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.

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

Regional changes in corneal thickness and shape with soft contact lenses

Purpose: To investigate the influence of soft contact lenses on regional variations in corneal thickness and shape while taking account of natural diurnal variations in these corneal parameters. Methods: Twelve young, healthy subjects wore 4 different types of soft contact lenses on 4 different days. The lenses were of two different materials (silicone hydrogel, hydrogel), designs (spherical, toric) and powers (–3.00, –7.00 D). Corneal thickness and topography measurements were taken before and after 8 hours of lens wear and on two days without lens wear, using the Pentacam HR system. Results: The hydrogel toric contact lens caused the greatest level of corneal thickening in the central (20.3 ± 10.0 microns) as well as peripheral cornea (24.1 ± 9.1 microns) (p < 0.001) with an obvious regional swelling of the cornea beneath the stabilizing zones. The anterior corneal surface generally showed slight flattening. All contact lenses resulted in central posterior corneal steepening and this was weakly correlated with central corneal swelling (p = 0.03) and peripheral corneal swelling (p = 0.01). Conclusions: There was an obvious regional corneal swelling apparent after wear of the hydrogel soft toric lenses, due to the location of the thicker stabilization zones of the toric lenses. However with the exception of the hydrogel toric lens, the magnitude of corneal swelling induced by the contact lenses over the 8 hours of wear was less than the natural diurnal thinning of the cornea over this same period.

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.

A review of rapid prototyping techniques for tissue engineering purposes

Rapid prototyping (RP) is a common name for several techniques, which read in data from computer-aided design (CAD) drawings and manufacture automatically threedimensional objects layer-by-layer according to the virtual design. The utilization of RP in tissue engineering enables the production of three-dimensional scaffolds with complex geometries and very fine structures. Adding micro- and nanometer details into the scaffolds improves the mechanical properties of the scaffold and ensures better cell adhesion to the scaffold surface. Thus, tissue engineering constructs can be customized according to the data acquired from the medical scans to match the each patient’s individual needs. In addition RP enables the control of the scaffold porosity making it possible to fabricate applications with desired structural integrity. Unfortunately, every RP process has its own unique disadvantages in building tissue engineering scaffolds. Hence, the future research should be focused into the development of RP machines designed specifically for fabrication of tissue engineering scaffolds, although RP methods already can serve as a link between tissue and engineering.

Photo-crosslinking of functionalised lactide oligomers for the fabrication of osteochondral tissue engineering scaffolds

In the fabrication of osteochondral tissue engineering scaffolds, the two distinct tissues impose different requirements on the architecture. Stereo-lithography is a rapid prototyping method that can be utilised to make 3D constructs with high spatial control by radical photopolymerization. In this study, biodegradable resins are developed that can be applied in stereo-lithography. Photo-crosslinked poly(lactide) networks with varying physical properties were synthesised, and by photo polymerizing in the presence of leachable particles porous scaffolds could be prepared as well.

Designed tissue engineering scaffolds prepared by stereolithography

For the fabrication of tissue engineering scaffolds, the intended tissue formation process imposes requirements on the architecture. The chosen porosity often is a tradeoff between volume and surface area accessible to cells, and mechanical properties of the construct. Interconnectivity of the pores is essential for cell migration through the scaffold and for mass transport. Conventional techniques such as salt leaching often result in heterogeneous structures and do not allow for a precise control of the architecture. Stereolithography is a rapid prototyping method that can be utilised to make 3D constructs with high spatial control by radical photopolymerisation. In this study, a regular structure based on cyclic repetition of cell units were designed through CAD modelling.. One of these structures was built on a stereolithography apparatus (SLA). Furthermore, a polylactide-based resin was developed that can be applied in stereolithography. Polylactide has proven before to be a well-performing polymer in bone tissue engineering. The final objective in this study is to build newly designed PDLLA scaffolds with a precise SLA fabrication technique to study the effect of scaffold architecture on mechanical and biological properties.

Development of a PDLLA-based stereolithography resin for making tissue engineering scaffolds

The use of porous structures as tissue engineering scaffolds imposes high demands on the pore architecture. Stereolithography is a rapid prototyping method based on photo-polymerisation, that can be utilised to make 3D constructs with high spatial control. In this study, biodegradable resins were developed that can find application in stereolithography. Poly(D,L-lactide) (PDLLA) oligomers were synthesised and functionalised with methacrylate end-groups. By mixing the resulting macromers with a diluent, photo-initiator and inhibitor, lowviscosity resins were obtained that were photocrosslinked to yield stiff and strong degradable poly(lactide) networks. Also, porous scaffolds were fabricated on a stereolithography apparatus (SLA) from a nondegradable resin.

Micro-CT analysis of tissue engineering scaffold architectures

A novel method was developed for a quantitative assessment of pore interconnectivity using micro-CT data. This method makes use of simulated spherical particles, percolating through the interconnected pore network. For each sphere diameter, the accessible pore volume is calculated. This algorithm was applied to compare pore interconnectivity of two different scaffold architectures; one created by salt-leaching and the other by stereolithography. The algorithm revealed a much higher pore interconnectivity for the latter one.