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Rationale: Despite direct immediate intervention and therapy, ST-segment–elevation myocardial infarction (STEMI) victims remain at risk for infarct expansion, heart failure, reinfarction, repeat revascularization, and death. Objective: To evaluate the safety and bioactivity of autologous CD34+ cell (CLBS10) intracoronary infusion in patients with left ventricular dysfunction post STEMI. Methods and Results: Patients who underwent successful stenting for STEMI and had left ventricular dysfunction (ejection fraction≤48%) ≥4 days poststent were eligible for enrollment. Subjects (N=161) underwent mini bone marrow harvest and were randomized 1:1 to receive (1) autologous CD34+ cells (minimum 10 mol/L±20% cells; N=78) or (2) diluent alone (N=83), via intracoronary infusion. The primary safety end point was adverse events, serious adverse events, and major adverse cardiac event. The primary efficacy end point was change in resting myocardial perfusion over 6 months. No differences in myocardial perfusion or adverse events were observed between the control and treatment groups, although increased perfusion was observed within each group from baseline to 6 months ( P P =0.05). At 1 year, 3.6% (N=3) and 0% deaths were observed in the control and treatment group, respectively. Conclusions: This PreSERVE-AMI (Phase 2, randomized, double-blind, placebo-controlled trial) represents the largest study of cell-based therapy for STEMI completed in the United States and provides evidence supporting safety and potential efficacy in patients with left ventricular dysfunction post STEMI who are at risk for death and major morbidity. Clinical Trial Registration: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01495364.

The aggressiveness of melanoma is believed to be correlated with tumor-stroma-associated immune cells. Cytokines and chemokines act to recruit and then modulate the activities of these cells, ultimately affecting disease progression. Because melanoma frequently metastasizes to the brain, we asked whether global differences in immunokine profiles could be detected in the cerebrospinal fluid (CSF) of melanoma patients and reveal aspects of tumor biology that correlate with patient outcomes. We therefore measured the levels of 12 cytokines and 12 chemokines in melanoma patient CSF and the resulting data were analyzed to develop unsupervised hierarchical clustergrams and heat maps. Unexpectedly, the overall profiles of immunokines found in these samples showed a generalized reconfiguration of their expression in melanoma patient CSF, resulting in the segregation of individuals with melanoma brain metastasis from nondisease controls. Chemokine CCL22 and cytokines IL1α, IL4, and IL5 were reduced in most samples, whereas a subset including CXCL10, CCL4, CCL17, and IL8 showed increased expression. Further, analysis of clusters identified within the melanoma patient set comparing patient outcome suggests that suppression of IL1α, IL4, IL5, and CCL22, with concomitant elevation of CXCL10, CCL4, and CCL17, may correlate with more aggressive development of brain metastasis. These results suggest that global immunokine suppression in the host, together with a selective increase in specific chemokines, constitute a predominant immunomodulatory feature of melanoma brain metastasis. These alterations likely drive the course of this disease in the brain and variations in the immune profiles of individual patients may predict outcomes.

In situ gelling constructs, which form a hydrogel at the site of injection, offer the advantage of delivering cells and growth factors to the complex structure of the defect area for tissue engineering. In the present study, visible light crosslinkable hydrogel systems were presented using methacrylated glycol chitosan (MeGC) and three blue light initiators: camphorquinone (CQ), fluorescein (FR) and riboflavin (RF). A minimal irradiation time of 120 s was required to produce MeGC gels able to encapsulate cells with CQ or FR. Although prolonged irradiation up to 600 s improved the mechanical strength of CQ- or FR-initiated gels (compressive modulus 2.8 or 4.4 kPa, respectively), these conditions drastically reduced encapsulated chondrocyte viability to 5% and 25% for CQ and FR, respectively. Stable gels with 80-90% cell viability could be constructed using radiofrequency (RF) with only 40s irradiation time. Increasing irradiation time up to 300s significantly improved the compressive modulus of the RF-initiated MeGC gels up to 8.5 kPa without reducing cell viability. The swelling ratio and degradation rate were smaller at higher irradiation time. RF-photoinitiated hydrogels supported proliferation of encapsulated chondrocytes and extracellular matrix deposition. The feasibility of this photoinitiating system as in situ gelling hydrogels was further demonstrated in osteochondral and chondral defect models for potential cartilage tissue engineering. The MeGC hydrogels using RF offer a promising photoinitiating system in tissue engineering applications.

Electrospun fibrous mats have gained popularity in bioengineering over the past decade, but few papers detail their degradative mechanisms. To address this, blends of hydrophobic poly(ε-caprolactone) (PCL) and hydrophilic PGA-PCL-PGA triblock copolymer were electrospun into aligned fibrous mats to assess the copolymers' mechanical and degradative properties. Increased hydrophilic triblock content led to enhanced morphological uniformity of fiber, tightening of fiber diameters, increased storage and Young's modulus, and decreased elongation. The corresponding decrease in hydrophobic PCL content led to faster hydrolytic degradation rate, as reflected by enhanced decrease in mass, molecular weight, and modulus loss at 25, 37, and 45°C. The activation energy for hydrolytic degradation for 15:85 PCL: triblock copolymer was approximately half that of 85:15 PCL: triblock copolymer. Detailed examination of fiber morphology and crystallinity revealed initial surface erosion followed by the evolution of crystalline lamellar stacks and bulk degradation at 37°C. Because of the high surface to volume and short diffusion length scale of the small diameter fibers, surface and bulk degradation may both contribute to the hydrolytic degradative behavior of these electrospun fibrous mats. Electrospun mats' distinct architecture that embodies high specific surface to volume and interfiber porous ultrastructures that lead to their unique degradative behaviors hold much potential for significant impact in the field of tissue engineering and controlled drug delivery.

Our knowledge of cells, extracellular factors and signaling pathways orchestrating angiogenesis, arteriogenesis, and vasculogenesis have greatly advanced since the first report describing angiogenesis in 1971. Early studies of angiogenic factor treatment yielded promising preclinical studies, which led to the investigation of cell therapies and angiogenic factors for peripheral arterial disease (PAD) in the clinical setting for the past two decades. Though early trials have provided evidence for clinical safety and bioactivity, a positive Phase 3 pivotal trial has yet to be conducted in the field. This chapter describes the clinical findings of cell therapy and angiogenic factors for the treatment of peripheral arterial disease, and discusses new approaches and insights into effective modulation of vascular response to ischemia for the treatment of peripheral artery disease.

The purpose of this study was to evaluate the biocompatibility and the efficacy in wound healing of a gelatin-based interpenetrating polymer network (IPN) containing poly(ethylene glycol) (PEG)-ylated RGD and soluble KGF-1 (RGD-IPN+KGF). IPNs were applied to full-thickness wounds on a rat model. Wound healing was assessed through histological grading of the host response and percent area contraction at 2 days, 1 week, 2 weeks, and 3 weeks. A control IPN containing unmodified gelatin (unmod-IPN) and a conventional clinical bandage were applied to similar wounds and also evaluated. During the first week of healing, the unmod-IPN and conventional dressing wound showed a greater amount of contraction than that of RGD-IPN+KGF. However, by 3 weeks the extent of wound contraction was comparable between treatments. The RGD-IPN+KGF treated wound demonstrated lower macrophage and fibroblast densities at 3 weeks as compared to unmod-IPN treated wounds. RGD-IPN+KGF acted as a tissue scaffold while preventing the entry of foreign bodies, advantages not seen with the conventional dressing. The extent of cellularity and extracellular matrix organization was higher for wounds healed with RGD-IPN+KGF than those healed with unmod-IPN. These results indicate that both soluble and immobilized bioactive factors can be incorporated into our IPN platform to enhance the rate and the quality of dermal wound healing.

We synthesized gelatin-based, interpenetrating network (IPN) scaffolds immobilized with fibronectin (FN)-derived peptides to assess monocyte-biomaterial interaction. Human primary monocytes were seeded onto peptide-grafted IPN or tissue-culture polystyrene (TCPS) pre-adsorbed with FN or FN-derived peptides. Monocyte cell density on both TCPS and IPN surfaces was higher in the presence of the arginine-glycine-aspartic acid (RGD) peptide. Pretreatment with anti-integrin beta1 or beta3 antibody decreased monocyte density on all ligand-modified TCPS and IPN. Interleukin-1 beta (IL-1beta) protein levels of cells on modified TCPS decreased over time. IL-1beta expression of monocytes in the presence of IPNs peaked at 24 h and then decreased through 168 h. Ligand identity did not affect IL-1beta expression in either TCPS or IPN samples. Pretreatment with anti-integrin beta1 or beta3 antibody reduced IL-1beta levels from both TCPS and IPN samples in a ligand-independent manner, particularly at 24 h. Monocytic IL-1beta mRNA expression in IPN samples without antibody pretreatment was highest at 2 h and decreased over time. IL-1beta mRNA expression in cells with anti-integrin beta1 or beta3 antibody pretreatment was similar to those without antibody pretreatment, except for methoxygrafted IPN samples. The change in IL-1beta mRNA expression did not correlate with changes in protein expression. The results indicate that monocyte adhesion was affected by the substrate and the RGD sequence and beta1 or beta3 containing integrin receptors. beta1- or beta3-containing integrin receptors were also involved in IL-1beta gene and protein expression in monocytes adhered to gelatin-based biomaterial surfaces.

Monocytes/macrophages and fibroblasts are recruited to the injury site and orchestrate the host response and tissue repair. We have previously shown that polyethylene glycol (PEG)-ylated arginine-glycine-aspartic acid (RGD) sequence grafted onto an extracellular matrix (ECM)-based semi-interpenetrating network (sIPN) enhances monocyte adhesion, and modulates subsequent gene expression and release of inflammatory and matrix remodeling factors. In this study, we investigate the direct influence of fibroblasts on monocyte response to this ECM mimic. Key wound-healing factors in inflammation, matrix remodeling, and regeneration were analyzed to gain insight into the interrelated role of regulation in fibroblast-monocyte interaction. Interleukin-1alpha/-1beta (IL-1alpha/-1beta), interleukin-6 (IL-6), tumor necrosis factor- alpha (TNF-alpha), monocyte inflammatory protein-1alpha/-1beta (MIP-1alpha/-1beta), transforming growth factor-alpha (TGF-alpha), monocyte chemoattractant factor (MCP-1), matrix metalloproteinase-2/-9 (MMP-2/-9), vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) were analyzed. Fibroblasts decreased monocyte adhesion onto the RGD-grafted sIPN while increasing monocyte GM-CSF on all surfaces over time except for on RGD and PHSRN-grafted sIPN at 96 h. Monocytes decreased initial fibroblast IL-1alpha and TGF-alpha, but drastically increased fibroblast MMP-2 and GM-CSF. Monocyte IL-1beta, TNF-alpha, MIP-1beta, MCP-1, MMP-9, and GM-CSF expression was increased over time in the presence of all sIPNs, and when the sIPNs were immobilized with ligands, a down-regulation of fibroblast IL-1beta, MIP-1alpha, MIP-1beta compared with unmodified sIPN was observed. When the ligand immobilized was RGD, monocyte TGF-alpha, MIP-1beta, and VEGF expression was increased while monocyte GM-CSF was decreased at selected time points. These results showed a dynamic monocyte response to selected ECM components in the presence of fibroblasts.

Ligands presented on biomaterials are a common method to facilitate and control the host response. In a gelatin and polyethylene glycol diacrylate (PEGdA) based semi-interpenetrating network (sIPN), the effects of extracellular matrix (ECM)-derived peptide amount on monocyte adhesion and subsequent protein and mRNA expression were examined. Peptide amount on the sIPN surface was controlled by varying the wt % ratio of the peptide-PEG grafted gelatin to PEGdA. We hypothesized that increasing bioactive peptide amount would modulate human blood-derived monocyte adhesion, cytokine expression, and gene regulation. Monocyte adhesion, release of gelatin degrading proteases matrix metalloprotease-2 (MMP-2), matrix metalloprotease-9 (MMP-9), and proinflammatory protein interleukin-1beta (IL-1beta), and mRNA expression of these proteins were evaluated. We found RGD-PEG grafted sIPNs with higher surface RGD concentrations showed increased adherent density. MMP-2 and IL-1beta protein release was also influenced by the ligand concentration, as initial increase in protein concentration was observed at higher ligand concentrations. MMP-9 protein showed an initial increase that subsided then increased. A decreased IL-1beta protein and mRNA expression was observed over time but MMP-2 mRNA was not detected at any time though MMP-2 protein concentrations showed an initial burst. Hence, monocyte behavior was modulated by surface ligand identity in tandem with ligand concentration.

In this study we synthesized gelatin-based, tissue-engineering, interpenetrating network (IPN) scaffolds immobilized with fibronectin (FN)-derived peptides to assess monocyte-biomaterial interaction. Human promonocytic U937 cells were seeded onto peptide-grafted IPN or tissue-culture polystyrene plate (TCPS) pre-adsorbed with FN or FN-derived peptides. The presence of RGD influenced U937 density on IPN. Interleukin-1 beta (IL-1beta) messenger ribonucleic acid (mRNA) expression in adherent U937 on treated TCPS was slightly upregulated at 4 h. Tumor necrosis factor alpha (TNF-alpha) and IL-1beta mRNA expression in adherent U937 on all IPNs was generally downregulated at 4 h. This downregulation of IL-1beta mRNA apparently varied in IPNs grafted with different ligand and was still present at 24 h. TNF-alpha and IL-1beta proteins released from U937 on treated TCPS were comparable with the control at 24 h, but TNF-alpha and IL-1beta protein expression in U937 on IPNs was lower at 24 h than on the TCPS control. The results indicate that the tissue-engineering substrate and the bioactive peptides modulate the initial U937 adhesion and the subsequent inflammatory cytokine gene and protein expression.

Macrophages are commonly observed at the biomaterial-tissue interface and interact with the extracellular matrix (ECM) mainly by integrin receptors to play a critical role in ECM turnover by secreting matrix metalloproteinases (MMPs). To investigate beta1 and beta3 containing integrin-mediated adhesion and subsequent MMP-2/-9 protein and gene expression in human blood-derived monocytes, biofunctional peptides immobilized onto flexible polyethylene glycol (PEG) arms were grafted onto a gelatin-based interpenetrating network (IPN). Adherent monocyte density was dramatically greater in the presence of RGD immobilized onto flexible PEG arms of the gelatin-based IPN. Pretreatment of monocytes with either anti-integrin beta1 or beta3 led to a significant decrease in adherent cell density on RGD-PEG-grafted IPNs. MMP-2 and MMP-9 protein and MMP-9 mRNA expression increased in the presence of IPNs initially, independent of ligand identity. Anti-integrin beta1 or beta3 antibody pretreatment of monocytes led to a general decrease in MMP-2/-9 protein expression. These results demonstrate the importance of beta1 and beta3 containing integrins in mediating monocyte adhesion onto RGD immobilized onto flexible PEG arms of the IPN. The results also reveal that MMP-2/-9 protein and gene expression is influenced by the presence of gelatin and not the ligands immobilized on the PEG arms of the IPN.