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This study presents the design, fabrication, and evaluation of a general platform for the creation of three-dimensional printed devices (3DPDs) for tissue engineering applications. As a demonstration, we modeled the liver with 3DPDs consisting of a pair of parallel millifluidic channels that function as portal-venous (PV) and hepatobiliary (HB) structures. Perfusion of medium or whole blood through the PV channel supports the hepatocyte-containing HB channel. Device computer-aided design was optimized for structural stability, after which 3DPDs were 3D printed in a polyethylene(glycol) diacrylate photoink by digital light processing and evaluated in vitro. The HB channels were subsequently seeded with hepatic cells suspended in a collagen hydrogel. Perfusion of 3DPDs in bioreactors enhanced the viability and function of rat hepatoma cells and were maintained over time, along with improved liver-specific functions. Similar results were observed with primary rat hepatocytes, including significant upregulation of cytochrome p450 activity. Additionally, coculture experiments involving primary rat hepatocytes, endothelial cells, and mesenchymal stem cells in 3DPDs showed enhanced viability, broad liver-specific gene expression, and histological features indicative of liver tissue architecture. In vivo implantation of 3DPDs in a rat renal shunt model demonstrated successful blood flow through the devices without clot formation and maintenance of cell viability. 3D printed designs can be scaled in 3D space, allowing for larger devices with increased cell mass. Overall, these findings highlight the potential of 3DPDs for clinical translation in hepatic support applications., Competing Interests: Competing interests statement:J.P.V. equity in 3D BioLabs, LLC. T.J.L., T.S., M.J.H., A.P.S., M.S.O., S.C.G., C.M.N., and J.P.V. hold patents and patent applications related to content in this work.

In vitro vascular wall bilayer models for drug testing and disease modeling must emulate the physical and biological properties of healthy vascular tissue and its endothelial barrier function. Both endothelial cell (EC)-vascular smooth muscle cell (SMC) interaction across the internal elastic lamina (IEL) and blood vessel stiffness impact endothelial barrier integrity. Polymeric porous track-etched membranes (TEM) typically represent the IEL in laboratory vascular bilayer models. However, TEM stiffness exceeds that of diseased blood vessels, and the membrane pore architecture limits EC-SMC interaction. The mechanical properties of compliant honeycomb film (HCF) membranes better simulate the Young's modulus of healthy blood vessels, and HCFs are thinner (4 vs. 10 μm) and more porous (57 vs. 6.5%) than TEMs. We compared endothelial barrier integrity in vascular wall bilayer models with human ECs and SMCs statically cultured on opposite sides of HCFs and TEMs (5 μm pores) for up to 12 days. Highly segregated localization of tight junction (ZO-1) and adherens junction (VE-cadherin) proteins and quiescent F-actin cytoskeletons demonstrated superior and earlier maturation of interendothelial junctions. Quantifying barrier integrity based on transendothelial electrical resistance (TEER), membranes showed only minor but significant TEER differences despite enhanced junctional protein localization on HCF. Elongated ECs on HCF likely experienced greater paracellular diffusion than blocky ECs on TEM. Also, larger populations of plaques of connexin 43 subunit-containing gap junctions suggested enhanced EC-SMC communication across the more porous, thinner HCF. Compared with standard TEMs, engineered vascular wall bilayers cultured on HCFs better replicate physiologic endothelial barrier integrity., (© 2023 Wiley Periodicals LLC.)

This study presents a novel surgical model developed to provide hematological support for implanted cellularized devices augmenting or replacing liver tissue function. Advances in bioengineering provide tools and materials to create living tissue replacements designed to restore that lost to disease, trauma, or congenital deformity. Such substitutes are often assembled and matured in vitro and need an immediate blood supply upon implantation, necessitating the development of supporting protocols. Animal translational models are required for continued development of engineered structures before clinical implementation, with rodent models often playing an essential early role. Our long-term goal has been generation of living tissue to provide liver function, utilizing advances in additive manufacturing technology to create 3D structures with intrinsic micron to millimeter scale channels modeled on natural vasculature. The surgical protocol developed enables testing various design iterations in vivo by anastomosis to the host rat vasculature. Lobation of rodent liver facilitates partial hepatectomy and repurposing the remaining vasculature to support implanted engineered tissue. Removal of the left lateral lobe exposes the underlying hepatic vasculature and can create space for a device. A shunt is created from the left portal vein to the left hepatic vein by cannulating each with separate silicone tubing. The device is then integrated into the shunt by connecting its inflow and outflow ports to the tubing and reestablishing blood flow. Sustained anticoagulation is maintained with an implanted osmotic pump. In our studies, animals were freely mobile after implantation; devices remained patent while maintaining blood flow through their millifluidic channels. This vascular anastomosis model has been greatly refined during the process of performing over 200 implantation procedures. We anticipate that the model described herein will find utility in developing preclinical translational protocols for evaluation of engineered liver tissue. Impact statement Tissue and organ transplantation are often the best clinically effective treatments for a variety of human ailments. However, the availability of suitable donor organs remains a critical problem. Advances in biotechnology hold potential in alleviating shortages, yet further work is required to surgically integrate large engineered tissues to host vasculature. Improved animal models such as the one described are valuable tools to support continued development and evaluation of novel therapies.

Monitoring the growth of salmon during their early marine phase provides insights into prey availability, and growth rates may be linked to risks of size-dependent mortality. However, the measurement of growth rate is challenging for free-living salmon in the ocean. Insulin-like growth factor (IGF)-I is a growth-promoting hormone that is emerging as a useful index of growth in salmon. In addition, laboratory-based studies using coho salmon have shown that one of circulating IGF-binding proteins (IGFBPs), IGFBP-1b, is induced by fasting and thus could be used as an inverse index of growth and/or catabolic state in salmon. However, few studies have measured plasma levels of IGFBP-1b in salmon in the wild. We measured plasma IGFBP-1b levels for postsmolt coho salmon collected in the Strait of Georgia and surrounding waters, British Columbia, Canada, and compared regional differences in IGFBP-1b to ecological information such as seawater temperature and stomach fullness. Plasma IGFBP-1b levels were the highest in fish from Eastern Johnstone Strait and relatively high in Queen Charlotte Strait and Western Johnstone Strait, which was in good agreement with the poor ocean conditions for salmon hypothesized to occur in that region. The molar ratio of plasma IGF-I to IGFBP-1b, a theoretical parameter of IGF-I availability to the receptor, discriminated differences among regions better than IGF-I or IGFBP-1b alone. Our data suggest that plasma IGFBP-1b reflects catabolic status in postsmolt coho salmon, as highlighted in fish in Eastern Johnston Strait, and is a useful tool to monitor negative aspects of salmon growth in the ocean.

Biological surgical scaffolds are used in plastic and reconstructive surgery to support structural reinforcement and regeneration of soft tissue defects. Macrophage and fibroblast cell populations heavily regulate scaffold integration into host tissue following implantation. In the present study, the biological host response to a commercially available surgical scaffold (Meso BioMatrix Surgical Mesh (MBM)) was investigated for up to 9 weeks after subcutaneous implantation; this scaffold promoted superior cell migration and infiltration previously in in vitro studies relative to other commercially available scaffolds. Infiltrating macrophages and fibroblasts phenotypes were assessed for evidence of inflammation and remodeling. At week 1, macrophages were the dominant cell population, but fibroblasts were most abundant at subsequent time points. At week 4, the scaffold supported inflammation modulation as indicated by M1 to M2 macrophage polarization; the foreign body giant cell response resolved by week 9. Unexpectedly, a fibroblast subpopulation expressed macrophage phenotypic markers, following a similar trend in transitioning from a proinflammatory to anti-inflammatory phenotype. Also, α-smooth muscle actin-expressing myofibroblasts were abundant at weeks 4 and 9, mirroring collagen expression and remodeling activity. MBM supported physiologic responses observed during normal wound healing, including cellular infiltration, host tissue ingrowth, remodeling of matrix proteins, and immune modulation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 716-725, 2018., (© 2017 Wiley Periodicals, Inc.)

Objectives: Creation of functional, durable vasculature remains an important goal within the field of regenerative medicine. Engineered biological vasculature has the potential to restore or improve human tissue function. We hypothesized that the pleotropic effects of insulin-like growth factor 1 (IGF1) would enhance the engineering of capillary-like vasculature., Materials and Methods: The impact of IGF1 upon vasculogenesis was examined in in vitro cultures for a period of up to 40 days and as subcutaneous implants within immunodeficient mice. Co-cultures of human umbilical vein endothelial cells and human bone marrow-derived mesenchymal stem cells in collagen-fibronectin hydrogels were supplemented with either recombinant IGF1 protein or genetically engineered cells to provide sustained IGF1. Morphometric analysis was performed on the vascular networks that formed in four concentrations of IGF1., Results: IGF1 supplementation significantly enhanced de novo vasculogenesis both in vitro and in vivo. Effects were long-term as they lasted the duration of the study period, and included network density, vessel length, and diameter. Bifurcation density was not affected. However, the highest concentrations of IGF1 tested were either ineffective or even deleterious. Sustained IGF1 delivery was required in vivo as the inclusion of recombinant IGF1 protein had minimal impact., Conclusion: IGF1 supplementation can be used to produce neovasculature with significantly enhanced network density and durability. Its use is a promising methodology for engineering de novo vasculature to support regeneration of functional tissue., (© 2017 John Wiley & Sons Ltd.)

Systemic delivery of therapeutic proteins to the central nervous system (CNS) is challenging because of the blood-brain barrier restrictions. Direct intrathecal delivery is possible but does not produce stable concentrations. We are proposing an alternative approach for localized delivery into the CNS based on the Transduced Autologous Restorative Gene Therapy (TARGT) system. This system was previously developed using a gene therapy approach with dermal tissue implants. Lewis rat dermal tissue was transduced to secrete human EPO (hEPO). TARGT viability and function were retained following cryopreservation. Upon implantation into the rat cisterna magna, a mild inflammatory response was observed at the TARGT-brain interface throughout 21-day implantation. hEPO expression was verified immunohistochemically and by secreted levels in cerebrospinal fluid (CSF), serum, and in vitro post explant. Detectable CSF hEPO levels were maintained during the study. Serum hEPO levels were similar to rat and human basal serum levels. In vitro, the highest hEPO concentration was observed on day 1 post-explant culture and then remained constant for over 21days. Prolonged incubation within the cisterna magna had no negative impact on TARGT hEPO secretion. These promising results suggest that TARGTs could be utilized for targeted delivery of therapeutic proteins to the CNS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Decellularized extracellular matrix (ECM) biomaterials are increasingly used in regenerative medicine for abdominal tissue repair. Emerging ECM biomaterials with greater compliance target surgical procedures like breast and craniofacial reconstruction to enhance aesthetic outcome. Clinical studies report improved outcomes with newly designed ECM scaffolds, but their comparative biological characteristics have received less attention. In this study, we investigated scaffolds derived from dermis (AlloDerm Regenerative Tissue Matrix), small intestinal submucosa (Surgisis 4-layer Tissue Graft and OASIS Wound Matrix), and mesothelium (Meso BioMatrix Surgical Mesh and Veritas Collagen Matrix) and evaluated biological properties that modulate cellular responses and recruitment. An assay panel was utilized to assess the ECM scaffold effects upon cells. Results of the material-conditioned media study demonstrated Meso BioMatrix and OASIS best supported cell proliferation. Meso BioMatrix promoted the greatest migration and chemotaxis signaling, followed by Veritas and OASIS; OASIS had superior suppression of cell apoptosis. The direct adhesion assay indicated that AlloDerm, Meso BioMatrix, Surgisis, and Veritas had sidedness that affected cell-material interactions. In the chick chorioallantoic membrane assay, Meso BioMatrix and OASIS best supported cell infiltration. Among tested materials, Meso BioMatrix and OASIS demonstrated characteristics that facilitate scaffold incorporation, making them promising choices for many clinical applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 585-593, 2017., (© 2015 Wiley Periodicals, Inc.)

In pre-clinical safety studies, drug-induced vascular injury (DIVI) is defined as an adverse response to a drug characterized by degenerative and hyperplastic changes of endothelial cells and vascular smooth muscle cells. Inflammation may also be seen, along with extravasation of red blood cells into the smooth muscle layer (i.e., hemorrhage). Drugs that cause DIVI are often discontinued from development after considerable cost has occurred. An in vitro vascular model has been developed using endothelial and smooth muscle cells in co-culture across a porous membrane mimicking the internal elastic lamina. Arterial flow rates of perfusion media within the endothelial chamber of the model induce physiologic endothelial cell alignment. Pilot testing with a drug known to cause DIVI induced extravasation of red blood cells into the smooth muscle layer in all devices with no extravasation seen in control devices. This engineered vascular model offers the potential to evaluate candidate drugs for DIVI early in the discovery process. The physiologic flow within the co-culture model also makes it candidate for a wide variety of vascular biology investigations.

Background: Adequate biomaterials for tissue engineering bone and replacement of bone in clinical settings are still being developed. Previously, the combination of mesenchymal stem cells in hydrogels and calcium-based biomaterials in both in vitro and in vivo experiments has shown promising results. However, results may be optimized by careful selection of the material combination., Methods: β-Tricalcium phosphate scaffolds were three-dimensionally printed with five different hydrogels: collagen I, gelatin, fibrin glue, alginate, and Pluronic F-127. The scaffolds had eight channels, running throughout the entire scaffold, and macropores. Mesenchymal stem cells (2 × 10) were mixed with each hydrogel, and cell/hydrogel mixes were dispersed onto the corresponding β-tricalcium phosphate/hydrogel scaffold and cultured under dynamic-oscillating conditions for 6 weeks. Specimens were harvested at 1, 2, 4, and 6 weeks and evaluated histologically, radiologically, biomechanically and, at 6 weeks, for expression of bone-specific proteins by reverse-transcriptase polymerase chain reaction. Statistical correlation analysis was performed between radiologic densities in Hounsfield units and biomechanical stiffness., Results: Collagen I samples had superior bone formation at 6 weeks as demonstrated by volume computed tomographic scanning, with densities of 300 HU, similar to native bone, and the highest compression values. Bone specificity of new tissue was confirmed histologically and by the expression of alkaline phosphatase, osteonectin, osteopontin, and osteocalcin. The bone density correlated closely with histologic and biomechanical testing results., Conclusion: Bone formation is supported best by β-tricalcium phosphate/collagen I hydrogel and mesenchymal stem cells in collagen I hydrogel., Clinical Question/level of Evidence: Therapeutic, V.

Advancement of engineered ear in clinical practice is limited by several challenges. The complex, largely unsupported, three-dimensional auricular neocartilage structure is difficult to maintain. Neocartilage formation is challenging in an immunocompetent host due to active inflammatory and immunological responses. The large number of autologous chondrogenic cells required for engineering an adult human-sized ear presents an additional challenge because primary chondrocytes rapidly dedifferentiate during in vitro culture. The objective of this study was to engineer a stable, human ear-shaped cartilage in an immunocompetent animal model using expanded chondrocytes. The impact of basic fibroblast growth factor (bFGF) supplementation on achieving clinically relevant expansion of primary sheep chondrocytes by in vitro culture was determined. Chondrocytes expanded in standard medium were either combined with cryopreserved, primary passage 0 chondrocytes at the time of scaffold seeding or used alone as control. Disk and human ear-shaped scaffolds were made from porous collagen; ear scaffolds had an embedded, supporting titanium wire framework. Autologous chondrocyte-seeded scaffolds were implanted subcutaneously in sheep after 2 weeks of in vitro incubation. The quality of the resulting neocartilage and its stability and retention of the original ear size and shape were evaluated at 6, 12, and 20 weeks postimplantation. Neocartilage produced from chondrocytes that were expanded in the presence of bFGF was superior, and its quality improved with increased implantation time. In addition to characteristic morphological cartilage features, its glycosaminoglycan content was high and marked elastin fiber formation was present. The overall shape of engineered ears was preserved at 20 weeks postimplantation, and the dimensional changes did not exceed 10%. The wire frame within the engineered ear was able to withstand mechanical forces during wound healing and neocartilage maturation and prevented shrinkage and distortion. This is the first demonstration of a stable, ear-shaped elastic cartilage engineered from auricular chondrocytes that underwent clinical-scale expansion in an immunocompetent animal over an extended period of time.

Objective: Our goal was to engineer cartilage in vivo using auricular chondrocytes that underwent clinically relevant expansion and using methodologies that could be easily translated into health care practice., Design: Sheep and human chondrocytes were isolated from auricular cartilage biopsies and expanded in vitro. To reverse dedifferentiation, expanded cells were either mixed with cryopreserved P0 chondrocytes at the time of seeding onto porous collagen scaffolds or proliferated with basic fibroblast growth factor (bFGF). After 2-week in vitro incubation, seeded scaffolds were implanted subcutaneously in nude mice for 6 weeks. The neocartilage quality was evaluated histologically; DNA and glycosaminoglycans were quantified. Cell proliferation rates and collagen gene expression profiles were assessed., Results: Clinically sufficient over 500-fold chondrocyte expansion was achieved at passage 3 (P3); cell dedifferentiation was confirmed by the simultaneous COL1A1/3A1 gene upregulation and COL2A1 downregulation. The chondrogenic phenotype of sheep but not human P3 cells was rescued by addition of cryopreserved P0 chondrocytes. With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated. Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks., Conclusions: Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo.

Surgical scaffold materials manufactured from donor human or animal tissue are increasingly being used to promote soft tissue repair and regeneration. The clinical product consists of the residual extracellular matrix remaining after a rigorous decellularization process. Optimally, the material provides both structural support during the repair period and cell guidance cues for effective incorporation into the regenerating tissue. Surgical scaffold materials are available from several companies and are unique products manufactured by proprietary methodology. A significant need exists for a more thorough understanding of scaffold properties that impact the early steps of host cell recruitment and infiltration. In this study, a panel of in vitro assays was used to make direct comparisons of several similar, commercially-available materials: Alloderm, Medeor Matrix, Permacol, and Strattice. Differences in the materials were detected for both cell signaling and scaffold architecture-dependent cell invasion. Material-conditioned media studies found Medeor Matrix to have the greatest positive effect upon cell proliferation and induction of migration. Strattice provided the greatest chemotaxis signaling and best suppressed apoptotic induction. Among assays measuring structure-dependent properties, Medeor Matrix was superior for cell attachment, followed by Permacol. Only Alloderm and Medeor Matrix supported chemotaxis-driven cell invasion beyond the most superficial zone. Medeor Matrix was the only material in the chorioallantoic membrane assay to support substantial cell invasion. These results indicate that both biologic and structural properties need to be carefully assessed in the considerable ongoing efforts to develop new uses and products in this important class of biomaterials., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Hepatic stellate cells (HSC) play a major role in the progression of liver fibrosis., Aim: The aim of our study was to investigate whether rat HSC cultured on a nanofiber membrane (NM) retain their quiescent phenotype during both short- and long-term culture and whether activated HSC revert to a quiescent form when re-cultured on NM., Methods: Rat HSC cultured for 1 day on plastic plates (PP) were used as quiescent HSC, while cells cultured for 1 week on PP were considered to be activated HSC. Quiescent or activated HSC were subsequently plated on PP or NM and cultured for an additional 4 days at which time their gene expression, stress fiber development, and growth factor production were determined. For long-term culture, HSC were grown on NM for 20 days and the cells then replated on PP and cultured for another 10 days., Results: Expression of marker genes for HSC activation, stress fiber development, and growth factor production were significantly lower in both quiescent and activated HSC cultured on NM than in those cultured on PP. After long-term culture on NM, activation marker gene expression and stress fiber development were still significantly lower in HSC than in PP, and HSC still retained the ability to activate when replated onto PP., Conclusions: HSC cultured on NM retained quiescent characteristics after both short- and long-term culture while activated HSC reverted toward a quiescent state when cultured on NM. Cultures of HSC grown on NM are a useful in vitro model to investigate the mechanisms of activation and deactivation.

Small facial skeletal muscles often have no autologous donor source to effect surgical reconstruction. Autologously derived muscles could be engineered for replacement tissue, but must be vascularized and innervated to be functional. As a critical step, engineered muscle must mimic the morphology, protein and gene expression, and function of native muscle. This study utilized a self-assembly process to engineer three-dimensional (3D) muscle from a statically strained muscle cell monolayer. Primary mouse myoblasts (PMMs) and mouse embryonic fibroblasts (MEFs) were separately proliferated and coseeded on a fibrin sheet with anchored sutures. Within 10 days of initiating PMM differentiation, the cell-gel layer contracted, lifted, and rolled into a cylindrical 3D structure around the tendon-like suture anchors; the myotubes longitudinally aligned along the lines of tensile force. The objectives of this study were to characterize these engineered muscles and to elucidate the role of the fibroblasts in the self-assembly process. Fibroblasts maintained myotube viability, mediated fibrin degradation, and assisted in muscle self-assembly. The optimal 1:1 PMM:MEF ratio resulted in tissue morphology remarkably similar to native muscle. Through gene and protein expression assays, the development and maturation of the engineered muscle tissue was demonstrated to recapitulate normal skeletal muscle development.

Decellularized dermis materials demonstrate considerable utility in surgical procedures including hernia repair and breast reconstruction. A new decellularized porcine dermis material has been developed that retains many native extracellular matrix (ECM) proteins and cytokines. This material has substantial mechanical strength with maximum tensile strength of 141.7 +/- 85.4 (N/cm) and suture pull through strength of 47.0 +/- 14.0 (N). After processing, many ECM proteins remained in the material including collagen III, collagen IV, collagen VII, laminin and fibronectin. Glycosaminoglycans, including hyaluronic acid, were also preserved. Among several cytokines whose levels were quantified, more vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-beta) were retained within this material than in comparable decellularized dermis materials. The retention of bioactivity was demonstrated in a cell culture assay. Because this decellularized porcine dermis material both retains significant strength and has substantial biological activity, it may promote rapid integration and repair in clinical applications., (Copyright 2010. Published by Elsevier Ltd.)

Mesothelium tissues such as peritoneum and pleura have a thin and strong layer of extracellular matrix that supports mesothelial cells capable of rapid healing. Decellularized porcine mesothelium was characterized for strength, composition of the matrix and biological activity. The tensile strength of the material was 40.65 +/- 21.65 N/cm. Extracellular matrix proteins collagen IV, fibronectin, and laminin as well as glycosaminoglycans were present in the material. Cytokines inherent in the extracellular matrix were preserved. Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and transforming growth factor beta (TGF-beta) were retained and the levels of VEGF and TGF-beta in the decellularized mesothelium were higher than those found in decellularized small intestinal submucosa (SIS). The decellularized mesothelium also stimulated human fibroblasts to produce more VEGF than fibroblasts grown on tissue culture plastic. Decellularized mesothelium is a sheet material with a combination of strength and biological activity that may have many potential applications in surgical repair and regenerative medicine., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The complex intricate architecture of the liver is crucial to hepatic function. Standard protocols used for enzymatic digestion to isolate hepatocytes destroy tissue structure and result in significant loss of synthetic, metabolic, and detoxification processes. We describe a process using mechanical dissociation to generate hepatic organoids with preserved intrinsic tissue architecture from swine liver. Oxygen-supplemented perfusion culture better preserved organoid viability, morphology, serum protein synthesis, and urea production, compared with standard and oxygen-supplemented static culture. Hepatic organoids offer an alternative source for hepatic assist devices, engineered liver, disease modeling, and xenobiotic testing.

Poly(glycerol sebacate) (PGS), a promising scaffold material for soft tissue engineering applications, is a soft, tough elastomer with excellent biocompatibility. However, the rapid in vivo degradation rate of PGS limits its use as a scaffold material. To determine the impact of crosslink density on degradation rate, a family of PGS materials was synthesized by incrementally increasing the curing time from 42 to 144 h, at 120 degrees C and 10 mTorr vacuum. As expected, PGS became a stiffer, tougher, and stronger elastomer with increasing curing time. PGS disks were subcutaneously implanted into rats and periodically harvested; only mild tissue responses were observed and the biocompatibility remained excellent. Regardless of crosslink density, surface erosion degradation was observed. The sample dimensions linearly decreased with implantation time, and the mass loss rates were constant after 1-week implantation. As surface erosion degradation frequently correlates with enzymatic digestion, parallel in vitro digestion studies were conducted in lipase solutions which hydrolyze ester bonds. Enzymatic digestion played a significant role in degrading PGS, and the mass loss rates were not a function of curing time. Alternative chemistry approaches will be required to decrease the enzymatic hydrolysis rate of the ester bonds in PGS polymers.

Regenerative technology promises to alleviate the problem of limited donor supply for bone or organ transplants. Most expensive and time consuming is cell expansion in laboratories. We propose a method of magnetically enriched osteoprogenitor stem cells, dispersed in self-assembling hydrogels and applied onto new ultra-high resolution, jet-based, three-dimensional printing of living human bone in a single-step for in situ bone regeneration. Human bone marrow-derived mesenchymal stem cells (hBMSCs) were enriched with CD 117+ cells, dispersed in different collagen I and RAD 16I hydrogel mixes, and applied onto three-dimensional printed btricalcium phosphate=poly(lactic-co-glycolic acid) scaffolds, printed from ultra-high-resolution volumetric CT images of a human thumb. Constructs were directly implanted subcutaneously into nude mice for 6 weeks. In vivo radiographic volumetric CT scanning and histological evaluations were performed at 1, 2, 4, and 6 weeks, and expression of bone-specific genes and biomechanical compression testing at 6 weeks endpoint. Time-dependant accumulation of bone-like extracellular matrix was most evident in CD 117+ hBMSCs using collagen I=RAD 16I hydrogel mix. This was shown histologically by Toluidine blue, von Kossa, and alkaline phosphatase staining, paralleled by increased radiological densities within implants approximating that of human bone, and confirmed by high expression of bone-specific osteonectin and biomechanical stiffness at 6 weeks. Human origin of newly formed tissue was established by expression of human GAPDH using RT-PCR. Statistical analysis confirmed high correlations between biomechanical stiffness, radiological densities, and bone markers. Bone tissue can be successfully regenerated in vivo using a single-step procedure with constructs composed of RAD 16I=collagen I hydrogel, CD 117+-enriched hBMSCs, and porous b-tricalcium phosphate=poly(lactic-co-glycolic acid) scaffolds.

Schwann cells and primary progenitor cells improve regeneration across peripheral nerve defects. This study examined the impact of immortalized neural precursor cells on regeneration of rat nerve defects. Across 10-mm gaps, neuromas formed without neural cables with C17.2- or RN33B-transplanted cells, but neural cables formed across 5-mm gaps seeded with RN33B cells. In vitro, dorsal root ganglia neurites elongated across Schwann and RN33B cells; RN33B cells induced neurite branching with shorter total outgrowth. Neural cable formation in vivo was likely determined by the balance of guidance and branch-inducing factors secreted by Schwann and transplanted precursor cells.

Interferences with the Olympus immunoturbidimetric assay for ferritin have been reported because the antibodies used in the immunoassay are derived from rabbits. Rabbits are familiar pets known to be a risk factor for developing heterophilic (or interfering) antibodies. This report shows how the current Olympus Ferritin assay has been improved to eliminate the interference from heterophilic antibodies.

Availability of grafts and morbidity at the donor site limit autologous transplantation in patients requiring bone reconstruction. A tissue-engineering approach can overcome these limitations by producing bone-like tissue of custom shape and size from isolated cells. Several hydrogels facilitate osteogenesis on porous scaffolds; however, the relative suitability of various hydrogels has not been rigorously assessed. Fibrin glue, alginate, and collagen I hydrogels were mixed with swine bone marrow-derived differentiated mesenchymal stem cells (MSCs), applied to 3-dimensionally printed porous beta-tricalcium phosphate (beta-TCP) scaffolds and implanted subcutaneously in nude mice. Although noninvasive assessment of osteogenesis in 3 dimensions is desirable for monitoring new bone formation in vivo, correlations with traditional histological and mechanical testing need to be established. High-resolution volumetric computed tomography (VCT) scanning, histological examination, biomechanical compression testing, and osteonectin (ON) expression were performed on excised scaffolds after 1, 2, 4, and 6 weeks of subcutaneous implantation in mice. Statistical correlation analyses were performed between radiological density, stiffness, and ON expression. Use of collagen I as a hydrogel carrier produced superior bone formation at 6 weeks, as demonstrated using VCT scanning with densities similar to native bone and the highest compression values. Continued contribution of the seeded MSCs was demonstrated using swine-specific messenger ribonucleic acid probes. Radiological density values correlated closely with the results of histological and biomechanical testing and ON expression. High-resolution VCT is a promising method for monitoring osteogenesis.

Trabecular bone is a material of choice for reconstruction after trauma and tumor resection and for correction of congenital defects. Autologous bone grafts are available in limited shapes and sizes; significant donor site morbidity is another major disadvantage to this approach. To overcome these limitations, we used a tissue engineering approach to create bone replacements in vitro, combining bone-marrow-derived differentiated mesenchymal stem cells (MSCs) suspended in hydrogels and 3-dimensionally printed (3DP) porous scaffolds made of beta-tricalcium-phosphate (beta-TCP). The scaffolds provided support for the formation of bone tissue in collagen I, fibrin, alginate, and pluronic F127 hydrogels during culturing in oscillating and rotating dynamic conditions. Histological evaluation including toluidine blue, alkaline phosphatase, and von Kossa staining was done at 1, 2, 4, and 6 weeks. Radiographic evaluation and high-resolution volumetric CT (VCT) scanning, expression of bone-specific genes and biomechanical compression testing were performed at 6 weeks. Both culture conditions resulted in similar bone tissue formation. Histologically collagen I and fibrin hydrogels specimens had superior bone tissue, although radiopacities were detected only in collagen I samples. VCT scan revealed density values in all but the Pluronic F127 samples, with Houndsfield unit values comparable to native bone in collagen I and fibrin glue samples. Expression of bone-specific genes was significantly higher in the collagen I samples. Pluronic F127 hydrogel did not support formation of bone tissue. All samples cultured in dynamic oscillating conditions had slightly higher mechanical strength than under rotating conditions. Bone tissue can be successfully formed in vitro using constructs comprised of collagen I hydrogel, MSCs, and porous beta-TCP scaffolds.

Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bgamma), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bgamma class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass=20 kDa; pI=5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean+/-standard deviation, n=3) of 3.13+/-0.27 and 3.43+/-1.0 micromol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.

Three non-ribosomal peptide synthetase genes, termed sidD, sidC and sidE, have been identified in Aspergillus fumigatus. Gene expression analysis by RT-PCR confirms that expression of both sidD and C was reduced by up to 90% under iron-replete conditions indicative of a likely role in siderophore biosynthesis. SidE expression was less sensitive to iron levels. In addition, two proteins purified from mycelia grown under iron-limiting conditions corresponded to SidD ( approximately 200 kDa) and SidC (496 kDa) as determined by MALDI ToF peptide mass fingerprinting and MALDI LIFT-ToF/ToF. Siderophore synthetases are unique in bacteria and fungi and represent an attractive target for antimicrobial chemotherapy.

The initiation of mammalian puberty requires the activation of hypothalamic neurons secreting the neuropeptide luteinizing hormone-releasing hormone (LHRH). It is thought that this activation is caused by changes in trans-synaptic input to LHRH neurons. More recently, it has been postulated that the pubertal increase in LHRH secretion in female animals also requires neuron-glia signaling mediated by growth factors of the epidermal growth factor (EGF) family and their astrocytic erbB receptors. Although it appears clear that functional astrocytic erbB1 receptors are necessary for the timely advent of puberty, the physiological contribution that erbB4 receptors may make to this process has not been established. To address this issue, we generated transgenic mice expressing a dominant-negative erbB4 receptor (DN-erbB4) under the control of the GFAP promoter, which targets transgene expression to astrocytes. DN-erbB4 expression is most abundant in hypothalamic astrocytes, where it blocks the ligand-dependent activation of glial erbB4 and erbB2 receptors, without affecting erbB1 (EGF) receptor signaling. Mice carrying the transgene exhibit delayed sexual maturation and a diminished reproductive capacity in early adulthood. These abnormalities are related to a deficiency in pituitary gonadotropin hormone secretion, caused by impaired release of LHRH, the hypothalamic neuropeptide that controls sexual development. In turn, the reduction in LHRH release is caused by the inability of hypothalamic astrocytes to respond to neuregulin (NRG) with production of prostaglandin E(2), which in wild-type animals mediates the stimulatory effect of astroglial erbB receptor activation on neuronal LHRH release. Thus, neuron-astroglia communication via NRG-erbB4/2 receptor signaling appears to be essential for the timely unfolding of the developmental program by which the brain controls mammalian sexual maturation.

To study the specification of inflow structures in the heart we generated transgenic animals harboring the human alkaline phosphatase (HAP) gene driven by the proximal 840 bp of a quail SMyHC3 promoter. In transgenic mice, the SMyHC3-HAP reporter was expressed in posterior heart precursors at 8.25 dpc, in sinus venosa and in the atrium at 8.5 and 9.0 dpc, and in the atria from 10.5 dpc onwards. SMyHC3-HAP transgene expression overlapped synthesis and endogenous response to retinoic acid (RA) in the heart, as determined by antibodies directed against a key RA synthetic enzyme and by staining of RAREhsplacZ transgenic animals. A single pulse of all-trans RA administered to pregnant mice at 7.5, but not after 8.5, dpc induced cardiac dismorphology, ranging from complete absence of outflow tract and ventricles to hearts with reduced ventricles expressing both SMyHC3-HAP and ventricular markers. Blockade of RA synthesis with disulfiram inhibited RA-induced transcription and produced hearts lacking the atrial chamber. This study defines a novel marker for atrial-restricted transcription in the developing mouse heart. It also suggests that atrial-specific gene expression is controlled by localized synthesis of RA, and that exclusion of RA from ventricular precursors is essential for correct specification of the ventricles.

Motor activity blocks the extrasynaptic expression of many genes in skeletal muscle, including those encoding ion channels, receptors, and adhesion molecules. Denervation reinduces transcription throughout the multinucleated myofiber, restoring the developmental pattern of expression, especially of the genes coding for the acetylcholine receptor. A screen for trans-acting factors binding to the enhancer region of the alpha-subunit gene of the acetylcholine receptor identified CTF4, a ubiquitously expressed and alternatively spliced chicken homologue of the human E protein transcription factor HTF4/HEB. Expression of the CTF4 locus closely parallels that of myogenin and acetylcholine receptor during development and maturation of skeletal muscle, but transcription is not similarly regulated by neuronal cues. Alternative splicing within the region encoding the transactivation domain generates two CTF4 isoforms with different tissue distributions, but similar binding affinities for the acetylcholine receptor alpha-subunit enhancer and similar transcriptional potential when complexed to myogenin. Direct injection of a myogenin, but not a MyoD, antisense expression vector into denervated skeletal muscle caused a significant decrease in the transcriptional activation of a depolarization-sensitive reporter gene. Similarly, injection of a CTF4, but less so of an E12, antisense expression vector impaired the denervation response, further implicating the involvement of a myogenin/CTF4 heterodimer in the expression of AChR genes in vivo.

Myogenic factor genes were found to respond differentially to electrical stimulation of denervated chick skeletal muscle. Myogenin gene activity declined rapidly (t1/2: approximately 2 min), comparable to the rate of acetylcholine receptor (AChR) gene inactivation, while other myogenic bHLH genes either lost activity more slowly (MyoD) or not at all (myf5, herculin). Protein kinase C (PKC) is known to couple membrane activity to AChR gene inactivation; myogenin gene transcription was also rapidly blocked by the PKC activator PMA, whereas electrostimulation remained without effect on myogenin gene activity in muscle that was either exposed to the kinase inhibitor staurosporine or chronically treated with PMA to deplete PKC. These results attest to a special role for myogenin in the activation of AChR genes in denervation supersensitivity.

1. We have prepared probes specific for the chicken myogenic determination genes MyoD, myogenin, myf5, and herculin and have investigated the expression of these genes in response to denervation and acute electrical stimulation in neonate chick muscle, using ribonuclease protection. 2. Upon denervation, herculin mRNA remains essentially unchanged, myf5 transcript levels approximately double, and MyoD message is up-regulated by two- to fivefold. In contrast, the message coding for myogenin, barely detectable in innervated muscle, rises dramatically (approximately 200-fold) on the second day after nerve section; in this respect it resembles acetylcholine receptor (AChR) alpha-, gamma- and delta-subunit mRNAs. Cohybridization experiments reveal that the increase in myogenin mRNA slightly precedes the rise in AChR alpha-subunit message. 3. Electrical stimulation of denervated muscle leads to an immediate decline in myogenin and AChR alpha-subunit mRNAs, with half-lives of less than an hour and approximately 4 hr, respectively; message stability measurements suggest that this is effected through a rapid shutdown of transcription. Messages coding for MyoD, myf5, and herculin decay much more slowly, as a result of slower turnover. 4. Previous experiments have indicated the involvement of a de novo induced (Tsay, H.-J., Neville, C. M., and Schmidt, J., FEBS Lett. 274:69-72, 1990) autocatalytic (Neville, C. M., Schmidt, M., and Schmidt, J., NeuroReport 2:655-657, 1991) transcription factor in the denervation-triggered up-regulation of AChR alpha-subunit expression; the denervation and electrical stimulation experiments reported here are compatible with the notion that myogenin is that factor.

Fish electric organ is a skeletal muscle homolog in which many muscle-specific genes are inhibited while acetylcholine receptor is expressed at high levels. The molecular mechanisms underlying this discoordinate regulation have not yet been explored. We have obtained partial sequences for MyoD, myogenin, and myf5 from Torpedo californica and have measured their mRNAs in several organs, using ribonuclease protection. We have found that MyoD and myf5 are expressed at comparable levels in muscle and electric organ, whereas myogenin transcripts could not be detected in either tissue. Acetylcholine receptor alpha subunit mRNA, on the other hand, is two orders of magnitude more abundant in electric tissue. We conclude that neither the loss of contractile proteins from, nor the enhanced expression of acetylcholine receptor genes in, the differentiating electrocyte is a simple consequence of the abundance of myogenic factor messages.

The effect of cycloheximide (CHX) on denervation-induced acetylcholine receptor (AChR) expression was investigated in chickens one day after nerve section, using probe excess solution hybridization to quantitate AChR alpha-subunit gene transcript levels and run-on analysis to measure subunit gene activity. The increase in alpha-subunit transcripts that normally follows denervation was prevented when drug treatment was commenced 2 h before or after denervation but was not blocked when CHX administration was begun 6 h after the operation. Drug-induced reduction of transcript levels results from decreased activity of genes coding for the alpha-, delta-, and gamma-subunits; in contrast, the transcription rates of several non-receptor genes are not affected by CHX. The results suggest that the de novo synthesis of a transcriptional activator is required as a mediating event in the signalling pathway linking the plasma membrane and AChR gene expression.