Hydrogel encapsulation to improve cell viability during syringe needle flow.

This work examines pluronic F-127 poloxamer for cell protection during injection through a syringe needle. Direct cell injection is a minimally invasive method for cell transplantation; however, it often results in poor cell viability. We proposed that encapsulating cells in this hydrogel would protect cells from detrimental mechanical forces during injection and increase cell viability. The hydrogel was tested at multiple weights and carbon nanobrush concentrations to determine how gel weight affects cell viability as well as to allow the gels to remain as electrically conductive scaffolds. This work assessed the ability of the hydrogel to prevent cell membrane bursting. We used D1 multipotent mouse bone marrow stromal precursor cells for this study. We found that the pressure drop increases with increasing weight of the gels. However, cell viability also increases as the weight of the gels increases. These results support the proposition that hydrogels can be used to protect cells during syringe needle injection. Since these hydrogels undergo a reverse phase transition, the gels can be used to transplant cells into the body in solution form through injection. The gels will then harden in situ to allow for cell proliferation and tissue regeneration at the desired site.

Engineering the extracellular matrix for clinical applications: endoderm, mesoderm, and ectoderm.

Tissue engineering is rapidly progressing from a research-based discipline to clinical applications. Emerging technologies could be utilized to develop therapeutics for a wide range of diseases, but many are contingent on a cell scaffold that can produce proper tissue ultrastructure. The extracellular matrix, which a cell scaffold simulates, is not merely a foundation for tissue growth but a dynamic participant in cellular crosstalk and organ homeostasis. Cells change their growth rates, recruitment, and differentiation in response to the composition, modulus, and patterning of the substrate on which they reside. Cell scaffolds can regulate these factors through precision design, functionalization, and application. The ideal therapy would utilize highly specialized cell scaffolds to best mimic the tissue of interest. This paper discusses advantages and challenges of optimized cell scaffold design in the endoderm, mesoderm, and ectoderm for clinical applications in tracheal transplant, cardiac regeneration, and skin grafts, respectively.

Tissue scaffold surface patterning for clinical applications.

Patterned scaffold surfaces provide a platform for highly defined cellular interactions, and have recently taken precedence in tissue engineering. Despite advances in patterning techniques and improved tissue growth, no clinical studies have been conducted for implantation of patterned biomaterials. Four major clinical application fields where patterned materials hold great promise are antimicrobial surfaces, cardiac constructs, neurite outgrowth, and stem cell differentiation. Specific examples include applications of patterned materials to (i) counter infection by antibiotic resistant bacteria, (ii) establish proper alignment and contractile force of regrown cardiac cells for repairing tissue damaged by cardiac infarction, (iii) increase neurite outgrowth for central nervous system wound repair, and (iv) host differentiated stem cells while preventing reversion to a pluripotent state. Moreover, patterned materials offer unique advantages for artificial implants which other constructs cannot. For example, by inducing selective cell adhesion using topographical cues, patterned surfaces present cellular orientation signals that lead to functional tissue architectures. Mechanical stimuli such as modulus, tension, and material roughness are known to influence tissue growth, as are chemical stimuli for cell adhesion. Scaffold surface patterns allow for control of these mechanical and chemical factors. This review identifies research advances in scaffold surface patterning, in light of pressing clinical needs requiring organization of cellular interactions.

Advances in biomaterials for the treatment of intervertebral disc degeneration.

In the past decade, the number of treatment methods for disc degeneration has dramatically increased due to advances in biomaterials. Disc degeneration is one of the leading causes of lower back pain in the adult population, and a large percentage of patients seek surgical solutions. Therefore, there is a great clinical need for biomaterials to alleviate pain associated with disc degeneration. Increased understanding of spinal physiology and pathophysiology has enabled the development of new surgical techniques as well as novel biomaterials for treatment. In this review, advances in biomaterials used for treating intervertebral disc degeneration are reviewed: cages and implants for spinal fusion, artificial discs for total disc arthroplasty, and emerging methods of synthetic nuclei and annulus repair. In addition, novel biomaterials are being explored to treat disc degeneration using a variety of treatment methods.

Regulation of inflammatory and lipid metabolism genes by eicosapentaenoic acid-rich oil.

Omega-3-PUFAs, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are associated with prevention of various aspects of metabolic syndrome. In the present studies, the effects of oil rich in EPA on gene expression and activation of nuclear receptors was examined and compared with other ω3-PUFAs. The EPA-rich oil (EO) altered the expression of FA metabolism genes in THP-1 cells, including stearoyl CoA desaturase (SCD) and FA desaturase-1 and -2 (FASDS1 and -2). Other ω3-PUFAs resulted in a similar gene expression response for a subset of genes involved in lipid metabolism and inflammation. In reporter assays, EO activated human peroxisome proliferator-activated receptor α (PPARα) and PPARß/γ with minimal effects on PPARγ, liver X receptor, retinoid X receptor, farnesoid X receptor, and retinoid acid receptor γ (RARγ); these effects were similar to that observed for purified EPA. When serum from a 6 week clinical intervention with dietary supplements containing olive oil (control), DHA, or two levels of EPA were applied to THP-1 cells, the expression of SCD and FADS2 decreased in the cells treated with serum from the ω3-PUFA-supplemented individuals. Taken together, these studies indicate regulation of gene expression by EO that is consistent with treating aspects of dyslipidemia and inflammation.

Pathway engineering strategies for production of beneficial carotenoids in microbial hosts.

Carotenoids, such as lycopene, ß-carotene, zeaxanthin, canthaxanthin and astaxanthin have many benefits for human health. In addition to the functional role of carotenoids as vitamin A precursors, adequate consumption of carotenoids prevents the development of a variety of serious diseases. Biosynthesis of carotenoids is a complex process and it starts with the common isoprene precursors. Condensation of these precursors and subsequent modifications, by introducing hydroxyl- and keto-groups, leads to the generation of diversified carotenoid structures. To improve carotenoid production, metabolic engineering has been explored in bacteria, yeast, and algae. The success of the pathway engineering effort depends on the host metabolism, specific enzymes used, the enzyme expression levels, and the strategies employed. Despite the difficulty of pathway engineering for carotenoid production, great progress has been made over the past decade. We review metabolic engineering approaches used in a variety of microbial hosts for carotenoid biosynthesis. These advances will greatly expedite our efforts to bring the health benefits of carotenoids and other nutritional compounds to our diet.

Carbon nanobrush-containing poloxamer hydrogel composites for tissue regeneration.

The objective of this study was to examine potential uses for electrically conductive hydrogel composites in tissue engineering and tissue regeneration, and to explore the composites as a growth matrix for clinically relevant cell lines. The composite was comprised of carbon nanobrushes embedded in a biocompatible poloxamer gel. In this study, we assessed the ability of such composite gels to support the growth of fibroblasts and myocytes and eventually serve as a matrix to stimulate wound closure. In such a model, fibroblasts and myocytes are seeded on the hydrogel and bathed in culture medium. The experimental model assesses the ability of fibroblasts and myocytes to grow into and adhere to the gel. The results of this study demonstrate that carbon nanobrushes can be dispersed within poloxamer gels and that fibroblasts and myocytes can proliferate within homogenously dispersed carbon nanobrush-containing poloxamer gels. We also examined the effects of carbon nanobrush content on the rheological properties of the poloxamer gel matrix; improvement occurred in several areas in the presence of carbon nanobrushes. Our future studies will investigate the effects of design parameters such as carbon nanobrush content and matrix structure on wound healing, as well as the growth of tendons and other cell lines within the hydrogel composites. In general, this work has relevance for tissue and cellular engineering and tissue regeneration in clinical medicine.

Derivation and application of a mathematical model for long bone growth.

The objective of this work was to develop a mathematical model of long bone growth and to gain insights regarding growth disorders. A cell balance (mass balance of moving cells) assessment was performed on the three regions of the growth plate, to determine the variables (including number of proliferating cells, and division rate of proliferating cells) that influence tibia growth rate. Once this relationship was established, clinical data were used to understand how tibia growth rate and number of proliferating cells change with time. These equations were then inserted into the model to determine how cell division rate changes with time. The model was utilized to determine the influence of growth time, and to measure changes in vitamin C deficiency, Indian hedgehog (IHH) expression, and bone morphogenetic protein-2 (BMP-2) implants on tibia length. According to the model, a 10-month discrepancy in growth time between the two tibias is required to produce clinically significant leg asymmetry. In addition, vitamin C deficiency, IHH overexpression, and BMP-2 implants can all affect tibia length. These bioactive molecules have the greatest effect on tibia growth rate when these perturbations occur early in life for extended periods of time. The results are significant for modeling and predicting the effects of perturbations, including bioactive implants, on long bone growth.

Metabolic engineering for the production of clinically important molecules: Omega-3 fatty acids, artemisinin, and taxol.

Driven by requirements for sustainability as well as affordability and efficiency, metabolic engineering of plants and microorganisms is increasingly being pursued to produce compounds for clinical applications. This review discusses three such examples of the clinical relevance of metabolic engineering: the production of omega-3 fatty acids for the prevention of cardiovascular disease; the biosynthesis of artemisinic acid, an anti-malarial drug precursor, for the treatment of malaria; and the production of the complex natural molecule taxol, an anti-cancer agent. In terms of omega-3 fatty acids, bioengineering of fatty acid metabolism by expressing desaturases and elongases, both in soybeans and oleaginous yeast, has resulted in commercial-scale production of these beneficial molecules. Equal success has been achieved with the biosynthesis of artemisinic acid at low cost for developing countries. This is accomplished through channeling the flux of the isoprenoid pathway to the specific genes involved in artemisinin biosynthesis. Efficient coupling of the isoprenoid pathway also leads to the construction of an Escherichia coli strain that produces a high titer of taxadiene-the first committed intermediate for taxol biosynthesis. These examples of synthetic biology demonstrate the versatility of metabolic engineering to bring new solutions to our health needs.

Sealing and healing of clear corneal incisions with an improved dextran aldehyde-PEG amine tissue adhesive.

PURPOSE: To create a non-cytotoxic, spontaneously curing tissue adhesive that is strongly bonding and persistent enough that 1-2 µL is capable of sealing a clear corneal incision throughout the first five days of healing. METHODS: A novel prototype delivery device capable of delivering 1-2 µL of a two-component adhesive delivered aqueous solutions of dextran aldehyde and star PEG amine, which mixed by diffusion and crosslinked to form an adhesive hydrogel. Adhesive hydrogels were tested for rates of degradation in phosphate-buffered saline, leak pressures when used to seal clear corneal incisions in enucleated rabbit eyes, and in vitro cytotoxicity when placed in contact with NIH3T3 fibroblast cells. Two formulations were used in vivo to seal clear corneal incisions in New Zealand White rabbits. Wound integrity after 1, 3, 5 and 7 days of healing was assessed by measuring the leak pressures of enucleated eyes. RESULTS: Tissue adhesives formed by combining aqueous solutions of dextran aldehyde (MW 10,000, 50% oxidized) and an 8-arm star poly(ethylene glycol) (MW 10,000) having two primary amine groups at the end of each arm gave mean leak pressures as high as 141 ± 35 mm Hg and exhibited no in vitro cytotoxicity. When 1-2 µL was used in vivo to seal clear corneal incisions in New Zealand White rabbits, the adhesive maintained an eye leak pressure of at least 120 mm Hg and remained visibly present at the wound site for 5 days. CONCLUSIONS: The combination of an 8-arm star poly(ethylene glycol), MW 10,000, having two primary amine groups at the end of each arm and dextran aldehyde (MW 10,000, 50% oxidized) forms a tissue adhesive that cures spontaneously, is non-cytotoxic, and is strongly bonding and persistent enough that 1-2 µL is capable of sealing a clear corneal incision through the first 5 days of healing.

A disease-centered approach to biomaterials education and medical device design.

This paper describes the development of a novel elective course in biomaterials which integrates clinical medicine with engineering principles. In this educational approach, students are first introduced to disease pathologies and clinical needs, and then exposed to engineering technologies that can fulfill unmet needs. The course is directed toward the question, "Where are clinical needs most urgent, and how can engineering be applied to meet those needs?" This clinically-oriented, disease-centered approach is valuable for science and engineering education, as it relays to students the centrality of engineering in solving the world's most pressing healthcare problems.

Tissue engineering for clinical applications.

Tissue engineering is increasingly being recognized as a beneficial means for lessening the global disease burden. One strategy of tissue engineering is to replace lost tissues or organs with polymeric scaffolds that contain specialized populations of living cells, with the goal of regenerating tissues to restore normal function. Typical constructs for tissue engineering employ biocompatible and degradable polymers, along with organ-specific and tissue-specific cells. Once implanted, the construct guides the growth and development of new tissues; the polymer scaffold degrades away to be replaced by healthy functioning tissue. The ideal biomaterial for tissue engineering not only defends against disease and supports weakened tissues or organs, it also provides the elements required for healing and repair, stimulates the body's intrinsic immunological and regenerative capacities, and seamlessly interacts with the living body. Tissue engineering has been investigated for virtually every organ system in the human body. This review describes the potential of tissue engineering to alleviate disease, as well as the latest advances in tissue regeneration. The discussion focuses on three specific clinical applications of tissue engineering: cardiac tissue regeneration for treatment of heart failure; nerve regeneration for treatment of stroke; and lung regeneration for treatment of chronic obstructive pulmonary disease.

Energetics of association in poly(lactic acid)-based hydrogels with crystalline and nanoparticle-polymer junctions.

We report the energetics of association in polymeric gels with two types of junction points: crystalline hydrophobic junctions and polymer-nanoparticle junctions. Time-temperature superposition (TTS) of small-amplitude oscillatory rheological measurements was used to probe crystalline poly(L-lactide) (PLLA)-based gels with and without added laponite nanoparticles. For associative polymer gels, the activation energy derived from the TTS shift factors is generally accepted as the associative strength or energy needed to break a junction point. Our systems were found to obey TTS over a wide temperature range of 15-70 °C. For systems with no added nanoparticles, two distinct behaviors were seen, with a transition occurring at a temperature close to the glass transition temperature of PLLA, T(g). Above T(g), the activation energy was similar to the PLLA crystallization enthalpy, suggesting that the activation energy is related to the energy needed to pull a PLLA chain out of the crystalline domain. Below T(g), the activation energy is expected to be the energy required to increase mobility of the polymer chains and soften the glassy regions of the PLLA core. Similar behavior was seen in the nanocomposite gels with added laponite; however, the added clay appears to reduce the average value of the activation enthalpy. This confirms our SAXS results and suggests that laponite particles are participating in the network structure.

Poly(vinyl alcohol) acetoacetate-based tissue adhesives are non-cytotoxic and non-inflammatory.

Polymer-based tissue adhesives composed of poly(vinyl alcohol) acetoacetate (PVOH acac) and cross-linking amines were investigated for their effects on cell survival and inflammatory cell activation using in vitro mouse cell cultures. Cytotoxicity of tissue adhesives was evaluated by placing adhesives in direct contact with 3T3 fibroblast cells. Tissue adhesives formulated from PVOH acac and 3-aminopropyltrialkoxysilane (APS) were non-cytotoxic to fibroblasts; adhesives formulated from PVOH acac and aminated poly(vinyl alcohol) (PVOH amine) were also non-cytotoxic to fibroblasts. In contrast, a commercial adhesive composed of 2-octyl cyanoacrylate was highly cytotoxic to fibroblasts. The inflammatory potential of tissue adhesives was evaluated by exposing J774 macrophage cells to adhesives, and measuring TNF-alpha release from macrophages. PVOH acac-based tissue adhesives did not elicit inflammatory TNF-alpha release from macrophages. These results suggest that PVOH acac-based tissue adhesives are non-cytotoxic and non-inflammatory. Such tissue adhesives represent a promising technology for a variety of medical applications, including surgical wound closure and tissue engineering, and the results are also significant in the design of in vitro cell culture systems to study biomaterials.

Effect of pharmaceuticals on thermoreversible gelation of PEO-PPO-PEO copolymers.

Pluronic F127, a triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), has generated considerable interest as a drug delivery vehicle due to its ability to gel at physiological temperatures. This work examines the gelation behavior of Pluronic F127 in the presence of a series of hydrophobic pharmaceuticals, to determine whether there is any correlation between gelation and physicochemical parameters of drug solutes. The study includes the local anesthetics dibucaine, lidocaine, and tetracaine; the pharmaceutical additives methyl paraben, ethyl paraben, and propyl paraben; the anti-cancer agents paclitaxel and baccatin III; and the anti-inflammatory agent sulindac. The results indicate that the presence of local anesthetics and pharmaceutical additives allows F127 solutions to form gels at lower copolymer concentrations; local anesthetics and pharmaceutical additives also shift gelation down to a lower gelation temperature. This behavior is strongly dependent on drug solubility; poorly soluble drugs (paclitaxel, baccatin III, sulindac) do not change the lower gelation temperature or minimum F127 concentration for gelation. An equation relating the decrease in gelation temperature to drug solubility is presented, and the equation fits the data well. The results have significant positive implications on the toxicity and economic issues related to use of Pluronic F127 in drug delivery.