaciers, acid mines and sewage ponds, suggesting that the genus has significant capacity for acclimation to micronutrient availability. Analysis of the draft genome indicates that metal homeostasis mechanisms in Chlamydomonas represent a blend of mechanisms operating in animals, plants and microbes. A combination of classical genetics, differential expression and genomic analysis has led to the identification of homologues of components known to operate in fungi and animals (e.g., Fox1, Ftr1, Fre1, Fer1, Ctr1/2) as well as novel molecules involved in copper and iron nutrition (Crr1, Fea1/2). Besides activating iron assimilation pathways, iron-deficient Chlamydomonas cells re-adjust metabolism by reducing light delivery to photosystem I (to avoid photo-oxidative damage resulting from compromised FeS clusters) and by modifying the ferredoxin profile (perhaps to accommodate preferential allocation of reducing equivalents). Up-regulation of a MnSOD isoform may compensate for loss of FeSOD. Ferritin could function to buffer the iron released from programmed degradation of iron-containing enzymes in the chloroplast. Some metabolic adjustments are made in anticipation of deficiency while others occur only with sustained or severe deficiency. Copper-deficient Chlamydomonas cells induce a copper assimilation pathway consisting of a cell surface reductase and a Cu(+) transporter (presumed CTR homologue). There are metabolic adaptations in addition: the synthesis of "back-up" enzymes for plastocyanin in photosynthesis and the ferroxidase in iron assimilation plus activation of alternative oxidase to handle the electron "overflow" resulting from reduced cytochrome oxidase function. Oxygen-dependent enzymes in the tetrapyrrole pathway (coproporphyrinogen oxidase and aerobic oxidative cyclase) are also increased in expression and activity by as much as 10-fold but the connection between copper nutrition and tetrapyrroles is not understood. The copper-deficiency responses are mediated by copper response elements that are defined by a GTAC core sequence and a novel metalloregulator, Crr1, which uses a zinc-dependent SBP domain to bind to the CuRE. The Chlamydomonas model is ideal for future investigation of nutritional manganese deficiency and selenoenzyme function. It is also suited for studies of trace nutrient interactions, nutrition-dependent metabolic changes, the relationship between photo-oxidative stress and metal homeostasis, and the important questions of differential allocation of limiting metal nutrients (e.g., to respiration vs. photosynthesis).

Genetic dissection of nutritional copper signaling in chlamydomonas distinguishes regulatory and target genes.

A genetic screen for Chlamydomonas reinhardtii mutants with copper-dependent growth or nonphotosynthetic phenotypes revealed three loci, COPPER RESPONSE REGULATOR 1 (CRR1), COPPER RESPONSE DEFECT 1 (CRD1), and COPPER RESPONSE DEFECT 2 (CRD2), distinguished as regulatory or target genes on the basis of phenotype. CRR1 was shown previously to be required for transcriptional activation of target genes like CYC6, CPX1, and CRD1, encoding, respectively, cytochrome c(6) (which is a heme-containing substitute for copper-containing plastocyanin), coproporphyrinogen III oxidase, and Mg-protoporphyrin IX monomethylester cyclase. We show here that CRR1 is required also for normal accumulation of copper proteins like plastocyanin and ferroxidase in copper-replete medium and for apoplastocyanin degradation in copper-deficient medium, indicating that a single pathway controls nutritional copper homeostasis at multiple levels. CRR1 is linked to the SUPPRESSOR OF PCY1-AC208 13 (SOP13) locus, which corresponds to a gain-of-function mutation resulting in copper-independent expression of CYC6. CRR1 is required also for hypoxic growth, pointing to a physiologically meaningful regulatory connection between copper deficiency and hypoxia. The growth phenotype of crr1 strains results primarily from secondary iron deficiency owing to reduced ferroxidase abundance, suggesting a role for CRR1 in copper distribution to a multicopper ferroxidase involved in iron assimilation. Mutations at the CRD2 locus also result in copper-conditional iron deficiency, which is consistent with a function for CRD2 in a pathway for copper delivery to the ferroxidase. Taken together, the observations argue for a specialized copper-deficiency adaptation for iron uptake in Chlamydomonas.

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.

Immunogenicity of H1N1 influenza virus-like particles produced in Nicotiana benthamiana.

The H1N1 influenza pandemic of 2009 stimulated interest in developing safe and effective subunit influenza vaccines using rapid and cost-effective recombinant technologies that can avoid dependence on hens' eggs supply and live viruses for production. Among alternative approaches to subunit vaccine development, virus-like particles (VLPs) represent an attractive strategy due to their safety and immunogenicity. Previously, we have produced a recombinant monomeric hemagglutinin (HA) protein derived from the A/California/04/09 (H1N1) strain of influenza virus in a plant-based transient expression system and demonstrated immunogenicity and safety of this monomeric HA in animal models and human volunteers. In an effort to produce higher potency influenza vaccine in plants, we have designed and generated enveloped VLPs using the ectodomain of HA from the A/California/04/09 strain and heterologous sequences. The resulting H1 HA VLPs (HAC-VLPs) elicited robust hemagglutination inhibition antibody responses in mice at doses lower than 1 µg in the presence or absence of Alhydrogel adjuvant. These results suggest enhanced immunogenicity of recombinant HA in the form of an enveloped VLP over soluble antigen.

Effects of biologic scaffolds on human stem cells and implications for CNS tissue engineering.

Biologic scaffolds composed of mammalian extracellular matrix (ECM) promote constructive remodeling of tissues via mechanisms that include the recruitment of endogenous stem/progenitor cells, modulation of the host innate immune response, and influence of cell fate differentiation. Such scaffold materials are typically prepared by decellularization of source tissues and are prepared as sheets, powder, or hydrogels. It is plausible that ECM derived from an anatomically distinct tissue would have unique or specific effects on cells that naturally reside in this same tissue. The present study investigated the in vitro effect of a soluble form of ECM derived from central nervous system (CNS) tissue, specifically the spinal cord or brain, versus ECM derived from a non-CNS tissue; specifically, the urinary bladder on the behavior of neural stem cells (NSCs) and perivascular stem cells. All forms of ECM induce positive, mitogenic, and chemotactic effects at concentrations of approximately 100 µg/mL without affecting stem cell viability. CNS-derived ECMs also showed the ability to differentiate NSCs into neurons as indicted by ßIII-tubulin expression in two-dimensional culture and neurite extension on the millimeter scale after 24 days of three-dimensional cultures in an ECM hydrogel. These results suggest that solubilized forms of ECM scaffold materials may facilitate the postinjury healing response in CNS tissues.

A plant-produced Pfs25 VLP malaria vaccine candidate induces persistent transmission blocking antibodies against Plasmodium falciparum in immunized mice.

Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based 'launch' vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20-30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the 'launch' vector technology for the production of VLP-based recombinant vaccines against infectious diseases.

A comprehensive protein expression profile of extracellular matrix biomaterial derived from porcine urinary bladder.

AIMS: To generate a comprehensive profile of the protein composition of xenogeneic biomaterial, derived from porcine urinary bladder matrix (UBM). MATERIALS & METHODS: Tunica layers and muscularis mucosa were removed from bladders using mechanical delamination. UBM was prepared using a solution of peracetic acid in ethanol, lyophilized then milled into powder. UBM biomaterial was subjected to tryptic digests and components separated using high-performance liquid chromatography with an ion trap mass spectrometer and identified through databases. RESULTS: A repertoire of 129 proteins with neurotrophic, antiangiogenic and tumor-suppressive activities and those associated with tissue remodeling and wound repair were identified. CONCLUSION: This study provides the first insight into the complex nature of the UBM and how its application may be tailored for specific applications in regenerative medicine. We propose that the UBM be further investigated for reconstructive and regenerative remodeling of cardiac and dermal tissues, as well as peripheral nerves.

Biologic scaffolds composed of central nervous system extracellular matrix.

Acellular biologic scaffolds are commonly used to facilitate the constructive remodeling of three of the four traditional tissue types: connective, epithelial, and muscle tissues. However, the application of extracellular matrix (ECM) scaffolds to neural tissue has been limited, particularly in the central nervous system (CNS) where intrinsic regenerative potential is low. The ability of decellularized liver, lung, muscle, and other tissues to support tissue-specific cell phenotype and function suggests that CNS-derived biologic scaffolds may help to overcome barriers to mammalian CNS repair. A method was developed to create CNS ECM scaffolds from porcine optic nerve, spinal cord, and brain, with decellularization verified against established criteria. CNS ECM scaffolds retained neurosupportive proteins and growth factors and, when tested with the PC12 cell line in vitro, were cytocompatible and stimulated proliferation, migration, and differentiation. Urinary bladder ECM (a non-CNS ECM scaffold) was also cytocompatible and stimulated PC12 proliferation but inhibited migration rather than acting as a chemoattractant over the same concentration range while inducing greater rates of PC12 differentiation compared to CNS ECM. These results suggest that CNS ECM may provide tissue-specific advantages in CNS regenerative medicine applications and that ECM scaffolds in general may aid functional recovery after CNS injury.

Macrophage phenotype as a predictor of constructive remodeling following the implantation of biologically derived surgical mesh materials.

Macrophages have been classified as having plastic phenotypes which exist along a spectrum between M1 (classically activated; pro-inflammatory) and M2 (alternatively activated; regulatory, homeostatic). To date, the effects of polarization towards an M1 or M2 phenotype have been studied largely in the context of response to pathogen or cancer. Recently, M1 and M2 macrophages have been shown to play distinct roles in tissue remodeling following injury. In the present study, the M1/M2 paradigm was utilized to examine the role of macrophages in the remodeling process following implantation of 14 biologically derived surgical mesh materials in the rat abdominal wall. In situ polarization of macrophages responding to the materials was examined and correlated to a quantitative measure of the observed tissue remodeling response to determine whether macrophage polarization is an accurate predictor of the ability of a biologic scaffold to promote constructive tissue remodeling. Additionally the ability of M1 and M2 macrophages to differentially recruit progenitor-like cells in vitro, which are commonly observed to participate in the remodeling of those ECM scaffolds which have a positive clinical outcome, was examined as a possible mechanism underlying the differences in the observed remodeling responses. The results of the present study show that there is a strong correlation between the early macrophage response to implanted materials and the outcome of tissue remodeling. Increased numbers of M2 macrophages and higher ratios of M2:M1 macrophages within the site of remodeling at 14 days were associated with more positive remodeling outcomes (r(2)=0.525-0.686, p<0.05). Further, the results of the present study suggest that the constructive remodeling outcome may be due to the recruitment and survival of different cell populations to the sites of remodeling associated with materials that elicit an M1 vs. M2 response. Both M2 and M0 macrophage conditioned media were shown to have higher chemotactic activities than media conditioned by M1 macrophages (p<0.05). A more thorough understanding of these issues will logically influence the design of next generation biomaterials and the development of regenerative medicine strategies for the formation of functional host tissues.

The effect of source animal age upon the in vivo remodeling characteristics of an extracellular matrix scaffold.

Biologic scaffolds composed of mammalian extracellular matrix (ECM) are routinely used for the repair and reconstruction of injured or missing tissues in a variety of pre-clinical and clinical applications. However, the structural and functional outcomes have varied considerably. An important variable of xenogeneic biologic scaffolds is the age of the animal from which the ECM is derived. The present study compared the in vivo host response and remodeling outcomes of biologic scaffolds composed of small intestinal submucosa (SIS)-ECM harvested from pigs that differed only in age. Results showed that there are distinct differences in the remodeling characteristics as a consequence of source animal age. Scaffolds derived from younger animals were associated with a more constructive, site appropriate, tissue remodeling response than scaffolds derived from older animals. Furthermore, the constructive remodeling response was associated with a dominant M2 macrophage response.

Partial characterization of the Sox2+ cell population in an adult murine model of digit amputation.

Tissue regeneration in response to injury in adult mammals is generally limited to select tissues. Nonmammalian species such as newts and axolotls undergo regeneration of complex tissues such as limbs and digits via recruitment and accumulation of local and circulating multipotent progenitors preprogrammed to recapitulate the missing tissue. Directed recruitment and activation of progenitor cells at a site of injury in adult mammals may alter the default wound-healing response from scar tissue toward regeneration. Bioactive molecules derived from proteolytic degradation of extracellular matrix (ECM) proteins have been shown to recruit a variety of progenitor cells in vitro and in vivo to the site of injury. The present study further characterized the population of cells accumulating at the site of injury after treatment with ECM degradation products in a well-established model of murine digit amputation. After a mid-second phalanx digit amputation in 6-8-week-old adult mice, treatment with ECM degradation products resulted in the accumulation of a heterogeneous population of cells, a subset of which expressed the transcription factor Sox2, a marker of pluripotent and adult progenitor cells. Sox2+ cells were localized lateral to the amputated P2 bone and coexpressed progenitor cell markers CD90 and Sca1. Transgenic Sox2 eGFP/+ and bone marrow chimeric mice showed that the bone marrow and blood circulation did not contribute to the Sox2+ cell population. The present study showed that, in addition to circulating progenitor cells, resident tissue-derived cells also populate at the site of injury after treatment with ECM degradation products. Although future work is necessary to determine the contribution of Sox2+ cells to functional tissue at the site of injury, recruitment and/or activation of local tissue-derived cells may be a viable approach to tissue engineering of more complex tissues in adult mammals.