Bioreactor Systems for Tissue Engineering

The hydrodynamic environment “created” by bioreactors for the culture of a tissue engineered construct (TEC) is known to influence cell migration, proliferation and extra cellular matrix production. However, tissue engineers have looked at bioreactors as black boxes within which TECs are cultured mainly by trial and error, as the complex relationship between the hydrodynamic environment and tissue properties remains elusive, yet is critical to the production of clinically useful tissues. It is well known in the chemical and biotechnology field that a more detailed description of fluid mechanics and nutrient transport within process equipment can be achieved via the use of computational fluid dynamics (CFD) technology. Hence, the coupling of experimental methods and computational simulations forms a synergistic relationship that can potentially yield greater and yet, more cohesive data sets for bioreactor studies. This review aims at discussing the rationale of using CFD in bioreactor studies related to tissue engineering, as fluid flow processes and phenomena have direct implications on cellular response such as migration and/or proliferation. We conclude that CFD should be seen by tissue engineers as an invaluable tool allowing us to analyze and visualize the impact of fluidic forces and stresses on cells and TECs.

Assimilating cell sheets and hybrid scaffolds for dermal tissue engineering

Cell sheets can be used to produce neo-tissue with mature extracellular matrix. However, extensive contraction of cell sheets remains a problem. We devised a technique to overcome this problem and applied it to tissue engineer a dermal construct. Human dermal fibroblasts were cultured with poly(lactic-co-glycolic acid)-collagen meshes and collagen-hyaluronic acid foams. Resulting cell sheets were folded over the scaffolds to form dermal constructs. Human keratinocytes were cultured on these dermal constructs to assess their ability to support bilayered skin regeneration. Dermal constructs produced with collagen-hyaluronic acid foams showed minimal contraction, while those with poly(lactic-co-glycolic acid)-collagen meshes curled up. Cell proliferation and metabolic activity profiles were characterized with PicoGreen and AlamarBlue assays, respectively. Fluorescent labeling showed high cell viability and F-actin expression within the constructs. Collagen deposition was detected by immunocytochemistry and electron microscopy. Transforming Growth Factor-alpha and beta1, Keratinocyte Growth Factor and Vascular Endothelial Growth Factor were produced at various stages of culture, measured by RT-PCR and ELISA. These results indicated that assimilating cell sheets with mechanically stable scaffolds could produce viable dermal-like constructs that do not contract. Repeated enzymatic treatment cycles for cell expansion is unnecessary, while the issue of poor cell seeding efficiency in scaffolds is eliminated.

The effectiveness of a short-term group music therapy intervention for parents who have a child with a disability

The relationship between the quality of parent-child interactions and positive child developmental trajectories is well established (Guralnick, 2006; Shonkoff & Meissels, 2000; Zubrick et al., 2008). However, a range of parental, family, and socio-economic factors can pose risks to parents’ capacity to participate in quality interactions with their children. In particular, families with a child with a disability have been found to have higher levels of parenting stress, and are more likely to experience economic disadvantage, as well as social isolation. The importance of early interventions to promote positive parenting and child development for these families is widely recognised (Shonkoff & Meissels, 2000). However, to date, there is a lack of evidence about the effectiveness of early parenting programs for families who have a young child with a disability. This thesis investigates the impact of a music therapy parenting program, Sing & Grow, on 201 parent-child dyads who attended programs specifically targeted to parents who had a young child with a disability. Sing & Grow is an Australian national early parenting intervention funded by the Australian Government Department of Families, Housing, Community Services and Indigenous Affairs and delivered by Playgroup Queensland. It is designed and delivered by Registered Music Therapists for families with children aged from birth to three years. It aims to improve parenting skills and confidence, improve family functioning (positive parent-child interactions), enhance child development, and provide social networking opportunities to socially isolated families. The intervention targets a range of families in circumstances that have the potential to impact negatively on family functioning. This thesis uses data from the National Evaluation Study of Sing & Grow from programs which were targeted at families who had a young child with a disability. Three studies were conducted to address the objectives of this thesis. Study 1 examines the effects of the Sing & Grow intervention on parent reported pre and post parent mental health, parenting confidence, parenting skills, and child development, and other parent reported outcomes including social support, use of intervention resources, satisfaction with the intervention and perceived benefits of and barriers to participation. Significant improvements from pre to post were found for parent mental health and parent reported child communication and social skills, along with evidence that parents were very satisfied with the program and that it brought social benefits to families. Study 2 explored the pre to post effects of the intervention on children’s developmental skills and parent-child interactions using observational ratings made by clinicians. Significant pre to post improvements were found for parenting sensitivity, parental engagement with child and acceptance of child as well as for child responsiveness to parent, interest, and participation in the intervention, and social skills. Study 3 examined the nature of child and family characteristics that predicted better outcomes for families while taking account of the level of participation in the program. An overall outcome index was calculated and served as the dependent variable in a logistic regression analysis. Families who attended six or more sessions and mothers who had not completed high school were more likely to have higher outcome scores at post intervention than those who attended fewer sessions and those with more educated mothers respectively. The findings of this research indicate that the intervention had a positive impact on participants’ mental health, parenting behaviours and child development and that level of attendance was associated with better outcomes. There was also evidence that the program reached its target of high risk families (i.e., families in which mothers had lower educational levels) and that for these families better outcomes were achieved. There were also indications that the program was accessible and highly regarded by families and that it promoted social connections for participants. A theoretical model of how the intervention is currently working for families is proposed to explain the connections between early parenting, child development and maternal wellbeing. However, more research is required to further elucidate the mechanisms by which the intervention creates change for families. This research presents promising evidence that a short term group music therapy program can elicit important therapeutic benefits for families who have a child with a disability.

Comparison of human alveolar osteoblasts cultured on polymer-ceramic composite scaffolds and tissue culture plates

The effects of medical grade polycaprolactone–tricalcium phosphate (mPCL–TCP) (80:20) scaffolds on primary human alveolar osteoblasts (AOs) were compared with standard tissue-culture plates. Of the seeded AOs, 70% adhered to and proliferated on the scaffold surface and within open and interconnected pores; they formed multi-layered sheets and collagen fibers with uniform distribution within 28 days. Elevation of alkaline phosphatase activity occurred in scaffold–cell constructs independent of osteogenic induction. AO proliferation rate increased and significant decrease in calcium concentration of the medium for both scaffolds and plates under induction conditions were seen. mPCL–TCP scaffolds significantly influenced the AO expression pattern of osterix and osteocalcin (OCN). Osteogenic induction down-regulated OCN at both RNA and protein level on scaffolds (3D) by day 7, and up-regulated OCN in cell-culture plates (2D) by day 14, but OCN levels on scaffolds were higher than on cell-culture plates. Immunocytochemical signals for type I collagen, osteopontin and osteocalcin were detected at the outer parts of scaffold–cell constructs. More mineral nodules were found in induced than in non-induced constructs. Only induced 2D cultures showed nodule formation. mPCL–TCP scaffolds appear to stimulate osteogenesis in vitro by activating a cellular response in AO's to form mineralized tissue. There is a fundamental difference between culturing AOs on 2D and 3D environments that should be considered when studying osteogenesis in vitro.

Tissue engineered prefabricated vascularized flaps

BACKGROUND.: Microvascular free tissue transfer has become increasingly popular in the reconstruction of head and neck defects, but it also has its disadvantages. Tissue engineering allows the generation of neo-tissue for implantation, but these tissues are often avascular. We propose to combine tissue-engineering techniques together with flap prefabrication techniques to generate a prefabricated vascularized soft tissue flap. METHODS: Human dermal fibroblasts (HDFs) labeled with fluorescein diacetate were static seeded onto polylactic-co-glycolic acid-collagen (PLGA-c) mesh. Controls were plain PLGA-c mesh. The femoral artery and vein of the nude rat was ligated and used as a vascular carrier for the constructs. After 4 weeks of implantation, the constructs were assessed by gross morphology, routine histology, Masson trichrome, and cell viability determined by green fluorescence. RESULTS: All the constructs maintained their initial shape and dimensions. Angiogenesis was evident in all the constructs with neo-capillary formation within the PLGA-c mesh seen. HDFs proliferated and filled the interyarn spaces of the PLGA-c mesh, while unseeded PLGA-c mesh remained relatively acellular. Cell tracer study indicated that the seeded HDFs remained viable and closely associated to remaining PLGA-c fibers. Collagen formation was more abundant in the constructs seeded with HDFs. CONCLUSIONS: PLGA-c, enveloped by a cell sheet composed of fibroblasts, can serve as a suitable scaffold for generation of a soft tissue flap. A ligated arteriovenous pedicle can serve as a vascular carrier for the generation of a tissue engineered vascularized flap.

Invention and business performance in the tissue-engineering industry

Tissue engineering is a young and interdisciplinary scientific discipline but it offers exciting opportunities to improve the quality of health care for hundreds of thousands of patients. Lured by its potential, several start-up companies, pharmaceutical corporations, and medical device enterprises alike are investing heavily in this sector. Invention is a key driver of competition in this sector. In this study, we aim to explain the variation in inventive output across the different firms in the sector. Our major premise is that firms that forge alliances will be able to tap into the expertise of their partners and thus improve their chances of inventive output. We further argue that alliances that enable technology acquisition or learning will enhance the inventive output of firms more than other kinds of alliances. We measure the inventive output of a company by the number of patents filed. On the basis of a preliminary analysis of seven companies, we find support for the hypotheses. We also argue that, to achieve commercial success, firms need to manage time to market (through alliances or otherwise), have a global outlook, nurture their financial resources, and attain critical mass through mergers.

Vitrification as a prospect for cryopreservation of tissue-engineered constructs

Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation

Effect of surgical approach on bone vascularisation, fracture and soft tissue healing : comparison of less invasive to open approach

Over the past ten years, minimally invasive plate osteosynthesis (MIPO) for the fixation of long bone fractures has become a clinically accepted method with good outcomes, when compared to the conventional open surgical approach (open reduction internal fixation, ORIF). However, while MIPO offers some advantages over ORIF, it also has some significant drawbacks, such as a more demanding surgical technique and increased radiation exposure. No clinical or experimental study to date has shown a difference between the healing outcomes in fractures treated with the two surgical approaches. Therefore, a novel, standardised severe trauma model in sheep has been developed and validated in this project to examine the effect of the two surgical approaches on soft tissue and fracture healing. Twenty four sheep were subjected to severe soft tissue damage and a complex distal femur fracture. The fractures were initially stabilised with an external fixator. After five days of soft tissue recovery, internal fixation with a plate was applied, randomised to either MIPO or ORIF. Within the first fourteen days, the soft tissue damage was monitored locally with a compartment pressure sensor and systemically by blood tests. The fracture progress was assessed fortnightly by x-rays. The sheep were sacrificed in two groups after four and eight weeks, and CT scans and mechanical testing performed. Soft tissue monitoring showed significantly higher postoperative Creatine Kinase and Lactate Dehydrogenase values in the ORIF group compared to MIPO. After four weeks, the torsional stiffness was significantly higher in the MIPO group (p=0.018) compared to the ORIF group. The torsional strength also showed increased values for the MIPO technique (p=0.11). The measured total mineralised callus volumes were slightly higher in the ORIF group. However, a newly developed morphological callus bridging score showed significantly higher values for the MIPO technique (p=0.007), with a high correlation to the mechanical properties (R2=0.79). After eight weeks, the same trends continued, but without statistical significance. In summary, this clinically relevant study, using the newly developed severe trauma model in sheep, clearly demonstrates that the minimally invasive technique minimises additional soft tissue damage and improves fracture healing in the early stage compared to the open surgical approach method.

Fumaric acid monoethyl ester-functionalized poly(D,L-Lactide)/N-vinyl-2- pyrrolidone Resins for the preparation of tissue engineering scaffolds by stereolithography

Polymer networks were prepared by photocross-linking fumaric acid monoethyl ester (FAME) functionalized, three-armed poly(D,L-lactide) oligomers using Af-vinyl-2-pyrrolidone (NVP) as diluent and comonomer. The use of NVP together with FAME-functionalized oligomers resulted in copolymerization at high rates, and networks with gel contents in excess of 90 were obtained. The hydrophilicity of the poly(D,L-lactide) networks increases with increasing amounts of NVP, networks containing 50 wt of NVP absorbed 40 of water. As the amount of NVP was increased from 30 to 50 wt , the Young's modulus after equilibration in water decreased from 0.8 to 0.2 GPa, as opposed to an increase from 1.5 to 2.1 GPa in the dry state. Mouse preosteoblasts readily adhered and spread onto all prepared networks. Using stereolithography, porous structures with a well-defined gyroid architecture were prepared from these novel materials. This allows the preparation of tissue engineering scaffolds with optimized pore architecture and tunable material properties.

A poly(D,L-lactide) resin for the preparation of tissue engineering scaffolds by stereolithography

Porous polylactide constructs were prepared by stereolithography, for the first time without the use of reactive diluents. Star-shaped poly(D,L-lactide) oligomers with 2, 3 and 6 arms were synthesised, end-functionalised with methacryloyl chloride and photocrosslinked in the presence of ethyl lactate as a non-reactive diluent. The molecular weights of the arms of the macromers were 0.2, 0.6, 1.1 and 5 kg/mol, allowing variation of the crosslink density of the resulting networks. Networks prepared from macromers of which the molecular weight per arm was 0.6 kg/mol or higher had good mechanical properties, similar to linear high molecular weight poly(D,L-lactide). A resin based on a 2-armed poly(D,L-lactide) macromer with a molecular weight of 0.6 kg/mol per arm (75 wt%), ethyl lactate (19 wt%), photo-initiator (6 wt%), inhibitor and dye was prepared. Using this resin, films and computer-designed porous constructs were accurately fabricated by stereolithography. Pre-osteoblasts showed good adherence to these photocrosslinked networks. The proliferation rate on these materials was comparable to that on high molecular weight poly(D,L-lactide) and tissue culture polystyrene.

Effects of the architecture of tissue engineering scaffolds on cell seeding and culturing

The advance of rapid prototyping techniques has significantly improved control over the pore network architecture of tissue engineering scaffolds. In this work we assessed the influence of scaffold pore architecture on cell seeding and static culturing, by comparing a computer‐designed gyroid architecture fabricated by stereolithography to a random‐pore architecture resulting from salt‐leaching. The scaffold types showed comparable porosity and pore size values, but the gyroid type showed a more than tenfold higher permeability due to the absence of size‐limiting pore interconnections. The higher permeability significantly improved the wetting properties of the hydrophobic scaffolds, and increased the settling speed of cells upon static seeding of immortalised mesenchymal stem cells. After dynamic seeding followed by 5 days of static culture, gyroid scaffolds showed large cell populations in the centre of the scaffold, while salt‐leached scaffolds were covered with a cell‐sheet on the outside and no cells were found in the scaffold centre. It was shown that interconnectivity of the pores and permeability of the scaffold prolongs the time of static culture before overgrowth of cells at the scaffold periphery occurs. Furthermore, novel scaffold designs are proposed to further improve the transport of oxygen and nutrients throughout the scaffolds, and to create tissue engineering grafts with designed, pre‐fabricated vasculature.

Mathematically defined tissue engineering scaffold architectures prepared by stereolithography

The technologies employed for the preparation of conventional tissue engineering scaffolds restrict the materials choice and the extent to which the architecture can be designed. Here we show the versatility of stereolithography with respect to materials and freedom of design. Porous scaffolds are designed with computer software and built with either a poly(d,l-lactide)-based resin or a poly(d,l-lactide-co-ε-caprolactone)-based resin. Characterisation of the scaffolds by micro-computed tomography shows excellent reproduction of the designs. The mechanical properties are evaluated in compression, and show good agreement with finite element predictions. The mechanical properties of scaffolds can be controlled by the combination of material and scaffold pore architecture. The presented technology and materials enable an accurate preparation of tissue engineering scaffolds with a large freedom of design, and properties ranging from rigid and strong to highly flexible and elastic.