Multi-peptide presentation and hydrogel mechanics jointly enhance therapeutic duo-potential of entrapped stromal cells.

The native extracellular matrix (ECM) contains a host of matricellular proteins and bioactive factors that regulate cell behavior, and many ECM components have been leveraged to guide cell fate. However, the large size and chemical characteristics of these constituents complicate their incorporation into biomaterials without interfering with material properties, motivating the need for alternative approaches to regulate cellular responses. Mesenchymal stromal cells (MSCs) can promote osseous regeneration in vivo directly or indirectly through multiple means including (1) secretion of proangiogenic and mitogenic factors to initiate formation of a vascular template and recruit host cells into the tissue site or (2) direct differentiation into osteoblasts. As MSC behavior is influenced by the properties of engineered hydrogels, we hypothesized that the biochemical and biophysical properties of alginate could be manipulated to promote the dual contributions of encapsulated MSCs toward bone formation. We functionalized alginate with QK peptide to enhance proangiogenic factor secretion and RGD to promote adhesion, while biomechanical-mediated osteogenic cues were controlled by modulating viscoelastic properties of the alginate substrate. A 1:1 ratio of QK:RGD resulted in the highest levels of both proangiogenic factor secretion and mineralization in vitro. Viscoelastic alginate outperformed purely elastic gels in both categories, and this effect was enhanced by stiffness up to 20 kPa. Furthermore, viscoelastic constructs promoted vessel infiltration and bone regeneration in a rat calvarial defect over 12 weeks. These data suggest that modulating viscoelastic properties of biomaterials, in conjunction with dual peptide functionalization, can simultaneously enhance multiple aspects of MSC regenerative potential and improve neovascularization of engineered tissues.

Delivery of anti-inflammatory peptides from hollow PEGylated poly(NIPAM) nanoparticles reduces inflammation in an ex vivo osteoarthritis model.

Targeted delivery of anti-inflammatory osteoarthritis treatments have the potential to significantly decrease undesirable systemic side effects and reduce required therapeutic dosage. Here we present a targeted, non-invasive drug delivery system to decrease inflammation in an osteoarthritis model. Hollow thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) nanoparticles have been synthesized via degradation of a N,N'-bis(acryloyl)cystamine (BAC) cross-linked core out of a non-degradable pNIPAM shell. Sulfated 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) was copolymerized in the shell to increase passive loading of an anti-inflammatory mitogen-activated protein kinase-activated protein kinase 2 (MK2)-inhibiting cell-penetrating peptide (KAFAK). The drug-loaded hollow nanoparticles were effective at delivering a therapeutically active dose of KAFAK to bovine cartilage explants, suppressing pro-inflammatory interleukin-6 (IL-6) expression after interleukin-1 beta (IL-1ß) stimulation. This thermosensitive hollow nanoparticle system provides an excellent platform for the delivery of peptide therapeutics into highly proteolytic environments such as osteoarthritis.

Controlled release of anti-inflammatory peptides from reducible thermosensitive nanoparticles suppresses cartilage inflammation.

Characterized by pain, cartilage degradation, and inflammation, osteoarthritis is often treated with anti-inflammatory therapies that provide short-term relief but can have adverse side effects; intra-articular drug delivery systems with controlled release of anti-inflammatory peptides using degradable poly(N-isopropylacrylamide) (pNIPAM) nanoparticles could prolong relief and minimize these side effects. Nanoparticles provide a biocompatible drug carrier that can protect encapsulated therapeutics from enzymatic degradation and increase payload delivery upon encountering a degradation stimulus. Here we demonstrate passive targeting of inflamed cartilage ex vivo by uptake of PEGylated pNIPAM nanoparticles with degradable disulfide crosslinks (abbreviated as NGPEGSS) into chondrocytes and subsequent intracellular release of an anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK). The KAFAK-loaded NGPEGSS treatment reduced ex vivo inflammation to a greater extent compared to its non-degradable counterparts. This study highlights a nanoparticle system that delivers therapeutics intracellularly with improved efficacy by triggered degradation and suppresses inflammation in multiple cell types within an inflamed joint.

Imaging of small animal peripheral artery disease models: recent advancements and translational potential.

Peripheral artery disease (PAD) is a broad disorder encompassing multiple forms of arterial disease outside of the heart. As such, PAD development is a multifactorial process with a variety of manifestations. For example, aneurysms are pathological expansions of an artery that can lead to rupture, while ischemic atherosclerosis reduces blood flow, increasing the risk of claudication, poor wound healing, limb amputation, and stroke. Current PAD treatment is often ineffective or associated with serious risks, largely because these disorders are commonly undiagnosed or misdiagnosed. Active areas of research are focused on detecting and characterizing deleterious arterial changes at early stages using non-invasive imaging strategies, such as ultrasound, as well as emerging technologies like photoacoustic imaging. Earlier disease detection and characterization could improve interventional strategies, leading to better prognosis in PAD patients. While rodents are being used to investigate PAD pathophysiology, imaging of these animal models has been underutilized. This review focuses on structural and molecular information and disease progression revealed by recent imaging efforts of aortic, cerebral, and peripheral vascular disease models in mice, rats, and rabbits. Effective translation to humans involves better understanding of underlying PAD pathophysiology to develop novel therapeutics and apply non-invasive imaging techniques in the clinic.

Release of anti-inflammatory peptides from thermosensitive nanoparticles with degradable cross-links suppresses pro-inflammatory cytokine production.

Pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) are mediators in the development of many inflammatory diseases. To demonstrate that macrophages take up and respond to thermosensitive nanoparticle drug carriers, we synthesized PEGylated poly(N-isopropylacrylamide-2-acrylamido-2-methyl-1-propanesulfonate) particles cross-linked with degradable disulfide (N,N'-bis(acryloyl)cystamine) (NGPEGSS). An anti-inflammatory peptide (KAFAK) was loaded and released from the thermosensitive nanoparticles and shown to suppress levels of TNF-α and IL-6 production in macrophages. Cellular uptake of fluorescent, thermosensitive, and degradable nanoparticles and therapeutic efficacy of free KAFAK peptide compared to that of KAFAK loaded in PEGylated degradable thermosensitive nanoparticles were examined. The data suggests that the degradable, thermosensitive nanoparticles loaded with KAFAK may be an effective tool to treat inflammatory diseases.

Thermo-responsive poly(N-isopropylacrylamide) grafted onto microtextured poly(dimethylsiloxane) for aligned cell sheet engineering.

Poly(N-isopropylacrylamide) (PNIPAAm)-grafted poly(dimethylsiloxane) (PDMS) offers an inexpensive, biocompatible, oxygen permeable, and easily microtextured thermo-responsive substrate for producing cell sheets. This study introduces a method of grafting PNIPAAm onto microtextured PDMS that is suitable for generating aligned vascular smooth muscle cell (VSMC) sheets. We examined a wide range of processing parameters in order to identify the conditions that led to acceptable sheet growth and detachment behavior. Substrates grafted under these conditions produced confluent cell sheets that fully detached in less than 10 min after lowering the culture temperature from 37 °C to 20 °C. The grafted layer thickness was determined to be 496±8 nm by atomic force microscopy. Surface characterization by Fourier transform infrared spectroscopy showed a relative grafting yield of 0.488±0.10, defined as the ratio of the PNIPAAm 1647 cm(-1) to the PDMS 2962 cm(-1) absorbance peaks. The water contact angle of the substrates was shown to change from 89.6° to 101.0° at 20 °C and 37 °C, respectively. We also found that cell behavior on PNIPAAm-grafted PDMS was not directly related to surface wettability or relative grafting densities.