In vivo effects of tailored laminin-332 α3 conjugated scaffolds enhances wound healing:a histomorphometric analysis

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides. In our previous work, we have shown the tethering of laminin-332 α3 chain to type I collagen scaffold using microbial transglutaminase (mTGase), promotes cell adhesion, migration, and proliferation. In this study, we evaluated the wound healing properties of tailored laminin-332 α3 chain (peptide A: PPFLMLLKGSTR) tethered to a type I collagen scaffold using mTGase by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide B: PPFLMLLKGSTREAQQIVM) or lysine (peptide C: PPFLMLLKGSTRKKKKG) in rat full-thickness wound model at two different time points (7 and 21 days). Histological evaluations were assessed for wound closure, epithelialization, angiogenesis, inflammatory, fibroblastic cellular infiltrations, and quantified using stereological methods (p < 0.05). Peptide A and B tethered to collagen scaffold using mTGase stimulated neovascularization, decreased the inflammatory cell infiltration and prominently enhanced the fibroblast proliferation which significantly accelerated the wound healing process. We conclude that surface modification by incorporating motif of laminin-332 α3 chain (peptide A: PPFLMLLK GSTR) domain and transglutaminase substrate to the laminin-332 α3 chain (peptide B: PPFLMLLKGSTREAQQIVM) using mTGase may be a potential candidate for tissue engineering applications and skin regeneration. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A:2788-2795, 2013. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Fabrication and characterization of macroporous poly(ethylene glycol) hydrogels generated by several types of porogens

Polymer scaffolds play an important role in tissue engineering applications. Poly(ethylene glycol) based hydrogels have received a lot of attention in this field because of their high biocompatibility and ease of processing. However, in many cases they do not exhibit proper tissue invasion and nutrient transport because of their dense structure. In the present work, several approaches were developed and compared to each other to produce interconnected macroporous poly(ethylene glycol) hydrogels by including different types of porogens in the photocrosslinking reaction. The swelling capacity of the resulting hydrogels was analyzed and compared to non-porous hydrogel samples. Moreover, the obtained materials were characterized by means of mechanical properties and porosity using rheometry, scanning electron microscopy, and mercury intrusion porosimetry. Results showed that interconnected and uniform pores were obtained when a porogen template was used during hydrogel fabrication by photocrosslinking. On the other side, when the porogen particles were dispersed into the macromer solution before matrix photocrosslinking the interconnexion was negligible. The templates must be dissolved before the hydrogel's cell-seeding in vitro, while the dispersed porogen can be used in situ in the in vitro seeding tests. Copyright © 2013 Taylor & Francis Group, LLC.

Enhanced anchorage of tissue-engineered cartilage using an osteoinductive approach

Articular cartilage injuries occur frequently in the knee joint. Several methods have been implemented clinically, to treat osteochondral defects but none have been able to produce a long term, durable solution. Photopolymerizable cartilage tissue engineering approaches appear promising; however, fundamentally, forming a stable interface between the tissue engineered cartilage and native tissue, mainly subchondral bone and native cartilage, remains a major challenge. The overall objective of this research is to find a solution for the current problem of dislodgment of tissue engineered cartilage at the defect site for the treatment of degraded cartilage that has been caused due to knee injuries or because of mild to moderate level of osteoarthritis. For this, an in-vitro model was created to analyze the integration of tissue engineered cartilage with the bone, healthy and diseased cartilage over time. We investigated the utility of hydroxyapatite (HA) nanoparticles to promote controlled bone-growth across the bone-cartilage interface in an in vitro engineered tissue model system using bone marrow derived stem cells. We also investigated the application of HA nanoparticles to promote enhance integration between tissue engineered cartilage and native cartilage both in healthy and diseased states. Samples incorporated with HA demonstrated significantly higher interfacial shear strength (at the junction between engineered cartilage and engineered bone and also with diseased cartilage) compared to the constructs without HA (p < 0.05), after 28 days of culture. These findings indicate that the incorporation of HA nanoparticles permits more stable anchorage of the injectable hydrogel-based engineered cartilage construct via augmented integration between bone and cartilage.^

Movement Effects on the Flow Physics and Nutrient Delivery in Engineered Valvular Tissues

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering (HVTE) owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by steady flow and scaffold flexure play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). In addition, scaffold flexure may enhance nutrient (e.g. oxygen, glucose) transport. In this study, we computationally quantified the i) magnitude of fluid-induced shear stresses; ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter, and iii) glucose and oxygen mass transport profiles. Noting that sample cyclic flexure induces a high degree of oscillatory shear stress (OSS), we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: 1) no flow, no flexure (control group), 2) steady flow-alone, 3) cyclic flexure-alone and 4) combined steady flow and cyclic flexure environments. We also coupled a diffusion and convention mass transport equation to the simulated system. We found that the coexistence of both OSS and appreciable shear stress magnitudes, described by the newly introduced parameter OSI-t , explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress. In addition, our simulations showed that mass transport of glucose and oxygen is enhanced by sample movement at low sample porosities, but did not play a role in highly porous scaffolds. Preliminary in-house in vitro experiments showed that cell proliferation and phenotype is enhanced in OSI-t environments.

Engineering Highly-functional, Self-regenerative Skeletal Muscle Tissues with Enhanced Vascularization and Survival in Vivo

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

(Table T1) Density, porosity, void ratio, diffusivity and permeability of ODP Site 185-1149 sediments

The subduction of oceanic plates regulates crustal growth, influences arc volcanism, and refertilizes the mantle. Continental growth occurs by subduction of crustal material (seawater components, marine sediments, and basaltic crust). The geochemical and physical evolution of the Earth's crust depends, in large part, on the fate of subducted material at convergent margins (Armstrong, 1968, doi:10.1029/RG006i002p00175; Karig and Kay, 1981, 10.1098/rsta.1981.0108). The crustal material on the downgoing plate is recycled to various levels in the subduction zone. The recycling process that takes place in the "Subduction Factory" is difficult to observe directly but is clearly illuminated using chemical tracers. Von Huene and Scholl (1991, doi:10.1029/91RG00969) and Plank and Langmuir (1993, doi:10.1038/362739a0) preliminarily calculated a large flux of subducted materials. By mass balancing the chemical tracers and measuring the fractionations that occur between them, the Subduction Factory work and the effect on the Earth's evolution can be estimated. In order to elucidate this mass balance, Ocean Drilling Program Leg 185 drilled two deepwater shales into the oceanic crust situated in the Mariana-Izu Trenches and recovered core samples of incoming oceanic crust. The calculations of mass circulation in the subduction zone, however, did not take into account the mass transfer properties within subducted oceanic crust, although the dewatering fluid and diffused ions may play an important role in various activities such as seismogeneity, serpentine diapiring, and arc volcanism. Thus, this paper focuses on the quantitative measurements of the physical and mass transfer properties of subducted oceanic crust.

Tissue-specific bioscaffolds for adipose-derived stem/stromal cell expansion and delivery

Decellularized adipose tissue (DAT) is a promising biomaterial for soft tissue regeneration, and it provides a highly conducive microenvironment for human adipose-derived stem/stromal cell (ASC) attachment, proliferation, and adipogenesis. This thesis focused on developing techniques to fabricate 3-D bioscaffolds from enzymatically-digested DAT as platforms for ASC culture and delivery in adipose tissue engineering and large-scale ASC expansion. Initial work investigated chemically crosslinked microcarriers fabricated from pepsin-digested DAT as injectable adipo-inductive substrates for ASCs. DAT microcarriers highly supported ASC adipogenesis compared to gelatin microcarriers in a CELLSPIN system, as confirmed by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, lipid accumulation, and endpoint RT-PCR. ASCs cultured on DAT microcarriers in proliferation medium also had elevated PPARγ, C/EBPα, and LPL expression which suggested adipo-inductive properties. In vivo testing of the DAT microcarriers exhibited stable volume retention and enhanced cellular infiltration, tissue remodeling, and angiogenesis. Building from this work, non-chemically crosslinked porous foams and bead foams were fabricated from α-amylase-digested DAT for soft tissue regeneration. Foams were stable and strongly supported ASC adipogenesis based on GPDH activity and endpoint RT-PCR. PPARγ, C/EBPα, and LPL expression in ASCs cultured on the foams in proliferation media indicated adipo-inductive properties. Foams with Young’s moduli similar to human fat also influenced ASC adipogenesis by enhanced GPDH activity. In vivo adipogenesis accompanied by a potent angiogenic response and rapid resorption showed their potential use in wound healing applications. Finally, non-chemically crosslinked porous microcarriers synthesized from α-amylase-digested DAT were investigated for ASC expansion. DAT microcarriers remained stable in culture and supported significantly higher ASC proliferation compared to Cultispher-S microcarriers in a CELLSPIN system. ASC immunophenotype was preserved for all expanded groups, with reduced adhesion marker expression under dynamic conditions. DAT microcarrier expansion upregulated ASC expression of early adipogenic (PPARγ, LPL) and chondrogenic (COMP) markers without inducing a mature phenotype. DAT microcarrier expanded ASCs also showed similar levels of adipogenesis and osteogenesis compared to Cultispher-S despite a significantly higher population fold-change, and had the highest level of chondrogenesis among all groups. This study demonstrates the promising use of DAT microcarriers as a clinically relevant strategy for ASC expansion while maintaining multilineage differentiation capacity.

Production of Polyhydroxyalkanoates by Pseudomonas mendocina using vegetable oils and their characterisation

Synthesis of Polyhydroxyalkanoates (PHAs) by Pseudomonas mendocina, using different vegetable oils such as, coconut oil, groundnut oil, corn oil and olive oil, as the sole carbon source was investigated for the first time. The PHA yield obtained was compared with that obtained during the production of PHAs using sodium octanoate as the sole carbon source. The fermentation profiles at shaken flask and bioreactor levels revealed that vegetable oils supported the growth of Pseudomonas mendocina and PHA accumulation in this organism. Moreover, when vegetable oil (coconut oil) was used as the sole carbon source, fermentation profiles showed better growth and polymer production as compared to conditions when sodium octanoate was used as the carbon source. In addition, comparison of PHA accumulation at shaken flask and fermenter level confirmed the higher PHA yield at shaken flask level production. The highest cell mass found using sodium octanoate was 1.8 g/L, whereas cell mass as high as 5.1 g/L was observed when coconut oil was used as the feedstock at flask level production. Moreover, the maximum PHA yield of 60.5% dry cell weight (dcw) was achieved at shaken flask level using coconut oil as compared to the PHA yield of 35.1% dcw obtained using sodium octanoate as the sole carbon source. Characterisations of the chemical, physical, mechanical, surface and biocompatibility properties of the polymers produced have been carried out by performing different analyses as described in the second chapter of this study. Chemical analysis using GC and FTIR investigations showed medium chain length (MCL) PHA production in all conditions. GC-MS analysis revealed a unique terpolymer production, containing 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid when coconut oil, groundnut oil, olive oil, and corn oil were used as the carbon source. Whereas production of the homopolymer containing 3-hydroxyoctanoic acid was observed when sodium octanoate was used as the carbon source. MCL-PHAs produced in this study using sodium octanoate, coconut oil, and olive oil exhibited melting transitions, indicating that each of the PHA was crystalline or semi-crystalline polymer. In contrast, the thermal properties of PHAs produced from groundnut and corn oils showed no melting transition, indicating that they were completely amorphous or semi-crystalline, which was also confirmed by the X-Ray Diffraction (XRD) results obtained in this study. Mechanical analysis of the polymers produced showed higher stiffness of the polymer produced from coconut oil than the polymer from sodium octanoate. Surface characterisation of the polymers using Scanning Electron Microscopy (SEM) revealed a rough surface topography and surface contact angle measurement revealed their hydrophobic nature. Moreover, to investigate the potential applicability of the produced polymers as the scaffold materials for dental pulp regeneration, multipotent human Mesenchymal stem cells (hMSCs) were cultured onto the polymer films. Results indicated that these polymers are not cytotoxic towards the hMSCs and could support their attachment and proliferation. Highest cell growth was observed on the polymer samples produced from corn oil, followed by the polymer produced using coconut oil. In conclusion, this work established, for the first time, that vegetable oils are a good economical source of carbon for production of MCL-PHA copolymers effectively by Pseudomonas mendocina. Moreover, biocompatibility studies suggest that the produced polymers may have potential for dental tissue engineering application.

Cimentos injetáveis à base de fosfatos de cálcio para vertebroplastia

O principal objectivo desta investigação foi o desenvolvimento cimentos de fosfatos de cálcio com injetabilidade melhorada e propriedades mecânicas adequadas para aplicação em vertebroplastia. Os pós de fosfato de tricálcico (TCP) não dopados e dopados (Mg, Sr e Mn) usados neste estudo foram obtidos pelo processo de precipitação em meio aquoso, seguidos de tratamento térmico de forma a obter as fases pretendidas, α− e β−TCP. A substituição parcial de iões Ca por iões dopantes mostrou ter implicações em termos de estabilidade térmica da fase β−TCP. Os resultados demonstraram que as transformações de fase alotrópicas β↔α−TCP são fortemente influenciadas por variáveis experimentais como a taxa de arrefecimento, a presença de impurezas de pirofosfato de cálcio e a extensão do grau de dopagem com Mg. Os cimentos foram preparados através da mistura de pós, β−TCP (não dopados e dopados) e fosfato monocálcico monidratado (MCPM), com meios líquidos diferentes usando ácido cítrico e açucares (sucrose e frutose) como agentes retardadores de presa, e o polietilenoglicol, a hidroxipropilmetilcelulose e a polivinilpirrolidona como agentes gelificantes. Estes aditivos, principalmente o ácido cítrico, e o MCPM aumentam significativamente a força iónica do meio, influenciando a injetabilidade das pastas. Os resultados também mostraram que a distribuição de tamanho de partícula dos pós é um factor determinante na injetabilidade das pastas cimentícias. A combinação da co-dopagem de Mn e Sr com a adição de sucrose no líquido de presa e com uma distribuição de tamanho de partícula dos pós adequada resultou em cimentos de brushite com propriedades bastante melhoradas em termos de manuseamento, microestrutura, comportamento mecânico e biológico: (i) o tempo inicial de presa passou de ~3 min to ~9 min; (ii) as pastas cimentícias foram totalmente injectadas para uma razão liquido/pó de 0.28 mL g−1 com ausência do efeito de “filter-pressing” (separação de fases líquida e sólida); (iii) após imersão numa solução durante 48 h, as amostras de cimento molhadas apresentam uma porosidade total de ~32% e uma resistência a compressão de ~17 MPa, valor muito superior ao obtido para os cimentos sem açúcar não dopados (5 MPa) ou dopados só com Sr (10 MPa); e (iv) o desempenho biológico, incluindo a adesão e crescimento de células osteoblásticas na superfície do cimento, foi muito melhorado. Este conjunto de propriedades torna os cimentos excelentes para regeneração óssea e engenharia de tecidos, e muito promissores para aplicação em vertebroplastia.

Scaffolds porosos à base de fosfatos de cálcio para regeneração óssea

Graças ao aumento da esperança média de vida do ser humano, a engenharia de tecidos tem sido uma área alvo de enorme investigação. A utilização de estruturas tridimensionais porosas e biodegradáveis, denominadas de scaffolds, como matriz para a adesão e proliferação celular tem sido amplamente investigada. Existem atualmente diversas técnicas para a produção destas estruturas mas o grau de exigência tem vindo a aumentar, existindo ainda lacunas que necessitam ser preenchidas. A técnica de robocasting consiste numa deposição camada a camada de uma pasta coloidal, seguindo um modelo computorizado (CAD) e permite a produção de scaffolds com porosidade tamanho de poro e fração de porosidade controlados, boa reprodutibilidade, e com formas variadas, as quais podem ser idênticas às dos defeitos ósseos a preencher. O presente estudo teve como objetivo produzir scaffolds porosos à base de fosfatos de cálcio através de robocasting. Para tal, foram estudadas duas composições de pós à base de β-TCP, uma pura e outra co-dopada com estrôncio, zinco e manganês. Inicialmente os pós foram sintetizados pelo método de precipitação química por via húmida. Após a síntese, estes foram filtrados, secos, calcinados a 1000ºC e posteriormente moídos até possuírem um tamanho médio de partícula de cerca de 1,5 μm. Os pós foram depois peneirados com uma malha de 40μm e caracterizados. Posteriormente foram preparadas várias suspensões e avaliado o seu comportamento reológico, utilizando Targon 1128 como dispersante, Hidroxipropilmetilcelulose (HPMC) como ligante e polietilenimina (PEI) como agente floculante. Por fim, e escolhida a melhor composição para a formação da pasta, foram produzidos scaffolds com diferentes porosidades, num equipamento de deposição robótica (3D Inks, LLC). Os scaffolds obtidos foram secos à temperatura ambiente durante 48 horas, sinterizados a 1100ºC e posteriormente caracterizados por microscopia eletrónica de varrimento (SEM), avaliação dos tamanhos de poro, porosidade total e testes mecânicos. Ambas as composições estudadas puderam ser transformadas em pastas extrudíveis, mas a pasta da composição pura apresentou uma consistência mais próxima do ideal, tendo originado scaffolds de melhor qualidade em termos de microestrutura e de propriedades mecânicas.

Scaffolds for controlled release of cartilage growth factors

International audience

Adipose-derived stem cell adhesion on laminin-coated microcarriers improves commitment toward the cardiomyogenic lineage

International audience

Survival, differentiation, and neuroprotective mechanisms of human stem cells complexed with neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo model of Parkinson's disease

International audience

Wood-derived biomaterials for biomedical applications

Driven by the global trend in the sustainable economy development and environmental concerns, the exploring of plant-derived biomaterials or biocomposites for potential biomedical and/or pharmaceutical applications has received tremendous attention. Therefore, the work of this thesis is dedicated to high-value and high-efficiency utilization of plant-derived materials, with the focus on cellulose and hemicelluloses in the field of biomedical applications in a novel biorefinery concept. The residual cellulose of wood processing waste, sawdust, was converted into cellulose nanofibrils (CNFs) with tunable surface charge density and geometric size through 2,2,6,6-tetramethylpiperidinyloxy (TEMPO)-mediated oxidation and mechanical defibrillation. The sawdust-based CNFs and its resultant free-standing films showed comparable or even better mechanical properties than those from a commercial bleached kraft pulp at the same condition, demonstrating the feasibility of producing CNFs and films thereof with outstanding mechanical properties from birch sawdust by a process incorporated into a novel biorefinery platform recovering also polymeric hemicelluloses for other applications. Thus, it is providing an efficient route to upgrade sawdust waste to valuable products. The surface charge density and geometric size of the CNFs were found to play key roles in the stability of the CNF suspension, as well as the gelling properties, swelling behavior, mechanical stiffness, morphology and microscopic structural properties, and biocompatibility of CNF-based materials (i.e. films, hydrogels, and aerogels). The CNFs with tunable surface chemistry and geometric size was found promising applications as transparent and tough barrier materials or as reinforcing additive for production of biocomposites. The CNFs was also applied as structural matrices for the preparation of biocomposites possessing electrical conductivity and antimicrobial activity by in situ polymerization and coating of polypyrrole, and incorporation of silver nanoparticles, which make the material possible for potential wound healing application. The CNF-based matrices (films, hydrogels, and aerogels) with tunable structural and mechanical properties and biocompatibility were further prepared towards an application as 3D scaffolds in tissue engineering. The structural and mechanical strength of the CNF matrices could be tuned by controlling the charge density of the nanocellulose, as well as the pH and temperature values of the hydrogel formation conditions. Biological tests revealed that the CNF scaffolds could promote the survival and proliferation of tumor cells, and enhance the transfection of exogenous DNA into the cells, suggesting the usefulness of the CNF-based 3D matrices in supporting crucial cellular processes during cell growth and proliferation. The CNFs was applied as host materials to incorporate biomolecules for further biomedical application. For example, to investigate how the biocompatibility of a scaffold is influenced by its mechanical and structural properties, these properties of CNF-based composite matrices were controlled by incorporation of different hemicelluloses (O-acetyl galactoglucomanan (GGM), xyloglucan (XG), and xylan) into CNF hydrogel networks in different ratios and using two different approaches. The charge density of the CNFs, the incorporated hemicellulose type and amount, and the swelling time of the hydrogels were found to affect the pore structure, the mechanical strength, and thus the cells growth in the composite hydrogel scaffolds. The mechanical properties of the composite hydrogels were found to have an influence on the cell viability during the wound healing relevant 3T3 fibroblast cell culture. The thusprepared CNF composite hydrogels may work as promising scaffolds in wound healing application to provide supporting networks and to promote cells adhesion, growth, and proliferation.

Movement effects on the flow physics and nutrient delivery in engineered valvular tissues

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering (HVTE) owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by steady flow and scaffold flexure play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). In addition, scaffold flexure may enhance nutrient (e.g. oxygen, glucose) transport. In this study, we computationally quantified the i) magnitude of fluid-induced shear stresses; ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter, and iii) glucose and oxygen mass transport profiles. Noting that sample cyclic flexure induces a high degree of oscillatory shear stress (OSS), we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: 1) no flow, no flexure (control group), 2) steady flow-alone, 3) cyclic flexure-alone and 4) combined steady flow and cyclic flexure environments. We also coupled a diffusion and convention mass transport equation to the simulated system. We found that the coexistence of both OSS and appreciable shear stress magnitudes, described by the newly introduced parameter OSI-:τ: explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress. In addition, our simulations showed that mass transport of glucose and oxygen is enhanced by sample movement at low sample porosities, but did not play a role in highly porous scaffolds. Preliminary in-house in vitro experiments showed that cell proliferation and phenotype is enhanced in OSI-:τ: environments.^