Search

Tendon healing is a complex coordinated series of events resulting in protracted recovery, limited regeneration, and scar formation. Mesenchymal stem cell (MSC) therapy has shown promise as a new technology to enhance soft tissue and bone healing. A challenge with MSC therapy involves the ability to consistently control the inflammatory response and subsequent healing. Previous studies suggest that preconditioning MSCs with inflammatory cytokines, such as IFN-γ, TNF-α, and IL-1β may accelerate cutaneous wound closure. The objective of this study was to therefore elucidate these effects in tendon. That is, the in vivo healing effects of TNF-α primed MSCs were studied using a rat Achilles segmental defect model. Rat Achilles tendons were subjected to a unilateral 3 mm segmental defect and repaired with either a PLG scaffold alone, MSC-seeded PLG scaffold, or TNF-α-primed MSC-seeded PLG scaffold. Achilles tendons were analyzed at 2 and 4 weeks post-injury. In vivo, MSCs, regardless of priming, increased IL-10 production and reduced the inflammatory factor, IL-1α. Primed MSCs reduced IL-12 production and the number of M1 macrophages, as well as increased the percent of M2 macrophages, and synthesis of the anti-inflammatory factor IL-4. Primed MSC treatment also increased the concentration of type I procollagen in the healing tissue and increased failure stress of the tendon 4 weeks post-injury. Taken together delivery of TNF-α primed MSCs via 3D PLG scaffold modulated macrophage polarization and cytokine production to further accentuate the more regenerative MSC-induced healing response. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:269-280, 2017.

Here, we describe an in vitro strategy to model vascular morphogenesis where human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are encapsulated in peptide-functionalized poly(ethylene glycol) (PEG) hydrogels, either on standard well plates or within a passive pumping polydimethylsiloxane (PDMS) tri-channel microfluidic device. PEG hydrogels permissive towards cellular remodeling were fabricated using thiol-ene photopolymerization to incorporate matrix metalloproteinase (MMP)-degradable crosslinks and CRGDS cell adhesion peptide. Time lapse microscopy, immunofluorescence imaging, and RNA sequencing (RNA-Seq) demonstrated that iPSC-ECs formed vascular networks through mechanisms that were consistent with in vivo vasculogenesis and angiogenesis when cultured in PEG hydrogels. Migrating iPSC-ECs condensed into clusters, elongated into tubules, and formed polygonal networks through sprouting. Genes upregulated for iPSC-ECs cultured in PEG hydrogels relative to control cells on tissue culture polystyrene (TCP) surfaces included adhesion, matrix remodeling, and Notch signaling pathway genes relevant to in vivo vascular development. Vascular networks with lumens were stable for at least 14 days when iPSC-ECs were encapsulated in PEG hydrogels that were polymerized within the central channel of the microfluidic device. Therefore, iPSC-ECs cultured in peptide-functionalized PEG hydrogels offer a defined platform for investigating vascular morphogenesis in vitro using both standard and microfluidic formats. Statement of Significance Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) cultured in synthetic hydrogels self-assemble into capillary networks through mechanisms consistent with in vivo vascular morphogenesis.

Here, we have developed a novel method for forming hydrogel arrays using surfaces patterned with differential wettability. Our method for benchtop array formation is suitable for enhanced-throughput, combinatorial screening of biochemical and biophysical cues from chemically defined cell culture substrates. We demonstrated the ability to generate these arrays without the need for liquid handling systems and screened the combinatorial effects of substrate stiffness and immobilized cell adhesion peptide concentration on human mesenchymal stem cell (hMSC) behavior during short-term 2-dimensional cell culture. Regardless of substrate stiffness, hMSC initial cell attachment, spreading, and proliferation were linearly correlated with immobilized CRGDS peptide concentration. Increasing substrate stiffness also resulted in increased hMSC initial cell attachment, spreading, and proliferation; however, examination of the combinatorial effects of CRGDS peptide concentration and substrate stiffness revealed potential interplay between these distinct substrate signals. Maximal hMSC proliferation seen on substrates with either high stiffness or high CRGDS peptide concentration suggests that some baseline level of cytoskeletal tension was required for hMSC proliferation on hydrogel substrates and that multiple substrate signals could be engineered to work in synergy to promote mechanosensing and regulate cell behavior. Statement of significance Our novel array formation method using surfaces patterned with differential wettability offers the advantages of benchtop array formation for 2-dimensional cell cultures and enhanced-throughput screening without the need for liquid handling systems. Hydrogel arrays formed via our method are suitable for screening the influence of chemical (e.g. cell adhesive ligands) and physical (stiffness, size, shape, and thickness) substrate properties on stem cell behavior. The arrays are also fully compatible with commercially available micro-array add-on systems, which allows for simultaneous control of the insoluble and soluble cell culture environment. This study used hydrogel arrays to demonstrate that synergy between cell adhesion and mechanosensing can be used to regulate hMSC behavior.

Because of the dynamics of spinal cord injury (SCI), the optimal treatment will almost certainly be a combination approach to control the environment and promote axonal growth. This study uses peripheral nerve grafts (PNGs) as scaffolds for axonal growth while delivering neurotrophin-3 (NT-3) via calcium phosphate (CaP) coatings on surgical sutures. CaP coating was grown on sutures, and NT-3 binding and release were characterized in vitro. Then, the NT-3-loaded sutures were tested in a complete SCI model. Rats were analyzed for functional improvement and axonal growth into the grafts. The CaP-coated sutures exhibited a burst release of NT-3, followed by a sustained release for at least 20 days. Functionally, the rats with PNGs + NT-3-loaded sutures and the rats treated with PNGs scored significantly higher than controls on day 56 postoperatively. However, functional scores in rats treated with PNGs + NT-3-loaded suture were not significantly different from those of rats treated with PNGs alone. Cholera toxin subunit B (CTB) labeling rostral to the graft was not observed in any controls, but CTB labeling rostral to the graft was observed in almost all rats that had had a PNG. Neurofilament labeling on transverse sections of the graft revealed that the rats treated with the NT-3-loaded sutures had significantly more axons per graft than rats treated with an NT-3 injection and rats without NT-3. These data demonstrate that PNGs serve as scaffolds for axonal growth after SCI and that CaP-coated sutures can efficiently release NT-3 to increase axonal regeneration. © 2016 Wiley Periodicals, Inc.

The gold standard for treating peripheral nerve injuries that have large nerve gaps where the nerves cannot be directly sutured back together because it creates tension on the nerve, is to incorporate an autologous nerve graft. However, even with the incorporation of a nerve graft, generally patients only regain a small portion of function in limbs affected by the injury. Although, there has been some promising results using growth factors to induce more axon growth through the nerve graft, many of these previous therapies are limited in their ability to release growth factors in a sustained manner and tailor them to a desired time frame. The ideal drug delivery platform would deliver growth factors at therapeutic levels for enough time to grow axons the entire length of the nerve graft. We hypothesized that mineral coated microparticles (MCMs) would bind, stabilize and release biologically active glial cell-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in a sustained manner. Therefore, the objective of this study was to test the ability of MCMs releasing growth factors at the distal end of a 10 mm sciatic nerve graft, to induce axon growth through the nerve graft and restore hind limb function. After sciatic nerve grafting in Lewis rats, the hind limb function was tested weekly by measuring the angle of the ankle at toe lift-off while walking down a track. Twelve weeks after grafting, the grafts were harvested and myelinated axons were analyzed proximal to the graft, in the center of the graft, and distal to the graft. Under physiological conditions in vitro, the MCMs delivered a burst release of NGF and GDNF for 3 days followed by a sustained release for at least 22 days. In vivo, MCMs releasing NGF and GDNF at the distal end of sciatic nerve grafts resulted in significantly more myelinated axons extending distal to the graft when compared to rats that received nerve grafts without growth factor treatment. The rats with nerve grafts incorporated with MCMs releasing NGF and GDNF also showed significant improvement in hind limb function starting at 7 weeks postoperatively and continuing through 12 weeks postoperatively when compared to rats that received nerve grafts without growth factor treatment. In conclusion, MCMs released biologically active NGF and GDNF in a sustained manner, which significantly enhanced axon growth resulting in a significant improvement of hind limb function in rats. The animal experiments were approved by University of Wisconsin-Madison Animal Care and Use Committee (ACUC, protocol# M5958) on January 3, 2018.

Translation of human pluripotent stem cell (hPSC)-derived therapies to the clinic demands scalable, cost-effective methods for cell expansion. Culture media currently used for hPSC expansion rely on high concentrations and frequent supplementation of recombinant growth factors due to their short half-life at physiological temperatures. Here, we developed a biomaterial strategy using mineral-coated microparticles (MCMs) to sustain delivery of basic fibroblast growth factor (bFGF), a thermolabile protein critical for hPSC pluripotency and proliferation. We show that the MCMs stabilize bFGF against thermally induced activity loss and provide more efficient sustained release of active growth factor compared to polymeric carriers commonly used for growth factor delivery. Using a statistically driven optimization approach called Design of Experiments, we generated a bFGF-loaded MCM formulation that supported hPSC expansion over 25 passages without the need for additional bFGF supplementation to the media, resulting in greater than 80% reduction in bFGF usage compared to standard approaches. This materials-based strategy to stabilize and sustain delivery of a thermolabile growth factor has broad potential to reduce costs associated with recombinant protein supplements in scalable biomanufacturing of emerging cell therapies.

DNA aptamers are integrated into synthetic hydrogel networks with the aim of creating hydrogels that undergo volume changes when exposed to target molecules. Specifically, single-stranded DNA aptamers in cDNA-bound, extended state are incorporated into hydrogel networks as cross-links, so that the nanoscale conformational change of DNA aptamers upon binding to target molecules will induce macroscopic volume decreases of hydrogels. Hydrogels incorporating adenosine triphosphate (ATP)-binding aptamers undergo controllable volume decreases of up to 40.3 ± 4.6% when exposed to ATP, depending on the concentration of DNA aptamers incorporated in the hydrogel network, temperature, and target molecule concentration. Importantly, this approach can be generalized to aptamer sequences with distinct binding targets, as demonstrated here that hydrogels incorporating an insulin-binding aptamer undergo volume changes in response to soluble insulin. This work provides an example of bioinspired hydrogels that undergo macroscopic volume changes that stem from conformational shifts in resident DNA-based cross-links.

Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes (iHEP) offer an attractive alternative to primary human hepatocytes (PHH) for drug toxicity studies, as PHHs are limited in supply, vary in their metabolic activity between donors, and rapidly lose their functionality in vitro. However, one of the major drawbacks with iHEP cells in drug safety studies is their decreased phenotypic maturity, with lower liver specific enzyme activity compared with that of PHH. Here we evaluated the effects of 3D culture and non-parenchymal cells on the maturation of iHEPs. We describe a serum-free, chemically defined 3D in vitro model using iHEP cells, which is compatible with automation and conventional assay plates. The iHEP cells cultured in this model form polarized aggregates with functional bile canaliculi and strongly increased expression of albumin, urea and genes encoding phase I and II drug metabolism enzymes and bile transporters. Cytochrome P450-mediated metabolism is significantly higher in 3D iHEP aggregates compared to 2D iHEP culture. Furthermore, addition of human liver sinusoidal endothelial cells (sECs) and iPS-derived endothelial cells (iECs) improved mature hepatocyte function and CYP450 enzyme activity. Also, ECs formed endothelial networks within the hepatic 3D cultures, mimicking aspects of an in vivo architecture. Collectively, these results suggest that the iHEP/EC aggregates described here may have the potential to be used for many applications, including as an in vitro model to study liver diseases associated with sinusoidal endothelial cells. STATEMENT OF SIGNIFICANCE: iPS-derived hepatocytes provide an inexhaustible source of cells for drug screening, toxicology studies and cell-based therapies, but lack mature phenotype of adult primary human hepatocytes (PHH). Herein, we show that 3D culture of iPS-derived hepatocytes and their co-culture with human sinusoidal endothelial cells (sECs) to improve their maturity.

Vascularization is an important strategy to overcome diffusion limits and enable the formation of complex, physiologically relevant engineered tissues and organoids. Self-assembly is a technique to generate in vitro vascular networks, but engineering the necessary network morphology and function remains challenging. Here, autofluorescence multiphoton microscopy (aMPM), a label-free imaging technique, is used to quantitatively evaluate in vitro vascular network morphology. Vascular networks are generated using human embryonic stem cell–derived endothelial cells and primary human pericytes encapsulated in synthetic poly(ethylene glycol)-based hydrogels. Two custom-built bioreactors are used to generate distinct fluid flow patterns during vascular network formation: recirculating flow or continuous flow. aMPM is used to image these 3D vascular networks without the need for fixation, labels, or dyes. Image processing and analysis algorithms are developed to extract quantitative morphological parameters from these label-free images. It is observed with aMPM that both bioreactors promote formation of vascular networks with lower network anisotropy compared to static conditions, and the continuous flow bioreactor induces more branch points compared to static conditions. Importantly, these results agree with trends observed with immunocytochemistry. These studies demonstrate that aMPM allows label-free monitoring of vascular network morphology to streamline optimization of growth conditions and provide quality control of engineered tissues., National Institutes of Health (U.S.) (Grant R01 HL093282-01A1), National Institutes of Health (U.S.) (Grant 1U H2TR000506-01), National Institutes of Health (U.S.) (Grant 3UH2TR000506-02S1), National Institutes of Health (U.S.) (Grant 4U H3TR000506-03), National Institutes of Health (U.S.) (Grant R01CA205101), National Institutes of Health (U.S.) (Grant R01CA185747), National Institutes of Health (U.S.) (Grant R01CA211082), National Institutes of Health (U.S.) (Grant R01CA226526), National Science Foundation (U.S.) (Grant (CBET-1642287), Entertainment Industry Foundation. Stand Up to Cancer Colorectal Cancer Dream Team (Grant SU2C-AACR-IG-08-16), Stand Up To Cancer (Grant SU2C-AACR-IG-08-16)

Mesenchymal stem cells (MSC), also called marrow stromal cells, are adult cells that have attracted interest for their potential uses in therapeutic applications. There is a pressing need for scalable culture systems due to the large number of cells needed for clinical treatments. Here, a tailorable thin polymer coating-poly(poly(ethylene glycol) methyl ether methacrylate-ran-vinyl dimethyl azlactone-ran-glycidyl methacrylate) [P(PEGMEMA-r-VDM-r-GMA); PVG]-to the surface of commercially available polystyrene and glass microcarriers to create chemically defined surfaces for large-scale cell expansion is applied. These chemically defined microcarriers create a reproducible surface that does not rely on the adsorption of xenogenic serum proteins to mediate cell adhesion. Specifically, this coating method anchors PVG copolymer through ring opening nucleophilic attack by amine residues on poly-l-lysine that is pre-adsorbed to the surface of microcarriers. Importantly, this anchoring reaction preserves the monomer VDM reactivity for subsequent functionalization with an integrin-specific Arg-Gly-Asp peptide to enable cell adhesion and expansion via a one-step reaction in aqueous media. MSCs cultured on PVG-coated microcarriers achieve sixfold expansion-similar to the expansion achieved on PS microcarriers-and retain their ability to differentiate after harvesting.

Engineered tissues offer great promise as engrafted therapies and in vitro models, but these tissues require a vascular network to retain viability at large scales. Significant efforts are focused on optimizing these in vitro vascular constructs, yet current evaluation methods require fixation and immunostaining. These destructive evaluation methods alter vascular network morphology, and cannot non-invasively monitor vascular assembly over time. Here, we demonstrate that autofluorescence multiphoton microscopy (MPM) can quantitatively assess the morphology of living 3D vascular networks without fixation, labels, or dyes. Autofluorescence MPM was used to non-invasively monitor the effect of culture conditions on 3D vascular network formation. Human embryonic stem (ES) cell-derived endothelial cells and primary human pericytes cultured in polyethylene glycol (PEG) hydrogels self-assembled into 3D vascular networks. Autofluorescence MPM of the metabolic co-enzyme NAD(P)H (excitation/emission wavelengths of 750 nm/400-460 nm) was used to quantify morphological parameters at day 6 of culture. Specifically, vessel diameter, vascular density, branch point density, and integration of endothelial cells into the network were quantified. Dynamic culture conditions (flow at 1μL/sec) led to vascular networks with higher mean vessel diameter compared to static culture (p

The inhibition of angiogenesis is a critical element of cancer therapy, as cancer vasculature contributes to tumor expansion. While numerous drugs have proven to be effective at disrupting cancer vasculature, patient survival has not significantly improved as a result of anti-angiogenic drug treatment. Emerging evidence suggests that this is due to a combination of unintended side effects resulting from the application of anti-angiogenic compounds, including angiogenic rebound after treatment and the activation of metastasis in the tumor. There is currently a need to better understand the far-reaching effects of anti-angiogenic drug treatments in the context of cancer. Numerous innovations and discoveries in biomaterials design and tissue engineering techniques are providing investigators with tools to develop physiologically relevant vascular models and gain insights into the holistic impact of drug treatments on tumors. This review examines recent advances in the design of pro-angiogenic biomaterials, specifically in controlling integrin-mediated cell adhesion, growth factor signaling, mechanical properties and oxygen tension, as well as the implementation of pro-angiogenic materials into sophisticated co-culture models of cancer vasculature.

Since hydroxyapatite and bone morphogenetic protein-2 (BMP-2) can regulate chondrogenesis and osteogenesis, their individual and combined effects on endochondral ossification within human bone marrow-derived stem cell (hMSC) aggregates were investigated. Hydroxyapatite was presented in the form of mineral-coated hydroxyapatite microparticles (MCM) capable of controlled BMP-2 delivery. Aggregates were treated with varied BMP-2 concentrations supplemented in the media and loaded onto MCM to examine the influence of BMP-2 amount and spatial presentation on regulating chondrogenesis and osteogenesis. MCM alone induced GAG and type II collagen production by week 5 for two of three donors, and BMP-2 may have accelerated MCM-induced chondrogenesis. ALP activity and calcium content of cells-only aggregates suggest that the BMP-2-induced osteogenic response may be concentration-dependent. Treatment with MCM and BMP-2 resulted in chondrogenesis as early as week 2, which may have promoted additional mineralization by week 5, suggesting the induction of endochondral ossification. Released BMP-2 had similar if not higher levels of bioactivity compared to that of exogenous BMP-2 with regard to chondrogenesis and osteogenesis. In addition to providing localized and sustained BMP-2 delivery, MCM incorporation within aggregates yields a self-sustaining system that may be injected or implanted more rapidly to heal bone defects through endochondral ossification without extended

The purpose of this study was to assess the potential for ultrasound shear wave elastography (SWE) to assess tissue elasticity and ultimate stress in both intact and healing tendons. The lateral gastrocnemius (Achilles) tendons of 41 New Zealand white rabbits were surgically severed and repaired with growth factor coated sutures. SWE imaging was used to measure shear wave speed (SWS) in both the medial and lateral tendons pre-surgery, and at 2 and 4 weeks post-surgery. Rabbits were euthanized at 4 weeks, and both medial and lateral tendons underwent mechanical testing to failure. SWS significantly (p

Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial.

Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues ("α/β-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

Scanning electron microscopy (SEM) was used to examine the external morphology of the head, legs, and postabdomen of adults of the Nearctic snailkilling fly Sepedon fuscipennis Loew (Diptera: Sciomyzidae). For both sexes, details are provided of the sensilla on the antenna, which has three sacculi on the first flagellomere. The number and arrangement of mechanosensilla on the aristae show sexual dimorphism. The black parafrontal spots are covered with microtrichia. Interfacetal mechanoreceptors were not numerous on the compound eyes, indication that sciomyzids are not major pollinators. Labellar hooks are evident in both sexes. A discussion is provided of various proposed functions. The shape of the head, especially the concave area housing the retractedmouthparts, suggests a novel explanation for the absence of a ptilinum in the genus Sepedon Latreille.

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF 165 -induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

Tendon healing is a complex coordinated event orchestrated by numerous biologically active proteins. Unfortunately, tendons have limited regenerative potential and as a result, repair may be protracted months to years. Current treatment strategies do not offer localized delivery of biologically active proteins, which may result in reduced therapeutic efficacy. Surgical sutures coated with nanostructured minerals may provide a potentially universal tool to efficiently incorporate and deliver biologically active proteins directly to the wound. Additionally, previous reports indicated that treatment with bone morphogenetic protein-12 (BMP-12) improved tendon healing. Based on this information, we hypothesized that mineral-coated surgical sutures may be an effective platform for localized BMP-12 delivery to an injured tendon. The objective of this study was, therefore, to elucidate the healing effects of mineral-coated sutures releasing BMP-12 using a rat Achilles healing model. The effects of BMP-12-releasing sutures were also compared with standard BMP-12 delivery methods, including delivery of BMP-12 through collagen sponge or direct injection. Rat Achilles tendons were unilaterally transected and repaired using BMP-12-releasing suture (0, 0.15, 1.5, or 3.0 μg), collagen sponge (0 or 1.5 μg BMP-12), or direct injection (0 or 1.5 μg). By 14 days postinjury, repair with BMP-12-releasing sutures reduced the appearance of adhesions to the tendon and decreased total cell numbers. BMP-12 released from sutures and collagen sponge also tended to improve collagen organization when compared with BMP-12 delivered through injection. Based on these results, the release of a protein from sutures was able to elicit a biological response. Furthermore, BMP-12-releasing sutures modulated tendon healing, and the delivery method dictated the response of the healing tissue to BMP-12.

Stem cell-derived organoids and other 3D microtissues offer enormous potential as models for drug screening, disease modeling, and regenerative medicine. Formation of stem/progenitor cell aggregates is common in biomanufacturing processes and critical to many organoid approaches. However, reproducibility of current protocols is limited by reliance on poorly controlled processes (e.g., spontaneous aggregation). Little is known about the effects of aggregation parameters on cell behavior, which may have implications for the production of cell aggregates and organoids. Here we introduce a bioengineered platform of labile substrate arrays that enable simple, scalable generation of cell aggregates via a controllable 2D-to-3D “self-assembly”. As a proof-of-concept, we show that labile substrates generate size- and shape-controlled embryoid bodies (EBs) and can be easily modified to control EB self-assembly kinetics. We show that aggregation method instructs EB lineage bias, with faster aggregation promoting pluripotency loss and ectoderm, and slower aggregation favoring mesoderm and endoderm. We also find that aggregation kinetics of EBs markedly influence EB structure, with slower kinetics resulting in increased EB porosity and growth factor signaling. Our findings suggest that controlling internal structure of cell aggregates by modifying aggregation kinetics is a potential strategy for improving 3D microtissue models for research and translational applications.

The physiological relevance of Matrigel as a cell-culture substrate and in angiogenesis assays is often called into question. Here, we describe an array-based method for the identification of synthetic hydrogels that promote the formation of robust in vitro vascular networks for the detection of putative vascular disruptors, and that support human embryonic stem cell expansion and pluripotency. We identified hydrogel substrates that promoted endothelial-network formation by primary human umbilical vein endothelial cells and by endothelial cells derived from human induced pluripotent stem cells, and used the hydrogels with endothelial networks to identify angiogenesis inhibitors. The synthetic hydrogels show superior sensitivity and reproducibility over Matrigel when evaluating known inhibitors, as well as in a blinded screen of a subset of 38 chemicals, selected according to predicted vascular disruption potential, from the Toxicity ForeCaster library of the US Environmental Protection Agency. The identified synthetic hydrogels should be suitable alternatives to Matrigel for common cell-culture applications.

Significance Generating fully functional arterial endothelial cells is a critical problem for vascular development and disease research. Currently, the arterial endothelial cells derived from human pluripotent stem cells lack the range of arterial-specific functions in vitro and the protective function for ischemic tissues in vivo. Here, we combine single-cell RNA sequencing and CRISPR-Cas9 technology to identify pathways for regulating arterial endothelial cell differentiation. We then manipulate these pathways and generate arterial endothelial cells that demonstrate unprecedented arterial-specific functions as well as improve survival of myocardial infarction. These findings facilitate the understanding of vascular development and disease and provide a source of cells that have broad applications for vascular disease modeling and regenerative medicine.

High-density mesenchymal stem cell (MSC) aggregates can be guided to form bone-like tissue via endochondral ossification in vitro when culture media is supplemented with proteins, such as growth factors (GFs), to first guide the formation of a cartilage template, followed by culture with hypertrophic factors. Recent reports have recapitulated these results through the controlled spatiotemporal delivery of chondrogenic transforming growth factor-β1 (TGF-β1) and chondrogenic and osteogenic bone morphogenetic protein-2 (BMP-2) from microparticles embedded within human MSC aggregates to avoid diffusion limitations and the lengthy, costly in vitro culture necessary with repeat exogenous supplementation. However, since GFs have limited stability, localized gene delivery is a promising alternative to the use of proteins. Here, mineral-coated hydroxyapatite microparticles (MCM) capable of localized delivery of Lipofectamine-plasmid DNA (pDNA) nanocomplexes encoding for TGF-β1 (pTGF-β1) and BMP-2 (pBMP-2) were incorporated, alone or in combination, within MSC aggregates from three healthy porcine donors to induce sustained production of these transgenes. Three donor populations were investigated in this work due to the noted MSC donor-to-donor variability in differentiation capacity documented in the literature. Delivery of pBMP-2 within Donor 1 aggregates promoted chondrogenesis at week 2, followed by an enhanced osteogenic phenotype at week 4. Donor 2 and 3 aggregates did not promote robust glycosaminoglycan (GAG) production at week 2, but by week 4, Donor 2 aggregates with pTGF-β1/pBMP-2 and Donor 3 aggregates with both unloaded MCM and pBMP-2 enhanced osteogenesis compared to controls. These results demonstrate the ability to promote osteogenesis in stem cell aggregates through controlled, non-viral gene delivery within the cell masses. These findings also indicate the need to screen donor MSC regenerative potential in response to gene transfer prior to clinical application. Taken together, this work demonstrates a promising gene therapy approach to control stem cell fate in biomimetic 3D condensations for treatment of bone defects.

Background: Efficient regeneration of denuded endothelial cells (ECs) is an important process that counteracts the pro-stenotic activities. Unfortunately, this natural healing process is frequently compromised by disease conditions such as diabetes. Protein kinase C-delta (PKCδ) plays a complex role in the arterial injury response by regulating apoptosis of vascular smooth muscle cells (SMCs) as well as production of chemokines capable of attracting resident and circulating cells. In this study, we explored whether SMCs participate in endothelial repair through a PKCδ-dependent paracrine mechanism. Methods and Results: Following balloon injury to the rat carotid, SMC-specific gene transfer of Prkcd accelerated reendothelialization compared to the empty vector, reflected by a larger area excluded from Evans blue measured 14 days post injury (59.60±5.01% vs 25.38±7.52%). In contrast, SMC-specific knockdown of endogenous Prkcd delayed reendothelialization compared to the non-targeting shRNA control (41.31±6.54% vs 70.31±5.97%). In vitro , media conditioned by AdPKCδ-infected SMCs increased endothelial wound healing without affecting their proliferation and viability. In addition, SMCs in a PKCδ-dependent fashion attracted circulating angiogenic cells (CACs), a cell population that promotes neovascularization via production of angiogenic factors. A PCR-based array analysis identified Cxcl1 and Cxcl7 among others as PKCδ-mediated chemokines produced by SMCs. Blocking CXCL7 or CXCR2 significantly inhibited endothelial wound healing and CAC migration in response to AdPKCδ-infected SMC conditioned media. In vivo , PKCδ overexpression in SMCs following balloon injury increased CXCL7 production and stimulated CACs recruitment to injured arteries. Furthermore, insertion of a Cxcl7 cDNA in the lentiviral vector that carries a Prkcd shRNA overcame the negative effects of Prkcd knockdown on reendothelialization. Conclusions: Regeneration of denuded endothelium involves multiple cell types from the vascular wall as well as circulation. SMCs stimulate reendothelialization in a PKCδ-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of ECs from uninjured endothelium and CACs from circulation.

In this study, we sought to improve ligament healing by modulating the inflammatory response after acute injury through the neutralization of Interleukin-17 (IL-17), which we hypothesized would decrease inflammatory cell infiltration and cytokine production. Administration of an Interleukin-17 neutralizing antibody (IL-17 NA) immediately following a rat medial collateral ligament (MCL) transection resulted in alterations in inflammatory cell populations and cytokine expression within the healing ligament, but did not reduce inflammation. Specifically, treatment resulted in a decrease in M2 (anti-inflammatory) macrophages, an increase in T cells, and an increase in the levels of IL-2, IL-6, and IL-12 in the MCL 7 days post injury. IL-17NA treatment, and subsequent immunomodulation, did not result in improved ligament healing, as measured by collagen composition and wound size.

Although calcium phosphate (CaP) binding peptides are used to link orthobiologics to orthopedic biomaterials, their binding stability in physiological environment is still unknown. In this study, we investigate the binding capability of a series of CaP-binding peptides and their binding stability in serum solutions, which are selected to resemble physiological conditions. The findings in this study may be applicable for designing robust orthobiologic delivery systems.

Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine.

To date, there is no periadventitial drug delivery method available in the clinic to prevent restenotic failure of open vascular reconstructions. Resveratrol is a promising anti-restenotic natural drug but subject to low bioavailability when systemically administered. In order to reconcile these two prominent issues, we tested effects of periadventitial delivery of resveratrol on all three major pro-restenotic pathologies including intimal hyperplasia (IH), endothelium impairment, and vessel shrinkage. In a rat carotid injury model, periadventitial delivery of resveratrol either via Pluronic gel (2-week), or polymer sheath (3-month), effectively reduced IH without causing endothelium impairment and vessel shrinkage. In an in vitro model, primary smooth muscle cells (SMCs) were stimulated with elevated transforming growth factor (TGFβ) and its signaling protein Smad3, known contributors to IH. TGFβ/Smad3 up-regulated Kruppel-like factor (KLF5) protein, and SMC de-differentiation which was reversed by KLF5 siRNA. Furthermore, TGFβ/Smad3-stimulated KLF5 production and SMC de-differentiation were blocked by resveratrol via its inhibition of the Akt-mTOR pathway. Concordantly, resveratrol attenuated Akt phosphorylation in injured arteries. Taken together, periadventitial delivery of resveratrol produces durable inhibition of all three pro-restenotic pathologies — a rare feat among existing anti-restenotic methods. Our study suggests a potential anti-restenotic modality of resveratrol application suitable for open surgery.

Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, "green" technology for regenerating large volume vascularized tissue mass.

Porousbiodegradable polymer scaffolds are widely utilized for bone tissue engineering, but are not osteoconductive like calcium phosphate scaffolds. We combine indirect solid freeform fabrication (SFF), ex vivo gene therapy, with biomineral coating to compare the effect of biomineral coating on bone regeneration for Poly (L-lactic acid) (PLLA) and Poly (ε-caprolactone) (PCL) scaffolds with the same porous architecture. Scanning electron microscope (SEM) and micro-computed tomography (μ-CT) demonstrate PLLA and PCL scaffolds have the same porous architecture and are completely coated. All scaffolds are seeded with human gingival fibroblasts (HGF) transduced with adenovirus encoded with either bone morphogenetic protein 7 (BMP-7) or green fluorescent protein (GFP), and implanted into mice subcutaneously for 3 and 10 weeks. Only scaffolds with BMP-7 transduced HGFs show mineralized tissue formation. At 3 weeks some blood vessel-like structures are observed in coated PLLA and PCL scaffolds, but there is no significant difference in bone ingrowth between the coated and uncoated scaffolds for either PLLA or PCL. At 10 weeks, however, coated scaffolds (both PLLA and PCL) have significantly more bone ingrowth than uncoated scaffolds, which have more fibrous tissue. Coated PLLA scaffolds have improved mechanical properties compared with uncoated PLLA scaffolds due to increased bone ingrowth.

Here we describe a strategy to model blood vessel development using a well-defined induced pluripotent stem cell-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats., National Institutes of Health (U.S.) (NIH 1UH2 TR000506-01), National Institutes of Health (U.S.) (3UH2 TR000506-02S1), National Institutes of Health (U.S.) (T32 HL007936-12), National Institutes of Health (U.S.) (RO1 HL093282), National Institutes of Health (U.S.) (R21 EB016381-01)

Vascular endothelial growth factor (VEGF) activity is highly regulated via sequestering within the ECM and cell-demanded proteolysis to release the sequestered VEGF. Numerous studies have demonstrated that VEGF activity mediates cellular events leading to angiogenesis and capillary formation in vivo. This has motivated the study of biomaterials to sustain VEGF release, and in many cases, the materials are inspired by the structure and function of the native ECM. However, there remains a need for materials that can bind to VEGF with high specificity, as the in vivo environment is rich in a variety of growth factors (GFs) and GF-binding moieties. Here we describe a strategy to control VEGF release using hydrogel microspheres with tethered peptides derived from VEGF receptor 2 (VEGFR2). Using biomaterials covalently modified with varying concentrations of two distinct VEGFR2-derived peptides with varying serum stability, we analyzed both biomaterial and environmental variables that influence VEGF release and activity. The presence of tethered VEGF-binding peptides (VBPs) resulted in significantly extended VEGF release relative to control conditions, and the resulting released VEGF significantly increased the expansion of human umbilical vein endothelial cells in culture. VEGF release rates were also strongly influenced by the concentration of serum. The presence of Feline McDonough Sarcoma-like tyrosine kinase 1 (sFlt-1), a serum-borne receptor fragment derived from VEGF receptor 1, increased VEGF release rates, although sFlt-1 was not sufficient to recapitulate the release profile of VEGF in serum. Further, the influence of serum on VEGF release was not due to protease activity or nonspecific VEGF interactions in the presence of serum-borne heparin. VEGF release kinetics correlated well with a generalizable mathematical model describing affinity-mediated release of VEGF from hydrogel microspheres in defined conditions. Modeling results suggest a potential mechanism whereby competition between VEGF and multiple VEGF-binding serum proteins including sFlt-1, soluble kinase insert domain receptor (sKDR), and α2-macroglobulin (α2-M) likely influenced VEGF release from microspheres. The materials and mathematical model described in this approach may be useful in a range of applications in which sustained, biologically active GF release of a specific GF is desirable.

Ligaments have limited regenerative potential and as a consequence, repair is protracted and results in a mechanically inferior tissue more scar-like than native ligament. We previously reported that a single injection of interleukin-1 receptor antagonist (IL-1Ra) delivered at the time of injury, decreased the number of M2 macrophage-associated inflammatory cytokines. Based on these results, we hypothesized that IL-1Ra administered after injury and closer to peak inflammation (as would occur clinically), would more effectively decrease inflammation and thereby improve healing. Since IL-1Ra has a short half-life, we also investigated the effect of multiple injections. The objective of this study was to elucidate healing of a medial collateral ligament (MCL) with either a single IL-1Ra injection delivered one day after injury or with multiple injections of IL-1Ra on days 1, 2, 3, and 4. One day after MCL injury, rats received either single or multiple injections of IL-1Ra or PBS. Tissue was then collected at days 5 and 11. Both single and multiple IL-1Ra injections reduced inflammatory cytokines, but did not change mechanical behavior. A single injection of IL-1Ra also reduced the number of myofibroblasts and increased type I procollagen. Multiple IL-1Ra doses provided no additive response and, in fact, reduced the M2 macrophages. Based on these results, a single dose of IL-1Ra was better at reducing the MCL-derived inflammatory cytokines compared to multiple injections. The changes in type I procollagen and myofibroblasts further suggest a single injection of IL-1Ra enhanced repair of the ligament but not sufficiently to improve functional behavior.

Pherbellia anitae Foote, sp. nov., from southeastern Arizona, is described, and the male postabdomen is illustrated.

Eighty-nine years after the only known specimen of Dictya disjuncta Orth was collected, in Mississippi in 1921, the species was rediscovered in Indiana, U.S.A. in 2010. A gravid female laid eggs that gave rise to two generations of flies. The egg, three larval stages, puparium, and adult female are described and illustrated for the first time, the life cycle is elucidated, the holotype male is redescribed, and the geographical distribution of the species is depicted.

Described herein are the immature stages (egg, three larval instars, and puparium) of Sepedon (Sepedomyia) nasuta Verbeke from specimens collected in Benin. Analyzed are larval life-cycle modalities and larval feeding behavior on freshwater gastropods. Rainfall appears to affect significantly the magnitude of S. nasuta populations in Benin. Sepedon nasuta is shown to be polyphagous. Its status as multivoltine remains questionable. The species appears to hold promise as a potential candidate for use as a biological control agent against freshwater snails serving as intermediate hosts of parasites transmitted to humans or domestic animals. We summarize herein published biological data on 12 of the 65 species of Sciomyzidae recorded from the Afrotropical Region.

Requirements of hydrogels for drug delivery, wound dressings, and surgical implantation can be extensive, including suitable mechanical properties and tailorable degradation time frames. Herein, an adaptable PEG-based hydrogel, whose mechanical properties and degradation rate can be systematically adjusted to meet these criteria by altering simple variables such as the PEG molecular weight, is described. The performance of these hydrogels in three physical manipulations (pushing, pulling, and folding), representative of manipulations that they may undergo during typical biomedical use, is also assessed. While not all of these formulations can withstand these manipulations, a subset did, and it is intended to further optimize these formulations for specific clinical applications. Additionally, the outcomes of the physical manipulation tests indicate that simply having a high modulus does not correlate with biomedical applicability.

Understanding the processes by which stem cells give rise to de novo tissues is an active focus of stem cell biology and bioengineering disciplines. Instructive morphogenic cues surrounding the stem cell during morphogenesis create what is referred to as the stem cell microenvironment. An emerging paradigm in stem cell bioengineering involves “biologically driven assembly,” in which stem cells are encouraged to largely define their own morphogenesis processes. However, even in the case of biologically driven assembly, stem cells do not act alone. The properties of the surrounding microenvironment can be critical regulators of cell fate. Stem cell-material interactions are among the most well-characterized microenvironmental effectors of stem cell fate, and they establish a signaling “context” that can define the mode of influence for morphogenic cues. Here we describe illustrative examples of cell-material interactions that occur during in vitro stem cell studies, with an emphasis on how cell-material interactions create instructive contexts for stem cell differentiation and morphogenesis.

Growth factors (GFs) are major regulatory proteins that can govern cell fate, migration, and organization. Numerous aspects of the cell milieu can modulate cell responses to GFs, and GF regulation is often achieved by the native extracellular matrix (ECM). For example, the ECM can sequester GFs and thereby control GF bioavailability. In addition, GFs can exert distinct effects depending on whether they are sequestered in solution, at two-dimensional interfaces, or within three-dimensional matrices. Understanding how the context of GF sequestering impacts cell function in the native ECM can instruct the design of soluble or insoluble GF sequestering moieties, which can then be used in a variety of bioengineering applications. This Feature Article provides an overview of the natural mechanisms of GF sequestering in the cell milieu, and reviews the recent bioengineering approaches that have sequestered GFs to modulate cell function. Results to date demonstrate that the cell response to GF sequestering depends on the affinity of the sequestering interaction, the spatial proximity of sequestering in relation to cells, the source of the GF (supplemented or endogenous), and the phase of the sequestering moiety (soluble or insoluble). We highlight the importance of context for the future design of biomaterials that can leverage endogenous molecules in the cell milieu and mitigate the need for supplemented factors.

Dictya behrmani Murphy, new species (Diptera: Sciomyzidae), from Indiana, U.S.A., is described and illustrated. A male collected near Belmont, Brown County, Indiana, U.S.A., represents the only known specimen of the new species. The most recent key to the genus Dictya is updated to include the two species de- scribed since publication of the key.

Optimization of non-viral gene delivery from biomaterials is of critical importance, as several material parameters are known to influence non-viral transfection efficiency. A series of previous studies have achieved screening of gene delivery vectors on two dimensional (2D) substrates, which have direct relevance to cell culture applications. There is an additional need to create screening systems that are 3-dimensional (3D), and can thus be applied to emerging tissue engineering applications. Here, we report an enhanced throughput, 3D scaffold platform to screen for the influence of mineral coating properties on stem cell transfection. Mineral coatings with a range of physicochemical properties were formed on the scaffolds within a 96-well plate format, while maintaining an interconnected macroporous scaffold structure. A series of general gene delivery parameters, including plasmid DNA amount, N/P ratio, and cell density, were efficiently screened in scaffolds using a luciferase-encoding plasmid as a reporter. In addition, human mesenchymal stem cell (hMSC) transfection with a plasmid encoding bone morphogenetic protein-2 (BMP-2) was successfully optimized by screening a library of mineral coatings, resulting in over 5-fold increases in BMP-2 production when compared to standard techniques. Notably, the majority of BMP-2 was incorporated into the mineral coating following secretion from the cells. The 3D mineral coated scaffold platform described here may accelerate gene delivery optimization and improve the predictability of the screening systems, which could facilitate translation of gene delivery to clinical applications.

A simple, degradable poly(ethylene glycol) (PEG) microsphere system formed from a water-in-water emulsion process is presented. Microsphere network degradation and erosion were controlled by adjusting the number of hydrolytically labile sites, by varying the PEG molecular weight, and by adjusting the emulsion conditions. Microsphere size was also controllable by adjusting the polymer formulation. Furthermore, it is demonstrated that alternative degradation and erosion mechanisms, such as proteolytic degradation, can be incorporated into PEG microspheres, resulting in mixed-mode degradation. Owing to the adaptability of this approach, it may serve as an attractive option for emerging tissue engineering, drug delivery and gene delivery applications.

Vascular endothelial growth factor (VEGF) was originally discovered as a tumor-derived factor that is able to induce endothelial cell behavior associated with angiogenesis. It has been implicated during wound healing for the induction of endothelial cell proliferation, tube formation and blood vessel remodeling. However, previous investigations into the biological effect of VEGF concluded that a particular range of growth factor concentrations are required for healthy vasculature to form, motivating recent studies to regulate VEGF activity via molecular sequestering to biomaterials. Numerous VEGF sequestering strategies have been developed, and they have typically relied on extracellular matrix mimicking moieties that are not specific for VEGF and can affect many growth factors simultaneously. We describe here a strategy for efficient, specific VEGF sequestering with poly(ethylene glycol) (PEG) microspheres, using peptides designed to mimic VEGF receptor type 2 (VEGFR2). By immobilizing two distinct peptides with different serum stabilities, we examined the effect of serum on the specific interaction between peptide-containing PEG microspheres and VEGF. We addressed the hypothesis that VEGF sequestering in serum-containing solutions would be influenced by the serum stability of the VEGF-binding peptide. We further hypothesized that soluble VEGF could be sequestered in serum-containing cell culture media, resulting in decreased VEGF-dependent proliferation of human umbilical vein endothelial cells. We show that soluble VEGF concentration can be effectively regulated in serum-containing environments via specific molecular sequestering, which suggests potential clinical applications.

Biomineral coatings have been extensively used to enhance the osteoconductivity of polymeric scaffolds. Numerous porous scaffolds have previously been coated with a bone-like apatite mineral through incubation in simulated body fluid (SBF). However, characterization of the mineral layer formed on scaffolds, including the amount of mineral within the scaffolds, often requires destructive methods. We have developed a method using micro-computed tomography (μ-CT) scanning to nondestructively quantify the amount of mineral in vitro and in vivo on biodegradable scaffolds made of poly (L-lactic acid) (PLLA) and poly (ε-caprolactone) (PCL). PLLA and PCL scaffolds were fabricated using an indirect solid freeform fabrication (SFF) technique to achieve orthogonally interconnected pore architectures. Biomineral coatings were formed on the fabricated PLLA and PCL scaffolds after incubation in modified SBF (mSBF). Scanning electron microscopy and X-ray diffraction confirmed the formation of an apatite-like mineral. The scaffolds were implanted into mouse ectopic sites for 3 and 10 weeks. The presence of a biomineral coating within the porous scaffolds was confirmed through plastic embedding and μ-CT techniques. Tissue mineral content (TMC) and volume of mineral on the scaffold surfaces detected by μ-CT had a strong correlation with the amount of calcium measured by the orthocresolphthalein complex-one (OCPC) method before and after implantation. There was a strong correlation between OCPC pre- and postimplantation and μ-CT measured TMC (R(2)=0.96 preimplant; R(2)=0.90 postimplant) and mineral volume (R(2)=0.96 preimplant; R(2)=0.89 postimplant). The μ-CT technique showed increases in mineral following implantation, suggesting that μ-CT can be used to nondestructively determine the amount of calcium on coated scaffolds.

"Biomimetic" inorganic coating on biomaterials has been an active area of research with the aim of providing bioactive surfaces that can regulate cell behavior. Previous studies have demonstrated that human mesenchymal stem cell (hMSC) behavior is differentially regulated by the physical and chemical properties of inorganic mineral coatings, indicating that modulation of mineral properties is potentially important in regulating hMSC behavior. However, the lack of an efficient experimental context, in which to study stem cell behavior on inorganic substrates, has made it difficult to systematically study the effects of specific mineral coating parameters on hMSC behavior. In this study, we developed an efficient experimental platform to screen for the effects of mineral coating morphology on hMSC expansion and differentiation. hMSC expansion on mineral coatings was regulated by the micro-scale morphology of these coatings, with greater expansion on small granule-like coatings when compared to plate-like or net-like coatings. In contrast, hMSC osteogenic differentiation was inversely correlated with cell expansion on mineral coatings indicating that mineral coating morphology was a key parameter that regulates hMSC differentiation. The effect of mineral coating morphology on hMSC behavior underlines the utility of this inorganic screening platform to identify optimal coatings for medical devices and bone tissue engineering applications.

cited By 1; International audience; Drug release from tissue scaffolds is commonly controlled by using coatings and carriers, as well as by varying the binding affinity of molecules being released. This paper considers modulating synthetic peptide incorporation and release through the use of interconnected microporosity in biphasic calcium phosphate (BCP) and identifies the microstructural characteristics important to the release using experiments and a model of relative diffusivity. First, the release of three modular peptides designed to include an osteocalcin-inspired binding sequence based on bone morphogenic protein-2 (BMP-2) was compared and one was selected for further study. Next, the incorporation and release of the peptide from four types of substrates were compared: non-microporous (NMP) substrates had no microporosity; microporous (MP) substrates were either 50% microporous with 5 μm pores (50/5), 60% microporous with 5 μm pores (60/5), or 50% microporous with 50 μm pores (50/50). Results showed that MP substrates incorporated significantly more peptide than NMP ones, but that the three different microporous substrates all incorporated the same total amount of peptide. NMP had a markedly lower release rate compared to each of three of the MP samples, though the initial burst release was the highest. The initial release and the release rate for the 60/5 samples were different from the 50/50, though they were not statistically different from the 50/5. The model indicated that the pore interconnection to pore size ratio, affecting the constriction between pores, had the greatest influence on the calculated relative diffusivity. While the model was consistent with the trends observed experimentally, the quantitative experimental results suggested that to attain an appreciable difference in release characteristics, both pore size and pore fraction should be changed for this system. These results contribute to rational scaffold design by showing that microstructure, specifically microporosity, can be used to modulate drug release. � 2016 Elsevier B.V.

Dynamic hydrogels undergo structural changes in response to specific external stimuli, and the structural modification is accompanied by changes in the physical and chemical properties of the hydrogel. The ability to alter these properties in a controllable manner has allowed hydrogels to be employed in a diverse set of biomedical applications: targeted drug delivery, tissue engineering, molecule sensing, and autonomous control in biomedical devices. Temperature, pH, and ligand binding are some of the environmental cues that have been used to trigger changes in hydrogels. The stimuli work by altering the balance of intermolecular and intramolecular forces, such as hydrogen bonding and hydrophobic interactions, between the chains in the hydrogel and the solvent. In some cases, the conformational change is reversible, while for other mechanisms, the polymer structure is irreversibly altered. A variety of polymers, peptides, and nanoparticles have been used to create responsive hydrogels. The availability of a diverse range of materials allows the properties of the hydrogel to be tailored to the application of interest. This chapter reviews the historical development of dynamic hydrogels, discusses the various mechanisms by which the structural changes take place, and presents select biomedical applications where these materials are being employed.

The styli, often called the aristae, of the antennae of adult Sepedon fuscipennis Loew (Diptera: Sciomyzidae) are shown to possess mechanosensilla, the number and arrangement of which differ between the sexes. It is suggested that the mechanosensilla provide sensory input to the female regarding the touching, or appositioning, of her styli by the male with his forelegs during copulation. Among the Sciomyzidae, S.fuscipennis males are unique in appositioning the antennae of the female during mating. Large clusters of pollen found on the styli of both sexes suggest that mechanosensilla and chemosensilla also provide sensory information about potential sources of food such as nectar (i.e., carbohydrates).